New Technologies in Aquaculture: Improving Production Efficiency, Quality and Environmental Management (Woodhead Publishing Series in Food Science, Technology and Nutrition)

New technologies in aquaculture

Related titles: Improving farmed fish quality and safety (ISBN 978-1-84569-299-5)

Fish farming enables greater control of product quality, but there have been concerns about the levels of contaminants found in farmed products. Their sensory and nutritional quality can also not equal that of wild-caught fish. This important collection reviews potential negative safety and quality issues in farmed fish and presents methods to improve product characteristics. The first part of the book discusses contaminants, such as persistent organic pollutants and veterinary drug residues and methods for their reduction and control. The second part addresses important quality issues, such as genetic control of flesh characteristics and the effects of feed on product nutritional and sensory quality. Improving seafood products for the consumer (ISBN 978-1-84569-019-9)

Consumer health and well-being may potentially be improved by consumption of health promoting, safe seafood products of high eating quality. This book provides a convenient review of significant findings in key areas of seafood research, with chapters authored by experts in the field. Consumer attitude to seafood products is first discussed, followed by chapters analysing important advances in the area of the health benefits of seafood, for example the relationship between omega-3 fatty acids and heart disease. Subsequent parts of the book discuss key topics in seafood safety, advances in processing technologies and methods to improve the quality of farmed fish. Shellfish safety and quality (ISBN 978-1-84569-152-3)

Shellfish are a very popular and nutritious food source worldwide and their consumption has risen dramatically. Because of their unique nature as compared to beef and poultry, shellfish have their own distinct aspects of harvest, processing and handling. Edited by leading authorities in the field, this collection reviews issues of current interest and outlines steps that can be taken by the shellfish industry to improve shellfish safety and eating quality. Opening chapters consider microbial, biotoxin, metal and organic contaminants of shellfish. Techniques to reduce contamination are then discussed, such as mitigation of the effects of harmful algal blooms. Chapters also address approaches to managing disease and other methods to improve quality, such as improved packaging methods and reduction of biofouling. Details of these books and a complete list of Woodhead’s titles can be obtained by: • visiting our web site at www.woodheadpublishing.com • contacting Customer Services (e-mail: [email protected]; fax: +44 (0) 1223 893694; tel.: +44 (0) 1223 891358 ext.130; address: Woodhead Publishing Limited, Abington Hall, Granta Park, Great Abington, Cambridge CB21 6AH, UK)

New technologies in aquaculture Improving production efficiency, quality and environmental management Edited by Gavin Burnell and Geoff Allan

Oxford

Cambridge

New Delhi

Published by Woodhead Publishing Limited, Abington Hall, Granta Park, Great Abington, Cambridge CB21 6AH, UK www.woodheadpublishing.com Woodhead Publishing India Private Limited, G-2, Vardaan House, 7/28 Ansari Road, Daryaganj, New Delhi – 110002, India Published in North America by CRC Press LLC, 6000 Broken Sound Parkway, NW, Suite 300, Boca Raton, FL 33487, USA First published 2009, Woodhead Publishing Limited and CRC Press LLC © Woodhead Publishing Limited, 2009, except Chapters 26 and 27 which are © The State of Queensland (through the Department of Primary Industries and Fisheries), 2009 The authors have asserted their moral rights. This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the authors and the publishers cannot assume responsibility for the validity of all materials. Neither the authors nor the publishers, nor anyone else associated with this publication, shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming and recording, or by any information storage or retrieval system, without permission in writing from Woodhead Publishing Limited. The consent of Woodhead Publishing Limited does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from Woodhead Publishing Limited for such copying. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. Library of Congress Cataloging in Publication Data A catalog record for this book is available from the Library of Congress. Woodhead Publishing ISBN 978-1-84569-384-8 (book) Woodhead Publishing ISBN 978-1-84569-647-4 (e-book) CRC Press ISBN 978-1-4398-0109-3 CRC Press order number: N10010 The publishers’ policy is to use permanent paper from mills that operate a sustainable forestry policy, and which has been manufactured from pulp which is processed using acid-free and elemental chlorine-free practices. Furthermore, the publishers ensure that the text paper and cover board used have met acceptable environmental accreditation standards. Typeset by SNP Best-set Typesetter Ltd., Hong Kong Printed by TJ International Limited, Padstow, Cornwall, UK

Contents

Contributor contact details......................................................................... xix Preface.......................................................................................................... xxix

Part I 1

2

Genetic improvement and reproduction ...................................

Genome-based technologies useful for aquaculture research and genetic improvement of aquaculture species .................................. Z. Liu, Auburn University, USA 1.1 Introduction.............................................................................. 1.2 DNA marker technologies ..................................................... 1.3 DNA sequencing technologies .............................................. 1.4 Gene discovery technologies ................................................. 1.5 Genome mapping technologies ............................................. 1.6 Genome expression analysis technologies ........................... 1.7 Acknowledgements ................................................................. 1.8 References ................................................................................ Genetic improvement of finfish ........................................................ G. Hulata, Agricultural Research Organization, Israel, and B. Ron, Israel Oceanographic & Limnological Research Ltd, Israel 2.1 Introduction: current status of aquaculture genetics ......... 2.2 Key drivers for genetic improvement of finfish .................. 2.3 Case studies – risks associated with selective breeding programs ...................................................................................

1

3 3 4 18 26 28 35 41 41 55

55 56 69

vi

Contents 2.4 2.5 2.6 2.7

3

4

5

6

Future trends ............................................................................ Sources of further information and advice .......................... Acknowledgement................................................................... References ................................................................................

Genetic variation and selective breeding in hatchery-propagated molluscan shellfish ........................................ P. Boudry, Ifremer, France 3.1 Introduction.............................................................................. 3.2 Monitoring genetic diversity and risks related to inbreeding ................................................................................. 3.3 Inheritance of traits important for aquaculture .................. 3.4 Current status of established molluscan shellfish breeding programs................................................................... 3.5 Present needs and future trends: use of marker assisted selection and genomics ........................................................... 3.6 References ................................................................................ Controlling fish reproduction in aquaculture ................................. C. Mylonas, Hellenic Center for Marine Research, Greece, and Y. Zohar, University of Maryland Biotechnology Institute, USA 4.1 Introduction.............................................................................. 4.2 The fish reproductive cycle and its control .......................... 4.3 Reproductive strategies and dysfunctions in captivity ....... 4.4 Hormonal therapies for the control of reproduction ......... 4.5 Induction of oocyte maturation and ovulation ................... 4.6 Induction of spermiation ........................................................ 4.7 Spontaneous spawning versus artificial insemination ........ 4.8 Future trends ............................................................................ 4.9 Sources of further information and advice .......................... 4.10 References ................................................................................ Producing sterile and single-sex populations of fish for aquaculture .......................................................................................... T. Benfey, University of New Brunswick, Canada 5.1 Introduction.............................................................................. 5.2 Sterile populations................................................................... 5.3 Single-sex populations ............................................................ 5.4 Future trends and further reading ........................................ 5.5 References ................................................................................ Chromosome set manipulation in shellfish ..................................... X. Guo, Y. Wang, Z. Xu, Rutgers University, USA, and H. Yang, Louisiana State University Agriculture Center, USA 6.1 Introduction..............................................................................

71 72 72 72 87 87 88 90 92 97 100 109

110 110 116 118 122 126 127 128 130 130 143 143 144 154 157 159 165

165

Contents 6.2 6.3 6.4 6.5 6.6 6.7 6.8

Part II 7

8

9

vii

Principles and methods of chromosome set manipulation ............................................................................ Triploid shellfish....................................................................... Tetraploid shellfish .................................................................. Gynogensis, androgenesis and aneuploids ........................... Summary and perspectives ..................................................... Acknowledgements ................................................................. References ................................................................................

166 174 183 187 187 188 188

Health ...........................................................................................

195

Advances in disease diagnosis, vaccine development and other emerging methods to control pathogens in aquaculture .......................................................................................... A. Adams, University of Stirling, UK 7.1 Introduction.............................................................................. 7.2 Key drivers to improve disease diagnosis and vaccine development ............................................................................. 7.3 Limitations of current diagnostic methods .......................... 7.4 Advances in methods of disease diagnosis (mainly for bacterial diseases) ............................................................. 7.5 Advances in vaccine development ........................................ 7.6 Other emerging methods to control pathogens .................. 7.7 Future trends ............................................................................ 7.8 Sources of further information and advice .......................... 7.9 References ................................................................................ Controlling parasitic diseases in aquaculture: new developments .............................................................................. C. Sommerville, University of Stirling, UK 8.1 Introduction.............................................................................. 8.2 Effects of parasitic disease in aquaculture........................... 8.3 Advances in the understanding of parasite biology and host–parasite interactions ............................................... 8.4 Advances in methods of identifying parasites..................... 8.5 Advances in methods of controlling parasites .................... 8.6 Future trends ............................................................................ 8.7 References ................................................................................ Controlling viral diseases in aquaculture: new developments...... T. Renault, Ifremer, France 9.1 Introduction.............................................................................. 9.2 Overview of viral diseases in aquaculture ........................... 9.3 Limitation of current management techniques ...................

197 197 198 198 199 203 207 208 209 211

215 215 216 218 220 221 237 237 244 244 245 248

viii

Contents 9.4 9.5 9.6

10

Diet and husbandry techniques to improve disease resistance: new technologies and prospects .................................... F. J. Gatesoupe, INRA-Ifremer, France 10.1 Introduction.............................................................................. 10.2 Fighting the pathogens............................................................ 10.3 Improving welfare ................................................................... 10.4 Improving feed......................................................................... 10.5 Concluding remarks ................................................................ 10.6 Sources of further information and advice .......................... 10.7 References ................................................................................

Part III 11

12

Advances in understanding of immunity of aquacultured species to viral diseases .................................. New methods to control viral diseases in aquaculture and future trends ..................................................................... References ................................................................................

249 254 259

267 267 268 273 276 289 290 291

Diet and husbandry ..................................................................

313

Fish larvae nutrition and diet: new developments ......................... S. Kolkovski, Dept of Fisheries, Australia, J. Lazo, Fish Nutrition Laboratory, Mexico, D. Leclercq, ACUI-T, France, and M. Izquierdo, Grupo de Investigación en Acuicultura, Spain 11.1 Introduction.............................................................................. 11.2 Determination of nutritional requirements of larvae ........ 11.3 Nutritional requirements of fish larvae ................................ 11.4 Feed identification and ingestion .......................................... 11.5 Ontogeny of digestive capacity in marine fish larvae .................................................................................. 11.6 Digestive system capacity ....................................................... 11.7 Diet manufacturing methods ................................................. 11.8 Microdiet characteristics......................................................... 11.9 Feeding system ......................................................................... 11.10 Dosage system ......................................................................... 11.11 Future directions ..................................................................... 11.12 References ................................................................................

315

Aquaculture feeds and ingredients: an overview ........................... R. Hardy, University of Idaho, USA 12.1 Introduction.............................................................................. 12.2 Sustainability of feed ingredients .......................................... 12.3 Safety of farmed fish products from harmful residues and pollutants...........................................................................

315 319 322 332 336 343 346 349 354 355 359 360 370 370 371 374

Contents 12.4

12.5 12.6 12.7 12.8

13

14

15

Categories of environmental pollutants and residues comprising risks to the safety of farmed fish products .................................................................................... Alternate protein and lipid sources ...................................... Future trends ............................................................................ Sources of further information and advice .......................... References ................................................................................

Ingredient evaluation in aquaculture: digestibility, utilisation and other key nutritional parameters ........................... B. Glencross, CSIRO Marine and Atmospheric Research, Australia 13.1 Introduction.............................................................................. 13.2 Characterisation and preparation of ingredients ................ 13.3 Defining ingredient digestibility ............................................ 13.4 Ingredient palatability............................................................. 13.5 Defining effects on growth and utilisation........................... 13.6 Ingredient functionality and feed technical qualities ......... 13.7 Frontier technologies for ingredient evaluation.................. 13.8 References ................................................................................

Quantifying nutritional requirements in aquaculture: the factorial approach ........................................................................ I. Lupatsch, Swansea University, UK 14.1 Introduction.............................................................................. 14.2 Quantification of nutritional requirements .......................... 14.3 Feed ingredient evaluation..................................................... 14.4 Feed formulation and feeding strategies .............................. 14.5 Future trends ............................................................................ 14.6 References ................................................................................

Advances in aquaculture nutrition: catfish, tilapia and carp nutrition ...................................................................................... D. Davis, Auburn University, USA, T. Nguyen, Nong Lam University, Vietnam, M. Li, National Warmwater Aquaculture Center, USA, D. M. Gatilin III, Department of Wildlife and Fisheries Sciences, USA, and T. O’Keefe, Aqua-Food Technologies, Inc., USA 15.1 Introduction.............................................................................. 15.2 Nutrient requirements ............................................................ 15.3 Sources of further information and advice .......................... 15.4 References ................................................................................

ix

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387

388 390 393 399 402 408 408 410

417 417 419 431 433 437 438

440

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x 16

17

18

19

Contents Advances in aquaculture feeds and feeding: basses and breams .... M. Booth, New South Wales Department of Primary Industries, Australia 16.1 Introduction.............................................................................. 16.2 Asian seabass ........................................................................... 16.3 Red sea bream and gilthead sea bream ............................... 16.4 Grouper .................................................................................... 16.5 Future trends ............................................................................ 16.6 References ................................................................................

459

Advances in aquaculture feeds and feeding: salmonids ................ S. Refstie, Nofima Marin and Aquaculture Protein Centre (APC), Norway, and T. Åsgård, Nofima Marin, Norway 17.1 Introduction.............................................................................. 17.2 Feed technology and formulation ......................................... 17.3 Digestive physiology ............................................................... 17.4 Nutritional requirements ........................................................ 17.5 Nutrition and health................................................................ 17.6 Dietary additives ..................................................................... 17.7 Species differences .................................................................. 17.8 Practical formulations ............................................................. 17.9 Feeding and feeding systems ................................................. 17.10 Future trends ............................................................................ 17.11 References ................................................................................

498

Monitoring viral contamination in shellfish growing areas ...................................................................................... F. S. Le Guyader and M. Pommepuy, Ifremer, France, and R. L. Atmar, Baylor College of Medicine, USA 18.1 Introduction.............................................................................. 18.2 Source of pollution .................................................................. 18.3 Methods .................................................................................... 18.4 Input and flux ........................................................................... 18.5 Strategies for reducing contamination ................................. 18.6 Other issues .............................................................................. 18.7 Future trends ............................................................................ 18.8 References ................................................................................ Impacts of harmful algal bloom on shellfisheries aquaculture .......................................................................................... Y. Matsuyama, National Research Institute of Fisheries and Environment of Inland Sea, Japan, and S. Shumway, University of Connecticut, USA 19.1 Introduction.............................................................................. 19.2 Global increase of harmful algal blooms (HAB) ...............

459 461 466 476 483 484

498 500 501 506 507 511 514 515 517 518 522

542

542 544 550 553 562 565 568 569

580

580 581

Contents 19.3 19.4 19.5 19.6 19.7 20

Advances in microalgal culture for aquaculture feed and other uses ............................................................................................. M. R. Tredici, N. Biondi, E. Ponis, L. Rodolfi, Università degli Studi di Firenze, Italy, and G. Chini Zittelli, Istituto per lo Studio degli Ecosistemi, Italy 20.1 Introduction.............................................................................. 20.2 Current status and new techniques for microalgae culture ....................................................................................... 20.3 Microalgae for aquaculture feed ........................................... 20.4 Microalgae as dietary supplements, animal feed and nutraceuticals ........................................................................... 20.5 Microalgae as source of pharmaceuticals and probiotics .................................................................................. 20.6 Wastewater reclamation and biofuel production by algae–bacteria consortia ......................................................... 20.7 Future trends ............................................................................ 20.8 Sources of further information and advice .......................... 20.9 References ................................................................................

Part IV 21

Impact of harmful algal bloom species on shellfisheries industries ........................................................... Prevention of harmful algal bloom threats .......................... Conclusions .............................................................................. Acknowledgements ................................................................. References ................................................................................

Environmental issues ................................................................

Predicting and assessing the environmental impact of aquaculture .......................................................................................... C. Crawford and C. MacLeod, University of Tasmania, Australia 21.1 Introduction.............................................................................. 21.2 Interactions between aquaculture and the environment ............................................................................. 21.3 Site selection and carrying capacity ...................................... 21.4 Considerations in developing an environmental monitoring and assessment program .................................... 21.5 Monitoring and assessment techniques ................................ 21.6 Recent technological advances and future trends .............. 21.7 Sources of further information and advice .......................... 21.8 References ................................................................................

xi

587 595 601 601 602

610

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679 681 684 686 690 696 699 700

xii

Contents

22

Spatial decision support in aquaculture: the role of geographical information systems and remote sensing ................. L. G. Ross, N. Handisyde, D.-C. Nimmo, University of Stirling, Scotland 22.1 The spatial planning context .................................................. 22.2 Database construction and project methodology ............... 22.3 Decision support systems and tools ...................................... 22.4 Selected applications and examples of geographical information systems in aquaculture............... 22.5 Case study: climate change .................................................... 22.6 Case study: multi-site coastal zone planning ....................... 22.7 Summary and future trends ................................................... 22.8 Acknowledgements ................................................................. 22.9 References ................................................................................

23

Zooremediation of contaminated aquatic systems through aquaculture initiatives ........................................................................ S. Gifford, G. R. MacFarlane, C. E. Koller, R. H. Dunstan, The University of Newcastle, Australia, and W. O’Connor, NSW Department of Primary Industries, Australia 23.1 Introduction.............................................................................. 23.2 Zooremediation of pollutants ................................................ 23.3 Zooremediation and pearl aquaculture: a case study .............................................................................. 23.4 Future trends ............................................................................ 23.5 Sources of further information and advice .......................... 23.6 References ................................................................................

Part V 24

Farming new species ...................................................................

Farming cod and halibut: biological and technological advances in two emerging cold-water marine finfish aquaculture species ............................................................................ V. Puvanendran and A. Mortensen, Nofima Marin, Norway 24.1 Introduction.............................................................................. 24.2 Atlantic cod .............................................................................. 24.3 Atlantic halibut ........................................................................ 24.4 Future trends ............................................................................ 24.5 Sources of further information and advice .......................... 24.6 Acknowledgements ................................................................. 24.7 References ................................................................................

707

707 711 718 720 726 739 745 746 747

750

750 751 758 761 764 764

769

771 772 774 789 794 797 798 798

25

26

27

28

Contents

xiii

Cobia cultivation................................................................................. E. McLean, Ministry of Fisheries Wealth, Sultanate of Oman, G. Salze, Virginia-Maryland Regional College of Veterinary Medicine, USA, M. H. Schwarz, Virginia Seafood AREC, USA, and S. R. Craig, Virginia Cobia Farms LLC, USA 25.1 Introduction.............................................................................. 25.2 Broodstock and spawning ...................................................... 25.3 Larval rearing........................................................................... 25.4 Juveniles and on-growing ....................................................... 25.5 Emerging issues and future trends........................................ 25.6 References ................................................................................

804

804 805 807 812 816 818

Advances in the culture of lobsters ................................................. C. M. Jones, Northern Fisheries Centre, Australia 26.1 Introduction.............................................................................. 26.2 Current situation and constraints .......................................... 26.3 Advances in culture ................................................................ 26.4 Production systems .................................................................. 26.5 Product issues: markets........................................................... 26.6 Future trends ............................................................................ 26.7 Sources of further information and advice .......................... 26.8 References ................................................................................

822

Advances in the culture of crabs ...................................................... B. D. Paterson, Queensland Department of Primary Industries and Fisheries, Australia 27.1 Introduction.............................................................................. 27.2 Current situation ..................................................................... 27.3 Product issues........................................................................... 27.4 Production systems .................................................................. 27.5 Future trends ............................................................................ 27.6 Sources of further information and advice .......................... 27.7 References ................................................................................

845

Aquaculture and the production of pharmaceuticals and nutraceuticals ...................................................................................... K. Benkendorff, Flinders University of South Australia, Australia 28.1 Introduction.............................................................................. 28.2 Marine pharmaceuticals ......................................................... 28.3 Marine nutraceuticals ............................................................. 28.4 Diversifying the aquaculture industry .................................. 28.5 Current case studies ................................................................

822 823 827 832 835 835 836 836

845 848 848 851 859 860 860

866

866 870 872 874 875

xiv

Contents 28.6 28.7 28.8 28.9

Steps towards commercialisation .......................................... Future trends ............................................................................ Acknowledgements ................................................................. References ................................................................................

884 886 887 887

Part VI Aquaculture systems design .....................................................

893

29

895

30

31

Opportunities and challenges for off-shore farming ..................... R. Langan, University of New Hampshire, USA 29.1 The context for off-shore farming ......................................... 29.2 Characterization and selection of off-shore sites ................ 29.3 Finfish species cultivated in off-shore cages ........................ 29.4 Off-shore mollusc culture ....................................................... 29.5 Environmental concerns ......................................................... 29.6 Future trends ............................................................................ 29.7 References ................................................................................ Advances in technology for off-shore and open ocean aquaculture .......................................................................................... A. Fredheim, SINTEF Fisheries and Aquaculture, Norway, and R. Langan, University of New Hampshire, USA 30.1 Introduction: historical development of fish farming technology ................................................................................ 30.2 Floating fish farm design ........................................................ 30.3 Current status and technical limitations............................... 30.4 Novel fish farm systems .......................................................... 30.5 Supporting technologies for off-shore and open ocean fish farming ............................................................................... 30.6 Sources of further information and advice .......................... 30.7 References ................................................................................ Advances in technology and practice for land-based aquaculture systems: tank-based recirculating systems for finfish production ................................................................................ T. Losordo, D. DeLong and T. Guerdat, North Carolina State University, USA 31.1 Introduction.............................................................................. 31.2 Components in recirculating systems design ....................... 31.3 Types of particulate waste solids ........................................... 31.4 Tank, water input manifolds, and drain design.................... 31.5 Settleable solids capture components................................... 31.6 Suspended solids capture components ................................. 31.7 Biological filtration .................................................................. 31.8 Oxygenation components and processes .............................

895 897 899 901 904 909 910

914

914 918 924 930 941 942 942

945

945 947 948 948 955 957 962 970

Contents 31.9 Sterilization components and processes ............................... 31.10 Comparing freshwater and marine systems design ............ 31.11 An example of a modern approach to a complete systems design .......................................................................... 31.12 References ................................................................................ 32

33

34

Advances in technology and practice for land-based aquaculture systems: ponds for finfish production......................... C. E. Boyd, Auburn University, USA, and S. Chainark, Phuket Rajabhat University, Thailand 32.1 Introduction.............................................................................. 32.2 Hydrologic types of ponds ..................................................... 32.3 Production methodology ........................................................ 32.4 Liming and fertilization .......................................................... 32.5 Feeds and feed management ................................................. 32.6 Dissolved oxygen management ............................................. 32.7 Pond amendments ................................................................... 32.8 Pond bottom treatments ......................................................... 32.9 Water quality monitoring ....................................................... 32.10 Pond effluents........................................................................... 32.11 Future trends ............................................................................ 32.12 References ................................................................................ Superintensive bio-floc production technologies for marine shrimp Litopenaeus vannamei: technical challenges and opportunities ............................................................ C. L. Browdy, J. A. Venero, A. D. Stokes and J. Leffler, Marine Resources Research Institute, USA 33.1 Introduction.............................................................................. 33.2 Superintensive bio-floc-based shrimp production systems .... 33.3 Components of superintensive bio-floc-based shrimp production systems .................................................................. 33.4 Current research priorities ..................................................... 33.5 Conclusions .............................................................................. 33.6 Acknowledgements ................................................................. 33.7 References ................................................................................ Traditional Asian aquaculture .......................................................... P. Edwards, Asian Institute of Technology, Thailand 34.1 Introduction.............................................................................. 34.2 Definitions and principles ...................................................... 34.3 Traditional aquaculture systems ............................................ 34.4 Recent changes to traditional practice ................................. 34.5 Research and development for improved traditional practice ......................................................................................

xv 973 975 977 979

984

984 985 988 989 992 994 997 1002 1003 1004 1006 1006

1010

1010 1011 1013 1017 1024 1025 1026 1029 1029 1030 1034 1037 1041

xvi

Contents 34.6 34.7 34.8 34.9 34.10

35

36

37

Recent development of semi-intensive aquaculture........... Bridging traditional and modern practice............................ Future trends ............................................................................ Sources of further information and advice .......................... References ................................................................................

1047 1050 1054 1056 1056

Use of information technology in aquaculture .............................. J. Bostock, University of Stirling, UK 35.1 Introduction.............................................................................. 35.2 Information and communications technology (ICT) for productivity and effectiveness ......................................... 35.3 ICT for quality and customer service ................................... 35.4 ICT in aquaculture innovation and learning ....................... 35.5 Conclusions .............................................................................. 35.6 Acknowledgements ................................................................. 35.7 Sources of further information and advice .......................... 35.8 References ................................................................................

1064

Inland saline aquaculture .................................................................. G. L. Allan and D. S. Fielder, New South Wales Department of Primary Industries, Australia, K. M. Fitzsimmons, University of Arizona, USA, S. L. Applebaum, Jacob Blaustein Institute for Desert Research BGU, Israel, and S. Raizada, Central Institute of Fisheries Education Rohtak Centre (ICAR), India 36.1 Introduction.............................................................................. 36.2 Saline groundwater from interception schemes to protect agriculture ................................................................... 36.3 Coal bed methane waste water ............................................. 36.4 Chemistry and remediation.................................................... 36.5 Case studies .............................................................................. 36.6 Future trends ............................................................................ 36.7 References ................................................................................

1119

Urban aquaculture: using New York as a model ........................... M. P. Schreibman and C. Zarnoch, City University of New York, USA 37.1 Introduction.............................................................................. 37.2 Goals ......................................................................................... 37.3 Technology................................................................................ 37.4 Potential urban aquaculture programs ................................. 37.5 The economics: siting, processing, and marketing for economic success ............................................................... 37.6 Marketing and competition.................................................... 37.7 The role of the university .......................................................

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1119 1121 1123 1126 1128 1144 1145

1148 1151 1152 1153 1157 1158 1159

Contents

xvii

37.8 Future trends ............................................................................ 1159 37.9 Acknowledgements ................................................................. 1161 37.10 References ................................................................................ 1161

Index ............................................................................................................. 1163

Contributor contact details

(* = main contact)

Chapter 1

Editors

Zhanjiang Liu The Fish Molecular Genetics and Biotechnology Laboratory Department of Fisheries and Allied Aquacultures and Program of Cell and Molecular Biosciences Aquatic Genomics Unit Auburn University Auburn AL 36849 USA

Gavin Burnell Director Aquaculture and Fisheries Development Centre University College Cork Cork Ireland E-mail: [email protected] Geoff L. Allan Port Stephens Fisheries Institute New South Wales Department of Primary Industries Locked Bag 1 Nelson Bay NSW 2315 Australia E-mail: [email protected]. gov.au

E-mail: [email protected]

Chapter 2 Gideon Hulata* Head, Institute of Animal Science Agricultural Research Organization The Volcani Center PO Box 6, Bet Dagan 50250 Israel E-mail: [email protected]

xx

Contributor contact details

Tetsuzan (Benny) Ron Aquaculture Program Coordinator Office of the Vice Chancellor for Research and Graduate Education University of Hawaii at Manoa 1960 East-West Road Biomed T-701A Honolulu HI 96822 Hawaii

Chapter 5

E-mail: [email protected]

Chapter 6

Chapter 3 Pierre Boudry Ifremer – UMR M100 Physiologie et Ecophysiologie des Mollusques Marins 29280 Plouzané France E-mail: [email protected]

Tillmann J. Benfey Department of Biology University of New Brunswick PO Box 4400 Fredericton New Brunswick E3B 5A3 Canada E-mail: [email protected]

Ximing Guo,* Yongping Wang, Zhe Xu Haskin Shellfish Research Laboratory Institute of Marine and Coastal Sciences Rutgers University 6959 Miller Avenue Port Norris NJ 08349 USA

Chapter 4

E-mail: [email protected]

Constantinos C. Mylonas* Institute of Aquaculture Hellenic Center for Marine Research PO Box 2214 Heraklion 71003 Crete Greece

Huiping Yang Aquaculture Research Station Louisiana State University Agriculture Center 2410 Ben Hur Road Baton Rouge LA 70820 USA

E-mail: [email protected] Yonathan Zohar Center of Marine Biotechnology University of Maryland Biotechnology Institute 701 E. Pratt Baltimore MD 21202 USA E-mail: [email protected]

Chapter 7 Alexandra Adams Institute of Aquaculture University of Stirling Stirling FK9 4LA Scotland UK E-mail: [email protected]

Contributor contact details

Chapter 8 Christina Sommerville Institute of Aquaculture University of Stirling Stirling FK9 4LA Scotland UK E-mail: [email protected]

Juan Pablo Lazo Fish Nutrition Laboratory Centro de Investigación Científica y de Educación Superior de Ensenada BC Organismo Descentralizado de Interés Público Km. 107 Carr. Tijuana-Ensenada Mexico

Chapter 9

E-mail: [email protected]

T. Renault Ifremer Laboratoire de Génétique et Pathologie 17390 La Tremblade France

Didier Leclercq ACUI-T 129 Bd St Aignan 44100 Nantes France

E-mail: [email protected]

Chapter 10 F. J. Gatesoupe INRA-Ifremer UMR1067 Nutrition Aquaculture et Génomique BP 70 F-29280 Plouzané France

E-mail: [email protected] Marisol Izquierdo Grupo de Investigación en Acuicultura ULPGC & ICCM PO Box 56 35200 Telde Las Palmas de Gran Canaria Spain E-mail: [email protected]

E-mail: [email protected]

Chapter 12 Chapter 11 Sagiv Kolkovski* Department of Fisheries Western Australia PO Box 20 North Beach WA 6920 Australia E-mail: [email protected]. gov.au

xxi

Ronald W. Hardy Director Aquaculture Research Institute University of Idaho 3059F National Fish Hatchery Road Hagerman ID 83332 USA E-mail: [email protected]

xxii

Contributor contact details

Chapter 13 Brett D. Glencross CSIRO Marine and Atmospheric Research PO Box 120 Cleveland QLD 4163 Australia E-mail: [email protected]

Chapter 14 Ingrid Lupatsch Centre for Sustainable Aquaculture Research Swansea University Singleton Park Swansea SA2 8PP UK E-mail: [email protected]

Chapter 15 Donald Allen Davis* Department of Fisheries and Allied Aquacultures Auburn University 203 Swingle Hall Auburn AL 36849-5419 USA E-mail: [email protected] Tri N. Nguyen Faculty of Fisheries Nong Lam University Thu Duc District Ho Chi Minh City Vietnam E-mail: [email protected]

Menghe Li National Warmwater Aquaculture Center 127 Experiment Station Road PO Box 197 Stoneville MS 38776-0197 USA E-mail: [email protected] Delbert M. Gatlin III Department of Wildlife and Fisheries Sciences 216 Heep Laboratory Building 2258 TAMUS College Station TX 77843-2258 USA E-mail: [email protected] Tim O’Keefe Aqua-Food Technologies, Inc. 3192 Matecumbe Key Road Punta Gorda FL 33955 USA

Chapter 16 Mark Booth New South Wales Department of Primary Industries Port Stephens Fisheries Institute Taylors Beach NSW 2316 Australia E-mail: [email protected]. au

Contributor contact details

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Chapter 17

Chapter 19

S. Refstie* and T. Åsgård Nofima Marin NO-6600 Sunndalsøra Norway

Yukihiko Matsuyama* Harmful Algal Bloom Division National Research Institute of Fisheries and Environment of Inland Sea Maruishi Hatsukaichi Hiroshima 7390452 Japan

S. Refstie Aquaculture Protein Centre (APC), CoE Norway E-mail: [email protected] [email protected]

Chapter 18 Françoise S. Le Guyader* Laboratoire de Microbiologie Ifremer BP 21105 44311 Nantes cedex 03 France

E-mail: [email protected] Sandra E. Shumway Department of Marine Sciences University of Connecticut 1080 Shennecossett Road Groton CT 06340 USA E-mail: sandra.shumway@uconn. edu

E-mail: [email protected] Monique Pommepuy Laboratoire de Microbiologie Ifremer BP 70 29280 Plouzané France E-mail: [email protected] Robert L. Atmar Departments of Medicine and Molecular Virology & Microbiology Baylor College of Medicine 1 Baylor Plaza MS BCM280 Houston TX 77030 USA E-mail: [email protected]

Chapter 20 Mario R. Tredici,* Natascia Biondi, Emanuele Ponis, Liliana Rodolfi Dipartimento di Biotecnologie Agrarie Università degli Studi di Firenze Piazzale delle Cascine 24 50144 Firenze Italy E-mail: [email protected] Graziella Chini Zittelli Istituto per lo Studio degli Ecosistemi CNR Via Madonna del Piano 10 50019 Sesto Fiorentino Firenze Italy

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Contributor contact details

Chapter 21 Christine Crawford* and Catriona MacLeod Tasmanian Aquaculture and Fisheries Institute University of Tasmania Nubeena Crescent Taroona Tasmania 7053 Australia E-mail: Christine.Crawford@utas. edu.au [email protected]

Chapter 22 Lindsay G Ross,* Neil Handisyde, Donna-Claire Nimmo Institute of Aquaculture University of Stirling Stirling FK9 4LA Scotland UK E-mail: [email protected]

Chapter 23 Scott Gifford, Geoff R. MacFarlane, Claudia E. Koller and R. Hugh Dunstan* School of Environmental and Life Sciences The University of Newcastle Callaghan NSW 2308 Australia E-mail: Hugh.Dunstan@newcastle. edu.au

Wayne O’Connor NSW Department of Primary Industries Port Stephens Fisheries Centre Private Bag 1 Nelson Bay NSW 2315 Australia E-mail: wayne.o’[email protected]. gov.au

Chapter 24 Velmurugu Puvanendran* and Atle Mortensen Nofima Marin Muninbakken 9-13 9291 Tromsø Norway E-mail: Velmurugu.puvanendran@ nofima.no [email protected]

Chapter 25 Ewen McLean* Ministry of Fisheries Wealth Marine Science and Fisheries Center PO Box 427 PC 100 Muscat Sultanate of Oman E-mail: [email protected] Guillaume Salze Virginia-Maryland Regional College of Veterinary Medicine Duck Pond Drive Blacksburg VA 24061-0442 USA

Contributor contact details Michael H. Schwarz Virginia Seafood AREC 102 S. King St Hampton VA 23669 USA Steven R. Craig Virginia Cobia Farms LLC 108 Battleground Road Saltville VA 24370 USA E-mail: [email protected]

Chapter 26 Clive M. Jones Department of Primary Industries and Fisheries Queensland Northern Fisheries Centre PO Box 5396 Cairns QLD 4870 Australia

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Chapter 28 Kirsten Benkendorff School of Biological Sciences Flinders University of South Australia GPO Box 2100 Adelaide SA 5001 Australia E-mail: Kirsten.benkendorff@ flinders.edu.au

Chapter 29 Richard Langan Atlantic Marine Aquaculture Center University of New Hampshire Gregg Hall, Suite 130 35 Colovos Road Durham NH 03824 USA E-mail: [email protected]

E-mail: [email protected]

Chapter 30 Chapter 27 Brian D. Paterson Bribie Island Aquaculture Research Centre Queensland Department of Primary Industries and Fisheries PO Box 2066 Woorim Bribie Island 4507 Australia E-mail: [email protected]. gov.au

Arne Fredheim* SINTEF Fisheries and Aquaculture Aquaculture Technology 7465 Trondheim Norway E-mail: [email protected]

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Contributor contact details

Richard Langan Atlantic Marine Aquaculture Center University of New Hampshire Gregg Hall, Suite 130 35 Colovos Road Durham NH 03824 USA E-mail: [email protected]

Chapter 31 Thomas M. Losordo, PhD,* Dennis P. DeLong, MSM, and Todd C. Guerdat, MS Department of Biological and Agricultural Engineering Campus Box 7625 North Carolina State University Raleigh NC 27695 USA

Chapter 33 Craig L. Browdy* Novus International 5 Tomotley Court Charleston South Carolina 29407 USA E-mail: craig.browdy@novusint. com Jesus A. Venero, Alvin D. Stokes and John W. Leffer Waddell Mariculture Center Marine Resources Research Institute South Carolina Department of Natural Resources 217 Ft Johnson Road Charleston South Carolina 29407 USA

E-mail: [email protected]

Chapter 34

Chapter 32

Peter Edwards 593 Lat Prao Soi 64 Bangkok Thailand 10310

Claude E. Boyd* Department of Fisheries and Allied Aquacultures Auburn University AL 36849 USA E-mail: [email protected] Suwanit Chainark Phuket Rajabhat University Faculty of Fisheries Technology 21 Tepknasatree Road Amphor Muang Phuket Province 83000 Thailand E-mail: [email protected]

E-mail: [email protected]

Chapter 35 John Bostock Senior Consultant Institute of Aquaculture University of Stirling Stirling FK9 4LA Scotland UK E-mail: [email protected]

Contributor contact details

Chapter 36 Geoff L. Allan* and D. Stewart Fielder Port Stephens Fisheries Institute New South Wales Department of Primary Industries Locked Bag 1 Nelson Bay NSW 2315 Australia E-mail: [email protected] Kevin M. Fitzsimmons Department of Soil, Water and Environmental Science University of Arizona 2601 E. Airport Drive Tucson AZ 85706 USA Samuel L. Applebaum Albert Katz Department of Dryland Biotechnologies The Bengis Center for Desert Aquaculture Jacob Blaustein Institute for Desert Research BGU Sede Boqer Campus Midreshet Ben-Gurion 84990 Israel

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Sudhir Raizada Central Institute of Fisheries Education Rohtak Centre (ICAR) Lahli Via Anwal Rohtak – 124 411 Haryana India

Chapter 37 M.P. Schreibman* Aquatic Research and Environmental Assessment Center (AREAC) Brooklyn College City University of New York 2900 Bedford Avenue Brooklyn New York NY 11210 USA E-mail: [email protected]. edu C.B. Zarnoch Baruch College City University of New York One Bernard Baruch Way Department of Natural Science 17 Lexington Avenue, Box A-0506 New York NY 10010 USA

Preface

Global aquaculture remains the fastest growing food industry with growth since 1970 of 8.8 % per annum.1 This growth compares with 1.2 % and 2.8 % for capture fisheries and terrestrial farmed meat production, respectively.1 Total global aquaculture production reached 59.4 million tonnes in 2004, worth an estimated US$ 70.3 billion, including 46 million tonnes of aquaculture product consumed for food. Global demand for seafood has continued to rise, fuelled by global population growth and an increase in per capita consumption due to increasing protein consumption in many developing countries and an increase in relative preference for seafood protein in many developed countries. Global population was estimated at 6.72 billion in November 2008 and, while growth has halved from the peak in around 1963, it is predicted to reach 9 billion by 2040.2 Seafood consumption has increased to 16.6 kg/person/y, the highest on record.1 To cater for global demands in 2020, an estimated 70 million tonnes of seafood will be required from aquaculture.1 Past production increases have come from new industries, new areas for production and intensification of production, mainly in developing countries. Since the early practice of capturing and holding animals, aquaculture has become much more sophisticated with most animals being bred in purpose-built hatcheries, cultured in ponds, cages or tanks, often with some control over the environment, harvested for a specific market and often processed to add value to the product. The massive production increase

1

FAO (2007) The State of World Fisheries and Aquaculture 2006, Rome, Food and Agriculture Organization of the United Nations. 2 http://www.census.gov/ipc/www/idb/worldpopinfo.html

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(from approximately 1 million tonnes in the 1950s to 59.4 million tonnes in 2004) arose from increases in the area under culture and the number of species cultured and from a shift in the reliance on natural feeds to the greater use of formulated feeds that increasingly met the species’ nutritional requirements. Improvements in other aspects of husbandry, such as health management, and better technology, such as cage and tank systems, also contributed. New technology is now needed to boost production, protect fragile environments and supply the highest quality product. The easy gains in production increase have now mostly been made and that to increase production in the next decade and a half to 70 million tonnes or beyond will require a major new improvement in technology. This is the subject of this book, Aquaculture: New Technologies. We have divided the contents of this book into six parts and selected international experts to contribute individual chapters. The first four parts deal with developments in new technologies for genetic improvement and reproduction, health, diet and husbandry and aquaculture system design. Aquaculture has lagged behind agriculture in applying genetic improvement techniques, but this is a rapidly changing field. We present chapters that detail advances in genetic improvement for finfish, shrimp and molluscs, controlling reproduction and gender, and sterility and genomics. Stress and disease accompany intensive animal production for all species, and managing health is a fundamental requirement for all aquaculture producers. We are regularly discovering new diseases for aquatic animals but, fortunately, we are also developing new treatments and therapies. In the Health part, advances in diagnosis, vaccine development and new methods to control viruses, parasites and other pathogens are presented. The major operating cost for all aquaculture species that are fed is diets and feeding costs. In the Diet and Husbandry part of the book, we have selected chapters that document advances in larval marine fish nutrition, challenges and opportunities with selection of dietary ingredients, bioenergetic modelling to estimate nutritional requirements and specific advances in nutritional science for salmonids; catfish, carps and tilapia; and seabasses and breams. There is also a chapter on microalgal culture – used as a feed for molluscs and other organisms and as a product for human and animal nutrition, in cosmetics and pharmaceutics, and for environmental applications. This part also has a chapter on the impact of harmful algal blooms on shellfish aquaculture. In the fourth part of the book, chapters describing the latest technology for off-shore and open ocean aquaculture, tank-based recirculating systems, land-based finfish and shrimp pond culture systems are presented. Chapters on inland saline aquaculture, urban aquaculture and traditional Asian aquaculture are also included. To conclude this section of the book we have included a chapter on the use of information technology in aquaculture.

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The final two parts of the book look at environmental issues and new species for aquaculture. Sustainability can be interpreted in many ways but, whatever your definition, there is universal agreement that all human activities should have a minimal impact on the environment. Therefore in the environmental part of the book, chapters are included on prediction and assessments of environmental impacts, advances in effluent treatment, reducing the impacts of escapees, the role of geographical information systems (GIS) in aquaculture and aquaculture for zooremediation. The new species part examines the latest developments for high-value species including cod, halibut and wolfish; cobia; lobsters; and the aquaculture of pharmaceuticals and nutraceuticals. Several figures from the book have additionally been included as colour plates in an eight-page section which appears between pages 576 and 577. An analysis of past advances in aquaculture gives optimism for the future. While the challenges are great, the adoption of new technology will facilitate rapid future increases in production. The potential of genetic improvement is still largely untapped, new health management strategies will reduce production costs and, while there are considerable constraints with the supply of feed ingredients, advances in our understanding of nutritional requirements will improve feed efficiency. New production systems will also allow production to increase and take production closer to the market for high-value species. The world is facing increasing challenges with food security, and aquatic protein will continue to play a major role in both the developing and developed world. Aquaculture is critical for the future supply of seafood and other aquatic products. Gavin Burnell and Geoff Allan

1 Genome-based technologies useful for aquaculture research and genetic improvement of aquaculture species Z. Liu, Auburn University, USA

Abstract: In spite of the relatively late start of aquaculture genome research, significant progress has been made in aquaculture genomics. Many of the genomic resources, tools, and technology developed from genomics have wide applications for aquaculture research and genetic improvements of aquaculture species. The major genome technologies include DNA marker technologies, novel sequencing technologies, gene discovery technologies, genome mapping technologies, and technologies for analysis of genome expression. This chapter was written to provide basic information such as theories behind the technologies, principles of the technologies, and potential applications of each of these genome technologies relevant to aquaculture. In some cases, research and technology development trends are discussed such that the readers are aware of the direction of future research. For instance, single nucleotide polymorphisms (SNP) are clearly the markers of choice in the future even though microsatellites are currently the predominant type of markers in aquaculture research and applications; the next generation of sequencing technologies will not only play a dominant role in sequencing, but also may replace the traditional genome expression analysis technologies such as microarrays. Key words: genome, amplified fragment length polymorphism (AFLP), microsatellites, single nucleotide polymorphisms (SNP), marker, quantitative trait loci (QTL), expressed sequence tag (EST), next generation sequencing technology, microarray, linkage mapping, radiation hybrid, comparative mapping, sequence tag.

1.1 Introduction Aquaculture, like animal husbandry, has a long history dating back some 3000 years when the Chinese began culturing carp. However, whereas genetic selection of animal livestock has been conducted for thousands of years, until very recently, most cultured aquatic animals were essentially

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wild. For thousands of years, aquaculture was limited to the harvesting of immature fish or shellfish and transferring them to an artificially created environment favorable to their growth. In spite of much earlier aquaculture practices in China, the documented artificial production of aquaculture seed was first introduced in 1733 when a German farmer successfully gathered fish eggs, fertilized them, and raised the fish that hatched. Initially this kind of aquaculture was limited to freshwater fish. In the 20th century, new techniques were developed to successfully breed saltwater species. Most brood stocks used in aquaculture were not genetically selected until the late 20th century. Since around the 1970s, major progress has been made in the genetic enhancement programs of many aquaculture species by using traditional selective breeding techniques, and it is only since the late 1990s that large-scale genome research of aquaculture species started to take off. Genomics, as a new field, has its own technologies and requires new sets of resources. To clarify the term used in the title of this chapter, genomebased technologies; this term is used to include technologies required to conduct genome research and technologies derived as a result of genome research for genetic improvement programs in aquaculture. As such, this chapter will include DNA marker technologies, sequencing technologies, genome mapping technologies, gene discovery technologies, and genome expression analysis technologies. This chapter will briefly introduce these technologies and discuss their applications in genome research and genetic improvements of aquaculture species. Readers with an interest in technical details of these technologies are referred to a recently published book, Aquaculture Genome Technologies (Liu, 2007a).

1.2 DNA marker technologies 1.2.1 Historical perspectives The genome compositions of each individual of the same species are similar but different at the level of DNA sequences and its encoding capacity, and thereby have different transcriptional activities, biological characteristics and performance. The entire task of DNA marker technologies is to provide the means to reveal such DNA level differences of genomes among individuals of the same species, as well as among various related taxa. Historically, these measurements relied on phenotypic or qualitative markers. Morphological differences such as body dimensions, size, and pigmentation are some examples of phenotypic markers. Genetic diversity measurements based on phenotypic markers are often indirect, and inferential through controlled breeding and performance studies (Parker et al., 1998; Okumus and Çifci, 2003). Because these markers are polygenically inherited and have low heritability, they may not represent the true genetic differences (Smith and Chesser, 1981). It is only when the genetic basis for these phenotypic markers is known, that some of them can be used to measure

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genetic diversity. Molecular markers including protein markers and DNA markers were developed to overcome problems associated with phenotypic markers. Much before the discovery of DNA markers, allozyme markers were used to identify broodstocks in fish and other aquaculture species (Kucuktas and Liu, 2007). Allozymes are different allelic forms of the same enzymes encoded at the same locus (Hunter and Markert, 1957; Parker et al., 1998; May, 2003). Genetic variations detected in allozymes may be the result of point mutations, insertions or deletions (indels). Allozymes have had a wide range of applications in fisheries and aquaculture, including population analysis, mixed stock analysis, and hybrid identification (May, 2003). However, they are becoming a marker type of the past due to the limited number of loci that in turn prohibits genome-wide coverage for the analysis of complex traits (Kucuktas and Liu, 2007). In addition, mutation at the DNA level that causes a replacement of a similarly charged amino acid may not be detected by allozyme electrophoresis. Another drawback is that the most commonly used tissues in allozyme electrophoresis are the muscle, liver, eye, and heart, the collection of which is lethal. Two specific technological advances, the discovery and application of restriction enzymes in 1973 and the development of DNA hybridization techniques in 1975, set the foundation for the development of the first type of DNA markers, restriction fragment length polymorphism (RFLP – for a recent review, see Liu, 2007b). Restriction endonucleases cut DNA wherever their recognition sequences are encountered. Therefore, changes in the DNA sequence due to insertions/deletions (indels), base substitutions, or rearrangements involving the restriction sites can result in the gain, loss, or relocation of a restriction site. Digestion of DNA with restriction enzymes results in fragments whose number and size can vary among individuals, populations, and species. Two approaches are widely used for RFLP analysis. The first involves the use of Southern blot hybridization (Southern, 1975), while the second involves the use of polymer chain reaction (PCR). Traditionally, fragments were separated using Southern blot analysis, in which genomic DNA is digested, subjected to electrophoresis through an agarose gel, transferred to a solid support such as a piece of nylon membrane, and visualized by hybridization to specific probes. Most recent analysis replaces the tedious Southern blot analysis with techniques based on PCR. If flanking sequences are known for a locus, the segment containing the RFLP region is amplified via PCR. If the length polymorphism is caused by a deletion or insertion, gel electrophoresis of the PCR products should reveal the size difference. However, if the length polymorphism is caused by base substitution at a restriction site, PCR products must be digested with a restriction enzyme to reveal the RFLP. The major strength of RFLP markers is that they are codominant markers, i.e., both alleles in an individual are observed in the analysis. The major disadvantage of RFLP is the relatively low level of polymorphism.

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In addition, either sequence information (for PCR analysis) or a molecular probe (for Southern blot analysis) is required, making it difficult and timeconsuming to develop markers in species lacking known molecular information. Due to these disadvantages, the application of RFLP markers in aquaculture and fisheries has been, and will be, limited. Mitochondrial genome evolves more rapidly than the nuclear genome. The rapid evolution of the mtDNA makes it highly polymorphic within a given species. The polymorphism is especially high in the control region (D-loop region), making the D-loop region highly useful in population genetic analysis. The analysis of mitochondrial markers is mostly RFLP analysis, or direct sequence analysis (Liu and Cordes, 2004). Due to the high levels of polymorphism and the ease of mitochondrial DNA analysis, mtDNA has been widely used as markers in aquaculture and fisheries settings. However, mtDNA is maternally inherited in most cases, and this non-Mendelian inheritance greatly limits the applications of mtDNA for genome research. In addition, most aquaculture-related traits are controlled by nuclear genes. For most aquaculture finfish species, their nuclear genome is at the level of a billion base pairs while their mitochondrial genomes are usually tens of thousands of times smaller than the nuclear genome. Clearly, in spite of their usefulness for the identification of aquaculture stocks, mitochondrial DNA markers will not be tremendously useful for aquaculture genome research and genetic improvement programs in aquaculture. However, some recent studies suggested that mitochondrial DNA could influence performance traits such as growth (Steele et al., 2008). When the Human Genome Project was launched in the mid-1980s, the capacity and capabilities of available DNA marker technologies seriously limited genome research. Such severe limits gave rise to pressure to develop more efficient marker systems for analysis of complex traits and genome organizations. At the end of the 1980s, the simple sequence repeats (SSR) or microsatellites were discovered; and they have since been used as one of the most preferred marker types because of their high levels of polymorphism, abundance, roughly even genome distribution, codominant inheritance, and small locus size that facilitate PCR-based genotyping (Tautz, 1989). Because of the importance of microsatellites for aquaculture genome research and genetic improvement programs, they will be discussed in more detail below. At the beginning of 1990s, efforts were also devoted to develop multiloci, PCR-based fingerprinting techniques. Such efforts resulted in the development of two marker types that were highly popular for a while: RAPD (random amplified polymorphic DNA, Welsh and McClelland 1990; Williams et al., 1990) and AFLP (amplified fragment length polymorphism, Vos et al., 1995). RAPD is a multi-locus DNA fingerprinting technique using PCR to randomly amplify anonymous segments of nuclear DNA with a single short

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PCR primer (8–10 bp in length) (for a recent review, see Liu, 2007c). Because the primers are short, relatively low annealing temperatures (often 36–40 ºC) must be used. Once different bands are amplified from related species, population, or individuals, RAPD markers are produced. RAPD markers thus are differentially amplified bands using a short PCR primer from random genome sites. Genetic variation and divergence within and between the taxa of interest are assessed by the presence or absence of each product, which is dictated by changes in the DNA sequence at each locus. RAPD polymorphisms can occur due to base substitutions at the primer binding sites or to insertions or deletions (indels) in the regions between the two close primer binding sites. The potential power for detection of polymorphism is reasonably high as compared to RFLP, but much lower than microsatellites; typically, 5–20 bands can be produced using a given primer, and multiple sets of random primers can be used to scan the entire genome for differential RAPD bands. Because each band is considered a bi-allelic locus (presence or absence of an amplified product), polymorphic information content (PIC) values for RAPDs fall below those for microsatellites. The major advantages of RAPD markers are their applicability to all species regardless of known genetic, molecular or sequence information, relatively high level of polymorphic rates, simple procedure, and a minimal requirement for both equipment and technical skills. RAPD has been widely used in genetic analysis of aquaculture species, but its further application in genome studies is limited by its lack of high reproducibility and reliability. In addition, RAPD is inherited as dominant markers and transfer of information with dominant markers among laboratories and across species is difficult.

1.2.2 Amplified fragment length polymorphism (AFLP) Alternatives to RAPD that overcome the major problems such as its low reproducibility have been actively sought, and AFLP (Vos et al., 1995) was the outcome of such efforts. AFLP is based on the selective amplification of a subset of genomic restriction fragments using PCR (for a recent review, see Liu, 2007d). Genomic DNA is digested with restriction enzymes, and double-stranded DNA adaptors with known sequences are ligated to the ends of the DNA fragments to generate primer binding sites for amplification. The sequence of the adaptors and the adjacent restriction site serve as primer binding sites for subsequent amplification of the restriction fragments by PCR. Selective nucleotides extending into the restriction sites are added to the 3′ ends of the PCR primers such that only a subset of the restriction fragments is recognized. Only restriction fragments in which the nucleotides flanking the restriction site match the selective nucleotides will be amplified. The subset of amplified fragments is then analyzed by denaturing polyacrylamide gel electrophoresis to generate the fingerprints.

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AFLP analysis is an advanced form of RFLP. Therefore, the molecular basis for RFLP and AFLP are similar. First, any deletions and/or insertions between the two restriction enzymes, e.g., between Eco RI and Mse I that are most often used in AFLP analysis, will cause shifts of fragment sizes. Second, base substitution at the restriction sites will lead to loss of restriction sites and thus a size change. However, only base substitutions in all Eco RI sites and roughly 1/8 of Mse I sites are detected by AFLP since only the Eco RI primer is labeled and AFLP is designed to analyze only the Eco RI–Mse I fragments. Third, base substitutions leading to new restriction sites may also produce AFLP. Once again, gaining Eco RI sites always leads to production of AFLP, gaining Mse I sites must be within the Eco RI–Mse I fragments to produce new AFLP. In addition to the common mechanisms involved in polymorphism of RFLP and AFLP, AFLP also scans for any base substitutions at the first three bases immediately after the two restriction sites. Considering large numbers of restriction sites for the two enzymes (250 000 Eco RI sites and 500 000 Mse I sites immediately next to Eco RI sites for a typical fish genome with one billion base pairs), a complete AFLP scan would also examine over two million bases immediately adjacent to the restriction sites. The potential power of AFLP in the study of genetic variation is enormous. In principle, any combination of a 6-bp cutter with a 4-bp cutter in the first step can be used to determine potential fragment length polymorphism. For each pair of restriction enzymes used in the analysis, e.g., Eco RI and Mse I, a total of approximately 500 000 Eco RI–Mse I fragments would exist for a genome with a size of 1 × 109 bp. Theoretically, 4096 primer combinations compose a complete genome-wide scan of the fragment length polymorphism using the two restriction enzymes if three bases are used for selective amplification. As hundreds of restriction endonucleases are commercially available, the total power of AFLP for analysis of genetic variation can not be exhausted. However, it is probably never necessary to perform such exhaustive analysis. Since over 100 loci can be analyzed by a single primer combination, a few primer combinations should display thousands of fingerprints. For genetic resource analysis, the number of primer combinations required for construction of phylogenetic trees/dendrograms depends on the level of polymorphism in the populations, but probably takes no more than five to ten primer combinations. AFLP combines the strengths of RFLP and RAPD. It is a PCR-based approach requiring only a small amount of starting DNA; it does not require any prior genetic information or probes; and it overcomes the problem of low reproducibility inherent to RAPD. AFLP is capable of producing far greater numbers of polymorphic bands than RAPD in a single analysis, significantly reducing costs and making possible the genetic analysis of closely-related populations. It is particularly well adapted for stock identification because of the robust nature of its analysis. The other

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advantage of AFLP is its ability to reveal genetic conservation as well as genetic variation. In this regard, it is superior to microsatellites for applications in stock identification. Microsatellites often possess large numbers of alleles, too many to obtain a clear picture with small numbers of samples. Identification of stocks using microsatellites, therefore, would require large sample sizes. For instance, if ten fish are analyzed, each of the ten fish may exhibit distinct genotypes at a few microsatellite loci, making it difficult to determine relatedness without any commonly conserved genotypes. In closely related populations, AFLP can readily reveal commonly shared bands which define the common roots in a phylogenetic tree, and polymorphic bands that define branches in the phylogenetic tree. The major weakness of AFLP markers is their dominant nature of inheritance. Genetic information is limited with dominant markers because essentially only one allele is scored; and at the same time, since the true alternative allele is scored as a different locus, AFLP also inflates the number of loci under study. As dominant markers, information transfer across laboratories is difficult. In addition, AFLP is more technically demanding, requiring special equipment such as automated DNA sequencers for optimal operations. AFLP has been widely used in aquaculture in areas such as analysis of population structures, migration, hybrid identification, strain identification, parentage identification, genetic resource analysis, genetic diversity, reproduction contribution, and endangered species protection (Jorde et al., 1999; Seki et al., 1999; Sun et al., 1999; Cardoso et al., 2000; Chong et al., 2000; Kai et al., 2002; Mickett et al., 2003; Whitehead et al., 2003; Mock et al., 2004; Campbell and Bernatchez, 2004; Simmons et al., 2006). AFLP has also been widely used in genetic linkage analysis (Kocher et al., 1998; Liu et al., 1998, 1999; Griffiths and Orr, 1999; Agresti et al., 2000; Robison et al., 2001; Rogers et al., 2001; Liu et al., 2003; Li et al., 2003), and analysis of parental genetic contribution involving interspecific hybridization (Young et al., 2001) and meiogynogenesis (Felip et al., 2000). In a study of the black rockfish (Sebastes inermis), Kai et al. (2002) used AFLP to distinguish three color morphotypes, in which diagnostic AFLP loci were identified as well as loci with significant frequency differences. In such reproductive isolated populations, it is likely that ‘fixed markers’ of AFLP can be identified to serve as diagnostic markers. Fixed markers are associated most often with relatively less migratory, reproductive isolated populations (Kucuktas et al., 2002). With highly migratory fish species, fixed markers may not be available. However, distinct populations are readily differentiated by difference in allele frequencies. For instance, Chong et al. (2000) used AFLP for the analysis of five geographical populations of Malaysian river catfish (Mystus nemurus) and found that AFLP was more efficient for the differentiation of sub-populations and for the identification of genotypes within the populations than RAPD although similar clusters of the populations were concluded with either analysis.

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In spite of its popularity, AFLP has two fundamental flaws that prohibit its wider applications in the future: the dominance inheritance and lack of information to link it to genome sequence information. In some cases, AFLP can be used as a rapid screening tool, and useful markers can then be converted to SCAR (sequence characterized amplified region) markers. However, genome scale applications of SCAR markers are unlikely.

1.2.3 Microsatellites Microsatellites are simple sequence repeats (SSRs) of 1–6 base pairs. The advantages of microsatellites as molecular markers include their abundance in genomes, even distribution, small locus size facilitating PCR-based genotyping, codominant nature of Mendelian inheritance, and high levels of polymorphism (for a recent review, see Liu, 2007e). Microsatellites are highly abundant in various eukaryotic genomes including all aquaculture species studied to date. In most of the vertebrate genomes, microsatellites make up a few percent of the genome in terms of the involved base pairs, depending on the compactness of the genomes. Generally speaking, more compact genomes tend to contain smaller proportion of repeats including simple sequence repeats, but this generality is not always true. For example, the highly compact genome of Japanese pufferfish contains 1.29 % of microsatellites, but its closely related Tetraodon nigroviridis genome contains 3.21 % of microsatellites (Crollius et al., 2000). During a genomic sequencing survey of channel catfish, microsatellites were found to represent 2.58 % of the catfish genome (Xu et al., 2006; Liu, 2007g). In fugu, one microsatellite was found for every 1.87 kb of DNA. For comparison, in the human genome, one microsatellite was found for every 6 kb of DNA (Beckmann and Weber, 1992). It is reasonable to predict that in most aquaculture fish species, one microsatellite should exist every 10 kb or less of the genomic sequences, on average. Dinucleotide repeats are the most abundant forms of microsatellites. For instance, in channel catfish, 67.9 % of all microsatellites are present in the form of dinucleotide repeats; 18.5 % are present as trinucleotide repeats; and 13.5 % as tetranucleotide repeats, excluding mononucleotide repeats which are not nearly as useful for molecular markers. Generally speaking, dinucleotide microsatellites are the most abundant, followed by tri-, or tetranucleotide repeats but, in some cases, tetranucleotide repeats can be more frequent than the trinucleotide repeats. Of the dinucleotide repeat types, (CA)n is the most common dinucleotide repeat type, followed by (AT)n, and then (CT)n (Toth et al., 2000; Xu et al., 2006). (CG)n type of repeat is relatively rare in the vertebrate genomes, partially because the vertebrate genomes are often A/T-rich. Of the trinucleotide repeats and tetranucleotide repeats, relatively A/T-rich repeat types are generally more abundant than G/C-rich repeat types. Microsatellites longer than tetranucleotide repeats (penta- and hexanucluotides) are much less abundant and

Genome-based technologies useful for aquaculture research

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therefore, are less important as molecular markers (Toth et al., 2000). It is important to point out that the definition of microsatellites limiting to repeats of six bases long is quite arbitrary. Technically speaking, repeats with seven bases or longer sequences are also microsatellites but, because they become rarer as the repeats are longer, they are less relevant as molecular markers. Microsatellites are distributed in the genome on all chromosomes and all regions of the chromosome. They have been found inside gene coding regions (e.g. Liu et al., 2001), introns, and in the non-gene sequences (Toth et al., 2000). The best known examples of microsatellites within coding regions are those causing genetic diseases in humans, such as the CAG repeats that encode the polyglutamine tract, resulting in mental retardation. In spite of their wide distribution in genes, microsatellites are predominantly located in non-coding regions (Metzgar et al., 2000). Only about 10–15 % of microsatellites reside within coding regions (Moran, 1993; van Lith and van Zutphen, 1996; Edwards et al., 1998; Serapion et al., 2004). This distribution should be explained by negative selection against frameshift mutations in the translated sequences (Metzgar et al., 2000; Li et al., 2004). Because the majority of microsatellites exist in the form of dinucleotide repeats, any mutation by expansion or shrinking would cause frameshift of the protein encoding open frames if they reside within the coding region. This also explains why the majority of microsatellites residing within coding regions have been found to be trinucleotide repeats, though the presence of dinucleotide repeats and their mutations within the coding regions do occur. Most microsatellite loci are relatively small, ranging from a few to a few hundred repeats. The relatively small size of microsatellite loci is important for PCR-facilitated genotyping. Generally speaking, within a certain range, microsatellites containing a larger number of repeats tend to be more polymorphic, though polymorphism has been observed in microsatellites with as few as five repeats (Karsi et al., 2002). For practical applications, microsatellite loci must be amplified using PCR. For best separations of related alleles that often differ from one another by as little as one repeat unit, it is desirable to have small PCR amplicons, most often within 200 bp. However, due to the repetitive nature of microsatellites, their flanking sequences can be quite a simple sequence as well, prohibiting the design of PCR primers for the amplification of microsatellite loci within a small size limit. Microsatellites are highly polymorphic as a result of their hypermutability and thereby the accumulation of various forms in the population of a given species. Microsatellite polymorphism is based on size differences due to varying numbers of repeat units contained by alleles at a given locus. Microsatellite mutation rates have been reported as high as 10−2 per generation (Weber and Wong, 1993; Crawford and Cuthbertson, 1996; Ellegren, 2000), which are several orders of magnitude greater than that of

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New technologies in aquaculture

non-repetitive DNA (10−9; Li, 1997). In several fish species, the mutation rates of microsatellites were reported to be at the level of 10−3 per locus per generation: 1.3 × 10−3 in common carp (Zhang et al., 2008), 2 × 10−3 in pipefish (Jones et al., 1999), 3.9–8.5 × 10−3 in salmon (Steinberg et al., 2002) and 2 × 10−3 in dollar sunfish (MacKiewicz et al., 2002). Microsatellites are inherited in a Mendelian fashion as codominant markers. This is one of the strengths of microsatellite markers in addition to their abundance, even genomic distribution, small locus size, and high polymorphism. Genotyping of microsatellite markers is usually straightforward. However, due to the presence of null alleles (alleles that can not be amplified using the primers designed), complications do exist. As a result, caution should be exercised to ensure that the patterns of microsatellite genotypes fit the genetic model under application. The disadvantages of microsatellites as markers include the requirement for existing molecular genetic information, a large amount of upfront work for microsatellite development, and the tedious and labor-intensive nature of microsatellite primer design, testing, and optimization of PCR conditions. Each microsatellite locus has to be identified and its flanking region sequenced for the design of PCR primers. Technically, the simplest way to identify and characterize a large number of microsatellites is through the construction of microsatellite-enriched small-insert genomic libraries (Ostrander et al., 1992; Lyall et al., 1993; Kijas et al., 1994; Zane et al., 2002). In spite of the variation in techniques for the construction of microsatelliteenriched libraries, the enrichment techniques usually include selective hybridization of fragmented genomic DNA with a tandem repeat-containing oligonucleotide probe and further PCR amplification of the hybridization products. In spite of the simplicity in the construction of microsatelliteenriched libraries and thereby the identification and characterization of microsatellite markers, for a large genome project, direct microsatellite marker development may not be the wisest approach. Recent progress in sequencing technologies with the next generation of sequencers will allow large numbers of genomic sequence tags to be generated that would include numerous microsatellites. Microsatellites can be identified and sequenced directly from genome sequence surveys such as bacterial artificial chromosome (BAC) end sequencing (Xu et al., 2006; Somridhivej et al., 2008), and from expressed sequence tag (EST) analysis from which many microsatellites can be developed into type I markers (Liu et al., 1999; Serapion et al., 2004). Caution has to be exercised, however, on microsatellites developed from ESTs. First, due to the presence of introns, one has to be careful not to design primers at the exon–intron boundaries. Second, the presence of introns would make allele sizes unpredictable. Finally, many microsatellites exist at the 5′- or 3′-untranstated region (UTR), making flanking sequences insufficient for the design of PCR primers. While introns are not a problem for microsatellites derived from BAC end sequencing, sequencing reactions often terminate immediately after the microsatellite

Genome-based technologies useful for aquaculture research

13

repeats, which also makes flanking sequences insufficient for the design of PCR primers. Microsatellites have recently become an extremely popular marker type in a wide variety of genetic investigations, as evidenced by the launch of the journal Molecular Ecology Notes, dedicated almost entirely to publishing primer and allele frequency data for newly-characterized microsatellite loci in a wide range of species. Since the late 1990s, microsatellite markers have been used extensively in fisheries research including studies of genome mapping, parentage, kinships, and stock structure. The major application of microsatellite markers is for the construction of genetic linkage and quantitative trait loci (QTL) maps. This is because of the high polymorphic rate of microsatellite markers. When a resource family is produced, the male and female fish parents are likely to be heterozygous in most microsatellite loci. The high polymorphism of microsatellites makes it possible to map many markers using a minimal number of resource families. There are other reasons for the popularity of microsatellites, one of which is that microsatellites are sequence-tagged markers that allow them to be used as probes for the integration of different maps including genetic linkage and physical maps. Communication using microsatellite markers across laboratories is easy, and the use of microsatellite across species borders is sometimes possible if the flanking sequences are conserved (FitzSimmons et al., 1995; Rico et al., 1996; Cairney et al., 2000; Leclerc et al., 2000). As a result, microsatellites can be also used for comparative genome analysis. If microsatellites can be tagged to gene sequences, their potential for use in comparative mapping is greatly enhanced. In spite of the popularity and great utilization of microsatellites, recent advances in molecular markers will have a major impact on the choice of DNA markers. In particular, the rapid progress in SNP, including its rapid identification and automation in genotyping, makes SNP the far more preferred marker system for genome studies as detailed below.

1.2.4 Single nucleotide polymorphism (SNP) SNP describes polymorphisms caused by point mutations that give rise to different alleles containing alternative bases at a given nucleotide position within a locus (for a recent review, see Liu, 2007f). Such sequence differences due to base substitutions have been well characterized since the beginning of DNA sequencing in 1977, but genotyping SNPs for large numbers of samples was not possible until several major technological advances in the late 1990s. SNPs are again becoming a focal point of molecular markers since they are the most abundant polymorphism in any organism, adaptable to automation, and reveal hidden polymorphism not detected with other markers and methods. SNP markers have been regarded by many as the markers of choice in the future.

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New technologies in aquaculture

Theoretically, a SNP within a locus can produce as many as four alleles, each containing one of four bases at the SNP site: A, T, C, and G. Practically, however, most SNPs are usually restricted to one of two alleles (quite often either the two pyrimidines C/T or the two purines A/G) and have been regarded as bi-allelic. They are inherited as codominant markers in a Mendelian fashion. In spite of its increasing popularity as the choice of markers for the future, SNP discovery is a daunting task. As defined by its definition, SNP discovery depends on sequencing. Several approaches have been used for the discovery of SNPs in humans and animals. Earlier efforts used approaches such as single-strand conformation polymorphism (SSCP) analysis (Gonen et al., 1999), heteroduplex analysis (Sorrentino et al., 1992), and direct DNA sequencing. However, several recently developed approaches provide greater efficiencies, two of which are described here in detail. The first and the simplest one is to conduct deep sequencing of reduced representation libraries (Altshuler et al., 2000; Van Tassel et al., 2008). In this method, genomic DNA from multiple individuals is mixed and digested with restriction endonuclease and subjected to electrophoresis through an agarose gel. The idea is that the allelic fragments from these individuals (with potential SNPs) should all migrate to the same gel location. The gel slice is cut and the DNA extracted for the construction of the reduced representation library. The reduced representation library is deeply sequenced to generate coverage of 20–30X using next generation DNA sequencers. The generated sequences can then be assembled for the identification of SNPs. Solexa sequencing can be used for species in which a draft genome sequence exists where assembly of the short sequence tags can be achieved. Longer sequences would be needed if a draft genome sequence is not yet available. In most aquaculture species, draft genome sequences are not yet available, and therefore 454 sequencing should be considered. The second strategy involves data mining from EST projects, if EST libraries were constructed using multiple individuals (each individual contains two sets of chromosomes so it is possible also to use just one individual, but a greater level of polymorphism is provided by multiple individuals). This approach is realistic because EST resources already exist, or are to be developed for the majority of important aquaculture species. In addition, EST-derived SNPs are coming from genes and therefore are type I markers. Mapping of gene-associate SNPs would allow analysis of association of SNPs with traits for the discovery of the ‘causing SNPs’ for the traits (Bader, 2001; Marnellos, 2003; Halldorsson et al., 2004; Stram, 2004). However, this approach has major limitations. Because of evolutionary restraint on mutations in coding regions, SNP rates are generally much lower in coding regions than in non-coding regions. The major problem of EST-derived SNPs could be related high sequence errors in EST sequenc-

Genome-based technologies useful for aquaculture research

15

ing resulting in pseudo-SNPs (Wang et al., 2008b). In order to avoid pseudoSNPs, two factors appeared to be crucial: the contig sizes and the lower sequence allele frequency. Contigs larger than four sequences with the minor sequence being represented at least twice seem to provide a high level of SNP validation rates (Wang et al., 2008). SNP genotyping requires special equipment. Many methods have been developed through the years to differentiate the alleles of SNPs. A lot of the earlier methods, in spite of being adaptable to individual laboratory situations, are not suitable for large-scale genome-wide applications. These include direct sequencing, single base sequencing (reviewed by Cotton, 1993), allele-specific oligonucleotide (ASO, Malmgren et al., 1996), heteroduplex analysis, denaturing gradient gel electrophoresis (DGGE, Cariello et al., 1988), single strand conformational polymorphism assays (SSCP, Suzuki et al., 1990), and ligation chain reaction (LCR, Kalin et al., 1992). Large-scale analysis of SNP markers, however, depends on the availability of expensive, cutting-edge equipment. Several options are available for efficient genotyping using the state of the art equipment. Particularly popular are methods involving MALDI– TOF (Matrix-assisted laser desorption ionization – time of flight) mass spectrometry (Ross et al., 1998; Storm et al., 2003), and the BeadArray technology developed by Illumina. The latter, as it can be adapted for largescale genome studies, is becoming the most popular SNP genotyping method. The BeadArray technology is based on 3-micron silica beads that self assemble in microwells on either of two substrates: fiber optic bundles or planar silica slides. When randomly assembled on one of these two substrates, the beads have a uniform spacing of ∼5.7 microns. Each bead is covered with hundreds of thousands of copies of a specific oligonucleotide that act as the capture sequences in one of Illumina’s assays (Fig. 1.1). The manufacturing process includes a sequential hybridization of every single array element (bead with oligos). This process, called decoding, allows validation of every feature of every array to ensure that each array element is present and functional. The allele discrimination at each SNP locus is achieved by using three oligos (P1, P2, and P3, Fig. 1.2), of which P1 and P2 are allele-specific and are Cy3- and Cy5-labeled. P3 is locus-specific designed several bases downstream from the SNP site. Upon allele-specific extension and ligation, the artificial, allele-specific template is created for PCR using universal primers. If the template DNA is homozygous, either P1 or P2 will be extended to meet P3; if the template is heterozygous, both P1 and P2 will be extended to meet P3, allowing ligation to happen (Fig. 1.2). P3 contains a unique address sequence that targets a particular bead type with complementary sequence to the address sequence. After downstream-processing, the single-stranded, dye-labeled DNAs are hybridized to their complement bead type through their unique address sequences. After hybridization, the BeadArray Reader is used to analyze fluorescence signal on the beadchip,

16

New technologies in aquaculture Optical fiber bundle Bead library each with a distinct oligonucleotide capture probe

cataaatattctggcaagga

cactatgaccttctggata cggcatcagtggactgggct atacaaaggcttgtactttt gaagggacttgcactgttgc tgcacaggtgaccaataccc

Fig. 1.1 Schematic illustration of the BeadArray technology.

which is in turn analyzed using software for automated genotype clustering and calling (http://www.illumina.com/). The Illumina platform is highly cost-effective and has been widely used for large-scale SNP analysis in the Human Genome Project. Among all the factors, the relatively low cost of the Illumina genotyping platform is the key for selection of an SNP genotyping platform. Currently, Illumina offers two platforms for the BeadArray technology: the GoldenGate® Platform for up to 7600 SNPs, and the iSelect® platform for 7600–60 000 SNPs. On the basis of cost per sample, the iSelect® platform is the most efficient with a cost of several cents per sample. In spite of its current low levels of application in aquaculture genome research, SNP markers should gain in popularity as more and more sequence information becomes available in aquaculture species. Equally important, once the genetic linkage maps are well constructed, genome scans for QTLs are expected to follow to study traits important to aquaculture, which then depends on the use of well-defined association analysis. As SNP markers are great markers for the analysis of trait–genotype associations, their application to aquaculture will become essential. It is clear that SNPs will

Genome-based technologies useful for aquaculture research

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A/G P1 P2 P1 P2

T C

Address P3

P3

Allele-specific extension and ligation PCR with P1, P2, and P3

Homozygous A/A Homozygous G/G Heterozygous A/G

Fig. 1.2 Allele discrimination using BeadArray technology. The allele discrimination at each SNP locus is achieved by using three oligos (P1, P2, and P3, of which P1 and P2 are allele-specific and are Cy3- and Cy5-labeled. P3 is locus-specific designed several bases downstream from the SNP site. Upon allele-specific extension and ligation, the artificial, allele-specific template is created for PCR using universal primers. If the template DNA is homozygous, either P1 or P2 will be extended to meet P3; if the template is heterozygous, both P1 and P2 will be extended to meet P3, allowing ligation to happen, generating templates for successful PCR. By measurements of red, green, or yellow of the fluorescence, the genotypes can be readily called.

become the major markers of choice for genome research and genetic improvement programs in aquaculture.

1.2.5 Trends in DNA marker technologies DNA marker technologies have become essential for aquaculture genetics research and the genetic improvement of aquaculture species. As a matter of fact, DNA markers, both the quality and quantity, have always been a limiting factor for in-depth genome research. Throughout the years, aquaculture geneticists have used various markers including allozyme markers, mitochondrial markers, RFLP markers, RAPD, AFLP, microsatellites, and SNPs. The overall trend, however, has been driven by: (i) the need for large numbers of markers for high-density coverage of the genomes, and (ii) the need for sequence-tagged markers for comparative genome analysis. Such demands have driven aquaculture genetic research away from using systems that do not offer a great number of markers such as RFLP and allozyme markers, and away from anonymous dominant markers such as RAPD and AFLP. Microsatellites, being codominant and sequence-tagged, have recently become very popular. However, with the draft genome sequence

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very soon becoming available for major aquaculture species, microsatellites are not without limitations. Their genotyping can be multiplexed, but the extent of multiplexing is limited. Automation of microsatellite genotyping is limited, thus prohibiting large-scale genome-wide applications. Mapping of thousands of microsatellites to the genome involves a lot of work, and analysis using tens or hundreds of thousands of microsatellites would be a daunting task, if not technically impossible for repeated analysis. This only leaves the SNP marker system to be viable. SNPs are the most abundant markers in genomes when compared to any other types of markers; SNPs are sequence-tagged and therefore would allow comparative mapping analysis; SNP genotyping is highly automated and therefore is adaptable to large-scale genome-wide analysis. Therefore, it is clear that SNP markers are the choice marker of the future. In spite of the current lack of draft whole genome sequences for aquaculture species, it is anticipated that they will soon become available for major aquaculture species. In addition, the availability of next generation sequencing technologies (see below) makes it unnecessary to have the whole genome draft sequences in order to develop a large number of SNP markers.

1.3 DNA sequencing technologies Two independent DNA sequencing technologies were originally invented in 1977, and they have been referred to as Sanger’s enzymatic method and Maxam-Gilbert chemical method (Maxam and Gilbert, 1977; Sanger et al., 1977). Since the 1980s, most DNA has been sequenced by the enzymatic method as the chemical method never gained popularity because of its use of toxic chemicals in the reactions. However, the Sanger’s method, once the golden standard, is rapidly losing ground as the next generation DNA sequencers are now emerging. As the principles and applications of original DNA sequencing technologies, especially those of the Sanger’s DNA sequencing technology, were well documented and most readers are familiar with them, this section will focus on the next generation of DNA sequencing technologies. Readers who are interested in the basic principles of the traditional Sanger’s sequencing are referred to a chapter in Aquaculture Genome Technologies (Liu, 2007i,j). Several major new sequencing platforms have been adopted recently, and they are collectively referred to as the next generation DNA sequencers. A common feature among the new generation of sequencing procedures is the elimination of the need to clone DNA fragments and the subsequent amplification and purification of DNA templates prior to sequencing. Instead, sequence templates are handled in bulk, and massively parallel sequencing allows the generation of numerous sequences simultaneously. Readers need to know that many sequencing platforms are being developed, and this field is one of the most active areas in technology

Genome-based technologies useful for aquaculture research

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Table 1.1 Comparison of three next generation sequencing platforms Platform

SOLiD

454

Solexa

Vendor Sequence tags per run Bases per run Potential applications

Applied Biosystems 240 million

454 Life Sciences 500 000

Illumina >20 million

6 × 109 bp Resequencing, gene expression analysis, microRNA discovery, chromatin immune-precipitation (ChIP)

1.2 × 108 bp Resequencing, gene expression analysis, microRNA discovery, ChIP, whole genome de novo sequencing

1 × 109 bp Resequencing, gene expression analysis, microRNA discovery, ChIP

development. The next section will focus only on the principles of three sequencing platforms: the SOLiDTM sequencing platform, and the Solexa sequencing platform, and the 454 sequencing platform (Table 1.1).

1.3.1 The SOLiD sequencing platform The SOLiD (Sequencing by Oligonucleotide Ligation and Detection) method utilizes ligation of fluorescently labeled 8-mer primers containing random bases at six of its eight positions and specific dinucleotides at the remaining two positions (the earliest version was the fourth and the fifth, but it can be the first and second, and that will be used for explanation here). The primers are fluorescently labeled with four specific dyes with each dye corresponding to four specific dinucleotides. Note that at each DNA base position, there are four base possibilities: A, C, G, or T. For dinucleotide at two consecutive base positions, a total of 16 dinucleotides (AA, AC, AG, AT, CA, CC, CG, CT, GA, GC, GG, GT, TA, TC, TG, and TT) should cover all possibilities. In order to make each of the four fluorescent dyes uniquely represent one specific nucleotide, a two-step decoding process is required. A random primer is ligated to the template only when the first and second nucleotides on the primer are complementary to those on the template. After visualizing the color, the fluorescent tag is removed by cleaving the primer between the fifth and sixth positions, removing bases 6, 7, and 8. The process is repeated; and in the second round of ligation, a new primer is ligated, with the new first and second nucleotides on the primer being complementary to those on the template, counting from the end of the previously ligated primer after cleavage (base 6 and 7 of the previous primer). Repeat this process and every first and second position is recorded. Next, the system is reset to generate the recording for every n − 1, n − 2, n − 3, and n − 4 position (Kate Marusina, http://www.

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New technologies in aquaculture

genengnews.com/articles/chitem.aspx?aid=1946) such that each base included in the dinucleotide is ‘sequenced’ twice to allow base calling. For example, the base at position 4 is sequenced in the dinucleotide base 3 and base 4, and it is sequenced again in the dinucleotide base 4 and base 5. As with all next generation sequencing platforms, the SOLiD sequencing does not require cloning of genomic DNA. It starts with the creation of a ‘library’ by ligation of two adaptors to sheared genomic DNA (Fig. 1.3). Once the adapters are ligated to the library, emulsion PCR is conducted using the common primers to generate ‘bead clones’ in which each contains a single nucleic acid species. Each bead is then attached to the surface of a flow cell (microscope slide) via 3′ modifications to the DNA strands (Fig. 1.4). Each microbead can be considered a separate sequencing reaction which is monitored simultaneously via sequential digital imaging.

Shear DNA

Ligate adaptors, P1 and P2

Fig. 1.3

Making ‘libraries’ of sheared segments by ligating to two adaptors as the first step of SOLiD sequencing.

+

P1 P2

+

Polymerase

Clonal PCR

+ P1 coated beads

Fig. 1.4 Clonal amplification of genomic DNA in SOLiD sequencing. Each genomic DNA segment with adaptors is absorbed to beads and amplified by using the adaptor primers to generate numerous clones ready for sequencing.

Genome-based technologies useful for aquaculture research

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SOLiD sequencing chemistry depends on specific ligation of a random primer to the existing primer only when the random primer harbors the specific dinucleotide that is complementary to the template DNA being sequenced. The actual base detection is no longer done by the polymerasedriven incorporation of labeled dideoxy terminators. Instead, SOLiD uses a mixture of labeled oligonucleotides and queries the input strand with ligase. In the early version, each oligo has degenerate nucleotide at positions 1–3 (N′s at 3′ first three bases), one of 16 specific dinucleotide at positions 4–5, and degenerate nucleotide at positions 6–8 that are fluorescently labeled (demonstrated in Fig. 1.3 with the first and second nucleotide being specific, and the remaining bases being degenerate). The sequencing reaction involves: (i) hybridization and ligation of a specific oligo whose 4th and 5th bases match that of the template; (ii) detection of the specific fluor associated with the specific dinucleotide; (iii) cleavage of bases 6–8; (iv) repeat, this time querying the 9th and 10th bases. Seven cycles of ligation would allow putative nucleotide identities at positions 4 and 5, 9 and 10, 14 and 15, 19 and 20, 24 and 25, 29 and 30, and 34 and 35 to be recorded. After seven cycles of this, a ‘reset’ is performed in which the initial primer and all ligated portions are melted from the template and discarded. Next a new initial primer is used that is N−1 in length. Repeating the initial cycling (steps 1–4) now generates an overlapping data set. In this manner, ligations using primer N generate sequences for bases 4 and 5, 9 and 10, 14 and 15, 19 and 20, 24 and 25, 29 and 30, and 34 and 35; ligations using primer N−1 generate sequences for bases 3 and 4, 8 and 9, 13 and 14, 18 and 19, 23 and 24, 28 and 29, and 33 and 34, and so on. After use of primers N, N−1, N−2, N−3, and N−4, every base is ‘sequenced’ twice using two primers (Table 1.2). Base calling is dependent on a two-step encoding procedure with known nucleotides in every fourth and fifth position of the primer (Fig. 1.5). For example, the dinucleotides CA, AC, TG, and GT are all encoded by the green dye. Because each base is queried twice, it is possible, using the two colors, to determine which bases were at which positions. For instance, if the sequence is TCGAACGTA . . . blue label is detected for the first ligation reaction using primer N that determines base composition at Table 1.2 Schematic illustration of the base positions sequenced by each ligation and using various primers. Each black bar represents the two bases sequenced by each ligation, and the numbers above the bars indicate the base positions to be sequenced. The primers N, N−1, N−2, N−3, and N−4 are indicated in the first column 0 1 2 3 4 5 6 7 8 9 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 N –1 –2 –3 –4

22

New technologies in aquaculture n–1

3′ A-T-n-n-n-z-z-z

1st base

2nd base A C G T

A-A-n-n-n-z-z-z

A

Ligase 3′

C G T

3′ Bead …… 5′

3′ 7x Ligation

Use n–1, n–2, n–3, and n–4 primers

3′

Primer

p5′

A-C-n-n-n-z-z-z

Adapter sequence

3′

A-G-n-n-n-z-z-z

Template sequence Record fluorescence

Primer A-C-n-n-n-z-z-z

Bead …… 5′

TG Adapter sequence Cleavage

3′

Primer

Template sequence

z-z-z

A-C-n-n-n p5′ Bead …… 5′

TG Adapter sequence

Template sequence

Fig. 1.5 Chemistry of SOLiD sequencing platform. The SOLiD sequencing utilizes ligation of fluorescently labeled 8-mer primers containing random bases at six of its eight positions. Shown here the first two bases are specific dinucleotide that are specifically labeled as displayed by the dotted circles, e.g., AA: blue, AC: green, AG: yellow, and AT: red. A random primer is ligated to the template only when the first and second nucleotides on the primer are complementary to those on the template. After visualizing the color, the fluorescent tag is removed by cleaving the primer between the fifth and sixth positions, removing bases 6, 7, and 8. The process is repeated; and in the second round of ligation, a new primer is ligated with the new first and second nucleotides on the primer being complementary to those on the template, counting from the end of the previously ligated primer after clevage (base 6 and 7 of the previous primer). Next, the system is reset to generate the recording for every n−1, n−2, n−3, and n−4 positions by using N−1, N−2, N−3, and N−4 primers.

base 4 and 5. There are still four possibilities with blue label: AA, CC, GG, and TT. However, in the first ligation reaction of primer N−1, yellow label is detected, which effectively calls the base at position 4 to be ‘A’. Similarly, with the second ligation reaction using N−4 primer (that determines base compositions at base 5 and 6), green should be detected that effectively calls the base at position 5 to be ‘A’ as well. Repeating this decoding process to include ‘sequencing’ of each base with two primers will provide unambiguous base callings.

Genome-based technologies useful for aquaculture research

23

As with any other type of technology, SOLiD sequencing technology is making rapid improvements allowing greater throughput and more applications. Several areas are under intense scrutiny to increase the efficiency and accuracy of the technology including: increasing the bead density, read lengths, and ability of multiplexing. Currently, SOLiD read length is around 35 bases, and it is anticipated to be 50 bases in 2009. The SOLiD sequencing technology has wide applications including, but not limited to, targeted resequencing, gene expression analysis by digital counting of sequence tags (e.g., Cloonan et al., 2008), microRNA discovery, chromatin immune-precipitation (ChIP), and whole genome sequencing. Due to its short read length, it is anticipated that its application to whole genome sequencing will be mostly applicable to whole genome resequencing. However, it is less amenable to de novo whole genome sequencing projects because the assembly of whole genome based on short sequence tags has proven to be a great challenge. Because the technology is quite new, there are no publications covering the use of the SOLiD sequencing platform in aquaculture species, to the author’s best knowledge.

1.3.2 The Solexa sequencing platform The Solexa sequencing platform depends on two of its core technologies: the Clonal Single Molecule ArrayTM technology that allows simultaneous analysis of hundreds of millions of individual molecules, and the reversible terminator technology that allows specific base calling based on sequencing by synthesis. Solexa sequencing starts with sheering of genomic DNA to small segments to which different adaptor sequences are ligated to either end. Upon binding single-stranded template DNA fragments with adaptors randomly to the inside surface of the flow cell channels, template DNA is amplified clonally through bridge PCR. DNA is sequenced by detection of fluorescently labeled dideoxynucleotide terminators. These specially created nucleotides, which also possess a reversible termination property, allow each cycle of the sequencing reaction to occur simultaneously in the presence of all four nucleotides (ddA, ddC, ddT, ddG). In the presence of all four nucleotides, the polymerase is able to select the correct base to incorporate, with the natural competition between all four alternatives leading to higher accuracy than methods where only one nucleotide is present in the reaction mix at a time (which require the enzyme to reject an incorrect nucleotide). Sequences where a particular base is repeated one after another (‘homopolymer repeats’) are dealt with as for any other sequence and with high accuracy; this avoids the problems of measuring intensity and deducing how many bases were present in the repeat that are the cause of uncertainty seen with ‘one base per reaction’ methods, as described with the 454 sequencing platform. Solexa sequencing technology achieves an unparalleled data density with highly accurate results. A typical Solexa sequencing run generates millions

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New technologies in aquaculture

of sequence tags with the capability to generate over a billion bases of DNA sequence per run. Currently, the read length is limited to about 35 bp. Like the SOLiD sequencing technology, the Solexa sequencing technology has wide applications including, but not limited to, gene expression analysis by digital counting of sequence tags, microRNA discovery, epigenetic studies, ChIP, and whole genome sequencing (Bentley, 2006; Butler et al., 2008; Chen et al., 2008; Cokus et al., 2008; Dolan and Denver, 2008; Glazov et al., 2008; Hillier et al., 2008). However, due to its short read length, it is anticipated that its application to whole genome sequencing will be mostly applicable to whole genome re-sequencing. As Solexa technology is also quite new, its application in aquaculture species is still limited, but the potential is tremendous. One of the earliest applications of the Solexa technology was actually conducted in oysters. Dennis Hedgecock’s group used the Solexa technology for the study of genes involved in heterosis of pacific oysters (Hedgecock et al., 2007).

1.3.3 The 454 sequencing platform 454 sequencing is based on pyrosequencing. During DNA synthesis, a pyrophosphate (PPi) is released when each base is incorporated. The released pyrophosphate can be converted to adenosine triphosphate (ATP) that generates a fluorescent signal upon the actions of luciferase in the presence of its substrates. Measurement of the light signal after sequential injection of A, C, G, and T would allow the determination of the base composition. After each base addition, the whole sequencing reaction system is reset by cleaning out all existing ATP and nucleotides with apyrase. When mononucleotide repeats are encountered in the sequence, the pyrosequencing reaction continuously incorporates the repeated nucleotide until it reaches a different nucleotide. The light signal produced is proportional to the number of mononucleotides incorporated–up to eight bases. Mononucleotide repeats greater than 8 bp cannot be accurately sequenced by pyrosequencing. The 454 sequencing platform uses microfabricated high-density picolitre reactors (Margulies et al., 2005). No cloning is necessary for 454 sequencing. The clonal DNA used for sequencing is obtained by clonal PCR amplification of a single molecule in emulsified water-in-oil microreactors. Preparation of the DNA library consists of a few simple steps. Genomic DNA is fractionated into smaller fragments (300–500 bp) that are subsequently filled in to polished ends (blunted), allowing ligation of adaptors to the genomic DNA for PCR amplification. In order to prevent intermolecular ligation of the genomic DNA fragments, the DNA fragments are dephosphorylated. Short adaptors (A and B) are then ligated onto the ends of the fragments. After ligation, the gap needs to be repaired, presumably using a DNA ligase. The adaptors provide priming sequences for both amplification and sequencing of the sample-library fragments. The two adaptors are

Genome-based technologies useful for aquaculture research

25

different. Adaptor B contains a 5′-biotin tag that enables immobilization of one strand of the library onto streptavidin coated beads. The nonbiotinylated strand is released and used as a single-stranded template DNA library. The single-stranded template DNA library is immobilized onto beads carrying short primers complementary to the adaptor A sequences by base pairing. The key element here is attaining the correct proportion of beads to DNA molecules such that only one molecule is captured by each bead. The beads containing a single molecule of the single-stranded template are emulsified with the amplification reagents in a water-in-oil mixture. Each bead is captured within its own microreactor where PCR amplification occurs. This results in bead-immobilized, clonally amplified DNA fragments. The single strand template DNA library beads are added to the DNA bead incubation mix (containing DNA polymerase) and are layered with enzyme beads (containing sulfurylase and luciferase) onto the PicoTiterPlate device. The device is centrifuged to deposit the beads into the wells. The layer of enzyme beads ensures that the DNA beads remain positioned in the wells during the sequencing reaction. Due to the size of the wells in relation to the beads, only one bead containing a specific clonally amplified genomic DNA segment should be placed into each well of the PicoTiterPlate device. The loaded PicoTiterPlate device is placed into the ‘454 sequencer’, the Genome Sequencer 20 Instrument or the FLX generation systems, which performs pyrosequencing-like reactions. Unlike a traditional pyrosequencing reaction, hundreds of thousands of beads, each with millions of copies of clonally amplified DNA, are sequenced in parallel. Each well of the PicoTiterPlate device is a separate pyrosequencing reaction. If a nucleotide complementary to the template strand is flowed into a well, the polymerase extends the existing DNA strand by adding a nucleotide(s). Addition of one (or more) nucleotide(s) results in a reaction that generates a light signal that is recorded by the CCD camera in the instrument. The signal strength is proportional to the number of nucleotides incorporated in a single nucleotide flow. Typically, over 200 000 reads can be achieved in a single run. Assuming generation of 250 bp by a single reaction, each run should generate 50 million bp or more of sequence in several hours using a single instrument. This is approximately a 10X coverage of a bacterial genome! The 454 sequencing platform holds much potential for its great applications such as gene expression profiling, epigenetic analysis, and whole genome sequencing (Patrick, 2007; Bekal et al., 2008; Hafner et al., 2008; Vera et al., 2008). The 454 sequencing platform is probably the most promising technology for de novo whole genome sequencing among the next generation sequencers as it produces sequences of approximately 250 bp, that is significantly shorter than the traditional Sanger sequencing (800– 1000 bp), but much longer those generated by SOLiD or Solexa sequencing

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New technologies in aquaculture

platforms. The current major problems prohibiting its application for the sequencing of complex genomes are its relatively short sequencing reads and difficulties in accurate determination of homopolymeric runs in the DNA. The short reads complicate genome sequence assembly, while the inability to determine the number of bases within a long homopolymeric run prohibits accurate sequencing of genomes. These problems are more significant for complex genomes with high levels of repeat structure. However, the technologies’ high throughput and low costs are very attractive, especially for aquaculture species. As the technology is perfected to minimize these drawbacks, the 454 sequencing platform will show even greater promise.

1.4 Gene discovery technologies Performance and production traits are controlled by genes, environments, and gene–environment interactions. In order to gain detailed understanding of performance and production traits, understanding the genes in aquaculture genomes become essential. Sequencing of ESTs has been the primary approach for the discovery of genes in aquaculture species, although several other approaches are also available such as serial analysis of gene expression (SAGE). Recently, however, the adoption of several novel sequencing platforms using next generation sequencers has allowed generation of expressed sequence tags through de novo sequencing of whole transcriptomes.

1.4.1 Expressed sequence tags and gene discovery ESTs are single pass sequences of random cDNA clones. They are partial cDNA sequences corresponding to mRNAs generated from randomly selected cDNA library clones (for recent reviews, see Liu, 2006, 2007h). EST analysis has traditionally been conducted by sequencing random cDNA clones from cDNA libraries. Such an approach is efficient at initial stages of gene discovery, but has proven to be inefficient in the gene discovery of rarely expressed genes. The rate of gene discovery usually drops precipitously soon after reaching a level of several thousand ESTs. By using regular cDNA libraries, the most abundantly expressed genes would have been sequenced many times before the most rarely expressed genes are sequenced just once. Clearly, EST sequencing from non-normalized libraries is inefficient for gene discoveries of rarely expressed genes. Normalization decreases the prevalence of clones representing abundant transcripts and dramatically increases the efficiency of random sequencing and rare gene discovery. Normalized cDNA libraries are cDNA libraries that have been equalized in representation to reduce the representation of abundantly expressed

Genome-based technologies useful for aquaculture research

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genes and to increase the representation of rarely expressed genes. While the details of how the subtraction is conducted may differ greatly, the basic principles behind normalization are the same, i.e. they all depend on the faster hybridization kinetics of abundantly expressed genes to form doublestranded complexes that can be removed by various means, whereas it takes a long time for the rarely expressed genes to reassociate. Several strategies have been developed for the normalization of cDNA libraries, but the fundamental principles behind all the normalization procedures are the same. We have used a strategy utilizing the Evrogen Trimmer-Direct Kit (http://www.evrogen.com/p3_2.shtml). This system is specially developed to normalize cDNA enriched with full length sequences (Zhulidov et al., 2004). The method involves denaturation–reassociation of cDNA, degradation of ds-fraction formed by abundant transcripts, and PCR amplification of the equalized ss-DNA fraction. The key element of this method is degradation of ds-fraction formed during reassociation of cDNA using duplex-specific nuclease (DSN) enzyme (Shagin et al., 2002). A number of specific features of DSN make it ideal for removing ds-DNA from complex mixtures of nucleic acids. DSN displays a strong preference for cleaving ds-DNA in both DNA–DNA and DNA–RNA hybrids, compared to ss-DNA and RNA, irrespective of the sequence length. Moreover, the enzyme remains stable over a wide range of temperatures and displays optimal activity at 55–65 ºC. Consequently, degradation of the ds-DNAcontaining fraction by this enzyme occurs at elevated temperatures, thereby decreasing loss of transcripts due to the formation of secondary structures and non-specific hybridization involving adapter sequences. EST analysis is one of the most rapid approaches for gene discovery. A small collection of ESTs in a species without any genome information can result in the rapid identification of a large number of genes. Gene discovery and identification is, therefore, the primary function of EST analysis. Because of the exceptionally high gene discovery rate of the EST approach, EST analysis has been extremely popular. The EST database dbEST has been one of the fastest growing databases at NCBI. As of May 30, 2008, there are 52 858 766 entries in the NCBI’s public EST database dbEST (dbEST release 053008, http://www.ncbi.nlm.nih.gov/dbEST/dbEST_ summary.html). Large EST resources are available for several major aquaculture species including Atlantic salmon, rainbow trout, catfish, oysters, and shrimps. ESTs provide the information and material basis for the development of microarrays for the analysis of genome expression as discussed below.

1.4.2 de novo sequencing of whole transcriptomes and gene discovery As detailed under sequencing technologies, all next generation sequencing platforms have the ability to generate hundreds of thousands (the 454 platform) to millions of expressed sequence tags (SOLiD and Solexa

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sequencing platforms) using RNA as the starting material. Clearly, such sequencing projects not only allow many genes to be identified, but, importantly, also allow expression profiling through digital counting of sequence tags, as will be further discussed in Section 1.6. As compared to traditional EST analysis, the next generation sequencing technologies can rapidly produce a large number of expressed sequence tags. They probably provide a much greater power in terms of expression profiling, as accurate estimation can be made based on the number of sequenced tags. In addition, relatively accurate counting of tags from various exons may also provide information concerning alternative splicing and alternative polyadenylation. However, it may be more difficult for the identification of short sequence tags for less conserved genes as no draft genome sequences are yet available from aquaculture species. This problem will soon be alleviated when the draft genome sequences become available. In comparison, the 454 sequencing platform perhaps provides a greater power for sequence identification because of its longer sequence reads, but at the expense of the number of sequence tags to be generated that would otherwise offer greater accuracy for expression profiling based on tag counting.

1.5 Genome mapping technologies Genome mapping and sequencing is the core of structural genomics. While no whole genome draft sequences are yet available for aquaculture species, genomes of many aquaculture species have been subjected to mapping. Genome mapping can be classified into several categories based on resource needs, technology requirement, and principles used in mapping, i.e., genetic linkage mapping, QTL mapping, physical mapping, cytogenetic mapping, radiation hybrid mapping, and comparative mapping (Liu, 2007a). While cytogenetic mapping is highly useful for the identification of chromosomes and physical mapping of genes to chromosomes, its resolution is low for large-scale genome-wide mapping analysis (this is covered in Chapter 2). Limited work has been conducted in comparative mapping of aquaculture species. A recent chapter in Aquaculture Genome Technologies provided excellent coverage on comparative mapping and positional cloning (Lee and Kocher, 2007). Interested readers are referred to the chapter for details on comparative mapping.

1.5.1 Genetic linkage mapping Genetic linkage mapping is an old technology. It is purely based on cosegregation of markers within a well-defined segregating population that is often referred to as the reference family or mapping population. Genetic mapping requires two major resources: the segregating population, and the molecular markers. The segregating population can be F2 population, back-

Genome-based technologies useful for aquaculture research

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cross progenies, or higher generation of intercrosses. In some cases, F1 can be used and treated as a pseudo-backcross because heterozygous markers are segregating in F1 population. However, for best results, three generation pedigrees are needed to provide non-ambiguous linkage phases. The number of individuals used for mapping analysis depends on the desired resolution. A population of 100 individuals can resolve markers that are 1 cM away (1 out of 100 are recombinant). If higher resolution is needed to detect rare recombinants, larger numbers of the mapping population can be used. Any molecular markers that are polymorphic and segregating in the mapping population in a Mendelian fashion can be used for linkage mapping. However, sequence tagged markers provide codominance and transferability across laboratories and possibly also across species borders allowing comparative mapping analysis (see below). The most often used marker type for linkage mapping is microsatellite, although SNP markers may soon dominate on linkage maps. As the advantages and disadvantages of various types of markers are detailed above, we will not repeat them here. Genetic linkage maps can be constructed upon analysis of marker segregation data within the mapping population. This is achieved by use of various software packages. Linkage maps have been constructed in over 30 major aquaculture species (Table 1.3). A recent review by Danzmann and Gharbi provided excellent details on linkage mapping, and summarized recent progress of linkage mapping in aquaculture species (Danzmann and Gharbi, 2007). It is clear, however, that the marker densities on the genetic linkage maps of aquaculture species are too low currently to provide sufficient coverage for efficient QTL analysis. The use of more markers, in particular the SNPs to be developed, should very soon change the situation.

1.5.2 Quantitative trait loci (QTL) mapping Most performance and production traits are controlled by multiple genes, and simple Mendelian genetic analysis is not therefore sufficient to provide answers as to how many genes are controlling the traits, and how they function. The multigene controlled traits are defined as quantitative traits and the loci controlling the traits are defined as QTL. QTLs can be mapped genetically by correlation of segregating markers with the traits, and such a process is referred to as QTL mapping. QTL mapping starts with creation of a population in which the traits and the markers are segregating. For instance, fish resistant to a particular disease can be crossed with susceptible fish to produce F1 fish. F2 fish can be produced from the F1 fish in which the disease trait is segregating. In a sense, QTL mapping is not any different from genetic linkage mapping except that the proper mapping population is needed in which the trait of interest is segregating along with DNA markers. QTL mapping is the core

Inter-population hybrids Interspecific haploids Interspecific hybrids Outcross

Walking catfish Japanese flounder European sea bass

Ayu Common carp Yellowtails Black tiger shrimp

Channel catfish

Brown trout Arctic charr Tilapia

AFLP, SSR SSR, genes SSR, genes SSR, AFLP, genes AFLP, SSR AFLP, SSR SSR, genes SSR, genes AFLP AFLP AFLP, SSR SSR, genes microsatellites AFLP, SSR SSR, genes, RAPD SSR AFLP

SSR, AFLP, genes

Inter-strain backcross

Outcross Inter-strain backcross Inter-strain backcross Inter-strain backcross Haploids Interspecific 3-way cross Interspecific F2 intercross Outcross Interspecific backcross Haploids Inter-strain hybrids Outcross

Atlantic salmon

AFLP, SSR, genes

Marker type(s)

Double haploids

Mapping panel(s)

Current status of linkage maps in aquaculture species

Rainbow trout

Table 1.3

527 64 302 327 174 292 552 293 506 146 463 174 369 195 272 200 673

1439

1359

Markers

Young et al., 1998 Nichols et al., 2003 Sakamoto et al., 2000 Danzmann et al., 2005 Moen et al., 2004 Gilbey et al., 2004 Gharbi et al., 2006 Woram et al., 2004 Kocher et al., 1998 Agresti et al., 2000 Lee et al., 2005 Waldbieser et al., 2001 Liu et al., 2003 Poompuang and Na-Nakorn, 2004 Coimbra et al., 2003 Chistiakov et al., 2005 Volckaert et al., 2007 Watanabe et al., 2004 Sun and Liang, 2004 Ohara et al., 2005 Wilson et al., 2002

Reference

Outcross Outcross Outcross Inter-population hybrids Outcross Inter-line double hybrids Inter-strain backcross Inter-population hybrids Inter-population hybrids Inter-population hybrids Interspecific hybrids

AFLP AFLP AFLP AFLP AFLP, SSR, genes SSR AFLP AFLP AFLP AFLP, RAPD, SSR AFLP AFLP, SSR AFLP, SSR SSR AFLP SSR

246 401 394 231–241 133–158 102 349 545 503 384 324–339 293 375 204 350 150

Moore et al., 1999 Li et al., 2003 Pérez et al., 2004 Li et al., 2006 Yu and Guo, 2003 Hubert and Hedgecock, 2004 Li and Guo, 2004 Li et al., 2005 Wang et al., 2005 Liu et al., 2006 Zhou et al., 2006 Shen et al., 2007 Ning et al., 2007 Bargelloni et al., 2007 Reith et al., 2007 Morishima et al., 2007

AFLP = amplified fragment length polymorphism, RAPD = random amplified polymorphic DNA, SSR = simple sequence repeats. Source: modified and amended from Danzmann and Gharbi, 2007.

Pacific abalone Sea urchin Guppy Yellow croaker Gilthead sea bream Atlantic halibut Loach

Zhikong scallop

White shrimp Chinese shrimp Eastern oyster Pacific oyster

Kuruma prawn

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New technologies in aquaculture

of aquaculture genomics as the ultimate practical goal of aquaculture genomic research is to provide tools for genetic improvements. Much progress has been made in QTL mapping in aquaculture species. A recent review by Korol et al. (2007) covered many of the examples of QTL mapping studies in aquaculture species, and interested readers are referred to this review. As a whole, QTL mapping in aquaculture species has fallen behind, and greater efforts should be devoted to this area. The practical application of QTL mapping is marker-assisted selection. There are only few examples of marker-assisted selection in aquaculture species (e.g., Fuji et al., 2007). Before the wider applications of markerassisted selection are developed, the trend is that selection will soon become whole genome-based (Meuwissen et al., 2001), as has already occurred in terrestrial livestock species. With whole genome selection, selection is performed on estimates of associations of phenotype with largest possible markers across the genome. This contrasts with the traditional markerassisted selection which is based on a small number of significant markers, thus limiting overall effectiveness. The application of whole genome selection involves using ‘training data’ to estimate ‘breeding values’ of SNP haplotypes or alleles.

1.5.3 Radiation hybrid mapping The concept of radiation hybrid mapping was initially derived from somatic cell hybrids. Back in the 1970s, technology was developed to fuse different types of cells to form hybrid cell lines. In 1990, Cox et al., resurrected the technology by fusing X-ray radiated cells (donor cells) with normal cells (recipient cells) (Cox et al., 1990). As the chromosomes of the X-ray radiated cells were broken, the cells cannot survive by themselves. However, upon fusion with a recipient cell, broken chromosomal segments from the donor cells can be fused into the recipient cells. In order to have a selection marker for the hybrid cells, a drug-resistant gene (e.g., neo) can be first inserted into the donor cell’s genome. Upon fusion of the cells, the selection drug G418 can be applied to select for hybrid cells as the recipient cells do not have the drug resistance and should be killed by the antibiotic. Fusion cells containing the neo gene would be selected for growth. Clonal expansion of such fused hybrid cells would create a panel of radiation hybrid cell lines with each containing a different segment of the broken chromosomes, along with the chromosomal segments containing the neo gene. Because radiation causes random chromosomal breakage, various chromosomal segments contained within the panel of radiated cell lines would collectively cover the entire genome, with some portions of the chromosomal segments overlapping one another. Such radiation panels are used to map the genome through radiation hybrid (RH) mapping.

Genome-based technologies useful for aquaculture research

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RH mapping is based on co-retention of markers. A recent chapter in Aquaculture Genome Technologies written by Dr Caird Rexroad provides an excellent explanation for RH mapping (Rexroad, 2007), in which he wrote: ‘RH mapping strategies are based on the concept that markers which are close together on chromosomes will frequently be co-retained in the same hybrids – the probability that irradiation will induce a chromosome break between two markers decreases as the physical distance between the two markers decreases’. To provide adequate statistical support for mapping, marker retention frequencies – the percentage of times a marker is scored positive in a RH panel – is critical. Optimal retention is 20–50 % (Walter and Goodfellow, 1993). RH mapping is calculated based on the co-retention of markers in fragments across the panel. The estimated frequency of breakage between two markers is θ, which ranges from 0 to 1 and is analogous to recombination frequencies (r) used in genetic mapping. A θ value of 0 means two markers are always co-retained, a value of 1 means they are co-retained at random. This raw value is then included in multipoint analyses and transformed into centiRays (cR) – the RH map unit – using map functions similar to those of Haldane or Kosambi which are used in genetic map construction. Hence, observation of chromosome breaks between two markers in RH mapping is analogous to observing recombination between two markers in genetic mapping. In fact, the term ‘linkage’ is often used in RH mapping. The frequency of chromosome breaks between two markers is not only due to their physical distance, but also to the intensity of the radiation used to create the panel. Siden and colleagues conducted experiments to observe the effects of different dosages of radiation on a segment of the human X chromosome (Siden et al., 1992). At 5000 rad 10 % of the clones retained the entire chromosome arm, 40 % had fragments of 3–30 MB, and 50 % had fragments less than 3 MB. At 25 000 rad only 6 % had fragments larger than 3 Mb. Therefore the radiation hybrid map–distance unit is annotated with a subscript stating the dosage used to create the panel in rads, i.e. cR3000. Retention of multiple fragments from a single chromosome in a hybrid cell line complicates analyses; therefore 100–300 cell lines must be scored for a panel to construct statistically significant maps. RH mapping was initially created to map the non-polymorphic markers. Back in the mid-1990s, polymorphic markers were limiting in most species. By fusing cells of the interest species to a rodent cell line, the background genes are usually not amplified by using PCR primers designed from the genes of the species of interest. Thus ESTs were mapped to the genome maps. Later, however, it was found that RH maps were critically important for guiding the whole genome assembly as they are essentially physical linkage maps. RH mapping was mostly applied to mammalian genome mapping, but less so in aquatic species. However, it has been used for the zebrafish, an

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New technologies in aquaculture

aquatic model organism used to study the genetics of development, growth, reproduction, and disease resistance (Kwok et al., 1999; Geisler et al., 1999; Hukriede et al., 1999). To date, the only aquaculture species in which RH maps have been constructed is the gilthead sea bream (Senger et al., 2006; Sarropoulou et al., 2007). One reason is that higher resolution can be achieved by BAC-based physical maps. In addition, the need to map nonpolymorphic markers is drastically reduced as many polymorphic markers have now become available, especially the SNP markers. Therefore, the application of RH mapping in aquaculture species is limited.

1.5.4 Bacterial artificial chromosome (BAC)-based physical mapping BAC-based physical maps are important for the understanding of genome structure and organization, and for position-based cloning of economically important genes. A well characterized physical map can often be an important foundation for whole genome sequencing. A BAC-based physical map would also allow exploitation of existing genomic information from maprich species using comparative mapping, thus accelerating genome research in the species of interest. The first step of BAC-based physical mapping is the construction of large-insert BAC libraries. BAC libraries are large-insert genomic libraries. A recent chapter in Aquaculture Genome Technologies (He et al., 2007) provided excellent technical details on construction and characterization of BAC libraries, and related chapters by Davidson (2007) and Xu et al. (2007a) had a great coverage on details of physical mapping. Interested readers are referred to these chapters. Briefly, large inserts contained in a BAC library were derived from multiple copies of the genome broken randomly by partial restriction enzyme digest. Therefore, the BAC library can be viewed as multiple genome copies broken randomly into segments that are overlapping one another. Because the genomic segments originally from the same genome locations harbor the same restriction sites, overlapping genome segments can be aligned by the presence of the same sets of restriction fragments. A set of overlapping genome segments aligned by overlapping restriction fingerprints are then defined as a contig [a contig (from contiguous) is a set of overlapping DNA segments derived from a single genetic source], and many contigs make up the entire genome, possibly with gaps (or short overlapping segments, not supporting statistical overlapping status). The efforts to make BAC contig-based physical maps in aquaculture species are a recent event. As a result, physical maps have been only constructed in Atlantic salmon (Ng et al., 2005), tilapia (Katagiri et al., 2005), and channel catfish (Quiniou et al., 2007; Xu et al., 2007b), and a physical map is under construction in rainbow trout (Yniv Palti, USDA ARS, personal communication). It is expected that physical maps will soon be constructed in many of the important aquaculture species.

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1.6 Genome expression analysis technologies The development of high throughput technologies for global or genomewide measurements of gene expression requires the availability of genome resources. Most often, such genome resources come in the form of whole gene sequencing or the availability of a large resource of expressed sequence tags such that the major fraction of the transcriptome is represented. Once the sequences representing all the genes of an organism or the vast majority of the genes of the organism are known, global or genome-wide measurements of gene expression can be made using either microarray technologies or sequence tag-based technologies.

1.6.1 Microarray technology While microarrays utilize several recent technological innovations, they are, at their core, simply a high-density dot blot. There are two primary approaches to microarrays, differing both in their construction and their sample labeling. Spotted arrays are constructed by spotting long oligos or cDNAs using a printing robot, whereas in situ arrays are constructed by synthesizing short oligos directly onto the slide by photolithography (for a detailed review, see Peatman and Liu, 2007). Spotted array technology encapsulates the printing of either PCR products or long oligos (60–70 mers). Traditionally referred to as cDNA arrays, spotted arrays are today just as likely to be long oligos, as the cost of synthesizing oligos continues to decline, and because the parallel PCR required to prepare for cDNA arrays is labor-intensive, costly, and requires having clones on hand. While these cDNA-associated difficulties can be overcome through hard work and collaboration among members of a species group, the printing of long oligos offers advantages in startup time, the purity of commercial oligo synthesis, easier clone tracking, and the ability to utilize all available sequences in public genetic databases for array construction. Readers are referred to Whitfield et al.’s (2002) EST sequencing and microarray research on honey bee using spotted cDNAs; Rise et al., (2004a) and von Schalburg et al. (2005) describe considerations taken in construction of salmonid spotted cDNA arrays; and Zhao et al. (2005) report validation of a porcine spotted oligo array. Operon Biotechnologies (http://www.operon.com/) is a leading provider of sets of synthetic oligos for microarray spotting, and their website provides an excellent resource for criteria used in gene selection and long oligo design. Additionally, The Institute for Genomic Research (TIGR), well known for its EST indices, provides 70-mer oligo predictions for genes in each of its indices that have been utilized by some groups (Zhao et al., 2005). Researchers should also decide in the design phase their array layout, feature duplication, and the controls to be spotted on the slide (Whitfield et al., 2002; Smyth et al., 2005).

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New technologies in aquaculture

A variety of microarray slides are available for printing, most are polyL-lysine and amino silane-coated (see Hessner et al., 2004 for surfacechemistry comparisons). Telechem (http://www.arrayit.com/Products/ Substrates/) and Erie Scientific (http://www.eriemicroarray.com/index. aspx) are leading providers of microarray slides. The actual robotic printing of microarrays is increasingly being outsourced to large university core labs or private companies which now have years of experience in the field. For groups that anticipate printing multiple array designs and batches and want increased printing flexibility, purchasing a spotting robot may be a good choice. Perkin Elmer (http://las.perkinelmer.com/) and Genomic Solutions (http://www.genomicsolutions.com) offer popular printing systems. In situ array technology relies on photolithography for microarray construction (Lipshutz et al., 1999), a technique often used in computer chip fabrication. In contrast to spotting nucleotide products on the slide surface, oligonucleotides are synthesized directly on the surface of the array, one base at a time. To achieve sufficient feature densities, unique physical lithographic masks are created for each array design, to either block or allow light to reach the slide. In the places the mask does not cover, light deprotects, or converts, a special protective group to a hydroxyl group. This allows the binding of a single oligo at that specific site by its phosphate group. This oligo also bears a protective group that must be deprotected before an additional oligo can be coupled to it. Through repeated cycles of deprotection and coupling, 25-mer oligos are synthesized directly on the slide at densities currently as high as 1.3 million features per array. Affymetrix (http://www.affymetrix.com/) is recognized as the developer and industry leader for in situ arrays. While their technology made genome-wide arrays a reality for model species and continues to expand the horizons of microarray research in biomedical fields, the technology has been prohibitively expensive for the smaller species groups including aquaculture species. Nimblegen Systems (http://www.nimblegen.com) has recently developed a ‘maskless’ version of the Affymetrix technology that uses digital mirrors to achieve the same effect (Nuwaysir et al., 2002) at a significantly lower startup cost, now making in situ arrays a feasible choice for aquaculture genomic research. The majority of array design considerations for in situ arrays overlap with those of spotted arrays. EST analysis, clustering, quality control, and probe selection are still necessary steps to arrive at the set of genes that will be synthesized on the array. The higher feature density allowed with in situ arrays means that more genes, duplicates, and/or controls may be included on the array, if desired. Because the per array cost is significantly higher for in situ arrays and project flexibility considerably less than for spotted arrays, researchers usually attempt to maximize the information that can be gained from each slide. Usually, desired sequences for the array are sent electronically to the company, which then carries out oligo probe selection (23–25 mers) and designs the array layout. Both Affymetrix and

Genome-based technologies useful for aquaculture research

37

Nimblegen use a perfect match (PM) and mismatch (MM) system that accounts for the majority of the features on in situ arrays. Mismatch probes, as their name suggests, contain one or more mismatched nucleotides in the PM probe sequence and are used to detect and screen out false background fluorescence resulting from non-specific cross-hybridization. Commonly, 10 PM and 10 MM probes are synthesized for each gene included on the array, and are believed to significantly increase the accuracy and sensitivity of gene expression detection (see Irizarry et al., 2003; Han et al., 2004; Chen et al., 2005 for more information on PM and MM probe theory). Spotted and in situ microarrays differ not only in their array construction but also in the procedures used to label and hybridize experimental samples (probes in the traditional sense) to them. Both array platforms require that you start with RNA sources. The RNA is extracted from the samples of interest. Each RNA sample is reverse transcribed to cDNA, after quantification and quality-checking by spectrophotometer measurement and agarose gel electrophoresis. From this step, differences in the procedure arise between the two microarray platforms. The cDNA samples for spotted arrays are labeled with two different fluorescent dyes, Cy3 and Cy5, which fluoresce ‘green’ and ‘red’, respectively, under two different wavelengths of light (633 nm and 543 nm). The control sample is labeled with one dye and the treatment sample with the other. Dye assignments should be swapped in replicates to avoid dyeassociated bias of hybridization (Churchill, 2002). Dye labeling is most commonly done either directly or through indirect aminoallyl labeling (see Manduchi et al., 2002; Badiee et al., 2003 for a comparison of labeling methods). The two labeled samples are hybridized simultaneously in equal amounts to the same array for 16–20 h. The hybridized array, after washing to remove unhybridized probes, is scanned under a laser scanner (e.g. Molecular Devices/Axon Instruments’ Axon 4000B) at both fluorescent wavelengths (or channels) for the two dyes. A digital image is acquired for both channels and, by overlaying the two images, a fluorescent signal ratio for each array feature is obtained. This fluorescent signal ratio indicates gene expression levels. Using the Cy3/Cy5 labeling system, yellow spots indicate approximately equal levels of mRNA from both the control and treatment samples (equal signals from the green Cy3 and the red Cy5). Features that appear red or green have hybridized a majority of mRNA from only one sample. Fluorescent intensity data for each feature are recorded, and the scanned image and data can be linked back to gene feature identities through programs such as Molecular Devices/Axon Instruments’ GenePix ProTM software. Background subtraction and normalization is customarily carried out at this point, followed by microarray analysis and validation of genes determined to be significantly differentially expressed after treatment. For in situ arrays, the RNA samples are reverse transcribed using a T7 promoter oligo-dT primer. The resulting cDNA is converted to a

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double-stranded template by a second strand synthesis reaction. After purification, these double-stranded cDNA samples (again control and treatment) are converted by in vitro transcription to biotin-labeled cRNA using a T7 RNA polymerase. The cRNA from each sample is fragmented and hybridized to its own slide (note: no mixing of samples). Streptavidinphycoerithryn is added as the fluorescent dye for both the control and treatment samples. To clarify, each biological sample for in situ arrays is hybridized to a different slide and labeled with a single dye. Differential expression is measured by comparing the fluorescent intensity measurement of a given gene on the control slide with a separate measurement for the same gene from the treatment slide. Labeling reactions and hybridizations of in situ arrays are commonly carried out by the array provider or core lab. Several groups have experimentally compared the precision and accuracy of the two platforms using the same biological samples. Their studies may prove helpful to those considering which system to implement in their own research (see Woo et al., 2004; Yauk et al., 2004; Meijer et al., 2005). Microarray research has advanced dramatically in recent years in aquaculture or aquatic species (summarized in Table 1.4). However, the field is still in its infancy and distribution of resources remains uneven. A number of microarrays have been developed from a variety of aquaculture species that has led to the publication of a special issue in the Journal of Fish Biology devoted entirely to the description of microarrays in aquatic species (Table 1.4). Interested readers are referred to this special issue (Journal of Fish Biology, Volume 72, issue 9, 2008). To date, most published microarray studies have used PCR-amplified spotted cDNA clones to fabricate the array. However, as microarray research typically takes several years from its inception to reach publication, the recent trends toward spotted oligos and in situ microarrays may not be reflected in the aquaculture literature for several years. A welldesigned microarray can be a valuable asset to an aquaculture species group, especially if the cost per slide can be minimized to the extent that researchers can integrate transcriptomic approaches into their already established research. Microarray studies are most successful when they are just one of several approaches used to answer biological questions. For example, salmonid researchers have implemented array technology in their study of reproductive development, toxicology, physiology, and repeat structures (Rise et al., 2004b; Ewart et al., 2005; Krasnov et al., 2005; Tilton et al., 2005; Vornanen et al., 2005; Martin et al., 2006; von Schalburg et al., 2006; Roberge et al., 2007; Eichner et al., 2008; Gahr et al., 2008; Jørgensen et al., 2008; Schiøtz et al., 2008; Wynne et al., 2008; Vanya et al., 2008; Young et al., 2008). In a similar effort, microarrays have been used to identify defense related genes in enteric septicemia of catfish (ESC)resistant blue catfish and ESC-susceptible channel catfish (Peatman et al., 2007, 2008).

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Table 1.4 Current status of microarray development in various aquaculture and aquatic species Species

Microarray platform

Reference

Salmonids

GRASP 3.5K cDNA GRASP 16K cDNA INRA 9K cDNA cGRASP 32K cDNA cGRASP 22K oligo (70) 16 950 cDNA 660 cDNA nylon 19K in situ oligo 28K in situ oligo

Rise et al., 2004a von Schalburg et al., 2005 Bonnet et al., 2007 Koop, in progress Koop, in progress Taggart et al., 2008 Ju et al., 2002 Li et al., 2006 Peatman et al., 2007, 2008; Liu et al., 2008 Jenny et al., 2007 Gracey et al., 2004 Williams et al., 2008 Douglas et al., 2008 Salem et al., 2008 Olohan et al., 2008 Geoghegan et al., 2008 Klaper et al., 2008; Kane et al., 2008 Villeneuve et al., 2008 Gracey, 2008 Diab et al., 2008 Garcia-Reyero et al., 2008 Sarropoulou et al., 2005 de la Vega et al., 2008; Robalino et al., 2007 Wang et al., 2006

Catfish

Oysters Carp Atlantic halibut Rainbow trout Three-spined stickleback Fathead minnow Goby European flounder Largemouth bass Sea bream Shrimps

5K cDNA 13.4K cDNA 13K–26K cDNA 9277 50mer oligo 37K oligo 21 500 oligo 9692 cDNA 15 000 oligo 4105 oligo 2000 oligo 12 661 cDNA 3336 cDNA 15 950 oligo 10K cDNA 3853 cDNA 2469 cDNA 3136

Due to low funding levels and a relatively small research community, aquaculture genomics stands today where the model species did almost ten years ago in the 1990s. In the same way, microarray research in aquaculture species is only in its infancy. Like researchers of humans and mice ten years ago, we are currently using microarrays to accelerate gene expression analysis under varied experimental conditions, to reveal novel functions in genes, and to discover possible gene interactions and networking through cluster analysis. To find future directions for microarray research in aquaculture species, we need only to observe microarray studies in model species today. The future looks especially promising for using microarrays for SNP analysis and QTL mapping to make tangible progress towards widespread marker-assisted selection (MAS) in aquaculture. In particular, merging positional candidate genes with expression candidate genes from microarray information may reveal QTL genes responsible for important performance traits (see Drake et al., 2006). Microarrays have, furthermore, evolved to allow studies of metabolomics and proteomics that will be

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important in development of fish vaccines (Cretich et al., 2006). A Veterinary Immune Reagent Network has already been established in the USA toward development of a set of antibodies for use in agricultural research including aquaculture (http://www.avma.org/onlnews/javma/jun06/060615b. asp). Microarrays are also being utilized in livestock disease diagnostics, a use easily adapted for detection of outbreaks of aquaculture pathogens (Schmitt and Henderson, 2005; Baxi et al., 2006). Much of the groundwork for practical microarray research has already been laid. It is up to the aquaculture community to exploit and adapt these advances for the advantage of their respective species. In spite of the bright outlook of microarray research, emerging next generation sequencing technologies may soon replace, at least in part, the capacity of microarrays. This is because sequence-based gene expression profiling can provide not only gene identities without any ambiguity, but also more accurate assessment of genome expression based on sequence tag counting.

1.6.2 Sequence tag-based technology Tag profiling is a revolutionary approach to gene expression analysis that generates expression profiles for any transcript from any organism. Using Solexa or SOLiD sequencing technologies, millions of expressed sequence tags can be generated from a single run allowing gene expression profiles to be accurately characterized based on sequence tag counting. The major advantage of tag profiling is its high ability to identify, quantify, and annotate expressed genes on the level of the whole genome without prior sequence knowledge. Because sequence tag-based genome expression analysis does not require any existing genome resources, it is much more adaptable to aquaculture species where whole genome sequences are lacking.

1.6.3

Comparison of the microarray technology with tag- or sequence-based technology In microarray experiments, hybridization signal intensities are used to generate abundance measurements that correspond to the amount of target mRNA that has hybridized to a specific probe; and relative measurements are determined by a comparison of two samples. Tag-based technologies measure the expression level of a gene by counting the abundance of a specific transcript in a sample. This count provides an abundance measure of each gene’s expression level within the sample. Recently, some studies have compared microarray and massively parallel signature sequencing (MPSS) technology. Their results suggested a moderate correlation between the two platforms. However, one platform often detects expression for some genes that are not measured by the other platform (Coughlan et al., 2004; Oudes et al., 2005; Liu et al., 2007), suggesting that using a combina-

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tion of transcription profiling technologies would provide more complete coverage of gene expression measurements. It is likely that different technology platforms will provide significant differences in the measurements of gene expression. Chen et al. (2007) recently found that RNA samples exhibited higher correlations within the technology platform used to measure RNA abundance rather than expected similarities due to the biological nature of the samples. In particular, the tag- or sequence-based platforms may be more variable in measuring RNA abundance than Affymetrix or Agilent microarray platforms. Therefore, comparison of RNA abundance across technology platforms requires exercise of caution. However, when relative expression between samples with different biological treatment is at issue, which most often is the most important question for aquaculture research settings, the samples were more closely clustered according to their biological nature than the technology platform (Chen et al., 2007).

1.7 Acknowledgements Research in my laboratory is supported by grants from USDA NRI Animal Genome and Genetic Mechanisms Program, USDA NRI Basic Genome Reagents and Tools Program, Mississippi-Alabama Sea Grant Consortium, Alabama Department of Conservation, USAID, National Science Foundation, and BARD. The author would like to thank Dr Huseyin Kucuktas for helping with drawings of the figures, and to thank Dr Hong Liu for her assistance with the references.

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woram r a, mcgowan c, stout j a, gharbi k, ferguson m m, hoyheim b, davidson e a, davidson w s, rexroad c and danzmann r g (2004) A genetic linkage map for Arctic char (Salvelinus alpinus): evidence for higher recombination rates and segregation distortion in hybrid versus pure strain mapping parents, Genome, 47, 304–15. wynne j w, o’sullivan m g, cook m t, stone g, nowak b f, lovell d r and elliott n g (2008) Transcriptome analyses of amoebic gill disease-affected Atlantic salmon (Salmo salar) tissues reveal localized host gene suppression, Mar Biotechnol (NY), 10, 388–403. xu p, wang s, liu l, peatman e, somridhivej b, thimmapuram j, gong g and liu z (2006) Channel catfish BAC-end sequences for marker development and assessment of syntenic conservation with other fish species, Anim Genet, 37, 321–6. xu p, wang s and liu z (2007a) Physical characterization of aquaculture genomes through BAC end sequencing, in Liu Z (ed.), Aquaculture Genome Technologies, Blackwell, Ames, IA, 261–74. xu p, wang s, liu l, thorsen j, kucuktas h and liu z (2007b) A BAC-based physical map of the channel catfish genome, Genomics, 90, 380–88. yauk c l, berndt m l, williams a and douglas g r (2004) Comprehensive comparison of six microarray technologies, Nucleic Acids Res, 32, e124. young w p, wheeler p a, coryell v h, keim p and thorgaard g h (1998) A detailed linkage map of rainbow trout produced using doubled haploids, Genetics, 148, 839–50. young w p, ostberg c o, keim p and thorgaard g h (2001) Genetic characterization of hybridization and introgression between anadromous rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki), Mol Ecol, 10, 921–30. young n d, cooper g a, nowak b f, koop b f and morrison r n (2008) Coordinated down-regulation of the antigen processing machinery in the gills of amoebic gill disease-affected Atlantic salmon (Salmo salar L.), Mol Immunol, 45, 2581–97. yu z and guo x (2003) Genetic linkage map of the eastern oyster Crassostrea virginica Gmelin, Biol Bull, 204, 327–38. zane l, bargelloni l and patarnello t (2002) Strategies for microsatellite isolation: a review, Mol Ecol, 11, 1–16. zhao s h, recknor j, lunney j k, nettleton d, kuhar d, orley s and tuggle c k (2005) Validation of a first-generation long-oligonucleotide microarray for transcriptional profiling in the pig, Genomics, 86, 618–25. zhang y, liang l, jiang p, li d, lu c and sun x (2008) Genome evolution trend of common carp (Cyprinus carpio L.) as revealed by the analysis of microsatellite loci in a gynogentic family, J Genet Genomics, 35, 97–103. zhou z, bao z, dong y, wang s, he c, liu w, wang l and zhu f (2006) AFLP lingkage map of sea urchin constructed using an interspecific cross between Strongylocentrous nudus (Venus) and S. intermedius (Mars), Aquaculture, 259, 56–65. zhulidov p a, bogdanova e a, shcheglov a s, vagner l l, khaspekov g l, kozhemyako v b, matz m v, meleshkevitch e, moroz l l, lukyanov s a and shagin d a (2004) Simple cDNA normalization using kamchatka crab duplexspecific nuclease, Nucleic Acids Res, 32, e37.

2 Genetic improvement of finfish G. Hulata, Agricultural Research Organization, Israel, and B. Ron, Israel Oceanographic & Limnological Research Ltd, Israel

Abstract: This chapter focuses on the major genetic approaches, technologies and methodologies that have shaped the aquaculture industry in recent years. Classic selective breeding programs (cross-breeding and hybridization) are the mainstream of finfish genetic improvement, and will continue to be the main engine driving the global finfish aquaculture industry forward. Breeding programs have been expanded, their design optimized and many new ones initiated since the late 1990s. Advances in application of biotechnology to fishes have provided tools that can be used to genetically change (improve) cultured populations using non-selective breeding methods through manipulations of genes and chromosomes (mainly triploidy). Cytological methodologies are useful tools helping with chromosomal gene mapping and with validation of aquacultured finfish species and hybrids. Modern technology has brought new types of molecular markers into play, and their application has allowed rapid progress into many aquaculture investigations. These include quantification of genetic variability and inbreeding, parentage assignments, species and strain identification, construction of high-resolution genetic linkage maps for aquaculture species and the detection of quantitative trait loci. They also offer the opportunity to include genomic information in breeding programmes (marker assisted selection; MAS). Advances have been made in developing genomic and bioinformatic tools. Although gene transfer technology has yielded promising products, their future is questionable due to severe controversies over public health and environmental issues. Use of embryonic stem (ES) cell lines is being investigated and may prove to be an alternative approach for gene transfer. Key words: Breeding programs, biotechnology, manipulations of genes and chromosomes, cytogenetics, quantitative trait loci (QTL), marker assisted selection (MAS), genomic and bioinformatic tools, gene transfer, embryonic stem cells.

2.1 Introduction: current status of aquaculture genetics The status and prospects of aquaculture genetics have been repeatedly reviewed during the last decade for specific groups of fish or for the industry in general (e.g., Gjedrem, 2000, 2002; Knibb, 2000; Lymbery et al., 2000;

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Dunham et al., 2001; Hulata, 2001; Myers et al., 2001; Fjalestad et al., 2003; Okamoto, 2005; Hershberger, 2006; Mair, 2007). A few relevant books, each covering a specific aspect of the field, have also been published (Hallerman, 2003; Dunham, 2004; Gjedrem, 2005; Liu, 2007a). Although classic selective breeding methods as well as emerging technologies are available and contribute to the progress of the industry, their application is not yet evenly spread over globally cultured species and culture areas (Hulata, 2001). How much of the nearly 48 million tonnes cultured globally (2005 estimate; FAO, 2007) stems from genetically improved stocks is hard to tell. Some five years ago, Gjedrem (2002) estimated that genetically improved stocks accounted for no more than 10 % of aquaculture production. ‘This figure is undoubtedly rising in developed countries, as the benefits of genetic improvement become apparent, but it is almost certainly much lower in most developing countries’ (Mair, 2007). With major producers such as China and other south-eastern Asian countries (FAO, 2007) lagging behind in application of genetic technologies in their aquaculture industries, it is unlikely to be more than 20 % at present. It is not our intention to duplicate the above-mentioned reviews, but rather to focus on the new approaches, technologies and methodologies that have shaped the field in recent years.

2.2 2.2.1

Key drivers for genetic improvement of finfish

Improving growth rate, disease resistance and other quality traits by selective breeding and other methods Classical breeding programs (selective breeding, cross-breeding and hybridization) are the mainstream of finfish genetic improvement (Bartley et al., 2001; Gjedrem, 2005). The impact of selective breeding programs on the aquaculture industry can be exemplified by the wide global distribution of the Donaldson strain of rainbow trout (Parsons, 1998), the success of the Norwegian Atlantic salmon breeding program (Gjedrem, 2000) and the progressing dissemination of the selectively bred Nile tilapia, known as genetically improved farmed tilapia – GIFT (Pullin, 2007). Breeding programs have been expanded, and many new ones initiated during the last decade – see examples in Table 2.1. The breeding goal in most of these programs was improving growth rate. Whereas in the past improving growth rate was the most common breeding goal, new traits have been incorporated more recently in breeding programs (Table 2.2). As fish welfare is becoming a crucial issue for the aquaculture industry (Ashley, 2007), attention has also been given recently to selection for sustainability and animal welfare-related traits (Olesen et al., 2000, 2003; Bentsen and Olesen, 2002). Attention is also given to the possible effects of selection on the social behaviour and growth pattern of the fish (Brännäs

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Table 2.1 Examples of recent breeding programmes Species

Reference

Atlantic cod (Gadus morhua) Atlantic salmon (Salmo salar) Common carp (Cyprinus carpio) Gilthead seabream (Sparus aurata) Hybrid striped bass (Morone chrysops × M. saxatilis) Lake Malawi tilapia (Oreochromis shiranus) Mediterranean sea bass (Dicentrarchus labrax L.) Nile tilapia (O. niloticus) Red sea bream (Pagrus major) Rohu carp (Labeo rohita)

Gjerde et al., 2004 Quinton et al., 2005; Kolstad et al., 2006 Vandeputte, 2003; Kocour et al., 2007 Gorshkov et al., 2004 Garber and Sullivan, 2006 Maluwa and Gjerde, 2006a,b, 2007; Maluwa et al., 2006 Saillant et al., 2006 Ponzoni et al., 2005; Li et al., 2006 Murata et al., 1996 Gjerde et al., 2002; Reddy et al., 2002

Table 2.2 Examples of production- and consumer-related breeding-goal traits in recent breeding programmes Trait Production-related Age at maturity Eliminating vertebral deformity Feed efficiency Reproductive traits Stress, disease and parasite resistance

Consumer-related Appearance Body composition Carcass quality

Species

Reference

Rainbow trout (On. mykiss) Atlantic salmon (S. salar) Atlantic cod (Gadus morhua) Atlantic salmon (S. salar) Coho salmon (On. kisutch) Rainbow trout (On. mykiss) Atlantic salmon (S. salar)

Kause et al., 2003a

Rainbow trout (On. mykiss) Rainbow trout (On. mykiss) Coho salmon (On. kisutch)

Kause et al., 2003b, 2004

Gjerde et al., 2005 Kolstad et al., 2006 Kolstad et al., 2004, 2005b; Kause et al., 2006b; Quinton et al., 2007 Gall and Neira, 2004; Gallardo et al., 2004a Pottinger and Carrick, 1999; Henryon et al., 2005 Kolstad et al., 2005a; Ødegård et al., 2006, 2007a,b

Tobin et al., 2006; Kause et al., 2007; Quillet et al., 2007 Neira et al., 2004

et al., 2005). Improvements have also been made in breeding programs through the introduction of new methodology for measuring complex traits, such as flesh color or feed efficiency [in rainbow trout (On. mykiss) – Helge Stien et al., 2006; Kause et al., 2006a].

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Advances in designing breeding programs in aquaculture Because broodstocks are limited in size, inbreeding is an inherent problem in many breeding programs (Gjedrem, 2005). Efforts have been made recently to optimize mating designs for reducing effects of inbreeding in breeding programs (Gjerde et al., 1996; Villanueva et al., 1996; Sonesson and Meuwissen, 2000, 2002; Sonesson et al., 2003; Gallardo et al., 2004b; Dupont-Nivet et al., 2006; Holtsmark et al., 2006, 2008; D’Agaro et al., 2007), and in improving the experimental designs and statistical models to enhance genetic gains (Sonesson et al., 2005; Hinrichs et al., 2006; Martinez et al., 2006a,b). In addition, emerging technologies based on molecular markers and genomic approaches are progressively rising in importance, and efforts are being made to involve molecular approaches in breeding programs (Fjalestad et al., 2003; Silverstein et al., 2006). A step further towards improving the design of breeding program was taken by Hayes et al. (2006) in their comparison of different strategies for using molecular marker information in order to maximize genetic diversity in the base population. Combining available phenotypic information for the traits of interest with marker data, they propose, would ‘ensure that as much genetic variance as possible, for as many traits as possible, is captured in the base population’.

2.2.2

Improving performance and other traits by non-selective breeding methods Advances in application of biotechnology to fishes (reviewed by Melamed et al., 2002) have provided tools that can be used to genetically change (improve) cultured populations using non-selective breeding methods through manipulations of genes and chromosomes (Rasmussen and Morrissey, 2007). The different approaches will be discussed in more detail below. Chromosome set manipulations (gynogenesis, androgenesis and polyploidy) Although chromosome-set manipulations, which have been heavily investigated since the 1970s, have not resulted in many commercial applications to advance the aquaculture industry (Khan et al., 2000; Hulata, 2001), these manipulations still attract interest (Arai, 2001; Felip et al., 2001a; Gomelsky, 2003; Komen and Thorgaard, 2007), and research has expanded to more species with emphasis on newly-cultured species (Table 2.3). The physiological effects of polyploidy are also being investigated (e.g., Peruzzi et al., 2005; Taylor et al., 2007; Maxime, 2008). The most common applications of these procedures are production of triploids to prevent reproduction and improve growth rate (reviewed by Tiwary et al., 2004), and cloning through gynogenesis and androgenesis (reviewed by Pandian and Kirankumar, 2003; Komen and Thorgaard, 2007). Triploidy is also used

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Table 2.3 Examples of chromosome manipulations in newly culture species Species

Reference

European sea bass (Dicentrarchus labrax) Sunshine bass (M. chrysops × M. saxatilis) Largemouth bass (Micropterus salmoides) Turbot (Scophthalmus maximus)

Felip et al., 2001a,b, 2002; Peruzzi et al., 2004; Bertotto et al., 2005 Kerby et al., 2002

Barfin flounder (Verasper moseri) Sturgeon (Acipenser sp.) Atlantic halibut (Hippoglossus hippoglossus)

Gomelsky et al., 2004; Neal et al., 2004 Piferrer et al., 2000, 2003, 2004; Cal et al., 2006 Mori et al., 2006 Flynn et al., 2006; Grunina et al., 2005, 2006 Tvedt et al., 2006

for genetic mapping (Nomura et al., 2006). Although resulting in high degree of sterility, triploidy does not always confer significant improvement of growth (e.g., Mori et al., 2006). Production of triploids would be more efficient by mating induced tetraploids with normal diploids; this has so far been restricted to rainbow trout, since in no other commercial finfish species have viable and fertile tetraploids been obtained (Arai, 2001; Babiak et al., 2002a,b). For other species, triploids are produced as all-female populations by using spermatozoa of artificially sex-reversed (often gynogenetic) males, so as to assure their complete sterility (e.g., Arai, 2000; Rothbard et al., 2000; Rothbard, 2006 and Fig. 2.1). This is because triploid males sometimes show gonadal development and often are not completely sterile (e.g., Pandian and Koteeswaran, 1998; Arai, 2000, 2001; Felip et al., 2001a; Oshima et al., 2005; Sousa-Santos et al., 2007). Commercial production and culture of (all-female) triploid fish is so far limited to brown trout, rainbow trout, Atlantic and Chinook salmon and European sea bass. Additionally, commercial production of triploid grass carp is carried out for stocking native bodies of water to control vegetation. Cytogenetics During the 1990s, the number of cytogenetic studies of marine fishes has increased. Its main application, however, has been in solving systematic and taxonomy issues rather than genetic improvement. Cytogenetic studies can help in predicting the success of inter-specific hybridization between species; successful hybridization occurs predominantly between closely-related species having identical chromosome structure and numbers, or at least identical number of chromosome arms (e.g., Kim et al., 1995). Resulting F1 are often reproductively viable, although in some cases they are sterile (in most cases due to being triploids).

60

New technologies in aquaculture ♂ Albino grass carp (XY)

♀ Wild-type grass carp (XX)

♀ Albino grass carp (XX)

♂ Common carp (XY)

Sperm (X & Y)

UV-irradiated eggs

Eggs (X)

UV-irradiated sperm

Fertilization

Fertilization

Shock Lateshock

♀♀ XX Female monosex (2nd Task)

♂♂ ♀♀ (YY) (XX) Homozygous progeny

MT sexinversion ♂♂ XX Gynogenetic males

Fertilization

Sperm bank

Sperm bank

Early-shock

♂♂ XY Male monosex (1st Task)

♀♀ XXX Female triploids (3rd Task)

Lateshock

♀♀ XXXX 4N-females (4th Task) 2N gametes Fertilization

♀♀ XXX Female triploids (5th Task)

Fig. 2.1 Schematic presentation of ploidy manipulations on grass carp to produce XXX female triploids (reproduced with permission from Rothbard, 2006).

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61

Another area where cytogenetics has played an important role is studies of sex determination (reviewed by Ezaz et al., 2006). Knowledge on sex determination may contribute to efficient production of monosex populations, which in turn may contribute to improved production efficiency in various cultured species (e.g., tilapias). Cytogenetics is also playing an important role in the verification of chromosome-set manipulations as well as for gene mapping (Gorshkova, 2006). Study of sex chromosomes: Ten sex-determination systems are known among fishes. The unusual cytology and exceptional evolution of sex chromosomes lead to numerous basic questions related to why and how sex chromosomes evolved. This was the source of a century-long debate from the time when H.J. Muller suggested that sex chromosome pairs evolved eventually from a pair of autosomes (Muller, 1914). With regard to cytological examination of sex chromosome evolution, fishes are the most fascinating of vertebrate groups. Fishes represent the largest vertebrate group which displays the widest diversity of sex determination and sex chromosomal systems, including gonochorism (separate sexes), hermaphroditism (individuals displaying both sexes) and unisexuality (all female-species). Using cytogenetic methodologies, among others, it was revealed that in gonochoristic fishes, the gender may be determined genetically – ranging from a single-allele determination to chromosomal sex determination. In addition, it was shown that polygenic sex determination and sex determination by genotype–environment interaction take place in fishes (see reviews by Devlin and Nagahama, 2002 and Ezaz et al., 2006). Cytogenetic studies play an important role in the identification of the various sturgeon species and hybrids. Inter-specific hybridization is well known in Acipenseriformes and can be advantageous in aquaculture (Steffens et al., 1990; Gorshkova et al., 1996; Fontana, 2002; Gorshkova, 2006). Lately, Fopp-Bayat et al. (2007) performed cytogenetic analysis by preparing chromosomes from the gill epithelium of Acipenser baeri × (Huso huso × Acipenser ruthenus) hybrids in both diploid and triploid states. Karyological inspection of Russian imported sturgeons, reared at the Kibbutz Dan fish farm, Israel, were conducted on hybrids of Russian sturgeon (Acipenser guldenstadti) and great (beluga) sturgeon (Huso huso). Results showed that the consistent mode of 2N was 181–190 and the karyotype consisted of 78 metacentric and submetacentric, 16 acrocentric, and about 88 micro-chromosomes. The intermediate origin of the imported sturgeon hybrid confirmation, based on different number of chromosomes, points at the polyploidy origin of the Acipenseridae, and allowed the geneticists to formulate recommendations concerning conservation and genetic management of cultured sturgeon stocks in Israel (Gorshkova et al., 1996; Gorshkova, 2006). During 1992–1995, Groshkova and co-workers conducted chromosome set manipulation research using the economically important marine finfish, gilthead seabream (Gorshkova et al., 1995; Knibb et al., 1998a, b). These

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studies led to the establishment of a protocol that provided gilthead sea bream gynogenetic and triploid progenies. In addition, these researchers achieved the production of meiotic gynogenetic sea bass progeny using heat shocks to eggs fertilized with UV-irradiated sperm (Gorshkova et al., 1995; Knibb et al., 1998a, b). As a result, a direct karyological confirmation concerning the hybrid nature and triploid origin of the offspring were described using karyotypes of chromosomally-manipulated forms with ‘marker’ chromosomes (Gorshkov et al., 1998). Another study was done during 1999, when Gorshkova and co-workers commenced cytogenetic inspection of white grouper (E. aeneus) early embryonic development (Gorshkova et al., 2002b). Although one of the most valuable fishes in the Mediterranean basin, white grouper has a severe limitation in commercial culture as a result of the unpredictable and often low quality of spawned eggs, embryos with low survival rates and escalating larvae mortality. The percentage of cytogenetically abnormal embryos carrying diverse types of chromosomal aberrations varied significantly amongst spawnings and ranged from 35.5–79 %. Continuous examination of subsequent spawning of different parental fishes using cytogenetic monitoring of the early embryonic stages would be of immediate interest for future genetic broodstock management of the white grouper (Gorshkova et al., 2002a). Aid in gene mapping: Cytological methodologies have been employed in chromosomal gene mapping of some aquacultured finfish species. This technique has been used mainly as a complementary approach to identify particular chromosomes (Phillips and Reed, 1996; Cnaani et al., 2007a, b; Phillips et al., 2006). Using fluorescence in situ hybridization (FISH) and linkage mapping, Cnaani and co-workers showed that in tilapia the sox2 and sox14 genes are on separate chromosomes. The rainbow trout (On. mykiss) genetic linkage groups have been assigned to specific chromosomes in the OSU (2N = 60) strain using FISH with bacterial artificial chromosomes (BAC) probes containing genes mapped to each linkage group. The set of BACs compiled for this research will be especially useful in construction of genome maps and identification of quantitative trait locus/loci (QTL) for important traits in other salmonid fishes (Phillips et al., 2006; see further discussion below). Molecular markers Since the 1960s, electrophoretic studies of proteins (allozymes, or allelic forms of isozymes) have provided the primary tool for studying genetic diversity in fisheries and aquacultured stocks (Liu and Cordes, 2004; Verspoor et al., 2005; Kucuktas and Liu, 2007). The main uses of allozyme electrophoresis have been for stock identification and management (in the wild as well as aquaculture), analysis of population genetics processes such as inbreeding and genetic drift, molecular tagging and parentage analysis and, to a much lesser extent, for genetic mapping (Liu and Cordes, 2004).

Genetic improvement of finfish

63

The major drawbacks of allozymes, apart from the need for fresh or frozen samples of relatively large quantities, are the limited variability and poor coverage of the genome. Nevertheless, allozyme analysis has made a significant contribution to our understanding of the genetic diversity and the structure of wild genetic resources in many species, and especially the Atlantic salmon (Verspoor et al., 2005). Since the mid-1980s, DNA-based analyses (e.g., Artamonova, 2007) that are characterized by higher genetic variation and polymorphism, as well as higher genome coverage, have gradually substituted for allozymes. Modern biotechnology has introduced into play new types of molecular markers, as well as other techniques that will be discussed below (see Chapter 1 in this volume for a detailed account). The new platform technologies have opened up vast possibilities to the aquacultural biotechnologist (Melamed et al., 2002).The various types of DNA markers – mitochondrial DNA, restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), microsatellite, single nucleotide polymorphisms (SNP) and expressed sequence tag (EST) markers – have been described in detail by Liu and Cordes (2004) and in Chapters 2–8 in Liu (2007a). The application of DNA markers has allowed rapid progress in aquaculture investigations of genetic variability and inbreeding, parentage assignments, species and strain identification and construction of high-resolution genetic linkage maps for aquaculture species (Liu and Cordes, 2004), as well as detection of QTL and enabling the use of genomic information in breeding programs (viz MAS). Chistiakov et al. (2006) state that in aquaculture research, microsatellites are the ‘workhorse markers’ and review the genomic distribution, function, evolution and practical applications of microsatellites to fish genetics and aquaculture. Gradually, SNPs are becoming the future markers of choice (Liu, 2007b), mainly because of the need for very high densities of genetic markers (SNPs by far exceed microsatellites in this respect), and the recent progress in genotyping techniques and detection of polymorphism. Genomic resources continue to be developed (e.g., Hayes et al., 2007; Somridhivej et al., 2008). These and the other new marker types paved the way for various applications that are re-shaping aquaculture breeding programs. Parental assignment and molecular pedigrees: DNA markers, notably microsatellites, have solved one of the major obstacles in breeding programs, namely the ability to individually mark small fish. In order to utilize maximum information available on relatives, and increase the accuracy of selection, fish should be marked individually so that pedigrees can be tracked across generations (Villanueva et al., 2002). Since newborn fish are too small to be physically tagged, families must be reared separately until the fish reach the size at which they can be safely tagged individually (Doyle and Herbinger, 1994; Herbinger et al., 1995). Apart from limiting the

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number of families that can be used, and the extra expenses incurred by rearing each family separately, this also introduces common-environment correlations that reduce the accuracy of selection. Furthermore, it allows applying family selection to fish species that are not easily reproduced by single-pair matings (e.g., gilthead sea bream; Knibb, 2000). The development of DNA markers has enabled solution of this problem, when it was shown that by using a series of polymorphic markers, each individual in a mix of several or many families can be uniquely assigned to its parents with nearly complete accuracy (e.g., Herbinger et al., 1995, 1999; Estoup et al., 1998; O’Reilly et al., 1998; Perez-Enriquez et al., 1999; Norris et al., 2000). The pioneering study by Herbinger et al. (1995) was designed to ‘assess the feasibility of establishing pedigrees in mixed aquaculture populations and of selection programs for commercial aquaculture operations based on genetic profiling data from microsatellite markers’, and in fact paved the way for the application of DNA markers in breeding programs. The Herbinger et al. (1995) study ‘showed that the pedigree of a mixed rainbow trout population could be satisfactorily determined using as few as four microsatellite markers even though the fish could have originated from 100 possible pairs (ten sires × ten dams). The ability to establish the pedigree of completely mixed fish from their single locus DNA profile means that this pilot study was able to take place in a production farm with practically no interference with the normal routine’. Using this approach, families produced separately can be mixed and reared communally from hatching, or pools of males and females can be bred in the same pond or tank and their progeny reared together, until a posteriori parentage assignment at a later stage, even just before selection of breeding candidates. Villanueva et al. (2002) attempted to answer the key questions related to this application – the number of loci and the level of information (i.e., the numbers of alleles per loci and their relative frequency) required for accurate assignment. Application to breeding programs is already under way, e.g., in the estimation of heritability with a microsatellite parentage assignment-based pedigree in common carp cultured under traditional pond conditions as demonstrated by Kocour et al. (2007). Another application of DNA markers that is becoming of major importance is for tracing live fish or fish products at any stage along the production chain. Hayes et al. (2005) compare and discuss three alternate traceability schemes using DNA markers. Linkage maps: Since the late 1990s, linkage maps have been developed for most commercially important finfish species (Table 2.4; see also Table 10.2 in Danzmann and Gharbi, 2007). Work is also underway to develop the necessary genomic resources to develop maps of barramundi (Lates calcarifer – Zhu et al., 2006) and striped bass (Morone saxatilis – Rexroad et al., 2006). Some maps are quite preliminary, although for a few species more advanced maps have already been

Genetic improvement of finfish

65

Table 2.4 Examples of linkage maps for commercially important species. Those already listed in Danzmann and Gharbi (2007) are marked in bold face Species

Reference

American catfish (Ictalurus punctatus ¥ I. furcatus) Arctic char (S. alpinus) Atlantic salmon (S. salar) Ayu (Plecoglosus altivelis) Baramundi (Lates calcarifer) Bighead carp (Aristichthys nobilis) Brown trout (S. trutta) Common carp (C. carpio) European sea bass (Dicentrarchus labrax) Gilthead sea bream (Sparus aurata)

Liu et al., 2003

Japanese flounder (Paralichthys olivaceus) Rainbow trout (On. mykiss) Silver carp (Hypophthalmichthys molitrix) Thai catfish (Clarias macrocephalus) Tilapia (O. niloticus ¥ O. aureus; F2) Yellowtail (Seriola spp.)

Woram et al., 2004 Moen et al., 2004 Watanabe et al., 2004 Wang et al., 2007 Liao et al., 2007 Gharbi et al., 2006 Sun and Liang, 2004 Chistiakov et al., 2005 Franch et al., 2006; Senger et al., 2006; Sarropoulou et al., 2007a,b; 2008 Coimbra et al., 2003 Nichols et al., 2003; Danzmann et al., 2005 Liao et al., 2007 Poompuang and Na-Nakorn, 2004 Lee et al., 2005 Ohara et al., 2005

obtained. The number of markers mapped range from 146 (C. Macrocephalus – Poompuang and Na-Nakorn, 2004) to over 1400 (On. mykiss – Danzmann et al., 2005) in the more advanced maps, and some contain genes as well (those of rainbow trout, brown trout, Atlantic salmon, Arctic charr, channel catfish, European sea bass, common carp and tilapia – for details see Table 10.2 in Danzmann and Gharbi, 2007). Apart from the Danzmann et al. (2005) map of rainbow trout and the preliminary map of common carp (Sun and Liang, 2004), all other maps have either more or less linkage groups (LG) than the haploid number of chromosomes (N) in the species. As long as no commercially important species has its genome fully sequenced, linkage maps constitute the basic prerequisite for detection of QTL (Korol et al., 2007) and fine mapping of genes through comparative mapping to sequenced genomes of model species, and for positional cloning (Lee and Kocher, 2007; Sarropoulou et al., 2008). QTL detection: A QTL is a segment of a chromosome with a significant effect on the expression (phenotype) of a trait of interest; this issue was recently reviewed by Korol et al. (2007). The chapter presents an overview of QTL detection, lists the four steps in the detection of QTL, and outlines their application in MAS; see below) – marker development; development

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of a linkage map of at least moderate density; mapping of QTL using genetic markers placed on the map, with development of a model for inheritance and expression of the trait; and finally, use of the identified association between marker(s) and QTL in practical breeding program (i.e., MAS). It then discusses recent advances in detection of QTL, and presents a list of traits for which QTL have been detected in various aquacultured species. Among the traits are growth rate and body shape traits, resistance to stress, pathogens and diseases, coloration, sex determination. These have been detected in Atlantic salmon, rainbow trout, coho salmon, Arctic char, tilapias and common carp (see references in Korol et al., 2007 and Sonesson, 2007a). Most recently, a QTL for early maturation was identified in rainbow trout (Haidle et al., 2008). Moen et al. (2004) present a testing strategy for detection of QTL affecting disease resistance in Atlantic salmon. Sonesson (2007a) reviewed the efforts to detect QTL in aquaculture species. Such studies yield knowledge of marker–QTL linkages and estimates of the effects of QTL alleles on the trait in the population. Komen and Thorgaard (2007) discuss the advantage of using double haploids for QTL mapping and present case studies for rainbow trout, but also mention the obstacles to implementation of this approach (related to yield, survival, fertility, quality control and sustained commitment of resources). They conclude that the biggest challenge is the extremely low yields of doubled haploids in experiments with a variety of fish species. MAS: The development of large numbers of genetic markers and genetic maps for many aquacultured species (as mentioned above), which enables detection of QTL, has further led to search for genes associated with commercial traits. Opportunities that were proposed about 25 years ago (Soller and Beckmann, 1982; Beckmann and Soller, 1983), namely the use of genetic markers for selection, are finally becoming a reality. And yet, ‘only a handful of cases demonstrating practical usefulness of MAS’ have been reported in livestock (Rothschild and Ruvinsky, 2007) and even fewer in aquacultured species. The most remarkable application so far is the breeding of a lymphocystis disease-resistant Japanese flounder (Paralichthys olivaceus) (Fuji et al., 2006, 2007; Sakamoto et al., 2006), although this is not a real selective breeding program. They identified a major locus that is mapped to LG15 of the Japanese flounder linkage map, which is highly associated with resistance to the lymphocyctis disease (LD). Specifically, one allele of this marker is tightly linked to LD resistance. LD resistance and the marker are inherited in a Mendelian fashion, with LD resistance behaving as a dominant trait. This inference enabled selection of resistant parents for establishing an LD-resistant stock. With the many projects of marker development, QTL and linkage mapping in progress (see for examples Liu, 2007a; Martinez, 2007), it is anticipated that the industry will adopt the MAS strategy in the near future (Rothschild and Ruvinsky, 2007). Sonesson (2007a) and Martinez (2007) reviewed the current status of aqua-

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culture breeding schemes and evaluated the possibilities for applying MAS. So far, MAS schemes have been mostly developed for livestock. Sonesson (2007b) is developing and optimizing models for combining MAS with the classical aquaculture breeding schemes using the best linear unbiased prediction (BLUP) model. Genomics Wenne et al. (2007) reviewed the current development of genomic technologies and their potential applications and implications for fisheries management and aquaculture. Full genome sequences are so far available only for a few model fish species such as zebrafish Danio rerio (http://www.sanger.ac.uk/ Projects/D_rerio/), fugu Takifigu rubripes (http://www.fugu-sg.org/), puffer fish Tetraodon nigroviridis (http://www.genome.gov/11008305), medaka Oryzias latipes (http://dolphin.lab.nig.ac.jp/medaka/index.php) and stickleback Gasterosteus aculeatus (http://www.genome.gov/12512292). A Tilapia Genome Sequencing Project is currently underway at the Broad Institute [a research collaboration involving the Massachusetts Institute of Technology (MIT) and Harvard University], and the release of a first high-coverage genome is expected before the end of 2009 (TD Kocher, University of Maryland, USA, pers comm). Sequenced genomes of the model species have been well exploited so far with bioinformatics analyses and molecular biology techniques. It is anticipated that integration of more traditional disciplines such as biochemistry and physiology and expanding the study to additional species such as carp, catfish, salmon, trout or tilapia will further exploit the potential of fish genomics. This will be accompanied by applications to environmental biology and aquaculture (Crollius and Weissenbach, 2007). Various aspects of fish genomics have recently been reviewed in great detail in a book edited by Liu (2007a). Therein, Davidson (2007) and Xu et al. (2007) discuss the importance and utility of BAC libraries as the key genomic resource required for building genetic maps and integrating them with the respective physical maps. Guo et al. (2007) reviewed the utility of FISH as a tool in genome mapping. Many FISH studies have been published, but the full potential of this tool for gene and genomic mapping as well as for comparative genomic analysis in aquacultured species has not yet been realized. Rexroad (2007) reviewed the construction of radiation hybrids (RH) and discuss the perspective of RH mapping for aquaculture species. Apart from zebrafish, the only RH map reported so far for an aquacultured species is for the gilthead sea bream (Senger et al., 2006; Sarropoulou et al., 2008). Lee and Kocher (2007) presented an example of how comparative mapping and positional cloning were employed in an attempt to identify the gene(s) underlying a QTL for sex determination identified on LG1 of Nile tilapia. Their conclusion is that ‘conservation of gene order among fish at scales of several Mbs allows the use of the relatively complete sequences of model fish species to accelerate gene discovery and positional cloning of these genes’.

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Bioinformatic tools already available and those that will be further developed enable prediction of genes in important aquaculture species using the genome sequences of the model fishes. The sequencing of the genome of Nile tilapia, expected to be completed during 2009 (TD Kocher, University of Maryland, USA, pers comm), will further boost the use of comparative mapping of aquacultured species. Chapters 20–24 in Liu (2007a) review and discuss the status and perspective of analyzing genome expression and gene function in fish using expressed sequence tags (EST) and microarrays. More details on the status of genome mapping and genomics in salmonids, cyprinids, catfish, tilapias, European sea bass and Japanese flounder can be found in the recently published book Genome Mapping and Genomics in Fishes and Aquatic Animals (Kocher and Kole, 2008). Transgenesis Gene transfer technology leading to the production of genetically modified organisms (GMOs) is probably the most controversial issue dealt with in this chapter. On one hand, its successful application in several aquaculture species has produced stocks with improved traits, such as enhanced growth rate. The most notable of these are in salmonids, e.g., Devlin et al. (2004) and Fletcher et al. (2004), mud loach, e.g., Nam et al. (2001, 2002, 2004), and tilapia, e.g., Martínez et al. (2000), Maclean et al. (2002) and Caelers et al. (2005). Another case is increased cold tolerance through expression of an antifreeze polypeptide that might potentially expand culture range of salmon (Devlin et al., 2004). Gene transfer also has the potential to contribute to disease resistance [e.g., enhanced bacterial disease resistance of cecropin-transgenic channel catfish (Ictalurus punctatus) – Dunham et al. (2002) – and resistance to Aeromonas hydrophila infection in human lactoferrin-transgenic grass carp (Ctenopharyngodon idellus) – Mao et al. (2004)] and other traits. On the other hand, GMOs pose environmental threats and have raised public concerns that, so far, prevent their commercial use by the aquaculture industry (e.g., Kapuscinski and Hallerman, 1990; Levy et al., 2000; NRC, 2002; Pew, 2003; Myhr and Dalmo, 2005; Rasmussen and Morrissey, 2007). Galli (2002) presented a quite comprehensive overview of the current status of modern biotechnology research in aquaculture. Directed to policy and decision makers, it highlights issues relevant to research and the potential for commercialization of genetically modified (GM) aquacultured organisms. Teufel et al. (2002) reviewed the research on transgenic trout and salmon, their potential and the constraints for implementation. The issues of public concerns and implications to the aquaculture industry have been further discussed by several scientists, e.g., Aerni (2004), Maclean (2003) and Millar and Tomkins (2007). A special volume (Kapuscinski et al., 2007) published recently has focused on the potential environmental risks (threats to biodiversity and natural ecosystems) and

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benefits of uses of aquacultured GMOs. It covers all aspects from the development of transgenic fish, through assessment of environmental risks from their use, to suggestions for risk management of transgenic fish. The main conclusion and message of the book is that the risks ‘must be honestly and accurately analysed and understood by society’ in order to allow the potential benefits of transgenic aquaculture research to be realized. It further suggests that ‘using this book’s chapters for guidance, countries can begin to approach the task of creating effective, scientifically sound and socially responsible biosafety policies for transgenic fish and other aquatic organisms’. Commercialization of transgenic fish, however, poses not only ecological, food safety and regulatory issues, but also animal welfare concerns. Hallerman et al. (2007) reviewed the effects of growth hormone transgenes on the welfare and behavior of four species: Atlantic salmon, coho salmon, tilapia and common carp. Various morphological, physiological and behavioral alternations occur in GH-transgenics that seem to negatively affect, among other traits, swimming ability and reproductive behavior. Possible means for reducing the welfare issues that arise are discussed, such as the use of weaker promoters in expression vectors and selection of transgenic lines with physiologically appropriate levels of GH expression. Furthermore, since GH-transgenic animals have higher energy demands than non-transgenic fish, optimizing their formulated diets may improve production potential of transgenic animals and help maintain their welfare. The production of transgenics in itself has met with various difficulties, one of which is lack of control over integration of a single copy of a transgene and its proper expression. In recent years, use of embryonic stem (ES) cell lines has been investigated as an alternative approach for gene transfer. Hong et al. (2000) have reviewed the status and perspectives of using ES for transgenesis in fish. Since then, more studies have focused on the model fishes medaka (Bubenshchikova et al., 2005) and zebrafish (e.g., Ma et al., 2001; Fan et al., 2004; Hong and Schartl, 2006; Alvarez et al., 2007; Chen et al., 2007), and in recent years, a few attempts have also been made in aquacultured species (Holen and Hamre, 2003; Parameswaran et al., 2007).

2.3 Case studies – risks associated with selective breeding programs Species or strains of many fish species have been translocated from their place of origin, or from places to which they have been introduced, and deliberately released for stocking or escaped from culture facilities, thereby affecting wild stocks (Cross, 2000). A few outstanding examples will be discussed.

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The farming of Atlantic salmon (Salmo salar), which has greatly expanded in the last 50–60 years, resulted in large numbers of escaped farm salmon invading native salmon populations throughout the North Atlantic (e.g., Fleming et al., 2000; Gilbey et al., 2005; Carr and Whoriskey, 2006; Hindar et al., 2006; O’Reilly et al., 2006). The nature of this interaction has been investigated by McGinnity et al. (2003, 2004), Weir et al. (2004, 2005) and others. Naylor et al. (2005) presented a thorough analysis of the problem in their assessment of the risks of escaped salmon from net-pen aquaculture, and listed various potential biological consequences of farm salmon escapes: risk of feral stock establishment; risks of competition with wild fish for mates, space and prey; risk of pathogen transmission; and, most relevant to this review, risks associated with genetic interactions with wild stocks. These were further discussed in the framework of the EU GENIMPACT project: Evaluation of genetic impact of aquaculture activities on native populations (Verspoor et al., 2006). Moreover, the culture of Atlantic salmon has been shown to genetically affect wild populations of other salmonids as well, e.g., sea trout (Salmo trutta) (Coughlan et al., 2006). Of even greater concern are the risks associated with the Atlantic salmon selective breeding programs. Since the 1980s, a series of corporate merger and spin-off through purchase and sale of Atlantic salmon breeding and/or growing companies has resulted in translocations of stocks among countries in Europe as well as North America and Chile. For example, the origin of Donegal Silver Irish salmon lies in the MOWI Norwegian broodstock, eggs from which were introduced at Fanad Fisheries in 1982. Marine Harvest, one of the largest salmon companies in the world resulting from these mergers, with branches in Ireland, Norway, Scotland, Canada and Chile, has moved stocks within Europe as well as from Europe to North and South America (A. Norris, Marine Harvest, pers comm; http://www. marineharvest.com/). The effects of cultured species on their respective wild populations is visible in the last two or three decades also with the Mediterranean gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax). These effects include interaction and competition for resources by accidentally escaping fish (whose numbers are increasing according to the records) and contribution of escaped fish to reproduction in the wild. Data suggest that the contribution of escaped spawners ‘is not negligible’ and that a decrease in mean size of fish caught in the longline fishery in Greek coastal lagoons has already been detected (Dimitriou et al., 2007). Naylor et al. (2005) predict that the rising production of two new marine species – cod and halibut – may lead to similar processes. Escaped hybrid catfish (female Thai walking catfish, Clarias macrocephalus × male African catfish, C. gariepinus) from farms in central Thailand may interbreed with C. macrocephalus individuals in the wild. Senanan et al. (2004) assessed genetic introgression of C. gariepinus genes into four wild and two broodstock populations of C. macrocephalus.

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Tilapias are a group of fish that have been widely spread around the world since the 1950s (Pullin et al., 1997). More recently, stocks of Nile tilapia (O. niloticus) were introduced from various regions in Africa into the Philippines and mixed with cultured (earlier – introduced) strains to form the base population for the GIFT breeding program carried out by the WorldFish Center (formerly ICLARM) and collaborators (Eknath et al., 1993, 2007; Eknath, 1995). Improved descendants from this program were disseminated to several countries in Southeast Asia for evaluation against local stocks, eventually leading to commercial culture of this introduced strain, which showed superior growth rate and survival relative to that of other strains used by farmers (De Silva, 2003). Since no native wild populations of tilapia existed in those countries, escapement did not result in any damage to wild populations. Upon termination of the GIFT research program, sub-samples were transferred to several countries in the region and served as founders for separate, parallel, further breeding programs (Gupta and Acosta, 2004). According to Ponzoni (2007, WorldFish Center, pers comm), the WorldFish policy has been not to transfer GIFT to Africa because of biodiversity considerations, namely, due to concerns that the fish could escape and cross with wild populations in Africa, thus contaminating their gene pool. Consequently, countries from which wild fish were sampled to initiate the breeding program (Egypt, Ghana, Kenya and Senegal) did not benefit from the genetic gain made, and have received nothing in return for their collaboration. The issue has been raised by some African representatives and it has re-kindled the debate on the matter. This has resulted in a recommendation to allow controlled introduction followed by a properly designed comparison of GIFT with relevant local strains to ascertain that there is a productivity advantage exhibited by GIFT. At present, WorldFish is finalizing the policy document that will define the circumstances under which the introduction of GIFT to an African country will be authorized. ‘Given a favorable outcome for GIFT from the above comparison, multiplication and dissemination of GIFT will be authorized. The multiplication and dissemination programs will be accompanied by a package of measures attempting to minimize the risk of escapes and to mitigate the impact in case these should occur’ (Ponzoni, 2007, WorldFish Center, pers comm).

2.4 Future trends Conventional breeding programs will continue to be the main engine driving the global finfish aquaculture industry forward. Efforts will persist to increase efficiency and optimize the design of breeding programs by maximising the use of pedigree information while using both established and cutting-edge technologies mentioned above. However, since these methods are less suitable for economically important traits that are

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difficult to measure on candidates for selection (such as carcass and disease traits), alternative approaches will have to be further developed and optimized. Here is where incorporation of recent biotechnological tools may come into play. The potential for accelerating breeding programs expected from applying these tools has yet to be realized in the aquaculture industry. Nevertheless, MAS and gene-assisted selection (GAS) methodologies, when mature, may eventually become practical in efforts towards identifying genes that underlie economically-important traits and towards combining quantitative and molecular data in breeding programs. A potential alternative breakthrough may arise from solving containment problems currently limiting the use of GM aquacultured organisms; once the public prefers education to regulation, antagonism to the use of GM may fade out.

2.5 Sources of further information and advice Several books devoted to various aspects and methodologies discussed in this chapter were published in recent years. These will obviously serve as the main sources for further information for the near future. Among them are Fingerman and Nagabhushanam’s (2000) Recent Advances in Marine Biotechnology: Aquaculture – Fishes, Beaumont and Hoare’s (2003) Biotechnology and Genetics in Fisheries and Aquaculture; Hallerman’s (2003) Population Genetics: Principles and Applications for Fisheries Scientists; Dunham’s (2004) Aquaculture and Fisheries Biotechnology: Genetic Approaches; Gjedrem’s (2005) Selection and Breeding Programs in Aquaculture; Liu’s (2007a) Aquaculture Genome Technologies; and Kocher and Kole’s (2008) Genome Mapping and Genomics in Fishes and Aquatic Animals.

2.6 Acknowledgement The authors would like to thank Eric Hallerman for his valuable comments and suggestions that helped shape the view-point expressed in the paper and improved its prose. This paper is contribution No. 526/08 from the ARO, The Volcani Center, Bet Dagan, Israel.

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pandian t j and koteeswaran r (1998) Ploidy induction and sex control in fish, Hydrobiologia, 384, 167–243. parameswaran v, shukla r, bhonde r and hameed a s s (2007) Development of a pluripotent ES-like cell line from Asian sea bass (Lates calcarifer) – an oviparous stem cell line mimicking viviparous ES cells, Mar Biotech, 9(6), 766–75. parsons j (1998) Status of genetic improvement in commercially reared stocks of rainbow trout, World Aquac, 29, 44–7. perez-enriquez r, takagi m and taniguchi n (1999) Genetic variability and pedigree tracing of a hatchery-reared stock of red sea bream (Pagrus major) used for stock enhancement, based on microsatellite DNA markers, Aquaculture, 173(1– 4), 413–23. peruzzi s, chatain b, saillant e, haffray p, menu b and falguière j-c (2004) Production of meiotic gynogenetic and triploid sea bass, Dicentrarchus labrax L.: 1. Performances, maturation and carcass quality, Aquaculture, 230(1–4), 41–64. peruzzi s, varsamos s, chatain b, fauvel c, menu b, falguière j-c, sévère a and flik g (2005) Haematological and physiological characteristics of diploid and triploid sea bass, Dicentrarchus labrax L., Aquaculture, 244(1–4), 359–67. pew initiative on food and biotechnology (2003) Future Fish? Issues in Science and Regulation of Transgenic Fish, Cambridge, MA, The Pew Charitable Trusts, www.pewtrust.org/our_work_report_detail.aspx?id=33350, accessed February 2009. phillips r b and reed k m (1996) Application of fluorescence in situ hybridization (FISH) techniques to fish genetics: a review, Aquaculture, 140(3), 197–216. phillips r b, nichols k m, dekoning j j, morasch m r, keatley k a, rexroad iii c, gahr s a, danzmann r g, drew r e and thorgaard g h (2006) Assignment of rainbow trout linkage groups to specific chromosomes, Genetics, 174, 1661–70. piferrer f, cal r m a, álvarez-blázquez b, sánchez l and martínez p (2000) Induction of triploidy in the turbot (Scophthalmus maximus). I. Ploidy determination and the effects of cold shocks, Aquaculture, 188(1–2), 79–90. piferrer f, cal r m, gómez c, bouza c and martínez p (2003) Induction of triploidy in the turbot (Scophthalmus maximus): II. Effects of cold shock timing and induction of triploidy in a large volume of eggs, Aquaculture, 220(1–4), 821–31. piferrer f, cal r m, gómez c, álvarez-blázquez b, castro j and martínez p (2004) Induction of gynogenesis in the turbot (Scophthalmus maximus): effects of UV irradiation on sperm motility, the Hertwig effect and viability during the first 6 months of age, Aquaculture, 238(1–4), 403–19. ponzoni r w, hamzah a, tan s and kamaruzzaman n (2005) Genetic parameters and response to selection for live weight in the GIFT strain of Nile tilapia (Oreochromis niloticus), Aquaculture, 247(1–4), 203–10. poompuang s and na-nakorn u (2004) A preliminary genetic map of walking catfish (Clarias macrocephalus), Aquaculture, 232(1–4), 195–203. pottinger t g and carrick t r (1999) Modification of the plasma cortisol response to stress in rainbow trout by selective breeding, Gen Comp Endocrinol, 116, 122–32. pullin r s v (2007) Genetic resources for aquaculture: status and trends, in Bartley D M, Harvey B J and Pullin R S V (eds), Workshop on Status and Trends in Aquatic Genetic Resources: a Basis for International Policy, FAO Fisheries Proceedings, No. 5, Rome, Food and Agriculture Organization of the United Nations, 109–43. pullin r s v, palomares m, casal c, dey m and pauly d (1997) Environmental impacts of Tilapia, in Fitzsimmons K (ed.), Tilapia Aquaculture: Proceedings of the Fourth International Symposium on Tilapia in Aquaculture, Publication No.

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NRAES-106, Ithaca, NY, Northeast Regional Aquacultural Engineering Services, 554–70. quillet e, leguillou s, aubin j, labbé l, fauconneau b and médale f (2007) Response of a lean muscle and a fat muscle rainbow trout (Oncorhynchus mykiss) line on growth, nutrient utilization, body composition and carcass traits when fed two different diets, Aquaculture, 269(1–4), 220–31. quinton c d, mcmillan i and glebe b d (2005) Development of an Atlantic salmon (Salmo salar) genetic improvement program: genetic parameters of harvest body weight and carcass quality traits estimated with animal models, Aquaculture, 247(1–4), 211–17. quinton c d, kause a, koskela j and ritola o (2007) Breeding salmonids for feed efficiency in current fishmeal and future plant-based diet environments, Genet Sel Evol, 39(4), 431–46. rasmussen r s and morrissey m t (2007) Biotechnology in aquaculture: transgenics and polyploidy, Comp Rev Food Sci Food Safe, 6(1), 2–16. reddy p v g k, gjerde b, tripathi s d, jana r k, mahapatra k d, gupta s d, saha j n, sahoo m, lenka s, govindassamy p, rye m and gjedrem t (2002) Growth and survival of six stocks of rohu (Labeo rohita, Hamilton) in mono and polyculture production systems, Aquaculture, 203(3–4), 239–50. rexroad c e iii (2007) Radiation hybrid mapping in aquatic species, in Liu Z (ed.), Aquaculture Genome Technologies, Ames, IA, Blackwell, 313–22. rexroad c, vallejo r, coulibaly i, couch c, garber a, westerman m and sullivan c (2006) Identification and characterization of microsatellites for striped bass from repeat-enriched libraries, Cons Genet, 7(6), 971–82. rothbard s (2006) A review of ploidy manipulations in aquaculture: the Israeli experience, Isr J Aquac – Bamidgeh, 58(4), 266–79. rothbard s, shelton w l, rubinshtein i, hinits y and david l (2000) Induction of all-female triploids in grass carp (Ctenopharyngodon idella) by integration of hormonal sex inversion and ploidy manipulation, Isr J Aquac – Bamidgeh, 52(4), 133–50. rothschild m f and ruvinsky a (2007) Marker-assisted selection for aquaculture species, in Liu Z (ed.), Aquaculture Genome Technologies, Ames, IA, Blackwell, 199–213. saillant e, dupont-nivet m, haffray p and chatain b (2006) Estimates of heritability and genotype-environment interactions for body weight in sea bass (Dicentrarchus labrax L.) raised under communal rearing conditions, Aquaculture, 254(1–4), 139–47. sakamoto t, fuji k, kobayashi k, hasegawa o, ozaki a and okamoto n (2006) Marker-assisted breeding for viral disease resistance in Japanese flounder (Paralichthys olivaceus), Isr J Aquac – Bamidgeh, 58(4), 384–6. sarropoulou e, kotoulas g, bargelloni l, power d m, franch r, louro b, magoulas a, patarnello t, senger f, galibert f and geisler r (2007a) A gene-based radiation hybrid map of the gilthead sea bream Sparus aurata, Aquaculture, 272(Suppl. 1), S308. sarropoulou e, franch r, louro b, power d m, bargelloni l, magoulas a, senger f, tsalavouta m, patarnello t, galibert f, kotoulas g and geisler r (2007b) A gene-based radiation hybrid map of the gilthead sea bream Sparus aurata refines and exploits conserved synteny with Tetraodon nigroviridis, BMC Genom, 8, 44–58. sarropoulou e, nousdili d, magoulas a and kotoulas k (2008) Linking the genomes of nonmodel teleosts through comparative genomics, Mar Biotechnol, 10(3), 227–33. sausa-santos c, collares-pereira m j and almada v (2007) Fertile triploid males – an uncommon case among hybrid vertebrates, J Exp Zool, 307A, 220–25.

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senanan w, kapuscinski a r, na-nakorn u, miller l m (2004) Genetic impacts of hybrid catfish farming (Clarias macrocephalus × C. gariepinus) on native catfish populations in central Thailand, Aquaculture, 235, 167–84. senger f, priat c, hitte c, sarropoulou e, franch r, geisler r, bargelloni l, power d and galibert f (2006) The first radiation hybrid map of a perch-like fish: the gilthead seabream (Sparus aurata L), Genomics, 87(6), 793–800. silverstein j t, weber g m, rexroad iii c e and vallejo r l (2006) Genetics and genomics – integration of molecular genetics into a breeding program for rainbow trout, Isr J Aquac – Bamidgeh, 58(4), 231–7. soller m and beckmann j s (1982) Restriction fragment length polymorphisms and genetic improvement, Proc. 2nd World Congress Genetics Applied to Livestock Production, VI, Madrid, Editorial Garsi, 396–404. somridhivej b, wang s, sha z, liu h, quilang j, xu p, li p, hu z and liu z (2008) Characterization, polymorphism assessment, and database construction for microsatellites from BAC end sequences of catfish: a resource for integration of linkage and physical maps, Aquaculture, 275, 76–80. sonesson a k (2007a) Possibilities for marker-assisted selection in aquaculture breeding schemes, in Guimarães E, Ruane J, Scherf B, Sonnino A and Dargie J (eds), Marker-Assisted Selection, Current Status and Future Perspectives in Crops, Livestock, Forestry and Fish, Rome, Food and Agriculture Organization of the United Nations, 309–28. sonesson a k (2007b) Within-family marker-assisted selection for aquaculture species, Genet Sel Evol, 39(3), 301–17. sonesson a k and meuwissen t h e (2000) Mating schemes for optimum contribution selection with constrained rates of inbreeding, Genet Sel Evol, 32(3), 231–48. sonesson a k and meuwissen t h e (2002) Non-random mating for selection with restricted rates of inbreeding and overlapping generations, Genet Sel Evol, 34(1), 23–39. sonesson a k, janss l l g and meuwissen t h e (2003) Selection against genetic defects in conservation schemes while controlling inbreeding, Genet Sel Evol, 35(4), 353–68. sonesson a k, gjerde b and meuwissen t h e (2005) Truncation selection for BLUPEBV and phenotypic values in fish breeding schemes, Aquaculture, 243(1–4), 61–8. steffens w, jähnichen h and fredrich f (1990) Possibilities of sturgeon culture in Central Europe, Aquaculture, 89, 101–22. sun x w and liang l q (2004) A genetic linkage map of common carp (Cyprinus carpio L.) and mapping of a locus associated with cold tolerance, Aquaculture, 238, 169–72. taylor j f, needham m p, north b p, morgan a, thompson k and migaud h (2007) The influence of ploidy on saltwater adaptation, acute stress response and immune function following seawater transfer in non-smolting rainbow trout, Gen Comp Endocrinol, 152(2–3), 314–25. teufel j, pätzold f and potthof c (2002) Scientific Research on Transgenic Fish With Special Focus on the Biology of Trout and Salmon, Berlin, Federal Environmental Agency. tiwary b k, kirubagaran r and ray a k (2004) The biology of triploid fish, Rev Fish Biol Fish, 14(4), 391–402. tobin d, kause a, mäntysaari e a, martin s a m, houlihan d f, dobly a, kiessling a, rungruangsak-torrissen k, ritola o and ruohonen k (2006) Fat or lean? The quantitative genetic basis for selection strategies of muscle and body composition traits in breeding schemes of rainbow trout (Oncorhynchus mykiss), Aquaculture, 261(2), 510–21.

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tvedt h b, benfey t j, martin-robichaud d j, mcgowan c and reith m (2006) Gynogenesis and sex determination in Atlantic halibut (Hippoglossus hippoglossus), Aquaculture, 252(2–4), 573–83. vandeputte m (2003) Selective breeding of quantitative traits in the common carp (Cyprinus carpio): a review, Aquat Liv Res, 16(5), 399–407. verspoor e, beardmore j a, consuegra s, garcía de leániz c, hindar k, jordan w c, koljonen m-l, mahkrov a a, paaver t, sánchez j a, skaala ø, titov s and cross t f (2005) Population structure in the Atlantic salmon: Insights from 40 years of research into genetic protein variation, J Fish Biol, 67(Suppl. 1), 3–54. verspoor e, olesen i, bentsen h b, glover k, mcginnity p and norris a (2006) Atlantic salmon – Salmo salar, in Crosetti D, Lapègue S, Olesen I and Svaasand T (eds), Genetic Effects of Domestication, Culture and Breeding of Fish and Shellfish, and Their Impacts on Wild Populations, GENIMPACT project: evaluation of genetic impact of aquaculture activities on native populations, A European network WP1 workshop, Viterbo, Italy, 12–17th June. villanueva b, woolliams j a and gjerde b (1996) Optimum designs for breeding programmes under mass selection with an application in fish breeding, Anim Sci, 63(3), 563–76. villanueva b, verspoor e and visscher p m (2002) Parental assignment in fish using microsatellite genetic markers with finite numbers of parents and offspring, Anim Genet, 33(1), 33–41. wang c m, zhu z y, lo l c, feng f, lin g, yang w t, li j and yue g h (2007) A microsatellite linkage map of barramundi, Lates calcarifer, Genetics, 175, 907–15. watanabe t, fujita h, yamasaki k, seki s and taniguchi n (2004) Preliminary study on linkage mapping based on microsatellite DNA and AFLP markers using homozygous clonal fish in ayu (Plecoglossus altivelis), Mar Biotechnol, 6(4), 327–34. weir l k, hutchings j a, fleming i a and einum s (2004) Dominance relationships and behavioural correlates of individual spawning success in farmed and wild male Atlantic salmon, Salmo salar, J Anim Ecol, 73(6), 1069–79. weir l k, hutchings j a, fleming i a and einum s (2005) Spawning behaviour and success of mature male Atlantic salmon (Salmo salar) parr of farmed and wild origin, Can J Fish Aquat Sci, 62(5), 1153–60. wenne r, boudry p, hemmer-hansen j, lubieniecki k p, was a and kause a (2007) What role for genomics in fisheries management and aquaculture?, Aquat Living Resour, 20, 241–55. woram r a, mcgowan c, stout j a, gharbi k, ferguson m m, hoyheim b, davidson e a, davidson w s, rexroad c and danzmam r g (2004) A genetic map for Arctic char (Salvelinus alpinus): evidence for higher recombination rates and segregation distortion in hybrid versus pure strain mapping parents, Genome, 47, 304–15. xu p, wang s and liu z (2007) Physical characterization of genomes through BAC end sequencing, in Liu Z (ed.), Aquaculture Genome Technologies, Ames, IA, Blackwell, 261–74. zhu z y, wang c m, lo l c, feng f, lin g and yue g h (2006) Isolation, characterization, and linkage analyses of 74 novel microsatellites in barramundi (Lates calcarifer), Genome, 49(8), 969–76.

3 Genetic variation and selective breeding in hatchery-propagated molluscan shellfish P. Boudry, Ifremer, France

Abstract: This chapter presents the current status of molluscan shellfish domestication and research made to plan and implement efficient breeding programs for the improvement of characters of aquaculture interest, notably yield-related and disease resistance traits. It presents the features of molluscan reproduction and hatchery rearing that have a bearing on the genetic composition of artificially bred and non-native populations, showing how they may hinder improvement but also offer possibilities for selection. The major mollusc breeding programs are reviewed with the methods they employ and progress achieved. Finally, recent developments in genomics and marker assisted selection are presented with their potential applications in the future improvement of molluscan shellfish. Key words: selective breeding, oysters, aquaculture, bivalves, genetics.

3.1 Introduction Despite the high proportion of world aquaculture production that molluscan shellfish represent, this group has benefited little from genetic improvement methods compared with other types of farmed species (Hulata, 2001). Unlike most finfish and shrimp production, molluscan shellfish aquaculture is largely based on the on-growing of wild seed. This is probably the main reason why there have only been small advances made in the development and adoption of genetically superior broodstock. Hatchery production is limited overall and relatively little investment has been made in genetics as a result. Among molluscan shellfish, oysters are the group of species where the most progress has been made so far in this respect (Sheridan, 1997). The first genetic improvements were initiated in countries where collection

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of wild seed is limited by environmental factors (e.g. Pacific oyster on the west coast of North America), or over-harvesting and subsequent low recruitment (e.g. abalone in New Zealand). It is easy to see how the demand for seed motivated the development of hatchery technology, offering in turn the possibility of controlled reproduction and planned breeding. Disease-related production constraints also strongly encourage work towards genetic improvement for disease resistance. The main genetic improvement made so far in marine molluscs has been based on polyploidy and, more specifically, the production of triploids (Chapter 6). Considering the results of quantitative genetics studies published since the mid-1990s, one might be led to think that selective breeding could vastly improve molluscan shellfish production in the near future. Several selective breeding programs for oysters have been initiated in recent years and have started to benefit the industry, notably in USA, Australia and New-Zealand (a review of these is made below). Selective breeding is indeed a slower way to genetically improve molluscan shellfish than the single-step improvement achieved with triploidy. It should, however, be seen as a logical long-term complement rather than as an alternative option, especially as it can target traits that cannot be modified by triploidisation. When selective breeding programs are correctly planned and well managed genetic gain is cumulative over successive generations, while triploidy is a single-step improvement. Triploidy presents the advantage of a relatively rapid return on investment but might limit or slow down further progress. Selective breeding of tetraploid broodstocks, commonly used in oysters to produce triploids by crossing with diploid parents (Guo et al., 1996), is known to be slower and more complex than for diploids because of the larger number and order of genetic interactions, as illustrated in many autotetraploid crop species (Gallais, 1981). Combining selective breeding and triploidy would therefore be a promising but challenging direction for genetic improvement (McCombie et al., 2005).

3.2 Monitoring genetic diversity and risks related to inbreeding One of the main characteristics of molluscan shellfish is their very high fecundity, highlighted by Williams in his ‘Elm-Oyster Model’ of reproductive strategy in evolution (Williams, 1975). An adult female Pacific oyster commonly produces more than 50 million eggs (Kang et al., 2003). As a result, a very small number of genitors can potentially produce huge numbers of offspring. However, populations based on small numbers of parents will have low effective sizes and inbreeding will occur in subsequent generations. Such loss of genetic diversity can be estimated from the decrease in mean number of alleles per locus, decrease in heterozygosity and/or genetic differentiation between populations (i.e. Fst estimates) (Li

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et al., 2004). More interestingly, effective population size (N(e)) can be estimated from temporal changes in the frequencies of selectively neutral alleles in isolated populations (Waples, 1989). This approach also makes it possible to obtain accurate estimates of present genetic effective population size in the wild for abundant species such as prawns (Ovenden et al., 2007) and oysters (Hedgecock et al., 2007a). Loss of genetic diversity due to genetic drift is a well known phenomenon in non-native species introduced into new geographic areas. Such introductions have frequently been made for the production of new species of molluscan shellfish. Several cases of inbreeding depression have been documented following introductions, especially for scallop species (for review see Beaumont, 2000). The bay scallop (Argopecten irradians) was introduced from USA to China in 1982. Only 26 individuals survived transportation and led to a commercial production of 200 000 metric tonnes in 1990 (Guo et al., 1999). The genetic effect of this bottleneck was documented using mitochondrial restriction fragment length polymorphism (RFLP) (Blake et al., 1997), allozyme (Xue et al., 1999) and microsatellite markers (Wang et al., 2007). Despite several recent introductions of new stocks aimed at expanding the gene pool, the genetic diversity remains limited, and clear effects of inbreeding depression have been demonstrated (Zhang et al., 2005). The Japanese scallop (Patinopecten yessoensis) was also introduced to China from Japan in 1982. Using six microsatellite loci, Li et al. (2007) showed that after about two decades of hatchery propagation, effective population sizes ranged from 26 to 70. Contrastingly, in several cases of unintentional introduction of non-native oyster species, little or no reduction of genetic variability was noted. This is the case for the Pacific oyster (Crassostrea gigas) following its introduction from Japan into Australia (English et al., 2000; Appleyard and Ward, 2006), New Zealand (Smith et al., 1986) and France (Huvet et al., 2004a). The same was observed for the Portuguese oyster thought to have been introduced from Taiwan into Portugal and then France (Huvet et al., 2001, 2004a). Similarly, feral populations of the Manila clam (Ruditapes philippinarum), introduced from Asia into Europe during the 1970s show similar levels of genetic variability to native clam species (Borsa and Thiriot-Quievreux, 1990; Moraga, 1986). Hatchery propagation can be a major cause of reduction in effective population size, mainly due to the practice of using a small number of parents to make crosses. Many shellfish broodstocks have been shown to have small effective size (i.e. low genetic variation due to random genetic drift), using either allozymes (Hedgecock et al., 1992) or microsatellite markers (Launey et al., 2001). Although the use of limited numbers of highly fecund genitors is the major reason for reduced effective size in hatchery-propagated molluscan shellfish, additional reasons have been identified. Gametic incompatibility (Gaffney et al., 1993) and differential survival during larval stage (Boudry et al., 2002; Taris et al., 2006) also

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contribute significantly to increasing variance in reproductive success between genitors. Reduction of effective population size leads to inbreeding in such isolated populations, resulting in a limited response to selection. In molluscan shellfish, inbreeding depression is much better documented than lack of genetic variance (Zheng et al., 2006). This is mainly due to the ease of comparing inbred progenies with outbred ones and the limited number of multi-generation selective breeding studies that have been made (see below). Inbreeding depression is due to unmasking of recessive deleterious alleles (i.e. genetic load). Very high levels of genetic load, revealed by high and frequent distortion of marker segregation ratios, have been reported in oysters (Bierne et al., 1998; Launey and Hedgecock, 2001). Culling, a common practice in shellfish hatcheries and nurseries, may contribute to masking inbreeding by selecting the fast-growing, more heterozygous genotypes (Taris et al. 2007). As a result, the development of homozygous lines, by making crosses between relatives, will be slower than in species with lower genetic load (homozygote disadvantage), as the most heterozygous individuals survive and grow better.

3.3 Inheritance of traits important for aquaculture With the increased interest in the potential for selective breeding in molluscs, much work has been focused on a variety of traits in these animals. The extent of the genetic basis of traits such as growth or disease resistance provides a direct indication of the potential for their improvement by selection. Such evaluations need to be considered in the context of environmental variation, so as to assess the extent of genotype × environment interaction and thus indicate what value a selected group could have under differing rearing environments of varied geographical areas. Selective breeding requires that observed phenotypic variation be partly due to genetic components, and the demonstration of this genetic basis is a prerequisite for any breeding program. Heritability estimates, genetic correlations between traits and genotype × environment interactions are commonly listed as essential information needed to set up a breeding program (Falconer and Mackey, 1996). There are, however, numerous examples of farmed molluscan shellfish species in which these key elements are still unknown, although some genetic information may be available. On one hand, some studies based on comparison between progenies reared in a common environment suggest local population adaptation (e.g. Ibarra et al., 1997; Soletchnik et al., 2002) or different levels of genetic variability (Zheng et al., 2004). Many published studies are based on a small number of families over just one or a limited number of generations (e.g. Ernande et al., 2003), limiting the validity of the genetic parameters estimated. On the other hand, a large number of studies report very encouraging results,

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showing that significant improvement could be obtained by selective breeding, even though these fail to provide heritability estimates, e.g. Bonamia ostreae resistance in the European flat oyster, Ostrea edulis (Naciri-Graven et al., 1998; Culloty et al., 2004), and resistance to Haplosporidium nelsoni (‘MSX’) (Ford and Haskin, 1987; Ford et al., 1990), Perkinsus marinus (‘Dermo’) (Encomio, et al., 2005) and Roseovarius crassostreae (Juvenile Oyster Disease) (Barber et al., 1998) in the American oyster, Crassostrea virginica (Farley et al., 1998). In these cases, resistant ‘lines’ or ‘strains’ have been produced, but the benefit for shellfish production has been lower than initially expected, either due to technical difficulties at the hatchery and/or nursery stages, or because the shellfish industry itself has not become involved. Among the numerous papers reporting heritability estimates for traits of aquacultural interest in farmed molluscan shellfish, two main types appear: (i) studies based on response to individual selection over one (e.g. Ibarra et al., 1999) or several generations (e.g. Zheng et al., 2006), and (ii) studies based on families using nested or diallel crossing designs. A few papers also report response to family selection (Langdon et al., 2003) and midparent–offspring regressions (Evans and Langdon, 2006a). The magnitude of interactions have been examined in several studies (Evans and Langdon, 2006b; Swan et al., 2007) and were limited in most cases. However, high plasticity of genetic correlations between reproductive effort and both survival and growth have been reported, underlining the influence of environmental conditions on genetic correlation estimations (Ernande et al., 2004). Heritability estimates of larval traits, such as growth or size at particular ages, have been made for mussel (Mallet and Haley, 1984; Toro and Paredes, 1996; Toro et al., 2004), oyster (Losee, 1978; Ernande et al., 2003) and scallop (Jones et al., 1996). Although such studies are relatively easy to perform due to the short time required to record these traits, they are often of limited aquacultural interest, and genetic correlations between larval and postlarval traits are usually low (Ernande et al., 2003). Additionally, heritability estimates often increase with age, e.g. in abalone (Jonasson et al., 1999), although it is not always the case as reported in mussel by Alcapan et al. (2007). Most molluscan on-growing is performed under extensive aquacultural conditions (relative to most finfish or agricultural species) along tidal or shallow coastal areas. As a result, artificial rearing conditions at early stages in the hatchery and nursery (where food availability and water temperature are controlled) might act as efficient domestication forces. The potential of strong (intentional or unintentional) selection at these early stages is therefore important in highly fecund molluscan species (Taris et al., 2007). Further research is needed to determine genetic correlations between traits recorded at spat size and in market size shellfish, which would allow us to gain a deeper understanding of what such approaches could offer (see Hedgecock and Davis, 2007).

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One of the most extensive heritability studies that has been conducted was made by Ward et al. (2005) on Pacific oyster. Based on a partially hierarchical crossing design (31 males by 31 females to produce 62 families), results provided estimates of heritabilities and genetic correlations for several traits: growth (total, shell, fresh and dry flesh weights), ornamentation and shell colour (from photographs), shell morphology (height, length and depth) and shape (flat, convex, concave or undulating, and presence/ absence of a hook). Estimates of genetic parameters showed a medium to high additive genetic basis for the studied traits and no dominance. In contrast, Hedgecock and Davis (2007) found strong evidence of heterosis for yield (i.e. growth, as the mortality component of yield was negligible in their experiments). To conclude, it is likely that most traits show significant additive but also non-additive variance in the Pacific oyster and many other molluscan shellfish. Special attention has been given to the heritability of resistance to summer mortality in oysters (Samain and McCombie, 2008). Following studies conducted in the USA in the early 1980s (Beattie et al., 1980; Hershberger et al., 1984), high heritability estimates of 0.83 ± 0.40 (Dégremont et al., 2007) were confirmed by the response of this trait to divergent selection (Boudry et al., 2008). Interestingly, broad-sense (Evans and Langdon, 2006b) and narrow-sense (Dégremont et al., 2007) heritability estimates for growth in C. gigas are lower than those for survival (see also Ernande et al., 2004).

3.4 Current status of established molluscan shellfish breeding programs Main work on genetic improvement has so far focussed on oyster species and green-lipped mussel. Although a number of breeding programs that have now been initiated for other molluscan shellfish, e.g. for the scallop Argopecten purpuratus in Chile and for the abalone Haliotis laevigata in Australia (Kube et al., 2007), these are not sufficiently established and/or documented to be described here. The breeding programs that are well described in the literature, and which have already led to significant transfer to industry, provide examples of methods followed and resources required. These selection schemes concern the Pacific oyster C. gigas in the USA, Australia and New Zealand, the Sydney rock oyster Saccostrea glomerata in Australia and the green-lipped mussel Perna canaliculus in New Zealand (Table 3.1). Common points between these programs are the collaboration of industry (farmers and/or hatcheries) and government (institutions, organising structures and funding), and the need for financial support to sustain the programs, at least though their early stages that may span several years.

CSIRO followed by ASI / Thoroughbred oysters New South Wales Department of Primary Industries, Select Oyster Company

Growth

Growth and quality

Growth and quality Resistance to diseases, growth

Pacific oyster

Pacific oyster, green lipped mussel Pacific oyster

Sydney rock oyster

Cawthron Institute

Yield

Pacific oyster

Molluscan Broodstock Program Taylor Shellfish Farms

Main targeted traits

Name of the organisation and/or operating company

New South Wales

Tasmania

New Zealand

West coast of USA West coast of USA

Location

Major selective breeding programs of molluscan shellfish

Species

Table 3.1

1990

1996

1998

1996

Starting year

http://www.oysterssa.com.au/ media/files/734.pdf http://www.dpi.nsw.gov.au/ research/updates/issues/ june-2006/ disease-resistant-oysters

http://www.cawthron.org.nz/ aquaculture/selective-breeding. html

http://hmsc.oregonstate.edu/ projects/mbp/index.htm http://www.fish.washington.edu/ wrac/pdfs/crossbreed_ completion.pdf

Dedicated website or information available on internet

Ward et al., 2005 Nell and Perkins, 2006

Langdon et al., 2003 Hedgecock and Davis, 2007 King et al., 2004

Main reference

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3.4.1 Selection of C. gigas in the USA Two major C. gigas breeding programs are in operation on the west coast of the USA. The Molluscan Broodstock Program (MBP) was initiated in 1996 (Hedgecock et al., 1996a). This is a collaborative program between Hatfield Marine Science Center, Newport (University of Oregon and USDA) and several commercial companies. Cohorts of 50–60 full-sib families are produced in Newport and evaluated at intertidal and subtidal commercial sites along the west coast. The main initial objective of the MBP was to improve yield (i.e. the combination of survival and growth) by selecting the heaviest 30 % of oysters in the 10 % of families with the best yield. Such a high among-family selective pressure scheme obviously requires a large number of families to be produced per generation (in this case 200–240 families corresponding to four cohorts), and hence the infrastructure necessary to achieve this. The first results published from the MBP concerned gain in yield, and showed an improvement of ≈10 % after one generation of selection (Langdon et al., 2003). More recently genotype × environment was studied in greater depth (Evans and Langdon, 2006b): separating survival and growth, and estimating their coheritability (Evans and Langdon, 2006a). High heritability estimates of mantle and shell pigmentation were also reported (Brake et al., 2004). The USDA Western Regional Aquaculture Center (WRAC) project is examining the potential of hybrid vigour (i.e. heterosis) for yield improvement, by cross-breeding inbred lines. A large genetic load (Launey and Hedgecock, 2001) both explains the frequently observed distortions of Mendelian ratios in inheritance studies (Bierne et al., 1998) and supports the dominance theory of heterosis in oysters (Hedgecock et al., 1996b). Inbred lines (with inbreeding coefficients ranging from f = 0.375 to 0.5) are obtained by mating within full-sib families produced by the MBP or from wild stocks. The oyster lines are reared and maintained by Taylor Resources, Inc., and field performance monitored by Taylor Shellfish Farms and Baywater, Inc. Diallel mating experiments allowed general and specific combining abilities (GCA, SCA) of the tested lines to be estimated (Hedgecock and Davis, 2007). Such an approach requires the production and testing of numerous inbred lines, a time- and labour-intensive process. Recent physiological (Pace et al., 2006) and transcriptomic (Hedgecock et al., 2007b) approaches could ease the selection of elite inbred lines for commercial seed production in the future. Although heterosis is now clearly demonstrated in C. gigas, the potential of cross-breeding relative to other breeding strategies based on additive genetic variance still remains to be explored in depth (see Ryan et al., 2006).

3.4.2 The Australian oyster selection programs Selection is performed on two different cupped oyster species in Australia: the native Sydney rock oyster (Saccostrea glomerata) and the non-native

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Pacific oyster (C. gigas). Research on C. gigas was initiated in 1993, with an initial phase aiming to estimate heritability and the magnitude of genotype × environment interactions for a number of traits. The value of different selection methods was also evaluated. An individual selection method with mass spawning and 20 % selection pressure, assisted by genetic fingerprinting, was compared with a family selection method based on the production of 40 families per generation using one male × two females. This research work was carried out at the TAFI (Tasmanian Aquaculture and Fisheries Institute) at the University of Tasmania and CSIRO (Commonwealth Scientific and Industrial Research Organization), in partnership with farmers in Tasmania and South Australia. In the second phase of this research (2000), the South Australian Oyster Growers’ Association (SAOGA), the Tasmanian Oyster Research Council and South Australian Oyster Research Council (SAORC), created the Australian Seafood Industry (ASI), a non-profit-making organisation to continue selection and rearing. In 2004, 20 % of Australian production benefited from this program. The first four generations (1996–2000) of individual selection for growth led to a gain of 60 % in live weight and a 50 % reduction in its heterogeneity (Ward and Thompson, 2005; Ward et al., 2005). These changes were accompanied by a 41 % decrease in allelic diversity at eight microsatellite markers, but none at 10 enzymatic loci studied nor in the observed heterozygocity (Appleyard and Ward, 2006). Family selection was initiated in 1997 (40–60 families per generation using one male × two females). Among the numerous results from this study (see Ward et al., 2005a), genotype × environment interactions were generally found to be low, with low genetic correlations between farms. The different selective breeding strategies were analysed, and it was concluded that the combination of moderate interfamily and strong intrafamily selection should maximise genetic gains. The native Sydney rock oyster (S. glomerata) commonly shows slower growth than introduced C. gigas, and its production is strongly affected by mortalities caused by two parasites Mikrocytos roughleyi (winter mortality) and, since 1994, Martelia sydneyi (QX disease). With the aim of improving this situation, researchers at the New South Wales (NSW) Department of Primary Industries initiated two breeding programs, selecting to improve growth and resistance to parasites in the Sydney rock oyster (Nell et al., 2000). These breeding programs started in 1990, formed part of the strategic plan for oyster farming development established through cooperation between the NSW Oyster Farmer’s Association and the Oyster Research Advisory Committee (ORAC). Australian oyster farmers reared the first selected S. glomerata in 2004. The first selection program, initiated in 1990 in Port Stephens, NSW, Australia, aimed to improve growth rate by individual selection of four independent lines. For each generation, each line was reproduced with a mass spawning of about 350 genitors. An overall pressure of 7 % was

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applied on each generation, and response to selection was estimated after each generation (Nell et al., 1996, 1999; Nell and Perkins, 2005). In 2001, after four generations of selection, the four lines were compared with two control lines at three sites. A mean weight gain of 40 % was observed by the fourth generation (i.e. +10 % per generation). No line × site interaction was observed either for growth (weight and length) or mortality (23 % on average over the whole cycle). The increase in growth rate allowed the rearing time to harvest to be reduced from 41 to about 29 months. The combined effect of selection for growth and triploidisation by cytochalasin B treatment was evaluated after two generations of selection (Hand et al., 2004). The results showed that growth improvements from selective breeding and triploidy were ‘at least additive’, as a positive interaction was observed between triploidy and improvement by selection. A parallel set of breeding lines was also established in 1990 at Georges River, NSW, to select for resistance to Mikrocytos roughleyi. This programme was reorganised and expanded following the outbreak of QX disease in 1994 (Nell et al., 2000; Nell and Hand, 2003). The evaluation of the third generation of selection at three sites (Nell and Perkins, 2006) showed that the three selected lines, which had been subjected to different disease pressures, had improved performances, but that genetic coheritability between resistances to the two diseases was low. Estimates of genetic correlation between resistance to parasites and growth are now needed to establish whether these traits can be improved together by individual selection or if a more complex breeding program is needed.

3.4.3

Selective breeding of Pacific oyster and green-lipped mussel in New Zealand In 1998, a prospective study concluded that it would be beneficial to the shellfish industry in New Zealand to develop selective breeding programs for its major commercial species. The Cawthron Institute undertook this task and is presently the sole hatchery in New Zealand, supplying the entire industry with hatchery seed. As in the MBP in the USA, the Cawthron Institute produces families of oysters (C. gigas), which are then reared on commercial farms and evaluated for their growth performances and quality (i.e. shell shape and uniformity). Following an initial cohort of nine families in 1999 (Janke et al., 2004), 60-family cohorts were then produced every two years from 2001 onwards (King et al., 2004). High within-family selective pressures were applied (20 oysters selected out of 1000), although results remain to be published. Using a similar strategy, cohorts of green-lipped mussel, Perna canaliculus (55–75 full-sib families) have been produced since 2002. The improvement objective in this species is to produce more uniform mussels with better growth rates for a number of different markets (whole live, ½ shell,

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fresh, frozen or processed). Because of the high influence that mussel density has on growth performance, a mixed family approach is being used, with external identity tags glued to the shells of the mussels from the age of nine months. As these tags are frequently lost during growth and harvest, genetic identification using microsatellite marker is now being considered (McAvoy et al., 2008).

3.5 3.5.1

Present needs and future trends: use of marker-assisted selection and genomics

Mixed-family approaches: the use of DNA fingerprinting for parentage assignment Practical limitations, including space constraints and the management demands of maintaining large numbers of rearing vessels at one time, increase the cost of breeding large numbers of families and represent major drawbacks to family-based selective breeding of molluscan shellfish. Highdensity flow-through larval rearing systems can, however, facilitate the development of such breeding programs (Janke et al., 2004). A mixedfamily approach, using physical tags or genetic markers and sorting, represents an alternative more practical strategy. Physical tagging is impossible at early stages in aquatic species and highly polymorphic polymer chain reaction (PCR)-based markers provide a suitable alternative for species where they are available. Typically, 10–20 variable genetic markers are needed to assign >95 % of individuals to single pairs of parents (e.g. Vandeputte et al., 2006). The very high levels of polymorphism observed at microsatellite markers in bivalves should theoretically make parentage assignment easier. However, high frequencies of null alleles are also commonly observed (Hedgecock et al., 2004), which can lead to difficulties in such analyses. Microsatellites are currently the type of marker most commonly used for parentage assignment (e.g. Herbinger et al., 1995; Fishback et al., 2002; Vandeputte et al., 2004), but amplified fragment length polymorphisms (AFLPs) have also been considered (Gerber et al., 2000). Single nucleotide polymorphisms (SNPs) are considered as a powerful tool for parentage inference that will potentially become important in the near future (Anderson and Garza, 2006), but which can be expensive to analyse and will therefore require the development of cost-efficient high-throughput genotyping methods (Hayes et al., 2005). As mean SNP density is very high in oysters (Curole and Hedgecock, 2005; Sauvage et al., 2007), and likely to be of a similar level in many other marine bivalves, the number of potential SNP markers is extremely high. Parentage assignment studies have been made using microsatellite markers in several mollusc species on an experimental level involving

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a limited number of genitors (Boudry et al., 2002; Li and Kijima, 2006; Taris et al., 2006; McAvoy et al., 2008). Microsatellites are also used on a routine basis to confirm parentage and pedigree of oyster broodstock (e.g. Hedgecock and Davis, 2007). However, the suitability of such approaches for mixed-family breeding programs remains to be demonstrated, despite the large number of markers already available (Li et al., 2003a). Other potential applications are walk-back selection (Li et al., 2003b; Sonesson, 2005) and pedigree-assisted selection methods (such as animal model-based methods (Lynch and Walch, 1998)). Walk-back selection is especially promising in species for which resources are limited (because the amount of genotyping is considerably lower than for the animal model-based breeding schemes), or for species were controlled reproduction is not optimised (i.e. species for which maturation techniques and/or strip-spawning are not yet well developed).

3.5.2 Quantitative trait loci mapping Mapping chromosome regions that influence phenotypic traits (i.e. quantitative trait loci; QTL) requires linkage maps of medium to high density. Maps are already available for some molluscan shellfish (Table 3.2), but to date only a few published papers report the identification of QTLs in these species. Yu and Guo (2006) identified QTLs for resistance to Perkinsus marinus in the American oyster C. virginica by comparing AFLP genotype frequency before and after mortality in two full-sib families. This method does not, however, evaluate the proportion of phenotypic variance associated with the identified QTLs. The other study described QTLs for shell, muscle, gonad, digestive gland and gill weight in Pacific abalone Haliotis discus hannai (Liu et al., 2007). In addition to these two published studies, positive results have recently been obtained in European Flat oyster Ostrea edulis for Bonamia ostreaea resistance (Lallias et al., 2009), and in the Pacific oyster C. gigas for summer mortality resistance (Sauvage, 2008) and heterosis for growth (Hedgecock et al., 2004b). Several other studies are currently in progress. This work will contribute to an improved understanding of genetic architecture of the studied traits and provide a basis for marker-assisted selection (MAS) programs (Sonesson, 2007; Wenne et al., 2007).

3.5.3 Candidate genes, genomics and marker-assisted selection The availability of molluscan shellfish selected for traits of aquacultural importance provides the opportunity to search for markers and/or assays indicative of different desirable traits. Such markers represent complementary or alternative phenotying tools and could subsequently be used in gene-assisted selection (GAS). For example, phenoloxidase assays have shown that oysters bred for QX resistance have significantly higher activi-

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Table 3.2 Published linkage maps for molluscan shellfish

Species

Number of linkage groups (LG), Number of markers, Total length (L), Mean distance between markers (D) Male

Crassostrea 10 LG 96 markers gigas L = 758 cM (n = 10) D = 9 cM 10 LG 88 markers L = 616 cM D = 8 cM Crassostrea 12 LG virginica 114 markers L = 647 cM (n = 10) D = 6 cM 9 LG Ostrea 104 markers edulis1 L = 471 cM (n = 10) D = 5 cM 14 LG Mytilus 116 markers edulis L = 825 cM, (n = 14) D = 8 cM 23 LG 166 markers L = 2468 cM D = 15 cM 20 LG Chlamys 197 markers farreri L = 1631 cM (n = 19) D = 9 cM 18 LG Haliotis 167 markers discus L = 702 cM hannai D = 5 cM (n = 18)

Type and number of markers scored (male / female)

Experimental Reference design

349 AFLP

2 F1 families

Female 11 LG 119 markers L = 1031 cM D = 9 cM 10 LG 86 markers L = 770 cM D = 10 cM 12 LG 84 markers L = 904 cM D = 13 cM 10 LG 117 markers L = 450 cM D = 4 cM 14 LG 121 markers L = 862.8 cM D = 8 cM 25 LG 198 markers L = 3130 cM D = 15 cM 19 LG 166 markers L = 1504 cM D = 10 cM 19 LG 160 markers L = 888 cM D = 6 cM

Li and Guo, 2004

115 microsatellites 3 F2 families

Hubert and Hedgecock, 2004

153 / 129 AFLP 3 microsatellites 1 / 2 EST

1 F1 family

Yu and Guo, 2003

235 AFLP 16 microsatellites

1 F2 family

Lallias et al., 2007a

791 AFLP

1 F1 family

Lallias et al., 2007b

603 AFLP

1 F1 family

Wang et al., 2005

667 AFLP

1 F1 family

Li et al., 2005

180 microsatellites 3 F1 families

Sekino and Hara, 2007

1

male and female parents could not be determined in the cross. AFLP = amplified fragment length polymorphism, EST = expressed sequence tag.

ties than the non-selected population, indicating that phenoloxidase may represent a specific QX disease resistance factor (Newton et al., 2004). Direct association between gene polymorphism and phenotypic variation in certain traits has recently been reported in oysters. (Prudence et al., 2006) showed that oyster genotypes with different alleles at the two Amylase

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gene loci had significant differences in growth. Physiological causes of such differences have also been investigated in relationship with feeding-related traits and specific amylase activity (Huvet et al., 2008). Differential mortality of genotypes at candidate genes have revealed an association between specific alleles of both glutamine synthetase (amino-acid metabolism) and delta-9 desaturase (lipid metabolism) genes with resistance to summer mortality (David et al., 2007). In addition to these a priori approaches, differential gene expression studies can provide new ‘candidate genes’. Differential gene expression between oysters selected to be resistant or sensitive to summer mortality (Huvet et al., 2004b) or exposure to pollutants (Boutet et al., 2004; Tanguy et al., 2005) has led to the identification of large numbers of candidate expressed sequence tags (ESTs). Until now, suppression subtractive hybridisation (SSH) has been the method most frequently used to identify genes differentially expressed between contrasting individuals. However, novel high-throughput transcriptome analysis methods such as microarrays (Jenny et al., 2007), massively parallel signature sequencing (MPSS) (Hedgecock et al., 2007b) or serial analysis of gene expression (SAGE) (Bachère, pers. comm.) are likely to increasingly contribute to the identification of genes of interest in the near future. The non-neutrality of allozyme markers has been strongly debated in bivalves, in relation to heterozygote deficiencies frequently observed in wild populations using these tools (Raymond et al., 1997) and the relationship between heterozygosity and fitness-related traits (Bierne et al., 2000). Evidence of departure from neutrality has been specifically shown for certain markers, both indirectly in a study examining patterns of isolation by distance (Launey et al., 2002) and more directly by measurements of specific activity of given enzymes (Pogson, 1991). However, contrasting results were found by Gardner and Lobkov (2005) and Wood and Gardner (2006). Although cross-breeding is currently proposed as an efficient method of commercial improvement in diploid oysters (Hedgecock and Davis, 2007) and higher multi-locus heterozygocity is associated with better performance in meiosis I triploids when compared with meiosis II (Magoulas et al., 2000), I am not aware of any selective breeding program targeting specific enzyme heterozygocity in any bivalve molluscs. The co-localisation of QTLs and candidate genes should be a mutual reinforcement of both approaches (Carlborg et al., 2005) that would benefit the ultimate progression of molluscan shellfish improvement programs based on such knowledge.

3.6 References alcapan a c, nespolo r f and toro j e (2007) Heritability of body size in the Chilean blue mussel (Mytilus chilensis Hupe 1854): effects of environment and ageing, Aquac Res, 38, 313–20.

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anderson e c and garza j c (2006) The power of single-nucleotide polymorphisms for large-scale parentage inference, Genetics, 172, 2567–82. appleyard s a and ward r d (2006) Genetic diversity and effective population size in mass selection lines of Pacific oyster (Crassostrea gigas), Aquaculture, 254, 148–59. barber b j, davis c v and crosby m a (1998) Cultured oysters, Crassostrea virginica, genetically selected for fast growth in the Damariscotta River, Maine, are resistant to mortality caused by Juvenile Oyster Disease (JOD), J Shellfish Res, 17, 1171–5. beattie j h, chew k k and hershberger w k (1980) Differential survival of selected strains of Pacific oysters (Crassostrea gigas) during summer mortality, Proc Natl Shellfish Ass, 70, 184–9. beaumont a (2000) Genetic considerations in transfers and introductions of scallops, Aquac Int, 8, 493–512. bierne n, launey s, naciri-graven y and bonhomme f (1998) Early effect of inbreeding as revealed by microsatellite analyses on Ostrea edulis larvae, Genetics, 148, 1893–906. bierne n, tsitrone a and david p (2000) An inbreeding model of associative overdominance during a population bottleneck, Genetics, 155, 1981–90. blake s g, blake n j, oesterling m j and graves j e (1997) Genetic divergence and loss of diversity in two cultured populations of the bay scallop, Argopecten irradians (Lamarck, 1819), J Shellfish Res, 16, 55–8. borsa p and thiriot-quievreux c (1990) Karyological and allozymic characterization of Ruditapes philippinarum, R aureus and R decussatus (Bivalvia, Veneridae), Aquaculture, 90, 209–27. boudry p, collet b, cornette f, hervouet v and bonhomme f (2002) High variance in reproductive success of the Pacific oyster (Crassostrea gigas, Thunberg) revealed by microsatellite-based parentage analysis of multifactorial crosses, Aquaculture, 204, 283–96. boudry p, dégremont l and haffray p (2008) The genetic basis of summer mortality in Pacific oyster spat and potential for improving survival by selective breeding in France, in Summer mortality of Pacific oyster Crassostrea gigas. The Morest Project, Quae Editions, Versailles, 153–96. boutet i, tanguy a and moraga d (2004) Response of the Pacific oyster Crassostrea gigas to hydrocarbon contamination under experimental conditions, Gene, 329, 147–57. brake j, evans f and langdon c (2004) Evidence for genetic control of pigmentation of shell and mantle edge in selected families of Pacific oysters, Crassostrea gigas, Aquaculture, 229, 89–98. carlborg o, de koning d j, manly k f, chesler e, williams r w and haley c s (2005) Methodological aspects of the genetic dissection of gene expression, Bioinformatics 21(10), 2383–93. culloty s c, cronin m a and mulcahy m f (2004) Potential resistance of a number of populations of the oyster Ostrea edulis to the parasite Bonamia ostreae, Aquaculture, 237, 41–58. curole j p and hedgecock d (2005) High frequency of SNPs in the Pacific oyster genome Plant and Animal Genomes XIII Conference, January 15–19, San Diego, CA, http://intl-pag.org/13/abstracts/PAG13_W026.html, accessed January 2009. david e, boudry p, dégremont l, tanguy a, quere n, samain j f and moraga d (2007) Genetic polymorphism of glutamine synthetase and delta-9 desaturase in families of Pacific oyster Crassostrea gigas and susceptibility to summer mortality, J Exp Mar Biol Ecol, 349, 272–83.

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hand r e, nell j a and thompson p a (2004) Studies on triploid oysters in Australia XIII. Performance of diploid and triploid Sydney rock oyster, Saccostrea glomerata (Gould, 1850), progeny from a third generation breeding line, Aquaculture, 233, 93–107. hayes b, sonesson a k and gjerde b (2005) Evaluation of three strategies using DNA markers for traceability in aquaculture species, Aquaculture, 250, 70–81. hedgecock d and davis j (2007) Heterosis for yield and crossbreeding of the Pacific oyster Crassostrea gigas, Aquaculture, 272, S17–S29. hedgecock d, chow v and waples r s (1992) Effective population numbers of shellfish broodstocks estimated from temporal variance in allelic frequencies, Aquaculture, 108, 215–32. hedgecock d, langdon c, blouin m and allen s (1996a) Genetic improvement of cultured oysters by selection, Coastal Marine Experiment Station Annual Report, Agricultural Experimental Station, Oregon State University, Corvallis, OR. hedgecock d, mcgoldrick d j, manahan d t, vavra j, appelmans n and bayne b l (1996b) Quantitative and molecular-genetic analyses of heterosis in bivalve mollusks, J Exp Mar Biol Ecol, 203, 49–59. hedgecock d, li g, hubert s, bucklin k and ribes v (2004a) Widespread null alleles and poor cross-species amplification of microsatellite DNA loci cloned from the Pacific oyster, Crassostrea gigas, J Shellfish Res, 23, 379–85. hedgecock d, li g m-l and voigt m l (2004b) Mapping heterosis QTL in the Pacific oyster Crassostrea gigas, Plant and Animal Genomes II Conference, January 10–14, San Diego, CA, http://www.intl-pag.org/12/abstracts/W06_ PAG12_19.html, accessed January 2009. hedgecock d, launey s, pudovkin a i, naciri y, lapegue s and bonhomme f (2007a) Small effective number of parents (N-b) inferred for a naturally spawned cohort of juvenile European flat oysters Ostrea edulis, Mar Biol, 150, 1173–82. hedgecock d, lin j z, decola s, haudenschild c d, meyer e, manahan d t and bowen b (2007b) Transcriptomic analysis of growth heterosis in larval Pacific oysters (Crassostrea gigas), Proc Natl Acad Sci USA, 104, 2313–18. herbinger c m, doyle r w, pitman e r, paquet d, mesa k a, morris d b, wright j m and cook d (1995) DNA fingerprint based analysis of paternal and maternal effects on offspring growth and survival in communally reared rainbow trout, Aquaculture, 137, 245–56. hershberger w k, perdue j a and beattie j h (1984) Genetic selection and systematic breeding in Pacific oyster culture, Aquaculture, 39, 237–45. hubert s and hedgecock d (2004) Linkage maps of microsatellite DNA markers for the Pacific oyster Crassostrea gigas, Genetics, 168, 351–62. hulata g (2001) Genetic manipulations in aquaculture: a review of stock improvement by classical and modern technologies, Aquaculture, 111, 155–73. huvet a, lapegue s, magoulas a and boudry p (2001) Mitochondrial and nuclear DNA phylogeography of Crassostrea angulata, the Portuguese oyster endangered in Europe, Mar Biotechnol, 1, 251–62. huvet a, fabioux c, mccombie h, lapegue s and boudry p (2004a) Natural hybridization between genetically differentiated populations of Crassostrea gigas and C. angulata highlighted by sequence variation in flanking regions of a microsatellite locus, Mar Ecol – Prog Ser, 272, 141–52. huvet a, herpin a, degremont l, labreuche y, samain j f and cunningham c (2004b) The identification of genes from the oyster Crassostrea gigas that are differentially expressed in progeny exhibiting opposed susceptibility to summer mortality, Gene, 343, 211–20. huvet a, jeffroy f, fabioux c, daniel j y, quillien v, van wormhoudt a, moal j, samain j f, boudry p and pouvreau s (2008) Association among growth, food

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consumption-related traits and amylase gene polymorphism in the Pacific oyster Crassostrea gigas, Anim Genet, 39, 662–5. ibarra a m, ramirez j l and garcia g a (1997) Stocking density effects on larval growth and survival of two Catarina scallop, Argopecten ventricosus (equals circularis) (Sowerby II, 1842), populations, Aquac Res, 28, 443–51. ibarra a m, ramirez j l, ruiz c a, cruz p and avila s (1999) Realized heritabilities and genetic correlation after dual selection for total weight and shell width in catarina scallop (Argopecten ventricosus), Aquaculture, 175, 227–41. janke a, king n, roberts r and kaspar h (2004) Selective breeding of the Pacific oyster Crassostrea gigas in New Zealand, Aquaculture 2004, Book of Abstracts, 311. jenny m j, chapman r w, mancia a, chen y a, mckillen d j, trent h, lang p, escoubas j m, bachere e, boulo v, liu z j, gross p s, cunningham c, cupit p m, tanguy a, guo x, moraga d, boutet i, huvet a, de guise s, almeida j s and warr g w (2007) A cDNA Microarray for Crassostrea virginica and C. gigas, Mar Biotechnol, 9, 577–91. jonasson j, stefansson s e, gudnason a and steinarsson a (1999) Genetic variation for survival and shell length of cultured red abalone (Haliotis rufescens) in Iceland, J Shellfish Res, 18, 621–5. jones r, bates j a, innes d j and thompson r j (1996) Quantitative genetic analysis of growth in larval scallops (Placopecten magellanicus), Mar Biol, 124, 671–7. kang s g, choi k s, bulgakov a a, kim y and kim s y (2003) Enzyme-linked immunosorbent assay (ELISA) used in quantification of reproductive output in the pacific oyster, Crassostrea gigas, in Korea, J Exp Mar Biol Ecol, 282, 1–21. king n, janke a, roberts r and kaspar h (2004) New Zealand oyster-breeding program seeks genetic improvements, Global Aquac Advocate, 7(3), 59–60. kube p d, appleyard s a and elliot n g (2007) Selective breeding greenlip abalone (Haliotis laevigata): Preliminary results and issues, J Shellfish Res, 26, 821–4. lallias d, beaumont a r, haley c s, boudry p, heurtebise s and lapègue s (2007a) A first-generation genetic linkage map of the European flat oyster Ostrea edulis (L.) based on AFLP and microsatellite markers. Anim Genet, 38, 560–8. lallias d, lapegue s, hecquet c, boudry p and beaumont a r (2007b) AFLP-based genetic linkage maps of the blue mussel (Mytilus edulis), Anim Genet, 38, 340–9. lallias d, gomez-raya l, haley c s, arzul i, heurtebise s, beaumont a r, boudry p and lapègue s (2009) Combining two-stage testing and interval mapping strategies to detect QTL for resistance to bonamiosis in the European flat oyster Ostrea edulis, Mar Biotechnol (in press). langdon c, evans f, jacobson d and blouin m (2003) Yields of cultured Pacific oysters Crassostrea gigas Thunberg improved after one generation of selection, Aquaculture, 220, 227–44. launey s and hedgecock d (2001) High genetic load in the Pacific oyster Crassostrea gigas, Genetics, 159, 255–65. launey s, barre m, gerard a and naciri-graven y (2001) Population bottleneck and effective size in Bonamia ostreae-resistant populations of Ostrea edulis as inferred by microsatellite markers, Genet Res, 78, 259–70. launey s, ledu c, boudry p, bonhomme f and naciri-graven y (2002) Geographic structure in the European flat oyster (Ostrea edulis L.) as revealed by microsatellite polymorphism, J Hered, 93, 331–8. li l and guo x m (2004) AFLP-based genetic linkage maps of the Pacific oyster Crassostrea gigas Thunberg, Mar Biotechnol, 6, 26–36. li q and kijima a (2006) Microsatellite analysis of gynogenetic families in the Pacific oyster, Crassostrea gigas, J Exp Mar Biol Ecol, 331, 1–8.

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li g, hubert s, bucklin k, ribes v and hedgecock d (2003a) Characterization of 79 microsatellite DNA markers in the Pacific oyster Crassostrea gigas, Mol Ecol Notes, 3, 228–32. li x, field c and doyle r (2003b) Estimation of additive genetic variance components in aquaculture populations selectively pedigreed by DNA fingerprinting, Biom J, 45, 61–72. li q, park c, endo t and kijima a (2004) Loss of genetic variation at microsatellite loci in hatchery strains of the Pacific abalone (Haliotis discus hannai), Aquaculture, 235, 207–22. li l, xiang j h, liu x, zhang y, dong b and zhang x j (2005) Construction of AFLPbased genetic linkage map for Zhikong scallop, Chlamys farreri Jones et Preston and mapping of sex-linked markers, Aquaculture, 245, 63–73. li q, xu k f and yu r h (2007) Genetic variation in Chinese hatchery populations of the Japanese scallop (Patinopecten yessoensis) inferred from microsatellite data, Aquaculture, 269, 211–19. liu x d, liu x and zhang g f (2007) Identification of quantitative trait loci for growth-related traits in the Pacific abalone Haliotis discus hannai Ino, Aquac Res, 38, 789–97. losee e (1978) Influence of Heredity on Larval and Spat Growth in Crassostrea virginica, Atlanta, GA, Louisiana State University Division of Continuing Education, Baton Rouge, LA. lynch m and walch b (1998) Genetics and Analysis of Quantitative Traits, Sunderland, MA, Sinauer Associates Inc. magoulas a, kotoulas g, gerard a, naciri-graven y, dermitzakis e, hawkins a j s and ll a (2000) Comparison of genetic variability and parentage in different ploidy classes of the Japanese oyster Crassostrea gigas, Genet Res, 76, 261–72. mallet a l and haley l e (1984) General and specific combining abilities of larval and juvenile growth and viability estimated from natural oyster populations, Mar Biol, 81, 53–9. mcavoy e s, wood a r and gardeur j n (2008) Development and evaluation of microsatellite markers for identification of individual GreenshellTM mussels (Perna canaliculus) in a selective breeding programme, Aquaculture, 274, 41–8. mccombie h, ledu c, phelipot p, lapegue s, boudry p and gerard a (2005) A complementary method for production of tetraploid Crassostrea gigas using crosses between diploids and tetraploids with cytochalasin B treatments, Mar Biotechnol, 7, 318–30. moraga d (1986) Genetic-polymorphism of cultivated populations of the Manila clam, Tapes philippinarum, Comptes Rendus Acad Sci Ser III-Sci Vie-Life Sci, 302, 621–4. naciri-graven y, martin a g, baud j p, renault t and gerard a (1998) Selecting the flat oyster Ostrea edulis (L) for survival when infected with the parasite Bonamia ostreae, J Exp Mar Biol Ecol, 224, 91–107. nell j a and hand r e (2003) Evaluation of the progeny of second-generation Sydney rock oyster Saccostrea glomerata (Gould, 1850) breeding lines for resistance to QX disease Marteilia sydneyi, Aquaculture, 228, 27–35. nell j a and perkins b (2005) Evaluation of progeny of fourth generation Sydney rock oyster Saccostrea glomerata (Gould, 1850) breeding lines, Aquac Res, 36, 753–7. nell j a and perkins b (2006) Evaluation of the progeny of third-generation Sydney rock oyster Saccostrea glomerata (Gould, 1850) breeding lines for resistance to QX disease Marteilia sydneyi and winter mortality Bonamia roughleyi, Aquac Res, 37, 693–700.

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4 Controlling fish reproduction in aquaculture C. Mylonas, Hellenic Center for Marine Research, Greece, and Y. Zohar, University of Maryland Biotechnology Institute, USA

Abstract: Industrial aquaculture is a new activity to most parts of the world and is looking for ways to establish a reliable and controlled system for the provision of seed stock for grow-out operations. Control of reproductive function can be achieved, in many fish species, by manipulating photoperiod, water temperature and spawning substrate. The reproductive cycle is separated in two phases – i.e., growth and maturation – which may be controlled by different reproductive hormones at the level of the pituitary and gonad. Although the first phase of reproductive development is concluded in captivity, the second stage of the reproductive cycle – i.e., oocyte maturation (OM) and ovulation in females, and spermiation in males – may require the employment of exogenous hormonal therapies. In some species, these hormonal manipulations are used only as a management tool to enhance the efficiency of egg production and facilitate hatchery operations, but in other fishes exogenous hormones are the only way to produce fertilized eggs at an industrial level. The reproductive cycle is controlled by the interactions of the hormones of the brain–pituitary– gonad axis. From the brain, gonadotropin releasing hormones (GnRHs) travel along neural axons and stimulate the gonadotroph cells of the pituitary to produce and secrete the two gonadotropins (GtH) follicle stimulating hormone (FSH) and luteinizing hormone (LH), which, in turn, act at the level of the gonad to induce steroidogenesis and the production of the androgens, estrogens and progestagens, which are the final effectors of reproductive function. Hormonal manipulations of reproductive function in cultured fishes have focused on the use of either exogenous LH preparations that act directly at the level of the gonad, or synthetic GnRH agonists (GnRHa) that act at the level of the pituitary to induce release of the endogenous LH stores, which, in turn act at the level of the gonad to induce steroidogenesis and the process of OM and spermiation. Key words: reproduction, induced spawning, final oocyte maturation, spermiation.

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4.1 Introduction As an industrial agricultural activity, aquaculture is quite new to most parts of the world, with the exception of the extensive carp culture (family Cyprinidae) in Asia and the more recent intensification of salmonid production (Oncorhynchus and Salmo spp) in Europe and North America. In essence, it is only since the 1970s that a truly worldwide industry has developed, lately focusing on marine fishes (Kirk, 1987), and this industry is looking for ways to establish a reliable and controlled system for the provision of seed stock for grow-out operations. Control of reproductive function can be achieved, in many fish species, by manipulating photoperiod, water temperature and spawning substrate. However, the conclusion of the final stage of the reproductive cycle – i.e., oocyte maturation and ovulation in females, and spermiation in males – may require the employment of exogenous hormonal therapies. In some species, these hormonal manipulations are used only as a management tool to enhance the efficiency of egg production and facilitate hatchery operations, but in other fishes exogenous hormones are the only way to produce fertilized eggs at an industrial level. This chapter provides a brief description of the reproductive biology of fishes, followed by a description of the major problems encountered in culture, and the hormonal methods developed in the last few decades to address these dysfunctions. Some consideration is also given to future trends in the spawning induction technologies.

4.2 The fish reproductive cycle and its control The reproductive cycle is separated in two phases – i.e., growth and maturation – which may be controlled by different reproductive hormones at the level of the pituitary and gonad (Fig. 4.1). In females, the first phase includes the growth of the primary oocytes and the accumulation of the yolk precursor, vitellogenin (vtg), in their cytoplasm (Fig. 4.2). At the completion of this phase, which is also referred to as vitellogenesis, the process of oocyte maturation (OM) includes both cytoplasmic and nuclear events that prepare the oocyte for its expulsion from the ovarian follicle (ovulation), its release to the environment during spawning and its fertilization by a single spermatozoon. In males, the growth phase is referred to as spermatogenesis and includes the mitotic proliferation of the spermatogonia into primary spermatocytes, their meiotic division into secondary spermatocytes and their differentiation to spermatids and flagellated spermatozoa. The process of maturation, better known as spermiation, includes the increase in seminal fluid production and the capacitation of the spermatozoa, which are now able to undergo forward motility once released in the water during spawning. The reproductive cycle is controlled by the interactions of the hormones of the brain–pituitary–gonad axis (Fig. 4.1). From the brain, gonadotropin releasing hormones (GnRHs) produced in specific neuroendocrine cells

Controlling fish reproduction in aquaculture

The reproductive axis in fish Environment (photoperiod, lunar cycle, temperature, rainfall)

Spermatogenesis & vitellogenesis

Follicle stimulating hormone (FSH)

Androgens Estrogens

Fully grown gametes

Brain GnRH +

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Gonad

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Final maturation DA Luteinizing hormone (LH)

17,20bdihydroxy Progesterone Fertile gametes

Fig. 4.1 Schematic representation of the reproductive axis in fish, its major components and phases, and its environmental and endocrine control.

(Gothilf et al., 1996, 1997; Holland et al., 2001) travel along neural axons and are released immediately at synapses with the gonadotropic cells of the pituitary gland (Yaron et al., 2003). The synthesis and release of the GnRHs is controlled by environmental and nutritional parameters in such a way that reproduction takes place under optimal conditions (Yu et al., 1997). In response to GnRH stimulation, the gonadotrophs produce and secrete the two gonadotropins (GtH) follicle stimulating hormone (FSH) and luteinizing hormone (LH), which, in turn, act at the level of the gonad to induce steroidogenesis (Rosenfeld et al., 2007) and the production of the androgens, estrogens and progestagens, which are the final effectors of reproductive function. In addition to the primary GnRH stimulatory system, neurons secreting dopamine (DA) exert an inhibitory action on both the brain (GnRH synthesis and release) and pituitary (down-regulation of GnRH-R and interference with the GnRH signal-transduction pathways) (Peter et al., 1993; Peter and Yu, 1997; Yaron et al., 2003; Dufour et al., 2005; Levavi-Sivan et al., 2004). As a result, DA inhibits both basal LH secretion and GnRHstimulated LH secretion from the pituitary. Although a dopaminergic inhibition on LH release has been demonstrated in all vertebrates, its intensity and temporal action may differ greatly among fishes. A strong dopaminergic inhibition of reproduction has been demonstrated in salmonids, cyprinids, silurids, tilapia (Oreochromis spp.), freshwater eel (Anguilla anguilla) and grey mullet (Mugil cephalus) (Saligaut et al., 1999; Silverstein et al.,

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A

B gv gv po y

po y l C

D

ca

y

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l gv

gv

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Fig. 4.2 Microphotographs of histological sections from ovaries. (A) Primary oocytes (po) of striped bass, having a centrally located germinal vesicle (gv) and peripheral nucleoli. (B) Oocytes at various stages of vitellogenesis from Atlantic bluefin tuna. Vitellogenic oocytes have various amounts of lipid droplets (l) and yolk vesicles (y). (C) Vitellogenic oocyte of white bass containing small numbers of lipid droplets. (D) Vitellogenic oocyte of striped bass with a very large percentage of lipid droplets. The periphery is occupied by cortical alveoli (ca). (E) Vitellogenic oocyte of American shad (Alosa sapidissima) showing no lipid droplets. (F) Oocyte of striped bass undergoing GV migration and lipid coalescence. (G) Oocyte of striped bass at the GV breakdown stage. (H) Ovulated egg from striped bass. Photographs are not at the same scale.

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1999; Yaron et al., 2003; Vidal et al., 2004). In contrast, a DA inhibitory system seems to be very weak or absent in most marine fishes (Copeland and Thomas, 1989; Zohar et al., 1995; Kumakura et al., 2003a; Prat et al., 2001).

4.2.1 Vitellogenesis, oocyte maturation and ovulation At the onset of vitellogenesis, the ovigerous lamellae of the ovary contain nests of primary oocytes (Fig. 4.2a), which are arrested at prophase I (Guraya, 1986; Wallace and Selman, 1990; Selman et al., 1993). After a period referred to as primary growth, or previtellogenesis, during which the appearance of the ovarian follicle (i.e., the granulosa and theca layers) takes place, vitellogenesis, or the secondary growth, begins (Fig. 4.2b). Vitellogenesis is a hormone-dependant process and its immediate effector is the estrogen 17β-estradiol (E2), produced from the androgen testosterone (T) by the ovarian follicle in a two-cell process involving both the theca and granulosa cell layers (Nagahama, 1994). Regulation of steroidogenesis and E2 production at this time is controlled by the pituitary GtH, in some species by FSH and in others by LH (Rosenfeld et al., 2007). As the name implies, the major characteristic of vitellogenesis is the production of vitellogenin (vtg), which takes place in the liver, and its sequestration in a pinocytosis-mediated process into the developing oocyte (Mommsen and Walsh, 1988; Tyler and Sumpter, 1996). At the start of vitellogenesis, the oocytes may be 150–250 μm in diameter (Fig. 4.2b) and, depending on fish species, at the end of the process the post-vitellogenic oocytes may have a diameter of 550 μm, as in shi drum (Umbrina cirrosa) (Mylonas et al., 2004a), 850 μm, as in striped bass (Morone saxatilis) (Mylonas et al., 1997e), 1400 μm, as in wreckfish (Polyprion americanus) (Fauvel et al., 2007), or up to 4 mm, as in Salmo and Oncorhynchus species (Bromage et al., 1992). Once sequestered into the oocytes, vtg is stored in the yolk globules (or granules), until the process of OM. Another type of nutrients accumulating into the growing vitellogenic oocytes are the lipids. Depending on the species of fish, lipids may be present in the form of triglycerides, phospholipids or wax esters (Lund et al., 2000), and the amount and type of lipid class dominating the cytoplasm determines the presence (Fig. 4.2b, c and d) or absence (Fig. 4.2e), and size and number of lipid droplets (Fig. 4.2c and d) (Mylonas et al., 1997e, 2004a; Corriero et al., 2007). Thus, at the end of vitellogenesis the oocyte has a large cytoplasm filled completely with yolk globules and, in most cases, lipid droplets, a centrally located nucleus (named germinal vesicle, GV), and thick zona radiata (the future chorion) and follicular layers (Fig. 4.2c and d). Oocyte maturation is the second phase of the gametogenic cycle in females, and it is regulated by an LH surge from the pituitary and the production of the maturation inducing hormone (MIH) by the ovarian follicle (Nagahama et al., 1994; Suwa and Yamashita, 2007). The MIH is a

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progestagen, and in most fishes it is 17α,20β-dihydroxy-progesterone (DHP), whereas in others it is 17α,20β,21-trihydroxy-progesterone (20β-S). Oocyte maturation begins with the resumption of meiosis, which has been arrested in prophase I, and the migration of the GV to the animal pole of the oocyte (Fig. 4.2f), underneath the micropyle (Mylonas et al., 1997b). At the same time, and depending on fish species, coalescence of the lipid droplets takes place, resulting in the production of a single lipid droplet (Fig. 4.2g) (Mylonas et al., 1997e). As OM progresses, the GV membrane dissolves (germinal vesicle breakdown, GVBD), chromosomes condense and the first polar body is expulsed from the oocyte. During this time, there is a chemical modification of the yolk, with the proteolysis of the vtg and the production of its major components, lipovitellin, phosvitin and β-component, and free amino acids (FAA) (Cerdá et al., 2007), which is apparent in the coalescence of the yolk globules and the change in staining properties of the yolk (Fig. 4.2g and h) (Mylonas et al., 1997e). Depending on whether the eggs produced are pelagic or bethic (i.e., positively or negatively buoyant), the extent of this proteolysis varies and results in the increase in the osmotic pressure of the oocyte, which drives a drastic uptake of water (Cerdá et al., 2007) with a many-fold increase in size. After hydration, the follicular wall ruptures and the oocyte is ovulated (Fig. 4.2h) into the ovarian cavity, in most fishes, or in the abdominal in others (e.g., Salmonidae and Acipenseridae).

4.2.2 Spermatogenesis and spermiation Spermatogenesis is the process of mitotic proliferation of the spermatogonia, their meiotic division into haploid spermatocytes, and their transformation into flagellated spermatozoa (Schulz and Miura, 2002). During mitotic proliferation, each spermatogonium goes through a species-specific number of divisions, which ranges between 5 and 15. During these divisions all daughter cells maintain direct cytoplasmic bridges between them; they are all contained within an individual spermatocyst, and undergo simultaneous development and maturation from spermatogonia to spermatocytes I and II, spermatids and finally flagellated spermatozoa (Fig. 4.3). Once spermatogenesis is completed, the Sertoli cells that surround each spermatocyst rupture and the spermatozoa are released into the testicular lumen. The presence and relative abundance of spermatocysts with the above maturation stage gametes are used as an indication of the degree of testicular development in fish. With the rupture of the spermatocyst starts the period of spermiation, during which the spermatozoa undergo maturation (capacitation) as they move along the tubular lumen of the testes and are gathered in the efferent ducts. Capacitation is the process by which the spermatozoa acquire the capacity for motility once released in the water, and thus the ability to fertilize the eggs (Billard et al., 1995; Miura and Miura, 2003). At the same

Controlling fish reproduction in aquaculture

115 B

A st sc

sg

sc

sz

E sg C

D sz sz sz

sc

Fig. 4.3 Microphotographs of histological sections from testes of European sea bass at various stages of maturation. (A) Before the onset of the reproductive season, containing only spermatogonia (sg). (B) At the early stage of spermatogenesis in November, containing spermatocyst with spermatocytes I and II (sc) and spermatids (st), including some spermatozoa (sz). (C) In December when spermiation begins, showing the extensive rupture of the mature spermatocysts and the aggregation of spermatozoa in the tubules. (D) In January at the onset of the females spawning season, with the tubules containing almost exclusively spermatozoa. (E) Spent testes at the end of the season, showing residual spermatozoa, new spermatogonia and hypertrophied somatic cells. Photographs are not in the same scale.

time, the testes produce large volumes of seminal fluid, in which the spermatozoa are transported and released to the environment during spawning. During the period of spermiation, gentle abdominal pressure can result in the release of milt (seminal fluid with spermatozoa) in most fishes, and the quality of the milt (density, percentage motility and survival) can be evaluated (Suquet et al., 1994; Billard et al., 1995; Fauvel et al., 1999). The general view of the involvement of pituitary GtHs in the male reproductive cycle, as with the female, is that FSH controls mainly the early stages of gametogenesis (Ohta et al., 2007), while LH regulates the process of spermiation (Schulz and Miura, 2002). Again, the final effectors of the GtHs on reproductive function are the gonadal steroids. Estrogens seem to induce the mitotic proliferation of the spermatogonia prior to the onset of

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gametogenesis (Miura and Miura, 2003). Then androgens (T and 11-keto T, 11-KT) regulate the whole process of spermatogenesis, to the production of flagellated spermatozoa (Miura et al., 1991b; Schulz and Miura, 2002). The maturation of the spermatozoa and the acquisition of motility capacity are controlled by DHP, the same progestagen that acts as an MIH in the female. This action involves the spermatozoa themselves, in a receptormediated process that results in an increase in seminal plasma pH (Miura and Miura, 2003). 4.2.3 Spawning Spawning is the release of the mature gametes (eggs and capacitated spermatozoa) to the external environment (in the vast majority of fishes), in order to produce the zygote. As spermatozoa lose their motility within seconds or minutes after release from the testes and contact with the water (Billard et al., 1995) and eggs absorb water resulting in the closure of the micropyle (Cerdá et al., 2007), spawning must be extremely synchronous in order to result in fertilized eggs. Therefore, fish employ both breeding rituals and pheromones in order to signal to the other sex their readiness for spawning (Liley, 1983; Stacey, 1984; Stacey et al., 1994; Zabala et al., 1997; Kobayashi et al., 2002). Spawning may take place in pairs (e.g., flounder, catfish), single females with a group of males (e.g., Salmonidae, Moronidae), or large groups of males and females (e.g., Sparidae, Thunnidae). Also, the eggs produced may be pelagic, demersal or may stick to each other and various substrates, such as rocks and vegetation. All these characteristics must be known for each fish of interest and be evaluated by the aquaculturist, in order to achieve the optimum results in terms of egg fecundity and fertilization success.

4.3

Reproductive strategies and dysfunctions in captivity

Being the largest vertebrate class with more that 27 000 species and a very long evolutionary history (Nelson, 2006), fishes exhibit an amazing diversity in reproductive biology and strategies. For the purpose of broodstock management and hormonal manipulations for the induction of OM, ovulation and spawning in aquaculture operations, female fish may be separated into two classifications: sychronous spawners (synchronous and single-batch group-synchronous) and asynchronous spawners (multiple-batch groupsynchronous and asynchronous) (Tyler and Sumpter, 1996). Synchronous spawners reproduce once in their lifetime or once during an annual reproductive season, and their ovary contains a single, uniform population of developing oocytes during the reproductive season (Fig. 4.4a). On the other hand, asynchronous spawners reproduce multiple times during every reproductive period. These spawns may be numerous and regular, e.g., daily or every other day for a period of 3–4 months; or can be few and irregular in

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B

Fig. 4.4 Microphotographs of histological sections from ovaries of striped bass, a synchronous fish (A), and of bluefin tuna, an asynchronous fish (B). Photographs are not in the same scale.

their timing, e.g. 3–7 spawns with an inter-spawn period of between 3 and 10 d. The ovaries of these species contain oocytes at all stages of development during the reproductive season (Fig. 4.4b), and different batches of oocytes mature during each OM, ovulation and spawning event. In terms of the males, the situation is somewhat simpler. Spermatogenesis and spermiation may be temporally separated, with spermiation occurring after the conclusion of spermatogenesis, and during the spawning season the testes may contain exclusively spermatozoa (Billard, 1986; Malison et al., 1994). In the majority of species, however, there is significant overlap between the two processes, with both spermatogenesis and spermiation taking place during the spawning season (Jackson and Sullivan, 1995; Mylonas et al., 2003; Rainis et al., 2003). Therefore, management of male reproduction in captivity and induction of spermiation utilizes very similar methods. Reproductive dysfunctions of captive fishes are often restricted to the females, since males do undergo complete maturation in captivity, albeit at times possibly producing a reduced amount of milt and of lower quality (Mylonas and Zohar, 2001a, 2007; Zohar and Mylonas, 2001b; Mañanos et al., 2008). The simplest reproductive problem in females is observed in salmonids (Onchorhynchus and Salmo spp.), which do undergo vitellogenesis, OM and ovulation, but fail to spawn their eggs when reared in captivity (Bromage and Cumaranatunga, 1988; Zohar, 1989), probably due to the

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lack of the appropriate spawning substrate to place their eggs. The most common reproductive dysfunction in captivity is the failure of OM upon completion of vitellogenesis. As a result there is no ovulation and no spawning of eggs (Berlinsky et al., 1996, 1997; Larsson et al., 1997; Mugnier et al., 2000; Mylonas and Zohar, 2001b; Barbaro et al., 2002; Duncan et al., 2003; Marino et al., 2003; Ibarra-Castro et al., 2004; Mylonas et al., 2004a; Yang and Chen, 2004; Chen, 2005; Agulleiro et al., 2006; Fauvel et al., 2007; Mylonas et al., 2007). The failure of captive females to undergo OM is due to dysfunctional release of LH from the pituitary. In striped bass, for example, the levels of various reproductive hormones were compared between cultured fish that fail to undergo OM during the spawning season and wild fish captured on their spawning grounds (Mylonas et al., 1997d, 1998b; Steven et al., 2000; Mylonas and Zohar, 2001b). In wild females, a plasma LH surge was observed during OM and ovulation, but in females reared in captivity plasma LH levels remained low at the end of vitellogenesis. However, LH was synthesized and stored in the pituitary during vitellogenesis, since levels of LH and its mRNA in the pituitary did not differ between wild and captive females, demonstrating that the problem is one of lack of release and not synthesis in captivity. In addition, mRNA levels of the pituitary receptor for the GnRH most relevant to pituitary LH synthesis were similar between wild and captive females. This suggests that the disruption in LH release from the pituitaries of captive fish is not due to a dysfunction in pituitary responsiveness, but may be related to the control of pituitary function by the reproductive brain. In fact, differences were observed between wild and captive females undergoing OM, when comparing the pituitary content of the endogenous GnRHs. The GnRH mRNA levels within the brain, however, were similar between the two groups, indicating that the altered pituitary content of GnRH in captive fish may be a result of altered release from the hypothalamus, rather than deficient synthesis (Steven, 2000; Steven et al., 2000). Similarly in males, lower plasma levels of LH during the spermiation period have been suggested as the cause of the reduced amount of milt produced by some fishes (Mylonas and Zohar, 2001b; Mañanos et al., 2002). As with females, the amount of LH in the pituitary or the ability of the pituitary to synthesize LH in response to treatment with exogenous GnRHa is not affected in these fishes, suggesting that again the reproductive dysfunction in the males may be identified in the brain control of GtH synthesis and/or release.

4.4 Hormonal therapies for the control of reproduction Based on the accumulating evidence that the failure of fishes to undergo OM and full spermiation in captivity is the result of diminished LH release

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♀♂

Hormonal induction of oocyte maturation & spermiation Brain

GnRH GnRHa

LH

Pituitary

LH

Gonads

Sex steroids

Oocyte maturation-spermiation

Fig. 4.5 Schematic representation of the dysfunction in the reproductive axis of cultured fishes, and the exogenous hormonal interventions for the induction of oocyte maturation and spermiation.

from the pituitary, manipulations of reproductive function in cultured fishes have focused on the use of either exogenous LH preparations that act directly at the level of the gonad, or GnRHa that acts at the level of the pituitary to induce release of the endogenous LH stores (Fig. 4.5). Endogenous LH, in turn, acts at the level of the gonad to induce steroidogenesis and the process of OM and spermiation.

4.4.1 Gonadotropin preparations Gonadotropin preparations include pituitary homogenates and pituitary extracts (PE) that contain LH (as well as other pituitary hormones), purified piscine LH or purified human chorionic gonadotropin (hCG), which has very strong LH activity (Lam, 1982; Donaldson and Hunter, 1983; Zohar, 1989; Zohar and Mylonas, 2001b). The main advantage of GtH preparations is that they act directly at the level of the gonad. Pituitary homogenates were the first type of exogenous hormonal treatments used by aquaculturists for the induction of maturation and spawning (Houssay, 1930; Von Ihering, 1937; Fontenele, 1955). Today, preparations of carp pituitary extract (CPE) and purified salmon GtH are available commercially and are used worldwide. These purified PE are more effective than the earlier pituitary homogenates, since they are purified to various extents and their activity is usually calibrated using bioassays (Yaron, 1995; Donaldson, 1973). Nevertheless, they still maintain the disadvantages of risking pathogen transmissions, as well as a high degree of species

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specificity, due to the significant differences in the primary structure of fish GtH (Rosenfeld et al., 2007). Treatments with pituitary homogenates and PE are usually split into a smaller priming dose (10–20 % of total) and a larger resolving dose given 12–24 h apart (Thalathiah et al., 1988; Parauka et al., 1991; Kucharczyk et al., 1997; Chen, 2005). Human CG has also been used extensively in hormonal manipulation of reproduction in fishes, as it has been available throughout the world for some time now, and it is purified and of clinical grade and standardized bioactivity. Unlike GtH preparations of piscine origin, hCG is most often effective in a single dose (100 and 4000 IU Kg−1), presumably due to its long residence time in circulation (Ohta and Tanaka, 1997). This is unrelated to its heterologous nature in fish, since it has been shown to have a significantly longer halflife compared to the pituitary GtHs both in fish (Fontaine et al., 1984) and humans (Ludwig et al., 2002). Recently, an hCG preparation has been approved for commercial utilization in commercial aquaculture (CHORULONTM, Intervet International bv, The Netherlands).

4.4.2 Gonadotropin-releasing hormone agonists The use of GnRHas for spawning induction therapies has important advantages over the use of GtH preparations. Firstly, being of synthetic nature, GnRHas do not pose a disease transmission threat, as pituitary homogenates or extracts may do. Secondly, GnRHa treatments are less speciesspecific than GtH ones, due to the high structural similarity of native GnRHs among fishes (Lethimonier et al., 2004). Thirdly, and perhaps most importantly, GnRHas stimulate the release of the endogenous GtHs and other necessary pituitary hormones (Le Gac et al., 1993; Weber et al., 1995; Cyr and Eales, 1996; Negatu et al., 1998), and thus they provide for a better integration of reproductive processes by acting at a higher level of the brain–pituitary–gonad axis. Although hundreds of different GnRHas are available, the only approved GnRHa for use in commercial aquaculture is Azagly-nafarelin (GONAZONTM, Intervet International bv, The Netherlands), which has so far been shown to be efficacious only in salmonids (Haffray et al., 2005). As mentioned earlier, in some fishes there is a strong inhibition of basal and GnRH-stimulated release of LH by DA. Therefore, administration of DA antagonists prior to the treatment with GnRHa removes the inhibition on the gonadotrophs and enhances the stimulatory effect of GnRHa on LH release. Currently, hormonal manipulations of reproduction using a combined GnRHa/DA antagonist treatment are used mostly in cyprinids (Yaron, 1995; Mikolajczyk et al., 2003, 2004; Kaminski et al., 2004), catfishes (Silverstein et al., 1999; Brzuska, 2001; Wen and Lin, 2004) and mullets (Glubokov et al., 1994; Arabaci and Sari, 2004; Aizen et al., 2005). There are several DA antagonists available in the market that

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proved to be useful for hormone treatments in aquaculture (i.e., domperidone, pimozide, reserpine and metoclopramide); these are usually administered as a liquid solution injected prior to, or at the same time as the GnRHa treatment.

4.4.3 Sustained-release delivery systems It was recognized almost from the first spawning induction experiment (Fontenele, 1955), that sustained administration of the hormone would result in improved efficacy. This is because the processes of OM and spermiation often require a prolonged treatment with exogenous hormones, given in multiple injections (Mylonas et al., 1992; Dabrowski et al., 1994; Slater et al., 1994, Carrillo et al., 1995, Pankhurst et al., 1996). Such repetitive handling of broodstock requires substantial labor, time and monitoring, and in situations where the broodfish are very large (groupers, amberjacks or tunas) or kept outdoors – in ponds or cages – it is very time-consuming and labor-intensive to crowd, capture, anaesthetize and inject the fish with hormones. Since the 1980s, a variety of hormone-delivery systems, almost exclusively for GnRHa, have been developed for use in a variety of fishes. The first such delivery system was prepared using cholesterol and was tested in Atlantic salmon (Salmo salar) (Weil and Crim, 1983). Cholesterol implants are prepared as solid, cylindrical pellets (3 mm in diameter) and are implanted intramuscularly using an implanter. The next type of GnRHadelivery system was fabricated in the form of microspheres (5–200 μm in diameter), using copolymers of lactic acid and glycolic acid (LGA) (Okada et al., 1994) or a copolymer of fatty acid dimer and sebasic acid (Fad-sa) (Mylonas et al., 1995). For treatment, the microspheres are suspended in a viscous vehicle and are injected into the muscle (Zohar, 1988; Breton et al., 1990; Chang et al., 1995; Mylonas et al., 1997c; Mylonas and Zohar, 2001b; Barbaro et al., 2002). The greatest advantage of biodegradable, microspheric delivery systems is that the same preparation can be used to treat fish with large variations in size. Also, since over time the microspheres degrade to their monomer constituents, which are all natural products – e.g., lactic acid, glycolic acid or sebasic acid – broodstock retired from production can be consumed for food without any concerns over harmful residual chemicals. Another type of GnRHa-delivery system used for spawning induction is prepared in the form of a solid implant, using a non-degradable co-polymer of ethylene and vinyl acetate (EVAc) (Zohar, 1996). In this delivery system, the GnRHa is mixed with an inert bulking agent, and the mixture is entrapped by the EVAc matrix. Upon application, the inert matrix dissolves, carrying with it the GnRHa. The EVAc implants are fabricated as disks 2 or 3 mm in diameter and are administered intramuscularly using an implanter (Mylonas et al., 2007), releasing GnRHa

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for periods from 2–5 weeks (Zohar, 1996; Mylonas et al., 1998b; Mañanos et al., 2002).

4.5 Induction of oocyte maturation and ovulation As mentioned earlier, for the purpose of broodstock management and hormonal manipulation of OM and spawning, fish are separated into two classifications: synchronous spawners (single-time and single-batch group-synchronous) and asynchronous spawners (multiple-batch groupsynchronous and asynchronous) (Tyler and Sumpter, 1996). These differences in reproductive strategies may necessitate the employment of different approaches in terms of hormonal therapy and egg acquisition. For example, single or double injections of GnRHa in liquid form may be effective in synchronous fish (Mylonas et al., 1992), which have all their oocytes developed at the same stage of maturation, but may not be the best approach to achieve maximum fecundity in asynchronous species with a long reproductive season (Zohar et al., 1995). Also, if required, strip spawning and artificial insemination is a good alternative to tank spawning in synchronous fishes, but will result in very poor fecundity in asynchronous species, since the fish ovulate only part of their total season production of vitellogenic oocytes, and the stripping process may damage the remaining oocytes. The use of GtH preparations in inducing OM, ovulation and spawning in synchronous fishes has been summarized well in previous reviews, which report also more extensive information on doses and treatment protocols (Donaldson, 1973; Lam, 1982; Donaldson and Hunter, 1983; Zohar and Mylonas, 2001a; Mañanos et al., 2008). Some more recent examples include the European catfish (Silurus glanis), which was successfully induced to ovulate using 4 mg Kg−1 CPE, though in a smaller percentage of females compared to a combined GnRHa/DA antagonist treatment (Brzuska, 2001). In the Japanese catfish (Silurus asotus), a single injection of 10 000 IU Kg−1 hCG induced OM and ovulation from June to September (Kumakura et al., 2003b). In the Brazilian catfish ‘cachara’ (Pseudoplatystoma fasciatum), both CPE and hCG were effective in inducing ovulation (Leonardo et al., 2004). Also, hCG at 1000 or 2000 IU Kg−1 was effective as a single injection in inducing ovulation in the Korean spotted sea bass (Lateolabrax maculatus) (Lee and Yang, 2002). Finally, in wild-caught ocellated puffer (Takifugu ocellatus), both single- and double-injections of 6 mg Kg−1 PE or 2500 IU Kg−1 hCG were very effective in inducing OM and ovulation (Chen, 2005), and in pikeperch (Sander lucioperca), either single or multiple injections of 200 IU Kg−1 hCG were effective in inducing ovulation (Zakes and Szczepkowski, 2004). The continued interest in the control of reproduction of the European eel has resulted in improved knowledge on the dysfunctions of this species

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in culture and has identified some blocks in the endocrine axis. The European eel undergoes gametogenesis during its long migration from European rivers to the Sargasso sea, off the coast of North America (Tesch, 2003). In captivity, the absence of this migration results in the complete absence of both oogenesis and spermatogenesis. Recent research has identified DA as the brain hormone responsible for the blocking of the reproductive axis and the absence of pubertal development (Vidal et al., 2004). Still, however, the only available practical method for the induction of gametogenesis in the freshwater eel is the weekly administration of fish gonadotropin extracts in the female (Ohta et al., 1997; Sato et al., 1997; Pedersen, 2003; Palstra et al., 2005) and one or two injections of the same gonadotropin or of hCG in the male (Miura et al., 1991a; Ohta et al., 1996; Ohta and Tanaka, 1997). Oocyte maturation is induced by the administration of the MIH, eggs and sperm are collected by stripping, and fertilization is accomplished artificially (Pedersen, 2004). A very promising new way to induce gametogenesis in the European eel is currently undergoing development (Guido van den Thillart and Herman Spaink, unpublished data), and employs genetically engineered zebrafish cell lines expressing the genes of zebrafish LH and FSH, controlled by a constitutive promoter. Once implanted subcutaneously to silver migrating eels, such cells are shown to produce continuously the two zf-GtHs, thus stimulating gonadogenesis. The development of this method will alleviate the need for multiple injections of heterologous hormones and will probably increase the effectiveness of maturation induction approaches in the freshwater eels. The synchronization of ovulation in salmonids was one of the very first applications of GnRHa in aquaculture (Donaldson et al., 1981; Crim and Glebe, 1984; Breton et al., 1990). The treatment is usually given in the form of two injections (10–100 μg Kg−1) spaced 3 d apart or a single application of a GnRHa-delivery system (10–50 μg Kg−1), given around two weeks before the onset of natural maturation of the broodstock. The two-injection (Van Der Kraak et al., 1985; Sullivan et al., 1989; Mylonas et al., 1992) and GnRHa-delivery system protocols (Crim et al., 1983; Crim and Glebe, 1984; Breton et al., 1990; Goren et al., 1995) induce ovulation in 100 % of the stock within two weeks after treatment. Single or multiple injections of GnRHa have also been used extensively in other synchronous fishes. When a two-injection protocol is used, GnRHa is given in a priming (5–10 %) and resolving dose (95–90 %), and if a DA antagonist is also used it is given with the priming dose. For example, in the ocellated puffer both single and double injections of 50 μg Kg−1 GnRHa were effective in inducing OM (Chen, 2005), while similar results were obtained using 2–4 injections of GnRHa in the bullseye puffer (Spoeroides annulatus) (Duncan et al., 2003). In the grey mullet, two injections of 30 μg Kg−1 GnRHa together with 15 mg Kg−1 metoclopramide were very effective in inducing spawning within 24 h (Aizen et al., 2005). Similarly, two injections of

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20 μg Kg−1 GnRHa with 5 mg Kg−1 pimozide induced ovulation in 95 % of treated common carp (Cyprinus carpio) (Mikolajczyk et al., 2004). Two injections of GnRHa in combination with a DA antagonist have been used successfully also in the koi carp (Cyprinus carpio) (Arabaci et al., 2004), lake mullet (Chalcalburnus tarichi) (Arabaci and Sari, 2004) and wild catfish (Silurus asorus) (Wen and Lin, 2004). Finally, a single injection of 20 μg Kg−1 GnRHa induced ovulation in tench (Tinca tinca) (Rodríguez et al., 2004). Sturgeon (Acipenser spp.) aquaculture for meat and caviar relies exclusively on the use of hormonal spawning induction methods and the artificial fertilization of the obtained eggs. Sturgeon females are evaluated for the completion of vitellogenesis and the extent of the migration of the nucleus (polarization index, PI) by laparoscopic (Hurvitz et al., 2007) or surgical removal of oocytes from the ovary and their in vitro processing (Williot et al., 1991; Conte et al., 1988). The selected mature females may be given sturgeon pituitary extract, carp pituitary extract or, more recently, GnRHa (Webb et al., 1999; Chebanov and Billard, 2001; Williot et al., 2001, 2002; Burtsev et al., 2002; Zhuang et al., 2002), usually in priming and resolving injections spaced 10–24 h apart, and ovulation is accomplished 24–50 h afterwards. Single treatments with carp pituitary homogenate have also been reported to be effective (Williot et al., 2005). Acquisition of eggs is done using caesarian surgery or, more recently, by inserting a scalpel into the abdominal pore and making a small incision at the basal part of the oviducts (Chebanov and Billard, 2001). Sperm can be used fresh or cryopreserved (Billard et al., 2004) and the amount of sperm produced may also be enhanced using GnRHa-based hormonal therapies (Williot et al., 2002). The greater efficacy of GnRHa-delivery systems in inducing OM in synchronous fishes has been demonstrated well since the mid-1990s (Mylonas and Zohar, 2001a, 2007). A GnRHa-delivery system was the only hormonal preparation able to induce spawning in the yaqui catfish (Ictalurus pricei), whereas combined sGnRHa/DA antagonist or catfish PE treatments were ineffective (Mylonas and Zohar, 2001a). In the tiger puffer (Takifugu rubripes), GnRHa-delivery systems (400 μg Kg−1) induced ovulation in 18 and 10 d in fish with mean oocyte diameter of 800–900 μm and 900–1000 μm, respectively (Matsuyama et al., 1997). Other examples of applications in synchronous fishes include the bullseye puffer (Duncan et al., 2003), cobia (Rachycentron canadum) (Kilduff et al., 2002), devil stinger (Inimicus japonicus) (Takushima et al., 2003) and common carp (Brzuska and Bialowas, 2002). GnRHa-delivery systems have been used in preference to liquid injections in a variety of asynchronous fishes. For example, GnRHa-delivery systems induced two consecutive spawns within 3 d in white bass (M. chrysops) (Mylonas et al., 1997b) and greater amberjack (Seriola dumenili)

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(Mylonas et al., 2004c), five spawns within 7 d in the barramundi (Lates calcarifer) (Almendras et al., 1988), five ovulations within two weeks in striped trumpeter (Latris lineate) (Morehead et al., 1998), one to four ovulations within 7 d in the black sea bass (Centropristis striata) (Watanabe et al., 2003) and seven ovulations within 10 d in the dusky grouper (E. marginatus) (Marino et al., 2003). The greatest potential, however, of sustained-release GnRHa-delivery systems is in the induction of OM in asynchronous fishes with a daily or almost daily ovulation/spawning frequency. For example, the red porgy (Pagrus pagrus), red seabream (P. major) and gilthead seabream (Sparus aurata) have an asynchronous mode of ovarian development and are capable of undergoing OM and spawning on a 24 h cycle for periods up to four months (Watanabe and Kiron, 1995; Zohar et al., 1995; Mylonas et al., 2004b). A single GnRHa injection in the gilthead seabream induced daily spawning in only 20 % of the stock, while a GnRHadelivery system induced daily spawning in >70 % of treated females. Similar results have been obtained with the other two sparids (Matsuyama et al., 1995; Zohar and Mylonas, 2001a). Thus, GnRHa-delivery systems result in significant increases in fecundity, by increasing the number of broodfish undergoing OM, and the number of ovulations per spawning season (Barbaro et al., 2002). The latest success of the GnRHa-delivery systems has been in the induction of OM, ovulation and spawning in cage-cultured Atlantic bluefin tuna (Thunnus thynnus) (Mylonas et al., 2007) and tankcultured Southern bluefin tuna (T. maccoyii) (M. Deichmann, Clean Seas Tuna Ltd, personal communication), which resulted in the production of fertilized eggs and viable larvae. Due to the inability of anaesthetizing bluefin tunas and the great difficulties in handling such large (60–120 Kg) and fast swimming pelagic fishes, GnRHa administration was done underwater in free swimming fish (Mylonas et al., 2007). It is expected that this method will continue to be used as the standard for the induction of spawning in captive-reared Atlantic bluefin tuna, until such time as land-based facilities are build or appropriate sea-cage sites are identified, which can reproduce the optimal environmental conditions necessary for reproductive maturation and spawning (e.g., temperature-photoperiod combination, water quality, etc.). Recently, the use of these GnRHa-delivery systems has induced spawning for four consecutive days in a captive-reared stock at Vibo Valentia, Italy, producing many millions of fertilized eggs, allowing the first larval rearing of Atlantic bluefin tuna in the Mediterranean Sea (G. Demetrio, unpublished data). Finally, GnRHa-delivery systems have been employed with great success in inducing multiple spawnings, often of improved quality compared to the few naturally spawning females, in various flatfishes. For example, GnRHadelivery systems induced daily ovulations in the greenback flounder (Rhombosolea tapirina) (Poortenaar and Pankhurst, 2000), and in wild-caught summer flounder (Paralichthys dentatus) GnRHa implants induced daily

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ovulations for 8 d (Berlinsky et al., 1997), whereas in fish maintained for more than a year in captivity the same treatment induced not only ovulation but also tank spawning (Watanabe et al., 1998). Similarly in turbot (Scophthalmus maximus), treatment with a GnRHa-delivery system induced multiple ovulations in 100 % of treated fish compared to 50 % of controls (Mugnier et al., 2000). Also, in the yellowtail flounder (Pleuronectes ferrugineus) different GnRHa-delivery systems induced an average of eight consecutive ovulations, compared to three in control fish, resulting in the production of twice as many eggs and of higher fertilization and hatching percentage than control females (Larsson et al., 1997). The same two GnRHa-delivery systems have also induced daily spawnings for up to two weeks in the Senegal sole (Solea senegalensis), though with very limited fertilization success (Agulleiro et al., 2006; Guzmán et al., 2008).

4.6 Induction of spermiation As mentioned earlier, the dysfunction observed in cultured male fishes is not the absence of any stage of testicular development, but rather a reduction in the spermiation process and the production of expressible milt. Due to the long-term nature of the process of spermatogenesis and spermiation – as opposed to OM in females – long-term hormonal therapies with GnRHa-delivery systems have proven more effective in enhancing milt production compared to acute treatments with either GtH preparations or GnRHas. For example, in the rabbitfish (Siganus guttatus) milt production increased significantly 24 h after GnRHa injection, but returned to pretreatment levels 48 h later (Garcia, 1991). Sustained elevation of sperm production was maintained for 5 d in carp by daily injections of GnRHa, but 3 d after the treatment was interrupted, milt volume decreased below pre-treatment levels (Takashima et al., 1984). In the winter flounder (Pleuronectes americanus) a single injection did not increase milt production, whereas two injections given 24 h apart induced a significant increase in total expressible milt (Harmin and Crim, 1993). Finally, in the European sea bass (Dicentrarchus labrax) a single injection of GnRHa at the end of the spawning season was effective in maintaining milt volume of stripped males for only 3 d, compared to 17 d of GnRHa implants (Rainis et al., 2003). These results underline the need for a long-term hormonal therapy, in order to induce sustained increases in milt production. Another disadvantage of single hormone injections is that they usually induce only a short-lived elevation of seminal plasma production, with a much smaller increase in spermatozoa production (Clemens and Grant, 1965; Garcia, 1991) and the increase in milt volume is accompanied with significant reductions in sperm density (Takashima et al., 1984; Garcia, 1991). In white bass, treatment with an hCG injection may restore milt

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release in males stripped completely of their milt, but the milt is extremely thin and contains mostly seminal fluid (Bayless, 1972). On the contrary, GnRHa-delivery systems increase milt production significantly, without any decrease in sperm density, motility or fertilizing ability of the spermatozoa (Mylonas et al., 1997a). Many different GnRHa-delivery systems have been used to enhance spermiation in cultured fishes and beginning with salmonid species such as Atlantic salmon (Zohar, 1996; Weil and Crim, 1983), rainbow trout (Oncorhynchus mykiss) (Breton et al., 1990), chinook salmon (O. tshawystcha) (Solar et al., 1995), coho salmon (O. kisutch) (Goren et al., 1995). GnRHa-delivery systems have also been very effective in basses of the Moronidae family. For example, in the European seabass at the peak of the spawning season, a single injection of GnRHa induced increases in milt production for 7 d only, whereas treatment with GnRHa-delivery systems resulted in increased milt production for 28–35 d (Mañanos et al., 2002). Also in the striped bass, GnRHa-delivery systems induced long-term increases in milt production, lasting for 14–20 d (Mylonas et al., 1997c; Mylonas et al., 1998a). GnRHa implants have also been used in Atlantic halibut (Hippoglossus hippoglossus) to enhance the quality of the sperm (Vermeirssen et al., 2003), in starry flounder (Platichthys stellatus) to increase milt volume and sperm density (Moon et al., 2003) and in greenback flounder to increase sperm volume (Lim et al., 2004). Still, in some species simple injections of GnRHa of GtH preparations have been employed for the successful enhancement of spermiation, such as the Siberian sturgeon (A. baerii) (Williot et al., 2002), the sterlet (Acipenser ruthenus) (Rzemieniecki et al., 2004), the precocious European sea bass (Schiavone et al., 2006) and the minnow (Rhynchocypris oxycephalus) (Park et al., 2002).

4.7 Spontaneous spawning versus artificial insemination Hormonal induction of OM and spermiation does not ensure spawning of the fish – i.e., release of their gametes – in a timely and synchronous way so that fertilized eggs are produced. This may be due to inappropriate tank size, lack of bottom substrate for the preparation of a nest or plant substrate for the adhesion of the eggs, and possibly other reasons that are not yet known. Therefore, for some species it is also necessary to employ artificial gamete collection and fertilization, using strip spawning. Such species include all salmonids, sturgeons and various carps, groupers and flatfishes. In these situations, it is important to establish with varying degrees of accuracy the time of ovulation after the hormonal treatment. This is because once the eggs are ovulated into the ovarian or abdominal cavity, they begin to lose their viability in a species- and temperaturedependent process that may last from minutes (e.g., Moronidae) to days

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(Salmonidae). Failure to strip the eggs within the appropriate time interval after hormonal stimulation will result in greatly reduced fertilization success. The same is not true for sperm collection, which can be done at any time after hormonal stimulation. In addition, sperm from most fishes can be kept viable without the use of cryopreservation or extenders from many hours (Rainis et al., 2003) to many days (Mylonas et al., 2003; Papadaki et al., 2008). Therefore, a typical artificial insemination protocol should plan for (i) collection and storage of sperm a few hours before the expected time of ovulation and (ii) stripping of the eggs at the appropriate time after hormonal therapy. This procedure will ensure optimal results in fertilization success.

4.8 Future trends Basic studies of fish reproductive physiology and endocrinology, in combination with functional genomics and modern tools of biotechnology, will lead to more precise and efficient control of reproduction in farmed fish and to better supplies of optimal quality seed. It is clear from the present review that GnRHs are of considerable importance to normal and induced gametogenesis in farmed fish. Understanding the environmental and endocrine regulation of GnRH gene expression is key to developing strategies for overcoming the GnRH failure that results in the lack of OM, ovulation and spawning in captive fish. More studies on the functional significance of GnRH multiplicity will lead to better tailored GnRH-based spawning induction technologies that will administer or manipulate the relevant, physiological combination of GnRH forms. In addition, the discovery and understanding of factors that control the early establishment of the GnRH system can be used to develop new approaches to induce sterility or precocious puberty in farmed fish. The introduction of zebrafish as a model for the study of the GnRH system (Steven et al., 2003; Palevitch et al., 2007) and the recent development of transgenic zebrafish expressing a green fluorescent protein (GFP) reporter gene under control of the GnRH promoter (Abraham et al., 2008) provide very powerful tools for improving our understanding of how the GnRH system is regulated in fish. Using these tools, simple manipulations of GnRH neuronal development and GnRH synthesis and release may be developed in the future to control the onset of puberty and induce oocyte maturation, ovulation and spawning. To be successful, exogenous application of GnRHa and other hormonal preparations in spawning induction therapies must be precisely synchronized with the acquisition of follicular maturational and ovulatory competence. Fish that are treated too early or too late will either not spawn or spawn unfertilizable or poor quality eggs. In most cases, prediction of

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‘readiness’ of the female for spawning induction is determined based on ovarian biopsy and measurement of oocyte diameter and/or microscopic observation of morphological characteristics of the oocytes, such as GV migration or occurrence of atresia. Although it has been standard practice for decades, the ovarian biopsy method is not ideal, as it is stressful to the fish, impossible to conduct on small (such as ornamental fishes) or very large (such as bluefin tuna) species, and not very accurate. The field of spawning induction needs non-invasive and more precise methods to determine the readiness of the female, as well as to predict and optimize the success of the spawning induction treatment. Many hormones and other factors are involved in the process of acquiring ovarian maturational competence, and these may be used as precise indicators of its progress (Patiño et al., 2001). Determining which factors are relevant and best suited as indicators will be greatly facilitated using genomics and proteomics information, and several recent studies have used this approach (Bobe et al., 2004; Aegerter et al., 2005; von Schalburg et al., 2005; Bonnet et al., 2007). More work needs to be done in order to fully exploit this approach in fish and develop DNA or molecule microarrays in order to efficiently and comprehensively assess spawning readiness in aquacultured species. In addition, significant effort has been devoted to developing methodologies that measure reproductive indicators, such as steroids and vtg, using mucous or muscle samples (Bridges et al., 2003; Susca et al., 2001). Application of novel and non-invasive sampling modalities, together with the development of fish ‘gene chips’ for reproductive factors, will undoubtedly lead to the future use of such approaches to optimize the timing and enhance the success of spawning manipulation in farmed fish. Finally, recent research has established a new paradigm for fish reproductive endocrinology. The simplistic partitional view of the brain– pituitary–gonadal axis has been replaced by a more complex and integrated web of endocrine interactions. The multiple brain GnRHs and their receptors were shown to be expressed locally in the pituitary (Mohamed et al., 2005) and gonads (Lin and Peter, 1996; Gray et al., 2002; Uzbekova et al., 2002; Soverchia et al., 2007). In both the ovary and testis of fish, GnRH has been demonstrated to directly affect gamete development and maturation (Habibi et al., 1988; Gazourian et al., 1997; Pati and Habibi, 2000; Soverchia et al., 2007). Likewise, the pituitary gonadotropins were shown to be expressed locally in the gonads, and this expression is regulated by GnRH (Wong et al., 2004). The occurrence of a complete GnRH–GtH–steroid axis within the fish gonads should be considered when hormonally manipulating reproduction and inducing spawning, as its balanced expression may be critical to the production of highest quality gametes and embryos. Thus, a more complete understanding of how the entire endocrine–reproductive axis is coordinated in fish is expected to lead to better application of

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spawning manipulation protocols and optimization of seed production in aquaculture.

4.9 Sources of further information and advice Further information in this subject can be obtained in recent reviews (Schulz and Miura, 2002; Miura and Miura, 2003; Mylonas and Zohar, 2007; Mañanos et al., 2008). Information on fish reproduction in general can also be obtained from the website REPROFISH (www.reprofish.eu), a website established currently in Europe, with the objective of acting as a portal for students, scientists and professionals interested in fish reproduction and its control. Through the site, the interested professional can access scientific articles and protocols related to the control of reproduction in fish under culture conditions. Also, articles related to fish reproduction and its control are published in the proceedings of the International Symposium on Fish Reproductive Physiology and the International Symposium on Fish Endocrinology, which take place once every four years. Journals such as Aquaculture, Aquaculture Research, Fish Physiology and Biochemistry, General and Comparative Endocrinology and Biology of Reproduction publish many articles related to fish reproduction.

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arabaci, m, cagirgan h and sari m (2004) Induction of ovulation in ornamental common carp (Koi, Cyprinus carpio L.) using LHRHa ([d-Ser(tBu)6, Pro9-NEt]LHRH) combined with haloperidol and carp pituitary extract, Aquac Res, 35, 10–14. barbaro, a, franceson a, bertotto d, bozzato g, di maria i, patarnello p, furlan f and colombo l (2002) More effective induction of spawning with long-acting GnRH agonist in the shi drum, Umbrina cirrosa L. (Sciaenidae, Teleostei), a valuable candidate for Mediterranean mariculture, J Appl Ichthyol, 18, 192–9. bayless, j d (1972) Artificial Propagation and Hybridization of Striped bass, Morone saxatilis (Walbaum), Columbia, SC, South Carolina Wildlife and Marine Resources Department. berlinsky, d l, king w v, smith t i j, hamilton r d, ii, holloway j, jr and sullivan c v (1996) Induced ovulation of Southern flounder Paralichthys lethostigma using gonadotropin releasing hormone analogue implants, J World Aquac Soc, 27, 143–52. berlinsky, d l, william k, hodson r g and sullivan c v (1997) Hormone induced spawning of summer flounder Paralichthys dentatus, J World Aquac Soc, 28, 79–86. billard, r (1986) Spermatogenesis and spermatology of some teleost fish species, Reprod Nutr Develop, 26, 877–920. billard, r, cosson j, crim l w and suquet m (1995) Sperm physiology and quality, in Bromage N R and Roberts R J (eds), Broodstock Management and Egg and Larval Quality, Oxford, Blackwell Science, 25–52. billard, r, cosson j, noveiri s b and pourkazemi m (2004) Cryopreservation and short-term storage of sturgeon sperm, a review, Aquaculture, 236, 1–9. bobe, j, nguyen t and jalabert b (2004) Targeted gene expression profiling in the rainbow trout (Oncorhynchus mykiss) ovary during maturational competence acquisition and oocyte maturation, Biol Reprod, 71, 73–82. breton, b, weil c, sambroni e and zohar y (1990) Effects of acute versus sustained administration of GnRHa on GtH release and ovulation in the rainbow trout, Oncorhyncus mykiss, Aquaculture, 91, 371–83. bonnet, e, fostier a and bobe j (2007) Microarray-based analysis of fish egg quality after natural or controlled ovulation, BMC Genomics, 8, 55. bridges c r, susca v, eicker j, corriero a, de metrio g, megalofonou p, de la serna j-m and kime d (2003) Fishy business in the Mediterranean – tuna, tonnara and testosterone, in Bridges C R, Gordin H and Garcia A (eds), Cahiers Options Méditerranéennes, Vol. 60: Domestication of the bluefin tuna Thunnus thynnus thynnus, CIHEAM, Zaragoza, 33–5. bromage, n r and cumaranatunga r (1988) Egg production in the rainbow trout, in Muir J F and Roberts R J (eds), Recent Advances in Aquaculture, London, Croom Helm/Timber Press Inc., 63–138. bromage, n, jones j, randall c, thrush m, springate j, duston j and barker g (1992) Broodstock management, fecundity, egg quality and the timing of egg production in the rainbow trout (Oncorhynchus mykiss), Aquaculture, 100, 141–66. brzuska, e (2001) Artificial spawning of European catfish Silurus glanis L.: differences between propagation results after stimulation of ovulation with carp pituitary and Ovopel, Aquac Int, 32, 11–19. brzuska, e and bialowas h (2002) Artificial spawning of carp, Cyprinus carpio (L.), Aqua Res, 33, 753–65. burtsev, i a, nikolaev a i, maltsev s a and igumnova l v (2002) Formation of domesticated broodstocks as a guarantee of sustainable hatchery reproduction of sturgeon for sea ranching, J Appl Ichthyol, 18, 655–8.

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duncan, n j, rodriguez m. de o. g a, alok d and zohar y (2003) Effects of controlled delivery and acute injections of LHRHa on bullseye puffer fish (Sphoeroides annulatus) spawning, Aquaculture, 218, 625–35. fauvel, c, savoye o, dreanno c, cosson j and suquet m (1999) Characteristics of sperm of captive seabass in relation to its fertilization potential, J Fish Biol, 54, 356–69. fauvel, c, suquet m, sévère a, mylonas c c and papandroulakis n (2007) Slowrelease GnRHa therapy prevented atresia during vitellogenesis and induced ovulation of captive wreckfish (Polyprion americanus), Cybium, 32, 191. fontaine, y a, dufour s, tanguy g, khan i a, cedard l and leloup-hatey j (1984) Clairances metaboliques de la gonadotropine chorionique humaine (hCG) et de la gonadotropin de carpe (cGtH) chez un poisson teleosteen, l’anguille, Colloque Physiologie des Poissons, INRA and IFREMER. fontenele, o (1955) Injecting pituitary (hypophyseal) hormones into fish to induce spawning, Prog Fish-Cult, 18, 71–5. garcia, l m b (1991) Spermiation response of mature rabbitfish, Siganus guttatus Bloch, to luteinizing hormone-releasing hormone analogue (LHRHa) injection, Aquaculture, 97, 291–9. gazourian, l, deragon k l, chase c f, pati d, habibi h r and sower s a (1997) Characteristics of GnRH binding in the gonads and effects of lamprey GnRH-I and -III on reproduction in the adult sea lamprey, Gen Comp Endocrinol, 108, 327–39. glubokov, a i, kouril j, mikodina e v and barth t (1994) Effects of synthetic GnRH analogues and dopamine antagonists on the maturation of Pacific mullet, Mugil so-iuy Bas, Aquac Fish Man, 25, 419–25. goren, a, gustafson h and doering d (1995) Field trials demonstrate the efficacy and commercial benefit of a GnRHa implant to control ovulation and spermiation in salmonids, in Goetz F W and Thomas P (eds), Reproductive Physiology of Fish, Austin, TX, Fish Symposium 95, 99–101. gothilf, y, muñoz-cueto j a, sagrillo c a, selmanoff m, chen t t, kah o, elizur a and zohar y (1996) Three forms of gonadotropin-releasing hormone in a perciform fish (Sparus aurata): complementary deoxyribonucleic acid characterization and brain localization, Biol Reprod, 55, 636–45. gothilf, y, meiri i, elizur a and zohar y (1997) Preovulatory changes in the levels of three gonadotropin-releasing hormone-encoding messenger ribonucleic acids (mRNAs), gonadotropin ß-subunit mRNAs, plasma gonadotropin, and steroids in the female gilthead seabream, Sparus aurata, Biol Reprod, 57, 1145–54. gray, s l, adams b a, warby c m, von schalburg k r and sherwood n m (2002) Transcription and translation of the salmon gonadotropin-releasing hormone genes in brain and gonads of sexually maturing rainbow trout (Oncorhynchus mykiss), Biol Reprod, 67, 1621–7. guraya, s s (1986) The cell and molecular biology of fish oogenesis, in Sauer H W (ed.), Monographs in Developmental Biology, Basel, Karger, 1–223. guzmán, j m, norberg b, ramos j, mylonas c c and mañanos e (2008) Vitellogenin, steroid plasma levels and spawning performance of cultured Senegalese sole (Solea senegalensis), Gen Comp Endocrinol, 156, 285–97. habibi, h r, van der kraak g, bulanski e and peter r e (1988) Effect of GnRH on reinitiation of oocyte meiosis in goldfish, in vitro, Am J Physiol, 255, R268–R273. haffray, p, enright w j, driancourt m a, mikolajczyk t, rault p and breton b (2005) Optimization of breeding of salmonids: GonazonTM, the first officially approved inducer of ovulation in the EU, Aquaculture Magazine, March, 52–6. harmin, s a and crim l w (1993) Influence of gonadotropin hormone-releasing hormone analog (GnRH-A) on plasma sex steroid profiles and milt production

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lethimonier, c, madigou t, munoz-cueto j-a, lareyre j-j and kah o (2004) Evolutionary aspects of GnRHa, GnRH neuronal systems and GnRH receptors in teleost fish, Gen Comp Endocrinol, 135, 1–16. levavi-sivan, b, safarian h, rosenfeld h, elizur a and avitan a (2004) Regulation of gonadotropin-releasing hormone (GnRH)-receptor gene expression in tilapia: effect of GnRH and dopamine, Biol Reprod, 70, 1545–51. liley, n r (1983) Hormones and reproductive behaviour in fishes, in Hoar W S, Randall D J and Donaldson E (eds), Fish Physiology, New York, Academic Press. lim, h k, pankhurst n w and fitzgibbon q p (2004) Effects of slow release gonadotropin releasing hormone analog on milt characteristics and plasma levels of gonadal steroids in greenback flounder, Rhombosolea tapirina, Aquaculture, 240, 505–16. lin x-w and peter r e (1996) Expression of salmon gonadotropin-releasing hormone (GnRH) and chicken GnRH-II precursor messenger ribonucleic acids int he brain and ovary of goldfish, Gen Comp Endocrinol, 101, 282–96. ludwig, m, felberbaum r e, diedrich k and lunefeld b (2002) Ovarian stimulation: from basic science to clinical application, Reprod Biomed, Online, 5, 73–86. lund, e d, sullivan c v and place a r (2000) Annual cycle of plasma lipids in captive reared striped bass: effects of environmental conditions and reproductive cycle, Fish Physiol Biochem, 22, 263–75. malison, j a, procarione l s, barry t p, kapuscinski a r and kayes t b (1994) Endocrine and gonadal changes during the annual reproductive cycle of the freshwater teleost, Stizostedion vitreum, Fish Physiol Biochem, 13, 473–84. mañanos, e, carrillo m, sorbera l s, mylonas c c, asturiano j f, bayarri m j, zohar y and zanuy s (2002) Luteinizing hormone and sexual steroid plasma levels after treatment of European sea bass with sustained-release delivery systems for gonadotropin-releasing hormone analogue, J Fish Biol, 60, 328–39. mañanos, e, duncan n and mylonas c c (2008) Reproduction and control of ovulation, spermation and spawning in cultured fish, in Cabrita E, Robles V and Herráez M P, Methods in Reproductive Aquaculture: Marine and Freshwater Species, Taylor and Francis Group LLC, Boca Raton, FL, CRC Press, 1–40. marino, g, panini e, longobardi a, mandich a, finoia m g, zohar y and mylonas c c (2003) Induction of ovulation in captive-reared dusky grouper, Epinephelus marginatus (Lowe, 1834) with a sustained-release GnRHa implant, Aquaculture, 219, 841–58. matsuyama, m, takeuchi h, kashiwagi m, hirose k and kagawa h (1995) Induced gonadal development and spawning of immature red sea bream Pagrus major with LHRH-a administration in different ways during winter season, Fish Sci, 61, 472–7. matsuyama, m, chuda h, ikeda y, tanaka h and matsuura s (1997) Induction of ovarian maturation and ovulation in the cultured tiger puffer Takifugu rubripes by different hormonal treatments, Suisanzoshoku, 45, 67–73. mikolajczyk, t, chyb j, sokolowska-mikolajczyk m, enright w j, epler p, filipiak m and breton b (2003) Attempts to induce an LH surge and ovulation in common carp (Cyprinus carpio L.) by differential application of a potent GnRH analogue, azagly-nafarelin, under laboratory, commercial hatchery, and natural conditions, Aquaculture, 223, 141–57. mikolajczyk, t, chyb j, szczerbik p, sokolowska-mikolajczyk m, epler p, enright w j, filipiak m and breton b (2004) Evaluation of the potency of azagly-nafarelin (GnRH analogue), administered in combination with different formulations of pimozide, on LH secretion, ovulation and egg quality in common carp (Cyprinus

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mylonas, c c, scott a p and zohar y (1997d) Plasma gonadotropin II, sex steroids, and thyroid hormones in wild striped bass (Morone saxatilis) during spermiation and final oocyte maturation, Gen Comp Endocrinol, 108, 223–36. mylonas, c c, woods l c, iii and zohar y (1997e) Cyto-histological examination of post-vitellogenesis and final oocyte maturation in captive-reared striped bass, J Fish Biol, 50, 34–49. mylonas, c c, woods l c, iii, thomas p, schulz r w and zohar y (1998a) Hormone profiles of captive striped bass (Morone saxatilis) during spermiation, and longterm enhancement of milt production, J World Aquac Soc, 29, 379–92. mylonas, c c, woods l c, iii, thomas p and zohar y (1998b) Endocrine profiles of female striped bass (Morone saxatilis) in captivity, during post-vitellogenesis and induction of final oocyte maturation via controlled-release GnRHa-delivery systems, Gen Comp Endocrinol, 110, 276–89. mylonas, c c, papadaki m and divanach p (2003) Seasonal changes in sperm production and quality in the red porgy Pagrus pagrus (L.), Aquac Res, 34, 1161–70. mylonas, c c, kyriakou g, sigelaki i, georgiou g, stephanou d and divanach p (2004a) Reproductive biology of the shi drum (Umbrina cirrosa) in captivity and induction of spawning using GnRHa, Isr J Aquac-Bamidgeh, 56, 75–92. mylonas, c c, papadaki m, pavlidis m and divanach p (2004b) Evaluation of egg production and quality in the Mediterranean red porgy (Pagrus pagrus) during two consecutive spawning seasons, Aquaculture, 232, 637–49. mylonas, c c, papandroulakis n, smboukis a, papadaki m and divanach p (2004c) Induction of spawning of cultured greater amberjack (Seriola dumerili) using GnRHa implants, Aquaculture, 237, 141–54. mylonas, c c, bridges c r, gordin h, belmonte ríos a, garcía a, de la gándara f, fauvel c, suquet m, medina a, papadaki m, heinisch g, de metrio g, corriero a, vassallo-agius r, guzmán j m, mañanos e and zohar y (2007) Preparation and administration of gonadotropin-releasing hormone agonist (GnRHa) implants for the artificial control of reproductive maturation in captive-reared Atlantic bluefin tuna (Thunnus thynnus thynnus), Rev Fish Sci, 15, 183–210. nagahama, y (1994) Endocrine regulation of gametogenesis in fish, Int J Dev Biol, 38, 217–29. nagahama, y, yoshikuni m, yamashita m and tanaka m (1994) Regulation of oocyte maturation in fish, in Sherwood N M and Hew C L (eds), Fish Physiology, San Diego, CA, Academic Press, 393–439. negatu, z, hsiao s m and wallace r a (1998) Effects of insulin-like growth factor-I on final oocyte maturation and steroid production in Fundulus heteroclitus, Fish Physiol Biochem, 19, 13–21. nelson, r j (2006), Fishes of the World, 4th ed, New York, Wiley. ohta, h and tanaka h (1997) Relationship between serum levels of human chorionic gonadotropin (hCG) and 11-ketotestosterone after a single injection of hCG and induced maturity in the male Japanes eel, Anguilla japonica, Aquaculture, 153, 123–34. ohta, h, kagawa h, tanaka h, okuzawa k and hirose k (1996) Milt production in the Japanese eel Anguilla japonica induced by repeated injections of human chorionic gonadotropin, Fish Sci, 62, 44–9. ohta, h, kagawa h, tanaka h, okuzawa k, iimura n and hirose k (1997) Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica, Fish Physiol Biochem, 17, 163–9. ohta, h, miyake h, miura c i, kamei h, aida k and miura t (2007) Follicle-stimulating hormone induces spermatogenesis mediated by androgen production in Japanese eel, Anguilla japonica, Biol Reprod, 77, 970–7.

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5 Producing sterile and single-sex populations of fish for aquaculture T. J. Benfey, University of New Brunswick, Canada

Abstract: Unlike terrestrial livestock, fish are highly amenable to manipulations leading to the production of single-sex and sterile populations, allowing producers to maximize output based on sex-specific characteristics of economic value while also addressing concerns about environmental impacts of fish which may escape from their farms. Induced triploidy is currently the only method available to produce commercial-scale numbers of sterile fish, and it is particularly useful when combined with all-female production. This approach, and the use of endocrine manipulations to generate broodstock capable of yielding all-female diploid offspring, are the most commonly applied manipulations for sex control used in commercial aquaculture. This chapter describes the methodology and rationale for using single-sex and sterile populations in aquaculture. Key words: androgenesis, gynogenesis, monosex, sex reversal, sterilization, triploidy.

5.1 Introduction This chapter addresses the approaches, rationale and limitations for using sterile and single-sex (i.e., all-female or all-male) populations of fish for aquaculture. Unlike traditional terrestrial livestock species, fish are remarkably amenable to such manipulations. The methods used are often very simple and, in some cases, mimic naturally-occurring exceptions to the typical vertebrate pattern of dioecious diploidy (Purdom, 1984; Devlin and Nagahama, 2002). As a result, some of the manipulations described in this chapter are already used in commercial aquaculture. There are three basic aquaculture applications for sterile populations: to prevent feral spawning of escaped farmed fish, to eliminate production losses associated with early sexual maturation and to protect investments made in developing novel genotypes. Single-sex populations can also be

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used to address concerns with the establishment of feral populations if the farmed species is not endemic to the culture location and cannot hybridize with native species. However, they are generally used to take advantage of sex-specific differences in production traits of economic value. For example, research has targeted the production of female populations of halibut (Hippoglossus hippoglossus) because of their faster growth as juveniles (Hendry et al., 2003), anguillid eels (Anguilla spp.) because of their larger ultimate size (Davey and Jellyman, 2005), and sturgeon (Acipenser spp.) and lumpfish (Cyclopterus lumpus) for the production of high-value caviar or cheaper equivalents (Flynn and Benfey, 2007, Martin-Robichaud et al., 1994, respectively).

5.2 Sterile populations The principal driving force currently behind using sterile fish in aquaculture is the mitigation of risks to wild populations associated with the escape of farmed fish. These risks can be divided into direct effects of the escaped individuals themselves (predation, displacement, disease transfer, etc.) as well as indirect effects of interbreeding between wild and farmed fish and/ or the establishment of feral populations of farmed fish. Sterilization addresses only these indirect effects, serving as a method of ‘genetic containment’ of any fish which escape from farms. Clearly, physical containment is the preferred option, both from the farmer’s standpoint and for the sake of protecting wild populations. Thus, sterilization serves as a back-up to effective physical containment. If physical containment could be assured, then there would be no need for sterile fish to address this issue. However, current aquaculture practices rarely allow for complete containment of farmed fish populations. A second important reason for using sterile fish in aquaculture is to prevent pre-harvest sexual maturation. Early maturation of farmed fish raises numerous production concerns for fish farmers because of the considerable energy invested by the fish in gamete production and spawning morphology/behaviour. Maturing fish lose flesh quality as muscle energy reserves are withdrawn for the production of gametes, especially in females. Maturing fish are also chronically stressed and, as a result, have reduced immunocompetence and are more susceptible to disease. Mature males also often show aggressive behaviour. Sterilization can effectively eliminate these problems for the fish farmer. However, they can also often be addressed through selection, as age at maturity is a heritable trait. A number of recent studies have also demonstrated photoperiod manipulation to be an effective method for delaying or suppressing maturation in a variety of fishes including salmonids (Peterson and Harmon, 2005; Unwin et al., 2005), sea bass (Dicentrarchus labrax) (Rodriguez et al., 2005; Felip et al., 2008),

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flatfish (Imsland and Jonassen, 2005; Garcia-Lopez et al., 2006) and gadoids (Davie et al., 2007a, b). Similar results have been achieved in salmonids through periodic reductions in feeding (Silverstein and Shimma, 1994; Duston and Saunders, 1999). Although such environmental manipulations do not render the fish sterile, they are likely more appealing to farmers and consumers as alternatives to the methods outlined below for permanent sterilization. A third reason for using sterile fish is to protect advances made through selection programs and ‘genetic engineering’ (by transgenesis), both of which are long-term, expensive approaches used for genetic improvement. The former is of critical importance to any type of farming, including aquaculture, whereas the latter has yet to be embraced by the fish-farming industry. In either case, having made such long-term investments in producing unique genotypes, there is clearly an interest in ensuring that producers cannot establish independent breeding programs from them. This is a classic problem in agriculture, and can be addressed through licensing agreements that establish breeding rights. However, sterilization can serve as insurance should such agreements not be possible. The fact that alternatives to sterilization exist to address problems associated with escapees, early maturation and the protection of breeding rights explains, at least to some extent, why sterile fish have not found greater use in aquaculture. However, these alternatives are not always adequate, and so there remains considerable interest in the development and use of effective sterilization techniques. Although there are a number of ways in which to sterilize fish, the use of female triploid populations is currently the only accepted method for achieving this on a commercial scale (Dunham, 2004; NRC Committee on Biological Confinement of Genetically Engineered Organisms, 2004; Johnstone, 2005; Devlin et al., 2006a).

5.2.1 Triploidy by direct induction Triploidy induction refers to the creation of individuals with three complete chromosome sets when the norm for that species would be some other number, this generally being two (i.e., diploid). Most vertebrates are diploid, with individuals inheriting one set of chromosomes from their mother and the other from their father. Individuals with an odd number of chromosome sets, such as triploids, are generally sterile, whereas those with an even number, such as diploids and tetraploids, are generally fertile. Triploidy is generally induced by interference with the completion of the second meiotic division, thereby yielding individuals with two sets of maternal chromosomes and one set of paternal chromosomes

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(Pandian and Koteeswaran, 1998). Germ cells (spermatogonia and oogonia) are the only cells which undergo meiotic division, a process necessary for reducing a cell’s chromosome complement by half (typically from diploid to haploid) so that the correct chromosome number (typically diploid) results from fertilization. Meiosis comprises two cellular divisions, and these are completed by the time of spermiation in males, yielding four haploid post-meiotic spermatozoa from each initial diploid premeiotic spermatogonium. The process is somewhat different in oocytes, which eliminate chromosomes through the production of ‘polar bodies’ that are shed as the oocytes develop, ultimately resulting in the production of a single egg from each diploid oogonium. The first meiotic division and polar body production occur early in ovarian development, but the second polar body, containing a single (haploid) set of chromosomes, is retained until after oocytes are fertilized. It is the physical penetration of the oocyte by the fertilizing spermatozoon that stimulates the oocyte’s second meiotic division to go to completion with release of the second polar body. Preventing this from happening results in triploidy (see Fig. 5.1). Thermal and pressure-induced retention of the second polar body has been used extensively for the production of triploid fish (Benfey, 1989; Pandian and Koteeswaran, 1998; Felip et al., 2001). Thermal treatments are relatively easy to apply with inexpensive equipment, but are less easily controlled, i.e., it is difficult to ensure that all eggs within a treatment batch are heated/cooled at the same rate and to the same temperature. The actual treatment temperature used is also dependent on pre-treatment incubation temperature, making it difficult to come up with a consistent, standardized treatment in the absence of adequate control of incubation temperature. These problems are avoided through the use of hydrostatic pressure treatment, which involves treating eggs within a sealed pressure vessel such that all eggs within a treatment batch are exposed to the identical treatment. Furthermore, the optimum pressure treatment is independent of incubation temperature. Systems for the hydrostatic pressure treatment of large numbers of eggs are commercially available and are used routinely by both commercial and government hatcheries for the production of triploids for aquaculture and fisheries management applications (e.g., Abiado et al., 2007). Triploidy induction by hydrostatic pressure treatment is considered to be highly reliable (Johnstone, 1993, 2005; Benfey, 2001). For instance, in an assessment of the aquaculture characteristics of triploid Atlantic salmon (Salmo salar), O’Flynn et al. (1997) applied a standard treatment of 5 minutes exposure to 65 500 kPa, beginning at 300 ºC minutes after fertilization, for triploidy induction in 86 families representing four different research strains of Atlantic salmon spawned over 5 consecutive years, as well as in 3 commercial runs over 2 consecutive years at an industry hatchery. Triploidy induction success was based on screening ten

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Fig. 5 1 Production of diploid (2n), triploid (3n) and tetraploid (4n) embryos. When ovulated, fish eggs are arrested at meiosis II, with one haploid maternal chromosome set (M) destined to become the egg pronucleus and another destined to become the second polar body (Step 1). The fertilizing spermatozoon carries one haploid paternal chromosome set (P). Its entry into the egg results in the completion of meiosis II with extrusion of the haploid second polar body (Step 2). This process is blocked for triploidy induction (X3n). The nuclear membrane then encloses either two (2n zygote) or three (3n zygote) chromosome sets (Step 3). The first cell division is preceded by mitotic duplication of all chromosomes within the zygote (Step 4). Cell division then occurs, giving the 2-cell embryo (Step 5). This process is blocked for tetraploidy induction (X4n). The subsequent mitotic cell division begins with a further duplication of all chromosomes (Step 6) and results in a 4n 2-cell embryo (Step 7).

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randomly selected fish from each family, and in all cases the tested fish were confirmed to be triploid. Although the number of fish tested per family was small, the uniform result across families, strains and year classes speaks to the reliability of this method. Similarly, Devlin et al. (2006b) found that triploidy induction success by hydrostatic pressure treatment in coho salmon (Oncorhynchus kisutch) can routinely exceed 99 %. The principal limitation of thermal/pressure-induced triploidy induction is that treatments are applied directly to the eggs destined to become triploid. This means that there is always the possibility for lower triploidy induction success based on inexperience and/or inadequate control over the key induction variables of pre-treatment duration (which is temperature dependent) and the duration and magnitude of the thermal/pressure treatment itself. Thus, although there is abundant evidence that triploidy induction can work with a high (effectively 100 %) rate of success, there is a need for standard operating procedures and quality assurance programs to ensure routine triploidy induction success. This is especially important for inexperienced users of this technology. Chemical inhibitors of microtubular formation, such as colchicine and cytochalasin B, have been used extensively to produce polyploid plants and bivalves, respectively. However, experiments with these chemicals have never yielded appreciable numbers of polyploid fish (Refstie et al., 1977; Allen and Stanley, 1979; Smith and Lemoine, 1979; Refstie, 1981; Bolla and Refstie, 1985). Exposure to hyperbaric nitrous oxide has shown more promise, with up to 100 % triploidy induction success in both rainbow trout (Oncorhynchus mykiss) (Shelton et al., 1986), and Atlantic salmon (Johnstone et al., 1989), but with reduced survival. This approach has not been further pursued, given the higher triploid yield obtained through hydrostatic pressure treatment (Johnstone et al., 1989). Chemical-induced triploidy suffers the same limitation as thermal and pressure-induced triploidy: treatments are applied directly to the eggs destined to become triploid, thus introducing the possibility of reduced efficacy due to inexperience and/or failure to ensure correct treatment. An additional limitation of this approach is that treating fish with chemicals known to interfere with cell division may affect consumer acceptance of the final product. The direct induction of triploidy is also possible through dispermic fertilization, whereby two spermatozoa enter a single egg to yield triploids with two sets of paternal chromosomes and one set of maternal chromosomes (Pandian and Koteeswaran, 1998). This method of triploidy induction has received far less attention than that described above for polar body retention. Polyethylene glycol and high pH/calcium solutions have been used to fuse spermatozoa prior to fertilization in rainbow trout, resulting

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in up to 33 % triploidy induction success (Ueda et al., 1986, 1988). The remaining eggs were fertilized with single (unfused) spermatozoa, yielding normal diploid offspring. The principal limitation of triploidy induction by dispermic fertilization is the high failure rate of spermatozoan fusion. These treatments have been shown to cause extensive fusion of spermatoan tails (in addition to heads), thereby affecting motility and reducing fertilization success (Araki et al., 1995). With further research it may be possible to develop a more reliable protocol to match the success rate of thermal/ pressure-induced triploidy, although this will be hampered by speciesspecific differences in optimum treatments (Araki et al., 1995). In any case, the same issues of inexperience and/or inadequate treatment control will apply.

5.2.2 Triploidy by tetraploid–diploid crosses As an alternative to the direct induction of triploidy, it is also possible to create triploids indirectly by crossing tetraploid individuals of one sex with diploids of the other (Pandian and Koteeswaran, 1998). Depending on which parent is the tetraploid, the offspring will either have two sets of maternal and one set of paternal chromosomes (tetraploid mother; similar outcome as direct induction of triploidy through retention of the second polar body) or one set of maternal and two sets of paternal chromosomes (tetraploid father; similar outcome as direct induction of triploidy through dispermic fertilization). In either case, this approach requires the direct induction of tetraploidy – rather than triploidy – and the subsequent incorporation of tetraploid broodstock into regular breeding programs. Tetraploidy is induced using the same thermal or hydrostatic pressure treatments employed for triploidy induction, but applied somewhat later after fertilization in order to block the first mitotic cell division of the zygote. Mitosis is a simpler form of cell division than meiosis, involving the duplication of all chromosomes followed by their separation into two chromosome complements of equal number, after which cell division occurs to leave the two resultant cells with one set of chromosomes each. Thus, prior to the first mitotic cell division in a normal diploid zygote, all the chromosomes have been duplicated to give it a tetraploid chromosome number for a short time. This is subsequently reduced to diploid with cell division to yield the twocell diploid embryo. Treatments that block this first mitotic cell division after chromosome duplication has occurred are effective for inducing tetraploidy (see Fig. 5.1). Although tetraploidy has been induced in numerous fish species (Solar et al., 1992; Pandian and Koteeswaran, 1998), their survival is generally very low – much lower than that of induced triploids. This has generally been related to difficulties with getting the treatment timing right to minimize mortality (e.g., Chourrout et al., 1986; Zhang et al., 2005), but there also

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appear to be inherent physiological problems with tetraploids that significantly reduce their viability (Sakao et al., 2003, 2006). However, several studies have demonstrated that it is feasible to create triploid populations of rainbow trout by crossing tetraploids with diploids (Chourrout et al., 1986; Blanc et al., 1987; Chourrout and Nakayama, 1987; Myers and Hershberger, 1991). There are a number of limitations with the use of tetraploid–diploid crosses as an approach for the production of all-triploid populations. Firstly, and critically, they do not generally yield all-triploid populations as predicted. For instance, although crosses between tetraploid male and diploid female rainbow give mostly triploid progeny, they also often yield small numbers of diploids and aneuploids (Chourrout et al., 1986). Similarly, rainbow trout crosses between tetraploid females and diploid males also yield mostly triploid offspring, but with the occasional haploids, diploids and mosaics (Chourrout and Nakayama, 1987). Similar observations have been made with mud loach (Misgurnus mizolepis) (Nam and Kim, 2004) and blunt snout bream (Megalobrama amblycephala) (Zou et al., 2004). There is some evidence from these studies that individual variation exists among tetraploids in their ability to produce triploid offspring, suggesting that careful tetraploid broodstock selection may ensure all-triploid progeny (e.g., Nam and Kim, 2004). However, based on the current level of experience with tetraploids it would be necessary to screen all the offspring from individual crosses to confirm their triploid status. Given this limitation to an otherwise theoretically ideal process for producing triploid populations, it is likely easier to use direct induction of triploidy for which treatment protocols are better defined. A second limitation of using tetraploid crosses with diploids, at least in rainbow trout, is that tetraploid females have delayed maturation (Chourrout et al., 1986) and tetraploid males have reduced fertility (Chourrout et al., 1986; Blanc et al., 1987, 1993; Myers and Hershberger, 1991). This latter effect is apparently because their relatively large spermatozoa – a consequence of increased chromosome number – have difficulty penetrating the micropyle of the egg (Chourrout et al., 1986). The micropyle is a small opening through which spermatozoa must pass to fertilize the egg in teleost fishes, and micropyle diameter is so closely matched to spermatozoan head diameter within a species that it cannot easily accommodate the 30 % increase in head width of spermatozoa from tetraploid males compared to those from diploids (Chourrout et al., 1986). A third limitation of this approach to producing triploids is that presumptive tetraploids may in fact be diploid-tetraploid mosaics. Such mosaics have been observed in several species following treatments designed to block the first mitotic division, including amago salmon (Oncorhynchus masou ishikawai) (Yamaki et al., 1999), rainbow trout

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(Zhang and Onozato, 2004a) and their hybrid (Zhang and Onozato, 2004b). Diploid–tetraploid mosaics have the potential to yield large numbers of diploid offspring (up to 100 %) when crossed to normal diploids (Yamaki et al., 1999; Yamaki and Arai, 2000; Zhang and Onozato, 2004b), and their occurrence in presumptive all-tetraploid populations may account for earlier observations of diploids and aneuploids arising from crosses between tetraploids and diploids (as described above: Chourrout et al., 1986; Chourrout and Nakayama, 1987; Nam and Kim, 2004; Zou et al., 2004). It is likely possible to overcome this limitation through refinement of tetraploidy induction protocols (e.g., Zhang and Onozato, 2004a) and screening of presumptive tetraploid broodstock before their use. However, this screening process would need to go beyond ploidy determination of presumptive tetraploid broodstock by standard techniques such as karyotyping or flow cytometric measurement of DNA content from blood cells because they may miss the existence of a mosaic genotype in the germ line. Rather, it would be necessary to screen the progeny resulting from tetraploid–diploid crosses between individual parents to confirm their all-triploid nature.

5.2.3 Genomic stability of triploids Ensuring the genomic stability of triploid individuals is critical when using induced triploidy as a means of achieving reproductive sterility. The specific concern with this is that triploid individuals do not reduce their chromosome number to diploid, either at the whole animal level or within populations of cells. Reversion to diploidy in the germ cells, whether limited to these cells alone or because of diploid reversion at the whole animal level, circumvents the reproductive sterilization aimed for through triploidy induction. Even if an animal remained triploid in all its somatic tissues, if any of the pre-meiotic germ cells (oogonia or spermatogonia) reverted to diploid they would yield viable haploid gametes as expected from a fertile diploid animal. When considering the possibility of triploid reversion to diploid, it is important to make a distinction between actual reversion from the triploid state to some other chromosomal configuration, as opposed to not having had true triploids in the first place. This latter possibility arises from the various limitations of triploidy induction protocols, and comes down to two possibilities: the direct triploidy induction process is not fully successful and results in a mix of triploid and diploid individuals, or the tetraploidy induction process is not fully successful and leads to the production of some mosaic individuals with the potential to produce diploid offspring. These limitations have been addressed above. The reversion of a truly triploid individual to the diploid state in some or all of its tissues could be envisaged to occur slowly, by the loss of one or a few chromosomes at a time, or quickly, by the loss of an entire

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haploid chromosome set at one time. The former would initially lead to aneuploidy (i.e., any chromosome number other than an exact multiple of haploid), but could ultimately lead to a reduction in chromosome number to a lower euploid state (i.e., an exact multiple of haploid – specifically diploid in this case). At the whole animal level, this would then lead to mosaic individuals, as would be the case if an entire haploid chromosome set was lost outright from individual cells. Such a scenario, which requires errors in, or modifications to, the typical processes of mitotic chromosome separation and cell division, has never been described in artificially produced triploid fish. However, concerns are frequently raised over just such a scenario, apparently based on its repeated observation in the Pacific oyster (Crassostrea gigas), where is has been seen not just in triploids (Allen et al., 1996, 1999), but also in tetraploids (McCombie et al., 2005) and even in diploids (Thiriot-Quiévreux et al., 1992; Zouros et al., 1996; Leitão et al., 2001a, b; Bouilly et al., 2005). Aneuploidy has also been observed in the pearl oyster (Pinctada martensii) (He et al., 2001, 2004). Unlike fish, oysters appear to be highly tolerant of aneuploidy and mosaicism (Wang et al., 1999; He et al., 2004). Given the greater number and longer history of publications on triploid fish compared to shellfish and the fact that reversion to diploidy has never been reported in triploid fish, it appears that it either does not happen or is lethal. Indeed, recent research by Poss et al. (2004) has demonstrated in zebrafish (Danio rerio) that vertebrate cells – and in particular germ cells – are highly sensitive to errors in chromosome separation and cell division that might lead to aneuploidy, with the result that such cells are unable to divide. Although not ‘reversion’ per se, another issue to consider is the fate of any post-meiotic germ cells that might develop in artificiallyproduced triploids. It is important to recognize that (i) artificiallyproduced triploid fish are capable of producing small numbers of post-meiotic gametes (reviewed by Benfey, 1999) and (ii) there are naturally occurring triploid ‘species’ (actually hybrids) which regularly do so because they have evolved atypical meiotic processes that allow them to circumvent triploid sterility and thereby maintain self-sustaining, reproductively viable populations (Purdom, 1984; Devlin and Nagahama, 2002). The production of gametes in artificially-produced triploids is more commonly reported in males than in females, likely for two related reasons (Benfey, 1999). Firstly, male fish typically produce far more gametes than females because spermatozoa are so much smaller than oocytes and gonads of a given size will therefore have far more pre-meiotic spermatogonia than oogonia. Thus, if a certain percentage of pre-meiotic cells are destined to complete meiosis in triploids – as appears to be the case (Benfey, 1999) – firstly, this will translate into much larger numbers of post-meiotic sperma-

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tozoa in males than post-meiotic oocytes in females. Secondly, triploid females do not produce sufficient levels of the ovarian steroid 17β-estradiol that stimulates the synthesis and uptake of yolk proteins by developing oocytes, resulting in slowed or arrested growth of those few oocytes that do progress through meiosis in triploid females (Benfey et al., 1989; Schafhauser-Smith and Benfey, 2001, 2003a, b). Although there have been occasional reports of ovulated eggs from triploid female salmonids, in no case have they yielded viable offspring (Johnstone et al., 1991; Benfey, 1996). The production of sperm (milt) by triploid males is more common (Benfey, 1999) but, as clearly demonstrated in rainbow trout, it comprises aneuploid spermatozoa with a modal DNA content halfway between haploid and diploid (Benfey et al., 1986). When used to fertilize eggs from normal diploid females, the offspring are aneuploid and do not survive (Lincoln and Scott, 1984; Ueda et al., 1987). This confirms the sterile nature of triploid fish, in the true sense of the word: an inability to produce viable offspring. Naturally occurring triploids that maintain self-sustaining populations all involve hybrids between two or more closely related species, and are called ‘allotriploids’ to distinguish them from the within-species artificial triploids (‘autotriploids’) that are the focus of this paper. Hybrid allotriploids invariably exist as all-female populations and rely on males of one of their progenitor species to provide sperm for the activation of development in their eggs (Vrijenhoek, 1994; Devlin and Nagahama, 2002). Maintaining reproductively viable populations of allotriploids is only possible through the evolution of atypical patterns of meiosis and fertilization that have never been observed in autotriploids.

5.2.4 Alternatives to triploidy for sterilization Detailed descriptions of the variety of alternatives to triploidy for producing sterile fish can be found elsewhere (e.g., Devlin and Donaldson, 1992; Maclean and Laight, 2000; Wong and Van Eenennaam, 2008), and are therefore only reviewed briefly. Surgical castration is probably the earliest technique to have been developed for sterilizing fish. It can be highly effective if no gonadal tissue remains after surgery. However, ensuring the complete removal of the gonads is difficult, especially when working with smaller fish. In any case, it is inconceivable that surgical castration can applied on a commercial scale because of the amount of time and handling involved in the procedure. High energy radiation (gamma- and X-ray) is well documented to have sterilizing effects on a wide range of organisms, including fish. However, permanent sterilization is difficult to achieve. Furthermore, the equipment used is heavy and cumbersome, and is therefore not amenable to transport

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to farm sites. Operator and environmental safety are clear concerns when using high energy radiation sources, and consumer acceptability of the final product is questionable. This technique is therefore not seen as suitable for commercial aquaculture. The use of chemicals, immunological manipulations or transgenic techniques to act upon the hypothalamic–pituitary–gonadal axis are approaches more likely to work on a commercial scale than surgical castration or high energy radiation, but as yet no effective treatments for permanent sterilization have been developed along these lines for any aquaculture species. Any approach which interferes with the production or release of gonadotropin releasing hormone (hypothalamus) or gonadotropins (pituitary) is potentially effective for ensuring sterilization. Unlike triploidy and most other sterilization approaches, one could envisage this approach as being reversible, allowing the breeding of top performing sterile fish by supplementation of the missing hormone(s) as a form of replacement therapy. This is a field of research worthy of pursuit for developing novel techniques for sterilizing fish. Lastly, androgen administration, through immersion or feeding, is well documented to be an inexpensive and effective method for the permanent sterilization of fish. It is easily applied on a commercial scale, although care must be taken to protect farm employees and the environment from exposure to high steroid doses. The steroid treatments are typically completed one or more years prior to fish reaching market size, by which time residual steroid levels have become non-detectable (Pandian and Kirankumar, 2003). The only apparent reason that this technique is not used for commercial production is because of concerns about consumer acceptance of a steroid-treated product, and this limitation is not likely to change.

5.3 Single-sex populations From a strictly production standpoint, there are often clear advantages of one sex over the other for commercial aquaculture. For most species of fish it is impossible to separate males and females as juveniles, and in many species this is even impossible as sexually mature adults. However, there are a number of different approaches that can be used to generate singlesex populations through simple genetic, endocrine or environmental manipulations.

5.3.1 Uniparental inheritance The activation of embryonic development with no contribution of the paternal genome (gynogenesis) or the maternal genome (androgenesis) is

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a simple genetic manipulation that can be used for the production of singlesex populations (Pandian and Koteeswaran, 1998; Felip et al., 2001; Komen and Thorgaard, 2007). Gynogenesis is usually accomplished by modifying or destroying the spermatozoan genome without affecting the motility of the spermatozoa and hence their ability to penetrate the egg and activate embryonic development. This is generally done using UV-irradiation, but is also possible with high energy radiation or certain chemicals. In some cases, the use of sperm from a different species has the same effect. Penetration of the treated spermatozoon into the egg stimulates the resumption of meiosis, which ends with extrusion of the haploid second polar body. The result is a haploid zygote with its genomic DNA derived solely from the egg’s pronucleus. Diploidy can be restored by using thermal or hydrostatic pressure treatments to either block extrusion of the second polar body itself, or by allowing this to occur but then blocking the first mitotic cell division after the chromosome complement has been duplicated. These treatments are identical to those used to induce triploidy or tetraploidy, respectively, after fertilization with intact spermatozoa. In this case, however, the resulting zygote has a normal diploid chromosome number but with all chromosomes inherited from the mother. Androgenesis works in the same way as gynogenesis, but requires irradiation of the eggs rather than the sperm, followed by fertilization with intact spermatozoa. Ordinarily this would yield androgenetic haploids, but again if the first mitotic cell division is blocked after pre-mitotic duplication of the paternal genome, then the result is androgenetic diploids. Considerably more research has been conducted on gynogenesis than on androgenesis in fish, principally because it is easier to effectively irradiate sperm than eggs. Gynogenesis can be used to create all-female populations but cannot yield all-male populations. For instance, in species having female homogamety, equivalent to the mammalian XX-female/XY-male system, exclusion of the paternal genome and duplication of the maternal genome through gynogenesis should result in all-female populations. This is well established for salmonid fishes (Ihssen et al., 1990) and has also been demonstrated for Atlantic halibut (Tvedt et al., 2006). In species with female heterogamety, equivalent to the avian WZ-female/ZZ-male system, gynogenesis yields populations comprising mostly normal males and ‘super-females’ (WW genotype), with the possibility of smaller number of normal females. Sex ratios recently obtained from gynogenetic populations of shortnose sturgeon (Acipenser brevirostrum) suggest that this species has male homogamety (Flynn et al., 2006). The creation of all-female populations in such cases requires the mating of normal males with super-females. Conversely, androgenesis results in all-male populations in species with male homogamety (WZ/ZZ-system) but cannot be used to make all-female populations in such species. For species

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having male heterogamety (XX/XY-system), androgenesis yields populations comprising mostly normal females and ‘super-males’ (YY genotype), with the possibility of smaller numbers of normal males. All-male populations can then be obtained by crossing normal females with super-males. Perpetuating all-female and all-male lines using such genetic manipulations is facilitated by endocrine manipulations. In species with female homogamety, functional sex reversal of gynogenetic females yields phenotypic males capable of generating all-female offspring when crossed with normal females. Similarly, in species with male homogamety, functional sex reversal of gynogenetic males yields phenotypic females capable of generating all-male offspring when crossed with normal males. Further details on the methodology for endocrine sex reversal are provided in Section 5.3.2. Uniparental inheritance results in reduced heterozygosity, an important consideration when designing breeding programs that incorporate gynogenetic or androgenetic fish. The pathways described above also assume simple (single locus) genetic control of sex determination, for which many exceptions exist among fishes (Devlin and Nagahama, 2002). Furthermore, because most fish lack discrete sex chromosomes or even much in the way of distinct sex-specific genes, the predicted sex ratio outcomes are not always realized due to segregation of recessive mutations affecting sex determination (Komen and Thorgaard, 2007). Thus, although uniparental inheritance is useful for determining the genetic basis to sex determination (e.g., Flynn et al., 2006; Tvedt et al., 2006; Komen and Thorgaard, 2007), it is not generally used for the direct production of single-sex populations of fish.

5.3.2 Endocrine sex reversal Although sex is ultimately under genetic control and is often determined – and fixed – at fertilization, the phenotypic expression of sex (i.e., gonadal differentiation into ovaries or testes) is mediated by sex steroids. Thus, estrogens and androgens have feminizing and masculinizing effects, respectively. These hormones are the natural ‘sex inducers’ in vertebrates, and their exogenous administration during gonadal differentiation can be used to change sex in fish (Devlin and Nagahama, 2002). Successful endocrine sex reversal therefore requires knowledge of the timing of gonadal differentiation. In some species this occurs prior to yolk absorption, requiring the immersion of embryos in steroid solutions. In species where gonadal differentiation occurs later in life, these steroids can be administered through the food, either via live prey such as steroid-enriched Artemia (e.g., lumpfish; Martin-Robichaud et al., 1994) or in prepared feeds (e.g., Atlantic halibut; Hendry et al., 2003).

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Despite the fact that it is possible to achieve ‘direct’ feminization or masculinization in this way, the marketability of such fish for human consumption is affected by their exposure to steroids (Pandian and Kirankumar, 2003). The preferred method for aquaculture applications is therefore ‘indirect’ feminization or masculinization. For instance, the appropriate administration of androgens to salmonid fishes results in all-male populations. If the sex-reversed genotypic females (‘neo-males’) within such populations are subsequently mated to normal females, all the resulting offspring are female but have not themselves been exposed to steroids. This method for the production of all-female populations for aquaculture is currently used for salmonid species such as rainbow trout and chinook salmon (Oncorhyncus tshawytscha) (Devlin and Nagahama, 2002), as well as for Atlantic halibut (Scotian Halibut Ltd, Nova Scotia, Canada, unpublished).

5.3.3 Environmental manipulation of sex ratio All steroid hormones are derived from cholesterol, with their biosynthesis controlled by specific steroidogenic enzymes. Chemical inhibition of such enzymes has been used to create single-sex populations of fish (Piferrer, 2001). Given that enzyme activity is sensitive to physical (e.g., temperature) and chemical (e.g., pH) conditions, it should also be possible to manipulate sex ratios in fish by manipulating the expression and/or activity of steroidogenic enzymes (Baroiller et al., 1999). The best candidate for such environmental manipulations is temperature, given the well documented effects of egg incubation temperature on sex ratios in reptiles and some species of fish. Such a protocol has obvious attractiveness for aquaculture because it represents a more consumer-friendly approach, given the absence of genetic manipulation or use of steroid hormones. Sex ratios have been altered by simple temperature manipulations in numerous aquaculture species, including Nile tilapia (Oreochromis niloticus) (Tessema et al., 2006), goldfish (Carassius auratus) (Goto-Kazeto et al., 2006) and several flatfish species (Goto et al., 1999, 2000; Yamamoto, 1999; Luckenbach et al., 2003), although not Atlantic halibut (Hughes et al., 2008). The number of fish species demonstrating temperature-sensitive sexual differentiation under experimental conditions is growing rapidly, and this may become a common approach to producing sex-biased populations for aquaculture.

5.4 Future trends and further reading As aquaculture continues to grow in importance as a source of high-quality food, there will be a greater focus on developing technologies to improve

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production efficiencies. Although aquaculture is rightly seen as an aquatic extension of terrestrial agriculture, there are some unique attributes of the animals farmed in aquaculture compared to more familiar terrestrial livestock because the latter focuses on mammals and birds, whereas in aquaculture the focus is on fish and invertebrates. The terrestrial paradigm of what is possible with respect to sex control does not apply to these aquaculture species. Given the ease with which single-sex and sterile populations of fish can be produced using simple manipulations that are not likely to be criticized by consumers, and in fact have added benefits to mitigating aquaculture impacts on the environment, it is likely that there will be widespread use of such populations in the future. The key is to not have one’s imagination constrained by what is (and is not) possible to do when farming mammals and birds. Given the fundamental differences between triploids and diploids with respect to genome size, gene dosage, cell size, etc. (Benfey, 1999), it is not surprising that triploid performance in aquaculture is not necessarily the same as for diploids. Triploid growth is, at best, generally no better than that of diploids (Benfey, 1999; Maxime, 2008), and they are less tolerant of chronic exposure to high temperature (Ojolick et al., 1995; Hyndman et al., 2003). Current research focuses on determining the limitations and best culture conditions for triploids, with respect to providing the optimum environment for letting them realize their potential (e.g., Atkins and Benfey, 2008). Through such research it should become possible to increase greatly the use of triploids in commercial aquaculture without affecting production efficiencies. At the same time, it is important to continue searching for effective alternatives to triploidy for providing sterile populations. Relevant advances made in other fields should be pursued for their applicability to aquaculture. For instance, the recent discovery of the key role of kisspeptin and its receptor (GPR54) in controlling puberty through the regulation of gonadotropin releasing hormone secretion in mammals (Dungan et al., 2006) should be investigated as a likely site for intervention for the control of gonadal development in fishes. With respect to the production of single-sex populations, the rapid increase in the number of species demonstrated to have thermal-labile sex determination – at least under culture conditions if not in nature – opens the door for widespread use of simple temperature manipulation at key stages of the life cycle as a way of altering sex ratio to the benefit of fish farmers. Advances in molecular biology will facilitate the integration of such manipulations into traditional breeding programs. For further reading on the topics covered in this chapter, the reader is recommended the comprehensive reviews by Pandian and Koteeswaran (1998), Benfey (1999), Felip et al., (2001), Devlin and Nagahama (2002), Koman and Thorgaard (2007) and Maxime (2008).

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5.5 References abiado m a g, penn m, dabrowski k, stafford j (2007) Evaluation of two commercially available pressure chambers to induce triploidy in saugeyes, N Am J Aquac, 69, 197–201. allen jr s k, stanley j g (1979) Polyploid mosaics induced by cytochalasin B in landlocked Atlantic salmon Salmo salar, Trans Am Fish Soc, 108, 462–6. allen jr s k, guo x, burreson g, mann r (1996) Heteroploid mosaics and reversion among triploid oysters, Crassostrea gigas. Fact or artifact, J Shellfish Res, 15, 514 (abstract). allen jr s k, howe a, gallivan t, guo x, debrosse g (1999) Genotype and environmental variation in reversion of triploid Crassostrea gigas to the heteroploid mosaics state, J Shellfish Res, 18, 293 (abstract). araki k, shinma h, nagoya h, nakayama i, onozato, h (1995) Androgenetic diploids of rainbow trout (Oncorhynchus mykiss) produced by fused sperm, Can J Fish Aquat Sci, 52, 892–6. atkins m e, benfey t j (2008) Effect of temperature on routine metabolic rate in triploid salmonids, Comp Biochem Physiol, 149A, 157–61. baroiller j-f, guiguen y, fostier a (1999) Endocrine and environmental aspects of sex differentiation in fish, Cell Mol Life Sci, 55, 910–31. benfey t j (1989) A bibliography of triploid fish, 1943 to 1988, Can Tech Rep Fish Aquat Sci, 1682, 1–33. benfey t j (1996) Ovarian development in triploid brook trout (Salvelinus fontinalis), in Goetz F W and Thomas P (eds), Proceedings of the 5th International Symposium on the Reproductive Physiology of Fish, Austin, TX, Fish Symposium, 95, 357. benfey t j (1999) The physiology and behavior of triploid fishes, Rev Fish Sci, 7, 39–67. benfey t j (2001) Use of sterile triploid Atlantic salmon (Salmo salar L.) for aquaculture in New Brunswick, Canada, ICES J Mar Sci, 58, 525–9. benfey t j, solar i i, de jong g, donaldson e m (1986) Flow-cytometric confirmation of aneuploidy in sperm from triploid rainbow trout, Trans Am Fish Soc, 115, 838–40. benfey t j, dye h m, solar i i, donaldson e m (1989) The growth and reproductive endocrinology of adult triploid Pacific salmonids, Fish Physiol Biochem, 6, 113–20. blanc j-m, chourrout d, kreig f (1987) Evaluation of juvenile rainbow trout survival and growth in half-sib families from diploid and tetraploid sires, Aquaculture, 65, 215–20. blanc j-m, poisson h, escaffre a m, aguirre p, vallée f (1993) Inheritance of fertilizing ability in male tetraploid rainbow trout (Oncorhynchus mykiss), Aquaculture, 110, 61–70. bolla s, refstie t (1985) Effect of cytochalasin B on eggs of Atlantic salmon and rainbow trout, Acta Zool (Stockholm), 66, 181–8. bouilly k, leitão a, chaves r, guedes-pinto h, boudry p, lapègue s (2005) Endonuclease banding reveals that atrazine induced aneuploidy resembles spontaneous chromosome loss in Crassostrea gigas, Genome, 48, 177–80. chourrout d, nakayama i (1987) Chromosome studies of progenies of tetraploid female rainbow trout, Theor Appl Genet, 74, 687–92. chourrout d, chevassus b, krieg f, happe a, burger g, renard p (1986) Production of second generation triploid and tetraploid rainbow trout by mating tetraploid males and diploid females – potential of tetraploid fish, Theor Appl Genet, 72, 193–206.

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davey ajh, jellyman d j (2005) Sex determination in freshwater eels and management options for manipulation of sex, Rev Fish Biol Fish, 15, 37–52. davie a, mazorra de quero c, bromage n, treasurer j, migaud h (2007a) Inhibition of sexual maturation in tank reared haddock (Melanogrammus aeglefinus) through the use of constant light photoperiods, Aquaculture, 270, 379–89. davie a, porter mjr, bromage n r, migaud h (2007b) The role of seasonally altering photoperiod in regulating physiology in Atlantic cod (Gadus morhua). Part I. Sexual maturation, Can J Fish Aquat Sci, 64, 84–97. devlin r h, donaldson, e m (1992) Containment of genetically altered fish with emphasis on salmonids, in Hew C L and Fletcher G L (eds), Transgenic Fish, Singapore, World Scientific, 229–66. devlin r h, nagahama y (2002) Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences, Aquaculture, 208, 191–364. devlin r h, sundström l f, muir w m (2006a) Interface of biotechnology and ecology for environmental risk assessments of transenic fish, Trends Biotech, 24, 89–97. devlin r h, biagi c a, sakhrani d, eom k-w (2006b) Assessment of pressure-shock induced triploidy for containment of transgenic salmon, in MacKinlay C and Busby C (eds), Book of Abstracts, 7th International Congress on the Biology of Fish, St. John’s, Newfoundland, Bethesda, MD, American Fisheries Society, 47. dungan h m, clifton d k, steiner r a (2006) Kisspeptin neurons as central processors in the regulation of gonadotropin-releasing hormone secretion, Endocrinol, 147, 1154–8. dunham r a (2004) Aquaculture and Fisheries Biotechnology: Genetic Approaches, Cambridge, MA, CABI. duston j, saunders r l (1999) Effect of winter food deprivation on growth and sexual maturity of Atlantic salmon (Salmo salar) in sea water, Can J Aquat Sci, 56, 201–7. felip a, zanuy s, piferrer f (2001) Induction of triploidy and gynogenesis in teleost fish with emphasis on marine species, Genetica, 111, 175–95. felip a, zanuy s, muriach b, cerda-reverter j m, carrillo m (2008) Reduction of sexual maturation in male Dicentrarchus labrax by continuous light both before and during gametogenesis, Aquaculture, 275, 347–55. flynn s r, benfey t j (2007) Sex differentiation and aspects of gametogenesis in shortnose sturgeon, Acipenser brevirostrum, Lesuere, J Fish Biol, 70, 1027–44. flynn s r, matsuoka m, reith m, martin-robichaud d j, benfey t j (2006) Gynogenesis and sex determination in shortnose sturgeon, Acipenser brevirostrum, LeSuere, Aquaculture, 253, 721–7. garcia-lopez a, pascual e, sarasquete c, martinez-rodriguez g (2006) Disruption of gonadal maturation in cultured Senegalese sole Solea senegalensis Kaup by continuous light and/or constant temperature regimes, Aquaculture, 261, 789–98. goto r, mori t, kawamata k, matsubara t, mizuno s, adachi s, yamauchi k (1999) Effects of temperature on gonadal sex determination in barfin flounder Verasper moseri, Fish Sci, 65, 884–7. goto r, kayaba t, adachi s, yamauchi k (2000) Effects of temperature on sex determination in marbled sole Limanda yokohamae, Fish Sci, 66, 400–2. goto-kazeto r, abe y, masai k, yamaha e, goto-kazeto r, abe y, masai k, yamaha e, adachi s, yamauchi k (2006) Temperature-dependent sex differentiation in

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goldfish: Establishing the temperature-sensitive period and effect of constant and fluctuating water temperatures, Aquaculture, 254, 617–24. he m, lin y, shen q, hu j, jiang w (2001) Production of aneuploid Pinctada martensii Dunker in tetraploid, Mar Sci Bull, 3, 63–8. he m, jiang w, huang l (2004) Studies on aneuploid pearl oyster (Pinctada martensii Dunker) produced by crossing triploid females and a diploid male following the inhibition of PB1, Aquaculture, 230, 117–24. hendry c i, martin-robichaud d j, benfey t j (2003) Hormonal sex reversal of Atlantic halibut (Hippoglossus hippoglossus), Aquaculture, 219, 769– 81. hughes v, benfey t j, martin-robichaud d j (2008) Effect of rearing temperature on sex ratio in juvenile Atlantic halibut, Hippoglossus hippoglossus, Env Biol Fish, 81, 415–19. hyndman c a, kieffer j d, benfey t j (2003) Physiology and survival of triploid brook trout following exhaustive exercise in warm water, Aquaculture, 221, 629–43. ihssen p e, mckay l r, mcmillan i, phillips r b (1990) Ploidy manipulation and gynogenesis in fishes: cytogenetic and fisheries applications, Trans Am Fish Soc, 119, 698–717. imsland a k, jonassen t m (2005) The relation between age at first maturity and growth in Atlantic halibut (Hippoglossus hippoglossus) reared at four different light regimes, Aquac Res, 36, 1–7. johnstone r (1993) Maturity control in Atlantic salmon, in Muir J F and Roberts R J (eds), Recent Advances in Aquaculture IV, Oxford, Blackwell Scientific, 99–105. johnstone r (2005) An overview of methods of control of maturation in salmonids, in Wild and Farmed Salmon – Working Together, Edinburgh, North Atlantic Salmon Conservation Organization, 32–6. johnstone r, knott r m, macdonald a g, walsingham m v (1989) Triploidy induction in recently fertilized Atlantic salmon ova using anaesthetics, Aquaculture, 78, 229–36. johnstone r, mclay h a, walsingham m v (1991) Production and performance of triploid Atlantic salmon in Scotland, Can Tech Rep Fish Aquat Sci, 1789, 15–36. komen h, thorgaard g h (2007) Androgenesis, gynogenesis and the production of clones in fishes: A review, Aquaculture, 269, 150–73. leitão a, boudry p, mccombie h, gérard a, thiriot-quiévreux c (2001a), Experimental evidence for a genetic basis to differences in aneuploidy in the Pacific oyster (Crassostrea gigas), Aquat Living Resour, 14, 233–7. leitão a, boudry p, thiriot-quiévreux c (2001b) Evidence of differential chromosome loss in aneuploid karyotypes of the Pacific oyster, Crassostrea gigas, Genome, 44, 735–7. lincoln r f, scott a p (1984) Sexual maturation in triploid rainbow trout, Salmo gairdneri Richardson, J Fish Biol, 25, 385–92. luckenbach j a, godwin j, daniels h v, borski r j (2003) Gonadal differentiation and effects of temperature on sex determination in southern flounder (Paralichthys lethostigma), Aquaculture, 216, 315–27. maclean n, laight r j (2000) Transgenic fish: An evaluation of benefits and risks, Fish Fish, 1, 146–72. martin-robichaud d j, peterson r h, benfey t j, crim l w (1994) Direct feminization of lumpfish (Cyclopterus lumpus L.) using 17ß-oestradiol-enriched Artemia as food, Aquaculture, 123, 137–51. maxime v (2008) The physiology of triploid fish: current knowledge and comparisons with diploid fish, Fish Fish, 9, 67–78.

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mccombie h, lapègue s, cornette f, ledu c, boudry p (2005) Chromosome loss in bi-parental progenies of tetraploid Pacific oyster Crassostrea gigas, Aquaculture, 247, 97–105. myers j m, hershberger w k (1991) Early growth and survival of heat-shocked and tetraploid-derived triploid rainbow trout (Oncorhynchus mykiss), Aquaculture, 96, 97–107. nam y k, kim d s (2004) Ploidy status of progeny from the crosses between tetraploid males and diploid females in mud loach (Misgurnus mizolepis), Aquaculture, 236, 575–82. nrc committee on biological confinement of genetically engineered organisms (2004), Biological Confinement of Genetically Engineered Organisms, Washington, DC, National Academies Press. o’flynn f m, mcgeachy s a, friars g w, benfey t j, bailey j k (1997) Comparisons of cultured triploid and diploid Atlantic salmon (Salmo salar L.), ICES J Mar Sci, 54, 1160–5. ojolick e j, cusack r, benfey t j, kerr s r (1995) Survival and growth of all-female diploid and triploid rainbow trout (Oncorhynchus mykiss) reared at chronic high temperature, Aquaculture, 131, 177–87. pandian t j, kirankumar s (2003) Recent advances in hormonal induction of sexreversal in fish, J Appl Aquac, 13, 205–30. pandian t j, koteeswaran r (1998) Ploidy induction and sex control in fish, Hydrobiologia, 384, 167–243. peterson r h, harmon p r (2005) Changes in condition factor and gonadosomatic index in maturing and non-maturing Atlantic salmon (Salmo salar L.) in Bay of Fundy sea cages, and the effectiveness of photoperiod manipulation in reducing early maturation, Aquact Res, 36, 882–9. piferrer f (2001) Endocrine sex control strategies for the feminisation of teleost fish, Aquaculture, 197, 229–81. poss k d, nechiporuk a, stringer k f, lee c, keating m t (2004) Germ cell aneuploidy in zebrafish with mutations in the mitotic checkpoint gene mps1, Genes Develop, 18, 1527–32. purdom c e (1984) Atypical modes of reproduction in fish, in Clarke J R (ed.), Oxford Reviews of Reproductive Biology, Oxford, Oxford University Press, 303–40. refstie t (1981) Tetraploid rainbow trout produced by cytochalasin B, Aquaculture, 25, 51–8. refstie t, vassvik v, gjedrem t (1977) Induction of polyploidy in salmonids by cytochalasin B, Aquaculture, 10, 65–74. rodriguez l, begtashi i, zanuy s, carrillo m (2005) Long-term exposure to continuous light inhibits precocity in European male sea bass (Dicentrarchus labrax L.): hormonal aspects, Gen Comp Endocrinol, 140, 116–25. sakao s, fujimoto t, tanaka m, yamaha e, arai k (2003) Aberrant and arrested embryos from masu salmon eggs treated for tetraploidization by inhibition of the first cleavage, Nipp Suis Gakk, 69, 738–48. sakao s, fujimoto t, kimura s, yamaha e, arai k (2006) Drastic mortality in tetraploid induction results from the elevation of ploidy in masu salmon, Oncorhynchus masou, Aquaculture, 252, 147–60. schafhauser-smith d, benfey t j (2001) The reproductive physiology of three age classes of adult female diploid and triploid brook trout (Salvelinus fontinalis), Fish Physiol Biochem, 25, 319–33. schafhauser-smith d, benfey t j (2003a) In vitro steroid production by triploid ovarian follicles, Gen Comp Endocrinol, 133, 279–86.

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schafhauser-smith d, benfey t j (2003b) The effects of long-term estradiol-17β treatment on the growth and physiology of triploid brook trout (Salvelinus fontinalis), Gen Comp Endocrinol, 131, 9–20. shelton c j, macdonald a g, johnstone r (1986) Induction of triploidy in rainbow trout using nitrous oxide, Aquaculture, 58, 155–9. silverstein j t, shimma h (1994) Effect of restricted feeding on early maturation in female and male amago salmon, Oncorhynchus masou ishikawae, J Fish Biol, 45, 1133–5. smith l t, lemoine h l (1979) Colchicine-induced polyploidy in brook trout, Prog Fish-Cult, 41, 86–8. solar i i, hajen w e, donaldson e m (1992) A bibliography of tetraploidy in fish (1964–1991), Can Tech Rep Fish Aquat Sci, 1901, 1–22. tessema m, muller-belecke a, horstgen-schwark g (2006) Effect of rearing temperatures on the sex ratios of Oreochromis niloticus populations, Aquaculture, 258, 270–7. thiriot-quiévreux c, pogson g h, zouros e (1992) Genetics of growth rate variation in bivalves: aneuploidy and heterozygosity effects in a Crassostrea gigas family, Genome, 35, 39–45. tvedt h b, benfey t j, martin-robichaud d j, mcgowan c, reith m (2006) Gynogenesis and sex determination in Atlantic halibut (Hippoglossus hippoglossus), Aquaculture, 252, 573–83. ueda t, kobayashi m, sato r (1986) Triploid rainbow trouts induced by polyethylene glycol, Proc Jap Acad Ser B, 62, 161–4. ueda t, sawada m, kobayashi j (1987) Cytogenetical characteristics of the embryos between diploid female and triploid male in rainbow trout, Jap J Genet, 62, 461–5. ueda t, sato r, kobayashi j (1988) Triploid rainbow trout induced by high-pH-highcalcium, Nipp Suis Gakk, 54, 2045. unwin m j, rowe d k, poortenaar c w, boustead n c (2005) Suppression of maturation in 2-year-old Chinook salmon (Oncorhynchus tshawytscha) reared under continuous photoperiod, Aquaculture, 246, 239–50. vrijenhoek r c (1994) Unisexual fish: model systems for studying ecology and evolution, Ann Rev Ecol Syst, 25, 71–96. wang z, guo x, allen jr s k, wang r (1999) Aneuploid Pacific oyster (Crassostrea gigas Thunberg) as incidentals from triploid production, Aquaculture, 173, 347–57. wong a c, van eenennaam a l (2008) Transgenic approaches for the reproductive containment of genetically engineered fish, Aquaculture, 275, 1–12. yamaki m, arai k (2000) Ploidies of gametes produced by putative tetraploid amago salmon induced by inhibition of the first cleavage, Bull Fac Fish Hokkaido Univ, 51(3), 135–52. yamaki m, satou h, taniura k, arai k (1999) Progeny of the diploid-tetraploid mosaic amago salmon, Nipp Suis Gakk, 65, 1084–9. yamamoto e (1999) Studies on sex-manipulation and production of cloned populations in hirame, Paralichthys olivaceus (Temminck et Schlegel), Aquaculture, 173, 235–46. zhang x, onozato h (2004a) Hydrostatic pressure treatment during the first mitosis does not suppress the first cleavage but the second one, Aquaculture, 240, 101–13. zhang x, onozato h (2004b) Allo-eudiploidy of the diploid cells in diploid-tetraploid mosaic hybrids between female rainbow trout Oncorhynchus mykiss and male amago salmon O. rhodurus, Fish Sci, 70, 924–6.

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6 Chromosome set manipulation in shellfish X. Guo, Y. Wang, Z. Xu, Rutgers University, USA, and H. Yang, Louisiana State University Agriculture Center, USA

Abstract: Chromosome set manipulation may produce phenotypic changes that are useful in aquaculture. Chromosome set manipulation in shellfish has led to the production of polyploid, gynogenetic and aneuploid animals. While studies on gynogenetic and aneuploid shellfish have been largely academic, research on polyploids has made significant contributions to shellfish aquaculture. Triploids have been produced and evaluated in many species. Triploid shellfish grow significantly faster than diploids in most species studied so far. Their sterility often results in improved meat quality and provides biological containment of cultured stocks. Triploids have become an important part of the oyster farming industry and may have similar potential in other shellfish. Tetraploids have been successfully produced and used for triploid production in oysters. Tetraploid induction in other species remains a major challenge that hinders commercial production of triploids. Key words: triploidy, tetraploidy, gynogenesis, sterility, shellfish, genome adaptation.

6.1 Introduction Shellfish are major aquaculture species. Shellfish species account for more than one third of the world total aquaculture production (FAO, 2007). Common aquaculture species include oysters, clams, scallops, mussels, abalone, shrimp, crawfish and crabs. They support major aquaculture industries in many countries. Various efforts have been made to genetically improve the performance of cultured shellfish. Selective breeding has been used to produce superior strains (see Chapter 3). Increasingly, biotechnical approaches are being applied to the genetic improvement of shellfish stocks (Guo, 2004).

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Chromosome set manipulation can be used to create new chromosome constructs that are potentially useful for aquaculture. Most animals including shellfish possess two sets of chromosomes with one set from the mother and the other from the father, a condition known as diploidy. We can alter the normal process of chromosome inheritance and create a range of variations in chromosome number and composition. Variations in chromosome number may involve partial or complete sets of chromosomes. The condition where a cell or organism has incomplete sets of chromosomes is referred to as aneuploidy, and the condition of having complete sets of chromosomes is called euploidy. Euploid conditions include normal diploidy with two sets of chromosomes, as well as haploidy with one and polyploidy with three or more sets of chromosomes. Triploidy and tetraploidy are common polyploid conditions with three and four sets of chromosomes, respectively. In addition to changes in chromosome number, the parental origin of chromosomes can also be changed. Uniparental inheritance can be obtained by excluding chromosomes from one of the parents. Uniparental inheritance based on maternal and paternal chromosomes is referred to as gynogenesis and androgenesis, respectively. The diploid chromosome complement of an organism represents rigid genome organization derived from millions of years of evolution and adaptation. Chromosome number within species or genera is usually conserved. For example, all oysters have a diploid number of 20 chromosomes, and most clams and scallops have a diploid number of 38 (Thiriot-Quiévreux, 2002; Wang and Guo, 2004; Wang et al., 2004). Changes in chromosome number are serious mutations that may have profound consequences. Chromosome mutations can be lethal or result in significant changes in phenotype, but some changes in phenotype can be beneficial for aquaculture. Triploid shellfish, for example, may be sterile and grow faster than normal diploids (Nell, 2002; Guo, 2004). Gynogenesis and androgenesis can potentially be used for the rapid production of inbred lines and for sex control. Because of these and other potential benefits, chromosome set manipulation has been investigated in many shellfish species. This chapter introduces basic principles and methods of chromosome set manipulation, examines available data and provides perspectives for future research and development.

6.2 Principles and methods of chromosome set manipulation 6.2.1 Reproduction in shellfish Most shellfish species are dioecious and reproduce sexually. Sexual dimorphism is evident in most crustaceans and some molluscs. In crustaceans and gastropods, sexual dimorphism is often expressed as differences in sexual

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organs and body size (Landau, 1992). In bivalve molluscs such as scallops and clams, female and male gonads may exhibit different colours. In the Chinese scallop Chlamys farreri, Japanese scallop Patinopecten yessoensis, Atlantic sea scallop Placopecten magellanicus, and dwarf surfclam Mulinia lateralis, female gonads are usually orange to red, while male gonads are milky white, and this ability to identify sex is advantageous in chromosome set manipulation because early and accurate separation of sexes can reduce the chance of uncontrolled fertilization or contamination. Most shellfish have exceptionally high fecundity. A two-year old female Pacific oyster Crassostrea gigas may produce 20–50 million eggs. Spawning in molluscan shellfish can be induced by thermal stimulation, feeding, ultraviolet (UV)-treated seawater and serotonin (Landau, 1992; Guo et al., 1999). During spawning, female and male shellfish release their gametes into surrounding water and fertilization takes place outside the body cavity, although there are some exceptions. Most crustaceans go through copulation, and males deposit spermatophores into females (Landau, 1992) and in this case fertilization occurs when the females release their eggs. Some molluscs such as Argopecten scallops and Ostrea oysters are hermaphrodites, although they may avoid self-fertilization by releasing sperm and eggs at different times. When released, eggs of most shellfish are resting at the prophase or metaphase of meiosis I (Gilbert, 1988). Fertilization triggers the resumption of meiosis I and II, which release polar body I (PB1) and II (PB2), respectively. Fertilized eggs go through rapid development and, depending on the temperature, develop into swimming larvae within 10–24 h. Most shellfish have a long planktonic larval stage (several weeks), during which larvae can disperse over long distances. Shellfish larvae usually go through some form of metamorphosis at the end of the larval period. High fecundity, external fertilization and free-living larvae make most shellfish good candidates for chromosome set manipulation, although some species such as crabs, freshwater shrimp and Ostrea oysters brood their eggs, making chromosome set manipulation more challenging.

6.2.2 Principles and approaches The objective of chromosome set manipulation is to create new chromosome combinations by altering the normal process of fertilization, meiosis, mitosis and zygote formation. Many types of manipulation are possible in shellfish. Unlike fish eggs that usually complete meiosis I before maturation, shellfish eggs rest at the metaphase of meiosis I (Gilbert, 1988), making both meiosis I and II accessible to manipulation. Meiosis I, II or both can be inhibited, causing the retention of polar bodies and therefore extra chromosome sets. The first mitosis of the zygote can be blocked, which may result in chromosome doubling, and gamete chromosomes from one of the sexes can be inactivated to produce uniparental inheritance which is called

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gynogenesis or androgenesis. The many types of manipulation and their combinations can produce a variety of new chromosome variants, although not all of them are viable or useful for aquaculture. Haploids, for example, can be easily created by gynogenesis, but they are not viable and of no practical benefit. Haploid embryos, however, are useful for certain genetic analysis. For shellfish genetics and breeding, the most relevant chromosome constructs are triploids, tetraploids, gynogenetic diploids and aneuploids. We will examine how these variants are created. The principles of chromosome set manipulation are illustrated in Fig. 6.1. Triploidy is produced by blocking meiosis II in normally fertilized zygotes. Blocking meiosis II retains PB2 which contains one set of chromosomes. During normal development, PB2 is released, leaving the egg with one set of maternal chromosomes. The maternal set of chromosomes unites with the paternal set from the sperm and forms a diploid zygote (Fig. 6.1). The retention of PB2 adds an extra set of maternal chromosomes to the diploid zygote, producing triploidy. Meiosis II blocking is widely used for the production of triploids in fish and shellfish species (Thorgaard, 1983; Beaumont and Fairbrother, 1991). The same process that produces triploids in normally fertilized eggs produces meiotic gynodiploids in gynogenetically activated eggs when sperm chromosomes are inactivated (Fig. 6.1). Inhibition of mitosis I leads to the doubling of chromosomes and formation of tetraploids in normally fertilized eggs or mitotic gynodiploids in gynogenetically activated eggs (Fig. 6.1). Mitotic gynodiploids, also known as double-haploids, are completely homozygous at all loci and particularly useful for the creation of inbred lines and genetic mapping (Thorgaard, 1983; Young et al., 1998). Tetraploids can also be created by blocking meiosis I and II in gynogenetically activated eggs (Fig. 6.1). Blocking meiosis I and II in normally fertilized eggs produces pentaploids (Fig. 6.1), which die before reaching D-stage (Cooper and Guo, 1989). While it is easy to understand that blocking meiosis II in normal zygotes produces triploidy (Fig. 6.1), blocking meiosis I is more complex. Blocking meiosis I was initially considered as a method for triploid induction. In the eastern oyster Crassostrea virginica, blocking meiosis I produced triploids as detected at adult stages (Stanley et al., 1981, 1984). In the Pacific oyster, however, meiosis I blocking resulted in predominantly tetraploids as detected at early embryonic stages by flow cytometry (Stephens, 1989). Chromosome counting of resultant embryos, however, revealed that blocking meiosis I produced not only triploids and tetraploids, but also high percentages of aneuploids (Guo et al., 1992a). Segregation analysis showed that blocking meiosis I significantly altered chromosome segregation during meiosis II (Guo et al., 1992b). It led to abnormal tripolar segregations in the majority of treated eggs and the formation of aneuploids (Fig. 6.2). Some studies have reported high percentages of triploids from meiosis I blocking as detected at adult stages (Stanley et al., 1984; Yang and Guo, 2006a), probably because most aneuploids are not viable and only triploids

Chromosome set manipulation in shellfish Normal fertilization

10

Gynogenesis

10

M1

M1

10

UV treated sperm

10 10

10 10 10

Blocking M1&2

PB1 M2 10

Blocking M1&2

Blocking M1 only

10

20

PB1

Abnormal segregation Aneuploids

20

10 M2

10

20 20

20 20

10 10 10

10

10 10

Blocking M2

Blocking M2 PB1

PB2

PB1

PB2

10 10

40

10

10

10

50

20

10 40

10

40

10

50

Pentaploid

20

30

40

20

Diploid

Tetraploid

Triploid

10

10

20

Blocking mitosis

Blocking mitosis

50

169

30

30

Triploid

Aneuploid

40

40

Tetraploid

10

10

Haploid

20

20

20

Meiotic Mitotic gynodiploid gynodiploid 100 % inbred Sex control

Fig. 6.1 Schematic presentation of chromosome set manipulation in shellfish. Left: in normal zygotes, blocking meiosis I (M1) and II (M2) produces pentaploids, blocking M2 produces triploids, blocking mitosis I produces tetraploids, and blocking M1 produces abnormal segregation and aneuploids. Right: in gynogenetically activated eggs, blocking M1 and M2 produces tetraploids, blocking M2 produces meiotic gynodiploids, and blocking mitosis I produces mitotic gynodiploids. Bottom: crossing diploids and tetraploids produces triploids, and crossing diploids and triploids produces aneuploids.

can survive. Because of the production of aneuploids and reduced survival, blocking meiosis I is no longer considered a good approach for triploid production. On the other hand, triploids produced from meiosis I blocking may grow faster than meiosis II triploids (Stanley et al., 1984; Beaumont and Kelly, 1989; Hawkins et al., 1994), and blocking meiosis I may produce some tetraploids (Yang et al., 2000b), which are desired for triploid production.

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10

MI

10 Blocking polar body I with cytochalasin B A

B 10 10

10 10

10

10

D

10

E

10 10 10

10 10

5

10 10

C

10 10

10 10

10

10

PB1 MII

7

20

7 10

10

10 10

20 20

10

10

= 20

PB2 10

10

5

6

10

10

10

2n Normal segregation

3n United bipolar

100 %

7%

10

13

13

14 13

15 15

10 10 10 10

10

10

10

= 13 14 13 10

= 10 15 15 10

3.7n/2.4n Randomized tripolar 68 %

4n/2.5n Unmixed tripolar

= 10/20 10/0 15 15 10

4n/3n/2n Separated bipolar 12 %

Fig. 6.2 Blocking meiosis I produces abnormal tripolar segregations of chromosomes during meiosis II and a wide range of aneuploids (from Guo et al., 1992b).

Once produced, tetraploids can be crossed with normal diploids to produce 100 % triploids, and triploids can be crossed with diploids to create aneuploids (Fig. 6.1). Gynogenetic diploids may be used to create inbred lines and monosex populations.

6.2.3 Methods for inhibiting meiosis and mitosis Meiotic and mitotic divisions can be blocked by a number of chemical and physical treatments. Cell division depends on the assembly of microtubules

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and microfilaments, and any agent that affects their synthesis and function can potentially block cytokinesis. In general, inhibiting meiosis is easier than blocking the first mitotic division, because the latter involves the complete cleavage of eggs. Several treatments have been tested for meiosis and mitosis inhibition in shellfish, showing varying degrees of effectiveness. These include heat shock, cold shock, pressure shock, cytochalasin B (CB), 6-dimethylaminopurine (6DMAP), caffeine, colchicine and nocodazole (Beaumont and Fairbrother, 1991; Guo, 1991; Guo et al., 1994; Scarpa et al., 1994; Dunstan et al., 2007). Among physical treatments, heat shock appears to be the most effective. In blue mussel Mytilus edulis, for example, a 10 min heat shock of 32 ºC applied at 20 min post-fertilization produced 97.4 % triploids (Yamamoto and Sugawara, 1988). In the dwarf surfclam Mulinia lateralis, a 35 ºC heat shock targeting meiosis I produced 82.5–98.5 % triploids at juvenile stages (Yang and Guo, 2006a). Heat shock is also effective in shrimp. Up to 100 % triploidy has been induced using heat shock in the Chinese shrimp Penaeus chinensis (Li et al., 2003b; Xiang et al., 2006). Cold shock and pressure shock have been tested in several shellfish species (Arai et al., 1986; Yamamoto and Sugawara, 1988; Guo, 1991; Liang et al., 1994; Yang and Guo, 2006b), but they are not as effective as heat shock. Among chemical treatments, CB and 6DMAP are clearly more effective than others. CB is a specific and reversible inhibitor of microfilaments, which are required for the formation of division furrow during cytokinesis. CB does not affect chromosome movement which is controlled by microtubules (Longo, 1972; Guo et al., 1992b). CB is probably the most frequently tested and used chemical for chromosome set manipulation in molluscan shellfish. It is highly effective in blocking meioses but less so in blocking mitosis I. CB can produce 70–100 % triploids by blocking meiosis II if treatments are applied correctly (Downing and Allen, 1987; Guo and Allen, 1994b). The effective dosage for CB ranges from 0.1 to 1.0 mg/l and, since CB is not water soluble, the stock solution is made by dissolving it in dimethylsulfoxide (DMSO) at 1 mg/ml. For triploid induction, the success rate depends on how synchronized the egg development is and how precisely the treatment is applied to target meiosis II. Typically, a 10–20 min treatment with 0.5 mg/l CB, applied when 60 % of the eggs release PB1 or as soon as PB2 is observed, gives good results. While CB works well in molluscs, it is not very effective in crustaceans and finfish, probably because eggs of crustaceans and finfish have a chitinous shell, which cannot be easily penetrated by CB. While CB is effective, it is also highly toxic and poses health risks for people who handle it. Double nitrile gloves should be used when working with CB. In the USA, the FDA has banned the use of CB in commercial shellfish hatcheries. Alternatively, 6DMAP can be used for triploid induction (Desrosiers et al., 1993) as it is water soluble and less toxic than CB. It is a kinase inhibitor, probably targeting enzymes required for cytokinesis.

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High percentages (>80 %) of triploids have been produced using 6DMAP in several species including Mytilus edulis (Brake et al., 2004), Haliotis rubra (Liu et al., 2004), Haliotis laevigata (Dunstan et al., 2007) and Penaeus japonicas (Sellars et al., 2006b; Coman et al., 2008). The effective dosage of 6DMAP ranges from 75–400 μM. A direct comparison finds that CB treatment results in higher percentages of triploids and lower mortality than 6DMAP (Nell et al., 1996). In another study, heat shock was highly effective in blocking mitosis I and producing tetraploidy (up to 98 %), while 6DMAP failed (Sellars et al., 2006a). It appears that 6DMAP is almost as effective as CB in blocking meiosis II, but not in inhibiting meiosis I and mitosis I. The results for caffeine are variable. In Mytilus galloprovincialis, caffeine treatments targeting meiosis II produced only 4.7–7.5 % triploids (Scarpa et al., 1993), while in Haliotis discus hannai it produced 91–100 % triploids by blocking meiosis I (Okumura et al., 2007). It is unknown if caffeine affects meiosis I and II differently, or whether the discrepancy is due to inherent inconsistence of the treatment. Further evaluation is needed.

6.2.4 Inactivation of gamete chromosomes Gynogenesis and androgenesis require the inactivation of gamete chromosomes. Gamete chromosomes can be inactivated with several methods including UV irradiation, X-ray, γ-ray and chemicals such as toluidine blue (Thorgaard, 1983; Guo, 1991). UV irradiation is an effective and popular method for inactivating sperm chromosomes. UV light has weak penetrating power, and sperm samples should be treated in a thin layer (1 mm). The dosage of UV varies greatly among studies and may be influenced by the age of the lamp, temperature and sperm concentration. It should be empirically determined for each species and experiment. As examples, sperm chromosomes have been successfully inactivated by a 5–6 min treatment at 1080 μW/cm2/s in C. gigas (Guo et al., 1993), 2 min treatment at 620 μW/cm2/s in Mytilus galloprovincialis (Scarpa et al., 1994) and 1.5 min treatment at 1400 μW/cm2/s in Mulinia lateralis (Guo and Allen, 1994b). Toluidine blue (5 μM for 29 min) can also inactivate sperm chromosomes of C. gigas, but the treatment is sensitive to light and often produces inconsistent results (Guo, 1991). In fish, X-ray or γ-ray is commonly used for the inactivation of egg chromosomes (Thorgaard, 1983) but, because shellfish eggs are small, their chromosomes can be inactivated with UV irradiation (Li et al., 2004).

6.2.5 Identification of polyploids and uniparental inheritance A key component of chromosome set manipulation is the identification or confirmation of new chromosome variants. Positive identification is

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173

essential for evaluating induction success and the performance of induced polyploids and aneuploids. Polyploids can be identified by chromosome counting, nucleus size and flow cytometry (FCM) analysis of DNA content. Chromosome counting is direct, accurate, but also labour intensive (Guo et al., 1992a). Nucleus size has also been used to identify polyploids in a few studies (Komaru et al., 1988; Utting and Child, 1994). FCM is probably the fastest and most reliable method for the identification of polyploids (Allen, 1983; Guo et al., 1996). It can accurately identify hundreds of samples in a day. However, FCM cannot detect small variations in DNA content or identify aneuploids. The identification of aneuploids requires chromosome counting. The success of inhibiting meiosis I or II can be verified with fluorescence staining of polar bodies and nuclei (Scarpa et al., 1994). Briefly, eggs are fixed in ethanol or formalin. Drops of embryos are placed on a slide and stained for 3–10 min with a fluorescence dye such as 4,6-diamidine-2phenylindole (DAPI). A coverglass is applied to the slide and gently pressed to flatten the eggs and remove excess stain. Segregation patterns and the presence of polar bodies can then be observed under an epifluorescence microscope. A fluorescence-stained egg is shown in Fig. 6.3A. Chromosome counting is easier with early embryos than with adults. Embryonic cells are actively dividing and provide abundant metaphases for analysis. With embryos, metaphases can be prepared using the squashing and orcein-staining method (Guo et al., 1992b). Since the orcein-staining method does not break cells, metaphases can be counted with confidence. More than one metaphase can be counted in most embryos (Fig. 6.3B). For adult shellfish, gill or gonad is often used for chromosome preparation. Animals are treated with colchicine (0.005 % for 4–8 h) to stop cells at metaphase (Guo et al., 2007). Tissues are dissected and treated with a hypotonic solution (0.075 M KCl) for 10–20 min before fixation in Carnoy’s fixative (3 : 1 of methanol : acetic acid). Metaphase spreads are made by airdrying and stained with Leishman’s stain. The air-drying and Leishmanstaining method produces high-quality metaphases with no or little background (Fig. 6.3C). However, the air-drying method may disrupt metaphases and create artificial chromosome loss. Care should be taken during screening, and only metaphases showing no obvious signs of chromosome loss are counted. For FCM, fresh tissue gives the best results. Fresh tissues can be frozen in the staining buffer that contains 10 % DMSO (Allen, 1983; Guo et al., 1996). When working with fresh samples is not possible, tissues can be fixed in ethanol (Yang et al., 2000a). Because FCM measures relative DNA content and fluorescence signals can vary considerably, it is important that diploids are always used as reference (Fig. 6.3D), and reference samples should be prepared in the same way as test samples. Success in gynogenesis and androgenesis must be genetically verified. Cross-contamination of experimental groups by normal gametes or larvae

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New technologies in aquaculture

A

C

B

1200 count

D

2

1000 800 600 1

400 200

3 0 0

50

100

150

200

250

300

Fig. 6.3 Cytogenetic techniques used for the verification of chromosome set manipulation in shellfish: A, a fluorescence-stained egg of C. gigas showing only polar body I after treatment; B, a triploid embryo of C. virginica showing two triploid metaphases (3n = 30) and PB1 only; C, two metaphases of C. virginica showing trisomy (2n + 1) with 21 chromosomes; and D, flow cytometry analysis of a triploid oyster (peak 2) using a diploid as reference (peak 1).

is common, especially when the survival of gynogenetic or androgenetic animals is low. Uniparental inheritance can be confirmed with the use of multiple codominant markers such as allozymes or microsatellites (Guo and Gaffney, 1993).

6.3 Triploid shellfish Triploid shellfish were first reported in the eastern oyster by Stanley et al. (1981). Since then, triploids have been induced in dozens of shellfish species. However, many studies are limited to reporting induction success and larval survival, and adult performance of triploid shellfish is not well documented in most species. Triploid shellfish can be produced with three methods: inhibiting meiosis II, inhibiting meiosis I and crossing diploids and tetraploids, and the three

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175

types of triploids may differ considerably in performance. So far, most of the performance data have been collected from triploids produced by inhibiting meiosis but, as we will see, natural triploids produced from tetraploids greatly out-perform induced triploids. Therefore, data from induced triploids may grossly underestimate the full potential of triploid shellfish.

6.3.1 Larval performance It is well documented that triploid induction reduces larval survival. Treated larvae often experience heavy mortality. For example, in Mulinia lateralis, the relative survival (with diploids as 100 %) of treated larvae to day 8 is 55.3 % (Guo and Allen, 1994b) and in C. gigas, the relative survival of treated larvae to day 2 ranges from 16.8–57.8 % depending on treatment temperature (Downing and Allen, 1987). In another study on the same species, the relative survival of the treated larvae to day 1 is 5.3 % (Guo et al., 1996), while that of natural triploids is 79.1 %. The difference in survival between natural triploids and diploids is not significant. Clearly, the reduced survival is not an inherent problem of triploidy, but is caused by induction treatments or genetic defects associated with induced triploids. Retention of PB2 may be deleterious because of homozygosity or imprinting. The larval growth of induced triploids is either faster or about the same as diploids (Downing and Allen, 1987; Guo et al., 1996). Larval growth is sensitive to culture density. As induced triploids experience high mortality, they may enjoy low culture density compared with diploid controls. Therefore, larval growth of induced triploids should be viewed with caution, unless equal densities are strictly maintained. There is no question, however, that in C. gigas, larvae of natural triploids grow significantly faster than normal diploids from day 1 (Guo et al., 1996). When reaching the eyedstage, diploid larvae are usually 250–260 μm in size, while natural triploid larvae are about 280–300 μm (Guo, unpublished).

6.3.2 Growth Post-larval growth of triploids has been evaluated in over 20 shellfish species and, in most of the species studied so far, triploids grow significantly faster than diploid controls (Table 6.1). The superior growth of triploids is most pronounced in oysters and, in fact, triploid oysters have shown superior growth in all species and studies reported to date. In early studies where triploids were produced by inhibiting meiosis I or II, triploids typically grew 10–40 % faster than diploids (Stanley et al., 1984; Allen and Downing, 1986; Barber and Mann, 1991; Maguire et al., 1995). In Saccostrea glomerata, triploids’ advantage in growth is remarkable and consistent, with four studies reporting 36–74 % growth increases in triploids, averaging 53 % (Nell et al., 1994; Hand et al., 1999, 2004; Troup et al., 2005). In C. virginica and Ostrea edulis, meiosis I triploids grow significantly faster than diploids, while

Scallop Argopecten irradians Argopecten ventricosus Chlamys farreri

Crassostrea madrasensis Crassostrea talienwhanensis Ostrea edulis Saccostrea glomerata

Crassostrea gigas

Oyster Crassostrea virginica

Species

CB, 0.05–0.10 mg/L CB, 0.5 mg/L CB, 0.5–0.9 mg/L

CB, 0.5 mg/L CB, 0.5 mg/L CB, 1.0 mg/L 4n 4n CB, 1.0 mg/L CB or 6DMAP CB, 0.5 mg/L CB, 1.0 mg/L CB, 0.5 mg/L; 4n 4n 4n 6DMAP, 100 μM Cold: 2–8 ºC CB, 1.0 mg/L CB, 0.5 mg/L CB, 1.25 mg/L CB, 1.0 mg/L CB, 1.25 mg/L

Method1

66–94 8–58 18

61–72 96 85 100 100 71–96 86 76 84 82–100 100 99–100 na 43–70 na 85 73–93 75–79 na

%3N

36, 73 (muscle) 55, 161 (muscle)3 5, 44 (muscle)

12–41 30 67 82–192 91–109 28 40 20–23 80–150 14; 26 25–51 159 128–2603 52 (length) 61 (PB1); −8 (PB2) 41 36–57 74 49

% increase2

1 1 1.2

3 2 1.4 1.3 1.5 1.5 0.8 2.3–3.2 1.5–2 1 0.8 1 1 1 1.25 2.5 2 1.3 3.2

Age (y)

Tabarini, 1984 Ruiz-Verdugo et al., 2000 Yang et al., 2000b

Stanley et al., 1984 Barber and Mann, 1991 Matthiessen and Davis, 1992 Guo et al., 2008 Allen, pers comm Allen and Downing, 1986 Garnier-Gere et al., 2002 Maguire et al., 1995 Akashige and Fushimi, 1992 Wang et al., 2002 Guo et al., 1996 Nell and Perkins, 2005 Mallia et al., 2006 Liang et al., 1994 Hawkins et al., 1994 Nell et al., 1994 Hand et al., 1999 Hand et al., 2004 Troup et al., 2005

Source

Table 6.1 Induction and post-larval growth of triploid shellfish, with growth measured as percent increase in body size (whole body weight unless otherwise noted) of triploids over diploid controls

Shrimp Fenneropenaeus chinensis Penaeus japonicus

3

2

43–100 73–93

28 Smaller

13–34 30 (tissue) −17 (length)

18–44

23–30 30–54 83 92

34, 63 (tissue) Larger (PB1)

8 0 72 −15 0 (length) 0 0 0

18, 42 (muscle) 40, 67 (muscle) −11, 13 (muscle)

83 26–67

30 62–100 21–100 44 56–85 35 50–80 70–77

75 44–90 48–95

0.5 0.5–1

0.33 3 0.5

2

1.9 0.1

4 1.5 0.3 1 0.06 1 1–3 0.25

1.2 1 1.8

Induction method: CB = cytochalasin B; 6DMAP = 6-dimethyaminopurine; 4n, tetraploid crossing with diploid. Percent increase: PB1 = blocking the release of polar body I; PB2 = blocking the release of PB2. Triploids are not verified.

Heat: 29–32 ºC 6DMAP, 150 μM

Abalone Haliotis discus hannai Haliotis laevigata Haliotis rubra

1

6DMAP, 75–150 μM 6DMAP, 100 μM CB, 0.5 mg/L

Pinctada martensii

CB, 1.0 mg/L CB, 1.0 mg/L CB, 0.5 mg/L CB, 1.0 mg/L CB, 1.0 mg/L CB, 0.5 mg/L CB CB, 0.5 mg/L

CB, 0.5 mg/L CB, 0.5 mg/L CB, 0.5–1 mg/L

6DMAP, 400 μM CB, 0.1, 0.5, 1 mg/L Heat: 25, 30 ºC CB, 0.75–1.0 mg/L

Mussel Mytilus edulis

Mulinia lateralis Mya arenaria Tapes dorsatus Tapes philippinarum

Clam Mercenaria mercenaria

Nodipecten subnodosus

Chlamys nobilis

Xiang et al., 2006 Coman et al., 2008

Zhang et al., 1998 Dunstan et al., 2007 Liu et al., 2004

Jiang et al., 1993

Brake et al., 2004 Beaumont and Kelly, 1989

Eversole et al., 1996 Yang and Guo, unpubl. Guo and Allen, 1994b Mason et al., 1988 Nell et al., 1995 Shpigel and Spencer, 1996 Ekaratne and Davenport, 1993 Utting and Child, 1994

Komaru and Wada, 1989 Lin et al., 1995 Maldonado-Amparo et al., 2004

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meiosis II triploids do not (Stanley et al., 1984; Hawkins et al., 1994). For natural triploids produced from diploid × tetraploid crosses, the increase in growth is even more impressive. Natural triploids grow up to 159 % faster than diploids in C. gigas (Nell and Perkins, 2005) and up to 190 % faster in C. virginica (Guo et al., 2008). The superior growth of natural triploids is remarkable even at three months of age (Fig. 6.4). It is also worth noting that in C. virginica natural triploids have shown significant improvements with successive generations of breeding of their tetraploid parents. Triploids produced from the first generation tetraploids grow 34 % faster

2n

3n

2n

3n

Fig. 6.4 Triploid (3n) C. virginica produced from tetraploids compares with their diploid (2n) controls at 3 (top) and 15 (bottom) months of age.

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than diploid (Wang et al., 2005), while triploids produced from the second generation tetraploids grow 88–190 % faster than diploids (Guo et al., 2008). Superior growth has also been demonstrated for triploid scallops (Tabarini, 1984; Komaru and Wada, 1989; Lin et al., 1995; Ruiz-Verdugo et al., 2000; Yang et al., 2000b). Triploid scallops show 5 %, 18 %, 36 %, 40 % and 55 % increases in whole body weight in five separate studies (Table 6.1). In these same studies, triploids show even greater increases in adductor muscle weight, by 44 %, 42 %, 73 %, 67 % and 161 %, respectively. In Nodipecten subnodosus, triploids are 11 % smaller than diploids in whole body weight, but still 13 % bigger than diploids in adductor muscle weight (Maldonado-Amparo et al., 2004). These observations point to an interesting phenomenon that triploid scallops have greatly enlarged adductor muscles. Since adductor muscle is the marketed product for most scallops, any increases in adductor muscle mean not only higher yield, but also higher per unit price. Therefore, triploid scallops may have tremendous potential in aquaculture. Triploid mussels, abalone and shrimp have also shown increased body size or growth (Zhang et al., 1998; Brake et al., 2004; Xiang et al., 2006), although the magnitude is not as impressive as in oysters. In mussels, the highest increase is 44 % in whole body weight observed in Pinctada martensii, a pearl oyster but closely related to mussels (Jiang et al., 1993), and 63 % in tissue weight in Mytilus edulis (Brake et al., 2004). Data from clams are interesting albeit less encouraging for aquaculture. In all but one study, triploid clams are either smaller or about the same size as diploids (Table 6.1). Mulinia lateralis is an exception, where triploids are 72 % bigger than diploids and show nearly normal gonadal development (Guo and Allen, 1994b). Overall, it seems that, for some unknown reasons, triploid clams do not grow faster than diploids. This observation is interesting and deserves further investigation. The growth of triploids is strongly influenced by environmental conditions. It has been shown in several species that triploids from the same cohort may perform very differently at different locations. In general, the triploid advantage in growth is highest at warmer and more productive sites (Davis, 1989; Brake et al., 2004). This finding is not surprising, as in nutrientlimiting environments triploids may not grow faster than diploids even if they have greater potential. This may explain why triploids do not show superior growth in some of the studies. Despite the poor showing of triploids in a few clams, it is clear that triploids grow significantly faster than diploids in most shellfish species, and the cause of their increased growth or body size has been the subject of much discussion. Three hypotheses have been proposed, attributing the increased body size of triploids to increased heterozygosity, sterility or enlarged cell size (Guo and Allen, 1994b; Wang et al., 2002). The heterozygosity hypothesis suggests that the extra set of chromosomes in triploids increases heterozygosity (Stanley et al., 1984), which is often positively

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correlated with growth in diploids. Supporting the heterozygosity hypothesis, triploids produced by blocking meiosis I, which may have higher levels of heterozygosity, grow faster than triploids produced by blocking meiosis II (Stanley et al., 1984; Hawkins et al., 1994). In addition, natural triploids produced from tetraploids are more heterozygous and also grow faster than induced triploids (Wang et al., 2002, 2005). A positive correlation between heterozygosity and growth is observed among diploids and two types of triploids (Fig. 6.5). There are also observations arguing against the heterozygosity hypothesis. First, meiosis I triploids do not show superior growth in some species including Mytilus edulis (Beaumont et al., 1995) and Pinctada martensii (Jiang et al., 1993). In the latter species, meiosis II triploids are slightly larger than meiosis I triploids. Secondly, although existing at the group level, the correlation between body size and multilocus heterozygosity is absent at the individual level within groups (Jiang et al., 1993; Beaumont et al., 1995; Garnier-Gere et al., 2002; Wang et al., 2002). In the absence of a highly positive and significant correlation at the individual level, the heterozygosity hypothesis has to be viewed with caution. The sterility hypothesis argues that triploids, because of their sterility, may allocate more energy to somatic growth rather than gonad development. Normal diploids spawn in summer and expend a considerable amount of energy on reproduction, while triploids do not. The sterility hypothesis is supported by studies in which triploids grow faster than diploids only after sexual maturation (Allen and Downing, 1986; Brake et al., 2004). However, energy relocation cannot explain the observation that in many cases triploids are significantly larger than diploids long before sexual maturation (Guo and Allen, 1994b; Guo et al., 1996). In C. gigas, triploid larvae are significantly larger than diploids on day 1, and triploid juveniles are 51 % larger than diploids at eight months of age or 3–4 months prior to sexual maturation (Guo et al., 1996). In C. virginica, natural triploids are 1.6

0.6

1.4 0.4 1.2

0.3 0.2

1

Meat weight (g)

Heterozygosity

0.5

0.1 0.8

0 2n

3nCB

Heterozygosity

3nDT Meat weight (g)

Fig. 6.5 A positive correlation between meat weight and multilocus heterozygosity in diploids (2n), triploids produced by inhibiting meiosis II (3nCB) and triploids produced from tetraploids (3nDT) in the Pacific oyster (based on Wang et al., 2002).

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165–180 % larger than diploids at 3 months of age or 8 months prior to their first reproductive season (Wang et al., 2006). In Mulinia lateralis, triploids are 72 % larger than diploids without showing signs of reduced gonadal development (Guo and Allen, 1994b). These data strongly suggest that sterility or energy relocation is not the major cause for the increased body size in triploids. Finally, the cell size hypothesis proposes that triploid cells are larger than diploids and automatically lead to increases in organ and body size (Guo and Allen, 1994b). Development in molluscs is mosaic, where increases in cell size are not compensated by a reduction in number (Gilbert, 1988). Supporting the cell size hypothesis, eggs from triploid C. gigas are about 54 % larger (Guo and Allen, 1994c), and adductor muscle cells of triploid Chlamys farreri are about 50 % larger (Yang and Guo, unpublished). The observation that natural triploids grow faster than induced triploids, seemingly against the cell size hypothesis, can be explained with potential genetic defects associated with meiosis inhibition. The cell size hypothesis also has its limitations as it cannot explain cases where triploids are more than 50 % bigger than diploids (Table 6.1), because the cell size increase in triploids should be proportional to the increase in DNA or about 50 %. Although all three hypotheses can explain some of the data, none of them can account for all the observations so a new synthesis is necessary. We propose here a unifying genome adaptation hypothesis to explain the greatly increased body size of natural triploids observed in oysters (Nell and Perkins, 2005; Guo et al., 2008). The genome adaptation hypothesis accepts that increased cell size and heterozygosity are primary and independent causes for the increased body size of triploids, and sterility contributes additional but secondary increases. Further, the hypothesis argues that newly established polyploid genomes are dynamic and have a greater potential to change and adapt. The great increase in body size of natural triploid eastern oysters (by 109–190 %, Guo et al., 2008) is the result of rapid genome adaptation, after two generations of selection on their tetraploid father. The rapid adaptation is possible because of the tremendous genetic variation created by the polyploid genome per se and possibly through non-Mendelian changes such as chromosome deletion, duplication and rearrangements due to multivalent formation (Guo and Allen, 1997) and unequal crossovers during meiosis. The genome adaptation hypothesis assumes no limits on body size increase and can explain the fact that natural triploids are larger than induced triploids, and become even larger when produced from the second generation tetraploids. In other words, natural triploid grow faster, because their tetraploid father has survived at least two generations of polyploidy (including their triploid grandmother), subjected to severe selection for growth and survival (relative to massive genetic variation in the polyploid genome), and become adapted to the polyploid condition. The genome adaptation hypothesis suggests that tetraploids can also be rapidly improved during the first few generations before reaching a new equilibrium for tetraploidy.

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6.3.3 Meat quality Because of their sterility, triploid molluscs may have improved meat quality during the spawning season (Allen and Downing, 1986). Most molluscs allocate a large portion (30–60 %) of their body to gonad production, and sexual maturation and reproduction often lead to a reduction in meat quality in normal diploids. For the half-shell oyster industry, animals with excessive gonadal material are considered as low-quality and oyster farms may have to stop or reduce production during the summer season. Because triploids have greatly reduced gonad development and are largely sterile, their meat quality is not affected by maturation and spawning during the reproductive season. Triploid molluscs therefore provide a high-quality product that can be sold year round, and it was the improved summer meat quality, not superior growth, that initially led to commercial production of triploids in the Pacific oyster (C. gigas) (Allen et al., 1989). On the negative side, triploid Pacific oyster cultured in warmer climates may develop brown spots in gonadal areas, but this phenomenon appears to be environment-specific (Nell, 2002; Davis, pers comm). Triploid Sydney rock oysters may also show brown coloration on the surface of their gonad (Hand and Nell, 1999); this coloration may be a response to aborted gametogenesis and high water temperature and it may disappear over time (Nell, 2002). We have not seen any brown coloration in triploid Pacific and eastern oysters cultured in New Jersey.

6.3.4 Survival The survival of adult triploids is variable. Triploid shellfish may have improved health or disease-resistance during reproductive seasons, and it has been shown that triploid oysters are less susceptible to summer mortalities caused by stress and energy depletion (Allen and Downing, 1986; G. Zhang, personal communication). However, there are also observations that triploids suffer from heavier mortality than diploids in summer at some locations (B. Eudeline, personal communication). Barber and Mann (1991) have shown that triploid and diploid eastern oysters are equally susceptible to Dermo, a parasitic disease caused by Perkinsus marinus. Another study has shown that triploid eastern oysters are less susceptible to MSX diseases (caused by Haplosporidium nelsoni) than diploids (Matthiessen and Davis, 1992). Similarly, in the Sydney rock oyster, mortality of triploids is two times lower than that of normal diploids under infection by the parasite, Mikrocytos roughleyi (Hand et al., 1998a,b). In the eastern oyster, triploids show better survival against ROD (Roseovarius oyster disease, caused by the bacterium Roseovarius crassostreae) than diploids (Guo et al., 2008). This improved survival is probably not due to specific resistance to Roseovarius crassostreae. ROD is a juvenile disease and primarily affects oysters smaller than 2.5 cm and, because triploids grow significantly faster, they may reach the refuge size earlier.

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6.3.5 Sterility and genome stability Triploids have three sets of chromosomes, and theoretically they cannot go through meiosis and produce viable gametes. This is true in most fish and some shellfish where triploid females do not produce mature eggs (Thorgaard, 1983; Allen et al., 1986; Li et al., 2003a). So the use of sterile triploids in aquaculture helps to reduce the concern that cultured stocks (often selectively bred) may escape and interbreed with wild populations. If sterile, triploids can provide effective biological containment, which is especially important for culturing non-native species. Unfortunately, in most molluscs, the triploids are not completely sterile. Triploids have greatly reduced gonadal development, but they do produce some mature and viable gametes. In the Pacific oyster, the fecundity of triploid females is about 2 % of normal diploids (Guo and Allen, 1994a). The survival of triploid × triploid crosses is about 0.04 126 % of diploid × diploid crosses. The reproductive potential of triploids, assuming unlimited sperm, is therefore 0.000 008 (2 % × 0.04 126 %) or 1 in 125 000. If we consider that only 7 % of the triploid × triploid progeny are diploids (Guo and Allen, 1994a), the relative chance of triploids producing diploid offspring becomes 0.000 008 × 0.07 = 0.00 000 056 or 1 in 1.8 million. If we assume that sperm are also limiting and triploid males produce 2 % of what diploids produce, then the reproductive potential becomes 0.000 000 011 or 1 in 89 000 000. It means that when diploids produce 89 million fertile diploids, triploids would produce one, which is extremely low. This calculation is based on only one study and can be influenced by many variables. For example, another study puts the relative fecundity of triploid C. gigas at 13.4 % (Gong et al., 2004). Further, the calculation assumes that the culture population is 100 % triploids, which is not always possible (see Section 7.4). Any residual diploids can greatly change the calculation, because the survival of triploid × diploid cross is much higher than triploid × triploid crosses (Guo and Allen, 1994a; Gong et al., 2004). Even if 100 % triploids can be produced, the stability of the triploid genome presents another concern. It has been shown that some triploids may revert to mosaics, which contain some diploid cells, although it appears that reversion occurs in a small fraction (5–15 %) of triploids and is primarily restricted to somatic tissues (Allen et al., 1999). Reversion of tetraploids is more frequent (Guo, unpublished), suggesting increased genome instability of tetraploidy. Reversion is not well understood at this time and further research is needed.

6.4 Tetraploid shellfish 6.4.1 Induction of tetraploids Although triploid shellfish can be readily produced by blocking meiosis II, direct induction of triploids has several disadvantages: (i) most induction

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treatments are toxic and detrimental to larval survival; (ii) the induction is rarely 100 % effective, which complicates hatchery management; and (iii) the retention of PB2 may have deleterious effects on the survival and growth of induced triploids (Chourrout et al., 1986; Guo et al., 1990). These problems can be eliminated by producing triploids from diploid × tetraploid mating (Guo et al., 1996). Tetraploidy is typically induced by inhibiting the first mitosis in fertilized eggs (Fig. 6.1). Viable tetraploids have been produced with mitosis I inhibition in fish (Chourrout, 1982; Myers et al., 1986) and amphibians (Fischberg, 1958; Reindschmidt et al., 1979). In shellfish, we have more ways to make tetraploids because meiosis I and II are both accessible. Tetraploid shellfish can be produced with at least the following approaches: inhibition of mitosis I, inhibition of meiosis I, inhibition of meiosis I and II in gynogenetically activated eggs, fusion of two diploid cells, and inhibition of meiosis I in eggs from triploids fertilized by normal sperm. All these approaches have been tested in shellfish with variable results (Table 6.2). Mitosis I inhibition has produced high percentages of tetraploid embryos, up to 80–90 %, in several species, but no tetraploids survived beyond metamorphosis in any of those studies, despite the diverse treatments used (Table 6.2). In C. gigas, blocking meiosis I and II in gynogenetically activated eggs produced 94.6 % tetraploids, none of which survived beyond larval stages (Guo, 1991), and neither zygote nor blastomere fusion produced viable tetraploids (Guo et al., 1994). Viable tetraploids were produced by meiosis I inhibition in several species, although the number of tetraploids that survived to juvenile stage was small (3–5) and no tetraploid lines were established (Scarpa et al., 1993; Allen et al., 1994; Yang et al., 2000b; Peruzzi and Guo, 2002; Yang and Guo, 2004). The poor survival of tetraploid shellfish may be caused by many factors. In many molluscs, the first cleavage is characterized by the formation of a polar lobe. The polar lobe contains morphogenic determinants that need to be precisely distributed (Gilbert, 1988). Therefore, inhibiting mitosis I may disrupt the distribution of morphogenic determinants and normal development. In fish and amphibians where viable tetraploids were produced by mitosis I blocking (Fischberg, 1958; Reinschmidt et al., 1979; Chourrout, 1982; Myers et al., 1986), the first cleavage is an equal division, and the development is regulative. The disruption of polar lobe formation cannot explain why tetraploid produced by blocking meiosis I and II in gynogenetically activated eggs are not viable, as the process does not affect the polar lobe. Guo (1991) hypothesizes that the poor viability of the induced tetraploids is caused by a cellnumber deficiency, and this deficiency arises from the cleavage of a normal egg by a large, tetraploid nucleus and the maintenance of a given nucleus/ cytoplasm ratio. In ‘mosaic’ development, a cell’s fate is programmed by the number of divisions and distribution of morphogenic determinants (Gilbert, 1988). A reduction in cell number at the end of cleavage may stop

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Table 6.2 Tetraploid (4n) induction in shellfish: approaches and treatments used, percent 4n embryos produced and number of viable 4n obtained after metamorphosis Approach/Species Mitosis I inhibition Crassostrea gigas

Treatment1

% 4n embryos

No. of viable 4n

Reference

Heat, 35–40 ºC Colchicine, 0.125 mM Nocodazole, 0.026 μM Heat, 35, 38 ºC Cold, 4, 7 ºC Nocodozle, 0.02–1.6 mg/L 6-DMAP CB KCI Heat, 33–34 ºC

45 5 10 63–86 23–31 0

0 0 0 0 0 0

Guo et al., 1994 Guo, 1991

50 55 80 22–90

0 0 0 0

Cui et al., 2004

Heat, 35–36 ºC Cold, 5 ºC 6-DMAP, 150 μM

33–67 15 0

0 0 0

Sellars et al., 2006a

CB, 1 mg/L

94.6

0

Guo, 1991

PEG, zygote fusion PEG, blastomere fusion PEG, sperm fusion

2.2 30

0 0

Guo et al., 1994

0

0

Meiosis I inhibition Crassostrea gigas Chlamys farreri Haliotis discus hannai Ostrea edulis

CB, 1 mg/L CB, 0.5 mg/L 6DMAP, 175–225 μM CB, 0.8 mg/L CB, 1 mg/L

28 26 20–23 25–33 40–53

0 5 0 0 0

Mulinia lateralis

CB, 0.75 mg/L

na

4

CB, 0.67 mg/L Heat, 35 ºC CB, 1 mg/L

40–90 0 18

3 0 5

Gendreau and Grizel, 1990 Perruzzi and Guo, 2002 Yang and Guo, 2004 Yang and Guo 2006a Scarpa et al., 1993

CB, 0.5 mg/L

na

3

Allen et al., 1994

Mercenaria mercenaria Eriocheir sinensis Fenneropenaeus chinensis Marsupenaeus japonicus Gynogenesis Crassostrea gigas Cell fusion Crassostrea gigas

Mytilus galloprovincialis Tapes philippinarum

Meiosis inhibition in 3n female × 2n male cross CB, 0.5 mg/L Crassostrea gigas CB, 0.5 mg/L Crassostrea gigas Crassostrea virginica CB, 1 mg/L CB, 0.25 mg/L Crassostrea ariakensis na Argopecten ventricosus CB, 0.5 mg/L Pinctada martensii 1

67 30–84 40–100 0–90

1970 – 4000 3120

Yang and Guo, 2006b

Li et al., 2003b

Guo, 1991 Yang et al., 2000b Zhang et al., 2000

Guo and Allen, 1994c Eudeline et al., 2000 Guo et al., 2002 Allen et al., 2005

na

5

Maldonado et al., 2003

17

2

He et al., 2000

CB = cytochalasin B, na = not available, PEG = polyethylene glycol, 6-DMAP = 6-dimethylaminopurine.

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development by retaining different morphogenic determinants in one cell, or by a lack of sufficient number of cells needed for further morphogenesis. The cell-number deficiency can be corrected by an increase in the egg volume. The eggs from triploids are significantly larger than that from normal diploids (Guo and Allen, 1994a). By blocking meiosis I in eggs from triploids fertilized with sperm from diploids, Guo and Allen (1994c) were able to produce a large number of viable tetraploids in C. gigas, a result that supports the cell-number deficiency hypothesis. Using the same method, viable tetraploids have been obtained in five bivalve molluscs and in three Crassostrea oysters, large numbers of tetraploids are produced and tetraploid lines established (Table 6.2). It has been shown recently that tetraploids produced by inhibiting meiosis II in eggs from diploids fertilized with sperm from tetraploids are viable (McCombie et al., 2005). This finding argues against the cell-number deficiency hypothesis, although the relative survival of tetraploids is not reported. The survival of a few tetraploids does not change the fact that the majority of tetraploids produced from eggs of diploids cannot survive. The cell-number hypothesis provides one explanation. The genome adaptation hypothesis (see Section 6.3.2) can also explain the improved survival of tetraploids derived from triploid females (Guo and Allen, 2004c) and tetraploid males (McCombie et al., 2005). By having survived at least one round of polyploidization, the triploid or tetraploid parents may have eliminated genes or gene complexes that are incompatible with polyploidy, giving their progeny an enhanced ability to survive tetraploidy.

6.4.2 Performance of tetraploids At present, large numbers of tetraploids and second generation tetraploids are only available in C. gigas (Guo and Allen, 1994c), C. virginica (Guo et al., 2002) and C. ariakensis (Allen et al., 2003). The first generation tetraploid C. gigas are larger than the diploids and triploids from the same treatment group (Guo and Allen, 1994c), but the diploids and triploids appear to be exceptionally small and possibly aneuploid. In C. virginica, the first generation tetraploids are also smaller than diploids and triploids (Guo et al., 2002). The first two to three generations of tetraploid C. virginica suffered heavy mortalities that appeared to be unique to tetraploids (Guo, unpublished data). Mortality occurred primarily in early spring when oysters are going through gonadal development. By the fourth generation, tetraploids were much healthier than in early generations and significantly larger than diploids (Guo et al., unpublished). This observation is in agreement with the genome adaptation hypothesis that tetraploids can go through rapid adaptation during the first few generations. Tetraploid oysters are fertile and have apparently normal development of gonads. The fecundity of tetraploid C. gigas is comparable to that of normal diploids (Guo and Allen, 1997), differing from the greatly reduced

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fecundity of triploids. Tetraploids have an approximately 1 : 1 sex-ratio at one year of age. In contrast to the abnormally high frequency of hermaphrodites among triploids, tetraploids have about the same level of hermaphrodites as normal diploids. During meiosis, all chromosomes are engaged in multivalent formation (Guo and Allen, 1997). In spite of massive multivalent formation, tetraploids produce primarily diploid sperm with some aneuploids (Guo and Allen, 1997; Wang et al., 1999). All-triploid populations have been produced in all three oyster species by mating diploid females with tetraploid males. Natural triploids produced from tetraploid are practically 100 % pure, with percentages typically between 99 and 100 % (Guo et al., 1996; Wang et al., 2006). The rare diploids found among natural triploids may be gynogenetic, androgenetic (Wang et al., 1999), or due to imperfect meiosis in tetraploids. It can also be the result of contamination by a diploid group.

6.5 Gynogenesis, androgenesis and aneuploids Gynogenesis has been successfully produced in several shellfish species including Haliotis discus hannai (Arai et al., 1984; Fujino et al., 1990), Mytilus edulis (Fairbrother, 1994), Mytilus galloprovincialis (Scarpa et al., 1994), C. gigas (Guo et al., 1993; Li et al., 2000), and Mulinia lateralis (Guo and Allen, 1994b). In Mulinia lateralis, all gynodiploids are females, providing evidence for XX-female and XY-male determination of sex (Guo and Allen, 1994b). In C. gigas, meiotic gynodiploids have been used for genecentromere mapping of allozyme loci (Guo and Gaffney, 1993). The mean recombination frequency over seven loci is 0.74, which is high but not unusual. Mitotic gynodiploids are extremely valuable for genetic analysis and breeding because they are completely homozygous at all loci. However, mitotic gynodiploids have not been reported in shellfish. Haploid androgenesis has been achieved in C. gigas (Li et al., 2004). Aneuploids can be created by crossing triploids with diploids with or without inhibition of meiosis (Guo and Allen, 1994a,c). The following chromosome numbers have been observed in adult oysters, demonstrating that they are viable. They include 2n − 1, 2n + 1, 2n + 2, 2n + 3, 3n − 2, 3n − 1, 3n, 3n + 1, 3n + 2, 3n + 3, 4n − 2, 4n − 1, 4n and 4n + 1 (Guo and Allen, 1994c; Wang et al., 1999; Gong et al., 2004). Preliminary data show that most aneuploid conditions negatively affect growth including trisomy, a condition of a diploid genome having one extra chromosome (2n + 1). Trisomic families have been produced in the Pacific oyster (Guo et al., 2000), which are potentially useful in gene mapping and other genetic analyses.

6.6 Summary and perspectives Chromosome set manipulation has made significant contributions to shellfish aquaculture through the development of triploid and tetraploid oysters (Nell,

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2002). Because of their superior growth, improved meat quality and sterility, triploids have become a desired product for oyster farming, and triploid oysters are now being used for commercial production in many countries including the USA, Australia, France, Chile, China and Korea. In the USA, about one third of the Pacific oyster production is from triploids. Triploid eastern oysters are also commercially cultured in the USA. Triploid C. ariakensis have been deployed for large-scale field trials in the USA, which would not be possible for a non-native species without the use of triploids. C. ariakensis is an Asian oyster that resists two devastating diseases (MSX and Dermo) of the eastern oyster. While the introduction of diploid C. ariakensis to US waters is controversial, triploids are being evaluated and considered as an alternative species for aquaculture. Tetraploids have played a critical role in the successful use of triploids in all three oyster species. On the other hand, the full potential of chromosome set manipulation has not yet been realized in shellfish. Research on uniparental inheritance and aneuploids remains largely academic, and triploids have not entered commercial production in most shellfish. Induced triploids have already demonstrated improved performance in scallops, mussels, abalone and shrimp, and natural triploids in the future should offer even greater improvements as has been shown in oysters. However, tetraploids which are needed for the production of natural triploids are only available in three species of oysters so the development of tetraploids in other major shellfish species should be a priority for the research community. Finally, chromosome set manipulation in shellfish is not only useful for aquaculture, but also important for our understanding of shellfish genomes. The creation of various chromosome variants provides unique opportunities to study some of the basic questions in genome biology. Research in shellfish has raised some interesting questions such as why triploids grow faster, why aneuploids grow slower, and why tetraploids have limited viability. Hypotheses have been advanced, and future studies may greatly improve our understanding of shellfish genomes.

6.7 Acknowledgements We thank Alison Guo for helping with graphics. This work is partly supported by grants from NOAA Sea Grant Oyster Disease Research Program. This is publication IMCS-2009-60 and NJSG-09-900.

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downing, s l and allen, s k, jr (1987) Induced triploidy in the Pacific oyster, Crassostrea gigas: Optimal treatments with cytochalasin B depend on temperature, Aquaculture, 61, 1–15. dunstan, g a, elliott, n g, appleyard, s a, holmes, b h, conod, n, grubert, m a and cozens, m a (2007) Culture of triploid greenlip abalone (Haliotis laevigata Donovan) to market size: Commercial implications, Aquaculture, 271, 130–41. ekaratne, s u k and davenport, j (1993) The relationships between the gametogenetic status of triploids or diploids of manila clams, Tapes philippinarum, and their oxygen-uptake and gill particle-transport, Aquaculture, 117, 335–49. eudeline, b, allen, s k, jr and guo, x (2000) Optimization of tetraploid induction in the Pacific oysters, Crassostrea gigas, using the first polar body as a natural indicator, Aquaculture, 187, 73–84. eversole, a g, kempton, c j, hadley, n h and buzzi, w r (1996) Comparison of growth, survival, and reproductive success of diploid and triploid Mercenaria mercenaria, J Shellfish Res, 15, 689–94. fairbrother, j e (1994) Viable gynogenetic diploid Mytilus edulis (L.) larvae produced by ultraviolet light irradiation and cytochalasin B shock, Aquaculture, 126, 25–34. fao (2007) The State of World Fisheries and Aquaculture 2006, Rome, Food and Agriculture Organization of the United Nations. fischberg, m (1958) Experimental tetraploidy in newts, J Embryol Exp Morphol, 6, 393–402. fujino, k, arai, k, iwadare, k, yoshida, t and nakajima, s (1990) Induction of gynogenetic diploid by inhibiting second meiosis in the Pacific abalone, Nippon Suisan Gakkaishi, 56, 1755–63. garnier-gere, p h, naciri-graven, y, bougrier, s, magoulas, a, heral, m, kotoulas, g, hawkins, a and gerard, a (2002) Influences of triploidy, parentage and genetic diversity on growth of the Pacific oyster Crassostrea gigas reared in contrasting natural environments, Mol Ecol, 11, 1499–514. gendreau, s and grizel, h (1990) Induced triploidy and tetraploidy in the European flat oyster, Ostrea edulis L, Aquaculture, 90, 229–38. gilbert, s f (1988) Developmental Biology, 2nd Edn, Sunderland, MA, Sinauer Associates Inc. gong, n, yang, h, zhang, g, landau, b j and guo, x (2004) Chromosome inheritance in triploid Pacific oyster Crassostrea gigas Thunberg, Heredity, 93, 408–15. guo, x (1991) Studies on Tetraploid Induction in the Pacific Oyster, Crassostrea gigas (Thunberg), Seattle, WA, University of Washington. guo, x (2004) Oyster breeding and the use of biotechnology, Bull Aquac Assoc Canada, 104(2), 26–33. guo, x and allen, s k, jr (1994a) Reproductive potential and genetics of triploid Pacific oyster, Crassostrea gigas (Thunberg), Biol Bull, 187, 309–18. guo, x and allen, s k, jr (1994b) Sex determination and polyploid gigantism in the dwarf surfclam (Mulinia lateralis Say), Genetics, 138, 1199–206. guo, x and allen, s k, jr (1994c) Viable tetraploids in the Pacific oyster (Crassostrea gigas Thunberg) produced by inhibiting polar body 1 in eggs from triploids, Mol Mar Biol Biotechnol, 3, 42–50. guo, x and allen, s k, jr (1997) Sex and meiosis in autotetraploid Pacific oyster, Crassostrea gigas (Thunberg), Genome, 40, 397–405. guo, x and gaffney, p m (1993) Artificial gynogenesis in the Pacific oyster, Crassostrea gigas: II. Allozyme inheritance and early growth, J Hered, 84, 311–15. guo, x, hershberger, w k and myers, j (1990) Growth and survival of intrastrain and interstrain triploids of rainbow trout, Oncorhynchus mykiss, J World Aquac Soc, 21, 250–56.

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guo, x, cooper, k, hershberger, w k and chew, k k (1992a) Genetic consequences of blocking polar body I with cytochalasin B in fertilized eggs of the Pacific oyster, Crassostrea gigas: I. Ploidy of resultant embryos, Biol Bull, 183, 381–6. guo, x, hershberger, w k, cooper, k and chew, k k (1992b) Genetic consequences of blocking polar body I with cytochalasin B in fertilized eggs of the Pacific oyster, Crassostrea gigas: II. Segregation of chromosome, Biol Bull, 183, 387–93. guo, x, hershberger, w k, cooper, k and chew, k k (1993) Artificial gynogenesis with ultraviolet irradiated sperm in the Pacific oyster, Crassostrea gigas: I. Induction and survival, Aquaculture, 113, 201–14. guo, x, hershberger, w k, cooper, k and chew, k k (1994) Tetraploid induction with mitosis-I inhibition and cell-fusion in the Pacific oyster (Crassostrea gigas Thunberg), J Shellfish Res, 13, 193–8. guo, x, debrosse, g and allen, s k, jr (1996) All-triploid Pacific oysters (Crassostrea gigas Thunberg) produced by mating tetraploids and diploids, Aquaculture, 142, 149–61. guo, x, ford, s and zhang, f (1999) Molluscan aquaculture in China, J Shellfish Res, 18, 19–31. guo, x, zhang, g, landau, b j, english, l and wang, y (2000) Aneuploidy in the Pacific oyster, Crassostrea gigas Thunberg and its effects on growth, J Shellfish Res, 19, 614. guo, x, wang, j, landau, b j, li, l, debrosse, g a and krista, k d (2002) The successful production of tetraploid eastern oyster, Crassostrea virginica Gmelin, J Shellfish Res, 21, 380–81. guo, x, wang, y and xu, z (2007) Genomic analyses by fluorescent in situ hybridization, in Liu, Z J (ed.), Aquaculture Genome Technologies, Ames, IA, Blackwell, 289–311. guo, x, wang, y, debrosse, g a, bushek, d and ford, s e (2008) Building a superior oyster for aquaculture, Jersey Shoreline, 25, 7–9. hand, r e and nell, j a (1999) Studies on triploid oysters in Australia XII. Gonad discolouration and meat condition of diploid and triploid Sydney rock oysters Saccostrea commercialis in five estuaries in New South Wales, Australia, Aquaculture, 171, 181–94. hand, r e, nell, j a and maguire, g b (1998a) Studies on triploid oysters in Australia. X. Growth and mortality of diploid and triploid Sydney rock oysters Saccostrea commercialis (Iredale and Roughley), J Shellfish Res, 17, 1115–27. hand, r e, nell, j a, smith, i r and maguire, g b (1998b) Studies on triploid oysters in Australia. XI. Survival of diploid and triploid Sydney rock oysters Saccostrea commercialis (Iredale and Roughley) through outbreaks of winter mortality caused by Mikrocytos roughleyi infection, J Shellfish Res, 17, 1129–35. hand, r e, nell, j a, reid, d d, smith, i r and maguire, g b (1999) Studies on triploid oysters in Australia: Effect of initial size on growth of diploid and triploid Sydney rock oysters, Saccostrea commercialis (Iredale & Roughley), Aquac Res, 30, 35–42. hand, r e, nell, j a and thompson, p a (2004) Studies on triploid oysters in Australia: XIII. Performance of diploid and triploid Sydney rock oyster, Saccostrea glomerata (Gould, 1850), progeny from a third generation breeding line, Aquaculture, 233, 93–107. hawkins, a j s, day, a j, gerard, a, naciri, y, ledu, c, bayne, b l and heral, m (1994) A genetic and metabolic basis for faster growth among triploids induced by blocking meiosis I but not meiosis II in the larviparous European flat oyster, Ostrea edulis L, J Exp Mar Biol Ecol, 184, 21–40. he, m, lin, y, shen, q, hu, j and jiang, w (2000) Production of tetraploid pearl oyster (Pinctada martensii Dunker) by inhibiting the first polar body in eggs from triploids. J Shellfish Res, 19, 147–51.

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jiang, w, li, g, xu, g, lin, y and qing, n (1993) Growth of the induced triploid pearl oyster, Pinctada martensii (D.), Aquaculture, 111, 245–53. komaru, a and wada, k t (1989) Gametogenesis and growth of induced triploid scallops Chlamys nobilis, Nippon Suisan Gakkaishi, 55, 447–52 (in Japanese with English abstract). komaru, a, uchimura, y, ievama, h and wada, k t (1988) Detection of induced triploid scallop, Chlamys nobilis, by DNA microfluorometry with DAPI staining, Aquaculture, 69, 201–10. landau, m (1992) Introduction to Aquaculture, New York, Wiley. li, q, osada, m, kashihara, m, hirohashi, k and kijima, a (2000) Induction of gynogenetic diploids and cytological studies in the Pacific oyster, Crassostrea gigas, Suisanzoshoku, 48, 185–91. li, f, xiang, j, zhang, x, zhou, l, zhang, c and wu, c (2003a) Gonad development characteristics and sex ratio in triploid Chinese shrimp (Fenneropenaeus chinensis), Mar Biotechnol, 5, 528–35. li, f, xiang, j, zhou, l, wu, c and zhang, x (2003b) Optimization of triploid induction by heat shock in Chinese shrimp Fenneropenaeus chinensis, Aquaculture, 219, 221–31. li, q, hisatsune, t and kijima, a (2004) Induction of haploid androgenesis in Pacific oyster by UV irradiation, Mar Biotechnol, 6, 291–7. liang, y, wang, r, tian, c, yu, r and qu, x (1994) A preliminary study on the triploid oyster Crassostrea talienwhanensis, J Fish China, 18, 237–40 (in Chinese with English abstract). lin, y, he, m, qing, n and jiang, w (1995) Triploid induction in the scallop, Chlamys nobilis and growth comparisons, Tropi Oceanol, 14, 84–9 (in Chinese with English abstract). liu, w, heasman, m and simpson, r (2004) Induction and evaluation of triploidy in the Australian blacklip abalone, Haliotis rubra: a preliminary study, Aquaculture, 233, 79–92. longo, f j (1972) The effects of cytochalasin B on the events of fertilization in the surf clam, Spisula solidissima. I. Polar body formation, J Exp Zool, 182, 321–44. maguire, g b, gardner, n c, nell, j a, kent, g n and kent, a s (1995) Studies on triploid oysters in Australia. 2. Growth, condition index gonad area, and glycogen content of triploid and diploid Pacific oysters, Crassostrea gigas, from oyster leases in Tasmania, Australia, Aquaculture, 137, 355–8. maldonado, r, ibarra, a m and ramirez, j l (2003) Induction to tetraploidy in catarina scallop, Argopecten ventricosus (Sowerby II, 1842), Cienc Mar, 29, 229–38. maldonado-amparo, r, ramirez, j l, avila, s and ibarra, a m (2004) Triploid lionpaw scallop (Nodipecten subnodosus Sowerby); Growth, gametogenesis, and gametic cell frequencies when grown at a high food availability site, Aquaculture, 235, 185–205. mallia, j v, muthiah, p and thomas, p c (2006) Growth of triploid oyster, Crassostrea madrasensis (Preston), Aquac Res, 37, 718–24. mason, k m, shumway, s e, allen, s k jr and hidu, h (1988) Induced triploidy in the soft-shell clam Mya arenaria: energetic implications, Mar Biol, 98, 519–28. matthiessen, g c and davis, j p (1992) Observations on growth rate and resistance to MSX (Haplosporidium nelsoni) among diploid and triploid eastern oyster [Crassostrea virginica (Gmelin, 1791)] in New England, J Shellfish Res, 11, 449–54. mccombie, h, ledu, c and phelipot, p (2005) A complementary method for production of tetraploid Crassostrea gigas using crosses between diploids and tetraploids with cytochalasin B treatments, Mar Biotechnol, 7, 318–30. myers, j m, iwamoto, r n and hershberger, w k (1986) Tetraploid induction in salmonids, J World Aquac Soc, 17, 1–7.

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7 Advances in disease diagnosis, vaccine development and other emerging methods to control pathogens in aquaculture A. Adams, University of Stirling, UK

Abstract: Disease is still regarded as a major constraint to aquaculture production globally. Rapid disease diagnosis and vaccination play a huge part in the control of bacterial diseases, and there has been significant progress in both of these areas. This chapter considers the limitations of existing methods and reviews recent advances made in pathogen detection technologies and vaccine development methodologies. Future directions are discussed, including nanotechnology and reversed vaccinology. Key words: fish disease, fish health, disease diagnosis, vaccine development, pathogen detection technologies.

7.1 Introduction Disease is still regarded as a major constraint to aquaculture production globally (Adams et al., 2005). As the industry continues to expand and diversify, the risk of new diseases emerging and old ones spreading to other geographical regions is a reality. The movement of eggs and fry between fish farms presents ideal circumstances for pathogens to adapt with their hosts and environment. Control of pathogens is complex and relies heavily on a combination of pathogen detection, disease diagnosis, treatment, prevention and general health management. Rapid disease diagnosis and vaccination play a huge part in this, and there has been significant progress in both of these areas since the 1980s. In the years since 2000 the pace has increased even more as methods developed for clinical and veterinary medicine are rapidly adapted and optimised for use in

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aquaculture. Novel diagnostic methods are published frequently and the success of vaccination in reducing the use of antibiotics has been realised, at least in some countries. Innovative alternative methods for the control of fish diseases are also being researched and applied. This chapter aims to provide a review of the recent advances made in disease diagnosis, vaccine development and other emerging methods to control pathogens in aquaculture.

7.2 Key drivers to improve disease diagnosis and vaccine development The main key driver to improve disease diagnosis and vaccine development is the continued significant losses to the industry caused by pathogens. Bacterial diseases cause a substantial economic burden to the aquaculture industry and, although antibiotics and chemotherapeutants are extensively used to control disease outbreaks, there is increasing concern about their use because of drug residues in food, the development of antimicrobial drug resistance and the detrimental effect on the aquatic microbial ecosystems and populations (Thompson and Adams, 2004). There is a concerted effort to move away from the use of antibiotics wherever possible. This was highlighted in September 2008 in Korea with the announcement that from next year the Korea Food and Drug Administration will ban the use of seven types of antibiotics in feed for livestock and fish raised in fish farms. In the UK, Norway and Japan there has already been a significant reduction in the use of antibiotics since the 1990s (Adams et al., 2005; Markestad and Grave, 1997). There is a real need to increase production of fish globally through aquaculture and therefore health and welfare must be given a high priority if targets set are to be reached. Some vaccines, in particular multivalent vaccines, have led to welfare concerns in the past few years due to the presence of adhesions on internal organs, thought to result from oil-based adjuvants in the vaccines (Berg et al., 2007). Farming of new species will play a significant role in increasing production. Persistent disease problems in cod (Vibriosis in particular) have played a large part in this new industry stagnating in Scotland. As cobia farming increases globally, and with the first farms being licensed in Brazil (Eric Routledge, Special Secretariat for Aquaculture and Fisheries, SEAP, Brazil; pers comm), there will be a need for farmers to be one step ahead of potential disease threats.

7.3 Limitations of current diagnostic methods Many of the current techniques for the detection of pathogens and diagnosis of diseases are actually very good. On the other hand, identification of

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certain pathogens is difficult to achieve and some of the methods developed may be too complicated to apply or interpret. Conventional pathogen isolation and characterisation techniques, alongside pathology, still remain the methods of choice for the diagnosis of many diseases. However, these traditional methods tend to be costly, labour-intensive, and slow, and might not always lead to a definitive diagnosis being made. The rapid progress made in biotechnology since the 1990s has enabled the development and improvement of a wide range of immunodiagnostic and molecular techniques (Cunningham, 2004; Adams and Thompson, 2006, 2008), and reagents and commercial kits have become more widely available. These rapid methods both complement and enhance the traditional methods of disease diagnosis. The Manual of Diagnostic Tests for Aquatic Animals (World Organisation for Animal Health, 2006) includes a variety of standardised methods (including traditional, immunological and molecular methods) for the identification of selected pathogens (causing notifiable diseases), and these will expand as new methods are developed and validated (Adams and Thompson, 2008). Most of these are, however, for viral diseases, and Renibacteriun salmoninarum and Piscirickettsia salmonis, the causative agents of bacterial kidney disease (BKD) and ricketsiosis, respectively, are the only bacterial pathogens included. The diseases caused by these pathogens do not meet the listing criteria, but they are included because reporting requirements for non-listed diseases apply in regard to significant epidemiological events. For those diseases not included in the Animal Health Code there are no set standards. It is important that reagents and methods used for detecting bacterial pathogens are standardised and rigorously tested for specificity and sensitivity. Commercial reagents and kit development (Adams and Thompson, 2008) have gone some of the way to achieving this, but there still is not a full range of reagents or kits available for use in aquaculture. The cost, speed, specificity and sensitivity of assays are all extremely important to end-users. The highest cost is often time, although labour costs do vary considerably between countries. Many of the new technologies require specialised equipment and highly skilled staff and few of the existing methodologies are suited to field testing, or use in rudimentary laboratories.

7.4 Advances in methods of disease diagnosis (mainly for bacterial diseases) Disease diagnosis is currently made using a variety of methods, as reviewed by Adams and Thompson (2008). Traditional bacteriology, whereby the pathogen is isolated and identified biochemically (e.g. using API® strips), and observation of histological sections from diseased fish are widely used. Rapid methods that specifically identify the pathogen using antibodies

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(immunodiagnostics) or by amplifying specific sequences of DNA or RNA using polymerase chain reaction (PCR) (i.e. molecular diagnostics) are also regularly used in many laboratories. In some instances molecular diagnostics has completely taken over from other methodologies. For many of the rapid methods live and dead pathogens cannot be distinguished; therefore, the inclusion of enrichment methods and the use of live/dead kits are useful supplementary methods (Vatsos et al., 2002). Interpretation of results using rapid methods of pathogen detection should be carefully considered with all the other clinical evidence, including histology and attempted culture of the pathogen. Immunodiagnostic methods such as immunohistochemistry (IHC), the fluorescence antibody test (FAT) and indirect fluorescence antibody test (IFAT) enable rapid specific detection of pathogens in tissue samples without the need to first isolate the pathogen. The IHC method is a simple extension of histology allowing specific identification of pathogens in formalin tissue fixed sections (Adams and Marin de Mateo, 1994; Steiropoulos et al., 2002), while FAT/IFAT is extremely rapid and sensitive as well as being specific but requires a fluorescent microscope to read the results (Adams et al., 1995; Miles et al., 2003; Klesius et al., 2006). Both IHC and FAT/IFAT are technically easy to perform, and examples of the results obtained using these methods are shown in Fig. 7.1. (p. 201, see also colour section.) A variety of other antibody-based methods have also been developed for use in aquaculture. Some are very simple to use, but usually require pathogen isolation prior to use and lack in sensitivity (e.g. agglutination), while others are more complex, but with the added advantage of pathogen quantification (e.g. enzyme-linked immunosorbent assay, ELISA), or detection and characterisation of specific pathogen antigens to their molecular weight, e.g. Western blot (Rose et al., 1989; Adams and Thompson 1990; Adams, 1992, 2004). The ELISA also has the advantage of high throughput and automated equipment is available. The ELISA can also be used for serology (detecting antibodies to specific pathogens). Although serology is an essential screening tool in clinical medicine and in most control programmes for the significant diseases of domestic animals (Palmer-Densmore et al., 1998; Yuce et al., 2001; Fournier and Raoult, 2003) it has not yet been validated for any bacterial diseases in fish. Serology is used effectively for detecting exposure to fish viruses, such as koi herpesvirus (KHV) (Adkinson et al., 2005; Adams and Thompson, 2008), but bacterial pathogens pose a much more complex picture with cross reactivities likely unless specific known proteins are used to coat the ELISA plates rather than whole pathogens. The use of molecular technologies for the detection of fish bacterial pathogens is rapidly increasing and a vast array of methods has already been developed (Karunasagar et al., 1997; Cunningham, 2004; Adams and Thompson 2006, 2008; Wilson and Carson, 2003). Molecular methods

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(c)

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Fig. 7.1 (See also Plate I) Examples of immunodiagnostic methods to detect fish pathogens in fish tissue. Detection of Renibacterium salmoninarum using indirect fluorescent antibody test, IFAT (a). Detection of Renibacterium salmoninarum using True Blue as substrate (b), Photobacterium damselae subspecies piscicida using 3,3′-diaminobenzidine (DAB) as substrate (c) and Streptococcus iniae using Fast Red as substrate (d) by immunohistochemistry, IHC. (Photographs (a)–(c) and (d) courtesy of Dr K D Thompson and Dr P Klesius, respectively)

generally have the highest sensitivity and are therefore particularly useful for detecting microorganisms that are present in low numbers or for those that are difficult to culture. In addition, molecular methods can be used for the identification of pathogens to species level (Puttinaowarat et al., 2000; Pourahmed, 2008) and in epidemiology for the identification of individual strains and differentiating closely related strains (Cowley et al., 1999). The PCR is the best known method, although there are many useful variations, including nested PCR, random amplification of polymorphic DNA (RAPD), reverse transcriptase-PCR (RT-PCR), reverse cross blot PCR (rcb-PCR) and RT-PCR enzyme hybridisation assay (Puttinaowarat et al., 2000; Wilson and Carson, 2003; Cunningham, 2004). Colony hybridisation has also been used successfully for the rapid identification of Vibrio anguillarum in fish (Aoki et al., 1989) and has the advantage of detecting both pathogenic and environmental strains (Powell and Loutit, 2004). Real-time PCR (also known as qPCR) offers quantification and high sample throughput. Real-

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time PCR methods have recently been developed for a variety of significant fish bacterial pathogens, such as Aeromonas salmonicida (Balcázar et al., 2007), Piscirickettsia salmonis (Karatas et al., 2008) Renibacterium salmoninarum (Jansson et al., 2008) and Edwardsiella ictaluri (US Patent 6951726), and it is likely that this range will expand rapidly. Polygenic sequencing following PCR of specific genes is being recommended for the identification of some pathogens where differentiation of closely related species is difficult. For example, Pourahmed (2008) found that sequencing of three different genes was necessary to classify certain mycobacteria species from fish. A variety of novel rapid diagnostic methods are currently being developed that have potential for future application in the diagnosis of aquatic animal health. These were recently reviewed by Adams and Thompson (2008), and comparisons were made between these methods and existing technologies with regard to their advantages and disadvantages. Loop-mediated isothermal amplification (LAMP) is an emerging technology with potential for detection of fish and shellfish pathogens, and research tests to detect Edwardsiella tarda, E. ictaluri, Nocardia seriolae, and Flavobacterium psychrophilum (bacterial pathogens that cause edwardsiellois, enteric septicemia of catfish and nocardiosis, respectively), Tetracapsuloides bryosalmonae (the parasite that causes proliferative kidney disease, PKD) and infectious haematopoitic necrosis virus (IHNV) in fish and white spot syndrome virus (WWSV) in shrimp have already been developed (Savan et al., 2005; Manji, 2008). A commercial LAMP test kit is available for WSSV. The LAMP is a relatively new method for amplifying DNA which relies on autocycling strand displacement DNA synthesis and, since it is carried out under isothermal conditions, it can be performed without the use of a thermocycler. The method uses Bst DNA polymerase and a set of four specially designed primers (two inner and two outer primers) to recognise a total of six distinct sequences on the template DNA (Notomi et al., 2000). The main advantages of the method are the speed with which it can be performed, its sensitivity, and the fact that the results are read by eye, and, although the test requires the use of pipettes and an incubator, it does not require any other specialised equipment. Figure 7.2 (p. 203, see also colour section) shows the results of a LAMP test carried out to identify Flavobacterium psychrophilum, where a simple colour change in the tube from orange to green indicates a positive. Simple rapid field tests using lateral flow technology are also in development, and rapid kits are currently commercially available to detect infectious salmon anaemia virus (ISAV) and WSSV in shrimp. Although there are none yet for bacterial pathogens in fish, this type of technology has been successfully developed for clinical (Gatta et al., 2004) and veterinary use (Bautista et al., 2002) and offers simple field test capabilities.

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Fig. 7.2 (See also Plate II) Example of a loop-mediated isothermal amplification (LAMP) test carried out using purified Flavobacterium psychrophilum DNA. Positive samples turn green and negative ones remain orange. LAMP is highly sensitive and specific, and is performed under isothermal conditions, needing minimal instrumentation. (Photograph courtesy of F. Manji)

There is also interest in the development of multiplex tests to simultaneously detect different pathogens in a single sample. Multiplex technologies such as the Luminex xMAPTM (a bead array) and microarray both have huge potential in this area, but these are currently expensive and labourintensive as assays are still being developed and optimised (Adams and Thompson, 2008). The xMap system theoretically offers simultaneous quantitative analysis of up to 100 different analytes from a single drop of sample in an integrated, 96-well formatted system (Dunbar, 2006). This is complex technology with huge potential as it can be used for vaccine development, through epitope mapping (Costa et al., 2007), as well as pathogen detection. This is flexible technology as antibodies, protein or DNA can be bound to the bead array. A number of research groups are also currently developing DNA and oligo microarray technology for diagnostics and these also will offer simultaneous detection of pathogens for the future (González et al., 2004; Matsuyama et al., 2006).

7.5 Advances in vaccine development A wide range of commercial vaccines is available against bacterial pathogens, with most targeting salmon and trout, with additional vaccines available for channel catfish, European seabass and seabream, Japanese

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amberjack and yellowtail, tilapia and Atlantic cod (Thompson and Adams, 2004; Adams et al., 2005; Sommerset et al., 2005). The salmonid market presently uses heptavalent vaccines containing Listonella (Vibrio) anguillarum serotypes O1 and O2, V. salmonicida, Moritella viscosa, Aeromonas salmonicida, the causative agents of vibriosis, Hitra disease, winter ulcer disease, furunculosis and infectious pancreatic necrosis, respectively, and infectious pancreatic necrosis virus (IPNV). Due to environmental and control concerns in most countries only two bacterial vaccines (Edwardsiella ictaluri and Flavobacterium columnarae, causing Columnarus, for Channel catfish in the USA) and one viral vaccine (KHV for Carp in Israel) are presently commercially available as ‘live attenuated’ vaccines (Adams et al., 2005). Most of the commercial vaccines are based on inactivated bacterial pathogens, with fewer available for viral vaccines and none against parasites yet. The major producers of fish vaccines are now Intervet-Schering Plough Animal Health (The Netherlands), Novartis Animal Health (Switzerland), Pharmaq (Norway) and Microtek International Inc. (Canada). There are a number of smaller companies producing vaccines locally (e.g. autogenous vaccines), in different countries. Vaccines used in Japan are mostly developed and distributed by Japanese companies. Many new vaccines are in development (Thompson and Adams, 2004; Adams et al., 2005). The primary considerations for vaccines for aquaculture are cost-effectiveness and safety. Vaccines need to provide long-term protection against diseases on commercial fish farms. All the serotypic variants of the disease agent need to be considered, the time/age when the animal is most susceptible to disease, the route of administration and the method of vaccine preparation (i.e. inactivated whole cell, attenuated, subunit, recombinant). In order to develop an effective vaccine the protective antigens need to be identified and their protective response confirmed in the host species. The latter may be antibody mediated, cell mediated or both depending on the vaccine components. A practical method of administration and an inexpensive method of vaccine production also need to be established. It is important in vaccine development to work with the antigens that are expressed during infection rather than antigens expressed in the laboratory. Many salmon vaccines from the past are based on inactivated (whole cell) cultures of the pathogenic organism (usually inactivated in formalin) grown in vitro. In these cases the vaccines gave good protection (e.g. Vibrio vaccine); however, many pathogens appear to switch off important protective antigens when cultured in vitro. In such cases alternative methods of culture (e.g. the inclusion of an iron-chelating agent) are required so that expression of the important ‘protective’ antigens is induced (Neelam et al., 1993). This can be achieved by modifying the culture medium of the pathogen in vitro, as shown by Bakopoulos et al., (2003) and Jung et al., (2007) for Photobacterium damselae subsp. piscicida,

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Fig. 7.3 Western blot analysis of Photobacterium damselae subspecies piscicida after culture of the bacterium in different media (1–8), showing that different proteins are expressed under different culture conditions. Blot (a) used rabbit serum while blot (b) was performed with fish serum where many fewer antigens were recognised. (Photographs courtesy of Dr V Bakopoulos)

the pathogen that causes pasteurellosis. The effects of altering the constituents of the media for culturing P. damselae subsp. piscicida in the laboratory on antigen expression are illustrated in Fig. 7.3, where differences are observed in the antigens expressed/recognised by fish sera between media 1, 7 and 8, for example (Fig. 7.3a, b). Some antigens appear to be up-regulated whilst others are down-regulated. This also serves to highlight the difference between mammalian and fish immune systems, as fish (Fig. 7.3b) recognise many fewer antigens than rabbits (Fig. 7.3a). An alternative approach is to place the pathogen of interest inside the peritoneal cavity of fish, enclosed in sealed chambers (that permit the exchange of small molecules only), so that antigen expression in vivo can be determined (Bakopoulos et al., 2004; Poobalane, 2007; Jung et al., 2008). Figure 7.4 shows how R. salmoninarum alters the expression of surface molecules when the bacterium is cultured in vivo or in vitro. Bacterial cells appears smooth and rounded when cultured in vitro in contrast to those cultured inside the host fish. Application of sera from fish (that have been infected with the disease of interest and then recovered) in Western blot analysis on one- or two-dimensional gels (immunoprotomics) can then pinpoint potential vaccine candidates that can then be identified and vaccines produced (e.g. a recombinant vaccine against Aeromonas hydrophila, Poobalane, 2007). Of course these antigens may be expressed and the fish may respond by producing antibodies to them, but they may or may not be protective. Thus, challenge of vaccinated and nonvaccinated fish is then performed to establish if the vaccine is actually protective.

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Fig. 7.4 Electron micrographs (EM) of Renibacterium salmoninarum cultured in vitro and in vivo, illustrating the differential expression of bacterial surface molecules when bacteria are cultured in the laboratory or inside fish. (Photographs courtesy of Dr K D Thompson)

Fish vaccines have become much more sophisticated since the mid1990s. Technologies such as recombinant and DNA vaccines are powerful tools for vaccine development (Leong et al., 1997; Smith, 2000) as these enable the isolation of potential protective antigens from suppressive ones. These are being developed because the simpler approach of using inactivated whole cell vaccines did not succeed for many of the important diseases, and attempts at attenuated vaccines in general have not been encouraged from a safety point of view (Benmansour and de Kinkelin, 1997). An IPNV vaccine based on a recombinant expressed viral protein has been developed (Frost and Ness, 1997) and has been on the market for several years for use in salmon in Norway, but the licensing of other recombinant vaccines has been slow. DNA vaccines for fish have been shown to be effective when based on DNA-sequences encoding for rhabdovirus glycoproteins (Lorenzen and La Patra, 2005) and the first DNA vaccine has been licensed in Canada against IHNV. Vaccines for fish can be administered by a variety of different methods, i.e. injection (intramuscular or intra-peritoneally), immersion (bath or dipvaccination) or orally. There is much interest in developing oral vaccines as this is the most practical method of administration. In the absence of natural exposure, booster vaccination is needed to maintain immunity. Oral vaccine boosters have been used successfully and are marketed commercially. For example, oral vaccines are available against enteric red mouth (ERM) and vibriosis in rainbow trout, and against furunculosis and IPNV in salmon (Meeusen et al., 2007). These vaccines employ an antigen protection vehicle to protect vaccines from the acid environment of the fish

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stomach, and improved delivery systems are currently being researched for a variety of vaccines.

7.6 Other emerging methods to control pathogens Close monitoring of stocks to ensure early detection of pathogens causing disease problems (and effective treatment) and vaccination to prevent infections are clearly not the only methods of disease control. Farmers can take a number of measures to manage the impact on healthy stocks, including improved nutrition/diet, and maintaining stocking densities at levels that optimise growth and avoid over-crowding that reduces the ability for the animal to resist infections. Immunostimmulants (e.g. glucans) added to diets have been reported to enhance the immune system of the fish in the short term, when applied either on their own or in vaccines as adjuvants, and are reported to be very affective at stimulating the nonspecific defense mechanisms of the animal (Thompson and Adams, 2004; Peddie and Secombes, 2005). Other dietary additives are also being used, sometimes to target specific diseases, e.g. addition of vitamin E to target salmon pancreas disease (SPD). A wide range of modified diets and immunostimulants is available commercially and many new products are being researched (Bricknell and Dalmo, 2005; Peddie and Secombes, 2005). In addition, probiotics are widely used in some countries (Birkbeck, 2004). Reducing the risk of exposure to, or impact on, healthy populations is also an effective alternative approach to pathogen control. Molecular methods (e.g. PCR) are ideal technologies to use for screening fish broodstock and eggs for the presence of pathogens, and as long as large enough sample sizes are tested (e.g. for eggs) this may prove to be an effective method of reducing the reservoir of specific pathogens on fish farms. Recently, Manji (2008) reported that, although the prevalence of Flavobacterium psychrophilum in rainbow trout eggs was very low (1–2.4 %) this still led to spread of disease (rainbow trout fry syndrome, RTFS) and mortalities when these eggs were grown on. In this study it was necessary to test at least 300 eggs per batch in the screening process. The long-term approach to disease control is through the selection of disease-resistant strains or families of fish, and much research is currently focused on fish genotype and disease susceptibility (Biacchesi et al., 2007). Selective breeding programs have been shown to have large beneficial effects on production and quality traits in cold-water fish species, but only a small percentage of aquaculture production is based on genetically improved fish and shellfish (Gjedrem, 2000). Resistance is a complex quantitative trait that is likely to be affected by many genes (Gjedrem

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et al., 1991; Grimholt et al., 2003). To select for disease resistance it is necessary to challenge a large number of animals and measure each family’s overall performance for the trait. Genes associated with disease and stress resistance are now being identified and their characteristics will be used as identifiers (markers) for selective breeding of disease- and/or stress-resistant individuals (Moen et al., 2004). This approach should continue to be pursued, bearing in mind that increased resistance to one pathogen can result in decreased resistance to other pathogens. As more nucleotide sequences become available for fish bacterial pathogens, this will provide opportunities to develop innovative alternative disease control methods. Recently, following the sequencing of R. salmoninarum, Sudheesh et al. (2007) described the identification of a new class of drug (phenyl vinyl sulfone) which inhibits the activity of a very important enzyme (sortase) in R. salmoninarum. Inhibition of this enzyme appears to dramatically reduce the virulence of the bacterium by interfering with the ability of the bacterium to adhere and colonise fish cells. This drug could offer a promising alternative to antibiotics to control bacterial kidney disease in fish, and this type of approach holds potential for future control of bacterial disease in general for fish.

7.7 Future trends Technologies to assist with disease control are moving at a rapid pace. Careful consideration must be given to selecting which methods to take forward and apply in aquaculture. Vaccines need to be cost-effective and safe, and pathogen detection methods should be robust yet sensitive. There are many innovative technologies that may fulfil these criteria and provide new vaccines and useful diagnostics tools. It is important, however, that the diagnostics methods already developed are standardised and fully validated, if they are considered useful, and that new technologies do not supersede these just because they are novel methods. They need to have clear advantages over the existing methods for use in aquaculture. Nanotechnology is an area being explored for the detection of pathogens in food (Kim et al., 2007) and in clinical and veterinary diagnostics, and this may prove extremely useful for application in diagnostics for aquatic animals. Nanotechnology is generally used when referring to materials of 0.1–100 nanometres; however, it is also inherent that these materials should display different properties from bulk (or micrometric and larger) materials as a result of their size. These differences include physical strength, chemical reactivity, electrical conductance, magnetism and optical effects. Such technology offers the ability to detect extremely low

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levels of pathogens very quickly, and progress has already been made for the identification of foodborne pathogens (Joseph and Morrison, 2006). Recently, the focus is on different types of sensors to detect pathogens and immunomagnetic reduction (IMR) technology has been reported for the detection of low levels of the H5N1 virus that causes bird flu (Yang et al., 2008). In this method magnetic nanoparticles were coated with antibody and a high-transition-temperature superconductive quantum interference device was used to sense the immunomagnetic reduction of the reagents. Nanotechnology is also being used in conjunction with proteomics (Marko et al., 2007) and may be a useful technology to identify markers of disease and vaccine antigens. Immunoprotomics, as discussed in Section 7.5, is also known as ‘reversed vaccinology’ and has recently been successfully used for the development of a recombinant vaccine against A. hydrophila for carp (Poobalane, 2007). Other technologies with the same approach are knockout technologies, which indicate whether specific antigens are essential or important for survival of the pathogen in the host. These methods have great potential for the future and include RNA interference where expression of certain genes is blocked by antisense RNA (Melamed et al., 2002), in vivo expression technologies, IVET (Rainey and Preston, 2000), and signature tagged mutagenesis (Saenz and Dehio, 2005). The information obtained from a variety of these techniques is combined with data from existing literature to identify potential vaccine candidate antigens for cloning and for recombinant expression (Adams et al., 2005). Delivery of DNA vaccines using bacteriophages has been reported to be successful in a number of animals including fish (Skurnik and Strauch, 2006). The phage particles used are non-infectious and only grow on specialised laboratory strains of bacteria; the phage coat protects the vaccine from degradation and allows the host’s immune system to process it more efficiently (March et al., 2004; Clark and March, 2006). This technology may be extremely useful for oral delivery of DNA vaccines for fish. Gene sequencing underpins many of the new technologies being developed both for diagnostics and vaccine development and, as gene sequences become available for both pathogens and the host fish species, microarrays can be developed. These will enable very rapid progress to be made in fish health control for the future.

7.8 Sources of further information and advice In addition to the references cited there are numerous websites that provide useful information and advice. These are summarised in Table 7.1.

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Table 7.1

Sources of further information and advice

Name of organisation or project

Website address

AquaFirst AquaNet CEFAS (Centre for Environment. Fisheries and Aquaculture Science) DipNet (An EU funded project investigating disease interaction and pathogen exchange between farmed and wild aquatic animal populations) EAFP (European Association of Fish Pathologists) EPIZONE – Network of Excellence for Epizootic Disease Diagnosis and Control EUROCARP FAO (Food and Agriculture Organisation of the United Nations) FAO oneFish Community Directory Project (aquaculture/diseases) FEAP (Federation of European Aquaculture Producers) FishEggTrade (An EU funded project appraising of the zoosanitary risks associated with trade and transfer of fish eggs and sperm) Fish Health Section of the American Fisheries Society Fish Health Section of the Asian Fisheries Society Fisheries Research Services IAAAM (International Association for Aquatic Animal Medicine) IMAQUANIM (EU funded project: Improved immunity of aquaculture animals) International Database for Aquatic Animal Diseases ISAAE (International Society of Aquatic Animal Epidemiology) NACA (Network of Aquaculture Centres in Asia-Pacific) OIE Aquatic Animal Health Standards Commission OIE Designated Experts and Reference Laboratories for Aquatic Animal Diseases PANDA (Permanent Advisory Network for Diseases in Aquaculture) WAS (World Aquaculture Society)

http://aquafirst.vitamib.com http://www.aquanet.ca http://www.cefas.co.uk http://www.dipnet.info

http://www.eafp.org http://www.epizone-eu.net http://eurocarp.haki.hu http://www.fao.org http://www.onefish.org/id/10752 http://www.feap.info/feap http://cordis.europa.eu/life/src/ control/qlk2-ct-2002-01546.htm http://www.fisheries.org/units/fhs/ http://afs-fhs.seafdec.org.ph/ http://www.marlab.ac.uk/ http://www.iaaam.org http://imaquanim.dfvf.dk/info/ http://www.collabcen.net http://www.isaaepi.org/modules/news/ http://www.enaca.org http://www.oie.int/aac/eng/en_fdc.htm http://www.oie.int/fdc/eng/Diseases/ en_reflabslist.htm http://www.europanda.net http://www.was.org

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7.9 References adams a (1992) Sandwich enzyme linked immunosorbent assay (ELISA) to detect and quantify bacterial pathogens in fish tissue, in Stolen J S, Fletcher T C, Kaattari S L and Rowley A F (eds), Techniques in Fish Immunology, Vol. 2, SOS Publications, Fair Haven, NJ, 177–84. adams a (2004) Immunodiagnostics in aquaculture, Bull Eur Assoc Fish Pathol, 24, 33–7. adams a and marin de mateo m (1994) Immunohistochemical detection of fish pathogens, in Stolen J S, Fletcher T C, Kaattari S L and Rowley A F (eds), Techniques in Fish Immunology, Vol. 3, SOS Publications, Fair Haven, NJ, 133–44. adams a and thompson k d (1990) Development of an ELISA for the detection of Aeromonas salmonicida in fish tissue, J Aquat Animal Health, 2, 281–8. adams a and thompson k d (2006) Biotechnology offers revolution to fish health management, Trends Biotechnol, 24, 201–5. adams a and thompson k d (2008) Recent applications of biotechnology to novel diagnostics for aquatic animals, Rev Sci Tech Off Int Epiz, 27, 197–209. adams a, thompson k d, morris d, farias c and chen s c (1995) Development and use of monoclonal antibody probes for immunohistochemistry, ELISA and IFAT to detect bacterial and parasitic fish pathogens, Fish Shellfish Immunol, 5, 537–47. adams a, aoki t, berthe f c j, grisez l and karunasagar i (2005) Recent technological advancements on aquatic animal health and their contributions towards reducing disease risks – a review, in Bondad-Reantaso M G, Mohan C N, Crumlish M and Subasinghe R P (eds), Disease in Asian Aquaculture VI, Proceedings of the sixth symposium on diseases in Asian aquaculture, Fish Health Section, Asian Fisheries Society, Manila, 71–88. adkison m a, gilad o and hedrick r p (2005) An enzyme linked immunosorbent assay (ELISA) for detection of antibodies to the koi herpesvirus (KHV) in the serum of koi Cyprinus carpio, Fish Pathol, 40(2), 53–62. aoki t, hirono i, de castro t and kitao t (1989) Rapid identification of Vibrio anguillarum by colony hybridization, J Appl Ichthyol, 5, 67–73. bakopoulos v, pearson m, volpatti d, gousmani l, adams a, galeotti m, richards r h and dimitriadis g j (2003) Investigation of media formulations promoting differential antigen expression by Photobacterium damsela ssp. piscicida and recognition by sea bass, Dicentrarchus labrax (L.), immune sera, J Fish Dis, 26, 1–13. bakopoulos v, hanif a, poulos k, galeotti m, adams a and dimitriadis g j (2004) The effect of in vivo growth on the cellular and extracellular components of the marine bacterial pathogen Photobacterium damsela subsp. piscicida, J Fish Dis, 27, 1–13. balcázar j l, vendrell d, de blas i, ruiz-zarzuela i, gironés o and múzquiz j l (2007) Quantitative detection of Aeromonas salmonicida in fish tissue by realtime PCR using self-quenched, fluorogenic primers, J Med Microbiol, 56, 323–8. bautista d a, elankumaran s, arking j a and heckert r a (2002) Evaluation of an immunochromatography strip assay for the detection of Salmonella sp. from poultry, J Vet Diagn Invest, 14, 427–30. benmansour a and de kinkelin p (1997) Live fish vaccines: history and perspectives, in Gudding R, Lillehaug A, Midtlyng P J and Brown F (eds), Fish Vaccinology Developments in Biological Standardization, Karger, Basel, 279–89. berg a, rødseth o m and hansen t (2007) Fish size at vaccination influence the development of side-effects in Atlantic salmon (Salmo Salar L.), Aquaculture, 265, 9–15.

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biacchesi s, le berre m , le guillou s, benmansour a, brémont m, quillet e and boudinot p (2007) Fish genotype significantly influences susceptibility of juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), to waterborne infection with infectious salmon anaemia virus, J Fish Dis, 30, 631–6. birkbeck t h (2004) Role of probiotics in fish disease prevention, in Leung K A (ed.), Current Trends in the Study of Bacterial and Viral Fish and Shrimp Diseases. Molecular Aspects of Fish and Marine Biology, Vol. 3, World Scientific, Singapore, 390–416. bricknell i and dalmo r a (2005) The use of immunostimulants in fish larval aquaculture, Fish Shellfish Immunol, 19, 457–72. clark j r and march j b (2006) Bacteriophages and biotechnology: vaccines, gene therapy and antibacterials, Trends Biotechnol, 24, 212–18. costa j z, adams a, bron j e, thompson k d, starkey w g and richards r h (2007) Identification of B-cell epitopes on the betanodavirus capsid protein, J Fish Dis, 30, 419–26. cowley j a, dimmock c m, wongteerasupaya c, boonsaeng v, panyim s and walker p j (1999) Yellow head virus from Thailand and gill-associated virus from Australia are closely related but distinct prawn viruses, Dis Aquatic Org, 36, 153–7. cunningham c o (2004) Use of molecular diagnostic tests in disease control: making the leap from laboratory to field application, in Leung K-Y (ed.) Current Trends in the Study of Bacterial and Viral Fish and Shrimp Diseases, Molecular Aspects of Fish and Marine Biology, Vol. 3, World Scientific, Singapore, 292–312. dunbar s a (2006) Applications of Luminex® xMAPTM technology for rapid, highthroughput multiplexed nucleic acid detection, Clin Chim acta, 363, 71–82. fournier p e and raoult d (2003) Comparison of PCR and serology assays for early diagnosis of acute Q fever, J Clin Micro, 41, 5094–98. frost p and ness a (1997) Vaccination of Atlantic salmon with recombinant VP2 of infectious pancreatic necrosis virus (IPNV), added to a multivalent vaccine, suppresses viral replication following IPNV challenge, Fish Shellfish Immunol, 7, 609–19. gatta l, perna f, ricci c, osborn j f, tampieri a, bernabucci v, miglioli m and vaira d (2004) A rapid immunochromatographic assay for Helicobacter pylori in stool before and after treatment, Alim Pharmacol Therapeut, 20, 469–74. gjedrem t (2000) Genetic improvement of cold-water fish species, Aquac Res, 31, 25–33. gjedrem t, salte r and gjoen h m (1991) Genetic-variation in susceptibility of Atlantic salmon to furunculosis, Aquaculture, 97, 1–6. gonzález s f, krug m j, nielsen m e, santos y and call d r (2004) Simultaneous detection of marine fish pathogens by using multiplex PCR and a DNA microarray, J Clin Microbiol, 1414–19. grimholt u, larsen s, nordmo r, midtlyng p, kjoeglum s, storset a, saebo s and ste r j (2003) MHC polymorphism and disease resistance in Atlantic salmon (Salmo salar); facing pathogens with single expressed major histocompatibility class I and class II loci, Immunogenetics, 55, 210–19. jansson e, lindberg l, säker e and aspán a (2008) Diagnosis of bacterial kidney disease by detection of Renibacterium salmoninarum by real-time PCR, J Fish Dis, 31, 755–63. jung t s, thompson k d, volpatti d, galeotti m and adams a (2007) Variation in the molecular weight of Photobacterium damselae subsp. piscicida antigens when cultured under different conditions in vitro, J Vet Sci, 8, 255–61. jung t s, thompson k d, volpatti d, galeotti m and adams a (2008) In vivo morphological and antigenic chracateristics of Photobacterium damselae subsp. piscicida, J Vet Sci, 9, 169–75.

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karunasagar i, nayak b b and karunasagar i (1997) Rapid detection of Vibrio parahaemolyticus from fish by polymerase chain reaction, in Flegel T W and MacRae L H (eds), Diseases in Asian Aquaculture III, Asian Fisheries Society, Manila, 119–22. karatas s, mikalsen j, steinum t m, taksdal t, bordevik m and colquhoun d j (2008) Real time PCR detection of Piscirickettsia salmonis from formalin-fixed paraffin-embedded tissues, J Fish Dis, 31, 747–53. kim g, om a s and mun j h (2007) Nano-particle enhanced impedimetric biosensor for detedtion of foodborne pathogens, J Phys: Conf Ser, 61, 555–9. klesius p, evans j, shoemaker c, yeh h, goodwin a e, adams a and thompson k d (2006) Rapid detection and identification of Streptococcus iniae using a monoclonal antibody-based indirect fluorescent antibody technique, Aquaculture, 258, 180–6. leong j c, anderson e, bootland l m, chiou p w, johnson m, kim c, mourich d and trobridge g (1997) Fish vaccine antigens produced or delivered by recombinant DNA technologies, in Gudding R, Lillehaug A, Midtlyng O J and Brown F (eds), Fish Vaccinology. Developments in Biological Standardization, Karger, Basel, 267–77. lorenzen n and la patra s e (2005) DNA vaccines for aquacultured fish, Rev Sci Tech Off Int Epiz, 24, 201–13. manji f (2008) Development of methods to determine prevalence of Flavobacterium psychrophillum in farm systems, PhD Thesis, University of Stirling, UK. march j b, clark j r and jepson c d (2004) Genetic immunisation against hepatitis B using whole bacteriophage lambda particles, Vaccine, 22, 1666–71. markestad a and grave k (1997) Reduction of antibacterial drug use in Norwegian fish farming due to vaccination, in Gudding R, Lillehaug A, Midtlyng O J and Brown F (eds), Fish Vaccinology. Developments in Biological Standardization, Karger, Basel, 365–9. marko n f, weil r j and toms s a (2007) Nanotechnology in proteomics, Expert Rev Proteomics, 4, 617–26. matsuyama t, kamaish t and oseko n (2006) Rapid discrimination of fish pathogenic Vibrio and Photobacterium species by oligonucleotide DNA array, Fish Pathol, 41, 105–12. meeusen e n t, walker j, peters a, pastoret p-p and jungersen g (2007) Current status of veterinary vaccines, Clinical Microbiology Reviews, 20, 489–510. melamed p, gong z, fletcher g and hew c l (2002) The potential impact of modern biotechnology on fish aquaculture, Aquaculture, 204, 255–69. miles d j c, thompson k d, lilley j h and adams a (2003) Immunofluorescence of the epizootic ulcerative syndrome pathogen, Aphanomyces invadans, using monoclonal antibodies, Dis Aquat Org, 55(1), 77–84. moen t, fjalestad k t, munck h and gomez-raya l (2004) A multistage testing strategy for detection of quantitative trait loci affecting disease resistance in Atlantic salmon, Genetics, 167, 851–8. neelam b, robinson r a, price n c and stevens l (1993) The effect of iron limitation on the growth of Aeromonas salmonicida, Microbios, 74, 59–67. notomi t, okayama h, masubuchi h, yonekawa t, watanabe k, amino n and hase t (2000) Loop-mediated isothermal amplification of DNA, Nucleic Acids Res, 28(12), 63. palmer-densmore m l, johnson a f and sabara m i (1998) Development and evaluation of an ELISA to measure antibody responses to both the nucleocapsid and spike proteins of canine coronavirus, J Immunoassay, 19, 1–22. peddie s and secombes c j (2005) An overview of fish immunostimulant research, Fish Vet J, 8, 1–31.

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poobalane s (2007) Aeromonas hydrophila vaccine development using immunoproteomics, PhD Thesis, University of Stirling, UK. pourahmed f (2008) Molecular detection and identification of aquatic mycobacteria, PhD Thesis, University of Stirling, UK. powell j l and loutit m w (2004) Development of a DNA probe using differential hybridization to detect the fish pathogen Vibrio anguillarum, Microb Ecol, 28, 365–73. puttinaowarat s, thompson k d and adams a (2000) Mycobacteriosis: detection and identification of aquatic Mycobacterium species, Fish Vet J, 5, 6–21. rainey p b and preston g m (2000) In vivo expression technology strategies: valuable tools for biotechnology, in Curr Opin Biotechnol, 11, 440–4. rose a s, ellis a e and adams a (1989) An assessment of routine Aeromonas salmonicida carrier detection by ELISA, Bull Eur Assoc Fish Pathol, 9, 65–7. savan r, kono t, itami t and sakai m (2005) Loop-mediated isothermal amplification: an emerging technology for detection of fish and shellfish pathogens, J Fish Dis, 28, 573–81. saenz h l and dehio c (2005) Signature-tagged mutagenesis: technical advances in a negative selection method for virulence gene identification, Curr Opin Microbiol, 8, 612–19. skurnik m and strauch s (2006) Phage therapy: facts and fiction, Int J Med Microbiol, 296, 5–14. smith p d (2000) Vaccines and vaccination – a widening choice, Fish Farmer, 23(6), 45–53. sommerset i, krossoy b, biering e and frost p (2005) Vaccines for fish in aquaculture, Future Drugs, Expert Review of Vaccines, 4, 89–101. steiropoulos n a, yuksel s a, thompson k d, adams a and ferguson h w (2002) Detection of Rickettsia-like organisms (RLOs) in European sea bass (Dicentrarchus labrax, L.) by immunohistochemistry, using rabbit anti-Piscirickettsia salmonis serum, Bull Eur Assoc Fish Pathol, 22(5), 428–32. sudheesh p s, crane s, cain k d and strom m s (2007) Sortase inhibitor phenyl vinyl sulfone inhibits Renibacterum salmoninarum adherence and invasion of host cells, Dis Aquat Org, 78, 115–27. thompson k d and adams a (2004) Current trends in immunotherapy and vaccine development for bacterial diseases of fish, in Leung K-Y (ed.), Current Trends in the Study of Bacterial and Viral Fish and Shrimp Diseases. Molecular Aspects of Fish and Marine Biology, Vol. 3, World Scientific, Singapore, 313–62. vatsos i n, thompson k d and adams a (2002) Development of an immunofluorescent antibody technique (IFAT) and in situ hybridisation to detect Flavobacterium psychrophilum in water samples, Aquac Rese, 33, 1087–90. wilson t and carson j (2003) Development of sensitive, high-throughput one-tube RT-PCR-enzyme hybridisation assay to detect selected bacterial fish pathogens, Dis Aquatic Org, 54, 127–34. yang s y, chieh j j, wang w c, yu c y, lan c b, chen j h, horng h e, hong c y, yang h c and huang w (2008) Ultra-highly sensitive and wash-free bio-detection of H5N1 virus by immunomagnetic reduction assays, J Virol Meth, 153, 250–2. yuce a, yucesoy m, genc s, sayan m and ucan e s (2001) Serodiagnosis of tuberculosis by enzyme immunoassay using A60 antigen, Clin Microbiol Infect, 7, 372–6. world organisation for animal health (2006) Manual of Diagnostic Tests for Aquatic Animals, 5th edn, OIE, Paris.

8 Controlling parasitic diseases in aquaculture: new developments C. Sommerville, University of Stirling, UK

Abstract: The expanding global aquaculture industry is urgently in need of more effective methods for the control of parasitic diseases. Advances in our understanding of parasite biology, host parasite interactions and improved diagnostic methods using new technologies which contribute to better management and control are highlighted in this chapter. There is an increasing volume of research pointing to effective control without the use of chemical interventions. The processes leading to increasing regulation and cost of drugs and chemicals are described. Their integration into a more sustainable pest management strategy is discussed and the imperative for the inclusion of resistance management principles in any strategy is emphasised. Key words: aquaculture, parasites, disease control, new developments, integrated pest management.

8.1 Introduction Aquatic parasites attracted little attention until the large-scale development of intensive aquaculture in the early 1970s created a surge of interest in parasitic diseases, resulting in an increase in our knowledge and understanding of parasites of aquatic organisms generally. The high fish density associated with culture intensification leads to increased chances of parasite/host encounters which inevitably results in a higher parasite prevalence and intensity. Parasitic diseases can impact on aquaculture systems in a number of ways which will determine their economic cost (Sommerville, 1998). Mortality has evident costs determined by the size and age of the fish but, more often, parasite infection causes morbidity and loss of appetite with a resultant waste of food, reduced food conversion efficiency and specific growth rate which, over a grow-out period in a population of fish, may account for a noteworthy proportion of the profits. In some cases

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parasite infections may be zoonotic, or reduce the market value owing to their large size or unaesthetic appearance, and this has been known to cause rejection of entire stocks of fish even where there was no discernible impact on the welfare of the fish; other parasitic infections may affect broodstock quality. The scarcity of taxonomists with expertise in aquatic parasites has always been a problem. It was, and is still, common to encounter undescribed parasites in fish and new methods are needed to aid non-specialists. It is necessary to underpin research into parasite control in aquaculture systems with studies on parasite biology and the host–parasite interactions for several reasons. In the first place, it is important to establish whether it is necessary to intervene as some parasites have no impact on the growth performance or wellbeing of the culture species and, secondly, what threshold level of infection is sustainable without treatment. Understanding the host–parasite interactions can lead to targets for drugs or other nonchemical treatments and knowledge of the life cycle of the parasite is key to making appropriate intervention at the right time. Many parasites have complex life cycles and knowledge of the location of transmission stages and vectors is essential for prevention of infection and management of the disease without the use of drugs or chemicals. However, commonly, the method of choice for the control of parasitic problems in fish has been chemotherapy, despite the availability of a range of non-invasive methods. The following discussion will illustrate how the potential of new methodologies to study the issues noted above may lead to more effective control.

8.2 Effects of parasitic disease in aquaculture In today’s aquaculture industry, parasite epizootics continue to have major economic consequences, and there is an urgent need to find more effective methods of control. The magnitude of the economic losses has increased as a result of the size of the operations, as in the salmon mariculture industry, and the greater part of the economic costs are currently due to only a small number of parasite pathogens. For example, the estimated cost to the industry of sea lice infections can be as high as 5 % of annual costs in growth alone (Rae, 2002). Drugs and chemical treatments have become an increasingly significant component of the production costs of aquaculture since the 1980s. Most of the early, cheaper, treatments are no longer available and the new treatments have been specifically tailored to aquaculture requirements and therefore are at higher cost. As a result of the widespread outbreaks of disease, attempts are now being made to regulate the aquaculture industry at governmental level, although this varies from country to country (Scarfe et al., 2006). The primary objective is to control the movements of fish across boundaries in a manner which minimises the risk of the spread of disease. A further major

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objective is the regulation of the use of drugs and chemicals which are used to treat disease with a view to developing good practices which will protect the consumer and the environment without compromising the welfare of the fish (see below). There is now a greater awareness of biosecurity in aquaculture on a global scale (Subasinghe and Bondad-Reantaso, 2006). Legislation is now becoming widespread and is coupled with better techniques for detection of low levels of chemical/drug contamination of food products (Reimschuessel, 2008). The impact of exotic parasites on native fauna has been recognised since the spread of rainbow trout, common carps, tilapias and eels around the globe. Indigenous species often have little defence against ‘foreign’ parasites and, without the benefits from evolution, may succumb rapidly. Unfortunately little control was exercised and efforts to contain further spread have often been too little, too late; many cosmopolitan pathogenic parasites have been recognised, e.g. the ciliate Ichthyophthirius multifiliis originated in the tropics of Asia and now extends into the sub-arctic regions (Valtonen and Keränen, 1981). Plans for quarantine systems to contain parasitic diseases have been identified since the early 1980s and taken up by governments and major agencies, e.g. FAO (Arthur, 1996; Subasinghe and Arthur, 1997; Subasinghe, 2004), but implementation at country or regional level for the most part seems to have been half-hearted. Thus, the transfaunation of fish parasite pathogens across global boundaries has been largely avoidable but has been facilitated through misjudgement, often founded on lack of knowledge of the parasite pathobiology. There has, to date, not been sufficient expertise available in vulnerable countries at a high enough level to understand the need for, and to implement effectively, preventative practices into the regulatory process. Expertise has been growing rapidly since the 1970s and most countries for which fish culture is a significant source of income and/or protein now have some diagnostic and advisory personnel who make recommendations on how to treat parasite problems (Scarfe et al., 2006). Organisations responsible for protection of wild stocks have been drawn into debates on the potential introduction and spread of parasite pathogens through aquaculture activities. These can be highly controversial, as in the case of sea lice. Concentration of research efforts on these problems has been ultimately beneficial to the aquaculture industry. For example, there has been widespread concern that mariculturalists do not effectively control sea lice infections and that these are the source of lice seen on wild salmonids, currently a major issue in Canada, see for example Krkosˇek et al. (2005). This has been hotly debated and is the cause of deep political division. Some have gone so far as to blame the decline in salmon and sea trout populations on sea lice infection from the farming activities to the extent that Canada, Norway and Scotland have invested considerable research effort in trying to demonstrate conclusively whether this is the case and it is very publicly debated in these countries (McVicar, 2004; Heuch et al.,

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2005; Krkosˇek et al., 2005, 2006, 2007; Boxaspen, 2006; Hilborn, 2006; Hume, 2008; Penston et al., 2008). A benefit of this controversy is the allocation of funding to try to find new and sustainable methods of control, some of which are now the subject of legislation. Similarly, the research associated with the protection of wild salmon parr from the monogenean Gyrodactylus salaris has contributed to our understanding of host–parasite interactions, parasite virulence, epidemiology and phylogeny of this genus, many species of which are common in culture systems worldwide (Bakke et al., 2007). G. salaris is a non-virulent parasite of Baltic salmon which, when introduced into Norwegian rivers, has been associated with mass decline of Atlantic salmon stocks on which it becomes more virulent. Research in non-endemic countries has also led governments to develop risk analyses and containment policies.

8.3 Advances in the understanding of parasite biology and host–parasite interactions Knowledge of the pathogenic processes of many aquatic parasites is now well established, as is the greater impact on a host of an exotic parasite with which it has not evolved, for example, whirling disease in rainbow trout (Hedrick et al., 1999; Rose et al., 2000; Gilbert and Granath, 2003), Anguillicola crassus in European eels (Kennedy and Fitch, 1990; Würtz et al., 1998) and Myxidium leei in Mediterranean mariculture (Diamant, 1998). Perhaps less well recognised and researched are the influences and relationships a parasite has with other pathogens within the same host and their combined impact on the host. The modulated susceptibility of a host in the presence of one or more pathogens has been studied for higher animals and rates more attention in fish (Booth et al., 2008). Describing the parasite population dynamics in wild fish allows for the assessment of the influence of environmental factors which are commonly associated with temperature and season but, in cultured fish, parasite populations are also affected by husbandry factors such as stocking time, size, age and reproductive state of the host, grading, transportation events, etc. and these, in turn, affect the parasite management strategy; an intervention at the inappropriate time is costly without having any benefit. Monitoring, informed by the population dynamics, allows intervention at an early stage when prevalence (percent of fish infected) and mean intensity are low. The life cycles of parasites are influenced by many aspects of the environment. During its life the parasite may have to contend with several potentially hostile environments, i.e. one or more hosts, which may constitute unrelated taxa, as well as the external aquatic environment during free-living stages. Climatic influences, host reproductive cycles and behaviour will be superimposed on these factors and determine when the parasite reproduces, leaves the host, becomes dormant, etc. Such epidemiological infor-

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mation is necessary both to prevent infection and to target any intervention (Giorgiadis et al., 2001). Mathematical models incorporating such data are increasingly being researched in order to analyse the risk of epidemics through the spread of pathogens across boundaries and to develop strategies for treatment and control, e.g. the risk of introducing Gyrodactylus salaris (Peeler and Thrush, 2004; Peeler et al., 2004). Most importantly, understanding the parasite biology and epidemiology has led to management solutions to a parasite problem without chemical intervention, for example, fallowing (Bron et al., 1993). Taylor et al. (2006) studied the complex interactions contributing to problem Argulus infections in stillwater trout fisheries in England and used risk analysis to identify contributing factors. A subsequent analysis of the risk factors pointed to low water clarity, slow stock turnover and high temperature influencing the abundance of Argulus, information which led to management of the infection without the use of chemicals. Knowledge of the biology and life cycle of a parasite pathogen is necessary for the efficient application of chemotherapeutants to the target stage. The need for targeted interventions is well exemplified with the use of various products which are useful against parasitic crustaceans. Crustacean parasites have a complex life history with numerous stages separated by moults. These stages are differentially susceptible (depending on the species) to the organophosphates (pre-adults) and the insect growth regulators (juveniles), while hydrogen peroxide, pyrethroids and emamectin benzoate have some activity against all stages. Excessive, ineffective use of pesticides with no economic benefit is harmful to the environment, the fish and the image projected by the industry to the public and the regulators, as well as contributing to the development of resistance (Jones et al., 1992). Mathematical models can help target effective control programmes, such as the life cycle model constructed by Tucker et al. (2002) to predict the outcomes of targeting treatments against different life stages of L. salmonis. Revie et al. (2005) used retrospective data on infection levels in Scottish fish farms with a view to advising the industry on a treatment strategy and Fenton et al. (2006) produced a population model of Argulus coregoni in Finnish trout ponds which pointed to variable egg hatching reducing the effectiveness of chemical control strategies in the field. Molecular epidemiology is applied to fish parasitic diseases but is not yet widespread. However, with the increase in genomics data and epidemiological studies, this is a promising area for future research in fish which has been successfully employed for the study of sea lice populations for example (Banks et al., 2000; Todd et al., 1997, 2004). A range of biochemical and immunohistochemical techniques have been important in elucidating host–parasite interactions. An exciting area for research which will ultimately result in new control strategies is the interplay between the immune responses of the fish to the parasite and the parasite’s immunomodulatory capability/activity. Whilst it reveals intrinsi-

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cally interesting host–parasite interactions, at the same time it may reveal targets for vaccine developments. For example, Bell et al. (2000) mapped the secretory glands of L. salmonis and demonstrated peroxidase secreting glands in the oral region. Butler et al. (2000) developed immunoassays to evaluate the immunosuppressive activity of secretory products produced in vitro by L. salmonis copepodids in an artificial skin model. Progress has been made on identifying some of the products of the interaction (see Firth et al., 2000; Ross et al., 2000; Johnson et al., 2002; Fast et al., 2003, 2004). Future research using proteomics and microarrays to study gene expression together with RT-PCR and real-time PCR for L. salmonis will form a model for future studies of many such parasite–host systems. The application of DNA probes to histological sections using in situ hybridisation is not yet widespread but helped elucidate the life cycle in the fish of the pathogenic myxosporeans, PKX (Tetracapsuloides bryosalmonae) and Sphaerospora trutta, which were unknown and would have remained so without the use of this technique to detect the parasite in blood and other organs where it was otherwise not visible (Morris et al., 1999; Holzer et al., 2003).

8.4 Advances in methods of identifying parasites Early diagnosticians of parasitic diseases often had to be content with identification to the generic or even family level. With the greater number of experts researching aquatic pathobiology and new methodologies, the situation has improved considerably. Unfortunately, there are still few taxonomists, but there are more keys and more helpful techniques. Morphology-based techniques are still largely used such as SEM and TEM (scanning and transmission electron microscopy) and are essential for determining protozoa and other small histozoic stages of parasites. Basic morphology is supported by specific staining techniques, e.g. chaetotaxy (Shinn et al., 1998), and newer technologies such as confocal microscopy (McGurk et al., 2005; Arafa et al., 2007) and image analysis. Developments in image analysis have made it possible to analyse digitally shapes to extract the key features. Such data can then be subjected to a statistical classifier (Shinn et al., 2000). This has been most advanced in the automation of the discrimination of Gyrodactylus salaris from other salmonid species of the genus (Shinn et al., 2000). The key characteristics of species of the gyrodactylids are the size, shape, number, etc. of the hard parts, the sclerites, and these lend themselves readily to digital image analysis. Recent developments have shown that these can be semi-automated, thereby relieving the need for specialist taxonomists (Kay et al., 1999; Harris et al., 2008). This is especially helpful where pathogen monitoring systems are dependent on non-specialists and where introduction of only a single specimen of a species is risky, as is the case of Gyrodactylus salaris.

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Parallel to these developments, and now having much wider potential, are PCR and DNA sequence analysis. The most common targets are the small sub-unit ribosomal RNA genes and the internal transcribed regions of rDNA loci. This technology has greatly increased our understanding of some groups such as the myxozoans which has made remarkable progress as a result. Prior to the molecular investigations of these parasites, this little-known group was thought to be protistan. However, they are now known to be highly specialised bilaterians close to the nematodes based on DNA sequences and ultrastructure. Further, it was demonstrated conclusively that a little-known group of sporozoan parasites of oligochaetes, the Actinosporea, are the alternate stages in the life cycle of the Myxosporea of teleosts, thus throwing the systematics into confusion. It is unlikely that these investigations would have been carried out if it wasn’t for the important myxozoan diseases in aquacultured fish, ‘Whirling Disease’, caused by Myxobolus cerebralis and ‘Proliferative kidney disease’ (PKD) caused by an unknown organism referred to as PKX. Subsequent studies on the Myxosporea have been able to link actinospore stages to myxospore stages, consequently elucidating the life cycles and reassigning them taxonomically using molecular phylogenetics. Molecular techniques are now so widely disseminated worldwide that descriptions of new parasite species are commonly accompanied by sequence data and there is a considerable gene bank resource to draw on. Molecular systematics is having a major effect on some taxa and it will be interesting to see how they overlay traditional taxonomy (Kent et al., 2001; Fiala, 2006; Holzer et al., 2007). Molecular techniques for the most part are more useful for parasitology than the immunodiagnostic techniques, e.g. IFAT and mAB probes, for reasons of simplicity, sensitivity and accuracy.

8.5 Advances in methods of controlling parasites 8.5.1 Non-chemical methods Aquaculturists have tended to use non-chemical methods only when chemotherapeutants were either inadequate or unavailable. Some nonchemical methods remain the most effective, such as manual removal of lymnaeid snails to control eye fluke and the introduction of a substrate to remove eggs of Argulus and leeches from ponds and lakes, although the modern adaptation of this two millennia-old Chinese method is to suspend plastic boards below the surface (Gault et al., 2002). Traditional horticultural methods, such as fallowing, were not introduced into salmon cage culture until the early 1990s (Bron et al., 1993). This, and single year class stocking, was shown to delay the build-up of sea lice populations until the fish are larger and less vulnerable to infection and, although it is largely a practice in maricultured salmon to minimise the effect of sea lice, it has benefits in relation to other diseases such as Furunculosis, as well as

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environmental benefits allowing some recovery of the sea bed below cages. The length of the fallow period was determined from knowledge gained from research into sea lice life cycles coupled with economic factors. Biological control has always been a popular concept, but its success has never lived up to the expectation. A common application which probably arose from some of the attempts to control schistosomiasis, but which did not successfully translate into fish culture ponds, was the stocking of mollusc-eating fish to control problematic digenea such as eye flukes and zoonotic heterophyids (Ben-Ami and Heller, 2001). Perhaps the most successful biological control application for fish parasites is the use of wrasse for control of sea lice (Sayer et al., 1996). Members of the wrasse family (Labridae) feed from surfaces such as rocks but will remove lice from the surface of salmon when confined in cages along with the salmon. This method was widespread in Norway and Scotland in the 1990s but has largely declined as there are a number of disadvantages with wrasse which are costly to surmount. In the main, these are associated with the lack of domestication of the species. There is potential for improvement, but little development of the use of wrasse has taken place since the mid-1990s. The best use of wrasse is in conjunction with a range of other methods, targeted to assist juvenile and newly stocked fish for the first few months at sea and fits in well with a coherent integrated pest management (IPM) strategy for sea lice. Biopesticides are based on pathogenic microorganisms, bacteria, fungi, viruses, protozoa, nematodes, etc. and are specific to a target pest. They have a high potential for being more ecologically sound but are still at an early stage of research. Preliminary studies of potential biopesticides have already been carried out for L. salmonis (Sommerville and Harper, unpublished). The natural pathogens have been identified (Freeman, 2002; Treasurer and James, 2002; Freeman et al., 2003) and studies on viral and bacterial pathogens advanced. Biological control, whilst highly favoured by conservationists and organic culture agencies, e.g. Soil Association in the UK, has not been taken up enthusiastically by the industry apart from wrasse. However, with further research it is easy to envision the development of a biopesticide. Although none so far have been developed for an aquatic production system, the problems to be overcome are similar to those for terrestrial ones such as specificity, large-scale production, stabilisation, storage, product application registration, etc. The use of semiochemicals has been explored for the control of L. salmonis. Semiochemicals are species specific odours (kairomones) which aid in host location. These have been studied for L. salmonis and shown to affect behaviour of motile stages. It is suggested that these chemicals, once identified, could form the basis for control of the parasite, e.g. by use of lures, but its practicability has yet to be established (Ingvarsdóttir et al., 2002). Early recognition of a strong positive phototaxis exhibited by L. salmonis free-living copepodids led to the invention of a light lure by a Scottish company in 1996. This rather complex and expensive piece of

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equipment was shown to be totally ineffective when suspended from sea cages (Gravil, 1996), illustrating the folly of imagining that what appears to be a relatively simple organism is responding to a single stimulus in its natural habitat. Understanding parasite biology is a very fertile field in the search for non-chemical controls. Ideally, these methods would be combined with an effective vaccination programme; however, the development of any commercial parasite vaccine has been elusive. Studies on fish immune responses to parasites abound and, from the early 1970s, were able to demonstrate humoral factors in response to parasite infection, although most studies failed to demonstrate protection. However, it is only with the greater understanding of the immune system in fish that more realistic approaches to parasite vaccine development have come about, such as those by Canadian workers, Ross et al. (2008), who have taken out a US patent for a recombinant vaccine against caligids (sea lice). Vaccine development has been most advanced for three parasite pathogens, namely Cryptobia salmositica, Ichthyophthirius multifiliis and Lepeophtheirus salmonis, through different approaches. Woo and colleagues developed a successful experimental live vaccine to an attenuated strain of Cryptobia salmositica and subsequently developed a recombinant DNA vaccine based on a 200 kDa metalloprotease (Tan et al., 2008). There is a substantial body of work describing the developments, summarised by Woo (2006). No useful attenuated strains of I. multifiliis have been described although controlled low dose administration of the parasite was shown to give protection as long ago as 1974 (Hines and Spira, 1974). Vaccine development has concentrated on immobilising antigens known as i-antigens which react specifically with ciliary membrane proteins. A number of IgG Class mAbs were identified which conferred protection (Dickerson and Clark, 1998) and are thought to be produced locally in the fish skin. Using molecular techniques, a substantial body of knowledge of the host parasite interactions has now been accrued which will ultimately lead to a vaccine. The development of a commercial vaccine has been delayed partly owing to the difficulty of axenic culture of the parasite in vitro on a commercial scale. Immobilisation antigens have been shown to produce protective immunity in channel catfish (Wang and Dickerson, 2002; Wang et al., 2002), and the cloned genes are expressed using a non-parasitic ciliate Tetrahymena thermophyla using plasmid vectors. A large amount of recombinant protein is produced which has been shown to produce protective immunity in channel catfish. The research associated with these developments is summarised by Dickerson (2006). Early attempts at vaccines against L. salmonis ranged from crude polyclonal antigens to recombinants (Raynard et al., 2002). Grayson et al. (1995) used a partially purified extract with limited success and Raynard et al. (1994) used recombinant louse proteins, although problems of funding halted progress of the research despite their identification of a B-galactoside

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fusion protein which reduced fecundity in the parasite. The more recent studies such as those outlined by Johnson and Fast (2004) are designed to avoid dependence on concealed gut antigens, and the way forward would be to block immunosuppressive products secreted by the louse. Husbandry There is commercial activity in the production of specific pathogen-resistant stocks which involves the selection of families with natural resistance. Most research activity has been against viruses although some progress has been made with stocks showing resistance to sea lice, for example, research into the heritability of resistance to sea lice infection (Mustafa and MacKinnon, 1999; Glover et al., 2005). Commercial research is underway following promising lab-based selection of families with high resistance and field trials are planned. Similar work is being carried out with amoebic gill disease. These are necessarily long-term studies; however, if genetic linkages are identified, selection for resistance can be facilitated by means of marker assisted selection (MAS) and should accelerate the process (see Jones et al. (2002) for review of different approaches). Glover et al. (2007) investigated polymorphism in MHC genes to resistance to lice and found a link between susceptibility and MHC Class II. Should transgenics become acceptable in food fish, there may be a role for disease-resistant fish, but parasite-resistant transgenic farm animals are, at present, at the early experimental stage of development. Transgenic strategies to confer resistance to specific diseases include the transfer of major histocompatibility – complex genes, T-cell receptor genes, immunoglobulin genes, genes that affect lymphokines – as well as specific disease resistance genes (Niemann et al., 2005). Transgenic crops such as Bt crops which incorporate a gene encoding for the Bacillus thuringiensis (Bt) toxin which is poisonous to insect pests, have become accepted in the USA, and this technology may have potential for fish. However, the claim that there is a reduction in pesticide use is only clear so far with Bt cotton. This technology is highly controversial but is considered to be a useful, environmentally friendly control by the US Environmental Protection Agency, and insect-resistant management plans are mandatory with their use (for useful information see Insecticide Resistance Action Committee (IRAC) http://www.irac-online.org). Interestingly, surveillance data shows that a significant proportion of farmers break the rules. There is potential for the engineering of culture systems to minimise outbreaks of disease, e.g. eliminating pathogens through filtration of incoming water is becoming more widespread and economic. Land-based marine systems have avoided sea-lice infection and net-pens could be designed to avoid deformations due to crowding, which alter fish density and dose determination. The advanced design of recirculation systems offers the potential for greater control of pathogens generally, and offshore cages would be expected to include a high degree of environmental control but poorer disease monitoring.

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Despite chemotherapy being the favoured control method by the aquaculture industry, improved husbandry to produce healthier, less stressed fish, improved diets and environmental conditions have contributed in a major way to disease control. Some diets are now available containing supplements which act as immunomodulators, improving the natural ability of the host to resist pathogens (Bricknell and Dalmo, 2005). Several in-feed immunomodulators, e.g. B-glucans, nucleotides, vitamins and yeast derivatives etc., have been tested in fish with parasite infection and apparently shown to have beneficial effects, e.g. sea lice (Burrells et al., 2001), Loma salmonae (Guselle et al., 2006 ). However, there are few fully scientific studies and data on the method of action are lacking. The results of research into light levels have also contributed to reduced stress.

8.5.2 Chemotherapy The search for a ‘quick fix’ has always been an industry goal. The ‘Medicine chest’ available in the beginnings of the current rapid expansion of intensive aquaculture was extremely limited in useful chemicals and dependent largely on extensive carp culture from the Middle and Far East and the ornamental fish trade. Some are still used in the ornamental trade and some, such as the highly toxic organochlorine insecticides, were banned from use in the 1970s and 1980s although may still be available and used in some parts of the world. Research efforts to find suitable treatment compounds were stepped up as new diseases emerged from the intensification process. Some 400 substances (including synonyms) are listed by Hoffman and Meyer (1974) of the Bureau of Sport Fisheries and Wildlife in the USA, accompanied by the minimum of advice and any available reference to their use. Almost all of these have fallen by the wayside since medicines have become heavily regulated for use in food fish in many countries, including the EU, the USA, Japan and Canada. The use of chemotherapy to control parasites in fish populations should take into consideration, as well as the treatment of the disease in the fish, the toxicity to the consumer and the impact on the environment. Unfortunately, there is no single agency which deals with all these aspects of products found useful for fish. The major controlling agencies are those which cover veterinary medicines which have produced a significant body of legislation based on quality, efficacy and safety. There has been a general trend for developed countries to continue to increase regulation of drugs and, as a result, farmers in these countries find they have access to a very limited number and range of products. Some of the most useful compounds have been banned for use in aquaculture, e.g. dimetranidazole (DMZ) for use against the ciliate protozoan Ichthyophthirius multifiliis and the flagellate Spironucleus spp., and have not been replaced. Malachite Green was a very useful chemical, well known for years to the ornamental fish trade but very effective against I. multifiliis in culture

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ponds and raceways when combined with formalin. On the other hand, many other countries have no licensing of drugs and are only concerned when there is a need to export to a highly regulated country. New drugs are rarely presented for aquaculture use as the pharmaceutical companies regard the market as too small and it has only really been worth their while to develop treatments for sea lice in salmonid culture, although the same products are useful against other crustacean ectoparasites, e.g. pyrethroids are used for isopod infection in Mediterranean mariculture and Argulus in freshwater. The data required for the authorisation of new products is voluminous and complex (Woodward, 1996). Schnick (1991) outlined the processes required for five continents (Africa, Asia, Australia, Europe and North America) and lists the approved drugs at the time. Most authorised products are derived from active compounds already used in mammalian or veterinary medicine. There are no new molecules designed to control fish parasites. For a marketing authorisation to be issued there are usually three factors to be taken into account. These are safety, efficacy and quality, although these are organised in different ways through a range of governmental organisations, depending on the country in question, see Schnick et al. (2005). Animal safety is determined through tolerance studies for adverse effects of single or repeat doses and all adverse reactions should be reported. There may be a change in the perception of an adverse reaction with the current increased awareness of animal welfare; this would also be informed by the current better understanding of stress in fish (Huntingford et al., 2006). With new information or unacceptable reactions, previously licensed products may be revoked. Consumer safety is effected through the establishment of maximum residue levels (MRLs) which are required to be set for a market authorization (MA). The details of how these are set in the EU are well outlined in Woodward (1996) and there are guidelines for how the studies should be carried out for registration purposes. Depending on the status, a drug may be placed in one of four annexes, and Annex IV includes substances which cannot be used on public health grounds and are effectively prohibited for use in food animals owing to the risk to the consumer. This categorisation is unlikely to be changed but may become accepted globally and incorporated into legislation worldwide. On the whole, the pattern over recent years has been to place compounds previously found to be useful into the Annex IV category, thereby reducing the number of available parasiticides to a mere handful. Data required for an MRL are extensive (Woodward, 1996) and extrapolation from mammalian levels is impossible because of the difference in metabolism of a poikilotherm; a complicating factor being the changes due to environmental temperature and salinity which must also be taken into account. There are some concessions which have been helpful to aquaculture, such as the now widely accepted Joint FAO/WHO Committee on Food Additives (JECFA) and the Centre for Veterinary

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Medicine Products (CVMP) old drug policy which allows substances such as formalin which have a long history of use, to be fast tracked by allowing safety experience to be taken into account (JECFA, 1993). It seems likely that this situation will only exist until data from new studies establish an acceptable risk. The MRL forms the basis for the determination of the withdrawal period which is determined by the pharmacokinetics and is mostly defined in terms of degree days. The MRL is also the yardstick used for surveillance, where surveillance is carried out, e.g. in the EU and USA. Policing the regulations in the interests of the consumer and the environment can feed the media with exposé stories. The ‘backlash’ in the finding of regulatory transgressions is very damaging to the aquaculture industry and can have serious adverse effects on the markets and consequently the economics, e.g. farmed salmon was added to the UK statutory residue surveillance programme in 2001 after the Food Standards Agency (FSA) of the UK forwarded anecdotal evidence it had received that the banned Malachite Green was being used in fish farming. There is now statutory surveillance for Malachite Green and the avermectin, Ivermectin©, at one time used to control sea lice. The result of the adverse publicity has been the tightening up of safety processes at grow-out sites and markets, and it can be said, therefore, to have had some beneficial effects. It is likely that surveillance will increase and include more molecules. This also has an indirect effect on overseas producers, for example, increasingly those from Asia who wish to import into the UK and other countries with effective surveillance. An alternative approach permits veterinarians to treat a condition in an animal by prescribing a product which does not have a specific MA for that species, provided only a small number of animals are involved, certain information is recorded and a standard withdrawal period of 500 degree days is used (Woodward, 1996). This facility, commonly referred to as ‘the cascade’ or ‘Off label’ system, is an important consideration for animal welfare which is increasingly recognised. Guidelines for fish welfare in aquaculture are currently being developed by a number of organisations including, for example, the European Food Standards Agency (2008), which are likely to form the basis of an EU Directive in this case and applied by regulatory authorities within the EU. Greater concerns are also shown for operator safety than previously in some regions, i.e. the EU, USA, Canada, etc., and this is now incorporated into the authorisation package; the hazards and how to deal with them are displayed on the packaging, and proper adherence to these directions may be monitored and subject to other legislation, e.g. Health and Safety. There are considerable extra costs involved in the form of lockable safety stores on site, equipment, such as respiratory masks, and protective clothing. Additional antidote kits and monitoring for acetyl cholinesterase is required in the case of organophosphates, which are expensive and have a relatively short life requiring constant updating.

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Environmental safety is now one of the most influential factors in chemotherapy in fish, especially in the marine environment. The public response to usage of chemicals in inshore waters, largely for the treatment of sea lice on salmon, has influenced the legislative controls almost as much as the scientific data, resulting in severe restrictions for use, particularly in Scotland, Norway and Canada. Ecotoxicological studies of the fate of treatments for sea lice, some of which are prolonged, have been retrospective, and therefore the precautionary principle has been applied and, indeed, demanded by conservation bodies. New technological developments in assessment of environmental impacts will eventually provide the rationale for regulation. Regulation and monitoring guidelines on procedure deal with a number of parasiticides (most being sea lice treatments) from which environmental quality standards (EQS) are set. Bath treatments result in the release of the product, after a fixed period, into the environment and, in some countries, such discharges require permission; this is one way by which treatments may be regulated, i.e. by controlling discharge consents. A further way in which regulations may be implemented is through a programme of monitoring. This seems to be a means favoured by regulators in Scotland, Ireland and Norway, and a monitoring programme goes hand in hand with authorisation. The implementation of threshold levels for sea lice would appear to be to satisfy the aquaculture critics whose concern is for wild fish populations, but this risks increasing the amount of pesticide used, with consequent resistance and environmental effects. It is likely that the environmental constraints on administration of bath treatments will increase, and make in-feed/oral treatments more favourable for development. Although in-feeds are still likely to be highly regulated, the impact is likely to be less, especially since mechanisms to minimise the amount of uneaten food are being developed as well as the collection of eliminated solid faecal waste in the interests of environmental quality. Validated spatial models are now in use to predict environmental impact and these may be used with geographic information systems (GIS) to great effect (Pérez et al., 2002). As has been indicated above, legislation abounds on drug regulation, largely arising from those (usually developed countries countries) with major aquacultural interests. It has been recognised that there is a need for harmonisation of the regulations, and some attempts have been made in this direction (Schnick, 2001). One example is the trilateral programme ‘International Co-operation on Harmonization of Technical Requirements for Regulation of Veterinary Medicinal Products (VICH)’, the parties to which are the EU, USA and Japan with Australia and New Zealand as observers. This has become a global issue. It is anticipated that the VICH recommendations should replace corresponding legal requirements. A major benefit will be that a common data package can be used for authorisation throughout the world. What is certain is that regulation will increase and controls

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and monitoring will tighten, further discouraging the pharmaceutical companies from processing products for authorisation. Already, the cost is such that it is only feasible to pursue products for high value species where the market size is large enough and the value of the product sufficiently high for the farmer to treat rather than destroy the stock or harvest early. This explains why most of the authorised products for fish parasites are those developed to treat sea lice on salmonids. During recent years new product licences have been issued or given appropriate governmental permits (see Table 8.1). Some of these have been withdrawn subsequently, e.g. Nuvan® 500EC (Ciba-Geigy Agrochemicals) (dichlorvos), and others have not found favour with the end-user, and market sales have declined. The use of hydrogen peroxide was highly favoured because it was a familiar compound used in the food industry and a search for other such compounds is highly desirable. The expansion of organic fish production is, at least partly, constrained by the lack of acceptable treatments for parasitic disease and the search for natural products which can be used is a very fruitful area for research. As can be seen from Table 8.1 there are now a number of oral parasiticides authorised for sea lice treatment. It is also evident from the table that the range of treatments available provide for different modes of action, and it should have been possible to avoid the development of resistance in sea lice with the appropriate rotation of use. However, even in the light of this knowledge, fish farmers have tended to persist in the use of single chemical treatments which has resulted in the development of resistance to some of the most effective compounds (Jones et al., 1992; Treasurer et al., 2000), the most recent one being emamectin benzoate which is under investigation (Lees et al., 2008). Furthermore, there is ample anecdotal evidence of cost cutting which has accelerated the development of resistance, e.g. use of less than the recommended dose. This was known in some cases to have been carried out through ignorance of the formulation concentration – a case of not reading the label carefully – but in many cases, it was under economic pressure. The costs of these therapeutants are very high due to high development costs which are passed on to the farmers. Fortunately, a few of the large pharmaceutical companies are continuing to search for new sea lice treatments. The ideal product would have all the characteristics shown in Table 8.2, and the further development of products with novel modes of action would be advantageous. Resistance to dichlorvos in sea lice populations was first reported in 1992 by Jones et al. (1992). Resistance development and its mechanisms have been recognised and largely understood since 1947 – when resistance to DDT in houseflies was detected within a few years of the introduction of synthetic organic insecticides – and are now being studied in sea lice (e.g. Fallang et al., 2004, 2005). With every new pesticide developed, resistance has appeared within 2–20 years after their introduction, leading to a

Organophosphate Organophosphate Organophosphate Organophosphate Oxidant Oxidant Synthetic pyrethroid Synthetic pyrethroid Avermectin Avermectin

Benzylphenylurea Benzylphenylurea

Nuvan® 500 EC Aquaguard®* Neguvon® Salmosan®* Paramove®* Salartect®* Excis®* Alphamax®* Ivomec® Slice®*

Calicide®* Lepsidon® Teflubenzuron Diflubenzuron

Dichlorvos Dichlorvos Trichlorfon Azamethophos Hydrogen peroxide Hydrogen peroxide Cypermethrin Deltamethrin Ivermectin Emamectin benzoate

Active ingredient

* Products having a marketing authorisation in the UK. Note: The dichlorvos products are no longer available.

Product type

Chemotherapeutants used against sea lice

Product name

Table 8.1

AChEase inhibitor in cholinergic nervous systems AChEase inhibitor AChEase inhibitor AChEase inhibitor Not established possibly mechanical Not established possibly mechanical Interferes with sodium channels in axon membranes Interferes with sodium channels in axon membranes Interferes with sodium channels in axon membranes Interferes with GABA recepters in the peripheral nervous system Chitin synthesis inhibitor Chitin synthesis inhibitor

Mode of action

Oral/in feed Oral/in feed

Bath Bath Bath Bath Bath Bath Bath Bath Oral/in feed Oral/in feed

Application method

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Table 8.2 The ideal chemotherapeutant For fish: • Low dose efficacy • Prolonged duration of efficacy • Single dose application • No effect on appetite • Wide toxicity margin • Effective against all stages of the parasite For humans: • Completely safe to handle in concentrated form • Rapidly metabolised in fish flesh to undetectable levels • Preferably used in the food industry and therefore acceptable to the public For the environment: • Specific toxicity to target species • Excreted metabolites in harmless form and rapid decomposition in the water • By-products which are already components of the natural environment and non-fouling For the product: • Cheap • Oral/in-feed administration • Prolonged shelf life • No evidence of cross-resistance

treadmill effect. Parasite pathogens in aquaculture are now firmly on that treadmill. The pattern is familiar as the selection pressure on the heritable resistance traits is accelerated by more frequent and intensive use of the pesticide applied by desperate aquaculturists. The rotation of pesticides with different modes of action interrupts this sequence and prolongs the active use of each pesticide. The speed of development is also influenced by the rate of reproduction of the parasite, the immigration and host range of the parasite, the proximity to resistant populations, the persistence of the product, its concentration and specificity and the rate, timing and number of applications made (see http://www. irac-online.org). More than 500 species of arthropods have already shown resistance to at least one class of insecticide. The resistance of sea lice to dichlorvos may have been delayed by the influx of unexposed populations of sea lice from wild salmonids into sites, which was possible owing to the distance between culture sites in the young industry and the growth and stocking cycles of the salmon; however, with multi-year class stocking and closer proximity of cage sites as the industry expanded, it became inevitable. For this reason, an untreated reservoir of the parasite is always desirable. The development of sea lice resistance has resulted in cycles of complacency during which there is widespread use of a single effective

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product, followed by pressure to seek new products when early stage resistance is recognised. With no new licensed products available, this is followed by crisis during which farmers are forced to seek out and use nonchemical methods of control. Since it is clear that parasites will always be present and there will always be a need for control, it is wise to take the long-term view and avoid the short-termism which leads farmers to reach for the current most effective product every time and hope to eliminate the parasite from the site. This will mean that farmers have to introduce an IPM strategy incorporating resistance management principles either voluntarily or by government mandate; the latter making it compulsory and coordinated on a regional or national basis. Resistance management principles are clear (Table 8.3, see also http://www.irac-online.org). The concept of an IPM strategy for sea lice control, incorporating resistance management principles, was put forward to the Scottish industry as long ago as 1995 (Sommerville, 1995) at a time when seven products were at either the licensing stage or advanced in their development with four different modes of action and a number of useful management/husbandry methods. The concept has slowly been taken up, but there is as yet little evidence of commitment to it by the aquaculture industry, save for a few enthusiasts. In some countries, small inroads have been made into an IPM for sea lice control, for example, in 1998 the Scottish industry introduced a scheme of area management agreements (AMA) as part of a National Treatment Strategy (Rae, 1999) which focused treatment events in the late winter, and obtained agreements to coordinate simultaneous treatments within a bay or at least with proximal sites. This is particularly important to avoid local dispersion of the treatment compound following release of tarpaulin enclosures around cages into

Table 8.3 • • • • • • • • • •

Resistance management principles

Always include husbandry and biological control methods. Do not rely on a single pesticide class; frequent use of the same pesticide will lead quickly to resistance. Rotate products from different classes based on modes of action i.e. use different classes of pesticide in alternation. Time the application against the most susceptible life stages but based on population size thresholds. Use only at the recommended rate and treatment interval. Do ensure the effective dose rate is achieved; for a bath treatment this means use enclosures. Always test efficacy post treatment. If treatment fails due to resistance, do not retreat with a pesticide of the same class. Do not treat floating cages piecemeal, treat a whole site. Synchronise treatments with adjacent sites.

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neighbouring sites. The persistent low dose into adjacent sites in such cases is thought to have accelerated the development of resistance to the organophosphate Aquagard®. Heuch et al. (2005) reviewed the Norwegian National Action Plan and outlined its shortcomings. However, this and other strategies have always fallen short of the full IPM by not incorporating rotation of chemicals, the main method by which resistance development can be avoided or delayed. Thus crisis management prevails. Government policy has favoured supervised control based on threshold levels of lice and expensive monitoring programmes have been put in place. However, action thresholds are difficult to design and use. In the case of sea lice there are seven stages of the life cycle living on the fish, the adult stage represented by both male and female. Since the adults and pre-adults are the most damaging to the fish, threshold levels have concentrated on numbers of these stages, together with a ‘zero gravids’ strategy. Not sufficient is known about the infra-population structure under different conditions to determine if this is reasonable or not. Additionally, the sampling method recognises only 40–50 % accuracy (Treasurer and Pope, 2000). The decision regarding the threshold level is arbitrary as the risk depends on a number of other factors and has not been clearly defined. With reliance on threshold levels, there is a danger of over-use of pesticides which, in the absence of any enforced rotation, is dangerous. In Scotland the Environment Agency retains the ability to control the number of treatment episodes in a given time period, but this will not necessarily engage farmers in rotation. Learning to live with parasites whilst minimising economic losses and guided by fish welfare issues is a much more sustainable way of satisfying all stakeholders. This is achievable by the development of epidemiological models and is the way forward for research into parasite control in order to develop non-chemical methods and interventions which will contribute to an IPM for any pathogen/host system in aquaculture. Whatever new methods of control are developed for parasites in aquaculture, these will only be sustainable if used within an IPM which incorporates resistance management principles.

8.5.3 Integrated pest management A good IPM will involve biological, cultural/husbandry, preventative, prophylactic as well as chemotherapy elements, all underpinned by monitoring, i.e. concerted multiple tactics based on knowledge. It would meet the goals of all the stakeholders, including farmers, consumers and government agencies, with the ultimate goal of reducing losses due to parasite pathogens at the same time as safeguarding against risks of environmental pollution, hazards to human health and reduced sustainability of the aquaculture industry. Thus it requires a multidisciplinary approach as political, social and economic perspectives have to be accommodated as well as husbandry,

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ecological and technical aspects. Importantly, the IPM in aquaculture will depend on knowledge of the parasite pathogens, life cycles, transmission route, etc. which, for some pathogens, is very scanty. Knowledge of the host species’ physiology, genetics, behaviour and interaction with the parasite is important and, again, in-depth knowledge is only known for a small handful of the major culture species. This information is taken into the context of the husbandry system and local management practices. A suggested IPM scheme, generalised for management and control of most parasite pathogens, is as follows and is illustrated in Fig. 8.1. Control of parasites starts with fish stock control. Many fish species are now progeny of home-grown broodstocks, and fish stocks available in the future will be specific pathogen-resistant, achieved either by selective breeding or by transgenics. They will be obtained from an approved source together with a health certificate which guarantees they are specific pathogen free. They will already be multi-vaccinated. The fish will be stocked into a licensed site which has been checked hydrographically to ensure appropriate environmental conditions are met, including depth, flushing action, etc. The site will not be too close to adjacent sites utilised for fish culture, and the wild fish migration paths in the vicinity will be known together with their seasonality so as to predict when there is likely to be an influx of parasite transmission stages. The site will have been fallowed for as long as possible and only a single year class will be stocked into it throughout the growth cycle. Much of this will be subject to regulatory

Yr 1 Fish stock • certified • resistant? • multivaccinate

Site • fallowed • single year class • hydrography • wild fish • proximity

R e g u l a t o r y

r e q u i r e m e n t s

Yr 2

Growth cycle

Screen Improve general health status

Stocking • max size • handling • lower density

Other infections • reject • treat • quarantine • prophylaxis

Improve transport conditions to minimise stress

H A R V E S T

Minimisation management • Hygiene: net • Stress reduction cleaning

monitoring

monitoring

TREAT? • Parasite population structure? • Treatment history? • Health status? • Temperature? • Cost-effectiveness? • Logistics? • Coordination possible?

• Environmental control monitoring

Biological/biopesticide

CHEMICAL/DRUG? Bath • Logistics? • Health status? • Site sensitivity? • Season/climate? • Temperature? • Site history? • Treatment history?

Fig. 8.1 Generalised IPM scheme for parasite control.

Oral

R O T A T I O N

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requirements. Fish would be screened prior to stocking by a reputable laboratory with skilled, expert staff equipped with appropriate molecular technology, and would be rejected if uncontrollable pathogens were found to be present or treated if appropriate treatment was available. Ideally these fish would then be quarantined and, where appropriate, given a prophylactic treatment. They would be tested post-treatment to ensure the parasite has been eradicated prior to stocking, and subjected to a risk analysis also prior to stocking. A knowledge of the ontogeny of the immune system of the particular species cultured is necessary both for this and for vaccination purposes, and to ensure that the size of the fish stocked gives the best opportunity to resist disease, etc. The transport conditions are crucial to the success of stocking new fish, and, by minimising stress and preventing multiplication of the parasites which reproduce rapidly under such conditions, e.g. flagellates and ciliates, the survival rate can be improved. There is ample room for improvement through technology if there was more investment in innovative engineering; for example, with the use of well-boats, mortality can be markedly reduced. Minimising handling stresses and using low stocking densities improves the quality of the new stock in the first few weeks in the new site. Constant monitoring of fish throughout the grow-out period will pay dividends, but this is often done in an ad hoc manner by taking bulk weights and giving a cursory glance to identify any clinical conditions. Using appropriate, statistically-based sampling techniques provides the opportunity to test muscle/fat levels, identify pathogens, etc. During the growth cycle, management aims are to reduce stress through environmental control, handling and hygiene which influence pathogen levels and may have welfare implications. It is likely that maintenance of fish welfare according to a benchmark set of recognised indices may become statutory in the near future in the EU. The grow-out period is continually at risk of disease outbreaks and, in the event of the appearance of a parasite pathogen, revealed in the routine samples, it is necessary to decide whether there is a need to intervene. This could be either chemical or biological, and would depend on a wide range of conditions. First choice would be a non-chemical control where these are available; often these are best used in concert if there is more than one. If the choice was chemical, rather than use threshold levels which are universally applied, a full assessment of the situation needs to be made, including bioassays to assess levels of chemical/drug resistance. Such a decision could readily be supported by appropriate software incorporating current knowledge. The technology for such software has been developed, and packages of variable usefulness are available. Such software would be frequently updated with local knowledge and with the results of experience as it became available. Important information would relate to the health status of the fish, environmental information such as temperature and salinity (which affects dose rate), the population structure of the parasite, its chemical sensitivity in a bioassay, the cost-effectiveness (would it

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be better to harvest, destroy, etc?). The logistics would be assessed, e.g. climatic conditions, ability to coordinate treatment with adjacent/regional sites, etc. The treatment history of the site would also be a significant factor. If the decision was that it was necessary to treat, the nature of treatment would need to be considered. In the event of a chemical treatment being the choice, the key questions depend on the availability of more than one product with different applications and modes of action. Only for sea lice is there currently such a choice. The current choices of an oral or a bath treatment may well be supplemented with a slow release drug implant in the near future. It may be also be that, as with emamectin benzoate, which has a prolonged activity time, these will be administered prior to stocking, e.g. to salmonids in freshwater before transfer to sea cages, thereby providing protection through the most vulnerable growing stages. The choice of whether to employ a bath or an oral treatment may depend on a number of factors such as the health status of the fish. Where the infection has already damaged gills, bath treatments themselves increase mortality. Early stages of disease are often first signalled by a reduced appetite, then there would be difficulties in achieving the correct dose level for an in-feed treatment and a bath treatment would be more appropriate. The season and climate would also have a considerable influence as this would affect both appetite and withdrawal times in temperate zones. The treatment will also be influenced by the salinity of a site, the flushing rate of a bath treatment, and, indeed, the feasibility of enclosures where fast tides or high winds may change the volume of the cage and prevent accurate dosing. The logistics may be such that a whole site cannot be treated at one time or that the size of the fish and the handling would, in themselves, increase mortality. Frequently it is simply a question of labour availability at the appropriate time or the stock may be too close to market, making a prescribed long withdrawal period uneconomic. The method for application of bath treatments is very basic and could be considerably improved by investment in some innovative engineering. Unfortunately the industry is not sufficiently profitable to engage entrepreneurs to take this on. Treatment tanks and swim-throughs have been tried which enable detoxification of chemical product prior to release but have so far proved to be too expensive. A key element of chemotherapy is post-treatment testing of efficacy, something which is often overlooked or forgotten. Following consideration of the pros and cons of different drugs and chemicals currently available, overriding all other considerations should be treatment history and the absolute requirement to rotate the treatments. Any treatment will be applied according to resistance management principles (Table 8.3) and may best be applied by mandate through regulatory processes in the future. The general principle should prevail that treatment with drugs or chemicals is a last resort rather than the method of choice as it is currently.

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8.6 Future trends The production of healthy fish is the best mechanism to assist fish in resisting disease, and this may be supported by transgenics and/or dietary immunostimulants in the future. It seems that there will always be a place for pharmaceuticals and, with the increased value of the global aquaculture industry, it is hoped that more investment in fish parasiticides with different modes of action will ensue. New applications are being sought for parasiticides; exciting possibilities for the near future are slow-release implants for antiparasitic drugs. However, a major constraint on the development of new and more effective parasiticides is the time (up to 10 years) to obtain a marketing authorisation for a new product. Pharmaceutical companies do not find this cost-effective given the size of the market and the lack of control over the patents exhibited in the Far East. The effect of this may, though, have benefits in persuading the farmers of the importance of pursuing non-chemical control methods. The current molecular tools available to find target molecules are very expensive; however, the benefits would be great. The more advanced studies of the major parasite pathogens such as those which use microarrays for L. salmonis can lead the way to find target molecules for both vaccines and chemotherapeutants. Vaccine research for C. salmositica, L. salmonis and for I. multifiliis is at the leading edge of parasite vaccine research, although they may be utilising different approaches. There is, as yet, little interest in investing in new biocontrol methods, but biopesticides, albeit slow to become of interest for control of fish disease, are gradually being accepted as part of the armoury in horticulture and elsewhere. The growth in predictive epidemiological modelling, and risk analysis to point to management and husbandry controls, will probably bring dividends more rapidly than any other approach by identifying and targeting the most effective interventions. Increased legislation and greater regulation can be expected, particularly on food safety, fish welfare and environmental issues. However, none of the measures will single-handedly control any specific pathogen, and it is clear that future sustainability can only be achieved through collaborative integrated management strategies.

8.7 References arafa sz, el-naggar mm, el-abbassy sa, stewart mt and halton dw (2007) Neuromusculature of Gyrodactylus rysavyi, a monogenean gill and skin parasite of the catfish Clarias gariepinus, Parasitology International, 56(4), 297–307. arthur jr (1996) Fish and shellfish quarantine: the reality for Asia-Pacific, in Subasinghe RP, Arthur JR and Shariff M (eds), Health Management in Asian Aquaculture, Proceedings of the Regional Expert Consultation on Aquaculture

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Health Management in Asia and the Pacific, FAO Fisheries Technical Paper No. 360, FAO, Rome, 11–28. bakke ta, cable j and harris pd (2007) The biology of gyrodactylid monogeneans: The ‘Russian-doll killers’, Advances in Parasitology, 64, 161–376. banks ba, taggart jb, shinn ap and sommerville c (2000) Microsatellite analysis of Scottish sea lice population, Acta Parasitologica, 45(3), 266. bell s, bron je and sommerville c (2000) Distribution of exocrine glands in Lepeophtheirus salmonis (Krøyer 1837) and Caligus elongatus Nordmann, 1832. (Copepoda: Caligidae), Contributions to Zoology, 69(1/2), 9–12. ben-ami f and heller j (2001) Biological control of aquatic pest snails by the black carp Mylopharyngodon piceu., Biological Control, 22(2), 131–8. booth m, graham a and viney m (eds) (2008) Parasitic co-infections: challenges and solutions, Preface, Parasitology, 13, 749. boxaspen k (2006) A review of the biology and genetics of sea lice, ICES Journal of Marine Science, 63(7), 1304–16. bricknell i and dalmo ra (2005) The use of immunostimulants in fish larval aquaculture, Fish Shellfish Immunology, 19, 457–72. bron je, sommerville c, wootten r and rae gh (1993) Fallowing of marine Atlantic salmon, Salmo salar L., farms as a method for the control of sea lice Lepeophtheirus salmonis (Krøyer, 1837), Journal of Fish Diseases, 16, 487–93. burrells c, williams pd and forno pf (2001) Dietary nucleotides: a novel supplement in fish feeds −1. Effects on resistance to disease in salmonids, Aquaculture, 199, 159–69. butler r, bowden tj, bron je, bricknell jr and sommerville c (2000) The use of an in vitro model to investigate suppression of Atlantic salmon cellular immune responses during infection of Lepeophtheirus salmonis, Acta Parasitologica, 45, 271, Abstract. diamant a (1998) Red drum Sciaenops ocellatus (Sciaenidae), a recent introduction to Mediterranean mariculture, is susceptible to Myxidium leei (Myxosporea). Aquaculture, 62(1/2), 33–9. dickerson hw (2006) Ichthyophthirius multifiliis and Cryptocaryon irritans, in Woo PTK (ed.) Fish ‘Diseases and Disorders’ Volume 1: Protozoan and Metazoan Infections, 2nd edn, CABI, Oxford, 116–53. dickerson hw and clark tg (1998) Ichthyophthirius multifiliis: a model of cutaneous infection and immunity in fishes, Immunogical Review, 166, 377–84. efsa (2008) Fish Welfare, Parma, European Food Standards Agency, http://www. efsa.europa.eu/EFSA/efsa_locale-1178620753812_1211902131969.htm, accessed January 2009. fallang a, denholm i, horsberg te and williamson ms (2005) Novel point mutation in the sodium channel gene of pyrethroid-resistant sea lice Lepeophtheirus salmonis (Crustacea: Copepoda), Diseases of Aquatic Organisms, 65, 129–36. fallang a, ramsay jm, sevatdal s, burka jf, jewess p, hammell kl and horsberg te (2004) Evidence for occurrence of an organophosphate-resistant type of acetylcholinesterase in strains of sea lice (Lepeophtheirus salmonis Krøyer), Pest Management Science, 60, 1163–70. fast md, burka jf, johnson sc and ross nw (2003) Enzymes released from Lepeophtheirus salmonis in response to mucus from different salmonids, Journal of Parasitology, 89, 7–13. fast md, ross nw, craft ca, locke sj and mackinnon sl and johnson sc (2004) Lepeophtheirus salmonis: characterization of prostaglandin E-2 in secretory products of the salmon louse by Rp-Hplc and mass spectrometry, Experimental Parasitology, 107, 5–13.

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fenton a, hakalahti t, bandilla m and valtonen et (2006) The impact of variable hatching rates on parasite control: a model of an aquatic ectoparasite in a Finnish fish farm, Journal of Applied Ecology, 43(4), 660–80. fiala i (2006) The phylogeny of Myxosporea (Myxozoa) based on small subunit ribosomal RNA gene analysis, International Journal for Parasitology, 36(14), 1521–34. firth k, johnson s and ross n (2000) Characterization of proteases in the skin mucus of Atlantic salmon (Salmo salar) infected with the salmon louse (Lepeophtheirus salmonis) and in whole-body louse homogenate, Journal of Parasitology, 86, 1199–205. freeman ma (2002) Potential biological control agents for the salmon louse Lepeophtheirus salmonis (Krøyer 1837), PhD Thesis, University of Stirling, UK. freeman ma, bell as and sommerville c (2003) A hyperparasitic microsporidian infecting the salmon louse Lepeophtheirus salmonis: An rDNA-based molecular phylogenetic study, Journal of Fish Diseases, 26(11–12), 667–76. gault nfs, kilpatrick dj and stewart mt (2002) Biological control of the fish louse in a rainbow trout fishery, Journal of Fish Biology, 60, 226–37. gilbert ma and granath wo (2003) Whirling disease of salmonid fish: life cycle, biology, and disease, Journal of Parasitology, 89(4), 658–67. giorgiadis mp, gardner ia and hedrick rp (2001) The role of epidemiology in the prevention, diagnosis and control of infectious diseases of fish, Preventive Veterinary Medicine, 1(48), 287–302. glover ka, aasmundstad t, nilsen f, storset a and skaala ø (2005) Variation of Atlantic salmon families (Salmo salar L.) in susceptibility to the sea lice Lepeophtheirus salmonis and Caligus elongatus, Aquaculture, 245, 19–30. glover ka, grimholt u, bakke hg, nilsen f, storset a and skaala ø (2007) Major histocompatibility complex (MHC) variation and susceptibility to the sea louse Lepeophtheirus salmonis in Atlantic salmon Salmo salar, Diseases of Aquatic Organisms, 76(1), 57–65. grayson th, john rj, wadsworth s, greaves k, cox d, roper j, wrathmell ab, gilpin ml and harris je (1995) Immunization of Atlantic salmon against the salmon louse: identification of antigens and effects on louse fecundity, Journal of Fish Biology, 47, 85–94. gravil hr (1996) Studies of the biology and ecology of the free swimming larval stages of Lepeophtheirus salmonis (Krøyer, 1838) and Caligus elongatus Nordmann, 1832 (Copepoda: Caligidae), PhD thesis, University of Stirling, UK. guselle nj, markham rjf and speare dj (2006) Intraperitoneal administration of β-1,3/1,6-glucan to rainbow trout, Oncorhynchus mykiss (Walbaum), protects against Loma salmonae, Journal of Fish Diseases, 29, 375–81. harris pd, shinn ap, cable j, bakke ta and bron je (2008) GyroDb: gyrodactylid monogeneans on the web, Trends in Parasitology, 24, 109–11. hedrick rp, mcdowell ts, mukkatira k, georgiadis mp and macconnell e (1999) Susceptibility of selected inland salmonids to experimentally induced infections with Myxobolus cerebralis, the causative agent of Whirling Disease, Journal of Aquatic Animal Health, 11(4), 330–39. heuch pa, bjørn pa, finstad b, holst jc, asplen l and nilsen f (2005) A review of the Norwegian ‘National Action Plan Against Salmon Lice on Salmonids’: The effect on wild salmonids, Aquaculture, 246, 79–92. hilborn r (2006) Salmon-farming impacts on wild salmon, Proceedings of the National Academy of Sciences USA, 103(42), 15277. hines rs and spira dt (1974) Ichthyophthiriasis in the mirror carp Cyprinus carpio L. V. Acquired immunity, Journal of Fish Biology, 6, 373–8.

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lees f, baillie m, gettinby g and revie cw (2008) The efficacy of emamectin benzoate against infestations of Lepeophtheirus salmonis on farmed Atlantic salmon (Salmo salar L) in Scotland, 2002–2006, PLoS ONE, 3(2), e1549. mcgurk c, morris dj, bron je and adams a (2005) The morphology of Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) spores released from Fredericella sultana (Bryozoa: Phylactolaemata), Journal of Fish Diseases, 28, 307–12. mcvicar ah (2004) Management actions in relation to the controversy about salmon lice infections in fish farms as a hazard to wild salmonid populations, Aquaculture Research, 35(8), 751–8. morris dj, adams a and richards rh (1999) In situ hybridization of DNA probes to PKX, the causative organism of proliferative kidney disease (PKD), Journal of Fish Diseases, 22(2), 161–3. mustafa a and mackinnon bm (1999) Genetic variation in susceptibility of Atlantic salmon to the sea louse Caligus elongatus Nordmann, 1832, Canadian Journal of Zoology, 77, 1332–5. niemann h, kues w and carnwath jw (2005) Transgenic farm animals: Present and future, Revue Scientifique et Technique–Office International des Épizooties, 24(1), 285–98. peeler ej and thrush ma (2004) Qualitative analysis of the risk of introducing Gyrodactylus salaris into the United Kingdom, Diseases of Aquatic Organisms, 62, 103–13. peeler ej, gardiner r and thrush ma (2004) Qualitative risk assessment of routes of transmission of the exotic fish parasite Gyrodactylus salaris between river catchments in England and Wales, Preventive Veterinary Medicine, 64(2/4), 175–89. penston mj, millar cp, zuur a and davies im (2008) Spatial and temporal distribution of Lepeophtheirus salmonis (Krøyer) larvae in a sea loch containing Atlantic salmon, Salmo salar L., farms on the north-west coast of Scotland, Journal of Fish Diseases, 31(5), 361–71. pérez om, telfer tc, beveridge mcm and ross lg (2002) Geographical information systems (GIS) as a simple tool to aid modelling of particulate waste distribution at marine fish cage sites, Estuarine, Coastal and Shelf Science, 54(4), 761–8. rae gh (1999) Sea lice, medicines and a national treatment for control, Fish Veterinary Journal, 3, 46–51. rae gh (2002) Sea louse control in Scotland, past and present, Pest Management Science, 58(6), 515–20. raynard rs, munro als, king j, ellis ae, bruno dw, bricknell ir, vahanakki p, wootten r, sommerville c, petrie a, vivers b, andrade-salas o, melvin w, amezega t, labus mb, coull jj, relly p, mulcahy mf, o’donoghue m and o’connell j (1994) Development of a vaccine for the control of sea lice (Lepeophtheirus salmonis and Caligus elongatus) in Atlantic salmon (Salmo salar L), F:17, International Council for Exploration of the Sea, Copenhagen. raynard rs, bricknell ir, billingsley pf, nisbet aj, vigneau a and sommerville c (2002) Development of vaccines against sea lice, Pest Management Science, 58, 569–75. reimschuessel r (2008) Assessing the human health implications of new veterinary drugs used in fish farming, in Lie Ø (ed.), Improving Farmed Fish Quality and Safety, Woodhead, Cambridge, 128–56. revie cw, robbins c, gettinby g, kelly l and treasurer jwa (2005) A mathematical model of the growth of sea lice, Lepeophtheirus salmonis, populations on farmed Atlantic salmon, Salmo salar L., in Scotland and its use in the assessment of treatment strategies, Journal of Fish Diseases, 28(10), 603–13.

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9 Controlling viral diseases in aquaculture: new developments T. Renault, Ifremer, France

Abstract: Looking at the level of aquaculture species production, viral disease control remains an important challenge. However, relatively little is known about what farmers can do to prevent and treat viral infections and how fish and shellfish fight viral diseases. Difficulties for control of viral infections in aquaculture mainly come from the lack of commercial vaccines and from the absence of specific therapeutic agents. Prevention and control first pass through control of fish and shellfish movements. Understanding complex interactions between animal, environment and pathogen also appears as a necessary avenue. In the long term, alternative treatments using antiviral drugs may be developed, but the most effective way for sustainable aquaculture production may certainly rely on the production of selected animals for disease resistance. Key words: viral diseases, aquaculture, disease control, immunity, vaccination, selection, transfer regulation.

9.1 Introduction World aquaculture has grown at an average annual rate of almost 8.8 % from 1950 to 2004 compared with 3 % for livestock meat and 1.6 % for capture fisheries production (FAO, 2006). Aquatic production (including plants) has steadily increased since the early 1950s (a million tonnes). By 1996, the total production of cultured finfish, shellfish and aquatic plants was 34.1 million tonnes which was valued at US$46.5 billion. Total world aquaculture production reached 59.4 million tonnes for a value of US$70.3 billion by 2004 (FAO, 2006). In this context, aquaculture is perceived as having the greatest potential to meet the growing demand for aquatic food, at time when the production from capture fisheries has reached a plateau, and most of the main fisheries are fully exploited.

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Aquaculture, however, has its own problems to contend with. From time to time populations of cultivated animals may suffer from severe mortality outbreaks. Among the possible causes there is the occurrence of infectious diseases due to an extended variety of pathogens. Resulting diseases arise partly from the high stocking density in production systems in conjunction with stress induced by environmental fluctuations and management practices. Viruses are among the most destructive pathogens encountered in aquaculture and are a limiting factor to the success of many aquaculture farms through (i) direct losses of fish and shellfish, (ii) indirect costs from reduced productivity and costs of disease management and (iii) loss of export markets related to imposition of trade restrictions. Although their management must be an integral part of farm husbandry, identification of viruses and investigation of viral diseases is highly specialized and requires special training and equipment. Despite the impact that viral diseases have on aquatic organisms, relatively little is known about what farmers can do to prevent and treat viral infections and how fish and shellfish fight viral diseases. Difficulties encountered in the control of viral infections in aquaculture mainly come from the lack of commercial vaccines and from the absence of specific therapeutic agents. Consequently, farmers are left with few resources other than the use of preventive measures. In the long term, alternative treatments such as antiviral drugs may be developed and the most effective way for sustainable aquaculture production will certainly rely on the production of selected animals for disease resistance. In a holistic overview of aquaculture species production, viral disease control remains an important challenge. The prevention and control of viral diseases ranges from the control of mollusc movements to understanding complex interactions between animal, environment and pathogens to the genetic selection for disease resistance.

9.2 Overview of viral diseases in aquaculture 9.2.1 Fish viruses As husbandry practices have improved in the past decades and bacterial diseases have been partially managed, viral diseases have emerged as continuing problems to the fish aquaculture industry (NASCO, 1993; Stephen and Iwama, 1997). Several major viral diseases such as infectious pancreatic necrosis (IPN), infectious haematopietic necrosis (IHN), viral haemorrhagic septicaemia (VHS) and infectious salmon anaemia (ISA) cause severe losses in fish farming (Table 9.1). Moreover, all the fish diseases notifiable to Office International des Epizooties (OIE, World Organisation of Animal Health) are viral infections indicating the importance of fish viruses worldwide (Table 9.2).

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Table 9.1 Main fish viral diseases Diseases

Type of virus

Fish species affected

References

Enzootic haematopoietic necrosis (EHN)

Iridovirus

Infectious haematopoietic necrosis (IHN) Spring viraemia if carp (SVC) Viral haemorrhagic septicaemia (VHS)

Rhabdovirus

Redfin perch, Perca fluviatilis, and rainbow trout, Oncorhynchus mykiss Salmonids

Langdon and Humphrey, 1987; Langdon et al., 1988 Bootland and Leong, 1999

Rhabdovirus

Cypriid fish

Fijan, 1999

Rhabdovirus

Rainbow trout, Oncorhynchus mikiss Trout and salmon

Wolf, 1988

Atlantic salmon, Salmo salar A wide range of species

Thorud and Djupvk, 1988 Frerichs et al., 1996

Channel catfish, Ictalutus punctatus Koi carps

Wolf, 1988

Infectious pancreatic necrosis (IPN) Infectious salmon anaemia (ISA) Viral encephalopathy and retinopathy (VER) or Viral nervous necrosis (VNN) Channel catfish virus disease (CCVD) Koi herpes disease (KHVD)

Table 9.2

Nodavirus

Herpes virus Herpes virus

Notifiable fish diseases

Notifiable viral diseases Enzootic haematopoietic necrosis (EHN) Infectious haematopoietic necrosis (IHN) Spring viraemia of carp (SVC) Viral haemorrhagic septicaemia (VHS) Infectious salmon anaemia (ISA) Red sea bream iridovirla disease Koi herpes disease (KHVD)

OIE, Health Code, 2007

Directive 2006/88/EC

X X X X X X X

X1 X2 X2* X2 X2 X2

1 Exotic disease in the Directive 2006/88/EU. 2 Non-exotic disease in the Directive 2006/88/ EU. 2* SVC removed from the Annex IV (Directive 2006/88/EU), Directive 2008/53/EU. Source: OIE, 2007; EC, 2006.

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9.2.2 Crustacean viruses Viruses are the most diverse and numerous of the infectious microbial agents described for crustaceans. Over 30 viruses have been reported from aquatic crustaceans (Bonami, 1976; Couch, 1981; Johnson, 1984; Sparks, 1985; Mari and Bonami, 1986), and in marine shrimp aquaculture, viruses cause the most economically significant biotic diseases. Management of viral diseases is particularly important to the success of semi-intensive shrimp farming. For example, in Taiwan, the production of Penaeus monodon decreased from about 90 000 tons in 1987 to 20 000 in 1989 and has still not recovered because of baculovirus infections. Since shrimp farming currently relies primarily on wild-caught stock, shrimp viral pathogens are repeatedly introduced into cultured systems. Additionally, these pathogens continue to be disseminated over wide geographical areas with movement of shrimp stocks for aquaculture purposes (Lightner et al., 1983; Colorni et al., 1987; Brock et al., 1993). The viruses affecting penaeid shrimp include DNA viruses and RNA viruses (Tables 9.3 and 9.4).

9.2.3 Mollusc viruses The discovery of viruses in marine bivalves is a fairly recent event. Although several viruses are only detectable in molluscs that are suffering from another disease or from environmental stress such as pollution, several massive mortality outbreaks have been correlated to viral infections. Mass mortalities of Portuguese oysters, Crassostrea angulata, among French

Table 9.3 Main crustacean viral diseases Crustacean species affected

Reference

Parvoviridae

Penaeus stylirostris

Lightner et al., 1983

Dicistroiridae

Penaeus vannamei Shrimp and other crustacean species Penaeus monodon Penaeid shrimp

Hasson et al., 1995 Lightner and Redman, 1998

Diseases

Type of virus

Infectious hypodermal and hematopoietic necrosis (IHHN) Taura syndrome (TSV) White spot syndrome disease (WSSV)

Nimaviridae

Yellowhead disease (YHD) Tetrahedral baculovirosis (Baculovirus penaei) Sperical baculovirosis (Penaeus monodontype baculovirus)

Roniviridae (Okavirus) Baculoviridae Baculoviridae

Penaeus monodon

Cowley et al., 2000 Lightner et al., 1983 Lightner et al., 1983

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Table 9.4

Notifiable crustacean diseases

Notifiable viral diseases Taura syndrome (TSV) White spot syndrome disease (WSSV) Yellowhead disease (YHD) Tetrahedral baculovirosis (Baculovirus penaei) Sperical baculovirosis (Penaeus monodon-type baculovirus) Infectious hypodermal and haematopoietic necrosis (IHHN)

OIE, Health Code, 2007

Directive 2006/88/EC

X X X X X

X1 X2 X1

X

1 Exotic disease in the Directive 2006/88/EU. 2 Non-exotic disease in the Directive 2006/88/ EU. Source: OIE, 2007; EC, 2006.

stocks between 1967 and 1973 were associated with irido-like virus infections (Comps and Duthoit, 1976, 1979; Comps and Bonami, 1977). Herpeslike virus and herpes virus infections have been identified in various marine mollusc species in different countries (Farley et al., 1972; Hine et al., 1992; Nicolas et al., 1992; Hine and Thorne, 1997; Renault et al., 2000, 2001; Arzul et al., 2001; Renault and Arzul, 2001; Vásquez-Yeomans et al., 2004; Chang et al., 2005; Friedman et al., 2005; Hooper et al., 2007). Other viruses described in bivalves are identified as members of the Papovaviridae, Togaviridae, Retroviridae, Reoviridae, Birnaviridae and Picornaviridae (Farley, 1976, 1978; Oprandry et al., 1981; Ramussen, 1986; Bower, 2001).

9.3 Limitation of current management techniques There are many transfers of aquatic species between the countries culturing them and many introductions of non-native species (alien species) in order to develop new farming activities (Goulletquer and Heral, 1997; Calvo et al., 2001; Anderson et al., 2004). Air transport and increased global trade facilitate transfers and introductions of live animals. However, although these movements and transfers have had a positive impact on the development of aquaculture, they are also identified as a threat to biodiversity and a major source of pathogens. Finally the aquatic environment makes it difficult to confine stock for the purpose of preventing disease spread and dissemination. Currently the available chemotherapy is highly restricted in aquaculture in order to control risks to consumer safety and to avoid the development of resistant pathogen strains. In this context, regulations on the use of

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antimicrobials, veterinary drugs and chemicals in aquaculture have been developed and implemented in many countries worldwide. Moreover, animals are often reared in open waters which strongly limits the use of drugs in relation to the quantity to be used and their impact on surounding ecosystems. Nevertheless, animals are continuously at risk of exposure to pathogens present in their environment. Chemotherapy may, however, be successfully applied in confined and controlled facilities such as hatcheries and nurseries. Although vaccination is used routinely to prevent viral diseases in veterinary medicine, it is not widely used in fish. At present, commercial vaccines have been developed for protection of fish from bacterial agents. Early prototype vaccines containing inactivated viruses developed for administration by immersion all resulted in insufficient protection. The use of attenuated or avirulent forms of viruses is regarded as unacceptable due to the residual virulence in targeted species and virulence in non-target species. In this context, DNA vaccines appear as promising tools for immunization of fish, and extended research has been developed for a number of viral fish diseases including rhabdovirus infections. DNA vaccination is based on the administration of the gene encoding the vaccine antigen. The host immune system is triggered by subsequent expression of the antigen after vaccination. Despite intensive research efforts since the late 1990s, there is only one DNA vaccine on the market targeting a fish viral disease. It encodes the glycoprotein G of IHNV (Apex-IHN®, Vical) and is used in salmon aquaculture in Canada (Hensley, 2005). Although promising results have also been reported for the vaccines against VHS, there are no commercial vaccines available to control VHS (Lorenzen et al., 2001; Utke et al., 2008). The mechanism through which resistance is conferred by these vaccines is unknown. However, non-specific protection is conferred by the vaccines at early stages post-infections suggesting that innate immunity in fish plays a key role in resistance to viral infections. Crustaceans and molluscs, like other invertebrates, lack an adaptative immune system and an immunological memory. They do not possess lymphocytes and do not produce antibodies. Hence, vaccination cannot be used to protect them against pathogens, and indirect diagnosis tests including serology are not available. The direct detection of infectious agents remains the only possible approach.

9.4 Advances in understanding of immunity of aquacultured species to viral diseases The vertebrate immune system displays both specific and non-specific (innate) mechanisms while invertebrates have only non-specific defence mechanisms. The innate system is the more ancient. This system relies on cells (i.e. phagocytes) and blood-borne molecules (i.e. complement). The

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fact that invertebrates make up greater than 90 % of all species on earth attests to the efficiency of their ‘primitive’ host defence systems, at least at the population level. Since the 1990s, it has become more and more evident that some of these innate mechanisms are conserved in invertebrates (e.g. Drosophila) and mammals, suggesting that discoveries are applicable to a wide range of phyla (Medzhitov et al., 1997; Means et al., 2000). In addition, it has been shown that the non-specific response is required to initiate the specific immune response in mammals. Thus, nonspecific immunity is increasingly perceived as being of fundamental importance. Various strategies are employed by invertebrates and vertebrates to kill invasive or opportunistic microorganisms, including phagocytosismediated killing, agglutination, encapsulation, release of microbicidal molecules and apoptosis. Antiviral innate immunity is important because it constitutes the first line of defence in vertebrates, and the only one in invertebrates. Host cells are stimulated to change their transcriptional profile, to produce antiviral molecules and immune mediators or to die in order to protect other cells. Therefore, innate immunity in fish, molluscs and crustaceans has been actively investigated in recent years in order to determine which innate defence mechanisms are triggered in viral infections. Several approaches including mRNA differential display, suppressive subtractive hybridization (SSH) (He et al., 2005), expressed sequence tag (EST) libraries (Jenny et al., 2002; Song et al., 2006) and gene arrays (microarray) (Dhar et al., 2003; Wang et al., 2006) have been developed in order to study differentially expressed genes after a viral infection in fish and shellfish. Through such studies, conserved mechanisms and pathways of the innate immunity have been identified. Characterization of innate immune responses has been carried out using different viral disease models including fish, crustacean and mollusc diseases. This permits interchange of knowledge and methodological progress in the field of non-specific immunity. Moreover, studies have been performed at the biochemical and genomic levels. Taken together, both approaches may lead to the characterization of antiviral molecules and mechanisms in invertebrates and lower vertebrates. This, in turn, is of benefit to the design of more potent vaccines in fish and antiviral therapeutic agents, and to the identification of new targets for preventive actions in different cultured aquatic species. Genomic and proteomic approaches may provide good opportunity to identify and exploit conserved pathways in different invertebrate phyla (molluscs and crustaceans) and vertebrates and to provide new insight into the evolution and flexibility of defence systems. The innate immune responses of fish, molluscs and crustaceans remains a vast domain to be explored and is very likely to present potential applications: (i) by the use of defined molecules as therapeutic agents, (ii) by the use of encoding genes as selection markers for improving resistance to infections and (iii) by the development of new vaccines in fish.

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9.4.1 Virus-induced genes Induction of host cell genes in response to viral infections constitutes a major step in the initiation of the host defence system. Viruses induce the up-regulation of various host genes (O’Shea, 1997; Welsh and Sen, 1997), some of which have antiviral activity. The interferon and interferon-induced genes are probably the best studied in mammalian models, but few data are available on fish and invertebrates. New virus-induced genes in nonclassical models such as fish and shellfish are of interest in studying the virus–host relationship in lower vertebrates and in invertebrates and in obtaining insights into the mechanisms involved in immune responses to viral stimuli. It is important to note that function characterization of conserved virus-induced genes may also add to discoveries in mammals. This is consistent with the current comparative approach to functional genomics and postgenomics, from bacteria to human. Some genes involved in the interferon-dependent response have been cloned in fish (Trobridge and Leong, 1997; Trobridge et al., 1997) and new fish genes (Vig-1 and Vig-2) induced by VHSV have been reported in rainbow trout, Oncorhynchus mykiss, cells (Boudinot et al., 1999, 2000). Vig-1 is homologous to a recently described human cytomegalovirusinduced gene (Zhu et al., 1997). The mouse homolog is very similar to the trout gene, and is also induced by viruses and lipopolysaccharides (LPS), showing that genes of this family are implicated in the non-specific response throughout the vertebrates (Boudinot et al., 2001). Vig-1 may be involved in the non-specific virus-induced synthesis of enzymatic cofactors of the nitric oxide (NO) pathway (Vasquez-vivar et al., 1998). This is significant because NO is an important compound of the innate immune response to viruses (Karupiah et al., 1993; Komatsu et al., 1996; Reiss and Komatsu, 1998; Zaragosa et al., 1998). An increased transcription of several immune related genes including interleukin 1β (Il-1β), transforming growth factor β (TGF-β) and IL-8 genes has also been reported in the rainbow trout after VHSV infection suggesting a role for these molecules in antiviral defence (Tafalla et al., 2005). Knowledge about antiviral defence at the molecular level has developed rapidly in recent years. Such approaches have been employed to identify differentially expressed genes and to find immune-relevant factors responsible for virus resistance in shrimp. For exmple, in WSSV-infected shrimp, Penaeus stylirostris, immune-related genes including the genes encoding lipopolysaccharide and β-1,3 glucan binding protein (LGBP), serine protease, C-type lectin, macrophage mannose receptor and low-density lipoprotein receptor were over-expressed in comparison to non-infected animals (Dhar et al., 2003). In a more recent study, genes encoding an interferon-like protein and a (2′-5′) oligo(A) synthetase-like protein, respectively, have been identified in WSSV-resistant shrimp, P. japonicus (He et al., 2005). These two proteins are key components of the interferon system in vertebrates. Wang et al. (2006) also demonstrated a differential

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profile of immune-related gene expression in response to WSSV infection in the Chinese shrimp, Fenneropenaeus chinensis. Although genes induced by viral infections and genes whose expression is related to the ability of shrimp to survive viral outbreaks have been identified, no significant insight into the antiviral mechanism in shrimp has been gained. A major concern in mollusc immunity is the fragmentary knowledge on effector proteins, regulatory pathways and related genes. However, various molecular technologies have been applied since the late 1990s in order to identify immunity conferring genes. These include genes encoding heat shock proteins, antimicrobial peptides, toll-like receptors and tumor necrosis factor (TNF) receptor associated proteins that have so far been identified in bivalves using EST libraries (Jenny et al., 2002; Guegen et al., 2003, 2006; Peatman et al., 2004; Tanguy et al., 2004; Song et al., 2006). Although such data may provide the basis for understanding the role of the innate immune system in molluscs, very little information is available on antiviral responses. During the course of an EU funded project (Avinsi, QLK2-CT2002-01691) SSH libraries have been constructed in order to study the antiviral response of adult Pacific oyster, Crassostrea gigas, to ostreid herpes virus 1 (OsHV-1) by identifying and studying virus-induced genes. Among the sequences that matched with the product of known genes, 9 % were related to putative immune functions (e.g. laccase, macrophage-expressed protein, molluscan defence protein, IK cytokine, myeloid differenciation factor 88, . . .) and the related oyster genes were totally sequenced by rapid amplification of cDNA ends polymer chain reaction (RACE PCR) (pers obs.). These immune-relevant genes have been used to construct a genetic map on the basis of a quantitative trait locus (QTL) approach during the course of another EU funded project (Aquafirst, Contract nº 513692) and have provided the basis for studying the role of the innate immune system in the immediate response to pathogens, especially viruses.

9.4.2 Antiviral molecules Biochemical characterization of antiviral molecules using tissue extracts from aquatic species has previously been reported. Several groups have carried out studies on induced proteins involved in the non-specific host defence response in invertebrates and have characterized a number of them including a large number of antimicrobial peptides (Gotz and Boman, 1985; Cociancich et al., 1994; Destoumieux et al., 1997; Bulet et al., 1999; Mitta et al., 1999; Relf et al., 1999). Although these molecules are characterized by a broad activity spectrum, affecting the growth of bacteria, fungi and yeast (Charlet et al., 1996; Mitta et al., 2000), antiviral effects have rarely been reported. However, many reports have been published about in vitro antiviral effects of extracts from aquatic species (algae, bacteria and plants) (Hasui et al., 1995; Garcia et al., 1999; Bergé et al., 1999; Matsuda et al.,

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1999; Yasin et al., 2000). These studies are based on the inhibition of the in vitro replication of different viruses. In recent years, some studies have been developed on antiviral drugs focusing on the regulation of the innate defence system (Falco et al., 2007, 2008).

9.4.3 Apoptosis During viral infections of multicellular organisms, induction of apoptosis is often observed and can be regarded as a primitive antiviral mechanism (O’Brien, 1998; Everett and McFadden, 1999; Koyama et al., 2000). After viral infection, cells that die by apoptosis limit the ability of the virus to replicate and spread. The detection of apoptosis in lower vertebrates and invertebrates indicates that programmed cell death may be a key defence against viral infections. There is, consequently, a selective advantage for viruses that subvert apoptotic processes. Several viruses carry genes that interfere with the host’s apoptotic machinery, and such genes have been found in mammalian herpes viruses (Henderson et al., 1993; Zhu et al., 1995) and insect baculoviruses (Huang et al., 2000). Indeed, many viruses have developed strategies to inhibit or delay apoptosis in target cells and to activate apoptosis in immune cells, thereby lowering the immune response (Roulston et al., 1999). Studies have shown that apoptosis is induced in fish by VHSV and IPNV (Bjorklund et al., 1997; Hong et al., 1998, 1999a,b; Eléouët et al., 2001). An increased transcription of two genes (ubiquitin conjugating enzyme 7 interacting protein and interferon induced with helicase C domain protein 1, which may be involved in apotosis and IFN regulation) has been reported in viral encephalopathy and retinopathy (VER) infected sea bream, Sparus aurata (Dios et al., 2007). Herpes viruses infecting oysters and clams have also been associated with apoptosis (Renault et al., 2000, 2001). Genes encoding proteins significantly related to the inhibiton of apoptosis proteins (IAPs) in mammalian and insect cells have been described in OsHV-1, a herpes virus infecting Crassostrea gigas oyster larvae (Arzul et al., 2001; Davison et al., 2005). Moreover, it has been suggested that apoptosis may be the cause of death in shrimp with lethal viral infections and that it may be an integral part of a process for adaptive tolerance to viruses in crustaceans (Flegel, 1997; Pasharawipas et al., 1997; Flegel and Pasharawipas, 1998; Flegel, 2007). DNA fragmentation of the type considered as a hallmark of apoptsis has been reported in cultured black tiger shrimp, Penaeus monodon, infected with white spot syndrome virus (Sahout et al., 2001). A BIR-like motif (baculovirus IAP repeats) was also recently found in TSV genome (Mari et al., 2002). A recent study focusing on gene expression profiles in the hepatopancreas of the WSSV-resistant and susceptible Pacific white shrimp, Litopenaeus vannamei, demonstrated that genes encoding apoptotic-related proteins were expressed at a higher level in the virusresistant shrimp (Zhao et al., 2007).

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9.4.4 RNA interference Double-stranded RNA (dsRNA), a virus-associated molecular pattern, is a potent inducer of antiviral responses. Specific RNA interference (RNAi) reponse, a phenomenon triggered by intracellular dsRNA, serves antiviral functions in vertebratres and invertebrates. RNAi relies on a series of gene silencing mechanisms; all of them are based on the processing of long dsRNA precursors into small interfering RNAs (siRNAs). The final effect is targeted degradation or translational repression of mRNAs that share sequence similiraty with the dsRNA inducer. In other words, RNAi is a sequence-specific, post-transcriptional process of mRNA degradation. RNAi has been reported as an effective mechanism to suppress viral infections or replication of many viruses. An effective RNAi machinery has been reported in fish and shrimp (Schyth et al., 2006; Robalino et al., 2007) and it may provide an efficient tool to fight viral diseases in aquaculture. Recently, it has been reported that exogenously synthetic long dsRNAs and siRNAs can inhibit viruses from different aquatic animals (Robalino et al., 2004; Tirasophon et al., 2005; Li et al., 2007; Wu et al., 2007, Xu et al., 2007). As an example, injection of sequence-dependent siRNA induces antiWSSV activity in Litopenaeus vannamei shrimp (Wu et al., 2007). Moreover, a sequence-specific dsRNA targeting shrimp β-integrin efficiently inhibits WSSV replication in experimentally infected shrimp P. japonicus (Li et al., 2007). The shrimp β-integrin has been demonstrated to act as a WSSV receptor (Li et al., 2007). Double strand (ds) RNA may also trigger innate antiviral immunity in fish and crustaceans (Dodd et al., 2004; Westenberg et al., 2005; Robalino et al., 2007). In fish, double-stranded DNA may induce a complex antiviral program mediated in part by interferons (IFN). Schyth et al. (2006) showed that VHSV glycoprotein siRNA efficiently inhibits VHSV multiplication. However, inhibition of SVCV, a heterologous rhabdovirus, has also been reported and an up-regulation of the interferon induced Mx gene observed. These results suggest that siRNAs induced a non-target specific antiviral effect. In the marine crustacean Litopenaeus vannamei, injection of dsRNA of diverse base composition is able to protect animals from mortality induced by two unrelated viruses, TSV and WSSV (Robalino et al., 2004). Shrimp immune system is able to recognize dsRNA as a virus-associated molecular pattern, resulting in the activation of an innate antiviral response.

9.5 New methods to control viral diseases in aquaculture and future trends 9.5.1 Control of animal movements Transfer regulations have been developed in order to avoid the introduction of animals from an enzootic area to a pathogen-free area. Specific viruses are included in lists of notifiable pathogens by the EU legislation

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(2006/88/EC, Annex IV) (Tables 1 and 2) and by the OIE (Manual of Diagnostic Tests for Aquatic Animals, 2006 and the Aquatic Animal Health Code, 2007) (Tables II and IV). Targeted viruses and related viral diseases are listed based on the fact that they (i) resist or respond poorly to therapy, (ii) have a restricted geographical range, (iii) induce major economic losses, and affect species that are traded internationally. Although most of the notifiable diseases are viral diseases in fish and crustaceans, no viral disease is listed at present for molluscs. In this context, defining the health status (free from infection or infected) of fish and shellfish stocks from a production site, a geographical zone or an entire country through health surveillance programmes is an essential prerequiste to allow or not allow animal movements and transfers. Hatcheries who supply seeds to growers as an alternative to wild-caught sources demonstrate a rapid and constant expansion. They may contribute to the development of a substantial international trade in gametes, larvae and juveniles, and the distribution of stocks improved through selective breeding. As an example, mollusc hatchery development significantly contributes to production worldwide and hatcheries therefore have an increased contribution to movements, transfers and introduction of live animals. In Europe, the global hatchery production of oyster larvae is up to three billion individuals, and a large part (around 30 %) of this production is exported outside the country of production. Animal transfers are currently recognized as a major cause underlying outbreaks of mortality, epidemics and spread of diseases. With this in mind, hatcheries should be recognized as a risky segment of the aquaculture sector. At the same time, hatcheries have closed facilities that enhance disease control capabilities and may produce certified pathogen free progeny, and so help to reduce the circulation of infected stocks. Moreover, they may have a pivotal role in the development and implementation of health management strategies based on improved resistance to infectious diseases. The involvement of hatcheries in the control of viral diseases using commercial diagnosis kits would offer producers the possibility to contribute proactively in the early detection of possible adverse conditions for aquacultured species growth and survival before a major problem occurs, contributing to the minimization of the effects of disease outbreaks on their business and subsequently to the enhancement of their competitiveness. Adequate control of larvae and juveniles from hatcheries in case of intensive rearing in controlled facilities may avoid costly epizootics. Moreover, the availability of efficient tests for detection of specific viruses may facilitate screening of broodstock, juveniles and larvae before commercial transactions and therefore constitute a guarantee of product quality for fish and shellfish producers. As another example, production of specific pathogen free (SPF) shrimp in hatcheries will assure the farmer in the grow-out ponds that the larvae that he is buying will not infect his pond or farm with a disease.

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It is also important to keep in mind that over-regulation could place unnecessary restrictions on free trade. Finally, at the administrative levels the impact of infectious diseases, especially non-notifiable diseases, is generally misunderstood or seriously underestimated, and it is not uncommon to find a lack of interest in their assessement. As an example, in mollusc hatcheries/nurseries, herpes virus infections have been frequently reported in larvae and juveniles and are involved in mortality outbreaks (Renault et al., 1994). The disease is not currently a notifiable disease subjected to specific control measures under EU or OIE legislation. However, the virus associated with these mortalities generates important economic losses and may jeopardize the sustainable development of this important socioeconomic activity in coastal regions.

9.5.2 Genetic improvment for disease resistance Enhancing fish and shellfish defense ability through their immunity is one of the important approaches to preventing and controlling infectious diseases. Studies were recently developed in order to provide valuable information for further understanding of the defense mechanisms in aquatic species and to define genetic markers of interest. In this context, selective breeding of aquacultured stocks appears as one of the most promising approaches for aquaculture development. Although there have been several fish breeding programs targeting the production of resistant animals to a particular viral disease worldwide since the 1990s, recent work has focused on positive correlation between resistance to several pathogens including bacteria and viruses. Odegard et al. (2007) assumed that efficient selection for improved resistance to both furonculosis, a bacterial disease, and ISA, a viral one, may be performed in Atlantic salmon (Salmo Salar). In shrimp, by serial cultivation of five successive generations of surviving P. stylirostris in captivity, an ‘IHHNVtolerant’ variety as been developed at the Ifremer center in Tahiti (French Polynesia) (Flegel, 2007). Taura syndrome virus (TSV)-tolerant shrimp stocks were also developed by serial selection of survivors from TSV challenges over several generations (Moss et al., 2005). However, TSV-tolerant families appeared to be not tolerant to WSSV, suggesting specific antiviral mechanisms (Moss et al., 2005). Based on recent data, research is iniated for developing Pacific oyster strains resistant or tolerant to OsHV-1 in France (T. Renault, pers. comm.). Data recently generated by genomic approaches may help in developing breeding programs by identification of genes of interest which can be used as genetic markers. Innate antiviral immunity and the RNAi pathways in fish and shrimp offer the possibility of genetic selection based on the identificaton of genes involved in these mechanisms. This information can be used to assist selection for antiviral resistance. Genetic engineering (gene transfer) can be a way to improve viral disease resistance in fish and shell-

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fish. However, this approach must be evaluated against the demands of society for environmentally responsible practices in agriculture and food safety.

9.5.3 Vaccination Although major advances in antiviral vaccine research in fish were promised by recombinant DNA technology, these vaccines are not easy to use in the field and cause security concerns. Long-term safety issues related to the environment and the consumer remain to be fully addressed. Other innovative technical approaches may also be developed. Liu et al. (2006) have recently reported the use of virus-like particles (VLPs) to protect grouper Epinephelus lanceolatus against grouper nervous necrosis virus (GNNV), a piscine nodavirus. Recombinant viral proteins produced in yeast also appear as promising tools for induction of a protective immune response in fish by delivery in feeds (Allnutt et al., 2007). This may be a feasible approach since yeast is already a componant of feeds and, moreover, its production is low cost and easily engineered.

9.5.4 Alternative methods of prevention and treatment The preventive or therapeutical use of chemotherapeutants is highly restricted since fish and shellfish farming is mostly carried out in the natural aquatic environment. No efficient therapeutic agents against fish and shellfish viruses have yet been developed and so alternative methods are urgently needed. Work with acute viral infections in shrimp has shown that administration of dsRNA based on the genomes of RNA and DNA viruses is protective (Robalino et al., 2005; Yodmuang et al., 2006). RNAi-based gene therapies in viral diseases appear clearly as a promising approach to silence viral gene expression and to inhibit viral replication in aquatic animals. Moreover, host genes such as genes encoding virus receptors may be also targeted (Li et al., 2007). However, the very short half-life of synthetic dsRNA after injection is a major concern: si RNAs may not stay long enough for complete protection. In this context, high expression vectors and better transfection techniques are needed. Moreover, it is important to keep in mind that some viruses have evolved strategies to inhibit gene silencing mechanisms. Antiviral factors including antimicrobial peptides (AMPs) produced early after a first encounter with a virus also appear promising tools to control viral infections in aquaculture. Falco et al. (2007) reported that synthetic human α-defensin-1 (HNP1) exhibits anti-VHSV activity. HNP1 inactivates virus particles and induces antiviral response in host cells (Falco et al., 2007). Morever, the same research team showed that a β-defensin-like peptide identified in the rainbow trout, O. mykiss, induces production of

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an antiviral activity and up-regulation of Mx, suggesting a type I IFNmediated antiviral mechanism (Falco et al., 2008).

9.5.5

Understanding complex interactions between animal, environment and pathogen for risk assessment Aquatic species are particularly challenged by their environment. They are frequently reared in environments subjected to fluctuations (temperature, salinity and pollution). Demonstration of the relationship between pollution and increase of susceptibility to infectious diseases has been carried out in aquatic species including bivalves. Chou et al. (1998) reported higher mortality rates in Meretrix lusoria contaminated with heavy metals and experimentally exposed to a birnavirus than in animals only contaminated or only infected. Summer seed mortality reported in Tomales Bay, California has been associated with extreme temperature, phytoplankton blooms and a herpesvirus (Burge et al., 2007). Reducing the impact of pathogens is likely to rely on knowledge of their biology.

9.5.6 Biosecurity Biosecurity appears as the favoured adopted approach. An efficient management of the sanitary status of fish and shellfish production relies on a significant involvement of the farmers who can be key players in disease control. Biosecurity has two main goals: to protect the facility and to protect the surrounding environment from the introduction of novel pathogens. Inherent to any biosecurity program is a record of the health history/status of all new animals entering a facility, control over movement of people and equipment, the ability to control water quality, a health monitoring program and standard operating procedures (SOPs) and current biosecurity measures at this facility. Measures must include: • • • •

health status of incoming animals; quarantine of brood animals; staff training; SOPs for all aspects of animal handling (e.g. daily checks, mortality reporting and response, handling of samples for disease diagnosis, etc.); • control measures: foot baths and footware of personnel; • handling of animals used in the development of their family lines. Moreover, a routine health sampling is needed to complement current protocols that examine animals only in response to problems. The utility of collecting samples for multiple analyses (such as histology and molecular analyses) appears as an important component of health monitoring for the facility particularly in light of the viral epidemic. The promotion of an integrated biosecurity approach and the development of rapid, reliable and sensitive diagnosis tools that meet the time

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constraints experienced under hatching conditions are of the utmost importance for efficient control of diseases in hatcheries.

9.6 References allnutt f c t, bowers r m, rowe c g, vakharia v n, lapatra s e and dhar a k (2007) Antigenicity of infectious pancreatic necrosis virus VP2 subvirla particles expressed in yeast, Vaccine, 25, 4880–8. anderson j, hedgecock d, berrigan m, criddle k r, dewey w, ford s, gouletquer p, hildreth r, paolisso m, targett n and whitlach r (2004) Non-native Oysters in the Chesapeake Bay, National Research Council, National Academy Press, Washington, DC. arzul i, nicolas j-l, davison a j and renault t (2001) French scallops: a new host for ostreid herpesvirus 1, Virology, 290, 342–9. bergé j p, bourgougnon n, alban s, pojer f, chermann j c, billaudel s, robert j m, durand p and franz g (1999) Antiviral and anticoagulant activities of a water soluble compound extracted from the marine diatom Haslea ostrearia, Planta Med, 65, 604–9. bjorklund h v, johansson t r and rinne a (1997) Rhabdovirus-induced apoptosis in a fish cell line is inhibited by a human endogenous acid cysteine proteinase inhibitor, J Virol, 71(7), 5658–62. bonami j r (1976) Viruses from crustaceans and annelids: Our state of knowledge, Proc Int Colloq Invertebr Pathol, 1, 20–3. bootland l m and leong j c (1999) Infectious hematopoietic necrosis virus, in Woo P K T and Bruno D W (eds), Fish Diseases and Disorders, volume 3, Viral, Bacterial and Fungal infections, CABI, Oxford, 57–121. boudinot p, massin p, blanco m, riffault s and benmansour a (1999) Vig-1, a new fish gene induced by the rhabdovirus glycoprotein, has a virus-induced homologue in humans and shares conserved motifs with the MoaA family, J Virol, 73, 1846–52. boudinot p, riffault s, salhi s, carrat c, sedlik c, mahmoudi n, charley b and benmansour a (2000) Vesicular stomatitis virus and pseudorabies virus induce a vig1/cig5 homologue in mouse dendritic cells via different pathways, J Gen Virol, 81, 2675–82. boudinot p, sahli s, blanco m and benmansour a (2001) Viral haemorrhagic septicaemia virus induces vig-2, a new interferon responsive gene of rainbow trout, Fish Shellfish Immunol, 11, 383–97. bower s m (2001) Synopsis of infectious diseases and parasites of commercially exploited shellfish: Assorted viruses detected in oysters and of unknown significance, Fisheries and Oceans Canada, Vancouver, BC, http://www.pac.dfo-mpo. gc.ca/sci/shelldis/pages/assortvirusoy_e.htm, accessed January 2009. brock j a, lightner d v and bell t a (1993) A review of four virus (BP, MBV, BMN and IHHN) diseases of penaeid shrimp with particular references to clinical significance, diagnosis and control in shrimp aquaculture, Proceedings of the 71st International Council for the Exploration of the Sea, C.M. 1983/Gen:10/1–18. bulet p, hetru c, dimarcq j l and hoffman j (1999) Antimicrobial peptides in insects: structure and function, Dev Comp Immunol, 23, 329–44. burge c a, judah l r, conquest l l, griffin f j, cheney d, suhrbier a, vadopalas b, olin p g, renault t and friedman c s (2007) Summer seed mortalities of the Pacific oyster, Crassostrea gigas Thunberg, grown in Tomales Bay, California, USA: the influence of oyster stock, planting time, pathogens and environmental stressors, J Shellfish Res, 26(1), 163–72.

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calvo g w, luckenbach m w, allen s k and burreson e m (2001) A comparative field study of Crassostrea ariakensis (Fujita 1913) and Crassostrea virginica (Gmelin1791) in relation to salinity in Virginia, J Shellfish Res, 20, 221–9. chang p h, kuo s t, lai s h, yang h s, ting y y, hsu c l and chen h c (2005) Herpeslike virus infection causing mortality of cultured abalone Haliotis diversicolor supertexta in Taiwan, Dis Aquat Org, 65, 23–7. charlet m, chernysh s, philippe h, hetru c, hoffman j and bulet p (1996) Isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc, Mytilus edulis, J Biol Chem, 271(6), 21808–13. chou h y, chang s j, lee h y and chiou y c (1998) Preliminary evidence for the effect of heavy metal cations on the susceptibility of Hard Clam (Meretrix lusoria) to clam birnavirus infection, Fish Pathol, 33, 213–19. cociancich s, bulet p, hetru c and hoffman j a (1994) Insect immunity and antibacterial proteins, Parasitol Today, 10, 132–9. colorni a, samocha t and colorni b (1987) Pathogenic viruses introduced into Israeli mariculture systems by imported penaeid shrimp, Bamidgeh, 39, 21–8. comps m and bonami j r (1977) Viral infection associated with mortality in the oyster Crassostrea gigas Thunberg, C R Acad Sci Hebd Seances Acad Sci D, 285, 1139–40. comps m and duthoit j l (1976) Infection virale associée à la maladie des branchies de l’huître portugaise (Crassostrea angulata lmk), C R Acad Sci Hebd Seances Acad Sci D, 282, 1991–3. comps m and duthoit j l (1979) Infections virales ches les huîtres Crassostrea angulata Lmk et Crassostrea gigas Th, Haliotis, 8, 301–7. couch j a (1981) Viral diseases of invertebrates other than insects, in Davidson E W (ed.), Pathogenesis of Invertebrates Microbial Diseases, Allanheld Osmun, Totowa, NJ, 127–60. cowley j a, dimmock c m, spann k m and walker p j (2000) Gill-associated virus of Penaeus monodon prawns: an invertebrate virus with ORF1a and ORF1b genes related to arteri- and coronaviruses, J Gen Virol, 81, 1473–84. davison a j, trus b l, cheng n, steven a c, watson m s, cunningham c, le deuff r m and renault t (2005) A novel class of herpesvirus with bivalve hosts, J Gen Virol, 86, 41–3. destoumieux d, bulet p, loew d, van dorsselaer a, rodriguez j and bachère e (1997) Penaeidins: A new family of antimicrobial peptides in the shrimp Penaeus vannnamei (Decapoda), J Biol Chem, 272(45), 28398–406. dhar a k, dettori a, roux m m, klimpel k r and read b (2003) Identification of differentially expressed genes in shrimp (Penaeus stylirostris) infected with White spot syndrom virus by cDNA microarrays, Arch Virol, 148, 2381–96. dios s, poisa-beiro l, figueras a and novoa b (2007) Suppression subtraction hybridization (SSH) and macroarray techniques reveal differential gene expression profiles in brain of sea bream infected with nodavirus, Molec Immunol, 44, 2195–204. dodd a, chambers s p and love d r (2004) Short interfering RNA-mediated gene trageting in the zebrafish, FEBS Lett, 561, 89–93. ec (2006) Council Directive 2006/88/EC of 24 October 2006 on animal health requirements for aquaculture animals and products thereof, and on the prevention and control of certain diseases in aquatic animals, Official Journal of the European Union, L328, 24 November, 14–56. eléouët j f, druesne n, chimlonczyk s, monge d, dorson m and delmas b (2001) Comparative study of In situ cell death induced by the viruses of Viral Heamorrhagic Septicemia (VHS) and Infectious Pancreatic Necrosis (IPN) in rainbow trout, J Comp Path, 124, 300–307.

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10 Diet and husbandry techniques to improve disease resistance: new technologies and prospects F. J. Gatesoupe, INRA-Ifremer, France

Abstract: This chapter examines the emergence of new products and methods that have been aimed at improving disease resistance in aquaculture, and the implications for health of other technologies developed for fish and shellfish husbandry and feeding. The first section deals with the direct inhibition of pathogens, and the second with ways of improving welfare in order to reduce stress. The third part is devoted to feed additives that can stimulate the defenses, and to dietary side effects important for fish and shellfish health. Key words: microbial management, biocontrol agents, neuroendocrine immune axis, feed additives, feed hazards.

10.1 Introduction Health is of concern for everybody. The geneticist can select resistant animals, the veterinarian can vaccinate, diagnose diseases and prescribe remedies, but their efforts would be ineffective without the constant awareness of the farmer, whose husbandry know-how is essential to preserve animal health. Before this, the nutritionist should have paved the way by designing healthy feeds and additives. Increasing knowledge allows the emergence of new products and methods, which can cope with sanitary issues within the framework of sustainable development. This section will examine such innovation, but also the implications for health of other technologies developed for fish and shellfish husbandry and feeding. For instance, intensive rearing and closed circuit systems may be stressful, and hence threaten health. Another challenge is to replace fish meal and oil with alternative sources of protein and lipids while still meeting nutritional

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Pathogens

Immunostimulants Probiotics Prebiotics

Stress

Microbiota

Physical barrier innate Immune system

adaptive (finfish)

Fig. 10.1 External factors affecting health and the immune response: interactions between pathogens, stress factors, feed, microbiota and the immune response. Hygiene maintenance is the first weapon against pathogens (Section 10.2). Welfare improvement should limit the causes of stress, which degrade the immune defenses (Section 10.3). Diet should be aimed at stimulating innate immunity and the balance of the gut microbiota which bar the way to infection and which ‘educate’ the immune system. The diet should also reduce the adverse effects of stress, while avoiding nutrient excess that could benefit pathogens (Section 10.4).

requirements, and stimulating the immune system. Furthermore, the role of intestinal microbiota in fish health and nutrition has been recently shown, and it is worth reconsidering the complex interactions between pathogens, stress factors, feed, microbiota and the immune response of the animal (Fig. 10.1). Two main strategies will be addressed to fish farmers, describing (i) how they can fight the pathogens directly, by adjusting hygiene practice, and the future trends in pathogen inhibition, then (ii) how they can improve welfare to reduce stress. The third part will be devoted to formulating the feed to optimize the defenses of fish and shellfish.

10.2 Fighting the pathogens The previous Chapters (7–9) described the means available to avoid infection with various pathogens. The maintenance of strict hygiene conditions is fundamental, but that does not signify that a completely sterile environment would be desirable. The association of gut microbiota with aquatic organisms has been recently requalified from commensalism to mutualism, inferring the importance of preserving this ecosystem as far as possible.

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That may be difficult in mollusc and shrimp hatcheries, where antibiotics are currently used to avoid mortality crashes, but alternative solutions are emerging (Riquelme et al., 2001; Garcia and Massam, 2005).

10.2.1

Understanding the importance of preserving intestinal microbiota The methods of gnotobiology have elucidated the roles of microbiota associated with aquatic animals (reviewed by Marques et al., 2006). Although bivalve larvae are certainly among the most sensitive organisms to bacterial diseases, Douillet (1989) noted poor growth and survival of bacteria-free oyster larvae (Crassostrea gigas) fed axenic algae. In aseptic conditions, most bacterial strains were harmful (Douillet and Langdon, 1993), but Douillet (1989) found three strains that improved survival and, among them, strain CA2 which also improved growth. In a similar experiment, Besse and Nicolas (1989) concluded that bacteria provide essential nutrients for bivalve larval growth. This was further confirmed by Douillet (1993), who demonstrated that oyster larvae digested and assimilated bacterial carbon from strain CA2. Antibiotic treatments may be also detrimental to water quality, as experienced by Andersen et al. (2000), who noted a build-up of ammonia in batches of great scallop larvae treated with chloramphenicol, hypothetically due to an alteration in microbial activity. In the absence of gnotobiological data, the role of microbiota has not been proved in penaeid larvae. However, Thompson et al. (1999) showed that São Paulo Shrimp (Penaeus paulensis) larvae fed bacteria in xenic conditions could survive longer than the starved control. Mohamed (1996) showed that relatively good results for development and survival of giant tiger prawn (Penaeus monodon) could be obtained with the partial substitution of dietary microalgae by bacterial biomass. The efficiency of bacteria as feed for brine shrimp (Artemia) was further studied in gnotobiotic conditions (Gorospe et al., 1996; Marques et al., 2006). The essential role of microbiota in fish gut ontogeny was demonstrated by Rawls et al. (2004, 2006), who compared the responses of axenic zebrafish (Danio rerio) larvae to those of germ-free fish inoculated post-hatch with bacterial strains or complex microbiota. These authors showed the role of gut bacteria in intestinal cell proliferation, immune response and nutrient metabolism, by quantifying the differential expression of marker genes. Microbiota originating from conventionally reared zebrafish were much more efficient than any of the isolates, especially in stimulating cell proliferation. The effect of microbiota on gut maturation in zebrafish was further evidenced by Bates et al. (2006), who observed in germ-free larvae several signs of poor differentiation of the intestinal epithelium, e.g. no detection of phosphatase activity in the brush border membrane, and lack of protein uptake by endocytosis.

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10.2.2 Improving hygiene Strict hygiene rules should accompany the preservation of intestinal microbiota, to avoid infection (Section 10.6). Every person, live organism and material entering the rearing area may introduce pathogens. Thus there is a need for appropriate precautions, which include the quarantine of animals of non-certified origin, and all the disinfection treatments that are reasonably feasible. Water disinfection is particularly important, and there are several methods available. The most common one is based on physical treatment by UV irradiation. Summerfelt (2003) preferred this method, which is less expensive and simpler than ozonation, but which cannot work in the case of turbid water. Mitigated results were obtained by the combination of sand filtration and UV sterilization in mollusc hatcheries (Ford et al., 2001; SainzHernández and Maeda-Martínez, 2005), and some negative effects were reported by Matson et al. (2006). The chemical methods of disinfection – for instance with ozone or chlorine – may generate toxic by-products. However, there is a growing interest in ozonation, which not only disinfects the water, but also improves its quality for recirculation systems (Tango and Gagnon, 2003). After initial disinfection, the microbial settlement is generally left to chance in the rearing system, based on the recurrence of ‘endemic bacteria’ (Van Rijn, 1996). This is made reliable by a biofilter that creates narrow niche specialization, with high stability in microbial function, but diversity of the competent strains (Skjermo et al., 1997; Cytryn et al., 2005; Tal et al., 2006). However, the introduction of ‘starter bacterial population’, and other bioremediation and biocontrol agents, may be a safe precaution (Gross et al., 2006; Sections 10.2.5, 10.3.2). During the course of rearing, the wastes should be properly treated, and animal health should be constantly surveyed. Compartmentalizing the rearing units is essential to reduce the risk of disease spread, as well as fallowing at the end of each production cycle.

10.2.3 New antimicrobial preparations and compounds Many extracts from plant and animal origin have been documented for their antibacterial activity against fish pathogens (Gatesoupe, 2008a). Herbal medicines seem particularly promising as alternatives to antibiotics, not only for fish but also for shrimps (Direkbusarakom, 2004), including the post-larval stages (Citarasu et al., 2003a,b). Inhibitory effects were noted against fish and shrimp pathogenic viruses (Direkbusarakom, 2004; Balasubramanian et al., 2007) and fungi (Xu et al., 1994; Harikrishnan and Balasundaram, 2005). These medicines may also have immunostimulatory effects on fish (Section 10.4.4), shrimps (Citarasu et al., 2006) and abalone (Xue et al., 2008). Such effects on the host could account for the broad spectrum of diseases that might be treated, including infestation with

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parasites (Auro de Ocampo and Jimenez, 1993; Direkbusarakom, 2004). However, the preventive or curative doses should be investigated carefully before application to a particular species, due to the risk of toxicity of some extracts, like that of neem (Azadirachta indica) for Indian carps (Das et al., 2002). This author has found no instances of plant extracts being used to treat bivalve larvae, which seem particularly susceptible to xenobiotics. It may be difficult to avoid the use of antibiotics in the case of great scallop (Pecten maximus) (Torkildsen et al., 2002). Smith (1994) suggested selecting microalgae with antimicrobial activity as shellfish food. That was effective for crustacean larvae – e.g. to control Vibrio spp. in shrimp hatcheries (Regunathan and Wesley, 2004) – but likely to be insufficient for molluscs, which require other sustainable treatments. Sun and Oliver (1994) suggested using diacetyl as antimicrobial agent to decrease the risk of human infection with Vibrio vulnificus from eating raw oysters, but the treatment was found effective only after shucking (Birkenhauer and Oliver, 2003). A solution applicable to larviculture was proposed by Takahashi et al. (2000a), who showed the inhibitory activity of ovoglobulins from hen eggs against Vibrio tubiashii, in experimental challenges of Pacific oyster (Crassostrea gigas) larvae. Other products from animal origin may have antimicrobial activity, and such antagonistic properties may be mainly expected from components of the innate immune system. Besides the natural defenses, some of these compounds extracted from other animals could be used, e.g. heterologous antimicrobial peptides. Ho et al. (2002) tested in vitro the antagonism of cecropins against shrimp pathogens. These peptides are unlikely to be suitable to treat marine shrimps, due to the minimum bactericidal concentration which was particularly high in seawater, close to the toxic dose for shrimp haemocytes. Jia et al. (2000) showed the need for a constant intraperitoneal supply of antimicrobial peptides to protect coho salmon (Oncorhynchus kisutch) against Vibrio anguillarum infection. The practical application of heterologous antimicrobial peptides would require finding the appropriate mode of introduction, such as transgenesis (Morvan et al., 1997; Cheng et al., 2001), or recombination in yeast (Dorrington, 2006). Among other animal products with antimicrobial activity, one could cite chitosan (Anas et al., 2005), and an extract from a marine sponge (Dendrilla nigra) (Selvin et al., 2004a), possibly due to symbiotic Streptomyces sp. (Selvin et al., 2004b). The use of inhibitors of bacterial quorum sensing has been also proposed by Defoirdt et al. (2004), with some practical applications to shrimps (Manefield et al., 2000; Defoirdt et al., 2006; Bai et al., 2008) and fish (Rasch et al., 2004, 2007). These inhibitors should be further tested, but caution should be exercised due their risk of toxicity for the host and microbiota. Moreover, slight changes in the biochemical structure may turn antagonistic molecules into agonists of quorum sensing (Geske et al., 2007). Another

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innovative treatment was proposed by Defoirdt et al. (2007), who showed the inhibitory effect of poly-β-hydroxybutyrate against Vibrio campbellii in a challenge test with Artemia franciscana. The compound is produced and accumulated by bacterial strains, which may be used as convenient vectors for aquaculture purposes (Halet et al., 2007).

10.2.4 New biocidal compounds The use and environmental impact of classical disinfectants were reviewed by the joint Group of Experts on the Scientific Aspects of Marine Environmental Protection (GESAMP, 1997), including chloramine T, formalin, hypochlorite, iodophores and quaternary ammonium compounds. There is a growing interest in bronopol (2-bromo-2-nitro-1,3 propanediol, Pycese®) to protect fish and eggs against pathogenic fungi (Pottinger and Day, 1999) and bacteria (Treasurer et al., 2005; Birkbeck et al., 2006). Bronopol seemed less efficient against parasitosis, like ‘white spot’ on rainbow trout (Oncorhychus mykiss) (Shinn et al., 2005). The compound was not suitable in preventing mortality in scallop larvae (Torkildsen et al., 2002).

10.2.5 Biocontrol agents Many microbes are antagonistic to other microbes, and some are specific pathogens of infectious agents encountered in aquaculture. These properties can be exploited for biological control, but the potential for several cross-relations between probiotics and pathogens has been under-explored, to my knowledge (Table 10.1). This approach should not be confused with the indirect action of microbes used to stimulate the immune response of the host, which will be considered in Section 10.4.6. The control of viral infection by recombinant virus was noted there, notwithstanding its indirect mode of action by killing specifically infected Table 10.1 Some examples of potential biocontrol agents in aquaculture; many cross relations are, to my knowledge, still unexplored Biocontrol agent Pathogen Virus

Bacterium

Fungus

Reviewed by Maeda, 2004

Unexplored

Reviewed by Gram and Ringø, 2005

Fungus

‘Virus against virus’ technology, unexplored Bacteriophage therapy (reviewed by Nakai and Park, 2002) Unexplored

Parasite

Unexplored

Suggested by Leaño et al., 2005 ‘Killer yeast’ (Wang et al., 2007) Unexplored

Virus Bacterium

Saprolegnosis control (Lategan et al., 2004) Unexplored

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cells of the host. First proposed by Schnell et al. (1997) against HIV, this promising technology for human medicine is unlikely to bring applications to aquaculture in the short term, but one could have the same opinion about bacteriophage therapy, which was successfully tested in fish (Nakai and Park, 2002) and shrimp (Vinod et al., 2006; Karunasagar et al., 2007). Other viruses are specific to pathogenic fungi and parasites, and they could be considered as possible biocontrol agents. For example, fungal hypoviruses were used against the chestnut blight fungus (Milgroom and Cortesi, 2004). The high specificity of viruses is a limiting factor in developing such approaches in connection with aquaculture. However, at least those targeting major diseases devoid of sustainable treatments should be worth investigating. The search for biocontrol of parasitism in aquaculture seems to be still lying fallow. There are few documented cases of applications of fungi, despite their great potential as anti-infective agents (Bhadury et al., 2006). Wang et al. (2007, 2008a) proposed to exploit a toxin produced by Pichia anomala against Metschnikowia bicuspidata, which was pathogenic in a Portunid crab (Portunis trituberculatus). Leaño et al. (2005) characterized fungi associated with shrimp culture in ‘green water’, and they suggested a possible involvement in the control of luminous Vibrio. The most documented candidates for biocontrol are certainly bacteria. Many strains have been proposed as probiotics, and some are commercially exploited in shrimp and fish farming. Specific antagonisms of probiotic bacteria have been listed in many reviews (e.g. Gram and Ringø, 2005; Gatesoupe, 2008a). Some bacteria may be used as antiviral agents (Maeda, 2004), while others are active against fungi. For example, Gil-Turnes et al. (1989) described the protection of shrimp embryos against pathogenic Lagenidium callinectes, due to the production of antifungal metabolite by symbiotic Alteromonas sp. Lategan et al. (2004) proposed to prevent saprolegniosis in eels by adding Aeromonas media cultures to tank water. However, even though these are attractive prospects, such treatments should be evaluated cautiously. Secord (2003) stressed the risk of attempting biocontrol in the aquatic environment, in view of the paucity of literature and experience, and Wang et al. (2000) raised a first warning on aquacultural application, by attributing the emergence of a new bacterial syndrome in Penaeus monodon to regular use of probiotic Bacillus subtilis.

10.3 Improving welfare Ashley (2007) identified health as a fundamental measure of fish welfare. The neurochemical response to stress interacts with the immune system, and deep distress may have a long-lasting effect on health (Huntingford et al., 2006). After a brief reminder of the knowledge of neuroimmunology

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in fish and shellfish, this section will introduce the main environmental and husbandry factors involved in stimulating the animal’s response.

10.3.1

Interactions between neuroendocrine and immune functions in fish and shellfish The turn of the 20th century has brought new understanding of the evolution of innate and adaptive immunity (Flajnik and Du Pasquier, 2004). The frontier between adaptive defenses – supposedly specific to vertebrates – and innate response has been mitigated with new findings. Arala-Chaves and Sequeira (2000) observed some secondary response in haemocyte proliferation of kuruma prawns (Penaeus japonicus), after stimulation with fungal antigens, and then Kurtz and Franz (2003) showed the reduction of infectivity of parasitic tapeworm in the copepod Macrocyclops albidus, after primary infection. Such responses – possibly mediated by adhesion molecules from the immunoglobulin superfamily – remain quite limited in comparison with those of vertebrates. Still speculative is the idea that the adaptive immune system could be an evolutionary offshoot of the vertebrate nervous system (Bayne, 2003), but there is growing evidence that both systems can communicate with cytokines and neuropeptides, in a similar way in vertebrates and invertebrates (Salzet, 2001; Engelsma et al., 2002; Fig. 10.2). Cortisol can depress some adaptive responses of the immune system in fish, while the activity of innate components is enhanced (Weyts et al., 1999). Many other hormones can modulate the immune response in fish, e.g. growth hormone, prolactin, reproductive hormones and melanotropins (Schreck, 1996; Harris and Bird, 2000; Yada, 2007). Acute stress increased the plasmatic concentrations of cortisol, adrenaline and lysozyme in rainbow trout, while chronic stress may reduce the efficacy the immune response (Demers and Bayne, 1997). Similar effects have been observed in invertebrates, for instance immunodepression in shrimps with dopamine (Cheng et al., 2005a; Li et al., 2005; Chang et al., 2007) and noradrenaline (Cheng et al., 2006). The stress response of Pacific oyster is also under adrenergic control (Lacoste et al., 2001a,b).

10.3.2 Improving water quality and bioremediation Among external signals that can elicit stress, many parameters of water quality have been identified in fish, like temperature, salinity, hardness, pH, nitrogenous compounds, dissolved oxygen and CO2 (Ellis et al., 2002; Håstein et al., 2005; Portz et al., 2006). Many immunoassays can be used to estimate the impact of environmental factors on fish immunity (Anderson, 1996). The effects of environmental factors on shrimp immunity have also been evaluated, mainly based on haemocyte counts and phenoloxydase activity (Le Moullac and Haffner, 2000). Smith et al. (2000) proposed to

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Stress Mollusca, Crustacea

Teleostei Hypothalamus CRH

DA

TRH Pituitary

ACTH CAs

α-MSH β-E

Cortisol Head kidney Cytokines

Immunocyte

Antibody formation

Encephalins

Fig. 10.2 Known similarities of mediators that interact between the neuroendocrine and immune systems in fish and shellfish, a tentative scheme after several sources (Ottaviani et al., 1998, 2004; Weyts et al., 1999; Salzet, 2001; Stefano et al., 2002). The grey zone indicates which hormones, or closely resembling molecules, have been identified both in invertebrates and vertebrates: CRH, corticotropin-releasing hormone; DA, dopamine; TRH, thyrotropinreleasing hormone; ACTH, adenocorticotropic hormone; α-MSH, α-melanocytestimulating hormone; β-E, β-endorphin; CAs, catecholamines.

measure nitric oxide in blue mussel (Mytilus edulis) as indicator of the immune status after environmental stress. For healthy stock, it is essential to monitor water quality and to avoid severe fluctuations. Physical processes can ensure water quality, but it may be worth considering the introduction of competent microorganisms, such as phytoplankters or other microbes. In pond aquaculture, the chemical equilibrium is dependent on microbial activities (Abraham et al., 2004), and bioremediation has been successfully applied to shrimp culture (Devaraja et al., 2002; Wang et al., 2005). Water treatment by live bacterial preparations seemed less efficient with fish in pond culture, but Taoka et al. (2006a) noted that the introduction of complex microbial mixture into closed recirculating system was more effective than oral administration to Japanese flounder (Paralichthys olivaceus). Fish growth indeed was only improved

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by water treatment, among other beneficial effects observed in both cases. Some Bacillus spp. strains also improved water quality in aquaria with common carp (Cyprinus carpio) (Lalloo et al., 2007).

10.3.3 Improving rearing conditions There is a general trend to maximize stocking density, for economical reasons. Such a practice may exert adverse effects on health, depending on the innate behavioural responses of each species and on the ability of the producer to maintain water quality. For example, Li et al. (2006) demonstrated the interacting effects of dissolved oxygen and stocking density on the immune status of Chinese shrimp (Fenneropenaeus chinensis). Subhash and Lipton (2007) observed that bacterial load increased with increasing stocking density of pearl oyster larvae (Pinctada margaritifera), while survival decreased. The response is not only species-specific, but sex ratio was also shown to interfere in Mozambique tilapia (Oreochromis mossambicus) exposed to chronic confinement (Binuramesh et al., 2005). Many other parameters in husbandry conditions can affect health, like photoperiod, possibly mediated by melatonin secretion from the pineal gland of fish during the dark phase (Ángeles Esteban et al., 2006; Cuesta et al., 2008). Stressful human interventions – like transportation, handling or netting – should be kept to a minimum. Acute stress, often experimentally reproduced by netting, increased the activity of cortisol, adrenaline and lysozyme in rainbow trout plasma (Demers and Bayne, 1997). Many other immunological indicators may be altered, including those related to adaptive immunity and complement pathways in gilthead sea bream (Sparus aurata) (Sunyer et al., 1995). Both environmental and husbandry factors may generate oxidative stress (Livingstone, 2001, 2003), which will be considered in the next section, since it can be prevented by adequate feed supply.

10.4 Improving feed The diet is mainly aimed at meeting the nutritional requirements of the animal, which will be considered in detail in the next Chapters (11–17). This section will deal first with the requirements to optimize the immune defenses, but the diet also has side effects important for fish health. In particular, pathogens could benefit from some nutrients brought in excess, whereas the gastrointestinal microbiota – the first defense line against pathogens – are also influenced by substrates available from feed. The dietary supply of specific products like probiotics and prebiotics may be a good precaution against health hazards due to microbial variability.

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10.4.1 Fatty acids and antioxidants The dietary fatty acid supply is particularly important for health, especially in terms of ratios between the n-3 and n-6 series, and between the highly unsaturated fatty acids (HUFA). The fatty acids requirements for growth are different between aquatic species. Warm freshwater species require both n-3 and n-6 series, unlike salmonids and marine fish that require mainly n-3 fatty acids, depending on bioconversion capabilities, which are generally greater in freshwater fish and salmonids than in marine species (Takeuchi, 1997). When considering the effects on disease resistance instead of growth, these trends should be moderated (Table 10.2). Dietary supplementation with vegetable oil rich in linoleic acid seems beneficial to the immune defense of many species, even when they do not require n-6 fatty acids for growth, while an excess of n-3 HUFA supply may be detrimental to the immune response. These requirements may result from several different biochemical pathways. 1. Dietary fatty acids can modulate the composition the phospholipid bilayer of the cell membrane, thus influencing membrane fluidity and toll-like receptors (Koch and Heller, 2005), which are involved in the immune response of both vertebrates and invertebrates (Beschin et al., 2001). 2. Dietary fatty acids also affect the production of eicosanoids by the cyclooxygenase pathway (Yaqoob, 2004). Several eicosanoids derived from arachidonic acid have been shown to modulate immunity in fish (Rowley et al., 1995; Van Anholt et al., 2004). Exogenous leukotriene B4 – extracted from blood leukocytes of European turbot (ScophthalTable 10.2 Comparisons of essential fatty acids requirement between fishes, considering either growth or disease resistance Fatty acids requirement for optimal Species Growth

Immune response

Cyprinus carpio Epinephelus malabricus

n − 3HUFA > LNA > LA (reviewed by Takeuchi, 1997) DHA > EPA (Wu et al., 2002)

Ictalurus punctatus Oncorhynchus mykiss Salvelinus alpinus

n − 3HUFA > LNA > LA (reviewed by Takeuchi, 1997) 1 %LNA or 0.5 %n − 3HUFA (reviewed by Takeuchi, 1997) LA + LNA? (Yang and Dick, 1994)

Improvement with n − 3HUFA (Pilarczyk, 1995) DHA > EPA (Wu et al., 2003) LA + n − 3HUFA > n − 3HUFA > LA (Lin and Shiau, 2007) Excess of n − 3HUFA detrimental (Li et al., 1994) 1 %n − 3HUFA detrimental (Kiron et al., 1995b) LA > LNA > n − 3HUFA (Lødemel et al., 2001)

n − 6 series: LA = linoleic acid. n − 3 series: LNA = linolenic acid; HUFA = highly unsaturated fatty acids; EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid.

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mus maximus) – inhibited the viral replication in rainbow trout cells infected with the virus causing hemorrhagic septicaemia (Tafalla et al., 2002). Prostaglandin E2 (PGE2) inhibited ulceration in gastric mucosa isolated from European eel (Anguilla anguilla) (Faggio et al., 2000). The parasitic copepod Lepeophtheirus salmonis secreted PGE2, likely inhibiting the immune response of its host, Atlantic salmon (Salmo salar) (Fast et al., 2004). The modulatory effect of PGE2 on cyclooxygenase 2 (COX-2) was found to be dose-dependent in macrophage-like cells from Atlantic salmon (Fast et al., 2005). Arachidonic acid and/or eicosanoid derivative(s) also stimulated the immune defenses of Pacific oyster (Delaporte et al., 2006). 3. Proinflammatory cytokines stimulate COX-2, and also the inducible nitric oxide synthase of fish macrophages (Secombes et al., 2001; Lindenstrøm et al., 2004; Buonocore et al., 2005). These two oxidative pathways are thus interacting to regulate macrophage respiratory burst (Novoa et al., 1996). Nitric oxide is important for normal physiological functions, and for the elimination of pathogens, but it can also contribute to oxidative stress by reacting with superoxide to form peroxynitrite, a powerful oxidant causing cellular damage (Beckman and Koppenol, 1996; Roch, 1999). 4. Animals activate NADPH-oxidase pathways in response to infective attack or to xenobiotic contaminants. They produce highly reactive oxygen species (ROS), such as superoxide and hydroxyl radicals, and hydrogen peroxide, which can kill the pathogen or detoxify the xenobiotic, but also generate oxidative stress, and damage host cells. This may occur especially when HUFA are supplied in excess, due to lipid peroxidation with ROS (Kelly et al., 1998; Roch, 1999; Zhang et al., 2007). Several minerals can catalyze these reactions, mainly iron (Sutton et al., 2006) and copper (Berntssen et al., 2000). These intricate relationships between oxidative pathways make it essential to counterbalance pro-oxidant activities by bringing sufficient amounts of antioxidant compounds into the diet, mainly vitamins C and E. Both these vitamins act in synergy, and they can be added at dietary doses that grossly exceed nutritional requirements. The effects of megadoses of vitamin C seem variable, depending on species and experimental conditions (e.g. Waagbø et al., 1993; Sealey and Gatlin, 2002; López et al., 2003; Wang et al., 2006; Gatesoupe, 2008a). Vitamin A also has antioxidant properties, but excess supply may be harmful, especially during larval stages (Ørnsrud et al., 2002; Fu et al., 2006). 10.4.2 Other nutrients important for health Besides fatty acid requirements, and the maintenance of the equilibrium between pro- and antioxidants in the organism, there are many other ways by which the diet can influence health and the immune system.

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The antioxidant vitamins have other physiological roles, like vitamin C, which acts as coenzyme for pro-collagen hydroxylation into collagen, and which is essential for tissue damage repairs and wound healing (Wahli et al., 2003). Other coenzyme vitamins have been documented to modulate the immune response of aquatic animals. For instance: pyridoxine – a key cofactor for amino acid metabolism – stimulates an immune response in disk abalone (Haliotis discus hannai) (Chen et al., 2005); folic acid – transformed to tetrahydrofolate, a coenzyme involved in the metabolism of amino acids and nucleic acids – seemed important for the resistance of channel catfish (Ictalurus punctatus) to Edwardsiella ictaluri infection (Duncan and Lovell, 1994). Metal ions, as common cofactors of many enzymatic reactions, are essential for the immune response. However, the dose needs to be adjusted cautiously (Lee and Shiau, 2002), to avoid excess that could increase the risk of peroxidation (Section 10.4.1), or benefit pathogens (Section 10.4.3). The organic forms of dietary metal supply are generally more efficient than inorganic minerals (Wang et al., 1997; Gatta et al., 2001). Li and Gatlin (2006) recently reviewed the effects of dietary nucleotides on fish immunity. Though nucleotides are not considered essential nutrients, de novo synthesis is costly, and it was not surprising to find many beneficial effects of their dietary supply on gene expression and enzymatic reactions involved in the immune and stress responses, not only in fish but also in shrimps (Fegan, 2004; Choudhury et al., 2005). However, the efficacy of dietary nucleotide supplementation remains to be demonstrated under fish farming conditions, where shrimps probably obtain a sufficient supply of nucleotides from the microbes that they ingest in large amounts (Li et al., 2007b). The same remark should also apply to bivalve molluscs, although genomic bacterial DNA was recently proposed as an immunostimulant for a mussel, Hyriopsis cumingii (Hong et al., 2006). Protein supply is also important for immunity. A protein-deficient diet reduced lysozyme activity and C-reactive protein response in rainbow trout (Kiron et al., 1995a), and sub-optimal dietary protein levels were detrimental to the immune status of whiteleg shrimp (Litopenaeus vannamei) (Pascual et al., 2004). The form of protein supply may have side effects, especially when fish meal is replaced by alternative protein sources, which may contain antigenic proteins and enzymatic inhibitors (Section 10.4.7). Gildberg et al. (1996) isolated peptidic fractions from Atlantic cod (Gadus morhua) stomach hydrolysate, which stimulated oxidative burst activity in Atlantic salmon leucocytes in vitro. These authors suggested applications of such protein hydrolysates as vaccine adjuvant and feed immunostimulants. The intraperitoneal injection of cod muscle hydrolysate confirmed that it was possible to stimulate respiratory burst in vivo (Bøgwald et al., 1996), but its use as a feed additive was inefficient (Gildberg et al., 1995;

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Gildberg and Mikkelsen, 1998). However, a dietary supply of 10–15 % of fish protein hydolysate stimulated humoral immunity in large yellow croaker (Pseudosciaena crocea) (Tang et al., 2008). Histones from cod milt seemed efficient as a feed additive to protect Atlantic cod challenged with V. anguilllarum, but it was not possible to elucidate the mechanism of action. Presumably, it either stimulated superoxide production in macrophages – as observed after peritoneal injection (Pedersen et al., 2003) – or directly inhibited the pathogen, due to antibacterial properties (Pedersen et al., 2004).

10.4.3

Nutritional competition between pathogens, gut microbiota and the host: iron as a key issue Although crucial for health, the competition for nutrients between host and microbes has seldom been studied. Before absorption, most nutrients remain freely available to the microbes present in the gastrointestinal tract. The pathogens that successfully invade can acquire all the nutrients they need from the host except iron, which is strongly bound (Ratledge and Dover, 2000). Natural iron chelators play a fundamental role in competition among prokaryotes and eukaryotes, especially in the iron-limited marine environment (Hutchins et al., 1999). The competition for iron is probably involved in the biocontrol of some fish pathogens by bacteria (Smith and Davey, 1993; Gram et al., 1999), and such chelators may be useful in inhibiting pathogens, like the oyster parasite Perkinsus marinus (Gauthier and Vasta, 2004) or in increasing disease resistance, like in European turbot challenged with pathogenic Vibrio (Gatesoupe, 1997). Kakuta and Murachi (1993) observed a sharp decrease in iron content of most tissues of Anguilla japonica (Japanese eel) infected with Aeromonas salmonicida, except at the infection site. Dietary iron supply must be sufficient to avoid anemia in channel catfish (ca. 30 mg kg−1, Lim and Klesius, 1997), but without excess, since a purified diet supplemented with 180 mg kg−1 inorganic iron caused increased susceptibility to Edwardsiella ictaluri (Sealey et al., 1997). The requirement for iron is conditioned by synergistic effects with vitamin C (Waagbø et al., 1993) and n-3 HUFA (Rørvik et al., 2003). Welker et al. (2007) demonstrated that the protective effect of dietary bovine lactoferrin on Nile tilapia (Oreochromis niloticus) challenged with Streptococcus iniae was related to a decrease in plasmatic iron, due to the iron-binding protein. The dietary supply of lactoferrin had variable effects, depending on species, rearing conditions and the immune parameters studied. It also reduced stress in common carp (Kakuta, 1998) and increased non-specific defenses and disease resistance in rainbow trout, Asian catfish (Clarias Batrachus) and the giant freshwater prawn (Macrobrachium rosenbergii) (Sakai et al., 1993; Kumari et al., 2003; Chand et al., 2006), but not in an experiment with Atlantic salmon (Lygren et al., 1999).

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10.4.4 Non-nutritive immunostimulants Several authors have reviewed the use of immunostimulants in fish farming. For instance, Sakai (1999) stressed that injection was the most effective mode of administration, though difficult to apply in practice, except as a vaccine adjuvant (Anderson, 1992). The efficacy of oral and immersion methods decreases with long-term administration (Sakai, 1999), but their use as a diet additive is more convenient for large-scale production applications (Gannam and Schrock, 1999). Dietary immunostimulants have various compositions, and there is no official classification. Their diversity is illustrated by some examples in Table 10.3, which deals with feed additives other than nutrients and living probiotics, since both of these categories are considered elsewhere (Section 10.4.1–3 and Section 10.4.6). Polysaccharides – the most important class of immunostimulants – will be described in Section 10.4.5. The most recently developed synthetic compound is levamisole, classified as a T cell stimulator by Anderson (1992). This compound was particularly efficient in stimulating immunoglobulin M (IgM) production in gilthead sea bream (Cuesta et al., 2004), but it also stimulated non-specific

Table 10.3 Some examples of immunostimulants used as feed additives for fish and shrimps Application to shrimp

Type

Product

Application to fish

Synthetic

Levamisole

Cuesta et al., 2004

β-glucans

Yeast glucan, laminaran, etc. Bacterin Peptydoglycan Lipopolysaccharides

Kumari and Sahoo, 2006 Sakai et al., 1995 Itami et al., 1996 O’Donnell et al., 1994

Whole cells

Ortuño et al., 2002

Mannan oligosaccharide Spray-dried

Staykov et al., 2007

Baruah and Prasad, 2001 Suphantharika et al., 2003 Azad et al., 2005 Itami et al., 1998 Takahashi et al., 2000b Sajeevan et al., 2006 Genc et al., 2007

Watanuki et al., 2006

Lee et al., 2003

Water-soluble extracts Alginate

Castro et al., 2006

Fu et al., 2007

Fujiki et al., 1994

Plant extracts

Medicinal herbs

Dügenci et al., 2003

Animal extracts

Chitin and chitosan

Gopalakannan and Arul, 2006

Cheng et al., 2005b Citarasu et al., 2006 Wang and Chen, 2005

Bacterial derivatives Yeast products

Spirulina platensis Seaweed derivatives

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immunity in fish (Anderson, 1992), as well as in shrimp (Baruah and Prasad, 2001). Among the natural immunostimulants, β-glucans are by far the most documented ones for aquaculture purposes, probably due to the limited risk of their having an impact on flesh quality and the aquatic environment (Gannam and Schrock, 1999; Section 10.4.5). As with most dietary immunostimulants, β-glucans tend to provoke mainly innate responses in fish (Kumari and Sahoo, 2006), and similar efficacy has been observed in shrimps (Suphantharika et al., 2003). These glucans can be obtained from various source organisms such as bacteria, fungi, and seaweeds. For example, the main cell wall component of yeast is (1,3/1,6)-β-D-glucan, and whole yeast cells are used as immunostimulants (Ortuño et al., 2002; Sajeevan et al., 2006). Besides bacterial preparations like bacterin, which are used as vaccine, other inactivated bacterial cells can be administered as simple immunostimulants, e.g. air-killed Clostridium butyricum, which increased the resistance of rainbow trout to Vibrio anguillarum (Sakai et al., 1995). Some authors spoke of ‘vaccination’ to describe the stimulation of the immune response observed in shrimps with formalin-killed Vibrio. Alabi et al. (1999) observed a protective effect of immersing larval Indian white prawn (Penaeus indicus) in a suspension of dead cells of Vibrio harveyi before challenge experiments, while the protection was not conferred by incorporating the lyophilized inactive pathogen into compound diet. However, Bohnel et al. (1999) obtained positive results with the oral route by using Artemia as ‘vaccine’ vehicle for post-larval Penaeus monodon. More recently, Azad et al. (2005) confirmed that heat-killed V. anguillarum could be efficiently administered via the feed of P. monodon post-larvae. Bacterial cell wall components were also used in fish and shrimps, for instance peptidoglycan from Bifidobacterium thermophilum (Itami et al., 1996, 1998) and lipopolysaccharides (Dalmo et al., 1998; Takahashi et al., 2000b), although Huttenhuis et al. (2006) warned about pathogen-derived lipopolysaccharides, which were suspected to cause immunotolerance. In addition, many other extracts from various materials have been put forward as potential immunostimulants, like alginate from seaweeds (Section 10.4.5). Many herbal medicines have been tested for their immunodulating properties in fish. Some may act as enzyme inhibitors, with possible side effects as antinutritional factors (Section 10.4.7). Among the other active preparations, one can cite for example: ginger extract (Dügenci et al., 2003); glycyrrhizin, the active ingredient of licorice root (Jang et al., 1995); aloe leaves (Kim et al., 2002); garlic bulbs (Sahu et al., 2007). Among animal products, chitin and its deacylated derivative, chitosan, seem particularly interesting in stimulating immunity. Chitosan was found more efficient than chitin in a challenge experiment of common carp with Aeromonas hydrophila (Gopalakannan and Arul, 2006), but the product may depress fish growth (Kono et al., 1987).

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10.4.5 Non-starch polysaccharides and oligosaccharides Among non-nutrient feed components, non-starch polysaccharides (NSP) are of particular importance. 1.

2.

3.

Polysaccharides may constitute a bulk in the intestine, especially with plant-derived diets, thus affecting transit and the physical properties of digesta. Transit duration can affect health due to nutrient availability and microbial activity. The incorporation of guar gum, a watersoluble galactomannan, in the diet of Nile tilapia and African catfish (Clarias gariepinus) increased digesta viscosity and reduced nutrients digestibility (Amirkolaie et al., 2005; Leenhouwers et al., 2006). Conversely, insoluble bulk agents like cellulose did not affect viscosity and digestibility, but increased faecal ejection time and recovery (Dias et al., 1998; Amirkolaie et al., 2005). Dietary NSP can directly stimulate the immune system of the animal. Several toll-like receptors seemed able to initiate the immune response to dietary carbohydrate molecules. In gilthead sea bream blood leucocytes, yeast whole cell phagocytosis was elicited by glucan receptors but not by mannose receptors (Esteban et al., 2004). However, another derivative from yeast cell wall, mannan oligosaccharide, seemed effective in various species (Sweetman and Davies, 2006; Genc et al., 2007; Staykov et al., 2007). The efficacy of alginates as immunostimulants may be related to their composition rather than the molecular weight of their constituents. For example, the proportion of β-1,4-D-mannuronate (M) to α-L-guluronate (G) residues was suspected to play a role (Fujiki et al., 1994; Vollstad et al., 2006). However, results are ambiguous since Fujiki et al. (1994) obtained good results on common carp with low-M alginates, unlike with high-M alginate from Lessonia nigrescens, whereas a high-M alginate from Durvillea antartica was preferred in Norwegian studies (Skjermo et al., 2006; Vollstad et al., 2006). Gastrointestinal microbiota of fishes have been documented for the fermentation of carbohydrates, resulting in the production of volatile fatty acids (VFA), and probable metabolic benefits accruing to the host (Clements, 1997). Kihara and Sakata (2001) observed in vitro the fermentation of different substrates by intestinal content extracted from rainbow trout fed a standard diet. Branched VFA were probably produced from nitrogenous compounds. The fermentation was significantly stimulated by chitin, and weakly by lactosucrose, while alginate tended to inhibit gas production. Soybean oligosaccharides and raffinose were fermented more intensively than other oligosaccharides in intestinal content from common carp fed a standard diet (Kihara and Sakata, 2002). The effects of feeding fish with different carbohydrates were studied in other experiments. When Siberian sturgeon (Acipenser baerii) were fed inulin – mostly linear β-2,1 linked fructans – their intestinal content

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New technologies in aquaculture produced in vitro a larger gas volume than when the fish were fed cellulose, but the highest gas production was observed with fish fed inulinderived fructooligosaccharides (FOS; Mahious et al., 2006a). Whether the fish were fed inulin or FOS, the production of VFA by their intestinal contents was slightly different, but significantly higher than the production from fish fed cellulose. When tilapia were fed α-starch, their intestinal content produced in vitro higher levels of VFA in comparison with fish fed NSP (Kihara and Sakata, 1997). That may explain why almost the same fermentation pattern was observed in the intestine of African catfish fed diets based on various cereal grains, which were more varied by their NSP than by their starch supply (Leenhouwers et al., 2007). Acetate, and probably other VFA, are actively absorbed through the intestinal epithelium of Oreochromis mossambicus (Titus and Ahearn, 1991). This might result in the reinforcement of the intestinal structure, with an increased thickness of the muscle layer in red sea bream fed lactosucrose and in Nile tilapia fed α-starch (Kihara et al., 1995; Kihara and Sakata, 1997). Refstie et al. (2006) also observed in Atlantic salmon fed inulin a higher intestinal mass than when the fish were fed a standard diet, whether or not the diet contained oxytetracycline. Consequently, the authors hypothesized a mechanical effect of dietary inulin, which acted as fibre that increased intestinal filling and peristaltic activity. Such interpretation could not account for the effect of dietary lactosucrose observed by Kihara et al. (1995). The water content of digesta was affected neither by lactosucrose in red sea bream (Pagrus major) nor by α-starch in Nile tilapia (Kihara and Sakata, 1997). Amirkolaie et al. (2006) confirmed this absence of physical influence of gelatinized starch (α-starch) on digesta, but these authors noted a decrease in hindgut fermentation due to gelatinization, in comparison with tilapia fed native starch. In summary, if some dietary carbohydrates can stimulate intestinal growth, the cause still remains uncertain, with opinion divided between a purely mechanistic phenomenon and the possible mediation of VFA. NSP, and especially their hydrolytic derivatives, may be used as prebiotics, which are food ingredients that beneficially affect the host by selectively stimulating the growth of and/or activating the metabolism of one or a limited number of health-promoting bacteria in the intestinal tract. The potential for applying this to fish intestinal microbiota were sketched out by Burr et al. (2005), but the practical results are still very limited. Presumptively beneficial microbes like Carnobacterium sp. and Bacillus sp. can use inulin and FOS, respectively (Ringø and Holzapfel, 2000; Mahious et al., 2006b). However, a high dose of inulin – incorporated as 15 % of the dry diet – damaged the ultrastructure of hindgut epithelial cells in Arctic charr (Salvelinus alpinus) reared in freshwater (Olsen et al., 2001). The number of bacterial cells

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adherent to the hindgut was reduced in Arctic charr fed inulin in comparison with those fed dextrin, and the species of Carnobacterium seemed different in each group (Ringø et al., 2006). In Atlantic salmon reared in seawater, the supply of inulin at 7.5 % of the dry diet did not damage the intestine, and a possible prebiotic effect was considered (Refstie et al., 2006). Inulin tended to decrease microbial diversity in Atlantic salmon (Bakke-McKellep et al., 2007), contrary to what was observed in piglets with several prebiotics, including inulin (Konstantinov et al., 2003; 2004). The incorporation of 2 % FOS in the diet for weaning turbot larvae improved growth, in comparison with a diet containing 2 % cellulose, inulin or lactosucrose (Mahious et al., 2006b). The dose of dietary prebiotics may be important, and the lowest level of FOS tested (0.4 %) was sufficient to obtain the best growth improvement in Litopenaeus vannamei (Zhou et al., 2007). A complex preparation of partially autolyzed yeast, dairy ingredients and fermentation products seemed to have a greater effect than that of FOS on VFA production and gut microbiota isolated from red drum (Burr et al., 2008). NSP purified from soy also affected intestinal microbiota in Atlantic salmon (Ringø et al., 2008).

10.4.6 Dietary probiotics Many probiotics have been experimentally introduced into aqua feeds, and a number of reviews on this subject are available (Ringø and Gatesoupe, 1998; Gatesoupe, 1999, 2005, 2007, 2008b; Gomez-Gil et al., 2000; Hansen, 2000; Verschuere et al., 2000; Irianto and Austin, 2002; Abidi, 2003; Maeda, 2004; Burr et al., 2005; Gram and Ringø, 2005; Ringø et al., 2005; Balcázar et al., 2006; Vine et al., 2006; Gómez and Balcázar, 2008; Kesarcodi-Watson et al., 2008; Tinh et al., 2008; Wang et al., 2008b). Bivalve larvae are highly sensitive to bacterial environment and water quality, and the most suitable means of introduction is to cultivate microalgal feed in association with the probiotic strain (Nicolas et al., 2007). Growth improvement was observed in pearl oyster (Pinctada fucata) spat fed with Lactobacillus acidophilius in addition to microalgae (Subhash et al., 2007). Macey and Coyne (2005, 2006) introduced into the compound diet for midas ear abalone (Haliotis midae) a microbial preparation composed of three strains isolated from the gut of the abalone: Vibrio midae, Cryptococcus sp. and Debaryomyces hansenii. The microbial consortium exerted several beneficial effects on the abalone, including increased resistance to Vibrio anguillarum. This correlated with increased phagocytic activity of haemocytes against the pathogen, and the maintenance of the number of circulating haemocytes after challenge, whereas it dropped in the control group. These probiotics also stimulated protease and amylase activity in the gastrointestinal tract, thus contributing to improving the health of abalone in different ways.

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An interesting feature of probiotics is their ability to combine several modes of action, from a direct antagonism to pathogens (Section 10.2.5) to more or less indirect effects on the immune and digestive functions of the host. This was also observed in shrimps (e.g. Li et al., 2007a; Wang, 2007; Castex et al., 2008) and in fish (e.g. Nikoskelainen et al., 2003; Frouël et al., 2008). The stimulation of the innate immune system by dietary probiotics may provide some protection against cutaneous and branchial parasites (Pieters et al., 2008; Reyes-Becerril et al., 2008). In most cases, the presence of living material did not seem essential for the probiotics to stimulate the immune and the digestive systems of fish (Villamil et al., 2002; Irianto and Austin, 2003; Díaz-Rosales et al., 2006; Salinas et al., 2006; Frouël et al., 2008). This point is important for practical application as feed additives in aquaculture because of the authorization for market release, which may be more easily obtained with dead microbial cells than with living probiotics. Nevertheless, the impact of germ viability on fish health should be investigated carefully since, for instance, the inactivation of a commercial probiotic consortium annihilated its protective effect against edwarsiellosis in Nile tilapia (Taoka et al., 2006b).

10.4.7

Alternative protein sources and feed hazards: antinutritional factors, mycotoxins The need to replace fish meal with alternative protein sources has brought new concerns about the health of aquatic animals. By-product feedstuffs from poultry or other land animals could constitute up to 40–50 % of the dietary protein supply without significant degradation of the immune status of Atlantic salmon (Bransden et al., 2001) and oriental river prawn (Macrobrachium nipponense) (Yang et al., 2004). It remains the case that the use of potentially contaminated animal by-products has been viewed negatively in the aquafeed industry (Amaya et al., 2007). The main effort is aimed at replacing fish meal by vegetable protein sources (Gatlin et al., 2007). For example, good results were obtained by Amaya et al. (2007) by feeding Litopenaeus vannamei a plant-based diet with 1 % squid meal as the only animal protein source. Although there was no negative effect on growth, it would be worth investigating whether there were any effects on health. Plant-based feeds may contain many antinutritional factors, whose effects on fish have been reviewed by Francis et al. (2001) Guillaume and Métailler (2001) and Gatlin et al. (2007). Information is seldom available for shrimps, which seem more resistant than fish to some compounds. For example, saponins are used to eradicate predatory fish from shrimp ponds (Nagesh et al., 1999). Soybean meals and soy protein concentrate should be introduced cautiously in salmonid feeds. High levels of incorporation caused a decline in macrophage activity and the disruption of epithelial integrity in the distal intestine of rainbow trout (Burrells et al., 1999; Ostaszewska et al., 2005).

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The resistance against Aeromonas salmonicida was also reduced (Neji et al., 1993). Soybean molasses, increased lysozyme activity and IgM production in Atlantic salmon, but decreased survival in challenge with A. salmonicida (Krogdahl et al., 2000). The main concern is enteritis provoked by dietary soybean products in Atlantic salmon (Bakke-McKellep et al., 2000, 2007). If the antinutritional or toxic factors can be identified, then adequate feed processing may solve the problem. The syndrome seemed to be caused by saponins – possibly in combination with disruption of the intestinal microbial balance, and with other factors like allergenic proteins (Knudsen et al., 2007). Soybean lectins may thus contribute to the toxic effect of soybean meal to salmonids (Buttle et al., 2001; Francis et al., 2001). Channel catfish can tolerate much higher levels of dietary soybean meal than salmonids (Peres et al., 2003). Raw soybean meal may contain a trypsin inhibitor, which can be destroyed with heat treatment, and Peres et al. (2003) improved feed efficiency and growth in channel catfish by autoclaving this feed ingredient for 40 mins. Adversely, the heat-treated meal decreased the disease resistance of the catfish challenged with Edwardsiella ictaluri. This example illustrates why it is important not to rely only on growth performances in assessing the dietary value of feedstuffs. SitjáBobadilla et al. (2005) observed a remarkable increase of complement alternative pathway activity in gilthead sea bream fed a half-and-half fish meal and plant protein mixture, but this immune response was depleted with higher rates of fish meal replacement. Some compounds classified as antinutritional factors may also have medicinal applications, when they are properly administered (Table 10.4). For example, saponins are known to increase permeability of the intestinal mucosa. This could account not only for their suspected role in enteritis of salmon fed soy products, but also for the interest in Quillaja saponin as an oral vaccine adjuvant to prevent edwardsiellosis in the Japanese flounder (Ashida et al., 1999), or to stimulate chemotaxis of yellowtail (Seriola quinqueradiata) leucocytes (Ninomiya et al., 1995). Lectins are sugar-binding proteins that may impair gut epithelial functions, but they can also antagonize fish parasites, as shown in vitro by Xu et al. (2001). These authors dissected fins from channel catfish, and they observed that the infection by Ichthyophthirius multifiliis was hindered when the parasite was immobilized by pre-incubation with plant lectins. Polyphenols, like tannins and gossypol, are toxic to fish and shrimps, but they may be exploited as antibacterial and immunostimulants in some cases, probably because of their antioxidant properties. Sodium phytate seemed a suitable source of phosphorous for Penaeus japonicus, but it depressed growth in Penaeus vannamei, as was observed in fish (Civera and Guillaume, 1989; Francis et al., 2001). In case of depletion of dietary calcium supply, phytic acid seemed detrimental even to P. japonicus (Civera Cercedo, 1994). The successful substitution of fish meal is, however, dependent on the selection of plant protein sources – possibly pre-treated – to minimize antinutritional factors and toxins.

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Table 10.4 Examples of antinutritional factors in plant protein sources; some of these factors could be used to improve the health of fish and shrimps Antinutritional factor Glucosidic factors Saponin

Glucosinolates

Effects on fish and shrimps Source

Quillaja saponaria

Brassica spp.

Proteinaceous factors Trypsin inhibitor Soya

Detrimental

Beneficial

High dose depresses salmonid growth, with abnormal intestinal morphology (Bureau et al., 1998) Thyroid hyperplasia in carp (Hossain and Jauncey, 1988)

Low dose increases carp growth (Francis et al., 2002); immunostimulant (Ninomiya et al., 1995; Ashida et al., 1999) –

May decrease dietary value (Peres et al., 2003) Soybean agglutinin binds to intestinal epithelium of salmonids, possibly causing damage (Buttle et al., 2001)

–

Lectins (agglutinins)

Ubiquitous

Polyphenols Tannic acid

Seeds

Toxic for tilapia and carp (Saha and Kaviraj, 1996; Becker and Makkar, 1999)

Gossypol

Cottonseed

Depresses growth in channel catfish (Yildirim et al., 2003)

Phytates

Seeds

Reduce protein digestibility and zinc availability in fish (Gatlin et al., 2007); unsuitable as phosphorous source in Penaeus vannamei (Civera and Guillaume, 1989)

Plant lectins bind to fish parasite Ichthyophthirius (Xu et al., 2001); wheat germ agglutinin proposed as immunostimulant for Penaeus orientalis (Xu et al., 1992); Antagonistic to fishpathogenic bacteria (Chung et al., 1995; Zhao et al., 1997); stimulates immune response in Labeo rohita (Prusty et al., 2007) Immunostimulant and antibacterial (Yildirim-Aksoy et al., 2004a,b) Suitable phosphorous source in Penaeus japonicus (Civera and Guillaume, 1989)

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Plant-based feed ingredients increase the risk of introducing mycotoxins (Spring, 2005), though such risk may be also encountered with fish meal (Encarnação, 2006). Even molluscs can be contaminated via fungi growing in sediments, and blue mussel stored gliotoxin in ‘meat’ (i.e. all tissues, except digestive gland; Grovel et al., 2003). Because mycotoxins are very stable, they may cause health risk for farmed animals, as well as for seafood consumers. For example, aflatoxin B1 is a potent hepatocarcinogen, which can cause damage in fish liver and shrimp hepatopancreas. It is also immunodepressing, and Ottinger and Kaattari (1998) showed that in winter – a period of decreased immune reactivity – rainbow trout leucocytes were 1000-fold more sensitive to the toxin than murine leucocytes. However, warm-water fish like channel catfish and tilapia seemed less sensitive (Spring, 2005; Encarnação, 2006). Generally, early life stages are more sensitive than adults, and the exposure of rainbow trout embryos to aflatoxin B1 resulted in more than two years of immune dysfunction in ongrowing stages (Ottinger and Kaattari, 2000). Other mycotoxins are also immunodepressive, like trichothecene T-2 (Smith et al., 1999a,b; Prearo et al., 2000) and fumonisin (Lumlertdacha and Lovell, 1995). Spring (2005) summarized the prevention strategies for the production of feed ingredients, e.g. by selecting fungal-resistant strains, to storage in dry conditions.

10.5 Concluding remarks Most of the husbandry practices in hatcheries and farms may affect health, more or less directly, and it was not possible to draw an exhaustive overview of all the methods that have been or that could be developed to improve disease resistance in aquaculture. The important thing is to keep in mind the complex mechanisms that coordinate homeostasis in animals and microbiota and, in this way, try to anticipate how each new technical change could interfere in the animal’s physiology. This is not an easy task, and innovation should be tested prudently before moving on to full-scale application. Among the new trends covered in this chapter, some are still highly experimental. Economical and regulatory constraints can cause long delays before novel commercial products are approved. This may often discourage R and D departments from designing special preparations when the potential market size is uncertain, which is generally the case in aquaculture. ‘A concerted approach to species or system-oriented health programmes is needed, particularly given the difficulties in achieving licenses for existing and new therapeutic products’, wrote Hough (2007) in a background document for the EATP (European Aquaculture Technology Platform) first stakeholders’ meeting. International and interprofessional organizations such as these need to play a vital role in turning scientific findings into practice.

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10.6 Sources of further information and advice 10.6.1 Hygiene rules Concerning hygiene and water treatment, the World Organisation for Animal Health (OIE) has inventoried the ‘methods for disinfection of aquaculture establishments’ (Manual of Diagnostic Tests for Aquatic Animals, 2006, Chapter 1.1.5., http://www.oie.int/eng/normes/fmanual/ A_00014.htm). 10.6.2 European directives It may be useful to refer to the European directive ‘on animal health requirements for aquaculture animals and products thereof, and on the prevention and control of certain diseases in aquatic animals’ (EC, 2006): The Panel on Animal Health and Welfare, created by the European Food Authority (EFSA), delivers opinions on current issues, in support of official directives (e.g. ‘possible vector species and live stages of susceptible species not transmitting disease as regards certain fish diseases’ (EFSA, 2007)). The European regulation for chemicals used in aquaculture has been reviewed by Costello et al. (2001), and Mortensen et al. (2006) have commented on the directive dealing with health risks caused by the movement of aquatic animals for farming purposes (EU, 1991). 10.6.3 Evaluation of safety of chemicals used in aquaculture The Food and Agriculture Organization (FAO) has promoted responsible practices ‘towards safe and effective use of chemicals in coastal aquaculture’ in the report by GESAMP (1997). More recently, the World Health Organization held an expert consultation on the risk of spreading antimicrobial resistance generated by aquaculture (Alday et al., 2006; FAO/OIE/ WHO, 2006). 10.6.4 Fish and shellfish diseases Specific information about fish and shrimp diseases is available with the national aquaculture program of the US Department of Agriculture’s Animal and Plant Health Inspection Service (APHIS; http://www.aphis. usda.gov/animal_health/animal_dis_spec/aquaculture/). A website has been dedicated to aquatic animal health with the support of the Fish Health Section of the American Fisheries Society (http://www.fisheries.org/units/ fhs/). There is also a virtual mollusc health laboratory moderated by Franck Berthe (http://vre.upei.ca/mhl/). 10.6.5 Dietary additives The dietary solutions to improve health can be documented from databases like that of ‘Level 1 Diet, the anti-inflammation health program’, which

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offers a search engine on more than one million health studies (http://www. level1diet.com/). Articles from Feed Mix dealing with aquafeeds and health can be browsed at http://www.allaboutfeed.net/article-database/ aquaculture/health.html.

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wang w n, wang y and wang a l (2006) Effect of supplemental l-ascorbyl-2polyphosphate (APP) in enriched live food on the immune response of Penaeus vannamei exposed to ammonia-N, Aquaculture, 256(1–4), 552–7. wang x h, chi z m, yue l x, li j, li m j and wu l f (2007) A marine killer yeast against the pathogenic yeast strain in crab (Portunus trituberculatus) and an optimization of the toxin production, Microbiol Res, 162(1), 77–85. wang l, yue l x, chi z m and wang x h (2008a) Marine killer yeasts active against a yeast strain pathogenic to crab Portunus trituberculatus, Dis Aquat Org, 80(3), 211–18. wang y b, li j r and lin j (2008b) Probiotics in aquaculture: challenges and outlook, Aquaculture, 281(1–4), 1–4. watanuki h, ota k, tassakka a c m a r, kato t and sakai m (2006) Immunostimulant effects of dietary Spirulina platensis on carp, Cyprinus carpio, Aquaculture, 258(1–4), 157–63. welker t l, lim c, yildirim-aksoy m and klesius p h (2007) Growth, immune function, and disease and stress resistance of juvenile Nile tilapia (Oreochromis niloticus) fed graded levels of bovine lactoferrin, Aquaculture, 262(1), 156–62. weyts f a a, cohen n, flik g and verburg-van kemenade b m l (1999) Interactions between the immune system and the hypothalamo-pituitary-interrenal axis in fish, Fish Shellfish Immunol, 9(1), 1–20. wu f c, ting y y and chen h y (2002) Docosahexaenoic acid is superior to eicosapentaenoic acid as the essential fatty acid for growth of grouper, Epinephelus malabaricus, J Nutr, 132(1), 72–9. wu f c, ting y y and chen h y (2003) Dietary docosahexaenoic acid is more optimal than eicosapentaenoic acid affecting the level of cellular defence responses of the juvenile grouper Epinephelus malabaricus, Fish Shellfish Immunol, 14(3), 223–38. xu b, ji w, xu h and shi j (1992) The immunologic competence of wheat germ agglutinin in Penaeus orientalis, J Ocean Univ Qingdao, 22(4), 29–34. xu b, xu h, chen q and shi j (1994) Growth conditions for Fusarium oxysporum, Trans Oceanol Limnol (Qingdao), 1, 30–36. xu d h, klesius p h and shoemaker c a (2001) Effect of lectins on the invasion of Ichthyophthirius theront to channel catfish tissue, Dis Aquat Org, 45(2), 115–20. xue j p, xu y p, jin l j, liu g, sun y x, li s y and zhang j c (2008) Effects of traditional Chinese medicine on immune responses in abalone, Haliotis discus hannai Ino, Fish Shellfish Immunol, 24(6), 752–8. yada t (2007) Growth hormone and fish immune system, Gen Comp Endocr, 152(2–3), 353–8. yang x and dick t a (1994) Arctic char (Salvelinus alpinus) and rainbow trout (Oncorhynchus mykiss) differ in their growth and lipid metabolism in response to dietary polyunsaturated fatty acids, Can J Fish Aquat Sci, 51(6), 1391–400. yang y, xie s, lei w, zhu x and yang y (2004) Effect of replacement of fish meal by meat and bone meal and poultry by-product meal in diets on the growth and immune response of Macrobrachium nipponense, Fish Shellfish Immunol, 17(2), 105–14. yaqoob p (2004) Fatty acids and the immune system: from basic science to clinical applications, Proc Nutr Soc, 63(1), 89–104. yildirim m, lim c, wan p j and klesius p h (2003) Growth performance and immune response of channel catfish (Ictalurus puctatus) fed diets containing graded levels of gossypol-acetic acid, Aquaculture, 219(1–4), 751–68. yildirim-aksoy m, lim c, wan p and klesius p h (2004a) Effect of natural free gossypol and gossypol-acetic acid on growth performance and resistance of channel catfish (Ictalurus punctatus) to Edwardsiella ictaluri challenge, Aquac Nutr, 10(3), 153–65.

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11 Fish larvae nutrition and diet: new developments S. Kolkovski, Dept of Fisheries, Australia, J. Lazo, Fish Nutrition Laboratory, Mexico, D. Leclercq, ACUI-T, France, and M. Izquierdo, Grupo de Investigación en Acuicultura, Spain

Abstract: Marine fish larvae fed microdiets have not, at this stage, matched the growth and survival performances demonstrated by larvae fed live feeds such as rotifers and Artemia. This chapter discusses the issues related to the use of microdiets as a sole or partial feed for marine fish larvae. The techniques and methods of manufacturing microdiet particles, chemical and physical properties and the relationship to the ingestion and digestion are described. The chapter also looks at the physiological development and the nutritional requirements of larvae. Advances in feeding regimes such as co-feeding and feeding systems are also reviewed. Key words: marine fish larvae, digestive enzymes, nutrition, feeding systems, microdiets.

11.1 Introduction During the past three decades, enormous efforts have been made to develop microdiets1 to replace live feed, both rotifers and Artemia, as complete or partial replacements for marine fish larvae (Koven et al., 2001; Kolkovski, 2004). While there have been substantial achievements in reducing the reliance on live feeds and weaning the larvae earlier onto microdiets, microdiets still cannot completely replace live feeds for most species. Although weaning the larvae from Artemia onto a microdiet can be achieved at metamorphosis in many species (Foscarini, 1988; Hardy, 1989; Koven et al., 2001; Curnow et al., 2006a,b), the early introduction of pre1

Microdiet is also referred to in the literature as formulated, inert, dry or weaning diet. It usually refers to first prepared (dry) diet fed to larvae. Usually particle sizes range between 150 μm and 800 μm.

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pared diets as the sole replacement for live food has met with limited success (Adron et al., 1974; Barnabe, 1976; Appelbaum and Van Damme, 1988; Kanazawa et al., 1989; Walford et al., 1991; Fernández-Díaz and Yúfera, 1997; Rosenlund et al., 1997). A clear example of the superiority of live food over commercial microdiets was demonstrated by Curnow et al. (2006a, Fig. 11.1). Barramundi (Lates calcarifer) larvae development was affected by rearing protocols, with co-feeding rotifers and commercial diet allowing complete replacement of Artemia. However, by including Artemia in the protocol with one of the commercial microdiets, survival was significantly improved. Furthermore, feeding protocols with earlier weaning from rotifers resulted in significantly reduced growth and survival (Curnow et al., 2006a). The efficiency of the utilization of feed particles (either live or inert) by marine larvae is affected by many external and internal factors (Kolkovski, 2001, 2004; Koven et al., 2001, Fig. 11.2). Primarily, the searching, identification and ingestion processes are influenced by physical and chemical factors including color, shape, size, movement and olfactory stimuli at a molecular level. Substances secreted by live food organisms that act to stimulate a feeding response belong to a group of chemicals known as ‘feed attractants’, and some have been specifically identified for larvae (Kolkovski et al., 1997b, 2001). Moreover, these physical and chemical factors affect the palate and influence the ingestion process, which is the precursor to the digestion process. Digestion involves secretion of enzymes, peristaltic movements and, after larvae metamorphosis, acid and bile salt secretions. The assimilation and absorption process begins after the food particle is digested and broken down into more simple molecules that can pass across the gut lining. This is further facilitated by the development of brush border and microvilli as well as protein transporters and other transport mechanisms (Zambonino-Infante and Cahu, 2001, 2007). Unsuccessful microdiet development is partially related to the limited knowledge of larval nutrition. Appropriated knowledge of the nutritional requirements of the larvae is necessary in order to design effective microdiets. However, the modulation of the biochemical composition of live preys is very limited, and this restricts research on larval nutrition. The lipidic component of live preys is more variable, easier to control and has a marked effect on larval performance. Hence more attention has been paid to lipids and the essential fatty acid requirements of fish larvae (Izquierdo et al., 2000). More recent, studies have focused on fat-soluble vitamins requirements and also the deleterious effect of their excess or inadequate molecular forms. Indeed, fat-soluble vitamin contents of microalgae and live prey vary greatly with culture conditions, frequently causing ‘hypo’ and ‘hypervitaminosis’. The development of experimental microdiets co-fed with very small quantities of live prey have allowed more precise studies of both fat- and water-soluble nutrients. Most reported water-soluble

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(a)

(b)

(c)

(d)

(e)

(f)

Fig. 11.1 Barramundi Lates calcarifer growth using different feeding protocols (Curnow et al., 2006). (a) 11 days rotifers, 9 days Artemia co-fed with Proton (INVE); (b) 11 days rotifers, 9 days Artemia co-fed with Micro-Gemma/Gemma (Skretting); (c) 11 days rotifers, Micro-Gemma/Gemma; (d) 7 days rotifers, Micro-Gemma/Gemma; (e) 3 days rotifers, Micro-Gemma/Gemma; (f) Micro Gemma/Gemma.

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Chemical factors feed attractants FAA, ammonium salts, etc. ‘smell’

Visual factors color shape size movement

Capsule proteins ingredients moisture

Ingestion size taste shape movement

Digestion digestive enzymes peristaltic movements digestive tract development acid secretion, bile salts

Assimilation/absorption brush borders microvilii transporters proteins

Fig. 11.2 Factors affecting food particle utilization.

vitamin requirements are much higher for larvae than for juveniles of the same species, although this may be related not only to the higher metabolic demand in the former, but also to the high ratio surface/volume in larval diets which makes nutrients more prone to oxidation and leaching (Yúfera et al., 2003). Very recently, it has been shown that live prey are also deficient in critical minerals. Modern encapsulation technology is allowing the development of new enrichment products including water-soluble nutrients such as vitamins and minerals, which in turn are improving our knowledge of the requirements of these nutrients for the developing larvae. Studies have also determined the effect of protein and amino acid requirements, frequently involving tube feeding trials (Rønnestad et al., 2000). Fast-growing fish larvae have a high demand for protein and require more elevated dietary contents than juveniles and adults. Requirements for all these nutrients and their utilization by the larvae will depend on a series of morphological and physiological changes which occur with larval development through complete metamorphosis. Moreover, interrelation among certain nutrients has also been shown to affect their optimum dietary levels. Very little attention was given to the feeding process of microdiets (Kolkovski, 2004), including feeding systems, specific design to deal with very small particles such as microdiet particles, dispersion of particles in the water column, feeding strategies (continuous vs. periodic), water hydrodynamics and the particle-larval interaction in the rearing tank. In many commercial hatcheries, manual feeding is still very common. A diet that supplies all the larva’s nutritional requirements will not achieve the best growth if the feeding frequencies, amounts and particle disposal are not optimized.

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This chapter reviews the aspects related to the utilization of microdiet by fish larvae from the feeding process, ingestion, digestion and nutritional requirements. It also reviews the chemical and physical properties of microdiet particles related to the particle behavior in the water and the interaction particle – larvae. Current production methods and feeding systems and protocols are also discussed.

11.2 Determination of nutritional requirements for larvae Despite the importance of clearly defining the specific nutritional requirements of fish larvae in order to formulate appropriate larval feeds, determination of larval requirements is one of the most complicated aspects of fish nutrition. Fish larval nutritional reserves are very limited at first feeding, and hence their survival dramatically depends on exogenous high-quality feeds that contain all the necessary nutrients to match larval requirements. Moreover, during larval development the fish will undertake several morphological and physiological changes, which in nature are simultaneous with changes in behavior and even habitat and type of prey that are not provided under culture conditions. All these changes affect nutrient availability and feed use by the larvae, in turn affecting their nutritional requirements. The requirement for a particular nutrient can be defined from a physiological point of view as the nutrient intake needed to fulfil a physiological role while the diet specification refers to nutrient content in the diet to supply the physiological requirements. Under the requirement for maintenance is the minimum amount of a nutrient or energy needed to keep the fish alive. For instance, we can consider the energy for maintenance as the energy needed to maintain the basal metabolism, plus the energy for involuntary activity, such as movement for body balance and buoyancy, and muscular activity. In larvae, the requirement for maximal growth or survival is utilized more frequently since both parameters are critical for mass fry production. Here, the relationships among larvae and larval diet and feeding have important effects in the determination of the quantitative needs. For instance, factors such as nutrient leaching or availability, diet production technology, feed stability or type of feeding will markedly affect the requirements. Least cost diet formulation, despite being broadly utilized for fish fry, has scarcely been studied in larval nutrition where feeds are generally expensive and feed production is relatively small. In juveniles, requirements for maximal growth are always higher than the requirements for least cost production. From a practical point of view, requirements in larvae have been also determined as requirement for health or stress resistance since, even though nutrient levels can be high enough in the diet to cover maintenance and growth requirements, they may be insufficient to promote maximum disease and stress resistance in

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larvae. Moreover, the requirements determined for these nutrients under optimal culture conditions increase when fish are exposed to unfavorable environmental conditions (poor water quality, stress, pathogens). Thus, for example, the definition of the requirement for vitamin E to improve immunological defenses is related to the production conditions applied. Finally, more recently, in larvae the requirements for highest fry quality are being considered in relation to the need of a nutrient to improve pigmentation and reduce bone deformities. Studies on determination of the nutritional requirements of larvae are not only scarce but their results and recommendations are difficult to generalize and apply in practice; this is due to the differences in the methodological approaches used and inexperienced in larval culture. Therefore, in order to further improve our knowledge on larval nutrition in a more effective way, studies aimed to determine the requirements for a given nutrient must fulfill a series of demands: • They should apply a practical point of view. • If optimal culture conditions for the tested species have been established, the requirements should be assayed in such conditions. If not, conditions should be established in relation to a review of what is known of this species in its natural conditions. • As far as possible, experimental conditions similar to those used at commercial scale should be used (feed preparation technique, water quality, photoperiod and larval stocking density, among others). • Due to the large interference between different nutrients, to establish the requirements for a particular nutrient only that nutrient should be varied in the diets, trying to avoid changes in the type of nutrient source. • At least triplicate or quadruplicate tanks of larvae should be used per dietary treatment, as one tank of fish represents a single block observation. • To determine quantitative requirements, it is important to consider different factors related to the species (e.g., larval size and developmental stage, broodstock origin and feeding), related to the culture medium (e.g., temperature, salinity, larval density, type of culture: extensive/ intensive, presence or absence of green water), related to the feeding strategy and feeding regime and related to the feed (e.g., type of inert feed or live preys, feed density, dietary energy content, nutrient availability in the ingredient source and interactions with other dietary nutrients or ingredients). • Complete feed ingredient descriptions should be provided, including international feed number (IFN), chemical composition and particle size, when reporting dietary formulations and the results of nutritional feeding trials. If a commercially prepared diet is used, the trade name and manufacturer should be indicated.

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• A standard diet or live prey protocol should be used as a control in addition to any local diet also designed as a control. In most cases the use of different control diets or live prey protocols complicates comparison of results among different authors. • A minimum of six dietary nutrient levels or treatments is recommended for nutrient requirement studies. • Larval body composition should be carried out at the beginning and at the end of the experiment, particularly for the nutrient tested. • An appropriate statistical analysis is always necessary. The most direct method to evaluate nutrient requirements for larvae is to feed them diets or live prey varying only in their content of a particular nutrient. For this purpose we must feed the larvae with enriched live prey or directly on microdiets containing different levels of the nutrient to be tested. However, this is not easy to achieve when live prey are used. Although it is possible to control the content of some nutrients such as fatty acids, the precise amount of certain nutrients such as total proteins, individual amino acids, vitamins and minerals is difficult to manipulate in live prey, whose own metabolism modifies the nutrients provided through the enrichments. In this sense, microdiets are a preferred method to determine nutritional requirements of larvae. Once several levels of the nutrient are provided through the diet, their effects on various parameters are studied: (i) growth rate, which is easily affected by some nutrients but not by others; (ii) survival rate, which is a very sensitive parameter to certain nutrients; (iii) resistance to stress (while resistance to stress is difficult to determine individually due to the small size of larvae, molecular markers of stress have recently been developed and should allow a more precise determination of the effects of certain nutrients); (iv) biochemical composition of the fish larvae. However, in the development of new species for aquaculture it is not always possible to conduct a dose–effect trial, and hence information about nutritional requirements of the larvae has been obtained by other methods such as: • Study of the biochemical composition of the eggs. Since marine fish eggs should contain all the nutrients that are essential for the embryo and the larvae development up to the stage of yolk-sac absorption, their biochemical composition should give us some information about which nutrients are required at this stage of development (Izquierdo et al., 1989; González et al., 1995). • Study of the evolution of a nutrient along the embryo and larval development. The depletion/deposition of a given nutrient during embryo and larval development, its utilization as energy yield or its incorporation into larval tissues can provide information about the relative importance of the nutrient for the growing larvae (Rodríguez et al., 1994a–c, 1998).

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• Study of enzyme activity and gene expression of molecules related with the metabolism of a particular nutrient or the physiological mechanisms in which this nutrient is implied (Izquierdo et al., 2000). • Behavior studies, related to the fast development of the central neural system and sensorial organs which occur in the larvae, can provide information about the importance of nutrients implied in this development, such as essential fatty acids, vitamin E, and amino acids (Benítez et al., 2007) . • Tube feeding combined with radio labeling studies, which provide accurate information about digestible and energetic utilization and deposition of dietary nutrients (Rønnestad et al., 2001). Thus, many authors have applied some of these methods to research on larval nutrition and have determined the requirements of a limited number of nutrients for certain species along larval development which will be reviewed in Section 11.3.

11.3 Nutritional requirements of fish larvae 11.3.1 Requirements for essential fatty acids (EFA) Essential fatty acids, particularly highly unsaturated fatty acids (HUFA) with 20 or more carbon atoms of the linolenic family (n−3 HUFA) including docosahexaenoic acid (DHA, 22:6n−3) and eicosapentaenoic acid (EPA, 20:5n−3), have long been recognized as an important nutritional factor affecting larval rearing success (Watanabe et al., 1983). Indeed both DHA and EPA, together with arachidonic acid (ARA, 20:4n−6) have a variety of very important functions in fish species, particularly in larvae. Despite the fact that freshwater fish seem to have sufficient Δ5 and Δ6 desaturases and elongases activities to produce ARA, EPA and DHA if their precursors linoleic (18:2n−6) and linolenic (18:3n−3) acids are present in the diet, such enzymatic activity is very restricted in marine fish larvae and, as a consequence DHA, EPA and ARA have to be included in the diet and are considered essential. Thus, although it was thought that Δ5 and Δ6 activity was lacking in marine fish, a Δ6 desaturase-like gene was finally isolated in larval gilthead sebaream (Sparus aurata) (Seiliez et al., 2003). More recently, it has been shown that the expression of this gene is affected by the larval diet (Izquierdo et al., 2008). For instance, substitution of fish oil by vegetable oils in enrichment emulsions for rotifers produced a six-fold higher relative expression of the Δ6 desaturase-like gene in gilthead seabream larvae, denoting the nutritional regulation of desaturase activity through its gene expression. Moreover, products of Δ6–Δ5 desaturases such as 18:2n−9, 18:3n−6, 20:3n−6 and 20:4n−6 significantly increased in tissues of larvae fed rotifers enriched with vegetable oils (Izquierdo et al., 2008). In fact, it is possible that the Δ6 desaturase present in seabream

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also has Δ5 activity as has been shown in zebrafish (Danio rierio) (Hastings et al., 2001). In practice, these findings imply a higher possibility of substitution of fish oils in first feeding diets and prey for marine fish larvae, if precursor fatty acids such as 18:3n−3 and 18:2n−6 are present. However, if dietary levels of these precursor fatty acids are too high it may significantly reduce the expression of desaturase gene (Izquierdo et al., 2008). Interestingly, expression of desaturases can be also affected by salinity. For instance, larvae of the eurihaline species Galaxias maculatus have been found to be higher in EPA, DHA and ARA acids when they were obtained from marine environments in comparison with those from freshwater (Dantagnan et al., 2005, 2007), denoting the important role of some of these fatty acids in osmotic regulation. Moreover, before first feeding, synthesis of those EFA was activated in larvae from the freshwater environment but not in those obtained in the estuary, suggesting the influence of environment salinity on activation of elongation and desaturation enzymes. Inadequate content of those EFA in live prey or microdiets gives rise to several biological symptoms in larvae such as reduced feeding, growth and swimming activities, and increasing mortality, fatty livers, hydrops, deficient swim bladder inflation, abnormal pigmentation and disaggregation of gill epithelia (Izquierdo, 1996, 2005). EFA requirement for gilthead seabream was very close to 1.5 % n−3 HUFA in dry matter when larvae were fed either live prey (Rodríguez et al., 1998) or microdiets (Salhi et al., 1999), regardless of dietary lipid level (Salhi et al., 1994). However, as in the other life stages, provided other nutrients such as antioxidants are also balanced, elevation of dietary n−3 HUFA up to 8 %, keeping balanced ratios among the different EFA further improves larval growth and survival (Liu et al., 2002). High n−3 HUFA requirements have been also estimated for red porgy (Pagrus pagrus) (3.39 %, Hernández-Cruz et al., 1999) and Dentex dentex (Mourente et al., 1999) despite the fact that in the latter the high EPA content in Artemia may have caused an over-estimation of the requirements as observed in gilthead seabream (Rodríguez et al., 1997). In contrast, carp larvae seem to require as little as 0.05 % n−3 fatty acids from cod liver oil (Radunz-Neto et al., 1994) to cover the essential fatty acid requirements during larval development.

11.3.2 Requirements for docosahexaenoic acid (DHA) The particular structure of DHA, with an extremely long chain and the highest number of double bonds, provides this fatty acid with special characteristics that allow it to carry out many important functions in fish metabolism. Its incorporation into cell membranes regulates membrane integrity and function, and it is an important component of phosphoglycerides, particularly phosphatidyl ethanolamine and phosphatidyl choline. It may be a substrate for some lypoxigenases, and several studies have shown that it has a greater potential as an EFA for marine fish larvae than EPA

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(Watanabe et al., 1989), its requirement being more limiting for growth and survival than those for other n−s HUFA (Izquierdo, 1996). Moreover, as for other fatty acids, DHA has been found to play an important role in gene expression regulation, affecting a variety of physiological functions in fish. It is specifically retained in starved or low-EFA fed larvae, possibly due to lower cell oxidation rates than other fatty acids (Madsen et al., 1999). DHA is predominantly important for neural tissue and sensorial organs, being accumulated in rod and cone photoreceptors in herring (Bell and Dick, 1993) and in the central nervous system and eyes in gilthead seabream (Benítez et al., 2004). Moreover, DHA has been found to increase eye diameter and density of photoreceptors in gilthead seabream larvae (Izquierdo et al., 2000) and, in agreement, visual capacity was found to be reduced in yellowtail (Seriola quinqueradiata) fed DHA-deficient diets (Masuda et al., 1999). Feeding gilthead seabream larvae with DHA-deficient rotifers has been found to delay for about 10 days the appearance of reaction after visual stimulus, in agreement with the minor DHA content in eyes and brains of these larvae, and suggesting a delay in the functional development of brain and vision (Benítez et al., 2007). DHA is necessary for growth, survival, disease prevention and flat fish pigmentation and metamorphosis (Izquierdo, 2005; Hamre and Harboe, 2008). DHA also appears to be important for bone development, and it has been found to reduce the incidence of opercular deformities in milkfish (Gapasin and Duray, 2001) and of cranial deformities, lordosis and vertebrae fusion in red porgy (Roo et al., 2009). In general, DHA requirements along larval development vary with reports of 0.5 % for Acanthochromys poliacanthus (Southgate and Kavanagh, 1999), 2.5 % for Atlantic halibut (Hippoglossus hippoglossus) (Hamre and Harboe, 2008) and 0.8 % for gilthead seabream larvae (Rodríguez et al., 1998; Salhi et al., 1999, Fig. 11.3).

24

% Survival

22 20 R2 = 0.7322

18 16 14 12 0

1

2

3 4 % DHA in microdiet

5

6

7

Fig. 11.3 Effect of dietary docosahexaenoic acid (DHA) on survival of gilthead seabream larvae (Izquierdo unpublished, 2007).

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In seabream larvae, high levels (5 % in dry basis) of dietary DHA in microdiets for gilthead seabream did not cause any excess problem, but further promoted growth and larval survival (Liu et al., 2002). However, under certain conditions a high level of dietary DHA may exert negative effects in the larvae. For instance, a higher incidence of cephalic and vertebral column deformities has been reported in European sea bass fed high levels of DHA and EPA adversely affecting fish growth and survival (Cahu et al., 2003). However, this negative effect of high dietary DHA content could be related to an insufficient presence of dietary antioxidants such as vitamin E. Thus, in the same species, increase in dietary DHA caused degeneration and breakage of the muscular fibre as well as an infiltration of mononuclear cells in the myosepta of sea bass larvae (Betancor et al., 2009), which could be related to the proliferation of free radicals derived from DHA, since the elevation of dietary vitamin E markedly reduced the incidence of these pathological signs.

11.3.3 Requirements for eicosapentaenoic acid (EPA) EPA also plays several general and particular roles in fish metabolism. It is a main component of polar lipids in larvae and it regulates membrane integrity and function, its incorporation into phosphoacylglycerides enhances fluidity of cell membranes to a degree higher than ARA (Hagve et al., 1998) but lower than DHA (Hashimoto et al., 1999). Moderate dietary levels of this fatty acid enhance DHA incorporation into larval phospholipids (PL) (Izquierdo et al., 2000, 2001), causing a sparing effect on such an important fatty acid. EPA is an important precursor of prostaglandins (PG) in marine fish and has a predominant role in immune regulation in certain marine fish species (Ganga et al., 2005). Indeed, in gilthead seabream, PGE3 derived from EPA is the major prostaglandin found in fish plasma (Ganga et al., 2005), and it is strongly correlated with plasma polar lipid concentrations of EPA. Nevertheless, PG production markedly differs among tissues of the same species. Moreover, EPA is also a main substrate for some lypoxigenases, being the main precursor for leukotriene synthesis in some species. Its competition with ARA for these two types of enzymes enables it to be an important regulator of eicosanoid synthesis. Dietary EPA is important for larval growth and survival (Watanabe et al., 1989). For instance, best growth and survival have been obtained in larval gilthead seabream with EPA dietary levels of 0.7–0.8 % on a dry weight basis (Rodríguez et al., 1998; Salhi et al., 1999; Fig. 11.4). However, increasing dietary EPA up to 1.8 % reduced growth when ARA levels are as high as 1.8 % and DHA/EPA about 1.3, denoting how the EFA value of EPA is dependent on the dietary levels of DHA and ARA as discussed later (Izquierdo et al., 2000). EPA also plays an important role in stress regulation in fish larvae. For instance, in gilthead seabream larvae, an increase of EPA up to 2.9 % on a dry weight basis significantly improved

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24 R2 = 0.8362

22 20 18 16 14 12 10 0

1

2 3 % EPA in microdiet

4

5

Fig. 11.4 Effect of dietary eicosapentaenoic acid (EPA) on survival of gilthead seabream larvae (Izquierdo unpublished, 2007).

resistance to both handling and temperature shock stresses (Liu et al., 2002). Indeed, EPA has been found to regulate the production of cortisol, the essential hormone in fish stress regulation, in ACTH-stimulated interrenal cells, together with ARA (Ganga et al., 2006). In general, EPA requirements described in the literature range from 0.7 % for gilthead seabream (Rodríguez et al., 1998; Salhi et al., 1999) to 1.3 % for Centropomus parallelus and 1.6 % for Dentex dentex (Mourente et al., 1999).

11.3.4 Requirements for arachidonic acid (ARA) ARA is a main component of a minor but very important polar lipid class, phosphatidyl inositol (PI). In vitro, ARA has been described as a preferred substrate for most cycloxigenases, being the main precursor for PG synthesis, whereas in vivo, at least in marine fish, EPA seems to be the main substrate, although this could be related to its high presence in the diet. ARA also constitutes a good substrate for several lypoxigenases, its derivative hydroxy-fatty acids having important physiological functions in marine fish. Its content in the PI of cell membranes regulates eicosanoid synthesis. In gilthead seabream larvae, an increase of ARA up to 1 % enhances survival and growth when DHA and EPA dietary contents are 1.3 and 0.7, respectively (Izquierdo, 1996; Bessonart et al., 1999; Fig. 11.6). Increase in ARA content in the rotifers also prevents post-stress mortality (Koven et al., 2001). Indeed, ARA has been found to regulate not only cortisol production in fish (Ganga et al., 2006) but also expression of stress-related genes such as HSP70 gene in gilthead seabream larvae (Negrín et al., 2009) (Fig. 11.5). ARA also seems to play important role in production of turbot juveniles (Castell et al., 1994) and in flatfish pigmentation (Estévez et al.,

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Cycloxigenase products Tissue fatty acids Lipoxygenase products

327

cAMPC

Stress-related genes expression

Fig. 11.5 Some mechanisms implicated in stress regulation by fatty acids (Izquierdo unpublished, 2007). 7.2 7

TL

6.8 6.6 6.4 6.2 6 5.8 0

0.05

0.1 ARA/EPA

0.15

0.2

Fig. 11.6 Effect of ARA/EPA on gilthead seabream larval growth (Izquierdo unpublished, 2007).

1997). Excess levels of ARA in live preys have been found to correlate with impaired pigmentation in turbot (Estévez et al., 1999), Japanese flounder (Estévez et al., 2001) and sole (Solea solea) (Lund et al., 2008). ARA requirements for fish larvae range from 0.6 % for gilthead seabream (Besonart et al., 1999) to 2.5 % for a freshwater fish larvae, the guppie (Poecilia reticulate) (Khozin-Goldberg et al., 2006).

11.3.5 Importance of balanced EFA ratios EFA requirements estimated in the literature are high, with EPA dietary contents being two or three times higher than those of DHA (Rodríguez et al., 1994, 1997). This is due to the very high incorporation of EPA into the larval polar lipids and the displacement of DHA from certain polar lipids (Izquierdo et al., 2000). Similarly, unbalanced EPA/ARA ratios seem

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TL

7

6.5

6

5.5 0

20

40 60 80 100 120 140 160 180 200 (EPA + DHA + ARA)*(DHA/EPA)/ARA

Fig. 11.7 Gilthead seabream larval growth in relation to dietary DHA + EPA + ARA)/DHA/EPA/ARA (Izquierdo unpublished, 2007).

to be detrimental for flatfish (McEvoy et al., 1998). Evidence of competition among two or more of these EFAs has been suggested for digestive enzymes, fatty acid binding proteins, phosphoacylglicerides synthetases, lypoxigenases and cyclooxigenases and, probably, in beta-oxidation as occurs in rats (Izquierdo, 2005). Not only absolute dietary values for each of these EFAs but also optimum dietary ratios among them must be defined since both will affect at least their incorporation into the tissue lipids and hence membrane fluidity and function, the energy values obtained from their betaoxidation and the production of metabolically active compounds. Thus, optimum DHA/EPA ratios seem to be around 1.5 for sparids (Rodríguez et al., 1997) and around 2 for flatfish (Reitan et al., 1994); whereas optimum EPA/ARA ratios seem to be around 5 for flatfish (Bell et al., 2003) and about 8 for sparids. Considering both the sum of the three EFAs and the ratios among them, a plot of the dietary value of the ratio (DHA + EPA + ARA)*DHA/EPA/ARA against growth in some recent studies (Fig. 11.7), yields a significant correlation. Application of the same equation to dietary fatty acids in other gilthead seabream studies (Rodríguez et al., 1994, 1998; Fernández et al., 1995; Salhi et al., 1999; Koven et al., 2001; Liu et al., 2002 and others), demonstrated that for ARA values higher than 0.5 % when the value of the equation (DHA + EPA + ARA)*DHA/EPA/ARA became closer to 50 growth performance was better.

11.3.6 Phospholipids (PL) Feeding larvae low dietary PL reduces growth and lipid transport from larval enterocytes to hepatocytes (Kanazawa, 1993; Izquierdo et al., 2000). For instance, feeding larval gilthead seabream diets without lecithin supplementation produces accumulation of lipidic vacuoles in the basal zone of the enterocyte and esteatosis in the hepatic tissue, both of them being

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markedly reduced by a 2 % addition of soybean lecithin, denoting an enhancement in the lipid transport activity in gut and liver (Liu et al., 2002). This reduction in lipid transport could be related to a limited capacity for ‘de novo’ synthesis of phospholipids in the larvae. Reacilation of phospholipids in the enterocyte is known to occur through the glycerol-3-phosphate pathway in both the rough and the smooth endoplasmic reticulum (Izquierdo et al., 2000). However, since marine fish larvae fed microdiets show enterocytes with a poor development of the endoplasmic reticulum and Golgi system, reacilation capacity may be limited in these larvae. Indeed, inappropriate dietary lipids have been found to markedly affect re-esterification pathways in seabream gut (Caballero et al., 2006a), modifying the type of lipoprotein formed (Caballero et al., 2006b) and hence lipid transport. In contrast, when gilthead seabream larvae were fed triglycerides (TG) of marine origin, rich in n−3 HUFA, an accumulation of lipid vacuoles in the basal zone of the enterocyte and hepatic steatosis was observed. This indicated good absorption of dietary TG but also a reduced lipid transport to peripheral tissues. Conversely, feeding with marine PL markedly reduced lipid accumulation in both type of tissues (Salhi et al., 1999). These results agree well with the higher incorporation into larval polar lipids of fatty acids from dietary polar lipids than from dietary triglycerides (Izquierdo et al., 2001). In studies with labeled fatty acids, dietary n−3 HUFA PL significantly improved the incorporation of free EPA, but not of free oleic acid, into larval polar lipids in comparison to n−3 HUFA-rich TG (Izquierdo et al., 2001). This specific tissue incorporation of EPA when dietary polar lipids are rich in n−3 HUFA could be related to the enhancement of lipid transport, mobilization and deposition in the peripheral tissues by n−3 HUFA rich dietary phospholipids. As a consequence, growth of larval gilthead seabream was improved when they were fed microdiets containing marine PL instead of marine TG despite the slightly lower dietary n−3 HUFA levels of the former (1.5 % versus 1.8 %, respectively) (Salhi et al., 1999). Incorporation of dietary free fatty acids seems to be even lower than that of TG. Thus, labeled oleic acid was better incorporated into both polar or neutral lipids of seabream larvae when it was provided in the diet esterified in a triglyceride than as a free fatty acid, suggesting again a limited capacity of reacilation or transport for dietary long-chain free fatty acids or its preferential utilization as an energy source in the enterocyte (Izquierdo et al., 2001). Enzymatic, histological and biochemical evidence suggests that marine fish larvae are able to digest and absorb n−3 HUFA-rich TG more efficiently than free fatty acids, but feeding with PL, particularly if they are rich in n−3 HUFA, will enhance PL digestion and especially lipid transport, allowing a better n−3 HUFA incorporation into larval membrane lipids and promoting fish growth. This confirms former studies which suggest that, in

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addition to the dietary level of essential fatty acids, the molecular form in which they are present in the diet is also important for good growth and survival of marine fish larvae (Izquierdo, 1996; Izquierdo et al., 1989a,b). Accumulation of lipidic vacuoles in the basal zone of the enterocyte caused by feeding diets without lecithin supplementation in gilthead seabream disappeared when 0.1 % PC was added regardless of its (squid or soybean) origin (Izquierdo et al., 2000). However, squid PC was more efficient in reducing hepatic steatosis than soybean PC, suggesting a combined effect of dietary PC and n−3 HUFA to further enhance hepatic lipid utilization. Indeed both types of molecules have been found to promote lipoprotein synthesis. Finally, increased PL concentration, particularly PI, reduced skeletal deformities in seabass (Cahu et al., 2003), and has been suggested to be related to an up-regulation in bone morphogenic genes.

11.3.7 Vitamins and minerals The content of water-soluble vitamins in most hatchery microalgae and live prey seems to match the requirements of water-soluble vitamins for fish larvae, except for the low levels of pyridoxine in enriched rotifers and thiamin in enriched Artemia (González, 1997). Both vitamins play essential roles in fish metabolism. Pyridoxine is necessary in several levels of amino acid metabolism, being also very important for immune regulation, whereas thiamin acts as a coenzyme cocarboxilase, being essential for the oxidative decarboxylation of alfa-keto acids and hence for obtaining energy from amino acids, sugars and lipids. Vitamin C also plays a very important role during larval development, particularly preventing opercular deformities as has been seen in milkfish. In microdiets, it is very important to determine optimum vitamin contents to match the larval requirements. In addition, most water-soluble vitamin requirements described for larvae are higher than for juveniles of the same species, and this could be related not only to the higher metabolic demand in the former, but also to the high ratio surface/volume in larval diets making the diets more prone to oxidation and leaching. Thus, whereas in juveniles vitamin premix accounts for about 2–3 % of the diet, in larval microdiets they may reach up to 6–8 % of the diet. In contrast, fat-soluble vitamin contents of microalgae and live prey vary greatly among sample batches and with culture conditions, frequently leading to hypo or hypervitaminosis. For instance, vitamin E decreases in seabream from fertilization to the onset of exogenous feeding and continues dropping down during enriched rotifer feeding until Artemia is introduced in the culture system. Increasing vitamin E content in microdiets for gilthead seabream or seabass from 300 up to 1500 or 3000 mg/kg markedly improves growth and stress resistance (Betancor et al., 2009). However, vitamin E efficacy is closely related to dietary vitamin C. For instance, the progressive elevation of dietary α-tocopherol acetate levels up to

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1500 mg/kg in larval seabream diets containing ascorbic acid in its free form significantly reduced larval survival, whereas the same increase in α-tocopherol when vitamin C was supplemented as ascorbic acid polyphosphate caused a significant improvement in larval growth without affecting survival, suggesting a pro-oxidative effect of α-tocopherol over vitamin C in the former. More attention has been recently paid to vitamin A requirements in fish larvae. In gilthead seabream total retinol contents in the larvae increase during rotifer feeding, whereas they are slightly reduced after feeding a non-retinol containing Artemia. Indeed, the carotenoid content in Artemia seems to be enough to cover halibut vitamin A requirements (Moren et al., 2004). Despite the fact that adequate levels of vitamin A are required for normal pigmentation of flatfish, enrichment of Artemia with all-trans retinoic acid increases retinol palmitate inducing a higher incidence of bone deformities and hyperpigmentation of the blind side in Japanese flounder larvae. Vitamin D has been also shown to be important for normal development of fish larvae but in much smaller quantities, since its accumulation can easily cause hypervitaminosis inducing bone deformities and malpigmentation in Japanese flounder. Minerals have been much less studied in fish larvae, despite recent studies showing the importance of P, and particularly I and Se for cod larval growth (Hamre, pers comm) and Zn and Mn for bone development (Sato, pers comm), encouraging its study in other species.

11.3.8 Protein and amino acid requirements Fast-growing fish larvae have a high demand for protein and require more than juveniles and adults, so microdiets designed for larvae contain between 50 and 70 % protein. From the 20 most common amino acids (AA), 10 have been found to be essential (EAA) or indispensable for all studied fish and are required for optimum growth: leucine, isoleucine, valine, threonine, phenylalanine, methionine, tryptophan, arginine, histidine and lysine. Another two amino acids, tyrosine and cystine, are only nonessential if phenylaline and methionine, respectively, are present in the diet. At least all those amino acids should also be supplied to marine fish larvae. The importance of other minor amino acids such as taurine, an important enhancer of growth and survival in several sparids larvae, should not be neglected. Methods to determine quantitative requirements of each of those AA in fish larvae include feeding microdiets with graded levels of one amino acid at a time in a test diet containing either all crystalline amino acids, a mixture of casein, gelatin and crystalline amino acids, or a semi-purified diet using an imbalanced protein (zein, corn gluten) formulated so that the amino acid profile is identical to the test protein except for the amino acid being tested. As studied by Kanazawa

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and co-workers for fish larvae of several species, diets are designed to contain protein levels at or slightly below the optimum protein requirement for that species to assure a maximum use of the limiting amino acid. Hence, quantitative requirements of several AA have been determined for red sea bream and Japanese flounder larvae (López-Alvarado, 1995). Relationships among AA, such as competition or common synthesis pathways, also need to be considered. AA leaching in microdiets that stay in the water for a relatively long time causes difficulties in accurately determining physiological requirements. Hence other methods previously utilized in juveniles have been applied to fish larvae. For instance, from the early 1980s it has been shown that there is no difference between the relative proportions of individual essential AA required in the diet and the relative proportions of the same 10 AA present in fish carcass. Since the essential AA profile of fish muscle protein does not differ greatly between individual fish species, the pattern of requirement for individual species should also be similar. Thus, analysis of the larval AA composition has frequently been used to predict its essential AA requirements (Watanabe and Kiron, 1994; Conceiçao et al., 1997). Comparison of live prey and fish larvae AA profiles would allow us to predict if such feed would cover the larval AA requirements. For instance, when turbot larvae and live food EAA profiles are compared, the profile of the latter seems to be deficient in some EAA such as leucine, arginine, threonine or methionine (Conceiçao et al., 1997), depending on the larval age and type of prey, whereas rotifers seem to be deficient in threonine and leucine for larval seabream. Other methods utilized in juveniles consider that when an EAA is deficient in a diet the major proportion will be used for protein synthesis and only a small fraction will be oxidized to carbon dioxide to obtain energy whereas, if that AA is supplied in the diet in excess, plasma levels will increase and it will be more available for oxidation. A force feeding method including labeled EAA has been recently developed for fish larvae (Conceiçao et al., 2003), denoting a high retention of labeled doses of EAA in the body (>60 %), and low catabolism as measured by liberated 14 CO2 (<25 %). In contrast, non-essential AA were catabolized faster (>40 %).

11.4 Food identification and ingestion The first interaction between food particle (live or inert) and larvae occurs in the water column. Following this interaction, the particle can be accepted or rejected. Therefore, it is essential that this interaction (i.e the feeding process) is maximized and optimized. There are many factors affecting this process including particle/organisms concentration, chemical and physical cues and many others.

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d

c b a

Fig. 11.8 The feeding process (modified from Mackie and Mitchell, 1985). (a) general and non specific reaction, initiation of search movement – chemical and electrical stimuli; (b) identification of the food particle location – chemical stimuli; (c) close identification of the food particle – chemical and visual stimuli; (d) tasting and/or actual feeding – chemical stimuli (taste buds).

The feeding process includes several steps in the larval process of finding and ingesting food particles (Fig. 11.8, modified from Mackie and Mitchell, 1985): 1. general and non-specific reaction, initiation of search movements involving chemical and electrical stimuli; 2. identification of the food particle location involving chemical stimuli; 3. close identification of the food particle, involving chemical and visual stimuli; 4. tasting and/or actual feeding requiring chemical stimuli (taste buds). Various substances, such as free amino acids, nucleotides, nucleosides and ammonium bases, are released from organisms that are prey for fish larvae and are potent inducers of feeding behavior in marine (Knutsen, 1992; Doving and Knutsen, 1993) and freshwater fish larvae. Generally planktonic organisms concentrate in ‘patches’ that attract the fish larvae. Kolkovski et al. (1997a, b) identified some of the active substances in Artemia rearing water and added these substances to the larvae-rearing tank. The authors then analyzed the effect that individual substances had on ingestion rates by eliminating one substance at a time and observing the differences in feeding activity. When microdiet ingestion rates dropped, the missing substance was regarded as being an active feed attractant. The

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authors found four amino acids which induced increased feeding activity; glycine, alanine, arginine and ammonium salt – betaine. Furthermore, a synergistic relationship was reported between the amino acids and betaine, which when combined produced a stronger effect than the sum of the individuals. These and other amino acids as well as other substances were also found to be active with other marine species (Table 11.1).

Table 11.1 Amino acids as feed attractant in marine organisms Rainbow trout (Salmo gairdineri) Atlantic salmon (Salmo salar) Sea bass (Dicentrarchus labrax) Pig fish (Orthopristis chrysopterus) Red sea bream (Chrysophrys major) Gilthead sea bream (Sparus aurata) Turbot (Scophthalmus maximus) Dover sole (Solea solea) Puffer (Fugu pardalis) Japanese eel (Anguilla japonica) Cod (Gadus morhua) Herring (Clupea harengus) Glass eel (Anguilla anguilla) Lobster (Homarus americanus) Western Atlantic ghost crab (Ocypode quadrata) Freshwater prawn (Macrobrachium rosenbergii) Abalone (Haliotis discus) Source: Kolkovski, 2001.

Mixture of L-amino acids Glycine Mixture of L-amino acids Glycine, betaine Glycine, betaine Glycine, alanine, lysine Valine, glutamic acid and arginine Glycine, betaine, alanine, arginine Inosine and IMP Glycine, betaine Glycine, inosine, betaine Glycine, betaine Glycine, arginine, alanine, proline Arginine Glycine, proline

Adron and Mackie, 1978 Hughes, 1990 Mackie and Mitchell, 1982 Carr et al., 1977, 1978 Goh and Tamura, 1980 Fuke et al., 1981 Ina and Matsui, 1980 Kolkovski et al., 1997 Mackie and Adron, 1978 Mackie et al., 1980 Metaillet et al., 1983 Ohsugi et al., 1978 Yoshii et al., 1979 Doving et al., 1994 Damsey, 1984

Glycine, arginine, alanine, proline Alanine, glycine, histidine, proline Glutamate, betaine, taurine, ammonium chloride Butanoic acid, carboxylic acid, trehalose, carbohydrates, homarine, asparagine Taurine, glycine, trimethylamine, betaine

Mackie and Mitchell, 1983 Kamstra and Heinsbroek, 1991 Corotto et al., 1992

Mixture of L-amino acid and lecithin

Harada et al., 1987

Trott and Robertson, 1984 Harpaz et al., 1987

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A practical way to increase the ingestion rates of microdiets would be to incorporate these substances as extracts or hydrolysates into the diet. Kolkovski et al. (2000) tested the effect of krill hydrolysate as a feed attractant on yellow perch (Perca flavescens) and lake whitefish (Coregonus clupeaformis), by coating commercial starter diet with 5 % krill hydrolysate. Fish fed the coated diet experienced similar growth to fish fed live Artemia and significantly higher growth than fish fed the control diet (Fig. 11.9) Furthermore, a recent experiment was conducted to determine whether the method of hydrolysate incorporation in microdiets affected growth of yellowtail kingfish (Seriola lalandi) larvae. Krill hydrolysate was compared coated or incorporated into the diet (Kolkovski, 2006a). Growth rates of larvae fed coated-diet were significantly higher than larvae fed krill hydrolysate incorporated diet. Both diets (incorporated and coated) preformed significantly better than the control diet with no hydrolysate (Fig. 11.10). Other hydrolystaes such as squid hydrolysate have also been found to be effective in increasing both ingestion and growth (Kolkovski et al., 1997a, 2009; Kolkovski and Tandler, 2000; Lian and Lee, 2003; Lian et al., 2008). It is assumed that inclusion of hydrolysates as partial protein replacement benefits the larvae in two ways: (i) higher ingestion rates (due to the hydrolysates feed-attractability properties) and (ii) higher assimilation due to the influx of free amino acid and short peptides. Currently, several commercial microdiets include different hydrolysates such as krill, fish and squid hydrolysates in their formulation. Due to the fact that hydrolysates are, in fact, a mixture of amino acids and peptides (as well as many other nutrients), it has been suggested that a mixture of known amino acids could be a better

Intake (μg diet larvae–1 60 min)

60 a 50 40

Smell

Smell and taste

30

a

a

20

a b

10 0

a

b

Starter

Starter + 5 % krill

Liquid krill

*Artemia

*Artemia dry weight calculated as 2 mg/nauplii

Fig. 11.9 Effect of krill hydrolysate on ingestion rates of yellow perch and whitefish (Kolkovski et al., 2000). Grey bars – yellow perch (average wet weight – 424 mg), white bars – whitefish (average wet weight – 13.52 mg).

336

New technologies in aquaculture 8000 7000 Wet weight (g)

6000 5000 4000 3000 2000 1000 0 Control

Krill incorporated

Krill coated

Treatments

Fig. 11.10 Effect of krill hydrolysate inclusion method on growth of yellowtail kingfish: incorporated krill – krill hydrolysate was mixed at 3 % (DW) with other ingredients; krill coated – krill hydrolystae was coated with MD particles.

solution (Kolkovski, 2001). Table 11.2 presents the pros and cons of each alternative.

11.4.1 Presentation of feed attractants to fish larvae There are several ways to introduce the attractants to the larvae. These include the following: • The addition of attractants directly into the water uses large amounts of these substances, but maintains a constant concentration. • Coating the diet particle results in unknown leaching rates, but can contribute to higher palatability and more specifically identifies particles as food. • Incorporation into the diet as part of the protein source also results in an unknown leaching rate (depending on the microdiet type); however, only a low amount of attractants is needed, part of the protein source in the diet is replaced and digestion and assimilation are improved.

11.5

Ontogeny of digestive capacity in marine fish larvae

The development of adequate compound microdiets to replace live foods in the culture of marine fish larvae requires a thorough understanding of the digestion processes occurring during ontogeny (Cahu and Zambonino Infante, 1997; Lazo et al., 2000a). This knowledge is required for overcoming the necessary use of live feeds in the rearing of marine fish larvae. The

Fish larvae nutrition and diet: new developments

337

Table 11.2 The use of marine organisms hydrolysates and free amino acids as feed attractants

Content

Nutritional value Formulation Activity

Concentrations

Synergism

Hydrolysate

Free amino acids

Digested protein (usually from marine organisms) components such as free amino acids and short peptides Can be used as partial protein replacement

Pure amino acids

Unknown and uncontrolled values of AA and peptides as well as other nutrients Krill, squid, fish and several crustaceans and molluscs hydrolysates found to be strong attractants. As a ‘general rule’, protein fraction weight between 1000 and 10 000 Dalton is found to have a positive effect on feeding Concentrations of extracts and/or hydrolysates made from aquatic animals are harder to quantify than amino acids. However, concentrations that are found to have a positive effect on feeding range from 10−2 to 10−10 g/l (when added to the water). In most cases, when incorporated into the diet, the concentration of hydrolysates and extracts released into the water was not determined No data available

Can be adjusted and balanced to the AA requirements Known amounts of AA Only the L-isomers have been found to be active as feed attractants

Increasing the concentration of amino acids (when added to the water) was found to have positive effects on feeding, range from 10−8–10−2 M

Synergistic effects were associated with many combinations of amino acids and other substances such as ammonium salts

AA = amino acids. Source: Kolkovski, 2006a.

lack of success in completely replacing live foods with compound microdiets since the onset of first feeding has been historically attributed to the presence of an undeveloped digestive system at the time of hatching and consequent low digestive capacity (Lauf and Hoffer, 1984; Munilla-Moran et al., 1990; Holt, 1993). Although most research to date indicates marine fish larvae have a very defined and specific digestive physiology that merits the development of specific diets and weaning protocols, studies also indicate that many marine fish larvae possess a differentiated and effective

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digestive system early in development (Sarasquete et al., 1995; Ribeiro et al., 1999; Lazo et al., 2000a; Zambonino-Infante and Cahu, 2001). The conventional approach used for assessing digestive capacity in marine fish larvae has typically involved characterizing the morphological development of the digestive system and associated organs while also quantifying digestive enzyme activities using biochemical, histochemical and molecular techniques (for an excellent review see Zambonino-Infante and Cahu, 2001). The morphological and functional development of the digestive system of fish larvae was first reviewed by Tanaka (1973) and Govoni et al. (1986) and more recently by Zambonino-Infante and Cahu (2001) and Hoehne-Reitan and Kjorsvik (2004). Briefly, at hatching, the stomach is typically undifferentiated and non-functional. Acid digestion and pepsin expression are lacking, and the proton pump used to secrete hydrochloric acid into the stomach lumen is not functional (Gawlicka et al., 2001; Rønnestad et al., 2001; Rust et al., 2002; Morais et al., 2005). Most species also lack functional mouth and jaws. Early larvae typically posses a simple tube-like alimentary canal that is closed at both ends and that is lined with columnar epithelium. The alimentary canal undergoes rapid transformations during the transition to exogenous feeding. By the onset of first feeding, the alimentary canal has already developed into its different functional regions, but it is still less elaborated than in juveniles. However, the liver, pancreas and gallbladder are usually present and functional (HoehneReitan and Kjorsvik, 2004). Digestion occurs in the midgut and hindgut, and nutrient absorption takes place through the apical region of the epithelium of each region, which is characterized by columnar cells named enterocytes. Alkaline proteases play a major role in protein digestion during the first days of feeding, while acid proteases became increasingly important toward the end of the larval period, concomitant with the appearance of a functional stomach (Lauf and Hoffer, 1984; Lazo et al., 2007). As the developmental process progresses, oxynticopeptic cells in the gastric glands become functional, as suggested by the production of hydrochloric acid through a functional proton pump, the expression of pepsinogen and it activation to pepsin (Gawlicka et al., 2001). From the perspective of the digestion system, the transformation to the juvenile stage is complete once the stomach is fully differentiated (Fig. 11.11). A high specific activity of digestive enzymes has been observed before the initiation of exogenous feeding in most species studied to date (Zambonino-Infante and Cahu, 2001). This suggests the process of enzyme production is initiated by underlying genetic mechanisms (Buddington and Diamond, 1989) rather than by the diet (Cahu et al., 1994; Lazo et al., 2000a). While it appears that during the early stages of development digestive enzyme activities are controlled by gene expression rather than by feeding activity, a diet’s composition can influence the maturation of the digestive system by triggering an onset or increase in the activity of some digestive enzymes (Zambonino-Infante and Cahu, 2001). Feeding nutri-

Fish larvae nutrition and diet: new developments MO

PA

YR

339

FS Lates calcarifer

MO

PA

PA

YR

FS

MO YR

PA

FS

MO

PA

0

FS Scophtalmus maximus 18 °C

1

2

3

4

FS

MO

5

6

Morone saxatilis 22 °C FS

7

17

Sparus aurata 19 °C

25

Dicentrarchus labrax 19 °C

40

Fig. 11.11 Comparison of developmental stages in the digestive tract of several marine fish species: PA – protease activity; MO – mouth opening; YR – yolk resorption; FS – functional stomach (from Moyano et al., 1996).

tionally unbalanced microdiets to marine fish larvae can disrupt the normal maturation process; the earlier the weaning onto unbalanced microdiets, the more negative the observed effect on maturation (Cahu and Zambonino, 1994; Lazo et al., 2000a). In contrast, some nutrients, such as polyamines, can enhance the maturation and differentiation of the enterocytes involved in nutrient absorption. For example, seabass larvae fed a diet containing 0.33 % dry weight of the polyamine spermine displayed a faster maturation of the enterocytes relative to those fed a similar diet lacking in the polyamine (Péres et al., 1997). Likewise, Tovar et al. (2002) included the polyamine-producing yeast (Debaryomyces hansenii HF1) in the diet of seabass larvae and observed an increase in digestive enzyme secretion and earlier maturation of the enterocytes that were mediated by spermine and spermidine. While most species can be effectively weaned onto microdiets before completion of metamorphosis, successful weaning during the early larval stages has proven more challenging (Kolkovski et al., 2001). Only a handful of species can be reared on microdiets from the time of mouth opening (i.e., red drum, Scieaenops ocellatus; Lazo et al., 2000b and seabass; Cahu and Zambonino-Infante, 2001). Most species cultured to date require the use of rotifers or Artemia at some point during development. As previously mentioned, early research suggested the problems associated with early weaning were attributed to low digestive enzyme activity or to the importance of live prey for aiding or triggering the digestive

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New technologies in aquaculture

process (Kolkovski, 2001). In contrast, recent studies indicate that enzymatic activity is high in early larvae, and that the potential contribution of digestive enzymes from the prey is negligible. Typically, enzymes for the luminal digestion of proteins (trypsin, chymotrypsin and elastase, among others), lipids (lipases and phospholipases) and carbohydrates (amylases and maltases) are present in larvae before exogenous feeding commences or shortly thereafter. Their activity tends to increase with age, length and type of feed (Alliot et al., 1980; Baragi and Lovell, 1986; Cousin et al., 1987; Moyano et al., 1996; Baglole et al., 1998; Izquierdo et al., 2000; ZamboninoInfante and Cahu, 2001; Lazo et al., 2007). Intracellular enterocyte digestive enzymes such as tri- and dipeptidases exhibit high levels of activity during the early larval stage and decrease as development progresses (Cahu and Zambonino-Infante, 1995; Lazo et al., 2007). In contrast, the activity of intestinal brush border membrane enzymes such as aminopeptidases and alkaline phosphatases are lowest at first feeding and subsequently increase with age. A decrease in intracellular peptidase activity concurrent to an increase in brush border peptidase activity is indicative of the full intestinal maturity of marine fish larvae (Cahu and Zambonino-Infante, 1994). The ratio between intestinal brush border membrane enzyme activity (i.e., leucine amino peptidase or alkaline phosphatese) and intracelular peptidase activity (i.e., leucine-alanine peptidase) is a good indicator of the normal maturation of the enterocytes in marine fish larvae, the higher the ratio the higher the degree of maturation. This ratio can be used to evaluate the effect of diet on digestive system maturation (Fig. 11.12). Thus, although not as complex as the juvenile digestive system, marine fish larvae posses a wide range of digestive enzymes that support the efficient digestion of

12.0 10.0

Ratio

8.0 Adequate diet Inadequate diet

6.0 4.0 2.0 0.0 0

10 20 30 40 Days after hatching

50

Fig. 11.12 Relationship between intestinal brush border enzyme activity and intracellular peptidase activity. Data were estimated from results of enzyme activities of seabass larvae fed nutritionally balanced and unbalanced diets reported by Cahu and Zambonino (1995).

Fish larvae nutrition and diet: new developments

341

nutrients if adequate feeds are provided (i.e., larvae can achieve very high growth rates in the wild and under culture conditions). It has been proposed that exogenous enzymes from live prey could directly aid in larval digestion or activate the zymogens present in larval gut, thus increasing digestion and growth rates (Dabrowski, 1979; Lauf and Hoffer, 1984; Kolkovski et al., 1993). The mechanisms through which exogenous enzymes could aid or stimulate the digestive process are not clearly understood. Moreover, the addition of exogenous enzymes to compound microdiets in the rearing of marine fish larvae has been shown to be beneficial for some species (such as sea bass and sea bream) while its benefits have not been conclusively demonstrated for other species (Kolkovski, 2001). Moreover, several authors have reported a lack of significant differences in levels of pancreatic and intestinal enzymes in fish larvae reared with live prey or microdiets (Baragi and Lovell, 1986; Cahu et al., 1995; Lazo et al., 2000a), which indicates that the ingestion of live prey does not stimulate enzyme production or secretion into the gut lumen. Kurokawa et al. (1998) estimated the relative contribution of exogenous enzymes to digestion in Japanese sardine (Sardinops melanotictus) larvae and determined that it was only 0.60 % of the total protease activity in the intestine, and therefore concluded that the contribution of the prey’s enzymes to digestion was minimal. Similarly, Diaz et al. (1997) using substrate-SDSpage to estimate protease activity in larval sea bream and their live prey (rotifers) failed to detect proteases from the prey within the digestive tract. They suggested that the contribution of exogenous enzymes was limited to an autolytic process of the prey in the larval gut. Based on this data, it appears that the contribution of exogenous digestive enzymes to the total digestive capacity of the larvae is negligible in most species. The lack of early weaning success cannot be attributed solely to the absence of a functional stomach and lower digestive enzyme production, so other factors have been conjectured to explain the lower performance of larvae fed on microdiets. These include low ingestion rates of the microdiets (Lazo et al., 2002) or the failure of microdiets to effectively stimulate digestive enzyme secretion (Kolkovski et al., 1997a, b; Cahu and Zambonino-Infante, 2001). The latter would lead to low levels of enzymes in the lumen to digest feed particles. In combination with the relatively fast gut transit time typical of marine fish larvae (Govoni et al., 1986), this would effectively reduce the ability of the larvae to absorb the dietary nutrients necessary for meeting the requirements for normal growth. Recent research has begun to shape a more comprehensive understanding of the development of the digestive system by focusing on the study of the hormonal mechanisms controlling the expression and secretion of digestive enzymes and their modulation through dietary nutrients (Rønnestad et al., 2007). For example, many compounds present in live feeds have the potential for influencing digestive enzyme activity in fish larvae. Polyamides, algal growth regulators which play multiple roles in

342

New technologies in aquaculture

stabilizing the intracellular conformation of nucleic acids and membranes (Mathews and Van Holde, 1990; García-Jiménez et al., 1998), have been shown to stimulate gut hormone, cholecystokinin (CCK) release in rats, which in turn mediates the release of pancreatic enzymes (Fioramonti et al., 1994). Most formulated diets designed for marine fish larvae contain large amounts of fish meal, which is naturally low in the polyamide spermine (Bardocz, 1993). The addition of spermine to microdiets fed to sea bass larvae has been shown to increase pancreatic enzyme secretion and induce earlier intestinal maturation (Peres et al., 1997). In addition, amino acids may increase the secretion of certain hormones, such as somatostatin and bombasin, which also stimulate the secretion of pancreatic enzymes (Chey, 1993; Kolkovski et al., 1997a, b). Live feeds contain large amounts of free amino acids, which may stimulate the secretion of trypsin (Dortch, 1987; Fyhn et al., 1993). Cahu and Zambonino-Infante (1995) reported increased trypsin secretion in sea bass larvae fed a mixture of free amino acids in their diets. Both neural and hormonal processes are involved in regulating the secretion of pancreatic enzymes (Fange and Grove, 1979). The sight, smell or presence of food triggers a nervous control mediated by the vagus nerve that results in the induction of pancreatic secretion. Hjelmeland et al. (1988) induced secretion of trypsinogen from pancreatic tissue into the intestine of herring (Clupea harengus) larvae by feeding polystyrene spheres with no nutritional value. Similarly, Pedersen and Andersen (1992) were able to enhance the secretion of pancreatic enzymes by increasing the size of the inert particles fed to herring larvae. Additionally, gastrointestinal hormones, such as CCK, play an important role not only in the stimulation of pancreatic enzyme secretion, but also in gallbladder contraction, intestinal peristalsis and gut transit time in fish larvae (Rønnestad et al., 2007), all of which are important factors regulating the digestion process. In first feeding larvae, CCK production seems to be genetically hardwired, but in older larvae it can also be regulated by dietary factors such as protein levels and chain length (Cahu and Zambonino Infante, 2001). However, distension of the gut wall is not a factor that triggers CCK production (Koven et al., 2002). This indicates that the secretion of pancreatic enzymes is regulated by mechanisms in addition to CCK production and requires further research. Kolkovski et al. (1997b) found a ten-fold increase in bombasine activity when larvae were fed live food (Artemia) compared to microdiet, suggesting that live food activated the digestive system significantly more than microdiet resulting in better digestion and assimilation. As we continue to move away from the paradigm of low digestive capacity as the main reason for the unsuccessful rearing of marine fish larvae on microdiets, researchers have begun to study the step following digestion in feed utilization, namely, the absorption and transport of ingested or digested nutrients. Recent research has shown that marine fish larvae have an

Fish larvae nutrition and diet: new developments

343

adequate but limited capacity to absorb and transport some of the digested nutrients. For example, Morais et al. (2004) fed 14C radio labeled Artemia to Senegalense sole (Solea senegalensis) larvae of different ages (12, 22 and 35 DPH) and found no significant differences (77–83 %) in Artemia utilization among ages. Surprisingly, the younger larvae were more efficient in retaining protein assimilated from the feed. Apparently, younger larvae have the ability to compensate for a moderate digestive capacity and a lower gut residence time by increasing the efficiency of retaining absorbed amino acids. The mechanism through which the larvae are capable of achieving an increased efficiency in the absorption of amino acids has yet to be established and warrants further research. Morais et al. (2007) investigated the effect of lipid level and fatty acid composition on ingestion, digestive enzyme activity and absorption and transport of digested nutrients using several species of marine fish larvae. The authors concluded that lipid transport after absorption may be more of a limiting factor than digestion of dietary lipids. Phospholipids were more easily digested than neutral lipids and their presence in the diet markedly improving lipid absorption and transport (Liu et al., 2002). Thus, large lipid droplets accumulate in the enterocytes of larvae fed a high-lipid diet in the absence of adequate phospholipids, which in turn may reduce the absorption of digested nutrients (including amino acids and free fatty acids) from the lumen (Morais et al., 2005). Collectively, the results of studies performed to date indicate the need for examining the inclusion of feed attractants and stimulants of the secretion of pancreatic enzymes in microdiets. In addition it is important to include an adequate balance of free amino acids, peptides and intact proteins, phospholipids, neutral lipid sources and free fatty acids in diets so as to develop an adequate replacement of live feeds from first feeding during the rearing of marine fish larvae.

11.6 Digestive system capacity Recent research evaluating the effect of specific nutrients on larval digestive physiology and characterizing the metabolic pathways of the assimilated nutrients has revealed an important role of the type (protein vs peptides and amino acids or triglycerides vs phospholipids), quantities (protein or lipid levels), ratios (DHA : EPA :ARA; or essential fatty acids vs other fatty acids for metabolic energy) and availability of a dietary nutrients (Morais et al., 2007; Rønnestad et al., 2007). Given the complexity the metabolic pathways involved, a more comprehensive approach is needed to further our understanding of the digestive process and nutrient requirements of developing marine fish larvae (Zambonino-Infante and Cahu, 2007). Similarly, more molecular research is needed to characterize nutrient transporters in the gut lumen throughout ontogeny,

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New technologies in aquaculture

so as to more thoroughly establish the assimilation capacity of developing larvae. To date, no routine assay has been developed to measure dietary protein digestibility in marine larval fishes. Although some interesting methods have been recently proposed (Alarcón, 1997; Lazo et al., 2002; Rønnestad et al., 2007; Muscato et al., 2009), a standard method applied among research laboratories has yet to be established. Results obtained through the characterization of digestive enzymes, such as the determination of protease activity and inhibition, optimum temperature and pH should be utilized to develop in vitro and in vivo assays to measure protein digestibility throughout the larval stage. In vitro tests can be used to evaluate digestibility of different proteins sources and ingredient-mediated enzyme inhibition. Enzyme extracts from larval guts, purified enzymes or readily-available commercial enzymes can be utilized for these assays (although the former enzymes are preferred). The effect of feed processing (i.e. temperature and drying) and ingredient pretreatment (i.e. enzyme hydrolysis) on digestibility of the protein sources can also be evaluated using in vitro assays (Alarcón et al., 1997; Lazo and Holt, 2001). Thus far, few studies have considered the changes in digestive capacity occurring within a specific stage of development such as the larval period (i.e., early, mid and late larvae or early juvenile). Information of changes in enzyme type and relative activity can be utilized to develop reliable in vitro digestibility assays that allow for the screening of potential dietary ingredients and formulated feeds, without the need for long, expensive and labor-intensive feeding trials. Recently, Lazo and Martinez (2007) evaluated the in vitro protein digestibility of potential dietary ingredients throughout ontogeny of digestive system in larvae of California halibut (Paralichthys californicus) using the pH-STAT technique to identify adequate protein sources for each stage of development. Highly significant differences (p < 0.001) in protein digestibility were found for some ingredients among the developmental stages evaluated (Fig. 11.13). Meals elaborated with typical live feeds for the rearing of marine fish larvae (rotifers and Artemia) showed the highest protein digestibility with a tendency to decrease as larval development progressed. Soybean meals were poorly digested, and this could be attributed to presence of antinutritional factors and/or differences in protein quality due to processing. Results from this type of studies warrant the importance of evaluating the digestibility of protein sources through larval development in order to formulate successful weaning diets for marine fish larvae (i.e., stage-specific diets). Information on the ontogeny of digestive enzymes will aid in the selection of more adequate ingredients for the successful design of adequate diets specific to each developmental stage. Although in vitro assays are simple, rapid and inexpensive techniques, the more accurate and comprehensive approach to assess nutrient digestibility is to evaluate digestibility using in vivo methods. Recently,

Fish larvae nutrition and diet: new developments 2.5

9 DPH 39 DPH

15 DPH 51 DPH

345

a

26 DPH ab ab

a

2.0

a

DH (%)

1.5

ab

a

a

bc

a a

a ab a

1.0

bb

b

0.5

bb

ab

ab

a

ab b

b b

a

a c

a abab

c bc

abc

c aa

b

bc

bc

b

b

bc c 0.0

Casein

Fish meal 1 Fish meal 2

Krill meal

Squid meal

Rotifers

Artemia

Soy meal Wheat gluten

Ingredients

Fig. 11.13 Changes in degree of hydrolysis (DH) of several ingredients through ontogeny of the digestive system in California halibut larvae: DAH – days post hatching (adapted from Martinez-Montaño et al., 2006).

Rønnestad et al. (2001) developed an improved in vivo method of the controlled tube feeding of radiolabeled nutrients originally conceived by Rust et al., 1993). In the improved technique, correct discrimination was made between unabsorbed labeled nutrients from gut evacuation and labeled nutrients from metabolism of the absorbed nutrients. With this technique, it is possible to quantify gut absorption, oxidation and retention of dietary nutrients and thus assess part of the functionality of the digestive system in marine fish larvae. Nevertheless, full digestion capacity is probably not realistically quantified since nutrients are ‘forced’ fed into the digestive system and many hormonal processes that may naturally occur in a feeding trial may not be taking place. How easily other laboratories will adopt this highly innovative method is yet to be seen since it requires sophisticated apparatus and uses radiolabeled compounds. A more simple approach was recently developed by Muscato et al. (2009), to determine in vivo protein digestion of microdiets in marine fish larvae. The new method uses FluoSpheres®, fluorescent microspheres, as an indirect marker to estimate protein digestibility. Additionally, the fluorescent microspheres can be used to quantify feed ingestion. The formulation and manufacture of adequate microdiets using new information will permit more nutritional studies to further our knowledge and understanding of the nutritional requirements of marine fish larvae and finally overcome the necessity of using live feeds in the rearing of marine fish larvae.

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New technologies in aquaculture

11.7 Diet manufacturing methods Several microdiet manufacturing methods are currently being used: • • • •

microbound diets (MBD) (Fig. 11.14a), microcoated diets (MCD) and micro-encapsulated diets (MED) (Fig. 11.14b) and, marumerization (MEM) (Fig. 11.14c).

(a)

(c) A

B

C

D

(b)

Fig. 11.14 Microdiets manufactured by different techniques: (a) MBD; (b) MED (photo Manuel Yúfera, CICS, Cediz, Spain); (c) MEM (photo Bernard Devresse, BernAqua, Belgium).

Fish larvae nutrition and diet: new developments

347

All have been used extensively in nutritional studies with finfish larvae and are commercially manufactured. 11.7.1 MBD Currently, the manufacturing process of MBDs is the simplest and most commonly used method of preparation. It consists of dietary components held within a gelled matrix or binder. They do not have a capsule, and it is suggested that this facilitates greater digestibility and increased attraction through greater nutrient leaching (Kolkovski, 2001; Yúfera et al., 2003; Kolkovski, 2006a,b). Some commercial microdiets are manufactured using extrusion and then crushed and sieved to the required particle sizes. All the ingredients are ground, mixed with a binder such as gelatine, alginate, zein, carrgeenan and carboxymethyl-cellulose, activated by temperature or chemically (López-Alvarado et al., 1994; Koven et al., 2001; Kolkovski, 2004, 2006b) and then dried (drum drying or spray drying), ground and sieved to the required size.

11.7.2 MCD The MCD method is based on coating or binding small MBD particles to reduce leaching (López-Alvarado et al., 1994; Baskerville-Bridges and Kling, 2000; Onal and Langdon, 2004). The coating layer is usually lipids or lipoproteins. This method is not often used in commercial processes.

11.7.3 MED MED particles are made using several different techniques. The particle usually has a membrane or capsule wall, which separates dietary materials from the surrounding medium (Fig. 11.14b,c). The capsule wall helps maintain the integrity of the food particle until it is consumed preventing leaching and degradation of the nutritional ingredients in the water. However, this attribute may restrict leaching of water-soluble dietary components and therefore reduce the larvae’s attraction to the food particles (Yúfera et al., 2003; Kvåle et al., 2006). The capsule wall is also thought to impair digestion of the food particle (Yúfera et al., 1998; Kolkovski, 2006a,b) (Fig. 11.14b). There are several methods for micro-encapsulation. These include chemical processes and mechanical processes. In chemical processes, the capsules are made within a liquid, usually stirred or agitated. The capsules are formed by (i) spraying droplets of coating material on core ingredients, (ii) capsulating liquid droplets containing the nutritional ingredients by spraying into gas phase, (iii) creating gel capsules by spraying droplets, containing the nutritional ingredients and a binder, into liquid solution that activate the binder or by polymerization reaction at a solid/gas or (iv) liquid interfaces. Protein cross-link (Yúfera et al., 1998, 2000) involves several stages of mixing and washing with organic solvents resulting in a very expensive diet

348

New technologies in aquaculture Arg Lys Glu His Asp

Pro Tyr Ser Gly Thr

Ala Cys Met

Leu Val lle

120 100

% Leaching

80 60 40 20 0 –6 Hydrophilic

–4

–2

0

2

4

6 Hydrophobic

Hydropathy index MB – microbound diet MC – microencapsulated diet MC-L – microencapsulated diet with lysine supplementation

Fig. 11.15 Percentage of FAA leached after 60 min of immersion in water in relation to the hydropathy index (Yúfera et al., 2002).

process, that is potentially toxic. These methods have never resulted in good growth rates due to the inability of larvae to digest and assimilate the particles as well as the high ratio of non-nutritional ingredients mainly the capsule, to the essential nutrients (Yúfera et al., 2005). Another method, complex coacervation, involves mixing and activating, using electrical charges, two liquid phases differing in their viscosity resulting in very small capsules. These capsules then bind to create a larger capsule containing hundreds or thousands of microcapsules (Thies, 2007) (Fig. 11.15c).

11.7.4 MEM Mechanical encapsulation involves processes such as spray drying, fluidized bed drying, cold micro-extrusion marumerization (MEM) and particleassisted rotational agglomeration. The last two techniques have gained attention in the past few years with commercially available diets produced using these methods. Initially developed for pharmaceutical processes, these methods involve purpose-built machines. MEM is a two-step process of cold extrusion followed by marumerization (spheronization). The process has the capability of producing particles from 500–1000 μm and greater. Particle-assisted rotational agglomeration (PARA), which is a single-step

Fish larvae nutrition and diet: new developments

349

process capable of producing particles from 50–500 μm that are lower in density than particles produced by the MEM method due to the fact that the extrusion step is avoided. The method is based on a spinning disk (marumerizer). A wet mash of the ingredients is put into the marumerizer with or without inert beads. The rotation movement of the disk breaks down the mash into smaller spherical particles. The diameter of the particles depends on several factors including the disk rotation speed, the inert beads and the raw ingredients (Barrows and Lellis, 1996, 2006).

11.8 Microdiet characteristics Although they have a very strong role in microdiet utilization, very little attention and research efforts have been donated to the identification of the chemical and physical properties of diet particles. There are only a handful of scientific works on leaching properties and even fewer on the buoyancy and behavior of the particles in the water column.

11.8.1 Leaching As mentioned above, one of the problems of MBD particles and most of the microdiet-type particles is the high leaching rate of amino acids. Kvåle et al. (2006) reported leaching of protein molecules (9–18 kD) after 5 min immersion in water (3 % NaCl, 12 ºC) at a rate of 80–98 %, 43–54 % and 4–6 % for agglomerated, heat coagulated and protein encapsulated microdiet. Yúfera et al. (2003) determined the rate of different amino acids leaching from both MBD and MED. The authors found contrary patterns between the two diet types. While hydrophilic amino acids leached the most from MBD, hydrophobic amino acids were found to leach from MED particles at a higher rate (Fig. 11.15). The leaching rates of the two diets were also significantly different. For instance, 70 % of free lysine leached from MBD particles after less than 5 min, while less than 7 % leached from MED particles after 60 min (Fig. 11.16). López-Alvarado et al. (1994) tested the leaching rates from several different microdiets made with different techniques and found similar results (Table 11.3). Heinen (1981) assessed water stability of formulated diets made from 11 different binders; MBD made from agar and alginate were amongst the most stable in terms of integrity, while carrageenan was amongst the poorest. A diet particle needs to achieve a fine balance between leaching amino acids and other nutrients to act as feed attractant and digestibility of the particle to suit the undeveloped larvae digestive system. A particle that will be hard and leach-resistant will also present a challenge to the larvae digestive system, whilst, a particle that will digest easily in the gut will also disintegrate relatively quickly in the water (Yúfera et al., 2000; Kolkovski, 2006a).

350

New technologies in aquaculture 120 a

100

(%) Leaching

80

a

a a

a

60

MC-L MC MB

20

10 c

0

a b ab b 0 10

d

c

c b

b 20

30 40 Time (min)

50

b 60

MB – microbound diet MC – microencapsulated diet MC-L – microencapsulated diet with lysine supplementation

Fig. 11.16 Leaching pattern of lysine during 60 min of immersion in water (Yúfera et al., 2002). Table 11.3 Leaching rates from different MD manufactured by different techniques MD type and binder type MBD, carrageenan MBD, alginate MBD, zein Protein MED Protein MED/lipid MCD Lipid walled (tripalmitin + triolein) Lipid walled (tripalmitin)

Leaching (%) 85 81 91 59 39 47 4

± ± ± ± ± ± ±

7 2 2 1 2 9 2

MBD = microbound diets, MCD = microcoated diets, MED = microencapsulate diet. Source: Lopez-Alvarado et al., 1994.

11.8.2 Buoyancy One of the most significant problems with microdiet particles is their negatively buoyant inert state. However, very few scientific studies have investigated this issue. In addition, MBD particles do not move like live zooplankton. This specific movement act as a visual stimulus for increased feeding activity (Kolkovski et al., 1997a,b). Furthermore, the particles sink to the bottom of the tank where they are no longer available to the larvae and accumulate there, leading to bacterial proliferation and deterioration of water quality. This further creates the need to effectively wean the larvae onto the MBD, in order to both modify their digestive capacity and their

Fish larvae nutrition and diet: new developments

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Accumulative diet return (%)

MicroGemma 150 MicroGemma 300

100

Kinko 0

80

Proton 2

60

Gemma 0.3

Proton 3 Kinko 1

40

Grow Best L3 20

Proton 4 NRD 4/6 2

4

6 8 Time (min)

10

12

14

NRD 5/8 Diet size

Fig. 11.17 Sinking patterns of commercial diets (Jackson and Nimmo, 2005).

feeding behavior. A change in behavior is illustrated by the larvae’s ability to recognize the inert particles as food and to more actively hunt for them during a relatively smaller window of opportunity, as the particles pass down through the water column. Figure 11.17 illustrates the sinking rates of several commercial microdiets (Jackson and Nimmo, 2005). Different attempts have been made to increase the amount of time the microdiet particle spends in the water column including increasing buoyancy by adjusting and modifying oil levels, manufacturing methods and also using rearing systems with up-welling currents (Kolkovski et al., 2004; Teshima et al., 2004). Knowledge of sinking and leaching rates of microdiets can and should be used to optimize feeding time in the larvae tank. The faster the diet particle sinks the shorter the feeding intervals should be coupled with smaller quantities of diet, in short, feeding less more often.

11.8.3 Weaning and co-feeding methods An important factor influencing the larvae’s acceptance of a microdiet, which affects both their growth and survival, is the weaning process. In the past, early weaning has led to poor growth and inferior quality larvae with an increased risk of skeletal deformities (Cahu and Zambonino Infante, 2001). Recent advances in microdiet formulation have considerably reduced the pre-weaning period, allowing the introduction of specific larval diets to marine finfish culture as early as mouth opening (Cahu and Zambonino Infante, 2001). ‘Co-feeding’ weaning protocols, simultaneously using inert and live diets, allow an earlier and more efficient changeover period onto microdiet from live feeds (Hart and Purser, 1996; Daniels and Hodson, 1999; Koven et al., 2001; Curnow et al., 2006,a,b; Rosenlund and Halldorsson, 2007). Co-feeding provides higher growth and survival than

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feeding solely live feeds or microdiets (Kolkovski et al., 1995). Early cofeeding of an appropriate microdiet will improve larval nutrition and can condition the larvae to accept microdiet more readily, thus preventing an adverse effect on subsequent growth following weaning (Rosenlund et al., 1997; Canavate and Fernandez-Diaz, 1999; Cahu and Zambonino Infante, 2001; Kolkovski, 2001, Saenz de Rodriganez et al., 2005). However, in spite of newer diet manufacture technique, better understanding of the digestive system ontogeny, complete live feed replacement has still not been achieved. In the past several years many experiments have been conducted looking at this problem and trying to wean larvae of different species at different ages to different microdiets with limited success. Curnow et al. (2006b) demonstrated the effect of different weaning and co-feeding treatments on growth and survival of barramundi larvae (Fig. 11.18). The authors found that early weaning before the larvae fully developed, as well as diet type and quality, influenced not only growth and survival but also the occurrence of cannibalism. They concluded that co-feeding barramundi larvae on microdiet should be started no earlier than three days prior to stomach differentiation and be continued post metamorphosis. Co-feeding improves growth by 25–30 % over the previous standard method (shorter and earlier weaning), and mortality during weaning was reduced from 5 % to 1 % (Bosmans et al., 2005). Similar results were found with many fresh water and marine species (Table 11.4). It is now clear that complete replacement of live food is still far from reality. Although replacement of Artemia has 25.00

Length (mm)

20.00

15.00

10.00

5.00

0.00 2

6

10

14

18

22

26

28

Larvae age in days post hatch G

G3

G7

G12

G12A

P12A

Fig. 11.18 Effect of various feeding protocols on barramundi larvae length average length, mm ± SE) (Curnow et al., 2006a). Treatments are denoted according to Table 11.5.

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Table 11.4 References to weaning protocols of different fish larvae species Fish species

Weaning protocol

Pikeperch (Sander lucioperca) (freshwater)

Weaning at hatch, 12 or 19 DPH

Senegaleresole (Solea senegalese)

Weaning protocols

Sea bass (Dicentrarchus labrax) Atlantic cod (Gadhus morhua)

Weaning period, 15, 20 and 25 DPH Weaning protocols, 0 %, 50 % and 100 % Artemia replacement with microdiet Larvae rearing protocols (review)

Atlantic cod (Gadhus morhua) Fat snook (Centropomus parallelus) Common sole (Solea sloea)

Weaning period

Tongue sole (Cynoglossus semilaevis) Dourado (Salminus brasiliensis)

Weaning protocols

Pacu (Piaractus mesopotamicus)

Weaning protocols

Sturgeon (Acipenser sturio) Barramundi (Lates calcarifer)

Weaning periods

Dover sole (Solea solea)

DPH = days post hatch.

Weaning diets comparison

Weaning time

Weaning protocols

Diet type and weaning time

Findings

Authors

Best growth, survival and lowest deformities, but high cannibalism at post-hatch weaning Artemia-fed larvae grew threefold less then fish fed an inert diet. Sudden weaning and co-feeding resulted in larger fish than late weaning Lowest growth and survival rates when weaned at 15 DPH, highest at 25 DPH Highest survival and growth achieved in treatments with Artemia (100 % and 50 %)iu

Kestemont et al., 2007

Weaning achieved at 22 with reduced growth, but higher growth achieved with late weaning (30 DPH) Successfully weaned at 35 DPH, but higher growth achieved at 40 DPH weaning Weaning at 30 DPH, one diet achieved comparable survival to Artemia treatment and better growth Co-feeding regimes preformed similar or better than Artemia regime Early weaning (3, 5 DPH) resulted in lower survival although length and weight was not affected Artemia-fed larvae showed the higest growth compared to diet-fed larvae Long weaning (21 days) resulted in better growth and survival then short weaning (3 days) Complete replacement of Artemia was achieved. However, better survival achieved when small amount of Artemia was added Early weaning (42 DPH) resulted at higher survival, but late weaning resulted at higher growth

Engrola et al., 2007

Suzer et al., 2007 Fletcher et al., 2007

Rosenlund and Halldorsson, 2007 Alves et al., 2005 Palazzi et al., 2006 Chang et al., 2006 Vega-Orellana et al., 2006 Tesser et al., 2005 Williot et al., 2005 Curnow et al., 2006a,b

Rueda-Jasso et al., 2005

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Table 11.5 Weaning protocols according to Curnow et al., 2006a Skretting Australia Diets Treatments

INVE Diets

G

Gemma Micro

G3

Gemma Micro

0.6

0.8

1.0

1.6

2.0

0.4

0.6

0.8

1.0

1.6

2.0

0.8

1.0

1.6

2.0

0.8

1.0

1.6

2.0

1.0

1.6

2.0

0.5

Gemma Micro

5 10 15 10

5

Gemma Micro 5 10 15

Rotifers G12A

0.4

5 10 15

Rotifers G12

15 10 5 0.6

Gemma Micro 5 10 15

Rotifers

0.8 15 10 5 2

Artemia P12A

0.6

Proton

0.8

10

6

1.0

2

Artemia 0

6

2 1.6

2.0

15 10 5

5 10 15

Rotifers

DPH

Gemma 0.3

Proton 3/5

Proton 2/3

Proton 1

Rotifers G7

Gemma Micro 300

Gemma Micro 150

5

10

6

10

15

6

2

20

25

30

DPH = days post hatch.

been achieved for some species such as red drum and European sea bass, replacing the Artemia is not yet considered to be realistic for most species. Replacement of rotifers is even more difficult. Weaning protocols are almost standard in terms of the weaning steps, i.e. rotifers, Artemia (nauplii and then enriched Artemia), Artemia– microdiet co-feeding and complete weaning. However, these protocols are varied according to the fish species, temperature, microdiet type, rearing system, feeding system and intervals. Even in fish species whose nutritional requirements are considered to be similar, such as gilthead sea bream and European sea bass, weaning protocols are different. While sea bass is one of the only marine fish species for which complete replacement of Artemia was achieved (Chau and Zambonino, 2001), sea bream protocol is still reliant on Artemia.

11.9 Feeding system The digestibility and nutritional qualities of the commercially available microdiets are now becoming much better due to continuous research and development, as mentioned above. However, none of these commercial

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microdiets is used solely without Artemia (not to mention without rotifers). Part of the reason is the microdiet distribution or the manner of delivery to the larvae. The best dry diet is as good as the method with which it is dispensed into the larvae tank. Compared to feeding systems and methods for on-growing fish, larvae feeding systems were not given much attention from both the scientific and commercial sectors. Only a handful of automated microdiet feeding systems exist and almost no scientific papers have been published (Papandroulakis et al., 2002). Hand feeding is the simplest and still the most widely used method. Hand feeding is usually undertaken using small devices (spoons, salt-boxes) with relatively long periods between feeding events (30–60 min). Covering long photoperiods is difficult due to labor and logistics involving long feeding periods, sometimes over 24 h. Due to the high larval metabolic rates and the demand for continuous feeding, the result is insufficient benefits from a relatively expensive product. It has been recognized that European hatcheries (and, in fact, any modern intensive hatcheries) have a strong need for automation in all the production processes. Not only would this generate labor savings, but it will also secure the production protocols and bring more repeatability to every step of the processes (Leclercq, 2004). With this in mind, plans were made to develop automatic dispensers of microparticles that would be precise in their distribution and easy to use in hatcheries.

11.10 Dosage system The first requirement from a mechanical microdiet dispenser concerns its capacity to deliver one stable quantity per feeding event. Well-known belt feeders (FIAP Aquaculture, Denmark), driven by a motor or by a clock, are not capable of splitting a daily ration in equal aliquots of feed. They are handy and cheap but not actually built for microdiet particles, resulting in the microparticle sticking to the belt, especially in humid conditions. Horizontal drums have also been developed (Arvotec, Finland). The small cavities on the drum external area can be loaded with, relatively, consistent quantities when the microparticle remains dry and not sticky. However, often, a thin layer of product accumulates between the rotating drum and its housing. This quickly becomes a contamination source, as well as a factor generating inconsistency in the distribution. Vertical hoppers with a rotating disk are also available commercially (Sterner, Norway). However, the self-compaction of the microparticles in the hopper coupled with a great deal of difficulty in cleaning the equipment limits its applicability. A different feeding mechanism was developed in Australia (‘AMD’ [Automatic Microdiet Dispenser], Department of Fisheries, Western

356

(a)

New technologies in aquaculture

(b)

Fig. 11.19 Automatic microdiet dispenser (AMD, Department of Fisheries, Western Australia): (a) side view; (b) bottom view, static plate secured with bolts while the moving plate above it is held by the solenoid piston.

Australia, Fig. 11.19a,b) with the dosage system based on the opening of a sluice-valve quickly moved by means of a simple solenoid allowing for a constant quantity of feed to be delivered at each feeding event. Cleaning the feeder is a very simple and quick process. The feeder uses air from the hatchery supply to supply a built-in spreader.

11.10.1 Delivery to the rearing tank Once a reliable dose is established, its repetition over the tank surface or in the tank volume is necessary to increase the larvae–particle interaction (i.e. before it sinks to the bottom of the tanks and become unavailable to the larvae). The dose delivery can take place directly above the water surface. However, there is a risk of aggregation of particles into small packs, sticking together and immediately sinking to the tank bottom. To avoid this, Raunes, a cod hatchery in Norway, has developed an intermediate vessel where the microdiet is mixed with water and further distributed into the water column at different points of the tank. These are commonly referred to as ‘spiders’. The vessel volume is only a few liters and the applied flow (part of the tank water intake) allows for 2–3 min of residence time in the vessel. In this vessel, the dose is delivered into the flow of water by any dispenser and the microparticles are separated and

Fish larvae nutrition and diet: new developments

357

dispersed by the strong water movements into the vessel. The suspended particles are then pushed into the tanks through the ‘spider legs’, these being made from a number (6–12) of small rigid plastic (2–3 mm internal diameter) pipes. The lower end of each of these is set into the water column (2–3 cm below the tank surface). This means of dispersing the microparticles is very efficient, especially in large tanks (4–10 m3 or more). It avoids trapping the microparticles in the surface skimmers, which are often used to capture the oil-film at the surface of the tanks. This way of dispersing the microdiet particles is most efficient; however, due to the strong water movement, it may also cause very strong leaching of the nutrients. The extent of this problem is yet to be determined. Another way of spreading the microdiet over the surface is by air. An air-blade is formed under the dosage point (where the microdiet dose is delivered by the dispenser) and blows the light particles over an area that can reach 30–90 cm. Once separated from each other by the air current, the particles do not tend to clump and conglomerate over the surface. This is simpler than the ‘spider’ device described above, but requires tanks without skimmers (only for <150 μm particles that tend to float rather then sink), which would trap the microparticles before they sink.

11.10.2 Fractioning of the daily ration into multiple events As mentioned above, fish larvae have a limited ‘window of opportunity’ to catch the microdiet particles before they sink to the bottom of the tank and become unavailable. It is also current practice in modern hatcheries to establish a long photoperiod during the larval production (from 16–24 h light, Leclercq, D pers comm). Therefore, it is extremely important that the distribution of the microdiet be split over time in very frequent feeding events. This will give the larvae a chance to catch fresh microparticles in the water column. In that respect, it is advantageous for the electronic control panels of the dispensing systems to include the ability to have frequent feeding events. This is a vital element in establishing the distribution regime and potential success of the microdiet feeding strategy. Knowing the sinking rates of specific diets and the minimum diet that can be dispersed at a feeding event will enable the design of an efficient and optimal feeding schedule that will allow continuous availability of microdiet particles to the larvae.

11.10.3 Sparing on the quantity of microdiet Microdiets are expensive (up to 200 ´/Kg) and are likely to remain so. Their production is difficult and the raw ingredients, in many cases, are very expensive. There is a relatively small market which does not allow for large-scale production in turn reducing the end cost. Therefore, it is necessary to optimize their use and the yield from such a product. In the

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New technologies in aquaculture

on-growing sector of fish farming, FCR (feed conversion ratio) is one of the most important factors farmers and feed producers are looking at. Using microdiet as a full or partial substitute for the live feed should lead to a close look at the FCR as a benchmark that can help hatchery managers improve their methods and efficiency. However, FCR figures for larvae feeds – live or formulated – do not exist. Estimating FCR with larvae is an extremely hard task. Although it is possible to estimate ‘commercial’ FCRs by knowing the amount of food (rotifers, Artemia and microdiets) specific larvae tanks received and the final biomass, measurements of these parameters during larvae production are always difficult to determine on a daily basis. During the past several years, the use of Artemia (per number of post-larvae produced) has dropped significantly (Moretti et al., 2005) due to better diets and weaning protocols. However, as mentioned previously, microdiet feeding is still far from optimal. Calculated microdiet FCRs (actual diet ingested/growth) could be as low as 0.6 or 0.8 : 1 due to high digestibility of the diet (Kolkovski, 2000; Leclercq D., pers comm). However, this is rarely if ever achieved because of spoilage of excess feed being distributed due to feeding systems and strategies. Currently, one should consider that actual FCR values above 3 : 1 are due to poor feed distribution strategies. Using the right feeding system to reach a precise and controlled distribution of feed will help to spare microdiet. Some European hatcheries quoted the figure of 20–40 % less feed being used when shifting from a hand distribution to automated. They also observed larvae survival and growth gain (Leclercq D, pers comm; Aquastream hatchery, France, pers observations).

11.10.4 Sparing cleaning time or cleaning efficiency In most cases, the majority of modern hatcheries still have to siphon the larvae tanks daily. Automation for continuous cleaning of the tank bottom (for example, cod hatcheries, Norway and some hatcheries in Japan), has not yet gained full acceptance by the majority of European hatcheries. Therefore, manual siphoning remains a time-consuming task and an important factor affecting labor. Changing from live feed to microdiet may impact negatively on cleaning demands, particularly if large time intervals are used with hand feeding or calibrated automatic feeding. In most cases, feed delivery is higher than necessary, with the intention of maintaining particles in suspension between events. If a good dispenser is used, efficient feeding can be obtained with less feed and generating far less deposition on the bottom of the tank. Reduction in siphoning and feed input will help, in turn, to increase water quality in the tank and will result in a positive effect on larvae survival. This was demonstrated by Fletcher et al. (2007) when comparing different weaning diets to Artemia feeding. The authors did not find significant differences between the treatments in

Fish larvae nutrition and diet: new developments

359

either water turbidity or bacteriology tests. The experimental system consisted of 80 l tanks, far smaller then commercial hatchery tanks. However, as mentioned above, with good feeding system, accurate feeding regimes adjusted to specific microdiet and hygiene protocols, using formulated diets can be as good if not better then using live food in terms of tank hygiene.

11.11 Future directions It is clear that complete replacement of rotifers and Artemia, as finfish larvae first food items, with microdiet has not been achieved commercially without reduced growth and survival performances. As described in the chapter, the reasons for this lack of success can be related to several factors and disciplines that need to be addressed using an integrative approach. First and foremost, the particles need to be attractive to the larvae. Therefore, feed attractants need to be incorporated or coated onto the particles. This should involve diet manufacture techniques that will limit leaching, particularly of amino acids. The microdiet particles should be available to the larvae at all times, while limiting fouling of the tank. This will require research into new feeding systems or optimizing existing systems and integrating knowledge of sinking rates of specific diets. Optimizing tank hydrodynamics will also contribute to a better food particle distribution. Following ingestion, easier to digest proteins and binders should be tested and adjusted to balance the amino acid requirements of specific species. This should be linked to diet manufacture methods that may increase or decrease the particle digestibility. Although the digestive system development has already been described for many marine species larvae, the digestive capability of larvae to break down hard, dry particles has not yet been defined completely. The ontogeny and the activity of the digestive system (both the neurohormones that trigger the enzymes secretion and the enzymatic activity itself) should be looked at from a microdiet digestibility perspective. To date, larval nutritional requirements are only partially identified and much is still unknown. With the introduction of better microdiets with higher attractability and better digestibility, the nutritional requirements of marine fish larvae can be defined more easily. Minerals, vitamins, specific proteins and amino acid balance should be looked at combined with FCR, both calculated and actual. This research will lead to better feeding strategies and will enable the use of nutritional tools, as is the case with fish nutrition. Finally, a better uniformity in the design and execution of nutritional trials will enable the comparison of data from different systems and different trials.

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11.12 References adron j w, blair a and cowey c b (1974) Rearing of plaice Pleuronectes platessa larvae to metamorphosis using an artificial diet, Fish Bull, 72, 353–7. alarc´ on f j (1997) Procesos Digestivos en Peces Marinos: Caracterizacion y Aplicaciones Practicas, PhD Dissertation, Universidad de Almeria. alliot e, pastoureaud a and trellu j (1980) Evolution des activities enzymatiques dans le tractus digestif au cour de la vie larvaire de la sole. Variations des proteinogrammes et des zymogrammes, Bichem Sys Ecol, 8, 441–5. alves t t, cerqueira v r and brown j a (2006) Early weaning of fat snook (Centropomus parallelus Poey 1864) larvae, Aquaculture, 253, 334–42. appelbaum s and van damme p (1988) The feasibility of using exclusively dry diet for rearing of Clarias gariepinus Burchell larvae and fry, J Appl Ichthyol, 4, 105–10. baglole c l, goff g p and wright g m (1998) Distribution and ontogeny of digestive enzymes in larval yellowtail and winter flounder, J Fish Biol, 53, 767–84. baragi v and lovell r (1986) Digestive enzyme activities in stripped bass from first feeding through larval development, Trans Am Fish Soc, 115, 478–84. bardocz s, grant g, brown d s, ralph a and pusztai a (1993) Polyamines in foodimplications for growth and health, J Nutr Biochem, 4, 66–71. barnabe g (1976) Elevage larvaire du loup Dicentrarchus labrax L.; Pisces Serranidae I’aide d’aliment seccompose, Aquaculture, 9, 237–52. barrows f t and lellis w a (1996) Diet and nutrition, in Summerfelt R C (ed.), Walleye culture manual, NCRAC Culture Series #101, NCRAC Publications Office, Iowa State University, Ames, IA, 315–21. barrows f t and lellis w a (2006) Effect of diet processing method and ingredient substitution on feed characteristics and survival of larval Walleye Sander vitreus, J World Aquac Soc, 37, 154–60. baskerville-bridges b and kling l j (2000) Early weaning of Atlantic cod (Gadus morhua) larvae onto a microparticulate diet, Aquaculture, 189, 109–17. bell m v and dick j r (1993) The appearance of rods in the eyes of herring and increased di-docosahexaenoyl molecular species of phospholipids, J Mar Biol Ass UK, 73, 679–88. bell j g, mcevoy l a, estévez a, shields r j and sargent j r (2003) Optimising lipid nutrition in first-feeding flatfish larvae, Aquaculture, 227, 211–20. benítez-santana t, masuda r, valencia a, hernández-cruz c m, carrillo e, ganuza e and izquierdo m s (2004) Effect of dietary lipids on larval gilthead sea bream (Sparus aurata, L.) behavior, European Aquaculture Society Special Publications, 34, 158–9. benítez t, izquierdo m, masuda r, hernández-cruz c, valencia a and fernándezpalacios h (2007) Modification of larval gilthead seabream swimming behaviour by dietary essential fatty acids, Aquaculture, 264, 408–17. bessonart m, izquierdo m s, salhi m, hernandez-cruz c m, gonzalez m m and fernandez-palacios h (1999) Effect of dietary arachidonic acid levels on growth and survival of gilthead sea bream (Sparus aurata L.) larvae, Aquaculture, 179, 265–75. betancor m b, atalah e, caballero m j, benítez-santana t, roo j, montero d and izquierdo m s (2009) Dietary α-tocopherol in weanning diets for European sea bass (Dicentrarchus labrax) improves survival and tissue damage caused by excess dietary DHA contents, Aquac Nutr (submitted). bosmans j m p, schipp g r, gore d j, jones b, vauchez f e and newman k k (2005) Early weaning of barramundi, Lates calcarifer (BLOCH), in a commercial, intensive, semi-automated, recirculated larval rearing system, in Hendry C I, Van Stappen G, Wille M and Srogeloos P (eds), Larvi ’05, 4th fish and Shellfish Lar-

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viculture Symposium, Gent, European Aquaculture Society Special Publication, 36, 46–9. buddington r k and diamond j m (1989) Ontogenic development of intestinal nutrients transporters, Ann Rev Physiol, 51, 601–19. caballero m j, gallardo g, montero d, robaina l, fernández a and izquierdo m s (2006a) Vegetable lipid sources affect in vitro biosíntesis of triacylglycerols and phospholipids in the intestine of sea bream (Sparus aurata), Br J Nutr, 95, 448–54. caballero m j, torstensen b, robaina l, montero d and izquierdo m s (2006b) Vegetable oils affect composition of lipoproteins in sea bream (Sparus aurata), Br J Nutr, 96, 830–39. cahu c l and zambinino infante j l (1994) Early weaning of sea bass (Dicentrarchus labrax) larvae with a compound diet: effect on digestive enzymes, Comp Biochem Physiol, 109, 213–22. cahu c l and zambonino infante j l (1995) Effect of the molecular form of dietary nitrogen supply in sea bass larvae: Response of pancreatic enzymes and intestinal peptidases, Fish Physiol Biochem, 14, 209–14. cahu c l and zambonino infante j l (1997) Is the digestive capacity of marine fish larvae sufficient for compound diet feeding? Aquac Int, 5, 151–60. cahu c and zambonino infante j l (2001) Substitution of live food by formulated in marine fish larvae, Aquaculture, 200, 161–80. cahu c, zambonino infante j l and barbosa v (2003) Effect of dietary phospholipid level and phospholipid: neutral lipid value on the development of sea bass (Dicentrarchus labrax) larvae fed a compound diet, Br J Nutr, 90, 21–8. canavate j p and fernandez-diaz c (1999) Influence of co-feeding larvae with live and inert diets on weaning the sole Solea senegalensis onto commercial dry feeds, Aquaculture, 174, 255–63. castell j d, bell j g, tocher d r and sargent j r (1994) Effects of purified diets containing different combinations of arachidonic and docosahexaenoic acid on survival, growth and fatty acid composition of juvenile turbot (Scophthalmus maximus), Aquaculture, 155, 149–64. chang q, liang m q, wang j l, chen s q, zhang x m and liu x d (2006) Influence of larval co-feeding with live and inert diets on weaning the tongue sole Cynoglossus semilaevis, Aquac Nutr, 12(2), 135–9. chey w y (1993) Hormonal control of pancreatic exocrine secretion, in Go V L W, Gardner J D, Brooks F P, Lebenthal E, Di Magno E P and Sheele G A (eds), The Pancreas; Biology, Pathology and Disease, Raven Press, New York, 403–24. conceiçao l e c, van der meeren t, verreth j a j, evjen m s, houlihan d f and fyhn h j (1997) Amino acid metabolism and protein turnover in larval turbot (Scophthalmus maximus) fed natural zooplankton or Artemia, Mar Biol, 129, 255–65. conceiçao l e c, grasdalen h and rønnestad i (2003) Amino acid requirements of fish larvae and post-larvae: new tools and recent findings, Aquaculture, 227, 221–32. cousin j c b, baudin-laurencin f and gabaudan j (1987) Ontogeny of enzymatic activities in fed and fasting turbot, Scophtalmus maximus L, J Fish Biol, 30, 15–33. curnow j, king j, partridge g, bosmans j and kolkovski s (2006a) The effect of Artemia and rotifer exclusion during weaning on growth and survival of barramundi (Lates calcarifer) larvae, Aquac Nutr, 12(4), 247–55. curnow j, king j, partridge g and kolkovski s (2006b) The effect of various co-feeding and weaning regimes on growth and survival in barramundi (Lates calcarifer) larvae, Aquaculture, 257, 204–13. dabrowski k (1979) The role of proteolitic enzymes in fish digestion, in StyczunskaJurewivcsk E, Jaspers T and Persoone E (eds), Cultivation of Fish Fry and its Live Food, Vol. 4, European Mariculture Society, Belgium, 107–26.

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tanaka m (1973) Studies in the structure and function of the digestive system of teleost larvae, D Agric Thesis, Kyoto University, Japan. teshima s i, ishikawa m, fudo y, alam m s, hernandez l h, koshio s and michael f r (2004) Effects of the formulation, sinking speed, and feeding method of diets on survival and growth of larval japanese flounder Paralichthys olivaceus and red sea bream Pagrus major fed microparticulate diets, World Aquaculture Symposium, Sydney. tesser m b, carneiro d j and portella m c (2005) Co-feeding of pacu, Piaractus mesopotamicus Holmberg (1887), larvae with Artemia nauplii and a microencapsulated diet, J Appl Aquac, 17(2), 47–59. thies c (2007) Microencapsulation of flavors by complex coacervation, in Lakkis J (ed.), Encapsulation and Controlled Release Technologies In Food Systems, Blackwell, Ames, IA, Chap 7. tovar-ramirez d, zambonino-infante j l, cahu c l, gatesoupe f j and vazquezsuarez r (2002) Dietary incorporation level of live yeast influences European sea bass (Dicentrarchus labrax) development, Aquaculture, 234, 415–27. vega-orellana o m, machado f d and kiyoko s (2006) Dourado (Salminus brasiliensis) larviculture: Weaning and ontogenetic development of digestive proteinases, Aquaculture, 252, 484–93. walford j, lim t m and lam t j (1991) Replacing live foods with microencapsulated diets in the rearing of seabass Lates calcarifer larvae: do the larvae ingest and digest protein-membrane microcapsules? Aquaculture, 92, 225–35. watanabe t and kiron v (1994) Prospects in larval fish dietetics, Aquaculture, 124, 223–51. watanabe t, kitajima c and fujita s (1983) Nutritional value of live organisms used in Japan for mass propagation of fish: a review, Aquaculture, 34, 115–43. watanabe t, izquierdo m s, takeuchi t, satoh s y and kitajima c (1989) Comparison between eicosapentaenoic and docosahexaenoic acids in terms of essential fatty acid efficacy in larval red seabream, Nippon Suisan Gakkaishi (Bull Japan Soc Scien Fish), 55(9), 1635–40. williot p, brun r, rouault t, pelard m and mercier d (2005) Attempts at larval rearing of the endangered western European sturgeon, Acipenser sturio (Acipenseridae), in France, Cybium, 29(4), 381–7. yúfera m, kolkovski s, fernandez-diaz c and thies, c (1998) Microencapsulated diets for fish larvae – current ‘state of art’, VII Bioencapsulation symposium – 20–23 November, Easton, MD. yúfera m, fernández-díaz c, pascual e, sarasquete m c, moyano f j, díaz f j, alarcón m, garcía-gallego m and parra g (2000) Towards an inert diet for firstfeeding gilthead seabream (Sparus aurata L.) larvae, Aquac Nutr, 6, 143–52. yúfera m, kolkovski s, fernandez-diaz c and dabrowski k (2003) Free amino acid leaching from protein-walled microencapsulated diet for fish larvae, Aquaculture, 214, 273–87. yúfera m, fernández-díaz c and pascual e (2005) Food microparticles for larval fish prepared by internal gelation, Aquaculture, 248, 253–62. zambonino-infante j l and cahu c (2001) Ontogeny of the intestinal tract of marine fish larvae, Biochem Physiol, 130C, 477–87. zambonino-infante j l and cahu c (2007) Dietary modulation of some digestive enzymes and metabolic processes in developing marine fish: applications to diet formulation, Aquaculture, 268, 98–105.

12 Aquaculture feeds and ingredients: an overview R. Hardy, University of Idaho, USA

Abstract: Aquaculture supplies half of the fisheries products consumed annually, and future global demand can only be supplied by increasing aquaculture production, which, in turn, requires more aquafeed. Fish meal and fish oil have traditionally been the primary ingredients in aquafeeds, but global production of these products is insufficient to support further growth of aquaculture at current use levels in feed formulations. Alternative ingredients must be increasingly used to supply significant proportions of protein and energy in aquafeeds, creating both challenges and opportunities for researchers and industry. The goal of researchers, industry, government organizations and non-government organizations is to promote sustainable aquaculture. To achieve this goal, sustainable feeds that support rapid, economical growth of farmed fish and result in safe, healthful fisheries products for consumers are required. Key words: sustainable aquaculture, fish feeds, fish meal, fish oil, alternative proteins.

12.1 Introduction Global aquaculture production increased by an annual percentage rate of 8.8 % between 1950 and 2004 (FAO, 2006), making it the fastest growing sector of animal production. Aquaculture production includes finfish, crustaceans, mollusks, amphibians, reptiles and plants; production of finfish in 2004 was 47.4 % of total aquaculture production of 59.4 million metric tons (mmt) or 28.156 mmt. During the same period, total fisheries landings from wild harvests averaged about 92 mmt. Of the total fisheries landings, an average of 64 mmt was used for direct human consumption and the remaining amount was used to produce fish meal and oil or used as feed for farmed fish without being made into fish meal. Between 1990 and 2005, an average

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of 25 mmt was used to produce fish meal and oil for use in animal and fish feeds, although it is important to note that yearly landings of fish for both direct human consumption and for fish meal and oil production vary from year to year. Aside from normal variations in landings from year to year, landings from capture fisheries have not increased since the mid-1990s and are not likely to increase beyond the current range of 89–98 mmt per year. Given the status of many of the world’s fisheries as fully or over-harvested, the only option to supply increasing consumer demand for fisheries products is by increasing aquaculture production. FAO estimates that aquaculture production may have to increase by as much as 40 mmt in the next 20+ years to maintain current world per-capita levels of fish consumption. While a large proportion of this increase is expected to involve species of fish not requiring direct feeding, a significant proportion (41.6 % according to FAO, 2006) will be in species of farmed fish that require feed inputs. Aquaculture feed production has increased from approximately 4 mmt in 1994 to 23 mmt in 2006, and is predicted to increase further to 36 mmt by 2015 (Tacon, 2009). This rate of increase is higher than the increase predicted for aquaculture production, the result of anticipated higher use of feed inputs in production of higher valued fish species. The significant increase in production of aquaculture feeds cannot occur without higher use levels of alternative protein sources in aquafeeds, both in absolute terms and in the percentages used in feed formulations. There is not enough fish meal produced each year to support the predicted amount of aquaculture feed needed in the future if the percentage of fish meal in aquaculture feeds is not reduced, and alternative protein sources supply the balance of protein that will be needed to support growth, health and welfare of farmed fish.

12.2 Sustainability of feed ingredients During the 1980s and 1990s, little thought was given outside of the research community to the issue of sustainability of feed ingredients used in aquaculture feeds. Researchers and feed manufacturers knew that annual global production of fish meal averaged about 6.2 mmt, but that in El Niño years, production could decrease by a million metric tons or more, depending upon the severity of the El Niño. In severe El Niño years, landings of anchovies in Peru and Chile, responsible for 30–40 % of global fish meal production, could drop severely. When production decreased, the price of fish meal increased, making it economically attractive to use higher percentages of alternative protein sources and lower percentages of fish meal in aquaculture feed formulations. However, when fish meal production increased after El Niño years and prices fell, use levels in aquaculture feeds returned to usual levels. This situation changed dramatically in 2006 when

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a combination of an El Niño-associated reduction in Peru’s fish meal production, decreased landings of several other species of fish used in fish meal production in other parts of the world associated with natural variability in stock abundance, and increasing purchases of fish meal by feed producers in China reduced supplies to the point where prices doubled from their former 30-year high price. This was a shock to aquaculture feed producers and a wake-up call to the aquaculture industry that the time had come to accept the fact that annual global production of fish meal was inadequate to sustain continued growth of aquaculture production unless fish meal use levels in feeds were lowered. Concerns about the dependence of finfish and crustacean producers on fish meal as the main protein source in aquaculture feeds have been expressed from many quarters for many years (Naylor et al., 2000), and researchers from around the globe have published literally hundreds of scientific papers dealing with alternative protein sources to fish meal for aquaculture feeds. As a result, a large body of knowledge exists to allow fish feed formulators to utilize a variety of alternative protein ingredients in aquafeeds to replace portions of fish meal. Nevertheless, the decision to replace portions of fish meal in aquaculture feeds with alternative protein sources is primarily an economic one. Until 2006, the price of fish meal coupled with its superior nutritional properties compared to alternatives favored the continued use of fish meal as the main protein source in aquafeeds despite well-publicized concerns about the ethics, sustainability or safety of doing so. Concerning ethics, many non-governmental organizations (NGOs) express concern about using fish to feed fish, by which they mean that fish meal used in aquafeeds is made from small pelagic fish obtained by commercial harvest. The issue is that for many species of farmed fish, the ratio of kg wild fish consumed to make fish meal to add to aquaculture feeds to the kg of farmed fish produced is between 2–3 : 1, depending on the species of farmed fish for which the comparison is being made and its life stage. When fish oil is included in the calculation, the ratio for species of fish fed high-lipid feeds, e.g., salmonids and marine fish, is even higher. Farming of these fish is seen as causing a net loss of fish in the world, with the implication that the poor suffer from this practice due to diversion of fish from human food to fish feeds used to raise expensive fish for consumers in developed countries where incomes are high. According to FAO (2006), the overall ratio across all finfish aquaculture of wild fish consumed to make fish meal to production of farmed fish is lower than 1 : 1. This is because a high proportion of farmed fish, such as carp, catfish and tilapia, are pond-raised fish that either consume natural food within ponds and therefore are not feed prepared feeds or are fed feeds that contain low levels of fish meal and very little fish oil. The ratio of wild fish consumed (via the feed) to production of fish exceeds 1 : 1,

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however, for salmon, trout, marine fish, shrimp and eels. Although the species of fish used to make fish meal are not major food sources for human consumption, there is increasing interest in using some species harvested to make fish meal for direct human consumption. On the other hand, according to FAO (2006), production of high-value fish through aquaculture production provides jobs and foreign exchange for many developing countries, and thus has a large, positive social impact. Ecological sustainability is another concern of NGOs who criticize aquaculture for its use of fish meal and oil from marine resources. Specifically, the issue expressed is that demands for fish meal to produce aquaculture feeds result in increasing and non-sustainable harvests of species of fish used to make fish meal, thus depleting these species of forage fish upon which many higher trophic level organisms in the marine environment depend. They emphasize the point that, although most of the fisheries which target forage fish for fish meal and oil production have strict harvest limits based upon calculations of sustainable yield, harvest limits do not consider large-scale ecosystem effects and the impacts of such harvests on higher trophic levels in the marine environment. On a global scale, landings of fish to produce fish meal have remained within a relatively narrow range since 1985, with the exception of El Niño years. This has occurred during a period of explosive growth of aquaculture production and increasing use of fish meal in aquaculture feeds, expressed on a metric ton basis. Since the 1980s, there has been little evidence that increased demand for fish meal by aquafeed producers has caused higher landings of fish species used in fish meal production, although there is evidence that landings of forage fish in SE Asia and China for direct feeding to farmed fish has increased in recent years (Tacon and Metian, 2008). Accurate information on the extent of this practice is difficult to obtain. Increased use of fish meal by the aquaculture feed sector has occurred because use levels by other sectors, mainly the poultry and swine feed industries, has declined. The net result has been diversion of fish meal from livestock and poultry feeds to aquaculture feeds. However, there is a general consensus that annual global production cannot supply enough fish meal for future needs unless the percentage of fish meal in aquafeed formulations decreases. Were fish meal levels in use in 2005 to remain unchanged, demand for fish meal in aquaculture feeds will reach annual production between 2015 and 2020 (IFFO, 2007). However, with increased fish meal substitution, annual production of fish meal will be sufficient to meet demand beyond 2020, at least. In some aquaculture systems, so-called trash fish are fed directly to farmed fish, and this has resulted in depletion of wild stocks in areas where such practices occur (Tacon et al., 2006). Clearly this practice is unsustainable. In fisheries, sustainability refers to the way in which harvests of wild stocks are managed to maintain stock abundance at healthy, sustained

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levels. Stocks of fish are subject to large variations in abundance unrelated to harvest pressure associated with environmental factors that determine spawning and year-class recruitment success (Hardy and Shepherd, 2009). As a result, harvest of such stocks must be managed conservatively to avoid compounding the effects of variable recruitment success with excessive harvest, thereby reducing stock abundance. Another factor that must be considered in stock management is the level of recruitment into spawning populations. Put simply, there must be sufficient escapement of maturing fish to perpetuate the stock. In the Western Hemisphere, Peruvian anchovy (Engraulis ringens) and gulf menhaden (Brevoortia patronus) are the two largest stocks harvested to produce fish meal and oil. Both stocks are managed to maintain sustainability by restricting harvest, and both have relatively long histories of stock assessment to document population abundance and variability in year-class strength (IFFO, 2007; Vaughan et al., 2007). Landings of the Peruvian anchovy are the highest of any species of fish in the world, ranging from 11 276 000 metric tons (mt) in 2000 to 6 204 000 mt in 2003, an Él Niño year. Extensive research on Peruvian anchovy stocks was conducted in the 1990s, leading to the establishment of strict harvest quotas and modified fishing techniques to reduce harvest of juvenile fish. Illegal fishing is minimized by aggressive surveillance methods and, as a result, stock biomass has remained healthy. Harvest rates of Peruvian anchovies have been adjusted to prevent overharvesting during fishing seasons when fish are less abundant (Shepherd et al., 2005).

12.3 Safety of farmed fish products from harmful residues and pollutants The safety of fisheries products is a topic of increasing interest to consumers and is a highly visible topic in the media. Concerns about the content of mercury in long-lived fish species, such as swordfish and large tuna, have resulted in recommendations that certain groups of consumers, e.g., pregnant women and children, limit intake of such fish (http://www.fda.gov/ fdac/features/2004/304_fish.html). Some species of fish have been found to contain relatively high levels of persistent organic pollutants (POPs) including PCBs, PBBs, and dioxin, especially fish from polluted areas, such as the Great Lakes and Puget Sound in Washington State (Cullon et al., 2008). Nearly all of the toxic compounds or elements found in wild or farmed fish are present as a consequence of high levels in their diet. Farmed fish are at risk of contamination if their feed contains contaminated ingredients, and studies have found elevated POP levels in farmed salmon from Northern Europe compared to levels present in wild salmon from Alaska (Hites et al., 2004). It is essential to understand this risk to ensure that feeds of farmed fish are formulated and produced to be virtu-

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ally free of these contaminants, thereby ensuring that farmed fish are among the safest products available to consumers compared to wild fish and other sources of protein. POPs are lipophilic compounds and are concentrated in residual lipid of fish meal and in fish oil, the two ingredients contributing nearly all POPs to farmed fish feeds. Strategies to lower POP levels in aquaculture feeds include sourcing fish meal and fish oil from areas where POP levels are low, such as Peru and Alaska, using fish meal and oil that has been stripped of POPs by cleaning with activated carbon, and/or replacing a portion of fish meal and fish oil in feeds with protein concentrates and oils sourced from grains or oilseeds. Recent studies indicate that farmed salmon from Canada contain low levels of POPs, similar to or less than wild salmon (Ikonomou et al., 2007), the result of using clean ingredients and substituting plant oils for a portion of fish oils in feeds for salmon.

12.4 Categories of environmental pollutants and residues comprising risks to the safety of farmed fish products Safety of fish products involves a number of potential hazards, some of which are common to both farmed and wild products. Hazards include (i) methylmercury, (ii) POPs, (iii) microbiological hazards, (iv) naturally-occurring toxins and (5) chemotherapeutics. Methylmercury and POP contamination of fish tissues occurs mainly via the feed, whereas microbiological hazards are primarily associated with consumption of uncooked fisheries products. Naturally-occurring toxins are almost exclusively a problem with wild fisheries products, mainly shellfish, whereas contamination of fisheries products with chemotherapeutants is mainly a problem with farmed fish products. In this chapter, attention will be directed to safety issues associated with farmed fish that are subject to control through choice of feed ingredients, e.g., methylmercury, POPs and chemotherapeutants.

12.4.1 Methylmercury Mercury exists in several forms in the environment, but the form of concern in foods, including fisheries products, is methylmercury. Methylmercury (CH3Hg+) is a product of bacterial synthesis that passes through the food chain to fish and other higher trophic level organisms. Methylmercury is concentrated through trophic levels, with the potential for bioaccumulation to relatively high concentrations in large, long-lived top marine carnivores. According to the US Food and Drug Administration (http://www. cfsan.fda.gov/~frf/sea-mehg.html), levels of methylmercury are highest in king mackerel, shark, swordfish and tilefish from the Gulf of Mexico (0.730, 0.988, 0.976 and 1.450 mg/kg, respectively). Levels are low in small, short-

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lived fish, such as anchovies (0.043 mg/kg), herring (0.044 mg/kg) and sardines (0.016 mg/kg), typical species used in fish meal production. Action levels for methylmercury in fish products vary among governmental entities. The FDA level is 1.0 mg/kg, whereas the EU action level is 0.5 mg/kg. The US Environmental Protection Agency (EPA) recommends 0.3 mg/kg as an action level based upon a reference dose of 0.1 μg/kg body weight per day of methylmercury intake for an average adult. To put this in perspective, an average adult could consume 273 kg of rainbow trout fillets per year without exceeding the EPA action level. Methylmercury levels in freshwater fish species are typically low, except in areas where pollution increases exposure. Such areas include lakes or reservoirs downwind from gold mines or coal-fired electrical plants. In such areas, longlived carnivorous fish can contain levels of methylmercury significantly above levels of concern. Farmed fish are fed feeds that are mainly composed of plant products, such as soybean meal, corn gluten meal and ground corn or wheat, plus fish meal, fish oil and rendered animal products. As mentioned above, fish meal is produced from small, short-lived pelagic fish, such as anchovies, sardines, capelin and menhaden, all of which are low in methylmercury content. None of the plant- or animal-derived feed ingredients used in aquaculture feeds is considered to be a major or even a minor source of methylmercury. Unsurprisingly, farmed fish contain negligible levels of methylmercury. The only way that fish feeds could conceivably contain significant amounts of methylmercury would be if they contained fish meal produced from trimmings of large tuna, marlin or swordfish, or if a localized population of freshwater fish from an area contaminated with methylmercury were used to produce fish meal.

12.4.2 Persistent organic pollutants (POPs) POPs are organic chemicals that persist in the environment, bioaccumulate and bioconcentrate up the food chain, and are toxic to humans, wildlife and the environment. Most are chlorinated compounds, such as polychlorinated biphenyls (PCB) or polybrominated diphenyl esters (PBDEs), derived from a brominated flame retardant. Some are pesticides, others are industrial chemicals and a few are by-products of burning or incineration. Pesticides include aldrin, chlordane, DDT, dieldrin, endrin, heptachlor, mirex and toxaphene. Industrial chemicals are hexachlorobenzine and PCBs, whereas those produced by combustion are dioxins (polychlorinated dibenzo-p-dioxins) and furans (polychlorinated dibenzo-p-furans). PCBs can also be produced by combustion. The compounds of concern in fisheries products are dioxins, dioxin-like compounds and PCBs. All are lipophilic, meaning that they are lipid-soluble and found in the lipid fraction of tissues. POPs are widespread in the environment and are present in detectable levels (ppb or ng/kg) in nearly all fish, birds and terrestrial animals.

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POPs are considered a potential threat to humans if levels in food are high. Due to the lipophilic nature of POPs, they tend to accumulate in fatty tissue of fish, such as the liver. Muscle tissue of most fish consumed by humans is relatively low in lipid, except for certain species, notably salmon, trout and sablefish among farmed species. The acute toxicity of POPs is well known, but the long-term effects of low exposure via the diet are not well characterized. Attempts to estimate risk are complicated by difficulties associated with epidemiological studies, establishing dose–effects relationships and/or threshold levels, and determining if adverse effects of low-level POP exposure are limited to pre-natal exposure or carry over to post-natal exposure. Another complication is that POPs such as PCBs and Polybrominated biphenyls (PBBs) each have a huge number of congeners, usually with varying degrees of unknown toxicities. Animal studies are helpful in identifying clinical effects of acute exposure, but results are not necessarily directly applicable to humans. Chronic exposure studies with animals are even more difficult to apply to human toxicity. The wisdom of limiting dietary intake of POPs is not in doubt, but the level of risk associated with POP intake at low levels over long periods is unclear. In regard to fisheries products, it is important to remember that POP intake is dictated not only by the concentration of POPs in food items, but also by intake (quantity consumed) of food items. In terms of average yearly accumulation of POPs by US citizens, the percentage accumulation that can be attributed to fish products is about 8 % for the general population. This contrasts with 37 % of yearly intake from meat and meat products, with the balance coming from dairy products and other foods. Annual intake levels of meat and dairy products are much higher than that of fish. Despite the relatively low contribution to the total yearly intake of POPs from fish for the average person, fish do present a risk in some situations because the concentration of POPs differs significantly among species and among locations, resulting in variable concentrations of POPs in fish. Thus, consumers eating large quantities of certain fish will have higher intakes of POPs than average. For example, salmon from the Great Lakes have significantly higher concentrations of POPs than do salmon from Alaska. Wild salmon caught in Puget Sound have higher concentrations of POPs than farmed salmon from North or South America. Among the five species of salmon, long-lived, large species like chinook salmon (average life span 4–5 years) have higher concentrations than shorter-lived, small species like pink salmon (two-year life cycle). Compared to common food items, however, neither species is near the top of the list with respect to PCB concentration. The same holds true for farmed salmon, but, in contrast to wild salmon, farmed salmon POP intake can be lowered by changing the ingredients used to produce their feed, thereby reducing POP levels in fillets to very low levels (Ikonomou et al., 2007).

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PCBs and other POPs are found in trace amounts in nearly all feed ingredients, but higher levels are found in marine protein and oil, notably oil. Therefore, as mentioned, the nutritional approach to reducing intake of POPs in farmed fish is to reduce levels of marine protein and oil in feed, or to use marine proteins and oils that are low in POPs as fish feed ingredients. Recently, new technology has been employed by the fish meal and oil industry to remove POPs from fish meal and oil using activated carbon (http://www.999.dk/). This approach reduces POP levels in fish meal and oil, and greatly reduces the exposure to farmed fish to POPs from these ingredients. Past studies concerning the presence of elevated levels of POPs in farmed salmon (Hites et al., 2004) do not reflect the current situation in farmed salmon (Ikonomou et al., 2007). Given the known benefits of fish consumption associated with omega-3 fatty acid intake and other dietary factors, the benefits of fish consumption have been estimated to outweigh the risks for most consumers by a factor of 100 to 1 (Mozaffarian and Rimm, 2006).

12.4.3 Chemotherapeutant residues In the USA and EU, the use of chemotherapeutics in aquaculture production is strictly limited and strongly monitored. As a result, farmed fish products are virtually free of chemotherapeutic residue. In other countries, however, chemotherapeutic use is largely unregulated in aquaculture production, and residues can occur (FAO, 2006). To the extent that many chemotherapeutics are administered via the feed, preventing residues is a feed issue. However, it is primarily a compliance issue rather than a production issue. For chemotherapeutics that are allowed, strict rules are in place in many countries concerning withdrawal periods prior to harvest to eliminate the risk of chemotherapeutic contamination of farmed fish products. In countries having well-developed and technologically advanced aquaculture industries, such as Norway, Scotland, Canada and the USA, disease prevention is the preferred method of health management and, as such, the use of vaccines and feed additives to stimulate the immune system of farmed fish has largely replaced antibiotic therapy. In this context, the use of immune stimulants and probiotics to enhance fish health deserves mention as a nutritional strategy to improve the quality of farmed fish products.

12.5 Alternate protein and lipid sources The list of candidate ingredients to replace most or all of the fish meal in aquaculture feeds includes protein concentrates from soybeans, canola (rapeseed), grains, peas and lupins, and also single-cell proteins (bacteria and yeasts) grown on carbon sources such as methane. Each of

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these candidate ingredients possesses characteristics, including cost, essential nutrient limitations, presence of antinutrients or presence of nonnutritive constituents, that have limited their use (Dong et al., 2000). The pros and cons of various plant protein ingredients were recently reviewed (Gatlin et al., 2007); the narrative below is an updated summary of that review. Rendered products are excluded from this discussion because they are not truly alternative ingredients, having been used for decades in aquafeeds.

12.5.1 Soybean products Soybean production increased tremendously since the 1980s, but, in the past few years, production has leveled off as farmers have planted more hectares of corn in the USA to supply the growing ethanol business, and production in other parts of the world has been affected by declining water tables (China) and poor weather conditions (Brazil and Argentina). Soybean meal is presently used in aquafeeds for many species of farmed fish (Storebakken et al., 2000). Omnivorous species, such as catfish and tilapia, have long consumed feeds in which soybean meal is the primary protein source, similar to poultry feeds. However, the use of soybean meal in feeds for carnivorous species of farmed fish has been limited by the presence of antinutrients and by the high amounts of non-digestible compounds in soybean meal. Currently, there are two main problems that limit the use of soybean meal in aquafeeds: (i) constituents that cause inflammation in the intestine; and (ii) non-soluble carbohydrates, e.g., stachyose and raffinose, that are not digestible by carnivorous fish and influence the water content of feces and rate of passage of feed in the gastrointestinal tract. The intestinal inflammation problem appears to be a food intolerance resulting in enteritis, with associated negative effects on nutrient assimilation, feed intake and growth rates of fish, although the etiology is not yet certain (Van den Ingh et al., 1991; Bæverfjord and Krogdahl, 1996; Bakke-McKellup et al., 2000). It is most commonly seen in salmon and trout. The non-soluble carbohydrate problem limits the amount of soybean meal that can be used in feeds. Soybean meal can contain up to 20 % non-soluble carbohydrates, and high inclusion levels of soybean meal constrain feed formulations by limiting bioavailable dietary energy content. Other problems with soybean meal, e.g., trypsin inhibitor activity, phytic acid, saponins, lectins, can generally be overcome by ingredient processing or use of supplements, such as the microbial enzyme phytase. A long-term solution to the non-soluble carbohydrate problem with conventional soybean meal may come about through the efforts of plant breeders. Work is underway to develop varieties of soybean that contain low levels of selected non-soluble carbohydrates.

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Soy protein concentrate is an excellent alternative potential substitute for fish meal in aquafeeds, as demonstrated by numerous research studies (Kaushik et al., 1995; Stickney et al., 1996; Mambrini et al., 1999). Until recently, the price of soy protein concentrate relative to that of fish meal prevented its use in aquaculture feeds, except certain specialty feeds. However, price relationships have changed with the increasing cost of fish meal, nearly double that of 2005. Currently the price per unit protein for soy protein concentrate is similar to that of fish meal. Soy protein concentrate does not cause distal enteritis in salmon or other fish, nor does it contain appreciable amounts of non-soluble carbohydrates. Aside from the relatively high level of phytic acid, which can be overcome by use of dietary phytase supplementation, there are no negative attributes of soy protein concentrate other than a low level of the essential amino acid methionine that prevents its use in aquafeeds as a partial substitute for fish meal. The methionine problem can be rectified by supplementing feeds with feed-grade DL-methionine or by formulating feeds to contain other protein sources that balance the amino acid profile of the finished feeds.

12.5.2 Corn products Corn gluten meal is widely used in salmon feeds, albeit at relatively low inclusion levels. Corn gluten meal contains a minimum of 60 % crude protein, mainly because other by-products of corn processing, e.g., fiber, starch overflows, resistant starch attached to fiber and condensed solubles from steep tanks, are added back to the protein fraction by processors. If these by-products are excluded, the resulting corn gluten meal contains 72–80 % crude protein, much more in line with the needs of the fish feed industry in terms of a fish meal-equivalent ingredient. Until recently cost has been an issue with this approach of making high-protein corn gluten meal. However, as mentioned above, pricing relationships among protein sources for aquafeeds have changed dramatically since 2006. A large commercial company recently began marking a high-protein corn gluten meal to the aquafeed industry. Corn gluten meal is, of course, deficient in lysine, making it a natural ingredient to blend with soy protein (deficient in methionine) to produce a more complete amino acid balance.

12.5.3 Wheat and barley Wheat and barley are similar in terms of nutritional content, but wheat is much more commonly used in aquafeeds than barley due to the relatively high fiber content of regular, hulled barley. Hull-less varieties of barley are much better suited as starting material from which to produce a protein concentrate similar to wheat gluten meal, an existing commodity

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mainly used as a component of human foods. As such, wheat gluten is too expensive to be considered for use in production aquafeeds, despite its high protein content, high protein digestibility and other positive attributes. A potential market exists for feed-grade wheat or barley gluten meal, providing it could be produced for a price that was competitive with fish meal.

12.5.4 Canola/rapeseed products Canola meal is used in feed formulations at limited levels where it is available. Canola meal contains 35 % crude protein, making it too low to be used in high percentages in high-energy, nutrient-dense aquafeeds. Rapeseed protein concentrate has been shown to be an excellent ingredient in feeds for salmonids and certain marine species, but it is not widely available (Teskeredzic et al., 1995; Kissil et al., 1997). Amino acids and palatability enhancing supplements are needed in feeds containing high amounts of rapeseed protein concentrate.

12.5.5 Distillers products Ethanol production in the USA is increasing rapidly, and this increase is generating large quantities of distiller’s by-products, known as distiller’s dried grains (DDG) and distiller’s dried grains with solubles (DDGS). DDG and DDGS contain 32–25 % protein but, unfortunately, they also contain high levels of fiber, limiting their use in aquafeeds to feeds for omnivorous species such as tilapia and catfish. New developments in ethanol production in which protein, starch and fiber are fractionated prior to ethanol production from the starch fraction provide the opportunity to recover the protein for use in livestock and aquafeeds. At present, this approach is not widely used, but recent dramatic increases in the cost of corn have altered the economics of ethanol production, making it necessary for ethanol producers to capture higher returns from the non-starch fraction of corn, making it likely that ethanol plants will be modified to add equipment to allow recovery of the protein and fiber fraction prior to starch fermentation, rather than after as is now the practice.

12.5.6 Single-cell protein products Microorganisms can be grown on a wide array of carbon sources, including methane, and efforts in Norway have led to development of a novel singlecell protein from bacteria grown on methane, called ‘bio-protein’. Feeding trials using feeds in which bio-protein has replaced fish meal have been conducted with Atlantic salmon with promising results (Skrede et al., 1998; Storebakken et al., 2004; Berge et al., 2005; Aas et al., 2006). Commercial

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products may appear in the next few years and, if commercialization is successful, their use in feeds for farmed salmon and other marine species is a certainty.

12.5.7 Peas and lupins Pea and lupin protein concentrates have been produced on a limited scale for experimental use by air-classification and wet milling-extraction. Feeding trials with these products with salmonids have been promising (Allan and Rowland, 1994; Thiessen et al., 2003; Glencross et al., 2004).

12.5.8 Seafood processing waste products Globally, the quantity of seafood processing waste generated each year is nearly equal to the amount of fish captured to produce fish meal and fish oil (Kilpatrick, 2003). In many parts of the world, processing waste is converted into fish meal and used in livestock and aquafeeds. Alaska generates tremendous quantities of seafood processing waste, and a large proportion of material recovered in land-based factories is converted into fish meal. A lower proportion of processing waste from shipboard processing is utilized. As demand for fish meal increases average prices, the economics of recovery and utilization of seafood processing waste will become more favorable, and much of this material will be used in aquafeeds, not necessarily as a primary protein source, but more likely in product forms designed to augment feeds based primarily on plant-derived feed ingredients (Hardy, 2003). Likely uses are as ingredients to overcome amino acid limitations in plant-derived feed ingredients, palatability enhancing materials, products to enhance growth and immunocompetence, and oil supplements to maintain high levels of omega-3 fatty acids in farmed fish products.

12.5.9 Plankton and krill The incredible biomass of copepods, Euphausids (krill) and amphipods in the sea has led to suggestions that this material be harvested to produce feed ingredients for use in aquafeeds (Langmyhr and Mjelde, 2005). Utilization of these resources is likely to stimulate controversy as it amounts to harvesting organisms from lower trophic levels than is presently practised and may be construed as being detrimental to marine food webs. Products made from plankton and krill are high in protein, essential fatty acids and astaxanthin, the carotenoid responsible for the pink-red color of salmon muscle. Further, they are likely to be highly palatable and thus suited for use in feeds containing high levels of oilseed proteins that typi-

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cally are lower in palatability than fish meal-based feeds for farmed marine species.

12.6 Future trends In mid-2008, prices for grains, oilseeds, fish meal and fish oil were at or near historical highs. For example, until 2006 the 30-year average price per ton of corn was $95. However, the price was $224 in mid-2008. Similar increases were seen in prices for soybeans, wheat and in products used in the feed industry produced from these commodities. Similarly, the price of fish meal, historically, ranged between approximately $350 and $750 per ton, but in the mid-2008 time period, the price was $1220. Fish oil was priced at $2200 per ton, up from $600 a few years ago. These disruptions in price have been challenging for the livestock and aquafeed industries, but they are also stimulating the development of alternatives as the new pricing structure relative to fish meal and fish oil makes alternatives more economically rewarding to producers. Until recent years, fish meal and fish oil were the ingredients of choice to supply protein and energy in aquafeeds. Now, their high cost is changing their role in aquafeeds from primary supplies of protein and energy to supplements that are added to balance amino acids and supply other essential nutrients, in the case of fish meal, and omega-3 fatty acids to maintain healthy levels in farmed fish products for the consumer in the case of fish oil. Future trends are likely to follow the current trend of reducing the percentages of fish meal and fish oil in aquafeeds, creating opportunities for a range of new aquafeed ingredients designed either to provide the majority of amino acids and energy in aquafeeds, to provide other nutritional needs of the fish, or to stimulate feed intake.

12.7 Sources of further information and advice • FAO: http://www.fao.org/fishery • USDA National Agricultural Statistics Service: www.nass.usda.gov/ index.asp • World Aquaculture Society: https://www.was.org/ • Aquafeed.com: http://www.aquafeed.com/ • International Aquafeed Information Portal: http://aquaculturenews. aquafeed.co.uk/ • AllAboutFeed.net: http://www.allaboutfeed.net/Aquaculture • Seafood Choices, Balancing Benefits and Risks edited by Nesheim and Yaktine (2007), and prepared by the Committee on Nutrient Relationships in Seafood: Selections to Balance Benefits and Risks, Food and

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Nutrition Board, Institute of Medicine of the National Academies. Available at www.nap.edu

12.8 References aas t s, grisdale-helland b, bendik f, terjesen b f and helland s j (2006) Improved growth and nutrient utilisation in Atlantic salmon (Salmo salar) fed diets containing a bacterial protein meal, Aquaculture, 259, 365–76. allan g l and rowland s j (1994) The use of Australian oilseeds and grain legumes in aquaculture diets, in Chou L M, Munro A D, Lam T J, Chen T W, Cheong L K K, Ding J K, Hooi K K, Khoo H K, Phang V P E, Shim K F and Tan C H (eds), The Third Asian Fisheries Forum, Manila, Asian Fisheries Society, 667–70. bæverfjord g and krogdahl å (1996) Development and regression of soybean meal induced enteritis in Atlantic salmon distal intestine. A comparison with the intestines of fasted fish, J Fish Dis, 19, 375–87. bakke mckellep a m, press c m, bæverfjord g, krogdahl å and landsverk t (2000) Changes in immune and enzyme histochemical phenotypes of cells of the intestinal mucosa of Atlantic salmon (Salmo salar L) with soybean-meal induced enteritis, J Fish Dis, 23, 115–27. berge g m, bæverfjord g, skrede a and storebakken t (2005) Bacterial protein grown on natural gas as protein source in diets for Atlantic salmon, Salmo salar, in saltwater, Aquaculture, 244, 233–40. cullon d l, yunker m b, alleyne c, dangerfield n j, o’neill s, whiticar m j and ross p s (2008) Persistent organic pollutants in Chinook salmon (Oncorhynchus tshawytscha): implications for resident killer whales of British Columbia and adjacent waters, Environ Toxicol Chem, 28, 148–61. dong f m, hardy r w and higgs d a (2000) Antinutritional Factors, in Stickney R R (ed.), Encyclopedia of Aquaculture, New York, Wiley, 45–50. fao (2006) State of world aquaculture 2006, FAO Fisheries Technical Paper No. 500, Rome, Food and Agriculture Organization of the United Nations. gatlin d m iii, barrows f t, bellis d, brown p, campen j, dabrowski k, gaylord t g, hardy r w, herman e, hu g, krogdahl å´ , nelson r, overturf k, rust m, sealey w, skonberg d, souza e, stone d, wilson r and wurtele e (2007) Expanding the utilization of sustainable plant products in aquafeeds – a review, Aquac Res, 38(6), 551–79. glencross b, evans d, hawkins w and jones b (2004) Evaluation of dietary inclusion of yellow lupin (Lupinus luteus) kernel meal on the growth, feed utilisation and tissue histology of rainbow trout (Oncorhynchus mykiss), Aquaculture, 235, 411–22. hardy r w (2003) Marine byproducts for aquaculture use, in Bechtel P J (ed.), Advances in Seafood Byproducts, Fairbanks, Alaska Sea Grant College Program (AK-SG-03-01), 141–52. hardy r w and shepherd c j (2009) Sustainable marine resources for organic aquatic feeds, World Aquac (in press). hites r a, foran j a, carpenter d o, hamilton m c, knuth b a. and schwager s j (2004) Global assessment of organic contaminants in farmed salmon, Science, 303, 226–9. iffo (2007) The production of fishmeal and fish oil from Peruvian anchovy, IFFO Update No. 183, Sept/Oct, St Albans, International Fishmeal and Oil Organization. ikonomou m g, higgs d a, gibbs m, oakes j, skura b, mckinley s, balfry s k, jones s, withler r and dubetz c (2007) Flesh quality of market-size farmed and wild British Columbia salmon, Environ Sci Technol, 41, 437–43.

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kaushik s j, cravedi j p, lalles j p, sumpter j, fauconneau b and laroche m (1995) Partial or total replacement of fish meal by soybean protein on growth, protein utilization, potential estrogenic or antigenic effects, cholesterolemia and flesh quality in rainbow trout, Oncorhynchus mykiss, Aquaculture, 133, 257–74. kilpatrick j s (2003) Fish processing waste: opportunity or liability, in Bechtel P J (ed.), Advances in Seafood Byproducts, Fairbanks, Alaska Sea Grant College Program (AK-SG-03-01), 1–10. kissil g wm, lupatsch i, higgs d a and hardy r w (1997) Preliminary evaluation of rapeseed protein concentrate as an alternative to fish meal in diets for gilthead seabream (Sparus aurata), Isr J Aquac-Bamidgeh, 49, 135–43. langmyhr e. and mjelde a (2005) Plankton as a new feed source for the aquaculture industry, Int Aquafeed, 8, 19–25. mambrini m, roem a j, cravedi j p, lalles j p, and kaushik s j (1999) Effects of replacing fish meal with soy protein concentrate and of DL-methionine supplementation in high-energy, extruded diets on the growth and nutrient utilization of rainbow trout, Oncorhynchus mykiss, J Anim Sci, 77, 2990–9. mozaffarian d and rimm e b (2006) Fish intake, contaminants, and human health: Evaluating the risks and the benefits, J Am Med Assoc, 296, 1885–99, abstract available at http://jama.ama-assn.org/cgi/content/short/296/15/1885, accessed January 2009. naylor r l, goldberg r j, primavera j h, kautsky n, beveridge m c m, clay j, folke c, lubchenco j, mooney h. and troell m (2000) Effect of aquaculture on world fish supplies, Nature, 405, 1017–24. nesheim m c and yaktine a l (eds) (2006) Seafood Choices: Balancing Benefits and Risks, Washington, D C, National Academies Press. shepherd c j, pike i h and barlow s m (2005) Sustainable feed resources of marine origin, Eur Aquac Soc Special Pub No. 35, 59–66. skrede a, berge g m, storebakken t, herstad o, aastad k g and sundstol f (1998) Digestibility of bacterial protein grown on natural gas in mink, pigs, chicken and Atlantic salmon, Salmo salar, in freshwater, Anim Feed Sci Technol, 76, 103–16. stickney r r, hardy r w, koch k, harrold r, seawrigh, d and massee k c (1996) The effects of substituting selected oilseed protein concentrates for fish meal in rainbow trout diets, J World Aquac Soc, 27, 57–63. storebakken t, refstie s and ruyter b (2000) Soy products as fat and protein sources in fish feeds for intensive aquaculture, in Drackley J K (ed.), Soy in Animal Nutrition, Savoy, I L, Fed Anim Sci Soc, 127–70. storebakken t, bæverfjord,g, skrede a, olli j j and berge g m (2004) Bacterial protein grown on natural gas in diets for Atlantic salmon, Salmo salar, in freshwater, Aquaculture, 241, 413–25. tacon a j g and metian m (2008) Global overview on the use of fish meal and fish oil in industrially compounded aquafeeds: trends and future prospects, Aquaculture, 285, 146–58. tacon a j g and metian m (2009) Fishing for aquaculture: non-food use of small pelagic forage fish – a global perspective, Rev Fish Sci, 17, 305–17. tacon a j g, hasan m r and subasinghe r p (2006) Use of Fisheries Resources as Feed Inputs to Aquaculture Development: Trends and Policy Implications, FAO Fisheries Circular No. 1018. Rome, Food and Agriculture Organization of the United Nations. teskeredzic a, higgs d a, dosanjh b s, mcbride j r, hardy r w, beames r m, jones j d, simell m, vaara t and bridges r b (1995) Assessment of undephytinized and dephytinized rapeseed protein concentrate as sources of dietary protein for juvenile rainbow trout (Oncorhynchus mykiss), Aquaculture, 131, 261–77.

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thiessen d l, campbell, g l and adelizi, p d (2003) Digestibility and growth performance of juvenile rainbow trout (Oncorhynchus mykiss) fed with pea and canola products, Aquac Nutr, 9, 67–75. van den ingh t s g a m, krogdahl å, olli j j, hendrix h g c j m and koninkx j g j f (1991) Effects of soybean containing diets on the proximal and distal intestine in Atlantic salmon (Salmo salar): a morphological study, Aquaculture, 94, 297–305. vaughan d s, shertzer k w and smith j w (2007) Gulf menhaden (Brevoortia patronus) in the U.S. Gulf of Mexico: fishery characteristics and biological reference points for management, Fish Res, 83(2–3), 263–75.

13 Ingredient evaluation in aquaculture: digestibility, utilisation and other key nutritional parameters B. Glencross, CSIRO Marine and Atmospheric Research, Australia Abstract: The evaluation of feed ingredients is a crucial part of the feed development process for most aquaculture species. In evaluating ingredients for use in aquaculture feeds there are several important knowledge components that should be understood to enable the practical use of any particular ingredient in a feed formulation. The three primary knowledge sets required are: (i) ingredient digestibilities, (ii) ingredient palatability, and (iii) ingredient nutrient utilisation potential and/or interference of utilisation potential. There is a range of methodological considerations for each of these three knowledge sets. This includes consideration of diet design, feeding strategies, the faecal collection methods and methods of calculation, all of which have important implications for the determination of the digestible value of nutrients from any ingredient. There are also several ways in which palatability of ingredients can be assessed, usually based on variable inclusion levels of the ingredient in question in a reference diet and feeding of those diets under an apparent satietal or self-regulating feeding regimes. However, the design of the diets, the parameters of assessment and the feeding regime can all be subject to variation depending on subtleties of the experimental design. Factors relating to feed intake are clearly the key performance criteria in palatability assessments, and it is important that such experiments maintain sufficient stringency to allow some self-discrimination of the test feeds by the fish. The third part tests the potential of fish to use nutrients from the test ingredient, or defines factors that interfere with that process. This is arguably the most complex and variable part of the ingredient evaluation process. However, despite its complexity, it is crucial to be able to discriminate effects on feed intake from effects on utilisation of nutrients from ingredients (for growth and other metabolic processes). To allow an increased focus on nutrient utilisation by the animals, there are several experimental strategies that can be adopted based on variations in diet design and feeding regime used. Other optional evaluation criteria include factors such as ingredient functionality, gene and/or protein expression, the influence on immune status and effects on sensory qualities which are also important considerations in determining the value of ingredients in aquaculture feed formulations. Key words: plant proteins, fishmeal replacement, raw materials, ingredients, methodology, techniques.

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13.1 Introduction To reduce the cost of diets for aquaculture species there is a need to be able to use a wide range of alternative ingredients, which can satisfy formulation constraints for the specific nutrient, energy and processing requirements of the intended diet type. However, prior to the use of any particular ingredient in any specific formulation it is critical for that ingredient to undergo evaluation to be able to consider its nutritional limitations. This nutritional evaluation process has several key facets that need to be considered in order to provide a clear indication of the potential that any ingredient may have for use in an aquaculture feed. This ingredient evaluation process has been reviewed in detail by Glencross et al. (2007a) and this chapter is largely an abridged version of that review. 13.1.1 Ingredient risk management Being too reliant on any one ingredient presents considerable risk to the feed formulation and manufacturing process. This risk includes supply and price volatility, and also quality variability and the risk of contaminants (Glencross et al., 2007a). As a strategy to reduce risk, the use of alternatives to fish meal and oil in aquaculture diets is an important option. Indeed, substantial effort has been spent evaluating a wide range of alternatives to fish meals and fish oils for use in aquaculture diets (Gatlin et al., 2007). Those ingredients can generally be classified into those being derived from either plant origin or terrestrial animal origin. Plant/grain-derived protein resources have had a substantially greater amount of evaluation undertaken on their applicability to aquaculture diets than other resources, principally because of the belief in their greater sustainability and lower health risks than other alternatives. Some of these include: soybean meals, protein concentrates and oils (Kaushik et al., 1995; Refstie et al., 1998; Glencross et al., 2004b), canola meals, protein concentrates and oils (Higgs et al., 1982; Burel et al., 2000; Glencross et al., 2004a) and lupin meals and protein concentrates (Burel et al., 1998; Glencross et al., 2005; Refstie et al., 2006). The extent of different plant protein options has been well reviewed by Gatlin et al. (2007). Terrestrial animal ingredients have also had a substantial amount of research on their application to aquaculture feeds. Some of the evaluated ingredients include resources such as rendered bovine, ovine and porcine meals (Stone et al., 2000; Sugiura et al., 2000; Williams et al., 2003a), blood meals (Bureau et al., 1999; Allan et al., 2000) and poultry meals (Bureau et al., 2000; Nengas et al., 1999). 13.1.2 Components to ingredient evaluation Primarily there are three essential components to ingredient evaluation which must be completed to provide a basis for being able to use a specific

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ingredient. These include assessments on the digestibility, palatability and nutrient utilisation (growth) responses of the animal to each ingredient (Glencross et al., 2007a). There are several other components that can also be considered to improve the confidence in the potential applicability of an ingredient, but these three components provide the foundation. Ingredient digestibility is the measurement of the proportion of energy and nutrients that an animal can obtain from a particular ingredient through its digestive and absorptive processes (Maynard and Loosli, 1969). There are several methods that have been used to determine diet and ingredient digestibilities in aquaculture species, and each of these methods has some key strengths and weaknesses that need to be considered. However, the principle act of feeding a diet to an animal with a defined component of a test ingredient is one of the most important experimental acts that can be undertaken to define the value of any ingredient. Digestibility should not be confused with availability, which is a measure of the amount of an absorbed (digested) nutrient or energy that an animal can effectively use for growth, and which is determined based on both digestibility and nutrient/energy retention studies. The determination of ingredient palatability is another key component of knowledge required about an ingredient before it can be successfully used. Irrespective of how digestible the nutrients and energy from an ingredient might be if the ingredient reduces feed intake then it may have limited value. A range of methods has been used to explore feed intake issues in aquaculture feeds and, by inference, diet and ingredient palatability (Juell, 1991; Helland et al., 1996; Boujard and Le Gouvello, 1997). Some of these methods are examined in this chapter. The determination of growth and nutrient utilisation as a function of the incorporation of any specific ingredient is perhaps the most complex step in the ingredient evaluation process. This complexity is largely related to the wide variety of factors that may impact on growth and nutrient utilisation (Cho and Kaushik, 1990; Booth and Allan, 2003; Glencross et al., 2007a). While studies examining the weight gain associated with different diets form the basis of most assessments, there are additional methods that can be employed to examine issues relating to nutrient utilisation.

13.1.3 Consolidating the evaluation process There are additional studies that can be used to reinforce the ingredientevaluation process beyond the three key elements of digestibility, palatability and utilisation. Examination of gene and protein expression as a consequence of ingredient use has exciting potential to define the molecular responses of an animal to those ingredients. This branch of nutritional science has been branded ‘nutrigenomics’ and is now beginning to show elements of its potential in fish nutrition research (Panserat et al., 2007).

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The use of biochemical, histological and sensory assays in expanding on the assessment of ingredient use has been undertaken for some time. This approach has shown things such as the inhibition of thyroid hormone production with the use of rapeseed meal, changes in cellular structure of the gut with the use of soybean meal, and changes in the sensory qualities of fish fed rapeseed oils instead of fish oil (Thommasen and Rosjo, 1989; Burel et al., 2001; Uran et al., 2007). Each parameter provides important additional information that contributes to the knowledge used to make decisions on feed ingredients. Often regarded as a functionality assessment, the effect that an ingredient can have on the physical qualities of a feed can be just as important as its nutritional properties in some cases (Thomas and Van der Poel, 2001; Hawkins et al., 2008). Indeed, some ingredients like wheat are largely used in modern fish feeds simply for the functional properties they contribute through their starch content, rather than any intrinsic nutritional property they have.

13.2 Characterisation and preparation of ingredients One of the primary limitations to many of the published studies on ingredient evaluation is the lack of detailed compositional information on samples of all the ingredients being considered. High levels of variability between common ingredients are well recognised, and this variability has been demonstrated to affect the nutritional value of ingredients when included in feeds (Glencross et al., 2008a). Therefore, one of the most important elements to manage that variability is its assessment prior to use of the ingredient, so that diets can be formulated on known nutritional parameters and not just those assumed (Jiang, 2001). 13.2.1 Characterising ingredients One of the key reasons for comprehensively characterising ingredients is so that the findings from the study can be used by others. Identification of factors such as the species of the ingredient, its genotype or cultivar classification (if relevant) and source should all be detailed where possible. Variability, even among ingredients from a single species, can be substantial (Glencross et al., 2008a). Identification of the origin of the ingredient being studied at least to country level should also be considered mandatory. This is especially important for plant ingredients as it is well known that soil type and climate can affect the composition of many grains, and significant differences in the nutritional value have been observed between the same ingredients, from different sources (Mwachireya et al., 1999; Burel et al., 2001; Glencross et al., 2004a). The processing of an ingredient sample prior to addition to the experimental diets also has important implications on its characterisation and the

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nutritional value likely to be attributed to it. Booth et al. (2001) noted clear differences in the chemical composition and also the nutritional value of a range of grain legume meals produced from either whole-seed or seed kernels. Glencross et al. (2007c) also showed in detail the effects of increasing levels of dehulling efficiency on the composition of lupins. Similarly, clear differences in canola meal produced through different oil extraction methods have also been noted (Glencross et al., 2004a). Accordingly, some details of what processing has been undertaken on the ingredient from its raw or natural state would be useful. In addition to the clear identification of the ingredient of concern, its origins and processing, it is imperative that a detailed analysis of the compositional characteristics of the ingredient is also provided. Ideally this analysis should be as comprehensive as possible, but key variables such as crude protein (nitrogen × 6.25), total lipids, ash, moisture and gross energy should be considered mandatory for all test ingredients. Crude fibre is another traditional element of proximate analysis that is losing favour to more useful analyses such as acid-detergent fibre and neutral-detergent fibre, which relate more closely to levels of cellulose, hemicellulose and lignins (Petterson et al., 1999). A guide to key compositional parameters to be considered in a range of ingredients is provided in Table 13.1. For studies examining lipid nutrition and/or fish oil replacement issues it is imperative that the fatty acid content of the diets and preferably the ingredients too be detailed. In the absence of specific details on the ingredient species, cultivar, origin and degree of processing, then the importance of the compositional analysis increases. Details of ingredient composition should include moisture content on an as-is basis, but other parameters should be expressed on a g/kg dry matter basis to help standardize ingredient information. This is because most feeds are prepared with the addition of water followed by a drying process, which dehydrates the feed to a relatively uniform dry matter content, irrespective of the initial dry matter content of the individual ingredients. Therefore, it is more practical to provide a standardized assessment of composition, such as that on a dry matter basis. Methods used for the composition analysis of ingredients should be consistent with those recommended by the Association of Official Analytical Chemists (AOAC, 2003). In addition to these standard methods, specific recommendations pertaining to the evaluation of grain products were made by Petterson et al. (1999) who identified several opportunities for improvement or modifications to methods that made them more suitable for grain products. The presence of antinutritional factors (ANF) or bioactive compounds also has important implications for potential ingredient use and, as such, forms an important part of the characterisation of some types of ingredients, particularly those derived from plant materials (Francis et al., 2001).

603 182 150 22.7

45 17 29 47 40 15 26 28 33

718 105 152 21.5

39 28 29 50 52 18 27 31 36

39 56 9 120 80 15 66 54 82

951 1 18 23

887

Blood meal

39 8 13 27 25 7 15 16 20

492 92 360 16.1

920

Bovine meal

10 6 11 17 17 3 8 8 12

255 11 34 17.0

920

Field pea

2

Chilean anchovetta meal. Narrow-leaf lupin Lupinus angustifolius (mixed cultivars) kernel meal. SE = solvent-extracted. Source: Data from Glencross unpublished and Allan et al. (2000).

1

920

Poultry meal

915

Fish meal1

47 10 15 29 14 3 16 16 14

415 53 33 20.4

885

Lupin kernel meal2

32 26 3 25 41 30 27 16 78

431 22 86 19.6

962

SE rapeseed meal

42 14 23 44 28 9 27 24 24

518 47 69 19.6

909

SE soybean meal

25 17 27 52 12 13 39 20 30

838 9 8 22.6

910

Wheat gluten

20 11 30 113 11 16 41 22 31

620 10 11 24.1

920

Corn gluten

5 2 4 8 3 2 5 3 5

122 10 20 18.3

920

Wheat

Composition of a range of commonly used aquaculture feed ingredients (values are g/kg DM unless otherwise detailed)

Dry matter content (g/kg) Crude protein Total lipids Ash Gross energy (MJ/kg DM) Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine

Nutrient

Table 13.1

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13.2.2 Ingredient preparation prior to evaluation To ensure that the assessment of ingredients is undertaken on a uniform and representative basis, it is important that consideration is given to the physical preparation of all the ingredients with respect to particle size. Fine grinding (to 200 to 300 μm particle size) is important to ensure homogeneity in the experimental diet. This particle size management extends beyond just the test ingredients to include all those used in any experimental diet. Particle size has been implicated as an important factor in affecting the ingredient evaluation process (Nir and Ptichi, 2001). A recommendation of 250 μm for maximum particle size was made by the National Research Council (NRC, 1993). However, application of this recommendation has not been widely adopted, with a maximum particle size of 600–800 μm being more typical of those that are reported (Burel et al., 2000; Glencross et al., 2005).

13.3 Defining ingredient digestibility Modern aquaculture diets are routinely formulated based on digestible nutrient and energy specifications (Kaushik, 1998; Glencross, 2008). Because of this, it is important to measure the digestible energy and nutrient content of prospective ingredients. Essentially this means measuring the amount of the energy or nutrient from a diet that is not excreted in faeces. In assessing diet digestibilities there are two key methodological approaches used: the direct and indirect assessment methods (Maynard and Loosli, 1969). In the direct assessment method a complete account of both feed inputs and faecal outputs is required. The digestible value of the feeds is then determined on a mass-balance basis. However, this method is fraught with problems, largely because of the difficulty and errors involved with collection of accurate data on feed intake and faecal production. The alternative method is via indirect assessment, where a representative sample of both the feed and faeces is required, and an indigestible marker is included in the diet. This method works primarily by measuring the ratio of the marker between the feed and faeces, which determines dry matter digestibility from which calculations for digestibility of energy and other nutrients can be extrapolated. This indirect assessment produces data that is referred to as ‘apparent digestibility’.

13.3.1 Feed issues in ingredient digestibility assessment Generally, diets for digestibility assessments use an ingredient substitution approach, where test diets comprise the test ingredient plus a reference diet component. The reference diets used for digestibility studies with most aquaculture species have usually been simple, practical diets. Typically, reference diets have been based on fish meal as a key protein

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and energy source, although other ingredients are also sometimes used. It could be argued that the specific formulation of the reference diet is not critical as it is just that, a reference. However, diets which provide a nutrient content similar to those likely to be used under practical circumstances provide some logic to the approach (Glencross et al., 2007a). There are two methods of ingredient inclusion for specific ingredient digestibility assessment. These are referred to as the diet replacement method (DRM) or the ingredient replacement method (IRM) (Aksnes et al., 1996). With the DRM method, a test ingredient is added to replace a portion of the reference diet (usually ∼30 %) to create a test diet (Glencross et al., 2005). The digestibility values for both the reference and test diets are then determined and, based on proportionality factors, the digestibility of the ingredient, or any of its nutrients, can be calculated. It is important to note that, with this method, the portion of the reference diet within any test diet must be fully representative of the complete reference diet. For example, all ingredients, including additives and the marker, must be included in the portion of the reference diet used and not added to the test diet at levels equivalent to those in the reference diet. The IRM also uses a reference diet, but differs in that the reference diet usually has a single, well-defined reference ingredient at a fixed, moderately high inclusion level (e.g. 50 %). This single ingredient is then replaced with the test ingredients to create the test diets, and the assessment of the digestibility of the test ingredients is thus based on the relative diet digestibility with regard the reference ingredient (Aksnes et al., 1996). The amount of the test ingredient that is included into a test diet usually varies from 20 % to 50 %, with 30 % being the most typical of digestibility studies. A study by Smith and Tabrett (2004) with shrimp showed improvements in the robustness of the determinations with increasing inclusion level; however, it has also been suggested that including the test ingredient at ‘typical’ commercial inclusion levels has more value (Gomes et al., 1995; Allan et al., 2000). There are also potential benefits from examining the use of a particular ingredient at more than one inclusion level as it allows the investigation of potential interactive effects of ingredients within a feed formulation (Allan et al., 1999; Smith and Tabrett, 2004). This is especially important for plant protein sources where certain carbohydrates have been shown to exert some effects on the digestibility of other dietary nutrients (Glencross et al., 2008a). A wide variety of marker types have been used in digestibility studies. While chromic oxide (Cr2O3) is perhaps the most commonly used marker, rare earth metal oxides such as ytterbium oxide, yttrium oxide and others are gaining favour (Austreng, 1978; Austreng et al., 2000). Studies focusing on lipid utilisation have found favour with the use of hydrocarbon markers such as cholestane (Ishikawa et al., 1996). Other endogenous markers such as acid-insoluble-ash and crude fibre have also been used, although they

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are somewhat less reliable and more prone to producing data with larger variance (Morales et al., 1999).

13.3.2 Collecting faeces for digestibility assessment There are basically three methods adopted by most researchers for faecal collection in aquaculture nutrition research: dissection, stripping and collection of voided faeces. Where faeces are collected by dissection or stripping (technically this should be called digesta, as it does not become faeces until it is voided by the animal), there is arguably the potential to underestimate digestibility because of incomplete digestion and potential contamination of digesta with endogenous material. In contrast, when faeces are collected from the water column or following settlement, there is the potential to overestimate digestibility because of leaching losses of organic matter. Austreng (1978) studied the changes in nutrient and energy digestibility when assessments were made from digesta collected from different parts along the gastrointestinal tract (GIT) of rainbow trout using a dissection approach. It was noted that substantial increases in digestibility occurred throughout the GIT except between the proximal and distal intestine. This observation provides strong support for the use of faecal stripping techniques, where gentle abdominal pressure is applied to the abdomen of the fish, approximately over the distal intestine, to expel its faecal contents as a means of obtaining unleached digesta from a standardised part of the digestion process. Arguably this method of sample collection is similar to that of ileal digesta collection which is used as a routine digesta collection process in pig nutrition (Reverter et al., 1999). However, it is important to note that it is not always possible to strip easily faeces from all species of fish, and it is clearly unsuitable for use with crustaceans. A comparison of the three faecal collection methods was published by Vandenberg and de la Noue (2001). The authors compared the digestibility of a practical diet containing a range of protein sources (e.g. fish meal, soyabean meal, whey, blood meal) when fed to rainbow trout. The findings suggested that there was essentially no difference in diet digestibility assessments between the methods of Cho and Slinger (1979) and Choubert et al. (1982), but that both of these settlement methods resulted in significantly different (higher) diet digestibilities than those determined from the faecal stripping collection method (Austreng, 1978). Furthering this comparison, Glencross et al. (2005) compared the ingredient digestibilites of a series of grain protein ingredients when faeces were collected using either settlement (Cho and Slinger, 1979 method) or stripping techniques (Austreng, 1978 method). Significant differences were observed between the two faecal collection methods as to their effects on ingredient digestibility. In particular, the differences in the digestibilities determined between the two methods were more pronounced with ingredients high in indigestible carbohydrates. Generally, it can be regarded that

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faecal stripping provides a more conservative estimate of both diet and ingredient digestibilities than that provided using settlement techniques. Assessment of the effect of repeated handling of the fish on the diet digestibility values determined from rainbow trout has also shown no significant effects, except on the digestibilities of lipids (Stone et al., 2008). It was suggested that a better approach to accumulate the required amount of faeces was to use a larger number of fish and to strip once only. The duration of the faecal collection period for most studies is usually dependent on obtaining sufficient sample to be able to undertake the required chemical analyses. It has been argued that a longer period of collection, e.g. > 5 days, will minimise variability in faeces composition and improve reliability of results (Wybourne and Carter, 1999), although arguably this can also be resolved by using a larger number of fish from which to collect faecal samples during a single collection period. Reduction in variance, however, has the potential to improve substantially the experimental power of digestibility studies and increase the capacity to detect significant effects and, irrespective of collection method, ingredient inclusion level or collection period duration, adequate replication is probably a far more important consideration (Searcy-Bernal, 1995).

13.3.3

Experiment management issues in ingredient digestibility assessment Experimental conditions can also have important effects on the determination of diet digestibility and need to be managed accordingly. Key considerations include environmental conditions, fish size and feeding ration structure. Water temperature has minor effects on digestibility. Windell et al. (1978) noted some influence of water temperature (7 ºC, 11 ºC and 15 ºC) on dry matter, protein, lipid, carbohydrate or energy digestibility of a diet fed to rainbow trout of three size classes (19 g, 207 g and 585 g), most notably at the lowest temperature and with the smallest fish. The ration size fed to the fish has also been shown to influence digestibility assessment, but only at the highest feeding rates. The study by Windell et al. (1978) also examined the effect of varying ration size on diet digestibility with rainbow trout. In this study the fish fed the highest feed ration produced significantly lower digestibility values for dry matter, carbohydrate and energy, but not for protein or lipid. Variability in the digestibilities of diets during the period when fish are first fed a new diet has also been noted (Wybourne and Carter, 1999). Because of this, a period of adaptation to new diets it has been suggested as necessary prior to faecal sample collection (Allan et al., 1999; Burel et al., 2000). The length of this period varies among researchers, although the data from Wybourne and Carter (1999) suggests that, as there was sufficient reduction in variability of digestibility assessments of most diets after four

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days, collection could be commenced from day five. Most researchers use a minimum of seven days as an acclimation period (Allan et al., 1999; Glencross et al., 2005).

13.3.4 Calculating diet and ingredient digestibilities The calculation of digestibilities (either diet or ingredient) allows the determination of the apparent digestibility, based on the ratio of marker in the diet and faeces. The apparent digestibility coefficient (ADCdiet) of a specific nutritional variable is based on the following formula (Eq. 13.1): ⎛ Marketdiet × Nutrient faeces ⎞ ADCdiet = 1 − ⎜ ⎝ Market faeces × Nutrient diet ⎟⎠

[13.1]

In this equation the terms Markerdiet and Markerfaeces represent the marker content of the diet and faeces respectively, and Nutrientdiet and Nutrientfaeces represent the nutritional parameter of concern (e.g. protein or energy) in the diet and faeces respectively. With this formula, values would typically range from 0 to 1. Values greater than 1 or less than 0 are indications of errors. To achieve a percent apparent digestibility the equation should be multiplied by 100. To calculate the digestibility of a test ingredient, as might be done with the IRM ingredient digestibility method, the following equation is used (Eq. 13.2): Nutr. ADingredient =

[ ADtest × Nutrtest − ( ADbasal × Nutrbasal × 70%)] 30% × Nutringredient

[13.2]

In Eq. 13.2, the Nutr.ADingredient is the digestibility of a defined nutrient from the test ingredient when included in the test diet at 30 %. The ADtest is the apparent digestibility of the test diet. The ADbasal is the apparent digestibility of the basal diet, which in this example makes up 70 % of the test diet. In Eq. 13.2 the NutrIngredient , Nutrtest and Nutrbasal are the level of the nutrient of interest in the ingredient, test diet and basal diet, respectively. This equation was published in the aquaculture nutrition literature by two separate authors around the same time, and, although the specific mathematics used by each author to express this equation was slightly different, they in essence presented the same concept (Sugiura et al., 1998; Forster, 1999). One of the assumptions with the calculation of ingredient digestibility coefficients is that they will fall between 0 and 100 %. This is not always the case, but, unlike for diet digestibilities, it is not necessarily indicative of errors, although analytical errors for markers or nutrients, poor mixing of the marker in the diet or ‘non-representative’ samples of diet or faeces or can be real sources of error. Another possibility is the interaction between ingredients (more specifically dietary nutrients) to produce non-additive effects. It is suggested that when these values are determined that they be reported as calculated, but for calculating the

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digestible energy or nutrient basis they should be rounded to 0 if negative or to 100 if greater than 100. There are several other assumptions used in the digestibility assessment process. As discussed earlier, it is assumed that digestible coefficients are additive, which means that if you sum the proportional digestibility values for each ingredient in a diet, then it should equal the measured digestibility of the diet. This assumption relies on there being no interactions among ingredients that differentially affect digestibility, and that changing the inclusion content of a particular ingredient does not change its digestibility either. However, neither of these assumptions holds true all the time. There have been several studies using a range of different ingredients where the assumption of additivity has been questioned (Wilson and Poe, 1985; Watanabe et al., 1996). While ingredients that are rich in ash, protein and/ or lipid usually produce results that are additive, the presence of carbohydrates (including different starch and fibre classes) adds a degree of complexity and potential departure from additivity (Glencross et al., 2007a). There is an increasing body of evidence that shows that certain fibre types can influence the digestibility process (Glencross et al., 2003a, 2008a). The inertness of the marker used to calculate apparent digestibility, being able to pass through the digestible tract without influencing digestion and at approximately the same rate as the rest of the digesta is another assumption. Chromic oxide has traditionally been the most commonly used marker. It has been claimed that its inclusion affects both carbohydrate and lipid digestibility (Ringo, 1995; Shiau and Liang, 1995); however, the effect was not consistent with all species as no affect on carbohydrate digestibility with gilthead sea bream was reported in a similar study by Fernandez et al. (1999). As a quality control mechanism the use of a standard reference ingredient in digestibility experiments is recommended. This also provides a useful way to standardise and assist in the assessment of the temporal and interlaboratory variance. Ingredients such as vitamin-free casein, enzymaticallyhydrolysed casein, wheat gluten and lupin kernel meals have previously been used for this purpose (Glencross et al., 2005, 2008a). Glencross et al. (2008a) demonstrated that use of such a reference ingredient and also reference diets was a good way to standardise digestibility assessments over time and managed to produce data with a coefficient of variation of 7.4 % and 4.2 % (for protein and energy digestibility, respectively) from seven experiments over a three-year period for a reference lupin kernel meal, while reference diet digestibility variance over the same experiments had even lower coefficients of variation of 1.3 % and 1.4 % (for protein and energy digestibility, respectively).

13.3.5 Species effects on the digestibility assessment process The comparability of digestibility data across species has shown contrasting results. A study comparing the diet and ingredient digestibility of a series

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of diets and ingredients fed to rainbow trout and Atlantic salmon found a high degree of correlation between energy digestibilities (Fig. 13.1), but somewhat less correlation between protein digestibilities (Glencross et al., 2004b). It was argued that the poorer correlation between the protein digestibilities was attributable to the low variability among protein digestibilities in either species. It was suggested that the diet and ingredient digestibilities of a species such as rainbow trout could be reasonably applied to other carnivorous species like Atlantic salmon. However, how broadly this could be applied to other fish species (including other carnivores or even omnivores), or even the same species but comparing fresh and salt water, remains to be tested. In contrast to the results between rainbow trout and Atlantic salmon, a comparison of the ingredient digestibilities between rainbow trout and black tiger shrimp (Penaeus monodon) showed poor correlation (Glencross et al., 2008b). Diet digestibilities in this case were not examined because different formulations were used for either species, but the same test ingredients were used in each series of diets (Fig. 13.2). This poorer correlation could be attributed to a wide range of factors including, but not limited to, species, diet formulation and faecal collection method differences.

13.4 Ingredient palatability If the ingredient has a negative effect on feed intake then, irrespective of how digestible the nutrients and energy from a particular ingredient might be, it is of limited use in a feed formulation. Although there may be strateDiet Atlantic salmon ADC

Atlantic salmon ADC

1.000 0.950 0.900 0.850 0.800 0.750 0.700 0.800 0.850 0.900 0.950 1.000 (a)

Rainbow trout ADC

(b)

Ingredient 1.400 1.300 1.200 1.100 1.000 0.900 0.800 0.700 0.600 0.600 0.700 0.800 0.900 1.000 1.100 Rainbow trout ADC

Fig. 13.1 Correlations among diet (a) and ingredient (b) digestibilities of the same diets when fed to either Atlantic salmon at 14 ºC or rainbow trout at 15 ºC. Shown are the nitrogen (•), energy ( ) digestibilities. Equations for regression functions are: diet nitrogen digestibilities: y = 0.7619x + 0.1996, R2 = 0.1406; energy digestibilities: y = 1.354x − 0.3263, R2 = 0.9845. Ingredient nitrogen digestibilities y = −2.0838x + 3.1163, R2 = 0.2931, energy digestibilities: y = 1.5431x − 0.3528, R2 = 0.8131. Data reproduced from Glencross et al. (2004b).

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New technologies in aquaculture 1.000

Shrimp ADC

0.900

0.800

0.700

0.600 0.500

0.600

0.700

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0.900

1.000

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Rainbow trout ADC

Fig. 13.2 Correlations among protein digestibilities of the same lupin kernel meals when fed to either black tiger shrimp or rainbow trout. Equations for regression function are: energy digestibility, y = 0.3191x + 0.5548, R2 = 0.6746; nitrogen digestibility, y = 0.0242x + 0.9155, R2 = 0.106. Data reproduced from Glencross et al. (2008b).

gies to avert or resolve palatability problems with certain feed ingredients using ingredient processing or feeding stimulants, clearly it is better if these problems can be avoided outright. For nutritional research to carry any credibility it has to be based on the actual ingestion of nutrients by an organism; therefore one of the key assessment criteria in research should be some demonstration or assessment of food intake by the animal (Jobling et al., 1995). Based on such an assessment it then becomes valid to base a measurement of a response by the animal relative to that feed intake. However, assessing feed intake, particularly for aquatic animals, is not necessarily straightforward or a simple parameter to measure. For an animal to demonstrate variability in feed intake to a diet, it must be given the opportunity to refuse feed. Therefore ensuring that the ration fed is above apparent satiety is important. Feed preference studies are one way of assessing affects on intake. A simple method published by Helland et al. (1996) provides an easy way of determining feed intakes in tanks of fish. By feeding to excess and simply collecting the uneaten feed and using compensation factors to account for solubilisation losses, a reasonable estimate of feed intake can be achieved. This method has strengths over fixed ration feeding regimes in that it allows for an element of self-discrimination of the feeds by the fish. A more advanced method, the use of self-feeding through computer-managed feedback response mechanisms, is another option that has been frequently used to allow discrimination of feeds by fish and certainly assists in removing human error from the feed intake assessment process (Juell, 1991; Boujard and Le Gouvello, 1997). In exam-

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45 40 35 30 25 20 15 10 5 0

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ining the feed intake response of fish to a novel diet, the use of appropriate control treatments, such as those with palatability inhibitors such as sulfamerazine sodium (Boujard and Le Gouvello, 1997; Glencross et al., 2006), provides an extra degree of confidence in the ability to discern feed intake variability (Fig. 13.3). Many experiments specifically designed to examine serial inclusion of a particular ingredient end up with no significant effects on either growth or feed intake. While this is often used to argue that the ingredient is palatable to the test animal up to the inclusion level studied, it can be difficult to determine the degree of confidence in such results when the experiments are run without controls designed to demonstrate a specific effect, such as a decrease in feed palatability. Feed intake variability over time can also be an important issue to consider (Fig. 13.3). It was noticed that adaptation to some diets occurred slower than others, and it was suggested that this was primarily a sensory discrimination by the animal against certain feeds until it had become accustomed to them (Glencross et al., 2006). To enable such an examination in feed intake variability an assessment of the daily feed intake of individual replicate tanks is advantageous. Ingredient inclusion studies are the simplest way to examine effects on feed intake. In this strategy an ingredient is included into a series of test diets at increasing inclusion levels and then the reference and test diets are fed to apparent satiety to replicate groups of fish for a period of time. Differences in feed intake between the reference and test diet, it can then be argued, are reflective of the apparent palatability due to the test ingredient. However, the issue of how much of a test ingredient should be included in test diets is somewhat subjective. Ideally a range of inclusion levels that cover what would be the practical inclusion levels should be used as this

Fig. 13.3 Daily feed intake (a) by tanks (n = 3) of rainbow trout (n = 20/tank) fed diets with either 0 %, 0.5 % or 1.0 % sodium sulfamerizine in the diet and the same dietary treatments represented as cumulative feed intake (b) per fish over a 28 day period. Data derived from Glencross et al. (2006).

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also allows examination of critical palatability levels or break-points in a practical context (Shearer, 2000).

13.5 Defining effects on growth and utilisation A third important issue to resolve for ingredient evaluation is in determining the capacity of the animal to use the digested nutrients for growth. However, there are many elements to be considered in defining growth. At its simplest this constitutes the gain in weight by an animal; it can also be extrapolated to other features such as protein and energy retention, feed use efficiencies and even molecular factors such as gene and/or specific protein expression.

13.5.1 Measuring growth The initial weight and size variability of the animals used in the study has an important bearing on the capacity of a study to determine significant effects. As a means of improving experimental power it is important to limit variance where possible. Front-end variance control can be managed by ensuring all fish weighed into a replicate (and across replicates) are from within one standard deviation of the mean. In addition to variance management controls, use of adequate replication or designs to provide significant experimental power to be able to discern effects being studied is another critical consideration (Searcy-Bernal, 1995). Generally, a minimum of three replicates should be considered mandatory for growth studies; however, if a regression relationship is being sought, then it may sometimes be more prudent to reduce the replicates to enable a greater number of treatments to be included. This is because in regression studies the power is derived from the range of treatments employed, not only the confidence of the assessment within each treatment (Shearer, 2000). Growth in nutritional experiments is generally defined as the difference between initial and final live-weights. More specifically this should be defined as live-weight gain. Live-weight gain is also often reported as percentage gain, which is usually expressed as a percentage of the final weight divided by the initial weight. For such measures as this, it is imperative that the specific initial weights of each replicate are used in any statistical analysis such as a covariance analysis. For a measure of growth to be considered a ‘growth rate’ it has to be time specific. The three most routinely used growth rate assessments are daily gain (DG), daily growth coefficient (DGC) and specific growth rate (SGR). Daily gain is the simplest of the three rates and is a measure of the live-weight gain over time. Daily growth coefficient in contrast is calculated based on a percentage of the one third root transformation of the final (Wf) and initial (Wi) live-weights over time (t) (Eq. 13.3) (Kaushik, 1998):

Ingredient evaluation in aquaculture

DGC = [(Wf1 3 − Wi1 3 ) t ] × 100

403 [13.3]

Thermal growth coefficient (TGC) is another growth rate parameter which is derived from the DGC, but the time component is expanded to be considered on a temperature basis. In this regard, the time component of the TGC is multiplied by the average temperature (ºC) over the period of the study (t) (Cho and Bureau, 1998) (Eq. 13.4): TGC = [(Wf1 3 − Wi1 3 ) ( t × °C )] × 100

[13.4]

A limitation to this descriptor is that it is only valid if used in the temperature range of the animal where its response to temperature is linear. As temperature approaches the animal’s optimum for growth, any further increases in temperature will not correspond to an actual increase in growth rate of the animal but should if based on a TGC. Because of this the temperature limits of TGC need to be recognised. Specific growth rate is another weight transformation often used to describe growth and is calculated based on a percentage of the natural logarithm transformation of the final and initial live-weights over time (Kaushik, 1998). However, the point of using a growth rate descriptor is to attempt to standardise the assessment and potentially allow for some comparability of performance across experiments. To achieve this, the growth rate assessment needs to provide some independence from fish size. Kaushik (1998) compared both DGC and SGR for a range of fish sizes and noted that SGR did not provide as good a transformation of growth rates when compared to that provided by using DGC. It was concluded that if such a growth rate descriptor is required, then DGC is more appropriate than SGR. However, if the initial weights of the animals are provided, then actual weight accrual as gain per day (DG) is perhaps just as, if not more, practical.

13.5.2 Survival Animal losses that occur during an experiment are usually expressed as a percentage survival. This survival is determined based on the number of individuals surviving at the end of a study relative to the number included in the study at the beginning. Unless the percentage is divided by the time of the experiment, survival should not be reported as a rate.

13.5.3 Feed conversion efficiency For an assessment to be made on the nutritional utilisation of a diet and, by referral, of an ingredient, there is a clear need to measure feed intake. Feed intake by fish is often reported as both an amount (g/fish) and rate (g/fish/d). However, truly accurate assessment of feed intake by fish is one of the more difficult aspects of aquaculture research to achieve. The

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efficiency of food use by fish is usually reported as either feed conversion efficiency (FCE: Eq. 13.6) or food conversion ratio (FCR: Eq. 13.7). These assessments are usually made on a dry weight of food and live-weight of fish basis. Because these variables rely on both live-weight gain and feed intake assessment they assume the errors of both assessments. FCE = (Weight gain) ( Feed consumed)

[13.5]

FCR = ( Feed consumed) (Weight gain)

[13.6]

Energy gain (kJ / kg0.8/ d)

13.5.4 Nutrient retention To determine the efficiency by which nutrients and energy are retained from feeds, an assessment of the nutrient and energy composition of both the feed and fish is required, and this is needed on an as-fed and live-weight basis respectively. However, efficiency data can be strongly influenced by animal size, with smaller animals typically being far more efficient at retaining both nutrients and energy than larger fish of the same species (Glencross, 2008). An advancement on this is the determination of the partial efficiencies of protein and/or energy utilisation. This examines the relationship between digestible nutrient/energy intake and somatic accretion of that nutrient/energy, the coefficient of that relationship equating the partial utilisation efficiencies (Fig. 13.4). Typically, this parameter has 200

R

150

H S

100 50 0 0

100

200

300

–50 –100 Digestible energy intake (kJ/ kg0.8/ d)

Fig. 13.4 Energy retention by rainbow trout with varying levels of digestible energy intake from diets with 300 g/kg inclusion levels of a lupin protein concentrate dried using either heat (H) or spray-drying (S) relative to a diet with fish meal (R) as the only protein source. Each data point is based on data derived from the mean of four replicates for each diet ration level. Data derived from Glencross et al. (2007b). Energy partial utilisation efficiencies above 100 kJ/ kg0.8/d intake for the R and S diets were estimated at 69.3 %. Energy partial utilisation efficiencies above 100 kJ/kg0.8/d intake for the H diet was estimated at 61.5 %.

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been determined to underpin bioenergetic models of fish growth and feed utilisation (Glencross, 2008). However, variants on this design have been used to examine the effect of ingredients on diet partial utilisation efficiencies (Glencross et al., 2007b). The forecast benefit of this is that it will better enable diet formulations with those ingredients to be used in models of nutrient and energy accumulation by fish. The apparent biological value (ABV) is a parameter derived from nutrient and energy retention values based on digestible nutrient and energy intake (Morales et al., 1994). Typically, ABV provides some assessment of the proportion of the nutrients or energy absorbed from the diet that is actually used for tissue growth. Clearly for this parameter to be estimated an assessment of the diet nutrient and energy digestibilities is required.

13.5.5

Factors likely to affect nutrient and energy utilisation of ingredients There are a range of factors that have the potential to affect nutrient and energy utilisation of ingredients. Among these are ANF, protein damage and amino acid limitations. ANF have the potential to cause significant problems to nutrient and energy utilisation by fish by interfering with digestion, palatability or even cellular function (Francis et al., 2001). In defining the effects of ANF on fish there have been a variety of experimental strategies examined, and these vary primarily based on the mode of action of the ANF being studied (Krogdhal et al., 1994; Refstie et al., 1998; Burel et al., 2001). For a detailed review on the variety and effects of ANF read Francis et al. (2001). The effect of protein damage on a range of lupin protein concentrates (LPC) dried using different techniques was examined by Glencross et al. (2007b). When excessive heat was used to dry an LPC it was shown that the ability of the fish to convert the digestible energy to retained energy was reduced compared to that from an LPC produced using less aggressive drying methods such as spray-drying (Fig. 13.4). The heat-damaged LPC was presumed to have had reduced nutritional value because of Mailliard reaction products, where carbohydrates condense with the free amino groups of lysine residues on the protein and render those amino acids unavailable for utilisation (Oste and Sjodin, 1984). Techniques can be developed to measure the degree of Mailliard products through a measuring the relative amounts of available lysine (Rutherfurd et al., 1997; McCafferty and Dods, 2008) (Fig. 13.5). Amino acid limitations can also limit the potential protein and energy utilisation by affecting the animal’s capacity to sustain growth potential. Specific ratios are required between the essential (indispensible) amino acids in the diet to allow protein synthesis to occur to its maximum potential (Kaushik, 1998). Typically, these ratios are referenced against the amount of dietary lysine, which is usually regarded as the first limiting amino acid

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4 Homoarginine (available lysine)

5 4 3

Lysine (unavailable lysine, freed by hydrolysis)

2 1 0 0

2

4

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Fig. 13.5 Chromatogram from a reactive lysine assay showing the homoarginine (available lysine content) and the lysine (unavailable lysine content) from a lupin protein concentrate sample. Data reproduced from McCafferty and Dods (2008).

in most practical diets. If an ingredient is included in a diet such that any one of the ten essential amino acids falls below the specific ratio required, then this amino acid becomes the limitation in protein synthesis and therefore growth. Concise experimental effects of such limitations due to an ingredient inclusion are rare, but have been demonstrated (Glencross et al., 2003b).

13.5.6 Gene and protein expression A modern aspect to the evaluation of nutritional effects on fish performance has been through the advent of nutrigenomics (Panserat et al., 2001, 2007). This aspect of nutritional evaluation has also had some application to the assessment of ingredients in fish diets, by allowing an examination of changes occurring at the molecular level (Kolditz et al., 2007; Lilleeng et al., 2007; Panserat et al., 2008). The two main streams of molecular science applied to ingredient assessment in fish diets have been genomics and proteomics, where gene and protein expression, respectively, are examined as a consequence of dietary treatments. Arguably proteomics has more direct relevance to animal function because it explores the actual functional changes occurring in protein expression within the cell (Vilhelmsson et al., 2004; Kolditz et al., 2008). In contrast, genomics explores the expression of genes through transcriptional processes, which still has to rely on transla-

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tional processes to allow its effects to be mediated upon the animal (Panserat et al., 2008). This branch of science also offers some interesting capabilities to the examination of the extent of the influence of microbial populations in the gut of fish as a function of ingredient choice (Pond et al., 2006; Ringo et al., 2006; Lilleeng et al., 2007).

13.5.7

Biochemical, histological and sensory factors in ingredient evaluation One of the primary biochemical evaluations undertaken in ingredient evaluation studies is the systematic comparison of the effects of dietary treatments on whole somatic or organ specific composition (Shearer, 1994; Booth and Allan, 2003). Notably, whole somatic composition analysis is required for the examination of nutrient/energy utilisation efficiency and/or apparent biological value assessments. In addition to this more standard biochemical assessment, other biochemical parameters such as changes in blood glucose levels or thyroid hormone levels (triiodothyronine and thyroxine) or enzyme activities have also been used to provide an indication of disruption to the metabolic function and nutrient utilisation by fish (Burel et al., 2001). There is a range of other hormone assays that can be examined, including hormones such as somatatropin and insulin-like growth factor 1, which have been used as a correlates of synthetic activity in fish (Dyer et al., 2004; Bakke-McKellup et al., 2007). An evaluation of immune responses and parameters associated with an immune challenge has also been effectively used in recent times to examine nutritional treatments, and further expansion in this area would add value (Krogdhal et al., 2000; Bakke-McKellup et al., 2007; Olsen et al., 2007; Stone et al., 2008). Assessment of tissue histology has also provided useful insights into examining some of the more long-term and chronic effects of ingredient and ANF inclusion in fish diets (Krogdhal et al., 2000; Bakke-McKellup et al., 2007; Uran et al., 2007). Some specific parameters, such as gastrointestinal enteritis problems associated with use of soybean meals, are a particular case in example (Krogdhal et al., 2000; Uran et al., 2007). Typically, studies use an arbitrary scoring system to rate degrees of cellular change, and this scoring system is then used to compare different dietary treatments. Sensory or organoleptic properties, while not a common assessment parameter in nutritional research on ingredients, have been used to evaluate the potential impact of novel ingredients on product quality aspects. Such assessments have been more prevalent with studies on fish oil replacement than for fish meal replacement, but some reports on influences of products such as rendered meat meals do exist (Thomassen and Rosjo, 1989; Williams et al., 2003b).

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13.6 Ingredient functionality and feed technical qualities Functionality of feed ingredients relates to their effects on the physical properties of the processed feed (Thomas and van der Poel, 2001; Hawkins et al., 2008). Irrespective of how good the nutritional value of an ingredient may be, if it cannot functionally be incorporated into a feed, or reduces the physical qualities of the feed, then its value as an ingredient is diminished. Key attributes sought are those where pellets produced from the formulation result in a product with properties that provide advantages for feeding aquatic species. These properties include aspects such as sinking rates, pellet durability, degree of starch gelatinisation and oil absorption capacity. Experimental extrusion processing is the most practical way to evaluate ingredient functionality, as the results have direct implications for a final product. In these studies, a hypothetical formulation including a test ingredient is run through an extruder and the properties of the pellets produced are compared against either a reference formulation or a series of target specifications (e.g. Fig. 13.6).

13.7 Frontier technologies for ingredient evaluation

Crush force (g)

As increasing pressure on fishery resources occurs there will be a shift towards the consistent use of alternative ingredients in most aquaculture feeds. Already there is substantial scope for the use of many plant and

9000

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Fig. 13.6 Pellet hardness (g force to crush) from extruded diets with increasing levels of soybean or kernel meals from Lupinus angustifolius and Lupinus luteus lupin varieties. Pellet hardness is one of several physical properties of benefit to extruded fish feeds. The data show that different ingredients when included in diets at similar inclusion levels can have dramatically different effects on the physical properties of feed pellets. Data derived from Hawkins et al. (2008).

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animal meals and oils as alternatives (Gatlin et al., 2007). This increasing use of alternatives will also introduce a greater array of challenges, the foremost being the reliable, accurate and rapid assessment of the nutritional value of each alternative nutrient source prior to its inclusion in a formulation. The use of rapid analysis techniques for ingredient composition, such as near-infrared spectroscopy (NIRS), has considerable potential to improve the basis for diet formulation from variable batches of raw ingredients. Although NIRS has gained almost routine use in many feed companies for the evaluation of crude protein, moisture and fat composition of ingredients and products, it has also begun to be used for the assessment of digestible nutrients and energy from ingredients and diets (Aufrere et al., 1996). Despite some good attempts at in vitro assessment of ingredient quality, routine assays using such technology are still to be adopted (Anderson et al., 1993; Carter et al., 1999). While such in vitro techniques do not offer the same potential turnaround as NIRS they are still significantly quicker and cheaper than in vivo studies. Accordingly further work on this area remains a priority. The use of nutritional modelling techniques to understand interactions among different compounds is another frontier to be explored. While recent studies have shown that specific interactions among compositional features of some plant ingredients affect their nutritional value, this is by no means the only source of variation in nutritional quality (Fairbairn et al., 1999; Glencross et al., 2008a). Furthermore it is likely that the causes of this variability in nutritional quality may vary between different ingredients and that, in many cases, there are also likely to be more than just two single factors that affect such nutritional qualities and therefore using modelling methods not only to identify them but also to describe them will be a significant advance forward in understanding ingredient limitations (Fig. 13.7). There are numerous other issues that also remain to be resolved. Improving our understanding of amino acid utilisation from different ingredients will improve the ability to formulate diets on an available amino acid basis (El-Haroun and Bureau, 2007). Further documentation on the critical threshold and biological effects of the various ANF is still required for many species (Francis et al., 2001). Similarly, the development of rapid assessment methods for the influence of ingredients on animal health is another need, and the use of molecular and biochemical techniques has much to offer in this regard (Aslaksen et al., 2007; Lilleeng et al., 2007). Improving our understanding of the nutritive values of ingredients will not only consolidate our confidence in using a broader range of ingredients, but also provide a more robust way of estimating the economic value on specific ingredients for inclusion in aquaculture feeds.

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50 45–50

45

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40 Lupin meal digestible protein (%)

35–40

35

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30

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Fig. 13.7 The modelled relationship between lupin kernel meal protein content and lignin content as it affects the digestible protein content of lupin kernel meals. Figure reproduced from Glencross et al. (2008a).

13.8 References aksnes a, hjertnes t and opstvedt j (1996) Comparison of two assay methods for determination of nutrient and energy digestibility in fish, Aquaculture, 140, 343–59. allan gl, parkinson s, booth ma, stone daj, rowland sj, frances j and warnersmith r (2000) Replacement of fish meal in diets for Australian silver perch Bidyanus bidyanus: I. Digestibility of alternative ingredients, Aquaculture, 186, 293–310. anderson jsl, anderson dm and mcniven ma (1993) Evaluation of protein quality in fish meals by chemical and biological assays, Aquaculture, 115, 305–23. aoac (2005) Official Methods of Analysis of the Association of Official Analytical Chemists 18th edn, Association of Official Analytical Chemists, Washington, DC. aslaksen ma, kraugerud of, penn m, svihus b, denstadli v, jorgensen hy, hillestad m, krogdahl a and storebakken t (2007) Screening of nutrient digestibilities and intestinal pathologies in Atlantic salmon, Salmo salar, fed diets with legumes, oilseeds, or cereals, Aquaculture, 272, 541–55. aufrere j, graviou d, demarquilly c, perewz jm and andrieu j (1996) Near infrared reflectance spectroscopy to predict energy value of compound feeds for swine and ruminants, Animal Feed Science and Technology, 62, 77–90. austreng e (1978) Digestibility determination in fish using chromic oxide marking and analysis of different segments of the gastrointestinal tract, Aquaculture, 13, 265–72. austreng e, storebakken t, thomassen ms, refstie s and thomassen y (2000) Evaluation of selected trivalent metal oxides as inert markers used to estimate apparent digestibility in salmonids, Aquaculture, 188, 65–78.

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bakke-mckellup am, koppang eo, gunnes g, sanden m, hemre g-i, landsverk t and krogdahl a (2007) Histological, digestive, metabolic, hormonal and some immune factor responses in Atlantic salmon, Salmo salar L., fed genetically modified soybeans, Journal of Fish Diseases, 30, 5–79. booth ma and allan gl (2003) Utilization of digestible nitrogen and energy from four agricultural ingredients by juvenile silver perch Bidyanus bidyanus, Aquaculture Nutrition, 9, 317–26. booth ma, allan gl, frances j and parkinson s (2001) Replacement of fishmeal in diets of silver perch: VI. Effects of dehulling and protein concentration on the digestibility of four Australian grain legumes in diets for silver perch (Bidyanus bidyanus), Aquaculture, 196, 67–85. boujard t and le gouvello r (1997) Voluntary feed intake and discrimination of diets containing a novel fluoroquinone in self-fed rainbow trout, Aquatic Living Resources, 10, 343–50. bureau dp, harris am and cho cy (1999) Apparent digestibility of rendered animal protein ingredients for rainbow trout (Oncorhynchus mykiss), Aquaculture, 180, 345–58. bureau dp, harris am and cho cy (2000) Feather meals and bone meals from different origins as protein sources in rainbow trout (Oncorhynchus mykiss), Aquaculture, 181, 281–91. burel c, boujard t, corraze g, kaushik sj, boeuf g, mol ka, van der geyten s and kuhn er (1998) Incorporation of high levels of extruded lupin in diets for rainbow trout (Oncorhynchus mykiss): nutritional value and effect on thyroid status, Aquaculture, 163, 325–45. burel c, boujard t, tulli f and kaushik s (2000) Digestibility of extruded peas, extruded lupin, and rapeseed meal in rainbow trout (Oncorhynchus mykiss) and turbot (Psetta maxima), Aquaculture, 188, 285–98. burel c, boujard t, kaushik sj, boeuf g, mol ka, van der geyten s, darras vm, kuhn er, pradet-balade b, querat b, quinsac a, krouti m and ribaillier d (2001) Effects of rapeseed meal glucosinolates on thyroid metabolism and feed utilisation in rainbow trout, General and Comparative Endocrinology, 124, 343–58. carter cg, bransden mb, van barneveld rj and clarke sm (1999) Alternative methods for nutrition research on the southern bluefin tuna, Thunnus maccoyii: In vitro digestibility, Aquaculture, 179, 57–70. cho cy and bureau dp (1998) Development of bioenergetic models and the FishPrFEQ software to estimate production, feeding ration and waste output in aquaculture, Aquatic Living Resources, 11, 199–210. cho cy and kaushik sj (1990) Nutritional energetics in fish: Energy and protein utilisation in rainbow trout (Salmo gairdnerii), World Reviews of Nutrition and Dietetics, 61, 132–72. cho cy and slinger sj (1979) Apparent digestibility measurement in feedstuff for rainbow trout, in Halver JE and Tiews K (eds), Finfish Nutrition and Fishfood Technology, Vol. 2, Heenemann GmbH, Berlin, 239–47. choubert g, de la noue j and luquet p (1982) Digestibility in fish: improved device for the automatic collection of feces, Aquaculture, 29, 185–9. dyer ar, barlow cg, bransden mp, carter cg, glencross bd, richardson n, thomas pm, williams kc and carragher jf (2004) Correlation of plasma IGF-I concentrations and growth rate in aquacultured finfish: A tool for assessing the potential of new diets, Aquaculture, 236, 583–92. el-haroun er and bureau dp (2007) Comparison of the bioavailability of lysine in blood meals of various origins to that of L-lysine HCl for rainbow trout (Oncorhynchus mykiss), Aquaculture, 262, 402–9. fairbairn sl, patience jf, classen hl and zijlstra rt (1999) The energy content of barley fed to growing pigs: Characterising the nature of its variability and

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developing predictive equations for its estimation, Journal of Animal Science, 77, 1502–12. fernandez f, miquel ag, martinez r, serra e, guinea j, narbaiza fj, caseras a and baanante iv (1999). Dietary chromic oxide does not affect the utilization of organic compounds but can alter the utilization of mineral salts in gilthead sea bream Sparus aurata, Journal of Nutrition, 129, 1053–9. forster i (1999) A note on the method of calculating digestibility coefficients of nutrients provided by single ingredients to feeds of aquatic animals, Aquaculture Nutrition, 5, 143–5. francis g, makkar hps and becker k (2001) Anti-nutritional factors present in plantderived alternate fish feed ingredients and their effect in fish, Aquaculture, 199, 197–227. gatlin dm, barrows ft, brown p, dabrowski k, gaylord tg, hardy rw, herman e, hu g, krogdahl a, nelson r, overturf k, rust m, sealy w, skonberg d, souza ej, stone d, wilson r and wurtele e (2007) Expanding the utilisation of sustainable plant products in aquafeeds: a review, Aquaculture Research, 38, 551–79. glencross bd (2008) A factorial growth and feed utilisation model for barramundi, Lates calcarifer based on Australian production conditions, Aquaculture Nutrition, 14, 36–73. glencross bd, boujard tb and kaushik sj (2003a) Evaluation of the influence of oligosaccharides on the nutritional value of lupin meals when fed to rainbow trout, Oncorhynchus mykiss, Aquaculture, 219, 703–13. glencross bd, curnow jg, hawkins we, kissil gwm and petterson ds (2003b) Evaluation of the feed value of a transgenic strain of the narrow-leaf lupin (Lupinus angustifolius) in the diet of the marine fish Pagrus auratus, Aquaculture Nutrition, 9, 197–206. glencross bd, hawkins we and curnow jg (2004a). Nutritional assessment of Australian canola meals. I. Evaluation of canola oil extraction method, enzyme supplementation and meal processing on the digestible value of canola meals fed to the red seabream (Pagrus auratus, Paulin), Aquaculture Research, 35, 15–24. glencross bd, carter cg, duijster n, evans de, dods k, mccafferty p, hawkins we, maas r and sipsas s (2004b) A comparison of the digestive capacity of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) when fed a range of plant protein products, Aquaculture, 237, 333–46. glencross bd, hawkins we, evans d, mccafferty p, dods k, maas r and sipsas s (2005) Evaluation of the digestible value of lupin and soybean protein concentrates and isolates when fed to rainbow trout, Oncorhynchus mykiss, using either stripping or settlement faecal collection methods, Aquaculture, 245, 211–20. glencross bd, hawkins we, evans d, mccafferty p, dods k, jones jb, sweetingham m, morton l, harris d and sipsas s (2006) Evaluation of the influence of the lupin alkaloid, gramine when fed to rainbow trout (Oncorhynchus mykiss), Aquaculture, 253, 512–22. glencross bd, booth m and allan gl (2007a). A feed is only as good as its ingredients – A review of ingredient evaluation for aquaculture feeds, Aquaculture Nutrition, 13, 17–34. glencross bd, hawkins we, evans d, mccafferty p, dods k, and sipsas s (2007b) Heat damage during some drying techniques affects nutrient utilisation, but not digestibility of lupin protein concentrates fed to rainbow trout (Oncorhynchus mykiss), Aquaculture, 265, 218–29. glencross bd, hawkins we, vietch c, dods k, mccafferty p and hauler rc (2007c) Assessing the effect of dehulling efficiency of lupin (Lupinus angustifolius) meals

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on their digestible nutrient and energy value when fed to rainbow trout (Oncorhynchus mykiss), Aquaculture Nutrition, 13, 462–70. glencross bd, hawkins we, evans d, rutherford n, mccafferty p, dods k, karopoulos m, veitch c, sipsas s and buirchell b (2008a) Variability in the composition of lupin (Lupinus angustifolius) meals influences their digestible nutrient and energy value when fed to rainbow trout (Oncorhynchus mykiss), Aquaculture, 277, 220–30. glencross bd, smith dm, carter cg (2008b) A comparison of the digestibility of lupin kernel meals when fed to rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and Black tiger shrimp (Penaeus monodon), in Glencross B (ed.), Aquaculture Feed Grains Program – Final Report, Department of Fisheries, Hillarys, WA, 453–70. gomes ef, rema p, kaushik sj (1995) Replacement of fish meal by plant proteins in the diet of Rainbow Trout (Oncorhynchus mykiss): digestibility and growth performance, Aquaculture, 130, 177–86. hawkins we, glencross bd, maas r, karopoulos m and hauler r (2008) Effect of lupin kernel meal inclusion on extruded salmonid pellet characteristics, in Glencross B (ed.), Aquaculture Feed Grains Program – Final Report, Department of Fisheries, Hillarys, WA, 471–95. helland s, grisdale-helland b and nerland s (1996) A simple method for the measurement of daily feed intake of groups of fish in tanks, Aquaculture, 139, 156–63. higgs da, mcbride jr, markert jr, dosanjh bs, plotnikoff md and clarke wc (1982) Evaluation of Tower and Candle rapeseed protein concentrate as protein supplements in practical dry diets for juvenile chinook salmon (Oncorhynchus tshawytscha), Aquaculture, 29, 1–31. ishikawa m, teshima s, kanazawa a and koshio s (1996) Evacuation of inert markers in digestibility determination, 5α-cholestane and chromic oxide, in the prawn Penaeus japonicus, Fisheries Science, 62, 229–34. jiang z (2001) Ingredient variation: Its impact and management, in van der Poel AFB, Vahl JL and Kwakkel RP (eds.), Advances in Nutritional Technology 2001. Proceedings of the 1st World Feed Conference, 7–8 November, Utrecht, Wageningen Academic, Wageningen, 47–56. jobling m, arnesen am, baardvik bm, christiansen js and jorgensen eh (1995) Monitoring feeding behaviour and food intake: methods and applications, Aquaculture Nutrition, 1, 131–43. juell je (1991) Hydroacoustic detection of food waste – a method to estimate maximum food intake of fish populations in sea cages, Aquacultural engineering, 10, 207–17. kaushik sj (1998) Nutritional bioenergetics and estimation of waste production in non-salmonids, Aquatic Living Resources, 11, 311–18. kaushik sj, cravedi jp, lalles jp, sumpter j, fauconneau b and laroche m (1995) Partial or total replacement of fish meal by soybean protein on growth, protein utilization, potential estrogenic or antigenic effects, cholesterolemia and flesh quality in rainbow trout, Oncorhynchus mykiss, Aquaculture, 133, 257–74. kolditz c, lefevre f, borthaire m and medale f (2007) Transcriptome and proteome analysis of changes induced in trout liver by suppression of dietary fish oil, FASEB J, 21(6), A1402-d-1403. kolditz c, borthaire m, richard n, corraze g, panserat s, vachot c, lefevre f, medale f (2008) Liver and muscle metabolic changes induced by dietary energy content and genetic selection in rainbow trout (Oncorhynchus mykiss), American Journal of Physiological Regulation and Integrated Comparative Physiology, 294, 1154–64.

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krogdhal å, lea tb and olli jj (1994) Soybean proteinase inhibitors affect intestinal trypsin activities and amino acid digestibilities in rainbow trout (Oncorhynchus mykiss), Comparative Biochemistry and Physiology, 107A, 215–19. krogdhal å, bakke-mckellep am, roed kh and baeverfjord g (2000) Feeding Atlantic salmon Salmo salar L. soybean products: effects on disease resistance (furunculosis), and lysozyme and IgM levels in intestinal mucosa, Aquaculture Nutrition, 6, 77–84. lilleeng e, froystad mk, vekterud k, valen ec and krogdahl å (2007) Comparison of intestinal gene expression in Atlantic cod (Gadus morhua) fed standard fish meal or soybean meal by means of suppression subtractive hybridisation and real-time PCR, Aquaculture, 267, 269–83. maynard la and loosli jk (1969) Animal Nutrition, 6th edn. New York, McGraw-Hill. mccafferty p and dods k (2008) Developing an in-vitro assessment method for heat damage of proteins and feed quality determination, in Glencross B (ed.), Aquaculture Feed Grains Program – Final Report, Department of Fisheries, Hillarys, WA, 290–6. morales ae, cardenete g, de la higuera m and sanz a (1994) Effects of dietary protein source on growth, feed conversion and energy utilisation in rainbow trout, Oncorhynchus mykiss, Aquaculture, 124, 117–26. morales ae, cardenete g, sanz a and de la higuera m (1999) Re-evaluation of crude fibre and acid-insoluble-ash as inert markers, alternative to chromic oxide, in digestibility studies with rainbow trout, Oncorhynchus mykiss, Aquaculture, 179, 71–9. mwachireya sa, beames rm, higgs da and dosanjh bs (1999) Digestibility of canola protein products derived from the physical, enzymatic and chemical processing of commercial canola meal in rainbow trout, Oncorhynchus mykiss (Walbaum) held in freshwater, Aquaculture Nutrition, 5, 73–82. nengas i, alexis mn and davies sj (1999) High inclusion levels of poultry meals and related by-products in diets for gilthead seabream Sparus aurata L, Aquaculture, 179, 13–23. nrc (national research council) (1993) Nutrient Requirements of Fish, National Academy Press, Washington, DC. nir i and ptichi i (2001) Feed particle size and hardness: Influence on performance, nutritional, behavioural and metabolic aspects, in van der Poel AFB, Vahl JL and Kwakkel RP (eds.), Advances in Nutritional Technology 2001. Proceedings of the 1st World Feed Conference, Utrecht, November 7–8. Wageningen, 157–86. olsen re, hansen ac, rosenlund g, hemre gi, mayhew tm, knudsen dl, eroldogan ot, myklebust r and karlsen o (2007) Total replacement of fish meal with plant proteins in diets for Atlantic cod (Gadus morhua L.) II – Health aspects, Aquaculture, 272, 612–24. oste re and sjodin p (1984) Effect of Maillard reaction products on protein digestion. In vivo studies in rats, Journal of Nutrition, 114, 2228–34. panserat s, plagnes-juanm e and kaushik s (2001) Nutritional regulation and tissue specificity of gene expression for proteins involved in hepatic glucose metabolism rainbow trout (Oncorhynchus mykiss), The Journal of Experimental Biology, 204, 2351–60. panserat s, kirchener s and kaushik s (2007) Nutrigenomics, in Nakagawa H, Sato M and Gatlin D III (eds), Dietary Supplements for the Health and Quality of Cultured Fish, CABI, Reading, 210–29. panserat sp, kolditz c, richard ng, plagnes-juan e, piumi f, esquerré d, médale f, corraze g and kaushik s (2008) Hepatic gene expression profiles in juvenile rainbow trout (Oncorhynchus mykiss) fed fishmeal or fish oil-free diets, British Journal of Nutrition, 100, 953–67.

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petterson ds, harris dj, rayner cj, blakeney ab and choct m (1999) Methods for the analysis of premium livestock grains, Australian Journal of Agricultural Research, 50, 775–87. pond mj, stone dm and alderman dj (2006) Comparison of conventional and molecular techniques to investigate the intestinal microflora of rainbow trout (Oncorhynchus mykiss), Aquaculture, 261, 194–203. refstie s, storebakken t and roem aj (1998) Feed consumption and conversion in Atlantic salmon (Salmo salar) fed diets with fish meal, extracted soybean meal or soybean meal with reduced content of oligosaccharides, trypsin inhibitors, lectins and soya antigens, Aquaculture, 162, 301–12. refstie s, glencross b, landsverk t, sørensen m, lilleeng e, hawkins w and krogdahl å (2006) Digestive function and intestinal integrity in Atlantic salmon (Salmo salar) fed kernel meals and protein concentrates made from yellow or narrow-leafed lupins, Aquaculture, 261, 1382–95. reverter m, lundh t, and lindberg je (1999) Ileal amino acid digestibilities in pigs of barley-based diets with inclusion of lucerne (Medicago sativa), white clover (Trifolium repens), red clover (Trifolium pratense) or perennial ryegrass (Lolium perenne), British Journal of Nutrition, 82, 139–47. ringø e (1995) Does chromic oxide (Cr2O3) affect faecal lipid and intestinal bacterial flora in Arctic charr, Salvelinus alpinus (L.)?, Aquaculture and Fisheries Management, 24, 767–76. ringø e, sperstad s, myklebust r, refstie s and krogdahl å (2006) Characterisation of the microbiota associated with intestine of Atlantic cod (Gadus Morhua L.): The effect of fish meal, standard soybean meal and a bioprocessed soybean meal, Aquaculture, 261, 829–41. rutherfurd sm, moughan pj and van osch l (1997) Digestible reactive lysine in processed feedstuffs: application of a new bioassay, Journal of Agricultural Food Chemistry, 45, 1189–94. searcy-bernal r (1995) Statistical power and aquacultural research, Aquaculture, 127, 371–88. shearer kd (1994). Factors affecting the proximate composition of cultured fishes with. emphasis on salmonids, Aquaculture, 119, 63–88. shearer kd (2000) Experimental design, statistical analysis and modelling of dietary nutrient requirement studies for fish: a critical review, Aquaculture Nutrition, 6, 91–102. shiau sy and liang hs (1995) Carbohydrate utilization and digestibility by tilapia Oreochromis niloticus x O. aureus, are affected by chromic oxide inclusion in the diet, Journal of Nutrition, 125, 976–82. smith dm and tabrett sj (2004) Accurate measurement of in vivo digestibility of shrimp feeds, Aquaculture, 232, 563–80. stone daj, allan gl, parkinson s and rowland sj (2000) Replacement of fish meal in diets for Australian silver perch, Bidyanus bidyanus. III. Digestibility and growth using meat meal products, Aquaculture, 186, 311–26. stone daj, gaylord tg, johansen ka, overturf k, sealey wm and hardy rw (2008) Evaluation of the effects of repeated fecal collection by manual stripping on the plasma cortisol leels, TNF-a gene expression, and digestibility and availability of nutrients from hydrolysed poultry and egg meal by rainbow trout, Oncorhynchus mykiss (Walbaum), Aquaculture, 275, 250–9. sugiura sh, dong fm, rathbone ck and hardy rw (1998) Apparent protein digestibility and mineral availabilities in various feed ingredients for salmonid feeds, Aquaculture, 159, 177–202. sugiura sh, babbit jk, dong fm and hardy rw (2000) Utilization of fish and animal by-product meals in low-pollution feeds for rainbow trout Oncorhynchus mykiss (Walbaum), Aquaculture Research, 31, 585–93.

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thomas j and van der poel afb (2001) Functional properties of diet ingredients: Manufacturing and Nutritional Implications, in van der Poel AFB, Vahl JL and Kwakkel RP (eds), Advances in Nutritional Technology 2001. Proceedings of the 1st World Feed Conference, Utrecht, November 7–8, Wageningen, 109–22. thomassen ms and rosjo c (1989) Different fats in feed for salmon: influence on sensory parameters, growth rate and fatty acids in muscle and heart, Aquaculture, 79, 129–35. uran pa schrama jw, rombout jhwm, obach a, jensen l, koppe w and verreth jaj (2007) Soybean meal-induced enteritis in Atlantic salmon (Salmo salar L.) at different temperatures, Aquaculture Nutrition, 13, 1–7. vandenberg gw and de la noue j (2001) Apparent digestibility comparison in rainbow trout (Oncorhynchus mykiss) assessed using three methods of faeces collection and three digestibility markers, Aquaculture Nutrition, 7, 237–45. vilhelmsson ot, martin sam, medale f, kaushik sj and houlihan df (2004) Dietary plant-protein substitution affects hepatic metabolism in rainbow trout (Oncorhynchus mykiss), British Journal of Nutrition, 92, 71–80. watanabe t, takeuchi t, satoh s and kiron v (1996). Digestible energy: methodological influences and mode of calculation, Fish Science, 62, 288–92. williams kc, barlow cg, rodgers lj and ruscoe i (2003a) Potential of meat meal to replace fish meal in extruded dry diets for barramundi, Lates calcarifer (Bloch). I. Growth performance, Aquaculture Research, 34, 23–32. williams kc, patterson bd, barlow cg, ford a and roberts r (2003b) Potential of meat meal to replace fish meal in extruded dry diets for barramundi, Lates calcarifer (Bloch). II. Organoleptic characteristics and fatty acid composition, Aquaculture Research, 34, 33–42. wilson rp and poe we (1985) Apparent digestible protein and energy coefficients of common feed ingredients for channel catfish, The Progressive Fish Culturalist, 47, 154. windell jt, foltz jw and sarokan ja (1978) Effect of body size, temperature and ration size on the digestibility of a dry pelleted diet by rainbow trout, Transactions of the American Fisheries Society, 107, 613–16. wybourne ba and carter cg (1999) The effect of plant meal inclusion on feed intake and nutritional adaptation by Atlantic salmon, Salmo salar L, in Fishmeal Replacement in Aquaculture Feeds for Atlantic Salmon, Project 93/120, Fisheries Research and Development Corporation, Deakin, ACT 100–26.

14 Quantifying nutritional requirements in aquaculture: the factorial approach I. Lupatsch, Swansea University, UK

Abstract: Growth and the feed required to sustain this growth is of major importance in aquaculture. To improve productivity and profitability within this industry we must provide feeds that supply adequate levels of energy and protein in order to sustain efficient growth. In view of the diversification of fish farming a general approach is needed to define energy and protein requirements so that each fish species can realize its full growth and economic potential. Growth is usually defined as deposition of new body components, which in fish is predominately protein and lipid. In addition to the maintenance requirement the feed must supply the precursors for new tissue production, as well as the energy necessary for synthesis of both protein and lipid. Thus, the quantification of energy and protein requirement in the growing fish is the sum of the needs for maintenance and growth. The significance of this approach is that energy and protein needs are quantified in terms of absolute requirements per unit of body weight and weight gain and only subsequently expressed as a percentage of the diet. As energy and protein intake is a function of feed consumption and its composition, it is necessary to foresee the amount of feed the fish is physically able to consume in order to adjust the energy and protein content of a potential feed. From the comparison of several fish species it can be concluded, that utilization of energy and protein for deposition of weight gain do not appear to be very different across the species examined. Differences are found, however, in the magnitude and the composition of the weight gain, which ultimately determines the amount of energy and protein required. Key words: factorial modelling, energy utilization, protein efficiency, bioenergetics.

14.1 Introduction To improve productivity and profitability of aquaculture we have to provide feeds that supply adequate levels of energy and protein to sustain optimal

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growth. Proper feed management is also crucial with regard to the aquatic environment, since feed that is neither consumed nor available to the fish will be lost to the effluent surroundings and will result in nutrient enrichment of the water body. Due to the shortage of fish meal and fish oil, alternative ingredients have to be chosen, but in order to utilize them appropriately the requirements of the fish have to be known. This can only be done by quantifying the key nutrient requirements of fish and evaluating the nutritional characteristics of the feed ingredients (composition, digestibility, limiting factors), so that least-cost formulations can optimize the balance between nutrient requirements and the cost of feeds. Nutrient requirements in fish are often quantified by dose–response relationships, where diets containing graded levels of a nutrient are fed and the resulting growth is measured. The quantitative requirement for the nutrient is then considered at the level below which the growth will be depressed or above which it will not increase (Zeitoun et al., 1976; Mercer, 1982). These methods, however, are time-consuming and limited in their broad application as the conditions under which the requirements were determined may not permit extrapolation (Baker, 1986). This fact is emphasized particularly in regards to effects of dietary protein and energy supply on performance in fish. Despite a vast body of information, results concerning the optimal protein requirement even for the same species are often not in agreement. Energy and protein requirements are very complex as they are closely linked. Without protein there is no growth, but neither is there growth without energy. Since protein can function as an energy source in addition to its essential role for growth, the optimal balance between the supply of dietary non-protein energy and protein should be determined. Considerable progress has been made in recent years in the study of the dietary nutrient requirements of fishes, mainly salmonids. Feeding charts for trout based on nutritional bioenergetics have been introduced by Cho and Kaushik (1990), Cho (1992) and Cho and Bureau (1998); however, information concerning the prediction of growth and energy and especially protein needs is still lacking for a number of fish species. Compared to terrestrial domesticated husbandry, which relies on a few selected species, aquaculture is utilizing an increasing variety of fish that show distinct differences with respect to feed requirements and conversion efficiencies. In addition, target species for aquaculture range in their feeding preferences from carnivorous, omnivorous to herbivorous. Thus, it is not always clear if differences in feed efficiencies that are reported for various species are due to true biological differences or the different conditions used in the various studies. One of the recurring questions in aquaculture is whether the farming of carnivorous species is sustainable. Carnivores are thought to require 45–50 % dietary protein while most omnivores and herbivores require only 24–32 % protein in their feeds. This gives the impression that herbivorous species are more efficient converters of protein into growth. However,

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protein requirements expressed solely on the basis of dietary inclusion levels are incomplete if feed intake is not considered. Protein intake is the product of the protein content of the feed and the total amount of feed consumed. As such, the protein demand per kg of fish produced will give a clearer picture of the overall efficiency of the species in question. The following is an attempt to quantify the nutritional requirements of fish that are very different in their natural feeding habitat, ranging from carnivorous to omnivorous, marine and freshwater. It focuses on species that are already well established in aquaculture like the gilthead sea bream (Sparus aurata), the European sea bass (Dicentrarchus labrax) and tilapia (Oreochromis sp.), and farmed all over the world in addition to new candidates like the white grouper (Epinephelus aeneus), the Asian sea bass (Lates calcarifer), also known as Barramundi, as well as the grey mullet (Mugil cephalus) (Lupatsch et al., 2001, 2003a,b; Lupatsch and Kissil, 2005). The factorial approach, which had been used in classical animal nutrition for decades to quantify requirements, is applied to assist in a better understanding of the underlying principles involved, and to point out not only the differences but also the common ground among fish species.

14.2 Quantification of nutritional requirements 14.2.1 Methodology Nutrient requirements are generally described for animals of a given age and for specific physiological functions, such as maintenance, reproduction or growth. In fish farming growth of fish flesh is one of the major goals of production. Growth itself is typically defined as deposition of new body components, which in fish consists mainly of protein, lipid and water. In addition to the requirement for maintenance, the feed must supply the precursors for synthesis of new tissue, and also the energy needed to deposit both protein and lipid. The following outlines the principles of the factorial approach for evaluating the energy and protein efficiencies for growth in fish. According to this, the energy and protein requirements are considered the sum for maintenance plus growth. The requirement for maintenance is mainly a function of the size of the fish and water temperature, and is proportional to the metabolic body weight in the form of a × BW(kg)b, where a is a constant, characteristic of a certain fish species at a set temperature and b is the exponent of the metabolic body weight. The requirement for growth is dependent on the amount and the composition of the weight gain, including the cost of energy to deposit the new growth. The actual requirement for dietary gross energy and protein must take into account the partial efficiency of utilisation of these nutrients for maintenance and for growth. The significance of this approach is that protein and energy needs are expressed primarily in terms of absolute

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demand per fish body mass and anticipated weight gain. As energy and protein consumption is the product of feed intake and feed composition the voluntary maximum feed intake as well as the composition of the feed have to be considered. Feeds can then be formulated and feeding tables established which are based on daily requirements for energy and protein dependent on anticipated growth. The following equation specifies the formal approach to those calculations: requirement = a × BW ( kg ) + c × gain b

( kg )b: Metabolic body weight where a is the constant for given conditions (activity, temperature) expressed in kJ per unit of metabolic weight and characteristic of a fish species, b is the exponent of the metabolic body weight and converts absolute weight to metabolic weight correcting for the decrease in metabolic rate per unit of body weight as fish grow and c is the cost in units of energy or protein to deposit new growth. The parameters to obtain for quantifying the requirements in fish are thus the following: • growth data – to describe daily potential weight gain along the growth cycle at different temperatures; • feed intake – a prediction of the maximum voluntary feed intake; • change in body composition along the growth cycle; • maintenance requirements for energy and protein at different temperatures; • efficiency of utilization of dietary energy and protein to deposit energy and protein as growth.

14.2.2 Growth and feed intake We might assume that different fish have a genetically determined asymptotic body size and that they are capable of adjusting their feed or energy intake to realize their genetic potential. Thus, a prerequisite for estimating feed requirements of a newly cultured fish species is to define its maximal potential for growth. This modelling requires growth data from trials, where feed supply in terms of energy and nutrients is not limiting and optimal growing conditions are met. Therefore, one of the first steps to determine energy and protein demand should be to establish a simple growth model described for various water temperatures. As an example, the growth potential of tilapia is illustrated in Fig. 14.1 and as expected daily weight gain is strongly dependent upon increasing water temperatures. The equation defining this relationship between daily weight gain, fish size as well as temperature appears later in this section [Eq. 14.1].

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421

Daily weight gain (g fish–1 day–1)

4.5 4.0 3.5 3.0 2.5 2.0 1.5

Temp 29 °C

1.0

Temp 26 °C Temp 24 °C Temp 22 °C

0.5 0.0 0

50

100

150 200 250 300 Fish weight (g)

350

400

450

Fig. 14.1 Daily weight gain (g) in relation to increasing body weights in tilapia fed to apparent satiation at increasing temperatures.

In contrast to terrestrial animals, fish seem to grow continuously. Growth does not cease and reaches an asymptote, which in aquaculture, however, might never be attained. Growth rates in aquaculture have been in the past typically described using the specific growth rate (SGR) or as absolute growth in g per day. As growth is affected by temperature like in all poikilotherms, it increases with increase in temperature up to an optimum above which growth decreases, until the upper lethal temperature is reached. Although SGR and absolute weight gain are dependent upon feed intake and water temperature, their main dependence is on the size of the fish, and as a result they cannot be compared among groups of fish having different weights. In studies with various fish species (Lupatsch et al., 2003a) growth along the growing cycle could be best described by using allometric equations relating the daily weight gain (y) dependent upon fish weight (x) with water temperature (T) as the additional variable. The voluntary feed intake in fish is expressed in a similar manner:

y = a × BW ( g ) × ec ×T b

This equation can be integrated as shown below and the weight BWt after t days can be predicted starting from BW0: BWt = [ BW0c 1 + c2 ⋅ ec 3⋅T ⋅ days]

c4

with c1 = (1 − b) c2 = (1 − b) ⋅ a

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New technologies in aquaculture c3 = temperature coefficient c4 = 1 (1 − b)

To describe the growth especially for salmonids the thermal-unit growth coefficient (TGC) as suggested by Cho (1992) and based on the model proposed by Iwama and Tautz (1981) has gained relatively wide acceptance in fish nutrition research by using the following equation: Wt = [W00.333 + ∑ (TGC ⋅T ⋅ days)]

3

This equation in its general form is equivalent to the formulae for fish used in the present study (eqs 14.1–14.7), which of course is not too surprising as biological principles of fish growth should be the same. Nonetheless, the main difference is that weight exponents other than 1 − b = 0.333 are used in the growth models described here in contrast to the TGC model, thus they should result in a better description of the growth pattern observed across the life cycle of a fish species. The growth potential itself (and therefore the coefficients of the equation) would of course be typical for different fish species, and even different genetic strains, as described in eqs 14.1–14.7. As energy and protein intake is a function of feed intake as well as content of feed, it is necessary to predict the amount of feed that the fish is physically able to consume; this is needed to adjust the energy and protein content of a potential feed. Feeds can then be formulated and feeding tables established which are based on daily requirements for energy and protein dependent on anticipated growth. Figure 14.2 depicts the relationship between daily weight gain (g), feed intake (g) and the body weight (g) of gilthead sea bream. The lines describe the relationships at an average water temperature of 23 °C for both feed intake and weight gain. As can be seen, absolute weight gain as well as the amount of food eaten increases with increasing weight, while feed intake increases at a higher rate than weight gain. Examples of the growth potential of several species are summarized in the equations below together with an estimation of voluntary feed intake (eqs 14.1–14.8). In those studies fish were fed to satiation in a controlled system and the feed intake, depending on size and water temperature, determined (Lupatsch, 2003a,b; Lupatsch, 2008). Tilapia Weight gain = 0.0113 × BW ( g )

0.547

× e0.090 ×T

[14.1]

or Wt = ( W00.453 + 0.00512 × e0.090 ×T × days]2.207 Feed intake = 0.0156 × BW ( g )

0.600

× e0.085×T

[14.2]

Weight gain and feed intake (g fish–1 day–1)

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423

5.0 Feed intake

4.5

Weight gain

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0

50

100 150 200 250 300 350 400 450 500 Fish weight (g)

Fig. 14.2 Daily weight gain (g) and feed intake (g) in relation to increasing body weight in gilthead sea bream fed to satiation.

Gilthead sea bream Weight gain = 0.024 × BW ( g )

0.514

× e0.060 ×T

[14.3]

or Wt = ( W00.486 + 0.01166 × e0.060 ×T × days]2.060 Feed intake = 0.017 × BW( g )

0.652

× e0.064 ×T

[14.4]

× e0.030 ×T

[14.5]

White grouper Weight gain = 0.062 × BW ( g )

0.558

or Wt = (W00.442 + 0.0274 × e0.030 ×T × days]2.262 Feed intake = 0.058 × BW ( g )

0.600

× e0.027 ×T

[14.6]

Asian sea bass Weight gain = 0.0051 × BW ( g )

0.508

× e0.135×T

[14.7]

or Wt = (W00.492 + 0.0025 × e0.135×T × days]2.033 Feed intake = 0.0057 × BW ( g )

0.576

× e0.121×T

[14.8]

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New technologies in aquaculture

14.2.3 Composition of weight gain As a large proportion of the energy and protein consumed by the fish is retained as growth, the composition of the gain is an additional factor determining the subsequent energy and protein requirement. When measuring whole body composition of fish at increasing sizes, each unit weight gain is assumed to equal the body composition at a certain size. As pictured in Fig. 14.3 for gilthead sea bream, moisture content and energy concentrations change with increasing fish weights. The protein content on the other hand is quite constant regardless of fish size and is on average 175 mg g−1. The fact that protein content remains quite stable and energy content is increasing with increasing fish size is typical for most fish (see Fig. 14.4). Therefore, in estimating requirements for tissue deposition and growth, wide variations between species in terms of energy needs are expected based on the differing tissue composition. For example, relatively energy dense gilthead sea bream require more dietary energy per unit of weight gain than leaner fish such as tilapia and grouper. It is noteworthy, however, that the demand for dietary protein per unit of weight gain remains similar irrespective of fish species (eqs 14.9–14.12). Gilthead sea bream Energy content of carcass ( kJ g −1 ) = 4.66 × BW ( g ) Protein content ( mg g −1 )

0.139

[14.9]

0.055

[14.10]

= 175

Tilapia Energy content of carcass ( kJ g −1 ) = 5.53 × BW ( g ) Protein content ( mg g −1 )

Moisture

700

Energy

Protein

16 14

600

12

500

10

400

8

300

6

200

4

100

2

0 0

Energy content (kJ g–1 fish)

18

800 Protein content (mg g–1 fish)

= 160

0 50 100 150 200 250 300 350 400 450 500 Fish weight (g)

Fig. 14.3 Proximate body composition (per g wet weight) of gilthead sea bream at increasing sizes and fed a standard diet to satiation.

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425

13

Energy content (kJ g–1 fish)

12 11 10 9 8 7 6 5 4

Gilthead seabream

3

Asian sea bass

Tilapia White grouper

2 0

50

100 150 200 250 300 350 400 450 500 Fish weight (g)

Fig. 14.4 Comparison of energy contents (kJ per g wet weight) of several species relative to fish size.

White grouper Energy content of carcass ( kJ g −1 ) = 5.01 × BW ( g ) Protein content ( mg g −1 )

0.056

[14.11]

0.104

[14.12]

= 169

Asian sea bass Energy content of carcass ( kJ g −1 ) = 4.51 × BW ( g ) Protein content ( mg g −1 )

= 170

14.2.4 Metabolic body weight Metabolic rate in fish is related to body weight and to temperature. Fish require energy for maintaining normal processes of life such as blood circulation, osmoregulation, excretion and movement, regardless of whether or not feed is consumed. Depending on the activity, several metabolic levels can be distinguished: standard, routine and active metabolism. Metabolic rate, at all levels of activity, depends largely on the size of the fish, and is proportional to the metabolic body weight in the form of a × BW(kg)b. Two major methods have been used to determine energy requirements in animals: direct and indirect calorimetry; however, most researchers have used indirect calorimetry in fish. The latter method estimates the energy demand of fish indirectly through measurements of oxygen consumption, but it can also include comparative carcass analysis.

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The comparative carcass analysis was employed in the present examples to measure the caloric value of the tissues utilized during starvation for fish of increasing sizes. This method was chosen as the most feasible and applicable. Fish could be kept in groups in a tank, move freely and the duration of each testing period was sufficiently long. The daily loss of energy as well as protein can be thus calculated in relation to body weight. The relationships between daily energy and protein loss and fish weight are not linear and results were fitted to ln–ln functions as have traditionally been used by animal nutritionists to express metabolic body weight (MBW). The antilog of these functions describes the allometric relationship common in biological measurements. The relationship between energy loss (kJ fish−1 day−1), protein loss (g fish−1 day−1) and fish weight (g) for gilthead sea bream are depicted in Fig. 14.5. For several fish species the daily loss of energy and protein respectively can be determined as follows: Gilthead sea bream Energy loss (kJ fish −1 day −1 ) = 41.5 × BW ( kg ) Protein loss ( g fish −1 day −1 ) = 0.40 × BW ( kg )

0.82

[14.13]

0.70

[14.14]

European sea bass Energy loss (kJ fish −1 day −1 ) = 34.6 × BW ( kg ) Protein loss ( g fish day −1

−1

) = 0.39 × BW ( kg)

0.79

[14.15]

0.69

[14.16]

White grouper Energy loss (kJ fish −1 day −1 ) = 26.3 × BW ( kg ) Protein loss ( g fish day

−1

) = 0.34 × BW ( kg)

[14.17]

0.70

[14.18] 0.00

0 –2 –4 –6 –8 –10 –12 –14 –16 –18 –20 –22

–0.05 –0.10 –0.15 –0.20 –0.25 –0.30 Energy Protein

–0.35 –0.40

Protein loss (g fish–1 day–1)

Energy loss (kJ fish–1 day–1)

−1

0.80

–0.45 0

50 100 150 200 250 300 350 400 450 Fish weight (g)

Fig. 14.5 Daily energy (kJ) and protein loss (g) in gilthead sea bream in relation to body size.

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427

Asian sea bass Energy loss (kJ fish −1 day −1 ) = 23.4 × BW ( kg ) Protein loss ( g fish day −1

−1

) = 0.24 × BW ( kg)

0.82

0.69

[14.19] [14.20]

The expressions of (kg)0.82, (kg)0.79, (kg)0.80 and (kg)0.82 can thus be described as the metabolic weights for gilthead sea bream, European sea bass, white grouper and Asian sea bass, respectively. No significant differences could be found among the fish species concerning the exponent b of the metabolic body weight (kg)b for energy, which was on average 0.80, giving a strong indication that this might be a common value for fish, as has been suggested already by Brett and Groves (1979). This also means that the rate of increase in energy metabolism with weight is higher in fish than in birds and mammals. The relationship between a fish’s protein metabolism and its body weight conforms also to the allometric equation: a × BW(kg)b. The exponent of the metabolic weight for protein metabolism in fish was shown to be on average b = 0.70. Data correlating protein metabolism to different fish weights are sparse, and most authors have assumed a common exponent for the relationship between energy or protein loss and body weight. However, the best fit between protein loss and body weight in fish was reached using a metabolic body weight with an exponent of b = 0.70. The similarities of these coefficients in various species indicate that protein and energy metabolic rate cannot be described by the same metabolic body weight. 14.2.5 Efficiency for energy and protein utilization Loss at starvation as described in eqs 14.13–14.20 is only an approximation of maintenance energy requirements. To quantify the maintenance requirements (zero energy and protein balance) and to define the efficiency of energy utilization for growth, fish are fed increasing amounts of known digestible energy and protein from zero feed up to maximum intake as described in detail by Lupatsch et al. 2001, 2003a,b and Lupatsch and Kissil, 2005. Figure 14.6 shows an example of growth trials with gilthead sea bream that were performed at three different temperature regimes using fish of various sizes. The experimental diets were based mainly on fish meal, fish oil and cornstarch and digestibility was determined beforehand. Fish meal was chosen as the primary protein source because of its balanced amino acid profile. To examine the relationship between dietary energy consumed and energy gained for different sized fish both DE intake (x) and energy retained (y) were expressed per metabolic body weight (MBW) of kg0.80. For the three trials performed at different temperatures, the following linear regressions were obtained (Fig. 14.6): Temp 20 °C

y = −30.2 + 0.69 x

[14.21]

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New technologies in aquaculture

Energy gain (kJ kg–0.80 day–1)

150 125

Temp 20.5 °C

100

Temp 24 °C

75

Temp 27 °C

50 25 0 –25 –50 0

50

100

150

200

250

300

Digestible energy fed (kJ kg–0.80 day–1)

Fig. 14.6 Daily energy retention per unit metabolic body weight of kg0.80 in gilthead sea bream fed increasing levels of digestible energy at different temperatures. Corresponding eqs 14.21–14.23 are presented in the text.

Temp 24 °C

y = −38.1 + 0.67 x

[14.22]

Temp 27 °C

y = −47.1 + 0.66 x

[14.23]

For the sea bream at zero energy retention (y = 0) the required intake of digestible energy (DE) can then be calculated as the daily maintenance requirement DEmaint at increasing temperatures: a = 43.7 kJ at 20.5 °C, 56.9 kJ at 24 °C and 71.4 kJ at 27 °C each describing the value a in the expression a × BW(kg)0.80. The efficiency of DE for growth is defined by the slope of the lines and is nearly identical at the different temperatures with an average value of 0.67. The reciprocal 1/0.67 = 1.49 describes the cost of DE (kJ) per unit of energy deposited (kJ). The relationship between dietary DP intake and protein retained, both referring to a MBW of (kg)0.70, shows a similar linear response in sea bream and the relationships can be described by the following equations: Temp 20 °C

y = −0.25 + 0.46 x

[14.24]

Temp 24 °C

y = −0.30 + 0.46 x

[14.25]

Temp 27 °C

y = −0.39 + 0.48 x

[14.26]

In the case of protein, the efficiency of utilization can again be defined by the slope of the lines and amounts to an average value of 0.47 at the three temperatures. Corresponding to the protein efficiency the reciprocal of 1/0.47 = 2.13 describes the cost of DP (g) per unit of protein deposited (g). It must be pointed out, however, that this value is only appropriate for

Quantifying nutritional requirements in aquaculture

429

a dietary protein with a balanced amino acid profile like fish meal as used in the present studies. In practical diets, which might be limiting in one or two amino acids, the protein efficiency will be reduced. Maintenance requirement for protein is dependent on temperature as well and can be determined as DPmaint = 0.54 g at 20 °C, 0.66 g at 24 °C and 0.81 g at 27 °C, each presenting the value a in the expression a × BW(kg)0.70 day−1. The maintenance requirement for energy increases at higher temperatures in sea bream as expected and shows a linear response from 43.7 kJ to up to 71.4 kJ × BW (kg)0.80 in the range between 20 and 27 °C (Fig. 14.7). The same can be said for the protein maintenance requirement and temperature; thus maintenance requirements in relation to temperature in gilthead sea bream can be well described by the following equations (only between 20 to 27 °C): Maintenance requirement for energy in kJ fish−1 day−1: DEmaint = ( 4.38 × T (°C ) − 45.65) × ( kg )

0.80

[14.27]

Maintenance requirement for protein in g fish−1 day−1: DPmaint = ( 0.045 × T (°C ) − 0.42) × ( kg )

0.70

[14.28]

90

1.10 Energy Protein

80

1.00

70

0.90

60

0.80

50

0.70

40

0.60

30

0.50 0.40

20 19

20

21

22

23

24

25

26

27

28

29

Constant ‘a’ in DPmaint = a × (kg)0.70 day–1

Constant ‘a’ in DEmaint = a × (kg)0.80 day–1

In growth studies with several fish species using the same methodology, the relationships between daily DE intake (x) and energy retained ( y) both expressed per unit of metabolic weight (kg)0.80 were obtained and are depicted in Fig. 14.8. The linear relationships between daily DE intake (x) and energy retained (y) both expressed per unit of metabolic weight (kg)0.80 as presented in Fig. 14.8 are shown below (at 27 °C):

Temperature (° C)

Fig. 14.7 Relationship between maintenance requirement expressed as a × BW(kg)b versus water temperature in gilthead sea bream.

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New technologies in aquaculture

Energy gained (kJ kg–0.80 day–1)

200 Gilthead sea bream Tilapia White grouper

150 100

Asian sea bass

50 0 –50 0

50

100

150

200

250

300

350

Digestible energy fed (kJ kg–0.80 day–1)

Fig. 14.8 Daily energy retention per unit metabolic body weight of kg0.80 in various fish species fed increasing levels of digestible energy at a temperature of 27 °C.

Gilthead sea bream y = −47.1 + 0.66 x

[14.29]

y = −37.2 + 0.64 x

[14.30]

Tilapia

White grouper y = −34.0 + 0.68 x

[14.31]

Asian sea bass y = −28.3 + 0.67 x

[14.32]

The efficiency of energy utilization (i.e. the slope of energy gain as a function of DE intake) is remarkably similar, ranging between 0.64 and 0.68 for the fish species, but differences are observed for maintenance requirements as depicted in Fig. 14.8. It supports the concept that the energy efficiency for growth is constant and independent of fish weight, feeding level and species, but maintenance requirement expressed per metabolic body weight is species specific. The similarities of energy efficiencies involving salmonids have been also demonstrated in rainbow trout where the utilization of DE for gain was 0.61 regardless of feeding level or temperature (Azevedo et al., 1998). This value is very close to the 0.68 of another study with rainbow trout (Rodehutscord and Pfeffer, 1999). Quantification of energy and protein requirements for maintenance and efficiency of growth for various fish species can thus be described according to the common equation:

Quantifying nutritional requirements in aquaculture

431

Requirements ( fish −1 day −1 ) = a × ( kg ) + c × gain b

and in detail for each specie: Gilthead sea bream DE ( kJ) = ( 4.38 × T − 45.65) × ( kg ) DP ( g ) = ( 0.045 × T − 0.42) × ( kg )

0.80

+ 1.49 × energy gain ( kJ )

[14.33]

0.70

+ 2.13 × protein gain ( g )

[14.34]

+ 1.61 × energy gain ( kJ )

[14.35]

Tilapia DE ( kJ) = ( 3.28 × T − 30.0) × ( kg )

0.80

DP ( g ) = ( 0.048 × T − 0.65) × ( kg )

+ 2.17 × protein gain (g )

[14.36]

0.80

+ 1.49 × energy gain (kJ )

[14.37]

0.70

+ 1.85 × protein gain ( g )

[14.38]

+ 1.48 × energy gain (kJ )

[14.39]

0.70

White grouper DE ( kJ) = ( 3.86 × T − 53.54) × ( kg ) DP ( g ) = ( 0.066 × T − 1.19) × ( kg ) Asian sea bass DE ( kJ) = ( 3.16 × T − 44.4) × ( kg ) DP ( g ) = 0.45 × ( kg )

0.70

0.80

+ 1.96 × protein gain ( g )

[14.40]

14.3 Feed ingredient evaluation Feed is the principal operating cost in the production of fish, and for aquatic feeds the main protein and energy source has traditionally been fish meal. However, there are limits to the continued expansion of aquaculture based upon feeds using fish meal and fish oils, which are costly and limited in supply. The production of successful fish feed formulae relying less on fish meal requires therefore accurate information on the nutritive value of more economical protein sources. In addition to quantifying energy and protein demands, one of the requirements for formulating practical feeds is determination of the digestibility of various feed ingredients. Ideally the nutrient requirements of fish and the nutrient concentration of a foodstuff should be expressed in units of digestible energy and protein so that least-cost formulations can optimize the balance between nutrient requirements and the cost of feeds. The nutritive value of compound diets will depend on the digestibility of the individual ingredients, and nutrient digestibility has been shown to be additive at least for energy, lipid and protein (Lupatsch et al., 1997). Apparent digestibilities of crude protein are on average high regardless of the origin of the protein. There seems to be more variation in protein digestibility of animal meal and their by-products than from plant proteins, possibly due to processing procedures. Some of the differences arise due to treatments such as heating, drum drying or spray drying. Based on digest-

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New technologies in aquaculture

ibility studies with several fish species it can be concluded, that the digestibility coefficients of protein and lipids are generally around 86 % and 92 %, respectively, regardless of fish species, while those for carbohydrates are more variable reflecting the quality or source of carbohydrates in the diets (Lupatsch et al., 1997; Sklan et al., 2004). Carbohydrate availability in trout and salmon has been shown to depend on the level of inclusion in the diet; however, omnivorous fish like, for instance, tilapia should be capable of digesting and absorbing relatively large amounts of carbohydrates. In general, it can be said that differences in digestibility data are mainly due (besides errors in the experimental design) to the ingredient itself, to processing procedures, mechanical or otherwise or, in regards to plant products, to the fibre content. There is less variation among various fish species in regard to protein digestibility of an ingredient than there is among different ingredients. In short, we can assume that protein digestibility is more a characteristic of a particular ingredient, less so of the fish species. For species with similar feeding habits (i.e. omnivores or carnivores), digestible energy values are also similar. Therefore, where there is a lack of actual data, digestibility values obtained from fish that consume similar feed items in nature can be safely adopted to describe the nutritional value of common feed ingredients. Table 14.1 provides average values of the DE and DP content of raw ingredients that are commonly used in practical feed formulations as determined in gilthead sea bream.

Table 14.1 Typical composition of practical feed ingredients (kg−1 on as fed basis)

Fish meal Meat meal Poultry meal Feather meal Fish oil Soybean meal Soya protein Full fat soya Corn gluten Canola protein Wheat gluten Lupin seed Pea seed meal Corn meal Wheat meal Corn starch

Digestible protein (g)

Dry matter

Crude protein (g)

910 930 930 920

637 595 627 828

560 446 502 521

910 910 900 920 920 930 930 940 900 910 910

445 630 351 630 590 778 396 247 110 123 –

394 570 289 567 543 746 356 173 82 98 –

Gross energy (MJ)

Digestible energy (MJ)

19.38 19.44 20.27 22.82 38.50 17.56 18.75 20.78 21.30 21.24 21.20 17.35 16.60 17.24 17.29 15.89

17.24 14.77 15.20 14.83 36.5 11.50 14.21 12.88 16.16 16.78 19.29 9.30 7.30 10.20 11.24 12.71

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433

14.4 Feed formulation and feeding strategies The results of the above mentioned allow calculation of the daily recommended intake for growing fish. By defining the fish’s demands for maintenance and growth a comprehensive energy and protein budget can be derived that essentially quantifies the energy and protein the fish needs to consume to achieve its anticipated growth at any specific temperature and part of its growth cycle as demonstrated for Asian sea bass in Table 14.2. Table 14.2 Calculations of daily energy and protein requirements in growing Asian sea bass (at 27 °C) Body weight, per fish

50 g

250 g

500 g

1.42

3.23

4.59

Energy requirement, kJ fish day Metabolic BW, kg0.80 DEmaint2 Energy gain3 DEgrowth4 DEmaint+growth5 Maintenance as % of total DE

0.091 3.72 9.65 14.28 18.01 20.7

0.330 13.50 25.84 38.24 51.74 26.1

0.574 23.50 39.49 58.45 81.95 28.7

Protein requirement, g fish−1 day−1 Metabolic BW, kg0.70 DPmaint6 Protein gain7 DPgrowth8 DPmaint+growth9 Maintenance as % of total DP

0.123 0.055 0.242 0.475 0.530 10.4

0.379 0.171 0.548 1.075 1.246 13.7

0.616 0.277 0.780 1.529 1.806 15.3

Weight gain1, g day−1 −1

Feed formulation DE content of feed, MJ kg−1 Required feed10, g fish−1 day−1 Feed conversion ratio DP content in feed11, g kg−1 DP DE−1 ratio, g MJ−1 1

−1

15 1.20 0.84 441 29.4

15 3.45 1.07 361 24.1

15 5.46 1.19 331 22.0

Predicted weight gain for Asian sea bass at 27 °C (eq. 14.7). Digestible energy required for maintenance – (3.16 × T − 44.4) × BW(kg)0.80. Expected energy gain = weight gain × energy content of gain (eq. 14.12). 4 Digestible energy required for growth = expected energy gain × 1.48 (cost in units of DE to deposit one unit of energy as growth). 5 Total DE required for maintenance and growth. 6 Digestible protein required for maintenance = 0.45 × BW(kg)0.70. 7 Expected protein gain = weight gain × protein content of gain (170 mg g−1). 8 Digestible protein required for growth = expected protein gain × 1.96 (cost in units of DP to deposit one unit of protein as growth). 9 Total DP required for maintenance and growth. 10 Required feed intake to meet daily requirements while using feeds containing 15 MJ kg−1 DE. 11 Required DP inclusion in feed to meet daily requirements of digestible protein. BW = body weight, DE = digestible energy, DP = digestible protein. 2 3

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The significance of the above mentioned approach is, that protein and energy needs are expressed primarily in terms of absolute demand per fish body mass and anticipated weight gain and only secondarily as a percentage or inclusion of feed. Due to the fact that protein and energy demands are constantly changing, different diets would have to be formulated for growing fish. However, on a practical basis it is unreasonable to expect that a large number of diets would be used to support production of any fish species. It is obvious from Table 14.2, that the proportion of total DE which is required for maintenance will increase with increasing body weight and with decreasing growth rate, thus influencing the feed conversion ratio (FCR). Also, the DP/DE ratio will decrease with increasing fish size and decreasing growth potential due to the changing ratio of energy to protein of the gain and the increasing proportion of energy used for maintenance with increasing fish size. Faster growing fish will usually display a better FCR, as do juveniles of any species. In addition, higher temperatures might have a positive effect on feed efficiency as demonstrated for tilapia in Table 14.3, where at 27 °C the potential for growth is much greater than at 22 °C and the FCR slightly improved. Even though maintenance requirements for energy increase with temperature, this increase is still minor compared to the advantage of higher weight gain, at least for the range of temperatures that are considered optimal for a certain fish species. Differences in nutrient requirements are largely due to the growth potential and the composition of the growth as demonstrated in Table 14.4 for grouper and mullet. On a daily basis grey mullet require more energy than grouper due to the high energy content of the weight gain and greater maintenance requirement. On the other hand, the daily protein requirement of grouper is greater due to the superior growth. The amount of energy and protein supplied to the fish is a function of the amount of feed consumed and the density of energy and protein in that feed. As shown in Table 14.4, it is possible for a formulator to come up with a series of feed formulations to meet the energy and protein requirements of a fish species. The absolute daily protein requirement of a fish is dependent on size and anticipated weight gain regardless of DE content. Therefore, as demonstrated in Table 14.4 the protein level expressed as a percentage of the feed will change according to the selected digestible energy content of 13 or 16 MJ kg−1 feed. However, it is important for the formulator to recognize that the fish has to be physically able to consume all the feed to acquire the energy and protein needed for maximum growth. For instance, a 300 g grey mullet that would grow 2 g day−1 would be able to consume 5.16 g feed on average (Table 14.4). White grouper, on the other hand, would be able to grow 3.36 g per day, but feed intake is just 3.68 g. As the energy requirement of grouper is relatively low and consequently the feed intake, the protein content has to be sufficiently high to satisfy the demand. Fish on a lower trophic level, like mullet or tilapia, could

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Table 14.3 Recommendations of DE and DP supply for tilapia grown at two water temperatures (22 °C and 27 °C) Body weight, per fish

50 g

300 g

Temperature Weight gain1, g day−1

22 °C 0.70

27 °C 1.09

22 °C 1.85

27 °C 2.91

Energy requirement, kJ fish−1 day−1 DEmaint2 Energy gain3 DEgrowth4 DEmaint+growth5 Maintenance as % of total DE

3.84 4.77 7.68 11.52 33.3

5.33 7.48 12.04 17.37 30.7

16.09 14.03 22.58 38.67 41.6

22.35 22.00 35.42 57.77 38.7

Protein requirement, g fish−1 day−1 DPmaint6 Protein gain7 DPgrowth8 DPmaint+growth9 Maintenance as % of total DP

0.050 0.111 0.241 0.291 17.1

0.079 0.175 0.379 0.458 17.3

0.175 0.297 0.643 0.818 21.4

0.278 0.465 1.009 1.287 21.6

Feed formulation DE content of feed, MJ kg−1 Required feed10, g fish−1 day−1 Feed conversion ratio DP content in feed11, g kg−1 DP DE−1 ratio, g MJ−1

13 0.89 1.27 329 25.3

13 1.34 1.23 343 26.4

13 2.97 1.61 275 21.2

13 4.44 1.53 290 22.3

1

Predicted weight gain for tilapia at 22 °C and 27 °C (eq. 14.1). Digestible energy required for maintenance – (3.28 × T − 30.0) × BW(kg)0.80. 3 Expected energy gain = weight gain × energy content of gain (eq. 14.10). 4 Digestible energy required for growth = expected energy gain × 1.61 (cost in units of DE to deposit one unit of energy as growth). 5 Total DE required for maintenance and growth. 6 Digestible protein required for maintenance = (0.048 × T − 0.65) × BW(kg)0.70. 7 Expected protein gain = weight gain × protein content of gain (160 mg g−1). 8 Digestible protein required for growth = expected protein gain × 2.17 (cost in units of DP to deposit one unit of protein as growth). 9 Total DP required for maintenance and growth. 10 Required feed intake to meet daily requirements while using feeds containing 13 MJ kg−1 DE. 11 Required DP inclusion in feed to meet daily requirements of digestible protein. DE = digestible energy, DP = digestible protein. 2

be fed low energy and protein diets because they are able to consume high amounts of feeds. When comparing energy and protein requirements per unit of fish produced, as shown in Table 14.5, white grouper and Asian sea bass prove to be the more efficient species compared to gilthead sea bream and even tilapia. To produce 1 kg of fish the need for crude protein is about 613 g for sea bream and only about 456 g for grouper. The energy required to produce 1 kg of grouper is as low as 20 MJ compared to 34 MJ for the sea bream.

Table 14.4 Predicted energy and protein requirement for grouper and mullet at 27 °C and proposed feed formulation while deciding on 13.0 or 16.0 DE MJ kg−1 feed

Body weight, per fish Weight gain, g day−1 Feed intake – voluntary, g day−1 Energy requirement, kJ fish−1 day−1 DEmaint Energy gain DEgrowth DEmaint+growth Protein requirement, g fish−1 day−1 DPmaint Protein gain DPgrowth DPmaint+growth Feed formulation DE content of feed, MJ kg−1 Required feed, g fish−1 day−1 DP content in feed, g kg−1 Feed conversion ratio DP DE−1 ratio, g MJ−1

White grouper

Grey mullet

300 g 3.36 3.68

300 g 2.00 5.16

19.34 23.17 34.53 53.87

27.84 20.78 36.37 64.21

0.255 0.568 1.051 1.306

0.319 0.321 0.713 1.033

13.0 4.14 315 1.23 24.2

16.0 3.37 388 1.00 24.2

13.0 4.94 209 2.47 16.1

16.0 4.01 257 2.01 16.1

Specifications for white grouper can be found in equations in text. Specifications for grey mullet (Lupatsch, unpublished). Weight gain (g day−1) = 0.0019 × BW(g)0.557 × e0.140×T Feed intake (g day−1) = 0.0026 × BW(g)0.659 × e0.142×T Energy content of carcass (kJ g−1) = 5.56 × BW(g)0.110 Protein content (mg g−1) = 161 Requirements: DE(kJ) = (5.3 × T − 70.2) × (kg)0.80 + 1.75 × energy gain (kJ) DP(g) = (0.086 × T − 1.58) × (kg)0.70 + 2.22 × protein gain (g) DE = digestible energy, DP = digestible protein.

Table 14.5 Comparison of energy and protein requirements for the production of 1 kg of fish of 300 g weight (forecast for gilthead sea bream and European sea bass at 23 °C, all others at 27 °C) Requirement (per kg fish produced) Energy Protein (g) (MJ) Gilthead sea bream European sea bass White grouper Asian sea bass Tilapia Grey mullet

Feed formulation CP1 GE1 content content (MJ kg−1 (g kg−1 feed) feed)

Production efficiency Feed conversion ratio (feed gain−1)

613

33.9

22.0

398

1.54

573

30.1

22.0

418

1.37

456 456 522 609

19.9 20.6 24.8 40.2

19.0 19.0 17.0 17.0

434 422 358 258

1.05 1.08 1.46 2.36

1 Requirements are based on gross energy (GE) and crude protein (CP) values assuming a digestibility of 85 % and 80 % for protein and energy, respectively.

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14.5 Future trends The challenges nutritionists are facing is to continually reduce feed costs, improve conversion efficiency and minimize environmental impact. One of the approaches is to accurately quantify nutrient requirements of the fish and choose feed ingredients according to least cost principles. Combining the protein and energy needs with digestibility data on available feed ingredients would then allow the formulation of feeds and establishment of proper feeding tables for each fish species. Once an optimal feeding strategy is in place, it can be used for predictions of fish production, feed demand, FCR and solid and soluble waste production and forecast of profitability. Regardless of whether fish are raised in cages, ponds or recirculation systems, a nutrient budget can be established by using the following mass balance: total food input = retention (growth) + faeces (solid waste) + excretion (dissolved waste). Table 14.6 provides an example of the total nutrient budget for gilthead sea bream and grouper based on daily requirements for energy and protein. The total nutrient input to produce one ton of fish is much lower for grouper, especially regarding carbon, but also concerning nitrogen, even although the protein content of the feed is 45 % for grouper and only 40 % protein for seabream. In accordance to the higher feed efficiency of grouper, the total waste production is considerably less compared to sea bream. By establishing a feeding regime based on nutritional bioenergetics of any fish species, a long-term forecast of production and nutrient flow can be made according to expected growth and production efficiencies. In conclusion, energy and protein requirements of fish are dependent on growth potential, composition of weight gain and demand for maintenance, regardless of whether they are carnivorous or herbivorous, marine or freshTable 14.6 Nutrient release for fish of 300 g (in kg ton−1 of fish produced) Gilthead sea bream

White grouper

Input Feed Carbon Nitrogen Phosphorus

1540 727.0 98.1 15.4

1050 431 72.9 12.6

Retention Carbon Nitrogen Phosphorus

262.3 28.0 7.2

161.5 27.0 7.5

Waste Carbon Nitrogen Phosphorus

Solid 145.1 14.7 5.4

Dissolved 319.8 55.3 2.8

Solid 89.0 10.9 4.4

Dissolved 180.9 35.0 0.70

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water fish. Additional differences between fish are mainly due to stomach capacity and the amount of feed that can be consumed. Hence, energy and protein requirements for each fish species can be calculated and feeds adapted to changing conditions for the duration of a growth period. Thus it is necessary to formulate a specific feed in combination with a suitable feeding regime. Using this approach to quantify energy and protein demands in various fish species, it is possible to estimate the biological and economical efficiency of different feeds and culture systems.

14.6 References azevedo p a, cho c y, leeson s and bureau d p (1998) Effects of feeding level and water temperature on growth, nutrient and energy utilisation and waste outputs of rainbow trout Oncorhynchus mykiss, Aquat Living Resour, 11, 227–38. baker d h (1986) Problems and pitfalls in animal experiments designed to establish dietary requirements for essential nutrients, Review J Nutr, 116, 2339–49. brett j r and groves t d d (1979) Physiological Energetics, in Hoar W S, Randall D J and Brett J R (eds), Fish Physiology, Vol. VIII, Academic Press, New York, San Francisco, CA, London, 279–352. cho c y (1992) Feeding systems for rainbow trout and other salmonids with reference to current estimates of energy and protein requirements, Aquaculture, 100, 107–23. cho c y and bureau d p (1998) Development of bioenergetic models and the fishPrFEQ software to estimate production, feeding ratio and waste output in aquaculture, Aquat Living Resour, 11, 199–210. cho c y and kaushik s j (1990) Nutritional energetics in fish: energy and protein utilisation in rainbow trout, World Rev Nutr Diets, 61, 132–72. iwama g k and tautz a f (1981) A simple growth model for salmonids in hatcheries, Can J Fish Aquat Sci, 38, 649–56. lupatsch i (2003a) Feeding regimes for Asian sea bass grown at high temperatures, Global Aquac Advocate, 6, 62–3. lupatsch i (2003b) Effect of water temperature on energy and protein requirements for maintenance and growth of Asian sea bass Lates calcarifer, International Conference of the World Aquaculture Society, Salvador, Bahia. lupatsch i (2008) Predicting growth, feed intake and waste production of intensively reared tilapia based on nutritional bioenergetics, Proceedings of the Seventh International Conference on Recirculating Aquaculture, 25–27 July, Roanoke, VA. lupatsch i and kissil g wm (2005) Feed formulations based on energy and protein demands in white grouper Epinephelus aeneus, Aquaculture, 248, 83–95. lupatsch i, kissil g wm, sklan d and pfeffer e (1997) Apparent digestibility coefficients of feed ingredients and their predictability in compound diets for gilthead seabream, Sparus aurata, Aquac Nutr, 3, 81–9. lupatsch i, kissil g wm and sklan d (2001) Optimization of feeding regimes for European sea bass Dicentrarchus labrax: a factorial approach, Aquaculture, 202, 289–302. lupatsch i, kissil g wm and sklan d (2003a) Comparison of energy and protein efficiency among three fish species: gilthead seabream (Sparus aurata), European seabass (Dicentrarchus labrax) and white grouper (Epinephelus aeneus): energy expenditure for protein and lipid deposition, Aquaculture, 225, 175–89.

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lupatsch i, kissil g wm and sklan d (2003b) Defining energy and protein requirements of gilthead seabream (Sparus aurata) to optimize feeds and feeding regimes, Isr J Aquac – Bamidgeh, 55, 243–57. mercer l p (1982) The quantitative nutrient-response relationship, J Nutr, 112, 560–6. rodehutscord m and pfeffer e (1999) Maintenance requirement for digestible energy and efficiency of utilisation of digestible energy for retention in rainbow trout, Oncorhynchus mykiss, Aquaculture, 179, 95–107. sklan d, prag t and lupatsch i (2004) Apparent digestibility coefficients of feed ingredients and their prediction in diets for tilapia Oreochromis niloticus × Oreochromis aureus, Aquac Res, 35, 358–64. zeitoun i h, ullrey d e, magee w t, gill j l and bergen w g (1976) Quantifying nutrient requirements of fish, J Fish Res Board Can, 33, 167–72.

15 Advances in aquaculture nutrition: catfish, tilapia and carp nutrition D. Davis, Auburn University, USA, T. Nguyen, Nong Lam University, Vietnam, M. Li, National Warmwater Aquaculture Center, USA, D. M. Gatlin III, Department of Wildlife and Fisheries Sciences, USA, and T. O’Keefe, Aqua-Food Technologies, Inc., USA

Abstract: Freshwater fish species account for almost 50 % of the world’s aquaculture production, with tilapia, carp and catfish representing the primary culture species. These species are cultured in numerous countries as they have adaptable feeding habits, respond well to a wide variety of culture technologies and are well accepted by consumers. All three species can be easily reared on commercially produced floating feeds. Feeds not only represent one of the primary production costs and source of nutrients but they are also the primary source of pollutants that contribute to poor water quality and disease occurrences. Understanding the nutrient requirements, optimizing commercial feed formulations and managing feed inputs are all critical to the continued success of the industry. This chapter will review current concepts on nutrient requirements, pre- and probiotics, the options of using incomplete feeds as well as current trends in feed manufacturing. Key words: catfish, tilapia, carp, nutrition, feed formulation.

15.1 Introduction Tilapia (Oreochromis and Tilapia sp.), carp (Cyprinid sp.) and catfish (Ictalurus, Clarias and Pangasius sp.) are freshwater fish which account for a considerable portion of the world production of cultured species (see Table 15.1 for a list of common species). Based on data from the Food and Agriculture Organization of the United Nations, Fisheries and Aquaculture Information and Statistics Service, freshwater aquaculture produced about 44 % of aquaculture products or around 27 757 935 tons of freshwater fish valued at 35.3 billion US dollars in 2005. As a percentage of freshwater production, carp, catfish and tilapia represent approximately 57.9, 4.5 and

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Table 15.1 Examples of commercially produced species of tilapia, carp, and catfish Family

Genus species

Cichlidae

Oreochromis mossambicus O. urolepis hornorum O. niloticus O. aureus Sarotherodon galilaeus S. melanotheron

Clariidae

Clarias gariepinus, C. macrocephalus (often hybridized with C. gariepinus)

Cyprinidae

Cyprinus carpio C. carassius Ctenopharyngodon idella Hypophthalmichthys molitrix H. nobilis Mylopharyngodon piceus Catla catla Labeo rohita Cirrhinus mrigala

Ictaluridae

Ictalurus punctatus I. furcatus

Pangasiidae

Pangasius hypophthalmus Pangasius bocourti

7.1 % of the production, respectively. These species are not necessarily high valued species; however, they are major contributors to world aquaculture production and are a critical source of high-quality protein in many countries. Hence, they can be viewed as staples of the food supply for many countries. Because these fish are sold more as commodities than as luxury food items, it is critical that production systems are cost-effective and efficient. Due to cost structures, modern production systems for these species have a number of similarities to that of terrestrial chicken production systems, albeit, somewhat less advanced. Because of their fast growth, adaptability to a wide range of environmental conditions, disease resistance, high-quality flesh which has a wide market appeal, ability to grow and reproduce in captivity and feed on relatively low trophic levels, these species could be considered ‘aquatic chickens’. All three groups of fish can be cultured in traditional extensive culture systems. As aquaculture has matured into a commercial industry, extensive production systems are generally not cost-effective and are being replaced by more modern intensive systems. The shift from extensive systems is most apparent in Asia where traditional carp polyculture systems based on organic fertilization and supplemental nutrient sources are being replaced by polyculture systems using complete feeds. The same trend has occurred

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with tilapia culture in every country that has a commercial industry. In semi-intensive farming systems, supplemental feeds that consist of locally available, low-cost single feedstuffs such as rice bran, copra meal, coffee pulp, brewery by-products and/or various combinations have been used as supplements to natural food (Lim, 1989). These systems are characterized by lower financial inputs but also poor returns on the investment. As stocking rate increases, the contribution of natural food decreases and nutritionally complete feeds are required. This increases the cost of production; however, it also improves the return on the investment, producing better profit margins for a commercial industry. Of course this is the catch. In intensive culture systems such as in ponds, raceways, cages and tanks, feed is the most expensive item, often accounting for 30–60 % of the total variable expenses (Lim and Webster, 2006a) as well as other variable costs. Therefore, the development of cost-effective feeds using high-quality, inexpensive and preferably locally available ingredients is critical to modern commercial aquaculture and to the continued sustainability of the industry. With improving cost effectiveness of the feed in mind, the industry has considerable challenges, especially as agriculture and food production systems adjust to new production models driven by increased world populations combined with higher per capita consumption of protein. This, combined with increased industrial uses of agriculture products as partial replacements for industrial products such as crude oil (e.g. carbohydrate sources for ethanol and oil seeds for biodiesel), has resulted in increases in prices and limited supplies. On the other hand, this industrial use of raw agriculture products has or will increase the availability of industrial byproducts such as distillers’ grains and oil seed meals that may be suitable for inclusion in animal feeds. In response to shifts in the availability of feed stuffs, the industry has invested considerable time and efforts in determining dietary requirements, optimizing the use of cost-effective ingredients and evaluating the potential of feed supplements that may enhance production through a variety of mechanisms. The biggest challenges to the feed industry are the redefining of agriculture as it shifts towards vertically integrated models and an increased use of by-products as cost-effective replacements for traditional ingredients. Once we have adjusted to new price structures and the availability of basic ingredients, research will continue to refine nutrient requirements and biological availability of nutrient from various ingredients. However, there will more likely be increased effort to tie economic and nutritional models together as production systems become more industrialized. Also, with the increasing use of molecular biology, including gene chips and progress in defining the genome of key culture species, we are likely to see genetic improvements for specific traits, whether it is for chain elongation for the production of essential fatty acids or a better utilization of specific ingredients.

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15.2 Nutrient requirements Nutrient requirement data are well published and there are numerous books and review papers that summarize data for these species. Examples of published resources include books such as NRC (1993), Webster and Lim (2002), Kelly and Silverstein (2005), Lim and Webster (2006a) and El-Sayed (2006). As with all animals, these species of fish require a well balanced diet containing protein, energy, lipids, vitamins and minerals. General requirement data have been summarized in a variety of sources; hence this chapter will primarily concentrate on more recent information and trends in research.

15.2.1 Protein requirements Protein (or amino acids) is the principal organic constituent of animal tissue and is the most expensive component in fish feeds. Providing a costeffective and suitably balanced protein source designed to promote efficient and cost-effective daily growth is critical. Considerable research efforts have been expended to determine essential amino acid requirements and dietary protein levels necessary to achieve optimum fish performance under various culture conditions. Optimum dietary protein levels vary depending on several factors including growth rate of the fish (which in turn is dependent on fish size and temperature), feed intake, amount of non-protein energy in the diet, protein quality (amino acid balance and digestibility), presence of natural foods and management practices. Grow-out diets for these species are often formulated with lower levels of protein as compared to other species. This is due to several reasons, including a moderate growth rate, the ability to consume relatively large quantities of feed as well as a reasonable use of carbohydrates as an energy source to spare protein. With suitable feed intake and protein sparing, the daily requirement of protein intake can be met from a variety of dietary protein levels. For example, the grow-out diets for channel catfish production are typically formulated to contain 28–32 % crude protein. However, researchers have examined a wide range of dietary protein levels (from 10 % to 40 %) in various studies and found no differences in weight gain in fish fed diets containing as low as 24 % protein when fish were fed to apparent satiation (Robinson and Li, 2007). In addition, fish fed 16–20 % dietary protein gain about 80–90 % of that of fish fed a 32 %-protein diet. However, as dietary protein decreases, the digestible energy/crude protein ratio increases beyond the optimum range (8.5–9.5 kcal digestible energy/g protein [36–40 kJ digestible energy/g protein]) resulting in an increase in body fat. This effect is quite dramatic in diets containing very low levels of protein. Fish fed a 16 %-protein diet contain about twice as much fillet fat as similar size fish raised on a 32 %-protein diet. It is clear that fish can be produced using a wide range of dietary protein levels.

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Even though a wide range of protein levels can be utilized to produce fish, one has to consider that commercially produced fish are sold for human consumption at a price that will provide an adequate return on investment to maintain the viability of the operation. Hence, the dress out (percentage of edible product) as well as the quality of the product is critical in determining market prices. The optimal protein level used by commercial operations is that which produces the best economic return based on feed inputs and final product quality. Hence it is critical that commercial operations match both feed intake and dietary protein levels. We often talk in terms of dietary protein levels, but we are really talking about a well balanced mixture of non-essential and essential amino acids (EAA). The essential amino acid requirements, expressed as % diet, % protein or % daily energy intake have been reasonably well established for carp, tilapia and catfish species. Albeit well studied, there are contradictory reports in the literature and quite often the terminology referring to the total sulfur amino acid and methionine requirements is not clearly distinguished. This is mainly because cystine can replace part of the methionine requirement. For example, the reported total sulfur amino acid requirement is 2.88 % of protein for Indian carp (Murthy and Varghese, 1998), 2.34 % of protein for channel catfish (Harding et al., 1977) and 3.21 % of protein for Nile tilapia (Santiago and Lovell, 1988). Schwarz et al. (1998) reported a methionine requirement of 2.13 % for common carp; however, the diet also contained 1.04 % cystine. In fact one of the advantages of these species is that they have relatively low requirements for methionine which makes formulating with alternative ingredients simpler than for other species. Albeit there is considerable information with regards to essential amino acid requirements there is limited data on the digestibility of essential amino acids from various feed ingredients. This is due to both the cost and difficulty of obtaining realistic values. As the industry moves towards increased cost efficiencies, feed formulations must become more precise, thus making the need for digestibility values even more critical. Unfortunately, as we reduce the cost of the feeds we also quite often move towards lower cost ingredients which may also have reduced digestibility. Hence, digestibility information is critical in terms of estimating cost effectiveness as well as providing critical information on the nutritional quality of an ingredient. When formulating feeds for fish, there is often a question as to whether it is better to use synthetic amino acids to balance the amino acid profile or to utilize intact protein sources. This is a debate that still goes on even in the poultry industry in terms of which is the more cost-effective method. When dealing with aquatic animals, there are additional complications such as leaching of water-soluble components. Consequently, the use of watersoluble pure amino acids is discouraged in larval, fry and early fingerling feeds where leaching is an issue. However, in grow-out diets the use of

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amino acid supplements is acceptable. The efficacy of crystalline and protein-bound amino acids has been evaluated (e.g. Williams et al., 2001). Unfortunately, the cost based on biological values is quite hard to determine; hence the cost effectiveness will always be debated.

15.2.2 Alternative protein sources The development of commercial feeds for aquaculture has been traditionally based on the use of fish meal as the main protein source (Gatlin et al., 2007). This is due to its high protein content and balanced EAA profile. Fish meal is also an excellent source of essential fatty acids, digestible energy, minerals and vitamins, making it an excellent ingredient. Because of its nutritive value, it is no surprise that fish meal is the most expensive protein source in animal feeds (Tacon, 1993). There is no question that marine feedstuffs such as fish meal, are exceptionally good sources of protein (EAA) and other essential nutrients. Supply and price considerations make the use of high levels of marine ingredients economically unjustified in grow-out diets for carp, tilapia or catfish. Due to nutritional and palatability requirements of fry and fingerling fish it often justified to use these ingredients in diet for early development stages. Practical grow-out diets designed for these species vary depending on local availability of ingredients but, in general, the most cost-effective formulations are based primarily on soybean meal in combination with other plant-based proteins and/or low levels of animal proteins to provide the desired level of protein with a balanced amino acid profile (Gatlin, 2003). As a group, these fish constitute one of the largest sectors of cultured fish and a major user of solvent extracted soybean meal. For example, it is not uncommon for commercial grow-out diets for these species to have 40–50 % soybean meal. Research has demonstrated that these protein sources may be replaced by other less expensive feedstuffs. For example, cottonseed meal is generally price competitive (on a protein basis) with soybean meal, so it is often used as a replacement. However, it does contain gossypol (a compound that can be toxic to fish), and has lower levels of lysine as well as processing problems that limit inclusion levels in practical diets. With regards to catfish, research has shown that about 50 % of soybean meal can be replaced with cottonseed meal (27 % of the diet) plus supplemental lysine in catfish diets without negatively affecting fish performance (Li and Robinson, 2006; Robinson and Li, 2008). There is considerable information with regards to alternative dietary protein sources for these species. For example, in a review presented by El-Sayed (1999) he noted that alternative sources of protein for the tilapia include fishery by-products, terrestrial animal by-products, oilseed plants, aquatic plants, single-cell proteins, grain legumes, plant protein concentrates and cereal by-products. Based on a wide variety of studies, there are advantages to retaining a low level of fish meal in fry and fingerling feeds;

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however, in production diets there are minimal difference in biological performance of well-balanced diets with or without fish meal. As noted by El-Sayed (1999) even in cases where biological performance is reduced, cost and profit indices of alternative protein sources were better than for fish meal-based feeds.

15.2.3 Energy Energy is often considered one of the most important components of the diet because protein, lipids and carbohydrates will all be degraded to meet energy demands prior to their use for growth. Furthermore, excesses of energy can reduce feed intake (limiting the intake of other nutrients) and produce fish that have higher levels of fat that may not be desired by the processor and/or consumer. As noted by Robinson et al. (2001), the most notable differences in the nutrition of fish as compared with other livestock concerns energy requirements. For example, less energy is required for protein synthesis in fish than other warm-blooded animals. The protein gain per megacalorie (Mcal) of metabolizable energy (ME) consumed is 47, 23, 9 and 6 g (= 197, 96, 38, 25 MJ) for catfish (ME estimated), broiler chickens, swine and beef cattle, respectively (Robinson et al., 2001). This means that the daily requirement for energy is relatively low and, with the exception of fry feeds, practical grow-out diets for these species are not likely to be limiting in energy. In fact most diets made with high-quality ingredients have an excess of energy, which results in an inefficient use of energy and fish with higher levels of fat than would be found in wild fish. Estimates of the dietary requirement for energy have generally been determined by measuring weight gain or protein gain of fish fed diets containing a known amount of energy. Energy requirements expressed as a ratio of digestible energy (DE) to crude protein (DE/P) range for the carp (9.7–11.6 kcal/g [= 40.6–48.6 kJ/g], Takeuchi et al., 1979), channel catfish (7.4–12 kcal/g [= 31.0–50.2 kJ/g], Robinson et al., 2001) and tilapia (8.13– 9.7 kcal/g [= 34.0–40.6 kJ/g], Lim and Webster, 2006b). Albeit energy requirements have been well studied, we have primarily approached dietary energy levels based on the need to spare protein and maximize growth rates. Current grow-out diets for these species have relatively low levels of protein, minimal levels of lipids (to keep energy levels down) and high levels of carbohydrates which result in relatively high energy to protein levels. High energy to protein ratios, poor inventory control and near satiation feeding using a floating feed all contribute to an inefficient energy conversion. Hence, one can only conclude that feeds and feeding practices are not optimized for cost efficiencies relative to dietary energy. This is a minor point today but, as our culture practices become more precise for these species, we will need to optimize costs relative to energy. Currently, there is basic information with regards to DE values for typical ingredients used in practical diet formulations (see Wilson, 1994 and

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Stone, 2003 for reviews). As with other species, lipids and protein are highly digestible whereas carbohydrates in general have lower digestibility values. Clearly the source of the carbohydrate, physical state and level of inclusion influence digestibility (Gray, 1992; Stone, 2003). Because carbohydrates are the least expensive source of energy we must expand the available information with regards to the influence of dietary inclusion level, processing conditions as well as ratios of specific types of carbohydrates or classes (soluble and insoluble) in order to better understand the cost benefits of using various ingredient processing combinations. Future research is needed to look at the cost effectiveness of various dietary energy levels in combination with feeding practices. In cases where the fish are fed to near satiation, it may be more cost-effective to allow the insoluble carbohydrate fraction to increase, thus reducing the level of available energy and providing a better energy to protein ratio in the diet. However, such concepts must be approached based on overall economics of the culture system as this will increase carbon loading on the culture system, possibly increasing overall costs of production. In cases where intake is controlled and energy from carbohydrates may be limited there are a variety of physical and chemical processes that may be used to enhance nutrient availability. The animal industry is often encouraged to utilize sub-optimal ingredients due to price considerations. Hence, improving nutrient availability could significantly improve performance. Poorly utilized ingredients can often be improved through processing conditions and/or enzyme supplementation. Both the pig and poultry industry have found that exogenous enzymes can be used to enhance starch utilization and improve the negative effects of the soluble fractions of the dietary non-starch polysaccharides associated with various plant ingredients. For example, work with chickens has also indicated that extrusion or enzyme treatment of barley improved performance (Vranjes and Wenk, 1995; Lazaro et al., 2003). There are limited examples of the use of enzyme supplements for a number of fish species. Data with fish are quite limited, although the same concept does seem to apply. For example, in work with lupin meal offered to rainbow trout it was demonstrated that removal of the oligosaccharides through enzyme degradation or alcohol extraction increased the nutritive values of the lupin meal (Glencross et al., 2003).

15.2.4 Lipids Dietary lipids are an important source of highly digestible energy and are the only source of essential fatty acids needed by the fish for normal growth, development and reproduction (Lim and Webster, 2006a). In terms of total lipids, fry feeds contain 8–12 % lipid to enhance the energy density of highprotein diets, whereas production diets typically have relatively low levels of protein and high levels of digestible carbohydrates and low (4–8 % diet)

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levels of lipids. In addition to a source of energy, they serve as a carrier for other fat-soluble nutrients, contribute to the palatability of the feed and provide lubrication to the feed manufacturing process as well as to help reduce feed dust by spraying oil in the finished extruded feed pellets. Dietary lipid levels are primarily adjusted to meet energy requirements and to facilitate processing. Dietary fatty acid content is often adjusted to meet the essential fatty acid requirements of the fish and, more recently, to improve the fatty acid profile of the fish tissue with respect to human nutrition. All three fish species discussed in this chapter have the ability to chain elongate and desaturate 18 : 2 n-6 and 18 : 3 n-3 to longer chain fatty acids of their respective families (Webster and Lim, 2002). As long as minimal levels of these fatty acids are provided essential fatty acid deficiencies are not likely to occur. These fish species are capable of producing arachidonic acid (ArA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from precursors. Although the lipid content of the fillet of these species is relatively low, they are a source for these essential fatty acids for the human consumer. Seafood is not only a major source of protein but it is also one of the best sources of essential fatty acids. Because of human health benefits, the fatty acid content of fish has come into play when marketing a number of products including fish. In the past fish oils were considered industrial products and sold on the market at very low costs. These oils were extensively used in aquaculture diets producing a final product high in essential fatty acids. However, supplies of marine fish oils are limited and the price of these oils has steadily increased making the use of alternative oils more common. A number of plant oils (e.g. soybean oil, linseed oil, rapeseed oil, sunflower oil and palm oil) have been shown to be partial or total replacements for fish oil (Ng et al., 2003; Bahurmiz and Ng, 2007). Although this has helped reduce feed costs, one also has to consider the implications to the final product. Hence, to ensure that our products remain a healthy food item of choice, a great deal of future research will be required to determine the most cost-effective ways to produce a final product that meets marketing requirements for human consumers.

15.2.5 Vitamins Vitamins are a group of organic substances that are required in small quantities in the diet for metabolism, immune function, growth and reproduction. Vitamins and minerals are critical components of any complete feed, and improper supplementation can have devastating effects on production. The dietary requirements for vitamins and minerals have been well studied in a number of species. However, there is a clear difference between determining a dietary requirement and being able to make clear recommendations under practical conditions. Vitamin and mineral premixes are a significant cost to a formulation but they can also dramatically influence

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production if proper supplementation is not achieved. In terms of vitamin requirements they have been well summarized by the NRC (1993) and more recently in books for each of the species. The practical application of dietary requirements for vitamins and minerals is often a complex issue for which there are no absolute solutions. Some fish are able to obtain certain vitamins to partially or completely meet their metabolic needs. For example, most fish species can synthesize enough inositol in the body to meet their requirement. Vitamin B12 can be synthesized by intestinal microorganisms in catfish, tilapia and carp to partially meet the requirement. Other vitamins such as choline are clearly required by the fish but have interactions with other nutrients. For example, choline has three major metabolic functions: as a component of phosphatidylcholine; as a precursor of neurotransmitter acetylcholine; and as a precursor of betaine, which acts as a source of labile methyl groups for methylation reactions (NRC, 1993). Consequently, the dietary methionine and betaine status as well as inherent levels in ingredients all influence the need for a choline supplement. Lecithin (a source of choline) is also often supplemented for a variety of nutritional and processing considerations, adding to the level of this vitamin. Additionally, vitamins can be destroyed during processing and storage; hence, the ‘required levels of dietary supplements’ become a complex issue for which it is often difficult to produce specific guidelines. In intensive systems where limited or no natural foods are available, feeds used for these species are typically supplemented with a vitamin premix that provides all essential vitamins in sufficient quantities to ensure optimum growth and health and to compensate for losses during feed manufacturing and storage. However, supplemental vitamins may not be necessary for tilapia stocked at moderate densities in fertilized ponds since natural foods can supply the vitamin needs of the fish (Shiau, 2002). There is also evidence that natural foods in channel catfish grow-out ponds supply some of the vitamin needs of the fish (Robinson et al., 1998). The extent to which natural food organisms contribute to the vitamin nutrition of intensively grown channel catfish is still unclear since the abundance of natural food organisms varies from pond to pond and from time to time. The concentrations of certain vitamins inherent in feedstuffs commonly used in fish feeds can be found in various feed ingredient tables, and can be used to estimate vitamin concentrations in formulated feeds. However, the actual concentration and the estimated value may vary considerably since tabular values are averages and the concentration of vitamins in a particular feed ingredient may vary from batch to batch. Also, since bioavailability for most vitamins present in feed ingredients is not known, this source of vitamins is usually not considered when formulating fish feeds, but it could be a significant source of vitamins for fish. Studies with channel catfish conducted in earthen ponds show that there were no differences in weight gain, feed consumption, feed conversion, survival or hematocrit values of fish regardless of whether or not the diet was supplemented with

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a vitamin premix or a specific vitamin (Robinson et al., 1998). The results suggest that vitamin requirements of pond-raised channel catfish are somehow met by sources other than supplemental vitamins, possibly by a combination of vitamin sources, such as those present in feed ingredients and natural food organisms. Vitamins, as well as certain other nutrients, are needed for the proper function of the immune system. There has been much attention given to the role of vitamins in the health of fish, the most notable being the use of megadoses of vitamin C in the diet to improve disease resistance. For channel catfish, published data seem to agree that under laboratory conditions immune response of the fish is depressed if an ascorbic acid-free diet is fed. However, the results from studies in which megadoses of ascorbic acid were fed to channel catfish to improve disease resistance are contradictory. The effects of other vitamins on immune function or disease resistance of these fish have also been investigated. However, there has been no conclusive evidence that increasing the concentrations of supplemental vitamins above the requirement level is beneficial to these fish species.

15.2.6 Minerals Mineral requirements of fish are similar to terrestrial animals other than functions in osmoregulation and the ability to directly obtain minerals from the water through absorption across exposed membranes. As a number of minerals can either be absorbed from the water (e.g. calcium, Ca) or are present in adequate levels in most diet formulations (e.g. potassium, K), dietary supplementation is generally not required. Of the 23 minerals which have been demonstrated to be essential for one or more species, practical diet formulations for fish typically only require phosphorus (P), magnesium (Mg), copper (Cu), iron (Fe), zinc (Zn), manganese (Mn) and selenium (Se) as supplements. The dietary requirements for both macro and trace minerals have been reviewed by Davis and Gatlin (1996) and Watanabe et al. (1997). Systematic evaluations of dietary mineral requirements, physiological functions and biological value of all biologically important minerals do not exist. There is also limited information on the biological value of minerals from various sources as well as potential interactions that can occur between minerals. Many of the studies that have been conducted have been driven by both economic and environmental concerns. For example, there is a fair amount of information on the bioavailability of phosphorus from both organic and inorganic sources (Robinson et al., 2001). Considerable research has also been conducted on the use of various forms of phytase to enhance P bioavailability from plants containing significant levels of phytate phosphorus. Such studies are becoming even more important as we move from animal protein sources, which are rich sources of minerals, towards plantbased diets that often do not contain adequate levels of minerals.

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As the aquaculture industry matures into a more cost-efficient industry and we shift feed formulations farther from animal-based ingredients, we will most likely need to re-evaluate mineral requirements, particularly for trace minerals. It is most likely that future research will be geared toward more cost-effective delivery mechanisms for minerals, whether through precise delivery of traditional inorganic sources or chelated minerals which can have higher biological availabilities but also have lower leaching rates from the feed once it is submersed in the water. Depending on consumer acceptance, we may even see trends for the enhancement of mineral in seafood products to ensure that they are excellent sources of minerals to meet human health concerns.

15.2.7 Prebiotics and probiotics If growth performance and feed efficiency are increased in commercial aquaculture, then the costs of production are likely to be reduced. Also if more fish are able to resist disease and survive until they are of marketable size, the subsequent cost of medication and overall production costs are reduced. There is a clear relation between nutritional status of an animal and their disease response (Lim and Webster, 2001). Additionally, there are numerous compounds that have the potential to enhance growth and survival through a variety of mechanisms. It has been documented in a number of food animals that their gastrointestinal microbiota play important roles in affecting the nutrition and health of the host organism. Thus, various means of altering the intestinal microbiota to achieve favorable effects such as enhancing growth, digestion, immunity and disease resistance of the host organism have been investigated in various terrestrial livestock as well as in humans. Dietary supplementation of prebiotics, which are classified as non-digestible food ingredients that beneficially affect the host by stimulating growth and/or activity of a limited number of health-promoting bacteria such as Lactobacillus and Bifidobacter spp. in the intestine, while limiting potentially pathogenic bacteria such as Salmonella, Listeria and Escherichia coli, have been reported to favorably affect various terrestrial species; however, such information is extremely limited to date for aquatic organisms. Based on a review by Burr et al. (2005), effects of probiotics, defined as live microbial feed supplements, on gastrointestinal microbiota have been studied in some fishes, but the primary application of microbial manipulations in aquaculture has been to alter the composition of the aquatic medium. In general, the gastrointestinal microbiota of fishes, including those produced in aquaculture, have been poorly characterized, especially the anaerobic microbiota. Therefore, more detailed studies of the microbial community of cultured fish are needed to potentially enhance the effectiveness of prebiotic and probiotic supplementation.

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15.2.8 Complete versus supplemental feeds It has been well established that using appropriate pond culture techniques in combination with a quality commercial feed with appropriate feed inputs results in the best overall performance and return on the investment. A wide variety of examples can be found at the Soy in Aquaculture website which presents results of various feeding trials in Asia (www.soyaqua.org). Another excellent example is the publication by Green et al. (1994) which presents economic results from using various levels of nutrient inputs that affect production, costs and returns. One advantage of carps, tilapia and, to a lesser extent, catfishes is that they are amenable to culture using sub-optimal feeds as they are capable of utilizing natural productivity. In terms of standing crop, production is increased as one moves from natural feeds enhanced with fertilization to single ingredients like rice bran in combination with inorganic fertilization, then to farm-made feeds and finally commercially produced feed. If one does a complete enterprise budget, in which all inputs, labor (including opportunity cost for sourcing and preparing materials), infrastructure (e.g. land and pond construction cost which are often neglected in budgets for subsistence farming) are taken into account, sub-optimal feed produces a negative net return. Furthermore, if performance and economic returns are compared for farm-made feed (for which all costs are accounted) as compared to a high-quality locally produced commercial feed, the high-quality commercial feed will out perform the on-farm feed in terms of return on investment. Importing a production diet can be cost-effective in some situations but, in general, in-country feed production using a combination of locally produced and imported ingredients is more cost-effective and the best solution. The exception to this rule is for hatchery diets which are often cost-effective to import. An interest in sub-optimal feeds comes about for a variety of reasons. Quite often the land was obtained at no cost, there is a lack of quality commercially produced feeds and/or inadequate financial resources to purchase feeds. This situation is often accompanied by a poor understanding of appropriate culture technologies, financial management and credit sources which further complicate the issues. In cases where only poor-quality feeds are available, the industry will benefit the most from improving feed technologies through the introduction of quality control standards, proper processing technologies and the production of floating feeds. This is best exemplified by the development of commercial aquaculture in Asia where a traditional fertilizer-based industry has shifted to high-quality commercially produced feeds. One can find numerous examples of the comparison of traditional culture technologies to modern feed technologies at the soy in aquaculture website (www.soyaqua.com). In the case where a commercial industry has not developed or in the case where the farmer does not have the financial resources to purchase feed he can graduate from one technology to the next if he is capable of goal-

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oriented financial management and has the proper training. If appropriate culture techniques are not developed simultaneously with improved feeding then no one will benefit from the advances. In such cases, the farmer who is utilizing high-nitrogen locally available ingredients (on-farm grasses, manures, etc.) would invest his money in the purchase of inorganic fertilizers to increase production and revenues. As his production is increased, he can then move to using simple mixes of ground cereal grains by-products (e.g. rice bran, wheat bran, corn bran, etc.) in combination with inorganic fertilizers. Again, as revenues increase, they can then graduate to locally produced feed using a simple mix of cereal grains and their by-products, vegetable and animal proteins that are then ground and mixed with a vitamin premix. Boiling water can then be blended into the dry ingredients to produce a moist dough which can be formed and dried. Unfortunately, in countries where commercially produced feeds are not available there is also a shortage of high-protein ingredients and vitamin premixes. The next advancement would be small-scale commercial production of feeds which are often pelleted and of poor quality due to a poor understanding of quality control and appropriate milling standards. As the industry develops, the final stage would be a commercial mill producing a high-quality feed with suitable quality control standards. To facilitate feed management this should be an extruded floating feed. As noted before, if one does a complete enterprise budget for operations using the lower levels of technology, these operations only make sense if infrastructure costs are free and there are no other options. In all cases, farmers must be educated in terms of appropriate culture technologies (stocking densities, water management, feeding protocols, etc.), financial management, record keeping, analysis and quite often marketing.

15.2.9 Complete feeds The commercial manufacturing of fish feeds has developed rapidly and represents one of the fastest growing components of feed manufacturing. With regards to grow-out diets for these species the production of a floating feed is the technology of choice. Given current market constraints the most costeffective diets are often plant-based with limited quantities of animal protein. Examples of plant-based juvenile and production diets are presented in Table 15.2, with feed specifications presented in Tables 15.3–15.6. Such feeds contain vitamin and mineral premixes to provide a complete diet. Floating feeds are most efficiently produced using a wet extruder designed for the production of expanded products. The ability to control the density of a pellet to produce a floating feed is clearly a major advantage for management. Extrusion processing is more expensive than the pelleting process but produces a feed that is more economical to use because of the benefits of using a floating feeds as a management tool. In cases where inventory control is very precise and feeding based on observation of the fish at the

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Table 15.2

Examples of generic production diets for carp, tilapia, and catfish

Ingredient (as is basis) Soybean meal 46 % protein Wheat flour 13.2 % protein Fish meal 64 % protein DDGS1 28.5 % protein Rice bran 13 % protein Corn gluten meal 60.7 % protein Fish oil, anchovy Blood meal 90 % protein Ca Phosphate Mono 21 % P Soy oil Vit PMX F-2 Min PMX F-1 Mold inhibitor Choline chloride 60 % Stay C 35 % active C Ethoxyquin 1

36/7 Fingerling

32/6 Grower

34.00 19.80 13.50 10.00 8.00 7.00 2.70 2.00 1.30 0.50 0.50 0.25 0.30 0.10 0.03 0.02

50.00 25.00 14.00 3.0 1.5 1.5 2.20 1.7 0.50 0.25 0.30 0.10 0.03 0.02

DDGS = distiller’s dried grains with solubles.

Table 15.3 General nutrient specifications for formulation of practical diets for tilapia, carp, and catfish Nutrient

Fry

Juvenile

Adult

Protein (%) DE/DP1 Fat (%) n3 (%) Fiber (%) Ash (%) Moisture (%)

45–41 9–8 12–11 1 min 2 max 9–7 11–10

40–36 9–8 10–7 0.7 min 2.5 max 6–8 11–10

35–30 9.5–8 6–5 0.5 min 3.5 max 5–8 11–10

1 DE, digestible energy in kcal/100 g of feed (9 kcal = 37.7 kJ); DP, digestible protein in g/100 g of feed.

surface is not critical, a properly pelleted feed is also quite adequate. Current trends in feed manufacturing include fine grinding and the production of very small pellets (1–2 mm). Such pellets are technically more difficult to produce but have numerous advantages over crumbled feeds. Another shift in mill management is that more attention is being paid to the reduction of energy costs. Quite often energy costs can be reduced by improving air assist systems, ensuring hammers on hammer mills are in good shape, as well as making a compromise between very fine grinding and the most energyefficient grind (which is typically larger). Additional savings can be made by

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Table 15.4 Recommended minimum amino acid levels in diets for tilapia, carp, and channel catfish Amino acid (available)

Tilapia

Carp

Arginine Histidine Isoleucine Leucine Lysine Methionine & cystine1 Phenylalanine and tyrosine Threonine Tryptophan Valine

4.2 1.7 3.1 3.4 5.1 3.2–3.0 5.7 3.7 1.0 2.8

4.3 2.1 2.5 3.3 5.7 3.1 6.5 3.9 0.8 3.6

Channel catfish 4.3 1.5 3.1 3.4 5.1 2.3 5.0 2.0 0.5 3.0

1

Minimum methionine level is generally considered to be 60 % (wt/wt) of the total Met + Cys value.

Table 15.5 Recommended vitamin fortification levels for warm water fish (adapted from Roche supplementation guidelines for optimum vitamin nutrition) Amount Vitamin A D E K Thiamine (B1) Riboflavin (B2) Pyridoxine (B6) Vitamin B12 Niacin Pantothenate Folic acid Biotin Vitamin C1 Choline

Units / kg of feed IU IU IU mg mg mg mg mg mg mg mg mg mg mg

Minimum

Optimum

4000 1000 100 3–5 10 15 8 0.02 80 40 4 0.5 50 0

8000 2000 300 10 20 20 12 0.05 120 50 7 1 300 600

1

Vitamin activity in the finished diet – amount depends on stability of the source. Only stabilized sources are recommended.

reducing the variability in drying to make more precise endpoints and optimizing pre-conditioning to facilitate starch gelatinization and reduce energy required to extrude the product. One clear trend in both pelleting and extrusion processing is the replacement of pre-conditioners with newer models that allow for longer retention times for better pre-cooking of the product.

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Units

Amount

Cobalt Copper Iodine Iron Manganese Selenium Zinc

mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg

0.025 10.0 3.5 25.0 25.0 0.3 100.0

15.3 Sources of further information and advice • American Feed Industry Association (2005) Feed Manufacturing Technology V, American Feed Industry Association, Arlington, VA. • Hertrampf J W and Piedad-Pascual F (2000) Handbook of Ingredients for Aquaculture Feeds, Kluwer Academic, Dordrecht. • Lim C E and Webster C D (eds) (2006) Tilapia: Biology, Culture and Nutrition, Food Products Press, New York. • Lim C E and Webster C D (2006b) Nutrient requirements, in Lim C E and Webster C D (eds), Tilapia: Biology, Culture and Nutrition, Food Products Press, New York. • NRC (National Research Council) (1993) Nutrient Requirements of Fish, National Academy Press, Washington, DC.

15.4 References bahurmiz o m and ng w k (2007) Effects of dietary palm oil source on growth, tissue fatty acid composition and nutrient digestibility of red hybrid tilapia, Oreochromis sp., raised from stocking to marketable size, Aquaculture, 262, 383–92. burr g, gatlin d m iii and ricke s (2005) Microbial ecology of the gastrointestinal tract of fish and the potential application of prebiotics and probiotics in finfish aquaculture, Journal of World Aquaculture Society, 36, 425–36. davis d a and gatlin d m iii (1996) Dietary mineral requirements of fish and marine crustaceans, Reviews in Fisheries Science, 4(1), 75–99. el-sayed a-f m (2006) Tilapia Culture, CABI Publishing, Cambridge, MA. el-sayed a-f m (1999) Alternative dietary protein sources for farmed tilapia, Oreochromis spp., Aquaculture, 179, 149–68. gatlin d m iii (2003) Use of Soybean Meal in the Diets of Omnivorous Freshwater Fish, United Soybean Board American Soybean Association, Chesterfield, MO/ Washington DC, 1–12. gatlin d m iii, barrows r t, brown p, dabrowski k, gaylord t g, hardy r w, herman e, hu g, krogdahl å, nelson r, overturf k, rust m, sealey w, skonberg d, souza e, stone d, wilson r and wurtele e (2007) Expanding the utilization of sustainable plant products in aquafeeds: a review, Aquaculture Research, 38, 551–79.

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glencross b d, boujard t and kaushik s j (2003) Influence of oligosacharides on the digestibility of lupin meals when fed to rainbow trout, Oncorbynchus mykiss, Aquaculture, 219, 703–13. gray g m (1992) Starch digestion and absorption in nonruminants, Journal Nutrition, 122, 172–7. green b w, teichert-coddington d t and hanson t r (1994) Summary of Freshwater Aquacultural Research Conducted from 1983 to 1992, International Center for Aquaculture and Aquatic Environments, Research and Development Series No. 39, Auburn University, Auburn, Al. harding d e, allen o w and wilson r p (1977) Sulfur amino acid requirement of channel catfish: L-methionine and L-cystine, Journal of Nutrition, 107, 2031–5. kelly a and silverstein j (eds) (2005) Aquaculture in the 21st Century, American Fisheries Society, Bethesda, Md. lazaro r m, garcia m j, aranibar and mateos g g (2003) Effect of enzyme addition to wheat-, barley- and rye-based diets on nutrient digestibility and performance of lying hens, British Poultry Science, 44, 256–65. li m h and robinson e h (2006) Use of cottonseed meal in diets of aquatic animals: a review, North American Journal of Aquaculture, 68, 14–22. lim c e (1989) Practical feeding – tilapias, in Lovell T. (ed.), Nutrition and Feeding of Fish, Van Nostrand Reinhold, New York, 163–83. lim c e and webster c d (eds) (2001) Nutrition and Fish Health, Food Products Press, New York. lim c e and webster c d (eds) (2006a) Tilapia: Biology, Culture and Nutrition, Food Products Press, New York. lim c e and webster c d (2006b) Nutrient requirements, in Lim C E and Webster C D (eds), Tilapia: Biology, Culture and Nutrition, Food Products Press, New York, 469–501. murthy h s and varghese t j (1998) Total sulphur amino acid requirement of the Indian major carp, Labeo rohita (Hamilton), Aquaculture Nutrition, 4, 61–5. ng w k, lim p k and boey p l (2003) Dietary lipid and palm oil source affects growth, fatty acid composition and muscle a-tocopherol concentration of African catfish, Clarias gariepinus, Aquaculture, 215, 229–43. nrc (national research council) (1993) Nutrient Requirements of Fish, National Academy Press, Washington, DC. robinson e h and li m h (2007) Catfish Protein Nutrition: revised, Bulletin No. 1159. Mississippi Agriculture and Forestry Experiment Station, Mississippi State University, MS. robinson e h, li m h and oberle d (1998) Catfish Vitamin Nutrition, Bulletin No. 1078, Mississippi Agricultural and Forestry Experiment Station, Mississippi State University, MS. robinson e h and li m h (2008) Replacement of soybean meal in channel catfish, Ictalurus punctatus, diets with cottonseed meal and distillers dried grains with solubles, Journal of the World Aquaculture Society, 39, 521–7. robinson e h, li m h and manning b b (2001) A Practical Guide to Nutrition, Feeds, and Feeding (second revision), Bulletin No. 1113, Mississippi Agriculture and Forestry Experiment Station, Mississippi State University, MS. stone d a j (2003) Dietary carbohydrate utilization by fish, Reviews in Fisheries Science, 11, 337–69. santiago c b and lovell r t (1988) Amino acid requirements for growth of Nile tilapia, Journal of Nutrition, 118, 1540–46. schwarz f j, kirchgessner m and deuringer u (1998) Studies on the methionine requirement of carp (Cyprinus carpio L.), Aquaculture, 161, 121–9. shiau s y (2002) Tilapia, Oreochromis spp., in Webster C D and Lim C (eds), Nutrient Requirements and Feeding of Finfish for Aquaculture, CABI, New York.

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tacon a g j (1993) Feed Ingredients for Warmwater Fish: Fish Meal and Other Processed Feedstuffs, FAO Fisheries Circular No. 856, Food and Agriculture Organization of the United Nations, Rome. takeuchi t, watanabe t and ogino c (1979) Optimum ratio of dietary energy to protein for carp, Nippon Suisan Gakkaishi, 45, 983–7. vranjes m v and wenk c (1995) The influence of extruded vs. untreated barley in the feed, with and without dietary enzyme supplement on broiler performance, Animal Feed Science and Technology, 54, 21–32. watanabe t, kiron v and satoh s (1997) Trace minerals in fish nutrition, Aquaculture, 151, 185–207. webster c d and lim c e (eds) (2002) Nutrient Requirements and Feeding of Finfish for Aquaculture, CABI, New York. wilson r p (1994) Utilization of dietary carbohydrate by fish, Aquaculture, 124, 67–80. williams k, barlow c and rodgers l (2001) Efficacy of cystalline and protein-bound amino acids for amino acid enrichment of diets for barramundi/Asian seabass (Lates calcarifer Bloch), Aquaculture Research, 32(1), 415–29.

16 Advances in aquaculture feeds and feeding: basses and breams M. Booth, New South Wales Department of Primary Industries, Australia

Abstract: The aquaculture species of bass and bream belong to the order Perciformes and come from five major family groups; Latidae, Sparidae, Serranidae, Moronidae and Lutjanidae. Well-known species include Asian seabass Lates calcarifer (Latidae), the common sea breams or porgies such as Pagrus pagrus, Pagrus major (= Pagrus auratus), gilthead sea bream Sparus aurata and common dentex Dentex dentex (all from the Sparidae), European sea bass Dicentrachus labrax, striped bass Morone saxatilus, white bass Morone chrysops and hybrid striped bass (Morone saxatilus × M. chrysops) (Moronidae) and the groupers Epinephalus sp. (Serranidae). All sea basses and sea breams are euryhaline and carnivorous and they generally command high market prices. Much of the research on basses and sea breams is dominated by basic nutrition and feeding research that aims to improve production efficiencies. To reflect recent advances in understanding nutrient requirements, use of alternative feed ingredients and feeding strategies for these species, new information is presented here for Asian seabass, red sea bream, gilthead sea bream and grouper. Future nutrition research with these species will continue to investigate increased use of alternative feed ingredients and reductions in the use of declining stocks of fish meal and fish oil. The impact of new ingredients and new diet specifications on extrusion and processing technology will need to be considered. The increase in demand for high-value species will demand research that improves our understanding of basic nutrient requirements for protein, amino acids, lipids or carbohydrates and a better understanding of the impacts of these factors, in conjunction with culture environmental conditions, on nutritional status, fish metabolism, health, and total farm productivity. Key words: Latidae, Asian seabass, barramundi, Lates calcarifer, Sparidae, red sea bream, Pagrus major, Pagrus auratus, gilthead sea bream Sparus aurata, Serranidae, grouper, Epinephalus, digestibility, requirements, replacement studies, feeding studies, alternative ingredients.

16.1 Introduction Important aquaculture species of bass and bream belong to the order Perciformes and come from five major family groups; Latidae, Sparidae,

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Serranidae, Moronidae and Lutjanidae. Within each of theses families is a diverse range of species and many have aquaculture potential. The best known representative from the Latidae is the Asian seabass Lates calcarifer, while the Sparids are represented by the common sea breams or porgies such as the red porgie Pagrus pagrus, Japanese red sea bream Pagrus major, also known as Pagrus auratus in Australia (Paulin, 1990), gilthead sea bream Sparus aurata and common dentex Dentex dentex (Pillay, 1993). Several emerging sparids include the sheepshead Archosargus probatocephalus (Tucker, 2008) and red banded seabream Pagrus auriga (Cardenas, 2008). The Moronidae are represented by European sea bass Dicentrachus labrax, striped bass Morone saxatilus, white bass Morone chrysops and hybrid striped bass (Morone saxatilus × M. chrysops). The Serranidae are probably best represented by the groupers (Epinephalus sp.) while the Lutjanidae include species such as red snapper Lutjanus campperchanus, mangrove red snapper Lutjanus argentimaculatus and mutton snapper Lutjanus analis. According to recent FAO data, Taiwan (15 000 t) and Thailand (6660 t) are the major producers of Asian seabass, but production in Malaysia (5550 t), Indonesia (2183 t) and Australia (2075 t) is increasing (Rimmer, 2003) and the species is even farmed in intensive recirculating systems in the USA and Europe. In Australia, the value of Asian seabass production has climbed to approximately $US15 million (ABARE, 2007). Japan produces nearly 71 000 t of red sea bream annually while Greece, Turkey, Spain and Italy produce about 43 900, 28 460 and 16 500 t of gilthead seabream, respectively (FAO Fisheries Statistics 1950–2006). Greece (34 000 t) and Spain (8300 t) are the major producers of European sea bass while the majority of mangrove red snapper are produced in Malaysia (4500 t). China was by far the major producer of grouper sp. (48 000 t valued at $US54 million), but production continues to escalate in Taiwan (9500 t), Indonesia (3100 t) and Thailand (3000 t) (FAO Fisheries Statistics 1950–2006). Common to all sea basses and sea breams are their euryhaline and carnivorous habits and the fact that they generally command high market prices (Pillay, 1993). Early publications on red sea bream (Foscarini, 1988) gilthead sea bream (Kissil, 1991), Asian seabass (Boonyaratpalin, 1991; Pillay, 1993), striped or hybrid bass (Brandt, 1991) and European sea bass (Landau, 1992) give some historical background on each species and describe their potential for aquaculture. More recently, reviews have been published on the basic nutritional requirements and feeding practices of Asian seabass (Boonyaratpalin and Williams, 2002), European sea bass (Kaushik, 2002), red sea bream (Koshio, 2002), gilthead sea bream (Koven, 2002) and hybrid striped bass (Webster, 2002). Review, discussion and evaluation of the development and constraints to aquaculture of sea bass, sea bream (Gasca-Leyva et al., 2002; Rad, 2007) and grouper (Pomeroy, 2007) have also contributed to the broader economic understanding of these species.

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Cited literature in the aforementioned nutritional reviews covered research up to about 2000. Henceforth there has been interest in new areas of research such as genetic and genomic (Wang et al., 2007) studies in fish nutrition. However, much of the research on basses and sea breams, like many aquaculture species, is dominated by basic nutrition and feeding research that aims to improve production efficiencies. This is the case whether industries are new (e.g. grouper) or mature (e.g. salmon). Recent advances in understanding nutrient requirements, use of alternative feed ingredients and feeding strategies are presented here for Asian seabass, red sea bream, gilthead sea bream and grouper. These species were selected for review because they are representative of the diverse range of basses and breams currently under culture and because nutritional research undertaken on these four species is similar to research undertaken on most of the other species related to their specific family groups.

16.2 Asian seabass Asian seabass are also known as giant sea bass or barramundi. They are widely distributed in the tropical and sub-tropical areas of Asia (Pillay, 1993). Their natural life cycle is considered biphasic, with the juvenile stages of growth occurring in freshwater followed by migration to sea for sexual maturation and spawning (Boonyaratpalin and Williams, 2002). They are amenable to culture in a variety of fresh or saltwater systems (cages, ponds or tanks) and are tolerant of turbidity and high stocking densities. Traditionally, Asian seabass were fed a ration of chopped low-value fish during the grow-out period, and this was often blended and extended with broken rice or rice bran (Pillay, 1993). This feeding strategy is still used in many Asian countries where this species is produced despite the emergence of highquality feeds manufactured using extrusion technology that dominate production in Australia, the USA, Europe and Taiwan.

16.2.1 Requirements Hitherto little was known about the quantitative protein or amino acid requirements of Asian seabass according to the recent review of Boonyaratpalin and Williams (2002). Like most carnivorous species, protein requirements were estimated to be between 40 and 50 % of the diet (NRC, 1993; Catacutan and Coloso, 1995). Early studies had elucidated the digestible energy demands of this species at different temperatures using a bioenergetic approach (Lupatsch, 2003) while recommended levels of dietary carbohydrate were given as 20 % in diets containing lipid levels ranging from 6 to 18 % (Catacutan and Coloso, 1997). Recently, Glencross (2006) has published a comprehensive review on the nutritional management of Asian seabass which covers nutritional advances on this species in some

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detail. The extensive nature of this document precludes repetition in this chapter and readers seeking an inclusive list of references, information on growth, dietary requirements, digestibility and feeding strategies are urged to consult this work. Glencross (2008) has also presented new and refined data on nutrient requirements, feed utilization and iterative diet formulation for Asian seabass determined from bio-energetic studies, partial validation of bio-energetic models for large fish (Glencross et al., 2008) and a wide ranging report on the cage culture of this species which includes information on growth performance, flesh quality, purging techniques and sensory analysis (Glencross et al., 2007). The potential of increasing dietary lipid in order to spare protein for growth of juvenile (80 g) or sub-adult (230 g) barramundi reared in freshwater was investigated by Williams et al. (2003a). In one factorial experiment, test diets contained 38–53 % crude protein with lipid concentrations of 7.0, 12.8 or 18.3 % and in another test diets varied in protein from 44–65 % with lipid concentrations of 13, 18 or 23 %. Regardless of fish size, feed conversion ratio (FCR) and growth rate improved linearly with increasing dietary crude protein content and improved step-wise with increasing dietary lipid content (Fig. 16.1). A small protein sparing effect was observed, but this was more pronounced in the smaller rather than the larger animals. Interestingly, an increase in dietary crude protein did not overly affect efficiency of nitrogen retention, but retention of dietary gross energy improved as dietary lipid increased; fat deposition was the major component of the retained energy. Their results showed that productivity can be markedly improved by increasing dietary protein and lipid concentrations, but that Asian seabass only has a limited capacity to use lipid as a 5.5 5.0

ADG

4.5 4.0 3.5 7.0 % lipid

3.0

12.8 % lipid 2.5

18.3 % lipid

2.0 35

38

40

43

45

48

50

53

55

58

Crude protein content of diet (%)

Fig. 16.1 Effect of dietary protein (air-dry) on average daily weight gain (ADG) of plate size Asian seabass fed diets that contained 7.0 %, 12.8 % or 18.3 % total lipid (air-dry). Data reproduced from Williams et al. (2003a).

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primary energy source. An earlier study found that for iso-nitrogenous diets, increases in dietary gross energy from 18 to 21 MJ kg−1 supplied as a mix of different lipid: carbohydrate ratios improved overall productive performance, a result indicative of protein sparing. However, there was little evidence that lipid (fish oil) or carbohydrate (autoclaved starch) was preferred as a protein sparing energy source at either of the dietary energy densities (Nankervis et al., 2000). Requirements for trypotophan, methionine, lysine and arginine were given as 0.5 %, 2.24 %, 4.5–5.2 % and 3.8 % of dietary protein, respectively (several authors cited in Boonyaratpalin and Williams, 2002). Based on growth response, the total sulphur amino acid demand of small fish (2.5 g) fed increasing levels of methionine (in the presence of cystine) was estimated to be 2.9 % of dietary protein which was similar to previous estimates (Coloso et al., 1999). New work has confirmed the tryptophan requirements of juvenile fish (5.3 g) to be 0.41 % of dietary protein (Coloso et al., 2004). Williams et al. (2001) conducted research into the efficacy of crystalline amino acids in diets for Asian seabass reared in freshwater by comparing their performance to that of fish fed protein bound amino acids (casein). They found that the efficacy of amino acid enrichment was dose dependent. At low dietary supplementation rates (i.e. <3.3 g lysine kg−1 for high-protein diets and up to 6.0 g lysine kg−1 for low-protein diets), crystalline amino acids were utilized as effectively as protein-bound amino acids; however, no further enhancement of fish productivity was induced by higher rates of crystalline amino acid supplementation. Requirements for lysine and arginine in fish weighing <15 g reared in seawater were studied by feeding a mix of intact protein sources (fish meal, zein, squid meal) and crystalline amino acids. On the basis of the growth response, survival and feed efficiency ratio, the lysine and arginine requirements were estimated to be 20.6 g kg−1 dry diet (4.5 % protein) and 18.2 g kg−1 dry diet (3.8 % protein), respectively (Murillo-Gurrea et al., 2001).

16.2.2 Digestibility A list of apparent digestibility coefficients (ADC) for Asian seabass was presented in an earlier review (Boonyaratpalin and Williams, 2002). These digestibility coefficients are reproduced in Table 16.1.

16.2.3 Replacement studies The replacement of fish meal and fish oil in diets for Asian seabass remains a priority as the production of this species increases. Experiments investigating the utilization of high and low ash meat meals (Williams et al., 2003b,c), solvent extracted soybean meal, extruded full-fat soybean meal, steamed full-fat soybean meal, soaked raw full-fat soybean meal (Boonyaratpalin et al., 1998), defatted soybean meal (Tantikitti et al., 2005)

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Table 16.1 Percent apparent protein and energy digestibility coefficients (ADC) of major protein feed ingredients in Asian seabass diets Digestibility coefficients (%)1 Feed ingredient Danish fish meal Tuna fish meal Poultry offal meal Meat meal (high ash) Meat meal (low ash) Soybean meal (full-fat) Soybean meal (solvent-extracted) Canola meal Lupin meal (dehulled) Groundnut meal Wheat-gluten meal

Protein

Energy

87.9 ± 1.0 92.3 ± 1.0 78.8 ± 3.5 53.9 ± 3.9 63.5 ± 3.4 84.8 ± 3.8 86.0 ± 0.8 81.0 ± 2.3 98.1 ± 1.3 91.9 ± 8.0 101.9 ± 1.6

83.3 ± 1.3 69.3 ± 1.3 76.7 ± 5.6 58.2 ± 6.5 66.5 ± 3.4 75.9 ± 7.8 69.4 ± 1.7 56.1 ± 3.0 61.5 ± 1.8 68.7 ± 5.0 98.8 ± 3.1

Mean ± standard error. Data derived from faeces collected either by hand-stripping or by intestinal dissection. Source: data reproduced from Boonyaratpalin and Williams, 2002. 1

and alternative lipid sources such as soybean oil, canola oil and linseed oil (Raso and Anderson, 2003) indicate that partial or even complete replacement of fish meal and fish oil is possible providing nutritional deficiencies (e.g. limiting amino acids, highly unsaturated fatty acids – HUFA, antinutrients, etc.) are carefully considered. The impacts of removing fish meal and fish oil from aqua feeds on the sensory and nutritional quality of fish destined for human consumption must also be considered.

16.2.4 Feeding studies As feed and labour costs continue to increase greater emphasis is being placed on optimizing feeding strategies to ensure feed waste is limited and productivity is maximized. This applies to all phases of farming, from nursery to grow-out. Recent studies indicate that under ambient conditions, feeding a 48 % crude protein diet twice daily (0800 h and 2000 h), as opposed to three (0800 h, 1400 h and 2000 h) or four times per day (0800 h, 1200 h, 1400 h and 2000 h) is adequate to promote optimal growth and FCR in small fish (40–110 g) (Salama, 2008). Others have investigated day versus night feeding response in juveniles fed on restricted rations (2, 4 or 6 % BW d−1) and how these regimes affect the activity of the brush border digestive enzymes such as sucrase, maltase, g-glutamyltranspeptidase and leucine-amino peptidase (Harpaz et al., 2005). Catch-up or compensatory growth has been tested in fish acclimated to different pre-feeding regimes. Fish were prior fed at 0 %, 25 %, 50 % or 75 % of satiation for two weeks before satiation feeding was imposed for the next five weeks. A control

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group was fed to satiation throughout the whole experimental period. Complete compensation was demonstrated in fish previously fed at 50 % and 75 % of satiation, weights matching the control fish after two and four weeks feeding, respectively. However, fish fed at 0 % and 25 % satiation did not catch up with the control fish during the experimental period (Tian and Qin, 2004). Theoretical feeding tables based on data derived from bioenergetic studies have recently been published for Asian seabass (Table 16.2) that cover the whole production cycle (Glencross, 2008). Due to their euryhaline nature, Asian seabass are being cultured in fresh and saltwater environments. In many countries the move to utilize inland saline water bodies for aquaculture has revealed that many of these water bodies are deficient in ions that are important for osmoregulation and homeostasis. Harpaz et al. (2005) demonstrated that the addition of 4 % salt to the diets of Asian seabass reared in freshwater resulted in a significant improvement in FCR, but under saltwater rearing conditions it had no effect. After measuring the activity of various enzymes in gut and intestine they postulated that the better FCR was a result of improved amino acid and glucose absorption since the absorption of these end-products is Table 16.2 Feed rations for Asian seabass fed either a 15 MJ DE kg−1 diet or a 18 MJ DE kg−1 diet and grown at different temperatures under Australian production conditions. Rations were calculated using bio-energetic models

20 °C

15 MJ DE

18 MJ DE

Temperature

Temperature

23 °C

26 °C

29 °C

32 °C

20 °C

23 °C

26 °C

29 °C

32 °C

Fish weight, g fish−1 10 0.23 0.38 50 0.61 1.00 100 0.93 1.54 500 2.57 4.24 1000 4.01 6.62 1500 5.21 8.62 2000 6.29 10.41 3000 8.21 13.59

0.50 1.34 2.07 5.70 8.92 11.61 14.02 18.31

0.59 1.56 2.40 6.63 10.37 13.50 16.30 21.30

0.59 1.58 2.43 6.70 10.47 13.63 16.45 21.49

0.19 0.51 0.78 2.14 3.34 4.34 5.24 6.84

0.31 0.83 1.28 3.53 5.52 7.18 8.67 11.33

0.42 1.12 1.72 4.75 7.43 9.67 11.68 15.26

0.49 1.30 2.00 5.53 8.64 11.25 13.59 17.75

0.49 1.32 2.02 5.58 8.72 11.35 13.71 17.91

Fish weight, %BW 10 2.29 3.76 30 2.03 3.34 100 0.93 1.54 500 0.51 0.85 1000 0.40 0.66 1500 0.35 0.57 2000 0.31 0.52 3000 0.27 0.45

5.04 4.48 2.07 1.14 0.89 0.77 0.70 0.61

5.86 5.21 2.40 1.33 1.04 0.90 0.82 0.71

5.93 5.27 2.43 1.34 1.05 0.91 0.82 0.72

1.91 1.69 0.78 0.43 0.33 0.29 0.26 0.23

3.13 2.78 1.28 0.71 0.55 0.48 0.43 0.38

4.20 3.73 1.72 0.95 0.74 0.64 0.58 0.51

4.88 4.34 2.00 1.11 0.86 0.75 0.68 0.59

4.94 4.39 2.02 1.12 0.87 0.76 0.69 0.60

Source: data reproduced from Glencross, 2008.

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dependent on the Na+/K+ATPase pump. Asian seabass have been successfully cultured in potassium-deficient saline groundwater although Partridge and colleagues (Partridge and Creeper, 2004; Partridge and Lymbery, 2008; Partridge et al., 2008) concluded performance and fish health were improved when potassium was added to the culture water. The effectiveness of adding potassium to feeds for Asian seabass grown in potassium-deficient water bodies is yet to be elucidated.

16.2.5 Miscellaneous studies Understanding the effects of water temperature on the metabolic demands of Asian seabass has been critical to improving our knowledge of its impacts on growth performance and nutritional status (Glencross and Felsing, 2006). In Australia, for example, Asian seabass are cultured under a range of temperatures (normally 20–30 °C), depending on seasonal influences and whether they are held in more or less controlled rearing systems. Extremes of temperature often approach thermal tolerance limits. They grow poorly and return high FCR at temperatures below 22 °C, and increased rearing times or over-wintering are often necessary to reach market sizes >3 kg (Williams et al., 2006b). Growth performance appears to be maintained at even very high temperatures (27–36 °C), but above 36 °C specific growth rate, protein and energy retention drop dramatically and reductions in dry matter, protein and energy content of whole body composition occur (Katersky and Carter, 2005). Williams et al. (2006b) hypothesized that the effects of low temperature (20 °C vs 29 °C) on growth and feed intake could be overcome by feeding diets where the nutrient and energy density of diets were greatly increased or the absolute and relative amounts of n-3 HUFA are increased. Increasing the digestible energy (DE) of diets from 15 to 19 MJ kg−1 in parallel with increases in other nutrients (e.g. protein) improved the daily growth coefficient and FCR of fish reared at 20 °C by nearly 56 % and 41 %, respectively; however, gains in production performance were offset by an increase in the fat content of the whole carcass. Increasing n-3 HUFA from 0.3 % to 2.0 % of diet while decreasing LOA from 2.7 % to 1.1 % of diet significantly improved FCR of fish at both water temperatures. Williams et al. (2006b) speculated that dietary levels of linoleic acid (LOA), EPA and DHA of 0.55 %, 0.60 % and 0.90 %, respectively, were probably adequate for barramundi. A dietary specification of at least 1.5 % n-3 HUFA is required for rapid growth and efficient feed conversion in barramundi reared at close to optimal water temperatures (30 °C).

16.3 Red sea bream and gilthead sea bream The majority of aquaculture research on red sea bream has been conducted in Japan, where this species has been reared experimentally since the early

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1900s (Foscarini, 1988) and cultured commercially since the mid-1960s (Watanabe and Vassallo-Agius, 2003). The Japanese red sea bream industry grew out of the increasing wealth of the Japanese consumer and their desire to consume high- rather than low-value fish. Consequently, the red sea bream industry was initially established by feeding lower-value fish diverted from other domestic uses (Foscarini, 1988). However, the expansion of this industry and the decline in the catch of low-value fish such as sardines, jack mackerel and sand lance necessitated the move towards semi-moist and eventually dry-based feeds to improve the economics of farming and to meet increasingly stringent environmental regulations (Watanabe and Vassallo-Agius, 2003). Today, the Japanese production of red sea bream demands approximately 180 kt of aquafeed per year (Koshio, 2002). Broodstock management, larval rearing and grow-out technologies are well developed for red sea bream (Foscarini, 1988; Koshio, 2002; Watanabe and Vassallo-Agius, 2003). The first nutritional studies commenced in the 1970s, and results were generally based on purified protein sources (Yone, 1976). Studies on the nutritional requirements of red sea bream prior to 1990 were mostly based on moist or semi-moist pellets (>30 % moisture), but the majority of work since then has been based on dry feeds (Koshio, 2002). Much of the diet research in Japan has been sponsored by private feed companies and, as such, feed formulations and production results are held in confidence. Publication of specific nutritional research on this species in the past was often limited or unavailable, but more manuscripts are now being published in readily accessible journals. General dietary requirements for red sea bream are estimated at 40–55 % crude protein, 10–15 % lipid, 10–15 % carbohydrate and 15–21 % ash (Foscarini, 1988; Koshio, 2002) and approximate requirements for other sparids such as the gilthead sea bream (Kaushik, 1997). The scale and success of the Japanese red sea bream industry, particularly once it had converted to using dry extruded feeds, prompted interest in the sea cage aquaculture of the same species in Australia (i.e. P. auratus). This led to significant advances in brood-stock management, larval rearing (Bell et al., 1991; Battaglene and Talbot, 1992; Battaglene and Allan, 1994; Fielder et al., 2002, 2005, 2008) and preliminary research on diet development (Quartararo et al., 1998a,b) for this species under Australian conditions. Henceforth, P. auratus is referred to as red sea bream (Paulin, 1990). Aquaculture of gilthead sea bream is more recent than that of red sea bream, and most of the research effort on this species occurred after 1980 (Kissil, 1991). Many of the early advances in the nutrition of gilthead were based on research conducted with red sea bream. As indicated earlier, Koven (2002) has recently presented a comprehensive review of the nutrient requirements and feeding practices for larval, broodstock and grow-out fish determined from studies up till 2001. His review also touches on alternative feed ingredients such as soy and rape seed protein concentrates, lupin meal, corn gluten meal, meat and poultry meal. Koven (2002) opined that, although the lipid and fatty acid requirements of larval fish were well

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studied, the lack of specialized microdiets for larval fish was restricting detailed investigation of other nutrient requirements. His review also indicated that there appeared to be discrepancies in published research about the ability of gilthead sea bream to utilize dietary lipid to spare protein.

Protein deposition (g kgBW–0.7 d–1)

16.3.1 Requirements Many of the basic requirements for protein, EAA, lipids, vitamins, minerals and energy during the juvenile or grow-out phase of growth have been previously elucidated for red sea bream (Takeuchi et al., 1991; Forster and Ogata, 1998; Koshio, 2002) and gilthead sea bream (Koven, 2002). Many requirements were determined using animals of one size fed diets in which the dietary crude protein, lipid or energy content was varied over a suitable range; changes in response criteria such as weight gain or protein retention were then measured. More recently, these types of studies have progressed to determine requirements on a digestible protein (DP) and DE basis. For example, Booth et al. (2007), using predetermined digestibility coefficients of dietary ingredients coupled with a summit/diluent approach, demonstrated that growth and protein retention in juvenile (30–90 g) red sea bream was closely linked to the DP : DE ratio of the diet. According to their results, diets for juvenile red sea bream should contain approximately 23 g DP MJ DE−1 to promote optimal weight gain and protein deposition (Fig. 16.2). This corresponded to practical diets containing dietary DP (g kg−1) and DE (MJ kg−1) contents of 460 : 20, 420 : 18 or 350 : 15, respectively.

1.50 1.25 1.00 0.75 High-energy series Mid-energy series Low-energy series

0.50 0.25 0.00 10

13

15

18

20

23

25

28

30

DP:DE ratio (g DP MJ DE–1)

Fig. 16.2 Effect of dietary digestible protein (DP) and digestible energy (DE) ratio on relative protein deposition in juvenile red sea bream (P. auratus). Data reproduced from Booth et al. (2007).

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Current practice has moved away from simple dose–response experiments to the use of bio-energetic or multivariate approaches (Shearer, 1995; Azevedo et al., 2005) to predict growth relationships or the DP and DE requirements of growing fish. This approach has been used to estimate the protein and energy requirements of gilthead sea bream (Lupatsch et al., 1998, 2001a, 2003b; Libralato and Solidoro, 2008; Lupatsch, 2008) and European sea bass (Lupatsch et al., 2001b) and to examine differences in energy utilization by sparids and grouper (Lupatsch et al., 2003a). However, no such work has been published on red sea bream. Similarly, extension of bio-energetic models to determine amino acid or other essential nutrient requirements is rare and warrants increased attention in the future. Amino acid requirements for lysine were extrapolated (A/E ratio) to determine the EAA for red sea bream in earlier work (Forster and Ogata, 1998). A recent experiment has elucidated the ideal amino acid pattern for juvenile red sea bream (1.6 g) by feeding a series of semi-purified diets containing 20 % crystalline amino acids to simulate the amino acid pattern of red sea bream egg protein, red sea bream larvae protein, red sea bream juvenile whole body protein or brown fish meal (Alam et al., 2005). The results suggest that red sea bream juveniles are able to utilize large amounts of crystalline amino acids in coated form and demonstrated that the highest weight gain was observed in fish fed the diet simulating the amino acid pattern of juvenile red sea bream. Takagi et al. (2001) found that supplementation of diets containing high levels of soy protein concentrate with crystalline lysine or methionine added alone or in combination improved growth in red sea bream juveniles (12 g) above that of fish fed a control diet having no added crystalline amino acids. However, the growth response of yearling red sea bream (180 g) fed similar diets was more equivocal leading these authors to suggest that the amino acid requirement of red sea bream might change according to growth rate or age. The essential amino acid pattern of juvenile gilthead sea bream (5 g) was recently updated using the amino acid dilution method. The levels, expressed as A/E ratios relative to lysine (= 100) were estimated as arginine, 108.3; threonine, 58.1; histidine, 36.8; isolouecine, 49.7; leucine, 92.7; methionine, 50.8; phenyalanine, 44.2; valine, 62.6; tryptophan, 14.6 (Oliva-Teles and Peres, 2008). As for red sea bream, these authors found the EAA requirement of gilthead sea bream correlated well with the EAA composition of fish tissue. Recently, many experiments have been conducted to investigate the efficacy of dietary taurine in ameliorating the effects of ‘green liver syndrome’ in red sea bream. This symptom is thought to be related to increases in dietary content of alternative feed ingredients such as soybean meal at the expense of fish meal content (Takagi et al., 2006). Early work hypothesized that taurine might in fact be an essential nutrient for larval and juvenile fish (Takeuchi et al., 2001), playing an important role in lipid metabolism (Matsunari et al., 2008b). Supplementing low fish meal diets with taurine has been shown to improve performance and reduce ‘green liver

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syndrome’ in yearling red sea bream. Red sea bream fed diets without taurine supplementation exhibited inferior feed performance, alteration of erythrocytes and significant reductions in hepato-pancreatic taurine concentration and elevations in hepato-pancreatic biliverdin concentration (Takagi et al., 2006). Recommended dietary inclusion levels of taurine have been suggested as 0.5 % (Matsunari et al., 2008a) and 0.52 % in terms of optimizing growth and 0.48 % in terms of optimizing feed efficiency (Matsunari et al., 2008b). Moderate elevation of dietary lipid is generally thought to enhance protein retention in gilthead sea bream, supporting a protein sparing effect in this species. However, the level of dietary protein, feeding regimes or activity level employed in different studies all appear to impact on the magnitude of this effect (Forster and Ogata, 1996; Vergara et al., 1996, 1999; Caballero et al., 1999; Company et al., 1999a,b; Santinha et al., 1999). Other research has found little evidence of protein sparing in sparids. For example, Schuchardt et al. (2008) recently reported that increases in the lipid content of iso-proteic diets from 10 % to 15 % did not induce a protein sparing effect in red porgy while lipid levels above 15 % negatively affected performance. In addition, Bonaldo et al. (2008a) found that although increasing the energy density of diets (i.e. from 16 % to 32 % crude fat) increased feed intake, it elevated FCR and did not improve weight gain of gilthead sea bream fed to apparent satiety. A similar outcome was reported by Velazquez et al. (2006a). Like lipid, carbohydrates are potential sources of dietary energy for marine fish despite the fact that fish have no requirement for carbohydrate per se. Recent research in this area is dominated by metabolic investigations on enzymes important to carbohydrate metabolism (Panserat et al., 2000, 2002; Caseras et al., 2002; Fernandez et al., 2007; Couto et al., 2008; Enes et al., 2008b) as well as simple studies on glucose regulation (Booth et al., 2006). Evidence of protein sparing is limited; however, Wu et al. (2007) found that protein productive value was improved by incorporating 20 % raw starch (corn starch, tapioca starch or potato starch) into the diets of juvenile yellowfin sea bream Sparus latus. Several studies have recommended including no more than 20–30 % carbohydrate in the diets of bass and sea breams (Booth et al., 2006; Fernandez et al., 2007; Wu et al., 2007; Rawles et al., 2008). Requirements for vitamin A have been recently estimated for small red sea bream (1.2 g) by feeding increasing dietary contents of retinol palmitate (i.e. 0, 300, 600, 1500, 3000, 4500, 6000, 15 000 or 30 000 retinol equivalent kg−1 diet). Specific growth rate tended to plateau around 6000 retinol equivalent kg−1 diet, beyond which a slight decrease in specific growth rate was observed. Besides a growth reduction in red sea bream fed diets with low levels of vitamin A, no other signs of deficiency were noted. Likewise, there were no signs of toxicity in fish fed the highest levels of vitamin A. Based on specific growth rate and liver retinol concentration the requirement of

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dietary vitamin A for juvenile red sea bream was estimated to be between 1500 and 6000 retinol equivalents kg−1 (Hernandez et al., 2004).

16.3.2 Digestibility Apparent digestibility coefficients for a diverse range of ingredients have been published for red sea bream (Yamamoto et al., 1998; Glencross and Hawkins, 2004; Glencross et al., 2004a; Booth et al., 2005, 2006) and gilthead sea bream (Nengas et al., 1995; Lupatsch et al., 1997). As for most carnivorous species, the digestibility of marine proteins and oils is high (Yamamoto et al., 1998; Booth et al., 2005). Newer studies have tended to focus on measuring digestibility of rendered animal by-product meals and plant products with higher levels of crude protein (Drew et al., 2007), such as improved varieties of lupin (Glencross et al., 2003a; Glencross and Hawkins, 2004) or differently processed canola meals (Glencross et al., 2004a). In the past it was common to measure the digestibility of alternative ingredients at one inclusion level (usually 30 %). However, concerns about the additivity of certain ingredients, especially carbohydrate sources, has driven research to investigate the effects of dietary inclusion content on digestibility. For example, red sea bream are highly efficient at digesting the protein from grains such as extruded wheat, but organic matter and gross energy digestibility decrease linearly as inclusion levels of wheat (Booth et al., 2005) or pregelatinized starch (Fig. 16.3) increase (Booth et al., 2006). In contrast, inclusion level had no affect on the protein and energy digestibility

Gross energy ADC (%)

105 95 85 75 65 55 45 150

250

350

450

Gelatinized wheat starch inclusion level (g kg–1 diet)

Fig. 16.3 Effect of increasing dietary inclusion content (g kg−1 diet) on percent apparent energy digestibility coefficients (ADC) of gelatinized wheat starch by juvenile red sea bream (P. auratus). Data reproduced from Booth et al. (2006).

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of meat meal and poultry meal by red sea bream (Booth et al., 2005) or soybean meal (Venou et al., 2006). Other researchers have reported similar issues with the additivity of carbohydrate apparent digestibility coefficients (ADCs) for gilthead seabream (Lupatsch et al., 1997). Alternate studies have confirmed that sparids are reasonably efficient at digesting the energy from wheat, provided inclusion levels are not excessive (Georgopoulos and Conides, 1999; Venou et al., 2003); however, there is some indication that the level of dietary fat can negatively affect starch and protein digestibility by interfering with the activity of alpha-amylase and protease in the digestive tract (Fountoulaki et al., 2005). It is clear that carbohydrate sources, excluding non-starch polysaccharides (NSP) have potential for use in sparid diets, but this potential will ultimately be governed by the protein and lipid requirements of individual species which in turn dictates the ‘formulation space’ remaining for carbohydrate inclusion. The aforementioned variability in carbohydrate digestibility underscores the need to determine the ADCs of carbohydrate sources over a wide range of inclusion levels in order to accurately formulate either research or commercial feeds. A list of apparent digestibility coefficients for different feed ingredients fed to red sea bream (P. auratus) is presented in Table 16.3.

16.3.3 Replacement studies There is a great deal of published research targeting the replacement of fish meal or fish oil in aquafeeds for sparids. Studies on the use of oilseeds, legumes and plant proteins in either their raw or processed forms dominate the literature (Takagi et al., 2000a; Pereira and Oliva-Teles, 2002, 2004; Venou et al., 2003, 2006; Glencross et al., 2004b; Kissil and Lupatsch, 2004; Biswas et al., 2007; Lozano et al., 2007; Martinez-Llorens et al., 2007a,b; Enes et al., 2008a). Many of these studies are aimed at understanding or overcoming the negative impacts of feeding plant proteins (e.g. low digestibility, antinutrients, palatability, HUFA profile, gut histology) on the nutritional or immune status of the fish (Sitja-Bobadilla et al., 2005) or implications for the consumer (Robles et al., 2008). By comparison, there are relatively few recent studies investigating the replacement of fish meal with animal by-product meals such as meat or poultry meals, blood meals or feather meals in aquafeeds for sparids (e.g. Martinez-Llorens et al., 2008), despite previous evidence that animal byproduct meals are suitable alternatives to fish meal (Quartararo et al., 1998a,b; Nengas et al., 1999; Takagi et al., 2000b). This may be related to the European ban on including some rendered animal by-product meals in animal diets, prompting more effort on use of plant proteins (MartinezLlorens et al., 2008). Australia is known to be free of BSE and no such legislative constraints are in place to preclude the use of rendered animal by-product meals in aqua-feeds. Booth et al. (2008a) found that the diets for juvenile red sea bream that are formulated on a digestible protein and

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Table 16.3 Percent apparent digestibility coefficients (ADC) for protein and energy of Australian ingredients fed to red sea bream Ingredient Fish meal1 Wheat gluten1 Lupin seed meal (Gungarru)1 Canola meal (solvent-extracted)2 Canola meal (expeller-extracted)2 Canola protein concentrate2 Solvent-extracted soybean meal2 Fish meal3 Fish oil3 Fish oil3 Extruded wheat3 Extruded wheat3 Extruded wheat3 Meat meal3 Meat meal3 Poultry meal3 Poultry meal3 Blood meal3 Haemoglobin powder meal3 Solvent-extracted soybean meal3 Expeller-extracted soybean meal3 Pregelatinized wheat starch4 Pregelatinized wheat starch4 Pregelatinized wheat starch4 Pregelatinized wheat starch4

Inclusion level (%) 42 na 30 30 30 30 30 50 15 25 20 30 40 30 50 30 50 15 15 30 30 15 25 35 45

Protein ADC (%) 87.5 102.0 98.7 83.2 93.6 52.6 79.2 94.3 na na 100.6 105.4 100.1 62.2 65.3 84.9 86.9 81.6 95.1 87.2 90.7 na na na na

Energy ADC (%) 87.8 84.3 56.3 43.9 61.6 73.7 58.3 99.2 100.5 98.3 80.5 76.9 74.4 72.0 70.5 91.4 91.4 81.3 79.5 66.8 64.3 89.2 73.9 70.2 55.2

1

Data from Glencross et al., 2003a. Data from Glencross et al., 2004a. Data from Booth et al., 2005. 4 Data from Booth et al., 2006. na = not applicable. 2 3

energy basis (25 g DP MJ DE−1) can contain as much as 36 % poultry meal, 35 % meat meal or 42 % solvent extracted soybean meal before weight gain and feed efficiency decline. In addition, commercially extruded diets that contained a blend of these feed ingredients were able to replace all but 16 % of the fish meal in similarly treated control diets. Various studies have found that gilthead sea bream will tolerate diets containing about 20 % carbohydrate (native or waxy maize starch, pregelatinized starch) before growth performance is reduced (Couto et al., 2008; Enes et al., 2008a). These tolerance levels are slightly lower than those cited for juvenile red sea bream (Booth et al., 2006). To date, replacement studies evaluating soybean meal have received the most attention. Soybean meal is known to impair growth and protein utili-

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zation and cause distinct morphological alterations to the intestine of Atlantic salmon and rainbow trout. However, its use, even at very high dietary levels (up to 30 %), does not appear to affect growth, feed intake, protein efficiency ratio or FCR in gilthead sea bream. Notwithstanding these results, at high inclusion levels it appears to induce subtle changes in the morphology of the distal intestine (i.e. presence of cellular infiltration of the submucosa and lamina propria) (Bonaldo et al., 2008b). Longer term studies have shown that suitable blends of plant proteins such as corn, wheat gluten, extruded peas, wheat and rapeseed meal can satisfactorily replace up to 75 % of fish meal in diets for gilthead sea bream without affecting growth, but fillet yield and n-3 fatty acid composition of fish fed plant protein-based diets was lower than fish fed a fish meal control diet (De Francesco et al., 2007). These studies have been extended to investigate the concurrent replacement of fish meal with plant proteins and fish oil with different types of vegetable oil (i.e. rapeseed: linseed: palm oils) with promising results (Benedito-Palos et al., 2007). Much attention is being focused on the use of plant oils in diets for cultured marine finfish, firstly because of their abundance in the face of decreasing stocks of fish oil (Bell and Tocher, 2008) and secondly because marine fish lack the ability to convert polyunsaturated fatty acids (PUFA) like LOA and linolenic acid (LNA) in vegetable oils to the essential fatty acids EPA, DHA or ArA. This issue becomes extremely important where dietary fish meal content is also significantly reduced (a good source of essential fatty acids), particularly when it is replaced with plant proteins (a poor source of EFA). Glencross et al. (2003b) has demonstrated that crude or refined canola oil and refined soybean oil can partially replace significant levels of fish oil in the diets of red sea bream before growth and FCR are negatively affected. However, they found a strong correlation between the level of fish oil substitution and the fatty acid composition of tissues, particularly an increase in the level of PUFA (especially LNA and LOA). Sensory assessment of fish fed these diets also indicated a preference for red sea bream fed refined canola oil > refined soybean oil > fish oil. These findings were supported in a later study that demonstrated that canola oil could replace up to 70 % of pollock oil in the diet of red sea bream without compromising growth performance (Huang et al., 2007). Generally, the judicious substitution of fish oils by plant oils has been shown to have dramatic impacts on lipid composition but relatively little impact on the production performance of sparids. Consequently, research has started to focus on the use of ‘finishing diets’ to restore or ameliorate the fatty acid composition of edible fish, particularly the n-3 HUFA. Such an approach was explored by Glencross et al. (2003c) who fed red sea bream for three months on canola or soybean oil diets before switching them back to diets containing fish oil. Moderate decreases in PUFA (18:2n-6, 18:3n-3) and increases in long-chain PUFA (20:5n-3, 22:6n-3) were recorded in tissue samples after one month, demonstrating the potential of this approach.

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These results were echoed in the study of Izquierdo et al. (2005) who found reductions in the muscle content of DHA, ArA and EPA of gilthead sea bream reared on diets containing rapeseed, linseed or soybean oil. Refeeding a fish oil-based diet for 60 days restored muscle contents of DHA and ArA, but contents of EPA were not recovered even after 90 days (Izquierdo et al., 2005). Although growth of marine fish fed vegetable oils, particularly blends of different oils, generally approximates that of fish fed fish oil, the use of these oils has implications for their immunological status and resistance to different stressors. Thus, increasing effort is being directed at understanding the impact of vegetable oils on the stress (Ganga et al., 2008) and immune response of gilthead sea bream, specifically the interferon system (Montero et al., 2008a,b). Readers are encouraged to consult the following publications for background on lipid research and replacement studies with red and gilthead seabream: Glencross et al. (2003c), Izquierdo et al. (2003) and Izquierdo et al. (2005).

16.3.4 Feeding studies Recent reviews have presented feeding tables for larvae, juvenile and adult red sea bream (Koshio, 2002), but at that time less information was available for gilthead sea bream (Koven et al., 2001). New work has recommended feeding rates of 2.3 % and 0.6 % BW day−1 as optimum and maintenance rations, respectively, for very small (3 g) gilthead sea bream (Mihelakakis et al., 2002), and numerous studies are now available that estimate the daily feed intake of gilthead sea bream based on the total energy or protein demands of growing fish (Lupatsch et al., 1998, 2003b; Lupatsch, 2004, 2008). Although bio-energetic models estimate total feed demand, much research on gilthead sea bream is still dedicated to exploring different feeding regimes and if these regimes can enhance feed intake and weight gain (Paspatis et al., 2000; Mihelakakis et al., 2002; Ibarz et al., 2003; SánchezMuros et al., 2003; Sitja-Bobadilla et al., 2003; Velazquez et al., 2006b). In comparison, there are far fewer feeding studies reported for red sea bream. Recent publications include a study on the interactive effects of photoperiod and feeding interval which demonstrated that weight gain of newly weaned red sea bream (0.14–1.80 g) could be maximized by rearing them under an 18L:6D photoperiod and feeding them at least 10 % of their body weight eight times per day (Tucker et al., 2006). This study was followed by an investigation of larger juveniles (5 vs 20 g) fed one of nine feeding regimes to apparent satiation: 1 feed early, 1 feed late, 2 feeds early, 2 feeds late, 4 feeds, 4 feeds early, 4 feeds late, 6 feeds or 8 feeds per day. The results indicated that optimum to maximum weight gain and FCR in juvenile red sea bream could be achieved by feeding fish to apparent satiation twice per day. In addition, the gastric evacuation rate of a single meal fed to small or large snapper proved to be similar (relative feed content = 2.733 ± 0.195 × exp(−0.139±0.013)), with approximately half the meal passed within 5 h and the

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whole meal cleared from the stomach within 16–20 h (Booth et al., 2008b). As for gilthead sea bream (Gines et al., 2004; Eroldogan et al., 2006a,b, 2008), there is renewed interest in evaluating the potential for compensatory growth in red sea bream (Oh et al., 2007) and the beneficial effects of extended photoperiod on feed intake (Biswas et al., 2005, 2006, 2008).

16.3.5 Miscellaneous studies The unnatural dark skin pigmentation (i.e. melanism) of cultured red sea bream, gilthead sea bream and red porgy has continued to affect the profitability of the sparid industry and a great deal of research effort has focused on developing practical nutritional or physical solutions to ameliorate this problem. Approaches have included the addition of natural or synthetic carotenoids to diets (Gomes et al., 2002; Gouveia et al., 2002; Booth et al., 2004; Kalinowski et al., 2007), alteration of the culture environment to reduce natural light intensity, background colour or alter certain wavelengths (Rotllant et al., 2003; Doolan et al., 2007, 2008a,b,c) and post-harvest treatment of fish with chemicals such as potassium or sodium chloride (Lin et al., 1998; Doolan et al., 2008a). Doolan et al. (2008b) recommended that red sea bream P. auratus should be held in white cages and fed diets containing 30 mg astaxanthin kg−1 for 50 days to increase skin lightness and red pigmentation. Pavlidis et al. (2008) have indicated the skin lightness of red porgy P. pagrus can be significantly improved by providing low light intensity, blue spectra, a water temperature of 19 °C and a white background. This suggests the problem is multifactorial in nature and indicates that for each species, a specific solution may be required for each particular culture situation.

16.4 Grouper Groupers are a diverse group of predatory reef fish comprising more than 90 species within five main genera of the family Serranidae (Williams, 2005). They are widely distributed in the tropical and subtropical seas of the world and are highly prized in the fish markets of Hong Kong and Singapore (Rimmer et al., 2004; Williams, 2005). Well known species include the orange spot grouper Epinephelus coioides, humpback grouper Cromileptes altivelis and coral trout Plectropomus spp. The preferred harvest size for groupers tends to be between 0.6 and 1.2 kg depending on the market, with species such as the orange spot grouper taking between 6 and 12 months to reach this weight under typical farm conditions. One of the greatest impediments to the aquaculture development of groupers, especially the higher value species (e.g. coral trout and humpback grouper), is the limited production of fry and fingerlings. This bottleneck is being addressed through advances in hatchery technology (Rimmer et al., 2004; Sim et al., 2005). Until recently,

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low-value fish was the preferred feed for the grow-out of groupers, especially in South East Asia. This practice has tended to place increased pressure on stocks of low-value fish (Shapawi et al., 2007) and can act as a potential vector for the introduction of diseases to farms. As issues surrounding hatchery bottlenecks for grouper have improved, grow-out production has escalated. This expansion can only continue if it is based on a move away from feeding low-value fish to feeding cost-effective, low polluting manufactured feeds. An excellent monograph listing advances in larval and grow-out technologies for grouper, including many papers on nutrition, has recently become available (Rimmer et al., 2004).

16.4.1 Requirements The gross nutrient requirements of farmed grouper appear to be quite similar (Williams, 2005), with fingerlings requiring a diet with high crude or digestible protein content (i.e. >44 % DP) (Luo et al., 2004; Williams et al., 2004; Tuan and Williams, 2007), medium levels of lipid (i.e. 7–16 %) (Lin and Shiau, 2003; Williams et al., 2004, 2006a; Tuan and Williams, 2007) and no more than 20 % cereal grains to promote good growth and feed conversion (Williams, 2005). A longer term feeding study with larger humpback grouper (150–400 g) found no interaction between increasing levels of dietary protein and lipid content and suggested diets for this slower growing species should contain a digestible protein content approaching 51 %, 10–12 % lipid and a DP : DE ratio of 31–32 g MJ−1 to promote optimal growth (Usman et al., 2005). Many studies have shown that growth rates of grouper continue to increase in response to increases in dietary protein while concomitant increases in lipid content have little impact apart from increasing adiposity (Cheng et al., 2006; Tuan and Williams, 2007), indicating that grouper rely heavily on dietary proteins as a source of metabolic energy (Williams et al., 2004). Further evidence for the use of dietary protein as an energy source for grouper can be found in the bio-energetic study of Lupatsch and Kissil (2005) who showed that white grouper (Epinephelus aeneus) has a relatively low feed intake coupled with a low energy requirement and thus the opportunity to spare dietary protein is limited. Lupatsch and Kissil (2005) presented iterative feed formulations for white grouper over the entire grow-out range which indicated that the DP : DE of aquafeeds for this species will decline from about 33 to 21 g DP MJ DE−1 as fish grow from 5 g to 750 g. Little is known of about the quantitative amino acid requirements of grouper. Recent work using diets containing graded levels of crystalline amino acids has determined the dietary lysine requirement of juvenile orange spot grouper to be 28.3 g kg−1 diet (e.g. 55.6 g kg−1 protein) (Luo et al., 2006b) and the dietary methionine requirement (in the presence of cystine) to be 13.1 g kg−1 diet (e.g. 27.3 g kg−1 dietary protein) (Luo et al., 2005). A similar study with the same species determined the dietary arginine

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requirement as 27 g kg−1 diet (e.g. 55 g kg−1 protein) (Luo et al., 2007). Other work which simulated the amino acid composition of different protein sources using a mix of intact proteins (i.e. fish meal and soybean meal) and crystalline amino acids suggests that diets with an amino acid pattern similar to white fish meal or juvenile grouper protein (E. coioides) are closer to the overall amino acid pattern required by this species than the amino acid pattern of proteins such as brown fish meal, red sea bream protein or hen egg protein (Luo et al., 2008). The type of fat appears to have an impact on its protein sparing capacity in grouper. For example, the addition of long-chain fatty acids (LCFA) in the form of olive oil was shown to induce a protein sparing effect (i.e. improved protein retention) when test feeds contained 15 % but not 30 % olive oil. No such sparing effect was apparent when medium-chain fatty acids (MCFA) in the form of coconut oil were included at 15 % of the diet while severe reductions in protein and energy retention were recorded in fish fed diets containing 30 % coconut oil (Williams et al., 2006a). High lipid levels (i.e. 30 %), regardless of the type of lipid, tended to reduce feed intake, but grouper fed diets containing high lipid-MCFA diets exhibited major reductions in feed intake. A concurrent radio labelling study by Smith et al. (2005) found that MCFA were more rapidly oxidized than LCFA and caused a general increase in respiration rate of grouper. These authors suggested that while MCFA appear to be a more utilizable form of energy than LCFA, the rapid oxidation of MCFA appears to trigger acidosis or ketogenic like responses that reduce appetite. Requirements for n-3 HUFA of juvenile grouper appear to be quite different for juveniles of different species with a minimum dietary content of 1 % suggested for humpback grouper and up to at least 2.5 % of diet for tiger grouper (Suwirya et al., 2005). Wu et al. (2002, 2003) have demonstrated that DHA is more important than EPA in promoting growth and elevating the immune response in E. malabaricus. The carbohydrate requirements of groupers have received little attention to date and much more research is needed in this area. Much of the work is equivocal. For example, Shiau and Lin (2001) demonstrated that starch and glucose were equally well utilized (i.e. weight gain, feed efficiency and protein efficiency ratio) at a high water temperature (29 °C), but that starch was better utilized than glucose at a lower water temperature (23 °C). More recent work has investigated the utilization and digestibility of diets containing 20 % glucose, sucrose, dextrin or starch in high protein (54 %), low lipid (11 %) diets of juvenile humpback grouper (8.0 g) and indicates that protein and energy retention was superior in fish fed glucose > dextrin > starch > sucrose (Usman et al., 2004). Not surprisingly, preliminary glucose tolerance tests also show that glucose is more rapidly absorbed into the plasma compartment; peaking approximately 3 h after feeding compared to 6 h for sucrose, dextrin or starch (Usman et al., 2004). No improvement in weight gain or feed efficiency was recorded in humpback

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grouper (8 g) fed isonitrogenous / isocaloric diets containing >14 % dextrin (Suwiyra et al., 2004). Requirements for vitamin C have been determined for E. malabaricus using diets containing graded levels of L-ascorbyl-2-monophosphate-Mg (C2MP-Mg) or L-ascorbyl-2-monophosphate-Na (C2MP-Na) esters. Percentage weight gain was maximized by feeding diets containing about 51 mg or 17 mg of C2MP-Mg or C2MP-Na ascorbic acid equivalent, respectively (Lin and Shiau, 2004). Requirements for other vitamin C derivatives such as L-ascorbyl-2-sulphate (C2S) or L-ascorbyl-2-polyphosphate (C2PP) are approximately 55 mg and 31 mg ascorbic acid equivalent, respectively (Lin and Shiau, 2005a). Vitamin A requirements for the greasy grouper E. tauvina have been reported as as 3101 IU vitamin A (as retinyl acetate) kg−1 diet (Mohamed et al., 2003) while the requirement for vitamin E (DL-atocopheryl acetate) appears to be between 50–100 mg kg−1 diet (Lin and Shiau, 2005b). A total dietary phosphorous requirement of 8.6 g kg−1 is recommended for juvenile orange spot grouper growing in floating cages (Zhou et al., 2004). New work on E. coioides has considered the complementary effects of dietary phosphorous (NaH2PO4 · 2H2O) and calcium (Ca-lactate) and indicates that adequate dietary phosphorous (about 11 g kg−1 diet) is essential for promoting weight gain and supporting mineralization of bone and scales in grouper; however, the beneficial effects of added dietary calcium were less evident and addition of this mineral reduced feed intake (Ye et al., 2006). Requirement for selenium (selenomethionine) E. malabaricus is about 0.8 mg kg−1 diet (Lin and Shiau, 2005c).

16.4.2 Digestibility Like many carnivorous species, grouper digest ingredients of animal origin better than those of plant origin. Protein digestibility of marine and terrestrial ingredients is generally high (>75 %) but is more variable for plant ingredients, being high for lupin and corn gluten for example but particularly low for ingredients high in fibre such as rice bran (Williams, 2005). A growing list of ADCs is now available for a diverse range of ingredients fed to grouper species. To date the majority of these data have been determined using indirect settlement techniques and selected values for humpback grouper (Laining et al., 2003) and orange spot grouper (Eusebio et al., 2004a; Lin et al., 2004) are reproduced in Table 16.4. Lin et al. (2004) found that the amino and fatty acid availability of test ingredients closely approximated values for protein and lipid digestibility, respectively, and that generally, fatty acid availability decreased as fatty acid chain length increased. There is some indication that apparent digestibility in grouper is negatively correlated with feed rations, possibly due to the increased passage rate of larger meals; however, reductions in ADCs were minor and more work is required to elucidate the effects of ration size on digestibility (Luo et al., 2006a).

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Table 16.4 Percent apparent digestibility coefficients (ADC) for dry matter, crude protein (CP) and energy for selected South East Asian ingredients fed to grouper Digestibility (%)1 Feed ingredient Dry matter

Crude protein (CP)

Marine product Acetes shrimp meal (72 % CP) Fish meal (Chilean, 65 % CP) Fish meal (local, 45 % CP) Fish meal (sardine, 65 % CP) Fish meal (tuna, 50 % CP) Fish meal (white, 69 % CP) Shrimp head meal (50 % CP) Squid meal (71 % CP)

76.0 ± 4.00 83.6 ± 3.09 59.1 ± 1.23 87.2 ± 2.53 75.4 ± 3.61 89.2 ± 1.69 58.5 ± 3.33 99.4 ± 0.95

95.0 ± 0.72 98.0 ± 0.07 82.4 ± 1.99 92.5 ± 1.40 76.2 ± 1.92 98.6 ± 0.31 78.0 ± 1.32 94.2 ± 0.21

Terrestrial animal product Blood meal (ring-dried, 84 % CP) Blood meal (oven-dried, 84 % CP) Blood meal (formic, 87 % CP)2 Blood meal (propionic, 84 % CP)2 Meat meal (Australian, 44 % CP) Meat meal (Philippine, 45 % CP) Meat solubles (Danish, 74 % CP) Poultry feather meal (67 % CP)

36.9 ± 0.98 48.1 ± 0.85 67.9 ± 1.63 61.7 ± 2.60 60.8 ± 0.80 77.7 ± 0.09 99.3 ± 0.45 74.3 ± 3.06

15.5 ± 2.01 55.2 ± 1.35 87.5 ± 0.55 84.2 ± 0.69 98.9 ± 1.32 83.8 ± 1.66 97.6 ± 0.08 81.8 ± 2.58

85.2 ± 2.81 94.0 ± 2.03 74.2 ± 3.14 56.0 ± 0.04 54.1 ± 1.24 45.3 ± 2.37 22.2 ± 1.52 68.5 ± 7.02 54.8 ± 2.72 75.7 ± 1.98

82.9 ± 4.71 99.5 ± 0.65 93.5 ± 1.22 78.8 ± 2.64 97.5 ± 3.65 80.5 ± 1.30 59.5 ± 1.41 42.7 ± 5.38 67.2 ± 1.29 96.0 ± 0.13

72.8 ± 0.85

96.0 ± 0.13

Terrestrial plant product Corn meal (8 % CP) Corn gluten meal (56 % CP) Cowpea meal (white, 24 % CP) Lucaena (ipil-ipil) meal (19 % CP) Lupin albus meal (26 % CP) Palm oil cake meal (11 % CP) Rice bran (11 % CP) Rice bran (14 % CP) Soybean meal (full-fat, 41 % CP) Soybean meal (solvent-extracted, 50–54 % CP) Wheat flour (9 % CP)

Energy

77.2 ± 1.91 85.2 ± 0.90 63.6 ± 0.89

40.4 ± 3.74 44.3 ± 0.97 51.1 ± 0.89

Mean ± SD. Oven-dried blood meal fermented using either formic or propionic acids. Source: data reproduced from Williams, 2005. 1 2

16.4.3 Replacement studies An increasing number of studies are beginning to evaluate alternative feed ingredients for use in farm-made and commercial feeds for grouper species; however, much of the work has used very small fish and further investigations with larger animals are warranted. Recent work includes a robust investigation of terrestrial proteins using predetermined ADCs of test and

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supplementary ingredients to formulate diets with a similar digestible protein content prior to evaluation (Eusebio et al., 2004b). Although this study used very small fish (3 g), it demonstrated that diets for E. coioides can include up to 20 % cowpea meal, 16–19 % meat and bone meal when fish meal is replaced on a digestible protein basis. Other ingredients such as corn gluten meal and soy protein concentrate also appear to be very promising (Eusebio et al., 2004b); however, no more than 10 % shrimp head meal is recommended in diets for humpback grouper (Rachmansyah et al., 2004). Millamena (2002) demonstrated that a 4 : 1 mix of processed meat meal and blood meal (both of Australian origin) could replace as much as 80 % of dietary fish meal (isonitrogenous basis) in a control diet without affecting the growth rate and feed conversion of E. coioides. Total replacement of fish meal with this mixed protein source resulted in significantly poorer growth rates and feed conversion, probably due to the declining content and availability of dietary essential amino acids such as lysine and methionine (Millamena and Golez, 2001; Luo et al., 2005, 2006b). In a similar study with E. coioides, highly digestible meat solubles (72 % crude protein; minced, pressed, sterilized and spray dried) were able to replace up to 40 % of fish meal protein without growth and performance being affected (Millamena and Golez, 2001). In contrast, Wang et al. (2008) found that performance of humpback grouper declined if more than 40 % of fish meal protein was replaced on an isonitrogenous/isocaloric basis with a blend of 50 : 20 : 20 : 10 poultry by-product meal:meat and bone meal:blood meal : fish meal. Based on weight gain alone, good quality sources of poultry by-product meal were able to replace more than 75 % of fish meal (sole protein source) in control diets for humpback grouper when formulated on an isoproteic/isolipidic basis (Shapawi et al., 2007). Caution should be exercized when reviewing replacement studies that do not account for the digestibility or availability of nutrients in ingredients a priori. This is especially so for animal by-product meals which often vary markedly in nutrient content and availability due to differences in the type and severity of the rendering process. Although the intensive culture of grouper is relatively recent, attention has already turned to investigating alternatives to fish oil. Shapawi et al. (2008) measured no difference in the performance of humpback grouper fed isolipidic test diets (10 %) containing 5 % crude or refined palm oil, refined soybean oil or canola oil compared to fish fed a diet containing the same level of cod liver oil. However, replacement of the marine oil with the vegetable oils significantly affected the fatty acid content and composition of tissue and liver; replacement of fish oil with vegetable oils resulted in reduced levels of total n-3 PUFA and an increased level of total n-6 PUFA in both muscle and liver tissues (Shapawi et al., 2008). Other research has found that E. malabaricus fed a 3 : 1 or 1 : 1 ratio of fish oil : corn oil (4 % total lipid) matched the weight gain and performance of fish fed a control diet containing a similar level of fish oil (Lin and Shiau, 2007). Like the

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previous study, tissue fatty acid composition was indicative of the fatty acid profile of the test diets. In addition, the authors of this study linked elevations in the leukocyte superoxide production ratio, plasma lysozyme and alternative complement (ACH50) activities of grouper (e.g. indicators of enhanced non-specific immune response) to test diets containing the blends of fish oil and corn oil. More clarification of these effects is warranted.

16.4.4 Feeding studies A very early study centred on feeding a mixture of low-value fish to wildcaught E. tauvina (60 g) held in floating cages. Tested feeding frequencies ranged from once every fifth, fourth, third or second day to once, twice or three times daily. Optimal growth and good FCR corresponded with a feeding frequency of once every two days and apparent feed intake was highly dependent on gastric evacuation time (Thia-Eng and Seng-Keh, 1978). Very small red spotted grouper E. akaara exhibited superior weight gain and feed conversion efficiency when feed dry pellets (52 % crude protein, 13 % lipid) between four and six times a day (Kayano et al., 1993), which is in line with more recent recommendations (Sim et al., 2005). Luo et al. (2006a) quantified the optimal and maintenance ration of juvenile E. coioides (10 g) fed a dry pellet (52 % crude protein, 9 % lipid, 18.8 MJ kg−1) as 2.5 % and 0.5 % BW day−1, respectively. Feed conversion ratio for these fish was optimized when the ration was nearer to 2.0 % BW day−1. Similarly, weight gain and feeding efficiency in juvenile (5 g) yellow grouper Epinephelus awoara continued to increase in a linear fashion as feeding rate was increased from 0.5 % BW day−1 to apparent satiation (2.5 % BW day−1) (Sun et al., 2007). The latter study incorporated useful information on faecal production and the fate of ingested nitrogen in order to develop an energy budget for juveniles of this species (i.e. 100C = 2F + 4U + 75R + 19G where C is food energy, F is faecal energy, U is excretory energy, R is metabolism energy and G is growth energy).

16.4.5 Miscellaneous studies As the production of grouper has intensified in South East Asia so too have problems caused by parasitic organisms and bacterial or viral diseases (Cheng et al., 2008). This has led to a push to improve the innate immunity of grouper species by feeding immunostimulants such as ascorbic acid (Lin and Shiau, 2004, 2005a). Several experiments have recently investigated the use of seaweed extracts such as sodium alginate to boost the non-specific immune response of grouper, with elevations in serum indicators such as alternative complement activity (ACH50), respiratory burst or phagocytic activity used as evidence of an increased immune response. For example, E.coioides recorded elevations in the aforementioned indicators after injection with 20 mg sodium alginate kg BW−1. In addition, the survival of

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injected fish subjected to a challenge with Vibrio alginolyticus was significantly higher than controls (Cheng et al., 2007). Similar improvements in the innate cellular and humoral response of brown marbled grouper (E. fuscoguttatus) was detected in fish fed diets containing 10 g sodium alginate kg−1 or 5 g carrageenan kg−1 for eight weeks. Fish fed these diets were also more resistant to a challenge with V. alginolyticus, recording significantly higher survival than controls after 168 h (Cheng et al., 2008). Lower dietary inclusion of sodium alginate (1–2 g kg−1 diet) also improved the non-specific immune response of juvenile E. fuscoguttatus and E. coioides and their resistance to infection with a pathogenic strain of Streptococcus sp. and a grouper iridovirus (Chiu et al., 2008; Yeh et al., 2008). At present the mechanisms underlying these improvements are not well understood; however, it is believed that seaweed polysaccharides such as sodium alginate and carrageenan upregulate phagocytic activity in much the same way as other immunostimulants such as glucans (Cheng et al., 2008; Chiu et al., 2008).

16.5 Future trends In the broadest sense research will continue to investigate increased use of alternative feed ingredients and reductions in the use of declining stocks of fish meal and fish oil (Allen Davis et al., 2005; Moriss, 2005). As such, the determination of digestibility coefficients for individual species should be the first step in any research strategy and will remain a priority well into the future as greater physical, economic and legislative demands are placed on traditional feed ingredients. Fundamental research that develops or improves our understanding of basic nutrient requirements for protein, amino acids, lipids or carbohydrates (Davies and Gouveia, 2004) will continue as will the need to develop or improve feeding strategies that maximize feed intake and reduce waste. Valuable marine species such as basses and breams are often cultured in areas where seasonal fluctuations in water temperature, salinity or photoperiod have dramatic impacts on feed intake, growth rates, organ function and immune status (e.g. winter syndrome in gilthead sea bream; Gallardo et al., 2003; Luzzana et al., 2003, 2005; Tort et al., 2004; Ibarz et al., 2005). The impacts of these factors on nutritional status, metabolism and health and how these factors affect farm productivity or management strategies is not well understood and will be the focus of future research in many species. Increased attention will also be directed at gaining greater understanding of the effects of using different types and blends of vegetable oils in diets for marine fish and how these lipid sources affect carcass composition. This in turn will generate further research on the judicious use of finishing diets aimed at returning the fatty acid composition of fish to levels that are acceptable to the consumer. For new species such as grouper, emphasis will focus on the development of cost-effective, readily available extruded feeds that reduce reliance on low-value fishery products.

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16.6 References abare (2007) Australian Bureau of Agricultural and Resource Economics, Australian Fisheries Statistics June 2006, Fisheries Research and Development Corporation, Deakin West, ACT. alam m s, teshima s, yaniharto d, sumule o, ishikawa m and koshio s (2005) Assessment of reference dietary amino acid pattern for juvenile red sea bream, Pagrus major, Aquaculture International, 13, 369–79. allen davis d, samocha t m and bullis r a (2005) Working towards the removal of marine ingredients in aquafeeds, International AquaFeed, 8(1), 8–11. azevedo p a, van milgen j, leeson s and bureau d p (2005) Comparing efficiency of metabolizable energy utilization by rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) using factorial and multivariate approaches, Journal of Animal Science, 83, 842–51. battaglene s c and allan g l (1994) Development of hatchery techniques for snapper, Pagrus auratus, in Australia, in Chou L M, Munro A D, Lam T J, Chen T W, Cheong L K K, Ding J K, Hooi K K, Khoo H W, Phang V P E, Shim K F and Tan C H (eds), Third Asian Fisheries Forum, Manila, Asian Fisheries Society. battaglene s c and talbot r b (1992) Induced spawning and larval rearing of snapper, Pagrus auratus (Pisces: Sparidae) from Australian waters, New Zealand Journal of Marine and Freshwater Research, 26, 179–83. bell j g and tocher d r (2008) Dietary oil sources: current position and future challenges, XIII International Symposium on Fish Nutrition and Feeding, June 2008 – Florianopolis, 12. bell j d, quartararo n and henry g w (1991) Growth of snapper, Pagrus auratus, from south-eastern Australia in captivity, New Zealand Journal of Marine and Freshwater Research, 25, 117–21. benedito-palos l, saera-vila a, calduch-giner j a, kaushik s and perez-sanchez j (2007) Combined replacement of fish meal and oil in practical diets for fast growing juveniles of gilthead sea bream (Sparus aurata L.): Networking of systemic and local components of GH/IGF axis, Aquaculture, 267, 199–212. biswas a k, seoka m, inoue y, takii k and kumai h (2005) Photoperiod influences the growth, food intake, feed efficiency and digestibility of red sea bream (Pagrus major), Aquaculture, 250, 666–73. biswas a k, seoka m, tanaka y, takii k and kumai h (2006) Effect of photoperiod manipulation on the growth performance and stress response of juvenile red sea bream (Pagrus major), Aquaculture, 258, 350–6. biswas a k, kaku h, ji s c, seoka m and takii k (2007) Use of soybean meal and phytase for partial replacement of fish meal in the diet of red sea bream, Pagrus major, Aquaculture, 267, 284–91. biswas a k, seoka m, ueno k, takii k and kumai h (2008) Stimulation of growth performance without causing stress response in young red sea bream, Pagrus major (Temminck & Schlegel), by photoperiod manipulation, Aquaculture Research, 39, 457–63. bonaldo a, fontanillas r, koppe w, fagioli p, mariani l, parma l and gatta p p (2008a) Effect of dietary energy level and ration on growth and feed utilisation of gilthead seabream (Sparus aurata): preliminary findings, XIII International Symposium on Fish Nutrition and Feeding, June, Florianopolis, 124. bonaldo a, roem a j, fagioli p, pecchini a, cipollini i and gatta p p (2008b) Influence of dietary levels of soybean meal on the performance and gut histology of gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.), Aquaculture Research, 39, 970–8. boonyaratpalin m (1991) Asian seabass, Lates calcarifer, in Wilson R P (ed.), Handbook of Nutrient Requirements of Finfish, Boca Raton, FL, CRC, 5–11.

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boonyaratpalin m and williams k (2002) Asian Sea Bass, Lates calcarifer, in Webster C D and Lim C E (eds), Nutrient Requirements and Feeding of Finfish for Aquaculture, New York, CABI, 40–50. boonyaratpalin m, suraneiranat p and tunpibal t (1998) Replacement of fish meal with various types of soybean products in diets for the Asian seabass, Lates calcarifer, Aquaculture, 161, 67–78. booth m a, warner-smith r j, allan g l and glencross b d (2004) Effects of dietary astaxanthin source and light manipulation on the skin colour of Australian snapper Pagrus auratus (Bloch & Schneider, 1801), Aquaculture Research, 35, 458–64. booth m a, allan g l and anderson a j (2005) Investigation of the nutritional requirements of Australian snapper Pagrus auratus (Bloch & Schneider, 1801): apparent digestibility of protein and energy sources, Aquaculture Research, 36, 378–90. booth m a, anderson a j and allan g l (2006) Investigation of the nutritional requirements of Australian snapper Pagrus auratus (Bloch & Schneider, 1801): digestibility of gelatinized wheat starch and clearance of an intra-peritoneal injection of d-glucose, Aquaculture Research, 37, 975–85. booth m a, allan g l and anderson a j (2007) Investigation of the nutritional requirements of Australian snapper Pagrus auratus (Bloch & Schneider, 1801): effects of digestible energy content on utilization of digestible protein, Aquaculture Research, 38, 429–40. booth m a, allan g l and anderson a j (2008a) Investigation of the nutritional requirements of Australian snapper Pagrus auratus (Bloch & Schneider, 1801): influence of poultry offal, meat or soybean meal inclusion level on weight gain and performance, in Booth M A, Allan G L, Fielder D S and Anderson A J (eds), Increasing the profitability of snapper farming by improving hatchery practices and diets; diet development, Aquafin CRC Project 1B3 & FRDC Project No. 2001/208, Final Report, Volume 1, Deakin, ACT, Fisheries Research and Development Corporation. booth m a, tucker b j, allan g l and fielder d s (2008b) Effect of feeding regime and fish size on weight gain, feed intake and gastric evacuation in juvenile Australian snapper Pagrus auratus, Aquaculture, 282, 104–10. brandt t m (1991) Temperate basses, Morone spp., and black basses, Micropterus spp., in Wilson R P (ed.), Handbook of Nutrient Requirements of Finfish, Boca Raton, FL, CRC, 161–8. caballero m j, lopez-calero g, socorro j, roo f j, izquierdo m s and fernandez a j (1999) Combined effect of lipid level and fish meal quality on liver histology of gilthead seabream (Sparus aurata), Aquaculture, 179, 277–90. cardenas s (2008) Perspectives for red banded seabream culture, Global Aquaculture Advocate, 11(3), 56–8. caseras a, meton i, vives c, egea m, fernandez f and baanante i v (2002) Nutritional regulation of glucose-6-phosphatase gene expression in liver of the gilthead sea bream (Sparus aurata), British Journal of Nutrition, 88, 607–14. catacutan m r and coloso r m (1995) Effect of dietary-protein to energy ratios on growth, survival and body composition of juvenile Asian seabass Lates calcarifer, Aquaculture, 131, 125–33. catacutan m r and coloso r m (1997) Growth of juvenile Asian seabass, Lates calcarifer, fed varying carbohydrate and lipid levels, Aquaculture, 149, 137–44. cheng a c, chen c y, liou c h and chang c f (2006) Effects of dietary protein and lipids on blood parameters and superoxide anion production in the grouper, Epinephelus coioides (Serranidae: Epinephelinae), Zoological Studies, 45, 492–502.

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cheng a c, tu c w, chen y y, nan f h and chen j c (2007) The immunostimulatory effects of sodium alginate and iota-carrageenan on orange-spotted grouper Epinephelus coicoides and its resistance against Vibrio alginolyticus, Fish & Shellfish Immunology, 22, 197–205. cheng a c, chen y y and chen j c (2008) Dietary administration of sodium alginate and kappa-carrageenan enhances the innate immune response of brown-marbled grouper Epinephelus fuscoguttatus and its resistance against Vibrio alginolyticus, Veterinary Immunology and Immunopathology, 121, 206–15. chiu s t, tsai r t, hsu j p, liu c h and cheng w (2008) Dietary sodium alginate administration to enhance the non-specific immune responses, and disease resistance of the juvenile grouper Epinephelus fuscoguttatus, Aquaculture, 277, 66–72. coloso r m, murillo-gurrea d p, borlongan i g and catacutan m r (1999) Sulphur amino acid requirement of juvenile Asian sea bass Lates calcarifer, Journal of Applied Ichthyology–Zeitschrift Fur Angewandte Ichthyologie, 15, 54–8. coloso r m, murillo-gurrea d p, borlongan i g and catacutan m r (2004) Tryptophan requirement of juvenile Asian sea bass Lates calcarifer, Journal of Applied Ichthyology, 20, 43–7. company r, calduch-giner j a, kaushik s and perez-sanchez j (1999a) Growth performance and adiposity in gilthead sea bream (Sparus aurata): risks and benefits of high energy diets, Aquaculture, 171, 279–92. company r, calduch-giner j a, perez-sanchez j and kaushik s j (1999b) Protein sparing effect of dietary lipids in common dentex (Dentex dentex): a comparative study with sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax), Aquatic Living Resources, 12, 23–30. couto a, enes p, peres h and oliva-teles a (2008) Effect of water temperature and dietary starch on growth and metabolic utilization of diets in gilthead sea bream (Sparus aurata) juveniles, Comparative Biochemistry and Physiology – Part A: Molecular & Integrative Physiology, 151, 45–50. davies s j and gouveia a (2004) Cereal processing & improved carbohydrate digestibility in over-wintering diets for juvenile gilthead sea bream, International AquaFeed, 8(2), 18–23. de francesco m, parisi g, perez-sanchez j, gomez-requeni p, medale f, kaushik s j, mecatti m and poli b m (2007) Effect of high-level fish meal replacement by plant proteins in gilthead sea bream (Sparus aurata) on growth and body/fillet quality traits, Aquaculture Nutrition, 13, 361–72. doolan b j, booth m a, jones p l and allan g l (2007) Effect of cage colour and light environment on the skin colour of Australian snapper Pagrus auratus (Bloch & Schneider, 1801), Aquaculture Research, 38, 1395–403. doolan b j, allan g l, booth m a and jones p l (2008a) Cage colour and postharvest K+ concentration affect skin colour of Australian snapper Pagrus auratus (Bloch & Schneider, 1801), Aquaculture Research, 39, 919–27. doolan b j, allan g l, booth m a and jones p l (2008b) Effect of carotenoids and background colour on the skin pigmentation of Australian snapper Pagrus auratus (Bloch & Schneider, 1801), Aquaculture Research, 39, 1423–33. doolan b j, allan g l, booth m a and jones p l (2008c) Effects of cage netting colour and density on the skin pigmentation and stress response of Australian snapper Pagrus auratus (Bloch & Schneider, 1801), Aquaculture Research, 39, 1360–8. drew m d, borgeson t l and thiessen d l (2007) A review of processing of feed ingredients to enhance diet digestibility in finfish, Animal Feed Science and Technology, 138, 118–36.

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enes p, panserat s, kaushik s and oliva-teles a (2008a) Growth performance and metabolic utilization of diets with native and waxy maize starch by gilthead sea bream (Sparus aurata) juveniles, Aquaculture, 274, 101–8. enes p, panserat s, kaushik s and oliva-teles a (2008b) Hepatic glucokinase and glucose-6-phosphatase responses to dietary glucose and starch in gilthead sea bream (Sparus aurata) juveniles reared at two temperatures, Comparative Biochemistry and Physiology a–Molecular & Integrative Physiology, 149, 80–6. eroldogan o t, kumlu m, kiris g a and sezer b (2006a) Compensatory growth response of Sparus aurata following different starvation and refeeding protocols, Aquaculture Nutrition, 12, 203–10. eroldogan o t, kumlu m and sezer b (2006b) Effects of starvation and realimentation periods on growth performance and hyperphagic response of Sparus aurata, Aquaculture Research, 37, 535–7. eroldogan o t, tasbozan o and tabakoglu s (2008) Effects of restricted feeding regimes on growth and feed utilization of juvenile gilthead sea bream, Sparus aurata, Journal of the World Aquaculture Society, 39, 267–74. eusebio p s, coloso r m and mamauag r e p (2004a) Apparent digestibility of selected ingredients in diets for juvenile grouper, Epinephelus coioides (Hamilton), Aquaculture Research, 35, 1261–9. eusebio p s, coloso r m and mamauag r e p (2004b) Evaluation of some terrestrial proteins in complete diets for grouper (Epinephelus coioides) juveniles, in Rimmer M A, McBride S and Williams K C (eds), Advances in Grouper Culture ACIAR Monograph 110, Canberra, Australian Centre for International Agricultural Research, 79–84. fernandez f, miquel a g, cordoba m, varas m, meton i, caseras a and baanante i v (2007) Effects of diets with distinct protein-to-carbohydrate ratios on nutrient digestibility, growth performance, body composition and liver intermediary enzyme activities in gilthead sea bream (Sparus aurata L.) fingerlings, Journal of Experimental Marine Biology and Ecology, 343, 1–10. fielder d s, bardsley w j, allan g l and pankhurst p m (2002) Effect of photoperiod on growth and survival of snapper Pagrus auratus larvae, Aquaculture, 211, 135–50. fielder d s, bardsley w j, allan g l and pankhurst p m (2005) The effects of salinity and temperature on growth and survival of Australian snapper, Pagrus auratus larvae, Aquaculture, 250, 201–14. fielder d s, allan g l and pankhurst p m (2008) Comparison of two environmental regimes for culture of Australian snapper, Pagrus auratus, larvae in commercialscale tanks, Journal of the World Aquaculture Society, 39, 364–74. forster i p and ogata h (1996) Growth and whole-body lipid content of juvenile red sea bream reared under different conditions of exercise training and dietary lipid, Fisheries Science, 62, 404–9. forster i and ogata h y (1998) Lysine requirement of juvenile Japanese flounder Paralichthys olivaceus and juvenile red sea bream Pagrus major, Aquaculture, 161, 131–42. foscarini r (1988) A review: Intensive farming procedure for red sea bream (Pagrus major) in Japan, Aquaculture, 72, 191–246. fountoulaki e, alexis m n, nengas i and venou b (2005) Effect of diet composition on nutrient digestibility and digestive enzyme levels of gilthead sea bream (Sparus aurata L.), Aquaculture Research, 36, 1243–51. gallardo m a, sala-rabanal m, ibarz a, padros f, blasco j, fernandez-borras j and sanchez j (2003) Functional alterations associated with ‘winter syndrome’ in gilthead sea bream (Sparus aurata), Aquaculture, 223, 15–27.

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ganga r, montero d, vaquero a, tort l and izquierdo m s (2008) Effect of feeding vegetable oils on growth and ACTH-induced cortisol release by interrenal tissue from gilthead seabream Pagris aurata, XIII International Symposium on Fish Nutrition and Feeding, June, Florianopolis, 51. gasca-leyva e, leon c j, hernandez j m and vergara j m (2002) Bioeconomic analysis of production location of sea bream (Sparus aurata) cultivation, Aquaculture, 213, 219–32. georgopoulos g b and conides a j (1999) Study on the effects of dietary starch on growth, food conversion and digestibility of adult gilthead sea bream (Sparus aurata L. 1758), Journal of Aquaculture in the Tropics, 14, 143–52. gines r, afonso j m, arguello a, zamorano m j and lopez j l (2004) The effects of long-day photoperiod on growth, body composition and skin colour in immature gilthead sea bream (Sparus aurata L.), Aquaculture Research, 35, 1207–12. glencross b (2006) The nutritional management of barramundi, Lates calcarifer – a review, Aquaculture Nutrition, 12, 291–309. glencross b d (2008) A factorial growth and feed utilization model for barramundi, Lates calcarifer based on Australian production conditions, Aquaculture Nutrition, 14, 360–73. glencross b d and felsing m (2006) Influence of fish size and water temperature on the metabolic demand for oxygen by barramundi, Lates calcarifer (Bloch), in freshwater, Aquaculture Research, 37, 1055–62. glencross b and hawkins w (2004) A comparison of the digestibility of lupin (Lupinus sp.) kernel meals as dietary protein resources when fed to either, rainbow trout, Oncorhynchus mykiss or red seabream, Pagrus auratus, Aquaculture Nutrition, 10, 65–73. glencross b, curnow j, hawkins w, kissil g w m and peterson d (2003a) Evaluation of the feed value of a transgenic strain of the narrow-leaf lupin (Lupinus angustifolius) in the diet of the marine fish, Pagrus auratus, Aquaculture Nutrition, 9, 197–206. glencross b, hawkins w and curnow j (2003b) Evaluation of canola oils as alternative lipid resources in diets for juvenile red seabream, Pagrus auratus, Aquaculture Nutrition, 9, 305–15. glencross b d, hawkins w e and curnow j g (2003c) Restoration of the fatty acid composition of red seabream (Pagrus auratus) using a fish oil finishing diet after grow-out on plant oil based diets, Aquaculture Nutrition, 9, 409–18. glencross b, hawkins w and curnow j (2004a) Nutritional assessment of Australian canola meals. I. Evaluation of canola oil extraction method and meal processing conditions on the digestible value of canola meals fed to the red seabream (Pagrus auratus, Paulin), Aquaculture Research, 35, 15–24. glencross b, hawkins w and curnow j (2004b) Nutritional assessment of Australian canola meals. II. Evaluation of the influence of the canola oil extraction method on the protein value of canola meals fed to the red seabream (Pagrus auratus, Paulin), Aquaculture Research, 35, 25–34. glencross b, percival s, jones b and hughes j (2007) Sustainable development of barramundi cage aquaculture at Lake Argyle, FRDC Final Report, KSRP Report – Project 2003/026 – Fisheries Research, Department of Fisheries, Hillarys, WA. glencross b, michael r, austen k and hauler r (2008) Productivity, carcass composition, waste output and sensory characteristics of large barramundi Lates calcarifer fed high-nutrient density diets, Aquaculture, 284, 167–73. gomes e, dias j, silva p, valente l, empis j, gouveia l, bowen j and young a (2002) Utilization of natural and synthetic sources of carotenoids in the skin pigmentation of gilthead seabream (Sparus aurata), European Food Research Technology, 214, 287–93.

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koven w, kolkovski s, hadas e, gamsiz k and tandler a (2001) Advances in the development of microdiets for gilthead seabream, Sparus aurata: a review, Aquaculture, 194, 107–21. laining a, rachmansyah ahmad t and williams k (2003) Apparent digestibility of selected feed ingredients for humpback grouper, Cromileptes altivelis, Aquaculture, 218, 529–38. landau m (1992) Introduction to Aquaculture, New York, Wiley. libralato s and solidoro c (2008) A bioenergetic growth model for comparing Sparus aurata’s feeding experiments, Ecological Modelling, 214, 325–37. lin y-h and shiau s-y (2003) Dietary lipid requirement of grouper, Epinephelus malabaricus, and effects on immune responses, Aquaculture, 225, 243–50. lin m f and shiau s y (2004) Requirements of vitamin C (L-ascorbyl-2-monophosphate-Mg and L-ascorbyl-2-monophosphate-Na) and its effects on immune responses of grouper, Epinephelus malabaricus, Aquaculture Nutrition, 10, 327–33. lin m f and shiau s y (2005a) Requirements of vitamin C (L-ascorbyl-2-sulphate and L-ascorbyl-2-polyphosphate) and its effects on non-specific immune responses of grouper, Epinephelus malabaricus, Aquaculture Nutrition, 11, 183–9. lin y-h and shiau s-y (2005b) Dietary vitamin E requirement of grouper, Epinephelus malabaricus, at two lipid levels, and their effects on immune responses, Aquaculture, 248, 235–44. lin y h and shiau s y (2005c) Dietary selenium requirements of juvenile grouper, Epinephelus malabaricus, Aquaculture, 250, 356–63. lin y h and shiau s y (2007) Effects of dietary blend of fish oil with corn oil on growth and non-specific immune responses of grouper, Epinephelus malabaricus, Aquaculture Nutrition, 13, 137–44. lin m q, ushio h, ohshima t, yamanaka h and koizumi c (1998) Skin color control of the red sea bream (Pagrus major), Food Science and Technology–LebensmittelWissenschaft & Technologie, 31, 27–32. lin h z, liu y j, tian l x, wang j t, zheng w h, huang j n and chen p (2004) Apparent digestibility coefficients of various feed ingredients for grouper Epinephelus coioides, Journal of the World Aquaculture Society, 35, 134–42. lozano n b, vidal a, martinez-llorens s, merida s, blanco j, lopez a, torres m and cerda m (2007) Growth and economic profit of gilthead sea bream (Sparus aurata L.) fed sunflower meal, Aquaculture, 272, 528–34. luo z, liu y j, mai k s, tian l x, liu d h and tan x y (2004) Optimal dietary protein requirement of grouper Epinephelus coioides juveniles fed isoenergetic diets in floating net cages, Aquaculture Nutrition, 10, 247–52. luo z, liu y-j, mai k-s, tian l-x, yang h-j, tan x-y and liu d-h (2005) Dietary l-methionine requirement of juvenile grouper Epinephelus coioides at a constant dietary cystine level, Aquaculture, 249, 409–18. luo z, liu y j, mai k s, tian l x, tan x y and shi j f (2006a) Effects of feeding levels on growth performance, feed utilization, body composition, and apparent digestibility coefficients of nutrients for grouper Epinephelus coioides juveniles, Journal of the World Aquaculture Society, 37, 32–40. luo z, liu y j, mai k s, tian l x, tan x y, yang h j, liang g y and liu d h (2006b) Quantitative L-lysine requirement of juvenile grouper Epinephelus coioides, Aquaculture Nutrition, 12, 165–72. luo z, liu y j, mai k s, tian l x, tan x y and yang h j (2007) Effects of dietary arginine levels on growth performance and body composition of juvenile grouper Epinephelus coioides, Journal of Applied Ichthyology, 23, 252–7. luo z, liu y j, mai k s, tian l x, tan x y, yang h j and liu d h (2008) Influence of dietary amino acid profiles on growth performance and body composition of

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juvenile grouper Epinephelus coioides, Journal of Applied Ichthyology, 24, 208–12. lupatsch i (2003) Israeli study examines feeding regimes for Asian seabass grown at high temperatures, Global Aquaculture Advocate, 6, 62–3. lupatsch i (2004) Gilthead seabream, AquaFeeds: Formulation & Beyond, 1(1), 16–18. lupatsch i (2008) By the numbers: nutritional bioenergetics for optimal feeding, Global Aquaculture Advocate, 11(3), 64–6. lupatsch i and kissil g w (2005) Feed formulations based on energy and protein demands in white grouper (Epinephelus aeneus), Aquaculture, 248, 83–95. lupatsch i, kissil g w, sklan d and pfeffer e (1997) Apparent digestibility coefficients of feed ingredients and their predictability in compound diets for gilthead seabream, Sparus aurata L., Aquaculture Nutrition, 3, 81–9. lupatsch i, kissil g w, sklan d and pfeffer e (1998) Energy and protein requirements for maintenance and growth in gilthead seabream (Sparus aurata L.), Aquaculture Nutrition, 4, 165–73. lupatsch i, kissil g w, sklan d and pfeffer e (2001a) Effects of varying dietary protein and energy supply on growth, body composition and protein utilization in gilthead seabream (Sparus aurata L.), Aquaculture Nutrition, 7, 71–80. lupatsch i, kissil g w and sklan d (2001b) Optimistion of feeding regimes for European sea bass Dicentrarchus labrax: a factorial approach, Aquaculture, 202, 289–302. lupatsch i, kissil g w and sklan d (2003a) Comparison of energy and protein efficiency among three fish species gilthead sea bream (Sparus aurata), European sea bass (Dicentrarchus labrax) and white grouper (Epinephelus aeneus): energy expenditure for protein and lipid deposition, Aquaculture, 225, 175–89. lupatsch i, kissil g w and sklan d (2003b) Defining energy and protein requirements of gilthead seabream (Sparus aurata) to optimize feeds and feeding regimes, Israeli Journal of Aquaculture-Bamidgeh, 55, 243–57. luzzana u, coutteau p, bavevi l, olak s, franzolini e, di giancamillo a and domeneghini c (2003) Nutritional solutions to winter syndrome in Gilthead sea bream; verification at a cage farm in Croatia, International AquaFeed, 6(3), 14–18. luzzana u, coutteau p, bavevi l, olak s, franzolini e, burlini a m, di giancamillo a and domeneghini c (2005) Optimization of feeding protocols for winter diet, International AquaFeed, 8(2), 36–40. martinez-llorens s, monino a v, vidal a t, salvador v j m, torres m p and cerda m j (2007a) Soybean meal as a protein source in gilthead sea bream (Sparus aurata L.) diets: effects on growth and nutrient utilization, Aquaculture Research, 38, 82–90. martinez-llorens s, vidal a t, monino a v, torres m p and cerda m j (2007b) Effects of dietary soybean oil concentration on growth, nutrient utilization and muscle fatty acid composition of gilthead sea bream (Sparus aurata L.), Aquaculture Research, 38, 76–81. martinez-llorens s, vidal a t, monino a v, ader j g, torres m p and cerda m j (2008) Blood and haemoglobin meal as protein sources in diets for gilthead sea bream (Sparus aurata): effects on growth, nutritive efficiency and fillet sensory differences, Aquaculture Research, 39, 1028–37. matsunari h, furuita h, yamamoto t, kim s-k, sakakura y and takeuchi t (2008a) Effect of dietary taurine and cystine on growth performance of juvenile red sea bream Pagrus major, Aquaculture, 274, 142–7. matsunari h, yamamoto t, kim s-k, goto t and takeuchi t (2008b) Optimum dietary taurine level in casein-based diet for juvenile red sea bream Pagrus major, Fisheries Science, 74, 347–53.

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mihelakakis a, tsolkas c and yoshimatsu t (2002) Optimization of feeding rate for hatchery-produced juvenile gilthead sea bream Sparus aurata, Journal of the World Aquaculture Society, 33, 169–75. millamena o m (2002) Replacement of fish meal by animal by-product meals in a practical diet for grow-out culture of grouper Epinephelus coioides, Aquaculture, 204, 75–84. millamena o m and golez n v (2001) Evaluation of processed meat solubles as replacement for fish meal in diet for juvenile grouper Epinephelus coioides (Hamilton), Aquaculture Research, 32, 281–7. mohamed j s, sivaram v, roy t s c, marian m p, murugadass s and hussain m r (2003) Dietary vitamin A requirement of juvenile greasy grouper (Epinephelus tauvina), Aquaculture, 219, 693–701. montero d, caballero m j, ganga r, mathlouthi f, tort l, robaina l and izquierdo m s (2008a) Replacing fish oil by vegetable oils in marine fish: an overview of the effects on fish health, XIII International Symposium on Fish Nutrition and Feeding, 2008, Florianopolis, 50. montero d, grasso v, izquierdo m s, ganga r, real f, tort l, caballero m j and acosta f (2008b) Total substitution of fish oil by vegetable oils in gilthead sea bream (Sparus aurata) diets: Effects on hepatic Mx expression and some immune parameters, Fish & Shellfish Immunology, 24, 147–55. moriss p (2005) The vegetarian trout: opportunities for replacing fishmeal and fish oil in feeds for rainbow trout, International AquaFeed, 8(1), 41–6. murillo-gurrea d p, coloso r m, borlongan i g and serrano a e (2001) Lysine and arginine requirements of juvenile Asian sea bass (Lates calcarifer), Journal of Applied Ichthyology, 17, 49–53. nankervis l, matthews s j and appleford p (2000) Effect of dietary non-protein energy source on growth, nutrient retention and circulating insulin-like growth factor I and triiodothyronine levels in juvenile barramundi, Lates calcarifer, Aquaculture, 191, 323–35. nengas i, alexis m n, davies s j and petichakis g (1995) Investigation to determine digestibility coefficients of various raw materials in diets for gilthead sea bream, Sparus auratus (L.), Aquaculture Research, 26, 185–94. nengas i, alexis m n and davies s j (1999) High inclusion levels of poultry meals and related byproducts in diets for gilthead seabream Sparus aurata L, Aquaculture, 179, 13–23. nrc (1993) National Research Council – Nutrient Requirements of Fish, Washington, National Academy Press. oh s y, noh c h and cho s h (2007) Effect of restricted feeding regimes on compensatory growth and body composition of red sea bream, Pagrus major, Journal of the World Aquaculture Society, 38, 443–9. oliva-teles a and peres h (2008) The optimum dietary essential amino acid pattern for gilthead seabream Sparus aurata, XIII International Symposium on Fish Nutrition and Feeding, 2008, Florianopolis, Brazil, 111. panserat s, medale f, blin c, breque j, vachot c, plagnes-juan e, gomes e, krishnamoorthy r and kaushik s (2000) Hepatic glucokinase is induced by dietary carbohydrates in rainbow trout, gilthead seabream, and common carp, American Journal of Physiology-Regulatory Integrative and Comparative Physiology, 278, R1164–R1170. panserat s, plagnes-juan e and kaushik s (2002) Gluconeogenic enzyme gene expression is decreased by dietary carbohydrates in common carp (Cyprinus carpio) and gilthead seabream (Sparus aurata), Biochimica Et Biophysica ActaGene Structure and Expression, 1579, 35–42.

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partridge g j and creeper j (2004) Skeletal myopathy in juvenile barramundi, Lates calcarifer (Bloch), cultured in potassium-deficient saline groundwater, Journal of Fish Diseases, 27, 523–30. partridge g j and lymbery a j (2008) The effect of salinity on the requirement for potassium by barramundi (Lates calcarifer) in saline groundwater, Aquaculture, 278, 164–70. partridge g j, lymbery a j and bourke d k (2008) Larval rearing of barramundi (Lates calcarifer) in saline groundwater, Aquaculture, 278, 171–4. paspatis m, maragoudaki d and kentouri m (2000) Self-feeding activity patterns in gilthead sea bream (Sparus aurata), red porgy (Pagrus pagrus) and their reciprocal hybrids, Aquaculture, 190, 389–401. paulin c d (1990) Pagrus auratus, a new combination for the species known as ‘snapper’ in Australasian waters (Pisces : Sparidae), New Zealand Journal of Marine and Freshwater Research, 24, 259–65. pavlidis m, karkana m, fanouraki e and papandroulakis n (2008) Environmental control of skin colour in the red porgy, Pagrus pagrus, Aquaculture Research, 39, 837–49. pereira t g and oliva-teles a (2002) Preliminary evaluation of pea seed meal in diets for gilthead sea bream (Sparus aurata) juveniles, Aquaculture Research, 33, 1183–9. pereira t g and oliva-teles a (2004) Evaluation of micronized lupin seed meal as an alternative protein source in diets for gilthead sea bream Sparus aurata L. juveniles, Aquaculture Research, 35, 828–35. pillay t v r (1993) Aquaculture Principles and Practices, Oxford, London, Blackwell Scientific. pomeroy r (2007) Public policy for sustainable grouper aquaculture development in Southeast Asia, in Leung P, Lee C S and O’Bryen P J (eds), Species & System Selection For Sustainable Aquaculture, Oxford, Blackwell, 461–76. quartararo n, allan g l and bell j d (1998a) Replacement of fish meal in diets for Australian snapper, Pagrus auratus, Aquaculture, 166, 279–95. quartararo n, bell j d and allan g l (1998b) Substitution of fishmeal in a diet for the carnivorous marine fish Pagrus auratus (Bloch and Schneider) from Southeastern Australia, Asian Fisheries Science, 10, 269–79. rachmansyah laining a and ahmad t (2004) The use of shrimphead meal as a substitute to fish meal in diets for humpback grouper (Cromileptes altivelis), in Rimmer M A, McBride S and Williams K C (eds), Advances in Grouper Culture, ACIAR Monograph 110, Canberra, Australian Centre for International Agricultural Research, 113–14. rad f (2007) Evaluation of the sea bass and sea bream industry in the Mediterranean, with emphasis on Turkey, in Leung P, Lee C S and O’Bryen P J (eds), Species & System Selection For Sustainable Aquaculture, Oxford, Blackwell, 445–9. raso s and anderson t a (2003) Effects of dietary fish oil replacement on growth and carcass proximate composition of juvenile barramundi (Lates calcarifer), Aquaculture Research, 34, 813–9. rawles s d, smith s b and gatlin d m (2008) Hepatic glucose utilization and lipogenesis of hybrid striped bass in response to dietary carbohydrate level and complexity, Aquaculture Nutrition, 14, 40–50. rimmer m (2003) Barramundi, in Lucas J S and Southgate P C (eds), Aquaculture Farming Aquatic Animals and Plants, Oxford, Blackwell, 364–81. rimmer m a, mcbride s and williams k c (eds) (2004) Advances in Grouper Culture, ACIAR Monograph 110, Canberra, Australian Centre for International Agricultural Research.

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robles r, ceulemans s, coutteau p and cremer m (2008) Soy products replace fishmeal, fish oil in gilthead seabream feed study, Global Aquaculture Advocate, 11, 69–70. rotllant j, tort l, montero d, pavlidis m, martinez m, bonga s e w and phm b (2003) Background colour influence on the stress response in cultured red porgy Pagrus pagrus, Aquaculture, 223, 129–39. salama a j (2008) Effects of different feeding frequency on the growth, survival and feed conversion ratio of the Asian sea bass Lates calcarifer juveniles reared under hypersaline seawater of the Red Sea, Aquaculture Research, 39, 561–7. sánchez-muros m j, corchete v, suárez m d, cardenete g, gómez-milán e and de la higuera m (2003) Effect of feeding method and protein source on Sparus aurata feeding patterns, Aquaculture, 224, 89–103. santinha p j m, medale f, corraze g and gomes e f s (1999) Effects of the dietary protein: lipid ratio on growth and nutrient utilization in gilthead seabream (Sparus aurata L.), Aquaculture Nutrition, 5, 147–56. schuchardt d, vergara j m, fernandez-palacios h, kalinowski c t, hernandez-cruz c m, izquierdo m s and robaina l (2008) Effects of different dietary protein and lipid levels on growth, feed utilization and body composition of red porgy (Pagrus pagrus) fingerlings, Aquaculture Nutrition, 14, 1–9. shapawi r, ng w k and mustafa s (2007) Replacement of fish meal with poultry by-product meal in diets formulated for the humpback grouper, Cromileptes altivelis, Aquaculture, 273, 118–26. shapawi r, mustafa s and ng w k (2008) Effects of dietary fish oil replacement with vegetable oils on growth and tissue fatty acid composition of humpback grouper, Cromileptes altivelis (Valenciennes), Aquaculture Research, 39, 315–23. shearer k (1995) The use of factorial modelling to determine the dietary requirements for essential elements in fishes, Aquaculture, 133, 57–72. shiau s y and lin y h (2001) Carbohydrate utilization and its protein-sparing effect in diets for grouper (Epinephelus malabaricus), Animal Science, 73, 299–304. sim s y, rimmer m a, williams k, toledo j d, sugama k, rumengan i and phillips m j (2005) A Practical Guide to Feeds and Feed Management for Cultured Groupers, Publication No. 2005-02 of the Asia-Pacific Marine Finfish Aquaculture Network. sitja-bobadilla a, mingarro m, pujalte m j, garay e, alvarez-pellitero p and perez-sanchez j (2003) Immunological and pathological status of gilthead sea bream (Sparus aurata L.) under different long-term feeding regimes, Aquaculture, 220, 707–24. sitja-bobadilla a, pena-llopis s, gomez-requeni p, medale f, kaushik s and perez-sanehez j (2005) Effect of fish meal replacement by plant protein sources on non-specific defence mechanisms and oxidative stress in gilthead sea bream (Sparus aurata), Aquaculture, 249, 387–400. smith d m, williams i h, williams k c, barclay m c and venables w n (2005) Oxidation of medium-chain and long-chain fatty acids by polka dot grouper Cromileptes altivelis, Aquaculture Nutrition, 11, 41–8. sun l h, chen h r and huang l m (2007) Growth, faecal production, nitrogenous excretion and energy budget of juvenile yellow grouper (Epinephelus awoara) relative to ration level, Aquaculture, 264, 228–35. suwiyra k, giri n a, marzuqi m and trijoko (2004) Utilisation of dietary dextrin by juvenile humpback grouper (Cromileptes altivelis), in Rimmer M A, McBride S and Williams K C (eds), Advances in Grouper Culture, ACIAR Monograph 110, Canberra, ACIAR, 107–9. suwirya k, giri n a and marzuqi m (2005) Effect of dietary n-3 HUFA on growth of humpback grouper (Cromileptes altivelis) and tiger grouper (Epinephe-

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17 Advances in aquaculture feeds and feeding: salmonids S. Refstie, Nofima Marin and Aquaculture Protein Centre (APC), Norway, and T. Åsgård, Nofima Marin, Norway

Abstract: Salmonid aquaculture is highly mechanised, thus depending on efficient feed utilisation, rapid growth, and short production cycles to maximise output of extensive facilities. This chapter addresses recent history, advances, practices, and future trends in feed ingredient choice, formulation, feed processing and manufacture, and feeding to obtain these goals. Chosen strategies and solutions are largely based on in-depth research on salmonid metabolism and nutritional physiology, which is used to reassess nutritional requirements, evaluate feed ingredients, and elucidate how physical properties of feeds, dietary nutrients or natural bioactive components, feed additives or contaminants, and malnutrition affects fish performance, health, and nutrient composition. Key words: salmon, trout, feed, nutrition, health.

17.1 Introduction By 2006 salmonid aquaculture produced more than 1.4 million metric tons of salmon (FAO, 2007) and probably about 300 000 tons of rainbow trout, consuming 2.5 million tons of feed in the process. These fishes are grown in both freshwater and seawater. Freshwater farming of portion sized salmonids developed from hatchery propagation programs during the late 1800s to spread worldwide through the temperate climate zones during the early 1900s (Jensen, 1975; Herschberger, 1992). This industry mainly produces rainbow trout (Oncorhynchus mykiss), which are grown in earth ponds, concrete dams, or raceway systems that have often implemented thorough water control and recirculation systems to minimise water use and effluent discharges.

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Successful sea farming of salmonids was first achieved in the late 1960s (reviewed by Jensen, 1975). This industry grows Atlantic salmon (Salmo salar) and Pacific salmonids of the Oncorhyncus genus up to 7 kg in pens with water volumes of up to 100 000 m3, holding up to 200 000 individual fish. Today the bulk of sea-farmed salmon and trout is produced in Norway and Chile, which have suitable climates and coastlines. This form of aquaculture depends on efficient feed utilisation, rapid growth, and short production cycles to maximise output of the extensive facilities. Current feeding systems are consequently highly mechanised and controlled. Moist or dry pelleted compound feeds were long the diets of choice for salmonids in freshwater. These were largely based on fish meal and oil, and were lean as a result of feed technological limitations. When attempts were made to farm salmonids in seawater they were initially fed moist diets made locally from scrap fish and animal by products (Åsgård and Austreng, 1985a, b, 1986, 1987). This was partly because it was difficult to feed dry feeds during cold winters, a problem that was solved when high-quality low-temperature (LT) dried fish meals (Pike and Hardy, 1997) were introduced. The 1980s saw the introduction of high-pressure moist extrusion technology, which paved the way for modern dry and durable highenergy salmon and trout diets containing up to 40 % lipid (Hillestad and Johnsen, 1994; Einen and Roem, 1997; Einen and Skrede, 1998; Hillestad et al., 1998; Hemre and Sandnes, 1999; Refstie et al., 2001). The period furthermore saw the introduction of synthetic carotenoid pigments in diets for sea-farmed salmonids (Foss et al., 1984; Choubert and Blanc, 1985; Storebakken et al., 1985, 1986, 1987), where pink flesh colour is a major quality criterion. This raised the share of the pigment costs to 20 % of the total raw material cost in commercial feeds. The 1990s then forced the aquaculture industries to look for alternative non-marine feedstuffs (Storebakken et al., 2000a; Francis et al., 2001; Bakke-McKellep et al., 2008), realising that further sustainable growth and stable ingredient supply depended on this. Thus, nutritional research in salmonids during the 2000s focused extensively on (i) defining raw materials that alone or in combination may legally and safely replace fish meal and oil in the diets without compromising fish health or productivity, (ii) defining pigment sources and means for more efficient flesh pigmentation, and (iii) avoiding producing less healthy foods when reducing the use of dietary fish oil. The in-depth focus and commitment of these efforts have significantly improved our understanding of salmonid nutrient requirements, digestive function, and metabolism, but also revealed nutritional knowledge gaps that need to be bridged by further research. Current status of salmonid nutrition may in many respects serve as a model for more recently domesticated fishes, pointing at risk factors that need to be investigated and controlled.

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Feed technology and formulation

17.2.1 Preferred technology: benefits and restrictions on formulation Current salmonid diets are manufactured by high-pressure moist extrusion technology (Fig. 17.1). The extrusion process expands starch in the feed mixture to produce wear-resistant pellets that do not crack or crumble easily, thus allowing efficient mechanical feed distribution. Due to the expanded starch matrix in the pellets, extruded feeds furthermore have high capacity for soaking up and holding oil. To reach the lipid levels in high energy diets (≥40 %), oil is sprayed onto the pellets in a vacuum coater and then pressed into the pellets by letting in air. High temperature (100–150 °C), pressure, and shear force during the extrusion process may cause oxidation that reduces protein quality and damages feed additives such as vitamins and carotenoid pigments (Camire et al., 1990; Haaland et al., 1993). High moisture content during extrusion protects the ingredients (Sørensen et al., 2002), but as water works as a lubricant in the extruder, it can not exceed 25–30 % (Rokey, 1994). The extrusion process must, thus, be carefully optimised to avoid nutrient losses. It follows that heat-sensitive feed additives for extruded feeds must be stabilised in beadlet matrixes (Gadient and Fenster, 1994; Killeit, 1994; Anderson and Sunderland, 2002) and/or added post-extrusion together with the oil. Thermally processed feedstuffs receive a secondary heating during extrusion, and this must be considered when determining quality criteria for such ingredients. The restriction on moisture in the feed mash furthermore limits the use of feasible wet ingredients such as fish silage and

Dry ingredients Fish meal,vegetable protein ingredients, vitamins, minerals, and carbohydrates

Pre-conditioning

Extrusion

Drying Moist ingredients Water, steam, oil, and fish silage Vacuum coating

Cooling

Fig. 17.1

Main principles of high-pressure moist extrusion feed manufacture.

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fermented vegetable feedstuffs, so extruded feeds are mainly made from dry meals.

17.2.2 Trends in feed formulation The salmon and trout feed industry has a strong focus on evaluating new feed ingredients to expand the base of safe ingredients. This is in part to reduce the dependency on marine feedstuffs derived from limited fisheries, thereby increasing the sustainability of the salmonid industry. Using a wide selection of feedstuffs also spreads risks related to batch variations in content of unwanted components in the feedstuffs, and ultimately allows true least cost formulation. Major feed companies optimise feeds based on digestible protein and energy content in the actual ingredient batches used, measured by near-infrared reflectance (NIR; Givens et al., 1997) or nuclear magnetic resonance (NMR; Makkar, 2008) spectroscopy. Thus, the feeds come with declared concentrations of digestible protein and energy as calculated by linear programming, while the chemical nutrient composition of the diets may vary.

17.3 Digestive physiology 17.3.1 Digestive physiology and regulation Salmonid fishes are strictly carnivorous species having short gastrointestinal tracts. These are functionally sectioned into a J-shaped muscular stomach, a pyloric intestine with 50–60 pyloric caeca, a mid-intestine of similar length and with similar morphology but without caeca, and a distinct distal intestine of similar length as the mid-intestine but with larger diameter and annular rings (Nordrum et al., 2000). The pyloric caeca substantially expand the absorptive surface of the intestine, contributing 70–80 % of the total absorptive capacity (Buddington et al., 1997). The bulk of nutrients are, thus, digested and absorbed in this intestinal section (Krogdahl et al., 1999; Denstadli et al., 2004; Refstie et al., 2006a). The entire length of the salmonid intestine is, however, capable of nutrient absorption, and the distal intestine also transports intact protein by endocytosis throughout the lifespan of the fish (Sire and Vernier, 1992; Buddington et al., 1997). Salmonids have a diffuse pancreas spread among and on the walls of the pyloric caeca. Endochrine, paracrine, and neural mechanisms are involved in regulating the enzyme secretion from this pancreatic tissue. Cholecystokinin (CCK) plays a major role, and products of nutrient hydrolysis are more potent stimulators than intact nutrients (Krogdahl and Sundby, 1999). Enzyme secretion and digestive capacity is regulated according to diet composition (Krogdahl and Sundby, 1999; Krogdahl et al., 2004; Santigosa et al., 2008; Bogevik et al., 2009), although the capacity to adapt secretion of α-amylase for starch hydrolysis is restricted (Spannhof and Plantikow, 1983; Krogdahl et al., 1999).

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17.3.2 Role of the intestinal microflora In homoeothermic terrestrial animals the physiological role of the microflora is dynamic and complex, including colonisation resistance against pathogens, metabolic conversion of dietary and endogenous compounds, and interaction with the host immune system (Cummings and Macfarlane, 1997; Lhoste et al., 2003). It has been generally accepted that fish have a stable intestinal microflora with lower bacterial density and simpler bacterial diversity (Cahill, 1990; Ringø et al., 1995; Kim et al., 2007). Some of these bacteria are considered transient and pass through the gut with the intestinal contents, while others are adherent to and associated with the intestinal mucosa (Ringø and Birkbeck, 1999; Ringø, 2008). Absence or inactivation of pathogenic intestinal bacteria is very important for good health in salmonid fish (Irianto and Austin, 2002; Jutfelt et al., 2008; Ringø et al., 2008). The role of the intestinal microflora regarding digestive function in fish is more uncertain. However, both total count of cultivable intestinal bacteria and bacterial species diversity is significantly changed when replacing fish meal by soybean meal (Heikkinen et al., 2006; Bakke-McKellep et al., 2007b) or krill meal (Ringø et al., 2006a) in diets for rainbow trout and Atlantic salmon, particularly in the distal intestine. The microflora is also altered by dietary cellulose, soy non-starch polysaccharides, or inulin (Bakke-McKellep et al., 2007b; Ringø et al., 2008, 2006b). Thus, just as in terrestrial animals, the microflora in salmonids is dynamic and modulated by diet, and probably affects both digestive function and health.

17.3.3 Feed and feedstuff related digestive function alterations Oil belching and pellet durability Regurgitation (belching) of oil is a significant problem in sea-farmed rainbow trout and Coho salmon, which are pacific salmonids of the genus Oncorhynchus, and may occasionally be observed in Atlantic salmon as well. It is manifested by continuous surfacing of oil droplets in the sea pens and an oil slick on the pen surface. In pacific salmoids oil belching often coincides with gastric dilation air sacculitis (GDAS; Staurnes et al., 1990; Anderson, 2006; Forgan and Forster, 2007), commonly known as bloat, which is characterised by an enlarged stomach, reduced abdominal wall thickness, and sublethal osmoregulatory stress. The stomach also contains up to six times more material than normal, largely consisting of water and accumulated dietary oil. The etiology of this condition is complex. With regard to lipid accumulation in the stomach, however, it appears related to the physical water stability and durability of the feed particles, and more specifically to how particles disintegrate and release oil. Extruded grower feeds for salmonids have a very high oil content, and pellets that rapidly disintegrate and release the feed oil cause oil separation and accumulation in the stomach

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(Baeverfjord et al., 2006a; Terjesen et al., 2008). Stressed fish will, thus, belch feed oil. It follows that optimal feeds for salmonids should not release feed oil while disintegrating. However, too durable feed is not feasible either. High pellet water stability and durability prolong gastric evacuation (Hilton et al., 1981), and too stable extruded diets depress appetite significantly (Terjesen et al., 2008). Thus, feed water stability, durability, and oil release are important quality criteria of salmonid diets that should be optimised carefully. Soybean meal-induced enteritis Salmoninds are intolerant to little refined soy products such as full-fat and extracted (de-oiled) soybean meals. The adverse effects are dose dependent (Krogdahl et al., 2003), and appetite and growth are significantly depressed at high dietary inclusion (>20 %; Olli and Krogdahl, 1994; Olli et al., 1994a, 1995; Krogdahl et al., 2003; Refstie et al., 2005). Consequently this restricts the use of this cost-efficient plant protein commodity in salmonid diets. With regard to appetite and growth depression, rainbow trout appears to have a higher tolerance for soybean meal than Atlantic salmon (Refstie et al., 2000). As repeatedly shown in both Atlantic salmon (van den Ingh et al., 1991, 1996; Baeverfjord and Krogdahl, 1996; Refstie et al., 2000, 2001, 2005, 2006a, b; Krogdahl et al., 2003; Sanden et al., 2005; Bakke-McKellep et al., 2007a, b, c; Knudsen et al., 2007, 2008; Urán et al., 2008) and rainbow trout (Rumsey et al., 1994; Burrells et al., 1999; Refstie et al., 2000; Ostaszewska et al., 2005; Heikkinen et al., 2006; Romarheim et al., 2006; Escaffre et al., 2007), the most severe digestive function alteration in response to dietary soybean meal is inflammation and histopathological changes in the distal intestine. Due to rapid regression following withdrawal of soybean meal from the diet, this condition is classified as a non-infectious and sub-acute enteritis (Baeverfjord and Krogdahl, 1996). It is characterised by infiltration of inflammatory cells in the basal membrane underlying the intestinal mucosa, shortening and clubbing of the mucosal folds, and loss of supranuclear vacuolisation of the enterocytes (Fig. 17.2; Baeverfjord and Krogdahl, 1996). The rate of cell renewal in the mucosa increases in response to losses of functional enterocytes (Bakke-McKellep et al., 2007b), probably resulting in an increasingly immature enterocyte population, as it corresponds with impaired digestive functionality and reduced relative weight of the distal intestine (Bakke-McKellep et al., 2000a, b, 2007b; Nordrum et al., 2000; Krogdahl et al., 2003; Refstie et al., 2006a, b). The condition also alters the local immune response in the intestine, resulting in increased susceptibility to bacterial infections entering the organism across the intestine (Krogdahl et al., 2000). The causative soy components are found in the alcohol extractable fraction of soybeans (van den Ingh et al., 1996; Knudsen et al., 2007; Yamamoto

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(b)

Fig. 17.2 Cross-sections of the distal intestine of Atlantic salmon with normal and undamaged intestine (a) or suffering from serious soybean-meal induced enteritis (b); pictures by Grete Baeverfjord. Note the infiltration of inflammatory cells in the basal membrane underlying the intestinal mucosa, shortening and clubbing of the mucosal folds, and loss of supranuclear vacuolisation of the enterocytes.

et al., 2008). This fraction is among other things rich in saponins (Anderson and Wolf, 1995; Knudsen et al., 2007), which in themselves do not induce the inflammatory reaction, but do increase the epithelial permeability of the distal intestine, thereby apparently exposing the immune system to foreign antigens from dietary components or intestinal bacteria (Knudsen et al., 2008). Soybean meal-induced enteritis appears T-cell-mediated (Bakke-McKellep, 2007c), suggesting a hypersensitivity to some soy peptide. It is plausible that altered intestinal microflora in response to soy (Heikkinen et al., 2006; Bakke-McKellep et al., 2007b) and probably most plant meals plays a role. Soybean meal-induced enteritis appears specific to salmonids, as it is not found in other investigated fish species (Grisdale-Helland et al., 2002a; Catacutan and Pagador, 2004; Evans et al., 2005; Ostaszewska et al., 2005; Refstie et al., 2006c; Romarheim et al., 2008a). The hypersensitivity

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furthermore appears specific to soy, as similar intolerance reactions are not observed when feeding legumes like lupin, field peas, and faba beans, oilseeds like sunflower and rapeseed, wheat and corn gluten, or cereal grains like wheat and oat to Atlantic salmon (Storebakken et al., 2000b; Refstie et al., 2006b; Aslaksen et al., 2007). Soybean meals for salmonids are upgraded by alcohol-washing to produce soy protein concentrates (Lusas and Riaz, 1995), which do not alter the distal intestine in these species (van den Ingh et al., 1991, 1996; Rumsey et al., 1994; Refstie et al., 2001; Escaffre et al., 2007). Impaired lipid digestion Inclusion of soybean meal in the diet reduces the digestibility of lipid in salmonids (Refstie et al., 1998, 2000, 2001, 2005). When comparing legumes, oilseeds, and cereal grains this effect also appears specific to soy (Refstie et al., 2006b; Aslaksen et al., 2007), and the causative agent(s) are found in the alcohol extractable soy fraction (Olli and Krogdahl, 1995; Yamamoto et al., 2008). This adverse response is associated with lower concentration of bile salts in the intestine (Refstie et al., 2006b; Romarheim et al., 2008b; Yamamoto et al., 2008), which is probably caused by altered cholesterol and bile acid metabolism and/or faecal drainage of bile acids in response to alcohol soluble soy component(s). It is neither a direct consequence of soybean meal-induced enteritis nor specific to salmonids, as dietary soybean meal significantly depresses the lipid digestibility in Atlantic cod without causing intestinal inflammation (Førde-Skjærvik et al., 2006; Refstie et al., 2006c). At low ambient temperature the digestibility of saturated fatty acids is reduced in Atlantic salmon and rainbow trout (Menoyo et al., 2003; Ng et al., 2003, 2004). This may potentially worsen the effects of soybean meal on lipid digestion. Dietary non-starch polysaccharides furthermore reduce digestibility of solid fat in e.g. poultry (Smulikowska and Mieczkowska, 1996; Langhout et al., 1997; Dänicke et al., 2000). Thus, the combination of fibre-rich plant meals with oils rich in saturated fatty acids may potentially impair lipid digestion in salmonids at low water temperature. Feed oils with low melting points should in general be avoided at low water temperature. High levels of plant oil (soy, rapeseed, linseed, and palm oil) in the diet may cause accumulation of oil droplets in the enetrocytes of salmonids (Olsen et al., 1999, 2000, 2003; Caballero et al., 2002). The lipid content in the mucosa is normalised by supplying palmetic acid (C16:0; Olsen et al., 2000), which stimulates synthesis of phospholipids, or by sypplying soy lecitin, which is the phospholipid fraction of soy oil (Olsen et al., 1999, 2003). The condition is, thus, interpreted as symptoms of phospholipid deficiency and insufficient capacity for synthesis of lipoproteins for lipid transport out of the intestinal mucosa.

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17.4 Nutritional requirements 17.4.1 Micronutrients The requirements of individual vitamins and vitamin-like components like inositol and choline (Woodward, 1994; Halver, 2002) and essential mineral elements (Lall, 2002) have long been established in salmonids. Thus, these are not further discussed here. It is, however, imperative that dietary minerals are available for digestion and absorption.

17.4.2 Macronutrients Protein and amino acids Protein and amino acid requirements are extensively investigated in Atlantic salmon and rainbow trout (Wilson, 2002). The optimum dietary protein to energy ratio decreases with fish size until the fish undergoes sexual maturation (Austreng et al., 1988; Einen and Roem, 1997). This is caused by escalating fattening while the body protein concentration remains constant (Shearer, 1984; Shearer et al., 1994), and by slower metabolism as the weight of the fish increases, so that a higher proportion of the dietary energy is used for maintenance (Jobling, 1994). Requirement values for all ten essential amino acids are established by dose–response studies for rainbow trout, Chinook salmon, and Coho salmon (Wilson, 2002). However, the growth responses in these studies were far inferior to what is achieved in modern commercial salmonid aquaculture. Adding to this, these values were generally established by broken line regression and/or analysis of variance, which frequently underestimate the requirement (Shearer, 2000). For Atlantic salmon requirement values based on dose–response experiments are only determined for lysine (Anderson et al., 1993; Berge et al., 1998), arginine (Lall et al., 1994; Berge et al., 1997), metionine (Rollin et al., 1994), and threonine (Bodin et al., 2008). Requirements values for the other essential amino acids are alternatively estimated based on optimal balance among essential amino acids of an ‘ideal reference’ protein expressed relative to lysine (Rollin et al., 2003). This model assumes that responses to dietary essential amino acids are well described by broken line regression, and that all essential amino acids are utilised with similar efficiencies. Thus, much work remains to update and establish amino acid requirement values for fastgrowing salmonids. Salmonids appear to have limiting capacity for converting proline to hydroxiproline (Aksnes et al., 2008), and to synthesise taurine from methionine and cysteine (Gaylord et al., 2006, 2007). These amino acids are, thus, conditionally essential in salmonids, and should be considered when formulating diets low in animal protein. Dietary supplementation of histidine above the required level for growth furthermore prevents osmotic cataract in Atlantic salmon genetically disposed for this disorder and/or grown in

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areas with variable salinity (Breck et al., 2003, 2005a; Bjerkås and Sveier, 2004). This is because N-acetyl-L-histidine and histidine containing derivates serve as molecular water pumps regulating hydration of lens tissue (Baslow, 1998; Breck et al., 2005b). Fatty acids Salmonids fishes require aracidonic acid (ArA; C20:4n-6), eicosapentaenoic acid (20:5n-3; EPA) (EPA; C20:5n-3), and docosahexaenoic acid (22:6n-3; DHA) (DHA; C22:6n-3) in the diet. The reason for this is limited Δ6 deasaturase activity for biosynthesis of C20 and C22 long-chained polyunsaturated fatty acids (PUFA) from C18 fatty acids (Storebakken et al., 2000a; Opsahl-Ferstad et al., 2003). The ArA requirement is low, and appears particularly important in broodstock to achieve reproductive success and good egg and fry quality (Bell and Sargent, 2003). Essential fatty acids are selectively retained in phospholipids by salmonids (Bell et al., 2001, 2003; Torstensen et al., 2004a, b), and requirement for EPA + DHA is about 1 % of dry feed (Sargent et al., 2002). The fatty acid profile of triglycerides in adipose tissue does, however, mirror the profile of the dietary oil. Carbohydrate As salmonid fishes have high capacity for synthesis of glucose by gluconeogenesis, they have no specific requirement for carbohydrate (Dabrowski and Guderley, 2002; Hemre et al., 2002). Growth is still promoted by low (∼5 %) dietary inclusion of starch (Hemre et al., 1995a). Due to limited capacity for starch digestion and glucose absorption, assimilation, and metabolism (Phillips et al., 1948; Spannhof and Plantikow, 1983; Wilson, 1994; Hidalgo et al., 1999; Krogdahl and Sundby, 1999; Krogdahl et al., 1999, 2004, 2005; Hemre et al., 1995a, b, 2002), use of starch in salmonid diets should be limited to what is necessary for feed technical purposes. For efficient digestion, this starch needs to be well gelatinised even in diets with moderate starch levels (Pfeffer et al., 1991). High glucose absorption leads to excessive glycogen deposition in the liver and ultimately liver dysfunction (Hilton and Dixon, 1982; Dixon and Hilton, 1985).

17.5 Nutrition and health 17.5.1 Unwanted dietary components and fish health Inherent bioactive components in protein feedstuffs The nutritional quality of fish meals is highly variable, depending on species composition, freshness of the raw materials, and processing conditions (Pike and Hardy, 1997). Spoiling of raw materials by microbial activity and autolysis starts immediately after catching, among other things facilitating oxidation of trimethylamineoxide (TMAO) to unpalatable TMA, and

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decarboxylation of amino acids to form biogenic amines. This process also gives rise to still unknown component(s) that induce pathological changes in the liver and intestine of Atlantic salmon (Opstvedt et al., 2000). Histamine produced from histidine and/or gizzerosine resulting from histidine reacting with lysine during heating furthermore cause stomach distension in rainbow trout (Fairgrieve et al., 1994), possibly preceding development of ulceration. Thus, raw material freshness is an imperative quality criterion for fish meals used for salmonids. Plant feedstuffs often contain bioactive components deposited to protect the plant from being eaten. These are often referred to as antunutritional factors (ANFs), which may broadly be divided into heat-labile and heatstabile factors. Heat-labile ANFs are generally of protein nature, and the most potent of these are inhibitors of digestive enzymes (Liener, 1980) and lectins that may bind to glycoconjugates (e.g. receptors) on animal cell membranes to disturb intestinal function and, if endocytosed, metabolism (van Damme et al., 1998). Although found in most legumes, high concentrations make these components particularly problematic in raw soybeans, which exhibit trypsin inhibitor activity (TIA) as high as 30 mg trypsin inhibited per g meal (Anderson and Wolf, 1995). Salmonids tolerate TIA up to 5 mg/g before digestive disturbances depresses appetite (Olli et al., 1989, 1994b). However, standard thermal processing when extracting soy (Lusas and Riaz, 1995) reduces TIA (Anderson and Wolf, 1995) to levels acceptable by salmonids and, in parallel, inactivates most lectins (Maenz et al., 1999). Heat-stable ANFs comprise a variable group of which the best characterised are tannins, saponins, phytoestrogens, goitrogens (i.e. glucosinolates), and plant structural or storage components such as non-starch polysaccharides (NSP), α-galactocide ologosaccharides, and phytic acid (inositol-6-phosphate; I6P). This group also includes ‘unknown’ components such as factor(s) involved in the etiology of soybean meal-induced enteritis, as described in Section 17.3.3. Saponins have become a topic of interest in salmonids due to their involvement in this ANF complex (Knudsen et al., 2008). With regard to plant carbohydrates, α-galactocide ologosaccharides appear to be utilised by salmonid intestinal bacteria (Refstie et al., 2005), potentially inducing digestive disturbances (Glencross et al., 2003). NSP also alter the intestinal microflora (Ringø et al., 2008), but appear less of a problem. As opposed to poultry, salmonids respond to dietary NSP by increasing the water content in the intestine, thereby avoiding digestive problems related to digesta viscosity (Refstie et al., 1999; Kraugerud et al., 2007). Salmonids appear to tolerate between 5 and 10 g phytic acid per kg diet (Spinelli et al., 1983; Storebakken et al., 1998; Denstadli et al., 2006a; Helland et al., 2006), above which appetite is depressed and mineralisation is compromised, particularly with regard to divalent metal ions like zinc.

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Isoflavonoid phytoestrogens do not affect growing salmonids (Milligan et al., 1998; Tollefsen et al., 2004; D’souza et al., 2005), but appear to alter reproductive performance in salmonid broodstock (Bennetau-Pelissero et al., 2001). Glucosinolates are found in cruciferous seeds like rapeseed and may induce severe goitre in salmonids, which are sensitive to these ANFs (Hilton and Slinger, 1986; Burel et al., 2000, 2001). An upper level of 1.4 mol/g diet is suggested (Tripathi and Mishra, 2007) but, as modern rapeseed varieties contain low levels of glucosinolates, this is normally unproblematic. Other heat-labile ANFs are not properly investigated with specific regard to salmonids. Contaminants in protein feedstuffs Due to the increased use of plant feedstuffs in feeds for salmonids, there is also a growing concern about mycotoxin in these species. Mycotoxins are toxins produced as secondary metabolites by fungi, and feed contamination results from fungal infection of crops. These components are a structurally diverse group, of which the most common are aflatoxins, fumonisins, and ochratoxins (Steyn, 1995; D’Mello and Macdonald, 1997; Binder et al., 2007). Mycotoxins have various acute and chronic adverse effects, and are highly carcinogenic (Hussein and Brasel, 2001; Speijers and Speijers, 2004; Santacroce et al., 2008). Rainbow trout appear highly responsive to mycotoxins, typically manifested by appetite depression, immuno suppression, liver cancer and dysfunction, and ultimately death (Bauer et al., 1969; Lee et al., 1971; Woodward et al., 1983; Rasmussen et al., 1986; Curtis et al., 1995; Ottinger and Kaattari, 1998, 2000; Carlson et al., 2001; Santacroce et al., 2008). Salmonella bacteria (Salmonella spp.) may contaminate feed and infect farmed animals, thereby infecting human consumers if the meats are not properly heated (Lax et al., 1995). Salmonella bacteria have been found in feed plants producing feeds for salmonids (Nesse et al., 2003, 2005a; Lunestad et al., 2007) and, despite extrusion processing, occasionally in ready-to-use salmonid feed (Lunestad et al., 2007). With a possible exception for Salmonella arizonale, salmonella bacteria are not fish pathogens (Kodama et al., 1987; Austin and McIntosh, 1991). Feedborne Salmonella bacteria may still penetrate the gut wall and infect internal organs of rainbow trout grown in freshwater (Hagen, 1966). This appears less of a problem in Atlantic salmon grown in saltwater (Nesse et al., 2005b), possibly due to different water environment and, thus, different modulation of the intestinal microflora. Contaminants in oils Lipid-soluble environmental toxins accumulate in the marine food web, and particularly Baltic and North Sea fish oils contain high levels of dioxin and polychlorinated biphenyls (PCBs). When feeding such oil, these components also accumulate in farmed salmonids. This problem is largely avoided

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when substituting such fish oil by cleaner oils such as plant oils in the diet (Bell et al., 2005; Berntssen et al., 2005).

17.5.2 Malnutrition and fish health The use of high-fat energy-dense diets and lack of exercise in pen culture causes high lipid accumulation in salmonids, especially around the viscera (Hillestad and Johnsen, 1994; Einen and Roem, 1997; Einen and Skrede, 1998; Hillestad et al., 1998; Hemre and Sandnes, 1999; Refstie et al., 2001). This excessive lipid deposition strongly implies a situation corresponding to human obesity. Increased replacement of marine feedstuffs in salmonid diets furthermore leads to reduced health promoting effects of marine n-3 PUFA, which are important regulators of numerous cellular functions, including those related to inflammation and immunity. Together these factors may push the fish towards a state similar to metabolic syndrome in humans and the ‘lifestyle related disorders’ that follow (Eckel et al., 2005). Adding to this, obesity gives rise to increased oxidative stress in the body and lowered stress tolerance (Kyrou and Tsigos, 2007). Although these phenomena are not described in fish, high mortality in slaughter size Atlantic salmon due to handling stress and heart failure is a growing problem, indicating that they are indeed occurring. In Atlantic salmon there is a high and variable frequency of fish suffering from skeletal malformations (Vågsholm and Djupvik, 1998; Kvellestad et al., 2000). The causes for this appear complex, but mineral deficiency clearly plays a role. Even marginal phosphorus deficiency causes critical reductions in whole body and vertebral mineral content of fast-growing salmon (Helland et al., 2005). Long-term feeding with diets marginally deficient in phosphorus and/or zinc induces a range of skeletal deformities commonly observed in the industry, which are mineralised and persist if the salmon is given sufficient mineral supply (Baeverfjord et al., 2006b). As described in Section 17.4.2, high dietary levels of digestible starch or glucose leads to excessive glycogen deposition in the liver and ultimately liver dysfunction in salmonids.

17.5.3 Salmon feed history and human health Cardioprotective effects of long-chain n-3 PUFAs of marine origin are well recognised (Connor and Connor, 1997). Because the fatty acid profile of salmonid adipose tissues mirrors that of the diet fed (Torstensen et al., 2004b; Seierstad et al., 2005), salmonid meats high in EPA and DHA may be produced by feeding diets high in fish oil. Such tailor-made Atlantic salmon has proved beneficial for patients with coronary disease, imposing favourable biochemical changes and reducing vascular inflammation (Seierstad et al., 2005). It follows that concentration of long-chain marine n-3 PUFAs is an important quality criterion for salmonid products marketed as healthy food.

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17.6 Dietary additives 17.6.1 Vitamins, minerals, and amino acids Premixes of stabilised vitamins are added to salmonid diets in accordance with long-established nutritional requirements (Woodward, 1994; Halver, 2002). As vitamins are destroyed during feed extrusion and storage, it is important to adjust and/or protect individual vitamins in the premix to account for this (Barrows et al., 2008a). With regard to mineral elements, premixes of iron, zinc, manganese, and copper salts are added to saltwater diets, and calcium, potassium, iodine, and selenium salts may furthermore be added to freshwater diets, in accordance with established requirements (Lall, 2002). Phosphorus (P) is added separately as readily available (Nordrum et al., 1997) calcium, sodium, or ammonium phosphates. Fish meals have high but variable content of P, typically ranging from 17–30 g P per kg depending on species composition and how much bone the meals contain (Sugiura et al., 1998). Bone-bound P and other mineral elements are, however, poorly available to salmonids (Nordrum et al., 1997; Sugiura et al., 1998; Vielma et al., 1999). Plant meals contain 5–10 g P per kg, but 30–60 % of this is bound as phytic acid (Lott et al., 2000), which is marginally digested by salmonids (Forster et al., 1999; Denstadli et al., 2006a) and furthermore binds essential divalent ions. Properly mineralised Atlantic salmon and rainbow trout contain 5 g P per kg during grow-out (Shearer, 1984; Shearer et al., 1994; Baeverfjord et al., 1998). Consequently, despite large P discharges from salmonid culture, there is still a need to fortify salmonid diets with digestible phosphate. Synthetic L-lysine x HCl, D,L-merhionine, and L-threonine are routinely used to optimise the amino acid profile in salmonid diets. 17.6.2 Carotenoids The unique pink flesh colour in salmonids is caused by deposition of carotenoids such as astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) and canthaxanthin (β,β-carotene-4,4′-dione) in the muscle protein fraction, associated with α-actinin (Matthews et al., 2006). Carotenoids are synthesised de novo by plants, algae, certain types of bacteria, and fungi, but all animals including fishes are unable to biosynthesise carotenoids and, thus, depend on dietary supply. Canthaxanthin and astaxanthin serve as vitamin A precursors in salmonids (Schiedt et al., 1985; Al-Khalifa and Simpson, 1988; Guillou et al., 1989; White et al., 2003), and also have beneficial effects on health, early growth, and fecundity, which is probably related to their effects as antioxidants and immunostimulants (Christiansen et al., 1994, 1995a, b; Torrissen and Christiansen, 1995; Christiansen and Torrissen, 1996; Palace et al., 1999; Stahl and Sies, 2003; Waagbø et al., 2003; Amar et al., 2004; Fraser and Bramley, 2004; Ahmadi et al., 2006). The share of the pigment is ∼10 % of the total raw material cost in commercial salmonid saltwater diets. Flesh colour is an imperative quality

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criterion in salmonids grown in sea pens, and the need for dietary carotenoids during the seawater grow-out phase is defined by the need to obtain sufficient colouring of the flesh. EU has posed a maximum level of 25 mg canthaxanthin per kg diet, and astaxanthin is consequently the main carotenoid used, at least in Europe. The most common carotenoid feed additives are synthetically manufactured and beadlet matrix stabilised (Foss et al., 1984; Choubert and Blanc, 1985; Storebakken et al., 1985, 1986, 1987), but also red yeast (Xanthophyllomyces dendrorhous, anamorph Phaffia rhodozyma; Storebakken et al., 2004a, b; Aarseth et al., 2006; Bjerkeng et al., 2007), which is a natural source of astaxanthin, is in use, particularly in Chile. Although less efficiently utilised, oil derived from crustaceous macrozooplankton may also be used as natural carotenoid sources (Hynes et al., 2009), particularly for organic farming of salmonids. Dietary carotenoids are, however, poorly utilised by salmonids. Thus, extra gain in flesh pigmentation is minor when applying dietary concentrations higher than 50–60 mg per kg (Bjerkeng et al., 1990; Torrissen et al., 1995; Forsberg and Guttormsen, 2006a). The carotenoid concentration in the muscle increases slowly with fish size in a curve-linear fashion when the dietary concentration is kept constant (Storebakken et al., 1987; Bjerkeng et al., 1992; Torrissen et al., 1995; Forsberg and Guttormsen, 2006a). The deposition of carotenoids ceases when the fish become sexually mature, when there is a mobilisation of carotenoids from muscles to skin and reproductive organs (Bjerkeng et al., 1992; Schiedt, 1998). The reasons for this low carotenoid utilisation are at least two-fold. First, the metabolic turnover of carotenoids by the intestine and/or liver appears high. This is indicated by very strong positive correlation between plasma astaxanthin concentration and astaxanthin deposition in muscle when injecting astaxanthin into the abdominal cavity of Atlantic salmon (Ytrestøyl and Bjerkeng, 2007a) and rainbow trout (Ytrestøyl and Bjerkeng, 2007b), showing that neither plasma transport capacity nor muscle binding capacity are limiting factors for flesh pigmentation. Second, the apparent digestibility of carotenoids in salmonid fishes is generally low, typically between 40 and 60 % (Bjerkeng et al., 1997; Bjerkeng and Berge, 2000; Ytrestøyl et al., 2005, 2006). It is furthermore depressed by increasing dietary pigment concentration (Choubert and Storebakken, 1989; Bjerkeng et al., 1990; Torrissen et al., 1990, 1995), increasing feed intake (Ytrestøyl et al., 2006), and falling ambient water temperature (Ytrestøyl et al., 2005). Thus, carotenoid digestibility and retention may vary with season, explaining the ‘spring drop’ in muscle pigmentation that is often observed in northern waters (Mørkøre and Rørvik, 2001). Both high content of lipid in the diet (Choubert et al., 1991; Torrissen et al., 1990; Bjerkeng et al., 1997, 1999, 2000; Hamre et al., 2004) and high proportion of PUFA in the dietary lipid (Waagbø et al., 1993; Bjerkeng et al., 1999, 2000; Regost et al., 2004; Rørå et al., 2005) increase carotenoid bioavailability.

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17.6.3 Feed enzymes Feed enzymes such as carbohydrases and phytases are routinely used as feed additives to improve nutrient utilisation and reduce phosphorus excretion in pigs and poultry. This practice is, however, practically difficult to apply to salmonids. First, feed enzymes are proteins most commonly of microbial origin, and are inactivated by high-pressure moist extrusion. Second, the salmonid gut is short and gastrointestinal transit rapid at high feed intake (Storebakken et al., 1999), giving limited time for added enzymes to work. Finally, salmonids are cold-blooded and cold-water fishes eat and grow at body temperatures far below the temperature optima of commercial feed enzymes. In line with this, addition of phytase post-extrusion to diets high in plant protein has shown little effect in Atlantic salmon and rainbow trout (Vielma et al., 2000; Denstadli et al., 2007). Pre-treatment of plant meals with phytase is, on the other hand, highly efficient in hydrolysing phytic acid to liberate essential divalent ions like zinc while rendering phytic acid-bound phosphorus available to salmonids (Storebakken et al., 1998; Vielma et al., 2002; Denstadli et al., 2006b, 2007). Thus, until extrudable enzyme products that work efficiently at low temperatures are developed, this appears the only feasible way to apply feed enzymes to salmonids.

17.6.4 Immunostimulants and pre-biotica Purified bakers’ yeast mixed link β-1.3/1.6-glucans and/or nucleotides are routinely used as dietary immunostimmulants when growing salmonids in seawater. Such diets are fed precautionary prior to stressful events such as handling, vaccination, saltwater transfer, anti-parasite treatments, and drastic environmental changes. Live yeasts are present and appear commensal in fish guts, possibly benefiting the immune and the digestive systems of the host (Gatesoupe, 2007). In line with this, dietary yeast β-glucans are shown to enhance nonspecific disease resistance (Robertsen et al., 1990; Robertsen, 1999; Sakai, 1999; Burrells et al., 2001a) and resistance to salmon lice (Ritchie, 2000) in Atlantic salmon. Dietary fortification of salmon diets with nucleotides give similar immunostimmulatory responses (Burrells et al., 2001a), and is furthermore reported to enhance growth and osmoregulatory capacity of Atlantic salmon following saltwater transfer, and to increase the mucosal surface area of the salmon gut (Burrells et al., 2001b). Mannan oligosaccharides (MOS) is another yeast cell wall derived feed additive. MOS are known to improve digestion and gut health in animals by binding to and blocking glycoprotein receptors on pathogens and/or function as a prebiotica, favouring growth of beneficial bacteria in the gut (Newman, 2001; Fernandez et al., 2002; Swanson et al., 2002). In line with this, dietary supplementation of purified MOS derived

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from bakers’ yeast cell walls are shown to improve immune status, gut development, and growth in rainbow trout (Staykov et al., 2007; Yilmaz et al., 2007), and to increase feed efficiency and reduce oxidative radical production and serum lysozyme activity in Atlantic salmon (Grisdale-Helland et al., 2008).

17.7 Species differences Carbohydrate utilisation Rainbow trout has significantly higher capacity for starch digestion than Atlantic salmon (Krogdahl et al., 2004). This appears to result from lower α-amylase synthesis and secretion and/or lower substrate affinity by this enzyme in Atlantic salmon (Frøystad et al., 2006). In consequence, the digestibility of even gelatinised starch is reduced in Atlantic salmon when the dietary level exceeds ∼10 %, and undigested starch appears to disturb the digestion of other nutrients (Hemre et al., 1995a; Krogdahl et al., 1999, 2004). A ‘side effect’ of this is that Atlantic salmon is more protected from metabolic disturbances following excessive glucose absorption than rainbow trout at moderate (10–25 %) inclusion of starch in the diet (Hemre et al., 1995b; Krogdahl et al., 2004). Oil belching and bloat Pacific salmonids of the Oncorhynchus genus (rainbow trout, Coho, and Chinook salmon) are susceptible to oil belching and bloat (Staurnes et al., 1990; Rørvik et al., 2000; Anderson, 2006; Forgan and Forster, 2007). Bloat is triggered by osmoregulatory failure, which may explain why this syndrome only rarely occurs in Atlantic salmon. Rainbow trout reared in fullstrength seawater have higher drinking rate than Atlantic salmon (Potts et al., 1970), and also grow faster in fresh and brackish water than in seawater, while the opposite is seen in Atlantic salmon (McKay and Gjerde, 1985; Austreng et al., 1987). Thus, rainbow trout and possibly other pacific salmonids spend more energy on hydromineral regulation, and are more susceptible to osmoregulatory stress. Bloat often coincides with accumulation of feed oil in the stomach. Slowed gastric evacuation in response to nutrient-dense diets is supposedly a major trigger of the condition (Anderson, 2006; Forgan and Forster, 2007). However, as stated in Section 17.3.3, gastric oil accumulation is rather caused by oil release from feed particles with poor water stability (Baeverfjord et al., 2006a; Terjesen et al., 2008). Oil belching then appears to result when these fish are stressed. Thus, an alternative and plausible explanation is that bloat is triggered by osmoregulatory stressors like abrupt temperature and salinity changes (Rørvik et al., 2000), while gastric oil accumulation and belching occurs concomitantly if the fish is fed diets releasing oil while disintegrating.

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Carotenoid utilisation Rainbow trout utilise dietary carotenoids more efficiently for muscle pigmentation than Atlantic salmon, taking colour faster and reaching higher muscle concentrations (Storebakken et al., 1986; March et al., 1990; March and MacMillan, 1996; Page and Davies, 2006). The mechanisms responsible for these differences are still unknown, but high correlation between plasma concentration and muscle deposition of astaxanthin (Ytrestøyl and Bjerkeng, 2007a, b) indicates differences in metabolic turnover of carotenoids by the intestine and/or liver.

17.8 Practical formulations 17.8.1 Choice of feedstuffs Proteins High-quality fish meals (Pike and Hardy, 1997) are still the protein raw materials of choice for salmonids when available at competitive prices. It has also proved difficult to successfully formulate fish-meal free salmonid feeds (Gomes et al., 1995; Espe et al., 2006), potentially due to insufficient knowledge about recognised nutrient requirements, ‘unknown’ essential nutrients in fish meals, and/or unwanted components in the alternative feedstuffs. Although there is, as described in Section 17.2.2, a drive towards fish meal replacement and use of multiple protein feedstuffs for salmonids, fish meals still constitute more than 15 % of salmonid commercial diets. From a nutritional point of view, animal by-products are obvious candidates for replacing fish meals in fish diets. However, EU and the European Economic Area have strong legislative restrictions on products of animal origin (Commission regulation (EC) No 1234/2003). Only hydrolysed nonruminant animal proteins with peptide sizes ≤10 kDa and non-ruminant blood products may be used in European fish feeds. In consequence, the European salmonid industries focus on dietary replacement of fish meals by plant protein sources. High use of plant protein is a challenging strategy, as plant protein sources are deficient in essential amino acids and, as described in Section 17.5.1, often contain ANFs (Storebakken et al., 2000a; Francis et al., 2001; Bakke-McKellep and Refstie, 2008). In Europe it is furthermore made difficult by legislative restrictions on use of DNA-containing feedstuffs originating from genetically modified (GM) crops, although GM plant feedstuffs may be authorised for use in feeds (EC No 1829/2003/EF). Thorough investigations have not been able to demonstrate negative responses (Sanden et al., 2004, 2005, 2006; Hemre et al., 2005, 2007; Bakke-McKellep et al., 2007a, 2008; Sagstad et al., 2007; FrøystadSaugen et al., 2008, 2009) or persistence of modified DNA (Sanden et al., 2004; Nielsen et al., 2005, 2006) in Atlantic salmon fed GM corn and soy. Despite this, European salmonid aquaculture is still reluctant to use GM feedstuffs out of fear of negative publicity and reduced consumer acceptance.

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Outside Europe, and particularly in Chile, GM plant meals are commonly used, and animal by-products such as blood meals and poultry byproducts are increasingly taken into the feeds. Animal by-products often have well-balanced amino acid profiles for fish, and contain few if any ANFs. Thus, carefully processed animal by-products have high nutritional value for salmonids (Fowler, 1990; Pfeffer et al., 1994; Steffens, 1994; Bureau et al., 1999, 2000; Hertrampf and Piedad-Pascual, 2000). However, although animal by-products are available in relatively large quantities at competitive prices, these protein sources are little standardised and often of highly variable nutritional quality. Oils At competitive prices, fish oils are the lipid sources of choice for salmonids. The fatty acid composition varies considerably among fish oils of different origin (Frankel, 1993; Hertrampf and Piedad-Pascual, 2000). North Atlantic fish oil typically contains 15–20 % EPA + DHA, while South American fish oils may contain 30–40 % of these fatty acids. South American fish oils are, thus, the fish oils of choice to cover the EPA + DHA requirements in salmonids at high dietary inclusion of plant oils. Plant oils are devoid of ‘marine’ long-chained polyunsaturated n-3 fatty acids, which are essential in salmonids and considered healthy for humans (Storebakken et al., 2000a; Opsahl-Ferstad et al., 2003). The fatty acid profile in triglycerides of salmonid adipose tissues furthermore reflect that of the feed oil, giving a ‘vegetable’ profile of the fat in salmonids fed plant oils (Caballero et al., 2002; Grisdale-Helland et al., 2002b; Bell et al., 2001, 2003, 2005; Torstensen et al., 2004a, b; Berntssen et al., 2005). Rapeseed oil is the preferred plant oil, as it contains little saturated fatty acids, like fish oil it is rich in oleic acid (C18:1n-9), but contains little linoleic acid (C18:2n-6), which leaves a ‘vegetable’ fatty acid ‘print’ when accumulating in the fish. Palm oil is rich in palmetic acid (C16:0), which may be beneficial for lipid transport across the intestine (Olsen et al., 2000), but contains too much saturated fatty acids and, thus, has too low a melting point for practical use in winter diets. Soy oil contains little saturated fatty acids, but contains up to 60 % of linoleic acid. A ‘marine’ fatty acid profile of fish fed plant oils may largely be restored by choosing plant oils with low levels of linoleic acid and using high levels of EPA + DHA during the last part of the growth period (fish oil ‘wash out’; Bell et al., 2003, 2005; Torstensen et al., 2004b; Berntssen et al., 2005).

17.8.2 Lifespan diets Seasonal diets As described in Section 17.4.2, growing salmonids grow increasingly fatter while their body protein concentration remains constant (Shearer, 1984; Shearer et al., 1994). In climate zones with large seasonal variations in day

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length and temperature, sea-farmed salmonids also show large cyclic variations in fattening, switching their metabolism to fat accumulation in the autumn to utilise this stored energy during the winter (Mørkøre and Rørvik, 2001; Roth et al., 2005). In response to this, commercial lifespan feed programs for sea-farmed salmonids are generally formulated with gradual increasing lipid and decreasing protein levels. Winter diets are furthermore formulated with more lipid and less protein than summer diets, but the time for recommended use of summer or winter diets varies at different latitudes. Within these restrictions typical commercial summer diets for sea-farmed rainbow trout and Atlantic salmon contain 32–40 % protein and 34–40 % lipid, while typical winter diets contain 30–37 % protein and 35–41 % lipid, depending on fish size and raw material use. Pigment strategies In sea-farmed Atlantic salmon the target carotenoid concentration is 6–7 mg per kg muscle, but may be higher in rainbow trout. To achieve this it is common practice to fortify seawater diets with 20–75 mg carotenoids per kg. Commercial feed companies have different models for flesh pigmenting and, thus, strategies for dietary carotenoid fortification, but in general smaller fish are given higher doses than larger fish. An example of such a mathematical programming model is given by Forsberg and Guttormsen (2006a). It predicts dietary astaxanthin concentrations giving adequately pigmented Atlantic salmon at minimum cost (Forsberg and Guttormsen, 2006b) as a function of fish size. Model simulations show that fish growing from seawater transfer to 2 kg should be fed 20–40 mg more pigment per kg diet than fish growing from 4–6 kg. Optimal strategy for dietary astaxanthin fortification depends on target muscle concentration, desired harvest size, and how fast the targeted muscle concentration should be reached. The model assumes that fish weight and dietary astaxanthin concentration are the only variables determining muscle pigmentation. In the light of Section 17.6.2, and knowing that physiological status and genetic disposition also affect the efficiency of carotenoid deposition (Torrissen et al., 1989; Storebakken and No, 1992), this assumption is obviously wrong. Still, such models have proved efficient tools for designing economically viable pigment strategies.

17.9 Feeding and feeding systems Salmonids are generally fed according to appetite, but meal frequencies in commercial operations vary with the feeding technology used. Stomach evacuation rate and, thus, optimal meal frequency varies with fish size and temperature (Handeland et al., 2008). In smaller operations and in landbased systems the fish may be fed continuously by automatic feeding

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systems. In larger sea pens the fish are often fed meals distributed by feeding canons, and the meal frequency will vary from one meal per day for large fish at low temperature to four times a day in small fish at high temperature. Feeding systems used in sea pens deliver feeds by mechanic and/or pneumatic action. Thus, they have in common the need for high physical durability of the feed particles to allow efficient mechanic feed distribution. Appetite feeding in sea pens is challenging due to deep pens, large biomasses and thus it is difficult to monitor feed intake (Alver et al., 2004). This can be controlled by video or hydroacoustic devices detecting uneaten pellets sinking through the bottom of the pens, signalling to stop feeding (Juell et al., 1993; Ervika et al., 1994; Parsonage and Petrell, 2003). Alternatively, attempts can be made to collect and recycle uneaten feed during the feeding periods (Ervika et al., 1994). When integrated with automatic feeding systems, these monitoring systems automatically regulate feeding according to apparent appetite.

17.10 Future trends 17.10.1 Raw materials Protein sources The salmonid feed industry has a declared goal to reduce their dependency on fish meals. The ultimate goal is to treat fish meal as any other protein feedstuff in least cost formulation. Thus, new feedstuffs will continuously be evaluated and adapted for use in fish feeds. With regard to increased use of vegetable protein, this will require updated requirement values for essential amino acids, as well as a better understanding about conditional requirements of amino acids and other components not found in plants (Aksnes et al., 2006a, b, 2008; Gaylord et al., 2006, 2007). Understanding soy intolerance (Baeverfjord and Krogdahl, 1996) is a holy grail in salmonid nutrition. Considering the progress made in this field, it is expected that the causative components will be identified and proper means developed to reduce or totally avoid soybean meal-induced enteritis. This will allow higher use of standard soybean meals for salmonids. As soybean meal is the major protein commodity for animal feeds, it will increase the competitiveness of salmonid aquaculture. The biofuel industry offer new possibilities for developing feedstuffs from extraction or fermentation residues. One promising candidate is soybean meal resulting from in situ transesterification to produce biodiesel directly from soybeans (Barrows et al., 2008b). Residues from fermentation of corn or wheat to produce bioethanol are nutrientwise interesting, but high contamination with mycotoxins in crops going into these processes is currently challenging.

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Increased harvesting of marine macrozooplankton like Antarctic krill (Euphausia superba) is offering new sources of marine protein. Marine crustacean macrozooplankton contain very high levels of fluoride (F; 1000– 6000 mg per kg). This is problematic for salmonids in freshwater, which absorb and deposit F in bones and scales (Yoshitomi et al., 2006). Dietary F is, however, not taken up by salmonids in seawater to any significant degree (Julshamn et al., 2004; Moren et al., 2007). The protective effect of water salinity indicates that ingested F like Cl is pumped out of the fish body across the gills by chloride cells. Thus, meals made from crustacean macrozooplanktoon have proved feasible feedstuffs for sea-farmed salmonids (Julshamn et al., 2004; Olsen et al., 2006; Suontama et al., 2007a, b). However, although the EU has accepted an upper level of 3000 g P per kg in krill, the upper level for complete diets is still 150 mg per kg (EU directive, 2005/87/EC). This is currently restricting the use of krill as a major protein source in European fish feeds. There is also a great potential for processing animal slaughter waste into by-products feasible for fish. This is a highly sustainable strategy, as it will allow recirculation of problematic wastes by converting them into healthy and high-quality seafood. However, due to the EU’s strong legislative restrictions on using products of animal origin as feedstuffs, this is currently difficult to achieve in Europe. Oils Fish and plant oils are already interchanged as energy substrates in salmonid feeds according to price fluctuations. As described in Section 17.8.1, a certain level of fish oil is still required in the diet to supply EPA and DHA. This may change when alternative sources of EPA and DHA become available. At the moment the most promising candidates are EPA- and DHA-rich plant oils originating from genetically modified (GM) oil seeds (Opsahl-Ferstad et al., 2003). Other sources may be GM yeast or bacteria. Harvesting of marine crustacean macrozooplankton also offers a new source of EPA- and DHA-rich marine oils. Although very rich in wax esters, such oils have proved feasible feed oils for Atlantic salmon (Olsen et al., 2004; Bogevik et al., 2008, 2009).

17.10.2 Feeds Functional feeds Functional feeds, i.e. feeds with added components stimulating specific physiological processes in the fish, are already used to some degree. Examples of such are feeds with immunostimulants used prior to stressful periods. As our understanding of fish physiology, immunology, and metabolism increases, new concepts will be developed. For example, increased awareness of the intestinal microflora in fish has spurred testing and development of prebiotica for salmonids (Refstie

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et al., 2006a; Bakke-McKellep et al., 2007b; Staykov et al., 2007; Yilmaz et al., 2007; Grisdale-Helland et al., 2008). This development is expected to continue and accelerate. Increased knowledge about bioactive components in feedstuffs may furthermore lead to proactive use of raw materials and/or isolated components to stimulate beneficial physiological responses. Focus on lipid metabolism and obesity in salmonids has likewise led to testing of dietary components that increase fatty acid oxidation. Examples of such are high doses of EPA and DHA (Ruyter et al., 1999; Vegusdal et al., 2005), or metabolic modulators such as conjugated linoleic acid (CLA; Berge et al., 2004; Kennedy et al., 2006, 2007) and tetradecylthioacetic acid (TTA; Moya-Falcon et al., 2004; Gjøen et al., 2007; Kennedy et al., 2007; Rørvik et al., 2007). Too high doses of EPA and/or DHA do, however, induce oxidative stress and reduce mitochondrial function in Atlantic salmon (Kjær et al., 2008; Todorcˇevic´ et al., 2008), so this approach requires great caution. Metabolic modulators such as TTA must be subjected to thorough biological testing before being legalised and commercialised for feed purposes. Physical feed quality Hardness and wear resistance are carefully controlled by commercial farmers and are the ever more important quality criteria of extruded feeds used for salmonids in seawater. This is driven by gradual enlargement of the sea pens, as shear physical durability allows more efficient mechanic distribution of the feeds. However, it is becoming clear that durable and water-stable feeds slow stomach evacuation rate and depress feed intake and growth in rainbow trout (Hilton et al., 1981; Terjesen et al., 2008), and probably in salmonids in general. Feed pellets that ‘collapse’ in the stomach while releasing feed oil may furthermore cause oil belching if the fish is stressed (Baevefjord et al., 2006a; Terjesen et al., 2008). Thus, it is necessary to reassess physical quality criteria for salmonid feeds, and to optimise them with respect to feasible disintegration in the fish stomach. In the light of this, currently adopted nutritional values of feedstuffs replacing fish meal in salmonid diets may actually be wrong. Feedstuffs for salmonids are traditionally evaluated in fish meal replacement studies. However, factors like type of feedstuffs (Olsen et al., 2006; Refstie et al., 2006b), feedstuff components such as cellulose (Hansen and Storebakken, 2007), as well as different processing conditions along the feed manufacturing line (Sørensen et al., 2002, 2005; Aarseth et al., 2006) all significantly affect the physical quality of extruded feeds. Changing physical feed quality is seldom considered in replacement studies, although these variables may actually cause the main responses when comparing extruded feeds. Thus, it may prove necessary to re-evaluate the nutritional value of important feed ingredients for salmonids in experimental setups considering or standardising physical feed quality parameters.

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17.10.3 Sustainability Sustainability in aquaculture is usually defined as low use of feedstuffs derived from limited and often threatened fisheries. As explained throughout this chapter, this is a major goal for several reasons in salmonid nutrition. Increased replacement of fish meals by other protein sources is part of the solution to achieve it. As shown in Fig. 17.3, the consumption of dietary fish protein will equal the production of farmed fish protein when reducing the dietary fish meal content to ∼25 %. At lower dietary fish meal inclusion salmonids will actually be net producers of fish protein. In this sustainability context, it is also problematic that valuable marine lipid resources are spent on producing visceral fat and visible muscular fat deposits which are lost as processing waste at slaughter and, thus, reduce

Spent marine protein 5,0

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10 % FM

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4,0 3,0 2,0 1,0 0,0 5 % FM (b)

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Fig. 17.3 Consumption of wild-caught fish (processed into fish meal – FM) and production of farmed Atlantic salmon when using different levels of fish meal in the diet, calculated as ton protein (a) or ton edible fish (b). The results are based on unpublished experimental data. When calculating edible proportions of the fish, filet yields of 30 % and 60 % were assumed for wild-caught fish and Atlantic salmon, respectively.

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harvest yields. Thus, preventing excessive lipid deposition in salmonids will have to be an important focus in future research. Part of the solution will be to regulate fattening of salmonids by dietary means. It is also becoming apparent that, like pigs, salmonids should be selectively bred for efficient but lean growth. When achieved, this will significantly spare lipid in salmonid aquaculture.

17.11 References aarseth k a, sørensen m and storebakken t (2006) ‘Effects of red yeast inclusions in diets for salmonids and extrusion temperature on pellet tensile strength: Weibull analysis, Anim Feed Sci Technol, 126, 75–91. ahmadi m r, bazyar a a, safi s, ytrestøyl t and bjerkeng b (2006) Effects of dietary astaxanthin supplementation on reproductive characteristics of rainbow trout (Oncorhynchus mykiss), J Appl Ichtyol, 22, 388–94. aksnes a, hope b, jonsson e, björnsson b t and albrektsen s (2006a) Sizefractionated fish hydrolysate as feed ingredient for rainbow trout (Oncorhynchus mykiss) fed high plant protein diets. I: Growth, growth regulation and feed utilization, Aquaculture, 261, 305–17. aksnes a, hope b and albrektsen s (2006b) Size-fractionated fish hydrolysate as feed ingredient for rainbow trout (Oncorhynchus mykiss) fed high plant protein diets. II: Flesh quality, absorption, retention and fillet levels of taurine and anserine, Aquaculture, 261, 318–26. aksnes a, mundheirn h, toppe j and albrektsen s (2008) The effect of dietary hydroxyproline supplementation on salmon (Salmo salar L.) fed high plant protein diets, Aquaculture, 275, 242–9. al-khalifa a r and simpson k l (1988) Metabolism of astaxanthin in the rainbow trout (Salmo gairdneri), Comp Biochem Physiol, 91B, 563–8. alver m o, alfredsen j o and sigholt t (2004) Dynamic modelling of pellet distribution in Atlantic salmon (Salmo salar L.) cages, Aquac Eng, 31, 51–72. amar e c, kiron v, satoh s and watanabe t (2004) Enhancement of innate immunity in rainbow trout (Oncorhynchys mykiss Walbaum) associated with dietary intake of carotenoids from natural products, Fish Shellfish Immunol, 16, 527–37. anderson c d (2006) A review of causal factors and controlling measures for bloat in farmed salmonids with a suggested mechanism for the development of the condition, J Fish Dis, 29, 445–53. anderson j s and sunderland r (2002) Effect of extruder moisture and dryer processing temperature on vitamin C and E and astaxanthin stability, Aquaculture, 207, 137–49. anderson r l and wolf w r (1995) Compositional changes in trypsin inhibitors, phytic acid, saponins and isoflavones related to soybean processing, J Nutr, 125, 581S–588S. anderson j s, lall s p, anderson d m and mcniven m (1993) Quantitative dietary lysine requirement of Atlantic salmon (Salmo salar) fingerlings, Can J Fish Aquat Sci, 50, 316–22. åsgård t and austreng t (1985a) Casein silage as feed for salmonids, Aquaculture, 48, 233–52. åsgård t and austreng t (1985b) Dogfish offal, ensiled or frozen, as feed for salmonids, Aquaculture, 49, 289–305. åsgård t and austreng t (1986) Blood, ensiled or frozen, as feed for salmonids, Aquaculture, 55, 263–84.

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small Atlantic salmon (Salmo salar) transferred into seawater as 0+ or 1+ smolts, Aquaculture, 250, 830–40. rumsey g l, endres j g, bowser p r, earnest-koons k a, anderson d p and siwicki a k (1994) Soy protein in diets of rainbow trout: Effects on growth, protein absorption, gastrointestinal histology, and nonspecific serologic and immune response, in Lim C E and Sessa D J (eds), Nutrition and Utilization Technology in Aquaculture, AOCS Press, Champaign, IL, 166–88. ruyter b, andersen o, dehli a, farrants a k o, gjøen t and thomassen m s (1999) Peroxisome proliferator activated receptors in Atlantic salmon (Salmo salar): effects on PPAR transcription and acyl-CoA oxidase activity in hepatocytes by peroxisome proliferators and fatty acids, Biochim Biophys Acta Lip Lip Metab, 1348, 331–8. sagstad a, sanden m, haugland ø, hansen a-c, olsvik p a and hemre g-i (2007) Evaluation of stress- and immune-response biomarkers in Atlantic salmon, Salmo salar L., fed different levels of genetically modified maize (Bt maize), compared with its near-isogenic parental line and a commercial suprex maize, J Fish Dis, 30, 201–12. sakai m (1999) Current research status of fish immunostimulants, Aquaculture, 172, 63–92. sanden m., bruce i j, rahman m a and hemre g-i (2004) The fate of transgenic sequences present in genetically modified plant products in fish feed, investigating the survival of GM soybean DNA fragments during feeding trials in Atlantic salmon, Salmo salar L., Aquaculture, 237, 391–405. sanden m, berntssen m h g, krogdahl å, hemre g-i and bakke-mckellep a m (2005) An examination of the intestinal tract of Atlantic salmon (Salmo salar L.) parr fed different varieties of soy and maize, J Fish Dis, 28, 317–30. sanden m, krogdahl å, bakke-mckellep a m, buddington r k and hemre g-i (2006) Growth performance and organ development in Atlantic salmon, Salmo salar L. parr fed genetically modified (GM) soybeans and maize, Aquac Nutr, 12, 1–14. santacroce m p, conversano m c, casalino e, lai o, zizzadoro c, centoducati g and crescenzo g (2008) Aflatoxins in aquatic species: metabolism, toxicity and prespectives, Rev Fish Biol Fish, 18, 99–130. santigosa e, sánchez j, médale f, kaushik s, pérez-sánchez j and gallardo m a (2008) Modifications of digestive enzymes in trout (Oncorhynchus mykiss) and sea bream (Sparus aurata) in response to dietary fish meal replacement by plant protein sources, Aquaculture, 282, 68–74. sargent j r, tocher d r and bell j g (2002) The lipids, in Halver J E and Hardy R W (eds), Fish Nutrition, Academic Press, San Diego, CA, 181–257. schiedt k (1998) Absorption and metabolism of carotenoids in birds, fish and crustaceans, in Carotenoids Vol. 3 Biosynthesis, Birkhäuser, Basel, 285–358. schiedt k, leuenberger f j, vecchi m and glinz e (1985) Absorption, retention, and metabolic transformations of carotenoids in rainbow trout, salmon and chicken, Pure Appl Chem, 57, 685–92. seierstad s l, seljeflot i, johansen o, hansen r, haugen m, rosenlund g, frøyland l and arnesen h (2005) Dietary intake of differently fed salmon; the influence on markers of human atherosclerosis, Eur J Clin Invest, 35, 52–9. shearer k d (1984) Changes in the elemental composition of hatchery reared rainbow trout (Salmo gairdneri) associated with growth and reproduction, Can J Fish Aquat Sci, 41, 1592–600. shearer k d (2000) Experimental design, statistical analysis and modelling of dietary nutrient requirement studies for fish: a critical review, Aquac Nutr, 6, 91–102.

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shearer k d, åsgård t, andorsdóttir g and aas g h (1994) Whole body elemental and proximate composition of Atlantic salmon (Salmo salar) during the life cycle, J Fish Biol, 44, 785–97. sire m f and vernier j-m (1992) Intestinal absorption of protein in teleost fish, Comp Biochem Physiol, 103A, 771–81. smulikowska s and mieczkowska a (1996) Effect of rye level, fat source and enzyme supplementation on fat utilization, diet metabolizable energy, intestinal viscosity and performance of broiler chickens, J Anim Feed Sci, 5, 379–93. sørensen m, ljøkjel k, storebakken t, shearer k d and skrede a (2002) Apparent digestibility of protein, amino acids, and energy in rainbow trout (Oncorhynchus mykiss) fed a fish meal based diet extruded at different temperatures, Aquaculture, 211, 215–25. sørensen m, storebakken t and shearer k d (2005) Digestibility, growth and nutrient retention in rainbow trout (Oncorhynchus mykiss) fed diets extruded at two different temperatures, Aquac Nutr, 11, 251–6. spannhof l and plantikow h (1983) Studies on carbohydrate digestion in rainbow trout, Aquaculture, 30, 95–108. speijers g j a and speijers m h m (2004) Combined toxic effects of mycotoxins, Toxicol Lett, 153, 91–8. spinelli j, houle c r and wekell j c (1983) The effect of phytates on the growth of rainbow trout (Salmo gairdneri) fed purified diets containing varying quantities of calcium and magnesium, Aquaculture, 30, 71–83. stahl w and sies h (2003) Antioxidant activity of carotenoids, Mol Aspects Med, 24, 345–51. staurnes m, andorsdottir g and sundby a (1990) Distended, water-filled stomach in sea farmed rainbow trout, Aquaculture, 90, 333–43. staykov y, spring p, denev s and sweetman j (2007) Effect of mannan oligosaccharide on the growth performance and immune status of rainbow trout (Oncorhynchus mykiss), Aquac Int, 15, 153–61. steffens w (1994) Replacing fish meal with poultry by-product meal in diets for rainbow trout, Oncorhynchus mykiss, Aquaculture, 124, 27–34. steyn p s (1995) Mycotoxins, general view, chemistry and structure, Toxicol Lett, 82/83, 843–51. storebakken t and no h k (1992) Pigmentation of rainbow trout, Aquaculture, 100, 209–29. storebakken t, foss p, austreng e and liaaen-jensen s (1985) II. Epimerization studies with astaxanthin in Atlantic salmon, Aquaculture, 44, 259–69. storebakken t, foss p, huse i, wandsvik a and berg lea t (1986) III. Utilization of canthaxanthin from dry and wet diets by Atlantic salmon, rainbow trout and sea trout, Aquaculture, 51, 245–55. storebakken t, foss p, schiedt k, austreng e, liaen-jensen s and manz u (1987) Carotenoids in diets for salmonids IV. Pigmentation of Atlantic salmon with astaxanthin, astaxanthin dipalmitate and canthaxanthin, Aquaculture, 65, 279–92. storebakken t, shearer k d and roem a j (1998) Availability of protein, phosphorus and other elements in fish meal, soy-protein concentrate and phytase-treated soy-protein-concentrate-based diets to Atlantic salmon, Salmo salar, Aquaculture, 161, 365–79. storebakken t, kvien i s, shearer k d, grisdale-helland b and helland s j (1999) Estimation of gastrointestinal evacuation rate in Atlantic salmon (Salmo salar) using inert markers and collection of faeces by sieving: evacuation of diets with fish meal, soybean meal or bacterial meal, Aquaculture, 172, 291–9.

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storebakken t, refstie s and ruyter b (2000a) Soy products as fat and protein sources in fish feeds for intensive aquaculture, in Drachley J (ed.), Soy in Animal Nutrition, Federation of Animal Sciences Society, Savoy, IL, 127–70. storebakken t, shearer k d, baeverfjord g, nielsen b g, åsgård t, scott t and de laporte a (2000b) Digestibility of macronutrients, energy and amino acids, absorption of elements and absence of intestinal enteritis in Atlantic salmon, Salmo salar, fed diets with wheat gluten, Aquaculture, 184, 115–32. storebakken t, sørensen m, bjerkeng b, harris j, monahan p and hiu s (2004a) Stability of astaxanthin from red yeast, Xanthophyllomyces dendrorhous, during feed processing: effects of enzymatic cell wall disruption and extrusion temperature, Aquaculture, 231, 489–500. storebakken t, sorensen m, bjerkeng b and hiu s (2004b) Utilization of astaxanthin from red yeast, Xanthophyllomyces dendrorhous, in rainbow trout, Oncorhynchus mykiss: effects of enzymatic cell wall disruption and feed extrusion temperature, Aquaculture, 236, 391–403. sugiura s h, dong f m, rathbone c k and hardy r w (1998) Apparent protein digestibility and mineral availabilities in various feed ingredients for salmonid feeds, Aquaculture, 159, 177–202. suontama j, kiessling a, melle w, waagbø r and olsen r e (2007a) Protein from Northern krill (Thysanoessa inermis), Antarctic krill (Euphausia superba) and the Arctic amphipod (Themisto libellula) can partially replace fish meal in diets to Atlantic salmon (Salmo salar) without affecting product quality, Aquac Nutr, 13, 50–58. suontama j, karlsen o, moren m, hemre g-i, melle w, langmyhr e, mundheim h, ringø e and olsen r e (2007b) Growth, feed conversion and chemical composition of Atlantic salmon (Salmo salar L.) and Atlantic halibut (Hippoglossus hippoglossus L.) fed diets supplemented with krill or amphipods, Aquac Nutr, 13, 241–55. swanson k s, grieshop c m, flickinger e a, healy h p, dawson k a, merchen n r and fahey jr g c (2002) Effects of supplemental fructooligosaccharides plus mannanoligosaccharides on immune function and ileal and fecal microbial populations in adult dogs, Arc Anim Nutr, 56, 309–18. terjesen b f, sigholt t, hillestad m, holm j, refstie s, baeverfjord g, aas t s, rørvik k-a and åsgård t (2008) The impact of physical pellet characteristics and environmental conditions on stomach content separation in rainbow trout, 7th International Conference on Recirculating Aquaculture Proceedings, 23–24 June, Roanoke, VA, 378–87. todorcˇ evic´ m, kjær m, djakovic´ n, vegusdal a, torstensen b e and ruyter b (2008) Effects of dietary n-3 fatty acids on fat deposition, mitochondrial function and oxidative stress in in Atlantic salmon visceral adipose tissue, Proc XIII ISFNF, June 1–5, 2006, Florianópolis, 53. tollefsen k e, øvrevik j and stenersen j (2004) Binding of xenoestrogens to the sex steroid-binding protein in plasma from Arctic charr (Salvelinus alpinus L.), Comp Biochem Physiol, 139C, 127–33. torrissen o j and christiansen r (1995) Requirements for carotenoids in fish diets, J Appl Ichthyol, 11, 225–30. torrissen o j, hardy r w and shearer k d (1989) Pigmentation of salmonidscarotenoid deposition and metabolism, CRC Crit Rev Aquat Sci, 1, 209–25. torrissen o j, hardy r w, shearer k d, scott t m and stone f e (1990) Effects of dietary canthaxanthin and lipid level on apparent digestibility coefficients for canthaxanthin in rainbow trout (Oncorhynchus mykiss), Aquaculture, 88, 351–62. torrissen o j, christiansen r, struksnæs g and estermann r (1995) Astaxanthin deposition in the flesh of Atlantic salmon, Salmo salar L., in relation to dietary astaxanthin concentration and feeding period, Aquac Nutr, 1, 77–84.

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torstensen b e, frøyland l and lie ø (2004a) Replacing dietary fish oil with increasing levels of rapeseed oil and olive oil – effects on Atlantic salmon (Salmo salar L.) tissue and lipoprotein lipid composition and lipogenic enzyme activities, Aquac Nutr, 10, 175–92. torstensen b e, frøyland l, ørnsrud r and lie ø (2004b) Tailoring of a cardioprotective muscle fatty acid composition of Atlantic salmon (Salmo salar) fed vegetable oils, Food Chem, 87, 567–80. tripathi m k and mishra a s (2007) Glucosinolates in animal nutrition: a review, Anim Feed Sci Technol, 132, 1–27. urán p a, schrama j w, rombout j h w m, obach a, jensen l, koppe w and verreth j a j (2008) Soybean meal-induced enteritis in Atlantic salmon (Salmo salar L.) at different temperatures, Aquac Nutr, 14, 324–30. vågsholm i and djupvik h o (1998) Risk factors for spinal deformities in Atlantic salmon, Salmo salar L., J Fish Dis, 21, 47–53. van damme e j m, peumans w j, pusztai a and bardocz s (1998) Handbook of Plant Lectins: Properties and Biomedical Applications, Wiley, Chichester. van den ingh t s g a m, krogdahl å, olli j, hendricks h and koninkx j (1991) Effects of soybean containing diets on the proximal and distal intestine in Atlantic salmon Salmo salar: a morphological study, Aquaculture, 94, 296–305. van den ingh t s g a , olli j j and krogdahl å (1996) Alcohol-soluble components in soybeans cause morphological changes in the distal intestine of Atlantic salmon, Salmo salar L., J Fish Dis, 19, 47–53. vegusdal a, gjøen t, berge r k, thomassen m s and ruyter b (2005) Effect of 18 : 1n-9, 20 : 5n-3, and 22 : 6n-3 on lipid accumulation and secretion by Atlantic salmon hepatocytes, Lipids, 40, 477–86. vielma j, ruohonen k and lall s p (1999) Supplemental citric acid and particle size of fish bone-meal influence the availability of minerals in rainbow trout Oncorhynchus mykiss (Walbaum), Aquac Nutr, 5, 65–71. vielma j, mäkinen t, ekholm p and koskela j (2000) Influence of dietary soy and phytase levels on performance and body composition of large rainbow trout (Oncorhynchus mykiss) and algal availability of phosphorus load, Aquaculture, 183, 349–62. vielma j, ruohonen k and peisker m (2002) Dephytinization of two soy proteins increases phosphorus and protein utilization by rainbow trout, Oncorhynchus mykiss, Aquaculture, 204, 145–56. waagbø r, sandnes k, torrissen o j, sandvin a and lie ø (1993) Chemical and sensory evaluation of fillets from Atlantic salmon (Salmo salar) fed three levels of N-3 polyunsaturated fatty acids at two levels of vitamin E, Food Chem, 46, 361–6. waagbø r, hamre, k, bjerkås e, wathne e, lie ø and torstensen b (2003) Cataract formation in Atlantis salmon, Salmo salar L., smolt relative to dietary pro- and antioxidants and lipid level, J Fish Dis, 26, 213–29. white d a, ørnsrud r and davies s j (2003) Determination of carotenoid and vitamin A concentrations in everted salmonid intestine following exposure to solutions of carotenoid in vitro, Comp Biochem Physiol, 136, 683–92. wilson r p (1994) Utilization of dietary carbohydrate by fish, Aquaculture, 124, 67–80. wilson r p (2002) Amino acids and proteins, in Halver J E and Hardy R W (eds), Fish Nutrition, Academic Press, San Diego, CA, 143–79. woodward b (1994) Dietary vitamin requirements of cultured young fish, with emphasis on quantitative estimates for salmonids, Aquaculture, 124, 133–68. woodward b, young l g and lun a k (1983) Vomitoxin in diets for rainbow trout (Salmo gairdneri), Aquaculture, 35, 93–101.

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18 Monitoring viral contamination in shellfish growing areas F. S. Le Guyader and M. Pommepuy, Ifremer, France, and R. L. Atmar, Baylor College of Medicine, USA

Abstract: Human and animal fecal wastes and urine contain a large number of different viruses that can enter the environment through the discharge of waste materials from infected individuals. Despite the high diversity of viruses that are introduced into the environment by human fecal pollution, only a few have been recognized to cause disease in association with consumption of contaminated shellfish. Viruses are present in shellfish in very low numbers. Nevertheless, they are still present in sufficient quantities to pose a health risk. This low level of contamination has made it necessary to develop highly sensitive viral extraction methods to ensure virus recovery from shellfish tissues. The most common route for accidental contamination is after heavy rainfall, leading to overflow and release of untreated sewage into the aquatic environment. To limit shellfish contamination the most desirable and effective option is to reduce the viral input. Key words: human enteric viruses, shellfish, sewage, persistence, flux.

18.1 Introduction Shellfish have been identified as a vector for human enteric pathogens for more than 150 years. Shellfish filter large volumes of water during their feeding, and in the process they concentrate small particles containing microalgae and microorganisms. The practice of consuming either raw or undercooked shellfish can lead to transmission of disease caused by human pathogens present in the shellfish. During the 1800s, outbreaks of typhoid fever and cholera were associated with shellfish consumption (Richards, 1985). Contamination of shellfish-growing waters with human sewage was recognized as a contributing cause of the outbreaks, leading to the development of bacteriologic criteria to assess the impact of sewage on shellfish and shellfish-growing waters.

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Most countries have endorsed sanitary controls on live bivalve shellfish. In the EU, these are covered by Council Directive 91/492/EEC (EU, 1991) and in the USA, by interstate trading agreements set out in the Federal Drug Administration National Shellfish Sanitation Program Manual of Operations (FDA, 1993). These regulations cover similar ground on the requirements for harvesting area classification, depuration, relaying, analytical methods, and provisions for suspension of harvesting from classified areas following a pollution or public health emergency. The legislation also requires that third country imports into the EU and USA have to be produced under the same standard as domestic products. Exporting nations have therefore developed programs for compliance with the regulations of their target export markets. A major weakness of these controls is the use of traditional bacterial indicators of fecal contamination, such as the fecal coliforms or E. coli, to assess contamination and hence implement the appropriate control measures. Fecal indicators are either measured in the shellfish themselves (EU requirement) or in the shellfish-growing waters (US FDA requirement). Levels of E. coli are used to categorize harvesting areas and prescribe levels of treatment required before they can be sold to consumers. These controls led to a significant decrease in the number of shellfish-associated outbreaks of bacterial infection, but a new problem emerged. Outbreaks of nonbacterial gastroenteritis and infectious hepatitis were recognized to be associated with shellfish consumption (Richards, 1987). Several reports described a lack of correlation between bacterial indicator microorganisms and viruses, and pathogenic viruses may be detected in shellfish from areas classified as suitable for commercial exploitation using fecal coliform criteria (Abad et al., 1997; Bosch et al., 2001; Butt et al., 2004; Le Guyader et al., 2006a, 2008). A number of examples of transnational outbreaks have recently been reported following trade between EU member states (Le Guyader et al., 2006a) and importation of shellfish from third countries into the EU and the USA (Bosch et al., 2001; Butt et al., 2004). In addition, the practice of depuration, a process by which shellfish ‘purify’ themselves of enteric bacteria by filtering clean waters, failed to eliminate the risk of viral-mediated disease (Richards, 1988). Given the failure of the current arrangements to protect public health fully, there is a clear need to develop better approaches to controlling this problem. European shellfish trade totals 460 M per year, and increases by approximately 7 % each year. The European production represents more than a third of the worldwide shellfish production (i.e., in 1991, 180 000 tons of live weight: 72 % of farmed bivalves, 28 % wild – Eurostat data), and 8500 companies currently employ around 23 000 workers. This activity is one of the major sources of employment in coastal areas (Ireland, France, Spain, The Netherlands). The costs of outbreaks of shellfish-associated viral disease have not been clearly defined, but they are likely to be substantial. In the USA, foodborne diseases are a major cause of morbidity and

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hospitalization, with about 325 000 hospitalizations and 5000 deaths per year (Butt et al., 2004). There are 76 million estimated cases of foodborne disease, 10–19 % of those for which a vehicle of transmission is identified seafood. Half of these are cases caused by viruses, and half of the illnesses are associated with shellfish consumption (Mead et al., 1999; Butt et al., 2004). In countries with higher seafood consumption, or where seafood is traditionally eaten raw, a larger percentage of foodborne illnesses are due to seafood consumption. For example, in Japan as much as 70 % of foodborne illness is associated with seafood consumption (Butt et al., 2004).

18.2 Source of pollution 18.2.1 Human enteric viruses Human and animal fecal wastes and urine contain a large number of different viruses that can enter the environment through the discharge of waste materials from infected individuals. These enteric viruses cause a wide spectrum of illnesses in humans including hepatitis, gastroenteritis, meningitis, fever, rash, and conjunctivitis. A brief description of the principal viruses that have been characterized either in outbreaks or in field studies is given below and their different characteristics are described Table 18.1. Hepatitis A virus (HAV) Infectious hepatitis, caused by the hepatitis A virus, is one of the most serious illnesses transmitted by shellfish. The hepatitis A virus belongs to the genus Hepatovirus of the family Picornaviridae, and is very stable in the environment, remaining viable for up to several weeks in water or on fomites (Abad et al., 1994; Arnal et al., 1998; Hollinger and Emerson, 2007). Hepatitis A virus infection has a long incubation period and is generally asymptomatic or associated with a mild illness in young children, while in older children and adults the illness is characterized by jaundice in more than 70 % of individuals (CDC, 2006). There is only a single serotype, and an effective vaccine is available for prevention of infection (CDC, 2006). Noroviruses (NoV) Noroviruses (NoV) are the most common infections currently associated with shellfish consumption. Norovirus is a genus in the family Caliciviridae, and the genus is divided into five genogroups (Zheng et al., 2006). Genogroups I, II and IV contain human strains, and the genogroups are further subdivided into genotypes based upon analyses of the amino acid sequence of the major capsid protein, VP1. Norovirus infection causes gastroenteritis characterized by the symptoms of vomiting and diarrhoea (Atmar and Estes, 2006). The prevalence of vomiting along with the short incubation period (1–2 days) and short clinical illness (1–3 days) has been used epidemiologically to identify probable outbreaks of NoV-associated

Adenovirus

70 nm Complex

dsDNA 35 900 bases Pair

3–10 days Gastroenteritis All year Young children

Size Capsid

Genome Size Genome

Incubation Illness Season Age

1–2 days Gastroenteritis All year Young adults

ssRNA 8251 bases

27–32 nm Icosahedral

Aichi virus

3–5 days Gastroenteritis Winter Children

ssRNA 6797 bases

27–32 nm Icosahedral

Astrovirus

Characteristics of the the main enteric viruses

Name

Table 18.1

2–3 days Gastroenteritis Winter All ages

ssRNA 7642 bases

27–32 nm Icosahedral

Norovirus

7–30 days Diverse Summer All ages

ssRNA 7200 bases

20–30 nm Icosahedral

Enterovirus

70 nm Triple layer Icosahedral dsRNA 11 genes (3302 to 667 bp) 3 days Gastroenteritis Winter Young children

Rotavirus

Up to 6 weeks Hepatitis All year All ages

ssRNA 7478 bases

27–32 nm Icosahedral

Hepatitis A virus

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gastroenteritis (Kaplan et al., 1982; Turcios et al., 2006). The virus is stable in the environment, and the infectious dose is estimated to be less than 20 virions (Teunis et al., 2008). Noroviruses are the major cause of epidemic non-bacterial gastroenteritis worldwide and have been identified as the cause of 73 % to more than 95 % of outbreaks (Atmar and Estes, 2006). Mead and colleagues estimated that there are 23 million NoV infections per year in the USA, and these viruses constitute 60 % of the illness burden caused by known enteric pathogens (Mead et al., 1999). Although some studies provide a good indication of the substantial illness burden that results from NoV infection, the true extent of disease may still not be fully known (Patel et al., 2008). Rotavirus Rotaviruses are the main etiological agent of viral gastroenteritis in infants and young children. They constitute a genus in the Reoviridae family (Estes and Kapikian, 2007). In developing countries the burden of rotavirus disease in children under five years of age has been estimated to be over 125 million cases annually, of which 18 million are severe cases (nearly half a million deaths) (Oh et al., 2003; Parashar et al., 1998). In the developed world, rotaviruses remain an important cause of morbidity and of hospitalization in young children, and they are also increasingly recognized as a cause of infectious diarrhea in adults as well (Anderson and Weber, 2004). Astrovirus Astroviruses are classified in genus Mamastrovirus (human and animal strains) within the family Astroviridae (Mendez and Arias, 2007). In most species astrovirus are found in association with gastroenteritis, although other manifestations have been described in avian species (Mendez and Arias, 2007). Enterovirus (EV) Human enteroviruses belong to the genus Enterovirus in the Picornaviridae family. Poliovirus is a species within the Enterovirus genus and its three serotypes each can cause a devastating neurological disease for which, despite vaccination campaigns, the goal of global eradication has not yet been completed. Other enterovirus species and serotypes cause a variety of other clinical syndromes, including respiratory infections, haemorrhagic conjunctivitis, and myocarditis (Pallansch and Roos, 2007). Aichi virus Aichi virus belongs to the Kobuvirus genus in the Picornaviridae family. Aichi virus was identified in several other outbreaks in Japan as a cause of gastroenteritis associated with shellfish consumption (Yamashita et al., 2000) and was recently recognized in an oyster-related outbreak in Europe in 2006 (Le Guyader et al., 2008).

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Adenovirus (ADV) Human adenoviruses belong to the genus Mastadenovirus in the Adenoviridae family (Wold and Horwitz, 2007). There are six species of human adenoviruses, and members of species F (formerly called subgroup F), consist of two serotypes, Ad40 and Ad41 causing diarrhoea and also referred to as enteric adenoviruses. Hepatitis E virus (HEV) Hepatitis E virus is a member of the Hepeviridae family. It is the primary cause of enterically transmitted non-A non-B hepatitis in tropical and subtropical developing countries, and it has an associated mortality rate of up to 20 % in pregnant women (Lu et al., 2006).

18.2.2 Animal viruses, potential zoonotic viruses A number of enteric viruses inducing gastroenteritis in humans have also been identified in animals. The predominant animals involved are porcine and bovine species. These observations raise the possibility that zoonotic transmission may occur. Such transmission has been best demonstrated to occur among rotaviruses. Bovine–human and porcine–human group A rotavirus reassortants have been identified in India, Italy, Slovenia, and Brazil, and a porcine group C rotavirus was identified in a child in Brazil (Estes and Kapikian, 2007; Gabbay et al., 2008; Martella et al., 2008; Steyer et al., 2008). There is also the potential for zoonotic transmission of noroviruses. Several porcine genogroup II norovirus strains have been characterized and, based upon the sequences of their major capsid protein (VP1), they have been classified in genotypes that are distinct from those of human strains (Wang et al., 2005; Zheng et al., 2006). Bovine strains are even less related to human strains and have been classified in a separate genogroup (genogroup III) (Oliver et al., 2003). However, the ability of human strains to replicate in pigs and cattle (Cheetham et al., 2006; Souza et al., 2008) has been demonstrated experimentally, and a recent study from Canada reported the presence of human-like GII.4 norovirus strains in pig feces and retail beef (Mattison et al., 2007). To date, no human infections with animal norovirus strains have been reported, but the simultaneous detection of human and animal enteric calciviruses in oysters samples collected from the markets suggests the potential for such infections to occur if humans can be infected with animal strains (Costantini et al., 2006; Symes et al., 2007). The significance of such an infection is the potential for the emergence of new strains through recombination events, as such events appear to be common among human strains (Bull et al., 2007). Enteroviruses are also important pathogens for cattle and swine, and high concentrations of these viruses may be detected in surface waters (Fong and Lipp, 2005). As for norovirus, shellfish contamination with

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animal and human enterovirus strains was demonstrated in oyster samples collected in an area impacted with both human and bovine sewage (Dubois et al., 2004). The stringency of the host specificity among human and bovine strains suggests that they can be good indicators for identifying human and non-human sources of fecal contamination of natural waters (Fong and Lipp, 2005). Hepatitis E virus is a potential emerging pathogen, and evidence of its global spread as a cause of disease in humans is increasing. Clear demonstration of sequence homology between strains detected among swine or other domestic animals has now been made in several countries (Renou et al., 2007; Rutjes et al., 2007; Kaci et al., 2008; Ward et al., 2008). However, transmission seems to occur primarily through direct contact with infected animal or food consumption (e.g., pig liver) (Renou et al., 2007; Lewis et al., 2008). Shellfish consumption has been reported as a risk factor for hepatitis E virus infection, but additional studies are needed to establish this link (Cacopardo et al., 1997; Koizumi et al., 2004).

18.2.3

Examples of outbreaks with a special emphasis on the source of virus contamination Despite the high diversity of viruses that are introduced into the environment by human fecal pollution, only a few have been recognized to cause disease in association with consumption of contaminated shellfish. Potential explanations for this observation include a lack of susceptibility of the persons consuming the shellfish to these viruses (i.e., pre-existing immunity), a requirement for exposure to higher doses than are present in the shellfish to establish infection, and a lack of recognition of disease either through under-reporting or the unavailability of sensitive diagnostic assays. The instigation of regulations to specify acceptable levels of bacterial enteric pathogens in shellfish tissues or in shellfish growing waters in Europe (European regulation, 91/492/EC) and the USA (National Shellfish Sanitation Program) and improvements in sewage waste treatment procedures were followed by the virtual elimination of shellfish-associated outbreaks of typhoid fever and cholera in the USA (Richards, 1985; Rippey, 1994). However, as shellfish-associated bacterial infection declined, outbreaks of non-bacterial gastroenteritis and infectious hepatitis were described in association with shellfish consumption (Butt et al., 2004). In many instances, the shellfish and shellfish-growing waters met regulatory criteria for fecal bacterial levels, suggesting an accidental contamination event rather than exposure to a continuous sewage discharge. The most common route for accidental contamination is after heavy rainfall, leading to overflow and release of untreated sewage into the aquatic environment. As mentioned above, untreated sewage is likely to be heavily contaminated by enteric viruses. Frequently the source of accidental events for shellfish contamination cannot be traced, but a number of reports have

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been able to elucidate the cause of human fecal pollution. For example, several clusters of gastroenteritis occurred in six states in the USA and were linked to oyster consumption. The oysters implicated in the outbreaks were all traced back to a single harvest area, and the epidemiological investigation showed that the outbreak resulted from disposal of human diarrheal stool from a single ill individual directly into the waters over the shellfish bed (Kohn et al., 1995). Another example was a large oyster-associated gastroenteritis outbreak that affected approximately 2000 persons during the summer of 1978 in Australia (Murphy et al., 1979). This outbreak was linked to sewage contamination of the oyster harvesting area near Sydney following a heavy rainfall. Runoff from heavy spring rains was also suspected to be responsible for 103 clusters of norovirus gastroenteritis involving more than 1000 persons after clam or oyster consumption in New York State in 1982 (Morse et al., 1986). In the south of France, heavy rainfall and sewage treatment plant failure were twice implicated as the cause of large gastroenteritis outbreaks due to consumption of oysters harvested from a single lagoon (Le Guyader et al., 2006a, 2008). The long incubation period of hepatitis A complicates linkage of this agent to particular food exposure incidents. However, linkage is still possible during large incidents. In 1988 in Shanghai, China, almost 300 000 hepatitis A cases were traced to the consumption of clams harvested from a sewage-polluted area (Halliday et al., 1991). A sizeable hepatitis A outbreak in the USA in 1973 was linked to Louisiana oysters. The harvesting areas were flooded by the Mississippi River, and there was evidence of sewage contamination based upon elevated fecal coliform levels that led to closure of the oyster beds. Subsequently, the oyster beds were re-opened to harvesting, but apparently the hepatitis A virus was retained in shellfish for at least six weeks following the contamination event. At the time of harvesting, oysters were fully compliant with the US sanitation program standard but still contaminated with the virus (Mackowiak et al., 1976). Many other hepatitis A outbreaks linked to bivalve shellfish have been reported, but the initiating fecal contamination event has been generally difficult to identify due to the protracted incubation period for this disease (Bosch et al., 2001). In summary, where a cause is ascribed, most contamination accidents are linked to failures or bypassing of treatment processes, often due to heavy rainfall (Fig. 18.1). The problem is that heavy rain causes the storage capacity of the sewage treatment plant to be exceeded. In combined sewer and rainfall systems this leads to storm spills. Such discharges result in the release of untreated effluent heavily contaminated with microorganisms. This may be particularly true during the ‘first flush’. Other causes are flooding of harvest areas with contaminated river water and disposal of feces from infected individuals on boats. It is interesting to note that although the efficiency of sewage treatment processes at fully removing viruses from effluents can be questioned, such effluents are not usually associated with

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Waste water treatment plant

Catchment area agriculture and urban activities

Shellfish farm wastes Spreading liquid manures

Fig. 18.1 Possible sources of contamination for shellfish growing in coastal area. (Source: Ifremer, www.ifremer.fr/envlit/)

bivalve mollusc disease incidents. However, because most enteric viruses retain their viability in the environment, they can persist for a much longer time in the marine environment than bacterial indicators – up to weeks or months (Wait and Sobsey, 2001; Lipp et al., 2002; Griffin et al., 2003). In addition, viral particles have been noted to persist for months in shellfish tissues, either via ionic binding or specific attachment (Burkhardt and Calci, 2000; Loisy et al., 2005a, Le Guyader et al., 2006b).

18.3 Methods 18.3.1 Rapid review Viruses are present in shellfish in very low numbers. Nevertheless, they are present in sufficient quantities to pose a health risk as presented above. This low level of contamination has made it necessary to develop highly sensitive viral extraction methods to ensure virus recovery from shellfish tissues. The observation that viruses are concentrated in digestive diverticulum tissues led to the development of a method that represented a major step in the improvement of extraction methodologies (Metcalf et al., 1980; Atmar et al., 1995). This observation was subsequently confirmed by detection of HAV (Romalde et al., 1994) as well as through the tissue-specific quantification of infectious enteric adenoviruses and rotaviruses in mussels previously contaminated by bioaccumulation of such viruses and similarly of Norwalk

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virus in oysters and clams (Abad et al., 1997; Schwab et al., 1998). Analysis of digestive tissues provides several advantages, including increased sensitivity, decreased processing time and decreased interference with reverse transcriptase-polymerase chain reaction (RT-PCR) by inhibitory substances (Atmar et al., 1995). Focusing the analysis of shellfish on the digestive tissues enhances assay performance by eliminating tissues (i.e. adductor muscle) that are rich in inhibitors (Atmar et al., 1995). The digestive tissues represent about one tenth of the total animal weight for oysters and mussels. With the exception of small species, such as clams or cockles, in which dissection may be technically difficult, most recent methods are based on dissected tissues and thus will be discussed here. Extraction of enteric viruses from shellfish is based on several steps: virus elution from shellfish tissues, recovery of viral particles, and then virus concentration. The weight analyzed generally ranges from 1.5–2 g of digestive tissues. Some recent methods propose larger weights for the first step but thereafter analyze only a fraction of the extracts (Boxman et al., 2006). Viruses are eluted from shellfish digestive tissues using various buffers (e.g. chloroform–butanol or glycine) before being concentrated either by polyethylene glycol precipitation or ultracentrifugation (Atmar et al., 1995; Nishida et al., 2003; Myrmel et al., 2004; Milne et al., 2007). Direct lysis of virus particles has also been used, including methods utilizing proteinase K or Trizol® to destroy shellfish tissues or Zirconia beads and a denaturing buffer for virus and/or nucleic acid elution (Jothikumar et al., 2005; LodderVerschoor et al., 2005; Kittigul et al., 2008; Lowther et al., 2008; Umesha et al., 2008). In addition to the in-house protocols that have been used for nucleic acids extraction and purification (Le Guyader and Atmar, 2007), a number of commercial kits can also be successfully applied to accomplish this task. Advantages of the commercial kits used for nucleic acid purification include their reliability, reproducibility and ease of use. Most of these kits are based on guanidium lysis followed by capture of nucleic acids on columns, beads or silica (Nishida et al., 2003; Lodder-Verschoor et al., 2005; Costafreda et al., 2006; de Roda-Husmann et al., 2007; Kingsley, 2007; Milne et al., 2007; Fukuda et al., 2008; Nenonen et al., 2008; Umesha et al., 2008). One of the goals of extraction methods is to remove inhibitors of the RT and PCR reactions sufficiently to allow detection of viral nucleic acids. Polysaccharides present in shellfish tissue are at least one substance that can inhibit the PCR reaction (Atmar et al., 1993). Elimination of inhibitors is difficult to evaluate and, depending on the time of the year and shellfish life, different compounds may be present (Di Girolamo et al., 1977; Burkhardt and Calci, 2000). Internal amplification control standards are used to detect the presence of significant sample inhibition, and the amount and frequency of sample inhibition has varied depending upon the shellfish tissue being analyzed (Atmar et al.,1995; Schwab et al., 1998; Le Guyader et al., 2000). Recent advances in food virology re-enforce the need for

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harmonization of methods as well as addressing quality assurance and quality control (Pinto and Bosch, 2008). The addition of an external virus to a shellfish sample has been proposed as a control to evaluate the extraction efficiency of molecular virus detection methods (Costafreda et al., 2006; Nishida et al., 2007; Lowther et al., 2008). An ideal candidate would have the following properties: (i) it would be an encapsidated RNA virus with properties similar to the enteric viruses contaminating shellfish; (ii) it would normally not be present in field samples (thus RNA phages may be problematic); and (iii) it would be nonpathogenic. Based on these considerations, Costafreda et al. (2006) proposed to use a mengovirus strain MC0 as a control for extraction efficiency. Mengovirus, a Picornaviridae family member, was initially proposed as a control in validation studies of HAV removal in blood products manufacturing by several agencies such as the European Agency for the Evaluation of Medicinal products and the American Food and Drug Administration (Pinto and Bosch, 2008). Advantages of mengovirus are that it is unlikely to contaminate shellfish naturally, it is non-pathogenic for humans and it can be grown in cell culture. The use of a single extraction control for different enteric viruses that may be detected in shellfish or other types of food is also considered to be important for method standardization (European working group CEN/Tag4) and for comparisons between different laboratories. Since the most important shellfish-borne viral pathogens (enteric hepatitis viruses A and E and noroviruses) are either non-culturable or grow only poorly in cell culture, RT-PCR and real-time RT-PCR have become the methods of choice for their detection. In addition to the problems posed by the presence of inhibitory substances in samples, there are other difficulties encountered when molecular analyses are performed for the detection of viruses in shellfish samples. These include low virus concentrations in the sample and genomic diversity of the contaminating viruses. The extraction– concentration procedure is not virus-specific, allowing the nucleic acid of several viruses to be extracted simultaneously. RT-PCR must be performed under stringent conditions and confirmed by hybridization. A number of reviews on RT-PCR methods are now available that address issues related to these methods, including assay specificity and sensitivity (Le Guyader and Atmar, 2007; Wyn-Jones, 2007). Real-time PCR assays allow the combination of RT, PCR and confirmatory hybridization assays in a single well, and these assays are now being used to detect enteric viruses in shellfish (Nishida et al., 2003; Jothikumar et al., 2005; Loisy et al., 2005b; Costafreda et al., 2006; Lowther et al., 2008; Le Guyader et al., 2009). This technology significantly shortens the time needed for virus detection by removing the need for gel electrophoresis and the additional hybridization step. When extraction, RT and PCR efficiencies are measured, virus quantification in the sample can be estimated (Costafreda et al., 2006; Le Guyader et al., 2009). The efficiency of the virus nucleic acids extraction is evaluated through the use of a model virus (as described above)

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while the efficiency of the RT-PCR reaction must be tracked by amplification of a RNA standard using the same combination of primers and probe used for virus detection (Pinto and Bosch, 2008). Such internal RNA controls have also been used for the detection of amplification inhibitors in qualitative assays (Schwab et al., 1998; Le Guyader et al., 2003).

18.3.2 Quantification The development of quantitative molecular assays for the analysis of shellfish has allowed estimates of the level of virus in naturally contaminated shellfish. However, relatively few data are currently available in this area. One report estimated the amount of HAV in coquina clams implicated in an outbreak to be between 7.5 × 103 to 7.9 × 105 genome copies per g of digestive tissues (Coastafreda et al., 2006). Another recent report used a semi-quantitative approach for norovirus detection to compare the levels of virus contamination between sites without describing the amount of shellfish analyzed (Lowther et al., 2008). Japanese investigators estimated the levels of norovirus contamination in oysters collected from two areas ranged from <100 copies to 7.9 × 103 genome copies per g of digestive tissues in two consecutive studies (Nishida et al., 2003, 2007). These estimates did not include an adjustment for extraction efficiency, as proposed by Costafreda et al. (2006). Similar data have been obtained for the levels of norovirus contamination in oysters from France (Le Guyader et al., 2008). Quantitative estimates of norovirus contamination in shellfish implicated in outbreaks are even more uncommon. By using most probable number (MPN) PCR or real-time RT-PCR, about 100 copies of genome/g of digestive tissues were found in oysters in France (Le Guyader et al., 2003, 2006a, 2008). This level of contamination is sufficient to cause human infection based upon the low infectious dose of the Norwalk virus (used as a prototype for norovirus) in a volunteer study (Teunis et al., 2008). The availability of quantitative assays offers the potential to perform risk assessments associated with the consumption of virus-contaminated shellfish.

18.4 Input and flux 18.4.1 Seasonal outbreaks The regular and predictable pattern of seasonal outbreaks dominates the epidemiology of many exclusively human pathogens (Dowell, 2001). The seasonal infection may vary between different pathogens, but the timing and characteristics of the annual outbreak of a single pathogen are remarkably consistent from year to year. It was shown that latitude has a clear influence on the timing and magnitude of outbreaks of rotavirus infections and poliomyelitis (Dowell, 2001). Data collected from different studies worldwide have also shown a clear seasonality for norovirus outbreaks. A

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clear peak of norovirus outbreaks occurs during cold weather months on several continents, with lack of UV, cold temperature, frequent runoff being some of the possible explanations of extensive transmission (Mounts et al., 2000). However, noroviruses continue to circulate endemically throughout the year and, although there is the theoretic possibility of zoonotic spread, currently there is no direct evidence of the existence of a reservoir for reintroduction into the human population (Lopman et al., 2008). Gastroenteritis from all causes predominates during colder months of the year, but it does not disappear during summer (Dowell 2001; Lopman et al., 2008). It is now evident that some viruses may be detected all year long, either in sporadic cases of illness or in untreated sewage (da Silva et al., 2007; Patel et al., 2008). The amount of virus shed by ill people may be very high (Atmar et al., 2008) (Table 18.2). Importantly post-symptomatic virus shedding may continue for some time, as demonstrated for enterovirus, hepatitis A, and norovirus (Hollinger and Emerson, 2007; Pallansch and Roos, 2007; Atmar et al., 2008). For example, norovirus shedding in an experimental human infection model lasted a median of 28 days, with a range from 13 to 56 days, and most subjects were no longer symptomatic by day 4 (Atmar et al., 2008). These data suggest that the impact of continued virus shedding from ill and post-symptomatic patients on sewage may be very significant. Enterically-transmitted hepatitis viruses are distributed worldwide with no clear season pattern. The endemicity in different parts of the world is closely linked to the level of socioeconomic development. In developing countries with poor sanitary and living conditions, transmission rates of HAV are high with a large number of subclinical cases shedding high concentration of viruses into the environment (Aggarwal and Naik, 2008). In contrast, the levels of HAV endemicity and transmission rates are much

Table 18.2 Titers of enteric viruses in stool specimens

Virus

HAV HEV Rotavirus NoV

SaV

Range of virus concentrations (RNA copies/g) Minimal

Maximal

3.4 × 105 <102 103 1.4 × 107 108 2.2 × 104 9 × 107 5 × 108 1.3 × 105

5.6 × 1011 107 1010 4.2 × 109 1012 1011 6 × 1010 1.6 × 1012 2.1 × 1010

Reference

Costafreda et al., 2006 Takahashi et al., 2007 Freeman et al., 2008 Ludwig et al., 2008 Lee et al., 2007 Chan et al., 2006 Kageyama et al., 2003 Atmar et al., 2008 Oka et al., 2006

HAV = hepatitis A virus, HEV = hepatitis E virus, NoV = norovirus, SaV = Sapovirus.

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lower in developed countries, and detection of viruses in the environment is less common so that introduction into the environment may be limited in time and localization (Pinto and Saiz, 2007).

18.4.2 Animal output As noted earlier, some enteric viruses can also be detected in animals. These animal infections can lead to contamination of the environment and can confound the identification of human strains, depending on the detection methods that are used. For example, rotavirus is a pathogen for young calves and pigs. Calves can excrete very high levels of rotavirus (up to 1011 viral particles/g of stool) for a week or longer. Most of the strains implicated in bovine disease belong to group A rotavirus, the most common cause of human disease (Dhama et al., 2009). Group A rotaviruses have also been detected in equine, swine, ovines, and caprines, and pet dogs and cats may also be infected with these viruses (Dhama et al., 2009). Norovirus also infect other species including swine and cattle. The prevalence of norovirus infection in these animals has varied in studies from different countries. One in 17 porcine fecal samples from Hungary was found positive for norovirus (Reuter et al., 2006) while in Italy none of the 209 stool samples was positive for norovirus (Martella et al., 2008). In contrast, 2 % of pigs’ stool samples in The Netherlands were NoV-positive (van der Poel et al., 2000), and the prevalence of porcine norovirus on farms in the USA was reported to range between 3 and 40 % (Wang et al., 2006). In Canada, about 25 % of swine fecal samples were found positive while only 1.6 % of bovine fecal samples contained noroviruses (Mattison et al., 2007). Bovine norovirus have also been detected in 9.1 % of diarrheic calves in Korea (Park et al., 2008). Other studies have identified bovine noroviruses in 72 % of veal calves from the USA (Smiley et al., 2003), in approximately a third of veal calf farm samples from The Netherlands (van der Poel et al., 2000) and in 8 % of calf diarrhoea samples from the UK (Oliver et al., 2003).

18.4.3 Enteric viruses in sewage and rivers A review of available information in the literature on the fluxes of human enteric viruses discharged into the environment clearly shows that the presence of pathogens in waters reflects the epidemiology of these viruses in the human population (Myrmel et al., 2006; Rohayem et al., 2006; da Silva et al., 2007; Schwab, 2007). The lack of reliable quantification methods (such as cell culture) for many of the enteric viruses has precluded accurate quantitative analyses of these viruses from environmental samples. Although the development of quantitative molecular assays now offers the promise to generate such data, they can be impacted by many factors such as virus concentration efficiency and amplification efficiency which many investigators fail to consider. In addition, molecular assays cannot evaluate virus

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infectivity, since they detect both live and inactivated virions. Consequently the molecular assay may lead to an overestimate of the amount of infectious virus present in a sample (Richards, 1999; Gerba, 2007). The concentration of noroviruses measured in natural waters and raw sewage has shown both regional and seasonal variability. In The Netherlands, noroviruses were detected on average at 2 × 105 PCR detectable units (PDU)/liter of raw sewage, with only one sample being negative (September) (Lodder and de Roda Husman, 2005). In the UK norovirus concentrations ranged from zero to 1.7 × 107 cDNA copies/L and in Germany approximately 106 copies/L were reported (Laverick et al., 2004; Pusch et al., 2005). Haramoto et al. (2006) described seasonal differences in norovirus concentrations in untreated sewage, from a peak of 1.9 × 106 RNA copies/L in December to a low of 2.4 × 103 copies/L in September. Similarly, da Silva et al. (2007) and Katayama et al. (2008) reported higher norovirus concentrations (about 105 genomes copies/L for both studies) in untreated sewage during winter months compared to spring or summer. All of these studies found both genogroup I and genogroup II norovirus strains. Genogroup II strains cause the large majority (80 to 90 % in most studies) of clinical cases seen by physicians (Atmar and Estes, 2006; Lopman et al., 2008). The consistent detection of genogroup I strains in raw sewage demonstrates that these viruses are circulating in the population (van der Berg et al., 2005; Haramoto et al., 2006; da Silva et al., 2007; Katayama et al., 2008). Rotaviruses and enteroviruses are also detected in raw sewage in approximately the same range of concentrations (Lodder and de Roda Husman, 2005; Pusch et al., 2005). Astrovirus may be detected as higher concentrations (up to 108 genome copies/L) (Le Cann et al., 2004; Pusch et al., 2005). The data for wastewater samples may reflect the presence of strains that circulate more widely in the population and as such may be a powerful and useful tool for public health surveillance (Hovi, 2006). Despite treatment of sewage to remove bacterial and viral pathogens, treated wasterwater contains enteric viruses that can potentially contaminate the environment (Table 18.3). Concentrations of hundreds to thousands of genome copies per liter of treated wastewater can be detected, and seasonal variability occurs similar to that seen for untreated sewage. Several studies have observed a higher frequency of genogroup I (GI) norovirus strains in treated effluent compared to genogroup II strains (van der Berg et al., 2005; Myrmel et al., 2006; La Rosa et al., 2007; da Silva et al., 2007). Haramoto et al. (2006) even reported that all treated sewage samples were positive for GI. The reason why GI noroviruses are more resistant to inactivation and removal during the sewage treatment process is not clear and should be studied in the future. Some differences in capsid proteins or binding properties may be one interesting hypothesis to analyze. That GI noroviruses are more often implicated in shellfish- and water-related outbreaks than GII may also be evidence in favor of higher resistance to

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Table 18.3 Average concentration of human enteric viruses detected in treated wastewater samples Virus

Positive sample (%)

Average concentration Reference PCR units/L

PFU/L

HAV

100

9 × 101–3.5 × 103

Brooks et al., 2005

NoV

93 7 53 100 100

<101–3 × 104 0–1.6 × 105 1.8 × 104–9.7 × 105 8.7 × 101–2.9 × 103 6 × 102–2.4 × 104

38 100

2.1 × 102–6 × 106 6–6.4 × 103

Van der Berg et al., 2005 Laverick et al., 2004 Pusch et al., 2005 Katayama et al., 2008 Lodder and de Roda Husman, 2005 Da Silva et al., 2007 Haramoto et al., 2006

58

0.5–1.8 × 102

AV

40 50 82

3.7 × 10 –1.2 × 10 1.1 × 103–6.2 × 105 8 × 102–5 × 105

EV

90 100

101–5.4 × 104

100

4 × 100–1.7 × 102

AdV

100

9 × 102–5 × 104

Reo

90

SaV

3

Haramoto et al., 2008 5

Pusch et al., 2005 El-Senousy et al., 2007 Le Cann et al., 2004 5–92

Schvoerer et al., 2001 Lodder and de Roda Husman, 2005 Katayama et al., 2008 Katayama et al., 2008

1–233 (MPN)

Sedmak et al., 2005

AdV = adenovirus, AV = astrovirus, EV = enterovirus, HAV = hepatitis A virus, MPN = most probable number, NoV = norovirus, PCR = polymerase chain reaction, PFU = plaque-forming unit, Reo = reovirus, SaV = sapovirus.

inactivation in the environment (Lopman et al., 2004; Blanton et al., 2006). Overall noroviruses are found in concentrations ranging from undetectable to 106 genome copies/L of treated wastewater (Table 18.3). The efficacy of different wastewater treatments for norovirus removal from wastewaters has varied from 0.5 to 4 log10 (Table 18.4). One of the most promising approaches utilizes ultrafiltration, such as membrane bioreactor (MBR) technology operating with biological treatment as well as physical separation of particles. Sano et al. (2006) reported complete elimination of noroviruses from sewage sludge and treated water, with greater than 4 log10 reduction values by ultrafiltration. The use of microfilters with pore sizes as large as 0.45 microns also effectively removed viruses, with log10 reduction values of more than 3.9 for sewage sludge and 2.9 for treated water. The microfilter membranes have permeability that is much larger than the diameter of viruses but can be effective since the viruses are embedded in or adsorbed to solids. Nevertheless, viral particles may be

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Table 18.4 Efficiency of different waste water treatment plant to remove norovirus log10 reduction value

Reference

Treatment

Genogroups

Physical treatment

GI GII GI GII GI & GII GI & GII GI & GII

1.82 (±0.61) 2.74 (±1.10) 0.45 (±0.49) 0.95 (±1.80) 0.50 (±0.84) 1.14 (±0.88) 2–2.7

Haramoto et al., 2006.

GI GII GI & GII

0–31 0–51 2.9–3.5

Da Silva et al., 2007

Chemical treatment (chlorination) Tertiary filtration Membrane bioreactor Activated sludge treatment Wastewater stabilized Pounds Membrane separation 1

Haramoto et al., 2006. Ottoson et al., 2006 Ottoson et al., 2006 van der Berg et al., 2005

Sano et al., 2006

Depending on the residence time.

detected in effluents after treatment with microfilters with larger pore sized, implying that such membranes are not an absolute barrier for the passage of viruses (Ueki et al., 2005; da Silva et al., 2007). Another approach is to utilize techniques that enhance the effectiveness of ultraviolet disinfection. Norovirus appears to be quite resistant to UV radiation (Hijnen et al., 2006; Ottoson et al., 2006) but genome destruction may be achieved using a highly photocatalytic material TiO2 (Titanium dioxide), in conjunction with UV (Kato et al., 2005). Addition of low concentration of TiO2U particles also increased the rate of inactivation of phage MS2, although the effect on murine norovirus (a surrogate for human noroviruses) was less (Lee et al., 2008).

18.4.4 Flux calculation from sewage Information on virus sources and virus levels in raw or treated wastewaters can be used to estimate the quantities of virus discharged into the environment. Relatively few data are available to allow the calculation of ‘virus base-flow’ or ‘event-flow’ discharges in river or in estuaries. Nevertheless recent data suggest that during non-epidemic periods less than 103–104 genome copies/L of norovirus are present in treated wastewaters. During the epidemic period (winter) the concentration is probably 100- to 1000fold higher. The rate of reduction of virus concentrations through sewage treatment processes is almost constant and independent of the concentration of viruses of the effluent (Myrmel et al., 2006; da Silva et al. 2007; Katayama et al., 2008). These data imply that viruses are discharged into environmental waters with a seasonal profile and raise questions about the frequency and duration of such peaks and the importance and impact of

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storm events that result in failure in wastewater treatment during a high flow and epidemic period. In the absence of precise information, calculations from epidemiological data suggest that 106 norovirus fluxes can be expected from a town of 60 000 population-equivalent when winter outbreaks are occurring (Pommepuy et al., 2004). Many environmental factors can have an impact, including currents, estuaries, and tide (Pommepuy et al., 2005). The persistence of viruses in the environment must be considered even in non-epidemic periods when risks of infection associated with shellfish consumption are estimated (Riou et al., 2007).

18.4.5 Surface water Viruses may be discharged into surface water from different sources, such as septic tank or sewer lines leaking, wastewater irrigation or sludge disposal. In a recent review, Gerba (2007) identified studies describing the occurrence of enteric viruses in surface freshwater and demonstrated that in all countries a number of different viruses can be detected. However, it is clear that data are still limited and information on virus concentration and infectivity is often lacking. Noroviruses are relatively resistant to inactivation compared to other enteric viruses, possibly explaining their predominant role in food-related outbreaks. Detection of infectious norovirus is not possible due to the lack of availability of an in vitro cell culture system or small animal model. Other culturable virus surrogates have been proposed to model virus persistence in environmental samples. Bae and Schwab (2008) used a murine norovirus strain to evaluate virus persistence in different types of surface waters compared with MS2 phage, poliovirus, and feline calicivirus in an infectivity assay. The murine norovirus infectivity persisted longer than feline calicivirus and as long as the other human norovirus surrogates. In addition, reduction of murine norovirus nucleic acids as measured by real-time RT-PCR was similar to that of a human norovirus strain in surface waters (0.08 and 0.09 log10/day, respectively) and in groundwaters (0.01 and 0.00 log10/day, respectively) (Bae and Schwab, 2008). Noroviruses are detected in surface waters less frequently than in wastewaters, probably due in part to dilution or sedimentation mechanisms during transport in the rivers. The reported frequency of norovirus detection has varied between studies from 5.8 % in a Brazilian river to 53 % in Japan (Haramoto et al., 2005; Miagostovich et al., 2008). Peak norovirus concentrations of up to 104 genome copies/liter of surface waters have been reported, suggesting that the risk of infection can vary quantitatively as well as qualitatively (Lodder and de Roda Husman, 2005; Westrell et al., 2006). The other human enteric viruses can also be detected to various concentrations as noted in a recent review by Gerba (2007). For example, hepatitis A virus is endemic in some Italian areas, and viral RNA has been detected at concentrations ranging from 75 to 730 genomes/L in Venetian canals (Rose et al., 2006).

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Fluctuations in virus concentrations may result from a combination of different factors, such as sewer overflows leading to untreated water being discharged in rivers and runoff from pastures. Detection of short-term fluctuations is important to predict the risk for water contamination, but few data are available. A two-year study on the Meuse river in The Netherlands found mean norovirus values to range from 4–4900 genome copies/L (Westrell et al., 2006). Within the winter seasonal peak, quantitative and qualitative fluctuations of norovirus presence in the river waters occurred over varying lengths of time, and the highest concentrations could lead to significant contamination of drinking waters if water treatment measures failed. There are models to study the impact of river and wastewater discharges on coastal waters and shellfish quality (Pommepuy et al., 2005; Riou et al., 2007). However, these models, even though they appear to be useful, can be improved with additional information, including a larger input database, data on short-term fluctuation frequencies, and a better understanding of viral behavior and persistence in the environment (e.g., in sediments and shellfish).

18.4.6 Potential indicators: phages, animal markers The current regulatory controls in most countries rely on fecal pollution indicator organisms, such as E.coli, to assess microbiological hazards. Methods to detect E.coli are inexpensive, standardized, and widely available (European regulation, 91/492/EC or United States National Shellfish Sanitation Program). However, it is now clear that fecal coliforms inadequately reflect the presence of human enteric viral contaminants (Butt et al., 2004). As no standardized methods are yet available for human enteric viruses in shellfish, a number of workers have proposed alternative indicators for better assessment of viral contamination. Various species of bacteriophages are some of the most frequently proposed indicators because of their physical and genomic similarity to human enteric viruses, their abundance in sewage effluents, and the availability of simple methods for their detection. Male-specific RNA (F-RNA) bacteriophages have been proposed as a candidate indicator for water pollution (Havelaar, 1993). However, although F-RNA bacteriophages share physical and genomic properties with the human enteric viruses, their distribution (similar to fecal coliform bacteria) is not restricted to human effluents. Studies related to bivalve shellfish contamination by human enteric viruses and phages have shown variable results (Table 18.5). The F-RNA bacteriophage appears to perform best in sites that are consistently polluted. A number of studies have shown that F-RNA bacteriophages were not detected when human enteric viruses were present. For example, data collected in an European survey demonstrated that shellfish collected from southern Europe were negative for phages but contained human viruses (Formiga-Cruz et al., 2003). Similarly, a study from

Oysters Mussels Mussels Mussels

Oysters Oysters Oysters Clams

UK Italy Norway Spain

Netherlands India

A A A A B B A B B

Class 134 36 681 7 36 11 22 100 74

ⱅ of samples

Note: phages and viruses expressed in %.

Shellfish 50 8 23.8 14 22 36 100 57 73

Phages 62 – 6.8 – – – 0 0 0

Norovirus – 36 0 25 9 27 37 45

0 22 18 0 0 0

Enterovirus

– 14

Hepatitis A virus

Human enteric viruses and phages detection in shellfish samples

Country

Table 18.5

Lodder-Verschoor et al., 2005 Umesha et al., 2008

Doré et al., 2000 Croci et al., 2000 Myrmel et al., 2004 Munian-Mujika et al., 2003

References

562

New technologies in aquaculture

France reported that the detection of F-RNA bacteriophages failed to predict the presence of human viruses (Miossec et al., 2001). A correlation was found between noroviruses and phage contamination of mussels in cold seawater, but more than half of the norovirus-positive samples were negative for F-RNA phages, and a positive F-RNA phage result was less than twice as common in samples with norovirus than in those without norovirus, raising the question as to whether to use F-RNA as an indicator (Myrmel et al., 2004). A one-year study in The Netherlands found that phages were present in 67 % of oyster samples analyzed, but no pathogenic viruses such as norovirus or hepatitis A virus were identified in the shellfish (LodderVerschoor et al., 2005). A discrepancy level of 55 % was observed between the detection of hepatitis A virus and F-RNA phages in association with a large outbreak of hepatitis A (184 human cases) from imported shellfish in the East of Spain in 1999 (Bosch et al., 2001). Phages may be used to classify areas for sanitary safety, but they have been unreliable indicators of viruscontamination of shellfish (Hernroth et al., 2002).

18.5 Strategies for reducing contamination 18.5.1 Resistance of viruses Many human enteric viruses are resistant to inactivation or removal from the environment. Nenonen et al. (2008) traced norovirus strains in the environment and were able to identify outbreak strains in mussels exposed to sewage three months after the outbreak. Similarly, Ueki et al. (2005) were able to follow clinical strains from stool to sewage, to river waters and ultimately to oyster samples. Similar results were reported more than 25 years ago for a number of picornaviruses (enterovirus, poliovirus and hepatitis A virus) (Metcalf, 1982). A characteristic of these enteric viruses is their prolonged survival in the environment. Temperature appears to be the most important factor in virus survival, with low temperatures being associated with increases in virus persistence. Virus survival for many months has been observed at freezing or near-freezing temperatures (Gerba, 2007). UV light may damage the nucleic acid causing dimer formation. Double-stranded DNA viruses like adenoviruses are more resistant to UV light inactivation than are enteroviruses (Gerba, 2007). In addition to these two principal environmental factors, other physical-chemical parameters such as pH, salts and organic matter may impact virus survival and distribution, but generally this is only in a limited manner (Gerba, 2007). Few data are available on the behavior of norovirus since most studies have only reported qualitative virus detection. Real-time RT-PCR allows a quantification approach as does the use of cultivable animal norovirus strains as surrogates, and these approaches can be used to evaluate virus resistance to different environmental factors such as temperature or UV light (Duizer et al., 2004; Bae and Schwab, 2008).

Monitoring viral contamination in shellfish growing areas

563

18.5.2 Persistence of viruses in shellfish tissues It was generally thought that oysters act as mere filters or ionic traps, passively concentrating particles such as bacteria or virus. However, unlike enteric bacterial species, enteric viruses persist in shellfish for an extended period of time, and it is this persistence that appears to result in its significant impact on public health. Viruses are principally concentrated in the pancreatic tissue, also called digestive diverticula. A number of different mechanisms have been suggested to explain differences in virus accumulation between different oyster species, including mechanical entrapment and ionic bonding (di Girolamo et al., 1977; Metcalf, 1982; Schwab et al., 1998; Burkhardt and Calci, 2000). Virus accumulation in oysters can also depend on factors such as water temperature, mucus production, glycogen content of the connective tissue, and gonadal development. The importance of secreted acid mucopolysaccharides in the concentration of poliovirus was first demonstrated 30 years ago (di Girolamo et al., 1977). Mucus present on gills was also suspected to be important for concentration of reovirus by oysters (Bedford et al., 1978). Later hepatitis A virus was demonstrated to persist for several weeks after bioaccumulation, with the infectious virus being detectable after three weeks and viral RNA still being detectable after six weeks using molecular assays (Kingsley and Richards, 2003). Infectious adenovirus was still detected in mussels for three weeks following bioaccumulation and in oysters for six weeks (Hernroth and Allard, 2007). Virus-like particles (VLPs) have also been used to study virus persistence in shellfish. Loisy et al. (2005a) used rotavirus VLPs in oyster bioaccumulation studies, and viral particles persisted in oyster tissues for one to three months depending on the initial input concentrations (Loisy et al., 2005a). We used VLPs of the prototype genogroup I Norwalk virus (rNV VLP) and native Norwalk virus for bioaccumulation of oysters (Crassostrea gigas) to study norovirus persistence. We observed no differences in virus distribution between the native Norwalk virus and the VLPs, confirming that VLPs are good surrogates of infectious virions for this type of study. Interestingly, virus particle and VLP binding was to specific cell types, and some viral particles were detected in phagocytes located either in the epithelium or in the connective tissue (Le Guyader et al., 2006b). This observation might reflect the process of virus elimination or of normal digestion, but it is unclear if the immunoreactive material detected in phagocytes corresponds to particles being degraded and digested or whether particles are able to escape digestion. Specific binding was observed to the main ducts in the digestive tract and may be a mechanism for many viral particles to avoid entering in the food circulation and thus being degraded (Le Guyader et al., 2006b). The existence of a specific attachment to oyster cells and the internalization into phagocytes could explain the difficulty in using depuration to rid oysters of virus. In our work, we observed no differences in the tissue distribution of VLP binding between samples collected in March or October, although additional studies during other seasons are needed.

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New technologies in aquaculture

Human susceptibility to norovirus infection depends upon the presence or absence of certain carbohydrates of the ABH, secretor, and Lewis histoblood group families (Tan and Jiang, 2007). Using tissue sections of oyster bodies, we observed that recognition of oyster digestive epithelial cells by rNV VLPs also involves carbohydrates. Similar to what was observed with human histo-blood group structures, the use of human saliva to inhibit VLP attachment to oyster tissues or the use of mutant VLPs that abolish VLP binding to histo-blood group antigens (alanine subtitution at positions H329A and W375A) prevent binding to oyster tissue. Additional studies showed that the oyster ligands are similar to histo-blood group A. Thus, norovirus binds to oyster tissues through an A-like carbohydrate structure, a binding site also used for attachment to carbohydrate on human epithelial cells (Tan and Jiang, 2007). Norovirus VLPs can also specifically bind to tissues of other oyster species (Crassostrea virginica, Crassostrea sikamea) or clams (Venerupis virginica) or mussels (Mytilis edulis) (Tian et al., 2007). Distinct norovirus strains belonging to both genogroup I and II exhibit various binding patterns with different carbohydrate structures of the histoblood group family, suggesting a possible co-evolution of this group of viruses and their host or carrier vector. The importance of these observations and the interaction with specific carbohydrates in shellfish is of primary importance to protect the consumer, and more research needs to be conducted on this subject. For example, it could be interesting to see if some bivalve mollusks less sensitive to contamination may be selected or if we can prevent the binding. The identification of the receptor may help to improve depuration.

18.5.3 Depuration Depuration processes are intended to eliminate or to purge live bivalves of the microbial contamination and are usually performed in tanks with clean seawater. European legislation does not stipulate a minimum length of time over which depuration should be performed, but approximately two days is probably the most widely used depuration time in Europe (Richards, 1988). Two days of depuration with clean seawater will efficiently eliminate contaminating enteric bacteria but, as mentioned above, this is an insufficient length of time to remove enteric viral pathogens efficiently. Depurated shellfish have been implicated as the cause of a number of virus illness outbreaks (Gill et al., 1983; Heller et al., 1986; Le Guyader et al., 2006a, 2008). Low levels of enterovirus contamination in soft shell clams following bioaccumulation were reported as often eliminated following depuration in early studies (Metcalf et al., 1979). However, more recent data obtained using bioaccumulated shellfish have shown that, although depuration may decrease virus concentration in shellfish tissues, it has usually been insufficient to completely remove viruses (Abad et al., 1997; Schwab et al., 1998;

Monitoring viral contamination in shellfish growing areas

565

Kingsley and Richards, 2003; Pommepuy et al., 2003; Loisy et al., 2005a). Different factors influence depuration efficiency. Increasing water temperatures can enhance virus removal, although an increase in depuration temperature may also result in increased shellfish mortality and induce some bacterial growth (Dore and Lees, 1995; Pommepuy et al., 2003). Poor depuration of enteric viruses can still occur at higher depuration temperatures as was reported in studies of Norwalk virus where only 7 % of Norwalk virus was depurated compared to a 95 % reduction in bacterial levels at a temperature of 22 °C (Schwab et al., 1998). Feeding naturally contaminated oysters at 22 °C increased depuration in semi-professional size depuration tanks compared to fed oysters at lower temperatures (Pommepuy et al., 2003). The large tank allowed depuration of up to 900 kg of oysters and showed the feasibility of performing this procedure in the setting of a shellfish farm using producer conditions. Phages have also been suggested as indicators of viral depuration as they are depurated less efficiently than E.coli (Doré et al., 2000). Feline calicivirus, another proposed surrogate, failed to be a good indicator of norovirus depuration from oysters (Ueki et al., 2007). Considering the differences in depuration observed among among various species of human enteric viruses (Abad et al., 1997) and differences observed in virus binding to shellfish tissues, extrapolating data from a phage to a human pathogen may be hazardous. The employment of appropriate surrogates remains a crucial issue in the evaluation of shellfish depuration.

18.6 Other issues Other approaches have been proposed to decrease the risk of shellfish for human consumption (Table 18.6). Cooking shellfish is one such approach, but failures of this strategy have also been reported. Following a large outbreak of viral gastroenteritis with cockles collected from the Thames estuary (England), a ‘double cooking’ was recommended (after an initial brief boil or steam treatment necessary for shell removal, cockle meats were to be boiled for a further 4 min). However, gastroenteritis and hepatitis A linked to cockle or mussel consumption were still reported (Millard et al., 1987). Frozen coquinas clams imported from Peru and served cooked in paella were responsible for hundred of cases of hepatitis A in Spain (Bosch et al., 2001). Croci et al. (2005) explored different approaches to kill the virus after bioaccumulation of hepatitis A virus in mussels, and these investigators showed that extended cooking times were needed to completely inactivate the virus (Table 18.6). Failures of cooking may be due to the inability to attain sufficiently high temperatures to inactivate the virus or to the relative resistance of some enteric viruses to heat inactivation and their low infectious dose (Koff and Sear, 1967; McDonnell et al., 1997). In food, several factors may influence viral behavior (Le Guyader and Atmar, 2008).

Oysters

Heat

Mussels

Shellfish

HAV HAV

Au gratin

NoV

HAV Rotavirus HAV

NoV

Poliovirus Poliovirus Poliovirus Poliovirus HAV

Virus

In hors d’oeuvre

Steamed

Steamed

Stewed in milk Fried/oil 177 °C Baked/oven 121 °C Steamed Boiling

Recipe

Bioaccumulated

Bioaccumulated

Seeded

Bioaccumulated

Seeded Seeded Seeded Bioaccumulated Seeded

Contamination

Effects of different physical factors on virus inactivation in shellfish

Treatment

Table 18.6

10 % survival after 8 min 10 % survival after 8 min 13 % survival after 20 min 7 % survival after 30 min No survival after 3 min for HAV 100 % persistence for NoV RNA 0.1 % survival after 5 min 0.1 % survival after 5 min No survival after 3 min for HAV 100 % persistence for NoV RNA Infectious viruses detected after 9 min1 Infectious viruses detected after 5 min1

Persistence of virus

Croci et al., 2005

Croci et al., 2005

Hewitt and Greening, 2006

Abad et al., 1997

Di Girolamo et al., 1970 Di Girolamo et al., 1970 Di Girolamo et al., 1970 Di Girolamo et al., 1970 Hewitt and Greening, 2006

References

HAV

Oysters

Infectious virus too low for quantification. HAV = hepatitis A virus, NoV = norovirus.

1

Murine NoV

Oysters

NoV

HAV

High pressure

Steamed and marination (pH 3.75)

Mussels

Bioaccumulated

Bioaccumulated

Seeded

Bioaccumulated

Poliovirus

Frozen (−17.5 °C)

Marinade

Bioaccumulated

Kept at 5 °C

Oyster

Bioaccumulated Bioaccumulated

HAV Poliovirus HAV Poliovirus

in tomato sauce Boiling

Cold

Cockles

No survival after 8 min No survival after 3 min No survival after 2 min 13 % survival after 1 month 10 % survival after 12 weeks 3 % survival after 4 weeks for HAV 100 % persistence for NoV RNA No survival after 5 min at 5 °C at 325 MPa 0.1 % survival after 1 min at 400 MPa

Calci et al., 2005

Kingsley et al., 2007

Hewitt and Greening, 2004

Di Girolamo et al., 1970

Di Girolamo et al., 1970

Croci et al., 2005 Millard et al. 1987

568

New technologies in aquaculture

High-hydrostatic pressure (HHP) processing has emerged as a promising technology for virus inactivation. HHP inactivates enteric viruses suspended in buffer, but the inactivation rates are affected by treatment temperature and virus strain (Chen et al., 2005; Kingsley et al., 2007). It has been applied to shellfish that have bioaccumulated HAV or a murine norovirus, and greater than 1000-fold reductions in viral titer was achieved with a treatment of ≤400 Megapascal (MPa) for 5 min at 5 °C (Calci et al., 2005; Kingsley et al., 2007). A potential disadvantage of this method is that changes in the character of the shellfish have been demonstrated in organoleptic studies, and some consumers prefer to eat live oysters (Cruz-Romero et al., 2004). The effect of marination (immersion in an acetic acid-based mixture) has been evaluated in mussels contaminated with norovirus or HAV. The commercial marination process is a two-stage procedure including a preliminary heat treatment (immersion in boiling water or steaming for 3 min) and then marination for several weeks. After four weeks of marination, the infectious titer for HAV decreased approximately 50-fold, and human norovirus RNA was still detected by real-time RT-PCR (Hewitt and Greening, 2004). These data suggest that marination alone is not sufficient to inactivate enteric viral pathogens in shellfish.

18.7 Future trends Although seafood can generally be regarded as a wholesome, safe, and nutritious food, it may occasionally pose consumer risks. Regulations are currently based on routinely monitoring shellfish for fecal bacteria to determine their sanitary quality. However, viral contamination of bivalve molluscs is currently recognized as one of the major causes of illness associated with seafood. Monitoring viral contamination is complex and must take into account different factors such as detection methods, technology, social demands, and the sustainable development of aquaculture. Recent advances in technology, especially in developing molecular tools, make it possible to look for pathogens directly in shellfish implicated in outbreaks. Evaluation of seafood for the presence of norovirus and hepatitis A virus for regulatory purposes has been recommended by experts involved in EU, FAO, and WHO working groups. Mandatory surveillance for virus may be implemented in the near future, but additional information including level of exposure and virus infectious dose are still needed to assess risks. Different actions may be considered to lower the contamination of shellfish bed in coastal area: • Reduction of fecal input in coastal areas: Water quality studies have demonstrated the feasibility of determining the main sources of fecal contamination and identifying the critical points in the catchments.

Monitoring viral contamination in shellfish growing areas

569

Hydrodynamical models when applied to these contaminants, even if they need further development and have to be validated by databases, could lead to rational bases for the choice of treatment levels based on results from screening purposes to limit the contamination. • Implementation of warning systems: in developed countries, some data are already available on outbreaks occurring in the population, so, associated with forecast information, salinity, sewage treatment plant failure, and other factors, a warning system could lead to real-time assessments of water quality in bathing or harvesting areas. The development of new tools for rapid pathogen detection (for example microarray) may help to collect additional information on the presence of pathogens in sewage or waters. • To limit shellfish contamination, the most desirable and effective option is to reduce the viral input. Villages, small towns, and dwellings must be equipped with small individual treatment tanks to comply with the regulations. New technologies may be needed to improve removal of viruses from sewage effluents (Shannon et al., 2008). Another solution could be the relocation of shellfish aquaculture away from the contamination sources. Other factors will also need to be considered to protect the consumer and to provide safe shellfish on the market. An important aspect of monitoring concerns the sustainable development of aquaculture. This development is closely linked to environmental quality in shellfish breeding areas. The nested-regulations set up for water quality, bathing areas, and shellfish growing areas, if well applied, should provide the guarantees and the management tools. There are promising examples of coastal management designed to reduce the fecal load which could make recovery of water quality feasible. Used in association with early warning systems, they could help ensure shellfish quality and increase consumer confidence and, thus, would greatly contribute to sustainable development of aquaculture. Shellfish have long been recognized as being beneficial to human health, and this benefit should also be taken into consideration in managing the coastal areas and preserving the water quality.

18.8 References abad f x, pinto r m, diez j m and bosch a (1994) Disinfection of human enteric viruses in water by copper and silver in combination with low levels of chlorine, Appl Environ Microbiol, 60, 2377–83. abad f x, pinto r m, gajardo r and bosch a (1997) Viruses in mussels: public health implications and depuration, J Food Prot, 60, 677–81. aggarwal r and naik s (2008) Enterically transmitted hepatitis, in Koopmans M, Cliver D O and Bosch A (eds), Food-borne Viruses Progress and Challenges, American Society for Microbiology, Washington, DC, 65–85. anderson e j and weber s g (2004) Rotavirus infection in adults, Lancet Infect Dis, 4, 91–9.

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arnal c, crance j m, gantzer c, schwartzbrod l, deloince r and billaudel s (1998) Persistence of infectious hepatitis A virus and its genome in artificial sweater, Zentralbl Hyg Umweltmed, 201, 279–84. atmar r l and estes m k (2006) The epidemiologic and clinical importance of norovirus infection, Gastroenterol Clin N Am, 35, 275–90. atmar r l, metcalf t g, neill f h and estes m k (1993) Detection of enteric viruses in oysters by using the polymerase chain reaction, Appl Environ Microbiol, 59, 631–5. atmar r l, neill f h, romalde j l, le guyader f, woodley c m, metcalf t g and estes m k (1995) Detection of Norwalk virus and Hepatitis A virus in shellfish tissues with the PCR, Appl Environ Microbiol, 61, 3014–18. atmar r l, opekun a r, gilger m a, estes m k, crawford s e, neill f h and graham d y (2008) Duration and magnitude of Norwalk virus shedding following experimental human infection, Emerg Infect Dis, 14, 1553–7. bae j and schwab k j (2008) Evaluation of murine norovirus, feline calicivirus, poliovirus, and MS2 surrogates for human norovirus in a model of viral persistence in surface water and groundwater, Appl Environ Microbiol, 74, 477–84. bedford a j, williams g and bellamy a r (1978) Virus accumulation by the rock oyster crassostrea glomerata, Appl Environ Microbiol, 35, 1012–18. blanton l h, adams s m, beard r s, wei g, bulens s n, widdowson m-a, glass r i and monroe s s (2006) Molecular and epidemiologic trends of caliciviruses associated with outbreaks of acute gastroenteritis in the United States, 2000–2004, J Infect Dis, 193, 413–21. bosch a, sánchez g, le guyader f, vanaclocha h, haugarreau l and pintó r m (2001) Human enteric viruses in coquina clams associated with a large hepatitis A outbreak, Water Sci Technol, 43, 61–6. boxman i l a, tilburg j j h c, te loeke n a j m, vennema , jonker k, de boer e and koopmans m (2006) Detection of noroviruses in shellfish in the Netherlands, Int J Food Microbiol, 108, 391–6. brooks h a, gersberg r m and dhar a k (2005) Detection and quantification of hepatitis A virus in seawater via real-time RT-PCR, J Virol Meth, 127, 109–18. bull r a, tanaka m m and white p a (2007) Norovirus recombination, J Gen Virol, 88, 3347–59. burkhardt w and calci k r (2000) Selective accumulation may account for shellfishassociated viral illness, Appl Environ Microbiol, 66, 1375–8. butt a a, aldridge k e and sanders c v (2004) Infections related to the ingestion of seafood. Part I: viral and bacterial infections, Lancet Infect Dis, 4, 201–12. cacopardo b, russo r, preiser w, benanti f, brancati g and nunnari a (1997) Acute hepatitis E in Catania (Eastern Sicily) 1980–1994. The role of hepatitis E, Infection, 25, 313–16. calci k r, meade g k, tezloff r c and kinsgley d h (2005) High-pressure inactivation of hepatitis A virus within oysters, Appl Environ Microbiol, 71, 339–43. cdc (2006) Prevention of hepatitis A through active or passive immunization, M M W R, 55 (RR7), 1–23. chan m c w, sung j j y, lam r k y, chan p k s, lee n l s, lai r w m and leung w k (2006) Fecal viral load and norovirus associated gastroenteritis, Emerg Infect Dis, 12, 1278–80. cheetham s, souza m, meulia t, grimes s, han m g and saif l j (2006) Pathogenesis of a genogroup II human norovirus in gnotobiotic pigs, J Virol, 80,10372–81. chen h, hoover d g and kinsgley d h (2005) Temperature and treatment time influence high hydrostatic pressure inactivation of feline calicivirus, a norovirus surrogate, J Food Prot, 68, 2389–94.

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costafreda m i, bosch a and pinto r m (2006) Development, evaluation, and standardization of a real-time TaqMan reverse transcription-PCR assay for quantification of hepatitis A virus in clinical and shellfish samples, Appl Environ Microbiol, 72, 3846–55. costantini v, loisy f, joens l, le guyader f s and saif l j (2006) Human and animal enteric caliciviruses in oysters from different coastal regions of the United States, Appl Environ Microbiol, 72, 1800–9. croci l, de medici d, scalfaro c, fiore a, divizia m, donia d, cosentino a m, moretti p and costanti g (2000) Determination of enteroviruses, hepatitis A virus, bacteriophages and Escherichia coli in Adriatic sea mussels, J Appl Microbiol, 88, 293–8. croci l, de medici d, di pasquale s and toti l (2005) Resistance of hepatitis A virus in mussels subjected to different domestic cooking, Int J Food Microbiol, 105, 139–44. cruz-romero m, smiddy m, hill c, kerry j p and kelly a l (2004) Effects of high pressure treatment on physicochemical characteristics of fresh oysters (Crassostrea gigas), Innov Food Sci Emerg Tech, 5, 161–9. da silva a, le saux j-c, parnaudeau s, pommepuy m, elimelech m and le guyader f s (2007) Evaluation of removal of noroviruses during wastewater treatment, using real-time reverse transcription-PCR: different behaviors of genogroups I and II, Appl Environ Microbiol, 73, 7891–7. de roda husman a-m, lodder-verschoor f, van der berg h h l j, le guyader f s, van pelt h, van der poel w h m and rutjes s a (2007) Rapid virus detection procedures for molecular tracing of shellfish associated with disease outbreaks, J Food Prot, 70, 967–74. dhama k, chauban r s, mahendran m and malik s v s (2009) Rotavirus diarrhea in bovines and other domestic animals, Vet Res Commun, 33, 1–23. di girolamo r, liston j and matches j (1970) Survival of virus in chilled, frozen, and processed oysters, Appl Environ Microbiol, 20, 58–63. di girolamo r, liston j and matches j (1977) Ionic binding, the mechanism of viral uptake by shellfish mucus, Appl Environ Microbiol, 33, 19–25. doré w j and lees d n (1995) Behavior of Escherichia coli and male-specific bacteriophage in environmentally contaminated bivalve molluscs before and after depuration, Appl Environ Microbiol, 61, 2830–4. doré w, henshilwood k and lees d n (2000) Evaluation of F-specific RNA bacteriophage as a candidate human enteric virus indicator for bivalves molluscan shellfish, Appl Environ Microbiol, 66, 1280–5. dowell s f (2001) Seasonal variation in host susceptibility and cycles of certain infectious diseases, Emerg Infect Dis, 7, 369–74. dubois e, merle g, roquier c, trompette a, le guyader f, cruciere c and chomel j-j (2004) Diversity of enterovirus sequences detected in oysters by RTheminested PCR, Int J Food Microbiol, 92, 35–43. duizer e, bijkerk p, rockx b, de groot a, twisk f and koopmans m (2004) Inactivation of caliciviruses, Appl Environ Microbiol, 70, 4538–45. el senousy w m, guix s, abid i, pinto r m and bosch a (2007) Removal of astrovirus from water and sewage treatment plants, evaluated by a competitive reverse transcription-PCR, Appl Environ Microbiol, 73, 164–7. estes m k and kapikian a z (2007) Rotaviruses, in Knipe D M, Howley P M (eds), Fields Virology, Lippincott Williams and Wilkins, Baltimore, MD, 1917–74. eu (1991) Council Directive 91/492/EEC of 15 July 1991 laying down the health conditions for the production and the placing on the market of live bivalve molluscs, Official Journal of the European Communities, L 268, 24 January, 1–14. fda (1995) National Shellfish Sanitation Program Manual of Operations, CFSAN/ FDA, Washington, DC.

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turcios r m, widdowson m a, sulka a c, mead p s and glass r l (2006) Reevaluation of epidemiological criteria for identifying outbreak of acute gastroenteritis due to norovirus: United States, 1998–2000, Clin Infect Dis, 42, 964–9. ueki y, sano d, watanabe t, akiyama k and omura t (2005) Norovirus pathway in water environment estimated by genetic analysis of strains from patients of gastroenteritis, sewage, treated wastewater, river water and oysters, Water Res, 39, 4271–80. ueki y, shoji m, suto a, tanabe t, okimura y, kikuchi y, saito n, sano d and omura t (2007) Persistence of caliciviruses in artificially contaminated oysters during depuration, Appl Environ Microbiol, 73, 5698–701. umesha k r, bhavani n c, venugopal m n, karunasagar i, krohne g and karunasagar i (2008) Prevalence of human pathogenic enteric viruses in bivalve molluscan shellfish and cultured shrimp in south west coast of India, Int J Food Microbiol, 122, 279–86. van der berg h, lodder w, van der poel w, vennema h and de roda husman a m (2005) Genetic diversity of noroviruses in raw and treated sewage water, Res Microbiol, 156, 532–40. van der poel w h m, vinje j, van der heide r, herrera m i, vivo a and koopmans m p g (2000) Norwalk-like calicivirus genes in farm animals, Emerg Infect Dis, 6, 36–41. wait d a and sobsey m d (2001) Comparative survival of enteric viruses and bacteria in Atlantic Ocean seawater, Wat Sci Tech, 43, 139–42. wang q-h, chang k-o, cheetham m g, souza m, funk j a and saif l j (2005) Porcine noroviruses related to human noroviruses, Emerg Infect Dis, 11, 1874–81. wang q-h, souza m, funk j a, zhang w and saif l j (2006) Prevalence of noroviruses and sapoviruses in swine of various ages determined by reverse transcriptionPCR and microwell hybridization assays, J Clin Microbiol, 44, 2057–62. ward p, muller p, letellier a, quessy s, simard c, trottier y-l, houde a and brassard j (2008) Molecular characterization of Hepatitis E viruses detected in swine farms in the province of Quebec, Can J Vet Res, 72, 27–31. westrell, t, teunis p, van den berg h, lodder w, ketelaars h, stenstrom t a and de roda husman a m (2006) Short- and long-term variations of norovirus concentrations in the Meuse river during a 2-year study period, Water Res, 40, 2613–20. wold w s m and horwitz m s (2007) Adenoviruses, in Knipe D M and Howley P M (eds), Fields Virology, Lippincott Williams and Wilkins, Baltimore, MD, 2395–436. wyn-jones p (2007) The detection of waterborne viruses, in Bosch A (ed.), Human Viruses in Water, Perspective in Medical Virology, Elsevier, Oxford and Amsterdam, 177–226. yamashita t, sugiyama m, tsuzuki h, sakae k, suzuki y and miyazaki y (2000) Application of a reverse transcription-PCR for identification and differentiation of Aichi virus, a new member of the picornavirus family associated with gastroenteritis in humans, J Clin Microbiol, 38, 2955–61. zheng d-p ando t, fankhauser r l, beard r s, glass r and monroe s s (2006) Norovirus classification and proposed strain nomenclature, Virology, 346, 312–23.

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19 Impacts of harmful algal blooms on shellfisheries aquaculture Y. Matsuyama, National Research Institute of Fisheries and Environment of Inland Sea, Japan, and S. Shumway, University of Connecticut, USA

Abstract: The impacts of harmful algal species on shellfish aquaculture were reviewed by Shumway (1990) and, while there were some specific studies available, much of the information was collated from anecdotal references in historical literature. The number of harmful algal species and blooms has increased globally since the 1960s, and their impacts on molluscan shellfish and aquaculture are profound. This chapter provides an updated account of harmful algal species and their impact on shellfish aquaculture. Key words: harmful algae, dinoflagellate, shellfish, aquaculture, phycotoxin, mitigation.

19.1 Introduction The global demand for marine products has increased dramatically since the 1990s, and overfishing of natural resources continues to be a serious problem (Pauly et al., 2003). In response, the expansion and contribution of aquaculture is expected to provide a sustained supply of marine products in the future (FAO, 2007). Shellfish cultivation relies on primary production in the sea, i.e. shellfish directly assimilate phytoplankton, which results in one of the most highly efficient protein production systems in the world. In addition, shellfish cultivation is a model of a low-cost, low-impact, and environmentally acceptable system. Several factors, including deterioration of farming beds due to excess load of organisms, settlement failure of natural spat, chemical pollution, contamination by bacteria and viruses of human origins, and excess regulations, as well as the increased incidence of harmful algal blooms (HABs) and associated toxins have hampered the development of shellfish aquaculture. Among these factors, HABs are one of the most serious problems for shellfisheries worldwide (Shumway, 1990; Hallegraeff, 1993). We summarize the impact of HABs on shellfisheries and

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discuss the introduction of some new technologies that may reduce the threat of HAB impacts on shellfisheries and aquaculture.

19.2 Global increase of harmful algal blooms (HAB) The rapid increase of certain algal species occasionally results in concentrations of several million cells per liter, and may form visible patches on the sea surface commonly referred to as ‘red tides’ or HABs. Red tides are a natural phenomenon and are described in the Old Testament (Exodus, 7:20–21), and in one of the oldest published records on 13 June 731 A.D. in Kii Channel, Japan (Okaichi, 2004). Some algal species produce natural toxins, which pose threats to humans or are detrimental to aquatic organisms. While the term ‘red tide’ has been used historically and generally to describe these phenomena, recent studies now refer to the occurrences as ‘harmful algal blooms (HABs)’ which have been associated with various types of shellfish poisonings and the co-occurrence of large-scale mortality of aquatic organisms (Shumway, 1990; Smayda, 1990; Hallegraeff, 1993). There has been a marked increase in harmful algal blooms since the 1960s (Shumway, 1990; Smayda, 1990; Hallegraeff, 1993; Anderson, 1994; Okaichi, 2004). Some have attributed the apparent increase to increased awareness, a greater number of observers, or prompt dissemination of information worldwide, but almost all scientists now agree that there is a very real increase in the frequency, intensity, duration, and geographic distribution of the blooms. Increased frequency of HABs has resulted in global public health hazards, economic losses, and destruction of aquatic ecosystem. Shellfisheries and shellfish aquaculture are particularly prone to the negative impacts of HABs because shellfish accumulate various phycotoxins through filter-feeding activities. Their biology, physiology, and survival are impacted as such, and studies on the impacts of HABs on shellfish have flourished since the early work of Shumway and collaborators (see Shumway, 1990; Landsberg, 2002). Numerous factors have been suggested as causes of the increase in HABs and are briefly discussed below – this is not meant to be an all-inclusive review, but rather a primer for the shellfish aquaculturist. While all of these factors have merit for some HAB species, there is no one cause to which we can attribute either the individual HABs or their global proliferation.

19.2.1 Climate changes Almost all toxic microalgal species are autotrophic, single-celled microbes and their growth is affected by numerous factors including water temperature, salinity, light intensity, coastal current and stratification, freshwater runoff, concentration and/or ratio of nutrients, grazing by zooplankters, competition with other phytoplankton species, and presence or absence of

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algicidal microbes. Drastic changes in these factors driven by global warming (Hallegraeff, 1993), marked changes in precipitation (Okaichi, 2004), eutrophication as a result of increased utilization of coastal areas (Hallegraeff, 1993; Okaichi, 2004), dispersal of toxic algal species by ships’ ballast water (Hallegraeff, 1998), dams (Humborg et al., 1997), and acid rain (Granéli and Haraldsson, 1993) all impact HAB species. Among these factors, the potential effects of climate change on phytoplankton behavior in coastal waters are well-known regulators of phytoplankton growth and species successions. In recent studies, it has been reported that the influence of global warming causes an increase in water temperature, which facilitates the expanding geographical ranges of the tropical toxic microalgal species: Alexandrium tamiyavanichii, Gymnodinium catenatum and Pyrodinium bahamense (Matsuoka and Fukuyo, 1994; Hashimoto et al., 2002; Phlips et al., 2006). The increased water temperature causes a tendency for non-swimming diatoms to sink from the water column, where phytoflagellate species remain in the surface layer, i.e. favorable for photosynthesis and succession, under the highly stratified conditions (Margalef et al., 1979). Long-term oceanographic surveys and monitoring of harmful algae have been carried out in Seto Inland Sea, Japan since 1972. These data show that the winter water temperature in this area has increased about 1–2 °C since 1988 (Matsuyama, 1999). Warm temperature events of the late 1980s facilitated the invasion of the tropical exotic species A. tamiyavanichii, proliferation of the inconspicuous indigenous flora, G. catenatum (responsible for paralytic shellfish poisoning – PSP), and the massive mortality of shellfish due to blooms of Heterocapsa circularisquama (Matsuyama, 1999). Further, increases and decreases of the magnitude or velocity of coastal currents due to hydrographical changes resulted in changes of the distribution of a toxic algal species, and/or increase of their bloom frequency. The influence of global warming on HABs is one factor that will undoubtedly continue to allow expansion of toxic algal species and their proliferation in hitherto unaffected areas.

19.2.2 Utilization of coastal water for shellfish aquaculture Urbanization and the associated concurrent increase of sewage effluents and industrial and agricultural waste can stimulate toxic algal blooms (Smayda, 1990; Hallegraeff, 1993; Okaichi, 2004). Since the 1960s, shellfish cultivation has expanded in many coastal areas and this may also have contributed to an increased awareness of toxic algal species and other impacts (Shumway, 1990). Shellfish cultivation relies upon the availability of natural food for consumption and bivalves discharge pseudofeces and feces directly into the natural habitat. Huge volumes of water are processed by shellfish filtration and the pesudofeces and feces are remineralized to produce new dissolved nutrients, which stimulate new phytoplankton

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growth (see review by Dame, 1996; Ferreira et al., 2007). Eutrophication due to finfish farming has had a serious impact on some coastal ecosystems (Gowen and Bradbury, 1987; Silvert, 1992; Yokoyama, 2002); however, the deposition rate of organic material in shellfish culture is generally accepted as lower than those of finfish farming (Yokoyama, 2002). Nevertheless, rapid unrestricted development of shellfish mariculture has the potential to induce coastal eutrophication (Gilbert et al., 1997) and concurrent toxic algal blooms in small embayments. Construction of large numbers of raft or farming pens for shellfish culture can significantly depress the flow of coastal currents and may aid in the accumulation of undesirable plankton. (Fig. 19.1). Shellfish cultivation involves consumption of large quantities of natural phytoplankton and can have profound impacts on the local ecosystem structure if not carefully monitored (Dame, 1996; Norén et al., 1999; Ward

Fig. 19.1 Large-scale cultivation of shellfish. Upper: Pacific oyster rafts covered by surface patches of the toxic dinoflagellate Heterocapsa circularisquama, Hiroshima Bay (1997). Lower: pearl oyster Pinctada fucata martensii in Bungo Channel, Japan.

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and Shumway, 2004). In addition, the filter-feeding bivalve molluscs can sort particles during filtration, which allows for preferential rejection of undesirable particles, such as inorganic particles and sometimes-toxic algae (Shumway et al., 1985; see Ward and Shumway, 2004 for review). Thus, culture of bivalve molluscs can control not only phytoplankton biomass, but also species composition in natural habitats. Figure 19.2 shows a photograph of the various species of plankton identified from the pseudofeces or feces collected from the cultured Pacific oyster, Crassostrea gigas. Although the phytoplankton flora in the natural habitat during sampling was composed of >80 % diatoms, there were almost no diatoms detected in feces with the exception of some benthic species. Motile cells and the resting cysts of dinoflagellate species were abundant and some appeared to be alive. Several authors have previously demonstrated that some species of phytoplankton, including dinoflagellates, can pass intact and viable through the digestive tract of bivalve molluscs (Scarratt et al., 1993; Shumway et al., 2006; Hégaret et al., 2008a). Most recently, Hégaret and co-workers have demonstrated that numerous species of harmful algae survive gut passage and are a potential vector of introduction of unwanted algal species through the transfer of shellfish to new environments (Shumway et al., 2006; Hégaret et al., 2007, 2008a). In addition, zooplankters (tintinnids, ciliates, and copepods), all of which are known to play roles as ‘top-down’ controllers of harmful algae in marine ecosystem, are also ingested by the oysters.

Fig. 19.2 Detection of phyto- and zooplankton species from ejected feces from the Pacifc oyster collected at Hiroshima Bay. A: alive; D: dead.

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In many coastal areas, shellfish culture is associated with a shift in phytoplankton community composition, from diatom-dominated communities to dinoflagellate-dominated communities. This suggests the potential proliferation of toxic algal species; however, there are limited experimental data available to clarify the effect of selective particle retention and ingestion of bivalve molluscs and toxic algal succession. Alternate community states resulting from intense grazing of filter-feeding bivalves depend on the intensity of cultivation. Although shellfish cultivation is part of the organic carbon cycle in ecosystems (Dame, 1996), it is necessary to choose cultivation methods that minimize the risk of HAB outbreaks.

19.2.3

Dispersal of harmful algal bloom species associated with shellfish transportation There is increasing discussion regarding toxic algal species being transported to new areas via ships’ ballast water or through infected shellfish species (Hallegraeff, 1998; Shumway et al., 2006; Smayda, 2007; Hégaret et al., 2008a). The International Maritime Organization (IMO) and other international agencies regulate the transfer of harmful aquatic organisms by ships through the implementation of the Global Ballast Water Management Programme. Such efforts are expected to reduce the potential dispersal of toxic algal species. The International World Health Organization lists the regulation of shellfish infected by specific pathogenic microbes for animal health. Toxic algal species are, however, still not included in this list. In shellfish culture, the mass transplant of seed stock or relaying of farm beds are carried out between geographically distant areas, and the transport of toxic algal species with shellfish is common (Hallegraeff, 1993; Scholin et al., 1995; Honjo et al., 1998; Shumway et al., 2006; Hégaret et al., 2008a). In the USA, oyster transplants are performed to improve the environment of shallow estuaries (e.g. Coen et al., 2007). This may also increase the risk of transferring toxic algal species. Shellfish species filter large volumes of water and can accumulate toxic algal species during continuous grazing on phytoplankton. Such grazing poses the risk of infecting a clean area with cysts or motile cells, making it possible to seed a future bloom (Honjo et al., 1998; Shumway et al., 2006; Hégaret et al., 2008a). Simulated experiments have demonstrated the range of toxic algal species that can be associated with shellfish transportation (Scarratt et al., 1993; Furuhata et al., 1996; Honjo et al., 1998; Shumway et al., 2006; Hégaret et al., 2008a). For example, the transfer of scallops and clams may result in the transport of the cyst of an Alexandrium spp. present in bottom sediment (Scarratt et al., 1993; Furuhata et al., 1996). The cyst exhibits high tolerance to mechanical damage and biological ingestion (Scarratt et al., 1993; Persson, 2000). Sendai Bay (SEN) and the Ohta River Estuary (OHT) are representative aquaculture areas for the Pacific oyster Crassostrea gigas in Japan, and the transplant of oyster spat is usually conducted between both areas

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(Nagai et al., 2007). During the domestic transplanting of spat attached to scallop shells (approximately 100 spat/shell) from the SEN to the OHT area (ca. 1200 km apart) in Japan (Fig. 19.3), oyster seed are transferred in trailers over land for 36 h and transplanted in a nursery near the OHT area upon arrival. It was shown that 720 motile cells of the toxic dinoflagellate Alexandrium tamarense had discharged from oysters on a single scallop shell. Nagai et al. (2007) analyzed the genetic structure of Japanese isolates of A. tamarense using microsatellite markers, and genetic isolation among populations occurred in accordance with geographic distance. No significant differences were detected between SEN and OHT area populations, suggesting that the genetic structure of A. tamarense populations between

135E

40N SEN OHT

Fig. 19.3 Transplantation of the Pacifc oyster Crassostrea gigas seed and associated transportation of the toxic dinoflagellate Alexandrium tamarense from SEN (closed circle) to OHT (closed triangle) site conducted via trailer by an oyster aquacultrist (c. 1000 km distance).

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SEN and OHT areas are disturbed. Disturbance may be due to an anthropogenic-mediated dispersal that has resulted in gene flow between geographically separate populations. Hégaret et al. (2008a) have demonstrated that some HAB species can be rendered non-viable if shellfish are held out of water for short time periods and that, in many cases, the mitigation strategy coincides with standard operating procedures and shipping practices. In order to minimize the risk of an anthropogenic transfer, shellfish transplants must be monitored until toxic algal species are not detected in either the seawater from the cultivation area or in the shellfish. Establishment of a global framework to reduce the potential dispersal risk of toxic algal species via shellfish transport, as well as potential dispersal risk of pathogenic microbes, should be considered.

19.3 Impact of harmful algal bloom species on shellfisheries industries Blooms of toxic algal species are common occurrences in shellfish culture areas worldwide, and pose various threats to public health and aquaculture economy. Here, we summarize three threats to shellfisheries.

19.3.1 Phycotoxin accumulation The marine toxins of algal origin, collectively known as ‘phycotoxins’, are a diverse group of biologically active compounds, many of which are acutely toxic to humans (Fig. 19.4). Filter-feeding shellfish accumulate toxic algal species during feeding, rendering them vectors in various forms of shellfish poisoning including: paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP), azaspiracid poisoning (AZP). A bloom of a toxic algal species and the subsequent potential threat to public health is the most serious risk to the shellfish industry. PSP is the most common shellfish toxin worldwide. Paralytic shellfish toxins (PSTs), such as saxitoxin, are neurotoxic alkaloids that block sodium channels in cells. Saxitoxin is so potent that one gram could be lethal to several thousand humans. Various derivatives of saxitoxin are produced by three genera of marine dinoflagellates, Alexandrium (Fig. 19.5a), Gymnodinium, and Pyrodinium (Shumway, 1990; Hallegraeff, 1993; Bricelj and Shumway, 1998). Composition and content of the toxin varies among species. Almost all bivalve molluscs, i.e. clams, mussels, oysters, and scallops, feed on these toxic dinoflagellates, and PST accumulates in their tissues. Some carnivorous gastropods and crabs also accumulate PSTs through the food web, i.e. feeding on toxic bivalve molluscs (Shumway et al., 1995; Oikawa et al., 2004). In some cases, the accumulated PST is chemically converted to

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Mechanism of shellfish poisoning Temperature, salinity, run-off, etc.

Consume products Feeding

Phycotoxin producing species Phycotoxin accumulation

Poisoning !

Germination and growth Resting cysts in sediment

Fig. 19.4 Mechanism of phycotoxin-related shellfish poisoning.

A

B

C

Fig. 19.5 Three representative toxic dinoflagellate species that hamper shellfisheries: A. Alexandrium tamarense; B. Karenia mikimotoi; C. Heterocapsa circularisquama.

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an alternate form in shellfish tissues (Oshima, 1995; Bricelj and Shumway, 1998), with concomitant fluctuations of toxicity. Some species (e.g. Saxidomus giganteus, Spisula solodissima) are known to harbor PST in their tissue for up to five years (Shumway et al., 1994; Shumway unpublished). Diarrhetic (DSP) toxins, including polyether okadaic acid (OA) and dinophysistoxin (DTX) derivatives, as well as pectenotoxin (PTX) and yessotoxins (YTX), are found in certain toxic algal species of several genera. DSP toxins, though non-lethal to humans, affect the gastrointestinal system through phosphatase inhibition (Haystead et al., 1989). The causative organisms are the dinoflagellates Dinophysis spp (OA, DTX, and PTX), Protoceratium reticulatum (YTX), Lingulodinium polyedrum (homoYTX), and a few epiphytic and benthic species of Prorocentrum lima. Since the 1970s, the production of DSP by Dinophysis has impacted the scallop and mussel aquaculture industries worldwide, particularly in Europe and Japan. Dinophysis does not have a resting stage, therefore, intensive plankton monitoring coupled with shellfish monitoring are carried out in infected areas. After conducting numerous trials in order to monoculture Dinophysis, several scientists have found that this phytoplankton is a mixotrophic dinoflagellate and can be grown in monoculture by providing the ciliate Myrionecta rubrum as prey in the laboratory (Park et al., 2007). These efforts have helped to clarify the ecophysiology of Dinophysis and the mechanism behind DSP occurrences in shellfish, and the ability to culture Dinophysis will make it possible to carry out controlled experiments with shellfish. ASP is caused by an amino acid that binds to a glutamic acid receptor of a superfluous nerve. Patients first experience gastrointestinal distress within 24 h of eating the contaminated shellfish and symptoms of dizziness, headache, disorientation, and permanent short-term memory loss, or death may occur. In 1987, three people died from eating ASPcontaminated mussels harvested from Prince Edward Island in Canada (Bates et al., 1989). Deaths associated with shellfish consumption are rare thanks to extensive and effective monitoring programs (Shumway, 1990) The pelagic diatoms Pseudo-nitzschia and a benthic species Nitzschia navis-varingica are major domoic acid producers (Bates et al., 1989; Kotaki et al., 2004). These diatoms have a cosmopolitan distribution and are common members of phytoplankton communities in coastal waters. Neurotoxic shellfish poisoning is caused by the dinoflagellate Karenia brevis (formerly Gymnodinium breve) with brevetoxin and its analogues accumulating in shellfish species from the Gulf of Mexico and the Atlantic coast of southern states. NSP has also been reported in New Zealand. Symptoms manifest 1–3 h after eating contaminated shellfish and include numbness, tingling in the mouth, arms and legs, incoordination, and gastrointestinal disturbances. AZP caused by azaspiracids (AZAs) was first identified from the mussel (Mytilus edulis) harvested in Ireland in 1995 (McMahon and Silke, 1996). The occurrence of this toxin is thought to be rare relative to other phyco-

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toxins; however, AZAs have been known to impact shellfish culture areas for extended periods of time around northeastern Europe (James et al., 2002).

19.3.2 Mass mortality and detrimental effects on molluscan shellfish Shellfish-borne phycotoxins usually do not cause shellfish die-offs; thus, shellfish harvesting can resume once the phycotoxins have depurated. In some instances, toxic algal species have had an adverse effect on shellfish physiology and intense blooms have devastated the cultures, i.e. caused a decrease in growth rate and declines in yield through inhibition of byssus production and filtration rates, increase of mortality rate, and suppression of reproduction due to recruitment failure (Ho and Zubkoff, 1979; Gainey and Shumway, 1988; Tracey, 1988; Gallager et al., 1989; Nielsen and Strömgren, 1991; Lesser and Shumway, 1993; Luckenbach et al., 1993; Wikfors and Smolowitz, 1995; Nagai et al., 1996; Bricelj and Lonsdale, 1997; Smolowitz and Shumway, 1997; Bricelj et al., 2001; Matsuyama et al., 2001a,b; Landsberg, 2002). The economic losses of local shellfish industries resulting from the direct impact of toxic algae on shellfish cultivation and natural stock are of great concern. Although some phycotoxins are shown to have weak effects on shellfish physiology (Gainey and Shumway, 1988), other phycotoxins, including some that remain uncharacterized, have potent adverse effects (Matsuyama et al., 1997; Bricelj et al., 2001; Lansberg, 2002). While these uncharacterized toxins are detrimental to shellfish physiology, they are not toxic to mammals and are, therefore, not considered a public health hazard. Loss of shellfish due to toxic algal species is a concern for the aquaculture industry, especially as blooms continue to increase in frequency and intensity, and hitherto non-toxic species continue to be identified. The dinoflagellate Karenia mikimotoi (formerly Gyrodinium aureolum, Gymnodinium cf. nagasakiense, Gymnodinium mikimotoi, Fig. 19.5b) is widely known as a shellfish-killing species (Tangen, 1977; Smolowitz and Shumway, 1997; Gentien, 1998; Matsuyama et al., 1999; Lansberg, 2002). The first report of a bloom of K. mikimotoi and associated shellfish death was mentioned in Nishikawa (1903). He found that red tide due to K. mikimotoi killed a number of pearl oysters (Pinctada fucata martensii) around culture grounds. Half of the individuals died within 44 h of exposure to K. mikimotoi. Oda (1935) also reported large-scale blooms due to K. mikimotoi and the associated death of pearl oysters from Gokasyo Bay and Nishikawa (1903). Since the 1960s, this species has formed massive blooms around European and East Asian coastal waters. These blooms have been associated with serious damage to finfish fisheries and shellfish cultivation (Cho, 1979; Honjo, 1994). Experimental efforts have shown that the mussel Mytilus galloprovincialis reduces filtration rate when exposed to concentrations greater than 5 × 105 cells/L of K. mikimotoi, and death occurs at

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concentrations greater than 5 × 106 cells/L (Widdows et al., 1979; Matsuyama et al., 1998). Recently, K. brevis, which is closely related to K. mikimotoi, has been shown to have various detrimental effects on bivalve molluscs (Leverone et al., 2007). The toxic effect of the genus Karenia on shellfish is thought to be common, but the detailed mechanism by which the species affects shellfish should be clarified in the future. In 1985, a massive bloom of the picoplankter Aureococcus anophagefferens occurred in the coastal bays of Long Island Sound, USA. It is recognized as a prolonged discoloration of the water commonly referred to as ‘brown tide’ (Cosper et al., 1987). The occurrence of brown tide continues to impact the commercially important bay scallop industry, resulting in severe mortality and drastically reduced recruitment of 80 % of the harvest (Cosper et al., 1990). Further, brown tide affected other commercially important industries, i.e. northern quahog or hard clam (Mercenaria mercenaria) and mussel (Mytilus edulis) farming (Tracey, 1988; Gallager et al., 1989; Shumway, 1990). Similar bloom events occurred concurrently in Narragansett Bay, RI, Barnegat Bay, and the Texas coast. These events were associated with massive die-offs of marine life (Tracey, 1988; Gallager et al., 1989; Shumway, 1990; Bricelj and Lonsdale, 1997). Laboratory experiments also revealed that exposure of the mussel to A. anophagefferens culture isolates resulted in significant inhibitory effects on ciliary movement (Gainey and Shumway, 1991) and filtration rate (Bricelj and Lonsdale, 1997). In another example, the 1988 toxic bloom of the ichthyotoxic flagellate Chrysochromulina polylepis extended over most of the Skagerrak and the entire Kattegat Seas, an area of about 75 000 km2, and lasted four weeks, from May to early June (Granéli et al., 1993). More recent blooms of a different species of Chrysochromulina bloomed along another European coast and caused fish farm mortalities, but there were no reported impacts on shellfish. The novel dinoflagellate Heterocapsa circularisquama (Fig. 19.5c) is the causal agent of red tide on the Japanese coast and has destroyed the shellfish aquaculture industries around the western part of Japan. This dinoflagellate exhibits detrimental effects on specific bivalve and gastropod molluscs. Figure 19.6 shows catastrophic death of shellfish during a bloom of H. circularisquama. Novel and persistent blooms of this species in Japanese coastal waters have adversely affected shellfish cultivation of the Pacific oyster Crassostrea gigas, Manila clam Ruditapes philippinarum, pearl oyster Pinctada fucata martensii, and abalones (Haliotis discus and H. diversicolor aquatilis). Economic losses to the shellfish aquaculture industry resulting from the direct mortality of marketable products were estimated at a minimum of 10 billion yen (approximately 93 million dollars) since the 1980s. Interestingly, there is no evidence that this species poses a threat to humans. Several hemolytic toxins have been characterized as the possible agents of shellfish mortality (Oda et al., 2001; Sato et al., 2001); however,

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1

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2

3

5

Fig. 19.6 Shellfisheries damage due to the toxic dinoflagellate Heterocapsa circularisquama: 1. pearl oyster Pinctada fucata martensii; 2–3. Pacific oyster Crassostrea gigas; 4. mytilid Musculista senhousia; 5. the mussel Mytilus galloprovincialis.

the principal toxin that causes a potent detrimental effect on shellfish species remains unclear. The ichthyotoxic raphidopyte Heterosigma akashiwo is a widespread species that remains a potential threat not only to fish farming (Honjo, 1994), but also to shellfish cultivation. Blooms of H. akashiwo have disrupted cultivation and the harvesting of wild stocks of shellfish in Canada, Portugal, and New Zealand (see Hegaret et al., 2007a). Exposure of commercially important shellfish to H. akashiwo under laboratory conditions shows detrimental effects, including damage to the hepatopancreas of Crassostrea virginica (Keppler et al., 2005), inhibitory effects on the early development of larvae in the scallop Argopecten irradians (Wang et al., 2006), and death of eye-spot larvae (Wang et al., 2006) and adult (Hégaret and Wikfors, 2007) bay scallops, Argopecten irradians. Toxic Alexandrium, responsible for PSP, also has an adverse effect on shellfish physiology (Gainey and Shumway, 1988; Matsuyama et al., 2001b;

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Yan et al., 2001; Yan et al., 2003; Ford et al., 2008). In these cases, the shellfish were affected by PST and/or uncharacterized toxins as some Alexandrium spp. showed PST-independent cytotoxic effects on shellfish physiology and function (Matsuyama et al., 2001b; Juhl et al., 2007; Ford et al., 2008). Recent studies imply that Alexandrium produces a cytotoxic substance different from PST that may have deleterious effects on shellfish species (Emura et al., 2004; Katsuo et al., 2007; Juhl et al., 2007; Ford et al., 2008). The effect of Alexandrium on shellfish is species-specific, and its toxicity varies significantly among the isolates (Ford et al., 2008; Hégaret et al., 2008b). Further investigation is still necessary to clarify the interaction between toxic Alexandrium species and shellfish species, especially with regard to new shellfish species being considered as candidates for aquaculture.

19.3.3 Halo effects or value degradation of products It is generally difficult to assess the total cost of damages to fisheries and aquaculture by a HAB event, except for direct mortality of shellfish by massive blooms such as red tides (Shumway, 1990). From an economic point of view, cost of prolonged shellfish closures should include indirect effects on the economy, i.e. declines in employment of fishers and secondary industries such as processing, shippers, and suppliers (Shumway, 1990; Anderson et al., 2000). Further, HAB events sometimes cause dramatic declines in the demand for a particular product. This is often a result of the distribution of misleading information and failure to distinguish between toxic and safe shellfish (Shumway, 1990; Matsuyama, 1999). For example, in 1988 a massive bloom of Heterocapsa circularisquama in Japan was associated with the catastrophic death of bivalves and gastropods, but did not actually cause any human illnesses or pose a risk to public health. The media broadcast this event with a number of pictures showing extensive patches of discolored water and dead shellfish for more than two weeks. The purchase and consumption of oysters and Manila clams virtually ceased and market price of this product dropped more than 30 %. In the raw shellfish market, the safety and freshness of the product is regarded as the key factor in the sustained price of shellfish. Despite the fact that quality and freshness of product is tested by microbiologists and laboratory technicians, consumers may still observe the unusual phenomena of red discoloration of the meat and unfavorable flavor. Discolored meat is often described as ‘blood oyster or scallop’ (Kat, 1984; Carver et al., 1996; Rhodes et al., 2001), and this can lead to degradation of the market value of the product. One cause is the ciliate Myrionecta rubrum (formerly Mesodinium rubrum) which also forms blooms. Its blooms account for approximately 5 % of the total occurrence of red tide in Japanese coastal waters (Oh et al., 2005). This ciliate harbors photosynthetic endosymbionts rich in phycobilins that reproduce by spores, and are associated with the red discoloration in some bivalve molluscs. The phycoerythrins are wine-colored, water-soluble

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pigments that accumulate in the digestive gland of the shellfish by feeding, and then gradually drip as if ‘bleeding’ during processing (Carver et al., 1996). Dinoflagellates may also be responsible for shellfish discoloration. Some dinoflagellate species contain a peridinin–protein complex (PCP, Haidak et al., 1966). PCP is a water-soluble pigment that may lead to the discoloration of shellfish. In general, this water-soluble pigment is concentrated in the gut; hence, when the bloom has ceased, shellfish species are usually free of unusual coloration. Persistence of reddish discoloration in the oyster, however, may be due to the haptophyte Chrysochromulina quadrikonta that occurs in Japanese coastal waters. This species posed a serious economic threat to Japan in 2000. In this case, the site of discoloration in the oyster was limited to the gills and labial palps, not to the gut, which is common among other algal species. C. quadrikonta forms blooms over short time periods (c. ten days) but, in this case, discoloration of the gills and labial palps persisted for several months or more. This resulted in a decrease in the harvest of more than 70 % as shown in Fig. 19.7.

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Fig. 19.7 Temporal change of the Pacific oyster Crassostrea gigas production (January) by three aquaculture companies located in Mie Prefecture, Japan. The haptophyte Chrysochromulina quadrikonta bloom and associated red discoloration in the oyster occurred from December 1999 through January 2000. During this incident, oyster dieoff and phycotoxin accumulation did not occur, but marketability was lost due to marked red discoloration of the gill. A slight decrease of production in 1998 was caused by mass mortality due to the unusually high water temperature during the summer.

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In Australia, the bitter taste of oysters was associated with the bloom of the diatom Rhizosolenia chunii (Parry et al., 1989) and, while it was reported that the market value was lost, no definitive values were reported. Rhodes et al. (2001) reported a ‘peppery taste’ in mussels harvested from a site experiencing a Heterosigma akashiwo bloom. Consumers sometimes report a strong sulfide-like smell during preparation and consumption of oysters farmed in Hiroshima Bay and other regions. These reports are often correlated to blooms of non-toxic dinoflagellates (Scrippsiella trochoidea, Heterocapsa triquetra, Prorocentruma spp.). The suspected causative agent is dimethylsulfoniopropionate (DMSP), which is widely produced by various phytoflagellate species (Townsend and Keller, 1996).

19.4 Prevention of harmful algal bloom threats Several studies have explored mechanisms for minimizing the impact of harmful algae and their phycotoxins on public health, aquaculture, fisheries resources, and marine ecosystems. Here, we discuss several techniques for mitigation of harmful algal blooms with respect to the shellfish industry.

19.4.1 Forecasts of the trajectory of the event by plankton monitoring As referenced above, harmful and toxic algal blooms cause outbreaks of shellfish poisoning and have adverse effects on shellfish cultivation. Thus, simply monitoring for the presence of toxic species is valuable in predicting trajectories of bloom events over time. Many toxic algal species form a resting cyst during non-bloom periods, which are considered inoculants of the initial population in the water column by germination (Anderson, 1994). The quantity of cysts in the farming bed profile reflects the outbreak history of toxic species. A number of efforts suggest that recurring outbreaks of PSP due to Alexandrium are linked to the existence of a dormant cyst stage in coastal sediment (e.g. Itakura and Yamaguchi, 2001; Harper et al., 2002; Kim et al., 2002). In order to determine the potential risk associated with an outbreak of toxic algal species, horizontal measurements of the distribution and density of cysts of toxic species around the farming bed and related area are recommended. These efforts may also help to identify other fishing grounds that are more suitable for cultivation. Toxic algal species are generally identified by microscopic observation of morphological features; however, identification of toxic Alexandrium and Pseudo-nitzschia species by this technique is not accurate due to subtle differences in morphological characteristics. The recent development of immunological and molecular detection methods has been proposed in order to identify these toxic algal species to the lowest possible taxonomic level on a real-time basis (Hosoi-Tanabe and Sako, 2005; Scholin et al., 1996; Culverhouse et al., 2006). Recent progress in developing detection

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techniques that do not use microscopic observation for identification of toxic algal species, e.g. rapid test kits, may become more popular in monitoring programs in the future. Currently, while the test kits are reasonably accurate, they are prohibitively expensive for use on aquaculture farms. Although many toxic algal species are distributed along the coast, some species, such as Karenia brevis, grow in open waters. Routine oceanographic surveys using a research vessel have determined the frequency and area of occurrence of some toxic algal species, and results show that they are rapidly moving and expanding along coastal current boundaries, eddies, and wind-driven currents (Raine et al., 2001). Toxic algal species have photosynthetic and accessory pigments, which make it possible to track their broad distribution by satellite images (Tester and Steidinger, 1997). Because the fluorescence profiles do not differentiate between toxic and non-toxic species, tracking by satellite is only possible in areas where the toxic species are dominant, such as the Gulf of Mexico. Enumeration of causative toxic species based on oceanographic survey and satellite images does not predict the sought-after level of toxicity. Further, the content and composition of a toxic algal cell can vary by an order of magnitude, due to environmental factors such as water temperature, salinity, and cell growth phase (White, 1986; Boyer et al., 1987; Ogata et al., 1987) and, for this reason, plankton monitoring must be performed concurrently with shellfish monitoring. Nevertheless, plankton monitoring still contributes to scientific knowledge and reduces the cost of shellfish monitoring (Rhodes et al., 2001). There is increasing evidence for the efficacy of another assay that measures shellfish toxicity (e.g. Garthwaite, 2000). Recent progress in chemical and immunochemical research on toxins is leading to the development of a replacement assay tool that will be inexpensive and easy to use. Highpressure liquid chromatography (HPLC) and/or liquid chromatography– mass spectrometry/mass spectrometry (LC–MS/MS) can be used to quantify toxicity of natural toxins present in suspended matter (Oshima, 1995; Suzuki et al., 1997). These methods require time for extraction and purification, as well as specialized equipment. However, enzyme immunoassay systems for phycotoxin detection have been developed (Jellett et al., 2002; Kawatsu et al., 2002), and these rapid test kits can be used as an immunoassay to determine contamination in shellfish based on the direct detection of phycotoxin in natural suspended matter or shellfish tissues.

19.4.2 Blanket closure during toxic algal blooms Safe marketing of shellfish is only feasible under a strict regulatory regime for monitoring of phycotoxins. The most effective means of quality control during outbreaks of toxic algae is either by blanket closure during certain times of the year or by instituting a shellfish toxicity monitoring program (Shumway et al., 1995). In general, cooking does not deactivate toxins.

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Therefore, shellfish should be eaten from areas known to be monitored regularly for the presence of toxins in compliance with an authorized public health agency. Since the 1980s, many regions of the world have established monitoring programs and the number of new programs continues to increase (Shumway, 1990; Shumway et al., 1995). It is common for such programs to be established in areas plagued by harmful algal blooms. It is, however, important for growers and harvesters to be aware of the potential for outbreaks of toxic algae even in areas not previously impacted. When monitored closely, early detection of blooms leads to minimized impacts on shellfish industries. In areas where shellfish aquaculture has not been hampered by the presence of toxic algal species (due to the success of monitoring programs), there are fewer public safety issues and minimal disruption of harvesting. Paralytic shellfish toxins are the most common among the shellfish poisons. Almost all countries still use the standard mouse bioassay of the Association of Official Analytical Chemists International (AOAC, 1990) as the method of toxin analysis (Shumway et al., 1995) and apply a level of 80 μgSTXeq/100 g (whole meat, see Shumway et al., 1995) as the safety threshold. PSP monitoring has been conducted using this method for over 50 years and, to date, the AOAC mouse bioassay remains the only method of toxin detection accepted by many countries. While this method has a low sensitivity (c. 32 μgSTXeq/100 g), development of more sensitive, rapid, and cost-effective methods is necessary to develop intensive toxin monitoring systems in shellfisheries and farming ground. Other methods, e.g. instrumental analysis (HPLC and LC–MS) and the enzyme-linked immunoassay (ELISA) method, have been developed (Lawrence et al., 1995; Jellett et al., 2002; Kawatsu et al., 2002), but the application of accurate methods for toxin analysis has also been hampered by the lack of pure analytical standards of the various toxins for validated analytical methodology. Further development and validation of toxic analytical methodology and reference materials for phycotoxins is highly desirable to regulate and control seafood safety and to make regular testing on individual farms simple and affordable.

19.4.3 Selection of aquaculture sites In order to minimize the impacts of toxic algal blooms, careful selection of aquaculture sites is essential. Areas prone to toxic algal blooms and zones where shellfish are either fished or cultured overlap with expansion of shellfish industries worldwide. The quantity or presence of the cyst stage of toxic species in the bottom sediment is a good indicator of past bloom history, and serves as valuable criteria for selection of nursery grounds. At finfish farming sites, aquaculturists place fish farming cages into the middle and bottom layer of the same farming ground in order to avoid exposure to the surface patchiness of (e.g. Rensel, 1995) raphidophytes such as Heterosigma akashiwo and the dinoflagellate Cochlodinium polykrikoides.

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Desbiens and Cembella (1993) investigated potential application of this vertical relaying technique to mussels in the water column as a means of minimizing PSP accumulation. Because shellfish cultivation usually takes place in shallow water, these methods are likely to be less effective, since the toxic species is vertically well mixed. Moreover, it is known that many toxic dinoflagellate species migrate vertically over the course of the day in coastal areas, e.g., the dinoflagellate Gymnodinium catenatum, a known PST-producer, swims up and down from the surface to approximately 20 m (Doblin et al., 2006). The dinoflagellate Karenia mikimotoi also exhibits similar patterns of vertical migration around farming sites (Koizumi et al., 1996), as do various species of Alexandrium. Therefore, in shellfish culture, it appears to be difficult to reduce damage from toxic algae by vertically moving cultured organisms. In shellfish cultivation areas that utilize tidal flats as farming beds, it is difficult, if not impossible, to avoid the impact of toxic algal blooms. Land-rearing technology is currently being developed and may serve to isolate the harvest products temporarily from infected habitat and avoid exposure to toxic algal species; however, the cost effectiveness of this tactic remains to be demonstrated.

19.4.4 Cultivation treatment: depuration of phycotoxins Depuration is a routine part of the cultivation process that reduces bacterial and viral contamination in shellfish (Shumway, 1990), and almost all bivalves commonly accumulate phycotoxins in their digestive gland. Toxins accumulated in the digestive gland are more readily detoxified than those toxins bound in other tissues (particularly in the muscle; see Bricelj and Shumway, 1988; Shumway et al., 1988; Cembella et al., 1994). In fact, many bivalves will gradually release the phycotoxins and detoxify themselves after the toxic algal bloom has ceased or after they have been transferred to a bloom-free site. The simplest method of detoxification is to transfer the contaminated shellfish to uninfected waters or to maintain them in flow-through seawater under controlled conditions (depuration). It is recognized, however, that transfer of cultivation pens or rafts during the occurrence of toxic algal blooms may cause expansion of the bloom (Hégaret et al., 2008a). Certain shellfish species exhibit a rapid detoxification rate of phycotoxins, and for these species, depuration could be effective. A number of previous trials reported detoxification of phycotoxincontaminated shellfish (Novaczek et al., 1992; Sekiguchi et al., 2001; Blanco et al., 2003; Choi et al., 2003; Lassus et al., 2000, Takata et al., 2008). It should also be noted that the accumulated phycotoxins in individual shellfish varies greatly. In general, oysters (Shumway et al., 1990) and mussels show rapid detoxification rates in comparison with other species, i.e. butter clams, surfclams, and scallops (Bricelj and Shumway, 1995; Shumway et al., 1995). A detoxification curve of the Pacific oyster Crassostrea gigas, highly contaminated by PST-producing Alexandrium tamarense (Takata et al., 2008),

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is shown in Fig. 19.8. Oysters release c. 62 % of PST within 48 h of being held in running seawater. In contrast to mussels and oysters, the available data suggest that among filter-feeding bivalve molluscs, several species of clams and scallops remain toxic for extended periods (Shumway, 1990). Scallop cultivation is a global aquaculture industry, and the effects of toxic algal blooms on scallop culture are extensive (Shumway and Cembella, 1993). Toxin retention in scallops can last up to several months from the cessation of the toxic algal bloom (Shumway, 1990), e.g. the sea scallop Placopecten magellanicus can remain toxic throughout the year (Bourne, 1965; Shumway and Cembella, 1993). To date, an available depuration method does not exist to eliminate the toxins in scallop tissue.

19.4.5 Clay scatter It is generally difficult to control blooms of toxic algal species in any waters, including shellfish farming areas. Aquaculture industries seek potential

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control strategies (mitigation) that reduce the risk of toxic algal blooms, and one of the most commonly employed is the use of clay (see Shumway et al., 2003 for summary). Preliminary studies suggest the efficacy of chemical substances and algicidal microbes to prevent the occurrence of toxic algal species (Nagasaki et al., 1999; Sun et al., 2004); however, chemical substances and algicidal microbes may have adverse effects on other aquatic organisms and their habitats, and an extensive environmental impact assessment of these mitigation strategies is required before considering introducing them to shellfish cultivation sites. Among previous efforts, clay scatter is the most effective and simple mitigation technique to remove toxic algal species. The use of sprayed clay was first proposed by Shirota (1989) to prevent the red tide threat due to the ichthyotoxic raphidophyte Chattonella and the dinoflagellate Cochlodinium polykrikoides. When certain clays were introduced to toxic algal blooms, the targeted algal cells flocculated, due to sudden change of pH and ion charge, and they formed larger particles with clay. These particles began to sink and removed toxic algal species from the water column. While spraying clay into red tide water, a strong increase in turbidity occurs, but the water recovers to a transparent state within several hours. In the countries of East Asia, such as Japan and South Korea, clay spraying is a routine process to prevent the threat of toxic algal species to finfish farming. In South Korea, this mitigation strategy is conducted on large spatial scales. Warehouses are used to store huge amounts of clay and the equipment that uniformly sprays clay into seawater (Fig. 19.9). The efficacy of this clay scatter method varies with different algal species.

Fig. 19.9 Clay spraying against surface patches of the toxic dinoflagellate Cochlodinium polykrikoides in Korean coastal waters.

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The impact of clay scatter on the environment needs careful consideration. Shirota (1989) emphasized that large particles which captured toxic algal species did not show acute toxicity to aquatic organism that were tested; however, a significant increase of organic matter to bottom sediments was observed. Laboratory rearing experiments demonstrated that some benthic organisms, such as a sea cucumber, showed higher growth rates when fed with a clay–toxic algal complex, and Shumway et al. (2003) and Seo et al. (2008) demonstrated a direct deleterious impact of highdensity exposure of clay on several filter feeding organisms. The effect of clay on invertebrates is species-specific and generally oysters showed a high tolerance to clay exposure (Shumway et al., 2003; Seo et al., 2008). In shallow water, however, a strong current driven by tidal movement and wind can easily resuspend clay and possibly transport the clay and clay–algal complexes to distant areas. There is also a report that excess and repeated clay loading has an adverse effect on the physiology of certain shellfish species (Shumway et al., 2003; Archambault et al., 2004). The use of clay as a strategy for mitigation of HABs should therefore be approached with extreme caution (Shumway et al., 2003).

19.5 Conclusions The impact of toxic algal species is a serious and major barrier in the global development of shellfish cultivation. Although control of harmful algal blooms appears to be difficult if not impossible, scientific-based prevention techniques have helped in reducing the threat of toxic algal species to shellfish culture. The cultivation of filter-feeding shellfish continues to grow internationally as a sustainable and environmentally acceptable form of aquaculture. As a result, research on the impacts of harmful algal species on shellfish and potential mitigation strategies is being actively pursued.

19.6 Acknowledgements We extend our appreciation to Dr N Hata of Mie Prefectural Fisheries Experimental Station for valuable information on the Chrysochromulina quadrikonta and associated red oyster incidence. Thanks are also due to Drs YT Park and HM Bae, National Fisheries Research and Development Institute (NFRDI) for providing the photograph of clay spraying. We are indebted to Kari Heinonen for editorial assistance and to Pam Shephard, librarian at the Bigelow Laboratory for Ocean Science for her ability to locate rare and esoteric references.

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19.7 References anderson d m, hoagland p, kaoru y, and white a w (2000) Estimated annual economic impacts from Harmful Algal Blooms (HABs) in the United States, Woods Hole Oceanographic Institution Technical Report, WHOI-2000-11, Woods Hole Oceanographic Institution, Woods Hole, MA. anderson d m (1994) Red tide, Sci Am, 271, 52–8. aoac (1990) Paralytic shellfish poison. Biological method. Final action, in Hellrich, K. (ed.), Official Method of Analysis, 15th ed, Association of Official Analytical Chemists, Arlington, Va, 881–8, Sec 959.08. archambault m c, bricelj v m grant j and anderson d m (2004) Effects of suspended and sedimented clays on juvenile hard clams, Mercenaria mercenaria, within the context of harmful algal bloom mitigation, Mar Biol, 144(3), 553–65. bates s s, bird c j, de freitas a s w, foxall r, gilgan m, hanic l a, johnson g r, mcculloch a w, odense p, pocklington r, quilliam m a, sim p g, smith j c, subba rao d v, todd e c d, walter j a, and wright j l c (1989) Pennate diatom Nitzschia pungens as the primary source of domoic acid a toxin in shellfish from eastern Prince Edward Island, Canada, Can J Fish Aquat Sci, 46, 1203–15. blanco j, fernandez m l, miguez a and morono a (2003) Depuration of okadaic acid (Diarrhetic Shellfish Toxin) in mussels, Mytilus edulis (Linnaeus), feeding on different quantities of nontoxic algae, Aquaculture, 218(1–4), 277–91. bourne n (1965) Paralytic shellfish poison in sea scallops (Placopecten magellanicus, Gmelin), J Fish Res Board Can, 22, 1137–49. boyer g l, sullivan j j, andersen r j, harrison p j and taylor f j r (1987) Effects of nutrient limitation on toxin production and composition in the marine dinoflagellate Protogonyaulax tamarensis, Mar Biol, 96, 123–8. bricelj m v and lonsdale d j (1997) Aureococcus anophagefferens: causes and ecological consequences of brown tides in U.S. mid-Atlantic coastal waters, Limnol Oceanogr, 42(5), 1023–38. bricelj v m and shumway s e (1998) Paralytic shellfish toxins in bivalve molluscs: occurrences, transfer kinetics, and biotransformation, Rev Fish Sci, 6, 315–83. bricelj m v, macquarrie s p and schaffner r a (2001) Differential effects of Aureococcus anophagefferens isolates (‘brown tide’) in unialgal and mixed suspensions on bivalve feeding, Mar Biol, 139, 605–16. carver c e, mallet a l, warnock r and douglas d (1996) Red-coloured digestive glands in cultured mussels and scallops: the implication of Mesodinium rubrum, J Shellfish Res, 15(2), 191–201. cembella a d, shumway s e and larocque r (1994) Sequestering and putative biotransformation of paralytic shellfish toxins by the sea scallop Placopecten magellanicus: seasonal and spatial scales in natural populations, J Exp Mar Biol Ecol, 180, 1–22. cho c h (1979) Mass mortalities of oyster due to red tide in Jinhae Bay in 1978, Bull Korean Fish Soc, 12, 27–33. choi m c, hsieh d p h, lam p k s and wang w x (2003) Field depuration and biotransformation of paralytic shellfish toxins in scallop Chlamys nobilis and greenlipped mussel Perna viridis, Mar Biol, 143(5), 927–34. coen l d, brumbaugh r d, bushek d, grizzle r, luckenbach m w, posey m h, powers s p and tolley s g (2007) Ecosystem services related to oyster restoration, Mar Ecol Prog Ser, 341, 303–7. cosper e m, dennison w c, carpenter e j, bricelj v m, mitchell j g, kuenstner s h, colflesh d and dewey m (1987) Recurrent and persistent brown tide blooms perturb coastal marine ecosystem, Estuaries, 10(4), 284–90.

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culverhouse p f, williams r, simpson b, gallienne c, reguera b, cabrini m, fonda-umani s, parisini t, pellegrino f a, pazos y, wang h, escalera l, moron˜o a, hensey m, silke j, pellegrini a, thomas d, james d, longa m a, kennedy s and del punta g (2006) HAB Buoy: a new instrument for in situ monitoring and early warning of harmful algal bloom events, African J Mar Sci, 28(2), 245–50. dame r f (1996) Ecology of Marine Bivalves; an Ecosystem Approach, CRC, Boca Raton, FL. desbiens m and cembella a d (1993) Minimization of PSP toxin accumulation in cultured blue mussels (Mytilus edulis) by vertical displacement in the water column, in Smayda T J and Shimizu Y (eds), Toxic Phytoplankton Blooms in the Sea, Elsevier, Amsterdam, 395–400. doblin m a, thompson p a, revill a t, butler e c v, blackburn s i and hallegraeff g m (2006) Vertical migration of the toxic dinoflagellate Gymnodinium catenatum under different concentrations of nutrients and humic substances in culture, Harmful Algae, 5(6), 665–77. emura a, matsuyama y and oda t (2004) Evidence for the production of a novel proteinaceous hemolytic exotoxin by dinoflagellate Alexandrium taylori, Harmful Algae, 3(1), 29–37. fao (2007) The State of World Fisheries and Aquaculture 2006, Food and Agriculture Organization of the United Nations, Rome. ferreira j g, hawkins a j s, monteiro p, service m, moore h, edwards a, gowen r, lourenco p, mellor a, nunes j p, pascoe p l, ramos l, sequeira a, simas t, and strong j (2007) SMILE – Sustainable Mariculture in Northern Irish Lough Ecosystems – Assessment of Carrying Capacity for Environmentally Sustainable Shellfish Culture in Carlingford Lough, Strangford Lough, Belfast Lough, Larne Lough and Lough Foyle, IMAR – Institute of Marine Research, Lisbon. ford s e, bricelj v m, lambert c and paillard c (2008) Deleterious effects of a non PST bioactive compounds from Alexandrium tamarense on bivalve hemocytes, Mar Biol, 154, 241–53. furuhata k, kakino j, miyama y and fukuyo y (1996) Elimination of cysts of the toxic dinoflagellates Alexandrium spp. contaminated in hard clam, Nippon Suisan Gakkaishi, 62(5), 813–14. gainey l f jr and shumway s e (1988). A compendium of the responses of bivalve molluscs to toxic dinoflagellates, J Shellfish Res, 7, 623–8. gainey l f jr and shumway s e (1991) The physiological effect of Aureococcus anophagefferens (‘brown tide’) on the lateral cilia of bivalve mollusks, Biol Bull, 181, 298–306. gallager m s, stoecker k d and bricelj m v (1989) Effects of the brown tide alga on growth, feeding physiology and locomotory behavior of scallop larvae (Argopecten irradians), in Cosper E M, Bricelj M V and Carpenter E J (eds), Novel Phytoplankton Blooms: Causes and Impacts of Recurrent Brown Tides and Other Unusual Blooms, Springer-Verlag, Berlin, 511–41. garthwaite i (2000) Keeping shellfish safe to eat: a brief review of shellfish toxins, and methods for their detection, Trends Food Sci Technol, 11(7), 235–44. granéli e and haraldsson c (1993) Can increased leaching of trace metals from acidified areas influence phytoplankton growth in coastal waters? Ambios, 22, 308–11. granéli e, paasche e and maestrini s y (1993) Three years after the Chrysochromulina polylepis bloom in Scandinavian waters in 1988: some conclusions of recent research and monitoring, in Smayda T J and Shimizu Y (eds), Toxic Phytoplankton Blooms in the Sea, Elsevier, New York, 23–32. gentien p (1998) Bloom dynamics and ecophysiology of the Gymnodinium mikimotoi complex, in Anderson D M, Cembella A D and Hallegraeff G M (eds),

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Physiological Ecology of Harmful Algal Blooms, NATO ASI Series, SpringerVerlag, Berlin, 155–73. gilbert f, souchu p bianchi m and bonin p (1997) Influence of shellfish farming activities on nitrification, nitrate reduction to ammonium and denitrification at the water-sediment interface of the Thau lagoon, France, Mar Ecol Prog Ser, 151, 143–53. gowen r j and bradbury n b (1987) The ecological impact of salmonid farming in coastal waters: a review, Oceanogr Mar Biol Ann Rev, 25, 563–75. haidak d j, christopher k, mathews c k and sweeney b m (1966) Pigment protein complex from Gonyaulax, Science, 152(3719), 212–13. hallegraeff g m (1993) A review of harmful algal blooms and their apparent global increase, Phycologia, 32, 79–99. hallegraeff g m (1998) Transport of toxic dinoflagellates via ships’ ballast water: bioeconomic risk assessment and efficacy of possible ballast water management strategies, Mar Ecol Prog Ser, 168, 297–309. harper f m, hatfield e a and thompson r j (2002) Recirculation of dinoflagellate cysts by the mussel, Mytilus edulis L., at an aquaculture site contaminated by Alexandrium fundyense (Lebour) Balech, J Shellfish Res, 21, 471–7. hashimoto t, matsuoka s, yoshimatsu s, miki k, nishibori n, nishio s and noguchi t (2002) First paralytic shellfish poison (PSP) infestation of bivalves due to toxic dinoflagellate Alexandrium tamiyavanichii, in the Southeast Coasts of the Seto Inland Sea, Japan, J Food Hyg Soc Jpn, 43(1), 1–5. haystead t a j, sim a t r, carling d, honnor r c, tsukitani y, cohen p and hardie d g (1989) Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism, Nature, 337, 78–81. hégaret h, wikfors g h and shumway s e (2007) Diverse feeding responses of five species of bivalve molluscs when exposed to three species of harmful algae, J Shellfish Res, 26(2), 549–59. hégaret h and wikfors g h (2007) Time-dependent changes in hemocytes of eastern oysters, Crassostrea virginica, and northern bay scallops, Argopecten irradians irradians, exposed to a cultured strain of Prorocentrum minimum, Harmful Algae, 4(2), 187–99. hégaret h, shumway s e, wikfors g h, pate s and burkholder j m (2008a) Potential transport of harmful algae through relocation of bivalve mollusks, Mar Ecol Prog Ser, 361, 169–81. hégaret h, wikfors g h, soudant p, lambert c, shumway s e, bérard j b and lassus p (2008b) Toxic dinoflagellates (Alexandrium fundyense and A. catenella) have minimal apparent effects on oyster hemocytes, Mar Biol, 152(2), 441–7. ho m s and zubkoff p l (1979) The effects of a Cochlodinium heterolobatum bloom on the survival and calcium uptake by larvae of the American oyster, Crassostrea virginica, in Taylor D L and Selinger H H Y (eds), Toxic Dinoflagellate Blooms, Elsevier, New York, 409–12. honjo t (1994) The biology and prediction of representative red tides associated with fish kills in Japan, Rev Fish Sci, 2, 225-53. honjo t, imada n, ohshima y, maema y, nagai k, matsuyama y and uchida t (1998) Potential transfer of Heterocapsa circularisquama with pearl oyster consignments, in Reguera B, Blanco J, Fernandez M L and Wyatt T (eds), Harmful Algae, Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, Paris, 224–6. hosoi-tanabe s and sako y (2005) Species-specific detection and quantification of toxic marine dinoflagellates Alexandrium tamarense and A. catenella by real-time PCR assay, Mar Biotech, 7(5), 506–14.

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20 Advances in microalgal culture for aquaculture feed and other uses M. R. Tredici, N. Biondi, E. Ponis, L. Rodolfi, Università degli Studi di Firenze, Italy, and G. Chini Zittelli, Istituto per lo Studio degli Ecosistemi, Italy

Abstract: The state of the art of microalgae biotechnology, particularly focusing on new culture techniques and actual and potential uses of microalgae in human and animal nutrition, in cosmetics and pharmaceutics, and for environmental applications, is described. Some examples of the world’s largest commercial plants in the field are presented. For the future, it is possible to foresee a huge increase in the demand for cultured algae, in terms of both quantity and diversity. For example, aquaculture will require new animal species and, consequently, new microalgae to fulfil their nutritional needs will be necessary. The production of algae for high-value markets (aquaculture, food supplements, nutraceuticals, pharmaceuticals) will be developed through the search for, isolation and cultivation of new algal strains endowed with the activity of interest. Algal biomass might become an important source of biofuels, especially if its production will be carried out in low-cost photobioreactors and associated with wastewater treatment and greenhouse gas abatement. Key words: microalgae, photobioreactors, aquaculture feeds, nutraceuticals, bioactive molecules, biofuels, wastewater treatment.

20.1 Introduction Edible blue–green microalgae or cyanobacteria, including Nostoc and Arthrospira (formerly Spirulina) species, have been part of the human diet for many centuries. However, the modern era of microalgal biotechnology begins in the early 1950s, when the first mass cultures were attempted. Since then, numerous technologies for mass cultivation and exploitation of microalgae have been suggested. Today, microalgae and cyanobacteria used for commercial exploitation are either harvested from natural habitats or obtained through more or less controlled cultivation processes in open

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ponds or photobioreactors (PBR) (Section 20.2). One of the most important applications of microalgae is in aquaculture, where large amounts of selected species with high nutritional value are required to feed molluscs, crustaceans, fish larvae and zooplankton (Section 20.3). Due to their great biodiversity, microalgae can produce an enormous variety of high-value compounds for human nutrition, medical applications, cosmetics and agrochemical industry (Section 20.4). Recent trends in drug research suggest that microalgae and cyanobacteria are the most promising groups to furnish novel pharmacologically active (including anticancer, antimicrobial and antiviral) substances (Section 20.5). An emerging use of microalgae is for wastewater remediation in combination with carbon sequestration and biofuel production (Section 20.6). This chapter aims to illustrate the state-of-the-art of microalgae biotechnology, particularly focusing on new culture techniques and actual and potential uses of microalgae in human and animal nutrition, in cosmetics and pharmaceutics, and for environmental applications, with some examples of the world’s largest commercial plants in the field.

20.2 Current status and new techniques for microalgae culture Nowadays, between 8000 and 10 000 tons (Pulz and Gross, 2004; Becker, 2007) of algae biomass are produced annually, mainly for use as human food supplements and animal feed. Table 20.1 shows the present status of commercial production and the main application areas of microalgae. The market size of microalgae products was estimated to have a retail value of d3000–4000 million per year, of which d800–1600 million generated by the health food sector, d900 million by the DHA [docosahexaenoic acid (22:6ω3)] market and d400 million from aquaculture products (Pulz and Gross, 2004). With the exceptions of few species (e.g. Crypthecodinium cohnii, Schizochytrium sp. and Ulkenia sp.) cultivated in fermenters, and the green stage of Haematococcus and, in a few cases, Chlorella grown in PBR, the commercial production of microalgae is mainly performed in open ponds (see Fig. 20.1a) or lagoons and is limited to species belonging to the genera Arthrospira, Chlorella and Dunaliella that take advantage of their high growth rate or of a selective growth medium that limits contamination (Richmond, 1999). The reason for this is that large open ponds are easier to operate, less expensive and more durable than large closed reactors (Tredici, 2004). However, the majority of microalgae does not require a specific growth environment or a selective medium, and can not be cultivated for prolonged periods in outdoor open systems because of contamination. Photobioreactors provide a close environment for the culture limiting direct fall-out and invasion by unwanted species and a better control of

Raceway ponds, natural blooms Natural bloom Arid, semi-arid soils Circular ponds, fermenters, PBR Raceway ponds, lagoons Raceway ponds, PBR Raceway ponds Fermenters (10–100 m3) Fermenters (10–100 m3) Fermenters (80 m3) Bags, PBR Tanks, cylinders, bags

3000 (d. wt)

500 (d. wt) 600 (d. wt) 2000 (d. wt) 1200 (d. wt)

300 (d. wt)

na 240 (oil)

10 (oil)

na

na 1000 (d. wt)

Arthrospira

Aphanizomenon Nostoc Chlorella Dunaliella

Haematococcus

Odontella Crypthecodinium

Schyzochytrium

Ulkenia

Porphyridium Various

Israel Worldwide

Germany

USA

France, Germany USA

USA Asia1, America Asia, Germany Australia, Israel, Asia USA, Israel

Asia, USA

Location

Dietary supplement ω3-PUFA (DHA) as dietary supplement, nutraceutical, feed additive ω3-PUFA (DHA) as dietary supplement, feed additive ω3-PUFA (DHA) as dietary supplement, nutraceutical Cosmetics, phycoerythrin ω6-PUFA (AA) Aquaculture feeds

Dietary supplements, aquaculture, astaxanthin

Dietary supplements, cosmetics, phycobiliproteins, feed additives Dietary supplement Health food Dietary supplements, cosmetics, aquaculture Dietary supplements, cosmetics, β-carotene

Application area/product

In China Nostoc harvesting is restricted. AA = arachidonic acid, d. wt = dry weight, DHA = docosahexaenoic acid, na = not available, PBR = photobioreactors, PUFA = polyunsaturated fatty acid.

1

Culture system

Production (tons y−1)

Commercially produced microalgae

Genus

Table 20.1

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A

B

C

D

E

Fig. 20.1 Examples of culture systems for microalgae: A. open raceway pond at NBT Ltd (Israel); B. polyethylene bags (sleeves); C. annular column; D. horizontal and vertical manifold reactors at Algatechnologies Ltd (Israel) (courtesy of Prof. S. Boussiba); E. disposable panel at Fotosintetica & Microbiologica Srl (Italy). (A, B, C, E, photograph by the authors)

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culture parameters (pH, temperature, pO2, etc.), ensuring the dominance of the desired species (Tredici, 2004). Currently, most of the research on culture systems is focused on cultivation of ‘new’ algal species and maximization of productivity in photobioreactors rather than on open ponds, which seem to have reached their technological limit. The main closed systems developed and their performance with different microalgal species are described in numerous reviews (Lee, 1986; Chaumont, 1993; Prokop and Erickson, 1995; Torzillo, 1997; Pulz and Scheibenbogen, 1998; Tredici, 1999, 2004; Pulz, 2001; Carvalho et al., 2006; Ugwu et al., 2008; Tredici et al., in press). In this chapter cultivation systems currently used in commercial plants or at pilot scale outdoors will be described.

20.2.1 Reactors and techniques for microalgae culture Sleeves and vertical columns Polyethylene bags (sleeves) suspended from a framework (see Fig. 20.1b) or supported within a mesh frame and mixed by air bubbling are the most common cultivation devices used in hatcheries for the production of algal biomass. From 50 to 500 L in volume, such reactors are mostly used indoors with artificial illumination. They are currently used by different companies (e.g. Ketura Kibbutz, Israel; GreenSea, France; NOVAgreen GmbH, Germany) for the cultivation of selected species for the cosmetic, food or pharmaceutical markets. Sleeves are also used by GreenFuel Technologies Corp (USA) for inocula production for biofixation–bioenergy applications (http://www.greenfuelonline.com). Although sleeve reactors are inexpensive, their low surface-to-volume ratio (S/V), biofouling and the need for a very large number of units for large-scale production limit their applications. Vertical columns, first devised by Cook (1950), are made of rigid transparent cylinders (typically 2–2.5 m in height and 30–50 cm in diameter), with mixing achieved by air bubbling or by an airlift. They are extensively used in hatcheries even if, because of their low S/V, volumetric productivities are rather low (typically below 0.1 g L−1 d−1) (Fulks and Main, 1991). Vertical cylinders internally illuminated, after the first design by Helm et al. (1979), have been developed and used to cultivate various microalgae and cyanobacteria at the University of Florence (Tredici et al., in press). The reactor, named annular column, is made of two concentric transparent cylinders of different diameter sealed at the base to form an annular culture chamber with higher S/V compared to completely filled columns. Lamps or fluorescent tubes can be placed inside the inner cylinder (see Fig. 20.1c). Using columns arranged so as to simulate a full-scale plant, high productivities (up to 38 g m−2 d−1) were attained outdoors with Tetraselmis suecica (Chini Zittelli et al., 2006). Annular columns of 30–230 L volume

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are currently marketed by Fotosintetica & Microbiologica Srl (Italy) (http://www.femoline.it), a spin-off company of the University of Florence. The small size and relatively high cost of this reactor limit its use for large-scale production. Recently, horizontal annular columns, in which mixing is performed by air bubbling, have been developed by Yamaha Motor Company Ltd (Japan) and used in the cultivation of Chaetoceros calcitrans (http://www.yamaha-motor.co.jp/global/news/2004/03/02/fukuroi. html). Tubular photobioreactors Most of the PBR adopted in commercial plants are of the tubular type. This category can be subdivided into three main groups: (i) serpentine, (ii) manifold and (iii) helical PBR. Serpentine photobioreactors, first designed by Tamiya in the 1950s (Tamiya et al., 1953), consist of straight tubes connected by U-bends to form a flat loop (the photostage) that may be arranged either vertically or horizontally. Gas exchange and nutrient addition normally take place in a separate vessel and culture circulation is achieved by a pump or an airlift. Serpentine horizontal PBR have been much studied and developed by the group of Gudin in the early 1980s (Chaumont, 1993) and later by Molina Grima and co-workers (Molina Grima, 1999). Fitoplancton Marino SL (Spain) uses a horizontal tubular reactor immersed in a pond for the production of different algae, which are sold mainly for aquaculture uses (http://www.easyalgae.com/ubicacion.asp). Following a design first developed by Torzillo et al. (1993), a 4000 L twolayer serpentine reactor made of 10 cm diameter Plexiglas® tubes has been set up by Cajamar for production of lutein-rich biomass of Scenedesmus almeriensis. Mera Pharmaceuticals, Inc (USA) utilizes a 25 000 L serpentine reactor made of large (38 cm in diameter) polyethylene tubes for culturing the green stage of Haematococcus pluvialis (Olaizola, 2003). The culture is used to inoculate ponds in which the cells are exposed to stresses (high irradiance, low nitrogen) that induce astaxanthin accumulation (‘red’ stage) (Olaizola and Huntley, 2003). A large-diameter (30–64 cm) serpentine photobioreactor, made of rigid plastic material, is marketed by AlgaeLink NV (The Netherlands) (http://www.algaelink.com). The reactor is pump-mixed, completely-controlled and self-cleaning. Manifold PBR are made of parallel tubes connected at the ends by two manifolds. The main advantages of these systems over serpentine reactors are the reduction of head losses and lower oxygen concentrations, two factors that facilitate scale-up to industrial size. A horizontal manifold reactor (see Fig. 20.1d) is used by Algatechnologies Ltd (Israel) for the production of the red stage of H. pluvialis. Tredici and co-workers developed the near-horizontal tubular reactor (NHTR), with tubes inclined from 5 to 20° to the horizontal so that mixing could be achieved by air bubbling (Tredici and Chini Zittelli, 1998; Chini Zittelli et al., 1999). A 20 m long NHTR was experimented with at the University of Hawaii (USA) (Szyper

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et al., 1998). Two small NHTR units are currently used by ENI SpA (Italy) to develop CO2 abatement strategies with microalgae (Pedroni et al., 2004). A similar design, the triangular airlift reactor, consisting of a series of riser tubes, gas separators and downcomer tubes arranged in a triangular configuration, was used by GreenFuel Technologies Corp (USA) for the abatement of greenhouse gases (Vunjak-Novakovic et al., 2005). Rather common, both at pilot scale and in commercial plants, are manifold photobioreactors arranged fence-like. The BioFence, developed by Applied Photosynthetics Ltd (UK) in the late 1990s, consists of an array of transparent tubes racked together in a fence-like structure in which the culture suspension is circulated by a centrifugal pump or by an airlift. BioFence systems are currently marketed by Varicon Aqua Solutions Ltd (UK) (http://www.variconaqua.com/bioreactors.htm). Biofence photobioreactors from 10 to 35 000 L are also distributed by B Braun Biotech International GmbH (Germany). Industrial-scale plants based on this design are operated by Bioprodukte Prof Steinberg GmbH (Germany) (http://www. bioprodukte-steinberg.de) for the production of Chlorella, by Algatechnologies Ltd (Israel) (http://www.algatech.com/astax.htm) for the growth of H. pluvialis (see Fig. 20.1d) and by Salata (Germany), which produces different algae for the cosmetic market. Helical photobioreactors (biocoils), consisting of small-diameter flexible tubes wound around an upright structure, have been the subject of much experimentation and have been marketed in the past. A flattened biocoil, similar to the one devised by Setlik et al. (1967), has been recently built in Nanchang (China) (http://www.newbioreactor.com). Flat photobioreactors Few flat photobioreactors (panels) have been used for mass cultivation of algae until recently. The first version of the alveolar panel developed by Tredici et al. (1988), in which mixing was achieved by a pump, was brought to industrial level by Pulz and co-workers (Pulz and Scheibenbogen, 1998). This reactor is marketed by B Braun Biotech International GmbH (Germany) (http://www.bbraunbiotech.com) in sizes varying from 10 to 2000 L. In the mid-1990s Richmond and co-workers developed glass panels (without alveoli) of various widths (Hu et al., 1996). The larger unit developed (500 L) was used for cultivation of Nannochloropsis (Zhang et al., 2001) and more recently for the green stage of H. pluvialis (Aflalo et al., 2007). Glass panels are highly transparent, easy to clean and resistant to weathering. However, weight, fragility and cost discourage their use for large-scale plants. A rigid plastic panel with a defined circulation path, that significantly enhanced productivity in comparison with randomly mixed bubble columns, has been developed and patented (Degen et al., 2001). A reactor of this type made of moulded plastic is marketed by Subitec GmbH (Germany) (http://www.subitec.com), a spin-off of the Fraunhofer-Institute for Interfacial Engineering and Biotechnology. These reactors are an

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interesting development of plastic panels; however, low scalability and high cost may limit their commercial application. In the early 2000s the concept of the ‘disposable panel’ was developed and patented by two groups working independently in Italy and Israel (Tredici and Rodolfi, 2004; Boussiba and Zarka, 2005). A disposable panel is a flat reactor consisting of a plastic culture chamber enclosed in a rectangular metal frame or cage. The main advantages of these systems are the low construction cost, the capacity to be scaled-up and a disposable culture chamber for the cultivation of those microalgae which suffer from contamination. These systems are successfully used in some hatcheries for microalgae production both in Israel and Italy. Using the disposable or ‘green wall’ panel (see Fig. 20.1e), as the reactor is called in Italy, productivities of about 20 and 30 g m−2 d−1 were attained with Nannochloropsis and T. suecica, respectively (Rodolfi et al., 2005, 2009). The ‘green wall’ panel is marketed by Fotosintetica & Microbiologica Srl (Italy) (http://www.femonline.it). Table 20.2 summarizes the main features of different PBR categories used at pilot or commercial scale. Culture systems for benthic diatoms The development of the above culture systems has increased the number of planktonic species that can be grown in reproducible and stable culture conditions, both indoors and outdoors, and under different climates. Very few studies have been made on culture systems for the production of benthic microalgae (Lebeau et al., 2000, 2002; Silva-Aciares and Riquelme, 2008), such as most of the pennate diatoms, which are of great importance for aquaculture (see Section 20.3). One photobioreactor recently developed for this purpose consists of a 30 L transparent acrylic column (1 m high and 19 cm in diameter) with a conical end for medium discharge. Inside the column a sort of large bottle brush bearing PVC bristles is placed, which occupies the whole column length and width and provides a large surface for cell adhesion. The medium circulation is obtained using an airlift system. With Amphora spp., Navicula spp. and Nitzschia ovalis this photobioreactor performed better than a bubble column having the same dimension and without bristles (Silva-Aciares and Riquelme, 2008).

20.2.2 Advances in techniques for microalgae culture Current methods for microalgae culture rely on batch, semi-continuous or continuous cultivation. Generally, as the need for high-quality biomass increases and the cultivation is performed in more specialized centres, semicontinuous and continuous techniques tend to become more diffuse. In fact, by growing microalgae at the optimal population density, the productivity is maximized and a biomass of constant composition can be produced. Most continuous and semi-continuous cultures are implemented in strictly

Horizontal/ vertical

Vertical/ horizontal

Annular columns

Tubular PBR Serpentine

Air bubbling/ airlift

Vertical

Vertical columns

Airlift/pump mixing

Air bubbling

Air bubbling

Paddle-wheel Rotating arm Air bubbling/ airlift

Mixing

Sleeves and vertical columns Sleeves Vertical

Horizontal Horizontal Horizontal

Arrangement

None None None

Temperature control

Very difficult Very difficult Very difficult

Unialgality maintenance

Low/high

Water spraying/cooling in separate tank/water jacket

Reasonable/good

None/indoor in Difficult/ thermoregulated room reasonable Low/high None/indoor in Difficult thermoregulated room/ internal cooling system High None/indoor in Reasonable thermoregulated room/ internal cooling system

Low/high

Poor Poor Poor

Gas transfer

Main features of the different cultivation systems for microalgae

Open systems Raceway ponds Circular ponds Tanks

Cultivation system

Table 20.2

Difficult

Very difficult

Very difficult

Very difficult

Easy Difficult Very difficult

Scale-up

High

High

High

Low/medium

Low Low/medium Low/medium

Cost per unit surface

Horizontal/ inclined/ vertical Inclined/ vertical Inclined/ vertical

Horizontal/ inclined/ vertical Vertical

PBR = photobioreactor.

Glass and rigid plastic panels Disposable panel

Flat PBR Alveolar panel

Helical

Manifold

Airlift/air bubbling Air bubbling/ pump mixing

Pump mixing/ air bubbling

Airlift/pump mixing/air bubbling Airlift/pump mixing

High

High

High Water spraying/internal cooling system Water spraying/internal cooling system

Water spraying/internal cooling system

Water spraying/cooling in separate tank/water jacket Low/high Water spraying/cooling in separate tank/water jacket

Low/high

Reasonable/good

Reasonable

Reasonable

Reasonable

Reasonable/good

Easy/reasonable

Difficult

Difficult

Difficult

Reasonable

Medium/ high

High

High

High

High

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controlled PBR to minimize introduction of airborne contaminants. This is due to the fact that continuous cultures tend to be operated over a much longer time period than typical batch cultures and thus the potential for contamination is increased. A ‘hydraulically integrated serial turbidostat algal reactor’ (HISTAR) has been proposed for continuous cultivation of Isochrysis for aquaculture uses (Rush and Christensen, 2003). In this system, two closed and tightly controlled tanks (turbidostats) provide a unialgal and contaminant-free inoculum and are hydraulically linked to six open continuous-flow stirred tanks, in which biomass production takes place. In a different system made of two vertical interconnected columns (6 L module), mixed by an airlift and artificially illuminated, a daily productivity of 16.4 million cells mL−1 at the steady state (lasting about one month) was obtained with Isochrysis T-ISO under continuous cultivation (Loubière et al., 2009). A 120 L prototype has been implemented and will be marketed by Jouin Solutions Plastiques® (France) (http://www.jouin.com). The concept of continuous culture has also been applied, at laboratory level, for the continuous production of high-value secondary metabolites, in order to lower their production cost. This involves continuous removal of the product (either extracellular or intracellular) without damaging the cells of the producing organism that may be reused. This is the case of the extraction of the water-soluble pigment marennin from the diatom Haslea ostrearia; the pigment released in the medium is continuously forced to pass through a membrane for separation (Rossignol et al., 2000). The method is also applied with the microalga entrapped in an agar gel layer (Lebeau et al., 2002). For the extraction of the intracellular β-carotene produced by stressed cells of Dunaliella salina, a biocompatible solvent is added directly in the PBR and the β-carotene is extracted selectively via continuous recirculation of the solvent through the aqueous phase containing the cells (Hejazi and Wijffels, 2004). The technique appears promising and applicable to other secondary metabolites. Continuous techniques may also be applied in wastewater treatment with microalgae. An interesting device proposed for this purpose is the corrugated inclined raceway (Craggs et al., 1997). The corrugations increase the surface area and form discrete microponds that increase the holding capacity of the system. An appropriate inclination allows the flow of wastewater down the raceway to be slowed down. This system is particularly efficient when applied to surface-adherent cells (like Phaeodactylum tricornutum in this study) because there is no need for continuous harvesting of the biomass. From the perspective of developing an integrated system for sustainable aquaculture, in which phytoplankton can be used as a trophic link between fish excretion and the culture of bivalves, natural phytoplankton populations were continuously cultured outdoors using fish-farm effluents as the source of nutrients (Lefevre et al., 2004). Similar experiments have been successfully carried out by using algae-dominated biofilms for the maintenance of

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water quality through uptake of ammonium and phosphate in intensive shrimp cultures (Thompson et al., 2002). Another advantage of the technique is that bacteria, microalgae and protozoa present in the biofilm are able to control pathogens by production of antibiotics and through grazing.

20.3

Microalgae for aquaculture feed

20.3.1 Introduction The culture of microalgae constitutes a fundamental step in different domains of aquaculture, with particular reference to hatchery production of molluscs, crustaceans and live prey for fish and shrimp larvae. These activities depend on the production of high-quality microalgal biomass in large amounts, a process requiring high human and economic investments. Microalgae production costs range from about d30 to more than d300 kg−1 of dry biomass depending on the facility size (Coutteau and Sorgeloos, 1992; Borowitzka, 1997), and represent one of the highest cost factors in many hatcheries. The food value of microalgae for aquatic animals depends on their ingestibility, digestibility and biochemical composition. It is difficult to evaluate and compare the food value of different microalgal strains on the basis of the results reported in different studies, because microalgae biochemical composition is strongly modulated by factors such as light quality and intensity, photoperiod, temperature, nutrient availability, growth phase and harvesting regimen (Brown et al., 1993; Carvalho and Malcata, 2000). Microalgae in late-logarithmic growth phase contain 15–50 % proteins, 5–20 % lipids and 5–20 % carbohydrates (Brown et al., 1997; Renaud et al., 1999). Stationary phase microalgae can double carbohydrate or lipid levels at the expense of proteins (Harrison et al., 1990; Brown et al., 1993, Muller-Feuga, 2003a). Most microalgae are richer in polar lipids during the exponential phase and accumulate triacylglycerols during the stationary phase (Dunstan et al., 1993) or under stress (Rodolfi et al., 2009). Generally, the use of multispecific diets, including microalgae of different classes, balances the biochemical composition of the food and offers a better nutritional package for larval animals than monospecific diets (Robert and Trintignac, 1997).

20.3.2 Microalgae for molluscs Traditionally, mollusc rearing depends on juvenile collection from the natural environment. However, spat production in hatcheries is increasing its importance. For the Pacific oyster (Crassostrea gigas), Muller-Feuga (2000) reports that hatchery production covered 80 % of juvenile requirements on the West coast of the USA, but only 10 % in France. More often than in the past, microalgae now have to be mass cultured because the natural phytoplankton population of the seawater used in

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hatcheries is insufficient to support optimum growth of reared animals. Moreover, in the case of larval culture, natural seawater is treated and filtered before use, and this reduces or eliminates its microalgal load. Cultured microalgae have been widely utilized as feed in bivalve hatcheries since the 1940s (Bruce et al., 1940). In mollusc hatcheries microalgae are cultured for larval, post-larval and juvenile production and for broodstock conditioning. Broodstocks are fed microalgae for up to two months. The daily feed dose, in dry weight of algae, is usually between 2 and 4 % of the dry meat weight of the adults at the start of the conditioning period (Helm and Bourne, 2004). Larval feeding in hatchery lasts for a period of few weeks (e.g. 3–4 weeks for Pacific oyster and Manila clam), depending on the reared species and on cultural and environmental conditions. The success of this delicate step is strictly dependent on the production of highquality microalgae, delivered to the larvae in batch or in continuous. The daily amount of food ingested per larva varies according to mouth size from 2800 to 50 000 equivalent cells (all diets are generally made equivalent in terms of feed volume, on the basis of microalgae, like Pavlova or Isochrysis, cellular volume) for the Pacific oyster and from 4400 to 15 000 equivalent cells for Manila clam (Helm and Bourne, 2004). After the metamorphosis, the amount of microalgae required increases significantly as the growth rate of spat becomes strongly dependent on the biomass provided. Due to the high cost of growing spat further, hatcheries prefer to deliver it at the smallest size possible. To be successfully used in mollusc rearing, microalgae should fulfil different criteria: (i) appropriate size to be efficiently ingested (0.5–20 μm for filter feeders; 10–100 μm for grazers); (ii) digestible cell wall; (iii) high nutritional value; (iv) easiness of culture in the different systems operated in hatcheries (Muller-Feuga et al., 2003b). Although more than 50 species of microalgae have been tested over the years as food for bivalves (Davis and Guillard, 1958; Walne, 1970; Brown, 1991; Brown et al., 1997), only a dozen species are routinely used in hatcheries (see Table 20.3). Moreover, in the case of molluscs which are highly demanding species in terms of nutrients, such as the Pacific oyster, this number is further reduced (Robert et al., 2004). In general, molluscs require a diet composed of 30–60 % protein and 5–30 % carbohydrate (Renaud et al., 1999). Several studies investigated the nutritional requirements of bivalves, clearly indicating the fundamental role of the polyunsaturated fatty acids (PUFA), with particular reference to EPA [eicosapentaenoic acid (20:5ω3)], DHA and AA [arachidonic acid (20:4ω6)] (Brown et al., 1997; Knauer and Southgate, 1999; Robert et al., 2004; Martínez-Fernández et al., 2006). However, it should be stressed that the ratios of DHA, EPA and AA might be more important than their absolute levels. While many algal species have moderate to high concentrations of EPA, relatively few are rich in DHA, e.g. Isochrysis aff. galbana (T-ISO), Pavlova lutheri and Rhodomonas salina. This latter cryptophyte has been successfully used as food for scallop (Pecten maximus) larvae and its addi-

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Table 20.3 Major classes and genera of microalgae used in aquaculture Class

Genus

Feeding target and frequency of use

Diatoms

Skeletonema Thalassiosira Phaeodactylum

BL (+++), BP (+++), PL (++) BL (++), BP (++), PL (+) FPL (+), PL (+), BL (±), BP (±), MZ (*) PL (+++), BL (++), BP (++), MZ (+) AL (+++); PL (+) AL (+++); PL (+) AL (+) BL (+++), BP (+++), MZ (+++), PL (++), FPL (++) BL (+++), BP (+++), MZ (+++), PL (+), FPL (+) MZ (+++), BL (+), BP (+), PL (+) BL (+), BP (+) BL (+), BP (+) MZ (*) FPL (*), FZ (*), MZ (*) FZ (*), MZ (*) MZ (+++) PL (± ), MZ (*)

Prymnesiophytes

Chaetoceros Navicula Nitzschia Amphora Isochrysis Pavlova

Prasinophytes Cryptophytes Chlorophytes Eustigmatophytes Cyanobacteria

Tetraselmis Pyramimonas Rhodomonas Dunaliella Chlorella Scenedesmus Nannochloropsis Arthrospira

* no data. AL = abalone larvae, BL = bivalve larvae, BP = bivalve postlarvae, FPL = freshwater prawn larvae, FZ = freshwater zooplankton, MZ = marine zooplankton, PL = penaeid shrimp larvae.

tion in mixed diet significantly improved growth performances and also metamorphosis competence, probably as a consequence of its high PUFA level (Tremblay et al., 2007). Sterols also play a major role in mollusc nutrition. The ability of bivalves to synthesize de novo or bioconvert sterols varies among different species, but it is generally low or absent. This implies that a dietary supply of sterols is essential for bivalve growth (Kanazawa, 2001; Muller-Feuga et al., 2003a). Due to the high cost of microalgae production in the hatchery, substitutes for fresh live microalgae have been sought and many experimental studies have been carried out to find alternatives, but the results have seldom been satisfactory (Coutteau and Sorgeloos, 1992; Robert and Trintignac, 1997; Knauer and Southgate, 1999; Heasman et al., 2000). An exception is represented by the commercial-scale test with mussel spat in which fresh algae were successfully substituted with a formulated supplement (Myspat®, INVE Technologies NV) (Nevejan et al., 2007). The gastropod abalone (Haliotis sp.) requires benthic diatoms as feed, and the maintenance of a suitable diatom film is a critical factor in the success of abalone hatcheries. The most used diatoms belong to the genera Nitzschia and Navicula. The growth and survival of post-larvae are affected

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by the ingestibility and, particularly, digestibility of the diatom which, in turn, depends on the species dominating the biofilm, on the strength of attachment to the biofilm itself, on cell size and morphology, and on frustule strength (Kawamura, 1996; Roberts et al., 1999). The relevance of these factors can change as post-larvae grow (Kawamura et al., 1998). A method for replacing live food with an inert diet has recently been assessed for production of Haliotis diversicolor aquatilis (Stott et al., 2004). Diatom biofilm (including bacteria and other microorganisms) serves not only as food for the more advanced stages of larval marine invertebrates, but also to provide settlement sites for larvae in the process of metamorphosis. Such a role has been reported for scallops (Placopecten magellanicus, Argopecten purpuratus) and abalone (Haliotis laevigata) (Pearce and Bourget, 1996; Daume et al., 1999; Avendan˜o-Herrera et al., 2003).

20.3.3 Microalgae for live prey Microalgae (see Table 20.3) play a major role for the culture of live preys for fish and shrimp larvae, providing energy, protein and other key nutrients such as vitamins, PUFA, pigments and sterols. Live preys used in aquaculture range in size from 0.1 to 2 mm. The most commonly reared zooplanktonic species are rotifers (Brachionus) and brine shrimps (Artemia). To a lesser extent cladocerans and copepods are also used (Duerr et al., 1998). Rotifers are indiscriminate filter feeders and may be fed with algae (generally Nannochloropsis, Tetraselmis, Chlorella or Isochrysis T-ISO), yeast, bacteria and microparticles up to approximately 25 μm in size. The fatty acid profile of rotifers is largely determined by the diet (Watanabe et al., 1983) as rotifers have limited capacity to synthesize PUFA (Lubzens et al., 1985). Rotifers fed with baker’s yeast are deficient not only in PUFA but also in ascorbic acid that is reported to have an essential role in reducing deformities and improving immune response as well as resistance to disease and stress in fish larvae (Merchie et al., 1997; Cahu et al., 2003). For these reasons rotifers are usually enriched with fresh microalgae, such as Nannochloropsis, Isochrysis and Pavlova, or with commercial emulsions alone or in combination, in order to pack their gut with high-quality compounds. Dried thraustochytrids (marine heterotrophs taxonomically aligned with heterokont algae) may also be used, being extremely efficient in DHA enrichment (Harel et al., 2002). Artemia are continuous, non-selective, particle filter-feeders. Different microalgae (e.g. Isochrysis, Chaetoceros, Arthrospira, Scenedesmus, Tetraselmis and Dunaliella) have been selected and used to culture Artemia for their high nutritional value (Sorgeloos et al., 1987). However, microalgal production is labour-intensive and expensive, and the adoption of ‘off-theshelf’ alternative diets has been considered (Coutteau et al., 1990; Intriago and Jones, 1993; Naegel, 1999; Abatzopoulos et al., 2002).

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20.3.4 ‘Green water’ and ‘pseudo-green water’ techniques Several studies indicate that some microalgae provide beneficial effects to the reared organisms (larval fish, shrimps) when delivered in intensive culture systems together with the zooplankton, leading to much better results in terms of survival, growth and transformation index of the reared animal compared with those obtained with the traditional technique (‘clear water’ technique) (Tamaru et al., 1994; Papandroulakis et al., 2001; Lio-Po et al., 2005). The ‘green water’ technique is defined as larviculture in an endogenous phytoplankton bloom together with zooplankton, while the ‘pseudo-green water’ is larviculture in a tank supplemented daily with exogenous phytoplankton and zooplankton (Papandroulakis et al., 2001; Muller-Feuga et al., 2003b). In the ‘pseudo-green water’ technique phytoplankton concentration remains constant through daily addition. This method is generally applied at the beginning of larval rearing, the most critical segment of the rearing process, after which the ‘clear water’ technique is adopted (Papandroulakis et al., 2001). The most popular algal species used for ‘pseudo-green water’ applications are Nannochlorospsis sp., Isochrysis galbana, Chlorella sp., T. suecica and Arthrospira platensis (Cahu et al., 1998; Can˜avate and Fernández-Díaz, 2001; Papandroulakis et al., 2001; Chuntapa et al., 2003; van der Meeren et al., 2007). For shrimps, an alternative method to limit dissolved nutrients in the rearing ponds consists of a biological treatment with a biofilm mainly composed of pennate diatoms, e.g. Amphora, Navicula, Synedra and Cylindrotheca (Thompson et al., 2002; Lebeau and Robert, 2003). ‘Green water’ may also be applied to extensive outdoor production systems, through fertilization to stimulate microalgal growth and, correspondingly, zooplankton production as feed for the larvae introduced into the ponds (Shields, 2001; Palmer et al., 2007). The reasons for the positive effects shown by these techniques are still unclear. These positive effects are probably due to the synergic action of different factors (Muller-Feuga et al., 2003b): • shading effect of microalgal biomass; • improvement of water quality due to oxygen production and nutrient (nitrogen and phosphorus) uptake by microalgae leading also to stabilization of pH fluctuations; • maintenance of a high nutritional quality of zooplankton by continuous feeding; • enhancement of larval performances and immune system by microalgal metabolites (vitamins, growth-promoting substances, unknown immunostimulants); • regulation of bacterial population (probiotic effect) by production of antibacterial substances (see Section 20.5); • induction of positive behavioural processes like prey catching. An example of the latter point is the selection by seabream larvae of larger preys (Artemia over rotifers) in the presence of microalgae (Rocha et al.,

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New technologies in aquaculture

2008). Seabream, seabass, turbot, striped mullet, flounders, Atlantic cod, halibut and Senegalese sole are reared with the ‘pseudo-green water’ technique, which guarantees better growth and survival compared to the ‘clear water’ technique (Tamaru et al., 1994; Cahu et al., 1998; Planas and Cunha, 1999; Cabrera and Hur, 2001; Papandroulakis et al., 2001; de Montgolfier et al., 2005; Can˜avate et al., 2007; van der Meeren et al., 2007; Rocha et al., 2008). The main drawbacks of the ‘green water’ technique depend on the slow development and seasonality of the endogenous food chain, that may lead to a shortage in food for the larvae and food chain deterioration followed by digestion problems and, possibly, by larvae mortality. These problems have not been observed with the ‘pseudo-green water’ technique, in which the quantity of phytoplankton and zooplankton introduced into the tanks is controlled (Papandroulakis et al., 2001).

20.3.5 Microalgae for shrimp culture Microalgae are commonly used in shrimp culture both as direct feed during the first larval and post-larval stages and as feed for zooplankton. After re-absorption of their yolk reserves shrimp nauplii molt and metamorphose into zoea. At this stage animals start the active feeding and their diet is composed of a combination of microalgae and zooplankton. Microalgae such as Chaetoceros, Skeletonema, Thalassiosira, Tetraselmis, Isochrysis and Chlorella (see Table 20.3) are considered the best feed for early shrimp larval stages (zoea and mysis), providing a balanced nutrition for larvae or for their live preys and contributing to improve larvae survival and growth, and enhance water quality (Muller-Feuga, 2004; Anon., 2007). For penaeid shrimp larvae microalgae concentrations are usually maintained at about 15 000 cells mL−1 throughout the larval cycle, adding zooplankton starting from mysis stage (Muller-Feuga et al., 2003b). For zoea, several alternative diets may be used. However, such products cannot completely replace live feeds and are generally used as supplements (Liao et al., 1993; Duerr et al., 1998; Anon., 2007). Shrimp larvae require about 30 % protein, 20 % lipid, while carbohydrate content in the diet can be more variable (Muller-Feuga et al., 2003b). Crustaceans, like other arthropods, are unable to synthesize de novo sterols and sufficient amounts of phospholipids. Thus, both cholesterol and phospholipids are essential nutrients for marine shrimps and freshwater prawns (Paibulkichakul et al., 1998; MullerFeuga et al., 2003b).

20.3.6 Microalgae for sea urchin culture The culture of sea urchin, of which roe (gonads) is a valuable food product, has been rapidly developing in some Asian and European countries, e.g. China, Japan, Ireland and France. Techniques for intensive cultivation have

Advances in microalgal culture for aquaculture feed and other uses

627

been established for different species (Grosjean et al., 1998; Kelly et al., 2000; Jimmy et al., 2003; George et al., 2004; Cárcamo et al., 2005; Liu et al., 2007). The type and amount of feed delivered as well as the feeding frequency during the larval phase influence larval development and, as a consequence, the success of metamorphosis. Sea urchin larvae are typically fed on live microalgae. In the larval culture of Loxechinus albus a mixed microalgal diet composed of I. galbana and Chaetoceros gracilis promotes larval development and metamorphosis, while C. gracilis alone seems to play an important role in maintaining high survival rate (González et al., 1987; Zamora and Stotz, 1994; Cárcamo et al., 2005). Experiments carried out on the use of alternative diets (formulated feeds, algal pastes) for sea urchin gave variable results. The complete development of larvae of Lytechinus variegatus fed on microencapsulated Dunaliella tertiolecta is reported by George et al. (2004). Microalgal paste (a mixture of Isochrysis sp., Pavlova sp., Tetraselmis sp. and Thalassiosira weissflogii) led to poor results in Paracentrotus lividus larvae metamorphosis (Liu et al., 2007).

20.3.7 Preserved microalgae for aquaculture Despite the recent advances in artificial feed formulations to replace live microalgae, on-site microalgal production remains a necessary step for most marine hatcheries. A possible alternative to on-site algal culture is the production of microalgae in specialized facilities and their delivery in a concentrated and preserved form to hatcheries. For this purpose suitable concentration methods and preservation techniques are required. To encourage their use in hatcheries, preserved microalgae diets have to: (i) be easily reconstituted into suspensions of unclumped cells; (ii) retain the nutritional quality for a reasonable period of time (spanning the hatchery rearing cycles); (iii) be suitable for storage within normal operating capabilities of hatcheries; (iv) be available with regularity; (v) be cost-effective. Many preservation techniques have been proposed and experimented with on a small scale, among which spray- or freeze-drying and freezing or refrigeration of algal concentrates (Lubzens et al., 1995; Montaini et al., 1995; Tredici et al., 1996; Heasman et al., 2000; Can˜avate and FernandezDiaz, 2001; Ponis et al., 2008). Among these methods, the storage at low positive temperatures of concentrates (pastes and slurries) has demonstrated to be able to preserve viability and nutritional quality in different microalgae (Montaini et al., 1995; Heasman et al., 2000; Chini Zittelli et al., 2003). These concentrates have shown potential as alternative diets to fresh algae for rotifers, prawn larvae and juvenile bivalves (Papandroulakis et al., 1996; Heasman et al., 2000; Hagiwara et al., 2001; Robert et al., 2001; Brown and Robert, 2002; D’Souza et al., 2002; Ponis et al., 2008). However, refrigeration may be limited by reduced biomass shelf-life, which strongly depends on the algal species and on the harvesting methods and associated post-

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harvest storage conditions (air-bubbling, addition of preservatives, biomass concentration, etc.). For example, the effects of preservatives on the quality and extended shelf-life of stored concentrates are unpredictable and highly species specific (Heasman et al., 2000; Ponis et al., 2008). A number of harvesting techniques, including sedimentation, filtration (Rossignol et al., 1999), electroflocculation (Poelman et al., 1997), ultrasound application (Bosma et al., 2003), foam fractionation (Csordas and Wang, 2004), chemical flocculation and centrifugation (Heasmann et al., 2000) has been evaluated to recover algal biomass. Of these, only centrifugation and chemical flocculation proved suitable to prepare algal concentrates as aquaculture feeds, in terms of efficiency of harvesting and of cell density (Heasmann et al., 2000). Although centrifugation is commonly applied for preparing concentrates, it may heavily damage algal cells, especially with naked flagellates such as Pavlova and Isochrysis and diatoms such as Chaetoceros (Heasmann et al., 2000). Centrifugation requires highcost equipment, and is not common outside large hatcheries. A promising flocculation technique based on pH adjustment has been recently developed at pilot scale (Knuckey et al., 2006). The process appears simple, inexpensive and readily applicable in hatcheries. Several companies are currently producing and selling microalgal products for aquaculture, that can be used ‘off-the-shelf’ (see Table 20.4). Reed Mariculture Inc. (USA) is among the major producers of algal concentrates. The company offers a range of stable (not viable) concentrated microalgae products (Instant Algae®) to meet the different nutritional needs of zooplankton, fish and shellfish species. These products are already distributed in 70 countries around the world. A shelf-life of three months is indicated for the refrigerated products. In Europe, freezedried Nannochloropsis grown autotrophically in photobioreactors (see Section 20.2.1) is distributed by two companies, Fitoplancton Marino SL (Spain) and Necton SA (Portugal), mainly for ‘pseudo-green water’ larval rearing and rotifer culture. Dried microalgal diets offer obvious advantages in terms of shelf-life (1–2 years), shipping costs and storage space requirements. Heterotrophically grown microalgae and microalgae-like organisms have recently become available on the market. In Japan, highly concentrated Chlorella suspensions enriched with vitamin B12 and ω3-PUFA (Hagiwara et al., 2001; Hayashi et al., 2001) are available in refrigerated form (see Table 20.4) and routinely used for ‘high-density’ mass culture of rotifers (Yoshimura et al., 1996). Several products based on spray-dried thraustochytrids of the genus Schizochytrium are currently marketed by Aquafauna Biomarine Inc. (USA) and are successfully applied as full or partial DHA enrichment for zooplankton (Harel et al., 2002). Although many alternative products are on the market, only few studies (Tang and Shields, 2002; Bonaldo et al., 2005; Espinosa and Allam, 2006) have been carried out to determine their suitability for aquaculture animals

Reed Mariculture Inc (USA) (www.reedmariculture. com) 180

90

90

Isochrysis 1800 (Isochrysis sp. CCMP1324)

Pavlova 1800 (Pavlova sp. CCMP 459)

184

Biomass concentration (g d. wt L−1)

Tetraselmis 3600 (Tetraselmis sp.)

Instant Algae® (marine microalgae concentrates) Nannochloropsis 3600 (Nannochloropsis oculata)

Marketed product/ microalgal species

3.3

4.6

1.5

68

(×109 mL−1)

37

51

8.3

369

(×109 g−1 d.wt)

Cell number

3 months/ refrigerated

3 months/ refrigerated

3 months/ refrigerated

3 months/ refrigerated 2 years/frozen

Shelf-life/ storage

Pseudo-green water, rotifer production and enrichment Bivalve shellfish post-larvae, shrimp larvae, rotifer production Bivalve shellfish larvae and postlarvae, shrimp larvae, rotifer enrichment Bivalve shellfish larvae and postlarvae, rotifer and Artemia enrichment

Feeding target

Commercial microalgae products for aquaculture currently available on the market (manufacturer’s data)

Manufacturer

Table 20.4

231–325

231–325

192–270

156–246

Price1 (× kg−1 d. wt)

Cont’d

Manufacturer

Table 20.4

0.32–46

2.0

na

90

180

Shellfish Diet 1800 (Mix: Isochrysis 25 % + Tetraselmis 20 % + Pavlova 20 % + Thalassiosira 30 % + Nannochloropsis 5 %) Rotifer Diet 2 (3600) (Mix: Nannochloropsis + Tetraselmis)

(×109 mL−1)

22

–

5.3–77

(×109 g−1 d.wt)

Cell number

60

Biomass concentration (g d. wt L−1)

Thalassiosira 1200 (Thalassiosira weissflogii CCMP 1051)

Marketed product/ microalgal species

3 months/ refrigerated

3 months/ refrigerated

3 months/ refrigerated

Shelf-life/ storage

Rotifer production

Bivalve shellfish and shrimp larvae and postlarvae, copepods and Artemia production Bivalve shellfish (all stages)

Feeding target

186

231–325

186–327

Price1 (× kg−1 d. wt)

Innovative Aquaculture Products Ltd (Canada) (www. innovativeaqua. com)

Greenwater Formula (Nannochloropsis oculata) Enrichment Formula (Mix: Nannochloropsis + diatoms) Spat Formula (Mix: Chaetoceros sp. + Phaeodactylum tricornutum)

Algae Paste (marine microalgae concentrates)

Reef Nutrition® Phyto-Feast (Mix: Nannochloropsis + Tetraselmis + Isochrysis + Pavlova + Thalassiosira + Amphora + Synechococcus) Rotifer Diet (Nannochloropsis)

30

6

10

386

146

18

50

227

na

na

68

16

132

360

–

1 year/ refrigerated 2 years/frozen at −5 °C

1 year/ refrigerated 2 years/frozen at −5 °C 1 year/ refrigerated 2 years/frozen at −5 °C

na/ refrigerated

na/ refrigerated

Bivalve postlarvae (>200 μm)

Rotifer, Artemia, copepod enrichment

Pseudo-green water

Zooplankton/coral

Filter feeders/ coral

520–648

196–246

334–418

748–998

na

Cont’d

2

64

PhytoBloom (Nannochloropsis) PhytoBloom Green Formula (concentrate)

Necton SA (Portugal) (www. phytobloom. com) 310

–

PhytoBloom Ice (paste)

PhytoBloom Prof (freeze-dried powder)

–

–

50–60

12

144

180

6

77

Nannochloropsis Starter Concentrate (Nannochloropsis sp.) Nannochloropsis Concentrate (Nannochloropsis sp.) T-Isochrysis Concentrate (Isochrysis T-ISO)

25

140

360

278

31

83

78

137

(×109 g−1 d.wt)

Cell number (×109 mL−1)

BlueBioTech GmbH (Germany) (www. bluebiotech. de.com)

Biomass concentration (g d. wt L−1) 182

Marketed product/ microalgal species

Starter Formula (Mix: Isochrysis + Pavlova + Nannochloropsis)

Manufacturer

Table 20.4

2 years/room temperature

1 year/frozen −20 °C

4 months/ refrigerated 0–4 °C

3 months/ refrigerated 5–10 °C

3 months/ refrigerated 5–10 °C

2 months/ refrigerated 5–10 °C

1 year/ refrigerated 2 years/frozen at −5 °C

Shelf-life/ storage

Pseudo-green water, rotifer production and enrichment Pseudo-green water, rotifer production and enrichment Pseudo-green water, rotifer production and enrichment

Pseudo-green water, rotifer production and enrichment Rotifer production and enrichment

Pseudo-green water, starter culture

Feeding target

na

na

na

750

528

766

417–521

Price1 (× kg−1 d. wt)

AlgaMac AlgaMac-3000 (Schizochytrium sp., fine spray-dried powder)

Aquafauna Bio-Marine Inc. (USA) (www.aquafauna. com)

AlgaMac-3050 (Schizochytrium sp., drum-dried cells in flake form)

Easyalgae® Freeze-dried microalgae (Nannochloropsis gaditana/ Tetraselmis chuii) Pastes and concentrates (Nannochloropsis/ Tetraselmis/ Chaetoceros/ Isochrysis/ Skeletonema/ Phaeodactylum/ Rhodomonas)

Fitoplancton Marino SL (Spain) (www.easyalgae. com/index_ ingles.asp)

–

–

–

na

na

–

–

–

2.5

2.5

–

na

2 years/ temperature <10 °C

2 years/ temperature <10 °C

na

2 years/not refrigerated

Artemia and rotifer enrichment, live algae replacement in larviculture Artemia enrichment, live algae replacement in larviculture

Pseudo-green water, starter culture, rotifer production and enrichment, bivalve culture

Pseudo-green water, rotifer production and enrichment

na

na

na

na

Cont’d

Fresh Chlorella-V12 (concentrate) (Chlorella sp. vitamin B12 enriched cells) Super Fresh Chlorella-V12 (concentrate) (Chlorella sp. EPA-DHA-vitamin B12 enriched cells)

AlgaMac-Enhance (Schizochytrium sp.+ Haematococcus, spray-dried powder)

Marketed product/ microalgal species

20 (Hagiwara et al., 2001) na

135

–

−1

(×10 mL )

9

−1

–

–

na

(×10 g d.wt)

9

Cell number

135

–

Biomass concentration (g d. wt L−1)

Price range refers to amount of product ordered. DHA = docosahexaenoic acid, EPA = eicosapentaenaic acid, na = not available.

1

Pacific Trading Co Ltd (Japan) (www.pacifictrading.co.jp)

Manufacturer

Table 20.4

20 days/ temperature <5 °C

20 days/ temperature <5 °C

1 year/ temperature <10 °C

Shelf-life/ storage

Rotifer enrichment

Rotifer production

Artemia and rotifer enrichment

Feeding target

na

38 (Hagiwara et al., 2001)

na

Price1 (× kg−1 d. wt)

Advances in microalgal culture for aquaculture feed and other uses

635

and most of the data from commercial hatcheries remain elusive or confidential. There is a considerable potential for these products, however, with few exceptions, such as Chlorella-V12 for rotifers in Japan (see Table 20.4), it has not been clearly demonstrated that they can succesfully substitute fresh algae. The challenge for the future will be to improve the quality and reduce the cost of these products, that in general is too high compared to that estimated for on-site algal production (see Section 20.3.1) (Coutteau and Sorgeloos, 1992; Duerr et al., 1998). The major commercial manufacturers of preserved marine microalgal products are located in Europe and the USA, so that significant shipping costs must be taken into account by users in other areas (e.g. Asia and Australia), where an important part of the world aquaculture production is located. The present cost of the preserved algal biomasses is nearly prohibitive for hatcheries where the profit is very low (e.g. shrimp farms in Asia, mainly at family scale). Considering the high potential of preserved microalgal products, a lowering of their production cost and wider availability is highly desirable.

20.4 Microalgae as dietary supplements, animal feed and nutraceuticals 20.4.1 Dietary supplements and animal feed additives Microalgae for human nutrition are mainly marketed as nutritional supplements, in the form of tablets and capsules. Microalgae and their extracts are also incorporated into pastas, snack foods, candy bars or gums and beverages (Liang et al., 2004). The market is dominated by few genera: Arthrospira, Chlorella, Dunaliella and Haematococcus, mainly because of their nutritive value and, not least, because they are easy to grow (Becker, 2004; Spolaore et al., 2006). Aphanizomenon and Nostoc have lesser importance. Arthrospira has a long history of human consumption (Abdulqader et al., 2000) and is known as a safe, nutritious microalga (Henrickson, 1997). It is considered as a valuable additional food source of macro- and micronutrients including high-quality protein, iron, γ-linolenic acid, carotenoids and vitamins B1 and B12 (Cohen, 1997). More recent studies have suggested possible health and therapeutic effects of Arthrospira (Gershwin and Belay, 2008) promoting this organism also for pharmaceutical and nutraceutical applications (Hu, 2004). The annual production of Arthrospira in the world exceeds 3000 tons (see Table 20.1), with most commercial producers located in Asia (Shimamatsu, 2004; Spolaore et al., 2006) and the USA. Cyanotech Corp (pond area 75 000 m2, 350 tons per year; http://www.cyanotech.com) and Earthrise Nutritionals LLC (pond area 150 000 m2, 500 tons per year; http://www.earthrise.com) in Hawaii and California (USA), respectively, are among the major producers of Arthrospira. Only Arthrospira grown in the USA has attained ‘GRAS’ (generally recognized as safe) status by the

636

New technologies in aquaculture

Food and Drug Administration for all food, beverage and supplement applications (US FDA, 2003). Chlorella is commercially produced by more than 70 companies for health food and mariculture feed. Although powder, pills or tablets are still the most popular Chlorella products, liquid extracts, such as the Chlorella Growth Factor, have now gained some share of the market. A novel immunostimulatory polysaccharide-peptide complex derived from Chlorella pyrenoidosa is offered by Ocean Nutrition Canada Ltd (Kralovec et al., 2007). Nowadays, the annual production of Chlorella exceeds 2000 tons (see Table 20.1), mainly obtained in open ponds (Spolaore et al., 2006) or by heterotrophic cultivation in fermenters (Iwamoto, 2004). The 700 m3 closed photobioreactor operated in Germany by Bioprodukte Prof Steinberg GmbH produces annually more than 80 tons of high quality Chlorella biomass for the health food market. Dunaliella is exploited for its high β-carotene content (Ben-Amotz, 2004). The global production of Dunaliella biomass is estimated at about 1200 tons per year (see Table 20.1). Open ponds and lagoons are the systems used in the commercial facilities, most of which located in Australia, Israel and China (Del Campo et al., 2007). The exploitation of Aphanizomenon flos-aquae (AFA) started in the early 1980s when natural blooms of this cyanobacterium were harvested from Upper Klamath Lake in Oregon (USA) (Carmichael et al., 2000). AFA is rich in proteins, polysaccharides, carotenoids, phycobiliproteins, vitamins and minerals. It is sold in the health food market as a nutrientdense food and dietary supplement. Beyond nutritional value, AFA has been claimed to improve overall wellbeing and to have antiinflammatory and immunomodulatory effects (Jensen et al., 2001). However, the potential production of neurotoxins by the genus Aphanizomenon and the presence of toxin-producing cyanobacteria in Klamath Lake, are of concern for the alimentary use of AFA (Saker et al., 2005). The Oregon Health Division and the Oregon Department of Agriculture have established a regulatory limit of 1 μg g−1 for microcystins in products containing blue green algae to prevent acute toxicity in humans (Gantar and Svircˇev, 2008). The exploitation of new microalgal species for alimentary purposes must be carefully evaluated regarding their potential toxicity and long-term effects on human health. Today, strict food safety regulations for placing novel products on the market have been established within the European Community (EC, 1997; Gantar and Svircˇev, 2008). In 2002, the newcomer marine diatom Odontella aurita, produced in open ponds by Innovalg SARL (France), has been authorized as food for human consumption in Europe (EU, 2003). The product, consisting of dried algae, is mainly used as dietary supplement. Arthrospira, Chlorella and Haematococcus are widely used as a feed supplement for cats, dogs, fish, ornamental birds, poultry and cows (Pulz and Gross, 2004; Spolaore et al., 2006).

Advances in microalgal culture for aquaculture feed and other uses

637

20.4.2 High-value molecules from microalgae Fatty acids Humans and animals lack the necessary enzymes to synthesize more than 18 carbon-long PUFA, and have to obtain them from external sources. In particular, EPA, DHA and AA are most valuable ingredients of feed and food, owing to their important effects on health. Many studies suggest that EPA and DHA support cardiovascular health, have a beneficial effect in several forms of cancer and also in inflammatory and autoimmune disorders, and play an essential role in the brain and retina development (Ruxton et al., 2005). Currently, the typical Western diet leads to an imbalanced intake of PUFA, with a large prevalence of ω6 over ω3 (15:1). This is thought to be responsible for increased incidence of cancer, heart disease, allergies, diabetes and other afflictions (Simopoulos, 2003). Although ω3-PUFA are essential, very high intakes of these fatty acids or an imbalanced ratio of DHA to EPA carry the risk of adverse effects on haemostasis (Takahata et al., 1998). General recommendations for daily dietary intakes of DHA + EPA are 0.5 g for infants and an average of 1 g for adults and patients with coronary disease (Ward and Sing, 2005). As fish oil fails to meet increasing PUFA demand, and there are also concerns about its use as supplement because of the possible presence of pollutants (Domingo et al., 2007), e.g. heavy metals, alternative sources are being sought (Ward and Singh, 2005). Production of microbial oil, also termed single-cell oil, is a relatively new concept (Ratledge, 2001). Several microalgae represent an interesting alternative to fish oil (see Table 20.5) since they contain high amounts of individual PUFA that may be more easily purified (Robles Medina et al., 1998). Microalgae for PUFA may be cultivated in bioreactors, under controlled phototrophic, mixotrophic or heterotrophic conditions (Ward and Singh, 2005), leading to a clean and safe oil. Despite the large number of EPA-containing microalgae known, only a few species have industrial production potential, among these Nannochloropsis sp., P. tricornutum and Monodus subterraneus (Chini Zittelli et al., 1999; Molina Grima et al., 2003) and the diatoms Nitzschia laevis and Nitzschia alba able to grow and produce EPA heterotrophically (Wen and Chen, 2003). The introduction of a specific gene into the obligate photoautotrophic diatom P. tricornutum, enables this microalga to use glucose and produce EPA in darkness (Zaslavskaia et al., 2001). DHA is the only algal PUFA commercially available. At present, Martek Biosciences Corp (USA) cultivates the heterotrophic dinoflagellate Crypthecodinium cohnii in controlled fermenters to manufacture an oil (life’s DHATM) that contains high (40–50 %) DHA levels (http://www.martek. com). Martek’s DHA oil is incorporated in infant formulas and in food and beverages for children and adults. It is also suitable as dietary supplement for pregnant and nursing women and for adults to support brain, eye and cardiovascular health. Martek’s DHA oil has been recognized as a safe (GRAS) ingredient in infant formulas (US FDA, 2001), and infant DHA-

– –` – 0.18 – –

– – – –

1.10

Thraustochytrids Schizochytrium sp. SR21 (H) Traustochytrium sp. G13 (H)

Diatoms Phaeodactylum tricornutum (P) Parietochloris incisa (P) Odontella aurita (P) Nitzschia laevis (H)

Eustigmatophytes Nannochloropsis sp. (P) Monodus subterraneus (P)

Prymnesiophytes Isochrysis galbana (P) Pavlova sp. (P)

Rhodophytes Porphyridium cruentum (P)

Cyanobacteria Arthrospira platensis (P) –

1.29

– –

0.68 0.55

– 6.61 – –

– –

–

20:4ω6 (AA)

–

1.27

0.08 1.80

4.64 3.80

2.00 0.23 4.00 2.80

– –

–

20:5ω3 (EPA)

–

–

1.58 1.32

– –

– – 0.50 –

27.70 11.40

17.40

22:6ω3 (DHA)

Hu et al., 1997

Rebolloso Fuentes, et al., 2000

Patil et al., 2007 Patil et al., 2007

Chini Zittelli et al., 1999 Hu et al., 1997

Sánchez Mirón et al., 1999 Goldberg et al., 2002 http://www.bluebiotech.de Wen and Chen, 2002

Sijtsma and Swaaf, 2004 Sijtsma and Swaaf, 2004

Sijtsma and Swaaf, 2004

Reference

AA = arachidonic acid, DHA = docosahexaenoic acid, d. wt = dry weight, EPA = eicosapentaenoic acid, GLA = γ-linolenic acid, H = heterotrophic growth, P = phototrophic growth, PUFA = polyunsaturated fatty acid.

–

18:3ω6 (GLA)

PUFA (% d. wt)

PUFA content in some microalgae and microalgae-like microorganisms

Dinoflagellates Crypthecodinium cohnii (H)

Organism

Table 20.5

Advances in microalgal culture for aquaculture feed and other uses

639

rich products are sold in over 70 countries and consumed by more than 24 million babies worldwide. Moreover, Martek Corp cultivates the marine protist Schizochytrium sp. to produce a low-cost oil, formerly produced by OmegaTech Inc. (USA) and known as DHAGold®, currently sold for nutritional supplements and animal feeds. Nutrinova GmbH (Germany), an operating unit of Celanese Corp (Germany), has developed a process to make DHA oil from the marine thraustochytrid Ulkenia sp. grown in 80 m3 fermenters, marketed under the brand name DHActiveTM (Ratledge, 2004). Pigments Microalgae are a natural source of a wide variety of pigments. The carotenoids and the phycobiliproteins are the most important from a commercial point of view. Carotenoids are a large family (over 700 types) of liposoluble pigments that are primarily produced by phytoplankton and plants (Lorenz and Cysewski, 2000). The worldwide market value of carotenoids is estimated at !630 million. β-carotene (!160 million) and the xanthophylls astaxanthin (!160 million) and lutein (!120 million) are the major carotenoids with commercial interest (Del Campo et al., 2007). Astaxanthin and canthaxanthin have been identified as the dominant pigment eliciting the pinkish-red hue in salmonids, being also responsible for the skin pigmentation of highly priced species such as seabreams (Chrysophrys major, Pagrus major) and for the desirable body colour of shrimps, lobsters and crayfish. In farmed animals, astaxanthin and canthaxanthin must be supplied with the diet, because of the animal inability to synthesize them de novo (Goodwin, 1984; Guerin et al., 2003). Synthetic astaxanthin represents about 95 % of the market. The natural pigment can be obtained from microorganisms such as the yeast Phaffia rhodozyma and the microalga H. pluvialis. When exposed to stress, this green microalga tends to form haematocysts in which astaxanthin can be accumulated to more than 3 % of the biomass dry weight (Olzaiola and Huntley, 2003). Canthaxanthin has been shown to negatively affect human retina, so allowed concentrations in animal feed have been recently reduced by the EU (EC, 2003). Carotenoids are widely used also in ornamental aquaculture, skin pigmentation being an important characteristic affecting market price of ornamental fish (Paripatanamont et al., 1999). Various non-algal products have been tested, but none has performed as effectively and consistently as natural pigments from microalgae such as Haematococcus, Arthrospira and Chlorella (Gouveia et al., 2003; Gouveia and Rema, 2005; Spolaore et al., 2006). In addition to their role in coloration, carotenoids act as provitamin A and as biological antioxidants, protecting cells and tissues from damaging effects of free radicals. Therefore, carotenoids are utilized for pharmaceuticals, health food, dietary supplements and cosmetics (Dufossé et al., 2005). A growing body of scientific literature indicates that natural astaxanthin is a more powerful antioxidant than other carotenoids and vitamin E (Dufossé

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et al., 2005) and may confer numerous health benefits (Guerin et al., 2003). H. pluvialis is cultivated for astaxanthin at industrial scale, mainly to provide a high-quality product for the nutraceutical and cosmeceutical markets (Oilazola and Huntley, 2003; Spolaore et al., 2006). The main commercial producers of natural astaxanthin from H. pluvialis are listed in Table 20.6. The dried microalgal biomass can be directly encapsulated or the astaxanthin extracted can be included in nutraceutical formulations. The most important process for natural production of β-carotene is the culture of the green halophilic flagellate D. salina that can accumulate this pigment up to 12 % of its dry weight in response to various environmental stresses (Ben-Amotz, 2004). Open ponds with no or scarce process control represent the conventional method used in commercial plants for Dunaliella production. The largest plant (c. 800 ha) is run by Cognis Nutrition and Health Co. (Australia), which produces β-carotene extracts and Dunaliella powder for human use and animal feed (see Table 20.6). Microalgal β-carotene has the advantage of supplying isomers in their natural ratio conferring superior bioavailability and antioxidant properties compared to synthetic forms (Becker, 2004). A daily intake of 6 mg of the xanthophyll lutein is recommended to prevent or reduce the effects of age-related cataract and macular degeneration (Olmedilla et al., 2003). Lutein is also used as drug and cosmetic dye as well as feed additive in aquaculture and poultry farming. Microalgae, where lutein usually appears in the free non-esterified form and does not require further chemical treatment, represent an interesting alternative to the conventional plant pigment. Among the microalgae that accumulate lutein, the most promising appear to be Muriellopsis sp. and S. almeriensis with a mean lutein content of about 0.5 % on a dry weight basis (Del Campo et al., 2007). An established commercial system for the production of lutein from microalgae does not yet exist. At pilot scale, the outdoor production of S. almeriensis is being tested by Cajamar (Spain) (Fernández Sevilla et al., 2006; Acién Fernández et al., 2007). The polyphenolic pigment marennin produced by the benthic diatom H. ostrearia, and released in the water (Pouvreau et al., 2006), is used in the terminal phase of the oyster rearing cycle to obtain the coloration appreciated by the consumers. It has been calculated that the greening of the mollusc increases the product market value by 40 % (Muller-Feuga, 2000). Cyanobacteria, red algae and cryptomonads are the only natural sources of phycobiliproteins (Glazer, 1999). The most widely used phycobiliproteins for commercial applications are phycoerythrin from Porphyridium cruentum (Román et al., 2002) and phycocyanin from Arthrospira sp. (Hu, 2004). Dainippon Ink & Chemicals Co. (Japan) has developed a series of products made from Arthrospira phycocyanin, specifically used as colorant in food and cosmetics. Pharmacological uses of phycobiliproteins are described in Section 20.5.1 and commercial products based on these pigments are reported in Table 20.6.

PBR (outdoor) PBR (indoor)

Maui-Hawaii, USA

Sweden

BioReal, Inc1 (http://www.bioreal.com/ BioRealHome.html) BioReal (Sweden) AB1 (http://www.bioreal.se)

PBR + raceway ponds (outdoor)

PBR1 + raceway ponds (outdoor)

Kona-Hawai, USA

Kona-Hawaii, USA

Unmixed ponds (158 ha)

Karratha, Australia

Mera Pharmaceuticals Inc (http://www.merapharma.com)

Astaxanthin from Haematococcus Cyanotech Corp (http://www.cyanotech.com)

Raceway ponds (10 ha)

Unmixed ponds (800 ha)

Culture system

Eilat, Israel

Whyalla, Hutt Lagoon, Australia

β-carotene from Dunaliella Cognis Nutrition and Health Co (http://www.cognis.com)

Nature Beta Technology Ltd (now owned by Nikken Sohonsha Corp) (http://www.chlostanin.co.jp) Aquacarotene Ltd (http://www.aquacarotene.com)

Location

Commercial producers of microalgae pigment products

Pigment and producing company

Table 20.6

AstaCarox® (crushed powder) AstaReal® (supercritical CO2 extracted oil)

na

Astafactor® (algal meal extract)

Naturose® (powder)

BioAstin® (extract)

Dry powder

Betatene® (natural mixed carotenoids, oil or dry powder) Spray-dried powder

Marketed product

Ingredient of all other products Nutraceuticals, cosmetics

Human dietary supplement (general health, suncare, rheumatoid arthritis, carpal tunnel syndrome and sports nutrition) Aquaculture, animal feed, pigments Dietary supplement, nutraceutical Feed supplement for animals na

Feed for fish and crustaceans

Health foods, pharmaceuticals, cosmetics

Nutritional supplement

Application area

Sakura, Japan

Dainippon Ink & Chemicals (DIC) Corp (http://www.dic.co.jp/en/index.html)

–

–

Raceway ponds

PBR (outdoor)

Culture system

PhycoProTM C-Phycocyanin PhycoProTM Allophycocyanin PhycoProTM Allophycocyanin (cross-linked) Lina Blue

APC, C-PC, B-PE, Cross-linked APC

AstaPureTM (supercritical CO2 extracted oleoresin with approximately 10 % astaxanthin) AstaPureTM (2.5 % astaxanthin beadlets)

AstaPureTM (crashed powder 4 % astaxanthin)

Food dye

Fluorescent pigments for medical diagnostics price ®3–10/mg Fluorescent pigments for medical diagnostics and research price ®4–32/mg

Nutraceuticals

Feed supplement for animals Feed supplement for horses Human dietary supplement containing AstaCarox® Food products or as animal feed Raw material for the extraction of astaxanthin Dietary supplement for humans, nutraceuticals, cosmetics

NOVASTAR® (powder) AstaEquus® AstaXin®

Application area

Marketed product

Subsidiary of Fuji Chemical Industry Co., Ltd., Japan (http://www.fujichemical.co.jp/english/). APC = allophycocyanin, B-PE = B-Phycoerithrin, C-PC = C-Phycocyanin, na = not available, PBR = photobioreactor.

1

CA, USA

ProZyme (http://www.prozyme.com)

Kona-Hawaii, USA

Ketura, Israel

Algatechnologies Ltd (http://www.algatech.com)

Phycobiliproteins from Arthrospira Cyanotech Corp (http://www.cyanotech.com)

Location

Cont’d

Pigment and producing company

Table 20.6

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20.4.3 Cosmetics Many compounds and extracts from microalgae are frequently used in cosmetics as thickening and water-binding agents and as antioxidants. Arthrospira sp., Chlorella vulgaris, Nannochloropsis oculata and D. salina are all used in cosmetics (Spolaore et al., 2006). Protulines® manufactured by Exsymol SAM (Monaco) incorporates Arthrospira, rich in γ-linolenic acid, to repair early skin aging. Codif St (France) produces a product called Dermochlorella® incorporating an extract of C. vulgaris to stimulate skin collagen synthesis (Spolaore et al., 2006). Pentapharm Ltd (Switzerland) (http://www.pentapharm.com) manufactures PEPHA®- TIGHT, a product containing a purified extract of N. oculata with skin-tightening properties and PEPHA®-CTIVE, a D. salina-based skin energy generator. Very promising for cosmetical applications are mycosporine and mycosporine-like aminoacids (MAAs), low-molecular-weight compounds absorbing UVradiation between 310 and 365 nm. Besides their UV screen role, it has been suggested that MAAs act as antioxidants, preventing cellular damage caused by reactive oxygen species (Oren and Gunde-Cinerman, 2007). MAAs have been commercially explored as suncare products, but at present only a product (HELIOGUARD®365) from the red macroalga ‘nori’ is available (Cardozo et al., 2007). Another strongly UV-A absorbing compound with potential application in cosmetics is scytonemin, a pigment of the outer sheath envelope of many cyanobacteria (Sinha and Häder, 2008). The microbial communities (mainly dominated by cyanobacteria) which naturally develop in hot spring waters are widely exploited for medical and cosmetic treatments in spas, though with inadequate scientific investigation.

20.5 Microalgae as source of pharmaceuticals and probiotics 20.5.1 Pharmaceuticals The pharmaceutical industry is in continuous search for novel molecules, to be used directly or to serve as leads for the development of analogues for new drugs. The goal is to obtain more effective chemotherapeutics for treatment of chronic and degenerative diseases and novel antibiotic principles to overcome the increasing problem of resistant pathogens. In this frame, screening organisms of unexplored taxonomic groups and habitats is of fundamental importance to reduce the probability of rediscovering known compounds. Since the mid-1970s, particular efforts have been devoted to screen for ‘bioactive molecules’, a term referring to substances able to affect life processes in a beneficial or in a harmful way at low concentrations (Skulberg, 2000), in two microbial groups traditionally poorly investigated (compared with actinomycetes and fungi): cyanobacteria and microalgae.

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The largest screening (>1000 strains) of cyanobacteria was carried out in the 1980s at the University of Hawaii and led to the identification of many interesting molecules. On the whole, 7 % of the strains showed antiviral (Patterson et al., 1993), 9 % antifungal (Moore et al., 1988) and 7 % antitumour activity (Patterson et al., 1991). Ghasemi et al. (2003) in a screening of 150 cyanobacteria from Iranian paddy fields found a frequency of 14 % for antibacterial and 9 % for antifungal activity. Piccardi et al. (2000) screened 50 strains of the genus Nostoc finding that 24 % were cytotoxic (Artemia salina test), 30 % showed antifungal and 16 % antibacterial activity. In screenings including 400 freshwater and 130 marine algal and cyanobacterial strains, antibacterial activity was found in 10 % of freshwater (Cannell et al., 1988b; Kellam et al., 1988) and 20 % of marine strains (Kellam and Walker, 1989), while antifungal activity was found in 1.5 % of freshwater and 14 % of marine strains (Kellam et al., 1988). Several screenings have focused on targets other than antimicrobial or cytotoxic activity. Cannell et al. (1987, 1988a) detected glycosidase and protease inhibition activity in about 40 strains out of the more than 500 screened. Lincoln et al. (1990) found 35 % of marine and 8 % of freshwater algae interfering with guinea-pig muscle activity. Haemoagglutinating activity towards human erythrocytes was detected in 27 out of 44 freshwater microalgae tested (Chu et al., 2004). Investigation of extreme environments is another possibility to find previously undiscovered substances. In a screening of Antarctic cyanobacteria, Biondi et al. (2008) found 29 % of strains endowed with antibacterial, 21 % with antifungal and 52 % with cytotoxic activity. Pawar and Puranik (2008) report a frequency of about 50 % for antifungal activity in halotolerant cyanobacterial strains. Although rather common, antibacterial activity in cyanobacteria has a low potency so it is difficult to forecast its application in the pharmaceutical field. More interesting levels of activity were found for antifungal compounds, several of which have been patented, though not yet exploited, e.g. cryptophycins (Hirsch et al., 1990) and scytophycins (Moore et al., 1991) isolated from Nostoc sp. and Scytonema hofmanni, respectively. Among the antitumoural compounds, the most promising are cryptophycins, a family of cyclic depsipeptides with a highly potent antimitotic activity against solid, even multi-drug-resistant, tumours. A synthetic analogue, cryptophycin-52, had reached phase II clinical trials before being discontinued for unsatisfactory in vivo activity. New synthetic analogues (cryptophycin 309 and 249) are now in preclinical trials (Liang et al., 2005). Curacin A, a thiazole lipid from Lyngbya majuscula interfering with tubulin, is in preclinical trials as an anticancer agent. In spite of its potency in vitro, the rather insoluble nature makes the in vivo application difficult, though recently a semisynthetic more soluble analogue has been obtained (Newman and Cragg, 2004). Dolastatin 10, a linear peptide acting on tubulin isolated from the marine gastropod Dolabella auricularia, was shown to be synthesized by the cyanobacterium Symploca, a component of the gastropod diet. The

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molecule reached phase II clinical trials as an anticancer agent before being discontinued. Many synthetic analogues from dolastatins 10 and 15 have been produced, three of which reached clinical trials: TZT-1027 now in phase I, synthadotin in phase II and cemadotin discontinued at phase II (Newman and Cragg, 2004; Simmons et al., 2005). Cyanovirin, a peptide from Nostoc ellipsosporum, is available as an experimental vaginal gel protective against HIV infection (Dunlap et al., 2007). New bioactive molecules reported since 2004 are listed in Table 20.7. Microalgae have been less investigated than cyanobacteria until recently. Dinoflagellates represent the most investigated group, especially for antitumoural compounds. Among the identified substances, amphidinolides, from the dinoflagellate Amphidinium, appear as an interesting family, showing a strong toxicity towards human and murine tumour cell lines (Kobayashi and Kubota, 2007). Strong antifungal activity was detected in a family of molecules called gambieric acids, from the dinoflagellate Gambierdiscus toxicus (Nagai et al., 1992). About 70 % of the diatom strains analyzed were able to produce polyunsaturated aldehydes (oxylipines), responsible for the reduced fertility of grazers owing to interference with cell division and toxic towards A. salina (Caldwell et al., 2003; Wichard et al., 2005). Few compounds from microalgae have been patented, among these the cytotoxic compound caribenolide I (Shimizu and Fairchild, 1996) produced by a dinoflagellate and the polysaccharide from the green alga Chlorella, showing antitumour activity (Umezawa and Komiyama, 1985). None of these compounds has been commercially developed. Brevenal is a polyether compound isolated from the dinoflagellate Karenia brevis responsible for red-tides and producer of the neurotoxin brevetoxin. Brevenal displaces the neurotoxin from its receptors (Bourdelais et al., 2005) and could be used to treat humans and animals affected by red-tide toxins (Potera, 2007). However, the most interesting feature of this molecule is its action on mucus clearance, which could be applied in the treatment of diseases such as cystic fibrosis, chronic obstructive pulmonary disease and asthma (Abraham et al., 2005; Baden et al., 2007). Bioactive molecules isolated from microalgae since 2004 are reported in Table 20.7. The best known algal molecules are toxins, of which cyanobacteria and dinoflagellates are the most important producers. Toxins are characterized by their potency and specificity of action. Toxin classes and the main molecules of each class are reported in Table 20.8. Besides their negative effect on the environment and on both humans and animals, algal toxins represent very interesting tools for pharmacological research. Their mechanism of action is generally known, so they are very useful to investigate the cell targets of other substances or the mechanism of action of other toxins (Skulberg, 2004; Garcia Camacho et al., 2007). Other algal metabolites useful as pharmacological tools are phycobiliproteins, derived from red algae or cyanobacteria, which are sensitive fluorescent (phycofluor) probes for immunological assays and fluorescent labels for

Anabaena sp.

Cyanobacteria

Ambigol C Ankaraholide A, B Hassallidin Pahayokolide A Aurilide B, C Jamaicamide Lyngbyabellin E-I

Freshwater

Freshwater

Marine

Soil Freshwater

Marine Marine

Marine

Hassallia sp. Lyngbya sp.

Lyngbya majuscula Lyngbya majuscula

Lyngbya majuscula

Peptide + polyketide

Peptide + polyketide Peptide + polyketide

Lipopeptide Peptide

Dichlorophenol

Alkaloid

Soil

Fischerella ambigua Fischerella ambigua Geitlerinema sp.

Polar lipids

Soil

Proteic pigment

C-phycocyanin Polar lipids

Phenolic compound

Iminotetrasaccharide

Type of molecule

Unknown

Iminotetrasaccharide

Molecule

Alkaline ponds Alkaline ponds Soil

Origin

Ambiguine isonitrile H-J Parsiguine

Arthrospira platensis Arthrospira platensis Chroococcidiopsis sp. Chroococcidiopsis sp. Fischerella sp.

Organism

Algal group

Antifungal Antibacterial, antialgal, cytotoxic Cytotoxic, anticancer Sodium channel blocking agent Cytotoxic

Han et al., 2006 Edwards et al., 2004 Han et al., 2005b

Andrianasolo et al., 2005 Neuhof et al., 2005 Berry et al., 2004

Antonopoulou et al., 2005b Antonopoulou et al., 2005b Raveh and Carmeli, 2007 Ghasemi et al., 2004 Wright et al., 2005

Hsiao et al., 2005

Thammana et al., 2006 Wu et al., 2005

β-glucoronidase inhibitor Antiproliferative Platelet aggregation inhibitor Platelet aggregation inhibitor Platelet aggregation agent Antibacterial, antifungal Antibacterial, antifungal Antibacterial, anti-protozoan Cytotoxic

Reference

Activity

Table 20.7 Bioactive molecules from cyanobacteria and microalgae reported since 2004. Molecules discovered before 2004 are extensively reviewed in Burja et al. (2001), Daranas et al. (2001), Shimizu (2003), Bhadury and Wright (2004), Harada (2004), Dunlap et al. (2007), Kobayashi and Kubota (2007) and Tan (2007)

Cyclic peptide

Wewakpeptin A-D Cyanopeptolin 954 Norharmalane Banyaside A, B Carbamidocyclophane A-E Nostocarboline Nostoflan Dihydroxybiphenyl Largamide D–G Venturamide A, B

Marine Freshwater

Freshwater

Freshwater

Soil

Freshwater

Soil

Freshwater

Marine

Marine

Freshwater

Freshwater

Lyngbya semiplena Microcystis aeruginosa Nodularia harveyana Nostoc sp.

Nostoc sp.

Nostoc sp.

Nostoc flagelliforme Nostoc insulare

Oscillatoria sp.

Oscillatoria sp.

Planktothrix sp.

Planktothrix rubescens

Planktocyclin

LPS-like

Dragonamide C, D

Marine

Cyclic peptide

Cyclic peptide

Dihydroxybiphenyl

Polysaccharide

Chlorinated paracyclophane β-carboline alkaloid

Peptide

β-carboline alkaloid

Cyclic depsipeptides Depsipeptide

Lipopeptide

Polychlorinated amides

Taveuniamides

Marine

Lyngbya majuscula/ Schizothrix Lyngbya polychroa

Cyclic depsipeptide

Trungapeptin A

Marine

Lyngbya majuscula

Bacterial endotoxin antagonist Protease inhibitor

Antibacterial, antifungal, antialgal Chymotrypsin inhibitor Antimalarial

Antibacterial, antifungal, cytotoxic Cholinesterase inhibitor Antiviral

Protease inhibitor

Cytotoxic Chymotrypsin inhibitor Antialgal

Cytotoxic

Cytotoxic

Ichthyotoxic

Kanekiyo et al., 2005 Volk and Furkert, 2006 Plaza and Bewley, 2006 Linington et al., 2007 Macagno et al., 2006 Baumann et al., 2007

Becher et al., 2005

Ploutno and Carmeli, 2005 Bui et al., 2007

Gunasekera et al., 2008 Han et al., 2005a von Elert et al., 2005 Volk, 2006

Bunyajetpong et al., 2006 Williamson et al., 2004

Unknown Spirolide G Amphezonol A Iriomoteolide-3a Karatungiol A

Marine

Marine

Marine

Marine Marine

Alexandrium ostenfeldii Alexandrium ostenfeldii Amphidinium sp.

Amphidinium sp. Amphidinium sp.

Dinoflagellates

Brunsvicamide B, C

Freshwater

Fucoxanthin

Malevamide E

Marine

Symploca laete-viridis Tychonema sp.

Marine

Micromide

Marine

Symploca sp.

Unknown

Guamamide

Marine

Symploca sp.

Marine

Peptide

Belamide

Marine

Chaetoceros muelleri Odontella aurita

Glycolipid

Soil

Macrolide Polyol

Polyhydroxyl

Imine

Unknown

Carotenoid

Lipidic fraction

DNA polymerase inhibitor Cytotoxic, antiviral Antifungal, antiprotozoan

Algicidal, antiprotozoan Toxic

Antibacterial, antifungal Cytotoxic, anticancer

Calcium channel inhibitor Protein phosphatase inhibitor in Mycobacterium tubercolosis

Peptide + polyketide Cyclic peptide

Cytotoxic

Cytotoxic

Platelet aggregation inhibitor Platelet aggregation agent Antimitotic

Activity

Alkaloid

Alkaloid

Glycolipid

Soil

Type of molecule

Scytonema julianum Scytonema julianum Symploca sp.

Molecule

Origin

Organism

Cont’d

Diatoms

Algal group

Table 20.7

Oguchi et al., 2008 Washida et al., 2006

Tillmann et al., 2007 MacKinnon et al., 2006 Kubota et al., 2006

Mendiola et al., 2007 Moreau et al., 2006

Müller et al., 2006

Antonopoulou et al., 2005a Antonopoulou et al., 2005a Simmons et al., 2006 Williams et al., 2004 Williams et al., 2004 Adams et al., 2008

Reference

19-epi okadaic acid Prorocentin Protoceratin Zooxanthellactone Symbioimine Zooxanthellamide Cs Zooxanthellamide D

Marine

Marine Marine

Marine

Marine

Marine

Marine

Prorocentrum belizeanum Prorocentrum lima Protoceratium cf. reticulatum Symbiodinium sp.

Symbiodinium sp.

Symbiodinium sp.

Symbiodinium sp.

F4 Fj1-3

Marine

Marine

Chattonella marina

Fibrocapsa japonica

Raphidophytes

Photosynthetic pigment derivative Polyunsaturated fatty acid

Polysaccharide/ protein complex Peptide

Freshwater CPH

Polysaccharide

Freshwater

Freshwater

Free fatty acids

Botryococcus braunii Chlorella pyrenoidosa Chlorella pyrenoidosa Chlorella pyrenoidosa

Green algae

Water-soluble fraction

Macrolide

Macrolide

Imine

Oxylipine

Polyketide Glycosidic polyether

Polyether

Polyether

Polyhydroxyl Polyene-polyhydroxy

Freshwater

Euglena sanguinea

Euglenophytes

Unknown

Brevenal

Marine

Freshwater

Lingshuiol Amphidinol 11–13

Marine Marine

Amphidinium sp. Amphidinium carterae Karenia brevis

Haemolytic

Haemolytic, cytotoxic

Haemoagglutinin

Immunomodulator

Antialgal, antiprotozoan Immunomodulator

Ichthyotoxic

Cytotoxic

Osteoclastogenesis inhibitor Vasoconstrictive

Cytotoxic

Antitumor Antifungal, haemolytic Brevetoxin antagonist Protein phosphatase inhibitor Anticancer Cytotoxic, anticancer

Fu et al., 2004

Kuroda et al., 2005

Kralovec et al., 2007 Chu et al., 2006

Yang et al., 2006

Chiang et al., 2004

Zimba et al., 2004

Onodera et al., 2005 Fukatsu et al., 2007

Lu et al., 2005 Konishi et al., 2004 Onodera et al., 2004 Kita et al., 2004

Huang et al., 2004 Echigoya et al., 2005 Bourdelais et al., 2005 Cruz et al., 2007

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Table 20.8 Main toxins from cyanobacteria and microalgae. Exhaustive reviews can be found in Daranas et al. (2001), Haider et al. (2003), Houdan et al. (2004), Codd et al. (2005), Friedman and Levin (2005), Wiegand and Pflugmacher (2005), Stewart et al. (2006a, b), Ibelings and Chorus (2007) and Tubaro and Hungerford (2007) Toxin

Toxic action

Producing organism

Anatoxin-a

Neurotoxic

Anatoxin-a(s) Omoanatoxin-a Kalkitoxin, antillatoxin, jamaicamide β-methylamine-L-alanine

Neurotoxic Neurotoxic Neurotoxic

Anabaena (C), Aphanizomenon (C), Cylindrospermum (C) Anabaena (C) Oscillatoria (C) Lyngbya (C)

Neurotoxic

Saxitoxin, neo-saxitoxin, gonyautoxins and derivatives

Neurotoxic (PSP)

Brevetoxin Domoic acid and derivatives

Neurotoxic (NSP) Neurotoxic (ASP)

Unknown

Neurotoxic (PEAS)

Ciguatoxin, maitotoxin, gambierol, palytoxin Azaspiracids

Gastrointestinal, cardiovascular and neurotoxic effect inducer (CFP) Neurotoxic, gastrointestinal and cardiovascular effect inducer (AZP) Gastric distress and tachycardia inducer Diarrhoetic (DSP), tumour promoter

Spirolides Okadaic acid and derivatives, dinophysistoxin, yessotoxin and dervatives, pectenotoxin Microcystins

Nodularin

Epatotoxic, tumour promoter

Epatotoxic, tumour promoter

Several cyanobacterial genera Anabaena (C), Aphanizomenon (C), Cylindrospermopsis (C), Lyngbya (C), Oscillatoria (C) Alexandrium (D), Gymnodinium (D), Pyrodinium (D) Karenia (D) Pseudonitzschia (B), Nitzschia (B), Amphora (B) Pfiesteria piscicida (D) (possibly) Gambierdiscus (D), Amphidinium (D), Prorocentrum (D), Ostreopsis (D) Protoperidinium (D) (possibly) Alexandrium (D) Dinophysis (D), Prorocentrum (D), Protoceratium (D), Coolia (D) Microcystis (C), Anabaena (C), Hapalosiphon (C), Nostoc (C), Oscillatoria (C) Nodularia (C)

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Table 20.8 Cont’d Toxin

Toxic action

Producing organism

Cylindrospermopsin

Epatotoxic

Lyngbyatoxin, aplysiatoxin and derivatives Lipopolysaccharides Prymnesin other unknown

Dermotoxic, tumour promoter Endotoxic Haemolytic, ichthyotoxic

PUFA

Haemolytic, ichthyotoxic

Cylindrospermopsis (C), Aphanizomenon (C) Lyngbya (C), Schizothrix (C) Cyanobacteria Prymnesium (P), Chrysochromulina (P) Fibrocapsa (R), Heterocapsa (R), Chattonella (R)

ASP = amnesic shellfish poisoning,AZP = azaspiracid poisoning, B = diatom (Bacillariophyceae), C = cyanobacterium, CFC = ciguatera fish poisoning, D = dinoflagellate, DSP = diarrhoetic shellfish poisoning, NSP = neurotoxic shellfish poisoning, P = prymnesiophyte, PEAS = possible estaurine associated illness, PSP = paralytic shellfish poisoning, PUFA = polyunsaturated fatty acids, R = raphydophytes.

cell-sorting (Bermejo Román et al., 2002) (see Table 20.6 for commercial applications). Bioactive molecules could also be applied as agrochemicals (Skulberg, 2000; Saxena and Pandey, 2001; Ördög et al., 2004). Many of the substances screened for pharmaceutical purposes, such as majusculamide C (Moore and Mynderse, 1982) and cryptophycins (Biondi et al., 2004), have proved to be active also against targets of agricultural importance. Antibacterial and antifungal activity are useful routes to produce leads for biopesticides, and toxicity towards eukaryotic cells indicates potential activity against insects and nematodes. Besides inhibitory activity, many microalgae and cynobacteria are endowed with plant growth promoting (phytohormonal) activity (Stirk et al., 2002). In the exploitation of bioactive molecules there are two main concerns besides their actual activity: (i) avoid loss of the active strain or of the activity following mutations in the encoding gene(s) (for this, cryopreservation seems to be the solution, although several strains lose viability with this treatment (Hédoin et al., 2006)); (ii) provide enough molecule to carry out clinical trials and commercial exploitation. This can be achieved in several ways, such as improvement of chemical synthesis, especially concerning raceme separation. When synthetic molecules are unavailable, there is the need to establish cultivation protocols aimed at maximizing the metabolite production (Borowitzka, 1999). These compounds are mainly secondary metabolites, and thus they are usually produced in small amounts and, often, during the stationary phase. To overcome this problem, besides physiological studies, genetic engineering (the so-called molecular farming) has been proposed. Genes encoding for the desired metabolite could be

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introduced in one of those species that are transformable, such as Chlamydomonas reinhardtii, D. salina or Arthrospira platensis (Hallman, 2007). This would allow the production of antibodies and vaccines and also the production or modification of bioactive molecules (Hallman, 2007). Of course, besides the dispute on transgenic organisms, to bring these systems to the market requires much work to find the genes involved in the synthesis of the bioactive molecules.

20.5.2 Probiotics in aquaculture The ‘green water’ technique used in aquaculture farms is based on the empirical observation of the positive effects that keeping microalgae in the rearing ponds brings in terms of lower sensitivity to pathogens and better growth results (see Section 20.3.4). Historically, this has been the starting point for the investigation of allelopathic activity in algae and cyanobacteria. Recently, the concept of probiotics has been introduced. In aquaculture this is slightly different from that referring to mammals, mainly because aquatic organisms live immersed in water, which bear potential pathogens not only in the intestinal tract but on the entirety of their skin, including the gills. So an aquaculture probiotic is considered to be a microorganism, or a part of it, able to ameliorate the general health of the host (Irianto and Austin, 2002). The probiotic in this case can also be dispersed in the water and not necessarily provided via feed. A probiotic can stimulate an immune response, alter enzymatic activity, exclude potential pathogens by antibiosis or nutrient, space or oxygen competition (Irianto and Austin, 2002). Besides the classical bacteria, Irianto and Austin (2002) proposed the green alga T. suecica, endowed with antibacterial activity towards aquaculture-important pathogens, as a probiotic in aquaculture. Recently the use of algae as probiotics in aquaculture ponds has been the object of a patent application (Kyle, 2006). The interactions between algae and other microorganisms are very complex and cannot be exposed here. They are based on the balance between stimulatory activities, due to production of exudates, or inhibitory activities, due to both antibiosis and other kinds of competition, of algae towards bacteria and the stimulatory or inhibitory activity of bacteria towards algae. In this sense, it is useful to introduce the concept of ‘phycosphere’, in parallel to the rhizosphere of plants, to indicate the space around the alga where these interactions take place (Tredici, 2008). It is somehow as if the alga chooses its own associated microflora. For example, Salvesen et al. (2000) found no Vibrio associated with algae used in larviculture, but a higher number of opportunistic bacteria were associated with diatoms compared to Tetraselmis. Makridis et al. (2006) demonstrated that bacteria isolated from cultures of Chlorella and Tetraselmis were able to inhibit Photobacterium damselae growth in vitro and to reduce bacterial load and Vibrio levels in Artemia metanauplii in vivo. It was also shown that

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Chaetoceros, Nitzschia and Leptolyngbya, associated with ‘green water’ practices in tiger shrimp (Penaeus monodon) farming, were able to prevent Vibrio harveyi outbreaks by reducing or completely halting growth of this pathogen when co-cultivated (Lio-Po et al., 2005). Similar prevention of Vibrio outbreaks was found in Fenneropenaeus indicus farmed with Tetraselmis in both larval and broodstock tanks (Reghunatan and Wesley, 2004). Marques et al. (2006) tested two Dunaliella cultures on gnotobiotic Artemia to evaluate their potential in the defence against Vibrio infections, showing that algae alone, especially good-quality cultures, were able to protect the crustaceans and appeared to be more efficient and stable than probiotic bacteria. The way in which the alga provides protection is still unclear. The possibilities are stimulation of Artemia digestion physiology or immune response, or antibiosis. Dunaliella extracts have also been reported to increase Penaeus monodon response against white spot syndrome virus (Supamattaya et al., 2005). Among the possible ways in which some algae could control associated bacteria and then, possibly, microbial community composition in the aquaculture ponds, there is the ability to produce mimics of the bacterial quorum sensing signal molecules, homoserine lactones (Defoirdt et al., 2004). Recently these type of substances have been detected in soil-isolated Chlamydomonas and Chlorella strains (Teplitski et al., 2004). Disrupting quorum sensing prevents bacterial biofilm formation and the onset of some virulence factors (Bauer and Robinson, 2002). This is a new field of investigation and much work is still necessary to clarify the exact interaction mechanism, the implications for microbial community ecology and the potential application of these substances as substitutes for antibiotics in aquaculture. Another way in which algae can be used to increase health of aquaculture species is as vectors of probiotic bacteria. In this case, bacteria should be useful to the host and useful or neutral to the microalga (KesarcodiWatson et al., 2008). A probiotic bacterium showing anti-Vibrio anguillarum activity was co-cultivated with axenic I. galbana, then the consortium was used to feed Argopecten purpuratum larvae, leading to an increased probiotic bacteria ingestion with respect to the bacteria fed alone (Avendan˜o and Riquelme, 1999).

20.6 Wastewater reclamation and biofuel production by algae–bacteria consortia Applications of mass algae cultures to wastewater treatment are very limited. The need for large land areas, the strong influence of climatic conditions on the process rate and efficiency, the expensive separation of the algal cells from the liquid and difficulty in finding an appropriate use for the algal biomass have strongly hampered the diffusion of this technology. The

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shortage of fossil fuels and growing concern that biofuel production from agriculture crops may jeopardize food security and increase agricultural commodities prices have changed this perspective. Algae cultivation in wastewater may be now viewed as a process able to economically produce a valuable source of renewable, carbon-neutral fuel (and possibly feed and fertilizer) not competing for resources with agriculture. Liquid wastes (human, agricultural, industrial) provide nutrients at no cost, and together with the disposal credits, may offer the possibility to bridge the gap between the current cost of algal biomass production in clean artificial media (d3–30 kg−1) and the very low cost required when the biomass is intended for biofuel production (
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Sunlight

O2 Organic matter

Bacterial oxidation

Algal photosynthesis

CO2 NH3 Phosphate Biomass

Fig. 20.2

Cycle for photosynthetic oxygenation of wastewater.

20.5.2). One main problem is that typically algae grow at slower rates than bacteria, and maintaining a stable consortium may be difficult. The complex interactions between the two groups and the factors responsible for the stability of the association must be investigated and fully understood for full exploitation of these systems. In the presence of sufficient light, HRAP can treat up to 35 g BOD m−2 d−1 (Mun˜oz and Guieysse, 2006), removing more than 90 % of the BOD (Oswald, 1988) and of the nitrogen and phosphorus content of the wastewater (Tredici et al., 1992), with retention times of a few days. This can be very productive in terms of biomass. In well-managed HRAP, the photosynthetically generated oxygen may suffice to support decomposition of complex polymers and hazardous organic pollutants.The efficacy of microalgae– bacteria consortia in the degradation of recalcitrant compounds (such as phenantrene, acetonitrile, phenol, salicylate) and detoxification of industrial wastewater has been proved (Mun˜oz and Guieysse, 2006). Other nonsecondary advantages are the absence of sludge accumulation, prevention of malodours and elimination of viruses and pathogenic bacteria. Provided the resulting biomass is separated, well-designed HRAP may attain a highquality effluent, and represent a valuable alternative to conventional systems based on activated sludge and trickling filters, with most of the energy for the process derived from sunlight. HRAP may achieve productivities that surpass that of algae cultures in clean media. Animal and human wastes typically contain all the nutrients required for algae growth, although carbon tends to become a limiting factor (Oswald, 1988). To fully achieve the potential productivity of HRAP (30–40 g m−2 d−1) addition of an exogenous carbon source is necessary. This may be provided by waste CO2. In Israel, with ponds of 300 m2, productivities of up to 100 g m−2 d−1 were measured in the summer, a value which is near the theoretical maximum. Many other studies, however, report variable and much lower productivities. However, interesting performances have been observed even in temperate and cold regions. It has been shown

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that, above a certain threshold, productivity becomes a linear function of solar radiation (Oswald, 1988; Tredici and Chini Zittelli, 1998), and in the Mediterranean countries an annual average productivity of 20 g m−2 d−1 should be achievable even at large scale. The high productivities found in HRAP may be explained by the fact that one third of the biomass is actually non-algal solids. A second factor which can significantly increase productivity is direct use of the organic soluble material by the algae (mixotrophy). Achieving maximum productivities depends mainly on location and characteristics of the waste. Despite their advantages, only a few full-scale HRAP plants are in operation (e.g. in St. Helena and Hollister, USA), because engineers are unaware of their efficiency and capability, and more complex and expensive systems are preferred (Oswald, 1988). There are also inherent drawbacks of this technology. The main limitation is that to be fully functional the algae cultures must receive sufficient light and not be limited by low temperatures. Thus, depending on the latitude, HRAP may be operative year-round or only part of the year. Other main limitations are lack of control of the composition of the algal population, algae susceptibility to infection by viruses, bacteria and fungi, and predation by protozoa, and the presence of compounds toxic to the algae. Land availability may be a severe limitation. When used for energy production, with water reclamation, given their much higher productivity and no requirement for arable land, HRAP present a clear advantage over conventional crops. The most difficult tasks will be maintaining a selected microalgal species in the culture, a necessary requisite when a specific use of the biomass (e.g. oil extraction) is pursued (Rodolfi et al., 2009), and developing efficient harvesting and processing techniques. Many variables, such as climatic conditions and wastewater composition, which affect algae and bacteria growth, cannot be controlled or are very difficult to optimize, especially in the shallow environment of the algal pond characterized by high thermal instability and strong chemical fluctuations due to evaporation and precipitations. It is the influence of many environmental, biological and operational factors and the complex interactions among algae and bacteria that make microalgae–bacteria consortia in wastewater very unstable systems. The cultivation of selected, fast-growing and resistant algal species of known requirements may help to improve the stability of the system, but will prove to be very difficult. Coupling closed bioreactors, in which active inocula of the selected species are produced, with open ponds for bulk cultivation may offer a solution (Rodolfi et al., 2009). Another major constraint is the difficulty of harvesting the algae biomass. This may be achieved by centrifugation, chemical flocculation followed by sedimentation or flotation or filtration. Centrifugation is very efficient, but its cost is prohibitive (in energy terms it was reported to be about 2700 kWh ton−1). Auto-flocculation, a natural process which occurs on sunny days when the increase in pond pH causes the algae to co-precipitate

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with carbonates and phosphates, is economically viable, but its dependability remains to be established. After separation, the biomass may be used directly as a slurry (e.g. for anaerobic digestion) or after dewatering and drying (e.g. for extraction of oil). If fermented to methane and converted to electrical energy, 1 kg of waste-grown algae can provide 1 kWh of electrical energy (Oswald, 1988). If dried and extracted, some microalgae will furnish 0.2–0.3 kg of oil per kilogram of biomass, with an oil productivity approaching, in the Mediterranean area, 10–12 tons per hectare per year (Rodolfi et al., 2009). Up to 0.6 kg of oil per kilogram of biomass and up to 20 tons oil per hectare per year can be obtained under nitrogen starvation (Rodolfi et al., 2009), which is, however, difficult to attain with wastewater. The greatest challenge will be producing the algal biomass at the low cost required for biofuel production. The high productivity of HRAP, no competition for crop land or clean water resources, and the possibility to combine biomass production with wastewater reclamation and fixation of CO2 and other hazardous combustion gases, such as NOx and SOx, justify the long-term R&D required to bring this technology to the market.

20.7 Future trends It is possible to foresee a huge increase in the demand for cultured algae, in terms of both quantity and diversity. The depletion of natural resources, such as fish stocks, will greatly amplify the necessity to produce seafood through aquaculture and, then, higher amounts of algal biomass will be required. The increasing number of people having the economic capacity to access aquaculture products will determine a widening of the market and thus an enhancement of production, not only in terms of quantity of product from already reared species, but also in terms of number of species to be reared. New animal species imply also new microalgae to fulfil their nutritional requirements. At the same time, the general perception that natural products are ‘healthier’ and more ‘friendly’ than synthetic ones is continuously increasing the necessity to produce algal biomass for dietary supplements and cosmetics. Both climate change, i.e. global warming, and globalization, that has magnified exchanges of goods and people movement throughout the world, have created the ideal situation for spreading infections. This is one of the factors, together with pathogen resistance to the presently used antibiotics, that will guide research in the pharmaceutical field. The search for chemotherapeutics to fight the most important diseases of the Western world will also continue to be a central focus for research. The poorly investigated microbial groups, like microalgae and cyanobacteria, will play an important role as sources of new lead compounds for drug development. Last, but surely not least, algal biomass might become an important source of biofuels, especially if its production can be associated

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with wastewater treatment and greenhouse gas abatement. In this latter case, the role of microalgae could be outstanding because of their ability to produce fuels without entering into competition with food production, as is the case of other crops like corn or sugarcane. The production of algae for high-value markets (aquaculture, food supplements, nutraceuticals, pharmaceuticals) will be developed through the search for, isolation and cultivation of new algal strains endowed with interesting activities or able to produce the desired compound more efficiently or in higher amounts. In-depth investigations on their physiology and genetics will be necessary in order to grow them and understand the cellular/molecular mechanisms that trigger the synthesis of the high-value compound, and allow the complete exploitation of their potential. These kinds of studies will also be useful for species which are already cultivated. For these applications, the reactor design is probably at its near-maximum potential, and no further great innovations are to be expected in the near future. To significantly decrease the cost of algal biomass for bioenergy two routes should be followed. First, the microalgae cultivated, either new isolates or already exploited strains, should be, as mentioned above, thoroughly studied to maximize their productivity in terms of biomass or of the desired component (e.g. triacylglycerols for biodiesel, sugars for ethanol). A great advantage might come from cultivation of thermophilic strains that will reduce the cost of biomass production in bioreactors by reducing cooling needs and the potential for contamination. The second way to be followed is the design of new reactors of very low cost, allowing inexpensive production of inocula and limiting the use of ponds to the last phase, where the accumulation of the ‘energetic’ compound is achieved. For a full exploitation of algal biotechnology, either with new or ‘old’ strains further research on the complex interactions between algae and associated bacteria is mandatory.

20.8 Sources of further information and advice Key books • Cohen Z (1999) Chemicals from Microalgae, London, Taylor and Francis. • Støttrup J and McEvoy L A (2003) Live Feeds in Marine Aquaculture, Oxford, Blackwell Science. • Richmond A (2004) Handbook of Microalgal Culture: Biotechnology and Applied Phycology, Oxford, Blackwell Science. • Barsanti L and Gualtieri P (2006) Algae: Anatomy, Biochemistry and Biotechnology, Boca Raton, FL, CRC Press, Taylor and Francis Group. • Flickinger M C, Wiley Encyclopedia of Industrial Biotechnology, New York, Wiley & Sons, in press.

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Relevant websites • Martek Biosciences Corp (Columbia, USA): http://www.martek.com • Cyanotech Corp (Hawaii, USA): http://www.cyanotech.com • Fotosintetica & Microbiologica Srl (Florence, Italy): http://www. femonline.it • Reed Mariculture Inc (California, USA): http://www.reed-mariculture. com

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baumann h i, keller s, wolter f e, nicholson g j, jung g, süssmuth r d and jüttner f (2007) Planktocyclin, a cyclooctapeptide protease inhibitor produced by the freshwater cyanobacterium Planktothrix rubescens, J Nat Prod, 70(10), 1611–15. becher p g, beuchat j, gademann k and jüttner f (2005) Nostocarboline: isolation and synthesis of a new cholinesterase inhibitor from Nostoc 78-12A, J Nat Prod, 68(12), 1793–5. becker w (2004) Microalgae in human and animal nutrition, in Richmond A (ed.), Handbook of Microalgal Culture: Biotechnology and Applied Phycology, Oxford, Blackwell, 312–51. becker e w (2007) Micro-algae as a source of protein, Biotechnol Adv, 25(2), 207–10. ben-amotz a (2004) Industrial production of microalgal cell-mass and secondary products – Major industrial species – Dunaliella, in Richmond A (ed.), Handbook of Microalgal Culture: Biotechnology and Applied Phycology, Oxford, Blackwell, 273–80. bermejo román r, alvárez-pez j m, acién fernández f g and molina grima e (2002) Recovery of pure B-phycoerythrin from the microalga Porphyridium cruentum, J Biotechnol, 93(1), 73–85. berry j p, gantar m, gawley r e, wang m and rein k s (2004) Pharmacology and toxicology of pahayokolide A, a bioactive metabolite from a freshwater species of Lyngbya isolated from the Florida Everglades, Comp Biochem Physiol C, 139(4), 231–8. bhadury p and wright p c (2004) Exploitation of marine algae: biogenic compounds for potential antifouling applications, Planta, 219(4), 561–78. biondi n, piccardi r, margheri m c, rodolfi l, smith g d and tredici m r (2004) Evaluation of Nostoc strain ATCC 53789 as a potential source of natural pesticides, Appl Environ Microbiol, 70(6), 3313–20. biondi n, tredici m r, taton a, wilmotte a, hodgson d a, losi d and marinelli f (2008) Cyanobacteria from benthic mats of Antarctic lakes as a source of new bioactivities, J Appl Microbiol, 105(1), 105–15. bonaldo a, badiani a, testi s, corso g, mordenti a l and gatta p p (2005) Use of centrifuged and preserved microalgae for feeding juvenile Manila clam (Tapes philippinarum): effects on growth and fatty acid composition, Ital J Anim Sci, 4, 375–84. borowitzka m a (1997) Microalgae for aquaculture: opportunities and constraints, J Appl Phycol, 9(5), 393–401. borowitzka m a (1999) Pharmaceuticals and agrochemicals from microalgae, in Cohen Z (ed.), Chemicals from Microalgae, London, Taylor & Francis, 313–41. bosma r, van spronsen w a, tramper j and wijffels r h (2003) Ultrasound, a new separation technique to harvest microalgae, J Appl Phycol, 15(2/3), 143–53. bourdelais a j, jacocks h m, wright j l c, bigwarfe jr. p m and baden d g (2005) A new polyether ladder compound produced by the dinoflagellate Karenia brevis, J Nat Prod, 68(1), 2–6. boussiba s and zarka a (2005) Flat panel photobioreactor, Patent WO 2005/006838. brown m r (1991) The amino-acid and sugar composition of 16 species of microalgae used in mariculture, J Exp Mar Biol Ecol, 145(1), 79–99. brown m and robert r (2002) Preparation and assessment of microalgal concentrates as feeds for larval and juvenile Pacific oyster (Crassostrea gigas), Aquaculture, 207(3/4), 289–309. brown m r, dunstan g a, jeffrey s w, volkman j k, barret s m and leroi j m (1993) The influence of irradiance on the biochemical composition of the Prymnesiophyte Isochrysis sp. (clone T-ISO), J Phycol, 29(5), 601–12. brown m r, volkman j k and dunstan g a (1997) Nutritional properties of microalgae for mariculture, Aquaculture, 151(1/4), 315–31.

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bruce j r, knight m and parke m w (1940) The rearing of oyster larvae on an algal diet, J Mar Biol Ass UK, 24(1), 337–74. bui h t n, jansen r, pham h t l and mundt s (2007) Carbamidocyclophanes A-E, chlorinated paracyclophanes with cytotoxic and antibiotic activity from the Vietnamese cyanobacterium Nostoc sp., J Nat Prod, 70(4), 499–503. bunyajetpong s, yoshida w y, sitachitta n and kaya k (2006) Trungapeptins A-C, cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula, J Nat Prod, 69(11), 1539–42. burja a m, banaigs b, abou-mansour e, grant burgess j and wright p c (2001) Marine cyanobacteria – A prolific source of natural products, Tetrahedron, 57(46), 9347–77. cabrera t and hur s b (2001) The nutritional value of live foods on the larval growth and survival of Japanese flounder, Paralichthys olivaceus, J Appl Aquac, 11(1/2), 35–53. cahu c, zambonino infante j, péres a, quazuguel p and le gall m (1998) Algal addition in sea bass (Dicentrarchus labrax) larvae rearing: effect on digestive enzymes, Aquaculture, 161(1/4), 479–89. cahu c, zambonino infante j and takeuchi t (2003) Nutritional components affecting skeletal development in fish larvae, Aquaculture, 227(1/4), 245–58. caldwell g s, bentley m g and olive p j w (2003) The use of a brine shrimp (Artemia salina) bioassay to assess the toxicity of diatom extracts and short chain aldehydes, Toxicon, 42(3), 301–6. can˜avate j p and fernández-díaz c (2001) Pilot evaluation of freeze-dried microalgae in the mass rearing of gilthead seabream (Sparus aurata) larvae, Aquaculture, 193(3/4), 257–69. can˜ avate j p, prieto a, zerolo r, sole m, sarasquete c and fernández-díaz c (2007) Effects of light intensity and addition of carotene rich Dunaliella salina live cells on growth and antioxidant activity of Solea senegalensis Kaup (1858) larval and metamorphic stages, J Fish Biol, 71(3), 781–94. cannell r j p, kellam s j, owsianka a m and walker j m (1987) Microalgae and cyanobacteria as a source of glycosidase inhibitors, J Gen Microbiol, 133(7), 1701–5. cannell r j p, kellam s j, owsianka a m and walker j m (1988a) Results of a largescale screen of microalgae for the production of protease inhibitors, Planta Med, 54(1), 10–14. cannell r j p, owsianka a m and walker j m (1988b) Results of a large-scale screening programme to detect antibacterial activity from freshwater algae, Br Phycol J, 23(1), 41–4. cárcamo p f, candia a i and chaparro o r (2005) Larval development and metamorphosis in the sea urchin Loxechinus albus (Echinodermata: Echinoidea): effects of diet type and feeding frequency, Aquaculture, 249(1/4), 375–86. cardozo k h m, guaratini t, barros m p, falcão v r, tonon a p, lopes n p, campos s, torres m a, souza a o, colepicolo p and pinto e (2007) Metabolites from algae with economical impact, Comp Biochem Phys C, 146(1/2), 60–78. carmichael w w, drapeau c and anderson d m (2000) Harvesting of Aphanizomenon flos-aquae Ralfs ex Born. & Flah. var. flos-aquae (Cyanobacteria) from Klamath Lake for human dietary use, J Appl Phycol, 12(6), 585–95. carvalho a p and malcata f x (2000) Effect of culture media on production of polyunsaturated fatty acid by Pavlova lutheri, Cryptogam Algol, 21(1), 59–71. carvalho a p, meireles l a and malcata f x (2006) Microalgal reactors: a review of enclosed system designs and performances, Biotechnol Prog, 22(6), 1490–506. chaumont d (1993) Biotechnology of algal biomass production: a review of systems for outdoor mass culture, J Appl Phycol, 5(6), 593–604.

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chiang i, huang w and wu j (2004) Allelochemicals of Botryococcus braunii (Chlorophyceae), J Phycol, 40(3), 474–80. chini zittelli g, lavista f, bastianini a, rodolfi l, vincenzini m and tredici m r (1999) Production of eicosapentaenoic acid by Nannochloropsis sp. cultures in outdoor tubular photobioreactors, J Biotechnol, 70(1/3), 299–312. chini zittelli g, rodolfi l, ponis e and tredici m r (2003) Cold preservation and use of microalgae aquaculture feed, Abstracts of the 5th European Workshop on Biotechnology of Microalgae, 23–24 June, Bergholz-Rehbrücke. chini zittelli g, rodolfi l, biondi n and tredici m r (2006) Productivity and photosynthetic efficiency of outdoor cultures of Tetraselmis suecica in annular columns, Aquaculture, 261(3), 932–43. chu c y, liao w r, huang r and lin l p (2004) Haemagglutinating and antibiotic activities of freshwater microalgae, World J Microbiol Biotechnol, 20(8), 817–25. chu c, huang r and ling l (2006) Purification and characterization of a novel haemagglutinin from Chlorella pyrenoidosa, J Ind Microbiol Biotechnol, 33(11), 967–73. chuntapa d, powtongsook s and menasveta p (2003) Water quality control using Spirulina platensis in shrimp culture tanks, Aquaculture, 220(1/4), 355–66. codd g a, morrison l f and metcalf j s (2005) Cyanobacterial toxins: risk management for health protection, Toxicol Appl Pharmacol, 203(3), 264–72. cohen z (1997) The chemicals of Spirulina, in Vonshak A (ed.), Spirulina platensis (Arthrospira): Physiology, Cell-biology and Biotechnology, London, Taylor & Francis, 101–15. cook p m (1950) Large-scale culture of Chlorella, in Brunel J, Prescott G W and Tiffany L H (eds), The Culturing of Algae, Yellow Springs, OH, Charles F. Kettering Foundation, 53–75. coutteau p and sorgeloos p (1992) The use of algal substitutes and the requirement for live algae in hatchery and nursery rearing of bivalve molluscs: an international survey, J Shellfish Res, 11(2), 467–76. coutteau p, lavens p and sorgeloos p (1990) Baker’s yeast as a potential substitute for live algae in aquaculture diets: Artemia as a case study, J World Aquac Soc, 21(1), 1–8. craggs r j, mculey p j and smith v j (1997) Wastewater nutrient removal by marine microalgae grown on a corrugated raceway, Water Res, 31(7), 1701–7. cruz p g, hernández daranas a, fernández j j and norte m (2007) 19- epi-okadaic acid, a novel protein phosphatase inhibitor with enhanced selectivity, Org Lett, 9(16), 3045–8. csordas a and wang j-k (2004) An integrated photobioreactor and foam fractionation unit for the growth and harvest of Chaetoceros spp. in open system, Aquac Eng, 30(1/2), 15–30. daranas a h, norte m and fernández j j (2001) Toxic marine microalgae, Toxicon, 39(8), 1101–32. daume s, brand-gardner s and woelkerling w (1999) Settlement of abalone larvae (Haliotis laevigata Donovan) in response to non-geniculate coralline red algae (Corallinales, Rhodophyta), J Exp Mar Biol Ecol, 234(1), 125–43. davis h c and guillard r r (1958) Relative value of ten genera of micro-organisms as foods for oyster and clam larvae, Fishery Bulletin of the Fish and Wildlife Service 136, 58, 293–304. de montgolfier b, audet c and lambert y (2005) Growth of early juvenile winter flounder (Pseudopleuronectes americanus Walbaum), Aquac Res, 36(16), 1595–601.

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21 Predicting and assessing the environmental impact of aquaculture C. Crawford and C. MacLeod, University of Tasmania, Australia

Abstract: Aquaculture can affect the environment in a number of ways, including organic enrichment around the farm, increased dissolved nutrients or chemicals in the growing area, escapees affecting native species, loss of habitat and loss of amenity. Likewise, the environment can affect aquaculture, through poor water quality (e.g. pollutants, high particulate matter), predators and nuisances species such as jellyfish or seals, or changing climate regimes. These impacts can be reduced by careful site selection, to provide the appropriate conditions for the species being cultured, and good farm management. New technologies are assisting in minimising these environmental impacts. Recent developments in remote sensing and seabed mapping have improved site selection. Monitoring and assessment of the environment around aquaculture farms is becoming more common, and this is also being supported by new technology. A range of probes is now available that can be used in the field to provide an immediate measure or placed in situ to continuously monitor environmental conditions. Other advances include the use of visual techniques with digital cameras and remotely operated or autonomous underwater vehicles. Computing capability and mathematical models are also becoming increasingly important to management, both for determining carrying capacity of growing areas and for prediction of impacts. These models are becoming increasingly sophisticated and are capable of linking across different temporal and spatial scales and across trophic levels and incorporating social and economic parameters. Key words: aquaculture, environmental impact, environmental monitoring, carrying capacity, site selection.

21.1 Introduction The very nature of aquaculture, whereby fish, invertebrates or plant species are grown at a density higher than occurs naturally, makes it likely that there will be some change to the existing natural environment. In many ways aquaculture is analogous to clearing the land for intensive agriculture. However, in today’s more environmentally aware world, farming the sea,

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rivers and lakes is often perceived as a major impost on natural ecosystems. As human populations continue to expand there may be conflicts with respect to water usage, especially freshwater. For example, the requirement for water for agriculture, industry, effluent disposal, transport, recreational use and for general aesthetic value, as well as for drinking water and maintenance of natural ecosystems and biodiversity, may be at odds with aquaculture requirements (Fig. 21.1). In order to inform debate and management decisions regarding these differing uses, it is essential to have reliable information on the impact of aquacultural activities on the social, economic and ecological environments. In addition, environmental monitoring and assessment is essential to ensure both environmental stewardship and industry sustainability (GESAMP, 2001). Although the term ‘environmental impact of aquaculture’ may encompass all three, it is commonly used to refer to ecological impact only, and it is this interpretation that is used in this chapter. Ecological impacts refer to any changes to the natural or pre-existing environment that arise as a result of aquaculture operations. However, the significance of ecological changes is typically assessed in relation to the social and economic conditions of a region or country. These can differ

Aquaculture Transport

Agriculture

Ecosystem sustainability

Industry Water resource

Aesthetic values

Effluent disposal

Urban water requirements

Recreational usage

Fig. 21.1 Diagram of potential conflicts in water usage.

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markedly between relatively wealthy developed countries and third world nations where a significant proportion of the population is struggling to produce enough food to survive. The effects of aquaculture on the environment will vary according to a number of factors, amongst which the most important are: • size and intensity of farming; • location of farm (e.g. ecological sensitivity and assimilative capacity of the area, hydrology, presence of other industries/polluters); • type of organism cultured (e.g. plants or animals, filter feeders or carnivores, endemic or introduced); • the farming method and management practices (e.g. open water cage or shore-based culture, subsistence or technologically advanced). Careful selection of a suitable site for farming operations (i.e. one which is well flushed, not affected by other human activities or sources of pollutants and with good growing conditions for the species concerned) is extremely important to maximise sustainable production (GESAMP, 2001).

21.2 Interactions between aquaculture and the environment There are several very good and comprehensive reviews that describe in detail the many and varied effects of aquaculture on the environment (e.g. Black, 2001; Davenport et al., 2003; Pillay, 2003). In this chapter we provide a summary of the more common and significant impacts (Fig. 21.2) in relation to various monitoring and assessment techniques. One of the more conspicuous impacts of aquaculture operations on the environment is organic enrichment of the local benthic habitat. This is

Seabird interactions

Impacts from industry, agriculture and other Native fish users on water interactions supply Escapees Effects of solid wastes

Loss of public amenity Effects of chemical therapeutics, biocides and medicines

Effects of soluble excretory products (N&P) Marine animal interactions

Fig. 21.2 Common environmental impacts both on and of aquaculture.

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particularly true in the case of intensive culture of higher trophic level fish which are fed an exogenous source of food. Under these conditions waste food and faeces can accumulate in the vicinity of the farming system leading to deterioration of environmental conditions, possibly to the extent that the sediments become anoxic (Iwama, 1991; Gowen and Rosenthal, 1993; Wu, 1995; Black, 2001). High-density shellfish culture can also result in organic enrichment but, as there is no extraneous food supply, significant anoxic events are unlikely (Crawford et al., 2003). In recent years there has been increasing concern about the environmental effects of dissolved nutrients from aquaculture wastes being released into the surrounding environment and dispersed into the wider ecosystem. In areas with poor flushing or naturally oligotrophic waters these nutrients can result in increased primary production, potentially leading to phytoplankton blooms or excessive growth of nuisance macroalgae (eutrophication) in shallow coastal waters (Christensen et al., 2000; Buschmann et al., 2006). These macro- or microalgal blooms can lead to rapid deterioration in environmental conditions and in some instances may be toxic. However, as with localised enrichment, the potential for eutrophication is aligned to the type and intensity of the aquaculture operation and will be strongly influenced by the nature of the receiving water body (WWF Salmon Aquaculture Dialogue, 2007). Intensive farming of any species increases the potential for disease outbreaks and for transference of diseases between cultured and wild stocks (Gross, 1998; Naylor et al., 2000; Pillay, 2004). Many intensive aquaculture operations use chemical therapeutics (e.g. antibiotics, antiparasitics, fungicides, herbicides and disinfectants) to maintain the health of the cultured organisms. However, these management options can have unfavourable effects on the environment as chemicals or therapeutics may accumulate in the sediment. The effects of these accumulations include changes in the faunal composition (Alderman and Hastings, 1998; Davenport et al., 2003). Biofouling is another significant issue for many aquaculture operations as algal growth on nets, lines, infrastructure and in pipes reduces water flow and may damage stock and equipment (Chau, 1992; Brzeski and Newkirk, 1998; Svane and Petersen, 2001; Davenport et al., 2003). This is often controlled using antifoulant paints, many of which contain chemicals that are toxic to aquatic flora and fauna even at low concentrations, such as copperbased antifoulants. Thus their use in the marine environment needs to be carefully monitored and managed. Loss or modification of habitat as a result of aquaculture activities has been recognised as a significant issue; for example, the conversion of wetlands and mangrove swamps to ponds for shrimp farming (Davenport et al., 2003; Siem et al., 1997). Aquaculture infrastructure can lead to changes in water flow patterns and the movement of sediments, which can alter the structure of habitats through sediment deposition or scouring. This change in habitat can have a significant impact on native species.

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The presence of farmed species may have effects on the behaviour of native species; e.g. changing the natural foraging behaviours and distribution patterns of seals and seabirds and causing aggregations of scavenging fish (Pillay, 2003; Dempster et al., 2005). Escaped shellfish can distribute widely and form biogenic reefs which strongly compete with native filter feeders (Smaal et al., 2003). Introductions of new species for aquaculture and the movement of farmed species and equipment between growing areas and markets have the potential for widespread dispersal of exotic farmed species as well as any introduced pests attached to the cultured organisms. In addition, where endemic species are cultured there is the added risk of genetic pollution of wild stocks (Kapuscinski and Brister, 2001), and impacts on the food chain ecology associated with escapees. As well as the direct effects outlined above there are several significant indirect environmental effects that may result from intensive aquaculture operations, including loss of amenity/resources due to the farming infrastructure and the potential for aesthetic pollution associated with the infrastructure and debris. This is likely to become increasingly problematic as human settlements in the coastal zone continue to increase. There are also several ways in which the environment can impact on aquaculture operations. The environmental condition of the water in which marine farming operations are being undertaken is critically important, but is prone to degradation due to human activities. The production of food from aquaculture relies on having a clean water supply to ensure that culture species are safe for human consumption. If the water becomes contaminated (for example with industrial chemicals, sewage or pesticides) this will likely show up in the eating quality (food safety) and/or in rates of growth, reproduction or physiological condition of the cultured species. Cultured shellfish have been described as the ‘canaries of the sea’ because monitoring programs for shellfish quality have alerted authorities to system-wide problems. These include the presence of toxic algae or unacceptably high bacterial levels in estuarine growing waters arising from faulty septic and sewage systems and from upstream agriculture effluent. Land-based activities, such as deforestation or coastal development, can result in high particulate matter levels in the receiving waters, which may in turn markedly reduce the feeding efficiency of filter feeders such as mussels and oysters (Wildish and Kristmanson, 1997). The survival and condition of cultured organisms can also be impaired by agricultural and industrial chemicals being washed from the catchments into rivers and estuaries. In many parts of the world the concentrations of nutrients in waterways are rising, especially nitrogen and phosphorous, as a result of agriculture and increased urban development, which is of particular concern to aquaculture. As already discussed, higher nutrient levels can result in eutrophication, with the possibility of concomitant reduction in light levels for plants and dissolved oxygen concentrations for animals; features which would ultimately reduce growth and survival rates of cultured species.

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Predator control is a significant issue for many marine farmers; e.g. seals, sharks, birds, crabs, fish or octopus preying on cultured fish, shellfish or crustaceans. Blooms of toxic jellyfish can also cause major mortalities of caged finfish (Purcell et al., 2007). An increase in the frequency and size of jellyfish aggregations has been linked to aquaculture activities (e.g. Lo et al., 2008), formation of red tides (Pitt et al., 2007), anthropogenic changes in habitat and to fishing of high trophic level predators (Purcell et al., 2007). Control methods generally focus on exclusion or eradication, and both these approaches have the potential to significantly influence the local ecology. Naturally occurring parasites may also proliferate under culture conditions and cause significant mortalities of the cultured organisms, e.g. sea lice on northern hemisphere salmon or gill amoeba on Tasmanian salmon (Davenport et al., 2003; Crosbie et al., 2005). In recent years, global climate change has increasingly been identified as having the potential to negatively affect aquaculture activities (Handisyde et al., 2006). Climate change can have a significant effect on reproductive strategies, growth, epidemiology and physiological response (Lehtonen, 1996; Kent and Poppe, 1998; Bondad-Reantaso et al., 2005). Climate change may also have indirect effects on the physical and ecological environment in which aquaculture operations are conducted, resulting in changes to infrastructure requirements and site locations and potentially affecting related ecosystem processes such as the availability of bait fish and other feed ingredients (Handisyde et al., 2006). Although advances in production technology in first world aquaculture operations may solve/mitigate many of these problems, particularly with land-based culture (see Chapter 31), many cultured species are still reliant on the ambient water quality and will continue to influence their local environmental condition. As a consequence, the precautionary principle would still advocate monitoring to ensure that optimal environmental conditions are maintained.

21.3 Site selection and carrying capacity Ensuring that the site selected is appropriate for the type of aquaculture proposed is one of the most significant ways to minimise impacts on the environment. Of particular importance is the hydrodynamic state; i.e. ensuring that there is sufficient water movement across the site to dilute and disperse wastes. Adequate flow of water to replenish food supplies for cultured filter feeders is also essential for production sustainability. Evaluation of hydrodynamic condition requires measurement of water flow, generally by the deployment of current meters for at least one lunar cycle in tidally influenced areas. Technological advances have resulted in current meters that are now relatively inexpensive and improvements in acoustic doppler current profiling (ADCP) technology enable much more

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accurate measurement of flow and allow for vertical profiles of flow characteristics. The recent development of remote sensing and seabed mapping techniques, incorporating accurate digital GPS positioning in conjunction with acoustic data from single beam echo sounders or multiple beam sonar, has also significantly enhanced the ability to select suitable sites for aquaculture (Nath et al., 2000; GESAMP, 2001). The seabed maps provide bathymetric contours and a characterisation of benthic habitats, which are presented visually using geographic information systems (GIS) software. This GIS technology has advanced rapidly in recent years such that accurate highresolution maps can now be readily produced. These maps are generally ground-truthed using underwater video, which also provides information on distributions of significant species and/or communities. Environmental and industry managers can use these maps to more accurately assess the suitability and/or the sensitivity of the region to aquaculture activities (e.g. Salam et al., 2003; Giap et al., 2005). As aquaculture moves offshore the role of seabed/habitat mapping in site selection will continue to increase (e.g. Perez et al., 2003). Determining the carrying capacity of an aquaculture growing area is critical for sustainable management. However, it is important to make the distinction between ‘production carrying capacity’, which is the maximum sustainable production from a growing area, and ‘ecological carrying capacity’, which is the level of culture that can be supported without significantly changing the ecology of the growing area (Jiang and Gibbs, 2005). Production carrying capacity only considers the species being cultured, whereas ecological carrying capacity takes into account the effects of aquaculture on other species, communities and ecological processes in the water body in which aquaculture is occurring. Thus for shellfish aquaculture, which relies on a natural source of particulate food supplied from the growing waters, the ecological carrying capacity is largely determined by the level of production that does not significantly limit food supplies to other filter feeders in the system and thus maintains the biodiversity of the growing area (Ferreira et al., 2008). For finfish farming, however, carrying capacity generally refers to the maximum production of fish that does not impact on the surrounding environment and, in this case, the assessment process is generally the same as for impact assessment. Advances in computing technology and availability of specialised software have greatly increased the ability to effectively assess carrying capacity (see review by McKindsey et al., 2006). Several models have been developed that can predict ‘production carrying capacity’ for shellfish based on rate of water replenishment, rate of supply of food (primary production) and rate of feeding (clearance time) (e.g. Carver and Mallett, 1990; Bacher et al., 2003). The most reliable models are, however, complex and require large quantities of site-specific data, which can make them expensive to produce. Modelling the ecological carrying capacity of an area for shellfish

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aquaculture is even more difficult, and few models have been developed (McKindsey et al., 2006; Ferreira et al., 2008). An alternate approach is to use functional performance indicators to assess the interaction between the culture operation and the surrounding environment and thereby estimate the carrying capacity of the system (Gibbs, 2007). Performance indicators for shellfish include the spatial extent of depletion of phytoplankton around farms, and ratios of growing area and shellfish parameters (total volume, tidal exchange volume, phytoplankton production, phytoplankton biomass, filtration rate of cultured shellfish and culture biomass) (Gibbs, 2007).

21.4 Considerations in developing an environmental monitoring and assessment program The specific monitoring and assessment approach employed in any given situation will be determined by a variety of factors including, but not limited to, culture species, biomass, location, type of feed and chemicals used and available technology. In establishing any environmental monitoring/assessment program it is an essential prerequisite that the objectives are clearly defined and that these specify (i) why the assessment is being conducted, (ii) the boundaries (spatial/temporal/ecological) of the assessment, (iii) the level of accuracy required and (iv) the expected outputs/outcomes from the assessment. Unfortunately these requirements are often overlooked with assessments being undertaken for a particular purpose that is not representative of all environmental issues, or monitoring programs are designed that are manifestly insufficient for their intention. An assessment of environmental impacts has to be able to detect changes due to aquaculture activities over and above those due to natural variation; thus the methods used and the design of the sampling program must be sufficiently robust to detect these changes (Fernandes et al., 2001). In order to establish this it may be necessary to undertake a pilot sampling program to determine the scale of natural variation and hence decide on the number of samples required to be able to detect a significant change in environmental condition (Green, 1989; Thrush et al., 2005). If a significant system-wide change in environmental condition occurs, it is important to be able to identify whether the change is due to aquacultural operations or some other anthropogenic or natural event (in the vicinity of the farm or higher in the catchment). The ability to determine change and infer causality as a result of aquaculture activities will require a well-designed monitoring program with a statistically relevant number of sampling sites at strategic locations in the growing area, including unimpacted reference sites. There is no single sampling design that will suit all situations, as each circumstance will have its own unique characteristics. A full description of how to structure statistically and environmentally relevant sampling strategies is beyond the scope of this text and we refer readers to one of the many very good references/

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texts in this area (e.g. Clarke and Green, 1988; Green, 1989; Schmitt and Osenberg, 1996; Underwood, 1997; Quinn and Keough, 2002). However, there are some fundamental premises that apply in virtually all circumstances. To evaluate the extent and degree of any impact accurately it is essential to understand the condition of the system without the impact. Consequently, collection of detailed baseline environmental data before any aquaculture activity occurs is essential. This should involve sampling at a number of sites both within the farm and remote from it (i.e. reference sites unaffected by farming activities) several times prior to farming commencing, to provide information on natural temporal and spatial variability (Thrush et al., 2000; Fernandes et al., 2001). Unfortunately, aquaculture activities often develop quickly once a site has been selected and approved by governing bodies and a detailed baseline assessment at one time only is often the norm. The significance of the baseline assessment may often be missed by industry, particularly the relevance of reference sites away from the farm. It is important for all stakeholders to recognise that baseline information is fundamental to determining whether changes have occurred or not and that multiple reference sites are essential (Underwood, 1990, 1991). Inclusion of reference sites decreases the risk of a type II statistical error (e.g. erroneously inferring no impact) and enables better attribution of causality, i.e. where changes have occurred at both farm and reference sites it is likely that the change is due to some extraneous environmental influence. A monitoring program including baseline assessment will generally include several indicators of water quality and ecological condition. Invariably no one environmental variable will provide sufficient information on the health of the environment around the aquaculture operation and a combination of variables is required; the selection of which will be dependent upon economic, social and ecological imperatives. The environmental variables to be measured should be SMART (specific, measurable, achievable, relevant and time-bound). Reviews of the application and suitability of potential indicators for estuarine, coastal waters and marine systems can be found in Bortone (2004), Niemi et al. (2004) and Rogers and Greenaway (2005), respectively. Indicators of the status of the system (‘system indicators’) are commonly used in monitoring programs, e.g. water quality indicators such as nutrient levels or dissolved oxygen concentrations (Table 21.1). These indicators demonstrate the current condition of the aquaculture growing area; however, they are generally from temporally and spatially isolated samples (e.g. water samples taken once a month from a few sites). Although this provides a valuable ‘snap shot’ of the physicochemical condition, it does not necessarily represent the biological response. Ecological indicators, which are similar to and sometimes referred to as value or impact indicators, can provide a more integrative assessment of overall condition spanning longer time periods. The distribution, abundance and community composition of

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Table 21.1 Common environmental condition indicators, method of use and comparative cost Indicator

Method

Reporting time

Cost range

Nutrient level (NOx, NH3, PO4) DO Redox Sulphide Turbidity Sediment particle size Organic matter Chl a

Lab analysis

Med–slow

$$

Field probe Field probe Field probe Field probe Lab analysis Lab analysis Field probe/lab analysis Field probe Lab analysis Lab analysis Lab analysis

Rapid Rapid Rapid Rapid Medium Medium Rapid–medium

$ $ $ $ $$ $$ $–$$

Rapid Med Slow Slow

$ $$ $$$ $$–$$$

Lab analysis

Slow

$$–$$$

Field & lab analysis

Med

$$

Field & lab analysis Video

Med Rapid

$$ $

pH Bacteria/pathogens Specific chemicals Benthic community (sp abundance & diversity) Microalgal community (sp. abundance & diversity) Macroalgal community (sp abundance & diversity) Seagrass (extent & cond.) Epibenthic community

$ = low, $$ = medium, $$$ = high cost of analysis

specific flora and/or fauna are commonly employed as ecological indicators (Table 21.1). It is generally recommended that some ecological indicators are included in any environmental assessment (Hellawell, 1986; Niemi et al., 2004; Devlin et al., 2007a). The European Union Water Framework Directive for water quality, for example, has shifted from targets based on chemistry to include those related to the ecological structure of natural systems. The ecological quality status of coastal and transitional waters is now assessed on biological, hydromorphological and physicochemical elements; with the biological elements considered being phytoplankton, macroalgae, benthos and fishes (Muxika et al., 2007). Around marine farms epibenthic and infaunal invertebrate communities have commonly been used as indicators of condition. These communities are preferred because they play a critical role in regulating sediment processes, such as denitrification and aeration of sediments and hence provide an integrative assessment of ecological condition (Dauvin et al., 2007). They are also relatively stationary, long-lived and generally contain a diverse

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range of species sensitive to different levels of fish farm effluents. For assessment of far field effects of aquaculture, such as nutrient enrichment, appropriate ecological indicators include phytoplankton communities (Devlin et al., 2007b; Painting et al., 2007) and, in specific areas, macroalgal communities (Wells, 2007; Scanlan et al., 2007; Pinedo et al., 2007) or seagrass distribution and abundance (Bortone, 2004; Borum et al., 2004; Koss et al., 2005). Levels of unacceptable impact need to be determined as part of any monitoring program. This may be through setting environmental quality standards (EQSs) in relation to environmental quality objectives (EQOs) (Fernandes et al., 2001), or as part of compliance monitoring regulations. The actions to be taken if the standards are breached or targets are not met must be clearly defined and agreed to by the industry participants and the regulators (GESAMP, 2001). Unfortunately, the resources available for assessment of the environmental effects of aquaculture are often limited. In this case the challenge is to develop the most rigorous assessment with the funds available so that when impacts do occur they are recognised and actions are taken to reduce the level of impact. This is particularly important in the aquatic environment where detrimental effects are often not obvious (i.e. ‘out of sight, out of mind’). In many countries, particularly those in the developed world, environmental risk assessment has become an increasingly common approach to examine and prioritise the potential effects of aquaculture on the environment; for example a qualitative risk assessment of the effects of shellfish farming on the benthic environment (Crawford, 2003). Standard procedures for conducting such assessments are available, e.g. Australian/New Zealand Standards (2004). Assessments are generally conducted by an expert panel containing representatives from scientific researchers, industry and governing bodies. Risk assessments consider all potential impacts of aquaculture on the environment, in terms of their likelihood of occurrence and level of consequence should they occur. Likelihood and consequence are combined to provide a risk factor (low, medium, high and catastrophic) of impact occurring. The significance of potential impacts of aquaculture on the environment can be evaluated against this ranking and resources allocated according to the risk identified. Prescribed management actions to remediate higher levels of risk are also agreed to and documented as part of the risk management process. Codes of practice and best management practices (BMPs) have been developed by many sectors of the aquaculture industry to improve their environmental performance (Fernandes et al., 2001; Boyd, 2003). These practices are considered to be the most practical means available to minimise negative environmental impacts and are generally developed in collaboration between industry representatives and environmental managers. They may be incorporated into environmental management programs, and are instigated either on a voluntary basis or as part of mandatory

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compliance regulations imposed by the regulators. Environmental management systems (EMSs), which aim to continually improve the social, economic and ecological performance of aquaculture industries through an adaptive management approach, are also being implemented by many seafood companies in developed countries as consumer demand increasingly requires environmental accountability. The most rigorous of these are certification programs that are independently audited, e.g. International organization for Standardization ISO 14001 EMS (http://www.iso.org/iso/ home.htm). EMSs specific to the seafood industry have also been developed, e.g. Seafood Services Australia (2005). However, the majority of world aquaculture production comes from developing nations where BMPs and EMSs are slow to develop (Boyd et al., 2007).

21.5 Monitoring and assessment techniques Standardised guidelines for monitoring both water quality and sediment conditions associated with aquaculture practices are now available in many countries. However, even within a country/region, specific requirements may vary based on the species farmed and size/location of the farming operations. The particular sampling approach required and the spatial and temporal frequency of sampling differs between monitoring programs, with many factors determined on a site by site basis by the local regulatory authorities. However, all programs require some measure of the natural and disturbance-related variability in water column and sedimentary physical, chemical and ecological parameters (Black, 2001; Davenport et al., 2003; Pillay, 2003).

21.5.1 Water quality Increased nutrient inputs, particularly nitrogen and phosphorous from waste food and excretory products, can lead to eutrophication (as defined by OSPAR, 2003). This can be monitored using several indicators including: (i) direct measurement of water column concentrations of the most biologically available nutrients – nitrates and nitrites, ammonium, phosphate, silicates (important for diatom production and hence phytoplankton composition) and of total nitrogen and total phosphorous (to assess loads of these elements into estuaries); (ii) measurement of biological effects such as chlorophyll a concentration as an indicator of phytoplankton biomass or the distribution and abundance of macroalgal blooms; (iii) measuring dissolved oxygen (DO) concentrations, especially in bottom waters. The oxygen requirements for bacterial breakdown of organic matter resulting from excessive primary production may be greater than that available, resulting in low DO. Reduced water quality may also occur as a result of increased particulate matter in the water column. This can be measured simply as

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turbidity or as water clarity (secchi disc). More complex measures involve filtering out the particulate matter and analysing for inorganic and organic composition. Recent technological advances have enabled most of these parameters to be easily measured using reliable, robust, inexpensive and accurate field probes, which have the added advantage of being able to provide results in real time. Even nutrients, which have traditionally been measured in specialised laboratories, can now be measured in situ. The standard ‘wet chemistry’ nutrient analyses can be conducted in miniature waterproof systems located on site and, although these systems are still relatively expensive, costs are decreasing rapidly. Recent developments have included a UV absorption method to measure nitrate and nitrite ions, organic carbon, turbidity, colour and chlorophyll a in situ (Johnson and Coletti, 2002; Johnson et al., 2007). These in situ monitoring systems have the potential to significantly improve our understanding of how aquaculture activities affect the natural water body in which they are located, and the relative impact of aquaculture compared with natural events such as storms and freshwater flooding. They can provide continuous sampling at much finer temporal scales (i.e. minutes, compared with traditional water quality sampling at weekly or, more commonly, monthly sampling scales). This increased frequency of sampling also provides very important information for farm management, such as the effect of stocking density on diurnal water quality and hence on the condition and growth rate of the cultured animals. Advances in telemetry technology mean that water quality results can be sent directly to a designated computer so that researchers and fish farmers can monitor minute by minute changes in water quality from their office.

21.5.2 Sediment condition Sediment condition can be measured in several ways, including assessment of specific chemical aspects (e.g. redox and sulphide) (Holmer and Kristensen, 1992; Hargrave et al., 1993) or a combination of physical, chemical and biological factors (e.g. infaunal community structure). Amongst the simpler indicators of sediment condition are those that reflect the oxic status of the sediments, such as redox and sulphide concentrations. These may be measured in the top layers of sediment using relatively inexpensive and straightforward field probes. Other measures of sediment condition directly reflect the organic enrichment and may commonly include measurement of total organic matter and/or total organic carbon. However, these measures are subject to the same constraints as the water quality indicators in that they only reflect the condition at the time when the sample was taken (although the sedimentary environment is not as transient as the pelagic environment). In addition, technology has not yet advanced to the extent that in situ telemetered sediment probes are commonplace.

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21.5.3 Characterisation of benthic biota Characterisation of the benthic infaunal community in the top layers of sediment is one of the most sensitive and reliable indicators of environmental status (Weston, 1990; Cairns et al., 1993; Johannessen et al., 1994; Codling et al., 1995; Dauer et al., 2000; Edgar et al., 2005). Changes in the infaunal community characterise the integrated effect of multiple environmental stresses (physical, chemical and biological) to which a system has been subjected. However, assessment of these ‘ecological’ indicators can be time-consuming and hence more costly to assess than either water column or geochemical parameters. Nevertheless, the integrated assessment of environmental condition generally justifies the additional time and expense. Where the potential for change in biodiversity is an issue it is preferable to identify to species level; however, identification to family or genus level is often sufficient to detect change due to farming activities (Lampardiou et al., 2005). Data on ecological indicators collected over several years have the added benefit that they may indicate environmental trends, and indicator species of specific conditions may become apparent (e.g. Pearson and Rosenberg, 1978; Holmer and Kristensen, 1992; Duplisea and Hargrave, 1996; Edgar et al., 2005; Macleod et al., 2006). Techniques for the analysis and interpretation of multivariate community data have also improved. The broader availability of purpose designed analytical software such as PRIMER (2005) and ECOSIM (Gotelli and Entsminger, 2001), in conjunction with rapid advances in computer technology, has enabled multivariate analysis of complex community datasets to be undertaken relatively easily and has greatly simplified the determination of relevant ecological indices (Magurran, 2004). Current advances in molecular techniques for assessment of biotic community change represent a significant development in monitoring and assessment approaches. The ability to identify key indicator species/taxa using gene probes and barcoding technology will greatly simplify ecological assessments and, once a reliable technique is found to proportionally represent or quantify communities, these approaches will be able to produce very rapid assessments of ecological impact. This is an active research area.

21.5.4 Visual assessment of sediment and epibenthic biota Recent advances in the technology for underwater visual assessments using underwater digital cameras and video photography, with a concomitant reduction in costs, have resulted in this equipment being used much more frequently to assess the condition of sediment and the status of the epibiotic community under and near fish farms. However, the information obtained is generally subjective, although recently developed semi-quantitative approaches for video evaluation have increased the value of video assessment (e.g. Crawford et al., 2003). In addition, specific techniques have been developed to improve local interpretation of video/photographic data; e.g.

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after years of detailed monitoring of benthic invertebrates on salmonid farms in Tasmania, a much simpler visual assessment of the changes in the epibenthic biota associated with salmon cage farming has been developed to enable farmers to assess the environmental condition around the fish farms (Macleod and Forbes, 2004). This has proven to be particularly useful for salmon farmers to manage sediment recovery within their leases more effectively. Remotely operated underwater vehicles (ROVs) and autonomous underwater vehicles (AUVs) with attached cameras are also increasingly being used on fish farms to monitor sediment condition in waters too deep or too dangerous for diving. These underwater platforms, with advanced photographic systems including fibre optics technology, enable highresolution digital stills and video imagery to be collected, while at the same time allowing the operator to directly observe the condition of the sea floor and manoeuvre the equipment around farm structures and uneven seabed terrain. This underwater photographic technology is now being used by many regulatory authorities to identify areas of major impact around marine finfish farms. It has the advantage of the results being available in real time, easily stored and reproduced and readily understood by nonscientific managers. Future visual assessment approaches may include stereo-video photographic techniques using highly-calibrated paired underwater cameras for accurate underwater measurement of animal/plant size or fine-scale distribution of particular habitats (Harvey et al., 2002). Another visual assessment technique that is increasingly being used is sediment profile imagery (SPI), which was originally developed to obtain a better understanding of the sediment environment of the deep sea and oceans, but was quickly identified as having potential for coastal resource managers and environmental regulators (see articles in special edition of Journal of Marine Systems (SPICE, 2006)). SPI involves photographing a slice of the top layers of sediment and then analysing the photographs for a number of attributes including evidence of fauna, depth of redox discontinuity layer and the depth of differing types and colours of sediment (e.g. Karakassis et al., 2002; Wildish et al., 2003; Munslow et al., 2006). It is now a feature of aquaculture monitoring programs in Europe, North America and Chile. The technology has advanced considerably in recent years, with the most advanced systems reportedly able to evaluate over 20 physical/ chemical and biological parameters. Recent technological advances, including the addition of planar optodes to profile in situ oxygen levels and the ability to deploy the systems for periods of time, will markedly increase the value of this approach to the aquaculture industry.

21.5.5 Predictive assessments Modelling is an increasingly important approach in all areas of environmental management and can be a powerful tool for predicting effects of

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aquaculture including (i) the effects of farm management on aquaculture production, (ii) the degree of impact of aquaculture wastes and (iii) the capacity of the water bodies to assimilate aquaculture wastes (Ervik et al., 1997; Silvert and Cromey, 2000; Henderson et al., 2001; Perez et al., 2002; McKindsey et al., 2006). This may involve developing specific individual models for different environments and/or impacts across different spatial scales and then linking these together. Clearly, these predictive impact models are closely connected to predictive carrying capacity models, which were discussed previously. Models that predict the shape and extent of waste deposition in the immediate vicinity of the farms can be used both in planning and development of aquaculture operations and in designing subsequent monitoring regimes. There are now several very good commercially available deposition models that provide an assessment of the likely distribution and impact of solid wastes from aquaculture operations (e.g. DEPOMOD, Cromey et al., 2002a,b). DEPOMOD was originally designed as a tool to enable farmers, consultants and regulators to objectively assess the localised impact of aquaculture operations and is probably one of the most advanced commercially available models. DEPOMOD is also unusual in that it has had relatively extensive field validation of many of the model parameters and outputs (Cromey et al., 2002b). Predicting the broader ecological effects of aquaculture is a much more complex task. Clearly the ability to simulate the broad range of inputs (natural and anthropogenic in origin) into an aquatic system would be of considerable value and interest to regional planners, environmental regulators and various user groups, such as aquaculture and the broader community. As a result, considerable research effort is now focussed on developing integrated coastal and estuarine catchment management models (GESAMP, 2001). There are several examples where coastal circulation and mixing models have been shown to be helpful in predicting broad-scale impacts (e.g. Panchang et al., 1997; Tomczak and Herzfeld, 1998; Hargrave, 2002; Foreman et al., 2006). However, for most systems there are not, as yet, the necessary data for full scale ecosystem models. Much of the ecosystem modelling effort has focussed on specific components of the system (e.g. Le Gall et al., 2000 on phytoplankton; AlveraAzcarate et al., 2003 on seaweeds) with relatively few multitrophic eutrophication models. There are several examples of broader environmental assessment packages that integrate various monitoring strategies, including modelling (e.g. CSTT (1997); National Estuarine Eutrophication Assessment (NEEA) (Bricker et al., 1999); Fjordenv (Stigebrandt, 2001; OSPAR, 2008) and ASSETS (2003/5 (Ferreira et al., 2007b)). Of these, the Assessment of Estuarine Trophic Status (ASSETS) screening model (Bricker et al., 2003; Nobre et al., 2005) is specifically focussed on providing management information through categorising the eutrophication status of estuarine and coastal systems into classes from high through to bad (Nobre

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et al., 2006). This model framework is still in development, though preliminary evaluations have been very promising (Ferreira et al., 2007a). Sustainable management of shellfish aquaculture is also being investigated by incorporating natural benthic biodiversity of suspension-feeding organisms into the model (Sequeira et al., 2008). The Farm Aquaculture Resource Management (FARM) model (Ferreira et al., 2007b) employs the ASSETS framework and has been designed to be used by the shellfish industry to explore differences in site location, culture practices and environmental impact, with a view to optimising sustainable production. An added advantage of this model is that combinations of species may be modelled, enabling an assessment of integrated nutrient management in coastal regions. A further extension of this work has been the development of an integrated framework for assessment of ecosystemscale carrying capacity for shellfish aquaculture (Ferreira et al., 2008). This framework incorporates various types of models from local farm to broadscale ecosystem models, enabling simulations over a range of temporal and spatial scales. It has also been used to investigate the effects of climate change on the culture of different species (Ferreira et al., 2008). More advanced models currently being developed link estuarine and coastal ecosystem dynamics, incorporating aquaculture activities, with catchment (basin or watershed) scale models. These models enable simulations of the effects of land-based activities, such as agriculture, on downstream water quality, ecosystem health and productivity (Ferreira et al., 2008). Other recent developments in environmental modelling include taking a more holistic environmental approach by incorporating social and economic factors as well as ecological parameters (McDonald et al., 2008). Such modelling approaches are important to sustainable aquaculture as they integrate indicators of ecosystem health and carrying capacity with the economics of production and with social issues, such as environmental values and employment. This approach has been termed ‘ecoaquaculture’ by Sequeira et al. (2007) and is likely to be increasingly important in the management of aquaculture. However, there are limitations to employing models or using modelled data. In a review of benthic and hydrodynamic models used in aquaculture in Europe, Henderson et al. (2001) pointed out that, although modelling is an extremely valuable and objective tool for optimisation of resource exploitation and is particularly useful for growth prediction, impact management and integrated coastal zone management, it should not be relied on in isolation. The usefulness of any given model is dependent on the quality and quantity of data available to populate it. Most currently available models have significant restrictions, and outputs should be used with caution as they can be highly sensitive to parameterisation (Fussmann and Blasius, 2004). In addition, it is also generally difficult to incorporate into a model irregular episodic events, such as storms, which may cause considerable environmental change (Silvert and Cromey, 2000; Henderson et al.,

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2001) and further work is required to be able to include interactions and feedback mechanisms into models (McKindsey, 2006; NWQMC, 2006). It is essential to collect reliable data through monitoring and assessment to ground-truth (validate) the models. Consequently, although the use of models as objective tools is to be encouraged, there is a need to standardise modelling approaches and ensure data validation (Henderson et al., 2001).

21.5.6 Other indicators Most intensive aquaculture operations will at some point have to rely on medicines and chemicals to manage detrimental health and environmental issues. These may include antibiotics, antiparasitics, fungicides, herbicides and disinfectants (Davenport et al., 2003). There is still insufficient information on the environmental impact of many of these chemicals (Costello et al., 2001). The main impacts depend largely on the toxicity and persistence of the specific chemical, although chemicals with the potential to impact on human health are amongst the most worrying. By virtue of their potential for human health effects and long-term environmental impacts, antibiotics are amongst the most well studied group of medicines used in aquaculture (Davenport et al., 2003). However, development of vaccines has significantly reduced the requirement for many medicines (e.g. furunculosis vaccine in salmonids) and research continues to make advances in this area. Generally the environmental effects of chemical residues are likely to be greatest for those organisms most closely related to the target organism (Black, 2001; Davenport et al., 2003). There is no universal standard or code of practice for the use of medicines and chemicals in aquaculture and, as a result, there are large variations between countries and even regions in maximum residue levels and regulatory requirements. Where chemicals/medicines have been administered, monitoring programs should include indicators to specifically assess any potential problems, both direct and indirect. This may require specific measurement of residue levels and persistence in the environment or in primary and secondary contact species. Laboratory-based ecotox experiments are helping to assess the environmental risk of many of these chemicals (Benton et al., 2007), providing much needed basic information on the ecological effects (see also Part II – Health).

21.6 Recent technological advances and future trends 21.6.1 Changes in types of aquaculture With an increasing demand for aquaculture products and greater diversification of aquaculture, into both new areas and species, there is a continued need to ensure the sustainability of operations through state-of-the-art environmental monitoring practices and technologies. There are now many intensive aquaculture operations in developed economies that are highly

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technologically advanced and, as the costs of such technology rapidly decrease, it is likely that automated real-time monitoring systems providing data on individual tank/cage environmental conditions will soon be a standard feature of all commercial aquaculture operations. Major advances in feed production and delivery technology (i.e. feed/ stock control), coupled with improved understanding of nutritional requirements, have resulted in greatly improved feed formulations with less wastage and therefore less localised environmental impact (see also Chapters 16 and 17). Technological improvements in feed delivery mechanisms continue to develop more efficient automated feeders which monitor feeding activity within the tanks/cages ensuring that feed wastage is minimised (Lekang, 2007). Advances in hydrodynamics, applied to tank and pond design, have enabled the development of closed/semi-closed systems, with recirculating technology maintaining optimal environmental conditions both within the aquaculture systems and in any effluent. Lekang (2007) provides an excellent review of this technology and its environmental potential. Recirculation technology is rapidly becoming an affordable option for many operators as it provides greater control of the intake water condition as well as having in-built monitoring systems enabling a much better understanding and control of effluent condition (Chapters 31, 32 and 36 deal with land-based culture). Engineering advances with technology adapted from offshore oilrig facilities have resulted in open ocean aquaculture systems that present a new and unique set of circumstances for environmental monitoring. Major advances in technology, particularly remote sensing and telemetry systems, should also make this easier (Chapters 29 and 30 deal with offshore farming). Polyculture, or integrated aquaculture, is where the growth of one commercial species is directly connected with another commercial species that benefits from the waste products, or where an associated species is used to mitigate the impacts of the cultured species (see Chapter 32 for details). Polyculture is commonly practised in developing countries, and in many circumstances is the primary means of impact management and mitigation. In developed countries polyculture is being revisited as an innovative and sustainable solution to increase profitability, at the same time managing the waste products and environmental impacts of intensive culture operations. The emphasis is on integrated multitrophic aquaculture (IMTA) systems, where farming of high trophic level organisms, such as carnivorous fish and shrimp, is integrated with lower trophic levels, e.g. filter feeders and seaweeds (Neori et al., 2007; Ridler et al., 2007). The extractive crops (shellfish and seaweeds) extract nutrients from the waste of the fed crops. In some cases the seaweed can be fed to another cash crop such as sea urchins or abalone (e.g. Matos et al., 2006; Troell et al., 2006). Modelling of sustainable shellfish polyculture is also progressing (e.g. Nunes et al., 2003). Genetically modified organisms (GMOs) are another major feature of recent technological advances. GMOs generally provide some commercial

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advantage in the cultured species, such as improvement in growth rate, increased disease resistance or tolerance to environmental conditions. However, there are risks associated with GMO production (see Dunham, 1999 and Maclean and Laight, 2001 for a complete review). To date, induction of polyploidy is the most common genetic manipulation undertaken in aquaculture, with triploidy regularly induced in shellfish and finfish to provide improvements in growth. Probably the greatest risks, with respect to the environment and our ability to monitor impacts, are the human health risks associated with any modification of DNA and the potential threat to local biodiversity from escapees. However, the nature of GMOs and the presence of specific gene markers mean that it would be relatively easy to establish whether individuals in the environment are of farmed origin. The primary challenge in this area will be to design systems that do not allow stock to escape. Another potential issue is market resistance by the consuming public from the perception of GMOs in food.

21.6.2 Changes in technology/monitoring approaches With technological advances and generally reducing costs in electronic environmental testing equipment and computer systems, as well as developments in material science providing more robust equipment suited to the rigours of the aquatic environment, monitoring in the future is likely to become more frequent but also more specialised and sophisticated. The improvements in underwater visual monitoring equipment, in particular, are removing some of the mysteries of the underwater environment and enabling farm operators, as well as environmental managers, to observe the condition of the underwater environment in real time. The development of in situ high-frequency water quality monitoring systems with results telemetered to laboratories also enables more timely assessment of environmental conditions and more prompt response to deteriorating conditions. New molecular techniques, such as simple field probes that can detect the DNA of specific biota, have the potential to provide hitherto specialised and complicated data relatively quickly and easily. However, the downside to this sophisticated equipment is that equipment failure can be much more problematic, requiring specialised servicing and potentially greater down time. A major advantage of this new technology to farmers and environmental managers, which is likely to lead to a change in monitoring approaches, is an increased ability to predict changes in condition and to respond in a more timely manner. The new technology is resulting in a better understanding of the temporal and spatial scale of ecological processes and the rates at which they occur in the aquatic environment. As a consequence, the capacity to track changing conditions and to take action in a more environmentally and economically effective manner is greatly enhanced. This is likely to be particularly important for field-based farms where

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rapidly changing climatic conditions or water quality can seriously impact on an aquaculture operation. Similarly, to be able to detect deteriorating environmental conditions before they reach an unacceptable level, such as low-level increases in nutrients before eutrophication occurs or before a build-up in sediment organic matter results in the release of toxic gases, and thus to have the ability to ameliorate the situation before it goes too far, is likely to lead to markedly improved environmental conditions. This is going to be particularly important in coastal areas where population pressures are rapidly rising and there is increasing conflict between the different users of the coastal and estuarine environments.

21.7 Sources of further information and advice Examples of environmental regulations and monitoring programs • Scottish Environmental Regulation – SEPA Aquaculture Homepage: http://www.sepa.org.uk/water/aquaculture/marine_aquaculture.aspx • Canada Environmental Regulation – DFO Aquaculture Homepage: http://www.dfo-mpo.gc.ca/aquaculture/aquaculture-eng.htm • US Environmental Regulation – EPA Aquaculture Homepage: http:// www.epa.gov/oecaagct/anaquidx.html • Australia – Regional planning and monitoring programs: { South Australia – http://www.pir.sa.gov.au/aquaculture { Tasmania – http://www.dpiw.tas.gov.au/inter.nsf/ThemeNodes/ ALIR-4YS2ZC?open { Dow A (2005). Norway vs British Columbia: A Comparison of Aquaculture Regulatory Regimes, Environmental Law Centre, University of Victoria, Victoria, http://www.elc.uvic.ca/projects/2004-02/ AquacultureReport.pdf, accessed January 2009.

Information related to undertaking selected monitoring techniques/analyses • Articles in Journal of Applied Ichthyology (2001), 17(4), MARAQUA special edition. • Articles in Marine Pollution Bulletin (2007), 55(1–6), Implementation of the water Framework Directive in European marine waters; (10–12), Measuring and managing changes in estuaries and lagoons. • Farm Aquaculture Resource Management (FARM) model: http://www. farmscale.org/ • GESAMP (IMO/FAO/UNESCO-IOC/WMO/WHO/IAEA/UN/UNRP Joint group of experts on the Scientific Aspects of Marine Environmental protection) (2001) Planning and management for sustainable coastal aquaculture development, Reports and Studies No. 68, Rome, FAO.

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• WWF Marine Program – Sustainable Aquaculture: http://www.panda. org/about_wwf/what_we_do/marine/our_solutions/sustainable_use/ aquaculture/index.cfm

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painting s j, devlin m j, malcolm s j, parker e r, mills d k, mills c, tett p, wither a, burt j, jones r and winpenny k (2007) Assessing the impact of nutrient enrichment in estuaries: susceptibility to eutrophication, Marine Pollution Bulletin, 55, 74–90. panchang v, cheng g and newell c (1997) Modelling hydrodynamics and aquaculture waste transport in coastal Maine, Estuaries, 20, 14–41. pearson t h and rosenberg r (1978) Macrobenthic succession in relation to organic enrichment and pollution of the marine environment, Oceanography and Marine Biology An Annual Review, 16, 229–311. perez o m, telfer t c, beveridge m c m and ross l g (2002) Geographical Information Systems (GIS) as a simple tool to aid modelling of particulate waste distribution at marine cages sites, Estuarine Coastal and Shelf Science, 54, 761–8. perez o m, telfer t c and ross l g (2003) On the calculation of wave climate for offshore cage culture site selection: a case study in Tenerife (Canary Islands), Aquacultural Engineering, 29, 1–21. pillay t v r (2004) Aquaculture and the Environment (2nd edn), Oxford, Blackwell. pinedo s, garcia m, satta m p, de torres m and ballesteros e (2007) Rocky-shore communities as indicators of water quality: a case study in the Northwestern Mediterranean, Marine Pollution Bulletin, 55, 126–35. pitt k a, kingsford m j, rissik d and koop k (2007) Jellyfish modify the response of planktonic assemblages to nutrient pulses, Marine Ecology Progress Series, 351, 1–13. primer (version 6.1) (2005) Plymouth Routines in Multivariate Ecological Research, Oxford, Primer-E Ltd. purcell j e, uye s and lo w (2007) Anthropogenic causes of jellyfish blooms and their direct consequences for humans: a review, Marine Ecology Progress Series, 350, 153–74. quinn g p and keough m j (2002) Experimental Design and Data Analysis for Biologists, Cambridge, Cambridge University Press. ridler n, wowchuk m, robinson b, barrington k, chopin t, robinson s, page f, reid g, szemerda m, sewuster j and boyne-travis s (2007) Integrated multi-trophic aquaculture (IMTA): a potential strategic choice for farmers, Aquaculture Economics & Management, 11, 99–110. rogers s i and greenaway b (2005) A UK perspective on the development of marine ecosystem indicators, Marine Pollution Bulletin, 50(1), 9–19. salam m a, ross l g and beveridge m (2003) A comparison of development opportunities for crab and shrimp aquaculture in southwestern Bangldesh, using GIS modelling, Aquaculture, 220, 477–94. scanlan c m, foden j, wells e, and best m a (2007) The monitoring of opportunistic macroalgal blooms for the water framework directive, Marine Pollution Bulletin, 55, 162–71. schmitt r j and osenberg c w (eds) (1996), The Design of Ecological Impact Studies: Conceptual Issues and Application in Coastal Marine Systems, San Diego, CA, Academic Press. seafood services australia (2005) Take your pick! – The Seafood EMS Chooser (2nd edn), Queensland, Seafood Services Australia Ltd, http://www.seafood.net.au/ intro/Seafood_EMS_Chooser.pdf/, accessed January 2009. sequeira a, ferreira j g, hawkins a j s, nobre a, lourenço p, zhang x l, yan x and nickell t (2008) Trade-offs between shellfish aquaculture and benthic biodiversity: a modelling approach for sustainable management, Aquaculture, 274, 313–28. siem w k, boyd c e and diana j s (1997) Environmental considerations, in Egna H S and Boyd C E (eds), Dynamics of Pond Aquaculture, Boca Raton, FL, CRC, 163–82.

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silvert w and cromey c j (2000) Modelling Impacts, in Black K D (ed.), Environmental Impacts of Aquaculture, Sheffield, Sheffield Academic Press, 154–81. smaal a, van stralin m and craeymeersch j (2003) Does the introduction of the Pacific oyster Crassostrea gigas lead to species shifts in the Wadden Sea?, in Dame R F and Olenin S (eds), The Comparative Roles of Suspension-Feeders in Ecosystems, Volume 47, Nato Science Series: IV: Earth and Environmental Sciences, Dordrecht, Springer, 277–89. spice (2006) Special edition – Sediment Profile Imagery Colloquium of Experts (SPICE), Journal of Marine Science, 62(3–4). stigebrandt a, aure j, ervik a and hansen p k (2004) Regulating the local environmental impact of intensive marine fish farming III. A model for estimation of the holding capacity in the modelling-ongrowing fish farm monitoring system, Aquaculture, 234, 239-61. svane i and petersen j k (2001) On the problems of epibioses, fouling and artificial reefs, a review, Marine Ecology, 22(3), 169–88. tomczak m and herzfeld m (1998) Pollutant pathways between Mururoa and other Polynesian islands based on numerical model trajectories, Marine Pollution Bulletin, 36, 288–97. thrush s f, hewitt j e, cummings v j, green m o, funnell g a and wilkinson m r (2000) The generality of field experiments: interactions between local and broadscale processes, Ecology, 81, 399–415. thrush s f, hewitt j e, hermann p m j and ysebaert t (2005) Multi-scale analysis of species-environment relationships, Marine Ecology Progress Series, 302, 13–26. troell m, robertson-andersson d, anderson r j, bolton j j, maneveldt g, halling c and probyn t (2006) Abalone farming in South Africa: An overview with perspectives on kelp resources, abalone feed, potential for on-farm seaweed production and socio-economic importance, Aquaculture, 257, 266–81. underwood a j (1990) Experiments in ecology and management: their logics, functions and interpretations, Australian Journal of Ecology, 15, 365–89. underwood a j (1991) Beyond BACI: experimental designs for detecting human environmental impacts on temporal variations in natural populations, Australian Journal of Marine and Freshwater Research, 42, 569–87. underwood a j (1997) Experiments in Ecology. Their Logical Design and Interpretation Using Analysis of Variance, Cambridge, Cambridge University Press. wells e, wilkinson m, wood p and scanlan c (2007) The use of macroalgal species richness and composition on intertidal rocky seashores in the assessment of ecological quality under the European Water Framework Directive, Marine Pollution Bulletin, 55, 151–61. weston d (1990) Quantitative examination of macrobenthic community changes along an organic enrichment gradient, Marine Ecology Progress Series, 61, 233–44. wildish d and kristmanson b (1997) Benthic Suspension Feeders and Flow, Cambridge, Cambridge University Press. wildish d j, hargrave b t, macleod c and crawford c (2003) Detection of organic enrichment near finfish net-pens by sediment profile imaging at SCUBAaccessible depths, Journal of Experimental Marine Biology and Ecology, 285–286, 403–13. wu r s s (1995) The environmental impact of marine fish culture – towards a sustainable future, Marine Pollution Bulletin, 31, 159–66. wwf salmon aquaculture dialogue (2007) Nutrient impacts of farmed Atlantic salmon (Salmo salar) on pelagic ecosystems and implications for carrying capacity, Final Report of the Technical Working group on Nutrients and Carrying Capacity of the Salmon Aquaculture Dialogue, http://www.worldwildlife.org/what/ globalmarkets/aquaculture/WWFBinaryitem8844.pdf, accessed January 2009.

22 Spatial decision support in aquaculture: the role of geographical information systems and remote sensing L. G. Ross, N. Handisyde, D.-C. Nimmo, University of Stirling, Scotland

Abstract: Geographical information systems (GIS) have developed rapidly, especially recently, in response to a burgeoning range of increasingly complex spatial management questions. Their adoption for decision support and planning in aquaculture has been slow but is now increasing, to the point where agencies are beginning to expect it to be used in project planning. GIS modelling can be used to great advantage both for management and research within the sector, and this chapter shows how GIS models and model collections are expert systems based around a virtual environment and illustrates the wide range of modelling that can be applied to aquaculture problems. Key words: geographical information systems, remote sensing, decision support systems, spatial modelling.

22.1 The spatial planning context 22.1.1 Introduction Geographical information systems (GIS) have developed rapidly, especially in recent years, in response to the burgeoning range of increasingly complex spatial management questions. Prior to the advent of GIS, spatial data was represented and managed principally through paper maps with the strong limitation that only a small amount of spatial data could be manipulated at one time and the process was very time-consuming. Development of GIS has been paralleled by a massive increase in low-cost computing power with the result that very comprehensive tools for handling spatial data are now available to a wide range of users. GIS is particularly suited to natural resource management of all kinds and at the simplest level can provide a data management and archiving system of unparalleled flexibility. Many different data sources and types can be used simultaneously and cartographic output is usually excellent.

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The real power of GIS, and the way that it can be distinguished from related technologies such as CAD, graphic design and database systems, is in its ability to draw together the many diverse and complex factors which may need to be considered to reach development and administrative decisions. Remote sensing and GPS are superb data collection methods, but it is GIS which gives us an excellent modelling tool for environmental issues as well as an excellent means of linking between biology, physiology, environment, production systems, socioeconomics and infrastructure. Models are goal-oriented and can use common database elements to address different issues and this, coupled with the facility to conduct time-series analyses and predict future scenarios, means that GIS is rapidly becoming an essential tool in all natural resource disciplines. Although GIS has been investigated for aquaculture support and actively promoted over the last 15 years (e.g. Nath et al., 2000; also see GISAP http:// www.aqua.stir.ac.uk/aqua/GISAP and FAO GISFish http://www.fao.org/fi/ gisfish/), its use in the sector has been taken up rather slowly. The scale of an investigation can vary greatly and GIS models can be based on very large or very small areas, with appropriately different spatial resolutions used for different purposes. Several mega-regional investigations of aquaculture potential have been made, particularly for Africa and Latin America, using relatively simple environmental and resource availability models (Kapetsky, 1994; Kapetsky and Nath, 1997; Aguilar-Manjarrez and Nath, 1998). A number of national or state level investigations have been conducted successfully, based on a wide range of data on environment, infrastructure, resource availability and socioeconomics (Aguilar-Manjarrez and Ross, 1993, 1995a,b). These meso-scale models are particularly useful for guiding national plans, for consideration of food security issues and for investigation of conflict and trade-off between different economic activities. Such studies are now being greatly facilitated by the rapidly increasing varieties and resolutions of digital, basic data that are becoming available. Site selection issues can range from meso-scale decisions to very local ones. GIS models based on environmental and system considerations have been shown to be an excellent tool for detailed facility location, once a preliminary choice of site has been made (Ross et al., 1993). In conjunction with remote sensing and direct data collection, GIS can also form the basis for continued monitoring of a site; for example, recent work has shown that dispersal of wastes from an aquaculture site can be modelled in GIS to great advantage (Corner et al., 2006).

22.1.2 Development of the software and hardware The core computational software began its development in the 1960s and 1970s and evolved in parallel with other emerging technologies, such as CAD, remote sensing and computer graphics, with which it shares many common features. Standards in GIS began to be developed during the 1980s,

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but comprehensive software packages were not commercially developed and available to end-users until the late 1980s. The range of operations available within a GIS package has expanded greatly, and the portability of images between software for image manipulation or cartographic work is now so easy that there are few limitations even for the average user. GIS is totally dependent upon computing; the technology could not have developed without it. Much of the early development work took place on mainframe machines, but later migrated to PCs, paralleling the astonishing rate of evolution of computing during the lifetime of GIS development. The processing power of CPUs has increased exponentially and the parallel decrease in cost and increase in performance of memory, graphics cards, displays and all other peripherals means that very high resolution images can easily be handled, at speed, and without excessive cost. The PC in particular has evolved so much that almost all aspects of GIS work can now be handled on an affordable machine. In parallel with this, there have been major changes in data media so that satellite images previously only available on large-format computer-compatible tapes are now conveniently available on CD or can be downloaded from the Internet. The availability and low cost of very large capacity hard discs and writeable double layer DVD has made storage problems of spatial databases a thing of the past. This, coupled with the development of high speed networking, means that very large digital datasets can be moved around extremely easily. While the hardware and software are obvious pre-requisites, the final critical component to the whole system is a competent human analyst. The task of the GIS modeller is to develop application-oriented expert systems tailored to the requirements of each project and ultimately to the needs of end-users and stakeholders.

22.1.3 Key geographical information systems (GIS) capabilities Typically, a GIS software suite can be expected to be able to deal with a series of steps in the overall process; data acquisition and encoding, data storage and retrieval, preliminary data processing and manipulation, spatial query and data analysis and finally graphical display and interaction with the outcomes. All commercial GIS packages have some elements of most of these features, but they differ in their relative strengths and costs. Data acquisition and encoding This includes the processes of converting data to a computer-readable form and writing the data to the GIS database. Geographic data can be derived from a variety of sources: satellite imagery, aerial photographs, internet databases, ground surveys, and tabular data. Most data collected today are encoded in a digital format and GIS packages are designed to handle varying forms of data input, different media types, data formats and file type conversions. Sometimes it may be necessary to digitise hard copy maps

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from a tablet or a scanner, and on-screen digitising also extends data input ability. A range of direct keyboard data entry and data editing methods is usually possible. Data storage and retrieval Spatial data can be represented and stored in different ways, but the two major data types in use are raster and vector. Raster data represents all spatial information as a grid of rows and columns, the spatial resolution depending on the number of rows and columns covering the selected study area. No special arrangements are made for representing points, lines or polygons on the ground and all such features are mapped to this grid so that a point is represented by one cell (a pixel), a line by multiple cells joined at the edges or corners and a polygon by a group of contiguous cells. The advantages of the raster structure are that overlaying maps is easy and ‘perfect’, there is excellent and straightforward integration of remotely sensed imagery, a huge variety of complex spatial analysis is supported and the software is generally cheaper and easier to learn. In vector data ‘points’ lines and polygons are handled as separate classes of data. The spatial accuracy is very good and map output is excellent. However, map algebra is less easy and conversion to raster is necessary for some modelling work. Data storage and retrieval focuses on building of a spatial database which will contain the required variables or ‘production functions’ as raster and/or vector spatial data often known as thematic ‘layers’. These may be accompanied by spatially referenced attribute data stored as databases, which may be textual or numerical in nature. The strength of a GIS package is in its ability to link descriptive and spatial information while maintaining the spatial relationships between features. Retrieval of selected categories of information at this stage mirrors the capabilities of relational databases, thus allowing simple extraction of certain classes of data that match certain criteria. Information in databases can be accessed directly or new data layers can be created using information in the tabular database. This enables one of the simple but very useful attributes of GIS, the database query, in which data matching certain numerical ranges can be extracted and displayed (e.g. show all fish farms producing over 200 tonnes per annum in waters of a particular salinity). Analysis The strength of a GIS lies in its analytical and modelling capabilities. The ability to perform spatial searches of subsets of the main study, together with a series of distance-based operations, is very important and immediately moves beyond the simpler database query. However, the core of GIS is ‘map algebra’, in which the digital data sets can be manipulated using a wide range of Boolean, logical, arithmetic and algebraic operators. Using a combination of processes including reclassification, overlay, distance, connectivity and neighbourhood operators, an accurate picture of the current

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landscape can be developed. This allows the development of a complex series of decisions applied to the spatial database which, when combined with decision support tools, can result in logical and realistic modelling from which decisions can be made and the possible effects of these decisions and future change analysed. The ability to extract statistical information from the results of overlay and modelling and to carry out correlations between layers further enhances the power of GIS. Display Output from GIS query or models can be statistical in the form of tables or graphs. However, the ability to display results in the form of specialised thematic output, either on a large screen or in hard copy, is one of the most powerful features of GIS output. Most GIS packages have this ability, including the facility to produce specialist cartographic output to a very high standard. Many systems will also allow viewing of data or outcomes as three-dimensional projections, usually from a variety of perspectives. This feature is a very powerful tool, aiding the understanding of many phenomena. As 3D computer graphics has developed, the ability to treat a spatial database as a virtual environment that can be travelled through and interrogated has become possible. Results developed from time-series models can be compiled into animations or videos, further aiding interpretation and understanding of different scenarios. Most of these core capabilities are available in GIS software such as IDRISI, ARC-GIS and other packages, although there is some variation in their ability to handle raster or vector data, processing capabilities and display options.

22.2 Database construction and project methodology 22.2.1 Setting the objectives The key stages in establishing and developing a GIS project are summarised in Fig. 22.1. GIS models will rarely have a single fixed outcome because the results can be varied by changing the parameters and rules within the models created. Consequently it is important at the outset to be clear about the objectives of the study and to be able to specify the required range of outcomes. Where raster data will be used, the level of detail and hence spatial resolution of the required imagery also needs to be established in advance.

22.2.2 Identifying data requirements and sources The information needed for any spatial decision process is varied and will usually consist of data describing the physical, chemical, economic, social and infrastructural environments. This data can come from a variety of sources ranging from primary data from the field or satellite scenes to all

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Fig. 22.1 Schematic diagram of the stages of development for a GIS project showing the stages where expert input is required, the interactive participative input and also the intervention of constraints and risks. MCE = multi-criteria evaluation.

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forms of secondary data, including paper maps and textual databases. Although sources such as satellite data are already in digital form, all others may require some work in order to prepare them for use in the spatial database. With the exception of satellite images, it is extremely costly and timeconsuming to collect field data first hand and for this reason GIS users attempt to locate the data they need from existing secondary sources, either in paper or digital form. A primary consideration is to identify what data are really needed specifically to model the activity in question, as distinct from the plethora of data that may be available. This is followed by attempts to source the data and considerations of its age, scale, quality and relative cost. The lingua franca of GIS is the thematic map in which particular attributes of, or activities in, the landscape are represented. Clearly, where such maps are available either in hard copy or digital form, they are directly usable in a GIS. The availability of thematic maps worldwide is very variable, as can be the currency of their content, and this must be considered where critical decisions are to be based upon such material. Thematic maps can be in the form of a chorochromatic, choropleth, isopleth or point map; all of these are useable, and a good example in terms of aquaculture is the hydrographic chart. The more common terrestrial topographic map frequently contains a compilation of thematic data which are of value to the GIS modeller, including elevations, water bodies, roads, cities, woodland, etc. These themes can be extracted at the stage of digitisation and established as separate layers in the spatial database. Such maps are usually quite up to date, although the spatial accuracy of some printed material can be suspect. This critical assessment and verification of source data quality is very important. It can often be the case that estimating one variable from another can create new layers which are more useful than the originals. Such data is referred to as ‘proxy’ data, and established relationships may exist for deriving useable output from such data. Good aquaculture examples are calculation of probable water temperatures from air temperatures, extraction of semi-quantitative soil texture from FAO soil association distribution maps, calculation of maximum dissolved oxygen levels from digital elevation and temperature data or calculation of maximum wave heights from wind direction, velocity and fetch (Scott, 2004). Satellite images provide a rich source of data in a form suitable for use in a spatial database. The information collected by the scanners on LANDSAT, SPOT and other satellite systems is aimed specifically at natural resource work, and the source data can be reprocessed in a variety of ways to reveal details of the environment which may not be apparent in the raw state. Most GIS packages have some tools to deal with this, including the ability to filter and clean up the image, make corrections for atmospheric variations and allow georeferencing of the image by rubber-sheet transformation of the image to known reference points. Images can then be

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classified based on spectral signatures of different features in the environment such as forest, grassland, water bodies, etc. so as to reveal different land uses, often in considerable detail. A number of widely used vegetation indices, such as normalised difference vegetation index (NDVI), can also be calculated and the resultant thematic data can then be extracted ready for use in a GIS. The digital nature of the product means that the data are easy to incorporate and the relative costs are low compared to primary data collection surveys; hence remotely sensed images are a common starting point for much GIS work. Digital databases are expanding worldwide. These now range from information on natural resources, e.g. data on soils, temperature or hydrology, to population census and land ownership data. In many cases such data may only be available in hard copy reports, etc., although much is now available on CD from where it can be easily extracted. A growing source of such data is the Internet, and a wide range of spatial data can be found in careful searches. Spatial information at varying resolution can be obtained in this way, good examples being the 1 km Global Land Cover and related and derived sets (drainage basins; slopes at 1 km) and the temperature, rainfall and other data at 5 minutes of arc resolution or less, multispectral MODIS data and a growing range of free Landsat imagery made available by the United States Geological Survey (USGS) on various websites. This free data is of immense value and should not be overlooked.

22.2.3 Verification Critical evaluation of the quality of all forms of input data is essential both before and after digitising, and the value of data verification cannot be overstressed. It is often the case that at least some of the layers required in a GIS database will not be of a high enough standard and verification in the lab may need to become verification by survey. Field work as part of a verification exercise is frequently referred to as ‘ground-truthing’ and this particularly applies where satellite images are used as data sources. The general approach to such work is identical to any field survey, and standard techniques for survey and environmental measurement are used. The main difference is in the sampling plan, and a verification exercise will typically be based on a series of sample points designed to cover the area. This coverage would normally be well distributed over the ground or water surface, but is often more efficient if a random stratified sampling pattern is used. This allows effort to be concentrated on ensuring that differences between different features in the landscape are assessed, rather than using a simple randomised pattern, which may repeatedly cover a large uniform area. Global positioning systems (GPS) have greatly aided the spatial accuracy of ground-truthing and field verification. Since the ‘selective availability’ previously imposed on the NAVSTAR system by the US Defence Department was removed in 2000, actual accuracy is frequently better than 10 m

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and the forthcoming European Galileo GPS system will have an accuracy of 1 m. By operating in differential mode, GPS can give real-time locations accurate to less than 1 m and with post-processing to within a few millimetres. The value of these systems has been rapidly developed by GIS researchers, and some GIS data acquisition systems will accept direct input from GPS so that locations can be displayed over a real satellite image of the study area. Waypoints can be marked and attribute data added with automatic attribute file creation for later incorporation into the spatial database.

22.2.4 Data rectification GIS packages allow preprocessing of source data including a wide range of data filtering, transformation and vector to raster conversion. A set of routines for geocorrection or georeferencing enables data to be fitted to different world co-ordinate systems by a process of rubber-sheet transformation. Facilities for restoration of satellite images can include image corrections and enhancements such as sharpening, softening and histogram stretching to maximise data value. Image classification and interpretation routines allow feature classification using spectral signatures and extraction of thematic data from satellite scenes.

22.2.5 Arithmetic operations Data can be preprocessed in a number of ways, but a useful precursor is the scalar operation in which arithmetic operations, such as dividing all values in an image layer by a constant or finding their square root, can be carried out on single data layers. An example of this could be to multiply population size of towns by a constant for annual fish consumption per capita to give estimations of potential consumption of aquaculture or fisheries products. It is frequently the case that overlay procedures involving simple arithmetic operations between a small number of layers will be needed in order to derive a further useful layer, for example subtraction of evapotranspiration and seepage from rainfall to give a net water balance. The overlay is a fundamental tool of GIS and a building block in data manipulation and model building.

22.2.6 Neighbourhood analysis Neighbourhood analysis analyses the relationship between an object and similar surrounding objects. A new data layer can be created by computing the value assigned to a location as a function of the independent values surrounding that location. This type of analysis is often used in image processing of remotely sensed data.

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22.2.7 Buffers A buffer in GIS is a polygon enclosing an area within a specified distance of a feature, e.g. a point, line or polygon. Buffers applied around any given feature give the opportunity to establish increasingly or decreasingly valued zones around specific features, for example areas of high biodiversity, initially scored in terms of distance. These can subsequently be reclassified as costs, travel times or difficulty of movement across a surface taking into account a range of frictional forces.

22.2.8 Reclassification It is almost always the case that the source data, either real or integer values, will need to be reclassified before further use. In this process, scores reflecting relevance to the activity being modelled are assigned to values or ranges of the values of the original variables. This ranking will often result in a reduction in the number of data classes in an image and care must be taken to preserve the appropriate level of detail needed for sensitive decision making at a later stage. This powerful reclassification process transforms all types of source data to integer values of suitability for the activity on a scale of, for example, 1–5, with 1 being the least suitable. Larger scoring ranges are possible but are not often necessary. It is important to note that this reclassification process should bring all data layers to the same scale; an essential prerequisite for further modelling.

22.2.9 Overlay Overlay allows a large range of mathematical operations between layers, and the map algebra that this enables is a very powerful component of GIS modelling. For example, following reclassification in terms of suitability for the application, a simple outcome could be reached if the contributing layers are overlain by addition. This could be further refined by overlaying by subtraction any constraint layers where no activity can be allowed. However, these layers are usually arranged to be Boolean in content, with disallowed areas defined as 0 and allowed areas defined as 1. In this way, multiplication of this constraint layer by any layer containing data will result in constraints being removed from the outcome image. A series of such logical reclassifications and overlays can be assembled as a tree of sequential choices, a decision tree, and this can often provide a sensible and logical outcome to a particular spatial problem.

22.2.10 Weighted overlay When it is clear that not all variables in the model have the same level of importance, an alternative approach to the decision tree must be taken. In this case, weighted overlay is used in which each layer can have a different

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weighting. In order to achieve this, each source layer is first reclassified onto a common scale and can then be multiplied by a weighting factor. The resulting values are used in further overlay operations to obtain an outcome, again applying Boolean constraints in the final stage if needed. As the number of layers (factors, variables or production functions) in a model increases, the logic of these processing steps can become confusing and so special routines to assist this process have been provided in certain GIS packages. For example, the Multi-Criteria Evaluation (MCE) module in IDRISI is based on the Analytical Hierarchy Process described by Saaty (1987) in which the scoring of all the relevant layers is first normalised so that they are all on the same scale. Weights are then developed in a matrix of these layers and the importance of each layer relative to all others is developed. A scoring system is used in which 9 signifies that the column layer is more important than the row layer and 1/9 signifies that the column layer is less important than the row layer. The derivation of weightings may be imagined to be an objective scientific matter. However, it is clear from a range of studies that even expert opinions on ranking of variables differ. Aguilar-Manjarrez (1996) showed that, from a list of variables, a group of experts with similar background would generally agree on which are the most relevant to use for any given decision process, but the ranking applied by these experts can differ. In general, from a list of ten selected variables different experts would be in agreement on the priority of the first three to four and of the last three, while those of middle priority, four to seven, would be dealt with quite differently. It is also known that experts with different backgrounds (e.g. aquaculturist, coastal zone planner or conservationist) bring differing agendas to the same problem, resulting in a range of outcomes. At worst, it is clear that without guidance, a range of prioritisations could be obtained which are cumulatively meaningless. Thus, overall, the GIS modeller will need to be sure that personal weightings are as objective as possible and that where expert group input is used, the basis for this input is fully explained and carefully analysed before application to a critical model. The MCE routine enables a structured, logical approach to weighted overlay with built-in cross-checking of the weighting process to ensure consistency of logic by the operator.

22.2.11 Hierarchical models Using a combination of these processes, it is possible to build hierarchical models which, following the scheme outlined in Fig. 22.1, develop the source data and then group the essential variables or ‘production functions’ into sub-models designed to address a particular issue. The design and scoring used in such an assembly of processes will be based on aspects of the species, farming system, social system and region under study. In any decisionmaking or planning procedure it is beneficial to predict the outcome of the action before the action is taken and well-constructed GIS allows the testing

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of scenarios in the digital landscape to predict a range of outcomes. Different variations and recombinations of these sub-models can be quickly assembled to depict a range of goal-oriented outcomes. This modelling capability of GIS can be very powerful, but is frequently underutilised.

22.2.12 Model verification Field verification as part of GIS work is absolutely essential, both for quality control of certain data sources and for testing of the outcomes of models. While an environment and an activity can be modelled in total isolation as an academic exercise, it is only through careful verification that the general applicability of results can be ensured. As well as verifying data and outcomes of models, field verification with participative input from stakeholders (Fig. 22.1) will provide feedback into the modelling process itself by allowing the modeller to understand, quantitatively and qualitatively, any errors of the assumptions used.

22.3 Decision support systems and tools 22.3.1 Introduction A decision support system (DSS) can take many different forms and there is no generally agreed definition, but overall a DSS is a computerised system for helping make decisions based on choices between alternatives based on estimates of the relative value of those alternatives. The key characteristics and capabilities of a DSS are to generate these alternatives and to provide support for decision makers, planners, managers and stakeholders in resolving problems. A DSS will be adaptable and flexible, will have a high cost– benefit ratio and will support modelling under the full control of the decision maker (Turban et al., 2005). It is easy to see that a well-designed GIS model may contain a number of decision support tools each of which may be freestanding and/or interlinked in the larger model and that, consequently, a GIS model is also a DSS. In addition, some GIS packages contain a range of powerful ready-made DSS tools.

22.3.2 Activity trade-off Once an activity has been modelled and quantified, it will invariably have some potential to conflict with other uses of the space or resources. This calls for trade-off decisions to be made so that activities can coexist, and such decisions will include aspects such as economic and social benefits of one activity against one or more others. DSS tools are available in some GIS packages to assist this process. The Multi-Objective Land Allocation (MOLA) tool in IDRISI is a good example and the input for this procedure

Spatial decision support in aquaculture

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is the result from weighted overlays. The approach allows the modeller to set limits on areas required for different land uses, and the method then uses an iterative process to successively reassign ranked cells to either one activity or another depending upon how closely they match the requirements for that activity.

22.3.3 Biodiversity tools In recent years attention has increasingly focused on the effects of aquaculture developments on environment and biodiversity. GIS can be used to model not only the current spatial distribution of a species but also to predict areas of likely occurrence of a species and its future status in the landscape. This interactive dialogue between current and future environment, future development and biology is a powerful predictive tool which is well-implemented in the Land Change Modeller routines developed for IDRISI and ArcView by Clark Labs.

22.3.4 Viewsheds Viewshed is a recently coined term used to indicate the entire area that an individual can see from a certain vantage point. Planners and landscape architects use viewsheds to identify the zone of visual influence of a feature or future development, so as to determine its visual impact. In many countries, aquaculture developments are only allowed subject to assessment of visual impacts, and this is a matter of increasing importance. GIS is particularly suited to developing such scenarios, and the capability is part of several software packages. Simple or complex viewsheds can be developed for a wide range of sites, with and without buildings, forest or other variable intervening features, allowing comprehensive assessment of this problem.

22.3.5 Additional internal or external modules Most GIS packages support macro programming and this can be used to automate complex sequences of processes. These macros can become important DSS tools in themselves and are often made available to the community via the Internet. Construction of additional capability using C+ +, DELPHI or Visual basic is a feature of several GIS suites. Working within the IDRISI environment, Corner et al. (2006) used DELPHI to develop a module for dispersion of fish cage wastes, the quantitative output of which is part of the spatial database. Some authors have developed specific modules or programmes external to the GIS package in order to achieve specific goals. Most GIS systems can easily handle input and output from and to such external programmes. A good example of this is the use of external but linked calculation of fish

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growth based on energetic models by Kapetsky and Nath (1997) and Aguilar-Manjarrez and Nath (1998). They extracted data on temperature, fish species and size and length of the growing season from a GIS and used it in the POND model (Bolte et al., 1995) to make useful predictions of aquaculture output at sites that had been pre-selected in a simple environmentally based GIS procedure. Del Campo Barquin (2003) used PowerSim Solver in conjunction with GIS to develop very detailed fishery and exploitation models for the red sea urchin Loxechinus albus in Chile. The further development of such specialist modules to accompany the more standard GIS tools can be expected.

22.3.6 Analytical scope and reporting One of the powerful features of GIS packages is that statistical summaries of layers, model stages or outcomes can easily be obtained. Statistical data can include area, perimeter and many other quantitative estimates, including reports of variance and comparison between images. A further powerful analytical tool that aids understanding of outcomes is visualisation of outcomes through graphical representation, for example in three dimensions. This can be applied to views of terrain, watersheds, viewsheds, etc. and can include overlay of quantitative data on such views. Decision support tools such as these are constantly being added to GIS packages and are of great value if used carefully. There is also currently some development of decision support tools which can be accessed via the Internet. Assuming that Internet bandwidth is available, and is not compromised by future expansion of use, considerable potential for such methods exists and it may be expected that real-time GIS via the Web will expand.

22.4 Selected applications and examples of geographical information systems in aquaculture 22.4.1 Detailed facility location, Scotland Data relevant to site selection can be conveniently arranged into four categories: the aquaculture system, the aquaculture species, socioeconomics and food safety/quality issues. In-shore marine environments are widely used for aquaculture and, because of their complexity and dynamics, present problems for the system developer in optimally locating facilities. GIS readily permits alternative site-selection scenarios to be explored and, in a simple early example, Ross et al. (1993) focused on aspects of the physical environment to determine suitable sites for salmonid cages at a fjordic site in Scotland using bathymetry, currents, wave heights, shelter and salinity variation. System decisions preceded species-specific considerations (Fig. 22.2, see also colour section).

Spatial decision support in aquaculture N

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Sea surface Secondary road Main road SUITABLE AREA Freshwater stream Buildings Survey points Marker buoys

Fig. 22.2 (See also Plate III) A simple GIS model of suitability for salmon cage location in Camas Bruaich Bay, Scotland. The bay is 800 m wide and the most suitable area based on the parameters modelled is shown in dark green (after Ross et al., 1993).

22.4.2 Shellfish scenarios, Brazil Models based on environment and infrastructure data were used to determine opportunities for culture of molluscs, principally mussel and oyster, in Baia de Sepetiba, Brazil (Scott et al., 1998). In this study, extensive use was made of specialist macros, particularly for generation of potential wave heights from wind and fetch data. This approach is essential in all such siting studies where system structures will be placed in the water. The outcome images (Fig. 22.3, p. 722, see also colour section) revealed that much of the bay could be exploited for mollusc culture, but that different areas are better for different culture systems. These results were partly verifiable from data on artesanal collection of molluscs in recent times. This is, of course, no real substitute for field verification of any model, but careful use of inventory data from fisheries departments and similar agencies enables at least partial verification of models during the creation process.

22.4.3 Mangroves and aquaculture, Bangladesh Salam (2000), Salam and Ross (1999, 2000), Salam et al. (2000) and Salam et al. (2003) investigated management scenarios for coastal development in the Khulna area of Southwestern Bangladesh which includes the Sunderbans mangrove forest, a coastal belt, part of the Bay of Bengal

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Km 15 A

Km 15 B

Fig. 22.3 (See also Plate IV) Shellfish culture scenarios in Baía de Sepetiba, Brazil showing opportunities for mussel culture in the outer bay (A) and on-shore shrimp culture (B). Green = most suitable, yellow = suitable, blue = marginal, red = unsuitable, mauve = urban areas (after Scott, 2004).

and several major rivers and their tributaries. They developed a series of GIS models in order to identify and prioritise the most suitable areas for brackish water shrimp and crab farming. Using qualitative and quantitative output from the models, MOLA was used, based on gross production, economic output and employment potential, to compare and trade-off relative benefits from such developments and to consider their economic and social impact. Comparisons were made of brackish water shrimp and crab culture with moderately saline-tolerant tilapia and prawn culture, freshwater carp culture and traditional rice production systems. Shrimp was identified as the most capital intensive and risky production system. The trade-

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Agriculture Meters Penaeus 25000 Crab

Fig. 22.4 (See also Plate V) Resolving the trade-off between the uses of land in south west Bangladesh for agriculture, Penaeus culture and crab culture using multi-objective land allocation tools (after Salam, 2000).

offs between shrimp and crab cultures are shown in Fig. 22.4 (see also colour section).

22.4.4 Sea urchin fishery management, Chile Del Campo Barquin (2002) used GIS coupled with external simulation models to depict spatially the current state of natural stocks of the red sea urchin Loxechinus albus and other important commercial benthic resources in a small area of the central coast of Chile. Coupled with data on the major physical characteristics of the coastal environment, such as bathymetry and seabed type, models were developed which optimised sites for restocking of hatchery-reared seed of red sea urchin and which accorded with the aims of the local area management plan (Fig. 22.5, p.724, see also colour section). By integrating the biological and socioeconomic outcomes in an external simulation model, the ultimate objective of the GIS-based model for restocking and exploitation of the red sea urchin fishery was achieved.

22.4.5 Waste dispersion models, UK Modern GIS offers a powerful modelling environment capable of handling large databases. It is a very suitable environment in which to develop a suite of tools designed for environmental management of aquaculture sites, including carrying capacity prediction, land–water interactions and

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LAND

SEA

Meters 500

Fig. 22.5 (See also Plate VI) Suitability of areas for fishery restocking in Quintay Bay, Chile. The image shows environmentally suitable areas for restocking cross-tabulated with existing populations of keyhole limpet Fisurella sp., the red sea urchin Loxechinus alba and the Chilean abalone Concholepas concholepas. Dark green = highly suitable areas for L. Alba restocking (after del Campo Barquin, 2002).

multisite effects. Perez-Martinez et al. (2002) developed a spreadsheetbased model of fish farm waste dispersion into a GIS form by using some preprocessing of the field dataset followed by automatic spatial assessment built up using a macro in IDRISI. Corner et al. (2006) developed and validated an IDRISI module which estimates waste input using the mass balance approach of Gowen et al. (1989) and takes account of variable bathymetry and variable settling velocity for feed and faecal components. The model also incorporates the effect of tidal cage movement on waste dispersion, the first such model to do so. They showed that highest deposition from particulate fish waste is under the cage and that incorporation of cage movement increased the effective area under a cage by 72 %. This reduced peak deposition measurements by up to 32 % and reduced the average modelled feed and faecal settlement at the cage centre by 23 % and 11 %, respectively. The model was validated by comparing model predictions with observed deposition measured using sediment traps. The mean ratio of observed to predicted waste deposition at 5–25 m from the cage

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Meters 100

725

0 1 2 3 5 6 7 8 9 10 11 12 14 15 16 17 18

Kg C/m2/year

Fig. 22.6 (See also Plate VII) Modelling solid waste dispersal as kg carbon.m2.y from an intensive salmon cage farm in Scotland, using GIS. Circles show the mean cage positions. The peak wastes are directly underneath the cages, reducing to background levels within a short distance from the cage group (after Corner et al., 2006).

centre ranged from 0.9–1.06, whilst under the cage the model slightly overpredicted. A typical output from the model (Fig. 22.6, see also colour section) shows a double row of individual fish cages and the quantified spread of waste material around them.

22.4.6 Aquaculture and tourism, Tenerife GIS has been useful for guiding decisions where aquaculture may be developed alongside an important tourism industry (Salam et al., 2000). PerezMartinez et al. (2003, 2005) used GIS and related technologies to build a spatial database using those criteria which were considered to have any influence in integrating marine fish-cage culture within the tourism industry in Tenerife. Criteria were grouped into three sub-models (distance to beaches, nautical sports and viewsheds), which were combined to generate a final output showing the most suitable areas for cage culture development in coexistence with this industry (Fig. 22.7, p.726, see also colour section). Most areas of the coastline of Tenerife were identified as being suitable (56 %) and very suitable (46 %), suggesting that marine cage aquaculture could be developed in the island in coexistence with the well established tourism industry.

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North

Units (m) 10000.00

0 [4.2 km2] 2 [36.3 km2] 3 [44.8 km2] 4 [35.9 km2] 5 [27.6 km2] 7 [1.2 km2] 8 [125.6 km2]

Fig. 22.7 (See also Plate VIII) Suitability of areas for aquaculture development in Tenerife, Canary Islands, Spain. The model combines environmental aspects of sites, the physical requirements of the on-growing systems and is optimised using viewshed analysis to minimise impacts on tourism. The highest scores, in blue, have the highest potential (after Perez-Martínez et al., 2005).

22.5 Case study: climate change 22.5.1 Introduction There is now a general consensus that climate change is a real and significant threat and that we are currently experiencing its early stages with an increasing body of work highlighting observed ecosystem changes on all continents and in most oceans (Parry et al., 2007). Globally the aquaculture sector has seen dramatic growth since the 1970s and has accounted for much of the recent increase in fish production. Per capita fish consumption has also risen and, in association with increasing per capita incomes, urbanisation and population growth, the future demand for fish is set to continue expanding (Brugère and Ridler, 2004). Various studies have sought to model potential future demand for food fish with estimates of 108–145 million tons by 2020 (Delgado et al., 2003), 126.5–183 million tons by 2030 (Wijkström, 2003), and 121.1 and 270.9 million tons for 2010 and 2050, respectively (Ye, 1999). With many capture fisheries near or at capacity and potentially vulnerable to climate change, aquaculture will play a vital role in meeting these demands. Many countries have plans in place to actively promote the aquaculture sector. If this is to be successful and aquaculture is to be able to reach its potential and meet future requirements then the ability to plan and sustainably develop aquaculture in relation to a changing climate will be vital.

Spatial decision support in aquaculture 22.5.2

727

Potential future climate change and its impact on aquaculture systems The ability to model future climate is steadily improving with the development of increasingly sophisticated general circulation models (GCMs). The situation is also helped by the increasing number of models and simulations that are becoming available, thus allowing for comparisons between models as well as the use of multimodel ensembles. Due to the constraints of current computing capacity GCMs typically operate at fairly low resolutions with grid cells equating to several hundred kilometres squared on the earth’s surface. As such, GCMs have a much greater ability to model long-term trends over large areas, such as average temperature increases, compared with localised events such as storms and localised intense precipitation. Along with uncertainties relating to the climate modelling process, such as positive and negative feedbacks from within the systems themselves, it is impossible to accurately predict future human activity and consequently emissions of green house gases that are driving climate change. This inherent uncertainty associated with climate change is normally tackled by using a range of potential scenarios. The Intergovernmental Panel on Climate Change (IPCC) has produced a range of emissions scenarios (IPCC, 2000, 2007) which represent different possibilities in terms of human population growth as well as economic and technological development, and which are commonly used to drive climate models. The degree of variation between the range of IPCC scenarios is demonstrated in terms of average global temperature increase and sea level rise in Table 22.1. Drivers of climate-related change in aquaculture production systems can largely be grouped as: changes in air and inland water temperatures, changes in solar radiation, changes in sea surface temperature, changes in other oceanographic variables (currents, wind velocity and wave action, etc.), sea level rise, increase in frequency or intensity of extreme events and water stress. These changes will in turn have physiological (growth, development, reproduction, disease), ecological (organic and inorganic cycles, predation, ecosystem services) and operational (species selection, site selection, sea cage technology, etc.) impacts (Handisyde et al., 2006). Table 22.2 outlines a range of potential pathways by which climate change may impact on aquaculture systems. When assessing these potential climate-related impacts it is often useful to consider the varied spatial and temporal scales over which different climate-related events operate. The terms climate trends, climate shocks and climate variation are often used in the literature and provide useful concepts when considering potential impact pathways. Climate trends can be considered as long-term changes in variables such as average temperature and precipitation which may have impacts such as gradually altering productivity at a given location and/or affecting the range of species and methods that are suitable for the area in question. Along with these gradual changes, changing climate trends may

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Table 22.1 Predicted global average surface warming and sea level rise by the end of the 21st century Temperature change (°C at 2090–2099 relative to 1980–1999)1

Sea level rise (m at 2090– 2099 relative to 1980–1999)

Scenario

Constant year 2000 concentrations2 B1 scenario A1T scenario B2 scenario A1B scenario A2 scenario A1FI scenario

Model-based range excluding future rapid dynamical changes in ice flow

Best estimate

Likely range

0.6

0.3–0.9

NA

1.8 2.4 2.4 2.8 3.4 4.0

1.1–2.9 1.4–3.8 1.4–3.8 1.7–4.4 2.0–5.4 2.4–6.4

0.18–0.38 0.20–0.45 0.20–0.45 0.21–0.48 0.23–0.51 0.26–0.59

1 These estimates are assessed from a hierarchy of models that encompass a simple climate model, several earth models of intermediate complexity (EMICs) and a large number of atmosphere-ocean global circulation models (AOGCMs). 2 Year 2000 constant composition is derived from AOGCMs only. NA = not applicable. Source: adapted from IPCC, 2007.

combine with other factors to create faster acting and more dramatic impacts, often associated with reaching thresholds within natural systems, for example, in November 2007 Northern Ireland’s only salmon cage farm lost more than a 100 000 fish to a swarm of jellyfish which was attributed to a combination of warmer sea temperatures, resulting in increased reproduction of the jellyfish, and a reduction in the number of the jellyfishes’ natural predators as a consequence of fishing pressure. Climate shocks take place over a shorter time period and include events such as inland flooding, coastal flooding as a result of storm surges, and direct damage from wind and wave action during storms. Losses of stock, facilities and infrastructure from these types of event can be very sudden and dramatic. Changes in climate variation, such as increasingly erratic precipitation patterns, are also likely to be significant in some areas and are likely to be associated with changing degrees of risk in relation to events such as floods, droughts and extremes of temperature. Aquaculture systems and aquaculture-related livelihoods do not operate in isolation from other sectors and, in addition to the direct effects of climate change outlined in Table 22.2, there will be many indirect impacts. For example, climate changes that affect the availability and price of inputs such as the ingredients of aquaculture feeds will influence production costs and methods, while the relative prices of aquaculture products in relation

Decreased flushing rate that can affect food availability to shellfish Alterations in water exchanges and waste dispersal Change in abundance and/or range of capture fishery species used in the production of fishmeal and fish oil

•

Change in other oceanographic variables (variations in wind velocity, currents and wave action)

Increase in harmful algal blooms that release toxins in the water and produce fish kills Decreased dissolved oxygen Increased incidents of disease and parasites Enhanced growing seasons Change in the location and/or size of the suitable range for a given species Lower natural winter mortality Enhanced growth rates and feed conversions (metabolic rate) Enhanced primary productivity (phostosynthetic activity) to benefit production of filter-feeders Altered local ecosystems – competitors and predators Competition, parasitism and predation from exotic and invasive species Damage to coral reefs that may have helped protect shore from wave action – may combine with sea level rise to further increase exposure

•

Sea surface temperature changes

• •

•

• •

•

• •

• • • •

Impacts on culture systems

• •

•

•

•

• •

Accumulation of waste under pens Increased operational costs

Increased chance of damage to infrastructure from waves or flooding of inland coastal areas due to storm surges

Changes in infrastructure and operation costs Increased infestation of fouling organisms, pests, nuisance species and/or predators Expanded geographic distribution and range of aquatic species for culture Potential for both increases and decreases in production as a result of changes in growth rates and/or growing season length

Operational impacts

Potential impact pathways of climate change on aquaculture systems and production

Drivers of change

Table 22.2

Reduced water quality especially in terms of dissolved oxygen Increased incidents of disease and parasites Enhanced primary productivity may benefit production Change in the location and/or size of the suitable range for a given species Increased metabolic rate leading to increased feeding rate, improved food conversion ratio and growth provided water quality and dissolved oxygen levels are adequate, otherwise feeding and growth performance may be reduced

•

Higher inland water temperatures (possible causes: changes in air temperature, intensity of solar radiation and wind speed

•

•

• •

Large waves Storm surges Flooding from intense precipitation Structural damage Salinity changes Introduction of disease or predators during flood episodes

• • • • • •

Increase in frequency and/or intensity of storms

•

•

• •

Seal level rise

Loss of areas available for aquaculture Loss of areas such as mangroves that may provide protection from waves/surges and act as nursery areas that supply aquaculture seed Sea level rise combined with storm surges may create more severe flooding Salt intrusion into groundwater

Impacts on culture systems

Cont’d

Drivers of change

Table 22.2

Loss of stock Damage to facilities Higher capital costs, need to design cages moorings, jetties, etc. that can withstand events Negative effect on pond walls and defences Increased insurance costs Changes in level of production Changes in operating costs Increase in capital costs, e.g., aeration, deeper ponds Change of culture species

• • •

• • • •

• •

• • •

Damage to infrastructure Changes in aquaculture zoning Competition for space with ecosystems providing costal defence services (i.e., mangroves) Increased insurance costs Reduced freshwater availability Impacts on the availability of wild stocks – availability of seed stock for aquaculture

• • •

Operational impacts

Salinity changes Reduced water quality Limited water volume

Decreased water quality leading to increased diseases Reduced pond levels Altered and reduced freshwater supplies – greater risk of impact by drought if operating close to the limit in terms of water supply

• • •

• • •

Drought (as an extreme event (shock), as opposed to a gradual reduction in water availability)

Water stress (as a gradual reduction in water availability (trend) due to increasing evaporation rates and decreasing rainfall)

Source: adapted from Handisyde et al., 2006.

Salinity changes Introduction of disease or predators Structural damage Escape of stock

• • • •

Floods due to changes in precipitation (intensity, frequency, seasonality, variability)

• • • • • •

• •

•

• • •

Costs of maintaining pond levels artificially Conflict with other water user Loss of stock Reduced production capacity Increased per unit production costs Change of culture species

Loss of stock Loss of opportunity – limited production (probably hard to insure against)

Loss of stock Damage to facilities Higher capital costs involved in engineering flood resistance Higher insurance costs

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to alternatives will influence demand. Capture fisheries have to be seen as highly significant both in terms of competing with aquaculture products in the market place and supplying inputs, typically in the form of fish meal and fish oil. Other types of agriculture may also play a significant role, supplying feeds and fertiliser as inputs along with producing competing products such as poultry (Delgado et al., 2003). The Peruvian anchovy industry provides a good example of a climate-related indirect impact on the aquaculture sector that is felt at the global scale. During El Nin˜o events the increased temperature of surface waters off the South American Pacific coast leads to reduced upwelling of nutrient-rich waters. This in turn leads to a significant drop in primary productivity, the effect of which extends through the food chain causing a considerable drop in anchovy numbers. The result is felt worldwide in the form of increasing fish meal and fish oil prices. Fish meal is used as a component of many feeds including those for terrestrial livestock. In many cases there are options for switching to alternative protein sources in response to increasing fish meal prices, but this is not necessarily the case for some aquaculture species. Currently there is a degree of dependence on fish meal and fish oil within the aquaculture industry when formulating feeds for species such as carnivorous marine and diadromous finfish which typically have higher requirements for highly unsaturated fatty acids. Livelihoods, including those linked with aquaculture, can be viewed as having a range of components or assets. The extent to which changes in climate impact on these livelihood components will be influenced by a broad range of socioeconomic, environmental, geographic and cultural variables. A generalised example that will be relevant in many cases is that of water availability where the degree to which inland aquaculture is susceptible to precipitation changes will be affected by competition for water resources from a broad range of agricultural, industrial and domestic users and, in many cases, a growing human population. Aquaculture systems themselves operate at a range of scales and intensities and in a variety of environments. These differences in turn dictate how climate-related impacts can affect these systems and ultimately those whose livelihoods are linked to them. For example, there are a great number of contrasts between a large-scale intensive marine cage site with considerable capital investment producing a high-value product destined for export and small-scale pond production that relies on little in the way of external inputs other than perhaps household and agricultural waste, and produces some additional protein and minimal cash income at the household or local community level. It is important to remember that not all impacts on aquaculture will be negative. Increasing average temperatures and changing rainfall patterns will mean that some areas will see improved production rates and/or longer growing seasons while in others new opportunities, such as the range of species that can be cultured, will arise.

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22.5.3

Adapting to climate change and the role of geographical information systems The previous section outlined how a complex range of variables, that include changes to the climate, interact to influence the potential success of a particular aquaculture strategy at a given location. This process is further illustrated here in schematic form in Fig. 22.8. If aquaculture is to adapt to a changing climate and reach its potential then a detailed understanding of how changing climate variables will interact with a diverse range of aquaculture production systems will be essential. The use of GIS has great potential for combining these broad-ranging variables in a multicriteria framework with the aim of predicting where and how aquaculture in its various forms may be negatively impacted and, importantly, where it may be successful. Such systems can be highly useful in terms of guiding decision making in relation to aquaculture development and ultimately its adaptation to future scenarios. Handisyde et al. (2006) used a GIS-based model, as part of a global level assessment, to indicate areas where aquaculture-related livelihoods

Climate changes e.g. − temperature − precipitation − variability − extreme events − sea level rise

Direct effects e.g. − damage to facilities − loss of stock − water availability − growth rates − suitable ranges − disease

Impacts on aquaculture that influence and are influenced by: − site selection − scale and intensity of production − culture methods − species choice

Indirect effects e.g. − availability and cost of inputs e.g. for fish feeds − value of aquaculture products in relation to alternatives

Other factors e.g. − socioeconomic − environmental − cultural − geographic / land use

Fig. 22.8 Schematic diagram outlining the complex interacting variables that influence how changes in climate may influence aquaculture activities.

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are likely to be vulnerable to climate change impacts. Modelling was based on the concept that vulnerability is a function of exposure to climate change, sensitivity to climate change and adaptive capacity, an approach that in terms of definition of vulnerability has been implemented in a number of other studies (Allison et al., 2004; O’Brien et al., 2004; Metzger et al., 2005; Schröter et al., 2005). Figure 22.9 shows the hierarchical structure of the model used as well as outlining its components. All layers were reclassified on a scale of 1–5 while combination of component layers and sub-models was achieved through MCE using a weighted linear combination. Eight different assessments of vulnerability were made to take account of differing aquaculture environments and climate threats (Fig. 22.9). This was achieved by altering the weightings, or levels of significance, assigned to the various model components. The choice of weightings used has a highly significant effect on the way in which the model behaves and ultimately its validity. Handisyde et al. (2006) used a consensus of expert opinion in order to help determine weightings. A focus group was invited to assign layer weightings via guided questionnaires where contributors were asked to rank variables in order of significance and then assigned weightings with the aid of pair-wise comparison matrices (Saaty, 1987). Consistency of logic between values in the matrices was checked through calculation of a consistency ratio (Saaty, 1987). The level of agreement between decision makers was also investigated through the calculation of Kendall’s coefficient of concordance (Siegel and Castellan, 1988). This approach to setting model weightings has been used successfully for a number of GIS- and aquaculture-based studies (e.g. Aguillar-Manjarrez, 1996; Salam, 2000). An example of the graphical output produced by Handisyde et al. (2006) is given in Fig. 22.10 (p. 736, see also colour section) and shows results for a general assessment of vulnerability of aquaculture-related livelihoods to climate change with the aim of including as many factors as possible. Asia features strongly with China, India, Bangladesh, Indonesia, The Philippines, Lao People’s Democratic Republic, Thailand, Vietnam and Cambodia considered vulnerable with scores of 4 for at least part of their area. Nigeria and Madagascar, Africa’s second and third largest aquaculture producers, are indicated as vulnerable along with Mozambique, while Egypt, the continent’s largest producer, is indicated as less vulnerable. However the authors note that the Nile delta area may be at risk in the longer term due to the effects of sea level rise but that the current model lacked the ability to incorporate sea level rise into the assessment and that should be a priority for future research. In the case of South and Central America, Guatemala, Honduras and Nicaragua have areas with scores of 4 for small sections of their range which the authors suggest are linked to areas of high population density. Table 22.3 gives a summary of the results indicating the top 20 countries in terms of vulnerability with scores of 4 or more for at least part of their area. Overall, many Asian countries with their

MCE

− Cyclone risk − Flood risk − Drought risk

Adaptive capacity sub-model

Exposure to extreme climatic events submodel

Exposure to climate trends submodel

Sensitivity sub-model

MCE

− Overall vulnerability − Vulnerability in terms of food security − Vulnerability based on economic importance − Vulnerability with emphasis on adaptive capacity − Vulnerability of freshwater aquaculture to inland flooding − Vulnerability of freshwater aquaculture to drought − Vulnerability of brackish water culture to cyclone − Vulnerability of mariculture to cyclone

Fig. 22.9 Schematic diagram of the GIS model used by Handisyde et al. (2006) to indicate vulnerability of global aquaculture to climate change. The model follows a hierarchical structure with all data reclassified on a scale of 1–5. Combination of component layers and sub-models was achieved through multi-criteria evaluation (MCE) using a weighted linear combination. It is possible to alter the weighting, or level of significance assigned to the various components within the model and, in this case, this technique was used to make eight different assessments of vulnerability with the aim of encompassing varied aquaculture environments and climate threats.

MCE

MCE

− Annual mean temperature change to 2050 − Annual mean precipitation change to 2050 (reclassified so that areas with increased precipitation are considered most favourable) − Annual mean precipitation change to 2050 (reclassified so that areas with decreased precipitation are considered most favourable) − Population density for the year 2000

− Education − Per capita GDP − Life expectancy − Governance

MCE

− Aquaculture production quantities as % of total fisheries production − Fish protein consumption as a % of total animal protein − Aquaculture production as a % of GDP − % of population undernourished − Freshwater aquaculture value as a % of GDP − Brackish water aquaculture as a % of GDP − Marine aquaculture as a % of GDP − Freshwater aquaculture production quantities as a % of total fisheries production − Brackish water aquaculture production quantities as a % of total fisheries production − Marine aquaculture production quantities as a % of total fisheries production

Fig. 22.10 (See also Plate IX) Vulnerability of aquaculture to climate change (from Handisyde et al., 2006). Areas with scores of 4 or more (orange and red) are considered vulnerable. Areas with a score of 3 (yellow) are considered to have moderate vulnerability and may be worthy of further investigation. Low scoring areas highlighted in green are considered to have low vulnerability. Vulnerability was assessed as a function of: sensitivity to climate change, i.e. how significant is the aquaculture industry at a given location?; exposure to climate change, i.e. the extent to which climate is set to change; and adaptive capacity, i.e. the ability of people to cope with changes that may take place. Variables were integrated within the model using multi-criteria evaluation (MCE) with weighted linear average.

Missing or incomplete data 1 Lowest vulnerability 2 3 4 5 Highest vulnerability

* * * * * * * * * * * * * * * * * * * *

*

* * * * * * * * * * * * * * * * *

Vulnerability in terms of food security

*

*

* * * * * * * * *

Vulnerability based on economic importance

*

* * * *

* * * * * *

* * * * *

Vulnerability with emphasis on adaptive capacity

* *

* * * * * * *

* * * * * * *

Vulnerability of freshwater aquaculture to inland flooding

* Countries scoring 4 or more for at least part of their area and thus considered vulnerable. Source: Handisyde et al., 2006.

Vietnam Bangladesh India Philippines Cambodia China Indonesia Korea, rep. of Laos Nepal Pakistan Uzbekistan Mozambique Nigeria Guatemala Nicaragua Thailand Madagascar Malawi Sudan

Country

Vulnerability general

Table 22.3 Top 20 countries in terms of vulnerability

* * * *

* * * * * * * * * * *

* * *

Vulnerability of freshwater aquaculture to drought * * * *

Vulnerability of brackish water culture to cyclone

*

*

*

*

*

*

*

Vulnerability of mariculture to cyclone

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substantial aquaculture sectors feature heavily while a number of African countries are notable in terms of their low adaptive capacity.

22.5.4 Conclusions and future direction At the current time, despite the global importance of the aquaculture sector as a source of food security and income and the wide acceptance that climate change poses a real and significant threat, there has been surprisingly little work linking these topics. With the exception of the vulnerability assessment discussed in the previous section (Handisyde et al., 2006), the use of GIS techniques and spatial data to address climate change issues for aquaculture is even rarer, and this would appear to represent a significant research gap. There are multiple examples in the literature of GIS being used to produce complex spatial models with the aim of guiding site selection for aquaculture. A number of these incorporate environmental variables related to present climate conditions such as water availability, salinity and temperature (e.g. Kapetsky and Nath, 1997; Aguilar-Manjarrez and Nath, 1998; Salam and Ross, 1999, 2000; Salam, 2000; Salam et al., 2003, 2005; van Brakel et al., 2003). In the case of terrestrial agriculture the use of climate model data and future scenarios to estimate production potential is more widely established (e.g. Fischer et al., 2002; Geerts et al., 2006; Hood et al., 2006). Based on this, there is clearly strong potential for GIS-based modelling of future scenarios for aquaculture production where the emphasis is not only on highlighting negative impacts but also indicating where aquaculture of various types could prosper under future scenarios and make best use of changing environmental and climatic conditions. Modelling such as this could provide highly useful decision support tools for those involved in aquaculture development and policy formation and, as such, should be seen as an important component of aquaculture’s adaptation to future climate change. By taking advantage of the ability of GIS to incorporate broad ranging data sources such as land use, demographic and socioeconomic variables along with those related to climate and the environment, there are possibilities for guiding site selection for aquaculture development with the aim of addressing particular livelihood-related issues such as poverty alleviation and food security. Another potential area of interest is the incorporation of observed data, or potentially future scenarios, for activities such as fisheries and terrestrial agriculture. By identifying areas where industries such as these are likely to be negatively affected and incorporating this information with projections of where aquaculture could be successful it may be possible to indicate areas where aquaculture itself could act as an adaptive option by providing alternative sources of food and income.

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22.6 Case study: multi-site coastal zone planning 22.6.1 Introduction Coastal aquaculture activities have many complex interactions as they include near-shore and on-shore environments. Although the potential conflicts arising from aquaculture activities are well known (Gowen et al., 1989; Black, 2001) their regulation is currently focused on a single-site approach which does not take the wider spatial context into account. Hunter et al. (2007a,b) developed GIS models that include simple waste dispersion, cage site suitability modelling, biodiversity sensitivity modelling and viewshed analysis. The conjunction of these approaches allows for a much more comprehensive approach to aquaculture development, and the framework and models developed aim to provide an insight into the strengths of using GIS to achieve the multisite, sustainable coastal aquaculture.

22.6.2 Approaches to model development A conceptual model (Fig. 22.11) was developed for aquaculture in the Western Isles, Scotland, in which four initial principal sub-models were selected: • A cage suitability sub-model to address the importance of siting different types of cage technologies based on their physical design capabilities. • A biodiversity sub-model which identifies ecologically sensitive habitats and both land and marine species of conservation concern. • A waste dispersion sub-model in the form of a footprint model appropriate for large-scale multisite analysis. • A viewshed model to identify areas of visual importance and where there is likely to be an unacceptable impact on visual receptors. These sub-models and the ability to combine them into an overall final model address many current issues in aquaculture cage siting in an informative and coherent way. With the appropriate data and the ability to integrate additional data and model aspects, specifically relevant to individual study areas, this approach is easily adaptable to other coastal areas.

22.6.3 Cage site suitability sub-model Defining the most appropriate areas for a cage based on its design and performance in the physical environment is of high importance so as to maintain long-term operational sustainability and safety. This sub-model incorporates assessments based on physical factors related to manufacturers’ specific cage designs. The approach was initially defined by Ross et al.

740

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Wave climate

Currents

Bathymetry Sediment type

Cage suitability model

Endangered species Species sensitive to aquaculture Habitat and species distribution Commercial fisheries

Biodiversity model

MARINE FISH CAGE SITE SELECTION

Waste dispersion model

Viewshed model

Bathymetry Currents Hydrological processes

PRIMARY DATA

PRIMARY DATA

Protected areas

Digital elevation model Viewpoints

PRIMARY DATA

Fig. 22.11 Conceptual model of the multi-site coastal decision support system for aquaculture in the Western Isles, Scotland. Each arm of the total model is free-standing and may contain a number of embedded models, such as maximum wave height prediction, within it. The four models can be further processed in the central decision support tool to give summary outcomes.

(1993) and further refined by Perez et al. (2005). Specifications for three differing Kames® cage types were considered; the C315 designed for hydrodynamically exposed conditions, the C250 designed for semi-exposed conditions and the LMS designed for sheltered conditions. Spatial data on numerous environmental factors were collated and modelled, including data on: • • • • •

a sub-model of wave amplitude; a sub-model of wave period; bathymetry; current velocity; sediment type.

The initial physical environmental factors were reclassified in terms of suitability for cage aquaculture and were then aggregated using an MCE approach within the GIS framework. Figure 22.12 (see also colour section) shows the outcome for the cage systems designed for semi-sheltered loca-

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Least suitable

Most suitable

Fig. 22.12 (See also Plate X) Cage site suitability model for LMS sea cages in sheltered environments. The expanded area shows Loch Roag. Highly suitable areas are indicated by the orange to dark red colours, while blue to dark green indicate unsuitable to least appropriate areas. This model was created by reclassifying submodels of the physical environmental factors in relation to manufacturers’ guidelines. The final model was derived by combining these factors in an MCE using weighted linear combination.

tions. For this type of cage design, the suitability model shows that ideal locations are restricted to in-shore sea lochs extending to an area of approximately 427 km2. By comparison, the models show that approximately 629 km2 of in-shore waters are suitable for the semi-exposed cage design and 707 km2 for the exposed cage design. The semi-exposed cage model was explored further as research showed that the majority of fish cages in use in the Western Isles are based on this design type. Overlaying the locations of current aquaculture farms with the cage suitability model for semiexposed cages showed that almost all current farms are located within areas that are suitable for this cage type. This initial sub-model is aimed at the pre-development and exploratory stages, and the suitability sub-model is based on the cage’s ability to endure varying environmental conditions. The resulting output models are expressed as suitability scores specific to the cage design being investigated. Arming regulators with suitability scores and highlighting the areas within management zones that could be appropriate for the cages to be sited allows a more robust approach than is often used in environmental impact assessments.

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22.6.4 Biodiversity sub-model The Western Isles not only has a substantial aquaculture industry but also has great diversity of habitats and wild species. This biodiversity is widely recognised and is protected by numerous national and international legislative instruments. It is consequently vital to ensure that aquaculture does not adversely impact on these conservation areas. Data on a wide range of physical and biological data were collated to create five initial biodiversity layers. Factors were reclassified in terms of biodiversity importance and then aggregated using a MCE approach within the GIS framework (Fig. 22.11) The biodiversity sensitivity layers were: • protected areas: special areas for conservation (SAC), special protection area (SPA), site of special scientific interest (SSSI), RAMSAR site, national scenic area (NSA); • commercially important fish spawning and nursery areas; • distributions of endangered species; • distributions of species sensitive to aquaculture; • habitat suitability and species distributions of species important to the Western Isles. The resulting model is shown in Fig. 22.13 (see also colour section) and it confirms areas previously known to have a high biodiversity (such as Loch Roag in the north-west of the islands), but also highlights other areas of

Lowest biodiversity

N

Highest biodiversity

25 km

Fig. 22.13 (See also Plate XI) Biodiversity model of species sensitive to aquaculture for the Western Isles. The highest biodiversity is indicated by the deep red to white areas. The expanded area shows Loch Roag. This model was created using the IDRISI Andes Land Changer Modeller tools.

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high biodiversity not previously considered. Many of these areas are important both for present and future development of aquaculture, and the biodiversity model developed here can be used to assess the ability of coastal sites to incorporate aquaculture activities while ensuring that the relevant biological criteria, such as species diversity, sensitive environments and species, and fishery nurseries are considered. The outputs from this model can be used in conjunction with other GIS-based models, to quantify the vulnerability of local biodiversities. This type of model has the potential for expansion in both geographical and information terms, allowing more spatial parameters or other relevant information to be included for other aspects of aquaculture production and development.

22.6.5 Waste dispersion sub-model The potential negative impacts from aquaculture on its surrounding environment are well known and the conditions which are favourable for a thriving aquaculture industry must be protected, especially from its own waste output. This will not only ensure that it does not significantly negatively affect the environment but will also improve sustainability and longevity of the activity. Currently, as with other aspects of the environmental impact assessment (EIA) process, wastes from fish farms are assessed on a site by site basis. In the Western Isles within some fjord systems there are numerous operational farms in close proximity to each other. Assessing these on individual levels may not give a realistic representation of the actual impact and current environmental conditions. While a detailed waste dispersion model can be used within GIS for each active or potential site (see above), for the purpose of wide area multiple site assessment a simple ‘footprint’ model was developed both for faeces and for waste food. All farms modelled can then considered together with their interactions (Fig. 22.14, p. 744, see also colour section). This type of large-scale model at a multisite level has not been developed previously and provides a major step forward in meeting the challenges for aquaculture in coastal environments. GIS software easily allows this type of modelling and provides an innovative and proactive way to model multisite solid waste dispersion for aquaculture. This model, though in its infancy, shows great potential as it highlights the current flaws of individual farm site modelling.

22.6.6 Viewshed sub-model The Western Isles coastline varies from sandy shores to rocky cliffs and has many scenic, highly valued areas currently protected by legislation. Visual and landscape impact assessments are a statutory requirement of an EIA for aquaculture, but the assessment method is not clearly defined. A submodel was developed to assess visual impact by considering areas that could

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Fig. 22.14 (See also Plate XII) Multiple waste footprints from marine farms in the Western Isles, modelled using GIS. The study area shows Loch Roag, identified in Fig. 22.12 (Plate X) and 22.13 (Plate XI), with expanded areas showing the footprint from multiple farm sites modelled using a simplified version of the tools used in Fig. 22.6 (Plate VII) for a single site.

be directly affected and the range of viewers (visual receptors) affected. IDRISI Andes has strong viewshed modelling capacity which can determine all cells which are visible from viewpoint cells, and this was utilised at two levels to create two different sub-models; first a simple Boolean layer of cells that are in view and cells that are not, and second a proportional output that shows the proportion of viewpoint cells from which the viewshed cell is visible. Lastly, the module has the ability to assess a viewpoint cell continuously, i.e. a road or path. A good-quality digital elevation model (DEM) is required for this process, and this was sourced from the UK Ordnance Survey Land Profile contour maps which have five metre interval contours at ±1 m height accuracy and, in the more mountainous areas, ten metre contours at ±1.8 m accuracy. The DEM was overlaid with the components of the built environment but, as trees are scarce or insignificant in the outer islands, these were ignored. Proportional and Boolean viewshed sub-models were created at a variety of distances from 47 key viewpoints (which had been identified in an earlier study (Benson et al., 2004), and which included a variety of important walks, popular viewpoints, dramatic coastlines and hostels)), as well as from all major roads and ferry routes. An example output from this structured approach to viewshed analysis is shown in Fig. 22.15, p. 745 (see also colour section). This GIS method can systemically identify visual impacts associated with any aquaculture development and also allows prediction of the level of impact from the viewers’ perspective. The viewshed sub-model presented here has a structure that can be implemented at a range of levels and that

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Low visibility areas

High visibility areas

Fig. 22.15 (See also Plate XIII) An example of proportional viewshed analysis for the Western Isles, Scotland, modelled within GIS. The model includes a range of viewpoints, including ferry routes, all roads, buildings, Scottish National Heritage and significant viewpoints up to a search distance of 1 km. The expanded area shows the viewshed analysis for the main city of Stornoway. Red indicates high visibility areas and dark blue indicates the low visibility areas.

is easily replicated in any study area. This level of clarity is missing from current viewshed analysis in an EIA. 22.6.7 Conclusions and future direction This multisite GIS-based DSS decision for coastal aquaculture can be easily adapted for other locations and to take account of current and future legislation, guidelines or environmental regulation policies. Although the four sub-models presented here reflect the particular needs of the Western Isles, they are flexible and have wide general application. In addition, other submodels may be added, to give a wider range of perspectives or a more refined outcome, as more data become available. Wider use of this kind of multifactorial approach could benefit both government regulators and the aquaculture industry as it can be used to maximise production in suitable areas, without impacting on environmentally-sensitive areas.

22.7 Summary and future trends In this chapter, the utility of spatial analysis to support complex decisionmaking for aquaculture has been illustrated. GIS is not a mapping tool;

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rather, the extensive GIS model-building capabilities and the range of decision support tools allow real decisions to be made and trade-off allocations of land, water and natural resource use and their benefits to be evaluated quantitatively. In addition to clear management benefits, the understanding of processes and events that this approach can engender is very powerful and means that GIS has a role in research as well as in end-user applications. The wide range of application of the GIS approach has recently been developed into aquaculture and fishery management, and this trend is now growing strongly. The strengths of GIS include the ability to handle a wide range of data sources and resolutions, speeding up work and allowing for easy updating of spatial databases and the ability to handle time-series analysis and to generate specialist output. Problems of sourcing the necessary data to create a spatial database and verifying its quality can be a big component in establishing a project. Similarly, there may be perceived difficulties in assigning financial benefit to the development time needed for any given application, as well as questions of ownership and access once the spatial database has been created. These matters can be overcome, however, and the effort of dealing with them will be rewarded by the benefits and power of using GIS models for natural resource modelling, management and decision making. Overall, GIS has an excellent future in the aquatic sector. The rapidly expanding access to digital datasets at low cost and, increasingly, via the Internet means that the time investment in establishment of spatial databases is reducing. Hardware is also developing rapidly and is becoming more powerful and affordable, and GIS and other spatial software is becoming more comprehensive in its capabilities. In general terms, GIS is now becoming more widely adopted, to the point at which some agencies are beginning to expect it to be used in project planning rather than treating it as an interesting add-on. There is, nevertheless, a real requirement for sound expertise in the techniques of GIS and remote sensing, and a proper understanding of the processes involved, in order to ensure good results. GIS should not be considered as a hardware- and software-based ‘point and click’ solution to decision making. It is particularly important to recall that a spatial database is created for an application-oriented reason, based on subject-specific rules. Consequently GIS models and model collections are expert systems based around a virtual environment and it is the manipulation of data and modelling of problems within that environment that is the real heart and philosophy of GIS.

22.8 Acknowledgements LGR wishes to acknowledge the major contribution of his many students to development of this sector over the last 20 years in the GISAP group at

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the Institute of Aquaculture, Stirling. Thanks are also due to Dr Malcolm Beveridge of WorldFish Center and to Dr Jim Kapetsky and Dr Pepe Aguilar of FAO, Rome, for useful discussion on many occasions. Further details of our work can be found at http://www.aqua.stir.ac.uk/aqua/GISAP/.

22.9 References aguilar-manjarrez j (1996) Process modelling and priorities in Geographical Information Systems for aquaculture, PhD Thesis, University of Stirling, UK. aguilar-manjarrez j and nath ss (1998) A strategic reassessment of fish farming potential in Africa, CIFA Technical paper, 32, Food and Agriculture Organization of the United Nations, Rome. aguilar-manjarrez j and ross lg (1993) Aquaculture development and Geographical Information Systems, Mapping Awareness and GIS Europe, 7(4), 49–52. aguilar-manjarrez j and ross lg (1995a) Geographical Information System (GIS) environmental models for aquaculture development in Sinaloa state, Mexico, Aquaculture International, 3, 103–15. aguilar-manjarrez j and ross lg (1995b) Managing Aquaculture Development: The role of GIS in environmental studies for aquaculture, GIS World, 8(3), 52–6. allison eh, adger wn, badjeck m-c, brown k, conway d, dulvy nk, halls a, perry a and reynolds jd (2004) Effects of climate change on the sustainability of capture and enhancement fisheries important to the poor: analysis of the vulnerability and adaptability of fisherfolk living in poverty [Draft], DFID, Fisheries Management Science Programme, London. benson jf, scott ke, anderson c, macfarlane r, dunsford h and turner k (2004) Landscape capacity study for onshore wind energy development in the Western Isles, Scottish Natural Heritage Commissioned Report No 042 (ROAME No FO2LC04), Scottish Natural Heritage, Inverness. black kd (ed.) (2001) Environmental Impacts of Aquaculture, Sheffield Academic Press, Sheffield. bolte jp, nath ss and ernst dh (1995) POND: A decision support system for pond aquaculture, 12th Annual Administrative Report, PD/A CRSP, Corvallis, OR, 48–67. brugère c and ridler n (2004) Global Aquaculture Outlook in the Next Decades: An Analysis of National Aquaculture Production Forecasts to 2030, Fisheries Circular No. C1001, Food and Agriculture Organization of the United Nations, Rome. corner ra, brooker a, telfer tc and ross lg (2006) A fully integrated GIS-based model of particulate waste distribution from marine fish-cage sites, Aquaculture, 258, 299–311. del campo barquin lm (2003) Restocking planning for sea urchin culture in Quintay bay, Chile, PhD Thesis, University of Stirling, UK. del campo barquin m (2002) Restocking A bio-socio-economic simulation model for management of the red sea urchin fishery in Chile, PhD Thesis, University of Stirling, UK. delgado cl, wada n, rosegrant mw, meijer s and ahmed m (2003) Fish to 2020: Supply and demand in changing global markets, International Food Policy Research Institute (IFPRI) and WorldFish Center, Washington, DC, Penang. fischer g, shah m and velthuizen hv (2002) Climate Change and Agricultural Vulnerability, a special report prepared by the International Institute for Applied

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Systems Analysis as a contribution to the World Summit on Sustainable Development, Johannesburg. geerts s, garcia m, del castillo c and buytaert w (2006) Agro-climatic suitability mapping for crop production in the Bolivian Altiplano: a case study for quinoa, Agricultural and Forest Meteorology, 139, 399–412. gowen rj, bradbury nb and brown jr (1989) The use of simple models in assessing two of the interactions between fish farming and the marine environment, in De Pauw N, Jaspers E and Wilkins N. (eds), Aquaculture – A Biotechnology in Progress, European Aquaculture Society, Oostende, 1071–80. handisyde nt, ross lg, badjeck m-c and allison eh (2006) The effects of climate change on world aquaculture: a global perspective, Final Technical Report, Stirling Institute of Aquaculture, Stirling, UK. hood a, cechet b, hossain h and sheffield k (2006) Options for Victorian agriculture in a ‘new’ climate: pilot study linking climate change and land suitability modelling, Environmental Modelling & Software, 21(9), 1280–9. hunter dc, telfer tc and ross lg (2007a) A GIS framework for the evaluation of aquaculture development in the Western Isles, Scotland: Modelling marine biodiversity to support net pen site selection, European Aquaculture Society Annual Meeting. Competing Claims, Istanbul, October. hunter dc, telfer tc and ross lg (2007b) A GIS framework for the evaluation of aquaculture development in the Western Isles, Scotland: Optimising site location based on physical environmental parameters and cage engineering design, European Aquaculture Society Annual Meeting. Competing Claims, Istanbul, October. ipcc (2000) Emissions Scenarios, Special Report of the Intergovernmental Panel on Climate Change, Nakicenovic N and Swart R (eds), Cambridge University Press, Cambridge. ipcc (2007) Climate Change 2007: The Physical Science Basis, Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change, Solomon S, Qin D, Manning M, Chen Z, Marquis M, Averyt KB, Tignor M and Miller HL (eds), Cambridge University Press, Cambridge and New York. kapetsky jm (1994) A strategic assessment of warm-water fish farming potential in Africa, CIFA Technical paper 27, Food and Agriculture Organization of the United Nations, Rome. kapetsky jm and nath ss (1997) A strategic assessment of the potential for freshwater fish farming in Latin America, COPESCAL Technical paper 10, Food and Agriculture Organization of the United Nations, Rome. metzger m, leemans r and schröter d (2005) A multidisciplinary multi-scale framework for assessing vulnerability to global change, International Journal of Applied Earth Observation and Geoinformation, 7, 253–67. nath ss, bolte jp, ross lg and aguilar-manjarrez j (2000) Applications of Geographical Information Systems (GIS) for spatial decision support in aquaculture, Aquacultural Engineering, 23, 233–78. o’brien k, leichenko r, kelkar u, venema h, aandahl g, tompkins h, javed a, bhadwal s, barg s, nygaard l and west j (2004) Mapping vulnerability to multiple stressors: climate change and globalization in India, Global Environmental Change, 14, 303–13. parry ml, canziani of, palutikof jp, van der linden pj and hanson ce (eds) (2007) Climate Change 2007: Impacts, Adaptation and Vulnerability, Contribution of Working Group II to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change, Cambridge University Press, Cambridge. perez-martínez om, telfer tc, beveridge mcm and ross lg (2002) Geographical information systems (GIS) as a simple tool to aid modelling of particulate waste

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distribution at marine fish cage sites, Estuarine, Coastal and Shelf Science, 54, 761–8. perez-martínez om, telfer tc and ross lg (2003) Use of GIS-based models for integrating and developing marine fish cages within the tourism industry in Tenerife (Canary Islands), Coastal Management, 34(4), 355–66. perez-martínez om, telfer tc and ross lg (2005) GIS-based models for offshore marine fish cage aquaculture site selection in Tenerife, Canary Islands, Aquaculture Research, 36, 946–61. ross lg, mendoza qm, ea and beveridge mcm (1993) The application of geographical information systems to site selection for coastal aquaculture: an example based on salmonid cage culture, Aquaculture, 112, 165–78. saaty rw (1987) The analytic hierarchy process–what it is and how it is used, Mathematical Modeling, 9(3), 161–76. salam ma (2000) GIS modelling of coastal aquaculture development in Khulna district, Sunderbans, Bangladesh, PhD Thesis, University of Stirling, UK. salam ma and ross lg (1999) GIS modelling for aquaculture in South-western Bangladesh: Comparative production scenarios for brackish and freshwater shrimp and fish, Proceedings of GIS’99, 13th Annual Conference on Geographic Information Systems, Vancouver, BC, 141–5. salam ma and ross lg (2000) Optimizing site selection for development of shrimp (Penaeus monodon) and mud crab (Scylla serrata) culture in Southwestern Bangladesh, Proceedings of GIS’2000, 14thAnnual Conference on Geographic Information Systems, Toronto, ONT, 13–16 March, 1–17. salam ma, ross lg and beveridge mcm (2000) Eco-tourism to protect the reserve mangrove forest of the Sundarbans and its flora and fauna, Anatolia, 11(1), 56–66. salam ma, ross lg and beveridge mcm (2003) A comparison of development opportunities for crab and shrimp aquaculture in South-western Bangladesh, using GIS modelling, Aquaculture, 220, 477–94. salam ma, khatun na and ali mm (2005) Carp farming potential in Barhatta Upazilla, Bangladesh: a GIS methodological perspective, Aquaculture, 245(1–4), 75–87. schröter d, polsky c and patt ag (2005) Assessing vulnerabilities to the effects of global change: an eight step approach, Mitigation and Adaptation Strategies for Global Change, 10(4), 573–95. scott p (2004) Aquaculture development interactions in Sepetiba Bay, Rio de Janeiro, Brazil. A GIS study, PhD Thesis, University of Stirling, UK. scott pc, cansado s and ross lg (1998) A GIS-assisted mollusc culture potential determination for Sepetiba Bay, Brazil, GIS Planet ’98, Lisbon, September, CD-ROM. siegel s and castellan jn (1988) Nonparametric Statistics for the Behavioral Sciences, 2nd, edn, McGraw-Hill, New York. turban e, aronson je and liang tp (2005) Decision Support Systems and Intelligent Systems, New Jersey, Pearson. van brakel ml, muir jf and ross lg (2003) Modelling for aquaculture related development, poverty and needs in the Mekong basin, Proceedings of the 2nd Large Rivers Conference, Phnom Penh, 11–14 February. wijkström un (2003) Short and long-term prospects for consumption of fish, Veterinary Research Communications, 27(Suppl. 1), 461–8. ye y (1999) Historical consumption and future demand for fish and fishery products: Exploratory calculations for the years 2015–2030, FAO Fisheries Circular No. 946, Food and Agriculture Organization of the United Nations, Rome.

23 Zooremediation of contaminated aquatic systems through aquaculture initiatives S. Gifford, G. R. MacFarlane, C. E. Koller, R. H. Dunstan, The University of Newcastle, Australia, and W. O’Connor, NSW Department of Primary Industries, Australia

Abstract: The ability of animals to act in a bioremediative capacity is not widely known. Animals are rarely considered for bioremediation initiatives due largely to ethical or human health concerns. Nonetheless, specific examples in the literature reveal that many aquatic species, including species employed in aquaculture, are effective remediators of metals, microbial contaminants, hydrocarbons, nutrients and persistent organic pollutants. We introduce zoological equivalents of the definitions used in the phytoremediation literature (zooextraction, zootransformation, zoostabilisation and animal hyperaccumulation), to serve as useful benchmarks in the evaluation of candidate animal species for zooremediation initiatives. Further, we present a case study assessing the deployment of pearl oysters to remove metals and nutrients from aquatic ecosystems. Key words: aquaculture, hyperaccumulation, metals, nutrients, organics, zooremediation, zooextraction, zootransformation, zoostabilisation.

23.1 Introduction Bioremediation involves the use of living organisms to remove or detoxify pollutants within a given environment. The methods used include: (i) bioextraction: the harvesting and treatment of pollutant-containing biomass; (ii) biostabilisation: the use of organisms to stabilise pollutants; and (iii) biotransformation/degradation: the use of organisms to degrade organic pollutants to less toxic compounds (Sursala et al., 2002). Most commonly, bacteria are employed in bioremediation, but the use of plants, ‘phytoremediation’, and algae, ‘phycoremediation’, is increasing (Reeves and Baker, 2000; Sursala et al., 2002; Olguin, 2003). Animals are less commonly considered for bioremediation, either because of ethical concerns or because

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many that are commercially cultured or harvested are bound for human consumption. Nonetheless, according to the above definitions, animals in aquatic ecosystems extract, stabilise or degrade pollutants, and this can be achieved through targeted aquaculture initiatives without human consumption as the economic incentive. Many animal species have simple life histories, are resistant to toxicity, and have the ability to generate an economic return following remediation activities. Indeed, the literature reveals that key aquaculture species, such as some bivalves, fish, polychaetes, and sponges, are suitable bioremediators (Newell, 1988; Haamer, 1996; Milanese et al., 2003; Mackenzie et al., 2004; Giangrande et al., 2005; Gifford et al., 2005; Stabili et al., 2006a) and could be used in this capacity in two ways. Pollutants may be extracted from an area by the introduction, culture, and harvest of animals (aquaculture), or the supplementation or maintenance of wild animal populations may lead to stabilisation or degradation of pollutants. While using animals for bioremediation initiatives has tentatively been termed ‘zooremediation’ (Gudimov, 2002) (pronounced zo-o-remediation), the concept is poorly developed and not widely recognised. The phycoremediative value of algal aquaculture has been acknowledged (Chung et al., 2002; McVey et al., 2002; Carmona et al., 2006) but, despite its sheer size (>48 million tonnes; FAO 2005), the zooremediative potential of animal aquaculture remains relatively poorly explored. This chapter outlines zoological analogies of botanical equivalents from the field of phytoremediation and thus the potential in aquatic ecosystems for (i) zooextraction, (ii) zoostabilisation and/or zoodegradation (Box 23.1 on p. 752). We discuss the utility of naturally occurring, supplemented and/or cultured aquatic animal taxa for potential reductions of contaminant load in aquatic receiving waters. We provide an aquaculture-specific case study, from our own research, for the potential of commercial pearl aquaculture to concomitantly reduce contaminant loads. In particular, we seek to broaden the consideration of zooremediation beyond traditional polyculture, and promote the paradigms of balanced ecosystem management (McVey et al., 2002) to encourage further consideration of economically viable aquatic remediation programs.

23.2 Zooremediation of pollutants 23.2.1 Zooextraction of nutrients and microorganisms The cultivation and harvest of animals to remediate nutrient and pathogenic microorganism pollution in aquatic systems is the most common form of zooremediation. The practice has a long history in aquaculture, where polyculture can reduce nutrient and microorganism pollution associated with some monocultures. The most common group of animals deployed is bivalve molluscs. Mussels and oysters have been co-cultured with salmon

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Box 23.1 Definitions Phytoremediation Phytoextraction: The harvest and treatment of pollutant containing above ground plant biomass. Focus is on plant species known to hyperaccumulate pollutants of interest. Phytostabilisation: The use of plant roots to inhibit pollutant migration. Phytotransformation/Phytodegradation: The use of plants to degrade organic pollutants to less toxic compounds. Plant hyperaccumulator: Plant species known to accumulate >100 mg kg−1 Cd, Cr, Co and Pb, >1000 mg kg−1 Ni, Cu, Se, As and Al, or >10 000 mg kg−1 Zn and Mn in their above ground biomass (dry weight). Zooremediation equivalents Zooextraction: The harvest and treatment of pollutant containing animal biomass. This would target animal species known to accumulate pollutants of interest. Sustainable harvest of wild populations and/ or aquacultured species. Zoostabilisation: The use of animals to inhibit pollutant migration. Involves the maintainance or supplementation of wild animal populations without harvesting of animal biomass. Zootransformation/Zoodegradation: The use of animals to degrade organic pollutants to less toxic compounds. Involves the maintainance or supplementation of wild animal populations without harvesting of animal biomass. Animal hyperaccumulator: Those animal species known to accumulate >100 mg kg−1 Cd, Cr, Co and Pb, >1000 mg kg−1 Ni, Cu, Se, As and Al, or >10 000 mg kg−1 Zn and Mn. For ethical reasons this field would be likely limited to invertebrates.

to reduce nutrient pollution from waste salmon feed (Stirling and Okumus, 1995; Neori et al., 2004). Oysters have been found to reduce the levels of nitrogen and phosphorus in shrimp effluent by 72 % and 86 %, respectively (Jones et al., 2001), while oysters and clams reduce turbidity and chlorophyll a concentrations in fish farm effluent by 68 % and 79 %, respectively (Shpigel et al., 1997). Accordingly, the deployment and harvest of shellfish has been proposed in Sweden (Haamer, 1996) and America (Rice, 2001) to mitigate anthropogenic nutrient input to coastal waters. In general, these bivalves have been cultivated with human consumption in mind, although this need

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not always be the case. At an estuary level, the cultivation and harvest of pearl oysters (Pinctada imbricata) could potentially balance the nitrogen input of a sewage treatment plant (Gifford et al., 2005) (see case study, Section 23.3). Recently, interest has arisen over the use of sponges for bioremediation of aquatic microorganism pollution (Milanese et al., 2003; Fu et al., 2006). Sponges have a renowned filtering capacity and, in large communities, filter the overlying water column in as little as 24 h (Reiswig, 1974), with high particle retention rates (Milanese et al., 2003) and potential for economic gains via bath sponge material (Stabili et al., 2006b) or novel metabolites (Hadas et al., 2005) (see Chapter 28). A recent European study demonstrated a successful trial of the marine sponge Chondrilla nucula as an environmental remediator of bacteria (Milanese et al., 2003). This study estimated that a 1 m2 patch of this sponge can retain up to 7 × 1010 E. coli cells and filter 14 L h−1 of water. A similar Chinese study investigated the potential of the marine sponge Hymeniacidon perleve to remediate E. coli and Vibrio anguillarum II, with the sponges filtering up to 8 × 107 E. coli cells h−1 per g fresh sponge (Fu et al., 2006). Polychaete culture is well established, providing fishing bait for anglers and feed used for fish and shrimp broodstocks in aquaculture (Oliver et al., 1991). Polychaetes have been suggested as environmental remediators of microbial pollution, with Sabella spallanzanii and Branchiomma luctuosum demonstrating retention efficiencies of 70 % and 98 % of Vibrio alginolyticus (Licciano et al., 2005). It was estimated that a standing stock of 250 000 worms (S. spallanzanii) could be used to remediate the waste suspended particulate matter from a 50 t y−1 fish farm, producing about 50 kg d.w. of worm material annually, suitable as bait or as the basis of fish feed (Giangrande et al., 2005). Food fish culture in wastewater has been widely practised for more than a century (Edwards and Pullin, 1990) but, more recently, several studies have investigated the feasibility of employing traditional practices for ornamental species to ameliorate wastewater. Referred to as integrated wastewater aquaculture (IWA), the viability of the culture of ornamentals such as rainbow fish (Melanotaenia fluviatilis; Kumar and Sierp, 2003) and goldfish (Carassius auratus; Gavine and Gooley, 2007) has been assessed. Kumar and Sierp (2003) found that rainbow fish can be cultured in wastewater and were more tolerant of higher nutrient levels (N) than some ‘food’ fish, e.g. silver perch (Bidyanus bidyanus). Gavine and Gooley (2007) suggested that the most suitable species among those studied for IWA is carp (Cyprinus carpio), due to greatest biomass increases, but free-range polyculture with goldfish is also feasible. Goldfish cultured in dams receiving wastewater inputs exhibited the fastest growth rates of all the species examined, but mortality was high. In the dams stocked with fish, numerous water quality parameters were improved compared with initial wastewater inputs. Most notably, significant decreases in coliforms, total phosphorus and total

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nitrogen were observed. Results suggest a productive use of urban wastewater, improvement in water quality, with increase in fish biomass for human consumption or alternative uses such as fish supply for the aquaria trade.

23.2.2 Zoostabilisation/degradation of nutrients and microorganisms Many filter-feeding animals act as benthic–pelagic couplers, where they actively transfer energy and nutrients from the water column to the benthos. In one of the most high-profile examples, Newell (1988) proposed that large-scale ecological changes in Chesapeake Bay due to eutrophication could have arisen from over-harvesting of oyster biomass. Newell contrasted the filtration capacity of the 1880 standing stock of oysters with that of 1988 and concluded it would have taken 3.6 days and 228 days, respectively, to filter the entire water column of the bay. This finding has led to a concerted effort to re-establish oyster bars in many areas of the USA for ecological reasons (Coen and Luckenbach, 2000; Kirby and Miller, 2005), with the largest, the ‘Chesapeake 2000 Agreement’, committing various stakeholders to a ten-fold increase in native oysters in the Chesapeake Bay by 2010, at a cost of US$100 million (CBPFAC, 2002). Recently the potential for success of the existing oyster restoration efforts has been questioned. Mann and Powell (2007) have argued that the remaining options for the Chesapeake oyster resource lies with husbandry, and that it is the economic deliverables accompanying aquaculture that are required rather than self-sustaining ecological goals. Other examples of the potential for filter feeders to act as ‘ecological engineers’ include the zebra mussel (Dreissenia polymorpha) and the Asiatic clam (Corbicula fluminea). Following the introduction of the zebra mussel, turbidity in Lake Erie decreased markedly, chlorophyll a concentrations reduced by 43 %, and mean sechi disc transparencies (a measure of turbidity) increased by 1.24 m (Leach, 1993). Meanwhile, Phelps (1994) reported that following establishment of the Asiatic clam in the Potomac River estuary, water quality improved substantially, with submerged aquatic vegetation that had been absent for 50 years reappearing. Subsequent fish and bird surveys revealed large increases in their respective populations. Following reductions in clam biomass, water quality declined and fish, bird and aquatic vegetation populations contracted. Evidence such as this has supported recent calls for the deliberate introduction of exotic bivalve mollusc species to aquatic ecosystems (Gottlieb and Schweighofer, 1996). In order to avoid the problems associated with the introduction of an invasive species, the use of native species is generally preferable unless it is certain that any exotic candidate species are non-invasive (see Section 23.4). In this regard, some debate has also surrounded the introduction of sterile (e.g. triploid) exotic species, although clear aquaculture and/or zooremediative incentives are required for continued maintenance of an effective population size.

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In addition to bivalve molluscs, zoostabilisation of nutrient and microorganism pollution is conceivable for polychaetes, sponges, and a variety of filter-feeding invertebrates. Here, maintenance or supplementation of wild populations of these organisms could be used to manage nutrient or microbial pollution in aquatic ecosystems. Recognition of the importance of these ecosystem services may aid in the conservation of these communities (Ostroumov, 2005). However, research in this area remains poorly developed in comparison to oyster reef conservation and would profit from increased endeavour.

23.2.3 Zooextraction of heavy metals While no definition has been suggested for an animal metal hyperaccumulator, we propose the use of plant definitions as a useful reference (Box 23.1). A recent review by Gifford et al. (2004), focusing on bivalve molluscs, identified species which satisfy the plant definition of a hyperaccumulator for Cu (Crassostrea virginica, 2013 mg kg−1 Cu), Pb (Mytilus edulis, 506 mg kg−1 Pb), Cd (Pinctada albina albina, 108 mg kg−1 Cd) and Al (Crassostrea rhizophorae, 2240 mg kg−1 Al) and approached this status for Zn (Crassostrea virginica, 9077 mg kg−1 Zn). This phenomenon of hyperaccumulation is well known, and many such animals are presently used in various large-scale environmental monitoring programs (O’Connor, 2002). Das and Jana (2003) investigated the potential for the freshwater bivalve Lamellidens marginalis as a biofilter of Cd pollution in India, demonstrating a bioconcentration factor (BCF, the ratio of concentration within the organism to the exposure concentration) for Cd of up to 347 and a dry weight Cd concentration exceeding 500 mg kg−1 (Jana and Das, 1997; Das and Jana, 2003). Some metal hyperaccumulating animals offer non-food economic returns. Gifford et al. (2005) demonstrated that each tonne of pearl oyster (Pinctada imbricata) harvested resulted in 703 g metals removed from an estuary on the east coast of Australia (see case study Section 23.3). In further work our research group investigated uptake of Pb and Zn by pearl oysters under controlled laboratory conditions. Pearl oysters exposed to 90 μg L−1 of each metal accumulated 601 mg kg−1 and 209 mg kg−1 Pb as well as 4421 mg kg−1 and 54 mg kg−1 Zn in the soft tissue and shell, respectively (Gifford et al., 2006). On-going work will assess the effects of selected pollutants on pearl quality, with the aim of developing a model that optimises environmental and economic outcomes. The sponges represent a diverse group of animals offering the potential for non-food economic returns that have yet to be explored as metal bioremediators. Sponges are exposed to many metal pollutants within aquatic ecosystems. Due to their renowned filtration capacity, they are known metal bioaccumulators (Bargagli et al., 1996; Cebrian et al., 2003), and have a history of use as reliable biomonitors of marine pollution (Hansen et al., 1995; Perez et al., 2005). Indeed, the little work carried out on sponges

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indicates that they meet the definition of hyperaccumulators for Cd: Halichondria panicea, 271 mg kg−1 (Cd, Cebrian et al., 2003) and Tedania charcoti, 15 000 mg kg−1 Cd (Capon et al., 1993). These characteristics, combined with recent interest in sponges as a source of novel pharmaceuticals and bioactive compounds (Licciano et al., 2005; de Voogd, 2007), indicate the possibility for a self-financing remediation program. Conceivably, other animals such as bryozoans, polychaetes, and ascidians, which are known to accumulate vanadium (Kawakami et al., 2006), could be used as environmental remediators of metals, and may also offer the potential for farming of novel chemical compounds (Box 23.2).

Box 23.2

Self-financing zooremediation models

The cost of environmental remediation programs can often be prohibitive, thus the development of ‘profitable’ remediation programs would enhance their use. Specialised animals that could function as a remediation model while at the same time producing a valuable economic product include: Pearl oysters: The pearls produced by pearl oysters are an ideal economic offset against the costs of remediation. They are easily stored, are not food products and have a high market value. Pearl oysters have been demonstrated to be effective nutrient remediators; however, further work is necessary to determine the effects of metals and organic pollutants on pearl quality prior to deployment against these pollutants. Sponges: As with pearl oysters, sponges provide an ideal opportunity for profitable zooremediation. Many sponge metabolites are in high global demand and fetch strong prices. For certain sponge taxa, bath sponge material offers an alternative economic return for programs aimed at nutrient and microorganism pollution (the use of chemically exposed bath sponge material is unlikely to be accepted in the market). However, culture of sponges is not as advanced as mollusc culture, and further work is required to demonstrate whether any effects exist on the metabolites of economic interest following pollutant exposure. Sponges have been successfully deployed as zooremediators of microbial pollution. Edible molluscs: Edible molluscs are well established as zooremediators of nutrient pollution. However, as products of human consumption, great care needs to be maintained to either depurate them of pollutants prior to sale, or to culture the organisms in estuarine locations not impacted by other pollution sources such as micro-organisms or metals. Nonetheless, increasing mollusc culture in estuaries is an economically advantageous method of nutrient stabilisation/reduction in estuaries suffering eutrophication.

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Fishes: ‘Integrated wastewater aquaculture systems’ or ‘integrated agriaquaculture systems’ have for some time made use of nutrient-rich wastewater sources such as sewage and animal wastes to produce fish as a food source. Systems involving species such as carp are common in Asia and areas of Europe where they significantly reduce nutrient loads and microorganisms in nutrient enriched ponds. In some cases contaminant concerns restrict this practice for food production; however, the potential for production of ornamental and aquarium fish is unaffected. Gastropod molluscs, bryozoans, ascidians: There is current interest within the bioprospecting field investigating the pharmaceutical value of novel gastropod secondary metabolites. It is possible that some of these economic molluscs may possess attributes amenable to concomitant bioremediation such as the ability to accumulate or break down pollutants.

As with phytoremediation, there is a need for adequate treatment of harvested metal-laden animal biomass. Fortunately, systems are presently in use for the recovery of Cd in waste scallop tissue (Ghimire et al., 2008). In scallops, only the muscle and the gonad are eaten, while the remainder of the organism preferentially accumulates natural sources of Cd from marine waters and this tissue is removed and discarded from the animal prior to sale. As such, there has been a need to develop systems to properly handle the estimated 400 000 t Cd contaminated scallop waste generated in Japan through scallop processing (Seki and Suzuki, 1997; Shiraishi et al., 2003). The Cd is harvested from the scallop waste prior to being reused in a nearby car battery plant, while the scallop tissue now free of Cd is used as fertiliser.

23.2.4 Zooextraction of organic pollutants While the deployment and harvest of animals that hyperaccumulate organic pollutants is yet to be trialed, the use of sponges and fish has potential. Spongia officinalis is known to concentrate many organic contaminants including PCBs to higher concentrations than bivalve molluscs (BCF of approximately 105) (Perez et al., 2003). Thus significant quantities of PCBs could potentially be removed from aquatic environments upon harvest of sponge tissue. Recently fish have been proposed for zooextraction of PCBs and DDT (MacKenzie et al., 2004). Here, the authors propose that by not discarding overboard fish waste such as cod liver, Baltic Sea fisheries could remove 31 kg y−1 PCBs from the Baltic ecosystem. This amount compares to an annual influx of some 260 kg of PCBs, and would remove more from

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the ecosystem than any other budgetary component (such as degradation in the water column).

23.2.5 Zoostabilisation/degradation of organic pollutants Several examples exist in the literature of the use of animals to degrade organic contaminants to less toxic by-products. Gudimov (2002) reported that degradation of oil was accelerated 10–20 times in the presence of Mytilus edulis. Similarly, the sponge Spongia officinalis is able to degrade the surfactant 1-(p-Sulfophenyl)nonane to its main degradation products, 3-(p-sulfophenyl)propionic acid and p-sulfobenzoic acid, ten times more rapidly than other taxa such as marine bacteria (Perez et al., 2002), the first evidence of pollutant degradation by a sponge. In addition, there is some evidence that this sponge is able to degrade the PCB CB138 (International Union of Pure and Applied Chemistry, IUPAC) (Perez et al., 2003). It is likely that many sponges are able to break down organic pollutants, particularly given their ability to produce and safely store many halogenated biomolecules within the cell. Recent research by our group has suggested a role for oysters in the reduction of estrogenic compounds in estuarine waters arising from a range of anthropogenic sources (Andrew et al., 2008). The oyster Saccostrea glomerata was capable of rapidly metabolising 17α-ethynylestradiol, when exposed to concentrations as great as 50 ng L−1, equivalent to the maximum level reported in UK sewage effluent (Desbrow et al., 1998). In addition, the differential accumulation of organic pollutants observed in molluscs could be used for zoostabilisation. The gastropod Austrocochlea constricta accumulated short-chain aliphatic hydrocarbons (C14-C18) in the soft tissue, whereas longer-chain aliphatic hydrocarbons (C20-C30) tended to accumulate in the shell (Walsh et al., 1995). The authors proposed that longer-chain compounds were isolated from metabolically active tissue and stored in the shell of the organism. As such, it is conceivable that certain contaminants could be remediated via isolation from trophic transfer by harvest and shell burial.

23.3 Zooremediation and pearl aquaculture: a case study Although zoodegradation is conceivable, perhaps the most appropriate form of remediation possible for commercial aquaculture is zooextraction of contaminants. Furthermore, aquaculture initiatives yielding commercial products which are not impacted by accumulated contaminants would be most desirable. In this manner, zooextraction occurs as a concomitant byproduct of the aquaculture process. Metal bioremediation of coastal waters has historically included the culture and harvest of various filter-feeding bivalve molluscs (Haamer, 1996;

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Soto and Mena, 1999; Rice, 2001; Landry, 2002). Pearl oysters have several advantages over other bivalve species that have led to our group proposing their use as an environmental remediation tool (Gifford et al., 2004). The cosmopolitan distribution of many species of pearl oyster, such as the Akoya Pearl oyster, Pinctada imbricata, Röding 1798 (Colgan and Ponder, 2002), allows the possibility of utilising an endemic species for remediation in many locales. The high protein content of P. imbricata (Suzuki, 1957; Seki, 1972) implies that a greater nitrogen yield per tonne oyster flesh is likely. The fact that the profit is obtained from the pearl, and the flesh is not necessarily bound for human consumption (unlike many cultured molluscs), allows deployment to potentially reduce nutrients, metals and other forms of recalcitrant pollution in waterways. Finally, the commercial return via pearling operations may be used to offset the costs of remediation (disposal or recycling of contaminated product). Our initial studies have assessed the nitrogen, phosphorus and metal content of pearl oyster tissue and shell harvested from a small commercial pearl farm and evaluated the potential to offset inputs to an estuary from a local sewage treatment plant (STP), in Port Stephens, Australia. In total, 7.5 kg N and 0.55 kg P was harvested per tonne of oyster material. As the total August harvest of 2003 was 9796 kg oyster material, the total amount of nutrients removed from the Port Stephens environment in the year 2003 via pearling operations was 73 kg N and 5.3 kg P. This harvest also removed approximately 1413 g of a suite of heavy metals from the estuary, including 539 g Zn and Al, 276 g Fe, 10 g As, and 6 g Cd in soft tissue. The yearly harvest removed approximately 5469 g metals from the waters of Port Stephens via pearl shell harvest, dominated by 4955 g of Sr within the shell matrix; however, some 257g Fe, 121 g Mn, 81 g Zn, and 42 g Al were also sequestered in the shell. In total 6882 g of selected metals were removed. As the 2003 oyster harvest was 9796 kg, this equates to approximately 0.7 kg metals per tonne oyster material harvested (Table 23.1). It is important to note that these removal rates do not indicate the maximum potential for pearl aquaculture as an environmental remediation technology, particularly in regard to metal loadings. The concentrations of metals in the oyster tissue recorded in this study represent site-specific loadings of the relatively uncontaminated Port Stephens environment. If oysters are deployed in metal impacted sites, tissue and shell loadings would be predicted to increase (Gifford et al., 2004). The oyster material could then be treated to recover the metals prior to further use or disposal, based on the scallop waste model mentioned previously (Section 23.2.3). Pearling operations could also assist in remediating the nitrogen inputs from the STP within Port Stephens. Given that the average nutrient inputs to the port from the STP for the years 1999–2000 to 2001–2002 were 3741 kg N and 2967 kg P (NSW EPA, 2000, 2001, 2002), the existing pearl farm harvesting 9.8 t y−1 oyster material would have to expand production approximately 51 times to 499 t y−1 to balance the nitrogen from the STP.

Shell (mg kg−1) 3900 ± 200 268 ± 18.4 0.01 ± 0.01 13.3 ± 1.92 42.25 ± 5.89 BDL BDL BDL BDL 0.86 ± 0.19 6.89 ± 2.32 814 ± 22.6 BDL 1.25 ± 0.15 19.9 ± 1.59 BDL

Tissue (mg kg−1)

98200 ± 700 7400 ± 500 1.08 ± 0.29 1076 ± 68.5 551 ± 33.9 BDL BDL 20.2 ± 1.19 11.9 ± 0.68 BDL 1076 ± 80.5 51.7 ± 6.02 BDL 1.36 ± 0.14 30.9 ± 2.72 1.15 ± 0.26 49 193 3707 0.63 539 276 BDL BDL 10.1 5.96 BDL 539 25.9 BDL 0.68 15.5 0.58 1413

Tissue remediation (g) 23 739 1631 0.06 80.9 257 BDL BDL BDL BDL 5.23 41.9 4955 BDL 7.61 121 BDL 5469

Shell remediation (g)

72 932 5338 0.69 620 533 BDL BDL 10.1 5.96 5.23 581 4981 BDL 8.29 137 0.58 6882

Gross remediation (g)

Note: Calculation for remediation is as follows: The wet tissue material is, on average, 37.8 % total weight of the animal. The dry weight of this material is 13.5 % (N = 20) of the mass of the wet weight. Therefore, every tonne of P. imbricata harvested will be composed of 622 kg of shell material and 378 kg of tissue. This 378 kg of tissue will have a dry mass of 51.19 kg. The 2003 harvest was approximately 9786 kg oyster material. BDL = below detection limit.

N P Cu Zn Fe Pb Ni As Cd Cr Al Sr U V Mn Se TOTAL (metals)

Pollutant

Table 23.1 Nitrogen, phosphorus and metal concentration of various dried tissues from Pinctada imbricata (N = 20) and the remediation resulting from 2003 oyster harvest

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An increase of this size would result in 272 kg P being removed from the port’s waters. Since 2003, additional lease area has been granted and pearl densities have increased so that the potential harvest has increased almost five-fold. Indeed, if as proposed, existing unused edible oyster lease areas were used for production of pearl oysters to a size suitable for pearl production, harvest could increase ten-fold. However, this remains small and sparsely stocked in comparison to many other commercial bivalve operations and would still be small in comparison to the estimated 591 t of edible oysters produced in the port. The efficiency of pearl oyster remediation is variable as a function of oyster condition, as nutrient content varies over the season and the oysters in our study were harvested in the middle of winter when flesh condition was low (O’Connor and Lawler, 2003). It is therefore likely that nutrient yield would be substantially improved by harvesting the oysters at a time prior to the Southern hemisphere winter, such as May. Provided pearl quality is satisfactory, some manipulation of pearl harvest dates is possible due to the ease of pearl storage, when compared with the hygiene issues surrounding edible bivalve harvest and storage. Further information is required on the tolerance of P. imbricata to various contaminants and contaminant mixtures; accumulation, equilibration, and depuration rates for contaminants of interest; temporal variation in accumulation of contaminants balanced with seasonal growth and deployment times required to produce quality pearls; and the potential effect of contaminants on pearl quality arising. Further, investigations are required to assess potential impacts of pearl oyster aquaculture on benthos beneath leases due to biodeposition of faecal and pseudofaecal material, at the stocking densities required to effectively decrease contaminant load. While acknowledging these unknowns, initial findings suggest that pearl aquaculture could form part of an overall coastal management strategy, particularly one aimed at maintaining water quality of coastal ecosystems whilst encouraging sustainable aquaculture.

23.4 Future trends Animals may be employed to extract or stabilise nutrient, microbial, heavy metal, and organic pollution. This can be achieved via the harvesting of wild populations or culture of animals to extract pollutants and/or the supplementation or maintenance of wild populations to stabilise/degrade pollutants. However, many questions remain to be addressed before zooremediation can be reliably adopted. The successful harvest of wild animal taxa for zooextraction of pollutants requires a clear understanding of the population dynamics of candidate taxa to ensure a sustainable harvest. The successful supplementation of wild animal populations for zoostabilisation with introduced species requires an understanding of the risk of candidate taxa

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perturbing local ecological communities. In addition, specific care must be exercised when contemplating the addition of exotic species in order to avoid the risk of introducing invasive species. A sound understanding of temporal accumulation dynamics (accumulation, equilibration, depuration) is paramount to optimising extraction efficiencies (Box 23.3). Furthermore, it is generally preferable to focus zooremediation initiatives on invertebrate taxa in order to minimise ethical concerns (Box 23.4). The use of animal taxa for zooremediation is likely to trigger significant community and governmental interest. To formulate sound decisions, quality scientific data must be available. A suitable model is the current debate surrounding the introduction of the non-native Asian oyster Crassostrea ariakensis to Chesapeake Bay, USA. Here, a whole of government approach and the commissioning of a report by the National Academy of Sciences (NRC, 2003) has recommended that too little is known about the biology of C. ariakensis to confidently assert that its introduction to US waters would be successful. Thus in reality, it is likely that the research

Box 23.3 Checklist for candidate zooremediation species Zoostabilisation

Zooextraction

Trait Accumulate pollutant Resistant to toxicity Non-invasive Rapid growth rate Relatively sedentary Ease of culture Population dynamics known Uptake dynamics known Knowledge of carrying capacity Disease risks understood

Nutrients

Metals

Organics

Nutrients

Metals

Organics

*

*

*

*

*

*

*

*

*

*

*

*

* *

* *

* *

* *

* *

* *

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

* Indicates that trait is required for sustainable zooremediation.

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Box 23.4 The ethics of employing animals for zooremediation The employment of animals for bioremediation initiatives is likely to trigger greater ethical concern from the community and decision makers than the use of plants or microorganisms. In this context, it is important to recognise that any employment of animals for zooremediation initiatives must be based on sound ethical principles. In many jurisdictions, the term animal refers to ‘all live non-human vertebrates’, thus most guidelines do not relate to invertebrate species. It is likely that the use of invertebrate animal species such as polychaetes, sponges and molluscs will meet little resistance and satisfy community ethical standards. However, this does not preclude the use of vertebrate species such as fish if it can be demonstrated that the ethics of such zooremediation programs conform to current best practice animal husbandry guidelines.

burden of introducing non-native species for zoostabilisation programs will support the use of native animal taxa in this role. Various forms of aquaculture (pearl oysters, sponges, gastropod molluscs, bryozoans, ascidians, and even edible molluscs in certain scenarios (Box 23.2)) hold greatest promise for zooremediation (zooextraction) of contaminated aquatic systems. However, the introduction, culture, and harvest of animal taxa for zooremediation requires detailed knowledge on the biological requirements for successful husbandry practices. In addition, knowledge of factors governing optimal carrying capacity, disease and parasitic risks, and impacts on surrounding ecology are necessary in order to successfully establish zooextractive aquaculture operations (Box 23.3). Zooremediation (specifically zooextraction) is perhaps most appropriate for aquaculture initiatives where the profit-generating product and its marketability are not impacted by accumulated contaminants, especially where the economic return is sufficient to offset the significant costs of postharvest remediation technologies (disposal, degradation, or recycling of contaminants within product). Pearl aquaculture is one such initiative where this approach may be feasible in the future, as initial studies suggest. Before such practice gains acceptance, further research is required to establish tolerance, temporal accumulation dynamics for optimal extraction efficiency, possible negative effects on pearl quality, and consumer perception. Finally, it may be some time before aquaculture ventures are financially rewarded for the zooremediative outcomes, but increased understanding and broader acceptance of the potential ecological benefits of specific aquaculture ventures is required. Further, the encouragement of suitable

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ventures could be more actively considered and incentives developed to include aquaculture as part of an overall pollutant remediative strategy.

23.5 Sources of further information and advice Aspects of this work were presented at the 3rd European Bioremediation Conference, 2005. This chapter is a modified version of earlier publications including Gifford et al. (2005) Marine Pollution Bulletin, 50, 417–22, and Gifford et al. (2007) Trends in Biotechnology, 25, 60–65. Reproduced with permission from Elsevier Press.

23.6 References andrew m n, dunstan r h, o’connor w a, van zwieten l and macfarlane g r (2008) Effects of 4-nonylphenol and 17α-ethynylestradiol exposure in the Sydney rock oyster, Saccostrea glomerata: Vitellogenin induction and gonadal development, Aquat Toxico, 88, 39–47. bargagli r, nelli l, ancora s and focardi s (1996) Elevated cadmium accumulation in marine organisms from Terra Nova Bay (Antarctica), Polar Biol, 16, 513–20. capon r j, elsbury k, butler m s, lu c c, hooper n a r, rostas j a p, o’brien k j, mudge l m and sim a t r (1993) Extraordinary levels of cadmium and zinc in a marine sponge, Tedania charcoti: inorganic chemical defense agents, Experientia, 49, 263–4. carmona r, kraemer g p and yarish c (2006) Exploring Northeast American and Asian species of Porphyra for use in an integrated finfish-algal aquaculture system, Aquaculture, 252, 54–65. cebrian e, marti r, uriz j m and turon x (2003) Sublethal effects of contamination on the Mediterranean sponge Crambe crambe: metal accumulation and biological responses, Mar Poll Bull, 46, 1273–84. cbpfac (2002) Recommendations on Suminoe oyster (Crassostrea ariakensis) aquaculture in Chesapeake Bay, Chesapeake Bay Program Federal Agencies Committee, Annapolis, MD. chung y h, kang c, yarish g, kraemer and lee j (2002) Application of seaweed cultivation to the bioremediation of nutrient-rich effluent, Algae, 17, 187–94. coen l d, and luckenbach m w (2000) Developing success criteria goals for evaluating oyster reef restoration: ecological function or resource exploitation, Ecol Eng, 15, 323–43. colgan d j and ponder w f (2002) Genetic discrimination of morphologically similar, sympatric species of pearl oysters (Mollusca: Bivalvia: Pinctada) in eastern Australia, Mar Freshw Res, 53, 697–709. das s and jana b (2003) In situ cadmium reclamation by freshwater bivalve Lamellidens marginalis from an industrial pollutant-fed river canal, Chemosphere, 52, 161–73. desbrow c, routledge e j, brighty g c, sumpter j p and waldock m (1998) Identification of estrogenic chemicals in STW effluent. 1. Chemical fractionation and in vitro biological screening, Environ Sci Technol, 32, 1549–58.

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de voogd n j (2007) The mariculture potential of the Indonesian reef-dwelling sponge, Callyspongia biru: growth, survival and bioactive compounds, Aquaculture, 262, 54–64. edwards p and pullin r s v (1990) Wastewater-fed Aquaculture, Proceedings of the International Seminar on Wastewater Reclamation and Reuse for Aquaculture, 6–9 December, 1988, Calcutta, Environmental Sanitation Information Center, Asian Institute of Technology, Bangkok. fao (2005). FishStat Plus, Food and Agriculture Organization of the United Nations, Rome, http://www.fao.org/fi/statist/fisoft/fishplus.asp, accessed January 2009. fu w t, sun l, zhang x and zhang w (2006) Potential of the marine sponge Hymeniacidon perleve as a bioremediator of pathogenic bacteria in integrated aquaculture systems, Biotechnol Bioeng, 93, 1112–22. gavine f m and gooley g j (2007) Adding value to recycled water through aquaculture – opportunities for large-scale implementation in Australia, Ozwater Convention & Exhibition, 4–8 March Sydney. ghimire k n, huang k, inoue k, ohto k, kawakita h, harada h and morita m (2008) Heavy metal removal from contaminated scallop waste for feed, Biores Technol, 99, 2436–41. giangrande a a, cavallo a, licciano m, mola e, pierri c and trianni l (2005) Utilisation of the filter-feeder polychaete Sabella spallanzii Gmelin (Sabellidae) as bioremediator in aquaculture, Aquac Int, 13, 129–36. gifford s, dunstan r h, o’connor w, roberts t and toia r (2004) Pearl aquaculture: profitable environmental remediation? Sci Total Environ, 319, 27–37. gifford s, dunstan r h, o’connor w and macfarlane g r (2005) Quantification of in situ nutrient and heavy metal remediation by a small pearl oyster (Pinctada imbricata) farm at Port Stephens, Australia, Mar Poll Bull, 50, 417–22. gifford s p, macfarlane g r, o’connor w a and dunstan r h (2006) Effect of the pollutants lead, zinc, hexadecane and octocosane on total and shell growth in the Akoya Pearl Oyster, Pinctada imbricata, J Shellfish Res, 25, 159–65. gottlieb s j and schweighofer m e (1996) Oysters and the Chesapeake Bay ecosystem: a case for exotic species introduction? Estuaries, 19, 639–50. gudimov a v (2002) Zooremediation, a new biotechnology solution for shoreline protection and cleanup, In Proceedings of the 25th Arctic and Marine Oilspill Program, 401–12. haamer j (1996) Improving water quality in a eutrophied fjord system with mussel farming, Ambio, 25, 356–62. hadas e, shpigel m and ilan m (2005) Sea ranching of the marine sponge Negombata magnifica (Demospongiae, Latrunculiidae) as a first step for latrunculin B mass production, Aquaculture, 244, 159–69. hansen i v, weeks j m and depledge m h (1995) Accumulation of copper, zinc, cadmium and chromium by the marine sponge Halichondria panicea Pallas and the implications for biomonitoring, Mar Poll Bull 31, 133–8. jana b and das s (1997) Potential of freshwater mussel (Lamellidens marginalis) for cadmium clearance in a model system, Ecol Eng, 8, 179–93. jones a b, dennison w c n and preston p (2001) Integrated treatment of shrimp effluent by sedimentation, oyster filtration and macroalgal absorption: a laboratory scale study, Aquaculture, 193, 155–78. kawakami n, tatsuya ueki t, matsuo k, gekko k, and michibata h (2006) Selective metal binding by Vanabin2 from the vanadium rich ascidian, Ascidia sydneiensis samea, Biochim Biophys Acta, 1760, 1096–101. kirby m x and miller h m (2005) Response of a benthic suspension feeder (Crassostrea virginica Gmelin) to three centuries of anthropogenic eutrophication in Chesapeake Bay, Estuar Coast Shelf S, 62, 679–89.

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kumar m s and sirep m (2003) Integrated Wastewater Treatment and Aquaculture Production, RIRDC Publication No 03/012 Project No MFR-16A, Rural Industries Research and Development Corporation, Barton, ACT. landry t (2002) The potential role of bivalve shellfish in mitigating negative impacts of land use on estuaries, in Cairns, D K (ed.), Effects of Land Use Practices on Fish, Shellfish, and their Habitats on Prince Edward Island, Canadian Technical Report of Fisheries and Aquatic Sciences, 2408, 155–7. leach j h (1993). Impacts of the zebra mussel (Dressena polymorpha) on water quality and fish spawning reefs in western Lake Erie, in, Nalepa T F and Schloesser D W (eds), Zebra mussels: biology, impacts, and control, Lewis, Boca Raton, FL, 381–97. licciano m, stabili l and giangrande a (2005) Clearance rates of Sabella spallanzii and Branchiomma luctuosum (Annelida: Polychaeta) on a pure culture of Vibrio alginolyticus, Water Res, 39, 4375–84. mackenzie b r, almesjo l and hansson s (2004) Fish, fishing, and pollutant reduction in the Baltic Sea, Environ Sci Technol, 38, 1970–76. mann r and powell e n (2007) Why oyster restoration goals in the Chesapeake Bay are not and probably cannot be achieved, J Shellfish Res, 26, 905–17. mcvey j p, stickney r, yarish c and chopin t (2002) Aquatic polyculture and balanced ecosystem management: new paradigms for seafood production, in Stickney R R and McVey J P (eds), Responsible Aquaculture, CABI, Oxford, 91–104. milanese m, chelossi e, manconi r, sara a, sidri m and pronzato r (2003) The marine sponge Chondrilla nucula Schmidt, 1862 as an elective candidate for bioremediation in integrated aquaculture, Biomol Eng, 20, 363–8. nrc (2003) Non-native oysters in the Chesapeake Bay, National Academies Press, Washington, DC. neori a, chopin t, troell m, bushmann a h, kraemer g p, halling c, shpigel m and yarish c (2004) Integrated aquaculture: rationale, evolution and state of the art emphasising seaweed biofiltration in modern mariculture, Aquaculture, 231, 361–91. newell r i e (1988) Ecological changes in Chesapeake Bay: Are they the result of overharvesting the American oyster, Crassostrea virginica?, in Lynch M P and Krome E C (eds), Understanding the Estuary: Advances in Chesapeake Bay Research, Chesapeake Research Consortium, Edgewater, MD, 536–46. nsw e p a (2000) Hunter water corporation environmental protection licence annual return Tanilba Bay wastewater treatment works, New South Wales Environmental Protection Agency, Sydney, NSW. nsw e p a (2001) Hunter water corporation environmental protection licence annual return Tanilba Bay wastewater treatment works, New South Wales Environmental Protection Agency, Sydney, NSW. nsw e p a (2002) Hunter water corporation environmental protection licence annual return Tanilba Bay wastewater treatment works, New South Wales Environmental Protection Agency, Sydney, NSW. o’connor t p (2002) National distribution of chemical concentrations in mussels and oysters in the USA, Mar Environ Res, 53, 117–43. o’connor w a and lawler n f (2003) Reproductive condition of the pearl oyster, Pinctada imbricata (Roding) in Port Stephens, NSW, Australia, Aquac Res, 35, 1–12. olguin e j (2003) Phycoremediation: key issues for cost-effective nutrient removal processes, Biotechnol Adv, 22, 81–91. oliver p j w, bury n, cowin p b d and smithard r r (1991) Commercial production of polychaetes for angling: implications for mainstream aquaculture, in De Pauw

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N and Joyce J (comps), Aquaculture and the Environment, Special Publication no. 14, European Aquaculture Society, Oostende, 241–2. ostroumov s (2005) Some aspects of water filtering activity of filter feeders. Hydrobiologia, 542, 275–86. perez t, sarrazin l, rebouillon p and vacelet j (2002) First evidences of surfactant biodegradation by marine sponges (Porifera): an experimental study with linear alkylbenzenesulfonate, Hydrobiologia, 489, 225–33. perez t, wafo e, fourt m and vacelet j (2003) Marine sponges as biomonitor of polychlorobiphenyl contamination: Concentration and fate of 24 congeners, Environ Sci Technol, 37, 2152–8. perez t, longet d, schembri t, rebouillon p and vacelet j (2005) Effects of 12 years’ operation of a sewage treatment plant on trace metal occurrence within a Mediterranean commercial sponge (Spongia officinalis, Demospongiae), Mar Poll Bull, 50, 301–09. phelps h l (1994) The Asiatic clam (Corbicula fluminea) invasion and system-level ecological change in the Potomac River Estuary near Washington, D. C., Estuaries, 17, 614–21. reeves r d and baker a j m (2000) Metal-accumulating plants, in Raskin I and Ensley B D (eds), Phytoremediation of Toxic Metals: Using Plants to Clean up the Environment, Wiley, New York, 193–229. reiswig h m (1974) Water transport, respiration and energetics of three tropical marine sponges, J Exp Mar Biol Ecol, 14, 231–49. rice m a (2001) Environmental impacts of shellfish aquaculture: filter feeding to control eutrophication, in Tlusty M, Bengtson D, Halvorson H O, Oktay S, Pearce J and Rheault R B Jr (eds). Marine Aquaculture and the Environment: A Meeting of Stakeholders in the Northeast, Cape Cod Press, Falmouth, MA, 77–86, seki h and suzuki a (1997) A new method for the removal of toxic metal ions from acid-sensitive biomaterial, J Colloid Interf Sci, 190, 206–11. seki m (1972) Studies on environmental factors for the growth of the pearl oyster, Pinctada fucata, and the quality of its pearl under the culture condition, Bulletin of the Mie Prefectural Fisheries Experimental Stations, 32–143 (in Japanese with English summary and figures). shiraishi t, tamada m, saito k and sugo t (2003) Recovery of cadmium from waste of scallop processing with amidoxime adsorbent synthesised by graftpolymerisation, Radiat Phys Chem, 66, 43–7. shpigel m, gasith a and kimmel e (1997) A biomechanical filter for treating fish pond effluents, Aquaculture, 152, 103–17. soto d and mena g (1999) Filter feeding by the freshwater mussel, Diplodon chilensis, as a biocontrol of salmon farming eutrophication, Aquaculture, 171, 65–81. stabili l, licciano m, giangrande a, fanelli g and cavallo r a (2006a) Sabella spallanzii filter-feeding on a bacterial community: ecological implications and applications, Mar Environ Res, 61, 74–92. stabili l, licciano m, giangrande a, longo c, mercurio m, marzano c n and corriero g (2006b) Filtering activity of Spongia officinalis var adriatica (Schmidt) (Porifera, demospongiae) on bacterioplankton: implications for bioremediation of polluted seawater, Water Res, 40, 3083–90. stirling h p and okumus i (1995) Growth and production of mussels (Mytilus edulis L.) suspended at salmon marine cages and mussel farms in two sea lochs on the west coast of Scotland, Aquaculture, 134, 193–210. sursala s v, medina f and mccutcheon s c (2002) Phytoremediation: An ecological solution to organic chemical contamination, Ecol Eng, 18, 647–58. suzuki k (1957) Biochemical studies on the pearl oyster (Pinctada martensii) and its growing environments. I. The seasonal changes in the chemical components of

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the pearl oyster, plankton and marine mud, Bulletin of the National Pearl Research Laboratories, 2, 57–62 (in Japanese with English summary). walsh k, dunstan r h and murdoch r n (1995) Differential bioaccumulation of heavy metals and organopollutants in the soft tissue and shell of the marine gastropod, Austrocochlea constricta, Arch Environ Contam Toxicol, 26, 367–73.

24 Farming cod and halibut: biological and technological advances in two emerging cold-water marine finfish aquaculture species V. Puvanendran and A. Mortensen, Nofima Marin, Norway

Abstract: Both Atlantic cod (Gadus morhua) and Atlantic halibut (Hippoglossus hippoglossus) have been considered new candidates for aquaculture in North Atlantic countries including Norway, Canada, the UK, Iceland, and the USA since the 1980s. Differences in cultivation methods existed between these two species mainly due to the differences in life stages and their requirements of nutrition, tank dynamics, and environmental conditions. Initially, extensive cultivation methods were employed for cod and halibut using wild zooplankton; however, decades of research on intensive culture methods using rotifer and Artemia resulted in development of improved hatchery protocols. Improvements have been made in recent years in broodstock management for both species, which resulted in improved egg quality. Year around production of eggs through photo-manipulation has been achieved for cod and halibut, which is a major boast for the industry. However, a bottleneck still exists regarding broodstock nutrition, which generally contributes to inconsistent quality of eggs and larvae. Breeding programs have already been initiated for cod in Norway, Canada, and Iceland, and Ireland will follow soon. No breeding programs are available for halibut; however, a genomics program has been initiated for halibut in Canada. Research activities in the early 1990s have resulted in improved egg and yolk sac larval survival and quality in halibut. Intensive larval rearing protocols are available for both species; however, consistent production of high-quality juveniles and survival are still elusive. Apart from poor broodstock management, dependence on cultured live feed, especially Artemia often contributed to poor larval and juvenile quality. However, attempts to replace live feed with formulated diets have been partly successful for cod, and currently most hatcheries in Norway are using only rotifers in their feed regime. Further improvements in feeds and feeding strategies are warranted to reduce the dependence on live feed further. Poor husbandry practices during larval stages could also result in major loss as the immune system of the larvae is underdeveloped. The on-growing phase of both species is more stable compared to the larval phase; however, disease outbreaks, both viral and bacterial, could provide major threats. More research should be conducted on developing vaccines for these disease problems. Escapes

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of cod from grow-out systems have become a major problem and more research is needed on cage technology and fish behaviour to minimize these escapes. Research is also needed to produce all-female stocks and sterile fish to avoid fertilized eggs escaping to the wild. Early maturation has been recognized as the major bottleneck in cod culture and light administration appears to have limited effectiveness in preventing early maturation. Production of sterile fish would be a solution not only for minimizing fish escapees interacting with wild fish but also for minimizing early maturation. Market development need to be addressed with an emphasis on product quality and food safety. Key words: alternate species, cod, halibut, production technology, broodstock management, larviculture.

24.1 Introduction Globally, aquaculture remains the fastest growing animal food sector (FAO, 2007), and over the decades, commercial aquaculture in the Northern hemisphere has primarily relied on salmon aquaculture. The most recent decline in salmon prices from 2001–2003 highlights the importance of diversification through introducing new species for culture. From the mid-1990s on, the Northern hemisphere has focused on developing indigenous species for diversification, with research concentrating on candidate species that have positive biological and economic attributes, e.g. simpler life stages, better flesh quality, established and/or higher market value (Le François et al., 2002). Several marine finfish species, such as cod, halibut, flounders, haddock and wolfish, were evaluated for their potential as appropriate aquaculture species since the 1990s. Most species were dropped and research since year 2000 has focused successfully on cod and halibut, which have a high market demand. The difficulties of the complicated life cycle of halibut, characterized by a very long yolk-sac development phase and early first feeding, are compensated for by its high market value, whereas cod, also has poorly developed newly hatched larvae but a relatively short yolk-sac stage (Fig. 24.1). The iconic status of cod as the premium white fish in the European and North American markets and declines in worldwide catches of cod have created a demand for product, which has accelerated cod production rapidly since 2003. The annual production of cultured cod is expected to be 20–30 000 tons in 2008 while the production of halibut is expected to reach 7–10 000 tons. Research on cod and halibut has been designed to progress towards commercialization from the initial fundamental research and pilot-scale commercial production. To achieve this, efforts have been undertaken by the scientific community with the support of the industry and funding agencies to develop the basic rearing protocol of these species. These efforts

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~15 days 5–6 °C 2–3 years (2–3 kg)

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~15 days 5–6 °C

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70–85 days

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Start feeding larval stage

Fig. 24.1 Life cycles of Atlantic cod and halibut in captivity. (Pictures of cod broodstock and eggs are provided by Lynn Lush of Department of Fisheries and Oceans, Canada and halibut pictures are provided by Brian Blanchard of Scotian Halibut Ltd., Canada)

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resulted in developing the basic production protocols for these two species; however, problems still exist in achieving consistent high survival of goodquality juveniles. The focus was then shifted to other important issues such as culture techniques, nutrition, selective breeding, cage technology, product quality, and marketing. In this chapter, we will discuss progress made by the scientific community and industry since the 1990s, starting from basic research to current technology.

24.1.1 Current and past research activities A review on cod aquaculture research activities from the mid-1980s until 1990 was presented by Tilseth (1990) while a special issue on halibut culture published in Aquaculture Research in 1998 (van der Meeren (ed.), Volume 29, issue 12) reviewed the halibut culture research activities from the 1970s to 1998.

24.2 Atlantic cod The current supply of Atlantic cod from capture fisheries has declined to one million tonnes per year from its peak of four million tonnes in 1969. All the cod stocks in the North Atlantic are either over exploited or in decline and quotas have been reduced in all jurisdictions. The Northern Cod stock fishery off Newfoundland has been under moratorium since 1992, with the stock recently being listed as a threatened species by the Committee on the Status of Endangered Wildlife in Canada (COSWIC). Since the recovery of cod stocks does not look promising and demand for white fish is increasing, interest in cod aquaculture has gained momentum. With the scarcity of wild cod in the market, the market price for cod increased, making intensive culture of cod more feasible compared to earlier attempts using capture-based aquaculture. Research on aquaculture of Atlantic cod started in Norway in the early 1980s concomitant with sharp declines in cod quotas. The first symposium on cod aquaculture was held on 1983 in Arendal, Norway. Initial attempts at rearing cod in an intensive environment met with limited success due to poor understanding of broodstock biology, environmental requirements, and larval nutrition. Subsequently, interest in cod aquaculture was reduced as the stocks rebuilt and by the mid-1990s focused research on cod aquaculture was also severely reduced. However, basic research on juvenile production still continued. Other than Norway, Newfoundland was the other major jurisdiction where cod aquaculture research has been an on-going activity since 1984. Initially in Newfoundland capture-based cod aquaculture was popular among cod fishermen from 1985 until 1995; however, this practice ceased due to lack of access to stock for grow-out. Early research focused on

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developing the economic parameters for successful cod aquaculture, including food and feeding, stocking density, disease management, vaccines, etc. With the decline in the fishery and the introduction of moratoria in the major areas where capture-based aquaculture was centred, the focus shifted to intensive aquaculture methods. Newfoundland researchers never experimented with either extensive or semi-intensive culture, believing that methods used in the Mediterranean for bass and bream would be easily adapted for use with cold-water species. The first commercial cod hatchery in Newfoundland was constructed in 1995 and produced 20 000 juveniles in 1996. Unfortunately this commercial activity ceased in 1997 when the hatchery was destroyed by fire. The success of intensive culture in Newfoundland spurred a significant investment in intensive cod aquaculture research in Norway, and subsequently significant investment in commercial production. Both Newfoundland and Scotland, the other major centre for cod aquaculture research, have fallen behind developments in Norway. Figure 24.2 shows the trend in cod juvenile production in Norway from 2002 to 2006 (Norwegian Fishery Directorate statistics 2007). The juvenile production, however, was expected to increase 5–6 fold in the next five years to 90 million juveniles (Rosenlund and Halldórsson, 2007). During this period of intensification, it became apparent that further research was needed in order to attain consistent production of cod fry in both numbers and quality. Although basic rearing protocols were developed, further research in broodstock, fine-tuning the existing production protocols, nutrition, disease management, product quality, on-growing, and market development are still needed.

16 14

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Cod aquaculture production in Norway from 2002 to 2006. (Source: Norwegian Fishery Directorate Statistics, 2007)

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24.2.1 Broodstock Most of the initial cod research focused on ensuring the mass production of juveniles for successful commercialization of cod aquaculture. Focus on broodstock research has received little attention, as egg supply is not limited. A single adult cod can produce millions of eggs in a breeding season. Cod are batch spawners that are easily maintained in tanks under light and temperature control, and spawn spontaneously in captivity. Floating eggs are collected via a surface out-flow using an egg collector situated externally to the tank, or in some cases collected within the broodstock tank using a suction collector. Cod generally spawn in March to June. For successful commercial production, eggs should be available throughout the year. Recent studies have shown that year-round egg production can be easily achieved by photoperiod manipulation alone (Penney et al., 2006a; van der Meeren and Ivannikov, 2006). In this method, the natural photoperiod is shifted so that the fish’s spawning may be advanced or delayed according to hatchery or environmental requirements. The quality of the eggs and larvae are not affected by this photomanipulation (Fig. 24.3; Penney et al., 2006a, b) which is a major advancement in cod aquaculture given that the cost involved is minimal but the return enormous. Currently, several cod hatcheries are using photomanipulation techniques to obtain year-round seed supply. Broodstock management is the bedrock of an aquaculture operation on which further production success depends. Until recently, it was a general 160

Spawning characteristics

140 120 100 80 60 40 20 0

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2003 Year

2004

Fig. 24.3 Comparison of number of egg batches, spawning duration and number of eggs produced (in millions) of photo-manipulated (PM) and ambient (A) spawning groups of Atlantic cod. Closed symbols denote PM group and open symbol denote the A group. 䊊 and 䊉 denote number of egg batches, 䉫 and 䉬 spawning duration and 䊐 and 䊏 number of eggs produced. (Data extracted from Figures 1 and 2 from Penney et al., 2006a)

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practice not to feed spawning cod during the spawning period on the expectation that, as they are stressed, they will not feed. The combined effects of spawning stress and starvation generally resulted in higher mortality, up to 50 %, in cod during spawning. A cod broodstock program initiated in 2002 at the Ocean Sciences Centre, Newfoundland, Canada, dramatically improved the egg fertilization and hatch rates of cod. Continued feeding during spawning season at a limited ration has reduced broodstock mortality to <1 %. Another important environmental condition that was neglected until recently was the broodstock holding temperature. Studies carried out in Newfoundland showed that a relatively consistent temperature (6 °C) throughout the year produced better quality eggs and larvae than eggs and larvae produced from broodstock held at variable (ambient) temperatures. Scientists in Newfoundland have suggested that the reduction of postspawning mortality could also be related to consistent holding temperature. Studies in Europe have also indicated the importance of consistent broodstock holding temperature in vitelogenesis and stress of other marine finfish species (Tveiten et al., 2001; Suquet et al., 2005; Brown et al., 2006). A few short-term studies have focused on the effects of stressful conditions (high temperatures, handling, and reduced ration) on gamete quality (Suquet et al., 2005; Brown et al., 2006). Although these studies provided some insight on stress and gamete quality, the effects of these stressors on later progeny (larvae/juveniles) were not monitored. Future studies that include a full life cycle should be carried out to provide a better picture of the long-term effects of these early stressors on later life stages. Once the juvenile production technology was developed, the importance of broodstock research became clearer (Fig. 24.4). About 50 % of the

Parental effect (genetics, broodstock nutrition)

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0

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Larval nutrition Husbandry Foraging environment

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Metamorphosis, weaning, grading, cannibalism 450

600

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Fig. 24.4 A schematic diagram of cod survival from hatching through weaning.

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mortality occurs before the yolk-sac is fully absorbed, which indicates the necessity for dedicated broodstock research. For a larval batch to experience 50 % mortality when they still have yolk-sac indicates genetics and broodstock nutrition could be the major cause for this mass mortality, barring major disease problems. Currently, many cod hatcheries achieve 5–10 % survival through weaning onto formulated diet while some reported having achieved 20 %. Recent figures indicate larval survival rates may be as high as 30 %; however, results are still variable. To date, approach to the cod broodstock nutrition is inconsistent. Those broodstock facilities that have wild fish as broodstock use a diet of fresh or frozen fish supplemented with vitamins and home-made moist diets. All these methods introduce potential threats from disease or poor nutrition. Those facilities with broodstock selected from cultured fish are now using newly available grower diets and specially formulated broodstock diets. Research on the impact of these diets on egg and larval performance is now being evaluated. It is clear that achieving a consistent survival higher than 10–20 %, will require focused research on broodstock nutrition, stress physiology, and genetics.

24.2.2 Breeding program The success of cod culture in Europe and North America depends on a supply of high-quality juveniles. Currently, most hatcheries rely on wildcaught broodstock captured from the wild and their genetic make-up and relatedness are unknown. In addition, the quality of the gametes may vary as it takes more time to condition the wild-caught adult, which could potentially reduce the growth, survival, disease resistance, and quality of the progeny. Furthermore, the use of wild broodstock results in extremely variable performance between individuals and the overall yield is negatively affected. In order to compensate for the variable performance, hatcheries have to high grade their fry, reducing the numbers available for ocean seeding. Selection of certain quantitative traits is the normal method used by breeding programs to overcome these problems. The main drawback of breeding programs is that they are very costly when viewed in isolation. However, breeding programs have become the cornerstone of modern agriculture, and this model is being applied to aquaculture. The huge expense of these programs has necessitated a partnership between government, universities, research institutes, and industry. The national cod breeding program in Norway was initiated in 2002, administrated by Nofima marin (previously Fiskeriforskning) in Tromsø, and funded by the Norwegian government. The National Cod Breeding Station in Tromsø is well equipped with facilities to produce about 300 families. At the same time, a private breeding program was started by Marine Breed AS in Sunndalsøra. Later, Iceland and Canada initiated their own cod breeding programs. The breeding goal of all these programs

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includes selection of important economic traits such as growth, disease resistance, flesh quality, and delayed maturation. Development and use of genetic markers such as microsatellite, SNPs (single nucleotide polymorphism) may be used to identify QTLs (quantitative trait loci) and implement marker-assisted selection (MAS) techniques to assist effective selection of elite broodstock. Breeding programs in Tromsø and Canada employed a strategy of spawning annually and producing parallel yearclasses while Marine Breed has a strategy to produce families every three years. Selective breeding programs for salmonids have contributed significantly to increased growth, survival, and productivity. Genetic gains in growth of ∼10–15 % per generation have been realized for Atlantic salmon, Salmo salar and rainbow trout, Oncorhynchus mykiss (Gjedrem, 2000), and similar gains have been realized for channel catfish (Ictalurus punctatus; Mickett et al., 2003). Early results from different cod breeding programs are encouraging with significant differences among families in growth (Fig. 24.5a), deformities

NE Arctic cod

Coastal cod

Body weight (%)

400 300 200 100

1

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Fig. 24.5 Difference in (a) body weight; (b) deformity among families originated from coastal and Arctic cod used in Norwegian National Cod Breeding Program. (Figures are provided by NOFIMA Marin, Tromsø, Norway)

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Post-challenge survival (%)

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Fig. 24.6 Differences in survival among families produced in Norwegian National Cod Breeding Program after during a challenge test against vibriosis. (Figures are provided by NOFIMA Marin, Tromsø, Norway)

(Fig. 24.5b), and post-challenge survival against vibriosis (Kettunen and Fjalestad, 2006; Fig. 24.6). Estimates of heritability for body weight varied between 0.4–0.6 (Kolstad et al., 2006; Kettunen and Fjalestad, 2007; Symonds et al., 2007) and disease resistance to vibriosis showed a moderate heritability (0.08–0.17; Kettunen et al., 2007). Heritability for sexual maturation (0.07; Kolstad et al., 2006) was estimated to be low; however, the low estimate may be due to the complexity of the trait. Nevertheless, these results indicate the potential of selecting progenies for increased growth rate and disease resistance in cod, which could provide the cod aquaculture industry an elite broodstock in the very near future.

24.2.3 Production technology, nutrition, and disease management Mortality during the larval stages of most marine fish is very high, including cod. Environmental variables such as prey density, temperature, and light regime affect the survival of cod larvae. Research on intensive production of cod juveniles has been on-going since the mid-1990s. With a goal of improving growth and survival of larval cod, a series of studies were undertaken to determine the optimal prey density and light regime. Results indicated that cod larvae grow and survive better at a density of 4000 prey l−1, 24 h photoperiod, and 2400 lux light intensity. Under these conditions, growth of six week-old larvae was increased by about 50 % while the survival improved from 5 % to 20 % compared to previous studies using lower light intensities, photoperiods, and prey densities. Similar research in Europe and North America has helped in developing reliable larval and juvenile

Farming cod and halibut: biological and technological advances Age (d)

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Fig. 24.7

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Larval rearing protocol for Atlantic cod. (Modified from Brown et al., 2003)

rearing protocols (Brown et al., 2003) and these protocols have been further refined in the last few years (Fig. 24.7). Live feed production is a vital part of the cod larval production and usually enriched rotifer and Artemia are used. Apart from these two zooplanktons, algae are also produced for green water culture. Several studies have shown the importance of live feed nutrition on the larval growth and survival, and many failures at this stage have been attributed to poor live feed nutritional quality (Rainuzzo et al., 1997). Lipid has been identified as one of the most important nutritional component that affects the larval performance (Sargent et al., 1999; Hamre, 2006; Garcia et al., 2008). Sargent et al. (1999) identified the importance of polyunsaturated fatty acids (PUFA), such as arachidonic acid (ARA; 20:4ω6), eicosapentaenoic acid (EPA; 20:5ω3), and docosahexaenoic acid (DHA; 22:6ω3), in neural development, pigmentation, growth, survival, and reproduction of marine finfish. It seems that both the concentrations of these highly unsaturated fatty acids (HUFA) and the proportion of these three HUFA in the live feed are very important (Park et al., 2006; Garcia et al., 2008). Cultured rotifers and Artemia lack several of these essential fatty acids in required amount and proportion and are thus enriched with commercially-available lipid emulsifiers. The importance of delivering these HUFAs in the form of phospholipids (PL) through live feed has also been emphasized in recent years. Furthermore, the presence of PL such as phosphatidylcholine (PC), phosphatidylinositol (PI), and phosphatidylethanolamine (PE) in cod larval natural

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prey (copepods) underlines the importance of these PL in cultured live feed (Sargent et al., 1999). However, practically it is very difficult to maintain the desired concentration and proportion of these HUFAs in live feed, mainly because rotifers and especially Artemia catabolize these fatty acids (Navarro et al., 1999). In addition, differences in body composition between different batches of these live feed make it difficult to administer standard enrichment regimes and procedures (Sorgeloos et al., 2001). This variability in live feed nutritional quality often results in unpredictable growth, survival, and quality of the juveniles. Apart from the difficulties in achieving consistent nutritional quality, live feed production is also expensive and labour-intensive. Thus, a significant body of research has been undertaken since the 1980s to reduce or remove dependency on live feed in marine finfish larviculture (Baskerville-Bridges and Kling, 2000; Callan et al., 2003). Early versions of these microparticulate diets have met with limited success, mainly due to poor acceptability of the feed by the larvae, low residence time in the water column, and relatively high nutrient leaching creating unfavourable hygienic conditions and microbial assemblages. The convergence of new feed manufacturing technologies, an improved understanding of nutritional requirements, and advances in larval rearing technologies has resulted in the some improvement in microparticulate diets. Presently, eliminating rotifers from the feeding regime seems to be difficult. However, attempts have been made to eliminate use of Artemia in cod culture (Baskerville-Bridges and Kling, 2000; Callan et al., 2003). Recently, Fletcher et al. (2007) demonstrated that Artemia could be eliminated from the feeding regime. It is a major boost to the industry because almost 80 % of the live feed cost is attributed to Artemia. Most of the commercial hatcheries in Norway are now producing cod juveniles without using Artemia. In this feeding regime, weaning starts earlier at 25 days posthatch (dph) using microparticulate diet. By this method, cod juveniles are fully weaned onto dry feed at 50–55 dph (Fletcher et al., 2007). Wold et al. (2009), in their study, started the weaning of larval cod onto dry feed at 17 dph and completed the weaning by 45 dph; however, the rotifers were used until 45 dph. Recently, while conducting a weaning trial for a new microparticulate diet, scientists at the National Cod Breeding Station in Tromsø have successfully weaned cod larvae in two days (co-feeding rotifer and formulated feed for two days) and all larvae were fully weaned at 23 dph with better growth and survival. Deformities have become a major issue in cod juvenile quality in the recent past. Several studies on other marine finfish species have suggested nutritional, genetic, disease, and husbandry practices could be responsible for the high deformity levels in juvenile fish (Takeuchi et al., 1998; Kolstad et al., 2006). Among the nutritional causes, deficiencies in phosphorus and vitamin C, excess vitamin A, and oxidative degradation of lipid have been suggested as possible reasons (Lall and Lewis-McCrea, 2007). In early 2000,

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it was reported that more than 50 % of the cod juveniles produced in Norwegian commercial hatcheries had skeletal deformities (Olsen et al., 2004). No published data are available on the current deformity levels of cod juveniles in the commercial hatchery production; however, the general consensus is that the quality of juveniles has been improved in the last couple of years. Data from Ocean Sciences Centre, Newfoundland indicate the deformity levels in their production system have been reduced below 10 % since 2002 (Fig. 24.8). They attributed this reduction in deformity to better broodstock management and larval nutrition and improved husbandry practices. Mass mortalities due to diseases are common in hatcheries and nurseries, but not all outbreaks are reported. Bacterial diseases are common and experimentally various strains of Vibrio (e.g. Vibrio anguillarum serotype 02β) have caused mortalities in Atlantic cod. Nevertheless, the vaccines available on the market are not effective against these strains (Bricknell et al., 2006). However, better husbandry practices in hatcheries and nurseries can help to keep the opportunistic pathogens such as Vibrio sp. and various strains of Aeromonas salmonicida under control. In addition to epizootics caused by these opportunistic pathogens, mass mortalities of cod in the hatcheries and nurseries in North America and Europe due to viral nervous necrosis (VNN) caused by nodavirus have been reported (Starkey et al., 2001; Johnson et al., 2002). Outbreak of nodavirus in hatcheries seems to be related to stressful conditions, especially higher temperatures. Recently, Patel et al. (2007) reported mass mortalities of cod juveniles between 5 and 24 g in a farm site in Norway caused by

25

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20 15 10 5 0 2001

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Fig. 24.8

Cod larval deformities at Ocean Sciences Center, Newfoundland, Canada during production years 2001–2004.

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nodavirus. Nylund et al. (2008) reported the presence of VNN in adult farmed and wild cod from southern and western Norway. This would have greater implications because broodstock from farmed and wild origin could act as reservoirs for nodavirus. Apart from horizontal transmission, the virus could also be vertically transmitted from the parents to the offspring. Recent unpublished data has shown that eggs and incoming water can effectively be treated with ozone to prevent this vertical transmission. No vaccines are currently available for nodavirus, and better screening of broodstock, ozone treatment for eggs, and better husbandry practices would help to prevent this disease in the hatcheries. Although several issues such as cage technology, cage husbandry, stocking density, and feeding regimes still need to be refined and resolved, the cod industry faces three major problems on the grow-out stage; fatty (enlarged) liver syndrome, early maturation, and diseases. Liver is the main lipid storage organ for cod and very little fat is stored in muscle. With unlimited high energy food (lipid) available throughout the year for the farmed cod, lipids are deposited in liver at higher rate, 11–16 % of the total body weight for farmed fish compared with 3–5 % for wild cod (Grant et al., 1998). It has been hypothesized that hyper-deposition of lipids to liver could affect growth. Several theories have been put forward to explain this phenomenon, such as high dietary lipid levels leading to higher deposition of lipid in the liver, reduced catabolism of deposited lipids in the liver, and difficulties in transporting these lipids to muscles, which suggest that enlarged liver syndrome in juveniles may not be reversible (Nanton et al., 2006). Morais et al. (2001) suggested that fatty acid profiles of cultured cod liver reflect slightly higher proportions of saturated fatty acids and linoleic acid but comparable PUFA levels to wild cod liver and so could be used for cod liver oil production. Due to the proportion that cod liver represents of the total carcass weight this resource could be a significant investment. Early maturation has also become an important issue for cod farmers (Dahle et al., 1999). Early maturation represents not only an increased cost for biomass lost during spawning but also increases in mortality, reduced size at harvest, or extended growth time to harvest. Farmed Atlantic cod in Norway have been reported to spawn for the first time around two years of age while wild cod sexually mature at 4–5 years (Karlsen et al., 1995; Berg and Pedersen, 2001). Favourable living conditions such as a steady supply of high-nutrient food enable the fish to grow faster, develop energy stores, and mature earlier. Early maturation causes the fish to use energy to develop gonads rather than somatic growth. This increases the length of culture time to achieve market size (Hansen et al., 2001). Several strategies have been investigated to prevent the onset of early maturation. Reduced ration, periodic starvation, and different photoperiod regimes have been examined as methods to delay or arrest the maturation until harvest (Karlsen et al., 1995; Hansen et al., 2001; Norberg et al., 2004;

Farming cod and halibut: biological and technological advances

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Davie, 2005; Taranger et al., 2006), and periodic starvation and reduced feeding levels were not effective (Karlsen et al., 1995). In recent years, photoperiod manipulation has been successfully used to allow broodstock in captivity to spawn year around (Penney et al., 2006a). This strategy has been expanded to delay maturation in cultured cod. Periods of continuous light have successfully prevented early maturation of cod in indoor tanks (Hansen et al., 2001). However, translating this knowledge to control early maturation in sea cages has presented some complications. The level of light needed and the diel rhythms created by natural photoperiod are not well understood and could not be controlled as effectively as in indoor tanks. Different theories regarding light intensity, the placement of the lights in or above the cage, the timing of light exposure, and the amount of natural light to be allowed into the cage have all been investigated as strategies of photoperiod manipulation (Davie, 2005). Results of these studies indicate that the percentage of early maturing animals in each case is highly variable. Moreover, the differences in latitude resulting in different day lengths, biological differences between species, and genetic factors within species may also bring further complications. In addition, the cost of supplying continuous high-intensity light in a cage environment is prohibitively expensive at present, although new LED lights may mitigate the costs. Samuelsen et al. (2006) reviewed the bacterial and viral diseases in Atlantic cod. Although vaccines are available for some bacterial diseases, no vaccines have been developed for viral diseases. Vibrio sp. can cause considerable loss in cod farming. Vaccines are available for cod against vibriosis, but effective application methods for these vaccines with regard to at what juvenile size they should be administrated, dosage, and methods of administration are yet to be developed. Recently, a bacterial disease known as francisellosis has caused major mortalities in some cod farms in southern and western Norway (Nylund et al., 2006). The pathogen has been identified as Francisella piscicida and lives intracellularly which makes any treatment difficult (Ottem et al., 2007). Infected fish display abnormally dark pigmentation, loss of appetite, and reduced swimming performance. Internally the fish develop swollen spleen, with the kidney and heart covered with and penetrated by white granulomas (Fig. 24.9). The proliferation of Francisella is aided by the warm summer temperatures in southern/western Norway, which causes mortalities in wild cod (Svåsand et al., 2007). The clinical signs are difficult to identify at early stages under farm environment and the condition of the fish become severe and irreversible; eventually the whole crop could be lost. In the absence of better diagnostics and screening and the lack of vaccines, setting up farms in areas where higher temperatures do not occur is the best strategy to mitigate the problem. Except for a few incidents in Norway (Patel et al., 2007; Nylund et al., 2008), mass mortalities of cod in grow-out phases due to viral infection have not been reported. Although nodavirus has been considered a major disease

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Fig. 24.9 Atlantic cod infected by Francisella piscicida. (A). Dark pigmented appearance (B). Internal organs showing the white granulomas cysts around the heart and kidney. (Pictures are provided by FARMAQ AS, Norway)

of early life-history stages, adult carriers can be identified in wild populations and in captive broodstock, and disease outbreaks can occur during grow-out. No vaccines have been developed against nodavirus and the diagnostic methods have not yet been perfected. In addition, the source of the infection, whether it is vertical, horizontal, environmental factor(s) or a combination of these, is not yet clear. Evidence suggests that the main source could be the broodstock. Hatcheries in the USA (Great Bay Aqua, New Hampshire, USA) and Canada have been using ozone for sterilizing eggs and successfully controlling this disease outbreak in nursery stage. Once they reach a certain size, the juvenile cod become immune to the disease but can act as carriers. Thus, more work is needed to understand the immune development of cod against VNN.

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The on-growing phase of Atlantic cod has benefited from the knowledge of the salmon aquaculture industry. Technologies used in salmon farming can be used to farm cod with little modification. Cage dimensions have been significantly increased in the last few years to more than 100 m circumference, which means that it is important to use the latest technologies such as underwater cameras and automatic demand feeders to monitor fish behaviour and to reduce feed wastage. Conflict of interest between various groups such as recreation, tourism, navigation, and the environment already exists. Thus, further expansion of aquaculture activities in coastal areas for new marine finfish species is likely to face tougher opposition. The search for new technologies such as submerged offshore cages is in progress, and cod, halibut, and haddock have been used to stock these cages (Fredriksson et al., 2003). Although submerged offshore cage aquaculture showed promising results, these ventures still need to be improved. Studies to improve the technology are continuing in New Hampshire, USA (Chambers and Howell, 2006).

24.2.4 Bottlenecks Despite the importance of cod broodstock nutrition, limited information is available on the role of different nutrients during vitellogenesis and reproduction (Izquierdo et al., 2001). Eggs from cultured broodstock had significantly lower fertilization rates, cell symmetry, and survival to hatching rates than the eggs obtained from wild broodstock (Salze et al., 2005). Although overall fatty acid contents and DHA were similar, eggs from cultured broodstock had lower ARA and astaxanthin concentration and were lacking some minor phospholipid classes compared to wild fish, which shows our poor understanding of cod broodstock nutrition and its effect on gamete quality. Future research should focus on the identification of specific nutrient requirements of broodstock and the relationship between reproductive performance and quality of the cod juvenile produced. Research on larval nutrition has increased in recent years, but dependence on live feed and lack of suitable microparticulate diets to replace live feed are bottlenecks. More research is needed to replace rotifers with microparticulate diets. Although incidence of skeletal deformities has been reduced, mainly through better husbandry, the causal mechanisms behind skeletal deformities in cod are poorly understood. A multidisciplinary approach involving genetics, nutrition, environmental factors, especially temperature and parental conditioning, is required to improve the juvenile quality. Early maturation has been considered the major bottleneck for the expansion of cod aquaculture. Limited success of light administration on preventing early maturation prompted a search for other ideas such as production of sterile fish; e.g. triploid fish (Svåsand et al., 2007). Sterile fish not only address the early maturation problem but also issues pertaining to

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fish escapes and cage spawning. Triploid fish could also ensure that better quality products are delivered to the market year-round because spawning events in normal diploid fish reduce the muscle quality during the spawning season. Unfortunately, while heralded as a cure for all these problems, experience from salmonids shows that triploid fish are less robust compared to diploid fish and are more prone to other diseases and deformities (Sadler et al., 2001). Techniques for induction of triploid cod are available (Peruzzi et al., 2007) and a triploid cod fish has been already produced by scientists at the St Andrews Biological Station, Canada with further investigations planned to follow this to adulthood. Intensified aquaculture activities may eventually create problems such as outbreaks of virulent infectious diseases if the culture operations are not properly managed. The infant cod aquaculture industry has already seen at least two localized disease outbreaks. Antibiotics use has been discouraged due to negative effects such as development of microbialresistant strains and residues in the product. Thus, preventive methods should be employed such as use of probiotics, development of vaccines, increased disease resistance through selective breeding, and better management practices.

24.2.5 Environmental issues Environmental issues such as fish escapes, disease outbreaks, parasite infestations, and nutrient sedimentation play a major role in determining successful aquaculture operations. The cod aquaculture industry can learn from the salmon industry and avoid making the same mistakes. There are reports of increasing cod escapes in sea cages in Norway since 2000 (Moe et al., 2007). The main reasons for these escapes appear to be behaviour of the cod, inferior technical standards of the nets, and increased predator activity around the sea cages. Studies have been already undertaken by different groups in Norway (e.g. How cod escape? Funded by Norwegian Research Council) to understand cod escape behaviour and to mitigate the technological and predatory issues. Another issue raised recently was the spawning activities of early maturing cod in sea cages. Studies carried out by scientists in the Institute of Marine Research, Bergen indicated an increase in spawning activities year to year that resulted in a 40 % increase in larval occurrence of farmed fish origin in adjacent fjords (Svåsand et al., 2007). However, what percentage of these larvae would managed to survive into adulthood is unknown and thus the long-term effect of the cage spawning on the wild population is unclear. The biggest problem with cod escapes and spawning in cages is the possibility of genetic interaction between cultured and wild cod stocks. More studies have already been initiated by the scientific community on this issue.

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24.2.6 Consumer preference for farmed cod Seafood quality is critical not only to support the increasing aquaculture demand but also to meet consumer preference. Consumers are concerned about product safety, environmental sustainability, and social responsibility, and they keep stressing the need to regulate and standardize the processes used to produce aquaculture products. In recent years, consumer preference for farmed cod has increased, mainly due to the freshness of the product, improvements in farming and slaughter methods, and the positive attributes of having higher amounts of omega 3 fatty acids from all seafood (Luten et al., 2002; Hemre et al., 2004). Consumers liked farmed and wild cod equally in terms of texture, taste, and aroma (Luten et al., 2002). Recently, Johnson Sea Farms Ltd in Scotland targeted the niche organic market and introduced ‘No Catch’ brand of cod. Although the company experienced problems in 2008 due to improper management practices, the ‘No Catch’ idea has been praised as one of the best marketing strategies. There were no differences in sample tests between wild and farmed fish products, and the perception scores showed slightly higher preferences towards wild fish products, especially among the age group 55 and older (Verbeke et al., 2007). Thus, consumers’ opinions about farmed fish are mainly based on perception and efforts should be undertaken to improve consumer awareness and factual knowledge of aquaculture.

24.3 Atlantic halibut Like cod, landings of Atlantic halibut have also been in decline and currently the annual wild catch is less than 4000 tonnes (FAO, 2006). Halibut is a difficult species to work with due to its unique early life history and problematic metamorphosis in culture conditions. However, it has a good market value and increased market demand which has intensified the efforts to culture this valuable species. Interest in halibut culture was initiated in the mid-1970s in Norway, and the first juvenile halibut were produced in 1980 at the Flødevigen Research Station, Arendal, Norway. During the 1980s most of the efforts were directed towards improving the gamete collection methods, egg incubation, and start-feeding. All those efforts at several institutes in Norway have resulted in better gamete collection techniques, increased fertilization rates, and better egg incubation techniques. However, the extended yolk-sac larval incubation period and lower feeding incidences of the start-feeding larvae remained problematic, and in the 1990s more efforts were directed toward developing technical competence in tank system configurations for egg incubation and start-feeding stages. During the mid-1990s sporadic success was evident. However, juvenile production was unreliable, mainly due to poor understanding of the biology of different life stages. Concerted efforts by several research institutes in

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Norway have led in standardizing production technologies and a viable halibut industry began to emerge in the early 2000s. At the early stages, like cod, halibut juvenile production was also carried out in extensive or semi-extensive systems. Both egg incubation and first feeding were performed in larger plastic bags submerged in fjords. During this time, zooplankton was collected from the lagoons and fed to the larvae. However, as in cod semi-extensive systems, uncontrollable environmental conditions and zooplankton densities resulted in variable survival and eventually efforts have been initiated to develop intensive juvenile production using Artemia.

24.3.1 Broodstock Halibut has a complicated early life history, and production of better quality juveniles is largely depended on acquiring high-quality gametes. Unlike cod, acquiring and raising halibut broodstock requires time and at least 2–5 years is needed to condition a wild-caught halibut. Initially, all halibut hatcheries relied upon wild-caught broodstock; however, since the late 1990s, cultured F1 adult fish have become a major part of the broodstock inventory. In general, broodstock are maintained at below 8–10 °C in summer and the temperature lowered to 5–6 °C from October/November to February/March to produce good-quality gametes (Brown et al., 2006). Photo-manipulation has proved to be successful in off-season spawning (Næss et al., 1996). Studies have shown a closer relationship between halibut broodstock nutrition and egg quality and that blastomere morphology and fertilization rates are affected by poor broodstock nutrition (Mazorra et al., 2003). In the late 1990s, most commercial broodstock holding facilities relied on fresh fish and home-made moist diets to feed the broodstock (Olsen et al., 1999). Currently, halibut broodstock diets are available. However, due to variable results, research efforts on the essential fatty acid profiles required to produce better quality gametes and larvae are continuing, and formulated diets of proper nutritional quality for broodstock could be available in the near future. Although halibut can spawn spontaneously in tanks, fertilization rates vary greatly. Thus, a manual stripping method is used to collect the gametes, which is very tedious and labour-intensive. Halibut is a batch spawner and releases eggs every three days. An observation on ovulatory rhythms and calculating the intervals between stripping times is essential in getting the quality gametes (Norberg et al., 1991). A mature female can produce several litres of eggs in 5–15 batches depending on the size of the fish and frequent physical inspection is required to ensure good-quality eggs are stripped (Fig. 24.10). This excessive handling during spawning usually leads to physical damage and stress in broodfish. Ultrasonic techniques and use of ultrasound equipment have been reported, but the cost and size of the equipment limit their usage (Shields et al., 1999). In the early stages of halibut aquaculture

Farming cod and halibut: biological and technological advances

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Fig. 24.10 Gamete collection from Atlantic halibut broodstock at the Scotian Halibut Ltd, Canada. (A). Broodstock identification, (B). Milt collection, (C). Egg collection and (D). Fertilization.

development, broodstock management was a problematic area and the fertilization rates were very low resulting in poor larvae and lower survival. However, almost ten years of research have refined and improved the broodstock conditioning and gamete collection methods and currently fertilization rates of 90 % are consistently reported.

24.3.2 Breeding program Initiation of an elaborate halibut selective breeding program is a costly venture due to lengthy maturation time. Thus, rather than starting complete selective breeding program involving several generations, genomics programs employing pedigree analysis and genetic linkage mapping to identify the genetic markers and genes associated with commercially important traits have been started recently in Canada, Norway, and Iceland.

24.3.3 Production technology, nutrition, and disease management Several review articles in Aquaculture Research (Volume 29, issue 12) discussed the halibut research activities from the 1970s to the late 1990s.

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Halibut eggs and yolk-sac larvae (YSL) have special requirements such as dark incubation, constant low temperatures of 5–6 (eggs)/6–8 °C (YSL) and up-welling water flow to minimize larval abnormalities. Hatching rates are relatively high but variable (60–80 %) and larvae hatch at a poorly developed stage. Poorly developed YSL require larger up-welling silos and good water quality to ensure proper development. Unfavourable conditions often result in poor larval quality, and the major larval quality issue at this stage is jaw deformity (Pittman et al., 1990); however, higher incidences of oedema in the yolk-sac sinus and pericardium is also seen as major problem (Ottesen and Bolla, 1998). Although several improvements in husbandry issues for this stage have been made since the 1990s, survival and larval quality at different commercial hatcheries are still unpredictable and generally range between 10 and 70 % with good egg batches surpassing 90 % survival. Larvae are transferred to start feeding tanks after 30–45 days of YSL incubation. First feeding tanks of halibut larvae need specific requirements to ensure successful start feeding. Tank dynamics, water current, aeration, and light regimes are adjusted to improve the start-feeding of the larvae (Harboe et al., 1998). Green water technology is used in halibut larval culture. Live feed regimes vary among hatcheries, and copepods and enriched Artemia and rotifers are used either in combinations or alone. In some commercial hatcheries, mass mortalities during first feeding larval stages have been reported. During this time, the larvae stop feeding and have increased mucus in the gut, and eventually die. Although the causes for these mortalities have not been identified, the amount and strains of bacteria introduced with the live feed is believed to be one of the reasons. Jelmert et al. (1995) described similar clinical symptoms in start-feeding halibut larvae (eight days post-start feeding). Larvae with these symptoms had white deposits in kidney tubules and urinary bladder, abdominal swelling, and long trailing faecal matters, but no evidence of bacterial or viral infections were found. Their finding suggested that the ultimate cause for the larval mortality was nephrocalcinosis. Jelmert et al. (1995) speculated that nutrition deficiency and elevated CO2 concentration could have caused nephrocalcinosis. This mortality is perceived to be a major problem and more research is continuing. Halibut larvae have a high requirement for ω3 HUFA, especially DHA, EPA, and ArA, in their diets, and any imbalance in these three fatty acids generally results in improper eye migration, pigmentation, and reduced growth and survival (Hamre et al., 2005, 2007). However, continuous research on live feed nutrition has resulted in major improvements in cultured live feed quality in recent years and the quality of halibut juveniles has been tremendously improved (Hamre and Harboe, 2008). Metamorphosis in halibut is a complex process, and in culture conditions this becomes more complicated due to improper husbandry, nutrition, and environmental conditions during larval stages (Hamre et al., 2005). Weaning onto formu-

Farming cod and halibut: biological and technological advances

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lated diets starts at 50–60 days after the initiation of first feeding and completes in 15–20 days. Survival through the larval stage in the late 1990s was inconsistent and low (0–20 %). With recent improvements in husbandry and larval nutrition, most hatcheries now produce halibut juveniles with a survival ranging from 20–30 % through first feeding stage. The survival of halibut is impressive once the weaning has been completed, exceeding 80–90 %. With improvements made in the early life stages, current efforts have been focused on finding cost-effective methods for halibut grow-out. However, the uniqueness of the flatfish benthic habitat offers challenges in both land and sea-based culture systems, with demands for a different technology than is used for rearing of typical round fish species. Initially, landbased grow-out has been seen as the only solution because halibut need more bottom surface area than the volume, which would be difficult to obtain in sea cage farming. However, an economic model comparing sea cage and land-based on-growing of halibut showed that sea cage operation would be more profitable than the land-based model (Penney, 1999). Landbased grow-out systems require major capital investment and operational expenditures, especially for the heating and cooling of the water. However, recent improvements in recirculation technology research on halibut ongrowing has rendered the land-based system more viable and profitable (Blanchard et al., 2002; McCallum, 2003; Tango and Gagnon, 2003). Research on on-growing stages of halibut in Norway and Canada showed that new technologies could be used to increase the stocking volume of halibut. Halibut, unlike salmon or cod, are very docile and rest most of the time, showing activity only during feeding time (Kristiansen et al., 2004), and this behaviour could be used in developing shelve systems. Aqualine®, a Norwegian company, invented and improved a shelve system for ongrowing stage which would increase the stocking densities of halibut. A few commercial operations in Norway and Canada have already started trials on these systems. With these recent developments, empirical studies on cost comparison of land-based and sea cage on-growing systems should be initiated. Halibut YSL are the most susceptible life stage to most bacterial and viral diseases, which is not surprising given their poor developmental stage (Bergh et al., 2001). Halibut larval and early juvenile stages have been susceptible to nodavirus and IPN virus. Mass mortalities due to nodavirus infection have been reported in the late 1990s in Norway (Grotmol et al., 1995), yet, no effective vaccines are available (Sommerset et al., 2001). While the epidemiology of this disease is not clear, evidence exists for both vertical (Grotmol and Totland, 2000) and horizontal transmission (Nerland et al., 2007). Cusack et al. (2001) reported mortalities among young halibut in a semi-commercial hatchery in Canada caused by aquareovirus. The clinical symptoms included slow movement, anorexia, enlarged abdomen, and consequent increased mortality. Vibrio sp. infection has also caused problems in young halibut, and the clinical signs included bacterial overgrowth in the

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intestines. Atypical furunculosis, a chronic problem in larger halibut, has been reported to be a waterborne disease. Poor husbandry practices often weaken the fish and subsequent secondary outbreaks occur (Hellberg and Colquhoun, 2005).

24.3.4 Bottlenecks The major bottleneck in halibut culture is inconsistent larval survival through first feeding stage, which could be related not only to diseases but also to improper nutrition of broodstock, live feed and larval stages, and poor husbandry. Addressing these issues should be prioritized to ensure the production of better quality juveniles. Improper eye migration and pigmentation also results in higher discard of juveniles during the nursery stage, which increases production cost. Another bottleneck has been identified as a lack of grow-out sites which stagnates the halibut juvenile production.

24.3.5 Environmental issues Unlike cod, no major issues such as escapees and spawning in cages are encountered in halibut farming. Diseases are, however, a major concern because outbreaks of viral diseases have been reported. Further, experimental cross-contamination of infectious pancreatic necrosis virus (IPNV) between salmon and halibut pathogens has been reported (Biering, 1997).

24.4 Future trends 24.4.1 Studies involving all life stages An increasing body of evidence is emerging in human and mammal research that events occurring in early life, often during conception and foetal development, can have a long-term ‘programming effect’ that lasts the lifetime of an animal. In agriculture, considerable interest has been directed towards this concept to find out how these events may affect the health, welfare, and productivity of farmed livestock. To date research on different life stages of most aquaculture species, including cod and halibut, is discrete and the long-term effects of early rearing conditions, for example, rearing temperature of broodstock, egg, and larvae, are unknown. Research involving the complete life cycle of these species should be initiated to study ‘programming effects’ of environmental and nutritional factors experienced by the early life stages on later life stages.

24.4.2 Improvements to current culture methods Despite the progress made both in cod and halibut research, reliable mass production of better quality juveniles is one of the major bottlenecks. Thus,

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studies should be carried out with an aim to improve the established basic culture methods and to develop new and innovative production strategies and procedures aiming at improving the juvenile quality and reducing the production cost. More research towards off-shore aquaculture activities and use of renewable energy should be encouraged to reduce the impact on the environment. Water recirculation systems (WRS) in marine aquaculture are a relatively new area and there is a growing interest in using WRS in intensive cod and halibut culture. The success of WRS depends not only on the technology but also on better husbandry techniques including feeding practices and disease management. Recent improvements in husbandry techniques, nutrition, disease prevention, water quality management, and technology development should help to provide better WRS for finfish aquaculture. If planned, constructed, and operated well, an intensive WRS may produce fish profitably. However, WRS requires high investment and operating costs and is economically vulnerable mainly due to management uncertainties. In addition, nitrogen poisoning due to inefficient biofilters and improper management (Svobodová et al., 2005) and increase in cataract in the eye of cod juveniles by improper use of UV to treat the water (Björnsson, 2004) have also been reported. While larval rearing of cod and halibut has been carried out intensively using flow-through systems, on-growing of cod juveniles has been carried out in sea cages due to the high cost of heating in land-based facilities. Use of live feed complicates using WRS in larval rearing; however, for ongrowing, WRS could be used profitably if the proper technology and husbandry practices are followed. To be successful, WRS should also be supportive of fish high stocking density. Björnsson and Ólafsdóttir (2006) and Foss et al. (2006) showed that juvenile cod can be reared at high stocking densities without reduction in growth rate. Most of the studies on economics of WRS are based on simulation studies and assumed best husbandry practices and maximum production. Thus, future studies based on real commercial data of cod and halibut from specific sites should be carried out to evaluate the economic value of WRS.

24.4.3 Nutrition Nutrition is an important part of all stages, but it is very important in broodstock and larvae. Most of the failures in first feeding stages have been correlated to the broodstock condition prior to and during spawning. Nutritional deficiencies not only lead to unreliable production but also to inferior quality of juveniles with abnormalities and susceptibility to stress and diseases. Considerable research that has been initiated in the recent past to identify the distinctive nutritional requirements of cod and halibut, especially the phospholipids, should be continued. Exploration of new feed resources and increased use of plant materials should be prioritized.

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Research on developing better microparticulate diets should be prioritized.

24.4.4 Selective breeding program It has been proven a successful approach for the salmon industry to invest in a selective breeding program. A similar approach has been started for cod in Canada, Norway, and Iceland and promising results have been reported. Continued long-term funding should be directed towards these programs to realize the full benefits.

24.4.5 Fish health Until recently, no major diseases or parasites were reported for cod and halibut in on-growing stages. However, recent outbreaks of francisellosis and VNN in some commercial operations in Norway saw the whole year’s production wiped out. Studies on disease control through better management practices, development of better diagnostic tools, development of vaccines, and selection of disease-resistant strains through selective breeding programs should be encouraged. Use of probiotics should be encouraged and more research should be carried out. Prevention is the best practice in combating diseases and healthy unstressed fish handle disease outbreaks well and survive through them. Better husbandry management practices and a balanced diet keep the fish healthy, and so the immune system of the fish is not compromised. More research should be conducted to understand the interaction between fish diseases and fish nutrition and their roles in modulating the immune system.

24.4.6 Environmental issues Interested parties (consumers, stakeholders) are demanding environmentally and socially responsible products; thus, sustainable and responsible aquaculture practices are a priority for aquaculture operations. With relatively rapid growth of marine finfish aquaculture, especially cod farming, it is important to understand consumer expectations on product quality and ethical farming practices. With the negative information on impacts of aquaculture on the environment and increasing customer awareness on this issue, more research and outreach activities should be carried out to minimize the environmental impacts and negate bad publicity. Escape of cultured fish has also become a hot topic and more technologies should be developed to mitigate this problem. All-female production could be seen as one solution as they have higher growth, and having only female fish in a sea cage would reduce the number of fertilized eggs escaping to the wild. The European Union recently categorized triploid fish as non-genetically modified organisms (non-GMO) and recommended that

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research on producing triploid fish should be prioritized. More research on triploid fish production technologies and their robustness should be carried out.

24.4.7 Industry driven research and marketing Currently, production protocols are in place and more efforts should be directed toward industry-driven research needs. To achieve this goal, coordinated research between teams from different countries should be initiated using a multidisciplinary approach on the above mentioned key areas. Research on marketing is a low priority at this time, mainly due to higher juvenile production cost, which should be reduced through improvements in culture methods and larval nutrition.

24.4.8 Consumer preference Today’s consumers recognize the importance of nutritional quality, texture, appearance, and taste; thus, enhancements of these qualities through proper nutrition, deformity reduction, stress reduction, and improved harvesting and slaughtering techniques should be developed. Buyer and consumer confidence should be boosted by using traceability techniques and properly conducted certification programs such as environmental, organic, and process certifications, which would also benefit the flow of safe goods into the marketplace. Research results on the health benefits of consuming fish and seafood products should be publicized such as the recent Harvard School of Public Health announcement, the most comprehensive medical study ever done on eating fish, which states, ‘Seafood is likely the single most important food one can consume for good health’.

24.5 Sources of further information and advice 24.5.1 Atlantic cod • Nofima, Norway specializes in selective breeding program, product quality, marketing and environmental issues, www.nofima.no. • Ocean Sciences Centre, Memorial University of Newfoundland, Canada, www.mun.ca/osc. • Genome Atlantic, Canada on cod selective breeding program, www. codgene.ca. • Institute of Marine Research (IMR), Bergen, Norway, www.imr.no. • Norwegian Seafood Centre, Bergen, Norway, www.sjomat.no. • Marine Research Institute, Reykjavik, Iceland, www.hafro.is. • National Institute of Nutrition and Seafood Research, Norway, www. nifes.no. • Norwegian Seafood Federation, www.fhl.no.

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24.5.2 Atlantic halibut • Brandal Havbruk AS: www.brandal-havbruk.no. • Fjord Halibut KS, 6475 Midsund, Norway. • Nordic Seafarms ASA: www.nordicseafarms.no. • Fiskey, Iceland on hatchery and nursery: www.fiskey.is. • Institute of Marine Research (IMR), Bergen, Norway: www.imr.no. • Scotian Halibut Ltd., NS, Canada on hatchery, nursery, on-growing (land-based and sea cages) and marketing: www.halibut.ns.ca. • St. Andrews Biological Station, NB, Canada on halibut broodstock, www.mar.dfo-mpo.gc.ca/sabs.

24.6 Acknowledgements We thank Jonathan Moir, Dr Anne Kettunen and Dr Geoff Allan for their constructive comments on the earlier version of the manuscript. We also thank Brian Blanchard, Lynn Lush and Yngve Lystad for providing photographs.

24.7 References baskerville-bridges b and kling l j (2000) Early weaning of Atlantic cod (Gadus morhua) larvae onto a microparticulate diet, Aquaculture, 189, 109–17. berg e and pedersen t (2001) Variability in recruitment, growth and sexual maturity of coastal cod (Gadus morhua L.) in a fjord system in northern Norway, Fish Res, 52, 179–89. bergh o, nilsen f and samuelsen o b (2001) Diseases, prophylaxis and treatment of the Atlantic halibut Hippoglossus hippoglossus: a review, Dis Aquat Organ, 48, 57–74. biering e (1997) Infectious pancreatic necrosis virus infections of farmed Atlantic halibut (Hippoglossus hippoglossus), DSc thesis, Department of Fisheries and Marine Biology, University of Bergen, Norway. björnsson b (2004) Can UV-treated seawater cause cataract in juvenile cod (Gadus morhua L.)?, Aquaculture, 240, 187–99. björnsson b and ólafsdóttir s r (2006) Effects of water quality and stocking density on growth performance of juvenile cod (Gadus morhua L.), ICES J Mar Sci, 63, 326–34. blanchard b, leblanc d, halldorsson o and scarratt d (2002) Development of a land based recirculation system for Atlantic halibut, Hatchery International, Sept/Oct, 30–2. bricknell i r, bron j e and bowden t j (2006) Diseases of gadoid fish in cultivation: a review, ICES J Mar Sci, 63, 253–66. brown j a, minkoff g and puvanendran v (2003) Larviculture of Atlantic cod (Gadus morhua): progress, protocol and problems, Aquaculture, 227, 357–72. brown, n p, shields r j and bromage n r (2006) The influence of water temperature on spawning patterns and egg quality in the Atlantic halibut (Hippoglossus hippoglossus L.), Aquaculture, 261, 993–1002.

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hamre k, moren m, solbakken j, opstad i and pittman k (2005) The impact of nutrition on metamorphosis in Atlantic halibut (Hippoglossus hippoglossus L.), Aquaculture, 250, 555–65. hansen t, karlsen ø´, taranger g l, hemre g i, holm j c and kjesbu o s (2001) Growth, gonadal development and spawning time of Atlantic cod (Gadus morhua) reared under different photoperiods, Aquaculture, 203, 51–67. harboe t, mangor-jensen a, naas k e and næss t (1998) A tank design for first feeding of Atlantic halibut, Hippoglossus hippoglossus L., larvae, Aquac Res, 29, 919–23. hellberg h and colquhoun d (2005) Health Situation of Farmed Fish 2005, National Veterinary Institute, Oslo. hemre g-i, karlsen ø, eckhoff k, tveit k, mangor-jensen a and rosenlund g (2004) Effect of season, light regime and diet on muscle composition and selected quality parameters in farmed Atlantic cod, Gadus morhua L, Aquac Res, 35, 683–97. izquierdo m s, fernandez-palacios h and tacon a g j (2001) Effect of broodstock nutrition on reproductive performance of fish, Aquaculture, 197, 25–42. jelmert a, rødseth o m and van der meeren t (1995) Nephrocalcinosis associated with mass mortality in cultured Atlantic halibut (Hippoglossus hippoglossus L.) larvae, J Fish Dis, 18, 365–9. johnson s c, sperker s a, leggiadro c t, groman d b, griffiths s g, ritchie r j, cook m d and cusack r r (2002) Identification and characterization of a piscine neuropathy and nodavirus from juvenile Atlantic cod from the Atlantic coast of North America, J Aquat Anim Health, 14, 124–33. karlsen ø´, holm j and kjesbu o s (1995) Effects of periodic starvation on reproductive investment in first-time spawning Atlantic cod (Gadus morhua L.), Aquaculture, 133, 159–70. kettunen a and fjalestad k t (2006) Resistance to vibriosis in Atlantic cod (Gadus morhua L.): First challenge test results, Aquaculture, 258, 263–9. kettunen a and fjalestad k t (2007) Genetic parameters for important traits in the breeding program for Atlantic cod (Gadus morhua L.), Aquaculture, 272, s276. kettunen a, serenius, t and fjalestad k t (2007) Three statistical approaches for genetic analysis of disease resistance to vibriosis in Atlantic cod (Gadus morhua L.), J Anim Sci, 85, 305–13. kolstad k, thorland i, refstie t and gjerde b (2006) Genetic variation and genotype by location interaction in body weight, spinal deformity and sexual maturity in Atlantic cod (Gadus morhua) reared at different locations off Norway, Aquaculture, 259, 66–73. kristiansen t s, fernö a, holm j c, privitera l, bakke s and fosseidengen j e (2004) Swimming behaviour as an indicator of low growth rate and impaired welfare in Atlantic halibut (Hippoglossus hippoglossus L.) reared at three stocking densities, Aquaculture, 230, 137–51. lall s p and lewis-mccrea l m (2007) Role of nutrients in skeletal metabolism and pathology in fish – an overview, Aquaculture, 267, 3–19. le françois n r, lemieus h and blier p u (2002) Biological and technical evaluation of the potential of marine and anadromous fish species for cold water mariculture, Aquac Res, 33, 95–108. luten j, kole a, schelvis r, veldman m, heide m, carlehög m and akse l (2002) Evaluation of wild cod versus wild caught, farmed raised cod from Norway by Dutch consumers, Økonomisk Fiskeriforskning, 12, 44–60. mazorra c, bruce m p, bell j g, davie a, alorend e, jordan n, rees j, papanikos n, porter m and bromage n (2003) Dietary lipid enhancement of broodstock reproductive performance and egg and larval quality in Atlantic halibut (Hippoglossus hippoglossus), Aquaculture, 227, 21–33.

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mccallum t (2003) Saltwater recirculation systems for land-based farming of Atlantic halibut (Hippoglossus hippoglossus), MSc thesis, University of New Brunswick, Saint John, Canada. mickett k, morton c, feng j, li p, simmons m, cao d, dunham r a and liu z (2003) Assessing genetic diversity of domestic populations of channel catfish (Ictalurus punctatus) in Alabama using AFLP markers, Aquaculture, 228, 91–105. moe h, dempster t, sunde l m, winther u and fredheim a (2007) Technological solutions and operational measures to prevent escapes of Atlantic cod (Gadus morhua) from sea cages, Aquac Res, 38, 91–9. morais s, bell j g, robertson d a, roy w j and morris p c (2001) Protein/lipid ratios in extruded diets for Atlantic cod Gadus morhua L.: effects on growth, feed utilisation, muscle composition and liver histology, Aquaculture, 203, 101–19. næss t, harboe t, mangor-jensen a, naas k e and norberg b (1996) Successful first feeding of Atlantic halibut larvae from photoperiod-manipulated broodstock, Prog Fish Cult, 58, 212–14. nanton d a, mcniven m a and lall s p (2006) Serum lipoproteins in haddock, Melanogrammus aeglefinus L, Aquac Nutr, 12, 363–71. navarro j c, henderson r j, mcevoy l a, bell m v and amat f (1999) Lipid conversions during enrichment of Artemia, Aquaculture, 174, 155–66. nerland a h, skaar c, eriksen t b and bleie h (2007) Detection of nodavirus in seawater from rearing facilities for Atlantic halibut Hippoglossus hippoglossus larvae, Dis Aquat Organ, 73, 201–5. norberg b, brown c l, halldorsson o, stensland k and björnsson b t (2004) Photoperiod regulates the timiNg of sexual maturation, spawning, sex steroid and thyroid hormone profiles in Atlantic cod (Gadus morhua), Aquaculture, 229, 451–67. norberg b, valkner v, huse j, karlsen i and lerøy grung g (1991) Ovulatory rhythms and egg viability in the Atlantic halibut (Hippoglossus hippoglossus), Aquaculture, 97, 365–71. norwegian fisheries directorate (2007) Statistics on cod aquaculture, Bergen, (http://www.fiskeridir.no/fiskeridir/english/statistics/norwegian_aquaculture/ aquaculture_statistics/cod), accessed January 2009. nylund a, karlsbakk e, nylund s, isaksen t e, karlsen m, korsnes k, handeland s, martinsen r, mork-pedersen t and ottem k f (2008) New clade of betanodaviruses detected in wild and farmed cod (Gadus morhua) in Norway, Arch Virol, 153, 541–7. nylund a, ottem k f, watanabe k, karlsbakk e and krossøy b (2006) Francisella sp. (Family Francisellaceae) causing mortality in Norwegian cod (Gadus morhua) farming, Arch Microbiol, 185, 383–92. olsen c, kjørsvik e, olsen a i and reitan k (2004) Effect of different phospholipid sources and phospholipid:natural lipid value in formulated diets on larval deformities of Atlantic cod (Gadus morhua), Spec Publ Eur Aquac Soc, 34, 621–2. olsen y, evjemo j o and olsen a (1999) Status of the cultivation technology for production of Atlantic halibut (Hippoglossus hippoglossus) juveniles in Norway/ Europe, Aquaculture, 176, 3–13. ottem k f, nylund a, karlsbakk e, friis-møller a and krossøy b (2007) Characterization of Francisella sp., GM2212, the first Francisella isolate from marine fish, Atlantic cod (Gadus morhua), Arch Microbiol, 187, 343–50. ottesen o h and bolla s (1998) Combined effects of temperature and salinity on development and survival of Atlantic halibut larvae, Aquac Int, 6, 103–20. park h g, puvanendran v, kellett a, parrish c c and brown j a (2006) Effect of enriched rotifers on the growth and survival of Atlantic cod (Gadus morhua) larvae, ICES J Mar Sci, 63, 285–95.

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25 Cobia cultivation E. McLean, Ministry of Fisheries Wealth, Sultanate of Oman, G. Salze, Virginia-Maryland Regional College of Veterinary Medicine, USA, M. H. Schwarz, Virginia Seafood AREC, USA, and S. R. Craig, Virginia Cobia Farms LLC, USA

Abstract: An assessment of the status of global cobia aquaculture is presented in this chapter. Published information on broodstock management and spawning, larval rearing and juvenile nutrition is reviewed. Brief synopses are provided on low-salinity culture of cobia, cold banking as a method for providing year-round supplies of juveniles and final product handling. The biology of disease and stress are concisely examined, while emerging issues of concern for commercial culture and areas for future research are highlighted. Key words: larvae, broodstock, nutrition, disease, genomics.

25.1 Introduction Cobia, Rachycentron canadum (L 1766), is the only member of the family Rachycentridae (Order Perciformes). It is a warm-water species found in tropical and subtropical waters and, other than in the Eastern Pacific, has a global distribution. There are reports of cobia in eastern Mediterranean waters (Golani and Ben Tuvia, 1986) and in this instance the species can likely be regarded as lessepsian. Nevertheless, although the intermingling of Red Sea and Mediterranean species, via the Suez Canal, has been remarked on for some time (Keller, 1882) the potential also exists for the migration of cobia into the Mediterranean via the Strait of Gibraltar (Golani et al., 2002). The global distribution of cobia means that for most areas suitable for cobia aquaculture there is limited or no need to import alien broodstocks, thereby reducing potential negative impacts on regional biodiversity through escapees and the importation of diseases. The first report of cobia larval rearing was that of Hassler and Rainville (1975), but the species was first farmed commercially in Taiwan during the early 1990s (Liao et al., 2004). Since that time, the industry has expanded to include commercial producers in the Bahamas, Belize, China, the

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Dominican Republic, Mexico, Panama, the Philippines, Puerto Rico, the USA and Vietnam. Feasibility studies for cobia culture are presently being considered or are underway in a number of other nations including Angola, Australia, Brazil, Burma, Cuba, Ecuador, Indonesia, Iran, Reunion, Saudi Arabia, the Sultanate of Oman and the Netherlands. Cobia experienced a 7000-fold increase in production between 1995 and 2005 (FAO, 2007), and this explosion in interest has resulted in no small part due to its impressive growth rates (Lunger et al., 2007a), with fish of 6–10 kg being grown over a 12 month period under ideal conditions. Cobia are amenable to rearing in ponds, net-pens and recirculating life support systems (Schwarz et al., 2006). Their flesh is of high quality (Duncan et al., 2007), and cobia readily accept commercial feed pellets (Craig et al., 2006); respond well to alternate dietary protein sources (Lunger et al., 2006, 2007b); and are passive under captive conditions. While exhibiting commonly encountered diseases of warm-water fishes (McLean et al., 2008), cobia appear to be hardy under intensive cultivation. Moreover, when compared against other farmed species of similar market size (salmon, halibut), their short generation time provides a rapid means to develop selective breeding programs. Even given the wealth of advantages that this candidate species offers, there nevertheless exist a number of technical and biological problems that must be resolved prior to anticipating the real potential that this carnivorous species offers global aquaculture.

25.2 Broodstock and spawning 25.2.1 Natural spawning cycles Cobia are batch spawners, and in US waters histological data have substantiated spawning from April through September (Brown-Peterson et al., 2001). In Taiwan, spawning peaks occur from February to May with intermittent spawns as late as October (Liao et al., 2001). A mean relative batch fecundity of 53.4 ± 9.4 g−1 ovary-free body weight has been established for US stocks (Franks and Brown-Peterson, 2002), with animals spawning once every 5–12 days (Brown-Peterson et al., 2001). Females may mature at 700 mm fork length, whereas males appear to mature at 1+ (Smith, 1995). Males exhibit spermatogenesis between February and August although the testes contain sperm year-round (Franks and Brown-Peterson, 2002). As with other species, ovarian development and maturation is associated with increased protein (49–55 %) and lipid (21–41 %) presence (Biesiot et al., 1994). Limited information is available on between-batch egg quality or age-related declines in fecundity in captive populations (Faulk and Holt, 2008), but similar studies in wild animals have not been undertaken. Clearly significant gains could be attained in terms of hatchery management with a more in-depth appreciation of differences in egg quality, fertilization rates and larval survival between batches.

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New technologies in aquaculture

25.2.2 Captive spawning Captive cobia have been reported to spawn spontaneously in seawater flowthrough ponds of 400–600 m2 and 1.5 m depth held at 23–27 °C (Liao et al., 2004). Brood fish were stocked at a male to female ratio of 1 : 1 and fed raw fish and squid once daily. Successful spawning has also been attained in fish held in tanks (Arnold et al., 2002). Cobia have been induced to spawn using intramuscular injection of human chorionic gonadotropin (275 IU kg−1 body weight; Caylor et al., 1994; Franks et al., 2001) and implantation of a slow-release preparation of 150 μg of salmon GnRHa (Kilduff et al., 2002). Success has also been attained using photothermal conditioning (Kaiser and Holt, 2004). In the latter case, eggs were available from March through November and expressed hatch rates of 80–90 % at salinities of 28–36 g L−1, a 12/13 : 12/11 photophase–scotophase and temperatures of 25–26 °C (Holt et al., 2007). Sex ratios of two males to one female, at tank stocking densities of 1–1.9 kg m3, have provided consistent success (Benetti et al., 2008a). Temperatures from 24–29 °C are considered appropriate for spawning which generally occurs during the late afternoon (Liao et al., 2001), sometimes following aggressive courtship behavior (Kaiser, 2004, pers. comm.). Anecdotal observations suggest enhanced larval production when eggs are derived from ‘natural’ rather than induced spawns, and this possibility has been confirmed using gene expression profiling of eggs from other species (Bonnet et al., 2007a). Of greater concern with respect to egg quality, however, are the negative consequences of environmental manipulation of spawning on oocyte gene expression profiles (Bonnet et al., 2007b). Whether similar detrimental effect would be anticipated for cobia will, however, have to await the development of a species-specific microarray. Batch fecundity of ∼18 kg females is approximately 1 million eggs per spawn (Faulk and Holt, 2008).

25.2.3 Eggs and collection Methods employed to collect fertilized eggs vary from the use of specialized side-plumbed egg collection tanks in recirculation systems through to seining in ponds. Eggs, the buoyancy of which is assisted by a single oil globule (see Holt et al., 2007 for further discussion), are cream-colored, transparent and spherical in form with a diameter of between 1.24 and 1.40 mm (Ditty and Shaw, 1992; Liao et al., 2001; Fig. 25.1). The egg’s perivitelline space is thin, embryos are pigmented and, as with other species, the duration of incubation is temperature and egg-size dependent; approximately 24 h at 29 °C and 30 h at 24–26 °C (Liao et al., 2001; Table 25.1). Cobia larvae hatch at about 2.5–3.0 mm (Fig. 25.1). As considered by Faulk and Holt (2008), the biochemical composition and quality of spawned eggs is dependent on a number of factors including genetic differences, broodstock diet, environmental factors, fish age, size and in all likelihood also their health status.

Cobia cultivation

807

Fig. 25.1 Photographs illustrating egg and larval cobia sizes at various points during the production cycle. Eggs were of approximately 1 mm diameter, while 1 day post-hatch (dph) larvae averaged 3 mm in length. Larvae more than doubled their length by 10 dph and continued impressive growth through to enforced weaning which, in this study, occurred at 22 dph. By 27 dph, weanlings were over ten times their hatch length.

25.3 Larval rearing 25.3.1 Feeding protocols, live feeds and enrichments The production of high-quality weanlings represents the greatest limitation to the more rapid expansion of cobia aquaculture on a global basis. Using contemporary rearing technologies and feeding protocols (Fig. 25.2), larval survival remains relatively poor when compared against other cultured marine fishes. Major larval die-offs are generally associated with shifts in feeding strategy during the production cycle, highlighting our limited knowledge of larval cobia nutrition. Few studies have been undertaken with respect to optimizing larval stocking densities and even fewer have examined optimum prey densities and feeding frequencies. This is surprising given that the culture of live feeds represents a major expenditure associated with the production of weanling cobia and may embody 80 % or more of total dietary costs. A future imperative, therefore, must be to determine elite stocking densities, optimal environmental parameters and feeding protocols for the species with an underlying theme of maximizing survival and production per unit of volume while concurrently reducing time to weaning. Several tactics have been considered with these goals in mind. Traditional live feeds (rotifers and Artemia) used by marine fish hatcheries have long been considered nutritionally inferior as prey items, and Hassler and Rainville (1975) confirmed this assumption for cobia larvae; superior growth performance was observed when larval cobia were offered wild

na = not applicable.

Flow-through ponds Flow-through ponds Flow-through tanks Flow-through tanks Recirculating Recirculating Recirculating Recirculating Recirculating Recirculating Recirculating Recirculating

22–31 26.5–30.7 29.4–31.8 29.4–31.8 28–28.5 26.5 ± 0.3 27 27 27.5 ± 0.4 27.4 ± 0.5 28–29.5 28–29

Temperature (°C) NA 0.06 5 10 10 3 5 10 14.7 8.7 10 10

Stocking density (larvae L−1) 5–10 5.3–8.5 31.4–34.9 17.5–19.2 22.2–28.2 4.4–15.6 12.7 ± 0.9 9.4 ± 0.7 10.4 13.2 21.8–28.2 21.8–28.2

Survival (%)

Salinity (g L−1) na na 26–34 26–34 24 34.5 ± 0.1 32.5 ± 0.5 32.5 ± 0.5 32–34 32–34 32–34 24

Final length (mm) ∼80 (45 dph) 124 (35 dph) 26.1 (21 dph) 20.4 (21 dph) 21 (25 dph) 11.8–15.2 (16 dph) 13.9 (21 dph) 14.1 (21 dph) 14.6 (22 dph) 14.7 (22 dph) 14.7 (22 dph) 19.7 (25 dph)

Liao et al., 2004 Weirich et al., 2004 Benetti et al., 2008b Benetti et al., 2008b Schwarz et al., 2006 Faulk and Holt, 2005 Hitzfelder et al., 2006 Hitzfelder et al., 2006 Faulk et al., 2007 Faulk et al., 2007 Holt et al., 2007 Salze et al., 2008

Reference

Summary results of various studies on cobia larviculture in ponds, flow-through tanks and recirculating life support systems

System employed

Table 25.1

Cobia cultivation

Length (mm)

Photoperiod:

24 h

809

20:4 h

20 15 10 5

Nanochloropsis1 Brachionus plicatilis2 Artemia AF naupli3 Artemia EG naupli4 Prepared diet 0

3

6

9

12

15

18 21 24 Days post-hatch

Fig. 25.2 Summary of feeding protocol used for production of weanling cobia. Green water, using 120 000 cells ml−1 Nannochloropsis sp. algal paste; 2Two L-type rotifers mL−1 water; 3Artemia at 1.5–3.5 individuals mL−1; algae and rotifers were added to the culture tanks simultaneously every 6 h. 3Non-enriched, small-sized (∼430 μm) improved HUFA profile AF-Artemia nauplii were fed every 6 h. Densities used were 1.5 nauplii mL−1 at 6 dph, 2.5 nauplii mL−1 at 7 dph and 3.5 nauplii mL−1 at 8–9 dph. 4From 8–11 dph, 1.5–3.5 EG-Artemia mL−1 were added to the tank followed by the identical density of enriched (24 h with DC DHA Selco) EG-Artemia nauplii from 12–21 dph every 6 h.

1

zooplankton. Such observations have led to the more involved management of live feeds in terms of their composition as well as prey size presented. Various enrichments of rotifers and Artemia, and especially those with docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (ARA), are employed to enhance their nutritional value and in attempts to speed the weaning process (Fig. 25.2). Other enrichments have also been considered in efforts to enhance live feed functionality. These include the use of nucleotides and probiotics, putative immunostimulants, essential amino acids, novel protein supplements and mineral sources. Salze et al. (2008) reported that enrichment of live feed with a mannan oligosaccharide derived from the yeast cell wall enhanced maturation of the cobia gut and increased the ability of larvae to withstand salinity challenge. The former response is deserving of future investigation with respect to examining whether such fish may be weaned at an earlier time point. The latter response could be of importance with respect to the increasing use of inland hatcheries that are less able to control salinity.

25.3.2 Microdiets and weaning In efforts to reduce dependence on live feeds and as a means to wean larvae earlier, a number of groups have examined the feasibility of their

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New technologies in aquaculture

replacement using microdiets. Several difficulties, however, have been encountered with these formulations. Problems associated with their palatability, size uniformity, water column residency time, digestibility and ingestion rates have resulted in poor growth and survival (Fletcher et al., 2007). The main factors accredited with reducing larval survival include nutritional inadequacies, erroneous feeding strategies (Yúfera and Darias, 2007) and digestive insufficiencies (Rønnestad et al., 2007) ultimately leading to starvation. To date no published information is available on the use of microdiets with cobia, but this area of investigation clearly warrants greater attention. A new strategy presently being addressed incorporates development of an optimized dry microparticulate reference weaning diet. It is anticipated that once preliminary studies have been completed this will aid sequential elimination of later live feed stages with the ultimate goal of complete live feed replacement. In addition to palatability, chemoattractant and buoyancy improvements, these new microparticulate diets will address the critical issue of nutrient leaching using a novel matrix binding manufacturing process. As with live feeds, experimental cobia weaning diets too have been fortified with nucleotides and probiotics (Didoha, 2004), putative immunostimulants (Salze et al., 2008), essential amino acids, novel protein supplements and mineral sources and feeding attractants. Although these diets have resulted in variable success, outputs of weaned fish of around 3 L−1 (Fig. 25.2; Salze et al., 2008) have materialized with such dietary manipulations. No studies have examined the relationship between optimal prey/feed size and mouth gape for cobia and, from an energetics perspective, this avenue of research might prove advantageous.

25.3.3 Systems design A critical issue with respect to larval rearing of any species is maintaining the developing animal in the optimal position in the water column. Subtle modifications to the hydrodynamic environment represent an additional approach for enhancing predator–prey and larvae–feed interactions while taking account of tank cleansing and mixing processes (Rasmussen et al., 2005). Problems relating to the maintenance of microdiets in the water column for extended periods might also be solved with judicious adjustments to tank hydraulics. The application of horizontal and vertical circulation patterns appears to have been successful in preserving larval cobia and their feed particles in a set ‘donut’ position within rearing tanks (Fig. 25.3). An added advantage of this hydrodynamic manipulation was an apparent reduction in physical damage due to wall and standpipe larval interactions and, until 17 dph, a reduction in cannibalism (Salze et al., 2008). It may be that cannibalism could be reduced with finer tuning of tank hydrodynamics, development of an effective larval grading system and/or by increasing dietary loading.

Cobia cultivation To blower Skimmer

Cut-off valve

811

Bead filter

Biofilter

Heater

Standpipe

Air line Primary feed line

uv

Air line

Position of larvae

Return line

1° Feed line Return line

Convectional flow

Mesh screen

Rotational flow

Fig. 25.3 Custom-designed cobia larval rearing system. The airline at the bottom of each tank provides vertical lift which, when combined with horizontal surface water inflow, creates a rotating cell in the upper third of the tank’s water column. Developing fish, together with live feed and/or prepared diets, are held in the rotating cell until individuals gain independent swimming and positioning ability. The rotating cell separates larvae, thereby reducing cannibalism while also preventing in-tank collisions of larvae with walls and standpipes.

25.3.4 Broodstock diets Upon weaning, high survival rates are the norm for cobia. Production of weanlings will inevitably increase, however, with the development of enhanced broodstock dietary formulations to ensure optimal fecundity, gamete quality and larval survival between egg batches, as has been attained with other species (Bromage, 1995; Watanabe and Vassallo-Agius, 2003). Elite nutrition, and especially that of the female brood, will be of particular importance in ensuring yolk quality and hence optimizing endogenous nutrition of developing larvae (Rainuzzo et al., 1997). Dietary deficiency in omega-3 fatty acids and protein, for example, are established to negatively impact gamete viability and larval survival (Kah et al., 1994), whereas inappropriate dietary ratios of polyunsaturated fatty acids (PUFA) affect circulating levels of androgens with the outcome of asynchrony of maturation between sexes (Cerdà et al., 1997). Enhancement of brood diets with vitamin C, perhaps due to its antioxidant capacity, improves sperm motility, concentration and fertility (Mangor-Jensen et al., 1994) while PUFA enrichment increases reproductive performance in terms of egg quality and larval development (Ma et al., 2005). However, for cobia no information is presently available on optimized broodstock diets, and this clearly represents an important production limitation which demands immediate attention.

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New technologies in aquaculture

Currently, broodstock are fed on diets of fresh and frozen cut fish and squid at approximately 4 % body weight day−1.

25.4 Juveniles and on-growing 25.4.1 Juvenile nutrition As with weanling production, the most expensive operating variable to which on-growing producers are exposed is feed costs (Bassompierre et al., 1997). Because cobia are a relatively new species to aquaculture, quantitative nutritional data for this fish have only started to accumulate recently. Clearly, therefore, while commercially formulated diets are readily accepted, it is highly likely that these diets are imperfect, and this is especially so for larger animals due to researchers concentrating on juvenile fish. Optimal dietary protein and lipid levels for juvenile cobia were determined by Chou et al. (2001) as 44.5 % and 5.76 % of dry diet, respectively. Conflicting reports have appeared, however, regarding the protein-sparing effect of dietary lipids. For example, Her et al. (2001) reported that cobia fed a diet containing 37 % protein and 15 % lipid exhibited statistically similar growth and feed conversion ratios to cobia fed diets containing 41 and 45 % protein along with high energy levels, indicating a potential protein-sparing effect by dietary lipid. Conversely, Craig et al. (2006) did not observe proteinsparing by lipid in a 3 × 2 factorial study with protein levels of 40 and 50 % crude protein and lipid levels of 6, 12 and 18 % total lipid (dry weight basis). There were no significant differences in weight gain or feed efficiency ratio values in these two trials, which were performed utilizing 44 and 12 g juvenile cobia. Wang et al. (2005) also investigated the impacts of dietary lipid on growth, feed utilization, lipid deposition and lipid metabolism of juvenile cobia. Results from this study showed that lipid levels higher than 15 % had a negative effect on growth and, as lipid level increased, feed intake declined. Results from the study of Wang et al. (2005), therefore, suggest that lipid levels above 15 % provided no production benefit due to fat accumulation and poorer growth and that no protein-sparing by lipid accrued. Nevertheless, it would appear that cobia are able to accept diets expressing higher lipid formulations, and these may be used to supply finished products for niche markets such as that seen for sashimi (thinly sliced raw fish traditionally served with soy sauce and wasabi). Future studies with high dietary lipids, however, should take account of animal health and welfare and environmental impact.

25.4.2 Replacement proteins Cobia appears well-suited and able to accept alternate proteins as replacements for fish meal. Indeed, dietary inclusion levels of alternate proteins (soybean meal, soy concentrate and isolate, hemp meal and yeast proteins)

Cobia cultivation

813

up to 40 % of dietary protein, without detrimental impacts upon production characteristics, have been reported (Chou et al., 2004; Lunger et al., 2006, 2007a; Zhou et al., 2005). Supplementation of soy protein-based diets with different amino acids appears to enhance juvenile cobia performance (Lunger et al., 2007a; Liou and Chen, 2007) and, inevitably, greater gains will be made with increased information on essential amino acid requirements for the species. Dietary lysine requirement for juvenile cobia has been determined as 2.33 % dry diet (Zhou et al., 2007) whereas that for methionine as 2.64 % dry diet (Zhou et al., 2006). The possible negative impacts of alternate proteins on final product quality of cobia have yet to be studied in detail, although Lunger et al. (2007b) reported higher texture measurements in fillets from cobia fed alternate plant proteins when compared to fish fed a fish meal control diet. Higher texture characteristics derived from fish meal substitution could become a major factor in the development of value-added products such as surimi or fish cakes/sticks (Duncan et al., 2007). In addition, there are reports of complete replacement of fish meal with organically-certifiable protein sources in juvenile cobia (Anon, 2007). The impacts of these types of formulations upon end-product quality must be determined and validated, and most likely, applied to specific markets for optimal economic returns (e.g. the organic sector).

25.4.3 Cold banking At the present time there exists a paucity of cobia hatcheries and it is not possible to obtain year-round supplies of weaned animals. Under ideal conditions grow-out facilities should have complete flexibility in terms of animal production to ensure that the systems used, whether tank, pond or net-pen, are always in production mode. The lack of weanling supply therefore, represents an impediment to production efficiencies and potential. Although experimental hatcheries have provided spawning animals between March and November using photothermal manipulations, until sufficient captive broodstocks become available to supply weanling demands yearround, alternative methods of maintaining uninterrupted supplies of juveniles must be examined. One method that may prove useful in this regard is cold banking which involves maintaining younger animals at a smaller size for use in later production without negatively impacting their overall performance characteristics. When fish are held at sub-optimal temperature, their growth becomes restricted due to the reduction in energy intake brought about by reduced feeding rates – the so-called ‘scope for growth’ concept (Brett, 1979). For juvenile cobia the median lethal low temperature has been reported as 12.1 °C (Atwood et al., 2004) whilst that for lethal upper temperatures is 37.7 °C (Shaffer and Nakamura, 1989). By holding larger cobia (∼300 g) at 18 °C Schwarz et al. (2007) reported that it was feasible to maintain fish at a basal metabolic level (i.e. no significant growth)

814

New technologies in aquaculture

for ten or more weeks without detriment to their future performance. Until the availability of high-quality weaned juvenile cobia becomes year-round in supply, this technique might prove commercially valuable as a stop-gap measure for supplying cobia out-of-phase in order to ensure consistent supplies of market-sized products. However, the impacts of cold banking on smaller cobia are deserving of further investigation since anecdotal information indicates that juveniles under 50 g may suffer from skeletal deformities and fin fraying when held at 18 °C for longer than 60 days.

25.4.4 Low-salinity culture The ability to rear cobia in low-salinity waters would give rise to an inland aquaculture industry, located miles away from expensive coastal properties and far from the damaging impacts of typhoons, hurricanes or Nor’Easters. While several marine species such as red drum and southern flounder have shown the ability to thrive in low-salinity conditions, the impacts of a lowsalinity environment have not been fully described in juvenile cobia. In a preliminary low-salinity trial, juvenile cobia were fed a control diet containing 47 % crude protein and 10 % lipid (dry weight basis) in identical recirculating life support systems. Five additional experimental diets were made by addition of various ions to the control diet, at the expense of cellulose. The ions of interest were calcium chloride (CaCl2), magnesium chloride (MgCl2), potassium chloride (KCl) and copper sulfate (CuSO4), both singly and in combination, and gave rise to the following diets: (i) control; (ii) CaCl2 @ 1 % of dry diet; (iii) MgCl2 @ 0.10 % of dry diet; (iv) CaCl2 and MgCl2 at the previous concentrations; (v) CaCl2, MgCl2 and KCl (1 % of diet); and (vi) CaCl2, MgCl2, KCl and CuSO4 (0.2 g kg−1 diet). The control diet was fed to cobia held in water of 20 g L−1 salinity while the experimental diets were fed to fish held at a salinity of 2 g L−1. The feeding trial was of six weeks duration. Growth, measured as percent increase from initial weight, was significantly impacted by the low-salinity environment (Fig. 25.4). Ion addition did not improve weight gain, although the diet containing Ca, Mg and K outperformed the remaining ion addition diets at lowsalinity conditions. Clearly, while juvenile cobia can survive and grow under low-salinity conditions, weight gain will be compromised unless further dietary refinements are made with respect to ion and/or salt additions to cobia feeds. Nevertheless, low-salinity technologies offer the potential to produce cobia to market size near inland cities while also offering production of juveniles for net-pen and pond stocking purposes. The optimal size of animal for net-pen operations has, however, yet to be determined.

25.4.5 Harvesting, processing and marketing Limited information is available on harvesting strategies used with cobia, but in Taiwan net-pen reared fish are cropped at around 6 kg wet weight

Cobia cultivation

Percent increase in weight

800

a

c

b

1

2

3

b,c

b

b,c

b

4

5

6

7

815

600

400

200

0

Diet

Fig. 25.4 Percent increase from initial weight of juvenile cobia (mean initial weight of 23 g) subjected to low-salinity conditions (2 g L−1). Fish fed diet 1 were held at 20 g L−1 and served as a control whereas the remaining diets were fed to cobia maintained at 2 g L−1. Fish fed diet 7 served as a negative control (i.e. diet 1 at low-salinity). Diet 2 was supplemented with CaCl2 (1 % of dry diet), diet 3 with MgCl2 (0.1 % dry diet), diet 4 with CaCl2 + MgCl2 (1 and 0.1 % diet, respectively), diet 5 with CaCl2 + MgCl2 + KCl (1, 0.1 and 1 % diet, respectively), diet 6 was identical to diet 5 but included CuSO4 at 0.2 g kg−1 diet. Significant differences are denoted by different letters as determined by ANOVA and, where appropriate, Duncan’s multiple range test (P < 0.0004).

which may be achieved in 10–12 months (Liao et al., 2004; Fig. 25.5). Incremental, selective and grading-based harvesting methods are used. In some areas, small numbers of pen-reared fish are selectively harvested year-round based on market demands. Methods for harvesting mimic that of the salmon industry, and it is inevitable that salmon-based harvest technologies will ultimately be employed by cobia net-pen operators (e.g., piscalators, fish pumps). Animals are fasted for 24 h before harvest after which they are bled and chilled prior to transportation to processing plants. Processed products include head-on/off, whole/gutted, fresh (iced)/frozen, smoked and fillet (skin on/off)-based fish. Product exported to Japan from Taiwan is mainly for sashimi. In the USA and EU, farmed cobia have entered the lucrative white table cloth sectors with high acceptability while gleaning premium prices. Product testing for the live market has been attempted and considered both in Asia and the USA with smaller individuals (1–2 kg). However, the predicted rapid expansion of cobia production globally will saturate most high-end and niche markets to ultimately drive product into mainstream distribution and eventually commodity sectors. Production of breaded cobia and cobia-based fish patties and sticks for institutional and

816

New technologies in aquaculture 6

kg

Cobia

Red drum

Salmon

3

0 10

20

30

Months

Fig. 25.5 At present, cobia are harvested at larger sizes (∼6 kg wet weight), although optimal size at harvest has yet to be established for different markets. Relative to other farmed species the growth rates of caged cobia are truly impressive.

conventional markets has been discussed at a number of levels. Survival in these sectors will mandate a high-quality, safe and wholesome product produced using environmentally sustainable methods with absolute traceability, and Taiwan has already commenced development of criteria for product traceability and supply chain management for cobia (Chen et al., 2007). A novel commodity derived from cobia is leather which is subtle and scaled similar to that of salmon. Cobia leather has been stitched as panels for the production of shoes and smaller leather goods.

25.5 Emerging issues and future trends 25.5.1 Diseases On an annual basis, disease costs the global aquaculture industry billions of dollars and losses due to pathogens have been reported with increasing incidence in farmed cobia. Cobia succumb to the usual array of parasites and diseases found in other warm-water species (McLean et al., 2008), and disease is recognized as the single most important factor limiting expansion of caged cobia operations (FAO, 2007). In Taiwan, serious photobacteriosis outbreaks in caged cobia, some of which resulted in 80 % mortalities, spurred interest in the development of vaccines (Chen, 2001; Lin et al., 2006) and application of dietary immunostimulants (Lean˜o et al., 2003; Chang et al., 2006). Significant success in reducing losses to photobacteriosis using these tactics should provide the impetus for the development and application of other existing and novel preparations for disease control. Neverthe-

Cobia cultivation

817

less, more stringent disease preventative and screening measures must be developed for hatchery and grow-out facilities throughout production. This might include use of disease-specific biomarkers for the development of functional diagnostic chips, breeding of specific pathogen-free broodstocks for the supply of disease-free eggs and greater use of biosecure recirculating life support systems for all stages of production. Larval fishes are exposed to microorganisms immediately after hatching. Throughout intensive cultivation, and especially during static culture phases, the level of larval exposure to microbes increases greatly. Many species encounter potentially pathogenic viruses and bacteria that are ubiquitous and simply representative of the rearing environment. In order to survive this hostile milieu, larval teleosts must possess an efficient immune system. The ontogeny of the larval cobia immune system, both in terms of the functional development of specific tissues (i.e., anterior kidney, thymus and spleen) and the ability to mount (non-) specific immune defenses, however, remains unknown. Because the point at which immunocompetence is achieved represents a vital factor in establishing precisely when developing larvae are most susceptible to disease and what treatments, if any, may be used to combat infections, greater research emphasis in this area is essential. Cnaani and McLean (2009) reported baseline levels of 1079 ± 139 U ml−1 and 1.19 ± 0.46 IU for circulating lysozyme and ceruloplasmin, respectively, in sub-adult cobia, and preliminary information indicates that hematologically they are replete with the usual battery of lymphocytes, granulocytes, monocytes and thrombocytes (McLean et al., 2007). Hypoxia of sub-adult animals caused a rapid increase in circulating cortisol, from baseline levels of 6.7 ng ml−1 to over 70 ng ml−1 1 h post-stress. This cortisol response was matched by disturbance in blood glucose levels, which rose from baseline values of 79 mg dl−1 to 179 mg dl−1 after 2 h (Cnaani and McLean, 2009). Likewise, ceruloplasmin levels also increased following hypoxic stress whereas lysozyme levels declined. These studies thereby indicate that cobia respond to stress in a manner that is similar to many other marine species and that ceruloplasmin and cortisol can be employed as physiological indicators of stress in cobia.

25.5.2 Aquanomics Genomic or ‘aquanomic’ (McLean and Craig, 2006; McLean et al., 2009) technologies will inevitably play an increasingly important role in all aquaculture sectors, and already transcriptomic, or microarray, techniques have impacted the formulation of in-house specialty cobia feeds. Other aquanomic technologies will, unavoidably, have significant impacts on selective breeding programs, enhancing our understanding of digestive physiology and hence the design of feeds for larvae, juveniles, adults and broodstocks. Aquanomic techniques will also play an increasingly important role in assisting selection of sustainable alternate protein and lipid supplies for

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New technologies in aquaculture

feeds and, as has been the case in terrestrial animals, these methods will aid detection and developing solutions for specific end-product quality issues inclusive of development of designer fish for specific markets (Cotter et al., 2008, 2009).

25.6 References anonymous (2007) Fishmeal-free cobia, Fish Farm Int, 34(1), 3. arnold c r, kaiser j b and holt g j (2002) Spawning of cobia (Rachycentron canadum) in captivity, J World Aquac Soc, 33, 205–8. atwood h l, young s p, tomasso j r and smith t i j (2004) Resistance of cobia, Rachycentron canadum, juveniles to low-salinity, low temperature, and high environmental nitrite concentrations, J Appl Aquac, 15, 191–5. bassompierre m, kjaer a and mclean e (1997) Simulating protein digestion on trout: a rapid and inexpensive method for documenting fish meal quality and screening alternative protein sources for use in aquafeeds, Ribarstvo, 55, 137–45. benetti d, orhun m r, sardenberg b, o’hanlon b, welch a, hoenig r, zink i, rivera j a, denlinger b, bacoat d, palmer k and cavalin f (2008a ) Advances in hatchery and grow-out technology of cobia Rachycentron canadum (Linnaeus), Aquac Res, 39, 701–11. benetti d, sardenberg b, welch a, hoenig r, orhun m r and zink i (2008b) Intensive larval husbandry and fingerling production of cobia Rachycentron canadum, Aquaculture, 281, 22–7. biesiot p m, caylor r e and franks j s (1994) Biochemical and histological changes during ovarian development of cobia, Rachycentron canadum, from the Northern Gulf of Mexico, Fish Bull, 92, 686–96. bonnet e, fostier a and bobe j (2007a) Microarray based analysis of fish egg quality after natural or controlled spawning, BMC Genomics, 8, 55. bonnet e, montfort j, esquerre d, hugot k, fostier a and bobe j (2007b) Effect of photoperiod manipulation on rainbow trout (Oncorhynchus mykiss) egg quality: a genomic study, Aquaculture, 268, 13–22. brett j r (1979) Environmental factors and growth, in Hoar W S, Randall D J and Brett J R (eds), Fish Physiology, Vol. VIII, Bioenergetics and Growth, Academic Press, New York, 599–675. bromage n r (1995) Broodstock management and seed quality – general considerations, in Bromage N R and Roberts R J (eds), Broodstock Management and Egg and Larval Quality, Blackwell Science, Oxford, 1–24. brown-peterson n j, overstreet r m, lotz j m, franks j s and burns k m (2001) Reproductive biology of cobia, Rachycentron canadum, from coastal waters of the southern United States, Fish Bull, 99, 15–28. caylor r e, biesiot p m and franks j s (1994) Culture of cobia (Rachycentron canadum): cryopreservation of sperm and induced spawning, Aquaculture, 125, 81–92. cerdà j, zanuy s and carrillo m (1997) Evidence for dietary effects on plasma levels of sexual steroids during spermatogenesis in the sea bass, Aqua Int, 5, 473–7. chang c f, yang j h and chang s l (2006) Application of dietary β-1,3-1,6-glucan in enhancing resistance of cobia (Rachycentron canadum) against Photobacterium damselae subsp. piscicida and Streptococcus iniae infections, J Taiwan Fish Res, 14, 75–87. chen f l, chuang c t, hu s h and nan f h (2007) Traceability and supply chain management for cage culture industry in Taiwan – the case of cobia, Development

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and Adoption of Traceability System for Fish and Fish Products in Asia, Denpasar, Bali, November 26–30. chen h e (2001) Evaluation of antigens from Photobacterium damselae subsp. piscicida using cobia (Rachycentron canadum) antiserum, MSc Thesis, National Cheng Kung University, Tainan, Taiwan (in Chinese). chou r l, her b y, su m s, hwang g, wu y h and chen h y (2004) Substituting fish meal with soybean meal in diets of juvenile cobia, Rachycentron canadum, Aquaculture, 229, 325–33. chou r l, su m s and chen h y (2001) Optimal dietary protein and lipid levels for juvenile cobia (Rachycentron canadum), Aquaculture, 193, 81–9. cnaani a and mclean e (2009) Time-course response of cobia (Rachycentron canadum) to acute stress, Aquaculture, 289, 140–142. cotter p a, mclean e and craig s r (2008) Hyperaccumulation of selenium in hybrid striped bass: a functional food for aquaculture? Aquac Nutr, 14, 215–22. cotter p a, mclean e and craig s r (2009) Designing fish for improved human health status, Nutr Health, 19, 1–9. craig s r, schwarz m h and mclean e (2006) Juvenile cobia (Rachycentron canadum) can utilize a wide range of protein and lipid levels without impacts on production characteristics, Aquaculture, 261, 384–91. didoha a v (2004) A review on the probiotics in finfish culture and preliminary research on the employment of probiotics in early developmental stages of cobia Rachycentron canadum, M Thesis, Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, FL. ditty j g and shaw r f (1992) Larval development, distribution, and ecology of cobia Rachycentron canadum (Family Rachycentridae) in the Northern Gulf of Mexico, Fish Bull, 90, 668–77. duncan m, craig s r, lunger, a n, kuhn d d, salze g and mclean e (2007) Bioimpedance assessment of body composition in cobia Rachycentron canadum (L. 1766), Aquaculture, 271, 432–8. fao (2007) Cultured aquatic species information program: Rachycentron canadum, Food and Agriculture Organization of the United Nations, Rome, http://www.fao. org/fi/website/FIRetrieveAction.do?dom=culturespecies&xml=Rachycentron_ canadum.xml, accessed January 2009. faulk c k and holt g j (2005) Advances in rearing cobia Rachycentron canadum larvae in recirculating aquaculture systems: live prey enrichment and green water culture, Aquaculture, 249, 231–43. faulk c k and holt g j (2008) Biochemical composition and quality of captivespawned cobia Rachycentron canadum eggs, Aquaculture, 279, 70–6. faulk c k, kaiser j b and holt g j (2007) Growth and survival of larval and juvenile cobia Rachycentron canadum in a recirculating raceway system, Aquaculture, 270, 149–57. fletcher r c, roy w, davie a, taylor j, robertson d and migaud h (2007) Evaluation of new microparticulate diets for early weaning of Atlantic cod (Gadus morhua): implications on larval performances and tank hygiene, Aquaculture, 263, 35–51. franks j s and brown-peterson n j (2002) A review of age, growth, and reproduction of cobia, Rachycentron canadum, from US waters of the Gulf of Mexico and Atlantic Ocean, Proc 53rd Ann Gulf Carib Fish Inst, 53, 553–69. franks j s, ogle j t, lotz j m, nicholson l c, barnes d n and larson k m (2001) Spontaneous spawning of cobia, Rachycentron canadum, induced by human chorionic gonadotropin (HCG), with comments on fertilization, hatching, and larval development, Proc Carib Fish Inst, 52, 598–609. golani d and ben-tuvia a (1986) New records of fishes from the Mediterranean coast of Israel including Red Sea immigrants, Cybium, 10, 285–91.

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golani d, orsi relini l, massutí e and quignard j p (2002) CIESM Atlas of Exotic Species in the Mediterranean, Vol. 1, Fishes, CIESM, Monaco. hassler w w and rainville r p (1975) Techniques for hatching and rearing cobia, Rachycentron canadum, through larval and juvenile stages, UNC Sea Grant College Program, UNC-SG-75–30, Raleigh, NC. her b y, chou r l, chen t i, su m s and liao i c (2001) Effects of protein/energy ratio on growth of juvenile cobia Rachycentron canadum, 6th Asian Fisheries Forum, Book of Abstracts, 94. hitzfelder g m, hold g j, fox j m and mckee d a (2006) The effect of rearing density on growth and survival of cobia, Rachycentron canadum, larvae in a closed recirculating aquaculture system, J World Aquac Soc, 37, 204–9. holt g j, faulk c k and schwarz m h (2007) A review of the larviculture of cobia Rachycentron canadum, a warm water marine fish, Aquaculture, 268, 553–7. kah o, zanuy s, pradelles p, cerdà j and carrillo m (1994) An enzyme immunoassay for salmon gonadotropin-releasing hormone and its application to the study of the effects of diet on brain and pituitary GnRH in the sea bass, Dicentrarchus labrax, Gen Comp Endocrinol, 95, 464–74. kaiser j b and holt g j (2004) Cobia: a new species for aquaculture in the US, World Aquac, 35, 12–14. keller c (1882) Die Fauna in Suez Canal und die Diffusion der mediterraneen und erythraischen Tierwelt, Naturweiss, 28, 39. kilduff p, dupaul w, oesterling m, olney j and tellock j (2002) Induced tank spawning of cobia, Rachycentron canadum, and early larval husbandry, World Aquac, 33, 35–7. lean˜o e m, chang s l, guo j j, chang s l and liao i c (2003) Levamisole enhances non-specific immune response of cobia, Rachycentron canadum, fingerlings, J Fish Soc Taiwan, 30, 321–30. liao i c, su h m and chang e y (2001) Techniques in finfish culture in Taiwan, Aquaculture, 200, 1–31. liao i c, huang, t s, tsai, w s, hsueh, c m, chang s l and leano e m (2004) Cobia culture in Taiwan: current status and problems, Aquaculture, 237, 155–65. lin j h y, chen t y, chen m s, chen h e, chou r l, chen t i, su m s and yang h l (2006) Vaccination with three inactivated pathogens of cobia (Rachycentron canadum) stimulates protective immunity, Aquaculture, 255, 125–32. liou b s and chen t i (2007) Effects of amino acids added to soy-protein replacement on the growth of juvenile cobia (Rachycentron canadum), J Taiwan Fish Res, 15, 55–61. lunger a n, craig s r and mclean e (2006) Replacement of fish meal in cobia diets using an organically certified protein, Aquaculture, 257, 393–9. lunger a n, mclean e, gaylord t g and craig s r (2007a) Taurine addition to alternative dietary proteins used in fish meal replacement enhances growth of juvenile cobia (Rachycentron canadum), Aquaculture, 271, 401–10. lunger a n, mclean e and craig s r (2007b) The effects of organic protein supplementation upon growth, feed conversion and texture quality parameters in juvenile cobia (Rachycentron canadum), Aquaculture, 264, 342–52. ma, a, chen c, lei j, chen s and zhimin z (2005) The effect of protein and n-3HUFA on the reproduction of turbot (Scophthalmus maximus), Mar Fish Res, 26, 7–12. mangor-jensen a, holm j c, rosenlund g, lie ø and sandnes k (1994) Effects of dietary vitamin C on maturation and egg quality of cod Gadus morhua L, J World Aquac Soc, 25, 30–40. mclean e and craig s r (2006) Nutrigenomics in aquaculture research: a key in the ‘Aquanomic’ revolution, in Jacques K and Lyons P (eds), Nutritional Biotechnol-

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ogy in the Food and Feed Industry: Delivering on the Nutrigenomics Promise, Nottingham, Nottingham University Press, 433–44. mclean e, harris h w, russell d, delbos b d, schwarz m h and craig s r (2007) Cobia Rachycentron canadum hematology, Aquaculture 2007, San Antonio, TX, 26 February–2 March, 587. mclean e, craig j c, dickerman a w and craig s r (2009) Aquanomics – the application of genomic technologies to aquaculture, in Thangadurai D (ed.), Genomics and Proteomics, Tamilnadu, Bioscience Publications, 195–206, in press. mclean e, salze g and craig s r (2008) Parasites, diseases and deformities of cobia, Ribarstvo, 66, 1–17. rainuzzo j r, reitan k i and olsen y (1997) The significance of lipids at early stages of marine fish: A review, Aquaculture, 155, 105–18. rasmussen m r, laursen j, craig s r and mclean e (2005) Do fish enhance tank mixing? Aquaculture, 250, 162–74. rønnestad i, kamisaka y, conceição l e c, morais s and tonheim s k (2007) Digestive physiology of marine fish larvae: Hormonal control and processing capacity for proteins, peptides and amino acids, Aquaculture, 268, 82–97. salze g, mclean e, schwarz m h and craig s r (2008) Dietary mannan oligosaccharides enhance stress resistance and gut development of larval cobia Rachycentron canadum (L. 1766), Aquaculture, 274, 148–52. schwarz m h, mclean e and craig s r (2006) Research experience with cobia: larval rearing, juvenile nutrition and general physiology, in Liao I C and Lean˜o E M (eds), Cobia Aquaculture: Research, Development and Commercial Production, Asian Fisheries Society, Manila, World Aquaculture Society, Baton Rouge, LA, 1–19. schwarz m h, mowry d, mclean e and craig s r (2007) Performance of advanced juvenile cobia reared under different thermal regimes: evidence for compensatory growth and a method for cold banking, J Appl Aquac, 16, 71–84. shaffer r v and nakamura e l (1989) Synopsis of biological data on the cobia, Rachycentron canadum (Pisces: Rachycentridae), FAO Fisheries Synopsis 153 (National Marine Fisheries Service/S 153), U.S. Department of Commerce, NOAA Technical Report, National Marine Fisheries Service 82, Washington, DC. smith j w (1995) Life history of cobia, Rachycentron canadum (Osteichthyes : Rachycentridae), in North Carolina waters, Rimleyana, 23, 1–23. wang j t, liu y j, tian l x, mai k s, du z y, wang y and yang h j (2005) Effect of dietary lipid level on growth performance, lipid deposition, hepatic lipogenesis in juvenile cobia, Aquaculture, 249, 439–47. watanabe t and vassallo-agius r (2003) Broodstock nutrition research on marine finfish in Japan, Aquaculture, 227, 35–61. weirich c r, smith t i j, denson m r, stokes a d and jenkins w e (2004) Pond culture of larval and juvenile cobia, Rachycentron canadum, in the southeastern United States: initial observations, J Appl Aquac, 16, 27–44. yúfera m and darias m j (2007) The onset of exogenous feeding in marine fish larvae, Aquaculture, 268, 53–63. zhou q c, mai k s, tan b p and liu y j (2005) Partial replacement of fishmeal by soybean meal in diets for juvenile cobia (Rachycentron canadum), Aquac Nutr, 11, 175–82. zhou q c, wu z h, tan b p, chi s y and yang q h (2006) Optimal dietary methionine requirement for juvenile cobia (Rachycentron canadum), Aquaculture, 258, 551–7. zhou q c, wu z h, chi s y and yang q h (2007) Dietary lysine requirement of juvenile cobia (Rachycentron canadum), Aquaculture, 273, 634–40.

26 Advances in the culture of lobsters C. M. Jones, Northern Fisheries Centre, Australia

Abstract: Commercial aquaculture of marine lobsters is an attractive proposition, as most species are high value with established market demand, and fishery production is static or diminishing. Nevertheless, achievement of commercial success will necessitate resolution of technical difficulties associated with on-growing of aggressive species (clawed lobsters) or with rearing the larvae, which for spiny and slipper lobsters is generally a painstaking and protracted process. Notwithstanding these technical challenges, increasing market demand for the product is driving a substantial research and development effort around the world to develop commercial lobster farming technology. This chapter reports on the status of that effort, the successes and obstacles. Key words: lobster, aquaculture, palinuridae, hatchery.

26.1 Introduction Aquaculture of lobsters has gained increased importance since the 1990s as demand for these premium crustaceans increases, and supply from traditional fishery sources remains static or diminishes. This stimulus is entirely market driven and, although the marketability of these crustaceans is high, this positive is somewhat countered by the biological challenges of cultivating and growing these species under controlled conditions. In comparison with most existing aquacultured species (fish, shrimp, molluscs), they are more difficult to cultivate, particularly through their larval phase which is generally very protracted (George, 2005). As an intermediate step to full life-cycle aquaculture, the collection of juveniles from the wild and their ‘ranching’ in cages, ponds or other enclosures, is relatively well established for several species. The sustainability of such practice is, however, of great concern. Clearly the future will demand aquaculture technologies that are independent of wild populations, and that are economically and environmentally sustainable. Development of lobster aquaculture is also advantaged by existing, well-established techniques for handling, holding and

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transport of live product, which have evolved as part of commercial fishery and marketing practices for a variety of species. This chapter will outline the current status of lobster culture, with a focus on hatchery-based aquaculture in addition to ranching. It will provide information on the specific biological challenges, their possible solutions and on-going problems regarding the development of commercial rearing technologies. Aspects of captive broodstock management and breeding, egg incubation and hatching, larval rearing and metamorphosis and grow-out will be presented. The future for the commercial production of these species is bright, although several significant challenges will need to be overcome. Prospects for lobster aquaculture have been reviewed several times and as recently as 2000 (Kittaka and Booth, 1994, 2000; Phillips and Evans, 1997), providing comprehensive literature review, particularly for Palinurid lobsters which are currently the most prized regarding aquaculture development. This chapter will therefore focus on the most recent of developments, and particularly those since 2000.

26.2 Current situation and constraints 26.2.1 Introduction Interest in the development of lobster culture has been prominent for many decades (Wickins and Lee, 2002), and several high-profile species have been the subject of comprehensive research and development programs aimed at establishing commercial aquaculture to supplement wild fishery catches, and to meet growing demand. Foremost among these are the two species of Homarus, H. americanus and H. gammarus, the clawed lobsters of the north-west and north-east Atlantic, for which aquaculture technology was keenly sought, but was unattainable due to biological constraints (primarily due to aggressive behaviour) (Conklin, 1983; Aiken, 1988; Addison and Bannister, 1994; Aiken and Waddy, 1995; Factor, 1995; Waddy et al., 1998; Tlusty, 2004). Species of other lobsters, some of which are of lesser market significance, have subsequently been investigated, although the only significant, established industries are based on ranching, that is, the grow-out of wild caught juveniles, a practice which is generally accepted to be unsustainable. The economic attraction of lobster aquaculture and prospects of successful commercial development have been heralded on a number of occasions (Phillips, 1985; Anon., 1989, 1994, 1997; Kittaka and Booth, 1994; Rahman and Srikrishnadhas, 1994; Jeffs and Hooker, 2000; Kittaka and Booth, 2000; Wickins and Lee, 2002; Jeffs and Davis, 2003). Although production is currently negligible, there are positive signs that substantial increase in lobster aquaculture may be imminent. 26.2.2 Species of interest The lobsters of interest for aquaculture development include the clawed variety (Homarus species), the spiny lobsters (Palinuridae) and the slipper

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lobsters (Scyllaridae and primarily Thenus species). Of these, Homarus has been the subject of considerable aquaculture R&D over many decades with no commercial outcome to date and none likely in the foreseeable future (Tlusty, 2004). In recent years, the focus of the research has been on enhancement programs for both European clawed lobster H. gammarus (Bannister, 1998; Browne et al., 1998; Wickins and Gendron, 1998; Knudsen and Tveite, 1999; van der Meeren, 2001) and American lobster H. americanus (Goldstein, 1998; Waddy et al., 1998; Tlusty, 2004; Fiore and Tlusty, 2005), although the economic viability of this appears questionable. This discussion will therefore focus on the species of contemporary priority within the families Palinuridae and Scyllaridae. Attempts to rear spiny lobster larvae date back to the mid-1900s (Kittaka and Booth, 1994, 2000) in Japan, where the foundations of spiny lobster culture were laid. Panulirus japonicus is an icon species of particular interest, although research also examined larval rearing for Jasus lalandii, Palinurus elephas, J. verreauxi and J. edwardsii (Phillips and Evans, 1997; Kittaka and Booth, 2000) over the past several decades. Elsewhere, isolated attempts were occasionally made to rear spiny lobster larvae such as for Panulirus interruptus in 1975 (Serfling and Ford, 1975), although no substantial, on-going program of aquaculture development occurred outside Japan, until the 1990s, when scientists in New Zealand successfully reared J. edwardsii (Illingworth et al., 1997) and J. verreauxi (Moss et al., 2000) from egg to puerulus. Since then, in Australia, pueruli of J. edwardsii and J. verreauxi have been reared in Tasmania and P. ornatus in Queensland and Western Australia. In addition to the spiny lobsters, there has also been some interest in the slipper lobsters (Scyllaridae) and particularly Thenus species (Mikami and Greenwood, 1997; Anon., 2007; Jones, 2007a).

26.2.3 Lobster ranching Although there is widespread interest and developmental industries in several countries, the only substantial production of lobsters by way of ranching is in Vietnam, where Panulirus ornatus is cultured in sea cages, from a supply of wild-caught post-larvae and juveniles (Thuy and Ngoc, 2004; Williams, 2007). In 2006 Vietnam produced approximately 2000 tonnes of 1 kg+ lobsters, valued at over $US60 million. Indonesia, Philippines, India and Australia have also expressed some level of interest in aquaculture of this species, although production to date is negligible. The interest is driven by market opportunity, specifically for large (1 kg+) P. ornatus lobsters, which are in great demand in China for banquet dining, as a sashimi product. In Vietnam the lobster grow-out industry has grown rapidly from its inception in 1996 to a substantial industry (Tuan and Mao, 2004). One of the advantages of the industry is that it is based on many individual, small-scale businesses, with low capital requirements, a lowtechnology approach and is village-based. There are currently some 35 000

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sea cages, utilising up to 2 million puerulus and post-puerulus collected per year. The lobsters are fed a diet consisting entirely of unprocessed fishery by catch, including small fish, molluscs and crustaceans. Lobsters can be grown from puerulus to 1 kg within an 18–24 month period. Of concern, however, is the sustainability of the industry, based as it is on wild caught seed. Further, over development of sea cage facilities with associated, inefficient feeding practices is leading to environmental issues and disease. Government intervention will be required to provide better industry practices that are more sustainable. Expansion of the industry is limited by the wild catch of seed, and the long-term viability of the industry will depend on successful development of larval rearing technology and a hatcherybased seed supply (see below). In Vietnam seed capture involves both active and passive methods to target either the swimming pueruli stage, or newly settled post-puerulus juveniles. Active methods employ lights to attract the swimming pueruli, which are most active during the night and particularly during the dark moon phase. Areas of high abundance have been identified where catch rates are superior, and these tend to be along the central Vietnam coast where northerly in-shore currents eddy off the southerly flowing South China Sea gyre, and are then further concentrated by coastal land forms, bays and islands (Yeung et al., 2001). Lights consist of fluorescent tubes mounted on floating frames or boats, with power supplied by gasoline generator, or via cables running from the coastal village out to the fishing site, some 100–500 m off the beach. Traps are deployed throughout the fishing site, consisting of bundled nets, timber poles with small holes drilled or seine nets mounted between floats. Swimming pueruli attracted to the lights will settle on the traps or nets, which are retrieved during daylight hours. Catch rates are generally low, between 1 and 10 pueruli per fisherman per night, but can on occasion reach as high as 50. Price per puerulus is market driven, and in 2007/08 averaged around $US 5.00–7.00 per piece. Passive methods are also employed using similar traps that are left in the water and regularly inspected by divers. This method often provides post-puerulus juveniles of up to a few grams in weight. In 2006/07 approximately 1.1 million P. ornatus seed were captured and sold for farming. One million were caught as swimming pueruli, and the remaining 10 % as settled juveniles. Development of sea cage culture of lobster in Vietnam was promoted on the basis of poverty alleviation for poor coastal villages. In this regard it has been an outstanding success, creating relative wealth for many (Hambrey et al., 1999, 2001). Of the several other tropical species of lobster, only P. homarus is farmed in any appreciable number, primarily because catches of pueruli are often as high or higher than for P. ornatus. This species achieves a much smaller harvest size (<500 g) and fetches a lower price in the market. Ranching of other tropical species is insubstantial due primarily to the availability of puerulus, and because demand and market price are much lower.

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In countries such as Australia and New Zealand, where fishery resources are intensively managed, the capture of seed stock for grow-out has been a much discussed issue. For lobsters, the issue of puerulus collection and its potential impact on the adult fishery have centred around the definition of biological neutrality (Phillips et al., 2003; Gardner et al., 2006) and estimations of how many seeds might be taken without impacting significantly on the adult fishery resource. Although some allocations of seed collection have been issued in New Zealand and Australia for Jasus verreauxi and J. edwardsii, they have not been widely supported and were discontinued. The matter of poor economic viability of ranching of wild caught seed for these species also contributed to the demise of this option for aquaculture (Jeffs and Hooker, 2000). At a research level, grow-out of P. cygnus has been examined (Johnston et al., 2006, 2007) although no commercial production appears likely in the short term. Minor sea cage production of P. homarus, P. versicolor, P. polyphagus and P. ornatus has been reported for India, Philippines and Taiwan (Kuthalingam et al., 1980; Radhakrishnan, 1996; Tan, 1997; Vijayakumaran et al., 2007) through the 1980s and 1990s. In India, it is understood that the tsunami of 2004 destroyed much of the cage facilities that existed and production in recent years has not been re-established (Vijayakumaran, pers. comm.). Grow-out of the Caribbean lobster P. argus from wild caught juveniles has been mooted (Miller, 1983; Brown et al., 1995; Jeffs and Davis, 2003; Jeffs et al., 2007), although no commercial development has yet occurred.

26.2.4 Hatchery-based lobster aquaculture There is as yet no significant, commercial aquaculture established for any lobster based on managed, hatchery-based larval production. At present there is a considerable research and development thrust to develop hatchery technology and to establish commercially viable hatcheries to supply seed lobsters to grow-out facilities. The following discussion provides information on the various aspects of hatchery technology as documented from the research. The bulk of larval rearing research for spiny lobsters has been conducted by the Japanese who first attempted phyllosoma culture in the late 1800s (Matsuda and Takenouchi, 2005) and who have intensively pursued it since the 1980s. In particular, the pioneering work of Jiro Kittaka established basic protocols for cultivation of puerulus from eggs for several species (Kittaka, 1988, 1994a,b, 1997a,b, 2000; Kittaka and Ikegami, 1988; Kittaka et al., 1988, 2001, 2005; Kittaka and Kimiua, 1989). Despite the body of knowledge and long research history, no commercial hatchery production of spiny lobster in Japan has yet been established, although production of pueruli is becoming more routine (Matsuda and Yamakawa, 2000; Matsuda

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et al., 2003; Matsuda and Takenouchi, 2005). In the mid-1990s New Zealand established a significant R&D effort and made great advances firstly with J. edwardsii and later with J. verreauxi (Booth, 1995; Illingworth et al., 1997; Moss et al., 1999, 2000, 2001; Tong et al., 2000b). Their program however was discontinued in the early 2000s as no commercial outcomes were identified. Australia has been the most recent country to invest heavily in development of hatchery technology for spiny lobsters. The research there is focussed on the same two temperate species as New Zealand, and on the tropical species P. ornatus. Pueruli of all three species have been produced in captivity from captive broodstock, although in small numbers and inconsistently (Bavage, 2004; Anon., 2006a,b; Calverley, 2006).

26.3 Advances in culture 26.3.1 Breeding Broodstock Because commercial hatchery technology has not yet been developed for most lobster species, sourcing of broodstock necessitates they be obtained from wild populations. Their selection therefore tends not to be based on any genetic consideration, but on general health and vigor. Because the bulk of lobsters of aquaculture interest are species for which commercial fisheries exist, the broodstock are most often taken from commercial fishing catches. Broodstock are generally chosen that are within their first year of maturity, as there appears to be no advantage in use of older and therefore larger individuals which require more resources to maintain and nurture. For tropical species like P. ornatus such individuals may be between one and two years of age and 1–2 kg in total weight (Bell et al., 1987; Sachlikidis et al., 2005). For temperate species such as Jasus edwardsii and J. verreauxi, broodstock are likely to be more than five years old and over 2 kg in size. Once hatchery technology for spiny lobsters is firmly established, and postlarval lobsters are routinely generated, broodstock will be selected from cultured stock and chosen on the basis of specified characteristics such as growth rate, colouration or disease resistance. The opportunity for significant gains in production and economic value through selective breeding for lobsters is likely to be high given expected high heritability of traits and ease of breeding. Broodstock populations are often skewed in favour of females. This maximises the egg/larval output from any given population and also appears more effective than using equal numbers of males and females (MacDiarmid and Kittaka, 2000). Ratios of two males to seven females have been reported for P. ornatus (Sachlikidis et al., 2005), 3 : 10 for P. japonicus (Matsuda et al., 2002) and 1 : 2 and 1 : 3 for J. verreauxi (Moss et al., 2004) for captive broodstock populations.

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Maturation and mating Managed maturation and mating of lobsters is currently done at a research level only, and has proven to be for several species one of the least problematic phases of their cultivation. A primary objective of lobster aquaculture development has been to enable year-round breeding that generates high-quality larvae on demand. For several species this has now been achieved. J. edwardsii and J. verreauxi are routinely stimulated to spawn in environmentally-controlled systems that facilitate maturation, mating and successful spawning, independent of natural seasonality of wild populations (Tong et al., 2000c; Moss et al., 2004; Smith and Ritar, 2007). Similarly, P. japonicus is bred year-round (Matsuda et al., 2002). For the tropical species P. ornatus, manipulations of water temperature and photoperiod are used to cue maturation and mate in any month of the year (Sachlikidis et al., 2005). Most lobster species of commercial interest are social animals (Atema and Cobb, 1980) for which the establishment of broodstock populations is straightforward. Small populations of between five and ten animals are easily maintained and, through technically simple methods of temperature and photoperiod control, they can be entrained to reproductive seasonality that meets the requirements of the hatchery. The morphological characteristics and behaviour associated with mating have been well described (Quackenbush, 1994; MacDiarmid and Kittaka, 2000; Nakamura, 2000; MacDiarmid and Sainte-Marie, 2006). Spawning Spawning of spiny lobsters has been well described by MacDiarmid and Kittaka (2000). From an aquaculture and hatchery management perspective, there are no specific requirements for spawning other than provision of shelter or other materials to enable the female to orient vertically. This accommodates their preference to hang vertically while the eggs are released. Females within managed broodstock populations are typically checked for spermatophores or fertilised eggs during regular (c. weekly) tank cleaning events or when eggs are evident in the broodstock system filters. Extra care must be taken when handling broodstock during this time as newly berried females may drop some or all of the eggs in a brood if the eggs have not had time to set properly on the pleopods. Smith and Ritar (2005) demonstrated that the viable fecundity and larval fitness of J. edwardsii is reduced significantly when animals are handled during the incubation period. This is likely to apply to all species. 26.3.2 Hatchery technology Incubation The incubation period for lobsters ranges from less than two weeks for tropical species to in excess of three months for temperate species

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(MacDiarmid and Kittaka, 2000). Although the incubation period of captive broodstock can be shortened with increasing temperature up to a thermal limit, minimising the period may have a deleterious effect on the quality of the eggs and subsequent larvae (Smith et al., 2002). For P. ornatus, a temperature of 26 °C will facilitate incubation over 30 days and generate highquality eggs, while at 29 °C incubation is reduced by seven days and larval quality drops significantly (Sachlikidis, unpublished data). Similarly, for J. edwardsii incubation at 12 °C was 99 days, and 67 days at 18 °C, but quality of larvae diminished significantly at the warmer temperature. From an aquacultural perspective, maximising output and minimising resources expended are commercial imperatives, but clearly must be balanced against quality of larvae produced and their likelihood of successful development. The fecundity of spiny lobsters is universally high (MacDiarmid and Kittaka, 2000; MacDiarmid and Sainte-Marie, 2006). All species have a capacity to generate hundreds of thousands of eggs per brood, and aquaculture will not be limited by egg availability. Generally, only a small proportion (<10 %) of a hatch will be retained for culture in accordance with capacity of tanks and systems available. Hatching Because lobster phyllosoma are small and delicate, the management of the hatching process is critical. Generally, spiny lobster larvae hatch at night (Ziegler and Forward, 2007). To maximise their condition and chances of subsequent survival, they must be gathered quickly after hatching and stocked to appropriate larval rearing systems. Estimation of likely hatch date for any given brood has become a key management tool and formulae have been developed that have a high degree of accuracy and precision. These have been developed for J. verreauxi (Moss et al., 2004), J. edwardsii (Tong et al., 2000c) and P. ornatus (Sachlikidis, unpublished data), and are based variously on known date of egg release, water temperature and metric characteristics of the developing embryo (eye index). Hatching often extends over several nights, although the bulk of larvae generally hatch on a single peak night, generally one or two nights after the first hatches occur. Larval handling and assessment Lobster larvae at hatch are around 2–3 mm in total length and extremely delicate. To minimise their disturbance and potential damage, they are quickly removed from tanks where hatching has occurred and often graded to isolate the strongest. Such grading may involve phototactic response whereby larvae that swim most strongly towards a light source are directed to a separate container for subsequent stocking and culture, while the remainder are discarded. The competency of the chosen larvae may be further assessed by way of a stress test of a sub-sample. Such competency

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tests enable a measure of the quality of larvae relative to other batches (Smith et al., 2003b). Larval culture Rearing of lobster larvae (phyllosoma) is technically challenging because of the protracted duration of larval development, the large number of molts involved and the delicate nature of the organism. At present there appear to be three primary areas of focus for technology development: systems, hygiene and nutrition. These are common to all aquaculture, but must be resolved specifically for lobsters and their particular requirements. Keep in mind that for all successful and established aquaculture species involving larval rearing, the duration of the larval phase is generally less than 30 days. The most abbreviated lobster larval duration, for a commercially relevant species, appears to be for Thenus species, a slipper lobster, for which larvae persist for 28 days (Mikami and Greenwood, 1997; Mikami and Kuballa, 2007) prior to metamorphosis. Within the spiny lobsters, species having the shortest larval duration reported within captive rearing are for tropical species P. ornatus, 120 days (MG Kailis pers comm) and P. argus 174 days (Goldstein et al., 2007), although in both cases these are more than four times longer than the larval duration for most commercially aquacultured shrimp and fish species. For temperate spiny lobsters, the duration is even longer, P. japonicus generally exceeding 250 days, and for some more than 350 days (Kittaka, 2000; Phillips and Melville-Smith, 2006). There is of course considerable variability in larval duration within species, due to environment, system and nutritional issues. Phillips and Melville-Smith (2006) point out that the larval development time for lobsters in captivity is often much less than in the wild, so prospects of further reduction through better husbandry and nutrition are exciting. Nevertheless, larval duration per se is not the most critical issue; it is the cumulative mortality over such a long period that makes culture of these organisms so demanding. Of those species for which the complete larval development in culture has been achieved, the number of pueruli produced has been generally less than 50, so there is some distance to go before thousands or tens of thousands of lobster pueruli are being generated. Larval systems The early success of the Japanese in rearing lobster larvae of several species through the entire development to puerulus can be attributed in great part to the exhaustive care and attention applied to individual larvae in solitary, glass bowls. These studies contributed significantly to biological knowledge of the larvae and their nurturing, but had no relevance for commercial application. Subsequently, development of small, mass-rearing systems has occurred that can support a few hundred and up to several thousand larvae (Illingworth et al., 1997; Kittaka, 2000; Ritar, 2001; Matsuda and Takenouchi,

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2005; Mikami and Kuballa, 2007). Further development of such systems will be a necessary part of commercial lobster aquaculture. Phyllosoma are planktonic animals that have limited capacity to swim and maintain position. Larval rearing systems development has therefore addressed hydrodynamic considerations in providing an environment that supports them in three-dimensional space, providing adequate spacing between individuals and access to food inputs, while enabling inflow and outflow of water from/to supply and waste lines. These have variously involved horizontal (annular) and vertical (upflow) flow patterns, pulsating flow and managed turbulence. Systems must also accommodate necessary cleaning, including removal of uneaten food, waste products and dead or dying phyllosoma. Because of the protracted larval duration, this necessitates periodic transfer of larvae to clean containers, to enable the used container to be rigorously cleaned and dried. Illingworth et al. (1997) achieved this by provision of transfer ports between adjacent chambers which facilitates the transfer of larvae while minimising their disturbance. In some instances, the rearing of lobster larvae has been successfully supported using a flow-through supply of filtered seawater. Recirculating systems have the advantage of enabling temperature control and other inputs, although they are disadvantaged by providing greater opportunity for microbial contamination. Partial recirculating systems, in which a substantial proportion of water is replaced regularly, provide a reasonable compromise. In tackling the hygiene issue which is of particular significance to long-lived lobster phyllosoma and more particularly because of the predominate use of live feeds (Artemia), use of ultraviolet irradiation, activated charcoal filtration and ozonation by-products is often applied (Ritar et al., 2006). Larval nutrition Although field data have suggested by means of spatial association that spiny lobster phyllosoma may consume soft-fleshy prey such as medusae and salps (Jeffs et al., 2004), it is only through recent morphological and physiological studies (Johnston and Ritar, 2001; Cox and Bruce, 2003; Cox and Johnston, 2003, 2004; Johnston et al., 2008a) that it has been confirmed that they are raptorial carnivores, with well-developed mouthparts at hatch, suggesting that they are capable of manipulating and spearing a wide range of potential prey items such as medusae, salps, fish larvae and small crustaceans. Most successful crustacean larval culture systems utilise live feeds. However, as few natural prey species can be cultured successfully in the laboratory or hatchery, their selection is typically based on their ease of culture rather than nutritional quality (Jones et al., 1997). For lobster larval culture, Artemia have become the live food of choice. Artemia are easy to culture and, although not ideally suited for phyllosomas, are the only readily

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available live prey that meet morphological and behavioural requirements of lobster larvae. To enhance the nutritional value of Artemia they are variously enriched, and considerable effort has been applied to optimising enrichment materials and methods (Tong et al., 1997, 2000a; Nelson et al., 2002; Ritar et al., 2002, 2003; Smith et al., 2004; Liddy et al., 2005). Development of manufactured diets to substitute or supplement Artemia is a focus of research attention (Johnston and Johnston, 2007; Johnston et al., 2008b). However, few such diets have yet been successfully formulated and those that have been trialled will require considerable further development. Larval health Ritar et al. (2006) declared that hatchery rearing of phyllosoma is often plagued by sudden high mortalities preceded by fouling of the exoskeleton as well as enteritis or septicaemia, termed ‘white-gut’ syndrome (Handlinger et al., 2000). It would appear from the experience of hatchery research and the few specific pathology studies (Shioda et al., 1997; Diggles et al., 2000; Evans et al., 2000; Igarashi and Kittaka, 2000; Bourne et al., 2004, 2006; Payne et al., 2006; Ritar et al., 2006), that the primary cause of mortality is bacterial infection, particularly by opportunistic species that proliferate when phyllosoma are compromised (Handlinger et al., 2000; Bourne et al., 2007). It is impossible to completely eliminate these bacteria, particularly over such long periods and when livefeeds are commonly used. Bourne et al. (2007) identified four compartments as potential sources of the infections: (i) the water column, (ii) biofilm on system surfaces, (iii) the feed organisms (Artemia) and (iv) the phyllosoma themselves. All need to be managed to minimise the extent of infections. The likely aetiology, as suggested by Bourne et al. for P. ornatus, involves initial infections by filamentous bacteria (Thiothrix- and Leucothrix-like organisms) of the mouthparts, which compromises food ingestion and facilitates nutritional decline. Phyllosoma are then highly susceptible to infection by pathogenic bacteria, particularly Vibrio spp., which quickly proliferate internally and kill the host. Management of larva health is primarily applied at a system level as discussed above.

26.4 Production systems 26.4.1 Nursery Ecological studies of lobsters have provided useful guidance regarding appropriate husbandry practices for the juvenile stages, from post-larval pueruli through to a size of around 30–50 g. For example, shelter preferences and social factors in wild populations (Eggleston et al., 1992; Dennis et al., 1997, 2004; Skewes et al., 1997; Dennis and Pitcher, 2001) have assisted research of P. ornatus aquaculture with the provision of artificial shelter and appropriate stocking densities under culture conditions. Booth and Kittaka

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(2000) provide a comprehensive review of background information pertinent to the potential aquaculture of several lobster species. Specific, aquaculture-based research has also been completed to define appropriate nursery methods. Such research has been applied to J. edwardsii (Hooker et al., 1997; Crear et al., 2000; Thomas, 2000; James et al., 2001; Radford and Marsden, 2005; Simon and James, 2007), J. lalandii (Dubber et al., 2004), Panulirus cygnus (Johnston et al., 2006) and P. ornatus (Jones, 2007b). Nursery culture of P. ornatus in Vietnam, although based on wild caught juveniles, provides a guide to how broader scale culture might develop when a hatchery supply of seed stock becomes available. A distinct nursery phase is recognised in Vietnam comprising the culture of puerulus and small post-puerulus juveniles over approximately three months to a size of around 10–30 g. They are maintained in submerged cages (approximately 1.5 m × 1.5 m × 1.0 m high) made from a steel frame, covered in fine mesh, and equipped with a feeding tube, through which finely chopped fishery by-catch (trash fish) is fed (Tuan and Mao, 2004). Research of P. cygnus (Johnston et al., 2006) suggests good grow-out potential and identifies important nursery husbandry protocols including use of shelter and appropriate stocking densities. From a nutritional standpoint, there is also good information now available as to feeding strategies, nutrient requirements and formulations for fresh and manufactured feeds for juvenile lobsters (James and Tong, 1997; Crear et al., 2000; Glencross et al., 2001; Sheppard et al., 2002; Thomas et al., 2002, 2003; Cox and Johnston, 2003, 2004; Smith et al., 2003a, 2005; Ward et al., 2003; Dubber et al., 2004; Perera et al., 2005; Williams et al., 2005; Cox and Davis, 2006; Nelson et al., 2006; Johnston et al., 2007; Jones, 2007b; Simon and James, 2007; Williams, 2007). The key issue for further development of manufactured diets will be reducing the cost of diets that are high in protein and most often based on a large proportion of expensive fish meal.

26.4.2 Grow-out Spiny lobster grow-out of commercial significance only exists in Vietnam at present. Booth and Kittaka (2000) have summarised the potential of several species and outlined key biological indicators that will contribute to the establishment of production systems. The most important prerequisite remains a reliable and sustainable supply of seedstock. Beyond that, several species have excellent prospects, although economics will play a major part in determining commercial viability (Hooker et al., 1997; Jeffs and Hooker, 2000). Grow-out research of lobsters, for stages beyond juveniles, has been very limited. Jones et al. (2001) examined density effects of large P. ornatus lobsters in tank systems, and Bryars and Geddes (2005) reported on diet effects on large J. edwardsii. Much of the recommended

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practice for lobster grow-out is based on extrapolations from juvenile studies (Hooker et al., 1997; Perera et al., 2005; Johnston et al., 2007).

26.4.3 Systems Technical design for large-scale grow-out of lobsters has not been subjected to specific research. In Vietnam where P. ornatus is produced in large volume, the production system consists of sea cages, either staked into the bottom in relatively shallow water, or, more commonly, supported by floating pontoons in sheltered, but deeper waters (Tuan and Mao, 2004; Williams, 2007). While this approach has been successful, unmanaged industry development has resulted in over-crowding of farms and poor feeding practices, generating collateral environmental damage to local water quality and sediment, and consequent health issues with the lobsters. These problems, however, are reversible, and sea cage grow-out is likely to remain a viable technology for lobster aquaculture. Small-scale, pond-based grow-out of lobsters has been reported in Taiwan (Chen, 1990; Wickins and Lee, 2002), although it is not clear whether this has persisted in recent years, as availability of wild stocks declines. Nevertheless, this approach suggests some species (e.g., P. homarus) may be suited to production in earthen ponds using methods similar to that for shrimp aquaculture. More intensive methods such as raceways and tanks could also have application to lobster aquaculture. For many species, marketing of live product has necessitated the development of live-holding systems (Crear and Forteath, 2001; Crear et al., 2003), in which survival over short periods and quality of stock is high. Such systems may require only minor modification to suit on-growing of lobsters.

26.4.4 Food and feeding Lobsters tend to be generally omnivorous, with a preference for slowmoving benthic invertebrates (Nelson et al., 2006). In many studies of captive lobsters they are fed fresh marine foods, and most often molluscs such as squid, mussels or other bivalves. They appear to survive and grow well on such diets, although clearly this practice is not sustainable for largescale aquaculture and manufactured diets will be necessary. In Vietnam, sea cage culture of P. ornatus employs fishery by-catch, so-called trash fish, as the source of food (Williams, 2007). This consists of a variety of fish, mollusc and crustacean species which have low value for human consumption. Typically this is supplied by middle-men who purchase from the fishers and transport it to the sea cage farms, where it is on-sold for around $US1 per kg. Although such a diet might be nutritionally adequate, due to a combination of factors including poor maintenance of quality and inappropriate feeding practices, the food conversion ratio is very high (25 : 50) and has

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contributed to environmental degradation and lobster health issues (Williams, 2007). For the on-going sustainability of the Vietnam lobster aquaculture industry and development elsewhere, formulation of pellet diets is essential. Recent studies of several species provide good baseline data for such formulation (Glencross et al., 2001; Smith et al., 2003a, 2005; Thomas et al., 2003; Ward et al., 2003; Perera et al., 2005; Williams et al., 2005; Nelson et al., 2006; Williams, 2007). Practical diets based on a high inclusion of commercial shrimp feed have proven effective (Jones, 2007b) while fundamental nutrient profile research continues (Williams, 2007).

26.5 Product issues: markets Lobsters are generally marketed as premium seafood products, supplied from wild fisheries. These fisheries are now understood to be fully or over exploited throughout the world, so any increased supply can only be generated by aquaculture (Kittaka and Booth, 2000). Because both demand and price are high, the interest in aquaculture production is primarily driven by the market. This is not necessarily a positive, as the most valuable or sought after species are not necessarily the most suited to aquaculture. For example the prolonged and comprehensive R&D effort in Japan has been stimulated to a large extent by demand for increased supply of the premium Japanese lobster P. japonicus. Their research, however, suggests that this species is one of the more difficult to culture (Kittaka, 2000). Booth and Kittaka (2000) identify that the Japanese market requirement is for live lobsters, 200–300 g in weight with sweet crisp flesh and deep red external colour. Although this derives from their historical exposure to P. japonicus, it may be satisfied by alterative species that meet the market requirement and are better suited to aquaculture production such as J. edwardsii. In China, a market exists for traditional banquet dining, which prefers a large (1 kg+), live lobster with pearly white flesh suited to raw (sashimi) consumption, and bright vibrant external colour. P. ornatus is currently the species of choice and is increasingly being supplied from aquaculture production. This species was previously in less demand as it is generally considered to be inferior for cooking.

26.6 Future trends Despite the optimism and attractive proposition of lobster aquaculture, substantial production is still some way off. For many years, aquaculture of clawed lobsters (Homarus spp.) was pursued, and yet it appears now it may never be commercially developed. The past few decades have seen great enthusiasm and some optimism for spiny lobsters, and yet the only commercial production is that of Vietnam, based at best on limited capacity and

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at worst on unsustainable practices of wild seed collection. Hatchery technology for lobsters has made huge advances, but the commercialisation of research outcomes has not yet occurred. It appears likely that the slipper lobster Thenus species will be the first to reach commercial aquaculture based on hatchery-generated seed because of its abbreviated larval life. A commercial hatchery and grow-out facility is currently being established in Australia (Mikami and Kuballa, 2007). Within the spiny lobsters, it is difficult to predict which species might first reach commercial production. In terms of puerulus production, albeit at research level, Jasus edwardsii, J. verreauxi and P. ornatus are the most advanced.

26.7 Sources of further information and advice The recently published research concerning lobster biology and production, with relevance to aquaculture is cited throughout this chapter. For comprehensive summaries, the reader is referred to the book of Wickins and Lee (2002) and to the edited volumes of Phillips and Kittaka (2000) and Phillips (2006).

26.8 References addison j t and bannister r c a (1994) Re-stocking and enhancement of clawed lobster stocks: a review, Crustaceana, 67, 2131–55. aiken d e (1988) Lobster farming: fantasy or opportunity, Proceedings of the Aquaculture International Congress and Exposition, Sept. 6–9, Vancouver, BC, 575–82. aiken d e and waddy s l (1995) Aquaculture, in Factor J (ed.), Biology of the Lobster Homarus americanus, New York, Academic Press, 153–75. anon. (1989) Rock lobster culture – is it a serious proposition? AustAsia Aquaculture, 3, 5–8. anon. (1994) Spiny lobster could go commercial, Fish Farmer, November/December, 15. anon. (1997) Aquaculture of rock lobster a national issue, R&D News, FRDC, 5(4), 2–4. anon. (2006a) Kailis’ babies make three, Fisheries R&D News, FRDC, 14, 2. anon. (2006b) Kailis rears tropical rock lobster eggs to juveniles, AustAsia Aquaculture, 20(5), 63. anon. (2007) Aquaculture potential, in Lavalli K L and Spanier E (eds), Crustacean Issues 17. The Biology and Fisheries of the Slipper Lobster, Boca Raton, FL, CRC, 325–58. atema j and cobb j s (1980) Social behaviour, in Cobb J S and Phillips B F (eds), The Biology and Management of Lobsters. Vol. 1. Physiology and Behaviour, New York, Academic Press, 409–50. bannister r c a (1998) Lobster Homarus gammarus stock enhancement in the United Kingdom: hatchery-reared juvenile do survive in the wild, but can they contribute significantly to ranching, enhancement, and management of lobster stocks? in Gendron L (ed.), Proceedings of a Workshop on Lobster Stock

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Enhancement held in the Magdalen Islands (Quebec) from October 29 to 31, 1997, Mont-Joli, QC, Department of Fisheries and Oceans, 23–32. bavage j (2004) Rock lobster farming looms, Burnie Advocate, 19 November. bell r s, channells p w, macfarlane j w, moore r and phillips b f (1987) Movements and breeding of the ornate rock lobster, Panulirus ornatus, in the Torres Strait and on the north-east coast of Queensland, Australian Journal of Marine and Freshwater Research, 38, 197–210. booth j (1995) Phyllosoma reared to settlement, The Lobster Newsletter, 8(1), 12. booth j and kittaka j (2000) Spiny lobster growout, in Phillips B and Kittaka J (eds), Spiny Lobsters: Fisheries and Culture, Oxford, Blackwell Scientific, 556–85. bourne d, hoj l, webster n, payne m, skindersoe m, givskov m and hall m (2007) Microbiological aspects of phyllosoma rearing of the ornate rock lobster Panulirus ornatus, Aquaculture, 268, 274–87. bourne d g, hoj l, webster n s, swan j and hall m r (2006) Biofilm development within a larval rearing tank of the tropical rock lobster, Panulirus ornatus, Aquaculture, 260, 27–38. bourne d g, young n, webster n, payne m, salmon m, demel s and hall m (2004) Microbial community dynamics in a larval aquaculture system of the tropical rock lobster, Panulirus ornatus, Aquaculture, 242, 31–51. brown p b, leader r, jones s and key w (1995) Preliminary evaluations of a new water-stable feed for culture and trapping of spiny lobsters Panulirus argus and fish in the Bahamas, Journal of Aquaculture in the Tropics, 10, 177–83. browne r, mercer j p and gendron l (1998) The European clawed lobster (Homarus gammarus): stock enhancement in the Republic of Ireland, in Gendron L (ed.), Proceedings of a Workshop on Lobster Stock Enhancement held in the Magdalen Islands (Quebec) from October 29 to 31, 1997, Mont-Joli, QC, Department of Fisheries and Oceans, 33–41. bryars s r and geddes m c (2005) Effects of diet on the growth, survival, and condition of sea-caged adult southern rock lobster, Jasus edwardsii, New Zealand Journal of Marine and Freshwater Research, 39, 251–62. calverley a (2006) Kailis rears tropical rock lobster eggs to juveniles, AustAsia Aquaculture, 20, 63. chen l (1990) Culture of the Spiny Lobster, in Chen L, Aquaculture in Taiwan, Oxford, Blackwell Scientific, 207–9. conklin d e (1983) Lobster culture: a perspective on intensive crustacean rearing, Proceedings of Oceans 83: Effective Use of the Sea Conference, 29 August–1 September, San Francisco, 875–9. cox s l and bruce m p (2003) Feeding behaviour and associated sensory mechanisms of stage 1-3 phyllosoma of Jasus edwardsii and Jasus verreauxi, Journal of the Marine Biological Association of the United Kingdom, 83, 465–8. cox s l and davis m (2006) The effect of feeding frequency and ration on growth of juvenile spiny lobster, Panulirus argus (Palinuridae), Journal of Applied Aquaculture, 18, 33–43. cox s l and johnston d j (2003) Developmental changes in the structure and function of mouthparts of phyllosoma larvae of the packhorse lobster, Jasus verreauxi (Decapoda: Palinuridae), Journal of Experimental Marine Biology and Ecology, 296, 35–47. cox s l and johnston d j (2004) Developmental changes in foregut functioning of packhorse lobster, Jasus (Sagmariasus) verreauxi (Decapoda: Palinuridae), phyllosoma larvae, Marine and Freshwater Research, 55, 145–53. crear b, cobcroft j and battaglene s (2003) Recirculating Systems for Holding Rock Lobsters. Guide for the Rock Lobster Industry No. 2, Hobart, TAs, Tasmanian Aquaculture and Fisheries Institute.

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crear b and forteath g (2001) Flow-rate requirements for captive western rock lobsters (Panulirus cygnus): effects of body weight, temperature, activity, emersion, daily rhythm, feeding and oxygen tension on oxygen consumption, Marine and Freshwater Research, 52, 763–71. crear b j, thomas c w, hart p r and carter c g (2000) Growth of juvenile southern rock lobsters, Jasus edwardsii, is influenced by diet and temperature, whilst survival is influenced by diet and tank environment, Aquaculture, 190, 169–82. dennis d and pitcher r (2001) Shelter preferences of newly-settled Panulirus ornatus, The Lobster Newsletter, 14, 6–7. dennis d m, skewes t d and pitcher c r (1997) Habitat use and growth of juvenile ornate rock lobsters, Panulirus ornatus (Fabricius, 1798), in Torres Strait, Australia, Marine and Freshwater Research, 48, 663–70. dennis d m, ye y, pitcher c r and skewes t d (2004) Ecology and stock assessment of the ornate rock lobster Panulirus ornatus population in Torres Strait, Australia, in Williams K C (ed.), Spiny Lobster Ecology and Exploitation in the South China Sea Region. Proceedings of a workshop held at the Institute of Oceanography, Nha Trang, Vietnam, July 2004, ACIAR Proceedings No. 120, Canberra, Australian Centre for International Agricultural Research, 29–40. diggles b k, moss g a, carson j and anderson c d (2000) Luminous vibriosis in rock lobster Jasus verreauxi (Decapoda: Palinuridae) phyllosoma larvae associated with infection by Vibrio harveyi, Diseases of Aquatic Organisms, 43, 127–37. dubber g g, branch g m and atkinson l j (2004) The effects of temperature and diet on the survival, growth and food uptake of aquarium-held postpueruli of the rock lobster Jasus lalandii, Aquaculture, 240, 249–66. eggleston d b, lipcius r n and miller d l (1992) Artificial shelters and survival of juvenile Caribbean spiny lobster Panulirus argus: Spatial, habitat, and lobster size effects, Fisheries Bulletin, 90, 691–702. evans l, jones j and brock j a (2000) Diseases of spiny lobsters, in Phillips B and Kittaka J (eds), Spiny Lobster: Fisheries and Culture, Oxford, Blackwell Scientific, 586–600. factor j r (ed.) (1995) Biology of the Lobster Homarus Americanus, New York, Academic Press. fiore d r and tlusty m f (2005) Use of commercial Artemia replacement diets in culturing larval American lobsters (Homarus americanus), Aquaculture, 243, 291–303. gardner c, frusher s, mills d and oliver m (2006) Simultaneous enhancement of rock lobster fisheries and provision of puerulus for aquaculture, Fisheries Research, 80, 122–8. george r w (2005) Evolution of life cycles, including migration, in spiny lobsters (Palinuridae), New Zealand Journal of Marine and Freshwater Research, 39, 503–14. glencross b, smith m, curnow j, smith d and williams k (2001) The dietary protein and lipid requirements of post-puerulus western rock lobster, Panulirus cygnus, Aquaculture, 199, 119–29. goldstein j s (1998) North American lobster culture (Homarus americanus), hatchery methods, and techniques: A tool for marine stock enhancement? in Howell W H, Keller B J, Park P K, McVey J P, Takayanagi K and Uekita Y (eds), U.S.–Japan Aquaculture Symposium, Durham, NH, 263–8. goldstein j s, matsuda h and butler m j (2007) Behavior of larval and postlarval Caribbean spiny lobster and implications for pan-Caribbean connectivity, Eighth International Conference and Workshop on Lobster Biology and Management, 23–28 September, Charlottetown, PEI, 73.

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hambrey j, tuan l, nho n, hoa d and thuong t (1999) Cage culture in Vietnam: how it helps the poor, Aquaculture Asia, 4, 15–17. hambrey j, tuan l and thuong t (2001) Aquaculture and poverty alleviation II. Cage culture in coastal waters of Vietnam, World Aquaculture, 32, 34–8. handlinger j, carson j, ritar a and crear b (2000) A study of diseases in cultured phyllosoma larvae and juveniles of southern rock lobster (Jasus edwardsii), Journal of Shellfish Research, 19, 676. hooker s h, jeffs a g, creese r g and sivaguru k (1997) Growth of captive Jasus edwardsii (Hutton) (Crustacea : Palinuridae) in north-eastern New Zealand, Marine & Freshwater Research, 48, 903–9. igarashi m a and kittaka j (2000) Water quality and microflora in the culture water of phyllosomas, in Phillips B and Kittaka J (eds), Spiny lobster: fisheries and culture, Oxford, Blackwell Scientific, 533–55. illingworth j, tong l j, moss g a and pickering t d (1997) Upwelling tank for culturing rock lobster (Jasus edwardsii) phyllosomas, Marine and Freshwater Research, 48, 911–14. james p j and tong l j (1997) Differences in growth and moult frequency among post-pueruli of Jasus edwardsii fed fresh, aged or frozen mussels, Marine and Freshwater Research, 48, 931–4. james p j, tong l j and paewai m p (2001) Effect of stocking density and shelter on growth and mortality of early juvenile Jasus edwardsii held in captivity, Marine & Freshwater Research, 52, 1413–17. jeffs a and davis m (2003) An assessment of the aquaculture potential of the Caribbean spiny lobster, Panulirus argus, Proceedings of the Gulf Of Carribean Fisheries Institute, 54, 413–26. jeffs a, davis m and lopez h (2007) Toward aquaculture of the Caribbean lobster from wild-caught seed, Eighth International Conference and Workshop on Lobster Biology and Management, 23–28 September, Charlottetown, PEI, 40. jeffs a and hooker s (2000) Economic feasibility of aquaculture of spiny lobsters Jasus edwardsii in temperate waters, Journal of the World Aquaculture Society, 31, 30–41. jeffs a g, nichols p d, mooney b d, phillips k l and phleger c f (2004) Identifying potential prey of the pelagic larvae of the spiny lobster Jasus edwardsii using signature lipids, Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 137, 487–507. johnston d, melville-smith r and hendriks b (2007) Survival and growth of western rock lobster Panulirus cygnus (George) fed formulated diets with and without fresh mussel supplement, Aquaculture, 273, 108–17. johnston d, melville-smith r, hendriks b, maguire g b and phillips b (2006) Stocking density and shelter type for the optimal growth and survival of western rock lobster Panulirus cygnus (George), Aquaculture, 260, 114–27. johnston d j and ritar a (2001) Mouthpart and foregut ontogeny in phyllosoma larvae of the spiny lobster Jasus edwardsii (Decapoda: Palinuridae), Marine and Freshwater Research, 52, 1375–86. johnston m, johnston d and knott b (2008a) Ontogenetic changes in the structure and function of the mouthparts and foregut of early and late stage Panulirus ornatus (Fabricius, 1798) phyllosomata (Decapoda: Palinuridae), Journal of Crustacean Biology, 28, 46–56. johnston m d and johnston d j (2007) Stability of formulated diets and feeding response of stage I Western spiny lobster, Panulirus cygnus, Phyllosomata, Journal of the World Aquaculture Society, 38, 262–71. johnston m d, johnston d j and jones c m (2008b) Evaluation of partial replacement of live and fresh feeds with a formulated diet and the influence of weaning

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Panulirus ornatus phyllosomata onto a formulated diet during early ontogeny, AQUACULTURE INTERNATIONAL, 16, 33–47. jones c m (2007a) Biology and fishery of the bay Lobster, Thenus spp., in Lavalli K L and Spanier E (eds), Crustacean Issues 17. The Biology and Fisheries of the Slipper Lobster, Boca Raton, FL, CRC, 325–58. jones c m (2007b) Feeding strategies for aquaculture of post-puerulus and juvenile tropical rock lobster P. ornatus, The Lobster Newsletter, 20, 16–20. jones c m, linton l, horton d and bowman w (2001) Effect of density on growth and survival of ornate rock lobster, Panulirus ornatus (Fabricius, 1798), in a flowthrough raceway system, Marine and Freshwater Research, 52, 1425–9. jones d a, yule a b and holland d l (1997) Larval nutrition, in D’Abramo L R, Conklin D E and Akiyama D M (eds), Crustacean Nutrition, Baton Rouge, LA, World Aquaculture Society, 353–89. kittaka j (1988) Culture of the Palinurid Jasus lalandii from egg stage to puerulus, Nippon Suisan Gakkaishi, 54, 87–93. kittaka j (1994a) Culture of phyllosomas of spiny lobster and its application to studies of larval recruitment and aquaculture, Crustaceana, 66, 258–70. kittaka j (1994b) Larval rearing, in Phillips B, Cobb S J and Kittaka J (eds), Spiny Lobster Management, Oxford, Blackwell Scientific, 403–23. kittaka j (1997a) Application of ecosystem culture method for complete development of phyllosomas of spiny lobster, Aquaculture, 155, 319–31. kittaka j (1997b) Culture of larval spiny lobsters: a review of work done in northern Japan, Marine & Freshwater Research, 48, 923–30. kittaka j (2000) Culture of larval spiny lobsters, in Phillips B and Kittaka J (eds), Spiny Lobster: Fisheries and Culture, Oxford, Blackwell Scientific, 508–32. kittaka j and booth j d (1994) Prospectus for aquaculture, in Phillips B F, Cobb J S and Kittaka J (eds), Spiny Lobster Management, Oxford, Blackwell Scientific, 365–73. kittaka j and booth j d (2000) Prospectus for aquaculture, in Phillips B and Kittaka J (eds), Spiny Lobster: Fisheries and Culture, Oxford, Blackwell Scientific, 465–73. kittaka j and ikegami e (1988) Culture of the Palinurid Palinurus elephas from egg stage to puerulus, Nippon Suisan Gakkaishi, 54, 1149–54. kittaka j, iwai m and yoshimura m (1988) Culture of a hybrid of spiny lobster genus Jasus from egg stage to puerulus, Nippon Suisan Gakkaishi, 54, 413–17. kittaka j and kimiua k (1989) Culture of the Japanese spiny lobster Panulirus japonicus from egg to juvenile stage, Nippon Suisan Gakkaishi, 55, 963–70. kittaka j, kudo r, onada s, kanemaru k and mercer j (2001) Larval culture of the European spiny lobster Palinurus elephas, Marine and Freshwater Research, 52, 1439–44. kittaka j, ono k, booth j d and webber w r (2005) Development of the red rock lobster, Jasus edwardsii, from egg to juvenile, New Zealand Journal of Marine and Freshwater Research, 39, 263–77. knudsen h and tveite s (1999) Survival and growth of juvenile lobster Homarus gammarus L. raised for stock enhancement within in situ cages, Aquaculture Research, 30, 421–5. kuthalingam m d k, luther g and lazarus s (1980) Rearing of early juveniles of spiny lobster Panulirus versicolor (Latreille) with notes on lobster fishery in Vizhinjam area, Indian Journal of Fisheries. Ernakulam, 27, 17–23. liddy g c, kolkovski s, nelson m m, nichols p d, phillips b f and maguire g b (2005) The effect of PUFA enriched Artemia on growth, survival and lipid composition of western rock lobster, Panulirus cygnus, phyllosoma, Aquaculture Nutrition, 11, 375–84.

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macdiarmid a b and kittaka j (2000) Breeding, in Phillips B F and Kittaka J (eds), Spiny Lobster: Fisheries and Culture, Oxford, Blackwell Scientific, 485–507. macdiarmid a b and sainte-marie b (2006) Reproduction, in Phillips B F (ed.), Lobsters: Biology, Management, Aquaculture and Fisheries, Oxford, Blackwell, 45–77. matsuda h and takenouchi t (2005) New tank design for larval culture of Japanese spiny lobster, Panulirus japonicus, New Zealand Journal of Marine and Freshwater Research, 39, 279–85. matsuda h, takenouchi t and yamakawa t (2003) Diel timing of molting and metamorphosis of Panulirus japonicus phyllosoma larvae under laboratory conditions, Fisheries Science, 69, 124–30. matsuda h, tekenouchi t and yamakawa t (2002) Effects of photoperiod and temperature on ovarian development and spawning of the Japanese spiny lobster Panulirus japonicus, Aquaculture, 205, 385–98. matsuda h and yamakawa t (2000) The complete development and morphological changes of larval Panulirus longipes (Decapoda, Palinuridae) under laboratory conditions, Fisheries Science, 66, 278–93. mikami s and greenwood j g (1997) Complete development and comparative morphology of larval Thenus orientalis and Thenus sp. (Decapoda: Scyllaridae) reared in the laboratory, Journal of Crustacean Biology, 17, 289–308. mikami s and kuballa a v (2007) Factors important in larval and postlarval molting, growth and rearing, in Lavalli K L and Spanier E (eds), Crustacean Issues 17. The Biology and Fisheries of the Slipper Lobster, Boca Raton, FL, CRC, 91–110. miller d l (1983) Shallow water mariculture of spiny lobster (Panulirus argus) in the Western Atlantic, Proceedings 1st International Conference on Warm Water Aquaculture-Crustacea, 9–11, February, Brigham Young University, HI, 238–45. moss g, james p and tong l (2000) Jasus verreauxi phyllosomas cultured, The Lobster Newsletter, 13, 9–10. moss g a, james p j, allen s e and bruce m p (2004) Temperature effects on the embryo development and hatching of the spiny lobster Sagmariasus verreauxi, New Zealand Journal of Marine and Freshwater Research, 38, 795–801. moss g a, tong l j and allen s e (2001) Effect of temperature and food ration on the growth and survival of early and mid-stage phyllosomas of the spiny lobster Jasus verreauxi, Marine and Freshwater Research, 52, 1459–64. moss g a, tong l j and illingworth j (1999) Effects of light intensity and food density on the growth and survival of early-stage phyllosoma larvae of the rock lobster Jasus edwardsii, Marine and Freshwater Research, 50, 129–34. nakamura k (2000) Maturation, in Phillips B and Kittaka J (eds), Spiny Lobster: Fisheries and Culture, Oxford, Blackwell Scientific, 474–84. nelson m, mooney b, nichols p, phleger c, smith g, hart p and ritar a (2002) The effect of diet on the biochemical composition of juvenile Artemia: potential formulations for rock lobster aquaculture, Journal of the World Aquaculture Society, 33, 146–57. nelson m m, bruce m p, nichols p d, jeffs a g and phleger c f (2006) Nutrition of wild and cultured lobsters, in Phillips B F (ed.), Lobsters: Biology, Management, Aquaculture and Fisheries, Oxford, Blackwell, 205–30. payne m s, hall m r, bannister r, sly l and bourne d g (2006) Microbial diversity within the water column of a larval rearing system for the ornate rock lobster (Panulirus ornatus), Aquaculture, 258, 80–90. perera e, fraga i, carrillo o, diaz-iglesias e, cruz r, baez m and galich g s (2005) Evaluation of practical diets for the Caribbean spiny lobster Panulirus argus (Latreille, 1804): effects of protein sources on substrate metabolism and digestive proteases, Aquaculture, 244, 251–62.

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phillips b f (1985) Aquaculture potential for rock lobsters in Australia, Australian Fisheries, 44, 2–7. phillips b f (ed.) (2006) Lobsters: Biology, Management, Aquaculture and Fisheries, Oxford, UK, Blackwell. phillips b f and evans l h (1997) Aquaculture and stock enhancement of lobsters: report from a workshop, Marine & Freshwater Research, 48, 899–902. phillips b f and kittaka j (eds) (2000) Spiny Lobsters: Fisheries and Culture, Oxford, Fishing News Books. phillips b f and melville-smith r (2006) Panulirus species, in Phillips B F (ed.), Lobsters: Biology, Management, Aquaculture and Fisheries, Oxford, Blackwell, 359–84. phillips b f, melville-smith r and cheng y w (2003) Estimating the effects of removing Panulirus cygnus pueruli on the fishery stock, Fisheries Research, 65, 89–101. quackenbush l s (1994) Lobster reproduction: a review, Crustaceana, 67, 82–94. radford c a and marsden i d (2005) Does morning as opposed to night-time feeding affect growth in juvenile spiny lobsters, Jasus edwardsii? Journal of the World Aquaculture Society, 36, 480–88. radhakrishnan e v (1996) Lobster Farming in India, Cochin, Central Marine Fisheries Research Institute. rahman m k and srikrishnadhas b (1994) The potential for spiny lobster culture in India, Infofish International, 1, 51–3. ritar a j (2001) The experimental culture of phyllosoma larvae of southern rock lobster (Jasus edwardsii) in a flow-through system, Aquacultural Engineering, 24, 149–56. ritar a j, smith g g, dunstan g a, brown m r and hart p r (2003) Artemia prey size and mode of presentation: effects on the survival and growth of phyllosoma larvae of southern rock lobster (Jasus edwardsii), Aquaculture International, 11, 163–82. ritar a j, smith g g and thomas c w (2006) Ozonation of seawater improves the survival of larval southern rock lobster, Jasus edwardsii, in culture from egg to juvenile, Aquaculture, 261, 1014–25. ritar a j, thomas c w and beech a r (2002) Feeding Artemia and shellfish to phyllosoma larvae of southern rock lobster (Jasus edwardsii), Aquaculture, 212, 179–90. sachlikidis n g, jones c m and seymour j e (2005) Reproductive cues in Panulirus ornatus, New Zealand Journal of Marine and Freshwater Research, 39, 305–10. serfling s a and ford r f (1975) Laboratory culture of juvenile stages of the California spiny lobster Panulirus interruptus (Randall) at elevated temperatures, Aquaculture, 6, 377–87. sheppard j, bruce m and jeffs a (2002) Optimal feed pellet size for culturing juvenile spiny lobster Jasus edwardsii (Hutton, 1875) in New Zealand, Aquaculture Research, 33, 913–16. shioda k, igarashi m a and kittaka j (1997) Control of water quality in the culture of early stage phyllosomas of Panulirus japonicus, Bulletin of Marine Science, 61, 177–89. simon c j and james p j (2007) The effect of different holding systems and diets on the performance of spiny lobster juveniles, Jasus edwardsii (Hutton, 1875), Aquaculture, 266, 166–78. skewes t d, dennis d m and pitcher c r (1997) Habitat use and growth of juvenile ornate rock lobsters, Panulirus ornatus (Fabricius, 1798), in Torres Strait, Australia, Marine and Freshwater Research, 48, 663–70.

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smith d, williams k, irvin s, barclay m and tabrett s (2003a) Development of a pelleted feed for juvenile tropical spiny lobster (Panulirus ornatus): response to dietary protein and lipid, Aquaculture Nutrition, 9, 231–7. smith d m, williams k c and irvin s j (2005) Response of the tropical spiny lobster Panulirus ornatus to protein content of pelleted feed and to a diet of mussel flesh, Aquaculture Nutrition, 11, 209–17. smith g, ritar a and dunstan g (2003b) An activity test to evaluate larval competency in spiny lobsters (Jasus edwardsii) from wild and captive oveigerous broodstock held under different environmental conditions, Aquaculture, 218, 293–307. smith g g, brown m r and ritar a j (2004) Feeding juvenile Artemia enriched with ascorbic acid improves larval survival in the spiny lobster Jasus edwardsii, Aquaculture Nutrition, 10, 105–12. smith g g and ritar a j (2005) Effect of physical disturbance on reproductive performance in the spiny lobster, Jasus edwardsii, New Zealand Journal of Marine and Freshwater Research, 39, 317–24. smith g g and ritar a j (2007) Sexual maturation in captive spiny lobsters, Jasus edwardsii, and the relationship of fecundity and larval quality with maternal size, Invertebrate Reproduction and Development, 50, 47–55. smith g g, ritar a j, thompson p a, dunstan g a and brown m r (2002) The effect of embryo incubation temperature on indicators of larval viability in Stage I phyllosoma of the spiny lobster, Jasus edwardsii, Aquaculture, 209, 157–67. tan l c (1997) Lobster culture in Guimaras (Philippines), Aquaculture Asia, 11, 35–7. thomas c, carter c and crear b (2002) Feeding strategies for rock lobster aquaculture, The Lobster Newsletter, 15, 12–14. thomas c w (2000) The effect of temperature on survival, growth, feeding and metabolic activity of the southern rock lobster, Jasus edwardsii, Aquaculture, 185, 73–84. thomas c w, carter c g and crear b j (2003) Feed availability and its relationship to survival, growth, dominance and the agonistic behaviour of the southern rock lobster, Jasus edwardsii in captivity, Aquaculture, 215, 45–65. thuy n, tb and ngoc n b (2004) Current status and exploitation of wild spiny lobsters in Vietnamese waters, in Williams K C (ed.), Spiny Lobster Ecology and Exploitation in the South China Sea Region. Proceedings of a workshop held at the Institute of Oceanography, Nha Trang, Vietnam, July 2004, ACIAR Proceedings No. 120, Canberra, Australian Centre for International Agricultural Research, 13–16. tlusty m (2004) Refocusing the American lobster (Homarus americanus) stock enhancement program, Journal of Shellfish Research, 23, 313–14. tong l j, moss g a, paewai m and pickering t d (1997) Effect of brine-shrimp numbers on growth and survival of early-stage phyllosoma larvae of the rock lobster Jasus edwardsii, Marine and Freshwater Research, 48, 935–40. tong l j, moss g a and paewai m p (2000a) Effect of brine shrimp size on the consumption rate, growth, and survival of early stage phyllosoma larvae of the rock lobster Jasus edwardsii, New Zealand Journal of Marine and Freshwater Research, 34, 469–73. tong l j, moss g a, paewai m p and pickering t d (2000b) Effect of temperature and feeding rate on the growth and survival of early and mid-stage phyllosomas of the spiny lobster Jasus edwardsii, Marine and Freshwater Research, 51, 235–41. tong l j, moss g a, pickering t d and paewai m p (2000c) Temperature effects on embryo and early larval development of the spiny lobster Jasus edwardsii and description of a method to predict larval hatch times, Marine and Freshwater Research, 51, 243–8.

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tuan l a and mao n d (2004) Present status of lobster cage culture in Vietnam, in Williams K C (ed.), Spiny Lobster Ecology and Exploitation in the South China Sea Region. Proceedings of a workshop held at the Institute of Oceanography, Nha Trang, Vietnam, July 2004, ACIAR Proceedings No. 120, Canberra, Australian Centre for International Agricultural Research, 21–5. van der meeren g i (2001) Effects of experience with shelter in hatchery-reared juvenile European lobsters Homarus gammarus, Marine and Freshwater Research, 52, 1487–93. vijayakumaran m, venkatesan r, murugan t s, kumar t s, jha d k, remany m c, thilikam l t, jahan s s, selvan k and kathiroli s (2007) Farming of spiny lobsters in sea cages, in Eighth International Conference and Workshop on Lobster Biology and Management, 23–28 September, Charlottetown, PEI, 38. waddy s l, aiken d e and gendron l (1998) Lobster (Homarus americanus) culture and resource enhancement: the Canadian experience, in Gendron L (ed.), Proceedings of a Workshop on Lobster Stock Enhancement held in the Magdalen Islands (Quebec) from October 29–31, 1997, Mont-Joli, QC, Department of Fisheries and Oceans, 9–18. ward l, carter c, crear b and smith d (2003) Optimal dietary protein level for juvenile southern rock lobster, Jasus edwardsii, at two lipid levels, Aquaculture, 217, 483–500. wickins j f and gendron l (1998) Lobster (Homarus gammarus) stock enhancement investigations in the United Kingdom from 1983 to 1993, in Gendron L (ed.), Proceedings of a Workshop on Lobster Stock Enhancement held in the Magdalen Islands (Quebec) from October 29–31, 1997, Mont-Joli, QC, Department of Fisheries and Oceans, 19–22. wickins j f and lee d o c (2002) Crustacean Farming. Ranching and Culture, Oxford, Blackwell Scientific. williams k c (2007) Nutritional requirements and feeds development for post-larval spiny lobster: a review, Aquaculture, 263, 1–14. williams k c, smith d m, irvin s j, barclay m c and tabrett s j (2005) Water immersion time reduces the preference of juvenile tropical spiny lobster Panulirus ornatus for pelleted dry feeds and fresh mussel, Aquaculture Nutrition, 11, 415–26. yeung c, jones d l, criales m m, jackson t l and richards w j (2001) Influence of coastal eddies and counter-currents on the influx of spiny lobster, Panulirus argus, postlarvae into Florida Bay, Marine and Freshwater Research, 52, 1217–32. ziegler t and forward r (2007) Control of larval release in the Caribbean spiny lobster, Panulirus argus: role of chemical cues, Marine Biology, 152, 589–97.

27 Advances in the culture of crabs B. D. Paterson, Queensland Department of Primary Industries and Fisheries, Australia

Abstract: Farmed crab production in 2005 reached 660 000 tonnes globally of which virtually all was produced in Asia. The freshwater Chinese mitten crab Eriocheir japonica sinensis accounts for two thirds of global crab production with the remainder estuarine portunid crabs such as Scylla species. Initially reliant upon harvest of wild juveniles, the adoption of hatchery methods to supply ‘seed’ makes a significant increase in aquaculture production possible. Many fundamental husbandry issues such as feeding and reproduction are only now receiving research attention. Key words: stock enhancement, broodstock maturation, manufactured diets, cannibalism, hatchery and nursery systems.

27.1 Introduction Reported farmed production of brachyuran crabs in 2005 reached 660 000 tonnes globally of which virtually all was produced in Asia. Total value was USD$ 2.8 billion (FAO, 2008). The freshwater mitten crab Eriocheir spp. accounts for two thirds of global crab production. The remaining third is mostly portunid crabs, mainly the mangrove/mud crabs Scylla spp. with various other swimming crabs (Portunus spp.) accounting for the rest. Crab aquaculture has appeared since the late 1980s primarily as a live holding or marketing strategy, to bring certainty to supply of high-value marine and freshwater crabs. The farmers ensure they will have ‘wild’ product for sale by buying wild caught ‘seed’ crabs and growing them to market size in ponds, lakes or reservoirs (FAO, 2008). This approach to aquaculture development had obvious drawbacks and risks, but one ‘drawback’ turned out to be a strength. While rapid expansion gave little opportunity for farmers to really come to grips with the best techniques for farming the crabs, a huge industry emerged so quickly that

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it commanded significant R&D assistance, the kind denied to a lone aquaculture pioneer. The obvious threat was that the rapidly escalating demand for seed would endanger the sustainability of local stocks. Hatchery methods had to be developed and introduced to make up the shortfall as a fishery stock-enhancement strategy that turned into aquaculture. For Eriocheir, the line between aquaculture and stock enhancement became quite blurred (Wang et al., 2006). Hatchery techniques have been used in Japan and elsewhere purely for stock enhancement of crab fisheries (Obata et al., 2006) – the latest example of this being with the blue crab in the USA (Zmora et al., 2005). The use of the plural ‘spp.’ for farmed crab genera is deliberate, and highlights the fact that the taxonomy of these commercially important groups is or has been controversial. The original ‘Scylla serrata’ monolith was broken up a decade ago; Keenan et al. (1998) showed that there were four distinct species, requiring particular attention to discriminate them as juveniles (Macintosh et al., 2002). In contrast, the taxonomy of the mitten crab Eriocheir remains unstable, with recent findings indicating that all commercial varieties may in fact be subspecies of E. japonica (Tang et al., 2003). In this chapter, unless stated otherwise, ‘mitten crab’ means the Chinese variety of mitten crab, which has otherwise been referred to as E. j. sinensis.

27.1.1 The Chinese mitten crab Known variously as ‘the most important crab in the world’ or as a ‘space invader’ the Chinese mitten crab has grown to dominate global farmed crab production since the late 1980s (FAO, 2008). It is not immediately obvious why freshwater mitten crabs should have flourished so much when farming of Asian marine crabs like Scylla spp. has languished. Mitten crabs appear to be extremely robust and mitten crab hatcheries can routinely produce huge quantities of post-larvae (FAO, 2008). The mitten crab is also an easily managed omnivore suited to growth in the ponds and paddy fields of coastal China, making it attractive to low-income farmers (FAO, 2008). In some respects the phenomenal growth of the industry is its greatest threat. Along with growth has come a steep learning curve – evidenced by questions about environmental deterioration, precocious maturation and variable quality of seeds (FAO, 2008). The recently recognised ‘shaking leg’ or tremor disease – caused by a spiroplasma bacteria – is a first for a crustacean (Wang et al., 2004). The dominance of hatchery-reared stocks and closure of the life cycle has led to unregulated movement and escapes of genetic stocks, and this has probably already blurred the original genetic variation in different catchments across China, threatening the integrity of already pressured wild stocks (Chang et al., 2006). Perhaps one day farmers will be regularly ‘calling in’ some of the overseas gene pool, albeit restricted as it is by its recent origins (Herborg et al., 2007), in order to keep local selection programs running.

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Eriocheir systematics is in a state of flux (Tang et al., 2003; Lee et al., 2004). In the past, a few widely recognised species, E. japonica and E. sinensis, and some more controversial minor ones were reported in China and neighbouring countries (Tang et al., 2003; Chan et al., 2005). Given the similarity of the species and the fact that the taxa known as E. japonica and E. sinensis readily interbreed it is not surprising that cytological and morphological schemes proposed to discriminate the different taxa have been difficult to apply (Chu et al., 2003; Tang et al., 2003). Recent molecular evidence confirms that the commercial species of Eriocheir are very closely related – to the point where some authors recommend that they are essentially conspecifics (Tang et al., 2003), an outcome where taxonomy conflicts with product branding! At this point, much of the regional variation observed within the genus may be attributed to subspecies, for example E. j. japonica and E. j. sinensis, according to broad geographic zones. Molecular research has revealed additional microsatellites that can be used to interpret the impacts of aquaculture ‘escapes’ on local populations (Chang et al., 2006) as well as insights into the non-marine radiation of the Grapsidae (Sun et al., 2005). The existence of isolated population structures is not surprising given that the crab’s riverine-estuary life history must restrict gene flow between distant estuaries (Yamasaki et al., 2006). As experience in Europe and North America shows, in order to cross oceans, Eriocheir needs people. Artificial translocation, interbreeding and escape of the diverse stocks in China may ultimately reverse the process of regional speciation.

27.1.2 Mangrove crabs Scylla spp. and other portunids Shrimp has usually been the most attractive prospect for coastal farmers in SE Asia, but recurring disease problems in that sector have stimulated interest in production of mangrove crabs in unused shrimp ponds (Christensen et al., 2004; Tri and Van Mensvoort, 2004). Mud crabs are asymptomatic carriers of white-spot syndrome virus (WSSV) (Hossain et al., 2001; Lavilla-Pitogo et al., 2007). The monospecific Scylla species complex was broken up, less controversially perhaps, several years ago (Keenan et al., 1998), with most Asian culture now involving Scylla paramamosain. Aside from studies to validate the new species mix, molecular fisheries studies have also selected microsatellites for identifying different stocks within a species (Gopurenko et al., 2002), an important precursor for broodstock management and selective breeding. Molecular studies are also examining genetic drift in Portunus pelagicus, another ubiquitous Indo-Pacific candidate for aquaculture (Bryars and Adams, 1999; Yap et al., 2002; Klinbunga et al., 2007), though the recent recognition of a formal lectotype (Holthuis, 2004), if nothing else, has cleared up confusion caused by the inclusion of three different swimming crab species in the original description! The relative ease with which mud crab juveniles could be harvested and sold led to a rapid expansion in the practice, threatening the sustainability

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of many local fisheries populations which were also directly threatened by human pressure on mangrove ecosystems. Since the Darwin conference in 1997 (Keenan and Blackshaw, 1999), a number of fruitful partnerships have been reported at international meetings at the University of the Philippines at Visayes in 1999 (see Asian Fisheries Science, volume 14, issue 2) and at the SEAFDEC Aquaculture centre at Iloilo in 2004 (Aquaculture Research, volume 38, issue 14) in order to improve the sustainability of the mud crab seed supply by developing hatchery methods for Scylla species. Since the late 1990s, the published proceedings of regular meetings of these research teams have provided a useful chart of the progress made. Now that practical hatchery methods are available (Marte, 2003) and commercial hatcheries are already appearing in places like Vietnam (Ut et al., 2007b), research is turning toward addressing the next likely constraints upon industry growth: including development and adoption of manufactured feeds (Pavasovic et al., 2004; Tuan et al., 2006) and improving production efficiency.

27.2 Current situation Crab farming has largely developed in advance of formal research programs. The current situation for product, grow-out, feeds and, finally, breeding of crabs is summarised in Table 27.1. There is probably still scope for significant improvements in production practices – particularly in the area of feed and genetics. The further progress made with mitten crab farming is evidenced by the emergence of genetics and broodstock quality as significant issues, while farming of portunid crabs has only achieved reliable hatchery production relatively recently. Subsequent sections examine product (Section 27.3) and these various production systems (Section 27.4) in further detail.

27.3 Product issues The market for farmed crabs is found largely in Asia, with the bulk of the mitten crab production consumed within China itself. There, the mitten crab is a culinary delicacy though the rapid expansion in farm production has put downward pressure on prices, taking some of the gloss off the sector (FAO, 2008). Farmed crabs are marketed and transported alive. Meat recovery from crabs is understandably low (∼15 % of harvest weight for Scylla serrata, Chiou and Huang 2003); however, the edible yield is often higher, for example 33 % for the mitten crab, (Chen et al., 2007). Edible recovery is increased by the marketing of mature adults, particularly females with ripe ovaries, which of course means that potential ‘broodstock’ are marketed.

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Table 27.1 Summary of current situation regarding aquaculture of crabs – for details and further reading refer to Sections 27.3 and 27.4 Mitten crabs (Eriocheir)

Portunid crabs (e.g. Scylla)

Ecology

Temperate freshwater omnivore with estuarine larval phase. Farmed in East Asia (especially China). Invasive species in USA and Europe.

Tropical/subtropical estuarine/ marine predators with estuarine/marine larval phase. Farmed in South Asia and SE Asia.

Product

150–250 g wet weight. Transported live, meat recovery low, but mature females with ripe ovaries prized. Lake-stocked product most valuable, leading to outbreaks of counterfeiting. Some caution required in cooking – host of a parasitic fluke.

300–500 g wet weight. Transported live, meat recovery low, but mature females with ripe ovaries prized. Some species also marketed live or frozen as ‘soft-shell’ crabs, though common methods of moult-induction (multiple limb autotomy) are inefficient.

Grow-out

Extensive – requires 2 years to grow. Wild juveniles or ‘seed’ crabs purchased and stocked for ‘nursery’ phase in first year (e.g. in rice paddies); in second year sold to operators of lake enclosures or earthen ponds. Instances emerging of poor growth rate (e.g. due to precocious maturation) and some diseases concerns (‘shaking leg disease’).

Extensive or semi-intensive. Harvested in less than one year. Wild juveniles or ‘seed’ crabs purchased and stocked directly to earthen ponds or mangrove enclosures. No significant disease outbreaks so far, but can be asymptomatic carriers of white-spot syndrome virus (WSSV).

Feeding

Agricultural/plant wastes. Manufactured feeds are becoming more common, but probably need improvement, especially for broodstock.

Low-value fish, manufactured feeds are becoming more common, but probably need improvement, especially for broodstock. Scylla species readily digest plant meals.

Breeding

Hatcheries introduced to ‘restock’ the industry with a reliable seed source. Quality of broodstock and the seed produced is increasingly under question, both in terms of broodstock diets and genetic management.

Was a significant bottleneck to industry growth, but now being introduced to reduce demand for wild juveniles and increase supply of ‘seed’ crabs. Requires a ‘nursery’ phase to ongrow metamorphosed crablets.

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While the consumer sees developed ovaries as a hallmark of ‘quality’, there is no guarantee that there is a simple correlation between maturation and flavour constituents so perhaps further work in this area is warranted (Chiou and Huang, 2003). Despite the ability of many farmed crabs to survive out of water for long periods, survival is not necessarily a guarantee of high quality or safety if the product is mistreated (Chiou and Huang, 2004). Bacterial cross-contamination is certainly possible within live seafood markets (Yano et al., 2006), and the mitten crab can be a classic host of a lung fluke – a potentially serious human pathogen if crabs are eaten uncooked (Liu et al., 2008).

27.3.1 Mitten crabs Intensively-reared pond crabs are viewed as inferior to lake-stocked product (Wu et al., 2007a) – while consumers may prefer the idea that the latter are ‘wild’ there may also be ways to change the characteristics of the pond crabs through improvements in selection, feeds and husbandry practices. The most prestigious mitten crabs reputedly come from the waters of Yang Cheng Lake in Jiangsu Province. It is widely reported in Chinese media and elsewhere that total sales of ‘Yang Cheng’ crabs regularly exceed the lake’s actual production figures. To counter a burgeoning trade in these fake or ‘counterfeit’ crabs, increasingly sophisticated labelling/tagging methods are being adopted by Yang Cheng growers to defend their premium sales. This labelling issue also has taxonomic overtones because the ‘purity’ of the Yang Cheng population is very likely vulnerable to interbreeding from related strains. Attempts to use microsatellites and other molecular tools to brand crabs from particular localities will also run foul of the controversial species structure within Eriocheir, a problem being exacerbated by uncontrolled translocation of stocks.

27.3.2 Portunid crabs Beyond the ready transportability of live Scylla, further processing of crabs (e.g. meat picking) is still largely the domain of the world’s high-volume crab fisheries. Processing or packaging of fresh or frozen farmed crabs is probably largely related to production of soft-shell crabs, which obtain a price premium. Limb-regeneration, nutrition and stress will influence moulting in crustaceans (Hartnoll, 1982, 2001). Multiple limb autotomy is sometimes used by farmers in SE Asia to induce moulting, but better knowledge of moulting mechanism may well improve efficiency (both in terms of shortening the period between moults and slowing the rate of hardening) and research is underway into the molecular basis of moulting (Kuballa et al., 2007a, b). Moulting crabs are housed in compartmentalised systems, such as baskets in ponds or arrays of containers (Paterson et al., 2007). Because of the

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intensive nature of these shedding systems, care has to be taken to maintain acceptable water quality parameters. In the USA, soft-shelled Callinectes sapidus sourced from wild pre-moults are normally marketed alive. The emerging trade in soft-shelled mud crabs in SE Asia currently requires freezing of packaged post-moults and our knowledge of quality constraints with this product are largely anecdotal.

27.4 Production systems 27.4.1 Grow-out Extensive culture is still generally the rule with all forms of crab farming where stocking densities are low (<1 crab m−2) and, if supplemental feed is supplied, it is usually of an opportunistic or ad hoc nature (e.g. low-value fish or agricultural wastes) (Christensen et al., 2004; FAO, 2008). Farmed crabs are so valuable that it is tempting to try to raise stocking densities, but cannibalism and reduced growth rates are significant challenges. Lowintensity practices can also be less risky and more sustainable in the long term if the alternative is unregulated accumulation of nutrients and wastes in water bodies and local ecosystems (FAO, 2008). Growth is central to issues of fisheries management, and there is a wealth of data on crab growth in wild fisheries. However, surprisingly, there is little research into changes in weight as brachyuran crabs grow. Most published research is fisheries modelling using carapace width as the indicator of size. The difficulty with this is that no-one buys crabs by the millimetre and recent experimental studies of crab growth have questioned the importance of carapace width as a measure of moult increment, and certainly as a meaningful measure of yield in aquaculture (Paterson et al., 2007). Changes in carapace width growth from moult to moult as crabs regenerate claws or as they mature simply tell us that the carapace width growth (a measure adopted for convenience by humans) is sacrificed in favour of weight increase in important parts of the body (Paterson et al., 2007). Moult by moult changes in weight by crabs and other crustaceans are most often measured when individuals are reared individually – such as in feeding trials (Cadman and Weinstein, 1988; Zhou et al., 1998; Josileen and Menon, 2005; Paterson et al., 2007). Mitten crabs The omnivorous habit of the mitten crab seemingly lends itself to rice–crab farming systems where the animals are largely able to fend for themselves as long as care in management is undertaken – particularly avoiding use of insecticides (FAO, 2008). In comparison with the breakneck individual growth rate in aquaculture by tropical and sub-tropical portunids like Scylla spp. and Portunus spp., the temperate mitten crab’s growth rate is quite modest, requiring two growing seasons to obtain a final crop, the last stage

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in net enclosures in lakes. Growth was not in question when the industry first exploded so, not surprisingly, relatively little is known about the growth dynamics of Eriocheir in comparison to extensive growth modelling in other crab fisheries (Zhang et al., 2001). However, slowed growth is now a major concern for this species; part of this is attributed to poor genetic control of broodstock and possibly inadequate nutrition (Wen et al., 2006; FAO, 2008), but seasonality is also an issue because some ‘seed’ crabs mature precociously if they are too large in their first autumn and all but cease growth as a consequence (Jin et al., 2001). Greater levels of precocity amongst seed crabs are observed at low stocking densities, when average final weights are the highest (Li et al., 2007a). Portunid crabs Mud crabs, grown at densities of <1 crab m−2 in earthen pond systems reach commercial size (200–500 g, depending upon the species) in around 4–9 months. The smaller commercial size of soft-shell crabs (40–80 g pre-moult) means that crops can be rotated in 3–4 months. Because of the premium associated with ripe mud crab females, the economics of monosex culture has been considered (Trino et al., 1999), but further work could probably be done to establish this as a more common practice. Mud crabs are also grown in some areas in mangrove-based pens or ponds (Trino and Rodriguez, 2002; Christensen et al., 2004). Before maturation begins, farm-reared portunids at least double their weight every time they moult. A portunid crab that is largest of its batch leaving the nursery will, all things being equal, have a good chance of being the largest crab on harvest day. In reality there will be a bit of slippage in rankings of crabs growing in ponds – and limb loss arising from agonistic encounters in ponds permanently sets back the growth of Portunus pelagicus and presumably other species as well (Paterson et al., 2007). The cost of limb regeneration and not genetics may be a major cause of size variation amongst crabs grown in ponds (Paterson et al., 2007). Where risks are high and seed relatively expensive, farmers inevitably stock ponds conservatively and there is little enthusiasm for pushing limits because of aggression and cannibalism amongst crabs. It is only really since the inception of routine crab nursery production that there has been an imperative to try to intensify stocking densities of early juvenile crabs – perhaps developments in this area will follow through into improved efficiency in grow-out itself. Laboratory experiments with P. pelagicus confirm that small individuals and crabs that are moulting are particularly vulnerable (Marshall et al., 2005). Container-based systems are an option, albeit a capital-intensive one, for rearing crabs. This is based upon the relative ease with which crabs can be reared individually, with zero cannibalism, in feed trials (Josileen and Menon, 2005; Paterson et al., 2007; Nicholson et al., 2008). For reasons of economy, crabs would have to be reared in the smallest container practical,

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both in terms of the wellbeing of the crab and the efficient operation of the system. One option for soft-shell crab production is to grow juvenile crabs in ponds until they are ready to move to the shedding facility. However, indoor containerised nurseries would probably also be feasible (Nicholson et al., 2008). These authors showed that juvenile three-spot crab P. sanguinolentus could be reared in containers so small that when ready for harvest the crabs can’t sit flat on the floor. As long as the crab had room to moult, growth rate was unaffected. While there is still anecdotal evidence that crabs grow faster when free in extensive ponds, intensive indoor systems probably have an advantage in terms of biomass surviving per square metre.

27.4.2 Food and feeding Crab farming began and thrived before a specific ‘crab feed’ became necessary. Much of what can be recognised as formal feed research for farmed crabs is only now being conducted. Thanks to hatcheries, the sector is less limited by seed supply which has signalled a switch away from supplemental feeding practices associated with low-intensity farming. At higher-density rearing, the crabs will rely upon the diet they are given and this requires a greater understanding of nutritional requirements of the species. While diets for growth and diets for maturation are to some extent separate issues, the distinction is clouded – crabs (Scylla spp. in particular) grow so fast that the ‘broodstock’ are actually on the menu. So while research may consider the important effects of diet on ovarian maturation and how this impacts on larvae quality of following generations, as the ovaries are a major selling point for both mitten crabs and mud crabs, the simultaneous impact of diet on flavour and marketability of the crabs also needs to be considered (Chiou and Huang, 2003). The natural diets of the freshwater and an estuarine species will receive a different level of input from terrestrial oils, so attention is being placed on the polyunsaturated and highly unsaturated fatty acid (PUFA and HUFA) levels as well as relative omega 3 and omega 6 ratios of broodstock and for larval rearing (Ying et al., 2006; Alava et al., 2007a, b). Mitten crabs The freshwater mitten crabs are omnivorous, and readily ingest plant material. Mu et al. (1998) recommended a dietary protein of around 40 % for E. j. sinensis (in experimental diets containing 7–11 % crude lipid), and recent protein digestibility experiments used amino acid availability measurements to recommend soy bean meal and Spirulina meal as substitutes for fish meals (Mu et al., 2000). Fine-tuning of manufactured diets for intensively-reared Eriocheir no doubt continues, seeking to bring the farmed crabs closer to the perceived qualities of lake-reared product and to fortify the reproductive capabilities of captive-reared populations. Recently, one manufactured diet received by pond-reared mitten crabs (38 % protein,

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4.5 % lipid) produced relatively poor HUFA levels, even though lipid level was higher in the hepatopancreas/midgut gland (and gonadosomatic index – GSI – was lower) than in lake-stocked crabs (Wu et al., 2007a). The total HUFA and PUFA was low across the board in hepatopancreas of pond reared specimens, which could have consequences for their role as broodstock. Portunid crabs The mud crabs as adults are generally viewed as predators of gastropods and small crabs (Hill, 1979) and, to date, they are typically fed low-value fish (‘trash-fish’) in farms. However, juvenile Scylla spp. are more opportunitistic and able to forage independently amongst the detritus and biota of the pond environment to the point that at typical low commercial densities supplemental feeding is unnecessary (Christensen et al., 2004). Specific research has even shown that S. serrata possess cellulases (Pavasovic et al., 2004) and can readily digest ingredients such as wheat flour, etc. (Catacutan et al., 2003; Tuan et al., 2006) so there is considerable interest in seeing the extent to which plant meals can be included in manufactured diets for this species. Perhaps Scylla spp. will also be able to utilise dietary carbohydrate (Tuan et al., 2006). Similarly, Portunus pelagicus is also beginning to look less an obligate predator in recent years. Originally held to be a benthic predator which ingested plant material by accident (Williams, 1982) the persistent reports of ingested seagrass (Edgar, 1990; de Lestang et al., 2000) in this species certainly warrant further investigation. Diet development is further advanced for Scylla serrata than other mud crabs, but advances for that species will likely speed developments with S. paramamosain and other species. Existing recommendations for S. serrata are that they receive 32–40 % dietary protein and 6–12 % lipid (Catacutan, 2002). Commercial shrimp feeds are routinely used as substitutes for specifically formulated crab diets (Mann et al., 2007; Paterson et al., 2007), although pellet size is an issue as the crabs grow. A previous study had shown that at constant dietary protein (∼47 %) growth is acceptable at dietary lipid levels as high as 13.8 %(Sheen and Wu, 1999). Holme et al. (2007), using soy lecithin, also noted that Scylla serrata megalops had an optimum dietary lipid content that is on the high side of crustacean range (∼10 %). Lipid accumulation is one of the recognised ‘problems’ with elevated dietary fat in crustaceans (D’Abramo, 1997) and not surprisingly, pond-reared Scylla broodstock have significantly more storage lipid than their wild counterparts (Alava et al., 2007b) which appears to reflect findings for lipid content in viscera of ‘farmed’ Eriocheir reported above. Whether this is a problem for consumption of the viscera remains to be seen. The current recommendation for the diet to contain less than 40 % protein was based upon changes in carapace width rather than body weight (Catacutan, 2002). This appears to point to morphometric shifts in crabs fed high-protein diets, and this may have implications in the market. Strictly speaking, where farmers sell

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product by weight, the caparace width is less meaningful as a measure of growth as changes in carapace size may not always reflect differences in weight (e.g. in the case of sexual maturation or limb regeneration, Paterson et al., 2007). Perhaps in future, particularly in respect to genetic selection programs, attention needs to be placed on carapace ‘depth’ and other measures. Studies on cholesterol requirement of S. serrata megalopa (Sheen, 2000) indicated that while the nutrient is probably essential for growth, optimum dietary levels may be lower for S. serrata than for previously studied crustaceans, particularly if elevated levels of dietary lecithin are provided (Holme et al., 2006a, 2007). 27.4.3 Breeding and hatchery technology Hatchery output is the major constraint to expansion of mitten crab and mud crab farming, but the issues and strategies to address this differ between sectors. For the better established mitten crabs, broodstock quality and nutrition are in question, while for Scylla, successful commercial hatcheryrearing methods have only been available for a few years and the technology is still needed in many areas. Much of the literature on Eriocheir is not available in English, but readers will find a recent review of hatchery practices for Eriocheir very helpful (Cheng et al., 2008). 27.4.4 Broodstock quality and nutrition Crabs can be reared from wild or pond-reared broodstock although, as questions remain about the quality and diet received by crabs maturing in captivity, this is an area of continuing research. Mitten crabs Stopping mitten crabs from breeding is high on the agenda where this species is an invasive pest, but no breakthroughs appear to have been made there. Interestingly, mitten crabs mate in intermoult and do not apparently rely as much upon pheromone attraction – at least at a distance (Herborg et al., 2006) – as Scylla and other crabs that ‘mate guard’ (where the successful male finds, protects and does not eat the moulting female) (Hayden et al., 2007). As is common with many decapods, mated female crabs extrude and retain the egg ‘sponge’ within the abdominal flap where the hatchery operator can monitor progress of embryo development. Perhaps related to the more extensive level of domestication already practised with mitten crabs, the quality of farmed broodstock is emerging as a significant issue limiting hatchery output. Maturation diets and their fatty acid profiles have been given significant attention (Wen et al., 2002; Wu et al., 2007b; Ying et al., 2006). Early stocked individuals of Eriocheir are capable of precocious sexual maturation, maturing in their first rather than their second autumn – which is a problem for grow-out rather

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than reproduction per se because the maturing crab’s growth suffers (Jin et al., 2001). Portunid crabs Broodstock quality is not so much of an issue for Scylla aquaculture while wild broodstock continue to be available. The crabs spawn readily in captivity and out-of-season spawning is a relatively straightforward practice (Zeng, 2007). The inception of hatchery-rearing means that use of farmreared broodstock will become more common. To this end, broodstock maturation diets are increasingly under the spotlight, with some element of ‘natural’ food still required to supplement artificial diets (Millamena and Quinitio, 2000; Alava et al., 2007a, b).

27.4.5 Hatchery practices Optimum control of hygiene is the first objective of the hatchery operator. Until recently, hatchery production of both mitten crab and mud crab larvae has relied on use of antibiotics at some point during development to sustain larval survival (Nghia et al., 2007a; Cheng et al., 2008). Preventative use of antibiotics in this manner is unacceptable because of the danger posed by build-up of antibiotic resistance in the environment – and in any case antibiotic-based methods do not necessarily produce healthy vigorous larvae (Cheng et al., 2008). Hatcheries keep the temperature as high as practical for the temperate mitten crab (22–25 °C) and tropical mud crab (28–30 °C) in the interests of efficient throughput of larvae. Possibly the relative ease of rearing Eriocheir larvae compared in particular to Scylla serrata relates to adaptation of larvae for entrainment within estuaries and migration into freshwater. However, recent observations indicate that except for the very early zoea, S. serrata larvae are not obliged to stay at marine levels of salinity (Nurdiani and Zeng, 2007). Mitten crabs Hatcheries for mitten crabs use either of two broad approaches: indoor intensive and controlled larviculture or outdoor extensive and less controlled larviculture (Cheng et al., 2008). The indoor approach is similar to that seen in other crustacean hatcheries with a combination of live feeds provided (Sui et al., 2008). Z1s are stocked at high densities (200–500 per litre) so that even if the percentage survival through to megalops is relatively low, at 5–10 %, the production output is still high (Cheng et al., 2008). Studies of larval nutrition in the area of fatty acid requirements and dietary protein have been undertaken (Pan et al., 2005; Sui et al., 2007). The method for outdoor culture in earthen ponds is simpler and less reliant on keeping the system on a technological knife-edge. This extensive approach, while demanding more land and not necessarily boosting survival of megalops, does apparently improve their quality and reduces the need to resort to

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antibiotics to contain bacterial pathogens (Cheng et al., 2008). During larval development, the outdoor ponds are still provided with typical live feeds such as Artemia and rotifers. One water quality problem recently identified in these systems involves larval mortality during outbreaks of calcium carbonate precipitation (Li et al., 2007b). Portunid crabs While water quality will deteriorate within culture media (SenerichesAbiera et al., 2007), bacterial influences appear to be the main problem restricting hatchery output of Scylla species. However, rather than addressing this through use of antibiotics as has been done in the past, future improvements may be possible in system design and operation, for example, use of ozone (Nghia et al., 2007a). The technical bottleneck limiting hatchery production of Scylla spp. has only been lifted in the last couple of years so it is worth considering this in more detail. Successful commercial hatchery methods are now either demonstrated or in use for both S. serrata and S. paramamosain (Rodriguez et al., 2007; Ut et al., 2007b) and similar methods have recently been applied to other portunids (Zmora et al., 2005; Parado-Estepa et al., 2007; Paterson et al., 2007). These methods are adaptations of common hatchery practices for carnivorous larvae and currently rely on ‘work horse’ live foods such as Artemia (all stages depending upon the larvae/post-larvae being fed) and in some cases rotifers for early stage larvae (Ruscoe et al., 2004; Davis et al., 2005). Supplements are already in use because of the uncertain nutritional value of these live feeds and promising research suggests that the costs and general complications associated with use of live feeds may be overcome in future using inert/microbound diets (Davis et al., 2005; Holme et al., 2006b). Clearly, successful microbound diets for Scylla larvae will have to be well bound so they will not fall apart when manipulated by larvae (Genodepa et al., 2007; Lumasag et al., 2007). The importance of omega-3 HUFA fatty acid supplementation of S. serrata larval diets has been demonstrated experimentally (Suprayudia et al., 2004), although demonstrating a benefit in hatchery practice amidst all the other factors at play is not straightforward (Mann et al., 2001) so a better understanding of HUFA levels during diet development may be required (Holme et al., 2007). High levels of HUFA in itself may not be desirable. A recent study examined HUFA and docosahexaenoic acid/eicosapentaenoic acid (DHA/EPA) ratios for larvae of S. paramamosain, finding effects on larval development rates rather than survival, particularly for low DHA/ EPA ratios (Nghia et al., 2007b). 27.4.6 Nurseries Nurseries are required to fully integrate artificial propagation into the crab farming sector. Until hatcheries appeared, crab farmers used rivers and

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estuaries as wild ‘nurseries.’ Ready transportability of early crab stages rather than megalops is also another important reason for establishing nurseries (Quinitio, 2000). The post-larvae and fry of many fish and shellfish aquaculture species must be transported in heavy bags of water. Mitten crab and mud crab juveniles can be transported in damp packaging (there is a reason why crab farmers put fences around their ponds!). Mitten crabs The slower, seasonally oriented growth of the temperate mitten crab has inevitably led to nursery practices that spread the risk during the first year of production; ongrowing megalopa to produce ‘seed’ or ‘coin-sized’ crabs of a more convenient size for final on-growing (Cheng et al., 2008; FAO, 2008). The seed crabs (5–10 g) are produced in a variety of tank and pond systems, with rice–crab systems being recommended (Li et al., 2007a). Premature maturation of seed crabs is a danger if the juveniles become too advanced in their first year (Jin et al., 2001; Zhang et al., 2001). High density stocking, which puts most strain on submerged vegetation, nevertheless reduces the incidence of precocity because of the increased competition and reduced growth rate (Li et al., 2007a). Portunid crabs With mud crabs, unfamiliarity with artificial seed has been a significant constraint, since farmers have required demonstration that the hatchery seed stock will perform as well as that from the wild (Ut et al., 2007b). Farmers comfortable with on-growing relatively large ‘seed’ crabs from the wild are understandably reluctant to venture into hatchery ownership or buy tiny post-settlement juveniles. A nursery that brings the hatchery seed to a more equal footing with wild juveniles is an important intermediate step in this confidence-building process (Ut et al., 2007a). The constraints applying to nursery or seed production are broadly the same as those for on-growing of large juveniles and sub-adult crabs (e.g. cannibalism and aggression). The subtle difference is that farmers require large numbers of seed crabs from nurseries to stock their ponds and, in turn, want those crabs to grow in weight. This inevitably makes high-intensity nurseries a sound proposition. These tank, or pond-based systems rear dozens of juveniles per square metre, feed them intensively and then harvest and sell them before the growing juveniles literally run out of space (Mann et al., 2007; Rodriguez et al., 2007; Ut et al., 2007a). When juvenile P. pelagicus are grown in ponds the largest individuals soon acquire the stable isotopic signature of cannibals (Møller et al., 2008). Survival can be improved by adding shelters or ‘habitat’ to the nursery environment (e.g. bricks or rolled fragments of net), apparently through a reduction in aggressive encounters and injury (Marshall et al., 2005; Mann et al., 2007; Ut et al., 2007a).

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27.5 Future trends Now that life cycles of farmed crabs have been closed and the process of refinement of existing ad hoc farming practices is underway, it is perhaps more instructive to use a more traditional order for topics to map out the work remaining in this sector.

27.5.1 Product Crab farming is succeeding to varying extents in filling the void between the increasing demand in SE Asia for crabs and the, at best, static production from the wild fisheries. When constraints to industry growth are lifted by routine hatchery production, a fall in price as supply increases is likely. This has already happened with mitten crabs (FAO, 2008). The mitten crab sector appears likely to remain segmented. Improvements in selection and husbandry of intensively-reared mitten crabs are needed to bring them on par with consumer preferences for ‘lake-stocked’ crabs. The traditional market celebrates the changes of the seasons, so there is an implied contradiction in the very concept of an intensively-farmed mitten crab. However, it wouldn’t be the first time that aquaculture broadened the market footprint of a sought-after product. The portunid crab sector, dominated by Scylla spp., has a broader market demand. Within that, the emerging ‘soft-shell’ crab sector will likely expand further but, in doing so, better techniques for efficient management and control of moulting should evolve. While simple, easily implemented methods will have most application throughout Asia, countries more inclined toward intensification and automation of the process may find that a more comprehensive understanding of molecular mechanisms of moulting may be one useful avenue to follow.

27.5.2 Breeding Better management of selected broodstock lines and separation from wild stocks is required for mitten crab culture, and will increasingly come to the fore with portunid culture now that routine life cycle closure is possible. Selected to suit the most cost-effective production models for each species, including their consumer characteristics and reproductive quality, these broodstock will also need suitable maturation diets that carry on from the regular grow-out diets. The edible yield for live-marketed farmed crabs (with the exception of soft-shelled juveniles) includes ripe gonads, blurring the distinction between quality of broodstock and product and between diets for grow-out and for maturation. This will also make protection of selected lines problematic.

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27.5.3 Hatchery methods While not the bottleneck that it has been, some further refinement of hatchery techniques can probably be expected in both sectors in coming years as methods developed locally become more widely adopted. Eventually, there may be a shift from use of rotifers and other live feeds to formulated larval diets as larval requirements become better understood (Holme et al., 2006b). While adoption of extensive practices has incidentally reduced the need for antibiotic intervention with mitten crabs (Cheng et al., 2008), it is possible that alternative water treatment methods may be found (Nghia et al., 2007a). 27.5.4 Nursery and grow-out The low stocking rates of individuals remain a brake upon productivity. Aggression and cannibalism amongst over-stocked crabs remain significant issues, as is food supply when the crop exceeds the carrying capacity of the environment (Wang et al., 2006; Cheng et al., 2008) and beyond that point manufactured feeds formulated to the crab’s dietary requirements must be available for semi-intensive production. Because of the various opportunities of using plant crops in ponds (e.g. Spirulina or rice), knowledge of shelter use and behaviour emerging in work with nursery-sized individuals need to be extended further into grow-out. Useful parallels apparently exist between this problem and ecological studies of shelter use and foraging by crabs in the wild (Hovel and Fonseca, 2005; Hayden et al., 2007). Crabs can probably be selected to grow fast, and monosex culture deserves more attention with Scylla spp.. Given that size is likely to be a major factor in aggression, practical methods of grading crabs should be examined both in nurseries and growout (Marshall et al., 2005).

27.6 Sources of further information and advice • FAO Cultured Species Information Program: http://www.fao.org/fishery/ culturedspecies/Eriocheir_sinensis • Culture and Management of Scylla Species (CAMS): http://inco-cams. seafdec.org.ph/

27.7 References alava v r, quinitio e t, de pedro j b, orosco z g a and wille m (2007a) Reproductive performance, lipids and fatty acids of mud crab Scylla serrata (Forsskal) fed dietary lipid levels, Aquac Res, 38, 1442–51. alava v r, quinitio e t, de pedro j b, priolo f m p, orozco z g a and wille m (2007b) Lipids and fatty acids in wild and pond-reared mud crab Scylla serrata (Forsskal) during ovarian maturation and spawning, Aquac Res, 38, 1468–77.

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bryars s r and adams m (1999) An allozyme study of the blue swimmer crab, Portunus pelagicus (Crustacea: Portunidae) in Australia: Stock delineation southern Australia and evidence for a cryptic species in northern waters, Mar Freshw Res, 50, 15–26. cadman l r and weinstein m p (1988) Effects of temperature and salinity on the growth of laboratory-reared juvenile blue crabs Callinectes sapidus Rathbun, J Exp Mar Biol Ecol, 121, 193–207. catacutan m r (2002) Growth and body composition of juvenile mud crab, Scylla serrata, fed different dietary protein and lipid levels and protein to energy ratios, Aquaculture, 208, 113–23. catacutan m r, eusebio p s and teshima s (2003) Apparent digestibility of selected feedstuffs by mud crab, Scylla serrata, Aquaculture, 216, 253–61. chan t y, ng p k l and ng n k (2005) The nomenclature and taxonomy of Eriocheir formosa Cahn, Hung & Yu, 1995 (Brachyura, Varunidae) from Taiwan: a rebuttal to Tang et al. (2003, 2004), Crustaceana, 78, 457–64. chang y m, liang l q, li s w, ma h t and he j g (2006) A set of new microsatellite loci isolated from Chinese mitten crab, Eriocheir sinensis, Mol Ecol Notes, 6, 1237–9. chen d w, zhang m and shrestha s (2007) Compositional characteristics and nutritional quality of Chinese mitten crab (Eriocheir sinensis), Food Chem, 103, 1343–9. cheng y, wu x, yang x and hines a h (2008) Current trends in hatchery techniques and stock enhancement for Chinese mitten crab, Eriocheir japonica sinensis, Rev Fish Sci, 16, 377–84. chiou t k and huang j p (2003) Chemical constituents in the abdominal muscle of cultured mud crab Scylla serrata in relation to seasonal variation and maturation, Fish Sci, 69, 597–604. chiou t k and huang j p (2004) Biochemical changes in the abdominal muscle of mud crab Scylla serrata during storage, Fish Sci, 70, 167–73. christensen s m, macintosh d j and phuong n t (2004) Pond production of the mud crabs Scylla paramamosain (Estampador) and S. olivacea (Herbst) in the Mekong Delta, Vietnam, using two different supplementary diets, Aquac Res, 35, 1013–24. chu k h, ho h y, li c p and chan t y (2003) Molecular phylogenetics of the mitten crab species in Eriocheir, sensu lato (Brachyura; Grapsidae), J Crust Biol, 23, 738–46. d’abramo l r (1997) Triacylglycerols and fatty acids, in D’Abramo LR, Conklin DE and Akiyama DM (eds), Crustacean Nutrition, Baton Rouge, LA, World Aquaculture Society, 71–84. davis j a, wille m, hecht t and sorgeloos p (2005) Optimal first feed organism for South African mud crab Scylla serrata (Forskal) larvae, Aquac Int, 13, 187–201. de lestang s, platell m e and potter i c (2000) Dietary composition of the blue swimmer crab Portunus pelagicus L. Does it vary with body size and shell state and between estuaries? J Exp Mar Biol Ecol, 246, 241–57. edgar g j (1990) Predator-prey interactions in seagrass beds. II. Distribution and diet of the blue manna crab Portunus pelagicus Linnaeus at Cliff Head, Western Australia, J Exp Mar Biol Ecol, 139, 23–32. fao (2008) Eriocheir sinensis: Cultured Aquatic Species Information Program, Rome, Food and Agriculture Organization of the United Nations, Fisheries and Aquaculture Department, http://www.fao.org/fishery/culturedspecies/Eriocheir_ sinensis, accessed January 2009. genodepa j, zeng c and southgate p c (2007) Influence of binder type on leaching rate and ingestion of microbound diets by mud crab, Scylla serrata (Forsskal) larvae, Aquac Res, 38, 1486–94.

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gopurenko d, hughes j m and ma j (2002) Identification of polymorphic microsatellite loci in the mud crab Scylla serrata (Brachyura: Portunidae), Mol Ecol Notes, 2, 481–3. hartnoll r g (1982) Growth, in Abele LG (ed.), The Biology of Crustacea Volume 2, New York, Academic Press, 111–96. hartnoll r g (2001) Growth in Crustacea – twenty years on, Hydrobiologia, 449, 111–22. hayden d, jennings a, muller c, pascoe d, bublitz r, webb h, breithaupt t, watkins l and hardege j (2007) Sex-specific mediation of foraging in the shore crab, Carcinus maenas, Horm Behav, 52, 162–8. herborg l-m, bentley m g, clare a s and last k s (2006) Mating behaviour and chemical communication in the invasive Chinese mitten crab Eriocheir sinensis, J Exp Mar Biol Ecol, 329, 1–10. herborg l-m, weetman d, van oosterhout c and hanfling b (2007) Genetic population structure and contemporary dispersal patterns of a recent European invader, the Chinese mitten crab, Eriocheir sinensis, Mol Ecol, 16, 231–42. hill b j (1979) Aspects of the feeding strategy of the predatory crab Scylla serrata. Mar Biol, 55, 209–14. holme m-h, zeng c and southgate p c (2006a) The effects of supplemental dietary cholesterol on growth, development and survival of mud crab, Scylla serrata, megalopa fed semi-purified diets, Aquaculture, 261, 1328–34. holme m-h, zeng c and southgate p c (2006b) Use of microbound diets for larval culture of the mud crab, Scylla serrata, Aquaculture, 257, 482–90. holme m-h, southgate p c and zeng c (2007) Assessment of dietary lecithin and cholesterol requirements of mud crab, Scylla serrata, megalopa using semipurified microbound diets, Aquac Nutr, 13, 413–23. holthuis l b (2004) The identity and lectotype of Portunus pelagicus (L., 1758), Crustaceana, 77, 1267–9. hossain m s, otta s k and karunasagar i (2001) Detection of white spot syndrome virus (WSSV) in wild captured shrimp and in non-cultured crustaceans from shrimp ponds in Bangladesh by polymerase chain reaction, Fish Path, 36, 93–5. hovel k a and fonseca m s (2005) Influence of seagrass landscape structure on the juvenile blue crab habitat-survival function, Mar Ecol Progr Ser, 300, 179–91. jin g, li z and xie p (2001) The growth patterns of juvenile and precocious Chinese mitten crabs, Eriocheir sinensis (Decapoda,Grapsidae) stocked in freshwater lakes of China, Crustaceana, 74, 261–73. josileen j and menon n g (2005) Growth of the blue swimmer crab, Portunus pelagicus (Linnaeus, 1758) (Decapoda, Brachyura) in captivity, Crustaceana, 78, 1–18. keenan c p and blackshaw a (1999) Mud Crab Aquaculture and Biology: Proceedings of an International Scientific Forum held in Darwin, Australia, 21–24 April 1997, ACIAR Proceedings No. 78, Canberra Australian Centre for International Agricultural Research. keenan c p, davie p j f and mann d l (1998) A revision of the genus Scylla de Haan, 1833 (Crustacea: Decapoda: Brachyura: Portunidae) (1998), Raff Bull Zool, 46, 217–45. klinbunga s, khetpu k, khamnamtong b and menasveta p (2007) Genetic heterogeneity of the blue swimming crab (Portunus pelagicus) in Thailand determined by AFLP analysis, Biochem Genetics, 45, 725–36. kuballa a v, guyatt k, dixon b, thaggard h, ashton a r, paterson b, merritt d j and elizur a (2007a) Isolation and expression analysis of multiple isoforms of putative farnesoic acid O-methyltransferase in several crustacean species, Gen Comp Endocrinol, 150, 48–58. kuballa a v, merritt d j and elizur a (2007b) Gene expression profiling of cuticular proteins across the moult cycle of the crab Portunus pelagicus, BMC Biol, 5, 45.

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lavilla-pitogo c r, de la pena l d and catedral d d (2007) Enhancement of white spot syndrome virus load in hatchery-reared mud crab Scylla serrata (Forsskal, 1775) juveniles at a low temperature, Aquac Res, 38, 1600–3. lee t-h, naitoh n and yamazaki f (2004) Chromosome studies on the mitten crabs Eriocheir japonica and E. sinensis, Fish Sci, 70, 211–14. li x, dong s, lei y and li. y (2007a) The effect of stocking density on Chinese mitten crab Eriocheir sinensis on rice and crab seed yields in rice-crab culture systems, Aquaculture, 273, 487–93. li x, lei y l, gao x, ma c and dong s (2007b) Calcium carbonate supersaturation and precipitation in Chinese mitten crab (Eriocheir japonica sinensis) larval ponds in China: mass mortality, crystal form analysis and safety saturation index, Aquaculture, 272, 361–9. liu q, wei f, liu w, yang s and zhang x (2008) Paragonimiasis: an important foodborne zoonosis in China, Trends Parasitol, 24, 318–23. lumasag g j, quinitio e t, aguilar r o, baldevarona r b and saclauso c a (2007) Ontogeny of feeding apparatus and foregut of mud crab Scylla serrata Forsskal larvae, Aquac Res, 38, 1500–11. macintosh d j, overton j l and thu h v t (2002) Confirmation of two common mud crab species (genus Scylla) in the mangrove ecosystem of the Mekong Delta, Vietnam, J Shell Res, 21, 259–65. mann d l, asakawa t, kelly b, lindsay t and paterson b (2007) Stocking density and artificial habitat influence stock structure and yield from intensive nursery systems for mud crabs Scylla serrata (Forsskal 1775), Aquac Res, 38, 1580–7. mann d l, asakawa t, pizzutto m and keenan c p (2001) Investigation of an Artemia-based diet for larvae of the mud crab Scylla serrata, Asian Fish Sci, 14, 175–84. marshall s, warburton k, paterson b and mann d (2005) Cannibalism in juvenile blue-swimmer crabs Portunus pelagicus (Linnaeus, 1766): effects of body size, moult stage and refuge availability, Appl Anim Behav Sci, 90, 65–82. marte c l (2003) Larviculture of marine species in Southeast Asia: current research and industry prospects, Aquaculture, 227, 293–304. millamena o m and quinitio e (2000) The effects of diets on reproductive performance of eyestalk ablated and intact mud crab Scylla serrata, Aquaculture, 181, 81–90. møller h, lee s y, paterson b and mann d (2008) Cannibalism contributes significantly to the diet of cultured sand crabs, Portunus pelagicus (L.): a dual stable isotope study, J Exp Mar Biol Ecol, 361, 75–82. mu y y, shim k p and guo j y (1998) Effects of protein level in isocaloric diets on growth performance of the juvenile Chinese hairy crab Eriocheir sinensis, Aquaculture, 165, 139–48. mu y y, lam t j, guo j y and shim k p (2000) Protein digestibility and amino acid availability of several protein sources for juvenile Chinese hairy crab Eriocheir sinensis H. Milne-Edwards (Decapoda, Grapsidae), Aquac Res, 31, 757–65. nghia t t, wille m, binh t c, thanh h p, van danh n and sorgeloos p (2007a) Improved techniques for rearing mud crab Scylla paramamosain (Estampador 1949) larvae, Aquac Res, 38, 1539–53. nghia t t, wille m, vandendriessche s, vinh q t and sorgeloos p (2007b) Influence of highly unsaturated fatty acids in live food on larviculture of mud crab Scylla paramamosain (Estampador 1949), Aquac Res, 38, 1512–28. nicholson s, mann d, fotedar r and paterson b (2008) The effects of holding space on growth and survival of individually reared three-spot crab (Portunus sanguinolentus), Aquac Eng, 39, 30–6. nurdiani r and zeng c (2007) Effects of temperature and salinity on the survival and development of mud crab, Scylla serrata (Forsskal) larvae, Aquac Res, 38, 1529–38.

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obata y, imai h, kitakado t, hamasaki k and kitada s (2006) The contribution of stocked mud crabs Scylla paramamosain to commercial catches in Japan, estimated using a genetic stock identification technique, Fish Res, 80, 113–21. pan l-q, xiao g-q, zhang h-x and luan z-h (2005) Effects of different dietary protein content on growth and protease activity of Eriocheir sinensis larvae, Aquaculture, 246, 313–19. parado-estepa f d, quinitio e t and rodriguez e (2007) Seed production of Charybdis feriatus (Linnaeus), Aquac Res, 38, 1452–8. paterson b, mann d, kelly b and barchiesi m (2007) Limb-loss in pond-reared blue swimmer crabs Portunus pelagicus (L.): effect on growth in an indoor shedding system, Aquac Res, 38, 1569–79. pavasovic m, richardson n a, anderson a j, mann d and mather p b (2004) Effect of pH, temperature and diet on digestive enzyme profiles in the mud crab, Scylla serrata, Aquaculture, 242, 641–54. quinitio e t (2000) Transport of Scylla serrata megalopae at various densities and durations, Aquaculture, 185, 63–71. rodriguez e m, parado-estepa f d and quinitio e t (2007) Extension of nursery culture of Scylla serrata (Forsskal) juveniles in net cages and ponds, Aquac Res, 38, 1588–92. ruscoe i m, williams g r and shelley c c (2004) Limiting the use of rotifers to the first zoeal stage in mud crab (Scylla serrata Forskal) larval rearing, Aquaculture, 231, 517–27. seneriches-abiera m l, parado-estepa f and gonzales g a (2007) Acute toxicity of nitrite to mud crab Scylla serrata (Forsskal) larvae, Aquac Res, 38, 1495–9. sheen s s (2000) Dietary cholesterol requirement of juvenile mud crab Scylla serrata, Aquaculture, 189, 277–85. sheen s s and wu s w (1999) The effects of dietary lipid levels on the growth response of juvenile mud crab Scylla serrata, Aquaculture, 175, 143–53. sui l y, wille m, wu x g, cheng y x and sorgeloos p (2008) Effect of feeding scheme and prey density on survival and development of Chinese mitten crab Eriocheir sinensis zoea larvae, Aquac Res, 39, 568–76. sui l, wille m, cheng y and sorgeloos p (2007) The effect of dietary n-3 HUFA levels and DHA/EPA ratios on growth, survival and osmotic stress tolerance of Chinese mitten crab Eriocheir sinensis larvae, Aquaculture, 273, 139–50. sun h, zhou k and song d (2005) Mitochondrial genome of the Chinese mitten crab Eriocheir japonica sinenesis (Brachyura: Thoracotremata: Grapsoidea) reveals a novel gene order and two target regions of gene rearrangements, Gene, 349, 207–17. suprayudia m a, takeuchia t and hamasaki k (2004) Essential fatty acids for larval mud crab Scylla serrata: implications of lack of the ability to bioconvert C18 unsaturated fatty acids to highly unsaturated fatty acids, Aquaculture, 231, 403–16. tang b, zhou k, song d, yang g and dai a (2003) Molecular systematics of the Asian mitten crabs, genus Eriocheir (Crustacea: Brachyura), Mol Phylo Evol, 29, 309–16. tri l q and van mensvoort m e f (2004) Decision trees for farm management on acid sulfate soils, Mekong Delta, Viet Nam, Austr J Soil Res, 42, 671–84. trino a t, millamena o m and keenan c (1999) Commercial evaluation of monosex pond culture of the mud crab Scylla species at three stocking densities in the Philippines, Aquaculture, 174, 109–18. trino a t and rodriguez e m (2002) Pen culture of mud crab Scylla serrata in tidal flats reforested with mangrove trees, Aquaculture, 211, 125–34. tuan v a, anderson a, luong-van j, shelley c and allan g (2006) Apparent digestibility of some nutrient sources by juvenile mud crab, Scylla serrata (Forskal 1775), Aquac Res, 37, 359–65.

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28 Aquaculture and the production of pharmaceuticals and nutraceuticals K. Benkendorff, Flinders University, Australia

Abstract: Marine organisms are a rich source of biologically active compounds of interest for development as pharmaceuticals and alternative medicines. Aquaculture could play a key role in supplying these marine drugs for clinical trials and commercialisation. Using several case studies, this chapter reviews the opportunities and some problems associated with the development of marine aquaculture for pharmaceuticals and nutraceuticals. Future collaborative research in marine biotechnology and aquaculture could help transform the aquaculture industry by value adding marine species for their health benefits in niche markets. Key words: marine drugs, functional foods, alternative medicine, sponge aquaculture, algal bioresources, green-lipped mussel extracts, Muricidae, Holothurians.

28.1 Introduction Since the dawn of modern medicine, health care in many western countries has been dominated by the pharmaceutical industry, with a commercial focus on chemically synthesised medications. The strict regulations required by Food and Drug Administrations for clinical testing and quality control of pharmaceuticals have led to widespread acceptance of their use by medical practitioners and consumers. However, throughout eastern countries, traditional medicines based on natural products have maintained an important share of the health market, and these ‘natural remedies’ are regaining popularity throughout the world as alternative or complementary medicines. Definitions and examples of the different types of natural products available for health care are summarised in Table 28.1. The term ‘nutraceutical’ has recently been adopted to encompass this wide range of herbal remedies, nutritional supplements, functional and medicinal foods, with some demonstrating pharmacological benefits. However, alternative remedies vary greatly with respect to their value, availability and the degree

A medication of licensed drug A product isolated or purified from foods with demonstrated protective or physiological benefits

Topical cosmetic with pharmaceutical properties intended to enhance the health and beauty of the skin Foods that support human health and wellbeing, providing health benefits beyond basic nutrition A form of alternative medicine that treats like with like in serial dilution Includes herbal and other natural products used by certain cultures for specific medicinal applications for thousands of years

Pharmaceutical

Cosmaceutical

Traditional medicine

Homeopathic remedies

Functional food

Nutraceutical

Definition Ara-A & Ara-C (sponge derivatives) Ziconitide (conotoxin) Squalene oil (shark liver) Condroitin sulphate (shark cartilage) Lyprinol® (NZ green lipped mussel) Mussel extract (NZ green lipped mussel) Eicosapentaenoic acid (microalgae) Astaxanthin (Haematococcus microalgae) Trepang extract (sea cucumber) Resilience® (gorgonia extract) Ampoule Marin (bladderwrack extract) Seaweed regeneration cream (algae) Bechitin®-W (crustacean shell chiton) Fish oils Chitosan Mussel powder Abalone powder Shark cartilage powder Spirulina Murex (purple section from Muricidae) Sepia (cuttlefish ink) Oyster shell (shell lysate) Sea cucumber Shi Jue Ming (abalone shell) Mu Li (oyster shell) Zhen Zhu Mu (pearl oyster shell)

Marine derived examples Not available Not available $660/kg $94–470/kg $15 000/kg $1500/kg $1900/kg $2500/kg $140/kg $2710/L $2980/L $88/L Not available $94–348/kg $200–600/kg $140/kg $660/kg $112–470/kg $225/kg $340/kg $340/kg $200–595/kg $27/kg $55/kg $48/kg $53/kg

Cardiovascular health Anti-oxidant Arthritis Dermatitis Skin care Skin regeneration Skin burn regeneration Cardiovascular health Lowers cholesterol Inflammation Anti-oxidant Inflammation Anti-oxidant Womens problems Depression Bone problems Health tonic Liver tonic and vision Headache, leukorrhea Sedative and conjunctivitis

Approx value (USD)

Antiviral agent Pain killer Anti-oxidant Antiinflammatory Chronic inflammation Arthritis

Primary application

Examples of marine medical and health care products and their associated economic values based on current retail prices

Health care product

Table 28.1

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of scientific substantiation supporting their health claims. Additionally, not all nutraceuticals are necessarily derived from traditional food sources, and such products should not be assumed to be non-toxic, especially at high concentrations. Consequently, the nutraceutical industry is currently subject to tightening regulatory requirements (Brower, 2005), and substantial research is required to support the efficacy, safety and quality of new nutraceutical products. While this research can be difficult, if used effectively it can also promote the new product, which should in turn lead to greater community awareness, acceptance and economic value. Biotechnology and research into the medicinal benefits of marine organisms provides the capacity to value-add the traditional low-tech seafood industry, transforming it into a high-technology industry for niche markets. The marine environment is renowned for high biological diversity, from which an incredible range of novel chemicals have been derived. Many of these marine natural products have interesting pharmacological properties (see reviews by Munro et al., 1999; Faulkner, 2000; Sennett, 2001; Blunt et al., 2006, 2007, 2008), with some demonstrating potent activity as neurotoxins, muscle relaxants and strong pain killers, as well as providing antiinflammatory, antiviral, antibacterial, antifungal, antiprotozoal and anticancer agents. In particular, targeting marine organisms for investigations into new anticancer agents has become the major focus of recent research (Fenical et al., 2003; Jimeno et al., 2004; Butler, 2008), with a number of promising candidates currently in clinical trials. Despite extensive documentation of the chemical diversity, bioactive properties and medicinal benefits associated with marine organisms, marinederived pharmaceuticals are currently under-represented in the health and drug industries. This is partly due to problems associated with sustainable supply of marine natural products (Mendola, 2003), but in several cases clinical trials have been discontinued due to apparent toxicity or lack of efficacy when tested in vivo (Butler, 2008). There is also a relative paucity of marine organisms used in traditional medicine. Ethno-medicinal history can facilitate public acceptance and attract entrepreneurial investment, both of which are required for the innovative development of marine resources. A range of marine nutraceuticals is commercially available as a result of sustainable production through aquaculture, and supply of marine natural products for pharmaceuticals is currently under investigation. This provides a precedent for attracting further consumer acceptance of medicinal marine products, and the current examples serve as useful models for the innovative development of new products. The development of medicinal marine industries requires strategic collaboration and financial investment in research and development to produce new and improved products using innovative production processes (e.g. Sankaran and Mouly, 2007). It is quite clear from the available literature that the development of high-value medicinal products from marine organisms is a risky business, requiring substantial financial investment (many

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millions of dollars) and long development times (usually > 10 y). However, many of the risks can be mitigated by adopting a strategic planning process, which should begin by making smart choices on which products to invest in and who to collaborate with. Two alternative approaches can be taken (Fig. 28.1), depending on whether the target is to (i) produce a pharmaceutical by developing new aquaculture methods for the source species; or (ii) develop a new nutraceutical/pharmaceutical from existing aquaculture species. In the later stages of development, the paths towards commercialisation may converge in order to gain the necessary consumer acceptance and approval for the new products (Fig. 28.1). However, the emphasis for research and development is entirely different in the early stages of product development, focusing on culture techniques for species with known pharmaceutical properties, and product value-adding for the existing aquaculture species. The justification for considering each of these approaches is outlined later in the chapter, followed by case studies to illustrate both the research hurdles and promising outcomes.

Select species for value adding New nutraceuticals from current aquaculture species

Culturing new species with pharmaceutical properties

Research bioactive properties for value – adding in niche markets

Develop sustainable aquaculture practices for large-scale production

Promote consumer awareness of functional food properties Cost–benefit analysis Optimise production and processing technologies (incl. formulation) Clinical testing FDA approval, licensing and registration Marketing and commercialisation

Fig. 28.1

Major research and development steps required for innovative aquaculture of species for medicinal resources.

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28.2 Marine pharmaceuticals One strategic approach for developing high-value marine resources through aquaculture could be to target new species with demonstrated medicinal properties. The development of new species culture for the supply of medicinal compounds is relatively risky because economic viability will be highly dependent on compound yields, alternative supply options and drug approval after the final stages of clinical testing. However, if successful, the financial returns on investment in aquaculture research and development could be very high, due to high market value, high demand and low competition for supply. The annual sales for pharmaceutical drugs of natural origin are currently in the order of $1 bill USD per annum (e.g. Taxol®, $0.9 bill; Zocar®, $3.9 bill). The chance of economic return on investment in aquaculture development for marine species that produce pharmaceutically useful compounds could be maximised by selecting species that have already passed through the first phases of clinical trials, but are held up at the final testing and commercialisation stages due to the lack of sustainable supply. Currently, the most significant impediment to the commercial development of new marine drugs is their limited supply (Munro et al., 1999; Faulkner, 2000; Sennet, 2001; Proksch et al., 2003; Mendola, 2003). Although the pharmaceutical industry would typically prefer chemical synthesis as a means of supply, many bioactive marine compounds are too complex to achieve successful or commercially-viable synthesis (Faulkner, 2000). Largescale harvest from natural populations of these often rare and ephemeral marine organisms is not economical or ecologically feasible, even for preclinical and clinical evaluation (Sennett, 2001). Consequently, aquaculture and mariculture (sea-farming) are being explored as a sustainable means for obtaining sufficient compound yields, at least as an intermediate measure until chemical synthesis or fermentation technology is developed. Bulk supply of the source organism for compound extraction is required to proceed through preclinical and human clinical trials (10–1000 kg) then, once approved through the development phases, large-scale commercial supply (1000s metric tonnes per annum) is required immediately to maximise investment returns on patents or licenses (Munro et al., 1999; Sennett, 2001). In recent years, there have been increased research efforts directed towards establishing aquaculture of marine invertebrates for marine compounds, and this is now a reality. Examples include the successful aquaculture of an ascidian Ecteinascidia turbinata for the anticancer compound ET-743 (YondelisTM), the bryozoan Bugula neritina for another anticancer agent bryostatin 1 (Faulkner, 2000; Mendola, 2003), as well as several sponge species in clinical trials (see case study, Section 28.5.1). Economic projections for Ecteinascidia turbinata and Bugula neritina suggest that in-sea culture could be a cost-effective option for the supply

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of these anticancer drugs, and this is currently the only option for Bryostatin 1 (Mendola, 2003). Despite the technical success of the ascidian aquaculture project in the Florida Keys, CalBioMarine Technonologies discovered that the high labour costs and insurance premiums associated with aquaculture enterprises in the USA priced them out of the world market for supply of ET-734 (Mendola, 2003). ET-743 is being developed by PharmaMar (Zeltia) in partnership with Johnson & Johnson Pharmaceutical Research & Development (Adis International Ltd, 2006) and has now been successfully synthesised (Fishlock and Williams, 2008). Nevertheless, the successful culture of both these marine invertebrates has enabled major advances in protocols that can now be applied to other related organisms to fast track their culture for future drug development purposes. To help finance the expensive clinical trials required for new pharmaceutical development (∼$230–500 mill USD), early financial returns on marine compounds may be gained through development as medical research tools. This requires a good understanding of the molecular and/or cellular targets of the compound, which can then also be used to infer mode of action and thus facilitate the drug approval process. A good example of this is provided by the conotoxins, which were available as research tools for targeting ion channels and receptors on nerve cells (Sigma $700–$1800 USD/mg), decades before being effectively developed as a pharmaceutical analgesic (Ziconitide licensed as Prialt® by Elan Pharmaceuticals). The conotoxins are peptides that can be chemically synthesised, thus precluding the need to culture the source cone snails. A number of other marine natural products are currently extracted and sold as fine chemicals for research by biotechnology companies, such as Calbiochem. These have included several high-value compounds sourced from sponges; the Bastadins from Lanthella basta enhance calcium ion release from muscle and non-muscle cells by modulating the RyR1/FKBP12 complex and were previously available from Calbiochem for $5800–$9040 USD per mg. Manoalide from Luffariella variabilis is licensed to Allergan Pharmaceuticals and, despite being withdrawn from clinical trials, it is commercially available as a probe for Phospholipase A2 inhibition. It was previously only available from Calbiochem for $20 400 UDS per mg, but is now also available from Sigma-Aldrich, presumably at a lower cost. Okadaic acid strongly inhibits protein serine/threonine phosphatase and was available from the sponge Halichondria okadai for >$4000 per mg (Calbiochem), but is also now available from the marine dinoflagellate Procentrum concavum at a lower cost (e.g. Sigma-Aldrich = $3960 and Sciencelab.com $2756). The market for fine chemicals may be too small and inconsistent to support an aquaculture venture to support this purpose alone. However, if compounds reach clinical trials for pharmaceutical development, any financial return from the medical research market can help mitigate the risks of heavy research investment. Alternatively, research into their pharmaceuti-

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cal application can lead to an understanding of the specific mode of action, thus enabling commercialisation as molecular probes to recover costs if clinical trials fail (e.g. manoalide). Another option for maximising early returns on investment in the research and development of marine pharmaceuticals is to consider the development of bioactive extracts as alternative medicines or cosmetics. Less stringent clinical testing is required for the approval of new products strictly intended for topical (skin) application, when compared to the internal delivery required for pharmaceuticals. One example of this is provided by the pseudopterosins from gorgonian coral (Pseudopterogorgia elisabethae), which have been under investigation by OsteoArthritis Sciences Inc. as potent antiinflammatory agents. Although they are not yet available as drugs, a partially purified extract from the gorgonian is available as an additive in the Estée Lauder cosmetic skin care product Resilience® (Kijjoa and Sawangwong, 2004). This commercial development has resulted in demand far exceeding the supply (Faulkner, 2000), which is based on controlled large-scale harvesting. Successful culture and carefully managed sea-farming could help ensure that this marine bioresource industry remains sustainable. However, significant advances have also been made in the chemical synthesis of pseudopterosins (Heckrodt and Mulzer, 2005).

28.3 Marine nutraceuticals In 1989, the term ‘nutraceutical’ was coined by DeFelice, the founder of the Foundation for Innovation in Medicine, to describe ‘a food or part of a food that provides medical or health benefits, including the prevention and/or treatment of disease’ (Brower, 2005). The concept that natural food products can contribute to medical practice has a history of well over 2000 years. For example, the Greek physician Hippocrates (460–377 BC) stated that ‘Our food should be our medicine. Our medicine should be our food’. Nevertheless, the term nutraceutical cleverly combines the idea of ‘nutritional’ and ‘pharmaceutical’ and was introduced to identify a rapidly growing area of biomedical research. This has effectively facilitated growing acceptance by public health authorities and health consumers in countries like the USA. At present, nutraceuticals represent the fastest growing segment of the food industry with an estimated market of $30 bill USD in 2002 growing at 5 % per annum (Andlauer and Fürst, 2002). Marine nutraceuticals currently share a small but growing portion of the natural health market (Molyneaux and Lee, 1998; Barrow and Shahidi, 2007). In comparison to marine-based pharmaceuticals, there is a wide range of commercially available marine nutraceuticals and alternative medicines currently on the market. This is most likely related to the fact that it has been historically easier to gain approval for the registration of a new food or natural extract, when compared to the many hurdles required prior to

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approval of a new pharmaceutical drug. As a consequence, there is often little research to support the health claims of these products, particularly when they are based on traditional (ethnomedical) use. Furthermore, the amount and stability of bioactive ingredients can vary greatly in nutraceuticals, resulting in unreliable outcomes in clinical trials (e.g. Cobb and Ernst, 2006). As a consequence, both the quality and associated value vary greatly between different types of natural health products (e.g. Table 28.1). These differences in value are primarily influenced by the availability of the source species, the amount of processing required and the degree of associated research to substantiate their health properties, in addition to market size and demand. For example, Chinese remedies based on powdered mollusc shells have a relatively low market value, as these are readily obtained as by-products of the fishing and aquaculture industry and only require lowtechnology processing. Perhaps surprisingly, the homeopathic products appear to have higher value despite less research to substantiate their use and the requirement for only tiny amounts of the source species. However, the preparation and formulation of homeopathic products requires specialist knowledge restricted to a few niche suppliers resulting in low market competition. Furthermore, the market for homeopathy is very small when compared to the large number of Asian consumers who still rely on cheap traditional medicines. By definition, nutraceuticals and functional foods offer the greatest opportunity for value-adding the aquaculture industry through the development of novel health products. The functional food market had an estimated value of between $30–50 bill USD and has been growing at a rate of 8–14 % per annum (Tapsell et al., 2005). At the extreme end, functional foods can be developed into ‘medicinal foods’. These are defined by the Food and Drugs Administration as foods ‘formulated to be consumed or administered internally under the supervision of a physician and intended for specific dietary management of a disease or condition for which distinctive nutritional requirements based on recognised scientific principles are established by medical evaluation’ (Molyneaux and Lee, 1998). Clearly, this requires rigorous and expensive research to meet regulatory approvals, similar to those imposed on pharmaceuticals, and should therefore ultimately attract equally high profit margins. However, nutraceuticals and medicinal foods should be cheaper to produce in large quantities, compared to the pure chemical agents required in pharmaceutical drugs. Nutraceuticals can be distinguished from pharmaceuticals by the fact that they contain a mixture of natural ingredients, which often work synergistically to confer a range of biological activities. This means that it can be harder to maintain and monitor their quality but, as demonstrated by the lyprinol case study (Section 28.5.3), strategic research alongside effective marketing can ultimately lead to successful returns on financial investments in new marine product development. Strategic investment by the New Zealand Government and seafood sector has been particularly successful in establishing

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innovative growth and emergent industries for high-value marine products (e.g. Sankaran and Mouly, 2007). One relatively low-risk approach for investing in marine biotechnology would be to concentrate on species already in aquaculture (Fig. 28.1) and optimise processes for concentrating or improving their known medicinal properties, or developing new extracts from their waste products. A prime example of this is the production of fish oils for omega-3 polyunsaturated fatty acids, which have been widely promoted for their health benefits in recent years, leading to remarkable increases in consumer demand. The Japanese fisheries industry has been particularly proactive in recovering commercially valuable natural products from marine sources (Ohshima, 1998), including over 200 fish oil products worth over $263 mill USD per year. The Japanese also extract protamine, an antibacterial agent used in the food processing industry, from fish gonads (a waste product) valued at over $47 mill USD per annum. Another example of valuable waste products from marine organisms is chitin and chitosan from crustacean shells (Table 28.1). Even a decade ago, the value of chitin from the Japanese Fisheries industry was estimated at $53 mill USD per annum (Ohshima, 1998). Demands for high-quality chitosan products are currently growing, particularly for incorporation into niche health foods, since these have been demonstrated to absorb fat and lower cholesterol. US researchers are developing ways to incorporate ground shellfish product into ‘nutraceutical snack foods’ (Obatulo et al., 2005). Processed chitin is also used to create a commercially-available film Bechitin®-W, used for regenerating burnt skin. Crustacean shells also provide an important source of glucosamine, which is reported to reduce inflammation and thus is incorporated into pet feed and human health products to treat the symptoms of arthritis. A wide range of other value-added marine products could be supplied through aquaculture, including marine-derived enzymes (see Ohshima, 1998), as well as processed mollusc shells and lipid extracts (e.g. Table 28.1), which are used as mineral supplements and natural remedies to promote health, respectively. Strategic research aimed at substantiating these health benefits will help increase consumer awareness and acceptance of these products, thus leading to larger market opportunities.

28.4 Diversifying the aquaculture industry To lower the risks associated with the aquaculture of new species for medicinal resources, it would be worthwhile considering options for diversifying existing aquaculture farms through co-culture or polyculture. The polyculture of different species, including those with medicinal properties, has several important advantages. First, different species may be more or less resilient to adverse environmental events, such as storms and disease outbreaks, thus reducing the chance that all cultured stock will be simultane-

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ously lost. Second, holding stock targeted towards different markets can help buffer problems associated with fluctuations in market demand and prices. Most important, however, is the potential for exploiting the complementary ecological roles of species in different trophic niches. In particular, nutrients can be effectively recycled within aquaculture systems by combining primary producers (e.g. algal, Neori et al., 2004) and/or detritivores (e.g. sea cucumbers, Kang et al., 2003) with herbivores (e.g. fish and abalones) or even secondary and tertiary predators. These approaches to polyculture have demonstrated ecological and economic benefits and, for this reason, integrated intensive aquaculture has been touted as the way of the future (Neori et al., 2004). Indeed a highly successful program of integrated multitrophic aquaculture has been established in Canada by Thierry Chopin’s interdisciplinary research laboratory (Chopin, 2006–2008 and references therein). A number of polyculture farms also exist in Europe and Asia, but these are mostly directed towards traditional food production. Nevertheless, there is much potential for combining traditional aquaculture species with species selected for high-value medicinal resources. For example, sponges grown on long lines could be integrated into mussel farms (Munro et al., 1999). A range of medically useful algal species can also be integrated into aquaculture farms, and there is much potential for future development of medicinal sea cucumbers and whelks through polyculture (see case study, Section 28.5.4).

28.5 Current case studies 28.5.1 Sponge aquaculture for pharmaceuticals Back in the 1950s, antiviral and antileukemic compounds isolated from a marine sponge provided a novel lead for the development of the first marine-derived pharmaceuticals, Ara-A and Ara-C. Since then, thousands of bioactive compounds have been documented from sponges, and many have proceeded into preclinical and clinical testing (see Sipkema et al., 2005) but, to date, no more have made it onto the market. One of the main reasons for this is the problem of cost-effective large-scale supply required for clinical trials, which significantly slows down the drug development process. Pharmaceutical companies will rarely prioritise compounds that are expensive or difficult to produce, whereas sponge secondary metabolites are often highly complex and may only be present in tiny amounts in the source species. Ara-A and Ara-C are comparatively simple structural derivatives from the sponge natural products that can be produced by microbial fermentation or chemically synthesised. As up to a kilogram of compound is required for drug development (Duckworth, 2001), the sponge metabolites that have entered into clinical testing are also typically amenable to synthesis (Sipkema et al., 2005). However in the last decade, attention has shifted towards alternative methods of supply, and in cases where

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the source organism is rare and/or has a limited distribution, aquaculture may be the only feasible option for preclinical and clinical work (Munro et al., 1999; Mendola, 2003). The farming of sponges is not a recent concept, as this has actually been in practice for the production of bath sponges since the early 20th century (Duckworth, 2001). However, much research is still required to optimise farming methods for bioactive metabolite production. Sponge aquaculture is facilitated by the ability of sponges to regenerate lost tissue. Small pieces, known as explants, can be cut from the sponges and transplanted onto rope, concrete disks, mesh structures or scallop lanterns for growth in their natural habitats. The growth and survivorship of explants can be influenced by the amount of tissue damage and the stress of transplantation (Duckworth et al., 1997). Interestingly, however, elevated biological activity was reported from two species after six months culture compared to wild sponges, presumably in response to the tissue damage (Duckworth, 2001). In other species the level of secondary metabolite production may be ‘hardwired’. For example, manoalide production could not be induced in the sponge Luffariella variabilis and was consistent across several geographic locations (Ettinger-Epstein et al., 2007). However, the variability in the levels of manoalide produced within populations suggests the production of this anticancer compound could be heritable, thus offering the potential for selective culture of high-yielding stocks (Ettinger-Epstein et al., 2007). The biosynthesis of halichondrins was reported to continue even after several years in culture (Munro et al., 1999). These results are promising for the supply of pharmaceutical compounds through sponge aquaculture, although in most cases this may only be viable for short- or medium-term supply of target compounds during the drug development phase (e.g. see Page et al., 2005 for the supply of mycalamide A, pateamine and peloruside A from the marine sponge Mycale hentscheli). Observations on species in their natural habitats can provide valuable clues for optimising productivity in aquaculture (Mendola, 2003). High growth rates and good survivorship have been reported for several sponges after many months in sea-based aquaculture (e.g. Duckworth et al., 1997; Munro et al., 1999; van Treeck et al., 2003; Page et al., 2005). However, some species cannot be successfully cultured using traditional approaches in mesh, and new substrate technologies are currently on trial using electrolysis to precipitate minerals from the seawater (van Treeck et al., 2003), which increases growth by increasing nutrient availability. Temperature, algal biomass and nutrients in suspended particulate matter, as well as depth and wave exposure sediment cover, appear to be some of the most important factors affecting sponge growth (Duckworth et al., 1997; Koopman and Wijffeks, 2008). Land-based aquaculture appears to offer the advantage of greater control over environmental conditions, such as seasonal variation and unpredicatable storm events. Recent progress has been made with the successful growth of sponges in closed aquarium systems and explants show

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high survivorship, reasonable growth rates and bioactivity similar to that of wild sponges after six months in culture (de Caralt et al., 2003). However, in general the growth rates in closed systems may not compare favourably to growth in the field. For most sponge species, a lack of knowledge on optimal environmental conditions, feeding regimes and reproductive strategies remains a major limiting factor for large-scale sponge aquaculture (Duckworth, 2001; Abdo et al., 2008). Recently, a cost–benefit analysis of alternative methods for the largescale production of pharmaceuticals has been undertaken for two marine sponges (Sipkema et al., 2005). Conflicting results were found for the two species, which was largely influenced by the amount of bioactive compound present in the sponge. Halichondrin B is an anticancer agent derived from a rare sponge Lissodendorxy sp. and is of interest as a potential drug to treat melanomas and leukaemia. Total synthesis of this compound requires at least 100 steps with an overall yield of less than 1 %, which is clearly not economically feasible. However, maximum growth rates of up to 5000 % in one month have been reported from cultured explants of this species, which appears promising for aquaculture. However, as only 400 μg of halichondrin B is present per kg wet weight of sponge, downstream processing is a major cost determining factor. Comparison of the economic potential with approved medicines revealed that treatment with halichondin B would be at least an order of magnitude more expensive to recover similar profits. Consequently, the production of halichondrin B through aquaculture does not appear to be economically viable. By comparison, the production of the paramedic medicine Avarol for treatment of psoriasis by aquaculture of the sponge Dysidea avara looks much more promising. Avarol can be chemically synthesised in 20 steps, although it has low yields (2 %) and requires several expensive substrates, and thus is not considered economically feasible. However, Avarol is naturally produced in quantities up to 3 g per kg sponge which, coupled with high growth rates, permits very reasonable production costs. The production of 150 kg of purified Avarol was estimated to yield an annual turnover of $34 mill USD, with $28 mill in profits and production prices of only $22 USD per patient per year. This can certainly compete economically with commercial antipsoriasis pharmaceuticals currently on the market. Overall, this in-depth study by Sipkema et al. (2005) illustrates the importance of comprehensive technical and economic analysis for assessing the feasibility of aquaculture for the production of marine pharmaceuticals.

28.5.2 Algal targets for pharmaceuticals and nutraceuticals Seaweed remedies have been traditionally used in a number of cultures for a wide range of applications, including pain, abscesses, menstrual problems, cancer and infection (Ruggieri, 1976). Research into the active ingredients of algae used in seaweeds therefore underlies an important area of drug

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discovery. Of particular interest are the sulphated polysaccharides from red algae, which have potent antiviral activity. Of these, carrageenans from Gigartina skottsbergii provide the active ingredient in the vaginal microbicide Carraguard®, which blocks HIV and other sexually transmitted diseases and is currently in Phase III clinical trials in Africa (Smit, 2004). Fucoidans are another type of algal sulphated polysaccharide with potential for the development of vaginal microbiocides with contraceptive properties, in addition to having demonstrated antitumor, antithrombic and anticoagulant properties in vivo (Smit, 2004). A range of other interesting drug leads has been identified from algae, but none are currently commercially available as pharmaceuticals. However, several algal compounds are available as medical research tools (reviewed by Smit, 2004). These include the phycobiliproteins used as fluorescent tags, lectins from Codium spp., which are used routinely in biochemical studies and toxic kainoids from dianoflagellates, which are used to investigate neurophysiological disorders. A review of phycobiliproteins has revealed 266 existing patents on their applications in medicine, biotechnology and fluorescence properties, as well as 55 patents on their production, with at least 11 companies currently producing and selling these algal proteins or their derivatives (Sekar and Chandramohan, 2008). Eriksen (2008) specifically reviews the production of phycocyanin. This phycobiliprotein is presently extracted from open pond cultures of the cyanobacterium Arthrospira platensis, but increased productivity may be obtained from high-density cultures of the red algae Galdieria sulphuraria. The kanoids have also been identified as high-value products that could provide a viable option for development through algal cultivation (Smit, 2004). In addition to their use in medical research, these algal toxins have insecticidal and antihelminthic activities. In fact many algae hold good potential as a source of environmentally-friendly pesticides, antiseptics, cleansing agents and agrochemicals based on widespread antibiotic, antifungal, algicidal and insecticidal activities. A number of cultivated seaweeds also have strong antibacterial activity against a range of fish pathogens, thus offering potential alternatives to the antibiotics currently used in aquaculture (Bansemir et al., 2006). The cultivation of high-value seaweeds in integrated aquaculture systems is being explored as part of a recent European Union project SEAPURA – Species Diversification and Improvement of Aquatic Production of Seaweeds Purifying Effluents from Integrated Fish Farms (Santos, 2006). Integrating seaweed culture into fish and shrimp farms has been recognised as a cost-effective and sustainable solution to reducing nutrient loads in aquaculture effluents but, to date, the most commonly-used biofilters are based on green algae with low market value. Mata et al., (2006) demonstrated that the red algae Asparagopsis armata was a viable alternative, with a high capacity to remove nutrients from fish waste, in addition to producing halogentated organic compounds for potential development as antibiotics, fungicides and skin cosmetics. Porphyra spp. also appear to be good candidates

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for integrated aquaculture, with high growth rates and efficient nutrient removal (Carmona et al., 2006). Porphyra provide a multi-billion dollar industry for the production of nori for human consumption, as well as providing a source of the phycobiliprotein R-phycoerythrin, which is used as a fluorescent conjugate for immunological detection in biomedical research. In the future, careful selection of seaweed species for integrated aquaculture based on their useful secondary metabolites could yield high economic returns, in addition to improving the public image through application of environmentally sustainable technologies (Santos, 2006). Microalgal biotechnology also provides opportunity for aquaculture development. Nutritional supplements are currently produced from cultured Chlorella, Dunaliella and Spirulina with a collective value suggested to be in the order of hundreds of million dollars (Apt and Behrens, 1999). More recent developments have focused largely on the production of polyunsaturated fatty acids, both as nutritional supplements and pharmaceutical agents. Whilst fish oils have long been recognised as a good source of these omega 3 polyunsaturated fatty acids, there are safety concerns regarding contamination by various toxins that accumulate in fish, as well as problems associated with instability and high purification costs. Omega 3 fatty acids from microalgae are reported to be of better quality, more stable and less expensive to obtain because their fatty acid profile is simpler than the marine animal lipids (Lebeau and Robert, 2003). Phaeodactylum tricornutum is a major source of high-quality eicosapentaenoic acid (EPA), which has been shown to prevent coronary heart disease and hypertriglyceridemia, lower blood cholesterol and reduce the risk of arteriosclerosis. EPA can be extracted in higher purity from P. tricornutum than other sources such as cod liver oil (Lebeau and Robert, 2003) resulting in a product that is five times more valuable (Table 28.1). Dinoflagellates are especially well suited to the production of the omega-3 Docosahexaenoic acid (DHA), which is required for postnatal brain development. DHA has been recently approved by the World Health Organisation for incorporation into commercial formulas for infant feed. Fish oils are not considered suitable for infant formulas because these are also rich in EPA, which can retard growth (Apt and Behrens, 1999). Instead, high-quality DHA containing oil from the dinoflagellate Crypthecodinium is currently incorporated into infant formulas available worldwide. Dunaliella is also cultured for the production of beta-carotene, another antioxidant marketed as a neutraceutical (NBT Ltd Eilat, Israel http://www.chlostanin.co.jp/english/e_top.html). The production of microalgae for high-value nutraceuticals and health food supplements is primarily achieved in open pond culture systems. Circular ponds first developed after World War II are still used in Japan, Taiwan and Indonesia, whereas raceway-shaped ponds are used in Israel, the USA, China and other countries (Lee, 2001). One of the main problems for open microalgal culture is the need to maintain monoculture and the potential for contamination by other microorganisms (Apt and Behrens, 1999; Lee,

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2001). Nevertheless, successful long-term monocultures have been achieved by maintaining extreme culture environments; high salinity for Dunaliella, high alkalinity for Spirulina and high nutrition for Chlorella (Lee, 2001). However, this approach greatly limits the range of microalgae that can be cultured. More recently, enclosed photobioreactors have been trialled with some success. Although these are generally more expensive to build than outdoor pond systems (Apt and Behrens, 1999), the cost can be justified for higher value microalgal products. For example, new developments in photobioreactor design for the dinoflagellate P. tricornutum have produced high cell densities and enhanced yields of EPA (Lebeau and Robert, 2003). Another problem with photobioreactors is that they can be difficult to sterilise efficiently due to the need for large surface area to volume ratios to supply light to the cultures (Lee, 2001). Stirred tank fermenters may therefore be the most viable option for producing high-grade pharmaceutical type products that require well-defined, pure microalgal cultures (Lee, 2001). Largescale commercial production of the dinoflagellate Crypthecodinium for DHA is achieved by fermentation (Apt and Behren, 1999). A large number of other microalgae are capable of heterotrophic growth (requiring organic carbon) and therefore should also be suited to fermentation technology. Another valuable compound obtained from the microalgae Haematococcus is astaxanthin (Higuera-Ciapara et al., 2006). Although this pigment can be chemically synthesised, consumer demand for natural products provides increasing opportunity for production by microalgal culture. Currently, astaxanthin has a worldwide market value estimated at $200 mill USD per annum, most of which is used as a pigment in aquaculture feeds (Lorenz and Cysewski, 2000). However, there is also increasing evidence to support the antioxidant benefits of astaxanthin and its role in enhancement of the immune system. Presently, the production of natural astaxanthin from Haematococcus is achieved in enclosed photobioreactors in Sweden and Hawaii (Lorenz and Cysewski, 2000). In Hawaii, open culture ponds have also been used for successful commercial production of Haematococcus. Once a sufficient density of the mircoalgal cells has grown, the cultures must be subjected to environmental and nutrient stress to induce the production of astaxanthin. Despite problems with the commercial scale-up and need for advanced technology with high setup costs, the production of astaxanthin provides further evidence that microalgal culture can become a commercial reality.

28.5.3 Green lipped mussel culture for nutraceuticals The development of the nutraceutical Lyprinol® from the New Zealand green-lipped mussel (Perna canaliculus) has been widely hailed as a success story for the New Zealand seafood sector (reviewed by Sankaran, 2005; Sankaran and Mouly, 2007). It began with a pioneer mussel farmer and two

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entrepreneurial brothers following up on a lead from Maori folklore which suggested that coastal dwelling communities who regularly consumed the mussel suffered much lower rates of arthritis than inland dwelling relatives. Initially, a freeze-dried mussel powder was developed as an anti-arthritic product. However, scientific investigations into the anti-inflammatory activity of the powder were inconclusive and attempts to isolate and identify the active compound failed. Rather than being deterred by these initial outcomes, the company persisted, seeking additional collaborations and financing further research to develop a stabilised lipid extract with reliable bioactivity. After many years and a financial investment of over $3.8 mill USD into research, the patented product Lyprinol® is now commercially available as a treatment for chronic inflammation. It has been independently tested by several research groups and shown to be effective in clinical trials for arthritis and asthma (Gibson, 2000; Halpern, 2000; Emelyanov et al., 2002). Due to the adverse effects associated with long-term use of conventional therapeutics, osteoarthritis patients spend more on alternative and complementary medicines than any other medical condition in the USA (Cobb and Ernst, 2006). In comparison to most pharmaceutical analgesics and non-steroidal antiinflammatory drugs, green lipped mussel preparations appear to have few side effects and possibly gastroprotective functions (Rainsford and Whitehouse, 1980). More recently, Lyprinol® has been shown to be effective in a preclinical rodent model for the prevention of inflammatory bowel disease (Tenikoff et al., 2005). Pharmaceutical preparations containing New Zealand green lipped mussel extracts have been patented for use in treating side effects, such as gastric ulcers caused by oral ingestion of analgesics (McFarlane and Croft, 1984) and provide good opportunities for combination anti-inflammatory therapy (Whitehouse and Butters, 2003). One of the difficulties associated with the commercial marketing of Lyprinol® is that there are a number of potentially competing green lipped mussel products available, which are also generally promoted as antiinflammatory agents, effective in halting the progression of joint and connective tissue problems and relieving the symptoms of arthritis. These include freeze-dried mussel powders and a mussel lipid extract, which is claimed to have up to five times greater anti-inflammatory properties (Aroma, 2007). However, a systematic review of clinical trials on these freeze-dried green lipped mussel preparations concluded that there was little consistent evidence supporting the therapeutic use of these products for rheumatoid or osteoarthritis (Cobb and Ernst, 2006). Conflicting results on products carrying the same trade name but sold by different manufacturers in different countries suggest there is much variation in the product potency, probably due to the lack of effective stabilisation and possible heat exposure, which degrades the bioactive lipids. This illustrates the need for quality control and regulated labelling of prod-

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ucts. However, ultimately, it is up to the consumer to investigate a product’s supporting evidence before outlaying considerable funds on the long-term use of nutraceuticals. In this case it appears that you get what you pay for, as there is a ten-fold increase in the price of green lipped mussel products, from powder to lipid extract to the stabilised and patented Lyprinol® (Table 28.1). The innovative development of Lyprinol® would never have come about without the relentless belief in the health benefits of the green lipped mussel by entrepreneurs who were willing to commit a substantial financial investment and strategically collaborate with appropriate researchers (Sankaran, 2005). The global market for Lyprinol® is now estimated at some tens of millions of dollars (NZ) and occupies a significant proportion of the sales in NZ GreenshellTM mussels (Sankaran and Mouly, 2007). The value chain for Lyprinol® appears to have created a market value of over 100 % compared to the relatively unprocessed green lipped mussels, resulting in the expansion of the company and even the need to procure mussels from other farms just to meet the demand (Sankaran and Mouly, 2007). This illustrates the clear benefits than can result from strategic investment in value addition in the aquaculture sector.

28.5.4 Polyculture potential for medicinal sea cucumbers and whelks Sea cucumbers (Holothurians) and the Muricidae family of whelks are two additional groups of marine invertebrates with current medicinal uses that are under-represented in aquaculture. These hold potential for polyculture on more traditional aquaculture farms for vastly different reasons. The sea cucumbers are benthic deposit feeders that can reduce ammonium and nitrate in wastewater. Recently, California sea cucumbers (Parastichopus californicus) were shown to successfully utilise the naturally-available biodeposits from the cultured Pacific oysters (Crassostrea gigas), indicating the feasibility of developing a co-culture system that would both reduce the amount of organic waste underneath shellfish farms and produce a secondary cash crop (Paltzat et al., 2008). In addition, the benefits of co-culturing sea cucumbers with abalone have been demonstrated, with significantly better growth and survival of the abalone in co-culture compared to controls grown without sea cucumbers (Kang et al., 2003). Sea cucumbers, also known as trepang and Beché-de-Mer, are considered an Asian culinary delicacy and are highly regarded by Chinese doctors as a general health tonic (Hei shen = sea ginseng). In addition to their use in traditional medicine, sea cucumber extracts are marketed as nutraceuticals (Table 28.1), particularly in Eastern Europe and Russia. Dried sea cucumbers are also marketed as a type of functional food. They contain a range of bioactive compounds, such as glycosides and prostaglandins, as well as providing a good source of vitamins and minerals. Clinical trials have revealed that sea cucumber extracts are effective antithrombotic

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agents (Li et al., 2000), and numerous cytotoxic (antitumor) glycosides have been isolated from their extracts (e.g. Zhang et al., 2006; Borsig et al., 2007; Liu et al., 2007). There is fairly high demand for sea cucumber products and, at present, the market is primarily reliant on wild fisheries. Therefore, further development of sustainable sea cucumber aquaculture could help supply the immediate demand and facilitate further research into value-added nutraceuticals by optimising processing procedures for quality control. The purple dye secretion from murex whelks (Muridicae; Caenogastropoda) is currently used as a homeopathic remedy to treat pain, hives and women’s problems, including uterine cancer, dysmenorrhoea, chronic endometritis, metrorrhagia, leucorrhoea, nymphomania, anxiety and melancholy disposition (Boericke, 2005; Cazalet, 2007). However, there are few data available to support the purported biological activity of homeopathic remedies in general (Straus, 2000) and the Murex remedy is no exception. Nevertheless, a minor constituent of the murex dye, 6,6’ dibromoindirubin, has been shown to inhibit cell proliferation (Meijer et al., 2003; Magiatis and Skaltsounis 2006), implying potential anticancer properties. The intermediate dye precursor tyrindoleninone also appears to have selective cytotoxicity towards lymphoma cell lines (Vine et al., 2007) and the oxidation product 6’bromoisatin is generally cytoxtoxic at reasonably high concentrations (Westley et al., 2006). Furthermore, the ultimate dye precursor is a salt of choline esters, which display potent neuromuscular blocking and muscle relaxing activity, as well as nicotinic action (Baker and Duke, 1976; Roseghini et al., 1996). These bioactive compounds could all potentially contribute to the homeopathic application of the Murex remedy, although it should be noted that serial dilution, characteristic of homeopathy, may significantly affect the likelihood of activity. Our ongoing research at Flinders University on semi-purified extracts from the Australian Muricidae Dicathais orbita indicates that there is good potential for developing an improved natural remedy from these marine snails with significant anticancer activity against a broad range of solid tumors and lymphomas (Benkendorff et al., 2008; 2009). Interestingly, the bioactive compounds in our Dicathais extracts appear to be retained after boiling, suggesting the potential exists for the use of these snails as a functional food, in addition to complementary medicines. At present, large numbers of these snails are required for preclinical safety and efficacy trials in rodent models (e.g. Benkendorff et al., 2008), which are largely supplied by abalone sea farms where the whelks are collected as pests, thus alleviating pressure on the natural populations. As muricid whelks are predatory, they are often regarded as pests on molluscan aquaculture farms, where they can occur in large numbers leading to high prey mortality (e.g. oyster drills, MacKenzie, 1961). However, some whelks are preferential scavengers (e.g. Woodcock and Benkendorff, 2008), suggesting there may be possibilities for using them to help maintain hygiene

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on molluscan aquaculture farms through the natural removal of moribund animals. Scavenging behaviour also facilitates supplementary feeding by the use of frozen food. We are currently undertaking research on the aquaculture potential of Dicathais orbita (e.g. Noble, 2006; Woodcock and Benkendorff, 2008), to help ensure a sustainable supply if and when the value-added (nutraceutical) products become commercially available.

28.6 Steps towards commercialisation The commercialisation of new marine resources for the health market requires investment in research and development at several stages (Fig. 28.1). Preferably, industry involvement is required across all stages, to transfer the relevant knowledge and technologies on a practical scale. For the development of new nutraceuticals, including functional foods from aquaculture species or their waste products, preliminary in vitro biological assays are required to scope the potential for marketing and development. For species that have already been identified as useful pharmaceutical leads, clinical testing is required, along with preliminary assessment of the potential to establish sustainable culture techniques for economical large-scale production. After these preliminary studies, a cost–benefit analysis should be undertaken in both cases before investing in production and processing technologies. These should consider the worst- and best-case scenarios predicted from the preliminary studies with consideration of potential for optimisation of production and processing. The costs associated with processing and purification of marine extracts, for pharmaceutical compounds in particular, can be quite significant (see Sipkema et al., 2005) and consequently research is required to optimise the yields. The product must then be formulated for delivery in a bioavailable form. For example, this may involve encapsulation technology for oral ingestion or incorporation into non-allergenic creams for topical application. Protection of the intellectual property must also be considered, e.g. through patents or secrecy agreements. It is possible to patent a novel marine extract or compound for specific pharmaceutical or nutraceutical applications providing there is no prior art published in the literature or available in the public domain (i.e. any information that has been made available in any form can be relevant to a patent’s claims of originality). It is also possible to patent a novel method of processing marine bioresources. The next stage of clinical testing is largely dictated by the requirements for product registration, which does differ between countries. Pharmaceuticals and nutraceuticals are controlled through the Food and Drug Administration (FDA) in many countries. Stricter regulatory requirements are usually required for the licensing of therapeutic drugs, whereas nutraceuticals are often regulated as foods. In the USA, the Dietary Sup-

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plementation Health and Education Act allows manufacturers, who are in the process of clinical trials for drug approval, to market marine nutraceuticals as dietary supplements, with appropriately labelled health claims (Molyneaux and Lee, 1998). However, in many other countries, nutraceuticals are treated as drugs and thus health claims are prohibited unless backed up by appropriate clinical research (Brower, 2005). Irrespective of the details in the legislation, both regulatory authorities and consumer groups are likely to evaluate the overall weight of scientific evidence qualifying the health claims for nutraceuticals. Substantiation for both the safety and efficacy of the product will therefore influence the allowable product labelling and consumer acceptance, providing a strong incentive to invest in clinical trials. In terms of product labelling and marketing, the laws in many countries prohibit specific health claims for non-pharmaceuticals, such as foods. However, third-party statements from researchers and medical doctors are permitted and can be highly respected by consumers (e.g. in Japan, Brower, 2005). Examples of the allowable health claims for functional foods in Australia are summarised in Table 28.2. In the USA, it is possible to make claims about the presence of bioactive compounds within nutraceuticals and their ability to affect the structure or function of the body, but medical claims are not allowed. However, the distinction between health and medical claims is not clear (Brower, 2005) and, whilst the FDA regulates food labelling, advertising in the USA is regulated by a separate body, the Federal Trading Commission (FTC). The FTC has been known to allow health claims in advertisements for new products prior to FDA approval for the claims (Sankaran, 2005). With the growing consumer reliance on nutraceuticals to improve and maintain health, regulatory authorities, along with consumer watch bodies and lawmakers are likely to work

Table 28.2 Health claims for functional food in Australia Type of claim

Generalised example

Regulatory requirements

Nutrient content

‘this product is a good source of X’ ‘this product contains X, which helps maintain blood vessels’ ‘this product contains X, which may reduce cholesterol levels’ ‘this product contains X, which prevents cancer’

Commonly used

Structure – function Health Therapeutic

Source: adapted from Tapsell et al., 2005.

Requires scientific substantiation and quality assurance Requires clinical trials for substantiation NOT permitted on foods

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together to ensure appropriate health claims are backed by appropriate scientific evidence.

28.7 Future trends There is no doubt that persistent research and commitment to commercialisation can lead to successful development of valuable medicinal marine products for niche markets. Aquaculture of marine invertebrates for medically useful compounds is now a reality and helping to solve the difficulties associated with supplying sufficient material for clinical testing and commercialisation. Worldwide interest in nutraceuticals is also growing rapidly, with some physicians starting to recommend natural products before prescribing pharmaceuticals (Brower, 2005). A wide range of natural remedies are available from marine organisms, and yet marine nutraceuticals currently represent only a small portion of the market. Given the widespread biological activities associated with aquatic organisms and the potential to farm them on a large scale through aquaculture, there is huge scope for value-adding this industry through the production of medically useful products. In the past, much of the funding for marine natural products chemistry has originated from health science agencies (Faulkner, 2000) and was undertaken by small independent research groups. Increasingly we are seeing the implementation of large-scale collaborative projects aimed at investigating new marine bioresources. For example, the National Cooperative Drug Discovery Group based in the USA involves three universitybased laboratories coupled with the Bristol-Myers Squibb Oncology Drug Discovery Group to discover and develop new anticancer agents from marine invertebrates, encompassing expertise in natural products chemistry, cancer pharmacology, drug development and marketing (Fenical et al., 2003). The European Commission has recently funded a Novel Marine Technologies project that ‘spans from baseline research, including the identification and extraction of bioactive compounds, through the application level dealing with the development and testing of prototypes for mariculture, to the dissemination and market implementation of new products and the evaluation of market opportunities’ (van Treeck et al., 2003). Similarly, the New Zealand Foundation for Research Science and Technology has funded a four-year project on ‘Determinants of Innovation and Growth in the Seafood Sector’ involving both the New Zealand Seafood Industry Council and the Crop & Food Research Institute (Sankarand and Mouly, 2007). These examples of large collaborative projects demonstrate growing interest in strategic investment in marine biotechnology, with the hope of transforming the aquaculture industry by value-adding seafood products for their health benefits. This should help to meet the growing consumer demands for scientifically substantiated natural alternatives to the currently available therapeutic drugs.

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28.8 Acknowledgements Professor Chris Battersill (Australian Institute for Marine Science) kindly provided a lead source for the retail of marine natural products as biotechnology research chemicals. Professor Rocky de Nys (James Cook University) provided many useful references and suggestions on the draft manuscript. Thanks also to Casey Campleman (Flinders University) for providing comments and editing the draft manuscript.

28.9 References abdo da, fromont j and mcdonald ji (2008) Strategies, patterns and environmental cues for reproduction in two temperate haliclonid sponges, Aquat Biol, 1, 291–392. adis international limited (2006) Trabectedin: Ecteinascidin 743, Ecteinascidin-743, ET 743, ET-743, NSC 684766, Drugs in R&D, 7, 317–28. andlauer w and fürst (2002) Nutraceuticals: a piece of history, present status and outlook, Food Res Int, 35, 171–6. apt k and behrens p (1999) Commercial developments in microalgal biotechnology, J Phycol, 55, 215–26. aroma new zealand ltd (2007) Company Profile and Product Specification, Aroma New Zealand, Christchurch, www.aromanz.com, accessed January, 2009. baker jt and duke cc (1976) Isolation of choline and choline ester salts of tyrindoxyl sulphate from the marine molluscs, Dicathais orbita and Mancinella keineri, Tetrahedron Letts, 15, 1233–4. bansemir a, blume m, schröder s and lindequist u (2006) Screening of cultivated seaweeds for antibacterial activity against fish pathogenic bacteria, Aquaculture, 252, 79–84. barrow cj and shahidi f (eds) (2007) Marine Nutraceuticals and Functional Foods, CRC Press, Boca Raton, FL. benkendorff k, mciver cm, westley cb and abbott ca (2008) Anticancer complementary medicines from muricid molluscs, NZMSS & AMSA Conference Abstracts, 6–10 July, University of Canterbury, Christchurch. benkendorff k, mciver cm and abbott ca (2009) Bioactivity of the Murex homeopathic remedy and of extracts from an Australian muricid mollusc against human cancer cells, Evid-Based Compl Alt Med, In press (accepted April 2009). blunt jw, copp br, munro mhg, northcote pt and prinsep mr (2006) Marine natural products, Nat Prod Rep, 23, 26–78. blunt jw, copp br, hu w-p, munro mhg, northcote pt and prinsep mr (2007) Marine natural products, Nat Prod Rep, 24, 31–86. blunt jw, copp br, hu w-p, munro mhg, northcote pt and prinsep mr (2008) Marine natural products, Nat Prod Rep, 25, 35–94. boericke w (1999) Murex, in Boericke W, Homeopathic Materia Medica, 9th edn, presented by Medi-T, Winhomeo Book. borsig l, wang l, cavalcante mc, cardilo-reis l, ferreira pl, mourao pa, esko jd and pavao ms (2007) Selectin blocking activity of a fucosylated chondroitin sulfate glycosaminoglycan from sea cucumber. Effect on tumor metastasis and neutrophil recruitment, J Biol Chem, 282, 14984–91.

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brower v (2005) A nutraceutical a day may keep the doctor away, EMBO reports, 6, 708–11. butler ms (2008) Natural products to drugs: natural product-derived compounds in clinical trials, Nat Prod Rep, 25, 475–516. carmona r, kraemer g and yarish c (2006) Exploring northeast American and Asian species of Porphyra for use in an intergrated finfish-algal aquaculture system, Aquaculture, 252, 54–65. cazalet s (2007) Materia Medica: Repertorium Homepathicum (Reversed Kent’s Repertory), Homeopathe International, http://www.homeoint.org/hidb/kent/ index.htm, accessed January 2009. chopin t (2006–2008) The Seaweed and Integrated Multi-Trophic Aquaculture (IMTA) Research Laboratory of Dr Thierry Chopin, University of New Brunswick, http://www.unbsj.ca/sase/biology/chopinlab/, accessed January 2009. cobb c and ernst e (2006) Systematic review of a marine nutraceutical in clinical trials for arthritis: the effectiveness of the New Zealand green-lipped mussel Perna canaliculus, Clin Rheumatol, 25, 275–84. de caralt s, agell g and uriz m (2003) Long-term culture of sponge explants: conditions enhancing survival and growth, and assessment of bioactivity, Biomol Eng, 20, 339–47. duckworth a (2001) Farming sponges for chemicals with pharmaceutical potential, World Aquac, June, 14–18. duckworth a, battershill c and bergquist p (1997) Influence of explant procedures and environmental factors on culture success of three sponges, Aquaculture, 156, 251–67. emelyanov a, fedoseev g, kranoschekova o, abulimity a, trendeleva t and barnes pj (2002) Treatment of asthma with lipid extract of New Zealand green-lipped mussel: A randomized clinical trial, Eur Respir J, 20, 596–600. eriksen n (2008) Production of phycocyanin – a pigment with applications in biology, biotechnology, foods and medicine, Appl Microbiol Biotechnol, 80, 1–14. ettinger-epstein p, tapiolas dm, motti ca, wright ad, battershill cn and de nys r (2007) Production of manoalide and its analogues by the sponge Luffariella variabilis is hardwired, Mar Biotechnol, 10, 64–74. faulkner d (2000) Marine Pharmacology, Antonie van Leeuwenhoek, 77, 135–45. fenical w, jensen p, kauffman c, mayhead s, faulkner d, sincich c, rao m, kantorowski e, west l, strangman w, shimizu y, li b, thammana s, drainville k, davies-coleman m, kramer r, fairchild c, rose w, wild r, vite g and peterson r (2003) New anticancer drugs from cultured and collected marine organisms, Pharm Biol, 41(Supplement), 6–14. fishlock d and williams rm (2008) Synthetic studies on ET-743. Assembly of the pentacyclic core and a formal total synthesis, J Org Chem, 73, 9594–600. gibson slm (2000) The effect of a lipid extract of the New Zealand green-lipped mussel in three cases of arthritis, J Alt Comp Med, 6, 351–4. halpern gm (2000) Anti-inflammatory effects of a stabilized lipid extract of Perna canaliculus (Lyprinol), Allerg Immunol, 32, 272–8. heckrodt tj and mulzer j (2005) Marine natural products from Pseudopterogorgia elisabethae: Structure, biosynthesis, pharmacology and total synthesis, Topics in Current Chemistry: Natural Products Synthesis II, 244, 1–41. hippocrates (460–377 BC) On ancient medicine, Trans, Adams F, 1849. higuera-ciapara i, félix-valenzuela l and goycoolea fm (2006) Astaxanthin: A review of its chemistry and application, Crit Rev Food Sci, 46, 185–96. jimeno j, faircloth g, sousa-faro j, scheuer p and rinehart k (2004) New marine derived anticancer therapeutics – a journey from the sea to clinical trials, Mar Drugs, 2, 14–29.

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kang kh, kwon jy, kim ym (2003) A beneficial coculture: charm abalone Haliotis discus hannai and sea cucumber Stichopus japonicus, Aquaculture, 216, 87–93. kijjoa a and sawangwong p (2004) Drugs and cosmetics from the sea, Mar Drugs, 2, 73–82. koopman m and wijffels r (2008) Seasonal growth rate of the sponge Haliclona oculata (Demospongiae: Haplosclerida), Mar Biotechnol, 10, 502–10. lebeau t and robert j (2003) Diatom cultivation and biotechnologically relevant products. Part II: current and putative products, Appl Microbiol Biotechnol, 60, 624–32. lee y (2001) Microalgal mass culture systems and methods: their limitation and potential, J Appl Phycol, 13, 307–15. li z, wang h, li j, zhang g and gao c (2000) Basic and clinical study on the antithrombotic mechanism of glycosaminoglycan extracted from sea cucumber, Chin Med J, 113, 706–11. liu bs, yi yh, li l, zhang sl, han h, weng yy and pan mx (2007) Arguside A: a new cytotoxic triterpene glycoside from the sea cucumber Bohadschia argus Jaeger, Chem Biodivers, 4, 2845–51. lorenz t and cysewski (2000) Commercial potential for Haematococcus microalgae as a natural source of astaxanthin, Trends Biotechnol, 18, 160–7. mackenzie jr cl (1961) Growth and reproduction of the oyster drill Eupleura caudate in the York River, Virginia, Ecology, 42, 317–38. magiatis p and skaltsounis, al (2006) From Hexaples trunculus to new kinase inhibitory indirubins, in Meijer L, Guyard N, Skaltsounis L and Eisenbrand G (eds), Indirubin, the Red Shade of Indigo, Life in Progress Editions, Roscoff, 147–56. mata l, silva j, schuenhoff a and santos r (2006) The effects of light and temperature on the photosynthesis of the Asparagopsis armata tetrasporophyte (Falkenbergia rufolanosa), cultivated in tanks, Aquaculture, 252, 12–19. mcfarlane sj and croft je (1984) Pharmaceutical preparations with gastro-protective action, Patent No. US4455298, accessed online esp@cenet database 20/02/2001. mciver c, abbott c and benkendorff k (2007) In vitro evaluation of bioactive muricid extracts and the murex homeopathic remedy in human solid tumors, Proceedings of the Australian Society for Medical Research Scientific Meeting, 6 June, Adelaide, SA. meijer l, skaltsounis a, magiatis p, polychronopoulos p, knockaert m, leost m, ryan x, vonica c, brivanlou a, dajani r, crovace c, tarricone c, musacchio a, roe s, pearl l and greengard p (2003) GSK-3-Selective inhibitors derived from tyrian purple indirubins, Chem Biol, 10, 1255–66. mendola d (2003) Aquaculture of three phyla of marine invertebrates to yield bioactive metabolites: process developments and economics, Biomol Eng, 20, 441–58. molyneaux m and lee cm (1998) The U.S. market for marine nutraceutical products, Food Technol, 52, 56–7. munro m, blunt j, dumdei e, hickford s, lill r, li s, battershill c and duckworth a (1999) The discovery and development of marine compounds with pharmaceutical potential, J Biotechnol, 70, 15–25. neori a, chopin t, troell m, buschmann ah, kraemer gp, halling c, shpigel m and yarish c (2004) Integrated aquaculture: rationale, evolution and state of the art emphasizing seaweed biofiltration in modern mariculture, Aquaculture, 231, 361–91. noble w (2006) Breeding and larval ecology of Dicathais orbita, a potential novel aquaculture resource, Molluscs 2006 Molluscs in Research, Conservation and the

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29 Opportunities and challenges for off-shore farming R. Langan, University of New Hampshire, USA

Abstract: It is widely acknowledged that future increases in seafood production will likely come from farming, not fishing. The growth of land-based and nearshore marine aquaculture in many developed countries is constrained by space, economics, and environmental concerns. Open ocean waters offer a tremendous potential for expansion of the marine farming sector and developments to date indicate that it is indeed feasible to install, maintain, and operate culture systems for finfish and shellfish in high-energy off-shore waters to produce high-quality seafood. Despite the evidence that off-shore farming is possible, production to date has been limited, and a number of technical, operational, economic, and political challenges must be addressed before large-scale production in true open ocean conditions can be realized. Key words: off-shore farming, site selection, finfish, molluscs, environmental effects.

29.1 The context for off-shore farming Population growth and consumer preference have resulted in a growing demand for seafood, a trend that is projected to continue into the future (FAO, 2006). Production from capture fisheries has leveled off and, by most projections, will remain stagnant or decline, depending on management and regulatory measures implemented by fishing nations (NOAA, 2005a; Worm et al., 2006). In contrast, aquaculture production has increased by approximately 10 % each year since 1980, and has played an important role in filling the gap between seafood supply and demand. Only a few decades ago, wild caught caught fish and shellfish supplied nearly all edible seafood although, with essentially flat growth since 1980 and the rise of aquaculture over the same time period, capture fishing now accounts for only about half of the total (FAO, 2006). In the most optimistic scenarios, wild caught fisheries production will remain stagnant (NOAA, 2005a),

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therefore, growth in the global seafood supply will continue to rely on aquaculture production. There are signs, however, that the rate of growth for global aquaculture may have peaked for land-based and near-shore marine culture due to political, environmental, economic, and resource constraints (FAO, 2006). Expansion of land-based culture is limited primarily by economics, particularly in developed countries where costs associated with land, capital equipment, and energy required to pump and filter water are prohibitive. For near-shore marine net-pen farming, available space is the primary limiting factor. Suitable sites for marine farming in protected coastal waters are for most countries quite limited to begin with, and those that do exist are used for a multitude of recreational and commercial activities with which aquaculture must compete for space. Expansion of large-scale finfish farming in coastal waters is also constrained by environmental concerns engendered primarily by the salmon farming industry. During a period of rapid growth of coastal salmon farming in the 1980s and 1990s, it became apparent that there were unintentional and undesirable environmental effects of net-pen culture. Incidents of seafloor pollution from uneaten feed and fish wastes (Hargrave et al., 1993), outbreaks of deadly diseases (Hovland et al., 1994), interaction with marine mammals and other predators (Nash et al., 2000), overuse of antibiotics and biocides, and escapement of fish from sea cages were documented. While the worst conditions were associated with inappropriate sites or poorly managed farms (NOAA, 2001), the growing opposition to net-pen culture was non-selective, and all salmon farming, and by way of extension, net-pen culture for many species, has been deemed environmentally ‘unsustainable’ by opponents of marine farming. Responding to criticism from environmental groups and pressure from regulatory agencies, the industry began to improve management practices to address environmental concerns. Better feed formulations and careful monitoring of the feeding response of fish resulted in improved feed conversion ratios and less waste (DFO, 2005). Vaccines drastically reduced the use of antibiotics (Knapp et al., 2007). More informed site selection led to a reduction in benthic impacts and fallowing cage sites allowed impacted sites to recover. While environmental performance has improved, public perception of coastal fish farming has not, and opposition to coastal salmon farms persists and, if anything, has increased in recent years. In the current climate, new permits to farm salmon or other species in coastal waters are difficult if not impossible to obtain. There are also concerns about environmental effects of near-shore bivalve mollusc culture, though they are minor in comparison to net-pen culture of finfish and are balanced by recognition of the ecosystem services such as enhanced habitat complexity and filtration capacity provided by molluscs (Shumway et al., 2003). It is rather the effect of environmental conditions on mollusc culture, and specifically the effects of pollution on

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product safety, that is limiting expansion in near-shore waters. Rapid coastal development and population growth and the resulting increase in human sources of pollution have affected the sanitary quality of near-shore waters, rendering shellfish grown there unsafe for consumption. As a consequence, many otherwise suitable sites for near-shore mollusc culture are off limits due to public health restrictions. In developed countries, conflict with coastal residents and tourist-related businesses over aesthetic values, primarily over water views from shorefront property, have also affected the permitting of new finfish and shellfish culture sites. As the demographics of coastal communities continues to change and new residents place more value on views and recreation than food production, these conflicts will only increase. Given the constraints on expansion of current methods of production, it is clear that alternative approaches are needed in order for the marine aquaculture sector to make a meaningful contribution to the world’s seafood supply. Farming in off-shore marine waters has been identified as one potential option for increasing production and has been a focus of international attention since the 1990s. Despite this global interest, development in offshore waters has been measured, primarily due to the significant technical and operational challenges posed by wind and wave conditions in most of the world’s oceans (Ryan, 2004). Farming in fully exposed off-shore waters requires a completely new engineering approach since equipment and methods currently used for fish and shellfish production in protected nearshore waters are largely unsuitable for the open ocean. In addition, the scale of investment required to develop and demonstrate new technologies and methods for off-shore farming is yet to be determined, although most engaged in this endeavor would agree that it will likely be substantial. Despite these challenges, there is sufficient rationale for pursuing the development of off-shore farming. Favorable features of open ocean waters include ample space for expansion, tremendous carrying capacity, reduced conflict with many user groups, lower exposure to human sources of pollution, the potential to reduce some of the negative environmental impacts of coastal fish farming (Ryan, 2004; Helsley and Kim, 2005; Ward et al., 2006; Langan, 2007), and optimal environmental conditions for a wide variety of marine species (Ostrowski and Helsley, 2003; Ryan, 2004; Howell et al., 2006; Benetti et al., 2006; Langan and Horton, 2003). For many countries, where cost, environmental concerns, limited space, and competing uses have restricted growth of land-based and near-shore marine farming, few other options for significant expansion exist.

29.2 Characterization and selection of off-shore sites 29.2.1 Definition Before discussing approaches to off-shore aquaculture development, it is important to first define what is meant by ‘off-shore’. For most engaged in

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New technologies in aquaculture Table 29.1 Classification of off-shore waters developed in Norway based on significant wave heights Site class 1 2 3 4 5

Significant wave height (m)

Degree of exposure

<0.5 0.5–1.0 1.0–2.0 2.0–3.0 >3.0

Small Moderate Medium High Extreme

Source: Ryan, 2004.

this sector, the term is generally accepted to mean farming in locations that are subjected to ocean waves and currents and removed from any significant influence of land masses rather than a set distance from shore. Clearly, a wide range of sea conditions falls under this broad definition. Ryan (2004) reported on a site classification system for marine waters developed in Norway that is based on significant wave height exposure (Table 29.1). While this classification method is instructive, knowledge of the full range of conditions that occur at a particular site is needed to design and test sufficiently robust engineered systems, and to develop safe and efficient operating procedures.

29.2.2 Site selection criteria and methods The suitability of sites for off-shore farming is dependent on a number of criteria, many of which are also considerations for near-shore sites. These include proximity to infrastructure such as ports, processing and distribution centers, as well as physical and biological criteria such as bathymetry, seabed characteristics and contour, current velocities, temperature profiles, dissolved oxygen, turbidity, and the frequency of occurrence of harmful algal blooms. For mollusc culture, the quantity and quality of phytoplankton is also an important consideration. The most important additional feature of off-shore sites is wave climate. Significant wave heights, wave periods, the frequency and duration of high-energy storm conditions, and combined forcing of waves and currents must be known in order to determine whether a site is suitable and, if so, what type of technology is required for farming. For example, some sites may be relatively calm most of the time and infrequently experience occurrences of severe weather such as tropical cyclones. Other sites may never have waves greater than 3 m, but they may experience short period waves in this range most of the time – conditions that would cause excessive wear and tear on equipment and

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make surface operations such as feeding and harvesting difficult. For the former scenario, technology operated at the surface with the option to submerge for short periods would be appropriate, while for the latter it is likely that submersible cages supported by automated technologies would be needed. It is imperative that a thorough evaluation that includes the parameters described above be conducted before proceeding with development of a site for farming. The requirements for data and subsequent analysis can be substantial; however, the use of advanced oceanographic technologies can greatly facilitate this task. Multibeam sonar and three-dimensional visualization can generate a wealth of data on seafloor contours and texture to inform mooring system design and placement. Collection of time-intensive data on temperature, salinity, dissolved oxygen, turbidity, and fluorescence can be greatly facilitated by strategic deployment of in situ instrumentation at appropriate depth intervals in the water column. Additional instrumentation should include acoustic Doppler current profiling (ADCP) current meters that can profile current velocity and direction throughout the water column, wave sensors that can give precise data on wave height, direction, and period, and meteorological sensors to measure air temperature and wind speed and direction. Many countries have buoy arrays in coastal waters that can provide long-tem data on regional climatology to aid site evaluation; however, collection of site-specific data is critical. Assessment of the potential for the effects of global climate change on critical parameters such as water temperature should also be considered. The data collection period required for site evaluation will vary, depending on local and regional environmental and meteorological conditions. Good baselines for some parameters can be established in a relatively short timeframe (one year); others such as the frequency, duration, and severity of storms or blooms of toxic algae are less predictable and it may take longer to determine the suitability of a particular site. In addition to physical, chemical, and biological characteristics of a site, other human uses in the vicinity such as shipping, fishing, and mining must be identified in order to avoid conflicts. Other factors such as use of the area by marine mammals, the likelihood of encounters with large predators, important spawning grounds, and proximity of sensitive biological communities must also be considered. Many countries require characterization of the benthic community and sediment quality to establish pre-operational baselines of environmental quality.

29.3 Finfish species cultivated in off-shore cages The following discussion of finfish species makes mention of cage and mooring systems in use for one or more species. Cages, mooring designs, and supporting technologies are described in detail in Chapter 30.

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29.3.1 Cold and cool temperate water species Fish cultivated in off-shore cages include warm and cool temperate as well as tropical and sub-tropical species, though at present, cool temperate species predominate. In terms of volume, the leading species are Atlantic salmon (Salmo salar), which are produced in gravity cages in Ireland, Scotland, Canada, Norway, and the Faeroe Islands. Sea bass (Dicentrarchus labrax) and sea bream (Sparus auratus) are grown in gravity, tension leg and submersible cages throughout the Mediterranean (Ryan, 2004). Fattening of Northern Bluefin tuna (Thunnus thynnus) in large high-density polyethylene (HDPE) collar gravity cages takes place in exposed locations throughout the Mediterranean, and has expanded dramatically in recent years. Other tuna fattening operations that use HDPE cages in exposed locations include southern bluefin tuna (Thunnus maccoyii) in South Australia and Pacific bluefin (Thunnus thynnus orientalis) and smaller quantities of yellowfin (Thunnus albacares) and bigeye tuna (Thunnus obesus) in Mexico. In the Northeast USA, the University of New Hampshire operates an experimental off-shore farm and has produced small quantities of Atlantic cod (Gadus morhua), haddock (Melanogrammus aeglefinis), and Atlantic halibut (Hippoglossus hippoglossus) in submerged Sea Station cages (Howell et al., 2006).

29.3.2 Tropical and sub-tropical species Warm temperate and tropical species produced in off-shore cages include milkfish (Chanos chanos) in the Philippines, yellowtail kingfish (Seriola lalandi) in Australia, Florida pompano (Trachinotus carolinus) in Belize, summer flounder (Paralichthys dentatus) in Mexico, and parrotfish (Oplegnathus faciatus) and olive flounder (Paralichthys olivaceus) in Korea. In Hawaii, Pacific threadfin (Polydactylus sexfilis) and amberjack (Seriola rivoliana) are being grown commercially in submerged cages at off-shore sites off Oahu and the Island of Hawaii. Another warm-water species of interest for off-shore farming is cobia (Rachycentron canadum), which is currently produced in Puerto Rico, the Bahamas, Panama, and Belize.

29.3.3 Species in development A number of species have been identified as having potential for off-shore production. In the USA, California yellowtail (Seriola dorsalis lalandi), striped bass (Morone saxatilis), California halibut (Paralichthys californicus), and tuna (Thunnus sp.) have been proposed as candidate species for off-shore culture in Southern California, while red drum (Sciaenops ocellatus), cobia, pompano, and tunas are being considered for the Gulf of Mexico. It is likely that many more species will come into production as off-shore farming technologies are further developed; however, it should be noted that aside from a few species such as Atlantic salmon, sea bass,

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and sea bream that are produced from established broodstock lines, many of the marine species under cultivation are the offspring of wild fish. Additional broodstock and hatchery work is needed to achieve desirable traits such as better and more uniform growth rates, improved food conversion ratios, delayed maturation, and disease resistance.

29.4 Off-shore mollusc culture 29.4.1 Drivers and limitations While most of the focus on off-shore development has been on finfish, there has also been growing interest in off-shore culture of bivalve molluscs. Some of the same drivers such as ample space and the opportunity to avoid user conflicts are identical to those for finfish culture although, perhaps more importantly, reduced risk of exposure to human sewage and industrial pollution presents a major advantage of open ocean waters over coastal locations. There are, however, possible limitations as well as advantages. Off-shore waters in many areas of the world are nutrient deficient, so careful attention must be paid during site selection to the quantity, quality, and seasonality of phytoplankton available for filter-feeding molluscs. Macro-scale information on primary productivity can be obtained from ocean color satellite data (e.g. SeaWiFS, MODIS), and site-specific data on concentration and composition can be generated by in situ fluorometry and microscopic analysis of the plankton community. Phytoplankton concentration at different depths is also an important factor as farmers will wish to maximize the use of vertical space for production in deeper off-shore waters. The frequency and duration of harmful algal blooms (HABs) is also a critical consideration for off-shore mollusc farming. In some locations, blooms of toxic algae originate and persist in off-shore waters (e.g. Alexandrium sp. in the Gulf of Maine, USA) and can result in extended public health closures with severe economic impact on producers.

29.4.2 Technologies for off-shore mollusc farming With the exception of bottom seeding of scallop spat in off-shore waters, a practice that takes place in several countries including Japan, New Zealand, and Canada, technologies for off-shore mollusc farming are essentially adaptations of suspension culture methods employed in protected nearshore waters. Designs and prototypes for submersible rafts have been developed (SubSea, 2004; Stanley, 2005); however, submerged longlines are the most commonly used method. Submerged longline technology was developed in Japan and has been in use there for many years for deep-water suspended scallop culture, though not in fully exposed open ocean conditions. The method was successfully adapted for mussel culture in Atlantic Canada where winter and spring drift ice can damage surface-referenced

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190 kg buoyancy

10 M 130 M

3500 kg concrete anchors 200 M

Fig. 29.1 A schematic of a submerged longline used for suspension culture of molluscs in open ocean environments.

longlines (Bonardelli, 1996). More recently, the technology has been shown to be effective for mussel production in very high-energy open ocean conditions (e.g. significant wave heights >10 m) in the northeast USA (Langan and Horton, 2003). The technology is quite simple and consists of relatively inexpensive materials. The structural stability of the longline is maintained by the opposing forces of submerged flotation at the ends of a single horizontal backbone, connected by lines set at a 45 ° angle to seafloor anchors (Fig. 29.1). Submergence depth of the backbone is dictated by site-specific wave climate and can range from 3–15 m. Surface floatation is minimized to prevent the transfer of wave-induced motion to the backbone, and consists of non-structural marker buoys for the anchor lines and a mid-backbone pick-up line that provides access to the crop from a service vessel. Anchors are generally spaced from 100–200 m apart and, depending upon the depth of the water and desired depth of submergence, the backbone length can range from 70–130 m. Cages of scallops or oysters and ropes or ‘droppers’ of mussels are suspended from the backbone, and additional submerged floatation is added as the crop gains mass during grow-out (Fig. 29.2). Service vessels are for the most part converted fishing boats, though large, seaworthy, specialized vessels will be needed to tend large off-shore farms.

29.4.3 Mollusc species cultivated in off-shore environments There are a number of species of bivalve molluscs that are and may potentially be farmed in off-shore waters; however, efforts to date have focused primarily on several mussel species and, to a lesser extent, scallops and oysters. Also, as this technology is relatively new, the sector is still in an early stage of development and production at individual farm sites is small by comparison with near-shore farms. Depending on the species, seed is sourced either from hatcheries or from the wild. Clam, oyster and, to a

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Fig. 29.2 A diagram of a submerged longlines showing the attachment of mussel growing ropes to the backbone and the placement of floatation added as the crop increases in mass during grow-out (from Langan and Horton, 2003).

lesser extent, scallop and mussel seed are produced in hatcheries. Mussel growers rely almost entirely on wild-caught seed, although this is considered a potentially limiting factor in some locations (Jeffs et al., 1999). Off-shore production of molluscs takes place in many countries around the world, and interest in development of this sector is increasing as the availability of near-shore sites diminishes. In North America, small quantities of blue mussels (Mytilus edulis) are produced in off-shore farms in New England (USA) and Atlantic Canada. Mediterranean mussels (M. galloprovincialis), Pacific oysters (Crassostrea gigas), and Manila clams (Tapes philippinarum) are being grown at an off-shore farm off the southern California coast (SB Mariculture, http://www.sbmariculture.com). In Europe, M. galloprovincialis are grown on submerged longlines at exposed locations off the Mediterranean coast of France (Brehemer et al., 2003), and culture trials have been initiated for M. edulis in the Baltic Sea off the coast of Germany (Buck, 2007) and in the Belgian North Sea (Van Nieuwenhove and Delbare, 2008). Other European countries, including Portugal, Spain, Italy, and Ireland, are developing strategies for off-shore mussel production. In New Zealand, where the near-shore greenshell mussel (Perna canaliculus) industry is well developed and highly mechanized, there is a great deal of interest in developing large-scale off-shore farms, as lease sites in near-shore waters have become difficult to obtain (Jeffs, 2003). Initial efforts at off-shore mussel farming involved moving the double longline surfacereferenced technology into more exposed sites where some success was achieved in wave conditions up to 2.5 m (Thompson, 1996). However, failure of surface-referenced systems in higher energy sites has led to the development of submerged technologies, and a small number of off-shore mussel farms are operating in New Zealand’s off-shore waters with many

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new farms proposed (Stevens et al., 2005). This scale of expansion is projected to provide a threefold increase production and in export earnings by 2020 (Plew et al., 2005). A progressive migration to more exposed sites has also taken place in Australia, where submerged longlines are used to produce Pacific oysters and small quantities of scallops (Thompson, 1996). Submerged longlines have been used for decades in semi-exposed sites in Japan to culture scallops (Patinopecten yessoensis) in suspended lantern cages and pearl nets.

29.4.4 Other considerations for off-shore mollusc culture Off-shore culture of single or multiple species of bivalve molluscs can be practised in isolation from other activities; however, there may be economic or environmental advantages to combining mollusc culture with off-shore fish farming or energy production. At a near-shore site in New Brunswick, Canada, Lander et al. (2004) demonstrated better growth rates for raftcultured mussels 100 m downcurrent of a salmon farm than at reference sites, and were able to document that organic wastes, primarily fine particulates from feed emanating from the salmon farm, contributed to the diet of the mussels. In off-shore sites, creating mollusc culture ‘zones’ in proximity to finfish farms may offset the effects organic loading to the environment (Langan, 2004). Energy installations may also provide structure for deployment of shellfish culture systems. Mussels (M. galloprovincialis) have been harvested from oil platforms in California for many years (Richards and Trevelyan, 2001), and there is interest in using decommissioned off-shore platforms for mussel culture in California and for oyster culture in the Gulf of Mexico. Buck et al. (2004) investigated the possibility of integrating suspension culture of mussels with off-shore wind platforms. It is unclear whether there are economic benefits for either the shellfish farmer or the energy producer; however, the net effect could be a reduction of overall footprint required for off-shore energy and food production facilities.

29.5 Environmental concerns Like all forms of food production, the culture of marine species, whether practised in land-based, near-shore, or off-shore locations, will have some effect on the environment. The effects can be both negative and positive and can vary depending upon the species, location, and farming practices. Adverse impacts from near-shore aquaculture of both molluscan shellfish and finfish have been documented since the 1980s, although most of the concerns and controversy are centered on finfish. In the discussion that follows, finfish and shellfish will be treated separately.

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29.5.1 Potential environmental effects of off-shore finfish farming Concerns over the potential impacts of off-shore finfish farming essentially mirror those of near-shore cage culture (Table 29.2); however, the degree of impact will likely be different, as will society’s perceptions. Due to the limited number and relatively small size of the farms operating in off-shore

Table 29.2

Potential environmental impacts from marine net pen farming

Effects 1. Increased organic loading

Sources • Particulate organic loading Fish fecal material Uneaten fish feed Debris from biofouling organisms Decomposed fish mortalities on the farm • Soluble organic loading Dissolved components of uneaten feed Harvest wastes (blood) • Nitrogen and phosphorus from fish excretory products • Trace elements and micronutrients (e.g., vitamins) in fish fecal matter and uneaten feed • Zinc compounds in fish fecal material • Zinc compounds in uneaten feed • Copper compounds in antifouling treatments • Indigenous parasites and pathogens • Exotic parasites and pathogens • Treatment by inoculation • Treatment in feed • Treatment in baths • Unplanned release of farmed fish • Unplanned release of gametes and fertile eggs • Cross-infection of parasites and pathogens • Planned release of cultured fish for enhancement or ranching • Entanglement with lost nets and other jetsam • Entanglement with nets in place, structures, and moorings, etc. • Attraction of wildlife species (fish, birds, marine mammals, reptiles) • Predator control • Buoyant fish containment structures and mooring lines • Anchors and moorings • Harvest of target and non-target species as larvae • Increased fishing pressure on the shoaling small • Increased fishing pressure on the shoaling small pelagic fish populations 䊊 䊊 䊊 䊊

䊊 䊊

2. Increased inorganic loading 3. Residual heavy metals 4. The transmission of disease organisms 5. Residual therapeutants 6. Biological interaction of escapes with wild populations 7. Physical interaction with marine wildlife

8. Physical impact on marine habitat 9. Using wild juveniles for grow-out juveniles and sub-adults 10. Harvesting industrial fisheries for fish feed Source: NOAA, 2005b.

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sites, scientific knowledge of possible effects on off-shore environments is not, as yet, well known. Data gathered to date would indicate that water column impacts from inorganic nutrients are unlikely (Helsley and Kim, 2005; Langan, 2007), although effects will vary depending upon production volume and the trophic status of receiving waters. Also, inputs from individual farms must be examined in the context of inputs from other sources in the region, including other farms. Benthic impacts from deposition of organic materials from waste feed feces may also be reduced due to greater dispersion by ocean currents. Ryan (2004) cited a 2001 Review of Benthic Conditions at Irish Fish Farms conducted by Aquafact International Services Ltd, which reported benthic impacts were greatly reduced, if not more or less absent, beneath farms at exposed sites. At the experimental off-shore farm off the New Hampshire (USA) coast, no significant differences have been observed in sediment organic content or benthic community diversity between projected impact and reference sites after seven years of multispecies farming (Ward et al., 2006; Langan, 2007). Alston et al. (2005) reported only minor changes to the benthos directly beneath cobia cages at an off-shore farm in Puerto Rico, and Rapp (2006) found no changes in the organic content of the sediments beneath the cages at the same farm. Due to the small size of the farms in these studies, the results should not be viewed as convincing evidence that the potential for benthic impacts from off-shore farms can be dismissed. A case in point is a benthic study conducted at an off-shore farm in Hawaii. Lee et al. (2006) reported an increase in opportunistic polychaete species and some loss of diversity in the benthic community, although these changes were spatially limited to areas immediately beneath the cages. As this study demonstrates, benthic impacts may be reduced in off-shore environments; however, an expectation of ‘no impact’ is unrealistic. The degree of environmental change that will be tolerated must be decided by governing bodies in their respective jurisdictions; however, it would be advantageous to establish international agreement on environmental performance standards with the caveat that some flexibility in the measured parameters is needed to account for site differences. There is no need to create standards from scratch. Based on existing knowledge of benthic impacts from near-shore farms, standards that have been developed for ecosystem protection in countries including Australia, Ireland, Norway, and Scotland, as well as for US state waters in Maine and Washington, have many common features and provide a good starting point for addressing environmental concerns. Models, particularly those that integrate hydrodynamic, physical and biological processes, can play a major role in predicting potential impacts of off-shore farming. They enable scenarios to be run to forecast possible effects at specific farm sites as well broader ecosystem effects. Farm-focused models such as AquaModel (Rensel et al., 2006) and DEPOMOD (Cromey et al., 2002) that use site-specific hydrodynamic, sediment and biological

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data to simulate water column and benthic effects can be used to evaluate different management and production strategies. Site-specific models are also useful for identifying depositional areas and locating monitoring stations. Cumulative effects must also be considered as the off-shore sector scales up, so ecosystem models will be needed for broad area management. It is reasonable to assume that monitoring will be required to ensure environmental compliance and to satisfy stakeholders that individual farms and the collective industry are operating in an environmentally responsible manner. Monitoring methods to determine the degree of organic enrichment vary in cost, clarity of interpretation, and the ability to standardize methods and establish meaningful performance standards. Detailed enumeration of benthic species obtained from sediment grab samples may be required during the initial site assessment to establish baselines, and during the first few stocking cycles; however, this type of monitoring is very expensive and time-consuming. Ideally, video or photographic analyses, coupled with some type of chemical measurement such as total organic carbon (TOC) or loss on ignition (LOI) that have been calibrated against this information would replace benthic monitoring and reduce costs for farm owners. Off-shore culture may offer some fish health benefits, which in turn may reduce the risk of transmission of parasites and diseases to wild populations as well as reduce the need for treatments and theraputins. Ryan (2004) reported lower incidence of sea lice at off-shore salmon farms in Ireland, which he attributed to dispersion of the planktonic stage of the ectoparasites. One species of sea lice, Lepeophtheirus salmonis, is known to aggregate at the mouths of estuaries; therefore, placing salmon farms further off-shore would greatly reduce exposure to this parasite (Costello et al., 2004). In addition, lower stress levels and better fish health observed at off-shore farms have been attributed to the more stable salinity regimes, as well as the higher oxygen concentration and reduced ammonium levels that result from the greater water exchange through cages (Ryan, 2004; Benetti et al., 2006; Howell et al., 2006). Bricknell (2006) concluded, ‘off-shore aquaculture offers many opportunities to reduce disease interactions between wild and farmed fish’. The high-energy conditions of off-shore sites increases the risk of fish escapement due to catastrophic equipment failure; therefore, the robustness of engineered systems (e.g. cages, moorings) must be carefully matched to site conditions. Some fish species under development for off-shore farming are prone to escapement. Cod, for example, will bite holes in netting material and escape from cages (Moe et al., 2007), so more durable alternatives to woven netting are needed. Also, since visual inspections of equipment by divers are more difficult and dangerous at off-shore sites, automated systems such as video surveillance or acoustic abundance estimators may be required to manage escapement. The major concern over

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salmon escapement from near-shore net-pens is biological interactions with wild salmon. Moving farms off-shore and away from salmon spawning rivers would greatly reduce this risk. Farming off-shore may reduce exposure to pinniped predators; however, exposure to other large predators like sharks may increase. This risk can be managed through careful site selection to avoid known aggregation areas of local predators, prompt removal of dead or moribund fish, and the use of more robust containment barriers. The chance of encounters with protected species (e.g. whales, sea turtles) may also increase; however, entanglement with the mooring lines typically used for off-shore cages is unlikely. Endangered whales are frequently sighted in close proximity to an off-shore farm off the coast of New Hampshire (USA) and no adverse interactions have occurred in nine years of farming at this site (Ward et al., 2005). Issues such as the use of wild juveniles, which has been standard operating procedure for tuna penning, and the use of industrial fish as feed ingredients are essentially the same for off-shore as for near-shore farming. There have been some advances in hatchery production of juvenile tuna species; however, it may be years before large quantities of hatchery produced juveniles are available for on-growing. Significant progress has been made in developing alternatives to fish meal and fish oil, and continued research and development is likely to produce economically and nutritionally viable substitutes.

29.5.2 Potential environmental effects of off-shore mollusc farming Mollusc culture is generally perceived as environmentally benign or even beneficial (Shumway et al., 2003); however, there have been environmental impacts from near-shore mollusc farming that merit consideration for development of the off-shore sector. As suspension culture will likely be the preferred method for off-shore farming, this discussion will focus on impacts of suspension culture on near-shore environments and the potential for adverse effects off-shore. Although molluscs feed on naturally occurring seston and no external feed is provided to the organisms, deposition of feces and pseudofeces can enrich bottom sediments beneath culture systems and impact benthic communities (Hatcher et al., 1994). Occurrences of sediment impacts have been associated with very dense culture in shallow embayments; therefore, if off-shore farms are sited in locations with sufficient depth and adequate water circulation to disperse wastes, enrichment of bottom sediments should not be an issue. High-density mollusc culture can also deplete the water column of planktonic food, affecting both the growth and fitness of the cultured organisms as well as naturally occurring filter-feeders in the system (Prins et al., 1997). This, too, is an impact associated with near-shore embayments and is unlikely to be an environmental issue in off-shore waters. However, in very large, high-density off-shore farms, depletion of food

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within the farm and reduced growth and condition of the stock may be an issue for producers. Hydrodynamic alteration is another environmental effect of bivalve culture that has been documented in embayments with high-density shellfish culture (Grant and Bacher, 2001) and has recently been an area of concern in New Zealand where large-scale off-shore mussel farming is in development. Plew et al. (2005) reported significant current and wave attenuation and strong water column stratification at a large (230 longline) offshore mussel farm in Golden Bay, New Zealand. The farm was located in relatively shallow water (10–12 m), and the culture organisms were suspended from the surface to a depth of 8 m, therefore occupying nearly the entire water column. As it is likely that off-shore development will use submerged culture in much deeper water (30–100 m) with ample space above and below the culture arrays, the severity of flow modifications as observed in this study are improbable. A legitimate environmental concern for off-shore mussel culture is entanglement of whales in seed collection lines (Lloyd, 2003). These collectors are either discrete lengths of line or one continuous length of rope suspended from the backbone to provide substrate for settlement of mussel larvae. As this sector develops, it is important to avoid deployment of seed collection lines in the migratory pathways of endangered marine mammals.

29.6 Future trends Developments in off-shore marine farming since the 1990s indicate that the culture of finfish and molluscan shellfish in open ocean environments is feasible and that opportunities exist for large-scale production of a wide variety of species. Conflicts with other uses can be significantly reduced, although they are not totally eliminated. Potential conflicts with capture fishing, navigation, and off-shore energy production must be considered when selecting sites for off-shore farming. There is also some evidence to support the premise that environmental impacts can be reduced by farming in off-shore environments; however, additional information on the potential effects of large-scale production is needed to inform the development of rational policies and counter the negative perceptions of marine farming. Ideally, development of off-shore farming should take place within the context of overall ocean management in order ensure compatibility with other uses and consistency with broader goals to restore and sustain the health, productivity, and biological diversity of the oceans. Significant progress has been made in the development of new marine species and engineered systems for off-shore farming; however, a number of technical challenges remain. Hatchery technology has been developed for a number of marine fish species, although additional time and investment is needed to establish broodstock lines and the infrastructure to

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produce and transport large quantities of robust fingerlings. Off-shore farms may need to be coupled with near-shore farms to provide appropriate sites for intermediate stages of stocks, as larger juveniles may be needed for offshore cages. Near-shore farms can also be used as holding facilities for market-ready stock when weather conditions prohibit safe harvesting at off-shore sites. There are a number of proven cage and mooring systems available; however, they need to scale up in order to achieve the production volumes required for economic viability. Further development of supporting technologies, including ocean-going service vessels for finfish and shellfish culture, automated feeders, and remote control and communication systems is needed. Therefore, substantial and sustained investment in R&D from public and private sectors must be secured. In particular, research should be focused on the development of highly mechanized and fully integrated off-shore farming systems to achieve greater efficiency and ensure worker safety in the conduct of routine operations. Until ‘turnkey’ systems that are essentially autonomous are available and economic viability of off-shore farming can be demonstrated, expansion of this sector in the near future will be limited. A logical next step to hasten the development of off-shore farming would be to establish commercial-scale off-shore demonstration farms supported by a combination of public and private funds where technologies can be tested at reduced financial risk for the private sector and a greater understanding the environmental effects of off-shore farming can be gained to inform a rational regulatory framework. Demonstration farms would also be useful for training vessel operators and farm personnel to work safely and efficiently in off-shore environments. Although the complexity and scale of the challenge for off-shore aquaculture development is formidable, the potential economic and health benefits of a sustainable off-shore industry are enormous. Realizing the vision for off-shore farming will require creativity and innovation supported by substantial investment, as well as close collaboration between nations and the business, government, and the research communities.

29.7 References alston de, cabarcas a, capella j, benetti d, keene-maltzof s, bonilla j and cortes r (2005) Environmental and Social Impact of Sustainable Offshore Cage Culture Production in Puerto Rican Waters, Final Report for NOAA Grant No. NA16RG1611, April 4, National Oceanic and Atmospheric Administration, Washington, DC. benetti d, o’hanlon b, brand l, orhun r, zink i, doulliet p, collins j, maxey c, danylchuk a, alston d and cabarcas a (2006) Hatchery, on growing technology and environmental monitoring of open ocean aquaculture of cobia (Rachycentron canadum) in the Caribbean, Aquaculture 2006, 10–13 May, Florence World Aquaculture Society, Baton Rouge, LA, abstract, http://www.was.org/meetings/ AbstractData.asp?AbstractId=10424, accessed January 2009.

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bonardelli j (1996) Longline shellfish culture in exposed and drift-ice environments, in Polk M (ed.), Open Ocean Aquaculture: Proceedings of an International Conference, 8–10 May, Portland, ME, New Hampshire/Maine Sea Grant College Program Rpt #UNHMP-CP-SG-96-9, 235–53. brehmer p, gerlotto f, guillard j, sanguinède f, guénnegan y and buestel d (2003) New applications of hydroacoustic methods for monitoring shallow water aquatic ecosystems: the case of mussel culture grounds, Aquatic Living Resources, 16, 333–8. bricknell i (2006) Disease implications of offshore aquaculture. Breaking the disease cycle? Offshore Mariculture 2006, 11–13 October, Malta. buck bh, krauseb g and rosenthal h (2004) Extensive open ocean aquaculture development within wind farms in Germany: the prospect of offshore comanagement and legal constraints, Ocean & Coastal Management, 47(3–4), 95–122. buck bh (2007) Experimental trials on the feasibility of offshore seed production of the mussel Mytilus edulis in the German Bight: installation, technical requirements and environmental conditions, Helgoland Marine Research, 61(2), 87–101. costello mj, burridge l, chang b and robichaud l (2004) Sea Lice 2003 – Proceedings of the sixth international conference on sea lice biology and control, Aquaculture Research, 35(8), 711–12. cromey cj, nickell td and black kd (2002) DEPOMOD – modelling the deposition and biological effects of waste solids from marine cage farms, Aquaculture, 214, 211–39. dfo (2005) Myths and Realities of Salmon Farming–Updated, Fisheries and Oceans Canada, Ottawa, ON, http://www.dfo-mpo.gc.ca/media/back-fiche/2005/salmoneng.htm, accessed January 2009. fao (2006) State of World Aquaculture 2006, FAO Fisheries Technical Paper No. 500, Food and Agriculture Organization of the United Nations, Rome. grant j and bacher c (2001) A numercial model of flow modification induced by suspended aquaculture in a Chinese Bay. Canadian Journal of Fisheries and Aquatic Sciences, 58, 1003–11. hargrave b, duplisea t, pdeiffer d and wildish e (1993) Seasonal changes in benthic fluxes of dissolved oxygen and ammonium associated with marine cultured Atlantic salmon, Marine Ecology Program Series 96, 249–57. hatcher a, grant j and schofield r (1994) Effects of suspended mussel culture (Mytilus spp.) on sedimentation, benthic respiration and sediment nutrient dynamics in a coastal bay, Marine Ecology Progress Series, 115, 219–35. helsley ce and kim jk (2005) Mixing downstream of a submerged fish cage: a numerical study, IEEE Journal Of Oceanic Engineering, 30, 12–19. hovland t, nylund a, watanabe k and endresen c (1994) Observations of infectious salmon anaemia virus in Atlantic salmon, Salmo salar L, Journal of Fish Disease, 17, 291–6. howell wh, watson wh and chambers md (2006) Offshore Production of Cod, Haddock and Halibut, CINEMar/Open Ocean Aquaculture Annual Progress Report for the period 1/01/05 through 12/31/05, Final Report for NOAA Grant No. NA16RP1718, interim Progress Report for NOAA Grant No. NA04OAR4600155, submitted January 23, National Oceanic and Atmospheric Administration, Washington, DC. jeffs ag, holland rc, hooker sh and hayden bj (1999) Overview and bibliography of research on the greenshell mussel, Perna canaliculus, from New Zealand waters, Journal of Shellfish Research, 18(2), 347–60. jeffs A (2003) Assessment of the Potential for Mussel Aquaculture in Northland, NIWA Client Report: AKL2003-057, NIWA Project: ENT03101, National Institute of Water & Atmospheric Research, Auckland.

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knapp g, roheim c and anderson j (2007) The Great Salmon Run: Competition Between Wild and Farmed Salmon, TRAFFIC North America, Washington DC.: World Wildlife Fund. lander t, barrington k, robinson s, macdonald b and martin j (2004) Dynamics of the blue mussel as an extractive organism in an integrated multi-trophic aquaculture system, Bulletin of the Aquaculture Association of Canada, 104(3), 19–29. langan r and horton cf (2003) Design, operation and economics of submerged longline mussel culture in the open ocean. Bulletin of the Aquaculture Association of Canada, 103(3), 11–20. langan r (2004) Balancing marine aquaculture inputs and extraction: combined culture of finfish and bivalve molluscs in the open ocean, Bulletin of the Fisheries Research Agency of Japan, Supplement No.1, 51–8. langan r (2007) Results of environmental monitoring at an experimental offshore farm in the Gulf of Maine: environmental conditions after seven years of multispecies farming, in Lee CS and O’Bryen PJ (eds), Open Ocean Aquaculture – Moving Forward, Oceanic Institute, Waimanalo, HI, 57–60. lee hw, bailey-brock jh and mcgurr mm (2006) Temporal changes in the polychaete infaunal community surrounding a Hawaiian mariculture operation, Marine Ecology Progress Series, 307, 175–85. lloyd bd (2003) Potential effects of mussel farming on New Zealand’s marine mammals and seabirds: a discussion paper, Department of Conservation, Wellington. moe h, dempster t, sunde lm, winther u and fredheim a (2007) Technological solutions and operational measures to prevent escapes of Atlantic cod (Gadus morhua) from sea cages, Aquaculture Research, 38, 91–9. nash c, iwamoto r and mahnken c (2000) Aquaculture risk management and marine mammal interactions, Aquaculture, 183(3–4), 307–23. noaa (2001) The Net-pen Salmon Farming Industry in the Pacific Northwest, Nash C. (ed.), NOAA Technical Memorandum NMFS-NWFSC-49, National Oceanic and Atmospheric Administration, Washington, DC. noaa (2005a) Fisheries of the United States – 2003, National Oceanic and Atmospheric Administration, Washington, DC, http://www.st.nmfs.gov/st1/fus/fus03/ index.html, accessed January 2009. noaa (2005b) Guidelines for Ecological Risk Assessment of Marine Fish Aquaculture, Nash CE, Burbridge PR and Volkman JK (eds), US Dept. of Commerce, NOAA Technical Memorandum, NMFS-NWFSC-71, National Oceanic and Atmospheric Administration, Washington, DC. ostrowski ac and helsley ce (2003) The Hawaii offshore aquaculture research project: critical research and development issues for commercialization, in Bridger CJ and Costa-Pierce BE (eds), Open Ocean Aquaculture: From Research to Commercial Reality, World Aquaculture Society, Baton Rouge, LA, 119–28. plew dr, stevens cl, spigel rh and hartstein nd (2005) Hydrodynamic implications of large offshore mussel farms, IEEE Journal Of Oceanic Engineering, 30(1), 95–108. prins tc, smaal ac and dame rf (1997) A review of the feedbacks between bivalve grazing and ecosystem processes. Aquatic Ecology, 31(4), 349–59. rapp p (2006) Measurement of the benthic loading and the benthic impact from an open-ocean fish farm in tropical waters, Preliminary report for NOAA grant no: NA040AR4170, submitted January 30, National Oceanic and Atmospheric Administration, Washington, DC. rensel je, buschmann ah, chopin t, chung ik, grant j, helsley ce, kiefer da, langan r, newell rie, rawson m, sowles jw, mcvey jp and yarish c (2006) Ecosystem-based management: models and mariculture, in McVey JP, Lee C-S

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and O’Bryen PJ (eds), Aquaculture and Ecosystems: An Integrated Coastal and Ocean Management Approach, World Aquaculture Society, Baton Rouge, LA, 207–20. richards jb and trevelyan ga (2001) Mussel culture, in California’s Living Marine Resources: A Status Report, California Department of Fish and Game, Sacramento, CA, 496–9. ryan j (2004) Farming the Deep Blue, Technical Report 82, Board Iascaigh Mhara, Westport. shumway se, davis c, downey r, karney r, kraeuter j, parsons j, rheault r and wikfors g (2003) Shellfish aquaculture – In praise of sustainable economies and environments, World Aquaculture, 34(3), 15–19. stanley s (2005) Development of a Submersible Raft for Shellfish Aquaculture, US Department of Commerce/National Oceanic and Atmospheric Administration Small Business Innovation Research (SBIR), Abstracts of Awards for Fiscal Year 2005, 12. stevens c, spigel r, plew d and fredricksson d (2005) A blueprint for better mussel farm design, NZ Aquaculture, issue 04, March/April, 8–9. subsea shellfish (2004) Biology and Innovation Primer Project, December, http:// www.freepatentsonline.com/EP1476011.pdf. thompson nw (1996) Trends in Australasian Open Water Aquaculture, in Polk M (ed.), Open Ocean Aquaculture: Proceedings of an International Conference, 8–10 May, Portland, ME, New Hampshire/Maine Sea Grant College Program Rpt #UNHMP-CP-SG-96-9, 223–34. van nieuwenhove k and delbare d (2008) Innovative offshore mussel farming in the Belgian North Sea, in Mees J and Seys J (eds), VLIZ Young Scientist’ Day, Brugge, Belgium, 29 February 2008: book of abstracts. VLIZ Special Publication, 40, Oostende, VLIZ, 67, www.vliz.be/imisdocs/publications/132623.pdf, accessed February 2009. ward lg, grizzle re and irish jd (2006) UNH OOA Environmental Monitoring Program, CINEMar/Open Ocean Aquaculture Annual Progress Report for the period 1/01/05 through 12/31/05, Final Report for NOAA Grant No. NA16RP1718, Interim Progress Report for NOAA Grant No. NA04OAR4600155, submitted January 23, National Oceanic and Atmospheric Administration, Washington, DC. worm b, barbier eb, beaumont n, duffy je, folke c, halpern bs, jackson jbc, lotze hk, micheli f, palumbi sr, sala e, selkoe ek, stachowicz jj and watson r (2006) Impacts of biodiversity loss on ocean ecosystem services, Science 314, 787–90.

30 Advances in technology for off-shore and open ocean finfish aquaculture A. Fredheim, SINTEF Fisheries and Aquaculture, Norway, and R. Langan, University of New Hampshire, USA Abstract: This chapter is a review of existing and new technologies used for sea-based fish farming. The first part is a presentation of the historical development and continues with a discussion of the characteristics and technical limitations of different commonly used fish farm systems. The chapter continues with technical challenges for off-shore and open ocean fish farming and a presentation of advances in technology with a discussion of pros and cons for different systems. Finally, challenges and advances for supporting equipment for off-shore fish farming are presented. Key words: off-shore and open ocean finfish aquaculture, fish farm system, floating fish farm, floating collar, net cage.

30.1 Introduction: historical development of fish farming technology The development of floating structures for fish farming in the sea started with the brothers Ove and Sivert Grøntvedt from Hitra Norway, who designed the first floating fish farm with a net cage and used it for farming salmon. Traditionally the fish were kept in place by a system of sticks or structures attached to the sea floor with a net pen attached, and penetrating the surface. This system was difficult to move and limited to shallow waters. To solve this, the Grøntvedt brothers developed a floating collar and used a purse seine from the catch-based fishing industry as a net cage for containment of the fish (Fig. 30.1). The floating collar was made of wooden beams and expanded polyester as floats. The fish farm was kept in place using ropes and anchors. It was a simple, yet ingenious and innovative, construction. This was the start of a technical and operational development which

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Fig. 30.1 The first floating fish farm, the ‘Grøntvedt’ fish farm, made of wood and expanded polyester.

Fig. 30.2 A circular high-density polyethylene pipe (HDPE) fish farm. (Photo by SINTEF Fisheries and Aquaculture)

has led to the multibillion dollar industry that fish farming has become in Norway and worldwide. The next major development in fish farm construction was the invention of the circular plastic collar fish farm, commonly known as polarcircle. This fish farm concept is basically high-density polyethylene (HDPE) pipes, often used for water or sewage transportation, turned into a circle and used for attachment of the net cage (Fig. 30.2). The early versions were small in size (typically 30–40 m of circumference) and single rim. Recent versions

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are constructions with two or three rims outside of each other, solid railing in either HDPE, steel or combination and with walkways. The circular plastic collar fish farm has some great advantages, but also disadvantages related to working conditions and the ability to attach equipment. Due to this, more sturdy and heavy fish farms in steel have been developed. Several conceptual designs exist, such as floating collars made of rigid steel tubes, interconnected hinged floating bridges (Fig. 30.3) and slender hulls connected by hinged bridges (known as catamaran fish farms). Some of the concepts also include integrated feed barges (Fig. 30.4). The

Fig. 30.3 Fish farm made of interconnected hinged floating bridges of steel. (Photo by SINTEF Fisheries and Aquaculture)

Fig. 30.4 Illustration of a large catamaran-type fish farm with integrated feed barge for storage and equipment. (Illustration by Feeding Systems)

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fish farms made of steel normally have more flotation than HDPE collars, making them easier and safer to work on. The fish farms of steel are often made able to support a fork lift and equipment to handle net cages. This is considered a great benefit, and in sheltered locations fish farms of steel of different designs are often preferred by the farmers. Due to its much more flexible characteristics, the circular plastic collar fish farm is much more durable and will hold much higher wave loads. The investment cost of a circular plastic collar fish farm is also much less than for a traditional fish farm of steel. The trend over the last years has been towards more use of fish farms made of HDPE, and it is fair to say that the HDPE collar fish farm is the present-day industry standard. Operational procedures, use of centralized feeding with feed barges (Fig. 30.5) and larger work boats capable of handling large net cages, have also contributed to this trend. Currently, standard fish farms for salmon and, to some degree, also sea bream and sea bass typically have collar or net cages of 120 m or, not uncommonly, 156 m circumference. For tuna fish farming, collars and net cages of 300 m circumference are in use. When floating fish farms became more common, the purse seines were exchanged for purpose-made net cages for aquaculture (Fig. 30.6). An important difference is the orientation of the netting; while the netting in most fishing gear has a diagonal orientation, a flag orientation is used in net cages for aquaculture. Since the initial attempts at fish farming in the sea, feeding methods and technology have developed from hand delivery of waste fish to the use of

Fig. 30.5 Centralized feeding systems used with a HDPE collar fish farm. (Photo by SINTEF Fisheries and Aquaculture)

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Fig. 30.6 Illustration of a modern net cage used for fish farming with ropes, weights, collar, etc.

large feed barges with control systems and remote distribution of feed pellets. These advances have, together with selective breeding and improvements in feed content, reduced the average biological feed conversion factor from 3 to approximately 1.2, and the biomass production per employee has increased from 50 metric tons in 1992 to 340 metric tons in 2004 (Fauske, 2005). Today, there is no other form of animal food production where such a high biomass is gathered in such a small area.

30.2 Floating fish farm design 30.2.1 Categorization of fish farm designs The substructure of a floating fish farm is often named a floating collar due to its shape and use. The main purpose of a floating collar is to be an attachment point for the net cage, contribute to maintaining the shape of the net cage, distribute forces to the mooring system and to be a working platform for daily operations. The floating collar is the main structural member of a floating fish farm and integrates all the other main parts of the farm. In addition to the floating collar, a floating fish farm consists of the net cage, a mooring system to keep the farm in position and normally some sort of flotation in the form of buoys on the mooring system. The main parts of different floating fish farms are illustrated in Figs 30.7, 30.8 and 30.9 for a circular plastic collar, interconnected hinged steel and a steel catamaran fish farm, respectively.

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Fig. 30.7 Illustration of HDPE collar fish farm. (Illustration by SINTEF Fisheries and Aquaculture)

Fig. 30.8 Illustration of an interconnected hinged steel fish farm. (Illustration by SINTEF Fisheries and Aquaculture)

Fig. 30.9 Illustration of a steel catamaran fish farm. (Illustration by SINTEF Fisheries and Aquaculture)

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Floating fish farms come in a large range of different shapes and constructional designs, from fully flexible structures to large rigid structures and with a large number of different attachment options for the net cages. Classification of fish farm constructions can be done in two ways, according to the containment system (Loverich and Gace, 1998) or from a constructional point of view. With regard to the containment system, it is rational to categorize the different systems and designs with respect to how the fish farm maintains the volume of the net cage. Based on this idea, fish farm designs are classified into five categories (Loverich and Gace, 1998): 1. Gravity fish farms: The shape of the net is maintained using different kinds of weights to make a downward vertical force. 2. Anchor tension fish farms: It is the tension and integrity of the mooring system that maintains the shape of the net as well as keeping the fish farm in place. 3. Semi-rigid fish farms: A frame made of a combination of ropes and rigid steel components makes up the integrity of the net cage and maintains the shape of the net. 4. Rigid fish farms: A rigid structure, usually of steel, but which could also be of plastic or other materials, maintains the shape of the net cage. 5. Other fish farm designs: Other types of fish farms that do not fit into the other categories or are a mixture of them. When considering loads on the fish farm and constructional analysis, this categorization has shortcomings, since it does not classify the hydrodynamical and constructional properties of the fish farm construction. From an engineering point of view, it will be more useful to categorize the floating fish farm with respect to its structural properties and behaviour in waves and current. Based on this, three categories are defined: 1. flexible systems; 2. hinged connected bridges; 3. rigid structures. In addition to the two systems of categories above, it is useful to distinguish between surface and submergible fish farms. Submergible fish farms can in addition be partly or fully submergible; some submergible fish farms are intended to be submerged all the time and others only during bad weather situations. Fish farm construction and systems in these different categories are presented in more detail later in this chapter. 30.2.2 The net cage As described earlier, the containment system used for fish farms in the sea is named a net cage and for a traditional fish farm is suspended inside a floating collar. A net cage for fish farming is constructed from a netting material, which is attached to a frame of ropes to take the forces. Weights are attached to maintain the correct shape and volume.

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The net cage can be either round, square, rectangular, six-sided or multisided in shape. It is normally made of a synthetic material, most commonly polyamide (often known under its brand name nylon). The material used should be both rigid enough to retain shape and flexible enough to minimize the effects of wind and wave forces. The netting can be made with or without knots. Presently, knotless or Rachel knitted netting are the most commonly used. More scientific information regarding fish nets and netting material can be found in Fridman et al. (1992) and, for net cages in particular, Moe et al. (2007). Some new fish farm and net cage designs use, modern high strength materials like ‘Ultra high molecular weight polyethylene’ (UHMWPE). These materials have different brand names such as Dyneema® and Spectra (Moe et al., 2005).

30.2.3 Mooring system The mooring system for a fish farm consists primarily of ropes, floats and anchors. In addition, several smaller components like shackles, connection plates, chains, rings, etc. are used in the mooring system to connect together these primary parts. The purpose of a mooring system is to secure the fish farm in the desired position. Mooring requirements are determined by the size and characteristics of the fish farm and conditions like bottom topography and weather conditions at the specific site. Computer programs for the design and dimensioning of mooring systems specifically for fish farms do exist and are commercially available (Fredriksson et al., 2004; Berstad and Tronstad, 2005). There are two principal systems for mooring a fish farm; either by independent separate lines directly to the floating collar or by a grid mooring system to which one or several floating collars is connected. The interconnected hinged bridge systems are usually moored using independent mooring lines. Figure 30.10 shows a single mooring line connected to a steel fish farm. A circular HDPE collar, due to low horizontal stiffness in the collar itself, needs to be moored using the grid system with bridles connecting the plastic collar to the mooring grid. The grid mooring system provides the necessary horizontal stiffness for the complete fish farm. An illustration of a grid mooring system is shown in Fig. 30.11. The grid mooring system is often kept at a depth of 5–10 m, to avoid ropes coming in contact with propellers of working boats and well-boats. Floats are used to keep the grid mooring system in place and at the desired depth.

30.2.4 Structural analysis of fish farms Which methods to use for structural analysis of a fish farm depends on its structural properties. Different methods and models need to be used if the system is made of plastic pipes, hinged bridges or long hull-shaped floaters

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Fig. 30.10

Detail of a mooring line connected to an interconnected hinged steel fish farm. (Photo by SINTEF Fisheries and Aquaculture)

Fig. 30.11 Illustration of a grid mooring system for HDPE collar fish farms. (Illustration by Aqualine)

(Berstad et al., 2005, Huang et al., 2006; Lader and Fredheim, 2006; Fredriksson et al., 2007; Jensen et al., 2007). Typically, the more flexible the construction is, the more wave loads it will withstand. Circular HDPE pipe fish farms are flexible and thus withstand more waves and are often used at locations exposed to more environmental forces than interconnected hinged fish farms made of steel. Hinged steel fish farms have hinges which only allow for rotation around one axis. This restriction in motion has implications for the behaviour and motion of the complete fish farm. A vertical motion in one point will

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introduce horizontal motions at other points, as horizontal motion of one bridge will introduce motions in other bridges. The behaviour and deformation of a hinged fish farm are non-linear and very complex, and calculation of the response in a realistic, irregular short-crested sea state is very difficult. Environmental forces inflict fatigue and damage due to excitation of motions and the forced displacements of parts of the construction. For a stiff hull-shaped design it will be important to consider sagging and hogging moments. This is a situation where the hull is either on a wave crest or in a wave trough. The ends of the hull or the mid part of the hull will then have less buoyancy than the rest of the hull, and potentially large moments are introduced (Faltinsen, 1990). There are three main environmental loads acting on a floating fish farm: wind, current and waves. The wind load will act on the parts of the fish farm above the surface; the current load acts on the net cage, the floating collar, flotation and mooring lines; while the wave loads will mainly act on the floating collar and upper part of the net cage. The current loads act on both the structural members of the floating collar and the net cage. Current loads will also act on mooring lines. Unless the mooring lines are very long the contribution from current loads on the mooring lines will normally be insignificant compared to the load on the net cage. The current force will be proportional to the exposed area of the structure and the drag coefficient and quadratic with current velocity. The drag coefficient will depend on the shape of the structural member and can be found from experiments. For standard shapes like cylinders and squares, several experimental results have been published and drag coefficients can be found from tables and figures (Hoerner, 1965). Calculations of current loads on net cages are more complicated. The net cage is a permeable surface; it changes shape under loading and the solidity will alter with biofouling (Swift et al., 2006) and loading. There are, in principle, two approaches towards calculating current loads on the net cages: (i) model individual twines and knots in the net and sum the drag forces on each of the members; or (ii) divide the net cage into panels and sum the drag forces calculated on each of the panels. The current force on each of the panels is normally calculated with empirical models based on experiments (Aarsnens et al., 1990; Løland, 1991; Lader et al., 2003; Fredheim, 2005). From a hydrodynamic point of view, the main characterization of a floating fish farm is the size of the different members of the structure compared to wave height and wave length. Depending on this ratio, different methods for calculating wave loads on the construction are used. A ship or an offshore floating oil platform will normally have large dimensions compared to relevant wave heights and wave lengths, and radiation–diffraction methods are often used. The members of a truss work will normally have small dimensions compared to relevant wave heights and wave lengths, and then simplified methods like the Morrison equation can be used. The

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structural members of floating fish farms will, on the other hand, often have neither large nor small dimensions compared to wave heights and lengths (Newman, 1989).

30.3 Current status and technical limitations In this section, existing and proven fish farm systems will be presented. All these fish farms are available from manufacturers and suppliers worldwide, and are in use for commercial industrial fish farming. Some of the presented fish farms are currently in use at semi-exposed in-shore and, to some degree, exposed off-shore locations. Some of the systems might also be applicable, with some modifications, for use at some open ocean sites. This is described further in Section 27.4 Novel fish farm constructions. In Norway, a regulatory framework requiring a technical certification from an independent accredited certification body is in place (Norwegian Directorate of Fisheries, 2005). Any floating collar, net cage, mooring system or feed barge used needs to be certified according to technical requirement for the dimensions of floating fish farms, given in the Norwegian standard NS 9415 ‘Marine fish farms: Requirements for design, dimensioning, production, installation and operation’ (Standard Norway, 2005). In this standard, a classification system with reference to wave height and current strength is given. Presently certified equipment for sites with significant wave heights up to 3 m and current strengths of 1.5 m/s is commercially available. The present trend in commercial fish farming is towards larger fish farms. Larger fish farms are believed to be more operationally efficient and thus more economical. However, increasing the size of the fish farm and net cages also involves new challenges with respect to keeping control of the volume, getting the feed distributed, keeping the net clean from biofouling, etc. Larger fish farms also involve larger well-boats and larger barges for storage and distribution of feed. A typical salmon fish farm can consist of eight collars in a grid system mooring, each collar 120 m in circumference and each net cage 30 m deep. Such a farm can produce more than 5000 tons of salmon in one production cycle (between 14 and 18 months). Each net cage will have an area of approximately 5000 m2 and a volume of 35 000 m3. In each net cage will there be more than 100 000 individual fish. This requires a high degree of reliability in the mooring system and construction, as well as presenting great challenges in operating such a farm. In an even larger, but not uncommon, fish farm of 156 m circumference with a net cage 15 m deep, it is possible to have more than 1000 tons of fish in one of these large net cages. To picture the extent of this large amount of biomass, the fish biomass can for instance be compared to a number of

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Fig. 30.12 Comparisons of the amount of biomass in one large net cage with using 2000 cows of 500 kg. (Illustration by Aqualine)

cows (Fig. 30.12). One thousand tons of fish will be equivalent to 2000 cows of 500 kg.

30.3.1 Circular high-density polyethylene (HDPE) collar fish farm As described earlier in the chapter, the circular plastic fish farm, often known as a polar circle after the brand name PolarCirkel® used by the first producer of this type of fish farm, is made of HDPE pipes, similar to the pipes used for sewage and water systems (Fig. 30.13). These fish farms are constructed by welding the pipes together into preferred lengths. The complete pipe length is then forced into a circle and the two free ends are welded together. These fish farms system are delivered as single, double or triple rim systems. The rims are connected together using a clamp, made of either HDPE or steel. The clamps are also often used for attachment of the net cage and mooring lines. Railings of either steel or HDPE are connected to the clamps. Some manufacturers also deliver walkways which can be attached on top of the pipes. A HDPE fish farm will normally be moored with a grid system mooring of ropes and floats (Fig. 30.14). The HDPE fish farm is a relatively low-tech product, which can be manufactured easily anywhere. HDPE pipes are available worldwide and the fish farm can be assembled on any relatively large flat space, often close to the location. Normal ring sizes can be 70, 90, 120 or 156 m. The diameter of the HDPE tube typically ranges from 250–400 mm. Due to the flexibility in the material and standard mooring system the HDPE fish farm withstands waves to a high degree and can be used at

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Fig. 30.13

HDPE pipes used for manufacturing circular plastic fish farms. (Photo by SINTEF Fisheries and Aquaculture)

Fig. 30.14 Circular HDPE collar fish farm in a grid mooring system. (Photo by AKVA Group)

semi-exposed locations. The investment costs are relatively low. The system has shown good production performance and is preferred by many farmers due to its layout and efficiency. Because there are several collars in the mooring system, the distance between the collars and net cages is relatively high, which ensures good flow of water between the net cages and available oxygen to the fish.

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The main disadvantage of the HDPE fish farm is the working conditions. Due to low freeboard, less space to move on and large movements of the collar in waves, the HDPE collar is difficult to work on. There is also little room on the collar for mechanical and auxiliary equipment. It is necessary to use workboats for operations such as, for instance, changing net when the size of the net cages increases.

30.3.2 Interconnected hinged steel fish farm As described in the introduction, the interconnected hinged steel fish farm is made up of bridges of steel with flotation connected together with hinges. Each bridge is normally 12 m in length, and the bridges are connected together into different configurations to make up the complete fish farm system. Flotation is normally made of expanded polyester covered with some protective material. The flotation is attached underneath the steel bridges (Fig. 30.15). The hinges allow for rotation around only one axis in the horizontal plane and not around any vertical axis. Standard sizes of each section for one net cage are 12 × 12 m, 24 × 24 m and even 36 × 36 m, arranged in different configurations, for example 2 × 3 or 1 × 6 sections. A typical interconnected hinged steel fish farm is shown in Fig. 30.16. The interconnected hinged steel fish farm gives a relatively stable system with large areas for walking and placement of equipment. The flotation can be controlled, and the design allows for heavy auxiliary equipment like feed blowers, equipment to handle net cages and fork lifts. Due to the working conditions, these fish farm systems are often preferred by workers.

Fig. 30.15 Expanded polyester flotation attached under a bridge of steel for a hinged steel fish farm. (Photo by SINTEF Fisheries and Aquaculture)

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Fig. 30.16 A typical interconnected hinged steel fish farm with attached feed blowers and a storage barge next to it. (Photo by SINTEF Fisheries and Aquaculture)

Due to its construction and because the bridges are connected together with hinges allowing for rotation around only one axis, this system has no or limited flexibility in the horizontal plane. This gives rise to structural problems when exposed to waves and current. Irregular short-crested sea is a particular problem for these types of fish farms. The waves will induce forced displacements of the different bridges and elements of the fish farm. In an irregular sea, these displacements will, to a large extent, be independent for the different parts of the structure. Due to the lack of horizontal flexibility, large forces and stresses are introduced into the construction. The result is that the interconnected hinged steel fish farms often have problems with fatigue in and around the hinges and are only suitable for sheltered locations. The investment cost of these fish farms is normally higher than for the HDPE circular fish farms.

30.3.3 Catamaran steel fish farm Catamaran steel fish farms are made up of steel hulls which are connected together with bridges and hinges into different configurations. The hulls are connected together to make up the complete fish farm systems. Flotation is given by the hulls. The hinges allow for rotation around only one axis in the horizontal plane, but not around any vertical axis. These fish farms are often large, with integrated feeding barges (Fig. 30.17), and are custom made. The Catamaran steel fish farms have much the same advantages as the interconnected hinged steel fish farms. The system has large areas for

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Fig. 30.17 A catamaran-type fish farm with integrated feed barge for storage and equipment. (Photo by Procean)

walking and placement of equipment and enough flotation to allow for heavy auxiliary equipment like feed blowers, equipment to handle net cages and fork lifts. In contrast to the interconnected hinged steel fish farm, the catamaran fish farm only has flotation along one axis. This is because the steel hulls are connected together with bridges that are not in contact with water. The hinges are still critical points but, compared to the interconnected hinged steel fish farms, these fish farms are not exposed to displacement forces to the same extent. The investment cost of these fish farms is even higher than for the interconnected hinged steel fish farms.

30.3.4 Rigid steel fish farm Rigid steel fish farms are a large category with several different designs. The most common has been fish farms made of steel pipes welded together into square collars but also fish farms made of truss work also exist (Fig. 30.18). These fish farms are often large, custom made and have integrated feed storage. The main advantages of these systems are stability and work platforms. To a large extent, rigid steel fish farms offer the same benefits as hinged steel fish farms. It is difficult to describe generic disadvantages due to the different shapes and constructional methods but, in principle, the more rigid or less flexible a structure, the more wave loads are introduced into it. Rigid steel fish farms often have very high investment costs.

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Fig. 30.18 An example of a rigid steel truss work fish farm design. (Photo by SINTEF Fisheries and Aquaculture)

30.4 Novel fish farm systems The first challenge for any aquaculture installation used at a truly open ocean off-shore site is to withstand the environmental loads due to waves, wind and current, otherwise, there will of course be no potential for farming. This can be achieved either by constructing a fish farm that has a shape that reduces the loads and a flexible body that deforms to reduce the impact of the loads, or by having a rigid structure strong enough to withstand the loads. The second challenge, and one that has proven to be more difficult than constructing the farm itself, is to make the farm operational for modern fish farming. This latter point includes economical aspects and the challenge of competing with large industrial-standard and cost-efficient farms at more sheltered locations. A few key operational aspects need to be in place before economical farming will be possible: regular feeding every day, getting fish in and out in large quantities (access by well-boats), treatment of diseases and parasites, change of nets and, to some extent, cleaning of the nets for biofouling. Moreover, all of these operations should be carried out without the use of divers most of the time. Technical challenges also include deformation of the net cage due to high currents and waves, making the use of gravity fish farms difficult. For submergible systems, another important technical challenge is control of the speed for submerging and, especially, elevating the fish farms, to allow the fish to compensate for the pressure changes by changing the volume of its swim bladder. Given the wide range of sea conditions that fall under the definition of ‘off-shore and open ocean aquaculture,’ no single fish farm technology can

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be considered ideal or even appropriate for use under all circumstances. Currently, the greatest production in exposed locations is achieved with flexible gravity fish farms. Circular flexible collar gravity fish farms are in use for salmon production in high-energy sites in Ireland, Scotland, the Faeroe Islands and New Brunswick, Canada; similar technologies are used in the Canary Islands and the Mediterranean for bass and bream culture. Tuna fattening operations in Australia, the Mediterranean and Mexico also use large HDPE collar cages in exposed sites. In this section, fish farm systems engineered specifically with the purpose of being used for off-shore and open ocean fish farming will be presented and discussed. The systems come in a large variety of designs. Some are completely new designs and others are more of an adaption of existing proven systems. Nearly all the systems are partly or fully submergible, either based on being submerged most of the time or on being submerged only during storm conditions. Both steel and HDPE and combinations of these material are used in the constructions. Several methods to retain the shape of the net cage are used. Most of the submergible novel fish farms use a solution with water and air in and out of different ballast chambers to submerge and elevate the construction. Only one of the presented fish farms has a solution for complete control of the elevation speed. Since the novel systems come in so many different shapes, they also have very different solutions, if any, for the operational challenges of farming fish at exposed locations. The systems presented will be described and discussed relative to the main challenges for an efficient and operable fish farm at an open ocean site. The first three systems described, the SeaStationTM, the Tension Leg Cage and the PolarCirkel® submergible fish farm, are all commercially available and installed at several sites, and fish are currently farmed using these systems. The other systems are at different stages of development, ranging from concepts barely built to prototypes with fish being farmed. Table 30.1 gives a summary of all the different fish farm systems presented in this chapter, including current fish farm design. The different fish farms are categorized according to the main structural characteristics and categories presented earlier.

30.4.1 SeaStationTM fish farm The SeaStationTM is the most common fish farm used for truly open ocean fish farming. This fish farm is developed and produced by OceanSpar LLC in the USA. SeaStations have been used successfully since the late 1990s in very rough conditions, including a site off the coast of New Hampshire in the Northwest Atlantic, where significant wave heights can exceed 9 m. There are currently 50 SeaStations deployed in 16 countries for grow-out of a wide variety of species.

Circular Square Square Square Conical

Circular

Circular Square

Ellipsoid

Circular/spherical

Circular/spherical

Circular

HDPE pipe Interconnected hinged steel Catamaran steel Rigid steel SeaStationTM

PolarCirkel® submergible

Tension Leg Cage Nautilus

OceanGlobe

Farmocean

Sadco

SubFish

HDPE = high-density polyethylene.

Main shape

Fish farm design

Gravity

Semi-rigid

Gravity

Rigid

Anchor tension Rigid

Gravity

Gravity Gravity Gravity Gravity Semi-rigid

Net cage

Flexible

Rigid

Rigid

Flexible/rigid

Flexible Rigid

Flexible

Flexible Hinged Hinged Rigid Flexible/rigid

Structure

Independent lines Grid system Independent lines Grid system Single-point

Single-point

Integrated Independent lines Single-point

Grid system

Grid system Independent lines Independent lines Independent lines Grid system

Mooring system

Surface Surface Surface Surface Submergible by buoyancy control Submergible by buoyancy control Submergible by tilting Submergible by buoyancy control and winch Submergible by buoyancy control Submergible by buoyancy control Submergible by buoyancy control Submergible by buoyancy control

Operational mode

Table 30.1 Technical properties of fish farm systems, categorized according to main structural characteristics and categories presented in Section 30.2.1 ‘Categorization of fish farm designs’

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This fish farm is conically shaped, with a solid steel cylinder in the middle, a rim of steel and high-strength netting and ropes connecting the centre pipe and the rim like spokes in a wheel. It is submerged and elevated by pumping water and air in and out of the centre pipe. New versions also feature the ability to have separate ballast chambers in the pipe and a system which makes the complete fish farm able to turn, while still being attached to the mooring systems. The fish farm is moored using a grid mooring system similar to that used for circular HDPE pipe collar fish farms. A picture of the fish farm is shown in Fig. 30.19. The conical shape and its submergibility make it very robust and suitable for high-energy environments. The SeaStation fish farm is designed to be fully submerged during normal operations. Because of this, it is necessary to use underwater feeding. Underwater feeding systems are or soon will be commercially available. When submerged only the top part of the fish farm and the floats in the grid mooring systems are visible, which is important in parts of the world where conflict with tourism and other recreational activities is possible. The main challenge and possible disadvantage of the SeaStation fish farm is that operational procedures and equipment are not yet developed for this type of fish farm (Rice, 2005). It is not possible to change the volume of the net cage when in operation, which makes it difficult to get fish out of the net cage. Also possible treatment of the fish for diseases or parasites with liquid, where the procedure requires use of a cover around the net cage, will be difficult. Operations and maintenance are, to a large extent, done using divers. Some of the operational aspects are partly solved with the ability to turn the fish farm in surface position, giving access to all parts

Fig. 30.19 SeaStationTM fish farm submerged. (Photo By OceanSpar LLC)

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of the net at surface and making it possible, for instance, to remove biofouling. The SeaStation fish farm faces the same challenge as most submergible fish farm designs with lack of control of speed when being elevated. Farming fish with a closed swim bladder, like cod for instance, puts very strict limits on the speed of elevating the fish farm. Farmers have experienced fish dying due to the net cage being taken to the surface too fast. To allow for proper compensation for the fish, it might take several days to move a net cage from 20 m to the surface. Even without a submergible fish farm, elevating the bottom of a net cage needs to be done over several days for the fish at the lower part of the net cage to be able to compensate. Existing SeaStation fish farms are of a moderate size, and it might be difficult to upgrade the fish farm to the commercial volumes of more than 30 000 m3 used for instance in salmon production. Due to the limited dimensions, the cost of the SeaStation fish farm per production volume is very high compared to existing commercial fish farms for in-shore and semiexposed in-shore locations.

30.4.2 PolarCirkel® submergible fish farm The PolarCirkel® submergible fish farm is in principle a HDPE circular fish farm as described earlier, with all its characteristics and the addition that it is submergible. It is developed and produced by the company PolarCirkel, now a part of the AKVA Group in Norway. This fish farm system is made submergible by introducing ballast chambers in the HDPE pipe collar. This makes it possible to pump water and air in and out of the ballast chambers to submerge and elevate the collar. The PolarCirkel submergible fish farm with hoses for water and air is shown in Fig. 30.20 during submergence. When the fish farm is submerged, it is hanging in floats which are a part of the surface mooring system. The floats can also be submerged if made incompressible. In principle, this fish farm could be produced in the size of any ordinary HDPE circular fish farm, but so far they have only been delivered at more moderate dimensions. Due to the relatively small alteration of a relatively low-cost commercially available system and the potential for this fish farm to be made with large dimensions, the PolarCirkel submergible fish farm have the lowest cost per volume of the open ocean fish farm designs. This fish farm is intended to be in a surface mode during normal operations. Its submergible systems are mainly intended to be used during storm situations and algae bloom or jelly fish attacks. The advantage of this submergible fish farm is its traditional main design which makes it, when in surface mode, easy and familiar to operate. The depth of the grid mooring system decides the depth of the fish farm when submerged. The submergence depth will be twice the depth of the grid mooring system. When submerged, both the fish farm and the fish will be out of the high-energy

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Fig. 30.20 A PolarCirkel® HDPE submergible fish farm being submerged. (Photo by SINTEF Fisheries and Aquaculture)

zone of waves. The size of the floats will be small compared to the wave lengths, and thus the forces transmitted to the fish farm will be small. The flexibility of the ropes in the mooring systems will also contribute to small forces being transmitted. This design is well suited for locations with high waves. With respect to the net cage, the PolarCirkel submergible fish farm is an ordinary gravity fish farm. Except for water current often being lower at deeper depths, this design does not perform any better with respect to current than traditional gravity fish farms. The PolarCirkel submergible fish farm faces the same challenge with lack of control of speed when being elevated as most submergible fish farm designs.

30.4.3 Tension Leg Cage fish farm This is a fish farm design where the net cage, collar, flotation and mooring systems are integrated into one construction. The fish farm is moored vertically and each fish farm is separate. The net cage maintains its shape due to the mooring system together with a small floating collar and floats. Illustrations of the fish farm in still water and exposed to current and waves are shown in Figs 30.21 and 30.22, respectively. The Tension Leg Cage fish farm is developed by and patents are held by SINTEF Fisheries and Aquaculture in Norway, and it is marketed in the Mediterranean by REFA Med in Italy. When exposed to current and waves, the fish farm will submerge due the vertical mooring system which introduces a tilting motion. The net cage and the fish will then be moved out of the high-energy wave zone. In addition, the Tension Leg Cage fish farm is a highly flexible structure. Both the vertical mooring system and the structural flexibility contribute towards the

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Fig. 30.21 Illustration of the Tension Leg Cage fish farm with vertical mooring system and floats in still water. (Illustration by MARINTEK)

Fig. 30.22 Illustration of the Tension Leg Cage fish farm exposed to current and waves. (Illustration by MARINTEK)

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ability of this fish farm to withstand environmental loads. A positive feature of this fish farm is that only a few parts are visible, and this can reduce potential conflict with tourism and other recreational activities. There are two challenges related to the design of this fish farm. First, it is not possible to control how much or to what depth the fish farm is submerged; and second in a situation with long periods of high currents the fish farm might be submerged for a long time period. However, there is no machinery or equipment involved when the fish farm submerges, reducing the probability of failure and also the cost. Due to the integrated construction of the net cage and the mooring system, there are a few operational challenges related to, amongst others, how to change nets, which might be necessary due to biofouling or when it is necessary to perform maintenance. It is also common to change the net cage to one with larger mesh size when the fish grow in size, ensuring good flow of water and oxygen. Due to its shape, only a small part of the net cage volume is accessible from the surface even when the fish farm is not submerged. This might be a challenge, for instance when getting fish in and out of the net cage. Also, possible treatment of the fish for diseases or parasites with liquid, where the procedure requires use of a cover around the net cage, will be difficult. It might also be necessary to have an underwater feeding system, but this is or soon will be commercially available.

30.4.4 Nautilus fish farm The Nautilus fish farm is an example of a rigid and fully submergible fish farm made of steel pipes and truss work. During normal conditions, the Nautilus fish farm operates on the surface, and it will be submerged only during rough weather conditions. Thus more or less common operational procedures can be used when at the surface. Due to the rigid design and attachment of net cages, they will more or less retain their volume 100 % in current. This fish farm can also be single-point moored, allowing it to turn and partly submerge when exposed to waves and current. An illustration is given in Fig. 30.23. In addition to its rigid construction, the main difference between the Nautilus fish farm and other submergible fish farms is in how it is submerged and elevated. The Nautilus fish farm has ballast chambers for which to pump water and air in and out, but these are intended to be used only for partly submerging the fish farm. In addition, the fish farm has an underwater hydraulically driven winch. A weight is attached to the end of cable of the winch, and the fish farm is submerged by the water and air system until the weight hits the bottom. The fish farm can then be pulled down by the winch system. This system is somewhat more complicated than those using only water in and out of ballast chambers, but the result is very good control of the elevation speed, making the fish farm usable for fish with closed swim bladders.

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Fig. 30.23 A computer simulation of the Nautilus fish farm exposed to waves. (Illustration by MARINTEK)

Due to the construction and necessary equipment for the control of submerging and elevation, the cost of the Nautilus fish farm per production volume is very high compared to existing commercial fish farms for in-shore and semi-exposed in-shore locations. The concept has been tried out with fish at a prototype stage, but to the knowledge of the authors no Nautilus fish farm has been sold commercially.

30.4.5 OceanGlobe fish farm The OceanGlobe fish farm is based on an ellipsoid net cage with the shape retained by a frame made of HDPE on the outside. Through the centre of the sphere there is a horizontal cylinder of steel. Connected to the two ends of the horizontal cylinder are a working platform made of steel truss work, stretching around one half of the net cage and frame at the surface like a rim. An illustration of the fish farm in maintenance position is shown in Fig. 30.24. The fish farm is submergible, with a surface position during normal conditions and submerged only when weather conditions are rough. When at the surface, 50 % of the net cage and frame is above water, leaving 50 % of the net cage volume for farming. The submergible system is based on pumping water and air in and out of ballast chambers, with the previously described limitation related to control of elevation speed. There are two main characteristics which make the OceanGlobe special. First, the fish farm is intended to be single-point moored, with a large float as a part of the single-point mooring system. Second, the fish farm is able to rotate around the horizontal cylinder, giving potential access to all parts and surface area of the net cage and frame for operations and maintenance. The OceanGlobe is a relatively complex construction with the frame of HDPE and the rim of steel truss work, in addition to the rotation and elevation systems. Due to this, it is expected that this fish farm design will have

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OceanGlobe Feed & energy station Vessel mooring Live fish carrier

Mooring buoy

Mooring line

Fig. 30.24 Illustration of the OceanGlobe fish farm in maintenance position with support vessel. (Illustration by BYKS)

a high cost per production volume compared to existing commercial fish farms for in-shore and semi-exposed in-shore locations. 30.4.6 Other novel fish farm systems In addition to the main designs described above, there are several other concepts and designs for open ocean fish farms. In this section a few of these are described briefly, but this list is by no means complete. Farmocean Farmocean is a partly submergible fish farm made of steel, consisting of a steel frame with flotation, ballast chambers and integrated feeding equipment. A gravity net cage is attached to the steel frame. The integrated feeder on top of the steel frame cannot be submerged, and this sets a limit to how deep the fish farm can be submerged. A picture of the fish farm completely elevated is shown in Fig. 30.25. SADCO The SADCO fish farm looks similar to the Farmocean fish farm, but the Sadco is completely submergible and also has a steel frame that supports the bottom part of the net cage. An illustration of the fish farm system without net cage is given in Fig. 30.26. SubFish The SubFish system is a single-point mooring fish farm. At the front towards the mooring is a stiff square steel frame, to which two long flexible HDPE

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Fig. 30.25

The Farmocean fish farm completely elevated. (Photo by Farmocean)

Fig. 30.26 Illustration of the SADCO fish farm with steel frame for net cage support. (Illustration by Sadco)

pipes are attached. Several circular HDPE collars are connected to these two pipes. Ordinary gravity net cages are attached to the circular collars. This fish farm has a normal surface mode and can be submerged by pumping water and air in and out of parts of the construction.

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Aquaculture Engineering Group (AEG) The New Brunswick, Canada, company Aquaculture Engineering Group (AEG) has developed a unique farming concept that integrates an array of modified gravity cages with an automated feeding system and current velocity deflector designed for use in the high-current open waters of the Bay of Fundy. The entire system is anchored to the seafloor using a single-point mooring and orients itself in the direction of the current.

30.5 Supporting technologies for off-shore and open ocean fish farming In addition to the challenge of developing sufficiently robust containment and mooring systems, off-shore farming presents additional challenges for nearly all aspects of day-to-day farm operation. Methods and equipment developed for routine operations such as feeding, harvesting, and monitoring at protected in-shore sites have been designed for calm sea conditions and, for the most part, cannot be directly transferred to the off-shore environment. Development of alternative operational systems has not kept pace with cage development, and farmers have struggled to integrate existing as well as new and unproven supporting technologies into off-shore installations. Of all off-shore operations, feeding is probably the most important. Inshore approaches, which include dispensing feed by cannons from a service vessel or automated feeding with blowers mounted on centralized feed barges, are severely hampered by rough seas. An ideal feeding system for off-shore aquaculture would be robust, remotely controlled, fully automated, able to accommodate the volume of food needed for a 2–3 week period, and be possessed of a hydraulic rather than pneumatic feed delivery system. It would also ideally be capable of wireless transmission of in-cage video, environmental monitoring data or other information critical to farm operation. Although no system as described currently exists, some progress has been made. The Scottish company Gael Force has developed the Sea Cap, a concrete feed barge that has operated successfully in exposed locations for several years. The University of New Hampshire (UNH) has developed two small (single cage), remotely operated feeders that have been in use since 2001 (Rice et al., 2003), and deployed a larger multicage feeder in 2007. Developed in conjunction with Ocean Spar, the feeder has four separate feed silos and can dispense feed to four submerged cages (Turmelle et al., 2006). It also incorporates a two-way remotely operated communications system that operates in-cage video cameras to monitor fish behaviour and response to feed introduction. The system will also house a unique acoustic tracking system that can continuously monitor behaviour and physiology of tagged fish for up to 40 days (Howell et al., 2006). Farm Ocean and SADCO have also integrated feeding systems into their cage

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designs, and the Canadian Company AEG has developed an automated hydraulic feeder that is integral to their single-point mooring cage array. Other routine off-shore operations such as grading, harvesting, biofouling control, and removal of mortalities are complicated by sea conditions, and additional strategies must be developed to make these practices safer and more efficient. Additional attention must also be paid to developing lower cost and reliable telemetry systems for transmission of data with large bandwidth requirements such as video.

30.6 Sources of further information and advice • There are two main research groups that have been working extensively with floating aquaculture constructions: { the Open Ocean Aquaculture group and Atlantic Marine Aquaculture Centre at University of New Hampshire (http://amac.unh.edu/); { the Department of Aquaculture Technology (www.sintef.no/fish) at SINTEF Fisheries and Aquaculture. { In Norway a centre for research-based innovation (CRI) in aquaculture technology has recently been established. The centre is jointly supported by industry partners, research partners and the Research Council of Norway. More information about the content of the centre and partners is available at www.sintef.no/create. • Aquaculture Engineering by Elsevier (www.sciencedirect.com) frequently publishes scientific articles related to aquaculture technology for off-shore and open ocean. • As a result of the Farming the Deep Blue conference (Ryan, 2004), an organization for promotion of off-shore aquaculture was established, the International Council for Offshore Aquaculture Development (ICOAD). More information is available through the web site of Bord Iascaigh Mhara – The Irish Sea Fisheries Board, www.bim.ie. • The European Research project, ‘Evaluation of the Promotion of Offshore Aquaculture Through a Technology Platform’, has established an open web site for information and exchange of interest related to offshore and open ocean aquaculture, www.offshoreaqua.com. • The Federation for European Aquaculture Producers (FEAP, www.feap. info) has developed a very extensive and useful web page for information and facts about aquaculture production in general, http://www. aquamedia.org.

30.7 References aarsnens j v, løland g and rudi h (1990) Current forces on cages, net deflection, in ‘Engineering for fish farming, Engineering for Offshore Fish Farming: Proceedings of the Conference Organized by the Institution of Civil Engineers, and Held

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in Glasgow on 17–18 October by Institution of Civil Engineers, Thomas Telford, London, 137–52. berstad a j and tronstad h (2005) Response from current and regular/irregular waves on a typical polyethylene fish farm, in Maritime Transportation and Exploitation of Ocean and Coastal Resources, Guedes Soares C, Garbatov Y and Fonseca N (eds), Taylor & Francis Group, London. berstad a j, tronstad h, sivertsen s and leite e (2005b) Enhancement of design criteria for fish farm facilities including operations, Proceedings of the 24th International Conference on Offshore Mechanics and Arctic Engineering, 12–17 June, Halkidiki. faltinsen o m (1990) Sea Loads on Ships and Offshore Structures, Cambridge University Press, Cambridge. fauske m (2005) Economic analysis on fish farming – salmon and trout, Norwegian Fisheries Directorate, Bergen. fredheim a (2005) Current Forces on Net Structures, Doctoral Thesis, Norwegian University of Science and Technology, Trondheim, Norway. fredriksson d w, decew j, swift m r, tsukrov i, chambers m d and celikkol b (2004) The design and analysis of a four-cage grid mooring for open ocean aquaculture, Aquacultural Engineering, 32, 77–94. fredriksson d w, decew j c, tsukrov i, swift m r and irish j d (2007) Development of large fish farm numerical modelling techniques with in situ mooring tension comparisons, Aquacultural Engineering, 36(2), 137–48. fridman a l (1992) Calculations for Fishing Gear Design, Fishing News Books Ltd., Farnham. hoerner s f (1965) Fluid-dynamic Drag, Hoerner Fluid Dynamics, Bakersfield, CA. howell w h, watson w h and chambers m d (2006) Offshore Production of Cod, Haddock and Halibut, CINEMar/Open Ocean Aquaculture Annual Progress Report for the Period from 1/01/05 to 12/31/05, Final Report for NOAA Grant No. NA16RP1718, interim Progress Report for NOAA Grant No. NA04OAR4600155, submitted January 23, National Oceanic and Atmospheric Administration, Washington, DC. huang c-c, tang h-j and liu j-y (2006) Dynamical analysis of net cage structures for marine aquaculture: Numerical simulation and model testing, Aquacultural Engineering, 35, 258–70. jensen ø, wroldsen a s, lader p f, fredheim a and heide m (2007) Finite element analysis of tensegrity structures in offshore aquaculture installations, Aquacultural Engineering, 36, 272–84. lader p f, enerhaug b, fredheim a and krokstad j r (2003) A full 3d model of net structures exposed to waves and current, The 3rd International Conference on Hydroelasticity in Marine Technology, 15–17 September, Oxford. lader p f and fredheim a (2006) Dynamic properties of a flexible net sheet in waves and current – a numerical approach, Aquacultural Engineering, 35, 228–38. loverich g f and gace l (1998) The effect of currents and waves on several classes of offshore sea cages, Proceedings of Open Ocean Aquaculture ’97, Charting the Future of Ocean Farming, April 23–25, Maui, HI, 131–44. løland g (1991) Current forces on and flow through fish farms, PhD thesis, Dept of Marine Hydrodynamics, The Norwegian Institute of Technology, Trondheim, Norway. moe h, fredheim a and heide m (2005) New net cage designs to prevent tearing during handling, IMAM 2005, Lisbon, 26–30 September. moe h, olsen a, hopperstad o s, jensen ø and fredheim a (2007) Tensile properties for netting materials used in aquaculture net cages, Aquacultural Engineering, 37, 252–65.

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newman n j (1989) Marine Hydrodynamics, MIT Press, Cambridge, MA. the norwegian directorate of fisheries (2005) New aquaculture regulations, http://www.lovdata.no/cgi-wift/ldles?doc=/sf/sf/sf-20031211-1490.html. rice g, stommel m, chambers m and eroshkin o (2003) The design, construction and testing of the University of New Hampshire feed buoy, in Bridger C J and CostaPierce B A (eds), Open Ocean Aquaculture: From Research to Commercial Reality, World Aquaculture Society, Baton Rouge, LA, 197–203. rice g (2005) Operation for submerged open ocean aquaculture, Open Ocean Aquaculture Workshop, 14–18 August, Torshavn, Faroe Islands, abstract. ryan j (2004) Farming the Deep Blue, Bord Iascaigh Mhara – The Irish Sea Fisheries Board, Dublin. standard norway (2005) Marin Fish farms: Requirements or design, dimensioning, production, installation and operation, Norwegian Standard NS 9415, Standards Norway, Lysaker. swift m r, fredriksson d w, unrein a, fullerton b, patursson o and baldwin b (2006) Drag force acting on biofouled net panels, Aquacultural Engineering, 35(3), 292–9. turmelle c, swift m, celikkol b, chambers m, decew j, fredriksson d, rice g and swanson k (2006) Design of a 20-ton Capacity Finfish Aquaculture Feeding Buoy, Proceedings of Oceans 2006, MTS/IEEE Conference, 18–21 September, Boston, MA.

31 Advances in technology and practice for land-based aquaculture systems: tank-based recirculating systems for finfish production T. Losordo, D. DeLong and T. Guerdat, North Carolina State University, USA

Abstract: Recirculating systems for aquaculture production are much talked about but often not very well understood. Proponents state that recirculating systems, systems that recycle water through a number of filters and components to clean the water and reuse it, are the way to a sustainable future for aquaculture. Detractors state with equal conviction that recirculating systems are too expensive to build and operate and are not and will not be an economically viable way to grow aquacultured products. As with most issues, there is no ‘black and white’ simple answer. This chapter seeks to highlight the required unit processes that must be dealt with in order to succeed when utilizing recirculating technology for tank-based finfish production. Additionally, examples of state-ofthe-art components are provided. Wherever possible, commercially available technology is referenced. Further, the chapter closes with the description of what the authors consider a well thought-out and implemented commercial-scale recirculating aquaculture production system. Key words: recirculating, aquaculture, intensive, tank-based, production systems.

31.1 Introduction Recirculating systems for aquaculture production are much talked about but often not very well understood. Proponents state that recirculating systems, systems that recycle water through a number of filters and components to clean the water and reuse it, are the way to a sustainable future for aquaculture. Detractors state with equal conviction that recirculating systems are too expensive to build and operate and are not and will not be an economically viable way to grow aquacultured products. As with most

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issues, there is no ‘black and white’ simple answer. Both groups are correct in some respects, and both groups are wrong in others. Let us begin this chapter by defining a recirculating system. Loosely defined, a recirculating system can be any production system that recycles water through some form of water treatment component or components. However, as recirculating systems have been under development since the 1970s (Broussard and Simco, 1976), most researchers, designers, and end-users of recirculating technology have come to understand recirculating systems to be defined and measured by the percent of the system water volume that is replaced per unit time, usually per day. Others have quantified a system’s level of recirculation as kg of feed fed to a system per unit of new water added. On a percent volume replacement basis, most recirculating technologies being employed in aquaculture today replace 5–20 % of the system volume per day. On some occasions no water is exchanged, except for making up for evaporation, and these systems are being termed ‘zero’ discharge. In actuality, many of these zero discharge systems do discharge water in the form of sludge (which is usually more than 80 % water). The fact is, almost all recirculating systems create a waste stream of some form and volume. To be truly environmentally friendly, a system design must incorporate the treatment of this waste stream. If the concentrated waste from a recirculating system is not properly treated, disposed of, or utilized, then, ultimately, that recirculating system is no ‘greener’ than a fish production system that uses large quantities of water and discharges a dilute waste stream into the environment. In fact, the discharge of highly concentrated organic and inorganic waste nutrients could be very harmful to a local environment. However, with the correct technology in place, recirculating technology can produce large quantities of fish with a small physical footprint and with very little environmental impact. From an economic perspective, those who suggest that recirculating technology is expensive to build and operate are correct when comparing it to more traditional pond-based or ocean-based technologies. However, in many areas of the world, the environmental impact of aquaculture effluent is a cost that is being directed back to the producer. For example, aquaculture producers in the Netherlands pay a ‘tax’ per kg of nitrogen and biochemical oxygen demand (BOD) or chemical oxygen demand (COD) discharged from the system or sent to a municipal treatment system. In some areas, effluent discharge regulations may severely limit the scale and location of more traditional aquaculture production systems. Therefore, there are situations where recirculating systems technology (that capture and treat or reuse waste effluents) can and should be used to produce aquacultured products. This chapter will endeavour to review advances that have been made in recirculating aquaculture technology since the late 1990s. While we will need to review some basics to frame the improvements that have occurred,

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we will try to focus on advancements that have been made in the industry as well as its future needs. The reader will be referred to some current URL addresses for various manufacturers. However, reference to or mention of a commercial product, specific trade name or component does not constitute endorsement of that product or component by the authors or by North Carolina State University.

31.2 Components in recirculating systems design With decades of experience in creating recirculating systems (and with a fair amount of trial and error), the industry is building more sophisticated systems. The technology of these systems is generally focused on two treatment ‘loops’. A primary loop filters tank water rapidly (usually at least once per hour) to remove waste solids, reduce ammonia–nitrogen through nitrifying filters, remove carbon dioxide, and add oxygen. Depending on the species being cultured, fine or dissolved solids are sometimes removed with foam fractionation (also referred to as protein skimming) or ozone contact, and ultraviolet light filters are employed to attempt some level of pathogenic bacteria and parasite control. The second loop, when properly designed and implemented, treats the waste stream from the primary recirculating filter loop. If this waste treatment loop is not used, the effluent from the primary filtration loop must be collected and disposed of in some other way. The waste from the primary loop is generally a very concentrated mixture of organic solids and inorganic nutrients. As noted previously, discharge of this waste directly to waterways will cause significant environmental degradation. Land application of freshwater waste from this loop is possible given the proper amount of land, the proper soil, a compatible cover crop, effluent storage capacity, and reasonable weather conditions (no prolonged rainy season). Disposal or treatment of waste from saltwater systems can be more difficult. Although little published information exists on the characteristics of sludge from marine systems, preliminary data from research conducted by the authors at NC State University suggests that waste from a production system operating at 24 ppt salinity has a chloride content in excess of 10 ppt. While some crops are salt tolerant, markets for these products are not expansive and could limit production. Clearly, more research into the characteristics and desalting, and further treatment of waste from marine production systems is required. Some who design commercial recirculating systems market their technology as proprietary in design. While there are some clever and sometimes unique components and innovative system designs, all successful systems have components that perform common and required unit processes. These processes include waste solids removal, biological filtration, oxygenation, and degassing of carbon dioxide. Remarkably, over the years (and even today) some systems being marketed as state-of-the-art, lack some of these

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components for these basic unit processes. However, the more successful systems have well-tested components that are sized correctly and placed within a system layout allowing for the energy-efficient movement of water. Therefore, as we investigate recent advancements in recirculating systems components and designs, a review of the basic unit processes and some of the typical components used in these processes will also be in order.

31.3

Types of particulate waste solids

Perhaps the most important process that must be accomplished in recirculating tank-based systems is the timely and efficient removal of waste solids. If waste solids are not removed from a tank as they are produced by the cultured organism, they are prone to degradation by physical and biological processes on-going within the tank. The biological degradation of particulate organic solids results in oxygen utilization and often ultimately in ammonia–nitrogen production. Waste solids are categorized into settleable, suspended, and dissolved. The reader is referred to Cripps and Bergheim (2000), Losordo and Westers (1994) and Chen et al. (1994) for a thorough review of these waste solids categories. However, in short, settleable solids are those that will congregate on the bottom of a tank, while suspended solids tend to be smaller with lower densities and remain in the water column under most culture conditions. Dissolved organic solids, which are more difficult to remove than the others, result from either a soft or liquid-like faecal pellet being excreted by the cultured species or the degradation of settleable or suspended solids within the system by mechanical sheer or heterotrophic biological activity.

31.4 Tank, water input manifolds, and drain design Culture tanks are categorized into raceways, tanks with flows from front to back, and tanks with circular flow. Tanks with circular flow are not always round in shape and can be square with rounded corners, hexagonal, or octagonal. The selection of tank shape and design is usually a function of the primary use of the tank. That is, while raceways can be more costly to build or buy than round tanks, they are often favoured for fingerling production, where subdividing the tank is possible and grading fish is easier with a simple linear bar grader. Conversely, round tanks are often favoured for on-growing fish to market due to reduced investment cost and ease of self-cleaning. Tanks for aquaculture are typically made of fiberglass, polyethylene, or concrete. In Europe, Hesy Aquaculture, B.V. (Bovendijk 35-Z, 2295 RV Kwintsheul, The Netherlands; www.hesy.com) has pioneered polyethylene tanks used within water reuse systems. Tanks manufactured by Hesy Aquaculture B.V. range in size from 1.9–10 m in diameter.

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In North America, steel panel tanks with a glass-fused surface have found their way into tank-based aquaculture. While previously pioneered by A.O. Smith Harvestore for agricultural silo construction (Engineered Storage Products Company, 345 Harvestore Drive, Dekalb, IL 60115, USA; www.engstorage.com), more recently these tanks have been used in largescale commercial aquaculture. The tanks are currently being marketed into the North American aquaculture industry by AquaCare, Inc. (P.O. Box 758, Bellingham, WA 98227, USA; www.aquacare.com). New to the North American market is a hybrid tank construction that combines a PVC form (that becomes the tank’s inner and outer surface) that has a reinforced poured concrete core. The inside PVC form provides a surface that is smooth and impervious to freshwater or saltwater intrusion. Marketed as OctaformTM (Suite 520, 885 Dunsmuir Street, Vancouver BC, Canada V6C1N5; www.octaform.com), tanks are fabricated on site and can be formed to create a large-scale raceway, round tank, square tank with corners, or octagonal tank. As noted before, a disadvantage of round tanks is the inefficient utilization of floor space. The inefficiency is most exaggerated in a system with multiple smaller tanks. This inefficiency has been overcome with the development of square tanks with rounded corners or tanks with an octagonal shape. An example of a tank system utilizing octagonal tanks has been developed and marketed by AquaOptima Norway AS (Brattøkaia 17B, 7010 Trondheim, Norway; www.aquaoptima.com). The tank systems, tradenamed Eco-Tank, are shipped flat-packed to aquaculture sites worldwide with the walls being assembled on site. Tank systems with volume from 10–200 m3 are available. Tank wall construction on tanks less than 10 m3 is foam-cored fiberglass panels with aluminium structural corners. Tanks greater than 40 m3 and larger are made from structural aluminium with the inside surface laminated with fiberglass. The octagonal tanks are sold in various combinations with shared walls. The tank layout can be tanks connected in a row, or some square or rectangular pattern (Fig. 31.1).

31.4.1 Raceway tanks Long rectangular tanks, referred to as raceways, have a long history in the farming of salmonids, particularly freshwater trout (Timmons and Summerfelt, 2007). Beginning as long earthen ponds, these production tanks evolved into concrete structures with vertical straight walls and, in smaller scale applications, rectangular fiberglass tanks and troughs. To withstand the water pressure exerted on straight walls, the structure requires significantly greater mass and reinforcement as compared to the design and construction of round tanks. As such, raceways and other tanks with straight walls may cost more to build or buy than round tanks. However, this may be offset by better utilization of space with a more efficient ‘footprint’ than round tanks.

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Fig. 31.1 Octagonal structural aluminum tanks with interior fiberglass liner grouped to maximize the utilization of floor space. (Photo provided courtesy of AquaOptima AS)

Traditional raceway designs with linear flow (inflow at one end and outflow at the other) require much higher flow rates to provide for selfcleaning as compared to circular flow tanks (Watten et al., 2000). To improve self-cleaning without major changes to raceway design or increased flow, researchers have investigated the use of moveable baffles. These devices move the length of the raceway, momentarily increasing the velocity of water at the bottom of the raceway at the location of the baffle (True et al., 2004). In systems being marketed today, to improve self-cleaning tank velocities, it is common to have tank turnover rates of four to six times per hour. Most of these units are designed with a ‘low head’ configuration (water is pumped with minimal pressure or elevation head), to reduce the energy requirement of moving large volumes of water. An example of this design philosophy can be found in a production system being marketed by a Danish company, INTER AQUA Advance A/S (Rosenholm Udviklingspark, Sortevej 40.DK-8543 Hornslet). In a marketing brochure (INTER AQUA Advance 2006; www.interaqua.dk), the corporation describes a large-scale aquaculture production facility that utilizes a compact design with side-by-side raceways having a low head recirculating water treatment scheme. The total production volume is 2100 m3 with a recycle flow rate of 8400 m3 per hour; hence, a four tank turnovers per hour recycle exchange rate. To overcome the need for high turnover rates or various types of baffles to achieve adequate self-cleaning velocities, research engineers have created various modifications in the design of water flow in raceways. Watten and

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Fig. 31.2 Cross-section of a ‘cross-flow’ raceway. Water flows in through an inlet manifold with jets and out through a similar drain manifold on the opposite side of the tank (after Colt and Watten, 1988).

Johnson (1990) developed a concept called cross-flow that was created as a modification for existing aquaculture raceway tanks. With an inflow manifold and outlet manifold placed at the bottom of the long sides of the raceway, the scouring velocities imparted to the flow across the bottom of the tank were shown to increase over tenfold compared with the velocities induced with the same flow applied at one end of the raceway (Fig. 31.2). More recently, Watten et al. (2000), with a similar goal of increasing selfcleaning velocities, developed a design that allows raceways to be operated with similar hydraulic characteristics to a tank with circular flow. The authors refer to the concept as the ‘mixed-cell’ raceway design. In this raceway modification, the rectangular tank is divided into equal individual square cells. A drain is placed in the center of each cell and vertical manifolds direct inflow jets of water such that a circular velocity is created in each cell of the raceway (Fig. 31.3). Most recently, the design was tested at a larger scale. Labatut et al. (2007) tested the hydraulic characteristics of three 30 m3 mixed cells which made up one 90 m3 raceway. This study determined that, with an exchange rate of 1.7 volumes per hour, the water velocities in the cells were in the optimum range to provide for self-cleaning of the cells and to promote good fish health and condition.

31.4.2 Circular flow tanks Round culture tanks are very common in land-based aquaculture. Today, tanks with diameters of greater than 10 m are becoming more common in the industry (Timmons et al., 1998). As noted previously, these tanks can be inexpensive to build, as the wall thickness and structural support in round tank design is a fraction of that required for a straight wall. In the past, the typical round tank had a single point of water entry to the tank at the

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Vertical manifold system for water distribution

Plan view

Circular velocity induced by manifold flow One central drain per raceway section

Raceway effluent

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Fig. 31.3 The mixed cell raceway design allows for circular flow in a raceway design. Central drains and vertical inlet manifolds provide multiple cells with circular flow in a rectangular raceway design.

surface and a single point water outlet in the center. The center drain has used either an internal standpipe (usually taking water from the surface) or an external standpipe that allowed for the water to be taken from any depth or at the bottom of the tank. Water commonly flows into the tanks tangentially from a single point at the surface side wall. This tangential flow creates a rotational water velocity in the tank. However, there are velocity profiles (differences in the velocity of the water in the tank and water that comes in contact with the surfaces of the tank), that are set up in the tank due to the interaction of the water molecules with the side walls and bottom surfaces of the tank. These velocity profiles, created by what is referred to as no-slip conditions at the tank surfaces, create a secondary flow within the tank that helps to move solids towards the center at the bottom of the tank. The secondary flow direction is outward from the center at the surface of the tank, downward at the side walls, and inward across the bottom of the tank towards the center. The combination of the rotational velocity and secondary flow causes solids deposited on the bottom to spiral towards the center of the tank. Tanks with circular flow easily provide self-cleaning velocities with fairly low turn over rates (0.75–1 tank volumes per hour). The reader should note that the self-cleaning benefits of the secondary flow velocities in a tank tend to decrease as the diameter to depth ratio of the tank approaches 5 : 1. A diameter to depth ratio of 3 : 1 is often considered as ideal, but may not be practical as the tank may become too deep to effectively work in during harvesting or sampling activities. As tanks have become larger in volume and in diameter, the inflow device (pipe) and drain

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design have become more important. In larger tanks, simple inflow pipes and drains may not promote the best tank mixing and water exchange. To meet this challenge, engineers have developed specialized inflow manifolds and double-drain outlet designs.

31.4.3 Water input manifolds Traditionally, water inlets to tanks are either an open pipe at the end (head) of a raceway or an elbow directing water flow tangentially at the side and surface of a tank with circular flow. This is not always the most effective way of introducing water to a tank. Designers of raceways and circular flow tanks have developed manifolds with multiple outlets to more evenly introduce water to a tank. As noted previously, cross-flow raceways are created by installing a water input manifold along the bottom of one side of the raceway with an outlet manifold at the other side. In circular flow tanks, however, water is introduced into the water column at various depths with a vertical manifold. A vertical manifold is a pipe extending from the surface of the water in the tank to the bottom where it is terminated with some form of closure; a cap or plug. Holes are drilled into the manifold, usually aligned vertically, spaced at approximately 10 cm on center. The outlet hole diameter is adjusted such that the head loss in the manifold is less than 30 cm. The manifold diameter is also sized larger than the water delivery pipe to reduce the water velocity (generally <1 m/s) to encourage equal flow of water from the top to the bottom of the manifold. An example of a commercially available vertical manifold is manufactured by AquaOptima AS (Fig. 31.4). The device, marketed as Eco-Flow, has a calibrated manometer tube that allows the user to estimate the flow through this outlet device.

31.4.4 Multiple drain systems Waste solids within a culture tank vary in size from heavier solids that tend to settle out of the water column quickly, to smaller and less dense solids that tend to remain suspended in the water column. These solids are referred to as settleable and suspended solids, respectively. Tanks with circular flow have traditionally had only one central drain. In this configuration, the settleable solids and suspended solids have been captured in the same flow stream and at the same time. Recognizing that settleable solids can be removed more quickly if taken separately, improvements in water quality have been realized with the development of double-drain systems. The original design for double drains included a small sump in the center of the tank with a small outlet from the sump and a large standpipe protruding vertically to remove water from the surface of the tank. In this configuration, approximately 80 % of the flow exits the tank via the surface drain and 20 % exits the smaller bottom sump

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R

Fig. 31.4 Vertical manifold for evenly distributing input water to an aquaculture production tank. The manifold shown has a calibrated clear manometer tube for measuring the flow through the manifold. Additionally, the height of the water in the manometer represents the head loss across the manifold. (Drawing courtesy of AquaOptima Norway AS)

drain. The smaller flow stream carries away a concentrated stream of the settleable solids. In an effort to further concentrate that flow, designs have been created to reduce this flow below 20 %. One notable design was created at the Norwegian Hydrotechnical Institute in Trondheim, Norway (Lunde et al., 1997). The double drain, referred to as a particle trap, removes the settleable solids from a circular flow culture tank with as little as 5 % of the water leaving the tank. As such, the concentration of solids in this waste stream tends to be higher than those in a larger flow stream, thus the solids removal device (usually some form of gravity settling device) can be smaller. Most recently, a novel approach to double drains has been pioneered by Dr Michael Timmons of Cornell University in the USA. Referred to as the Cornell dual-drain, this design employs a small center drain but removes the majority (80–90 %) of tank water from the side-box on the culture tank (see Fig. 31.5, Timmons and Summerfelt, 2007). This drain was developed

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Elevation view Center drain settleable solids flow 20 % of total Side drain suspended solids flow 80 % of total

Side drain suspended soids flow 80 % of total Center drain settleable solids flow 20 % of total

Fig. 31.5 The Cornell dual-drain, employs a small center drain but removes the majority (80–90 %) of tank water from a side box drain on the culture tank.

to overcome the negative consequences of the strong vortex that can develop around a single or dual drain located in the tank center when exchange rates exceed once per hour. Use of a dual drain (either central or side) provides for a smaller, more concentrated solids waste stream from the tank. As such, components should be smaller and less costly to capture solids in this smaller and more concentrated waste stream (Timmons et al., 1998).

31.5 Settleable solids capture components Early design work in the development of intensive tank-based aquaculture followed the precepts of wastewater engineering. As such, these early designs sometimes included settling basin technology in the water recycle process stream (Broussard and Simco, 1976; Miller and Libey, 1984; Provenzano and Winfield, 1987). While settling basin technology may be adequate for cool and cold-water systems, in warm-water systems (temp > 25 °C), the degradation of sludge within the settling basins usually causes the sludge to float (gas accumulation) and ammonia–nitrogen and nitrite–nitrogen can be released to the recycle flow-stream. To improve performance of recirculating systems, designers have implemented the double-drain technologies described above, having the smaller more concentrated flow stream directed to a smaller settling unit. Two designs have been popular; swirl separators and radial flow settlers.

31.5.1 Swirl separator Swirl separators, sometimes referred to as hydrocyclones, operate by introducing water tangentially at the sidewall of a conical tank (Fig. 31.6). As in circular tank flow described above, the primary rotation inside the tank

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Clarified effluent

Elevation view Influent

Waste sludge

Fig. 31.6 Swirl separator that works in conjunction with a double drain to settle solids from the smaller more concentrated wasteflow stream (after Hobbs et al., 1997).

and no-slip conditions create a secondary radial flow towards the center (Davidson and Summerfelt, 2004). Swirl separators are usually used to remove heavier solid wastes from a flow-stream before entering a second device to remove smaller or lighter solids. Multiple tanks can flow to one larger swirl separator or each tank in a system can be fitted with a smaller swirl separator. The small size of these devices, as compared with settling basins, allows for the settled solids to be wasted more often in a smaller flow-stream, thus saving water. By disposing of the solids more often, the operator can eliminate or reduce floating sludge and the associated nutrient releases. In the study of a single largescale tank system by Davidson and Summerfelt (2005), the associated swirl separator was found to retain 37 % of the solids that entered the unit. In an earlier study, Twarowska et al. (1997) reported the mean removal efficiency for four swirl separators connected to individual particle traps in four fish culture tanks to be 80 %. Veerapen et al. (2005) studied changes in swirl separator efficiency with changes in design. The authors determined that swirl separators were mainly gravity-driven; thus low inlet velocities achieved higher solids removal efficiencies. Additionally, this study showed that outlet design was very important, with a center disc drain functioning

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Wasted sludge

Fig. 31.7 A radial flow separator may be used to improve solids removal efficiency from the central drain of a dual-drain tank system (after Davidson and Summerfelt, 2005).

much better than a single outlet or cut-away pipe section (a pipe with the top half cut away spanning the device). 31.5.2 Radial flow separators In an effort to develop a more efficient means of removing settleable solids from double-drain systems, Davidson and Summerfelt (2005) compared the solids removal characteristics of a radial flow settler to those of a more traditional swirl separator. Radial flow separators, also referred to as center feed sedimentation basins, were originally used at the large-scale in domestic wastewater. In this design, water enters the center in an upwards flow direction, but must exit below the input location due to a circular central baffle (Fig. 31.7). Water exits via a peripheral weir much like, if not identical to, those employed in the swirl separator design. In this study, the authors determined that the radial flow separator was more efficient at removing solids than the swirl separator. The authors noted that removal efficiency with the swirl separator was strongly (positively) correlated to increasing influent solids concentration. The highest removal efficiency was just less than 70 % with influent solids concentrations of 25–30 mg/L. This was not the case with the radial flow separator, where the removal efficiency (70–80 %) was not correlated to the influent concentration. As such, this study appears to point to the radial flow separator as the more reliable design choice.

31.6 Suspended solids capture components In tank systems with single drains, all of the solids, settleable and suspended, exit the tank together and usually travel to a solids removal device. When

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using systems with dual-drain configurations, the clarified flow (after removal of the settleable solids, described above) and the larger flow carrying suspended solids will re-combine and enter the suspended solids removal component. Components used today for this process fall into two general categories; screen filters and expandable bed filters.

31.6.1 Screen filters Screen filters utilize some form of fine mesh material, usually stainless steel or polyester, through which effluent passes while the suspended solids are retained on the screen. Solids are usually removed by rotating the clogged screen surface past high-pressure jets of water. These waste solids are carried away from the screen in a small stream of waste water. Two types of screen filters are used in aquaculture; drum screen filters and incline belt filters. Drum screen filters The most common screen filter is the drum screen filter (Fig. 31.8). In this configuration, water enters the open end of a drum and passes through a screen attached to the circumference of the drum. In most systems, the drum rotates only when the filter mesh becomes clogged with solids, and

Wasted sludge

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Clarified flow to culture tanks Waste effluent Inflow from culture tanks

Fig. 31.8 Typical drum screen filter (shown with a cut-away and expanded mid-section) for waste solids removal from aquacultural recycle flow streams. (Drawing provided by and used with permission of PRA Manufacturing, Nanaimo, B.C.)

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high-pressure jets of water (located outside of the drum) wash the solids off the screen and into an internal collection trough leading to a waste drain. Drum screen filters are made of various combinations of stainless steel and fiberglass. A wide variety of sizes of drum screen filters are commercially available worldwide including, but not limited to, those manufactured by Hydrotech (Hydrotech AB, Mejselgatan 6, 235 32 Vellinge, Sweden; www. hydrotech.se), PR Aqua (PR Aqua, 1631 Harold Road, Nanaimo, British Columbia, V9X 1T4, Canada; www.praqua.com), Atlantech Systems and Equipment (Atlantech Systems & Equipment, 89 Hillstrom Ave, West Royalty Business Park, Charlottetown, PEI, Canada, C1E 2C8; www. atlantech.ca), and the Faivre Company (7 rue de l’Industrie, 25110 Baume-les-Dames, France; www.faivre.fr). Properly designed commercially available units have been shown to be a reliable means of removing solids greater than 40 microns in size. With screens much smaller than 40 microns, excessive amounts of backwash water would be required to keep the filters from clogging. Drum screen filters with mesh sizes of between 40 and 60 microns will utilize on average 7.5–15 % of the system volume per day for backwashing. Incline belt filters Incline screen filters are also referred to as belt filters. These units resemble conveyor belts placed on an incline (Fig. 31.9). Water passes through the screen where suspended solids are retained and lifted out of the water on the inclined screen. The solids are either scraped off or sprayed off with high-pressure water in a cleaning process similar to that of the drum screen filters. Large-scale incline belt filters are manufactured by Hydrotech AB in Sweden, Atlantech Systems and Equipment in Canada, and Salsnes AS, in Norway (Salsnes Filter, PO Box 279, 7801 Namsos, Norway; www.salsnesBelt screen filter

1

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10 8 Waste sludge

3 Front

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Fig. 31.9 Side view of an incline belt filter manufactured by Salsnes Filter AS. (Drawing reproduced with permission from Salsnes Filter AS)

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Fig. 31.10 Incline belt filters treating recycled system water in a large scale barramundi production facility. (Photo provided by AquaOptima AS)

filter.no). These types of filters were initially used in aquaculture to capture and concentrate solids in the waste stream of drum screen filters. More recently, they have been used as replacements for drum screen filters within recirculating systems. An example of this can be found in a very large-scale barramundi (Lates calcarifer) production facility designed by AquaOptima Norway in the UK. The system, owned and operated by the AquaBella Group plc. (Aquabella Group plc, 53 Lafone Street, London SE1 2LX, UK), utilizes 12 large incline belt filters to process the recycled system water (Fig. 31.10). An example of a smaller scale use of incline belt filters has been demonstrated by Cell Aquaculture in Australia (Cell Aquaculture Ltd, PO Box 251, South Fremantle, Western Australia 6162; www.cellaqua.com). These recirculating production systems are also designed to raise warmwater fish such as the barramundi. The system is made up of groups of smaller circular tanks with effluent flowing to a single small-scale incline belt filter for solids removal. Large production systems are created by installation of multiple smaller production systems. 31.6.2 Expandable bed filters Expandable bed filters remove solids by passing water through a bed of granular media. While sand filters have been used for years in other industries, they have not proven successful for aquaculture due to the excessive volume of water required to backwash a typical sand filter. Additionally, the concentration of biologically active solids often causes beds of sand to clog and become ‘gelled’ with bacteria. Since the late 1990s, various types of plastic media have been used in the development of these types of filters for the aquaculture industry. These filters differ not just in the shape of the

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media but, more importantly, in how they are backwashed and solids removed. The following is a brief description of the latest expandable media filter technologies. The development of floating bead filters has been pioneered by Dr Ronald Malone at Louisiana State University and has been well documented in the literature (Malone et al., 1993, 1996, 1998; Malone and Beecher, 2000). This upflow filter technology has been under development since the 1980s. While the basis for the design of each filter is a bed of floating plastic beads, the filters differ mostly in how the bead bed is expanded and backwashed. The two original designs utilized plain round beads that were backwashed when agitated by a mixing propeller or mixing the beads with bubbles of air while draining the beads through a washing ‘throat’, or narrowing of the bottom of the bead chamber. These filters are referred to as ‘prop-washed’ bead filters and ‘bubble-washed’ bead filters, respectively. In both filter designs, flow to the filter has to be stopped and a settling period provided as part of the backwashing cycle. In the most recent design, the bead media are backwashed by rapid air injection and mixing into the bed with a concurrent vertical displacement or dropping of the bed. Initially these filters were referred to as ‘drop’ filters. Currently, however, these filters are marketed under the name ‘PolygeyserTM’ by several manufacturers. Aquaculture Systems Technologies, LLC (108 Industrial Avenue, New Orleans, LA 70121 USA; www.beadfilters.com) produced the first, small, commercially available unit which contains 85 l of bead media (Fig. 31.11).

Fig. 31.11

First commercially available PolygeyserTM bead filter. (Drawing courtesy of Aquaculture Systems Technologies, LLC)

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Currently, larger units are being manufactured. Examples of these larger Polygeyser filters are manufactured by International Filter Solutions (International Filter Solutions, Inc., 13636 W IH-10. Marion, TX 78124, USA; www.ifsolutions.us). IFS manufactures Polygeyser filters with bead media volumes of up to 1.41 m3. The advantages of the Polygeyser design over previous floating bead filters include: (i) no interruption of flow to the filter during backwashing, and (ii) no time is required to settle the waste during the backwashing cycle. Wasting of the sludge from the bottom of the filter is independent of the backwashing cycle, thus, backwashing can be done as often as needed to keep the head loss across the filter to a minimum. As such, this filter can be run with a low head pumping system, including airlift pumps. A drawback to this and other floating bead filter designs is that some waste solids are released to the culture tank after backwashing unless a significant volume of water is passed through the bead bed and diverted away from the flow to the tank.

31.7 Biological filtration In recirculating aquaculture production systems, ammonia–nitrogen comes from two main sources: the cultured species and microbial processes. The primary source of ammonia is from excretion by the cultured species as a result of the metabolism of feeds. Additionally, but to a lesser extent, the aerobic heterotrophic oxidation of organic matter contributes to the ammonia production within RAS (Henze et al., 1995). Using biological filters in recirculating aquaculture production systems, the concentration of ammonia–nitrogen can be maintained at acceptable limits through the nitrification process. Biological nitrification, the primary process that occurs within biological filters, is an aerobic process by which autotrophic nitrifying bacteria oxidize ammonia–nitrogen (NH3 + NH4+–N) to nitrate–nitrogen (NO3–N) with nitrite–nitrogen (NO2–N) as the intermediate component. The general half-reaction equations (Eqs 31.1 and 31.2) show the basic conversions occurring in autotrophic nitrification (US-EPA, 1984). When the dissolved and particulate organic concentration to ammonia–nitrogen ratio (COD/N) is high enough, ammonia–nitrogen may also be consumed when organic waste products are assimilated by heterotrophic bacteria within the culture system and converted directly into microbial biomass (Ebeling et al., 2006; Schneider et al., 2007). In most recirculating systems described in this chapter, the production systems are designed to keep the water as clean as possible, thus significantly limiting the heterotrophic activity within the system. Ebeling et al. (2006) derived Eqs 31.3 and 31.4 through stoichiometry to predict the autotrophic nitrification process summary and heterotrophic removal of ammonia-nitrogen in aerobic systems, respectively.

Recirculating systems for finfish production NH +4 + 1.5O2 → 2 H + + H 2 O + NO2− − 2

NO + 1.5O2 → NO

− 3

963 [31.1] [31.2]

NH +4 + 1.83O2 + 1.97 HCO−3 → 0.024C 5 H 7 O2 N + 0.98NO−3 + 2.90H 2 O + 1.86CO2

[31.3]

NH +4 + 1.18C 6 H12 O6 + HCO−3 + 2.06O2 → C 5 H 7 O2 N + 6.06 H 2 O + 3.07CO2

[31.4]

CO2 + H 2 O ↔ H 2 CO3 ↔ H + + HCO3

[31.5]

There are some important points that should be observed with reviewing the equations listed above. These include that the nitrification process consumes both oxygen and alkalinity in the water. For every gram of ammonia– nitrogen converted to nitrate–nitrogen in the nitrification process, 4.18 g dissolved oxygen (DO) and 7.04 g alkalinity as CaCO3 are consumed. Additionally, 0.19 g of cellular biomass (C5H7O2N) and 5.85 g carbon dioxide (CO2) are produced. The first step (Eq. 31.1) of the nitrification process produces hydrogen ions (H+). Once in solution, CO2 maintains equilibrium with carbonic acid (H2CO3) (Eq. 31.5). The combination of these two processes effectively makes nitrification an acid-producing process lowering the pH of the system water. The ‘take home’ message here is that the operator of recirculating production systems must continuously monitor pH and alkalinity with the understanding that some source of alkalinity must be added back to the system water. If this is not done, the pH will decline to a point that nitrification will cease. Nitrification can be measured and quantified by the rate of removal of total ammonia nitrogen (TAN), the sum of NH4+ and NH3, from a system (Zhu and Chen, 1999). The oxidation of ammonia effectively reduces the TAN concentration, part of which is toxic to fish (un-ionized ammonia; NH3) (Meade, 1985), thereby providing a means for reuse of system water within a production system. To maintain a constant concentration of TAN within the production system, the biofilter must be sized to remove TAN from the system at a rate equal to the production of TAN within the system. Changes to and within the system will affect water quality parameters and, potentially, the ability of the biological filter to remove TAN. Operational changes that may influence the performance of a biological filter include harvesting, changes in feed quality and quantity, and changes in mechanical filtration. The diurnal variability in feeding creates variability in waste production which affects the system water quality (Eding et al., 2006). Although feeding regimes vary by species, more constant feeding schemes can be used to reduce variability in the waste production and maintain more consistent water quality conditions. As a rule of thumb, the rate of TAN excretion (creation) is approximately 3 % (by weight) of the daily feed rate (Wheaton et al., 1994). Higher protein feeds will create more TAN. As feed inputs and/or protein content change to maximize growth

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rates, the TAN production rate in the system will also increase. As such, the rate of inputs such as oxygen and some form of alkalinity will be required to maintain proper oxygen concentrations within the filter and culture system and buffer the pH to avoid decline. To reduce the amount of alkalinity required for pH buffering, a degassing tower may be employed for the removal of CO2 from the system water.

31.7.1 Quantifying the nitrification rate Perhaps one of the most important criteria to measure and report in comparing biofilters is the removal rate of TAN from system water. In the past, many studies discussed and reported nitrification rates based upon the media specific surface areas (SSA) (Zhu and Chen, 1999, 2001; Ling and Chen, 2005; Chen et al., 2006; Eding et al., 2006). Biofilter media has long been valued by its inherent specific surface area, with value placed on the highest SSA. The theory behind such a valuation is that the greater the SSA, the more bacteria living space and this ideally translates into the higher nitrification rates. In actuality, the bacteria create a biofilm that effectively covers the media, possibly layering over structural and topographical features of the media designed to increase surface area. This covering of the media topography essentially creates new media topography, reducing the actual media surface area used by the bacteria. Thus, in the past, the theoretical nitrification capacity of a particular filter media has not always been reflective of the actual nitrification rates achieved by biofilters in real-world use. Recently, it has been suggested that biofilter nitrification rates be measured and reported based upon ammonia removal per unit of unexpanded biofilter media volume (Malone and Beecher, 2000; Drennan et al., 2006; Malone and Pfeiffer, 2006). Referred to as the volumetric TAN conversion rate, or sometimes the volumetric nitrification rate, this measures the rate at which TAN is removed from the bulk solution through the biological filter and converted to either nitrite- or nitrate-nitrogen. Typical units for this standard measure of nitrification are g TAN removed per cubic meter of biofilter media per day (g TAN/m3/d). As noted above, un-ionized ammonia nitrogen is toxic to most aquatic organisms. The concentration of un-ionized ammonia–nitrogen becomes even more important as the designers move to develop marine recirculating systems. Saltwater species, in many cases, are more sensitive to un-ionized ammonia–nitrogen than many commonly cultured freshwater species (Person-Le Ruyet et al., 1995). Thus the concentration of ammonia– nitrogen should be kept lower for some species (marine) than others (freshwater). It is widely understood that the rate of nitrification in biofiltration is greatly influenced by a number of water quality variables, most importantly, the concentration of TAN that is present in the filter, water temperature, and the organic carbon concentration in the system water.

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31.7.2 Effect of total ammonia nitrogen (TAN) concentration Under most aquaculture conditions, where the TAN concentration is below 2–3 mg/L, there is a linear relationship between the TAN removal rate and TAN concentration (Ling and Chen, 2005). Above 2–3 mg TAN/L, there is usually no further change in the rate of nitrification. The slope of the linear relationship and peak of the TAN removal rate are filter media and system water quality specific. As noted before, the level of dissolved organic carbon in the water is an important factor in determining the biofilter nitrification performance.

31.7.3 Effect of organic carbon concentration Organic carbon is derived from solid waste excreted by fish and also results from the degradation of uneaten feed and dead bacteria (Leonard et al., 2002; Steeby et al., 2004). Heterotrophic bacteria primarily degrade and metabolize dissolved and particulate organic matter. Autotrophic and heterotrophic bacteria occupy the space on biological filter media, thus creating a biofilm. The sharing of media surface causes competition for nutrients and oxygen between the two types of bacteria, resulting in a stratified biofilm structure (Nogueira et al., 2002). Faster growing heterotrophic bacteria ultimately occupy the outer layer of the stratified biofilm, where substrate concentration and detachment rates are both higher, resulting in the slower growing autotrophic nitrifying bacteria occupying the inner layer of the biofilm (Ohashi et al., 1995; Satoh et al., 2000; Nogueira et al., 2002; Lee et al., 2004; Chen et al., 2006; Michaud et al., 2006). Heterotrophic bacteria have been shown to have a maximum growth rate five times greater than that of autotrophic bacteria (Zhu and Chen, 2001). By covering the nitrifying bacteria, heterotrophic bacteria inhibit the diffusion of nitrogenous substrate and dissolved oxygen to the autotrophic nitrifying bacteria, thus negatively affecting the rate of nitrification (Nogueira et al., 2002; Chen et al., 2006). Higher levels of dissolved organic carbon-containing compounds result in decreased nitrification rates and associated undesirable affects on water quality (i.e., higher TAN concentrations). The biomass and growth of heterotrophic bacteria increase on biofilter media as the organic carbon to ammonia–nitrogen (C/N) ratio increases (Zhu and Chen, 2001; Leonard et al., 2002; Nogueira et al., 2002; Ling and Chen, 2005; Michaud et al., 2006). Zhu and Chen (2001) demonstrated a 70 % reduction in TAN removal rate at C/N = 1.0 as compared to C/N = 0. Heterotrophic bacteria concentrations are also linked to the amount of organic matter contained within the overall system. Faeces, uneaten feed particles, and other sources of organic carbon should all be removed from the process flow prior to entering the biological filter. Leonard et al. (2002) found that the most effective means of eliminating heterotrophic bacteria from the system was to remove the particulate organic matter (POM) by

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way of mechanical filtration. Failing to remove the POM from the waste stream before the biological filter will result in accumulation in the biofilter, and heterotrophic bacterial growth will increase and successfully outcompete the autotrophic bacteria on the biofilter media (Leonard et al., 2002). Placement of the mechanical filter directly before the biological filter will result in maximum POM removal, ultimately improving the biological filter nitrification rate.

31.7.4 Effect of temperature on nitrification The influence of temperature on nitrification in a fixed biofilm is more complicated than that of a suspended growth (Zhu and Chen, 2002). In a suspended growth reactor, higher biological reaction rates are observed as the system reaches an optimum temperature, above which rates decrease due to the denaturing of enzymatic proteins (Sawyer et al., 1994; Henze et al., 1995). Addressing the effects of temperature on a fixed film bioreactor is much more difficult. Zhu and Chen (2002) demonstrated that diffusional mass transport is of primary importance to the nitrification rate in a fixed film. Their study showed that the effect of temperature on nitrification in a fixed film biofilter was less significant than previous studies had estimated. Dissolved oxygen may become a limiting factor in higher temperature systems due to the diffusion process limitation of oxygen flux into the fixed biofilm (Zhu and Chen, 2002). As noted above, in stratified biofilms, competition for DO exists between the inner layer of autotrophic nitrifiers and outer layer of heterotrophic bacteria. The DO saturation concentration in water decreases as temperature increases. Thus, Zhu and Chen (2002) determined that in a fixed film biofilter oxygen limitation usually has more impact and that at temperatures between 14 and 27 °C, temperature variation does not significantly change the nitrification performance of a biofilter. Similarly, Ling and Chen (2005) also showed that the TAN removal rate was not significantly affected by temperature in cool water aquaculture systems (15 and 20 °C). Perhaps the most important message to take away from the above discussion is that attention should be paid to organic carbon concentration build-up in recirculating system water. Since commercial RAS operates at low TAN concentrations and high feed rates, the resulting organic loads play a much larger role in biofilter performance than temperature or almost any other water quality parameter. There are many biological filter options available to designers of recirculating systems. Many of these, such as trickling filters and fluidized bed filters, are well known and well defined. The reader is referred to Eding et al. (2006) for a complete review of trickling filter design and Summerfelt (2006) for fluidized sand bed filter design. A brief description follows of three types of biological filters that have been developed or have become widely used in aquaculture since the late 1990s.

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31.7.5 Floating bead filters Floating bead filters have been described in Section 28.6.2. Referred to by Dr Malone as ‘bio-clarifiers’, these filters have been shown to provide both waste solids capture and nitrification as a biofilter. When used as a bio-clarifier, Malone and Beecher (2000) suggests a design volumetric nitrification rate (rate of ammonia conversion to nitrate based on the volume of the biofilter media) of 140–350 g TAN/m3/d. Drennan et al. (2006) suggests a design nitrification rate for bead filters with ‘enhanced’ media of 530 g/m3/d as readily achievable. Enhanced media is a crushed form of beads that have additional surface area created due to the deformation of the bead. Approximately 3–4 % (depending on the feed protein content) of feed becomes ammonia–nitrogen in recirculating systems. If we use 3.5 % of the feed rate for an average ammonia–nitrogen production rate, then we can estimate that 1 m3 of floating bead media should be able to remove solids and ammonia created by approximately 15 kg of fish feed (0.53 kg TAN ÷ 0.035 kg TAN per kg feed = 15.14 kg feed). The reader should note that these numbers have been developed in systems with fresh water. Saltwater systems may be less (see below). The attraction of floating bead filters is the simplicity and relatively small ‘foot-print’ of the component. Having solids removal and nitrification in one compact unit is attractive. Part of the facility design process needs to factor in both purchasing and operating costs. The PolygeyserTM bead filter design offers the desirable attributes of low water loss when wasting sludge and low energy use in pumping water through the filter. 31.7.6 Moving bed filters The moving bed reactor process for wastewater treatment, including nitrification, was developed at the Norwegian Institute of Technology, Center for Scientific and Industrial Research in Trondheim, Norway (Odegaard et al., 1991). Originally developed for use in domestic and industrial wastewater treatment, this technology has found its way into use in the aquaculture industry worldwide. Moving bed filters are made up of thousands (sometimes hundreds of thousands) of small structured media (Fig. 31.12). With a thin coating of bacteria, the media is just slightly negatively buoyant. Placed within an open top reactor, the media is mixed vigorously with air or water. While claims for nitrification rates of up to 2 g TAN/m2/d exist, no data or evidence has been presented in the peer-reviewed literature to substantiate these rates. In fact, while these units are widely used, little has been published with regard to nitrification rates in freshwater aquaculture for this reactor type. AnoxKaldnes, Inc. (Providence, RI 02903, USA) in Drennan et al. (2006) suggests that for an influent concentration of 1.5 mg TAN/L, and an estimated reactor effluent concentration of 0.9 mg TAN/L, a design VTR (volumetric TAN removal rate) for Kaldnes media would be 604 g TAN/m3/d. Water Management Technologies, Inc. (Baton Rouge, LA 70896, USA) in Drennan et al. (2006) suggests using a design volumetric

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Fig. 31.12 Moving bed media and filters come in many shapes and sizes. The top section of the photo above is a rectangular moving bed, agitated by air. The lower section shows three types of commercially available media. (Photo by Marc Hall, NC State University)

nitrification rate of 400 g TAN/m3/d. Tal et al. (2003) characterized the microbial community and nitrogen transformation processes associated with moving bed bioreactors in a closed recirculating mariculture system. The authors reported a maximum nitrification rate of 378 g TAN/m3/d. Based on this information, and until more data are presented in the literature, a maximum nitrification of 500 g TAN/m3/d would be reasonable to expect in fresh water. As such, moving bed filters should be able to process the ammonia generated by approximately 14.3 kg of feed per m3 of (nonexpanded) media bed. Key advantages of the moving bed reactor are its compact size, low energy requirements, and stable nitrification process. The filter component is generally run with very little head loss across the filter (usually 10–20 cm). Typical installations will have effluent being clarified by some form of screen filter with the water flowing by gravity to the moving bed filter. Water is then pumped from the moving bed to the next process, usually oxygenation and/or UV sterilization. While some commercial providers offer standard moving bed units (Multivis Waterbehandeling B.V., J.P. Santeeweg 1, 9312 PB Nietap, The Netherlands, www.multivis.nl; Water Management Technologies, Inc., PO Box 66125, Baton Rouge, LA 70896 USA, www.w-m-t.com; Aquatic Eco-

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Systems, Inc., 2395 Apopka Boulevard, Apopka, FL 32703 USA, www. aquaticeco.com), a large number of vendors offer the various moving bed medias as well as custom made filters worldwide. 31.7.7 Microbead filters Microbead filter technology is finding its way into the aquaculture industry in North America and Australia. Micro-bead filters were first described and evaluated for aquaculture by Greiner and Timmons (1998) and later applications were described in Timmons et al. (2006). The microbead filter combines the operational characteristics of trickling filters and bead filters into what can best be described as trickling bead filters. The biofilter media are highly buoyant polystyrene beads that are 1–3 mm in diameter and have a bulk density of 16 kg/m3. Water, filtered to remove particulate solids, is distributed over the top of the bed of beads. The water trickles through the beads and accumulates beneath the bed. The bed of beads floats on the treated effluent and is generally less than 45 cm deep and not more than 1.25 m in diameter. Fig. 31.13 is a drawing of a generic microbead filter design.

Red. bush’g

6’-5”+

Poly tank Blower Check valve Union Red. bush’g Water level

Spray diffuser Orifice plate Mist eliminator

Micro bead media

Red. bush’g Spraybar

Stand Side box Submersible pump

Fig. 31.13

Drawing of a generic microbead filter. (Courtesy of JLH Consulting, Courtenay, BC, Canada, 2004)

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At the time of this writing, only one line of commercially pre-built microbead filters is available. Holder-Timmons, Inc. (606 Evergreen Ave. Courtenay, BC, Canada V9N7N5; www.holdertimmons.com) offers a ‘proprietary floating microbead filter’ referred to as the HTE BiofilterTM. While no other commercial ‘off-the-shelf’ units are available, numerous research facilities and some commercial farms in North America have utilized this technology with on-site fabricated components. Greiner and Timmons (1998) estimated that the specific surface area of the microbead filter media was 3936 m2/m3. From the data presented by these authors, the area nitrification rate of this type of media at TAN concentrations of 1.5 mg/L would be approximately 0.25 g TAN/m2/d. As such, a volumetric nitrification rate of approximately 984 g TAN/m3/d could be expected. In warm-water commercial systems, volumetric nitrification rates of 1000 g TAN/m3/d have been verified (Timmons et al., 2006). With the same assumptions as above, we would expect the microbead filter to be able to process the ammonia generated by approximately 28.5 kg of feed per m3 of (non-expanded) media bed.

31.8 Oxygenation components and processes Unlike tank designs, solids removal components, and biofiltration components, there have not been any great leaps forward in oxygenation technology in intensive tank-based aquaculture since the late 1990s. As such, this section will provide a brief review of the most common technologies used by the industry and refer the reader to the most current publication(s) on the subject matter. Oxygenation systems can be broadly categorized by the pressure at which they function. High-pressure systems, those that inject oxygen at greater than 1.5–2 bar (20–30 psi), have been used widely in the freshwater trout industry. Tank-based recirculating technology has utilized either medium-head or low-head technology.

31.8.1 Oxygen cones Perhaps the most common medium head (pressures to 1.5 bar; 20 psi) component used in the industry is the down-flow bubble oxygen contactor, also referred to as the oxygen cone (Fig. 31.14, Summerfelt et al., 2000). The oxygen cone of today, usually made of fibreglass, is the ‘descendant’ of the steel (usually stainless) Speece Cone developed for the water and wastewater industry in the early 1970s (Speece et al., 1971). Due to the fibreglass construction, pressure within the unit is kept below 1.5 bar. Commercial units are manufactured with the capacity to transfer 0.2–4.9 kg O2/h at flow rates of up to 2300 L/min and outflow DO concentrations of up to 25 mg/L (Timmons and Vinci, 2007). Commercially produced oxygen cones

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Fig. 31.14

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Commercially available fiberglass oxygen cone. (Photo courtesy of Aquatic EcoSystems Inc.)

are available worldwide. A few examples of these oxygen cones are available from PR Aqua, Water Management Technologies, Inc., Aquatic EcoSystems, Inc., and Marine Biotech, Inc. (54A West Dane Street, Beverly, MA 01915 USA; www.marinebiotech.com) in North America. Similarly, examples of these components are available in Europe from companies such as UNI-Aqua A/S (Teknikervej 14, 7000 Fredericia, Denmark; www. uni-aqua.com), Oxymat A/S (Fasanvej 18–20, DK-3200 Helsinge, Denmark; www.oxymat.dk), and AquaOptima Norway AS.

31.8.2 Low head oxygenator (LHO) While oxygen cones are effective and efficient at dissolving oxygen to water, supersaturating the water with oxygen in this manner results in extra energy

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costs. To pump large volumes of water at pressures of up to 2 bar requires a significant energy input via water pumps. The low head oxygenator (LHO) was originally developed to provide pure oxygen to the flow between raceways in the trout industry. These raceways had small elevation drops between each raceway. In essence, the LHO provided pure oxygen additions where before there was only a waterfall type aerator. Created and first described in a patent by Watten (1989), the LHO breaks the process water flow into multiple sections or chambers. The water flows onto a distribution plate (a platform with numerous holes) where it falls as droplets through to multiple chambers below and is exposed to a highly oxygenated atmosphere. Oxygen gas passes from chamber to chamber and, as oxygen is dissolved into the water, the concentration of oxygen gas in the chambers declines. As such, the oxygen concentration exiting the last chamber is low and usually discharged below the surface of the water through a small pipe or hose. The original design of the LHO configured for raceway use can be seen in Fig. 31.15. LHOs are particularly well suited for oxygenating large flows of water to oxygen concentrations not greatly exceeding saturation. Wagner et al. (1995) determined that an LHO could raise the oxygen concentration of water at 17 °C to 12–13 mg/L with an oxygen absorption efficiency of 70–90 %. More recent LHO designs have been developed for use in recirculating tank-based systems. These LHO components sit within a basin or pumping

Water in low oxygen

Oxygen in

Off gas out

Water level

Water out high oxygen

Fig. 31.15 Low head oxygenator design for raceway applications. The front plate has been removed in this drawing to reveal the internal sections (after Losordo, 1997).

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sump rather than between tanks. Water from the recycle flow stream is pumped or flows by gravity to the top of the LHO, then enters the basin or pumping sump. These LHOs can be square, rectangular, or round in design. LHOs are licensed for manufacture in North America by PR Aqua, Water Management Technologies, and Aquaculture Systems Technologies.

31.9 Sterilization components and processes Under intensive culture conditions, viruses and bacteria leading to disease outbreaks can become a serious problem in recirculating production systems. Ultraviolet light and ozone have become the two most popular methods to control pathogenic organisms.

31.9.1 UV light Ultraviolet (UV) light has become the process of choice in aquaculture when water sterilization is desirable or required. The reader should note that there is a significant difference between sterilization for water disinfection and sterilization for pathogen control. In many cases, the latter may be possible and desirable while the former may not be economically viable. A primary requirement for UV light to work effectively is that the water should not have significant turbidity. As such, UV light should be applied where the water will be most free of particulate solids. It should be noted that if a significant suspended solids load is present in all locations in the process stream, UV light will not be effective for this application (Liltved and Summerfelt, 2007). While the effect of the UV dose is heavily influenced by the turbidity of the water, all things being equal, the lethal dose also depends on the target organism. Liltved and Summerfelt (2007) provide a comprehensive review of the dose of UV radiation required to kill bacteria, virus, mould spores, and yeasts. UV sterilizer units are marketed to provide a given dose of UV light at a given flow rate. Typically, manufacturers market units that have an effective dose of 15 000–30 000 micro-Watt seconds per square centimetre (μ-Ws cm−2). It is important to note that any effective dose can be obtained by reducing the flow rate through or past the UV unit. That is, the dose can be doubled by halving the flow rate. The most common type of UV component is the pressurized ‘tube in shell’ unit where the water flows into the unit pipe or shell and past the UV light or lights that are encased in a quartz tube or sleeve. These units, sold worldwide, are made from materials ranging from plastic to stainless steel depending on the pressure requirements of the system and the liquid being processed. Examples of a broad line of commercial stainless steel UV sterilizers are those marketed by ITT-Wedeco (WEDECO AG, Boschstrasse 4, 32051 Herford, Germany; www.wedeco.com). Similarly, Emperor Aquatics, Inc. (2229 Sanatoga Station Road, Pottstown, PA 19464 USA; www.

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Fig. 31.16 Examples of the traditional tube in shell pressurized design (left) and more recent drop-in open channel UV units that are now available to the industry. (Drawing and photo provided by Emperor Aquatics, Inc.)

emperoraquatics.com) produces stainless steel and plastic units that have been marketed widely to the aquaculture industry in North America. A recent innovation since the late 1990s has been the development of the open channel UV unit. First developed as a grid of UV lights that could be submerged in tanks or troughs, the design has migrated to a drop-in unit that is installed from the top in an open channel. Both designs are shown in Fig. 31.16.

31.9.2 Ozone contact Ozone, while used widely in aquaculture to clear the water and reduce the organic load on the biofilter, is not often used in commercial recirculating aquaculture production systems for sterilization purposes. This is mainly due to the fact that it is difficult to maintain the required residual concentration for an adequate period of time in an aquaculture treatment system to assure sterilization (Liltved and Summerfelt, 2007). According to Wedemeyer (1996), disinfecting water requires that a residual concentration of 0.1–2.0 mg/L be maintained in a contact chamber for 1–30 min. The variation in residual concentration and length of contact time is microorganism specific. Keep in mind that the ozone demand of the organic constituents and nitrite in the water must be overcome (oxidized) before a residual concentration can be maintained. In recirculating systems, the ozone demand will vary according to the concentration of dissolved and particulate organic solids that are present in the water and with the rate of

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feed additions. Higher feed rates and more dissolved and particulate organic solids will create a higher ozone demand that must be overcome. Liltved et al. (2006) noted that proper ozone applications will reduce heterotrophic bacterial populations, improve the removal of colloids and smaller particles by other filtration components, increase the biodegradability of organic compounds and increase the oxygen content of the treated water. Liltved and Summerfelt (2007), based on previous studies by Bullock et al. (1997) and Brazil (1996), and experience with commercial aquaculture recirculating systems provides as a ‘rule of thumb’ that water quality can be improved and fish health maintained with ozone additions of 13–24 g per kg of feed added to the system. Sharrer and Summerfelt (2007) determined that ozone application with short term residuals (0.1–0.2 mg/L for 1 min) followed by UV radiation (50 mJ/cm2) was effective in bringing the bacterial population of the flow-stream to near zero. This system combination has the added benefit of ensuring residual ozone destruction with UV light before the water enters the culture tank. During the ozonation process, some intermediate organic and inorganic compounds can be formed. Often referred to as ozonation by-products, these compounds can sometimes be toxic to aquatic organisms. Of particular concern in treatment of saltwater are brominated compounds. Bromide concentrations in natural seawater range from 60–70 mg/L. Ozone applied to natural saltwater reacts with bromide (Br−) to form hypobromous acid (HOBr) and hypobromite ions (OBr−) (Tango and Gagnon, 2003; Summerfelt, 2003; Liltved et al., 2006). Both of these compounds are relatively stable and can be toxic to fish and shellfish (Huguenin and Colt, 1989). Fisher et al. (1999) reported the LC50 of the hypobromite ion to be 0.068 mg/L. Prolonged ozonation can also oxidize hypobromite further to the bromate ion (BrO−3) which is another stable but less toxic compound (Liltved et al., 2006; Summerfelt, 2003). Hutchinson et al. (1997) reported BrO3− to have a 96 h LC50 of 31 mg/L. However, with bromate having been shown to be carcinogenic in animals (Kurokawa et al., 1986), concerns of bioaccumulation have been raised (Liltved et al., 2006). While residual bromine and brominated compounds can be removed with an activated carbon filter, the creation of toxic compounds in marine recirculating systems can be avoided by using commercially available bromide-free artificial seawater. Readers are directed to publications by Summerfelt (2003), Sharrer and Summerfelt (2007), Tango and Gagnon (2003), and Liltved et al. (2006) for more complete reviews of UV and ozonation technologies in freshwater and marine aquaculture applications.

31.10 Comparing freshwater and marine systems design There are a few obvious differences in design requirements for freshwater and saltwater applications. The main requirement of saltwater or brackish

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water applications is in materials compatibility. Needless to say, more plastic and fibreglass should be used in a saltwater system. However, where metal is required, a high-quality (316) stainless steel is usually the most economical and durable material to use. The less obvious differences are in water chemistry and biological reaction rates.

31.10.1 Fine and dissolved solids control Fine suspended solids (<30 micrometers) have been shown to contribute more than 50 % of the total suspended solids in some recirculating systems. Fine suspended solids increase the oxygen demand of the system and cause gill irritation and damage in finfish. Dissolved organic solids (protein) can contribute significantly to the oxygen demand of the total system. Fine and dissolved solids cannot be easily or economically removed by sedimentation or most mechanical filtration technologies used in aquaculture today (Cripps and Bergheim, 2000). Foam fractionation (also referred to as protein skimming) is often employed in trying to remove these solids. Foam fractionation, as employed in aquaculture, is a process of introducing air bubbles at the bottom of a closed column of water that creates foam at the air/water interface at the top of the column. As the bubbles rise through the water column, solid particles attach to the bubbles’ surfaces, forming the foam at the top of the column. The foam build-up is then collapsed into a solids-rich liquid and channelled out of the fractionation unit through pipe. Solids concentration in the waste tank can be five times higher than that of the culture tank. The efficiency of the foam fractionation process is subject to the chemical properties of the water. In general, in well-designed freshwater systems where dissolved solids concentrations tend to be low, the foam fractionation is not an efficient process and, with some water exchange (5–10 % per day), is not necessary. Summerfelt and Vinci (2007) describe foam fractionation as an unpredictable and erratic process. This statement is especially true for freshwater applications. Conversely, the process is much more efficient and effective in saltwater systems. Finer bubbles can be created in saltwater increasing the air-to-water interface and reducing the rise velocity which increases the effectiveness of the foaming process. Additionally, the process tends to be more effective in water with the higher pH values found in marine systems (Summerfelt and Vinci, 2007). For this reason, the application of foam fractionation as a treatment process should be more carefully considered in marine or brackish water systems while they may not be in freshwater recirculating systems designs.

31.10.2 Changes in nitrification rates As noted before, marine species tend to be more sensitive to elevated levels of ammonia–nitrogen concentrations. Therefore, a saltwater system may

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need to be designed to maintain lower levels of TAN. There also appears to be a relative abundance of nitrification data in the peer-reviewed literature for freshwater systems as opposed to seawater systems. Currently there is some debate as to the effect of seawater on nitrification. Nijhof and Bovendeur (1990) presented data suggesting that nitrification in saltwater systems is significantly lower than in freshwater. Chen et al. (2006) reports similar results with saltwater nitrification being 37 % less than freshwater rates. Rusten et al. (2006) reported that in commercial marine fish farming operations the nitrification rates for moving bed filters were approximately 60 % of the rate that would be expected in a similar freshwater biofilter. Thus, when designing a marine system, one must take into account both the lower level of tolerance of TAN that marine species exhibit, and the fact that biofilters have been shown to have reduced capacity in saltwater. As such, it is safe to say that marine biofilters should be larger than freshwater biofilters for the same rate of feed applications. Unless data are available from systems that have grown the species that are under consideration, it is wise to develop a prototype system first to determine the correct rates of oxygen consumption, CO2 and ammonia–nitrogen production, and expected nitrification rates.

31.11 An example of a modern approach to a complete systems design Because the private sector often considers details of a production system design as proprietary, it is difficult to provide a detailed design example of a commercially available system. There are, however, some systems that have been developed over the past few years that can provide good examples of the thought process that is required to develop a state-of-the-art recirculating fish production system. One such system was developed at Mote Aquaculture Park in Sarasota, FL, USA (Mote Marine Laboratory; www.mote.org). The system described here was developed to demonstrate the scientific and economic feasibility of rearing sturgeon for caviar production in a recirculating system. Sturgeon production for caviar is unique in the aquaculture industry in that the product (caviar) takes approximately seven years or more to reach the caviar market. An important aspect then of the design of the production unit for this product is energy efficiency. Given the length of time that the system must run without market income, low energy use and associated costs are key to the development of this type of purpose-built system. The system described below is also described in Hamlin et al. (2008). The Mote Aquaculture sturgeon system is based on a four tank module (Fig. 31.17). The farm is designed with multiple production modules within each building and more than one building to provide some level of biosecurity and a reduction in risk. The tanks are semi-square with dimensions of

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Feeder Tank 3

Bottom drain

Side drain

Inlet manifold

Feeder Tank 3

Bottom drain

Side drain

Inlet manifold

Drum filter

Feeder Tank 1

Bottom drain

Side drain

Moving bed biofilter 1

Inlet manifold

Moving bed biofilter 2

Bottom drain

Side drain

Inlet manifold

Oxygenator Aeration sump

Oxygenator

Fig. 31.17 Plan view of the four-tank module for sturgeon production at Mote Aquaculture Park. (Drawing by D.P. DeLong, courtesy of Mote Marine Laboratory)

7.62 m × 7.62 m × 1.52 m deep (25 ft × 25 ft × 5 ft deep) and manufactured by Dolphin Fiberglass Products, Inc. (26800 SW 202 Avenue, Homestead, FL 33031 USA; www.aquaculturetanks.com). Each tank, with a working volume of 70 m3, has a bottom drain and a Cornell-style side drain. Each drain flows to an open fibreglass channel via a water level controlling standpipe. The height of each standpipe controls the relative percentage of flow leaving the tank from each drain point. A drain channel was selected over a piping system to provide for ease of cleaning the drain system. Bacterial growth on drain pipes and organic accumulations can lead to off-flavour in fish and eventually in the end product of caviar. The combined flow from each tank flows by gravity to a drum screen filter with 60 micron screens manufactured by PR Aqua (Rotofilter Model 4872) and then exits to a ‘D-ended’ raceway that provides biofiltration, aeration and CO2 degassing, and oxygenation. As the water enters the filtration unit, it moves through two biofiltration chambers containing a total of 25 m3 of moving bed media (non-expanded volumes). The media is an extruded plastic media marketed as AMBTM Bio Media and is manufactured by EEC Global Operations LLC (537 Newport Center Drive #613, Newport Beach, CA 92606 USA; www.eecusa.com). The media and water is aerated with numerous inflatable diffusers (Model 84P, Environmental Dynamics, Inc., 5601 Paris Road, Columbia, MO 65202 USA; www.diffuserexpress.com). A total of 3.9 m3/min of low-pressure air is diffused into the water within the moving bed filters. An aeration chamber is available after the moving bed filter but is not currently used, as the moving bed aeration is enough to add oxygen and remove carbon dioxide. The air is provided by half of the capacity of a regenerative blower that is utilizing 3.7 kW of electricity under the operational load conditions (one Rotron Model DR858, 7.5 hp, services two production systems). The process water

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then falls over a curved weir into a pumping sump. Within the pumping sump, two floating FASTM ‘hooded’ oxygenators are positioned and operated to raise the oxygen content of the water as needed. The oxygenators (Turboxygene Model LR200, F.A.S. S.r.l., via Lasta, 11/a – 37030, Vago di Lavagno, (VR) Italy; www.fas.vr.it/fas1/fas_ing.html) are hooded paddlewheels that agitate water in an oxygen-rich atmosphere and move approximately 200 m3/h to the terminal end of the pumping sump. Each of the two units utilizes approximately 0.75 kW of electricity and is designed to deliver approximately 2.4 kg of oxygen per hour to the process water. The water from the pumping sump is then lifted into a large-diameter pipe network to deliver the water back to each tank via an inlet vertical manifold. The large pipe diameter was designed into the system to reduce the energy requirements for pumping. Water is lifted only 60 cm, to be returned to the tanks at a total flow of 7570 litres per minute. The recycled water is pumped by a 5.3 kW (5 hp) variable speed vertical turbine type pump (Model AJ 10, American Marsh Pumps, 185 Progress Road, Collierville, TN. 38017 USA; www.american-marsh.com). With a total tank volume of 280 m3, system water can be processed through the filtration process approximately every 40 min. As designed, the system has the capability of filtering the waste from a maximum of 100 kg high-protein extruded pellet feed per day. Eight four-tank systems have been built at Mote Aquaculture Park and have had operational characteristics as expected. The system has been shown to maintain water quality conditions for excellent growth as demonstrated by the harvesting and marketing of caviar for worldwide distribution. The Mote sturgeon system is a very good example of a well thought-out, well implemented and efficient modern recirculating system design.

31.12 References brazil b (1996) Impact of ozonation on system performance and growth characteristics of hybrid striped bass (Morone chrysops x M. saxatilis) and tilapia hybrids (Sarotherodon sp.) reared in recirculating aquaculture systems, PhD Dissertation, Virginia Polytechnic Institute and State University, Blacksburg, VA. broussard m and simco b (1976) High-density culture of channel catfish in a recirculation system, Progress in Fish Culture, 38, 138–41. bullock g, summerfelt s, noble a, weber a, durant m and hankins j (1997) Ozonation of a recirculating rainbow trout culture system. I. Effects on bacterial gill disease and heterotrophic bacteria, Aquaculture, 158, 43–55. chen s, stechey d and malone r (1994) Suspended solids control in recirculating aquaculture systems, in Timmons M and Losordo T (eds), Aquaculture Water Reuse Systems: Engineering Design and Management, Elsevier, Amsterdam, 61–100. chen s, ling j and blancheton j (2006) Nitrification kinetics of biofilm as affected by water quality factors, Aquacultural Engineering, 34, 179–97. colt j and watten b (1988) Application of pure oxygen in fish culture, Aquacultural Engineering, 7, 397–441.

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cripps s and bergheim a (2000) Solids management and removal for intensive land-based aquaculture production systems, Aquacultural Engineering, 22, 33–56. davidson j and summerfelt s (2004) Solids flushing, mixing, and water velocity profiles within large (10 and 150 m3) circular ‘Cornell-type’ dual-drain tanks, Aquacultural Engineering, 32, 245–71. davidson j and summerfelt s (2005) Solids removal from a coldwater recirculating system–comparison of a swirl separator and a radial-flow settler, Aquacultural Engineering, 33, 47–61. drennan d, hosler k, francis m, weaver d, aneshansley e and beckman g (2006) Standardized evaluation and rating of biofilters. II. Manufacturer’s and user’s perspective, Aquacultural Engineering, 34, 403–16. ebeling j, timmons m, bisogni j (2006) Engineering analysis of the stoichiometry of photoautotrophic, autotrophic, and heterotrophic removal of ammonia–nitrogen in aquaculture systems, Aquaculture, 257, 346–58. eding e, kamstra a, verreth j, huisman e and klapwijk a (2006) Design and operation of nitrifying trickling filters in recirculating aquaculture: a review, Aquacultural Engineering, 34, 234–60. fisher d, burton d, yonkos l, turley s and ziegler g (1999) The relative acute toxicity of continuous and intermittent exposure of chlorine and bromine to aquatic organisms in the presence and absence of ammonia, Water Research, 33, 760–68. greiner, a and timmons, m (1998) Evaluation of nitrification in microbead and trickling filters in an intensive recirculating tilapia production facility, Aquacultural Engineering, 18, 189–200. hamlin h, michaels j, beaulaton c, graham w, dutt w, steinbach p, losordo t, schrader k and main k (2008) Comparing denitrification rates and carbon sources in commercial scale upflow denitrification biological filters in aquaculture, Aquacultural Engineering, 38, 79–92. henze m, harremoës p, la cour jansen j and arvin e (1995) Wastewater Treatment: Biological and Chemical Processes, Springer Verlag, Berlin, Heidelberg. hobbs a, losordo t, delong d, regan j, bennett s, gron r and foster b (1997) A commercial, public demonstration of recirculating aquaculture technology: the CP&L/EPRI Fish Barn at North Carolina State University, in Timmons M B and Losordo T M (eds), Advances in Aquacultural Engineering. Proceedings from the aquacultural engineering society technical sessions at the fourth international symposium on tilapia in aquaculture, NRAES-105, Northeast Regional Agricultural Engineering Service, Ithaca, NY, 151–8. huguenin j and colt j (1989) Design and Operating Guide for Aquaculture Seawater Systems, Elsevier, Amsterdam. hutchinson t, hutchings m and moore k (1997) A review of effects of bromate on aquatic organisms and toxicity of bromate to oyster (Crassostria gigas) embryos, Ecotoxicology and Environmental Safety. 38, 238–43. kurokawa y, takayama s, konishi y, hiasa y, asahina s, takahashi m, maekawa a and hayashi y (1986) Long-term in vivo carcinogenicity tests of potassium bromate, sodium hypochlorite, and sodium chlorite conducted in Japan, Environmental Health Perspectives, 69, 221–35. labatut r, ebeling j, bhaskaran r and timmons m (2007) Hydrodynamics of a largescale mixed-cell raceway (MCR): Experimental studies, Aquacultural Engineering, 37, 132–43. lee l, ong s and ng w (2004) Biofilm morphology and nitrification activities: Recovery of nitrifying biofilm particles covered with heterotrophic outgrowth, Bioresource Technology, 95, 209–14.

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leonard n, guiraud j, gasset e, cailleres j and blancheton j (2002) Bacteria and nutrients – nitrogen and carbon – in a recirculating system for sea bass production, Aquacultural Engineering, 26, 111–27. liltved h, vogelsang c, modahl i and dannevig b (2006) High resistance of fish pathogenic viruses to UV irradiation and ozonated seawater, Aquacultural Engineering, 34, 72–82. liltved h and summerfelt s (2007) Ozonation and UV-Irradiation, in Timmons M and Ebeling J (eds), Recirculating Aquaculture, NRAC Publication No. 01-007, Cayuga Aqua Ventures, Ithaca, NY, 439–82. ling j and chen s (2005) Impact of organic carbon on nitrification performance of different biofilters, Aquacultural Engineering, 33, 150–62. losordo t m (1997) Tilapia culture in intensive recirculating systems, in Costa-Pierce B and Rakocy J (eds), Tilapia Culture in the Americas, Volume 1, World Aquaculture Society, Baton Rouge, LA, 185–208. losordo t and westers h (1994) System carrying capacity and flow estimation, in Timmons M and Losordo T (eds), Aquaculture Water Reuse Systems: Engineering Design and Management, Elsevier, Amsterdam, 9–60. lunde t, skybakmoen s and schei i (1997) Particle Trap, United States Patent and Trademark Office, Department of Commerce, Washington, DC, No. 5,636,595. malone r, chitta b and drennan d (1993) Optimizing nitrification in bead filters for warmwater recirculating aquaculture systems, in Wang J (ed.), Techniques for Modern Aquaculture, ASAE Publication 02-93, American Society for Agriculture Engineers, St Joseph, MI, 315–25. malone r, rusch k and christensen j (1996) Design of recirculating crawfish systems employing expandable granular biofilters, in Aquacultural Engineering Society Proceedings II, Successes and Failures in Commercial Recirculating Aquaculture, NRAES-98, Northeast Regional Aquaculture Engineering Service, Ithaca, NY, 2, 467–80. malone rf, beecher le and delosreyes jr aa (1998) Sizing and management of floating bead bioclarifiers, in Libey GS and Timmons MB (eds), Proceeding of the Second International Conference on Recirculating Aquaculture, 16–19 July, Virginia Polytechnic Institute and State University, Roanoke, VA, 319–41. malone r and beecher l (2000) Use of floating bead filters to recondition recirculating waters in warmwater aquaculture production systems, Aquacultural Engineering, 22, 57–73. malone r and pfeiffer t (2006) Rating fixed film nitrifying biofilters used in recirculating aquaculture systems, Aquacultural Engineering, 34, 389–402. meade j (1985) Allowable ammonia for fish culture, Progressive Fish-Culturist, 47, 135–45. michaud l, blancheton j, bruni v and piedrahita r (2006) Effect of particulate organic carbon on heterotrophic bacterial populations and nitrification efficiency in biological filters, Aquacultural Engineering, 34, 224–33. miller g and libey g (1984) Evaluation of a trickling biofilter in a recirculating aquaculture system containing channel catfish, Aquacultural Engineering, 3, 39–57. nijhof m and bovendeur j (1990) Fixed film nitrification characteristics in seawater recirculating fish culture systems, Aquaculture, 87, 133–43. nogueira r, melo lf, purkhold u, wuertz s and wagner m (2002) Nitrifying and heterotrophic population dynamics in biofilm reactors: effects of hydraulic retention time and the presence of organic carbon, Water Research, 36, 469–81. odegaard h, paulsrud b, bilstad t and pettersen j (1991) Norwegian strategies in the treatment of municipal wastewater towards reduction of nutrient discharge to the North Sea, Water Science and Technology, 24, 179–86.

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ohashi a, viraj de silva d, mobarry b, manem j, stahl d and rittman b (1995) Influence of substrate C/N ratio on the structure of multi-species biofilms consisting of nitrifiers and heterotrophs, Water Science and Technology, 32, 75–84. person-le ruyet j, chartois h and quemener l (1995) Comparative acute ammonia toxicity in marine fish and plasma ammonia response, Aquaculture, 136, 181–94. provenzano a and winfield j (1987) Performance of a recirculating fish production system stocked with tilapia hybrids, Aquacultural Engineering, 6, 15–26. rusten b, eikebrokk b, ulgenes y and lygren e (2006) Design and operations of Kaldnes moving bed biofilm reactors, Aquacultural Engineering, 35, 322–31. satoh h, okabe s, norimatsu n and watanabe y (2000) Significance of substrate C/N ratio on structure and activity of nitrifying biofilms determined by in situ hybridization and the use of microelectrodes, Water Science and Technology, 41, 317–21. sawyer c, mccarty p and parkin g (1994) Chemistry for Environmental Engineering, 4th edn, McGraw-Hill, New York. schneider o, sereti v, eding e and verreth j (2007) Heterotrophic bacterial production on solid fish waste: TAN and nitrate as nitrogen source under practical RAS conditions, Bioresource Technology, 98, 1924–30. sharrer m and summerfelt s (2007) Ozonation followed by ultraviolet irradiation provides effective bacteria inactivation in a freshwater recirculating system, Aquacultural Engineering, 37, 180–91. speece r, madrid m and needham k (1971) Downflow bubble contact aeration, Journal of the Sanitary Engineering Division, ASCE, 97, 433–41. summerfelt s, vinci b and piedrahita r (2000) Oxygenation and carbon dioxide control in water reuse systems, Aquacultural Engineering, 22, 87–108. summerfelt s (2003) Ozonation and UV irradiation – an introduction and examples of current applications, Aquacultural Engineering, 28, 21–36. summerfelt s (2006) Design and management of conventional fluidized-sand biofilters, Aquacultural Engineering, 34, 275–302. summerfelt s and vinci b (2007) Solids capture, in Timmons M and Ebeling J (eds), Recirculating Aquaculture, NRAC Publication No. 01-007, Cayuga Aqua Ventures, Ithaca, NY, 171–219. steeby j, hargreaves j, tucker c and kingsbury s (2004) Accumulation, organic carbon and dry matter concentration of sediment in commercial channel catfish ponds, Aquacultural Engineering, 30, 115–26. tal y, watts j, schreier s, sowers k and schreier h (2003) Characterization of the microbial community and nitrogen transformation processes associated with moving bed bioreactors in a closed recirculating mariculture system, Aquaculture, 215, 187–202. tango m and gagnon g (2003) Impact of ozonation on water quality in marine recirculation systems, Aquacultural Engineering, 29, 125–37. timmons m, holder j and ebeling j (2006) Application of microbead biological filters, Aquacultural Engineering, 34, 332–43. timmons m, summerfelt s and vinci b (1998) Review of circular tank technology and management, Aquacultural Engineering, 18, 51–69. timmons m and summerfelt s (2007) Culture units, in Timmons M and Ebeling J (eds), Recirculating Aquaculture, NRAC Publication No. 01-007, Cayuga Aqua Ventures, Ithaca, NY, 115–70. timmons m and vinci b (2007) Gas transfer, in Timmons M and Ebeling J (eds), Recirculating Aquaculture, NRAC Publication No. 01-007, Cayuga Aqua Ventures, Ithaca, NY, 397–438. true b, johnson w and chen s (2004) Reducing phosphorous discharge from flowthrough aquaculture II: Hinged and moving baffles to improve waste transport, Aquacultural Engineering, 32, 145–60.

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twarowska j, westerman p and losordo t (1997) Water treatment and waste characterization of an intensive recirculating fish production system, Aquacultural Engineering, 16, 133–47. us-epa (1984) Methods for the chemical analysis of water and wastewater, EPA600/4-79-020, US Environmental Protection Agency, Office of Research and Development, Environmental Monitoring and Support Laboratory, Cincinnati, OH. veerapen j, lowry b and couturier m (2005) Design methodology for the swirl separator, Aquacultural Engineering, 33, 21–45. wagner e, bosakowski t and miller s (1995) Evaluation of the absorption efficiency of the low head oxygenation system, Aquacultural Engineering, 14, 49–57. watten b (1989) Multiple stage gas absorber, United States Patent and Trademark Office, Department of Commerce, Washington, DC, No. 4,880,445. watten b, honeyfield d and schwartz m (2000) Hydraulic characteristics of a rectangular mixed-cell rearing unit, Aquacultural Engineering, 24, 59–73. watten b and johnson r (1990) Comparative hydraulics and rearing trial performance of a production scale cross-flow rearing unit, Aquacultural Engineering, 9, 245–66. wedemeyer g (1996) Physiology of Fish in Intensive Culture, International Thompson Publishing, New York. wheaton f, hochheimer j, kaiser g, malone r, krones m, libey g and easter c (1994) Biological filter design methods, in Timmons M B and Losordo T M (eds), Aquaculture Water Reuse Systems: Engineering Design and Management, Elsevier, Amsterdam, 101–71. zhu s and chen s (1999) An experimental study on nitrification biofilm performances using a series reactor system, Aquacultural Engineering, 20, 245–59. zhu s and chen s (2001) Effects of organic carbon on nitrification rate in fixed film biofilters, Aquacultural Engineering, 25, 1–11. zhu s and chen s (2002) The impact of temperature on nitrification rate in fixed film biofilters, Aquacultural Engineering, 26, 221–37.

32 Advances in technology and practice for land-based aquaculture systems: ponds for finfish production C. E. Boyd, Auburn University, USA, and S. Chainark, Phuket Rajabhat University, Thailand

Abstract: Ponds are the major grow-out system for freshwater finfish. The trend towards increasing production intensity in ponds requires greater use of feeds and mechanical aerators and less dependence upon manures, agricultural by-products, and fertilizers. More emphasis must be placed on avoiding or correcting site limitations and designing ponds with regard to specific culture requirements. Moreover, attention to bottom soil condition increases in importance as culture intensity increases. Research should be conducted to improve the efficiency with which land, water, fishmeal and other feed ingredients, and energy for aeration and other purposes are used in culture systems. Findings of this research will lead to greater profitability, lessen negative environment impacts, and make pond aquaculture more sustainable. Key words: finfish culture, ponds, water quality, pond bottom soils, aquaculture effluents, sustainable aquaculture.

32.1 Introduction The FAO fisheries statistics for 2004 revealed that finfish aquaculture accounted for 28.1 million tonnes or 47.3 % of total aquaculture production of 59.4 million tonnes. Freshwater fish production was 23.4 million tonnes or 83.2 % of finfish aquaculture production. Three groups of fish species dominated freshwater fish production as follows: carps, 16.1 million tonnes; unspecified fishes, 1.8 million tonnes; tilapias, 1.6 million tonnes. China was by far the largest producer of freshwater fish, but other major producers were India, Philippines, Indonesia, Japan, Vietnam, and Thailand.

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Ponds are the most common grow-out units for land-based, finfish aquaculture. Ponds have been used for fish production for at least 2000 years, but the application of science to pond aquaculture has occurred mostly during the past 100 years. Initially, research focused on developing technology for spawning, fingerling production, and grow-out of different species. A relatively small number of species were acceptable for pond culture, and efforts were made to increase the production of these species. Application of manures and chemical fertilizers were first used to increase fish production in ponds. Later, manufactured feed was found to increase production above that possible with manures and fertilizers. Finally, mechanical aeration to supplement the natural supply of dissolved oxygen allowed greater feed inputs and higher fish production in ponds. The tendency towards increasingly greater production is continuing. Increases in aquacultural production have been possible through technological advances in several areas, and one of the most critical areas has been water quality management. Boyd and Tucker (1998) presented a thorough discussion of water quality management in aquaculture production systems. This chapter briefly reviews land-based aquaculture systems for freshwater finfish and their management, discusses improvements made since the late 1990s, and identifies research needs. It is, however, significant to note that there is considerable interest in the cultivation of brackish water and marine finfish in saline, inland waters (Fielder et al., 2001). Most of the issues discussed for freshwater finfish culture in this chapter also apply in salinewater finfish culture in inland areas.

32.2 Hydrologic types of ponds 32.2.1 Watershed ponds A watershed pond is formed by building an embankment across a portion of a watershed to capture runoff. These ponds also are called rain-fed ponds or terrace ponds. Some watershed ponds remain full all year because they receive permanent inflow from springs or streams or because water is added from wells or other external sources. Most have seasonal fluctuations in water levels because of differences in frequencies and amounts of rainfall. Water balance in a watershed pond may be expressed as follows: P + R + Ia = E + S + O + I d + C + ΔH

[32.1]

where P = precipitation, R = storm runoff and inflow from springs or streams, Ia = intentional additions, E = evaporation, S = seepage, O = overflow, Id = intentional discharge, C = consumptive use, and ΔH = change in storage. Consumptive use rarely occurs in aquaculture ponds. Because ponds have a free water surface, they may increase evapotranspiration from

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watersheds. Inflow in excess of storage volume overflows, and the stored water is discharged when ponds are drained. Seepage from ponds either infiltrates downward to the water table or it seeps through embankments to evaporate or pass downstream. Thus, ponds do not greatly alter water yields from watersheds, but they may influence stream flow patterns by diminishing the hydrograph peak (Schoof and Gander, 1982; Yoo and Boyd, 1994).

32.2.2 Embankment ponds Embankment ponds are made in level land by excavating enough surface soil to build embankments to enclose an area in which water is impounded. Watersheds for embankment ponds consist of the inside slopes and tops of embankments. Runoff is, therefore, much less than for watershed ponds. For most purposes, runoff can be neglected in water budgets for embankment ponds (Boyd, 1982). There are few locations where rain falling directly into ponds will keep them full. Water to supply embankment ponds is taken from streams, wells, lakes, reservoirs, estuaries, and the sea. The water balance of an embankment pond may be expressed as follows: P + Ia = E + S + O + I d + ΔH

[32.2]

Embankment ponds are favored for aquaculture, because water levels can be controlled. Depths can be established through design and construction, and bottoms can be sloped to facilitate complete draining. At harvest, water can be quickly discharged through gates or pipes. In the USA, embankment ponds are usually operated by the drop-fill method. Water levels are allowed to fall about 15 cm and then partially restored by water additions. This retains storage volume to capture rain falling into ponds (Cathcart et al., 1999). If the drop-fill method is combined with seine harvest of fish without drawdown of water levels, ponds only discharge after large rainfall events. This practice greatly reduces the discharge of nutrients, organic matter, and suspended solids from ponds (Boyd et al., 2000).

32.2.3 Excavated ponds This type of pond is constructed by excavating a basin in which to store water. Where the water table is high, groundwater seeps into excavated ponds filling them to the level of the water table. Ponds also may be filled by overland flow from the surrounding area and by rain falling into them. The water level in excavated ponds is usually uncontrollable. They seldom overflow, and they must be emptied by pumping out the water. The water budget equation for excavated ponds is: P + R = E + S + I d + ΔH

[32.3]

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Excavated ponds are less suitable than watershed or embankment ponds for aquaculture because of the lack of water level control. They also tend to be small because of the difficulty and expense of removing earth to form them. Nevertheless, excavated ponds are widely used in India, Bangladesh, and some other Asia countries for small-scale aquaculture.

32.2.4 Water reuse Water reuse is becoming increasingly popular in pond aquaculture. Fish may be produced at high density in small production units and water recycled through larger ponds for treatment. A high degree of water level control is possible in grow-out units. Fish are confined in a small area to facilitate feeding, mechanical aeration, observation, and health management. Reuse also conserves water and lessens effluent volume. The land area needed for production in water reuse systems is roughly the same as for normal pond aquaculture because one or more external ponds must provide the waste treatment service (Boyd et al., 2007a).

32.2.5 Seepage and erosion control Areas with relatively impermeable soils are favored for aquaculture ponds because seepage loses will not seriously affect water balance at such sites. However, improvements in membrane technology have made it possible to install impermeable membranes in ponds where soils are highly permeable. This practice is limited to highly-intensive culture of highly-valued fish because of the high expense of membranes. The use of membranes also results in challenges to water quality management. Pond bottom soils are a sink for phosphorus resulting from feed inputs, and lined ponds have much greater concentrations of phosphorus than unlined ponds (Leonard, 1995). Dense phytoplankton blooms that develop in lined ponds tend to be unstable and exhibit periodic ‘booms and crashes.’ Moreover, dead phytoplankton cells accumulate in pond bottoms and do not decompose as rapidly as in ponds with earthen bottoms (Leonard, 1995). The result of dense but unstable phytoplankton communities is wide fluctuations in dissolved oxygen concentration and other water quality variables. Pond embankments are subject to erosion from waves generated by wind and water currents caused by mechanical aerators, and eroded soil accumulates in pond bottoms. Ultimately, ponds must be drained, sediment removed, and embankments renovated at a considerable cost (Steeby et al., 2001, 2004). Pond liners prevent erosion and allow higher levels of mechanical aeration than possible in unlined ponds (McIntosh, 1999). Research on less expensive ways of reducing erosion through better construction techniques, grass cover on embankments, partial lining of embankments, and aerator placement is needed.

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32.2.6 Pond shape There has been little research on pond shape. It has long been recognized that ponds should not have areas of shallow water because aquatic weed infestations develop in waters less than 50–60 cm deep (Yoo and Boyd, 1994). The ideal depth of ponds depends upon culture species and intensity, but ponds should be at least 1 m average depth but not over 2 m in average depth (Mezainis, 1977; Yoo and Boyd, 1994). Shallow ponds are subject to aquatic weed infestations, and their waters may warm excessively on hot days. Deeper ponds stratify thermally, and the bottom layer often becomes anaerobic. Sudden thermal destratification may cause water quality deterioration and fish stress or mortality. Larger ponds are not as expensive to build as smaller ones because less earthwork is required per unit storage area (volume). Management of larger ponds is more difficult, for feeds, fertilizers, liming materials, and other inputs cannot be applied from shore. Also, it may require several days to drain and harvest large ponds. This can lead to fish stress and deterioration of fish quality. Shell (1966) referred to the need for research to determine the best pond size and shape for culture of different species, but little has been done on this topic. Several geometric shapes have been used for ponds to include rectangles, squares, and circles. Water mixing and circulation in ponds is important and a narrow rectangular shape does not favor this process. Rectangular ponds usually should not be more than twice as long as wide. The benefit of circular ponds over square ponds is not great. A square pond and a circular pond are quite similar – especially when it is considered that the bottom of a square pond is sloped in the four corners. A circular pond is superior only in that small areas of dead water which occur in the corners of square ponds are eliminated. It is doubtful that this difference justifies the greater cost of constructing round ponds.

32.3 Production methodology There are no reliable statistics on the proportion of freshwater fish produced in ponds, but it is likely that over 75 % of production is from ponds. There are several methods for increasing production above that possible from natural productivity of pond waters. In the simplest method (manuring), human or animal excrement, agricultural by-products, or grass clippings are applied to ponds. Fish may eat the organic matter directly but, more often, the organic matter is eaten by zooplankton and other small aquatic animals that serve as fish food. Decomposition of organic matter also releases nitrogen, phosphorus, and other nutrients that stimulate phytoplankton which provides the base of the natural food web. It is likely that the majority of freshwater fish are produced in ponds fertilized with organic matter.

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Chemical fertilizers also may be used to stimulate phytoplankton productivity and increase fish production in ponds. In some cases, both chemical fertilizers and manures are applied. This combination often provides higher production than either manuring or chemical fertilization alone. Feeds provide a more reliable source of nutrition for fish allowing greater and more predictable production. Moreover, when used responsibly, feeds usually cause fewer problems with water and sediment quality than do manures. Fish produced with feeds are also more acceptable to consumers in developed nations than fish from manured ponds. High rates of feed input can lead to water quality deterioration and especially low dissolved oxygen concentration in ponds. Water exchange may be used to flush phytoplankton and wastes from ponds. Mechanical aeration also may be applied to supplement dissolved oxygen supplies and allow greater feed input and production. Most fish are produced in single-batch systems with ponds being drained at the end of the grow-out period to facilitate harvest. Some ponds cannot be drained and fish must be seine harvested and ponds restocked. In channel catfish culture in the USA, a multibatch system is used in which larger fish are harvested with a grading seine and small fish are restocked. Ponds may be operated for 5–10 years without draining. Culture methods not requiring ponds to be drained for harvest conserve water. However, the large variation in fish size in multibatch systems results in inefficient use of feed (J. Chappell, pers comms).

32.4 Liming and fertilization 32.4.1 Liming Limestone is pulverized to make agricultural limestone; it is burned to make burnt lime, or burned and hydrated to make hydrated lime. Limestone is usually a mixture of calcium and magnesium carbonate. The burned products consist of oxides and hydroxides of calcium and magnesium, respectively. Liming materials have traditionally been applied to ponds to neutralize acidic bottom soil and increase total alkalinity concentration in water (Hickling, 1962; Boyd and Tucker, 1998). In addition, lime may be applied to empty ponds between crops to increase soil pH and destroy fish disease organisms and their vectors (Boyd, 1995). The quality of liming materials is related to neutralizing value (ability to neutralize acidity) and particle size. Pure CaCO3 has a neutralizing value of 100 %; the values for pure CaO and pure Ca(OH)2 are 174 % and 136 %, respectively. Recent improvements in manufacturing technology have led to finely pulverized agricultural limestone that will react faster and more completely than coarser material. Silapajarn et al. (2004) developed a new system for evaluating the particle size distribution (fineness rating) in

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agricultural limestone to account for the greater percentage of smaller particles in modern liming materials. The use of liming materials is essential in most intensive aquaculture systems because of large inputs of ammonia nitrogen from feeding waste and fish metabolites. Ammonia nitrogen is oxidized by nitrifying bacteria according to the following equation: NH +4 + 2O2 → NO−2 + 2 H + + H 2 O

[32.4]

Acidity from nitrification can decrease total alkalinity and pH in pond water. Boyd (2007) gave the following equation for estimating the amount of agricultural limestone needed to neutralize the potential acidity from feeding based on feed input and percentage crude protein in feed: CaCO3 Equivalence of feed ( kg ) kg Feed input ×% Crude protein in feed × 0.01285

[32.5]

In commercial aquaculture systems, total alkalinity should not fall below 75 mg/L. Thus, Eq. (32.5) can be used to estimate agricultural limestone inputs at weekly or less frequent intervals to prevent total alkalinity concentration from declining. Interest in ensuring that aquaculture products are safe has led to a reduction in the use of antibiotics, drugs, and other chemicals in ponds. In the past, some producers treated pond bottoms with chlorine compounds, short-lived pesticides, and certain other harsh chemicals to kill wild fish in puddles and disinfect soils. There is increasing use of burnt lime and hydrated lime for killing unwanted organisms in puddles and for disinfecting soil. The minimum application rate of lime to pond bottoms for this purpose should be at least 1000 kg/ha, and better results can be achieved at rates of 1500– 2000 kg/ha (Boyd and Tucker, 1998). Pond soil must be moist so that the lime dissolves and raises pH. An effective method is to spread lime over the pond bottom and then add a few centimeters of water over the bottom. After about one week, the pH will decline and the pond can be filled and culture species stocked.

32.4.2 Fertilization Like liming, fertilization is an old practice that has changed slowly over time. Major advances in pond fertilization were development of ‘high analysis’ fertilizers (fertilizers with high percentages of plant nutrients) in the 1950s and 1960s, and the adoption of liquid fertilizers since the 1980s (Boyd and Tucker, 1998). The use of manure for pond fertilization is declining and chemical fertilizers are used more widely. Nevertheless, aquaculture is intensifying, and fertilizer-based culture is declining as feed-based culture increases.

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Pond fertilization research has traditionally been conducted according to techniques analogous to those used in agronomy (Boyd and Tucker, 1998). Several fertilizer treatment rates and an unfertilized control were replicated three or more times, and fish production assessed statistically through analysis of variance techniques. In agronomy, it is easy and inexpensive to have many treatments and replicates, because a field can be divided into many small plots. In aquaculture, each replication of a treatment requires a pond, and the requirement for ponds severely limits the number of treatments that can be used in pond fertilizer research. Wudtisin and Boyd (2006) and Boyd et al. (2008a) applied fertilizer nutrients to 12, unreplicated ponds and analyzed the results by regression analysis. In two growing seasons, they established optimum nitrogen and phosphorus fertilizer application rates in bluegill (Lepomis macrochirus) ponds. These rates were essentially the same as those that had required several years to determine in traditional, replicated pond fertilization experiments (Swingle and Smith, 1947; Boyd, 1990). The rates were 6 kg N and 3 kg P2O5/ha per application in new ponds. In ponds that have been fertilized with N and P2O5 for five years or more, nitrogen fertilization is usually not necessary and fertilization with 3 kg P2O5/ha will normally suffice. Redfield et al. (1963) reported that the ratio of carbon : nitrogen : phosphorus in marine phytoplankton was usually 103 : 16 : 1. The Redfield Ratio is often applied to freshwater phytoplankton. One of the greatest mistakes in both pond fertilization research and practical pond fertilization is the assumption that fertilizers should provide a nitrogen to phosphorus ratio of about 6 : 1 as suggested by the Redfield Ratio. Phosphorus applied to ponds in fertilizers and not quickly absorbed by phytoplankton is sequestered by bottom soil through various chemical reactions with aluminum and iron in acidic soils and calcium in neutral or basic soils. Phosphorus adsorbed by sediment is largely unavailable to phytoplankton (Masuda and Boyd, 1994a). Conversely, fertilizer nitrogen tends to remain in water longer and nitrogen in organic matter that settles to the pond bottom is recycled through microbial activity. The fertilizer N : P ratio should be less than that desired in the phytoplankton biomass. In new ponds at Auburn, Alabama the ideal fertilizer N : P ratio was 4.6 : 1 (6 kg N and 3 kg P2O5/ha), and older ponds do not need nitrogen fertilizer (Boyd et al., 2008a). Many pond fertilization trials have been conducted by the Aquaculture Collaborative Research Support Project of the United States Agency for International Development (ACRSP/USAID). In these trials, investigators often applied N and P in a 7 : 1 ratio. In order to supply enough phosphorus to obtain good phytoplankton growth, a great excess of nitrogen was applied. The resulting fertilizer recommendations were wasteful of nitrogen (Boyd and Tucker, 1998). Moreover, urea was used as a source of nitrogen, and the authors suspect that use of the fertilizer recommendations from the ACRSP effort will result in ammonia concentrations great enough to stress

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or even kill fish. Boyd et al. (2008a) found that ammonia nitrogen applications above 8 kg/ha at two-week intervals resulted in a decline in bluegill reproduction. The ACRSP fertilizer trials often had application rates exceeding 100 kg/ha ammonia nitrogen. There also is a general belief that nitrogen fertilization is more important than phosphorus fertilization in ponds filled with brackish water or seawater. Boyd and Tucker (1998) disputed this belief, and a recent review of nitrogen and phosphorus limitation in freshwater, marine, and terrestrial ecosystems (Elser et al., 2007) revealed that nitrogen and phosphorus limitations were equally important in the three types of ecosystems. There is still interest in using animal manures and other plant wastes for pond fertilizers in developing countries. Organic fertilizers cause poor water quality in ponds, and lead to accumulation of organic matter that must be removed periodically. Chemical fertilizers can be as efficient as organic fertilizers, and they are less likely to cause water and bottom soil problems. Animal manures and other organic, agricultural wastes can be disposed more effectively on agricultural land than in fish ponds. In the culture of some fish species, fry or small fingerlings need zooplankton as a food source. Chemical fertilizers are not as effective as organic matter in quickly establishing a zooplankton bloom (Boyd and Tucker, 1998). However, high-quality organic matter such as fish offal meal or plant meals should be used instead of animal manures in stimulating zooplankton production.

32.5 Feeds and feed management The quality of aquaculture feeds has been steadily improved through research, and nutritionally balanced feeds are available for many species. Methods of producing, packaging, and handling feed pellets have also improved, and feeds have less fines, greater water stability, and longer storage life than in the past. There is a great need, however, to reduce the use of marine fish meal and fish oil in aquafeeds, to produce feeds with lower phosphorus concentrations, and to use feed efficiently (Naylor et al., 2000; Boyd et al., 2007a).

32.5.1 Fish meal and fish oil Environmentalists emphasize that aquaculture requiring more marine fish to make the feed than returned in production detracts from world fisheries production (Naylor et al., 2000). The issue often is expressed simply as the fish in : fish out ratio. Environmentalists desire that this ratio not exceed 1. There is a continuing effort to use less marine fisheries products in aquafeeds feeds, a good example of which is channel catfish culture in the USA. In the early 1970s, grow-out feeds had crude protein concentrations of 38–42 %

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and contained 12–14 % fish meal. Present-day feeds contain about 28 % crude protein and only 2–6 % fish meal. Fish meal can be entirely left out of catfish feed and replaced with other meat meals or plant meals (Boyd and Tucker, 1995). Feeds for tilapia and most other common species cultured in ponds also have relatively low marine fishery product inclusion rates. The calculation of the fish in : fish out ratio will be illustrated. Suppose fish are produced in a pond at an feed conversion ratio (FCR) of 1.7 using a feed containing 6 % fish meal, 2 % fish oil, and made without inclusion of fishery by-products. Each kilogram of fish in this example requires 1.7 kg feed containing 0.102 kg fish meal (0.06 × 1.7) and 0.034 kg fish oil (0.02 × 1.7). The ratios of live fish to fish meal and fish oil are 4.5 : 1 and 12 : 1, respectively (Boyd et al., 2007a). However, fish oil is a by-product of fish meal manufacturing, and each unit of fish meal results in about 0.375 unit of fish oil. It will take 0.459 kg live fish to make the fish meal for the feed needed for 1 kg of the culture species. This amount of live fish will yield 0.049 kg fish oil when reduced to fish meal – more than needed in the feed for 1 kg of the culture species. Thus, the ‘fish in’ component is only fish for making fish meal, and the fish in : fish out ratio (0.459) is less than 1. Fish processing wastes are used to make offal meal and offal oil. These products are sometimes substituted for fish meal and oil. When this is done, the marine fishery product inclusion rate of aquaculture feeds may be estimated with the following equation: Adjusted marine product inclusion rate (%) = % Total marine meal + % Total marine oil − % Offal meal − % Offal oil − [( Total marine meal − Offal meal ) × 0.54 ]

[32.6]

Much of the concern over fish meal and fish oil use has resulted from high inclusion rates of these products in feeds for salmon, trout, and marine shrimp. With these species, the fish in : fish out ratio typically is more than 1 and sometimes 3 or greater (Boyd et al., 2007a).

32.5.2 Nitrogen and phosphorus Nitrogen and phosphorus are contained in plant and animal meals used as feed ingredients, and dicalcium phosphate is often added to fish feeds as a phosphorus supplement. Nitrogen and phosphorus often limit growth of phytoplankton and other aquatic plants, and they are responsible for excessive phytoplankton blooms in ponds. These two nutrients also may contribute to eutrophication in water bodies receiving aquaculture effluents. Nitrogen and phosphorus concentrations in feed should not be greater than necessary for optimum growth of the culture species. Low nitrogen and phosphorus feeds are made by substituting plant meal for a portion of

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the fish meal, eliminating or lessening the inclusion of dicalcium phosphate, and lowering crude protein to the optimum concentration. Such feeds often are marketed as ‘environmentally friendly’ or ‘non-polluting’ but, in reality, they only reduce the amount of nitrogen and phosphorus entering the waters of aquaculture ponds.

32.5.3 Feed management The objective of good feed management is to maximize production per unit of feed, i.e., to lower the FCR. A lower FCR reduces feed cost, and it lessens the pollution potential of fish feeding. One of the most important ways of reducing feed input is to not apply more feed than fish will eat in a reasonable amount of time. This amount will be less than the fish will eat if fed to satiation. The practice requires both careful application of feed and observation of the feeding behavior of the fish. For species that will feed at the surface, use of an extruded, floating feed is more efficient than use of a sinking feed. The feed should be spread over a large area to facilitate distribution to all fish. In research ponds, it is not unusual to obtain FCR values lower than achieved by commercial producers. For example, researchers often obtain a FCR of 1.4–1.6 in channel catfish culture, but many commercial producers do not feed conservatively and obtain FCR values of 2–3. Large fish do not convert feed as efficiently as smaller fish. Thus, the culture system should be operated to produce fish of the optimum marketable size. A mixture of large and small fish in a pond leads to inefficiency because the larger fish are able to outcompete the smaller fish for feed.

32.6 Dissolved oxygen management Feeding in aquaculture ponds results in additions of nutrients to the water, and this stimulates phytoplankton production. An abundance of phytoplankton results in a high concentration of dissolved oxygen during daylight hours, but the concentration falls during the night reaching its lowest level near dawn. Where there is an ample supply of oxygenated water, some farmers flush ponds when dissolved oxygen concentrations are undesirably low. Water exchange replaces pond water with water of higher dissolved oxygen concentration and flushes out nutrients and phytoplankton. This practice can be effective in improving water quality and preventing low dissolved oxygen where there is enough water to exchange 50 % or more of pond volume daily (Leonard, 1995). However, most production facilities do not have enough water to rely on water exchange for avoiding water quality deterioration. Water exchange also reduces water retention time lessening the waste assimilation capacity of ponds and increasing the pollution potential of effluents.

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Mechanical aeration is the most effective practice for increasing the availability of dissolved oxygen in pond waters (Boyd and Tucker, 1998). Enhancing the dissolved oxygen supply prevents respiratory stress in culture animals and supplies dissolved oxygen for aerobic decomposition, nitrification, and other processes. As a general rule, if there is plenty of dissolved oxygen in pond waters, feeding wastes will be oxidized efficiently and water quality will be adequate for good survival and growth of fish. Mechanical aeration also circulates pond water to avoid thermal stratification and prevent zones with unusually high or low concentrations of water quality variables. Movement of oxygenated water over the bottom prevents anaerobic conditions at the sediment–water interface. During much of the day, waters in ponds are usually supersaturated with dissolved oxygen, and aeration at the surface increases oxygen loss to the air. Thus, aerators often are operated from early evening until mid-morning. There has been some use of axial-flow water circulators during late morning and afternoon to blend oxygen supersaturated surface waters with deeper water to conserve dissolved oxygen produced by photosynthesis (Tucker and Steeby, 1995).

32.6.1 Pond aeration devices There are two basic types of aerators – devices that splash water into the air to provide greater surface air for adsorption of oxygen from the atmosphere, and those that release air bubbles into the water to produce a greater surface area through which oxygen in air of the bubbles enters the water (Boyd, 1998). The driving force causing oxygenation is the difference in the pressure of dissolved oxygen between the air and the water. Aerators function best when the dissolved oxygen concentration in the water is low. Several types of aerators are used in pond aquaculture to include vertical pumps, propeller-aspirator-pumps, paddlewheels, and diffused-air systems. Paddlewheel aerators have been more widely used than other types in ponds of 0.5 ha or larger. The other types have been used mostly in smaller ponds and especially in small ponds on research stations. Considerable effort was devoted in the USA to design and performance testing of aerators for aquaculture during the 1980s and early 1990s (Ahmad and Boyd, 1988; Boyd and Watten, 1989; Moore and Boyd, 1992). A highly efficient design for a floating, electric paddlewheel aerator was developed (Boyd, 1998); aerators based on this design usually transfer more than 2 kg O2/kW⋅h. Paddlewheel aerators of this type are used almost exclusively in catfish farming in the USA. Typical oxygen-transfer rates for other types of aerators used in fish farming are as follows: propeller-aspirator-pumps, 1.6 kg O2/kW⋅h; vertical pumps, 1.4 kg O2/kW⋅h; diffused-air systems, 1.0 kg O2/kW⋅h. The Taiwan-style, floating, electric, paddlewheel aerators have been widely used in Asia. These devices will transfer 1.2–1.6 kg O2/kW⋅h, and

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their service life is less than that of paddlewheel aerators used in channel catfish farming in the USA. In a modification of the Taiwan paddlewheel aerator called a ‘long-arm’ aerator, the paddlewheel is floated in the pond and powered by a small diesel engine or electric motor positioned on the pond bank. There has been little effort to improve aerator design in Asia, but the aerators used there possibly could be improved greatly by modifying paddlewheels based on previous research in the USA.

32.6.2 Water circulators Several types of water circulators also have been used in aquaculture. The most common ones are axial-flow, propeller pumps directed downward or horizontally, and air-lift pumps. Both types of circulators cause increased oxygenation by creating turbulence at the water surface, and air-lift devices also release bubbles of air into the water. The amount of aeration affected by axial-flow and air-lift devices is modest when compared with aerators (Boyd, 1998).

32.6.3 Pond aeration and circulation Aeration is widely used in channel catfish culture in the USA, and it is sometimes used in intensive tilapia culture in several nations. There is some use of mechanical aeration in pond fish culture in Asia, but most production is from unaerated ponds. This probably is because freshwater fish production in Asia is mainly for domestic markets where prices are too low to allow investment in aeration. In channel catfish culture, production of about 3000 kg/ha is possible without aeration. However, tractor-powered paddlewheel aerators are used if dissolved oxygen concentration is too low. Floating aerators are used to increase production to a greater level. It usually is assumed that 1 kW of mechanical aeration with floating, electric, paddlewheel aerators will allow 700–800 kg fish production above that possible in unaerated ponds. Aeration at about 6 kW/ha is commonly used and standing crops of fish may reach 7000 or 8000 kg/ha. A 7.5 kW aerator costs about $666/kW installed, maintenance is about $67/kW/y, and a service life of 10 years is expected. Aerator purchase, installation, and maintenance amortized over 10 years is $134/kW/y or $804/ha/y. Aerators are usually operated about 8 h/night from May through October (1224 h or 7344 kW⋅h/ha). Electricity costs about $0.075/kW⋅h or $550.80/ha. The total aeration cost is $1354.80/ha/y, and cost of aeration per kilogram is roughly $0.30/kg for a resulting increase in production of 4500 kg/ha. The farm gate value of channel catfish is typically $1.40–1.60/kg. Aeration cost is roughly 18–21 % of the value of the extra production resulting from aeration. Water circulation devices have potential for improving the efficiency of aeration by conserving dissolved oxygen from photosynthesis within the

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water column. Research has provided conflicting results, and the cost benefit ratio of circulators and aeration versus aeration alone has not been established (Tucker and Steeby, 1995). Few commercial fish producers are willing to invest in water circulators. Automated devices for stopping and starting aerators in response to dissolved oxygen concentrations are a possible energy savings technique. A number of channel catfish farmers in the USA have installed such devices, but the economy of this practice has not been documented. In channel catfish farming, all aerators are usually positioned at one location in the deep end of ponds to direct water along the long axis. In smaller ponds, several aerators may be positioned to cause circular water movement. Research is needed to ascertain the best way of positioning aerators in ponds. The ideal positioning would allow fish to readily access oxygenated water, ensure that water entering the aerator is relatively low in dissolved oxygen concentration, and avoid erosion of pond embankments by aerator-generated currents.

32.7 Pond amendments The main techniques for increasing aquaculture production and enhancing water quality in ponds are fertilization and liming, feeding, and mechanical aeration. A few other treatments, however, may be applied in efforts to improve water quality. The most common are oxidants, algicides, probiotics, mineral amendments, and flocculants.

32.7.1 Oxidants Potassium permanganate (KMnO4) has been recommended for oxidizing substances and enhancing dissolved oxygen concentration (Lay, 1971). This practice is ineffective and often counterproductive (Boyd and Tucker, 1998). Although no longer used to combat dissolved oxygen depletion, certain fish diseases are treated with potassium permanganate. Calcium hypochlorite [Ca(OCl2)], has been applied to ponds to thin algal populations, oxidize dissolved organic matter, and improve water quality. Only small doses can be applied because the resulting hypochlorous acid is highly toxic to fish. Studies revealed the amount of calcium hypochlorite that could be safely applied to ponds was too low to kill phytoplankton or oxidize dissolved or suspended organic matter (Potts and Boyd, 1998). Chlorination of ponds during grow-out is seldom practised anymore, but some farmers may treat newly-filled ponds with 20–30 mg/L calcium hypochlorite to kill disease organisms (Potts and Boyd, 1998). The hypochlorous acid will dissipate within a few days and fish can be stocked. Sodium nitrate (NaNO3) may be applied to pond waters to maintain nitrate in water in contact with bottom soil. Nitrate is used by denitrifying

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bacteria as a source of oxygen for decomposing organic matter. Nitrate is converted to nitrogen gas and lost to the atmosphere. Denitrification often is illustrated by the following equation in which methanol (CH3OH) is the source of organic matter: 6 NO−3 + 5CH 3OH → 5CO2 + 3N 2 + 7 H 2 O + 6OH −

[32.7]

The presence of nitrate at the soil–water interface will poise the redox potential at a level sufficient to prevent hydrogen sulfide and other reduced, potentially-toxic microbial metabolites from diffusing into the water column (Boyd, 1995). Hydrogen peroxide (H2O2) can be used as an emergency supply of dissolved oxygen: 2 H 2 O2 → O2 + 2 H 2 O

[32.8]

Hydrogen peroxide decomposes spontaneously in water to release molecular oxygen. The yield of dissolved oxygen is 47 % of the weight of hydrogen peroxide or 2.12 mg/L of H2O2 will provide 1 mg/L dissolved oxygen. Hydrogen peroxide concentration varies from about 1.5 % in household disinfectant to 70 % or more in rocket propellant. Because it is highly unstable, concentrations above 10–20 % are hazardous to handle. Thus, rather large applications of hydrogen peroxide solution must be applied for emergency aeration. Mechanical aeration is a cheaper source of dissolved oxygen but, where aeration devices are not available, hydrogen peroxide treatment can prevent fish kills. Calcium peroxide (CaO2) and other more stable peroxides have also been used as oxidizing agents in ponds. Calcium peroxide releases oxygen as follows: 2CaO2 + 2 H 2 O = 2Ca(OH )2 + O2

[32.9]

The yield of oxygen from calcium peroxide is about 22.2 % by weight.

32.7.2 Herbicides Problems with aquatic macrophytes in aquaculture ponds usually result from shallow areas, bad management, or both. Underwater weed problems can usually be prevented if ponds have no areas less than 30 cm deep, and fertilizers are applied to establish plankton turbidity immediately after filling. This procedure prevents macrophytes by creating shade to prevent light penetration to the pond bottom. Macrophytes that float on the surface are not affected by shading. Macrophytes can often be controlled by adding herbivorous fish to ponds but, in some situations, herbicides must be used (Boyd and Tucker, 1998).

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Herbicides should be used as a last resort, and instructions on the product label should be followed strictly. Macrophytes will regrow quickly in shallow or clear water, and herbicide treatment is often a temporary solution. Likewise, algicides will not provide a lasting reduction in phytoplankton where there is an abundant supply of inorganic nutrients. Problems with off-flavor in fish resulting from blue-green algae typically are treated with algicides. The most common compound is copper sulfate. It usually is applied at 1/100 of the total alkalinity concentration. Chelated copper compounds are much more expensive than copper sulfate, but copper sulfate can be as effective as chelated compounds if used at a slightly greater concentration (Masuda and Boyd, 1993). Copper sulfate is quickly lost from pond water and sequestered in sediment (Han et al., 2001; McNevin and Boyd, 2004). Sediment in aquaculture ponds usually has a large capacity to absorb and retain copper (Silapajarn and Boyd, 2006). Thus, frequent use of copper compounds for controlling algae responsible for off-flavor is not likely to cause copper pollution in pond waters or in water bodies receiving pond effluents. Fish do not absorb and store large amounts of copper following copper algicide treatments. The herbicide diuron also has been used to control algae responsible for off-flavor in channel catfish ponds (Boyd and Tucker, 1998).

32.7.3 Microbial products Microbial products used in aquaculture include cultures of living bacteria, bacteria mixed with enzymes, and enzyme preparations. Vendors of these materials make claims about water quality benefits of microbial products as follows: greater decomposition of organic matter, improved nitrification and denitrification, higher dissolved oxygen concentration, less phytoplankton growth, fewer blue-green algae, etc. These claims are questionable because microorganisms are ubiquitous. Factors controlling their growth are warmth, favorable pH, moisture, suitable substrate, carbon : nitrogen ratio, and availability of dissolved oxygen (Boyd and Tucker, 1998). If substrate is present in the pond environment and not utilized efficiently by the microorganisms, is it for lack of microorganisms or extracellular enzymes? Might not the reason be that an environment factor, e.g. pH, dissolved oxygen concentration, C : N ratio, etc., is not within the optimum range for microbial activity? Many companies make microbial products for aquaculture, and some scientists advocate use of these products. Use of microbial products is especially widespread in Asia (Gräslund et al., 2003). Studies have not demonstrated that microbial products improve pond water quality or bottom soil condition (Boyd and Silapajarn, 2006). Mischke (2003) sums up the results of studies on microbial products as follows: ‘Currently, the most efficient way to keep the pond environment suitable for microbial processes is through normal pond management. Maintaining adequate dissolved oxygen

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levels through aeration and careful feeding practices are more viable strategies for water quality maintenance than adding expensive commercial formulation.’

32.7.4 Mineral amendments Sodium chloride It is not common to increase salinity in ponds for freshwater species and, in some cases, brackish water species may be grown in low-salinity water at inland locations. Sodium chloride (NaCl) from granular salt or brine solutions from coastal, seawater evaporation basins have been used to increase salinity in ponds (Limsuwan et al., 2002). The main use of sodium chloride in freshwater aquaculture ponds has been to increase chloride concentration to counteract nitrite toxicity (Boyd and Tucker, 1998). Chloride and nitrite are transported across gill membranes and released into fish blood by the same carrier mechanism. Greater chloride concentration in pond water allows fish to tolerate a higher nitrite concentration. Addition of sodium chloride to provide a chloride : nitrite–nitrogen ratio of 20 : 1 will avoid nitrite toxicity (Boyd and Tucker, 1998). In Alabama, farmers typically apply enough salt (98 % NaCl) to ponds once a year to increase chloride to 100 mg/L. This usually maintains chloride concentrations above 50 mg/L for a year (Tavares and Boyd, 2003). Calcium sulfate Some well waters on coastal plains contain high concentrations of total alkalinity (mainly from bicarbonate) but low concentrations of calcium (Hem, 1970). This results when water infiltrates through limestone into an aquifer with a geological matrix high in sodium. Calcium in infiltrating water exchanges for sodium in the geological matrix leaving a water high in bicarbonate (alkalinity) and low in calcium (hardness). Photosynthesis in such waters can cause pH to rise to 12 or more because carbonate does not precipitate at low calcium concentration. Calcium sulfate (CaSO4⋅2H2O) treatment will increase calcium concentration to precipitate carbonate and limit the rise in pH (Wu and Boyd, 1990). Usually, enough calcium sulfate is added to increase hardness to the same concentration as alkalinity. The molecular weight ratio of CaSO4⋅2H2O : CaCO3 is 172 : 100. Thus, the treatment rate may be calculated as follows: Gypsum dose ( mg L ) = ( Total alkalinity − Total hardness) 1.72

[32.10]

Potassium and magnesium salts There is a growing interest in producing brackish water or marine fish in inland ponds supplied by brackish water from saline aquifers or other

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sources. Proportionality among major ions is often different in inland, saline waters than in seawater (Boyd and Thunjai, 2003). Potassium and magnesium concentrations in particular may be too low for survival and growth of fish (Fielder et al., 2001). Shortages of these two ions can be overcome by additions of mineral amendments (Boyd et al., 2007b) such as muriate of potash (KCl), potassium magnesium sulfate sold under the trade name KMag® (18 % K, 11 % Mg, and 22 % S), and magnesium sulfate MgSO4⋅7H2O. Currently, most producers attempt to adjust potassium and magnesium concentrations to those that would be expected in normal seawater diluted to the salinity of water in the particular culture system (Boyd and Thunjai, 2003; Boyd et al., 2007b). Research is needed to better define the dissolved potassium and magnesium requirements of shrimp and finfish species grown in inland, saline-water systems. Flocculants Gypsum and aluminum sulfate or alum [Al2(SO4)3⋅14H2O] may be applied to ponds to lessen turbidity by flocculating suspended clay particles and causing them to precipitate. Colloidal clay particles have a net negative charge. Like charges repel, and colloidal particles remain suspended against the force of gravity. The charge on colloidal clay particles can be neutralized by increasing the concentration of cations, especially divalent or trivalent ones, to neutralize the negatively charged clay particles. Uncharged particles bump together and flocculate. The resulting floc will quickly settle from relatively still water. The two most popular flocculants are gypsum and alum, but other compounds such as lime, aluminum chloride, ferric sulfate, and ferric chloride have been used (Boyd and Tucker, 1998). The treatment rate for flocculants is usually determined in a laboratory test in which the lowest concentration necessary to clear turbid water is ascertained. Usual treatment rates for gypsum and alum are 200–300 mg/L and 20–30 mg/L, respectively (Boyd and Tucker, 1998). Gypsum treatment increases calcium concentration in water providing residual protection against turbidity, but alum has no residual effect. The best approach to clearing ponds of clay turbidity is to eliminate the source of turbidity, treat soft waters with agricultural limestone for this may clear turbidity, and use coagulants only if turbidity remains. Alum must be used carefully in ponds because it creates acidity: Al 2 (SO4 )3 ⋅ 14 H 2 O = 2 Al 3+ + 3SO24 − + 14 H 2 O

[32.11]

2 Al 3+ + 6 H 2 O = 2 Al(OH )3 ↓ + 6 H +

[32.12]

One molecular weight of alum (590 g) can produce 6 H+ equal to three molecules of CaCO3 or 300 g of total alkalinity. Thus, an alkalinity reduction of about 0.5 mg/L can be expected when 1 mg/L alum is applied. Low pH

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can be avoided by limiting the alum treatment rate to a concentration no greater than the total alkalinity concentration. Aluminum and iron compounds can precipitate phosphorus from water because Al3+ and Fe3+ react with phosphate to form insoluble aluminum phosphate. Calcium additions also favor precipitation of insoluble calcium phosphate from water – especially if pH is high. Research showed that the application of aluminum and iron sulfates and chlorides and calcium compounds reduced phosphorus concentrations in ponds (Masuda and Boyd, 1994b; Rowan, 2001). This practice has not been adopted by commercial producers, but it should be evaluated.

32.8 Pond bottom treatments The bottom of a new aquaculture pond usually consists of subsoil, for the top soil was removed and used in forming the embankments or dam. A new pond bottom typically has a low concentration of organic matter, nitrogen, phosphorus, and other nutrients. As soon as aquaculture is initiated, the pond begins to develop an aquatic soil consisting of sediment resulting from erosion of the pond embankments, external inputs of particles eroded from watersheds, and deposition of organic matter added for fertilizer, dead plankton, uneaten feed, and fish feces (Munsiri et al., 1995). The depth of sediment increases in the deeper waters of ponds at rates of 0.5–1 cm per year in ponds supplied with clear water to several centimeters per year in ponds with highly turbid water supplies (Tepe and Boyd, 2002). The concentration of organic water in sediment usually reaches an equilibrium level of 2–4 % organic carbon after two or three years, but the amount of carbon in the bottom continues to increase over time because the layer of organically-enriched sediment increases in thickness (Munsiri et al., 1995). Nitrogen concentration is a function of organic carbon concentration, because most nitrogen in sediment is in organic combination. Concentrations of phosphorus in sediment increase over time, but sediment phosphorus is tightly bound and not highly soluble (Masuda and Boyd, 1994a). Nitrification and denitrification tend to be linked in ponds and to occur mainly in sediment (Hargreaves 1997, 1998). The main concerns about pond bottom condition are excessive sediment accumulation, high organic carbon concentration, and low pH. The most common management procedures for pond bottoms are dry-out following draining to stimulate decomposition of reactive organic carbon, tilling of dry pond bottoms to enhance soil aeration, liming of acidic soils, and removal of sediment. Application of these practices to tilapia, carp, and catfish ponds in Thailand has maintained good bottom soil quality for 20–40 years (Thunjai et al., 2004; Wudtisin and Boyd, 2006). Sediment removal is effective in improving bottom soil quality. Yuvanatemiya and Boyd (2006) showed that concentrations of organic

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carbon were reduced by about one-fourth, but the reactive organic carbon was reduced 10-fold. Concentrations of phosphorus and other nutrients were reduced almost to original levels by sediment removal. Sediment removed from pond bottoms should be used to repair erosion on embankments or disposed of outside of ponds in a manner to avoid erosion by rainfall and turbidity and sedimentation in nearby waters.

32.9

Water quality monitoring

The concentration of suspended soil particles in source water is important in ponds. Excessive concentrations of fine suspended solids limit light penetration leading to low phytoplankton productivity. High concentrations of coarse suspended solids result in excessive sedimentation. Other critical variables related to the water supply are the degree of mineralization of the water (total dissolved solids), pH, concentrations of total alkalinity and total hardness, and possible presence of toxic pollutants. During culture, phytoplankton abundance, availability of dissolved oxygen, and concentrations of carbon dioxide, ammonia nitrogen, nitrite nitrogen, and hydrogen sulfide may be of concern. In regions with a history of aquaculture, producers know whether or not waters are suitable for aquaculture and rely on management practices that have been successful in the past. This approach has deficiencies, for producers may not be aware of limitations in water quality that could be corrected through modern management techniques. Nevertheless, water analysis requires a substantial investment in equipment and a knowledge of the relationship between water analysis results and production. Small-scale producers often do not have the financial ability or knowledge necessary to employ water analysis in pond culture. Water analysis is used widely by large-scale producers worldwide. For example, in the USA, channel catfish producers often measure dissolved oxygen concentration daily, nitrite concentration weekly, and total ammonia nitrogen occasionally. In sportfish production in the USA, pond owners measure total alkalinity and Secchi disk visibility to provide information needed for effective liming and fertilization. Tilapia producers often measure dissolved oxygen, Secchi disk visibility, and total ammonia nitrogen concentration in ponds. There is increasing interest in reducing pollution from aquaculture and, in some nations, governments may require measurements of effluent quality. There is also a growing number of eco-label certification programs for aquaculture. The programs usually require effluent monitoring for several water quality variables. Portable meters should be used to measure specific conductance, temperature, turbidity, dissolved oxygen, and pH. Water analysis kits are available for determination of total alkalinity, total hardness, and potentially

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toxic metabolites. Government agencies may require effluent analyses to be made by governmental or commercial laboratories, but most certification programs allow producers to make effluent analyses on site using portable instruments or water analysis kits. If a problem with toxic pollutants in incoming water is suspected, samples should be sent to a commercial laboratory for analysis.

32.10 Pond effluents Efficient use of resources to lower production costs is the underlying theme of this chapter. Feed, particularly, should be used efficiently because it is often the largest production cost. However, the conversion to fish flesh of organic carbon, nitrogen, and phosphorus added to ponds is relatively low. Usually, not more than 5–10 % of organic carbon and 20–40 % of nitrogen and phosphorus applied to ponds is recovered in fish biomass (Boyd et al., 2007a). Nutrients not recovered in harvest biomass enter the pond water. Suspended organic carbon is food for filter-feeding animals, microorganisms degrade organic carbon to carbon dioxide, and carbon accumulates in sediment (Gross et al., 2000). Some nitrogen is deposited in organic matter, but most is converted to ammonia through metabolism of the culture species and heterotrophic microorganisms. Ammonia may diffuse into the atmosphere (Gross et al., 1999), but it is also oxidized to nitrate by nitrifying bacteria. Nitrate is reduced to nitrogen gas by denitrifying bacteria in anaerobic zones of sediment. Much phosphorus is adsorbed by bottom soil (Masuda and Boyd, 1994a). Nevertheless, natural processes cannot remove all of the nutrients, and concentrations of dissolved and particulate carbon, ammonia nitrogen, nitrate, soluble and particulate organic nitrogen, and soluble and particulate phosphorus increase in pond water. Nutrients stimulate phytoplankton growth, and this depresses night time dissolved oxygen concentration. Mechanical aeration used to supplement natural supplies of dissolved oxygen suspends solids in pond water. Activity of some culture species also resuspends sediment particles, and sediment is resuspended when ponds are harvested. Pond waters contain higher concentrations of organic carbon, total nitrogen, total phosphorus, total suspended solids, and biochemical oxygen demand than normally found in waters into which ponds discharge. Concentrations of these variables are especially elevated in draining effluent (Tucker and Hargreaves, 2008). Environmental advocacy groups have expressed concern about water pollution caused by aquaculture effluents (Goldburg and Triplett, 1997; Naylor et al., 1998, 2000). In response, governments are developing aquaculture effluent regulations, and aquaculture producer associations and advocacy groups are working with producers to encourage pollution

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reduction practices. Consumers are more concerned than in the past about environmental issues, and supermarkets and restaurants are seeking aquaculture products produced by environmentally-responsible methods. Some buyers are developing production standards and seeking producers willing to comply with their standards. Aquaculture eco-label certification programs have also been initiated, and some major buyers seek certified aquaculture products. Most developed nations and many developing countries have made aquaculture regulations. The aquaculture effluent regulation for the USA (Federal Register, 2004) is an example. This rule states that all concentrated aquatic animal production (CAAP) facilities must have a National Pollutant Discharge Elimination System (NPDES) permit. A warm-water CAAP facility is defined as one that produces more than 45 454 kg per year and discharges 30 days or more annually. However, days in which discharge is the result of excess runoff are not considered discharge days. Cold-water (CAAP) facilities include those that produce more than 9090 kg per year and discharge 30 days or more per year excluding excess runoff. A definition of excess runoff was not provided but, in Alabama, excess runoff has been defined based on daily rainfall amounts (Boyd et al., 2008b). The US aquaculture effluent rule does not specify effluent limitation guidelines (ELGs), but it recommends the use of best management practices (BMPs) to improve the quality and reduce the volume of effluents from CAAP facilities. Many aquaculture support groups, including governmental aquaculture agencies, promote BMPs to lessen water pollution by aquaculture. These BMPs usually focus on prevention of erosion in and around ponds, conservative fertilization, use of high-quality feeds with optimum nitrogen and phosphorus levels, conservative feed management, reduction in water exchange, measures to lessen overflow after rains, harvest methods that minimize discharge and sediment resuspension, effluent treatment by sedimentation, and water reuse. The majority of the BMP programs for voluntary adoption and for marine shrimp, cage culture of salmon, culture of trout in flow-through systems, and marine, bivalve shellfish. Few have been prepared specifically for pond culture of freshwater species. The movement towards lessening the water pollution by aquaculture is well underway. In the future, there will be increasing use of BMPs to allow compliance with effluent standards imposed by governmental agencies, buyers, and eco-label certification programs. Pond aquaculture is becoming increasingly intensive. Prevention of water pollution will require a major effort by producers, and it will increase production cost. It is regrettable that buyers seem unwilling to pay more for aquatic animals produced by responsible methods. Most of the cost of reducing water pollution in aquaculture will probably be borne by producers, and their only benefit will be market access. Another unfortunate aspect of the responsible aquaculture movement is that it focuses on species produced for export. There is little interest in promoting responsible aquaculture by small-scale farmers

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producing for the domestic market – the majority of aquaculture in many nations.

32.11 Future trends Pond aquaculture is the major method for increasing the world supply of finfish, and it will probably continue to be the dominant source of production. Moreover, the tendency towards more intensive production will likely continue. This will require greater use of feeds and less dependence upon manures, agricultural by-products, and fertilizer. Mechanical aeration will be applied more widely and intensively. More attention to bottom soil condition also will be necessary. There are several areas in which research is especially needed as listed below: • • • •

optimum pond shape and size; improvements in water reuse; feed management to lower feed inputs and improve the FCR; more efficient aerators, optimum operating schedules, and better positioning of aerators; • protection of embankments from erosion; • optimization of production intensity with respect to profitability; • use of water quality amendments – especially bacterial products. Improvements in production practices would improve the efficiency and profitability of pond culture. The sustainability of aquaculture also is an important consideration that cannot be achieved through greater production efficiency alone (Boyd et al., 2007a). There should be more emphasis on siting and operating aquaculture facilities to avoid destruction of wetlands and other sensitive habitats, to lessen negative impacts on biodiversity, and to reduce water pollution. Greater efficiency in use of land, water, energy, fish meal, and other resources also will be necessary for the development of sustainable pond aquaculture.

32.12 References ahmad t and boyd c e (1988) Design and performance of paddle wheel aerators, Aquacultural Engineering, 7, 39–62. boyd c e (1982) Hydrology of small experimental fish ponds at Auburn, Alabama, Transactions of the American Fisheries Society, 111, 638–44. boyd c e (1990) Water Quality in Ponds for Aquaculture, Alabama Agricultural Experiment Station, Auburn University, AL. boyd c e (1995) Bottom Soils, Sediment, and Pond Aquaculture, New York, Chapman and Hall.

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boyd c e (1998) Pond water aeration systems, Aquacultural Engineering, 18, 9–40. boyd c e (2007) Nitrification important process in aquaculture, Global Aquaculture Advocate, 10, 64–6. boyd c e and silapajarn o (2006) Effluence of microorganisms on water and sediment quality in aquaculture ponds, in Ray R C (ed.), Microorbial Biotechnology in Agriculture and Aquaculture, Enfield, NH, Science Publishers, 261–85. boyd c e and thunjai t (2003) Concentrations of major ions in waters of inland shrimp farms in China, Ecuador, Thailand, and the United States, Journal of the World Aquaculture Society, 34, 524–32. boyd c e and tucker c s (1995) Sustainability of channel catfish farming, World Aquaculture, 26, 45–53. boyd c e and tucker c s (1998) Pond Aquaculture Water Quality Management, Boston, MA, Kluwer Academic. boyd c e and watten b j (1989) Aeration systems in aquaculture, Reviews of Aquatic Science, 1, 425–72. boyd c e, queiroz j, lee j, rowan m, whitis g n and gross a (2000) Environmental assessment of channel catfish Ictalurus punctatus farming in Alabama, Journal of the World Aquaculture Society, 31, 511–44. boyd c e, tucker c, mcnevin a, bostick k and clay j (2007a) Indicators of resource use efficiency and environmental performance in fish and crustacean aquaculture, Reviews in Fisheries Science, 15, 327–60. boyd c a, boyd c e and rouse d b (2007b) Potassium budget for inland, saline water shrimp ponds in Alabama, Aquacultural Engineering, 36, 45–50. boyd c a, pengseng p and boyd c e (2008a) New nitrogen fertilization recommendations for bluegill ponds in the southeastern United States, North America Journal of Aquaculture, 70, 308–13. boyd c e, pine h, boyd c a and hulcher r (2008b) Excess runoff contribution to daily discharge frequency of channel catfish farms in Alabama, Journal of World Aquaculture Society, 39, 490–9. cathcart t p, pote j w and rutherford d w (1999) Reduction of effluent discharge and groundwater use in catfish ponds, Aquacultural Engineering, 20, 163–74. elser j j, bracken m e s, cleland e e, gruner d s, harpole w s, hillebrand h, ngai j t, seabloom e w, shurin j b and smith j e (2007) Global analysis of nitrogen and phosphorus limitation of primary producers in freshwater, marine and terrestrial ecosystems, Ecology Letters, 10, 1135–42. federal register (2004) Effluent limitation guidelines and new source performance standards for the concentrated aquatic animal production point source category: final rule, Federal Register, 69(162), Washington, D C, Office of Federal Register, National Archives and Records Administration. fielder d s, bardsley w j and allan g l (2001) Survival and growth of Australian snapper, Pagrus auratus, in saline groundwater from inland New South Wales, Australia, Aquaculture, 201, 73–90. goldburg r and triplett t (1997) Murky Waters: Environmental Effects of Aquaculture in the US, Washington, DC, Environmental Defense Fund. gräslund s, holmström k and wahlström a (2003) A field survey of chemicals and biological products used in shrimp farming, Marine Pollution Bulletin, 46, 81–90. gross a, boyd c e and lovell r t (1999) Effects of feed protein concentration and feeding rate combinations on quality of pond water and effluent in channel catfish culture, Israeli Journal of Aquaculture/Bamidgeh, 51, 47–57. gross a, boyd c e and wood c w (2000) Nitrogen transformations and balance in channel catfish ponds, Aquacultural Engineering, 24, 1–14.

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han f x, hargreaves j a, kingery w l, huggett d b and schlenk d k (2001) Accumulation, distribution, and toxicity of copper in sediments of catfish ponds receiving periodic copper sulfate applications, Journal of Environmental Quality, 30, 912. hargreaves j a (1997) A simulation model of ammonia dynamics in commerical catfish ponds in the southeastern United States, Aquacultural Engineering, 16, 27–43. hargreaves j a (1998) Nitrogen biogeochemistry of aquaculture ponds, Aquaculture, 166, 181–212. hem j d (1970) Study and Interpretation of the Chemical Characteristics of Natural Water, Reston, Via, US Government Printing Office. hickling c f (1962) Fish Culture, London, Faber & Faber. lay b a (1971) Applications for potassium permanganate in fish culture, Transactions of the American Fisheries Society, 100, 813–15. leonard s e (1995) Water quality factors influencing striped bass (Morone saxatilis) production in lined ponds, PhD dissertation, Auburn University, AL. limsuwan c, somsiri t and silarudee s (eds), (2002) The appropriate salinity level of brine water for raising black tiger prawn under low-salinity conditions, Aquatic Animal Health Research Institute Newsletter, Department of Fisheries, Bangkok. masuda k and boyd c e (1993) Comparative evaluation of the solubility and algal toxicity of copper sulfate and chelated copper, Aquaculture, 117, 287–302. masuda k and boyd c e (1994a) Effects of aeration, alum treatment, liming, and organic matter application on phosphorus exchange between soil and water in aquaculture ponds at Auburn, Alabama, Journal of the World Aquaculture Society, 25, 405–16. masuda k and boyd c e (1994b) Phosphorus fractions in soil and water of aquaculture ponds built on clayey ultisols at Auburn, Alabama, Journal of the World Aquaculture Society, 25, 379–95. mcintosh r p (1999) Changing paradigms in shrimp farming I. General description, Global Aquaculture Advocate, 2, 40–7. mcnevin a a and boyd c e (2004) Copper concentrations in channel catfish Ictalurus punctatus ponds treated with copper sulfate, Journal of the World Aquaculture Society, 35, 16–24. mezainis v e (1977) Metabolic rates of pond ecosystems under intensive catfish cultivation, MS thesis, Auburn University, AL. mischke c c (2003) Evaluation of two bio-stimulants for improving water quality in channel catfish, Ictalurus punctatus, production ponds, Journal of Applied Aquaculture, 14, 163–9. moore j m and boyd c e (1992) Design of small paddle wheel aerators, Aquacultural Engineering, 11, 55–69. munsiri p, boyd c e and hajek b f (1995) Physical and chemical characteristics of bottom soil profiles in ponds at Auburn, Alabama, USA and a proposed system for describing pond soil horizons, Journal of the World Aquaculture Society, 26, 346–77. naylor r l, goldburg r j, mooney h, beveridge m, clay j, folke c, kautsky n, lubchenco j, primavera j and williams m (1998) Ecology: Nature’s subsidies to shrimp and salmon farming, Science, 282, 883–4. naylor r l, goldburg r j, primavera j h, kautsky n, beveridge m c m, clay j, folke c, lubchenco j, mooney h and troell m (2000) Effect of aquaculture on world fish supplies, Nature, 405, 1017–24. potts a c and boyd c e (1998) Chlorination of channel catfish ponds, Journal of the World Aquaculture Society, 29, 432–40.

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redfield a c, ketchum b h and richards f a (1963) The influence of organisms on the composition of sea-water, in Hill M H (ed.), The Sea, New York, Wiley, 26–77. rowan r (2001) Chemical phosphorus removal from aquaculture pond water and effluent, PhD dissertation, Auburn University, AL. schoof r r and gander g a (1982) Computation of runoff reduction caused by farm ponds, Water Resources Bulletin, 18, 529–32. shell e w (1966) Comparative evaluation of plastic and concrete pools and earthen ponds in fish-cultural research, Progressive Fish-Culturist, 28, 201–5. silapajarn o and boyd c e (2006) Copper adsorption capacity of pond-bottom soils, Journal of Applied Aquaculture, 18, 85–92. silapajarn k, boyd c e and silapajarn o (2004) An improved method for determining the fineness value of agricultural limestone for aquaculture, North American Journal of Aquaculture, 66, 113–18. steeby j, kingsbury s, tucker c and hargreaves j (2001) Sediment accumulation in channel catfish production ponds, Global Aquaculture Advocate, 4, 54–6. steeby j a, hargreaves j a and tucker c s (2004) Factors affecting sediment oxygen demand in commercial channel catfish ponds, Journal of the World Aquaculture Society, 35, 322–34. swingle h s and smith e v (1947) Management of farm fish ponds, Bulletin 254, Alabama Agriculture Experiment Station, Alabama Polytechnical Institute, Auburn, AL. tavares l h s and boyd c e (2003) Possible effects of sodium chloride treatment on quality of effluents from Alabama channel catfish ponds, Journal of the World Aquaculture Society, 34, 217–22. tepe y and boyd c e (2002) Sediment quality in Arkansas bait Minnow ponds, North American Journal of Aquaculture, 64, 284–9. thunjai t, boyd c e and boonyaratpalin m (2004) Bottom soil quality in Tilapia ponds of different age in Thailand, Aquaculture Research, 35, 698–705. tucker c s and hargreaves j a (2008) Environmental Best Management Practices for Aquaculture, Oxford, Blackwell. tucker c s and steeby j a (1995) Daytime mechanical water circulation of channel catfish ponds, Aquacultural Engineering, 14, 15–27. wu r and boyd c e (1990) Evaluation of calcium sulfate for use in aquaculture ponds, Progressive Fish-Culturist, 52, 26–31. wudtisin i and boyd c e (2006) Physical and chemical characteristics of sediments in catfish, freshwater prawn and carp ponds in Thailand, Aquaculture Research, 37, 1202–14. yoo k h and boyd c e (1994) Hydrology and Water Supply for Pond Aquaculture, New York, Chapman and Hall. yuvanatemiya v and boyd c e (2006) Physical and chemical changes in aquaculture pond bottom soil resulting from sediment removal, Aquacultural Engineering, 35, 199–205.

33 Superintensive bio-floc production technologies for marine shrimp Litopenaeus vannamei: technical challenges and opportunities C. L. Browdy, J. A. Venero, A. D. Stokes and J. Leffler, Marine Resources Research Institute, USA

Abstract: Since the late 1990s, researchers in the USA have greatly increased stocking densities in zero exchange shrimp production systems reaching harvest biomass levels almost five times higher than typical intensive pond production rates. These increasing production densities reduce the overall cost per unit area and allow for intensification of management inputs. These developments have generated much interest in the dynamics of controlled shrimp production in reduced or zero exchange systems. Components include: (i) use of nursed high-quality genetically improved broodstocks; (ii) high-quality dense feeds applied in a controlled feeding program; (iii) engineered systems which efficiently meet oxygen demands of superintensive culture, maintain optimal growing temperature and water quality, and integrate emergency back-up systems and waste treatment; and (iv) bio-floc-based microbial communities managed to cycle wastes while enhancing shrimp growth. The present paper looks at lessons learned from application of bio-floc production systems technologies for marine shrimp Litopenaeus vannamei focusing on opportunities, technical constraints and research priorities. Key words: shrimp, superintensive, bio-floc, raceway, microbial ecology.

33.1 Introduction In 2006, penaeid shrimps represented the 6th largest aquaculture commodity produced in the world by volume, ranking second overall in terms of value (FAO, 2007). According to the FAO Aquaculture production database, in 2005 penaeid shrimp aquaculture was valued at over 10.6 billion dollars (FAO Fishstat Plus). In the USA alone, shrimp imports were valued at 3.9

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billion dollars, accounting for over 28 % of total edible fishery product imports into the country in 2007 (NOAA, 2008). Per capita consumption of shrimp in the USA has increased over 27 % from 3.2 lbs (1.451 kg) in 2000 to a record 4.4 lbs (1.996 kg) per person per year in 2006, fueled by increasing world aquaculture production and declining shrimp prices. Pond culture technology for production of marine shrimp has advanced quickly. Beginning in the 1970s and 1980s with demonstration of feasibility and opportunities for intensification, the shrimp farming industry expanded rapidly. As a result of this fast growth and relative lack of regulatory controls, by the early 1990s, issues with environmental sustainability and disease control began to surface. Partially in response to these problems, technologies based on reduction or elimination of water exchange were applied in research settings and then in pilot commercial scale with great success (see Browdy et al., 2001a for review). Reduction or elimination of water exchange was shown to have many advantages. It improved stability of pond microbial communities reducing problems with algal blooms and crashes which had complicated management strategies based on pumping and flushing (Clifford, 1994). It reduced problems with self-pollution resulting from eutrophication of receiving waters. In fact, research demonstrated that much of the waste in the system was mineralized in situ during the cycle such that total pollutants released at harvest were much lower than ponds managed with water exchange (Hopkins et al., 1995, 1996). Although reducing water exchange in intensive pond culture necessitated increased aeration to meet oxygen demand from pond microbial communities, aeration cost was shown to be lower than that of pumping and resulted in improved efficiencies (Hopkins et al., 1995). Similarly, growth factors in the natural productivity in the ponds improved production and feed nitrogen conversion efficiencies and corresponding return on investment. Finally, drastic reductions in water exchange allowed for improved biosecurity as introduction of pathogens was reduced, incoming water treatment options became more feasible, and release of contaminated water was reduced (Jahncke et al., 2002). It was perhaps the need to reduce or eliminate introduction of viral pathogens that drove widespread industry reductions in pumping over the 1990s.

33.2 Superintensive bio-floc-based shrimp production systems Since the late 1990s, researchers in the USA have greatly increased stocking densities in zero exchange shrimp production systems (Browdy and Moss, 2005). US producers applying traditional production technologies have been unable to compete on commodity markets as shrimp prices declined. The cost of producing shrimp in the USA is high due to land, labor, energy and effluent compliance costs. Growing seasons are limited, constraining

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consistency in product availability. Regulatory burdens have also contributed to reduced competitiveness of the US shrimp aquaculture industry. Strategies aimed at increasing densities and improving management control in engineered systems have been applied, demonstrating production levels of almost 92 000 pounds per acre per crop (103 000 kg/ha) with reduced water exchange (Otoshi et al., 2007) and up to 62 000 lbs/acre (69 200 kg/ ha) with zero water exchange (Venero et al., 2009). These harvest biomass levels are almost 10 times higher than typical intensive pond production rates and are 6.5 times higher than the most successful current intensive pond production levels. This increase in production density reduces the overall cost per unit area and allows for intensification of management inputs. It also allows for enclosure of production systems in greenhouses or buildings, which is crucial for biosecure year-round production (Fig. 33.1). It is this continuous production which can further multiply annual production levels by a factor of four to almost 368 000 lbs (327 520 kg/ha) per acre per year. Most importantly, consistent year-round production allows for staggering of crops and consistency in supply of fresh product that can be differentiated from commodity imports. In the longer term, research innovation will be directed at significantly reducing production costs to allow shrimp from these systems to compete on commodity markets. These developments have generated much interest in the dynamics of shrimp production in reduced or zero exchange systems. Be they pondbased or superintensive raceways, the establishment and management of microbial bio-flocs which develop in the systems is crucial. The present chapter looks at lessons learned from application of bio-floc production

Fig. 33.1 Greenhouse enclosed raceway system at the Waddell Mariculture Center during winter operation.

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systems technologies for marine shrimp Litopenaeus vannamei focusing on opportunities, technical constraints and research priorities.

33.3 Components of superintensive bio-floc-based shrimp production systems Production of marine shrimp in superintensive systems close to large population centers in the continental USA offers unique opportunities for developing new markets for consistent high-quality fresh local shrimp products (Wirth and Davis, 2001a,b). Consistent production of shrimp on a continuous basis in proximity to large markets can offer opportunities for product differentiation and brand development. At present there are no reliable domestic sources for fresh-never-frozen shrimp, available on a consistent basis. However, achieving consistent year-round production, while maintaining control over production costs, remains a significant technological challenge. High costs of land, labor and energy require increasingly complex engineering systems to achieve high production intensities. Similarly, maintaining high growth rates and consistent survivability requires improved seed stocks and cost-effective specially designed feeds. Finally, environmental sustainability requires innovative approaches to water reuse and waste management. Key components of the present superintensive production system include: (i) availability of improved specific pathogen-free (SPF) shrimp stocks, facility biosecurity and maintenance of the health of the target crop; (ii) high-quality feeds and efficient feeding practices; (iii) advanced systems engineering for heating, oxygenation, particle filtration, water quality monitoring, alarm systems, automated back-up systems and waste management; and (iv) development and management of biofloc communities to support system water quality and boost shrimp performance.

33.3.1 Shrimp stocks Increased availability of high-quality, genetically improved, diseaseresistant post larvae (PL) has resulted in greater consistency in shrimp growth, size at harvest and survival of stocked animals (Browdy, 1998). Economic and biosecurity concerns have forced shrimp producers to consider protection of cultured animals through more stringent production methods – both to avoid diseases and to prevent spread of diseases (Bullis and Pruder, 1998; Pruder, 2005). Since the early 1990s research by the US Marine Shrimp Farming Program has demonstrated the advantages of the stocking of SPF shrimp as the basis for intensive culture systems (Wyban et al., 1992; Browdy, 1998). Since the late 1990s, production has shifted to SPF stocks of Litopenaeus vannamei worldwide. As shrimp prices have declined, producers have been forced to improve efficiency, and reliance on

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high-quality sources of brood stocks has been an important strategy for the most successful producers. Genetic selection has played an important role in improving the growth and survivability of these SPF lines (Argue et al., 2002). Increasingly sophisticated breeding programs have contributed to continuing improvements in growth and resistance to Taura Syndrome Virus (Moss et al., 2005, Argue et al., 2002). At the same time, aquaculture producers have increasingly applied principles of biosecurity, which form the cornerstone of modern agricultural production systems (Moss, 1998, 2000; Moss, et al., 1998; Bratvold and Browdy, 1999; Lotz and Lightner, 2000; Jahncke et al., 2002). The present superintensive production systems technologies rely upon the best fastgrowing SPF stocks available on the market produced in enclosed systems managed under stringent biosecurity protocols. Production efficiencies of the system have been improved by including a nursery phase (Samocha et al., 2003). This provides several advantages including assurance of stock quality and animal health prior to stocking, better control over shrimp stocking numbers, survivability and system biomass estimates during the grow-out cycle, more efficient use of production area by stocking shrimp at higher densities during the nursery phase and shortening the grow-out cycle by stocking animals well into the linear growth portion of the shrimp life cycle (Fig. 33.2). 33.3.2 Feeds and feeding It is well recognized that feed inputs are the major driver of the dynamics of intensive zero exchange shrimp culture systems (Hopkins el al. 1992;

Fig. 33.2 Configuration of Waddell Mariculture Center pilot scale raceway system for nursery production.

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Browdy et al., 2001b). Control of feeding rates is one of the most important factors in assuring optimal shrimp growth while minimizing waste production and controlling variable production costs. Feed formulation is also a critical consideration in terms of feed costs and shrimp growth rates. In general, financial analyses show that small increases in direct costs such as feed can be more than offset by incremental increases in growth. However, inefficient feeding practices or increases in costs without consistent increases in growth have a strong negative effect on financial viability. Recent studies have indicated that controlled application of nutrient-dense high-protein feeds can improve growth performance when compared to lower protein diets offered on an iso-nitrogenous basis (Kureshy and Davis, 2002; Davis and Venero, 2005; Venero et al., 2007, 2008). As discussed below, the formulation of feeds can have an important indirect effect on the system microbial community. Inputs of carbon and nitrogen must be considered as part of overall management of the bio-floc communities upon which system stability depends. Thus, some researchers suggest a lower protein feed formulation strategy to encourage heterotrophic bio-floc production (Avnimelech, 1999; McIntosh, 2000, 2001; Ebeling et al., 2006). Feeding strategies will rely upon careful control of application rates and use of nutrient-dense highprotein feeds with highly digestible ingredients.

33.3.3 Systems engineering The present production system technologies are based on well-mixed raceways to assure resuspension of particulate matter. Flow and mixing are accomplished by aeration and/or injection of recirculated water in grow-out raceways and by use of aeration in nursery raceways. High feeding rates in these systems require significant supplemental aeration in nursery tanks and addition of supplemental oxygen to meet demand of the target crop and of the water column microbial community. In addition to efficient and reliable delivery systems, high rates of oxygen demand necessitate monitoring, alarm and back-up systems to assure oxygen supply in the event of power or equipment failure. One of the most important engineering components is the design of the enclosures and supplemental heating which will be critical for maintenance of optimal growing temperatures year round in the USA. The high carrying capacities projected for the systems require external particle filtration, fractionation or settling to reduce total suspended solid loads. Economically efficient operation of these systems requires near continuous operation year round, yielding 3.5–4 crops per year for each raceway. This necessitates the ability to harvest a system and restock it with juvenile shrimp ideally within 24 h while maintaining intact the bio-floc community critical for the system’s success. This requires reliable mechanical systems and back-up systems that can be serviced and repaired over long periods without necessitating delays or abortion of grow-out operations. Sustain-

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ability and continuous operation of the systems away from the coast necessitates waste collection, treatment and return of treated water. Treatment systems should be designed to dewater and digest volatile solids; to promote denitrification, reducing nitrate levels and restoring alkalinity; and to recover salts (Boopathy et al., 2005). By returning treated wastewater and supplementing for lost salts and trace minerals, system water quality can be maintained while minimizing the need for fresh seawater inputs and solid waste disposal. Any solid or liquid waste that might result should be of such quality that it causes no negative environmental impacts and which ideally could provide an auxiliary source of revenue. 33.3.4 Bio-floc microbial communities When shrimp are reared at high densities without water exchange a biofloc develops in the ponds that has several advantages for shrimp culture. These include increased recycling of waste and mineralization of nutrients within the system and improved water quality stability (Hopkins et al., 1996). The diverse microbial community in the bio-floc-dominated systems is thought to increase competition with potentially pathogenic microbes including vibrios, reducing problems with non-excludable pathogens (Bratvold and Browdy, 1999). In addition, the natural productivity associated with these bioflocs has been shown to provide growth-enhancing factors which improve shrimp production (Moss et al., 1992; Moss, 1995; Moss and Pruder, 1995; Decamp et al., 2002; Moss, 2002; Burford et al., 2004, Wasileski et al., 2006). Additional research has been conducted on the dynamics of the microbial communities in these systems, methods for measuring microbial activity and some techniques for manipulation of the make-up of these communities (Bratvold and Browdy, 1998; 1999, 2001; Browdy et al., 2001a,b; Burford et al., 2003; Decamp et al., 2003; Ebeling, 2006). Our research demonstrated that, typically, ponds undergo an algal bloom and crash followed by the establishment of a community characterized by fairly stable and efficient processes for removing ammonia and nitrite wastes. Generally bio-flocs include an active heterotrophic community and varying levels of photoautotrophic activity. The relative mix of bacteria and algae with very high feed inputs into the system is dependent upon intensity of cropping of bio-floc particles, resulting light levels, and nutrient balances within the system. Chemoautotrophic nitrifiers colonizing the bio-floc particles and other surfaces make up a third important component of the community in bio-floc systems. Nitrification is a significant part of the overall microbial activity serving to control ammonia and nitrite levels in the system once the community becomes established. High levels of nitrification necessitate supplementation or regeneration of system alkalinity. The relative balance between these community components may vary depending upon different philosophies regarding inputs, solids removal and management protocols.

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33.4 Current research priorities 33.4.1 Integration of research and commercialization efforts Some of the most important factors in reducing production costs are economies of scale. On the other hand, scale-up of current technologies will involve significant opportunities for modifications in current experimental or pilot-scale applications. The development of large-scale commercially viable demonstration projects supported by experts in current state-of-theart research-scale systems has often been the missing link in public and private investments in technology development. This has, at times, resulted in lack of focus in experimental efforts aimed at promoting commercial-scale technology development and in ‘reinventing the wheel’ in some commercial-scale operations. A new paradigm is needed targeting scientific research coupled with commercial-scale applications applying (adopting) a robust and diverse funding base. This could lead to solving of short-term problems associated with scale-up while improving systems applications for improved competitiveness in the medium and long term. Fundamental protocols for the management of superintensive bio-floc systems must be identified, agreed upon and disseminated. Areas where consensus is not achieved should be identified as research priorities and appropriate resources dedicated to solve those problems. While subject to modification in light of new research and local conditions, such protocols nevertheless provide the necessary foundation for successful commercialization. These protocols must be based on achieving clearly defined benchmarks and must use pragmatic indicators to guide management actions towards reaching those benchmarks. Guided by appropriate indicators and benchmarks, a blending of economic costs and biological decisions needs to be made. Modeling efforts which integrate economics and biological responses may provide a comprehensive framework and should be supported. Researchers need to identify critical benchmarks to reduce costs while concurrently determining key biological factors limiting responses and the appropriate indicators for understanding interactions between the two.

33.4.2 Economics and marketing Economic modeling and planning is a key component of developing strategies to improve competitiveness. Accurate prediction of fixed and variable costs combined with effective sensitivity analyses will allow prioritization of efforts aimed at reducing production costs and increasing productivity with the goal of quantifying and optimizing return on investments. As described above, the price of commodity shrimp has been on a downward trend for several years. This can be attributed to increasing supplies of imported white shrimp due to decreasing production costs as production efficiencies improve. While these trends have led to growing global markets

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and per capita consumption rates, dropping commodity prices affect competitiveness of investments in more technology-intensive production methods. Efforts to determine the magnitude of specialty fresh product markets and/or the scope of potential branding or certification premiums are needed. These efforts can be facilitated by measurement and certification of quality in terms of improved environmental sustainability of bio-floc production systems, better flesh quality, food safety and/or higher human health benefits. Credible eco-labeling efforts, organic certification and effective product branding offer opportunities for marketing premiums which can improve competitiveness.

33.4.3 Health management The maintenance of the health of the shrimp under culture is a critical prerequisite to profitability. Health assurance begins with use of SPF stocks and control of facility biosecurity. Tools for the evaluation of health status on an on-going basis to see sub-acute indications of declines in fitness are needed. Existing tools for rapid diagnosis and longer-term confirmation of acute pathologies should be refined. Emerging non-listed pathogens and disease caused by non-excludable pathogens will continue to be a problem. New pathogens will need to be studied to develop diagnostics and improve basic knowledge. Methods to avert disease, immunostimulation and approved chemotheraputants for prophylaxis or to treat disease outbreaks from non-excludable pathogens (including vibriosis) will be needed. This will necessitate better understating and control of microbial communities as addressed below. Improved appropriate applications for probiotics and better understanding of how to encourage growth of non-pathogenic microbes in the systems is necessary. Fundamental to these efforts are tools to measure immune status, fitness and health of the target crop on an ongoing basis.

33.4.4 Seed supply Genetics and breeding A stable year-round supply of healthy genetically improved fast growing robust PL is a critical prerequisite to industry competitiveness. Basing production of food shrimp on L. vannamei provides an important advantage in that much effort has gone into the closing of the life cycle and genetic selection for this species. Today there are commercial companies in the USA devoted to development of SPF, disease-resistant breeding stocks selected for fast growth. These efforts are currently devoted to producing stocks for shrimp production in open pond systems. A focused breeding effort targeting performance in these biosecure closed systems would speed improvement. This would also avoid potential selection for traits which may be of significance in open pond systems in Asia but which could be nega-

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tively correlated with growth and other critical performance characteristics in the more biosecure superintensive systems. Efforts related to selection for new traits relative to survivability and growth under very high densities or production-related characteristics such as dress-out percentage could also improve competitiveness if new strains are protected and used only in domestic production systems. In the long-term, improvement of genomic enablement for the species will allow for discovery science that could lead to important breakthroughs enhancing production and fitness-related traits. Measurement technologies in the area of genetics and breeding will include objective assessments of heritability, genetic gains, pedigree tracking and inbreeding coefficients. Development of molecular tools should include markers for traits of interest and for stock identification, genome maps and functional genomic tools such as microarrays. Seed production Maturation and hatchery technologies are well developed for L. vannamei although further attention to scalability issues and development of closed system hatcheries have implications for vertical integration, especially during early phases of industry establishment. Nauplii quality and systems efficiencies could be increased by improving maturation systems technologies, eliminating reliance on fresh feeds and otherwise maximizing reproductive performance. Larval culture systems improvements in areas of systems engineering could focus on physical characteristics such as mixing, settling rates, microbiology and water reuse technologies. Systems still rely on live feeds, and continuing incremental improvements in developing nutritious artificial diets could improve production efficiency and consistency. Developments in additives like pre- and probiotics, disinfectants and nutrients to enhance microbial activity could be beneficial. Husbandry improvements in quantifying and assessing larvae and PL, harvest and counting methods and transport techniques could be envisioned. Measurement technologies in the area of maturation and larval rearing could focus on determination of water quality conditions and organic loading, fitness of the broodstock, nauplii, larvae and PL, fecundity in terms of oocyte and sperm development, mating, spawning, egg production, fertilization, hatch and metamorphosis rates.

33.4.5 Production systems Systems engineering for both pond and raceway-based applications of highly intensive closed system shrimp production technologies is one of the most important technical gaps to commercial competitiveness. It involves a highly complex set of interrelated issues many of which are highly sensitive to changes in scale. For enclosed systems, the size and type of enclosure is of paramount importance in terms of capital costs, energy efficiencies and secondary effects on systems operations. Use of barn-type structures, green-

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houses or combinations of the two is a fundamental issue which relates back to importance of light, energy efficiencies and initial investment costs. Interrelationships between economic factors that vary with geographic location and biological factors that vary with operational methodologies complicate optimization. Second, optimal raceway and pond designs, dimensions and materials may also vary according to specifics of the production system, relative cost and availability of materials and management philosophies. Alternative operational systems (automated where appropriate) for aeration and circulation, water heating, systems monitoring and control, filtration, waste treatment, water conditioning and reconditioning and waste disposal offer many different potential options and solutions all of which have important implications for production efficiencies. Areas for which measurements could be applied to drive innovation aimed at simplifying operations and reducing costs include: efficiencies of water use, mixing and oxygen transfer efficiencies, heat transfers and energy efficiencies, accuracy of monitoring and control systems, effectiveness and cost of filtration alternatives and waste treatment efficiencies.

33.4.6 Feed programs Feed is one of the most important components of variable costs. The feed is the driver of the system both in terms of microbial community dynamics as well as shrimp performance. Feed effects on shrimp growth and production, both direct and indirect, are a major component of system financial returns. Feed nutrient quality, nutrient density and physical properties should be optimized to promote cost effectiveness, shrimp growth and condition and water quality stability. Management of feed inputs is critical to maximize growth while preventing water quality deterioration from overfeeding, and optimizing nitrogen conversion efficiencies from feed to shrimp. This would include the amount of feed offered, method of feed distribution, frequency and timing of feeding and opportunities for automation. Critical to achieving these goals is improved measurement technologies to assess shrimp population numbers, distribution throughout the culture system and behavioral response to feeding. Serious difficulties associated with low-visibility bio-floc systems include inaccurate estimation of shrimp mortality and current population size, as well as inadequate understanding of shrimp location within the system. These factors make overfeeding and underfeeding much more likely. These issues must be addressed to improve feeding efficiencies with resultant economic benefits. Feed additives including immunostimulants, gut microflora stimulants (prebiotics) and agents to alter the bio-floc microbial community (probiotics) may offer opportunities to improve shrimp growth, condition and feed conversion efficiency. Feeds and feeding must also be considered in the framework of overall system sustainability. The ratio of live weight unit of forage (reduction) fisheries used to produce one live weight unit of product

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is an important measure of long-term sustainability of global fishery resources. Development of fish meal and fish oil replacements are important in this regard to both sustainability and economic concerns and will become a prerequisite to organic certification. Finally, consideration of the flavor, appearance and human health characteristics of the final product depend upon feed inputs and may offer opportunities for development of specialty finishing diets. Application of measurement science to optimization of a feeds program in terms of costs, financial returns and sustainability of the technology involves both traditional and new approaches. Methods for determination of digestibility, leaching, attractability, nutrient densities, nutrient toxicities, feed conversion efficiencies and shrimp performance are well documented. Further efforts are necessary to better measure feed performance under the conditions in high-density bio-floc systems to improve formulation of feeds specific for these applications. Quantification of contributions from bio-floc natural productivity in small-scale systems can lead to optimizing feed formulations to take advantage of these supplements and not duplicate unnecessary ingredients in the feed. More problematic is measurement of long-term impacts of feeds in systems reusing water for multiple crops. Accumulations of excess nutrients, potentially toxic trace metals and antinutritional factors must be measured. New measures of marine protein conversion efficiencies coupled with benchmarks relevant to both nutritional and sustainability concerns can enhance responsible shrimp culture. Finally, measures of product palatability, appearance and quality in terms of human health effects can offer opportunities for optimizing diets and development of finishing diets with an eye towards improving competitiveness through product differentiation.

33.4.7 Husbandry Some of the most significant challenges to stability and efficiency of production in superintensive bio-floc-based systems relate to husbandry. The optimal stocking density in such systems has yet to be determined, but has significant economic ramifications. The ability to grow shrimp at very high densities through technological innovation is critical to the economic viability of these technologies. Density effects on growth rates, behavioral changes, disease susceptibility, dissolved oxygen delivery, waste management and stability of the microbial community are important considerations that must be addressed before an optimal density can be established. Maintenance of optimal water quality is a challenge in that systems must be designed to maintain growing conditions without any significant use of seawater exchange. At inland locations, protection of aquifers and waste disposal are significant issues. Management of water quality must be accomplished through the exchange of water within the system and through the proactive maintenance of optimal bio-floc communities in the systems over

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individual growing cycles and between multiple cycles over time. It is this management of bio-floc communities which can present one of the greatest opportunities for improvement in competitiveness and one of the greatest risks to consistency over time. Optimizing microbial communities will require improved understanding of both the underlying ecological mechanisms and the relative advantages and disadvantages of the heterotrophic bacterial growth, the blooms and crashes of different algal species, the activity of chemoautotrophs responsible for nitrification, the dynamics of anaerobic denitrifying microbes, as well as the effects of ciliate and rotifer grazers and other bio-floc-associated organisms. Once strategies targeting specific community compositions and levels of microbial activity are designed, development of management protocols to achieve these benchmarks will be necessary. Pragmatic indicators need to be identified or developed that will permit commercial operators to routinely monitor their bio-floc communities as they guide them toward the desired benchmark community structure. Techniques must be optimized for indirect management of the bio-floc community through control of nutrients, solids removal and light quality, and might be developed for direct application of probiotics and algal inoculations. Management of long-term water quality profiles must take into consideration build-up of waste materials, potentially toxic or growth-inhibiting substances, nutrients from feed and evaporative water inputs. Supplementation of micronutrients that may be depleted over time might also be required. Clearly, optimization of husbandry and system management will depend upon accurate measurement and analysis of traditional water quality parameters in real time. Innovation to optimize bio-floc and water management for system productivity, profitability and sustainability will require new strategies for measuring community composition and activity. Better metrics for description or quantification of floc densities and improved understanding of the significance of measures like total suspended solids (TSS), volatile suspended solids (VSS), turbidity, light extinction and chlorophyll are needed. Community composition can be measured through direct microscopic observation and application of more indirect methods such as quantification of algal pigments, or analysis of bacterial strains using DGGE techniques. Measurement of activity will require accurate determination of oxygen fluxes due to respiration and photosynthesis or indirect measurements of changes in nitrogen species. Once accurate estimates are made, models can be developed to better understand carbon and nitrogen fluxes within the system. Similarly, correlations can be made with shrimp growth and fitness to optimize productivity if techniques can be developed to accurately determine shrimp survival, condition and growth in real time. Moreover, it is essential to move beyond research tools used to understand the fundamental dynamics of bio-floc systems to practical, relatively

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inexpensive parameters that can be used routinely to manage production systems on a daily basis. Major innovation in measurement science is still necessary to identify the critical indicators, interpret them in relation to established benchmarks, and then translate those readings into management actions that will optimize shrimp productivity. Development of reliable, easily operated, targeted monitoring systems will be crucial to enable managers to react quickly to changes in water quality.

33.4.8 Stocking and harvesting of nursery systems Nursery systems can contribute to increased profitability in a number of ways. They can provide a buffer to accept PL from hatcheries as they become available. Better facility utilization, short-cutting grow-out cycles, allows for higher production rates per unit area per year. A nursery can allow quarantine and elimination of poorly performing batches of PL in a timely manner, particularly if a pre-stocking acclimation station is included in facility designs. This is particularly important when PL are procured from outside sources. Survivability of juveniles can be more predictable than that of direct stocked PL, improving management during the production cycle. Benefits of nursery systems are maximized by efficient and rapid transfer of juveniles from nursery to grow-out. Optimizing design and operation of nursery systems depends upon appropriate sizing of production units coupled with efficient scheduling of stocking and harvest to allow for efficient cycling of on-growing systems. Development of more accurate methods to estimate survivability and performance of juveniles during this phase would improve management efficiency. Improved techniques for rapid accurate measurement of harvest biomass and condition of juveniles integrated with more automated and efficient transfer technologies could improve control during subsequent grow-out.

33.4.9 Harvesting of grow-out systems Improving overall competitiveness requires appropriate design and husbandry of grow-out systems as described above along with successful harvest strategies. Harvesting systems should be automated and should facilitate water recovery while assuring product quality. Effective postharvest handling techniques for live or fresh shrimp could improve quality delivered to the customer. Growers will need to design harvest timing strategies based on market demands and to develop alternative partial harvest techniques. These efforts will require good models of system economics, of risks associated with delayed harvest and of key market segments. Better control of molt synchrony could improve the percentage of high-quality product. Measurement of compensatory growth and density effects on system productivity can help optimize harvest strategies in

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general and partial harvest strategies in particular. Accurate assessment of product quality is a prerequisite to improvement of harvest and postharvest product handling.

33.5 Conclusions In February 2008, a workshop sponsored by the US Department of Commerce, National Institute of Standards and Technology and National Oceanographic and Atmospheric Administration was convened to identify research priorities. Much of the information in this article was prepared by the authors and reviewed by a committee of experts in the field to provide a basis for facilitated workshop discussions. At the workshop a large number of participants from academia, government, industry and nongovernmental organizations shared ideas in breakout sessions and prioritized research objectives. Priorities were ranked based on the following: • Feasibility: An evaluation of the scientific and technical potential for overcoming technological barriers. What is technically feasible? What can be accomplished through research? How difficult is it to overcome the barrier? • Importance/relevance/urgency: How pressing is the need? How critical to overall success is overcoming this particular gap? • Socioeconomic impact: Projections of expected benefits and consequences. Are results broadly applicable or narrowly focused? What is the relative return on investment made to overcome a gap? The following is the prioritized summary of technology gaps developed after the workshop. • System engineering and life-support systems: Improve the costeffectiveness of life-support technology. Improve system energy efficiency, particularly for maintaining water temperature. Establish standards and specifications for system engineering design, with the goal of improving production efficiency as measured by shrimp growth and production potential. Establish standard management techniques to establish, manage and maintain the structure, abundance and activity of stable bio-floc communities that maximize contributions to shrimp growth and water quality management. Improve methods to collect, dewater, digest and dispose of waste solids. Improve methods for denitrifying, desalting and treating water to reclaim minerals for reuse. • Genetic improvement: Continue to invest in robust selective breeding programs for penaeid shrimp. Develop new and use existing molecular tools to understand the genetic basis of shrimp production performance and disease resistance and apply discoveries to improve selection. Develop methods for monosex female production.

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• Feeds and feeding: Develop feeds that maximize the efficiency of fish meal use, minimize the ecological footprint of all ingredients, improve water quality and maximize the contribution from bio-floc to shrimp growth. Improve techniques for monitoring shrimp density and survival throughout grow-out in order to optimize feeding rates. • Health and biosecurity: Improve understanding of the factors affecting shrimp health and fitness. Develop diagnostic tools that permit rapid assessment of stress and disease. Develop standard biosecurity protocols, including pathogen monitoring and disease control systems. Establish protocols for management response to early warnings of stress or disease. • Value-added products: Develop products that can provide added value or some other premium quality over competitive products and can be distinguished in the market through branding or labeling. Examples include fatty acid enrichment, organic certification, and marketing live or fresh-never-frozen shrimp. • Bio-economic models: Develop accurate, flexible and user-friendly financial models that include sensitivity and risk analysis. Apply marketing studies to determine market depth for value added products. • Larval culture: Develop replacements for live foods in larval culture. Establish standards for post-larval quality and fitness. Develop noninvasive methods to accurately count PL and juveniles. It is hoped that by defining and prioritizing technology gaps, national and international multidisciplinary collaborations which include industry, academia, governmental and non-governmental participants will be facilitated which can continue to improve the environmental sustainability, consistency of production and economic profitability of biofloc based production systems for marine shrimp.

33.6 Acknowledgements The authors would like to acknowledge with our thanks, the input from the technical advisory group and NIST-NOAA aquaculture workshop participants and proceedings editor John Hargreaves for their invaluable contributions to this manuscript. We are grateful to the USDA CSREES US Marine Shrimp Farming Program and Integrated Organic Program, NIST, NOAA, and the South Carolina Department of Natural Resources for financial support. The concept for the workshop and funding for our efforts to improve measurement of key parameters in biofloc systems was developed based on financial and intellectual input from the leadership of the Hollings Marine Laboratory in Charleston SC and the hard work of the staff of the Waddell Mariculture Center in Bluffton SC. This is contribution number 642 from the Marine Resources Research Institute, Marine Resources Division of the South Carolina Department of Natural Resources.

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33.7 References avnimelech y (1999) Carbon/nitrogen ratio as a control element in aquaculture systems, Aquaculture, 176, 227–35. argue b a, arce s m, lotz j m. and moss s m. (2002) Selective breeding of Pacific white shrimp (Litopenaeus vannamei) for growth and resistance to Taura Syndrome Virus, Aquaculture, 204, 447–60. boopathy r, fontenot q and kilgen m b (2005) Biological treatment of sludge from a recirculating aquaculture system using a sequencing batch reactor, Journal of the World Aquaculture Society, 36, 542–5. bratvold d and browdy c l (1998) Simple electrometric methods for estimating microbial activity in aquaculture ponds, Aquaculture Engineering, 19, 29–40. bratvold d and browdy c l (1999) Disinfection, community establishment and production in a prototype biosecure shrimp pond, Journal of the World Aquaculture Society, 30, 422–32. bratvold d and browdy c l (2001) Effects of sand sediment and vertical surfaces (AquaMats™) on production, water quality, and microbial ecology in an intensive Litopenaeus vannamei culture system, Aquaculture, 195, 81–94. browdy c l (1998) Recent developments in penaeid broodstock and seed production technologies: improving the outlook for superior captive stocks, Aquaculture, 164, 3–21. browdy c l and moss s m (2005) Shrimp Culture in Urban, Superintensive Closed Systems, in: Costa Pierce B A (ed.), Urban Aquaculture, Blackwell Science, Oxford, 173–86. browdy c l, bratvold d., stokes a d and mcintosh r p (2001a) Perspectives on the application of closed shrimp culture systems, in Browdy C L and Jory D E (eds), The New Wave: Proceedings of the Special Session on Sustainable Shrimp Culture, Aquaculture 2001, World Aquaculture Society, Baton Rouge, LA, 20–34. browdy c l, bratvold d, hopkins j s, stokes a d and sandifer p a (2001b) Emerging technologies for the mitigation of environmental impacts associated with shrimp aquaculture pond effluents, Asian Fisheries Science, 134, 255–67. burford m a, thompson p j, mcintosh r p, bauman r h, pearson d c (2003) Nutrient and microbial dynamics in high-intensity, zero-exchange shrimp ponds in Belize, Aquaculture, 219, 393–411. burford m a, thompson p j, mcintosh r p, bauman r h, pearson d c (2004) The contribution of flocculated material to shrimp (Litopeaeus vannamei) nutrition in a high intensity, zero-exchange system, Aquaculture, 232, 525–37. bullis r a and pruder g d (1998) Controlled and Biosecure Production Systems. Evolution and Integration of Shrimp and Chicken Models, Oceanic institute, Waimanalo, HI. clifford h c iii (1994) Semiintensive sensation: a case study in marine shrimp pond management, World Aquaculture, 25, 6–12, 98–104. davis d a and venero j a (2005) Rethinking feeding for cultured shrimp, Global Aquaculture Advocate, 10, 78–81. decamp o, conquest l, forster i, tacon a g j (2002) The nutrition and feeding of marine shrimp within zero-water exchange aquaculture production systems: role of eukaryotic microorganisms, in Lee C S and O’Bryen P, (eds), Microbial Approaches to Aquatic Nutrition within Environmentally Sound Aquaculture Production Systems, World Aquaculture Society, Baton Rouge, LA, 79–86. decamp o, cody j, conquest l, delanoy g, tacon a g j (2003) Effect of salinity on natural community and production of Litopenaeus vannamei (Boone), with experimental zero water exchange culture systems, Aquaculture Research, 34, 345–55.

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ebeling j m, timmons m b and bisogni j j (2006) Engineering analysis of the stoichiometry of photoautotrophic, autotrophic, and heterotrophic removal of ammonia– nitrogen in aquaculture systems, Aquaculture, 257, 346–58. fao (2007) The State of World Fisheries and Aquaculture 2006, Food and Agriculture Organization Of The United Nations, Rome. hopkins j s, stokes a d, browdy c l and sandifer p a (1992) The relationship between feeding rate, paddlewheel aeration rate and expected dawn dissolved oxygen in intensive shrimp ponds, Aquacultural Engineering, 10, 281–90. hopkins j s, sandifer p a and browdy c l (1995) A review of water management regimes which abate the environmental impacts of shrimp farming, in Browdy C L and Hopkins J S (eds), Swimming Through Troubled Water: Proceedings of the Special Session on Shrimp Farming, Aquaculture ‘95., World Aquaculture Society, Baton Rouge, LA, 66–75. hopkins j s, sandifer p a, browdy c l and holloway j d (1996) Comparison of exchange and no-exchange water management for the intensive culture of marine shrimp. Journal of Shellfish Research, 13, 441–5. jahncke m l, browdy c l, schwarz m h, segars a, silva j l, smith d c and stokes a d (2002) Application of Hazard Analysis Critical Control Point (HACCP) Principles as a Risk Management Tool to Control Viral Pathogens at Shrimp Production Facilities, Publication Number VSG 02 10, Virginia Sea Grant, Charlottesville, VA. kureshy n and davis d a (2002) Protein requirement for maintenance and maximum weight gain for the Pacific white shrimp, Litopenaeus vannamei, Aquaculture, 204(1–2), 125–43. lotz j m and lightner d v (2000) Shrimp biosecurity: pathogens and pathogen exclusion, in Bullis R A and Pruder G D (eds), Controlled and Biosecure Production Systems. Proceedings of a Special Session – Integration of Shrimp and Chicken Models, Oceanic Institute, Waimanalo, HI, 67–74. mcintosh r p (2000) Changing paradigms in shrimp farming: V. Establishment of heterotrophic bacterial communities, Global Aquaculture Advocate, 3(6), 52–4. mcintosh r p (2001) High rate bacterial systems for culturing shrimp, in Summerfelt, S T et al. (eds). Proceedings from the Aquacultural Engineering Society’s 2001 Issues Forum, Aquaculture Engineering Society, Shepherdstown, WV, 117–29. moss s m (1995) Production of growth-enhancing particles in a plastic-lined shrimp pond, Aquaculture, 132, 253–60. moss s m (1998) U.S. Marine Shrimp Farming Program Biosecurity Workshop, Oceanic Institute, Waimanalo, HI. moss s m (2000) Biosecure shrimp production: Emerging technologies for a maturing industry. Global Aquaculture Advocate, 2(4/5), 50–52. moss s m, (2002) Dietary importance of microbes and detritus in penaeid shrimp aquaculture, in Lee C S and O’Bryen P (eds), Microbial Approaches to Aquatic Nutrition within Environmentally Sound Aquaculture Production Systems, World Aquaculture Society, Baton Rouge, LA, 1–18. moss s m and pruder g d (1995) Characterization of organic particles associated with rapid growth in juvenile white shrimp, Penaeus vannamei Boone, reared under intensive culture conditions, Journal of Experimental Marine Biology, 187, 1715–91. moss s m, pruder g d, leber k m and wyban j a (1992) Relative enhancement of Penaeus vannamei growth by selected fractions of shrimp pond water, Aquaculture, 101, 229–39. moss s m, reynolds w j and mahler l e (1998) Design and economic analysis of a prototype biosecure shrimp growout facilityk, in Moss S M (ed.), U.S. Marine

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Shrimp Farming Program Biosecurity Workshop, Oceanic Institute, Waimanalo, HI, 67–74. moss s m, doyle r w and lightner d v (2005) Breeding shrimp for disease resistance: challenges and opportunities for improvement, in Walker PJ, Lester RG and Bondad-Reantaso MG (eds), Diseases in Asian Aquaculture V: Proceedings of the Fifth Symposium on Diseases in Asian Aquaculture, Asian Fisheries Society, Manila, 379–93. noaa (2008) Fisheries of the United States 2007, National Marine Fisheries Service, Office of Science and Technology, Silver Spring, MD. otoshi c a, naguwa s s, falesch f c and moss s m (2007) Commercial scale RAS trial yields record shrimp production for Oceanic Institute, Global Aquaculture Advocate, 10(6), 74–6. pruder g d (2005) Biosecurity: application in aquaculture, Aquacultural Engineering, 32, 3–10. samocha t m, gandy r l, mcmahon d z, mogollón m, smiley r a, blacher t s, de wind a, figueras e and velasco m (2003) The role of shrimp nursery systems to improve production efficiency of shrimp farms, in Jory D E (ed.), Responsible Aquaculture for a Secure Future: Proceedings of a Special Session on Shrimp Farming. World Aquaculture 2003. World Aquaculture Society, Baton Rouge, LA, 179–95. venero j a, davis d a and rouse d b (2007) Variable feed allowance with constant protein input for the Pacific white shrimp Litopenaeus vannamei reared under semi-intensive conditions in tanks and ponds, Aquaculture, 269, 490–503. venero j a, davis d a and rouse d b (2008) Effect of the dietary radio of digestible energy to crude protein on growth and feed conversion in juvenile Pacific white shrimp Litopenaeus vannamei under similar levels of daily protein consumption, North American Journal of Aquaculture, 70, 43–9. venero j a, mcabee b j, lawson a, thomas b, stokes a, browdy c and leffler j (2009) Greenhouse-enclosed super-intensive shrimp production systems. Are they the sustainable alternative to traditional pond shrimp farming in the United States? Global Aquaculture Advocate, 12(1), 61–4. wasilesky w, atwood h l, stokes a d and browdy c l (2006) Effect of natural production on brown water super-intensive culture system for white shrimp Litopenaeus vannamei, Aquaculture, 258, 396–403. wirth f f and davis k j (2001a) Seafood Restaurant Shrimp Purchasing Behavior and Attitudes Toward Farm-Raised Shrimp, Staff Paper 01-19, Food and Resource Economics Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL. wirth f f and davis k j (2001b) Assessing Potential Direct Consumer Markets for Farm-Raised Shrimp, Staff Paper 01-13, Food and Resource Economics Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL. wyban j a, swingle j s, sweeney j n, pruder g d (1992) Development and commercial performance of high health shrimp using specific pathogen free SPF broodstock Penaeus vannamei, in Wyban J (ed.), Proceedings of the Special Session on Shrimp Farming, World Aquaculture Society, Baton Rouge, LA, 254–60.

34 Traditional Asian aquaculture P. Edwards, Asian Institute of Technology, Thailand

Abstract: Traditional inland aquaculture is socially and environmentally compatible with the local landscape as it uses on-farm and locally available wastes and by-products as nutritional inputs for farmed aquatic organisms. Integrated agriculture/aquaculture systems (rice/fish, crop/livestock/fish and feedlot livestock/fish), integrated peri-urban aquaculture systems (wastewater-fed aquaculture) and integrated fisheries aquaculture (low-value, trash fish fed systems) are described as well as recent changes to traditional practice. Researchbased guidelines for improved traditional practice are outlined. Recent examples of the development of semi-intensive aquaculture are given. Examples are also given of how the principles of traditional practice are being used to reduce the adverse environmental impact of intensive pellet-fed aquaculture through reduction of wastes in situ and treatment of intensive aquaculture effluents. Key words: integrated agriculture/aquaculture systems, integrated fish farming, semi-intensive aquaculture, treatment of aquaculture effluents.

34.1 Introduction Traditional inland aquaculture, developed and disseminated by farmers and local communities using on-farm and/or locally available resources, comprises diverse systems. It continues to dominate production in Asia, although science/industrial-based aquaculture technology using agro-industrially formulated feed (FF) is expanding rapidly, both geographically and in production. Traditional integrated farming systems and their intensities of production are defined. Examples of traditional practice and recent changes to such practice are given. The scientific basis to traditional practice is outlined and design criteria are presented. However, the distinction between traditional and modern aquaculture technologies is becoming increasingly blurred: traditional practice is being widely disseminated through projects; farmers involved in traditional practices continue to experiment, including use of agro-industrial chemical fertilizers (CF) and FF; science-based technology is improving traditional

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practice; and some of the principles of traditional aquaculture are being used to reduce the adverse environmental impact of modern industrial aquaculture (Edwards, 2004; Milstein, 2005; Azim and Little, 2006). Examples of recent developments in traditional practice and application of its principles to intensive aquaculture using FF and CF are also given.

34.2 Definitions and principles 34.2.1 Intensity of production Extensive, semi-intensive and intensive are commonly used terms for the degree of intensification of production through nutrition in aquaculture. As these terms are used in varying ways, they are defined below for the purpose of this study (Edwards, 1993; Tacon and De Silva, 1997; Allan, 2004) (Fig. 34.1). Extensive systems Organisms farmed in extensive systems depend on natural food produced within the system without nutritional inputs provided intentionally by humans. Natural food consists of plankton suspended in the water column (bacterioplankton; phytoplankton; and zooplankton) and benthos in sediments (insect larvae and adults; snails; and worms) and is usually high in protein (50–70 % dry matter). Aquatic macrophytes do not occur in wellmanaged ponds because high phytoplankton-based turbidity, which usually ‘greens’ the water, prevents the light necessary for growth of rooted aquatic macrophytes from reaching the sediments, although they may be present in extensive and poorly managed semi-intensive systems. Extrapolated annual

Rate of increase in the yield (Y) Natural food supply (NFS)

NFS

Increasing protein level in food

Y CSC

Standing crop

Fig. 34.1 The critical standing crop (CSC) at which there is a need for an increasing protein level in supplementary feed. (Source: Tacon and De Silva, 1997)

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Table 34.1 Approximate relationship between extraploted annual yield (tonnes/ha/) and intensity of culture Extrapolated annual yield (tonnes/ha) Intensity of culture 0–1 Extensive No nutritional inputs

1–5

5–10

10–20

20–100

100–1000

+

+

+

Semi-intensive Low-quality manure, macrophytes, supplementary feed High-quality manure, supplementary feed Inorganic fertilization, pelleted feed Intensive Trash fish/offal FF, static water FF, aeration FF, recirculation FF, raceway

+ + +

+

+

+ + + +

FF = formulated feed. Source: modified from Edwards, 1993.

fish yields are usually less than 1 tonne/ha (Table 34.1). Examples of extensive aquaculture are traditional rice/fish culture in China and traditional pond culture in the Indian sub-continent. Project introduced examples are cage culture in eutrophic lakes in Nepal and community-based fisheries in lakes and reservoirs and in rice field floodplains. Semi-intensive systems Farmed organisms in semi-intensive systems depend on intentional fertilization to produce natural food in situ and/or on the addition of supplementary feed to complement high-protein natural food. Natural food is also a source of minerals and vitamins (De Silva and Davy, 1992; De Silva, 1993). Natural food provides a significant amount of nutrition for fish in semiintensive systems and may be increased traditionally by organic fertilization with human, livestock or green manure (vegetation) or CF such as urea to provide N (nitrogen) and triple superphospate (TSP) to provide phosphorus (P). There is also a residual fertilizer effect from uneaten fish feed and fish excretory products and faeces. Traditional supplementary feeds are locally available plants and agricultural by-products, often with a low protein content (<20 % dry matter) that nutritionally complement high-protein natural food (Hepher, 1988): rice

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bran, broken rice and waste vegetables; domestic waste food from households or restaurants; volunteer (wild) or cultivated terrestrial vegetation, e.g. grass and weeds; wild or cultivated aquatic macrophytes, e.g. duckweed, water spinach and pond weed; agro-industrial by-products, e.g. rice bran, broken rice and oil cakes; and also food processing wastes such as waste noodles and confectionary produce. Supplementary feed is traditionally fed as single ingredients, unprocessed and uncooked in pond culture in China and Southeast Asia (Edwards and Allan, 2004), although feeding practices in more recently developed aquaculture may involve mixed ingredients offered to fish on trays or in perforated sacs in some countries such as Bangladesh and India (Tacon and De Silva, 1997). Extrapolated annual fish yields range from 1–5 for low-quality fertilizers and feeds, 5–10 for high-quality fertilizers and feeds and 10–20 tonnes/ha for fertilized ponds supplemented with FF (Table 34.1). Examples of traditional semi-intensive systems are most integrated agriculture–aquaculture systems (IAAS) and some wastewater-fed integrated peri-urban-aquaculture systems (IPAS). Chemical fertilization is widely practiced in IAAS; and in small irrigation reservoirs stocked with carps in China (Miao and Liang, 2007). Intensive systems Fish farmed in intensive systems depend on nutritionally complete feed with little to no contribution from natural food. Examples of complete feeds in traditional systems are trash fish (small or low-value marine or freshwater fish, ‘naturally’ complete diets for carnivorous fish); slaughterhouse waste such as chicken bones and offal; and moist or dry feed formulations, e.g. trash fish and rice bran. Use of farm-made feeds is common in traditional intensive aquaculture. Intensive aquaculture increasingly uses FF. Most cage, raceway and recirculation systems depend on FF for almost all their nutrition. There may be an overlap between semi-intensive and intensive modes of production. As increasing amounts of supplementary feed are provided to growing fish in a semi-intensive pond, the proportion of nutrition derived from natural food declines markedly relative to that of added feed so that the system increasingly resembles an intensive one in the later stages of the culture cycle. Extrapolated annual fish yields range from 5–20 for carps and tilapia up to 50–100 tonnes/ha for airbreathing fish such as striped catfish (Pangasius hypophthalmus), hybrid walking catfish (Clarias gariepinus × C. macrocephalus) and striped snakehead (Channa striata) in static water ponds, and up to 1000 tonnes/ha for fish fed FF in raceways (Table 34.1). Examples of traditional intensive culture are striped catfish in cages in Cambodia and striped catfish and hybrid walking catfish in ponds in Thailand.

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34.2.2 Integrated aquaculture Traditional aquaculture is integrated with other human activity systems and these provided the only sources of nutritional inputs for farmed aquatic organisms in the past, before the relatively recent manufacture of CF and FF. Most traditional integrated aquaculture probably originated in China, developed empirically by trial-and-error by farmers over centuries, although integrated fisheries aquaculture systems (IFAS) may have been developed first in Cambodia (Edwards et al., 1997). Integrated aquaculture may involve only on-farm integration with aquaculture linked to plant crop and/or livestock sub-systems on a farm. Its main physical features are waste or by-product recycling which provide the nutrition for the fish; and improved space utilization with crops grown on pond dikes or on frames extending over the pond water surface and/or livestock raised on the pond dike or over the pond water surface. In some societies household nightsoil is a traditional pond input (Edwards, 1998). Traditional integrated aquaculture may also be indirect and use off-farm wastes (by-products) or products of diverse human activity systems such as agro-industry, fisheries and sanitation from local rural or peri-urban areas (Edwards, 1998). Thus, some form of transportation is involved as inputs and production are not directly linked spatially. Three major types of traditional integrated aquaculture systems have been recognized (Edwards, 2002a): • IAAS with on-farm or local sources of vegetation, manures and agricultural by-products as nutritional inputs, e.g. rice/fish; crop/fish; livestock/ fish. These are semi-intensive. • IPAS use wastes of cities and agro-industry. These include wastewater (human sewage or agro-industrial effluents); waste vegetables from markets; waste food from canteens and restaurants; and factory processing wastes from the food industry, including offal from slaughterhouses and fish processing factories. Fertilized IPAS are semi-intensive systems, but those fed with large amounts of food and factory processing wastes may approach an intensive mode of production or be intensive if stocked with carnivorous fish fed with trash fish, or livestock or fish processing offal. • IFAS use freshwater or marine trash /small low-value fish as feed. These are traditional intensive systems. The principles of traditional Chinese aquaculture have been characterized as integration with other human activities systems such as agriculture, animal husbandry, sanitation and local agro-industry; polyculture of fish with complementary spatial and feeding niches in the pond; waste or byproduct reuse such as terrestrial or aquatic vegetation, livestock manure, nightsoil, brans and oil cakes, and food and drink manufacturing residues; nutrient and water reuse and multiple use between farm sub-systems or

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enterprises and the pond for the production of high-protein natural food in situ as well as an aquatic environment for fish (Edwards, 2004).

34.3

Traditional aquaculture systems

34.3.1 Integrated agriculture/aquaculture systems (IAAS) Farms with traditional aquaculture are usually crop dominated with limited numbers of livestock (Edwards, 1993). Rice/fish integration Rice/fish culture is perhaps the most basic and may also be the oldest type of IAAS as recent archaeological evidence indicates possible co-evolution of agriculture and aquaculture 8000 years ago in China (Edwards, 2004). Capture and culture of fish in rice fields have both diminished since the intensification of agriculture due to pesticides. However, culture of fish in rice fields has never been widespread although it is common in some areas of Asia, e.g. mountainous Southwest China and West Java, Indonesia (dela Cruz et al., 1992). A trench or ditch is usually dug in the rice field with excavated soil used to strengthen and raise the height of the dike to hold more water for fish. The trench provides deeper and cooler water in hot weather for fish and a refuge when the field is drained for weeding, pesticide application and harvesting rice. Water sources are screened to prevent loss of fish. Traditional rice/fish culture is mostly extensive, with fish consuming natural food that develops in the rice field. Two basic types of rice/fish culture are concurrent, with rice and fish raised together, and rotational with alternating fish and rice crops. Commonly cultured fish are herbivorous/omnivorous species such as common carp (Cyprinus carpio), silver barb (Barbodes gonionotus) and Nile tilapia (Oreochromis niloticus). Extrapolated annual yields range from 100–250 kg/ha and with supplementary feed to 1 tonne/ha or higher if the trench is much greater than 10 % of the total area of the field. Rice yields are reported to be increased by about 15 % when fish are stocked; reasons usually given for the increase in rice yields are fertilization from fish faeces and fish consuming rice pests, but the observed increase may be due more to farmers managing the water better in rice fields stocked with fish. A red coloured variety of common carp is raised in stream-fed terraced rice fields in mountainous Qingtian County, Zhejiang Province, China, an example of a traditional rice/fish system with a documented history of 1200 years (Edwards, 2006; Lu and Li, 2006). Fish are bred in trenches and fry directly released into rice fields of small farms averaging 1300–1700 m2. Livestock manure is provided as a basal fertilizer for the rice, but the fry are otherwise raised extensively without further addition of fertilizer or

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feed for 2–3 years until they reach table size of 350–400 g. Fish production is low, 600–1200 kg/ha, although the fish have a high market value because of their special taste. Crop/livestock/fish integration in China Integrated farming has a long history in China where traditional familylevel integrated farms were widespread in the Yangtze and Pearl River basins before the development of communes and collective agriculture in the 1950s (Hoffmann, 1934). There is a voluminous literature on IAAS, including in China (Pullin and Shehadeh, 1980; NACA, 1989; Edwards, 1993; Symoens and Micha, 1995; Edwards et al., 1997; Prein, 2002). The traditional Chinese system of carp polyculture had several on-farm linkages. Fertilizers included human manure or nightsoil for crops and pond; livestock manure, mainly pig, for crops and pond; vegetation or green manure for crops and pond; and pond mud for crops. Supplementary feed for fish was provided by wild grass from on and around the farm; and by waste from vegetables grown for humans and pigs. Mulberry, which used to be grown for leaves to feed silkworms and silkworm pupae, was a traditional high-protein fish feed in both China and Japan. Nightsoil and pig manure were used to fertilize rice, vegetables and fish pond in decreasing importance; feed requirements of pigs were met before feeding fish. Pond mud was used to fertilize terrestrial crops grown on pond dikes as ponds fertilized with green manure and fed grass as green fodder for grass carp developed thick deposits of organic-rich sediments. Few on-farm resources were used for the fish pond due to intense competition from crops and pigs on resource-poor, small-scale farms. The main input to the pond was wild grass of low nutrient value so extrapolated annual fish yields were low, probably only 1–2 tonnes/ha. Similar traditional integrated systems occur in two other densely populated area: West Java, Indonesia and the Red River Delta, Vietnam. Livestock/fish integration IAAS have been developed to varying degrees since the 1960s in many other countries, especially in South and Southeast Asia. The most common type of IAAS in Southeast Asia is feedlot livestock/fish integration: especially pig/fish, chicken/fish and duck/fish integration (Little and Edwards, 1999, 2003). Livestock such as pigs and poultry usually scavenge for their food on small-scale farms so it is difficult and labour-intensive to collect manure for aquaculture. If livestock are confined to permit manure collection for aquaculture, they have to be fed with feed, manually collected or purchased which may not be possible for most resource-poor small-scale farming households. Large ruminants, e.g., buffalo and cattle, are usually penned/corralled at night near the house to prevent theft so it is possible to collect manure. It may also be possible to collect some manure of scavenging poultry when they are confined at night. Small-scale ponds may be

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fertilized with livestock manure, but there is usually insufficient as most farms are resource-poor and there are often alternative uses as a crop fertilizer or as fuel in the Indian sub-continent. Most livestock/fish integration depends largely on manufactured formulated feed for the livestock so it is intensive, although pig farms often depended in the past on wastes from food factories or waste food from canteens. Nutrient-rich high-quality manure, often with spilled livestock feed, leads to high extrapolated annual fish yields, up to 8–10 tonnes/ ha. Feedlot livestock/fish integration is most commonly located in periurban areas with ready access to markets for inputs and produce and in rural areas associated with rice mills with rice bran to feed both livestock and fish.

34.3.2 Integrated peri-urban aquaculture systems (IPAS) The use of human excreta as a fertilizer to produce fish and aquatic plants has a long history in several countries in East, South and South East Asia, especially in China and Vietnam (Edwards and Pullin, 1990; Edwards, 1992, 2000, 2005a). Fresh excreta or nightsoil may fall directly into ponds from overhung latrines; nightsoil and septage may be transported by various means from rural households as well as urban areas to ponds; and waterborne faecal matter in surface waters such as rivers, and domestic wastewater or sewage may flow or be pumped into ponds. Traditional IPAS were commonly developed in low-lying peri-urban areas that received the flow of city liquid wastes by gravity flow but have come under pressure from urban development recently as areas less prone to flooding have been built on. Fish or aquatic vegetables such as water spinach for direct human consumption are most commonly grown in these systems, but fish seed (fingerlings) and feed for fish and livestock may also be farmed (Edwards, 2000, 2005a). Fish may be cultured in ponds, cages or pens; and plants in ponds or staked in surface waters. Examples of countries with traditional wastewater-fed aquaculture are Cambodia, China, India, Indonesia and Vietnam, most of which were also densely populated in the past with a ‘nutritional imperative’ to use whatever resources were available for food production (Edwards, 1992). There is growing reluctance or opposition to waste-fed aquaculture with improving social and economic status, even in societies where it is traditional practice. Various factors may be involved: reduced availability of nightsoil as sanitation improves; increasing contamination of domestic wastewater with industrial sewage which reduces fish growth and taints the produce with chemical off-flavours; rapid urban expansion with rising land prices; intensification of aquaculture with reduced demand for fish pond fertilizer; and increasing consumer demand for high-value fish rather than low-value fish from waste-fed ponds (Edwards 2000, 2005a).

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34.3.3 Integrated fisheries aquaculture systems (IFAS) These systems use marine or freshwater trash fish or low-value small fish as feed. Several freshwater species are traditionally fed mainly marine trash fish in Asia. Eel (Anguilla japonica) used to be fed marine trash fish in China, Japan and Korea, but most intensive eel farming now uses artificial commercial feed, a moist paste for larval glass eels and steam pressed or extruded pellets for later stages (Ottolenghi et al., 2004). Striped snakehead and hybrid walking catfish (Jantrarotai and Jantrarotai, 1993) are traditionally raised in ponds and marble goby (Oxyeleotris marmoratus) in cages and fed trash fish in Thailand. Soft shelled turtle (Trionyx sinensis) is raised in ponds in China, Thailand and Vietnam using a marine trash fish-based diet. Striped catfish is traditionally raised on a diet of marine trash fish and rice bran in cages and, more recently, in pens and ponds, in the Mekong delta in southern Vietnam (Le et al., 2007; Nguyen et al., 2007). Use of small freshwater fish for feed is not common as their sale for human food is usually more profitable than using them as feed for carnivorous fish (APFIC, 2005). Striped catfish is raised on small freshwater fish in Cambodia when they are abundant, otherwise rice bran (ICCILMB, 1992). Striped catfish raised in cages in the Mekong River Delta in Vietnam used to be fed small freshwater fish before they became scarce and their price rose, after which marine trash fish were used. Giant snakehead (Channa micropeltes) used to be farmed in cages in Cambodia, but the practice has recently been banned by the government (see Section 34.4.4). There is limited production of giant and striped snakehead in Nam Ngum reservoir in Lao PDR using small freshwater pelagics (APFIC, 2005).

34.4 Recent changes to traditional practice 34.4.1 Integrated agriculture/aquaculture systems Rice/fish integration Only about 1 % of the world’s rice fields are stocked with fish (Halwart and Gupta, 2004). The practice is constrained by intensification of rice culture: high-yielding varieties of rice which require water less than 20 cm deep and have a shorter growing period than traditional rice varieties make it difficult for fish to attain marketable size; and increased use of pesticides. A further constraint is theft of fish as rice fields are often considered as a common property resource and are difficult to guard due to their large size and location often distant from the farmer’s dwelling. It is also labour-intensive to modify rice fields and manage water for fish, ensuring sufficient water during the dry and preventing flooding with loss of fish during the rainy season. While there is a relatively low return on producing small fish in shallow water rice/fish systems, such fish may provide an important source

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New technologies in aquaculture

of animal protein, healthy fats, vitamins and minerals for poor farming households (Halwart, 2006). Culture of high-value crustaceans has been developed recently in rice fields with produce such as fingerlings and high-value crustaceans. Postlarvae of giant river prawn (Macrobrachium rosenbergii) are initially nursed in trenches in rice fields in southwest Bangladesh and then raised concurrently with rice as rains flood the field (Nandeesha, 2003). Some farmers in the Mekong River Delta in southern Vietnam culture low-value fish such as common carp, kissing gourami (Helostoma temmincki), rohu (Labeo rohita), silver carp (Hypophthalmichthys molitrix) and Nile tilapia concurrently with rice, but there is a trend to culture river prawns for sale in modified rice fields in rotation with rice, with prawns stocked and fed in flooded rice fields following the harvest of a dry season rice crop (Nguyen et al., 2006). Rice field aquaculture is reported to occur on a massive scale in China with over 1.5 million ha in 2001, including high-value river prawns (M. nipponensis and M. rosenbergii) and Chinese mitten-handed crab (Eriocheir sinensis) grown concurrently with rice in trenches connected to the rice field (Fang, 2003). However, partial or complete conversion of rice fields to fish ponds is increasing in Asia. While some countries have policies restricting conversion because of concerns about the national food staple, rice, these are being relaxed to allow farmers to diversify as rice usually does not provide an adequate income. Thousands of hectares subject to flooding and able to produce only one rice crop annually have recently been converted to fish ponds in the Red River Delta, Vietnam. Rice-based aquaculture is considered as a low-cost and low-risk entry point for farmers to carry out aquaculture without jeopardizing the sustainability of rice production in China, but farmers have been reported to abandon rice farming and convert their fields to ponds (Miao et al., 2007). Crop/livestock/fish integration in China The most complex IAAS were developed in China during the era of communes and they reached their peak of complexity in the 1980s with intensification through direct and indirect integration (Chen et al., 1995). Improved grasses such as elephant and rye grass were cultivated on pond dikes to feed grass carp. Nutrient flows were greatly increased through manure from increased numbers of integrated livestock and transport of urban nightsoil as crop and pond fertilizers, harvest of wild aquatic feed organisms such aquatic macrophytes and snails and use of wheat and rice bran, soybean and rapeseed cakes as well as CF and FF. Carp production in China surged following the introduction of a marketdriven economy in the mid-1980s because it was more profitable than terrestrial farming, but production exceeded market demand with a decline in aquaculture profitability (Ye, 2002). Large-scale integrated farms became less integrated following privatization, with a reduction in the number of

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sub-systems. Crops have been eliminated except for grass for relatively high-value grass carp. Small-scale livestock integrated with aquaculture is being replaced by more efficient and profitable large-scale, industrial livestock farms; pond fertilization during grow-out has declined with increasing intensification so less manure is required as the ponds have excess nutrients from the residual fertilizer effects of feed (Miao, 2007; Miao and Liang, 2007). The centuries old integrated mulberry dike/pond system has also disappeared from the Pearl River Delta with an increase in intensive monoculture of high-value fish, giant river prawn and soft shelled turtle from the late 1980s (Yee, 1999). Semi-intensive carp polyculture still contributed an estimated 50–58 % of total inland production in 2005 in China, but the trends are to introduce new species and intensify production, driven by consumer demand for higher-value species and farmer pursuit of increased returns (Miao and Liang, 2007). Farmers are moving increasingly to a feed-based production system with FF to improve fish feeding efficiency (Ye, 2002). As extrapolated, annual yields of intensive monoculture of fish such as common carp are up to 30–40 compared to 12–15 tonnes/ha for traditional polyculture, the former ‘becomes the choice of farmers for higher production and profit’ (Miao, 2007). Livestock/fish integration The practice is also declining in several countries in Southeast Asia. Industrial-scale livestock farms are increasingly run as separate, highly specialized enterprises. Concerns about biosecurity to minimize the risk of disease preclude integration of large-scale livestock with pond aquaculture. There is no evidence that poultry/fish integration has been involved in any outbreak of highly pathogenic avian influenza (HPA1), commonly known as bird flu, but after the 2003/2004 bird flu outbreak contract farming companies in Central Thailand stopped providing day-old chicks to broiler farmers operating open systems, including those integrated with fish ponds (Edwards and Mohan, 2007). Broiler farming is exclusively on a contract basis with large companies providing day-old chicks and feed and buying back birds at harvest, much for export. Only layer farms appear to have been able to continue integration with fish in Central Thailand as they operate independently and eggs are sold on the local market. Chicken manure is now often only available from large, biosecure industrial farms in Thailand, with indirect integration with aquaculture (Thongrod, 2007).

34.4.2 Integrated peri-urban aquaculture systems Wastewater reuse through aquaculture is declining even although it provides a low-cost system to treat municipal wastewater and benefits the poor through employment and income for peri-urban farmers and low-cost fish for urban consumers (Bunting, 2004; Little and Bunting, 2005). It has

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declined drastically in China with the longest tradition and until recently the greatest extent of practice. The area occupied by the largest system in Vietnam in peri-urban Hanoi (Vo and Edwards, 2005) has recently been declared an urban area, with a marked increase in land value and rapid urbanization. The largest single wastewater-fed aquaculture system in the world, the East Kolkata Wetlands (EKW) in Kolkata, India, with almost 4000 ha of ponds may continue as it has been declared a protected area under the Ramsar Convention on Wetlands based on the wise use of the area through the traditional low-cost sewage treatment with agriculture as well as aquaculture (Bunting et al., 2005). The EKW provides social benefits for the poor in peri-urban and urban areas of the city and environmental benefits through low-cost wastewater treatment as well as a haven for wildlife.

34.4.3 Integrated fisheries/aquaculture systems There have been major recent changes in IFAS in the Mekong River Delta in both Cambodia and Vietnam with a decline in the use of trash fish/lowvalue small fish. The government of Cambodia banned the culture of giant snakehead in floating cages from 2005 (Edwards, 2008). Snakehead farming was initially a secondary occupation of small-scale fishers who caught both wild seed and feed, but the practice became dominated by large-scale fish farms. Wild snakehead seed were abundant, but the practice required the harvest of a large amount of small wild fish to feed carnivorous snakehead, with a high food conversion ration of 4–6 : 1 on a fresh weight basis. The practice was also constrained by the seasonal availability of fish for feed as their capture was prohibited during the closed fishing season from June to September. Thus snakehead culture encouraged poaching as well as use of illegal fine-mesh fishing gear such as mosquito netting. Major concerns were catching the young of commercially important species and naturally small fish used to make fermented fish paste, a national dietary staple. The massive use of trash fish is also a major constraint to snakehead farming in the Mekong River Delta in Vietnam (Le et al., 2007). Traditional cage culture of striped catfish started in the Mekong River Delta in Vietnam in the 1960s, followed by that of pen and pond culture in the 1990s (Nguyen et al., 2007). The explosive development of catfish farming is driven by the increasing demand for white fillets marketed in more than 80 countries with production of about 1 million tonnes in 2007 (Nguyen T.P. and Le T.H., pers comm). The recent availability of relatively cheap hatchery raised seed is also a contributing factor. Caged fish were traditionally fed trash fish as well as rice bran, but most production is now from ponds and to a lesser extent from pens based on FF (Le et al., 2007; Nguyen et al., 2007). Farmers are changing to pond culture because it is more economic with the current high price of wood; and farmers have an increased ability to control water quality in ponds compared to cages

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installed in the river, with river water becoming increasingly polluted from factories and fish processing plants discharging effluents into the river as well as agricultural and urban runoff. It is expected that pond culture of striped catfish fed with FF will soon be the predominant culture system (Le et al., 2007). Airbreathing striped catfish is now being stocked at high densities of 10–20 individual 10–15 cm fingerlings/m2 in 1.5–6.0 m-deep earthen ponds converted from rice fields. Pond water is exchanged daily in the second half of the culture cycle at 20–30 % of pond water volume with the Mekong River. Yields after 8–12 months of culture are 150–350 tonnes/ha/crop of 0.8–1.5 kg fish. There is no treatment of pond effluents so all wastes are flushed into the river. The long-term effects of catfish farming, which has occurred without any government guidelines, recommendations or regulations (Nguyen, 2006), are unknown.

34.5 Research and development for improved traditional practice Research has provided a scientific basis for traditional aquaculture and designs for improved practice. However, optimum design criteria should only be used as guidelines due to large variations in the nature of inputs and the variable responses of aquatic systems. Design criteria should be used as broad guidelines with system response (bioassays) used to guide management of the system. Farmers traditionally use two bioassays to determine rates of fertilization and feeding, and water quality: observation of the type and amount of phytoplankton in the water from the colour and the intensity of the colour of pond water, and the depth of penetration of light into the pond water; and the degree of fish surfacing to gulp air after dawn and in the early morning when pond dissolved oxygen (DO) is at its lowest. Pond water ideally should be light to medium green in colour, indicating adequate phytoplankton to feed fish as well as sufficient night-time DO; dark green water reveals excess phytoplankton which removes DO from the water during the night and which could suffocate the fish.

34.5.1 Fertilization Pond fertilization has a long and confusing history concerning the types and amounts of nutrients required and the relative merits of organic and chemical fertilization (Colman and Edwards, 1987). The basic biological, chemical and physical mechanisms and interrelations of pond fertilization, often referred to as pond dynamics, are now well understood (Boyd, 1990; Egna and Boyd, 1997; Knud-Hansen, 1998). Manure provides natural food for fish through the release of soluble nutrients by decomposition by microbial activity that supports light-induced

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autotrophic production through phytoplankton; and by providing substrates for bacteria, protozoa and invertebrates or heterotrophic production (Colman and Edwards, 1987). Fish may consume manure, but it is a poor direct feed for fish because of low amounts of metabolizable nutrients unless it contains spilled livestock feed. Both autotrophic and heterotrophic food chains operate simultaneously in manured fish ponds, but phytoplankton dominate and usually stain pond water green. Nitrogen and P are the major nutrients in aquatic fertilization. Unlike agriculture, potassium or other trace minerals have not been shown to be required. Adequate carbon (C) is usually provided by bacterial breakdown of organic matter and release of CO2, but ponds should be limed to provide C if total alkalinity is <20 mg/l as CaCO3. Optimal fixed fertilization rates are 4 kg N and 1 kg P/ha/day (2 kg P/ha/day if pond sediment and water contain high concentrations of aluminium, calcium and iron which precipitate P as insoluble phosphates). An algal bioassay may more accurately determine phytoplankton nutrient requirements and thus more efficient pond fertilization (Knud-Hansen, 1998; Knud-Hansen et al., 2003). Although pond fertilization based on the algal bioassay yielded 20 % less fish than the conventional fixed fertilization input strategy, it required almost 20 % less N and almost 90 % less P, with a corresponding 45 % lower cost of production of fish. Livestock manure is usually an effective pond fertilizer as 60–90 % of the nutrients in livestock feed are potentially recoverable in the manure (faeces and urine) (Taiganides, 1979). Manure nutrient content depends on species of livestock and diet, inclusion of urine and foreign matter, and methods of handling and storage. Poultry manure has more nutrients than that of pigs which in turn has more than cattle manure. Feedlot animals fed formulated diets have nutrient-richer manure than scavenging animals. Urine as well as faeces should be used, as urine usually contains more N than faeces. Water, soil and litter or bedding decrease and spilled feed increases the nutrient content of manure. Fresh manure contains the most nutrients because decomposition releases ammonia; loss of N can be considerable, 20–90 % depending on the method of collection and handling, temperature and degree of protection from wind and leaching by rain. Loss of P is less pronounced because its compounds are non-gaseous. Experimental extrapolated fish yields from livestock manured ponds range from about 2–10 tonnes/ha/year (Edwards et al., 1997; Little and Edwards, 2003). Feedlot duck manure with a relatively high N content of about 4 % on a dry matter basis and a relatively low C : N ratio of 11 gave a higher fish yield than that of small-scale farm buffalo manure with a relatively low N content of about 1 % on a dry matter basis and a relatively high C : N ratio of 26 (AIT, 1986). Fish production in ponds integrated with ducks was increased by spilled feed from the ducks penned over the pond; and that from buffalo-manured ponds was reduced by tannins which stained the pond water brown, reducing light penetration and inhibiting phyto-

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plankton growth (Shevgoor et al., 1994). Maximum numbers of livestock (full grown animals) recommended for integration with fish, based on manure loading rates of 100 kg manure dry weight/ha/day, are 2000 chickens or ducks weighing 2 kg each, 100 pigs of 100 kg or 15 cattle of 500 kg (Little and Edwards, 2003). It is commonly believed that organic fertilizers are cheaper than CF, but the cost of nutrients in dry chicken manure was seven times more than urea as a source of available N and more than four times more than TSP as a source of available P (Knud-Hansen, 1998). As nutrients in CF are concentrated, only 10 kg each of urea and TSP together provided an amount of available N and P equivalent to about 1 tonne of chicken manure. As CF are usually the cheapest form of N and P, increased pond productivity on small-scale farms may be achieved by using relatively small amounts of CF to supplement limited on-farm pond organic matter inputs (Edwards et al., 1996).

34.5.2 Supplementary feeding There are complex interrelationships between the production of natural food by fertilization and supplementary feeding practices, and these are often unpredictable due to varying organismic (pond natural food as well as fish), chemical (C, N and P cycles and DO) and physical (temperature, water movement) processes in pond water, or pond dynamics. An important concept in supplementary feeding is that of the critical standing crop (CSC) of natural food and its relationship with the standing crop of cultured fish (Hepher, 1988; De Silva, 1993). At the CSC of natural food, there is a need to provide an increasing amount of food nutrients and especially protein in the supplementary feed to make up for its deficit in the natural food supply as the fish standing crop or biomass increases with time (Fig. 34.1). A dynamic model showed that supplementary feeding compensates for natural food deficiences as fish grow and that protein supplements are required to increase fish production in fertilized ponds (Li and Yakupitiyage, 2003). Semi-intensive systems have been divided into four stages based on the amount of natural food present and the type of supplementation of natural feed required as the fish biomass increases (Table 34.2) (Yakupitiyage, 1993; Edwards et al., 2000). Natural food, increased by addition of fertilizers, predominates in stage 1. In the following three stages, supplementary feed is added to complement the nutritional value of the natural food present in terms of quality and quantity required for growth of the fish. As natural food has a high protein content, the first and most logical supplementary feed is a relatively inexpensive energy supplement (semi-intensive, stage 2). As the fish biomass increases with growth, or at high stocking density, there is a need for more food as the fish cannot eat enough natural food and, furthermore, the quantity of natural food is insufficient; thus, in stage 3 there is a need to give denser feedstuffs with both energy and protein supple-

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New technologies in aquaculture Table 34.2 Relationship between nutritional input and intensity of system Input

Type of system

None Fertilizer Energy Energy + protein Energy + protein + phosphorus Complete diet

Extensive Semi-intensive stage 1 2 3 4 Intensive

Source: based on Yakupitiyage, 1993; Edwards et al., 2000.

ments. There may also be a need to add P if plant meals are used as the protein supplement instead of fish meal (semi-intensive stage 4).

34.5.3 Integrated agriculture/aquaculture systems Rice/fish integration There has been considerable R & D to better understand and promote rice/ fish since the 1980s in both irrigated areas and in rainfed areas (dela Cruz, 1992; Halwart and Gupta, 2004). It has been promoted as a technique for integrated pest management as farmers who stock fish in their rice fields reduce or eliminate the use of pesticides, especially in Bangladesh, but the extent of a sustained adoption by farmers appears to be limited (Nandeesha, 2004). Research has also indicated the feasibility of nursing common carp and Nile tilapia fry in irrigated rice fields in Northwest Bangladesh (Barman and Little, 2006). A newly developed ‘system of rice intensification’ which conserves water is likely to be a constraint to rice/fish farming as flooding the rice plants is avoided to enhance plant root growth and the activities of soil organisms (Uphoff, 2007). It is being widely adopted in several countries in which rice/ fish occurs as it leads to a doubling or tripling of rice yields. However, a marked increase in the production of rice could encourage farmers to diversify their rice-based farm by converting part of the field to a pond to product higher-value fish or crustaceans. Crop/livestock/fish integration Small-scale farms have limited on-farm resources so intensification of aquaculture depends on off-farm inputs of fertilizers and/or supplementary or even complete feeds. Intensification of livestock would provide significant manure for pond fertilization, but attempts to introduce scaled-down feedlot livestock integration with fish have met with little success because resource-poor farmers are usually unable to continue to maintain feedlot

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livestock following withdrawal of project support (Edwards et al., 1996; Edwards, 1998). Research aimed to increase the productivity of the traditional household-level grass carp (Ctenopharyngodon idella) which dominated carp polyculture system in the Red River Delta, Vietnam and which had reached a maximum extrapolated annual yield of about 3–5 tonnes/ha (Luu et al., 2002). Increased input of grass was technically unsustainable because a higher input of grass caused poor water quality, stressed the fish and may have caused red spot disease with occasional mass mortality of fish; and it was socially unsustainable by placing a heavy burden on women who collected fresh grass daily which had become scarcer as aquaculture expanded and intensified. A monoculture of Nile tilapia in ponds fertilized with pig manure and inorganic fertilizers was recommended for farmers wishing to intensify fish production.

34.5.4 Wastewater-fed aquaculture Revised guidelines have recently been published for the safe use of wastewater and excreta in aquaculture (WHO, 2006). Although reuse of various forms of human waste is a traditional practice in some countries in Asia, there has been little to no introduction of formally designed and engineered wastewater reuse systems (Edwards, 2000, 2005a,b). Research has provided a scientific basis for the key parameters in wastewater-fed fish culture; a design for its incorporation with sewage treatment in stabilization ponds (Mara et al., 1993); and designs for the production of plants (duckweed) and fish (tilapia) as high-protein animal feed (Edwards, 2000, 2005a). Various hazards are associated with waste-fed aquaculture: excretarelated pathogens (bacteria, helminths, protozoans and viruses), skin irritants, vectors that transmit pathogens and toxic chemicals (WHO, 2006). Fish and plants passively accumulate microbial contaminants on their surfaces, but they rarely penetrate into edible fish flesh or muscle except for trematodes (parasitic tissue flukes). The relative risk of disease from bacteria, e.g. Salmonella, Protozoa, e.g. Giardia and viruses, e.g. hepatitis is low to medium. Some of these microbes may be present in the gut of fish. Crosscontamination of foods at the marketplace or in the kitchen is the greatest risk which is reduced by hygienic processing and cooking. Soil-transmitted helminths, e.g. Ascaris, present low to high risks for producers and consumers, which are lower from fish than plants. For foodborne trematodes, e.g. liver flukes, and schistosome trematodes (blood flukes), e.g. Schistosoma, the risk ranges from nil to high as they have restricted geographical ranges. For the former the risk is where they are endemic and fish or plants are eaten raw; for the latter where they are endemic and transmitted through water contact. The transmission of trematode parasites is of particular concern in aquaculture as trematode-associated diseases are associated with high morbidity. Although seldom fatal, they are highly debilitating and

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complications may lead to death. The risk from skin irritants causing contact dermatitis, possibly through microbes or chemicals, is from medium to high. The risk from vector-borne pathogens, e.g. Plasmodia spp., causing malaria, is nil to medium, with no specific risk associated with aquaculture. Regarding the risks from chemicals, the risk from antibiotics is nil to low as they are not usually used in waste-fed aquaculture. The risk from heavy metals is low; although they may accumulate in fish or aquatic plants, concentrations of heavy metals from fish raised in waste-fed aquaculture do not usually exceed levels recommended by the Codex Alimentarius Commission. The risk from halogenated hydrocarbons is low as they are generally in low concentrations in wastewater and excreta and fish raised in wastewater usually show only low concentrations. A range of health protection measures in different combinations can be used to reduce health risks for product consumers, workers and their families, and local communities. Risk management strategies for waste-fed aquaculture involve constructing ‘multiple barriers’ to prevent exposures to pathogens and toxic chemicals with combinations of various interventions such as waste treatment to remove pathogens; application of wastes to allow die-off periods; produce restriction; control of trematode intermediate hosts; prevention of cross-contamination; post-harvest processing; food hygiene; and cooking food. Most waste use involves production of fish or plants for direct human consumption, but waste may be used in aquacultural nurseries to produce seed or fingerlings which are then grown out to full-size table fish in separate systems without the use of wastes. Waste may also be used to raise high-protein animal feed such as duckweed or small tilapia, to be subsequently used as feed for fish or livestock raised in separate systems without the use of wastes. The benefits of so-called produce restriction are: a reduced public health risk; and lengthening the food chain which may increase social acceptability of waste use. Conventional designs for wastewater treatment in sewage stabilization ponds recommend complete wastewater treatment (anaerobic, facultative and maturation ponds in series) prior to effluent use in fish ponds, but the high degree of treatment minimizes the reuse of nutrients prior to fish culture. An improved design introduced the concept of minimal wastewater treatment in stabilization ponds prior to fish culture for maximal production of microbially safe fish (Mara et al., 1993). The design takes into consideration the extremely rapid die-off of enteric bacteria and viruses in ‘green water’ fish ponds so that there is a need for only minimal pretreatment in anaerobic and facultative ponds before the partially treated effluent is fed into fish ponds for further treatment and reuse. The improved design was compared, using bioeconomic modeling, with a series of conventional wastewater stabilization ponds and with complete treatment before using the effluent for fish culture (Bunting, 2007). The improved design would require a larger area than conventional wastewater treatment, but financial

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returns, nutrient retention and fish production would be higher for the former than the latter. Regarding produce restriction, duckweed is effective in wastewater treatment through shading the water column and nutrient uptake, with extrapolated high annual yields of 10–40 tonnes dry matter/ha; the duckweed had a high crude protein content of 25–45 % on a dry matter basis and was readily consumed by Nile tilapia (Edwards, 2000). Pilots have been run in Mirzapur and Khulna, Bangladesh but are unlikely to be maintained or introduced to other areas as the integrated technology is land intensive. Extrapolated annual yields of almost 7 tonnes/ha of small tilapia were produced by seining septage-fed ponds stocked with a breeding population of tilapia; the septage-raised tilapia was as effective in raising hybrid catfish as marine trash fish, fed either fresh or dried in a formulated fish diet. Waste use schemes, if properly planned and managed, can have a positive environmental impact as they reduce surface pollution which would otherwise occur; and they also lead to conservation or more rational use of freshwater resources, especially in arid and semi-arid areas (WHO, 2006). Waste-fed aquaculture may be considered a low-cost waste treatment system, with the cost of waste treatment offset by sale of fish or aquatic plants; a model to calculate the revenues for wastewater-fed aquaculture in tropical and sub-tropical areas has been developed in Lima, Peru. 34.5.5 Periphyton Possibly inspired by the traditional acadia system of Africa in which tree branches are placed in water to attract fish and possibly increase their growth through the development of periphyton, research has aimed to increase natural food in the form of periphyton on hard materials such as bamboo inserted into the pond water column (Azim et al., 2004). The nutritional value of periphyton is comparable to phytoplankton and fish yields 30–210 % higher have been reported from ponds with bamboo substrates compared to ponds without. However, farmer adoption may be constrained by the availability, alternative uses and cost of bamboo (Azim et al., 2004). Artificial substrates in ponds can increase production of giant river prawn by almost 25 % (D’Abramo, 2006).

34.6 Recent development of semi-intensive aquaculture Three examples are given of the development and dissemination of IAAS, two highly successful initiatives from South Asia (India and Bangladesh) and a less successful example from sub-Saharan Africa (Malawi). 34.6.1 Carp polyculture in India Traditional carp polyculture in India has a long history but was extensive with wild seed of native carps (mainly catla, Catla catla; rohu; mrigal,

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Cirrhina mrigala) stocked in ponds without nutritional inputs (Bhimachar and Tripathi, 1967). Native and introduced carps (common carp, grass carp and silver carp) were bred in captivity following scientific research carried out in the 1950s and fish hatcheries were established to produce a more reliable source of seed. Indian scientists also developed so-called ‘composite culture’, a polyculture of the six species of native and exotic species, with eradication of predatory and weed fish, liming, stocking large fingerlings, fertilization with cow dung and CF, supplementary feeding with a 1 : 1 mixture of groundnut or mustard oil cake and rice bran or wheat bran, and provision of aquatic or terrestrial vegetation for grass carp The technological package was widely disseminated in India with the majority of farmers attaining extrapolated annual yields as high as 6–7 tonnes/ha (Tripathi and Ranadhir, 1982). Farmers in the Kolleru Lake region of Andhra Pradesh, which has been transformed into the ‘fish bowl’ of India, have developed a much simplified semi-intensive carp technology with just two species, rohu as the dominant species and catla at a ratio of 80–90 : 10–20 %, respectively (Nandeesha, 2001; Ramakrishna, 2007). As rohu has the highest market demand, farmers increased its stocking level from less than the 20 % recommended to more than 80 % of stocked fish. Fish are mainly fed so-called ‘farm-made feeds’ (Suresh, 2007), although in reality these are only a mixture of mainly de-oiled rice bran and oil cake meal fed to fish in perforated sacs suspended in the pond, a technique developed by farmers (Nandeesha, 2003). Extrapolated annual yields range from 7.5–12.5 tonnes/ha with total annual carp production of 0.45 million tonnes from about 60 000 ha of ponds in Andhra Pradesh. The use of FF could improve the efficiency and profitability of carp culture in India with FCRs reduced from 3 : 1 to nearly 1 : 1 (feasible with FF in ‘green water’ ponds), less labour for feed preparation and feeding and better water quality compared to use of farm-made feeds (Suresh, 2007). Farmers in Punjab, India were reported to be gradually adopting FF as the FCR was better at 1.6 than that of 2.1 for a farm-made mix of rice bran and oil cake, with similar costs to the farmer (Debnath et al., 2007).

34.6.2 Carp polyculture in Bangladesh Traditional polyculture of Indian major carps in Bangladesh was also extensive but semi-intensive fish culture has been introduced through projects (ADB, 2005). Farmers now attain extrapolated annual fish yields of 3–5 tonnes/ha in well-managed ponds using on-farm and locally purchased manures, brans and oil cakes although CF are widely used. Important introductions of exotic fish to the traditional polyculture of indigenous carps are grass carp fed with on-farm vegetation such as grass, banana leaves and duckweed, and filter-feeding silver carp which grows rapidly in green water ponds and comprises a major part of the harvest. Bangladesh IAAS are

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relatively simple compared to the traditional pond IAAS of China and Vietnam. Monogastric livestock are not commonly raised on or near the pond; nor are vegetables commonly raised on pond dikes, although cultivation of dike crops has developed recently in some areas (Ahmed et al., 2007; Edwards, 2007). There has been a dramatic increase in the production of intensive pond monoculture of striped catfish and Nile tilapia in the Greater Mymensingh area since about the year 2000 with both farm-made feed as well as FF (Ahmed, 2007; Ahmed and Hassan, 2007; Edwards and Karim, 2007). Catfish can be stocked in ponds at a much higher density than carps while maintaining a fast growth rate with average annual yields of over 8 tonnes/ha, almost three times greater than that of about 3 tonnes/ha for carps.

34.6.3 Small-scale farm integration in Malawi Pond-based IAAS are not traditional in sub-Saharan Africa and have been introduced through projects. Aquaculture development is considered to have stalled in the region despite decades of interventions and support programs from regional and international development agencies (FAO, 2006). In 10 countries of sub-Saharan Africa there are believed to be only about 110 000 ‘non-commercial’ farmers defined as small-scale subsistence, small-scale artisanal or integrated aquaculture normally practised by resource-poor farmers. A major reason for the limited contributed of aquaculture to fish consumption in Africa is undoubtedly the poor resource base of most small-scale African farms. WorldFish Center sought to make traditional farming systems more sustainable through integration of aquaculture into agriculture in Malawi (Brummett and Noble, 1995). However, the low productivity of aquaculture in these systems has severely constrained its contribution to rural development although pond water is also used to cultivate crops on or near the pond dikes (NASP, 2005). The average fish farmer in Malawi has one to two, 200 m2 ponds fertilized with vegetation from compost cribs in the pond and occasionally with limited amounts of livestock manure from mainly free-ranging livestock; and fed with maize or rice bran if available (Chimatiro and Chirwa, 2007). Extrapolated annual yields range from 0.5–2.3 tonnes/ha with an average of only 1.4 tonnes/ha. The cost of an adequate quantity of livestock manure to fertilize their ponds is too high for most small-scale fish farmers and CF are rarely used due to their high price (Hecht, 2007). The total annual national production from fish ponds in Malawi has reached only about 1000 tonnes. Rather than promoting aquaculture to provide greater food security at the family level in Malawi, it has now been recognized that farmers are motivated by profit and not raising fish for direct subsistence (NASP, 2005; FAO, 2006; Moehl et al., 2006). A paradigm shift in donor and lead agency

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support towards entrepreneurship rather than subsistence is required to increase the fish supply in many countries in sub-Saharan Africa with support targeted at zones with income earning potential such as sites well endowed with water or a peri-urban location with good access to markets for inputs and produce (Moehl et al., 2006; Brummett et al., 2008).

34.7 Bridging traditional and modern practice Aquaculture systems are being developed that incorporate one or more of the principles of traditional aquaculture to reduce the adverse environmental impact of industrial aquaculture in which a single target organism is fed with FF in monoculture (Edwards, 2004; Milstein, 2005; Azim and Little, 2006). Examples are outlined below that involve reduction of wastes in situ and treatment of effluents through integration.

34.7.1 Reduction of wastes in situ 80 : 20 system A pond feed-based system has been developed that combines production of high-value fish such as crucian carp, grass carp or tilapia fed with FF with traditional Chinese polyculture (Ye, 2002). In the ‘80 : 20 pond fish culture’ system about 80 % of the harvest weight is a high-value species and the other 20 % is a ‘service species’ such as the filter-feeding silver carp to remove phytoplankton and the carnivorous mandarin fish (Siniperca chuatsi) to control breeding in tilapia stocked ponds and also to consume wild fish and other competitors. Feeding a high-value target species with a high physical quality extruded and nutritionally complete FF leads to faster growth, higher production, better feed conversion and therefore higher profits than traditional polyculture technology with less adverse environmental impact. Following 17 years of experience of trials and demonstrations in China, the American Soybean Association’s International Marketing (ASA-IM) Program, in conjunction with the Chinese Extension Service, has recently promoted the 80 : 20 system in India, Indonesia, the Philippines and Vietnam (Manomaitis and Cremer, 2007). Bio-floc technology The principle behind bio-floc technology is addition of low-value carbohydrate-rich supplementary feed to constantly aerated intensive culture of fish fed with FF to stimulate N uptake by heterotrophic bacteria which form suspended microbial flocs of bacteria, fungi, microalgae and organic detritus in the culture system water (Avnimelech et al., 1994; Avnimelech, 2006; Serfling, 2006). Bio-flocs convert most wastes into natural food organisms which can be consumed by filter-feeding freshwater fish such as tilapia and shrimp, in contrast to conventional biofilter systems which treat water in

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recirculation systems by removing particulate waste. Bio-flocs reduce excess N in the system and both waste disposal costs and feed costs. Bio-flocs constituted almost 50 % of the conventional feed ration for tilapia (Avnimelech, 2007). A large commercial-scale tilapia farm using bio-flocs was developed in California, USA, in the 1980s and a similar tilapia farm was developed during the 1990s in Jordan which still operates (Serfling, 2006). A bio-floc system has also been developed in Israel (Avnimelech, 1998). There appears to be limited commercial application of bio-floc technology to date in inland aquaculture, possibly because of the high cost of maintaining particles in suspension. Evaluation of ‘activated sludge technology’ (AST) for intensive tilapia production in indoor tanks with a commercial tilapia producer in Thailand indicated that the closed system would not be a commercially viable alternative to tilapia culture in a conventional recirculating aquaculture system (Azim et al., 2008; Murray and Little, 2009). There was marked inhibition of feeding and growth associated with bio-floc accumulation in the closed system, challenging the claims of improved feeding efficiency in AST. Commercial pellets as supplementary feed Chemical fertilizers are widely used in small-scale semi-intensive aquaculture, e.g. Banagladesh and India (Sections 34.6.1 and 34.6.2). A case was made for their use to lower the cost of large-scale intensive culture of tilapia (Edwards et al., 2000). Chemical fertilizers are a cheaper source of N and P than nutrients contained in FF; and, furthermore, by using FF feed as a supplementary rather than as a complete feed in a chemically fertilized pond system, the use of FF and the FCR can be reduced considerably. Optimal pond fertilization produces Nile tilapia of only about 200–250 g in 5 months and subsequent growth slows considerably as fertilizer-induced phytoplankton is insufficient to sustain a high growth rate. Nile tilapia fed a daily supplementary feed ration with FF (commercial 30 % crude protein floating pellet) at 50 % satiation level in an optimally chemically fertilized pond produced growth as high as fish fed a 100 % satiation ration because of substantial use of natural food by the fish (Fig. 34.2) (Diana et al., 1994). It was most economical to grow fingerlings from 15 to 50–100 g for about 3 months in chemically fertilized ponds before starting to also give supplementary FF at a 50 % satiation rate (Diana et al., 1996). Higher-value fish of at least 500 g were harvested in another 3.5 months, with a FCR of only 1.0 due to considerable nutrition from natural food with an annual average extrapolated net yield of 21.0 tonnes/ha, almost double that of 8–11 tonnes/ ha with chemical fertilizer alone (Fig. 34.3). Pond hydroponics Promising hydroponic trials have cultivated terrestrial (Chinese cabbage, Brassica campestris cv pekinensis; Romaine lettuce, Lactuca sativa) and aquatic (water spinach, Ipomoea aquatica) vegetables in various susbstrates

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New technologies in aquaculture 500

Mean weight (g)

400

Fertilized 1.00 Feed 0.75 Feed 0.50 Feed 0.25 Feed

300

200

100

0 Jun

Jul

Aug

Sep

Oct

Nov

Dec

Fig. 34.2 The growth of Nile tilapia in fertilized ponds and with additional fractions of satiation fed with commercial pelleted feed. (Source: Diana et al., 1994) 700 50 g 600

100 g 150 g

Mean weight (g)

500 400

200 g 250 g

300 200 100 A B C D E 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Fig. 34.3 The growth of Nile tilapia in fertilized ponds with additional feeding to 50 % satiation by commercial pelleted feed. Five treatments (A–E) of date of first addition of pelleted feed are indicated above x axis. (Source: Diana et al., 1996)

on polystyrene rafts in eutrophic fish pond water in hybrid walking catfish and Nile tilapia ponds in Thailand (Pantanella, 2008). Hydroponics of vegetables through uptake of nutrients in fish ponds could enable farmers to get premium prices for soil-less produce which may also be considered to be organically grown as chemical fertilizers are not used.

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34.7.2 Treatment of intensive aquaculture effluents Intensive pond effluents for semi-intensive pond culture This involves integrated nutrient and water reuse through discharge of nutrient-rich effluents of intensive aquaculture ponds into semi-intensive ponds as a fertilizer where they are treated and converted into phytoplankton and grazed by filter-feeding fish. Hybrid walking catfish is raised intensively in Thailand on slaughterhouse offal mixed with rice bran or FF; some farms discharge the nutrient-rich effluents into ponds stocked with Chinese carps, Indian major carps and tilapia (Little and Griffiths, 1992). Integrated intensive–semi-intensive pond culture has also been reported with tilapia in Israel and the USA (Hargreaves, 2006). Many commercial intensive tilapia farms in Israel exchange their water with a nearby reservoir or fish pond in which semi-intensive polyculture of common carp and tilapia is carried out with the latter serving as a source of water as well as a treatment system for intensive culture effluents (Milstein, 2005). Water from intensive 1000 m2 fish ponds on a farm in Israel was reported to be exchanged five times daily with that in larger ponds which functioned as treatment reservoirs as well as semi-intensive fish ponds (Avnimelech, 1998). The integrated system required a large area of land as the ratio of semi-intensive to intensive ponds was at least 10 : 1. Research on the use of intensive aquaculture effluents to fertilize fish ponds has also been carried out in Hungary (Gal et al., 2003). Integration of intensive and semi-intensive aquaculture is similar to the concept of integrated multitrophic aquaculture (IMTA) in which species from different trophic or nutritional levels are incorporated into the same system in the coastal environment, e.g., mussels and seaweeds to utilize nutrients released from salmon cage farms (Ridler et al., 2006). Intensive cage culture within semi-intensive pond culture Wastes from intensive cage culture of tilapia were treated and recycled in the static water semi-intensive pond in which the cages were suspended, with simultaneous nursing of fingerlings to stock the next cage culture cycle (Yi et al., 1996; Yi, 1999). Tilapia of about 100 g were stocked in the cages and fed with FF until they reached a marketable size of at least 500 g; and tilapia fingerlings were nursed until they were about 100 g in semi-intensive culture in the same pond, feeding solely on natural food produced by fertilization of the pond with caged fish wastes. Large-size tilapia of relatively high-market value could be raised in 3 months in the integrated system. Aquaponics Aquaponics, the integration of aquaculture recirculating systems with hydroponics (the cultivation of vegetables without soil), has been run on a pilot scale since the mid-1970s, but few have been fully commercialized. The best known is a commercial-scale system run as a prototype at the Univer-

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sity of the Virgin Islands (UVI) (Rakcocy and Bailey, 2003; Rakocy et al., 2006). Tilapia are reared in tanks, fed with FF and the effluents are used to fertilize vegetables such as basil, lettuce and okra on floating sheets of polystyrene in hydroponic tanks. The system occupies 500 m2 of land and can produce 4.2–4.8 tonnes of tilapia and 5 tonnes of basil, 2.9 tonnes of okra or 1400 cases (24–30 heads per case) of leaf lettuce annually. The immediate potential is for niche markets in which consumers are willing to pay a higher price for high-quality fish and vegetables. Aquaponic systems based on the UVI design have been constructed and perform well at temperate sites in Australia, Canada and the USA and in the tropics in India, Mexico and Thailand (J.E. Racocy, pers comm). Partitioned aquaculture system (PAS) A partitioned aquaculture system (PAS) incorporates high-rate microalgal culture to fish culture (Brune et al., 2003; Hargreaves, 2006). Fish are confined at high density in a rearing tank and fed FF, with the water circulated through a shallow high-rate phytoplankton pond by a low-energy paddle to treat the fish wastes. Low-speed paddle wheels move large volumes of water at low velocities uniformly throughout the pond with filter-feeding tilapia reducing algal biomass in water produced by residual fertilization from channel catfish (Ictalurus punctatus) raised in adjacent raceways and fed FF. The PAS has the potential to reduce total water usage per unit of fish produced by 90 %. A less intensive PAS has subsequently been tested with channel catfish to benefit from fish stock management from growing fish at higher density while reducing the need for intensive system management (C.S. Tucker, pers comm). A PAS-type system has been adapted to an existing earthen pond with a channel catfish harvest of about 20 tonnes/ha. A commercial-sized 2.5 ha PAS system is about to be tested.

34.8 Future trends Traditional small-scale IAAS usually have such a low resource base that, in most cases, aquaculture in rice fields and ponds provides mainly household subsistence. It has been claimed that IAAS, in particular the Chinese carp polyculture system, are a relatively closed ecological cycle and thus an ecologically sustainable system which could be a model for aquaculture development elsewhere (Ruddle and Zhong, 1988; Folke and Kautsky, 1992; Korn, 1996). However, high-yielding IAAS are driven by a large import of nutrients from outside the system, mainly in FF for integrated livestock (Edwards, 1993). Attempts to introduce feedlot livestock to increase smallscale farm fish production have usually been unsuccessful and large-scale livestock farms are increasingly stand-alone biosecure operations, a trend which has been reinforced by avian influenza. Small-scale IAAS have a

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continuing role to play in providing small but significant contributions towards relatively poor farming household nutrition and income; they also provide an almost risk-free safety mechanism through which farmers may gain aquaculture experience before deciding if they wish to intensify production and profitability through use of off-farm inputs. As farmers throughout the developing world are increasingly motivated by income, direct on-farm integration of aquaculture has limited potential for major expansion, as indicated by experience in sub-Saharan Africa. Small-scale farmers on nutrient-poor farms aiming to develop aquaculture as a major occupation usually need to import nutrients from off-farm to intensify production. This invariably leads to specialization with a reduction of farm sub-systems or enterprises and on-farm integration as recent Chinese experience shows. Semi-intensive aquaculture based on indirect integration with locally available off-farm resources such as livestock manure, brans and oil cakes has huge potential for expansion as agriculture and livestock farming are also intensifying with greater amounts of wastes and by-products, as exemplified by recent experiences in South Asia (Bangladesh and India). The transfer of semi-intensive technology, including use of CF, to the Indian sub-continent is one of Asian inland aquaculture’s success stories. Indirect integration is also likely to increase fish production in sub-Saharan Africa. Traditional IPAS, as wastewater-fed aquaculture, is declining in most countries, e.g. China and Vietnam, and appears to have limited future relevance, with a possible exception of Kolkata, India. It was socially acceptable in some pre-industrial and early industrial societies because of high population pressure and scarce resources but appears to be a transient phenomenon (Edwards, 2005a, b). Once development starts to expand, several factors constrain the practice: rising value of peri-urban land; increasing contamination of wastewater with industrial effluents, leading to declines in yield and quality of produce; and increasing demands of more affluent consumers for larger and higher value species. Wastewater-fed aquaculture may have potential as a component of low-cost wastewater treatment, especially in arid and semi-arid climates where there is increasing necessity to recycle water (WHO, 2006). Traditional IFAS with direct use of trash and small-value fish to feed carnivorous fish is declining as indicated by experience in Cambodia and Vietnam. The practice is unlikely to be sustainable in the long term (APFIC, 2005). Semi-intensive aquaculture probably still dominates inland aquaculture in Asia, but there has been a spectacular rise in the use of intensive aquaculture based on FF, with growing concern about eutrophication of surface waters caused by farm effluents. The technologies outlined in this paper that incorporate one or more of the principles of traditional aquaculture to reduce nutrients in intensive farm effluents are mostly at the pilot scale and thus have had limited impact to date. However, they are likely to become

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of increasing importance alongside more conventional waste management and treatment strategies to ensure that inland aquaculture has minimal adverse environmental impact (Tucker et al., 2008).

34.9 Sources of further information and advice • For resource books on IAAS see Integrated Agriculture–Aquaculture, a Primer (IIRR and ICLARM, 1992) and Utilizing Different Aquatic Resources for Livelihoods in Asia (IIRR et al., 2001). Integrated Livestock–Fish Farming Systems is the title of a book by FAO (Little and Edwards, 2003). Recent conference proceedings on research and development in IAAS are Rural Aquaculture (Edwards et al., 2002) and Fishponds in Farming Systems (van der Zijpp et al., 2007). • For wastewater-fed aquaculture see the chapter in the UNEP International Source Book on the topic (Edwards, 2002b) and A Users Manual for the Cultivation of Commercially Important Edible Aquatic Plants in and around 4 Cities in Se Asia (PAPUSSA, 2002). • Pond fertilization is covered in depth in Dynamics of Pond Aquaculture (Egna and Boyd, 1997) and in a simplified way in the small book, Pond Fertilization: Biological Approach and Practical Applications (KnudHansen, 1998). FAO recently carried out studies on fertilizers and feeds in several countries (Hasan, 2007; Hasan et al., 2007). • Topics on IAAS, rice/fish, integration of poultry, pigs and large ruminants with fish, fertilizers, IFAS and traditional aquaculture in various countries can be found in the CABI Aquaculture Compendium (CABI, 2006). • The theory and considerations for design and operation of photosynthetic suspended growth systems that are involved in several systems to incorporate the principles of traditional practice into modern intensive aquaculture are reviewed by Hargreaves (2006).

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Brussels, Technical Centre for Agricultural and Rural Co-operation, Wageningen, and Food and Agriculture Organization of the United Nations, Rome. tacon agj and de silva ss (1997) Feed preparation and feed management strategies within semi-intensive fish farming systems in the tropics, Aquaculture, 151, 379–404. taiganides ep (1979) Wastes are . . . resources out of place, Agricultural Wastes, 1, 1–9. thongrod s (2007) Analysis of feeds and fertilizers for sustainable aquaculture development in Thailand, in Hasan MR, Hecht T, De Silva SS and Tacon AGJ (eds), Study and analysis of feeds and fertilizers for sustainable aquaculture development, FAO Fisheries Technical Paper 497, Food and Agriculture Organization of the United Nations, Rome, 309–30. tripathi sd and ranadhir m (1982) An economic analysis of composite fish culture in India, Aquaculture Economics Research in Asia, Proceedings of a Workshop Held in Singapore, 2–5 June 1981, IDRC-193e, Ottawa, ONT, 90–6. tucker cs, hargreaves ja and boyd ce (2008) Better management practices for freshwarer pond aquaculture, in Tucker CS and Hargreaves JA (eds), Environmental Best management Practices for Aquaculture, Wiley-Blackwell, Ames, IA, 151–226. uphoff n (2007) The system of rice intensification (SRI): using alternative cultural practices to increase rice production and profitability from existing yield potentials, IRC Newsletter, No. 55, Food and Agriculture Organization of the United Nations, Rome. van der zijpp aj, verreth jaj, le qt, van mensvoort mef and beveridge mcm (2007) Fishponds in farming systems, Wageningen Academic, Wageningen. vo qh and edwards p (2005) Wastewater reuse through urban aquaculture in Hanoi, Vietnam: status and prospects, in Costa-Pierce B, Desbonnet A, Edwards P and Baker D (eds), Urban Aquaculture, CABI, Wallingford, 103–17. who (2006) Guidelines for the Safe Use of Wastewater, Excreta and Greywater. Volume 3, Wastewater and Excreta Use in Aquaculture, World Health Organization, Geneva. yakupitiyage a (1993) On-farm feed preparation and feeding strategies for carps and tilapias, in New MB, Tacon AGJ and Csavas I (eds), Farm-made Aquafeeds. Proceedings of the FAO/AADCP Regional Expert Consultation on Farm-made Aquafeeds, 14–18 December 1992, Bangkok, FAO-RAPA/AADCP, Bangkok, 87–100. ye jy (2002) Carp polyculture system in China: challenges and future trends, in Eleftheriou M and Eleftheriou A (eds), Proceedings of the ASEM Workshop AQUACHALLENGE, April 27–30, Beijing, ACP-EU Fish Res Rep 14, 27–34. yee awc (1999) New developments in integrated dike-pond agriculture-aquaculture in the Zhujiang Delta, China: ecological implications, Ambio, 28(6), 529–33. yi y (1999) Modelling growth of Nile tilapia (Oreochromis niloticus) in a cagecum-pond integrated culture system, Aquacultural Engineering, 21, 113–33. yi y, lin ck and diana js (1996) Influence of Nile tilapia (Oreochromis niloticus) stocking density in cages on their growth and yield in cages and in ponds containing the cages, Aquaculture, 146, 205–15.

35 Use of information technology in aquaculture J. Bostock, University of Stirling, UK

Abstract: Continuing advances in information and communications technologies (ICT) are providing increasing computing power in smaller units and at lower cost. Of particular significance is the rise of the Internet, and the increasingly ubiquitous access it is providing to information and global communication. This chapter explores how ICT is being applied in aquaculture to help producers reduce costs whilst improving quality and customer service, and to meet higher standards of environmental and social responsibility. Key topics include monitoring and control, management information systems and product traceability. The role of ICT in supporting sector innovation is also explored. Key words: ICT, software, Internet, communications, traceability.

35.1 Introduction 35.1.1

Information and communications technology (ICT) in aquaculture development Continuing advances in information and communications technologies (ICT) are providing increasing computing power in smaller units and at lower cost. Of particular significance is the rise of the Internet and the increasingly ubiquitous access it is providing to information and global communication. ICT is helping to drive many industrial and social changes, so this chapter explores how ICT is being applied in aquaculture, and prospects for the future. It must be noted at the outset that aquaculture is very diverse with respect to species produced, technologies employed, scale and type of enterprise, physical, social and economic environments. Many aquaculture products are from traditional extensive or semi-intensive systems that appear relatively untouched by modern ICT. At the other end of the scale is the salmon industry, where continuing consolidation has led to the emergence

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of substantial vertically integrated international corporations, where the use of sophisticated ICT has become essential. This chapter focuses mainly on the direct use of ICT by aquaculture enterprises and on specialist software and services designed to support the sector. The use of standard software for business administration is only briefly covered. Most aquaculture enterprises will also be benefiting indirectly from the use of ICT by their suppliers (goods, services and utilities), and possibly by their customers. However, boundaries need to be drawn, so these will only be considered where there are specific linkages to the activities and performance of aquaculture producers. Almost all aquaculture enterprises operate in a competitive environment. Even in subsistence aquaculture, there is competition from other potential smallholder activities that might yield better returns on the efforts and resources employed. Investment decisions for ICT, as with other technologies, will therefore depend on consideration of costs, benefits and risks. ICT investments that rapidly reduce overall cost of production and increase competitiveness with minimal risk should prove attractive and be readily adopted. Such technologies might be considered transformational when enterprises that do not adopt the new technology are no longer able to compete and go out of business, or when they enable novel products or production systems that can open up new markets or production environments. However, relatively few technologies are truly transformational, particularly in the short term, and analysis of costs and benefits must take a broad approach (Table 35.1). Especially in smaller enterprises with few formal procedures for decision making, adoption may be more strongly influenced by cultural and practical factors. The first uses of ICT in aquaculture enterprises are usually generic and widely used tools such as mobile phone text (SMS) messaging or an office computer running accounts software (Nieto, 2005).1 ITC may also be introduced through control systems for aquaculture equipment such as automated feeders, aerators and oxygenators and alarm systems, although integration with wider management information systems (MIS) is still limited. The use of computers for core stock management activities is more common, and increasing requirement for full production chain traceability is driving further adoption. Better access to the Internet and its use for non-business purposes is also lowering the barriers for adoption of Internetbased commerce, learning and communications.

35.1.2 The functions of information and communications technology Information and communications technology provide a wide variety of tools to support all kinds of human endeavour. Systems are commonly described in terms of hardware (the computers and network cables, etc.) and software (programs that run on the computers). Taking a reductionist approach, the core functions of most ICT systems involve the collection,

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Table 35.1

Factors affecting adoption of ICT in aquaculture

Factors affecting ICT adoption

Examples

Regulatory

•

Regulatory requirements concerning data recording and reporting

Economic

•

Overall cost–benefit analysis considering capital cost, financing cost (if applicable), maintenance, operating and training costs and risks of cost escalation, etc. considering expected cost savings, potential for increased sales or prices, risks that these targets will not be achieved Capital cost in relation to available finance or availability of loans (i.e. there may be financial barriers even if cost–benefit analysis is positive) 䊊

䊊

•

Practical

• • •

Availability and applicability of ITC solutions for different types of aquaculture system and enterprise Robustness of system in relation to environment where it will be deployed Ease of use by target operators intellectual level required language localisation if outdoors, accessibility whilst wearing gloves or visibility in bright sunlight or at night whether it makes core tasks easier or longer and more complex ease of correcting errors in data entry Reliability and ease of access to maintenance or support services Health and safety implications 䊊 䊊 䊊

䊊

䊊

• • Social and cultural

• •

Level of understanding of issues that ICT solutions are addressing Existing attitudes to ICT: degree of trust or mistrust in technology or its promoters perceptions about people who sit in offices and use computers (intellectual or gender prejudices) openness to learning Peer pressure from other enterprises, including competitors, suppliers and customers Perceived impact on employment or working practices, e.g. on job numbers or job specifications on workload or responsibilities on scrutiny of staff performance on job satisfaction 䊊

䊊

䊊

• •

䊊 䊊 䊊 䊊

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storage, manipulation, transmission and display of data. Whilst often not obvious to users, a database is at the heart of most computer applications. These store data elements in a structured way that allows interrogation and retrieval and often further manipulation. A basic example from aquaculture is the daily collection of data on feed fed to each batch of fish and the usually less frequent sampling of the batch to measure average weight. The data may be collected and entered manually into a computer, or captured directly through computerised monitoring systems. Combining these data elements through a simple calculation provides information on the food conversion ratio (FCR) – one measure of production efficiency. The collection and storage of data is usually of little benefit and, indeed, can be distracting and confusing until it is analysed and turned into useful information. That information may also be of little value until it is properly communicated to the right person at the right time and they have acted appropriately on it. Computers and the software they run can play an important part in those later processes, providing ways of presenting and visualising the information, sorting and highlighting the most significant according to built-in rules. The receiver of the information needs to evaluate it in relation to their existing knowledge, interpreting and incorporating it into their understanding of the situation in order to decide what action is necessary. Since people vary in their knowledge, experience and approach, the same information may be treated differently, perhaps leading to opposite decisions. Taking the human element out of the process is to some degree possible through the use of expert systems. However, this is rarely the goal. ICT is usually implemented to support rather than replace human decision making. In the following sections the direct role of ICT in decision support is examined first, and then the wider issues of ICT in support of knowledge building, learning and innovation.

35.2 Information and communications technology (ICT) for productivity and effectiveness 35.2.1 Principles of monitoring, control and automation Management often involves making a plan for activities and targets, communicating that to everyone involved, monitoring actual progress in relation to planned performance and taking controlling actions when deviations from the plan are detected. These functions can all be supported by ICT, especially where the required control actions can be determined by reference to a simple set of rules. A simple example of automated monitoring and control is sometimes found in aeration systems. An oxygen probe monitors dissolved oxygen (DO) concentration in a fish tank or other strategic location. The signal is monitored by a microprocessor device and if the value drops below a preprogrammed level, an aeration device is started. When DO levels have risen

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above a set level, the device is stopped. Additional rules may be added, such as to trigger an alarm if DO levels fall below a second lower value (indicating a problem with the aeration device), or to add adjustable hysteresis (width of ‘dead’ or ‘trigger’ band) to prevent too frequent starting and stopping of the device. Similar types of control systems can be used for temperature or pH control, maintaining water volumes and correct salinity or regulating ozonation. Generically they are all closed feedback systems where equipment is controlled via actuators depending on values obtained from sensors and compared with programmed decision rules. Stand-alone process control equipment of this type are generally built around a control box in the form of a PID (proportional-integral-derivative) controller with integrated LED or LCD display and simple push-button or touch-screen controls. Such basic monitoring and control systems are usually mounted close to the monitoring point and display current measured values and perhaps maximum and minimum values recorded since last reset. Decisions concerning the parameters to monitor have been examined by Timmons et al. (2001),2 and the equipment available by Lekang (2007).3 It is often desirable to group these data and control facilities by area, or bring them together in a central place, especially for a large farm or where there are few staff. This may be done through the use of one of more customised control panels which use a programmable logic controller (PLC) to process data from multiple inputs and route instructions to selected outputs. These may be further linked through a PC-based ‘distributed control system’ (DCS) or ‘supervisory control and data acquisition’ (SCADA) system. The terms are broadly interchangeable, although DCS usually refers to localised industrial applications, whereas SCADA systems refer to widely distributed systems using a mix of communications links. In DCS/SCADA systems the local control units have appropriate communication facilities built in and are referred to as remote terminal units (RTUs). The main computer (which may be a PC, server or industrially mounted computer with touch-screen panel display) is the master station, which normally runs a graphical software application, ‘human–machine interface’ (HMI), displaying the status of each monitored component or process. In addition to increased convenience, advantages include the addition of data logging to a central database for further analysis and the centralisation of alarm functions, perhaps with further options added such as telephone list dialling or SMS messaging (Fig. 35.1). The most important design feature, however, is that the DCS/SCADA system sits on top of the individual monitoring and control systems, providing greater functionality without compromising the robustness of industrial process controllers. If the computer running the DCS/SCADA software crashes, the individual RTUs continue to work as normal. Cheaper multichannel monitoring and optionally control systems can be built using a PC fitted with an appropriate data acquisition card or USB module directly linked to sensors and perhaps

Use of information technology in aquaculture

A

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B Fish tanks/raceways/ponds Farm Patrol Setup Diagram

Oxygen probe

Oxygen probe

Remote access by PC using web browser via broadband or dial-up connection

Farm Patrol unit can have up to 256 inputs

220 Vac power supply Farm Patrol unit with local touch-screen and Telephone line alarm beacon/sounder Standard network cable link up to 100 m

Remote access by mobile phone using web browser

Oxygen, temperature & pH probes

Transmitter unit A radio unit can Twisted pair be used where cable link up the office is more to 750 m than 250 m from the Farm RS232/485 Patrol unit convertor

Receiver unit Local access by PC using network connection

C

Fig. 35.1 (A–C) Farm Patrol monitoring and control system from Pisces Engineering. (With kind permission from Pisces Engineering Ltd.)

actuators. This can be a useful approach for research aquariums, where cost is a major factor, and flexibility in configuration is also an advantage. However, without serious back-up systems, it is not robust enough for commercial applications. Building complex integrated monitoring and control systems has usually required a good understanding of the different signal types generated by

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the sensors (transducers), knowledge of PCL programming languages, data communications infrastructures (e.g. RS-485 or Ethernet networking used between the RTUs and computer(s)) and protocols (such as Modicon MODBUS, IEC 60870-5-101 or 104, IEC 61850, Profibus, DNP3 and many other proprietary protocols). The value of such monitoring equipment can be increased by recording the collected data for more detailed analysis. Some instruments include data logging facilities, but a SCADA system enables a wider range of variables to be monitored, compared and displayed in real time. An increasingly common enhancement is for the SCADA system to run on an Internet connected server providing an interface that can be accessed via a standard browser from any Internet connected computer. However, the security risks of doing this need to be carefully assessed before implementation.

35.2.2 Sensors and monitoring tools for aquaculture stock The examples used above to illustrate process control in an intensive hatchery or recirculated aquaculture system borrow heavily from chemical process control or wastewater treatment system engineering. The sensors measure parameters relating to water quality and flow rates, and control equipment designed to maintain these within set parameters. Farmers, however, are also very interested in monitoring the status of their stocks, including numbers, size, feed consumption, health and behaviour. A range of more specialist sensors and applications have been developed to help with this. Counting stock An accurate assessment of stock numbers is essential for good management, but can be difficult to achieve, especially when individuals are small and easily damaged by handling. Manual sampling is slow and often not particularly accurate. Automated counting, especially if it helps to avoid significant stress or damage, is very desirable. One of the earlier approaches was a simple infrared beam across a pipe opening. As fish (or possibly other type of stock) pass across the detector, the beam is momentarily broken and the fish counted. The main problem with this approach was that if fish are crowded together, several may cross the detector together, but the beam is interrupted only once, leading to an underestimate of the numbers (Shardlow and Hyatt, 2004).4 More recent pipeline counters (e.g. by the Icelandic company Vaki) use a more sophisticated system where both a vertical and horizontal axis outline image of each fish is captured as it passes through a digital camera-based sensor unit containing a mirror (Fig. 35.2). By using dual images, the analysis software can detect and count the fish even when two or more overlap as they pass through the counter (Fig. 35.3). Accuracies of over 98 % are claimed if the system is operated under optimal conditions for fish weights between 0.2 and 400 g. In practice, the bunching

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Fig. 35.2 Vaki pipeline fish counters. (With kind permission from Vaki)

Fig. 35.3 Record from Vaki fish counter, showing image of salmon smolts as they pass through the counter. (With kind permission from Vaki)

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Fig. 35.4 AquaScan fish counters installed on Norwegian well boat graders. (With kind permission from AquaScan & Sølvtrans)

Fig. 35.5 Laptop computer running AquaScan fish counting software. (With kind permission from AquaScan and Sølvtrans)

of fish can still be a problem, and accuracy can also be reduced by debris in the water and some double counting of fish that do not pass smoothly through the detector. However, these counting systems (and functionally similar equipment from the Norwegian company AquaScan) have now become standard equipment, particularly in the salmon industry, where efficiency is paramount (Figs 35.4–35.6). Another approach that has been tested in cage aquaculture (Bjordal et al., 1993)5 and is used in fisheries biology is sonar systems (Fisheries and

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Fig. 35.6 AquaScan fish counter detail. (With kind permission from AquaScan)

Oceans Canada, 2008).6 Previous generations of split-beam sonar were more or less able to discriminate individual fish, allowing software to count the number present within the radius of the sonar beam. Newer multibeam high-frequency sonar provides a more detailed image and hence more accurate count. Estimating weight and biomass Biomass estimations are essential for feed management and are derived from multiplying numbers by average weight. Knowledge of both average weights and the distribution of weights can also be important for grading and harvesting operations. The accuracy of sample weights depends on having sufficient numbers, and weighing individual fish is time-consuming and stressful. Approaches to solving this problem generally involve techniques to estimate the fish size, and then convert these dimensions to weight via equations derived from empirical measurements. The two main approaches in use are sampling frames and stereo cameras.

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Fig. 35.7 Vaki biomass frame. (With kind permission from Vaki)

Sampling frames are manufactured by Vaki (Fig. 35.7) and Storvik (model developed by AquaMetric). These consist of a rectangular frame with a grid of infrared beams. As a fish swims through the frame, the beams are broken for varying lengths of time from which software is able to build up an image of the fish shape. The Vaki system calculates the length and depth of the fish, whilst the Storvik model also adds width. From this, the weight and condition factor can then be derived from constants according to species. The frame is usually deployed in a tank or cage for between four and 24 hours to generate a large enough sample size. Analysis can be carried out on a hand-held computer on site, or downloaded to a desktop computer for analysis and incorporation into other management information systems.

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Stereo vision camera systems (e.g. Akva Vicass) are able to capture images at a higher rate and use analysis software to determine fish size depending on the exact orientation of the fish to the cameras (Costa et al., 2006).7 Sonar systems have also been tested for measuring fish size (Knudsen et al., 2004)8 but have not yet proved sufficiently accurate or cost-effective for commercial use. Feed management In many systems, the cost of feed is the largest single component of direct operating costs. Optimal management of feed is therefore a critical factor for profitability. Technologies to help with this have mostly been developed for marine cage systems, where depth makes it difficult to monitor feed consumption by eye. Where feed is controlled manually (e.g. dispensed by feed cannons) underwater video cameras have become popular to help farmers evaluate the feeding response and adjust feed rate and amount (Fig. 35.8). Manual air-lift pellet collectors can also be used to check for feed wastage. On sites where feeding is fully automated, sensors are required that can provide feedback for the control system. The most sophisticated currently available is the AkvasmartTM system. This has a choice of two technologies for detecting waste feed that is falling through the bottom of the cage net. The first is an infrared beam detector, mounted at the base of a collector cone and positioned towards the bottom of the fish cage. The second is a sonar-based feed pellet detector, with integrated video camera to help observe fish feeding activity. The Akvasmart feeders use feedback from the pellet detectors to determine when to halt feed input in order to prevent waste. The control system is also programmed with the expected feed requirement of the fish, and can take additional inputs from temperature, current and oxygen sensors to adjust further both total feed quantity and to ensure it is delivered when conditions are optimal and fish are actively feeding. Earlier work using sonar to assess feeding behaviour (Bjordal et al., 1993)5 showed promise, but has not yet proved commercially successful. Fish identification and individual monitoring It is routine practice to tag individual fish used in aquaculture breeding programmes. Tagging is also common in restocking programmes such as those for Pacific salmon in Canada, and many other branches of fisheries research. External visual tags such as T-tags, VI alpha tags or visible implant elastomer tags (e.g. Northwest Marine Technology) have advantages in some circumstances, but PIT (passive integrated transponder) tags are now widely used. These are small microchips that can be injected into the body cavity of quite small fish (e.g. 2 g for European perch – Baras et al., 20009). When the chipped fish comes into the range of a PIT tag reader (less than 20 cm), low-frequency radio waves from the reader excite the transponder

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Top of the line underwater and surface video camera. SmartEye can pan 360°, and tilt 90°.

Robust and waterproof connection plug.

An extra bottom camera checks for morts. New Super low-light bottom camera.

A SmartEye system flow chart:

Dual SmartWinch

SmartBox

Video Base Receiver

SmartController

Monitor

Portable PC

SmartEye Cameras B

Fig. 35.8 (A and B) AkvasmartTM SmartEye camera system. (With kind permission from Akva)

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inside the PIT tag causing it to transmit its unique identification code to the reader. PIT tag readers are usually hand-held, or can be mounted in fish passes or fish pump systems. Data from the readers can be readily integrated into other computer applications. Where it is desirable to track and identify individuals over a greater range, tags are available with either low-power radio transmitters, or sonic transmitters (Thorsteinsson et al., 2002).10 Radio tags do not work well in seawater, and usually have a more limited range than acoustic tags, but can be more cost-effective for some applications. Acoustic tags transmit a sound signal (ping), including a unique identity code, which can be detected with a hydrophone receiver up to 1 km in seawater. By using an array of hydrophones, the 3D position of the tag can be calculated and software used to create real-time images or movement tracks. The tags are larger than PIT tags due to the requirement for an integrated battery. However, many are small enough for insertion into the body cavity, or may be attached externally. The delay between pings is determined by the nature of the research. A short delay provides more detailed track data, but results in shorter tag life (due to heavier battery use) and reduces the number of fish that can be tagged (due to increased risk of ping collisions which prevents the receiver from identifying individual tag locations). More sophisticated acoustic encoding techniques, such as that used in Lotek MAP acoustic products can help to overcome this limitation (Fig. 35.9). Combined radio and acoustic tags for fish that migrate between fresh and saltwater are also available. A further advance is the use of telemetry tags, which include sensors and data recording facilities. For instance an accelerometer to measure swimming activity or detect mortality; sensors for pH, temperature, pressure or

Fig. 35.9

Lotek acoustic tags. (With kind permission from Lotek)

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New technologies in aquaculture

daylength; a heart rate monitor or tail beat monitor; and a compass to detect swimming direction. One system by Star-Oddi11 also allows global positioning system (GPS) location data to be recorded when the fish pass within range of a vessel or station transmitting this data via sonar. Data can be collected and stored on the tag and either recovered when the fish is caught or some can upload data to the listening station when in range (e.g. a buoy with facilities for relaying the data on via global system for mobile communication (GSM) or satellite communications). For large fish, ‘pop-up’ satellite telemetry tags have been used, which detach from the fish after a preset time, or when specific conditions are met, and float to the surface, where they communicate with a satellite (e.g. Microwave Telemetry Inc., which use a transmission frequency of 401.650 MHz ± 36 kHz ) to upload their data. A further related development is ‘business card tags’ which include an acoustic transmitter and receiver. When fish with these tags come into close proximity, they exchange identity data and record the time of the encounter (Dagorn and Holland, 2008).12 As the capability of these tags increases, and sizes decrease, it seems likely that they will be more commonly used in aquaculture research, or even as live monitors of conditions and behaviour within cages, tanks or ponds.

35.2.3 Stock management systems For most aquaculture operations the stock (mainly livestock, but also feed and pharmaceuticals) is the most valuable current asset. Optimising production efficiency requires careful monitoring of stock status and key variables such as feed usage, growth and mortality rates. Forward feed and harvest planning can be greatly assisted by software capable of stock projections using various modelling techniques. Analysis of past data can provide information useful for management and trend data for incorporation in growth and feed utilisation models. Many farms develop their own computer-based stock management systems using spreadsheet software. The primary objective is usually to track changes in the numbers and weight of individual stock batches, the feed utilised for the batch and ultimate harvest data. For most farms, there is also the issue of which stock batch is held in which rearing unit. This can be relatively straightforward in some systems, or can be highly complex where stock is frequently graded, split or mixed between many units. Using spreadsheets, managers can set up a system that suits their own farm and allows forward projections using growth equations or calculations based on tables from feed manufacturers. However, there can also be serious drawbacks to using spreadsheets. Since the data are stored in a format also used for analysis and display, performing different analysis requires a reworking of the data, or an increasingly complex spreadsheet structure. Spreadsheets also have limited data storage capacity so, in the case of large farms, new spreadsheets are required each month with others used for analysing summarised longer-term data. In smaller farms, it is often only the person who

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developed the spreadsheet application who is able to use it properly, causing significant difficulties if they leave the company. It is normally preferable to store data within a database which can be accessed and analysed as necessary using customised forms and reports. Popular database management applications such as Microsoft Access provide tools for this, but require considerable expertise to develop an application sophisticated enough for most farms. Commissioning a bespoke database application from an external company is usually too expensive for smaller farms and raises questions about long-term maintenance. A more popular solution has been the development of specialist aquaculture applications, which have either been marketed commercially, or provided as a service by feed companies to their customers. Early packages developed in the 1980s included Pro Manager from Island Science (USA) and Farmcontrol from the feed company Ewos. The latter has been continually developed, although by different owners, most recently by Maritech, which has itself been purchased by Akva. This company already markets one of the most sophisticated stock management systems, namely FishTalkTM (developed after the company’s take over of Superior Systems). This is actually a suite of applications that extend the functionality of the core stock management module to include links with feed controllers, value chain planning, traceability and equipment maintenance (Fig. 35.10). Also worthy of note

Overview of status on site with optional definition of column.

Company portal and site portal giving quick overview of status and shortcuts to the most common routines.

The user can easily adjust the information on the screen to each user or role.

Easy to select site or unit in organisation tree.

A

Presentation of key numbers when using dashbard.

Visual overview of all registrations in the period.

Fig. 35.10 (A and B) Akvasmart FishTalkTM software. See also p 1080. (With kind permission from Akva)

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New technologies in aquaculture

FishTalk CV displays chosen information in simple sections.

Detailed overview of feed types in use and when the fish were fed.

Graphical presentation of size distribution.

Flexible selection of the traceability information to be presented in the report.

B

Fig. 35.10 Cont’d

is Mercatus Aqua Farmer. This is a new generation of software built around secure central server applications which can be accessed by web browsers for data entry and reporting (often referred to as cloud computing – Chappell, 2008).13 Centralisation of data is important as the salmon industry in particular becomes more corporate and production needs to be coordinated across tens if not hundreds of sites. The data in Mercatus Aqua Farmer and Farmcontrol is held in Microsoft SQL databases allowing ready integration with other systems and analysis using a wide variety of standard

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tools. All packages have some facilities to export data to spreadsheets. This allows both users of commercial software or spreadsheet-based systems to use more advanced business modelling tools such as @RISK (probabilistic) or EVOLVERTM (deterministic) from Palisade. These can help with optimisation of production schedules, feed purchases and other analysis to find lowest costs or best strategies to maximise revenue. Many other commercial packages have been developed, but have not proved commercially successful. A summary of available stock management software is shown in Table 35.2. Much of the recent development has been driven by the increasingly corporate structure of the salmon industry with requirements to coordinate production across many sites and different countries. Traceability has become a particularly important element, requiring systems that exchange data with both suppliers and customers.

35.2.4 Business information systems Aquaculture enterprises have many business processes in common with other industries. These include managing sales, purchasing and accounts, personnel and payroll, health and safety, asset and maintenance records, etc. Commercial packages are available for many of these, especially accounts and payroll. For other record keeping, customised databases are generally more suitable than spreadsheets, although smaller companies may well use the latter as they are simpler to develop and analyse. Few specialised tools for aquaculture exist, although Akva market a separate database for maintaining service logs for nets and equipment (Fishtalk Service Log). A key problem when multiple systems are in use is the risk of data duplication and mismatch. For instance, elements of staff personal data may be held in a payroll system, a health and safety system and a customer relations system. When an element of that changes (e.g. name change through marriage), it is necessary to update three different software systems. If that is not done, problems may occur when using information from one database to form queries for another. There is also an inherent inefficiency if, for instance, details of each sale need to be separately recorded in a stock management system, the sales and customer relations system, the transport management system, traceability system and the finance system. Linking different package databases together is sometimes possible, for instance using ODBC (open database connectivity), which is a feature of Microsoft Office and some other software. Other solutions are also available, but usually require considerable IT expertise if working with systems from different vendors. The ideal is for all the company information to be held in a single database, with each department updating the records for which it is responsible, and reports being extracted as required for management. This is the target of ERP (enterprise resource planning) software, which aims to provide an

AquaMaximaTM Production-IT

Cowex, Denmark http://www.cowex.com/ aquamaxima.aspx

Flexible

Salmon

An Internet-based application that is readily scalable for multiple sites and users. Data can be exported as OLAP cubes for analysis in Excel, Business Objects or other compatible programs. It can be integrated with fish counting and feeding systems. Advertised as a ‘management execution system’ this software from a process control specialist, integrates production management, supervision and alarm, recirculation control and automatic feeding. Analysis and reporting tools assist with production optimisation and batch level traceability.

Stock management program for pond-based shrimp farming. Particular features include tools for comparing the results of different treatments and conditions, and an economic forecasting tool to help with planning pond harvests.

Shrimp

Aqua-In-Tech, USA (Originally developed by Naturisa, Ecuador) http://www.tropical-fishfood.com/computer_ software_for_pond_ manag.htm http://aqua-in-tech.com/ shrimp_tank_farm_ software_a_po.htm Mercatus, Norway http://www.mercatus.no/

Aqua Farmer

A range of stock management systems for different species with comprehensive data collection and management facilities including orders, harvesting and sales. Data input via hand-held computer is available, and numerous reports including traceability records.

Salmon, trout, tuna, abalone, pearl, processing

Aqua Assist Pty Ltd, Australia http://www.aquaassist.com/

Abalone Assist, Manufacture Assist, Hatchery Assist, Process Assist, Salmon Assist, Tuna Assist, Pearl Assist & Monitoring Assist AP/1 (or ‘Shrimp Tank’)

Description

Species

Developer or supplier

Examples of aquaculture stock management programs

Package

Table 35.2

Maritech (Part of Akva Group), Norway, UK http://www.wisefish.com/

Craig Treat, USA http://fishapps.com/ FishApps/Hatcheus_ Manax.html Softmakers, Italy http://www.fishmakers.com/

Akva Group, Norway, Chile, UK http://www.akvagroup.com/

FarmControl

FishApps Hatchery Manager

FishTalkTM (Modules: Control; CV; Service Log; & Value Chain Planner)

Fishmakers

Skretting, UK http://www.djournal.dk/

Djournal

Originally developed for salmon, but now used for more than 10 species

Sea bass, sea bream

Designed for salmonid hatcheries

Salmon, trout, seabass, seabream, cobia and several other species

Salmon, trout

A modular system geared towards the management of production, organisation and rationalisation processes. Includes stock movements, feeds and records of medications. Basic cost information can also be entered to calculate cost of production. Companion software – Fishmakers Tables – allows the creation and distribution of feed tables. A suite of modular programs that has been developed to meet the needs of major Norwegian aquaculture companies. FishTalk Control is the main stock management module with facilities for recording and reporting a wide range of variables. The program integrates well with Akva adaptive feed systems and allows interrogation at site or company level. FishTalk CV integrates with Control to provide a full traceability record. FishTalk Service Log allows detailed recording of equipment, service and maintenance. FishTalk Value Chain Planner is a simulation-based production planning tool.

Originally developed in Denmark for trout, it was adopted by Trouw (subsequently part of Skretting) as part of their customer service package. It remains exclusively available for Skretting customers. One of the more sophisticated packages using growth models based on feed tables and expected feed conversion ratios due to previous ownership by the Ewos feed company. The system uses Microsoft SQL database technology, which makes it easy to integrate with other systems or analysis tools. Provides a customised record keeping system for hatcheries, including egg inventories and calculators for feed and treatments.

Department of Agricultural Economics, Mississippi State University, USA http://www.agecon.msstate. edu/software/fishy.php

L.G.V Systems Ltd, Israel http://www.meydag.co.il/

De Hann Automatisering BV, Netherlands http://www.dha-software. com/ Venture Farms Pte Ltd, Singapore (Originally Applied Information Management Systems Ltd (AIMS)) http://members.tripod.com/ jimmyaolim/id79.htm

FISHY

Meydag

Poseidon Aquaculture Module Shrimp

Adaptable

Any species or system

Catfish

Species

Stock management system developed for shrimp farming in Asia. Provides facilities for multiple farms

FISHY is a program for catfish production management written in Visual FoxPro. Fishy records and reports historical fish farming events including stocking, feeding, harvesting, moving and losing fish. It can also simulate fish growth into the future, enabling its users to predict future harvest dates, numbers and weights, as well as future feed needs. The program is based around six modules: the cage/ pond module maintains records of stock, weights, numbers, movements and water quality data; the feed module maintains feed inventory data and usage in each cage or pond; the marketing module records sales actions; the financial module tracks and analyses expenditure; the reporting module has 26 standard reports and facilities for custom reports; and the system module is used to define system constants and variables. A relatively simple aquaculture stock management package that integrates into the company’s wider Poseidon suite of software for full chain traceability

Description

Note: There are several other packages that have been important in the past, but do not show up as currently available in web searches.

Samakia

Developer or supplier

Cont’d

Package

Table 35.2

Use of information technology in aquaculture

1085

integrated solution for all normal major business functions. The market leaders are SAP and Oracle Applications, although there are many others including Microsoft and Lawson. The latter has provided an ERP solution to The New Zealand King Salmon Company (iStart, 2007).14 The stated aim being to improve the company’s ability to respond to customer demand through better integration of sales, production and distribution data, and to improve customer service including shorter order times and the introduction of out-of-hours e-sales. A major challenge in this instance has been development of the stock management module, as aquaculture is fundamentally different to most manufacturing processes. This highlights one of the key issues with ERP software, which is the degree to which it can be adapted to meet the exact needs of a company, rather than forcing the company to use standard systems. However, as international aquaculture companies continue to develop, it seems likely that more ERP software companies will offer solutions to the industry, and that companies such as Akvasmart will build up their range of integrated offerings to encompass the full needs of the industry. Consideration should also be given to more general business communications at this point. Mobile telephones are at the centre of many companies’ day-to-day operations, usually backed up by voice, text and e-mail messaging. Electronic calendar and contacts directories are also widely used. There is increasing linkage and convergence between mobile devices (e.g. web-enabled cell phones) and office systems, such that staff can access a wider range of information and communication channels. Organisations will need to continually assess the costs and competitive advantages of implementing these new technologies and the extent to which they need to be integrated with other ERP solutions.

35.2.5 Planning and design There are many commercial software packages designed to help with business planning, particularly financial projections and the construction of a business plan suitable for submitting to a bank or other investors. However, these do little to help generate the basic data on aquaculture production and systems that are likely to be required. Some of the software tools previously introduced for stock management have basic modelling tools for production planning, which provide the basis for system design. There are also a small number of specialist planning and design packages for different aquaculture systems. These include at least one or more of the following functions: • growth model for the selected species to calculate expected biomass at any point in the future and as a basis for calculating holding requirements and harvesting schedules;

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New technologies in aquaculture

• calculation of key operating parameters for system design (e.g. oxygen requirements, feed input, solids and ammonia production, required water flows, etc.); • tools to help with sizing and costing of the holding facilities and associated equipment (e.g. pumping and water treatment plant); • tools to help with financial appraisal of new projects or system designs. Examples of specific aquaculture planning and design software are given in Table 35.3. Others have used generic modelling software packages, or spreadsheets, perhaps with add-on optimisation or analysis tools. For instance Halachmi et al. (2005)15 used ARENA® simulation software (by Rockwell Automation) to model the design and management of recirculated aquaculture systems, whilst Nunes and Parsons (2006)16 used STELLA® (by ISEE Systems) to model feeding and growth of Southern brown shrimp in aquaculture. This package has also been used by many other researchers, such as Neill et al. (2004)17 for modelling fish growth or McCausland et al. (2006)18 for examining bio-economic consequences of aquaculture and fisheries development. Spreadsheets and @RISK and EVOLVER modelling software from Palisade have been used for production optimisation of tilapia in Costa Rica (Alvarez, 2008).19 These software packages are part of the wider category of decision support tools, which include a range of more specialist functions that also rely on software models. Some of these are covered in more detail elsewhere in this publication, and include for instance: • environmental modelling tools to predict impacts (e.g. used by industry regulators) such as DEPOMOD, KK3D, MOM (see http://www.ecasatoolbox.org.uk/); • geographic information systems (GIS) models (used by planners to help manage development and assess potential in a broader context, for instance, Hunter et al., 2006);20 • tools for risk assessment, either whole business or specialist areas such as biosecurity, hazard analysis and critical control points (HACCP) or health and safety (e.g. Risk Aid by Risk Reasoning Ltd; HACCP Now; Norback, Ley & Associates LLC HACCP software; SHE Software, etc.); • market models to help analyse sales and predict future demand patterns in relation to price, marketing activities, weather and many other factors (e.g. Marketing Analytics Inc.; Marketing Management Analytics; and Aprimo); • business models to support company or industry strategy decisions (e.g. Matrix by Market Modelling Ltd) or develop business plans (e.g. PaloAlto Software; Rosetta IT Solutions Ltd). Once an aquaculture enterprise is operational, one of the more important tasks is that of harvest planning. This is particularly the case for larger companies with multiple sites and many different customers with varied

Table 35.3

Software for aquaculture planning and design

Package

Developer or supplier

Species/system

Description

AquaFarm

Doug Ernst, USA www.AquaFarm. com

Configurable for many species and systems

Aquafarmer

Mintec, Australia http://www. mintec.com.au/

Various species in recirculated aquaculture systems

FARMTM and WinShell

Longline Environmental Ltd, UK http://www. farmscale.org/ and http://www. longline.co.uk/ winshell/ Griffen and Treece, USA http://aquanic.org/ images/interact/ shrimp_an.htm West Virginia University, USA http://www.caf. wvu.edu/afmdp/ disciplines/ engineering/ chemsoftware. shtml

Various bivalve species (grow-out only)

A simulation, data management and decision-support system for aquaculture system design and management planning. AquaFarm can be applied to a wide range of plant and animal aquaculture production facilities, including static and recirculating green-water pond systems, intensive water reuse and recirculation systems and flow-through systems. Developed with the Victoria Department of Primary Industry in Australia. A bioeconomic model, particularly of recirculated aquaculture systems, using growth, FCR and mortality models to specify hardware requirements. These underpin financial models used to determine overall feasibility. Web-based simulation model for calculating bivalve growth based on food availability, bivalve density, farm size and environmental conditions.

Pond-based shrimp farming in Texas

A spreadsheet with seven tables providing a bioeconomic model of a shrimp farm and tools for investment appraisal.

Trout in flowthrough raceways

An Excel spreadsheet with Visual Basic for Applications front-end for data entry and model set-up. Allows calculation of fish growth, feed requirements, oxygen requirements and whether preset conditions (such as stocking density or minimum oxygen concentration) are likely to be exceeded at any time.

A guide to the financial analysis of shrimp farming RDSS (Raceway design and simulation system) (Yin-Han Wang)

FCR = feed conversion ratio. Source: Adapted from http://www.aquafarm.com/aquaculture-software-index.htm and http://aquanic. org/images/interact/software.htm excluding some packages that are no longer available and with some additional sources.

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requirements. In terms of programming, the problem presents a number of constraints and variables that it is desirable to optimise. Constraints, for instance, may include the need to keep total biomass within environmental regulatory limits, or stocking density below welfare limits. Variables to be optimised might include ensuring the mean harvest size and spread of sizes is as close as possible to customer requirements, or scheduling production for when demand and market price are highest. Commercial software such as Akva FishTalkTM Value Chain Planner addresses this function by enabling multiple production scenarios to be set up and easily compared to select the optimum. Mathematical optimisation tools may also be used to determine optimum time of harvest (Bjørndal et al., 2004;21 Hernandez et al., 200722) or to optimise dietary pigment concentrations through the growout period in salmon to reach the desired flesh colour at minimum cost (Forsberg and Guttormsen, 2006).23

35.3 ICT for quality and customer service 35.3.1 Quality management Aquaculture production companies are increasingly expected to operate in accordance with codes of practice and quality management frameworks. These may for instance include: • • • •

HACCP – implemented independently or as part of a wider scheme; ISO 9001 – quality management systems; ISO 14001 – environmental management systems; ISO 22000 – food safety management systems (including ISO 22003 on food safety and ISO 22005 on food traceability); • industry-specific quality standards and codes of good practice; • other certified quality or ethical standards such as organic, welfare or fair trade. Each of these will have a requirement for written standards of performance and expected procedures (e.g. standard operating procedures) that need to be maintained and any alterations recorded in a way that allows for subsequent audit. Typically there will be a collection of documents associated with standards management maintained in a secure yet readily accessible document database which maintains archive copies of altered files and details of the changes made. A system of forms to request and authorise alterations to key documents is also common. Associated with maintenance of the standard will be the requirement to monitor and record compliance. This will range from training records for individuals involved in the specified operations, to logs of product temperature at critical points in the processing chain. Whilst it is not necessary for all this information to be held in a central database, it is usually desirable for it to be readily accessible from a central location in order to combine and analyse the data for traceability, management and reporting purposes.

Use of information technology in aquaculture

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There are some generic software packages marketed to help companies implement HACCP or ISO schemes, and companies such as Maritech have products that service this area; e.g. WiseFishTM Production, which is mainly aimed at processors. Some companies have been able to adapt generic software such as Lotus Notes, Microsoft Exchange or Sharepoint Services to provide the required functionality, whilst others use customised database or spreadsheet applications. 35.3.2 Market chain and traceability The safety of food products, especially those from intensive production systems, has come under closer scrutiny since the BSE crisis in the 1990s. The Atlantic salmon industry has been affected by reports about the levels of dioxins and polychlorinated biphenyls (PCBs) in salmon flesh due to the use of fish meal and oil from contaminated northern capture fisheries, whilst markets for Chinese aquaculture products were severely affected in 2006 when high levels of malachite green and other carcinogenic chemicals such as nitrofuran antibiotics were found in cultured turbot, mandarin fish and dace. In order to help prevent blatant contamination, and allow for the investigation and rapid recall of products that might be contaminated, the EC, US and other jurisdictions have introduced regulations requiring trading companies to keep clear records of all materials purchased for use in food production, and all sales of food or materials used in the production of food. In particular they need to record supplier and customer contact details, quantities, dates and batch identification codes. Although food safety is the primary driver for traceability, and the only reason for its original introduction into EC law, the scope of traceability is broadening, with other drivers appearing. Whilst some European countries have always paid great attention to food quality, for others, this is a developing trend as societies become more affluent and consumers can afford to be more discerning. The market for premium, higher value, usually branded foods has grown considerably in countries such as the UK and Germany. Some aspects of quality can be readily discerned by consumers, particularly taste and texture, whilst others, such as where and how it was produced, require clear labelling. Once products are labelled, several issues arise. Firstly standardisation of label descriptions and, where these represent a method of production for instance, standardisation of underlying practice. Secondly, verification (or certification) that the standards claimed on the label are being observed. Thirdly, product auditability – having documentary evidence to support the claims on the label. There are an increasing number of ‘labels’. Most encompass one or more of the following issues: • quality labels – defined with respect to freshness, texture, colour, and increasingly, means of production and place of origin; • organic labels – produced to various organic standards;

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New technologies in aquaculture

• eco-labels – certifying production that minimises damage to the environment and/or sustainable production; • ethical labels – certifying adherence to ethical standards of production, such as on animal welfare, or fair trade. To have credibility, it is important that claims made on labels are independently verified and certified. The certifying body may be a branch of government or, more frequently, a private organisation, perhaps accredited by government. These organisations will need to inspect company premises and production systems, but will also need to carry out occasional spot checks to ensure products are compliant. This assurance process becomes easier and cheaper if a comprehensive traceability scheme is introduced. In Europe, a relatively small number of large supermarket chains have increasing control over food retailing. Some appear to have a policy that their own brand should be sufficient reassurance of product quality, and have discouraged independent certificated labels. Others have encouraged independent labels, particularly for premium product lines. Whatever the approach to labelling, however, there is an increased insistence on the implementation of production standards and product traceability. Looking to the future, it seems inevitable that there will be some consolidation in standards and certification as consumers are left confused by the proliferation of labels and messages. Some governments are also concerned about the trend toward privatisation of functions they believe belong to them. A further driver for traceability is concern over bioterrorism, particularly in the USA. With the concept and practice of traceability well established, traceability systems and technologies are rapidly developing as companies increasingly recognise the benefits for process management and cost control, and governments see the potential for monitoring compliance with other regulations. European Community legislation on traceability was introduced in Regulation EC/178/200224 and, in particular, Article 18 of the Regulation, which came into full force on 1st January 2005. This affects all producers and processors of food for human consumption, and all producers of feeds or feed materials used for the production of food for human or pet consumption. The basic regulation requires companies to keep records of the suppliers of all foods, ingredients and feeds that they purchase or use, and of the customers for their products (the so-called ‘one up and one down’ principle). These records should be available on demand to the relevant national authorities, to enable tracing of materials forwards or backwards through the production chain. Since most companies would in any case record quantities purchased and from which company in their accounts and, likewise, quantities sold to which customer, the most significant new element is the recording of batch information. This is specified rather vaguely in Article 18 itself, as ‘adequately labelled or identified to facilitate its traceability’. This is also known as ‘the traceable unit’ or ‘trade unit’.

Use of information technology in aquaculture

1091

A variety of secondary regulation and guidance has developed to further specify this. In the case of farmed mammals, individual animals must be identified and tagged separately. For most other products, a batch number can be assigned. The size of the batch is up to the company concerned. The use of large batches simplifies record keeping, whilst the use of small batch sizes means that if any problems do occur, the quantity of product that may need to be withdrawn from the market, or recalled, is smaller. In companies where product flows are not properly reflected in accounts – for instance when goods are distributed to several different sites, but only invoiced to a central office – delivery notes and other product movement documents must be incorporated into the traceability system. Flexibility is also required in the batch recording system to enable splitting or merging trade units, which can happen at various points in the production and distribution system. Many companies use batch numbers and record data from supplier labels. However, information is easily lost if only selected properties are recorded. Where trade units are properly recorded, any of the item properties can be retrieved by reference to the trade unit batch code. In practice, a variety of codes are used to properly trace items. Products may be identified by reference to individual items, the type of product, and the tradable unit (batch or lot). Additional codes may be used to identify locations in the production and distribution chain, and logistics units such as pallets, where lots may be combined or split (Fig. 35.11). The primary legislation only requires chain traceability – i.e. what is coming into or going out of a company. It does not require internal traceability, i.e. utilisation of product within the company. However, all but the smallest companies would also choose to implement internal traceability. For instance, without internal traceability, if a fish farm were supplied with a batch of contaminated feed, which was subsequently detected, it would be required to destroy all potentially affected stocks. If there are no records of which stocks were fed that feed, all stocks would need to be destroyed. Internal traceability and smaller batch sizes help to limit the quantity of product that would need to be withdrawn or recalled in the event of a contamination incident. The amount of information that needs to be recorded and passed on in traceability records is gradually increasing. For instance, Commission Regulation 2065/200125 introduced a labelling requirement for fishery and aquaculture products which includes the requirement for the information to be passed on as necessary through the market chain. This requires the species (recognised trade name and optionally scientific name) to be recorded; whether the product is from marine or freshwater capture fisheries or has been cultured, and the country or sea area where it was caught or produced. Regulation (EC) 852/200426 on the hygiene of foodstuffs has further implications for traceability. It requires food producers to maintain appropriate records documenting adherence to appropriate standards and procedures for hygiene control – e.g. the HACCP approach or other relevant standards.

1092

New technologies in aquaculture >> Market chain –passing information along the chain to allow tracing backwards from final product >> Location ID (Site/tank/pond/cage)

Hatchery Product (record by flow > batch)

Process line/ Shift/sequence

Grow-out (record by batch – often combined or split)

Feed and medicines (record by batch/treatment)

< Batch attributes and process identifiers

Vehicle ID/ Journey ID

Processing/ Wholesale (split batches to trade units)

Ice and chemical inputs (record by batch/treatment)

Transport (aggregate trade units into logistics units)

Retail (record by sales unit)

< Ingredient and treatment sources and use

Labels on trade units, logistics units and usually sales units provide essential information, but most importantly, a key to the detailed product history via a unique identification (ID) code

Product name Product origin Farm/site/ batch ID Quantity Presentation Storage instructions 1

2 2 3 4 56 78 9 0 1

2 3 4 5 6>

Fig. 35.11 Example elements of traceability systems.

This, for instance, involves monitoring and recording product temperature throughout processing and distribution (‘the chill chain’). The records must be made available to relevant national authorities when required and, more significantly, must be available to the receiving food business on request. For businesses rearing animals, and this includes aquaculture, records must also be kept on the use of veterinary products. Regulation (EC) 1830/200327 adds further specific requirements for the traceability of genetically modified materials. Whilst implementing traceability is often perceived as a burden, many companies have found that greater monitoring, particularly if it is properly analysed, can result in significant efficiency gains and savings. This is particularly the case with respect to internal traceability – the factors over which the company has direct control. Downstream companies such as processors are also finding that analysis of traceability data passed to them can provide useful information for improving product quality or minimising waste.

Use of information technology in aquaculture

1093

Information and communication technologies that support traceability continue to become more powerful and cheaper to implement. The basic elements are a means of labelling batches with a code number, a database to record details about the batch and, critically, a means of linking related batch records so that the database can be queried to provide a full history (if tracing backwards), or to identify final products linked with a particular batch of feed ingredients. This is relatively straightforward in any modern multitable relational database. Often confused with traceability is product tracking, which is essentially quite separate, but frequently combined with traceability functions. Traceability records the origins of a product and the processes through which it has passed, whilst tracking is recording of the specific path taken by products through the production and distribution chain. This will normally include spatial and temporal data, often allowing the exact location to be determined in real time. Both tracking and traceability data are useful for management and are often linked in terms of collection and storage. The most effective systems make full use of automation where possible to minimise the amount of information that has to be entered into the computer system manually; and also make best use of networking technologies to link different elements together. Typically a tracking and traceability system might use: • Real-time (or sometimes batch) links between the traceability application and other management information tools, such as stock management – this eliminates the need for retyping and reduces potential transcription errors. • Automated data capture – such as bar code labels and scanners, or the more sophisticated radio frequency identification (RFID) tags and readers (Text Box 35.1) to reduce the need for manual input of batch

Box 35.1

RFID tags

Radio frequency identification (RFID) tags build on the functionality of bar codes in at least two important ways. First, they do not need to be scanned individually by an optical device; instead they are interrogated via radio signals, meaning that many tags can be scanned virtually simultaneously as they pass within range of the detector. Second, in addition to transmitting a code number, they can also store and transfer additional data. This may be product data that would normally be on printed labels but, through the coupling of RFID tags with temperature sensors for instance, could also allow product temperature to be interrogated remotely. Most RFID tags contain at least two parts: first, an integrated circuit for storing and processing information, handling the modulation and

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New technologies in aquaculture

demodulation of the radio signal and other specialised functions; second, an antenna for receiving and transmitting the signal. There are at least three general types of RFID tag. • Passive RFID tags: Passive RFID tags have no internal power supply. These receive just enough electrical current from the incoming radio signal to power the integrated circuit and transmit a response. The range of passive tags is between 10 cm and several metres depending on the chosen radio frequency and design of the antenna. Passive RFID tags tend to be smaller and cheaper than other types, making them most suitable for tagging final individual products. Designs are also available for inserting under the skin of animals (or humans) (see earlier discussion of PIT tags). • Active RFID tags: Active RFID tags have an internal power source and can typically transmit data over several hundred metres with greater reliability than passive tags. They can include more sophisticated functionality (extended capability) such as incorporation of sensors for temperature, humidity, shock/vibration, light, radiation and specific gas detection. Logging facilities for these can also be included so that time series datasets can be retrieved from the tags. Battery life on such tags can be up to 10 years. As these are larger and more expensive, they are best suited at present for incorporation in containers or cartons rather than at the level of individual product. • Semi-passive RFID tags: Semi-passive RFID tags are similar to Active tags, but the power source is only used for the microchip (integrated circuit) and not for the broadcast signal. Most of the advantages of active RFID chips are retained, but battery life is improved with only some loss of range and sensitivity. For all types of tags, readers can be small portable devices, or built into other equipment. Some hand-held units have built-in displays and data logging facilities for subsequent download to a computer. Most, however, will be connected to computers and networks to directly load information into central databases. Further information: http://en.wikipedia.org/wiki/RFID

identification data into the computer system. This can be extended to include the use of other data logging instruments such as fish counters, weighing machines, feed dispensers, temperature probes, GPS devices, etc. As product moves through a process, such devices can capture and record relevant data automatically. • Network technologies for linking elements – for instance data can be passed from point of capture to a central database using a range of wired or wireless technologies; likewise the central data can also be accessed

Use of information technology in aquaculture

1095

from many locations to provide management information wherever it is needed. Increasingly, tracking and traceability applications are Internetbased, allowing company employees and sometimes suppliers and customers to access customised reports or data input forms via standard web browsers. Companies providing tracking and traceability applications for aquaculture and fisheries include AkvaSmart, Maritech, AquaAssist, TraceAll, De Haan Automatisering (DHA), Marel Food Systems and Olrac. These are often extensions to packages originally developed for internal monitoring and control. Custom-built applications are also commonly in use, especially by larger vertically integrated companies. In general, tracing and tracking information is used within the market chain with only minimal information normally passed to the consumer (Text Box 35.2). However, in Taiwan, Tekho Company Ltd is using recyclable passive RFID tags on individual grouper sold through the An Pin Live fish Centre in Tainan to provide full traceability to restaurant customers. The system uses Microsoft BizTalk RFID platform which integrates database servers, web modules and hand-held devices. In the restaurant, a

Box 35.2

Case study – tracking technology in action

TraceAll, a UK company, provides tracking solutions for fisheries. Data loggers on fishing vessels collect position data from GPS devices and record corresponding data from sensors to detect the deployment of fishing gear, the temperature in the fish holds and the weight of fish caught. Operators can add additional data concerning species and observations on fishing conditions. The data are regularly uploaded via satellite telephone, cellular telephone or radio communication links, to a central database, providing shore-based managers with useful real-time information for marketing and fleet management. Once landed, boxes of live product such as Nephrops (langoustine) or pallets of chilled fish (e.g. in boxes) can be tracked in a similar way via sensors, data loggers and uploading via cellular telephone networks. The hardware consists of a small black box that can be easily hidden in packaging or transport containers. This uploads location, temperature, shock and, perhaps, other data from on-board sensors to the central server, either continuously or at set time intervals. Company employees or authorised customers can use a web browser to view the location of the product on real-time maps (e.g. Google maps) and view reports on the data collected. This has often proved invaluable, identifying exactly when and where products are spoilt or even stolen, but otherwise providing assurance of good practice. Further information: http://www.traceall.co.uk/

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New technologies in aquaculture

waiter can scan a tag and print out a certificate showing the basic history and qualities of the fish. Consumers can also enter the fish code number into a website and obtain the full traceability history (Swedberg, 2008 and Microsoft, 2007).28,29 Having a variety of companies providing tracking, tracing and management information solutions provides competition and choice, but would lead to major inefficiencies if data cannot be easily transferred from one system to another. Much of the value of computerised traceability data would be lost for instance if downstream companies have to rekey data, or refer to paper records. This issue was addressed in the EU TraceFish project, which looked only at chain traceability, and developed draft standards for exchanging traceability information through the market chain. There are two key elements to this. The first is the use of a common standard for identifying products, locations and shipping containers. This currently exists in the form of GS1 (Global Standard One), formally EAN.UCC (European Article Numbering-Uniform Code Council). This is a system for allocating unique bar codes so that physical items can be labelled and immediately related to information about the item held in a computer database by reading the bar code with a hand scanner. For legal chain traceability purposes purchasers need only record the bar codes into their own systems. If there is a need to trace a product, the barcode will provide the link backwards through the chain. Barcodes are not very informative without the support of computer systems, however, so labels will normally include other human readable information. The barcode system on its own also does not allow for the passing on of some of the essential secondary information that is now required under EC legislation. For that, additional data must be passed on associated with the barcode. The TraceFish project promoted the use of XML (eXtensible Markup Language) as the universal standard for exchanging data between different computer systems, over the older EDI (electronic data interchange) standard. The work of TraceFish has been further developed under the EC TraceFood project which is developing ‘TraceCore’ XML standards for all food products. These have already been adopted by several software providers, including Olrac, for instance in the EC SHEEL project, which is developing ways for exchanging fishing boat logbook data. This is a complex and developing area, however. GS1 also has standards for the automatic transmission of electronic data between trading partners, a global data synchronisation network for ensuring all partners have consistent data in their systems, and is working on a standard RFID system for tracking items. The company TraceTracker is working closely with the EC Seafood Plus and Trace projects, as well as GS1 standards to provide a ‘global traceability network’ as a subscription service to enable companies with different information systems to easily exchange traceability data. The aquafeed company Skretting joined this system in March 2008 (Skretting, 2008).30

Use of information technology in aquaculture

1097

35.3.3 Marketing and sales Marketing is concerned with discerning customer wants and focusing the company’s products or services to meet them. Ensuring customer awareness and encouraging them to purchase the company’s products or services follows on from this. Sales is the function of optimally matching supply and demand through customer communications and individual sales deals. Although related, marketing and sales are functionally different and often form separate departments in larger organisations. Many smaller aquaculture enterprises utilise the service of agents to handle sales, and rarely assign much resource to marketing. There are exceptions in companies that seek to differentiate their product and hence achieve a price premium. Aquaculture sales commonly involve telephone negotiation with customers with orders confirmed by email or fax. Accounting and stock control software will commonly handle the transaction once an order has been placed. For busier sales offices there can be considerable value in having linked sales and customer relations management software. Separate packages are available, or add-on modules to popular accountancy programmes. These allow sales people to record all their interactions with customers, note expected forward orders, share details with colleagues and analyse longer-term sales patterns. The task of matching actual production with customer orders as well as taking into account distribution logistics and costs is a complex one and often customised software solutions are required to achieve full integration with other systems. As far as possible, companies try to ensure they have firm orders for all production is before it is harvested, with long-term contracts quite common. However, production estimates may be inaccurate and adjustments need to be made to orders, or additional customers found, once final harvest details are available. Once deals are finalised, financial transactions are usually conducted through electronic fund transfer systems facilitated by the company’s bank. In the capture fisheries sector, fish auctions are common. Aquaculture producers might also use them when stock needs to be sold quickly and other channels to market are not available. These are increasingly being updated to ‘electronic fish auctions’ where bidding is via an electronic system, which is faster and provides electronic documentation for buyers, sellers and authorities. In some places, these have also been connected to the Internet, providing wider access for trading, or simply for monitoring market conditions. For some aquaculture producers, especially those marketing directly to consumers, the Internet may provide a suitable channel for direct sales. Establishing a commerce-enabled website is relatively straightforward, particularly if the company is already approved by their bank for receiving credit card payments. More technically complex is linking an Internet sales

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system with existing accounts and stock management systems, although this is also becoming easier using tools available from leading accountancy software suppliers.

35.3.4 Public relations Many aquaculture companies find it increasingly necessary to develop a public relations strategy. This may be for product promotion, investor relations, or to communicate with the wider public on issues such as food safety and environmental responsibility, where misinformation is commonly disseminated by other parties. Such communication strategies may use a number of channels, but developing a company web site is one of the most common and cost-effective. Designing and creating a site that communicates well is quite difficult and usually requires specialist advice and assistance. Maintaining a site to provide up-to-date news and information requires particular attention. In general this is an area where the aquaculture industry has much to learn, with the Internet providing opportunities for communication. These include the potential for developing interactive farm guides, quizzes for children, audio podcasts, slideshows and video clips to help educate the public, or packaged information for news media and politicians.

35.4 ICT in aquaculture innovation and learning 35.4.1 Linking innovation, research and learning It is widely recognised that innovative organisations are better able to respond to changes in the external environment and compete in the increasingly globalised economy. Innovation is therefore good for local and national economies and is invariably supported in many ways at the policy level. Central to this is the creation of strong linkages between commerce and research, and the fostering of lifelong learning. Exploration of the relationship between government, academic and commercial research, and economic development and performance is beyond the scope of this chapter. However, it is worth noting that the emergence and use of the Internet is helping to break down some of the traditional barriers for companies wanting to access research results or participate in research projects. This includes the development of online research repositories, either freely accessible or providing instant access to research papers for a fee. These are supported by online bibliographic search tools that offer much greater accessibility than the traditional printed versions. Presentations made at conferences are now much more commonly distributed from event or organisation websites. Access to researchers and debate on current issues has also been opened up by the use of public email

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and web discussion forums and blogs. To a more limited extent there has also been experimentation with ICT-based expert systems, to provide tailored knowledge and advice to non-experts depending on their responses to structured questions. For instance veterinary diagnostic software such as fish-vet (Zeldis and Prescott, 2000)31 and T-Vet (Li et al., 2006)32 or the earlier Hawaii Aquaculture Module Expert System for tilapia (Brock and Itoga, 1993).33 Lifelong learning is an important component as individuals within organisations who are open and engaged with learning are more likely to be oriented to problem solving and creative thinking. Furthermore, they will be better equipped to effect change through possession of latest knowledge and understanding. Lifelong learning may be formal, non-formal or informal. Formal learning generally involves taught courses, or at least supervised learning that is assessed and leads to formal qualifications. Non-formal learning is structured and organised, but does not lead to a qualification (e.g. in-service training). Informal learning involves working experience, discussion with colleagues and contacts, and knowledge acquired through books and other media.

35.4.2 Applications of ICT in aquaculture education and learning Once again, the Internet is central to many of the current developments in learning and teaching. One feature is the increasing volume of information or ‘learning resources’ that are available through the Internet. Whilst there are legitimate concerns about the quality of much of the information on the Internet (e.g. Keen, 2007),34 the steep change it brings in access is too great to be ignored. A rural aquaculture worker can now access from home a far larger knowledge base than has previously been available from the best academic libraries. Whilst the latter are by no means replicated by the Internet, the need for large physical repositories of printed materials will gradually decrease as electronic documents become the more prevalent format. The low cost of Internet publishing is one of the key drivers, lowering the barriers for anyone with information or opinions that they wish to disseminate. The quality control that has traditionally been exercised through academic peer review, specialist publishing companies or material selection by librarians does not generally exist on the Internet. This places more onus on users of the information to assess and verify its quality. It is also leading to new models of collaborative quality control such as that exercised by the Wikipedia encyclopaedia service. That said, the Internet is also allowing many people with valid, but very specialised, knowledge and experience to publish it without facing the barriers that may be encountered when dealing with other commercial or academic bodies. ICT is not only facilitating access to material, it is also providing lowercost tools for authoring. Word processors, presentation software and

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desk-top publishing applications enable individuals to be both author and publisher. Cost barriers are also falling for audio and video productions such that they are becoming an increasingly common way to communicate information and ideas. The Internet is also providing new channels of communication, again at lower cost and especially internationally. So far the predominant technology is text-based: email, bulletin boards and discussion blogs. However, with increasing bandwidth and computing power, video conferencing and messaging is becoming more common. This could progress further as mobile devices become more powerful and Internet connections faster and cheaper. Equally significant is the development of virtual communities facilitated by these technologies and the trend for people to spend increasing amounts of their time working and socialising online. Bringing together access to both knowledge and communications from virtually any location opens up new possibilities, especially higher and adult education. In particular it can be made available to people who might not previously have been able to access it, due to its location within college campuses. The emerging ICT platform to support these developments is the ‘virtual learning environment’ (VLE), which can be used for distance learning courses or for supporting face-to-face teaching on campus. These are essentially tools for creating and managing a website suitable for educational activities. They provide a content management system (CMS) that makes it easy for academic staff to upload and organise teaching materials, and tools such as email and bulletin board forums for interaction with and between students. Other facilities vary, but most have tools for tutors to set and students to upload assignments, and they usually have course management tools for tracking student progress. Other terms in common usage include learning management system (LMS), learning content management system (LCMS), course management system (CMS) or managed learning environment (MLE). These all have slightly different focus, and overlap to varying degrees. The term MLE is promoted as including all information systems involved in the delivery and administration of learning. The more sophisticated VLEs link with institutions’ other management information systems, such as student record systems and computer services user management. Direct linkages between VLEs and library systems are also under development. There are over 100 different VLE software packages available, although a process of consolidation is evident, with Blackboard now the leading commercial package and Moodle emerging as the most common Open Source solution. One of the challenges in implementing comprehensive and fully integrated VLEs has been the variety of structures in use. Educational provision is often organised into elements such as courses, programmes, electives, modules, units, classes, lectures, practicals, tutorials, etc. These can be

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arranged in a hierarchy according to length of time or academic credit accumulated. An important element in this ‘granularity’ of organisation is the ‘learning object’. This is generally taken as being the smallest identifiable component of teaching that still has structure with respect to learning objectives and pedagogy. Learning objects would include presentations and student exercises, for instance. Sub-components of these would be text, video or graphic objects, etc. The digitisation of learning objects for delivery via VLEs has opened up several major issues for the educational sector. Firstly, teaching materials often have to be significantly reworked or extended to be made available in digital format. This has drawn attention to the amount of duplication of effort that exists at institutional and, even more so, national or international levels. It would clearly be more efficient if learning objects can be shared, with new or adapted materials added to learning object repositories that can be accessed by all concerned. This should be readily feasible at institutional level, and has attractions at national and international levels. Issues of copyright, competition, economics and language create significant barriers, but these are being challenged and addressed through the emerging movement for open educational resources. Many learning object repositories have been established, which led to the early realisation that much of the value of these would be lost if there were not common access standards in place. The key elements of this are: • Metadata: Learning content and catalogue offerings must be labelled in a consistent way to support the indexing, storage, discovery (search) and retrieval of learning objects by multiple tools across multiple repositories. Various interim standards exist with the most important probably the Learning Object Metadata (LOM) standards developed by the Learning Technology Standards Committee (LTSC) of the IEEE. • Content packaging: The goal of content packaging specifications and standards is to enable organisations to transfer courses and content from one learning system (e.g. VLE) to another. Content packages include both learning objects and information about how they are to be put together to form larger learning units. They can also specify the rules for delivering content to a learner. The key standard in use is SCORM (Sharable Content Object Reference Model), primarily developed by ADL, a US government sponsored organisation that researches and develops specifications to encourage the adoption and advancement of e-learning. • Learner profiles: These standards allow different system components to share information about learners across multiple system components. Learner profile information can include personal data, learning plans, learning history, accessibility requirements, certifications

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and degrees, assessments of knowledge (skills/competencies). In addition, systems need to communicate learner data to the content, such as scores or completion status. Standards for learner profiles are less well developed, but are emerging out of initiatives such as European Qualification Framework, EUROPASS, European CV and the Diploma Supplement. In the UK, a draft British Standard has been developed (DD 8788-3:2006 UK lifelong learner information profile – UKLeaP), but is not regarded as comprehensive and is expected to interact with emerging European or wider international standards and be further developed.

All these standards could eventually become subsets of wider schemes, such as the emerging ‘semantic web’ and allied development of the Web Ontology Language. Much of the thrust of developments to date has been in terms of adapting existing teaching practices and pedagogical models to the Internet age. As new technologies mature and become more accessible, the balance of teaching approaches might change substantially. For aquaculture teaching there is substantial potential for the greater use of computer simulations – interactive software that allows students to test their understanding and skills and further develop their knowledge through problem-based learning and case studies. ICT can provide software models for student interaction, facilitate interactions between students and tutors and deliver appropriate information according to student needs. Virtual reality software could greatly enhance such simulations in the future, providing valuable visual and spatial elements that are easily lost in other representations. The Internet service ‘second life’ is pioneering virtual reality representations and interactions, including, for instance, virtual college courses. In addition to new opportunities, the use of ICT in education also poses a number of problems. One of the most widely discussed is that of plagiarism. It has become much easier for students to cut and paste material from other sources, or even access entire essays via the Internet. This is an issue that can largely be addressed within the education process and through altering the means by which assignments are set and assessed. However, there are also a number of online plagiarism detection services such as Turnitin, available to help tutors identify prior work within written materials. A related issue for assessment is authentication that work submitted, tests completed or contributions made online are entirely the work of the registered student. The use of video tutorials and video monitored tests may help in this respect. Biometric identifiers, including analysis of keyboard input patterns, voice recognition and fingerprint verification may also become more common. In the long term, there may be greater specialisation by individuals, institutions and Internet services with respect

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to developing and delivering teaching materials, coaching, tutoring or otherwise supporting learners, and assessing and accrediting learning outcomes.

35.4.3 Role of ICT in the innovation process There are many definitions of innovation, but in the sphere of commerce and economics these commonly emphasise the development and adoption of new technologies, services or management practices. We have already discussed the importance of linking industry and research and how this is being achieved through open research repositories and government funded research, and similarly, how widening access to education is regarded as a tool for stimulating and enabling innovation. A third leg is the promotion of collaboration between organisations, particularly small and medium scale enterprises (SMEs). The rationale for this is that smaller companies will rarely have all the knowledge and resources required to implement innovation, or even recognise opportunities for innovation. By improving networking between organisations, particularly at the personal level, new collaborations can be stimulated, leading to greater rates of innovation. A variety of web-based tools are being developed and deployed to facilitate this: • Benchmarking tools: These collect performance data from individual companies and compare them with the calculated mean so that users can see if their performance is above or below average. Benchmarking for aquaculture typically includes feed conversion, growth and mortality rates, although it could also include other management metrics such as production per person employed or cost per unit produced. Benchmarking tools are used internally by large companies to compare performance between sites or units. Their use in a wider context depends on some level of trust and collaboration existing between companies that are otherwise competitors. This is being fostered in the Sentinel Farms project for UK trout farmers (Turnbull, 2008).35 Farmers can upload mortality and FCR data manually or directly from their stock management systems to a central database. This allows for more or less real-time aggregation and analysis of the data, providing farmers with feedback on their performance compared with other farms of a similar type, and at the same time of year. It also means that participating farmers can be quickly alerted to changes in average mortality rates for instance, which may be indicative of emerging disease issues. The most common use of benchmarking, however, is to help identify the need for innovation among companies that are not performing so well.

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• Partnering tools: These are usually web-based databases where companies record their competences and needs with respect to products and services they either offer or require. Algorithms then match companies within the database and provide recommended contacts. Their usefulness depends greatly on the quality of data and number of companies involved. • Personal networking tools: Recognising that collaborations start with personal contacts and relationships, web-based services such as LinkedIn provide tools for individuals to present themselves and build a network of colleagues and associates who are also participating in the service. This effectively builds chains of personal links enabling people to find contacts with the skills and experience they need via personal contacts who are able to provide recommendations or other information. • Knowledge bases: There are numerous commercial, academic and nonprofit initiatives to create structured knowledge bases relevant for aquaculture. For instance the CABI Aquaculture Compendium, FishBase, LarvalBase and AquaNIC. Wikipedia has demonstrated the value of collaboration to create a global encyclopaedia based on the contributions of anyone with knowledge to share. It may be that the breadth and depth of Wikipedia increases to cover all aspects of the aquaculture sector in great detail (e.g. through the WikiBooks initiative). Alternatively, more specialist services, perhaps with greater time-sensitive content, could develop around a similar model. • Discussion list communities: email discussion lists and website discussion forums have been available for many years (e.g. the Yahoo groups for shrimp and tilapia or the SARNISSA project for Africa). Relatively few achieve widespread use, but those that do become invaluable tools for those involved, providing a peer group community for asking questions and sharing knowledge and advice. This can lead either directly to innovation or, at least, the establishment of further collaborations.

35.5 Conclusions Information and communications technologies are now used throughout the aquaculture sector from production of the feed ingredients to presentation of recipes to busy consumers. Although many aquaculture sectors are small scale and traditional in structure, they are nevertheless benefiting from ICT through better availability of technical information and access to markets or supplies. More industrialised branches of aquaculture such as salmon farming are highly technology dependent with computerised monitoring and control systems integral to most operations. Aquaculture customers are increasingly demanding customised products that meet their specific

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requirement. ICT systems will help companies to achieve this by enabling effective management of smaller batches with better product tracking. For the future, several trends are already apparent. Firstly the growth of the Internet will not stop at linking personal computers. Mobile phones now commonly have Internet connections and an increasing number of electronic devices will link directly to the net. This will make it much easier and cheaper to collect data. The value of that, however, will depend on how it is subsequently analysed and used to support management and improve control over production and distribution. From an ICT perspective, the key challenge will be in writing the Internet server-based applications that integrate input from many data sources, provide real-time analysis and output to a similarly diverse range of client access devices for reporting and control.

35.6 Acknowledgements The author would like to thank the following people for their assistance in providing information or illustrations used in preparing this chapter: Bob Bawden (Pisces Engineering Ltd), Alan Steel (Traceall Ltd), Professor Karim Erzini (Algarve University), Chris Hempleman (Maritech), Jorge Arturo Alvarez (Private Consultant), Sonia Tsai (Tekho Company Ltd), Philip Bodington (Selonda Aquaculture SA), Dr Carlos Mazorra (Tinamenor SA), Torbjorn Kvassheim (AquaScan AS), Frank Herr (Lotek Wireless Inc.), Kjetil Opshaug (Sølvtrans), David Jarron (Vaki), Sissel Wiedenmann, Trond Severinsen, and Einar Helsoe (Akva Group ASA). Responsibility for any errors or omissions remains entirely with the author.

35.7 Sources of further information and advice Information and communications technology (ICT) in aquaculture development (Section 35.1.1) • Information and Communication Technology in Aquaculture, A summary of different presentations made at MARSOURCE Seminars and Workshops for the ‘Maritime Information Society’ organised in Genoa, Bilbao and Sassnitz in 1998: http://www.feap.info/feap/ presentations/itechaq_en.asp • FAO (2006) Information and communication technologies benefit fishing communities, New Directions in Fisheries – A Series of Policy Briefs on Development Issues, No. 9, United Nationals Food and Agriculture Organization, Rome: http://www.sflp.org/briefs/eng/09.pdf • The e-Aqua project: http://www.e-aqua.org/ • Development Gateway Community for Information and Communications Technologies: http://ict.developmentgateway.org/

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• OECD Home Page for Information and Communication Technologies: http://www.oecd.org/topic/0,3373,en_2649_37441_1_1_1_1_37441,00. html

The functions of information and communications technology (Section 35.1.2) • Kotelnikov V (2007) Small and Medium Enterprises and ICT, AsiaPacific Development Information Programme, United Nations Development Programme, Bangkok: http://www.apdip.net/publications/ iespprimers/eprimer-sme.pdf

Principles of monitoring, control and automation (Section 35.2.1) • Hughes T A (2006) Measurement and Control Basics, 4th edn, The Instrumentation, Systems and Automation Society, Research Triangle Park, CA: http://www.isa.org/Template.cfm?Section=Books3&template=/ Ecommerce/ProductDisplay.cfm&ProductID=8879 • Cooper D J (2000) Practical Process Control Using Control Station, Department of Chemical Engineering, University of Connecticut: http:// www.engr.uconn.edu/~ewebhk/buttons/data/data1.html • MIT Open Courseware, Chemical Engineering, 10.450 Process Dynamics, Operations, and Control, Massachusetts Institute of Technology, Cambridge, MA: http://ocw.mit.edu/OcwWeb/Chemical-Engineering/ 10-450Spring-2006/CourseHome/index.htm • PAControl.com: http://www.pacontrol.com/

Sensors and monitoring tools for aquaculture stock (Section 35.2.2) Programmable logic controllers • Automation.com (2009) Programmable Logic Controller (PLC): Products, News, Articles & Resources, Automation Resources, Inc., Pelham, AL: http://www.automation.com/portals/programmable-logiccontroller-plc • Jack H (2008) Automating Manufacturing Systems with PLCs; Version 5.2, Grand Valley State University, Grand Rapids, MI: http://sites. google.com/site/automatedmanufacturingsystems/ • Bolton W (2006) Programmable Logic Controllers: an Introduction, Newnes, Oxford/Burlington MA. Communications protocols • Fundamentals of Communications, Alcatel Internetworking Inc., Agoura Hills, CA: http://www.ind.alcatel.com/fundamentals/index2. html?pass=true

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• Introduction to Data Communications, Eugene Blanchard: http://www. techbooksforfree.com/intro_to_data_com/ • Data Communication Fundamentals, Kennesaw State University, Kennesaw, GA: http://science.kennesaw.edu/~cxu/21_8040/07_funda/07_ funda_files/frame.htm • Scada Primer: http://members.iinet.net.au/~ianw/primer.html • Controllers and SCADA: http://www.pacontrol.com/Controller.html • Fieldbus: http://www.pacontrol.com/Fieldbus.html • Industrial Ethernet: http://www.pacontrol.com/Ethernet.html Process control equipment suppliers • Cowex a/s: http://www.cowex.com/ • Craig Ocean Systems Inc.: http://www.cos-inc.com/ • Aquadyne: http://www.aquadyne.com/ • Pisces Engineering: http://www.pisces-aqua.co.uk/farmpatrol.htm • SEDIA (Société d’Etude et Développement en Informatique et Automatismes): http://www.sedia.org/html/gb/aqualarm-supervision. html • Aqua Systems (UK) Ltd: http://www.aquasystems.co.uk/files/contents/ frame.htm • Automated Aquarium Systems: http://www.automatedaquariums.com/ • Invensys Eurotherm: http://www.eurotherm.co.uk/ • ABB Group: http://www.abb.com/ • Schneider Electric: http://www.schneider-electric.com/sites/corporate/ en/products-services/automation-control/automation-control.page • Rockwell Automation: http://www.rockwellautomation.com • Mitsubishi Electric: http://global.mitsubishielectric.com/bu/automation/ products/auto/index.html • Honeywell Automation and Control Solutions: http://www.honeywell. com/sites/acs/ • Siemens Industrial Automation Systems (SIMATIC): http://www.automation.siemens.com/simatic/portal/index_76.htm • Omron: http://www.omron.com/products/indu.html • GE Fanuc: http://www.gefanuc.com/ • Panasonic Industrial Solutions: http://industrial.panasonic.com/ • Campbell Scientific: http://www.campbellsci.com/index.cfm • Moore Industries: http://www.miinet.com/ • Omega Engineering Inc.: http://www.omega.com/ • Process Aquatics International: http://www.processaquatics.com/ Feeding system controllers • AkvaGroup: http://www.akvagroup.com/index.cfm?id=202355 • Pisces Engineering Ltd: http://www.pisces-aqua.co.uk/feeder_ controllers.htm

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• SEDIA: http://www.sedia.org/html/gb/aquaculture.html • Feeding Systems: http://www.feeding-systems.com/ • Arvo-Tec: http://www.arvotec.fi/ Fish counters and biomass estimators • Aquascan: http://www.aquascan.com/ • Vaki: http://www.vaki.is/ • Storvik: http://www.storvik.no/gml/index_english.html • AkvaGroup: http://www.akvasmart.com/index.cfm?id=202355 Video and security systems • Orbit Aquacam: http://www.orbitaquacam.com/ • Precision Aquaculture: http://precisionelectronics.co.uk/ • Integrated Aqua Systems: http://www.iasproducts.com/aCUvideomon. html • VideoRay: http://www.videoray.com/missions/9 • IndigoVision: http://www.indigovision.com/ Fish tags • Floy Tags: http://www.floytag.com/ (traditional visual tags) • Biomark: http://www.biomark.com/ (RFID/PIT tags) • Vemco: http://www.vemco.com/ (acoustic tags) • Hydroacoustic Technology Inc.: http://www.htisonar.com/ (acoustic tags) • LotekWireless Inc.: http://www.lotek.com/ • Star-Oddi.: http://www.star-oddi.com/ Other useful reading • Anon (undated) Acoustic fish tags, University of Rhode Island, Office of Marine Programmes, Narrangansett, RI: http://www.dosits.org/ gallery/tech/of/aft1.htm (acoustic tags) • Askgaard J M B (2008) Sea Cage Gateway – A Distributed Sensor Management Network in ActorFrame, MSc thesis, Norwegian University of Science and Technology, Trondheim, Norway: http://urn.ub.uu.se/ resolve?urn=urn:nbn:no:ntnu:diva-1010 • Dagorn L, Pincock D, Girard C, Holland K, Taquet M, Sancho G, Itano D and Aumeeruddy R (2007) Satellite-linked acoustic receivers to observe behavior of fish in remote areas, Aquatic Living Resources, 20(4), 307–12: http://www.ifremer.fr/docelec/doc/2007/publication-3731. pdf • Grødal, J A and Paaske F G (2008) Context-Aware Services in Aquaculture: FiFaMoS – Fish Farm Monitoring System, MSc thesis, Norwegian

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University of Science and Technology, Trondheim, Norway: http://urn. ub.uu.se/resolve?urn=urn:nbn:no:ntnu:diva-1275 Halfdanarson B (1997) Estimating the future growth of salmon by using the Vaki Biomass Counter and Biomest Aquaculture Software, Aquaculture Trondheim 1997: Cultivation of Cold-Water Species: Production, Technology and Diversification, 13–16 August, Trondheim. (World Meeting Number 973 5006). Hateley J and Gregory J, Evaluation of a multi-beam imaging sonar system (DIDSON) as fisheries monitoring tool: Exploiting the acoustic advantage, Technical Report, Environment Agency, Warrington: http:// www.soundmetrics.com/NEWS/REPORTS/UKEA_DIDSON_Report. pdf Lines J A, Tillett R D, Ross L G, Chan D, Hockaday S and McFarlane N J B (2001) An automatic image-based system for estimating the mass of free-swimming fish, Computers and Electronics in Agriculture, 31, 151–68. Mills D J, Gardner C and Johnson C R (2006) Experimental reseeding of juvenile spiny lobsters (Jasus edwardsii): Comparing survival and movement of wild and naive lobsters at multiple sites, Aquaculture, 254(1–4), 256–68. Petrell R J, Shi X, Ward R K, Naiberg A and Savage C R (1997) Determining fish size and swimming speed in cages and tanks using simple video techniques, Aquacultural Engineering, 16(1–2), 63–84. Treasurer J (2002) Application of sonar systems in aquaculture, Fish Farmer, 25(4), 37–9. University of Tasmania project on determining salmon size in aquaculture facilities using underwater video: http://www.cis.utas.edu.au/ external/research/marineICT/projects/marinevideo/5.html

Stock management systems (Section 35.2.3) Relevant companies are listed in Table 35.2.

Business information systems (Section 35.2.4) • Lotus Notes & Domino: http://www-306.ibm.com/software/lotus • Microsoft Exchange Server: http://www.microsoft.com/exchange/default. mspx • Microsoft SharePoint Server: http://www.microsoft.com/sharepoint/ default.mspx • Business Link web site advice for business on IT and e-commerce: http:// www.businesslink.gov.uk/bdotg/action/layer?r.l1=1073861197&topicId =1073861197&r.l2=1075422920&r.s=b

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Business integration/enterprise resource planning (ERP) software • Wailgum T and Koch C (2007 – updated 2008) ERP definition and solutions, CIO, Framingham, MA: http://www.cio.com/article/40323/ ERP_definition_and_solutions • ERP vendors: http://en.wikipedia.org/wiki/List_of_ERP_vendors Further reading on background technologies (Sections 35.2.3 and 35.2.4) • Connolly T M Begg C E (2005), Database Systems: A Practical Approach to Design, Implementation and Management (4th edn), Pearson Education, Harlow. • Microsoft SQL: http://www.microsoft.com/sqlserver • Microsoft ODBC: http://support.microsoft.com/kb/110093

Planning and design (Section 35.2.5) The main aquaculture packages are listed in Table 35.3. Web-based software • FARMTM: http://www.farmscale.org/ • WinShell: http://www.longline.co.uk/winshell/ Environment • ECASA Project: http://www.ecasatoolbox.org.uk/ Business planning and strategy suppliers and software • Market Modelling Ltd: http://www.market-modelling.co.uk/ • Palo Alto Software Ltd: http://www.paloalto.co.uk/ • Rosetta IT Solutions Ltd: http://www.rosetta-it.com/ • Palisade: http://www.palisade.com • Arena simulation software by Rockwell Automation: http://www. arenasimulation.com/ • STELLA software by ISEE Systems: http://www.iseesystems.com/ Other useful reading • Corner R A, Brooker A J, Telfer T C and Ross L G (2006) A fully integrated GIS-based model of particulate waste distribution from marine fish-cage sites, Aquaculture, 258(1–4), 299–311. • Gifford J A, Benetti D D and Rivera J A (undated) National marine aquaculture initative: Using GIS for offshore aquaculture siting in the U.S. Caribbean and Florida, NOAA/NMFS, CARR 102 Cabo Rojo, PR 00623, National Oceanographic and Atmospheric Adminsitration, Washington, DC: http://www.lib.noaa.gov/docaqua/reports_noaaresearch/ nmaifinalreportgis.pdf

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• Kiefer D A, O’Brien F and Rensel J E J (2007) Modeling water column and benthic effects of fish mariculture of cobia (Rachycentron canadum) in Puerto Rico: Cobia AquaModel, Report for Ocean Harvest Aquaculture Inc. and The National Oceanic and Atmospheric Administration, Systems Science Applications Inc., Pacific Palisades, PA: http://www.lib. noaa.gov/docaqua/reports_miscellaneous/noaa_cobia_final_report_ May_2007.pdf • Kodra B (2007) Risk Analysis of tilapia recirculating aquaculture systems: A monte carlo simulation approach, MSc Thesis, Virginia Polytechnic Institute and State University, Blacksburg, VA: http://scholar.lib.vt.edu/ theses/available/etd-04232007-125139/ • Nunes A J P and Parsons G J (2006) A computer-based statistical model of the food and feeding patterns of the Southern brown shrimp Farfantepenaeus subtilis under culture conditions, Aquaculture, 252(2–4), 534–44. • Wang Y-H, Turton R and Semmens K (2007) Software development for predicting fish growth in trout raceways, Poster Presentation, Aquaculture Forum 20 January, West Virginia University Extension Service: http://www.wvu.edu/~agexten/aquaculture/07Forum/posters/Aqua_ forum_07.ppt • Wang Y-H (2006) Model and software development for predicting fish growth in trout raceways, MSc Thesis, Department of Chemical Engineering, West Virginia University, Morgantown, WV: http://www.caf. wvu.edu/afmdp/disciplines/engineering/publications/Wang%20 Yin-Han.pdf (Software model available at http://www.caf.wvu.edu/ afmdp/disciplines/engineering/chemsoftware.shtml) • Ferreira J G, Hawkins A J S and Bricker S B (2007) Management of productivity, environmental effects and profitability of shellfish aquaculture – the Farm Aquaculture Resource Management (FARM) model, Aquaculture 264, 160–74: http://www.fojo.org/papers/farm/farm.pdf

Quality management (Section 35.3.1) • Code of Conduct for Responsible Fisheries, Food and Agriculture Organization of the United Nations, Rome: http://www.fao.org/fishery/ccrf/1 • European Aquaculture Code of Conduct, Federation of European Aquaculture Producers, Liege: http://www.feap.info/consumer/codes/ feapintro_en.asp • Code of Good Practice for Scottish Finfish Aquaculture, Scottish Salmon Producers Organisation, Perth: http://www.scottishsalmon.co. uk/aboutus/codes.asp • Codes of Practice for Responsible Shrimp Farming, Global Aquaculture Alliance, St Louis, MO: http://www.gaalliance.org/code.html • GLOBALGAP Integrated Farm Assurance, GLOBALGAP, Cologne: http://www.globalgap.eu

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ISO Management Standards • International Organization for Standardization, Geneva: http://www.iso. org/iso/management_standards.htm Risk assessment and HACCP software suppliers and software • Risk Reasoning Ltd: http://www.riskreasoning.co.uk/ • Norback Ley and Associates: http://www.norbackley.com/ • She Software Ltd: http://www.shesoftware.com/ • HACCP Now: http://www.haccpnow.co.uk/ • HACCPHelp! TM: http://www.haccphelp.com/haccphelp_software.htm Example quality management suppliers and software (including HACCP compliance) • SoftExpert Ltd: http://www.softexpert.com/en/ • Qualsys Ltd: http://www.qualsys.co.uk/ • Lennox Hill Ltd: http://www.lennoxhill.co.uk/ • Pilgrim Software, Inc.: http://www.pilgrimsoftware.com/ • The Harrington Group: http://www.harrington-group.com/ • The Interax Group, Inc.: http://www.interaxgrp.com/ • Qudos: http://www.qudos-software.co.uk/

Market chain and traceability (Section 35.3.2) • EC (2007) Food Traceability, Fact Sheet, European Commission DG Health and Consumer Protection, Brussels: http://ec.europa.eu/food/ food/foodlaw/traceability/factsheet_trace_2007_en.pdf • EAN (2002) EAN Fish Traceability Guidelines, EAN International, Brussels: http://www.gs1.org/docs/traceability/GS1_fish_traceability.pdf • Codex Alimentarius (International Standards): http://www. codexalimentarius.net/web/index_en.jsp • GS1: http://www.gs1.org/ • Good Traceability Practice, TraceFood Framework: http://www. tracefood.org/index.php/GTP Example traceability suppliers and software (fisheries, processing and distribution) • DHA BV: http://www.dha-software.com/ • Traceall Ltd: http://www.traceall.co.uk/ • AkvaSmart TM: http://www.akvasmart.com • Innova: http://www.marelfoodsystems.com/Products/Innova • Tracetracker®: http://www.tracetracker.com/ • Olfish: http://www.olfish.com/ • WiseFishTM: http://www.wisefish.com/

Use of information technology in aquaculture

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Coordination of traceability • Tracetracker®: http://www.tracetracker.com/ • Traceregister TM: http://traceregister.com/ (North American) EU projects on traceability • Fish-Tracenet: http://www.fishtracenet.org/ • FOODTRACE: http://www.eufoodtrace.org • TraceFood: http://www.tracefood.org/ • Trace: http://www.trace.eu.org/ • SEAFOOD plus: http://www.seafoodplus.org/ • E-passport for frozen shrimp, VietNamNet Bridge, 28/8/08: http:// english.vietnamnet.vn/tech/2008/08/801022/

Marketing and sales (Section 35.3.3) • CRM Landmark: http://www.crmlandmark.com/ Electronic fish auction companies • Pefa: http://www.pefa.com/ • Mercapesa: http://www.mercapesca.net/ • Aucxis Trading Solutions: http://www.aucxistrading.com/ Market models • Marketing Analytics, Inc.: http://www.marketinganalytics.com/ • Marketing Management Analytics: http://www.mma.com/ • Aprimo, Inc.: http://www.aprimo.com/

Public relations (Section 35.3.4) • The Institute for Public Relations, Gainesville, FL: http://www. instituteforpr.org/ Example PR software • DNA 13: http://www.dna13.com/ • Bluevizia: http://www.bluevizia.com/ • Glide Technologies: http://www.glidetechnologies.com/ • Vocus: http://www.vocus.com/ • Solcara: http://www.solcara.com/

Linking innovation, research and learning (Section 35.4.1) • Li D L and Fu Z (2002) Knowledge warehouse: A Web-based integrated information system for freshwater aquaculture, Proceedings of the Third

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New technologies in aquaculture

Asian Conference for Information Technology in Agriculture, 26–28 October, Beijing, Agricultural Scientech Press, Beijing, 379–83: http:// zoushoku.narc.affrc.go.jp/ADR/AFITA/afita/afita-conf/2002/part4/ p379.pdf • EC Competitiveness & Innovation Framework Programme, European Commission DG Enterprise and Industry, Brussels: http://ec.europa.eu/ cip/index_en.htm • OECD Science and Innovation, Organization for Economic Cooperation and Development, Paris: http://www.oecd.org/topic/0,3373,en _2649_37417_1_1_1_1_37417,00.html • The Science Business Network: http://www.sciencebusiness.net/

Applications of ICT in aquaculture education and learning (Section 35.4.2) • AQUA-TNET Project: http://www.aquatnet.com/ • AQUA-TNET (2008) Draft report on innovation in aquaculture teaching and learning: http://docs.google.com/View?docid=dc8mztpb_2gjbsrx • OECD (2007) Giving Knowledge for Free: The Emergence of Open Educational Resources, Organization for Economic Development and Cooperation, Paris: http://www.oecd.org/document/41/0,3343,en_2649_ 37455_38659497_1_1_1_37455,00.html • IEEE Learning Technology Standards Committee Workgroup on Learning Object Metadata: http://ltsc.ieee.org/wg12/ • Dublin Core Metadata Initiative: http://dublincore.org/ • CanCore Learning Resource Metadata Initiative: http://www.cancore. ca/ • List of learning object repositories: http://www.uwm.edu/Dept/CIE/ AOP/LO_collections.html • Centre for Educational Technology Interoperability Standards: http:// zope.cetis.ac.uk/ • SCORM (Sharable Content Object Reference Model): http:// adlcommunity.net/course/view.php?id=25 • IMS Global Learning Consortium, Inc., Content Packaging Specification: http://www.imsproject.org/ • IEE Standards: http://ieeeltsc.org/ • The Higher Education Academy, Managing Effective Student Assessment: http://www.heacademy.ac.uk/ourwork/learning/assessment/mesa • Global Development Learning Network: http://www.gdln.org/ • Turnitin®: http://turnitin.com/ • Blackboard: http://www.blackboard.com/ • Moodle: http://moodle.com/ • European Qualifications Framework: http://ec.europa.eu/education/ lifelong-learning-policy/doc44_en.htm • EUROPASS: http://europass.cedefop.europa.eu/

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Semantic web • WrC: http://www.w3.org/2001/sw/ • The Semantic Web: http://semanticweb.org/ • Stojanovic´ N, Staab S and Studer R (2001) eLearning in the Semantic Web, WebNet2001-World Conference on the WWW and Internet, October 24–27, Orlando, FL: http://www.aifb.uni-karlsruhe.de/~sst/Research/ Publications/WebNet2001eLearningintheSemanticWeb.pdf Web ontology language • Costello R L and Jacobs D B (2003) A Quick Introduction to OWL Web Ontology Language, MITRE Corporation, Bedford, MA: http://www. iro.umontreal.ca/~lapalme/ift6281/OWL/CostelloQuickIntroOwl.pdf • W3C OWL web ontology overview: http://www.w3.org/TR/owlfeatures/ Virtual learning environment • Second Life: http://secondlife.com/

Role of ICT in the innovation process (Section 35.4.3) • EC, ICT for Competitiveness and Innovation, European Commission DG Enterprise and Industry, Brussels: http://ec.europa.eu/enterprise/ ict/index_en.htm • Aquaculture Innovation Network: http://www.aquainnovation.net • LinkedIn: http://www.linkedin.com/ • Itoga S and Brock J (1995) Hawaii Aquaculture Module Expert System for Macintosh Computers, CTSA#119, Center for Tropical and Subtropical Aquaculture, Waimanalo, HI: http://praise.manoa.hawaii.edu/ software.php • FishGuts – A multimedia guide to the art and science of fish anatomy, health and necropsy, developed at the University of Maryland Aquatic Pathobiology Centre, College Park, MD: http://aquaticpath. umd.edu/fg/ • CABI Aquaculture Compendium: http://www.cabi.org/compendia/ac/ index.asp • SARNISSA Project: http://www.sarnissa.org/ • AquaNIC: http://www.aquanic.org/ • FishBase: http://www.fishbase.org/ • LarvalBase: http://www.larvalbase.org/ • Yahoo shrimp discussion group: http://finance.groups.yahoo.com/group/ shrimp • Yahoo tilapia discussion group: http://tech.groups.yahoo.com/group/ tilapia/ • Other Yahoo groups can be found at http://groups.yahoo.com/

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New technologies in aquaculture

35.8 References 1 nieto d (2005) Observatory and survey result report, EC E-aQua project. http:// www.e-aqua.org/ 2 timmons m b, ebeling j m, wheaton f w, summerfelt s t and vinci b j (2001) Recirculating aquaculture systems, Northeastern Regional Aquaculture Center, Ithaca, NY, Cayuga Aqua Ventures. 3 lekang o-i (2007) Aquaculture Engineering, Oxford, Blackwell. 4 shardlow t f and hyatt k d (2004) Assessment of the counting accuracy of the vaki infrared counter on chum salmon, North American Journal of Fisheries Management, 24(1), 249–52. 5 bjordal a, juell j e, lindem t and fernö a (1993) Hydroacoustic monitoring and feeding control in cage rearing of Atlantic salmon (Salmo salar L.), in Reinertsen H, Dehle L A, Jorgensen L and Tvinnereim K (eds), Fish Farming Technology, Rotterdam, Balkema, 203–8. 6 fisheries and oceans canada (2008) Echo-Sounding to Count Pacific Fish, Ottawa, ONT, http://www.dfo-mpo.gc.ca/science/Publications/article/2005/0209-2005-eng.htm, accessed January 2009. 7 costa c, loy a, cataudella s, davis d and scardi m (2006) Extracting fish size using dual underwater cameras, Aquacultural Engineering, 35(3), 218–27. 8 knudsen f r, fosseidengen j e, oppedal f, karlsen o and ona e (2004) Hydroacoustic monitoring of fish in sea cages: target strength (TS) measurements on Atlantic salmon (Salmo salar), Fisheries Research, 69(2), 205–9. 9 baras e, malbrouck c, houbart m, kestemont p and melard c (2000) The effect of PIT tags on growth and physiology of age-0 cultured Eurasian perch Perca fluviatilis of variable size, Aquaculture, 185(1–2), 159–73. 10 thorsteinsson v, arnold g, davenport j and maoiléidigh n o (2002) Tagging Methods for Stock Assessment and Research in Fisheries, Report of Concerted Action FAIR CT.96.1394 (CATAG), Reykjavik, Marine Institute Technical Report (79), http://www.hafro.is/Bokasafn/Timarit/catag.pdf, accessed January 2009. 11 star-oddi, GPS Fish Positioning System, Reykjavik, Star-Oddi, http://www.staroddi.com/Temperature_Recorders/GPS_fish_tag/, accessed January 2009. 12 dagorn l and holland k (2008) Development of ‘Business Card’ Tags: Interindividual Data Transfer, Pelagic Fisheries Research Program, University of Hawaii, Honolulu, HI, http://www.soest.hawaii.edu/PFRP/biology/dagorn_ business_tags.html, accessed January 2009. 13 chappell d (2008) A Short Introduction to Cloud Platforms, David Chappell & Associates, San Francisco, CA, http://www.davidchappell.com/ CloudPlatforms–Chappell.pdf, accessed January 2009. 14 istart (2007) Salmon company reels in a solution, Auckland, http://www.istart. co.nz/index/HM20/PC0/PVC197/EX232/CS2210, accessed January 2009. 15 halachmi i, simon y, guetta r and hallerman e m (2005) A novel computer simulation model for design and management of re-circulating aquaculture systems, Aquacultural Engineering, 32(3–4), 443–64. 16 nunes a j p and parsons g j (2006) A computer-based statistical model of the food and feeding patterns of the Southern brown shrimp Farfantepenaeus subtilis under culture conditions, Aquaculture, 252(2–4), 534–44. 17 neill w, brandes t, burke b, craig s, dimichele l, duchon k, edwards r, fontaine l, gatlin d. iii, hutchins c, miller j, ponwith b, stahl c, tomasso j and vega r (2004) Ecophys. Fish: A simulation model of fish growth in time-varying environmental regimes, Reviews in Fisheries Science, 12(4), 233–88. 18 mccausland w d, mente e, pierce g j and theodossiou i (2006) A simulation model of sustainability of coastal communities: Aquaculture, fishing, environ-

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21 22 23

24

25

26

27

28 29

30 31

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ment and labour markets, Ecological Modelling, 193, 271–94, http://www.abdn. ac.uk/marfish/pdfs/McCausland2006.pdf, accessed January 2009. jorge arturo alvarez (2008) pers comm. hunter d c, telfer t c and ross l g (2006) Development of a GIS-based tool to assist planning of aquaculture developments. A report to The Scottish Aquaculture Research Forum, SARF-003, University of Stirling, http://www.aqua.stir.ac.uk/ GISAP/pdfs/SARF003_Full.pdf, accessed January 2009. bjørndal t, lane d e and weintraub a (2004) Optimal research models and the management of fisheries and aquaculture: A review, European Journal of Operational Research, 156(3), 533–40. hernandez j m, leon-santana m and leon c j (2007) The role of the water temperature in the optimal management of marine aquaculture, European Journal of Operational Research, 181(2), 872–86. forsberg o i and guttormsen a g (2006) Modeling optimal dietary pigmentation strategies in farmed Atlantic salmon: Application of mixed-integer nonlinear mathematical programming techniques, Aquaculture, 261(1), 118–24. ec (2002) Regulation (EC) No. 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety, Official Journal of the European Communities, L31, 1 February, 1–24, http://eur-lex.europa.eu/ pri/en/oj/dat/2002/l_031/l_03120020201en00010024.pdf, accessed January 2009. ec (2001) Regulation Commission (EC) No. 2065/2001 of 22 October 2001 laying down detailed rules for the application of Council Regulation (EC) No 104/2000 as regards informing consumers about fishery and aquaculture products, Official Journal of the European Communities, L278, 23 October, 6–8, http://eur-lex. europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2001:278:0006:0008:EN:PDF, accessed January 2009. ec (2004) Regulation (EC) No. 852/2004 of the European Parliament and of the Council of 29 April 2004 on the hygiene of foodstuffs, Official Journal of the European Communities, L139, 30 April, 1–54, http://eur-lex.europa.eu/ LexUriServ/LexUriServ.do?uri=OJ:L:2004:139:0001:0054:EN:PDF, accessed January 2009. ec (2003) Regulation (EC) No. 1830/2003 of the European Parliament and of the Council of 22 September 2003 concerning the traceability and labelling of genetically modified organisms and the traceability of food and feed products produced from genetically modified organisms and amending Directive 2001/18/ EC, Official Journal of the European Communities, L268, 18 October, 1–5, http:// europa.eu/eur-lex/pri/en/oj/dat/2003/l_268/l_26820031018en00240028.pdf, accessed January 2009. swedberg c (2008) Taiwanese seafood producer tracks fish to the dish, RFID Journal, 10 March, http://www.rfidjournal.com/article/articleview/3964/1/1/, January 2009. microsoft (2007) Case Study: Tekho Company Ltd. Individual Fish Traced from Farm to Restaurant with BizTalk RFID, http://www.microsoft.com/canada/ partnersolutionmarketplace/CaseStudyDetail.aspx?casestudyid=4000000640, accessed January 2009. skretting (2008) Skretting invites the seafood industry to join the global traceability network, Company press release, Stavanger, 4 March 2008 http://www. tracetracker.com/cgi/doc.cgi?id=174, accessed January 2009. zeldis d and prescott s (2000) Fish disease diagnosis program – problems and some solutions, Aquacultural Engineering, 23(1–3), 3–11.

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32 li d, zhu w, duan y and fu z (2006) Towards developing a tele-diagnosis system on fish disease, Artificial Intelligence in Theory and Practice, 217, 445–54, http:// zoushoku.narc.affrc.go.jp/ADR/AFITA/afita/afita-conf/2002/part4/p374.pdf, accessed February 2009. 33 brock j and itoga s (1993) Hawaii Aquaculture Module Expert System (HAMES), Pacific Regional Aquaculture Information Service, University of Hawaii, Honolulu, HI, http://praise.manoa.hawaii.edu/software.php?download, accessed January 2009. 34 keen a (2008) The cult of the amateur. How today’s Internet is killing our culture, Doubleday, New York. 35 turnbull j (2008) pers comm.

36 Inland saline aquaculture G. L. Allan and D. S. Fielder, New South Wales Department of Primary Industries, Australia, K. M. Fitzsimmons, University of Arizona, USA, S. L. Applebaum, Jacob Blaustein Institute for Desert Research BGU, Israel, and S. Raizada, Central Institute of Fisheries Education Rohtak Centre (ICAR), India

Abstract: Increasing demand for aquaculture has led to the development of new production systems. Inland saline aquaculture, defined here as land-based aquaculture using saline groundwater, occurs in several countries including Israel, the USA, India and Australia. A number of species are cultured, or are being evaluated for their potential, including finfish such as tilapia, Asian sea bass and trout, shrimp and oysters. Sources of saline groundwater include ephemeral and permanent saline lakes, saline water extracted with coal seam gas and saline groundwater extracted from aquifers. Saline groundwater is extracted in some areas to protect the root zone of plants. Characteristics of saline-affected land are described, with particular focus on Australia and India. Another emerging source of saline groundwater is the coal bed methane gas industry. Saline water accompanies extraction of the gas and, while it can be a major environmental problem for the gas industry, it presents an opportunity for aquaculture. Saline groundwater can differ in chemistry compared with coastal seawater and adjusting the chemistry or choosing species that are tolerant to the differences is one of the major challenges for expansion of inland saline aquaculture. The chemistry of different sources of water is described and common methods of adjusting the chemistry described. Finally, case studies of inland saline aquaculture are presented for Australia, India, Israel and the USA. Novel food production methods, such as inland saline aquaculture, are needed to increase aquaculture production and meet increasing demands for seafood. Key words: inland saline aquaculture, desert aquaculture, Australia, India, Israel, USA.

36.1 Introduction Demand for seafood throughout the world is increasing (FAO, 2007) while production from capture fisheries is static or declining. To cater for global demands in 2020, an estimated 163 million tonnes of fish will be required

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and, given static production from capture fisheries, aquaculture will need to supply 70 million tonnes (FAO, 2007). Expansion of coastal aquaculture is limited in many areas because of other land and water use activities. Inland aquaculture already makes the greatest contribution to total aquaculture (approximately 60 %: FAO, 2007), but inland saline aquaculture also offers the potential to increase production of euryhaline and marine species. Inland saline aquaculture is defined here as land-based aquaculture using saline groundwater. Sources of saline groundwater include ephemeral and permanent saline lakes, saline water extracted with coal seam gas and saline groundwater extracted from aquifers, including shallow water tables where saline groundwater is extracted to protect the root zone of plants. Inland saline water can differ in chemistry compared with coastal seawater, and adjusting the chemistry or choosing species that are tolerant to the differences is one of the major challenges for expansion of inland saline aquaculture. A range of species have been evaluated for culture in saline groundwater including euryhaline finfish (e.g. Lates calcarifer, Sparus auratus, Dicentrarchus labrax, Argyrosomus japonicus), crustaceans (e.g. Penaeus monodon, Litopenaeus vannamei, Marsupenaeus japonicus) and molluscs (e.g. Saccostrea glomerata), diadromous species such as salmonids (e.g. Oncorynchus mykiss) and salt-tolerant freshwater species such as finfish (e.g. Oreochromis niloticus, Bidyanus bidyanus) and crustaceans (e.g. Macrobrachium rosenbergii). Different farming systems are used including earthen or plastic-lined ponds, tanks (including with recirculation technology) and raceways. Partridge et al. (2008) described the range of pond, tank and tank–pond hybrid systems that have been used for inland saline aquaculture. Commercial production using saline groundwater occurs in the USA, Israel, India and Australia. In Texas, farming shrimp (Litopenaeus vannamei) commenced in the 1970s after quarry operators attempted to culture shrimp using saline quarry water. Inland saline shrimp culture has been evaluated in Texas, Arizona, Arkansas, Alabama and Florida. In Arizona, saline groundwater (2–5 ppt) is used to grow marine shrimp in ponds and raceways with effluents used to irrigate olives, dates and other agricultural crops. In Florida and Alabama, saline aquifers are also tapped to provide water for pond culture. In Israel, inland saline aquaculture, known as ‘desert aquaculture’, began operating commercially in the late 1980s and is characterised by raising finfish in brackish geothermal water from deep aquifers, discovered in the 1940s. The depth of these aquifers varies, depending on the topography, between 450 and 1000 m below the surface. These aquifers contain huge quantities (billions of cubic meters) of brackish (3–7 ppt TDS – total dissolved solids) geothermal (∼40 °C) pollutant-free, ancient water. This socalled ‘artesian desert water’ rises by its own artesian pressure to the height of about sea level and therefore only has to be pumped up for use from an average depth of 400 m below the surface. Several farms in the Israeli

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Negev desert (∼13 000 km2) are producing finfish in this ‘desert water’, Asian sea bass (Lates calcarifer) being the major species with an annual production of around 200 tonnes, followed by red drum, striped bass, sea bass, sea bream, tilapia and catfish, the total of which, 400–500 tonnes annually, are consumed domestically fresh. The white leg shrimp (Litopenaeus vannamei) was successfully cultured for several years, but production has ceased due to limited demand on the local market. In addition to the edible fish farming, a number of farms in the desert are using the brackish water, occasionally in combination with freshwater, to produce a variety of ornamental hobby fish, all of which are exported to Europe. The system operations for desert aquaculture in Israel are mostly based on water recirculation that includes mechanical and biological water filtration. However, a significant amount of fish culture effluent is diverted into irrigation systems providing fresh water rich with dissolved fish metabolites that benefit a variety of crops such as olives, dates, jojoba and hay for cattle. In India, culture of the giant freshwater prawn (Macrobrachium rosenbergii) occurs in freshwater ponds in the northwestern states, including Haryana. The practice has relied on transport of postlarvae (PLs) from coastal regions until recently after pioneering research by the Central Institution of Fisheries Education developed techniques for hatchery production using saline groundwater. In Australia, small-scale production of rainbow trout (Oncorynchus mykiss) occurs using saline groundwater in southwestern Western Australia (Trendall, 2008), and an Asian sea bass (Lates calcarifer) farm using deep saline aquifer water was operating but closed for a number of reasons including regulatory difficulties with expansion to achieve economies of scale. Research with rainbow trout demonstrated this species was suitable for culture using saline groundwater pumped into evaporation ponds to protect the root zone in southwestern New South Wales, but commercial development of that research has stalled because of the severe drought in the Murray–Darling River system in Australia (Allan et al., 2008a). In this chapter, key sources of saline groundwater from interception schemes and waste associated with the extraction of coal seam gas will be described and the chemistry and methods of remediating saline groundwater for culture discussed. Examples of inland saline aquaculture will be presented, including desert aquaculture in Israel, aquaculture using groundwater interception schemes in Australia, giant freshwater prawn culture in India and shrimp farming in the USA.

36.2 Saline groundwater from interception schemes to protect agriculture Increasing soil salinity is a global issue, and occurs in many semi-arid to arid regions where large-scale irrigated agriculture is practised. Rising saline

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water tables and the associated environmental degradation are problems in developed countries, e.g. Australia, Israel and the USA, and developing countries, e.g. India, Pakistan, China, Thailand and countries in the middleeast. Globally, an estimated 380 million ha of land is unusable for agriculture because of salinisation of soils and groundwater (Lambers, 2003). A commonly practised engineering solution to rising saline groundwater is the construction and operation of large-scale subsurface drainage systems, which collect saline groundwater and dispose of it in ponds or canals. These engineering schemes are very expensive to construct and operate. Incorporation of aquaculture into the design of these engineering schemes can provide a beneficial use of the otherwise waste water and a partial economic return on the initial investment. In the future, this may assist with raising finance for more engineering schemes to protect valuable arable land for agriculture and towns and cities from the ravages of salinity. For aquaculturists, a sure water supply and disposal through terminal evaporation ponds provide critical and expensive infrastructure. The situation in India and Australia illustrates the problem. Inland salinity in those countries is a major environmental problem that threatens productive agricultural enterprises, fragile ecosystems, valuable infrastructure as well as roads and buildings (including homes) in many country areas (Agarwal and Roest, 1996; Ingram et al., 1996; Qureshi and BarrettLennard, 1998; Allan et al., 2001; Anon, 2001). In Australia, some 5.7 million ha of land are under high risk of salinisation through dryland salinity with predictions that this may increase to over 17 million ha in 50 years (Anon, 2001). There are 11 saline groundwater subsurface drainage schemes, which incorporate more than 6000 ha of evaporation ponds operating in the Murray–Darling Basin (MDB) river system. A further eight schemes are planned for construction in the MDB. Adelaide, the capital of South Australia and home to over 1.1 million people, is threatened by salinity in the domestic water supply with extraction of salt through saline groundwater interception schemes along the Murray River as the primary response. In addition, 76 other towns in Australia were identified as being threatened by salinity, and groundwater schemes with evaporation ponds are being considered in many of these (Allan et al., 2001). Approximately 8.7 million ha of land is salt-affected in India and about 40 % of this salt-affected land is concentrated in the north-western, semiarid/arid states of Haryana, Punjab, Uttar Pradesh and Rajasthan. Disposal of saline drainage effluent from irrigated land may introduce significant environmental problems (Dr S. Raizada, CIFE, Rohtak Centre, pers comm, 2001). Haryana is a leading agriculture producing state and, since the 1980s, agricultural production has increased from 2.6 to 10.5 million tonnes. Most of this increase is associated with a concurrent large-scale increase in canal irrigation and an increase in the number of shallow tubewells from 25 000 to 550 000. Consequently, the area of irrigated land increased from 1.3 to

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2.6 million ha. However, creation of intensive irrigation facilities and inadequate and inefficient on-farm water management infrastructure has led to waterlogging and soil salinity. This problem is exacerbated by other factors such as impeded drainage conditions, topography, high salt content of parent material, poor water management practices, brackish and saline groundwaters (which as such are not suitable for irrigation) and, importantly, the arid and semi-arid climate. Groundwater tables are rising in almost 50 % of Haryana and 0.52 million ha of land is salt-affected (saline, sodic or both). A four-fold increase in salt-affected land has been predicted over the next 30 years, unless preventative measures are taken (Agarwal and Roest, 1996). In India, at least 21 pilot projects have been undertaken in Haryana, Rajasthan and Gujarat to demonstrate the efficacy and evaluate the feasibility of installing subsurface drainage schemes (Pal et al., 1999). A joint 10-year pilot-scale project to investigate several aspects of drainage and salt management was recently completed in Haryana by the Dutchbased International Institute for Land Reclamation and Improvement, Indian Council of Agricultural Research (ICAR) and CCS Haryana Agricultural University. Installation of other large-scale tile drainage schemes is now planned, but disposal of the saline groundwater will pose a serious problem. This may be solved by incorporation of evaporation ponds in the system.

36.3 Coal bed methane waste water A relatively new potential source of inland saline water for aquaculture is the water extracted during harvest of coal bed methane (CBM). CBM, also called coal seam gas and coal seam methane, is methane, gas produced with the formation of coal (http://www.australianminesatlas.gov.au/aimr/ commodity/coal_bed_methane.jsp). CBM is similar to conventional natural gas and can be used to generate electricity and, directly, to power domestic and industrial appliances such as water heaters, stoves, etc. Water accompanies the extraction of methane but, in contrast to conventional oil and gas production, the volume of water produced when CBM is extracted is greatest in the early stages of production and decreases as production of CBM increases (Veil et al., 2004). The chemistry of the water varies with the original conditions occurring during deposition, the depth of burial and the type of coal (Jackson and Myers 2002, cited in Veil et al., 2004) (see Table 36.1 for examples from Australia and the USA). There are significant reserves of CBM in the USA, Canada, China and Australia with reports of probable deposits elsewhere (e.g. Russia). In the USA, proven reserves were estimated at 19 620 billion cubic feet in 2006 (= 20 601 Petajoules; PJ) (Energy Information Administration, http://tonto. eia.doe.gov/dnav/ng/ng_enr_cbm_a_EPG0_r51_Bcf_a.htm). In the 1980s,

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Table 36.1 Water chemistry for inland saline water Cl

Country

Location

Salinity ppt

pH

Alkalinity mg/l

Seawater Australia

Coastal (east coast) Wakool (New South Wales) Undera (Victoria) Wakerie (South Australia) Wannamal (Western Australia) nr (Western Australia)

Cl/salinity

SO4

ion/Cl ratio

mg/l

ion/Cl ratio

% ion/Cl ratio relative to seawater

35.0 35.3

8.1

114.0

19 000.0 20 000.0

542.9 566.6

1.0 1.0

885.0 2500.0

0.0466 0.1250

100.0 268.4

19.6

7.9

195.0

11 000.0

561.2

1.0

1100.0

0.1000

214.7

10.0

nr

nr

4 130.0

413.0

1.0

1280.0

0.3099

665.4

16.0

7.0

nr

9 410.0

588.1

1.0

1600.0

0.1700

365.0

32.0

nr

nr

15 800.0

493.8

1.0

1614.0

0.1022

219.3

45.0

nr

nr

25 000.0

555.6

1.0

1400.0

0.0560

120.2

5.0

7.5–8.0

nr

700.0

140.0

1.0

nr

nr

nr

1.2

8.0–9.0

nr

590.0

491.7

1.0

5.0

nr

nr

nr

nr

Quirindi (CBM) Surat Basin (CBM) (min) Surat Basin (CBM) (max) Alabama

4.3

8.0–9.0

nr

1 900.0

441.9

1.0

10.0

3.9

nr

nr

2 274.0

583.1

1.0

3.0

0.0013

2.8

Arizona

7.1

nr

nr

2 016.0

283.9

1.0

2369.0

1.1751

2522.8

Florida

2.9

nr

nr

1 023.0

352.8

1.0

640.0

0.6256

1343.1

14.0

nr

nr

3 479.0

248.5

1.0

4775.0

1.3725

2946.7

Powder River Basin Jiangsu Province

0.9

nr

nr

13.0

15.1

1.0

2.4

0.1846

396.3

0.5

nr

nr

43.0

86.0

1.0

122.0

2.8372

6091.2

Ecuador

Guayas Province

2.5

nr

nr

1 344.0

537.6

1.0

125.0

0.0930

199.7

India

Haryana

25.0

8.4

148.7

9 738.4

389.5

1.0

129.7

0.0133

28.6

8.2

8.3

277.0

3 701.0

451.3

1.0

nr

nr

nr

30.0

nr

nr

21 300.0

710.0

1.0

nr

nr

nr

2.0

7.3

nr

1 225.0

612.5

1.0

USA

Texas

China

Haryana (Rothak) Rajasthan

Israel

CBM = coal bed methane; nr = not recorded.

480.0

0.3918

841.2

Inland saline aquaculture

Ca

Mg

K % ion/Cl ratio relative to seawater

mg/l

ion/Cl ratio

% ion/Cl ratio relative to seawater

mg/l

ion/Cl ratio

400.0 364.0

0.0211 0.0180

100.0 85.5

1350.0 1000.0

0.0711 0.0500

100.0 70.4

380.0 0.0200 365.0 0.0180

504.0

0.0460

218.5

820.0

0.0750

105.6

350.0

0.0847

402.5

470.0

0.1138

347.0

0.0369

175.2

439.0

592.0

0.0375

178.0

740.0

0.0296

10.0

1125

Na % ion/Cl ratio relative to seawater

% ion/Cl ratio relative to seawater

mg/l

ion/Cl ratio

100.0 90.0

10 500.0 9 470.0

0.5526 0.4740

100.0 85.8

9.2 0.0010

5.0

4 210.0

0.3830

69.3

160.2

25.0 0.0061

30.3

2 700.0

0.6538

118.3

0.0467

65.7

81.6 0.0087

43.4

5 240.0

0.5569

100.8

1 537.0

0.0973

136.9

80.0 0.0051

25.3

8 026.0

0.5080

91.9

140.6

2 800.0

0.1120

157.6

28.0

12 000.0

0.4800

86.9

0.0143

67.9

30.0

0.0429

3.0

0.0051

24.2

1.0

9.0

0.0047

22.5

86.0

0.0378

494.0

mg/l

ion/Cl ratio

Reference

Spotte, 1979 Fielder et al., 2001 Fielder et al., 2001 Ingram et al., 1996 Flowers and Hutchinson, 2004 Prangnell & Fotedar, 2006 Partridge & Creeper, 2004 Dutney, pers comm Anon, 2004

140.0

0.0056

60.3

nr

nr

nr

3 000.0

4.2857

775.5

0.0017

2.4

nr

nr

nr

300.0

0.5085

92.0

3.0

0.0016

2.2

nr

nr

nr

1 700.0

0.8947

161.9

Anon, 2004

179.6

21.0

0.0092

13.0

8.0 0.0035

17.6

1 393.0

0.6126

110.8

0.2450

1 163.9

86.0

0.0427

60.0

11.0 0.0055

27.3

2 013.0

0.9985

180.7

74.0

0.0723

343.6

83.0

0.0811

114.2

55.0 0.0538

268.8

865.0

0.8456

153.0

961.0

0.2762

1312.1

581.0

0.1670

235.0

51.0 0.0147

73.3

2 996.0

0.8612

155.8

36.0

2.7692

13 153.8

16.0

1.2308

1732.2

305.0

23.4615

4245.4

Boyd & Thunjai, 2003 Boyd & Thunjai, 2003 Boyd & Thunjai, 2003 Boyd & Thunjai, 2003 Veil et al., 2004

44.0

1.0233

4 860.5

17.0

0.3953

556.4

9.0 0.2093

1046.5

105.0

2.4419

441.9

147.0

0.1094

519.5

79.0

0.0588

82.7

10.0 0.0074

37.2

496.0

0.3690

2 030.2

0.2085

990.2

1987.9

0.2041

287.3

322.0

0.0870

413.2667

477.0

0.1289

181.3924

10.6 0.0029

14.3

2 406.0

0.6501

661.3

0.0310

147.4730

377.9

0.0177

24.9699

76.4 0.0036

17.9

6 011.6

0.2822

127.0

0.1037

492.4490

92.0

0.0751

105.6992

27.0 0.0220

700.0

0.5714

nr

nr

nr

nr

nr

nr

110.2041

nr

nr

Boyd & Thunjai, 2003 66.8 Boyd & Thunjai, 2003 nr Barman et al., 2005 117.6 Raizada, pers comm. 51.0710 Shakeeb-UrRahman et al., 2005 103.4014 Appelbaum, pers. comm.

1126

New technologies in aquaculture

this resource was not considered a target yet, today, there are over 200 wells in Montana alone, producing over US$300 million/y and total CBM production now accounts for 8 % of USA natural gas production (Anon, 2004). In Australia, as at December 2007, there were proven and probable reserves estimated at 7500 PJ producing 107 PJ (95 % in Queensland, with the remainder in NSW), and estimates that the potential increase in production could supply 50 % of Australia’s demand for the east coast by 2020 (http://www.australianminesatlas.gov.au/aimr/commodity/coal_bed_ methane.jsp). The large volume of water produced has made disposal of the water a priority topic. For example, the estimates for total water production from CBM harvest in Queensland, Australia currently is in excess of 4000 ML/y, and this is predicted to exceed 42 000 ML/y by 2010 (Anon, 2001). Opportunities for beneficial use of the water produced have included re-injection to assist oil or gas recovery, industrial uses (e.g. dust control), use by animals, including for stock or wildlife watering, irrigation, creation of recreation areas, constructed wetlands, fisheries and aquaculture (Fitzsimmons, 1988; Anon, 2004; Veil et al., 2004).

36.4 Chemistry and remediation Table 36.1 lists ionic composition of saline groundwater sources. While the chemistry differs greatly across locations there are some similarities. Most saline groundwater is deficient in potassium with the lowest levels recorded in the Wakool region of the MDB in Australia where potassium was present at 5 % of the concentration found in equivalent salinity seawater. With the exception of CBM waste water from Queensland Australia, calcium in saline groundwater was higher than in seawater by a multiplication factor of between 1.4 and 130 (highest was CBM waste water from Powder River Basin, USA). Sulphate was also in relative excess at all locations except Alabama and Haryana, India. Magnesium was deficient at some locations (lowest in CBM wastewater in Queensland, Australia) and in excess at others (highest in CBM wastewater in Powder River Basin). Not surprisingly, considerable research has been conducted on methods to ameliorate deficiencies in water chemistry. Boyd and Thunjai (2003) summarised deficiencies in a number of water sources around the world used for culturing shrimp and provided a list of mineral sources that could be used as sources for major cations. Given the physiological importance of potassium, this ion has probably received the most attention. Fielder et al. (2001) found red sea bream (Pagrus auratus) lost equilibrium and became moribund within three days of transfer to saline groundwater from Wakool (see Table 36.1 for ionic composition) but, provided potassium was added to the water, survival and growth was similar to that in controls with coastal seawater. Potassium addition has also been found useful for other

Inland saline aquaculture

1127

species cultured in saline groundwater (Flowers and Hutchinson, 2004; Prangnell and Fotedar, 2005, 2006; Doroudi et al., 2006; Tantulo and Fotedar, 2006; Partridge and Lymbery, 2008). Doroudi et al. (2006) found that survival and growth of mulloway (Argyrosomus japonicus) in saline groundwater was similar to that recorded in sea cage or tank trials with coastal seawater, provided potassium concentration was adjusted to 40 % or more of the equivalent concentration in coastal seawater. Additions of magnesium to the water have also been investigated. Forsberg and Neill (1997) found survival of red drum (Scieanops ocellatus) improved in low-salinity water with the addition of magnesium chloride. Similarily, Roy et al. (2007) recorded an improvement in survival of L. vannamei when magnesium chloride was added to magnesium-deficient water but neither study recorded significant improvements in growth. Boyd et al. (2006) added potassium chloride (muriate of potash) and potassium magnesium sulfate (Kmag) to ponds in Alabama used for L. vannamei culture to increase potassium, magnesium and sulfate concentrations to about 40, 25 and 60 mg/L, respectively. This treatment was successful in increasing shrimp survival and production. Calcium imbalances are also common. Forsberg and Neill (1997) improved survival, growth and feed efficiency of red drum with addition of calcium chloride to low-salinity groundwater in west Texas. The CBM waste water from Queensland, Australia is deficient in potassium and calcium (Table 36.1 and Dutney et al., 2008). Dutney et al. (2008) reported difficulties with adjusting calcium because of the formation of calcium carbonate. Reducing the pH was effective, but this was expensive and impractical. Most other sources of saline groundwater have excessive calcium compared with seawater. In Haryana, saline groundwater is high in calcium and magnesium compared with equivalent salinity seawater and this was recognised as a problem with larval production of Macrobrachium rosenbergii. Scientists addressed this by a clever use of ion-exchange resin incorporated into practical, largescale filters (Dr S. Riazada pers comm, 2008). Using a filter containing approximately 0.5 m3 of ion exchange resin, 2500 L/d of saline groundwater can be treated, sufficient to operate a large Macrobrachium hatchery capable of producing 1.5 million PLs per year (Dr S. Raizada, pers comm). Remediation of culture water can be expensive and ions are lost from ponds in overflow after rains, through soil adsorption and water exchange. Pond sediments in particular adsorb potassium because the native soils contain clays that fix potassium between adjacent tetrahedral layers (Boyd et al., 2006). The efficacy of adjusting potassium deficiency in saline groundwater through dietary manipulation has been investigated as an alternative or complementary approach but has not been effective for finfish (Gong et al., 2004; Saoud et al., 2007; Allan et al., 2008b). Gong et al. (2004) did record an improvement in osmoregulatory capacity of L. vannamei reared in low-salinity groundwater in Arizona when diets were supplemented with magnesium, potassium, phospholipids and cholesterol.

1128

New technologies in aquaculture

There are numerous other imbalances in ionic composition and possible contaminants in saline groundwater. For many of these ions, site-specific studies will be needed. Most studies have focussed on large deviations of single ions but, as production from saline groundwater sources increases, it is likely that chronic effects will become apparent. Research to understand how to construct artificial mixed-salt environments and physiological responses of aquaculture species in low-salinity water will be valuable in helping to understand these relationships (see for example Cheng et al., 2005; Sowers et al., 2006; Zhu et al., 2006). In addition to ionic composition per se, sub-optimal pH has also negatively impacted on potential for aquaculture using saline groundwater. Acidic conditions can arise from the presence of acid sulphate soils, use of acid-forming fertilisers on agricultural lands, ferrolysis and elevated concentrations of dissolved carbon dioxide (Partridge et al., 2008). Research in South Australia (Hutchinson, 2008) found concentrations of dissolved carbon dioxide were regularly in excess of 160 mg/L and fan-forced, packed column degassing columns were installed both prior to water storage and prior to water entering culture tanks. In the Powder River Basin of Wyoming, residual volatile organics were also stripped in degassing columns that had the added benefit of introducing dissolved oxygen, which was very low in the raw CBM water.

36.5 Case studies 36.5.1 Case study: Australia There has been significant national interest in inland saline aquaculture in Australia since the late 1990s (see http://www.australian-aquacultureportal. com/saline/saline.html) and a national R&D Plan and resource inventory have been published. The grow-out of marine species in shallow aquifers (including evaporation basins) was identified as currently having the highest commercial prospect. Partridge et al. (2008) has thoroughly reviewed development of inland saline aquaculture in Australia, including a description of the water sources and composition, facilities used and culture methods. They concluded that while developing these industries could provide significant benefits, the goal of sustainable development had not yet been realised. Key challenges were the suitability of the available species to the farming environment and the quantity, quality and consistency of available saline groundwater. The MDB is one of the largest sources of saline groundwater in Australia. It extends from southern Queensland in the north, through New South Wales and Victoria in the south and across South Australia in the west. Research into the aquaculture potential of saline groundwater within the MDB was initiated in Victoria when different species were held in saline groundwater (Ingram et al., 1996). Oyster and penaeid shrimp species did

Inland saline aquaculture

1129

not survive and only a couple of the eight species of finfish survived, probably because of ionic deficiencies, particularly potassium (Table 36.1). The aquaculture potential of saline groundwater within the MDB has also been studied at two sites within South Australia (Flowers and Hutchinson, 2004; Hutchinson, 2008) and one site in New South Wales. The research approach and development toward a commercial industry at the location in New South Wales is presented here as a case study. The Wakool–Tullakool Sub-Surface Drainage Scheme (WTSSDS) in New South Wales is the largest subsurface drainage scheme in Australia. It disposes of up to 35 000 ML of saline groundwater each year. The WTSSDS consists of 60 bore pumps for salt interception and 1600 ha of ponds for evaporation and disposal of the saline groundwater. It is estimated that the WTSSDS has helped return 60 000 ha of unproductive salt-degraded land back to productive farming. NSW Department of Primary Industries established the Inland Saline Aquaculture Research Centre in 2002 (Fig. 36.1). Potassium concentration in groundwater from the WTSSDS was only 5 % of the concentration found in marine water of the same salinity and, as such, was not suitable for survival and growth of marine species. Addition of potassium in the form of potash fertiliser increased potassium concentration in the saline groundwater, and small-scale experiments demonstrated that marine species survived and grew in potassium-fortified groundwater at similar growth rates reported for the species in marine water (Fielder

Fig. 36.1 ISA Research Centre facilities, Wakool, NSW.

1130

New technologies in aquaculture

et al., 2001). A list of potential candidate species was established which included red sea bream (Pagrus auratus), black tiger prawn (Penaeus monodon), mulloway (Argyrosomus japonicus), silver perch (Bidyanus bidyanus) and rainbow trout (Oncorynchus mykiss). Early trials focused on determining optimal salinity and potassium concentrations for red sea bream, silver perch, mulloway and black tiger prawn. The key finding was that marine species required potassium to be fortified in saline groundwater to above 50 % of the concentration found in marine water of the same salinity. Growth rates of red sea bream, mulloway and black tiger prawn during bioassays were comparable to published rates in marine water. Silver perch, a salt-tolerant native freshwater species, grew well in raw groundwater and did not require potassium adjustment (Fielder et al., 2001; Doroudi et al., 2006, 2007; Allan et al., 2008a). Upon completion of the bioassays, pilot-scale commercial production of black tiger prawns, snapper, silver perch, mulloway and trout was undertaken in plastic-lined ponds. Cool winters and fluctuating daily water temperatures up to 0–5 °C limited the growth of red sea bream, silver perch and black tiger prawns. Pilot-scale commercial production showed that mulloway grew well when water temperature exceeded 16 °C. Average pond water temperature was generally greater than 16 °C during the period of November–May in the region. Mulloway appeared stressed when water temperature was less than 12 °C and, when water temperature was less than 10 °C (typically June and July), some fish died (Fig. 36.2).

Temperature covered

350 Temperature uncovered

250 200

Weight covered

Covers destroyed

150

Weight uncovered

100

20

10

50 0

12

-M a 11 y-0 -J 5 un 11 -05 -J u 10 l-0 5 -A ug 9- -05 Se p 9- -05 O ct 8- -05 N ov 8- -05 D ec 7- -05 Ja n 6- -06 Fe b 8- -06 M ar 7- -06 Ap r 7- -06 M ay 6- -06 Ju n6- 06 Ju l 5- -06 Au g06

0

Date

Fig. 36.2 Water temperature and wet weight of mulloway in uncovered and covered ponds at ISA Research Centre, Wakool, NSW, Australia.

Water temperature (°C)

Wet weight (g)

300

30

Inland saline aquaculture

1

1131

2 3

Fig. 36.3 Three floating solar covers positioned on a 0.05 ha experiment pond. Table 36.2 Water quality of uncovered and covered ponds during experiment at ISA Research Centre, Wakool, NSW, Australia

CO2 (mg/L) NH4 (mg/L) pH DO (mg/L) Salinity (ppt)

Covered pond

Uncovered pond

0–15 0–0.4 7.3–8.5 >5.0 17–28

0–5 0–0.2 7.4–8.6 >5.0 17–29

The use of floating solar covers on mulloway ponds was investigated as a novel way to increase water temperatures (Fig. 36.3). Three floating polythene strips were constructed to allow complete coverage of a 500 m2 pond. Subsurface aeration was supplied with an Aero-2 aspirator pump aerator which was positioned between two adjacent solar covers. Access to the fish for feeding and sampling was done by retracting one cover to expose a small area of the surface water. The floating solar covers increased mean minimum winter and summer water temperatures by 1.5 °C and 3.0 °C, respectively, and had little effect on the major water quality parameters compared with those of uncovered, ambient ponds (Table 36.2). After 12 months of growout, mulloway cultured in solar covered ponds were 20 % heavier than fish cultured in uncovered ponds. Trout survival and growth was excellent when pond water quality was high and fortification of groundwater with potassium was unnecessary. However, build-up of organic matter in static ponds (minimal water exchange) reduced growth.

1132

New technologies in aquaculture

600 500 Average trout weight (g) 2004

Weight (g)

400

Average weight (g) of G&G sale trout 2004

300

Average trout weight (g) 2005

200

Average weight (g) of G&G sale trout 2005

100

5-Dec

5-Nov

5-Oct

5-Sep

5-Aug

5-Jul

5-Jun

5-May

0

Date

Fig. 36.4 Growth of rainbow trout in saline groundwater ponds at ISA Research Centre, Wakool, NSW, Australia (2004 & 2005).

The ambient pond water temperatures in the region were found to be potentially suitable for salmonid production for at least seven months of the year from April to October. Growth performance of the trout produced during three successive years was outstanding and after three months fish stocked at 40 g had an average wet weight of >310 g (Fig. 36.4). Survival was near to 100 % during winter but began to decrease as pond temperatures exceeded 21 °C in summer, indicating that 60 % exchange of pond water each day was inadequate to reduce heating of ponds during summer. Average feed conversion ratio (FCR) during the production period was 1.1 : 1. Despite increasing pond water temperatures market-size fish were continually harvested until late December. The rainbow trout cultured in saline groundwater were very popular with consumers due to the fresh, salty flavour compared with freshwater cultured rainbow trout and consequently returned up to two dollars per kg more than freshwater trout as they were considered a superior product. Economic analyses of rainbow trout culture in raceways was completed using an economic model (Johnston, 2008) and confirmed by independent investment analysis. These analyses indicated that a 200 t/y rainbow trout farm could return an attractive rate of return on investment. However, the biggest constraint to commercial development of inland saline aquaculture in southern NSW currently is the deficit of saline groundwater as a consequence of the severe, long-term drought in the MDB system. The extended drought has meant that little or no fresh, irrigation water has been available for irrigated cropping and significant rain has not fallen in the MDB for many years. As a consequence, the groundwater table has not been recharged and the saline groundwater table is very deep. Pumping of saline ground-

Inland saline aquaculture

1133

water is therefore not needed and, as it is expensive, has been reduced. The volume of saline groundwater at the WTSSDS has decreased from an average of approximately 35 ML/d in ‘normal’ years to 4–5 ML/d in ‘drought’ years. The limited availability of saline groundwater has highlighted the need for on-going research to identify viable, commercial methods for reuse of saline groundwater in rainbow trout production as well as effluent disposal from evaporation basins.

36.5.2 Case study: India, culture of Macrobrachium rosenbergii Management of irrigation salinity has been a major issue in India for many years. For example, since 1966 the state of Haryana has experienced a major increase in development of new irrigation schemes. Approximately 70 % of Haryana’s net sown area, or 2.6 million ha, is irrigated from canals (54 % of area) or tubewells (46 % of area). However, major land degradation through rising groundwater tables, accumulation of soluble salts in the soil profile and reduced crop yields and, in extreme cases, abandonment of cultivation, is occurring on a wide scale – almost 50 % of the state. Agricultural production loss due to waterlogging and salinity in Haryana in 2000 was estimated at approximately US$22.5 million (Agarwal and Roest, 1996). Waterlogging and salinity problems are exacerbated in Haryana due to a topographical depression or basin, which covers an area of 1.7 million ha in the centre of the state. Groundwater, which in 55 % of the state is unfit (too saline) for irrigation, flows towards the centre of the basin and accumulates as it cannot be discharged through natural drains. As in Australia, the value of subsurface drainage systems for reducing water tables and improving productivity has been demonstrated in a number of pilot projects and more are being constructed or planned. For example, two large-scale subsurface projects (1000 ha each) in Haryana have recently been established in two clusters of villages in Gohana and Kalayat areas, with the aim to assess social, technical and economical parameters in farmers’ fields. Not only has agricultural productivity increased but also land value has increased and waterborne diseases and malaria were perceived to have reduced after the system was installed (Pal et al., 1999). Although the benefits of installing subsurface drainage systems appear to be high and environmentally sound, sustainable disposal of the collected saline groundwater is paramount. Evaporation basins offer a solution to this problem. This is recognised by Indian authorities, and installation of evaporation basins will be necessary as part of saline groundwater mitigation schemes. The potential to use saline groundwater for aquaculture in India has been identified as a high-priority research area. Research and education facilities have been constructed in Haryana (e.g. Central Institute of Fisheries Education (CIFE) at Rohtak) aimed specifically to evaluate the potential for aquaculture using saline groundwater and to educate farmers in developed technology.

1134

New technologies in aquaculture

Recent research at the CIFE, Rohtak Centre has focussed on development of technology for hatchery and grow-out production of Macrobrachium rosenbergii, in shallow water table, saline groundwater of low salinity (4–12 ppt). This research was initiated for several reasons. Firstly, a small industry had developed in Haryana for growout of Macrobrachium in ponds using high-quality freshwater. Macrobrachium are not endemic to Haryana and PLs were sourced from coastal hatcheries, which was expensive due to costs of airfreight and, importantly, availability was often uncertain and delayed well into the summer growing season, thus reducing the grow-out period and potential return/ha. Production from local hatcheries would therefore improve availability and possibly reduce the purchase cost of PLs. Secondly, a great deal of saline groundwater was available for aquaculture, but little was known about pond management and the production performance of Macrobrachium in saline groundwater. Initial attempts to culture larvae in raw saline groundwater (12 ppt) were unsuccessful and led to investigation of the need for water chemistry amendment. Low potassium (11.7 % of seawater (SW)) and high magnesium (132 % of SW) and calcium (316 % of SW) concentrations in the groundwater compared with that of similar salinity seawater were found to cause total mortality of larvae within days of hatching. A simple and cheap method of filtering the groundwater through a zeolite, sodium–aluminium– orthosilicate, was used to reduce the calcium concentration to that of equivalent salinity SW. The process also decreased magnesium concentration which subsequently required addition of magnesium, as magnesium chloride, to restore the magnesium : calcium to 2.5 : 1. Addition of potassium chloride (or potash) increased the potassium level similar to that found in the same salinity seawater (Table 36.3). Once these ionic amendments were made, growth and survival of prawn larvae was high (routinely >40 % survival) and 100s of thousands of PLs were produced in an experiment-scale hatchery. Management of broodstock to provide gravid females in spring was also addressed because maintenance of prawns in outdoor ponds during winter

Table 36.3 Water chemistry of diluted coastal seawater (12 ppt) and inland saline groundwater (12 ppt) from Haryana, before and after amendment for larval rearing of Macrobrachium rosenbergii Ion Magnesium Calcium Potassium 1

Coastal sea water diluted1 (mg/L)

Raw saline groundwater (mg/L)

Amended saline groundwater (mg/L)

339.9 123.7 124.0

616.0 440.0 10.9

275–304 148–160 118

Adapted from Fielder et al., 2001.

Inland saline aquaculture

1135

months from December to March was not possible due to sub-optimal temperatures. Broodstock were successfully held indoors in heated tanks during winter; however, the small number of prawns held and the high cost or, at times, unavailability of electricity precluded this as an option for a large-scale hatchery. Alternatively, polyhouses were constructed over two 450 m2 ponds and each was stocked in December with approximately 650 mature prawns. The minimum water temperature in the covered ponds was 19.5 °C, 7.5 °C greater than the minimum temperature in uncovered ponds, and approximately 90 % of prawns survived with 70 % of the female prawns becoming gravid by March. Polyhouses therefore effectively maintained suitable water temperature during winter for large-scale broodstock management and allowed timely supply of larvae for hatchery production at the start of spring. The semi-arid, inland environment of Haryana is characterised by extreme hot summers and cold winters which restrict the growout of Macrobrachium in ponds to nine months from April to mid-December. Therefore early stocking of juvenile prawns is essential to maximise the period of growth and subsequently produce the greatest volume of large, more valuable prawns. Macrobrachium are generally cultured in freshwater and their production in saline groundwater was not well understood. Laboratory and pilot-commercial scale experiments in ponds demonstrated that Macrobrachium grew well in raw saline groundwater up to 4 ppt, and production of approximately 1200 kg/ha was reliably achieved after 100 day grow-out. However, at salinities of 5–10 ppt, performance of Macrobrachium in raw saline groundwater was reduced and this was caused by low potassium concentration. The addition of potassium to the groundwater to provide 50–100 % of that found in similar salinity seawater significantly improved growth. Investigations at the Rohtak Centre are now concentrating on evaluation of the benefits of polyculture of Macrobrachium with carp species compared with prawn monoculture.

36.5.3 Case study: Israel, Desert aquaculture The availability of freshwater in Israel is limited due to climatic conditions with rainfall usually only in the winter and mainly in the northern and central areas of the country (Table 36.4). Though national projects have been undertaken to ease this shortage and to achieve a more rational water distribution and management policy throughout the country (Fig. 36.5) due to the continued water shortage, agriculture and aquaculture have been developed to high standards in terms of output per unit of water used. Fish farming in the country began in the 1940s. Today aquaculture is practised in 73 Kibbutz farms located mostly in the northern and central parts of the country. Total annual production, including the catch in the Sea of Galilee, the only freshwater lake in the country, amounts to ∼25 000 tonnes. The main species of fish produced are tilapias, carps, mullet and

1136

New technologies in aquaculture

Table 36.4

Israel’s freshwater resources and average annual use

Resource Coastal aquifer Mountain aquifer Sea of Galilee and the Jordan River system West Galil aquifer Carmel aquifer Negev and Arava aquifer Total potential of replenishable water

Potential utility (million m3)

Addition potential (million m3)

250 600 640 170 40 55

90 million lost into the Dead Sea 55 million diverted to Jordan

1755

Treated sewage

270

Total potential

2025

Total

2400

145 230 million lost into the Mediterranean 375

trout, reared in earthen ponds and reservoirs filled with spring and rain water. The total annual fish consumption in Israel has reached over 75 000 tonnes (nearly 11 kg per capita). One third of this comes from local production and catch and two thirds are imported. Consumption forecasts for the years 2010 and 2020 are 86 000 tonnes and over 100 000 tonnes, respectively, demanding an increase in production and importation. Israel has to continue developing and expanding aquaculture to meet its increasing demand for fish. However, the continuous domestic freshwater shortage in Israel (Table 36.4) and its high costs, accompanied with intensive urbanisation and rising land prices, particularly along the Israeli coastal belt where fish farms have been traditionally operating, limit the expansion of aquaculture, highlighting the need for alternative water resources and sites. Two thirds of Israel is covered by desert, an area of ∼13 000 km2 which receives an annual rainfall of only 60–100 mm and is inhabited by just 2–3 % of the population. Shortly after the establishment of the state of Israel (1948), a 100 m deep drilling project, in search for freshwater in the Negev Highland district, the largest district of the Negev desert, was undertaken with disappointing results. However, in the late 1950s, further drilling for freshwater down to a depth of 1000 m led to the discovery of the ‘Nubian Sand Stone’ (300 m and below) and the ‘lower Cenomanian Turonian’ (800–1000 m) aquifers, containing huge reserves of billions m3 of unpolluted, brackish geothermal water – the ‘desert water’. In the Negev Highland district there are at present eight combined wells (600–750 m deep) supplying 6.3 million m3 of brackish geothermal

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Mediterranean Sea West Galil Aquifer

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Sea of Galilee System Sea of Galilee

Hiafa Carmel Aquifer

Schem - Gilboa Aquifer

Tel Aviv Jordan River Jerusalem System Mountain Aquifer

Coastal Aquifer

Dead Sea

Beer Sheva

Arava Aquifer Negev Aquifer

Eilac

Fig. 36.5 The national water carrier: water transfer from wet to arid areas.

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New technologies in aquaculture Table 36.5 Mineral composition of desert brackish geothermal water compared with seawater and freshwater

pH EC mS Cl mg/L SO4 mg/L NO3 mg/L Br mg/L NH4 mg/L K mg/L Ca mg/L Mg mg/L Na mg/L HCO3 mg/L F mg/L Sr mg/L Fe mg/L Mn mg/L Ba mg/L SiO2 mg/L P mg/L Al mg/L Cu mg/L TOC TDS mg/L

Brackish water

Seawater

Freshwater

7.25 4.43 1225 480

– – 19 000 885 – – – 380 400 1350 10 500 – – – – – – – – – – – –

7.45 0.988 128 58.5 16.5 ND 0.144 5.54 64.3 27.9 85.0 260 – – – – – – – – – – 646

– – – 27 172 92 700 189 1.19 5.34 ND ND 0.03 17.04 ND 0.06 ND 6.00 ∼2919

ND = not determined; TDS = total dissolved solids; TOC = total organic carbon.

(38–40 °C) ‘desert water’ per annum to the farms of five major settlements in this district of ∼400 000 ha, with a population of about 5000 settlers in total. For the last 40 years this brackish water (3–7 ppt, TDS) has been successfully used for irrigation of agricultural crops and, since the late 1980s, has also been in use for aquaculture. This ‘desert water’, though resting deep in the ground (400–1000 m), is easily accessible as it rises by artesian pressure to nearly sea level and has just to be further raised to ground level from an average suction depth of about 400 m. The fact that this water is available at warm temperatures all year round (around 40 °C when reaching the surface) provides an excellent and realistic potential for the expanding national fish farming industry. Many studies have proven that growth rate, metabolic rate, feed intake, feed conversion and survival in fish are influenced largely by water salinity. This subsurface ‘desert water’, with a salinity ranging from 3–7 ppt (TDS) (Table 36.5), has been found most suitable for fish culture and is currently in use for raising freshwater and marine fish species in the Israeli desert. Species cultured include: tilapia (Oreochromis

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niloticus); barramundi (Lates calcarifer); gilthead sea bream (Sparus aurata); hybrid striped bass (Morone saxsatilis x M. chrysops); red drum (Sciaenops ocellatus); and catfish (Clarias gariepinus). Desalination of seawater or brackish water is one of the major steps towards combating the severe water shortage in Israel. Brine resulting from the process of desalination of seawater is easily returned to the sea. However, brine from desalination of inland water away from the sea is disposed of by using evaporation ponds. The desalination plant in Ramat Negev district, near the Israeli–Egyptian border, produces 3.5 millions m3 of freshwater annually, while producing as a by-product thousands of m3 of brine. This brine can be used for aquaculture, being particularly suitable for the reproduction of aquatic species that can grow in low-saline water but only breed in water of higher salinity such as seawater. Fish farming in the desert benefits from the following significant advantages: • the existence of large amounts of accessible subsurface unpolluted brackish geothermal water, the so-called ‘desert water’; • the moderate salinity of the water provides an osmoregulatory advantage for a number of species; • the geothermal ‘desert water’ provides constant temperatures favourable for faster fish growth and more economic production; • the expense to the contrary of the ‘desert water’ is lower than that of freshwater in the country; • land for aquaculture in the desert is readily available and much more accessible compared with the high priced land in other regions of the country; • the pollutant-free ‘desert water’ has the unique potential of yielding high quality fish products for marketing. Culture of ornamental fish has gained enormous popularity worldwide and interest appears to be continuously growing, making it potentially a very profitable global component of international trade worth more than US$10 billion annually. The culture of ornamental and tropical hobby fish in Israel for export started during the 1970s and is expanding due to continuous high demand. Today there are about 20 tropical fish farms, most of which are located in the Israeli desert area. The size of the farms is typically from 0.1–0.3 ha, each farm operating in separate hot-houses isolated from the others, with no common water system. Among the major ornamental fish species cultured in the desert farms in Israel are: guppy (Poecilia reticulate); platy (Xiphophorus maculates); swordtail (Xiphophorus helleri); angelfish (Pterophyllum scalare); and catfish (Corydoras). The routine use of saline groundwater for agricultural irrigation, together with the realisation that commercial production of fish reared in saline water in ponds and hothouses is not only feasible but also economical, has enabled Israeli fish farms in the desert to successfully combine aquaculture

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and agriculture into integrated farming systems in the sense that the effluent from fish ponds, rich with organic waste, is used for field and orchard irrigation making more rational use of the ‘desert water’. There are currently about 15 commercial fish farms operating in the Israeli desert which produce edible and ornamental fish as well as ornamental crustaceans (see example of a typical desert fish farm; Fig. 36.6). All edible fish that are produced in the desert are sold on the domestic market while most of the ornamental fish produced are exported. In order to expand its aquaculture activities it has become necessary for Israel to increase the use of available marginal water, i.e. existing brackish ‘desert water’, as well as desalinated sea and brackish water. Expansion of aquaculture in the Israeli Negev desert, adapting and developing technologies for intensive fish culture, especially in integrated operations with agriculture, is a matter of necessity. Thus the intensive utilisation of brackish geothermal water in the Israeli desert for integrated agriculture/aquaculture is enabling the continued expansion of Israel’s aquaculture industry while significantly easing the pressure on Israel’s scarce freshwater resources. Figure 36.7 shows the distribution of brackish geothermal water collected from several bores from a well head. The bulk of the brackish water flows directly to a reservoir, but part of the 40 °C brackish water goes directly to heat greenhouses before exiting to the reservoir. Another part flows to covered fish ponds providing clean and warm brackish water on demand. The fish ponds and the reservoir form a recirculation water system that provides a growing environment for fish culture. Freshwater from the reservoir and the effluent-rich water from the fish ponds is used for irrigation of open fields and orchards. Experiments for the use of saline water for agricultural irrigation began in the late 1940s. Ongoing research has led to widespread use of ‘drip irrigation’ systems and to the current use of saline water for irrigation. The use

Fig. 36.6 Typical Israeli desert fish farm for edible fish.

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Well head brackish geothermal water ~40°C

Geothermally heated greenhouses

Cattle

Effluent enriched water

Alfalfa

Fig. 36.7

Covered fish ponds

Water reservoir

Selected vegetables

Pomegranates Olives

Layout of integrated desert aqua/agriculture operation.

of saline water for commercial production of wheat, cotton and fodder began in 1972. Since then, sweet tomatoes, onions, sweet potatoes, alfalfa, jojoba, Salicornia, pomegranates and olives are also being produced with saline water. Recently saline water is being used for irrigation experimentally in vineyards and for growing almonds. Research programs which have been carried out at the Bengis Center for Desert Aquaculture (The Albert Katz Department of Dryland Biotechnologies of the Institutes for Desert Research at the Ben-Gurion University of the Negev) have proved that this brackish ‘desert water’, due to its moderate salinity, mineral composition, constant warmth, purity and availability regardless of the weather conditions, is highly advantageous for culturing warmwater aquatic species (e.g. tilapia; Fig. 36.8). Desert aquaculture is not a technological revolution; it is rather an innovative approach that differs from conventional fish farming. Arid or desert lands with subsurface water resources have huge potential for developing and sustaining aquaculture and agricultural products. Research findings continue to show that the possibility of using inland brackish water for farming aquatic species is a promising realistic alternative to many of the traditional operations. Further development of Israeli aquaculture will have to go hand in hand with the expansion of the existing domestic desert aquaculture. Technologies applied in arid or desert lands must minimise negative impacts on the unspoiled environment and should maximise the preservation of the land as well as the efficiency of water use. This can ideally be achieved by integrating aquaculture with agriculture, allowing the conservation of water through the expansion of the chain of users utilising the same

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Fig. 36.8 Happy red tilapia in an Israeli desert fish farm.

water source. The steadily growing consumer market for high-quality aquaculture products and the vast amounts of unpolluted brackish geothermal water accessible beneath the Israeli desert suggest the production of thousands of tonnes of fish and other aquatic organisms in the Israeli desert to be a reality forecast for the not too distant future. In the Israeli aquaculture development and expansion, the Negev Desert, associated with and guided by local applied research, will hold an increasingly dominant position (Applebaum, 1995, 1998).

36.5.4 Case study: USA, inland marine shrimp aquaculture Inland culture of marine shrimp in the USA began in the 1990s in two parallel activities. First, as a specialty enterprise devoted to production of pathogen free (SPF) broodstocks. Second, as commercial farms for grow-

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out of penaeid shrimp for domestic markets. Early SPF broodstock efforts were in cooperation with the University of Arizona with small projects, mostly in conjunction with tilapia farms using saline groundwater along the Salton Sea in Southern California and over saline aquifers in Southwestern Arizona. These efforts, while technically successful, led to only minor commercial activity selling SPF broodstock to hatcheries in the USA and abroad. Also in the 1990s, commercial activity began on production farms in the Pecos Valley of West Texas, in western Alabama, in Southern Florida and in the Hyder Valley of Southwestern Arizona. The goal of these farms was to produce high-quality marine shrimp for US consumer markets. By targeting a premium market with locally grown shrimp, the farmers hoped to cover the higher costs of production in the USA compared to imported shrimp. In practice, the farms have managed to develop niche markets to sell most of their shrimp. These include sales of 5–10 g shrimp to large public aquaria to feed display animals or to fishermen for bait, 15–20 g live shrimp to Oriental restaurants to put in live tanks for diners’ selection and, finally, 15–20 g, fresh and frozen shrimp for on-farm or direct delivery to local restaurants. At one time in the early 2000s there were at least two farms in Alabama, two in West Texas, four in Arizona and one in Florida. By 2008, only one farm in Alabama and one in Arizona appeared to be operating. The low prices of foreign shrimp and limited market appeal for higher cost domestic shrimp appeared to be the greatest obstacle. High labour and water costs were major contributors along with the higher than expected capital and operating costs. One of the major expenses was the costly acclimation procedure to adapt PLs to the salinity levels found on most of the inland farms. PLs typically arrived at the farms in 30 ppt water while the local aquifer water tends to be 2–5 ppt. At most of the farms, a recirculating water system was constructed to receive PLs and hold them while they are fed and farm water is added to the high-salinity water in which they arrived. The acclimation process often covered 2–4 weeks before the animals were fully acclimated and could be stocked into local ponds or raceways with saline groundwater. Other significant costs were adjustments to the feed and/or water amendments to account for differences in mineral constituents between the groundwater and oceanic water. The groundwaters used at the farms, while saline, usually had ratios of mineral salts that were somewhat different from the ratios in marine waters. In some instances, changes were made in the diets, especially mineral premixes to improve shrimp growth and survival (Gong et al., 2004). In other cases, it was easier to add commercially available minerals in bulk to the water (McIntosh and Fitzsimmons, 2003; Boyd et al., 2006). Once the shrimp are acclimated to the local low-salinity waters and stocked into the grow-out units, growth to harvest size of 15–25 g typically occurs over 3–5 months. At some farms, effluents are reused to irrigate salt-tolerant crops including olives, dates and

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Fig. 36.9 Effluent to olives.

wheat (Fig. 36.9). In other instances the saline effluent is blended with low-TDS water from adjacent wells to irrigate less salt-tolerant crops (McIntosh et al., 2003).

36.6 Future trends Inland saline aquaculture encompasses a number of culture species, systems and water types. The potential for expansion of these production systems is almost unlimited. With the increasing demands on potable water and marine coastal locations, use (or re-use) of inland saline waters provides a critical resource for high-quality seafood production using otherwise unproductive, or even detrimental, resources. Integration of aquaculture with conventional, halophytic or even seaweed culture would further increase the efficiency and sustainability of these food production systems (Riley et al., 1997; Brown et al., 1999). Basic and applied research into practical management systems for these systems is rapidly providing us with the knowledge of how to turn these into profitable farming ventures. Additional research into the physiological stresses of rearing more valuable species in inland saline systems and practical integration of aquaculture effluents for plant crop production are high priorities. The food and fuel crises of 2008 will inevitably be followed by another water crisis, impacts of climate change on conventional crops, fertilizer shortages and other consequences of increasing populations competing for limited resources. Novel food production methods are needed to further improve global wellbeing and inland saline aquaculture is bound to be a most valuable tool.

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36.7 References agarwal mc and roest cjw (1996) Towards improved water management in Haryana State, Final report of the Indo-Dutch operational research project on hydrological studies, Hisar, Haryana. allan gl, banens b and fielder ds (2001) Developing commercial inland saline aquaculture in Australia: Part 1 R&D Plan, Final report to FRDC Project No. 98/335, NSW Fisheries Final Report Series No. 31, NSW Fisheries, Cronulla, NSW. allan gl, blackburn j and fielder ds (2008a) Toward commercialisation of inland saline aquaculture in the Murray Darling Basin, Skretting Australasian Aquaculture Conference, 3–6 August, Brisbane. allan gl, heasman h and bennison s (2008b) Development of industrial-scale inland saline aquaculture: coordination and communication of R&D in Australia, Final report to FRDC Project No. 2004/241, NSW Department of Primary Industries – Fisheries Final Report Series No. 100, Cronulla, NSW. anon. (2001) Dryland salinity in Australia: A summary of the National Land and Water Resources Audit – extent impacts, processes and management options, Australian Natural Resources Atlas, Department of the Environment, Water, Heritage and the Arts, Canberra, http://www.anra.gov.au/topics/salinity/pubs/ national/salinity_summary.html, accessed January 2009. anon. (2004) Coal seam gas (CSG) water management study, Australian Department of Natural Resources, Mines and Energy, Contract NROOO11, Parsons Brinkerhoff, Brisbane, QLD. appelbaum s (1995) Technology for desert aquaculture, Journal of Arid Land Studies, Abstract No. 5S, 207–10. applebaum s (1998) Desert aquaculture – a new opportunity for world aquaculture production, Journal of Arid Land Studies, Abstract No. 7S, 101–3. barman uk, jana sn, garg sk, bhatnagar a and arasu art (2005) Effect of inland water salinity on growth, feed conversion efficiency and intestinal enzyme activity in growing grey mullet, Mugil cephalus (Linn.): field and laboratory studies, Aquaculture International, 13, 241–56. boyd ca, boyd ce and rouse db (2006) Water quality issues related to inland shrimp farming in Alabama (USA), Skretting Australasian Aquaculture Conference, 27–30 August, Adelaide, SA. boyd ce and thunjai t (2003) Concentrations of major ions in waters of inland shrimp farms in China, Ecuador, Thailand, and the United States. Journal of the World Aquaculture Society, 34, 524–32. brown jj, glenn ep, fitzsimmons km and smith se (1999) Halophytes for the treatment of saline aquaculture effluent, Aquaculture, 175, 255–68. cheng km, hu cq, liu yn, zheng sx and qi xj (2005) Dietary magnesium requirement and physiological responses of marine shrimp Litopenaeus vannamei reared in low salinity water, Aquaculture Nutrition, 11, 385–93. doroudi ms, fielder ds, allan gl and webster gk (2006) Combined effects of salinity and potassium concentration on juvenile mulloway (Argyrosomus japonicus, Temminck and Schlegel) in inland saline groundwater, Aquaculture Research, 37, 1034–9. doroudi ms, webster gk, allan gl and fielder ds (2007) Survival and growth of silver perch, Bidyanus bidyanus, a salt-tolerant freshwater species, in inland saline groundwater from southwestern New South Wales, Australia, Journal of the World Aquaculture Society, 38, 314–17. dutney l, burke m, willet d and collins a (2008) Evaluation of the potential for aquaculture in cotton catchments using coal seam gas water. Skretting Australasian Aquaculture Conference, 3–6 August, Brisbane.

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fao (2007) The State of World Fisheries and Aquaculture 2006, Rome, Food and Agriculture Organization of the United Nations. fielder ds, bardsley wj and allan gl (2001) Survival and growth of Australian snapper, Pagrus auratus, in saline groundwater from inland New South Wales, Australia, Aquaculture, 201, 73–90. fitzsimmons km (1988) Status of aquaculture in the state of Arizona, Geothermal Heat Center Bulletin, 11(1), 23–4. flowers tj and hutchinson w (2004) Preliminary studies towards the development of an aquaculture system to exploit saline groundwater from salt interception schemes in the Murray Darling Basin, SARDI Aquatic Sciences, Adelaide, SA. forsberg ja and neill wh (1997) Saline groundwater as an aquaculture medium: physiological studies on the red drum, Sciaenops ocellatus, Environmental Biology of Fishes, 49, 119–28. gong h, jiang dh, lightner dv, collins c and brock d (2004) A dietary modification approach to improve the osmoregulatory capacity of Litopenaeus vannamei cultured in the Arizona desert, Aquaculture Nutrition, 10, 227–36. hutchinson wg (2008) SARDI – Application for demonstration facility and summary of R&D progress, in Allan GL, Heasman H and Bennison S (eds), Development of Industrial-scale inland saline aquaculture: Coordination and communication of R&D in Australia, Final Report to FRDC Project no. 2004/241, NSW Department of Primary Industries – Fisheries Final Report Series 100, Cronulla, NSW, 38–52. ingram ba, mckinnon lj and gooley gj (1996) Growth and survival of selected aquatic animals in two saline groundwater evaporation basins: an Australian case study, Aquaculture Research, 33, 425–36. johnston b (2008) Profit model consultancy: Economic models for inland saline aquaculture of finfish, prawns and recirculation culture, in Allan GL, Heasman H and Bennison S (eds), Development of industrial-scale inland saline aquaculture: Coordination and communication of R&D in Australia, Final report to FRDC Project No. 2004/241, NSW Department of Primary Industries – Fisheries Final Report Series 100, Cronulla, NSW, 191–210. lambers h (2003) Dryland salinity: A key environmental issue in southern Australia, Plant and Soil, 257, v–vii. mcintosh d and fitzsimmons k (2003) Characterization of effluent from an inland, low-salinity shrimp farm: what contribution could this water make if used for irrigation, Aquacultural Engineering, 27, 147–56. mcintosh d, fitzsimmons kaguilar j and collins c (2003) Toward integrating olive production with inland shrimp farming, World Aquaculture, 34(1), 16–20. pal s, sharma br and parshad r (1999) Social audit on reclamation of salt affected soils in India, Indian Council of Agricultural Research, New Delhi. partridge gj and creeper j (2004) Skeletal myopathy in juvenile barramundi, Lates calcarifer (Bloch), cultured in potassium-deficient saline groundwater, Journal of Fish Diseases, 27, 523–30. partridge gj and lymbery aj (2008) The effect of salinity on the requirement for potassium by barramundi (Lates calcarifer) in saline groundwater, Aquaculture, 278, 164–70. partridge gj, lymbery aj and george rj (2008) Finfish mariculture in inland Australia: A review of potential water sources, species, and production systems, Journal of the World Aquaculture Society, 39, 291–310. prangnell di and fotedar r (2005) The effect of potassium concentration in inland saline water on the growth and survival of the western king shrimp, Penaeus latisulcatus Kishinouye, 1896, Journal of Applied Aquaculture, 17, 19–33. prangnell di and fotedar r (2006) Effect of sudden salinity change on Penaeus latisulcatus Kishinouye osmoregulation, ionoregulation and condition in inland

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saline water and potassium-fortified inland saline water, Comparative Biochemistry and Physiology A-Molecular & Integrative Physiology, 145, 449–57. qureshi rh and barrett-lennard eg (1998) Saline Agriculture for Irrigated Land in Pakistan: A Handbook. Monograph No. 50, Australian Centre for International Agricultural Research, Canberra. riley jj, fitzsimmons km and glenn ep (1997) Halophyte irrigation: An overlooked strategy for management of membrane filtration concentrate, Desalination, 110, 197–211. roy la, davis da, saoud ip and henry rp (2007) Effects of varying levels of aqueous potassium and magnesium on survival, growth, and respiration of the Pacific white shrimp, Litopenaeus vannamei, reared in low salinity waters, Aquaculture, 262, 461–9. saoud ip, roy la and davis da (2007) Chelated potassium and arginine supplementation in diets of Pacific white shrimp reared in low-salinity waters of west Alabama, North American Journal of Aquaculture, 69, 265–70. sowers ad, tomasso jr, browdy cl and atwood hl (2006) Production characteristics of Litopenaeus vannamei in low-salinity water augmented with mixed salts, Journal of the World Aquaculture Society, 37, 214–17. shakeeb ur r, jain ak, reddy ak, kumar g and raju kd (2005) Ionic manipulation of inland saline groundwater for enhancing survival and growth of Penaeus monodon (Fabricius), Aquaculture Research, 36, 1149–56. spotte s (1979) Fish and Invertebrate Culture: Water Management in Closed Systems, Wiley, Chichester. tantulo u and fotedar r (2006) Comparison of growth, osmoregulatory capacity, ionic regulation and organosomatic indices of black tiger prawn (Penaeus monodon Fabricius, 1798) juveniles reared in potassium fortified inland saline water and ocean water at different salinities, Aquaculture, 258, 594–605. trendall j (2008) Inland saltwater aquaculture saltwater trout: a case study in supply chain development, in Allan GL, Heasman H and Bennison S (eds), Development of industrial-scale inland saline aquaculture: Coordination and communication of R&D in Australia, Final Report to Fisheries Research and Development Corporation Project No. 2004/241, NSW Department of Primary Industries – Fisheries Final Report Series No. 100, Cronulla, NSW, 191–210. veil ja, puder mg, elcock d and redweik rj (2004) A White Paper Describing Produced Water from Production of Crude Oil, Natural Gas, and Coal Bed Methane, prepared by Argonne National Laboratory, Argonne, Illinois for the U.S. Department of Energy, National Energy Technology Laboratory. zhu cb, dong sl, wang f and zhang hh (2006) Effects of seawater potassium concentration on the dietary potassium requirement of Litopenaeus vannamei, Aquaculture, 258, 543–50.

37 Urban aquaculture: using New York as a model M. P. Schreibman and C. Zarnoch, City University of New York, USA

Abstract: Here we discuss the major ramifications of urban aquaculture development using New York City as a model and template. We define the parameters essential for successful aquaculture, as well as reflecting on the problems that we face as we move urban aquaculture from concept to practice and to ultimate success. We envision closed, water-reuse systems (recirculating aquaculture systems; RAS) as key to environmentally responsible, sustainable, intensive, and economically feasible aquaculture in metropolitan areas. We suggest that research must may play a major role in the development and application of technology, biological principles, and socioeconomic feasibility, each one of which is an important entity for successful urban aquaculture development. Furthermore, the future success of urban aquaculture is likely to depend upon support in the form of grants and subsidies, research, and access to capital. Key words: New York City, recirculating aquaculture systems, environmentally responsible, metropolitan area, technology.

37.1 Introduction The United Nations estimates that by 2025 the urban population of the world will increase to 5.1 billion people – the population size of the entire Earth in 1930 (Costa-Pierce and Desbonnet, 2005). It is also estimated that by the year 2020, 80 % of the population of the USA will live in a coastal city or within a one hour drive of an ocean or bay. There will be demand for aquaculture to supply 49 million Mt of product (16 million Mt higher than the 1999 levels) to maintain the current per capita consumption for a projected world population of 6.8 billion people in 2010 for a total demand of 142 million Mt (Timmons, 2005). These projections, coupled with an increasing demand for high-quality protein derived from seafood products, the drastically dwindling supplies of our natural fishing stores. The socioeconomic issues spawned by growing metropolises around the world,

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such as ecosystem stability, employment, and fuel costs will demand new approaches to address these issues. What novel technologies will enable intensive, sustainable production of aquatic organisms in populous areas? Urban aquaculture can respond to these needs using the water-reuse systems that have been developed and refined during recent years, coupled with responsible ecosystem management and creative economic planning. The term aquaculture need not be defined for the audience that will study the contents of this book. The term ‘urban’, however, may need some discussion. Our perception of what is urban and what is rural would no doubt be influenced by where we live – the new world, the old world, or in developing countries. In their discussion of what is meant by ‘urban’, Little and Bunting (2005) suggest that rural areas are those that are under agriculture, forest and woodland, and wild tracts of land that have never been manipulated. They conceptualize that urban areas are characterized by nucleated settlements, labor organizations, and markets and with connecting links. Thus, urban centers serve as major distribution hubs with an available labor force and potential sources of investment capital. While ‘urban’ centers may differ around the world, they are generally characterized by larger numbers of consumers who want high-quality, low-cost and diversified food sources, which are free from pollutants and chemical contamination. In our discussion, we will use the term ‘urban aquaculture’ to mean the rearing of aquatic organisms under controlled conditions in or near a populous area. These organisms could be consumed, adored as pets, used for research, used for stock enhancement, or used as bait by anglers. Conducting aquaculture in an urban environment has many incentives. In this chapter, we have chosen to use New York City as a template to discuss the scope, promises, and problems of urban aquaculture, knowing full well that New York is a unique environment. New York City typifies the characteristics needed for successful aquaculture but it also demonstrates the many problems that we face as we move urban aquaculture from concept, to practice, and to ultimate success. A New York aquaculture industry is feasible, profitable, and long overdue. Why choose New York City as model for urban aquaculture? The people of New York eat considerably more seafood than the US norm (Timmons et al., 2001). New York City serves as a large distribution hub for fish and aquatic products from around the country and the world. Unfortunately, little product is grown locally. The Hunts Point Market in the borough of the Bronx accounts for about one-third of the value of all wholesale seafood activity in the entire state (Sea Grant, 2001). The waters in proximity to New York City are home to many novel species for potential aquaculture development. New York City also has a large multicultural population for consuming these new products. In addition, there are a wide variety of ethnic neighborhoods in dynamic flux with distinctive seafood tastes and needs that are currently not being satisfied (Schreibman and Zarnoch,

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2005). Many of these features, increased demand for seafood, ability to generate product in great quantities in restrictive and limited space and where land and water are limited, have led to successful ventures in urban aquaculture in South and Southeast Asia, India, Vietnam, and select cities in the Americas. Uses of urban waters, sitings to dense population areas, seeking solutions to urban sanitary problems, and the need to improve economics have been some of the stimuli for success. Among the many parameters essential to an urban aquaculture industry, New York City provides more than 580 miles of land–water interface that can provide a network of water passages, in addition to the extensive rail and highway routes, for product distribution. New York City is central to a distribution network that stretches through bordering counties and neighboring states – all within a ‘few hours’ drive. New York’s superb interstate roadway system links suburban markets closely to New York through a large trucking and transportation industry. With two international airports and two major national airports in proximity with the inherent major freight businesses located on or near their properties, fresh cargo can reach remote markets quickly. New York’s port serves to transport properly packed cargo to other port cities. However, New York City experiences, as do many cities, blatant needs to improve its economic climate by developing new industries, creating jobs, and providing training, especially for socially disadvantaged and economically challenged citizens. Similarly, a vibrant aquaculture industry could help along these lines in many parts of the world. The USA imported more than $10 billion of seafood at the turn of this century. The total trade deficit in seafood, according to the US Department of Commerce is $6.2 billion. New York City relies heavily on aquaculture product imports from elsewhere in the nation and the world. In 1999, the largest source of fish and seafood purchased by the New York seafood industry was imports from outside the USA. The state’s seafood industry and others purchased an estimated $786 million worth of fish and seafood products from foreign sources. Shrimp, almost all of which is frozen, accounted for 42 % of the value of fish and seafood imported to New York in 1999. The New York seafood industry purchased an estimated $535 million worth of fish and seafood products from sources in other states in 1999. This is in addition to purchases from other countries. Even in a depressed economy there is an ever-growing plethora of restaurants which makes the greatest economic contribution from among the seafood consuming industry segments. This contribution is attributable to the substantial value added by restaurants to the fish and seafood products they purchase and from the great number of jobs generated in restaurants (Sea Grant, 2001). New York City and other metropolitan areas worldwide are not generally short on restaurants! Major universities are needed to participate in this growing industry by conducting research and development of the essential technologies,

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providing professional expertise and training opportunities, arranging for community outreach, and developing a strong aquaculture curriculum at all levels of education. Our own aquaculture programs and the use of fish in basic and applied science have been based at Brooklyn College, the City University of New York for more than 40 years. In more recent years we have been able to expand our aquatic research activities, especially those related to aquaculture and to the environment, by creating a two million dollar center of excellence, the Aquatic Research and Environmental Assessment Center (AREAC). Universities and other research and educational agencies can play a vital role in developing a sustainable urban industry, especially since private venture is unlikely or unwilling to sustain their own R & D programs.

37.2 Goals There are several vital components to be considered if successful urban aquaculture is to be achieved. Generally, success is measured by the production of a product that is in demand and one that generates a positive cash flow. The driving goal should be to create these products through environmentally responsible, sustainable practices. More specifically, the several key issues that need to be addressed for attaining success include determining when to start (acquiring the capital and gathering knowledge and support teams for both the aquaculture and the commerce components), assessing the technology to be employed, siting (where to place the facility), selecting the best candidates to grow, and determining how to market your product. It would be foolhardy not to be thoroughly educated in each of these areas. In addition, establishing collaborations with leading experts to help with biological aspects, business plans, engineering design, and implementation is essential. Timmons et al. (2002) reviewed the failure of several aquaculture ventures in the USA and noted several commonalities. These included a lack of experience with indoor production, labor-intensive technology, poor engineering, choice of sensitive species, and a lack of commitment from investors. This clearly reiterates our point in that a new aquaculture venture must recognize and address all of the major issues. Bioshelters, Inc., located in Amherst, MA, USA, is an example of an aquaculture venture that appears to have addressed all of these issues and enjoys success. Bioshelters is an integrated production system that grows basil along with tilapia in a closed loop RAS (termed ‘aquaponics’). The key principals of the company include experts in aquaculture production and economics as well as experts in marketing and sales. This combination of expertise has led to creative and effective marketing that creates demand for a high-quality product reliably and sustainably produced in their

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integrated system. Further innovative use of energy, labor, and space has also led to their success (www.bioshelters.com).

37.3 Technology We are strong proponents, with an ever-growing band of supporters, of indoor, water-reuse systems – the so called ‘recirculating aquaculture systems’ (RAS). (The reader is referred to Timmons et al., 2002 and Losordo, this volume) for an in-depth discussion of the design and utilization of RASs) Recirculating aquaculture systems hold the key to the economic success of urban aquaculture for they permit high-density growth of aquatic organisms in facilities that could be located in most parts of the city, including low value real estate value areas and brownfields. Essential to its utility is that RAS are environmentally friendly, adding only minimal amounts of water as a by-product of the production process. RAS may take different forms to accommodate different species and most phases of their life cycle, from spawning to preparation for market. The following lists the most significant advantages of RAS that dictate their use: • closed systems facilitating the control of disease, eliminating predators, and making them biosecure; • flexible in design to accommodate different organisms; • self-cleaning because of their circular design and hydrodynamic properties; • easy to monitor and maintain; • permit high-density growth and intensive stocking of fish; • expandable; • guarantee safety and quality of fish; • useful and uniform production all year through controlled environment; • environmentally friendly; • use municipal or potable water; • facilitate disease management; • require less land and water than extensive or semi-intensive aquaculture (conventional tilapia aquaculture requires 500 times more land area and 1000 times more water per unit of fish than a RAS-produced fish). Consumers have become sensitized to environmental and health concerns related to finfish production due to aggressive media coverage. Small producers in foreign countries, extensive or semi-intensive aquaculture practices, are difficult to regulate and no predominant player has emerged to set standards for healthy, sustainable seafood production and processing. And yet, there is a trend in the USA in retail trade towards healthy, environmentally safe, and organic foods that are produced under very strict standards. This provides a prime opportunity for future aquaculture

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endeavors to provide a certified healthy food product through the use of the RAS.

37.4 Potential urban aquaculture programs Urban aquaculture programs could develop along several lines that include finfish for food, ornamental organisms for hobbyists, the production of organisms used in research, and the rearing of baitfish. The market and the geographic location and structural flexibility of your facility will dictate your choice of aquaculture program and candidate species. Potential candidates come from a variety of water types (e.g., salt, fresh, and brackish water) and extremes of water temperatures. Commercial entities will likely decide on their program and candidate species early in the planning and development process, for the luxury of converting from one production system to another is prohibitive in cost. 37.4.1 Finfish for consumption The number of potential candidates for culture has increased significantly in recent years for both fresh and marine species, driven by consumer demand, profit margins, and development of new technologies. This has led to successful culturing of marine species, even exotic ones, with more rewarding market prices than the traditional freshwater species (Zohar et al., 2005). AREAC’s finfish aquaculture activities have varied over time as dictated by our specific research and development programs and have included the commercially important walleye (Stizosledion vitreum), summer flounder (Paralichthys dentatus), winter flounder (Pseudopleuronectes americanus), and tilapia (Oreochromis niloticus). Our most intensive culturing program, and one that is currently drawing major commercial attention, is the grow-out of tilapia – a highly recommended candidate for urban aquaculture. Focusing on a single species has distinct advantages since genetic and nutritional research programs can be directed more sharply. Fingerlings obtained from a local producer are brought into our facility at approximately 1–3 g in weight. These fingerlings, reared in a 10 m3 fiberglass tank, can be brought to market size (approximately 600 g) after 6–7 months of culturing. Tilapia are fast growers, resistant to disease, tolerant to over-crowding, highly compatible, and excel in activity and growth in RAS. Commercial entities could and do set up their own nurseries to control production which can reduce costs. Tilapia are prolific and easy to produce, with low mortality. Genetic programs are available to produce mainly male offspring – the desired gender for this industry – thus skirting the need for treatment of fry with sex steroids to produce the same desired end results. The restrictive aspects of maintaining a hatchery include the necessity of additional dedicated facilities, contending with another step in

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the life cycle and, most significantly, obtaining and maintaining a genetically reliable and preferred broodstock (i.e., a selective breeding program). It is our belief that tilapia, coupled with RAS technology, could be the keystone to the successful establishment of a major tilapia urban aquaculture industry, one billion pounds per year, in New York State (Timmons et al., 2001). The intention of this effort is to make tilapia the ‘poster fish’ for New York State urban aquaculture in very much the same way that salmon and catfish are identified with certain regions of the USA. The global seafood market is large and growing. The worldwide retail market is $200B and the US share is $27B. Within this market, tilapia sales from producers to distributors alone are estimated at $350M, with the fresh market being $150M. Retail sales of tilapia are at least double this amount. The market opportunity for tilapia is robust; consumption is growing at 35 % per year, with the fresh portion growing at a rate of 21 % annually. Tilapia is already the 3rd or 4th most consumed fish in the USA and growing, up from relative obscurity several years ago. A New York tilapia industry can model itself after the successful catfish industry in the southern USA. The catfish industry has developed a 273 million kg per year production base since the 1990s, adding nearly 46 million kg of production in just the last two or three years. In large measure, the catfish industry was patterned after the chicken broiler industry. The success of both these industries is attributed to their vertical integration of breeding, growing, processing, and distribution operations under a single business structure (Timmons, 2005). The success of the ‘tilapia plan’ is augmented by the use of RAS. In contrast to outdoor pond and net-pen systems, indoor fish production using RAS is sustainable, infinitely expandable, environmentally responsible, and has the ability to guarantee both the safety and the quality of the fish produced throughout the year (Timmons et al. 2001). Site selection for this type of aquaculture industry is aided by the flexibility of the RAS. These can be situated in close proximity to the market on underutilized low-value real estate such as brownfields (Schreibman and Zarnoch, 2005) and poorquality farmland (Doupé et al., 2003). Close proximity to the market will reduce transportation costs and also allow for selling a fresh product which generally demands twice the price of frozen products (Timmons et al., 2002). In addition to creating a food product, there is also the spin-off of creating a new job market. It has been estimated that an indoor aquaculture industry will create jobs in three primary areas: (i) growing the fish, (ii) processing the fish, and (iii) associated jobs including selling, general and administration, feed production, and distribution. According to available figures, each million pounds of production will require five people in the production facility – the processing facility will add an additional 10 people (Schreibman and Zarnoch, 2005). Therefore, there are 15 jobs created for every 450 000 kg of production, or 15 000 jobs will be generated by a 450 000 000 kg/year industry in New York State.

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The association of fish species with a particular country is evident at the international level. Commitment to single species has ignited the growth of economic development and employment of countries either through aquaculture or utilization of wild fisheries. Consider the development of the salmon industry in Norway and Chile, the culturing of turbot in Scotland, Patagonian toothfish in southern South America, shrimp culture in Ecuador, and the recent expansion of bluefin tuna farming in Mexico. While the growth of a single species using the technology we propose may not be applicable to urban aquaculture in some countries, the concept would be worth pursuing.

37.4.2 Ornamental aquaculture The ornamental industry presents a scenario similar to the consumable aquaculture industry – challenged natural fisheries resulting from overfishing and habitat destruction resulting from an increased demand for product by an expanding populace. The US ornamental retail aquarium market is valued at approximately $1 billion/year which demonstrates the significant market potential of these organisms. In addition, over 15 million ornamental fish are imported into the USA each month worth about $50 million/ year. Although many species are still collected from tropical lakes and streams of South and Central America, Asia, and Africa, imports are now beginning to come from aquaculture farms in Thailand, Malaysia, Singapore, Hong Kong, and Germany (Davenport, 1996; Corbin et al., 2003). In the USA, production of ornamental fish is valued at $52 million/year and most fish are produced in Florida (Chapman et al., 1994, 1997; Rowland and Cox, 2003). Research and development needs to continue in an attempt to ascertain the technical and marketing feasibility of bringing successful ornamental aquaculture ventures to non-traditional parts of the USA, most notably to New York and the Northeast, as well as to the rest of the world. The application of sustainable, ecologically responsible production of ornamental aquatic organisms by those countries that have provided them for so long would help to protect and restore their unbalanced ecosystems and increase the living standards of their people. Restoration of coral reefs, which have been so brutalized by an unregulated industry in the past, could be restored through responsible aquaculture and at the same time serve to stimulate the tourist industry, an industry that has waned in Belize and Australia because of ecosystem devastation. Highly valued ornamentals that bring very attractive market prices can be cultured in a wide range of rearing equipment. In our facility over 800 aquaria, ranging in size between 9.5 and 284 liters are used in the culture of ornamental organisms, both vertebrates and invertebrates. Some aquaria are RAS while others are balanced, static systems containing snails and aquatic plants (also of considerable market value) in addition to the fish creating a multitrophic aquaculture system. Additionally, much less space

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is required to establish a commercially successful ornamental fish entity compared to the dimensions and scope required for a viable food-fish business. Couple this with a comparison of price per kilogram of ornamental versus food fish and we can expect to see more commercial ventures springing up in the near future.

37.4.3 Aquaculture to produce research organisms AREAC has used aquatic animals in basic and applied research for almost 50 years, beginning in the early 1960s, when the live-bearing teleosts, platyfish and swordtails, were used to study such diverse physiological topics as osmoregulation, cancer, maturation, development, reproductive system structure and function, aquatic toxicology, and aging. Our program to study the impact of space travel on the neuroendocrine regulation of physiological processes, especially reproduction, reached its pinnacle on two space shuttle flights (STS-89 and STS-90) when adult and juvenile swordtails (Xiphophorus helleri) were flown in a closed equilibrated biological aquatic system (CEBAS) developed at Ruhr University in Germany (Bluem et al., 1994) in a cooperative program between NASA and DARA, the German space agency. This ‘closed equilibrated system’ could truly be called the first orbiting RAS. In more recent years, scientific research has moved away from the use of mammals as research models. Concerns of animal rights groups and more stringent guidelines for use of animals in academia and pharmaceutical companies have stimulated the search and application for unusual animal models to carry out research programs. In some cases these research organisms are grown by the investigators themselves. However, living resource centers are expensive to establish and maintain and it becomes more economical to procure these plants and animals from commercial biological supply houses. And, as we have witnessed in other areas that have depended on collection rather than culturing of aquatic organisms, depleted and challenged natural ecosystems will dictate the application of aquacultured technology to meet research demands.

37.4.4 Baitfish production The recreational fishing industry in New York State is important, thriving, and has significant economic momentum. However, New York imports almost all of the baitfish sold within its borders, importing primarily from Arkansas and Midwestern states. Precise economic data are not available, but wholesale value is estimated at $3–5 million per year; total retail sales of baitfish in the USA is around $1 billion, but farmgate sales are less than $100 million. Assuming that the size of the recreational fishery industry will remain fairly static, the potential for baitfish sales is somewhat bracketed into the $3–5 million range.

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37.5 The economics: siting, processing, and marketing for economic success Since we are proposing reliance on indoor, water-reuse systems, urban aquaculture facilities may be placed anywhere; therefore, siting need not be limited by environmental concerns or land constraints or proximity to water. However, location can have a large impact on economic viability as affected by local energy costs, a major factor for consideration in siting. To be successful, an urban aquaculture program will need to approach an energy cost of $0.02/KWH – not always easy to attain in this time of soaring energy costs and the present economic climate. Alternative sources of energy should be explored and could be used to help achieve manageable energy costs. For these reasons cogeneration plants, wastewater treatment systems, landfills, composting facilities, and producers or purveyors of manure have become popular sitings for joint ventures. Asia, which is a major contributor to the world aquaculture market, has had almost from its inception multiple roles for aquaculture in their rural and peri-rural farms. They have long accrued benefits from urban aquaculture by dealing with issues of waste disposal and treatment by coupling it to food production (Costa-Pierce and Desbonnet, 2005; Edwards, 2005; Phan Van and DePauw, 2005; Quy Hoan and Edwards, 2005). The concept of raising a single aquaculture product is being markedly modified with the introduction of the concept of coordinating the growth of multiple organisms where there was only one. This is especially attractive in the spatial confines of the metropolis. Aquaponics, the practice of growing fish and plants in the same closed system, and other multitrophic cocultured organisms, is receiving special attention. It has augmented the better known principle of hydroponics (soiless growth of plants with the addition of chemical fertilizers) which itself is now enjoying increased appreciation and application. Furthermore, the concept of expanded aquaculture systems, those that go beyond just raising fish to one that produces energy, has received special consideration. The energy generated by these systems can be combined with, and drive other, integrated food systems (as aquaponics) and even have residual energy to provide to the local community. Such ‘fuel cell systems’ (Timmons, 2005) would be well suited for large urban areas to deal with multiple municipal needs while at the same time assisting in achieving broader sustainable practices. Calculations suggest that for an urban (New York City) aquaculture entity to be highly competitive and successful it would need to produce 2 700 000 kg (of tilapia) per year. Additionally, this facility would need to be coupled to an operational and cost-effective processing plant and have direct costs of production close to $4.40 a kg (Timmons, 2005). This could be achieved by a single farm or by employing the methods of cooperatives, as used by dairy farmers in former years. In the cooperative model, farmers with much lower growing abilities can couple their products with other

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‘small farms’ to produce product and maintain quality control, distribution, and pricing through unity. The cooperative model helps to address the challenge of acquiring a large enough tract of land for the successful production levels that we have suggested in a metropolitan area. The Northeast USA has a competitive advantage because of its ability to grow the highest quality fish where consumer demand is most significant (Timmons, 2005). There are few, if any, filleting facilities in the USA, and most consumers prefer it in this form. Almost all fresh tilapia fillets marketed in the USA are imported from Central and South America and the Far East. These producers face significant transportation costs which give US, urban-based entities a distinct advantage. According to Timmons (2005), there are no commercial RAS operations in the USA that are of sufficient scales of production or processing to compete with large-scale aquaculture or off-shore commercial markets.

37.6 Marketing and competition There are a number of foreign countries that currently satisfy the demand for tilapia in the USA. Production in proximity to consumer, as in urban aquaculture, can have a great impact on providing better regulation of growth conditions and delivering a fresher product to the consumer. Of the current markets, there is, first, the international frozen market segment. These competitors are predominantly based in Southeast Asia and represent the lowest cost competitors. Their products are not fresh because they are frozen and shipped by sea to the USA, resulting in long lead times. Low-cost shipping combined with low-cost labor and land makes this segment the cheapest choice for tilapia. However, many of the aquaculture techniques they use are harmful to the environment and produce fish that were, until recently, not subject to extensive quality control (Islam et al., 2004). The World Health Organization (WHO) has established standards for aquaculture production using wastewater to address the human health risks associated with this practice (WHO, 2006). A second segment is the international fresh tilapia market. These producers, who are the primary competition for the US fresh tilapia market, are located in the South and Central America, and ship their fish by air to various locations in the USA overnight. The cost of shipping is offset by a price premium for fresh fish as well as low-cost labor and land in these regions. These producers generally use traditional aquaculture methods instead of RAS technology. A third, and final, segment is the domestic live market. These competitors are small and generally serve the ethnic restaurant market with live fish. Because production occurs in the USA (Florida, Texas, Missouri, California, New York, Virginia) and the product must be shipped live, there are the additional costs of shipping to consumers in excellent condition.

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This market has been saturated for over ten years and there is no reason to believe that it will expand; it may in fact decrease (Timmons, 2005). Currently, there are no domestic competitors in New York, urban or rural, who operate at a very large scale.

37.7 The role of the university 37.7.1 Research and development Universities and other government agencies, especially those with existing infrastructure, facilities and faculty, are better able to indulge in these expensive activities for they are better positioned to obtain grants and develop these programs on a smaller, less expensive level that can be up scaled for industry at a later date. There is also heuristic value when curriculum can be developed in conjunction with this research and development to satisfy increasing demands by students for this curriculum.

37.7.2 Education Educational programs that are built on an aquaculture foundation have expanded in the last ten years. They include the development of aquaculture and aquatic science curricula from pre-elementary to higher educational levels. Promoting interest and education of young minds and the general public can only have a positive impact of the future of aquaculture. 37.7.3 Community outreach University-based urban aquaculture programs can lead to a number of exciting community outreach activities. In an example from one of our more successful programs, several thousand pounds of tilapia raised for our studies have been given to homeless shelters. Tilapia have been donated to community programs, political rallies, and environmental awareness events. These are all positive expenditures of energy for they serve to familiarize citizens with the technology, needs, and current status of urban aquaculture. Community outreach programs develop interests and incite advocacy on the part of the participants on such major global issues, as sustainability, climate trends, and ecosystem stability.

37.8 Future trends The initiation of large-scale urban and peri-urban aquaculture industries, whether in New York State or elsewhere in the world, will require strong government support and coordination. The socioeconomic improvement that will be realized from the creation of such industries would justify the

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short-term assistance needed. Success will require support in at least three areas, as follows: • Grants and subsidies: Indoor aquaculture requires numerous expenses including electricity, heat, equipment, and real estate. Indoor aquaculture is already cost-competitive with outdoor systems and ocean-caught fish if done at large scale, e.g., 1000 metric ton per year of production (Timmons et al., 2004). However, to be as competitive as possible, costs must be reduced with access to low-cost electricity, heat for water (for instance waste heat from cogeneration or manufacturing facilities), equipment, and real estate (i.e., help in using abandoned or underutilized buildings, or brownfield sites). • Research: Aquaculture would benefit greatly from more comprehensive research related to the nutrition, genetics, animal health management, animal husbandry, and fundamental physiology. • Access to capital: Every pound of production capacity will require approximately $1.50 of capital investment for equipment and facilities (Timmons et al., 2004). Government loan guarantee programs or access to capital through state-backed industrial revenue bonds would assist potential farmers to get started. Growing urban aquaculture entities requires a collective effort among, farmers, research institutions, and political leaders. In the final analysis success in the aquaculture industry, especially urban-based ones, would be difficult without support in the form of government subsidies. Most other industries in the USA receive this form of support – why has aquaculture been ignored? So, what is the current status of urban aquaculture in New York and the northeast? Lots of potential energy but very little kinetic energy! Yet, for all of its potential and resources, New York City, does not currently have a single successful major aquaculture venture. The inertia and obstacles in moving successful urban aquaculture programs from discussion to reality must be overcome to face the ever-growing needs imposed by overfishing, pollution, and increasing demands for aquaculture products. These demands must be met locally and statewide by way of effective, dynamic, and significant initiatives. As a member of a university family, we firmly believe that educational collaborations within New York State can move towards these ends. The New York State public and private universities are numerous and scattered throughout the state with concentrations in the metropolitan area. They must partner and collaborate to make the case known for the importance of developing a statewide urban aquaculture industry. They must trumpet the important reasons for economic development, job training, education, and environmental stability to the policy makers and land users and to the individuals with capital who can support these essential ventures. A collaboration of universities can and must lead to the development and enhancement of effective urban aquaculture practices, programs, and ven-

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tures in New York City, as well as nationwide. In those municipalities that lack a university presence, the business sector needs to couple with the policy makers to move these endeavors along.

37.9 Acknowledgements Portions of this manuscript were taken from our article, ‘Urban Aquaculture in Brooklyn, New York, USA’ which appeared in Urban Aquaculture, B. Costa-Pierce, A. Desbonnet, P. Edwards and D. Baker (eds), CAB International Publishing, Cambridge, MA., 285 pp., 2005. Our programs have been funded by: United States National Park Service, United States Army Corps of Engineers, the Department of Environmental Conservation, Con Edison, NASA, New York Sea Grant, The New York City Board of Education, and the City University of New York. We acknowledge, with many thanks, the support over the years from the Gateway National Recreation Area and its former Chief of Natural Resources, Dr John T. Tanacredi. They provided the funds for our first small recirculating system. We are also grateful to our students at Brooklyn College, and students at every level of education who have participated in our programs as well as to the AREAC staff for their dedication and support of our aquaculture programs.

37.10 References bluem v andriske m, eichhorn h, kreuzberg k and schreibman m p (1994) A controlled aquatic ecological life support system (CAELSS) for combined production of fish and higher plant biomass suitable for integration into a lunar or planetary base, Acta Astronautica, 37, 361–71. chapman f a, fitz-coy s, thunberg e, rodrick j t, adams c m and andre m (1994) An Analysis of the United States of America International Trade in Ornamental Fish, University of Hawaii Sea Grant, Honolulu, HI. chapman f a, fitz-coy s a, thunberg e m, adams c m, rodrick j r and andre m (1997) United States of America trade in ornamental fish, J World Aqua Soc, 28, 1–10. corbin j s, cato j c and brown c l (2003) Marine ornamentals industry 2001: Priority recommendations for a sustainable future, in Cato JC and Brown CL (eds), Marine Ornamental Species. Collection, Culture and Conservation, Iowa State Press, Ames, IA, 3–10. costa-pierce b and desbonnet a (2005) A future urban ecosystem incorporating urban aquaculture for wastewater treatment and food production, in Costa-Pierce B, Desbonnet A Edwards P and Baker D (eds), Urban Aquaculture, CABI, Cambridge, MA, 1–14. davenport k e (1996) Characteristics of the current international trade in ornamental fish, with special reference to the European Union, OIE Revue Scientifique et Technique, 15, 436–43. doupé r g, lymbery a j and starcevich m r (2003) Rethinking the land: The development of inland saline aquaculture in Western Australia, Int J Agri Sustain, 1(1), 30–37.

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edwards p (2005) Development status of, and prospects for, wastewater-fed aquaculture in urban environments, in Costa-Pierce B, Desbonnet A, Edwards P and Baker D (eds), Urban Aquaculture, CABI, Cambridge, MA, 45–60. islam m s, chowdhury m t h, rahman m m and hossain m a (2004) Urban and periurban aquaculture as an immediate source of food fish: Perspectives of Dhaka City, Bangladesh, Urban Ecosystems, 7, 341–59. little d c and bunting s w (2005) Opportunities and constraints in urban aquaculture, with a focus on South and Southeast Asia, in Costa-Pierce B, Desbonnet A, Edwards P and Baker D (eds), Urban Aquaculture, CABI, Cambridge, MA, 25–44. phan van m and depauw n (2005) Wastewater-based urban aquaculture systems in Ho Chi Minh City, Vietnam, in Costa-Pierce B, Desbonnet A, Edwards P and Baker D (eds), Urban Aquaculture, CABI, Cambridge, MA, 77–102. quy hoan v and edwards p (2005) Wastewater reuse through urban aquaculture in Europe and North America, in Costa-Pierce B, Desbonnet A, Edwards P and Baker D (eds), Urban Aquaculture, CABI, Cambridge, MA, 103–18. rowland l w and cox l j (2003) Opportunities in Ornamental Aquaculture, National Coastal Resources Research and Development Institute, Portland, OR. sea grant (2001) The Economic Contribution of the Sport Fishing, Commercial Fishing and Seafood Industries to New York State, New York Sea Grant Publication Number NYSGI-T-01-001, Stony Brook, NY (prepared for SG by TECHLAW Inc.). schreibman m p and zarnoch c b (2005) Urban aquaculture in Brooklyn, New York, USA, in Costa-Pierce B, Desbonnet A, Edwards P and Baker D (eds), Urban Aquaculture, CABI, Cambridge, MA, 207–22. timmons m b, regenstein j m, schreibman m p and warner p (2001) Creating a Tilapia Industry for New York: 15 000 Jobs and One Billion Pounds, A White Paper Prepared for the New York AgriDevelopment Corporation. timmons m b, ebeling j m, wheaton f w, summerfelt s t and vinci b j (2002) Recirculating Aquaculture Systems, 2nd ed, Cayuga Aqua Ventures, Ithaca, NY. timmons m b, rivara g, baker d, regenstein j m, schreibman m p, warner p, barnes d and rivara k (2004) New York Aquaculture Industry: Constraints and Opportunities, A White Paper, Cornell Aquaculture Program, Cornell University, Ithaca, NY. timmons m b (2005) Competitive potential for USA urban aquaculture, in Costa-Pierce B, Desbonnet A, Edwards P and Baker D (eds), Urban Aquaculture, CABI, Cambridge, MA, 137–58. who (2006) Guidelines for the safe use of wastewater, excreta and greywater. Volume 3: Wastewater and excreta use in aquaculture, World Health Organization, Geneva. zohar y, tal y, schreier h, steven c, stubblefield j and place a (2005) Commercially feasible urban recirculating aquaculture: Addressing the marine sector, in Costa-Pierce B, Desbonnet A, Edwards P and Baker D (eds), Urban Aquaculture, CABI, Cambridge, MA, 159–72.

Index

a priori, 100 abalones, 591 acetate, 284 Acipenser spp., 124 acoustic Doppler current profiling, 684, 899 activated sludge technology, 1051 Aero-2 aspirator pump aerator, 1131 Aeromonas salmonicida, 783 aflatoxin, 509 Akva FishTalk Value Chain Planner, 1088 Akva Vicass, 1075 AkvaSmart, 1075, 1095 Alexandrium, 587, 595, 598 Alexandrium sp, 901 Alexandrium tamarense, 598 allozyme electrophoresis, 62–3 allozymes, 5 alum, 1001 aluminium sulfate, 1001 AMB Bio Media, 978 Amphidinium, 645 amphidinolides, 645 amplified fragment length polymorphism, 7–10 genetic linkage analysis, 9 genetic variation, 8 An Pin Live fish Centre, 1095 Analytical Hierarchy Process, 717 androgen, 154 androgenesis, 155, 187

aneuploids, 187 animal safety, 226 annular column, 614 antimicrobial peptides, 257–8 antinutritional factors, 287, 508 plant protein sources, 288 antiviral molecules, 252–3 Apex-IHN, 249 Aphanizomenon, 635 Aphanizomenon flos-aquae, 636 apoptosis, 253 apparent digestibility coefficients, 463 AquaAssist, 1095 aquaculture, 751 advances, xxix advances in catfish, tilapia and carp nutrition, 440–56 advances in disease diagnosis, vaccine development & other pathogen detection technologies, 197–209 advances in diagnostic methods (for bacterial diseases), 199–203 advances in vaccine development, 203–7 future trends, 208–9 key drivers for improvement, 198 limitations of current methods, 198–9 other pathogen control methods, 207–8

1164

Index

controlling fish reproduction, 109–30 factorial approach in quantifying nutritional requirements, 417–38 genome technology for genetic improvement, 3–41 global, xxvii inland saline (see inland saline aquaculture) integrated, 697 interactions with environment, 681–4 marine medical and health care products and their associated economic values, 867 monitoring and assessment techniques, 690–6 characterisation of benthic biota, 692 other indicators, 696 predictive assessments, 693–6 sediment condition, 691 visual assessment of sediment and epibenthic biota, 692–3 water quality, 690–1 new development for controlling parasitic diseases, 215–37 effect on the industry, 216–18 new development for controlling viral disease, 244–59 new technology, xxviii–xxix pharmaceuticals and neutraceuticals production, 866–86 algal targets for pharmaceuticals and nutraceuticals, 877–80 diversifying the aquaculture industry, 874–5 future trends, 886 green lipped mussel culture for nutraceuticals, 880–2 health claims for functional food in Australia, 885 major research and development steps required, 869 marine neutraceuticals, 872–4 marine pharmaceuticals, 870–2 polyculture potential for medicinal sea cucumbers and whelks, 882–4 sponge aquaculture for pharmaceuticals, 875–7 steps towards commercialisation, 884–6

predicting and assessing the environmental impact, 679–99 common condition indicators, method of use and comparative cost, 688 common environmental impacts both on and of aquaculture, 681 considerations in monitoring and assessment program development, 686–90 effects on environment, 681 potential conflicts in water usage, 680 site selection and carrying capacity, 684–6 production, xxvii–xxviii Rachycentron canadum cultivation, 804–18 recent technological advances and future trends, 696–9 changes in technology/monitoring approaches, 698–9 changes in types of aquaculture, 696–8 role of GIS in spatial decision support, 707–46 shellfish, impact of harmful algal blooms, 580–601 sterile and single-sex fish populations, 143–58 future trends, 157–8 use of ICT, 1064–105 Aquaculture Collaborative Research Support Project, 991 Aquaculture Engineering Group, 941 aquaculture feeds advances for basses and breams, 459–83 advances for salmonids, 498–522 advances in microalgal culture, 610–58 ingredient evaluation using digestibility, utilisation and nutritional factors as parameters, 387–410 aquaculture feeds and ingredients, 370–83 alternate protein and lipid sources, 378–83 canola/rapeseed products, 381 corn products, 380 distillers products, 381

Index peas and lupins, 382 plankton and krill, 382–3 seafood processing waste products, 382 single-cell protein products, 381–2 soybean products, 379–80 wheat and barley, 380–1 environmental pollutants and residues categories, 375–8 chemotherapeutic residues, 378 methylmercury, 375–6 farmed fish product safety, 374–5 future trends, 383 sustainability of ingredients, 371–4 Aquagard, 233 AquaMetric, 1074 AquaModel, 906 AquaNIC, 1104 aquanomics, 817–18 AquaOptima AS, 953 aquaponics, 1053–4, 1151, 1157 Aquatic Animal Health Code, 255 ‘aquatic chickens,’ 441 Aquatic Research and Environmental Assessment Center, 1151 ARA. see arachidonic acid Ara-A, 875 Ara-C, 875 arachidonic acid, 326–7 ARA/EPA on gilthead seabream larval growth, 327 stress regulation mechanisms by fatty acids, 327 ARC-GIS, 711 ArcView, 719 ARENA simulation software, 1086 Argyrosomus japonicus, 1127, 1130 Artemia, 624, 831, 857 Artemia salina, 645 Artemia salina test, 644 ‘artesian desert water,’ 1120 Arthrospira, 610, 624, 635, 636, 639, 640 Arthrospira platensis, 625, 878 Asian marine crabs, 846 Asian seabass advances in aquaculture feeds and feeding, 461–6 apparent digestibility coefficients, 464 digestibility, 463 effect of dietary protein on average daily weight gain, 462

1165

feeding studies, 464–6 miscellaneous studies, 466 replacement studies, 463–4 requirements, 461–3 theoretical feeding tables based on data from bioenergetic studies, 465 composition of weight gain, 425 energy and protein loss, 427 growth potential, 423 Asparagopsis armata, 878 Assessment of Estuarine Trophic Status, 694 Association of Official Analytical Chemists, 391 Association of Official Analytical Chemists International, 597 astaxanthin, 511–12, 515, 517, 615, 639, 880 asynchronus spawners, 116–17 Atlantic cod, 774–89 aquaculture production in Norway, 775 bottlenecks, 787–8 breeding program, 778–80 broodstock, 776–8 consumer preference for farmed cod, 789 difference in breeding programs, 779 differences in survival among families during test against vibriosis, 780 effect of photomanipulation on quality of eggs and larvae, 776 environmental issues, 788 infected by Francisella piscicida, 786 larval deformities, 783 larval rearing protocol, 781 production technology, nutrition, and disease management, 780–7 survival from hatching through weaning, 777 Atlantic halibut, 789–94 bottlenecks, 794 breeding program, 791 broodstock, 790–1 environmental issues, 794 gamete collection, 791 production technology, nutrition, and disease management, 791–4 Atlantic salmon. see Salmo salar

1166

Index

Aureoumbra lagunesis, 591 Australia oyster selection programs, 94–6 family selection, 95 Austrocochlea constricta, 758 automatic microdiet dispenser, 356 autonomous underwater vehicles, 693 Avarol, 877 azaspiracids, 589 bacterin, 282 barramundi. see Asian seabass; Lates calcarifer bastadins, 871 Beché-de-Mer, 882 Bechitin-W, 874 betaine, 449 β-glucans, 513 Bidyanus bidyanus, 753 Bif idobacter spp, 451 bioactive molecules, 643 biochemical oxygen demand, 946 biocidal compounds, 272 bio-clarifiers, 967 biocoils, 616 biocontrol agents, 272–3 agents used in aquaculture, 272 BioFence, 616 bio-floc technology, 1050–1 biological control, 222 biopesticides, 222 bio-protein, 381–2 biosecurity, 258 Bioshelters Inc., 1151–2 Blackboard, 1100 bloat, 502–3 blue crab, 846 bluegill. see Lepomis macrochirus Branchiomma luctuosum, 753 bream, Red sea bream and gilthead sea bream advances in aquaculture feeds and feeding, 466–76 ADC for protein and energy of Australian ingredients, 473 dietary digestible protein and digestible energy effects on relative protein deposition, 468 digestibility, 471–2 effect of increasing dietary inclusion content on ADC of gelatinised wheat starch, 471

feeding studies, 475–6 miscellaneous studies, 476 replacement studies, 472–5 requirements, 468–71 brevenal, 645 brevetoxin, 589, 645 brown tide, 591 bryostatin 1, 870, 871 ‘bubbled-washed’ bead filters, 961 Bugula neritina, 870 buoyancy, 350–1 business card tags, 1078 C. gigas, 94–6 CABI Aquaculture Compendium, 1104 calcium hypochlorite, 997 calcium peroxide, 998 calcium sulfate, 1000 Callinectes sapidus, 851 canola meal, 381 canthaxanthin, 511–12, 639 Carassius auratus, 753 carp. see also freshwater fish species commercially produced species, 441 general nutrient specifications for formulation of practical diets, 454 generic production diets, 454 recommended minimum amino acid levels in diets, 455 supplemental mineral levels recommended for practical diets, 456 Carraguard, 878 catamaran steel fish farm, 919, 928–9 with integrated feed barge for storage and equipment, 929 large, 916 catfish. see also freshwater fish species commercially produced species, 441 general nutrient specifications for formulation of practical diets, 454 generic production diets, 454 recommended minimum amino acid levels in diets, 455 supplemental mineral levels recommended for practical diets, 456 cell size hypothesis, 181 cemadotin, 645 centre feed sedimentation basins, 957 ceruloplasmin, 817

Index Chaetoceros, 624, 626, 628 Chaetoceros calcitrans, 615 Channa micropeltes, 1037, 1040 channel catfish. see Ictalurus punctatus Chattonella, 600 chelated copper compounds, 999 chemical oxygen demand, 946 chemotherapeutic residues, 378 chemotherapy, 225–9, 231–3 chemotherapeutants used against sea lice, 230 ideal chemotherapeutants, 231 resistance management principles, 232 Chinese mitten crabs, 846–7 product issues, 850 production systems broodstock quality and nutrition, 855–6 food and feeding, 853–4 grow-out, 851–2 hatchery practices, 856–7 nurseries, 858 chitin, 282, 874 chiton, 874 chitosan, 282, 874 Chlorella, 616, 624, 626, 628, 635, 636, 639, 645, 653, 879 Chlorella sp, 625 Chlorella-V12, 635 choline, 449 Chondrilla nucula, 753 CHORULON, 120 chromosome counting, 173 chromosome set manipulation, 58–9, 165–88 grass carp ploidy manipulations, 60 gynogenesis, androgenesis and aneuploids, 187 newly culture species, 59 principles and methods, 166–74, 167–70 blocking meiosis I, 170 cytogenetic techniques for verification, 174 gamete chromosome inactivation, 172 methods for meiosis and mitosis inhibition, 170–2 polyploids and uniparenteral inheritance identification, 172–4 schematic presentation, 169

1167

shellfish reproduction, 166–7 summary and perspectives, 187–8 tetraploid shellfish, 183–4, 186–7 triploid shellfish, 174–5, 178–83 Chrysochromulina polylepis, 591 Chrysochromulina quadrikonta, 594 circular flow tanks, 951–3 circular HDPE collar fish farm, 925–7 in grid mooring system, 926 circular HDPE pipe fish farm, 915 clay scatter method, 600 ‘clear water’ techniques, 625 closed equilibrated biological aquatic system, 1156 cloud computing, 1080 coal bed methane, 1123, 1126 coal seam gas, 1126 coal seam methane, 1126 cobia. see Rachycentron canadum Cochlodinium polykrikoides, 597, 600 Codex Alimentarius Commission, 1046 Codium spp, 878 colchicine, 148 cold banking, 813–14 cold-water marine finfish aquaculture biological and technological advances, 771–97 Atlantic cod, 774–89 Atlantic halibut, 789–94 current and past research activities, 774 future trends, 794–7 consumer preference, 797 environmental issues, 796–7 fish health, 796 improvements to current culture methods, 794–5 industry driven research and marketing, 797 nutrition, 795–6 selective breeding program, 796 studies involving all life stages, 794 life cycles of Atlantic cod and halibut in captivity, 773 Commission regulation (EC) No 1234/2003, 515 Committee on the Status of Endangered Wildlife in Canada, 774 comparative carcass analysis, 426 composite culture, 1048 concentrated aquatic animal production, 1005

1168

Index

conjugated linoleic acid, 520 consumer safety, 226 content management system, 1100 copper sulfate, 999 Corbicula fluminea, 754 corn gluten meal, 380 Cornell dual-drain, 954 Cornell-style side drain, 978 cortisol, 274 cottonseed meal, 445 Council Directive 91/492/EEC, 543 course management system, 1100 crab feed, 853 crabs aquaculture advances, 845–60 current situation, 848 Chinese mitten crab, 846–7 current situation, 849 future trends, 859–60 breeding, 859 hatchery methods, 860 nursery and grow-out, 860 product, 859 Mangrove crabs Scylla spp and other portunids, 847–8 product issues, 848–51 mitten crabs, 850 portunid crabs, 850–1 production systems, 851–8 breeding and hatchery technology, 855 broodstock quality and nutrition, 855–6 food and feeding, 853–5 grow-out, 851–3 hatchery practices, 856–7 nurseries, 857–8 Crassostrea ariakensis, 762 Crassostrea gigas, 621, 882 Crassostrea rhizophorae, 755 Crassostrea virginica, 175, 178, 755 critical standing crop, 1043 cross-flow raceway design, 951 crustacean viruses, 247 main crustacean viral diseases, 247 notifiable crustacean diseases, 248 Crypthecodinium, 879, 880 cryptophycin-52, 644 cryptophycin 249, 644 cryptophycin 309, 644 cryptophycins, 651 curacin A, 644 Cyanotech, 635

cyclooxygenase pathway, 277–8 Cyprinus carpio, 753 cytochalasin B, 148, 171–2 cytogenetics, 59, 61 daily loss of energy and protein, 426 De Haan Automatisering, 1095 decision support systems, and tools, 718–25 activity trade-off, 718–19 additional internal or external modules, 719–20 analytical scope and reporting, 720 biodiversity tools, 719 viewsheds, 719 DELPHI, 719 DEPOMOD, 694, 906, 1086 depuration, 564–5, 598 Dermochlorella, 643 desert aquaculture, 1120 desert water, 1136 DGGE techniques, 1022 DHA. see docosahexaenoic acid DHActive, 639 DHAGold, 639 diagnostic methods advances, 199–203 immunodiagnostic methods for fish pathogen detection, 201 loop-mediated isothermal amplification test, 203 key drivers for improvement, 198 limitations, 198–9 Diagnostic Tests for Aquatic Animals, 199 Dicathais orbita, 883–4 dichlorvos, 229, 231 diet feed improvement, 276–87, 289 alternative protein sources and feed hazards, 286–7, 289 antinutritional factors in plant protein sources, 288 dietary probiotics, 285–6 fatty acids and antioxidants, 277–8 non-nutritive immunostimulants, 281–2 non-starch polysaccharides and oligosaccharides, 283–5 nutritional competition, 280 other nutritional requirements, 278–80

Index diet replacement method, 394 dietary nucleotides, 279 dietary probiotics, 285–6 Dietary Supplementation Health and Education Act, 884–5 digestibility, 393–9 calculating diet and ingredient digestibilities, 397–8 collecting faeces for digestibility assessment, 395–6 correlation among protein digestibilities, 400 correlation of diet and ingredient digestibilities, 399 diet and ingredient digestibilities, 399 effects of species on assessment process, 398–9 experiment management issues, 396–7 feed issues in ingredient digestibility assessment, 393–5 digital elevation model, 744 6-dimethylaminopurine, 171–2 dinophysistoxin, 589 dinucleotide repeats, 10–11 dioxin, 509 Diploma supplement, 1102 disease resistance, 256–7 new technologies and prospects in diet and husbandry techniques, 267–91 complex interaction between pathogens, stress factors, feed, microbiota and immune response, 268 feed improvement, 276–87, 289 pathogen control, 268–73 welfare improvement, 273–6 distiller’s dried grains, 381 distiller’s dried grains with solubles, 381 ‘distributed control system,’ 1068 DNA fingerprinting, 97–8 DNA marker technologies, 4–18 amplified fragment length polymorphism (AFLP), 7–10 historical perspectives, 4–7 microsatellites, 10–13 single nucleotide polymorphism (SNP), 13–17 trends, 17–18

1169

DNA sequencing technologies, 18–26, 221 454 sequencing platform, 24–6 comparison of next generation sequencing platforms, 19 Solexa sequencing platform, 23–4 SOLiD sequencing platform, 19–23 DNA vaccine delivery, 209 DNP3, 1070 docosahexaenoic acid, 323–5 effect on survival of gilthead seabream larvae, 324 Dolabella auricularia, 644 dolastatin 10, 644 double-stranded RNA, 254 down-flow bubble oxygen contractor, 970 Dreissenia polymorpha, 754 ‘drip irrigation’ system, 1140 Dunaliella, 624, 635, 636, 879 Dunaliella salina, 620 Dunaliella tertiolecta, 627 Dysidea avara, 877 E. j. sinensis, 846, 853 Earthrise Nutritionals, 635 East Kolkata Wetlands, 1040 EC Seafood Plus and Trace project, 1096 EC SHEEL project, 1096 Eco-Flow, 953 ecological carrying capacity, 685 ecological sustainability, 373 ECOSIM, 692 Eco-Tank, 949 Ecteinascidia turbinata, 870 effluent limitation guidelines, 1005 eicosapentaenoic acid, 325–6 effect on survival of gilthead seabream larvae, 326 gilthead seabream larvae larval growth in relation to DHA + EPA + ARA, 328 electronic fish auctions, 1097 embankment ponds, 986 enteric viruses characteristics, 545 human, 544, 546–7 average concentration detected in wastewater samples, 554 and phages detection in shellfish samples, 561 in sewage and rivers, 555–8

1170

Index

titers, 554 enterprise resource planning, 1081 Environmental management systems, 690 environmental quality objectives, 689 environmental quality standards, 228, 689 environmental safety, 228 enzyme-linked immunoassay, 200, 597 EPA. see eicosapentaenoic acid Epinephelus aeneus, 477 Epinephelus coioides, 478, 481, 483 Epinephelus fuscoguttatus, 483 Epinephelus malabaricus, 478, 481 Eriocheir japonica, 846, 847 Eriocheir sinensis, 847 Eriocheir spp, 845 Escherichia coli, 451, 543 ET-743, 870, 871 Ethernet networking, 1070 ethnomedical, 873 EU TraceFish project, 1096 Euphausia superba, 519 EUROPASS, 1102 European Article Numbering-Uniform Code Council, 1096 European CV, 1102 European Galileo GPS system, 715 European Qualification Framework, 1102 European regulation 91/492/EC, 548, 560 European sea bass, energy and protein loss, 426 European Union Water Framework Directive, 688 EVOLVER, 1081, 1086 excavated ponds, 986–7 expandable bed filters, 960–2 eXtensible Markup Language, 1096 eye index, 829 faecal stripping techniques, 395–6 Farm Aquaculture Resource Management, 695 Farmcontrol, 1079 Farmocean, 939 fish farm completely elevated, 940 FAS ‘hooded’ oxygenator, 979 fatty acids, 322–3 fatty acids and antioxidants, 277–8 growth/disease resistance in essential fatty acids requirement, 277

fecundity, 88 feed conversion efficiency, 403–4 feed conversion ratio, 434, 462, 993, 1132 feed ingredients characterisation and preparation of ingredients, 390–1, 393 aquaculture feed ingredients composition, 392 ingredient identification, 390–1 preparation prior to evaluation, 393 defining ingredient digestibility, 393–9 calculating diet and ingredient digestibilities, 397–8 collecting faeces for digestibility assessment, 395–6 correlation among protein digestibilities, 400 correlation of diet and ingredient digestibilities, 399 diet and ingredient digestibilities, 399 effects of species on digestibility assessment process, 398–9 experiment management issues, 396–7 feed issues in ingredient digestibility assessment, 393–5 frontier technologies for evaluation, 408–9 growth and utilisation effect, 402–7, 408 biochemical, histological and sensory factors in evaluation, 407–8 energy retention by rainbow trout, 404 factors affecting nutrient and energy utilisation, 405–6 feed conversion efficiency, 403–4 gene and protein expression, 406–7 measuring growth, 402–3 nutrient retention, 404–5 reactive lysine assay chromatogram, 406 survival, 403 ingredient functionality and feed technical qualities lupin kernel meal, 410 pellet hardness, 408

Index ingredient palatability, 399–402 daily feed intake, 401 introduction, 388–90 components to ingredient evaluation, 388–9 consolidating the evaluation process, 389–90 ingredient risk management, 388 feed intake, 420–3 feline calicivirus, 565 Fenneropenaeus indicus, 653 fertilisation, 990–2, 1041–3 finfish genetic improvement, 55–72 future trends, 71–2 key drivers, 56–69 growth rate, disease resistance, and other quality traits improvement by selective breeding methods, 56–8 performance and other traits improvement by non-selective breeding methods, 58–9, 61–9 production- and consumer-related breeding-goal traits, 57 recent breeding programmes, 57 selective breeding programs risks, 69–71 fish health improvement, 273–6 mediators in neuroendocrine and immune system interaction, 275 neuroendocrine and immune function interaction, 274 rearing condition, 276 water quality and bioremediation, 274–6 fish farming current status and technical limitations, 924–9 catamaran steel fish farm, 928–9 circular HDPE collar fish farm, 925–7 comparisons of amount of biomass in one large net cage, 925 expanded polyester floatation attached under a bridge of steel, 927 HDPE pipes used for circular plastic fish farm manufacturing, 926

1171

interconnected hinged steel fish farm, 927–8 rigid steel fish farm, 929 floating fish farm design, 918, 920–4 categorisation, 918, 920 detail of mooring line connected to interconnected hinged steel fish farm, 922 grid mooring system for HDPE collar fish farms, 922 HDPE collar fish farm, 919 interconnected hinged steel fish farm, 919 mooring system, 921 the net cage, 920–1 steel catamaran fish farm, 919 structural analysis of fish farms, 921–4 historical development of technology, 914–18 centralised feeding systems used with HDPE collar fish farm, 917 circular high-density polyethylene pipe fish farm, 915 fish farm made of wood and expanded polyester, 915 interconnected hinged floating bridges of steel fish farm, 916 large catamaran-type fish farm, 916 modern net cage, 918 novel systems, 930–1, 933–5, 937–41 computer simulation of Nautilus fish farm exposed to waves, 938 Nautilus fish farm, 937–8 OceanGlobe fish farm, 938–9 PolarCirkel submergible fish farm, 934–5 SeaStation fish farm, 931, 933–4 submerged SeaStation fish farm, 933 Tension Leg Cage fish farm, 935–7 off-shore and open ocean advances in technology for, 914–42 supporting technologies, 937–41 other novel systems, 939–41 Aquaculture Engineering Group, 941 Farmocean, 939

1172

Index

Farmocean fish farm completely elevated, 940 SADCO, 939 SubFish, 939–40 technical properties of systems, 932 fish larvae nutrition and diet development, 315–59 diet manufacturing methods, 346–9 digestive system capacity, 343–5 dosage system, 355–9 factors affecting food particle utilisation, 318 feeding system, 354–5 food identification and ingestion, 332–3, 332–6 future directions, 359 Lates calcarifer, 317 microdiet characteristics, 349–52, 354 nutritional requirement, 322–32 nutritional requirement development, 319–22 ontogeny of digestive capacity, 336–43 fish meal, 372–3 fish pumps, 815 fish reproduction, 109–30 future trends, 128–30 reproductive endocrinology, 129–30 hormonal therapies for reproductive control, 118–22 gonadotropin preparations, 119–20 gonadotropin-releasing hormone agonists, 120–1 reproductive axis dysfunction & oocyte maturation & spermiation induction, 119 sustained-release delivery systems, 121–2 oocyte maturation and ovulation induction, 122–6 reproductive cycle control, 110–11, 113–16 major components and phases, environmental and endocrine control of the fish reproductive axis, 111 microphotographs of histological sections from ovaries, 112 spawning, 116

spermatogenesis & spermiation, 114–16 testes microphotographs, 115 vitellogenesis, oocyte maturation & ovulation, 113–14 reproductive strategies and captivity dysfunctions, 116–18 striped bass ovaries microphotographs, 117 spermiation induction, 126–7 spontaneous spawning versus artificial insemination, 127–8 fish vaccines, 203–7 fish viruses, 245 main fish viral diseases, 246 notifiable fish diseases, 246 FishBase, 1104 FishTalk, 1079 Fishtalk Service Log, 1081 fish-vet, 1099 flat photobioreactors, 616–17 floating bead filters, 967 floating fish farm design, 918, 920–4 flocculants, 1001–2 fluorescence antibody test, 200 foam fractionation, 947, 976 food conversion ratio, 1067 Francisella piscicida, 785 francisellosis, 785, 796 freshwater fish species advances in aquaculture nutrition, 440–56 nutrient requirements, 443–55 alternative protein sources, 445–6 complete feeds, 453–5 complete vs supplemental feeds, 452–3 energy, 446–7 lipids, 447–8 minerals, 450–1 prebiotics and probiotics, 451 protein, 443–5 vitamins, 448–50 recommended vitamin fortification levels for warm water fish, 455 fuel cell systems, 1157 fumonisins, 509 Gadus morhua, 771. see also Atlantic cod Galdieria sulphuraria, 878

Index Gambierdiscus toxicus, 645 gambieric acids, 645 gamete chromosomes, 172 gametogenesis, 123 gamma ray irradiation, 172 gene discovery technologies, 26–8 de novo sequencing of whole transcriptomes and gene discovery, 27–8 expressed sequence tags and gene discovery, 26–7 gene mapping, 62–3 gene sequencing, 209 general circulation models, 727 genetic drift, 89 genetic engineering, 256–7 genetic linkage mapping, 28–9 status of linkage maps in aquaculture species, 30–1 genetic variation and selective breeding in hatcherypropagated molluscan shellfish, 87–100 genetically modified organisms, 68 genome expression analysis technologies, 35–41 microarray technology, 35–40 development status in various aquaculture and aquatic species, 39 dye labeling, 37 vs tag- or sequence-based technology, 40–1 sequence tag-based technology, 40 genome mapping technologies, 28–9, 32–4 bacterial artificial chromosomebased physical mapping, 34 genetic linkage mapping, 28–9 quantitative trait loci (QTL) mapping, 29, 32 radiation hybrid mapping, 32–4 genome technology genetic improvement in aquaculture research, 3–41 DNA marker technologies, 4–18 DNA sequencing technologies, 18–26 gene discovery technologies, 26–8 genome expression analysis technologies, 35–41 genome mapping technologies, 28–9, 32–4

1173

genomic stability, 151–3 genomics, 67–8 geographical information systems, 685, 1086 climate change, 726–38 adapting to climate change and role of GIS, 733–4, 738 conclusion and future direction, 738 potential future climate change and its impact on aquaculture systems, 727–8, 732 database construction and project methodology, 711, 713–18 arithmetic operations, 715 buffers, 716 data rectification, 715 hierarchical models, 717–18 identifying data requirements, 711, 713–14 model verification, 718 neighbourhood analysis, 715 overlay, 716 reclassification, 716 setting the objectives, 711 verification, 714–15 weighted overlay, 716–17 decision support systems and tools, 718–25 activity trade-off, 718–19 additional internal or external modules, 719–20 analytical scope and reporting, 720 biodiversity tools, 719 viewsheds, 719 influence of climate change on aquaculture activities, 733 key capabilities, 709–11 analysis, 710–11 data acquisition and encoding, 709–10 data storage and retrieval, 710 display, 711 model to indicate vulnerability of global aquaculture to climate change, 735 modelling solid waste dispersal, 725 multi-site coastal zone planning, 739–45 approaches to model development, 739 biodiversity sub-model, 742–3

1174

Index

cage site suitability sub-model, 739–41 conclusion and future direction, 745 viewshed sub-model, 743–5 waste dispersion sub-model, 743 potential impact pathways of climate change on aquaculture systems and production, 729–31 predicted global average surface warming and sea level rise by the end of the 21st century, 728 role in spatial decision support in aquaculture, 707–46 selected applications and examples in aquaculture, 720–5 aquaculture and tourism, Tenenife, 725 detailed facility location, Scotland, 720 mangroves and aquaculture, Bangladesh, 721–3 sea urchin fishery management, Chile, 723 shellfish scenarios, Brazil, 721 waste dispersion models, UK, 723–5 shellfish culture scenarios in Brazil, 722 simple model of suitability for salmon cage location, 721 spatial planning context, 707–11 software and hardware development, 708–9 stages of development, 712 suitability of areas for aquaculture development in Spain, 726 suitability of areas for fishery restocking in Chile, 724 summary and future trends, 745–6 trade-offs between tilapia/carp and shrimp/crab cultures, 723 giant sea bass. see Asian seabass giant snakehead. see Channa micropeltes Gigartina skottsbergii, 878 gilthead sea bream. see also bream; Sparus aurata composition of weight gain, 424 energy and protein loss, 426

growth potential, 423 GIS. see geographical information systems global positioning system, 714–15 Global Standard One, 1096 global traceability network, 1096 glucosamine, 874 gnotobiology, 269 gonadotropin-releasing hormone agonists, 120–1 GONAZON, 120 gossypol, 445 green liver syndrome, 469 green water techniques, 625–6 green-lipped mussel. see Perna canaliculus greenshell mussel. see Perna canaliculus grouper advances in aquaculture feeds and feeding, 476–83 ADC for crude protein and energy for selected South East Asian ingredients, 480 digestibility, 479 feeding studies, 482 miscellaneous studies, 482–3 replacement studies, 480–2 requirements, 477–9 growth, 420–3 daily weight gain in relation to increasing body weights in gilthead sea bream, 423 daily weight gain in relation to increasing body weights in tilapia, 421 Gymnodinium, 587 Gymnodinium catenatum, 598 gynogenesis, 155–6, 187 gypsum, 1000, 1001 Haematococcus, 635, 636, 639, 880 Haematococcus pluvialis, 616 Halichondria okadai, 871 Halichondria panicea, 756 halichondrin B, 877 halichondrins, 876 Haliotis discus, 591 Haliotis sp., 623 harmful algal blooms clay sprinkling against surface patches of toxic dinoflagellate, 600

Index detection of phyto- and zooplankton species from ejected faeces from the Pacific oyster, 584 global increase, 581–7 climate changes, 581–2 coastal water utilisation for shellfish aquaculture, 582–5 dispersal associated with shellfish transportation, 585–7 large-scale cultivation of the shellfish, 583 impact on shellfisheries industries, 587–95 halo effects or value degradation products, 593–5 mass mortality and detrimental effects on molluscan shellfish, 590–3 phycotoxins accumulation, 587, 589–90 mechanism of phycotoxins-related shellfish poisoning, 588 and shellfisheries aquaculture, 580–601 shellfisheries damage due to toxic dinoflagellate, 592 temporal change of pacific oyster production, 594 temporal changes of toxicity in PST-contaminated oysters, 599 toxic dinoflagellate species that hampered shellfisheries, 588 treat prevention, 595–601 aquaculture sites selection, 597–8 blanket closure during the toxic algal bloom, 596–7 clay scatter, 599–601 cultivation treatment: phycotoxins depuration, 598–9 forecasts of the trajectory of the event by plankton monitoring, 595–6 Haslea ostrearia, 620, 640 hatcheries, 255 hatchery propagation, 89 Hawaii Aquaculture Module Expert System, 1099 hazard analysis and critical control points, 1086, 1088, 1091 HDPE collar fish farm, 919 Hei shen, 882

1175

HELIOGUARD 365, 643 hepatitis A virus, 544 herbal medicine, 270–1 herbicides, 998–9 heritability estimates, 90–2 larval traits, 91 Hesy Aquaculture, 948 Heterocapsa circularisquama, 591, 593 Heterocapsa triquetra, 595 Heterosigma akashiwo, 592, 595, 597 heterozygosity hypothesis, 179–80 high energy radiation, 153–4 high rate algal ponds, 654 high-hydrostatic pressure, 568 high-pressure liquid chromatography, 596, 597 Hippoglossus hippoglosus, 771. see also Atlantic halibut holothurians, 882 Homarus americanus, 823 Homarus gammarus, 823 host-parasite interactions, 218–20 immunomodulation, 219–20 HTE Biofilter, 970 Human Genome Project, 6 husbandry, 224–5 techniques and diet to improve disease resistance, 267–91 hydraulically integrated serial turbidostat algal reactor, 620 hydrocyclones. see swirl separator hydrogen peroxide, 998 hydroponics, 1157 hydrostatic pressure, 146 Hymeniacidon perleve, 753 ICT. see information and communications technology Ictalurus punctatus, 779 IDRISI, 711 IEC-60870-5-101, 1070 IEC-60870-5-104, 1070 IEC 61850, 1070 immunohistochemistry, 200 immunological manipulation, 154 In situ array technology, 36 in situ fluorometry, 901 inbreeding depression, 90 indirect fluorescence antibody test, 200 infectious pancreatic necrosis virus, 794 information and communications technology in aquaculture development, 1064–5

1176

Index

in aquaculture innovation and learning, 1098–104 applications of ICT in aquaculture education and learning, 1099–103 linking innovation, research and learning, 1098–9 role of ICT in innovation process, 1103–4 factors affecting adoption in aquaculture, 1066 farm patrol monitoring and control system from Pisces Engineering, 1069 functions, 1065, 1067 for productivity and effectiveness, 1067–88 Akvasmart FishTalk software, 1079, 1080 aquaculture stock management programmes, 1082–4 business information systems, 1081, 1085 planning and design, 1085–6, 1088 principles of monitoring, control and automation, 1067–70 software for aquaculture planning and design, 1087 stock management systems, 1078–81 for quality and customer service, 1088–98 elements of traceability systems, 1092 market chain and traceability, 1089–96 marketing and sales, 1097–8 public relations, 1098 RFID tags, 1093–4 active RFID tags, 1094 passive RFID tags, 1094 semi-passive RFID tags, 1094 sensors and monitoring tools for aquaculture stock, 1070, 1072–5, 1077–8, 1085–6 Akvasmart SmartEye camera system, 1076 AquaScan fish counter detail, 1072 AquaScan fish counters installed on Norwegian well boat graders, 1071 counting stock, 1070, 1072–3

estimating weight and biomass, 1073–5 feed management, 1075 fish identification and individual monitoring, 1075, 1077–8 laptop computer running AquaScan fish counting software, 1071 Lotek acoustic tags, 1077 record from Vaki fish counter, 1073 Vaki biomass frame, 1074 Vaki pipeline fish counter, 1072 tracking technology in action, 1095 use in aquaculture, 1064–105 ingredient replacement method, 394 inland saline aquaculture, 1119–44 case studies, 1128–44 Australia, 1128–33 effluent to olives, 1144 floating solar covers positioned on experiment pond, 1131 growth of rainbow trout in saline groundwater ponds at ISA Research Centre, 1132 happy red tilapia in an Israeli desert fish farm, 1142 India, culture of Macrobrachium rosenbergii, 1133–5 ISA Research Centre facilities, 1129 Isaraeli desert fish farm for edible fish, 1140 Israel, desert aquaculture, 1135–6, 1138–42 Israel’s freshwater resources and average annual use, 1136 layout of integrated desert aqua/ agriculture operation, 1141 mineral composition of desert brackish geothermal water vs seawater and freshwater, 1138 national water carrier: water transfer from wet to arid areas, 1137 USA, inland marine shrimp aquaculture, 1142–44 water chemistry of diluted coastal seawater and inland saline groundwater from Haryana, 1134 water quality of ponds at ISA Research Centre, 1131

Index water temperature and wet weight of mulloway in ponds at ISA Research Centre, 1130 chemistry and remediation, 1126–8 coal bed methane wastewater, 1123, 1126 future trends, 1144 saline water from interception schemes to protect agriculture, 1121–3 water chemistry for inland saline water, 1124–5 innate immunity, 250 Innovalg SARL, 636 inositol, 449 Instant Algae, 628 integrated agriculture/aquaculture systems, 1033 recent changes to traditional practice, 1037–9 crop/livestock/fish integration in China, 1038–9 livestock/fish integration, 1039 rice/fish integration, 1037–8 research and development for improved traditional practice, 1044–5 crop/livestock/fish integration, 1044–5 rice/fish integration, 1044 traditional aquaculture systems, 1034–6 crop/livestock/fish integration in China, 1035 livestock/fish integration, 1035–6 rice/fish integration, 1034–5 integrated fisheries aquaculture systems, 1033 recent changes to traditional practice, 1040–1 traditional aquaculture systems, 1037 integrated multitrophic aquaculture, 697, 1053 integrated peri-urban aquaculture systems, 1033 recent changes to traditional practice, 1039–40 traditional aquaculture systems, 1036 integrated pest management, 233–6 IPM scheme for parasite control, 234 integrated wastewater aquaculture, 753 INTER AQUA Advance A/S, 950

1177

interconnected hinged steel fish farm, 919, 927–8 with attached blowers and storage barge, 928 detail of connected mooring line, 922 IPM. see integrated pest management iron, 280 ISA Research Centre facilities, 1129 growth of rainbow trout in saline groundwater ponds, 1132 uncovered and covered ponds water quality, 1131 water temperature and wet weight of mulloway, 1130 ISO 9001, 1088 ISO 14001, 1088 ISO 22000, 1088 ISO 22003, 1088 Isochrysis, 624, 626, 628 Isochrysis galbana, 625, 627 Isochrysis T-ISO, 620, 622, 624 isoflavonoid phytoeostrogens, 509 ITT-Wedeco, 973 Ivermectin, 227 Jasus edwardsii, 826, 827, 828, 829, 833, 835, 836 Jasus lalandii, 824, 833 Jasus verreauxi, 824, 826, 827, 828, 829, 836 JECFA. see Joint FAO/WHO Committee on Food Additives Joint FAO/WHO Committee on Food Additives, 226–7 kairomones, 222 Kames cage, 740 Karenia brevis, 589, 596, 645 Karenia mikimotoi, 590, 598 KK3D, 1086 KMag, 1001 Lactobacillus, 452 Lamellidens marginalis, 755 Land Change Modeller, 719 LANDSAT SPOT, 713 Lanthella basta, 871 LarvalBase, 1104 Lates calcarifer, 317, 960, 1121 leaching, 349

1178

Index

learning content management system, 1100 learning management system, 1100 Learning Object Metadata, 1101 Learning Technology Standards Committee, 1101 lecithin, 449 Lepeophtheirus salmonis, 907 Lepomis macrochirus, 991 levamisole, 281–2 liming, 989–90 linkage maps, 64–5 commercially important finfish species, 65 LinkedIn, 1104 liquid chromatography-mass spectrometry/mass spectrometry, 596, 597 Lissondendorxy sp., 877 Listeria, 451 Litopenaeus vannamei, 1010–25, 1120, 1121, 1127 lobsters advances in aquaculture, 822–36 breeding, 827–8 broodstock, 827 maturation and mating, 828 spawning, 828 current situation and constraints, 823–7 hatchery-based lobster aquaculture, 826–7 lobster ranching, 824–6 species of interest, 823–4 future trends, 835–6 hatchery technology, 828–32 hatching, 829 incubation, 828–9 larval culture, 830 larval handling and assessment, 829–30 larval health, 832 larval nutrition, 831–2 larval systems, 830–1 product issues: markets, 835 production systems, 832–5 food and feeding, 834–5 grow-out, 833–4 nursery, 832–3 systems, 834 ‘long-arm’ aerator, 996 loop-mediated isothermal amplification, 202

loss on ignition, 907 Lotek MAP, 1077 Lotus Notes, 1089 low head oxygenator, 971–3 ‘lower Cenomanian Turonian’ aquifers, 1136 Loxechinus albus, 720, 723 Luffariella variabilis, 871, 876 Luminex xMAP, 203 Lyngbya majuscula, 644 Lyprinol, 880–2 lysozyme, 817 Macrobrachium rosenbergii, 1120, 1121, 1127, 1134 macronutrients, for salmonids, 506–7 carbohydrate, 507 fatty acids, 507 protein and amino acids, 506–7 magnesium salts, 1000–1 majusculamide C, 651 male-specific RNA, 560 managed learning environment, 1100 management information systems, 1065 mannan oligosaccharides, 513–14 manoalide, 871, 872, 876 Manual of Diagnostic Tests for Aquatic Animals, 255 map algebra, 710, 716 Marel Food Systems, 1095 marennine, 620, 640 mariculture, 870 Marine Shrimp Farming Program, 1013 Maritech, 1089, 1095 marker assisted selection, 66–9, 224, 779 genomics, 67–8 transgenesis, 68–9 marumerization, 348–9 massively parallel signature sequencing, 100 mathematical models, 219 Matrix, 1086 maximum residual level, 226–7 medicinal foods, 873 Mekong River Delta, 1040 melanism, 476 Melanotaenia fluviatilis, 753 membrane bioreactor technology, 557 mengovirus, 552 Mercatus Aqua Farmer, 1080

Index Mercatus Aqua Farmer and Farmcontrol, 1080 metabolic body weight, 419–20 metabolic rate, 425 metal ions, 279 methionine, 449 methylmercury, 375–6 microalgae advances in culture for aquaculture feed and other uses, 610–58 aquaculture feed, 621–35 commercial products currently available on the market, 629–34 ‘green water’ and ‘pseudo-green water’ techniques, 625–6 live prey, 624 major classes and genera used in aquaculture, 623 molluscs, 621–4 preserved microalgae for aquaculture, 627–8, 635 sea urchin culture, 626–7 shrimp culture, 626 commercial producers of microalgae pigment products, 641–2 commercially produced, 612 cosmetics, 643 current status and new techniques for culture, 611–21 advances in techniques, 617, 620–1 culture systems, 613 main features of different cultivation systems, 618–19 and cyanobacteria bioactive products, 646–9 main toxins, 650–1 cycle for photosynthetic oxygenation of wastewater, 655 dietary supplements and animal feed additives, 635–6 future trends, 657–8 high-wave molecules from algae, 637, 639–40 fatty acids, 637, 639 pigments, 639–40 and microalgae-like microorganisms PUFA content, 638 probiotics in aquaculture, 652–3 reactors and techniques for microalgae culture, 614–17 culture systems for benthic diatoms, 617

1179

flat photobioreactors, 616–17 sleeves and vertical columns, 614–15 tubular photobioreactors, 615–16 source of pharmaceuticals, 643–5, 651–2 wastewater reclamation and biofuel production by algae–bacteria consortia, 653–7 microbead filters, 969–70 microbound diets, 347 microcoated diets, 347 microcystins, 636 microencapsulated diets, 347–8 micropyle, 150 microsatellite-enriched small-insert genomic libraries, 12–13 microsatellites, 10–13 polymorphism, 11–12 Microsoft Access, 1079 Microsoft BizTalk RFID platform, 1095 Microsoft Exchange, 1089 Microsoft SQL databases, 1080 Microwave Telemetry Inc., 1078 Mikrocytos roughleyi, 96 mitochondrial genome, 6 ‘mixed-cell’ raceway design, 951 Model AJ 10, 979 Model 84P, 978 Modicon MODBUS, 1070 MODIS, 901 molecular farming, 651 molecular markers, 62–3 mollusc viruses, 247–8 Molluscan Broodstock Program, 94 molluscan shellfish current status of established breeding programs, 92, 94–7 C. gigas selection, 94 major selective breeding programs, 93 oyster selection programs in Australia, 94–6 Pacific oyster and green-lipped mussel selective breeding in New Zealand, 96–7 genetic variation and selective breeding, 87–100 monitoring genetic diversity and inbreeding risks, 88–90 present needs and future trends, 97–100

1180

Index

candidate genes, genomics and marker-assisted selection, 98–100 DNA fingerprinting, 97–8 quantitative trait loci mapping, 98 traits inheritance, 90–2 heritability estimates, 90–2 MOM, 1086 Moodle, 1100 mooring system, 921 connected to interconnected hinged steel fish farm, 922 and floats in still water, with Tension Leg Cage fish farm, 936 grid, circular HDPE collar fish farm, 926 grid mooring system for HDPE collar fish farms, 922 Morrison equation, 923 Mote Aquaculture Park in Sarasota, 977, 979 Mote Aquaculture sturgeon system, 977, 979 moving bed filters, 967–9 mulloway. see Argyrosomus japonicus Multi-Criteria Evaluation module, 717 Multi-Objective Land Allocation tool, 718, 722 multiple drain systems, 953–5 Murex whelks, 883 Muriellopsis sp., 640 mycalamide A, 876 Mycale hentscheli, 876 mycotoxins, 289 Myrionecta rubrum, 589, 593 Myspat INVE Technologies, 623 Mytilus edulis, 755, 758 NADPH-oxidase pathway, 278 Nannochloropsis, 624, 625, 628 nanotechnology, 209 National Estuarine Eutrophication Assessment, 694 National Pollutant Discharge Elimination system, 1005 national scenic area, 742 National Shellfish Sanitation Program, 548, 560 Nautilus fish farm, 937–8 Navicula, 623 NAVSTAR system, 714 near-horizontal tubular reactor, 615 near-infrared spectroscopy, 409

nephrocalcinosis, 792 Nile tilapia. see Oreochromis niloticus nitrification, 1016, 1022 Nitzschia, 623 Nitzschia navis-varingica, 589 nodavirus, 783 non-chemical methods, 221–5 non-nutritive immunostimulants, 281–2 as feed additives, 281 non-starch polysaccharide, 508 nori, 643 normalised difference vegetation index, 714 noroviruses, 544, 546 Northwest Marine Technology, 1075 Norwegian standard NS 9415, 924 Nostoc, 610, 635 Nostoc sp, 644 ‘Nubian Sand Stone,’ 1136 nutraceuticals, marine, 872–4 nutrient retention, 404–5 nutrigenomics, 389 Nutrinova, 639 nutrition determination, 319–22 diet manufacturing methods, 346–9 marumerization, 348–9 microbound diets, 347 microcoated diets, 347 microdiets using different techniques, 346 microencapsulated diets, 347–8 digestive system capacity, 343–5 degree of hydrolysis, 345 dosage system, 355–9 automatic microdiet dispenser, 356 cleaning time or cleaning efficiency, 358–9 delivery to the rearing tank, 356–7 fractioning of the daily ration into multiple events, 357 sparing on the quantity of microdiet, 357–8 feeding system, 354–5 fish larvae, 322–32 arachidonic acid requirements, 326–7 balanced EPA ratio, 327–8 docosahexaenoic acid requirement, 323–5 eicosapentaenoic acid requirement, 325–6

Index essential fatty acids requirements, 322–3 phospholipids, 328–30 protein and amino acid requirements, 331–2 vitamins and minerals, 330–1 food identification and ingestion, 332–3, 335–6 amino acids as feed attractant, 334 effect of krill hydrolysate inclusion method on growth of yellowtail kingfish, 336 feed attractants presentation, 336 feeding process, 333 krill hydrolysate effect on ingestion rates, 335 marine organism hydrolysates and free amino acids as feed attractants, 337 future directions, 359 microdiet characteristics, 349–52, 354 buoyancy, 350–1 Curnow et al. weaning protocols, 354 feeding protocols on barramundi larvae, 352 leaching, 349 leaching rates, 350 lysine leaching pattern, 350 references to weaning protocol, 353 sinking patterns of commercial diets, 351 weaning and co-feeding methods, 351–2, 354 ontogeny of digestive capacity in marine fish larvae, 336–43 comparison of digestive tract developmental stages, 339 intestinal brush order enzyme and intracellular peptidase activity, 340 nutritional requirements, 417–38 feed formulation and feed strategies, 433–5 comparison of energy and protein requirements in fish production, 436 daily energy and protein requirements calculation, 433 digestible energy and protein required for growth, 435

1181

grouper and mullet predicted energy and protein requirement, 436 feed ingredient evaluation, 431–2 future trends, 437–8 nutrient release, 437 quantification, 419–31 composition of weight gain, 424–5 energy and protein utilisation efficiency, 427–31 growth and feed intake, 420–3 metabolic body weight, 425–7 methodology, 419–20 Nuvan, 229 NZ Greenshell mussels, 882 Ocean Sciences Centre, 783 OceanGlobe fish farm, 938–9 in maintenance position with support vessel, 939 ochratoxins, 509 Octaform, 949 Odontella aurita, 636 off-shore farming characterisation and selection of sites, 897–9 definition, 897–8 site selection criteria and methods, 898–9 classification of off-shore waters based on wave heights, 898 context for off-shore farming, 895–7 environmental concerns, 904–9 finfish species cultivation, 899–901 cold and cool temperate water species, 900 potential environmental effects, 905–8 species in development, 900–1 tropical and sub-tropical species, 900 future trends, 909–10 mollusc culture, 901–4 drivers and limitations, 901 other considerations, 904 potential environmental effects, 908–9 species cultivated in off-shore environments, 902–4 technologies for off-shore mollusc farming, 901–2 opportunities and challenges, 895–910

1182

Index

potential environmental impacts form marine net pen farming, 905 submerged longline showing attachment of mussel, 903 used for molluscs suspension culture, 902 oil belching, 502–3 okadaic acid, 871 Olrac, 1095, 1096 Oncorhynchus mykiss, 148, 498–522, 779. see also salmonids Oncorynchus mykiss, 1121 oocyte maturation, 113–14 open database connectivity, 1081 Oracle applications, 1085 Oreochromis niloticus, 1034, 1038, 1044, 1045, 1047, 1049, 1051, 1052, 1138–9, 1153 organic carbon concentration, 965–6 oxidants, 997–8 oxygen cones, 970–1 oxylipines, 645 ozonation, 270 ozone contact, 947, 974–5 P. homarus, 825, 826, 834 Pacific oyster. see C. gigas paddlewheel aerators, 995 Pagrus auratus, 467, 1126 palatability, 399–402 Palinurus elephas, 824 Palo-Alto Software, 1086 Panulirus argus, 824, 830 Panulirus cygnus, 826, 833 Panulirus interruptus, 824 Panulirus japonicus, 824, 827, 835 Panulirus ornatus, 824, 825, 827, 829, 830, 832, 833, 834, 835, 836 Panulirus polyphagus, 824, 826 Panulirus versicolor, 826 Paralichthys dentatus, 1153 parasitic disease new development for control in aquaculture, 215–37 advances in parasite biology and host-parasite interactions, 218–20 advances in parasite control methods, 221–9, 231–6 advances in parasite identification methods, 220–1 effect on the industry, 216–18

future trends, 237 Parastichopus californicus, 882 parentage assignment-based pedigrees, 63–4 partitioned aquaculture system, 1054 passive integrated transponder tags, 1075 pateamine, 876 pathogen control, 268–73 biocontrol agents, 272–3 agents used in aquaculture, 272 hygiene improvement, 270 intestinal microbiota preservation, 269 new antimicrobial preparation and compounds, 270–2 new biocidal compounds, 272 Pavlova, 628 Pavlova lutheri, 622 PCB CB138, 758 peas and lupins, 382 Pecten maximus, 622 pectenotoxin, 589 peloruside, 876 PEPHA-CTIVE, 643 PEPHA-TIGHT, 643 peptidoglycan, 282 peridinin–protein complex, 594 periphyton, 1047 Perna canaliculus, 96–7, 880, 903–4 persistent organic pollutants, 376–8 pesticides, 376–7 polybrominated biphenyls, 377 polybrominated diphenyl esters, 376–7 polychlorinated biphenyls, 376–8 pesticides, 376–7 Phaeodactylum tricornutum, 620, 879, 880 pharmaceuticals, marine, 870–2 phospholipids, 328–30 phosphorus, 511 photobacteriosis, 816–17 Photobacterium damselae subspecies piscicida, 204–5 photobioreactors, 611 photoperiod manipulation, 144–5 photosynthetic oxygenation, 654 pH-STAT technique, 344 phycobilins, 593 phycobiliproteins, 640, 645, 878 phycocyanin, 640 phycoerythrin, 593, 640

Index phycofluor, 645 phycoremediation, 750 ‘phycosphere,’ 652 phytohormonal, 651 phytoremediation, 750, 757 Pinctada albina albina, 755 Pinctada imbricata, 753, 755, 759, 761 piscalators, 815 plankton and krill, 382–3 Plexiglas tubes, 615 PolarCirkel submergible fish farm, 934–5 being submerged, 935 polychlorinated biphenyls, 509 polyculture, 697 polyether okadaic acid, 589 Polygeyser, 961, 962, 967 polymerase chain reaction, 201, 221 pond dynamics, 1041 80:20pond fish culture, 1050 pond hydroponics, 1051–2 POND model, 720 ponds, for finfish production advances in technology and practice, 984–1006 amendments, 997–1002 herbicides, 998–9 microbial products, 999–1000 oxidants, 997–8 bottom treatments, 1002–3 dissolved oxygen management, 994–7 pond aeration and circulation, 996–7 pond aeration devices, 994–6 water circulators, 996 effluents, 1004–6 feeds and feed management, 992–4 feed management, 994 fish meal and fish oil, 992–3 nitrogen and phosphorus, 993–4 future trends, 1006 hydrologic types, 985–8 embankment ponds, 986 excavated ponds, 986–7 pond shape, 988 seepage and erosion control, 987 water reuse, 987 watershed ponds, 985–6 liming and fertilisation, 989–92 fertilisation, 990–2 liming, 989–90 mineral amendments, 1000–2

1183

calcium sulfate, 1000 flocculants, 1001–2 potassium and magnesium salts, 1000–1 sodium chloride, 1000 production methodology, 988–9 water quality monitoring, 1003–4 ‘pop-up’ satellite telemetry tags, 1078 Porphyra spp, 878, 879 Porphyridium cruentum, 640 portunid crabs, 847–8 product issues, 850–1 production systems broodstock quality and nutrition, 856 food and feeding, 854–5 grow-out, 852–3 hatchery practices, 857 nurseries, 858 Portunus pelagicus, 847, 852, 854, 858 Portunus sanguinolentus, 853 Portunus spp, 845, 851 poster fish, 1154 potassium permanganate, 997 potassium salts, 1000–1 PowerSim Solver, 720 prebiotics, 284–5, 451, 1020 Prialt, 871 PRIMER, 692 Pro Manager, 1079 probiotics, 451, 1020 Procentrum concavum, 871 production carrying capacity, 685 Profibus, 1070 programmable logic controller, 1068 proinflammatory cytokines, 278 proportional integral derivative, 1068 ‘prop-washed’ bead filters, 961 Prorocentruma spp., 595 protamine, 874 protein, 279 and amino acid, 331–2 protein content, 424 protein skimming, 947, 976 Protulines, 643 ‘pseudo-green water’ techniques, 625–6 Pseudo-nitzschia, 589, 595 Pseudopleuronectes americanus, 1153 Pseudopterogorgia elisabethae, 872 Pycese, 272 Pyrodinium, 587

1184

Index

QTL detection, 65–6 quantitative trait loci, 98, 252, 779 linkage maps for molluscan shellfish, 99 raceway tanks, 949–51 Rachycentron canadum aquaculture cultivation, 804–18 broodstock and spawning, 805–6 captive spawning, 806 eggs and collection, 806 natural spawning cycles, 805 custom-designed larval rearing system, 811 egg and larval cobia sizes, 807 emerging issues and future trends, 816–18 aquanomics, 817–18 diseases, 816–17 feeding protocol used for production of weanling cobia, 809 harvested at larger sizes, 816 juveniles and on-growing, 812–16 cold banking, 813–14 harvesting, processing and marketing, 814–16 juvenile nutrition, 812 low-salinity culture, 814 replacement proteins, 812–13 larval rearing, 807–12 broodstock diets, 811–12 feeding protocols, live feeds and enrichments, 807, 809 microdiets and weaning, 809–10 systems design, 810 larviculture in ponds, flow-through tanks and recirculating life support systems, 808 percent increase from initial weight of juvenile cobia, 815 radial flow separators, 957 radiation hybrid mapping, 32–4 gilthead sea bream, 34 zebrafish, 33–4 radio frequency identification tags, 1093–4 rainbow trout. see Oncorhynchus mykiss; Oncorynchus mykiss rain-fed ponds, 985–6 Ramsar Convention on Wetlands, 1040 RAMSAR site, 742 random amplified polymorphic DNA, 6

raster data, 710 recirculating aquaculture systems, 697, 1152 advances in technology and practice for land-based aquaculture systems, 945–79 biological filtration, 962–70 floating bead filters, 967 microbead filters, 969–70 moving bed filters, 967–9 organic carbon concentration, 965–6 quantifying the nitrification rate, 964 temperature on nitrification, 966 total ammonia nitrogen concentration, 965 commercially available fibreglass oxygen cone, 971 the Cornell dual-drain, 955 ‘cross-flow’ raceway, 951 definition, 946 design components, 947–8 drum screen filter for waste solids removal, 958 freshwater vs marine systems design, 975–7 changes in nitrification rates, 976–7 fine and dissolved solids control, 976 generic microbead filter, 969 incline belt filter manufactured by Salsnes Filter AS, 959 treating recycled system water, 960 low head oxygenator design, 972 mixed cell raceway design, 952 modern approach to a complete systems design, 977–9 moving bed media and filters, 968 octagonal structural aluminium tanks with interior fibreglass, 950 oxygenation components and processes, 970–3 low head oxygenator, 971–3 oxygen cones, 970–1 plan view of four-tank module for sturgeon production, 978 Polygeyser bead filter, 961 radial flow separator, 957 settleable solids capture components, 955–7

Index radial flow separators, 957 swirl separator, 955–7 sterilisation components and processes, 973–5 ozone contact, 974–5 UV light, 973–4 suspended solids capture components, 957–62 expandable bed filters, 960–2 screen filters, 958–60 swirl separator, 956 tank, water input manifolds, and drain design, 948–55 circular flow tanks, 951–3 multiple drain systems, 953–5 raceway tanks, 949–51 water input manifolds, 953 traditional tube in shell pressurised design and more recent drop-in open channel UV units, 974 types of particulate waste solids, 948 vertical manifold for evenly distributing water input, 954 Red River Delta, 1045 red sea bream. see bream; Pagrus auratus red tide. see harmful algal blooms Redfield Ratio, 991 Regulation EC/178/2002, 1090 Regulation EC/852/2004, 1090 Regulation EC/1830/2003, 1092 remote terminal units, 1068 remotely operated underwater vehicles, 693 Renibacterium salmoninarum, 205 Resilience, 872 reverse transcriptase – polymer chain reaction, 551 RFID. see radio frequency identification tags Rhizosolenia chunii, 595 Rhodomonas salina, 622 rice–crab farming systems, 851, 858 rigid steel fish farm, 929 truss work design, 930 @RISK, 1081, 1086 RNA interference, 254 RNAi-based gene therapies, 257 R-phycoerythrin, 879 RS-485, 1070 RyR1/FKBP12, 871

1185

S Lyprinol, 882 Sabella spallanzanii, 753 Saccostrea glomerata, 94–6, 758 SADCO, 939 fish farm with steel frame for net cage support, 940 Salmo salar, 498–522, 779. see also salmonids salmon vaccines, 204–5 Salmonella, 451 Salmonella Arizonale, 509 salmonids advances in aquaculture feeds and feeding, 498–522 choice of feedstuffs, 515–16 oils, 516 proteins, 515–16 consumption of dietary fish protein and production of farmed fish protein, 521 cross-sections of distal intestine of Atlantic salmon, 504 dietary additives, 511–14 carotenoids, 511–12 feed enzymes, 513 immunostimulants and pre-biotica, 513–14 vitamins, minerals and amino acids, 511 digestive physiology, 501–5 and regulation, 501 role of intestinal microflora, 502 feed and feedstuff related digestive function alterations, 502–5 impaired lipid digestion, 505 oil belching and pellet durability, 502–3 soybean meal-induced enteritis, 503–5 feed technology and formulation, 500–1 benefits and restrictions, 500–1 high-pressure moist extrusion technology, 500 trends, 501 feeding and feeding systems, 517–18 future trends, 518–22 feeds, 519–20 raw materials, 518–19 sustainability, 521–2 lifespan diets, 516–17 pigment strategies, 517 seasonal diets, 516–17

1186

Index

main principles of high-pressure moist extrusion feed manufacture, 500 nutrition and health, 507–10 malnutrition and fish health, 510 salmon feed history and human health, 510 nutritional requirements, 506–7 macronutrients, 506–7 micronutrients, 506 practical formulations, 515–17 species differences, 514–15 carbohydrate utilisation, 514 carotenoid utilisation, 515 oil belching and bloat, 514 unwanted dietary components and fish health, 507–10 contaminants in oils, 509–10 contaminants in protein feedstuffs, 509 inherent components in protein feedstuffs, 507–9 SAP applications, 1085 saponins, 508 SARNISSA project, 1104 saxitoxin, 587 scallop waste model, 759 scanning electron microscopy, 220 Scenedesmus, 624 Scenedesmus almeriensis, 640 Schizochytrium, 628, 639 Scieanops ocellatus, 1127 ‘scope for growth’ concept, 813 screen filters, 958–60 drum screen filters, 958–9 incline belt filters, 959–60 Scrippsiella trochoidea, 595 Scylla paramamosain, 847, 854, 857 Scylla serrata, 846, 848, 854, 857 Scylla spp, 845, 851, 860 Scytonema hofmanni, 644 scytonemin, 643 scytophycins, 644 sea-farming, 870 seafood processing waste products, 382 Seafood Services Australia, 690 SEAPURA, 878 SeaStation fish farm, 931, 933–4 submerged, 933 SeaWiFS, 901 secchi disc, 691, 754, 1003 sediment profile imagery, 693 segregation analysis, 168–9

selective breeding and genetic variation in hatcherypropagated molluscan shellfish, 87–100 selective breeding programs, 69–71 Sentinel Farms project, 1103 454 sequencing platform, 24–6 epigenetic analysis, 25 gene expression profiling, 25 whole genome sequencing, 25 serial analysis of gene expression, 100 serpentine photobioreactors, 615 sewage treatment plant, 759 sex chromosomes, 61–2 Sharable Content Object Reference Model, 1101 Sharepoint Services, 1089 shellfish chromosome set manipulation, 165–88 schematic presentation, 169 monitoring of viral contamination in growing areas, 542–69 shrimp, superintensive bio-floc production technologies, 1010–25 single nucleotide polymorphism (SNP), 13–17, 779 allele discrimination using BeadArray technology, 17 BeadArray technology, 16 direct DNA sequencing, 14 genotyping, 15 heteroduplex analysis, 14 single-strand conformation polymorphism, 14 single-cell oil, 637 single-sex and sterile population of fish for aquaculture endocrine sex reversal, 156–7 environmental manipulation of sex ratio, 157 future trends, 157–8 uniparenteral inheritance, 154–6 site of special scientific interest, 742 Skeletonema, 626 Skretting, 1096 SMART, 687 sodium chloride, 1000 sodium nitrate, 997–8 Solexa sequencing platform, 23–4 SOLiD sequencing platform, 19–23

Index base position sequenced by ligation and with various primers, 21 chemistry of sequencing platform, 22 clonal amplification of genomic DNA, 20 creation of ‘libraries’ of sheared segments, 20 Soy in Aquaculture website, 452 soy protein, 380 soybean meal, 379–80, 445, 853 Sparus aurata, 470 Sparus latus, 470 spawning, 116 special areas for conservation, 742 special protection area, 742 Speece Cone, 970 spermatogenesis, 110, 114 spermiation, 110, 114–15 spiroplasma bacteria, 846 Spirulina, 879 Spirulina meal, 853, 860 Spongia officinalis, 757, 758 STELLA, 1086 sterile and single-sex fish population in aquaculture, 143–58 direct induction, 145–6, 148–9 future trends, 157–8 genomic stability of triploids, 151–3 tetraploid-diploid crosses, 149–51 triploidy alternatives, 153–4 sterility hypothesis, 180–1 steroidogenesis, 111 Stizosledion vitreum, 1153 STS-89, 1156 STS-90, 1156 sturgeon. see Acipenser SubFish, 939–40 submerged longline technology, 901 summer flounder. see Paralichthys dentatus superintensive bio-floc production technologies, 1011–13 components, 1013–16 bio-floc microbial communities, 1016 feeds and feeding, 1014–15 shrimp stocks, 1013–14 systems engineering, 1015–16 current research priorities, 1017–24 economics and marketing, 1017–18 feed programs, 1020–1

1187

harvesting of grow-out systems, 1023–4 health management, 1018 husbandry, 1021–3 integration of research and commercialisation efforts, 1017 production systems, 1019–20 stocking and harvesting of nursery systems, 1023 for marine shrimp, technical challenges and opportunities, 1010–25 prioritised summary of technology gaps, 1024–5 bio-economic models, 1025 feeds and feeding, 1025 genetic improvement, 1024 health and biosecurity, 1025 larval culture, 1025 system engineering and lifesupport systems, 1024 value-added products, 1025 seed supply, 1018–19 genetics and breeding, 1018–19 seed production, 1019 Waddell Mariculture Center configuration of pilot scale raceway system for nursery production, 1014 greenhouse enclosed raceway system during winter operation, 1012 ‘supervisory control and data acquisition’ system, 1068 suppression subtractive hybridisation, 100 surgical castration, 153 sustained-release delivery systems, 121–2 cholesterol implants, 121 EVAc solid implant, 121–2 microspheres, 121 swirl separator, 955–7 Sydney rock oyster. see Saccostrea glomerata Symploca, 644 synchronus spawners, 116 synthadotin, 645 system of rice intensification, 1044 tagging, 1075 Taiwan paddlewheel aerator, 995–6

1188

Index

Taura Syndrome Virus, 1014 Taxol, 870 Tedania charcoti, 756 telemetry tags, 1077 telemetry technology, 691 Tension Leg Cage fish farm, 935–7 exposed to current and waves, 936 with mooring system and floats in still water, 936 terrace ponds, 985–6 tetradecylthioacetic acid, 520 tetraploid shellfish, 183–4, 186–7 induction, 183–4, 186 tetraploid induction, 185 performance, 186–7 tetraploid-diploid crosses, 149–51 Tetraselmis, 624, 626, 652 Tetraselmis suecica, 614, 625 Thalassiosira, 626 ‘the chill chain,’ 1092 the net cage, 920–1 The New Zealand King Salmon Company, 1085 thematic layers, 710 Thenus, 824, 830, 836 thermal pressure, 146 Thierry Chopins interdisciplinary research laboratory, 875 tilapia. see also freshwater fish species; Oreochromis niloticus commercially produced species, 441 composition of weight gain, 424 daily weight gain in relation to increasing body weights, 421, 423 general nutrient specifications for formulation of practical diets, 454 generic production diets, 454 growth potential, 422 recommended minimum amino acid levels in diets, 455 supplemental mineral levels recommended for practical diets, 456 Tilapia Genome Sequencing Project, 67 tilapia plan, 1154 titanium dioxide, 558 total ammonia nitrogen, 963 concentration, 965 total organic carbon, 907 total suspended solids, 1022

TraceAll, 1095 ‘TraceCore’ XML, 1096 traditional Asian aquaculture, 1029–56, 1033–4 bridging traditional and modern practice, 1050–4 CSC where an increasing protein level in supplementary feed is needed, 1030 definitions and principles, 1030–4 integrated aquaculture, 1033–4 intensity of production, 1030–2 extrapolated annual yield and intensity of culture, 1031 future trends, 1054–6 growth of Nile tilapia in fertilised ponds with additional feeding to 50% satiation by commercial pelleted feed, 1052 with additional fractions of satiation feeding with commercial pelleted feed, 1052 recent changes to traditional practice, 1037–41 integrated agriculture/aquaculture systems, 1037–9 integrated fisheries/aquaculture systems, 1040–1 integrated peri-urban aquaculture systems, 1039–40 recent development of semiintensive aquaculture, 1047–50 carp polyculture in Bangladesh, 1048–9 carp polyculture in India, 1047–8 small-scale farm integration in Malawi, 1049–50 reduction of wastes in situ, 1050–2 80:20 system, 1050 bio-floc technology, 1050–1 commercial pellets as supplementary feed, 1051 pond hydroponics, 1051–2 relationship between nutritional input and intensity of system, 1044 research and development for improved traditional practice, 1041–7 fertilisation, 1041–3

Index integrated agriculture/aquaculture systems, 1044–5 periphyton, 1047 supplementary feeding, 1043–4 wastewater-fed aquaculture, 1045–7 traditional aquaculture systems integrated agriculture/aquaculture systems, 1034–6 integrated fisheries/aquaculture systems, 1037 integrated peri-urban aquaculture systems, 1036 treatment of intensive aquaculture effluents, 1053–4 aquaponics, 1053–4 intensive cage culture within semi-intensive pond culture, 1053 intensive pond effluents for semi-intensive pond culture, 1053 partitioned aquaculture system, 1054 transgenesis, 68–9 transmission electron microscopy, 220 trepang, 882 triangular airlift reactor, 616 trimethylamineoxide, 507 triploid shellfish, 174–5, 178–83 growth, 175, 178–81 induction and post-larval growth, 176–7 positive correlation between meat weight and multilocus heterozygosity in diploids & triploids, 180 triploid C. virginica, 178 larval performance, 175 meat quality, 182 sterility and genome stability, 183 survival, 182 triploidy, 58–9 triploidy induction, 145–6, 148–9 diploid, triploid & tetraploid embryo production, 147 Trizol, 551 trypsin inhibitor activity, 508 T-tags, 1075 tubular photobioreactors, 615–16 Turboxygene Model LR200, 979 Turnitin, 1102

1189

T-Vet, 1099 TZT-1027, 645 UKLeaP, 1102 uniparenteral inheritance, 154–6 Upper Klamath Lake, 636 urban, vs rural, 1149 urban aquaculture description, 1149 the economics: siting, processing, and marketing, 1157–8 future trends, 1159–61 goals, 1151–2 marketing and competition, 1158–9 New York as a model, 1148–61 potential programs, 1153–6 role of university community outreach, 1159 education, 1159 research and development, 1159 technology, 1152–3 USDA Western Regional Aquaculture, 94 UV irradiation, 172, 270 UV light, 973–4 vaccination, 257 vaccine development, 223–4 advances, 203–7 electron micrographs of Renibacterium salmonarum, 206 western blot analysis Photobacterium damselae, 205 key drivers for improvement, 198 vector data, 710 VI alpha tags, 1075 Vibrio alginolyticus, 483, 753 Vibrio anguillarum, 783 Vibrio anguillarum II, 753 vibriosis, 780 viewshed sub-model, 744 Vig-1, 251 Vig-2, 251 viral contamination characteristics of the main enteric viruses, 545 detection methods, 550–3 quantification, 553 rapid review, 550–3 effects of different physical factors on virus inactivation in shellfish, 566–7

1190

Index

efficiency of different waste water treatment plant to remove norovirus, 558 human enteric viruses, 544, 546–7 adenovirus, 547 Aichi virus, 546 astrovirus, 546 enterovirus, 546 hepatitis A virus, 544 hepatitis E virus, 547 noroviruses, 544, 546 rotavirus, 546 input and flux, 553–62 animal output, 555 enteric viruses in sewage and rivers, 555–8 flux calculation from sewage, 558–9 potential indicators, 560, 562 seasonal outbreaks, 553–5 surface water, 559–60 monitoring of shellfish growing areas, 542–69 future trends, 568–9 other issues, 565, 568 possible sources for shellfish growing in coastal area, 550 reduction strategies, 562–5 depuration, 564–5 persistence of viruses in shellfish tissues, 563–4 virus resistance, 562 sources of pollution, 544–50 animal viruses, potential zoonotic viruses, 547–8 outbreaks with emphasis on the source of virus contamination, 548–50 titers of enteric viruses in stool specimens, 554 viral disease new developments for control in aquaculture, 244–59 advances in immunity of aquacultured species to viral diseases, 249–54 limitation of current management techniques, 248–9 new methods for disease control and future trends, 254–9 overview, 245, 247–8 viral nervous necrosis, 783, 796 ‘virtual learning environment,’ 1100

virus-induced genes, 251–2 virus-like particles, 563–4 visible implant elastomer tags, 1075 vitamin A, 331 vitamin B12, 449 vitamin C, 331, 450 vitamin D, 331 vitamins, 279 vitamins and minerals, 330–1 vitellogenesis, 110, 113 volatile suspended solids, 1022 volumetric nitrification rate, 964 volumetric TAN conversion rate, 964 Waddell Mariculture Center raceway system configuration of pilot scale for nursery production, 1014 enclosed, greenhouse during winter operation, 1012 walleye. see Stizosledion vitreum wastewater-fed aquaculture, 1045–7 water disinfection, 270 water input manifolds, 953 water recirculation system, 795. see also recirculating aquaculture systems watershed ponds, 985–6 weaning, 351–2, 354 Web Ontology Language, 1102 ‘wet chemistry’ nutrient analyses, 691 wheat and barley, 380–1 whirling disease, 221 white grouper composition of weight gain, 425 energy and protein loss, 426 growth potential, 423 ‘white-gut’ syndrome, 832 white-spot syndrome virus, 847 WikiBooks, 1104 winter flounder. see Pseudopleuronectes americanus WiseFish Production, 1089 xenobiotics, 271 Xiphophorus helleri, 1156 X-ray irradiation, 172 ‘Yang Cheng’ crabs, 850 yellowfin sea bream. see Sparus latus yessotoxins, 589 Yondelis, 870

Index Ziconitide, 871 Zirconia beads, 551 Zocar, 870 zooremediation, 751 of aquatic systems, 750–64 checklist for candidate species, 762 definitions, 751–2 the ethics of employing animals, 763 future trend, 761–4 nitrogen, phosphorus and metal content of dried tissues from Pinctada imbricata, 760 and pearl aquaculture, 758–9, 761 of pollutants, 752–8

1191

zooextraction of heavy metals, 755–7 zooextraction of nutrients and microorganisms, 752–4 zooextraction of organic pollutants, 757–8 zoostabilisation/degradation of organic pollutants, 758 zoostabilistaion/degradation of nutrients and mircoorganisms, 752–5 self-financing models, 756–7 phytoremediation, 756 zooremediation equivalents, 752