Advances in Parasitology Volume 44

Advances in PARASITOLOGY

VOLUME 44

Editorial Board C. Bryant Division of Biochemistry and Molecular Biology, The Australian National University, Canberra, ACT 0200, Australia

M. Coluzzi Director, Istituto di Parassitologia, Universita Degli Studi di Roma ‘La Sapienza’, P. le A. Moro 5, 00185 Roma, Italy C. Combes Laboratoire de Biologie Animale, Universitt de Perpignan, Centre de Biologie et d’Ecologie Tropicale et MCditerranCenne, Avenue de Villeneuve, 66860 Perpignan Cedex, France

D.D. Despommier Division of Tropical Medicine and Environmental Sciences, Department of Microbiology, Columbia University, 630 West 168‘h Street, New York, NY 10032, USA W.H.R. Lumsden 16A Merchiston Crescent, Edinburgh, EHlO 5AX, UK J.J. Shaw Instituto de Ciincias Biomtdicas, Universidade de Siio Paulo, av. Prof. Lineu Prestes 1374,05508-900, Cidade Universitaria, Siio Paulo, SP, Brazil

Lord Soulsby of Swaflham Prior Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 OES, UK

K. Tanabe Laboratory of Biology, Osaka Institute of Technology, 5-16-1 Ohmiya, Asahi-Ku, Osaka 535, Japan

P. Wenk Falkenweg 69, D-72076 Tubingen, Germany

Advances in PARASIT0LOGY Edited by

J.R. BAKER Royal Society of Tropical Medicine and Hygiene, London, England

R. MULLER International Institute of Parasitology, St Albans, England and

D. ROLLINSON The Natural History Museum, London, England VOLUME 44

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CONTRIBUTORS TO VOLUME 44 N . BOULTER,Department of Biology, PO Box 373, University of York, York YO1 5 Y W , U K L A . CHISHOLM, Department of Parasitology, The University of Queensland, Brisbane, Queensland 4072, Australia E. HANDMAN,The Walter and Eliza Hall Institute of Medical Research, Post Ofice Royal Melbourne Hospital, Victoria 3050, Australia R. HULL, Department of Biology, PO Box 373, University of York, York YO1 5 Y W , UK H . MONE, Laboratoire de Biologie Animale, U M R no. 5555 du CNRS, Centre de Biologie et d'Ecologie tropicale et mhditerrane'enne, Universith, Avenue de Villeneuve, 66860 Perpignan Cedex, France S. MORAND, Laboratoire de Biologie Animale, U M R no. 5555 du C N R S , Centre de Biologie et d'Ecologie tropicale et me'diterrane'enne, Universite', Avenue de Villeneuve, 66860 Perpignan Cedex, France G. MOUAHID,Laboratoire de Biologie Animale, U M R no. 5555 du C N R S , Centre de Biologie et d'Ecologie tropicale et me'diterranhenne, Universite', Avenue de Villeneuve, 66860 Perpignan Cedex, France A.W. PIKE, Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, and Marine Harvest McConnell, Lochailort, Inverness-shire PH38 4 L Z , U K K . ROHDE,School of Biological Sciences, Division of Zoology, University of New South Wales 2351, Australia S.L. WADSWORTH,Department of Zoology, University of Aberdeen. Tillydrone Avenue, Aberdeen AB24 2 T Z , and Marine Harvest McConnell, Lochailort, Inverness-shire PH38 4 L Z , UK I.D. WHITTINGTON, Department of Parasitology, The University of Queensland, Brisbane, Queensland 4072, Australia

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The volume opens with a review of the cell biology of the flagellate protozoan genus Leishmania by Emanuela Handman (Walter and Eliza Hall Institute of Medical Research, Victoria, Australia). Infection with any of about ten species of this genus in humans causes one of three main clinical manifestations: cutaneous, mucocutaneous or visceral leishmaniasis. The World Health Organization estimates that there are currently from 3 to 5 million cases in the world and the prevalence is rising. Visceral leishmaniasis is usually fatal if not treated and is reckoned to have killed about 75 000 people in one year alone in recent outbreaks in India and Sudan. The author reviews recent progress made in understanding the cell biology of this fascinating parasite, which invades macrophages, the very cells which should protect against invading organisms, as it shuttles between the mammalian tissues and the gut of the sandfly intermediate host. The host and parasite molecules that facilitate the establishment of infection, parasite survival in the two hosts and its transmission from one to the other are given special emphasis. She forecasts that the complete genome will be sequenced in the coming decade, and the challenge will be to identify the function of the genes and then to understand the whole organism better. The volume continues with an account by Nicola Boulter and Roger Hall (University of York, UK) of the immune response of cattle to the apicomplexan protozoan parasites Theileria annulata, T. parva and T. sergenti. The global cost of these parasites to agriculture is estimated to be over one billion (lo9) US dollars annually. Immunity to T. annulata and T. parva is predominantly cell mediated (by cytostatic macrophages and cytotoxic lymphocytes, respectively), while that against T. sergenti, although less understood, appears to be mainly humoral. Control of these infections is currently directed against the parasites, by chemotherapy or vaccination, and against the vectors (ticks). Neither the use of acaricides nor chemotherapy is particularly effective, and both are expensive. Vaccination against T. annulata gives over 90% homologous protection and is often cross-protective against the other two species also. However, it involves the use of a live, attenuated vaccine with the attendant need for constant refrigeration and the risk of inadvertent transfer of other pathogens with the vaccine. Current research is directed towards the production of effective,

viii

PREFACE

stable and cheap subunit vaccines requiring only a single application, and the authors predict that success will eventually be achieved with ‘naked DNA’ vaccines containing cytokine genes as immunopotentiators. Ian Whittington and Leslie Chisholm (University of Queensland, Australia) and Klaus Rohde (University of New England, Australia) have contributed the first detailed review of the larvae (oncomiracidia) of the class Monogenea for over 30 years (Advances in Parasitology 1, 1963, and 6, 1968). Members of this group have direct life cycles and some are economically important parasites of fishes, particularly in aquaculture. The authors have examined in detail the general morphology and behaviour of many examples, and also the structure of the epidermis, ciliated cells, haptorial sclerites, glands, digestive system, protonephridia and sense organs in particular. In 1957 a new classification of the group based on larval rather than adult characteristics was proposed and it was realized that the phylogenetic relationship to the Digenea was more remote than previously thought. Recently, there has been controversy about whether the subclasses Monopisthocotylea and Polyopisthocotylea have a monophyletic origin or whether the many similar characteristics are due to convergence brought about by similar selection pressures. The authors have not been able to elucidate their phylogenetic relationships conclusively but indicate which larval characters, in addition to adult features and molecular data, must be combined to provide a comprehensive data set. They also point out many areas where there is still a lack of knowledge and this should act as a stimulus to further studies. The distribution of schistosomiasis reflects in part the distribution of potential intermediate host species. A knowledge of the intermediate snail hosts of schistosomes is essential for the recognition of transmission foci and allows an assessment of the risk of the disease spreading to new areas. In this review article Helene Mone, Gabriel Mouahid and Serge Morand (University of Perpignan, France) examine the distribution of Schistosoma bovis and consider the reported interactions occurring between parasites and snails. This species has a wide intermediate host spectrum and naturally infected molluscs belong to two genera, Planorbarius and Bulinus. The authors recognize three major groups of S . bovis populations (Iberian, Mediterranean and south Sahara) and propose a possible local adaptation to the parasite in the Iberian Peninsula. The article provides fresh insights into the biogeography of S. bovis and complements the recent review in Volume 41 by Jan De Bont and Jozef Vercruysse on schistosomiasis in cattle. The increasing demand for fresh salmon has given rise to a dramatic increase in high-density farming of fish in cages in Scotland, Norway and North America. Parasitologists are well aware that large groupings of hosts inevitably lead to disease problems and one of the most serious parasitic

PREFACE

ix

diseases in salmonid aquaculture is due to crustacean ectoparasites known as sealice. In 1998 the costs resulting from sealice damage in Scotland alone were estimated at &15-30 million. Not surprisingly there has been a considerable increase in research concerning sealice. In this detailed review on both biology and control, Alan Pike (University of Aberdeen, UK) and Simon Wadsworth (Marine Harvest McConnell, UK) pay particular attention to Lepeophtheirus salmonis and Caligus elongatus, the two common species of sealice. Much recent research has been directed towards finding new treatments and methods of control, and this review emphasizes the need to understand the basic biology of the parasite and identifies those research areas in need of further investigation. J.R. Baker R. Muller D. Rollinson

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CONTRIBUTORS TO VOLUME 44 . . . . . . . . . . . . . . . . . PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V

vii

Biology of Leishmania E. Handman Abstract . . . . . .

. . . . . . . . . . . . . .. . . . . . . . . . . . . . .. 2 . The Interaction of Leishmania with the Macrophage . . 3. The Interaction of Leishmania with the Sandfly . . . . . 4. Concluding Remarks . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . 1 . Introduction

. . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 2 5 21

26 21 21

Immunity and Vaccine Development in the Bovine Theilerioses N. Boulter and R . Hall

1. 2. 3. 4. 5. 6.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Theileria annulata . . . . . . . . . . . . . . . . . . . . . . . Theileriaparva . . . . . . . . . . . . . . . . . . . . . . . . Theileria sergenti . . . . . . . . . . . . . . . . . . . . . . . Comparative Aspects . . . . . . . . . . . . . . . . . . . . . TheFuture . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

42 43 51

61 11

80 82 83 83

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CONTENTS

The Distribution of Schistosoma bowis Sonsino. 1876 in Relation to Intermediate Host- Parasite Relationships H. Mone. G . Mouahid and S. Morand 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Collection of Data . . . . . . . . . . . . . . . . . . . . . . The Natural Mollusc Intermediate Host Spectrum . . . . . . Geographical Distributions of the Mollusc Intermediate Hosts Geographical Distribution of S . bovis . . . . . . . . . . . . The Experimental Mollusc Intermediate Host Spectrum . . . Compatibility in the Mollusc-S . bovis Association . . . . . . Three Main Populations of S . bovis . . . . . . . . . . . . . Paleobiogeographical Scenario of S . bovis . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

100 100 102 . 113 . 113 . 124 . 125 . 125 . 128 . 130 132 133 133

The Larvae of Monogenea (Platyhelminthes)

.

I.D. Whittington. L.A. Chisholm and K Rohde

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . General Morphology . . . . . . . . . . . . . . . . . . . . . Haptoral Sclerites. . . . . . . . . . . . . . . . . . . . . . . Ciliated Cells . . . . . . . . . . . . . . . . . . . . . . . . . Epidermis . . . . . . . . . . . . . . . . . . . . . . . . . . Terminal Globule . . . . . . . . . . . . . . . . . . . . . . . Glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protonephridia . . . . . . . . . . . . . . . . . . . . . . . . Sense Organs . . . . . . . . . . . . . . . . . . . . . . . . . Nervous System . . . . . . . . . . . . . . . . . . . . . . . Digestive Tract . . . . . . . . . . . . . . . . . . . . . . . . Parenchyma . . . . . . . . . . . . . . . . . . . . . . . . . Behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

140 141 142 146 153 159 163 169 177 183 198 200 201 203 215 218 218

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CONTENTS

Sealice on Salmonids: Their Biology and Control A.W. Pike and S.L. Wadsworth 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Species, Morphology, Host Range and Geographical Distribution The Reproductive System and Reproduction . . . . . . . . . Life Cycles of Sealice. . . . . . . . . . . . . . . . . . . . . Epidemiology of Sealice Infections . . . . . . . . . . . . . . Physiology of Sealice . . . . . . . . . . . . . . . . . . . . . Pathological Effects of Sealice on Salmonids . . . . . . . . . Treatment and Control of Infection . . . . . . . . . . . . . . Economics of Sealice Infection . . . . . . . . . . . . . . . . Priority Areas for Future Sealice Research . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF VOLUMES IN

THISSERIES . . . . . . . . . . . . .

234 234 238 245 261 268 279 286 292 311 317 318 318 339 349

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Cell Biology of Leishmania Emanuela Handman

The Walter and Eliza Hall Institute of Medical Research. Post Ofice Royal Melbourne Hospital. Victoria 3050. Australia Abstract ....................................................................... 2 1. Introduction ................................................................. 2 2 . The Interaction of Leishmania with the Macrophage ........................... 5 2.1. Promastigote Invasion and Establishment of Infection ..................... 5 2.2. Promastigote to Amastigote Differentiation in the Macrophage ........... 15 2.3. Mechanisms of Parasite Persistence ..................................... 18 3. The Interaction of Leishmania with the Sandfly ............................... 21 3.1. Parasite Differentiation in the Blood Meal ................................ 21 3.2. Establishment of Infection in the Sandfly ................................ 22 3.3. Metacyclogenesis ...................................................... 23 3.4. Transmission to the Mammalian Host ................................... 24 4. Concluding Remarks........................................................ 26 4.1. Cell Biology in the Post-genome Era .................................... 26 Acknowledgements ........................................................... 27 References.................................................................... 27

ADVANCES IN PARASITOLOGY VOL 44 ISBN 0-12-031744-3

Copvrrghr 2000 Academic Press A / / righrs of rrprodurrron in ang fonn rr.wrvd

2

E. HANDMAN

ABSTRACT

Leishmania are digenetic protozoa which inhabit two highly specific hosts, the sandfly, where they grow as motile flagellated promastigotes in the gut, and the mammalian macrophage, where they survive and grow intracellularly as non-flagellated amastigotes in the phagolysosome. Leishmaniasis is the outcome of an evolutionary ‘arms race’ between the host’s immune system and the parasite’s evasion mechanisms, which ensure survival and transmission in the population. The diverse spectrum of patterns and severity of disease reflect the varying contributions of parasite virulence factors and host responses, some of which act in a host protective manner while others exacerbate disease. This chapter describes the interaction of the Leishmania with their hosts, with emphasis on the molecules and mechanisms evolved by the parasites to avoid, subvert or exploit the environments in the sandfly and the macrophage, and to move from one to the other.

1. INTRODUCTION

Leishmania parasites were first noticed by Cunningham in 1885, and described subsequently by Leishman in 1900 and Donovan in 1903 (quoted in Peters, 1988). Leishmaniasis is endemic in the tropical regions of Africa and the Americas, in the Indian subcontinent, and in the Mediterranean and South-west Asian regions. It is a group of diseases with a spectrum of clinical manifestations ranging from self-healing cutaneous ulcers to severe disease with massive tissue destruction and even death. Despite the varied clinical manifestations and the homing of the organisms to different organs, all leishmaniases are caused by infection with the protozoan parasite Leishmania. Moreover, all infections start with the introduction of the organisms into the skin by the bite of an infected sandfly. However, there is considerable diversity within the genus, with at least ten species of Leishmania that are pathogenic for humans. Different Leishmania species also display significant preference, if not absolute specificity, for particular sandfly species. Traditionally, leishmaniasis has been classified into three groups according to the clinical manifestations of disease. Cutaneous leishmaniasis is, by and large, a self-limiting, but chronic skin ulcer developing at the site of the sandfly bite, which may take months to heal. Mucocutaneous leishmaniasis initially causes similar skin ulcers that heal, but subsequently lesions reappear, primarily in the mucous tissue of the nose and mouth.

CELL BIOLOGY OF LEISHMANIA

3

These are often accompanied by secondary infections and massive tissue destruction. Visceral leishmaniasis is a very severe systemic disease, with the organisms homing to the liver, spleen and bone marrow. Visceral leishmaniasis is usually fatal if not treated. The global number of infected individuals cannot be determined with accuracy, but the World Health Organization (WHO) estimates that there are at least 3-5 million clinical cases among the 12 million infected individuals from a total population of about 350 million living in endemic areas (Modabber, 1993). Of the 1.5 million new cases each year, it is estimated that 500 000 are visceral leishmaniasis. The most recent epidemics of visceral leishmaniasis in India and the Sudan are estimated to have killed about 75 000 people in 1991 alone. Currently, a new wave of epidemic visceral leishmaniasis is sweeping through many parts of the world (McGregor, 1998). The general prevalence of leishmaniasis has increased significantly over the last decade or so, as a result of wars, environmental degradation and unplanned urbanization. Another significant development in leishmaniasis has been the reactivation of subclinical asymptomatic infection into full-blown disease in acquired immunodeficiency syndrome (AIDS) patients (Altges et al., 1991; WHO, 1995). All species of Leishmania are transmitted by sandfly vectors, either of the genus Phlebotomus (in the Old World) or Lutzomyia (in the New World), and it is generally accepted that all Leishmania are obligatory intracellular parasites in mammalian macrophages (Alexander and Russell, 1992) (Figure 1). The factors that determine the varied clinical manifestations and severity of leishmaniasis have been an area of intense study and much speculation. It is clear that the species of parasite initiating the infection is important, but equally important is the genetic susceptibility of both the insect vector and mammalian host (Convit et al., 1972; Wu and Tesh, 1990a; Liew and O'Donnell, 1993; Blackwell, 1996). Control of leishmaniasis has been hampered by the fact that the disease is primarily a zoonosis with large reservoirs of rodents, dogs or other animals. In addition, it is now apparent that asymptomatic infection is quite common and represents a potential reservoir in its own right (Aebischer, 1994). Organic pentavalent antimonials have formed the mainstay of treatment for the last half century (Pentostam, Wellcome or Glucantime, Rhone Poulenc). Other drugs such as amphotericin B, allopurinol and aminosidine (paromomycin) have not proved as useful as initially hoped (Olliaro and Bryceson, 1993). Control through vaccination, although the most costeffective form of disease eradication and a long-term quest of the WHOTDR Programme, has only been attempted for cutaneous leishmaniasis (Handman, 1997). Significant progress has been achieved in this area but

4

E. HANDMAN Intracellular amaatlaote " Transtormatlo

Mammalian host

Transformation

Proliferation in the midgut

Figure I A schematic representation of the Leishmania life cycle in the mammalian host and sandfly vector.

there is still a long way to go before production of a successful vaccine for mass administration in the field (WHO, 1998). Leishmaniasis, like many infectious diseases, is the aftermath of a protracted evolutionary 'arms race' between the host defence mechanisms and the parasite virulence factors. The host has evolved an innate rapid

CELL BIOLOGY OF LEISHMANIA

5

deployment defence system, such as the complement system, which is not organism-specific and can be activated without delay. On the other hand, the parasite has developed strategies to overcome the innate immune system, and in so doing it can exploit the very system whose function it is to eliminate the parasites. In this chapter, a review of the progress made in understanding the cell biology of the Leishmania parasite as it shuttles between the sandfly gut and the mammalian tissues is presented. Special emphasis will be given to host and parasite molecules that facilitate the establishment of infection, the parasite survival in the two environments and its transmission from one host to the other.

2. THE INTERACTION OF LHSHMANlA WITH THE MACROPHAGE 2.1. Promastigote Invasion and Establishment of Infection

2.1.1. Promastigote Entry into the Mammalian Host As mentioned earlier, Leishmania are digenetic organisms shuttling between a flagellated promastigote, living in the midgut and foregut of the female sandfly, and an intracellular amastigote in the mammalian macrophage (Figure I). Sandflies generate a small pool of blood by the secretion of saliva into the wound from which they feed (Schlein, 1993). Leishmania promastigotes are deposited by the sandfly into this pool of blood. By analogy to the end-stage developmental forms of African trypanosomes, the developmental stage of the parasite introduced into the mammalian host has been named ‘metacyclic’ and the dividing, immature form present in the fly has been named ‘procyclic’ (Sacks, 1989). A detailed description of the process of metacyclogenesis and the structural changes involved are presented in Section 3.3. The first hurdle the promastigotes encounter in the new environment is the need to escape the lytic effects of serum complement. While the complement system has a central role in host defences against many microorganisms, pathogenic microbes have evolved mechanisms to evade it and, in some cases, such as in Leishmania, to exploit it (Figure 2). There are three mechanisms which activate the complement cascade: the classical pathway, which is primarily activated by immune complexes; the alternative pathway, which is activated by direct binding of the complement component C3 to the microbe surface; and the lectin pathway, which is initiated by binding of the mannose-binding protein to terminal mannose residues on microbial surfaces (Figure 2).

6

E. HANDMAN

Mannan-binding lectin pathway

Pathogen surfaces

binds mannose on pathogen surface

-

I

~3conveflase

I

~5 convertase

I

Membrane-attack complex

Figure 2 The main pathways and components of the complement activation system. (Adapted from Taylor, P. et al., 1998, Current Biology 8, R259-261.)

Early in vivo studies suggested that most promastigotes introduced into the host are killed rapidly and, until recently, killing was presumed to be via the activation of the alternative pathway of complement (Zuckerman, 1975; Alexander and Russell, 1992). Subsequent in vitro studies showed that promastigotes do indeed activate the alternative pathway (Mosser and Edelson, 1984). It was shown that the infectious stage of promastigotes, the metacyclic promastigotes, are much more resistant to lysis than the immature procyclic organisms despite the fact that both forms bind

CELL BIOLOGY OF LEISHMANIA

7

significant amounts of C3b (Joiner, 1988; Puentes et al., 1988). Resistance to complement appears to be due to the inability of the membrane attack complex to penetrate the dense phosphoglycan coat on the parasite surface (Puentes et al., 1989, 1990). A major contributor to the resistance of metacyclic promastigotes to complement is the proteolytic activity of a membrane protease, the ‘leishmanolysin’ or gp63, which cleaves C3b to a form that cannot fix the membrane attack complex (Brittingham et al., 1995; Brittingham and Mosser, 1996). An added advantage to the parasite from the hydrolysis of C3b is the generation of the chemotactic peptides C3a and C5a, which attract monocytes to the area (Brittingham and Mosser, 1996). Newly arrived monocytes that are low in major histocompatibility complex (MHC) class I1 molecules cannot present antigen and are quiescent hosts for the parasites during the early phase of lesion formation (Murray, 1994). A totally new perspective on the mechanism of host cell invasion by promastigotes has been provided recently by Dominguez and Torano (1999), who showed that, in humans, the classical complement pathway plays a much more important role than the alternative pathway. In an in vitro system, promastigotes were shown: to bind natural IgM antibodies present in human blood; to attach to complement receptor CR1 on erythrocytes within seconds of contact with the blood; and to invade neutrophils, where they are destroyed, and also macrophages, where they survive (Dominguez and Torano, 1999). 2.1.2. Phagocytosis of Promastigotes by Macrophages Phagocytosis is an important effector mechanism for the eradication of micro-organisms, and is performed by ‘professional phagocytes’ such as polymorphonuclear cells and macrophages. Paradoxically, the macrophage is both the home of the parasite and also the means of its destruction. Phagocytosis transports the microbes into a cellular compartment where they can be killed and degraded. The macrophage also signals the presence of the intracellular microbe to cells of the adaptive immune system which can in turn activate the macrophage to destroy the parasites through mechanisms involving, in part, signalling via the receptor for y-interferon. Many microbes, including Mycobacterium tuberculosis and Leishmania, have developed mechanisms to subvert the macrophage microbicidal activity and have made it their preferred host cell. Macrophages therefore act both as host cells for the invading parasite and as antigen presenting cells to immune T cells, turning on the Th 1, macrophage-activating responses necessary for parasite destruction (Mauel, 1996). Macrophages are the final

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E. HANDMAN

effector cells which kill the intracellular organisms once a protective T-cell immune response has been established. Some intracellular parasites such as Toxoplasma gondii and Trypanosoma cruzi can establish infection in a variety of cell types, both phagocytic and non-phagocytic. For this purpose they have developed mechanisms actively to invade their host cells. Leishmania, on the other hand, does not seem to contribute actively to the invasion process but rather relies on the phagocytic activity of the macrophage to gain entry. Phagocytosis comprises two linked events: attachment and internalization. It has been known for a long time that promastigote binding and phagocytosis are receptor-mediated events (Chang and Dwyer, 1978; Alexander and Russell, 1992; Mauel, 1996). Initial studies assumed that uptake was carried out by the classical ‘zipper’-type phagocytosis. According to this mechanism, the initial attachment of the microbe to receptors on the phagocyte triggers the recruitment of additional receptors from the surrounding membrane with a concurrent rearrangement of the cytoskeleton. This enables the extension of a pseudopod, which advances along the particle like a zipper engulfing it into a phagosome (Rittig et al., 1998). Recently, a process termed ‘coiling phagocytosis’, which involves asymmetrical occurrence of pseudopodia coils and other multilayered pseudopod stacks, has been suggested as an additional mechanism for parasite uptake (Rittig et a/., 1998). In both processes the complement receptors CRl and CR3 play major roles and may act cooperatively to amplify the effect (Rosenthal et al., 1996). However, uptake by coiling phagocytosis may target the organisms to a cytoplasmic compartment and alter their survival capability (Bogdan and Rollinghoff, 1999). The best characterized interaction of the parasite with the macrophage involves the complement receptors. Leishmania can bind to the complement receptors in three different ways: in the presence of serum by activating C3 directly and binding through C3bi to CR3, through the direct serumindependent binding of the surface protease gp63 to the CR3, and finally via direct binding of lipophosphoglycan to the lectin-like site on CR3 and to CRl (Alexander and Russell, 1992; Mauel, 1996). Engagement of the complement receptors does not trigger the respiratory burst (Wright and Silverstein, 1983) and indeed opsonization by complement increases the survival of L. major in macrophages (Mosser and Edelson, 1987; Mosser and Brittingham, 1997). In humans, the major mechanism for invasion appears to be through the engagement of CR1 on erythrocytes and the classical complement pathway (Dominguez and Torano, 1999). This process does not seem to be present in animal models of disease, in the natural animal reservoir. Macrophage receptors other than CR3/CR 1 have also been implicated in the initial attachment of the parasite, including complement receptor CR4,

CELL BIOLOGY OF LEISHMANIA

9

as well as receptors for fibronectin, mannose receptor and advanced glycosylation end products (Alexander and Russell, 1992). With the availability of ‘knock-out’ mice lacking FcyR, the scavenger receptor and the complement receptor, it should be possible to re-evaluate the contribution of individual host receptors to parasite invasion for each Leishmania species. However, it is likely that, in vivo, multiple receptorligand interactions occur simultaneously depending on the activation state of the macrophage and its microenvironment. 2.1.3. Promastigote Ligands for Host Macrophages The two major promastigote surface molecules, the major protease gp63 or leishmanolysin and the phosphoglycans, are also ligands for attachment to macrophages (Alexander and Russell, 1992). This is the case for both the serum-dependent and the direct binding. L. major gp63 is a zinc metaloprotease, which is abundant on the surface of promastigotes. It is a glycoprotein with unusual N-glycosylation, which suggests that it may be a potential target for the development of a parasitespecific drug (Olafson et al., 1990). The protein is encoded by a family of seven genes. Six of these genes are constitutively expressed in promastigotes. The expression of the seventh gene is developmentally regulated and the protein is produced exclusively in infective metacyclic promastigotes and amastigotes (Joshi et al., 1993). The genes encoding gp63 are situated in a cluster that is present on a similar size chromosome in all species examined (Button et al., 1989). Although very similar in general terms, the gene organization and expression of gp63 in other Leishmania species is somewhat different (Ramamoorthy et al., 1992, 1995; Roberts et al., 1995). Northern blot analysis from both promastigotes and amastigotes revealed a 3-kilobase (kb) mRNA indicating expression in both parasite life stages (Frommel et al., 1989; Medina-Acosta et al., 1989, 1993). Biosynthesis of gp63 is complex, and involves a cleavable N-terminal signal sequence which guides it across the endoplasmic reticulum membrane, and a C terminal hydrophobic signal sequence that is cleaved 25 amino acids from the carboxyl terminus and is replaced by a glycosylphosphatidylinositol (GPI) anchor (Voth et al., 1998). The biological significance of this complex series of events is not yet understood. Surface proteases that are homologous to gp63 have been described for other trypanosomatids including Crithidia which is a parasite of insects (Bouvier et al., 1987; Russell et al., 1991; El-Sayed and Donelson, 1997). The presence of gp63 in these organisms indicates that the protein predates the divergence of Leishmania to become pathogens in vertebrate hosts and suggests an ancestral role for the protein in the insect.

10

E. HANDMAN

Recently, the three-dimensional structure of gp63 has been obtained. It reveals three domains, two of which have folds that have not been previously described for similar enzymes, suggesting another opportunity to exploit gp63 as an antiparasite drug target (Schlagenhauf et al., 1998). The literature describing the mechanism by which gp63 binds to macrophages and that describing the macrophage receptors involved in the process is confusing. Much of the existing literature is summarized elegantly in Mosser and Brittingham (1997). Early data indicated that CR3 was the main receptor for gp63, binding via iC3b as well as directly via the sequence SRYD, in a manner similar to the binding of integrins to the sequence RGDS. More recent studies have suggested that fibronectin receptor rather than CR3 may be the main receptor for SRYD on gp63. With new insights into the biology of complement receptors and the availability of several of the gp63 genes and gp63 gene knock-out parasites, it may be timely to re-examine these issues using site directed mutagenesis and binding assays. 2.1.4. Phosphoglycans The Leishmania phosphoglycan family of molecules comprises glycolipids and glycoproteins containing repeating units of Gal(/31-4)Man(a 1-)PO4 with or without additional glycan side chains (Mengeling et al., 1997; Haynes, 1998). The family includes lipophosphoglycan (LPG), phosphoglycan (PG) as well as the proteins secreted acid phosphatase and proteophosphoglycan (PPG) (Figure 3). 2.1.5. Lipophosphoglycan Lipophosphoglycan is a complex glycolipid present on the surface of all Leishmania promastigotes examined to date. It forms a dense glycocalyx over the entire surface including the flagellum. The history of LPG is instructive in terms of the unexpected and tortuous path of scientific discovery. Studies were originally undertaken by Schnur to explain the observation that promastigotes grown in the presence of immune serum agglutinate and are morphologically abnormal (Adler and Theordor, 1926; Noguchi, 1926; Schnur et al., 1972). From these studies it became apparent that each Leishmania species produces and secretes into the medium a serologically distinct ‘excreted factor’ (Schnur et al., 1972). Excreted factor was said to be ‘haptenic in nature’, sharing antigenic determinants with the whole parasites from which it was derived (Schnur and El-On, 1974; El-On et al., 1979). Serological analysis of excreted factor allowed the identification and classification of isolates of Leishmania based on intrinsic characteristics

11

CELL BIOLOGY OF LEISHMANIA

Membrane

I cap HGal - Man -@H Glycan core H GPI anchor

a LPG

rl

1 cap H G a i - Man -81

PG

rl Protein

---(Gal

- Man -81

EthN

4 GPI anchor

( Serine )-

Protein

44

GPI- anchored PPG Secreted PPG SAP

Figure 3 Schematic representation of the Leishmania phosphoglycans, the GPIanchored lipophosphoglycan (LPG), the related water-soluble phosphoglycan (PG), which lacks the anchor and the glycan core present in LPG, the GPI-anchored proteophosphoglycan (PPG) and its related, secreted PPG. Another proteophosphoglycan is the secreted acid phosphatase (SAP). Abbreviations: EthN, ethanolamine; Gal, galactose; Man, mannose; GPI, glycosylphosphatidylinositol. (Adapted from Beverley S.M. and Turco S., 1998.)

of the organisms rather than on the clinical manifestations of the patient from which they were isolated (Schnur et al., 1973, 1981; Schnur, 1982a). Turco et al. (1984) characterized an acidic glycoconjugate in L. donovani, which behaved like excreted factor. Our laboratory was the first to describe its presence on the promastigote membrane and demonstrate its amphipathic properties (Handman et al., 1984). These properties were subsequently shown to be due to the presence of a covalently attached lipid anchor, which was absent from the soluble form (McConville et al., 1987; McConville and Ferguson, 1993). Early data suggesting the presence of sulphate in LPG (Handman et al., 1984) have not been confirmed. The molecule has no sulphate. This molecule, initially called lipopolysaccharide because of its superficial similarity to bacterial lipopolysaccharide, eventually became known as lipophosphoglycan (Handman et al., 1984; Turco et al., 1984; Handman and Coding, 1985). With the advent of monoclonal antibodies to parasite surface antigens, functional studies examining their role in invasion became possible (Handman and Hocking, 1982; Greenblatt et al., 1983). Monoclonal antibodies to LPG inhibited attachment of the parasite to macrophages, and it was shown that the L. major LPG is one of the parasite ligands

12

E. HANDMAN

interacting specifically with the mammalian macrophage (Handman and Goding, 1985). Subsequent studies on L. donovani confirmed these observations and extended the functional characterization of LPG by showing that it is also a major ligand for the sandfly gut epithelium (Turco, 1988b; McNeely et al., 1990; McNeely and Turco, 1990; Sacks et al., 1994; Beverley and Turco, 1998). Structural analysis revealed that LPG contains four basic domains conserved in all species examined: a 1-0-alkyl-2-lyso-phosphatidylinositol lipid anchor, a glycan core, and a backbone made of repeating phosphodiester linked disaccharide units of Gal(P1-4)Man(a 1-)-PO4 terminating in a neutral oligosaccharide cap (McConville et al., 1990; Turco and Descoteaux, 1992) (Figure 3). The cap structure varies significantly among species, as do the presence or absence of oligosaccharide side chains on the backbone repeat. L. tropica LPG is the most complex, with more than 19 different glycan side chains (McConville et al., 1995). This is followed in complexity by L. major, while the simplest of the cutaneous organisms is L. mexicana with a single 0-linked glucose residue (Ilg et al., 1992; McConville and Ferguson, 1993). The simplest of all the LPGs is that of L. donovani, which contains only the disaccharide backbone and has no side chains (Turco et a[., 1987). In addition to the interspecies and intraspecies polymorphism of LPG, the structure of the molecule is developmentally regulated during the life cycle of the parasite. As will be described in Section 3.3, this is particularly striking in L. major, where LPG undergoes major structural changes in the transition from the immature procyclic to the infective metacyclic promastigotes. The side-chain structure changes, with reduced numbers of galactose residues and an increased number of terminating arabinose residues. Moreover, the number of disaccharide repeating units almost doubles (McConville et al., 1992). This elongation of LPG seems to be important in protection against complement lysis of metacyclic promastigotes. The structure of LPG detected on promastigotes grown in culture is to some extent dependent on the medium used and the culture conditions. The culture conditions also affect the ability of the promastigotes to fulfil the criteria defining metacyclic parasites, such as agglutination by peanut agglutinin (E. Handman, unpublished observations). In the L. major amastigote, LPG is about a 1000-fold less abundant; it becomes a larger but more sparsely substituted molecule (Glaser et al., 1991; Turco and Sacks, 1991; Moody et al., 1993). Some of the side chains are longer containing as many as 10-12 galactose residues (Moody et al., 1991, 1993; Turco and Sacks, 1991). There is a significant heterogeneity in the level of expression of LPG in amastigotes of different species of Leishmania; L. donovani and L. mexicana amastigotes do not seem to express it at all (McConville and Blackwell, 1991; Bahr et al., 1993).

CELL BIOLOGY OF LNSHMANlA

13

LPG has a distinct GPI anchor structure compared to the Leishmania protein GPIs, and these are in turn distinct from mammalian protein GPIs (McConville and Ferguson, 1993). Another unusual feature of the Leishmania LPG molecule is the presence of a galactofuranose residue in the glycan core. This sugar is not present in mammalian glycoconjugates. The biological significance of the GPI anchors and the presence of the galactofuranose is not clear, but their uniqueness makes them potential targets for specific drug design. LPG is a virulence factor essential for parasite survival in both the insect vector and mammalian macrophage. In the case of L. major promastigotes, studies by Handman and Goding (1 985) and subsequent work identified the galactose-containing side chains Gal( /31-3)Gal( /31-3)Ga1(/31-3)Gal(/314)Man(al-)P04 of LPG as the domain involved in the interaction with host cells (Kelleher et a[., 1992). Amastigotes also use LPG to bind to macrophages but in this case the epitope includes the disaccharide repeats of the backbone, which is more accessible in the sparsely substituted amastigote LPG (Kelleher et al., 1993, 1995). As originally described by Handman and colleagues (Handman et a[., 1984; Handman and Goding, 1985), the glycoconjugate exists in two forms: a membrane-bound amphipathic form and a hydrophilic form found in parasite culture supernatant. The structural differences between these forms has now been established. The water-soluble fragment of LPG is known as phosphoglycan (PG), and is released into the culture medium by promastigotes (Figure 3 ) . PG consists of the LPG repeating units and the terminating cap structure, but does not have hydrophobic properties and lacks a GPI anchor (Handman et al., 1984; Greis et al., 1992; Ilg et al., 1994). However, it is still not known whether PG is a hydrolysis product of LPG, or whether it is synthesized separately and secreted from the flagellar pocket. Nor is it known whether PG is also produced in vivo in the sandfly. While it is quite clear that PG is one of the main components of the original excreted factor described above, it may not be the only one. With the discovery that glycan chains are shared by LPG and several proteophosphoglycans, much of the literature will have to be revisited (see below). No function has been proposed for PG, but LPG glycans have been shown to appear on the surface of macrophages soon after invasion by promastigotes (Handman, 1990; Tolson et al., 1990). Some of these molecules are the hydrophobic form of LPG, and others are hydrophilic and may be PG. Studies on L. donovani LPG have demonstrated that it is also involved in macrophage binding and it acts as a virulence factor (reviewed by Turco and Descoteaux, 1992: Beverley and Turco, 1998). The critical role of LPG in virulence for the mammalian host is supported by the fact that L. major and L. donovani mutants lacking LPG are avirulent (Elhay et al., 1990; McNeely

14

E. HANDMAN

and Turco, 1990; Opat et al., 1996). Moreover, intercalation of LPG into the surface membrane of the mutant organisms restored their virulence to some extent (Handman et al., 1986; McNeely and Turco, 1990). More recently, using elegant genetic approaches, mutant genes from several LPG-null mutants have been identified and cloned (Ryan et al., 1993; Beverley and Turco, 1995, 1998). Wild-type genes were capable of restoring the production of LPG. In some of the mutants, transfection with the wildtype gene restored virulence, thus fulfilling Koch’s postulates (Beverley and Turco, 1998). The functions of these genes have not been fully elucidated. Based on their overall structure, some may be glycosyltransferases, although this awaits definitive biochemical characterization. In addition to its role in attachment to macrophages and invasion, LPG has been shown to have many immunomodulatory activities. It scavenges hydroxyl radicals and superoxide ions which are normally released upon activation of NADPH oxidase by phagocytosis (Mauel, 1996; Bogdan and Rollinghoff, 1998). Intact L. donovani promastigotes have been shown to block protein kinase C activity, and purified promastigote LPG has also been shown to have this activity (McNeely and Turco, 1987; McNeely et al., 1989; Descoteaux and Turco, 1993). 2.1.6. Proteophosphoglycans While the first phosphoglycans to be described were the glycolipid LPG and the polysaccharide PG, a family of phosphoglycan-modified proteins has now been added to the list (Mengeling et al., 1997). To date, three protein members of this family have been characterized. These are the secreted acid phosphatase (SAP) from L. mexicana, a high molecular weight filamentous PPG secreted by promastigotes of L. major (pPPG), and a smaller and structurally distinct PPG secreted by amastigotes of L. mexicana (aPPG). SAP is secreted from the flagellar pocket, the specialized site for secretion and endocytosis (Stierhof et al., 1994). The enzyme is monomeric or oligomeric in structure depending on the organism (Ilg et al., 1991; Stierhof et a[., 1994). In L. mexicana the enzyme is encoded by two genes, and both products are enzymatically active. SAP is a serine- and threonine-rich molecule, and many of its serine residues have a novel type of modification, phosphoglycosylation, the role of which is not yet known (Haynes, 1998). Many of the glycans present on SAP are shared with LPG. SAP is present in most Leishmania species but its biological function is still not understood. SAP is not present in L. major, and ablation of the L. mexicana SAP genes has no effect on parasite growth in vitro or virulence in vivo (Wiese, 1998). Promastigotes of many Leishmania secrete a filamentous proteophosphoglycan which forms gel-like networks and is seen at the centre of

CELL BIOLOGY OF LElSHMANlA

15

parasite rosettes in vitro (Stierhof et al., 1994; Ilg et al., 1996). pPPG from L. major is a large and highly glycosylated mucin. Reminiscent of vertebrate proteoglycans, pPPG has a predominance of carbohydrate (76%) and only 4% amino acids. About half the amino acids are serine, which, together with alanine and proline, form over 80% of the protein backbone (Ilg et al., 1996). The majority of the serines are phosphoglycosylated with LPG-like phosphodiester-linked PG chains. In a striking parallel to LPG and PG, PPG also is found in two distinct forms, a water-soluble secreted form and a GPI-anchored membrane-bound form (A. Piani et al., unpublished data). A gene encoding the membranebound pPPG has been isolated (Ilg et al., in press). The deduced amino-acid sequence contains a hydrophobic C-terminal domain but no cytoplasmic tail, consistent with GPI anchorage. Adjacent to this region is a nonrepetitive domain, while the main body of the open reading frame consists of a large number of repeats of a basic unit of 12- 15 amino acids rich in serine, alanine and proline. At the amino-terminal region there is a second nonrepetitive domain of about 600 amino acids. While the definitive demonstration of the function of pPPG awaits the characterization of gene knock-out organisms, it is already clear that watersoluble pPPG plays a role in the interaction of the parasite with the sandfly (Y.-D. Stierhof, T. Ilg, Y. Schlein and R.L. Jacobson, unpublished data; see also Section 3.2). Purified pPPG binds to macrophages, is internalized and can be detected in the lysosomal compartment (Piani et al., 1999). PPG can also be detected in amastigotes and in parasite-free vesicles in infected macrophages. In view of the striking structural similarities between LPG and PPG, it will now be important to re-examine their relative contribution to a variety of functions previously attributed to LPG, in particular in the amastigote, which displays much less LPG on its surface. A particularly intriguing question is why evolution has led to the production of two polymers with similar or identical side chains, but with such different backbones (phosphodiester-linked sugars versus amino acids).

2.2. Promastigote to Amastigote Differentiation in the Macrophage

2.2.1. Parasitophorous Vacuole Formation and Microbicidal Mechanisms

The first stage in Leishmania infection involves the uptake of promastigotes into a membrane-bound phagosome, which is contiguous with the outer plasma membrane of the macrophage. At this stage the parasite is still topographically in the extracellular environment. The phagosome then becomes modified by fusion with secondary lysosomes, resulting in the

16

E. HANDMAN

phagolysosome or parasitophorous vacuole (PV) (Chang, 1983; Chang and Fong, 1983). The PV is an acidic compartment, rich in microbicidal peptides and hydrolytic enzymes (Antoine et al., 1998). With the transition from the sandfly to the mammalian host, the promastigotes face two major environmental changes, a temperature shift to 35537°C and a pH shift to around pH 5. The organisms sense this new environment and transform into the obligatory intracellular amastigotes (Antoine et al., 1998) with loss of flagellum, closing off of the flagellar pocket, drastic reduction in size and major changes in gene expression. The details of how this transition is triggered and carried out are unclear, but seem to involve unknown factors in addition to the change in pH and temperature. The availability of the complete Leishmania genome sequence may soon provide some tools to help elucidate this process (Blackwell, 1997; Ivens and Smith, 1997; Foote et al., 1998). The elucidation of the mechanisms by which the transition between promastigote and amastigote is mediated will require much more knowledge than just the sequences of the genes. The transition may be regarded as a process of differentiation, and the key questions are how individual genes are activated and inactivated, how a stable phenotype is maintained, and how the changed environment triggers a genetic reprogramming. Although all PVs containing Leishmania share many features, such as mildly acidic pH and the presence of hydrolases and lysosomal membrane markers such as LAMP-1 and LAMP-2, there are significant differences between the PVs produced by different Leishmania species. For example, L. mexicana and L. amazonensis produce large PVs containing many amastigotes arranged around the periphery and attached to the membrane, while L. major and L. donovani produce small PVs with little space around the amastigotes. Some differences in the PVs may be related to the life-cycle stage of the parasite producing it. For example, the aPPG produced by L. mexicana amastigotes induces the formation of large vacuoles in macrophages in the absence of parasites (Peters et al., 1997). In contrast, pPPG from L. major promastigotes causes only modest vacuolation in macrophages (J.-C. Antoine, personal communication). Although the mechanism involved in the formation of Leishmania PV and the role of the intracellular pathogen in its development are not well understood, there are some similarities with the vacuole formation induced by Helicobacter pylori (Antoine et al., 1998). In the case of H. pylori, the toxin Vac A produces large vacuoles in many cell types by inhibiting phosphatidylinositide 3-kinase, an enzyme required for the fusion of late endosomes (Antoine et al., 1998). Vac A was shown to bind to, and to be internalized by, the target cells (Garner and Cover, 1996; Massari et al., 1998).Vac A has also been shown to have immunomodulatory effects and to interfere with antigen presentation by B cells (Molinari et al., 1998).

CELL BIOLOGY OF LElSHMANlA

17

In contrast to PPG, which, in the case of L. mexicana and L . amazonensis induces large vacuole formation in macrophages, promastigote LPG has been shown transiently to prevent the fusion of phagosomes with lysosomes (Desjardins and Descoteaux, 1997). Whether the transient sojourn of the parasite in this type of the phagosome provides the trigger for the initiation of transformation to amastigotes is not known. Infection by Leishmania seems to alter some but not all the microbicidal processes of the macrophage. The vacuolar pH is maintained, the hydrolases are targeted to the PV normally and vesicular traffic does not seem to be disturbed (Russell et al., 1991, 1992; Russell, 1995; Antoine et al., 1998). On the other hand, infection inhibits the production of superoxide (02) and H202, which is one of the major macrophage microbicidal effector mechanisms (Murray, 1986). In addition, infection of cells by Leishmania seems to induce the rapid and transient production of a subset of chemokines. Among these is the monocyte chemoattractant protein 1, which may be important in attracting into the lesion ‘safe targets’ in the form of immature monocytes. These can be infected but, because they express little MHC class I1 on their surface, they present antigen poorly and do not kill the parasites (Racoosin and Beverley, 1997). 2.2.2. Amastigote Survival in the Macrophage What are the biochemical changes that make the amastigote so well adapted to the hostile intracellular environment of the PV with its acidic pH and abundance of hydrolases? Early work from our laboratory suggested that the membrane proteins of the amastigotes were more resistant to proteolysis compared to promastigote membrane proteins (Handman and Curtis, 1982). The amastigote metabolism is adapted to an acidic pH (Glaser et al., 1988) and amastigotes are thought to exploit the proton gradient across their membrane formed under acidic conditions to drive the active transport of glucose and amino acids (Zilberstein, 1991; Zilberstein and Shapira, 1994). This mechanism may actually contribute to the maintenance of the acidic pH in the PV. Another metabolic change, noted by Janovy (1967), was a drastic reduction in the rate of respiration suggesting a switch to anerobic metabolism. Several amastigote-specific gene products have been identified, such as a 3’-nucleotidase (Bates, 1993), the amastigote-specific protein A2 of L. donovani (Zhang and Matlashevski, 1997) and a mitogen-activated protein (MAP) kinase in L . mexicana (Wiese, 1998). An amastigote-enriched histone H1 gene has also been identified (Fasel et al., 1994). In addition, members of multigene families, such as the parasite surface antigen 2 (PSA-2) or gp63 of L . major are differentially expressed in

18

E. HANDMAN

amastigotes (Handman et al., 1995). The structure of the GPI anchor of the amastigote PSA-2 polypeptide is different from that of the three PSA-2 polypeptides expressed by promastigotes. In contrast to the promastigote forms, the amastigote GPI is resistant to hydrolysis by phosphatidyl inositol-specific phospholipase C from B. thuringiensis (E. Handman, unpublished data). When the amastigote PSA-2 gene is expressed in promastigotes by transfection, the GPI anchor is susceptible to hydrolysis, suggesting a stage-specific control mechanism in anchor biosynthesis (E. Handman, unpublished data). It would seem a reasonable hypothesis that some of these amastigote-specific gene products contribute to the ability of the amastigotes to establish in the macrophage and to evade its microbicidal activity, but at present direct evidence is lacking. An intriguing characteristic of the amastigote surface is the increased ratio of glycolipids to proteins. The endogenous glycolipids and the host-derived sphingolipids incorporated into its membrane represent a significantly larger proportion of the surface components compared to promastigotes (McConville and Ferguson, 1993). Among them, the glycoinositol phospholipids (GIPLs) are the most abundant, forming a densely packed and morphologically distinct protective coat. The GIPLs are structurally related to LPG but distinct from it. They possess a unique GPI anchor, containing lipids which are different from both protein GPIs and LPG (Ralton and McConville, 1998). It has been suggested that they play a role in the regulation of the physicochemical properties of the amastigote membrane by making it more resistant to enzymatic attack in the PV (Ralton and McConville, 1998). In addition, GIPLs have been shown to contribute to amastigote survival in the macrophage by directly inhibiting microbicidal activities such as NO production (Winter et al., 1994; Proudfoot et al., 1995). 2.3. Mechanisms of Parasite Persistence

2.3.1. Invasion of Macrophages and Dendritic Cells by Amastigotes Although the initiation of infection is due to the promastigote, the maintenance of infection in the mammalian host relies on the amastigotes and their ability to replicate in macrophages, and to exit and re-infect new host cells. Much progress has been made in the elucidation of the host and parasite molecules, and of the mechanisms involved in early promastigote attachment and uptake by macrophages. Much less is known about the interaction of the obligatory intracellular amastigote and its host cells in an already established infection.

CELL BIOLOGY OF LEISHMANIA

19

In vitro studies indicate that LPG is a major ligand on L. major amastigotes, but this cannot be the case for Leishmania species that do not express significant amounts of LPG on their surface. For those it will be interesting to explore the role of PPG. In the case of L. amazonensis amastigotes, an undefined heparin-binding molecule has been implicated in the attachment to macrophages (Love et ul., 1993), as have amastigotespecific glycosphingolipids (Straus et al., 1993). On the host side, recent data implicate the Fc receptor for IgG as a major contributor to infection of macrophages by L. mexicana amastigotes in vivo. The complement receptor CR3 and the mannose receptor are also important (Peters eta]., 1995). It has been suggested that the Fc receptor may also play a role for L. major amastigotes. A major pathogenic role for Fc receptor in infection is difficult to reconcile with the very low amount of antibody present in the tissue of L. major-infected individuals and with the massive infection of hypothymic nude mice that lack IgG antibodies. It is likely that, as part of the ‘arms race’ between host and parasite, multiple receptorligand interactions have evolved to allow parasitism. There is now abundant evidence that a host cell carries amastigotes from the initial site of infection in the skin to the draining lymph nodes, where the antigen presentation to the naive T cells occurs and where parasites persist indefinitely (Aebischer et al., 1993; Moll, 1993a,b,c; Moll et al., 1993, 1995a,b). The cell that ferries the amastigotes from the skin to the lymph node appears to be a dendritic cell (Langerhans cell). The receptor on the dendritic cells responsible for the interaction with amastigotes is not known nor is the ligand on the amastigote. Dendritic cells are required to initiate primary T-cell responses (Caux el al., 1995). Macrophages can only present antigen to T cells that have already been primed (Caux et al., 1995). In contrast to the infected macrophages, which seem to be impaired in antigen presentation (see below), the infected dendritic cells are competent to present antigen and initiate T-cell immune responses to the parasite (Moll, 1993~). An intriguing question in leishmaniasis is the homing of the different Leishmania species to different organs. What is the contribution of the parasite and what is the contribution of the host? Are dendritic cells involved in this migration? How early in infection does it occur? 2.3.2. Evasion and Subversion of the Host Immune Response Leishmaniasis is a chronic disease; the infection is slow in turning on the host protective macrophage-activating immune responses. The parasites have evolved numerous ways to interfere with the host immune responses, including modulating cytokine production, inhibiting antigen presentation

20

E. HANDMAN

and turning off co-stimulatory molecules necessary for activation of antigenspecific T cells. L. mexicana, L. major and L. braziliensis trigger the production of transforming growth factor /3 (TGF-/3) and interleukin 10 (IL-lo), which inhibit killing of the intracellular organisms (Bogdan and Rollinghoff, 1998, 1999). Recovery from infection has been shown to be critically dependent on the macrophage-activating cytokine IL-12 (Reiner and Locksley, 1995), and infection with promastigotes inhibits production of IL-12 and TNF-a (Reiner et al., 1994). Some of the parasite molecules involved in modulating the immune response of the host have been identified but the mechanisms by which they act on the macrophage biology are mostly not understood. For example, LPG and PPG suppress IL-1 and TNF-a production in response to bacterial LPS. LeIF, a Leishmania homologue of the initiation factor 4A has been shown to modulate IL-12, IL-10 and TNF-a expression in monocytederived antigen presenting cells (Probst et al., 1997). Parasite-driven mechanisms operating at the level of the PV also have direct effects on the recognition of the parasite by T cells. The parasite causes a reduction of host MHC class I1 molecules available for binding to parasite antigens (Handman et al., 1979; Reiner et al., 1987; Antoine et ul., 1991; Lang et al., 1994a,b), which presumably helps prevent its detection in the macrophage by T cells. Interestingly, in the case of L. amazonensis and to some extent L. major, this seems to be achieved by selective and active sequestration and degradation of class I1 antigens by the amastigotes (Antoine et al., 1998). In L. amazonensis, the MHC class I1 molecules seem to accumulate in amastigote-specific organelles known as megasomes, and it is possible that amastigote-specific proteases are involved in the hydrolysis of the MHC molecules. An additional mechanism which reduces antigen presentation by infected macrophages has been described in L. mexicana. The availability of parasitederived antigens for presentation to the immune system seems to be restricted to macrophages containing dead organisms (Wolfram et al., 1995, 1996). This might imply that the death of the parasite allows the release of parasite antigens which then find their way to the macrophage surface. More interestingly, it could suggest the existence of active suppressive mechanisms that require the parasite to be alive. These immune evasion mechanisms mean that many, if not all, infected macrophages remain immunologically silent. This could provide an explanation for the slow development of the host protective effector mechanisms and possibly for the long-term persistence of the parasite in the immune individual (Aebischer, 1994; Bogdan et al., 1996). The mechanisms allowing indefinite persistence of the parasite in the presence of an otherwise host protective immune response are still poorly

CELL BIOLOGY OF LEISHMANIA

21

understood (Bogdan et al., 1996). One effect of the parasite on the macrophage described recently is inhibition of apoptosis through induction of ‘pro-survival’ cytokines such as macrophage colony-stimulating factor (M-CSF), tumour necrosis factor a (TNF-a) and IL-6 (Moore and Matlashewski, 1994; Moore et al., 1994; Antoine et al., 1998). This phenomenon may be responsible for the persistence of amastigotes by extending the life of the host cell. It is also possible that the persistent organisms reside in cells other than macrophages, for example, dendritic cells or fibroblasts (Bogdan et al., 1996).

3. THE INTERACTION OF LElSHMANlA WITH THE SANDFLY 3.1. Parasite Differentiation in the Blood Meal

When feeding on blood as opposed to fruit, the female sandfly is a pool feeder that uses its mandibles and maxillae to cut a wound in the host skin, and sucks up the blood that accumulates. Infected macrophages are taken up from that pool. The blood meal becomes enclosed in a sac-like peritrophic membrane, which is secreted by the midgut epithelium and consists of chitin embedded in a protein-carbohydrate matrix (KillickKendrick, 1990a,b). Amastigotes continue to undergo a few cell divisions in the blood meal but the new environment is sensed by the organisms, and metabolic changes are set in train leading to the transformation of the non-motile, aflagellar amastigote into the motile flagellated promastigote (Killick-Kendrick, 1990a). Just as the amastigote has evolved mechanisms to survive in the mammalian macrophage, so the promastigote has evolved mechanisms to promote life in the insect host. The nomenclature used for the different developmental stages of Leishmania in the sandfly has been adopted from the nomenclature of the African trypanosomes, and includes the procyclic or midgut form and the metacyclic or mature infective form (Vickerman and Preston, 1976; Sacks, 1988). The early procyclic promastigotes are eliptical in shape, measure only 6-8 pm in length, divide rapidly and, after a few days, escape from the disintegrating peritrophic membrane and migrate forward to the thoracic midgut and the cardiac valve (Schlein, 1993). Procyclic promastigotes continue to divide and attach to the microvilli of the midgut, particularly in the thoracic section and the cardiac valve. Later, some invade the oesophagus and the pharynx where they attach to the cuticle lining with their flagella, which form plaques called hemidesmosomes (Warburg et al., 1989; Lang et al., 1991; Schlein, 1993). Surprisingly, it seems that the flagella

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may even penetrate into the epithelial cells, presumably increasing the strength of the parasite anchorage (Walters et al., 1987). A parasite molecule present along the flagellum and at its tip has been implicated in this specific interaction (Warburg et al., 1989). Further differentiation and maturation of the promastigote population occurs from day 5-8 onwards, and is reflected by major changes in morphology and biochemistry. At this stage, the metacyclic form of the promastigote becomes dominant in the population (Sacks and Perkins, 1984; Sacks, 1989). The metacyclic promastigotes are slender, highly motile organisms with small bodies and long flagella. The metacyclic promastigotes are now ready to be delivered by the sandfly to the mammalian host at the next blood meal.

3.2. Establishment of Infection in the Sandfly

The sandfly is not just a vector for delivery of organisms to mammalian hosts. It is itself a host and, as such, the interaction of the parasite with the sandfly is just as complex as its interaction with the mammalian host. The parasites undergo a series of developmental modifications which on the one hand allow them to survive in the gut environment and on the other hand make it possible for transmission to the mammalian host. There is evidence for significant specificity in the interaction of the parasite with the sandfly. Certain species of Leishmania can be transmitted only by particular species of Phlebotomus. What determines this hostparasite specificity? Studies dissecting the genetics of P. papatasi susceptibility to a single isolate of L. major showed that individual flies varied in susceptibility (Wu and Tesh, 1990b) and that this variation was due to multiple genes (Wu and Tesh, 1990a). Species-specific differences in vectorial competence have been correlated with the ability of the parasites to establish in the gut. The parasites have to withstand the effects of the proteases secreted by the fly in response to the blood meal. They also have to anchor themselves in the peristaltic environment of the gut to prevent expulsion through the anus. LPG and the related water-soluble polysaccharide PG are thought to protect the parasites from the gut hydrolytic environment (Schlein, 1993; Pimenta et al., 1994). There seems to be some specificity in the ability of these molecules to protect the parasites. L . major but not L. donovani PG was able to inhibit the proteolytic activity of the gut enzymes, and to increase survival of the L. major but not L . donovani parasites in P. papatasi, the natural host of L. major. This sandfly is not a host for L. donovani (Schlein and Romano, 1986; Schlein, 1993).

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One of the parasite molecules implicated in the anchorage to the gut is the LPG coat, which forms a thick glycocalyx on the promastigote surface. The receptor on the gut epithelium of P. papatasi, which is specific for L. major, appears to recognize galactose residues on the side chains of promastigote LPG. Side chains are absent from L. donovani LPG, which consists of only the backbone Gal(,B1-4)Man(a l-)PO4 terminating with a neutral cap. The structural differences in LPG may explain the inability of L. donovani to colonize this particular sandfly species. The importance of the galactosecontaining side chains for the establishment of L. major infection in this particular sandfly has been unequivocally demonstrated by the use of a L. major mutant lacking the gene for the ,Bl,3-galactosyltransferase(Butcher et af., 1996). This mutant, which produces an LPG similar to L. donovani devoid of side chains, cannot bind to the midgut and cannot sustain a successful infection in P. papatasi. As described earlier, the L . major promastigotes display a surface-bound PPG, which is decorated with carbohydrate side chains similar in structure to those present on LPG (Ilg et al., 1996; A. Piani et al., unpublished data). This molecule may also contribute to the binding to gut epithelium. The next stage of parasite development is accompanied by the escape from the peritrophic membrane. The haemoglobin in the blood meal is digested, possibly by the parasite major surface protease gp63 (Schlein, 1993), following which the membrane is lysed by parasite chitinases (Schlein et al., 1991; Schlein, 1993). The cues for the parasites’ migration to the thoracic midgut and cardiac valve and for their subsequent differentiation remain to be elucidated. Turco (1988a) has made the interesting suggestion that sugar meals that are taken into the sandfly crop and are delivered into the gut may facilitate the parasite migration by competing for binding to the receptors for LPG on the epithelial cells. However, parasites can complete their differentiation even in the absence of sugar meals (Schlein, 1993). Certain bacteria possess a sophisticated signalling strategy called ‘quorum sensing’ in which the density of the population is sensed and virulence genes are expressed when the population reaches a certain size. The quorum sensing signal is triggered by the concentration of a secreted microbial molecule (Straws and Falkow, 1997). It would be interesting to investigate the existence of quorum sensing as a mechanism for the changes in the parasite developmental programme in the gut.

3.3. Metacyclogenesis

The parasite morphotypes present in the thoracic midgut and proboscis have always been considered strong candidates for the initiation of infection because of their proximity to the wound (Adler, 1964). The presence of

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different morphotypes along the digestive tract of the fly during the differentiation of the parasites from the amastigote to the promastigote form also suggested potential differences in the ability of individual forms to infect the mammalian host. Sacks and Perkins (1984) provided the definitive demonstration that the mature or metacyclic non-dividing promastigotes present in the midgut are the infective stage of the organism by showing that promastigotes taken from sandflies 3 days after infection were avirulent, whereas those taken after 7-8 days were infective (Sacks and da Silva, 1987). Subsequently, they showed that metacyclic promastigotes are also present in in vitro cultures in a stationary phase of growth but not in the logarithmic phase (Sacks, 1989). The small population (2- 10%) of metacyclic organisms can be isolated from these cultures by negative selection for agglutination by the galactose-binding lectin peanut agglutinin (PNA; Sacks, 1989). Procyclic or immature promastigotes bind PNA and are agglutinated by the lectin, whereas the metacyclic promastigotes remain in suspension. It should be noted, however, that the parasites that are not agglutinated by PNA still bind the lectin, and the precise basis for the agglutination by PNA remains to be determined. Metacyclogenesis is accompanied by ultrastructural and biochemical changes in the parasite, in particular at the cell surface. One of the most significant changes is thickening of the glycocalyx owing to modifications in the structure of LPG (Pimenta et al., 1989, 1991, 1992, 1994). Changes in glycosylation of LPG lead to increases in the length of its backbone and in the masking of galactose residues on its side chains, the targets for PNA binding, by arabinose (McConville et al., 1992; McConville and Ferguson, 1993). A second surface molecule whose expression is upregulated during metacyclogenesis is the major promastigote surface protease, gp63 (Sacks and Perkins, 1984; Russell and Wright, 1988; Kweider et al., 1989). Not only is gp63 more abundant but in L. braziliensis there seems to be a developmentally regulated isoform expressed specifically in metacyclic promastigotes (Kweider et al., 1987) correlating with their increased virulence. Although most of the work on metacyclogenesis has been done in L. major, metacyclic forms have also been observed in L. donovani (Howard et al., 1987) and L. panamensis (Walters et al., 1989a,b). Since these parasites lack the galactose-containing side chains that bind PNA and define metacyclic forms, the molecular basis for the classification of these forms as metacyclic is not clear.

3.4. Transmission to the Mammalian Host

Infection with Leishmania causes havoc in the sandfly, most obviously to its ability to feed. Infected flies seem to probe multiple times until successful

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feeding is achieved. It may be significant that each of these ‘unproductive’ bites is infective (Killick-Kendrick, 1979; Jefferies et al., 1986; Warburg and Schlein, 1986). A hypothesis has been put forward by Killick-Kendrick (1979) that multiple probing is due to the blockage of the fly midgut, cardia and stomodeal valve by a plug of carbohydrate-rich gel-like material. There is now convincing evidence that this plug is formed by the secreted promastigote PPG, which forms large filaments containing masses of promastigotes (Y.-D. Stierhof, T. Ilg, E. Handman and Y. Schlein, unpublished data). Metacyclic promastigotes appear mainly anterior to this plug and their motility is unimpeded by the gel (Lawyer et al., 1990). Backflow from the plug into the wound during unsuccessful feeding would carry the parasites into the wound. An important factor in the mechanics of transmissibility of the parasites by the fly is the damage to the cardiac valve induced by parasite-derived molecules (Schlein, 1993). In infected flies the damaged valve remains open and the suction of the food pump occurs in both directions, with the gut content including parasites, being drawn back, thus mixing with the newly drawn blood meal. As the pump contracts, the mixture is released in both directions. 3.4.1. The Role of Sandfly Saliva in Promastigote Virulence During the feeding process, sandflies inject saliva into the skin of the mammalian host, as do many blood-sucking insects. The saliva facilitates the location of suitable blood vessels and prevents blood clotting (Lehane, 1991). In addition, the sandfly saliva contains potent vasodilating compounds which presumably increase blood flow to the bite (Ribeiro et al., 1989). The sandfly saliva also acts as a virulence factor for the Leishmania promastigote. It facilitates infection by increasing both ‘the lesion size and the parasite survival (Titus and Ribeiro, 1988). Parasite survival is critical to the establishment of infection because, unlike the situation in the laboratory where large numbers of organisms are injected into the host, in nature, sandflies introduce only 10- 100 organisms at each bite (Warburg and Schlein, 1986). Insect saliva does not act directly on the parasite, but rather through the modulation of macrophage-killing mechanisms, possibly via inhibition of the ability of interferon (IFN-y) to activate macrophages to produce NO, one of the major effector molecules responsible for parasite killing (Hall and Titus, 1995). Recent data indicate that the mechanism of downregulation of NO production may involve inhibition of protein phosphatase activity, blocking signal transduction pathways (Waitumbi and Warburg, 1998). The importance of IFN-y and NO in the early stage of infection is not clear but the effect of the saliva is

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surprisingly long lasting. The components of saliva that mediate these effects on macrophages have not been isolated, but one appears to be low molecular weight, soluble in ethanol and resistant to proteases, DNAse and RNAse. Another important bioactive component in sandfly saliva is the vasodilator and erythema-inducing peptide maxadilan (Lerner and Shoemaker, 1992). Studies by Warburg et al. (1994) indicate that maxadilan may influence the clinical manifestations of disease, possibly by modifying the spreading of the parasites from the skin to the viscera.

4. CONCLUDING REMARKS 4.1. Cell Biology in the Post-genome Era

At the time of writing, at least 13 bacterial genomes have been completely sequenced, 49 are currently in progress in the public domain and several others are held in private companies (Saunders and Moxon, 1998). For more complex organisms such as protozoa, the pace has been slower. The Plasmodium fakiparum genome project is the most advanced, but other parasites such as the African Trypanosomes and Leishmania are on the way (Blackwell, 1997). As a prelude to the genome project, a karyotype map for L. infantum and L. major have been prepared (Wincker et al., 1996) and a physical map has been developed by Ivens and Smith (Blackwell, 1997). Mapping studies have suggested that the overall chromosomal structure and gene order have been maintained in all Old World Leishmania species and therefore a single strain of L. major was chosen for sequencing (Blackwell, 1997). What would the complete sequence of the Leishmania mean to the cell biologist? We will have the sequence of every enzyme and structural protein, all the virulence factors and all the potential drug targets (Foote et al., 1998). However, the availability of the sequence will not translate immediately into function. How should we go about exploiting the knowledge of the Leishmania genome? How can we identify the function of important genes, find virulence determinants, stage-specific genes and specific drug targets? Homology to known genes and analysis of clusters of orthologous groups of genes are obvious approaches, but some genes may be missed because of evolutionary divergence in sequence or because specialized genes may have evolved to exploit the intracellular environment of the parasitophorus vacuole or the sandfly gut (Strauss and Falkow, 1997; Saunders and Moxon, 1998). From the completed sequence of bacteria, yeast and the nematode C . elegans, it is becoming clear that a significant proportion of open reading

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frames have no identifiable homologues or known function. Characterization of these sequences will require ingenuity, luck and novel approaches. Gene knock-out is relatively straightforward in Leishmania. Recently, the value of transposon mutagenesis for identification of gene function has been demonstrated in Leishmania (Gueiros-Filho and Beverley, 1997). Modifications to the in vivo expression technology, pioneered in the Sabnonella system, may facilitate the study of differential gene expression in the Leishmania amastigote (Saunders and Moxon, 1998). Finally, the development of the chip microarray technology promises to revolutionize the way we think and approach gene expression and function, in particular the coordinated expression of multiple genes. At present, microchip technology lacks the sensitivity to detect transcripts that are of low abundance but may nonetheless be very important. The last decade or so in molecular parasitology has been dedicated to the reduction of complex systems to individual components. The next decade should see the complete sequence of many genomes, including the Leishmania genome. The challenge will be to identify the function of these genes, to put the pieces together, and to make sense of the total organism.

ACKNOWLEDGEMENTS The work from the investigator’s laboratory has been supported by the Australian Health and Medical Research Council and the UNDP/WORLD BANK/WHO Special Programme for Research and Training in Tropical Diseases. I am particularly grateful to my long-standing collaborators Jim Goding and Tony Bacic for helpful discussions and critical input.

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Voth, B.R., Kelly, B.L., Joshi, P.B., Ivens, A.C. and McMaster, W.R. (1998). Differentially expressed Leishmania major gp63 genes encode cell surface leishmanolysin with distinct signals for glycosylphosphatidylinositol attachment. Molecular and Biochemical Parasitology 93, 3 1-41. Waitumbi, J. and Warburg, A. (1998). Phlebotomus papatasi saliva inhibits protein phosphatase activity and nitric oxide production by murine macrophages. Infection and Immunity 66, 1534- 1537. Walters, L.L., Modi, G.B., Tesh, R.B. and Burrage, T. (1987). Host-parasite relationship of Leishmania mexicana mexicana and Lutzomyia abonnenci (Diptera: Psychodidae). American Journal of Tropical Medicine and Hygiene 36, 294-3 14. Walters, L.L., Chaplin, G.L., Modi, G.B. and Tesh, R.B. (1989a). Ultrastructural biology of Leishmania ( Viannia) panamensis ( = Leishmania braziliensis panamensis) in Lutzomyia gomezi (Diptera: Psychodidae): a natural host-parasite association. American Journal of Tropical Medicine and Hygiene 40, 19-39. Walters, L.L., Modi, G.B., Chaplin, G.L. and Tesh, R.B. (1989b). Ultrastructural development of Leishmania chagasi in its vector, Lutzomyia longipalpis (Diptera: Psychodidae). American Journal of Tropical Medicine and Hygiene 41, 295-3 17. Warburg, A. and Schlein, Y. (1986). The effect of post-bloodmeal nutrition of Phlebotomus papatasi on the transmission of Leishmania major. American Journal of Tropical Medicine and Hygiene 35, 926-930. Warburg, A,, Saraiva, E., Lanzaro, G.C., Titus, R.G. and Neva, F. (1994). Saliva of Lutzomyia longipalpis sibling species differs in its composition and capacity to enhance leishmaniasis. Philosophical Transactions of the Royal Society of London 345, 223-230. Warburg, A., Tesh, R.B. and McMahon, P.D. (1989). Studies on the attachment of Leishmania flagella to sand fly midgut epithelium. Journal of Protozoology 36, 613-617. Wiese, M. (1998). A mitogen-activated protein (MAP) kinase homologue of Leishmania mexicana is essential for parasite survival in the infected host. EMBO Journal 17, 2619-2628. Wincker, P., Ravel, C., Blaineau, C., Pages, M., Jauffret, Y . , Dedet, J.-P. and Bastien, P. (1996). The Leishmania genome comprises 36 chromosomes conserved across widely divergent human pathogenic species. Nucleic Acids Research 24, 1688-1694. Winter, G., Fuchs, M., McConville, M.J., Stierhof, Y.-D. and Overath, P. (1994). Surface antigens of Leishmania mexicana amastigotes: characterization of glycoinositol phospholipids and a macrophage-derived glycosphingolipid. Journal of Cell Sciences 107, 2471-2482. Wolfram, M., Ilg, T., Mottram, J.C. and Overath, P. (1995). Antigen presentation by Leishmania mexicana-infected macrophages: activation of helper T cells specific for amastigote cysteine proteinases requires intracellular killing of the parasites. European Journal of Immunology 25, 1094- 1100. Wolfram, M., Fuchs, M., Wiese, M., Stierhof, Y.-D. and Overath, P. (1996). Antigen presentation by Leishmania mexicana-infected macrophages: activation of helper T cells by a model parasite antigen secreted into the parasitophorous vacuole or expressed on the amastigote surface. European Journal of Immunology 26, 31533162. World Health Organization (1995). Report on the Consultative Meeting on Leishmania/HIV Co-infections, September, 1994, pp. 6-7. Rome: Istituto Superiore di Sanita/Geneva: World Health Organization.

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World Health Organization (1998). Advances in battle against leishmaniasis. T D R News 57. Wright, S.D. and Silverstein, S.C. (1983). Receptors for C3b and C3bi promote phagocytosis but not the release of toxic oxygen from human phagocytes. Journal of E.xperimenta1 Medicine 158, 2016-2023. Wu, W.K. and Tesh, R.B. (1990a). Genetic factors controlling susceptibility to Leishmania major infection in the sand fly Phlebotomus papatasi (Diptera: Psychodidae). American Journal of Tropical Medicine and Hygiene 42, 329-334. Wu, W.K. and Tesh, R.B. (1990b). Selection of Phlebotomus papatasi (Diptera: Psychodidae) lines susceptible and refractory to Leishmania major infection. American Journal of Tropical Medicine and Hygiene 42, 320-328. Zhang, W.W. and Matlashevski, G. (1997). Loss of virulence in Leishmania donovani deficient in an amastigote specific protein A2. Proceedings of the National Academy of Sciences of the USA 94, 8807-881 1. Zilberstein, D. (1991). Adaptation of Leishmania species to an acidic environment. In: Biochemical Protozoology ( G . Coombs and M . North, eds), pp. 349-358. London: Taylor and Francis. Zilberstein, D. and Shapira, M . (1994). The role of pH and temperature in the development of Leishmania parasites. Annual Review of Microbiology 48,449-470. Zuckerman, A. (1975). Current status of the immunology of blood and tissue protozoa. I . Leishmania. Experimental Parasitology 38, 370-400.

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Immunity and Vaccine Development in the Bovine Theilerioses Nicola Boulter and Roger Hall* Department of Biology, PO Box 373, University of York, York, YO1 5 Y W , UK

Abstract ................................ ................................... 42 1. Introduction.. ........................ ................................... 43 1.1. Bovine Theilerioses .... ........................................... 43 1.2. Clinical and Pathological Features.. ...... ........................... 48 1.3. Control Measures .......................................... . . . . . . . 49 2 . Theileria annulata .......................................................... 51 51 2.1. Immune Responses .................................................... 2.2. Vaccination ............................................................ 62 3. Theileria parva ............................. ............................ 67 3.1. Overview .................................................. 3.2. Immune Responses.. ...................................... 3.3. Vaccination ............................................................ 73 4. Theileria sergenti. ........ .......................... 77 4.1. Classification . . . . . . . . .......................... 77 4.2. Clinical Features and Control .... .................... 4.3. Immune Responses.. . . . . . . . . . . . ................................... 78 4.4. Vaccination with Non-living Comp n t s . . .............................. 79 5. Comparative Aspects ....................................................... 80 6. The Future ............................... ............................ 82 Acknowledgements ............................................ . . . . . . . . . . 82 References. ........................... ..................................... 83

*Corresponding author. ADVANCES IN PARASITOLOGY VOL 44 ISBN 0- 12-031744-3

Cupl.rr~hr11.. 2000 Academic Press

A / / right., oJ' rrprodut rioi: in any liirnl rrs~~ri'ei!

42

N. BOULTER AND R. HALL

ABSTRACT There are three economically important bovine Theileria species: Theileriu annulata, which causes tropical theileriosis and occurs across north Africa and most of central Asia; Theileria purva, which causes East Coast fever and is found in East and Central Africa; and Theileria sergenti, which is predominantly a problem in Japan and Korea. Theileriu annulata preferentially infects macrophages in vivo. It is controlled largely by means of live, attenuated vaccines, which are produced by prolonged tissue culture of the schizont-infected cells. The immunity induced in animals, which have either recovered from an infection or have been vaccinated (with an attenuated vaccine), is broad, solid and cell mediated. It is considered that the main effector cells are cytostatic macrophages that produce nitric oxide. Subsidiary roles for bovine leucocyte antigen (BOLA)-restricted, transiently appearing, cytotoxic T cells, and possibly also natural killer (NK) cells, have been identified. Cytokines such as tumour necrosis factor a (TNF-a) may have important roles, particularly in the induction of pathology. Matrix metalloproteinases have been implicated in the metastatic behaviour of schizont-infected cells. The nature of the protective schizont target antigens remains unknown. Attempts to develop a subunit vaccine have focused upon a sporozoite antigen (SPAG-1) and a merozoite antigen (Tamsl). Both SPAG-1 and Tams I have given partial protection using different delivery systems and adjuvants, but further vaccine development will probably require identification of a range of other antigens, especially from the schizont stage. Theileria parva has a tropism for T cells. Vaccination is currently by the ‘infection and treatment’ method, which involves challenging with a controlled dose of sporozoite stabilate and the simultaneous administration of long-acting tetracyclines. The immunity thus induced is mediated by BOLA-restricted cytotoxic T cells, which recognize polymorphic schizont antigens. These antigens have not been characterized at the molecular level. However, the polymorphic nature of the target antigens underlies the fact that the immunity is very strain specific - a situation that distinguishes T. parva from T . annulata. Interestingly, it is not possible to produce an attenuated vaccine to T . parva, as T . parva requires up to two orders of magnitude more schizonts in order to achieve transfer to the new host. A suggested reason for this is that the macrophage targets of T. unnulata are phagocytes and thus the schizont has a natural, efficient route of entry whilst the preferred host of T . parva is the non-phagocytic T cell. Analysis of the cytotoxic T-cell response has revealed evidence of BOLA haplotype dominance plus competition between parasite epitopes. Subunit vaccination using a recombinant sporozoite antigen (p67) has proved very promising,

IMMUNITY IN THE BOVINE THEILERIOSES

43

with levels of protection of the order of 70% being achieved. A proportion of the protected calves exhibits complete sterile immunity. Interestingly, the basis for this immunity is not clear, since there is no correlation between the titre of antibodies that inhibit sporozoite penetration of lymphocytes and protection. Similarly, there is no significant T-cell response that distinguishes the protected and susceptible animals. These data are very encouraging, but other components, particularly those derived from the schizont, need to be identified and characterized. The mild Theileria species of Japan and Korea (termed T . sergenti in the literature) cause fever and severe chronic anaemia. The schizont stage of the life cycle is very rare and the host cell type is not known. The pathology is associated with chronic piroplasm infection. Immunity can be induced by immunizing with crude piroplasm extracts. Serological analysis of immune sera reveals that the immunodominant antigen is a polypeptide of 3033 kDa, which corresponds to the protective T . annulata polypeptide Tams1 . Passive immunity can be transferred with a monoclonal antibody to this 30-33 kDa molecule. Active immunization with a synthetic p32 antigen afforded protection against clinical symptoms and parasitaemia, confirming the importance of this antigen. A subunit vaccine against T . sergenti will therefore almost certainly include the 30-33 kDa antigen. As yet there is no subunit vaccine sufficiently protective against any of the Theileria species to allow widespread field usage. The focus of research, particularly for T . annulafa and T . parva, is the identification of the protective schizont antigens. The use of naked DNA delivery systems is also likely to be the method of choice, since the full spectrum of immunity, including T-cell responses, can be provoked. The vectors will most probably include selective immunopotentiators such as cytokine genes. It is likely that the vaccines that emerge will be multicomponent, being composed of several antigens from each life-cycle stage. Much of the research into vaccine development will, however, be empirical.

I. INTRODUCTION 1.1. Bovine Theilerioses

1.1.1. Background and Scale of the Problem

Theileria species are tick-borne protozoan parasites belonging to the phylum Apicomplexa. They are characterized by having an intracellular schizont stage that multiplies in leucocytes. This property distinguishes Theileria from true Babesia species, as the latter develop exclusively within

44

N. BOULTER AND R. HALL

erythrocytes (Mehlhorn and Schein, 1984). There are many species of Theileria that affect domestic and wild animals (Mehlhorn and Schein, 1984; Irvin and Morrison, 1987; Dolan, 1989). For the purpose of this review, only three pathogenic species affecting cattle, T . annulata, T . parva and T . sergenti, will be considered in detail (Table 1). Theileria annulata (Dschunkowsky and Luhs, 1904) is transmitted by ixodid ticks of the genus Hyalomma, principally H . anatolicum anatolicum, and is the causative agent of the extremely debilitating and often fatal disease known as tropical theileriosis. Former synonyms include Mediterranean Coast fever, tropical gonderiosis and tropical piroplasmosis (Neitz, 1957). The true economic impact of tropical theileriosis is difficult to assess, but it threatens several hundreds of millions of cattle in a massive area encompassing North Africa, southern Europe, the Middle East and Central Asia, including the Indian subcontinent (Robinson, 1982). In India, where much effort is directed towards cross-breeding programmes aimed at increasing the milk yield of the national herd, potential losses as a result of tropical theileriosis are estimated to be US$ 800 million per annum or 10% of the gross national product (Brown, C.G.D., 1990; Devendra, 1995). In endemic areas, the indigenous cattle are usually infected as calves, when they show a milder clinical reaction. If they recover, they develop a persistent carrier state and become resistant to reinfection (Gautum, 1981; Brown, C.G.D., 1990). However, exotic breeds of cattle, particularly imported taurine dairy cattle, and cross-bred cattle are extremely susceptible, and the disease can cause mortality rates of 40-60% in these groups (Brown, C.G.D., 1990). Thus the need to develop efficient control measures against T. annulata is becoming ever more important as the practice of improving local dairy and beef herds by cross-breeding becomes more popular. Theileria parva causes the extremely virulent disease known variously as East Coast fever, Corridor disease or January disease. It is transmitted by the three-host tick Rhipicephalus appendiculatus in areas of East and Central Africa, where it is a major constraint on livestock production. The mortality for Bos taurus exotic breeds exceeds go%, whilst it is less often fatal but none-the-less extremely debilitating in local Boran cattle. Interestingly, it is only mildly pathogenic in the African buffalo, Syncerus caffer (see Irvin and Morrison, 1987). Total losses due to T . parva are estimated to be US$ 168 million per annum (Muhkebi, 1992). There is much confusion and debate over the nomenclature of the so-called benign Theileria species of Asia, Australia and Europe (Sugimoto et al., 1991, 1992; Kawazu et al., 1992a,b; Tanaka et al., 1993; Fujisaki et al., 1994; Katzer et al., 1994, 1998; Shiels et al., 1995; Kubota et al., 1996; Stewart et al., 1996). These are currently known as T . sergenti in Japan and Korea, T . buffeli in Australia and T. orientalis elsewhere (Fujisaki et al., 1994), although they are commonly collectively called the T . sergenti/orientalis/buffeli group. They are

IMMUNITY IN THE BOVINE THEILERIOSES

45

all transmitted by ticks of the genus Haemaphysalis. Since the purpose of this review is principally immunological, not taxonomic, we intend to use the term T . sergenti, as this is how the Japanese and Korean species, upon which most of the relevant immunological work has been done, are described in the primary literature. It should be pointed out that T . sergenti is often pathogenic, but not in all cases, and rarely fatal (0.2-0.4% mortality), to ‘western’ breeds (e.g. Holstein) and less so to indigenous (Japanese Black) breeds, causing chronic anaemia and serious loss of productivity. The economic impact to Japan is estimated at US$15 million per year (Sugimoto, 1997). In passing, it is worth mentioning that the benign species of Theileria of Africa, namely T . mutans and T . velifera (also possibly found in the Caribbean), can be differentiated from the other benign species of Theileria as they are transmitted by ticks of the genus Amblyomma (see Young, 1990). Note also T . taurotragi, usually apathogenic but occasionally fatal, which infects cattle, sheep, goats, eland and other bovids in Africa and is transmitted by ticks of the genus Rhipicephalus (see Dolan, 1989). 1.1.2. Life Cycle The life cycle is essentially the same for all Theileria species and has been extensively documented elsewhere (Barnett, 1977; Mehlhorn and Schein, 1984; Higuchi, 1986, 1987; Morrison et al., 1986; Irvin and Morrison, 1987; Kawamoto et al., 1990; Tait and Hall, 1990; Stewart et al., 1996). Development of the parasite takes place within the vertebrate and invertebrate hosts, with asexual reproduction by schizogony and merogony * in the bovine host followed by sexual reproduction and sporogony in the tick vector. Theileria annulata sporozoites preferentially invade major histocompatibility complex (MHC) class I1 positive cells (monocytes and B cells) in vitro (Glass et al., 1989; Spooner et al., 1989; Campbell et a[., 1994) and the host cells in vivo are now established to be monocytes (Forsyth, 1997; Forsyth et al., 1997, 1999). In contrast, T . parva sporozoites can infect and transform B and T cells at similar frequencies in vitro, but the majority of parasitized cells in the tissues of infected cattle are cx/p T cells (Baldwin et al., 1988; Dolan, 1989; Morrison et al., 1996). The detailed phenotype of the host cell type(s) transformed by T . sergenti schizonts has not been documented. They are, however, very rare, a feature which clearly distinguishes T. sergenti from T . annulata or T . parva, and the host cell is very enlarged (Kawamoto et a[., 1990; Minami et al., 1990; Kawazu et al., 1991; Sato et al., 1993, 1994).

* ‘Schizogony’ refers to the macroschizont and ‘merogony’ to the microschizont.

P 0)

Table 1 Comparative aspects of Theileria annulata, Theileria parva and Theileria sergenti. Thderia parva

Theileria onnulata

Theileria sergen ti

Location

Southern Europe, North Africa, Middle East, Central Asia, India, China

East and Central Africa

Japan, Korea, possibly world-wide'

Tick vector genus

Hyalomma

Rkipicephalus

Haemaphysalis

Estimated cost per annum

US$800 million in India

US$168 million total

U S 1 3 million in Japan

Mortality to exotic

40-60Y0

> 90%

Rare

Host cell type invaded and transformed by sporozoitc in vivo

Macrophage/monocy te

T cell

Not known but resides in lymph nodes and liver

Stage of life cyclc causing most pathology

Schizont and piroplasm

(Bos taurus) cattle

Z m

0 C

i Schizont

Piroplasm

P I

Disease

Tropical theileriosis

East Coast fever

Japanese bovine theileriosis

F

I-

Major clinical and pathological features

Live vaccine

Listlessness, anorexia, cachexia, diarrhoea, leucopenia, dyspnoea, petechial haemorrhages in major organs, ulcerated lesions of the gut, widespread dissemination of schizont-infected cells to major organs, anaemia Yes. Attenuated macroschizonts. Made by long-term tissue culture. > 90% effective. Cross-protective

Listlessness, anorexia, cachexia, diarrhoea, leucopenia, dyspnoea, petechial haemorrhages in major organs, ulcerated lesions of the gut, widespread dissemination of schizont-infected cells to major organs. Most virulent of the bovine Theileria species

Chronic relapsing anaemia

No; but can use ‘infection and treatment’ method. Naturally avirulent strains may have potential. Protects against homologous challenge only

Yes. Blood vaccine has been used. Not currently recommended

r

I C z_

2Z -I

I rn

m _.

2z

rn -I

I

! r

rn

Main effectors in immune animals

Cytostatic macrophages. Transient BOLA-restricted CTL and non-restricted NK cells

BOLA-restricted CD8+ CTLs

Antibodies to the merozoite/piroplasm?

Progress in subunit/recombinant vaccine research

Partial protection with recombinant SPAG-1 (sporozoite surface) and Tams (merozoite surface) proteins in some trials

Complete protection in a proportion of animals and partial protection in a proportion using p67 (sporozoite surface) protein in several trials with a range of delivery systems. One trial showed cross-protection against heterologous stocks

Partial protection with recombinant p32/34 (merozoite surface) proteins in one trial. Passive protection using anti-p33/34 monoclonal antibody

’

-

Theileria sergenti is a taxonomically controversial term and hence it is difficult to comment accurately on its distribution. BOLA = bovine leucocyte antigen; CTL = cytotoxic T lymphocyte; NK = natural killer.

z $ rn v)

48

N. BOULTER AND R. HALL

1.2. Clinical and Pathological Features

The clinical aspects of tropical theileriosis have been well characterized and documented (Neitz, 1957; Barnett, 1977; Srivastava and Sharma, 1981; Eisler, 1989; Forsyth, 1997; Forsyth et al., 1997, 1999). The course of the disease varies depending on the parasite strain, the host’s susceptibility and the quantity of sporozoites inoculated. It is well known that the severity of the disease is directly proportional to the initial inoculum of sporozoites (Uilenberg, 1981; Preston et al., 1992b). Time to detection of the initial symptoms varies from 4 to 14 days after the host is bitten by an infected tick. Swelling of the lymph node adjacent to the site of the tick bite is quickly followed by generalized lymphadenopathy. Schizonts can often be found in the lymph nodes at this time (Srivastava and Sharma, 1981). Hyperpyrexia is a hallmark of the disease and usually persists until death or recovery (Srivastava and Sharma, 1981). As the illness progresses the animal becomes anorexic, and there is a rapid loss of weight and condition. Other symptoms which accompany the disease are listlessness, mucous discharge from the eyes and nostrils, diarrhoea and dyspnoea. In all severe cases, petechial haemorrhages of the serous and mucous membranes occur and ulcers of the abomasum are also occasionally seen on post-mortem examination (Srivastava and Sharma, 1981). During the terminal stages there is usually oedema of the lungs, which causes severe respiratory distress, leucopenia, marked haemolytic anaemia, bilirubinuria, bilirubinaemia and jaundice (Neitz, 1957). The anaemia is thought to be a result of removal of erythrocytes by phagocytosis rather than parasite-induced lysis, although it has been suggested that autoimmune responses may contribute (Uilenberg, 1981). Tumour necrosis factor a (TNF-a) may also play a role (see Section 2.1.3, p. 56). Death usually occurs within 2-4 weeks from the onset of infection. Cattle that survive may undergo a prolonged period of recovery, which, in severe cases, may be incomplete, and animals may remain permanently debilitated and unproductive. In such cases a carrier piroplasm state ensues (Irvin and Morrison, 1987). The disease characteristics are similar for T. parva but some important differences must be noted. For example, in T.parva piroplasm parasitaemias are lower and anaemia is usually slight, with the main pathological effect arising from the lymphodestructive stage (Morzaria and Nene, 1990). In contrast, in other Theileria species, including T. sergenti, the severity of symptoms relates to the piroplasm infection rate and the resultant anaemia (Barnett, 1957; Jura and Losos, 1980; Morrison et al., 1986; Shimizu et al., 1990; Kawamoto et al., 1991; Yagi et al., 1991). T. sergenti causes chronic anaemia and one characteristic, resulting from a persistent carrier state, is relapsing parasitaemias, which can be induced by stress, and such episodes may be fatal.

IMMUNITY IN THE BOVINE THEILERIOSES

49

Occasionally, T. parva, T. taurotragi and T. mutans infections are associated with a syndrome known as turning sickness (Irvin and Morrison, 1987). The condition is usually associated with the presence of large numbers of free schizonts and infected lymphoid cells in brain capillaries and large haemorrhagic necroses in cerebral tissues. Affected animals show nervousness, ataxia and circling and may die during convulsions in this form of the disease, which usually occurs several months, and even years, after the initial theilerial disease (Irvin and Morrison, 1987).

1.3. Control Measures

Current control measures against bovine theilerioses are fourfold: tick control; chemotherapy; vaccination with attenuated lines; and vaccination by ‘infection and treatment’ (Brown, C.G.D., 1990; Musisi, 1990; Singh, 1990; Tait and Hall, 1990; Pipano et al., 1991; de Castro and Newson, 1993; Stewart et al., 1996). Each method will be briefly discussed separately. 1.3.1. Tick Control The conventional method of tick control is using acaricide in dips or sprays (Chizyuka and Mulilo, 1990; Musisi, 1990). More recently, acaricideimpregnated ear tags, slow-release rumen boluses and ‘pour-ons’ have been used (de Castro and Newson, 1993). Although these latter methods are more ‘user friendly’, disadvantages still exist including cost, residual contamination of meat and milk, and acaricide resistance of the ticks (Young et al., 1988). Continuous use of acaricides can also result in the breakdown of anti-tick immunity, causing a loss of equilibrium or ‘endemic stability’, and leave the cattle more susceptible to infection (Tatchell, 1981; Young et a[., 1988; Pipano and Grewal, 1990). Problems of susceptibility also occur when highly productive dairy cattle are introduced into an endemic area. One solution is to maintain these herds as ‘tick free’, involving the confinement of cattle on pasture in isolation, or in yards or barns to which tick-free fodder is transported, under a regime of regular short-interval application of acaricides (Lawrence, 1990). T h s system is obviously very expensive and it is prone to breaking down with the subsequent infection of many, if not all, cattle. Resistance of cattle to ticks can be produced by controlled infestations and taurine cattle immune to Rhipicephalus appendiculatus have been documented (Cunningham, 1981). Vaccination against many species of ticks, using crude antigenic extracts, is partially successful (reviewed by Kay and Kemp, 1994). A significant recent development is the production of a commercial subunit vaccine against Boophilus microplus, the vector for Babesia bigemina and

50

N. BOULTER AND R. HALL

B. bovis (see Cobon and Willadsen, 1990; Willadsen, 1990; Willadsen et al., 1995). A similar vaccine against the ticks responsible for the transmission of any of the bovine Theileria species would obviously be a useful control tool. 1.3.2. Chemotherapy Chemotherapy has not been widely used for the treatment of T. annulata, but it is an important component in the control of T. parva and T. sergenti infections (Purnell and Chang Rae, 1981; Stewart et al., 1990a,b; Tait and Hall, 1990; Hagiwara et al., 1993; Stewart et al., 1996). Halofuginone, a febrifugine, and the hydroxynapthoquinones parvaquone and buparvaquone, are all effective against Theileria infections (McHardy et al., 1985; Stewart et al., 1990a,b; Hagiwara et al., 1993). Buparvaquone has much higher anti-theilerial activity than parvaquone (Hashemi-Fesharki, 199la). It appears that the actions of these drugs are different; parvaquone is active against all stages of Theileria, but halofuginone and buparvaquone are active only against the schizont stage (Young et al., 1988). Halofuginone, despite being relatively cheap, is no longer a preferred drug because it has a narrow therapeutic range. Oxytetracyclines are also used to limit parasite development in the so-called ‘infection and treatment’ method as described in Section 1.3.4 below. 1.3.3. Vaccination with Attenuated Parasites The most widespread control measure against T. annulata is the inoculation of animals with an attenuated cell line vaccine. This involves inoculation of schizont-infected cells derived from a continuously growing tissue culture in vitro and is discussed in detail in Section 2.2.1 (p. 62). The production and use of schizont tissue culture vaccines has been described by many authors including Pipano (1981), Hall (1988), Brown, C.G.D. (1990) and Tait and Hall (1990). The cell culture vaccine protects most breeds of cattle against homologous challenge and often against heterologous challenge (Pipano, 1981). A single vaccination is usually sufficient but exotic breeds may require a second vaccination with a heterologous strain to provide full protection. A blood vaccine has been used against T. sergenti but it is no longer recommended. 1.3.4. Vaccination by ‘Infection and Treatment’ Another method of vaccination is the infection and treatment method (discussed in detail in Section 3.3.1, p. 73). This method was initially

IMMUNITY IN THE BOVINE THEILERIOSES

51

designed for use against T. parva and, as the name suggests, cattle are deliberately infected with a defined dose of sporozoites and then treated with a chemotherapeutic agent (Morzaria and Nene, 1990). Long-acting oxytetracyclines are the drugs of choice for this method when applied to T . parva, but buparvaquone is preferred in the case of T. annulata (see Hashemi-Fesharki, I99 la). The timing of treatment is important. Giving chemotherapy immediately after infection might clear the parasites before they have had the chance to establish themselves in the host lymphocytes, resulting in a lack of immunity to challenge. On the other hand, chemoprophylaxis must be given early enough to prevent the development of clinical symptoms (Morzaria and Nene, 1990). This type of vaccination provides solid immunity to challenge with homologous parasites and, depending on source of infection, frequently against heterologous challenge. 1.3.5. The Need for New Vaccines

There are several actual and theoretical problems associated with the use of live parasites. The infection and treatment method uses virulent stocks of parasites for the immunization regime. T h s method also results in the presence of piroplasms in the immunized animals, which can aid the spread of the disease by ticks to unprotected cattle (Tait and Hall, 1990). In addition, the drugs used are very expensive. Reversion to virulence of attenuated parasites used in the cell culture vaccine is always a worrying possibility, but there has been no indication of this happening so far. Drawbacks related to the use of either vaccine involve the need for a ‘cold chain’ from the point of production to the location of use, as well as the potential for transmitting other pathogens with the immunizing parasites (Tait and Hall, 1990). All of these problems could be avoided if non-living, effective, stable, cheap, single application subunit vaccines for these pathogens could be developed. To achieve this objective requires a combined molecular, genetic and immunological approach. A balance between fundamental and empirical research is also required. Eventually the ‘new’ safer vaccines will form part of integrated disease control programmes (Young et al., 1988).

2. THElLERlA ANNULATA

2.1. Immune Responses

The bovine immune system is subjected to different antigenic determinants at each stage of the parasite’s life cycle, and this results in a heterogeneous

52

N. BOULTER AND R. HALL

nocyte/macrophage

Neutralizing

, +

e.g. anti-SPAG-1 antibodies

f

% @ Invasion

1 Cytostatic macrophage

I

development 24 hours

TNF-a and IL-ip, etc

.

._.._I._

Inhibition by cytostasis

4

antibodiese.g. anti-p32/33

Figure 1 Summary of the proposed immune response against Theileria annulata. The figure is drawn to emphasize the proposed distinctive roles for the innate immune response (on the right) and the adaptive immune response (on the left). The thickness of the arrows is intended to reflect the relative importance of the relevant immune mechanisms. The major effector depicted is the cytostatic macrophage and the prime effector mechanism is nitric oxide-mediated inhibition of the schizont-infected macrophages. Note that TNF-a inhibits development from the trophozoite to the

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response comprising humoral and cellular components. In all cases, animals that recover from infection are solidly immune to homologous challenge and often to heterologous challenge (for a review, see Hall, 1988). Immunity is acquired irrespective of the mode of primary infection, whether this be by the natural route via injection of sporozoites by a feeding tick, or by artificial immunization with sporozoites followed by chemotherapy, or by vaccination with attenuated schizont-infected cell lines (Pipano, 198 1). Immunity has been shown to last for approximately 3 years or longer if the animal is challenged (Pipano, 1995). The immune response to each stage of the life-cycle will be discussed separately and the overall situation is summarized in Figure 1 and by Preston et al. (1999). 2.1.1. The Sporozoite From the outset it should be stated that the very existence of an effective attenuated schizont vaccine (see Section 2.2.1, p. 62) means that immunity to the sporozoite stage is not essential to achieve protection. On the other hand, even a partially protective response against sporozoites might well prove beneficial by reducing the severity of the ensuing disease. This line of thinking is based on the established correlation between sporozoite dose and virulence of the disease (Preston et al., 1992b) plus the fact that live sporozoite immunization provides better protection against heterologous challenge than do attenuated cell lines (Sergent et al., 1945; Preston and Brown, 1988). Since the sporozoite is extracellular, intuition suggests that effective anti-sporozoite immunity might be humoral, and specifically might be mediated by neutralizing antibodies. Evidence for such antibodies comes from the work of Gray and Brown (1981), who clearly demonstrated that sera taken from animals, after infection with a stabilate of T. annulata sporozoites (either Hissar or Ankara strains), contained activity that neutralized the infectivity of sporozoites for bovine peripheral blood mononuclear cells (PBMs) in vitro. These sera were also capable of inhibiting invasion by sporozoites from a heterologous parasite stock, which suggests that parasites from geographically distinct regions share some common antigenic determinant(s). It should, however, be pointed out

schizont stage but not the multiplication of schizonts. Natural killer cells also play a role in lysing the schizont-infected macrophage. Nitric oxide is also able to inhibit the ability of sporozoites to invade the host cell in vitro. In the adaptive immune response, a central role for BOLA-restricted cytotoxic T-cells targeted against the schizontinfected cells is depicted. Neutralizing antibodies against the sporozoite and merozoite stages are also thought to be important. APC = antigen presenting cell; CTL = cytotoxic T lymphocyte; IL = interleukin; M@ = macrophage/monocyte; NK = natural killer cell; NO = nitric oxide; rbc = red blood cell.

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that these experiments used whole serum and thus do not formally prove a role for antibodies. Proof of antibody-mediated sporozoite neutralization is provided by the demonstration that several different monoclonal antibodies (mAbs) to sporozoite antigens can also abrogate infectivity. One such mAb, 1A7, blocks sporozoite invasion effectively and also reacts positively in immunofluorescent (IFA) tests against formalin-fixed sporozoites, but not against the schizonts or piroplasms (Williamson, 1988; Williamson et al., 1989). mAb 1A7 also recognizes sporozoites from three geographically distinct stocks (from Morocco, India and Turkey), which are known to differ by several other criteria, including reactivity to a panel of anti-schizont mAbs and genotypically (Shiels et al., 1986; Katzer et al., 1999). Recent work has focused on characterizing and assessing the immunological significance of the sporozoite antigens defined by the neutralizing mAbs (Knight, 1993; Boulter et al., 1995; Knight et al., 1996, 1998). The gene for the sporozoite surface antigen (SPAG-1) of T. annulata, recognized by mAb lA7 and derived from a T. annulata Hissar complementary deoxyribonucleic acid (cDNA) library, has been well characterized. SPAG- 1 has been shown to be located on the surface of the sporozoite by immunogold electron microscopy (Knight, 1993). There is a single open reading frame extending for 2721 nucleotides encoding a predicted 91.9 kDa polypeptide of 907 amino acids (Hall et al., 1992). A number of structural and immunological features are summarized in Figure 2. Perhaps of most interest are two blocks of repetitive motifs of PGVGV and VGVAPG (single-letter amino-acid notation). The former of these is identical to repeat structures found in bovine elastin. The latter is also found in elastin and has been demonstrated to be chemotactic and a ligand for the elastin receptor (Davidson, 1987). The presence of the VGVAPG motifs led to the suggestion that they might be involved in host cell recognition (Hall et al., 1992). However, this idea has since been weakened by the discovery that some variants of SPAG-1 lack the VGVAPG motif and also that sporozoites invade cells lacking the elastin receptor (Campbell et al., 1994; Hall, 1994; Katzer et al., 1994). There are several immunodominant sites on the SPAG-1 molecule, as determined by reactivity to various subfragments of SPAG-1 in Western blots with a panel of bovine immune sera, and these are summarized in Figure 2 (Boulter, 1996; Knight et al., 1996). Furthermore, SPAG-1 has been shown to carry neutralizing determinants in the C-terminus defined by immune calf sera, which are distinct from the 1A7 epitope (Williamson, 1988; Williamson et al., 1989; Hall et al., 1992; Boulter et al., 1994, 1995; Boulter, 1996). Partial protection has been achieved with recombinant SPAG-1 (Boulter et al., 1995, 1998, 1999) and this will be described in detail in Section 2.2.2 (p. 64). In addition to antibody responses, SPAG-1 has also been shown to elicit T-cell responses, although these are very variable

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Figure 2 Comparison of the sporozoite surface proteins from Theileria annulata (SPAG-1) and T . parva (p67) shown diagramatically to emphasize their structural and immunological similarity. The numbers above the boxes refer to amino-acid residues in SPAG-1. The anti-SPAG-1 and anti-p67 epitopes were defined by reacting bovine antisera (to recombinant SPAG-1 and p67) with short regions of SPAG-1 (Knight et al., 1996).

(Boulter ef al., 1995, 1998, 1999). Recent experiments have shown that extremely strong CD4+ memory T-cell responses can be generated to the N terminus of the SPAG-1 molecule, in the presence of interleukin 2 (IL-2) from the PBMs of immune animals (J.D. Campbell, personal communication). T-cell clones, specific to the N terminus, have now been generated and are the first T cells to be isolated in vitro specific for a T. annulata-derived peptide. An interesting observation is the cross-reactivity of SPAG-1 and the T. parva homologue, p67 (Figure 2; Knight et al., 1996). There is a 56% identity between the C-terminal regions of SPAG-1 and p67 (Figure 2; Katzer et al., 1994). mAb 1A7, which reacts to a C-terminal epitope of SPAG-1, cross-reacts with p67 and is also capable of neutralizing T. parva sporozoite infectivity with 100% efficiency. In addition, seven continuous amino acids from the predicted sequence recognized by lA7 are present in the p67 sequence and would be sufficient to form a common epitope (Knight et al., 1996). The potential importance of the C-terminal regions of these

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molecules is supported by the evidence that antisera raised against recombinant p67 block T . annulata sporozoite invasion in vitro and vice versa. Furthermore, vaccination trials in which animals have been immunized with SPAG-1 and challenged with T . parva sporozoites, or immunized with p67 and challenged with a T. annulata sporozoite stabilate, provide some evidence of cross-protection to the heterologous challenge (Boulter et al., 1998, 1999, unpublished data; see Section 2.2.2, p. 64). Hence there is a possibility of designing a subunit vaccine containing either SPAG-1 or p67, which will be effective against both T . annulata and T.parva sporozoites. Two different antigens (called SPMl and SPM2), both recognized by sporozoite-neutralizing mAb 4B11, have been characterized (Knight, 1993; Knight et a[., 1998). Unlike SPAG-1, these two antigens are also expressed on the schizont and piroplasm stages and, other than being recognized by immune calf serum, their protective relevance remains obscure. Apart from antibodies, nitric oxide (NO), produced by activated macrophages, has been shown to inhibit sporozoite invasion into host cells in vitro (Visser et al., 1995). Whether this is effective in vivo remains to be elucidated. 2.1.2. The Trophozoite Theileria trophozoites are susceptible to factors in immune serum (Preston and Brown, 1985). Thus when cultures, containing parasites just established within the host cells, were incubated with T . annulata immune sera, the development of trophozoite-infected cells into schizont-infected cells was suppressed (Preston and Brown, 1985). Initially, it was postulated that this activity was antibody-dependent and was mediated via sporozoite antigens left on the surface of the invaded lymphocyte after entry, but further work indicated that it is more likely that other serum factors, probably cytokines, are responsible (Preston et al., 1992a). To this end, a comprehensive study of the effect of various recombinant cytokines on the development in vitro of T . annulata and T. parva trophozoite-infected cells showed that TNF-a, interferon y (IFN-y), IFN-a, IL-1 and IL-6 were inhibitory (Figure I; Preston et al., 1992a). However, the mode of inhibition by these cytokines remains obscure, although they may be inducing NO production (Visser et al., 1995).

2.1.3. The Schizont (a) Protective immunity. Cell-mediated responses to the schizontinfected cell are generally thought to be responsible for protective immunity as well as for much of the underlying pathology (Rehbein et al., 1981a,b;

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Preston et al., 1983; Ahmed et al., 1989; reviewed by Tait and Hall, 1990; Kachani and Spooner, 1992; Chaudhri and Subramanian, 1992). Currently, both the innate and adaptive arms of the immune system are believed to play important effector roles. In particular, roles for cytotoxic T cells, natural killer (NK) cells, helper T-cells and macrophages have been identified and are elaborated below (Figure 1). Preston et al. (1983) demonstrated that, when immune cattle are challenged, two peaks of cytotoxic cell activity were observed in the blood and lymph nodes. The cells in the first of these peaks, appearing one week after challenge, were bovine leukocyte antigen (BoLA) (MHC class I) restricted, whereas those in the second peak, observed three weeks after challenge, were BoLA restricted in some, but not all, animals. The MHCrestricted components are cytotoxic T-cells (CTLs), whilst the non-restricted class are designated as NK cells. In the same study it was shown that calves undergoing a primary infection, but destined to recover, also developed two peaks of cytotoxic cells. In contrast, calves that did not recover rarely showed such responses. In a more recent study, Chaudhri and Subramanian (1992) produced similar but not identical results. They demonstrated that three calves that survived a primary infection induced by sporozoites, or a virulent schizont-induced infection, developed a single wave of cytotoxic cells that killed schizont-infected autologous cells. These reached a peak three weeks after infection, which correlated with a peak of T cells defined by the ability to form E rosettes with sheep erythrocytes. Calves that failed to recover from the primary infection had no, or only very weak, cytotoxic responses. A second peak of cytotoxic cells was induced after sporozoite challenge. In addition, T cells from these calves produced macrophage migration inhibition factor (MIF), in response to both schizont and piroplasm antigens. Innes et al. (1989b) showed that animals infected with an allogeneic T. annulata cell line exhibited very mild clinical reactions compared to the very severe response in animals infected with an autologous cell line. In the allogeneic group, there were two distinct waves of cytotoxic activity, the first being directed against the foreign BoLA antigens and the second aimed at the parasite. However, animals infected with the autologous cell line developed cytotoxicity only against the parasite, as expected. In both cases the anti-parasite response involved both BOLA-restricted and non-restricted elements. All cattle became immune to heterologous sporozoite challenge, during which they developed a cytotoxic response, which was MHC restricted and cross-reactive. The non-restricted cytotoxic response seen in T. annulata infections has been postulated to be due to NK cells (Preston et al., 1983). NK cells have been implicated as effectors in other intracellular parasitic infections such as those due to Trypanosoma cruzi (see Cardillo et al., 1996) and Leishmania

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(see Tizard, 1992). They are characterized as large, granular, non-adherent, non-phagocytic, non-T and non-B cells that originate in the bone marrow and will kill xenogeneic tumour cell lines (Tizard, 1992). In model systems, NK cells are powerful producers of numerous cytokines including IFN-y, granulocyte-macrophage colony-stimulating factors (GM-CSFs), TNF-a, IL-3 and IL-8 (reviewed by Scott and Trinchieri, 1995). The activities of NK cells (cytotoxicity, proliferation and production of cytokines) are regulated by various cytokines. It is believed that NK cells usually become effective soon after an infection because many pathogens stimulate the production of cytokines critical for NK cell activation. The most powerful NK cell enhancers are IL-12, IFN-a and IL-2 (Scott and Trinchieri, 1995). In this regard it is interesting that schizont-infected cells have been shown directly to produce IFN-a (Entrican et al., 1991; Forsyth et al., 1999). However, interestingly, in the work of Preston et al. (1983) cited above, contrary to dogma, the adaptive BoLA-restricted CTL response preceded the innate NK response. Other innate effectors of the immune system are involved in protection against T. annufata, including cytostatic macrophages, which are believed to have an absolutely pivotal role (Figure 1). Preston (1981), Preston and Brown (1988) and Preston et al. (1993) clearly showed that macrophages, isolated from the peripheral blood of calves immunized with sporozoites or schizont-infected cell lines, exhibited strong cytostatic effects on schizontinfected cells. These cytostatic cells were active against allogeneic and autologous cell lines. Macrophage activity declined after a few weeks but rose again after challenge, with the same kinetics as before. The macrophages were shown spontaneously to produce TNF-a (Preston et af., 1993) and NO (Visser et af., 1995), and the levels produced were enhanced upon exposure of the cells to IFN-y, which suggests a role for antigen-specific helper CD4+ T cells. The cytostasis was shown to be mediated via a soluble factor, which was not TNF-a, IL-1 or IFN-a, and was probably NO, as discussed below (Preston et al., 1992a). In fact recombinant TNF-a (and IL-2) consistently enhanced the proliferation of schizont-infected cells (Preston et al., 1992a). This suggests that these cytokines may play a role in the pathogenesis of the disease (see below). The synthesis in vivo of IFN-y and TNF-a by host cells in response to T. annulata infection has been demonstrated (Preston et al., 1993). NO is a reactive metabolite of leucocytes, and in particular macrophages, and has been implicated in immunity to many protozoa such as Leishmania species (Tizard, 1992). The production of NO by these cells is promoted by IFN-y and TNF-a, and thus Visser et al. (1995) undertook a study to see if NO could be a mediator of macrophage anti-Theileria activity. PBMs from calves undergoing an infection spontaneously produced NO in vitro. However, PBMs from immune calves did not produce NO unless exposed

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to IFN-y, indicating that NO production and release depend upon macrophages being both primed and triggered (Visser et a/., 1995). PBMs from an immune calf were stimulated to produce NO by co-cultivation with schizont-infected cells. These results, together with the fact that schizontinfected cells can stimulate lymphocytes to secrete IFN-y and TNF-a in vivo, suggested that these infected cells may initiate the production of macrophage-derived NO (Visser et a/., 1995). NO has been implicated as an effective mediator of protective immunity, in vivo, against trophozoite- and schizont-infected cells (Visser et al., 1995). Furthermore, NO has been shown by Richardson et al. (1998) to inhibit the proliferation of schizontinfected cells and to cause intracellular schizonts to disappear and host cells to become apoptotic. By extrapolation, these authors suggested that merozoites may also be susceptible to the effects of NO. In contrast to this protective role, it is postulated that NO, in excessive amounts, could well play a prominent role in the pathogenesis of Theileria; there is evidence that it plays a role in the pathogenesis of malaria (Mendis and Carter, 1995). (b) Immunopathology. It is well established that inappropriate immune responses can lead to pathology and disease. Recent evidence has indicated that primary infection with T. annulata induces aberrant T-cell activation, which in turn results in a failure to mount an effective immune response (Campbell et al., 1995, 1997; Campbell and Spooner, 1999). The consequence of this is an uncontrolled acute lethal infection. A manifestation of this inappropriate T-cell activation is the fact that T. annulata schizont-infected cells are able to induce non-specific proliferation of autologous resting T cells in vitro [dubbed the 'autologous mixed lymphocyte reaction' (MLR)] in the absence of added antigen (Preston, 1981; Campbell ef a/., 1995). In fact, this type of autologous MLR in vitro was first described for T. parva by Pearson et al. in 1979, but it has not been implicated in the pathogenesis of East Coast fever to date. Other indicators of naive T-cell activation by T. annulata-infected cells are the induction of IL-2 receptor (IL-2R) and MHC class I1 molecules on the surface of CD4+ and CD8' T cells from naive animals (Campbell et al., 1995).This activation appears to be mediated via a combination of direct cell contact and cytokine effects. Thus in vitro-derived schizont-infected cells express messenger ribonucleic acid (mRNA) for IL-la, IL-ID, IL-6, IL-10, TNF-a (Brown, D.J. et af., 1995) and IL-12 (J.D. Campbell, personal communication) and the levels of IL- 1a and IL-6 correlate with the level of induced proliferation. Resting CD4+ or CD8' T cells are stimulated by schizont-infected cells irrespective of their overall phenotype or memory status, although contactdriven activation is directed only at CD4+ cells (Campbell et al., 1997). Both cup and yS T cells are activated by schizont-infected cells. The autologous MLR phenomenon has been likened to a superantigen effect but there are differences. In particular, the proportion of T cells activated is much greater

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in the autologous MLR than would be expected for a superantigen, so this may be an entirely different phenomenon. According to Campbell, the counterpart in vivo of the autologous MLR results in the following sequelae (Campbell and Spooner, 1999). First, within 48 hours, large numbers of infected macrophages develop within the medulla of the lymph node draining the site of infection. Macrophages are professional antigen-presenting cells and, when infected with T. annulata, their capacity to present third-party antigens, via MHC class 11, to antigenspecific CD4+ T cells is augmented (Glass and Spooner, 1990). As a result, within 4 days, large numbers of CD4+ T cells are inappropriately activated in the medulla instead of the paracortex of the lymph node, where normal T-cell interactions should occur; i.e., during infection, T cells are not being primed in normal anatomical sites. Thus, instead of antigen-driven selection of a small number of T cells that would subsequently undergo clonal expansion, there results a rapid polyclonal expansion of IL-2Rf T cells that also quickly leave the node. The overall effect is ablation of the ability to generate an effective immune response. Additionally, it has been shown in some cases that infection with T. annulata results in a modification of the BOLA class I antigens on the surface of the transformed host cells (Oliver and Williams, 1996). These authors postulated that this may be a mechanism that the parasite uses to evade the host immune response. TNF-a may have a major role in the pathogenic effects (pyrexia, cachexia, anorexia, depression and anaemia) seen in T. annulata infection (Preston et al., 1993; Brown et al., 1995; Adamson and Hall, 1997; Forsyth et al., 1999). In fact, many of these symptoms are produced in cattle administered recombinant TNF-a (Bielefeldt Ohman et al., 1989). Macrophages harvested from cattle undergoing a primary or challenge infection of T. annulata spontaneously produce TNF-a and the levels produced are enhanced by exposure to IFN-7 (Preston et al., 1993). Abnormally high levels of IFN-y are seen in lethal T. annulata infections and thus may result in excessive amounts of TNF-a being produced (Campbell et al., 1997). Also high levels of matrix metalloproteinases produced by schizont-infected cells may enhance the conversion of pro-TNF-a to the mature species (Adamson and Hall, 1997). Furthermore, 76 T cells, which are stimulated to proliferate by autologous T. annulata-infected cells in the presence of IL-2, express an mRNA transcript for TNF-(u (Collins et al., 1996). TNF-a acts directly on the temperature-regulating centre in the hypothalamus to cause a fever (Tizard, 1992). This in turn may help the parasitic transition from schizonts to merozoites (Glascodine et al., 1990). Weight loss and cachexia in animals suffering from a chronic parasitic infection can be attributed, at least partly, to this cytokine. It inhibits the synthesis of lipoprotein lipase, acetyl coenzyme A, carboxylase and fatty acid synthetase. As a result it prevents the uptake of lipids by preadipocytes and causes mature adipocytes to lose

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stored triglycerides. TNF-a also acts on muscle cells and hepatocytes to stimulate their catabolism. In addition, TNF-a suppresses haematopoietic progenitors and thus decreases red blood cell production, and also reduces their life span, resulting in anaemia (Tizard, 1992). TNF-a has also been implicated in both the pathology (cerebral malaria, anaemia and fever) and protection (control of parasite multiplication) in Plasmodium infections (reviewed by Taverne, 1994; Jakobsen et al., 1995; Mendis and Carter, 1995), and in the cachexia associated with Trypanosoma cruzi infection in mice (Truyens et al., 1995). The pathology of tropical theileriosis is partly associated with the metastatic behaviour of the schizont-infected cell and the lesions induced in the invaded tissues (Forsyth et al., 1999). It is quite likely that the expression of matrix metalloproteinases induced by this organism are necessary, but probably not sufficient, to explain the mechanisms of tissue invasion and tissue damage (Baylis et al., 1992, 1995; Adamson and Hall, 1996, 1997; Somerville et al., 1998a). 2.1.4. The MerozoitelPiroplasm The merozoite, like the sporozoite, is an extracellular stage and is therefore also a potential target for a protective humoral immune response. Work on the merozoite stage has been hampered because of difficulties in obtaining sufficient amounts of merozoites for molecular analysis and the lack of an assay in vitro for erythrocyte invasion. The former problem has now been overcome by utilizing the fact that schizont-infected cell lines can be induced to differentiate into merozoites by culturing at 41 "C (Glascodine et al., 1990). This differentiation is associated with a change in mAb reactivity profile, with epitopes being detected on the merozoite and piroplasm which were absent from the schizont. One of these stage-specific antigens, recognized by mAb 5E1, was shown to have a molecular mass of 30 kDa (Glascodine e f al., 1990). This antigen was further characterized by Dickson and Shiels (1993). It was strongly recognized by serum from an immune cow and was shown to exist in two forms with molecular masses of 30 kDa and 32 kDa. The two molecules were shown to be related, and are in fact the products of alternative alleles, but only the 30 kDa form was recognized by mAb 5E1. Molecules with similar characteristics have been identified in other species of Theileriu with molecular masses ranging from 30 to 34 kDa (reviewed by Dickson and Shiels, 1993). The genes encoding for the 30132 kDa proteins from T . annulata, Tamsl-1 and Tamsl-2, respectively, have been isolated and characterized (Shiels et al., 1994, 1995). Sequence analysis of many alleles of Tams have shown that they encode very polymorphic molecules, particularly within a region that

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contains a number of putative N-linked glycosylation sites (Katzer et al., 1999). It is postulated that this level of antigenic diversity may indicate selection of variable glycosylation sites or amino-acid epitopes in order to evade the bovine immune system (Shiels et al., 1995). A number of other polypeptides has been immunoprecipitated from surface-iodinated piroplasms by immune cow serum (Shiels et al., 1989). These polypeptides probably originate from the merozoite and give some indication that an immune response might be mounted to this stage. In addition, Ahmed et al. (1988) provided evidence that immune serum contained antibodies that specifically opsonized free merozoites. Antibodyindependent complement lysis was also shown to occur. Thus, to summarize, infection with T. annulata stimulates both the innate and adaptive arms of the immune response. Activated macrophages producing TNF-a and NO, NK cells, cytotoxic T cells and antigensensitized CD4+ cells are all at work as the disease progresses. It is likely that the cytokines produced by the parasitized cells themselves modulate the immune response. However, there is a delicate balance between the induction of a protective and a pathological response. These considerations have important implications in subunit vaccine design. It is expected that the most effective immunity will be induced by vaccines that are able to stimulate CD4+ T-cell-mediated macrophage activation (Richardson et al., 1998). The inclusion of antigens that promote specific macrophage activation should result in the adaptive immune response being driven towards a protective Thl response and the generation of cytotoxic T cells, cytokines (predominantly TNF-a, and controlled levels of IFN-y) and NO, which have parasiticidal properties. 2.2. Vaccination

2.2.I . Attenuated Cell Line Vaccine The most widespread control measure taken against T. annulata is the vaccination of animals with an attenuated cell line vaccine. This involves inoculation of cattle with schizont-infected cells derived from a continuously growing tissue culture in vitro. The production and use of schizont tissue culture vaccines have been described by many authors including Pipano (1981), Hall (1988), Brown, C.G.D. (1990) and Tait and Hall (1990). Long-term culture attenuates schizont-infected cells so that their pathogenicity is reduced but their infectivity is retained (Darghouth et al., 1996; Sutherland et al., 1996; Somerville et al., 1998b). In addition, the ability to produce the merozoite stage is reduced both in vivo and in vitro once the cell line is attenuated. The number of passages required to achieve

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attenuation is dependent on the isolate: some require as few as 20 passages whilst others require up to 300 passages. Virulence is tested periodically by inoculating the culture into susceptible cattle (Hall, 1988). Once attenuation has been achieved, aliquots of the infected cells can be cryopreserved in liquid nitrogen. These can then be resuscitated and subcultured or used directly for immunization (Pipano, 1981), although the shelf-life of the infected cells once thawed is limited. This vaccine is effective because the schizont establishes itself in the recipient cells, an essential requirement if it is to engender protective immunity. The mechanism by which the schizont transfers from the donor to recipient cells is unknown, but Forsyth et al. (1997) suggested a process involving opsonization with complement. Schizont-infected cells express CDI Ib, the membrane receptor for C3bi, which is phenotypically a reflection of their macrophage origin. C3bi is a component of the complement cascade and an opsonin. It is suggested that free schizonts are opsonized by complement and then transferred to macrophages bearing the C3bi receptor. Attachment of the parasite-complement complex would thus enable phagocytosis of the parasite by the cell. Innes et al. (1989a) have shown that as few as 100 allogeneic cells can be used to immunize against T. annulata, whereas very high doses of allogeneic cells (> lo8) are required to immunize against T. parva. The normal vaccine dose is about lo6 cells. Since both parasite species are introduced in allogeneic cells, histoincompatibility between cell line and recipient cannot be the main influence on the successful establishment of the parasite within the new host. The different outcomes are probably a result of their preferences for different cell types and, specifically, the fact that T. annulata shows a distinct macrophage/monocyte tropism (Glass et al., 1989; Spooner et al., 1989). The mechanism of attenuation is not known and studies are currently under way to attempt to define the markers of attenuation. A number of genes has been identified that are either upregulated or downregulated on attenuation (Somerville, 1997; Somerville et al., 1998b). In addition, marked changes in the host matrix metalloproteinase profile of T. annulata-infected cells have been observed in some cell lines (Baylis et al., 1992, 1995; Somerville, 1997; Somerville et al., 1998b). Another interesting observation is the selective expression of the schizont antigen recognized by mAb EU106 (Sutherland et al., 1996). The antigen is stage-specific and is also expressed on the surface of infected host cells (Preston et al., 1998), but only in virulent cell lines; the expression diminishes upon attenuation. Attenuation, at least in some instances, is accompanied by clonal selection of a minor subpopulation or single genotype and/or altered parasite gene expression (Baylis et al., 1995; Darghouth et al., 1996; Sutherland et al., 1996; Somerville, 1997; Preston et al., 1998; Somerville et al., 1998b; Hall et al., 1999). The cell culture vaccine protects most breeds of cattle against

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homologous challenge and usually against heterologous challenge also (Pipano, 1981). A single vaccination is usually sufficient but exotic breeds may require a second vaccination with a heterologous strain to provide full protection (Nichani et al., 1997). The schizont vaccine has the advantage that it does not result in the production of erythrocytic stages in the immunized animal and therefore does not allow further transmission (Tait and Hall, 1990). However, it does not induce sterile immunity and cannot prevent the formation of piroplasms after natural tick challenge, and cannot therefore be used as a means of eradicating the disease (Pipano et a[., 1991). Nevertheless, this type of vaccine has been used successfully against T. annulata infection in many countries. These include: Israel, where it has been used for over 30 years (Pipano, 1995); Iran, in which a long-term vaccination campaign has been undertaken (Hashemi-Fesharki, 1988, 1991b); India, where the vaccine is produced commercially (Singh et al., 1993); the former Soviet Union (Zablotsky, 1991); and Turkey (Sayin et al., 1997). It is in various stages of development in other countries such as Iraq (Khdier and Latif, 1991), Tunisia (Darghouth et al., 1996, 1997), Morocco (Ouhelli et al., 1997), China (Gu et al., 1997; Shirong, 1997) and, most recently, Spain (Viseras et al., 1998). 2.2.2. Subunit Vaccine Development (a) General considerations. Although the cell line vaccines are highly efficacious, a number of drawbacks compromises their use (see Section 1.3.5). Testing and the long culture period needed to produce a vaccine, which make it expensive, are limitations. It is also unknown whether the parasite can revert to virulence in vivo. Furthermore, there are concerns about the likelihood of transmitting other pathogens with the immunizing parasite and there is also a requirement for a ‘cold chain’ from the point of production to the location of use (Tait and Hall, 1990). As a result of these limitations, research is currently aimed at producing a recombinant subunit vaccine that will obviate the problems. It is anticipated that such a vaccine will comprise components of the three major life-cycle stages, i.e. the sporozoite, schizont and merozoite/piroplasm. To date, vaccine trials in cattle have been performed with two recombinant antigens: SPAG-1, a sporozoite surface antigen (Hall et al., 1992), and Tams1 from the merozoite (Shiels et al., 1994, 1995). In most cases the recombinant antigens induced partial protection to sporozoite or blood challenge, respectively, but the trials showed that efficacy depended on the delivery system. (b) SPAG-1 trials. Five SPAG-1 trials have been performed to date, with various outcomes (Williamson, 1988; Knight, 1993; Boulter et al., 1995,

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1998, 1999). The first two, utilizing the C-terminal domain of SPAG-1 (SRl region, Figure 2) as a P-galactosidase fusion and the full-length SPAG-1 molecule expressed as a glutathione-S-transferase fusion, respectively, yielded disappointing results in that there was little evidence of protection against sporozoite challenge (Williamson, 1988; Knight, 1993; Boulter et al., 1999). However, sporozoite neutralizing antibodies were obtained and this observation gave the encouragement needed to investigate alternative ways of delivering the antigen in order to enhance the protective immune response. The first trial to provide evidence of protection used hepatitis B core antigen (HBcAg) as a powerful source of stimulating T-helper cells. Four calves were immunized with recombinant HBcAg with the SR1 region fused as a surface loop structure (HBcAg-SRl), and four control animals were immunized with native HBcAg (Boulter et al., 1995). Saponin was used as the adjuvant and five immunizations were given at monthly intervals. After sporozoite challenge, all the calves became infected, but the immunized animals showed a marked reduction in the number of schizonts and piroplasms, and also had significantly longer prepatent periods (length of time until schizonts were seen in lymph node smears) compared to the controls. Furthermore, very high titres of anti-SR1 antibodies (> 1 in 10 240) were apparent only 14 days after the first immunization, and sera collected between the third and fourth immunizations exhibited very strong sporozoite neutralizing ability (84% inhibition of sporozoite invasion into host cells when used at a dilution of 1 in 250). Strong CD4' T-cell responses were observed, although evidence of a T-suppressor element within the SR1 region was also noted. Importantly, all the test calves survived challenge whereas two of the control calves did not (Boulter et al., 1995). The subsequent trial utilized the full-length SPAG- 1 molecule, expressed with a his6 tag (an N-terminal run of six histidine residues) to facilitate purification, formulated with RWL@, a proprietary adjuvant from SmithKline Beecham, or incorporated into immunostimulatory complexes (ISCOMs) (Boulter et al., 1998, 1999). Six animals were immunized three times with SPAG-1-RWL and six animals with SPAG-1 in ISCOMs, whilst controls were immunized with phosphate-buffered saline (PBS) and adjuvant (PBS-RWL) or empty ISCOMs. A further group was immunized with p67-RWL, the SPAG-1 homologue in T. parva, to see if any crossspecies protection could be induced with a T. annulata challenge. On sporozoite challenge all animals developed classical symptoms of tropical theileriosis but of varying intensities. Briefly, the SPAG-1 -RWL group were the best protected as assessed by an increase in the prepatent and incubation periods, and the observation that three of six animals survived challenge whereas all 12 controls (six PBS-RWL and six empty ISCOMs)

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did not. Again, significant levels of anti-SPAG-1 antibodies, sporozoite neutralizing antibodies and T-cell proliferative responses were obtained, although there was no correlation between these and the level of protection induced (Boulter et a/., 1998, 1999). The SPAG-1-ISCOM immunized group exhibited very limited anti-SPAG- 1 antibodies and T-cell responses, but two did survive challenge. Interestingly, the p67-RWL group also showed some protection to T . annulata challenge as indicated by the fact that three of six survived. Significant levels of anti-SPAG-1 antibodies recognizing determinants in the extreme N- and C-termini and sporozoiteneutralizing antibodies were also produced by this group, indicating that there is some level of cross-species protection induced (see also Figure 2). The reciprocal trial, in which animals were immunized with SPAG-1RWL but then challenged with T . parva sporozoites, was subsequently conducted (N. Boulter et a/., unpublished data). Preliminary results indicated that, whilst all animals became infected, four of ten animals immunized with SPAG-1 -RWL were protected against a 70% lethal dose of T. parva, whereas four of seven animals immunized with p67, and only one in seven of the control group, were protected. The existence of cross-reactive epitopes between SPAG-1 and p67 is well documented (Knight et a/., 1996; Figure 2), but these studies raised the possibility of the development of a single vaccine that would be effective against both parasites. (c) Tams trial. In the trial with Tams, two allelic forms of the major merozoite surface antigen (Tamsl- 1 and Tams 1-2) were included. Tams 1- 1 and 1-2 proteins were produced. They were expressed in Escherichia coli with his6 tag. They were also expressed in Salmonella typhimurium aroA vaccine strain SL3261 (d’oliveira et al., 1996). Naked DNA constructs with Rous sarcoma virus long terminal repeats (LTRs) as the promoter were also prepared. Five groups of three cattle were used as follows: one group was immunized with his6 tagged Tams 1- 1/ 1-2 incorporated into ISCOMs; a second group was immunized with naked DNA plasmids encoding the antigens; a third and fourth group were immunized with recombinant S . typhimurium via the subcutaneous or oral routes, respectively; and the fifth group contained unimmunized controls. The immunizing regimen differed between the groups but all animals were challenged 4 weeks after the last immunization with a stabilate made from blood taken from an infected animal with 30% piroplasm parasitaemia (d’oliveira et a/., 1997). The Tams-ISCOM immunized group developed anti-Tamsl-1 and 1-2 antibodies by the time of challenge and were protected against blood challenge as designated by a parasitaemia of 1 % or less (compared to an average of 16% in the controls) and a maximum rectal temperature less than 41 “C. Two of the three DNA-immunized animals survived challenge despite the absence of detectable anti-Tams antibodies. In contrast, none of the Salmonella immunized animals was

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protected against challenge, and two of the animals died. All three controls died (d’oliveira et al., 1997). Whilst one must, of course, be cautious about the interpretation of such small-scale vaccine trials, the results provided some encouragement concerning the feasibility of developing a subunit vaccine against tropical theileriosis. Obviously, further work needs to be conducted in order to try to enhance the degree of protection by utilizing alternative delivery systems and adjuvants, and by including more antigens, particularly from the schizont stage.

3. THElLERlA PARVA 3.1. Overview

Animals recovered from a T. parva infection are solidly immune to a homologous lethal challenge (reviewed by Irvin and Morrison, 1987; Morrison and McKeever, 1998). However, similar animals usually succumb to heterologous challenge owing to antigenic polymorphism, a feature which distinguishes T. parva from T. annulata. Live vaccines based on the administration of oxytetracycline with a sporozoite stabilate (‘infection and treatment method’) produce a mild or subclinical infection and induce strong protection (see Sections 1.3.4, and 3.3.1; Radley, 1981; Irvin and Morrison, 1987; Dolan, 1989). Most research to date is consistent with the view that immunity induced by infection with T. parva is cell mediated (Figure 3). A body of evidence exists which implicates MHC-restricted cytotoxic T cells as important effectors in this immunity (Section 3.2.2, p. 69; Morrison and McKeever, 1998). However, other processes may well be important, and it is the purpose of this section to explore what is known and to speculate about what may be significant with respect to vaccine design. Immune responses to the sporozoite, whilst probably not important in the immunity induced by infection and treatment, can be significant. The strongest evidence to support this statement is the fact that protection against sporozoite needle challenge can be engendered in a proportion of animals by vaccinating with recombinant p67 protein, a sporozoite surface antigen (see Sections 3.3.3 and 3.3.4 p. 75, 76; Musoke et al., 1992). The basis for this immunity is not known but there is no correlation with neutralizing antibody titre, whilst antigen-reactive T cells are difficult to detect. Nevertheless, this is very encouraging and field trials to evaluate this antigen under natural challenge are being undertaken. A final subunit vaccine formulation will almost certainly require inclusion of a schizont component.

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Unknown anti-sporozoite effector induced by p67 vaccination

t

Trophozoite development 24 hours

CD4+

I

* Cytos atic rnacrophage

Inhibition by cyiostasis p

BOLA-restricted

Figure 3 Summary of the proposed immune response against Theileriu parvu. The figure is drawn to emphasize the proposed central role for BOLA-restricted CTLs directed against schizont-infected T cells. An important role for anti-sporozoite immunity (of unknown character) induced by immunization with recombinant p67 is also stressed. Potential roles for neutralizing anti-sporozoite antibodies as well as effectors of the innate immune system are suggested. Abbreviations as in Figure 1.

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3.2. Immune Responses

3.2.1. Sporozoite Naive animals which have recovered from a single acute experimental infection with T . parva will not have significant serum antibodies to the sporozoite stage but they will be immune to homologous sporozoite challenge. The basis for this immunity is almost certainly a cell-mediated response to the schizont-infected lymphocyte (Figure 3). However, animals under persistent field challenge do acquire sporozoite neutralizing antibodies, as do animals hyperimmunized with sporozoite lysates (Musoke et al., 1982, 1984). One very important aspect of this neutralization activity is that it is largely directed at highly conserved epitopes (Musoke et al., 1984). These sera recognize a range of polypeptides, detected by immunoblotting, ranging in size from 150 to 24 kDa with one very prominent species of 67 kDa (Iams et al., 1990a). This antigen, dubbed p67, was also defined by several neutralizing monoclonal antibodies (Musoke et al., 1982, 1984, 1992; Dobbelaere et al., 1985a,b). As discussed later, this antigen in recombinant form is a prime vaccine candidate and induces complete or partial immunity in a proportion of animals. The only other fully characterized antigen from the sporozoite is a microneme/rhoptry antigen of 104 kDa (Iams et al., 1990b), but antibodies to this molecule do not neutralize sporozoite invasion in vitro. In addition, vaccination with this antigen does not induce any protection (S. Morzaria, personal communication). 3.2.2. Trophozoite and Schizont (a) The cell-mediated response - a perspective. As stated earlier, the main focus of protective immunity to T . parva is the schizont-infected cell (Morrison and McKeever, 1998). T . parva schizonts are thought to reside primarily in T cells in vivo (cf. T . annulata, which resides principally in macrophages/monocytes), although the sporozoite can invade different lymphocyte lineages in vitro (Baldwin et al., 1988; Glass et al., 1989; Spooner et al., 1989; Campbell et al., 1994; Morrison et al., 1996; Forsyth, 1997; Forsyth et al., 1997, 1999). The effector mechanism(s) against the schizont is (are) undoubtedly cellular. This assertion is based partly on the fact that serum from infected/immune animals, whilst containing anti-schizont antibodies, will not passively confer immunity on a naive recipient (Theiler, 1907; Muhammed et al., 1975). More importantly, T cells can, however, successfully transfer immunity from an immune twin to the non-immune identical partner (Emery et al., 1981; McKeever et al., 1994). All the available evidence is consistent with the main effector mechanism being a

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cytotoxic response mediated by MHC class 1-restricted CD8+ T cells (Figure 3; Morrison and McKeever, 1998). The first indication of this mechanism was provided by Pearson et al. (1979), who demonstrated that PBMs from immune cattle underwent a proliferative response to schizontinfected autologous cells - the so-called ‘autologous MLR’. Furthermore, they showed that the resulting population of primed cells contained cytotoxic activity against schizont-infected autologous targets and, interestingly, also some lesser activity against schizont-infected allogeneic cells. The detailed kinetics of the MHC-restricted response were first documented by Emery et al. (1981) and Eugui and Emery (1981), who showed that CTLs were transiently detectable in the peripheral blood leucocytes of calves recovering from an infection and treatment regimen for 2-3 days at the remission period. MHC-restricted CTLs also appear transiently in immune animals following challenge (Morrison et al., 1987). In contrast, calves undergoing a lethal reaction produce totally unrestricted cytotoxic responses late in the infection (Emery et al., 1981), which may underlie the massive destruction of lymphocytes that is a major pathological feature of this disease. The MHC-restricted CTLs are CD8+ and are highly specific, as they recognize only T. parva schizont-infected cells. Depending on the immunization protocol, these CTLs can be specific to a particular parasite strain (Morrison et al., 1987, 1995; Morrison and Goddeeris, 1990). Typing and blocking with monoclonal antibodies against class 1 MHC has confirmed that the response requires a minimum of one shared specificity between target and donor (Morrison et al., 1987). Very strong evidence that CD8+ cells have a role in vivo has been obtained by adoptive transfer. The population of cells obtained by draining a lymph node of an immune animal responding to challenge was highly enriched for the CD8+ cells by depleting the y6 T cells, B cells and CD4+ cells using specific monoclonal antibodies and complement. These were transferred to one member of pairs of naive twins which had been infected 1-3 days previously with a lethal sporozoite inoculum. This resulted in recovery of these animals, whereas the controls (the other member of each pair) succumbed. Whilst these data are compelling, it must be noted that the depletion left a substantial proportion of cells that were negative for CD4, CD8, B and y6 T markers, which cannot formally be discounted. However, this criticism is allayed to a large extent since one calf received cells which had also been depleted of CD8+ cells and this showed no protection (McKeever et al., 1994). Immune animals clearly retain memory in their CTL pool for schizont antigens, as demonstrated by the increase in precursor frequency from around 1 in 10 000 to 1 in 30 in the efferent lymph around day 6 after sporozoite challenge (Taracha et al., 1992, 1995a; McKeever et al., 1994). Morrison and McKeever (1998) reported that the frequency of around 1 in

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10 000 CTL precursors persisted from 5-6 weeks after immunization in animals that were not challenged, but they did not say for how long they were monitored. The role of other effectors, such as components of the innate immune system like macrophages, may be important but they have been given relatively little attention. CD4+ cells also probably have a role to play, even if it is a subservient one, as helper cells to the CD8+ effectors (Baldwin et al., 1987; Brown, W.C. et al., 1989a,b, 1990). Grab et al. (1992) characterized a 24 kDa schizont antigen, which was specifically recognized by two CD4+ clones reactive with schizont-infected cells. The contribution of such clones could involve cytotoxic properties as well, since one report described a cloned CD4+ line with the ability to kill schizont-infected cells (Baldwin et al., 1992). However, since the bulk of the literature is about analysing the CD8+ CTL response, we shall concentrate on this theme. (b) The cell-mediated response - antigenic polymorphism. In crosschallenge experiments, frequently no protection is afforded in a proportion of animals, demonstrating that there is variation in the protective antigens (Radley et a/., 1975a,b). Immunizing with mixtures of as few as three isolates, however, led to wider cross-protection against several isolates, suggesting that the extent of the antigenic diversity may be limited (Radley et al., 197%). One important observation is that cross-immunity is not always reciprocal. This is exemplified by the Muguga and Marekibuni stocks of T . parva: Marekibuni invariably protects against both itself and Muguga, whereas Muguga protects only a proportion of animals challenged with Marekibuni (Irvin et al., 1983). By several independent criteria, including molecular profiling, the Marekibuni isolate is clearly a mixture of genotypes, whereas the Muguga isolate is homogeneous (Morrison, 1996). However, the Marekibuni stock has been cloned (Marekibuni 3219) and, when this is used as a vaccine, it protects only a proportion of calves against Muguga challenge (Taracha et al., 1995a). This type of observation suggests that, as well as the protective antigens exhibiting polymorphism, cross-reactive epitopes exist even on clones but that there is a host-determined selection in the response. In other words, although the Marekibuni clone contains both specific and cross-reactive epitopes, some cattle will ‘prefer’ the crossreactive epitopes to the exclusion of the specific ones and these will resist cross-challenge. However, the converse can also occur when the genotype of the cow selects the stock-specific epitope and is therefore unable to resist cross-challenge. This will be explored in detail below. The importance of the CTL response as a mediator of protection is supported by the fact that there is complete concordance between CTL parasite specificity and protection. Thus the Muguga-immunized calves that resist Marekibuni challenge have CTLs that kill Marekibuni-infected cells in an MHC-restricted manner, whereas those that succumb to challenge exhibit only Muguga-specific CTLs (Taracha et al., 1995a). This cross-reactivity is exhibited by individual T-cell

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clones, indicating that the phenomenon is due to shared epitopes and, furthermore, CTLs raised against Muguga will kill a Marekibuni clone that is otherwise genetically distinct, indicating that the phenomenon is not due to a Muguga-like genotype in the Marekibuni stock. (c) The cell-mediated response - MHC restriction, dominance and epitope competition. The phenomena described above suggest that different MHC class 1 elements select different parasite epitopes to present to the CTLs. If so, one could predict that there would be a correlation between MHC type and parasite specificity. To address this directly, Goddeeris et al. (1990) examined 3 1 CTL clones derived from four Muguga-immunized calves. They tested their specificity against a panel of Marekibuni-infected target clones that were half-matched at MHC class 1 locus. The restricting element of the clone determined the pattern of targets recognized and all CTLs restricted by the same MHC type had identical recognition patterns. Thus, epitope selection is dictated by the host MHC class 1 restriction element and hence the degree of cross-reactivity, all other things being equal, will be determined by the preference of the host MHC. A distinct phenomenon is that, in MHC heterozygous animals, which are the majority, there is a dominance hierarchy such that certain haplotypes act as restriction elements in preference to the allelic counterpart - a form of allelic exclusion (Morrison et al., 1987; Taracha et al., 1995b; Morrison, 1996). Moreover, a similar dominance is observed between class 1 molecules expressed by the same haplotypes (most haplotypes have two expressed class 1 genes). A specific example is afforded by the BOLA A10 and KN104 determinants: when they are both expressed on the same haplotype, the response to the Muguga strain of T . parva is always restricted by the KN104 element. (d) The cell-mediated response - a role for cytokines? Is the effector response solely due to direct action by CTL or do soluble mediators such as cytokines have an effect? Established schizont-infected cell lines are not inhibited by any cytokine tested to date - IL-1, IL-2, IL-4, IL-6, IL-10, IFN-a, IFN-y and TNF-a (Morrison and McKeever, 1998). In fact, TNF-a and IL-2 actually enhance the growth of established schizont-infected cell lines, whilst, interestingly, TNF-a, IFN-y, IFN-a, IL-1 and IL-2 inhibit the trophozoite (i.e. the preschizont, post-sporozoite) stage (De Martini and Baldwin, 1991; Preston et al., 1992a; Visser et al., 1995). Interestingly, using a multiplex polymerase chain reaction (PCR), McKeever et al. (1997) tested 11 parasitized lines for IL6, TNF-a, IL2Ra, IL-10, IL-lp, IFN-y, IL-2 and IL-4. No line produced IL-lp or IL-4, whilst all lines produced IL-2Ra and IL-10. Each line, however, produced a unique profile. The upregulation of IL-10 could have a detrimental effect, as this cytokine dampens down CD4' cells, which could explain why no CTL is present in naive cattle undergoing a lethal infection.

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All things considered, proof of a direct role for cytokines in protective immunity to T. parva is largely lacking, although indirect effects such as effector cell recruitment cannot be ruled out and are indeed quite probable. (e) The cell-mediated response - immunopathology. Naive cattle infected with T. parva exhibit lymphadenopathy, i.e. the draining lymph node enlarges about fourfold. This enlargement takes only 1 or 2 days, and occurs after schizont-infected cells are first detectable, at just over a week after infection on average (Morrison et al., 1981). Analysis of the node and efferent lymph revealed 25% lymphoblasts, most of which were uninfected - indeed, parasitosis is of the order of only 1% at this time (Emery, 1981b). These cells have been reported to be predominantly a,O T cells with the profile CD8+, CD2-, CD3+, a phenotype rare in healthy cattle. T-cell receptor VP analysis indicates that these cells are polyclonal (F. Houston and W.I. Morrison, personal communication). These cells do not possess any measurable effector function. Additionally, it is not possible to detect CTLs at any stage in a primary infection (Emery et al., 1981; Taracha et al., 1992). It has been speculated that this anomalous T-cell proliferative response results in paracrine stimulation of growth and division of parasitized cells, thereby contributing to the pathology. This phenomenon may be similar in some respects to the aberrant T-cell proliferation observed in T. annulata (see Section 2.1.3(b), p. 59). Preliminary analysis demonstrated that these cells secrete IL-10 and that IL-10 potentiates growth in vitro (Morrison and McKeever, 1998). Since, as already mentioned, parasitized cells express IL-10 in their own right, IL-10 could be pivotal to the immunopathology, especially since it would also suppress the development of a Thl CTL response, which is required for immunity. An interesting contribution to the understanding of pathogenesis was provided by the work of Morrison et al. (1996). These authors infected CD8+, CD4+ and B cells with sporozoites in vitro and then inoculated the resulting schizont-transformed lines back into the cow from which they had been derived. Infected CD4+ and CD8+ cells produced severe pathology, whereas B cells caused mild self-limiting reactions. The basis for this interesting observation remains obscure.

3.3. Vaccination 3.3.1. Live Vaccines

-

The ‘Infection and Treatment’ Method

Early work (Lowe, 1933; Wilson, 1950; Barnett, 1957) suggested that the number of infectious particles (sporozoites) was proportional to the disease severity, and this was confirmed by Cunningham et al. (1974) and Radley et al. (1974), who performed titration experiments using ground tick

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suspensions containing sporozoites. Since those animals that recovered were solidly immune to homologous challenge, it was realized that the basis for a live vaccine was at hand. However, variability in clinical outcome was too great to allow the use of titrated stocks of sporozoites as vaccines in their own right, and tetracyclines were used to control the disease progression. Initially, these were short acting and had to be given continually to limit the infection (Neitz, 1953; Brocklesby and Bailey, 1965), but the method was improved by using modified longer acting tetracyclines. Thus, first, N-pyrrolidinomethyl tetracycline (Cunningham et al., 1973; Brown, C.G.D. et al., 1977) was introduced (given for 3 days from the time of infection) and then this ‘infection and treatment’ method was refined by Radley (1981), who used a single dose of oxytetracycline co-administered with the sporozoites. However, not all stocks can be controlled by this regime and some require administration of the drug for up to 6 days. Clinical breakthrough of the vaccinating strain(s) has been reported in Malawi (Mbogo et al., 1996) and Kenya (Mutugi et al., 1991). Animals vaccinated by infection and treatment can resist challenge with a 1000-fold lethal sporozoite dose and can remain immune without challenge for more than 3 years. Owing to antigenic polymorphism, the ‘field’ protection afforded by any one stock is limited and in practice combination ‘cocktail’ vaccines are used to give wider coverage. However, this is a controversial practice as there is concern that such vaccines actually enhance the spread of novel variants into the areas where they are used and, for this reason, the alternative practice of preparing local vaccines is recommended in some countries (Dolan, 1987). A very important point, worth re-emphasizing, is that it is not possible to produce an attenuated cell line vaccine against T. parva that is equivalent to those used so successfully against T. annulata. The principal reason for this is apparently the poor ability of the T. parva schizonts to transfer from donor host cells to recipient host cells. Indeed, the efficiency of T. parva transfer is at least two orders of magnitude lower than that achieved by T. annulata (see Irvin and Morrison, 1987). The reason for this difference is unknown but is probably connected with the difference between the host cells preferentially invaded by the two species. It is established that, in vivo, T. annulala resides predominantly in monocyte/macrophages, whilst T. parva is found largely in T cells (Morrison et al., 1996; Forsyth et al., 1997). Inoculation of a recipient with infected cells of either species will lead to an allogeneic graft rejection response, thus releasing the schizonts. Presumably this rejection and schizont release is equally efficient in the case of both T. parva and T. annulafa. Therefore, it is likely that the difference in transfer efficiency is at the level of entry into the new recipient host cells. As the recipient host cells of T. annulata are phagocytes (monocytes), whilst those of T. parva are not, a mechanism for enhanced transfer of T. annulata is

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provided. This argument, of course, assumes that the preferred host cell invaded by the transferring schizont is the same as that invaded by the sporozoite, which we doubt has been established. 3.3.2. Live Vaccines - Mild Strains of T. parva as Vaccines An alternative method to infection and treatment, which, as stated above, can lead to clinical disease, is the use of naturally occurring mild strains of T. parva (see Barnett and Brockelsby, 1966a; Koch et al., 1988; Mbogo et al., 1996). In their 1996 study, Mbogo et al. demonstrated that one mild T.parva strain (Lanet, NVRC stabilate 263), inoculated as a sporozoite stabilate, cross-protected against five virulent stocks of T . parva, including one buffalo-derived isolate. The protection against the four cattle-derived isolates was complete, whereas three of five animals challenged with buffalo isolates died but the other two were completely protected. These are promising results and this type of vaccine has real potential, although the possibility of reversion to virulence after tick passage, as was demonstrated by Barnett and Brockelsby (1966b), is a serious counter consideration. 3.3.3. Dead Vaccines

-

p67, a Recombinant Sporozoite Antigen

For many reasons, it would be desirable to replace the current live vaccines with non-living material. This would have the advantages of being noninfective, free of carrier pathogens and, hopefully, more stable. Attempts to induce protection using killed parasites or parasite extracts from other life-cycle stages of the parasite result in the production of serum antibodies but no protection is engendered. However, with the sporozoite stage, it is possible to induce neutralizing antibodies, and this observation in principle means that it should be possible to reduce the sporozoite inoculum and limit the infection. This line of thinking led to the search for sporozoite antigens with neutralizing determinants and a major candidate was defined using mAbs (Musoke et al., 1984; Dobbelaere et al., 1985a,b). This molecule, called p67, was shown to be recognized by immune bovine sera, highly conserved, and surface-located (see Section 3.2.1., p. 69; Dobbelaere et al., 1985a,b). The gene for this antigen has been cloned, and contains 709 codons and is homologous to the SPAG-1 gene of T. annulata, particularly at the N and C termini (Figure 2; Knight et al., 1996). Interestingly, the p67 and SPAG-1 antigens each contain the elastin motif PGVGV, although this occurs only once in p67 and 17 times in SPAG-1 (Nene et al., 1992). The two antigens have also been shown to cross-react immunologically, and sera and mAbs against the two antigens cross-neutralize (Figure 2).

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The p67 antigen has been expressed in a number of systems, including E. coli, baculovirus, vaccinia and Salmonella dublin (see Musoke et al., 1992; Nene et al., 1995; Heussler et al., 1998; Honda et al., 1998). In the E. coli system, the antigen has been used in vaccination trials when expressed as a C-terminal fusion to the non-structural protein 1 (NS1) of influenza virus and as a his6 tag product. The first published trial was using the NS1 fusion: nine indigenous Boran (BOSindicus) cattle were immunized five times with 3% saponin as the adjuvant (Musoke et al., 1992). Six of the animals were protected from a 70% lethal dose (LD70) homologous challenge with T.parva Muguga. Protection of four of the six was complete, showing no evidence of clinical reaction, whilst the other two underwent a mild reaction. The other three calves immunized with p67 had severe reactions, as did all ten controls. The mechanism of protective immunity was unclear. All the nine animals developed neutralizing antibodies but the titres did not correlate with protection. The authors reported that two of the non-reactors were PCR-negative using specific T. parva primers on lymph node biopsy material obtained 60 days after the challenge, suggesting that sterilizing immunity had been induced. An important follow-up investigation reproduced the initial results by observing protection in 7 of 12 calves (two of which were completely protected) against homologous challenge (Nene et al., 1996). More importantly, the authors reported that they protected 6 of 11 calves (one completely) against a heterologous challenge with a stock (T. parva Marekibuni) which is not cross-protective by infection and treatment. The same group of authors has evaluated a number of other expression and antigen delivery systems. Baculovirus-derived p67 was produced in an attempt to obtain ‘native’ antigen, but the material produced was unfortunately only weakly glycosylated and the bulk of it was produced as a series of partially processed forms, most of which failed to react with a mAb (TPM12) that recognizes native p67. None the less, four of six Boran cattle were protected by this material against homologous challenge (Nene et al., 1995). Recently, it was reported that, in trials using 86 cattle in total, 70% protection was obtained using p67 (Morrison and McKeever, 1998).

3.3.4. Recombinant Vaccines - Live Delivery Systems The protection induced by recombinant p67 relies on multiple vaccinations with antigen in adjuvant. Clearly this is not an ideal delivery strategy from a practical viewpoint and simpler systems would be preferable. In addition, it has proved difficult to detect significant T-cell responses using p67 formulated in adjuvant and lack of this type of immunity may be compromising the effectiveness of p67 as a protective immunogen. Hence two live delivery vehicles, Salmonella and vaccinia, have been evaluated in the context of p67.

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In the first trial, Heussler et af. (1998) immunized a group of seven calves intramuscularly on 3 monthly occasions with 2 x lo9 colony-forming units of S. dubfin, composed of an equal mixture of three populations of expression transformants. These consisted of high, medium and low copy number expression plasmids; in addition, the high copy plasmid exported the antigen to the periplasmic space, whilst the others retained it in the cytoplasm. Controls consisted of seven naive calves and seven calves immunized with S . dubfin alone. Six of the experimental animals underwent mild or moderate reactions on challenge, whilst one animal was completely protected. The controls were less protected, with two calves in each group experiencing severe theileriosis. Statistically, the differences were significant and indicated protection. In a follow-up study, Gentschev e f af. (1998) attempted to compare the effect of cytoplasmically located p67 with secreted p67 using S. dubfin as the live host. Oral and intramuscular routes of delivery were also assessed. Again, multiple inoculations were used. The secreted p67 was made by attaching the C-terminal E. coli haemolysin secretion signal sequence. The results indicated that secreted antigen engendered better protection than the internally located antigen and that intramuscular delivery was marginally superior to the oral route. However, the results must be regarded as preliminary owing to the small numbers (three) of animals per group. Recombinant p67 delivered via vaccinia virus has been evaluated by Honda et af. (1998). Again, this system is capable of inducing T-cell responses, particularly CD8+ cells. The p67 vaccinia construct given alone to seven animals (two inoculations) did not have any protective effect. However, when given in conjunction with a vaccinia construct engendering production of IL2, five of seven calves were protected. A co-administered vaccinia construct stimulating IL-4 enhanced production of antibody to p67 but had no effect on protection. T h s live delivery approach has much potential merit, particularly if the system could be reduced to a one-shot application. The antigen p67 is a very remarkable molecule, as it was unexpected that a single sporozoite component would induce significant protection on its own. Also, its partially protective effect against T. annulata is intriguing (Boulter et al., 1999). Research to elucidate the mechanism of immunity in completely protected animals should be a priority.

4. THElLERlA SERGENTI 4.1. Classification

Taxonomically, there is a debate about the classification of the relatively mild species of Theileria that constitute what can be loosely described as the

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T. buflelilorientalislsergenti group (Fujisaki et al., 1994; and see Section 1.1.1, p. 43). These organisms (or this organism) are (is) characterized by being relatively non-pathogenic and inducing symptoms associated with the piroplasm stage of the life cycle. One of the defining characteristics is the rarity of the schizonts, which occur in markedly enlarged host cells, and have been observed in the draining lymph node, liver and spleen and, sometimes, extracellularly (Minami et al., 1990; Kawazu et al., 1991; Sat0 et al., 1993, 1994). For the purposes of this review, with its focus on immunity, we shall use the term T. sergenti to describe the organism(s) that infect cattle in Japan and Korea, where they cause chronic anaemia. The Japanese and Korean parasites are transmitted by the tick Haemaphysalis longicornis. 4.2. Clinical Features and Control

As stated above, the major pathological phase of the life cycle of T. sergenti is the piroplasm stage, which leads to severe chronic anaemia, particularly in susceptible ‘western’ breeds of cattle such as Holsteins. Indigenous breeds such as native Korean cattle and Japanese Black calves are relatively resistant (Baek et al., 1992a; Terada et al., 1995). One interesting feature of the disease is that a carrier state is established, with the consequence that stress-induced relapses occur (Sugimoto, 1997). Recovery from infection induces immunity to homologous challenge and a live blood vaccine has been used as a control measure, but is not now recommended. The major method of control is using anti-theilerial drugs.

4.3. Immune Responses

Analyses of the immune response in T. sergenti-infected calves are very limited (see Figure 4). There is a humoral response, first described by Takahashi et al. (1972), who used an indirect fluorescent antibody (IFA) assay to demonstrate antibodies to the piroplasm by day 5, which reached a peak on day 20 and persisted until day 60. Convalescent sera reacted with piroplasm antigens of 23, 29, 32 and 67 kDa in Western blots (Ohgitani et al., 1987). mAbs directed against the 32 kDa polypeptide (p32) (Kobayashi et al., 1987) are able passively to confer resistance against merozoite challenge (Tanaka et al., 1989). Protection against challenge is afforded by vaccination with synthetic p32, as described in more detail below. Cell-mediated immunity is also detectable in calves undergoing a clinical infection. Yasutomi et al. (1991) observed T-cell proliferation of PBMs directed against merozoites in infected blood. The peak of proliferation occurred about 3 weeks after infection and the response was ablated by

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Sporozoites

Neutralizing antibodies

Innate immunity

Cell-mediated immunity e.g. CTL

. macrophage?

e.g. anti-p32/33 Piroplasm

+ Figure 4 Summary of the proposed immune response against Theileria sergenri. This diagram highlights a role for neutralizing antibodies directed at the merozoite/piroplasm stages. The other suggested effectors are speculative and clearly indicate our general state of ignorance of the immune mechanisms involved in modulating T . sergenti infections. Abbreviations as in Figure 1.

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anti-T cell mAbs and by autologous infection serum. However, interestingly, stimulation of proliferation resulted from inclusion of a mAB against merozoites. The same authors detected cytotoxic cells, reaching a peak at weeks 7-8 after infection, which killed a bovine leukaemia cell line and were probably NK effectors. Co-culture with merozoites caused a slight enhancement of the degree of cytotoxicity, whereas further addition of autologous serum (taken at weeks 5-6 after infection) caused a marked reduction. Asaoka et al. (1991) conducted a detailed study of macrophage activation in calves infected with T. sergenti. Specifically, they studied the oxidative burst induced either specifically by merozoites or non-specifically by zymosan. Six calves produced activated macrophages that responded, for up to one month after inoculation, to merozoites, particularly those that were opsonized with homologous or heterologous antisera. However, the parasites were not eliminated at the time of the peak oxidative burst in vivo, suggesting that this mechanism may not be fully effective. On the other hand, Ishii and co-workers (1992) demonstrated that the inhibition of activated monocytes in vivo by prednisolone correlated with an increase in parasitaemia, suggesting an important effector role for these cells.

4.4. Vaccination with Non-living Components

Vaccination with non-living components has been reported by two groups in Korea and Japan (Baek et al., 1992a,b, 1994; Onuma et al., 1997). Baek and co-workers in Korea prepared crude soluble extracts by sonication of merozoites and centrifugation at 20 000 g, and then used this material to vaccinate calves. Freund’s complete adjuvant was used in the primary injection and Freund’s incomplete adjuvant in the booster dose 4 weeks later; 100 mg of crude antigen were given per animal per injection. Nine weeks after the boost, calves were challenged with 5.6 x lo6 erythrocytes at 40% parasitaemia with the homologous parasite. The vaccinated animals and the controls both developed parasitaemia, but the vaccinated did not become anaemic, whereas the controls did. The serum of vaccinated animals predominantly recognized the 33 kDa antigen. The same authors inoculated 20 Holstein calves and placed them under heavy natural tick challenge together with 20 control calves. All 20 controls needed chemotherapy (five requiring blood transfusions), whereas only six of those vaccinated required treatment. It is probable that the effect was mediated primarily by an immune response directed to the dominant 33 kDa antigen. The 33 kDa antigen is thus a candidate for a subunit vaccine. The gene was therefore cloned and shown to be a highly polymorphic system (Kawazu et al., 1991; Matsuba et al., 1993a,b). Recombinant antigen has been produced in the baculovirus system and synthetic multiple antigenic peptides

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(MAPS)have been made containing a KEK motif. Animals were vaccinated four or five times with these antigens, using a range of doses and Freund’s complete adjuvant or liposomes as adjuvants. Protection against both parasitaemia and clinical symptoms was achieved (Onuma et al., 1997). The prospects for producing a recombinant vaccine against T. sergenti, where a strong humoral anti-merozoite response is probably all that is required, must be better than those for either of the other two species considered.

5. COMPARATIVE ASPECTS

All three species have much in common biologically. However, it is the differences that are perhaps worth stressing (Table 1). From an immunological point of view, the differences are marked. To begin with, it is possible to immunize against T. annulata with attenuated schizont-infected cells, whereas this is not possible with T . parva (because the efficiency of parasite transfer is too low) or T. sergenti (because the schizont stage is too rare). The influence of the host cell type in which the schizont resides probably underlies the differential ability of T. annulata to transfer at a 100 times greater frequency than T. parva. Specifically, as T. annulata inhabits a phagocyte (the macrophage), it is provided with an efficient process for schizont uptake after the donor host cell has been destroyed by allograft rejection. T.parva, residing in T cells that are non-phagocytic, is not availed of the same process. The immunity induced against the schizont-infected cell is known to be cell mediated in T. parva and T. annulata infection, but no data exist for T. sergenti (Figures 1 , 3 and 4). With T. parva the main effector is the MHCrestricted CTL, whereas for T. unnulata the most important effector is the macrophage with a secondary role for CTLs. The secreted products of macrophages, such as NO and TNF-a, are thought to be important in protective immunity to T. annulata. The attenuated T. annulata vaccine is claimed to have efficacies of more than 90% and is cross-protective against different field isolates. The infection and treatment regime for T. parva protects against homologous challenge only. Presumably this is because T. parva exhibits polymorphism of the target antigens recognized by the CTLs. The CTL response against T. parva exhbits complex phenomena, such as epitope competition and dominance of certain BOLArestriction elements. NK cells probably play a role in T. parva and T. annulata infections. Passive immunization with a mAb against the major merozoite antigen indicates a key role for antibody in immunity to T. sergenti. This is largely due to the fact that the only pathogenic phase of the life cycle is the piroplasm. This compares with T. annulata in which pathogenesis is associated with both the schizont

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and piroplasm phases, and contrasts with T. parva infection, in which the pathology is solely a function of the schizont stage. Both T. parva and T. annuluta induce polyclonal autologous T-cell proliferation. In the case of T. annulata, this is thought to damage the normal immune response by effectively depleting and/or overwhelming the antigen-specific T-cell pool. Vaccination with the major sporozoite surface antigen p67 gives sterilizing immunity against T. parva sporozoite challenge in a proportion of animals and partial immunity in others, whilst some are fully susceptible. The immunological and genetic bases for this phenomenon are not understood, but it is not observed with T. annulata when immunizing with the homologous antigen SPAG-1 (with which only partial immunity is obtained). The major merozoite antigen (30-33 kDa) provides protection against T. sergenti and also against a blood challenge with T. annulata. It is considered unlikely that this component will be needed or effective against T. parva, in which the piroplasm plays a secondary role.

6. THE FUTURE

Future immunological and vaccine research in bovine theilerioses must be aimed at usable, effective products within the next decade. This is a tall order, especially if we demand certain standards, such as that a T. annulaiu vaccine must be as effective as the current attenuated ones. It is highly likely that the vaccines that emerge will be of the ‘naked’ DNA type and will include selective immunopotentiators in the form of cytokine genes. The focus of attention for T. sergenti is likely to remain surface proteins of the merozoite, although, if the methodology became available, schizont constituents would be useful components also as, in principle, merogony could be inhibited. Of paramount importance is the need to define protective schizont antigens for T. parva and T. annuluta, which we think are going to be fundamental in the production of effective vaccines. For T. parva and T. annuluta, multistage, multicomponent vaccines are likely to be preferred, but this may create difficulties about whether the required spectra of immune responses are fundamentally compatible. Much basic immunological research and empirical vaccine testing lies ahead.

ACKNOWLEDGEMENTS We thank Drs Rachel Adamson, John Campbell, Ivan Morrison and Pat Preston for critically reading this manuscript. Thanks also to Duncan

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Brown and his team for all their support over the years. We wish Duncan a happy retirement. Original work referred to from the authors’ laboratory was funded by the Biotechnology and Biological Sciences Research Council, the European Union (contract numbers CT95 0003 and CT91 0019) and the Wellcome Trust (grant numbers 0312219 and 040179).

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and T . parva infect and transform different bovine mononuclear cells. Immunology 66, 284-288. Srivastava, A.K. and Sharma, D.N. (1981). Studies on the occurrence, clinical features and clinicopathological and pathomorphological aspects of theileriasis in calves. Veterinary Research Journal 4, 22-29. Stewart, N.P., de Vos, A.J. and Shiels, I. (1990a). Elimination of Theileria buffeli infections from cattle by concurrent treatment with primaquine phosphate and halofuginone lactate. Tropical Animal Health and Production 22, 109- 11 5. Stewart, N.P., de Vos, A.J., McHardy, N. and Standfast, N.F. (1990b). Elimination of Theileria buffeli infections from cattle by concurrent treatment with buparvaquone and primaquine phosphate. Tropical Animal Health and Production 22, 116-122. Stewart, N.P., Uilenberg, G. and de Vos, A.J. (1996). Review of Australian species of Theileria, with special reference to Theileria buffeli of cattle. Tropical Animal Health and Production 28, 81 -90. Sugimoto, C. (1997). Economic importance of theileriosis in Japan. Tropical Animal Health and Production 29 (suppl.), 49s. Sugimoto, C., Kawazu, S., Kamio, T. and Fujisaki, K. (1991). Protein analysis of Theileria sergentilbuffelilorientalis piroplasms by two-dimensional polyacrylamide gel electrophoresis. Parasitology 102, 341 -346. Sugimoto, C., Kawazu, S., Sato, M., Kamio, T. and Fujisaki, K. (1992). Preliminary biochemical characterization of ‘veil’ structure purified from Theileria sergenti-, T. buffeli- and T . orientalis-infected bovine erythrocytes. Parasitology 104, 207-213. Sutherland, I.A., Shiels, B.R., Jackson, L.A., Brown, D.J., Brown, C.G.D. and Preston, P.M. (1996). Theileria annulata: altered gene expression and clonal selection during continuous in vitro culture. Experimental Parasitology 83, 125133. Tait, A. and Hall, F.R. (1990). Theileria annulata: control measures, diagnosis and the potential use of subunit vaccines. Revue Scientifique et Technique, OfJice International des Epizooties 9, 387-403. Takahashi, K., Yamashita, S . , Isayama, Y. and Shimizu, Y. (1972). Serological response to the indirect fluorescent antibody test of cattle infected with Theileria sergenti. British Veterinary Journal 132, 112- 117. Tanaka, M., Ohgitani, T., Okabe, T., Kawamoto, S., Takahashi, K., Onuma, M., Kawakami, Y. and Sasaki, N. (1989). Protective effect against erythrocytic merozoites of Theileria sergenti infection in calves by passive transfer of monoclonal antibody. Japanese Journal of Veterinary Science 52, 63 1-633. Tanaka, M., Onoe, S., Matsuba, T., Katayama, S . , Yamanaka, M., Yonemichi, H., Hiramatsu, K., Baek, B., Sugimoto, C. and Onuma, M. (1993). Detection of Theileria sergenti infection in cattle by polymerase chain reaction amplification of parasite-specific DNA. Journal of Clinical Microbiology 31, 2565-2569. Taracha, E.L.N., Goddeeris, B.M., Scott, J.R. and Morrison, W.I. (1992). Standardization of a technique for analysing the frequency of parasite-specific cytotoxic T lymphocyte precursors in cattle immunized with Theileria parva. Parasite Immunology 14, 143- 154. Taracha, E.L.N., Goddeeris, B.M., Morzaria, S.P. and Morrison, W.I. (1995a). Parasite strain specificity of precursor cytotoxic T cells in individual animals correlates with cross-protection in cattle challenged with Theileria parva. Infection and Immunity 63, 1258-1262. Taracha, E.L.N., Goddeeris, B.M., Teale, A.J., Kemp, S.J. and Morrison, W.I.

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(1995b). Parasite strain specificity of bovine cytotoxic T cell responses to Theileria parva is determined primarily by immunodominance. Journal of Immunology 155, 4854-4860. Tatchell, R.J. (1981). Current methods of tick control with special reference to theileriosis. In: Advances in the Control of Theileriosis (A.D. Irvin, M.P. Cunningham and A.S. Young, eds), pp. 148-159. The Hague, Boston, London: Martinus Nijhoff Publishers. Taverne, J. (1994). Transgenic mice and the study of cytokine function in infection. Parasitology Today 10, 258-262. Terada, Y., Ishida, M. and Yamanaka, H. (1995). Resistibility to Theileria sergenti infection in Holstein and Japanese black cattle. Journal of Veterinary Medical Science 57, 1003-1006. Theiler, A. (1907). Experiments with serum against East Coast Fever. Journal of Tropical Veterinary Science 2, 249-260. Tizard, I.R. (1992). Immunology: an Introduction, 3rd edn. Philadelphia: Saunders College Publishing. Truyens, C., Torrico, F., Angelo-Barrios, A., Lucas, R., Heremans, H., De Baetselier, P. and Carlier, Y. (1995). The cachexia associated with Trypanosoma cruzi acute infection in mice is attenuated by anti-TNF-a, but not by anti-IL-6 or anti-IFN-y antibodies. Parasite Immunology 17, 561-568. Uilenberg, G. (1981). Theileria infection other than East Coast fever. In: Diseases of Cattle in the Tropics (M. Ristic and I. McIntyre, eds), pp. 41 1-427. The Hague, Boston, London: Martinus Nijhoff Publishers. Viseras, J., Garcia-Fernandez, P. and Adroher, F.J. (1998). Development of an experimental tissue culture vaccine against Mediterranean theileriosis in Spain. Journal of Veterinary Medicine 45, 19-24. Visser, A.E., Abraham, A,, Bell-Sakyi, L.J., Brown, C.G.D. and Preston, P.M. (1995). Nitric oxide inhibits establishment of macroschizont-infected cell lines and is produced by macrophages of calves undergoing bovine tropical theileriosis or East Coast fever. Parasite Immunology 17, 9 1 - 102. Willadsen, P. (1990). Perspectives for subunit vaccines for the control of ticks. Parassitologia 32, 195-200. Willadsen, P., Bird, P., Cobon, G.S. and Hungerford, J. (1995). Commercialisation of a recombinant vaccine against Boophilus microplus. Parasitology 110, S43-S50. Williamson, S.M. (1988). A Theileria annulata Sporozoite Surface Antigen as a Potential Vaccine for Tropical Theileriosis. Ph.D. thesis, University of Edinburgh. Williamson, S . , Tait, A., Brown, D., Walker, A., Beck, P., Shiels, B., Fletcher, J. and Hall, R. (1989). Theileria annulata sporozoite surface antigen expressed in Escherichia coli elicits neutralizing antibody. Proceedings of the National Academy of Sciences of the USA 86, 4639-4643. Wilson, S.G. (1950). An experimental study of East Coast Fever in Uganda. 1. A study of the type of reaction produced when the number of infected ticks is controlled. Parasitology 40, 195-234. Yagi, Y., Ito, N. and Kunugiyama, I. (1991). Decrease in erythrocyte survival in Theileria sergenti-infected calves determined by non-radioactive chromium labelling method. Journal of Veterinary Medical Science 53, 391 -394. Yasutomi, Y., Asaoka, H., Takahashi, K., Kawakami, Y. and Onuma, M. (1991). Proliferation of lymphocytes in Theileria sergenti-infected calves in vitro. Journal of Veterinary Medical Science 53, 161- 162. Young, A.S. (1990). Control of Theileria species (other than East Coast Fever and

IMMUNITY IN THE BOVINE THEILERIOSES

97

Theileria annulata infection), Ehrlichiu and tick toxicosis: present situation and proposals for future control strategies. Parassitologia 32, 41 -54. Young, A S . , Groocock, C.M. and Kariuki, D.P. (1988). Integrated control of ticks and tick-borne diseases of cattle in Africa. Parasitology 96, 403-432. Zablotsky, V.T. (1991). Specific prevention of bovine theileriosis in Soviet Union. In: Proceedings of the Second EEC Workshop on Orientation and Co-ordination of Research on Tropical Theileriosis (D.K. Singh and B.C. Varshney, eds), pp. 9-10. Anand, India: National Dairy Development Board.

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The Distribution of Schistosoma bovis Sonsino. 1876 in Relation to Intermediate Host Mollusc-Parasite Relationships Helene Mone. Gabriel Mouahid and Serge Morand

Laboratoire de Biologie Animale. U M R n"55.55 du CNRS. Centre de Biologie et d'kcologie tropicale et me'diterrankenne. Universitk. Avenue de Villeneuve. 66860 Perpignan Cedex. France Abstract ..................................................................... 100 1. Introduction ............................................................. 100 102 2. Collection of Data ........................................................ 3. The Natural Mollusc Intermediate Host Spectrum .......................... 113 4 Geographical Distributions of the Mollusc Intermediate Hosts...............113 124 5. Geographical Distribution of S. bovis ...................................... 6. The Experimental Mollusc Intermediate Host Spectrum ..................... 125 7. Compatibility in the Mollusc-S . bovis Association ......................... 125 8 Three Main Populations of S bovis ....................................... 128 9 Paleobiogeographical Scenario of S. bovis................................. 130 10. Conclusion .............................................................. 132 Acknowledgements .......................................................... 133 References................................................................... 133

. . .

ADVANCES IN PARASITOLOGY VOL 44 ISBN 0-12-031744-3

.

Copyright Q 2000 Academic Press A / / rights o/reproducrion in any form reserved

100

ti. MONE ETAL.

ABSTRACT Schistosoma bovis is a digenean platyhelminth that is responsible for a parasitic disease called schistosomiasis or bilharziasis in bovines. It has a natural wide mollusc intermediate host spectrum and is compatible, experimentally, with a wide range of species. Our working hypothesis is that the Mediterranean Sea and the Sahara were two physical barriers that could have separated the populations of S. bovis in three parts and may have played a role in gene flow. Experimental data were collected from earlier published studies, and the different intermediate host spectra and the mollusc-parasite geographical compatibilities were compared between the North Mediterranean zone, the South Mediterranean zone and the South Saharan zone. From our results, the three major groups of S. bovis populations that could be determined were the Iberian, the Mediterranean and the South Saharan populations. Our tested hypothesis was thus not confirmed concerning the Mediterranean sea barrier but was confirmed with the Saharan one. A paleogeographical scenario of S. bovis is proposed following three major steps from a South Saharan origin to a possible local adaptation of the parasite in the Iberian Peninsula.

1. INTRODUCTION

Different species of animals coexisting in the same environment exert on each other reciprocal influences which can lead to coevolutionary processes (Futuyama and Slatkin, 1983). Parasites, as any other animals, are also under the selective pressures of the environment. The peculiarity of animal parasites is that they are closely associated with one or several species of animals called hosts. The host-parasite associations will coevolve under both the influences of the parasite on its host and of the host on the parasite (Frank, 1991). In addition to the morpho-anatomical, biochemical or behavioural markers, which may reveal an interpopulational genetic diversity, both host spectrum and host-parasite compatibility constitute useful markers as regards the knowledge of the transmission foci and of the risks of extension of the parasitism. The intermediate mollusc host spectrum corresponds to the number of different species of hosts a parasite may infect. This may range from a reduced host spectrum, where the parasite develops in only one species of host, to a wide host spectrum, where the parasite develops in many species of hosts.

DISTRIBUTION OF SCHlSTOSOMA BOVlS

101

The host-parasite compatibility can be defined as the result of both host susceptibility and parasite infectivity (Combes, 1985, 1995), and may show considerable variation among the population host-population parasite systems. It is genetically determined (Vkra et al., 1990; Richards et al., 1992) and constitutes the expression of the genetic variability of the host confronted to the genetic variability of the parasite. The host-parasite compatibility corresponds to the success of infection. It is usually expressed as the number of molluscs infected divided by the number of molluscs exposed to the miracidia (prevalence). This will be different depending on the geographical origin of both the parasite and the host, and on the species of host the parasite will use (Vkra et al., 1990; Ebert, 1994; Gandon et al., 1996; Morand et al., 1996). It will express the quality of the relationship existing between the parasite and its host. Schistosoma bovis Sonsino, 1876 (Platyhelminth, Digenean) was used as the parasite model. This parasite is the agent of a bovine debilitating disease called schstosomiasis or bilharziasis. It has a complex life cycle including two obligatory hosts: a vertebrate definitive host, and a mollusc intermediate host. Some authors suggest that the digeneans are primitive parasites of molluscs (Gibson, 1987; Rohde, 1994), whereas others suggest they are primitive parasites of vertebrates (Brooks and McLennan, 1991). It is generally agreed that the digenean-mollusc association is an ancient one. This association extends for at least 400 million years (Gibson, 1987). A synthesis of the biology and the ecology of S. bovis transmission, especially the patterns of the mollusc-parasite association, has been published recently (Mouahid, 1994). The geographical distribution of S. bovis is wide and covers different geographical zones, which are isolated by two major horizontal natural barriers: the Mediterranean Sea, and the Saharan Desert. These two barriers, even though not impassable, delimit the North-South geographical distribution of S. bovis in three major zones that we term the North Mediterranean zone, the South Mediterranean zone and the South Saharan zone, respectively. In other respects S. bovis presents large differences in both host spectrum and host-parasite compatibilities according to the geographical origins of both the host and the parasite. We took into account these peculiarities of S. bovis to analyse the different host-parasite geographical intermediate host spectrum and compatibilities in these three geographical zones. Our working hypothesis was that the natural barriers (Mediterranean Sea and Saharan Desert) have played a role in the gene flow between the different S. bovis populations. As no molecular data are available at the intraspecific interpopulational level of S. bovis, and as the host-parasite compatibility is the expression of the genetic relationship between the host and the parasite, we assumed that the data concerning the mollusc-S. bovis compatibility reflect the host-parasite coevolution in such a way as to propose a scenario of the biogeographical history of S. bovis.

102

H. MONE ETAL.

2. COLLECTION OF DATA

Data concerning the natural S. bovis-mollusc associations came from 45 published studies (Table 1). Data concerning the experimental S. bovismollusc associations were collected from 20 papers (Tables 2-6). The experimental results obtained using multimiracidial infections were considered. The number of miracidia used per snail varied from 2 to 100 but 83% of the prevalences concerned infections using 5 miracidia per snail. We made sure that the numbers of miracidia used for the multimiracidial infection tests were not correlated to the numbers of infected molluscs; the available data set (260 observations) showed such a bias did not exist (v = 29.85 + 0 . 3 7 ~ ; r = 0.09; 95% confidence interval - 0.03 and 0.21; P > 0.05). For each host-parasite association, the following parameters were checked: the species of mollusc; its geographical origin; the geographical origin of the schistosome; when possible, the number of miracidia used per mollusc during the exposure, the number of surviving or exposed molluscs which have been used for the infection test; and the number of infected molluscs on the number of surviving or exposed molluscs ( = prevalence, expressed in percentage). Mean prevalences were used when several data were recorded from the same population host-population parasite association. The albino populations of molluscs were considered as different populations from non-albino ones. All the prevalences were then expressed in proportions (p) and arcsine transformed (Zar, 1984). The ANOVA-factorial test together with the Scheffe’s F post-hoc Table I Bibliographic data on the natural geographical distribution Schistosoma hovis. Geographical zone

Country

Authors

of

Year

North Mediterranean zone

France (Corsica) Italy (Sardinia) Italy (Sicily) Spain

Brumpt Deiana Grassi and Ravelli Ramajo-Martin

1930 1954 1898 1972

South Mediterranean zone

Egypt Iraq

Sonsino MacHattie and Chadwick Al-Barrak et al. Arfaa et al. Lengy Blanc and Desportes

1876 1932

Iraq Iran Israel Morocco

1977 1965 1962 1936 (continued)

103

DISTRIBUTION OF SCHlSTOSOMA BOWS

Table I Continued. Geographical zone

Country Morocco Morocco Tunisia

South Saharan zone

Burkina Faso Cameroon Central African Republic Chad Congo Ethiopia Ethiopia The Gambia Ghana Guinea Guinea-Bissau Kenya Kenya Kenya Kenya Kenya Mali Mali Mauritania Niger Nigeria Nigeria Rwanda Senegal Somalia Sudan Sudan Sudan Tanzania Tanzania Tanzania Tanzania Tanzania Togo Uganda Zaire Zaire

Authors Dazo and Biles (cited by Southgate and Knowles) Freton et al. Anderson and Gobert Gillet (cited by Pitch ford) Ngonseu et al. Ngendaha yo Ngendahayo Fain and Lagrange Graber and Daynes Lo and Lemma Smithers Edwards and Wilson Gillet (cited by Pitchford) Gillet (cited by Pitchford) Southgate and Knowles Jelnes Ouma and Waithaka Southgate et al. Southgate et al. Ngendahayo Rollinson et al. Marill Vtra Cowper Ndifon et al. Wery (cited by Pitchford) Diaw and Vassiliades Sobrero Malek Bushara et al. Majid et al. Kinoti Dinnik and Dinnik Southgate et al. Kassuku et al. M wambungu Dogba Berrie Schwetz Chartier et al.

Year

1975a 1989 1934 1977 1991 1989 1989 1952 1974 1975 1956 1958 1977 1977 1975b 1983 1984 1985a 1989 1989 1990 1961 1991 1963 1988 1977 1987 1965 1969 1978 1980 1964b 1965 1980 1986 1988 1976 1964 1955 1990

A

0 P

Table 2 Snail infection experiments in PIanorbarius metidjensis.

Snail species

P. metidjensis

Snail Origin

Spain

Spain

S. bovis origin

Spain

Spain

Number of miracidia per snail

2 3 4 5 5 5 5 8 9 10 10 10 10 10 15 50 5, 10 or 20 5, 10 or 20 5

5 10

Number of surviving or exposed' snails

87 46

21 50 39 40 43 19 43 18 7 15 24 92 4 3 258' 109' 25' 25' 25'

% snails infected with S. bovis from

NMZ' 17.2 17.6 14.3 44 28.2 17.5 11.5 52.5 72 28 100 73 83.3 75 25 100 69.4 44.2 20

64 24

SMZ3

References

SSZ4 Ramajo-Martin (1972)

Ramajo-Martin (1978)

I

r

0

Sampaio-Silva et al. (1975)

z rn

rn ' I b

!-

5,

Portugal

Morocco

Spain

Portugal Portugal Morocco

Sardinia (Italy) Morocco Iran Sudan

Spain Spain

Sudan Kenya

Portugal

'

Spain

Number of exposed snails. *North Mediterranean zone. South Mediterranean zone. South Saharan zone.

5, 5, 5, 5,

10 20 20 10 or 20 5 5 10 10 20 20 10 or 20 10 or 20 10 or 20 10 or 20 5 10 5

5 5 5 10 5, 10 or 20 5, 10 or 20

25' 25' 25' ?

25' 25' 25' 25' 25' 25' ? ? ? ?

94 71 ? ? ?

170 71 ? ?

72 8 32 73.3 68 12 52 24 80 20 62.9 78.6 29.2 14.3 45.7 86 0

Southgate et al. (1984) Sampaio-Silva et al. (1975)

m c i 0

z

Ramajo-Martin (1978) Southgate et al. (1984) Touassem and Jourdane (1986) Southgate and Knowles (1975a)

0 0 5.9 35.2 0 0

Touassem and Jourdane (1986) Southgate et al. (1984)

5 s cn

A

Table 3 Snail infection experiments in the genus Bulinus of the truncatusltropicus group.

0 Q)

Snail species

B. natalensis

B. octoploidus

Snail origin

South Africa Ethiopia

S. bovis origin

Ethiopia Ethiopia

B. permembranaceus

Kenya

Morocco

B. trigonus

Ethiopia Kenya Mauritania Uganda Uganda Kenya Uganda Kenya Sudan Tanzania

Sardinia (Italy) Sardinia (Italy) Sardinia (Italy) Sardinia (Italy) Morocco Iran Iran Kenya Kenya Tanzania

Uganda

Kenya

Number of miracidia per snail

5 15-20

Number of surviving or exposed' snails

0 6.3

16 48

5-6

?

3

? ? ?

23 25 25 28 20-25' 20-25' 20-25' 20-25' 25

Loand Lemma (1975)

0 8.3

15

41 47 24

References

NMZ2 SMZ3 SSZ4

44

10-15 20

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

% snails infected with S . bovis from

Frandsen (1979)

0

Southgate and Knowles (1975a)

21.9 12.7 95.7 0 0 0

0

30.4 20 32 14.3 13 38 17 50 12

Southgate et al. (1980) Mutani et al. (1983)

Southgate and Knowles (1975a)

I rn

' I

b !-

B. tropicus

South Africa South Africa South Africa Zimbabwe

Sardinia (Italy) Morocco Iran Kenya

Tanzania

Tanzania

Zambia

Tanzania

Ethiopia Kenya

Tanzania Tanzania

South Africa

Tanzania

5 5 5 5 5 5 5 4 10 30 5 5 5 5 5 5

5 B. truncatus Mediterranean zone

Portugal

Spain 5, 5,

Egypt

Spain

Libya

Spain

5, 5,

5 10 20 10 or 20 10 or 20 5 10 20 10 or 20 10 or 20

? ? ?

0

251 20-251 20-251 20-251

9

0 0 0 0 0 0 0 0 0 0 0 0 0

9

30 24 20-251 20-251 20-251 20-251 20-25' 20-251 20-251 25' 251 25' 168' 90' 251 25' 251 1501 331

0 0 0 0

52.1 100 85 67.3 74.4 55.5 70 94.4 55.1 50

W

Southgate et al.

c

Mutani et al. (1983) (1985a)

0 Z

=!

4 Kassuku et al. (1986) Kinoti (1964a) Mutani et al. (1983)

$ 2 0 $

5

Simon-Vicente et al. (1975) Ramajo-Martin (1978) Simon-Vicente et al. (1975) Ramajo-Martin (1978) (continued)

~

0 4

a

Table 3 Continued.

UI

Snail species

Snail origin

S. bovis origin

Tunisia

Spain

Israel Egypt Iraq Libya Morocco Sardinia (Italy) Iran Israel Egypt Iraq Libya

Sardinia (Italy) Sardinia (Italy) Sardinia (Italy) Sardinia (Italy) Sardinia (Italy) Sardinia (Italy) Morocco Morocco Morocco Morocco Morocco

Morocco

Morocco

Israel

Israel

Iran Portugal

Iran Iran

Number of miracidia per snail 5 10 5 5 5 5 5 5 5 5 5 5 5 6 5 5 5, 10, 15 or 20 5, 10, 15 or 20 60 2-4 2-4

Number of surviving or exposed' snails 57 53 ?

20 18 23 43 99 ? ?

14 45 101 24 54

YOsnails infected with S. bovis from

NMZ2 SMZ3 SSZ4 Touassem and Jourdane (1986) Southgate and Knowles (1975a)

61.4 56.6 0 15 5.6 60.9 30.2 21.2

0 0 7.1 4.5 32.7 91.6 31.5

?

90 36

?

58.6

?

87 53.3 60

30

30 20

References

Frandsen (1979) Southgate and Knowles (1975a) Frandsen (1979) Lengy (1962)

r 0 Arfaa ef al. (1967)

z rn rn -4 L

r

630ch3.3d Southgate and Knowles (1975a)

B. truncatus South Saharan zone

Iraq

Iran

5

59

50.8

Israel Libya Morocco Sardinia (Italy) Egypt Egypt

Iran Iran Iran Iran Iran Ethiopia

5 5 5 5 5 20

43 234 28 64 40

15

Israel Libya Morocco Sardinia (Italy) Tunisia

Kenya Kenya Kenya Kenya Sudan

5 5 5 5 5 10

23 17 25 20 160 53

13 47.1 16 45 15 41.5

Chad

Spain

5 10 20 5, 10 or 20

25' 25' 25' 130'

Sudan Ethiopia Kenya Mauritania Malawi Senegal

Morocco Morocco Morocco Morocco Morocco Morocco

86 22.2 25 11 0

?

?

30 50 106 91 40 57

25 33.5

Lo and Lemma (1975) Southgate and Knowles (1975a) Touassem and Jourdane (1986) Simon-Vicente et al. (1975)

100

64.5

0 23.3 18 0.9 86.4 87.5 93

Ramajo-Martin (1978) Southgate and Knowles (1975a) Frandsen (1979)

(continued

Table 3 Continued.

Snail species

B. coulboisi (syn. B. truncatus)

Snail origin

S. bovis origin

Zimbabwe Uganda

Morocco Morocco

Uganda Uganda Uganda Tanzania Kenya

Sardinia (Italy) Morocco Iran Tanzania Tanzania

Ethiopia

Tanzania

Sudan Zambia Gabon

Tanzania Tanzania Senegal

Senegal

Senegal

Uganda

Kenya

Number of miracidia per snail

Number of surviving or exposed' snails

5 5 or 6

28

5 5 5 4-5

25 24 19

5 5 5 5 5 5 5

% snails infected with S. bovis from

NMZ'

40

SMZ3 SSZ4

3.6 0

?

20-25l 20-25l 20-25l 20-25l 20-25l 20-25l 50

References

Southgate and Knowles (1975a)

24 4 10.5 0 50

Kinoti (1 964a) Mutani et al. (1983)

60 0 19 0 0 48

3-4

19

63.6

5

23

8.7

Southgate et al. (1985b) Diaw and Vassiliades (1987) Southgate and Knowles (1975a)

?

5 Z

rn

B. guernei (syn. B. truncatus)

Senegal

Senegal

5

89

64

Southgate et al. (1985b)

rn

-i

b !-

B. sericinus (syn. B. truncatus)

The Gambia

Ethiopia

5

45

Ghana Nigeria Cameroon Ghana Nigeria Chad Cameroon Ghana Cameroon Ghana

Sardinia (Italy) Sardinia (Italy) Morocco Morocco Morocco Iran Iran Iran Kenya Tanzania

5 5 5 5

59 14 27 98 38

5

41 95 39 15

Saudi Arabia Saudi Arabia Saudi Arabia Saudi Arabia

Sardinia (Italy) Morocco Iran Kenya

5 5 5 5

17 18 25 21

5 5 5 5 5

2.2 57.6 100

Loand Lemma (1975) Southgate and Knowles (1975a)

7.4 59.2 42.1 100 12.2 46.4

5

33.3 53.3 82.3

Southgate et al. (1980) Southgate and Knowles (1975a)

11.1 20

23.8

Number of exposed snails. North Mediterranean zone. South Mediterranean zone. South Saharan zone.

A A A

Table 4 Snail infection experiments in the genus Bulinus of the reticulatus group.

Snail species

B. reticulatus

B. wrighti

Snail origin

Kenya Kenya Kenya Kenya

Sardinia (Italy) Morocco Iran Kenya

Saudi Arabia Saudi Arabia Saudi Arabia Saudi Arabia Yemen Oman Yemen

Sardinia (Italy) Morocco Iran Kenya Kenya Tanzania Tanzania

Oman

'

S. bovis origin

Number of exposed snails. 'North Mediterranean zone. South Mediterranean zone. South Saharan zone.

Senegal

Number of miracidia per snail

Number of surviving or exposed' snails 8 12 8 7

5 5 5 5

5 5 5 5 5 5

30 86 8 25 16' 14 11 20-25' 20-25' 19

% snails infected with S. bovis from

Nh4Z'

SMZ3

References

SSZ4

50

Southgate and Knowles (1975a)

8.3 25 100

80 34.9 62.5 56

12.5 100 36.4 92 84 47.4

Southgate et al. (1985a) Southgate et al. (1980) Mutani el al. (1983) Southgate et al. (1985b) ?

z

0 z rn

m

-i

b

r

Table 5 Snail infection experiments in the genus Bulinus of the forskalii group. Snail species

Snail origin

S. bovis origin

Number of miracidia per snail

Number of surviving or exposed' snails

% snails infected with

NMZ'

SMZ3

SSZ4

Madagascar Madagascar Madagascar

Sardinia (Italy) Morocco Iran

5 5 5

3 2 40

33.3

Cameroon Cameroon Cameroon Cameroon

Sardinia (Italy) Morocco Iran Kenya

5 5 5 5

25 22 23 3

80

B. canescens

Zambia

Tanzania

5

20-25'

0

B. cernicus

Mauritius

Kenya

15

43

53.5

B. crystallinus

Angola Angola Angola Angola Angola

Sardinia (Italy) Morocco Iran Kenya Tanzania

5 5 5 5 5

24 79 39 13 48

Cameroon Nigeria Senegal

Sardinia (Italy) Sardinia (Italy) Sardinia (Italy)

5 5 5

? ?

B. bavayi

B. camerunensis

B. forskalii

25

References

S. bovis from

Southgate and Knowles (1975a)

50 47 100 69.9 100

11.9

Rollinson and Wright (1984) Southgate and Knowles (1975a)

1.3 17.9

30.8 31.3 0 0 12

Mutani et al. (1983)

Southgate et al. (1980) Southgate and Knowles (1975a)

continued

Table 5 Continued.

Snail species

B. scalaris

Snail origin

S. bovis origin

Kenya Nigeria Senegal QYPt Cameroon Nigeria Senegal Cameroon Ethiopia Senegal Zaire

Morocco Morocco Morocco Morocco Iran Iran Iran Kenya Kenya Kenya Tanzania

Tanzania

Tanzania

Tanzania

Tanzania

Gabon Senegal

Senegal Senegal

Zimbabwe Zimbabwe Zimbabwe

Sardinia (Italy) Morocco Iran

' Number of exposed snails. 'North Mediterranean zone. South Mediterranean zone. South Saharan zone.

Number of miracidia per snail

5 5 5 5 or 6 5 5 5 5 5 5 5 5 5

Number of surviving or exposed' snails

% snails infected with S. bovis from

NMZ'

SMZ3

?

45 25 17 12 25 20-25' 20-25' 19 17 21 24 9 96

5 5 5

? ? ?

0

0 0

P

Frandsen (1979) Southgate and Knowles (1 975a) 30.8 100 68 100 85 52.9 63.2 64.7 100 41.7 66.7 48.9

?

4- 5 5 5 5 5 3-4

a a

SSZ4

0 0 37.5 0 0 6.7 8

? 24 ?

References

Mutani et al. (1983) Southgate et al. (1980) Kinoti (1964a) Mwambungu (1988) Southgate et al. (1985b) Diaw and Vassiliades (1987) Southgate and Knowles (1975a)

I

5

P v)

i W

c

-I

0

z

Table 6 Snail infection experiments in the genus Bulinus in the africanus group.

Snail species

B. abyssinicus B. africanus

Snail origin

S. bovis origin

Ethiopia Somalia

Ethiopia Tanzania

South Africa Ethiopia South Africa Ethiopia South Africa Ethiopia Ethiopia Kenya

Sardinia (Italy) Sardinia (Italy) Morocco Morocco Iran Iran Ethiopia Tanzania

Tanzania

Tanzania

Number of miracidia per snail 5

5

5 5 5 5 5 5 20 5 5 5 5 5 5 4 4-6

Number of surviving or exposed' snails

% snails infected with S. bovis from

? ? ? ? ? 20 4 20-25' 20-25' 20-25' 20-25' 20-25' ? 35

References

0

a

0

8

62.5 0

Lo and Lemma (1975) Mutani et al. (1983) Southgate and Knowles (1975a)

0 100 67 83 83 100 100 83.3 65.7

Lo and Lemma (1975) Southgate et al. (1980) Mutani et al. (1983)

0 0

cn

-i

N M Z ~ SMZ~ SSZ~

8 20-25' ?

%

0 0 0 0

Kassuku et al. (1986) Kinoti (1964a) continued

51D

0

s

Table 6 Continued

Snail species

B. globosus

Snail origin

S . bovis origin

Zimbabwe

Number of surviving or exposed' snails

Number of miracidia per snail

Sardinia (Italy)

Nigeria South Africa Uganda Zaire Zimbabwe Zimbabwe albino Uganda South Africa Zambia Zimbabwe

Sardinia (Italy) Sardinia (Italy) Sardinia (Italy) Sardinia (Italy) Morocco Morocco Morocco Morocco Morocco Iran

Nigeria South Africa Uganda Zaire Zimbabwe Zimbabwe Zimbabwe albino

Iran Iran Iran Iran Ethiopia Kenya Kenya

6 8-9 10-12 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

5 5

% snails infected with S. bovis from

Nh4Z2

SMZ3

20 20 23 ?

47 ? ? ? ? 37

Southgate and Knowles (1975a)

0 29.8 0 0 0 0

27 ? ? ? ?

0 0 0 0

13 24 9

SSZ4 95 100 100

10.8 80 0 0 0 0 68.2

5 ? ? ? ?

References

Frandsen (1979) Southgate and Knowles (1975a)

I

Lo and Lemma (1975) 8.3 Southgate and Knowles 11.1 (1975a) 0

I 0 z rn

3 b

r

South Africa Ghana Uganda Tanzania

Kenya Kenya Kenya Tanzania

5 5 5 10-12 5 5

Zimbabwe South Africa Kenya Kenya albino

Tanzania Tanzania Tanzania Tanzania

B. jousseaumei

Senegal

Senegal

B. nasutus

Tanzania

Tanzania

5 5 5 5 5 5 5 5 5 5 5 5 5 4-5 10-12 15 100 5

5

13 17 23 24 ?

20-251 20-25' 20-25' 22 23 20 25 14 25 20-251 20-251 20-25' 20-251 ?

6 29 28 17

30.8 11.8 33 4.1 Kinoti (1964a) 20 Southgate and Knowles (1975a) Mutani et al. (1983) 0 5 4

63.6 0 0 20 100 32 71 28 40 67 0

?

0 0 0 0 0

20-251

0

Mwambungu (1988) Southgate et al. (1980) Mutani et al. (1983)

Southgate et al. (1985b) Kinoti (1964a)

Southgate and Knowles (1975a) Mutani et al. (1983) continued

--.

Table 6 Continued.

Snail species

B. obtusispira

B. ugundae

Snail origin

Number of miracidia per snail

OD

Number of % snails infected with surviving S. bovzk from or exposed’ snails NMz’ S M Z ~ S S Z ~

Madagascar Madagascar Madagascar

Sardinia (Italy) Morocco Iran

5 5 5

? ? ?

0

Uganda

5 5 5

? ? 25

0

Uganda

Sardinia (Italy) Morocco Kenya

Senegal

Senegal

5

22 15

Uganda B. umbilicatus

S. bovis origin

2

3-4

References

Southgate and Knowles (1975a)

0 0

Southgate and Knowles (1975a)

0 100

4.5 Southgate et ul. (1985b) 13.3 Diaw and Vassiliades (1987)

Number of exposed snails.

’North Mediterranean zone. South Mediterranean zone. South Saharan zone.

6z

rn

m

-i

b

r

119

DISTRIBUTION OF SCHlSTOSOMA BOWS

Table 7 The species of Planorbarius and Bulinus, and their relationships as natural and experimental hosts for Schistosoma bovis of the North Mediterranean zone (NMZ), of the South Mediterranean zone (SMZ) and of the South Saharan zone (SSZ). The mollusc species are named alphabetically within each group. Natural host Mollusc Genus Planorbarius P. corneus P. metidjensis Genus Bulinus (truncatus/ tropicus group) B. angolensis B. depressus B. hexaploidus B. Iiratus B. natalensis B. nyassanus B. octoploidus B. permembranaceus B. succinoides B. transversalis B. trigonus B. tropicus B. truncatus B. yemenensis Genus Bulinus (reticulatus group) B. reticulatus B. wrighti Genus Bulinus uorskalii group) B. barthi B. bavayi B. beccarri B. browni B. camerunensis B. canescens B. cernicus B. crystallinus B. forskalii B. scalaris B. senegalensis Genus Bulinus (africanus group) B. abyssinicus

Experimental host

NMZ

SMZ

SSZ

NMZ

SMZ

SSZ

NF Yes

NL No

NL NL

NT Yes

NT No

NT Yes

NL NL NL NL NL NL NL NL NL NL NL NL Yes NL

NL NL NL NL NL NL NL NL NL NL NL NL Yes NL

NF NF NF NF NF NF Yes NF NF NF NF Yes Yes NF

NT NT NT NT NT NT NT NT NT NT Yes No Yes NT

NT NT NT NT NT NT NT No NT NT No No Yes NT

NT NT NT NT Yes NT Yes NT NT NT Yes No Yes NT

NL NL

NL NL

NF NF

Yes Yes

Yes Yes

Yes Yes

NL NL NL NL NL NL NL NL NL NL NL

NL NL NL NL NL NL NL NL NF NL NL

NF NF NF Yes NF NF NF NF Yes NF Yes

NT Yes NT NT Yes NT NT Yes Yes No NT

NT Yes NT NT Yes NT NT Yes Yes No NT

NT NT NT NT Yes No Yes Yes Yes NT NT

NL

NL

Yes

NT

NT

Yes

(continued)

120

H. MONE ETAL.

Table 7 Continued.

Natural host Mollusc B. africanus B. globosus B. hightoni B. jousseaumei B. nasutus B. obtusispira B. obtusus B. ugandae B. umbilicatus

Experimental host

NMZ

SMZ

SSZ

NMZ

SMZ

SSZ

NL NL NL NL NL NL NL NL NL

NL NL NL NL NL NL NL NL NL

Yes Yes NF NF NF NF NF Yes NF

No Yes NT NT NT No NT No NT

No Yes NT NT

Yes Yes NT No No NT NT Yes Yes

NT No NT No NT

NF = the species of mollusc has not been found infected yet in that zone; NL = the species of mollusc do not live in that zone; NT =the host-parasite association has not been tested.

Figure I Geographical distribution of Planorbarius metidjensis (shaded area). (Data obtained from Brown, 1994.) 0 = Countries in which Planorbarius metidjensis has been found naturally infected by S. bovis.

Figure 2 Geographical distribution of Bulinus belonging to the truncatus (shaded area)/tropicus (hatched area) group. (Redrawn with permission from Brown, 1994, Figures 126 and 127.) 0 = Countries in which the molluscs of the truncatusltropicus group have been found naturally infected by S. bovis.

Figure 3 Geographical distribution of the molluscs of the Bufinus reticulatus group (shaded area). (Data obtained from Brown, 1994.)

Figure 4 Geographical distribution of the molluscs of the Bulinus forskalii group (shaded area). (Redrawn with permission from Brown, 1994, Figure 128.) O=Countries in which the molluscs of the forskalii group have been found naturally infected by S.bovis.

Figure 5 Geographical distribution of the molluscs of the Bulinus africanus group (shaded area). (Redrawn with permission from Brown, 1994, Figures 124 and 125.) 0 = Countries in which the molluscs of the africunus group have been found naturally infected by S. bovis.

DISTRIBUTION OF SCH/STOSOMA BOWS

123

test or the unpaired t-test were applied on the arcsine-transformed data. For each prevalence, one set of arcsine-transformed data was calculated.

3. THE NATURAL MOLLUSC INTERMEDIATE HOST SPECTRUM

The natural intermediate host spectrum of Schistosoma bovis is wide: S. bovis may naturally infect molluscs belonging to two genera, Planorbarius and Bulinus. Within these genera, one species of Planorbarius (P. metidjensis) and ten species of Bulinus may act as intermediate hosts, showing a wide host spectrum tolerance of S. bovis for mollusc intermediate hosts. The species belonging to the genus Bulinus are usually divided into four groups: truncatusltropicus, reticulatus, forskalii and africanus. In each group, the recent classification of Brown (1994) was used. S . bovis may infect Bulinus species belonging to three of the four groups (Table 7). The comparative intermediate host spectra of S. bovis between the three geographical zones showed that: the North Mediterranean populations of S. bovis use two species of mollusc, P. metidjensis and B. truncatus; the South Mediterranean populations of S . bovis use only B. truncatus; and the South Saharan populations use ten species of Bulinus. B. truncatus is the only species acting as an intermediate host in the three zones.

4. GEOGRAPHICAL DISTRIBUTIONS OF THE MOLLUSC

INTERMEDIATE HOSTS

The geographical distribution of P. metidjensis is presented in Figure 1. It is limited to the two Mediterranean zones, the North Mediterranean zone to the Iberian peninsula, and the South Mediterranean zone to Morocco and Algeria. The geographical distributions of the Bulinus species are presented in Figures 2-5. The truncatusltropicus group occurs in the three geographical zones, and it has a Mediterranean (North and South) and Pan-African distribution (Figure 2). The reticulatus group has a sparce geographical distribution in the South Mediterranean zone (Arabia peninsula) and in the South Saharan zone (Figure 3). The forskalii group has a sparce geographical distribution in the South Mediterranean zone (Egypt) but is widely present in the South Saharan zone, where it has an Afro-intertropical geographical distribution extending to Yemen and some islands of the Indian Ocean (Figure 4). The africanus group has a strictly South Saharan Afro-intertropical distribution including Madagascar (Figure 5).

124

H. MONE ETAL.

5. GEOGRAPHICAL DISTRIBUTION OF S. BOWS

The natural geographical distribution of S . bovis is presented in Figures 1-5, according to the mollusc group, and in Table 7. It runs from the latitude 43 ON crossing the North of Corsica, France, to the latitude 13 "S crossing the very south of Zaire, and from the longitude 17.7"W crossing the Green Cap in Senegal to the longitude 63.2"E crossing the very east of Iran. It has been found in countries in all three zones (3 countries in the North Mediterranean zone, 4 countries in the South Mediterranean zone and 12 countries in the South Saharan zone). The number of regions where S . bovis is present is smaller than the number of regions where the mollusc intermediate hosts are found. In some other regions, we may just suppose an actual transmission of S . bovis because its presence in cattle does not imply that the definitive host has been infected locally (Figure 6 ) .

Figure 6 Countries where S. bovis is probably present (0).In these countries, S . bovis was found in vertebrate definitive hosts whose geographical origin was uncertain.

DISTRIBUTION OF SCH/STOSOMA BOWS

125

6. THE EXPERIMENTAL MOLLUSC INTERMEDIATE HOST SPECTRUM

The experimental intermediate host spectrum of S. bovis is wider than the natural one: among the 24 species of mollusc tested, S . bovis may experimentally infect one species of Planorbarius ( P . metidjensis) and 16 species of Bulinus belonging to the four groups (Table 7). The comparative intermediate host spectra of S . bovis between the three geographical zones showed that the North Mediterranean populations of S. bovis may use ten species of molluscs belonging to two different genera and to the four groups of Bulinus, the South Mediterranean populations of S. bovis may use eight species of molluscs all belonging to the four groups of the genus Bulinus, and the South Saharan populations may use 16 species of molluscs belonging to two different genera and to the four groups of Bulinus. B . truncatus for the truncatusltropicus group, B. reticulatus and B. wrighti for the reticulatus group, B . camerounensis, B. crystallinus and B. forskalii for the forskalii group, and B. globosus for the africanus group have been experimentally infected with S . bovis populations from the three zones.

7. COMPATIBILITY IN THE MOLLUSC-S. BOWS ASSOCIATION

A total of 294 prevalences were available, including 188 population hostpopulation parasite systems. Experimental prevalences 44, 124, 14, 39 and 73 obtained for P . metidjensis, and the truncatusltropicus, reticulatus, forskalii and africanus groups, respectively, are presented in Tables 2-6 for the three geographxal zones. For each species of mollusc, the order in which these prevalences are presented was always the same: firstly, the prevalences concerning the S. bovis populations of the North Mediterranean zone; then the prevalences concerning the S. bovis populations of the South Mediterranean zone; and, finally, the prevalences concerning the S. bovis populations of the South Saharan zone. Transformed prevalences (p’) 9, 88, 11, 34 and 46 obtained for each population host-population parasite system for P . metidjensis, and the truncatusltropicus, reticulatus, forskalii and africanus groups, respectively, were used for the analysis of the host-parasite compatibilities. The mean transformed prevalences are presented in Table 8 together with the ANOVA comparisons. When considering the influence of the mollusc group on compatibility, the results showed that the compatibilities were significantly different among the

Table 8 Transformed prevalences of Schistosoma bovis from the North Mediterranean zone (NMZ), the South Mediterranean zone (SMZ) and the South Saharan zone (SSZ) according to the mollusc group.

Mollusc group

Genus Planorbarius P. metidjensis Genus Bulimus truncatusltropicus group reticulatus group forskalii group ajiricanus group ANOVA

NMZ

ssz

SMZ

N

Mean f SE

N

Mean fSE

N

Mean fSE

4

0.57 f0.19

2

0.00 f 0.00

3

0.16 f0.16

20 0.62 f 0.09 2 0.95 f 0.16 7 0.35 f 0.16 9 0.04 f0.04 F= 5.746; P = 0.002 S

39 4 15

0.45 f 0.07 0.59 f 0.13 0.39 f 0.12 17 0.12 f0.07 F = 2.72; P = 0.036 S

27 0.37 f0.06 5 1.02 f0.20 12 0.89f0.13 20 0.58f0.12 F= 5.93; P = 0.0004 S

ANOVA

F = 2.75; P = 0.14 F = 2.45; P = 0.09 S F= 1.48; P=0.28 F=5.00; P=O.O13 S F=8.79; P=0.0006 S

S = significance. I

DISTRIBUTION OF SCHISTOSOMA BOVIS

127

groups whatever the S. bovis geographical zone: 1. for the North Mediterranean populations of S. bovis, the reticulatus, and truncatusltropicus groups and P . metidjensis showed the highest compatibilities; 2. for the South Mediterranean populations of S. bovis, the compatibilities were the highest for the reticulatus, truncatusltropicus and forskalii groups and null for P . metidjensis; 3 . for the South Saharan populations of S. bovis, the reticulatus, forskalii and africanus groups showed the highest compatibilities. When considering the influence of the geographical origin of S. bovis on compatibility, the results showed that the compatibilities were significantly different only in the forskalii and the africanus groups as shown below. 1. Despite the few data available, P . metidjensis was highly compatible with

2. 3. 4.

5.

the North Mediterranean population of S. bovis but refractory with the South Mediterranean populations, and slightly compatible with the South Saharan populations (see Table 8). In the truncatusltropicus group, the highest prevalence was found with the North Mediterranean populations of S. bovis. The reticulatus group was highly compatible with the three geographical populations of S. bovis. The forskalii group was compatible with the three geographical populations, with a significant highest prevalence in the South Saharan populations of S. bovis. The significant difference came from the comparison between the South Saharan populations of S. bovis and the South Mediterranean populations (post hoc Scheffe comparison procedure: P = 0.03). Notice that the prevalences between the two Mediterranean zones, with only the Sardinian population as member of the North Mediterranean zone, were not significantly different (see Tables 5 and 8). The africanus group was compatible with the South Saharan geographical populations of S. bovis, but only slightly compatible with the populations from the other two zones. The difference was highly significant for the comparison between the South Saharan populations of S. bovis and the North Mediterranean populations (post hoc Scheffe comparison procedure: P = 0.006), and for the comparison between the South Saharan populations of S. bovis and the South Mediterranean populations (post hoc Scheffe comparison procedure: P = 0.004). Notice that the prevalences of the two Mediterranean zones, with only the Sardinian population as member of the North Mediterranean zone, were very low and not significantly different (see Tables 6 and 8).

When considering the North Mediterranean zone, the populations of S. bovis, peculiarly, were very heterogeneous in their compatibilities towards

128

H. MONE ETAL.

P . metidjensis, and may be separated into the highly compatible Spanish populations (0.76 f 0.005) and the refractory Sardinian populations (see Table 2). Similarly, for the truncatus-tropicus group, the Spanish populations (0.91 f 0.05; N = 5) had a higher compatibility than the Sardinian populations (0.52 f 0.13; N = 15) but the difference was just below being significant ( t = 2.08; P = 0.05) (see Table 3). For the other three groups, reticulatus, forskalii and africanus, only Sardinian populations of S. bovis were tested for the North Mediterranean zone (see Tables 4-6) and no comparison with the Spanish populations could be made with our data set.

8. THREE MAIN POPULATIONS OF S. BOWS

The natural and experimental intermediate host spectrum and host-parasite compatibility results between S. bovis and its intermediate hosts showed that our hypothesis, according to which the two natural barriers (the Mediterranean Sea and the Sahara) played a role on the genetic flow between the different populations of S. bovis, has to be rejected for the Mediterranean barrier but that it can be accepted for the Saharan barrier. The intrazone variability was clearly high for the North Mediterranean populations confronted with P. metidjensis, and suggests a separation between the highly compatible Spanish populations and the refractory Sardinian populations, which behaved like South Mediterranean populations. The interzone variabilities between the North Mediterranean zone and the South Mediterranean zone were similar enough to reject the hypothesis concerning the role played by the Mediterranean barrier. The interzone variabilities between the South Mediterranean zone and the South Saharan zone were high enough to accept the separation according to the Saharan barrier. Thus, three main geographical populations of S . bovis exist but they are different from those proposed by our hypothesis: from the North to the South, we find the Iberian populations (Spanish and Portuguese), the Mediterranean populations (including the Sardinian populations), and, finally, the South Saharan populations. The Iberian populations of S. bovis use exclusively P . metidjensis in the natural conditions; their host spectrum is thus restricted to one species. At the same time, the reanalysed compatibilities according to these three new geographical zones (Figure 7) showed that the Iberian populations are as compatible with B. truncatus as with P . metidjensis, and both compatibility values are high. This result agrees with those of Southgate and Knowles (1975~)whose results could not be included in our data set because the origins of the snails were not precise. However, these authors were the sole authors who showed experimentally that the Spanish population of S. bovis was not

DISTRIBUTION OF SCHlSTOSOMA BOWS

129

1.m 1.20 1.00

0.80 0.60 0.40

0.20 0.00

Figure 7 Comparative compatibilities of S. bovis from the Iberian zone (a),the Mediterranean zone (0)and the South Saharan zone @) for the different groups of molluscs.

compatible with the africanus group, that it had a very poor compatibility with the forskalii group and that it had a reasonable good compatibility with the reticulatus group. For our results, the deletion of the Sardinian populations from our previous North Mediterranean zone made the prevalences higher for the Iberian populations (compare Table 8 and Figure 7). The Mediterranean populations, including the Sardinian populations of S. bovis, use exclusively B. truncatus in natural conditions; their natural host spectrum is also restricted to one species. In experimental conditions, they are refractory to P . metidjensis (Figure 7 ) ; however, they are compatible with all other groups of Bulinus: the compatibility is the highest with the truncatusltropicus group and weaker with the species belonging to the africanus group which proves to be restricted to the S. bovis populations of the South Saharan zone. The inclusion of the Sardinian populations in the new redefined Mediterranean zone did not alter the pattern (compare Table 8 and Figure 7). The South Saharan populations of S. bovis benefit from a wide natural host spectrum: the natural hosts belong to three groups of Bulinus the truncatus/tropicus, forskalii and africanus groups. In experimental

130

H. MONE ETAL.

conditions, S. bovis successfully infected some species from all groups of molluscs (Figure 7), but preferentially the reticulatus, the forskalii and the africanus groups. The compatibility is the lowest with the genus Planorbarius, which appears to be restricted to S. bovis from the Iberian zone. This situation is very different from that occurring in the Mediterranean zone where the truncatusltropicus group plays the lead role in the transmission of S. bovis. The South Saharan populations of S. bovis are slightly compatible with P.metidjensis.

9. PALEOBIOGEOGRAPHICALSCENARIO OF S. BOVlS

From these results, one possible scenario of the natural paleobiogeography of the S. bovis species complex could be proposed (Figure S), despite the lack of any fossils of schistosomes and the wide vertebrate definitive host spectrum (see Mouahid, 1984), creating possible lateral transfers between hosts favoured by animal migrations (Combes, 1995). The time scale of the history of S. bovis could not be determined, but the knowledge on both intermediate host spectrum and compatibility allows us to propose a three-step but probably continuous history of this species. The first step is the South Saharan origin of S. bovis, which is not in doubt, even though the exact local origin is unknown (Combes, 1990; Combes et al., 1991; Despres et al., 1992). It will be assumed that the origin was in the West of Africa. As early as its origin, S . bovis was confronted with a wide spectrum of molluscs belonging to the genus Bulinus and consequently coevolved with them. The extension of the geographical distribution to the South, East and West did not alter the intermediate host spectrum. The very low compatibilities between the South Saharan populations of S. bovis and Planorbarius may be explained by the absence of this mollusc in the South Saharan region. The second step, occurring in a more recent period, is the extension of S. bovis to the Mediterranean zone. The North migration was accompanied by a drastic change in the host spectrum, which was reduced to one species of mollusc. As this species, B. truncatus, also existed in the South Saharan zone, S. bovis did not necessarily have to adapt to it, as suggested by the similar compatibilities existing between the Mediterranean and the South Saharan populations of S. bovis with B. truncatus. The low compatibilities between the Mediterranean populations of S. bovis and the mollusc species of the africanus and forskalii groups corroborate the existence of an adaptation of the Mediterranean populations to a host spectrum restricted to one species. In this second step, the North-West and the North-NorthEast migrations have to be analysed separately.

DISTRIBUTION OF SCHlSTOSOMA BOVlS

131

Figure 8 The three steps of the paleobiogeographical scenario of S. bovis.

The North-West extension of S. bovis stopped in Morocco owing to the natural barrier of the Gibraltar Strait. Indeed, the few data available on the time-scale history of S. bovis are compatible with a recent origin of S. bovis in this region. According to recent schistosome molecular phylogenies, S. bovis emerged as a new species between 1 and 4 and between 1 and 7 million years ago, respectively, based on nuclear and mitochondria1 DNA (Despres et al., 1992). Recently, a study on the relative phyletic relationships in schistosomes using RAPD markers has even suggested a very recent origin of S. bovis, that is, the possibility that this species has been transferred from man to livestock (Barral, 1996). These results from molecular biology thus suggest it is unlikely that S. bovis originated in Africa before or even during the Messinian period (also called the ‘Salt crisis’), which lasted from 6 to 5.5 million years ago, and where the Mediterranean Sea level dropped more than 1000 metres, thus blocking the connection between the Mediterranean

132

H. MONE ETAL.

Sea and the Atlantic Ocean (Van der Zwaan, 1982) at the Strait of Gibraltar. Furthermore, during this period, the Iberian peninsula-North West African interchange of land vertebrates was followed by massive extinctions of these vertebrates, removing the possibility for them to act as important hosts for any parasite evolution (Jaeger et al., 1987; Jaeger and Hartenberger, 1989). It is important to note there that the Moroccan populations of S . bovis have been always detected in B. truncatus despite the presence of Planorbarius. The North-North-East extension of S . bovis used the Middle Eastern passage to invade the Southern part of Europe and Sardinia. This extension could have been favoured by human migration with cattle to Europe and to the Mediterranean islands. For instance, humans are known to have introduced domestic species in some Mediterranean islands as early as 7000 BP (Blonde1 and Vigne, 1993). The third, last and more recent step was the colonization of the Iberian peninsula by S. bovis. The intermediate host spectrum of the schistosome ‘reopened’ and S. bovis captured a new host belonging to a different genus, the genus Planorbarius. This capture of a new genus would have been harder than the capture of any other new species in the same genus owing to its remote phylogenetic position from the molluscs of the genus Bulinus (Hubendick, 1955). The strict non-compatibility between the Mediterranean populations of S. bovis and P. metidjensis, and the slight compatibility between the South Saharan populations of S. bovis and P. metidjensis, compared to the high level of compatibility between the Iberian populations of S. bovis and P. metidjensis, suggest the existence of a local adaptation of the Iberian S. bovis strain to Planorbarius, which isolated the Iberian populations of S. bovis from the remainder of the Mediterranean and Afrotropical populations. This local adaptation has been proposed by Mouahid and Thtron (1987) comparing the cercarial production of a Spanish population of S . bovis among B. truncatus, B. wrighti and P. metidjensis. These authors showed that, with a lower productivity compensated by a lengthening of the production period, S . bovis was better adapted to P. metidjensis than to the species belonging to the genus Bulinus because the parasite exploited this mollusc host optimally.

10. CONCLUSION

From our results, the three major groups of S. bovis populations that could be determined were the Iberian, the Mediterranean and the South Saharan populations. The Saharan barrier may have played a role in the gene flow between the different populations of S. bovis but not the Mediterranean one.

DISTRIBUTION OF SCHISTOSOMA BOVlS

133

More work should be done with the different populations of S . bovis, especially with those existing around the Mediterranean Sea, because not all of the interzone combinations have been tested. Despite the data originating from different authors working under different conditions, we believe that the host-parasite compatibility may constitute a tool for researchers to extend knowledge on the evolutionary biogeography of hosts and parasites.

ACKNOWLEDGEMENTS We gratefully acknowledge Drs V. R. Southgate (The Natural History Museum, London, UK), S. Mas-Coma (University of Valencia, Spain) and C. Combes (University of Perpignan, France) for their comments and constructive criticism. This work received financial support from the UNDP World Bank WHO Special Program for Research and Training in Tropical Diseases and the CNRS (Sciences de la Vie).

REFERENCES Al-Barrak, N.S., Wajdi, N. and Fadhil, S. (1977). Observations on schistosomes parasitic in cattle in Iraq. Iraqi Journal of Biological Sciences 5, 69-81. Anderson, C.W. and Gobert, E. (1934). Note sur la prksence, en Tunisie, de Schistosoma bovis, infection naturelle de Bulinus contortus. Bulletin de la SociPtP de Pathologie Exotique 27, 850-853. Arfaa, F., Sabbaghian, H. and Bijan, H. (1965). Studies on Schistosoma bovis in Iran. Transactions of the Royal Society of Tropical Medicine and Hygiene 59, 68 1-683. Arfaa, F., Massoud, J. and Chu, K.Y. (1967). Susceptibility of Portuguese Bulinus contortus to Iranian strains of Schistosoma haematobium and S. bovis. Bulletin de I’Organisation Mondiale de la SantP 37, 165-166. Barral, V. (1996). Identification, Relations PhylPtiques, Processus d’lsolement et Structuration des Populations Naturelles chez les Schistosomes: Approche par I’Etude de Marqueurs GPnomiques (RAPD). Thesis, University of Perpignan, France. Berrie, A.D. (1964). Observations on the life-cycles of Bulinus (Physopsis) ugandae Mandahl-Barth, its ecological relation to Biomphalaria sudanica tanganycensis (Smith) and its role as an intermediate host of Schistosoma. Annals of Tropical Medicine and Parasitology 58, 457-466. Blanc, G. and Desportes, C. (1936). La bilharziose bovine au Maroc. Comptes Rendus des SPances de la SociPtP de Biologie 12, 766-767. Blondel, J. and Vigne, J.-D. (1993). Space, time, and man as determinants of diversity of birds and mammals in the Mediterranean region. In: Species Diversity in Ecological Communities: Historical and Biogeographical Perspectives (R.E. Ricklefs and D. Schlester, eds), pp. 135-146. Chicago: University of Chicago Press.

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Futuyama, D.J. and Slatkin, M. (1983). Coevolution. Sunderland, MA: Sinauer Associates. Gandon, S., Capowiez, Y., Dubois, Y., Michalakis, Y. and Olivieri, I. (1996). Local adaptation and gene-for-gene coevolution in a metapopulation model. Proceedings of the Royal Society of London, Series B, Biological Sciences 263, 1003-1009. Gibson, D.I. (1987). Questions in digenean systematics and evolution. Parasitology 95,429-460. Graber, M. and Daynes, P. (1974). Mollusques vecteurs de TrCmatodoses humaines et animales en Ethiopie. Revue d’Elevage et de Mkdecine Vktkrinaire des Pays Tropicaux 27, 307-322. Grassi, G.B. and Ravelli, G. (1898). La bilharzia in Sicilia. Atti della Accademia Lincei 55, 799. Hubendick, B. (1955). Phylogeny in the Planorbidae. Transactions of the Zoological Society of London, Vol. 28, pp. 453-542. London: Taylor and Francis. Jaeger, J.-J. and Hartenberger, J.-L. (1989). Diversification and extinction patterns among Neogene perimediterranean mammals. Philosophical Transactions of the Royal Society of London B 325, 401 -420. Jaeger, J.-J., Coiffait, B., Tong, H. and Denys, C. (1987). Rodent extinctions following Messinian faunal exchanges between western Europe and northern Africa. MPmoires de la Sociktk gkologique de France 150, 153-158. Jelnes, J.E. (1983). Bulinus browni Jelnes, 1979 (Gastropoda: Planorbidae), a member of the forskalii group, as intermediate host for Schistosoma bovis in western Kenya. Transactions of the Royal Society of Tropical Medicine and Hygiene 77, 566. Kassuku, A., Christensen, N.O., Monrad, J., Nansen, P. and Knudsen, J. (1986). Epidemiological studies on Schistosoma bovis in Iringa region, Tanzanie. Acta Tropica 43, 153-163. Kinoti, G. (1964a). A note on the susceptibility of some gastropod molluscs to Schistosoma bovis and S. mattheei. Annals of Tropical Medicine and Parasitology 58, 270-275. Kinoti, G. (1964b). Observations on the transmission of Schistosoma haematobiurn and Schistosoma bovis in the Lake Region of Tanganyika. Bulletin de I’Organisation Mondiale de la SantP 31, 815-823. Lengy, J. (1962). Studies on Schistosoma bovis (Sonsino, 1876) in Israel. I. Larval stages from egg to cercaria. Bulletin of the Research Council of Israel 10, 1-36. Lo, C.T. and Lemma, A. (1975). Studies on Schistosoma bovis in Ethiopia. Annals of Tropical Medicine and Parasitology 69, 375-382. MacHattie, C. and Chadwick, C.R. (1932). Schistosoma bovis and S. mattheei in Iraq with notes on the development of eggs of the S. haematobium pattern. Transactions of the Royal Society of Tropical Medicine and Hygiene 26, 147-156. Majid, A.A., Marshall, T.F. de C., Hussein, M.F., Bushara, H.O., Taylor, M.G., Nelson, G.S. and Dargie, J.D. (1980). Observations on cattle schistosomiasis in the Sudan, a study in comparative medicine. I. Epizootiological observations on Schistosoma bovis in the White Nile Province. American Journal of Tropical Medicine and Hygiene 29, 435-441. Malek, E.A. (1969). Studies on bovine schistosomiasis in the Sudan. Annals of Tropical Medicine and Parasitology 63, 50 1-513. Marill, F.G. (1961). Enseignements d’une premiere enquCte sur 1’CpidCmiologie de la bilharziose a Schistosoma haematobium en Mauritanie. Mkdecine Tropicale 21, 373-386. Morand, S., Manning, S.D. and Woolhouse, M.E.J. (1996). Parasite-host

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coevolution and geographic patterns of parasite infectivity and host susceptibility. Proceedings of the Royal Society of London B 263, 119-128. Mouahid, A. (1984). Schistosoma bovis (Trematoda, Schistosomatidae): Chronobiologie et Production Cercariennes dans DiFkrentes Conditions Expe'rimentales. Thesis, University of Montpellier 11, France. Mouahid, A. (1994). Biologie et Ecologie de la Transmission dans le Mod& Schistosoma bovis: Implications dans le Contrde Biologique. Thesis, University of Marrakech, Morocco, and University of Perpignan, France. Mouahid, A. and ThCron, A. (1987). Schistosoma bovis: variability of cercarial production as related to the snail hosts: Bulinus truncatus, B. wrighti and Planorbarius metidjensis. International Journal for Parasitology 17, 1431- 1434. Mutani, A., Christensen, N.O. and Frandsen, F. (1983). Studies on the relationship between Schistosoma and their intermediate hosts. V. The genus Bulinus and Schistosoma bovis from Iringa, Tanzanie. Zeitschrft fur Parasitenkunde 69, 483487. Mwambungu, J.A. (1988). Transmission of Schistosoma bovis in Mkulwe (Mbozi District, Mbeya Region, Southern Highlands of Tanzania). Journal of Helminthology 62, 29-32. Ndifon, G.T., Betterton, C. and Rollinson, D. (1988). Schistosoma curassoni Brumpt, 1931 and S. bovis (Sonsino, 1876) in cattle in northern Nigeria. Journal of Helminthology 62, 33-34. Ngendahayo, L.D. (1989). Zdentijication Spkcifique des Schistosomes a m f s a Eperon Terminal d'Afrique Occidentale et Centrale. Thesis, University of Montpellier, France. Ngonseu, E., Greer, G.J. and Mimpfoundi, R. (1991). Dynamique des populations et infestation de Bulinus globosus en zone soudano-sahklienne du Cameroun. Annales de la SociPtP Belge de Mkdecine Tropicale 71, 295-306. Ouma, J.H. and Waithaka, F.T. (1984). Bulinus tropicus (Krauss, 1848) from Kenya found naturally infected with Schistosoma bovis. Annals of Tropical Medicine and Parasitology 78, 341 -342. Pitchford, R.J. (1977). A check list of definitive hosts exhibiting evidence of the genus Schistosoma Weinland, 1858 acquired naturally in Africa and the Middle East. Journal of Helminthology 51, 229-252. Ramajo-Martin, V. (1972). Contribucion a1 estudio epizootiolbgico de la esquistosomiasis bovino (Schistosoma bovis) en la Provincia de Salamanca. Revista Zbkrica de Parasitologia 32, 207-242. Ramajo-Martin, V. (1978). Observaciones acerca de la receptividad de diversas poblaciones de Planorbarius metidjensis, Bulinus (B.) truncatus y Biomphalaria glabrata a Schistosoma bovis de Espaiia. Revista Ibkrica de Parasitologia 38, 537549. Richards, C.S., Knight, M. and Lewis, F.A. (1992). Genetics of Biomphalaria glabrata and its effects on the outcome of Schistosoma mansoni infection. Parasitology Today 8, 171- 174. Rohde, K. (1994). The origins of parasitism in the platyhelminthes. International Journal for Parasitology 24, 1099- 1 1 15. Rollinson, D. and Wright, C.A. (1984). Population studies on Bulinus cernicus from Mauritius. Malacologia 25, 447-463. Rollinson, D., Southgate, V.R., Vercruysse, J. and Moore, P.J. (1990). Observations on natural and experimental interactions between Schistosoma bovis and S. curassoni from West Africa. Acta Tropica 47, 101- 1 14. Sampaio-Silva, M.L., Simon-Vicente, F., Avelino, I.C. and Ramajo-Martin, V.

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(1975). Susceptibility of Planorbarius metidjensis from Portugal and Spain to Schistosoma bovis from Salamanca (Spain). Revista IbPrica de Parasitologia 35, 131- 137. Schwetz, J. (1955). Recherches sur la bilharziose des bovides (Schistosoma bovis) dans le Haut-Ituri. Bulletin Agricole du Congo Belge 46,143-1454, Simon-Vicente, F., Sampaio-Silva, M.L., Ramajo-Martin, V. and Avelino, I. (1975). Susceptibility of Bulinus truncatus from Portugal and other origins to a strain of Schistosoma bovis of Salamanca (Spain). Revista Ibkrica de Parasitologia 35, 98103. Smithers, S.R. (1956). On the ecology of schistosome vectors in the Gambia with the evidence of their role in transmission. Transactions of the Royal Society of Tropical Medicine and Hygiene 50, 345-365. Sobrero, R. (1965). Bulinus (Physopsis) abyssinicus, ospite intermedio di Schistosoma bovis in Somalia. Ricostruzione del ciclo di vita del parassita. Parassitologia 7 , 4144. Sonsino, P. (1876). Intorno ad un nuova parassito del bue (Bilharzia bovis). Rendiconti della Accademia ScientiJca de Napoli 15, 84-87. Southgate, V.R. and Knowles, R.J. (1975a). Observations on Schistosoma bovis (Sonsino, 1876). Journal of Natural History 9 , 273-314. Southgate, V.R. and Knowles, R.J. (1975b). The intermediate hosts of Schistosoma bovis in Western Kenya. Transactions of the Royal Society of Tropical Medicine and Hygiene 69, 356-357. Southgate, V.R. and Knowles, R.J. (197%). Studies on Schistosoma bovis from different geographical areas. Proceedings of the Second European Multicolloquy of Parasitology, Trogir, pp. 135- 142. Southgate, V.R., Rollinson, D., Ross, G.C. and Knowles, R.J. (1980). Observations on an isolate of Schistosoma bovis from Tanzania. Zeitschrijt fur Parasitenkunde 63, 241-249. Southgate, V.R., Wright, C.A., Laaziri, H.M. and Knowles, R.J. (1984). Is Planorbarius metidjensis compatible with Schistosoma haematobium and S. bovis? Bulletin de la SociPtP de Pathologie Exotique 77, 499-506. Southgate, V.R., Brown, D.S., Rollinson, D., Ross, G.C. and Knowles, R.J. (1985a). Bulinus tropicus from Central Kenya acting as a host for Schistosoma bovis. Zeitschrijt fur Parasitenkunde 71, 61-69. Southgate, V.R., Rollinson, D., Ross, G.C., Knowles, R.J. and Vercruysse, J. (1985b). On Schistosoma curassoni, S. haematobium and S. bovis from Senegal: development in Mesocricetus auratus, compatibility with species of Bulinus and their enzymes. Journal of Natural History 19, 1249- 1267. Southgate, V.R., Brown, D.S., Warlow, A,, Knowles, R.J. and Jones, A. (1989). The influence of Calicophoron microbothrium on the susceptibility of Bulinus tropicus to Schistosoma bovis. Parasitology Research 75, 38 1-39 1. Touassem, R. and Jourdane, J. (1986). Etude de la compatibilite de Schistosoma bovis du Soudan et d'Espagne vis a vis de Bulinus truncatus de Tunisie et Planorbarius metidjensis du Maroc. Analyse comparke des tests de compatibilite utilisks. Annales de Parasitologie Humaine et ComparPe 61, 43 - 54. Van der Zwaan, G.J. (1982). Paleoecology of late Mediterranean foraminifera. Utrecht Micropaleontological Bulletins 25. Vera, C. (1991). Contribution ci I'Etude de la Variabilite GPnPtique des Schistosomes et de Leurs Hbtes IntermPdiaires: Polymorphisme de la CompatibilitP entre Diverses Populations de Schistosoma haematobium, S. bovis et S. curassoni et les Bulins Hstes Potentiels en Afrique de I'Ouest. Thesis, University of Montpellier, France.

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VCra, C . , Jourdane, J., Selin, B. and Combes, C . (1990). Genetic variability in the compatibility between Schistosoma haernatobium and its potential vectors ,in Niger. Epidemiological implications. Tropical Medicine and Parasitology 141, 121-224. Zar, J.H. (1984). Eiostatistical Analysis. Englewood Cliffs, New Jersey: Prentice-Hall, Inc.

The Larvae of Monogenea (Platyhelminthes) Ian D. Whittington’a2. Leslie A . Chisholm’ and Klaus Rohde3

’Department of Parasitology. The University of Queensland. Brisbane. Queensland 4072 Australia; Heron Island Research Station of The University of Queensland. Great Barrier ReeA via Gladstone. Queensland 4680. Australia; School of Biological Sciences. Division of Zoology. University of New England. Armidale. New South Wales 2351. Australia

Abstract ................................................................... 1. Introduction .............................................................. 2. General Morphology ..................................................... 2.1. Monopisthocotylea ................................................. 2.2. Polyopisthocotylea .................................................. 3. Haptoral Sclerites ........................................................ 3.1. Summary .......................................................... 4. Ciliated Cells ............................................................. 4.1. Monopisthocotylea ................................................. 4.2. Polyopisthocotylea .................................................. 4.3. Summary .......................................................... 5. Epidermis ................................................................ 5.1. Monopisthocotylea ................................................. 5.2. Polyopisthocotylea .................................................. 5.3. Summary .......................................................... 6. Terminal Globule ......................................................... 6.1. Monopisthocotylea ................................................. 6.2. Polyopisthocotylea .................................................. 6.3. Possible Functions .................................................. 6.4. Summary .......................................................... 7. Glands................................................................... 7.1. Monopisthocotylea ................................................. 7.2. Polyopisthocotylea .................................................. 7.3. Function ........................................................... 7.4. Summary .......................................................... ADVANCES IN PARASITOLOGY VOL 44 ISBN 0-12-031744-3

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8. Protonephridia . . . . . . . . , . , . , . . . . . . . , . . . . . . . . . . . . . , . . . . . . . . . . . . . . . 177 8.1. Monopisthocotylea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.78 8.2. Polyopisthocotylea.. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 180 8.3. Summary .......................................................... 182 . . . . . .. 183 9. Sense Organs.. . . . . . . . . . .. .. . . . . , , , . . .. . .. .. . .. .. .. . . . 9.1. Eyes ............................................................... 183 9.2. Other Sense Organs.. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. 191 10. Nervous System.. , . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. 198 11. Digestive Tract ........................................................... 200 201 12. Parenchyma.............................................................. 13. Behaviour................................................................ 203 13.1. Role of the Oncomiracidium.. . . . . . . . . . . .. . . . . . . . . 203 13.2. Emergence of Oncomiracidia from Eggs . . . .. . . . . . . . . . . . . . 205 207 13.3. Swimming Behaviour.. ............................................ 13.4. Host Finding and Host Recognition . . . . . . . . . . . , . . . . . . . . . . . . . . 211 13.5. A Dispersal Phase? ................................................. 213 , . . . . . . . . . . . , . , 213 13.6. Invasion of Hosts by Oncomiracidia.. . . . . , , . 13.7. Summary ........... .............................................. , 215 14. Conclusions .........................,.............................. ...... 215 218 Acknowledgements .......................................................... References.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , , , . . . . . . . . . . . 218

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ABSTRACT There has been no comprehensive review of the infective larval stage (oncomiracidium) in the direct life-cycle of monogeneans since Llewellyn (1963, 1968). In the last 30 years, knowledge of the general anatomy and morphology of oncomiracidia has increased significantly as has information on swimming behaviour and egg-hatching strategies that may enhance chances of host infection. Nevertheless, oncomiracidia are known for only a small proportion of monogenean species described. This review consolidates established, and summarizes new, knowledge since Llewellyn’s work and integrates light- and electron-microscopy studies including unpublished data. Currently there is considerable debate, fuelled largely by phylogenetic studies using molecular techniques, about whether or not the class Monogenea (comprising subclasses Monopisthocotylea and Polyopisthocotylea) is monophyletic. This challenges established views that Monopisthocotylea and Polyopisthocotylea form a single clade based on two larval characters: two pairs of rhabdomeric eyes; three bands of ciliated cells. In an attempt to reveal further synapomorphies for the entire Monogenea (or provide evidence against its monophyly) or possibly for the Monopisthocotylea and Polyopisthocotylea only, we review the following larval features: haptoral sclerites; ciliated cells; epidermis; terminal globule; gland, protonephridial and nervous systems; sense organs; digestive tract; parenchyma;

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and behaviour. Conclusions are equivocal but indicate that further larval studies, especially ultrastructural, are necessary to assess: the presence or absence of ‘false’ vertical rootlets of epidermal cilia; tapering epidermal cilia; the protonephridial system; the presence or absence of a terminal globule; glands and their secretions; and the embryology and chemical composition of haptoral sclerites. Future integration of light- and electron-microscopy studies are likely to be particularly informative.

1. INTRODUCTION

The Monogenea are parasitic flatworms most of which inhabit the gills, skin and fins of fish. Some have adopted a mesoparasitic way of life infecting, for example, the urinary bladders and cloaca1 bursae of turtles or the digestive tract or coelom of fish (Kearn, 1994). Monogenea are hermaphroditic and have a direct life-cycle, although circumstantial evidence suggests that two marine species of Polyopisthocotylea may have indirect life-cycles using small pelagic fish as intermediate hosts (Bychowsky and Nagibina, 1967). Because of the direct life-cycle, many species of monogeneans are of great economic importance, particularly in aquaculture. The oncomiracidium is the larval stage of monogeneans and has the task of locating, attaching and establishing itself on the host. For this reason, studies of its morphology and behaviour are important. The monograph by Bychowsky (1957, English translation 1961) contains a section on oncomiracidia and, although larvae of less than 30 species had been described at the time, it remains a useful source of information. Numerous studies on oncomiracidia were carried out between 1955 and 1963, and two excellent reviews by Llewellyn (1963, 1968) compiled information about the larvae of more than 100 species. Llewellyn (1972; see also Kearn, 1981) reviewed the behaviour of oncomiracidia. Even three decades since the last major reviews of the estimated 3000-4000 species of Monogenea that have been described (Whittington, 1998) and the 20 000 or more species that have been estimated to exist (Rohde, 1996; Whittington, 1998), only a small proportion of their larvae have been studied. Undoubtedly this paucity of information on the larvae of monogeneans relates to the relative difficulties in obtaining live adult parasites, and collecting and hatching their eggs. Even when larvae are hatched, the majority are minute (<300 pm in length) and cilia, which are usually present, can propel them rapidly. Therefore, mounting them for examination on a microscope, fixing them for ultrastructural studies or observing their behaviour can be a frustrating and time-consuming process. Despite these difficulties, patient investigators have persevered and our knowledge on the morphology, ultrastructure and

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behaviour of oncomiracidia has expanded considerably since the reviews of Llewellyn (1963, 1968, 1972). The major aim of this review is to summarize our accumulated knowledge of oncomiracidia and indicate where future studies will fill the gaps in our present understanding. We include some unpublished data, especially on ultrastructure. The classification used in this review mostly follows that of Yamaguti (1963) and the numbering of hooklets follows the scheme proposed by Llewellyn (1963). One family of monogeneans, the Gyrodactylidae, comprises 22 genera, 18 of which are viviparous. An ‘oncomiracidium’ of only a single species of oviparous gyrodactylid, Oogyrodactylus farlowellae, has been described (Harris, 1983). Viviparous gyrodactylids produce juveniles that resemble the adults. We discuss only a few aspects of this family because of their phylogenetic interest. Recent DNA studies have shown almost consistently that the two major groups within the Monogenea, the Monopisthocotylea and Polyopisthocotylea, may not be monophyletic (Blair, 1993; Rohde et al., 1993; Mollaret et al., 1997; Litvaitis and Rohde, 1999; Littlewood et al., 1999). Ultrastructural studies of sperm and spermiogenesis have also failed to find evidence for their monophyly (Justine, 1991a,b). This contradicts the established view of the monophyly of Monogenea based on two synapomorphies: the presence of two pairs of rhabdomeric eyes and three bands of ciliated cells in oncomiracidia. Another goal of this review which summarizes previous, and offers new, information is to uncover, perhaps, further synapomorphies for the Monogenea as a whole (or provide evidence against the monophyly of the group), and possibly for Monopisthocotylea and Polyopisthocotylea.

2. GENERAL MORPHOLOGY 2.1. Monopisthocotylea

The oncomiracidium of the capsalid monogenean Entobdella soleae (see Kearn, 1963a, 1974a) illustrates the basic morphology of larval monopisthocotyleans (Figure IA). The haptor is armed with seven pairs of hooklets, all with a domus, a pair of accessory sclerites, one pair of anterior hamuli and one pair of posterior hamuli. Three distinct bands of ciliated epidermal cells, which contain refringent droplets, are present: one on the anterior and median portions of the body proper, and one on the haptor. A terminal globule is absent. Four anteromedian gland cells that contain granular secretion open at the anterior tip of the larva (Figure IA). Immediately posterior to these cells but anterior to the eyes is a large mass of gland cells which contain needle-like secretion; ducts lead from these gland

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Figure I A Oncomiracidium of Entobdella soleae (Monopisthocotylea: Capsalidae), ventral view. Scale bar = 100 pm. (Redrawn and modified from Kearn, 1974a).

cells and open on the anterolateral margin of the head. Ducts from groups of gland cells, which contain needle-like secretion, are located on either side of the pharynx and they also open on the anterolateral margin of the larva (Figure 1A). Post-pharyngeal glands containing granular secretion are located around the anterior part of the gut and ducts from these glands lead into the posterior part of the pharynx. There are two pairs of lateral body gland cells that contain granular secretion in the posterior part of the body proper. Two additional pairs of gland cells in the most posteromedian part of the body contain granular secretion and have ducts that extend into and open on to the haptor (Figure 1A). The larva of E. soleae has a total of nine pairs of flame bulbs: two pairs anterior to the eyes, two pairs lateral to the pharynx, one pair lateral to the gut and posterior to the excretory bladders, and four pairs, not distributed symmetrically, in the haptor. Excretory bladders open laterally at a level posterior to the pharynx

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Figure 1 B Oncomiracidium of Plectunocotyle gurnardi (Polyopisthocotylea: Plectanocotylidae), ventral view. Scale bar = 50 pm. (Redrawn and modified from Whittington and Kearn, 1989). ag = anterior gland cells; ah = anterior hamulus; alg = gland cells associated with gut; amg = anteriomedian gland cells; acc = accessory sclerite; b = small body (eye lacking pigment shield?); bgc = body gland cell; bl = excretory bladder; c = cilia; cc = ciliated cone; ce = ciliated epidermis; ci = ciliary eye; d = domus; e = pigment-shielded eye; ep = excretory pore; f = flame bulb; g = gut; h = hooklet; h I = hooklet I; h VI = hooklet VI; ha = haptor; lgc = lateral gland cells; m = mouth; ph = pharynx; phg = pharyngeal gland cells; poh = posterior hamulus; pmg = posterior median gland cells; rd = refringent droplets; tg = terminal globule. Note that the dotted connection between the posterior capillaries of the protonephridial system in P. gurnardi denotes that the transverse commissure was not always visible in live larvae.

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(Figure IA). Note that the left and right anterior capillaries are fused and the posterior ones are connected by a transverse commissure (Figure IA). The larva possesses two pairs of pigment-shielded eyes situated dorsally. The anterior pair are smaller than the posterior pair and each eye has a permanent crystalline lens (Figure 1A). The mouth is located just anterior to the pharynx. The gut has two branches which are fused posteriorly to form a ring. Refringent droplets are distributed throughout the body and haptor (Figure 1A). The distribution, morphology and number of sclerites in the haptor of monopisthocotylean larvae is extremely variable (see p. 146). Hamuli and/or accessory sclerites may be present or absent and the number of hooklets varies (Llewellyn, 1963; see p. 148). The larvae of some monopisthocotyleans are unciliated and eyes may also be absent (see p. 185). The distribution and contents of gland cells differ (see p. 170), and the number and distribution of flame bulbs are variable (see p. 178). The gut can be unbranched or perhaps absent altogether, although it is more likely that, in some oncomiracidia, the gut is especially difficult to see. Oliver (1994) and Kearn et al. (1993) described some abnormalities in monopisthocotylean larvae (abnormal number of hooks, abnormal eyes), but reports are rare.

2.2. Polyopisthocotylea

The oncomiracidium of Plectanocotyle gurnardi (see Whittington and Kearn, 1989) illustrates the general characteristics of polyopisthocotylean larvae (Figure 1B). This larva is armed with six pairs of hooklets; pairs II-VI have a domus and are similar in shape and size, while pair I is larger with no domus. The ciliated epidermal cells, which do not contain refringent droplets, form two bands: one continuous along the lateral margins of the body proper, and the other, on the posterior border of the haptor, forms a ciliated cone (Figure 1B). The cone terminates in a non-ciliated terminal globule. Ducts from two pairs of clusters of gland cells lateral to the pharynx lead to the anterior margin. Small gland cells were seen in the pharynx of some specimens. The above gland cells all produce granular secretion. There is a total of three pairs of flame bulbs: one pair just posterior to the eyes, one pair lateral to the pharynx and one pair located in the haptor. They are connected to excretory capillaries which, in turn, open into the large excretory ducts (Figure 1B). Note that the anterior capillaries are not fused, whereas the posterior capillaries of both sides of the body are thought to be connected by a transverse commissure, although in P. gurnardi this was not always visible (I.D. Whittington, unpublished observation; Figure 1B). Excretory pores open laterally at the level of the posterior end of the

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pharynx. One pair of medially fused pigment-shielded eyes is present. Each eye has a spherical refringent droplet associated with it. In some specimens a second smaller refringent droplet is located between the larger droplet and the lateral margin of the larva (Figure 1B). The mouth is terminal, and no connection between it and the pharynx was seen. A sac-like gut, which is sometimes lobed, is present. Smaller refringent droplets were seen throughout the posterior part of the body and in the haptor (Figure 1B). The distribution, morphology and number of sclerotized structures, including the presence of clamps, in the haptor of polyopistocotylean larvae is extremely variable depending on the species and family (see below) and Llewellyn (1963) discussed these differences in detail. Polyopisthocotylean larvae may be unciliated and sometimes a group of long, non-motile cilia (sensory?) may be present at the anterior end (see p. 195). The terminal globule is absent in some species (see p. 167). Eyes are absent in some species but in others, two pairs of pigment-shielded eyes are present (see p. 188). Also, the anterior excretory capillaries may be fused (Rajonchocotyle emarginata and Hexabothrium appendiculatum; see Whittington, 1987a).

3. HAPTORAL SCLERITES

Opinions differ about whether some of the hard parts on the haptor of Monopisthocotylea and Polyopisthocotylea are homologous (e.g. Pariselle and Euzet, 1995; and explained further below). For convenience, however, monopisthocotyleans and polyopisthocotyleans are dealt with together here. The presence of ‘hooks’ (hard proteinaceous ‘sclerites’) distinguishes the oncomiracidia of monogeneans (onkos is Greek for ‘hook’) from the hookless miracidia of digeneans. These sclerotized elements are present in the form of ‘marginal hooklets’, accessory sclerites, hamuli (Llewellyn, 1963, 1968) and, in some polyopisthocotyleans, oncomiracidia may hatch with one pair of clamps already developed (e.g. Bovet, 1959, 1967; Owen, 1970; Thoney, 1986; Roubal and Diggles, 1993). There has been considerable discussion among specialists about the terminology of the haptoral sclerites. The most common haptoral sclerites in oncomiracidia are the ‘marginal hooklets’ (Llewellyn, 1963, 1968) but, here, we prefer and recommend use of the word ‘hooklet’ because not all of these sclerites remain at the margin of the haptor in larvae and adults (e.g. Llewellyn, 1963, 1970). It is important to note that different authors have used different schemes to number the hooklets (Lambert, 1980a) but the most widely accepted scheme is numbering them using Roman numerals in a posterior to anterior direction (Figure 2), as proposed by Llewellyn (1963) and adopted at ICOPA IV (Euzet and Prost, 1981). The central haptoral elements in capsalids are

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Figure 2 Numbering of hooklets of oncomiracidia in a posterior to anterior direction using Roman numerals according to Llewellyn (1963).

called accessory sclerites and are considered to be modified hooklets of pair I (Kearn, 1964). Hamuli (singular: hamulus) are the large sclerites that occur in one or two pairs, and in adult monogeneans they occupy a position between hooklets I1 and I11 (Llewellyn, 1963, 1968, 1970). The hamuli are primarily post-oncomiracidial developments but they are present in some larvae between hooklets I1 and I11 (e.g. in capsalids, Kearn, 1974a; in some dactylogyrids, Lambert, 1975, 1977; in microcotylids, Llewellyn, 1963; in axinids, Euzet and Ktari, 1970a; in polystomatids, Llewellyn, 1963, 1968). Among oncomiracidia of some polystomatids, one of the two pairs of sclerites interpreted as hamuli in Protopolystoma xenopodis (see Thurston, 1964; Tinsley and Owen, 1975) and Polystomoides spp. (see Lambert et al., 1978; Lambert and Kulo, 1982) and a single pair of sclerites interpreted as hamuli in Concinnocotyla australensis ( S . Pichelin and I.D. Whittington, unpublished observation) are present between hooklets I and 11. Further investigations on the haptors of larval polystomatids are required to resolve this apparent departure from the schemes proposed and refined by Llewellyn (1963, 1968, 1970, 1982). Sclerites which may be hamuli have been reported in other oncomiracidia (e.g. in the monocotylid Dictyocotyle coeliaca by Kearn, 1970) but these structures may fail to persist in adults. The appearance and development of hamuli in post-oncomiracidia was referred to by Llewellyn (1968) and has been described by, for example, Kearn (1968a,b), Lambert (1975, 1977) and Cone (1979a). The migration of the hooklets in the haptor of Pseudodactylogyroides marmoratae (Ancyrocephalidae) described by Lim (1995) differs from accounts in other monogeneans and requires more study. Alternate terminology for oncomiracidial haptoral sclerites was proposed recently by Pariselle and Euzet (1995): they suggested the term

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‘uncinuli’ (singular: ‘uncinulus’) for hooklets which they claim persist in adult monopisthocotyleans but which supposedly disappear in adult polyopisthocotyleans; the large, hook-like sclerites in the larvae of polyopisthocotyleans (modified ‘marginal’ hooklets I of Llewellyn, 1963) are termed ‘hamulus/hamuli’ and these persist in the adults of some species on the terminal lappet; ‘gripus’ is used for the ‘large hooks’ (hamuli of Llewellyn, 1963) of the Dactylogyridea that descend from the posterior part of the body proper in post-oncomiracidia (e.g. Lambert, 1977; Cone, 1979a). However, Pariselle and Euzet (1995) state that further studies of post-oncomiracidial development may indicate that ‘gripus’ should be applied to the large sclerites ( = hamuli of Llewellyn, 1963) of all monopisthocotyleans. It is the opinion of Pariselle and Euzet (1995), therefore, that the ‘large median sclerites’ ( = hamuli of Llewellyn, 1963) in the Monopisthocotylea and Polyopisthocotylea are unlikely to be homologous because their origin, structure and development between the two groups may differ. The modified hooklets I of some polyopisthocotyleans, i.e. modified ‘marginal hooklets’ I of Llewellyn (1963) and the ‘hamuli’ of Pariselle and Euzet (1995), are usually larger and a different shape from the other hooklets (Diclidophora spp., see Macdonald, 1977; Heteraxine heterocerca, Kearn et al., 1992a; Plecanocotyle gurnardi, see Whittington and Kearn, 1989, Figure 1B; Microcotyle sebastis and Heteraxinoides xunthophilis, see Thoney, 1986, 1988). They lack a domus (see below) but Owen (1970) stated that the relatively large hooklets of the larva of Discocotyle sagittata ‘appear also to possess a domus’. Generally, however, all of the hooklets of polystomatid larvae (e.g. Llewellyn, 1963; Tinsley and Owen, 1975; Lambert et al., 1978; Tinsley, 1978) and of the Monopisthocotylea possess a domus. Further studies are needed to examine the embryology and the ontogeny of hooklets in monogenean larvae. In particular, the possible importance and significance of hooklets without a domus must be assessed because consistent differences may support the concept of Pariselle and Euzet (1995) about the non-homology of some of these structures. The number of hooklets on the haptor of oncomirdcidia is of evolutionary and taxonomic significance (Bychowsky, 1957; Llewellyn, 1963) and ranges from 10 (e.g. in hexabothriids, Whittington, 1987a) to 14 (e.g. in capsalids, Kearn, 1974a, Figure 1A; monocotylids, Chisholm and Whittington, 1996a) up to 16 (e.g. in acanthocotylids, Kearn, 1967a; enoplocotylids, Kearn, 1993a; polystomatids, Pichelin, 1995a). Some controversial interpretations claim that selected taxa may have 18 hooklets (Malmberg, 1990) but this scheme is not widely accepted. Llewellyn (1963) has given a detailed account of the hooklets of larval monogeneans which remains useful and to which we refer the reader.

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Hooklets and hamuli are scleroproteins (not chitinous) of a keratinous nature (Lyons, 1966). The hooklets are the first sclerites to form during embryology (Lyons, 1966). The little evidence available on hooklet embryology indicates that they form intracellularly inside specialized cells called oncoblasts (reported in a capsalid, a polystomatid, a diclidophorid, a gyrodactylid and a hexabothriid; Wiskin, 1970). However, the tanned egg shell and the presence of vitelline cells around the embryonating larva has undoubtedly restricted studies on embryogenesis. Further studies on the embryonic development of hooklets and, indeed, other larval haptoral sclerites are desirable. Several monopisthocotylean families possess especially characteristic hooklets that are 'hinged'. The junction between the shaft and the 'sickle' (i.e. combined hooklet tip, blade and guard; Llewellyn, 1963) of the hooklet forms a hinge allowing the sickle to move through more than 180". Families that have hinged hooklets are the Acanthocotylidae (see Kearn, 1967a), Anoplodiscidae (see Ogawa and Egusa, 198la), Bothritrematidae (see Malmberg, 1990), Enoplocotylidae (see Kearn, 1993a), Gyrodactylidae (see Harris, 1983), Sundanonchidae (see Malmberg, 1990) and Tetraonchoididae (see Malmberg, 1990). The significance of hinged hooklets is unknown but a controversial evolutionary scheme of the Monogenea (by Malmberg, 1990) grouped these families together as the subclass Articulonchoinea. It should be noted that the oncomiracidia of the Bothritrematidae, Sundanonchidae and Tetraonchoididae are unknown. Hooklets are usually independently active and can flex (Figure 3) which would force these small sclerites into host epithelial tissue to secure attachment by the oncomiracidium to the host (Figure 4). The muscles and associated structures that must operate the hooklets have been termed an ogive or more commonly a domus (Llewellyn, 1963) but there has been a surprising lack of attention paid to these structures. Llewellyn (1963) considered that the domus was a dome-shaped sclerite, but this is probably because the hooklets, and especially the blade, are likely to be surrounded in a sheath of musculature that appears sclerotized. Few ultrastructural studies have been made on hooklets. Kearn and Gowing (1990) used transmission electron microscopy (TEM) to demonstrate the presence of hooklets in the oncomiracidium of the microbothriid Leptocoryle minor [compare Llewellyn (1963) and Llewellyn (1968) for discussions about haptoral sclerites in the Microbothriidae]. Each hooklet consists of a solid rod of homogeneous electron-dense material enclosed in one or more cells that may be oncoblasts. El-Naggar (1992) made a detailed electron microscopic study of the hooklets of the adult monopisthocotylean Cichlidogyrus halli typicus (Dactylogyridae). It is likely that they are identical with the hooklets of the oncomiracidium and the findings are therefore discussed here briefly. Hooklets consist of a handle, a curved blade with a tapering guard and an

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Figure 3 Hooklets of Kuhnia sprostonae (Polyopisthocotylea: Mazocraeidae) showing how the shaft can flex. Scale bar = 10 pm. (I.D. Whittington, original photograph.)

accessory domus. Most hooklets studied by El-Naggar (1992) had a long extension attached to the proximal end of the handle. The handle has three layers: the guard and the proximal region of the blade lack the outer electron-dense layer. The dense core is lacking in the rest of the blade. The different hooklet layers may reflect differences in chemical composition (El-Naggar, 1992), a possibility that has not been explored and chemical staining of the kind used by Lyons (1966) may be useful. The hooklet extension in C. halli typicus is of a fibrous nature. A distinct cavity partly lined by a syncytium containing secretory granules is found around each hooklet. The domus consists of the thickened edges of a single gutter-shaped sclerite. El-Naggar (1992) considered that this sclerite may provide insertion points for myofibres, but the musculature and functional morphology have not been studied. Our own TEM studies of hooklets (K. Rohde, unpublished data) in the larvae of the monopisthocotyleans Encotyllabe chironemi (Capsalidae) and Neoheterocotyle rhinobatidis (Monocotylidae)

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Figure 4 Oncomiracidium of Acanthocotyle lobianchi (Monopisthocotylea: Acanthocotylidae) attached to host ray skin by hooklets (arrowhead) within minutes of hatching. Scale bar = 25 pm. (I.D. Whittington, original photograph.)

(Figure 9, and the polyopisthocotylean Zeuxapta seriolae (Axinidae) have shown that the hooklets and domus are similar ultrastructurally to that described above. Further studies may show whether there are differences between various taxa, and between the Monopisthocotylea and Polyopisthocotylea.

3.1. Summary

The above account of oncomiracidial hooklets, and larval sclerites in general, demonstrates that there is still much to determine about their origin, embryology, development, chemical nature, functional morphology and homology. To test the hypothesis of Pariselle and Euzet (1999,

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Figure 5 Transmission electron micrograph of the haptor of a larval Neoheterocotyle rhinobatidis (Monopisthocotylea: Monocotylidae). Note hooklets (h) with granular core (c), domus (d) and cavities around hooks. Also note densely packed electron-lucent granules (g) and some scattered electron-dense secretory granules (s). Scale bar = 1 pm.(K. Rohde, original photograph.)

additional studies on the embryology of oncomiracidia, and on postoncomiracidial development of monopisthocotyleans and polyopisthocotyleans are necessary. All of these areas must be explored further if we are to address homologies between the Monopisthocotylea and Polopisthocotylea, and identify possible synapomorphies for these subclasses and/or for the Monogenea as a whole.

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4. CILIATED CELLS

The distribution of the ciliated cells on larvae has proven to be a useful character to distinguish between monogenean taxa. Ciliated cells can be revealed by staining live larvae with silver nitrate, which has an affinity for the boundaries between epidermal cells (Figure 6 ) (see also Lambert, 1980a,b). Staining freshwater species is a relatively easy process but studies of marine species are more difficult because silver nitrate reacts with

Figure 6 Ventral surface of silver stained oncomiracidium of Neoheterocoryfe rhinobaridis (Monopisthocotylea: Monocotylidae) showing ciliated cells, spaces between cells (arrow) and mouth (arrowhead). Scale bar = 50 pm.(L.A. Chisholm, original photograph.)

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chloride ions in seawater to form a thick precipitate of silver chloride, making small larvae almost impossible to locate. To combat this problem, marine larvae must be washed rapidly in distilled water before the stain is added (Lambert, 1980a) and rinsing must be completed quickly because the stain is ineffective on dead larvae. Llewellyn (1963) identified only 11 monogenean species (ten monopisthocotyleans and one polyopisthocotylean) whose distribution of larval ciliated cells had been mapped and, not surprisingly, the majority of these were freshwater representatives. It is due primarily to the careful work of Lambert and colleagues (e.g. Lambert, 1980a,b and references therein, 1981, 1988; Le Brun et al., 1986) on a range of taxa and for polystomatids by Tinsley and colleagues (e.g. Tinsley and Owen, 1975; Tinsley, 1976, 1981, 1983; Cable and Tinsley, 1992) that the number and distribution of larval ciliated cells of over 60 species are now known.

4.1. Monopisthocotylea

The ciliated cells are distributed in three major zones: the anterior zone, two symmetrical regions around the median zone of the body, and the haptoral zone (e.g. Figure 6). Lambert (1980b) summarized the findings of his earlier studies and listed the number of ciliated cells of 14 species of monopisthocotyleans (representing seven families) showing the striking similarity between the number of ciliated cells in each zone. The Ancyrocephalidae, Calceostomatidae, Dactylogyridae, Diplectanidae and Tetraonchidae all have 27 cells in the anterior zone, 10 cells on each side of the median zone and 12- 14 cells in the posterior zone, and the ciliated cells are not joined (Figure 7A) (see also Cone, 1979b; Vladimirov and Gusev, 1986). Larvae in the Capsalidae (Benedenia rnonticelli, EntobdelEa soleae and Trochopus pini, Lambert, 1980b; Nitzschia sturionis, Gusev and Timofeeva, 1986) also have 27 cells in the anterior zone and 10 cells on each side of the median zone, but there are 17 cells in the posterior zone and the cells within each zone are joined (Figure 7B). Gusev and Timofeeva (1986) also saw an additional pair of cells on the ventral surface of N . sturionis near the mouth that have not been described in any other capsalid. Lambert (1980b) chose the larva of Calicotyle kroyeri (Calicotylinae) to represent the Monocotylidae and illustrated 25-28 cells in the anterior zone, 11 on either side of the median zone and 17 in the posterior zone (Figure 7C). As reported for the Capsalidae, the cells abut in each region. Recently, Chisholm (1998) examined seven additional species of monocotylids (from five subfamilies) and discovered that, although the distribution and number (17) of ciliated cells are consistent in the posterior zone, the number and pattern of ciliated cells in the anterior

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I

I -1

0

I 17

I -1

12

A

0 -

- 14

B

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Figure 7 Ciliated cells of larval Monopisthocotylea. Dorsal (top) and ventral (bottom) views. A. Ancyrocephalidae, Calceostomatidae, Dactylogyridae, Diplectanidae and Tetraonchidae. B. Capsalidae. C. Monocotylidae. (Redrawn from Lambert, 1980b.)

and/or the median zones are unique for some subfamilies (Figure 8). Additional species from each subfamily need to be examined to determine whether these differences are consistent. Chisholm (1998) also found a separate pair of anterior ciliated cells on the dorsal surface of each monocotylid species but similar cells were not described in C . kroyeri (compare Figures 7C and 8). These cells probably support the non-motile cilia, which have been described at the anterior end of many monocotylid larvae (see Figures 8 and 15) (Chisholm and Whittington, 1996a), but were not described in C . kroyeri (Kearn, 1970). Furthermore, Chisholm (1998)

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A

w w B

C

Figure 8 Ciliated cells of larvae representing five subfamilies of the Monocotylidae (Monopisthocotylea). Dorsal (top) and ventral (bottom) views. A. Dasybatotreminae, Decacotylinae and Heterocotylinae. B. Merizocotylinae. C. Monocotylinae. Arrow indicates space between ciliated cells. (Redrawn from Chisholm, 1998.)

discovered small spaces between some of the ciliated cells on both the dorsal and ventral surface of the anterior zone (Figures 6 and 8), but these spaces were not noted in C. kroyeri or in other larvae where the ciliated cells abut. The distribution of ciliated cells on the larva of the enigmatic Euzetrema knoepfleri (Iagotrematidae) differs considerably from other monopistocotyleans and, interestingly, shows convergence with the Polystomatidae (see below). There are a total of 62 ciliated cells distributed in seven groups (Lambert, 1980b).

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4.2. Polyopisthocotylea

The ciliated cells of the Polyopisthocotylea are much more diverse in both numbers and distribution. Lambert (1980b) identified three general ciliated cell types for the Polyopisthocotylea. The first type is found in the parasites of teleosts including Gastrocotyfe trachuri (Gastrocotylidae) (Figure 9A), Mazocraes sp. (Mazocraeidae) (Figure 9B), Cycfocofyfabellones (Diclidophoridae) (Figure 9C) and Diplozoon paradoxurn (Discocotylidae) (Figure 9D). The second type is characteristic of the Hexabothriidae and the third type is typical of the Polystomatidae. In the first type, there is a group of three or four ciliated cells that are not joined (which may be absent in Mazocraes sp.) in the anterior zone. In the median zone, a group of usually 12 ciliated cells, which are joined, extend down each side of the larva. A group of 12 or 13 cells, all joined, are found in the posterior zone. Further variation of this arrangement is seen in some species in the Microcotylidae, in D . paradoxurn (Diplozooidae) and in Discocotyle sagittata (Discocotylidae) (Owen, 1970),

Figure 9 Ciliated cells of larval Polyopisthocotylea from teleosts. Dorsal (top) and ventral (bottom) views. A. Gastrocotyle trachurii. B. Mazocraes sp. C . Cyclocotyla bellones. D. Diplozoon paradoxurn. (Redrawn from Lambert, 1980b.)

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where the lateral cells in the median zone are further divided into two groups, the anteromedian and posteromedian groups (Figure 9D). Gusev and Slusarev (1986) described ciliated cells of Diclybothrium armatum (Diclybothriidae), which are unlike all other polyopisthocotylean larvae. None of the ciliated cells are joined and there is an additional group of small ciliated cells on the mid-ventral surface. They suggested that other polyopisthocotyleans, which were considered primitive (e.g. the Chimaericolidae), should be examined. Lambert (1980b) stated that the oncomiracidium of Callorhynchicola multitesticulatus (Chimaericolidae) was unciliated but Beverley-Burton et al. (1993) demonstrated, using scanning electron microscopy (SEM), that one anterior, four median and one posterior groups of ciliated cells are present. Closer examination of the electron micrographs has shown that a similar zone of cilia is present on the mid-ventral surface of the larvae of C. multitesticulatus as described by Gusev and Slusarev (1986) for D . armatum but, unfortunately, the number and distribution of ciliated cells cannot be determined (L.A. Chisholm and M. Beverley-Burton, unpublished data). Silver staining of larvae of C. multitesticulatus to reveal the ciliated cells was unsuccessful (L.A. Chisholm, unpublished data). The ciliated cells of only a single species of hexabothriid, Erpocotyle catenulata (Figure lOB), have been examined so it is difficult to draw conclusions regarding its relationship to other polyopisthocotylean taxa despite the fact that Lambert (1980b) distinguished this as a distinct type. Its pattern of ciliated cells is similar to the Polystomatidae (Figure 10A) because the cells in the median zone extend transversely on to the dorsal surface. However, unlike the Polystomatidae, the ciliated cells are joined except for a single cell in the anterior and median zones (Lambert, 1980b; Figure 10B). Whittington (1987a), however, used SEM to visualize the distribution of the

’ B

Figure 10 Ciliated cells of larval Polyopisthocotylea. Lateral views. A. Polystomatidae. B. Hexabothriidae. (Redrawn from Lambert, 1980b.)

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ciliated cells on the larva of another hexabothriid, Rajonchocotyle emarginata. He reported that the ventral surface of the larva is ciliated and that the haptor bears an extensive covering of ciliated cells, therefore differing from that described for E. catenulata. Whittington (1987a) suggested that the distribution of ciliated cells in the Hexabothriidae may be more heterogenous than Lambert (1980b) implied. These results also highlight the dangers of using a single exemplar species to characterize an entire family. The distribution of ciliated cells in 10 of the 17 genera of the Polystomatidae have been examined (Ozaki, 1935; Bychowsky, 1957; Combes, 1968; Thurston, 1968; Lamothe-Argumedo, 1973; Maeder, 1973; Tinsley and Owen, 1975; Tinsley, 1976; Vande Vusse, 1976; Combes et al., 1978; Lambert and Bourgat, 1978; Lambert et al., 1978; Salami-Cadoux, 1978; Lambert and Kulo, 1982; Tinsley, 1981, 1983; Cable and Tinsley, 1992; Pichelin, 1995a,b). The ciliated cells are not joined (Figure 10A) and the total number varies from 55 to 64. They are distributed in five distinct groups and Tinsley (1981) outlined the cell patterns in each of the genera. He concluded that the distribution of ciliated cells is a conserved character because the pattern of ciliated cells on the larva does not distinguish taxa that are clearly different using adult morphological characters. Among polystomatids, there are reports that adults of Eupolystoma alluaudi (Fournier and Combes, 1979) and Polystomoides nabedei (Lambert and Kulo, 1982) lay two types of eggs that may give rise to ciliated or unciliated oncomiracidia.

4.3. Summary

Silver staining of live larvae is a difficult technique and it is not surprising that, in most cases, only a few species have been chosen to represent each family. However, results from Chisholm (1998) and Whittington (1987a) indicate that examination of more species within a single family may reveal additional characters, which will enhance further the trends discovered by Lambert (1980b) within the Monopisthocotylea and Polyopisthocotylea, respectively.

5. EPIDERMIS

Studies on the outer body covering of adult monogeneans show that they, like other Neodermata, have a living surface layer known as the tegument or neodermis (Tyler and Tyler, 1997). The outer body covering of oncomiracidia is an epidermis which is often ciliated. Few TEM studies have been made of the body surface of monogenean larvae. The only published

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accounts of the epidermis of monopisthocotylean larvae include those on Entobdella soleae (Capsalidae), Euzetrema knoepfleri (Iagotrematidae), Monocotyle spiremae and Neoheterocotyle rhinobatidis (Monocotylidae), and those on polyopisthocotylean larvae are confined to a few species of Polystomatidae and Zeuxapta seriolae (Axinidae). 5.1. Monopisthocotylea

Lyons (1973a; see also the brief review by Threadgold, 1984) gave a detailed description of the epidermis in the oncomiracidium of Entobdella soleae (Figure 11). The ciliated cells are thin (1.5-2.0 pm) and are approximately 20 pm in diameter. They contain droplets of triglyceride but only little glycerine. The cilia are 16 pm long, are located in shallow pits and have long rootlets which are anteriorly directed. The mitochondria are well developed and they occupy a zone between the rootlets (Figure 11). Nuclei of the epidermal cells are lost at hatching (Figure 11C). The cytoplasm between the ciliated cells is considered to be probably syncytial and its nuclei are perhaps lost as well. During development, cells in the parenchyma send cytoplasmic processes that form the tegument of the adult beneath the ciliated cells and may fuse with the interciliary syncytial cytoplasm (Figure 1IC). The ciliated cells are shed within 30 seconds of the oncomiracidium infecting its host (Kearn, 1967b). The discontinuous subepidermal tegument now forms a continuous syncytium around the post-oncomiracidium. Fournier (1976) examined the larva of Euzetrema knoepfleri from the bladder of an amphibian and found nucleated ciliated cells. Studies of the oncomiracidia of two monocotylids, Neoheterocotyle rhinobatidis and Monocotyle spiremae by Rohde et al. (1998) showed that the cilia on the epidermal cells have two rootlets, a well-developed horizontal rootlet and a much less-developed vertical rootlet. However, contrary to the vertical rootlets of turbellarians, which originate from the basal bodies (Rohde et al., 1998), in these monocotylids they originate from the basal parts of the horizontal rootlets. It was considered unlikely that they were homologous in the two groups and were therefore termed ‘false vertical rootlets’ in the monocotylid species (Rohde et al., 1998). The epidermis of the larva of Encotyllabe chironemi has been shown by SEM to have an elaborate system of wrinkles and microvilli on the surface (Figure 12C) (K. Rohde, unpublished data). 5.2. Polyopisthocotylea

Dupouy (1975) examined the ultrastructure of the epidermis of larval Polystoma integerrimum, but few details were shown. Fournier (1979, 1980)

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A

B

C

Figure I1 Diagram showing the series of events in the embryogenesis of the epidermis in Entobdelfu soleue (Monopisthocotylea: Capsalidae). A. 9- 10-day stage showing nucleated ciliated cells and (syncytial?) interciliary region containing a nucleus and Golgi body. B. 13-16-day stage. The ciliated cells have flattened greatly and cell bodies of the presumptive adult layer are differentiating in the parenchyma. The basement lamina and muscle layers are clearly differentiated but not fully formed. Nuclei in the interciliary cytoplasm project right out from the surface and may be lost. C. Hatched larva. The ciliated cells have lost their nuclei. Cells in the parenchyma have sent cytoplasmic processes beneath the epidermis to form a ‘presumptive adult’ layer that is discontinuous but seems to make contact with the interciliary cytoplasm (now anucleate). b = basement lamina; cb = ‘cell’ body; ce = ciliated epidermal cell; ic = interciliary cytoplasm; m = muscles; nu = nucleus; pal = presumptive adult layer. (Redrawn from Lyons, 1973b.)

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Figure 12 Scanning electron micrographs of the epidermis. A, B. Zeuxapta seriolae (Polyopisthocotylea: Axinidae). Note tapering locomotory cilia, large surface projections of non-ciliated epidermis and small microvilli (arrows) between cilia (c) of ciliated cell. C. Encotyllabe chironemi (Monopisthocotylea: Capsalidae). Note the irregular surface projections. Scale bars = 2 pm. (K. Rohde, unpublished photographs.)

made electron microscopic studies of the epidermis of the oncomiracidia of Polystoma integerrimum and P . pelobatis. The most important findings are that the epidermis has nucleated ciliated cells attached to a syncytial tegument whch is also nucleated. Nuclei, in contrast to those of Monopisthocotylea,

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are shed only at a later stage, i.e. in the post-larva attached to the tadpole. Fournier (1979) explains t h s by the neotenic development of polystomes. Rohde (1998~) used SEM and TEM to study the epidermis of the oncomiracidium of Zeuxapta seriolae. The epidermal cilia do not have the ‘false’ vertical rootlets described in the larvae of two species of monopisthocotyleans. However, bundles of fine filaments extend from the basal bodies into the cytoplasm of the epidermal cells, ‘straddling horizontal rootlets of cilia in the same longitudinal rows’. The non-ciliated surface has many irregular folds and the ciliated cells bear many short microvilli between the cilia (Figure 12B). The cilia taper towards their tips owing to a reduction in the number of axonemal microtubules (Figure 12A) and the apical parts of the cilia have a diameter of about a quarter of that of the more basal parts. Both the ciliated and non-ciliated cells are nucleated, and ciliated cells are separated by lateral cell membranes, i.e. they are not syncytial. Since 2. seriofae does not have neotenic development, it cannot explain the delay in the elimination of epidermal nuclei as suggested by Fournier (1979) for Pofystoma (see above). Scanning electron micrographs show short microvilli between the cilia of larval Pofystomaaustralis (see Du Preez and Kok, 1987) and Zeuxapta seriofae (Figure 12B). Using SEM, many small pores were reported on the epidermis of larval Rajonchocotyfe emarginata by Whittington (1987a). Zhang and Lang (1990) examined the development of the tegument of Diplorchis hangzhounensis in various life-cycle stages.

5.3. Summary

The epidermis of so few larval monogeneans has been studied that no substantial conclusions can be drawn. Studies of monopisthocotyleans include only four species, one of which, Euzetrema knoepfleri, is highly unusual and adapted to living in the bladder of a urodele. For the polyopisthocotyleans, most studies have focused on polystomatids, which are highly adapted to parasitic life in amphibians and reptiles. In this respect, however, it is interesting to note the similarities between the epidermis of E. knoepfleri and polystomatids. Further studies of more species from both subclasses are required that better represent the monogeneans as parasites of fishes.

6. TERMINAL GLOBULE

Frankland (1955) illustrated and referred to a ‘small, clear vesicle’ projecting from the posterior tip of the ciliated cone in the larva of Dicfidophora

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denticulata (Polyopisthocotylea: Diclidophoridae) but mentioned it no further. The same structure was illustrated but not labelled by Llewellyn (1957) on a schematic drawing of the oncomiracidium of the superfamily Diclidophoroidea and was referred to as a ‘drop of viscous material’ secreted by the ciliated cone. This structure was labelled as a drop of ‘glandular secretion’ by Llewellyn (1963) in Gastrocotyle trachuri (Polyopisthocotylea: Gastrocotylidae) but he stated that the gland had not been identified. Kingston et al. (1969) referred to this structure in two monopisthocotylean and many polyopisthocotylean oncomiracidia as a ‘globular terminal excrescence’ but did not discuss it further. We call this terminal structure a ‘terminal globule’ after Whittington and Kearn (1989, 1990a). 6.1. Monopisthocotylea

Although illustrations in Kingston et al. (1969) suggest that a terminal globule is present in at least two species, Ancyrocephalus parvus (Dactylogyridae) and Heterocotyloides pricei ( = Monocotyle pricei) (Monocotylidae) (Table l), it is most likely that the structures illustrated are artefacts. There is strong light-microscopic evidence that the terminal globule is absent in oncomiracidia of many species belonging to several families, for example: a Acanthocotylidae - Acanthocotyle lobianchi (see Kearn, 1967a); a Capsalidae - Capsala martinieri (see Kearn, 1963b), Entobdella spp. (see Kearn, 1974a), Benedenia spp. (see Kearn et al., 1992b; Whittington and Kearn, 1993; Whittington et al., 1994), Encotyllabe spp. (see Whittington and Kearn, 1992); a Dactylogyridae - Neocalceostomoides brisbanensis (see Whittington and Kearn, 1995); a Microbothriidae - Leptocotyle minor (see Kearn, 1965); 0 Monocotylidae - Merizocotyle sp. (see Kearn, 1968a), Dictyocotyle coeliaca and Calicotyle kroyeri (see Kearn, I970), Clemacotyle australis (see Beverley-Burton and Whittington, 1995), Dendromonocotyle ardea (see Chisholm and Whittington, 1999, Monocotyle helicophallus, M . spiremae, Merizocotyle australensis, M . icopae, Neoheterocotyle rhinobatidis and Troglocephalus rhinobatidis (see Chisholm and Whittington, 1996a), Heterocotyle capricornensis (see Chisholm and Whittington, 1996b), N . rhinobatis and N . rhynchobatis (see Chisholm and Whittington, 1997), Decacotyle j7oridana (see Chisholm and Whittington, 1998).

Terminal globules are also absent from a range of larvae of other species studied by light microscopy belonging to the families Dactylogyridae, Dionchidae, Diplectanidae and Monocotylidae (L.A. Chisholm and I.D.

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Table 1 Species in which a terminal globule has been illustrated. Family Mono pisthocot ylea Dactylogyridae Monocotylidae Polyopisthocot y lea Axinidae

Diclidophoridae

Discocotylidae Gastrocotylidae Macrovalvitrematidae

Mazocraeidae

Microcotylidae

Plectanocotylidae Protomicrocotylidae

Species

Reference

Ancyrocephalus parvus' Heterocotyloides pricei'

Kingston et al., 1969 Kingston et al., 1969

Heteraxine heterocerca Heteraxinoides hannibali H. xanthophilis Nudaciraxine gracilis Zeuxapta seriolae Diclidophora denticulata D. luscae D. merlangi Heterobothrium okamotoi Pedocotyle minima Vallisia striata

Kearn et al., 1992a Euzet and Ktari, 1970a Kingston et al., 1969; Thoney, 1988 Kingston et al., 1969 Rohde, 1998a Frankland, 1955; Macdonald, 1977 Gallien, 1934; Macdonald, 1977 Macdonald, 1977 Ogawa, 1998 Kingston et al., 1969 Euzet and Raibaut, 1961; Llewellyn, 1963 Llewellyn, 1957, 1963 Kingston et al., 1969

Gastrocoiyle trachuri Macrovalvitrematoides micropogoni Neomacrovalviirema argentinensis Neopterinotrematoides avaginaia Grubea cochlear Kuhnia scombri K. sprostonae Mazocraeoides hargisi Aspinatrium gallieni Cynoscionicola heieracantha Metamicrocotyla cephalus Microcotyle mugilis M . poronoti M . sebasiis Polylabris diplodis Polylabroides mullispinosus Plectanocotyle gurnardi Protomicrocotyle ivoriensis

Suriano, 1975 Suriano. 1975 Whittington and Kearn, 1990a Whittington and Kearn, 1990a Whittington and Kearn, 1990a Kingston et al., 1969 Euzet and Ktari, 1971 Kingston et al., 1969 Euzet and Combes, 1969 Euzet and Combes, 1969 Kingston et al., 1969 Thoney, 1986 Euzet and Cauwet, 1967 Roubal and Diggles, 1993 Whittington and Kearn, 1989 Wahl, 1972

'

Presence of a terminal globule in live monopisthocotyleans appears doubtful and illustrations supposedly showing the globules may be of artefacts due to pressure from the coverslip (see also Figure 13A).

Whittington, unpublished data). The possibility that, when reported, the terminal globule in monopisthocotyleans may be an artefact is supported by observations that when some live larvae are compressed beneath a coverslip, leakage of material, possibly from the ciliated epidermal cells, may occur

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and can form ‘blebs’ reminiscent of the terminal globule described in some polyopisthocotylean larvae (Figure 13A; L.A. Chisholm and I.D. Whittington, unpublished data). That these structures in monopisthocotylean larvae are artefacts is supported by the ultrastructural observation that such globules are absent in the oncomiracidium of the monocotylid, Neoheterocotyle rhinobatidis (K. Rohde, unpublished data). 6.2. Polyopisthocotylea

A terminal globule (e.g. Figure 1B) has been illustrated, but not always discussed, for a number of polyopisthocotylean oncomiracidia belonging to Axinidae, Diclidophoridae, Discocotylidae, Gastrocotylidae, Macrovalvi-

Figure 13 Terminal globule. A. Light micrograph of Neoheterocotyle rhinobatidis (Monopisthocotylea: Monocotylidae). Arrow indicates ‘false’ terminal globule and arrowheads indicate ‘blebs’ caused by leakage from epidermal cells (L.A. Chisholm, unpublished photograph). B. An unciliated gastrocotylid larva (Polyopisthocotylea) (L.A. Chisholm and I.D. Whittington, unpublished photograph.) C. Light micrograph of Callorhynchicola multitesticulatus (Polyopisthocotylea: Chimaericolidae) (M. Beverley-Burton, L.A. Chisholm and F.R. Allison, unpublished photograph.) D. Scanning electron micrograph of C . muititesticulatus showing trilobed terminal globule (M. Beverley-Burton, L.A. Chisholm and J. McKenzie, unpublished photograph.) Scale bars = 10 pm.

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trematidae, Mazocraeidae, Microcotylidae, Plectanocotylidae and Protomicrocotylidae (Table 1). Its origin and nature, however, is somewhat of a curiosity. Whittington (1987a) did not describe a terminal globule or a posterior ciliated cone on the haptor of the oncomiracidium of Hexabothrium appendiculatum (Hexabothriidae) but did report that seven of 46 live larvae studied under controlled compression on a slide beneath a coverslip developed a structure resembling the terminal globule described among some other polyopisthocotylean oncomiracidia. The ‘terminal globules’ in H . appendiculatum were significantly smaller and less obvious than those reported in other species, and during examination of one larva, similar globules arose between the ciliated cells on the lateral margins of the body proper (I.D. Whittington, unpublished observation). This indicates, perhaps, that the ‘terminal globules’ in H . appendiculatum, at least, may be the result of leakage from the ciliated epidermis. The terminal globule reported in the oncomiracidia of Plectanocotyle gurnardi (Plectanocotylidae; Figure 1B) and in three species of mazocraeids (Whittington and Kearn, 1989, 1990a, respectively) was, however, reported consistently but no evidence was found to support a glandular nature for these structures. The ‘terminal globule’ of Heteraxine heterocerca (Axinidae), was referred to as a ‘refringent terminal droplet attached externally at the apex of the posterior ciliated cone’ (Kearn et al., 1992a). No mention was made as to whether this droplet was considered to be lipid. Unpublished observations (M. Beverley-Burton, L.A. Chisholm and J . McKenzie) show that the oncomiracidium of Callorhynchicola multitesticulatus (Chimaericolidae) has a trilobed terminal globule (Figure 13C and D). The oncomiracidium of an undescribed polyopisthocotylean (Gastrocotylidae) from the gills of a haemulid fish at Heron Island, Queensland, Australia, may also bear a terminal globule (L.A. Chisholm and I.D. Whittington, unpublished observation) but this discovery confuses the issue because the larva is unciliated (Figure 13B). The possible origin, therefore, of a terminal globule from the ciliated epidermal cells of oncomiracidia and a function related to swimming becomes obscure. Rohde (1998a) has made the only ultrastructural study of a terminal globule. In larval Zeuxapta seriolae (Axinidae), the structure is joined to the tip of the posterior end by a septate junction (Figure 14). Electron-dense granules and mitochondria are found in the proximal part and the middle part is strongly vacuolated (Figure 14). The vacuoles have conspicuous aggregations of electron-dense material and the distal tip contains some densely packed nuclei with little cytoplasm (Figure 14). No evidence of nervous or sensory structures was found. The dense material in the vacuoles is suggestive of heavy material perhaps functioning to weigh the posterior end down during swimming.

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Figure 14 Transmission electron micrograph of terminal globule of Zeuxuptu seriolae (Polyopisthocotylea: Axinidae). Note septate junction (j), two nuclei (n) and large vacuoles (v) containing some conspicuous electron-dense structures (arrowheads). Scale bar = 1 pm. (K. Rohde, unpublished photograph.)

6.3. Possible Functions

Whttington and Kearn (1990a) suggested that its function may relate to the swimming behaviour of the larvae and/or may be sensory in function because the terminal globule is deciduous and is jettisoned when the oncomiracidia shed their ciliated epidermal cells. The single ultrastructural study to date presents no evidence for nervous connections or sensory structures (Rohde, 1998a), and no experimental or behavioural evidence for the function of the terminal globule among polyopisthocotylean larvae is available. Rohde (1998a) has also made the following suggestions: a stabilizing function during swimming by elongating the body; stabilizing forward swimming by keeping cilia at the posterior end separate; the prevention of turbulence or the use of turbulence to make locomotion more effective; a function as a statocyst; and a function as an organ of ballast to keep the posterior end down during swimming. Further investigations of the fine structure of this enigmatic organ among larvae of polyopisthocotyleans are required to assess its likely role.

6.4. Summary

Studies so far have revealed the consistent presence of terminal globules only in polyopisthocotyleans; strong evidence presented here indicates that the

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few reports of a terminal globule among monopisthocotylean larvae are artefacts. Terminal globules may be a synapomorphy for the Polyopisthocotylea but, so far, only species belonging to the Microcotyloidea and Diclidophoroidea have been examined. Further studies using light and electron microscopy are required, therefore, to determine whether they are present more widely. At this stage, however, terminal globules cannot be considered to be a synapomorphy for the Monogenea as a whole.

7. GLANDS

Llewellyn (1963) noted that oncomiracidial gland cells had received little attention and that, while they may be depicted in drawings, often the path of ducts leading from gland cells and the type of secretion they contain is disregarded. These incomplete observations are related to the difficulty in seeing these structures which are best observed in live larvae using phasecontrast optics but they may also be difficult to find for other reasons. The problems in finding glands or their ducts may simply be because the secretory products within them have been depleted. Whittington (1987a) noted that 43 larvae of Rajonchocotyle emarginata (Hexabothriidae) had to be examined before the position and contents of the anterior gland cells and their ducts could be determined. Unfortunately, large numbers of larvae are not always available, particularly for polyopisthocotyleans, which tend to lay small numbers of eggs with lower hatching success (I.D. Whittington and L.A. Chisholm, unpublished data). Glands could not be traced in Clemacotyle australis (Monocotylidae) because extensive pigmentation in the larva obscured them (Beverley-Burton and Whittington, 1995). Nonetheless, several comprehensive studies have been made on the glands of oncomiracidia (e.g. Calicotyle and Dictyocotyle, see Kearn, 1970; Heterocotyle, see Chisholm and Whittington, 1996b; Entobdella spp., see Kearn, 1974a; Rajonchocotyle and Hexabothrium, see Whittington, 1987a; Plectanocotyle, see Whittington and Kearn, 1989; Kuhnia, see Whittington and Kearn, 1990a). Recent interest in the distribution, ultrastructure, chemical composition and function of the anterior glandular secretions in adult monopisthocotylean monogeneans (Cribb et al., 1997, 1998; Whittington and Cribb, 1998) has sparked further interest in the distribution, composition and function of the gland cells in their larvae, especially of the Monocotylidae (see Chisholm and Whittington, 1996a,b, 1997, 1998). The examples listed below are by no means exhaustive, but they have been chosen to show the variation in the number, distribution and contents of glands that occurs between different species.

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7.1. Monopisthocotylea

The distribution of gland cells varies but the majority of larvae have gland cells in the anteromedian region, the mid-body region lateral to the pharynx and in the posterior region of the body proper near the haptor. The gland cells of the oncomiracidium of the monocotylid Heterocotyle capricornensis were examined in detail by Chisholm and Whittington (1996b), and this species is used to illustrate their distribution and contents. The larva of H . capricornensis has two anteromedian gland cells, which contain dense granular secretion, that open on the anterior margin of the head (Figure 15). A group of five gland cells which contain needlelike secretion are found on either side of the pharynx (Figure 15). Three

Figure 15 Glands of Heterocotyle capricornensis (Monopisthocotylea: Monocotylidae). agc = anteromedian gland cell containing granular secretion; lgc = lateral gland cell containing granular secretion; lnc = lateral gland cell containing needlelike secretion; pgc = posterior gland cell containing granular secretion. Scale bar = 50 pm. (Modified from Chisholm and Whittington, 1996b.)

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narrow ducts from these gland cells lead anteriorly and open on the lateral margin of the head lateral to the duct openings of the anteromedian gland cells. Two gland cells containing less dense granular secretion are present on each side of the body immediately posterior to the gland cells containing needle-like secretion, and ducts from these lead anteriorly and open on the anterior margin (Figure 15). A single gland cell which contains granular secretion is located on each side of the body near the haptor. Ducts or pores associated with these gland cells were not observed but there was no indication that ducts extended into the haptor. Chisholm and Whittington (1996b) demonstrated the homology between glands in the larva of H . capricornensis and those in the adult. The distribution and content of gland cells in the larvae of 20 species of monocotylids is similar to that described above (Chisholm and Whittington, 1996a, 1997, 1998). Some monocotylid larvae have an additional pair of glands that contain granular secretion and enter the posterior portion of the pharynx (Chisholm and Whittington, 1996a). Some specific examples follow where variation occurs between larvae in the Monopisthocotylea but these examples are certainly not exclusive. Kearn (1974a) found a transverse band of gland cells (not seen in monocotylid larvae) posterior to the anteromedian gland cells in Entobdella hippoglossi, E. diadema and E. soleae (Capsalidae) and these contain needlelike secretion (e.g. Figure IA). Ducts from these glands open on the lateral borders of the anterior end. The larvae of these Entobdella spp. also have glands lateral to the pharynx which contain needle-like secretion but the lateral gland cells with granular secretion seen opening on the anterior margin of monocotylid larvae are not present. Instead, there are numerous glands termed ‘body glands’ that lie along the lateral margins of the body and have ducts that open on the ventral or ventrolateral surface. These body glands are more numerous in E. hippoglossi and E. diadema (5-7 pairs) than in E. soleae (two pairs). There are also four gland cells which lie in the median posterior part of the body and a duct from each cell runs into the haptor (e.g. Figure 1A). Kearn (1974a) describes gland cells in the wall of the pharynx of E. hippoglossi and E. soleae. There is an outer ring of 40-50 narrow gland cells and ducts from an inner ring of about 12 larger gland cells open via papillae into the pharyngeal lumen. Urocleidus adspectus (Ancyrocephalidae) has a pair of anteromedian gland cells with granular secretion, a group of five or six gland cells, which contain needle-like secretion on either side of the pharynx, that open on the anterior margin, and a group of four cells with granular secretion are found immediately anterior to the haptor and each has a duct which leads to the margin of the haptor (Cone, 1979b). The unciliated larvae of Acanthocotyle lobianchi (Acanthocotylidae) and Enoplocotyle kidakoi (Enoplocotylidae) are conspicuous in that the anteromedian glands are absent - see Kearn

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(1967a) and Kearn (1993a) respectively. Leptocotyle minor has welldeveloped gland cells in the posterior region of the body (Kearn, 1965). There is only one published account of the ultrastructure of the anterior secretions in a larval monopisthocotylean. El-Naggar and Kearn ( 1983) described three adjacent adhesive sacs on each side of the head of the larva of Entobdella soleae (Capsalidae) supplied with two kinds of secretion. The most abundant secretory type is rod-shaped bodies which open through ducts with multiple apertures (‘pepperpots’). A second spherical secretory type is less abundant and opens via ducts with a single aperture. The gland ducts carrying each secretory type intermingle at each anterior adhesive sac, permitting the two types of secretions to mix (El-Naggar and Kearn, 1983). TEM studies of the anterior region of Neoheterocotyle rhinobatidis (K. Rohde, unpublished data) also reveals the presence of rod-shaped (Figure 16A, C, D) and spherical secretions (Figure 16A, B). The rods originate from gland cells on either side of the pharnyx (compare with Figure 15). There appear to be two different types of spherical secretion, one large and one small. Large granules (Figure 16A, B) likely correspond to the secretion from the anteromedian glands (cf. Figure 15) and the smaller granular secretions appear to be produced by the lateral gland cells (cf. Figure 15). Chisholm and Whittington (1996a) commented that the anteromedian granular secretion in the monocotylid larvae they examined appeared to be darker and denser than the lateral granular secretion, and the confirmation of two different sizes of spherical secretion revealed by this preliminary TEM study warrants further investigation. That the anteromedian granular secretion appears to be different from the lateral granular secretion may also support the possibility that the anteromedian glands in monocotylids serve a function other than attachment - see Kearn (1970) and further discussion below. 7.2. Polyopisthocotylea

Glands of polyopisthocotylean larvae are less well documented. Whittington and Kearn (1989) illustrated the larva of Plectanocoryle gurnardi (Plectanocotylidae) with two sets of glands on either side of the pharynx. These glands contain granular secretion and have ducts that run anteriorly and open on either side of the head near the mouth (Figure 1B). No anteromedian glands or posterior glands were observed. A similar arrangement of anterior gland cells containing granular secretion was described in Diclidophora spp. (Diclidophoridae) (Macdonald, 1977). The larvae of Heteraxine heterocerca (Axinidae) (Kearn et al., 1992a) and Heterobothrium okamotoi (Diclidophoridae) (Ogawa, 1998) also have a similar arrangement of anterior gland cells; however, the type of secretion they contain was not described in the text but was depicted in figures as ‘granular’. Kingston et al. (1969) depicted

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Figure 16 Transmission electron micrographs of oncomiracidium of Neoheterocoryle rhinobatidis (Monopisthocotylea: Monocotylidae). A. Three types of gland secretion: large granules (g), rod-shaped bodies (r) and small granules (d). B. Openings of gland ducts with rod-shaped bodies (r) and small granules (d). C. Section through anterior end showing gland ducts with rod-shaped bodies (r), and perikaryon of gland cells producing large granular secretion (arrowhead). D. Section at level of pharynx. Note large gland cells with rod-shaped bodies (r) and pharynx (p). Scale bars = 2 pm. (K. Rohde, unpublished photographs.)

Heteraxinoides xanthophilis (Axinidae) with three pairs of anterior gland cells which open anteriorly, but Thoney (1988) found four groups of anterior gland cells which contained granular secretion and led to the anterior margin. He did not show ducts but rather a line of many gland cells in the larva of the same species extending from the level of the eyes to the anterior margin. Based on observations of other larvae, it is likely that these lines of gland cells are actually winding ducts which open anteriorly. The chimaericolid Callorhynchicola multitesticulatus has a pair of anteromedian glands, three lateral glands on either side of the pharynx, which open anteriorly, and two

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pairs of glands in the haptor; all contain granular secretion (M. BeverleyBurton and L.A. Chisholm, unpublished data). The hexabothriids Rajonchocotyle emarginata and Hexabothrium appendiculatum have a pair of anterior glands located immediately posterior to the mouth that open, via short ducts, on the anterior margin of the head (Whittington, 1987a). One pair of glands are located anterolateral to the pharynx and another pair are located just posterior to the pharynx. Ducts from these two pairs of glands also run anteriorly and open on the anterior margin of the larva (Whttington, 1987a). A fourth pair of glands are located in the posterior part of the body with ducts that lead into the haptor. All of the glands in the larvae of these two species contain granular secretion. In addition, peripheral ducts which contain granular secretion open on the margin of the haptor near the hooklets in H. appendiculatum (see Whittington, 1987a). The mazocraeid larvae of Kuhnia scombri, K . sprostonae and Grubea cochlear have a set of gland cells on either side of the pharynx and another set on either side of the body posterior to the pharynx (Whittington and Kearn, 1990a) but, contrary to the polyopisthocotyleans described above, all of these gland cells contain needle-like secretion. The unciliated larva of Tonkinopsis transfretanus (Byckowskicotylidae) has a single gland cell which contains granular secretion on either side of the body, just anterior to the pharynx (Chisholm et al., 1996). A single gland cell is located on either side of the body lateral to the pharynx and it contains needle-like secretion as does a third cell, which is located laterally on either side of the body posterior to the pharynx. Surprisingly, this appears to be the only report at the level of the light microscope of a polyopisthocotylean larva with two different types of anterior secretion, but Chisholm et al. (1996) stated that the presence of needle-like secretion in T. transfretanus must be confirmed. Apart from T. transfretanus and the mazocraeid larvae cited above, the larvae of all other polyopisthocotyleans appear to have glands which produce granular secretion. There is a striking dearth of knowledge about the glands of larval polystomatids despite the fact that many species have been examined (see p. 159). TEM of the the larva of Zeuxapta seriolae (Axinidae) has revealed that there are two different types of anterior secretion (K. Rohde, unpublished data). There are two pairs of ducts that contain a large granular secretion on either side of the body and open at the anterior end (Figure 17A). A single duct, which contains a different, smaller secretion, is also present on either side of the body (Figure 17A). This secretion also opens anteriorly and may comprise either small granules or short rods (Figure 17B). Unfortunately, there are no light microscope observations of the larva of Z. seriolae to confirm these preliminary studies. A single posterior median duct with electron-lucent ‘granules’ quite different from the anterior secretions is also present in this species (Figure 17C).

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Figure I7 Transmission electron micrograph of larval Zeuxapta seriolae (Polyopisthocotylea: Axinidae). A. Section near anterior end showing gland ducts (g) with large electron-dense ‘granules’ and smaller duct (d) with small electron-dense granules. B. Section through anterior end showing duct openings with large granules (g) and small granules or short rods (d). C. Section at level of haptor showing posterior median duct @g) with electron-lucent granules and cross-sections through hooklets (h). Scale bars = 2 pm. (K. Rohde, unpublished photographs.)

7.3. Function

Much attention has focused on the haptor of oncomiracidia and its role in attachment to the host, but there is evidence that secretions from some anterior glands also play a role in attachment. Larvae of Entobdella spp. (Kearn, 1974a), many species of monocotylids (Chisholm and Whittington, 1996a, 1996b, 1997, 1998), Plecanocotyle gurnardi (see Whittington and Kearn, 1989), Hexabothrium appendiculatum and Rajonchocotyle emarginata (see Whittington, 1987a) have all been observed to attach rapidly to glass

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surfaces via their anterior end and then sever this attachment just as rapidly. As noted above, monopisthocotylean larvae have glands with both granular and needle-like secretion, identifiable clearly using the light microscope, that open anteriorly and perhaps these different secretions mix to form a glue. On the other hand, the majority of polyopisthocotyleans that have been studied with the light microscope seem to have only a single type of secretion that opens anteriorly (except, perhaps, for Tonkinopsis transfretanus and Z . seriolae using TEM; see above), which may be responsible for adhesion. For the larvae with two secretory types, one may be an adhesive while the other may sever attachment or perhaps detachment is a mechanical event. It is also possible that one of the secretions may not function in attachment at all. Kearn (1970) speculated that the anteromedian gland cells (which contain granular secretion) in the oncomiracidia of monocotylid monogeneans could soften the opercular cement of the egg before hatching. Our finding that the anteromedian secretion of N . rhinobatidis is different ultrastructurally from other anterior secretions may support this hypothesis. The function(s) of the gland cells in the posterior part of the body are also unknown. Kearn (1970) again speculated that their close relationship with the haptor in monocotylids indicated a role in attachment but Kearn (1974a) stated that there was no indication that the posterior body glands which opened in the haptor of Entobdella spp. had an adhesive function. He has hypothesized that the body glands may be responsible for the production of pheromones in adult worms. Among polyopisthocotyleans, Whittington (1987a) considered that the posterior gland cells in the hexabothriids R. emarginata and H . appendiculatum may have an adhesive function, and in H . appendiculatum because strands of material, probably secreted by the posterior gland cells, were seen in larvae that had attached to surfaces in the presence of host secretions. Glands in the wall of the pharynx of adult E. soleae produce proteolytic secretion (Kearn, 1963c) and it is likely that the function is the same for the larva. Glands lying outside the pharynx may also produce digestive enzymes (Kearn, 1974a).

7.4. Summary

Kearn (1974a) identified three ‘avenues for future work on the gland cells of monogeneans’. First, he stated that it was important to know more about the gland cells in oncomiracidia from various families because this may lead to a better understanding of relationships within the Monogenea. Second, he said that histochemical studies may help to resolve the functions of the different gland cells. Finally, he considered that assessing the importance of the gland cells in post-oncomiracidia and adult monogeneans may help determine the roles of the glands in monogeneans. Twenty-five years later,

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these deficiencies in our knowledge remain to be addressed, although an ongoing study of glands and their secretions in adult monogeneans intends to shed light on the function, chemistry and interactions of these secretions (Cribb et al., 1997, 1998; Whittington and Cribb, 1998). Our review of the literature shows that, at the level of the light microscope, the larvae of most monopisthocotyleans have two different types of secretion (needle-like and granular) which open anteriorly, whereas the majority of polopisthocotylean larvae appear to have only a single anterior secretion (either needle-like or granular). However, ultrastructural studies currently in progress on the anterior secretions of larvae reveal that there could be three different types of secretion (rods, large granules and small granules) in monopisthocotyleans and two different types (large granules, and either small granules or short rods) in polyopisthocotyleans (K. Rohde, unpublished data). More species from both subclasses must be examined to resolve the potential differences in anterior secretions and, most importantly, ultrastructural studies need to be done in conjunction with light microscopic observations to verify results.

8. PROTONEPHRIDIA The protonephridial system of flatworms assists in excretion and may also serve a role in osmoregulation, although evidence for the latter function is not conclusive (Hertel, 1993). Llewellyn (1963) remarked that the protonephridial system of few oncomiracidia had been described. This situation has been remedied considerably (see below) but, as noted for the ciliated cells and the gland cells, the protonephridial system is difficult to study and can only be examined effectively by observing live oncomiracidia. When studying live larvae beneath a compound microscope, an otherwise indistinct protonephridial system can suddenly become clear for a short period before again becoming difficult to see. Many papers refer to the fact that the description of the protonephridial system may be incomplete because it is difficult to see flame bulbs, ducts, capillaries and bladders clearly in every specimen examined. In particular, the excretory bladders can be very difficult to see: Llewellyn (1963) commented that ‘occasionally no bladder is evident in specimens belonging to species known to have a bladder’. This is certainly our experience and it applies also to the flame bulbs, ducts and capillaries whose prominence varies depending on the activity of, and volume of, fluid in the protonephridial system (I.D. Whittington and L.A. Chisholm, unpublished data). The flame bulb formula 2[(a + b) + (c d)] used in the following section describes the distribution of flame bulbs in oncomiracidia, where the number

+

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2 in front of the square brackets indicates that the system is duplicated on the left and right sides of the body, a is the number of flame bulbs in the head region, b is the number in the pharyngeal region, c is the number in the posterior region of the body proper, and d is the number in the haptor. Thus, the larva of Entobdella soleae (Figure 1A) has a flame bulb formula of 2[(2 2) + (1 + 4)] and that of Plectanocotyle gurnardi (Figure 1B) has a flame bulb formula of 2[(1 + 1) + (0 l)].

+

+

8.1. Monopisthocotylea

A description of the protonephridial system of the larvae of many monopisthocotyleans belonging to several families is known (Table 2). Euzet et al. (1995) made a detailed comparison of the protonephridial systems both of the Monopisthocotylea and the Polyopisthocotylea based on these studies. The main difference between the subclasses is the fusion of the anterior capillaries in the former, but complete separation of the anterior ducts and capillaries in the latter (compare Figures 1A and 1B). However, Kearn (1974a) has shown that the anterior capillaries in the capsalid Entobdella hippoglossi are commonly separate and only sometimes fused. They are always fused in the related species E. diadema and E. soleae. Different ‘types’ of Monopisthocotylea differ in the number of flame bulbs but the basic pattern is the same, and Euzet et al. (1995) distinguished several subtypes of protonephridial systems on the basis of differences in the numbers of flame bulbs: 0 0 0 0 0

Type 1 with Type 2 with Type 3 with Type 4 with Type 5 with

+ + +

a flame cell formula of 2[(1 1) (1 l)]; 2[(1 2) (1 l)]; 2[(1 2) (1 2)]; 2[(2 2) (1 2)]; 2[(2 2) (1 + 4)] or 2[(2 + 2) + (3 + 4)].

+ + + +

+ + + + + + +

There are clearly exceptions to these formulae because at least 32 flame bulbs have been counted in the haptor of the larva of an undescribed species of Entobdella (Capsalidae) from a ray at Heron Island, Queensland, Australia (I.D. Whittington, unpublished observation). Our interpretation of the flame bulb formula for the acanthocotylid, Acanthocotyle lobianchi, is 2[(1 2) (1 O)] (Kearn 1967a), and for the enoplocotylid, Enoplocotyle kidakoi, it is 2[(0 2) (2 O)] (Kearn, 1993a), although Euzet et al. (1995) stated that it was difficult to determine. Only a single ultrastructural study of the protonephridial system of a larval monopisthocotylean has been published. Rohde (1998b) examined the larva of Encotyllabe chironemi (Capsalidae). As in all neodermatans examined, flame bulbs are formed by two interdigitating cells, the terminal

+ + +

+ + +

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Table 2 Families for which the protonephridia of the oncomiracidia have been described.

Family Monopisthocotylea Acant hocoty lidae Calceostomatidae Capsalidae

Dact ylogyridae Diplectanidae Enoplocotyldae Gyrodacty lidae (juveniles) Microbothriidae Monocotylidae

Polyopisthocotylea Axinidae Bychowskicotylidae Diclidophoridae Diplozoidae Discocotylidae Gastrocotylidae Hexabothriidae Hexostomatidae Macrovalvitrematidae Mazocraeidae Microcotylidae

Plectanocotylidae Protomicrocotylidae Pterinotrematidae

Reference Kearn, 1967a Euzet and Ktari, 1970b; Whittington and Kearn, 1995 Jahn and Kuhn, 1932; Kearn, 1963a, 1963b, 1971, 1974a; Llewellyn, 1963; Kearn et al., 1992b; Suriano, 1971; Whittington and Kearn, 1992, 1993; Whittington et al., 1994; Euzet et al., 1995 Malmberg, 1956; Kingston et al., 1969; Lambert, 1977; Cone, 1979b Kingston et al., 1969; Suriano, 1971; Oliver, 1987 Kearn, 1993a Malmberg, 1956, 1970 Kearn, 1965 Kingston et al., 1969; Kearn, 1968a, 1970; Suriano, 1977; Beverley-Burton and Whittington, 1995; Chisholm and Whittington, 1995, 1996a, 1996b, 1997, 1998 Kingston et al., 1969; Ktari, 1971a; Thoney, 1988; Kearn et al., 1992a Chisholm et al., 1996 Kingston et al., 1969; Euzet and Suriano, 1975; Macdonald, 1977; Ogawa, 1998 Bovet, 1959, 1967; Euzet and Lambert, 1971; Lambert and Denis, 1982 Euzet and Raibaut, 1961; Owen, 1970 Llewellyn, 1963; Euzet and Wahl, 1970; Ktari, 1971a Maillard, 1966; Suriano and Incorvaia, 1982; Whittington, 1987a Ktari, 1971a Kingston et al., 1969; Suriano, 1975 Kingston et al., 1969; Ktari, 1971a; Whittington and Kearn, 1990a Llewellyn, 1963; Euzet and Cauwet, 1967; Euzet and Combes, 1969; Kingston et al., 1969; Ktari, 1969, 1970, 1971a, b; Euzet and Ktari, 1970a, 1971; Euzet and Marc, 1963; Euzet and Noisy, 1979; Thoney, 1986; Oliver, 1989 Whittington and Kearn, 1989 Wahl, 1972 Suriano, 1975

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and the proximal canal cell. The filtration apparatus (weir) is formed by outgrowths of the terminal cell, the internal ribs (rods), and by outgrowths of the proximal canal cell, the external ribs, connected by a ‘membrane’ of extracellular matrix. Some basal bodies are located basal to the plate of basal bodies of the ciliary flame. The flame, i.e. a bundle of cilia arising from the terminal cell, contained 34 cilia (Rohde, 1998b). Many additional outgrowths, the so-called internal leptotriches, extend into the lumen of the flame bulb, but there are few or no external leptotriches, i.e. outgrowths that extend into the tissue spaces surrounding the flame bulbs. A septate junction extends along the proximal canal (capillary) of the flame bulb and a similar junction is also found in the wall of larger capillaries. Some cilia (lateral flames) are also present along the capillaries. The wall of the capillaries is strongly vacuolated but no lamellae were seen. Two excretory bladders are located dorsolaterally and their nuclei are located in the ventral part of their wall (Rohde, 1998b). The lumen of the bladders is filled with concretions and granular inclusions and the surface area of the wall is increased by some lamellae. A terminal excretory duct connects each bladder to the dorsal surface, its wall thick and filled with numerous electron-lucent and electrondense vacuoles. Some lamellae extend around the excretory opening. Electron-microscopic observations of the oncomiracidium of Neoheterocotyfe rhinobatidis (Figure 18) have shown a similar structure for the flame bulb, although the maximum number of cilia seen was only 12 (K. Rohde, unpublished data). Capillaries have a septate junction and, in contrast to those of Encotyffabe (Rohde, 1998b), they contain lamellae (Figure 18B). Lateral flames consist of few cilia (the maximum seen was four) and distal canals join the excretory bladders by short narrow ducts surrounded by a well-developed muscular sphincter (Figure 18C). Both the wall of the distal canals and the bladders contain long lamellae (Figure 18C). Three nuclei were seen in the bladder wall but no ducts connected the bladders to the outside (Figure 18C). Thus, the possibility exists that the protonephridia of larval Neoheterocotyfe do not open to the exterior until after infection of the host.

8.2. Polyopisthocotylea

The distribution of the protonephridia of a considerable number of larval polyopisthocotyleans belonging to several families is known (Table 2). While the polyopisthocotyleans generally have separate anterior ducts (Euzet et al., 1995; Figure 1B), Whittington (1987a) illustrates fused anterior capillaries in the hexabothriids Rajonchocotyle emarginata and Hexabothrium appendiculatum. In the Polyopisthocotylea, the two excretory pores are located anterolaterally; they are connected to a longitudinal main duct

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Figure I8 Transmission electron micrographs of protonephridial system of larval Neoheterocotyle rhinobatidis (Monopisthocotylea: Monocotylidae). A. Cross-section through flame bulb. Note internal (i) and external (e) ribs connected by a filtration ‘membrane’ (arrows), and 12 cilia (c). B. Cross-section through capillary. Note nucleus (n), cilia of lateral flame (c) and lamellae (1). C. Section through distal canal (d) with cilia (c), lamellae (l), junction 6)and excretory bladder (b) with two nuclei (n). Arrowhead indicates sphincter between distal canal and bladder. Scale bars = 1 pm. (K. Rohde, unpublished photographs.)

on each side of the body into which the two anterior capillaries open, each terminating in a single flame bulb. Posteriorly, at the level of the anterior margin of the haptor, the main ducts are connected by a transverse canal and, typically (Type l), two capillaries on each side of the body open at the junction of the transverse and the main longitudinal duct. Each of these

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capillaries terminates in a single flame bulb, one located in the mid-body, the other in the haptor. In some species there is a greater number of flame bulbs. On the basis of such differences, Euzet et al. (1995) distinguished a number of subtypes of protonephridia:

+ + + + + +

Type 1 with a flame bulb formula of 2[(1 1) (1 I)]; Type 2 with a formula of 2[( 1 1) (1 2)]; 0 Type 3 with 2[(1 1) (1 4)], 2[(1 1) (2 4)] or 2[(1 0 Type 4 with 2[(2 2) (2 2)].

0 0

+ + + + + +

+ + +

+ 1) + (3 + 411;

In the oncomiracidium of Tonkinopsis transfretanus (Bychowskicotylidae), Chisholm et al. (1996) illustrated only two anterior and one posterior flame bulb on each side of the body, and the larva of Plectanocotyle gurnardi (Plectanocotylidae) has the same number (Figure 1 B). Callorhynchicola rnultitesticulatus (Chimaericolidae) has the flame bulb formula 2[(2 + 1) + (1 l)] (M. Beverley-Burton and L.A. Chisholm, unpublished data). These variations again suggest that the types distinguished by Euzet et al. (1995) are not exhaustive. Many species of polyopisthocotyleans have not yet been examined. It is especially noteworthy that there are no accounts of the protonephridial system for the larvae of polystomatids. The larva of Zeuxapta seriolae is the only polyopisthocotylean whose protonephridial system has been examined ultrastructurally (Rohde, 1997). The flame contains few (not more than five) cilia. The weir has the same structure described above for the monopisthocotylean E. chironemi and there are few external leptotriches. The wall of the proximal canal cell is vacuolated (reticulate) and closes in on itself, the ends connected by a septate junction (Rohde, 1997). The wall of the protonephridial capillary further away from the flame bulb has a surface increased by lamellae (sheetlike outgrowths of the surface membrane). Some cilia of ‘lateral flames’ are found along the capillaries but they are few in number. The terminal part of the distal capillary is in contact with the surrounding epidermis by a septate junction (Rohde, 1997). No well-defined excretory bladders were identified, although the terminal parts of the distal capillaries can perhaps swell temporarily. Comparison between the flame bulbs of adult polyopisthocotyleans shows that flame bulbs of the oncomiracidium of Zeuxapta are smaller and have fewer cilia than those of adults (Rohde, 1997).

+

8.3. Summary The gross structure of the protonephridial system of the oncomiracidia of both the Monopisthocotylea and Polyopisthocotylea shows remarkable similarities: anteriorly located excretory pores are connected to main ducts with branches running anteriorly and posteriorly, each of them splitting into

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a number of capillaries terminating in flame bulbs. The posterior ducts of both sides of the body are connected by a transverse commissure, and the left and right anterior ducts, typically, are fused in the Monopisthocotylea but not in the Polyopisthocotylea. These differences are considered minor and, on the whole, the structure of the protonephridial system can be considered to be a synapomorphy for both subclasses. Distinction of a variety of patterns incorporating the number and distribution of flame bulbs is useful but, as indicated above, there are certainly more types than those listed by Euzet et al. (1995). It is surprising that there have been no experimental or physiological studies to determine that the protonephridial system does indeed function in excretion and perhaps osmoregulation. Larval and adult monogeneans may be useful animals for such work. At the ultrastructural level, the protonephridial system of larval Monopisthocotylea and Polyopisthocotylea examined differ in a number of characters, but very few species have been studied. Flame bulbs of the oncomiracidium of the polyopisthocotylean Zeuxapta seriolae have fewer cilia than those of the monopisthocotyleans (probably because of the smaller size of larval Zeuxapta; see Rohde, 1997) and, most importantly, species of both groups differ markedly in the structure of the distal part of the system. No well-developed bladder was identified in Zeuxapta (although it is likely that parts of the excretory ducts temporarily swell when filled with liquid), whereas well-developed bladders were present in the two larval monopisthocotyleans examined, as well as in the adult monopisthocotylean, Anoplodiscus cirrusspiralis (see Rohde et al., 1992). Further studies on the larvae of additional species are likely to show whether these are genuine differences.

9. SENSE ORGANS

The larvae of monogeneans are provided with a battery of presumed sensory structures which have been reviewed previously by Lyons (1972), Kearn (198 l), Lambert (198 1) and Fournier (198 1). Among oncomiracidia, these sensory structures either open through the epidermis or terminate beneath it. 9.1. Eyes

The organs reviewed in this section are collectively termed ‘eyes’, but we stress that these structures probably simply detect the presence/absence of light or perhaps detect differing light intensities. The pigment-shielded eyes are also likely to detect light from particular directions because the orientation of the pigment shield blocks light from other directions. These

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‘eyes’ are certainly incapable of forming images ( = vision; Fournier, 1981) and, from a functional viewpoint, they may better be termed ‘photoreceptors’. For convenience, however, we refer to them as ‘eyes’. The socalled pigmented, or pigment-shielded, eyes of oncomiracidia are especially prominent and, when present, are often the first organs to be seen in studies even at the level of a low-power stereodissecting microscope. Not all oncomiracidia, however, possess pigment-shielded eyes. Reviews by Bychowsky (1957) and Llewellyn (1963, 1968) covered the general arrangement of the pigment-shielded eyes in monopisthocotyleans and polyopisthocotyleans, but no ultrastructural studies were made before that by Lyons (1972). Investigations of oncomiracidial fine structure have also revealed inconspicuous eyes without pigment shields in several taxa and these are also reviewed briefly below. References to eyes are also made in Section 13 (p. 203) on behaviour. 9.1.1. Monopisthocotylea Most oncomiracidia studied possess two pairs of pigment-shielded eyes located anterodorsally. Each pigment-shielded eye comprises a pigment cup that encloses a photosensitive cell or cells (Lyons, 1972), which have terminal, light-sensitive microvilli called rhabdomeres. The four pigment-shielded eyes in most taxa are each provided with a crystalline lens that fractures radially on application of considerable coverslip pressure (e.g. Kearn and Baker, 1973; Whittington and Kearn, 1995; Chisholm and Whittington, 1996a). Twitching movements are reported for the pigment-shielded eyes of Entobdella soleae (Capsalidae; Lyons, 1972; Kearn and Baker, 1973). The pigment-shielded eyes of monopisthocotyleans have a characteristic arrangement: the pigment cup and lens of the smaller, anterior, pair of eyes are directed posterolaterally whereas those of the larger, posterior, pair of eyes are directed anterolaterally (e.g. see Figures IA, 8 and 15). Few exceptions to this configuration have been reported. For example, pigment of the posterior eye in Capsala onchidiocotyle (Capsalidae) does not form a regular cup but has an irregular, diffuse outline (Euzet, 1958). Oncomiracidia of Entobdelfa diadema (Capsalidae) have no well-developed crystalline lenses but small oval bodies next to the pigment cups of each eye may represent lens remnants (Kearn, 1974a). Furthermore, Kearn (1974a) observed a single vesicle between the pigment cup, and lens remnant in the anterior eyes and two vesicles in the same location in the posterior eyes of E. diadema that he considered were parts of the rhabdomeric cells. Pigment-shielded eyes probably serve their role almost entirely in larvae (see p. 210). Pigment cups of the eyes in larvae of many monopisthocotyleans may, however, persist in adult parasites but the lenses may disappear

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(e.g. in the capsalids E. soleae and Trochopus pini; Kearn, 1971) or may persist albeit as smaller lens remnants, e.g. in the capsalids Benedenia lutjani and B. rohdei; see Whittington and Kearn (1993) and Whittington et al. (1994), respectively. In adult E. soleae, the rhabdomeres are known to persist (Kearn, 1993b), which suggests that they may remain functional, but this is not so for Euzetrema knoepfleri (Iagotrematidae) where the rhabdomere degenerates in adults (Fournier, 1975). The converse occurs in larvae of the capsalids Encotyllabe caranxi and E. caballeroi: Whittington and Kearn (1992) reported that pigment-shielded eyes were absent in the oncomiracidia, although juvenile and adult specimens are known to possess them. Furthermore, adult specimens of E. caballeroi are known to display a strong response to light on the pharyngeal tooth pads of their fish hosts (Kearn and Whittington, 1992). Despite the high degree of similarity among the appearance, configuration of lenses and directional arrangement of the two pairs of pigment-shielded eyes within larvae of the Monopisthocotylea, larvae of some species are known to lack them, e.g. Acanthocotyle lobianchi (Acanthocotylidae; Kearn, 1967a), Amphibdella torpedinis (Dactylogyridae; Euzet and Raibaut, 1962), Enoplocotyle kidakoi (Enoplocotylidae; Kearn, 1993a), Leptocotyle minor (Microbothriidae; Kearn, 1965), and Dictyocotyle coeliaca and Empruthotrema raiae (Monocotylidae; Kearn, 1970, 1976a). The ultrastructure of the pigment-shielded eyes of only two species of larval monopisthocotyleans has been studied (Justine, 1998). Kearn and Baker (1973) determined that the smaller, anterior pigment-shelded eyes of Entobdella soleae each contained a single rhabdomere, whereas the larger, posterior, pigment-shielded eyes each contained two rhabdomeres. The pigment inside the pigment cell that forms the pigment cup was identified as melanin (Kearn and Baker, 1973). The parallel microvilli of each rhabdomere in the posterior eyes lie at right angles to the microvilli of the adjacent rhabdomere. The entire structure was termed a ‘refracting eye spot with lens’ by Fournier and Combes (1978). Pigment-shielded eyes in Euzetrema knoepfleri resemble closely those of Entobdella soleae, but they lack a lens (Fournier, 1975) and have been termed a ‘refracting eye spot without a lens’ (Fournier and Combes, 1978). TEM of the pigment-shielded eyes of larval Neoheterocotyle rhinobatidis (Monocotylidae) (see Rohde et al., 1999) shows an arrangement like that in E. soleae: two pairs of eyes, each with a pigment cup and lens, the anterior pair with a single rhabdomere and the posterior pair with double rhabdomeres (Figure 19). The microvilli of the rhabdomeres are approximately hexagonal in cross-section, are densely packed and contain some dense material (Figure 19B). Importantly, however, the presence of mitochondria1 cristae in the periphery of the lens (Figure 19C and D) indicates that it is of mitochondria1 origin. Furthermore, the lens is part of the pigment cup cell, indicated by cytoplamic processes continuous between

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Figure 19 Transmission electron micrographs through the pigment-shielded eye of a larval Neoheterocotyle rhinobatidis (Monopisthocotylea: Monocotylidae). A. Anterior eye with single rhabdomere (r). Scale bar = 2 pm. B. Rhabdomere showing microvilli with dense material (arrows). Scale bar = 0.2 Fm. C . Posterior eye with double rhabdomeres and mitochondria1 cristae (c) in periphery of lens. Scale bar = 2 pm. D. Mitochondria1 cristae (c) in periphery of lens. Scale bar = 0.5 pm. Note the lens (1) and pigment cup (p) with large electron-dense granules in each eye. Arrowheads show cytoplasmic processes. (K. Rohde, unpublished photographs.)

lens and pigment cup (Figure 19A and C). This is the first time mitochondrial lenses have been shown to exist in a neodermatan. Lyons (1972) reported and described the ultrastructure of oval structures containing cilia on the head of the oncomiracidium of E. soleae (Figure 20) and ascribed to these a photoreceptive role because of their similarity to presumptive ciliary photoreceptors in molluscs, annelids and ctenophores. Kearn et al. (1992b) have since reported that these bodies (= ‘ciliary eyes’) are visible using phase-contrast light microscopy in live oncomiracidia of E. soleae, and lie between the pigment-shielded eyes and the margins of the head (see Figure 1A). A special search was made with the light microscope

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m

Figure 20 Reconstruction of the putative ‘ciliary eye’ of the larva of Entobdella soleae (Monopisthocotylea: Capsalidae). bb = basal bodies of cilia; c = cilia; lam = lamellate whorls; m = mitochondria; np = nerve process; nt = neurotubules; nu = nucleus. (Redrawn from Lyons, 1972.)

for similar structures in the head region of the larva of Benedenia seriolae (Kearn et al., 1992b) and in the larvae of several other capsalid species (I.D. Whittington, unpublished data) but these searches have been unfruitful. According to Boeger et al. (1994), synapomorphies that support their concept of the Gyrodactylidae (including the oviparous genera) include the absence of ‘eyes’ (meaning pigment-shielded eyes) in larvae, juveniles and adults. Nevertheless, photoreceptors have been reported in a Gyrodactylus sp. Watson and Rohde (1994) described two pairs of anteriorly located subsurface ciliary aggregations in a species of Gyrodactylus. The more anterior pair has several basal bodies without rootlets as well as modified cilia with a single microtubule projecting into an extracellular cavity. This pair may have a pressure/contact or photoreceptor function. Each of the second pair has about 12 basal bodies without rootlets in the periphery of a cytoplasmic cavity. Flattened ciliary membranes extend from the basal bodies into the interior, forming several whirls. Because of its similarity with presumed photoreceptors in other flatworms, this type of receptor in this Gyrodactylus sp. was considered to be a photoreceptor (Watson and Rohde, 1994). 9.1.2. Polyopisthocotylea The majority of oncomiracidia studied from polyopisthocotylean parasites from the gills of fishes have either a single pair of pigment-shielded eyes (e.g.

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Bychowsky, 1957; Llewellyn, 1963, 1968) or pigment-shielded eyes are absent, e.g. Diclidophora spp. (Diclidophoridae; see Macdonald, 1977), Rajonchocotyle emarginata and Hexabothrium appendiculatum (Hexabothriidae; see Whittington, 1987a), and Grubea cochlear (Mazocraeidae; see Whittington and Kearn, 1990a). More rarely, the larvae of some polyopisthocotyleans from fishes possess two pairs of pigment-shielded eyes, e.g. Hexastoma thynni (Hexastomatidae) and Axine belones (Axinidae) (see Euzet, 1955), and Axine spp. and Diclybothrium armatum (Diclybothriidae) (see Bychowsky, 1957). In the most common condition, the single pair of anterodorsal, pigment-shielded eyes occur close to each other, backto-back, and the pigment cups themselves are directed laterally (e.g. see Figure IB). Some larvae appear to have, and are often drawn with, a single median eye, e.g. Mazocraes alosae (Mazocraeidae; see Bychowsky, 1957), Discocotyle sagittata (Discocotylidae; see Owen, 1970), and Serranicotyle labracis (Microcotylidae; see Oliver, 1989), but Bychowsky suggested that this arrangement may be due to fusion of a pair of eyes near the mid-line. Studies by Whittington and Kearn (1989) on Plectanocofyle gurnardi and by Whittington and Kearn (1990a) on two species of mazocraeid oncomiracidia support this theory because, under compression, a cleavage line appears and delineates two closely apposed pigment cups. There are very few reports of crystalline lenses associated with the pigment-shielded eyes in larvae of polyopisthocotyleans (see, for example, Diclybothrium armatum in Llewellyn, 1963), but Gusev and Slusarev (1986) did not illustrate lenses in their figures of the larva of D . armatum. Llewellyn (1957) suggested that prominent droplets thought to be oil (or lipid) may function as lenses in the larvae of many polyopisthocotyleans by concentrating available light on the rhabdomere(s). This possibility is likely, for example, in the oncomiracidium of P . gurnardi (see Whittington and Kearn, 1989; Figure 1B) but is less likely in the larvae of Kuhnia spp. because Whittington and Kearn (1990a) reported that oil droplets close to their pigment-shielded eyes did not lie in the same optical axis. A different arrangement of pigment-shielded eyes is reported for most oncomiracidia in the Polystomatidae, the group of polyopisthocotyleans that have radiated principally in amphibian and reptilian hosts. Oncomiracidia of polystomatids have two pairs of anterodorsal eyes in the same configuration as those described earlier for monopisthocotyleans but lenses are absent. Ultrastructurally, however, the eyes of polystomatids differ fundamentally from the eyes of nearly all platyhelminths (see below). Ultrastructural studies of the pigment-shielded eyes and unpigmented eyes of polyopisthocotyleans are restricted to the oncomiracidia of Diplozoon paradoxum (see Kearn, 1978) Zeuxapta seriolae (K. Rohde, unpublished data) and seven species of polystomatids (Fournier and Combes, 1978;

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Zhang, 1987; Cable and Tinsley, 1991a). In D . paradoxum, the single pair of median laterally directed pigment shields each contains a single rhabdomere and no lens is evident (Kearn, 1978). Muscle fibres have a close association with the eyes but no eye movements similar to those reported above for the larva of Entobdella soleae were reported in D . paradoxum (see Kearn, 1978). The ultrastructure of the pigmented eyes of larval Z . seriolae corresponds with that for D . paradoxum. The fundamental difference in the ultrastructure of the larval eyes of polystomatids is that the dense pigment granules of the pigment cup are absent and, instead, the supportive cell comprises a system of regular platelets of an electron-dense, reflective substance arranged in about 15 concentric rows (Fournier, 1981). The structure of the platelets in the supporting cells of the eyes of polystomatids (those studied are Polystoma integerrimurn, P . pelobatis, Eupolystoma alluaudi and Polystomoides ocellatum; see Fournier and Combes, 1978) indicates that they function like the concave mirror of a reflecting telescope and concentrate light on the rhabdomere by reflection (Fournier, 1981) rather than by refraction, which operates in those larvae that have pigment-shielded eyes with or without a lens. Such an arrangement is considered to have the ability to amplify available light at low intensities (Fournier, 1981). In living polystomatid larvae, these reflecting eyes shine like mirrors as intense pinpoints of light and are very different from the dark black pigmentshielded eyes of other monogeneans (Kearn, 1998). In addition to the single pair of pigment-shielded eyes in the oncomiracidium of D . paradoxum, Kearn (1 978) described the ultrastructure of two other rhabdomeric eyes, each near the lateral border of the head. However, these lacked pigment shields and were, therefore, hard to see with the light microscope. As noted by Kearn (1978), the photoreceptors lacking pigment shields in D . paradoxum may be homologous with the posterolateral pigment-shielded eyes reported in the larvae of some other polyopisthocotyleans from fish (Euzet, 1955; Bychowsky, 1957) and possibly with the posterior pair of eyes in polystomatids. Interestingly, circular translucent areas, which may be photoreceptors lacking pigment shields, were seen using the light microscope in a similar location to the unpigmented photoreceptors of D . paradoxum, and have been illustrated for oncomiracidia of Metamicrocotyla cephalus and Microcotyle mugilis (Microcotylidae; see Euzet and Combes, 1969), of Heteraxinoides hannibali (Axinidae; see Euzet and Ktari, 1970a), and of Aspinatrium gallieni (Microcotylidae; see Euzet and Ktari, 1971). Similar structures have since been described at the level of the light microscope in Rajonchocotyle emarginata (Hexabothriidae; see Whittington, 1987a), Plectanocotyle gurnardi (Plectanocotylidae; see Whittington and Kearn, 1989; Figure 1B) and Heteraxine heterocerca (Axinidae; see Kearn et al., 1992a), and deserve further ultrastructural study.

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Kearn (1984a) found a pair of multiciliate, saccular structures containing multilayered lamellar bodies in the head region of freshly hatched larvae of a Sphyranura sp. (Sphyranuridae). These bodies resembled the putative ciliary eyes described by Lyons (1972) in the larva of Entobdella soleae (see above), but the lamellar bodies in Sphyranura sp. were less densely packed and the lamellae arose clearly from cilia (Kearn, 1984a). 9.1.3. Functional Morphology It is widely considered that the pigment-shielded eyes and also the reflector eyes of polystomatids function to help the oncomiracidia orientate themselves with respect to light (Lyons, 1972; Kearn, 1973, 1978; Fournier, 1981). For a detailed account of the relative merits of the dorsal arrangement and the characteristic configuration of two pairs of eyes, see Lyons (1972). Those larvae that possess pigment-shielded eyes respond strongly to light at some stage in their short, free-swimming life, e.g. E. soleae (see Kearn, 1980), P . gurnardi and Kuhnia spp. (see Whittington and Kearn, 1989, 1990a, respectively), and Diplectanum aequans (I.D. Whittington, unpublished observation) (see Table 6). Oncomiracidia that lack pigment-shielded eyes fail to respond in a directional sense to light, e.g. Diclidophora spp. (see Frankland, 1955; I.D. Whittington, unpublished observation), Rajonchocotyle emarginata (see Whittington and Kearn, 1986), Hexabothrium appendiculatum and Leptocotyle minor (see Whittington, 1987b) (see p. 210). Nevertheless, some oncomiracidia that lack pigment-shielded eyes can still demonstrate a response to light because eggs of Diclidophora spp. (see Macdonald, 1975) and R . emarginata (see Whittington and Kearn, 1986) hatch rhythmically at specific times in the daily illumination cycle (see Table 3). The putative unpigmented photoreceptors of a ciliary nature described by Lyons (1972) for E. soleae and by Kearn (1984a) for Sphyranura sp. and the unpigmented but rhabdomeric eyes described by Kearn (1978) in Diplozoon paradoxum may be responsible for monitoring day or night length, and may control hatching rhythms (e.g. Kearn, 1973, 1978; Whittington and Kearn, 1986; Ernst and Whittington, 1996). A clear body identified with the light microscope that could be an unpigmented photoreceptor of some kind was seen by Whittington (1987a) in the larva of R. emarginata and may also be responsible for monitoring day length. However, much remains to be determined about the photobiology and chronobiology among monogeneans because Ernst and Whittington (1996) demonstrated a hatching rhythm in Benedenia lutjani and B. rohdei (see Table 3) even when eggs were laid and incubated in total darkness. Eggs exposed to natural illumination for as little as 4-12 hours during laying (when the embryos within were likely to be without a nervous system and photoreceptors) still hatched rhythmically. This

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prompted Ernst and Whittington (1996) to suggest that ‘biological clocks’ may operate at a cellular or even at a molecular level. Larvae of E. diadema and P . gurnardi respond to shadows (Kearn, 1982; Whittington and Kearn, 1989 respectively), and photoreceptors that may ‘sense’ shadows are likely to lack pigment shields and may be ciliary or rhabdomeric structures. There is clearly a need not only to determine more about the diversity and structure of ‘eyes’ in the larvae of monogeneans, but also to investigate more about their range of ‘photobehaviours’. 9.1.4. Summary Two pairs of pigment-shielded eyes in the characteristic configuration described above (see Figures lA, 8, 15) may be the primitive condition in monogeneans (Lyons, 1972) and occur in larvae of most monopisthocotyleans and in some polyopisthocotyleans from fish. The same configuration occurs in larvae of polystomatids (Polyopisthocotylea) but their eyes are the platelet, reflecting type rather than pigment-shielded (Fournier, 1981). Among the larvae of the remaining polyopisthocotyleans that have been examined, there is more variability. However, the evidence suggests that the number of pigmented eyes has reduced from two pairs to one pair but the second posterior pair may be retained as an unpigmented rhabdomeric eye. The presence of four eyes in the oncomiracidium is one of two synapomorphies used previously to support the monophyly of the Monogenea (e.g. Ax, 1987; Blair et al., 1996). The review of eyes above demonstrates that significant, albeit conflicting, data are available at the level of the light microscope but studies at the ultrastructural level are wanting. Nowhere is this better argued than by Justine (1998) who points out that, for monopisthocotyleans, ultrastructural literature on eyes concerns only two species, one of which, Euzetrema, is an aberrant freshwater species adapted to life in a tetrapod. For the polyopisthocotyleans, Justine (1998) concludes that published ultrastructural studies are based on the eyes of several polystomatid larvae (freshwater parasites adapted to life in tetrapods) and Diplozoon, an aberrant, freshwater monogenean that fuses with a partner to form a pair. Further studies on the ultrastructure of the eyes of larval polyopisthocotyleans from marine fishes, like those reported above for Zeuxapta seriolae, are required.

9.2. Other Sense Organs

Knowledge of the number and distribution of the array of sensory receptors other than ‘eyes’ or photoreceptors found in oncomiracidia has advanced

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significantly in the last 30 years but precise information about their specific function(s) is still lacking. Llewellyn (1963) conceded that ‘nothing appears to be known of sense organs other than eyes in oncomiracidia’ but commented that ‘ciliated tufts’ at the anterior end of two polyopisthocotylean oncomiracidia that were not shed with the locomotory cilia were likely to be sense organs. Silver staining in addition to revealing the ciliated epidermal cells (see p. 153), also reveals symmetrical patterns of individual ciliated sensilla of unknown function. Use of this technique on the larvae of three species of polystomatids by Combes (1968) was heralded by Llewellyn (1968) as a technique of potential taxonomic and phylogenetic significance among the Monogenea provided that precipitation problems with sea water were resolved. Lambert (1980a) presented such a technique to stain oncomiracidia from freshwater and marine hosts, and provided a synthesis of his results (Lambert, 1980b). Chisholm (1998) has described an effective modification to this technique that may further facilitate silver impregnation of larvae from marine hosts. 9.2.1. Monopisthocotylea Lambert (1980b) demonstrated that the distribution of sensilla among oncomiracidia of monopisthocotyleans is generally more variable than among the polyopisthocotyleans and three types were distinguished: one for Dactylogyridea, one for Capsalidea and one for Euzetrema knoepfleri (Figure 21; Combes et al., 1974). Differences between these chaetotaxal types depend most notably on the number of dorsal sensilla in various groupings that form a longitudinal row (Figure 21). It is possible, however, to arrive at different interpretations for the numbers of these dorsal sensilla in longitudinal rows (Figure 21). There are twice as many sensilla in juvenile Gyrodactylus as found in the ciliated oncomiracidia of other monogeneans (Lambert, 1979). Gyrodactylus possess three ‘post-ocular’ sensilla, which suggests a close affinity to the ciliated oncomiracidia of the Polyopisthocotylea (Section 9.2.2). Lambert (1979) and recently Shinn et al. (1997) used the distribution of sensilla to distinguish between some species of Gyrodactylus. Chisholm (1998), however, found that the arrangement of sensilla on the dorsal surface of larval monocotylids is generally the same and attributed minor variations to difficulties in seeing these structures. Studies by Lambert (1980b) of the chaetotaxy of one species of unciliated monopisthocotylean larvae, Udonella caligorum [see Littlewood et a f .(1998), for the status of Udonella as a monogenean], has demonstrated that dorsal sensilla are absent and this is discussed further below. Chisholm (1998) found an additional pair of ciliated cells at the anterior end of monocotylid larvae, which are presumed to correspond to the non-motile tufts of cilia

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n

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Figure 21 Pattern of sensilla (chaetotaxy) of ‘larval’ Monopisthocotylea. A. Dactylogyridae. B. Capsalidae. C. Euzetrema knoepfleri. D. Gyrodactylus sp. Note the differences in the number of ‘dorsal sensilla’ (SD) and different interpretations indicated by the line. A cluster of sensilla in Gyrodactylus sp. is interpreted as corresponding to the ‘post-ocular sensilla’ (SPO) of Polyopisthocotylea (see Figure 23). (Redrawn from Lambert, 1980b.)

seen at the anterior end of many monocotylid oncomiracidia - see Chisholm and Whittington (1996a), Figs 8 and 15 and p. 155). Ultrastructural studies have characterized three ciliated receptors other than photoreceptors among the oncomiracidia of monogeneans: single uniciliate, compound uniciliate, and compound multiciliate (Lyons, 1972; Fournier, 1981). Lyons (1969a,b, 1972, 1973b) examined single and compound receptors of the oncomiracidium of Entobdella soleae using electron microscopy. She found uniciliated receptors whose cilium lacks a basal body and so-called cone sensilla and ciliated pits. The cone sensilla were later renamed grouped receptors because there is little evidence that they correspond to conical projections seen using the light microscope (Lyons, 1972). They are multiciliary with highly modified cilia, which, for most of their length, have many microtubules. The base of each cilium is surrounded by a nerve collar. The ciliated pits are located laterally, anterior to the eyes. Each pit has an opening 2-5 pm wide containing at least five cilia with many microtubules. In larval Encotyllabe chironemi, multiciliate and uniciliate receptors of various types have been found near the anterior end (Figure 22) and uniciliate receptors along the body and on the haptor (K. Rohde, unpublished data). Likewise, larval Neoheterocotyle rhinobatidis has large numbers of multiciliate and uniciliate receptors, mainly at the anterior end (K. Rohde, unpublished data).

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Figure 22 Sensory receptors of larval Encotyllabe chironemi (Monopisthocotylea: Capsalidae). A. Scanning electron micrograph. Note uniciliate (u) and multiciliate (arrowhead) receptors, and tegumental microvilli (t). B. Transmission electron micrograph. Note uniciliate (u) and multiciliate (arrowhead) receptors of various types. In one type, there are numerous microtubules inside the cilia (double arrowheads). Note electron-dense collars (arrows). Scale bars = 2 pm. (K. Rohde, unpublished photographs.)

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Gyrodactylids have so-called spike sensilla. They comprise a compound (uniciliate) receptor but have only been studied in an adult Gyrodactylus sp. (Lyons, 1969b). Further studies are required to examine the ultrastructure of the spike sensilla in newborn (i.e. ‘juvenile’) gyrodactylids. Watson and Rohde (1994) found two pairs of subsurface ciliary aggregations in adult Gyrodactylus sp. and these sense organs are likely to be present in juvenile specimens as well. 9.2.2. Polyopisthocotylea Lambert (1980b) reported a remarkable similarity between the distributional patterns of sensilla ( = chaetotaxy) among the oncomiracidia of polyopisthocotyleans, especially with respect to the three pairs of dorsal, postocular sensilla. Small differences, however, permitted the distinction of three types embracing ciliated larvae from species that infect teleosts, the Polystomatidae and the Hexabothriidae (Lambert, 1980b; Figure 23). The distribution of sensilla, especially the dorsal sensilla, has also been reported using SEM (e.g. Whittington, 1987a). Lambert (1980b) showed that dorsal sensilla are absent in the unciliated Epicotyle torpedinis (Hexabothriidae) larva as in an unciliated monopisthocotylean larvae (see above); this is discussed further below. Individual ‘hairs’, small groups of presumed

SPO SPO SPO

Figure 23 Pattern of sensilla (chaetotaxy) of larval Polyopisthocotylea. A. Parasites of teleosts. B. Polystomatidae. C. Hexabothriidae. Note three pairs of dorsal ‘post-ocular sensilla’ (SPO) but in each type there are differences in the number of other sensilla. (Redrawn from Lambert, 1980b.)

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sensilla or crowns of cilia have been illustrated and/or described from studies on live oncomiracidia of the following taxa: Diclidophora merlangi (see Macdonald, 1977), Rajonchocotyle emarginata and Hexabothrium appendiculatum (see Whittington, 1987a), Serranicotyle labracis (see Oliver, 1989), and in three species of mazocraeids (Whittington and Kearn, 1990a). In some of these species, it was found that these presumed sensilla do not move with the same vigour as the locomotory cilia and some were seen to twitch occasionally (e.g. Macdonald, 1977; Whittington, 1987a). According to Fournier (1 98l), ultrastructural studies of single uniciliate and compound multiciliate receptors in polyopisthocotyleans were only of receptors in adults. This situation has only been rectified lately with the report of uniciliate receptors around the mouth of a recently emerged larva of Pseudodiplorchis americanus (Polystomatidae) (Cable et al., 1998). In recent unpublished studies (K. Rohde) on the oncomiracidium of Zeuxapta seriolae, both uniciliated and multiciliated receptors were found, particularly at the anterior end (Figure 24). 9.2.3. Functional Morphology The different types of ciliated receptors that can be identified ultrastructurally cannot be distinguished by silver impregnation (Lambert, 1981). Furthermore, Lyons (1972) stressed, and it is re-emphasized here, that the

Figure 24 Transmission electron micrograph of anterior sensory receptors of larval Zeuxapta seriolue (Polyopisthocotylea: Axinidae). Note uniciliate (u) and multiciliate (arrowhead) receptors of various types and electron-dense collars (d). Scale bar = 1 Fm.(K. Rohde, unpublished photograph.)

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sensory functions attributed to sensilla in monogeneans are based purely on a comparative morphological approach, i.e. their similarity with structures known to be sense organs in other organisms. The tiny size of these organs in the Monogenea and also of the presumed photoreceptors discussed earlier, precludes electrophysiological recordings, a technique that in other animal groups such as insects can reveal the roles of sensilla (van der Pers and Minks, 1997). As suggested by Lambert (1981), a better knowledge of the role(s) of sensilla may follow behavioural, distributional, ultrastructural and electrophysiological studies, although most of these areas of research have advanced little for flatworms in the past 15 years. Lyons (1972) refrained from ascribing a function to the compound (uniciliate) receptors recorded on the head of the oncomiracidium of Entobdella soleae. She commented that without electrophysiological recordings, it would be very difficult to identify which of the many compound (multiciliate) receptors found inside pits at the anterior end of the larva of E. soleae are chemosensory. The spike sensilla of Gyrodaczylus sp. is suggested to be a chemoreceptor (Lyons, 1969b), and the two pairs of subsurface ciliary aggregations in adult Gyrodactylus sp. that are likely to be present in juveniles as well have been suggested to have a pressure/contact or photoreceptory function (first pair) and a photoreceptive function (second pair) (Watson and Rohde, 1994; see above). Some clues about the possible functions of sensilla among the oncomiracidia of monogeneans can be derived from the important observation that some sensilla disappear when the larvae attach to a host whereas others remain and proliferate as the post-oncomiracidium develops and establishes itself for parasitic life on the host (Lambert, 1980b, 1981). This change involves loss of the dorsal sensilla of monopisthocotyleans (SD sensilla of Lambert, 1980b) and of polyopisthocotyleans (SPO sensilla of Lambert, 1980b) and these structures never reappear. Lambert (1981) discussed the need to examine the ultrastructure of these ‘deciduous’ sensilla in the Monopisthocotylea and the Polyopisthocotylea. The three dorsal pairs of sensilla in polyopisthocotyleans are generally larger than other sensilla, and each sensilla group may comprise two or three separate sensory cilia to form multiciliate receptors. This is supported by the observations of Whittington (1987a) using SEM on Rajonchocotyle ernarginata. Multiciliate sensilla have never been recorded, however, from the dorsal region from which sensilla are lost in monopisthocotyleans (Lambert, 1981). Comparisons between the ultrastructure of these dorsal sensilla are critical because mechanoreceptors are usually considered to be uniciliate sensilla and chemoreceptors are usually considered to be multiciliate (Lyons, 1972; Fournier, 1981; Lambert, 198 I). Thus, on the basis of comparative morphology, are the deciduous dorsal sensilla of monopisthocotyleans mechanoreceptors and are the deciduous dorsal sensilla of polyopisthocotyleans chemoreceptors (Lambert, 198l)?

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Receptors of such fundamentally different appearance and structure deserve further study. As noted by Whittington (1987a), the position of the dorsal sensilla of oncomiracidia in general may indicate a possible role in detecting water currents in swimming larvae. The importance of dorsal sensilla during the free-swimming period of the oncomiracidia of monopisthocotyleans and polyopisthocotyleans is highlighted further by the discovery that a species with an unciliated larva from each group lacks dorsal sensilla (Lambert, 1980b; see above). The ciliated sensory receptors of monogenean larvae seem likely to be involved in detecting environmental stimuli, such as mechanical disturbances, water currents and chemicals. Sense organs will also be considered briefly in the behavioural section (see p. 203). 9.2.4. Summary Until technology advances sufficiently to allow the recording of nervous activity from tiny, presumed sensory organs such as uniciliate, compound uniciliate and compound multiciliate receptors in larval monogeneans, their specific functions will remain unknown. The ultrastructure of these organs for so few larvae is known, especially among the polyopisthocotyleans, that few conclusions can be drawn about possible homologies even though significant data are collated about their spatial distribution.

10. NERVOUS SYSTEM

There are few studies on the nervous system of any monogenean larvae. We therefore discuss the monopisthocotyleans and polyopisthocotyleans jointly. Lyons (1972), Tinsley and Owen (1975) and Venkatanarsaiah (1981) demonstrated the presence of cholinesterase in the nervous system of the oncomiracidia of the monopisthocotyleans Entobdella soleae (Capsalidae) and Acanthocotyle lobianchi (Acanthocotylidae), and of the polyopisthocotyleans Protopolystoma xenopodis and Pricea multae (Polystomatidae), respectively. In P . multae, the cholinesterase staining activity was more intense in the cerebral ganglia and the ‘ganglionic masses’ at the base of the haptor, although nerves were stained as well. No cholinesterase activity was found in the nerve cells. Tinsley and Owen (1975) reconstructed the nervous system of Protopolystoma xenopodis (Figure 25). The brain is located dorsal to the pharynx between the anterior and posterior eyes. A pair of nerves extends anteriorly from the brain to form a ring around the mouth; two pairs of posterior nerves unite in front of the haptor (Figure 25). The posterior nerves are connected by some complete and incomplete commissures and

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Figure 25 Nervous system of the oncomiracidium of Protopolystoma xenopodis (Polyopisthocotylea: Polystomatidae) based on cholinesterase activity. Scale bar = 50 pm. (Redrawn and modified from Tinsley and Owen, 1975.)

nerves radiate from the point of junction in front of the haptor into the haptor (Figure 25). The cerebral ring (brain) in P . multae consists of two cerebral ganglia and the cerebral commissure, from which a pair of lateral and ventral nerves run anteriorly and which are linked by a commissure in front of the brain (Venkatanarsaiah, 1981). There are three pairs of posterior nerves connected by several commissures, of which the lateral and dorsal pairs terminate at the base of the haptor. The ventral pair forms the ‘ganglionic mass’ and enters the haptor as two haptoral nerves. The latter gives rise to lateral branches which innervate the larval and median hooks (Venkatanarsaiah, 1981). The brevity of this section on the nervous system of oncomiracidia reflects the lack of attention paid to this field. Immunohistochemistry using an antibody against acetylated tubulin on capsalid and monocotylid larvae inside eggs at various stages during their embryology, however, shows

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considerable promise in staining developing neurons, and will help to chart the development of the nervous system in monogenean oncomiracidia (V. Hartenstein, A. Younossi-Hartenstein and I.D. Whittington, unpublished data).

11. DIGESTIVE TRACT

Bychowsky (1957) and in particular Llewellyn (1963) illustrated the pharynx and intestine of the larvae of several species of both the Monopisthocotylea and the Polyopisthocotylea, as seen using the light microscope. Illustrations of the digestive tract can also be found in descriptions of larvae given by numerous authors. In freshly hatched larvae, the intestine may be sac-like, bifurcate or sometimes confluent posteriorly, but it is often difficult to see. In some species, the intestine may be absent but it should be noted that even in species in which an intestine cannot be seen, it may well be present. Many descriptions at the level of the light microscope have referred to the presence of numerous refringent droplets inside the gut (e.g. Kearn, 1974a; Whittington and Kearn, 1989; Figure lB), but similar droplets are reported from elsewhere in the body proper, haptor and ciliated epidermal cells. There is a need for embryological studies to determine whether refringent droplets that occur in the gut of oncomiracidia are remnants of lipids or similar material remaining from the vitelline cells that nourish developing embryos. Although the larvae of monogeneans are unable to feed until they find and attach themselves to a host, there is strong evidence that the digestive tract of oncomiracidia is ready to assume its function. Some larvae are known to be capable of attaching to their host immediately after hatching (e.g. the capsalid Entobdella soleae; Kearn, 1981) and the gut of others is reported to contain host blood within about a day after alighting on the gills (e.g. the microcotylid Polylabroides multispinosus; Roubal and Diggles, 1993). No ultrastructural studies of the digestive system of oncomiracidia have been published but TEM of the digestive system of the oncomiracidium of the monopisthocotylean Encotyllabe chironemi has revealed the presence of discrete epithelial cells joined by septate junctions (Figure 26) (K. Rohde, unpublished data). The cells are strongly vacuolated and contain many, small, electron-dense granules as well as some larger dense bodies that are possibly lipid (Figure 26). The surface of the cells is enlarged by lamellae (Figure 26). Viviparity among gyrodactylids is well known, but the nutritional interplay between mothers and daughters has received little attention. Among oviparous monogeneans, however, Cable and Tinsley (1991b) have

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Figure 26 Transmission electron micrograph through intestine of a larval Encotylluhe chironemi (Monopisthocotylea: Capsalidae). Note discrete epithelial cells separated by junctions (j), nucleus of epithelial cells (n), lipid droplets (Id), electron-dense granules (e), and numerous vacuoles and lamellae (I) near the lumen. Scale bar = 2 pm. (K. Rohde, unpublished photographs.)

reported cytoplasmic connections between the surface of larvae and the thin membraneous sac that envelops embryos of Pseudodiplorchis americanus (Polystomatidae). These cytoplasmic extensions were thought to play a placenta-like function for nourishment of the developing oncomiracidia during hibernation of the desert toad host of P . americanus.

12. PARENCHYMA

Characteristic of larvae of both the Monopisthocotylea and the Polyopisthocotylea is the dense packing of parenchymal cells with little cytoplasm surrounding the nuclei (Figure 27). Large lipid globules are found between the parenchymal cells and in the capsalid monopisthocotylean Capsala martinieri extensive pigmentation was reported owing to

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Figure 27 Transmission electron micrograph showing parenchyma of monopisthocotylean and polyopisthocotylean larvae. A. Posterior part of larval Zeuxapta seriolue (Polyopisthocotylea: Axinidae) showing lipid droplets (Id), ciliated epidermal cells (c) and posterior gland ducts (pg). B. Anterior part of larval Neoheterocofyle rhinobatidis (Monopisthocotylea: Monocotylidae) showing cross-section of oesophagus (0).Note densely packed nuclei of parenchymal cells in both species. Scale bars = 2 pm. (K. Rohde, unpublished photographs.)

small, yellowish brown granules similar to those in the pigment cups of the eye spots (Kearn, 1963b). Such body pigment is uncommon among larvae but was also evident in Clemacotyle australis (Monocotylidae; BeverleyBurton and Whittington, 1995) ramifying throughout the body but no studies to locate the pigment were made. Unfortunately, the parenchyma is not considered to be a particulqrly important, variable o r informative

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203

feature in oncomiracidia and the poor state of our knowledge of it reflects this.

13. BEHAVIOUR

Llewellyn’s (1972) review on the ‘behaviour of monogeneans’ concentrated heavily on eggs and oncomiracidia, and he concluded that little was known about egg hatching, larval behaviour and invasion of hosts. Reviews by Kearn on oncomiracidial behaviour (Kearn, I98 I), monogenean eggs (Kearn, 1986a) and chemically induced egg hatching (Kearn, 1986b) summarized later knowledge. However, work by Whittington (1987b) and Whittington and Kearn (1986, 1988, 1989, 1990a) extended considerably the range of taxa for which behavioural information on swimming larvae was available, and, in particular, included studies on larvae of polyopisthocotyleans. The Monopisthocotylea and Polyopisthocotylea are covered together in this section. However, they are distinguished in tables, and major differences between the two groups are highlighted throughout and in the summary. 13.1. Role of the Oncomiracidium

The task of the oncomiracidium appears at first sight to be formidable. These small (usually between 100 and 300 pm long) larvae are short lived - longevity is up to 48 hours and possibly considerably less, especially in warm waters, but see Ogawa (1998) for reports of a life span of 9.1 days for the larva of Heterobothrium okarnotoi (Diclidophoridae). Oncomiracidia are slow swimming (relative to their hosts; between 1 and 5 mm s-’) but must find their specific host species, attach and then establish themselves to continue the life cycle. Llewellyn (1972) described the large differences in speed between the typical fish hosts of monogeneans and their oncomiracidia. To put this into perspective, tuna, for example, can swim at speeds of more than 40 kmh-’ (Bone and Marshall, 1982) and yet are parasitized successfully by monogeneans. To compound the problems of relatively slow-swimming monogenean larvae finding and then attaching to fast-swimming fish hosts, many fishes may also display diurnal (e.g. vertical movements in the water column) and/or seasonal (e.g. large-scale, oceanic movements) migrations. A few monogeneans have removed completely the tenuous link of a free-swimming oncomiracidium from the life cycle and attach their eggs directly to the host (Llewellyn, 1972; Kearn, 1986a; Whittington, 1997). For many

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monogeneans, however, the constraints described above have exerted considerable selection pressures on the timing of oncomiracidial emergence and on their behaviour during their limited free-swimming period to enhance the chances of contact between larvae and potential hosts (Kearn, 1981; Whittington, 1997). It is likely that adaptations developed by monogenean eggs and oncomiracidia to increase opportunities for host invasion reflect a long association between parasites and hosts. This prolonged (ancient?) association has permitted coevolution so that the biology and behaviour of parasites and hosts have become closely matched. In the most extreme case known among the Monogenea (and arguably among helminths), oncomiracidia of the polystomatid Pseudodiplorchis americanus invade their amphibian desert toad hosts success-

Table 3 Species of monogeneans reported to hatch from their eggs with a rhythm.

Family Monopisthocotylea Capsalidae

Monocot y lidae

Polyopisthocotylea Axinidae Diclidophoridae

Diplozoidae Discocotylidae Hexabothriidae Polystomatidae

Species Entobdella soleae E. hippoglossi Benedenia seriolae B. lutjani B. rohdei Merizocotyle icopae Neoheterocotyle rhinobatidis Troglocephalus rhinobatidis Heteraxine heterocerca Diclidophora denticulata D. Iuscae D . merlangi Diplozoon paradoxum Discocotyle sagittata Rajonchocotyle emarginata Polystoma integerrimum

Reference Kearn, 1973 Kearn, 1974b Kearn ef al., 1992c Ernst and Whittington, 1996 Ernst and Whittington, 1996 L.A. Chisholm and I.D. Whittington (unpublished data) L.A. Chisholm and I.D. Whittington (unpublished data) L.A. Chisholm and I.D. Whittington (unpublished data) Kearn et al.. 1992c Macdonald, 1975 Macdonald, 1975 Macdonald, 1975 Macdonald and Jones, 1978 Gannicott and Tinsley, 1997 Whittington and Kearn, 1986 Macdonald and Combes, 1978

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THE LARVAE OF MONOGENEA (PLATYHELMINTHES)

fully despite the fact that the window of opportunity for infection may be less than 24 hours annually (Tinsley and Earle, 1983).

13.2. Emergence of Oncomiracidia from Eggs

Studies indicate that rhythmical hatching of monogenean eggs appears to be a relatively widespread strategy (Table 3) and has already been mentioned briefly because the phenomenon may be monitored and controlled by unpigmented photoreceptors (see Section 9.1.3). This hatching response represents a close match between parasite and host behaviour. Other hatching strategies include emergence of larvae in response to ‘shadows’ (light intensity fluctuations) and mechanical disturbance (Table 4) and to host mucus or tissue (Table 5). A few monogeneans retain the ability to hatch rhythmically even though they respond to host mucus, e.g. Entobdella soleae (Kearn, 1973, 1974c) and Discocotyle sagittata (Gannicott and Tinsley, 1997), but most other species listed in Table 5 adopt a ‘sit-and-wait’

Table 4 Species of monogeneans reported to hatch in response to ‘shadows’ (light intensity fluctuations) and mechanical disturbance.

Hatching stimulant

Family

‘Shadows’

Monopisthocotylea Capsalidae Polyopisthocotylea Plectanocotylidae

Mechanical disturbance

Species

Reference

En tobdella diadema

Kearn, 1982

Plectanocotyle gurnardi

Whittington and Kearn, 1989

Dendromonocotyle kuhlii

Kearn, 1986a

Diclidophora luscae Diplozoon paradoxum

Whittington & Kearn, 1988 Bovet, 1967

Neonchocotyle pastinacae Microcotyle salpae

Ktari and Maillard, 1972

Monopisthocotylea Monocoty lidae Polyopisthocotylea Diclidophoridae Diplozoidae Hexabothriidae Microcotylidae

Ktari, 1969

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Table 5 Species of monogeneans reported to hatch in response to chemical hatching factors (either mucus or direct application of tissue) from the host.

Family Monopisthocotylea Acanthocotylidae

Capsalidae Enoplocotylidae Microbothriidae Monocotylidae

Polyopisthocotylea Discocotylidae Hexabothriidae

Microcotylidae

Species

Host group

Reference

Acanthocotyle greeni A . lobianchi

E

Entobdella soleae Enoplocotyle kidakoi Leptocotyle minor Empruthoirema n. sp.

T T

Macdonald and Llewellyn, 1980 Macdonald, 1974; Kearn and Macdonald, 1976; Whittington and Kearn, 1990b Kearn, 1974c Kearn, 1993a

E

Whittington, 1987c

E

L.A. Chisholm and I.D. Whittington (unpublished observation)

Discocotyle sagittata Hexabothrium appendiculatum Squalonchocotyle torpedinis Microcotyle salpae

T

Gannicott and Tinsley, 1997

E

Whittington, 1987c

E

Euzet and Raibaut, 1960

T

Ktari, 1969

E

E = elasmobranch: T = teleost.

strategy and hatch only in response to host chemical cues. This is particularly the case for Acanthocotyle spp., Enoplocotyle kidakoi, Squalonchocotyle torpedinis and Microcotyle salpae because their oncomiracidia are unciliated and therefore rely on close physical contact between eggs and host for infection to proceed. The rapidity of hatching in Acanthocoryle spp. in response to ray mucus (larvae emerge within 2-4 seconds; Macdonald, 1974) and in Enoplocotyle kidakoi in response to physical contact of eggs with fresh moray eel skin (larvae emerge ‘within a few seconds’; Kearn, 1993a) ensures that the oncomiracidia can capitalize on brief periods of contact between eggs and host. It was mentioned earlier (Sections 9.2.1, 9.2.2 and 9.2.3) that dorsal sensilla, characteristic of free-swimming monopisthocotylean and polyopisthocotylean oncomiracidia (Lambert, 1980b), are absent in the few unciliated larvae studied. It is apparent, however, that unciliated oncomiracidia do indeed possess other presumed sensory receptors (e.g. Figure 28) and it is likely that some of these sensilla

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may be responsible for mediating egg hatching in response to chemical stimulation.

13.3. Swimming Behaviour

The hatching strategies outlined above have evolved to maximize opportunities for infective oncomiracidia to co-occur, spatially and temporally, with a potential host. The majority of oncomiracidia are ciliated and are able to swim. References to the swimming behaviour of oncomiracidia are scattered and often amount to only a few lines of description in larger studies. Early investigations were summarized by Llewellyn (1972) and Kearn (198 1). More detailed studies that focused specifically on larval swimming behaviour are those of Kearn (1980), Whittington (1987b) and Whittington and Kearn (1986, 1989, 1990a). Many oncomiracidia display special behaviours, such as phototaxis, geotaxis, rheotaxis and chemotaxis, and may respond to shadows and mechanical disturbances. Table 6, although not an exhaustive list, attempts to

Figure 28 Scanning electron micrograph of Acanthocotyle lobianchi (Monopisthocotylea: Acanthocotylidae) hatching from egg; operculum of egg has been removed. Note abundant non-motile cilia (sensilla?) on anterior end of larva which lacks locomotory cilia. Scale bar = 20 pm. (I.D. Whittington, unpublished photograph.)

Table 6 Some swimming characteristics and some behaviourai responses detected in different species of monogenean larvae to various environmental stimuli. Family

Species

Speed

Rotation Spiral Phototaxis Geotaxis Rheotaxis Chemotaxis Longevity’

Reference

(mm S d ) Monopisthocotylea Benedenia lutjani Capsalidae

Dactylogyridae Dactylogyridae Diplectanidae Microbothriidae

Ernst and Whittington, 1996 Ernst and Whittington, 1996 Hoshina, 1968 Kearn, 1982

J

B. rohdei

J

B. seriolae EntobdelIa dindema E. hippoglossi E. soleae

J J

5

Y

J

J

Nitzschia sturionis

J

Various dactylogyrids Urocleidus adrpectus Diplectanwn aequans Leptocotyle minor

J

J J

J

24 h+ 9-30 h

Cone and Burt. 1981

J

1-5

Y

J

J

J

Y

1-3

J

J

x

?

Y

Kearn, 1974b Kearn, 1967b; 1980; 1981 Bychowsky, 1957; Llewellyn, 1972 Bychowsky, 1957

30 h ?

30 min’

Whittington, unpublished data Whittington, 1987b

Polyopisthocotylea Diclidophoridae Diplozoidae Discocotylidae Hexabothriidae

Mazocraeidae

Microcotylidae

Diclidophora denticulata Diplozoon paradoxum Discocotyle sagittata Hexabothrium appendiculatum Rajonchocotyle emarginata Grubea cochlear

Y

Y

12-24 h

Y

10 h

Y

4-6 h

3

J

J

J

0.4

Y

J

J

1-4

J

J

Y

?

Y

?

30 min’

1-2

J

J

Y

J

Y

J

24-40 h

J

Y

J

36 h

Kuhnia scombri

J

J

J

15-36 h

K . sprostonae

J

J

J

15-48 h

Microcotyle sebastis Plectanocotylidae Plectanocotyle gurnardi Polystoma Polystomatidae integerriwn I

J

0.03-4.6 3-4

J

J

J

J

J

J

12-24 h J

24-52 h

J

J

J

Frankland, 1955 Bovet, 1967; Llewellyn, 1972; Kearn, 1981 Owen, 1970; Paling, 1969; Llewellyn, 1972 Whittington, 1987b Whittington and 1986 Whittington and 1990a Whittington and 1990a Whttington and 1990a Thoney, 1986

Kearn, Kearn, Kearn, Kearn,

Whittington and Kearn, 1989 Llewellyn, 1957, 1968, 1972; Combes, 1966a

Longevity is temperature dependent. For details, refer to original papers.

’Longevity assessed in the presence of hatching stimulants.

J = Characteristic present or behavioural response detected; Y = Characteristic absent or behavioural response not detected; ? = behavioural response present but weak: neither a J nor a Y denotes that no information is available.

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I.D. WHllTlNGTON ETAL.

summarize current knowledge of swimming and behavioural responses (in the absence of host tissue) detected in those oncomiracidia that have been studied most thoroughly. In general, oncomiracidia swim erratically and abrupt direction changes are frequent; sometimes larvae follow straight lines but rarely for more than 1-2 cm (Whittington and Kearn, 1986) after which they may swim in a spiral. Spiral swimming can be tight (Whittington and Kearn, 1986) or open (Whittington, 1987b). There are reports of oncomiracidia swimming by spinning on the spot in a tight circle (e.g. Kearn, 1967b; Owen, 1970) but this could be due to ciliary damage (Kearn, 1967b). Swimming behaviour may change with age. Kearn (1980) reported that oncomiracidia of Entobdella soleae (which had hatched with no chemical stimulation) spent more time in a photopositive phase, perhaps dispersing widely from the eggs. With increasing age, however, the larvae spent more time in a photonegative phase, a behaviour that is more likely to help them find their specific host fish. Behavioural changes with age were also reported for larvae of Rajonchocotyle emarginata (Whittington and Kearn, 1986) and three species of mazocraeids (Whittington and Kearn, 1990a). There are also some reports of larval behaviour that is interpreted as conserving energy. Larvae of species of Diclidophora display periods of active upward swimming alternating with periods of passive sinking (Macdonald in Kearn, 1981; I.D. Whittington, unpublished data). Similar behaviour was reported in R . emarginata (see Whittington and Kearn, 1986). Oncomiracidia of some species are reported to attach themselves regularly to observation vessels (e.g. Plectanocotyle gurnardi; Whittington and Kearn, 1989) by the anterior end (presumably using adhesive secretions; see Section 7, p. 175) and this behaviour may also conserve valuable energy resources. Attached larvae of P. gurnardi detached themselves when shaded or when disturbed by vibration, but some attached oncomiracidia also detached themselves spontaneously (Whittington and Kearn, 1989). Estimates of longevity vary greatly but are dependent on temperature (Table 6): in general, oncomiracidia in warmer climates are likely to live for shorter periods than those in cooler regions. From the relatively limited studies made so far some generalizations can be made. Oncomiracidia of species with pigment-shielded eyes display a directional response to light whereas those with no pigment-shielded eyes show no detectable directional response to light (see also Section 9.1.3). Comment was made earlier, however, that oncomiracidia with no pigmentshielded eyes can hatch rhythmically (see Section 9.1.3). Monopisthocotylean larvae with two pairs of pigment-shielded eyes do not appear to rotate about their longitudinal axis as they swim, whereas the majority of polyopisthocotylean larvae with or without pigment-shelded eyes do rotate while swimming (Table 6). This swimming characteristic may relate to the orientation of the eyes but the larvae of many more species need to be studied.

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21 1

Strong evidence exists for a response by many oncomiracidia to gravity (Table 6) and the merits of geotaxis include: enabling larvae to escape from eggs buried in sediment; orientation of swimming larvae with pigmentshielded eyes in darkness (Kearn, 1980); orientation of swimming larvae that have no pigment-shielded eyes and display no directional response to light during periods of light and darkness (Whittington and Kearn, 1986). Presently, however, no sense organ which may be a gravity receptor (i.e. a statocyst) has been identified in any oncomiracidium, although it has been suggested that the terminal globule may play a role in gravity responses (Rohde 1998a; Section 6.3). Organs believed to be statocysts have been described from many free-living ‘turbellarians’ (e.g. Hyman, 1951; Rohde and Watson, 1995, and further references therein). Oncomiracidia of some species have been observed to respond to water currents (rheotaxis; Table 6), and exposed sensory receptors such as the dorsal sensilla identified in monopisthocotyleans and polyopisthocotyleans by Lambert (1980a,b; Section 9.2.3) seem particularly well-located to respond to such stimuli, although sensilla in these locations may also serve as chemoreceptors. The likely significance of behavioural changes by larvae in response to water currents are discussed in Section 13.6.

13.4. Host Finding and Host Recognition

Behavioural responses by oncomiracidia hatching from eggs or by oncomiracidia during their swimming period to environmental stimuli, such as photoperiod, light intensity and direction, gravity, shadows, disturbances and water currents, simply direct larvae to or help to ensure that larvae congregate, spatially and temporally, in places where potential hosts are llkely to occur. However, these behavioural responses by larvae are not specific and the strict host specificity displayed by most monogeneans must be controlled by other factors. Kearn (1967b) established that a specific substance secreted from host epidermis is probably important in inducing attachment of EntobdelZu soZeue oncomiracidia to host skin and also stimulates shedding of the ciliated epidermal cells. Chemotaxis or chemoperception was implicated in this response (Kearn, 1967b). However, more than 30 years later the identity of the chemical(s) is still unknown. How much host ‘recognition’ is active discrimination by the oncomiracidia, and how much may be trial and error is not understood, but the low numbers of infective stages produced by most monogeneans in comparison with other parasitic platyhelminths indicates that monogenean larvae are highly successful at finding hosts (Whittington, 1997) and chemotaxis is likely to play a significant role. Whether oncomiracidia can follow chemical gradients emanating from the host, or whether host identification and invasion depends on contact chemoperception

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remains to be determined. Many experiments or observations that have sought to determine whether oncomiracidial behaviour may change in the presence of the host or host tissue, or whether larvae are ‘attracted’ to host tissues are equivocal. Experiments using the larvae of E. soleae (see Kearn, 1967b), E. hippoglossi (see Kearn, 1974b) and Rajonchocotyle emarginata (see Whittington and Kearn, 1986) suggest that they may change behaviour (Table 6) and can attach to host tissue, but behaviour changes are not marked and oncomiracidia were not observed to swim directly to host tissue. In the presence of host elasmobranch tissue, a chemotactic response by larvae of Leptocotyle minor and Hexabothrium appendiculatum was present but weak (Whittington, 1987b; Table 6). Studies by Frankland (1955), Bovet (1967) and Paling (1969) suggested that larvae of Diclidophora denticulata, Diplozoon paradoxum and Discocotyle sagittata, respectively, showed no response to host fish tissue (Table 6). Monogeneans are considered to be among the most host-specific of parasites but, other than the work of Kearn (1967b) cited above, details on factors that may play a role in determining host specificity, especially at the oncomiracidial stage, are lacking. Experiments by Combes (1966b, 1968) demonstrated that larvae of three species of Polystoma from Europe would not infect tadpoles of the ‘wrong’ host. Similar studies by Kok and Du Preez (1987) and Du Preez and Kok (1997) also demonstrated a strong preference for the natural host among African polystomatids at the oncomiracidial/ tadpole stage, but not all the parasite species displayed the same degree of host specificity. Furthermore, Du Preez et al. (1997) investigated the behaviour of the oncomiracidia of three species of African polystomatids in the presence of natural and ‘substitute’ host tadpoles and found that host recognition did indeed occur at this stage in the life cycle, and was the basis for host specificity among these polystomatids. For monogeneans of fish, it is known that ‘serum antibodies’ in fish skin mucus and local antibody production may play a role in the host specificity of monogeneans (Kearn, 1976b). Recently, Buchmann and Bresciani (1 998) wrote that ‘monogenean invasion of fish epithelia primarily involves an attraction of the parasite to the host’ and this has been demonstrated experimentally at the oncomiracidial stage at least for Entobdella soleae (Kearn, 1967b). However, Buchmann (1998) considered that host specificity involved several factors: in addition to attraction by specific substances, the host provides appropriate nutrients, cues and factors to signal to the parasite that it can maintain itself, but that some hosts may produce substances that harm or kill some parasites species. It is contemporary studies such as these that, while presently applied primarily to adult monogeneans, may shed light on the kinds of signals that must operate between the oncomiracidium and its host for successful parasitism to occur. These areas offer exciting challenges and, hopefully, intriguing answers for the future!

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13.5. A Dispersal Phase?

Kearn (1981) made reference to Bychowsky’s (1957) hypothesis that oncomiracidia have two distinct behavioural phases: an early photopositive period when the larva is unable to attach, which amounts to a dispersal period, and a later infection phase lasting the rest of the free-swimming life of the oncomiracidium, which is often characterized by loss of photopositive behaviour and sometimes accompanied by development of photonegative behaviour. Work by Bovet (1967) on Diplozoon paradoxum and by Paling (1969) on Discocotyle sugittata lent support to this hypothesis, but studies by Kearn (1980, 1981), Whittington and Kearn (1986) and Whittington (1987b) suggest strongly that the behaviour of monogenean larvae is not stereotyped as Bychowsky (1957) had proposed. The great diversity of egg-hatching strategies and oncomiracidial behaviour in monogeneans probably reflects the diversity of the habits and habitats of their hosts, and indicates adaptation on the part of the parasites to the behavioural habits of their specific hosts. The observations that not all larvae are photopositive and that larvae of E. soleae can attach to their host immediately after hatching (Kearn, 1980, 1981) contradicts Bychowsky’s hypothesis. While not proven experimentally, chemically induced hatching of larvae from eggs (Table 5) implies that attachment to hosts immediately after hatching may be relatively common. 13.6. Invasion of Hosts by Oncomiracidia

Whatever mediates host specificity, the adaptations described above appear to be highly successful in placing oncomiracidia near their specific host. Evidence on the invasion of fish hosts by monogeneans indicates that there are large differences between the routes taken by monopisthocotylean and polyopisthocotylean larvae. Not surprisingly, larvae of typical ‘skin-parasitic’ monopisthocotyleans, such as the capsalids Entobdella soleae (see Kearn, 1984b), Benedenia hoshinai (see Ogawa, 1984) and B. lufjani (I.D. Whttington and I. Ernst, unpublished data), invade host skin whereas the oncomiracidia of the gill-parasitic capsalid, Trochopus pini, appear to invade the gills directly (Kearn, 1971). There are interesting movements by post-oncomiracidia of many species that are beyond the scope of this review. The oncomiracidia of monopisthocotyleans that, when adult, typically inhabit the gills of fishes also tend to invade the s h n and then migrate to reach the gills, e.g. a dactylogyrid, diplectanid and tetraonchid (Kearn, 1968b), Actinocleidus oculutus and A . recurvatus (see Lambert, 1975), Ergenstrema mugilis (see Lambert, 1977), and Urocleidus adrpectus (see Cone and Burt, 1981). Observations by Izjumova (1956), Paperna (1963), Gusev and Kulemina (1971) and Lambert (1980a)

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suggest that invasion by dactylogyrids using the cutaneous route may be dependent on host size. Larger fish are more likely to be infected via the inhalent current, which is more typical for the gill-dwelling polyopisthocotyleans (see below), but this is not the case for U.adspectus (see Cone and Burt, 1981). A response by larvae to water currents (rheotaxis) was described above (see also Table 6) and this behaviour may be significant for larvae of polyopisthocotyleans. Bovet (1967) observed that larvae of Diplozoon paradoxum stopped swimming when they fell into the cone of influence of the inhalent current of their host, and were drawn into the mouth and branchial cavity - see Llewellyn (1968) for an account of this. A similar phenomenon was reported for Discocotyle sagittata by Paling (1969) and these species are thought to invade the gills of their hosts directly by means of the inhalent water current. Direct invasion of the gills by oncomiracidia of polyopisthocotyleans is also implicated, but has been observed rarely, in several other species (e.g. Diclidophora denticulata, see Frankland, 1955; Heteraxine heterocerca, see Ogawa and Egusa, 1981b; Rajonchocotyle emarginata, see Whittington and Kearn, 1986; Bivagina tai, see Ogawa, 1988; Plectanocotyle gurnardi, see Whittington and Kearn, 1989; Polylabroides multispinosus, see Roubal and Diggles, 1993) because parasites the same size as freshly hatched larvae have been discovered on the gills of their hosts. The apparent difference in the invasion behaviour between the oncomiracidia of some gill-inhabiting adult monopisthocotyleans and polyopisthocotyleans of fish seems likely to be driven by food, but probably reflects also the evolutionary progression of these parasites. Dactylogyrids are epidermal feeders, and epidermis is abundant on the skin and in the gill chamber. A cutaneous phase in dactylogyridsmay reflect their ancestry as ‘skin parasites’. Polyopisthocotyleans of fishes are principally blood feeders (Halton, 1997), which is not readily available or accessible via the epidermis on the body surfaces of fishes. Since oncomiracidia probably need to feed almost as soon as they alight on a host, a cutaneous phase for larval polyopisthocotyleans is an unlikely option. Furthermore, the larvae of some polyopisthocotyleans hatch precociously and already bear a pair of sclerotized clamps specialized for attachment to secondary gill lamellae. The haptor of these polyopisthocotylean larvae is unlikely to be suitable for attachment to epidermis on the outer surface of a fish host. The Polystomatidae have evolved a bewildering array of strategies by which they may invade their hosts (generally amphibians or chelonian reptiles). Oncomiracidia of Polystoma integerrimum were reported by Llewellyn (1957) to detect ‘slack water’ between bursts of exhalant flow from the operculum of tadpoles, a clear but different demonstration of rheotaxis (see Table 6). An excellent recent review by Euzet and Combes (1998) of invasion by oncomiracidia of polystomatids is recommended for a synthesis of the complex strategies involved in this family.

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13.7. Summary

Tables 3-6 summarize important information on the behaviour of the oncomiracidia of those monogeneans that are best studied. Relatively few larvae, however, have been investigated in detail following the kinds of procedures developed by Kearn (1980), Whittington (1987b) and Whittington and Kearn (1986, 1989, 1990a). Nevertheless, much is to be learned from simple studies on the swimming behaviour of oncomiracidia. For instance, do most polyopisthocotylean larvae rotate along their longitudinal body axis when swimming while monopisthocotyleans appear not to (Table 6), and is this associated with the number and orientation of the pigmented eyes (e.g. Kearn, 1978)? A particular area that requires further examination is the mechanism (or mechanisms?) involved in the rapid hatching by oncomiracidia in response to host mucus or host skin. Kearn and Macdonald (1976) considered that either the egg shell or the opercular cement must be permeable to small molecules, such as urea, but the nature and mechanism of the activity stimulated in the larva in response to chemicals (a rapid neuromuscular response?) is unknown. The chemoresponsiveness of monogenean oncomiracidia to their specific host(s) is also a field that deserves attention (Whittington, 1997). There is clearly much to be gained from an integrated study of oncomiracidial morphology and physiology, their sensory apparatus and behaviour using whole, live organisms. Focusing in detail on particular features at the cellular level (e.g. sense organs) using electron microscopy and perhaps at the molecular level (e.g. chemotaxis and immunology) for host recognition may also yield fascinating results.

14. CONCLUSIONS

This review has highlighted that there is still much that we do not know about monogenean larvae. We know very little about terminal globules, glandular secretions, protonephridia, the functions of sensory receptors and lipid droplets in the parenchyma, and we know nothing about the mechanism by which ciliated cells are shed. Thus, physiological and behavioural studies and, where possible, an integration of these approaches are of foremost importance. Are terminal globules, which have been reported from the larvae of many polyopisthocotyleans, also found in larvae of some Monopisthocotylea? What is the detailed composition of the parenchyma and are there, for example, different types of parenchymal cells? We know practically nothing about the early development of most larval organs and tissues. Of particular importance in this respect is a study of the ontogeny and embryology of the

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haptoral sclerites, and also of sensory receptors. Such studies may contribute to our future understanding of possible homologies between the Monopisthocotylea and the Polyopisthocotylea. On the basis of current knowledge, the presence of two pairs of rhabdomeric eyes in a characteristic configuration (see Figure lA), as reported in most larval Monopisthocotylea but only in some Polyopisthocotylea, is a complex character unique in the invertebrates. This character is not found in the infective stages of digeneans, aspidogastreans and cestodes, all of which have, when present, a single pair of rhabdomeric eyes. It is, therefore, unlikely to be plesiomorphic for all the neodermatans. Two pairs of eyes are likely to be a synplesiomorphy of the monogeneans. The presence, typically, of three zones of ciliated cells in both the Monopisthocotylea and the Polyopisthocotylea may be a further synapomorphy, although there is much variation in the arrangement of these cells, especially among the Polyopisthocotylea (see Section 4, p. 157). The pattern of ciliated cells, therefore, does not support the monophyly of the Monogenea as strongly as the presence of two pairs of rhabdomeric eyes. The protonephridial system of the oncomiracidia of both subclasses shows remarkable similarities: anteriorly located excretory pores are connected to main ducts with branches running anteriorly and posteriorly, each of them splitting into a number of capillaries terminating in flame bulbs. The posterior ducts of both sides of the body are connected by a transverse commissure, and the left and right anterior ducts, typically, are fused in the Monopisthocotylea but not in the Polyopisthocotylea. However, in one monopisthocotylean species, Entobdella hippoglossi, they are not fused in most individuals, and in two polyopisthocotylean species, Rajonchocotyle emarginata and Hexabothrium appendiculatum, they are fused. Thus, the differences are not consistent and, on the whole, the structure of the protonephridial system is very similar in both subclasses and can be considered to be a further synapomorphy for the Monogenea as a whole. There are few ultrastructural studies on oncomiracidia and conclusions regarding the usefulness of certain characters for phylogeny would be premature, although some studies may have important phylogenetic implications. Rohde et al. (1998) have shown that two species of Monopisthocotylea, Neoheterocotyle rhinobatidis and Monocotyle spiremae (Monocotylidae), have ‘false’ vertical rootlets of the epidermal cilia, arising not from the basal bodies but from the horizontal rootlets. Such rootlets are absent in the polyopisthocotylean Zeuxapta seriolae and have, in fact, never been found in other platyhelminths. This character may be, therefore, a synapomorphy of the Monopisthocotylea, although clearly larvae of species representing different families must be studied. Alternatively, if such rootlets were found in the larvae of other species of polyopisthocotyleans, another synapomorphy for the Monogenea would be provided. Rohde (1998~)has

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demonstrated the presence of epidermal cilia in a monopisthocotylean and in a polyopisthocotylean larva that taper strongly towards the tips, the result of a reduction in the number of axonemal microtubules. This character is not known from other platyhelminths and, although this must be verified by examining several additional species, it may well be another synapomorphy of the Monogenea. Convergence of this feature between the two subclasses cannot be ruled out owing to strong selection pressure for fast and effective swimming. Likewise, ultrastructural studies of the protonephridia of few larval monogeneans have been made. Therefore, the observation that monopisthocotyleans have well-developed excretory bladders [oncomiracidia of Encotyllabe chironemi and Neoheterocotyle rhinobatidis, and adult Anoplodiscus cirrusspiralis (see Rohde, 1998b and Rohde et al., 1992, respectively], whereas the polyopisthocotyleans do not (Rohde, 1997), needs to be verified for other species before excretory bladders can be considered to be a synapomorphy of the Monopisthocotylea. However, demonstration of similar bladders in both groups would further strengthen the view that monogeneans may be monophyletic. Some support for the view that Monopisthocotylea and Polyopisthocotylea form one clade comes from the observation that the pattern of sensilla of gyrodactylids shows certain similarities with that of the Polyopisthocotylea, and that the pattern of ciliated cells in the monopisthocotylean Euzetrema knoepfleri resembles that of polystomes (Lambert, 1980b). Alternatively, however, such similarities in sensilla and ciliated cells may represent convergence between the Monopisthocotylea and Polyopisthocotylea because selection pressures exerted on both groups are likely to be similar. Terminal globules have so far been found beyond doubt only in the Polyopisthocotylea and may be apomorphic for that taxon. Another potentially useful character is the type of anterior glandular secretions. At the level of the light microscope, the larvae of most monopisthocotyleans have two different types of secretion (needle-like and granular), which open anteriorly, whereas the majority of polyopisthocotylean larvae appear to have only a single type of anterior secretion (either needle-like or granular). However, preliminary ultrastructural studies have shown that there could be three different types of secretion (rods, large granules and small granules) in monopisthocotyleans and two different types (large granules and either small granules or short rods) in the polyopisthocotyleans. This avenue needs to be explored further. Traditionally, the arrangement of sclerites on the haptor of larval and adult monogeneans has been used extensively to develop hypotheses about their evolution (e.g. Llewellyn, 1957, 1963, 1968, 1970, 1982). Further studies on the haptor of larval monogeneans are warranted, especially on the embryology and chemical composition of the sclerites. In particular, the ideas of Pariselle and Euzet (1995) regarding the non-homology of some

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haptoral elements deserves further study. Possible convergence between monopisthocotyleans and polyopisthocotyleans was mentioned above with reference to a number of characters. If monopisthocotyleans and polyopisthocotyleans are shown, in the future, to be paraphyletic, then nowhere will the power of convergence be better exemplified than among the evolution of similarly shaped and similarly arranged haptoral sclerites in these two groups. For a clearer picture of the monophyly or otherwise of the Monogenea, however, many larval characters, in addition to adult features and molecular data, must be combined to provide a comprehensive data set.

ACKNOWLEDGEMENTS Financial support for each aut.,or was provided by the Australian Research Council. We thank the Director and staff of the Heron Island Research Station of The University of Queensland for their assistance and use of facilities, and Peter Garlic for use of facilities at the Electron Microscopy Unit at The University of New England ( W E ) . We appreciate the assistance of Nikki Watson (UNE) for preparing specimens for electron microscopy, Rick Porter (UNE) for developing and Zoltan Enoch (UNE) for printing most electron micrographs. Maureen Heap and Craig Hayward ( W E ) helped rear and hatch larvae of Zeuxupta seriolue. We are especially indebted to Louise Percival (UNE) for redrawing and modifying many of the figures.

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Roubal, F.R. and Diggles, B.K. (1993). The rate of development of Polylabroides multispinosus (Monogenea: Microcotylidae) parasitic on the gills of Acanthopagrus australis (Pisces: Sparidae). International Journal for Parasitology 23, 87 1-875. Salami-Cadoux, M.-L. (1978). Les stades larvaires de Polystoma africanum Szidat, 1932 (Monogenea, Polystomatidae), parasite de Bufo regularis Reuss. Bulletin du Muskum National d’Histoire Naturelle, 514, Zoologie 353, 22 1-226. Shinn, A.P., Gibson, D.I. and Sommerville, C. (1997). Argentophilic structures as a diagnostic criterion for the discrimination of species of the genus Gyrodactylus von Nordmann (Monogenea). Systematic Parasitology 37, 47-57. Suriano, M. (197 1). Systkmatique et Biologie des MonogPnes du Genre Ancyrocephalus Parasites des Muges de Mkditerranke Occidentale. Thbe de 3e cycle de biologie animale, parasitologie et pathologie des invertkbrks, Universitk des Sciences et Techniques du Languedoc, Montpellier, France. Suriano, M. (1975). Sistematica, biologia y microecologia de tres Monogenea, Polyopisthocotylea parasitos de las branquias de Micropogon opercularis (Quoy y Gaimard) y Umbrina canosia Berg (Pisces, Sciaenidae) del ocean0 atlantico sudoccidental. Physis, Buenos Aires 34, 147- 163. Suriano, D.M. (1977). Parasitos de elasmobranquios de la region costera de Mar del Plata (Monogenea-Monopisthocotylea). Neotropica 23, 161- 172. Suriano, D.M and Incorvaia, I.S. (1982). Sistematica y biologia de ‘Callorhynchocotyle marplatensis’ gen. et sp. nov. (Monogenea: Polyopisthocotylea) parasita de las branquias de Callorhynchus callorhynchus (Linne, 1758) Garman, 1904 (Pisces: Holocephali) de la region costera de Mar del Plata. Communicaciones del Museo Argentino de Ciencias Na turales ‘Bernardino Rivadavia’ e Instituto Nacional de Investigacion de las Cienias Naturales 2, 19-32. Thoney, D.A. (1986). The development and ecology of the oncomiracidium of Microcotyle sebastis (Platyhelminthes: Monogenea), a gill parasite of the black rockfish. Transactions of the American Microscopical Society 105, 38-50. Thoney, D.A. (1988). Morphology of the oncomiracidium of Heteraxinoides xanthophilis (Monogenea), a gill parasite of Leiostomus xanthurus (Sciaenidae). Transactions of the American Microscopical Society 107, 345-354. Threadgold, L.T. (1984). Parasitic Platyhelminths. In: Biology of the Integument. 1 Invertebrates. (J. Bereiter-Hahn, K., Matoltsy and K.S., Richards, eds), pp. 112131. Berlin: Springer. Thurston, J.P. (1964). The morphology and life cycle of Protopolystoma xenopi (Price) Bychowsky in Uganda. Parasitology 54, 441 -450. Thurston, J.P. (1968). The larva of Oculotrema hippopotami (Monogenea: Polystomatidae). Journal of Zoology, London 154, 475-480. Tinsley, R.C. (1976). Oncomiracidial morphology and evolutionary relationships within the Polystomatidae (Monogenoidea). Parasitology 73, 25. Tinsley, R.C. (1978). Oviposition, hatching and the oncomiracidium of Eupolystoma anterorchis (Monogenoidea). Parasitology 77, 121- 132. Tinsley, R.C. (1981). The evidence from parasite relationships for the evolutionary status of Xenopus (Anura Pipidae). Monitore Zoologico Italiano, N. S. Supplemento 15, 367-385. Tinsley, R.C. (1983). Ecological and phylogenetic specificity amongst polystomatid rnonogeneans. Parasitology 87, 11- 12. Tinsley, R.C. and Earle, C.M. (1983). Invasion of vertebrate lungs by the polystornatid monogeneans Pseudodiplorchis americanus and Neodiplorchis scaphiopodis. Parasitology 93, 451 -469. Tinsley, R.C., and Owen, R.W. (1975). Studies on the biology of Protopolystoma

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xenopodis (Monogenoidea): the oncomiracidium and life-cycle. Parasitology 71, 445 -463.

Tyler, S. and Tyler, M.S. (1997). Origin of the epidermis in parasitic platyhelminths. International Journal for Parasitology 27, 7 15-746. van der Pers, J.N.C. and Minks, A.K. (1997). Measuring pheromone dispersion in the field with the single sensillum recording techniques. In: Insect Pheromone Research. New Directions (R.T. Card6 and A.K. Minks, eds), pp. 359-371. New York: Chapman and Hall. Vande Vusse, F.J. (1976). Parapolystoma crooki sp. n. (Monogenea: Polystomatidae) from Rana magna in the Phillipines. Journal of Parasitology 62, 552-555. Venkatanarsaiah, J. (1981). Detection of cholinesterase in the nervous system of the oncomiracidium of a monogenean, Pricea multae Chauhan, 1945. Parasitology 82, 241 -244. Vladimirov, V.L. and Gusev, A.V. (1986). The ciliary cover and sensillae of the swimming larva of Dactylogyrus vastator Nybelin, 1924 (Monogenea) (in Russian). Trudy Zoologicheskogo Instituta Morfologiya Sistematika i faunistika Paraziticheskikh Zhivotnykh 155, 50-54. Wahl, E. (1972). Protomicrocotyle mirabilis (MacCallum, 1918) Johnston et Tiegs 1922 et P. ivoriensis n. sp., monogines parasites de Caranx hippos dans la lagune Ebrii (Cbte d'Ivoire). Zeitschrift fur Parasitenkunde 38, 3 19-332. Watson, N.A. and Rohde, K. (1994). Two new sensory receptors in Gyrodactylus sp. (Platyhelminthes, Monogenea, Monopisthocotylea). Parasitology Research 80, 442 -445. Whittington, I.D. (1987a). A comparative study of the anatomy of the oncomiracidia of the hexabothriid monogeneans Rajonchocotyle emarginata and Hexabothrium appendiculatum. Journal of the Marine Biological Association of the United Kingdom 67, 757-772. Whittington, I.D. (1987b). Studies on the behaviour of the oncorniracidia of the monogenean parasites Hexabothrium appendiculatum and Leptocotyle minor from the common dogfish, Scyliorhinus canicula. Journal of the Marine Biological Association of rhe United Kingdom 67, 773-784. Whittington, I.D. (1987~). Hatching in two monogenean parasites from the common dogfish (Scyliorhinus canicula): the polyopisthocotylean gill parasite, Hexabothrium appendiculatum and the microbothriid skin parasite, Leptocotyle minor. Journal of the Marine Biological Association of the United Kingdom 67, 729-756. Whittington, I.D. (1997). Reproduction and host-location among the parasitic Platyhelminthes. International Journal for Parasitology 27, 705-714. Whittington, I.D. (1998). Diversity 'down under': monogeneans in the Antipodes (Australia) with a prediction of monogenean biodiversity worldwide. International Journal for Parasitology 28, 148 1- 1493. Whittington, I.D. and Cribb, B.W. (1998). Glands associated with the anterior adhesive areas of the monogeneans, Entobdella sp. and Entobdella australis (Capsalidae) from the skin of Himantura fai and Taeniura Iymma (Dasyatididae). International Journal for Parasitology 28, 653-665. Whittington, I.D. and Kearn, G.C. (1986). Rhythmical hatching and oncorniracidial behaviour in the hexabothrid monogenean Rajonchocotyle emarginata from the gills of Raja spp. Journal of the Marine Biological Association of the United Kingdom 66, 93- 111. Whittington, I.D. and Kearn, G.C. (1988). Rapid hatching in response to mechanical disturbance in the eggs of the monogenean gill parasite Diclidophora luscae, with

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observations on sedimentation of the egg bundles. International Journal for Parasitology 18, 847-852. Whittington, I.D. and Kearn, G.C. (1989). Rapid hatching induced by light intensity reduction in the polyopisthocotylean monogenean Plectanocotyle gurnardi from the gills of gurnards (Triglidae), with observations on the anatomy and behaviour of the oncomiracidium. Journal of the Marine Biological Association of the United Kingdom 69, 609-624. Whittington, I.D. and Kearn, G.C. (1990a). A comparative study of the anatomy and behaviour of the oncomiracidia of the related monogenean gill parasites Kuhnia scombri, K. sprostonae and Grubea cochlear from mackerel, Scomber scombrus. Journal of the Marine Biological Association of the United Kingdom 70, 21-32. Whittington, I.D. and Kearn, G.C. (1990b). Effects of urea analogs on egg hatching and movement of unhatched larvae of monogenean parasite Acanthocotyle lobianchi from skin of Raja montagui. Journal of Chemical Ecology 16,3523-3529. Whittington, I.D. and Kearn, G.C. (1992). The eggs and oncomiracidia of Encotyllabe spp. and the relationship between encotyllabines and other capsalid monogeneans. Parasitology 104, 253-261. Whittington, I.D. and Kearn, G. (1993). A new species of skin-parasitic benedeniine monogenean with a preference for the pelvic fins of its host, Lutjanus carponotatus (Perciformes: Lutjanidae) from the Great Barrier Reef. Journal of Natural History 27, 1-14. Whittington, I.D. and Kearn, G.C. (1995). A new calceostomatine monogenean from the gills and buccal cavity of the catfish Arius graeffei from Moreton Bay, Queensland, Australia. Journal of Zoology, London 236, 21 1-222. Whittington, I.D., Kearn, G.C. and Beverley-Burton, M. (1994). Benedenia rohdei n. sp. (Monogenea, Capsalidae) from the gills of Lutjanus carponotatus (Perciformes, Lutjanidae) from the Great Barrier Reef, Queensland, Australia, with a description of the oncomiracidium. Systematic Parasitology 28, 5- 13. Wiskin, M. (1970). The oncomiracidium and post-oncomiracidial development of the hexabothriid monogenean Rajonchocotyle emarginata. Parasitology 60,457-419. Yamaguti, S. (1963). Systema Helminthum. Volume IV, Monogenea and Aspidocotylea. London: Interscience Publishers. Zhang, S. (1987). Ultrastructure of ocellus in the oncomiracidium of Diplorchis sp. (Monogenea, Polystomatidae) (in Chinese). Acta Zoologica Sinica 33, 380. Zhang, S. and Lang, S. (1990). Tegument of Diplorchis hangzhouensis (Monogenea: Polystomatidae) in various stages of life cycle (in Chinese). Acta Zoologica Sinica 36, 217-221.

Sealice on Salmonids: Their Biology and Control A.W. Pike and S.L. Wadsworth

Department of Zoology. University of Aberdeen. Tillydrone Avenue. Aberdeen AB24 2TZ. and Marine Harvest McConnell. Lochailort. Inverness-shire PH38 4LZ. UK Abstract ..................................................................... 234 1. Introduction ............................................................. 234 1.1. What are sealice? .................................................... 235 1.2. Histon/ and Present Status of the Problem ............................ 236 1.3. Aims of this Review.................................................. 237 2 . Species, Morphology. Host Range and Geographical Distribution ...........238 2.1. Species of Sealice on Salmonids ...................................... 238 2.2. Host Range.......................................................... 239 2.3. Distribution on the Host .............................................. 240 2.4. Geographical Distribution of Infections on Wild and Farmed Salmonids . 242 2.5. Morphology of Lepeophtheirus salmonis and Caligus elongatus ........243 2.6. Other Species ....................................................... 245 3. The Reproductive System and Reproduction ............................... 245 3.1. Structure of the Reproductive System ................................. 245 250 3.2. Mating .............................................................. 3.3. Oviposition .......................................................... 254 3.4. Larval Development .................................................. 258 4 . Life Cycles of Sealice ..................................................... 261 4.1. General Pattern ...................................................... 261 4.2. Transmission Biology ................................................ 261 4.3. Generation Times .................................................... 266 4.4. Adult Life Span ...................................................... 266 267 4.5. Seasonal Effects ..................................................... 4.6. Laboratory Maintenance .............................................. 268 5 . Epidemiology of Sealice Infections ........................................ 268 5.1. Wild Salmonids ...................................................... 269 5.2. Cage-cultured Salmon ................................................ 273 5.3. Hydrographical Effects on Copepodid Dispersion ...................... 275 5.4. Interaction between Wild and Farmed Salmonids ...................... 276

ADVANCES IN PARASITOLOGY VOL 44 ISBN 0-12-031744-3

Copyright

0 1999 Academic Press

A// rights of reproduction in any form reserved

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6. Physiology of Sealice..................................................... 6.1. Nutrition ............................................................ 6.2. Osmoregulation ..................................................... 6.3. Endocrine Control .................................................... 6.4. Sensory Biology ..................................................... 7. Pathological Effects of Sealice on Salmonids .............................. 7.1. Mechanical Damage Caused by Infection .............................. 7.2. Pathophysiological Effects of Infection ................................ 7.3. Host Responses...................................................... 7.4. Transmission of Pathogens by Sealice ................................ 8. Treatment and Control of Infection ........................................ 8.1. Chemotherapeutic Treatments ........................................ 8.2. Wrasse .............................................................. 8.3. Management ........................................................ 8.4. Future Control Strategies ............................................. 9. Economics of Sealice Infection ............................................ 9.1. Treatment Costs of Sealice Infection in Scotland ....................... 10. Priority Areas for Future Sealice Research ................................. Acknowledgements .......................................................... References...................................................................

279 279 282 284 284 286 287 290 291 292 292 292 303 304 306 311 311 317 318 318

ABSTRACT

Lepeophtheirus salmonis and Caligus elongatus are the two common species of sealice responsible for serious disease problems in salmonid aquaculture . L . salmonis in particular is the most serious parasitic infection on Atlantic salmon farms in the Northern Hemisphere and is the best-known species. This review examines the voluminous literature on the biology and control of sealice and brings together ideas for developing our knowledge of these organisms . Research on the distribution. host range. structure. life cycle. epidemiology. laboratory maintenance. reproductive biology. physiology and pathogenesis is reviewed in depth . The control strategy and economic cost to the industry is discussed. The interactions between wild and cultured salmonids are examined .

.

1 INTRODUCTION

Crustacean parasites of fish present an impressive diversity of morphological and functional adaptations for life mainly on. but occasionally inside. their hosts . These intriguing examples of evolutionary flexibility are thoroughly reviewed elsewhere (Kabata. 1970. 1981. 1984). Crustacean parasites.

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belonging principally to the Copepoda, but also to the Branchiura, Isopoda, Amphipoda, Cirripedia and Ostracoda (in descending order of importance), can be extremely damaging to their hosts Kabata (1984). Among the most harmful are those that, although ectoparasitic, nevertheless reach the internal organs by means of elaborate and invasive attachment organs through which they obtain nutrients. These sedentary species differ significantly from the more mobile species, such as those found in the Caligidae, that restrict their feeding activities largely to the host’s epidermis. The majority of species from fish occur in the marine environment but the smaller number from freshwater include some species that are important in aquaculture. Salmonids (Salmonidae of the genera Salmo, Salvelinus and Oncorhynchus) are known hosts to only seven genera of crustacean parasites (six copepods and one branchiuran), two of which currently represent the greatest threat to salmonid culture in the marine environment. They have achieved a justifiable notoriety over the last three decades because of the damage caused on, predominantly, Atlantic salmon farms first in Europe but, subsequently, also in North America. Kabata (1973) anticipated such problems but his timely warning was unlikely to have averted subsequent events. However, it is still worth reminding fish-farming enterprises around the world, where both salmonids and other non-salmonid species are being raised, of the threat posed by ectoparasites in general and sealice in particular. 1.1. What are sealice?

1.1.1. Common names Sealice are Copepoda of the family Caligidae. This review is devoted to species belonging to the genera Caligus Miiller 1785 and Lepeophtheirus Nordmann 1832, and, in particular, to C. elongatus Nordmann 1832 and L . salmonis K r ~ y e r 1837, the two species causing the greatest problem, although C. teres Wilson 1905 is currently causing serious problems on Atlantic salmon farms in Chile (C. Wallace, personal communication) together with an indescribed Caligus species. L . Salmonis is sometimes referred to as the salmon louse, because of its predilection for salmon of various species, but since it also naturally inhabits sea trout and other species, it is not appropriate to use this term. The Norwegians use the vernacular term ‘lakselus’ for Lepeophtheirus salmonis - see Berland and Margolis (1983) for an interesting account of its derivation and of the early records of sealice. Johnson and Margolis (1994) give white spot and summer lesion syndrome as additional North American names for sealice.

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1.1.2. IdentiJication The history, systematics and taxonomy of the family Caligidae have been reviewed comprehensively by Kabata (1979). The two common species of sealice, C . elongatus and L. salmonis are easy to identify and to distinguish, although earlier difficulties with specific identifications were resolved by Parker (1969) and Margolis (1958), respectively. In North America, however, additional species of Caligus exist which are not easily distinguishable to the unaided eye. Johnson and Margolis (1994) provide a key to these species.

1.2. History and Present Status of the Problem

The increase in salmon production has been dramatic. In 1998 world production was expected to reach around 690 000 tons (Table l), of which half will be from Norway alone. Production has increased 3-4-fold in Norway and Scotland over the last decade, but Chile has tripled its production in just 5 years. The first outbreaks of sealice infection caused by L. salmonis occurred on Norwegian Atlantic salmon farms during the 1960s soon after cage culture began. Similar events overtook the Scottish Atlantic salmon farming industry in the mid-1970s when the annual production was only

Table I World-wide Atlantic salmon production (thousands of tons). Country

Year of production 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998'

-

Norway UK Chile Canada USA Iceland/Faroes Ireland Sweden Total

74 17

110 28

130 30

155 41

130 36

180 49

n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 6 4

10 6

16 6

21 9

21 10

17 12

n.d. n.d. n.d. n.d. n.d.

n.d. -

207 249 64 72 54 34 30 32 17 14 15 8 12 12 9 8 384 453

292 316 83 100 77 97 34 46 18 20 17 21 14 16 11 10 546 626

345 115 108 50 21 22 17 12 690

Data from Institut FranCais de Recherche pour 1'Exploitation de la Mer and Lindsay Laird, personal communication. n.d. = no data. Expected production

'

SEALICE ON SALMONDS

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500 tonnes (Rae, 1979). In North America, Atlantic salmon farming on the Atlantic coast began to experience similar problems in the late 1980s owing initially to infections of C . elongatus. The early epizootics initially stimulated research into sealice biology and control, and rapidly led to successful use of the organophosphates trichlorphon (Brandal, 1979; Brandal and Egidius, 1979) and dichlorvos (Rae, 1979) for their control. With the benefit of hindsight, this early success, although vital to the subsequent expansion of the industry, was nevertheless counterproductive because further research into sealice biology declined until disease problems escalated, with the subsequent immense increase in salmon production. The corresponding increase in use of these treatments has succeeded in avoiding decimation of the industry but has provoked acrimonious debate with the environmental organizations about their potential effects on non-target marine organisms. The economic cost of sealice infections is considerable. In Scotland alone, the costs due to sealice are estimated at f15-30 million in 1998. Norway’s costs are put at Nk 500 million for 1997. Canada estimated its costs at $20 million for 1995. 1.3. Aims of this Review

With a remarkable degree of prescience, Kabata (1981) stated ‘All in all, the next two decades promise substantial progress in our understanding of parasitic Copepoda’. Although it may not be true of copepods in general, it is certainly true of sealice biology. For some areas, the research effort into sealice biology has increased enormously in the last decade. Much of it has been directed to finding new treatments but also, significantly, to increase fundamental knowledge of the parasites as a basis for developing more widely accepted methods of control. The commercial importance of sealice on salmon farms is such that most recent knowledge of these organisms is based on studies and material from commercial rather than wild sources. Furthermore, the industry, in Scotland at least, funds significant amounts of research not directed solely to treatment and control. Many of the developments in control strategies have come from commercial organizations owing to the economic pressures of sealice infection on their industry. Our aim is to examine the sealice literature critically, to summarize what is known and to highlight areas of research for which there is a need. The majority of papers are about L. salmonis because this species is the most common and causes the greatest damage to cage-culture populations, but information is also included, where available, on C. elongatus and other species of both genera. Not all the literature quoted originates in

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conventional peer-reviewed journals. To invoke this restriction would omit contemporary thinking, and good practice, that may never find its way into the conventional literature because it is practice evolved on farms rather than in laboratories.

2. SPECIES, MORPHOLOGY, HOST RANGE AND GEOGRAPHICAL DISTRIBUTION 2.1. Species of Sealice on Salmonids

2.1.1. Lepeophtheirus salmonis and Caligus elongatus Lepeophtheirus salmonis Krrayer is the only member of this genus, containing 90 species, that is found naturally on salmonids and to which it is almost exclusive (Kabata, 1973, 1979). About 200 species of Caligus have been described, all but one of which parasitize marine fish (Kabata, 1979). C . elongatus is found on over 80 species of fish (Parker, 1969; Kabata, 1979).

2.1.2. Other Species One other species of Lepeophtheirus, L . cuneifer Kabata, has been reported on farmed salmonids (Johnson and Albright, 1991~).The many papers on L . pectoralis, from mainly pleuronectid flatfish, that examine its reproductive biology (Anstensrud, 1990a,b,c,d, 1992) and taxonomy and ecology (Boxshall, 1974a,b,c,d,e,f, 1976, 1977; Boxshall and Bellwood, 1980) are also of interest with regard to the study of sealice on salmonids. Other species of Caligus that infect salmonids in the Northern Hemisphere are C. clemensi Parker & Margolis 1964, C . curtus Miiller 1785 and C . orientalis Gussev 1951. C . teres and C . longicaudatus Brady 1899 have been reported from salmonids in the Southern Hemisphere. Owing to the fact that wrasse are used in salmonid culture to control sealice, it is worth noting that C . centrodonti Baird 1850 has been recorded on ballan, goldsinny and rock cook wrasse (Bron and Treasurer, 1992; Costello et al., 1996; Karlsbakk et al., 1996), although this species does not seemingly invade salmonids (Bron and Treasurer, 1992). C . labracis Scott 1902 has been recorded from ballan wrasse (Costello et al., 1996). C. elongatus occurs only rarely on wrasse (Bron and Treasurer, 1992; Costello et a/., 1996).

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239

2.2. Host Range

2.2.1. Lepeophtheirus salmonis and Caligus elongatus L. salmonis is essentially a parasite of salmonids and has been recorded on Salmo salar L., Atlantic salmon, S . trutta L., sea trout, Salvelinus fontinalis Mitchill, brook trout, S. alpinus L., Arctic charr (A.W. Pike, unpublished observations), S. leucomaenis Pallas, white-spotted charr, Oncorhynchus clarki Richardson, cutthroat trout, 0. gorbuscha Walbaum, pink salmon, 0. keta Walbaum, chum salmon, 0. kisutch Walbaum, coho salmon, 0. masou Brevoort, masu salmon, 0. mykiss Walbaum, rainbow or steelhead trout, 0. nerka Walbaum, sockeye salmon and 0. tshawytscha Walbaum, chinook salmon (Margolis and Arthur, 1979; Nagasawa et al., 1987, 1994; Johnson and Margolis, 1994). Other, non-salmonid, host records are given in Kabata (1979), Margolis and Arthur (1979) and Johnson and Margolis (1994). Information about these hosts is available in Laird and Needham (1988). C . elongatus has a very wide host range according to Parker (1969) and Kabata (1979). Over 80 species of both elasmobranch and teleost fishes belonging to 43 families have been recorded (Kabata, 1979).

2.2.2. Other Species

L. cuneifer from a variety of elasmobranch and teleost fish in southeastern Alaska (Kabata, 1974a) has been found by Johnson and Albright (1991~)on farmed Atlantic salmon and rainbow trout. C . clemensi, first recorded from the pink, coho and chum salmon by Parker and Margolis (1964), has been recorded on sockeye salmon and rainbow trout (Margolis and Arthur, 1979) and is thought to infect chinook salmon (Johnson and Margolis, 1994). Interestingly Parker and Margolis (1964) believe that the parasite is specific more to the environment than the host, this being sheltered coastal waters where it can colonize juvenile salmon, in particular. C . curtus is regarded by Kabata (1979) as a primarily gadid parasite but with a range of other hosts including elasmobranchs. It has also been found on Atlantic salmon by Hogans and Trudeau (1989b) who nevertheless regard it as a rare event and of little significance commercially. C . longicaudatus has been found only on farmed sockeye salmon, which Jones (1988) rightly considers to be a secondary host for the parasite. C. orientalis reported by Urawa and Kato (1991) as severe infections on farmed rainbow trout also occurs widely on other, nonsalmonid, species. C. teres was found on farmed coho salmon by Reyes and Barvo (1983a,b).

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2.3. Distribution on the Host

2.3.1. Lepeophtheirus salmonis Jaworski and Holm (1992) examined the overall distribution of preadult and adult L. salmonis on Atlantic salmon in cage culture, and recorded their findings using a more refined method of describing distribution. For this, they calculated the surface area of delineated zones of the salmon’s body surface, and calculated the ratio of sealice per zone to total infection for young (24-44 cm) and older (44-75 cm) salmon (Figure 1). The anterior distribution of sealice on the young salmon is noticeable compared to older salmon, where a more variable distribution is evident with many around the post-anal region. Larger fish harboured more sealice and treatment did not affect distribution, except for those post-anally on larger salmon. Using experimentally infected single-stage and sex populations on 300 g post-smolts, Pike et al. (1993~)found that mobile stages of sealice tend to position themselves dorsally and anteriorly, aggregating mainly just behind the head of the fish, but a more complex pattern existed with mixed sex and stage populations. Laboratory experiments with both manipulated infections of mixed mobile stages and single-cohort copepodid infections of sealice on Atlantic salmon suggested that adult males might be strongly implicated in this distribution pattern. When present, adult males tend to occupy the preferred anterior dorsal sites with other stages occupying lateral and posterior locations (Hull et al., 1996). Prior to the appearance of adult males, single cohort infections of preadult I and I1 stages were found evenly mixed on the dorsal surfaces of the host. As the adult males appeared, preadult stages of both sexes tended to be found in ventral and posterior locations. It has been suggested that this could be ‘refuge seeking’ behaviour by the other stages to avoid frequent disturbance by the more active adult males (M.Q. Hull, personal communication). The exceptions to this rule are preadult and unmated adult females, which are also found in this dorsal anterior region when pairing with adult males. Furthermore, it appears that the density of sealice on the surface of the fish is linked to the distribution pattern, with more mobile stages being found in more ventral and posterior locations, as the intensity of infection increases (Hull et al., 1996; Hull, 1997). Attached stages have a different distribution from mobile stages. Most of the copepodids settle on the skin, at the base of the dorsal fins but some may be found in all regions of the body (Johnson, 1993; Finstad et al., 1994; Dawson et al., 1997). The gills have been reported as settlement sites by Bron et al. (1991) and Johnson (1993), but Dawson et al. (1997) found very few in this location.

41

a.a. 27.2%

29.5%

I

51

0.

h.

a.b.

b.p.

a.a.

a.p.

p.a.

c.f.

Fish body regions

Figure I Percentage surface area of Atlantic salmon body regions and the distribution of mobile L. salmonis, by body region, for (a) small and (b) larger salmon. a.a = anterior abdomen; a.p. = posterior abdomen; a.b. = anterior back; b.p. = posterior back; c.f. = caudal fin; h. = head; 0.= operculum; p.a. = postanal. Cross hatched bars before treatment or untreated, open bars after treatment. (Adapted with permission from Jaworski and Holm, 1992.)

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A.W. PIKE AND S.L. WADSWORTH

2.3.2. Caligus elongatus C. elongatus adults are not restricted to any area of the host’s skin according to Hogans and Trudeau (1989b), but they note that more adults were found on the head and anterior abdomen than elsewhere on the body of cultured Atlantic salmon. Hogans and Trudeau (1989a) found that the ventro-lateral surfaces near the pelvic fins, around the anus and at the base of the dorsal fin, were preferred sites of attachment on cultured smolts and market-sized Atlantic salmon. The mobility of this species and its tendency to leave the host when disturbed makes reliable recording of distribution difficult. Hogans and Trudeau (1989b) found attached stages anywhere on the body of cultured Atlantic salmon. 2.4. Geographical Distribution of Infections on Wild and Farmed Salmonids

2.4.1. Lepeophtheirus salmonis and Caligus elongatus Experience bears out what has been stated in the literature (Kabata, 1979): L. salmonis is restricted to the Northern Hemisphere, where it is considered to be circumpolar in distribution, whereas C. elongatus is very widely distributed in both the Northern and Southern Hemispheres. In terms of species representation, Caligus is considered to be more tropical and subtropical than Lepeophtheirus, which is typical of temperate latitudes (Kabata, 1979). L. salmonis is the dominant cage-culture species on the northern Pacific and Atlantic coasts of Canada and the USA (Smith, 1998), and the Pacific coast of Japan (Nagasawa and Sakamoto, 1993), in Ireland, Norway and Scotland. C. elongatus has a local and often seasonal significance as a cageculture species in Ireland and Scotland. It is not common in Norwegian farms. Initially, it was the dominant species on Atlantic salmon farms in Atlantic Canada, accounting for over 97% of all sealice found in New Brunswick (Hogans and Trudeau, 1989a,b), but its importance has been eclipsed by the appearance of L. salmonis. It is not regarded as a problem species on the Pacific coast of Canada. The combination of world-wide distribution, broad host range and widely recognized propensity for leaving the host to become temporarily planktonic (Neilson et al., 1987) should make C. elongatus a serious threat to salmonid culture world-wide. Why then is C. elongatus so conspicuous in its absence from Norwegian (Bristow and Berland, 1991) and Pacific coast Canadian cage-culture systems? Furthermore, with some localized exceptions, L. salmonis is dominant in cage culture where both species are known to

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co-occur, raising the question, why is C . elongatus less common in these circumstances? One explanation for this apparent inconsistency is that there is competition between the species, whereby C. elongatus is displaced by L. salmonis, for which salmonids are the natural and almost exclusive hosts. There is little evidence for this possibility, however. Another aspect of geographical distribution relates to the spread of the farming of Atlantic salmon to parts of the world where it does not occur naturally, namely, the Southern Hemisphere. It might be reasonable to conclude, from the foregoing, that Atlantic salmon farms in these areas would be exposed mainly to C . elongatus infections, although there could be geographical strains of C . elongatus that are more or less deleterious than those to which Atlantic salmon are currently exposed. However, if L. salmonis was inadvertently introduced into farms in the Southern Hemisphere, could this species survive and establish itself to become as serious a problem as elsewhere? Notwithstanding assumptions about the distribution of the species (Kabata, 1979), it may be that its distribution is determined as much by host distribution as environmental determinants. There is general acceptance that the introduction of host species to new habitats does expose that host to new parasites that could establish and create disease problems; in this instance, not only copepods but also other crustacean parasites. 2.4.2. Other Species C . clemensi is a Pacific species that has been reported on salmonid and other teleost fish from Pacific Canadian waters (Kabata, 1988) but not from elsewhere. Johnson and Margolis (1994) cite an outbreak with this species on cage-cultured salmonids in British Columbia. C . curtus has been reported only once from Atlantic Canada (Hogans and Trudeau, 1989b) and not at all from Europe, where it is also common on non-salmonids (Kabata, 1979). C. orientalis has not been reported outside Japan. Similarly, C . longicaudatus is known only from New Zealand. L. cuneifer is known only from Pacific Canadian waters.

2.5. Morphology of Lepeophtheirus salmonis and Caligus elongatus 2.5.1. Adults

Detailed morphological descriptions and drawings of adult C. elongatus and L. salmonis (see Table 2) are given by Kabata (1979), Johnson and Albright (1991b) and Schram (1993). Adults of C. elongatus develop directly from chalimus IV (Piasecki, 1996), whereas those of L. salmonis develop through

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A.W. PIKE AND S.L. WADSWORTH

two preadult stages. The prominent sexual dimorphism in L. salmonis, where the females are approximately twice the size of the males, is absent in C . elongatus, where females are only slightly larger than males. The adults of the two species are readily distinguished: C . elongatus is smaller, the young adult females being 4.93 f 0.26 mm long and the males 4.33 f 0.25 mm long (Piasecki, 1996). Both sexes are light brown in colour and with a pair of prominent lunules (sucker-like organs) situated antero-laterally on the ventral cephalothorax. L. salmonis is light to dark brown and without lunules. The females are up to 11.52 f 0.07 mm long, excluding the long, light-coloured, trailing egg strings, whereas the males are up to 6.22 f0.29 mm long according to Schram (1993) (but see below). Considerable variation in size of adult L. salmonis has been reported associated with the source of the specimens, i.e. farmed or wild, location and season. L. salmonis from wild salmon are larger than those from farmed salmon collected at similar times in the year (Jackson and Minchin, 1992; Tully and Whelan, 1993). Sharp et al. (1994) give other data on the cephalothorax lengths of L. salmonis from farmed salmon and wild sea trout that reveal a large variability in size between and within sites. Tully (1989) has shown that there is a negative correlation between body length and water temperature for adult male L. salmonis and gravid female C . elongatus: body length increases with declining water temperatures. Ritchie et al. (1993) showed that there were seasonal changes in adult female cephalothorax length whereby size increased in winter and decreased in summer. Temperature was negatively correlated with cephalothorax length whereas there was no significant effect owing to photoperiod. Further investigation of body size in sealice would be worthwhile, although the problems of obtaining reliable samples from wild sources and of avoiding artefacts in farmed samples should not be underestimated. The particular relevance of such studies lies in the fact that body size varies with season and this affects egg output (Ritchie et al., 1993). This will be an important parameter in modelling the dynamics of sealice populations. Preadult L . salmonis possess the body shape of the adults except for the shape and size of the genital complex and urosome. These differences, together with other details of their morphology, are summarized in Table 2. The internal anatomy of caligid copepods is described in detail by Wilson (1905). 2.5.2. Larvae The number of larval stages is the same for both L. salmonis and C. elongatus, that is, two nauplius, one copepodid and four chalimus.

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Johannessen (1978) studied the early stages of L. salmonis. The larvae of L. salmonis have been described fully by Johnson and Albright (199 1b) and Schram (1993). In both cases the stages are described from eggs laboratoryreared at 10-12"C, the former from Pacific Canada and the latter from Norway. The two papers are complementary in that Johnson and Albright (1991b) provides very detailed anatomical descriptions, whereas Schram (1993) gives information on pigmentation and other features that assist with the practical task of distinguishing stages (Table 2). The morphology of C. elongatus larval stages has been studied in detail by Piasecki (1996) using specimens grown on Arctic charr at 10°C and Pike et al. (1993~)described the development of the early stages of this species from eggs of gravid adults on rainbow trout reared in sea water. Larvae can be sexed from chalimus IV according to Schram (1993). 2.6. Other Species

The morphology of other species of Caligus and Lepeophtheirus are given in the papers that have been mentioned earlier. Johnson and Margolis (1994) give a key to the species infecting salmonids in Canadian waters. There are detailed accounts of all stages in the life cycle of C. clemensi; the adults were described by Parker and Margolis (1964) and the larval stages by Kabata (1972). Descriptions of the adults and larvae of other species of Caligus and Lepeophtheirus are reviewed by Kabata (198 1).

3. THE REPRODUCTIVE SYSTEM AND REPRODUCTION

Published information relates mainly to species of Lepeophtheirus and the descriptions that follow are based on L . salmonis unless stated otherwise. 3.1. Structure of the Reproductive System

Early descriptions of the reproductive system of caligid copepods were made by Scott (1901) and Wilson (1905). Subsequently, Ritchie et al. (1996b) described the morphology and histology of the reproductive system of L . salmonis using light and electron microscopy. The reproductive organs of both sexes are paired with the ovaries or testes situated in the cephalothorax behind the level of the eyes. Oviducts and vasa deferentia arise from their respective gonads and pass posteriorly into the genital complex ( = segment). In females, there are paired receptaculum seminis organs and

A.W. PIKE AND S.L. WADSWORTH

246

Table 2 Developmental stages of Lepeophtheirus salmonis,copepodid to adult, to show features useful for identification. Data from Johnson and Albright (1991b) and Schram (1993). (Figures reproduced with permission of Taylor & Francis from Schram, 1993.) Feature

Lengthlwidth fSD mm (n) Johnsonand Albright (1991b) Lengthlwidth fSD mm (n) Schram (1993)

Body shape

Appendages

Chalimus I

Copepodid

0.70f0.01/0.28f0.01 (25)

1.21f0.05/0.50rt0.02 (12)

0.68f0.02/0.23i0.01 (15)

1.06f0.0210.48f0.02 (21)

Cephalothorax slender, oval, formed from cephalic and first 2 thoracic segments; posterior region with 4 segments. Dorsal shield present

Cephalothorax broader, includes 3d thoracic segment (in Pacific larvae); posterior margin with 2 indentations. Frontal filament present.

Well-developed rostrum; antennae flexed ventrally.

Fourth leg a short bulbous outgrowth

~

Pigmentation

Body colour black and brown due to distribution of pigment. Dark red pigmented eyespots.

Brown, distributed throughout cephalothorax; unpigmented area around eyespots. Black pigment absent.

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Table 2 Continued. Chalimus I1

Chalimus 111

Chalimus IV

2.20M.0811.e0.06(11) 2.773J0.18/1.34+0.11(12) 1.78M.l6/0.78*0.07(13) 2.293J.14/1.05+0.07(39) 1.28M.08/0.52M.04(15)Dlder chalimus 111 2.06M.14/0.95*0.07(44) Cephalothorax with partially incorporated fourth thoracic segment. Segmentation indistinct. Urosome segment boundaries absent.

Second leg with interpodal bar; third leg broad, plate-like; fourth leg two segmented. Similar to chalimus I; ventral sparse but scattered brown spots.

Cephalothorax anteriorly pointed, midpoint widest, includes 3 pedigerous segments; cuticular sutures H-shaped. Segmentation regained 3n telescoped urosome (illustrated). Older larva urosome normal Fourth leg further developed. Fifth leg short bulbous outgrowth Brown, widespread; concentrated around suture lines. None anterior cephalothorax; Urosome sparse.

Cephalothorax more opaque, dorso-ventrally flattened, widest posteriorly; lateral margins convergent. Segment boundaries indistinct in urosorne and between genital complex Second leg membrane on posterior margin. Fourth leg sympod larger than exopod. Fifth leq two seqmented. Similar to Chalimus 111.

A.W. PIKE AND S.L. WADSWORTH

Table 2 Continued. Pre-adult I female

Pre-adult II female

Ovigerous female

5.40M.51/3.24*0.16(8) 9.96*1.55/4.46*0.35(26) 3.59f0.I 2/1.89M.05( 75)

5.18 M .55/2.91 f0.31 (26) 8.39fl.1 8/4.02f0.27( I 8)

Cephalothorax as adult, marginal membrane broad, frontal plates fully developed. Ovoid genital complex cuticular folding antero-laterally. Urosome segmented, separated from genital complex.

Cephalothorax as preadult I. Genital complex larger with cuticular folds antero-laterally; lobed postero-laterally. Urosome narrows near genital complex; indistinct separation.

Cephalothorax round to oval; fifth thoracic segment narrower than genital complex. Latter ovoidlelongate with straight sides. Urosome cylindrical, same length as genital complex.

Fifth leg a bulbous outgrowth of genital complex.

Fifth leg on posterolateral aspect of genital complex.

Fifth leg ventral surface of genital complex lateral to oviduct openings.

Brown, widely scattered, esp. along suture lines and laterally on genital segment. Not posterior abdomen or caudal rami.

Brown widely scattered, esp. along suture lines and laterally on genital segment. Not posterior abdomen or caudal rami.

Light brown to terracotta to copper or venetian red; Darker pigmentation some distance from rim of genital complex.

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SEALICE ON SALMONDS

Table 2 Continued. Pre-adult I male

2.90*0.45/1.81k00. 16(10)

Pre-adult I1male

Adult male

4.27+0.52/2.6Ok0.40(6) 5.40kOo.48/3.25*0.35(15)

3.35*0.16/1.58kO0.O5(33)4.31 +0.14/2.17f0.07( 34) 5.06*0.24/2.72*0.12( 32) Similar to pre-adult I female. Genital complex barrel shaped to ovoid as it ages. No cuticular folds.

Fifth and sixth leg growths visible; former more anterior than in female.

but none on lateral genital complex.

Similar to but narrower than pre-adult II female. Genital complex longer and ovoid. Urosome constricted laterally.

Cephalothorax round to oval. Genital complex larger, ovoid. Urosome cylindrical, shorter than genital complex and constricted anteriorly.

I Fifth and sixth legs incomplete; four and three setae respectively.

Fifth and sixth legs complete; four and three setae respectively.

and caudal rami.

red. Heavy on genital complex and first

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A.W. PIKE AND S.L. WADSWORTH

cement glands. Both open into the genital antrum where they respectively furnish sperm to unfertilized ova and cement to form the egg sac. The receptaculum seminis organs also communicate with the genital orifice, through which they receive sperm via the spermatophore tubes that grow out from the spermatophores after insemination by the male. In males, each vas deferens is divided into anterior and posterior regions, the latter entering the spermatophore sac in the genital complex. A pair of cement glands is situated in the genital complex and discharge into the spermatophore sacs. Ritchie et al. (1996b) described the development of the gametes from histological and ultrastructural preparations, and additional unpublished information on the reproductive system may be found in Ritchie (1993).

3.2. Mating Mating in sealice is a complex process that was first described in elegant detail by Anstensrud (1990a,b,c,d, 1992) who worked on L. pectoralis on flounder. This and subsequent work on L. salmonis on Atlantic salmon (Hull, 1997; Hull et al., 1998) has been done using experimentally infected hosts to provide known age cohorts of sealice. Often, experiments have required sealice to be transferred from fish to fish to create particular combinations of sex and/or age of sealice. Until recently, the use of fish anaesthetics (MS222 or benzocaine) for immobilizing fish and manual placement of sealice on the host’s skin has been a part of the protocol for these experiments, although their effect on sealice is largely unknown. In more recent experiments, the use of anaesthetics has been avoided (Hull, 1997; Hull et al., 1998). In all experiments requiring transfer of sealice from one host to another, the possibility that sealice will fail to resettle has been recognized and every effort made to minimize the transfer times. Hull (1997) and Hull et al. (1998) developed techniques whereby mobile sealice were removed and then placed in the water column near the host, and allowed to resettle actively without intervention. Resettlement rates of over 80% were routinely achieved if the parasite was separated from the host for less than 24 hours, although the most consistent results were achieved with separation times of less than 30 minutes. It has been shown that mobile stages exhibit a range of survival times off the host with smaller ones lasting for the shortest times and larger, more mature stages lasting longer. Adult males can survive off the host for as long as 21 days and adult females for 22 days. Some adult males can also settle on the host after 5 days but subsequent survival was significantly reduced after 72 hours. M.Q. Hull (personal communication) also regarded separation times of longer than 30 minutes as potentially affecting the behaviour of resettled sealice. In many experiments

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251

where behaviour of the parasite is critical, it has been preferable to grow lice on the host and avoid transfer altogether. 3.2.1. Pair Formation Single-age infections of L. salmonis show that the development of male sealice is one stage ahead of the females (Hull et al., 1998). The first adult males appeared on day 15 after infection and adult females were present a day later. Adult males mate with newly moulted, virgin, adult females and will pair with these in preference to more numerous preadult I1 lice. It is not fully understood how pair formation is established. It is clear that intraspecific interaction and relative distribution of the mobile stages are important in the pair-formation process. Ritchie et al. (1996a) observed the formation of pairs and noticed that the preadult female secretes a temporary frontal filament. Pike et al. (1993~)demonstrated that the adult males stages were by far the most active on the surface of the host and that the motile activity of a single-sex population of adult males increased significantly in an experimental observation chamber when preadult females were allowed to settle on the host. This was further explored and males were observed forming pairs with females that were not bound by temporary filaments. However, it was observed that motile preadult females actively avoided pairing attempts by males and would lift the genital segment out of reach of the male when it was in the vicinity, and move to lateral and ventral locations on the fish where the adult males were not present. This presents the conundrum of how the precopular pairs are formed in the anterior dorsal region when the females appear actively to avoid both the males and this area. Hull et al. (1996) suggested that some, second, overriding factor must modify the behaviour of the female so that it enters into the area where the males are present and that this could be linked to preparation for moulting. The sensory mechanism by which males locate and identify females has not been characterized, but Hull (1997) has investigated this and his data are discussed in Section 6.4, p. 284. Mate choice is hierarchical for both L. pectoralis (Anstensrud, 1992) and L. salmonis (Hull et al., 1998). The former show a hierarchy in order of preference of newly mature virgins, preadult I or 11, older virgins with an expanded genital complex; the latter have a hierarchy of virgins, preadult 11, preadult I. L. salmonis would also mate with older virgins in which the genital complex had expanded. The basis for this selection is not clear but mechanoreception and chemoreception signals may be involved (Anstensrud, 1992). Hull et al. (1998) concluded that this preference was maintained in the presence of an adequate number of appropriate females but broke down once the ratio became adverse.

252

A.W. PIKE AND S.L. WADSWORTH

3.2.2. Mate guarding by males Mate guarding, in which male copepods hold the female for extended periods before spermatophore transfer, is common in copepods (Boxshall, 1990) and is found in both L. salmonis (Ritchie et al., 1996a) and C. elongatus (Piasecki and MacKinnon, 1995). Once a suitable female has been located, the male attaches to the preadult I1 female’s genital segment where

I

,

mp a2

s12

:

I

.

mp a i

:

nip a2

mp a2

mp a2

Figure 2 Mate guarding and copulation in L. salmonis (a) precopula, (b-e) copula, and (f) postcopula. Arrows represent movements of appendages. a2 = antennule; mp = maxilliped; s12 = second swimming leg; m = male; f = female. (Reproduced with permission from Ritchie et al., 1996a.)

253

SEALICE ON SALMONDS

it remains until the final moult is completed (Figure 2). This mate guarding, described for L. salmonis by Ritchie et al. (1996a), is almost identical to that described by Anstensrud (1990b) for L. pectoralis. The distribution of precopula pairs of sealice varies according to the conditions in which the salmon host is maintained. Ritchie et al. (1996a) found that, in tank experiments with a water flow of 2 L min-’, the majority of pairs were on the head, whereas with flow rates of 8.5 L min-’, or in sea cages, the main concentration of precopula pairs was in the posterior dorsal region (Figure 3). Hull et al. (1996) also observed pairs in the anterior dorsal regions under similar conditions, but suggested that the females in newly formed pairs migrate more posteriorly and ventrally on the host after the pair has been established.

Dorsal 1

I I I

I

I

0%

Ventral 1

I I

Ventral 2

I

I I

Figure 3 Distribution of precopula pairs of L. salmonis on Atlantic salmon smolts: (a) with a water flow of 2 L min-’ and (b) with a water flow of 8.5 L min-’. (Reproduced with permission from Ritchie et al., 1996a.)

254

A.W. PIKE AND S.L. WADSWORTH

3.2.3. Copulation The sequence of events ending in the application of the male’s spermatophores to the female’s genital orifices is shown in Figure 2. The process begins approximately 24 hours after precopula is established and takes approximately 7 minutes. Transfer of spermatophores is completed in a matter of seconds by the male who uses the second swimming legs for the purpose. Postcopula, where the precopula position is re-established, lasts for about 3 hours (Ritchie et al., 1996a), after which the male moves away. In the initial minutes of this phase, the male holds the newly transferred spermatophores in place with its maxillipeds. Male sealice already contain new spermatophores within the spermatophore sac whilst inseminating a female and Hull et al. (1998) have shown that multiple mating can occur within 48 hours of a previous mating. Where mating of L. salmonis was artificially delayed, by removing adult males from a mixed-sex population of sealice, mating resumed with reintroduced males without extended mate guarding (Hull et al., 1998). All females in the sample were mated, with 80% success (for correct attachment of both spermatophores), despite the morphological changes that take place to the genital complex. Previously mated female L. pectoralis that lost their spermatophores have been shown to mate a second time but less than 50% became inseminated (Anstensrud, 1990d). There is no information at present about female L. salmonis multiple mating. Piasecki and MacKinnon (1995) found that adult, male, C. elongatus numbers declined rapidly in their experimental infections and concluded that males die after copulation. They do not indicate if males are capable of more than one insemination.

3.3. Oviposition 3.3.1. Post-mating Behaviour of Females Female L. salmonis relocate to the dorsal adipose or anal fin areas to produce egg sacs (or egg strings) according to Ritchie (1993), although it is possible to find ovigerous females on other parts of the salmon’s body including the head. Since ovigerous females lay several batches of egg sacs without further male contact (see below), avoiding disturbance by mateseeking adult males seems to take priority over remaining in an anterior dorsal location (Hull et al., 1996). Mated females repositioned themselves in the presence of adult males but remained in an anterior dorsal location when males were absent.

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3.3.2. Production of Egg Sacs

The twin egg sacs that are extruded from the genital atria on the posterior edges of the genital complex of L. salmonis may be more than twice the body length, but vary greatly. Each fertilized egg is extruded through the external opening of the oviduct at which time it is enveloped in secretions of the cement gland (Wilson, 1905). Repetition of this process leads to the formation of the egg sacs. Egg sacs are chains of fertilized eggs enclosed in a layer of cement that binds the sac together. There is no outer cuticular membrane and this suggests that, once extruded, the eggs are functionally isolated from the female. Egg sacs are elongate, straight and tubular when produced normally but, in some conditions, experimentally raised females produce egg sacs that are partially coiled. It may be that extrusion of normal-shaped egg sacs is dependent upon water flow. The egg sacs remain attached to the female during incubation of the embryos but there is no evidence to suggest that this is a necessary prerequisite to successful eclosion, at least for egg sacs in the later stages of development, because nauplii within egg sacs detached from the female will continue to develop and hatch (Johannessen, 1978). However, it has been suggested (Z. Kabata, personal communication) that egg sacs fail to develop if removed from the female at an early stage in development. Whether this is due to loss of some component supplied by the female or to infection of the egg sacs with saprophytic micro-organisms is not clear. Piasecki and MacKinnon (1995) in their study of the C. elongatus life cycle make the valid point that oxygen content is a critical aspect of eclosion. They stated that female C . elongatus keep their egg sacs off the surface of the fish to achieve maximum exposure to oxygenated water. It is normal to see both oviducts filled with eggs inside the genital complex in gravid females, of both species, already incubating egg sacs. The rate of production has not been recorded but Johannessen (1978) showed that, for L. salmonis, egg sacs were replaced within 24 hours of the previous pair releasing all their nauplii, and Piasecki and MacKinnon (1995) stated that, for C. elongatus, oviposition occurs within ‘a few hours’ of the previous generation completing hatching. 3.3.3. Number of Egg Strings Produced per Female per Mating

Although control measures are designed to prevent the appearance of gravid females on cage-cultured salmon, inevitably the logistics of doing so mean that this can rarely be achieved. For epidemiological reasons, it is desirable to know what capacity female sealice have for producing more than one set of egg sacs. This information is probably only obtainable from experimental

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A.W. PIKE AND S.L. WADSWORTH

infections, which were completed by Ritchie (1993). This study showed that as many as six sets of egg sacs were produced by individual, single-mated females over a period of 50 days when maintained at 14°C and 6:18 h L/D. Since survival of sealice in experimental infections is limited (50% survival was recorded between 12 and 22 days in this experiment), it is conceivable that the number of sets produced was limited by the survival rate rather than ageing. Piasecki and MacKinnon (1995) state that adult female C.elongatus produce at least two sets of egg sacs but give no details of timing. 3.3.4. Length of Egg Sacs and Number of Eggs per Sac Egg sac length and number of eggs per sac are related, and vary widely between individuals. Figures for L. salmonis produced by Johannessen (1978) were for the range 100-500 eggs per sac (source unspecified) and Wootten et al. (1982) gave a figure of up to 350 eggs per sac for farmed Atlantic salmon of unspecified size. Differences in number of eggs per female between wild and farmed Atlantic salmon have been noted by Jackson and Minchin (1992). They found that L. salmonis from wild salmon, from the west of Ireland, produced egg sacs with 965 f 30.1 eggs per louse compared to salmon from cage-culture sites where the counts were 758 f 39.4 for untreated salmon and 297 f 19.1 for salmon that were routinely treated (sizes unspecified). Johnson (1993) found that the number of eggs produced by L. salmonis grown experimentally on mature Atlantic and chinook salmon were significantly different. The adult females from the two hosts did not differ in size, but the numbers of eggs per female were, respectively, 879.2 f 112.5 and 430.0 f 100.0. Ritchie (1993) demonstrated a significant correlation between egg-sac length and number of eggs per sac for three of the six sets of egg sacs produced in experimental infections with Atlantic salmon smolts (Figure 4). In these experiments, the first two generations produced a large number of non-viable eggs (Figure 5). Tully (1989) reported that mean numbers of eggs per sac in L . salmonis were significantly greater in January (315 f 135) than in August (107 f 19) on farmed Atlantic salmon smolts. Ritchie et al. (1993) studied infections on farmed Atlantic salmon, and also recorded seasonal changes to egg-sac length and number of eggs in which mean egg-sac length increased significantly from October to March and then decreased to August. During the observation period, egg-sac length was negatively correlated with temperature but not with photoperiod. Egg numbers per sac increased from 147 in October to 246 in March and then declined to 175 in August. During the same period, mean egg length also changed, declining from October until February and increasing progressively thereafter until August. The change in length in autumn preceded that of change in egg number, which Ritchie

257

SEALICE ON SALMONDS

+Q*

150

*o*

*+*+ +o

P

.-

.E

hg

100

5? 3

50

.

..

a "

1 0 0

4

6

10

8

12

Egg string length (mm)

Figure 4 Relationship between egg sac length and number of eggs per sac for six batches of eggs produced by adult female L. salmonis in an experimental infection. r 2 values are from linear regression. = batch 1 ( r 2 = 0.12); 0 = batch 2 ( r 2 = 0.09); = batch 3 ( r 2 = 0.75); 0 = batch 4 ( r 2 = 0.95); = batch 5 ( r 2 = 0.79); + = batch 6 ( r 2 = 0.70). (Reproduced with permission from Ritchie, 1993.)

+

8o

1

1

2

3

4

5

6

Batch Number

Figure 5 Change in mean percentage of egg sac length containing non-viable eggs in successive generations of egg sacs. (Reproduced with permission from Ritchie, 1993.)

2 58

A.W. PIKE AND S.L. WADSWORTH

et al. (1993) interpreted as indicative of being stimulated by factors other than temperature. It is worth noting, in relation to the data on egg numbers per egg sac, that Johnson (1993) stated that egg counts differed between sealice grown on mature and immature coho salmon. This has not been taken into account in these reports, so they should be viewed with caution until it is established if there are similar differences between mature and immature Atlantic salmon. Egg sacs of C . elongatus contain a mean of 89 eggs per sac according to Hogans and Trudeau (1989b). 3.4. Larval Development

The ontogenetic processes that take the fertilized ovum from the egg sac to chalimus IV on the host's skin are largely unknown, as are the physical and biological factors that control them. There are many reports on the developmental stages of both L. salmonis and C . elongatus, but these are concerned almost entirely with changes in morphology obtained from point sampling from experimental or cage-culture populations of sealice. The available information is reported here, together with data on the effect of physical factors on rates of development. 3.4.1. Egg Development Egg sacs of L. salmonis are cream coloured when first extruded from the genital complex. As development proceeds, they darken, in part owing to the appearance of pigment in the embryos. Fully developed egg sacs, ready for eclosion, are dark brown. Pike et al. (1993~)report a similar change in coloration of the egg sacs of C. elongatus. Development times from extrusion to eclosion are temperature dependent. Johnson and Albright (1991a) recorded development times of 17.5, 8.6 and 5.5 days at temperatures of 5, 10 and 15"C, respectively, for L. salmonis, whereas Johannessen (1978) recorded much longer development times of between 30 and 40 days at 9°C and around 10-12 days at 11.5"C. Piasecki and MacKinnon (1995) report development times for C . elongatus of 8 days at 10°C. 3.4.2. Eclosion The onset of eclosion is predictable from the colour of the egg sacs and the activity of the nauplii within, but what controls the onset is not clear. Davis (1968) reviewed eclosion in free-living copepods and concluded that changes

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in the permeability of the egg membranes occurs, which may or may not be accompanied by a change in osmotic pressure of the fluid within the egg membrane. Mature nauplii within the egg membranes are only intermittently active until they emerge from their compartment within the egg sac. At this point, the nauplius, still within the egg membrane, becomes much more active. There are no published descriptions of how the nauplius escapes from the egg membranes for L. salmonis or C . elongatus, but Lin and Ho (1993) described and illustrated the process for C . epidemicus. Eclosion begins at the distal end of the egg sacs when they are attached to the female, as observed by Johannessen (1978) and by Johnson and Albright (1991a) for L. salmonis, by Pike et al. (1993~)and Piasecki and MacKinnon (1995) for C . elongatus, and by Lin and Ho (1993) for C . epidemicus. Where egg sacs are incubated in isolation of the female, eclosion occurs at any point on the egg sac (Johannessen, 1978; Johnson and Albright, 1991a). The time scale for completion of eclosion varies widely. Johnson and Albright (1991a) report a range of 18-65 hours for L. salmonis at 10°C and Pike et al. (1993~)found the mean hatching period for C . elongatus to be 3.25 f 0.22 hours. 3.4.3. Development of Nauplius Stages The nauplius I and I1 of both L. salmonis and C . elongatus are non-feeding stages that depend upon internal reserves for nutrients. Both are active although intermittent swimmers and are photopositive (Johannessen, 1978; Pike et al., 1993). Nauplii of both species become less active before moulting (Johannessen, 1978; Pike et al., 1993). The mean duration of the nauplius I of L. salmonis is 52.0, 30.5 or 9.2 hours at 5, 10 or 15°C respectively, and the duration of the nauplius I1 is 170.3, 56.9 or 35.6 hours at 5, 10 or 15"C, respectively (Johnson and Albright, 199la). Similar variations in development times have been reported for C . elongatus. Pike et al. (1993) found that the duration of the nauplius I was 36.9,25.2 and 16.6 hours at 5, 10 and 15"C, respectively, and Piasecki and MacKinnon (1995) report times of 24 and 67 hours for nauplius I and 11, respectively, at 10°C. The wide differences probably reflect the greater developmental processes that take place between nauplius I1 and copepodid, compared to that occurring between nauplius I and 11. The late nauplius I1 becomes much less active and contains the clear outline of the copepodid within the body (Pike et al., 1993a). As Boxshall (1974a) observed for L. pectoralis, this is the first of two major metamorphoses. The moult to copepodid marks the end of the planktonic form to one that, although still planktonic, now possesses the capability of infecting the fish host. We know next to nothing about this transformation.

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3.4.4. Development of Copepodid Stage The copepodid is a more active swimmer than either of its predecessors. No further development takes place until it settles on the host’s skin; presumably the stimulus to proceed with the on-host phase of the life cycle is provided by the host itself, although there is no evidence one way or the other. The copepodid is positively phototactic and remains non-feeding, although a functional gut, including the external mouthparts, has developed. Survival of the copepodid is dependent upon the energy reserves left within the yolk sac. The process of host location and attachment is energetically demanding so depleted reserves at this point in the development process are critical. Pike et al. (1993~)have shown that survival rates for C . elongatus nauplius to copepodid are 90% at 15°C and decline to only 60% at 5°C. Whether this is associated with energy depletion is not known but might be worth investigating, since it could have an influence on the seasonal population dynamics of sealice. 3.4.5. Development of Chalimus Stages I-IV The second major metamorphosis in the life cycle of L. salmonis is that in which the planktonic copepodid transforms into a parasitic organism after settling on, and attaching to, the host’s skin. It will spend the rest of its life on the salmonid host. This transient phase is, nevertheless, a crucial step in the life cycle and again little is known about the processes that control it. After the copepodid has attached itself to the host’s skin, it is able to feed because a functional mouth has developed in the free-swimming phase. The copepodid will moult, in due course, to produce the chalimus I stage and thereafter chalimus 11-IV at a rate largely determined by the physical environment. There is no evidence that chalimus stages are found detached from the host and there would be more to be gained from quickly reestablishing on the host. Body shape gradually changes during development and comes to resemble that of the adult at the chalimus IV stage (Johannessen, 1974). The sexes can be distinguished morphologically from chalimus IV. Two further stages precede the adult: the preadult I and 11. Sexual maturation occurs at some time during these later stages but there is no information on the physiological mechanism by which it is achieved. However, it may be assumed that, in common with other crustaceans, it is hormonally and environmentally controlled (Adiyodi, 1985). An interesting study by Kuperman and Shulman (1977) on the seasonality of reproduction in Ergasilus sieboldi on the pike, Esox lucius, suggests that photoperiod plays a part in regulating maturation of the females. A similar study should be conducted on the sealice of salmonids.

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4. LIFE CYCLES OF SEALICE 4.1. General Pattern

All salmon and some trout migrate to sea for varying periods before returning to spawn in their home rivers. Some salmon migrate immense distances and are therefore oceanic for considerable periods of their life. According to data obtained experimentally (Sections 3.3.3 and 3.3.4), female sealice are not outstandingly fecund: females produce only a few thousand eggs. Nevertheless, there is plenty of evidence to show that wild salmon on the high seas are commonly infected with sealice and that prevalence rates are often high, although intensities are low (Nagasawa, 1985, 1987; Nagasawa and Yanagisawa, 1992; Nagasawa and Sakamoto, 1993; Nagasawa and Takami, 1993; Nagasawa et al., 1993). In all the available records, infections on salmon taken from the high seas are almost entirely of adult parasites, suggesting that infection is an intermittent event and possibly that adult sealice are long-lived. Examination of data on coastal salmonids, in particular sea trout from areas where there is no cage culture, shows a similar pattern of infection to high-seas catches (Tingley et al., 1997). These data do not suggest that transmission of sealice to salmonids is restricted to, or more likely in, shallower coastal waters where it might be expected that there would be greater densities of fish and sealice copepodids. The evidence points to a low level but consistent transmission of fish in coastal and oceanic waters. Nevertheless, this seems extraordinary in view of the limited output of larvae from females, the ephemeral nature of the infective stage as well as the purging effect of freshwater on lice infections. It is a matter of some interest that sealice are so successful given the unusual biology of their hosts. The situation on salmon in cage culture could not be more different. Here, the whole age and development range is frequently present, although the composition of the population varies according to the type and effectiveness of control measures in use.

4.2. Transmission Biology

Transmission is dependent solely upon the copepodid, which, like most freeliving infective stages, is time limited by endogenous energy supplies. In such circumstances, it is to be expected that the copepodid will exhibit a range of behaviours that will compensate for the limited life span by enhancing transmission success. Such behaviours have been identified from laboratory

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and field experiments, but knowledge of the copepodid in its natural environment is limited because it has been remarkably elusive in spite of determined efforts to find it. The few successful recoveries of copepodids by Costelloe et al. (1996a,b) were more concerned with dispersal and are discussed later (see Section 5.2, p. 273). Paradoxically, field experiments in which sentinel cages of salmon are deployed at various depths and locations produce substantial infections within 7 days - from around 20-65 (median c.38) sealice per fish at a depth of 0-4 m with artificial light supplementation (Hevrnry et al., 1997). Infections without artificial light but at the same depth were substantially lower at around 11-40 (median c. 19) sealice per fish. 4.2.1. Host Location Transmission-enhancing behaviour may be divided into two aspects: that which orientates the infective stage towards the host’s natural environment, and that which assists the infective stage to respond to and/or recognize a potential host. Identification of the preferred habitat of Atlantic salmon in coastal waters has been attempted by several researchers (Holm et al., 1982; Westerberg, 1982; Dnrving et al., 1985; Dutil and Coutu, 1988). The evidence from these studies suggests that salmon frequent surface waters down to 3 m, especially at night. Caged fish occupy the surface waters when feeding and at night (Ferns et al., 1995). On this basis it might be concluded that infective copepodids should orientate in surface waters at night through a generally upward movement in the water column. Host finding after orientation towards its natural environment might then require responses that stimulate movement in response to chemical signals and/or turbulence caused by fish. There is limited published research on the behaviour of copepodids upon which to create a model for host location and selection. Vertical movement in a water column has been investigated by Heuch et al. (1995) who discovered that copepodids of L. salmonis perform a significant die1 vertical migration when confined within 6 m deep plastic enclosures suspended in the sea. In these experiments, copepodids migrated to the surface during daylight and moved into deeper water at night. No effects were associated with changes in salinity or temperature. Nauplii were found to be located in deeper water and to move smaller distances. Based on these results, copepodids of L. salmonis do not orientate towards the hosts’ preferred location but rather cross over. As Heuch et al. (1995) conclude, it may be that this crossing over, as copepodids migrate upwards and salmon move downwards during daylight, allows transmission to take place. Hevrsy et al. (1997) showed that, for both artificially illuminated and naturally illuminated control sentinel fish in cages at depths of 0-4,4-8 and 8-12 m

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depth, infections were substantial in fish held at 0-4 m, but became vanishingly small at the greater depths. This confirms the presence of copepodids near the surface but also shows that fish held at greater depths acquire fewer parasites than might be expected if copepodids were moving to deeper layers at night. There is a clear need for these interesting studies to be extended. Heuch (1 995) established, from laboratory experiments, that copepodids , and aggregate in step salinity gradients. In single-step gradients of ~ Y w5%0 15%0over 34%0,the greatest aggregation response, with 80% accumulating just below the halocline, was observed at 15Y6 in illuminated conditions. More copepodids were in the upper layers where the gradients were lower but significant differences were obtained in the gradient sections compared to the same section of controls. In linear salinity gradients, peak numbers of copepodids were found at ~ 2 0 % in the light. The possibility that copepodids are merely avoiding low salinities is rejected and the possibility raised that copepodid responses to salinity gradients may orientate them in locations frequented by feeding salmon. Laboratory experiments, on a variety of species of both Caligus and Lepeophtheirus, have shown that copepodids react positively to directional light (Lewis, 1963; Johannessen, 1975; Boxshall, 1976; Bron et al., 1993a; MacQnnon, 1993; Pike et al., 1993~).This is also true of infective stages of other parasites, including the cercariae of Digenea, where similar hostfinding requirements exist. Interpreting this response in terms of behaviour in the natural environment is difficult as has been cautioned by Forward (1988) and Pike (1990), as it could be artefactual. Taken in conjunction with the work of Heuch et al. (1995), however, it seems reasonable to conclude that a positive phototaxis is part of the functional behavioural repertoire of sealice. Bron et al. (1993a) found that the L. salmonis copepodid response to light was positively correlated to intensity and that peak sensitivity was to light of wavelength 550 nm. There was no shadow response, a common feature of active infective stages, such as cercariae. No responses were elicited to chemical cues derived from a variety of body fluids and tissues in either flow through or static chambers. This conclusion was reached because of the observed lack of direct response of copepodids to the target stimulus. However, miracidia of Digenea often behave in a similar negative fashion even though it is accepted that chemoreception is part of their host-finding behaviour . Observations by Heuch and Karlsen (1997) showed that copepodids of L . salmonis were sensitive to vibrations and that vibrations of 3 Hz, with a threshold intensity of 5.1 x ms-2, produced the highest response. The stimulation induced some copepodids to swim c.9 cm in the first second of stimulation compared to a background of 2 cm. These responses are

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interpreted as being likely to induce swimming activity near a swimming fish to facilitate host location. Progress has been made in identifying copepodid behaviours, some of which seem to enable environmental orientation and others to allow response to fish. Nevertheless, we are far from understanding the role played by behaviour in securing the appropriate host species so it is worth revisiting this area of copepodid biology, given its central importance to maintenance of the life cycle. Despite what has been found about copepodid responses, there appears to be no careful description of normal behaviour in approximately natural environmental conditions, which would seem to be a prerequisite for comparison with elicited behavioural responses. 4.2.2. Attachment and Settlement The period of infectivity of the copepodids is time limited and temperature dependent. Wootten et al. (1982) suggested 4 days at 12°C for L. salmonis copepodids and rightly cast doubt on the longevity claimed for the copepodid by Johannessen (1978). Boxshall (1976) has given a maximum duration for the copepodid stage of L. pectoralis of 6 days and Voth (1972) gave a survival time of 4-6 days at 15°C for L . hospitalis. When the copepodid encounters a suitable host during this brief, free-living period, initial attachment to the host is achieved with the prehensile antennae and the maxillipeds followed by a more durable connection via a frontal filament. This structure persists throughout the larval development of sealice and in some instances has been found as a temporary structure in preadults. The frontal filament, a feature of most, if not all, siphonostomatoid copepods (Kabata, 1981) differs markedly between the two major species, C . elongatus and L. salmonis in both its morphology and origin. In the former it is a long slender structure, and in the latter it is short and stumpy. In both cases, however, it is finally attached directly to the fish scale by a basal plate after the epidermis has been disrupted by mechanical activity of the copepodid in C . elongatus (Piasecki and MacKinnon, 1993) and by the copepodid/chalimus I in L. salmonis (Jones et al., 1990; Bron et al., 1991). The literature on species of Caligus shows that there is a consistent pattern regarding the origin of the frontal filament. It is preformed within the body of the copepodid of C . elongatus (Piasecki and MacKinnon, 1993; Pike et al., 1993a), C . centrodonti (Gurney, 1934), C . epidemicus (Lin and Ho, 1993) and C . spinosus (Isawa, 1969). Piasecki and MacKinnon (1993) stated that the filament is deployed when the pocket, in which it is accommodated, evaginates. Although no details are given, it seems that the filament is simply extruded and cemented to the fish scale. Gurney (1934), studying C .

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centrodonti, noted that the filament is retained during the moult and that a new basal portion is added on each occasion, adding an increment to the length of the filament. The series of discrete, rounded bases produced was considered a reliable way of ageing the larvae. This has been confirmed subsequently for C. elongatus (Piasecki and MacKinnon, 1993). Bron et al. (1991) described the settlement and attachment of copepodids of L. salmonis and reported that the filament is secreted on to the surface of the fish scale. Neither Bron et al. (1991) nor Pike el al. (1993a) found any evidence of a preformed frontal filament within the copepodid body, but Bron et al. (1991) noted a region comprising three cell types that were identified as filament-producing glands. The ultrastructure of the frontal filament of C. elongatus has been described by Piasecki and MacKinnon (1993) and Pike e f a / . (1993a). The filament is constructed of tightly packed rows of fibres inside a sheath. The fibres have a helicoid arrangement. Anstensrud (1990b) resolved the debate about the function of a widely reported frontal organ on the anterior margin of the cephalothorax of caligid copepods. Kabata (1981) originally ascribed a chemosensory function on the basis of the presence of a microvillous surface on the organ, but Oldewage and Van As (1989) contradicted this view, on the basis of the absence of any neural organization associated with the organ. In a set of excellent scanning electron micrographs, Anstensrud (1990b) showed that the organ in L. pectoralis, which has the same structure as those already described (Kabata, 1981; Oldewage and Van As, 1989) was the origin of the frontal filament. An interesting postscript to this debate is the comment by Heegaard (1947) who, in reference to earlier discussion of the so-called ‘medial sucker’, said ‘This frontal medial sucker is nothing but the last remnant of the cement gland and the frontal filament’. Anstensrud (1990b) reported that a frontal filament was also produced by L. pectoralis as a temporary holdfast, just before moulting, during the preadult to adult stages. Until the publication of this report, it had been widely believed that the filament was only present, as a functional organ, in the larval stages. Wootten et al. (1982) stated that the preadult males of L. salmonis were attached initially but the females are rarely so, but Ritchie et al. (1996a) discovered that a frontal filament was temporarily formed during the moult from preadult to adult in female L. salmonis. Hogans and Trudeau (1989b) found ‘several gravid females of C. elongatus that still possessed fully developed frontal filaments’. This last observation has not been confirmed by Piasecki and MacKinnon (1993) but they did find inseminated adult females still attached to the host by a frontal filament. It has been reported that preadults of both sexes of C. epidemicus can produce filaments and that mating may be attempted between two attached individuals (Lin and Ho, 1993). The presence and function of temporary

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frontal filaments in adult sealice seems to be established, but available evidence does not confirm their importance or universal presence. 4.3. Generation Times

The first reported estimate of generation time for L. salmonis was that of Wootten et al. (1982), who used cage-culture data to produce figures of 42 days at temperatures between 9 and 12°C. Johnson and Albright (1991a) subsequently completed the life cycle in the laboratory and recorded generation times at 10°C of 40 days for males, and 52 days for females. In both cases, Atlantic salmon were the hosts. Again based on laboratory infections, Hogans and Trudeau (1989b) determined the generation time of C. elongatus from laboratory infections maintained at 10°C to be ‘approximately 5 weeks’. This was calculated from newly hatched nauplius I to adult female. Piasecki and MacKinnon (1995) reported a generation time of 43.3 days for C . elongatus at 10°C; they grew the parasites on Arctic charr. Tully (1989) estimated generation times for both L. salmonis and C . elongatus from data collected on untreated, caged salmon between July and January in Ireland. Both species show a highly significant correlation with water temperature. Based on the observed or estimated generation times, various authors have calculated the expected number of generations likely to be completed per year. According to Wootten et al. (1982), this would be three or four for L. salmonis, between May and October, but subsequent work by Wootten (1985) demonstrated that the cycle could continue at low water temperatures so the number of cycles could be up to six per year. Tully (1989) estimated that there were three generations of C. elongatus between July and January, which could translate into five or six in a year. Tully (1992) subsequently calculated 5-7 generations with a time scale of 120 days in winter but only 23 days in summer. Hogans and Trudeau (1989b) estimated that there could be between four and eight generations of C. elongatus per year on salmon on the Canadian Atlantic coast. The higher of these estimates seems unrealistic in view of their comments on water temperature. 4.4. Adult Life Span

The natural life span of adult L. salmonis has not been determined and it is difficult to see how this could be achieved reliably. Wootten et al. (1982) found that adults survived for at least 21 days in freshwater, and Johnson and Albright (1991a) kept adult L. salmonis alive for up to 18 days in sea

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water at 9-10°C and a salinity of 30-31%. In laboratory experiments using 200 g Atlantic salmon smolts maintained at 14°C and 18:6 L/D, adult L. salmonis survived 50 days after insemination, a total life span of up to 75 days (Ritchie, 1993). A prematurely aborted experiment at 6°C indicated an even longer life span at this lower temperature. This is confirmed by Nordhagen (1997) who recorded survival of L. salmonis for up to 191 days (egg to adult) at 7.4"C. Survival on wild hosts could be higher given that survival in laboratory conditions is generally low and, indeed, Jacobsen and Gaard (1997) present good circumstantial evidence that adult female L. salmonis can overwinter on wild salmon in the Norwegian Sea, perhaps extending the potential life span beyond 6 months. Piasecki and MacKinnon (1995) recorded survival of adult female C. elongatus on a single Arctic charr maintained in ambient overwinter conditions in a temperature range of 2.2- 12°C and found that lice survived for up to 260 days with a 60% survival at around 240 days. Conversely, they reported that male C. elongatus on Atlantic salmon in cage culture experience poor survival in winter, declining from up to 21-40% in October to 0% in February to April. Hogans and Trudeau (1989b) found a somewhat similar change in cultured Atlantic salmon with proportions of males in the population of around 38% in July falling to around 3% in November but rising to around 25% in December. Overwintering females on cage-cultured Atlantic salmon contained up to 80% of non-viable eggs in extruded egg sacs (Piasecki and MacKinnon, 1995). Females were torpid at low temperatures and the authors speculate that this may be a period of diapause. There are no similar observations published for L. salmonis, although poor development success to copepodid seems to be a feature during the winter months.

4.5. Seasonal Effects

Most salmon cage culture takes place in the Northern Hemisphere, where there are marked seasonal changes in temperature and photoperiod. It is here also that the life cycle of L. salmonis is naturally maintained on wild salmonids. Temperature inevitably has a major effect on development rates for both on and off host stages of the life cycle. The role, if any, of photoperiod is less clear, although it influenced egg size (Ritchie et al., 1993), which in turn may have implications for survival. Ritchie (1993) and Wadsworth (1998), for example, have shown that successful nauplius development to copepodid is depressed in winter. The effect of season on egg number (Section 3.3.4) has already been mentioned but another variable associated with season is investment in biochemical constituents of the egg by females (Ritchie, 1993).

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4.6. Laboratory Maintenance

Sealice are now routinely maintained in laboratory culture. It is most convenient to remove pigmented egg sacs from gravid female sealice to minimize the time to hatching. Nauplii can be maintained in clean, aerated seawater at any convenient temperature and allowed to moult to infective copepodids. These can be concentrated by use of directional light and collected for use in infections. The fish hosts are best confined in a restricted volume of static aerated water to allow the copepodids to settle on the skin. After around 2 hours the water supply can be re-established at maintenance levels. Successful establishment of copepodids can be detected in a few days because black pigment spots appear at the site of infection. Further details on maintenance are given by Johnson and Albright (1991a) and Hull et al. (1998).

5. EPIDEMIOLOGY OF SEALICE INFECTIONS

Historically, wild salmon have not been available for study because of their sport value. Consequently, the data on their sealice infections are generally sparse. Even where data sets are available, their shortcomings are evident in that either no information on sex or age of the sealice is given or only partial information has been collected, and no reference is made to that which was not collected. The situation with respect to sea trout is much better, partly because the well-publicized debate about the causes of sea-trout stock collapse has increased the availability of samples and the number of scientists devoting time to gathering the data. The quality of data on seatrout infections is superior to anything available for salmon. Nevertheless, for both species, the data available should be treated with caution because the fishing methods, their effect on sealice on the skin, and the time delay between a fish being caught and subsequently processed may significantly affect recoveries of sealice. Sealice infections on salmonids were common before the establishment of salmon cage culture but nothing is known about the dynamics of transmission. Wild salmon, of various species, and steelhead trout have been captured on the high seas with gravid females on their skin so that the potential for transmission clearly exists. This potential must be realized because many fish have an ocean age in excess of the probable life span of L. salmonis. Since it is a specific parasite to salmonids, it is unlikely to have come from other fish species. Sea trout are normally short-term inhabitants of coastal waters with a residence time of up to 3-4 months but with an overwintering period for

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some that rarely exceeds 6-8 months. This migration is repeated annually so that the population of sea trout in the sea will be mixed age. It may be assumed that they mix with returning prespawning salmon and with newly smolted parr emerging from their natal river, although under favourable conditions newly smolted salmon will quickly begin their migration away from coastal waters and returning prespawners will enter freshwaters and so lose their sealice. The great unknowns are the distribution and survival characteristics of the infective copepodids in oceanic waters. It seems likely that the best opportunities for transmission are presented when salmon are not actively migrating. The limited survival of copepodids, known from laboratory data, otherwise must reduce the chances of finding a host. Recruitment to the copepodid population is equally uncertain, since the life span and fecundity of female sealice is not known, with any accuracy, for wild populations. The population dynamics of L. salmonis on Pacific species of salmon are equally uncertain. Substantial data on the prevalence and intensity of infection with L. salmonis adult females are available (see below), which suggest that transmission may occur in a similar way to that for Atlantic salmon. Certainly many species of Pacific salmon spend long periods on the high seas, including those that are regarded as the most important in maintaining the life cycle. 5.1. Wild Salmonids

5.1.1. Salmon

Pacific salmon have been studied extensively by Nagasawa (1985, 1987), Nagasawa et al. (1 993), and Nagasawa and Takami (1 993) from commercial and research vessel catches by both gill netting and long lining. The latter technique produced recoveries that are more accurate. Nagasawa (1985) compared recoveries of L. salmonis from fish caught by netting or long lining, and found very large reductions in the former. Figures for long lining were a 98.2% prevalence and mean intensity 13.0 (range 1-65), compared to netting, which returned a prevalence of 42.1% and mean intensity of 3.2 (range 1- 18). The least affected area of the body was the post-anal region. Most fish were mature with a sea age of at least 1 year but usually were much older. Only adult females were counted, and generally prevalence and intensity (or abundance) increased with host length and ocean age. Nagasawa (1987) examined 61 15 gill-netted specimens of five species of Pacific salmon and of steelhead trout during May/June 1982- 1983 from the North Pacific Ocean. Most fish were between 30 and 99 cm in length, and

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varied between 1 and 5 years’ ocean age, but with most between 3 and 4 years. Peak prevalences (and abundances) were 46.0% (1.67) for chinook salmon and 28.1 % (0.44) for steelhead trout but, since their representation in the catches were only 0.6 and 0.2%, respectively, they were not considered as major hosts for L. salmonis. Pink and chum salmon comprised 69.8% of the catch and contributed 91.2% of all sealice collected even though the prevalences (and abundances) were only 19.1% (0.32) and 14.7% (0.26), respectively. Sockeye salmon, which formed over a third of the total sample, contributed only 1.1% of sealice. In a further study, using long lining, Nagasawa et al. (1993) examined 2545 specimens of the same host species collected in June/July 1991 from the Bering Sea and North Pacific Ocean. The length range was 20-79 cm and ocean age range 1-5 years. Pink salmon were most infected, prevalence 91.8% and mean intensity 5.83 (range 1-33). Chum salmon had a prevalence of 15.9% and mean intensity of 2.28 (range 1-26). Together these hosts accounted for 94.2% of all lice on the hosts. It was notable that the pink and coho salmon caught were of ocean age 1. Sockeye were again the least infected. Sealice populations were overdispersed. Records of sealice on Atlantic salmon are mostly very poorly documented. The early records of sealice on Atlantic salmon are reviewed by Berland and Margolis (1983) and suggest that as early as 1753 salmon were adversely affected. Subsequently, White (1 940, 1942) recorded severe infections and mortalities caused by L. salmonis on Atlantic salmon in Canada. More recent reports by Berland (1993), Berland and Bristow (1993), Holst et al. (1993), Bristow and Berland (1994, 1995), Finstad et al. (1994) and Bristow et al. (1996) document the presence of L. salmonis and C. elongatus on Atlantic salmon in Norwegian waters. With the exception of Bristow et al. (1996), none of these reports detail the sex or maturity of infections. Bristow et al. (1996), reporting on 21 Atlantic salmon from Tanafjorden, Norway found 90% prevalence with L. salmonis. Ovigerous females were present on 81% of the sample and 19% had adult males. They also found 43% harboured juveniles and 29% chalimus, but the distinction between these is not clear. A further exception is a very useful paper by Jacobsen and Gaard (1997), who documented infections of wild and escaped cage-culture Atlantic salmon with all stages of L. salmonis in the Norwegian Sea north of the Faroes. The most informative of the earlier contributions is that of Berland (1993), who reproduces data from Johannessen (1975) together with two data sets from later years. Johannessen (1975) gill-netted wild Atlantic salmon in June/July 1975 before the cage culture of salmon was expanding and showed that they were 100% infected with a mean intensity of 11.7 (range 1-37). The salmon were also infected with C . elongatus to a prevalence of 45.6% and mean intensity of 1.42 (range 1-8). A similar

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gill-netted sample of 157 fish taken from the same location in June 1988 revealed a 93% prevalence and mean intensity of 7.44 (range 1-40) for L. salmonis, and 17% prevalence and mean intensity of 1.42 (range 1-3) for C . elongarus. A final sample of 45 fish caught in a weir in June/July 1991, and from 50-99 cm in length, were infected with both L. salmonis and C . elongatus with prevalences and mean intensities of 97% and 20.18 (range 194), and 97.7% and 23.9 (range 2-171), respectively. This last sample is clearly indicative of quite intense infections on wild hosts, but it should be noted that the difference between this last sample and the previous two is due to the sampling technique. The samples of Atlantic salmon taken on long lines by Jacobsen and Gaard (1997) between November and March at sea temperatures of 7°C down to 3°C consisted of 1 and 2 sea winter (SW) fish. Wild 1SW were significantly smaller than 1SW escapees but this difference did not persist in 2SW fish. Overall prevalence and mean intensity were 99.2% and 29.7 (range 1-187). The proportion of adults was 90%, of which 72% were ovigerous females. The remaining lice were represented by all stages down to chalimus 111. Escaped 1SW salmon had significantly higher abundances than wild fish of the same age, but this difference did not persist in 2SW fish. Abundance increased with sea age for wild salmon but not escapee salmon. The importance of this study lies in the demonstration of transmission at the feeding grounds based on extrapolations from sea-temperature profiles and known development rates of the life-cycle stages. There appear to be no data on immediate post-smolted salmon from coastal waters, which may be an indication of a short residence time. The data sets from mature salmon caught in coastal waters might reflect a true picture of the infection levels, if it is assumed that the hosts have spent little time in coastal waters where they would be exposed to new infection. 5.1.2. Sea Trout Sea trout have received much attention over the past 5 years because of the precipitate decline in catches. Early records of sea trout infected with sealice include those of Pemberton (1976) and Boxshall (1974b). The latter found 81% of 31 sea trout, from the east coast of England, infected with L. salmonis with a mean intensity of 4.0 (range 0- 12) and 74% infected with C . elongatus with a mean intensity of 9.8 (range 0-32). Subsequent records have been made after 1990. They began with Tully et al. (1993), who reported high prevalences and mean intensities of infection with L. salmonis amongst post-smolt sea trout sampled from 14 sites on the west coast of Ireland. Prevalences were from 14.3% to 100% and mean intensities from 7.0 to 104.8 on fish of length 16.4-27.3 cm. The majority of sealice were

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chalimus but all stages were present. The highest numbers of preadult and adult sealice coincided with the lowest prevalences of chalimus. At five sites, chalimus larvae accounted for over 80% of the sealice population. Dawson (1998) reported a broadly similar situation in a sample of 51 sea trout smolts from two areas on the West Coast of Ireland. Records of sealice from sea trout in Scotland are from 1991 to 1992 (Sharp et al., 1994) and 1994 (MacKenzie et al., 1998). The 1991 sample contained fish from a wide area of Scotland, most of which were infected with sealice of varying age. The percentage of chalimus in the infections varied from 0% to 54.6%. The 1994 sample comprised 622 fish from 17 locations in Scotland of which a third were infected with L. salmonis. Prevalence varied widely as did abundance and proportion of chalimus larvae in the samples. The most heavily infected fish from all areas harboured the highest proportions of chalimus larvae. The largest samples of fish came from the area around Dunstaffnage and provided a time series from May to August. This revealed a clear sequence of infections with high numbers of copepodids in May leading to peak adult numbers in July. The highest levels of infection were also found in fish from the West and Northwest of Scotland. The patterns of infection observed in Ireland, with large proportions of the populations as chalimus larvae, were not repeated in the Scottish samples, except for a few fish from the Dunstaffnage area. Tingley et al. (1997) collated all available records for sealice infections of sea trout on the East Coast of England and produced a similar picture to the situation in Scotland, except that intensities of infection were somewhat lower and fewer chalimus were recovered. Using data for 1972, 1973, 1992 and 1993, they found that prevalence was between 66.7% and 88.6% with mean intensities of 1.75-5.47. A subsample of 34 fish from the 1992 catch, examined in more detail to record all sealice stages, revealed that only 15% contained sealice below adult and of these only one included chalimus larvae. C.elongatus were also present at prevalences between 16.7% and 74.2% with mean intensities of 2.00-9.78. The authors conclude that seatrout infections in the area studied are stable over the long term and that the virtual absence of chalimus larvae on the fish suggests very low transmission rates. The pattern they record is consistent with many other host parasite systems; where juvenile stages are only rarely seen in the parasite populations except at certain times of the year. The first Norwegian investigation of sealice on sea trout was conducted in 1992 by Birkeland (1996a,b). Observation of sea trout ascending and descending a river on the West Coast of Norway documented movement of post-smolts and older migrants. Mean lengths of the two groups were 17.4 f 2.1 cm and 37.4 f 7.3 cm, respectively. Prevalence of infection with L. salmonis on ascending post-smolts was 96% with a mean intensity of

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272.4 and for older migrants 87%, mean intensity 103.0. The composition of the sealice populations on the two groups was broadly similar, with over 70% of sealice at copepodid or chalimus stages. Older migrants had significantly more adult sealice than post-smolts. Two other studies of sea trout by Mo and Heuch (1998) and Schram et al. (1998) report on sealice infections from sea trout taken from Southeast Norway, where there is no salmon cage culture. Mo and Heuch (1998) sampled fish from the inner Oslo Fjord and an adjacent river. River-caught fish were smaller and less frequently infected, but supported higher mean intensities of lice than fish from the fjord. In general, the proportion of chalimus larvae in the populations was less than 50%. Schram et al. (1998) examined 502, largely mature, fish between 1992 and 1995, and found distinct seasonal trends of infection, which peaked in late summer or autumn. The proportion of chalimus larvae never exceeded 15% of the total.

5.2. Cage-cultured Salmon

Sealice infections are regularly monitored, partly to enable decisions about treatment times. This monitoring programme also provides longterm intelligence about the status of sealice as pathogens on different farm sites. In general, this information is considered confidential to the company and therefore unavailable for use by the scientific community. Variation exists between countries with regard to lice-count methodology and, although a universally applied protocol is regarded as desirable, there is no agreement on such a protocol. The protocols used for sealice monitoring are often based on samples of around five fish, which, for statistical purposes, is too few to obtain reliable data to examine population dynamics (Anonymous, 1997b). Treasurer and Grant (1998) reported the system in use by Marine Harvest McConnell for monitoring sealice populations on their sites, and, in Ireland, a National Sealice Monitoring Programme was set up in 1991 to monitor sealice infection levels in cage-culture systems. Apart from the study by Tully (1989), which provided the opportunity to examine the population changes in caged salmon without chemotherapeutic intervention, all other published work is based on cage-culture systems subjected to the normal sealice treatment procedures (Jaworski and Holm, 1992; Tully, 1992; Jackson and Minchin, 1993; Jackson et al., 1997). Such studies are difficult to compare not only because of the physical and biological differences between sites, but also because of differences in chemotherapeutic control, which can significantly alter the composition of the sealice populations (Jaworski and Holm, 1992).

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Published information on prevalence and intensity of infection with sealice is surprisingly sparse for cage-cultured salmon, considering the frequency with which the parasites occur. Despite their common occurrence, sealice are not uniformly distributed. There is geographical variation as well as differences between proximate farms, and even within individual farms and between cages. Some farms experience regular, severe sealice epidemics, others are barely affected. The reasons for such variation are equally diverse, but will include the physical and biological conditions pertaining to the particular location and, secondarily, the animal husbandry practices of the farm, including disease management. Atlantic salmon spend up to 2 years at sea before harvest and, so far as is possible, one and two sea winter fish are stocked separately to avoid transfer of infection. The population structure on the two age classes is predictably quite different and follows the pattern for wild salmonids, where rising infection levels reflect duration and extent of exposure. Hogans (1995) and Tully (1989) are useful studies in that they provide baseline information on parasite prevalence and intensity. It is an unfortunate coincidence, however, that both studies report on infections that are predominantly C. elongatus, with L. salmonis accounting for a smaller proportion of the total infection. On most, although not all, commercial European farms, L. salmonis predominates. The study by Tully (19891, in particular, is important because it was conducted on a commercial site but fish in the experimental cage remained untreated for the duration of the work. The overall prevalence of infection with C . elongatus and L. salmonis between July 1987 and January 1988 was 93%, but varied widely during the period of study. Intensity of infection was slightly more consistent with a peak for both species in August, followed by a decline in September/October and recovery thereafter. The ratio of C . elongatus to L . salmonis in this study was 2-3:l. In contrast, the data of Hogans and Trudeau (1989b), obtained on three, commercial, cage-culture sites on the Canadian Atlantic coast during the same time period, show a peak of infection, for both prevalence and intensity, during September/October, with a subsequent decline over the winter period. The authors have correlated this pattern of infection with sea temperature, an interpretation that does not coincide with Tully's (1989) results, where infections decline during the period of maximum sea temperature. Wootten (1985) has also shown that L . salmonis continued to multiply on the west coast of Scotland at temperatures down to 3.5 "C, only marginally higher than the minimum recorded by Hogans and Trudeau (1989b). There is clearly a considerable need for more extensive epidemiological studies on sealice infections, particularly to elucidate the natural cycles of sealice generations unperturbed by routine treatment.

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5.3. Hydrographical Effects on Copepodid Dispersion Hydrographic factors are recognized as important in sealice transmission between cage-culture systems because good flushing of the site will minimize reinfection and vice versa. In enclosed bays, there may be several sites not all belonging to the same company, so that interchange of infection between the farms may not be acted upon in a coordinated way. Despite many attempts, relatively little is known about the movement of nauplii and copepodids in and around cage-culture sites. There are obvious technical difficulties to separating sealice larvae from other plankton in samples that are likely to contain very small numbers of sealice nauplii and copepodids. The dispersion of planktonic stages from ovigerous females on wild salmonids is also of considerable interest in understanding the dynamics of transmission of sealice in wild populations, particularly in areas free of cage culture. There are innumerable technical and logistic problems in obtaining reliable data for this purpose. These include: obtaining large enough numbers for analysis; accurate identification of nauplii and copepodids of L. salmonis from other related species, especially C. elongatus; and the timing of sampling to optimize catches given the differing behaviour of the planktonic stages and the aggregation of definitive hosts. The only reported successful recoveries of sealice are those by Costelloe et al. (1996a) and Costelloe et al. (1998). Costelloe et al. (1996a) took plankton samples from within, and at 10 m to 1 km from, commercial salmon cages and found the highest densities of larvae (presumed to be nauplii and copepodids) (maximum of 66.1 m-3) within the cages, and decreasing numbers with distance outside the cages (maxima at 10 m and 1 km were 4.8 mP3and 0.4 mP3,respectively). The data sets for inside and outside the cage were from dates a month apart and no indication is given of sealice treatment between the sample dates that could affect the results. It is also unclear if the data are single samples or means of several samples as no descriptive statistics are given for the in-cage samples. Counts are given as larvae per cubic metre but total volumes of water sampled are not given, so reliability of the counts can not be estimated. Costelloe et al. (1998) conducted a surface, plankton-sampling programme in Killary Harbour during spring/summer 1995- 1996. The highest recorded number of larvae (nauplii and copepodids) was recovered in 1996. The maximum density of 16.05 m-3 was recorded in April, but was exceptional compared to regular counts of between 0.04 and 2.59 mP3. Counts within sites vary widely over time and, since the sample volume and number of replicates have not been given, it is difficult to evaluate the significance of the results.

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5.4. Interaction between Wild and Farmed Salmonids

For Atlantic salmon from areas where cage culture is absent, there is evidence for the existence of two foci for transmission of L. salmonis, one coastal and driven by mixed-age populations of salmon and sea trout, and the other maintained by long-lived adult females on shoaling salmon on the oceanic feeding grounds. Depending on the speed of migration to the feeding grounds, transmission may or may not take place to any extent during this period. It seems probable that regular mixing of L. salmonis populations can occur between the two locations. In the presence of cage culture, there is clearly a large but variable population of infective stages available for transmission to both cultured and potentially to wild fish. The degree to which copepodids from cultured fish infect wild salmonids will depend on: the degree of overlap between cage-culture establishments and migratory routes, and natural distributions of salmon and sea trout; the dispersion rate and survival of copepodids from cultured fish; and the behaviour of potential host and copepodid with regard to orientation in the environment. Although it must be the case that the generation of copepodids from cageculture sources greatly exceeds that from wild hosts, when farms are fallowed for several months, sealice reappear eventually to reinfect the site. Clearly, this can be from another farm but the availability of wild salmonids in the area must also be an alternative source. Quantifying the role of wild versus cultured fish reliably will be a difficult task unless reliable estimates of migratory salmon and trout populations can be obtained. Tully and Whelan (1993) calculated figures based on populations of sealice on cultured fish and population estimates of salmon and their sealice. They concluded that 95% of available nauplii would come from cage-culture sources but accepted that the model had a number of weaknesses. Nevertheless, their paper highlights the need for reliable data on which to model sealice populations in order to understand better the population dynamics in both wild and cultured fish populations. 5.4.1. Decline of Wild Salmonid Populations Long-term fluctuations in wild salmonid populations, based on the interpretation of catch statistics, have been known for many years. In the UK, fisheries statistics have been collected since 1952, and show short-term fluctuations of increase and decrease, but overall showing a declining trend. With the emergence of salmonid cage culture in the late 1960s, followed by its dramatic expansion, concern was expressed about its biological impact on wild stocks. In 1989, the recovery of ascending sea trout in Irish rivers, whch

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were frequently and often heavily infected, mainly with sealice chalimi, kindled a debate, which became heated, passionate and acrimonious, and which involved anglers, fish farmers, government scientists and politicians at all levels. The arguments have continued until the present and have generated as much heat as they have light. The absence of baseline epidemiological data and information on the pathogenicity of sealice for sea trout has hindered proper scientific evaluation of the perceived problem. Some progress has been made to gather such data as are reported here (Section 7.2), but progress is slow and establishing cause and effect notoriously difficult. Anglers and riparian owners are understandably exercised by the inability of the scientific community to provide answers because, in some cases, sea trout populations are alarmingly depleted. However, as Northcott and Walker (1995) point out, there are many other possible explanations for the decline including coniferous afforestation, reduced food availability, increased predation, overfishing, escaped farm salmon and unusual climatic conditions. Drawing the wrong conclusions from inadequate data will serve no useful purpose and, in the long term, would hinder progress. A question that needs to be addressed is, what could be the biological basis for the phenomenon that has been described as premature return to freshwater? Although the answer, so far as sea trout are concerned, has been accepted by many as the fish’s need to rid itself of sealice, there seems no rational explanation for this. How would this mechanism have evolved? Presumably selection pressure might be considered but this would probably have to be related to some other disease because the problem with sealice appears to be recent. The problem that might arise in using this argument is that fish in poor condition are more likely to succumb to fungal infections on returning to freshwater; it hardly seems a good idea to move back to freshwater when debilitated, and presumably more prone to fungal attack, especially since more food is available in the sea. The published work on sealice infections of sea trout was reported earlier (Section 5.1.2, p. 271). The steep decline of sea-trout populations initially observed in 1989-1990 in Ireland (Anonymous, 1991, 1993, 1994b, 1995a; Whelan, 1991; Tully et al., 1993; Whelan and Poole, 1996) was a widespread occurrence in the British Isles with declines reported in Scotland, England and Wales (Anonymous, 1994a; Walker, 1994). Subsequently, a Scottish Salmon Strategy Task Force was established in 1995 and reported its findings in 1997 (Anonymous, 1997a). So far as sealice are concerned, this amounted to recommendations to reduce levels of infection through the availability of appropriate medicines; clearly, this is also the aim of the industry in general. At the same time, the West Highland Sea Trout and Salmon Group published their Report and Action Plan (Anonymous, 1995b), which also encouraged the development of sealice research with the aim of reducing the impact of disease.

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In 1996, the International Council for the Exploration of the Sea (ICES) convened a workshop on the interaction between salmon lice and salmonids to review the literature on L. salmonis and salmonids from the point of view of assessing the effects of salmon lice on salmonid stocks (Anonymous, 1997b). A subsequent ICES/NASCO (North Atlantic Salmon Conservation Organisation) symposium held in 1997 looked at the wider aspects of interactions between salmon culture and wild stocks of Atlantic salmon. The report of this symposium recognized inter alia that ‘There is an urgent requirement for rigorous experimentation to test hypotheses on possible relationships between salmon farming and parasite (especially sealice) infestation levels in wild salmonids’ (Youngson et al., 1998). It is equally desirable to be able to evaluate the potential impact of such infections in comparison with other pressures on wild salmonid populations. The unusual age profile of sealice on the returning post-smolts in Irish rivers in 1989-1990, where infections were largely chalimus stages, was also recorded in Norway but only partly reproduced in Scotland, and not in areas of Scotland, England or Norway where cage culture is not practised. This phenomenon clearly requires explanation because newly smolted sea trout were exposed to seemingly large populations of copepodids of L. salmonis within around 12-20 days of re-entering the estuary from which they emerged. The origin of such populations of copepodids is a priority for investigation. 5.4.2. Genetic Studies on Sealice One aspect of the controversy over the alleged involvement of sealice originating from salmon in cage culture in the decline of sea-trout populations has been the provenance of the parasites found on returning sea trout. Many salmon farms share sea lochs inhabited by sea trout, but the degree of mixing of the sealice populations between wild and cultured fish is not known. Inevitably, the planktonic phase of the life cycle increases the probability of mixing of the two populations, even if they are indeed separate. A recent study by Todd et al. (1997) has investigated the genetic differentiation of populations of L. salmonis collected from wild and farmed salmon, and sea trout and rainbow trout from around the Scottish coast using RAPD analysis. Sixteen samples of sealice were taken from various sources around Scotland from the River Tay, on the East coast, to Argyll on the Southwest coast. With one exception, the sealice were collected over the period from July to November 1995 and analyses performed on female sealice. Allozyme electrophoresis revealed two highly polymorphic enzymes, fumarate hydratase and glutamate oxaloacetate transaminase, suitable for further analysis and which indicated that sealice from sea trout and wild and

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farmed salmon are ‘genetically homogeneous and therefore comprise a single undifferentiated panmictic population’ (Todd et al., 1997). A subsequent in-depth examination was made of samples from all hosts and from the whole of the Scottish coastline using PCR and RAPD analysis. This revealed genetic differentiation between farmed salmonids from different farms, which was considered to be due, in part, to founder effect but principally to selection pressure, perhaps caused by chemotherapeutic treatments. The farmed salmon sealice were also considered genetically distinct from those from wild salmonids and the wild salmonid sealice formed a genetically homogeneous group. A small number of sealice from sea trout in two locations carried markers that might have indicated a farm origin for the sealice. Banks et al. (1998) reported a study in Scotland using RAPD analysis of starved L. salmonis from various farm sites as well as wild Atlantic salmon and sea trout. They concluded that the sealice samples examined can be divided into two clusters comprising sealice from wild salmonids plus sealice from two outlying farms, and the second cluster comprising sealice on farmed salmon. Shinn et al. (1998) sequenced L. salmonis nucleotides. They obtained partial sequences from the 18s ribosomal RNA gene, the cytochrome oxidase gene and the ribosomal internal transcribed spacer to distinguish sealice from wild and farmed salmonids.

6. PHYSIOLOGY OF SEALICE

There has been little study of the physiology of sealice in general, partly because of the technical problems of working with them but also because of other priorities.

6.1. Nutrition

6.1.1. The Digestive System There are only two studies of the alimentary canal of sealice, based on L. salmonis (Nylund et al., 1992; Bron et al., 1993b). These papers deal with the adult and larval stages respectively. Nylund et al. (1992) include a table of publications on gut structure in both free-living and parasitic copepods. In general, the alimentary canal is broadly similar in all cases, with the greatest difference in structure being associated with the midgut region, which may be divided both morphologically and histologically.

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The alimentary canal of adult L. salmonis comprised a short tubular foregut (or oesophagus), large wide midgut with forwardly directed blindending caecum and short tubular hindgut (Nylund et al., 1992). The foregut-midgut junction is identifiable by the presence of a papilla provided with a pair of valves. The midgut is not morphologically divided. Both foregut and hindgut are cuticle lined and all sections are supplied with muscle fibres. The histology of the gut varies according to nutritional state, hence the study was based on fed individuals. The highly folded foregut is lined with a simple squamous epithelium. The midgut histology is more complex, comprising a lining of columnar cells divisible into three morphological types designated type I, I1 and 111. The midgut lining is highly folded at irregular intervals along its length. Type I cells are identifiable on the basis of large amounts of rough endoplasmic reticulum, mitochondria and smooth vesicles, an apically situated layer of zymogen granules and densely packed microvilli. Type I1 cells are elongate, microvillous cells that are easily identifiable by the presence of large lipid droplets. Cells, intermediate in structure between type I and 11, are also found which the authors suggest indicates that cell types I and I1 may be simply developmental stages of the same cell type. Together these two cell types and the intermediaries account for 90% of the cells lining the midgut. Type 111cells are also columnar with an invaginated apical surface provided with less dense microvilli. Apical invaginations may connect with electronlucent cytoplasmic inclusions. Cytoplasm also contains electron-dense inclusions. There was no discernible stratification of cell types within the midgut. The hindgut is lined with cuboidal to squamous cells the transition occurring posteriorly. Bron et al. (1993b) identified a similar overall gut morphology from their study of copepodids and chalimus stages except that they divided the midgut into anterior and posterior zones, on the basis of a constriction between the two parts, and identified a disjunction between midgut and hindgut occupied by a collar of enlarged cells. They also recognized a muscular sphincter in front of these cells. They did not identify a valve between foregut and midgut. It seems unlikely that the structures clearly identified in the larval and juvenile stages would not be present in the adult and vice versa. The histology of the foregut and hindgut appear to be essentially as described for the adults. The authors identified three cell types in the midgut epithelium, designated as microvillous, vesicular and basal cells, all of which occurred in the anterior midgut, but only the first named were present in the posterior midgut. None of the midgut cells appears to be columnar in form as compared to those in the adult midgut. Microvillous cells possess a pronounced brush border of varying height. Vesicular cells contain periodic acid-Schiff (PAS)-positive vesicles and have a rugose apical membrane. Basal cells are small and possess a brush border.

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Since the histological description of the larval and juvenile stages was based on light microscopy and that of the adults on electron microscopy, it is impossible to reliably identify the two sets of midgut cells with respect to each other. Although vesicular cells would appear to be similar to cell type 11, the nature of the vesicles seems to be different. Cells containing lipid droplets were identified from copepodids and were regarded as likely to be microvillous cells. The lipid inclusions in midgut cells of copepodids presumably represent energy stores provided for the non-feeding stage, whereas those in adults presumably accumulate from feeding on host tissues and therefore are not homologous. Reconciliation of the midgut cell types described for larval, juvenile and adult stages is probably only possible by examination of a time-course series of stages at the ultrastructural level. 6.1.2. Feeding Mechanism The feeding apparatus that is responsible for damaging the host skin is the mouth tube described from studies on C . curtus, a parasite of gadids, by Parker and Margolis (1964) and subsequently elaborated by Kabata (1974b). The structure of this organ seems to be identical in the species of sealice known to infect salmonids. The mouth tube first appears in the freeswimming copepodid stage and, although its structure has not been investigated in any of the larval stages, it may be reasonable to assume that there are no major changes during the developmental process (Lewis, 1963). It is a muscular tube surrounding the foregut and its mode of operation has been theoretically interpreted by Kabata (1974b). Its potential for disrupting the fish’s skin is connected with the presence of a toothed ridge, the strigil, which lies inside the mouth and across the entrance to the foregut. It is this organ which Kabata has identified as the means whereby the parasite abrades the fish’s epidermis; the tissue so removed is conveyed to the foregut by another ridged plate, the mandible, lying further inside the mouth tube. 6.1.3. Feeding and Digestion All stages from chalimus I to adult feed by the same means as described above. The difference is that the chalimus stages I-IV are attached by the frontal filament and are consequently restricted in the area that they can brouse. Mobile preadults and adults feed in the same way, but are unrestricted in where, on the host, they feed. Nylund et al. (1992) reported peristalsis in the gut of L. salmonis as had been observed earlier for L. pectoralis (Scott, 1901).

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The structure of the feeding organ is consistent with sealice being epidermal brousers but inevitably, because of the epidermal structure, its disruption may lead to haemorrhage from damage to epidermal capillaries and consequent ingestion of blood by sealice. It is widely recognized that adult sealice, in particular, ingest blood especially as the blood-filled gut is readily identified on infected salmon. Juveniles are much less commonly found to have blood in the gut contents. Brandal et al. (1976) were the first to identify blood in the contents of adult L. salmonis and recorded that 42% and lo%, respectively, of adult females and male L. salmonis contained blood in their gut, which was subsequently identified spectrophotometrically. Haji Hamid et al. (1998) confirm that adult females were more often found with blood in the gut and that this was not simply a size effect. They found that adult females containing blood were significantly larger than those without. It is unclear how they were able to confirm that those adult females without blood had not taken it on earlier occasions. There could be a selective advantage to females taking a high-protein diet, especially during oviposition, if as Haji Hamid et al. (1998) report, bloodfeeding individuals produce longer egg sacs. What is not obvious is a mechanism by which sealice could selectively take blood as a nutrient, given the structure of their feeding apparatus. This tends to reject suggestions that females may require a blood meal in order to oviposit. Digestion has not been investigated. Nylund et al. (1992) confirm the absence of a peritrophic membrane and speculate on the possible role of the midgut cells in digestion and absorption by comparison with other crustaceans. They draw no firm conclusions about how digestion occurs and whether it is intracellular or extracellular. The existence of proteases in L. salmonis and C. elongatus has been confirmed by Ellis et al. (1990) who identified seven bands for C. elongatus and five for L. salmonis on electrophoretic gels from whole-body homogenates. The latter species required up to eight times the protein concentration to obtain identifiable bands, which was suggested to be related to the host range used by the two species, C. elongatus utilizing a wide range of host species. Further studies using artificially fed sealice are needed in order to elucidate the role of the midgut cells in digestion and whether enzymes are secreted into the midgut or are retained within the midgut cells. The particular need for this clarification is for research into recombinant vaccines that are being targeted at gut enzymes. 6.2. Osmoregulation

The life cycles of sealice take place entirely within the marine environment and, although Atlantic-salmon and sea-trout anglers regard the presence of

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sealice as indicative of a fresh-run fish, there is some evidence to suggest that this might not be true in all cases. Thus Hutton (1923), Ashby (1951) and Wootten et al. (1982) report instances of L. salmonis on Atlantic salmon 14, 25 and 21 days, respectively, after entering freshwater. Sea trout have been caught, still bearing sealice, 70 km from the sea (A.F. Walker, personal communication). Similarly, Kabata (1981) reported Oncorhynchus nerka bearing live L. salmonis up to 96 km from the sea. It was impossible to estimate the time the fish had been in freshwater in either of these last two reports. Calderwood (1907) observed that L. salmonis survived on grilse in tanks of freshwater at 1 1.6"C for around 5 days. McLean et al. (1990) kept Atlantic salmon grilse naturally infected with adult male and female (gravid and non-gravid) L. salmonis in freshwater at temperatures of 12.8- 16°C and found over 50% were lost within 24 hours. Less than 10% survived more than 48 hours but some gravid females remained alive and attached for up to 5 days in freshwater. In contrast, Finstad et al. (1995) demonstrated experimentally that naturally acquired L. salmonis could survive on Arctic charr for up to 21 days in freshwater at temperatures of 9.1-11.7"C. Furthermore, survival rates were higher at c.60°/0 after 7 days and, even after 14 days, 21% of the sealice remained. Berger (1970) conducted experiments on isolated adults and nauplii of L. salmonis in freshwater to determine survival rates at 14- 15°C. Adults died rapidly below 12%0but above this value survival did not change, although all were dead after 12 hours. All nauplii died within 48 hours at a salinity of 12%0and above, whereas at 8%0, survival was reduced to 32 hours. Johnson and Albright (1991a) found that isolated adults of L. salmonis at 10°C survived for 9.5 days at 10%0salinity. They also incubated eggs at different salinities and found that, although eggs hatched successfully in salinities as low as 15%0, survival was nil. Survival improved at 20-25%0 but development to the copepodid was negligible. Complete development was only achieved at salinities of 30%0and, even then, it varied widely. Experimental evidence of the ability of L . salmonis to survive in freshwater comes from the study by Hahnenkamp and Fyhn (1985). They investigated the survival characteristics of adult females (males were excluded for size reasons), both attached and free-swimming, in fullstrength sea water, 37% sea water and fresh water. The parasites osmoconformed in full-strength sea water but were hyperosmotic in 37% sea water, irrespective of whether they were attached or not. However, when exposed to fresh water, the parasites attached to the skin survived for up to 7 days, during which time the haemolymph osmolality and chloride concentration declined slowly for the first 8 hours but remained constant thereafter until death. Free-swimming parasites, on the other hand, survived no longer than 8 hours, during which the haemolymph osmolality and chloride concentrations declined dramatically.

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Explanations suggested by Hahnenkamp and Fyhn (1985) for this marked difference in survival on and off the host relate, in part, to the possible protective value of the host mucus, either through being consumed or by forming a covering layer over the sealouse. They propose that the consumption of mucus, and/or tissue fluids, replace the salts lost through exchange with a hypo-osmotic environment. Another suggested explanation is that, by being situated within the mucus layer, the sealice reduce the rate of loss of salts to the surrounding freshwater. This work is interesting because it highlights the close relationship between the host skin and the parasite, particularly with regard to the possible protective function of the host mucus. In this context it would be very interesting to compare the survival characteristics of the larval stages of sealice subjected to the same environmental variables, since they are much smaller and therefore more likely to benefit from any protection offered by the mucus layer. Undoubtedly a better understanding of the host-parasite relationship will come from an improved awareness of the subtle interactions occurring in the microhabitat occupied by the sealice. Sealice are able to survive better on than off the host. Small changes in salinity are likely to be tolerated and even large fluctuations seem to be tolerated for extended periods of several days at least. The nauplii are more susceptible to salinity reductions and are unlikely to develop in brackish water. The copepodid has a slightly greater survival rate at lowered salinities but survives best above 15%0salinity. 6.3. Endocrine Control

Crustacea in general have complex endocrine and neurosecretory systems but these are far from being well understood. Moulting is controlled by the endocrine and neurosecretory systems, and juvenile hormone has been tentatively implicated in adult metamorphosis of some crustaceans. Little is known about the endocrine system of sealice and nothing has been published apart from the conference paper by Bron et al. (1998) on ecdysis in L. salmonis. Ironically, the first chemotherapeutic treatment for sealice based on the insect growth regulator teflubenzuron, to be marketed as Ektobann@ or Calicide@,has been developed. There is a need for further research on the endocrine control of moulting and reproduction. 6.4. Sensory Biology

All stages in the life cycle of sealice bear paired pigmented eyespots, which have been described for the nauplius of L. salmonis (Bron et al., 1993) and

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the copepodid stage of C . elongatus (MacKinnon, 1993). The latter study showed that the pigmented eye in this species comprised a pigmented cup cell containing a spherical, anterolaterally orientated lens contained within a lens cell and surrounded by six rhabdomeric sensory cells. Between these cells and the pigment cup cell are a number of platelet cells. A third lensless eye situated between the two pigmented eyes consists of two rhabdomeric sensory cells and at least two platelet cells. The nauplius eyes of L. salmonis are broadly similar. There are two dorsal eyes containing lenses surrounded by nine retinular ( = rhabdomeric) cells and a tapetum comprising two cells (= platelet). There is a third lensless ventral eye comprising ten retinular cells. The role of photoreception in the copepodid has already been discussed (Section 4.2.1, p. 262) but no role has been ascribed to these structures in the adults. It is evident from observation of the responses of adult sealice to shadowing that the eyes remain functional but how important vision is at this stage remains unknown. Recent studies on the mating biology of a variety of free-living copepods show the importance of chemosensory ability in mate finding (Boxshall, 1998). There is little hard evidence of a similar ability amongst parasitic copepods, although the circumstantial evidence is accumulating (Anstensrud, 1990a, 1992; Pike et al., 1993b; Ritchie et al., 1996a; Hull, 1997; Hull et al., 1998). The evidence for chemosensory capability in male L. salmonis reported by Ritchie et al. (1 996a) included ‘mate-searching’ behaviour, increased activity in the presence of preadult females and male clustering around preadult females. Although there is now considerable behavioural evidence for the use of the senses during the processes of pair formation and mating (Hull, 1997; Hull et al., 1998), there is less direct experimental evidence of the use of chemical stimuli. Pike et al. (1993b) also demonstrated increased activity of males in the presence of preadult females as well as responses to salmon-conditioned water in bioassays in v i m . Fraile (1989) elicited a chemotactic response from the copepodid of C . minimus to host odours, and Hull (1997) proposed a sensory basis for differential settlement of L. salmonis copepodids on chemically and physically altered Atlantic salmon epidermal and scale assemblages. The capacity to detect and identify the fish host and for the males to detect the presence of the female is of major significance to the mobile stages of sealice. Studies of the sensory organs of adult sealice are few. Gresty et al. (1993) and Laverack and Hull (1993) have described some of the structure of the antennulary sensors of the copepodid of L. salmonis and the adult male of C . elongatus, respectively. Both species bear an apical tuft of 13 setae on the distal segment of the antennule. Some of these setae are believed to be mechanoreceptors. In L. salmonis, this would include 1 1 of the I3 setae based on the presence of dendrites packed with microtubules and a scolopale. The two aesthetascs identified on the antennule of L. salmonis are believed to be

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chemoreceptors (Gresty et al., 1993). Similar conclusions were reached regarding the setae on adult male C. elongatus (Laverack and Hull, 1993). Hull (1997) made an extensive study of the ultrastructure and innervation of the sense organs of adult L. salmonis, concentrating in particular on the antennules. The study divided the sensory structures of the antennule into six different types. This strongly supported the modality of the two aesthetascs and some of the mechanosensory structures previously described in the apical tuft, but also provided evidence for a number of bimodal contact chemosensors on both the apical tuft and the proximal article of the appendage. Hull et al. (1998) studied the effect of ablating the distal tip of the antennules of male sealice on host resettlement, pair formation and mating success. Ablating the antennules of males significantly reduced resettlement on the salmon host compared to control individuals, but there was no effect on long-term survival of those sealice that did resettle. In the presence of adult females, ablated male sealice established significantly fewer pairs for the first 28 hours of the experiment; thereafter the difference in number of pairs compared to controls persisted but was not significant. When the time to mate was compared, the first mated female in the control group appeared after 8 hours, whereas with ablated males this was delayed until 75 hours. Caution was urged in interpreting the results because, although the putative chemoreceptors were removed during ablation, so too were the putative mechanoreceptors that may have interfered with swimming behaviour. Experimental demonstration of the chemosensory basis for host-finding and mate-finding by adult male L. salmonis is now a priority because ‘interference with the chemosensory basis of mate location and mate recognition could provide information of great value in the development of novel control methods’ (Boxshall, 1998).

7. PATHOLOGICAL EFFECTS OF SEALICE ON SALMONIDS

The disease processes associated with sealice infections are, in common with many other parasitic infections, dynamic interactions between host and parasite in which parasite stage, and number present, host age, species, genetic strain, physiological condition and position in the population hierarchy all contribute to determining the nature and extent of the effects. Both common species of sealice cause injury to salmonid hosts, but that caused by L. salmonis is more severe, and more is known about its effects on both wild and cultured fish. Neither species is life threatening unless the infection levels increase beyond the host’s ability to compensate. In farmed salmon this is a continual threat with L. salmonis but is rarely so in infections with C. elongatus.

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As far as is known, development of pathology associated with sealice is due exclusively to the mechanical effects of feeding by the parasitic stages. It has been suggested that opportunistic infections, such as Vibrio, could be associated with sealice infection (Hgstein and Bergsjo, 1976; Wootten et al., 1982) but there is no experimental evidence, and only limited clinical evidence, to support this interpretation. Histein and Bergsjo (1 976) found that Atlantic salmon brood stock developed aggressive fungal infections on being returned to fresh water. Birkeland (1996a) found that whereas most (> 96%) early returning post-smolt sea trout survived, older migrants developed fungal infections after returning to fresh water and suffered mortalities of almost 20%. Erosion of the epidermis may have profound effects on osmoregulatory homeostasis. The effect will increase with magnitude of infection and size of the parasites that increase. the surface area of damaged epidermis. The consequential imbalance in plasma osmolality and electrolyte concentrations can be regulated, in the short term, by physiological adaptation to counteract the changes, but it is energetically demanding and stressful. If the condition is allowed to continue, the salmon may succumb in part owing to its inability to maintain homeostasis. It is probable, however, that osmoregulatory stress alone is not the sole cause of mortalities because the skin represents only 20% of the total surface area over which osmoregulation takes place, the other 80% being the gills. Epidermal erosion will also allow the loss of body fluids including blood and, importantly, lymph as well as proteins and electrolytes. Furthermore, epidermal lesions elicit hormonal responses, such as the response to stress, with consequent release of cortisol and its immunosuppressive effects. In this scenario, morbidity and subsequent mortality are consequences of multifactorial perturbations to the host’s physiology that finally exceed its homeostatic capabilities. Natural epidemics of sealice infection on salmon have occurred only rarely according to the available literature (White, 1940; Johnson et al., 1996). The more recent problems associated with sea-trout post-smolt mortalities were discussed earlier. The potential for mass mortalities in cultured fish exists but effective management of the disease means that only in exceptional circumstances, such as severe weather delaying or interrupting treatments, is this potential likely to be realized. 7.1. Mechanical Damage Caused by Infection

7.1.1. Larval Stages Newly attached copepodids of L. salmonis cause a local cellular response visible to the naked eye as a small black spot formed by an accumulation of

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melanocytes. Once attached by the frontal filament, which is achieved after disruption of the epidermis, the chalimus begins to cause further damage to the epidermis through its feeding activity. This produces a very limited area of erosion around the site of attachment. Rather more damage has been reported on sea trout owing to aggregation of copepodids around and on the dorsal fin, where they cause epidermal erosion sufficient to expose the fin rays (Tully et al., 1993; Dawson, 1998). Dawson (1998) examined ascending sea-trout post-smolts and estimated dorsal-fin erosion in four categories of increasing surface area. The heaviest infections, with a mean of 140 chalimus per fish, suffered the highest erosion with over 66% of the fin affected. Dawson et al. (1997) used the same method to compare L. salmonis infections on experimental sea trout and Atlantic salmon, and found no difference between the two species with regard to severity of effect. Extensive damage to the fins, including complete removal of fingerling 0. gorbuscha by C . clemensi chalimus, has been reported several times (Parker and Margolis, 1964; Kabata, 1972). C. elongatus juveniles have not been reported to cause severe injury. 7.1.2. Adults

Adult sealice, despite their mobility, spend much of their time grazing the epidermis of the host. To do this requires close physical contact, which is achieved by a combination of morphology and intermittent activity of the paired thoracic appendages, as described by Kabata and Hewitt (1971). In effect, the body of the parasite behaves rather like a suction cup, with the edge of the cephalothorax providing a seal. Thus secured, the parasite can graze the skin undisturbed. The mouth tube is not an intromittent organ such as is found in many ectoparasitic insects; rather it can be regarded as a non-selective rasping apparatus, more akin to the molluscan radula. Because of its position on the body and its relative restriction of application to the skin, it seems likely that it is adapted as a browsing organ. This is important because sealice do ingest blood (Johannessen, 1975; Brandal et al., 1976; Haji Hamid et al., 1998) and it may be tempting to consider that this is a primary food supply, selectively taken by the parasite. This is unlikely to be true and it is more probable that sealice take blood opportunistically as a result of damaging superficial epidermal capillaries exposed by extensive grazing of the skin. This is more likely to happen in conditions where infections are well established and have created patches of erosion of the skin. Under these circumstances, further grazing of these areas will expose underlying tissues, including blood vessels. Adult L. salmonis occur commonly on the head, dorsal surface and postanal area. The frequent presence, at least, on the head of the host has been

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explained by Histein and Bergsjo (1976) based on the thinness of the skin and absence of scales. It is also the area where mating takes place. The aggregation of sealice on the head causes the most obvious pathology, which has been depicted in many papers on sealice infection (Johannessen, 1975; Histein and Bergsjo, 1976; Egidius, 1985; Pike, 1989; Johnson et a[., 1996; MacKinnon, 1997). All of these refer to farmed salmon infections but there are accounts of wild salmon displaying extreme pathology (Kabata, 1970; Johnson et al., 1996), in which the subepidermal musculature and even the cranial cartilages had been eroded through to the cranial cavity. In another case, a major epidemic occurred in which large numbers of salmon died, in part due to L. salmonis infections (White, 1940) but also, in a separate incident, due to high ambient temperatures (Huntsman, 1942). Severe cranial pathology caused by L. salmonis infection has led to exposure and erosion of underlying myotomes, sometimes reaching the cranium (Wootten et al., 1982), but it is more common to see fish in which there is extensive epidermal and submucosal erosion with associated haemorrhaging. Similar pathology can occur elsewhere on the body but is less commonly seen (Johnson et al., 1996). Subepidermal oedema may also be associated with infection. The clinical picture in fish infected with C . elongatus has not been described, perhaps because its prevalence on commercial farms is usually much lower. In view of its smaller size, it might be expected that the severe pathology associated with L. salmonis is absent. A brief account by Hogans and Trudeau (1989b), however, does suggest that, on Canadian salmon farms, a similar pathology can develop and Wootten et al. (1982) refer to several epidemics in which mortalities occurred. Our experience of severe infections on farmed Oncorhynchus mykiss is that there are few signs of severe erosion, but fish of varying ages suffer numerous small haemorrhages over the general body surface and the subepidermal tissues become excessively oedematous. Heavily infected fish were also grey in colour, which may be due to an increase in the amount of mucus on the skin. Categorization of skin lesions has been attempted by several authors to describe the nature and extent of adult sealice damage. Dawson et al. (1997) used a five-category scale to examine lesions caused by L. salmonis on experimentally infected sea trout and Atlantic salmon based on severity of damage to the skin. Johnson et al. (1996) used a more complex system in a detailed study to categorize lesions on wild sockeye salmon severely infected with L . salmonis. The capacity of the sealice to cause pathology is therefore related to age (through increase in size), mobility and severity of infection. Larvae of sealice are comparatively small and attached to the skin. Their potential for causing pathology is correspondingly limited compared to the adult sealice, which are much larger, especially in the case of L. salmonis, and able to

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move freely over the body surface. There is evidence also of overdispersion (aggregation) in adult parasite populations (Nagasawa, 1987), which would tend to focus lesions, thereby causing more intensive and persistent damage to the host’s skin.

7.1.3. Behavioural Effects of Infection Parasite-induced increase in irritability may also lead to changes in behaviour, but little is known about this. Furevik et al. (1988) showed that there is a correlation between intensity of infection with sealice and frequency of leaping activity (fish breaks water surface with most of its body above the water), and report a significant decrease in leaping in caged salmon after delousing treatment. Unfortunately, it is not possible to distinguish in these experiments between the effect of sealice removal and the direct effects of the Neguvon@ used to treat the fish. Grimnes and Jakobsen (1996) also reported increased leaping and rolling (fish quietly breaks the water surface) by Atlantic-salmon post-smolts experimentally infected with L. salmonis copepodids. Such activity was six times higher in infected than uninfected fish, both during and after initial exposure to infection. Inappetance is also evident in fish with moderately severe infections (Dawson, 1997).

7.2. PathophysiologicalEffects of Infection

Several studies have been completed to examine the effects of sealice on the physiological condition of the host from both wild and cultured sources. 1.2.1. Wild Salmonids Dawson (1998) examined 51 ascending sea trout smolts (length 20-22 cm) caught in Irish rivers, and recorded skin lesions and blood biochemical changes. Significant reductions in total protein, serum albumin and cholesterol were found in sea trout infected with mobile sealice compared to those with chalimus or without infection. 1.2.2. Experimentally Infected Salmonids Two studies have been completed on the physiological effects of L. salmonis infection on experimentally infected salmonids. Grimnes and Jakobsen (1996) studied 40 f 6.3 g post-smolt Atlantic salmon infected with variable

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numbers of L. salmonis ranging from 30 to 215 at day 11 after infection. Until mobile sealice appeared on the skin, the effect of infections was minimal, both in terms of skin erosion and physiological effects. Thereafter, both skin erosion and blood parameters changes significantly with time. Total serum protein, serum albumin and haematocrit values decreased throughout the experimental period up to 31 days after infection. Plasma chloride levels increased over the same period. All infected fish became moribund during the experimental period and it was concluded that infection levels above 30 sealice larvae per fish had fatal consequences for post-smolts of the size used. Bjerrn and Finstad (1997) conducted a similar experiment on 93.1 f 11.2 g post-smolt sea trout infected with c.80-90 parasites per fish. As the infection progressed to the appearance of mobile stages, fish became moribund and mortalities occurred thereafter. Plasma chloride levels again increased with time and haematocrit levels declined. Plasma cortisol measurements increased with time up to day 24 after infection and lymphocyte to leucocyte ratios deciined with time from day 7 after infection, i.e. during the time when the infection comprised only chalimus larvae. These studies show that measurable osmoregulatory changes take place during the course of infection, and that anaemia and hypoproteinaemia are additional effects of infection. Furthermore, hormonal changes occurred at least so far as cortisol levels are concerned and these may have affected lymphocyte numbers in infected fish. 7.3. Host Responses

Atlantic salmon have very limited cellular and humoral responses to L. salmonis infection. Johnson and Albright (1992a) showed that there was a species-specific variability in cellular response to infection with copepodids of L. salmonis by naive Atlantic, chinook and coho salmon. Coho were shown to be the most resistant to infection characterized by extensive epidermal hyperplasia and a well-developed inflammatory response. As a consequence, chalimus stages were eliminated from the gills and markedly reduced in number on the fins of coho salmon at 10 days after infection. In comparison, Atlantic salmon showed a limited cellular response in which chalimus stages remained on both the gills and fins for the period of the experiment. Chinook salmon had a response level intermediate between the two extremes. The main cell type in all species was the neutrophil and macrophages were common in the fin tissue of coho salmon at 10 days after infection, but there were few lymphocytes present. Another form of cellular response to sealice infection is that of tissue repair at the site of infection. The process of wound healing in fish is known

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to be extremely rapid. This may represent an advantage to the chalimus, which is only able to graze the skin within the radius of its attachment site. If epidermal replacement did not occur rapidly, it is presumably possible that the parasite would exhaust its food supply. Dawson et al. (1998) investigated the possibility of identifying increased mitotic activity in the vicinity of attached sealice by means of the bromodeoxyuridine staining technique. The technique revealed active cells in the epidermis but further study is necessary in order to quantify the effect. Grayson et al. (1991) reported a weak serum response to L. salmonis by naturally infected Atlantic salmon. One antigen recognized by infected salmon had a mass in excess of 200 kDa and was associated with the gut epithelium of the parasite. Johnson (1993) speculated that the difference in rate of development of L. salmonis on Atlantic and chinook salmon could be due to a non-specific host response. 7.4. Transmission of Pathogens by Sealice

Similarly, questions have been raised concerning the possibility that sealice could act as vectors of disease in the way that many insects do. Although this is perhaps unlikely because feeding is mechanical with, so far, no evidence of egested secretions from the parasite, Nylund et al. (1991, 1993) have discussed the potential for transmission by sealice of the furunculosis bacterium Aeromonas salmonicida and the virus that causes infectious salmon anaemia (ISA). Rolland and Nylund (1998) have examined infected fish tissues and shown that ISA is present in large amounts in blood and mucus. They consider it possible in these circumstances that L. salmonis could transmit the infection. It is possible for transmission of pathogens to take place by peripatetic lice, but this has not been shown yet to be of epidemiological importance or to contribute to the overall pathology associated with sealice infection.

8. TREATMENT AND CONTROL OF INFECTION

8.1. Chemotherapeutic Treatments

8.1.1. Dichlorvos Until recently, sealice on salmon farms have been controlled predominantly with dichlorvos (DDVP), a topically applied organophosphate registered as Aquagard SLT@ (Novartis). The development and use of dichlorvos has

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been extensively reviewed by Jackson and Costello (1991) as well as Roth et al. (1993a). The use of dichlorvos for sealice control has declined recently. Dichlorvos is effective against all mobile stages of L. salmonis at concentrations of 1 mg L-I for 1 hour between 5°C and 16"C, but is ineffective against chalimus (Rae, 1979; Wootten et al., 1982). All stages of sealice have acetylcholinesterase (AChE) and it is still not understood why chalimus appear unaffected by organophosphates (Walday and Fonnum, 1989). Treatments cannot begin until chalimus IV have moulted through to preadult and this represents a serious disadvantage in sealice control. Chalimus can cause significant stress and reduction in feeding response (Grimnes, 1994). At higher temperatures the final moult to mobile stage can occur in a matter of hours (Johnson and Albright, 1991a), generating large numbers of preadults that can cause extensive grazing damage, physiological stress and mortalities (Grimnes and Jakobsen, 1996). Treatments need to be repeated frequently, especially at higher temperatures and there is no effective clearance of the parasite from the fish (Boxaspen, 1994). Recently, lack of efficacy against mobile stages has been observed at a number of production sites. Lice populations taken from previously untreated sites had much lower 'resistance ratios' than lice taken from sites that had been treated regularly over the previous 10 years (Jones et al., 1992). The resistance mechanism is poorly understood but may be related to the development of organophosphate detoxifying esterase enzymes. In addition to the development of lice resistance, fish exposed to repeated treatments suffered increasing mortalities. Up to an 80% reduction of brain AChE levels in Atlantic salmon occurred following treatment (Horsberg et al., 1989). Salmon that died showed brain AChE activity down to 4% of unexposed controls (Salte et al., 1987). Effects may be non-reversible, cumulative and increase with age. In healthy, naive salmon the therapeutic margin of DDVP is estimated at four times the recommended dose (Roth et al., 1993a). Following repeated exposure, this therapeutic margin may well decline. The actual volumes of water contained in the tarpaulins vary significantly and this exacerbates the small therapeutic margin. Wells et al. (1990) observed DDVP concentrations within the treatment pen varying from 0.55 mg L-' to 3.5 mg L-l because of poor stratification and mixing. DDVP does not persist in fish tissues and is biodegradable (H0y and Horsberg, 1990). At 4"C, following exposure to 1.0 mg L-' DDVP for 1 hour, residues of up to 0.017 pg L-' and 0.054 pg L-l were detected in muscle and liver tissue 3 days after treatment. At 12°C residues in muscle tissue could be detected directly after treatment but only at trace levels in liver tissue up to 6 days after treatment. The longer retention times of DDVP in the liver tissue was thought to be due to individual variation as well as to excess fat in the liver, resulting in a slower metabolic rate (H0y and Horsberg, 1990). Current withdrawal periods for fish treated with

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DDVP are 14 days in Norway (H0y and Horsberg, 1990) but 4 days in Scotland (Roth et al., 1993a). Roth et al. (1993a) concluded that the use of dichlorvos appeared to be a perceived threat rather than an actual problem with respect to environmental impact. Field studies indicated that the compound appeared to have little, if any, effect on non-target organisms, which, under laboratory conditions, were relatively sensitive to the compound. McHenery (1990) observed mortalities of the lobster Homarus gammarus inside pens during treatment but individuals outside pens were unaffected. The World Health Organization (WHO) found that the proper use of DDVP constituted neither a human health nor environmental hazard (WHO, 1989). Exposure of pelagic organisms to high concentrations of DDVP is transient (McHenery et af., 1996), although concerns have been raised over the effect of repeated exposure (McHenery et al., 1991). Lobster larvae were able to tolerate and recover from five 1-hour exposures to DDVP at concentrations of 50 pg L-' (McHenery et al., 1996). Aquagard has been reclassified under UK law as a prescription medicine for the treatment of lice on fish. Licences for fish medicines are issued on the recommendation of the Veterinary Products Committee. To give this recommendation, the Committee required significant new data to be collected about the effects of DDVP in the marine environment. Control of the release of fish medicines to the environment is by powers under the UK Control of Pollution Act 1974. Discharges from farms are controlled by consents issued by the relevant government body. In Scotland, this falls to the remit of the Scottish Environmental Protection Agency (SEPA). The basis for control of its use depends upon its toxicity and behaviour in the marine environment. Using this information an Environmental Quality Standard (EQS) is established in order to determine a discharge consent (McHenery et af., 1992). An EQS for DDVP of an annual mean concentration of 40 ng L-' and 0.6 pg L-' for the period 24 hours after release has been proposed. If DDVP concentrations following treatment are at 0.6 pg L-' or below, then any effect on sensitive species such as lobsters will be expected to be minimal and reversible (McHenery et al., 1996). Field studies on dispersion and dilution of DDVP following treatments have recorded concentrations ranging from 0.015 pg L-' to 0.35 pg L-' (Wells et af., 1990). Lobster larvae have survived and recovered from prolonged exposure of 23 days to DDVP at 1.23 pg L-l (McHenery et al., 1996). In summary, DDVP has played a major role in the amelioration of sealice infections on salmon production sites since the 1970s and continues to play a limited role in the UK. Legislation has reduced discharge consents and limited its use on production sites in recent years. Additional factors such as increasing sealice resistance, fish susceptibility to toxic effects, low therapeutic margins and lack of effect against chalimus stages has also

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contributed to its reduction in use. Other organophosphates have been assessed for sealice control that are less toxic to fish and have wider therapeutic margins. 8.1.2. Azamethiphos Azamethiphos (Salmosan@, Novartis) is another topical organophosphate used in the control of sealice. Its mode of activity is the same as dichlorvos (Roth et al., 1996). The compound was highly efficacious against adult and preadult L. salmonis at a concentration of 0.01-0.05 mg L-' for 1 hour exposure, but there was no effect against chalimus (Roth and Richards, 1992; Roth et al. 1993a, 1996). Hodneland et al. (1993) reported a lower efficacy of 79% mortality of mobile lice at 0.01 mg L-' azamethiphos for a 40-minute exposure. Azamethiphos appears to have a wider therapeutic margin than dichlorvos and is less toxic to salmon (Roth el al., 1996). Unlike dichlorvos, AChE depression from repeated exposure did not appear to be cumulative (Roth and Richards, 1992). Despite having lower toxicity to fish, there are still a number of concerns associated with its use. Lower efficacy against L. salmonis populations resistant to dichlorvos have been observed and it appears that cross-resistance to both organophosphates has occurred (Roth et al., 1996). No reduction in efficacy was observed with the use of azamethiphos in Canada where L. salmonis populations have not been pre-exposed to organophosphate compounds (O'Halloran and Hogans, 1996). Environmental impact is believed to be similar to DDVP with relatively low toxicity to mussels and oysters, but as toxic to lobster larvae (Roth et al., 1993a). Salmosan@ is licensed in the UK but its use has been restricted with limited discharge consents issued by SEPA (B. Belwood, personal communication). It is licensed and widely available in Norway and use has been permitted in Canada under a time-limited registration (O'Halloran and Hogans, 1996). 8.1.3. Hydrogen Peroxide In the UK, the use of organophosphates has been largely superseded by hydrogen peroxide (Treasurer and Grant, 1997). Hydrogen peroxide (H202) is a strong oxidizing agent that readily breaks down into water and oxygen. It has been used as a bacteriostat, bactericide and fungicide as well as a treatment for ectoparasites on freshwater fish (Thomassen, 1993). The mode of action against bacteria is thought to be due to the formation of hydroxyl radicals and its attack on DNA. Molecular oxygen liberated from Hz02 as a result of catalase activity caused death in Protozoa and Monogenea (Thomassen, 1993). The mode of action against sealice is not fully understood

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but large amounts of oxygen are liberated inside sealice that have been exposed to H202. Gas bubbles were observed in the haemolymph and gut causing the sealice to float to the surface (Thomassen, 1993; Bruno and Raynard, 1994).Thomassen (1993) reported that H202 removed 85-100% of mobile sealice at a concentration of 1.5 g L-' with an exposure time of 20 minutes at 8-12°C. Some variable effects on chalimus levels after treatment were also observed. Johnson et al. (1993a) found that 80% of mobile stages were removed following a 20-minute treatment at 1.5 g L-' and 11°C. However, there was no significant effect on the intensity of infection with the attached chalimus stages. Up to 57% of egg sacs exposed to 1.5 g L-' H202 for 20-40 minutes failed to develop. No copepodids were recovered from the developed egg sacs (Johnson et al., 1993a). Bruno and Raynard (1994) observed that copepodids exposed to 1.25 g L-' H202 died within 19 hours after treatment. It was suggested that there was an initial sublethal effect resulting in weakening of the sealice. Numerous workers have reported a large percentage of sealice recovering following treatment (Hodneland et al., 1993; Holm, 1993; Johnson et al., 1993a; Thomassen, 1993; Bruno and Raynard, 1994; Treasurer and Grant, 1997). Johnson et al. (1994) found that, even at treatment regimes that cause significant mortalities of both chinook and Atlantic salmon, high proportions (84-96%) of chalimus, preadult and adult stages of L. salmonis recovered. Holm (1993) observed that 36% of preadult and adult sealice removed from the surface following treatment were capable of reattaching to salmon in tanks. However, Thomassen (1993) and Treasurer and Grant (1997) did not observe reinfection of preadult or adult stages on production sites following treatment. Hydrogen peroxide is highly toxic to fish with acute lethal toxicity increasing with dose, temperature and exposure (Johnson et al., 1993a; Bruno and Raynard, 1994). At 6°C the LCso (60 minutes) was 2.5 g L-I; LCso (30 minutes) was 8.8 g L-' (Thomassen, 1993). Atlantic salmon exposed to a therapeutic dose of 1.23 g L-' for 20 minutes at 133°C suffered a 35% mortality within 2 hours (Bruno and Raynard, 1994) and 100% mortality at 2.58 g L-' for 20 minutes (Kiemer and Black, 1997). Smaller fish appear more susceptible to toxic effects than larger fish (Bruno and Raynard, 1994). Atlantic salmon Salmo salar are less sensitive to H202 than chinook salmon (Johnson et al., 1994). Goldsinny wrasse Ctenolabrus rupestris were unaffected by a treatment of 1.26 g L-' H202 for 20 minutes at 10°C (Bruno and Raynard, 1994). At low temperature (6°C) the therapeutic ratio for Atlantic salmon is estimated to be 5; however, at higher temperature (14°C) the ratio approaches 0 (Roth et al., 1993a). Subacute toxicity occurred to the gills at concentrations above 3.7 g L-' for 30 minutes at 6"C, with no damage being observed to the cornea or oesophagus. Extended exposure of 60 minutes at 1.6 g L-' and 6°C also resulted in histopathological changes to the gill (Thomassen, 1993).

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Hydrogen peroxide is in use extensively in Scotland, Canada and the Faroe Islands (Bruno and Raynard, 1994). It is not widely used in Norway or Ireland owing to the availability of other, more effective compounds. In the UK it has a full product licence and unrestricted access from SEPA owing to its very limited ecotoxicological effect. There are two licensed products available, Salartect@ (Solvay-Interox Ltd) and Paramove@ (Brentag) (Rae, 1998). Problems have been encountered with its use owing to the large volumes required, safety implications for operators, low therapeutic margin, variable and low efficacy against mobile stages as well as the lack of efficacy against chalimus. Treatment with H202 is not recommended above 14°C (Thomassen, 1993); however, sea-water temperatures regularly reach 16°C in Scotland during the summer months (Kiemer and Black, 1997). Owing to the lack of alternative compounds in the UK, and the reduced discharge consent of organophosphates, hydrogen peroxide treatments have had to continue at higher temperature. During 1997, large numbers of fish were lost during treatments in Scotland over the summer months (S.L. Wadsworth, personal observation). Surviving fish suffered from inappetance, physical damage and rapid recruitment from unaffected attached chalimus stages. More effective and less toxic compounds have been identified, but these are not available for the majority of sites in the UK (Rae, 1998). 8.1.4. Pyrethroids Pyrethrum, the petroleum extract derived from the flowerheads of chrysanthemum (Chrysanthemum cinerariaefolium), the naturally derived precursor to the pyrethroid compounds (Boxaspen and Holm, 1991) was the first agent in the group to be assessed for use in sealice control (Jakobsen and Holm, 1990). An oil containing 4% Py-sal25* (Norsk Pyrethrum A.S.) (1'YOpyrethrum) and 4% piperonyl butoxide was dispensed on to the surface of an Atlantic salmon cage. A floating collar around the surface of the cage retained the oil layer (0.24 mm m-2). As the salmon jumped through the oil, it was presumed that the pyrethrum would penetrate the sealouse cuticle, but not the water-soluble mucus layer of the fish. By 25 days after treatment, there was a significant reduction in the number of all sealice stages between the treated and untreated controls. No detailed observations of the effects on larval stages were given (Jakobsen and Holm, 1990). Boxaspen and Holm (1991) reported an efficacy of 82-96% against mobile stages of L. salmonis when Atlantic salmon were passed through a pipe containing a 1.2 m oil layer with 1% pyrethrin. Effects on chalimus numbers were not recorded. Hogans (1994) reported a 76-99% reduction in mobile C . elongatus following a 1 hour immersion treatment with 0.01 g L-' pyrethrin.

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Although the lice were removed from the fish, they survived for up to 7 days after treatment. There was no effect observed on the chalimus survival or rate of development by 5 days after treatment. Roth et al. (1993b) reported an efficacy of 90% against mobile stages of L. salmonis following a 1-hour bath treatment with resmethrin at concentrations ranging from 0.0 1 to 0.1 mg L-'. Lamda-cyhalothrin was also found to be highly efficacious at a concentration of 0.005 mg L-' (l-hour bath treatment). Neither of the formulations appeared to be significantly toxic to the larval stages at the concentrations that were found to effectively kill adults and preadults. Hart et al. (1997) applied a novel formulation of cypermethrin (Exis@)as a bath treatment on production sites for 1 hour at 5 pg L-'. Statistically significant reductions (> 90%, P < 0.01) in the number of both mobile and chalimus III-IV were observed. No significant reductions were observed on the numbers of chalimus 1-11. Recent laboratory studies have confirmed efficacy of cypermethrin against all chalimus stages with a significant reduction in mean numbers (83%, P
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Pyrethroids such as resmethrin or lambda-cyhalothrin show improved therapeutic margins between lice and fish toxicity over currently used sealice chemotherapeutants. The estimated therapeutic margin for Aquagard is four, compared to a margin of five for lambda-cyhalothrin and ten for resmethrin (Roth et al., 1993b). Initial target animal safety studies for cypermethrin show a therapeutic margin of 200 (unpublished data). Exis@ is currently available for use in Norway and restricted use in Canada and Ireland. A further pyrethroid compound deltamethrin, (Alphamax@, Alpharma as.) is also available in Norway and is widely used. Exis@ is awaiting a full product licence for the UK. Availability and use of pyrethroids in the UK will ultimately be determined by discharge consents issued by SEPA. A licensed medicine that has good efficacy against all stages of L. salmonis and has low toxicity to fish represents a major advance over currently available topical compounds. All topical treatments involve the enclosure of large volumes of water prior to the administration of the compound. Variability in the enclosed volume of water can significantly affect the final dose and may result in reduced efficacy (Roth et al., 1996). Other disadvantages of topical treatments include high associated labour costs, vulnerability to environmental conditions and stress to fish (Costello, 1993). The recent increase in the size of fish pens (Anonymous, 1998) has rendered the enclosure of larger water volumes increasingly difficult. Some operators have resorted to the use of skirts rather than fully enclosed tarpaulins. This may expose the lice to sublethal concentrations of the compounds and increase the risk of resistance (Costello, 1993). Efficacy of treatments is also reduced with the use of skirts (Roth et al., 1996). Compounds that are administered orally overcome many of the difficulties of topical treatments. 8.1.5. Ivermectin Efficacy against L. salmonis was initially assessed by Palmer et al. (1987). Ivermectin administered orally at a dose of 0.2 mg kg-' body weight at intervals of 14-2 1 days produced significant reductions in populations of L. salmonis and C . elongatus. Damage to the host by the copepods was also prevented. Reinfection by juvenile copepods occurred during the trial but these did not survive to adult. Smith et al. (1993) recorded a 93% reduction in lice numbers following an oral administration of 0.05 mg kg-' body weight. At a dose of 0.02 mg kg-' body weight there was a 46% drop in numbers of sealice suggesting that this treatment regime was close to or below the lowest effective dose. No fish mortalities were observed at 0.2 mg kg-' body weight over prolonged use under commercial conditions. Johnson and Margolis (1993) fed 0.05 mg kg-'

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body weight every third day until fish had received between three and six doses. They observed a significant reduction of all stages of L. salmonis. There was also a significant reduction in the rate of development of chalimus on treated fish, as well as the fish being protected from reinfection following termination of the treatment. The duration of protection remained to be determined (Johnson and Margolis, 1993). Palmer et al. (1987) found that ivermectin had a narrow margin for safety with fish mortalities occurring at twice the recommended mammalian dosages. Cumulative mortalities of > 80% were reported for Atlantic salmon fed three doses of ivermectin at 0.20 mg kg-I body weight or one dose at 0.50-1.0 mg kg-' body weight (Johnson et al., 1993b). Atlantic salmon were more susceptible to the toxic effects of ivermectin than chinook or coho, which were the most resistant. The surviving fish also showed a loss of equilibrium, reduction in feeding activity and darkened in colour. Histological examination of the major organs showed no pathological changes that could be associated with ivermectin toxicity (Johnson et al., 1993b). The LDSOfor a single oral intubation of Atlantic salmon with ivermectin was recorded as 0.5 mg kg-' and 17 pg L-' for a 96-hour immersion (Kilmartin et al., 1996). H0y et al. (1990) followed the distribution of orally intubated [3H]ivermectinat 0.2 mg kg-' body weight. They found high concentrations in the central nervous system, indicating a poorly developed blood- brain barrier in salmon compared to mammals. This may explain the relatively low margin of safety for this drug towards fish species (H0y and Horsberg, 1991). The excretion of the drug was very slow, with the treatment dose being reduced to 19% after 28 days. The drug was mainly excreted unchanged via the bile (Hay et af., 1990). Kennedy et al. (1993) treated Atlantic salmon orally with 0.05 mg kg-I body weight twice weekly for 2 months. At 0 degree days withdrawal, the concentration of ivermectin in brain, gill, kidney, skin and spleen were very similar (47-87 ng g-I). The ivermectin concentration in muscle was lower than that detected in any other tissue (35.2 ng gg'), whereas that in the liver was higher (459.8 ng g-I). The half-life ( t 1 / 2 ) of ivermectin in liver, muscle and skin ranged from 89 to 98 degree days (OD). Roth et al. (1993~)found longer retention times in the skin (t112= 188.1'0) than for the muscle ( t 1 / 2 = 120.4"D) following an oral treatment of 0.05 mg kg-' once a week for 9 weeks. Residues of 7.2 f 4.7 pg kg-I could be detected in muscle tissue following 500 "D withdrawal and in the skin following 750"D withdrawal (5.2 f 1.5 pg kg-I). No levels were detected after 1000"D withdrawal (67 days at 15°C). Environmental risks of terrestrial use of ivermectin and abermectin are usually considered to be low as the compounds are rapidly photolysed, have a high affinity to particles and are not bioaccumulated (Grant and Briggs,

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1998a). Use of ivermectin in the marine environment may lead to a longer residence time as the rate of breakdown is dependent upon the amount of light present and temperature (Grant and Briggs, 1998b). Owing to the potentially long residence time in sediment, concern has been expressed over the impact of ivermectin to benthic organisms below cages. Black et al. (1 997) found ivermectin had a significant effect on the polychaete Capitella sp. at applications of 8 1 and 8 10 pg mP2.This concentration was an order of magnitude greater than would be expected from a single treatment. The shrimp Crangon septemspinosa was unaffected by ivermectin in water at 21.5 pg L-I, but sensitive when fed on salmon pellets containing ivermectin (96-hour LD50 = 8.5 pg g-' food). The NOEC value was 2.6 pg g-' (Burridge and Haya, 1993). Although ivermectin has not been fully licensed for use in any country (Smith et al., 1993), it has been used extensively in Ireland (Costello, 1993). Small-scale trial use has been permitted at a limited number of sites by SEPA in Scotland, after research to determine ecotoxicology (Anonymous, 1996). 8.1.6. DiJlubenzuron Diflubenzuron is an established insecticide belonging to the insect growth regulator (IGR) group (Hay and Horsberg, 1991). During the intermoult period, the effect of the pesticide on crustaceans was negligible (Horsberg and H0y, 1991). The compound is registered as Dimilin@ (T. H. Agricultural & Nutrition Company) in the USA for control of the gypsy moth and in Canada for the control of larval mosquitoes (WHO, 1996). The effect against L. salmonis was examined by H0y and Horsberg (1991) who found that an oral dose of 75 mg kg-' over 14 days produced a significant reduction in adult and larval stages of sealice. This dose was considered high when compared to other compounds such as ivermectin (0.05 mg kg-' for 2 days) to achieve the same result (Roth et al., 1993a). Further trials have shown 98-100% efficacy against L. salmonis after 14 days of oral treatment at a much lower dose range of 2.26-4.54 mg kg-I, within the temperature range of 7-17°C (Erdal, 1997). It is efficacious against all developmental stages of L. salmonis, although the effect against adults is low because the moulting sequence has been completed. Some chitin deposition is still thought to occur especially during deposition of egg sacs. Efficacy is increased if treatments are targeted against the early chalimus stages and would be particularly useful in strategic management (Erdal, 1997). Diflubenzuron was poorly adsorbed by the gut of salmon with peak concentrations representing only 3.75% of the administered dose, found in

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the blood, muscle, liver and kidneys 12 hours after administration. A proportion of the compound was found to accumulate in the cartilaginous tissues (Horsberg and Hay, 1991). Despite low adsorption from the intestine, approximately 4 pg g-' of diflubenzuron was detected in the skin mucus layer 48 hours after administration. Up to 37% of the compound was rapidly metabolized and excreted via the bile within 6 hours of administration. Varying concentrations of 14C-labelled diflubenzuron (and/or metabolites) were present in the bile for 10 days, after which they began to decrease significantly. Diflubenzuron as (Lepsidon@;Ewos as.) is available for use in Norway as a formulated feed pellet. Its use is currently controlled under a conditional exemption where use is restricted to smolts in the first year at sea. A conditional exemption is granted for a 2-year period. A full market authorization licence in Norway is expected in the near future (C. Wallace, personal communication). 8.1.7. Tejlubenzuron Other IGRs are also being assessed in the control of sealice. These include teflubenzuron (Ektobannm-Skretting a s . in Norway and Calicide@in the UK). An oral treatment of 10 mg kg-' body weight daily for 7 days resulted in a reduction in lice levels of 90%, 7 days after treatment (Grantvedt, 1997). Treated sealice were attached more loosely than controls, were less mobile, a smaller size and had a slower rate of development. The cuticle lacked organization, was only half as thick and the basal membrane was absent. Greater changes were found in sealice collected from the gills than from the skin. This may be due to those on the gills being more exposed to the compound from the richer supply of blood (Grantvedt, 1997). In the control group, 90% of sexually mature females had developed egg sacs, with none being observed in the treated group. In field trials, some ovigerous females had developed prior to the treatment. The egg sacs subsequently exposed to teflubenzuron had major deformations and defects (Grantvedt, 1997). Further toxicological studies on non-target species and environmental impact assessments are currently being conducted in order to determine the viability of use of IGRs in salmon production (Ritchie, 1995). Teflubenzuron is currently in use in Norway and controlled under a 2-year conditional exemption. IGRs will have to be used in conjunction with compounds that are effective against adult stages such as pyrethroids. Their controlled, targeted use will be especially useful during periods of increased larval settlement on production sites such as spring (Wadsworth et al., 1997).

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8.2. Wrasse

Initial aquarium studies found that four species of wrasse, namely goldsinny Ctenolabrus rupestris, rock cook Ctenolabrus exoletus, corkwing Crenilabrus melops and, to a lesser extent, cuckoo Labrus mixtus removed lice from salmon (Bjordal, 1988, 1990, 1991). Wrasse have also been observed removing mobile L. salmonis and C . elongatus from salmon in production cages (Costello and Donelly, 1991; Treasurer, 1991, 1994, 1996a; Darwall et al., 1992). There was some evidence for removal of chalimus stages (Costello, 1993), although this has not been reported in other studies. In field trials, cages with wrasse stocked at 1:lOO salmon had mean lice numbers ranging from 3 to 12. Lice numbers in the controls cages rose to 46 per fish. Wrasse were transferred to these cages to avoid treating them with dichlorvos and there was an immediate reduction to 12 lice per fish. Once the wrasse were removed, the numbers of lice rose to 45 per fish within 8 days (Treasurer, 1993). The number of lice consumed by an individual goldsinny was in the range of 26-46 lice per day, wet weight 349-907 mg, representing 1.2-2.7% body weight for a fish of 30 g (Treasurer, 1993). This cleaner fish technology has been widely adopted in Norway and Scotland. By 1994 over 130 farm sites in Norway were using 1.5 million wrasse and 30 farm sites in Scotland were using 150 000 wrasse (Costello, 1996). These fish were all wild caught from inshore coastal waters (Sayer et al., 1996; Treasurer, 1996b). Concern has been expressed over the impact on local stocks if this effort continues or increases (Danvall et al., 1992; Varian et al., 1996). Long-term or widespread effects on wrasse populations are unlikely owing to their early maturity, high fecundity, abundance in areas distant from farms and capacity for rapid recruitment (Darwall et al., 1992; Costello, 1993). Preliminary trials have been conducted on culturing wrasse intensively and the initial results appear encouraging (Sherwood, 1990; Skiftesvik et al., 1996; Stone, 1996). There are difficulties associated with the use of wrasse in sealice control that have led to a reduction in the number of wrasse deployed in recent years (J.W. Treasurer, personal communication). Both typical and atypical Aeromonas salmonicida have frequently been isolated from wild-caught wrasse (Treasurer and Cox, 1991; Frerichs et al., 1992; Costello, 1993; Treasurer and Laidler, 1994; Costello et ul., 1996; Karlsbakk et al., 1996). Wrasse are susceptible to both typical and atypical strains of A . salmonicida as well as Vibrio anguillarum (serotypes 01 and 02), resulting in very high mortality of the introduced stock (Gravningen et al., 1996). Although the atypical strain was non-pathogenic to salmon (Frerichs et al., 1992), it represents a significant risk to the industry owing to the limited number of licensed antibiotics and vaccines available to combat A . salmonicida (Evelyn, 1997). There is also the potential for viral transmission from wild-caught

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wrasse (Gibson and Sommerville, 1996). However, many of these potential health risks could be alleviated by using cultured wrasse, reared in protected conditions.

8.3. Management The coordination of fallowing and the stocking of single-year class fish within designated management areas has led to a significant reduction in lice infestations in Ireland (Jackson et al., 1997), Scotland (Grant and Treasurer, 1993) and Norway (Boxaspen, 1997). Bron et al. (1993a) found that smolts introduced into a multiyear class site in April quickly became infected with lice and needed to be treated by June. Smolts introduced into a single-year class site, after the site had been fallowed, were not heavily infected with lice and no treatments were needed until the following winter. A number of sites were examined with varying length of fallow periods. It was found that the longer the fallow period, the longer the refractory period before lice numbers rise to a level where treatment becomes necessary. The numbers of C.elongutus were not influenced by a fallow period. Grant and Treasurer (1993) observed that smolts transferred to a multiyear class site were infected with copepodids of L. salmonis after 3 days and required treatment within 4 weeks of transfer. The minimum fallow period recommended was 30 days over the period February to March, although survival of females off host 32 days in the laboratory according to Grant and Treasurer (1993) should be taken into account. In 1997, 166 (49.7%) production sites in Scotland incorporated a fallow period. On the sites that did not fallow, the practice of stocking with multiple-year classes of fish was still common (SOAEFD, 1997). The increasing use of photoperiod (SOAEFD, 1997) for smolts will have to be carefully managed to ensure adequate fallow periods remain within loch systems. Understanding the epidemiology of sealice infestations in relation to these management and treatment strategies is essential for implementing effective control measures (Tully, 1989). Wootten et ul. (1982) examined the succession of generations of L. salmonis and recommended treatment at the preadult stage prior to the development of ovigerous females owing to the ineffectiveness of dichlorvos to remove larval stages. Bron et al. (1993d) examined the influence of dichlorvos treatments on the epidemiology of L. salmonis. A general rise in the intensity of infection was observed and this was temporarily interrupted by treatments. On multiyear class sites, numbers of sealice built up far more rapidly than on single-year class site, despite treatment. Where treatments were conducted against a population sensitive to dichlorvos, a large proportion of the mobile stages were removed as well as a reduction in the number of settled larval stages observed

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following treatment. It was concluded that, owing to the fall in the number of larvae following treatment, the sites were principally self-infecting. It was suggested that, in order to reduce the pool of infection for a site as a whole, all groups on a farm needed to be treated within a short space of time. At some production sites, the initial infection of L. salmonis is derived from an external source (S.L. Wadsworth, personal observation). Dispersal and transmission of infective stages of L. salmonis is not fully understood, but is likely to be influenced by a number of factors including distance, temperature and hydrography between sites (Heuch, 1995; Costelloe et al., 1996a; Boxaspen, 1997; Jackson et al., 1997). Extending treatment areas to coordinate between farms within a loch system has also increased efficacy (Wadsworth et al., 1997). By instigating a series of coordinated, synchronous, strategic treatments throughout the loch system during the winter, initial chalimus levels during the spring were significantly reduced by 90% (P< 0.001). Lice numbers for the rest of the production cycle were significantly lower (P< 0.01). There was also a reduction in the number of treatments needed, an increase in the interval between treatments, reduced fish mortalities and improvements in fish harvest quality (Wadsworth et al., 1997). Larger geographical areas and a greater number of production sites were incorporated into the treatment strategy during 1998 and coordinated by the Scottish Salmon Growers Association (SSGA). A number of these sites throughout the west coast of Scotland have shown beneficial effects (Rae, 1998). Coordination of treatments and common winter delousing has also been attempted in Norway (Melingen, 1997) with positive effects (C. Wallace, personal communication). Winter and spring delousing strategies have been based upon the seasonal variation observed at many sites in the epidemiology of L. salmonis. Low recruitment of L. salmonis during the late winter (January-early March) followed by increased larval settlement observed during the spring have been reported by a number of authors (White, 1940, 1942; Wootten et al., 1982; Hogans and Trudeau, 1989b; Jackson et af., 1997). High lice numbers have also been observed during the winter (Wootten, 1985; Bron et al., 1993c) but this may be due to milder temperatures occurring during the earlier winter periods (Boxaspen, 1997). Hogans (1995) observed that the proportion of ovigerous females to non-ovigerous females was higher towards the end of winter. The production of egg sacs by mature females was relatively constant until the end of March when a significant increase was noted. The rate of infection by copepodids only increased with rising water temperature ( r 2 = 0.82) at the end of March. Rising water temperature has an important effect upon the intensity of infection. The ratio between the period during which a cohort is spawned and during which it matures is dependent upon the direction and degree of changes in water temperature (Durban and

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Durban, 1992). From this it may be predicted that the reproductive output of L . salmonis increases in the spring and reaches a peak in early summer (Tully, 1992). The intensity and timing of this peak in output may vary between years depending upon the rate of change of temperature (Tully, 1992). Total output of planktonic stages of L . salmonis from both farmed and wild salmon in some regions of Ireland is highest during the spring and early summer (Tully and Whelan, 1993). Thus it is essential to remove ovigerous L . salmonis populations from production sites prior to this period. Other Caligid parasites such as Lepeophtheirus pectoralis exhibit distinct seasonal variability in abundance (Boxshall, 1974e). Low numbers of L . pectoralis were observed over winter and the population comprised mainly adults that had survived from the previous year. The overwintering females produced 2-3 batches of eggs in April and died soon afterwards. By May over 50% of the population comprised infective copepodids from the winter female population. The seasonal variability of Lepeophtheirus sp. may be related to the density of host populations (Boxshall, 1974e; Pemberton, 1976; Tully and Whelan, 1993). 8.4. Future Control Strategies

8.4.1. Integrated Pest Management

In recent years, there has been an increasing understanding of the epidemiology of L . salmonis, the availability of more effective compounds as well as improvement in management and coordination of treatments between sites. Further improvements could be attained with the development of an integrated pest management (IPM) scheme for L. salmonis as suggested first by Pike (1989) and, more recently, repeated by Sommerville (1995). There has been increasing concern over the development of pest resistance as well as environmental impact. Discharge has been limited over the past few years and alternatives sought to reduce organophosphate use. Recent use of insect growth regulators have proved more effective as well as more compatible with biological controls (Valentine et al., 1996). Efficacy against agricultural pests has been increased further by targeting treatments to specific periods as well as using available pesticides in rotation with other controls within an IPM scheme (Marsula and Wissel, 1994; Hall and Barry, 1995; Pickett et al., 1997). Similar IPM schemes could also be adopted for salmonid aquaculture. An ideal IPM scheme would incorporate loch fallowing, stocking of single-year class fish along with hatchery-reared wrasse. The gradual increase in lice numbers during the first year at sea would be intensively monitored and the information

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freely exchanged between relevant bodies. Targeted, planned treatments could be coordinated and synchronous between sites within the management areas. Compounds available could be used on rotation to reduce the risk of the development of resistance as well as environmental impact. It is expected that an effective adoption of an IPM scheme will reduce the overall number and increase the interval between treatments. This should also reduce any adverse environmental impact. Other stakeholders should be included in any IPM scheme, such as representatives of crustacean and wild salmonid fisheries, as well as environmental groups. Rational concerns could then be identified and strategies adapted to ameliorate any impact. 8.4.2. Husbandry Basic husbandry practices could be adapted to ameliorate L. salmonis infection. It has been found that, whilst fish were hungry, they congregated near to the surface to await feed (Huse and Holm, 1993). As they were fed, they gradually descended to greater depth where they remained whilst satiated (Fern0 et al., 1995). It has been shown that fish maintained in 0-4 m will have up to 40 times more lice than fish maintained at lower depth (Hevray et al., 1997). With the advance of recent feeding technology, such as demand feeding systems, it may be possible that lice settlement could be reduced by keeping fish well satiated and maintained at a depth below the optimum for settlement of L. salmonis. 8.4.3. StresslDisease It has been demonstrated that physiological stress such as osmoregulation significantly increase susceptibility of Atlantic salmon to infection by L. salmonis (Grimnes et al., 1996; Grimnes and Jakobsen, 1996; Dawson, 1997). Thus care should be taken to transfer smolts, which are well adapted to sea water, into sites that have been fallowed and are free of L. salmonis. Routine husbandry practices such as grading and sample weighing are likely to stress and physically damage the fish, making them more susceptible to settlement. Methods and technology could be further adapted to reduce these stressors. Additional factors will also affect susceptibility to L . salmonis, such as sexual maturity (Johnson, 1993) and disease status. Fish affected by A . salmonicida had up to 70% more L. salmonis chalimus than unaffected fish (Wadsworth, 1998). Thus immune/physiological status of the fish should be an important factor in future lice control strategies (Firth et al., 1999).

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8.4.4. Immune Modulation

The immune system of fish is capable of producing a variety of responses that are capable of protection against parasitic infections (Woo and Shariff, 1990; Woo, 1992). The potential exists for immune modulation of salmonids to lead to a reduction in susceptibility to L. salmonis (Woo, 1997). An innate response by coho salmon to L. salmonis infection was characterized by acute inflammatory response and hyperplasia. In severe cases, the chalimus were completely encapsulated and the surrounding infiltrate comprised neutrophils, macrophages, lymphocytes and necrotic tissue. No reaction, or only limited response, was observed in coho salmon injected with cortisol (Johnson and Albright, 1992b). Non-specific, innate defence mechanisms were responsible for the effective control of L. salmonis on coho salmon and suppression of these responses significantly increased the levels of infection (Johnson and Albright, 1992b). A more limited response to L. salmonis infection has also been observed for Atlantic salmon. The types of proteases collected from the mucus of Atlantic salmon infected and non-infected with L. salmonis were identical. However, mucus from the infected fish exhibited substantially higher quantities of protease activity, especially serine and metalloproteases (Firth et af., 1999). These proteases affect the production of mucus as well as innate immune reactions in fish (Firth et al., 1999). Incubation of naive control mucus with sealice extract did not show any elevation of proteases. Incubation of naive control mucus with live adult sealice significantly increased protease activity, including the production of additional proteases, which could have been derived from the sealice (Firth et a[., 1999). Arctic charr and Atlantic salmon fed iodine had lower blood cortisol levels and lower numbers of C. elongatus after challenge than fish implanted with cortisol (Mustafa and MacKinnon, 1993). Variability in lice infection between individual fish was attributed to differences in genetic make up, handling and disease status (MacKinnon, 1998). Immunosuppression by cortisol has been shown to elevate infection for a range of fish pathogens including bacteria, viruses and parasites in addition to L. salmonis (Anderson, 1990; Houghton and Matthews, 1990; Espelid et al., 1996; Bly et af., 1997). Crude antigen of L. salmonis and C. elongatus injected into rainbow trout produced antibodies that reacted to antigens of mobile chalimus and eggs of lice (Stone, 1989; Wadsworth, 1989; Grayson et al., 1991). Immunohistochemical screening of monoclonal antibodies produced to L. salmonis revealed binding to oviducts, ovaries, cuticle, haemolymph and brush border of the gut epithelium (Andrade-Salas et al., 1993; Grayson et al., 1995). Naturally infected Atlantic salmon and rainbow trout have produced antibodies to various antigens of L. salmonis, including the gut (Grayson et al., 1991). Immune activity against the gut wall of the cattle tick Boophilus

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microplus has shown some reduction in tick survival and fecundity (Opdebeeck et al., 1988; Willasden et al., 1989). Potential vaccine formulations against L. salmonis have also concentrated on gut antigens. Atlantic salmon immunized with purified gut antigens of L. salmonis showed a significant difference ( P < 0.01) in the number of ovigerous females and these sealice possessed fewer eggs per egg sac. There was no difference observed in the viability of the eggs between the groups nor was there any difference in the number of other mobile stages observed (Grayson et al., 1995). Recent investigations have concentrated on the larval stages of L. salmonis, which offer increased potential for immune control, owing to their more intimate association with host tissues than mobile stages (Pike et al., 1993a). Antigenic differences were observed between chalimus and mobile L. salmonis, with antibodies being produced to both stages in rainbow trout (Grayson et al., 1991). Survival and development of L. salmonis chalimus on salmonid species may be dependent upon either masking the epitopes of exposed antigens, especially the hold fast, or suppressing the host’s immune system. It appears that the ability to mount an effective immune response against L. salmonis is highly variable between salmonid species. Atlantic salmon and sea trout are especially vulnerable to L. salmonis infection, whilst coho and chinook salmon appear relatively resistant (Johnson and Albright, 1992a; Grimnes, 1994; Grimnes and Jakobsen, 1996; Dawson et a/., 1997). Plasmacortisol levels have been shown to increase naturally in salmonids following infection with L . salmonis with significant variation between Arctic charr, Atlantic salmon and sea trout. Highest plasmacortisol levels were observed in sea trout following infection, which induced immunosuppression (reduction of lymphocytes) and elevated levels of L. salmonis in relation to the other species (Grimnes et al., 1996). Other species of Lepeophtheirus are specifically adapted to certain host species: L. hippoglossus on halibut Hippoglossus hippoglossus (Kabata, 1979); L. pectoralis on plaice Pleuronectes platessa (Boxshall, 1974e); L. thompsoni on turbot Scophthalimus maxima; and L. europaensis on brill Scophthalimus rhombus (De Meeiis et al., 1993). This suggests that certain Lepeophtheirus species have evolved mechanisms for defeating the immune responses mounted by their respective hosts. In contrast to L. salmonis, C. elongatus has an extensive range of hosts, including some 80 species of fish world-wide (Kabata, 1979). C. elongutus was found to contain a greater number and quantity of both serine and non-serine proteases than L. salmonis. This was related to the more diverse array of biochemical substrates present in the food (Ellis et al., 1990). Potential mechanisms for defeating the immune systems of fish by sealice are not fully understood and a variety of factors may be involved. Parasitic copepods have a higher number of exocrine glands present on the dorsal

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surface than non-parasitic copepods (Bell et al., 1998). Both larval and mobile stages of L. salmonis had numerous exocrine glands that stained positive for peroxidase, although other compounds may also have been present (Bell et al., 1998). The saliva of adult, female, lone star ticks Amblyomma americana contained a cocktail of pharmacologically active compounds, e.g. immunosuppressants, analgesics, anticoagulants, antiplatelet aggregatory compounds (Bowman et al., 1996). Prostaglandin (PGE2) possesses many of these activities, which are believed to be extremely important to tick infection and survival on the host. The synthesis of prostaglandins and other eicosanoids have been reported for protozoan, trematode, cestode and nematode parasites (Bowman et al., 1996). It is not known whether L. salmonis secretions include, or are able to lead to the synthesis of prostaglandins, but elevated PGE2 levels have been observed in a limited number of Atlantic salmon sera following infection with L. salmonis (unpublished data). Isolation and characterization of eicosanoids involved in immunosuppression could lead to effective immunization of Atlantic salmon. It is unlikely that any form of immune modulation would be effective as a ‘stand-alone’ control and would need to be incorporated into an overall IPM strategy including antisealice medicines. 8.4.5. Stock Selection Since 1975, breeding programmes have been established for Atlantic salmon and rainbow trout in Norway and Scotland, Selection has focused on increased growth rates, reduced frequency of early maturation, improved flesh quality and disease resistance. The programmes have dramatically improved growth, survival and production efficiency of the aquaculture industry (Refstie, 1995; Gjedrem, 1997). There have been good results to date in the selection for resistance as fish immune responses appear to have a high heritability (Fjalestad et al., 1993; Schreck, 1996). Variable resistance by fish populations have been demonstrated to bacteria, such as Vibrio anguillarum, A . salmonicida and Renibacterium salmoninarum (Beacham and Evelyn, 1992; Marsden, 1993; Lund et al., 1995). It has been estimated that selective breeding could be expected to reduce stock mortality to furunculosis by 34% per generation and that this may correspond to increased resistance to other diseases (Gjedrem, 1997). Resistance in rainbow trout to the myxosporidian Ceratomyxa shasta varied significantly between different populations (Ibarra et al., 1994) as did brook trout to the haemoflagellate Cryptobia salmositica (Woo, 1992). Variation in settlement of Caligus elongatus has been observed between different families of Atlantic salmon and it was suggested that

SEALICE O N SALMONDS

31 1

resistance to sealice might be a heritable trait. However, the mechanisms of resistance were not understood (MacKinnon et al., 1995). Higher mean numbers of C. elongatus were also observed on certain families of rainbow trout but the difference was not significant. Epidermal thickness varied between the trout families, and it was thought that a thinner epidermis could allow easier access to the dermal layers for feeding on host tissues and fluids (G. Ritchie, personal communication). Significant differences in mean numbers of L. salmonis were observed after challenge on different stocks and families of Atlantic salmon (Wadsworth, 1998).

9. ECONOMICS OF SEALICE INFECTION

Sealice were the most serious threat to salmonid aquaculture in terms of economic losses (until the appearance of infectious salmon anaemia). Even where vigilance and good fortune minimize stock losses, the regular indirect wastage from treatment will translate, during a production cycle, into significant loss of profitability. These include, depending upon farm practice and severity of infection, pre- and post-treatment loss of growth from withholding feed, and inappetance due to infection and the treatment. 9.1. Treatment Costs of Sealice Infection in Scotland

This analysis of treatment costs was contributed by Scott Peddie as an abbreviated version of Peddie (1997). It attempts to quantify the enormous cost to the Atlantic salmon cage culture industry using data from Scottish sources. Whilst every effort has been made to present an accurate picture, no single model can account for all the variables and the data used are those that we believe to be reasonably representative. Sealice control can constitute a substantial proportion of variable costs so it follows that choice of treatment is vital to profitability. Not only that, the legislative choices made by the regulatory authorities such as the SEPA and the Veterinary Medicines Directorate (VMD) can have wide-ranging implications for the economics of salmon culture in Scotland. It would be tempting to simplify the issue by comparing each treatment option based on treatment cost alone, but this would be a gross oversimplification. In reality, there are many parameters to consider that can be categorized into those relating to the composite costs of treatment and those pertaining to the savings made as a result of treatment (Figure 6). Either all or a combination of these categories can be incorporated into the

312

A.W. PIKE AND S.L. WADSWORTH

Losses due to side effects

COSTS

t

f

MODEOFTREATMENT

) SAVINGS

Figure 6 The relationship between treatment costs and savings for L. salmonis infections of Atlantic salmon. (Reproduced with permission from Peddie, 1997.)

following three comparative economic tools: 0 0 0

cost per fish; break-even points; treatment margins.

Each of these will be used to facilitate a financial appraisal of Cypermethrin@, hydrogen peroxide, IvermectinB, AquagardB and wrasse as methods for controlling sealice. To do this, a hypothetical farm with a 27 000 m3 capacity and a target production of 540 tonnes will be used, with fish harvested after either one or two sea winters. The figures used for cost of treatments are best estimates and might not accurately reflect actual current costs. 9.1.1. Cost per Fish

Quantifying cost per fish necessitates considering the cost of the treatment (or vaccinated/screened wrasse in the case of biological control), the cost of labour involved in treatment, the value of fish lost due to side-effects and the reduction in fish growth. Additional costs, such as oxygen costs and treatment tarpaulins for bath treatments, must also be taken into account.

313

SEALICE ON SALMONDS

As a function of actual treatment cost, labour costs and reduced fish growth subsequent to treatment, the analysis indicates that sequential hydrogen peroxide bath treatments are the most expensive, regardless of the duration of the growout cycle (Table 3). Conversely, the in-feed application of Ivermectin@will minimize the financial impact, largely due to the absence of a requirement for oxygen, tarpaulins, additional labour and periods of starvation. Moreover, although wrasse are the second least expensive form of treatment, practical experience in Scotland suggests that farmers only use them for the period subsequent to smolt transfer until the first sea winter is reached. 9.1.2. Break-even Point Cost per fish only addresses one side of the equation; it does not take into consideration the savings accrued from applying the treatment in terms of reduced mortality and increased growth. To do this, it is necessary to use break-even point analysis. The break-even point occurs when the savings associated with using a particular treatment equal the costs (Figure 7). The area to the right of the intercept represents the region where the particular treatment is cost effective, i.e. savings are greater than costs; the non-cost-effective area is where costs outweigh savings. The x-axis shows the percentage of fish mortalities attributable to sealice infection. This can be related to the savings made because of treatment. Although the former may vary between sites, it is reasonable to assume that, if they are left unchecked, sealice will cause 100% mortality. Theoretically, the x-axis of the model should also incorporate reduction in growth because of infection, but as no reliable data exist to quantify this reduction, it is excluded. The break-even point can be calculated using a formula adapted from a model developed by Lillehaug, (1989) for evaluating the cost effectiveness of

Table 3 Cost per fish (Atlantic salmon) per treatment for a 540-tonne enterprise where fish are harvested after either one or two sea winters (SW). -~

~

Treatment method Cypermethrin H202

Ivermectin Aquagard Wrasse

Cost per fish for salmon sold after 1SW (pence)

Cost per fish for salmon sold after 2SW (pence)

45.53 50.01 1.62 33.68 5.61

122.69 147.66 5.79 64.82 n.a.

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A.W. PIKE AND S.L. WADSWORTH Saving when the site is at risk from 100% sealice-induced mortality

,

Savings

Coststsavings Area where treatment is cost-effective

costs Area where treatment is not cost-effective

*100% % Mortalities attributable to sealice infection

Figure 7 Conceptual break-even point graph used to evaluate L. salmonis treatments on Atlantic salmon. (Reproduced with permission from Peddie, 1997.)

vibriosis vaccines. It can be formalized as follows:

Labour

Treatment

7

Value per fish

costs: Hme, Wh

Ptreat Cadd

The man-hours required for the treatment method concerned Workers’ hourly wage The price of treatment over the entire cycle, or the cost of wrasse (at a ratio of 1 wrasse: 50 salmon) Additional costs such as necessary equipment, the value of fish lost due to side effects and reduced growth. For wrasse, additional costs include screening a sample of fish for pathogens, identification of pathogens, stress tests and vaccinating the population against vibriosis and furunculosis.

Savings: FCR Mno

Feed conversion ratio (averaged over the entire production cycle) The expected or actual mortality because of infection

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SEALICE ON SALMONDS

The relative percentage efficacy for the treatment method The mean weight of fish at slaughtering The price obtained for the fish per kilogramme The price of food per kilogramme (averaged over the entire production cycle).

RPE,,, Wfish

PkkPfd

Table 4 highlights the fact that the relatively high break-even points for hydrogen peroxide in both 1- and 2-sea-winter fish, in conjunction with the lowest levels of savings for application of a treatment, make it the least economically viable option. In contrast, only 1 YOoverall stock mortality has to occur to make IvermectinB treatment financially viable. Moreover, the relatively high efficacy of the product, concomitant with low purchase costs and minimal 'additional costs', means that savings are maximized by the exclusive use of IvermectinB. Furthermore, despite the fact that CypermethrinB is the second most expensive treatment to administer, its high level of efficacy ensures that savings will be maximized by using it strategically. Usage of another bath treatment, AquagardB, results in intermediate break-even points and savings. Although wrasse become economically viable at 6% sealice-induced mortality, the savings at 100% mortality are lower for wrasse than for any other treatment. This is a function of their relatively low efficacy levels.

9.1.3.Treatment Margins Treatment margins can be utilized to evaluate the impact of treatment costs on the entire farm; the calculation used to determine these margins is: Gross margin = Output - variable Costs

(2)

Table 4 Break-even points and savings at 100% L. salmonis-induced mortality for different treatments. Treatment

Break-even point (1SW fish) (Yo)

Savings at 100% sealice induced mortality ( 1 SW fish) (€1

Break-even point (2SW fish) (X)

Savings at 100% sealice induced mortality (2SW fish) (€1

18 44

650 000 325 000

24 58

315000 275 000

1 25 6

680 000 375 000 280 000

1 23 n.a.

580 000 325 000 n.a.

Cypermethrin Hydrogen peroxide Ivermectin Aquagard Wrasse

SW

=

sea winters.

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A.W. PIKE AND S.L. WADSWORTH

The output side of the equation is equal to theoretical salmon sales minus the value of fish lost due to side effects minus reduction in fish growth. In the case of sealice treatments, variable costs consist of the cost of the chemical plus labour costs plus ‘additional costs’ plus the cost of smolts. By applying this equation to the range of sealice control methods under consideration, the results obtained are presented in Table 5. The difference in margin obtained on actual production of 510 tonnes is estimated to be as much as El53 000 between hydrogen peroxide and IvermectinB for 2-seawinter fish; equivalent to 30p per kilogram of salmon produced, based on calculations made in 1997. Although the economics of sealice control will vary somewhat from site to site, the following generalizations can be made: Ivermectin is the most economical treatment on the basis of cost per fish, break-even point and treatment margins; hydrogen peroxide is the least economic treatment; Cypermethrin is relatively expensive to apply, but its high levels of efficacy make it the most financially attractive form of bath treatment available, as reflected in the break-even point analysis; Aquagard is the least expensive bath treatment in terms of cost per fish; this is also reflected in the treatment margins. However, the break-even point and savings are intermediate in relation to the other modes of lice control; wrasse appear advantageous from a financial point of view in l-seawinter fish, but their relatively low efficacy impose financial restrictions where the risk of sealice-induced mortality is high. Despite the above, it is important to appreciate the fact that the financial aspect of sealice control cannot be considered in isolation. The farmer must also take into account the simplicity of treatment application, operator safety, withdrawal period, environmental impact of usage and licensing. For

Table 5 Treatment margins (before overheads) for Atlantic salmon sold after either 1 or 2 sea winters (SW). Treatment

Hydrogen peroxide Cy permethrin Aquagard Ivermectin Wrasse

Margin before overheads $7000 Fish sold after 1SW

Fish sold after 2SW

352 370 391 483 472

333 360 422 486 n.a.

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SEALICE ON SALMONDS

example, although expensive to use, hydrogen peroxide has the major advantage of being environmentally benign. Consequently, its use may prove economic if a premium price can be achieved for fish grown under an ‘organic’ regime. Conversely, although Ivermectin appears to be an attractive option from a financial point of view, its widespread and longterm usage is precluded in Scotland. This is for two main reasons: 0 0

the manufacturers do not support its use in aquaculture, therefore it will not be licensed for use in the marine environment; and it can only be prescribed under the ‘option cascade’, thus ensuring that Ivermectin use can only be under specific circumstances where no effective alternatives exist. Moreover, even this form of restricted use may cease when another in-feed compound is authorized.

Another approach has been taken by Sinnott (1998) who has calculated losses based on the effects of starvation, losses during treatment, mark down of harvested fish due to skin damage, losses due to secondary diseases, loss of stock and cost of treatment. The losses due to sealice on a hypothetical 764-tonne production range from a low of cX94 000 to a worst case of E200 000 per year.

10. PRIORITY AREAS

FOR FUTURE SEALICE RESEARCH

As with most parasites, establishing a laboratory maintenance system is crucial to progress. This has been achieved for both major species. What has not been established are protocols enabling the culture of larval stages on artificial substrates. It would be very useful to be able to monitor the development processes of the copepodid and chalimus larvae off-host. One of the key stages in parasite life cycles is the infective stage. Research into disrupting the life cycle is directed frequently at preventing infection and, in this respect, little is known about copepodid behaviour: where it is in the environment, how it orientates, and how it locates and identifies the preferred host. Research into the defence mechanisms of the salmonid host might exploit, or strengthen, latent defences either through selective breeding programmes [a new research programme just funded will assess the heritability and variability of resistance between different stocks of Atlantic salmon to L. salmonis (C.J. Secombes, personal communication)] or by further examination of naturally occurring resistance mechanisms, such as that identified by Johnson and Albright (1992a) for coho salmon. Understanding the general ecology of sealice is important both in the context of cage-culture populations and in wild salmonids. At present there are no mathematical models to describe the population dynamics of sealice,

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although a new project has just been funded in Scotland to address this deficiency (G. Gettinby, personal communication). If sealice can not be prevented from establishing on the host, then the next option is to interfere with their reproductive success. This could be through the use of pest-control strategies aimed at preventing males from locating and mating with females (Pickett et al., 1997) (a new project has been funded recently to investigate this). Alternatively, reproductive output might be inhibited if some means could be found to interfere with ovarian development. Research into developing a vaccine against sealice has been in progress for many years and will continue to attract funding as the preferred option for long-term control of sealice infection in cage culture. This programme needs to be evaluated and broadened to improve its chance of success by examining the early, attached stages that may be vulnerable as already mentioned. National monitoring schemes do exist but in Scotland this is done independently by the companies themselves. Apart from national monitoring programmes being encouraged, there should be international agreement on a protocol that would allow direct comparison between countries to be possible.

ACKNOWLEDGEMENTS We wish to thank the following for their generous assistance with various parts of this review: Mark Hull, Scott Peddie, Thomas Schram, Brian Stewart.

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in wild and cultured fish populations. Invertebrate Reproduction and Development 22, 91-102. Tully, 0. and Whelan, K.F. (1993). Production of nauplii of Lepeophtheirus salmonis, (Krnyer) (Copepoda: Caligidae) from farmed and wild salmon and its relation to the infestation of wild sea trout (Salmo trutta L.) off the west coast of Ireland in 1991. Fisheries Research 17, 187-200. Tully, O., Poole, W.R., Whelan, K.F. and Merigoux, S. (1993). Parameters and possible causes of epizootics of Lepeophtheirus salmonis (Kreryer) infesting sea trout (Salmo trutta L.) off the west coast of Ireland. In: Pathogens of Wild and Farmed Fish: Sea Lice (G.A. Boxshall and D. Defaye, eds), pp. 202-218. Chichester: Ellis Horwood. Urawa, S. and Kato, T. (1991). Heavy infections of Caligus orientalis (Copepoda: Caligidae) on caged rainbow trout Oncorhynchus mykiss in brackish water. Gyobyo Kenkyu 26, 161-162. Valentine, B.J., Gurr, G.M. and Thwaite, W.G. (1996). Efficacy of the insect growth regulators tebufenozide and fenoxycarb for lepidoptran control in apples and their compatibility with biological control for integrated pest management. Australian Journal of Experimental Agriculture 36, 501-506. Varian, S.J.A., Deady, S. and Fives, J.M. (1996). The effect of intensive fishing of wild wrasse populations in Lettercallow Bay, Connemara, Ireland: implications for the future management of the fishery. In: Wrasse: Biology and Use in Aquaculture (M.D.J. Sayer, J.W. Treasurer and M.J. Costello, eds), pp. 100-118. Oxford: Fishing News Books. Voth, D.R. (1972). Life history of the caligid copepod Lepeophtheirus hospitalis Fraser, 1920 (Crustacea: Caligoida). Dissertation Abstracts International, B. Sci. Eng. 33, 5547-5548. Wadsworth, S.L. (1989). An Investigation into the Development of an Effective Vaccine against the Salmon Louse Lepeophtheirus salmonis. University of Plymouth. Wadsworth, S.L. (1998). The Control of Lepeophtheirus salmonis (Copepoda: Caligidae) (Kreyer 1837) on Atlantic Salmon Salmo salar L . Production Sites. Ph.D. thesis, Department of Zoology, University of Aberdeen. Wadsworth, S.L., Grant, A.G. and Treasurer, J.T. (1997). Strategic approach to lice control. Fish Farmer 21, 8-9. Wadsworth, S.L., Fraser, N.F. and Braidwood, J.C. (1999). The efficacy of cypermethrin upon the survival and rate of development of chalimus stages of Lepeophtheirus salmonis (Copepoda: Caligidae). Aquaculture (in press). Walday, P. and Fonnum, F. (1989). Cholinergic activity in different stages of sealice (Lepeophtheirus salmonis). Comparative Biochemistry and Physiology 93C, 143147. Walker, A.F. (1994). Sea trout and salmon stocks in the Western Highlands. In: Problems with Sea Trout and Salmon in the Western Highlands (R.G.J. Shelton, ed.), pp. 6- 18. Pitlochry: Atlantic Salmon Trust. Wells, D.E., Robson, J.N. and Finlayson, D.M. (1990). Fate of Dichlorvos ( D D V P ) in Sea Water Following Treatment for Salmon Louse, Lepeophtheirus salmonis, Infestation in Scottish Fish Farms. Aberdeen: Department of Agriculture and Fisheries for Scotland, 13/90. Westerberg, H. (1982). Ultrasonic tracking of Atlantic salmon (Salmo salar L.) I . Movements in coastal regions. Report of the Institute of Freshwater Research Drottningholm 60, 81-101. Whelan, K.F. (1991). Disappearing sea trout: decline of collapse? The Salmon Net XXIII,24-31.

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Index

Page numbers in italics refer to figures and tables. Acanthocotyle lobianchi, oncomiracidium attachment to host 151 hatching from egg 207 acaricides, tick control 49 AChE depression, organophosphates on salmon 293, 295 adaptive immune response, T. annulata 52 adult life span, sealice 266-7 alimentary canal, L. salmonis 280 Alphamax (Alphapharma) 299 amastigote survival in macrophage, Leishmania 17-18 to promastigote transformation in blood meal, Leishmania 21 Amblyomma, tick species 45 apoptosis, inhibition, Leishmania infected macrophage 21 Aquagard (Novartis) 292, 299, 3 15, 3 16 Atlantic salmon, sealice infections cost of control 313, 314, 316 epidemiology 270, 270- 1 cage cultured 274 L. salmonis distribution on host 241, 253 response to 291, 308 attachment monogenean oncomiracidia 149, 151, 197 and settlement, sealice on salmonids 262-4 attenuated cell line vaccine T . annulata 50, 62-4 T. parva, impossibility of producing 74-5 azamethiphos 295

behaviour, monogenean oncomiracidia 203-15 dispersal 213 emergence from eggs 205-7 ADVANCES IN PARASITOLOGY VOL 44 ISBN 0-12-031744-3

host finding and recognition 21 1 12 invasion 213-14 role 203-4, 205 summary 215 swimming behaviour 207- 1 1 behavioural effects, sealice infection 290 bilharziasis in bovines 100 blood feeding, sealice 282, 288 meal, parasite differentiation in, Leishmania 21 -2 body size and water temperature, sealice 244 BoLa restricted cytotoxic T-cells 42, 57 bovine theileriosis 43-7 background and scale of problem 43-5 carrier states 44, 48, 78 chemotherapy 50 clinical and pathological features 48-9 control measures 49-51 see also Theileria annulata; Theileria parva; Theileria sergenti break-even point, control of sealice 313- 15 Bulinus africanus group geographical distribution 113, 120-1, 123 mollusc-S. bovis association, compatibility in 125, 126, 127 snail infection experiments 116- 19 transformed prevalences, S. bovis 126 forskalii group geographical distribution 113, 120, 123 mollusc-S. bovis association, compatibility in 125, 126, 127 snail infection experiments 114-15 transformed prevalences, S. bovis 126 -

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INDEX Bulinus (continued) reticulatus group geographical distribution 113, 120, 122 mollusc- S. bovis association, compatibility in 125, 126, 127 snail infection experiments 112 transformed prevalences, S. bovis 126 truncatusltropicus group geographical distribution 113, 120, 122 mollusc-S. bovis association, compatibility in 125, 126, 127, 128 snail infection experiments 106-11 transformed prevalences, S.bovis 126 buparvaquone 50, 51

cage cultured salmonids egg sac length and number of eggs per sac, L. salmonis 256 hydrographic factors, sealice transmission 275 interaction with wild salmonids, transmission of sealice 276-9 sealice infection epidemiology 273-4 Caligus centrodonti frontal filament 264-5 on wrasse 238 Caligus clemensi 243 host range 239 Caligus curtus geographical distribution 243 mouth tube 281 Caligus elongatus 234, 235, 238 adult life span 267 copepodid stage 260, 285 distribution on host 242 epidemiology Atlantic salmon, wild 270, 27 1 cage cultured salmon 274 sea trout, wild 271, 272 frontal filament 264, 265 generation times 266 geographical distribution, farmed and wild salmonids 242-3 host range 239 morphology adults 243-4 larvae 244-5 nauplius stages, development of 259 pathological effects of infection 289 reproduction copulation 254 egg development 258 egg sac production 255 mate guarding by males 252-3 water temperature and body size 244

Caligus epidemicus, frontal filament 264- 5 Caligus longicaudatus geographical distribution 243 host range 239 Caligus orientalis 238 geographical distribution 243 host range 239 Caligus teres 235, 238 host range 239 Cullorhynchicola multitesticulatus oncomiracidia, terminal globule 166, 167 Capsala martinieri oncomiracidia, parenchyma 201, 202 carrier state, theileriosis 44, 48, 78 cell biology in the post-genome era 26-7 culture vaccine, theileriosis 42, 50, 51 cell-mediated response, Theileria T. annulata cell-mediated immunity 42, 56-9 immunopathology 59-60 T. parva 67 trophozoite and schizont 69-73 T. sergenti 78-9 chaetotaxy, polyopisthocotylean oncomiracidia 195 chalimus I-IV, sealice development 260 feeding and digestion 28 1 gut morphology 280 chemoresponsiveness hatching factors, monogenean oncomiracidia species responding to 206 monogenean oncomiracidia 21 1- 12, 21 5 sealice 285 chemotherapy bovine theileriosis 50 sealice on salmonids 292-302 diflubenzuron 30 1-2 hydrogen peroxide 295-7 ivermectin 299-301 organophosphates 292-5, 295, 304-5, 306 pyrethroids 297-9 teflubenzuron 302 chinook salmon, sealice infection epidemiology 270 response 291 cholinesterase, oncomiracidial nervous system 198 chum salmon, sealice infection epidemiology 270 Cichlidogyrus halli typicus, hooklets 149, 150

ciliary eyes, oncomiracidia 186-7

INDEX ciliated cells, oncomiracidia 153-9, 216 monopisthocotylea 154-6 polyopisthocotylea 157-9 ciliated receptors 192-6 functional morphology 196-8 monopisthocotylea 192-5 polyopisthocotylea 195-6 classical complement pathway 6, 7 Clemacotyle australis, oncomiracidium, parenchyma 202 coevolutionary process 100 coho salmon, sealice infection 291, 308 ‘coiling’ phagocytosis 8 cold chain, vaccines 51, 64 complement activation 5, 6 Leishmania, promastigote entry into mammalian host 5-7 receptors, Leishmania binding to 8-9, 10 convergence, monopisthocotyleans and polyopisthocotyleans 217, 218 copepodid stage, sealice development 260 dispersion, hydrographical effects 275 frontal filaments 264-5 gut morphology 280 sensory biology 284-6 transmission biology 261 -2 attachment and settlement 264-6 host location 262-4 vibrations, sensitivity to 263-4 copulation, sealice 254 Corridor disease see East Coast fever cost, sealice infections 237 treatment cost in Scotland, analysis 31 1 - 17 cross-immunity, T. parva Muguga and Marekibuni stocks 71-2 cross-reactivity, SPAG-I and p67 55-6, 75 cross-species immunization trial, theileriosis 65-6 crustacean parasites of fish 234-5 see also sealice cutaneous leishmaniasis 2 cypermethrin (Exis) 298, 299, 315, 316 cytokines immune response subversion, Leishmania 20, 21 immune response, T. parva schizont 72-3 T. annulata infections 58 immunopathological effects 60- I dactylogyrids 213, 214 dead vaccines, T. parva 75-6 deltamethrin 299 dendritic cells, Leishmania amastigote invasion 19

341 dichlorvos 237, 292-5 L. salmonis, effects on epidemiology 304-5 diflubenzuron (Dimilin) 301 -2 digestive system Encotyllabe chironemi 200, 201 monogenean oncomiracidia 200- 1 sealice 279-81 Dimilin (T. H. Agricultural & Nutrition Company) 301 Diplozoon paradoxum oncomiracidia eyes 188, 189 rheotaxis and host invasion 214 dispersal period, monogenean oncomiracidia 213 domus, monogenean oncomiracidia 148, 149 dorsal sensilla, oncomiracidia 197-8 East Coast fever 42, 48 eclosion, sealice 258-9 economics of sealice infection 3 1 1 - I7 egg development, sealice 258 sacs, sealice length and number of eggs per 256-8 production 255 Ektobann 284, 302 Encotyllabe chironemi, oncomiracidium digestive system 200, 201 sensory receptors 193, 194 ultrastructural study, protonephridial system 178, 180 endocrine control, sealice 284 Entobdella soleae, oncomiracidium 142-3, 145 chemotaxis 21 I epidermis 160, 161 eyes ciliary 186, 187 pigment shielded 185 glands 171, 172 grouped receptors 193 host specificity 212 Entobdella spp., glands 171, 175, 176 environmental impact abermectin 300-1 ivermectin 300- 1 organophosphates 306 azamethiphos 295 dichlorvos 294 Epicotyle torpedinis oncomiracidia, sense organs 195 epidemic viseral leishmaniasis 3 epidemiology, sealice infections 268-79 epidermal erosion, sealice on salmon 287, 288 epidermis, monogenean oncomiracidia 159-63

342 Euzetrema knoepjleri oncomiracidia, pigment-shielded eyes 185 Exis see cypermethrin experimental infection. sealice on salmonids 250- I pathophysiological effects 290- 1 intermediate host spectrum, Schisrosoma bovis 120-1, 125 prevalences, compatibility in molluscSchistosoma bovis association 104-5, 106-11, 112, 114-15, 116-19, 125 eyes, monogenean oncomiracidia 183-91 functional morphology 190- 1

fallow period, fish farm management 304 Fc receptor for IgG, macrophage infection, Leishmania 19 febrifugine 50 feeding and digestion, sealice 281 -2 pathological effects 287, 288 fish farm management, sealice control 304-6 medicines, licences for 294 flame bulbs 180, 183 formula 177-8 Monopisthocotylea 178, 180, 181 Polyopisthocotylea 181-2 freshwater, premature return to, sea trout 277 frontal filaments, sealice, copepodid stage 264-5 generation times, sealice 266 genetic studies, sealice 278-9 geographical distribution Leishmania 2 Schistosoma bovis 120-1, 124 bibliographic data 102-3 mollusc intermediate hosts 113, 121-3 sealice on salmonids 242-3 geotaxis, monogenean oncomiracida 21 I gill dwelling monopisthocotyleans 213- 14 polyopisthocotyleans 2 14 glands, monogenean oncomiracidia 169-77, 217 function 175-6 Monopisthocotylea 170-2 Polyopisthocotylea 172-5 summary 176-7 glycoinositol phospholipids (GIPLs), Leishmania amastigote 18 gp63 7, 9-10 gripus 148 gut morphology, L . salmonis 280

INDEX

Gyrodact ylidae ciliary eyes 187 spike sensilla 195, 197 viviparity 142 Haemaphysalis longicornis 78 Haemaphysalis, tick species 45 halofuginone 50 hamuli, haptoral sclerites 147, 148 haptoral sclerites, monogenean oncomiracidia 146-52, 217- 18 terminology 146-7 alternate 147-8 hatching methods, monogenean oncomiracidia 21 5 rhythm, monogenean oncomiracidia 190-1, 204, 205 Helicobacter pylori, vacuole formation 16 Heteraxinoides xanthophilis oncomiracidia, glands 173 Heterocotyle capricornensis oncomiracidia, glands 170-1 Hexabothrium appendiculatum oncomiracidia glands 174 terminal globule 167 hooklets, oncomiracidia 146, 148, 149, 151 -2 embryology 149 independent activity 149, 150 numbering of I47 host specificity Leishmania and sandfly 2, 22 monogenea 211, 212 humoral response, Theileria T . annulaia sporozoite 53-6 T . sergenti 78 husbandry, sealice control 307 Hyalomma anatolicum anatolicum 44 hydrogen peroxide 295-7, 3 13, 3 16, 3 17 hydrographical effects on copepodid dispersion 275 hydroxynapthoquinones 50

Iberian populations, Schistosoma bovis 100, 128-9, 132 IL-I0 upregulation, T . parva 72, 73 immune evasion mechanisms, Leishmania 19- 21 memory, T . parva schizont antigens 70- 1 modulation, control of sealice on salmonids 308- 1I responses T . parva 69-73 T . annulata 51-62 T . sergenti 78-9 Theileria species, comparative aspects 80, 82

INDEX immunopathology T. annulata 59-61 T. parva 72, 73 India, tropical theileriosis 44 infection and treatment, T. parva 42, 50-1, 67, 73-5 innate immune response, T. annulata 52 insect growth regulator (IGR) 301-2, 306 integrated pest management, sealice on salmonids 306-7 International Council for the Exploration of the Sea 278 ivermectin 299-301, 315, 316, 317 costs 313 January disease see East Coast fever Japan, economic impact, theileriosis 45 Kuhnia srombri, glands 174 Kuhnia sprostonae glands 174 hooklets 150 laboratory maintenance, sealice 268 lakselus see Lepeophtherius salmonis lambda-cyhalothrin 299 larval development, sealice 258-60 mechanical damage to salmonids, sealice 287-8 Leishmania life cycle 4 macrophage, interaction with 5-21 parasite persistence mechanisms 18-21 promastigote invasion and infection 5-15 promastigote to amastigote differentiation IS- 18 nomenclature, developmental stages 21 parasitophorous vacuoles, inter-species differences 16 sandfly, interaction with 21-6 blood meal, differentiation in 21-2 establishment of infection 22-3 mammalian host, transmission to 24-6 metacyclogenesis 23 -4 sequencing 26-7 see also leishmaniasis Leishmania amazonensis immune response evasion 20 parasitophorous vacuoles 16. 17 Leishmania donovani LPG 13 parasitophorous vacuoles 16 Leishmania major gp63 9-10 immune evasion 20

343 LPG 12, 13 parasitophorous vacoules 16 pPPG 14, 15 Leishmania mexicana aPPG 14 immune response evasion 20 parasitophorous vacuoles 16 SAP 14 leishmaniasis 2, 4-5, 19-20 asymptomatic infection 3 clinical manifestation, factors determining 3 control 3, 4 cutaneous 2 epidemic visceral 3 geographical distribution 2 global number of infected individuals 3 host defence system 4-5 see also complement infection establishment of 5-15 first stage 15- 16 prevalence 3 see also Leishmania leishmanolysin see gp63 Lepeophtheirus cuneifer 238 geographical distribution 243 host range 239 Lepeophtheirus hospitalis, period of infectivity 264 Lepeophtheirus pectoralis 238 frontal filament 265 mating 251, 254 period of infectivity 264 seasonal variability 306 Lepeophtheirus salmonis 234, 235, 238 adult life span 266-7 attachment to host 264 body size 244 chalimus stages I-IV, development 260 chemosensory ability 285 costs and savings of treatment 312, 314, 315

delousing strategies 305-6 developmental stages 246-9 digestive system 279-81 distribution on host 240-1 epidemiology Atlantic salmon, wild 270, 270- 1 cage cultured salmon 274 sea trout, wild 271-2, 272-3 feeding and digestion 282 freshwater survival 283-4 frontal filament 265 generation times 266 genetic differentiation, populations from wild and farmed salmonids 278-9

344 Lepeophtheirus salmonis (continued) geographical distribution, infection on wild and farmed salmonids 242-3 host immune system evasion 309 location 262-3 range 239 response to 291, 292, 308 infection, first outbreaks of 236-7 morphology adults 243-4 larvae 244-5 nauplius stages development of 259 pigmented eye spots 284, 285 pathological effects of infection adult stages 288-9, 289-90 larval stages 287-8 period of infectivity 264 precopula pairs, distribution on Atlantic salmon smolts 253 reproduction copulation 252, 254 eggs and egg sac 255, 256, 257, 258 mate guarding 252-3 pair formation 251 post-mating behaviour 254 reproductive system, structure 245, 250 sensory organs 284, 285-6 light, response to L. salmonis copepodid 263 monogenean oncomiracidia 210 see also phototaxis lipophosphoglycan (LPG) 10- 14 L. major amastigote ligand 19 parasite protection in sandfly gut 22, 23 structural analysis 12- 13 live larvae, study of monogenean 153, 154, 177 vaccines T. annulafa 42, 62-4 T. parva 73-5 loch system, coordinated treatment, sealice 305 longevity, monogenean oncomiracidia 203, 210 Lufzomyia, sandfly 3

macrophages Leishmania interaction with mammalian 5-21 parasite persistence mechanisms 18-21 promastigote invasion and establishment of infection 5- 15 promastigote to amastigote differentiation 15- 18 T. annulata, protection against 58

INDEX

mammalian host, Leishmania transmission to 24-6 mapping studies, Leishmania 26 mating, sealice 250-4 copulation 254 mate guarding 252-3 pair formation 25 1 maxadilan 26 Mediterranean Coast fever see tropical theileriosis Mediterranean populations, Schistosoma bovis 100 compatibilities, mollusc intermediate hosts 129 merozoite/piroplasm, T. annulafa 61 -2 metacyclic promastigotes, Leishmania 5, 6-7 metacyclogenesis, Leishmani, sandfly host 23-4 MHC class 11, Leishmania immune response evasion 20 MHC-restricted CTLs, immune response, T. parva schizont 70 mollusc intermediate host, Schistosoma bovis 100 host spectrum 113, 120-1, 125 see also experimental: prevalences, compatibility in molluscSchistosoma bovis association monoclonal antibodies T . annulafa,antibody-mediated sporozoitk neutralization 54 to LPG, Leishmania attachment to macrophages 11 - 12 monocyte chemoattractant protein I, Leishmania infection 17 Monogenea, oncomiracidia 140- 1 behaviour 203- 15 ciliated cells 153-9 conclusions 215-18 digestive tract 200- 1 epidermis 159-63 general morphology 142-6 glands 169-77 haptoral sclerites 146-52 host finding and recognition 21 1- 12 invasion 213- 14 nervous system 198-200 parenchyma 201-3 protonephridia 177-83 sense organs 183-98 species hatching in response to chemical factors 206 terminal globule 163-9 see also Monopisthocotylea; Polyopisthocotylea

INDEX Monopisthocotylea ciliated cells 154-6 epidermis 160, 161 eyes 184-7 general morphology 142-3, 145 glands 170-2 hooklets, TEM studies 150-1 host invasion 213-14 nervous system 198 other sense organs 192-5 protonephridial system 178-9, 180 terminal globule 164-6, 169 mucocutaneous leishmaniasis 2-3 nauplius stages, sealice development 259 pigmented eye spots 284, 285 Neoheterocotyle rhinobatidis, oncomiracidium false terminal globule and blebs 166 flame bulb 180, 181 glands 172, I73 haptor 152 parenchyma 202 pigment-shielded eyes 185-6 stained 153 nervous system, monogenean oncomiracidia 198-200 NK cells response to T. annulata 57-8 NO, macrophage anti-Theileria activity 58-9 North Mediterranean zone, Schistosoma bovis 101 geographic distribution, bibliographic data 102 mollusc hosts 113, 128 transformed prevalences 126, 127 oncomiracidia, Monogenea see Monogenea, oncomiracidia Oncorhynchus nerka, freshwater survival 283 oral treatments, sealice on salmonids 299-302 organophosphates see azamethiphos; dichlorvos; trichlorophon osmoregulation salmon, pathological effects of sealice 287 sealice 282-4 oviposition, sealice on salmonids 254-8 egg sacs length and number of eggs per sac 256-8 production 255 egg strings produced per mating, number of 255-6 female post-mating behaviour 254 oxytetracycline, infection and treatment method, T. parva 51, 74

345 p67 42-3,55, 67 cross-reactivity with SPAG-I 55-6, 75 T. parva vaccine 75-6 live delivery systems 76-7 Pacific salmon 269, 269-70 pair formation, sealice 25 1 paleobiogeographical scenario, Schistosma bovis 130-2 parasitophorous vacuole formation and microbicidal mechanisms, Leishmania 15- 17 parenchyma, monogenean onchomiracidium 201-3 parvaquone 50 pathological effects, sealice on salmon 286-92 behavioural effects 290 host response 291-2 mechanical damage 287-90 pathophysiological effects 290- 1 transmission of pathogens 292 Phlebotomus, sandfly 3 phosphoglycans (PG), Leishmania 9, 10- 15 photoperiod, sealice life cycle, effects on 267 maturation of females 260 phototaxis, sealice copepodids, host location 263 pigment-shielded eyes, monogenean oncomiracidia 183-4 directional light response 190 Monopisthocotylea 184-6 Polyopisthocotylea 187-9 pink salmon, sealice epidemiology 270 piroplasm stage, T. sergenti 78 Planorbarius meridjensis geographical distribution 113, 120, 121 transformed S. bovis prevalences 126 intrazone variability 128 mollusc-S. bovis association, compatibility in 125, 126, 127, 127-8 snail infection experiments 104-5 Plectanocotyle gurnardi oncomiracidium 144, 145-6 flame bulbs 182 glands 144, 172 pigment-shielded eyes 188 terminal globule 144, 167 Polyopisthocotylea 2 17 ciliated cells 157-9 epidermis 160, 162-3 eyes 187-90 general morphology 144, 145-6 glands 172-5 host invasion 214 nervous system 198, 199

346 Polyopisthocotylea (continued) other sense organs 195-6 protonephridia 180-2 families in which described 179 terminal globule 166-8, 168-9 species identified in 165 Polystoma integerrimum oncomiracidia epidermis 162-3 host invasion 214 Polystoma pelobatis oncomiracidia, epidermis 162-3 polystomatids, oncomiracidia eyes 188 host invasion 214 population decline, wild salmon 276-8 post-mating behaviour female sealice 254 Pricea mulrae oncomiracidia, nervous system 198, 199 procyclic promastigotes, Leishmania 21 -2 promastigotes, Leishmania entry into mammalian host 5-7 ligands for host macrophages 9-10 metacyclic and procyclic 5, 6-7 phagocytosis by macrophages 7-9 promastigote to amastigote differentiation in macrophage 15- 18 proteophosphoglycans (PPG) 14- 15, 23 protonephridia, oncomiracidia 177-83, 216 families in which described 179 Monopisthocotylea 178-9, 180 Polyopisthocotylea 179, 180-2 Protopolystoma xenopodis oncomiracidia, nervous system 198, 199 Pseudodiplorchis americanus oncomiracidia cytoplasmic connections, larvae and embryos 201 and host coevolution 204, 205 pyrethroids 297-9 ‘quorum sensing’ 23 Rajonchocotyle emarginata oncomiracidia glands 174 hatching rhythm 190 recombinant vaccines T. parva 75-6 live delivery systems 76-7 refringent droplets, oncomiracidia 200 resmethrin 299 rhabdomeric eyes, oncomiracidia Monopisthocotylea 143, 216 Polyopisthocotylea 189, 216 rheotaxis, monogenean oncomiracidia 21 1, 214 Rhipicephalus, tick species 45 rhythmic hatching, monogenean oncomiracidia 190, 204, 205

INDEX

Salartect (Solvay-Internox Ltd) 297 salinity copepodid response to 263 and survival rates, L. salmonis 283-4 salmon cage cultured see cage cultured salmonids louse seecaligus teres migration 261 production 236-7 species of sealice on 238 wild see wild salmonids Salmosan (Novartis) 295 sandfly, Leishmania host 2, 3, 5 blood meal, parasite differentiation in 21-2 establishment of infection 22-3 feeding habits and mammalian infection 5, 24-5 metacyclogenesis 23-4 promastigote transition to mammalian host 16 sandfly saliva, promastigote virulence, role in 25-6 specificity 2, 22 Schistosoma bovis 99- 133 collection of data 102-13 compatibility, mollusc-S. bovis associations 125-8 experimental intermediate host spectrum 120-1, 125 geographical distribution 101, 124 mollusc intermediate hosts 113, 121-3, 124 natural mollusc intermediate host spectrum 113 paleobiogeographical scenario 130-2 Planorbius metidjenensis see Planorbarius metidjenensis three main populations 128-30 schistosomiasis 100 schizont stage, Theileria 43-4 T. annulata 56-61 tissue culture vaccines 62-4 T. parva 42 immune responses 69-73 Scottish Salmon Strategy Task Force 277 sea trout, wild ascending Irish rivers, sealice infections 276-7 decline of populations 277 sealice infection epidemiology 268-9, 271-3 sealice 233-318 common names 235 economics of infection 3 1 1- 17 epidemiology of infections 268-79 experimental infection, problems with 250- 1 genetic studies on 278-9

INDEX

geographical distribution 242-3 history and present status of problem 236-7 host distribution on 240-2 immune system evasion 309- 10 location 262-4 range 239 identification 236 infection, natural epidemics 287 life cycles 261-8, 262-8 morphology 243-5 pathological effects 286-92 physiology 279-86 reproductive system and reproduction 245-60 research 237 priority areas for future 3 17- 18 salmonids, resistance to infection 309, 310-11 species on salmonids 238 transmission of pathogens by 292 treatment and control 292-31 1 see also Caligus elongatus; Lepeophtheirus salmonis seasonal effects, sealice 256, 267 secreted acid phosphatase (SAP), Leishmania mexicana 14 sense organs, monogenean oncomiracidia 183-98 sensilla, monogenean oncomiracidia 192, 193, 195, 197 sensory biology, sealice 284-6 silver nitrate staining, live monogenean larvae 153, 154, 192 skin parasitic monopisthocotyleans 213 snail infection experiments Bulinus africanus group 116-19 forskalii group 114- 15 reiiculaius group 112 truncatusltropicus group 106- I I Planorbarius metidjenensis 104-5 sockeye salmon 270 South Mediterranean zone, Schistosoma hovis 101, 128 geographical distribution, bibliographic data 102-3 mollusc hosts 113 transformed prevalences 126, 127 South Saharan zone, Schistosoma hovis 100, 101, 128 geographical distribution, bibliographic data 103 mollusc hosts 113, 129-30 transformed prevalences 126, 127 origins of 130

347 Southern hemisphere salmon farming 243 SPAG-I 42, 54, 55 cross-reactivity with p67 55-6, 75 subunit vaccine development 64 trials 64-6 spike sensilla, gyrodactylids 195, 197 sporozoites surface proteins see p67; SPAG-I T. annulata 45, 53-6 T. parva, immune responses 69 stock selection, control of sealice on salmonids 310-11 stress/disease, susceptibility, salmon to sealice 307 subunit vaccines T. annulaia 64-7 T . parva 42-3 T . sergenti 43 summer lesion syndrome see sealice swimming behaviour, monogenean oncomiracidia 207- 1 I , 21 5 environmental stimuli, responses to 208-9

T-c& immune response, T. parva 69-71 inappropriate activation, T. annulata 59-60 Tams1 42, 61-2 subunit vaccine development 64 trials 66-7 teflubenzuron 284, 302 temperature effects on sealice 267 egg sac length 256, 258 size 244 terminal globule, monogenean oncomiracidia 163-9 Monopisthocotylea 164-6 Polyopisthocotylea 165, 166-8 possible functions 168 species identified in 165 tetracyclines, infection and treatment method, T . parva 74 Theileria benign species, nomenclature 44-5 life cycle 45 T. anulata, T. parva, T . sergenti, comparative aspects 46-7, 80-2 Theileria annulaia 42, 46- 7 , 51 -67 immune responses 51-62 merozoite/piroplasm 61 -2 schizont 56-61 sporozoite surface proteins see SPAG-1 sporozoites 45, 53-6 trophozoite 56 vaccination 62-7 attenuated cell line vaccine 50, 62-4 subunit vaccine development 64-7 see also tropical theileriosis

348 Theileria parva 42, 44,46-7, 67-77 immune responses 69-73 sporozoite surface proteins see p67 sporozoites, donor host to recipient host transfer 45, 74-5 vaccination 73-7 live, infection and treatment method 42-3, 50-1, 67, 73-5 live, mild strains 75 recombinant vaccines 75-7 see also East Coast fever Theileria sergenti 42, 43, 46-7, 77-80 clinical features and control 48, 78 immune responses 78-9, 81 vaccination with non-living components 79-80 Theileriosis see bovine theileriosis tick control 49-50 Tonkinopsis tranfretanus oncomiracidia, glands 174 topical treatments, sealice on salmonids 292-9 Toxoplasma gondii 8 transmission biology, sealice 261 -6 treatment margins, control of sealice on salmonids 315, 316-17 trichlorophon 237 trophozoite T. annulata 56 T. parva, immune responses 69-73 tropical theileriosis 42 clinical and pathological features 48-9 Trypanosoma cruzi 8 turning sickness 49 Udonella caligorum, sense organs 192 ultrastructural studies, monogenean oncomiracidia, phylogenic implications 216- I7 uncinuli 148 Urocleidus adspectus oncomiracidia, glands 171

vaccination L. salmonis, potential development against 309

INDEX leishmaniasis control 3, 4 T. annulata 50, 62-7 T. parva 42-3, 50-1, 67, 73-7 T. sergenti 79-80 Theileria species comparative aspects 80, 82 the future 82 tick control 49-50 vibration, sensitivity to, L. salmonis copepodids 263 -4 virulence factors, Leishmania 13- 14, 25-6 visceral leishmaniasis 3 West Highland Sea Trout and Salmon Group 177 white spot see sealice wild salmonids decline of populations 276-8 egg sac length and number of eggs, L. salmonis 256 interaction with farmed salmonids, sealice transmission 276-9 sea trout, sealice infection epidemiology 268-9, 271-3 sealice infection epidemiology 268, 269-71 pathophysiological effects 290 patterns, coastal waters and high seas 26 1 World Health Organization, leishmaniasis estimates 3 world-wide salmon production 236 wrasse, control of sealice 303-4 Caligus centrodonti 238 economic viability 315, 316 Zeuxapta seriolae, anterior sensory receptors 196 Zeuxapta seriolae oncomiracidia epidermis 162, 163 glands 174, 175 parenchyma 202 pigment-shielded eyes 188, 189 protonephridial system 182 terminal globule 167, 168 ‘zipper’-type phagocytosis 8

Contents of Volumes in This Series Volume 31 Parasitic Infections in Women and their Consequences . . . . . . . . L. BRABINAND B. J . BRABIN The Pathophysiology of Malaria . . . . . . . . . . . . . . . . . N. J. WHITEAND M. HO The Interaction of Leishmania Species with Macrophages . . . . . . . J. ALEXANDER AND D. G. RUSSELL The Effects of Trypanosomatids on Insects . . . . . . . . . . . . G. A. SCHAUB Echinococcus multilocularis Infection: Immunology and Immunodiagnosis . B. GOTTSTEIN Nematodes as Biological Control Agents: Part I1 . . . . . . . . . . I. POPIEL AND w . M. HOMINICK

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Volume 32 Blasfocysris in Humans and Animals: Morphology, Biology, and Epizootiology . . . . P. F. L. BOREHAM AND D. J. STENZEL Giardia and Giardiasis . . . . . . . . . . . . . . . . . . . . . . . . . J. A. REYNOLDSON AND A. H. W. MENDIS R. C. A. THOMPSON, Immunology of Leishmaniasis . . . . . . . . . . . . . . . . . . . . . . F. Y.LIEWAND c . A. ODONNELL Transport of Nutrients and Ions across Membranes of Trypanosomatid Parasites. . . D. ZILBERSTEIN The Biology of Fish Coccidia . . . . . . . . . . . . . . . . . . . . . . A. J. DAVIESAND S. J. BALL The Sexuality of Parasitic Crustaceans . . . . . . . . . . . . . . . . . . . A. RAIBAUTAND J. P. TRILLES

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26 1 293 367

Volume 33 The Treatment of Human African Trypanosomiasis . . . . . . . J. PEPIN AND F. MILORD Plasmodium Species Infecting Thamnomys rurilans: a Zoological Study I. LANDAU AND A. CHABAUD Metacercarial Excystment of Trematodes . . . . . . . . . . . B. FRIED The Minor Groups of Parasitic Platyhelminthes . . . . . . . . K. ROHDE Sarcoptes scabiei and Scabies. . . . . . . . . . . . . . . . . I. BURGESS

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91

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235

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CONTENTS OF VOLUMES IN THIS SERIES

Volume 34 Molecular Studies for Insect Vectors of Malaria . . . . . . . . . . . . . . . . J. M. CRAMPTON The Ribosomal RNA Genes of Plasmodium . . . . . . . . . . . . . . . . A. P. WATERS Molecular Mimicry. . . . . . . . . . . . . . . . . . . . . . . . . . . R. HALL Relationships Between Chemotherapy and Immunity in Schistosomiasis. . . . . . P. J. BRINDLEY Regulatory Peptides in Helminth Parasites . . . . . . . . . . . . . . . . . D. W. HALTON,C . SHAW, A. G. MAULEAND D. SMART Bait Methods for Tsetse Fly Control . . . . . . . . . . . . . . . . . . . . C. H. GREEN

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Volume 35 Chemotherapy of Nematode Infections of Veterinary Importance, with Special Reference to Drug Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . GEORGEA. CONDER AND WILLIAM c . CAMPBELL Parasites as Indicators of Water Quality and the Potential Use of Helminth Transmission in Marine Pollution Studies. . . . . . . . . . . . . . . . . . . . . . . . K. MACKENZIE, H. H. WILLIAMS,B. WILLIAMS, A. H. MCVICARAND R. SIDDALL Variation in Echinococcus: Towards a Taxonomic Revision of the Genus . . . . . . R.C . A. THOMPSON,A. J . LYMBERYAND c. C . CONSTANTINE How Schistosomes Profit From the Stress Responses They Elicit in Their Hosts. . . . . MARIJKEDE JONG-BRINK Myiasis of Humans and Domestic Animals. . . . . . . . . . . . . . . . . MARTINHALLAND RICHARDWALL Parasitism and Parasitoidism in Tarsonemia (Acari: Heterostigmata) and Evolutionary Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . MAREKKALISZEWSKI, FRANCOISE ATHIAS-BINCHE AND EVERTE. LINDQUIST

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335

Volume 36 Rare, New and Emerging Helminth Zoonoses. . . . . . . . . . J. D. SMYTH Population Genetics of Parasitic Protozoa and Other Microorganisms M. TIBAYRENC The Biology of Fish Haemogregarines . . . . . . . . . . . . A. J. DAVIES The Taxonomy and Biology of Philophthalmid Eyeflukes . . . . . P. M. NOLLENAND I. KANEV Human Lice and Their Management . . . . . . . . . . . . . . I. F. BURGESS Ticks and Lyme Disease . . . . . . . . . . . . . . . . . . . C. E. BENNETT

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27 I

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343

351

CONTENTS OF VOLUMES IN THIS SERIES

Volume 37 Nitric Oxide and Parasitic Disease. . . . . . . . . . . . . . . . . . . . . I. A. CLARKAND K. A. ROCKETT Molecular Approaches to the Diagnosis of Onchocerciasis . . . . . . . . . . J. E. BRADLEYAND T. R. UNNASCH The Evolution of Life History Strategies in Parasitic Animals . . . . . . . . . R. POULIN The Helminth Fauna of Australasian Marsupials: Origin and Evolutionary Biology . I . BEVERIDGE AND D. M. SPRATT Malarial Parasites of Lizards: Diversity and Ecology, . . . . . . . . . . . . J . J. SCHALL

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57

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135

. 255

Volume 38 Intracellular Survival of Protozoan Parasites with Special Reference to Leishmania spp., Toxoplasma gondii and Trypanosoma cruzi. . . . . . . . . . . . . . . . . . J. MAUEL Regulation of Infectivity of Plasmodium to the Mosquito Vector . . . . . . . . . R. E. SINDEN, G. A. BUTCHER,0. BILLKERAND s. L. FLECK Mouse-Parasite Interactions: from Gene to Population . . . . . . . . . . . . c . MOULIA,N. LE BRUNAND F. RENAUD Detection, Screening and Community Epidemiology of Taeniid Cestode Zoonoses: Cystic Echinococcosis, Alveolar Echinococcosis and Neurocysticercosis. . . . . . . P. S. CRAIG,M. T. ROGANAND J. c. ALLAN Human Strongyloidiasis. . . . . . . . . . . . . . . . . . . . . . . . . D. I. GROVE The Biology of the Intestinal Trematode Echinosroma caproni . . . . . . . . . . B. FRIED AND J. E. HUFFMAN

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169 25 1 31 1

Volume 39 1 Clinical Trials of Malaria Vaccines: Progress and Prospects . . . . . . . . . . . C. A. FACER AND M. TANNER Phylogeny of the Tissue Cyst-forming Coccidia . . . . . . . . . . . . . . . . 69 A. M. TENTERAND A. M. JOHNSON 141 Biochemistry of the Coccidia . . . . . . . . . . . . . . . . . . . . . . . G. H. COOMBS,H. DENTON,S. M. A. BROWNAND K.-W. THONG Genetic Transformation of Parasitic Protozoa . . . . . . . . . . . . . . . . 221 J. M. KELLY The Radiation-attenuated Vaccine against Schistosomes in Animal Models: Paradigm 271 for a Human Vaccine? . . . . . . . . . . . . . P. S. COULSON

352

CONTENTS OF VOLUMES IN THIS SERIES

Volume 40 Part I Cryptosporidium parvum and related genera

Natural History and Biology of Cryptosporidium parvum . . . . . . . . . . s. TZIPORIAND J. K. GRIFFITHS Human Cryptosporidiosis: Epidemiology, Transmission, Clinical Disease, Treatment, Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. K. GRIFFITHS Innate and Cell-mediated Immune Responses to Cryprosporidiurn parvum . . . . C. M. THEODOS Antibody-based Immunotherapy of Cryptosporidiosis. . . . . . . . . . . . J. H. CRABB Cryptosporidium: Molecular Basis of Host-Parasite Interaction . . . . . . . . H. WARD AND A. M. CEVALLOS Cryptosporidiosis: Laboratory Investigations and Chemotherapy . . . . . . . S. TZIPORI Genetic Heterogeneity and PCR Detection of Cryptosporidium parvum . . . . . G. WINDMER Water-borne Cryptosporidiosis: Detection Methods and Treatment Options . . . C. R. FRICKERAND J. H. CRABB

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Part 2 Enterocytozoon bieneusi and Other Microsporidia

Biology of Microsporidian Species Infecting Mammals . . . . . . . . . . . . . 283 E. S. DIDIER,K. F. SNOWDENAND J. A. SHADDUCK Clinical Syndromes Associated with Microsporidiosis . . . . . . . . . . . . . . 321 D. P. KOTLERAND J. M. ORENSTEIN Microsporidiosis: Molecular and Diagnostic Aspects . . . . . . . . . . . . . . 35 1 L. M. WEIS AND C. R. VOSSBRINCK Part 3 Cyclospora cayetanensis and related species Cyclospora cayetanensis . . . . . . . . . . . . . . . Y . R. ORTEGA, C. R. STERLING AND R. H. GILMAN

399

Volume 41 Drug Resistance in Malaria Parasites of Animals and Man . . . . . . . . . . . W. PETERS Molecular Pathobiology and Antigenic Variation of Pneumocystis carinii . . . . . . Y . NAKAMURA AND M. WADA Ascariasis in China. . . . . . . . . . . . . . . . . . . . . . . . . . . PENG WEIDONG, ZHOU XIANMIN AND D.W. T. CROMPTON The Generation and Expression of Immunity to Trichinella spiralis in Laboratory Rodents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R. G. BELL Population Biology of Parasitic Nematodes: Applications of Genetic Markers . . . . T. J. C. ANDERSON, M. S. BLOUINAND R. M. BEECH Schistosomiasis in Cattle. . . . . . . . . . . . . . . . . . . . . . . . . J. DE BONT AND J. VERCRUYSSE

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149 219 285

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CONTENTS OF VOLUMES IN THIS SERIES

Volume 42 The Southern Cone Initiative Against Chagas Disease . . . . . . , . . . . , c. J. SCHOFIELD AND J. c . P. DIAS Phytomonas and Other Trypanosomatid Parasites of Plants and Fruit . . . . . , E. P. CAMARGO Paragonimiasis and the Genus Paragonimus . . . . . . . . . . . . . . . , . D. BLAIR,Z.-B. XU AND T. AGATSUMA Immunology and Biochemistry of Hymenolepis diminuta . . . . . . , . . , . . J. ANREASSEN, E. M.BENNET-JENKINS AND C. BRYANT Control Strategies for Human Intestinal Nematode Infections . . . . , . . . . . M. ALBONICO, D. W. T. CROMPTON AND L. SAVIOLI DNA Vaccines: Technology and Applications as Anti-parasite and Anti-microbial Agenis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. B. ALARCON.G. W. WAINE AND D. P. MCMANUS

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Volume 43 Genetic Exchange in the Trypunosomufidue . . . . . . . . . . . . . W. GIBSONA N D J. STEVENS The Host-Parasite Relationship in Neosporosis . . . . . . . . . . . A. HEMPHILL Proteases of Protozoan Parasites . . . . . . . . . . . . . . . . . P. J. ROSENTHAL Proteinases and Associated Genes of Parasitic Helminths . . . . . . . . J. TORT,P. J. BRINDLEY,D. KNOX,K. H. WOLFEAND J. P. DALTON Parasitic Fungi and their Interactions with the Insect Immune System . . . A. VILCINSKAS AND P. GOTZ

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Volume 44 Cell Biology of Leishmania . . . . . . . . . . . . . . E. HANDMAN Immunity and Vaccine Development in the Bovine Theilerioses. N. BOULTERAND R. HALL The Distribution of Schistosoma bovis Sonsino, 1876 in Relation Mollusc-Parasite Relationships. . . . . . . . . . . . . H. MoNE, G. MOUAHIDAND S. MOUND The Larvae of Monogenea (Platyhelminthes). . . . . . . . I. D. WHITTINGTON, L. A. CHISHOLM AND K.ROHDE Sealice on Salmonids: Their Biology and Control . . . . . . A. w.PIKE AND s. L. WADSWORTH

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