Chemoecology of Insect Eggs and Egg Deposition

Monika Hilker Torsten Meiners (Editors)

Chemoecology of Insect Eggs and Egg Deposition

Monika Hilker Torsten Meiners (Editors)

Chemoecology of Insect Eggs and Egg Deposition With 46 Figures and 17 Tables

Blackwell Publishing

1st English-language edition: 0 2002 by Blackwell Verlag GmbH Berlin . Vienna A Blackwell Publishing Company

Editorial Offices: Blackwell Publishing (Holdings) Ltd Osney Mead, Oxford, OX2 OEL, UK 108 Cowley Road, Oxford, OX4 lJF, UK Blackwell Publishing, Inc. 350 Main Street, Malden, MA 02148-5018, USA Blackwell Publishing Asia Pty Ltd 550 Swanston Street, Carlton Victoria 3053, Australia Blackwell Verlag GmbH Kurfiirstendamm 57,10707 Berlin, Germany Firmiangasse 7,1130 Wien, Austria

Editors' addresses: Prof. Dr. Monika Hilker Dr. Torsten Meiners Department of Applied Zoology/ Animal Ecology Free University of Berlin Berlin, Germany

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Contents

List of Contributors ..................................................................................................

xi1

Acknowledgements...................................................................................................

XIV

Chemoecology of Insect Eggs and Egg Deposition: An Introduction Monika Hilker and Torsten Meiners ..........................................................................

XV

Chemoecology of Insect Eggs Chapter 1 Novel Morphological and Physiological Aspects of Insect Eggs loannis P . Trougakos and Lukas H . Margaritis ......................................................... 1.1 Introduction ........................................................................................... 1.2 Structure, Microstructure and Physiology of Eggs and Eggshells in Various Insect Orders .................................................... 1.2.1 Eggshell Layers ...................................................................................... Physiological Functions of the Eggshell ............................................ 1.2.2 1.3 Cell Types that Participate in Egg Formation: Panoistic and Meroistic Ovarioles ...................................................... 1.4 Formation of Egg Polarity .................................................................... Vitellogenesis: How Does the Yolk Get into the Egg? ................... 1.5 1.6 Eggshell Morphogenesis ...................................................................... Formation of the Vitelline Membrane ............................................... 1.6.1 1.6.2 Formation of Chorion Layers .............................................................. 1.7 Eggshell Composition and Assembly ................................................ 1.7.1 Chemistry and Molecular Events ....................................................... 1.7.2 Hardening of the Eggshell ................................................................... 1.8 Nurse Cells and Follicle Cells Programmed Cell Death ................. Concluding Remarks ............................................................................ 1.9 1.10 Acknowledgements .............................................................................. 1.11 References ..............................................................................................

3 4

5 5 9 13 16 17 19 22 22 24 24 28 29 30 30 31

VI Contents ...................................................................................................

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

Chapter 2 Insect Accessory Reproductive Glands: Key Players in Production and Protection of Eggs Cedric Gillott ............................................................................................................... 2.1 Introduction ........................................................................................... 2.2 Development and Structure of Accessory Reproductive Glands (= ARG) .................................................................................................. 2.2.1 Male ARG ............................................................................................... 2.2.2 Female ARG ........................................................................................... 2.3 Functions and Biochemistry of ARG Products ................................. 2.3.1 Male ARG Products .............................................................................. 2.3.2 Female ARG Products .......................................................................... 2.4 Concluding Remarks ............................................................................ 2.5 Acknowledgements .............................................................................. 2.6 References ..............................................................................................

37 38 39 39

40 43 43 47 54 55 55

Chapter 3 Chemical Protection of Insect Eggs Murray S. Blum and Monika Hilker .......................................................................... 3.1 Introduction ........................................................................................... 3.2 Defensive Components of Intrinsic Origin ..................................... 3.2.1 Autogenously Produced Defensive Components Applied onto the Eggs ......................................................................................... 3.2.2 Autogenously Produced Defensive Components within the Eggs ...................................................................................... 3.3 Defensive Components of Extrinsic Origin ...................................... 3.3.1 Chemically Defensive Plant Material Covering Eggs ..................... 3.3.2 Sequestered Defensive Components within the Eggs .................... 3.4 Chemical Defence and Egg Cannibalism .......................................... 3.5 Concluding Remarks ............................................................................ 3.6 Acknowledgements .............................................................................. 3.7 References ..............................................................................................

67 73 73 73 81 82 83 83

Chapter 4 Paternal Investment in Egg Defence Thomas Eisner. C a m e n Rossini. Andrks Gonza'lez. Vikram K . Zyengar. Melody V. S . Siegler and Scott R . Smedley ............................................................... 4.1 Introduction ........................................................................................... 4.2 Enemies of Insect Eggs ......................................................................... 4.3 Types of Egg Defences ......................................................................... 4.3.1 Fireflies .................................................................................................... 4.4 Chemical Defences: Cantharidin ........................................................ 4.4.1 Meloid Beetles ........................................................................................ 4.4.2 Cantharidiphiles ....................................................................................

91 92 92 93 93 96 96 97

61 62

64 64

Contents

Chemical Defences: Pyrrolizidine Alkaloids .................................... Arctiid Moths ......................................................................................... Danaine Butterflies ............................................................................... Ithomiine Butterflies ............................................................................. Chemical Defences: Cucurbitacins ..................................................... The Issue of Paternity ........................................................................... When Defence Backfires ...................................................................... Other Paternal Contributions ............................................................. Puddling ................................................................................................. Concluding Remarks ............................................................................ Acknowledgements .............................................................................. References ..............................................................................................

100 100 103 104 105 106 107 107 109 110 111 111

Chapter 5 Brood Protection in Social Insects Manfred Ayusse and Robert J. Paxton ........................................................................ 5.1 Introduction ........................................................................................... 5.2 Interspecific Brood Defence ................................................................ 5.2.1 Mechanical Defence .............................................................................. 5.2.2 Chemical Defence ................................................................................. 5.3 Intraspecific Brood Defence ................................................................ 5.3.1 Defence against Non-Nestmates ........................................................ 5.3.2 Defence against Nestmates: Kin Conflict and Egg Cannibalism 5.3.3 Queen-Queen Conflict and Brood Defence ..................................... 5.3.4 Worker-Worker Conflict and Brood Defence .................................. 5.3.5 Queen-Worker Conflict and Brood Defence .................................... 5.3.6 Compliant Brood Cannibalism: Diploid Males ................................ 5.4 Concluding Remarks ............................................................................ 5.5 Acknowledgements .............................................................................. 5.6 References ..............................................................................................

117 118 121 121 124 129 129 130 132 133 134 139 140 141 141

4.5 4.5.1 4.5.2 4.5.3 4.6 4.7 4.8 4.9 4.10 4.11 4.12 4.13

Chapter 6 The Role of Microoganisms for Eggs and Progeny Rupert L . L . Kellner ..................................................................................................... 6.1 Introduction ........................................................................................... 6.2 Insect Adaptations towards Transmission of Symbiotic Microorganisms ..................................................................................... 6.2.1 Transmission upon the External Surfaces of the Eggs .................... 6.2.2 Transovarial Transmission ................................................................... 6.2.3 Transmission Bypassing the Egg in Viviparous Insects ................. 6.2.4 Larval Uptake of the Symbionts ......................................................... 6.3 Infections which Reduce Survival of the Host ................................. 6.3.1 Cytoplasmic Incompatibility and Male Killing ................................ 6.3.2 Egg Pathogens .......................................................................................

149 150 150 150 151 153 153 155 155 155

VII

Vlll

Contents

6.3.3 6.3.4 6.4 6.5 6.5.1 6.5.2 6.6 6.7 6.8

.........

Oviposition by Pathogen Infected Females ...................................... Defences against Invasion of Pathogenic Microorganisms ............ Attraction of Gravid Females towards Oviposition Sites by Cues of Microorganisms ...................................................................... Beneficial Effects of Microorganisms for Eggs and Progeny ......... Nutritional Interactions ....................................................................... Defensive Substances .......................................................................... Concluding Remarks ............................................................................ Acknowledgements .............................................................................. References ..............................................................................................

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

156 157 158 160 161 162 163 164 164

Chemoecology of Egg Deposition Chapter 7 Plant Chemical Cues Important for Egg Deposition by Herbivorous Insects Erich Stadler ................................................................................................................ Introduction ........................................................................................... 7.1 “Insect Active“ Compounds in Plants ............................................... 7.2 7.2.1 Different Plant Parts ............................................................................. 7.2.2 Leaf Surface ............................................................................................ Plant Interior .......................................................................................... 7.2.3 Variability within the Plant Species ................................................... 7.2.4 Variability between the Plant Species ................................................ 7.2.5 Distribution of Oviposition-Relevant Chemicals in the Plant 7.2.6 Kingdom ................................................................................................. Characteristics of “Insect Active” Plant Compounds ...................... 7.3 Primary versus Secondary Metabolites ............................................. 7.3.1 7.3.2 Volatiles and Non-Volatiles ................................................................. Polarity .................................................................................................... 7.3.3 Insects‘Responses to Plant Chemicals ............................................... 7.4 Methods of Investigation ..................................................................... 7.4.1 7.4.2 Orientation by Ovipositing Females to Host Plant ......................... Host Selection Behaviour ..................................................................... 7.4.3 ”Host Plant Search Image”: The Essence of Multi-Component 7.4.4 Mixtures .................................................................................................. 7.4.5 Interactions between Plant Compounds .......................................... Specialization ......................................................................................... 7.4.6 Prior Experience and Learning ........................................................... 7.4.7 Host Selection Related to Different Stages ....................................... 7.4.8 Oviposition Related to Performance of the Progeny ...................... 7.4.9 Oviposition and Chemoreception ...................................................... 7.5 Chemosensory Coding in General ..................................................... 7.5.1 7.5.2 Coding of Quality .................................................................................

171 172 173 173 174 176 177 179 179 185 185 185 186 186 186 187 188 188 189 190 191 192 192 193 193 193

Contents

7.5.3 7.5.4 7.6 7.7 7.8

Coding of Quantity Independent of Quality ................................... Perception of Mixtures ......................................................................... Concluding Remarks ............................................................................ Acknowledgements .............................................................................. References ..............................................................................................

Chapter 8 The Plant's Response towards Insect Egg Deposition Monika Hilker. Odette Rohfritsch and Torsten Meiners ........................................... Introduction ........................................................................................... 8.1 Plant Tissue: Changes in Response towards Oviposition ............. 8.2 Plant Tissue: Changes in Response towards Oviposition by 8.2.1 Non-Galling Insects ............................................................................. Plant Tissue: Changes in Response towards Oviposition by 8.2.2 Gall Insects ............................................................................................ 8.3 Plant Surface Chemicals: Changes in Response towards Oviposition ............................................................................................ 8.4 Plant Volatiles: Changes in Response towards Oviposition .......... 8.4.1 Local and Systemic Plant Response towards Oviposition by Emission of Volatiles ............................................................................. 8.4.2 Eliciting Mechanisms of Induction of Plant Volatiles by Oviposition ............................................................................................. Role of Jasmonic Acid in Induction of Plant Volatiles by 8.4.3 Oviposition ............................................................................................. Chemistry of Released Plant Volatiles Induced by Oviposition .. 8.4.4 Concluding Remarks ............................................................................ 8.5 Acknowledgements .............................................................................. 8.6 References .............................................................................................. 8.7

195 196 196 197 197

205 206 208 208 214 220 221 223 223 225 225 226 227 228

Chapter 9 Oviposition Pheromones in Herbivorous and Carnivorous Insects

Peter Anderson ............................................................................................................ 9.1 9.2 9.2.1 9.2.2 9.2.3 9.3 9.4 9.5 9.6 9.7 9.7.1

Introduction ........................................................................................... Oviposition Deterring Pheromones (= ODPs) ................................ ODPs Deposited by Females ............................................................... ODPs Associated with Eggs ................................................................ ODPs from Larvae ................................................................................ Oviposition Stimulating Pheromones ............................................... Chemical Structure of Oviposition Pheromones ............................. Detection of Oviposition Pheromones .............................................. Intraspecific Effects of ODPs on Behaviour other than Oviposition ............................................................................................. Interspecific Effects .............................................................................. Response of Competitors towards ODPs .........................................

235 236 236 240 245 246 248 248 251 253 253 253

IX

X Contents ................................................................................................................................................................................................................................................................

9.7.2 9.8 9.9 9.10 9.11

Response of Carnivores towards ODPs of their Prey ..................... Applications ........................................................................................... Concluding Remarks ............................................................................ Acknowledgements .............................................................................. References ..............................................................................................

Chapter 10 Chemoecology of Oviposition in Insects of Medical and Veterinary Importance Philip J. McCall ........................................................................................................... Introduction ........................................................................................... 10.1 Oviposition Attraction Pheromones .................................................. 10.2 Mosquitoes and Blackflies ......................... Aquatic Environment . 10.2.1 Terrestrial Environment . Sandflies. Tsetse Flies and Bugs .......... 10.2.2 On the Host ............................................................................................ 10.2.3 Oviposition Attraction Allelochemicals and their Interaction 10.3 with Pheromones and Other Sensory Information ........................ Aquatic .................................................................................................... 10.3.1 Terrestrial ............................................................................................... 10.3.2 On the Host ............................................................................................ 10.3.3 Oviposition Repellents ......................................................................... 10.4 Cues from Plants ................................................................................... 10.4.1 Predator and Parasite-Associated Cues ............................................. 10.4.2 The Influence of Experience on Responses to Oviposition Cues . 10.5 Responses to Plant Mimics and Plant Allelochemicals ................... 10.6 Concluding Remarks ............................................................................ 10.7 10.8 Acknowledgements .............................................................................. References .............................................................................................. 10.9 Chapter 11 Chemoecology of Parasitoid and Predator Oviposition Behaviour Johannes L . M. Steidle and Joop J . A . van Loon ........................................................ 11.1 Introduction ........................................................................................... 11.1.1 Parasitoids .............................................................................................. 11.1.2 Predators: Oviposition Site Location and Acceptance .................... 11.2 Theories on the Use of Infochemicals for Location of Oviposition Sites by Natural Enemies ............................................... 11.2.1 The Reliability-Detectability Problem ................................................ 11.2.2 The Variable Response Model ............................................................ 11.2.3 Dietary Specialization and Infochemical Use in Natural Enemies 11.2.4 Infochemical Use in Koinobiont and Idiobiont Larval Parasitoids 11.2.5 Plant-Entomophage Mutualism ......................................................... 11.3 Concluding Remarks ............................................................................ 11.4 Acknowledgements .............................................................................. 11.5 References ..............................................................................................

254 255 255 256 256

265 266 268 268 272 274 274 274 276 276 279 279 279 280 281 282 282 283

291 292 293 298 300 301 303 306 309 309 310311 311

................................................................................................................................................................................................................................................................3

Contents

Chapter 12 Evolutionary Ecology of Oviposition Marking Pheromones Thomas S. Hoffmeister and Bernard D . Roitberg ...................................................... 12.1 Introduction ........................................................................................... 12.2 Four Major Questions on the Use of Oviposition Marking Pheromones .......................................................................................... 12.2.1 Why Use Oviposition Marking Pheromones? ................................. 12.2.2 Where Should Insects Deposit their Mark? ..................................... 12.2.3 When Should an Insect Mark its Oviposition Site? ........................ 12.2.4 What Information Should Oviposition Marking Pheromones Convey? ................................................................................................. 12.3 How Might Natural Selection Shape the Use and Chemistry of Oviposition Marking Pheromones? ................................................... 12.3.1 Should Marking Be Circumstance-Dependent? ............................. 12.3.2 What Is the Optimal Persistence of an Oviposition Mark? ........... 12.3.3 Should Marking Pheromones Be Variable or Invariant among Individuals? ............................................................................................ Concluding Remarks ............................................................................ 12.4 Acknowledgements .............................................................................. 12.5 12.6 References ..............................................................................................

331 339 340 340

Chapter 13 Evolutionary Ecology of Oviposition Strategies Niklas Janz ................................................................................................................... 13.1 Introduction ........................................................................................... 13.2 Phylogenetic Patterns ........................................................................... 13.3 Oviposition Site Selection .................................................................... 13.3.1 Searching, Finding and Accepting ..................................................... 13.3.2 Maternal Care ........................................................................................ 13.3.3 The Pros and Cons of Laying Egg Clutches ..................................... 13.3.4 Genetics, Plasticity and Learning ....................................................... 13.4 Specialization ......................................................................................... 13.4.1 Internal vs. External Factors ................................................................ 13.4.2 The Cost of Information ....................................................................... 13.5 Preference-Performance Correlations ............................................... 13.5.1 Why is the Correlation not Always Perfect? ..................................... 13.6 Concluding Remarks ............................................................................ 13.7 Acknowledgements .................................................. .: .......................... 13.8 References ..............................................................................................

349 350 350 352 353 354 356 357 359 360 361 364 364 366 367 367

Subject Index .............................................................................................................

377

Taxonomic Index ......................................................................................................

384

319 320 322 322 324 326 326 328 329 329

XI

XI1 ...........................................................................................................................................................................................................................

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

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

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List of Contributors

Peter Anderson, Swedish University of Agricultural Sciences, Department of Crop Sciences, Chemical Ecology, 230 53 Alnarp, Sweden Manfred Ayasse, Universitat Wien, Abteilung Evolutionsbiologie, Biozentrum, Althanstr. 14,1090 Wien, Austria Murray S. Blum, University of Georgia, Department of Entomology, 413 Biological Sciences Building, Athens, Georgia 30602-2603, USA Thomas Eisner, Cornell University, Department of Neurobiology and Behavior, Ithaca, New York 14853, USA Cedric Gillott, University of Saskatchewan, Department of Biology, 112 Science Place, Saskatoon, SK S 7 N 5E2, Canada Andre3 Gonzalez, Universidad de la Republica, Facultad de Quimica, General Flores 2124, Montevideo, CC 1157, Uruguay Monika Hilker, Freie Universitat Berlin, Institut fur Biologie, Haderslebener Str. 9, 12163Berlin, Germany Thomas S. Hoffmeister, Christian-Albrechts-Universitat Kiel, Zoologisches Institut, Okologie, Am Botanischen Garten 1-9,24098 Kiel, Germany Vikram K. lyengar, Cornell University, Department of Neurobiology and Behavior, Ithaca, New York 14853, USA Niklas lanz, Stockholm University, Department of Zoology, 106 91 Stockholm, Sweden Rupert L. L. Kellner, Universitat Bayreuth, Lehrstuhl f i r Tierokologie 11,95440 Bayreuth, Germany Lukas H . Margarifis, University of Athens, Faculty of Biology, Department of Cell Biology & Biophysics, Panepistimiopolis,Zografou 15784, Athens, Greece

Philip 1.McCall, Liverpool School of Tropical Medicine, Division of Parasite & Vector Biology, Pembroke Place, Liverpool L3 5QA, United Kingdom

List of Contributors

Torsten Meiners, Freie Universitat Berlin, Institut fLir Biologie, Haderslebener Str. 9, 12163 Berlin, Germany Robert I. Puxton, Universitat Tiibingen, Institute of Zoology, Auf der Morgenstelle 28,72076 Tiibingen, Germany Odette Rohfritsch, CNRS, Institut de Biologie Moleculaire des Plantes, 12 rue du General Zimmer, 67084 Strasbourg, Cedex, France Bernard D. Roitberg, Simon Fraser University, Department of Biological Sciences, 8888 University Drive, Burnaby, B.C. V5A 1S6, Canada Carmen Rossini, Universidad de la Republica, Facultad de Quimica, General Flores 2124, Montevideo, CC 1157, Uruguay Melody V. S. Siegler, Emory University, Department of Biology, Atlanta, Georgia 30322, USA Scott R. Smedley, Trinity College, Department of Biology, Hartford, Connecticut 06106, USA

Erich Stiidler, Eidgenossische Forschungsanstalt, Wadenswill, Schloss 334, Postfach 185,8820 Wadenswill, Switzerland Johannes Steidle, Freie Universitat Berlin, Institut f i r Biologie, Haderslebener Str. 9,12163 Berlin, Germany

Iounnis P. Trougukos, National Hellenic Research Foundation, Institute of Biological Research & Biotechnology, Lab. Molecular and Cellular Aging, 48, Vas. Constantinou Ave., 11635Athens, Greece

loop 1.A. van Loon, Wageningen Universiteit, Department of Entomology, Binnenhaven 7,6709 PD Wageningen, The Netherlands

XIV

Acknowledgements

We owe deep thanks to so many people who inspired, encouraged, contributed to, improved and produced this book, We are very grateful to all authors for their stimulating contributions, inspiring e-mail-discussions during the growth of the chapters, their cooperation and their patience in putting up with demands and deadlines. Many thanks are also due to those who critically reviewed the chapters: Anurag A. Agrawal, Reginald Chapman, Konrad Dettner, Marcel Dicke, Wolf Engels, Frank E. Hanson, Rudiger Hartmann, Klaus H. Hoffmann, Cesar R. Nufio, Hans Joachim Poethke, Dan T. Quiring, Louis Schoonhoven, John N. Thompson, Ted Turlings,JacquesM. Pasteels, Joachim Ruther, Eric Waijnberg, and Louise E.M. Vet. Especially grateful thanks are due to Marcel Dicke, who so thoroughly and critically read all the chapters. We highly appreciate the work of the Blackwell Verlag Berlin, especially the encouraging co-work with Olaf Kahl and Cornelia Dippel. We are also very grateful to Urte Kohlhoff and Frank Miiller of our lab who helped to find all the typewriting errors and prepare figures and to Eva Haffner for her drawings of insect eggs. We thank our friends and families for their patience with us while working on the book. Special thanks are due to Egbert Matzner for all his encouragement to go ahead in initiating the book.

xv

Chemoecology of Insect Eggs and Egg Deposition: An Introduction Monika Hilker and Torsten Meiners

Insect chemical ecology usually addresses chemistry and ecology of natural products relevant for the life and survival of larval and adult stages of insects. Several excellent books considering this topic have been written during the last decades (e.g. Sondheimer and Simeone, 1970; Bettini, 1978; Blum, 1981; Bell and Carde, 1984;Roitbergand Isman, 1992; Eisner and Meinwald, 1995; Carde and Bell, 1995). In contrast, information on chemical ecology focusing on the highly vulnerable insect eggs is scattered. Most insect species start life outside their mother’s body in the egg stage. As immobile eggs they face numerous problems such as predation, parasitization, and pathogen infection. How about the role of natural products for defence and protection of this so vulnerablestage duringinsect life? When looking at the intense predation upon eggs by vertebrate and invertebrate animals, Orians and Janzen asked about 30 years ago “Why are embryos so tasty?” (Oriansand Janzen, 1974).After analysisof this question they came to the conclusion that “evidence for palatability of eggs is almost entirely anecdotal or at best inconclusive”.They stated ”’it is possible to be toxic if one is an embryo, but under most circumstances it isn’t worth the price”. A few years later, Hinton (1981) described in one of the chapters of his excellentand fascinatingthree-volume work on the “Biology of Insect Eggs” the protection of eggs of several insect species by toxins. Today, a considerable range of defensive chemicals isolated from insect eggs is known, which may be provided by the parents, by endosymbionts or by the developing embryo itself. Whether these chemicals render the eggs unpalatable to predators and parasitoids has been thoroughly studied for several insect species during the past decades. However, survival of insect eggs does not depend only on whether or not the egg is tasty and palatable, but also on the mother’s choice of a “safe” and suitable oviposition site. Most insect species hide their eggs in plant or animal tissue or interstitia of the soil or stones. Thus, avoidance of enemies by laying eggs at concealed sites is one strategy to protect the offspring. Furthermore, recognition of eggs as prey or host may be hindered by covering the eggs with hairs, scales and faecal matter, by resemblance of eggs to e.g. plant structures, or by disruptive coloration, which destroys the appearance of the egg’s form (Hinton, 1981).When considering the mother’s choice for a proper site to deposit the offspring, chemicals

XVI Chemoecology of Insect Eggs and Egg Deposition: An Introduction ................................ .......................................... ................................................. ............................. ..............................................................................

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from this site may significantly affect the choice. Volatile and contact chemicals from plants affect egg deposition of herbivorous insects, prey and host chemicals influence oviposition of predators and parasitoids, and cues released by conspecific and interspecific competitors at potential oviposition sites influence the behaviour of gravid females. This book is intended to elucidate the plethora of chemoecological aspects of insect egg deposition and egg protection. It is organized in two sections. Part I addresses the chemoecology of the eggs themselves, and part I1 focuses on intraand interspecific chemoecologicalinteractions associated with egg deposition. The first chapter gives up-to-date information on the morphology and physiology of insect eggs. Since it is almost unknown how toxins find their way into eggs, studies on vitellogenesis can shed light on how maternally provided toxins get inside insect eggs. Furthermore, molecular and physiological knowledge on formation of the eggshell provides information on how the outer shell is stabilized. The second chapter focuses on both female and male accessory glands. The secretions of these glands can modulate oviposition behaviour by regulating the female’s fecundity and receptivity. Furthermore, many of these secretions are known to provide egg coatings and protectants. Thus, the glandular secretions of accessory glands can be considered as key players in the production and protection of eggs. Chapter 3 addresses the wide array of defensive and protective components of insect eggs that range from simple fatty acids over complex terpenoids, polyketids, and steroidsto numerous different nitrogen-containingcomponents such as cyanoglycosides, alkaloids and toxic peptides. Several of these components have been tested for their efficacy against predators and parasitoids. In parasitoids, even protective egg coatings with virus-like particles are known that enable eggs to circumvent immune responses of larval and adult hosts. Chapter 4 shows the significant contribution of males to chemical egg defence. Males of several insect species are able to transfer protective chemicals to the female by seminal infusion, and the mother adds these compounds to the eggs. The female’s mate choice for males with a rich prospective paternal investment enables her to provide high protection for herself and the offspring. A female may furthermore dupe and attract non-conspecific males by deceitful signals, in order to kill them and to utilize their defensive chemicals for the protection of her eggs. In chapter 5, special emphasis is given to defence of eggs and brood in eusocial insects. Social bees and wasps, ants, and termites owe their success in many terrestrial ecosystems to a large extent to their sophisticated defensive devices, which they employ to protect their offspring. Defensive chemicals and pheromones mediating protection of brood are discussed in an inter- and intraspecific context. Insect eggs may suffer pathogen infection and need devices to cope with this danger. On the other hand, insect eggs act as endosymbiotic interface between

Chemoecology of Insect Eggs and Egg Deposition:An Introduction

adults and larvae to ensure transfer of endosymbiotic microorganismsfrom one generation to the next. In chapter 6, both beneficial and pathogenic microorganisms associated with insect eggs and ovipositionare addressed with special emphasis to chemoecological aspects. Part I1 of the book addresses the chemoecology of egg deposition and considers insects with different feeding habits such as herbivory, carnivory, necrophagy, and haematophagy. Chapter 7 reviews variability, distributionand characteristics of plant chemical cues important for egg deposition by herbivorous insects. The female’sbehavioural responses towards these chemicalsas well as chemosensory aspects are addressed. However, not only are egg laying herbivorous insect females able to respond to plant chemical cues, but also vice versa - the plant may respond towards oviposition by herbivorous insects. This is well known for the plant’s response towards gall insects, but has also been shown for egg depositions by non-galling insects. Several of these plant responses towards non-galling insects result in the production of plant defensive devices that act directly against the herbivore by deterring oviposition or dropping the eggs from the plant. Other plant responses induced by insect egg deposition act indirectly against the herbivores by producing plant volatiles that attract egg parasitoids. Chapter 8 describes these phenomena and reviews knowledge on the mechanisms of these plant responses induced by oviposition. Ovipositing insects are known to respond to pheromonal marks released by other females or larvae at potential oviposition sites. These marks may function as deterrents (e.g. to avoid competition) or as stimulants (to indicate suitable sites). Chapter 9 reviews current knowledge on chemistry and perception of oviposition pheromones of herbivorous and entomophagous insects. The function and evolution of oviposition pheromones is discussed to a larger extent in chapter 12.Another recent review by Nufio and Papaj (2001)placed oviposition pheromones in the context of recent theory in the field of animal communication. Oviposition pheromones are also known to affect egg deposition by insect species that feed upon blood or tissue of vertebrates. Furthermore, oviposition of several insects that feed upon living or dead vertebrate tissue is influenced by the typical volatiles of decaying meat. Chapter 10 addresses those chemical cues relevant for egg deposition by haematophagous and carnivorous insects of medical and veterinary importance (mainly Diptera, but also hemipteran species). While foraging of parasitoids for hosts usually ends up with oviposition on or into the host, foraging of predators for prey is not so tightly connected with egg deposition, even though predators are expected to deposit their eggs where prey is available. Chapter 11 reviews the knowledge on chemical information important for egg deposition by predators and parasitoids and places it into a common theoretical frame of foraging behaviour of these two types of carnivorous insects.

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xvlll Chemoecology of Insect Eggs and Egg Deposition: An Introduction

The last two chapters of this book focus on evolutionary ecology of egg deposition. Chapter 12 picks up evolutionary aspects of oviposition marking pheromones by discussing costs and benefits of chemical oviposition marks. When, where, why and how to mark an ovipositionsite are some of the questions addressed here. A novel concept for the evolution of individual, specific marks is developed. Finally, chapter 13 places the chemical cues important for egg protection and egg deposition by herbivorous insects into a broader context. A complex pattern of trade-offs between abiotic and biotic factors affects the oviposition strategy of an insect and may explain, “Why mother doesn’t know best” when females deposit eggs at sites that are not optimal for offspring development (Courtney and Kibota, 1990).This chapter also addresses issues like the influence of larval preconditioning on oviposition behaviour (compare Barron, 2001), as well as egg clustering and aposematism in the context of the evolution of unpalatability of eggs. In this book we did not devote a special chapter to parental care in terms of egg guarding by sub- and semisocial insects. Evolution of maternal and paternal care in arthropods has been a matter of exciting debates for many years (Trivers, 1972; Wilson, 1975; Maynard-Smith, 1977; Zeh and Smith, 1985; Choe and Crespie, 1997; Tallamy, 1999,2001;Tallamy and Brown, 1999).However, chemoecologicalaspects of egg guarding in insects have hardly been investigated up to now. For example, no analyses are available which consider chemical defence efficacy of parents guarding their offspring.Furthermore, it is unknown whether parentally guarded eggs are more palatable or less chemically protected against oophages than nonguarded eggs of closely related species. To include such chemoecological aspects in thoughts about the costs and benefits of egg guarding might provide in the future a further valid facet to hypotheses on the evolution of parental care in insects. When considering chemoecology of insect eggs and egg deposition behaviour from an autecological and ecophysiologicalperspective, the interplay between the female’s experience with environmental factors ( e g availability and quality of food), her hormonal physiology, her oviposition behaviour and investment in chemical defence of eggs needs further studies to elucidate the plasticity of insect reproduction (e.g. Bailey and Ridsdill-Smith, 1991; Wheeler, 1996; Davey, 1997; Trumbo, 1997; Moehrlin and Juliano, 1998).Additionally, since the female’s mate may also significantly affect ovipositionbehaviour and egg protection (see chapter 2 and 4), the interference of mating and ovipositionstrategies needs more attention. From a multitrophic point of view, chemical bottom-up-information emitted by sites occupied with eggs as well as chemical top-down-cues released by potential predators and parasitoids of the offspring might be future research fields for those interested in the question how the choice of insects for oviposition sites is influenced by the pressure to avoid both competition and predatiodparasitization. While the presence of chemical cues released by competitors has been intensively studied for its effects on ovipositionbehaviour of herbivorous and entomophagous insects

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(see Chapter 9), the influence of the presence of predators or parasitoids on insect oviposition behaviour has hardly been analysed up to now (but see Dicke and Grostal, 2001). In Chapter 10 several carnivorous and haematophagous insect species are mentioned which have been observed to avoid egg deposition at those sites where predators are present. Future studies need to elucidate whether this avoidance behaviour is mediated by chemical information. Biological control aimed at insect eggs is a promising approach to managing insect pests since the target is a highly vulnerable stage, which cannot try to escape behaviourally.The release of egg predators and egg parasitoids is common practice (e.g. Wajnberg and Hassan, 1994). The chapters of this book considering basic knowledge on chemoecology of insect eggs and egg deposition might encourage further studies examining how this basic information can be utilized to control pest insects in forest and agricultural systems as well as insects of medical and veterinary importance (Chapter 10). For example, future studies need to analyse whether induced plant responses towards oviposition may be utilized in pest control as a preventive defence strategy of the plant against herbivore feeding (Chapter 8) (Agrawal et al., 1999). Also the use of oviposition pheromones is promising (see Chapter 9) and modifying chemical plant characteristicsmight be a way to prevent oviposition of agricultural pest insects (Chapter 7). Several open questions will remain at the end. We hope that the information, thoughts and hypotheses compiled in this book promote research in the field of chemoecology of insect eggs and egg deposition to get deeper insight into the chemical information that is relevant and so crucial for the phase where insect life starts.

References Agrawal, AA, Tuzun, S, Bent, E (eds). 1999. Induced Plant Defenses Against Pathogens, and Herbivores. APS Press, St. Paul. Bailey, WJ, Ridsdill-Smith, J (eds). 1991. Reproductive Behaviour of Insects. Individuals and Populations. Chapman and Hall, London. Barron, AB. 2001. The life and death of Hopkin’s host-selection principle. J. Insect Behav. 14: 725-737. Bell, WJ, Card@,RT (eds). 1984. Chemical Ecology of Insects. Chapman and Hall, London. Bettini, S (ed). 1978. Arthropod Venoms. Handbook of Experimental Pharmacology. Vol. 48. Springer Verlag, Berlin. Blum, MS. 1981. Chemical Defense of Arthropods. Academic Press, New York. Carde, RT, Bell, WJ (eds). 1995. Chemcial Ecology ofInsects II. Chapman and Hall, New York. Choe, JC, Crespie, BJ, (eds). 1997. The Evolution of Social Behavior in Insects and Arachnids. Cambridge University Press, Cambridge. Courtney, SP, Kibota, TT. 1990. Mother doesn’t know best: Selection of hosts by ovipositing insects. In: Bernays, EA (ed.).Insect-Plant Interactions. Pp. 161-168.CRC Press, Boca Raton. Davey, KG. 1997. Hormonal control on reproduction in female Heteroptera. Arch. Insect Biochem. Physiol. 35: 443-453. Dicke, M, Grostal, P. 2001. Chemical detection of natural enemies by arthropods: An ecological perspective. Ann. Rev. Ecol. Syst. 32: 1-23. Eisner, T, Meinwald, J (eds). 1995. Chemical Ecology. The Chemistry of Biotic Interactions. National Academy Press, Washington D.C. Hinton, HE. 1981. Biology of Insect Eggs. Vol. 1-3. Pergamon Press, Oxford.

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Maynard Smith, J. 1977. Parental investment: A prospective analysis. Animal Behav. 15: 1-9. Moehrlin, GS, Juliano, SA. 1998. Plasticity of insect reproduction: Testing models of flexible and fixed development in response to different growth rates. Oecologia 115: 492-500. Nufio, CR, Papaj, DR. 2001. Host marking behavior in phytophagous insects and parasitoids. Entomol. Exp. Appl. 99: 273-293. Orians, GH, Janzen, DH. 1974. Why are embryos so tasty? Am. Nat. 108: 581-592. Roitberg, BD, Isman, MB (eds). 1992. Insect Chemcial Ecology. A n Evolutionary Approach. Kluwer Academic Press, Boston. Sondheimer, E, Simeone, JB (eds). 1970. Chemical Ecology. Academic Press, New York. Tallamy, DW. 1999. Child care among the insects. Sci. Am. 280: 72-77. Tallamy, DW. 2001. Evolution of exclusive paternal care in arthropods. Ann. Rev. Entomol. 46: 139-165. Tallamy, DW, Brown, WP. 1999. Semelparity and the evolution of maternal care in insects. Animal Behav. 57: 727-730. Trivers, RL. 1972. Parental investment and sexual selection. In: Campbell, B (ed.) Sexual Selection and the Descent of Man. Pp. 136-179.Aldine-Atherton, Chicago. Trumbo, ST. 1997. Juvenile hormone-mediated reproduction in burying beetles: From behavior to physiology. Arch. Insect Biochem. Physiol. 35: 479490. Wajnberg, E, Hassan, SA. 1994. Biological control with egg parasitoids. Oxford University Press, Oxford. Wheeler, D. 1996. The role of nourishment in oogenesis. Ann. Rev. Entomol. 41: 407431. Wilson, EO. 1975. Sociobiology: The New Synthesis. Harvard University Press, Cambridge, MA. Zeh, DW, Smith, RL. 1985. Paternal investment by terrestrial arthropods. Am. Zool. 25: 785-805.

Che moecoIogy of Egg Deposition

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Chapter 1 Novel Morphological and Physiological Aspects of Insect Eggs loannis P. Trougakos and Lukas H. Margaritis

Table of Contents 1.1 1.2 1.2.1 1.2.1.1 1.2.1.2 1.2.2 1.2.2.1 1.2.2.2 1.2.2.3 1.3 1.4 1.5 1.6 1.6.1 1.6.2 1.7 1.7.1 1.7.2 1.8 1.9 1.10 1.11

Introduction Structure, Microstructure and Physiology of Eggs and Eggshells in Various Insect Orders Eggshell Layers In General Taxon Specificities.of Chorionic Layers Physiological Functions of the Eggshell Spermatozoon Entry: Micropyle Apparatus Oxygen Entry: Aeropyles and Plastron Eggshell Structures for Larval Hatching Cell Types that Participate in Egg Formation: PanoisticandMeroisticOvarioles Formation of Egg Polarity Vitellogenesis: How Does the Yolk Get into the Egg? Eggshell Morphogenesis Formation of the Vitelline Membrane Formation of Chorion Layers Eggshell Composition and Assembly Chemistry and Molecular Events Hardening of the Eggshell Nurse Cells and Follicle Cells Programmed Cell Death Concluding Remarks Acknowledgements References

a Abstract

Insect eggs consist of the oocyte surrounded by an epithc ial layer of somatically derived follicle cells. In some insects the oocyte is also associated with nutritive cells called nurse cells (or trophocytes). A fertilized egg accomodates in two ways for the embryo it will enclose. Firstly, the oocyte is provisioned with nutritive substances to support the embryo until it can obtain its own food and thus a central event in egg maturation is the extra-ovarian synthesis of the yolk proteins and

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their selective uptake by the developing oocyte. Secondly, the oocyte contains developmental instructions that result in the establishment of egg polarity and proper embryonic pattern formation through the function of maternally acting genes and localized signals. Nurse cells provide the developing oocyte with RNA and proteins that are necessary for proper development and during late oogenesis they degenerate by apoptosis. On the other hand the follicle cells, which also die apoptotically following completion of oogenesis, separate into subpopulations, move to specific positions, modify their surfaces and participate in the oocyte polarity formation. During the late stages of oogenesis they secrete the constituent proteins of the eggshell, which is a highly organized supramolecular structure featuring both radial and regional complexity. Different orders of insects have eggshells of widely different composition and structure, presumably reflecting different physiological needs during embryonic development. Eggshell allows and facilitates sperm entry, provides elasticity to allow oviposition, protects the embryo from environmental hazards and liberates the larva. Finally, the eggshell accomplishes the mutually antagonistic functions of encouraging gas exchange and discouraging water loss.

1.1

Introduction

A key event in biology is the continuity of life expressed in the germ cells. Oviposition of the major insect lineages (see also Chapters 10,11,13), as described by Hinton (1981), occurs in a wide range of cryptozoic (in-litter), endogean (inground), subcortical (under-bark), endophytic (within plant), endozoic (within animal),exophytic (on-plant),exozoic (on-animal),aquatic (in-water),semi-aquatic (periodically submerged) and other exposed habitats. Parental care should be particularly costly for insects. According to the "insect egg" hypothesis proposed by Zeh and Zeh (1989),the initial evolution of an assembly of eggrelated characters, comprising a complex eggshell architecture, the amnion and the ovipositor, provided the Insecta with the potential for rapid and extensive phyletic divergence and enabled them to colonize successfully almost every existing environment on earth. Despite the enormous variability seen in the maturation period, the architecture and the egg size among the various species of Insecta, the basic molecular events that lead to egg (otherwise termed follicle) development in the ovary remain largely common.Key developmental events during oogenesis are the establishment of the polarity axes, the accumulation of the yolk into the oocyte and the construction of the eggshell. All these incidents are under tight sex-, stage-, cell-, and steroid-specific temporal and spatial regulation. Their molecular details have been analysed mainly in moths (Lepidoptera) and fruit flies (Diptera). We will attempt to summarize our current knowledge about these developmental strategies on a comparative basis hoping that some of the ideas that will be discussed here will provoke their analysis in many other insect orders where our knowledge

Structure, Microstructure and Physiology of Eggs and Eggshells in Various Insect Orders

remains extremely limited. Understanding the function and physiology of the variable structures (e.g. eggshell) seen in insect eggs and the molecular developmental events of oogenesis would be extremely useful for analysing the ecology of the insect eggs and their progeny (e.g.their relation to and dependence on the oviposition substrate).

1.2

Structure, Microstructure and Physiology of Eggs and Eggshells in Various Insect Orders

Insect egg shapes vary significantly ranging from cylindrical with rounded or pointed poles to spherical or hemispherical, or irregular. They also exhibit surface projections and a variable number of appendages originating from the anterior, the posterior or both poles (Figure 1-la-f). Their size also varies considerably, ranging from the tiny eggs of the dipteran Acnemiu umoenu (Mazzini and Santini, 1983) with about 140 pm diameter to the more than 10 mm long eggs of the orthopteran S u p pedo (Mazzini, 1976).

1.2.1

Eggshell Layers

1.2.1.1 In General The insect eggshell in the majority of the species studied so far is constituted by several successive layers (radial complexity)which appear conserved in terms of their nature and arrangement. Since terminology describing these layers varies significantly,in order to avoid confusion, we will describe these layers by adopting the terminology used for Drosophilidae species (Margaritiset al., 1980; Margaritis, 1985a).In these species the eggshell layers seen in the main body of the mature egg were divided into two major overlays, which from the oocyte outwards are the vitelline membrane (otherwise termed the vitelline envelope) and the chorion. Furthermore, in the chorion four successive layers were recognized, namely the plaques of the wax layer, the crystallineinnermost chorionic layer, the amorphous tripartite endochorion and the fibrous exochorion. The organization of the vitelline membrane and wax layer appears quite conserved and they both have been found almost invariably in most insect species studied so far. More specifically, the vitelline membrane represents a continuous amorphous (Figure 1-lg, h, l), porous (Figure 1-li), granular (Figure 1-lj) or perforated layer (Chauvin and Barbier, 1979), with regular (Figure lg, lh, 11) or irregular shape (Figure 1-li, j) covering the entire oocyte. Similarly, the wax layer that according to permeability studies should exist in most insect eggshells is organized in 4 (Figure 2a - Papassideri and Margaritis, 1986) to 7 (Mazzini et al., 1987) planes of overlapping hexagonally shaped plaques that are squeezed one upon the other followingcompletion of oogenesis due to oocyte expansion against the rigid endochorion. No wax layers have been observed in the parasitic hymenopteran species, presumably because these eggs never develop in a dry substrate (Margaritisand Mazzini, 1998).

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Novel Morphological and Physiological Aspects of Insect Eggs

The endochorion can b e further subdivided into the perforated i nner endochorion, th e pillars creating a n e t w o r k of cavities (otherwise termed the trabecular layer) a n d th e continuous layer of t he outer endochori on (Figure 1-lg). The described configuration of the eggshell layers seen in the m a i n body o f the egg i s significantly altered across the extremely variable specialized structures (Figure 1la-f) seen across the anterio-posterior axis o f the insect egg. In Drosophila species these regions constitute the so-called regional complexity (Figure 1-3i) a n d i ncl ude the posterior pole, the m a i n body, th e operculum, the collar, the micropyle a n d the respiratory appendages (see also Margaritis a n d Mazzini, 1998). The endochorionic layers v a ry significantly even in v e r y closely related insect species a n d show h a r d l y a n y specific features within a larger taxon (Kambysellis, 1993; Kalantzi-Makri et al., 1999). This variability coul d b e attributed to the fact that t he endochorion i s directly affected by the micro-environment conditions of the ovipositional site a n d thus s h o u ld exhibit u n i q u e features to accommodate specific physiological functions during embryogenesis. Figure 1-1Insect eggs and eggshells. (a-f) External morphology and shape of selected insect eggs. (a) Scanning Electron Microscope (SEM) micrograph of laid egg of dipteran Drosophila virilis. Anterior pole with micropyle (m) and the four respiratory filaments (rf) are indicated. (b) SEM of laid egg of the lepidopteran silkmoth Antheraea polyphernus showing a central localization of the micropyle (m). (c) SEM of laid egg of lctinogornphus australis (Odonata) with narrowcollar(c) anteriorlyand cone offine filaments posteriorly(courtesyofJ.Trueman). (d) SEM micrograph of laid egg of Kathroperlaperdita (Plecoptera) showing wide collar (c) on top and surface projections organized in rows (courtesy of B. Stark). (e) Whole mount S E M view of laid Eurytorna arnygdali egg (Hymenoptera) showing tw o filaments, the short one being the micropyle (m) (courtesy of D. Mouzaki). (f) SEM of laid egg of Dacusoleae (Diptera). The anterior cup with the micropyle (m) is indicated. (g-I) Fine structure of the eggshell. (g) Three dimensional representation ofthe radial complexity seen a t the main body of Drosophila virilis eggshell. Thefibrous exochorion (ex),thetripartite amorphous endochorion (consisting of the outer endochorion (oe), the pillars (p) and the perforated inner endochorion (ie)), the crystalline innermostchorionic layer(icl),the hexagonal waxplaques(w1) andtheamorphous vitelline membrane (vm) are indicated. (gJ High magnification TEM micrograph showingthe successive layers (arrows-periodicity about 10 nm) of Drosophila virilis crystalline innermost chorionic layer. Arrowhead indicates a transition pointtoanother crystallite (g, g,-Trougakos, IP and Margaritis, LH, unpubl. results). (h) TEM micrograph of a thin section at the eggshell of the dipteran Ceratitis capitata. Two consecutive trabecular layers (tll, tl2) are seen in the endochorion. The vitelline membrane (vm), the exochorion (ex) and the oocyte (oc) are also indicated. (i) TEM of thin section through the eggshell of the coleopteran Leptinotarsa decernlineata.The irregular porousvitelline membrane(vm) iscovered bythecrystallineinner chorioniclayer(ic1)andthegranulatedfibrousouterchorionic layer(ocl).(iJ High magnification of Leptinotarsa decernlineata inner chorionic layer revealing fine transverse striations with a periodicity of 10 nm (arrows) (i, il- Papassideriet al., unpubl. results). (i)TEM of thin section through the eggshell of the lepidopteran Bornbyx rnorithat consists ofthe irregular electron translucent vitelline membrane (vm), the trabecular layer (tl) and the lamellate inner (il) and outer(o1)zones;a portionofafolliclecell (fc) isalsoshown.(k)TEM micrograph ofthechorion organization ofthe lepidopteran Trichophaga tapetzellashowing the trabecular layer (tl) and the crystalline outer endochorion (oe) with honeycomb-like arrangement of i t s components (courtesyof G. Chauvin and R. Barbier).(I)TEM ofthin sectionthrough the eggshell of Eurytorna arnygdali. The oocyte (oc), the thin vitelline membrane (vm) and the three chorionic layers, namely the translucent (tl),the granular (gl) and the columnar (cl) layer are shown (courtesy of D. Mouzaki). Bars, (a, c,d, e,f) 100 pm, (b) 1000 pm, (g, h, i,I) 1pm, (g,) 80 nm, (il, k) 100 nm, (i) 5 pm.

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Structure, Microstructure and Physiology of Eggs and Eggshells in Various Insect Orders .................................. ... .............. . . ..............,,,,.................................................................................................................................

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Chorion organization in higher dipteran species follows the rather typical configuration described above for the Drosophilu species with species-specific adaptations like the two exochorion layers seen in the Mediterranean fruit fly Cerutitis capitutu and the cherry-tree fly Xhugoletis cerusi (Mouzaki and Margaritis, 1991a;Mouzaki and Margaritis, 1991b),or the two trabecular layers seen in Cerutitis cupitutu (Figure l-lh) (Mouzakiand Margaritis, 1991a)and the Muscidae (Diptera) species (Hinton, 1981). 1.2.1.2 Taxon Specificities of Chorionic layers

Diptera. In most of the eggshells a continuous crystalline layer has been found to line up the wax layer, similar to the innermost chorionic layer (Figure 1-lg, g,) described in Drosophilu species (Margaritis et al., 1991) and other Diptera, like Cerutitis cupitutu (Mouzaki and Margaritis, 1991a)and the olive fruit fly Dacus oleue (Mouzakiet al., 1991).Interestingly, in the dipteran species Alloconturina sorghicolu, no trabecular layer is found and the chorion is constituted by a continuous layer covered by exochorion (Isidoro and Lucchi, 1989).By comparing ultrastructure in 8 mosquito species (lower Diptera) from the genera Aedes, Anopheles, Culex and Toxorhynchites, Sahlen (1996) found that the endochorion always includes a lamellate layer (with at least one lamellae) and (except in Toxorhynchites) tubercles of varying size and shape. Coleopteru. In all coleopteran species studied thus far, the major character of the eggshell appears to be a thick crystalline chorionic layer lying below a rather thick fibrilar exochorion (Figure 1-li). In the Colorado potato beetle Leptinotursu decemlineutu the crystalline layer shows fine transverse striations with a periodicity of about 10 nm (Figure l-lil). Lepidopteru. In contrast, the Ditrysian lepidopteran chorion consists of a trabecular layer that is covered by several layers of helicoidally arranged fibril lamellae (Figure 1-lj)(Regier et al., 1982).The major differencesbetween the silkmoth species appear in the outer region which, in Bombyx mori for example, containsa unique osmiophilic layer with 15 to 20 lamellae and very narrow aeropyles (Papanicolaou et al., 1985). In contrast, the outer region of Antherueu polyphemus exhibitsvery wide aeropyleswith extremely tall regionally specific crowns (Figure 1-2g, gl),not seen in Bombyx mori or Hyulophoru cecropia (Hatzopoulosand Regier, 1987).Noteworthy is the spectacular chorion of the lepidopteran species Trichophugu tupetzellu (Chauvin and Barbier, 1972) that consists of a trabecular zone covered by an outer endochorion exhibitinga honeycomb-like crystalline arrangement of its structural components (Figure 1-lk). Hymenoptera. In the hymenopteran species [like the almond wasp Eurytomu amygdali (Mouzakiand Margaritis, 1994)],the chorion consists of an amorphous translucent layer, a granular layer including large and small electron-dense granules and a columnar layer very similar to other layers found in insects of the same or other orders (Figure 1-11). Similar chorion configurations have also been seen in the hymenopteran species Hubrobruconjuglandis (King and Cassidy, 1973)and Nusoniu vitripennis (Richards, 1969).

9 Structure, Microstructure and Physiology of Eggs and Eggshells in Various Insect Orders .................................. .

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Orthopteru. A very complex chorion is seen in the orthopteran species Eyprepocnemis ploruns (Viscusoet al., 1990).In this species the endochorion has a spongy structure constituting of plate-like structures, as well as other structures of fibrilar, granular and crystalline appearance. Comparable endochorionic organization has been found in most of the locusts species analysed so far (Hartley, 1961; Viscuso et al., 1984). ”Apterygotu”. In the most primitive insects (Apterygota),the eggshell is very thin and structure-less. In the Campodea species (Apterygota: Cumpodeidue) the egg is covered by only one envelope, built of fine granular material (Bilinskiand Larink, 1989).

1.2.2

Physiological Functions of the Eggshell

It is conceivable that since the major insect lineages oviposit in a wide range of exposed habitats (Hinton, 1981),the physiology of an insect eggshell should largely depend on the extend to which ambient conditions are consistent with requirements for embryogenesis.The eggshell thus accomplishes several functions of the reproductive machinery; it protects the embryo from environmental hazards such as temperature fluctuations, humidity, dryness and bacterial attack, provides elasticity to accommodate oviposition through a narrow ovipositor, allows and facilitates sperm entry through the micropyle apparatus, ensures an adequate oxygen supply for proper embryo development and finally liberates the larva (Margaritis, 1985a; Margaritis and Mazzini, 1998). Occasionally, several other species-specific functions are attributed to the eggshell such as the one achieved by the “anterior cup” in mosquito eggs which serves as a hydrating device to hold the eggs upright on the water surface (Hinton, 1981) or the bristles and projections in the eggshells of mallophagans and anoplurans (Phthiraptera) that facilitate attachment to feathers of the host (Zawadzka et al., 1997).Water loss in insect eggs is restricted by the wax layer plaques (Papassideri and Margaritis, 1986) that cover and seal the entire oocyte (Figure 1-2a).Elasticity and rigidity are especially needed in species like the olive fruit fly Ducus oleue where the egg has to pass a narrow and long ovipositor during oviposition, which is accomplished via crosslinking of the eggshell constituent proteins that ensure its adequate hardening (see Section 1.7.2). 1.2.2.1 Spermatozoon Entry: Micropyle Apparatus

Spermatozoon entry is facilitated by the micropyle apparatus. The structural organization, the number of micropyles and the micropyle apparatus location on the egg varies significantly among insect species (Figure 1-2b-f). When only one micropyle apparatus is present it is usually located at the anterior pole of the egg, as seen in dipteran Drosophilidae (Figure 1-2b) and Tephritidae (Figure 1-2c, f ) . It appears to be a protrusion that occasionally can be very long, as in the case of the hymenopteran Eurytomu umygduli (Figure 1-2e).Micropyle apparatus have similar

10

Novel Morphological and PhysiologicalAspects of Insect Eggs

architecture in all Drosophilidae studied so far, consisting of a n external orifice (the micropyle) and a micropylar canal that connects the micropyle with the oocyte (Zarani a n d Margaritis, 1991). The micropyle apparatus i s believed t o block polyspermy by restricting passage to a single sperm (Perotti, 1974). In contrast, in Lepidoptera, the micropyle apparatus forms a depression and i s usually surrounded by a rosette consisting of numerous petal-shaped imprints (Figure 1-2d). In the lepidopteran Bombyx mori and Antherueu polyphemus (Figure 1-2d) the number of canals range from 2 to 6 per egg, radiating asymmetrically from a common external orifice (Margaritis, 1985a). The micropyle apparatus of the stickinsect Curuusius morosus has two micropylar canals (Godecke and Pijnacker, 1984) whereas in the anopluran Pediculus humanus cupitis numerous micropyles are located at the anterior pole (Mazzini and Ermini, 1978). The micropyle apparatus i s located at the dorsal side of the egg in stick insects (Mazzini et al., 1993), while in the orthopteran Eyprepocnemis ploruns about 40 micropyles located posteriorly can be seen arranged in a regular ring-like manner (Viscus0 et al., 1990). I t i s w o r t h mentioning that in some Apterygota n o micropyle apparatus has been reported (Larink and Bilinski, 1989). Nevertheless, despite the vast micropyle apparatus heterogeneity, it seems that this structure has been acquired early ininsect evolution since i t i s present even in the most primitive insect orders.

Figure 1-2 Eggshell physiology. (a) Egg waterproofing by wax. TEM micrograph of freezefractured Drosophila melanogaster eggshell revealing the four consecutive layers (star) o f squeezed wax plaques (courtesyof I.5. Papassideri).(b-f) Micropylarapparatus-spermatozoon route for fertilization. (b) SEM front view of the micropylar protrusion (m) of Drosophila rnelanogaster lying a t the apical region of the collar (c) and the operculum (op). (c) SEM side view of Dacus oleae micropyle (m) a t the anterior inverted cup. (d) SEM apical view of the Antheraea polyphemus micropylar apparatus (m) forming a depression and surrounded by concentriccircles of cell imprints.(e) Schematic representationofthe hymenopteran Eurytoma arnygdali micropyle (see also Figure 1-le). The micropylar canal (c) that reachesthe vitelline membrane (vm) is formed by the follicle cells (fc) that secrete the material of the successive chorionic layers (En) (courtesy of F. Zarani). (f) SEM side view of Rhagoletis cerasi (Diptera) micropyle (m) located a t the anterior tip (courtesy of F. Zarani). (g-m) Respiration through the eggshell. (g) S E M side view of filler-less (removed during sample preparati0n)Antheraea polyphemusfractured chorion (ch), revealingconically shaped aeropyles (arrow) surrounded by tall crowns (cr). (gJ SEM apical view of Antheraea polyphernus egg surface showing the aeropyles (arrow) and the crowns. (h) TEM thin section micrograph of Drosophilagrimshawi endochorion(en)transversed by an aeropyle (arrow).(i)SEM view of a respiratoryappendage from the Hawaiian species Drosophila mimica. The network of modified pillars (arrow) i s shown. (i)SEM view of lctinogomphus australis main body surface reticulation (courtesy of J. Trueman). (k) SEM apical view of the polygonal chorionic patterns a t the main body of the Hawaiian species Drosophila setosimentum (i,k - Margaritis et al., unpubl. results). (I, m) The route of gas exchange through the eggshell in the air (left) or when the eggs are submerged (right) in the dipteran Drosophila melanogaster(1)equipped with respiratoryappendagesand in the lepidopteran Bombyx mori (m) having only aeropyles (see also text). (n, 0). Hatching. (n) SEM micrograph revealing Drosophilafasciculisetae larva emerging from the eggshell a t thecollar region (arrow).(o)Whole mountviewshowinga Ceratitiscapitata(Diptera)eggshell with a tear (arrow)atthe main body and a larva after hatching. Bars, (a) 500 nm, (b, c,f, g, g, k) 10 pm, (d, 0),100 pm, (e, j) 20 pm , (h) 1 pm, (i) 5 pm , (n) 50 pm.

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Structure, Microstructure and Physiology of Eggs and Eggshells in Various Insect Orders

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Novel Morphological and Physiological Aspects of Insect Eggs

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1.2.2.2 Oxygen Entry: Aeropyles and Plastron One of the most important physiological functions of the eggshell is to allow respiratory gas molecules to diffuse freely in and out during a passive process based on diffusion and governed by the co-efficient and the relative partial pressure of oxygen on either side of the eggshell (Hinton, 1981).To meet embryonic oxygen the chorion in some species is perforated by aeropyles, ranging in diameter from 0.2 to 2.5pm, an order of magnitude greater than the mean free path of oxygen in air. Aeropyles (Figure 1-2g, g,, h) connect to a gas-filled meshwork in the endochorion. Oxygen uptake occurs by diffusion down a partial pressure gradient generated by embryonicmetabolic activity (Figure 1-21,1-2m-left panels),although passive gas uptake in eggs with thin chorion could also directly occur from the egg surface that often appears rough, revealing in most cases imprints of the follicle cells that participate in eggshell production and secretion (Figure 1-2j, k). Prolonged submersion (a common incident in Hawaiian Drosophilu species),on the other hand, requires more elaborate eggshell modifications to enable oxygen absorption through plastron respiration (i.e. retention of a film of gas around a submerged body by means of hydrofuge structures)like the respiratory appendages seen in Drosophilidae (Figure 1-2i). These structures are tubular chorionic extensionsserving as physical gills when the egg is submerged under water, where an extensive water-air interface is established. They are seen in many insect orders and their number could be as many as 26 in the Borborphilus prirnitivu (Hemiptera), whereas their length in some Nepidue can be as much as 10 mm (Hinton, 1981). Drosophilu respiratory appendages are impermeable to water due to their hydrophobic properties and surface texture and they extract oxygen from the ambient water, transporting it by means of diffusion via partial pressure gradients towards the oocyte (Figure 1-21, right panel). In Drosophilidae the specialized regions of the operculum and the posterior pole (Figure 1-21, right panel) may also function as plastrons. In insect eggs bearing aeropyles the water-air interface across the aeropyles themselves acts as a plastron (Figure 1-2m, right panel). In these species the efficiency of the plastron can be enhanced by increasing the number and the width of the aeropyles.Plastron-bearingchorions are also prevalent among species ovipositing in decaying animal matter (such as carrion and dung) where water surface tension is reduced by the organic solutes. 1.2.2.3 Eggshell Structures for larval Hatching

Hatching is accomplished through regions of weakness that differ substantially in structure from the rest of the eggshell. In Drosophilu species larva hatching at the end of embryogenesis is facilitated through the collar region (Figure 1-2n) that consists of two discontinuous endochorionic halves held together by a common layer of fragde inner endochorion (Margaritiset al., 1980).On the other hand, eggs of specieslike the fruit flies Ducus oleue or Cerutitis cupitutu feature no visible structural specializationinvolved in hatching and in these species hatching is accomplished by random tearing of the eggshell at the main body (Figure 1-20). The lack of

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weakness lines is apparently related to the extreme stretching that these eggs receive during their passage through the very narrow ovipositor. Some of these eggshellrelated physiological properties, however, like elasticity, might represent evolutionary remnants since eggshell elasticity in Drosophfidae is rather useless as compared to species like Ducus oleue (see above).

1.3

Cell Types that Participate in Egg Formation: Panoistic and Meroistic Ovarioles

In insects, the germ cells that receive the information necessary for the continuation of life arise from pole cells that segregate from the rest of the single cell layered embryo very early in development. They migrate to the interior as development progresses and become associated with a group of mesodermal cells to form the ovary. The roughly sphericalovary, consistingof two lobes, increases in cell number during the larval stages and differentiates at the pupal stage, resulting in the appearance of cylindrical pillars of cells that will eventually develop into ovarioles (Postlethwait and Giorgi, 1985). Ovarioles are linear arrays of progressively more mature ovarian follicles (Figure 1-3a-c). The number of follicles in each ovariole varies significantly between species, ranging from 3000 in higher termites, more than 50 in Bombyx mori (Figure1-3b),6-7in D. melunoguster (Figure1-3c)(Margaritis, 1985a; Spradling, 1993), to 1 in some aphids. Two types of ovarioles can be distinguished in insects, namely the panoistic and meroistic (Postlethwait and Giorgi, 1985) (Figure 1-3a). In a panoistic ovariole (found in cockroaches, crickets and certain other groups), each follicle consists of an oocyte surrounded by a layer of follicle cells. Each ovariole matures a single oocyte at a time and the necessary materials for development are produced through vast and selective gene amplification in the nucleus of the oocyte (Cave, 1982). In contrast, in the meroistic ovariole, three cell types cooperate in egg chamber assembly, namely the oocyte, the nurse cells and the follicle cells. In this ovarian type it is the nurse cells that supply the oocyte with the RNA and proteins necessary for later development. Meroistic ovaries themselves are of two subtypes. In the one subtype (found in true bugs and some beetles),called the telotrophic meroistic ovary (Figure 1-3a),the cluster of nurse cells remains at the tip of each ovariole in a tropharium which is connected to the oocyte as it moves down to the ovariole by means of ever-lengthening slender trophic cords. In the second subtype, namely the polytrophic meroistic ovary (found in flies,bees, moths and in some Dermaptera and Psocoptera) (Figure 1-3a-c), the nurse cells are directly connected to the oocyte by means of short open cytoplasmicbridges (also called ring canals)through which nurse cells transfer their cytoplasmic contents to the oocyte during a process known as cytoplasmic“dumping” (Mahajan-Miklosand Cooley, 1994).In the lepidopteran follicle (e.g. Hyulophoru cecropia)an 8-cell cluster (Figure 1-3bl)arises through only three sets of synchronous mitoses and three nurse cells are bridged directly to the oocyte forming a central ring canal or fusome (De Loof, 1983).

13 ....

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Novel Morphological and PhysiologicalAspects of Insect Eggs

In the Drosophilu species, germline cystoblasts located at the anterior germarium (Figure 1-3c) undergo four mitotic divisions to generate 16 cystocytes connected by ring canals. Only two cells are interconnected to 4 others by a ring canal and it is one of these which will become the oocyte (Figure 1-3d). The other 15, which are also connected through ring canals with the oocyte (Robinsonet al., 1994)(Figure 1-3e),become the nurse cells. Both cell types are initially surrounded by a monolayer of follicle cells (Figure 1-3c, stage 6) that in more developmentally advanced follicles migrate posteriorly and cover only the oocyte (Figure 1-3c, 1-3f). Oogenesis in D. melanogaster has been divided into 14 developmental stages (King, 1970), where the stage 1follicle represents the 16cell syncytium immediately after encapsulation by the follicle cells and stage 14 is the mature follicle where the nurse cells have degenerated and the eggshell is completed. The majority of the molecular, biochemical and morphological developmental events occurring in the ovariole during D. melunoguster follicle maturation (discussed throughout the current chapter) are schematically summarized in Figure 1-3g. On the basis of the macroscopical and fine structural changes occurring in the choriogenic follicles of Drosophilidae, choriogenesis has been further subdivided into several developmental stages (Figure 1-3h).Moreover, the follicular epithelium is seemingly composed of distinct follicle cell subpopulations engaged in the formation of the various specialized eggshell regions (Figure 1-3i) (see also Margaritis et al., 1980; Margaritis, 1986). Figure 1-3 Follicle development in the ovary. (a-g) Insect ovarian types, cell types that participate in the follicle assembly and follicle maturation in the ovariole of Drosophila rnelanogaster.(a) Diagrammatic representation ofthe three types of insect ovarioles (modified from Postlethwait and Giorgi, 1985). (b) Single ovariole of the lepidopteran Bornbyx rnori containing follicles (arrow) in a arrayed sequence of development. (b,) Schematic representation of the lepidopteran immature follicle with the oocyte and 7 nurse cells. (c) Whole mount confocal microscopy of Drosophila virilis ovarioles following nuclear staining with Hoechst (a DNA-binding dye). Developmentally advanced follicle stages exiting germarium (g) are indicated. A t stage 6, follicle cells (fc) cover both the oocyte (oc) and nurse cells (nc) ((n) nurse cell nucleus), while following completion of posterior migration a t stage lothey are locatedovertheoocyte.(d)Schematicrepresentation ofthefour incomplete mitotic divisions (Dl-D4) of a stem cystoblast (c) t o a 16-cell cluster in which the cells are interconnected by ring canals. One of the tw o cells with 4 ring canals (dark shading) will differentiate t o oocyte. (e) Whole mount confocal microscopy of a phalloidin stained (for filamentous actin) Drosophila rnelanogaster stage 10 follicle, demonstrating the nurse cell ring canals (rc); arrowhead indicates border cells. The oocyte (oc) and the follicle cells (fc) are also indicated (courtesy of I. P. Nezis). (f) Whole mount confocal microscopy of a Drosophila virilis stage 10 follicle following Hoechst nuclear staining. Follicle cells (fc) have complete posterior migration, leaving only a few stretched cells (arrow) between the nurse cells (nc). (g) Diagrammatic representationof a Drosophilarnelanogasterovariolesummarizingthe major developmental events occurring during follicle maturation (see text). (h) Schematic representation of the successive morphogenic events during the 11choriogenic stages of Drosophila virilis. (i) Schematic illustration of the follicle cell subpopulations participating in eggshell formation of Drosophila virilis. The number of arrows in each eggshell specialized region indicate the number of cell subpopulations that participate in i t s morphogenesis (h, i - Trougakos, IP and Margaritis, LH, unpubl. results). (i) Diagrammatic representation of the key point events during anterio-posterior (A-P) and dorso-ventral (D-V) polarityformation in Drosophila rnelanogaster(see text - composed from Deng and Bownes, 1998; van Eeden and S t Johnston, 1999; Dobens and Raftery, 2000). Bars, (b) 5 mm, (c, e,f,) 50 pm.

Cell Types that Participate in Egg Formation: Panoistic and Meroistic Ovarioles

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Formation of Egg Polarity

In all insect eggs (with only a few exceptions) the embryonic anterior-posterior and dorso-ventral axes are already established before fertilization. These axes are apparent in the external morphology of the eggshell (e.g. localization of the micropyle apparatus) and the location of the nurse cells or the trophic cords. The bulk of our current knowledge concerning the genetics and the molecular basis of pattern formation during folliclematuration in the ovariolerefers to the model insect Drosophilu melunoguster; a diagrammatic representation of the major events during follicle polarity formation in the D. melunoguster ovariole is shown in Figure 1-3j. In that species once the oocyte is determined it must be positioned at the extreme posterior of the cyst as this determines the anterior-posterior polarity for the rest of the development (Gonzalez-Reyesand St Johnston, 1994).Polar follicle cells are determined in the germarium by the hedgehog gene (Dobens and Raftery, 2000), while establishment of terminal cells is Notch dependent and occurs before oogenic stage 7 (Larkin et al., 1999).The follicle cell layer becomes polarized along both the anterior-posterior and dorso-ventral axis through two rounds of gurken signaling from the oocyte. At stages 4-6, when oocyte nucleus is still localized posteriorly, the early gurken signals to the follicle cells adjacent to the oocyte to induce them to adopt a posterior fate (Gonzalez-Reyes et al., 1995).Dorso-ventral follicle cells and embryonic polarity arise at stage 8 followinga microtubule directed migration of the nucleus to an asymmetric position in the anterior of the oocyte where it determines where gurken mRNA is translated and localized (lategurken signaling), resulting in the induction of two prominent dorsal-anterior fates, the operculum and the dorsal appendages (Ray and Schupbach, 1996). The polarized microtubule cytoskeleton is also required for creating an oocyte anterior-posterior polarity (that is eventually transmitted to the embryo) by directing the localization and localized translation of bicoid mRNA anteriorly and oskar mRNA-staufen protein posteriorly (Gonzalez-Reyes and St Johnston, 1998). Gurken signaling by activating the Epidermal Growth Factor Receptor (EGFR) signaling pathway in the dorsal follicle cells (rhomboid, vein, kekkon and sprouQ), restricts pipe expression to the ventral follicle cells, defining thus the ventral side of the embryo. High level EGFR activation induces the dorsal expression of the EGFR inhibitor urgos and this may split the single peak of EGFR activation into two lateral peaks (van Eeden and St Johnston, 1999). The establishment of follicle polarity is also accompanied by five impressive migration events of small groups (or subpopulations) of follicle cells (Dobens and Raftery, 2000). The first major follicle celkmigration is that from the wall of the germarium to surround the 16-cellgerminal cysts in germarium region two (Figure 1-3c).During stage 8 a group of anterior follicle cells (border cells) separate from the follicularepithelium, migrate posteriorly through the nurse cell cluster (second migration) and eventually locate at the nurse cell-oocyteborder at stage 10 (Figure 1-3e). During late stage 9 most follicle cells retract from the nurse cell cluster to cover only the oocyte (third migration), leaving only a few stretched cells to be

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Vitellogenesis: How Does the Yolk Get into the Egg?

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associated with the nurse cells (Figure 1-3f).At stage 10b concurrently with nurse cell "dumping" the anterior most columnar cells migrate centripetally along the nurse cell-oocyteborder to cover the anterior end of the oocyte (fourth migration). Finally, two groups of columnar follicle cells in the dorsal-anterior region of the follicle migrate anteriorly to produce the respiratory appendages (Deng and Bownes, 1998).

1.5

17.

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Vitellogenesis: How Does the Yolk Get into the Egg?

Insect eggs are provisioned by the female with huge amounts of nutritive substances to support the embryo until it can obtain its own food, using a process called the "box lunch strategy (term introduced by Postlethwait and Giorgi (1985)),otherwise called vitellogenesis or yolk deposition. Insect vitellogenesis can be accomplished by two general mechanisms. The first, in which the follicle produces its own yolk, is called autosynthetic vitellogenesis and has been reported in only a few species of the most primitive insects, the Apterygota (Bilinski, 1979).In the second, called heterosynthetic vitellogenesis and used by almost all advanced species, the follicle obtains the bulk of its yolk from an extra-ovarian source (the fat body) via the maternal blood stream through a hormonally (juvenile hormone andor 20hydroxyecdysone)regulated process (Postlethwaitand Giorgi, 1985;Bownes, 1994). The major yolk components in insect eggs are protein granules containing mainly yolk proteins, lipid droplets whose contents (neutral lipids) are probably synthesized by the nurse cells and glycogen rich particles synthesized by the oocyte itself (Postlethwait and Giorgi, 1985). Since insect vitellogenesis is almost exclusively a heterosynthetic process, the major-female specific protein sequestered from the blood, modified co- and posttranslationally by the addition of glycosyl, phosphate and sulfate residues (Izumi et al., 1994) and stored in yolk granules, is defined as vitelline and its soluble counterpart in the haemolymph as vitellogenin. Insect yolk proteins may be divided into five categories. (1)Vitellines, found in most insect species, such as moths, bees, wasps, locusts and lower flies such as the mosquitoes. These proteins derive from a vitellogenin precursor molecule that is synthesized in the fat body in a female specific manner, cleaved in a large and small subunit (e.g.the vitellogeninsof the cicada Gruptopsultriu nigro@scutu (Homoptera) (Lee et al., 2000), or the mosquito Aedes uegypti (lower Diptera) (Raikhel and Dhadialla, 1992)), secreted into the haemolymph and sequestered in the developing oocytes. Reported cases where no vitellogenin precursor cleavage occurs refer to higher Hymenoptera (suborder Apocrita) (Nose et al., 1997)and two species of Homoptera (Tu et al., 1997),while vitellogenin can be also cleaved into three or four subunits, as occurs in Locustu migrutoriu and in sawflies (Izumi et al., 1994; Takadera et al., 1996). (2) Yolk proteins of the higher Diptera, including fruit flies, flesh fly and house fly, which are synthesized in both the female fat body and ovarian follicle cells and are incorporated into developing oocytes; there are no differencesbetween the

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yolk proteins originating in the fat body and those originating in the follicle cells. In D. melunoguster three major yolk proteins have been isolated with molecular weight of 4446 kDa, encoded by single copy genes (Bownes, 1994), while in Ducus oleue and Cerutitis cupitutu yolk proteins are represented by only two polypeptides with molecular weight ranging between 45 and 49 kDa (Rina and Mintzas, 1988;Trougakos et al., 1999).Among Diptera, vitellogenin synthesis in the ovary has been documented for D. melunoguster (Brennan et ul., 1982), Ducus oleue (Trougakos et al., 1999), Culliphoru erythrocephulu (Martinez and Bownes, 1994),Muscu domesticu (White and Bownes, 1997)and Cerutitis cupitutu (Rina and Mintzas, 1988). (3) Proteins such as the egg-specific protein of Bomlryx mori (Ire and Yamashita, 1983), the paravitellogenin of Hyulophoru cecropia (Telfer et al., 1982), the two proteins isolated from Munducu sextu eggs (Tsuchida et al., 1992) and a follicle cell-specific protein recently isolated from Rhodnius prolixus (Melo et al., 2000). These proteins are synthesized only in the ovarian follicle cells and sequestered into the oocytes, but appear to be distinct from the vitellogenins produced in the fat body. (4) Proteins such as the 30 kDa protein of Bombyx mori, the microvitellogenin found in Munducu sextu and Hyulophoru cecropia, the 21 kDa protein of Locustu migrutoriu and the female specific 53 (a proenzyme of vitellogenic carboxypeptidase) and 44 kDa (40KP)proteins of Aedes uegypti (Raikheland Snigirevskaya, 1998).These proteins are synthesized in a non sex-specific manner in the fat body cells and appear to be distinct from the major vitellogenins. They are however secreted into the haemolymph and sequestered in the developing oocytes. (5) The cochineal yolk protein in the homopteran Ductylopius confusus (Ziegler et al., 1996). The yolk proteins secretory pathway in the fat body of the mosquitoes (Raikhel and Snigirevskaya, 1998) and the ovary of fruit flies (Trougakos et al., 1999; Trougakos et al., 2001) has only recently been analysed by means of transmission electron microscope (TEM) immunolocalization and appear to be quite similar. In the fruit fly folliclecells, however, which at the same time are engaged in the vitelline membrane proteins secretion, the yolk proteins are differentially sorted from the co-secreted vitelline membrane proteins either in distinct parts of biphasic vesicles (Figure 1-4b)or in entirely distinct vesicles (Figure 1-4c).Following secretion, yolk proteins, along with their haemolymph originating counterparts (Figure 1-4a), diffuse through permeable channels in the vitelline membrane maintained by the extended follicle cell microvilliand finallyreach the plasma membrane of the oocyte (Figure 1-4d). Yolk sequestration in the oocyte follows a similar pathway in all insects studied so far and is accompanied by two major changes in the egg chambers (Raikhel and Dhadialla, 1992), namely follicle cell "patency" (referring to formation of large intercellular spaces between the adjacent follicle cells) and the appearance of coated pits on the surface of the oocyte (Figure 1-4e) that is related to receptor mediated endocytosis (Sappington and Raikhel, 1998; Schonbaum et al., 2000).

Eggshell Morphogenesis

Analysis of the Aedes uegypti (Sappington et al., 1995) and D. rnelunoguster (Schonbaum et al., 1995) yolk protein receptors revealed that they appear to be homologous in spite of the very different ligand they bind. A similar vitellogenin receptor has also been isolated from the locust Locustu migrutoriu and was found to be a large membrane protein of 156kDa (Ferenz, 1993).Fine immunocytochemical analysis of the yolk protein oocyte endocytic pathway in Aedes uegypti (Raikheland Snigirevskaya, 1998)and the fruit flies Ducus oleue and D. melunoguster (Trougakos et al., 1999; Schonbaum et al., 2000; Trougakos et al., 2001) demonstrated that this pathway consists of coated pits, coated vesicles and endosomes (Figure 1-4e)that later on fuse to form a transitional yolk sphere. Yolk spheres are specialized lysosomes (whose degradative activity is postponed until embryogenesis (Schonbaum et al., 2000)) that finally transform into a maturp yolk sphere; in Drosophilu and the mosquito this organelle contains crystallineparts (Figure 4g). In mosquitoes, the crystalline part of the mature yolk sphere is likely to be composed exclusively of vitelline, while the other yolk proteins, such as the vitellogenic carboxypeptidase and the 40KP, are segregated from the crystalline vitelline and are localized together in the non-crystalline matrix (Snigirevskaya et al., 1997). In contrast, in D. melunoguster yolk proteins distribute over the entire organelle (Figure 1-4g) (Trougakos et al., 2001). In Ducus oleue yolk proteins are segregated (as in the mosquito) in distinct parts of the mature yolk sphere (Figure 1-4h)although in that species the mature organelle is devoid of crystallineregions (Trougakoset al., 1999).Finally, the stored yolk proteins accomplish their nutritive role following proteolytical cleavage during the early stages of embryogenesis. Proteases have been shown to participate in yolk degradation in Bombyx mori (Indrasith et al., 1988),D. melunoguster (Medina and Vallejo, 1989)and Aedes uegypti (Cho et al., 1991).

1.6

Eggshell Morphogenesis

Almost invariably in the insect eggshell type, its formation and morphogenesis is a gradual process involvingintense synthetic and secretory activity of the follicular epithelium. Consequently, eggshell formation has been divided into 9 successive stages in D. rnelunoguster (Margaritis, 1986; see also Figure 1-3h) or 10 in Cerutitis cupitutu (Mouzakiand Margaritis, 1991a)and the 1epidopteranAntherueupolyphemus (Paul et al., 1972). The time period needed for eggshell formation varies greatly among species and seems to be related to the size of the final structure, since the 60 pm thick chorion of Antherueu polyphemus is formed during a period of about 51 hrs, while morphogenesis of the 0.8 pm thick-chorion of D. melunoguster requires about 6.5 hrs (Margaritis,1985a).Similarly,the number of follicle cell subpopulations engaged in eggshell formation is species-dependent (even in the same order) ranging from 12 to 13 in Drosophilu species (Margaritis, 1986; Trougakos and Margaritis, unpubl. results; see Figure 1-3i)to 2 in Ducus oleue (Mouzaki et al., 1991). The production of a functional eggshell (a self-assembly process) features: (1)Continuous apposition of secreted material onto pre-formed structures.

19

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(2) Differentiation of follicle cell subpopulations in response to ovarian signals. (3) Directed migrations o f th e follicle cells within the developing egg chamber. (4) Expression of th e eggshell structural genes by the follicle cells in a defi ned t em p o ra l a n d spatial order a n d possibly differential synthesis of regional specific proteins a m o n g th e follicle cell subpopulations. (5) Post-depositional modifications of the eggshell proteins i n c l u d i n g several temporally regulated proteolytic cleavage events. (6) Regulated trafficking of several eggshell proteins in the assembling structure.

Figure 1-4 Vitellogenesis and eggshell morphogenesis. (a-g) Vitellogenesis in Drosophila melanogaster (a) lmmunohistochemical localization of yolk proteins over a longitudinal ovarian section showing positive immuno-reaction a t haemolymph (h), trapped between the follicles and in the oocyte (oc). (b) High magnification of a cryosectioned follicle cell bipartite vesicle, triple immunolabelled for vitelline membrane proteins (large and median gold particles-double arrow) and yolk proteins (small gold particles-arrow). The differential distribution of the antigens in the vesicle is apparent. (c) Cryosedion of a vitellogenicfollicle cell (fc) double immunolabelledforavitellinemembrane protein (small gold particles-double arrow) and yolk proteins (big gold particles-arrow), revealing differential distribution of the antigens in distinct secretory vesicles. (d) Confocal immunofluorescence localization of yolk proteins in a vitellogenic follicle revealing positive immuno-reaction in the follicle cells (fc) secretory vesicles (double arrow-(n) follicle cell nucleus) and in the gaps (arrow) of the incomplete vitelline membrane (vm). (e) TEM cryosedion a t the vitelline membrane (vm)oocyte plasma membrane interface,double immunolabelledforavitelline membrane protein (small gold particles) and yolk proteins (big gold particles). Yolk protein related gold particles are distributed in the oocyte endocytotic pathway consisting of coated pits (cp), coated vesicles (cv) and endosomes (end). (f) TEM immunogold labelling of yolk proteins over cryosectionedendosomes (end) seen a t the cortical ooplasm (oc). (g) TEM cryosedion showing yolk protein immunogold localization in the mature yolk spheres (a,-y); gold particles are seen over both the crystalline (star) and the amorphous regions of the organelle. (h) lmmunogold localization of Dacusoleaeyolk proteins in the mature yolk spheres (az-y)ofthe oocyte (oc). lmmunogold labelled portions over the organelle contrast with morphologically distinct unlabelled regions; no crystalline yolk is seen. (i-n) Morphogenesis of eggshell layers bythe folliclecells oroocyte microvilli and the flocculent material (or filler). (i)TEM micrograph of the Leptinotarsa decemlineata vitelline membrane (vm) during the completion of i t s morphogenesis. Irregularities are caused by the oocyte microvilli (arrows); in the follicle cells (fc) numerous secretoryvesicles (sv) are seen (Papassiderie t al., unpubl. results). (i)TEM thin section micrograph during Drosophila melanogastervitellogenesis,showing follicle cells (fc) extended microvilli (mv) that inhibit the coalescence of the vitelline membrane (vm) bodies, thus enablingyolk uptake by the oocyte (oc). (k)Thin section through mid-choriogenic stage in a follicle of Ceratitis capitata. Long follicle cell (fc) microvilli (arrows) insert into the endochorionic material cavities (ch), thus creating a perforated endochorion; flocculent material intheendochorioniccavitiesis indicated bya star.Thecompletedvitelline membrane (vm) and the oocyte (oc) are also indicated. (I) TEM micrograph of respiratory appendage morphogenesis in Drosophila virilis. Abundant flocculent material (star) is seen between the secreted chorionic material (ch) inhibiting itscoalescence,thusenablingtheformationofthe vacantspacesseeninthe matureappendage.(rn)TEMthin section micrograph ofa Drosophila virilis follicle at late choriogenesis, revealing flocculent material absorption (arrow) over the pillar (p). The completed innermost chorionic layer (icl), the wax layer (wl) and the vitelline membrane (vm) are also shown (I, m - Trougakos and Margaritis, unpubl. results). (n) TEM micrograph of a thin cross section cut through a forming aeropyle channel in the eggshell of Bombyx mori. A bundle of follicle cell microvilli (mv) is transversing the chorion and along with filler (f)forms the future air channel. Bars, (a) 50 pm, (b, c, e, f, g, h) 100 nm, (d) 5 pm, (i, k) 1pm, (j,I, m, n) 500 nm.

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22

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(7)Differential sorting of the secreted proteins in the follicle cells. (8)Active involvement of the major morphogenic factors, namely the follicle cell microvilli and the ”filler”,in cases of chorion with cavities. (9) Secondary structural modifications in the various layers which involve alteration in thickness (i.e. vitelline membrane) or organization and condensation status (i.e. innermost chorionic layer crystallization) in order to accommodate the continuously increasing oocyte volume. (10)”Hardening” or insolubilization of the eggshell through the generalized activation of a “hardening factor” (e.g. di-sulphide bonding or enzyme).

1.6.1 Formation of the Vitelline Membrane The first layer to be formed by the secretory activity of the follicle cells during eggshell morphogenesis is the vitelline membrane. In hymenopteran species (like Eurytorna arnygdali), vitelline membrane formation includes uniform secretion by the follicle cells of a loose material at the oocyte-follicle cell interface and active involvement of the oocyte and follicle cell microvilli (Mouzaki and Margaritis, 1994) whose withdrawal after completion of secretion results in the formation of a uniformly sized continuous layer. A similar mode of vitelline membrane formation is seen in some orthopteran (e.g. Locustu rnigrutoriu(Goltzene, 1979))and coleopteran (e.g. Leptinotarsa decernlineatu (De Loof, 1971)) species, although in these cases the extended oocyte microvillido not withdraw after completion of vitelline membrane formation, so that a mature vitelline membrane irregular in thickness and shape is produced (Figure 1-4i). Very impressive involvement of oocyte microvilli in vitelline membrane formation is seen in Korscheltellus lupulinus (Lepidoptera) where a perforated mature vitelline membrane is formed following material apposition in a symmetrical three-dimensional matrix created by the vertically extended oocyte microvilli (Chauvin and Barbier, 1979). Interestingly, in the primitive insect Tetrodontophora bielanensis (Collembola)the vitelline membrane seems to be formed during early embryogenesis, apparently by the oocyte (Krzysztofowiczand Kisiel, 1989).In Diptera (e.g. fruit flies and mosquitoes), the vitelline membrane is formed following the localized apposition of vitelline membrane material called “vitelline bodies” on the oocyte-follicle cell interface (Mahowald, 1972; Edwards, 1996). Extended and spatially organized follicle cell and oocyte microvilli prevent fusion of the layer, accommodating thus the synchronous uptake of yolk proteins by the oocyte (Figure 1-4j).Following the completion of yolk uptake, microvilli withdraw and the vitelline bodies fuse to make a thick, highly elastic membrane that gradually thins down due to the continuous increase in the oocyte volume.

1.6.2

Formation of Chorion Layers

After vitelline membrane morphogenesis, follicle cells are engaged in the secretion and morphogenesis of the successive chorion layers (see Section 1.2.1) featuring both radial and regional complexity. In D.rnelanoguster, a wax layer is formed

Eggshell Morphogenesis

through the secretion by the follicle cells of large electron transparent vesicles (oil droplets)which are deposited on the surfaceof the vitelline membrane (Papassideri and Margaritis, 1986)and are finally squeezed one upon the other to form 4-5 wax plaques that seal the oocyte (Figure 1-2a).The subsequent morphogenic processes of the remaining chorion layers largely depend on the type of structure and follows quite a complicated pattern. In insect species where chorion features vacant spaces (e.g. trabecular layers, aeropyles or respiratory appendages), chorion morphogenesis includes active involvement of two major morphogenic structural factors, namely the follicle cell microvilli (Figure 1-4k, 1-4n)and a flocculent material (or filler) (Figure 1-4k, 1-4n). In dipteran species like the fruit flies (Margaritis, 1986; Mouzaki and Margaritis, 1991a; Mouzaki et al., 1991) or the viviparous fly Glossina austeni (Huebner et al., 1975),the flocculent material inhibits the fusion of the secreted chorionic material and along with the extended follicle cell microvilli(Figure1-4k)lead to the formation of the cavities seen in the trabecular layers (Figure 1-1) or the Drosophilidae respiratory appendages (Figure 1-41). The flocculent material is finally absorbed on the surfaces of the chorion (Figure 1-4m).Interestingly, flocculent material can also be seen during the formation of the Acrididae chorion, where it intercalates between the secreted structures of crystalline appearance, thus resulting in the formation of the endochorionic sponge-like meshwork (see above) (Lebouvier et al., 1985).Similarly, aeropyles in lepidopteran species form around a closely packed bundle of extended microvilli surrounded by filler (Figure 1-4n) that functions as a mold for channel formation (Reger et al., 1982; Margaritis, 1985a; Papanicolaou et al., 1985). The function of the two morphogenic factors mentioned above is accompanied by localized (i.e. pillar formation in Drosophilidae and Tephritidae-see, Figure 13h) or uniform secretion (i.e. lamellate chorion layer in moths-see Figure 1-lj)of the main structural components of the chorion itself in a secretory pattern that is usually differentiated across the regional complexity of the eggshell. It is worth noting that the secretionpattern during chorion morphogenesis in the follicle main body of Drosophilidae switches from localized during pillar formation to uniform during morphogenesis of the outer endochorion (Figure 1-3h-stage 12d). In the silkmoths and the gypsy moths initial lamellogenesis or framework formation is followed by intercalation of newly synthesized proteins, which leads to a nearly twofold increase in overall chorion density with no significant increase in overall chorion thickness (Mazur et al., 1989; Leclerc and Regier, 1993).Finally, differential secretion and assembly of the surface specialized structures (where existing) take place (Regier et al., 1982; Mazur et al., 1980).In some lepidopteran species, however, no lamellar expansion occurs and the minimal lamellar framework is added throughout lamellogenesis (Regier and Vlahos, 1988). In chorions like the ones seen in the almond wasp Eurytornu arnygdali (Hymenoptera) (Figure 1-11),(Mouzakiand Margaritis, 1994),Allocontarinasorghicolu (Diptera) (Isidoro and Lucchi, 1989)and the potato beetle Leptinotarsa decernlineuta (Coleoptera)(Figure1-lm) (DeLoof, 1971),consistingof homogeneous thick layers

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(crystalline,amorphous, granular, or fibrous) featuring no vacant spaces, filler is absent and morphogenesis occurs through uniform material secretion with minimum involvement of the follicle cell microvilli. Where crystalline layers are seen (Figure l-lgl, 1-li,, 1-lk), crystallization appears to be a late event during choriogenesis triggered by an unknown factor and not directly related to any morphogenic process. Finally, it is worth mentioning that the morphogenic processes described above could vary significantly during formation of the chorionic specialized regions (Margaritis, 1985a).

1.7

Eggshell Composition and Assembly

1.7.1 Chemistry and Molecular Events Consecutiveinsect eggshelllayers are largely proteinaceous structures (Regierand Kafatos, 1985),the only exceptionsbeing the wax layer that appears to contain only lipids (Papassideri and Margaritis, 1986) and the fibrous exochorion which is composed mainly of polysaccharides (Trougakosand Margaritis, unpubl. results). In D. melanogaster, a limited number of six proteins has been proven to represent structural components of the vitelline membrane and most of the genes encoding for these proteins have been cloned (Spradling, 1993; Waring, 2000). Analysis of the amino acid sequence of the known structural vitelline membrane proteins revealed the existence of a highly conserved hydrophobic domain of 38 amino acids in length which have been termed vitelline membrane specific domain and is probably implicated in late assembly events occurring within this layer (Scherer et al., 1988). In the mosquito Aedes aegypti, three recently cloned vitelline envelope genes encode for proteins that contain a conserved region overlapping with the Drosophila vitelline membrane protein hydrophobic specific domain (Edwards et al., 1998). In lepidopteran moths such as Bombyx mori the vitelline membrane appears to be constituted by a relatively high number of protein molecules (Regier and Kafatos, 1985).Similarly, chorion in flies and moths is highly proteinaceous. In both cases distinct groups of chorion proteins are deposited in overlapping succession according to a developmental program which is presumably responsible for the intricate morphogenesis of the chorion. As many as 20 distinct polypeptides can be seen following two-dimensional electrophoresis of purified D. melanogaster (Waring and Mahowald, 1979) or Drosophila virilis chorion (Figure 1-5a).Among these, six proteins represent the major chorion proteins, being encoded by differentiallyamplified genes and classified as developmentally "early" (s38, s36), "middle" (s19, s16) and "late" (s18, s15) on the basis of their developmentally regulated expression profile (Figure 5a) (Orr-Weaver, 1991; Spradling, 1993).The expression of the chorion genes is asynchronous in the various follicle cell subpopulations and initiatesfor all genes studied so far from the anterior pole (Parks and Spradling, 1987; Trougakos and Margaritis, 1998b). Comparative biochemical and molecular studies in other dipteran species

Eggshell Composition and Assembly

distantly related to Drosophilidae,such as Rhagoletis cerasi (Mouzakiand Margaritis, 1991b),Dacus oleae (Margaritis,198513)and Ceratitis capitata (Mouzakiand Margaritis, 1991a; Konsolaki et al., 1990; Vlachou et al., 1997) revealed a conserved chorion composition in terms of protein number, expression pattern and sequence of the major chorion proteins. In Drosophila species, constituent proteins of the vitelline membrane and chorion are also the multiple products of the dec-2 locus which are synthesized in distinct temporal patterns and moreover undergo a series of complicated post-translational and positional modifications during chorion maturation (Noqueron and Waring, 1995;Noqueron et al., 2000). On the other hand, the lepidopteran chorion proteins are characterized by high complexity (almost 100 distinct spots are detectable on two-dimensional gels), widely differing abundance levels, small sizes, slightly acidic isoelectric points and complicated developmental expression pattern (Mazur et al., 1980; Regier et al., 1982; Leclerc and Regier, 1993) (Figure 1-5b). The shift in synthesis from one protein subset to another [A, B, C , and D] correlates with observable changes in modes of chorion morphogenesis related to formation of the predominant lamellar substructure and to its subsequent modification. In Drosophila, no specific proteins have been found to be related with specific specialized eggshell regions, except a high molecular weight protein that appears to be highly enriched in the respiratory appendages of Drosophila virilis (Trougakos and Margaritis, 1998a). Region-specific proteins in lepidopteran silkmoths represent the crown (Regier et al., 1993) and filler specific proteins (Mazuret al., 1980; Regier et al., 1984).The nature and composition of the crystalline layers seen in insect eggshells is poorly understood although it is expected that these layers should be composed exclusively of protein as indicated by the identificationof eggshell peroxidase or one of the dec-2 locus products as structural components of the innermost chorionic layer in Drosophila (Mindrinos et al., 1980; Noqueron et al., 2000). Similarly, by analysing pure fractions of the coleopteran Leptinotarsa decemlineata crystalline chorionic layer (see Figure 1-li, l-lil) we confirmed its proteinaceous nature and identified its components (Papassideri IS et al., unpubl. results). Another component of the chorion layers in some insects orders is an enzyme, namely the eggshell peroxidase that is involved in crosslinking the chorion proteins through di-, tri-tyrosine bonding following the completion of oogenesis (Figure 15c). This enzyme has been detected in the chorion of all Drosophila species studied so far (Mindrinos et al., 1980; Trougakos and Margaritis, 1998a),as well as in the chorion of other higher (e.g. Ceratitis cupitutu and Dacus oleae (Margaritis, 1985b)) and lower (e.g. Aedes aegypti (Li et al., 1996))dipteran species. Eggshell peroxidase has also been found in the chorion of the hymenopteran Eurytoma amygduli (Mouzakiand Margaritis, 1994).Enzyme activation and chorion crosslinkingin D. melanogaster and Aedes aegypti seems to be related to H202secretionby the follicular epithelium (Margaritis, 1985c; Han et al., 2000a).Recently, eggshellperoxidase was purified and cloned from the mosquito (Han et al., 2000b) and found to be a 63 kDa hemoprotein with an enormously high specific reaction to tyrosine.

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Novel Morphological and Physiological Aspects of Insect Eggs

The molecular events that lead to the assembly of a functional eggshell from i t s constituent proteins have been analysed in detail only in Drosophila species. The term ”assembly” refers to the sequence of molecular events during and following the completion of eggshell morphogenesis that result in the fully functional supramolecular structures seen in the mature chorion of D. melanogaster (Figure 15d) a n d Bombyx mori (Figure 1-5e); the success of the ”structural functions” performed by the various eggshell layers and regions (see Section 1.2.2) depends entirely u p o n the proper assembly of the component molecules. InDrosophila, until recently, studies o n the assembly of the multilayered eggshell body were limited to the realm of electron microscopy autoradiography (Giorgi, 1977). According to these data i t was assumed that the chorion was built inzones by protein apposition. However, recent studies with specific antibodies against vitelline membrane and chorion proteins (Noqueron and Waring, 1995; Pascucci et al., 1996; Trougakos and Margaritis, 1998a, Trougakos and Margaritis, 1998b; Noqueron et al., 2000) have allowed the fates of individual eggshell proteins to be analysed in the assembling and completed structure. The early s38/36 proteins were immunolocalized apart from the endochorion in the vitelline membrane (Figure 1-5f) that i s formed before the onset of s38/36 secretion and n o t in the innermost chorionic

1

Figure 1-5 Eggshell composition, assembly and nurse cell apoptotic death. (a-c) Eggshell composition. (a) Two-dimensionalelectrophoreticfractionation of Drosophila virilis chorion proteins.The 8 major chorionic proteinsare indicated;about 15 additional minor components were resolved in this preparation. Decreasing molecular weight (mw) and pH are indicated by large arrows (Trougakos and Margaritis, unpubl. results). (b) Numerous proteins are seen following differential fractionation (I, II, 111, T) (upper panel) and electrophoretic analysis (lower panels) of Antheraea polyphemus chorions (courtesy of C. Rodakis). (c) Cytochemical localization of ESP in the endochorion (en) of the dipteran Dacus oleae. No electron dense reactionisseen inthevitelIinemembrane(vm)andtheoocyte (oc).(d,e)Chorion organization. (d) Freeze fractured-etched TEM view of a Drosophila melanogaster endochorionic pillar revealingi t s fine structure consistingofnumerous spherical particles(arrows)interconnected byfine fibers (doublearrows).(e)TEM ofthin section through Bombyxmorichorion showing helicoidally arranged lamellae (dashed line) composed of fine fibers (arrow). (f-h) TEM immunogold localization of the early s38/36 and the late s15 chorion proteins over the forming and matureeggshe1I of Drosophila virilis. (f)At mid-choriogenic stagest he early s38/36 chorion proteins are distributed (gold particles-arrow) over the forming endochorion (en) and the vitelline membrane (vm) whose morphogenesis was completed before the onset of s38/36 secretion by the follicle cells (fc). (g) A t the mature chorion the s38/36 proteins (gold particles-arrow) are distributed solely in the endochorion (en); the exochorion (ex) and the innermost chorionic layer (icl) are devoid of immunogold particles. (h) The s15 late chorion protein secreted bythefolliclecells duringthevery late stages ofendochorion morphogenesis was immunolocalized (immunogold particles - arrow) over the entire endochorion (en) of the mature eggshell. No gold particles are seen overthe exochorion (ex). (i-I) Demonstration of nursecell apoptoticdeath.(i)TEM micrographshowing Drosophilamelanogaster nursecell nuclei (n) undergoingchromatin condensation during late oogenesis. (i)Confocal microscopy following TUNEL labelling of a Drosophila melanogaster stage 1 3 follicle revealing nurse cell (nc) DNA fragmentation; an autofluorescent elongating respiratory filament (rfl is also indicated. (k)TEMmicrograph showingDacusoleaenursecellnuclei (n)condensation.(I)TUNEL confocal image revealing nurse cell (nc) DNA fragmentation in a Dacus oleae late oogenic follicle (i-I, courtesy of I. P. Nezis). Bars, (c) 1pm, (d) 30 nm, (e) 250 nm, (f, g, h) 100 nm. (i,k) 500 nm, (i)50 pm , (I) 100 pm.

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Eggshell Composition and Assembly

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Novel Morphological and PhysiologicalAspects of Insect Eggs .....................................................

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layer formed during their secretion (Figure 1-5g).The s18 and s15 chorion proteins, secreted very late in choriogenesis, distribute throughout the entire endochorion (Figure 1-5h). Similarly,immunolocalizationof the dec-2 proteins and their derivatives showed that they contribute to all three proteinaceous layers of the eggshell (i.e. vitelline membrane, innermost chorionic layer and endochorion) and seem to play a crucial role in the stabilizationof these layers (Waring,2000).Moreover, it became apparent from these studies that the main products of the dec-2 locus (Noqueron and Waring, 1995), as well as some vitelline membrane proteins undergo stage specific extracellularproteolyticprocessing and stage specificsequestration and maturation (Pascucciet al., 1996; Noqueron et al., 2000) during chorion assembly. Interestingly, dec-2 homologs have recently been isolated from the olive fruit fly Ducus oleue (Douroupi, F, pers. comm.) demonstrating that the unique functions of the locus are conserved among dipteran species. These complicated distribution patterns revealed unexpected dynamics, complex extracellular processing events and molecular trafficking of the various components between the different eggshell layers and provided definite evidence of the existence of intercalation events during eggshell formation in Drosophilidae. Chorion proteins intercalation has also been demonstrated in the huge chorion of the lepidopteran Antherueu polyphernus by autoradiography, where it was shown that a substantial proportion of the chorion proteins reach their final destination by permeating a previously constructed framework (Blau and Kafatos, 1978).

1.7.2

Hardening of the Eggshell

Following all these rather complicated intercalation events seen during eggshell formation the final step in producing a fully functional assembled eggshell is the process of its insolubilization(or "hardening"). Severalmodes of eggshell hardening (occurringseparatelyor in combination)have been evolved in insect eggs, including di-sulphide bridges, di-, tri-tyrosine bonds and sclerotization through quinone tanning. The chorions of eggs of Coleoptera and silkmoths are stabilized by disulphide and hydrogen bonds, while in dragonflies (Odonata) eggshell hardening seems to require di-sulphidebonds in chorion and di-tyrosine in vitellinemembrane (Margaritis, 1985a). In higher Diptera [i.e. Drosophila species (Margaritis, 1985c) Cerutitis cupitutu (Mouzakiand Margaritis, 1991a)or Ducus oleae (Margaritis, 1985b) and lower Diptera (i.e.Aedes aegypti (Han et al., 2000b)) chorion hardening occurs mainly through eggshell peroxidase mediated di-, tri- tyrosine bond formation among the chorion constituent proteins. Since no eggshell peroxidase activity was detected in the vitelline membrane of Drosophila we can assume that the conserved motifs of the vitelline membrane proteins could provide a structural conformation that is favourable to the formation of cysteine bridges (Scherer et al., 1988).It has also been suggested that Drosophilu eggshell layers could be stabilized through glutamyl-lysine cross-links of the dec2 locus derivatives or the chorion proteins catalyzed by transglutaminases

Nurse Cells and Follicle Cells Programmed Cell Death

(Noqueron and Waring, 1995).In Aedes aegypti, an additional biochemical event, namely phenol oxidase-dopa decarboxylase catalyzed chorion melanization, contributes to the formation of a rigid and protective chorion (Li et al., 1996).

1.8

Nurse Cells and Follicle Cells Programmed Cell Death

Programmed cell death, or apoptosis, denotes a genetically regulated process occurring mainly during development in most, if not all, organisms as a way of removing unwanted cells. The major effector molecules of the apoptotic program in D. melanogaster are eight cystein proteases (or caspases) (Vernooy et al., 2000). On the other hand, three negative regulators of apoptosis DIAP1, DIAP2 and Deterin (DIAP: Drosophila Inhibitor of Apoptosis) have been found in Drosophila (Hay et al., 1995; Jones et al., 2000), all being differentially expressed in a stagespecificpattern during oogenesis (Foleyand Cooley, 1998).Programmed cell death of D. rnelanogaster nurse cells is a developmentally regulated asynchronous phenomenon that normally occurs during late oogenesis following completion of nurse cell dumping and it is probably stimulated by the massive depletion of the apoptosis-inhibitingfactors (i.e. DIAPl and DIAP2) from the nurse cells due to the rapid transport of nurse cell cytoplasm into the oocyte (Nezis, 2000). In some cases, however, apoptosis can be activated even if the cytoplasm is retained in the nurse cells (Cavaliere et al., 1998).At late stage 12 the depleted nurse cells reorganize their actin cytoskeleton and eventually degenerate, displaying typical apoptotic features such as nuclear condensation (Figure1-5i)and DNA fragmentation (Figure 1-5j)followed by phagocytosisfrom the neighboring follicle cells (Foleyand Cooley, 1998; Nezis et al., 2000). An additional pattern of in vivo nurse cell apoptosis occurs sporadically and is spontaneously induced during the early oogenic stages 7 and 8, representing a potent protective mechanism throughout Drosophila oogenesis that results in programmed cell death of damaged, spontaneously mutated or superfluous follicles (Nezis et al., 2000). Interestingly, early nurse cell apoptosis (during check point stages 7-9) depends on the female mating status (Soller et al., 1999) and can be induced either by destruction of the follicle cell layer integrity (Chao and Nagoshi, 1999)or by altering the juvenile hormone and 20-hydroecdysonehormone balances (Solleret al., 1999).On the basis of morphological observationsduringlate oogenesis in higher Diptera (Trougakos,IP and Margaritis LH, unpubl. results) and the recent demonstration of identical nurse cell programmed cell death patterns (Figure 5k, 51) and underlying cytoskeleton alterations during Dacus oleae (Diptera) oogenesis (Nezis et al., 2001), we suggest that nurse cell apoptosis represents a highly conserved phenomenon in the meroistic polytrophic ovary. Follicle cells, on the other hand, must also be specifically eliminated from the follicle following eggshell formation, through a genetically regulated process, since mature eggs that have been deposited carry no follicular epithelium and follicle cell degeneration in various dipteran species is apparent following the completion of oogenesis (Trougakos and Margaritis, unpubl. results). Interestingly, Nezis et al.

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(2000)did not observe massive folliclecell apoptosis in D. rnelunoguster mature stage 14 follicles. Thus, up to now no specific molecular mechanism has been proposed as being responsible for follicle cell apoptotic death and it is not known where exactly (in the ovary or during oviposition) the apoptotic death and phagocytosis of the follicle cells occurs.

1.9

Concluding Remarks

During the past two decades significant progress has been achieved in our understanding of egg structure and the developmental physiology of various insect orders, while a growing number of recent studies have elucidated many of the molecular pathways that regulate insect oogenesis. Although the bulk of the existing data (especially at the molecular level) still refer to D. rnelanoguster, data from other insect orders are rapidly accumulating. Additional data from phylogenetically divergent specieswill help to address many outstanding questions that remain to be solved. Briefly, future efforts should include further analysis of (1)the supramolecular assembly of the eggshell components and layers (these layers are directly exposed to the physical and biotic pressures associated with oviposition substrates), (2) the intracellular sorting and secretion mechanisms used by the follicle cells, (3) the crystalline layer components and organization, (4) the apoptotic pathways expressed during late oogenesis, (5) the molecular mechanisms of folliclecell migrations, communication and pattern formation, (6) the genes that trigger differential eggshell-related morphogenic activities in follicle cell subpopulations, (7) the development of egg polarity, as well as (8) hormonal vitellogenesis regulation and the vitellogenin receptors. It is certain that the answers to these questions will be the basis for more exciting developments in many basic biological aspects of cellular and developmental biology. Moreover, such studies could also help to understand how insect females incorporate toxins into the eggs (see Chapter 3) and symbiotic microorganisms onto and into the eggs (see Chapter 6), while in parallel they will provide more insight into the ecology of insect eggs and their progeny.

1.10 Ac knowledegment s We are grateful to all our colleagues in Prof. L. H. Margaritis’ laboratory for communicating unpublished results and contributing original illustrations and to Drs J. Trueman, B. Stark, G. Chauvin and R. Barbier for the contribution of valuable illustrations of their work. Prof. L. H. Margaritis wishes also to express his gratitude to all current and former colleagues, collaborators and undedpost-graduate students (F. Kafatos,M. Kambysellis,W. Petri, D. Brandon, M. Cans, S. Hamodrakas,

References

K. Komitopoulou, I. Papassideri, V. Galanopoulos, N. Messini, F. Zarani, D. Mouzaki, M. Kalantzi-Makri, K. Keramaris, I. Trougakos, F. Douroupi, I. Nezis, R. Konstanti, D. Stravopodis, N. Santama, V. Petrou, I. Mamali, G. Dranos, G. Stefas and G. Apostolou)who have contributed extensively, for almost 30 years, to some of the results and ideas discussed and who have shared the excitementof analysing insect egg structure and physiology. We hope colleagues will accept our sincere apologies for omitting, due to space limitations,many citations andor illustrations of important contributions to the field. Our published or unpublished data have been supported by numerous research grants, the current ones being grants to L.H.M. from the Special Account for Research Grants of Athens University and from the European Union (TMR Network, Contract No: ERBFMXCT-980200).

1.11 References Bilinski, S. 1979. Ultrastructural study of yolk formation in Porcellio scaber Latr. (Isopoda). Cytobios. 26: 123-130. Bilinski, SM, Larink, 0.1989. Fine structure of the egg envelope and the supporting stalk in the dipluran Campodea (Apterygota: Campodeidae). Int. J. Insect Morphol. Embryol. . . 18: 199-204. Blau, HM, Kafatos, FC. 1978. Secretory kinetics in the follicular cells of silkmoths during eggshell formation. J. Cell Biol. 78: i31-151. Bownes, M. 1994. The regulation of the yolk protein genes, a family of sex differentiation genes in Drosophila melanogaster. BioEssays 16: 745-752. Brennan, MD, Weiner, AJ, Goralski, TJ, Mahowald, AP. 1982.The follicle cells are the major site of vitellogenin synthesis in Drosophila melanogaster. Dev. Biol. 89: 225-236. Cavaliere, V, Taddei, C, Gargiulo, G. 1998. Apoptosis of nurse cells at the late stages of oogenesis of Drosophila melanogaster. Dev. Genes Evol. 208: 106-112. Cave, MD. 1982.Morphological manifestations of ribosomal DNA amplificationduring insect oogenesis. In: King, RC, Akai, H. (eds.) Insect Ultrastructure. Vol. 1, Pp. 86-117. Plenum Press, New York. Chao, S, Nagoshi, RN, 1999. Induction of apoptosis in the germline and follicle layer of Drosophila egg chambers. Mech. Dev. 88: 159-172. Chauvin, G, Barbier, R. 1972,Permkabilitk et ultrastructures des oeufs de deux Lkpidoptkres Tineidae: Monopis rusticella et Trichophaga tapetzella. J. Insect Physiol. 18: 1447-1462. Chauvin, G, Barbier, R. 1979. Morphogenese de l'enveloppe vitelline, ultrastructure du chorion et de la cuticule serosale chez Korscheltellus lupulinus L. (Lepidoptera: Hepialidae). Int. J. Insect Morphol. Embryol. 8: 375-386. Cho, WL, Deitsch, KW, Raikhel, AS. 1991. An extraovarian protein accumulated in mosquito oocytes is a serine carboxypeptidase activated in embryos. Proc. Natl. Acad. Sci. USA 88: 10821-10824. DeLoof, A. 1971. Synthesis and deposition of oocyte envelopes in the Colorado beetle, Leptinotarsa decemlineata Say. Z. Zellforsch. 115: 351-360. DeLoof, A. 1983. The meroistic insect ovary as a miniature electrophoretic chamber. Comp. Biochem. Physiol. 74A: 3-9. Deng, WM, Bownes, M. 1998: Patterning and morphogenesis of the follicle cell epithelium during Drosophila oogenesis. Int. J. Dev. Biol. 42,541-552. Dobens, LL, Raftery, LA. 2000. Integration of epithelial patterning and morphogenesis in Drosophila ovarian follicle cells. Dev. Dynam. 218: 80-93. Edwards, MJ. 1996. The vitelline membranes of Aedes aegypti and Drosophila melanogaster: a comparative review. Inv. Reprod. and Develop. 30: 255-264. Edwards, MJ, Severson, DW, Hagedorn, HH. 1998. Vitelline envelope genes of the yellow fever mosquito, Aedes aegypti. Insect Biochem. Mol. Biol. 28: 915-925.

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37

Chapter 2

Insect Access0 ry Reproduc t ive G Ia nds : Key Players in Production and Protection of Eggs Cedric Gillott

Table of Contents 2.1 2.2 2.2.1 2.2.2 2.3 2.3.1 2.3.1.1 2.3.1.2 2.3.1.3 2.3.2 2.3.2.1 2.3.2.2 2.3.2.3 2.3.2.4 2.3.2.5 2.3.2.6 2.3.2.7 2.4 2.5 2.6

Introduction Development and Structure of Accessory Reproductive Glands (= ARC) Male ARC Female ARC Functions and Biochemistry of ARC Products Male ARC Products Fecundity-EnhancingSubstances (= FES) Receptivity-Inhibiting Substances (= RIS) Other Male-Transferred Substances Female ARC Products Egg Coverings Egg Adhesives Chemical Egg Protectants Call Initiators Venoms and Polydnaviruses Oviposit ion Pheromones Other Products of Female ARC Concluding Remarks Acknowledgements References

Abstract

Accessory reproductive glands (ARG) (defined as secretory components of the internal reproductive system whose products play a role in a species’ reproductive strategy) have an array of forms, reflecting the morphological diversity of the Insecta. In addition to specialized collateral glands, all parts of the reproductive tract may have glandular cells. The secretions produced by ARC have a wide variety of both pre- and post-copulatory functions, including nourishment of gametes, packaging and transport of sperm, induction of behavioural and physiological changesin the mated female (notablychanges in female receptivity and oviposition behaviour), protection, transfer, and attachment of fertilized eggs to a substrate or

38

Insect Accessory Reproductive Glands: Key Players in Production and Protection of Eggs

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host, gall initiation, and manipulation of host by parasitoids. This review will first summarize the structure and development of male and female ARG. It will then examine current knowledge of the functions and biochemical nature of ARG products that affect the production, deposition and survival of eggs.

2.1

Int roduction

As a key event in the life history of insects, egg deposition must occur at the right time and place to optimizethe offspring’s chances of survival.Firstly,because sperm entry occurs as an egg passes along the common oviduct en route to the exterior, a female must be “aware” that she has been inseminated before oviposition is induced. The eggs must be protected from desiccation, predation, and parasitism. Further, as most juvenile insects are relatively immobile, it is imperative that the eggs are deposited on or adjacent to a food source. Sometimes, it is necessary to space out eggs so as to avoid having too many conspecific juveniles in close proximity when resources are limited.For parasitoids, there are particular problems. Notably, the host’s physiology must be controlled so that it does not reject the invader and provides a suitable milieu for the developing parasitoid (Beckage,1997, 1998). From the male perspective, mechanisms that ensure paternity of the offspring have evolved. Thus, any copulation-induced mechanism that increases the rate of egg development, promotes egg laying soon after mating, plugs the female’s reproductive tract preventing further sperm entry, or renders a female unwilling to remate, will increase a male’s chances of paternity (compare Chapter 4). In most insects the solution to the requirements noted above involves the participation of secretionsproduced within the reproductive system. The glandular regions andor discrete tubular diverticula, whose products play a direct role in the reproductive process, are collectivelycalled accessory reproductive glands (ARG). In females, secretions of the ovaries, lateral oviducts, common oviduct and bursa copulatrix may affect egg deposition and protection. In males of some species, testicular and ejaculatory duct secretions may have a role in oviposition. And for both sexes, secretions of discrete paired tubular structures, the collateral glands, may play major roles in egg production, deposition and protection. Encompassing reviews of ARG have been provided by Chen (1984),Gillott (1988),and Kaulenas (1992). These reviews focus on the structure and development of ARG and the functions of ARG secretions such as maintenance and transport of sperm (seminal fluid and spermatophore production), sperm capacitation, and sperm activation within the female reproductive tract. The present chapter summarizes former and recent studies of the morphology, development, physiology, and biochemistry of male and female ARG with special respect to functions of the ARG for egg deposition.

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2.2

Development and Structure of Accessory ReproductiveGlands

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Development and Structure of Accessory Reproductive Glands (= ARC)

2.2.1 Male ARC As noted above, the testes, ejaculatory duct and collateral glands may produce materials that influence egg deposition. The testes arise from mesoderm or primordial germ cells. The ejaculatory duct in most species is an ectodermal structure, typically a single median tube. However, in some exopterygotes and lower endopterygotes, the structure may bifurcate anteriorly. In other insects where the ejaculatory duct is bifurcated, the anterior arms are formed from the backward growth of mesodermal material (Matsuda, 1976). The collateral glands of the great majority of male insects are mesodermal (and known as mesadenia), developing from the swollen posterior end of the embryonic vasa deferentia. However, in those groups in which there is anterior bifurcation of the ejaculatory duct, the collateral glands may be ectodermal (ectadenia) or may include both mesadenial and ectadenial components. Collateral glands are primitively absent in male Thysanura, Ephemeroptera, most Odonata, Plecoptera and Dermaptera, and secondarily lost in many Diptera (where their functions may be taken over by the ejaculatory duct). The male collateral glands remain undeveloped until the final juvenile stage when, under the influence of striking changes in the juvenile hormone (JH)ecdysteroid balance, they grow and differentiate.Their final form is almost as diverse as the morphology and habits of the insects themselves (Figure 2-1). Indeed, they have been used occasionally in taxonomic studies ( e g , Singh, 1978).In most species the collateral glands are uni-paired (Figure 2 - 1 D); ~ ~in others they are bi- or tripaired (Figure2-lb). In most Acrididae there are 16 pairs of tubules (Figure2-la), and the conglobate glands of cockroaches comprise very numerous tubules. Collateral gland tubules of mesodermal origin have been the subject of numerous histological and fine-structural studies (reviews by Happ, 1984; Gillott, 1988; Kaulenas, 1992). A typical tubule comprises a one-cell thick epithelial layer, resting on a basal lamina, outside which is a muscle layer of varied composition. The glandular epithelium has the characteristics of cells producing protein for export, namely, large amounts of rough endoplasmic reticulum, many Golgi complexes, generally a large nucleus with prominent nucleoli, and an apical membrane much folded into microvilli. Within this general pattern, however, individual tubules, regions, or even cells may have their own distinct characteristics. By contrast, studies of ectadenia are comparatively rare. However, tubules of the conglobate gland of Periplunefuamericuna have the fine structure typical of insect ectodermal glands; that is, each secretory unit includes a central secretory cell, in the core of which is a cuticular end apparatus. Leading from the end apparatus is a cuticle-lined duct generated by the epidermal cell that also produces the intima of the tubule (Beams et al., 1962). During sexual maturation, the collateral glands accumulate secretory material within their lumen. In most species the synthesis of secretory material is under

39

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, ,

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,.

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

.

C

Figure2-1 Representativemalereproductivesystems(nottosca1e) toshowvarietyofcollateral gland forms. (a) Melanoplus sanguinipes (Orthoptera); (b) Lytta nuttali (Coleoptera); (c) Anagasta kuhniella (Lepidoptera);(d) Drosophila mehogaster (Diptera). In M. sanguinipes 16 pairs of tubules make up each collateral gland (CG). There are4 white tubules (WT), 10 short hyaline tubules (SHT), and a long hyaline tubule (LHT). The sixteenth tubule serves as a seminalvesicle(SV).InL.nuttallitherearethreetubulesineachcollateralgland;a spiral tubule (SpT), short tubule (ST), and a long tubule (LT). Otherabbreviations: CED, cuticular ejaculatory duct; EB, ejaculatory bulb; ED, ejaculatory duct; LVD, lower vas deferens; MED, mesodermal ejaculatory duct; T, testis; TF, testis follicle; UVD, upper vas deferens; VD, vas deferens. (a Original; b from Gerber, GH, Church, NS, Rempel, JG. 1971. The anatomy, histology, and physiology of the reproductive systems of Lyfta nuttalli Say (Coleoptera: Meloidae). 1. The internal genitalia. Can. J. 2001.49: 523-533. By permission of the National Research Council ofcanada; cfrom a diagram supplied byDr.J.G. Riemann;dfrom Kubli, E. 1996.TheDrosophila sex-peptide: A peptide pheromone involved in reproduction. Adv. Dev. Biochern. 4: 99-128. By permission of JAI Press Inc.)

h o r m o n a l control. Probably, b o t h J H a n d p-ecdysone are i n v o l v e d a n d regulate the synthesis of specific proteins within the secretion. However, their molecular site of action remains unclear (see Gillott, 1996).

2.2.2

Female ARC

The ovaries, lateral a n d c o m m o n oviducts a n d collateral glands m a y produce secretions that are i m p o rta n t ine g g laying. The ovaries arise during embryogenesis from either somitic m e s o d e rm or p r i m o r d i a l g e r m cells. In m o r e p r i m i t i v e

Development and Structure of Accessory Reproductive Glands

a

Figure 2-2 Representative female reproductive systems (not to scale). (a) Melanoplus sanguinipes (Orthoptera);(b) Rhodnius prolixus (Hemiptera);(c) Periplaneta americana (Dictyoptera);(d) Nasonia vitripennis (Hymenoptera).In M . sanguinipes the collateral glands (pseudocolleterialglands [PCG]) are extensions of the lateral oviducts (LO).In P. americana the left colleterial gland (LCG) is much larger than the right (RCG).Nasonia vitripennis has collateral glands (CG), a venom gland (VG) and a Dufour’s gland (DG).Other abbreviations: BC, bursa copulatrix; CA, calyx; CO, common oviduct; OV, ovariole; SP, spermatheca; SPG, spermathecal gland; VGR, venom gland reservoir. (a, c, d Originals; b from Ruegg, RP. 1981. Factors influencing reproduction in Rhodnius prolixus (Insecta:Hemiptera).PhD Thesis. York

University, Canada.) exopterygotes, the lateral oviducts develop solely from mesodermal material; however, in more advanced exopterygotes and lower endopterygotes, the mesodermal component tends to be replaced by the growth cephalad of the common oviduct. The latter develops either as an ectodermal invagination usually between the eighth and ninth sterna or, in advanced endopterygotes, from imaginal discs in the posterior abdominal segments (Matsuda, 1976). Collateral glands are found in female Thysanura, many exopterygotes and most endopterygotes. They are absent from female Ephemeroptera, Orthoptera, Plecoptera, Psocoptera,Heteroptera, and most Coleoptera (Matsuda, 1976).Where

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Insect Accessory ReproductiveGlands: Key Players in Production and Protection of ERRS

Figure 2-3 A secretory unit in the cement gland of Rhodnius prolixus. This arrangement is typical of most ectodermally derived accessory reproductive glands. Abbreviations: CL, cuticular lining of cement gland; D, ductule; DC, ductule cell; EA, end apparatus; SC, secretory cell (from Lococo, D, Huebner, E. 1980.The ultrastructure of the female accessory gland, the cement gland, in the insect Rhodnius prolixus. Tissue Cell 12:557-580. Reproduced by permission of Harcourt Publishers Ltd.).

/cL D /

DC

they occur, the collateral glands are almost always ectodermal, arising either as evaginations of the common oviduct or from imaginal discs. Exceptionally, the collateral glands of grasshoppers and locusts (Acridoidea: Orthoptera) develop as anterior extensions of the lateral oviduct and are thus mesodermal (Figure 2-2a). In Hydrophilidae (Coleoptera),the collateral glands are highly modified ovarioles (Hinton, 1981). Growth and differentiation of the female reproductive tract, including collateral glands, are regulated by the interplay of JH and ecdysteroids, principally at metamorphosis when major changes in the titres of these hormones occur. There is some evidence that JH, at least, is important in controlling the synthesis and/or accumulation of secretory material within the glandular tissues (see Gillott, 1988). Mature female collateral glands take on a range of forms (Figure 2-2). In most insects they are paired structures that open into the common oviduct caudad to the spermathecal opening. In other species they are bi- or multipaired structures, though the tubules usually fuse prior to entering the common oviduct. In cockroaches the left gland is much larger than the right gland (Figure 2-2c). Typically, female collateral glands are referred to as colleterial or cement glands, when their secretion is used for coating the eggs and/or as a glue for sticking the eggs to a substrate. In tsetse flies, which are viviparous, the collateral glands are known as ”milk glands” on account of the nutrient fluid they produce. In most aculeate Hymenoptera, there are distinct forms of collateral glands: the venom gland and Dufour’s gland (Figure 2-2d).

Functions and Biochemistrv of ARC Products

Despite this variety of morphology, and the array of secretionsthat they produce (see Section 2.3.2), the glands have a remarkably consistent ultrastructure. There appear to be two distinct patterns. In the simpler form there is a single layer of secretory epithelial cells lying beneath a thin cuticular intima and set on a basal lamina. The apical cytoplasm of the cells is formed into numerous microvilli. More commonly, the epithelial component includes two or more distinct cell types that form a secretory unit. Each unit comprises columnar cells that contain a cuticlelined end apparatus and produce the accessory gland secretion, and one or more surrounding cells that synthesize the overlying intima and the duct running from the end apparatus (Figure2-3).The internal architectureof the secretory cells reflects their principal role of synthesizing materials (principallyproteins) for export, with much rough endoplasmic reticulum and many Golgi complexes in evidence. Exceptionally, cells of the Dufour’s gland contain only a few Golgi complexes and free ribosomes, their cytoplasm being filled with much smooth endoplasmic reticulum, numerous mitochondria, and membranous inclusions. These features are associated with the production and export of aliphatic hydrocarbons, reflecting the pheromone-producing role of these structures (Cavil1 and Williams, 1967; Jackson et al., 1989).

2.3

Functions and Biochemistry of ARC Products

Secretions of the ARG are reported to have a range of functions in relation to egg production and deposition. However, for only a few specific systems have biochemical characterization and experimental studies of individual components been undertaken. This is not a criticism of the studies carried out on other parts of the reproductive system; rather, it is intended to emphasize the difficulty of investigating such microstructures.

2.3.1

Male ARG Products

Functions of the male ARG products associated with egg production include enhancement of fecundity (the lifetime number of eggs laid), stimulation of ovulation or oviposition, induction of behavioural and physiological mechanisms to guarantee paternity, and protection of the deposited eggs. 2.3.1.1 Fecundity-EnhancingSubstances (= FES)

The first demonstration of a FES was by Kummer (1960)in the paragonia (collateral glands) of Drosophilu melunoguster. Since then, the existence of FES in representatives of Orthoptera, Lepidoptera, Hemiptera, and Coleoptera, as well as in other Diptera has been confirmed (see Gillott and Friedel, 1977; Gillott, 1988). In most species studied the source of the FES is the collateral glands, though in species lacking these structures ( e g ,M u m domesticu) the ejaculatory duct is the site of production. In a few species the gonads appear to produce an FES, though it is usually unclear whether the source is the sperm per se or glandular tissue within the testes. Identification of these organs as the sites of FES production was established

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Insect Accessory Reproductive Glands: Key Players in Production and Protection of Eggs

................................................. ................................................................................................,,,,,.,............ ,,,,,,,.............

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

,

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principally by experiments in which the tissues were implanted, or homogenates were injected, into the body cavity of virgin females whose egg production then increased to levels near that of mated individuals (see Gillott, 1988). It should be recognized that such experiments are subject to two criticisms: 1. The ARG substances enter the female in a non-natural way rather than by the reproductive tract; and 2. homogenization of ARG mixes materials that, under normal circumstances, would not meet, opening the possibility that the results may be due to unnatural causes. Biochemical studies have revealed that the FES are typically either peptides (MW 0.5-3 kilodaltons) or proteins (MW 13-100 kilodaltons). The “sex peptide” of D. melanogaster contains 36 amino acid residues. It is synthesized as a 55 amino acid precursor that includes a 19 amino acid hydrophobic signal sequence at the Nterminus. Although other species of Drosophila have peptidic FES, these have distinct amino acid sequences, and it is only within sibling species groups that cross reactivity can occur (see Chen, 1991,1996; Kubli, 1992,1996).For example, injection of FES of D.funebris does not stimulate egg laying in virgin D. melanogaster, and vice-versa (Chen, 1991). Complementing the work of Chen, Kubli and colleagues has been a series of studies on the genes that regulate production of D. melanogaster paragonial secretionsby Wolfner and colleagues (seeWolfner, 1997; Wolfner et al., 1997).More than 20 Acp genes (Accessory gland protein genes) have been identified, one of which codes for a prohormone-like protein Acp26Aa. The Acp26Aa, a 36-41 kilodalton glycoprotein (Park and Wolfner, 1995),appears to be a second Drosophila FES. It is transferred during mating, then cleaved in the female tract (by other Acp gene products) to become an egg-laying stimulant (see below). In Aedes aegypti a protein ”matrone” has been identified, separable into a and p fractions, with molecular weights of 60 and 30 kilodaltons, respectively. Early work indicated that a-matrone was a FES, but later studies showed that matrone exerts many and varied effects. It is likely that reanalysis would lead to the identification of additional active components in the collateral gland secretion.Indeed, Klowden (1999)believes that the term matrone has outlived its usefulness because it implies a single component is mediating the diverse effects of male mosquito collateral gland material. In Melanoplus sanguinipes and Locustu migratoria the FES are 60 and 13 kilodaltons, respectively. In the crickets Acheta domesticus and Teleogryllus commodus the FES is an enzyme (or complex of enzymes),prostaglandin synthetase, which when transferred to the female reproductive tract promotes the production of prostaglandins, the precursors for which are also male-derived (references in Gillott, 1988; Yi and Gillott, 1999). A potential FES of a totally different chemical nature is JH. Several studies have reported that the collateral glands of male moths and mosquitoes synthesize and store large amounts of JH (see Borovsky et al., 1994; Park et al., 1998).Clarification of the role of the transferred JH is awaited, though naturally it is tempting to speculate that, like endogenously produced JH in female insects, it will enhance egg production (and influence receptivity).

Functions and Biochemistry of ARC Products

potentially, there are several modes of action for the FES, including stimulation of egg development and egg laying, so that over the adult female’s life span, more eggs are produced. Some early reports provided circumstantial evidence that the FES promotes egg development, though it remained unclear whether the effect was direct (on the ovary) or indirect ( e g , modulation of digestion, vitellogenin production in the fat body, hormone release, or ovarian uptake of vitellogenins). In D. melunoguster sex peptide specifically enhances synthesis of yolk proteins by the ovarian follicle cells, though as whole flies were used, it is not possible to say precisely where the molecule acts (Soller et al., 1997). In Helicoverpu zeu, also, accessory gland material appears to promote egg maturation as well as oviposition (Bali et al., 1996). By far the most commonly demonstrated effect of FES is the stimulation of egg laying. Many authors (see Gillott, 1988; 1995)have reported that injection of male ARG material (typicallytissue homogenates but occasionally the purified FES) into the female body triggers egg laying in virgin females. For species that lay eggs singly or a few at a time, the FES promotes ovulation, oviposition occurring soon after. In species that lay eggs in batches, ovulation may occur some time before egg laying, the mature eggs being stored temporarily in the lateral oviducts. In this situation the FES induces oviposition, specifically contraction of the oviductal muscles. However, the manner in which FES exert these ovulation- or ovipositioninducing effects varies among species. In Rhodnius prolixus and Hyulophoru cecropiu the FES stimulates the female reproductive tract to release a hormone that causes the neuroendocrine system to produce a myotropic factor. The latter triggers contractions of either the ovarian sheath or the oviduct. In crickets, the prostaglandin-synthetase complex stimulates the wall of the female reproductive tract to produce prostaglandins that induce oviposition (see Gillott, 1988).In other species, the FES passes through the wall of the reproductive tract into the haemolymph to act elsewhere. The sex peptide of Drosophilu appears to be multifunctional (see also Section 2.3.1.2). It may act within the brain (on neurosecretory cells?)to induce ovulation (Kubli,1992,1996)and also stimulates JH production by the corpora allata (Moshitzky et al., 1996).Wolfner (1997) has speculated that while sex peptide may thus promote egg production, Acp26Aa may be a true myotropin, stimulating contraction of the reproductive tract. Myotropic factors have been purified from male ARG material of locusts, grasshoppers and Munducu sextu (see Yi and Gillott, 2000). However, in these species these factors cannot be the entire story as mating (hence transfer of FES) is not always followed immediately by egg laying. Circumstantialevidence suggests that the FES primes the brain to release a myotropin at the appropriate time (when mature eggs are in the lateral oviducts).Perhaps the role of ARG myotropic factors is to modulate the action of the brain myotropin (Yi and Gillott, 2000).

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Insect Accessory ReproductiveGlands: Key Players in Production and Protection of Eggs ..........................................................................................................................................................................................................

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2.3.1.2 Receptivity-InhibitingSubstances (= RIS)

Advantages accrue to both sexes when, as a result of mating, the female becomes refractory, that is, unwilling to remate. For the successful male, paternity of the fertilized eggs is assured. For the female, it may cause a switch in behaviour, from mate-seeking to food- and oviposition-site location. For some insects, the physical presence of a spermatophore in the bursa induces refractoriness. In others, notably > 20 species of Diptera and some moths, a RIS originating in the collateral glands, ejaculatoryduct, or testes induces the switch in female behaviour (see Gillott, 1988). In H. zea and probably some other moths the RIS causes both “calling” and pheromone production to cease (Kingan et al., 1993). Fan et al. (2000) reported that D. rnelunogaster sex peptide inhibited in vitro pheromone synthesis by the pheromone glands of H. armigera, suggesting that the glands may be a direct target for putative sex peptide-likefactors.However, calling was not terminated in moths injected with sex peptide. As noted in Section 2.3.1.1, caution is urged in the interpretation of data from experimentsinvolvingimplantation of ARG or injection of whole ARG extracts into the female’s body cavity. Chemically, the RIS are similar to, sometimes identical with, the FES; that is, they are peptides or small proteins. Thus, the sex peptide of D. rnelunogaster serves as both a FES and a RIS (Chen et al., 1988).Likewise, the RIS and FES of M. domestica may be the same molecule. Both ct and p matrone were originally reported to be necessary to induce refractoriness in A. aegypti. However, this is at odds with more recent work showing that receptivity in A. aegypti (Lee and Klowden, 1999) and Culex tarsalis (Young and Downe, 1987) is inhibited by peptides with molecular weights of 7,600 and c. 2000, respectively. The site and mode of action of RIS are not well understood for any species. In M. domestica experiments involving decapitation, decerebration, or neck ligation of virgin flies strongly suggest the brain as the site for RIS action (Leopold et al., 1971). This is also likely for the sex peptide of Drosophila (Kubli 1992,1996). Ottiger et al. (ZOOO), using radiolabelled sex peptide, have shown that the molecule binds strongly to a number of sites in the nervous system, particularly afferent connections. This suggests that sex peptide may modulate sensory input to exert its receptivity-inhibiting function. It should be noted, however, that whereas injection of sex peptide induces only a transient (24 h) inhibition of receptivity, mating renders females refractory for 7-9 d. Thus, it is likely that the presence of sperm and/or seminal fluid in the spermatheca is also important in regulating receptivity, as originally suggested by Manning (1962). Confirmation of the importance of the spermatheca in regulating refractory behaviour has been shown by the elegant work of Hartmann and Loher (1996, 1999) on the grasshopper Gornphocerus rufus. It was observed that injection of minute quantities of white tubule secretion directly into the spermathecal bulb of receptive females caused these insects to become refractory (Hartmann and Loher, 1996). In contrast, material placed directly in the body cavity did not affect receptivity. Hartmann and Loher (1999) reported the existence of a large number

Functions and Biochemistryof ARC Products

of bristles at the entrance of the spermathecal duct, some of which are morphologically similar to contact chemoreceptors. White tubule 1 has been identified as the source of the RIS; however, it seems that the active component is initially bound to a protein, and it is only after proteolysis within the spermatheca that it can stimulate the chemoreceptors and induce refractory behaviour (Hartmann and Loher, 1999). 2.3.1.3 Other Male-Transferred Substances

In addition to, or instead of, using RIS as a means of guaranteeing paternity, male insects may use other ARG materials to improve the chances that eggs are fertilized by their sperm. The large spermatophore produced by some species, notably Orthoptera, may remain in situ for several days, thereby preventing other males from mating. Another strategy is to produce a mating plug from secretions of the collateral glands which harden in the lower part of the female reproductive tract. Such structures have been reported in Orthoptera, Lepidoptera, mosquitoes, and Apis melliferu (see Gillott, 1988).While offering temporary protection against subsequent insemination, the plugs typically dissolve or are expelled by the female within a day or two, and perhaps a more plausible function is to prevent sperm loss. A more subtle way of ensuring paternity involving male ARG substances has recently been demonstrated at the level of sperm competition in D.melanoguster. The protein Acp36DE, cotransferred with sperm during insemination, ensures that sperm accumulate in the spermathecae and seminal receptacle (Neubaum and Wolfner, 1999).Sperm from Acp36DE-deficientmutant males cannot be properly stored and are thus rapidly lost (Chapman et al., 2000).A further mating, to a normal male, will thus ensure that the second male’s sperms are used in fertilization (compare also Chapter 4). In some species chemicals supplied by the male during insemination are used to provide the eggs with protection against predators, parasites and pathogens. This phenomenon is covered in Chapter 4.

2.3.2

Female ARC Products

A variety of egg laying-related functions have been ascribed to products of the female ARG. Unfortunately, with rare exceptions,the detailed studies seen for some male ARG systems have no parallel in investigations of female ARG. Commonly, several functions may be ascribed to a given secretion, though no attempt has been made to study the nature and role of individual components. 2.3.2.1 Egg Coverings

Insects from many orders coat their eggs with material produced in the collateral glands (seeHinton, 1981).This coatingis presumed to provide eggs with protection against predators, parasites, microorganismsand, perhaps, desiccation. The coating may range in nature from a soft jellylike material to a hard dry froth or a discrete thin-walled but hard case.

47

48 Insect Accessory Reproductive Glands: Key Players in Production and Protection of Eggs ................................... ................................... .................................. ............................... ......................... ................................................................... .........................................................................................

.

Cockroaches and mantids deposit their eggs within a thin-walled case (cockroaches)or a hardened mass of froth (mantids) known as an ootheca, formed from secretions of the collateral glands. The mechanical and biochemical events leading to ootheca formation have been studied in oviparous cockroaches where there is a single pair of collateral glands, the left being much larger than the right (Figure 2-2c). Each gland is composed of many individual tubules. The left gland produces the structural materials for ootheca production and the enzyme phenoloxidase; the right gland secretes only the enzyme, P-glucosidase. Within the left gland, each tubule is differentiated into at least two regions with respect to the materials produced. The distal region secretes protocatechuicacid glucoside (a phenolic precursor), structural proteins and calcium oxalate, while the proximal region releases the phenoloxidase.During ootheca formation,secretions from both glands are mixed into a thick paste that is moulded into shape by the ovipositor. The protocatechuic acid glucoside is first hydrolysed by the P-glucosidase into glucose and protocatechuic acid. The latter is then oxidized by the phenoloxidase, forming an orthoquinone which can crosslink the amino residues of the structural proteins. There are at least eight structural proteins in the secretion (Adiyodi, 1968; Pau et al., 1971),and though their individual roles have not been studied, collectively they contain a high proportion of tyrosine residues. Oxidation of the tyrosines could enable autotanning to occur, in addition to the crosslinking involving the orthoquinone. The role of the calcium oxalate, which is found only in the oothecae of oviparous species, remains obscure. It may strengthen the ootheca or provide antibiotic protection (Stay et al., 1960).In oviparous species the ootheca is typically hard and dark in colour.In contrast, in ovoviviparous and viviparous species, which carry the ootheca in a brood sac, it is thin-walled and soft, and may not fully enclose the eggs. Comparative studies on the biochemistry of ootheca formation in ovoviviparous and viviparous species have been neglected. Preliminary studies of ootheca formation in mantids suggest that the process is similar to that in cockroaches.In mantids there is one pair of long tubular and one pair of short tubular glands, which correspond in function to the left and right glands, respectively, of cockroaches.Crude extracts of cockroach right gland cause melanization of extracts of mantid long tubules, and vice versu. Also, an extract of mantid right gland hydrolyses the glucosidein the cockroach's left gland, indicating the presence of a glucosidase in mantid right gland. However, mantids incorporate calcium citrate rather than calcium oxalate into the ootheca.The citrate is produced in the form of crystals in a separate accessory gland that lies beneath the left and right collateral glands. In Acridoidea (short-horned grasshoppers and locusts) the frothy mass that encloses the eggs is formed from the secretion of the lateral oviducts, including their anterior extensions, the collateral glands (Figure2-2a).Lauverjat (1965)showed histochemicallythat the secretion contained a variety of amino acid types, together with polyphenols and mucopolysaccharides. However, no biochemical studies of froth formation have taken place. Oothecae resembling those of cockroaches are produced from secretions of the

Functions and Biochemistryof ARC Products

collateral glands in certain beetles (Chrysomelidae, subfamily Cassidinae). The oothecae are typically attached to the host plant and may contain many or a few eggs. In some cassidines, especially those in which the ootheca contains only few eggs, faecal particles are incorporated. In other chrysomelid subfamilies,ovariolar pedicels, calyces and lateral oviducts appear to produce the egg coating (Hinton, 1981; Hilker, 1994). Eggs of the blister beetle Lyttu nuttulli are held within a loose clump of a frothy mucilage produced, not by the collateral glands, but by the epithelium of the oviducts and calyces (Gerber et al., 1971). No information is availableon the chemical nature of this froth, though it is reported to be hygroscopic and may prevent desiccation of the eggs (Sweeny et al., 1968).In Hydrophilidae, femalesprotect their eggs in a silken structure, the egg cocoon. The silkis produced by highly modified ovarioles that have taken on a glandular function. As the liquid silk is released from the vagina, it is pulled by the female’s styli and spun into the thread from which the cocoon is formed (Hinton, 1981). Plecoptera,some Odonata,Ephemeroptera,Chironomidae(Diptera),Trichoptera, and some Coleoptera coat their eggs with a gelatinous secretion produced by the collateral glands. This material, spumaline, swells on contact with water. Typically, females of these groups lay their eggs on an underwater substrate. However, some Trichoptera attach their eggs to overhanging vegetation, vegetation in dried up seasonal pools, or rarely, as in Enocylu pusillu which has terrestrial larvae, damp moss. In these species the spumaline is hygroscopic. Also, the alder leaf beetle, Agelusticu ulni (Chrysomelidae, Galerucinae), lays eggs not on an underwater substrate, but on leaves of alders that usually grow close to water. The gelatinous substance covering the egg mass of A. alni effectively protects the eggs against predators and parasitoids by gluing up the mouth parts or legs of the attackers (Hilker, personal observation). Embryonic development and even hatching may occur within the spumaline. Hinton (1981) suggests that the spumaline imprisons the larvae until such time as there is abundant moisture to soften the material. Escape at this time will enhance the chances of the larvae encountering a suitable body of water. Almost nothing is known of the chemical nature of spumaline. Hinton (1981)cites data indicating that the spumaline contains <0.1% protein on a dry weight basis, with complex polysaccharides apparently the major constituents. Some Lepidoptera also encase their eggs in a polysaccharide-rich spumaline (Chew & Robbins, 1984). 2.3.2.2 Egg Adhesives

Production of a cement (also referred to as spumaline) for attaching eggs to each other and to the substrate occurs in several orders of insects, including Hemiptera, Phthiraptera, Neuroptera, Mecoptera, Lepidoptera, Diptera, Hymenoptera, and Coleoptera (Hinton, 1981).The collateral glands are the presumed site of cement production, hence the commonly used alternate name “colleterial” glands. However, the evidence for this function is largely circumstantial. For example, in Lepidoptera it includes comparison of gland size between species laying eggs ”loosely”and species laying eggs in large masses, the absence of colleterial glands

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Insect Accessory Reproductive Glands: Key Players in Production and Protection of Eggs .. . . .................................

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in speciesthat simply drop their eggs in flight, and histochemicalsimilarity between colleterial gland contents and the material covering laid eggs. In the sunflower beetle, Zygogramma exclamationis (Chrysomelidae), secretion from the oviducts sticks the eggs to the plant (Gerber et al., 1978).Virtually nothing is known with respect to the chemical makeup of the cement. A little work done more than 40 years ago indicates that the colleterial gland secretion in Lepidoptera is a mixture of protein and lipid (Hinton, 1981). 2.3.2.3 Chemical Egg Protectants

Eggs of numerous species of Lepidoptera, Hemiptera, Orthoptera, and Coleoptera contain toxins (Hinton, 1981; Hilker, 1994). These toxins either may have been transferred from the male to the female during mating (see 2.3.1.3, this chapter, and also Chapter 4) or may be provided by the female (see Chapter 3).In numerous species the female produces the toxins de novo and incorporates them into the eggs as, for example, leaf beetles of the genus Chrysolina that produce cardiac glycosides de novo from dietary steroids (Van Oycke et al., 1997). Females of other species sequester plant toxins and transfer them into or onto the eggs. For example, the arctiid moth Utetheisa ornatrix sequesters pyrrolizidine alkaloids from its host plants. These plant-derived alkaloids are also present in the eggs (Dussourd et al., 1988). The leaf beetle Pyrrhalta viburni uses toxic triterpenes (amyrins) from the host plant (Viburnum spp.) and deposits them in a faecallayeronto the eggs (Hilker, 1992). Amyrins are well-known as emetics for vertebrates and may thus protect the eggs from being eaten by birds. 2.3.2.4 Call Initiators

Especially in several Hymenoptera, gall induction may result from oviposition (Shorthouse and Rohfritsch, 1992).Less certain is whether induction is due to the incision made by the female prior to oviposition, by substances of the egg surface, or the collateralgland fluid secreted with the egg. These questionswill be addressed in Chapter 8 of this book. 2.3.2.5 Venoms and Polydnaviruses

In the Hymenoptera and Diptera are a large number of parasitoids that lay their eggs on or within the body of their host. Among the potential challenges that an ovipositing parasitoid faces are preventing the host from moving into an inhospitable environment, suppressing the host’s immune response to an egg deposited internally, integrating the host’s and the larval parasitoids development, and ensuring that metabolites are readily available to the developing parasitoid (Vinson and Iwantsch, 1980).For ectoparasitoids, it is also critical that shedding of the cuticle does not occur once the eggs have been deposited on the host’s integument. Except in Braconidae and Ichneumonidae (see below), hymenopteran venom produced in the collateral glands and injected at ovipositioninduces a multiplicity of responses to ensure that the above challenges are met. In ectoparasitoid species

Functions and Biochemistry of ARC Products

the initial action of the venom, which may be injected at consistent sites in the host’s body, is generally to permanently paralyse the host. In some endoparasitoids the venom has a paralysing effect that is almost immediate, but lasts only a very short time, facilitating oviposition. However, in egg parasitoids and most endoparasitoids paralysis does not ensue. Thus, a post-embryonic host continues to move and feed, obtainingresources that can be used by the developingparasitoid. In these endoparasitoids, the venom affects the host’s immune, endocrine and metabolic systems (Beckage, 1993, 1997, 1998; Coudron, 1991; Lawrence and Lanzrein, 1993; Strand and Pech, 1995). Some evidence suggests that proteins in the venom of some endoparasitoids are similar enough to those produced by the host that when they coat the parasitoid egg, the egg is not recognized as foreign and therefore is not encapsulated. Alternatively, the venom proteins may directly attack the haemocytes involved in encapsulation, causing them to either lyse or lose their ability to spread over the antigenic surface (Coudron, 1991; Strand and Pech, 1995). Additionally, venom may alter levels of JH and P-ecdysone, so that the host no longer is able to initiate a moult cycle. It remains unclear how venom acts, though Weaver et al. (1997)have shown that venom proteins of Eulophus pennicornis cause the ecdysteroidpeak seen in non-parasitized host Lucunobiu oleruceu (tomato moth) to be delayed until it is too late to induce a moult cycle.Venom also induces major changes, both qualitative and quantitiative, in proteins, carbohydrates and lipids, with concomittant alterations to normal body weight gain, morphology, and appearance of host tissues (Vinson and Iwantsch, 1980).However, it remains unclear whether venom acts directly or indirectly on the affected tissues. Considerableeffort has been put into elucidation of the sites and modes of action of the polydnavirus/venom system. To date, this system has been found only in Braconidae and Ichneumonidae, which are endoparasitoids of eggs and immature stages of a wide range of insect hosts, especially Lepidoptera and symphytan Hymenoptera, including many major pests. Polydnaviruses have segmented, double-stranded DNA genomes, and are transferred vertically through germ line tissue as proviral DNA integrated in the parasitoids chromosomalDNA (seeWebb, 1998). However, the polydnaviruses replicate only in nuclei of epithelial cells in the calyx region of the parasitoid ovary. Virus particles accumulate in the calyx fluid and, together with venom, are injected into the host along with an egg. Following injection of the polydnavirus/venom into a host larva, three major effects may be observed, namely, suppression of the host’s immune response, perturbation of hormone levels, and changes in the production, concentration and storage of metabolites (Stoltz, 1993; Lavine and Beckage, 1995). To date, most attention has been focussed on the activities of the polydnavirus which, in some systems, can induce all the effects observed in the absence of venom. The polydnavirus typically inhibits encapsulation of the parasitoid egg and reduces the spreading ability of the host haemocytes. Further, in some systems, there is a reduction in haemocyte numbers and changes in haemocyte morphology and ultrastructure (Stettler et al., 1998).Generally, it is found that venom enhances the

51

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Insect Accessory Reproductive Glands: Key Players in Production and Protection of Eggs ...

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

..,.........................

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,,

.,,..............................................................

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effect of the virus, by facilitating the entry and survival of the virus in the haemocytes. A second feature of the polydnavirus/venom system is that it typically prevents the host from moulting. Thus, much attention has been focussed on changes in the host’s endocrine system induced by the polydnavirus and venom (Lawrence and Lanzrein, 1993). In some species the polydnavirus alone can arrest development; in others both virus and venom are necessary, and occasionally both components plus teratocytes (specializedparasitoid embryonic cells) are required. Effects on both JH and ecdysteroids in the host system have been observed, including elevated JH titres, decreased JH-esterase activity, decreased sensitivity or degeneration of the prothoracic gland, lowered ecdysteroid levels, and inhibition of prothoracicotropic hormone (PTTH) synthesis or secretion (Lawrence and Lanzrein, 1993). Currently, most attention seems to be focussed on the PTTHprothoracic gland axis which is upstream of the other effectsnoted, though it should be stressed that the polydnavirus/venom complex could have several sites of action (Grossniklaus-Burginet al., 1998; Pennacchio et al., 1998).Pennacchio et al. (1997, 1998) suggest that in the Cardiochiles nigriceps/Heliothis virescens system, the polydnavirus disrupts the PTTH signal transduction pathway at the prothoracic glands, specifically at the level of the CAMP-dependent protein kinase. Further, they have obtained evidence for the occurrence of protein kinase inhibitor-like genes within the polydnavirus genome. The polydnavirus/venom system induces major changes in the host insect’s metabolism and quality and quantity of specific nutrients. The changes fall into two categories: (a) alterations to levels and storage of existing host metabolites, and @) production of parasitism-specific materials (Beckage, 1993; Stoltz, 1993). Several studies have shown that parasitized hosts have elevated levels of haemolymph trehalose and proteins, the latter arising from failure of the fat body to act as a storage depot. This presumably makes the nutrients more readily available to the developing parasitoid (Stoltz,1993).Parasitic castration (premature gonad atrophy) may be another strategy for diverting nutrients to the benefit of the parasitoid, though it is not clear whether this is induced by the polydnavirushenom system (Junnikkala, 1985).The appearance of novel proteins in the haemolymph of polydnavirus/venom injected hosts has been demonstrated in many studies (Beckage, 1993). Some of these proteins may appear late in the parasitoids life within the host, so that involvement of teratocytes or production by the parasitoid per se cannot be ruled out. However, others can be detected very early in parasitoid development, implying that polydnavirus genome transcription is responsible. For example, in the Cumpoletis sonorensislH. virescens system, polydnavirus-specific transcription begins as soon as 2 h after oviposition and continues for up to 9 d (Fleming and Krell, 1993). The multiple effects of the polydnavirus imply that the viral particles can enter a variety of host tissues in order that the viral genome can be expressed, but evidence for this is scarce. Strand (1994) observed electron microscopically that polydnavirus entered all types of haemocytes. Though polydnavirus has not yet

Functions and Biochemistry of ARC Products 53 ............... .. ................. . ..................... . ................. . .................. ............................................................................................................................... ................................................... ..................... .

been seen in other tissues, changes in size and structure of the prothoracic glands of parasitized hosts have been reported (Pennacchioet al., 1997;Cusson et al., 2000), and Varricchio et al. (1999) reported that a polydnavirus gene was expressed in the prothoracic glands of mature H. virescens larvae parasitized by C. nigriceps. 2.3.2.6 Oviposition Pheromones

Pheromones released by egg-laying females to inform conspecifics about the suitability of the oviposition site are known both in herbivorous and carnivorous insects (see Chapters 9,lO and 12).In several species, glandular cells of the female reproductive tract are known to be the site of production of these oviposition pheromones. For example, the oviposition-stimulatingpheromone of the sandfly Lutzomyiu longipulpis, dodecanoic acid (Dougherty and Hamilton, 1997),is produced in the collateral glands (Dougherty et al., 1992). It has been suggested that this type of pheromone would lead females to valuable resources, may reduce predation and parasitism on the offspring of individual females, reduce the risk of desiccation and facilitate synchronized mass emergence and therefore reproductive success (McCall and Cameron, 1995).A pheromone that attracts conspecific gravid female desert locusts (Schistocercu greguriu) to a common egg-laying site is released from the egg-pod froth produced by the collateral glands (Sainiet al., 1995).Such group oviposition, perhaps induced by similar pheromones, is seen in other locust species and assists in ensuring the temporal and spatial cohesiveness of the offspring populations in these gregarious insects. In Pieris brussicue, the oviposition-deterring pheromone has long been thought to be associated only with the collateral gland secretion attaching the eggs to the cabbage leaf (Rothschildand Schoonhoven, 1977).Indeed, Blaakmeer et al. (1994b) identified three avenanthramide alkaloids (miriamides) in the collateral gland secretion of P. brussicue and suggested that these are components of the ovipositiondeterring pheromone. However, Blaakmeer et al. (1994a)showed that oviposition deterrency is retained by the plant even after egg batches are removed. Thus, Blaakmeer et al. (1994a)hypothesize that the host plant produces an allomone in response to egg deposition, while the original purpose of the miriamides may be to protect the eggs against predators or fungal infection. 2.3.2.7 Other Products of Female ARC

It has been known for some time that, in response to feedingby herbivorousinsects, the host plant may release volatile chemicals (synomones) that attract parasitoids and predators of the herbivore (e.g., Karban and Baldwin, 1997; Agrawal et al., 1999).However, only recently has it been shown that an herbivore’s oviposition activity may exert a similar effect. Thus, the field elm (Ulmus minor) releases a synomone (of unknown identity) when the elm leaf beetle (Xunthogulerucu luteolu) oviposits on the leaf surface. The synomone attracts the eulophid egg parasitoid Oomyzus gul2erucue (Meiners and Hilker, 1997). Meiners and Hilker (2000) have confirmed that the elicitor is a component of the oviduct secretion which glues

54 Insect Accessory Reproductive Glands: Key Players in Production and Protection of Eggs ....................... . .................................. .................................. ........................... ..................................................................... ................................................................. ................

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eggs to the leaf. A synomone is also present in the oviduct secretion of the pine sawfly, Diprion pini, which elicits the emission of volatiles by the host tree, Pinus sylvestris. The volatiles attract the eulophid egg parasitoid, Chrysonotornyia ruforum (Hilker et al., 2002) (see Chapter 8). Antibacterial peptides are produced by the accessory glands of the medfly, Cerutitis capitata (Marchini et al., 1993).The peptides, named ceratotoxins A and B, are expressed only in sexually mature insects (Rosetto et al., 1996) and were presumed initially to protect the genital tract from infection during insemination. However, Marchini et al. (1997)have shown that the peptides are released on the egg’s surface during oviposition. Thus, a role for the peptides in egg protection seems more likely (see Chapter 6). For members of a large number of insect orders, there are reports of the existence of symbiotic intracellular microorganisms (endocytobiotes)that are presumed to supply the insect partner with supplementary nutrients such as vitamins and amino acids (see Schwemmler and Gassner, 1989).Generation-to-generationtransmission of the endocytobiotes may occur in various ways. Notably, in some Coleoptera, the female collateral glands serve as repositories for the endocytobiotes which are smeared over the eggs during oviposition (De Marzo and De Stradis, 1994). The progeny are infected when they eat the egg shell at hatching. Hagele et al. (2000) have shown that the accessory glands of crowded female desert locusts produce an agent that induces gregariousbehaviour in the hatchlings of such females.Thus, ligation of the glands or washingfreshly-laideggs of crowded females results in a shift in the behaviour of hatchlings towards solitarious characteristics.Conversely,application of an extract of the egg-pod foam (produced by the accessory glands) on eggs restores gregariousness in the hatchlings (McCaffery et al. 1998).

2.4

Concluding Remarks

This review has demonstrated that, despite their rather uniform cellular ultrastructure,the ARG produce an array of secretions,for a wide range of functions. Some of these functions (e.g., egg protectants and cements) are common across diverse insect orders, while others (most obviously, the polydnavirus/venom system) may be restricted to a few groups within a single order. Also striking is the ”imbalance”in attention that has been paid to discrete functions. At one extreme (e.g., for the sex peptide and other paragonial products of Drosophila), considerable progress has been made in the resolution of the nature and mode of action of the secreted materials. Yet, for some widespread functions (e.g., egg cements and coverings), there is little information on the biochemistry and formation of the components. In the former situation, much use has been made of modern analytical and molecular genetic techniques. There is no reason why similar methods cannot also be applied to help us understand these more ”mundane”, yet equally important, processes related to egg laying.

References

2.5

Acknowledgement s

I wish to thank Dr. Monika Hilker for her original invitation to contribute this chapter, the writing of which has opened my mind to previously unfamiliar, yet fascinating, areas of insect egg biology. I thank also many colleagues, too numerous to mention individually, who have provided information and suggestions about the content of the chapter. Thanks go to Dr. Martin Erlandson who commented on the chapter, Mr. Dennis Dyck for assistance in preparing the figures, and Mrs. Joan Virgl who largely prepared the reference list. Original work of the author cited in this review was supported by grants from the Natural Sciences and Engineering Research Council of Canada.

2.6

References

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Insect Accessory Reproductive Glands: Key Players in Production and Protection of Eggs 1996. Sex peptide activates juvenile hormone biosynthesis in the Drosophilu melunogaster corpus allatum. Archs Insect Biochem. Physiol. 32: 363-374. Neubaum, DM, Wolfner, MF. 1999. Mated Drosophilu melunoguster females require a seminal fluid protein, Acp36DE, to store sperm efficiently. Genetics 153: 845-857. Ottiger, M, Soller, M, Stocker, RF, Kubli, E. 2000. Binding sites of Drosophilu rnelunoguster sex peptide pheromones. J. Neurobiol. 44: 57-71. Park, M, Wolfner, MF. 1995. Male and female cooperate in the prohormone-like processing of a Drosophilu melunoguster seminal fluid protein. Devl Biol. 171: 694-702. Park, YI, Shu, S, Ramaswamy, SB, Srinivasan, A. 1998. Mating in Heliothis virescens: Transfer of juvenile hormone during copulation by male to female and stimulation of biosynthesis of endogenous juvenile hormone. Archs Insect Biochem. Physiol. 38: 100-107. Pau, RN, Brunet, PCJ, Williams, MJ. 1971. The isolation and characterization of proteins from the left colleterial glands of the cockroach, Periplunetu umericunu (L.). Proc. R. SOC. B. 177: 565-579. Pennacchio, F, Falabella,P, Sordetti, R, Varricchio, P, Malva, C, Vinson, SB. 1998.Prothoracic gland inactivation in Heliothis virescens (F.) (Lepidoptera: Noctuidae) larvae parasitised by Cardiochilesnigriceps Viereck (Hymenoptera: Braconidae).J. Insect Physiol. 44: 845-857. Pennacchio, F, Sordetti, R, Falabella, P, Vinson, SB. 1997. Biochemical and ultrastructural alterations in prothoracic glands of Heliothis virescens (F.) (Lepidoptera: Noctuidae) last instar larvae parasitised by Curdiochiles nigriceps Viereck (Hymenoptera: Braconidae). Insect Biochem. Molec. Biol. 2 7 439450. Rosetto, M, Manetti, AGO, Giordano, PC, Marri, L, Amons, R, Baldari, CT, Marchini, D, Dallai, R. 1996. Molecular characterization of ceratotoxin C, a novel antibacterial femalespecificpeptide of the ceratotoxin family from the medfly Cerutitis cupitutu. Eur.J. Biochem. 241: 330-337. Rothschild, M, Schoonhoven, LM. 1977. Assessment of egg load by Pieris brussicue (Lepidoptera: Pieridae). Nature 266: 352-355. Saini, RK, Rai, MM, Hassanali, A, Wawiye, J, Odongo, H. 1995. Semiochemicals from froth of egg pods attract ovipositing female Schistocercu gregariu. J. Insect Physiol. 41: 711-716. Schwemmler, W, Gassner, G (eds.).1989.Insect Endocytobiosis:Morphology, Physiology, Genetics, Evolution. CRC Press, Boca Raton. Shorthouse, JD, Rohfritsch, 0 (eds.). 1992. Biology of Insect-Induced Gulls. Oxford University Press, New York. Singh, T. 1978. The male accessory glands in Bruchidae (Coleoptera) and their taxonomic significance. Entomol. Scand. 9: 198-203. Soller, M, Bownes, M, Kubli, E. 1997. Mating and sex peptide stimulate the accumulation of yolk in oocytes of Drosophilu melunoguster. Eur. J. Biochem. 243: 732-738. Stay, B, King, A, Roth, LM. 1960. Calcium oxalate in the oothecae of cockroaches. Ann.. Entomol. SOC.Am. 53: 79-86. Stettler, P, Trenczek, T, Wyler, T, Pfister-Wilhelm, R, Lanzrein, B. 1998. Overview of parasitism associated effects on host haemocytes in larval parasitoids and comparison with effects of the egg-larval parasitoid Chelonus inunitus on its host Spodopteru littoralis. J. Insect Physiol. 44: 817-831. Stoltz, DB. 1993.The polydnavirus life cycle. In: Beckage, NE, Thompson, SN, Federia, BA (eds.) Parasites and Pathogens ofInsects. Vol 1(Parasites).Pp. 167-187. Academic Press, San Diego. Strand, MR. 1994. Microplitis demolitor polydnavirus infects and expresses in specific morphotypes of Pseudoplusiu includens haemocytes. J. Gen. Virol. 75: 3007-3020. Strand, MR, Pech, LL. 1995. Immunological basis for compatibility in parasitoid-host relationships. Ann. Rev. Entomol. 40: 31-56. Sweeny, PR, Church, NS, Rempel, JG, Gerrity, RG. 1968. The embryology of Lyttu viridunu LeConte (Coleoptera: Meloidae). 111. The structure of the chorion and micropyles. Can. J. Zool. 46: 213-217. Van Oycke, S, Randoux, T, Braekman, JC, Daloze, D, Pasteels, JM. 1987. Cardenolide biosynthesis in chrysomelid beetles. Experientia 43: 460462. Varricchio, P, Falabella, P, Sordetti, R, Graziani, F, Malva, C, Pennacchio, F. 1999.Curdiochiles nigriceps polydnavirus: Molecular characterization and gene expression in parasitised Heliothis virescens larvae. Insect Biochem. Molec. Biol. 29: 1087-1096.

References

Vinson, SB, Iwantsch, GF. 1980.Host regulation by insect parasitoids. Q. Rev. Biol. 55: 143-165. Weaver, RJ, Marris, GC, Olieff, S, Mosson, JH, Edwards, JP. 1997. Role of ectoparasitoid venom in the regulation of haemolymph ecdysteroid titres in a host noctuid moth. Archs Insect Biochem. Physiol. 35: 169-178. Webb, BA. 1998. Polydnavirus biology, genome structure, and evolution. In: Miller LK, Ball, LA (eds.) The Insect Viruses. Plenum, New York. Wolfner, MF. 1997. Tokens of love: Functions and regulation of Drosophila male accessory gland products. Insect Biochem. Molec. Biol. 27: 179-192. Wolfner,MF, Harada,HA, Bertram,MJ, Stelick,TJ,Kraus,KW,Kalb, JM,Lung,YL,Neubaum, DM, Park, M, Tram, U. 1997. New genes for male accessory gland proteins in Drosophila melanogaster. Insect Biochem. Molec. Biol. 27: 825-834. Yi, S-X, Gillott, C. 1999. Purification and characterization of an oviposition-stimulating protein of the long hyaline tubules in the male migratory grasshopper, Melanoplus sanguinipes. J. Insect Physiol. 45: 143-150. Yi, S-X, Gillott, C. 2000. Effects of tissue extracts on oviduct contraction in the migratory grasshopper, Melanoplus sanguinipes. J. Insect Physiol. 46: 519-525. Young, ADM, Downe, AER. 1987. Male accessory gland substances and the control of sexual receptivity in female Culex tarsalis. Physiol. Entomol. 12: 233-239.

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Chapter 3 Chemical Protection of Insect Eggs Murray 5. Blum and Monika Hilker

Table of Contents 3.1 Introduction 3.2 Defensive Components of Intrinsic Origin 3.2.1 Autogenously Produced Defensive Components Applied onto the Eggs 3.2.1.1 Toxic Hairs Covering the Eggs 3.2.1.2 Defensive Secretions Coating the Eggs 3.2.2 Autogenously Produced Defensive Components within the Eggs 3.2.2.1 Cantharidin Produced by Meloid and Oedemerid Beetles 3.2.2.2 Oleic Acid as Repellent 3.2.2.3 Reactive Anthraquinones as Defensive Allomones 3.2.2.4 Self-produced Cardenolide Diversity 3.2.2.5 Autogen o us ly Produced Cya noglycosides as Pro-Toxins 3.2.2.6 Alkaloidal Deterrents Produced de novo 3.2.2.7 lsoxazolinones as Chrysomelid Defensive Components 3.2.2.8 Betaine in Eggs of the “Femmes Fatales” 3.2.2.9 Pederin as Endosymbiotic Product 3.2.2.10 Cajin - a Toxic Polypeptide in Ardia caja 3.3 Defensive Components of Extrinsic Origin 3.3.1 Chemically Defensive Plant Material Covering Eggs 3.3.2 Sequestered Defensive Components within the Eggs 3.3.2.1 Cantharidin Sequesteredfrom Prey 3.3.2.2 Cucurbitacins - Bitter Triterpenes in Diabroticite Beetles 3.3.2.3 Salicin -Toxins in Eggs and Precursorsfor Larval Defence 3.3.2.4 Bitter Aristolochic Acids 3.3.2.5 Abundant Cardenolides Sequesteredfrom the Host Plant 3.3.2.6 Lucibufagins Sequestered from Prey 3.3.2.7 Plant Derived Cyanogenic Components 3.3.2.8 N-Oxide Alkaloids as Pro-Toxins 3.3.2.9 Cycasin as a Nontoxic p-Glycoside 3.3.2.10 Mustard Oil in Eggs of Pieris 3.4 Chemical Defence and Egg Cannibalism 3.5 Concluding Remarks 3.6 Acknowledgements 3.7 References

62

Chemical Protection of Insect Eggs

Abstract

A dazzling variety of natural products, some of which are highly reactive (e.g., anthraquinones) or present as pro-toxins (e.g., cyanoglycosides),is known from insect eggs to constitute efficacious defensive activity against a wide range of predators, parasitoids, pathogens, or even against host immune responses in the case of endoparasitoid eggs. These protective agents may be synthesized de ~ O U O by either the mother or the father, or in some insect species contributed by both, after introduction into the female as a copulatory "bonus". On the other hand, the female may protect her eggs with natural products that are of dietary origin. Several species are known which load their eggs both with autogenously produced components and dietary sequestered ones. The protective chemicalscan be applied onto the eggs by e.g. hairs or secretions, or they may be incorporated into the egg yolk or egg shell. An overview of the protective chemicals associated with insect eggs with respect to their origin and efficacy will be given. Also egg cannibalism and its ecological significance particularly in toxic eggs will be considered.

3.1

Introduction

Insect eggs are not mobile. They cannot flee in the presence of a hungry oophage. Once placed on the substrate by the mother, the egg is vulnerable to attack by a variety of predators whose numbers can dramatically increase by recruitment if, for example, clustered eggs are discovered by foraging ants. Indeed, a large variety of predators have been identified as oophages and these include vertebrate and invertebrate animals. Especially, birds, ants, Hemiptera, coccinellid adults and larvae, syrphid fly larvae, and neuropteran larvae are frequently identified as egg predators (Pasteels et al., 1986). Also in spiders oophagy may be more common than usually thought (Nyffeler et al., 1990).Furthermore, eggs as immobile stages are exposed to numerous egg parasitoids which may be highly specialized and able to detect very well hidden eggs. Losses of insect eggs due to predation and parasitization are often high (Orians and Janzen, 1974). In addition to the risk of being consumed by carnivorous animals, eggs may also suffer from microbial disease, especially when laid in humid microhabitats where conditions for fungal growth are optimal. The vulnerability of the egg notwithstanding, it is nevertheless true that insect eggs may possess certain characteristicsthat can play a role in blunting the attacks of oophages and pathogens. Numerous insects are known to make their eggs less accessible for enemies by hiding them in plant tissue or by covering them with faeces, hairs, scales or secretion. Hinton (1981) describes a wide variety of egg coatings and coverings in an excellent and outstanding overview of the biology of insect eggs. However, in addition to very effectivephysical protective devices, eggs are also known to bring up a wide variety of natural products that act against their enemies. The growing embryo within the eggs has to be protected against the toxicity of natural products within the eggs. Such protection may be provided either

Introduction

Figure 3-1(a) Egg mass ofthe leaf beetle Castrophysa viridula. The eggs are brightly coloured and contain oleic acid (seeSection 3.2.2.2).(b) Eggs of the leaf beetle Cl'ra which are coated by pieces of faeces that function as a barrier. (c) Eggs of the leaf beetle Calerucella lineola which contain anthraquinones and carry pieces of faeces (see section 3.2.2.3).The function of the tinyfaecel pellets is unknown. (d) Eggs ofthe alder leaf beetle Agelastica alnicovered with a sticky secretion that immobilises a small parasitoid which is sticked to the secretion.

by adaptation of the embryo to the toxin or by incorporation of the toxin as pretoxin or by applying the toxins onto the egg surface. Aposematic colouring of eggs may signal egg toxicity to avoid predation and parasitization (Stamp, 1980)(Figure3-la). However, chemical signalling of toxicity has also been suggested for coccinellid eggs which contain toxic alkaloids inside but display a "toxin label" consisting of certain hydrocarbons on their surface (Hemptinne et al., 2000b).The presence of toxins in eggs has been suggested to be advantageous especially for eggs laid in clusters. Aggregated eggs frequently have a longer incubation period than singly laid ones (Stamp, 1980).Thus, their longer exposure to enemies might have favoured incorporation or application of noxious components for deterrence. Even though eggs laid in masses may provide a rich meal for oophages encountering them, such an egg "bonanza" may be denied if the eggs contain a high enough concentration of noxious components to deter the attacker after one or two eggs have been sampled (Stamp, 1980).However, pressure

63

64

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

Chemical Protection of Insect Eggs .....................

.

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

. ..........

.. .........

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

.

. ..........

from predation and parasitization is not the only selective force for laying eggs in clusters, as has been discussed by e.g. Stamp (1980)and Clark and Faeth (1998). This chapter is intended to give an overview of those natural products associated with insect eggs that may protect them against predators, parasitoids, microbial disease, and - in the case of endoparasitoids - against host immune responses. In several insects it is known that not only the mother but also the father contributes to egg defence by transfer of protective chemicals to the female during mating. These male nuptial gifts are addressed in detail in Chapter 4, but will also be briefly touched on in this chapter to complete the picture. The wide range of protective chemicals in insects eggs, ranging from terpenoids over steroids to alkaloids and other N-containing natural products, will be addressed here with respect to their efficacy and to the question whether they are produced autogenously by the insect or sequestered from their food plant or prey.

3.2

Defensive Components of Intrinsic Origin

Insects are able to produce autogenously a wide variety of natural products for defensive purposes (e.g. Blum, 1987).Also, protective components of the eggs are known to be produced de novo to render the eggs unpalatable or toxic to predators and parasitoids (Table 3-1).

3.2.1

Autogenously Produced Defensive Components Applied onto the Eggs

Numerous insects are known to cover their eggs with faecal matter (Figure 3-lb, c), hairs, scales or secretions from glands of the reproductive tract, e.g. the oviduct, the ovariole pedicel, or accessory (collateral)glands (compare Chapter 2; Hinton, 1981). These egg coverings or coatings often provide a physical barrier against enemies rather than a chemicalone. Even though the physically defensive function of such covering is often intuitively assumed rather than precisely proved, a few studies compare predation or parasitization of eggs with and without coverings (e.g. Damman and Cappuccino, 1991; Tsukada, 2000). 3.2.1.1 Toxic Hairs Covering the Eggs

Covering of eggs by hairs is well-known in numerous lepidopteran species. Lepidopteran adults emerging from the cocoon may take up the hairs from inside the cocoon where the larvae have released them (Kawamota and Kumada, 1984). If no noxious larval hairs are left in the cocoon, the covering of eggs with hairs just provides a physical barrier against small egg predators and parasitoids. However, in the cocoons of several lepidopteran species larvae have left venomous hairs which are well known to cause inflammation and severe irritation of human skin (e.g. Delgado Quiroz, 1978).While adults of many Lepidoptera lose the irritating hairs at emergence, females of a few taxa are able to take up the poisonous hairs and cover their eggs with them.

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

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

Defensive Components of Intrinsic Origin

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

Table 3-1 Defensive chemicals of intrinsic origin known from insect eggs. For references please see text. “Insect taxon”: for one or several species of the taxon listed the respective chemical has been detected in the eggs Origin of Compounds

Chemicals

Insect Taxon

Applied onto the eggs Irritating hairs

Histamine, enzymes

Lepidoptera: Lymantriidae: Euprodis, Acyphas Saturniidae: Hylesia Thau meto poeidae: Thaumetopoea

Secretion

Antibacterial peptides

Diptera: Tephritidae: Ceratitis capitata Neuroptera: Chrysopidae: Ceraeochrysa smithi

INTRINSIC ORIGIN

Aldehydes, fatty acids with deterrent activity against ants Immunosuppressive virus-like particles

65.

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

Numerous endoparasitoids of insect larvae

Within the eggs Cantharidin

Coleoptera: Meloidae Oederneridae Oleic acid Coleoptera: Gastrophysa cyanea Anthraquinones, Coleoptera: ant h rones Calerucinae, Galerucini Coccidae: e.g. Dactylopius confusus Cardenolides Coleoptera: Chrysomelidae: Chrysolinina Cya nogenic Lepidoptera: components Zygaenidae Nymphalidae: Acraeinae and Heliconius Coleoptera: Chrysomelidae: Paropsis atomaria Alkaloids Coleoptera: Coccinellidae lsoxazolinone derivates Coleoptera: Chrysomelidae: Chrysornelinae Betaine Coleoptera: Lampyridae: Photuris Pederin Coleoptera: (endosymbiotic origin) Staphylinidae: Paederus Toxic polypeptide Lepidoptera: Arctiidae: Ardia caja

Females of the lymantriid genera Euproctis and Acyphas cover their eggs with venomous hairs obtained from the cocoon (Pesce and Delgado, 1971; Kawamoto and Kumada, 1984).The hairs of Euproctis are known to contain histamine (Figure and enzymes such as esterase, protease, and phospholipase A2 (Bleumink 3-2, [l]), et al., 1982; de Jong et al., 1982; Kawamoto and Kumada, 1984).No studies on the

66

Chemical Protection of Insect Eggs

protective value of the hairy egg coverings in these species are available.In contrast, the chemistry and defensive value of noxious haemolymph components of lymantriid larvae, their osmeterial glandular secretion, and their ballon hairs in first instars have been studied intensively (Deml and Dettner, 1995,1997). Thaurnetopoea females (Lepidoptera) cover their egg masses mainly with scales, but a few poisonous larval setae may be among them (Hinton, 1981).Ziprkowski and Rolant (1972)reported that the thaumetopoeid venom consists of polyhalogen compounds, carbohydrates, phospholipids, and toxic proteins. Females of the saturniid Hylesia spp. also cover their eggs with poisonous larval setae (Hinton, 1981),however, the toxic chemicals are unknown. 3.2.1.2 Defensive Secretions Coating the Eggs

Secretions may just be smeared onto the eggs and make them so sticky that small predators or parasitoids attackmg the eggs are glued to them (Figure 3-ld). Also the mouthparts of oophages may be disabled by the egg glue. However, several insects build complex oothecae, for example mantids. They hide their eggs in a very elaborated ootheca formed by proteinous secretions of the collateral glands (Chapter 2; Hinton, 1981).In addition to such physical barrier function of secretions covering eggs, several examples of chemical defensive functions are known. Protection of eggs by antibacterial secretion coating the eggs is known in the tephritid Ceratitis capitata. The antibacterial activity is ascribed to so-called ceratotoxin peptids. The secretion is produced in the accessory glands and may be recovered from the egg surface as clustered droplets (Marchini et al., 1997).Also, the so-called miriamides detected in both the accessory glandular secretion and the eggs of Pieris brassicae have been suggested to have antifungal activity (Blaakmeeret al., 1994a,b), since the structurally related avenanthramides are known to be strong fungicides (Niemann, 1993).However, this suggestion still needs to be proved. In earwigs, the eggs are not known to be covered by an antibacterial secretion, but the females frequently manipulate the eggs with their mouthparts. This "licking" behaviour is important for egg survival, since eggs are attacked by mould when not treated by the females (Lamb, 1976) (compare Chapter 6). The egg stalks of the chrysopid Ceraeochrysa srnithi are coated by a protective secretion that repels ants (Eisner et al., 1996). For egg deposition, female Chrysopidae (Neuroptera) release a droplet of accessory glandular secretion from the end of the abdomen onto the substrate, The female then raises her abdomen from this droplet, thereby pulling a tiny stalk. The egg is placed onto the tip of this stalk (Jakobs and Renner, 1988).Several studies show that the egg stalkin chrysopids serve as protection against predation and also cannibalism (Chen and Young, 1941; Ruzicka, 1997).In C. srnithi the secretion was found to contain saturated aldehydes of different chain lengths ranging from butanal to tetracosanal in trace amounts (0.2 to 1.2 ng per stalk). Several of these aldehydes are known as defensive components of e.g. hemipteran secretions (Blum, 1981).Fatty acids such as myristic acid, palmitic, linoleic, oleic, stearic, and docasaenoic acid were found in larger amounts. About 30 ng of oleic acid were quantified per stalk (Eisner et al., 1996).

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

Defensive Components o f Intrinsic Origin

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

The vast majority of studies which have shown defensive functions of secretion on eggs address the question how parasitoid eggs avoid immune responses of the host. Several hymenopteran endoparasitoids attacking insect larvae coat their eggs with a secretion that prevents the encapsulation of their eggs by host blood cells and avoid subsequent melanization. The secretion from ovarian glands contains virus-like particles as immunosuppressants. The virus-like particles are known as polydnaviruses with viral DNA sequences integrated throughout the genome of the parasitoid. The process of virus replication takes places in the parasitoids ovary. When the parasitoid lays its egg in the host, the virions associated with the secretion of the eggs penetrate host tissues, including fat body and haemocytes. The viral infection may alter expression of host proteins or initiate expression of virus encoded proteins. Thus, host haemocytes may be destroyed or haemocyte functions changed so that they fail to spread and attach to the parasitoids egg. Thereby, encapsulation with haemocytes is prevented. The viral infection may also cause inhibition of host haemolymph peroxidase activity, thus avoiding formation of melanin (Beckage, 1997; 1998a,b, and references therein). However, the virus-like particles injected into the host larvae along with the parasitoids egg affect not only the immune system of the host but also larval development and feeding activity. Only eggs of larval parasitoids are known to be covered with a secretion containing virus-like particles. Egg parasitoids and parasitoids of adult insects lack the viruslike particles in their ovarian secretion (Beckage, 1998a) (compare also Chapter 2).

3.2.2

Autogenously Produced Defensive Components within the Eggs

3.2.2.1 Cantharidin Produced by Meloid and Oedemerid Beetles

Cantharidin is a unique monoterpene anhydride only known to be produced autogenously by blister beetles (Meloidae) and oedemerid beetles (Oedemeridae) (Figure 3-2, [2]).This chemical is highly toxic for numerous organisms due to its inhibitory activity towards protein phosphatases, mainly of type 2A (Knapp et al., 1998).It acts as feeding deterrent towards predacious insects such as ground beetles (Carrel and Eisner, 1974), spiders (Smedley et al., 1995/6), Heteroptera, and ants (Dettner, 1997).Racoons initially consume meloids, but then they quickly become reluctant to take them up (Carrel, 1999).However, other vertebrates such as frogs (Eisner et al., 1990; Kelling et al., 1990)and invertebrates such as the southern house spider Kukulcuniu hibernulis (Carrel, 1999)are not harmed by cantharidin and readily take up cantharidin-containing insects. The antifungal activity of cantharidin is considered in Chapter 6. Furthermore, insects of numerous families have even been shown to be attracted by cantharidin (see Section 3.3.2.1; Dettner, 1997; and references therein). In the meloids, males are mostly the ones who biosynthesize cantharidin from a methyl farnesoate intermediate (McCormick and Carrel, 1987), even though cantharidin synthesis was observed in both sexes of Epicnutu and Meloe larvae (Meyer et al., 1968; Carrel et al., 1993).Males of the genera Lyttu and Epicnuta are

67 ..... .

68

Chemical Protection of Insect Ems

0

I

&&&p&a 1 Histamine

OH

0

2 Cantharidin

OH

OH

3 Oleic acid

0

\

0

/

0

a

b

d

C

4 Anthraquinones (a,b). Anthrones (c.d) glucose

O+O

OH

n

a

8 lsoxazolinone a R=H b R=CO-CH2-CH2-N02

b

6 Prunasin(a) (R)-rnandelonitrile(b)

5 Sanentogenin glycoside

9 Betaine

7 Precoccinelline(a), Coccinelline(b)

10 Pederin

N-Methylquinolinium Zcarboxylale

Figure 3-2Examples of defensive egg componentsof intrinsic origin (i.e.produced de novo).

known to transfer cantharidin to the female with their sperm during mating (Sierra et al., 1976; Carrelet al., 1993;Dettner, 1997).The meloid females deposit cantharidin into the eggs, thus protecting their progeny. In essence, male-derived cantharidin is highly adaptive for the female since it enables her to dedicate her energetic resources to reproduction while utilizing the anhydride as a proven defensive shield for herself and the offspring. Thus, utilizing the semen as a copulatory "bonus" for transferring protective natural products to the female is clearly of great selective value (Blum, 1981). Both sexes of adult Oedemeridae produce cantharidin (Frenzel and Dettner, 1994; Holz et al., 1994). No transfer of male cantharidin to the females could be detected. However, the eggs also contain cantharidin, most probably provided by the female (compare Chapter 4). 3.2.2.2 Oleic Acid as Repellent

At a glance, the egg masses of the chrysomelid Gustrophysu cyuneu are eminently aposematic. The brilliant coloration of the eggs is identified with a high concentration of p-carotene,which however, is certainly not completelyresponsible

Defensive Components of Intrinsic Origin

for their pronounced repellency to ants (Howard et al., 1982a). It has been demonstrated that a very high concentration of oleic acid in the eggs functions as an effective feeding deterrent for the hungry formicids (Figure 3-2, [3]).A single egg of G. cynaea contains about 40 pg of oleic acid. In comparison, the eggs of the closely related species G. viridula only contain at maximum 0.065 pg of oleic acid (Howard et al., 1982a). 3.2.2.3 Reactive Anthraquinones as Defensive Allomones

Anthraquinones are regarded as both eminently toxic and reactive (e.g. Muller, 1988; Teuscher and Lindequist, 1994). Thus, it is rather surprising to encounter them as the defensive mainstay of chrysomelid beetles in the tribe Galerucini, subfamily Galerucinae. Anthraquinones and anthrones have been identified in eggs and larvae of species of the genera Xanthogaleruca, Galeruca, Galerucella, Pyrrhalta, Hydrogaleruca, and Lochmaea (Hilker and Schulz, 1991; Hilker, 1992; Hilker et al., 1992a; Howard et al., 1982b)(Figure3-2, [4a-d]).Trace amounts of these components were also detected in the haemolymph of adults. The anthraquinones and anthrones occurring in the Galerucini as well as its derivatives have been shown to act as feeding deterrents against both ants (Hilker and Schulz, 1991; Howard et al., 198213) and birds (Hilker and Kopf, 1995; Avery et al. 1997). Autogenously produced anthraquinones are unusual for insects. Apart from their presence in the Galerucini,they are only known in coccids (Eisneret al., 1980;Kayser, 1985;Thomson, 1987).Eggs and embryos of the coccid Dactylopius confusus contain a glycosidated anthraquinone, carminic acid, which deters ants from feeding (Eisner et al., 1980). In contrast, anthraquinones are very common in plants (Hegnauer, 1959; Thomson, 1987).However, the host plants of the Galeruciniand the coccids do not contain these components. Whether the anthraquinone-containing chrysomelids and coccids themselves have enzymes that enable them to produce anthraquinones or whether they harbour endosymbionts with these abilities is unknown up to now (Howard et al., 1982b). Anthraquinones have also been detected in the haemolymph of leaf beetles of the genus Timarcha (Petitpierre, 1995))but analysis of eggs has not been reported. Since the fluid of Timarcha eggs has the same bright orange-reddish colour typical for these anthraquinones as the haemolymph of the adults has, the presence of anthraquinones also in eggs of Timarcha is very likely. However, several Timarcha species feed upon plants containing anthraquinones (Rubiaceae,Galium spp.) and might sequester the anthraquinones from their host plants (Teuscher and Lindequist, 1994). 3.2.2.4 Self-Produced Cardenolide Diversity

For a long time, insect associations with cardenolides had been limited to insect species that feed on cardenolide-rich plants such as are found in species of Asclepiadaceae.Selectivesequestration of these compounds by aposematic species generally provided the conspicuous insects with a powerful chemical defence predicated on the availability of a deterrent cardenolide arsenal (compare Section

69

70

Chemical Protection of Insect Eggs

3.3.2.5).Numerous cardenolides are bitter tasting. They are also known to disrupt the ionic balance of differentanimal cells (Teuscherand Lindequist, 1994).However, the monarch butterfly has been shown to be insensitive towards dietary cardenolides (Holzinger and Wink, 1996). Cardenolide-containing insect prey often evokes emesis in predators (Brower, 1984). Herbivorousinsects are not only able to sequester cardenolides from food plants; some are known to be able to produce these defensive components autogenously. Severalchrysomelidsof the taxon Chysolinina have been recognized as cardenolide chemists. They biosynthesize the cardenolides by themselves by using plant phytosterols as precursors (Van Oycke et al., 1987).Adults release the cardenolides from exocrine defensive glands located on the pronotum and elytra. The eggs of Chrysolina coerulans, C. polita, and C.fuliginosa were shown to contain cardenolides (Daloze and Pasteels, 1979; Hilker et al., 1992b).In C. coerulans and C. polita, the pattern of cardenolides found in the eggs was not as complex as the one found in the adult secretion (Daloze and Pasteels, 1979).In C. fulginosa, the same cardenolides were detected in adult secretion and eggs (Hilker et al., 1992b) (Figure 3-2, [5]). Till now, no studies have examined whether the cardenolide concentration in the egg is sufficient to act as a successful predator deterrent. It remains to be determined whether the cardenolides in the eggs have a maternal origin rather than being synthesized by the embryo in the egg. 3.2.2.5 Autogenously Produced Cyanoglycosides as Pro-Toxins

In essence, cyanoglycosidesand cyanohydrins constitute pro-toxins that are capable of releasing very volatile toxins ( e g , HCN), on demand, as a consequence of the presence of P-glucosidases that hydrolyze cyanogens, or because of the instability of cyanohydrins. HCN acts as respiratory inhibitor in animals by inhibition of cytochrome oxidase (Davis and Nahrstedt, 1985; Teuscher and Lindequist, 1994). Eggs, larvae, pupae, and adults of the Australian chrysomelid Paropsis atomaria contain both the cyanogenic glycoside prunasin and the cyanohydrin (R)mandelonitrile which liberates about 80% of the HCN detected (Nahrstedt and Davis, 1986) (Figure 3-2, [6]).The beetle feeds upon Eucalyptus trees which do not contain the cyanogens. Thus, the cyanogenic components have been suggested to be produced by the beetles themselves. The pro-toxins present in the eggs provide a cyanogenic source for the larvae. The larvae defend themselves against enemies by releasing HCN from the exocrine secretion emitted by glands located on the 7th and 8th abdominal tergite. Eggs of several zygaenid moths such as Zygaena filipendula, Z . lonicera, Z. trifolii and Porcris geryon have been found to liberate HCN when crushed (Jones et al., 1962; Davis and Nahrstedt, 1985). These moths feed upon plants that contain cyanogenic components. However, Z. trifoliifeeding upon the cyanogenic food plant Lotus corniculatus (Fabaceae) has been shown to be able to gain cyanogenic components in two ways. This speciesis able to sequester the cyanogenicglycosides from the host plant and to biosynthesize them de novo from valine and isoleucine (Davis and Nahrstedt, 1985; Nahrstedt, 1988,1989).

Defensive Components of Intrinsic Origin

Also the eggs of nymphalid species of the Acraeinae subfamily (Brown and Francini, 1990)and Heliconius spp. (Davis and Nahrstedt, 1985)contain cyanogenic components that are biosynthesized by the moths. While the food plants (Passifloraceae)of the Heliconius butterflies contain cyanogenic components, host plants of the Amercian Acraeinae of the genera Actinote and Altinote are Asteraceae which do not contain cyanogenic components, but instead toxic pyrrolizidine alkaloids (PAS).However, no PAS are sequestered by these moths (Brown and Francini, 1990). A selective force driving herbivores to biosynthesize cyanogenic components by themselves instead of sequestering these or other toxins from the host plant may be emancipation from the availability of the toxins in the host plant. 3.2.2.6 Alkaloidal Deterrents Produced de nova Adult ladybird beetles have been proven to be a treasure trove of distinctive natural products which have often been demonstrated to be eminently efficacious as deterrents towards vertebrate and invertebrate predators (Frazer and Rothschild, 1961; Eisner et al., 1986;Aganvala and Dixon, 1992; Daloze et al., 1995; Hemptinne et al., 2000a). Alkaloids belonging to more than six chemical classes have been characterized in the haemolymph of these coleopterous “chemists” and in some cases these proven repellents have been introduced into eggs. Adults may release the toxins by reflex bleeding when disturbed. Even though a few of the predaceous coccinellid species are known which sequester their alkaloids from the prey, other species have been shown to produce the alkaloids autogenously. For example, Coccinella septempunctata feeding on aphids colonizing Senecio jacobea containing pyrrolizidine alkaloids (= PAS) have been found to sequester the PAS from the aphids in large amounts (Witte et al., 1990). The studies on the autogenous biosynthesis of alkaloids in ladybird beetles suggest a polyketidfatty acid origin of the alkaloids (Daloze et al., 1995). Eggs of several coccinellids have been shown to contain alkaloids. For example, the ladybirds Adalia bipunctata and A. decernpunctata produce two related alkaloids, the homotropane adaline and a minor constituent, the piperidone adalinine, which were shown to be present in adults and eggs (Lognay et al., 1996). The alkaloids coccinelline and its N-oxide known from adults of Coccinella septernpuncta are also present in the eggs (Pasteels et al., 1973; Daloze et al., 1995) (Figure 3-2, [7a,b]). Furthermore, eggs of Exochomus 4-pustulatus and E. varivestis are known to contain alkaloids (Dalozeet al., 1995).Alkaloids present in the eggs and other juvenile stages have been found to be either the same as in the adult haemolymph (Lognay et al., 1996) or to differ from adult alkaloid pattern (Attygale et al., 1993; Proksch et al., 1993). 3.2.2.7 lsoxazo Iinones as C hrysomeIid Defensive Component s Adult chrysomelids of the genera Chrysornela, Gastrophysa, Linaeidea, Phrafora, Plagiodera, Phaedon, and Phaedoniu are known to release two isoxazolinone glucosides from exocrine glands when disturbed. For several of these species, the two isoxazolinones have also been shown to be present in the eggs (Hilker, 1994;

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Chemical Protection of Insect Eggs

Pasteels et al., 1986, 1988, 1994) (Figure 3-2, [8]).The major isoxazolinone, which contains a nitropropionate group, is a very effective deterrent for aggressive ants (Pasteels et al., 1986). At the concentration found in the eggs, workers in contact with the major isoxazolinone derivative quickly retreat while at the same time dragging their mouthparts on the substrate. The nitropropionate-containing isoxazolinone present in the fluid content of the egg, might constitute a powerful deterrent against generalist predators which pierce eggs and subsequently suck the fluid content. The isoxazolinonederivatives are biosynthesized from L-aspartic acid (Randoux et al., 1991). 3.2.2.8 Betaine in Eggs of the “Femmes Fatales”

Female fireflies (Coleoptera,Lampyridae) of the genus Photuris are well-known as the “femmes fatales” among insects. They are able to attract male fireflies of the genus Photinus by imitating the flash signals of Photinus females, which are important for mate finding. The duped Photinus male arriving at a Photuris female will become her prey (Lloyd, 1965).In addition to components obtained from their male prey (see Section 3.3.2.6), Photuris females produce autogenously the betaine N-methylquinolinium 2-carboxylate. Photuris larvae are also able to produce this component (Gonzaleset al., 1999a,b). Females transfer the betaine to the eggs where it deters the ant Leptothorux longispinosus and coccinellid larvae of the species Hurrnoniu uxyridis from feeding (Gonztileset al., 1999a,b). 3.2.2.9 Pederin as Endosymbiotic Product

Pederin, the most structurally complex nonproteinaceous compound identified in insects, is a powerful vesicant present in the haemolymph of staphylinids of the genus Puederus (Pavan and Bo, 1953; Cardani et al., 1965a,b; Matsumoto et al., 1968; Pavan, 1975)(Figure3-2, [lo]).Kellner and Dettner (1996)showed that wolf spiders are deterred by prey containing pederin. The component is produced by endosymbionts within the female staphylinids (Kellner, 1999). Even though aposymbiotic females, so called (-) females, were found in the field, females with the respective endosymbionts, i.e. (+) females, dominate (Kellner, 1997).Only the eggs of (+) females contain pederin, whereas (-) females lay eggs devoid of this substance (Kellner,2001).When larvae hatching from (-) eggs feed upon (+) eggs, they take up both the pederin from the eggs and the endosymbionts necessary for pederin production in the adult stage (Kellner and Dettner, 1995).Thus, they may become (+) females although they have hatched from (-) eggs. If larvae feed upon sterilized (+) eggs, they are unable to produce pederin in the adult stage. This result clearly shows pederin as a defensive substance traceable to endosymbionts (Kellner, 1999). 3.2.2.10 Cajin - a Toxic Polypeptide in Arctia caja

Some species of arctiid moths positively bask in the glory of their aposematism and toxicity. Endowed with brilliant colours, they flaunt their pronounced aposematism and if tactilely stimulated, discharge an often colourful thoracic froth

... .

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

Defensive Components o f Extrinsic Origin

that is enriched with choline esters and histamine (Bisset et al., 1960). Internally, they contain toxic alkaloids that have been sequestered from food plants and in addition, autogenously produced compounds that are stored in the female reproductive organs and eggs (Marsh and Rothschild, 1974).Clearly, these insects do not invite ingestion. The garden tiger moth, Arctiu cuju, produces toxins that are toxic to both mammals and insects by injection. One of the compounds is a polypeptide with a molecular weight of about 1,000 (Rothschild et al., 1979)and it has been assigned the epithet cajin. Cajin is reported to stimulate muscles directly and it is suggested that it acts as a calcium ionophore or possibly an analogue of an insect neurotransmitter. Also extracts of gravid females and eggs of the zygaenid moth Zyguenu trifolii are reported to kill mice in two to three minutes when they are injected (Marsh and Rothschild, 1974).

3.3

Defensive Components of Extrinsic Origin

Insect phytophages feed on a great diversity of plant species which are often endowed with an incredible variety of natural products, many of which are of pronounced toxicity. Ultimately the herbivores turn these plant effronteries against potential adversaries, and they have thus appropriated the plant’s chemical defences. Insect eggs clearlybenefit from the ability of their mother - and sometimes their father - to ingest and sequester plant toxins that can be channeled to their eggs (e.g. Blum, 1992, and references therein) (Table 3-2).

3.3.1

73

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

Chemically Defensive Plant Material Covering Eggs

Numerous insects are known to insert their eggs into plant material, thereby hiding the eggs and making them less conspicuous. In addition to such physical protection, the plant may also chemically protect the eggs if the plant material harbouring the eggs contains noxious components. For example, the leaf beetle Pyrrhaltu viburnilays the eggs in the twigs of Viburnum spp. For egg deposition, the female gnaws a cavity into the twig, deposits several eggs inside, and finally covers the eggs with faeces and chewed plant material. This egg covering contains the triterpenes a-and P-amyrin (Hilker, 1992) which are also known as constituents of Viburnum spp. (Hegnauer, 1989),but seem to be These triterconcentrated as free terpenes in the egg covering (Figure 3-3, [ll]). penoids are toxic and emetic towards vertebrates (Teuscher and Lindequist, 1994).

Sequestered Defensive Components within the Eggs 3.3.2.1 Cantharidin Sequestered from Prey 3.3.2

Insects of several families are known to be attracted towards cantharidin. These canthariphilic insects are obviously able to sequester cantharidin from their cantharidin-containing prey. Anthicid and pyrochroid beetles as well as ceratopogonids are known to transfer this sequestered cantharidin to the eggs (Dettner, 1997, and references therein) (for details see Chapter 4).

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Chemical Protection of Insect ERRS

Table 3-2 Defensive chemicals of extrinsic origin known from insect eggs. For references please see text. "Insect taxon": for one or several species of the taxon listed the respective chemical has been detected in the eggs

Origin of Compounds

Chemicals

Insect Taxon

wand p-Amyrin

Coleoptera: Chrysomelidae: Pyrrhalta viburni

Cantharidin

Canthariphilous insects with cantharidin proved to be present in the eggs: Coleoptera: Anthicidae Pyrochroidae Diptera: Ceratopogonidae Coleoptera: Chrysomelidae: Diabroticina Coleoptera: Chrysornelidae: Chrysomela spp., Phratora vitellinae Lepidoptera: Papilionidae: Atrophaneura alcinous Lepidoptera: Nymphalidae: e.g. Danaus plexippus Orthoptera Pyrgornorphidae: e.g. Poecilocerus bufonius Heteroptera: Lygaeidae: e.g. Oncopeltusfasicatus Coleoptera: Larnpyridae: Photuris Lepidoptera: Zygaenidae Papilionidae: Parnassius phoebus Lepidoptera: Arctiidae Nymphalidae: Ithorniinae, Danainae Coleoptera: Chrysomelidae: e.g. Oreina elongata Lepidoptera: Lycaenidae: Eumaeus atala Lepidoptera: Pieridae: Pieris brassicae

EXTRINSIC ORCIN Applied onto the eggs

Within the eggs

Cucurbitacins Salicin

Aristolochic acids Cardenolides

Lucibufagi ns Cyanogenic components Alkaloids

Cycasi n Mustard oils

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

.. ........... . . .. ................................................................................ . ,.............

11 a-Amyrin

Defensive Components o f Extrinsic Origin 75 ......... . ..................................... ........... ......................... .. .......,..............

12 23,24-DihydrocucurbitacinD

,,,,,,,,

,

13 Salicin 0

AcO AcO

14 Aristolochic acids

15 Calotropin HO 1

OH

16 Lucibufagin

OH 1

/OH Glu-0

C-N CHFCHCHZ-NCS

17 Sarrnentosin

18 Monocrotaline-N-oxide

19 Cycasin

20 Allylisothiocyanate

Figure 3-3 Examples of defensive egg components of extrinsic origin (i.e.sequestered from

food). 3.3.2.2 Cucurbitacins

- Bitter Triterpenes in Diabroticite Beetles

Diabroticite rootworm beetles (Chrysomelidae) have developed a very close relationship with species of Cucurbitaceae, often utilizing these cucurbits as their major food plants. Many cucurbit species are fortified with cucurbitacins, oxygenated tetracyclic triterpenoids which are very bitter and highly toxic (Ferguson et al., 1985).The cucurbitacins are sequestered by larvae and adults of diabroticites.Females transfer these triterpenoids from their haemolymph into the eggs (Ferguson et al., 1985). Thus, from the egg perspective, the haemolymph becomes the toxin source and the egg becomes the toxin sink. The aspect of male contribution to cucurbitacin content in diabroticite eggs is considered in detail in Chapter 4. Utilizing radiolabelled cucurbitacin B as a metabolic paradigm, the fate of this compound in five diabroticite species was determined (Ferguson et al., 1985).All species produced an identical array of metabolites although specialists (Aculymmu vittutum and Diubroticu virgiferu virgiferu) excreted more unchanged triterpene than the three polyphagous Diubroticu species, D. undecimpunctutu howurdi, D. bulteutu, and D. cristutu. On the other hand, the three generalists,in contrast to D. v.virgiferu and A. vittutum, produced a higher concentration of polar end products than the specialists.The more polar metabolites predominated in the guts of all speciesexcept

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Chemical Protection of Insect Eggs

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

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

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

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

...........

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

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

.

A. vittaturn. A single cucurbitacin metabolite accumulated in the haemolymph of all Diabrotica species and was retained in the blood throughout development. Large amounts of metabolites are transferred to the eggs by females of D. u. howardi, D. balteata, and A. vittaturn,probably from cucurbitacins fortifying the haemolymph (Ferguson et al., 1985).Feeding radiolabelled cucurbitacin C to adults of five species of diabroticites, with different host plant preferences, resulted in essentially the same array of metabolites in the species studied (Ferguson et al., 1985).Almost all of the metabolites were more polar than cucurbitacin C itself, suggesting that the metabolic transformations constituted detoxification products. Adult diabroticites that fed upon squash containing cucurbitacin B and D were rejected by mantids, while those beetles that had been reared on a cucurbitacinfree diet were readily consumed by the mantid (Ferguson and Metcalf, 1985).Larvae and eggs obtained from individuals of D. u. howardi, that fed on cucurbitacin B and D-rich food were shown to be significantly less often attacked by the entomopathogen fungus Metarhiziurn anisopliae (Tallamy et al., 1998; compare Chapter 6). 23,24-Dihydrocucurbitacin D has been isolated from the two Brazilian leaf beetles D. speciosa and Ceratorna arcuata as major substances causing bitterness (Figure 3-3, [12]).It is not known whether these substances are selectively sequestered from the host plants or whether they are metabolites of the cucurbitacins B and D occurring in the plant. The dihydro-form of cucurbitacin D was shown to repel sparrows (Nishida and Fukami, 1990).However, eggs obtained from diabroticite females that fed upon a cucurbitacin C-containing cucumber variety failed to deter carabid beetles, predatory mites, and centipeds from predation (Brust and Barbercheck, 1992).Similarly, adults of D. u. howurdi fed with cucurbitacin C-rich cucumber seedlings did not deter mice, quails, and toads from feeding (Gould and Massey, 1984).Feeding upon host plants rich in cucurbitacins D or E did not protect diabroticite larvae from attack by nematodes, but instead feeding upon cucurbit hosts made them even more susceptible to nematode infection (Barberchecket al., 1995; Eben and Barbercheck, 1997). Thus, evidence for the protective efficacy of cucurbitacins is somewhat equivocal. 3.3.2.3 Salicin: Toxins in Eggs and Precursors for Larval Defence

The leaves of many species of willows and poplars (Salicaceae) contain salicin, a phenolglucoside that is reported to be toxic to nonadapted herbivores (Pasteels et al., 1986; Rowell-Rahier and Pasteels, 1986). Several chrysomelid species feeding upon salicin- and salicortin-containing Salicaceae have been shown to use the phenolglycosides of their host plants for chemical defence in the larval and egg stage. Larvae of several Chrysornela species as well as of Phratom vitellinae have been shown to hydrolyse salicin and to oxidize the resulting aglycone to salicylaldehyde, which is then released as major volatile component from their exocrine glandular secretions (Pasteels et al., 1988).Salicylaldehyde acts as a potent repellent against ants (e.g. Pasteels et al., 1983; Blum, 1994). Several of these species using the phenolglycosides as precursor for production of defensive secretion in the larval

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

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

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

Defensive Components of Extrinsic Origin

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

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

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

77

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

stage have been shown to use salicin also for defence in the egg stage (Figure 3-3, [13]).Females incorporate salicin into the eggs, where it acts both deterrent and toxic to ants (Pasteels et al., 1986).Thus, salicin acts as defensive component of the egg stage and as precursor of larval chemical defence. Larvae hatching from salicin-containingeggs use the phenolglycoside of the eggs to produce the major volatile component of their defensive secretion (Pasteels et al., 1983).Thus, they are already provided with the precursor of their defensive secretion prior to feeding on the host plant. The presence of salicin in the egg has been exploited so that the effective protector of the egg is converted to an immediate larval protector, at a time when autogenously synthesized repellents are ordinarily not yet produced. As a bonus, these chrysomelid larvae produce and utilize glucose as a product of the transformation of salicin to salicylaldehyde, providing them with an important nutritional resource (Pasteels et al., 1983; Rowell-Rahier and Pasteels, 1986). 3.3.2.4 Bitter Aristolochic Acids

Larvae of many swallowtail butterflies (Papilionidae)feed on plant species in the genus Aristolochiu (family Aristolochiaceae) which are a source of bitter tasting, genotoxic nitro compounds of the aristolochicacid series (Teuscherand Lindequist, 1994; Nishida, 1995; Klitzke and Brown, 2000). Larvae of Atrophaneuru alcinous sequester aristolochic acids which are transferred to the adults. The females incorporate them into the reddish orange eggs (Nishida and Fukami, 1989a, b). The toxic acids are reported to be present in the egg yolk and egg coating as a readily available deterrent to predators (Figure 3-3, [14]). 3.3.2.5 Abundant CardenolidesSequestered from the Host Plant

A wide range of insects, such as Orthoptera, Heteroptera, Homoptera, and several Lepidoptera, are known to sequester cardenolides from the host plants and use them for their own defence (Rothschild, 1985; Malcolm, 1991,1995; Wink and von Nickisch-Rosenegk, 1997 and references therein) (compare Section 3.2.2.4). However, while usually storageof plant cardenolidesin larval and adult body tissue is known, the eggs have not been analysed in all cardenolide sequestering species. The pioneering studies on sequestration of plant cardenolides by herbivorous insects have been conducted by Brower, Reichstein, and Rothschild, who showed that the monarch butterfly, Dunuus plexippus, stores the cardenolides obtained from its food plants, AscZepius spp., in all life stages, including the eggs (Reichstein et al., 1968; Brower and Glazier, 1975; Rothschild et al., 1975). A very aposematic species of Orthoptera, Poecilocerus bufonius, sequesters cardenolides from milkweeds (Asclepiadaceae)in body tissues and defensive glands which can eject these steroids. For the most part, this species of Pyrgomorphidae contained two cardenolides which were found in different life stages including the eggs (von Euw et al., 1967) (Figure 3-3, [15]).Also the painted grasshopper PoeciZocerus pictus feeds on poisonous cardenolide-rich milkweed (Culotropis gigunteu). The metathoracic scent gland, the ovary, and the eggs of the

78

Chemical Protection of Insect Eggs

grasshopper have been found to contain high concentrations of cardenolides (Pugalenthi and Livingstone, 1995). Many heteropterans are associated with toxic plants and that certainly is the case for the lygaeid Cuenocoris nerii which feeds on the highly toxic Nerium oleunder. Four very toxic cardiac glycosides have been identified in the adults and larvae of C. nerii, and these compounds are also present in the eggs (von Euw et al., 1971). Eggs of the lygaeid OncopeZtusfusciutus feeding upon seeds of AscZepius syriucu also contain cardiac glycosides (Duffey and Scudder, 1974). Host-derived cardenolides appear to be secondarily sequestered by parasites and predators that have attacked hosts that contain sequestered cardenolides. Tachinid parasites obtain cardenolides from monarch butterflies (Reichsteinet al., 1968), and lacewings (Chrysopu spp.), and the ladybird CoccineZZu undecirnpunctutu appears to obtain these compounds from the aphid Aphis nerii feeding on cardenolide-rich oleander (Rothschild et al., 1973). 3.3.2.6 LucibufaginsSequestered from Prey

As mentionend above (see Section 3.2.2.8), female fireflies of the genus Photuris prey upon male fireflies of the genus Photinus. Males of this genus contain steroidal pyrones called lucibufagins which are not present in the Photuris females (Figure 3-3, [16]). The "femmes fatales" sequester the lucibufagins from their prey and endow their eggs with these components (Eisner et al., 1997). Lucibufagins act as feeding deterrents against coccinellid and formicid predators (Gonzdes et al., 1999a). Thus, eggs of the Photuris fireflies are protected both by de novo produced betaine (see Section 3.2.2.8) and by the sequestered lucibufagins. The purpose of such an "overload with defensive components may be to provide hatching larvae also with the defensive lucibufagins (Gonziles et al., 1999a) (see also Chapter 4). 3.3.2.7 Plant Derived Cyanogenic Components

As already mentioned in Section 3.2.2.5, zygaenid moths may sequestercyanogenic components from their host plants and in addition being able to produce them autogenously (Davisand Nahrstedt, 1985). The percentage of uptake of cyanogenic glucosides has been estimated in Zyguenu trifoZii at 20 to 45% (Nahrstedt, 1988). While eggs of the zygaenid species and the nymphalids that are able to autogenously produce cyanogens contain cyanogenic components, the other lepidopteran species known to sequester cyanogeniccomponents from their food plants have hardly been studied for the presence of cyanogens in the eggs (Nahrstedt, 1988; Seigler, 1991; Jones, 1998). An exception is the study on the aposematicpapilionid Purnussius phoebus, which sequesters sarmentosin (Figure 33, [17]), a bitter cyanoglucoside from the food plant Sedum stenopetulurn (Nishida and Rothschild, 1995). Adults contain > 400pg of sarmentosinand each oviposited egg contains 1pg of this cyanoglucoside.Sarmentosin epoxide, a product of another Sedum species, liberates HCN in aqueous media, and a similar reaction may occur with sarmentosin if the double bond is enzymatically epoxidized.

Defensive Components of Extrinsic Origin

3.3.2.8 N-Oxide Alkaloids as Pro-Toxins

Multifarious aposematic insect species feed on a large diversity of alkaloidcontaining plants belonging primarily to the families Asteraceae, Boraginaceae, Fabaceae, and Orchidaceae. The insect phytophages feeding on these potentially toxic plants are, for the most part, eminently aposematic, and generally constitute a dazzling number of species in the Lepidoptera order as well as species of Orthoptera, Hemiptera, Homoptera, Coleoptera, and Hymenoptera. In general, the insects in these orders are very effective in sequestering the alkaloids they encounter in their food plants (Boppr6,1990;Schaffner et al., 1994;Hartmann, 1995a, b; Trigo et al., 1996; Hartmann, 1999).In addition to uptake of alkaloids with the food, several so-called pharmacophagous species (Boppre, 1986; Tallamy et al., 1999) are known to obtain the alkaloids from plants that are not intended for food uptake but just for exploitation of plant alkaloids (e.g. Trigo et al., 1996). Herbivorous insects may use the alkaloids sequestered from the plant for different purposes: (a) for defence of the feeding stage (eg. Aplin et al., 1968; Hartmann, 1999;Dobler et al., 2000), (b) for defence of the adult stage when transferred from the larva to the adult (e.g. Trigo and Motta, 1990; Trigo et al., 1996), (c) for production of courtship pheromones (e.g. Eisner and Meinwald, 1987; Boppr6,1990; Dussourd et al., 1991; Schulz et al., 1993; Weller et al., 1999), (d)for defence of the offspring when transferred either from the copulating male to the female into the eggs (compare Chapter 4) or directly from the female into the eggs (e.g.Brown, 1984a,b; Dussourd et al., 1988,1989;von Nickisch-Rosenegk et al., 1990). Pyrrolizidine alkaloids (PAS)have been shown to deter spiders (Brown 1984a,b; Eisner and Meinwald, 1987; Trigo et al., 1996).PASrender insect eggs deterrent to coccinellid predators (Dussourd et al., 1988), and chrysopids (Eisner et al., 2000). However, PASdo not protect eggs from fungal infection (Storeyet al., 1991, compare Chapter 6). Herbivorousinsects feedingupon plants containing PASstore these components mainly as the nontoxic N-oxides which, if ingested by a predator, are reduced in the gut to the tertiary alkaloid that manifests its pronounced toxicity (Figure 3-3, [l8]). The tertiary PA is toxic for any organism with an accessible microsomal multisubstrate cytochromeP-450 (Hartmann, 1999).Insects also have the capability of converting plant-derived PAS into idiosyncratic PAS which are probably more suitable for long-term sequestration than at least some of the alkaloids ingested as part of the plant diet (Hartmann, 1995a). The ability of insects to exploit pyrrolizidine alkaloids (PAS)adaptively has been demonstrated to be of great selective value, in a variety of contexts, for species in severalorders. Transfer of plant alkaloids into the eggs has been shown in numerous lepidopteran species belonging to the Arctiidae, severalIthomiinae, and Danainae (see references above under [d]).In several of these lepidopteran species the male

79

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Chemical Protection of Insect Eggs

........

transfers his PAS to the female, which incorporates the paternal gift and her own PASinto the eggs (for details see Chapter 4). In addition to lepidopteran species sequestering plant PAS, several Chrysomelidae feeding on PA-containingplants have alsobeen studied intensively (Pasteels et al., 1996, and references therein). While several species of the genus Oreinu release cardenolides produced de novo from their exocrine defensive glands on the elytra and pronotum, several other Oreinu species sequester PASfrom their food plant and store them in the body and secretion as N-oxides. Three species, Oreinu intricuta, 0. speciosissirnu, and 0. elonguta, add sequestered PAS to their cardenolide-rich secretion, increasing its deterrency considerably (Pasteels et al., 1996).Another species, 0.cucaliue, does not produce any cardenolides, but instead releases PASas the only defensive components with the secretion. In the oviparous species 0. elongata, the female protects her eggs with de novo-synthesized cardenolides plus PAS sequestered from food plants. The PAS are clearly the dominant defensive compounds (4.5 pg/egg) in comparison to the autogenous cardenolides (0.2 to 0.5 &egg ). Curiously, females of the larviparous 0. cuculiue do not transfer the alkaloids to their offspring (Dobler and Rowell-Rahier, 1994). Till now it is unknown whether the larviparous females of 0. speciossirnu and 0. intricutu transfer sequestered plant alkaloids to their progeny. Analysis of a tritrophic scenario involving PAS demonstrates that these compounds may be transferred from a PA-containingfood plant over the herbivore into the third trophic level. Three Senecio species are readily fed upon by the aphid Aphis jucobaeue and the PA pattern of the insects corresponds very closely with those of its food plants (Witteet al., 1990).The aphid sequesters PASwith great efficiency, the alkaloidal concentration reaching levels of 3.5 mg/g fresh weight. Although the PA-rich aphids should be eminently distasteful and in addition very toxic, they are actively preyed upon by the ladybird Coccinella septernpunctutu. This coccinellid beetle bioconcentrates the PAS so efficiently that alkaloidal concentrations of 4.9 mg/g fresh weight are reached. Sequestration is not selective,the PA pattern of the ladybird mirroring those of the aphid and the plant (Witte et al., 1990).Whether or not the female ladybird endows its eggs with its abundant PAS has not been determined, although it would seem to be highly adaptive to exploit this rich pool of readily available predator deterrents. 3.3.2.9 Cycasin as a Nontoxic B-Clycoside

Larvae of the Atala hairstreak, Eurnaeus utula, a lycaenid butterfly, sequester the Pglycoside cycasin from the cycad leaves on which they feed (Rothschild, 1992). Although the aglycone of cycasin, methylazoxymethanol,is quite toxic, conversion to the P-glycosideresults in a compound that can be safely sequestered in structures including the eggs (Figure 3-3, [19]). 3.3.2.10 Mustard Oil in Eggs of Pieris

Pieris butterflies are closely associated with plants of the family Cruciferae which serve as food plants for their larvae. Many species of cruciferous plants produce

Chemical Defence and Egg Cannibalism

glucosinolates.The glucosinolates and their hydrolysis products (mustard oils) are known as irritants, deterrents, and toxins towards non-adapted animals (Louda and Mole, 1991).Sinigrin is a well-known glucosinolate of cabbage. The hydrolysis product allylisothiocyanate (mustard oil) provide the typical smell of cabbage. Allylisothiocyanate has been detected in eggs, pupae, and adults of the white butterfly Pieris brussicue (Figure 3-3, [ZO]).The glucosinolate sinigrin has only been detected in pupae of P. brussicue, not in eggs and adults (Aplin et al., 1975).

3.4

Chemical Defence and Egg Cannibalism

Egg cannibalism is not rare among insects (Polis, 1981).Both larvae and adults are known to feed upon conspecific eggs (Crespi, 1990; Dickinson, 1992; Mafra-Net0 and Jolivet, 1996). Cannibalism may have detrimental effects when, for example, pathogens are taken up from the victims. However, the major advantages that have promoted the evolution of cannibalistic behaviour may be reduction of competition by killing intraspecific competitors and acquisition of nutrients when availabilityand quality of food plants or interspecific prey is low (Elgar and Crespi, 1992; Mafro-Net0 and Jolivet, 1996).Egg cannibalism in socialinsects may contribute to maintenance of social dominance hierarchies (Kukuk, 1992; Heinze et al., 1996). Furthermore, egg cannibalism by neonate larvae may provide the larvae with (additional)endosymbiontsthat have been added to the eggs (seee.g. Section3.2.2.9 and Chapter 6.).Neonate larvae, which hatch from chemically defended eggs and use egg chemicals for their own defence, are known to feed upon eggs of conspecifics, probably in order to enhance and fortify their own defence. For example, larvae of the papilionid Atrophaneuru ulcinous feed upon egg masses of conspecifics and, thus, gain toxic aristolochic acid from the eggs (Nishida and Fukami, 1989a). Larvae of the arctiid moth Utetheisa that have been reared on an alkaloid-free diet obviously long for alkaloids, which they usually sequester from the host plant. Such alkaloid-free larvae readily feed upon conspecific eggs containing these defensive chemicals (Eisner et al., 2000). The first oedemerid larvae hatching from an egg batch feed upon conspecific eggs, probably in order to enhance their cantharidin concentration by the cannibalizing on cantharidincontaining eggs (Holz et al., 1994). Thus, egg cannibalism seems to occur frequently even when eggs contain toxins. However, physical barriers associated with eggs may be good devices to prevent cannibalism. For leaf beetles, no egg cannibalism has been reported for those species that cover their eggs with faeces or hide them in primitive oothecae or just in plant tissue. While egg clustering might favour cannibalism (Clark and Faeth, 1998), scattered distribution of eggs is reported as a behavioural trait to prevent cannibalism (Stamp, 1980; Mafra-Net0 and Jolivet, 1996). An interesting study of chemical prevention of cannibalism and intraguild predation has been provided by Hemptinne and co-workers (Hemptinne et al., 2000b). The alkaloid-containing eggs of the coccinellid Aduliu bipunctu tu contain hexane-soluble components on their surface that prevent both conspecifics and

81

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the intraguild predator Coccinellu septernpunctutu from feeding. Nearly 90% of the cuticular compounds are hydrocarbons in their eggs. For A. bipunctatu it has been shown that larvae avoid cannibalism and intraguild predation on eggs of C. septernpunctu. On the other hand, eggs of C. septernpunctutu were not protected against cannibalism, but protection against intraguild predation by A. bipunctutu was observed (Hemptinne et al., 2000b). The alkane signal emanating from the eggs of A. bipunctutu is nearly the same as those present in the larval tracks and on the adult elytra. The hydrocarbons function as important semiochemicals,first as larval tracks that deter females from ovipositing in patches of prey that are already under attack by their larvae. In addition, the elytral alkanes play an important role in mate recognition, further documenting the pronounced parsimonious utilization of the hydrocarbon blend (Hemptinne et al., 2000b).

3.5

Concluding Remarks

A large variety of insects, feeding on toxic plants containing eminently eclectic natural products, sequester these compounds and transfer them to the eggs. In these species, the idea that eggs may be considered as a toxin "sink" has become a topic of increasing attraction. The great significance of determining the fate of a plant natural product after ingestionby adapted insect phytophages is emphasized by Ferguson et al. (1985). Radiolabelled cucurbitacin B was administered to five diabroticitebeetles and it was possible to follow the fate of this tetracyclic triterpene throughout adult life. The availability of the radiolabelled compound eventuated in easy detection of a key polar metabolite which persisted in the haemolymph as a source of a repellent toxin which was transferred to eggs even if the adults had not recently fed on cucurbitacins. Thus, important insights into the rnodus operundi of chemical defence in diabroticite beetles became possible only because of the availability of radiolabelled plant natural product utilized in an excellent research program. It would seem very desirable to use radiolabelled plant allelochemicals more frequentlyin order to ascertain the fates of these compounds with a fair degree of exactitude. As was shown by the excellent studies by Hartmann and coworkers (reviewedin e.g. Hartmann, 1999),such radiolabelingof plant allelochemicals may provide detailed insight into the transfer, storage and metabolism of these components within herbivorous insects (Hartmann, 1999). However, numerous insect species are known not to use noxious plant components, but instead to produce autogenously chemicals that protect them and their eggs. The role of endosymbioticmicroorganisms for production of these components has hardly been studied till now. Insects appear to contain a plethora of endosymbionts. If Puederus species are not exceptional in utilizing microorganisms for the biosynthesis of a key natural product, pederin (see Section 3.2.2.9), then a number of insect defensive compounds may be candidates for studies of their biosynthetic origins. Indeed, several strainsof microorganismswere isolated from the foregut of the dytiscid beetle Agubus guttutus and it was demonstrated

References

that pregnenolone was converted to three steroids found in the defensive prothoracic glands (Jungnickel and Dettner, 1997).If the chemicalecology of insects vis-a-vis their apparently autogenously produced natural products is to be understood, it may be necessary to examine these arthropods as microbial chambers in which significant compounds are biosynthesized for their insect hosts. In addition to questions of the origin of defensive components of insect eggs and the fate of plant components on their way from the feeding insect into the egg, future studies also need to consider how the egg endowed with toxins prevents autotoxicity and how the developing embryo deals with the toxins. The utilization of egg hydrocarbons to “label”unpalatabilityof eggs (Hemptinne et al., 2000b; see Section 3.4) raises the question of whether these hydrocarbons may play more widespread defensive roles in the chemical ecology of insect eggs. Hydrocarbonsare normal constituentsof the insect cuticle and it will not necessarily prove surprising if this class of compounds has been adapted to subserve the role of chemical signalling agents (compare also Section 5.3.3 in Chapter 5).

3.6

Ac know1edgements

We wish to thank Dr. Miriam Rothschild, Professor Thomas Hartmann, Professor JacquesPasteels, Dr. Ritsuo Nishida, and Dr. Reinhold Deml for valuable comments and resource material. Many thanks are also due to Frank Muller for drawing the figures.

3.7

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Muller, K. 1988. Aktive Sauerstoffspezies. Bedeutung fur Krankheit und Gesundheit. Pharmazie in unserer Zeit 17: 71-80. Nahrstedt, A. 1988. Cyanogenesis and the role of cyanogenic compounds in insects. In: Ciba Foundation (ed.) Symposium 140. Cyanide Compounds in Biology. Pp. 131-143. Wiley, Chichester. Nahrstedt, A. 1989. The significance of secondary metabolites for interactions between plants and insects. Planta Medica: 55: 333-338. Nahrstedt, A, Davis, RH. 1986. (RjMandelonitrile and prunasin, the sources of hydrogen cyanide in all stages of Paropsis atomariu (Coleoptera: Chrysomelidae). Z . Naturforsch. 41c: 928-934. Niemann, GJ. 1993. The anthranilamide phytoalexins of the Caryophyllaceae and related compounds. Phytochemistry 34: 319-328. Nishida, R. 1995. Sequestration of plant secondary compounds by butterflies and moths. Chemoecology 6: 127-138. Nishida, R, Fukami, H. 1989a. Ecologicaladaptation of an Aristolochiaceae-feedingswallowtail butterfly, Atrophneura ulcinous, to aristolochic acids. J. Chem. Ecol. 15: 2549-2563. Nishida, R, Fukami, H. 1989b. Oviposition stimulant of an Aristolochiaceae-feeding swallowtail butterfly, Atrophaneura alcinous. J. Chem. Ecol. 15: 2565-2575. Nishida, R, Fukami, H. 1990. Sequestration of distasteful compounds by some pharmacophagous insects. J. Chem. Ecol. 16: 151-164. Nishida, R, Rothschild, R. 1995. A cyanoglucoside stored by a Sedum-feeding butterfly, Purnussius phoebus. Experientia 51: 267-269. Nyffeler, M, Breene, RG, Dean, DA, Sterlin, WL. 1990. Spiders as predators of arthropod eggs. J. Appl. Entomol. 109: 490-501. Orians, GH, Janzen, DH. 1974. Why are embryos so tasty. Am. Nat. 108: 581-592. Pasteels, JM, Braekman, J-C, Daloze, D. 1988. Chemical defence in the Chrysomelidae. In: Jolivet, P, Petitpierre, E, and Hsiao, TH. (eds.). Biology of the Chrysornelidae. Pp. 233-252. Kluwer, Dordrecht, Netherlands. Pasteels, JM, Daloze, D. 1977. Cardiac glycosides in the defensive secretion of chrysomelid beetles: evidence for their production by the insects. Science 197 70-72. Pasteels, JM, Daloze, D, Rowell-Rahier,M. 1986. Chemical defence in chrysomelid eggs and neonate larvae. Physiol. Entomol. 11: 29-37. Pasteels, JM, Deroe, C, Tursch, B, Braekman, JC, Daloze, D, Hootele, C. 1973. Distribution et activites des alcaloides des coccinelles.J. Insect Physiol. 1 9 1771-1784. Pasteels, JM, Rowell-Rahier, M, Braekman, JC, Daloze. 1994. Chemical defence of adult leaf beetles. In: Jolivet, PH, Cox, ML, and Petitpierre, E. (eds.). NoueZ Aspects of Biology of Chysomelidae. Pp. 263-276. Kluwer Academic Publishers, Dordrecht. Pasteels, JM, Rowell-Rahier, M, Braekman, JC, Dupont, A. 1983. Salicin from host plant as precursor of salicylaldehyde in defensive secretion of chrysomeline larvae. Physiol. Entomol. 8: 307-314. Pasteels, JM, Rowell-Rahier, M, Ehmke, A, Hartmann, T. 1996. Host-derived pyrrolizidine alkaloids in Oreinu leaf beetles: Physiological, ecological and evolutionary aspects. In: Jolivet,PHA, Cox, ML. (eds.).Chysomelidae Biology, Vol. 2. Ecological Studies. Pp. 213-225. SPB Academic Publ., Amsterdam. Pavan, M. 1975. Sunto delle attuali conoscenze sulla pederina. Pubblicazioni dell'Istituto di Entomologia Agrania dell' Universita di Pavia. pp. 1-35. Pavan, M, Bo, G. 1953. Pederin, toxic principle obtained in the crystalline state from the beetle Paederus fuscipes Curt. Physiol. Comp. Oecol. 3: 307-312. Pesce, H, Delgado, A. 1971. Poisoning from adults moths and caterpillars. In: Buecherl, W, Buckley, E. (eds.) Venomous Animals and Their Venoms. Vol. 111. Pp. 119-156. Academic Press, New York. Petitpiem, E. 1995.Presence of anthraquinones in the hemolymph of Timarcha.Chrysomela30.4. Polis, GA. 1981. Evolution and dynamics of intraspecificpredation. Ann. Rev. Ecol. Syst. 1 2 225-251. Proksch, P, Witte, L, Wray, V, Hartmann, T. 1993. Ontogenic variation of defensive alkaloids in the Mexican bean beetle EpiZuchnu uuriuestis (Coleoptera:Coccinellidae).Entomol. Gen. 18: 1-7.

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Pugalenthi, P, Livingstone, D. 1995.Cardenolides (heart poisons) in the painted grasshopper Poicolocerus pictus F. (Orthoptera: Pyrgomorphidae) feeding on the milkweed Calotropis gigantea L. (Asclepiadaceae).J. NY Entomol. SOC.103: 191-196. Randoux, T, Braekman, JC, Daloze, D, Pasteels, JM. 1991. De nom biosynthesis of delta3isoxazolin-5-one and 3-nitropropanoic acid derivates in Chrysomela tremulae. Naturwiss. 78: 313-314. Reichstein, T, von Euw, J, Parsons, JA., Rothschild, M. 1968. Heart poisons in the monarch butterfly. Science 161: 861-866. Rothschild, M. 1985. British aposematic Lepidoptera. In: Heath, J (ed.). The Moths and Butterflies of Great Britain and Ireland. Vol. 2. Cossidae-Heliodinidae.Pp. 9-62. Harley Books, Colchester. Rothschild, M. 1992.Egg protection by the Atala hairstreak butterfly (Eumaeus atalaflorida). Phytochemistry 31: 1959-1960. Rothschild,M, von Euw, J, Reichstein, T. 1973. Cardiac glycosides in a scale insect (Aspidiotus), a ladybird (Coccinella) and a lacewing (Chrysopa). J. Ent. (A) 48: 89-90. Rothschild, M, von Euw, J, Reichstein, T, Smith, DAS, Pierre, J. 1975. Cardenolide storage in Danaus chysippus (L.) with additional notes on D. plexippus (L.). Proc. R. SOC.London B 190: 1-31. Rothschild, M, Keutmann, H, Lane, NJ, Parsons, J, Prince, W, Swales, LS. 1979.A study on the mode of action and composition of a toxin from the female abdomen and eggs of Arctia caja (L.) (Lep. Arctiidae): An electrophysiological, ultrastructural and biochemical analysis. Toxicon 17: 285-306. Rowell-Rahier, M, Pasteels, JM. 1986. Economics of chemical defence in Chrysomelinae. J. Chem. Ecol. 12: 1189-1203. Ruzicka, Z . 1997. Protective role of the egg stalk in Chrysopidae (Neuroptera). Eur. J. Entomol. 94: 111-114. Schaffner, U, Boevb, JL, Gfeller, H, and Schlunegger, UP. 1994. Sequestration of Veratrum alkaloids by specialist Rhadinoceraea nodicornis Konow (Hymenoptera, Tenthredinidae) and its ecoethological implications. J. Chem. Ecol. 20: 323S3250. Schulz, S, Francke, W, Bopprk, M, Eisner, T, and Meinwald, J. 1993. Insect pheromone biosynthesis: stereochemical pathway of hydroxydanaidal production from alkaloidal precursors in Creatonotus transiens (Lepidoptera, Arctiidae). Proc. Natl. Acad. Sci., USA 90: 6834-6838. Seigler, DS. 1991. Cyanide and cyanogenic glycosides. In: Rosenthal, GA, Berenbaum, MR. (eds.) Herbivores. Their lnteractions with Seconday Plant Metabolites. Vol. 1. The Chemical Participants. Pp. 35-78. Academic Press, San Diego. Sierra, JR, Woggon, WD., Schmid, H. 1976. Transfer of cantharidin during copulation from the adult male to the female Lytta vesicatoria ("Spanish flies"). Experientia 32: 142-144. Smedley, SR, Blankespoor, CL, Yuang, Y, Carrel, J, Eisner, T. 1995/6. Predatory response of spiders to blister beetles (family Meloidae). Zoology-Jena 99: 211-217. Stamp, NE. 1980. Egg deposition patterns in butterflies: Why do some species cluster their eggs rather than deposit them singly? Amer. Nat. 115: 367-380. Storey, GK, Aneshansley, DJ, Eisner, T. 1991. Parentally provided alkaloid does not protect eggs of Utetheisa ornatrix (Lepidoptera: Arctiidae) against entomopathogenic fungi. J. Chem. Ecol. 17: 687-694. Tallamy, DW, Mullin, CA, Frazier, JJ. 1999. An alternate route to insect pharmacophagy. The loose receptor hypothesis. J. Chem. Ecol. 25: 1987-1997. Tallamy, DW, Whittington, DP, Defurio, F, Fontaine, DA, Gorski, PM, Gothro, PW. 1998. Sequestered cucurbitacins and pathogenicity of Metarhizium anisopliae (Moniliales: Moniliaceae) on spotted cucumber beetle eggs and larvae (Coleoptera: Chrysomelidae). Environ. Entomol. 27: 366-372. Teuscher, E, Lindequist, U. 1994. Biogene Gifte. Gustav Fischer, Stuttgart. Thomson, RH. 1987.Naturally Occurring Quinones. 111. Recent Advances. Chapman and Hall, London. Trigo, JR, Motta, PC. 1990. Evolutionary implications of pyrrolizidine alkaloid assimilation by danaine and ithomiine larvae (Lepidoptera: Nymphalidae). Experientia 46: 332-334. Trigo, JR, Brown, KS, Witte, L, Hartmann, T, Ernst, L, and Barata, LES. 1996. Pyrrolizidine

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alkaloids: different acquisition and use patterns in Apocynaceae and Solanaceae feeding ithomiine butterflies (Lepidoptera: Nymphalidae). Biol. J. Linnean SOC.58: 99-123. Trigo, JR, Witte, L, Brown, KS, Hartmann, T, Barata, LES. 1993. Pyrrolizidine alkaloids in the arctiid moth Hyulurgu symu. J. Chem. Ecol. 19: 669-679. Tsukada, M. 2000. Frass-cover on egg masses decreases parasitisation in the andromeda lace bug, Stephanitis fukeyui (Heteroptera: Tingidae). Entomol. Sci. 3: 303-307. Von Euw, J, Fishelson, L, Parsons,JA., Reichstein, T, Rothschild, M. 1967. Cardenolides (heart poisons) in a grasshopper feeding on milkweeds. Nature 214: 35-39. Von Euw, J, Reichstein, T, Rothschild, M. 1971. Heart poisons (cardiac glycosides) in the lygaeid bugs Cuenocoris nerii and Spilostethus pundurus. Insect Biochem. 1: 373-384. Von Nickisch-Rosenegk, E, Schneider, D, Wink, M. 1990. Time course of pyrrolizidine alkaloid processing in the alkaloid exploiting arctiid moth, Creutonotus frunsiens. Z. Naturforsch. 45c: 881-884. Van Oycke, S, Braekman, JC, Daloze, D, Pasteels, JM. 1987. Cardenolide biosynthesis in chrysomelid beetles. Experientia 43: 460-462. Weller, S, Jacobson, N, Conner, WE. 1999. The evolution of chemical defences and mating systems in tiger moths (Lepidoptera, Arctiidae). Biol. J. Linn. SOC.68: 557-578. Wink, M, von Nickisch-Rosenegk, E. 1997. Sequence data of mitochondria1 16s rDNA of Arctiidaeand Nymphalidae: Evidence for a convergent evolution of pyrrolizidine alkaloid and cardiac glycoside sequestration. J. Chem. Ecol. 23: 1549-1568. Witte, L, Ehmke, A, Hartmann, T. 1990. Interspecific flow of pyrrolizidine alkaloids from plants via aphids to ladybirds. Naturwissenschaften 77: 540-543. Ziprkowski, L, Rolant, F. 1972. Study of the toxin from the poison hairs of Thaumefopoeu wilkinsoni caterpillars. J. Invest. Dermatol. 46:439445.

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Chapter 4 Paternal Investment in Egg Defence Thomas Eisner, Carmen Rossini, Andres Conzalez, Vikram K. lyengar, Melody V. 5. Siegler and Scott R. Smedley

Table of Contents 4.1 Introduction Enemies of Insect Eggs 4.2 Types of Egg Defences 4.3 4.3.1 Fireflies Chemical Defences: Cantharidin 4.4 4.4.1 Meloid Beetles 4.4.2 Cantharidiphiles Chemical Defences: Pyrrolizidine Alkaloids 4.5 4.5.1 Ardiid Moths 4.5.2 Danaine Butterflies 4.5.3 lthomiine Butterflies Chemical Defences: Cucurbitacins 4.6 The Issue of Paternity 4.7 When Defence Backfires 4.8 Other Paternal Contributions 4.9 4.10 Puddling 4.11 Concluding Remarks 4.12 Acknowledgements References 4.13

w Abstract

Defensive chemicals bestowed upon insect eggs may stem in part or entirely from the father, rather than the mother. Chemicals of paternal origin are transmitted to the mother by seminal infusion. The chemicals may be produced by the fathers themselves (as, for example, cantharidin in meloid beetles), or may be sequestered by the fathers from exogenous sources (as, for example, cantharidin in cantharidiphilic insects, pyrrolizidine alkaloids in arctiid, danaine, and ithomiine Lepidoptera, and cucurbitacinsin chrysomelid cucumberbeetles).In arctiidsand danaines there is evidence that the male, in courtship, advertises his pyrrolizidine alkaloid-giving capacity by way of a pheromone, and that the female exercises mate choice on the

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basis of that signal. Recent work on Drosophila is reviewed demonstrating maleto-female transmissionof peptides that affect male reproductive success (antibiotics; factors affecting sperm storage, female receptivity, and oviposition).

4.1

Introduction

Eggs are an extension of the self. They are the direct products of the mother but, to a greater extent than implied by the contribution of sperm, may be part of the legacy of the father as well. In insects that legacy may find different forms of expression. Males may offer physical commitment to eggs, as by attending them (Ralston 1977; Scott 1990) or carrying them upon their own body (Smith 1976),or they may bestow gifts upon the eggs, either overt giffs, presented in courtship and benefiting the eggs indirectly, or covert gifts, transmitted seminally and more directly routed to the eggs. The literature on overt gifts, or nuptial offerings as they are often called, has been well summarized (Vahed, 1998), and is exemplified by such classic cases as prey presentations by males to females in bittacid Mecoptera (Thornhill, 1976)and empidid flies (Cumming,1994).The topic of seminal bestowal of gifts, of covert gifts as one might call them, has received increasing attention recently, particularly as regards bestowal of nutrients (Boggs,1995).But males may do more than transmit nourishment with their sperm. They may also bestow minerals and, to a greater extent than is perhaps realized, defensive substances. We here address the topic of seminal transmission of protective chemicals, which has not been reviewed.

4.2

Enemies of Insect Eggs

Insect eggs are threatened by three categories of enemies - predators, parasitoids, and microbial pathogens. The relative magnitude of the threats posed by these enemies to the eggs of any one species of insect is doubtless variable, and could depend on many factors, including the physical conditions of the microhabitat in which the eggs are laid, the season in which they are deposited, their incubation time, and their degree of clustering. Their vulnerability, in turn, is a function of their defences, which one would imagine could be diversified and serve for protection against any combination of enemies. In the present discussion we will be concerned almost exclusively with paternally transmitted factors that defend against predation. At the end of the chapter we will give consideration also to some recent work dealing with factors contributed by the father that protect eggs or gametes against microbes, or sperm against sperm competition. We exclude from consideration the subject of egg defence against parasitoids.

Types of Egg Defences

4.3

Types of Egg Defences

There are many ways in which insect eggs may be protected against predation (Figure 4-1). An older but insightful review of the topic is that by Hinton (1981). Means of defence are often physical. Thus, eggs may be hidden, as by certain cockroaches, which bury their eggs (McKittrick, 1964) (Figure 4-lc); or provided with cover, as by certain moths which coat their eggs with scales (Koshio, 1996) (Figure 4-ld); or made inaccessible, as by chrysopids, which lay their eggs at the tip of stalks (Chen and Young, 1941)(Figure 4-lf). But as is becoming increasingly apparent, eggs may also be protected chemically,by noxious substances. Chemical provisioning of eggs is not always self-evident and may be demonstrable only by way of bioassays (in other words, by proving that the eggs are unacceptable to predators), but there is no question that it is of widespread occurrence. It is also becoming clear that the defensive chemicals may be produced by the insects themselves or acquired from an exogenous source (Figure4-lg, h). The assumption, usually, is that the providing parent is the female, which of course is now known not always to be the case. Another assumption, that for the eggs of any one species, the defensive chemicals are either of endogenous or exogenous origin, and never from both sources, is also open to question, as is the usual presumption that the exogenous source must be botanical. In fact, in matters pertaining to insect egg defence, our imagination appears not to have kept up with reality.

4.3.1

Fireflies

Take the case of Photuris fireflies.Although the example is one in which the provider is the female, it is presented here because it is illustrative of the levels of complexity that provisioning systems can achieve. Photuris fireflies are nocturnal and they court by use of their light organs. The males fly about emitting trains of light pulses, species-specificin pattern, to which the females answer with a single flash of their own. The female response is timed to follow the male's pulse train with a specific delay, thereby enabling the conspecific male to recognize the signallingfemale as a potential mate (Lloyd,1966). As is now well established, the female Photuris also uses her light organ to hunt male fireflies of the genus Photinus. When these males fly within range, emitting their own characteristically patterned pulses of light, the female Photuris answers them, timing her reply to match the reply interval of the Photinus female. Fooled by the imitation, the male Photinus flies toward the Photuris female and is devoured (Lloyd, 1965) (Figure 4-2). As is also now established, the Photuris female, or "femme fatale" as she is sometimes called, derives more than nutrient from her catch. Photinus males are laden with lucibufagins (for example I) (Eisner et al., 1978), pyrone steroids of defensive potential, which the Photuris themselves do not produce. By eating Photinus, the Photuris female incorporates the lucibufagins systemically and is as a consequence rendered unacceptable to predators such as spiders (Eisner et al., 1997). The female Photuris also transmits lucibufagins to the eggs, which, as has

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Types of Egg Defences

4 Figure 4-1 Egg protection in arthropods. (a) Egg of an unidentified coreid bug (right) imitative of a dew drop. (b) Femalegeophilid centipede (Orphnaeus brasilianus)guarding her eggs. When disturbed the female holds her ground and emits a cyanogenic secretion (Jones e t al. 1976). (c) Female of the cockroach, Eurycotisjoridana, digging a trench in which she will bury the egg case that projects from her rear. (d) Eggs of a noctuid moth (Litoprosopus futilis) protected by a loose cover of scales provided by the mother. (e) Egg of the chrysomelid beetle, Hemisphaerota cyanea. The single egg is encrusted with some of the mother’s fecal pellets. (f) Eggs of the chrysopid, Ceraeochrysa smithi. Chrysopids ordinarily lay stalked eggs, butC.smithiis unusual inthat itdepositsdropletsofa repellentsecretion onthestalks(Eisner et al., 1996a). The fluid provides protection against ants and nourishment for the emergent larvae, which embibe the droplets when they descend along the stalks after hatching. (g) Female of the reduviid bug, Apiomerusflaviventris, harvesting resin droplets from the leaf surfaceofthecompositeplant, Heterothecapsammophi/a.Thefemaleisshown applyingresin to the ventral surface of the abdomen with a hindleg. The resin is eventually applied by the female to her eggs (h), which are laid in clusters and are protected by the coating (Eisner, 1988).

Figure 4-2 The firefly, Photuris versicolor, feeding on i t s prey, Photinus ignitus. By so doing the predator acquires defensive lucibufagins. been shown by laboratory experiments with ants a n d coccinellid beetles, are themselves protected as a result (Gonzalez et al., 2000). The female Photuris i s not totally defenceless without lucibufagins. She i s in possession of a quinoline derivative, N-methylquinolinium 2-carboxylate (11), a compound not k n o w n from another natural source (Gonzalez et al., 1999a), which she apparently herself produces (Gonzalez, 1999), and which she also shares with the eggs. Judgingfrom the deterrency of this compound to ants and coccinellid beetles, the eggs must benefit from the endowment (Gonzhlez et al., 2000).

I1

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Paternal Investment i n Egg Defence . .................... ..

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Chemical Defences: Cantharidin

4.4.1 Meloid Beetles The notion that defensive substances might be transmitted seminally in insects was slow to take hold. One of the early suggestions of its occurrence was by Leidy (1860),who had been studying the notorious beetles of the family Meloidae, known as blister beetles (Figure 4-3) on account of the vesicating toxin, cantharidin (111), they contain. Wanting to know about the distribution of cantharidin in the body of the beetles, Leidy had been applying the various organs of these insects to his skin and keeping track of which ones induced blistering. He obtained strongest reactions with the accessory glands of the male reproductive system, and with a “pyriform sac attached to the generative apparatus of the female” (doubtless the spermatheca). He found both to contain white granular matter, which he judged to be the vesicating principle (now known to be cantharidin). However, since the pyriform sac was at times empty, he concluded that its contents, when present, had been delivered by the male. He also noted, on the basis of the blistering assay, that the eggs contained vesicating principle, but he fell just short of concluding that the eggs received their chemical endowment from the male. Actual demonstration of seminal cantharidin transmission in meloids came later when it was shown that the females of Lyttu vesicutoriu failed to produce cantharidin themselves from injected radiolabelled mevalonate, but ended up containing radiolabelled cantharidin if they had mated with males that had been so injected (Meyer et al., 1968; Schlatter et al., 1968; Sierra et al., 1976).The demonstration that cantharidin thus received by the female is eventually bestowed in part upon the eggs came still later (unpublished data, cited by McCormick and Carrel, 1987). Cantharidin had been shown to be deterrent to ants and carabid beetles at submillimolar concentrations (Carrel and Eisner, 1974), so it seemed reasonable to assume that the eggs benefited from the grft. But actual proof that meloid eggs with cantharidin fare better vis u vis predators than cantharidin-free control eggs has apparently not been obtained. At any rate, it appears that cantharidin synthesis in adult meloidsis restricted to the male (Meyer et al., 1968; Schlatter et al., 1968; Carrel et al., 1993)and that meloid eggs therefore,

Figure 4-3 Meloid beetle

respondingto disturbance by emitting cantharidin-laden blood droplets from its knee joints.

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Chemical Defences: Cantharidin

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

to the extent that they contain cantharidin, receive that endowment from the father. However, cantharidin appears to be produced by larvae of both sexes in at least some meloids (Carrel et al., 1993), so some question remains as to why the larval cantharidin is not retained in adulthood, at least in the female, since without the compound she is made dependent on receipt of cantharidin from the male. Quite extraordinary are the amounts of cantharidin produced by the adult meloid male. In Epicuu tufunebris, for instance (formerly E. pestifera), the adult male biosynthesizes about 17 mg cantharidin, amounting to 10% of his live weight! In E. funebris, unlike in other meloids, little of that quantity finds its way to the eggs (McCormick and Carrel, 1987; Carrel et al., 1993),raising the question whether the female in that species uses the cantharidin exclusively for self-protection. Facts such as these suggest the last word on cantharidin utilization in meloids may not have been spoken as yet. A question of some interest is whether the possession of cantharidin can also impose some risks on the meloid egg. Cantharidin is an attractant to many insects (see next, 4.4.2) and a cantharidin-laden meloid egg could be an easy-to-find ”nugget”for cantharidiphilic predators. Interestingly, larvae of some meloid beetles may themselves prey on meloid eggs (Selander, 1981,1982) Cantharidin is also produced by beetles of the Oedemeridae, a family closely related to the Meloidae (Carrel et al., 1986). Both sexes in oedemerids contain cantharidin and there appears to be no (or at most minimal) seminal transfer of the compound. The adult female contains higher amounts of cantharidin than the male, and the larvae are able to produce the compound; the eggs too contain cantharidin, received evidently from the mother alone (Holz et al., 1994).

4.4.2

97.

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

Cant haridiphiles

A number of insects have an affinity for cantharidin, evidenced by their attraction to the compound. Such cantharidiphilic insects include members of a number of families of the orders Hemiptera, Coleoptera, Diptera, and Hymenoptera. The phenomenon, which in some cases manifests itself also by actual attraction of the insects to meloid beetles (both dead and alive) in nature, has been the subject of reviews (Young, 1984a, b; Dettner, 1997).Gornitz (1937),in a seminal paper on the subject, pointed out that in many species the attracted individuals were mostly or exclusively males, thereby raising the possibility that the compound serves the attracted insects in some reproductive capacity. Studies with a number of cantharidiphiles have now shown that cantharidin is ingested by the attracted males, and subsequently transmitted by way of the female to the eggs.

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Figure 4-4 (a) Head of the pyrochroid beetle, Neopyrochroaflabellata, showing the deep glandular clef3 on the frons. (b) Cantharidin crystals in secretion from cephalic glandular cleft of a N.j/abe/latamale that ate cantharidin (polarized light) (bar = 50 pm).

In the pyrochroid beetle Neopyrochrouflubellu tu (Figure 4-4 and 4-5) (Eisner, 1988; Eisner et al., 1996b, c), the cantharidin ingested by the male finds itself transferred in substantial quantity to the large accessory glands of its reproductive system, and from there, at mating, and presumably as part of the spermatophore, to the spermatheca of the female. The female allocates the gift in large measure to the eggs, but instead of all at once, in gradually rising amounts, so that peak egg endowments (cu. 40 ng/egg) are not achieved until 10-12 days after mating. Predation tests with a coccinellidbeetle showed cantharidin-endowed eggs to have higher survivorship than cantharidin-free controls (Eisner et al., 1996~). When male N.flubelZutu ingest cantharidin, they do not convey the entire quantity to the accessory glands but shunt a fraction to a gland in the head, which secretes the cantharidin into an external cranial depression (Figure 4-4). Prior to mating, as part of the precopulatory ritual, the female reaches into this depression with the mouthparts, thereby testing the secretion for cantharidin content. If the secretion is cantharidin-laden, the female proceeds immediately to mate with the male, but if the secretion is cantharidin-free, indicating that the male did not have access to cantharidin, she rejects him. The male evidently provides an honest advertisement of his cantharidin-donating capacity by way of the cephalic secretion, and the female pays heed to the message by mating preferentially with males that fed on cantharidin. Indeed, cantharidin-unfed males can be rendered acceptable to females if cantharidin is added to their cephalic glands (Eisner et al., 1996b).As will become apparent from some of the examples that follow, the theme of male advertisement in courtship, of the capacity to donate chemicals for the protection of eggs, is a recurrent one with insects.

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Chemical Defences: Cantharidin 99 . ................., .............. . ............... ........................... ........................... . ,,

Figure 4-5 Use of cantharidin (arrows) by Neopyrochroapabellata. The male ingests the chemical (a) and stores it in the cephalic gland, and in the large accessory glands of the reproductive system (b). The female partakes of the secretion of the male’s cephalic gland (b) and as a sequel allows the male to copulate with her. In the course of insemination(c) the female receives cantharidin from the male’s accessory glands and stores the chemical in the spermathecae.Theacquired cantharidin iseventually bestowed bythe female upontheeggs (d) (from Eisner et al., 1996~). In the pyrochroid Schizotus pectinicornis the male also transfers acquired cantharidin to the female at mating, and this cantharidin is transmitted in part to the eggs, where one assumes it functions in defence. Deuterium-labelled cantharidin from males is detectable even in the emergent larvae. Interestingly, the male of this pyrochroid also has a cephalic exocrine gland that secretes cantharidin after ingestion of the compound by the male (Holz et al., 1994; Holz, 1995; Dettner, 1997). In the tiny beetles of the familyAnthicidae (Abdullah, 1969),cantharidin ingested by the male is also destined for partial incorporation into the eggs, and the male has a pair of cantharidin-secreting elytral glands that the female samples in courtship, presumably to test the male for cantharidin content (Schutz and Dettner, 1992; Dettner, 1997). Much remains to be learned about cantharidiphilic insects. It is not even certain in most cases where the cantharidiphiles get their cantharidin. While the principal sources may well be meloids or oedemerids (or their carcasses, eggs, or excreta), the possibility cannot be excluded that the chemical is available from additional sources as well. It is also uncertain that cantharidiphiles invariably transmit cantharidin to their eggs in amounts adequate for defence. Dettner’s review (1997) addresses some of these issues.

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4.5

Chemical Defences: Pyrrolizidine Alkaloids

Many insects sequester secondary metabolites from plants (Rosenthal and Berenbaum, 1991)and in some cases utilize these, in part at least, for protection of eggs. Moreover, in some instances it has been established that the chemicals in the eggs stem in at least some measure from the male parent. Best documented are the cases involving use of pyrrolizidine alkaloids (henceforth referred to as PA or PAS), a group of highly toxic compounds (for instance, monocrotaline, IV) of sporadic distribution in plants (Bull et al., 1968; Mattocks, 1986). The general literature on PA sequestration by insects has been the subject of more or less comprehensive reviews (Boppre,1986,1990;Eisner and Meinwald,l987; Hartmann and Witte, 1995). Emphasis will be given here to examples in which paternal allocation of PA to the eggs has been proven or is likely to occur.

IV

4.5.1

Arctiid Moths

Two moths of the family Arctiidae have been the subject of intensive study, Utetheisa ornafrix, a New World species, and Creatonotos transiens, from Asia. Utetheisa (Figure 4-6) obtain their PA as larvae from their leguminous foodplants (Crotalaria spp.). They retain the toxins through metamorphosis and are protected thereby against spiders, both as larvae and adults (Eisner, 1982; Eisner and Meinwald, 1987; Eisner and Eisner, 1991).At mating, the males transmit PA to the female, most probably through seminal infusion (Dussourd et al., 1988).The female herself may benefit from the male’s gift, in that if she is deficient in PA at the time of mating, she is rendered protected (against spiders) by the gift, from the moment she uncouples from the male (Gonzhlez et al., 1999b).The female also transmits a part of the gift, together with some of her own supply of PA, to the eggs. These are therefore biparentally endowed with defensive substance (Dussourd et al., 1988). Indeed, the PA endowment has been shown to protect the eggs against coccinellid beetles (Dussourd et al., 1988), ants (Hare and Eisner, 1993), and chrysopid larvae (Eisner et al., 2000) (Figure4-6e). While, strictly speaking, the male’s PA contribution to the eggs is merely a supplement (the female seems to contribute the larger share of the eggs’ PA), that supplement, in itself, has been shown in tests with coccinellid beetles to convey upon the eggs a significant degree of protection (Dussourd et al., 1988).

Chemical Defences: Pyrrolizidine Alkaloids

Figure 4-6 The arctiid moth, Utetheisa ornatrix. (a) Adults mating. (b) Larva chewing its way into a seed pod of Crotalaria mucronata. (c) Spermatophore (bar = 1 mm). (d) Scent brushes (coremata)of male (bar = 1 mm). (e) Chrysopid larva (Ceraeochrysa cubana) feeding on Utetheisa eggs. If the eggs contain pyrrolizidine alkaloid, the chrysopid samples a few eggs (as shown here) and then rejects the remainder of the cluster (Eisneret al., 2000).

The male Utetheisa has a pair of scent brushes (coremata) that it everts during close-range precopulatory interaction with the female (Figure 4-6d). The brushes bear a volatile substance, hydroxydanaidal (V),which is of such structure as to leave no doubt that it is derived chemically from PA (Conner et al., 1981).Indeed, the compound is absent in males raised on PA-free diet. Ordinarily, in males that fed as larvae on PA-containing diet, it is produced in quantities proportional to the male’s systemic PA content (Dussourd et al., 1991), and - as was early proposed (Eisner, 1980; Conner et al., 1981) - could therefore serve as the means by which the male proclaims his PA content to the female. Since the amount of PA he transmits to the female is proportional to his PA content (Dussourd et al., 1991), the hydroxydanaidal message could actually provide the female with a measure of the male’s alkaloidal giving capacity, a parameter upon which she could base mate choice. Existing evidence indicates that the female does favour males having higher corematal levels of hydroxydanaidal (Conner et al., 1981; Iyengar et al., 2001) and

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that by so choosing she receives larger alkaloidal gifts. She is thereby enabled to provide better protection for self and eggs, advantages that can be viewed as constituting phenotypic benefits. By choosing as she does in favour of males rich in hydroxydanaidal, the female also receives genetic benefits. High hydroxydanaidal levels in male Utetheisu correlate not only with high systemic PA content (Dussourd et al., 1991),but with large body size (Conner et al., 1990), a trait that is heritable in the moth (Iyengar and Eisner, 1999a). Thus, by mating preferentially with hydroxydanaidal-rich males, the female accesses genes that enable her to have larger sons, sons which in turn will have an edge in courtship and an increased ability to provide for the chemical defence of their mates and eggs (Iyengar and Eisner, 1999b).Accessing genes for largeness will also insure that the female has larger daughters, which by virtue of larger size produce more eggs (LaMunyon, 1997; Iyengar and Eisner, 1999b). The mate choice strategy in Utetheisu is thus seen to revolve around the issue of paternal contribution of both PA and "good genes" to the eggs. Creutonotos also acquires its PA from the larval diet (Schneider, 1992) and presumably derives protection from the acquisition. The female also obtains PA from the male at mating, and she bestows both part of her own PA and part of that received from the male upon the eggs (Niclusch-Rosenegk et al., 1990). The eggs presumably are protected by the endowment. Creutonotos males also produce hydroxydanaidal, in amounts that are a reflection of their PA intake (Bopprk and Schneider, 1985), and they air the compound from a pair of coremata (Wunderer et al., 1986). Remarkably, in Creutonotos, the coremata vary enormously in size. Largeness of the organs correlates directly with systemic PA load (Schneider et al., 1982; Boppre and Schneider, 1985), so that males rich in PA have both large coremata and high levels of hydroxydanaidal. Somewhat unexpectedly, corematal size does not correlate with mating success in the males, at least not in the laboratory (Schneider, 1992). And there is the puzzling fact that field-collected males may have large coremata even when almost devoid of PA (Nickisch-Rosenegk et al., 1990). One would hope that future data will establish not only the natural diet of Creutonotos, but exactly how the males make use of their coremata. A quantitative analysis of the courtship, and of the male aggregation behaviour that seems to be a component of it, would be highly desirable. This is not to detract from the fact that paternal contribution to egg defence has been proven for this elegant insect. Other arctiid moths may also make use of PAS for egg defence. Some species are attracted to PA-containing plants as adults (Pliske, 1975a), an attraction that appears to be mediated specifically by the PAS, since crystalline samples of PASare also attractive (Krasnoff and Dussourd, 1989).Such evidence as exists suggests that the moths feed at the plants, indicating that they are imbibing PAS. Visitation to the plants may involve males, females, or both sexes. One is tempted to predict that the acquired PA eventually finds its way to the eggs of these moths, certainly in part, and that paternal donation of PA to the eggs occurs in those cases where the visitation is by males or both sexes. One is also inclined to predict that mate choice in these moths may revolve around the issue of the PA donation by the

Chemical Defences: Pyrrolizidine Alkaloids

males, and it is conceivable even that in cases where the females alone acquire PA, the male is the discriminating partner. Whatever the truth, mate choice in arctiids may be based on more than chemical criteria, since sound production appears to be a central component of the courtship ritual in a number of species (Comer, 1999; Weller et al., 1999).

V

4.5.2

VI

Danaine Butterflies

Utilization of PAS by butterflies of the nymphalid subfamily Danainae has been well documented. Typically it is the danaine male that procures the PA, and it does so by visitation of PA-producing plants as an adult and feeding from the excrescences oozing from senescent or injured parts of the plants (Edgar and Culvenor, 1975; Pliske, 1975a; Boppre, 1983).The subsequent fate of the acquired PA, and the pheromonal utilization by the male of a derivative of the PA in courtship, bears some similarity to what occurs in the arctiid moths discussed above. The first demonstration that a male danaine transmits acquired PA to the female, and that the female bestows part of the gift upon the eggs, was achieved experimentally with Danaus gilippus, the queen butterfly. The queen in central Florida, where its behaviour had been studied (Brower et al., 1965), has access to a number of PA-producing plants, including Crotalaria specfabilis, whose principal PA is monocrotaline (IV).Males of the queen routinely visit this plant to feed from

Figure 4-7 Male of the queen butterfly, Danaus gilippus, feeding on crystalline monocrotaline.

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Paternal Investment in Egg Defence

the surface of its seedpods, thereby presumably acquiring monocrotaline. Given crystalline monocrotaline (1mg, in honey water) in the laboratory, males consume the offering in its entirety (Figure 4-7), and if subsequently mated, transfer 64% of the acquired quantity to the female. The female in turn bestows an astonishing 93% of the gift upon the eggs (Dussourd et al., 1989).It is assumed, judging from the predation data with Utetheisa eggs, that the gift protects the queen’s eggs as well. Queen males possess abdominal coremata, which were shown by Brower et al. (1965) to be everted by the males during courtship, while fluttering beside the female. Chemical work proved the coremata to bear a compound, danaidone (VI) (Meinwald et al., 1969),which like hydroxydanaidal in arctiids, could be expected to be derived from PAS (Meinwald and Meinwald, 1966). Courtship experiments showed danaidone to have pheromonal capacity. Males lacking the compound were less successful in achieving matings than males that bore it, and males without the compound could be rendered successful if their coremata were experimentally endowed with danaidone (Pliske and Eisner, 1969). It seems possible, therefore, that the corematal pheromone of the queen butterfly has very much the same role as its counterpart in arctiids, namely to serve for “advertisement” of the male’s PA content (and PA-giving capacity) and as a basis for the female’s assessment of the male. In other danaines the courtship strategy may be similar (Seibt et al., 1972), although neither the pheromonal role of corematal products nor the seminal transfer of PASappears to have been established for species other than the queen. Brown (1984a, b) found high levels of PA in the spermatophore of a species of Lycorea and presence of PA in the eggs of a number of danaine species, suggesting that paternal PA transferral to eggs may be widespread in that group of butterflies. All in all, PA acquisition and utilization in danaines and arctiids are strikingly illustrative of the degrees of similarity that can be achieved by convergent evolution. The courtship story may be complicated in danaines by the presence in males of some species of special scent organs in the wings, which the males may bring into physical contact with the abdominal scent brushes. What precisely is accomplished by such contact is not always clear, although in Danaus chrysippus the contact appears to be a prerequisite for danaidone biosynthesis (Bopprk, 1984). By the same token it is unclear to what extent cardenolides, which many danaines sequester from their asclepiad food plants, and which are known to provide the adults with protection against birds, also play a role in egg protection. In the monarch butterfly, Danaus plexippus, for instance, cardenolides appear to be present in the eggs (Brower,1984).It would be interesting to know whether this endowment stems from both parents or the mother alone.

4.5.3

lthomiine Butterflies

Butterflies of the nymphalid subfamily Ithomiinae also utilize PAS (Pliske et al., 1976; Brown, 1984b; Trigo et al., 1996). Long known as the supposed models of

Chemical Defences: Cucurbitacins

neotropical lepidopteran mimicry complexes, ithomiines were assumed to derive their protection from secondary metabolites sequestered from their larval food plants (mostly poisonous Solanaceae). However, extensive experimentation with the orb-weaving spider Nephila claviceps have shown ithomiines to be acceptable to this predator upon emergence from the pupa, but not after they have obtained PASby visitation of PA-containing plants. The PAS are available to them from the nectar in some plants (mostly Eupatorieae), and from the wilting foliage of others (Boraginaceae).Nephila treats PA-laden ithomiines by cutting them from the webs (Brown,1984a, b), just as it had been noted to treat PA-laden Utetheisa (Eisner, 1982). As already noted by Pliske (1975a,b) ithomiine visitation of PA-containing plants is strongly male-biased. This finding, coupled with the fact that males allocate particularly large amounts of PA to the spermatophore (verified by analysis of spermatophores obtained from mating pairs) (Brown, 1984b), strongly suggested that there is male-to-female transmission of PASin these butterflies. Given that the eggs themselves contained PA, it seemed logical to conclude that at least part of the eggs’ PA endowment came from the father (Brown, 1984b). Male ithomiines possess a pair of scent organs on the forewings, consisting of patches of modified scales, which play a role in courtship (Pliske, 1975b). Interestingly, these scent organs contain compounds most probably derived from PAS,providing a third example, besides that of arctiids and danaines, of pheromone production from such alkaloids. In ithomiines, the pheromonal products are butyrolactones (for example VII) that are derived presumably from the acid moiety that esterifies the pyrrolizidine nucleus. Not enough is known about how the ithomiine scent organs are used in courtship to justify proposing that the organs provide the chemical means by which the female assesses the male’s PA content, although it is certainly tempting to advance such a suggestion. Pliske (1975b) proposed that the organs could also serve in male-male interaction. A detailed analysis of ithomiine courtship is evidently very much in order.

4.6

Chemical Defences: Cucurbitacins

Cucurbitacins, oxygenated tetracyclic triterpenes (for example VIII) produced in roots, seeds, leaves and fruit of Cucurbitaceae, are among the most bitter substances known. Parts per billion are detected by humans (Metcalf et al., 1980; Metcalf, 1986). Interestingly, the compounds are not only tolerated but used by chrysomelid cucumber beetles (genus Diabrotica among others). These beetles may or may not ordinarily feed on cucurbits,but in either case have a strong affinity for cucurbitacins

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and will consume crystalline samples of the compounds if p e n the opportunity. In nature, these beetles may obtain cucurbitacins by visitation of cucurbits as adults (Metcalf et al., 1980; Tallamy et al., 1998). Tallamy et al. (2000), working with Diubrotica undecimpuncfafuhowurdi in the laboratory, showed males to transmit the acquired cucurbitacin in part to the female, by way of the spermatophore at mating (the compounds are about 8 times more concentrated in the spermatophore than elsewhere in the male’s body). The females transfer nearly 80% of the gift to the eggs. Whether the male-derived cucurbitacin is sufficient to protect the eggs is open to question. The total amount of cucurbitacin of male origin allocated to the eggs is small (at most 1pg per 250 eggs), certainly relative to the circa 8 times greater quantity bestowed by the female. The female, moreover, does not apparently accrue multiple loads of cucurbitacins through repeated matings, since she is reluctant to accept additional males after a successful first mating. That first mating is sometimes preceded by a number of unsuccessful matings, but the female receives no cucurbitacins in these failed attempts (Tallamy et al., 2000). There is evidence that in nature the male may contribute larger quantities of cucurbitacins to the eggs. Adult D. u. howardi seeking cucurbitacin sources in the field are largely males, suggesting that the female may contribute little if any cucurbitacin for egg protection (Tallamy et al., 2000). The issue of egg defence in Diubroticu beetles is somewhat open to question. While given the extreme bitterness of the cucurbitacins one would expect the compounds to play an anti-predatory role, the data on vulnerability of Diubroticu eggs to predators is inconclusive (for review of literature see Tallamy et al., 1998, 2000). There is however persuasive data to the effect that the cucurbitacins can act by virtue of antimicrobial capacity. Both eggs and larvae of D. u. howurdi proved less vulnerable to infection by the entomopathogenic fungus Meturhizium unisopliue if they contained cucurbitacinsthan if they were cucurbitacin-free (Tallamyet al., 1998). 0

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4.7

I

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The Issue of Paternity

An important question, pertinent to any situation where a male insect bestows defensive chemicals upon a female for eventual transfer to the eggs, is whether that gift will in fact be transmitted to eggs of his siring. Interestingly, so far as these situations have been studied in detail, males do not always have that assurance.

Other Paternal Contributions

In D. u. howardi, as we have seen, where the female may mate only once and receives cucurbitacins from the inseminator only, the male has no problem (Tallamy et al., 2000). In N.flabellata, the female is unable to accommodate another spermatophore for several days after a previous mating. A male is therefore certain of being the father and source of cantharidin for all eggs laid during that period, but only if he was the female’s first partner. Whether males mating with non-virgins have similar assurance remains unknown, since the details of sperm competition have yet to be ascertained for N.flabellata (Eisner et al., 1996~). In Utetheisa the situation is more complicated. Females mate on average with 4-5 males during their life span (Pease, 1968) and there is no last-male sperm precedence. Instead, it is the sperm of larger spermatophores (produced by larger males) that ”win out” (LaMunyon and Eisner 1993,1994).Males therefore stand a chance of being cuckolded by any competing inseminator of larger size. Only as inseminators of virgins, and only with respect to offspring that the female produces prior to her second mating, can males have assurance of paternity and of having provided the eggs’ PA. For the male, therefore, the window of reproductive opportunity may be a small one. For the female, in contrast, every mating delivers at least some PA (and nutrient) that she can use to enhance the supply at hand (Eisner and Meinwald, 1995).

4.8

When Defence Backfires

Substances that in one context provide for protection may in another constitute a handicap. As already mentioned, eggs laden with cantharidin, while protected against ants and other predacious insects ordinarily deterred by the chemical, could well be vulnerable to cantharidiphilic predators. In Utetheisa the risk to the eggs is from cannibalism. The larvae of Utetheisa have been shown to feed on Utetheisa eggs in laboratory tests (Bogner and Eisner, 1991). They do so only when they are systemically deficient in PA, and they direct their attacks preferentially against PA-laden rather than PA-free eggs. Experiments with Utetheisa eggs staked out in nature confirmed the greater vulnerability to larval attack of the PA-endowed eggs. It is suggested that under conditions when PAS are in short supply - as when the Crotalaria food plants are immature or for other reasons short of seeds -larval cannibalization of eggs could be a real factor.A father’s PA contribution to the eggs, like the mother’s, at times when larvae are underendowed with PA, could thus be a liability (Bogner and Eisner, 1991).

4.9

Other Paternal Contributions

In the preceding discussion we focused almost exclusively on paternal contribution of defensive factors effective against predators. With the single exception of the work on Diabrotica (Tallamy et al., 1998), we ignored cases in which the father contributes antimicrobial factors. While there is little literature on paternal contribution of antibiotics, two cases are worthy of mention (compare Chapter 6).

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The first example is intriguing because it involves not direct transmission of antibiotics by the male but induction by the male of antibiotic production in the female. The insect in question is the medfly Ceratitis capitata, in which the female produces a group of antibacterial peptides named ceratotoxins. Expression of these chemicals is restricted to the accessory glands of the female reproductive system and is not affected by bacterial infection. It is, however, enhanced by mating (Marchini et al., 1995; Rosetto et al., 1996,1997).Given their site of production, the ceratotoxins could serve for protection of the female genital tract during fertilization. But they could also serve for protection of the eggs themselves, since laid eggs have been shown to bear a coating of ceratotoxins (Marchini et al., 1997). Exactly how the induction of ceratotoxin production is triggered, whether physically by the act of mating or chemically by a factor transmitted by the male, appears to be open to question. The second example is pioneering in that it demonstrates actual transmission of antimicrobialfactors from male to female. The insect here is that jack-of-all-trades, Drosophila melanogaster. Males of this fly had been shown to produce an antimicrobial peptide, andropin, expressed exclusively in the ejaculatory duct (Samakovliset al., 1990).In an elegant study, based in part on the use of transgenic flies, Lung et al. (2000) have now shown that three male-derived antibacterial proteins, two produced by the ejaculatory duct (and including very probably andropin) and one produced by the accessory glands, are all transferred, at least in part, to females at mating. The chemicals could evidently contribute to the maintenance of asepsis in both the male and the female genital tracts. Sperm and eggs could both be the beneficiaries, the eggs certainly for the hour or so following mating. Since the chemicals do not endure beyond that time, it is unlikely that the eggs derive protection from them after deposition. Drosophila is doubtless not the only insect in which antibiotics are transferred with the sperm. The search for further examples is bound to pay off, and may even yield information of medicinal interest. Evidence to the effect that males, by way of bestowed seminal factors, can influence both the behaviour and physiology of their sexual partners is extensive and the subject of considerable current interest. By mating, males may induce oocyte maturation, accelerated oviposition, inhibition of the female’s remating, and other effects (Eberhard, 1996), all beneficial ultimately to the male himself in that they lead to his increased reproductive success (compare Chapter 2). They are therefore part of the overall package of male strategies that include the chemical protection of eggs, and they could for that reason have been included for discussion in the present chapter. Suffice it to say, there are developments in this area of research that are of enormous interest, particularly from studies of Drosophila, where it has been possible to tease apart the molecular underpinnings of some of the phenomena. In Drosophila, several protein components of the seminal fluid are produced by the male accessory glands and transferred to the female upon mating. These components contribute uniquely, or in combination with sperm or other accessory gland proteins (Acps), to the constellation of post-mating changes in the female.

Puddling

One such component is the so-called “sex peptide” (Acp 70A), a 36 amino acid peptide hormone (Chen, 1996; Kubli, 1996).Upon transmission to the female, this peptide presumably moves into the haemolymph and, possibly by way of multiple targets, elicits two main events: increased egg laying, and short-term decreased female receptivity. Both effects can be induced experimentally, either by injection of synthetic sex peptide into females or by ectopic expression of the peptide in transgenic females under the control of heat shock or tissue-specificdrivers (Aigaki et al., 1991; Schmidt et al., 1993).Another effect of the sex peptide, the stimulation of vitellogenesisand oocyte development, may be evoked indirectly through action of the peptide on the corpora allata, in other words, by induction of increased juvenile hormone production. Indeed, the effect can be duplicated by application of juvenile hormone analogue (Moshitzky et al., 1996; Soller et al., 1999). Another effect of Acps in Drosophilu is the promotion of sperm storage. Males bearing a transgenic construct that drives the selective destruction of certain accessory gland cells virtually lack Acps and have low fertility, even though they produce normal numbers of sperm. Sperm transfer occurs in the absence of Acps, but sperm storagein the seminal receptacle and spermatheca is profoundly affected. Females mating with Acps-impaired male partners store only about 10% of the normal number of sperm (Tram and Wolfner, 1999).The effect has been traced to one of the Acps, Acp36DE, a large glycoprotein that normally becomes tightly associated with sperm in the female reproductive tract (Neubaum and Wolfner, 1999). As might be expected, Acp36DE, through its effect on sperm storage, alters the outcome of sperm competition in Drosophilu. In an elegant series of experiments, Chapman et al. (2000) showed that as second mates, males lacking Acp36DE sired significantly fewer offspring than normal controls, apparently because the sperm of such males are poorly stored. They showed further that in the absence of sperm co-transfer, Acp36DE cannot displace first-male sperm. Acp36DE requires sperm, therefore, to effect its action. Interestingly, Acp36DE may persist in the female’s reproductive tract and aid in the storage of any sperm, including that received from later-mating males. These findings are of broad implication, well beyond what they tell us about a single species of insect. They redefine the concept of paternal contribution to progeny defence, inasmuch as they highlight the importance of chemical factors produced by males in defence and promotion of their sperm. Research in this area is bound to blossom.

4.10 Puddling The sight of butterflies and moths, aggregated by the hundreds, drinking at water sources, has long been a familiar one to naturalists. The phenomenon occurs most commonly at the edge of puddles and has appropriately been called “puddling”. The behaviour is heavily sex-biased and involves for the most part males (Figure 4-8). It had long been suspected that puddling serves for sodium intake (Poulton,

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Figure 4-8 Male of the moth, Gluphkia septentriones, ejecting a n anal squirt while puddling.

1917; Arms et al., 1974).Recent evidence has confirmed this (Smedley and Eisner, 1995), and has shown that the males transfer sodium to females upon mating (Pivnickand McNeil, 1987;Smedley and Eisner, 1996),and that the females bestow the gift upon the eggs (Smedley and Eisner, 1996). One cannot help wondering whether puddling might serve also for uptake and transference of other factors. The possibility that puddlers obtain nutrients by puddling, amino acids and proteins perhaps, has received some recent support (Armset al., 1974;Lederhouse et al., 1990;Becket al., 1999).We feel that it is possible also that the males obtain secondary plant metabolites by drinking, compounds which upon transference to the female could be invested in egg defence. Puddles, under the right circumstances, could be veritable infusions of plant metabolites, and for adult insects that imbibe their foods in liquid form, an ideal source of the compounds. Furthermore, moths and butterflies are reported to imbibe various fluids produced by vertebrates, including perspiration (Collenette, 1934), blood (Banziger, 1975), and lachrymal fluid (Turner et al., 1986; Banziger, 1990), as well as faeces and urine (Norris, 1936; Downes, 1973; Sevastopulo, 1974).In addition to sodium and nitrogenous nutrient, these liquids could contain metabolites with possible defensive activity. Thus, in cases where such drinking is done by males, the stage is set for the potential contribution of these vertebrate-derived substances to female and offspring. The increasingly sensitive techniques now available in analytical chemistry should make possible an investigation of such metabolite incorporation into insect eggs.

4.11 Concluding Remarks Additional cases are bound to be discovered of males transmitting defensive chemicals to their mates, for incorporationinto the eggs. Moreover,one could easily envision such males being put to the test by the females in courtship, to see whether they are sufficiently endowed with the chemicals to be able to bestow adequately large gifts. By the same token, it seems likely that many male insects will be found that manipulate their mates chemically, as Drosophilu males do, by injecting factors with the sperm which, in one way or another - as by triggering oviposition,

References

inhibiting female receptivity, facilitating sperm storage, and promoting asepsis enhance the fitness of the male. But more than anything else, we feel that only the barest essentials are so far known regarding paternal contribution of defensive substances to eggs in insects.

4.12 Acknowledgements Our studies have been supported by grant A102908 from the National Institutes of Health (T.E.), a Fogarty International Research Collaboration Award (National Institutes of Health) (T.E, C.R. and A.G.), Johnson and Johnson fellowships (C.R. and A.G.), and grant R01-NS32684 from the National Institutes of Health (M.V.S.S.). We thank Maria Eisner for preparing the illustrations, and Mariana F. Wolfner (Department of Molecular Biology and Genetics, Cornell University) and James E. Carrel (Department of Zoology, University of Missouri) for helpful input and comments.

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Pliske, TE, Edgar, JA, Culvenor, CCJ. 1976. The chemical basis of attraction of ithomiine butterflies to plants containing PAS.J. Chem. Ecol. 2: 25.5262. Pliske, TE, Eisnir, T. 1969. Sex Fheromone of the queen butterfly: biology. Science 164: 1170-1 172. Poulton, EB. 1917. Salt (chloride of sodium) probably sought by the Hesperidae [sic]. Proc. Entomol. SOC.Lond. 65: 77-80. Ralston, JS. 1977. Egg guarding by male assassin bugs of the genus Zelus (Hemiptera: Reduviidae). Psyche 84: 103-107. Rosenthal, GA, Berenbaum, MR. (eds.) 1991. Herbivores: Their Interaction with Seconduy Metabolites, 2nd edition. Academic Press, San Diego. Rosetto, M, De Filippis, T, Manetti, AG, Marchini, D, Baldari, CT, Dallai, R. 1997. The genes encoding the antibacterial sex-specific peptides ceratotoxins are clustered in the genome of the medfly Cerutitis cupitatu. Insect Biochem. Mol. Biol. 27: 1039-1046. Rosetto, M, Manetti, AG, Giordano, PC, Marri, L, Amons, R, Baldari, CT, Marchini, D, Dallai, R. 1996.Molecular characterizationof ceratotoxin C, a novel antibacterial female-specificpeptide of the ceratotoxinfamily from the medfly Cerutitis cupitutu.Eur.J. Biochem.241:330-337. Samakovlis, C, Kylsten, P, Kimbrell, DA, Engstrom, A, Hultmark, A. 1990. The andropin gene and its product, a male-specific antibacterial peptide in Drosophila melunogaster. EMBO 10: 163-169. Schlatter,C, Waldner, EE, Schmid, H. 1968.Zur Biosynthese des Cantharidins. I. Experientia 22: 994-995. Schmidt, T, Choffat, Y, Klauser, S, Kubli, E. 1993. The Drosophilu rnelanoguster sex-peptide: a molecular analysis of structure-function relationship. J. Insect Physiol. 39: 361-368. Schneider, D. 1992. 100 years of pheromone research: an essay on Lepidoptera. Naturwissenschaften 79: 241-250. Schneider, D, BopprC, M, Zweig, J, Horsley, SB, Bell, T. W, Meinwald, J, Hansen, K, Diehl, EW. 1982. Scent organ development in Creutonotos moths: regulation by pyrrolizidine alkaloids. Science 215: 1264-1265. Schutz, C, Dettner, K. 1992. Cantharidin secretion by elytral notches of male anthicid species (Coleoptera: Anthicidae). Z . Naturforsch. 47c: 290-299. Scott, MP. 1990. Brood guarding and the evolution of male parental care in burying beetles. Behav. Ecol. Sociobiol. 26: 31-39. Seibt, VU, Schneider, D, Eisner, T. 1972. Duftpinsel, Flugeltaschen und Balz des Tagfalters Danaus chrysippus (Lepidoptera: Danaidae). Zeitschr. Tierpsych. 31: 51S530. Selander, RB. 1981. Evidence for a third type of larval prey in blister beetles (Coleoptera: Meloidae).J. Kansas Entomol. SOC.54: 757-783. Selander, RB. 1982.Further studies of predation of meloid eggs by meloid larvae (Coleoptera). J. Kansas Entomol. SOC.55: 427-441. Sevastopulo, DG. 1974. Lepidoptera feeding at puddle margins, dung and carrion. J. Lepidopterists’ SOC.28: 167-168. Sierra, J. R, Woggon, W. D, Schmid, H. 1976. Transfer of cantharidin 1. during copulation from the adult male to the female Lyttu vesicatoriu (“Spanishflies”).Experientia 32: 142c143. Smedley, SR, Eisner, T. 1995. Sodium uptake by puddling in a moth. Science 270: 1816-1818. Smedley, SR, Eisner, T. 1996. Sodium: a male moths gift to its offspring. Proc. Natl. Acad. Sci. USA 93: 809-813. Smith, R. L. 1976. Male brooding behavior of the water bug Abedus herberti (Hemiptera: Belostomatidae).Ann. Entomol. SOC.Am. 69: 740-747. Soller, M, Bownes, M, Kubli, E. 1999. Control of oocyte maturation in sexually mature Drosophilu females. Dev. Biol. 208: 337-351. Tallamy, DW, Gorski, PM, Burzon, JK. 2000. Fate of male-derived cucurbitacins in spotted cucumber beetle females. J. Chem. Ecol. 26: 413-427. Tallamy, DW, Whittington, DP, DeFurio, F, Fontaine, DA, Gorski, PM, Gothro, PW. 1998. Sequestered cucurbitacins and pathogenicity of Metarhizium unisopluiae (Moniliales: Moniliaceae) on spotted cucumber beetle eggs and larvae (Coleoptera: Chrysomelidae). Environ. Entomol. 27: 366372. Thornhill, R. 1976.Sexual selection and nuptial feeding behavior in Bittucus upiculis (Insecta: Mecoptera). Am. Nat. 110: 529-548.

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Paternal Investment in Egg Defence .. . ............................................................................................................

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Tram, U, Wolfner, MF. 1999. Male seminal fluid proteins are essential for sperm storage in Drosophila melanogaster. Genetics 153: 837-844. Trigo, JR, Brown, KS, Witte, L, Hartmann, T, Ernst, L, Barata, LES. 1996. Pyrrolizidine alkaloids; different acquisition and use patterns in Apocyanceae and Solanaceaefeeding ithomiine butterflies (Lepidoptera: Nymphalidae). Biol. J. Linn. SOC.58: 99-123. Turner, JRG, Andrews, M, McGregor, A. 1986. Drinking crocodile tears: the only use for a butterfly? Antenna 10: 119-120. Vahed, K. 1998. The function of nuptial feeding in insects: a review of empirical studies. Biol. Rev. Camb. Philos. SOC.73: 43-78. Weller, SJ,Jacobson,NL, Conner, WE. 1999. The evolution of chemical defenses and mating systems in tiger moths (Lepidoptera: Arctiidae). Biol. J. Linn. SOC.68: 557-578. Wunderer, H, Hansen, K, Bell, TW, Schneider, D, Meinwald, J. 1986. Male and female pheromones in the behaviour of two Asian moths, Creatonotos (Lepidoptera: Arctiidae). Exp. Biol. 46: 11-27. Young, DK. 1984a. Cantharidin in insects: an historical review. Great Lakes Entomol. 1 7 187-194. Young, DK. 1984b. Field records and observation of insects associated with cantharidin. Great Lakes Entomol. 17: 195-199.

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Chapter 5 Brood Protection in Social Insects Manfred Ayasse and Robert J. Paxton

Table of Contents 5.1 5.2 5.2.1 5.2.1.1 5.2.1.2 5.2.1.3 5.2.1.4

5.2.2 5.2.2.1 5.2.2.2 5.2.2.3 5.2.2.4 5.3 5.3.1

5.3.2 5.3.3 5.3.4 5.3.4.1 5.3.5 5.3.5.1 5.3.5.2 5.3.6 5.4 5.5 5.6

Introduction lnterspecific Brood Defence Mechanical Defence Appropriate Nest Site and Camouflage Entrance Turrets Elaborate Enclosure of Nest Entrance and Burrow Plugs Removal of Parasitized or Dead Individuals, Grooming and Hygienic Behaviour Chemical Defence Chemical Barriers Alarm Pheromones Chemical Weapons and Defence Secretions Antiseptic Chemicals lntraspecific Brood Defence Defence against Non-Nestmates Defence against Nestmates: Kin Conflict and Egg Cannibalism Queen-Queen Conflict and Brood Defence Worker-Worker Conflict and Brood Defence Parthenogenic Laying of Female Eggs by Workers Queen-Worker Conflict and Brood Defence Conflict over the Sex Ratio Conflict over Male Production Compliant Brood Cannibalism: Diploid Males Concluding Remarks Acknowledgements References

w Abstract Socialinsects (allants and termites, somebees and wasps) are an amazingsuccessful group, dominating in terms of biomass and energy turnover many terrestrial ecosystems. Though adult social insects are often well protected from predators,

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their brood is not only highly vulnerable but also usually concentrated within the nest, making it a nutritious target for many predators, pests and diseases.A defining feature of truly social (eusocial)organisms is their co-operative care of offspring, which extends to the defence of their brood. We provide an overview of the diversity of predators, pests and parasites of social insect brood and the means by which hosts defend their brood from these enemies.Mechanicalor physical defence is a common means by which adult social insects protect their brood, though they also possess subtle chemical defence mechanisms, often directed against specific parasites or pathogens. Insect eggs lack cellular defence against antigens, and there is no indication of protective chemicals within social insect eggs or attached to them. Brood pheromones used in intraspecific recognition and defence may also mediate a range of intraspecific interactions among adults and, in particular, we review their role in underpinning intracolonial or kin conflict. We suggest future directions for research on the chemical ecology of social insect brood, emphasising the role of experimental manipulations in clarifying the causal basis for brood recognition and protection.

5.1

Introduction

The social insects are those that live together, thereby forming a colony, usually in a physical structure termed a nest, and often for the purposes of producing offspring.True sociality, or eusociality, has been further defined as existingin those species in which there is an overlap of generations of adult individuals, cooperative brood care and reproductive division of labour, i.e. queens and workers (Batra 1966; Wilson 1971).Social insects show a great diversity in their life cycles, nesting habitats, and reproduction. In terms of biomass, they are the dominant group of arthropods in most terrestrial habitats and consequently have a great impact on many ecosystems. There are about 20,000 species of social insects. Most of them are found in the order Hymenoptera, that is, in the ants (all are eusocial; Holldobler and Wilson, 1990),bees (some are social, including eusocial; Michener, 1974,2000;Roubik, 1989) and wasps (several species are social, including eusocial; Ross and Matthews, 1990).Furthermore, all species of Isoptera are eusocial (Krishna and Weesner, 1969, 1970; Abe et al., 2000).During the last decade, eusocialspecies have been also found in other insect orders (see Choe and Crespi, 1997). Social insect colonies with their brood (i.e. eggs and later juvenile stages), with their adults and food stores are highly attractive for many organisms that act as parasites, predators, or even competitors. For example, there are hundreds of organisms ranging from viruses to vertebrates that try to enter a honey bee (Apis) colony to consume part or all of it (Morse, 1978; Seeley, 1985).Social hymenopteran and isopteran eggs lack a cellular defence against invading antigens (Askew, 1971) and, contrary to the situation in other insect orders, do not possess protective

Introduction

chemicals (see Chapter 3 and 4). Nest defence is therefore of extreme importance to social insects, and various mechanisms are thought to have evolved to enhance colony survival (for references see Deligne et al., 1981; Hermann and Blum, 1981; Spradbery, 1991; Bourke and Franks, 1995; Schmid-Hempel, 1998).The other side of the coin is that sociality itself is thought to have evolved in part because of the benefits it provides in terms of brood defence (Lin, 1964; Lin and Michener, 1972). A hallmark of all eusocial insects is their division of labour, most keenly seen in that over reproduction (Engels, 1990), in which the queen lays all or most of the eggs and the workers engage in foraging, colony maintenance and brood care (Wilson, 1971). Many social insect colonies consist of hundreds to millions of workers, and an intruder that enters into a colony may be attacked by them before it can harm brood or the reproductive individuals. Primarily in the wasps and termites but also in other groups of social insects, passive defence by the use of physical nest structures and barriers is found. Chemical defence includes the use of alarm-recruitment pheromones, inciting mass worker attacks, mostly by individuals more or less specialized in nest defence. In some termites and ants, individuals with morphological features, called soldiers, are responsible for these attacks, but also for protecting food trails. Soldiers in termites show enlarged mandibles, a strong sclerotizationof the head, stopper-like heads and glands that produce large quantities of defence secretions (Wilson, 1971; Engels, 1990).In ants, there is a comparable situation. In many species, worker subcastes specialized for nest defence are morphologically different from other worker groups and often show disproportionately large heads (major workers, Wilson, 1971). In bees and wasps, morphologicalworker castes are generallyweakly developed or non-existent (review in Wilson, 1971; Engels, 1990).Division of labour is rather based on worker age (Gerstung, 1891-1926), with underlying genetic predispositions (Page and Robinson, 1991). In honey bees, each individual passes through a regular progression of tasks called the age polyethism (Free, 1965),with the speed of progression of an individual through the polyethism schedule dependent upon colony needs in an apparently adaptive fashion (Robinson, 1992). Behaviouralchanges are accompanied by differing activity of exocrine glands. For example, guarding the nest entrance is usually performed before initiatingforaging, between the ages of 12 and 25 days, when a worker’s mandibular glands produce peak amounts of 2-heptanone and when it is maximally sensitive to this alarm pheromone (Boch and Shearer, 1967).In the genus Vespula,workers older than 30 days spend most of their time near the nest entrance acting as guards. In other groups of social wasps like Polybia, nest guarding is also performed by mid-aged workers. Chemical weapons and defence secretionsare additional features inflicting pain or death on intruders, acting as allomones against predators or as fungicides and bactericides against parasites (Blum, 1981). Schmidt (1990) has suggested that, without venoms, social Hymenoptera would probably not exist due to the almost insurmountable problem of vertebrate predation. Social insect colonies are targets for invertebrates and vertebrates acting as

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Brood Protection in Social Insects

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

..

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

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

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

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

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Table 5-1 Mechanisms of brood protection in social insects. (A) Mechanical defence. (B) Chemical defence (A) Mechanical defence

Appro- Camou- Entrance Closure Cell Abdom- Guards Grooming, inal hygienic priate flage turrets, of nest walls, nest site pedicels entrance, cell cups bursting be haviou r burrow Plugs Ants x XA X XB X Bees x X X X X X X Wasps x X X X X X X Termites x XA X XB X

Taxon

(8)Chemical defence Taxon Chemical barriers

Ants Bees

Alarm

pheromones

Chemical weapons, defence secretions

Antiseptic chemicals

x x

Wasps x Termites x phragmotic heads; morphological soldier castes; C bite; D sting

parasites and predators. Various pathogen and parasite groups of social insects such as viruses, bacteria, fungi, nematodes and mites have been recently reviewed by Schmid-Hempel (1998). However, brood is also endangered by conspecific individuals of other nests that may enter a colony and steal brood. Intraspecific brood parasitism is found in all groups of social Hymenoptera (Wilson, 1971)and, even within a colony, conflicts over reproduction may arise amongst nestmates and influence the survival of eggs and larvae (see Section 5.3). Here we review the various mechanisms of brood protection in social insects (Table 5-1). We focus on the major groups of social insects, the ants, wasps, bees and termites. Because of the breadth of this topic, this review is far from complete. However, we have aimed to give examples of the major mechanisms of brood defence, and refer to recent reports for more detailed information of specific topics. In Section 5.2 we will focus on different forms of interspecific defence and then, in Section 5.3, give an overview of intraspecific defence in which we emphasise intracolonial brood defence. Brood may require protection from conspecific nestmates, and we present the arguments for brood recognition and defence within the conceptual framework of kin conflict, a subject area that has been intensively studied during the last decade from both theoretical and empirical perspectives (e.g. Bourke and Franks, 1995; Crozier and Pamilo, 1996; Queller and Strassmann, 1998). Chemical ecology of brood recognition and protection likely plays a major role therein; however, pheromones that act as recognition signals or discriminators (Holldobler and Michener, 1980) have rarely been identified.

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5.2

Interspecific Brood Defence

5.2.1

Mechanical Defence

lnterspecific Brood Defence

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121.

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Mechanical defence shows different forms and generally protects colonies of social insects from both biotic and physical factors. Utilization of a nest in which to interact with and rear offspring is universal among social insects and represents, in the first place, a form of physical defence for the brood. Social insects such as Eciton army ants that are nomadic and therefore merely form a daily bivouac nevertheless choose protected sites beneath fallen trees or between the buttresses of trees in which to establish a temporary nest (Holldobler and Wilson, 1990). Mechanical defence of the nest or the brood may include: 1)the construction of nest envelops and cell walls and the formation of cell caps (wasps and bees); 2) burrow plugs and other nest entrance blockages; 3) the presence of pedicels (wasps), entrance turrets and tunnels to the nest (bees); 4) a camouflaged appearance of the nest; and 5) removal of parasitized and/or dead individuals, grooming and hygienic behaviour (Hermann and Blum, 1981; Spradbery, 1991; Schmid-Hempel, 1998). 5.2.1.1 Appropriate Nest Site and Camouflage

Choosing an appropriate nest site may be the simplest principle of mechanical defence in social insects. In termites, a suitable site consists of a cavity dug into wood or the ground. The most essential function of a nest is defence of the society against vertebrate and invertebrate predators (Deligne et al., 1981).In many species, the central part of the nest containing brood chambers and food storage rooms is surrounded by an outer wall which is particularly compact and harder than the rest of the nest. In many species of social Hymenoptera, subterranean nests are built in pre-formed cavities or in holes in trees and even within empty seed shells (Spradbery, 1991) or within the nests of more defensive social insects, as when stingless bees nest inside ant and termite nests (Roubik, 1989).The honey bees Apis rnelliferu and Apis cerunu usually nest within enclosed cavities, presumably as a defence against powerful predators such as honey badgers and bears (Seeley,1985). Jeanne (1991) considered predation by ants as a major force shaping wasp nest evolution. In wasps there are two major types of nests (Jeanne, 1975). (1)Open nests without envelopes, as found in the paper wasps, Polistes, Ropulidiu, Mischocyt turus, Belonoguster and Purupolybiu. They are attached to the substrate with a single pillar or petiole that protects nests from ants more easily than those built directly onto the substrate. Often the pedicel is coated by a repellent secretion from abdominal sternal glands (Jeanne, 1970; Hermann and Dirks, 1974; Dani et al., 1996; see Section 5.2.2.1). (2) The second basic nest structure is the closed nest with a surrounding envelope, which restricts access to a single entry. This entrance hole is often guarded by workers, which plug the entrance with their heads or abdomens (see Section 5.2.1.3). Subterranean nests may be covered by a multi-layered envelope which separates the brood combs from soil and detritus of the nest cavity, and presumably also from pathogens and soil-dwelling pests.

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Brood Protection in Social Insects ....... ...... ..................

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

5.2.1.2 Entrance Turrets In certain ground nesting bees, nest entrance turrets are thought to function as a defence mechanism against parasites, other invading bees or insect predators (Stephen et al., 1969).Some stingless bees have long and intricate entrance turrets made of resin and line the inner surface of their entrance turrets with sticky secretions(Michener,1974;Roubik, 1989).For example, species of Plebeiu, Tetragonu, Trigonu, and Tetrugoniscu deposit wax mixed with vegetable gums and resins in the entrance tube against invading insects, mostly ants. Entrance turrets also occur in nests of some South American swarming Polistinae (Jeanne, 1975).

5.2.1.3 Elaborate Enclosure of Nest Entrance and Burrow Plugs Nest entrance blockage is a method of protecting the nest and the brood against damage by water or as means of stabilising humidity and temperature within the nest. However, it may also be an effective defensive mechanism against parasites and predators (Stephen et al., 1969). Many ground-nesting bees plug their nest entrances with soil or with parts of their bodies. For example, many social halictine bees use their heads or the flattened posteriodorsal part of their abdomen to block the nest entrance, and stinglessbees use cerumen (a mix of wax and resin) to close the nest entrance at night (Michener, 1974), during rainfall (W. Engels, personal communication) or when attacked (Roubik, 1989).Pogonomyrmex ants have been observed blocking the entrance of their nests with sand and leaves (Gentry, 1974). Ants with enlarged and flattened "phragmotic" heads that can block and thereby seal a narrow nest entrance from predators (Wilson, 1974)are best demonstrated in subgenera of the formicine genus Cumponotus (especially Colobopsis spp.). Other species of ant possess phragmotic gasters (Brown, 1967). Convergent structures are described in soldiers of the isopteran genus Cryptotermes (Buren et al., 1977; Wilson, 1971). 5.2.1.4 Removal of Parasitized or Dead Individuals, Grooming and Hygienic Behaviour Defence against parasites by social insects also involves behavioural strategies (Schmid-Hempel,1998).In honey bees, several traits are thought to have evolved in order to prevent the spread of diseases and parasites within colonies (Seeley, 1985).For example, guards attack workers infected with chronic bee paralysisvirus more aggressively than healthy bees (Drum and Rothenbuhler, 1985).In the termite Coptotermes, nestmates infectedby nematodes are walled off by workers, essentially sealing them and their parasites off from the rest of the colony (Fuji, in Klein, 1990). Honey bee colonies quickly detect and remove larvae infected with the sacbrood virus (Seeley, 1985).Resistance against the honey bee brood diseases chalkbrood, whose causative agent is the fungus Ascosphaeru, or foulbroods, whose causative agents are the bacteria Melissococcus pluton (European foulbrood, EFB) and Puenobucillus larvae (American foulbrood, AFB), depends in part on the hygienic behaviour of workers who uncap and remove infected brood from their cells

IntersDecific Brood Defence

(Milne, 1983; Bailey and Ball, 1991). Rothenbuhler (1964a) suggested that these hygienic behaviours are genetically based on at least two loci. In the case of AFB, the bacterium usually infects honey bee larvae less than two days old. Larvae become more resistant later on. Infective spores are only produced after the larvae reach the age of 11to 12 days, after which some 2500 million spores per larva may be formed (Bailey,1981).Interestingly, diseased brood is often removed by day 12 of larval life (Woodrow and Holst, 1942; Rothenbuhler, 1964b), at a time before larvae begin to fill with spores. Such infected larvae are either eaten by workers or removed from the nest. Cannibalism or removal from the nest of dead or diseased individuals by nestmate workers is common among all social insects (Wilson, 1971).Within the honey bee nest, a small group of workers, the undertakers, specialise in the task of removing dead adults (Visscher, 1983; Robinson and Page, 1988).A consequence of undertaking is that nests rarely contain dead adult bees. Undertakers probably recognise dead bees by their changed olfactory cues. Pogonomyrmex ants recognise dead and diseased nestmates because they develop an odour rich in oleic acid; even healthy individuals who are experimentally daubed with oleic acid are removed from the nest (Wilson, 1971). In honey bees, the proventriculus forms a filtering device, located between a bee's crop (food storage chamber) and the ventriculus (digestive chamber) (Snodgrass, 1956). Its main function is extraction of pollen grains (5-100 pm in diameter)from the crop for later digestion in the ventriculusby proteolytic enzymes. But it also removes smaller particles such as spores of parasites (Seeley,1985).When workers suck up fluids or chew faeces which contain spores of AFB or Nosema, the spores are quickly concentrated in their hindguts from which they may be defecated away from the nest and thereby removed from susceptible brood (Schmid-Hempel, 1998).In ants, the infrabuccal cavity functions similarly and may prevent infection by microparasites such as microsporidia and bacteria (Eisner and Happ, 1962; Glancey et al., 1981; Sanchez-Pena et al., 1993). Grooming behaviour can help to reduce infections by parasites (Farish, 1972). The termite Reticulitermes frequently becomes infected by the entomophagous fungus Metarhiziium anisopliue (Kramm et al., 1982).Whereas infected workers are most attractive to groomers,individuals that have already been killed by the fungus are normally avoided and not groomed by healthy workers. Honey bees (Apis spp.) infected with ectoparasitic Varroa mites solicit by a special dance grooming from nestmates, and tolerance to these mites by Apis cerana has in part been considered a function of the grooming ability of this host bee (Peng et al., 1987).Grooming of a nestmate may remove parasites from the cuticle surface of a groomed individual. However, at the same time, the grooming individual itself could become infected. Therefore, grooming behaviour could also lead to the transmission of pathogens or parasites.

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5.2.2

Chemical Defence

Chemical defence of the brood is particularly well developed in social Hymenoptera and Isoptera (Whitman et al., 1990).The various forms of chemical defence include chemical barriers, alarm pheromones, chemical weapons and defence secretions acting as allomones against predators or as fungicides and bactericides against parasites large and small. 5.2.2.1 Chemical Barriers

In many cases it is difficult to distinguish between mechanical and chemicalbarriers since the enemy may be mechanically hindered to attack brood by a barrier, but may also additionally be repelled or deterred by a chemical of the barrier. Furthermore, chemical barriers may contain antimicrobial components as will be outlined below. Barriers such as sticky fluids, with or without a repellent function, are described for all groups of social Hymenoptera (Stephen et al., 1969; Hermann and Blum, 1981). In social wasps they can often be found around the entrance to nests or around the pedicel connecting nest to substrate in the early stages of colony establishment. Since these nests are protected by single females only, the risk of predation is extremely high (Spradbery, 1991).In situations when the female has to leave the nest to forage for food or nest building materials, the nest is unprotected and vulnerable to predation. Ants are the dominant predators in the lives of social wasps, which are thought to have evolved specific anti-ant defences. Nests of the wasp Purischnoguster nigricans s e m i are attached to roots (Turillazziand Pardi, 1981). Several rings of a gelatinous, pearly white substance around the nest pedicel protect their nests from ants which are unable to pass over the treated area. The secretion is exuded from the tip of the abdomen and transferred to hind legs, middle legs and thence to the mouth. From here, the secretion is added to the base of the nest and adjacent substrate. In non-swarming Polistinae the eponymous van der Vecht’s organ (Van der Vecht, 1968) is used to apply secretions to the substrate. Many other species of social wasp which build exposed nests suspended from a substrate by pillars also use chemical ant repellents that are applied to the nest support (Jeanne, 1970,1996; Turillazzi and Ugolini, 1979; Kojima, 1983). Interestingly, the pillar-rubbing behaviour is far higher in single-female nests than in nests with multiple foundresses. Rubbing behaviour becomes most intense in nests with mature larvae and less so when only eggs or early-stage larvae are present. After the emergence of the first worker brood, the frequency of application of ant repellency decreases. Post et al. (1984) identified the active compound in Polistes fuscatus as methyl palmitate. In several other species of Polistinae, unsaturated carboxylic acids (palmitoleic acid, linoleic acid) produced in the sternal glands have an ant repellent effect (Dani et al., 1996). Bumble bees, honey bees and stingless bees use sticky fluids as tools in colony defence. Invading insects are immobilized by honey and propolis, a mix of plant

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resins and wax (Stephen et al., 1969; Michener, 1974; Roubik, 1989). Honey bees furthermore use propolis to plug cracks and holes in their nest’s walls, reduce their nest’s entrance opening to render it more weather-tight and easier to defend, embalm the carcasses of invaders (such as mice or wax moths) which they have killed but cannot carry outside, and build a smooth, clean coating over the nest cavity’s walls (Seeley, 1985).Besides filling the purely mechanical roles of glue or space-fillingcement, propolis also serves in colonial defence against fungi, bacteria, and viruses (see Section 5.2.2.4). Stingless bees use plant resins similarly. For example, many species use deposits of wax mixed with vegetable gums and resins as entrance blocking substances (Kerr and de Lello, 1962) to keep ants out. In ants, sticky fluids are released from the mandibular glands (Camponotus [Colobopsis] saundersi, Maschwitz and Maschwitz, 1974) or metapleural glands (Crematoguster influfa, Maschwitz, 1974).The secretion is employed as a defensive fluid, which functions by immobilising predators. 5.2.2.2 Alarm Pheromones

Alarm behaviour mostly occurs as the result of an external threat to a colony or as a result of a signal (chemical or otherwise) produced by nestmates (Landolt et al., 1998).Therefore the most important role of alarm behaviour is to protect the colony and its brood from predators. Alarm pheromones occur in all groups of social insects and are employed in or near the nest. However, in ants and termites, pheromones to elicit alarm behaviour are also emitted outside of the nest when predators or enemies attack workers that are following a food trail. Alarm pheromones in social Hymenoptera are usually produced in defensive glands, often in close connection with their main weapons, mandibles and sting (Blum, 1969; Maschwitz, 1964). Therefore alarm pheromones may have evolved from defensive compounds (Wilson and Regnier, 1971; Holldobler and Wilson, 1990), a reflection of the great semiochemical parsimony of insects (Blum, 1996).Alarm pheromones have been shown to be highly volatile, and are normally produced in large amounts (Blum, 1969). In honey bees, alarm pheromones are produced primarily by cells of the setose membrane surrounding the sting shaft base (Ghent and Gary, 1962; Maschwitz, 1964). Chemical analysis of the volatile compounds extracted from the sting apparatus of guarding workers reveals about 70% isopentyl acetate, with the remainder a complex blend of mostly C4 to Clo alcohols and their acetates (Boch et al., 1962; Boch and Shearer, 1966; Blum et al., 1978; Collins and Blum, 1982,1983; Schmidt, 1998).The mandibular glands in the honey bee worker’s head produce Sheptanone, a compound that is at least 20 times less potent than isopentyl acetate in signalling alarm (Shearer and Boch, 1965; Boch et al., 1970).The alarm pheromone is released from autotomized stings imbedded into attacked animals, thereby creating a chemical signal which guides other guards to the enemy (Seeley, 1985). Alarm pheromones of honey bees and stingless bees can mobilise masses of individuals and orchestrate probably the best insect defence on earth (Roubik, 1989; Schmidt, 1998).

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Alarm pheromones have been identified from seven of the 11extant subfamilies of ants. They are variously produced in pygidial glands, mandibular glands, Dufour’s glands, rectal glands and poison glands (Holldobler and Wilson, 1990; Vander Meer and Alonso, 1998). Alarm pheromones represent a wide range of chemical structures including terpenoids, alcohols, aldehydes, ketones, esters, nitrogen heterocycles, and sulphur containing compounds. These chemicals are volatile and most have a boiling point between 151-263°C (Blum, 1969).As in other groups of social insects, ant alarm pheromones have been proven to be the least species-specificof all pheromone classes; the same alarm pheromones can be found in a number of ant species (see Table 7.4 in Holldobler and Wilson, 1990). For example, undecane is used as an alarm pheromone in many Formica species such as F. aquiloniu and F. rufu. Pheromonal communication of alarm is widespread in the social Vespidae; however it is not universal (Landolt et al., 1998). In Mischocytturus drewensi, the release of alarm pheromones is accompanied by wing buzzing and abdomen wagging, shaking and bending behaviour (Jeanne, 1972).The normal response to an enemy is a mass stinging attack. Alarm pheromones are mostly produced in the poison glands and are therefore released during stinging, when venom is expressed. Alarm pheromones have been isolated and identified in three species of social vespine wasps: Vespa crubro, Vespulu squumosu, and Vespulu maculifrons. Whereas N-3-methylbutylacetamide was identified in both species of Vespulu (Landolt and Heath, 1987; Landolt et al., 1995),2-methyl-3-butene-2-01elicited alarm behaviour in Vespu crubro (Veith et al., 1984).Behavioural experiments have provided evidence for the occurrence of alarm pheromones in several species of Polistinae wasps (reviewed in Landolt et al., 1998).The alarm pheromones within the venom of Polybiu occidenfulis have also recently been chemically characterized (Dani et al., 2000). In termites, defence reactions can be released by various signals from nestmates that include vibratory movements and head banging, or by chemical compounds, i.e., trail and alarm pheromones (Pasteels and Borderau, 1998).The occurrence of alarm pheromones in termites was first demonstrated by Ernst (1959) in a Nusutifermes species. The alarm pheromones in many species have since been chemically identified (reviewed in Pasteels and Borderau, 1998).They are mostly produced in the frontal glands of soldiers and consist of volatile monoterpenes. Soldiers and workers have a complementary role in defence and respond differently during defence recruitment. Soldiers are first recruited by alarm pheromones that act as short-range attractants. Workers are recruited later by trail pheromones or soldier defensive secretions and thereafter participate in defence. Workers bite enemies as well as covering them with faecal material. According to Deligne et al. (1981),defensive secretions in termites have evolved primarily as deterrents against ants. Many of theses exudates are rich in monoterpene hydrocarbons, compounds that have long been known to be selectively neurotoxic to insects (Richards and Weygandt, 1945).

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Interspecific Brood Defence

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5.2.2.3 Chemical Weapons and Defence Secretions According to their mode of action, defence secretions can be classified into three categories (Pasteels et al., 1983). First, sticky, slimy, or entangling secretions can act mechanically rather than chemically (compare Section 5.2.2.1) (Blum, 1981). Second, non-specific irritants acting as repellents can exert their effect via the enemy’s olfactory sense, possibly inactivating or overstimulating its receptors. Third, true poisons act at specific sites or interfere with specific physiological processes in enemies. All categories can be found in social insects. In direct interactions with ants, termites are handicapped by the slowness of their reactions and the clumsiness of their movements (Eisner et al., 1976).Probably as a consequence, they have therefore evolved a great variety of chemical and mechanical defence weapons. Soldiers are mainly responsible for a termite colony’s defence (Howse, 1984; Prestwich, 1984). In some species, they attack and hurt enemies with their sharp mandibles while squirting them with toxic monoterpene hydrocarbons or ketones derived from the frontal gland, salivary gland or cibarial gland (review in Deligne et al., 1981; Pasteels et al., 1983; Prestwich, 1984; Whitman et al., 1990).So-called ”nasutes” secrete and squirt white latex that is rather sticky and consists of a complex mixture of compounds. The secretions fall into three main chemical groups: terpenoid, acetate-derived and compounds derived from amino acid and carbohydrate metabolism (Prestwich, 1984).Soldiers of Globitermes exhibit suicidal defence behaviour. During an interaction with an enemy, they explode by violently contracting their abdomens and a sticky salivary secretion is released from their hypertrophied cephalic glands which, in this genus, extend into the abdomen (Noirot, 1969). In the social Hymenoptera, venom delivered with a sting is an effective weapon in nest and brood defence, acting against invertebrates and vertebrates alike. An overview of the chemistry and pharmacology of hymenopteran venoms can be found in Piek (1986). Stinging may result in sting autotomy in some ants and the honey bees, when the sting and associated structures become embedded in the attacker and continue to pump venom into it after the defending individual has departed (Hermann and Blum, 1981). Unlike termites, most social Hymenoptera do not possess topical allomones or, with a few exceptions among the ants, powerful mandibles (Schmidt, 1990). Those groups of ants which possess most effective allomones and mandibles yet lack defence venoms are derived from ancestors that possessed potent stings (Taylor, 1978). Excluding venomous constituents, no defensive allomones have been identified in social wasps. In social bees, workers of stingless bees use chemicals for defensive-communicative functions (Blum et al., 1970; reviewed in Roubik 1989).Defensive compounds have been identified in various groups of ants (Hermann and Blum, 1981). 5.2.2.4 Antiseptic Chemicals

Social insects coexist with a great number of parasites (reviewed in Schmid-Hempel, 1998). These parasites may be transferred from outside the colony and into the

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nest and from one brood cell to another on contaminated food, nest material and workers. Social insects produce a rich variety of chemical compounds that are important for chemical communication and nest building but also for defence of the brood against such microparasites (compare also Chapter 6). The use of antibiotics by all groups of social insects has been proven beyond doubt (Schmid-Hempel, 1998; Rosengaus et al., 2000). Antibiotics are variously produced in the metapleural, Dufour’s, salivary and venom glands of different social insects and are also present in rectal fluids and body exudates. The fungistatic, bacteriostatic, and nematocidal properties of these compounds have been repeatedly demonstrated (see Rosengaus et al., 2000). Fungicides and bactericides are found in the secretions of mandibular glands and Dufour’s glands of many bees, both solitary and social (Cane et al., 1983; Hefetz, 1987; Roubik, 1989). In soil-nesting species, these secretions are used to line the inner surface of brood cells. The linings are either cellophane-likeand transparent (Colletidae) or waxy (Halictidae, Andrenidae, Anthophoridae). They function variously to provide chemical cues for nesting, as a food source, to control humidity and to defend against microbial infection (Duffield et al., 1984).Females of Philunthus triungulurn, a member of the solitary sphecid wasps that are taxonomically closely allied to bees, adds antifungal chemicals to the cuticle of its prey items with which it mass provisions its offspring, though the source of the antibiotics is not known (Strohm and Linsenmair, 2001). Honey bees also produce antimicrobialsubstances that are present in larval food (Rose and Briggs, 1969),in the royal jelly, the food given to queen larvae (McCleskey and Melampy, 1939),and in one of their storage products, honey (White et al. 1963); honey itself has wide-ranging antibacterial properties (Molan, 1992).One product of the mandibular glands of worker honey bees, 10-hydroxy-2-decenoicacid, is the major component of the lipid fraction of royal jelly which also functions as an antiseptic compound (Blum et al., 1959). Honey bees and stingless bees, in addition to producing glandular secretions, gather plant resins from various species which they mix with wax, and possibly other secretions,and which they use in colony defence against fungi, bacteria and viruses (Seeley, 1985).The chemical composition of propolis has been the subject of several reviews (Ghislaberti, 1978; Marcucci, 1995; Walker and Crane, 1987; Greenaway et al., 1991; Bankova et al., 2000). It consists of 70% resin (natural polymers), 25% beeswax, and 5% volatile oils. Many of the compounds in the volatile oil fraction, mainly flavonoids, exhibit antiseptic properties. For example, pterostilbene is an inhibitor of various fungal species (Lyr, 1961),as are ferulic and caffeic acids, which also possess antibacterial activity against many gram-positive and gramnegative bacteria (Cizmarik and Matel, 1970, 1973).A comprehensive review on the biological activity of propolis has recently been published (Burdock, 1998). Wasps have received little attention with regard to antibioticsecretions (Jeanne, 1996),although their nests are subject to infection by microbes in the same way as those of other social insect groups. Female wasps can often be observed licking or rubbing their gasters against cell walls of nests. This may serve to spread

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antimicrobial secretion to protect the brood and nest from invasion by parasites. In Vespula pennsylvanicu and V. vulgaris, larval saliva showed antibiotic properties that may have a function as an antiseptic (Gambino, 1993). In many ant species the metapleural gland secretes antiseptic substances that protect the body surface and the nest against microorganisms (Maschwitz, 1974; Attygalle et al., 1989; Holldobler and Wilson, 1990; Veal et al., 1992).Metapleural glands are well developed in the Ponerinae,Myrmiciinae,in Nothomyrmeciu mucrops, the only living species of the primitive subfamilyNothomyrmeciinae, and possibly in the extinct Sphecomyrmu (Holldobler and Wilson, 1990). The relatively few diseases found in ants are probably a consequence of the effectiveness of their metapleural gland defence secretions(Holldoblerand Wilson, 1990).In Attu sexdens, the active compound of the metapleural gland was identified as phenylacetic acid. In Cremutoguster deformis, the metapleural gland contains a mixture of phenols, including mellein (Attygalle et al., 1989), that has a dual function as an antiseptic and as a repellent against other ants (Holldobler and Wilson, 1990). Fire ant (Solenopsis)venoms were found to contain antimicrobialand antifungal compounds (Jouvenaz et al., 1972; Cole, 1974) that function as antiseptics and repellents, as does the venom of C. deformis (Obin and Vander Meer, 1985). Fungi-growing termites and leaf-cutter ants may use fungicides to get rid of unwanted fungal infections. The antimicrobal function of frontal gland secretions of Nusutitermes costulis and N. nigriceps soldiers has recently been demonstrated by in vitro assays (Rosengauset al., 2000).Alpha-pinene and limonene reduced spore germination of the fungus Meturhizium unisopilue through direct and indirect (vapour) contact. Since both components are widespread in secretions of social insects, they may have a similar function in other groups, too. Social insects also employ beneficial micro-organisms to combat unwanted micro-organisms, particularly those that spoil food sources or reserves within the nest. For example, Neotropical attine leafcutter ants carry Streptomyces bacteria on their bodies which have an antibiotic effect on destructive Escovopis fungi that can invade the underground fungus garden that these ants cultivate as a food source (see Wilkinson, 1999).

5.3

lntraspecific Brood Defence

5.3.1

Defence Against Non-Nestmates

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Brood defence against conspecifics from other nests is likely a problem for those social insects that are insectivorous (some ants and many wasps) or that, like many ants, engage in the stealing of brood (reviewed in Holldobler and Wilson, 1990). Intraspecific parasitism, in which a female enters the nest of another and replaces the owner’s egg with her own, is also widespread amongst bees and wasps (Field, 1992).In these cases, some of the interspecific defences described in Section 5.2 may also function equally well in intraspecific brood defence, or may even have explicitly evolved in response to brood loss to conspecifics.

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A potentially serious threat to the social insect colony is intraspecificusurpation, in which the resident queen or queens are ejected from the nest by a usurping queen. Nest usurpation is common amongst eusocial Hymenoptera with independent colony founding in the phase before workers emerge (Field, 1992). At this stage of the colony cycle, a resident queen has no workers to support her defence of nest and developing brood and thus must often rely upon physical aggression, though pheromone signalling of dominance (large size) has been implicated in the sweat bee Lasioglossurn malachururn (Smith and Weller, 1989). A usurping queen may gain the nest site or physical structure of the nest if successful in usurpation. Moreover, any brood that she inherits may represent a valuable resource; for the bumble bee Bornbus hypnorurn, usurping queens head colonies comprising more workers than non-usurping queens (Paxton et al., 2001). However, any advantages that accrue to a usurping queen through the inheritance of worker brood may be lost through increased intracolonial conflicts (see Section 5.3.2) that arise from her acquisition of genetically unrelated nestmates.

5.3.2

Defence against Nestmates: Kin Conflict and Egg Cannibalism

Socialinsect workers are highly altruistic in their co-operativebrood care and colony defence, an extreme example being the kamikaze defence by stinging honey bees that results in the defending individual's death. These self-sacrificial traits have probably been favoured by enhancing the fitness of the entire colony, lending support to the concept of the colony as a "superorganism" (Moritz and Southwick, 1992). Notwithstanding this view, it has become increasinglyclear over the past decade that social insect colonies are at the same time a hot-bed of strife amongst nestmates, particularly over reproduction. Though workers in many ants and stingless bees produce non-viable eggs that are consumed by nestmates (termed "trophic" or "alimentary" eggs, Engels and Imperatriz-Fonseca,1990; Crespi, 1992),cannibalism on viable eggs by nestmates is also widespread across the social insects (e.g.Wilson, 1971; Kukuk, 1992), yet it presumably reduces colony efficiency and is therefore difficult to reconcile with the superorganismal view of altruistic workers labouring solely for the good of the colony. Social insect nestmates within one colony are generally not genetic clones of each other (Figure 5-1). Thus it is understandable that the fitness interests of a colony's queen(s) and workers, though aligned, are not necessarilyidentical, and may lead to evolutionaryconflictsamongst nestmates. These so-called intracolonial or kin conflicts have attracted considerable theoretical attention, in particular for the social Hymenoptera. Good overviews are givenby Bourke and Franks (1995)and Crozier and Pamilo (1996).Reproductive skew models broaden and therefore generalise the inclusive fitness arguments by integrating them with ecological and social selection pressures to give a cohesive framework in which to describe social evolution (reviewed in Keller and Reeve, 1999; Johnstone, 2000; Reeve and Keller, 2001). Theoretical models of kin conflict thus have a firm theoretical base and they are rich in predictive power over the

IntrasDecific Brood Defence

a

b

y4

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’f ............. 0.25

*

g

0.25

Figure 5 - 1 Haplodiploid pedigree of nestmates within a monogynous social hymenopteran colony in which the queen has mated with one male (a) or several males (b). Solid lines show the physical transmission of gametes t o offspring whilst dashed lines link collateral kin. Va Iues a re “I ife-for-life” coefficients of relatedness.Conf/ict between workers over the patriline ofnew queens: in (b), workers are more closely related t o full-sisters of the same patriline (0.75)compared t o half-sisters of other patrilines (0.25)and are expected t o favour rearing of full-sisters as new queens, in conflict with half-sisters’ fitness interests. Queen-worker conflict over the sex ratio: under Trivers and Hare’s (1976)original hypothesis, workers in (a) prefer a 3:l (fema1e:male) sex investment ratio because they are three times more closely related t o sisters versus brothers but, in (b), they prefer an even ratio; there is a queen-worker conflict in (a) because the queen is equally related to daughters and sons, regardless of her mating frequency, and so she always prefers an even sex ratio. Alternatively, in (a), workers’ relative relatedness asymmetry (RRA) t o femaleversus male sexual offspring is high and they should favour investing more heavily in female reproductive offspring whilst in (b) their RRA is low and so they should favour investing relatively more in males. Queen-worker conflict over male production: in (a), workers are more closely related to nephews than brothers and so should raise nephews. A conflict exists with the queen; she prefers sons over grandsons to be raised as she is more closely related t o the former. In (b), both queen and workers are on average more closely related t o the queen’s sons versus another worker’s sons; workers are expected t o police other workers‘ reproduction.

occurrence a n d extent of kin conflict (potential or actual sensu Ratnieks a n d Reeve, 1992),usually by assuming that individualsbenefit in h a v i n g n e w sexual offspring raised that are m o r e closely related to themselves. These models have been widely applied to the experimental investigation a n d interpretation of social insect traits (e.g. Queller a n d Strassmann, 1998). G i v e n that kin conflicts are generally played out amongst a colony’s nestmates within the confines of the nest, the olfactory m o d e of communication is likely t o

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be of primary importance in mediating interactions between brood and adults. We now sketch some of the major kin conflicts and highlight the role therein of the chemical ecology of intracolonial brood recognition and defence. For discussion of another kin conflict, that over caste determination, see Bourke and Ratnieks (1999).

5.3.3

Queen-Queen Conflict and Brood Defence

There are a number of cases in which two or more queens share the same nest and lay eggs. Competition amongst them for domination in egg-laying seems a likely outcome, and predictions of the degree of conflict from reproductive skew models have been largely vindicated in empirical studies (Reeve and Keller, 2001). Brood protection may clearly be a necessity for individual queens to guarantee their reproduction, particularly for queens that found nests pleometrotically, i.e. with other, often unrelated queens. Multi-female or pleometrotic associations arise during independent nest founding by queens of many eusocial species, including several ants and wasps plus a few bees (Michener,1974; Holldobler and Wilson, 1990; Ross and Matthews, 1991; Turillazzi and West-Eberhard, 1996).Termite queens generally found nests haplometrotically, without the assistance of other queens (Wilson, 1971).For the majority of pleometrotic ants, the nest founding associationis generally of unrelated individuals, but lasts only for as long as the first workers emerge, soon after which fighting amongst queens eliminates all but one (reviewed in Bernasconi and Strassmann, 1999).During the pre-worker phase, polygynous queens within a nest actively engage in egg cannibalism (e.g. for the ant Messor pergundei, Cahan, 2001). Benefits would accrue to queens that could recognise their own eggs and selectively eat those of others, or selectively feed their own brood. Queens of the pleometrotically nest-founding fire ant Solenopsis invictu are known to add exocrine products associated with the reproductive apparatus onto their eggs duringlaying, of which the alkaloid cis-2-methyl-6-undecylpiperidine derived from the queen’s venom gland is a significant component (Vander Meer and Morel, 1995).Eggs of the single dominant reproductive worker (gamergate) of the queenless ponerine ant Dinoponeru quudriceps are coated in the hydrocarbon 9-hentriacontene (9-C31:1), likely derived from the gamergate’s cuticle; they are not eaten by the gamergate whilst other workers’ (unfertilized) eggs, which are not marked by 9-C31,are eaten (Monnin and Peeters, 1997).Thus these two ants possess a variety of exocrineglands and tissues that label eggs, as do other insects (see Chapter 3). In both S. invictu and D. quudriceps, the pheromone apparently signals that the egg is derived from the queen or dominant egg-layer, respectively, and apparently functions similarly within all colonies as a signal of queen presence or dominance, respectively (Keller and Nonacs, 1993).It is not known how widespread egg-marking is amongst ants. For species that do mark their eggs, analysis of the variation amongst pleometrotic queens in the odour they deposit on eggs and its functional significance are now warranted.

lntraspecific Brood Defence

Pleometrotic Polistes wasps are often related females (but see Queller et al., 2000), though usually only one queen dominates in egg laying; oophagy and eggreplacement by other queens can be intense (Field et al., 1998).Subordinate Polistes fuscutus queens react aggressively toward the dominant queen if eggs are experimentally removed from the nest (Reeve and Nonacs, 1992).Queens probably coat their eggs with Dufour’s gland product during laying; when eggs were rubbed with Dufour’s gland product of subordinates, they were consumed by the dominant (Downing, 1991),suggesting this gland as one source of queen-derived egg-marking pheromone. Dominant queens can distinguish eggs of own versus others’ nests (Downing, 1991), so Dufour’s gland products do not act merely as a marker of queen dominance. It remains an open question as to whether queens can monitor their own and their nestmates’ reproduction, for example via individual-specific odour cues on eggs. Long-term polygynous associations are found in numerous ants (Holldobler and Wilson, 1990)and a number of facultatively social bees (Allodapini, Ceratinini and Xylocopini, see Michener, 1974; Hogendoorn and Velthuis, 1999) and wasps (e.g. Gervet et al., 1996; Strassmann et al., 1997). Polygynous queens of the ant Leptothorux ucervorurn do not discriminate between their own eggs and those of other nestmate queens when engaged in oophagy, though in experiments they preferentially eat non-nestmate eggs over nestmate eggs (Bourke, 1994).Thus, despite the fact that eggs carry a colony-specificodour, either queens cannot mark their own eggs with a long-lasting individual-specific label or they do not use such labels to discriminate amongst nestmate eggs, which is the situation they encounter in natural colonies.Alternatively, as ants store brood in clusters, surface-borne chemicals may easily transfer from one brood member to another; the marking of or recognition of individual brood by ants may not be feasible. Chemical analysis of the pheromonal bouquet of individual eggs from queens of the same colony could help distinguish amongst these possibilities.

5.3.4

Worker-Worker Conflict and Brood Defence

In polygynous eusocialnests or in monogynous nests in which the queen has mated with more than one male, workers would theoretically enhance their inclusive fitness through nepotism, for example by favouring the production of new sexual offspring of, respectively, the same matriline or patriline (see Figure 5-1). Though queen number varies considerably in ants (Holldobler and Wilson, 1990),as does queen mating frequency in social Hymenoptera (Boomsma and Ratnieks, 1996), worker-worker conflicts seem to be rare, at least with respect to the rearing of new queens in polygynous nests of the fire ant S. invictu (DeHeer and Ross, 1997) and in the well studied monogynous and polyandrous honey bee Apis rnelliferu (Tilley and Oldroyd, 1997; Visscher, 1998; Osborne and Oldroyd, 1999). In other social Hymenoptera too (Bourke and Franks, 1995,pp. 225-227), worker nepotism over queen production has not been conclusively demonstrated, suggesting a lack of or suppression of worker-worker conflict, presumably because

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of the costs of discrimination or costs of intracolonial conflict on total colony fitness (Keller, 1997).It has also been pointed out on theoretical grounds that selection is likely to reduce any genetically based patriline-based differences in odour cues (Ratnieks,1991).Moreover, either queen or workers might benefit from scrambling matriline or patriline-specificsignals (Keller,1997; Keller and Reeve, 1999).Analysis of the pheromonal bouquet of larvae coupled to their genetic analysis is needed to determine whether they possess matriline or patriline-specific odours and, if so, whether they are used by workers in their preferential feeding of larvae or, alternatively, in brood cannibalism. 5.3.4.1 Parthenogenic Laying of Female Eggs by Workers

In the Cape subspecies of the honey bee, A. rnelliferu cupensis, workers can lay eggs parthenogenetically that develop into females (workers or gynes, see Velthuis et al., 1990),termed thelytoky. Workers from just a few patrilines predominate in the laying of parthenogenic eggs (Moritz et al., 1999),even when the colony becomes queenless (Moritz et al., 1996).The lack of patriline-biased discrimination of brood by workers is analogous to both the apparent lack of nepotism in queen rearing discussed above and also male-egg laying by “anarchistic” patrilines and the subsequent rearing of worker-laid males in honey bee colonies containing anarchistic patrilines (see Section 5.3.5.2 and Barron et al., 2001). Typically, honey bee worker-laid (haploid) eggs are eaten by other workers in the presence of the queen (Ratnieks and Visscher, 1989; see Section 5.3.5.2). How do cupensis workers avoid their (diploid) eggs being eaten by nestmates? Parthenogenic egg-layers develop the mandibular pheromonal bouquet of a queen (Velthuis et al., 1990).Such cupensis workers might also be able to mimic putative pheromones added by a queen when she lays an egg. One potential pheromonal source is the Dufour’s gland (Ratnieks, 1995).That ovipositing worker honey bees of other subspecies develop a Dufour’s gland odour profile similar to that of the queen’s, rich in wax type esters (Katzav-Gozansky et al., 1997),lends weight to the view that this exocrine gland is the source of cues added to eggs and recognized by workers. However, complementary analyses of egg odours suggest that other exocrine glandular sources are additionally or alternatively used to mark eggs in honey bees (Katzav-Gozansky et al., 2001).

5.3.5

Queen-Worker Conflict and Brood Defence

For the social Hymenoptera, haplodiploidy leads to asymmetric coefficients of relatedness of workers to a colony’s sexual offspring (Figure 5-l), with interesting consequences for queen-worker conflicts. These potential reproductive conflicts are an active field of research, and there have been rapid advances both in the theory underlying them and in empirical testing of hypotheses (reviewsin Bourke and Franks, 1995; Crozier and Pamilo, 1996; Queller and Strassmann, 1998; see also Bourke and Ratnieks, 1999).

lntraspecific Brood Defence

5.3.5.1 Conflict over the Sex Ratio

Social hymenopteran workers are predicted to favour a more female-biased sex ratio than that favoured by the queen (Figure 5-1; Trivers and Hare, 1976; Boomsma and Grafen, 1990,1991).Indeed, analysis of social Hymenoptera sex allocation ratios, in particular their variation among colonies within a population, is now considered a powerful approach to evaluate the role of kin selection in shaping their societies (Chapuisat and Keller, 1999), though not all studies support predictions (Heinze and Keller, 2000). It is implicit in the logic of the theory that workers can both (see below, Section Assessing Relative Relatedness Asymmetry) assess their relative relatedness asymmetry (RRA) to the colony’s female versus male offspring (Figure 5-1) and (see Section Distinguishing Between Male versus Female Brood) distinguish between male versus female brood so as to preferentially rear one, possibly against the fitness interests of the queen or queens. Odour cues are the signals most likely used in these tasks. What evidence is there in support of the two assumptions?

Assessing Relative Relatedness Asymmetry RRA is most likely evaluated indirectly by social hymenopteran workers through the diversity of odour cues amongst nestmates rather than via direct assessment of self to a specific egg or larva (cf. Visscher, 1986).In the primitively eusocial bee, Lasioglossurn zephyrurn, there is a genetic basis to adult worker odour bouquet (Smith and Wenzel, 1988),and workers can recognise and preferentially allow entry to their nest of other workers who are genetically more closely related to them (Greenberg, 1979).In principle, then, genetically more heterogeneous colonies ( e g those headed by a queen mated to several males) should have a greater diversity of worker-derived odour cues by which workers could assess a low RRA compared to genetically more homogeneous colonies (e.g. those headed by a queen mated to one male, see Ratnieks, 1990). For social Hymenoptera that live in colonies comprising hundreds to thousands of workers, environmentally acquired odour cues are important in nestmate discrimination (e.g. in the honey bee, Downs and Ratnieks, 1999).However, workers in these species may also have endogenous, genetically based differences in odour (e.g. Arnold et al., 1996) that would allow indirect assessment of RRA. Myrrnica tahoensis worker ants appear to use larval cues in RRA assessment and adaptive sex ratio biasing (Evans, 1995). The experimental manipulations of Evans (1995) involved mixing nestmate with non-nestmate larvae. Chemical identification of the odour cues used in RRA, whether they be located on brood or adults, and their variation amongst individuals from the same colony is a potentially fruitful avenue of further research. Experimental manipulation of odour cue diversity within colonies that exhibit split sex ratios (or other adaptive shifts in sex ratio) would then be possible, allowing finer analysis of the mechanistic basis for RRA assessment by workers.

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Distinguishing Between Male versus Female Brood By recording the sex of eggs laid by queens and the sex of the sexual adults raised by workers from those eggs, it has become clear that social hymenopteran workers can preferentially raise one sex, to their own inclusive fitness benefit (reviewed in Chapuisat and Keller, 1999). Formica exsecta is a good example, an ant whose workers preferentially eliminate males late in larval development (Chapuisat et al., 1997).However, that F. exsecta workers cannot or do not distinguish the sex of offspring at the egg or early larval stage is surprising as early recognition of sex would be advantageous (Nonacs, 1993).First, early recognition would reduce the waste in time and food resources given to brood of one sex that is subsequently destroyed. Second, eggs and young larvae can be eaten, thus recycling some of the energy they contain, whereas late stage larvae and pupae are often only ejected from the colony, therefore representing a far greater cost. Workers of the Argentine ant Linepithema humile, in contrast, practise fratricidesoon after larvae have hatched (Passera and Aron, 1996). In the honey bee, volatile odour differences exist among male and female brood in cuticular esters and hydrocarbons, though these differences are small and quantitative rather than qualitative (Trouiller et al., 1992, 1994; Aumeier and Rosenkranz, 1999; Santomauro, 2000).Moreover, male honey bee larvae are reared in cells that differ in size and odour from those used to rear female larvae (Free, 1987),giving workers additional cues by which to distinguish sex of brood in this species. It is almost universal among ants that their brood is sorted and grouped within the nest by size classes (Wilson, 1971); thus worker ants probably do not have cues other than those produced endogenously by a larva with which to gauge its sex. Do female and male eggs or young larvae have different odours in F. exsecta and, if so, why do workers apparently not use such cues to eliminate male larvae? What are the odour cues used by this and other social hymenopteran workers in their differential elimination of brood of one sex? An intriguing possibility is that the queen scrambles endogenous brood odour differences so that workers cannot distinguish the sex of egg or young larva, thus forcing workers to raise sexual offspring of the queen’s preferred sex ratio (Nonacs and Carlin, 1990; Crespi ,1992; Nonacs, 1993; see Bourke and Franks, 1995, pp. 240-241). Male brood would be in ”agreement” with odour scrambling as they would avoid being eaten. Support for such an evolutionary scenario demands a better understanding of the chemical ecology of brood discrimination. 5.3.5.2 Conflict over Male Production

Though most social hymenopteran queens monopolise egg-laying, the generally unmated workers can often lay haploid eggs which develop into males (Bourke, 1988).An individual female, either queen or worker, is more closely related to its own sons than those of other females (queens or workers) and therefore would be expected to prefer its own sons to be raised (Figure5-1).However,it is often assumed

lntraspecific Brood Defence

that an individual worker living amongst hundreds or thousands of nestmates cannot defend its own male-eggs against attack by other workers. Hence workers are united into a ”community of interest” (Ratnieks and Reeve, 1992), and an individual worker’s fitness is enhanced if it selectivelyrears either the queen‘s maleeggs when the queen‘s effective mating frequency is greater than two and those of self or other workers when the queen’s effective mating frequency is less than two (Figure 5-1; Starr, 1984; Woyciechowski and Lomniki, 1987; Ratnieks, 1988). In the latter case, there is a conflict between queen and workers over the source of the male-eggs. Remarkable empirical support for this theory comes from the wasp Dolichovespula saxonica whose queens are facultatively polyandrous; in colonies headed by a polyandrous queen, workers eat other workers’ eggs and hence the queen is the source of most male-eggs whereas in colonies headed by a monandrous queen, workers do not consume other workers‘ eggs and hence raise many worker-derived males (Foster and Ratnieks, 2000). The proximate basis of this lun conflict may rest on adults being able to identify the caste of the egg-layer. Can and do they do this?

Laying of Male Eggs by Workers For monogynous social Hymenoptera with a queen mating frequency less than two, for example many bumble bees (Bombus spp., see Paxton et al., 2001) and queenless ponerine ants (see Monnin and Ratnieks, 2001), both queen (or gamergate) and workers may engage in egg-laying and egg cannibalism as well as displaying overt aggression toward ovipositing members of the other party, in agreement with theoretical expectations (Figure 5-1).The outcome of this conflict is variable; in some species the queen or gamergate seems to produce most or all of the male-eggs, to the fitness costs of the workers (e.g. the hornet Vespa crabro, Foster et al., 2000; Bombus hypnorum, Paxton et al., 2001; the ponerine ants Dinoponeru quadriceps, Monnin and Peeters, 1997, and Diacamma sp., Kikuta and Tsuji, 1999) whereas in others the workers probably lay some or many of the male-eggs, to the fitness costs of the queen (e.g. the ant Formica rufa, Walin et al., 1998; Dolichovespula social wasps, Foster and Ratnieks, 2001). Odour is likely to be the primary means by which queen and workers distinguish own from other’s male-brood. Ayasse et al. (1999)have recently demonstrated that eggs of the bumble bee Bombus terrestris have a complex volatile bouquet rich in hydrocarbons. Queen-laid (combined haploid and diploid) and worker-laid (haploid only) eggs from the same colony differed in volatiles, especially in the relative proportions of alkenes and alkadienes in the range CZ3-C3,.These compounds were also found in subtly differing proportions in the Dufour’s gland of the same females whose eggs were analysed, thus this exocrine gland is probably an important source of egg odour cues. Moreover, individuals from the same colony differed in their Dufour’s gland odour profile (Ayasse et al., 1999), giving the possibility of individual-specific egg marking. A queen B. terrestris only needs to differentiate between its own eggs and those of other nestmates (workers) to selectively remove others’ eggs and enhance its

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fitness. A worker, on the other hand, may need to differentiate among queen-laid diploid brood, worker-laid haploid brood and queen-laid haploid brood and selectivelyreplace only the latter to enhance its fitness, a more exactingrequirement. It will now be interesting to determine if and how the queen’s male-eggs differ from workers’ male-eggs and from the queen’s diploid-eggs in pheromonal bouquet, and whether queens or workers can use any odour differences in selective oophagy. For ants whose eggs are gathered into a pile within the nest, workers may shuffle their male-eggs into the egg pile to avoid their detection by the queedgamergate or other workers (seeMonnin and Ratnieks, 2001). For B. terrestris, Bourke and Ratnieks (2001) conclude that workers are unable to discriminate between queen-laid male and female brood until they have developed well into the larval stage. There could be selection for the party in control of male-egg production to mark their male-eggs with a specific signal and for the other party to disguise its maleeggs to smell like those of the party in control. As noted above (Section 5.3.5.1), a queen may alternatively scramble the odours of her male and female eggs as a means of ensuring that her male eggs are raised by workers. Bioassays will therefore be needed to determine if queen and workers can use putative brood odour differences in selective eating of the other party’s eggs, and should provide deep insight into the role of kin conflict in shaping patterns of male production. Worker Policing In monogynous social Hymenoptera with high queen mating frequency, both queen and workers may be expected to agree on the queen as the source of maleeggs, and “worker policing” is expected (Woyciechowski and Lomniki, 1987; Ratnieks, 1988); that is, workers are expected to limit the reproduction of other workers, for example by eating their eggs (Figure 5-1). For the monogynous and polyandrous honey bees (Apis spp.), the queen is generally the source of all maleeggs (reviewed in Barron et al., 2001).Additionally, there is empirical support for worker policing in Apis; workers differentiallyeat worker-produced male-eggs but not those of the queen (Barron et al., 2001). If queen and workers do evolutionarilyagree on the queen as the source of maleeggs, selection might be expected to lead to a distinguishable label on queen-laid male-eggs so that workers could selectively rear them and eat worker-laid maleeggs; both signal producer (queen) and signal receiver (worker police) would thereby benefit (Ratnieks and Reeve, 1992). Bioassays using A. rnelliferu have demonstrated that the Dufour’s gland and/or adjacent associated exocrine glands and structures of the queen reproductive apparatus are the source of a putative egg odour, and that workers use this odour in worker policing (Ratnieks, 1995; Katzav-Gozanskyet al., 2001; see Section 5.3.4.1). Odours adhering to queen-laid male-eggs are rich in hydrocarbons with smaller quantities of esters and aldehydes, many of which were not detected in the queen Dufour’s gland (Katzav-Gozansky et al.,2001); odours adhering to worker-laid male-eggsawait chemicalidentification.

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lntraspecific Brood Defence

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Anarchistic Workers Within a very small minority of honey bee (A. melliferu) colonies, workers from one or a few "anarchistic" patrilines may lay male eggs which are successfully raised by other workers, even when the colony is queen-right (Oldroyd et al., 1994; reviewed in Barron et al., 2001), in contradiction to the hypothesis of worker policing. It appears that workers from anarchistic patrilines avoid policing of their (male)eggs through a variety of mechanisms, including (see Section Laying of Male Eggs by Workers) greater acceptability of eggs laid by an anarchistic patriline worker and (see Section Worker Policing) reduced policing abilities of workers in colonies containing anarchistic patrilines, i.e. reduced ability to discriminate between queen-laid versus worker-laid male-eggs (Oldroyd and Ratnieks, 2000). As we have pointed out above, chemical identification of the odours added by workers to their male-eggs is lacking, though would be very helpful in designing manipulative experiments to unravel how and why policing functions in the majority of honey bee colonies, yet can be circumvented by anarchistic patrilines.

5.3.6

Compliant Brood Cannibalism: Diploid Males

For the haplodiploid Hymenoptera (females are diploid, males are haploid), peculiarities of their mechanism of sex determination mean that diploid males are sometimes produced (reviewed in Cook, 1993; Cook and Crozier, 1995). Such diploid males have a broad taxonomic occurrence within the Hymenoptera, including social species (e.g. Paxton et al., 2000 and references therein), and they generally have low to zero fitness. They therefore represent a cost, a genetic load, to the colony. For colonies that produce diploid males, one possibility is for these individuals to be consumed at an early developmental stage, thus reducing their burden on colony resources. This might benefit all colony members, including diploid males themselves who have little or no reproductive potential, because resources may be spared and reinvested into new kin: gynes, workers or haploid males. Conflict between colony members over the fate of diploid male brood would therefore be absent, and selection should favour diploid male brood giving an honest signal to nestmates that they should be eaten, a cannibalism cue. Rather than brood protection, this is its corollary, namely compliant brood advertisement. Honey bee workers cannot differentiate among diploid male versus female eggs but they consume all diploid male larvae in the 24 hours following larval hatching whilst feeding the equivalent aged diploid female larvae, suggesting that diploid male larvae possess a cannibalism signal (Woyke, 1963).This signal is most likely a self-produced pheromone, though its chemical identification has remained elusive. The odour bouquet of freshly hatched diploid larvae comprises a series of alkanes, alkenes and esters in the range C23-C29,though there is no qualitative differencein bouquet between diploid female and diploid male larvae; rather, larvae differ quantitatively in bouquet and diploid male larvae have a total quantity of odour higher than that of female but lower than that of haploid male larvae

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(Santomauro, 2000). Additional biotests are required to determine which compounds, ratio of compounds or concentrations represent the cannibalism cue in honey bees. The presence of adult diploid males in other social Hymenoptera (e.g. some Formica ant species, Pamilo et al., 1994) suggests that cannibalism cues are not universal.

5.4

Concluding Remarks

It is clear that social insects possess a variety of defence mechanisms to protect their colony and its brood from interspecific attack by a wide range of pests, predators and pathogens (Schmid-Hempel, 1998; Table 5-1). Many of these mechanisms are underpinned, directly or indirectly, by odours used variously as pheromones, allomones and kairomones; indeed, social insect workers are superlative chemists, utilisingboth self-synthesizedand environmentally acquired compounds in defence (Blum, 1981).Understanding of the functional significance of alarm pheromones has been enriched through the identification of their active constituents and their use in field bioassays (Free, 1987; Holldobler and Wilson, 1990; Landolt et al., 1998).For other chemical-based defence mechanisms such as the daubing of the nest pedicel with abdominal secretions, the biologically active compounds await detection in most species. Moreover, the functional role of chemistry is not always fully apparent in these instances. Compounds may serve functions in addition to defence within the colony and therefore their use may be exposed to a variety of selectivepressures. Identificationof putative active substances and their use in manipulative experiments will allow their functional significance in interspecific brood defence to be evaluated more directly and convincingly. We foresee considerable advances being made in the coming years in the field of intraspecific defence. Brood defence in social insects takes on an additional dimension when considered in the light of kin conflict, a body of theory which provides general, plausible and predictive hypotheses to guide future research on social insects (e.g.Queller and Strassmann, 1998).Egg and larval recognition odours (pheromones or discriminators) undoubtedly play a major proximate role in intracolonial conflict. Yet little is known of their source and composition (cf. Ratnieks, 1995; Katzav-Gozanskyet al., 2001). There are some relevant outstanding questions which we hope will inspire further study. What is the chemical identity of putative pheromones on brood that act as recognition signals? Do they originate from the egg-layer or are they endogenously produced by the brood itself? If the former is the producer of such pheromones, then from which glandular source do they originate? Given that brood communication is likely to be rife with deception, manipulation and cheating, appropriate bioassays will also be required to determine whether and how pheromones influence brood recognition and defence by adults.

References

O u r contention is that a better understanding of proximate mechanisms will provide a more comprehensive account of the role of kin selection in shaping social insect societies and, at the same time, make possible manipulative experiments that examine the ultimate factors shaping reproductive conflicts.

5.5

Acknowledgements

We wish to thank A. Bourke, W. Engels, A. Hefetz a n d P. DEttore for providing helpful comments o n this chapter. We further gratefully acknowledge the help of E. Zellinger in its preparation. We were supported by grants from the FWF Austria a n d the DFG.

5.6

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Chapter 6 The Role of Microorganisms for Eggs and Progeny Rupert L. L. Kellner

Table of Contents Introduction 6.1 insect Adaptations towards Transmission of Symbiotic Microorganisms 6.2 6.2.1 Transmission upon the External Surfaces of the Eggs 6.2.2 Tra nsova rial Transmission 6.2.3 Transmission Bypassing the Egg in Viviparous Insects 6.2.4 Larval Uptake of the Symbionts Infections which Reduce Survival of the Host 6.3 6.3.1 Cytoplasmic Incompatibility and Male Killing 6.3.2 Egg Pathogens 6.3.3 Oviposition by Pathogen Infected Females 6.3.4 Defences against invasion of Pathogenic Microorganisms Attraction of Gravid Females towards Oviposition Sites by Cues 6.4 of Microorganisms Beneficial Effects of Microorganismsfor Eggs and Progeny 6.5 6.5.1 Nutritional Interactions 6.5.2 Defensive Substances Concluding Remarks 6.6 Ac k nowIedgeme nt s 6.7 References 6.8

rn Abstract Insects are associated with a large variety of microorganisms having a wide range of different functions for eggs and the resulting progeny. A main objective during female egg deposition is the maintenance of an existing mutualistic relationship between insect and microorganism for the future generation. Endosymbiotic microorganisms are applied onto the egg surface or transmitted transovarially into the oocyte. The larvae themselves often play an active part in taking up symbionts from the eggshell or preying on conspecifics. Indeed, egg cannibalism might be a mechanism to fulfil larval requirements for microorganisms. Ovipositing females have been shown to be attracted by chemical lures of microbial origin. While the insect serves as host for the symbiont, the microorganism may have positive effects

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.

on the insect by providing nutrients or defensive chemicals. However, not only mutualistic microorganismsbut also pathogenic ones may be transmitted vertically and horizontally to the eggs. But the eggs and progeny are not unprotected against infections as they may be provisioned with defensive substances by their mother.

6.1

Introduction

The environment of insects is largely determined by macroscopically visible organisms, but unicellular microorganisms are present almost everywhere. Mutualistic symbionts may be essential for insect life as they live inside the host insect and provide digestive enzymes, nutrients or defensive chemicals. On the other hand, numerous pathogenic microorganisms are known to attack insects. Thus, the insects have to cope with hazards on the one extreme and to preserve benefits on the other. During the insect life cycle, the egg stage is particularly vulnerable as it is immobile and depends on resources provisioned by the mother. As it is supplied with plenty of nutrients that suffice until hatching of the larva (Chapter l),it provides a good substrate for pathogenic microorganisms. The risk of infection can, however, be reduced by antibiotic compounds or by the females choosingappropriate oviposition sites.While pathogens might be quite ubiquitous in certain localities (e.g. the soil), the occurrence of mutualists is much more restricted as they may be confined to one host species.Therefore, mechanisms have evolved for securemaintenance of such valuable microorganismsduring successive generations.

6.2

Insect Adaptations towards Transmission of Symbiotic Microorganisms

As non-social insects usually do not meet their progeny and may even die soon after reproduction, the females are responsible for supply of their eggs with mutualistic microorganisms that are not readily available in the environment. In these cases of vertical transmission, the egg is a link between stages of completely different morphology that often depend physiologically on the biosynthetic capabilitiesof these microorganisms.However, microorganismsthat do not benefit the insect harbouring them are known to exploit these transmission paths, as will be outlined below for Wolbuchiu (see Section 6.2.2).

6.2.1 Transmission upon the External Surfaces of the Eggs Symbionts that inhabit the gut lumen do not really enter the insect’s body and generally are transmitted outside of the eggs. In this mode of transmission only the eggs’ external surfaces are contaminated with symbionts. Extracellular as well as intracellular endosymbionts can be transmitted by inoculation of the external surfaces of the eggs.

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Insect Adaptations towards Transmission of Symbiotic Microorganisms

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Smearing of the eggs’ surfaces with faeces containing microorganisms from the digestive tract is regarded as the most primitive mode of symbiont transmission (Buchner, 1953).In its basic form, this may only ensure permanent associations of an insect with one type of gut bacteria, as reported from the Western flower thrips, Frunkliniellu occidentulis (de Vries et al., 2001). Nevertheless, in some insects, the terminal parts of the genital and digestive tracts are specifically structured to ensure transmission of the symbionts to the progeny. This may be illustrated by three examples: (1)The endosymbioticbacteria of the olive fruit fly, Buctroceru oleue (formerlyDucus oleue), are mainly contained in midgut caeca during larval stages. They survive metamorphosis in a bulbous diverticulum of the esophagus and, after eclosion, they recolonize the gut. For transmission to the eggs, great numbers are harboured in saclike evaginations of the hindgut. That part of the hindgut is separated from the oviduct only by a membrane containing a slit opposite to the evaginations’ openings which facilitates smearing of the individual eggs with bacteria. Oviduct and hindgut have a common opening at the base of the ovipositor (Steinhaus, 1946; Buchner, 1953). (2) The chrysomelid Cussidu species (e.g. Cussidu viridis) possess 2 to 3 tubular pouches that apply a cap of bacteria to the eggs passing by through the vagina (Buchner, 1953). (3) In Donuciu species (e.g. Donuciu sernicupreu) two of the six malpighian tubules have specialized for multiplication of the symbionts that are then delivered via the hindgut (Buchner, 1953). Symbionts may also be harboured in mycetocytes, large and often polyploid cells that enclose numerous endosymbionts. These specialized cells may be located together in an organ, the mycetome. Symbionts inhabiting midgut mycetocytes that are smeared onto the eggs occur, for example, in the brown-winged green bug, Pluutiu stuli, a pentatomid (Abe et al., 1995).Yeasts are involved in mycetocyte formation in certain beetles. Although harboured in larval mycetomes encircling the midgut, the yeast symbionts of cerambycid beetles (e.g. Lepturu rubru) are not retained in mycetocytes during the adult stage. While the larvae rely on the symbionts for polysaccharide digestion due to their xylophagous habit (e.g. cellulose, Campbell, 1989), the adults do not need them for purposes other than transmission to the progeny. The females keep their symbionts in intersegmental pouches of the ovipositor. At the beginning of oviposition, the vaginal pockets, which are other integumental folds near to the genital opening, are filled with the symbionts that are then smeared over the eggs (Steinhaus, 1946; Buchner, 1953).

6.2.2

151

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Transovarial Transmission

While the intracellular symbionts found along the alimentary canal often leave their host for transmission to the progeny and thereby obviously recapitulate a mode of transmission developed early in their symbiotic association with the insect, intracellular symbionts inhabiting mycetomes in the body cavity are

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transmitted directly into the egg. Cockroaches, for example, contain flavobacteria in mycetocytes of their fat body (Bandi et al., 1994). In the German cockroach, Blattellagermanica, amoeboid mycetocytes migrate from the fat body to the ovarioles and are partially embedded in the epithelium of the ovariole sheath. By exocytosis, symbionts leave the mycetocytes, pass the follicular epithelium mainly between neighbouring cells and are concentrated in the space between oolemma and follicle cells. Covering the whole oocyte they are in contact with microvilli of the oolemma. After vitellogenesis, they are actively phagocytosed by the mature oocyte (Sacchi et al., 1985; Sacchi et al., 1988). Other transovarial infections are confined to one pole of the oocyte, especially to the posterior one. In species harbouring more than one endosymbiont, they are often transmitted simultaneously or successively via the same route. Symbionts released from the mycetomes are transported through the haemolymph and reach the ovarioles (Buchner, 1960). In viviparous morphs of aphids, they traverse the follicle through gaps at the posterior pole of the oocyte and enter the blastula via the blastopore. In leafhoppers, however, special follicularcells at the posterior pole are first entered that release the symbionts to the other side (Houk and Griffiths, 1980). The anterior pole is used for infection of the oocyte in curculionid beetles and coccid homopterans. Early in embryo development of curculionids (e.g. Hylobius abietis) symbionts are taken up into the germ cell line, where they survive only in the nurse cells of the female sex. From these cells they are transported through the nutritive cords to the oocyte (Buchner,1953).In the bamboo pseudococcid, Antonina crawii, two endosymbionts belonging to the y- and P-Proteobacteria, respectively, are harboured in the same mycetocytes. They are embedded in peculiar mucous spherules in the cytoplasm. Free spherules transmit the endosymbionts to the nutritive cord junction between the oocyte and its nurse cells that is specifically infected (Fukatsu and Nikoh, 2000). Recent research in another pseudococcid, the citrus mealybug, Planococcus citri, has shown that the mucous spherules may indeed be identical to the P-Proteobacteria. These are the primary endosymbionts of mealybugs with internal y-Proteobacteria, which thus may be regarded as own endosymbionts of the P-Proteobacteria (von Dohlen et al., 2001). Intracellular endosymbiontsoften are pleomorphicand reside in the mycetocytes in a non-dividing vegetative form. Dividing infectious forms, which occur mostly extracellularly, are released from parental mycetocytes and are passed to the oocyte. Both a- and t-symbiontsof leafhoppers have three membranes in vegetative form but only two in infectious form. The third membrane of the vegetative form is supplied by the host (Houk and Griffiths, 1980). Specialized parasitic microorganisms are able to invade the ovarioles and use similar modes of transmission. Wolbachiaare parasitic intracellulara-Proteobacteria that are rather widespread in insects. Reported infection rates range from 16%in neotropic or neotemperate insects over 50% in Indo-Australian ant species up to 76% in selected arthropods, mainly insects, and various insect orders are affected (Werren, 1997; Wenseleers et al., 1998; Jeyaprakash and Hoy, 2000). Like other

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Insect Adaptations towards Transmission of Symbiotic Microorganisms

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intracellular endosymbionts they are transmitted transovarially.This is exemplified by Wolbuchiu in Aphytis aphelinid wasps: They are concentrated in the nurse cells and move to the oocyte, where they cluster around the pole cells at the posterior pole opposite to the nurse cells and micropyle (Zchori-Fein et al., 1998). Wolbuchiu is transmitted transovarially also in tsetse flies (Aksoy et al., 1997), however, these glossinid flies show additional ways of transmitting further microorganisms (see Section 6.2.3).

6.2.3

Transmission Bypassing the Egg in Viviparous Insects

A special case of symbiont transmission that skips the egg stage is found in hippoboscid and glossinid flies, whose larvae develop in the female uterus where they are fed with milk gland secretions (Buchner, 1953).In their gut tissue, tsetse flies (e.g. Glossinu morsitans) harbour two endosymbionts belonging to the y-Proteobacteria: Primary (P-) endosymbionts, Wigglesworthiu glossinidiu, inhabit specialized mycetocytes in the anterior portion of the gut and secondary (S-) endosymbionts, Sodulis glossinidius, occupy midgut epithelial cells. While the Pendosymbionts’ mode of transmission remains unknown (but is non-transovarial), the S-endosymbionts are obviously transmitted through the milk gland (Aksoy et al., 1997; Dale and Maudlin, 1999).

6.2.4

153.

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Larval Uptake of the Symbionts

Intracellular symbionts that are transmitted inside of the oocyte already develop in the embryo, while all symbionts transmitted on the egg’s surface need to be taken up by the larva. If the microorganisms are allowed to enter the egg through the micropyle as in the olive fruit fly, Buctroceru oleue, they are present inside the gut at hatching of the larva (Steinhaus, 1946). Some symbionts are ingested as a by-product of the hatching procedure: Larvae of the chrysomelid reed beetle Donacia semicupreu need to eat through a gelatinous egg coating that contains a distinct accumulation of bacteria near to the head. Likewise, other insect larvae like that of the cerambycid and chrysomelid beetles mentioned above (see Section 6.2.1)eat (part of) the eggshellinoculated with symbionts (Steinhaus,1946; Buchner, 1953). More activity of the larvae is needed in the plataspid bug Coptosomu scutellatum: neonate larvae have to search for bacteria-containing capsules that are dispersed among the eggs of one batch. A capsule is pierced and the content is consumed before the larva will feed on the host plant (Buchner, 1960). Symbionts that are not obtained in sufficient numbers from the own eggshell can also be acquired if the larva preys on conspecifics. Pederin biosynthesis, which is an indicator of endosymbionts’ presence in so-called (+)-females of Puederus staphylinid beetles, is easily lost from one generation to the next. Symbiont-free larvae fed with (+)-eggs derived from (+)-females take up symbionts and thereby acquire biosynthetic capabilities (Kellner, 1999,2000).Although symbiont-free, socalled (-)-females are found in field-collected females of most species (Kellner,1997),

154

The Role of Microorganisms for Eggs and Progeny

100

n=132

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279

0

1

2

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18

80

60

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0 No. of (+)-eggs eaten -

Figure 6-1Percentage of (+)-females (with 95% confidence interval) in laboratory-reared Paederus sabaeus offspring of (+)-femalesthat are enabled by their endosymbionts to biosynthesizepederin (n: number of females reared), as compared to collected females (Coll.,

n: number of females collected).During the rearing, larvae either had no access to (+)-eggs or they consumed 1-3 (+)-eggspresented to them. Several larvae of individual (+)-mothers were analysed and any possible, although unproved, bias ofthe outcomedue tothe individuals was reduced by as large a number of mothers per test as possible (no eggs fed: 39 mothers, 1egg: 17 mothers, 2 eggs: 111mothers, and 3 eggs: 7 mothers). Furthermore,the (+)-eggs fed to individual larvae were most often descendedfrom other (+)-femalesand not from the own mother. The tests were performed during a four-year period. The resulting ratio of (+)and (-)-females(incapableof biosynthesis)clearly depends on the number of (+)-eggseaten b2 = 56.2, df= 3, P < 0.001).

the predominance of (+)-females in natural populations appears to be based on intraspecific cannibalism (Figure 6-1), which is the only known means of retaining such high infection rates (compare Section 6.5.2) Egg cannibalism is generally considered in terms of its nutritional benefit or the accompanyingreduction of competition. Although these may be important criteria, deficient numbers of endosymbionts could also be supplemented by oophagy. Especially in herbivorous insects, there are intriguing reports of egg cannibalism. For example, after hatching pierid butterfly larvae (Pieris r a p e crucivora and P. melete) first eat their own eggshell and then proceed with nearby eggs (Watanabe and Yamaguchi, 1993; Watanabe and Ohura, 1997).In that case and numerous other ones, potential requirement for symbionts that could be transmitted upon the eggshell is a new hypothesis that would merit examination (compare Chapter 3).

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6.3

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Infections which Reduce Survival of the Host

Infections which Reduce Survival of the Host

As opposed to mutualistic endosymbionts, various microorganisms clearly have detrimental effects on those insects that they successfully colonize. Pathogenic bacteria and fungi may infect females or the eggs and consecutively cause death of the specimen. Some bacteria with detrimental effects on their hosts live in long term association with them, as is well known for Wolbuchiu.

6.3.1

Cytoplasmic Incompatibility and Male Killing

The already described transovarial transmission of Wolbuchiu (see Section 6.2.2)is extraordinary for bacteria that have detrimental effectson their hosts. The negative effects exerted by Wolbuchiu upon infected insects have recently been reviewed in detail (Werren, 1997; Stouthamer et al., 1999) and only their impairment of the eggs is considered here. Wolbuchiu is able to manipulate host reproduction in two ways: (1)cytoplasmic incompatibility of the parents and (2) killing of male eggs. Hence, the eggs appear sterile. In cytoplasmic incompatibilitythe father is infected by a certain Wolbuchiu strain while the mother is either uninfected or infected with a different strain. Although the eggs are fertilized, the paternal chromosomes are lost resulting in haploid embryos. In diploid species the embryos die whereas a male-biased sex ratio is produced in haplodiploid wasps as the haploid embryosdevelop into normal males. If the father is uninfected, however, normal progeny is produced whether the mother is infected or not. Cytoplasmic incompatibility is known from all major insect orders (Stouthamer et al., 1999). Male-killingWolbuchiu have only been reported from two beetle species (the two spot ladybird, Aduliu bipunctutu, and the black flour beetle, Tribolium mudens) and a nymphalid butterfly, Acrueu encedon (Hurst et al., 1996; Jiggins et al., 1998; Hurst et al., 1999; Fialho and Stevens, 2000). The maternally inherited infection considerably lowers egg hatch rates but the reasons for embryo death are unknown. Male-killing Wolbuchiu might be more common in insects and perhaps this ability of Wolbuchiu is easily evolved (Stouthamer et al., 1999; Fialho and Stevens, 2000). Other bacteria like Spiroplusmu and Rickettsia are also known as agents of male killing showing that it is not confined to Wolbuchiu (Stouthamer et al., 1999; Jiggins et al., 2000). Although they reduce the number of certain females’ offspring, Wolbuchiu, which can only be transmitted maternally, thereby increase the infection rate of a population, which enables them to spread (Werren, 1997; Stouthamer et al., 1999).

6.3.2

155

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Egg Pathogens

Other microorganisms are true pathogens as they infect the deposited eggs from outside and finally cause their death. Such pathogens can be isolated from soils and include both fungi as Beuuveriu bussiunu (Hyphomycetes)and bacteria as Bacillus sphuericus (gram-positive bacteria). The latter is a heterogenous group of bacilli containing insecticidal strains, which produce spherical endospores. A parasporal

156

The Role of Microorganisms for Eggs and Progeny

crystal is composed of two protein toxins that are active against mosquito larvae, particularly Culex (Boucias and Pendland, 1998). One of these highly larvicidal strains has been reported to effectively inhibit hatching of phlebotomine (Phlebotomus duboscqi and Sergentomyia schwetzi)sand fly eggs. Whether B. sphaericus toxins cross the eggshell is unknown (Robert et al., 1998). The enterobacterium Serratia marcescens is an example for an opportunistic entomopathogen (Boucias and Pendland, 1998) that may be present harmlessly within the gut but soon causes death after having entered the haemolymph. It is frequently found in insectaries.For example, in the noctuid Heliothis virescens it is transmitted both horizontally and vertically (mainly by external contamination of the eggs) and it reduces adult longevity. Infected females may deposit a small number of eggs with acute infection that turn red (Sikorowskiand Lawrence, 1998). Several entomopathogenic fungi can penetrate insect cuticular barriers (Hajek and St. Leger, 1994; Boucias and Pendland, 1998),which makes it possible for them to also invade the eggs. However, only Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces lilacinus (Hyphomycetes) have been shown explicitly to possess pathogenic potential during the egg stage (Storey et al., 1991; Tallamy et al., 1998). Scarcity of reports on effects of ubiquitous pathogenic bacteria and fungi on insect eggs may be related to their inability to infect that stage (see Section 6.3.4). The performance of the egg and its comparison with other stages, however, have not been analysed systematically.Egg hatch of Colorado potato beetle, Leptinotarsa decemlineata, was not affected by spraying with Beauveria bassiana conidia, nor did the hatched larvae suffer from higher mortality (Long et al., 1998).On the other hand, eggs of the silverleaf whitefly, Bemisia argentifolii, were found to be immune to Verticillium lecanii (Hyphomycetes) infection, although mortality of nymphs hatched from treated eggs was very high (Gindin et al., 2000).

6.3.3

Oviposition by Pathogen Infected Females

Treatment of insects with pathogens has been shown to reduce both their fecundity and fertility. In the Mediterranean fruit fly, Ceratitis capitata, fewer eggs are laid by females after exposure to various pathogenic fungi. Furthermore, the percentage of hatching larvae is reduced, especially after application of Metarhizium anisopliae, while Penicillium chrysogenum and Verticillium lecanii exhibit moderate effects (Castillo et al., 2000). Vertically transmitted bacteria may also be able to reduce fecundity, as reported from a Rickettsia (a-Proteobacteria) in the pea aphid, Acyrthosiphon pisum. In that case, the reduction at 20°C depended on the host plant while the facultativebacterium could even have an ameliorative effect on the severe effect of an elevated temperature of 25°C (Chen et al., 2000). When using pathogens for pest control, the interaction of the applied pathogens with naturally occurring microorganismsneeds consideration. For example, in the grasshopper Melanoplus sanguinipes, where adults and nymphs are susceptible to Beauveria bassiana infections, the soil was inoculated with conidia to investigate whether the fungal infection of the soil affects mortality rates of females depositing

Infections which Reduce Survival ofthe Host

into the soil. However, the soil microflora of non-sterile field soils adversely affects the efficacy of B. bassiana (Inglis et al., 1998). Decreased viability of eggs laid by infected femalesmay be a result of insufficient nutrient supply. In the grasshopper Melanoplus bivittatus no differences were found in the total lipid content of eggs from control and infected females which had been treated with cysts of MaZamoeba Zocustae (Rhizopoda), but relative abundance of unsaturated fatty acids varied. Although the fat bodies were affected by the pathogen, female metabolism apparently operated sacrificiallyto maintain the lipid level of eggs (Jackson et al., 1968). In contrast to the expected negative effects of pathogens on fecundity of females, increased ovipositionby infected females has also been reported. The increase was elicited by injectionof Serrutia marcescens into the haemolymph of the house cricket, Achetu domesticus, resulting in a potentially fatal bacterial infection. In view of a low probability of future reproduction, the adaptive value of increased reproductive output at the expense of survival was suggested as an explanation of this paradox. However, it is unknown whether the increased oviposition is related to egg production or is due to accelerated laying of already mature eggs (Adamo, 1999). In the latter case the females would have made the most of their time left and overall fecundity might be reduced nevertheless.

6.3.4

Defences against Invasion of Pathogenic Microorganisms

Although immobile and dependent on the mother’s choice of oviposition site, the eggs are not unprotected against attacks by pathogens. An important protective device is obviously the secretion of an eggshell that is an efficient barrier against invasion (Chapter 1).Furthermore, various insect females apply substances upon or into their eggs that protect them against attack by predators, parasitoids, and pathogenic microorganisms. Such compounds are known to be of different origin. They may be produced either de novo by the female or be sequestered from the mother’s diet (Chapter 3) or they originate from nuptial gifts provided by the mate (Chapter 4). A phytochemical with defensive activity against pathogens of the eggs is sequestered by the chrysomelid cucumber beetle Diabrotica undecimpunctuta howardi from its cucurbit host plants. Females incorporate cucurbitacinsobtained from their plants into the eggs and, thus, effectively reduce the pathogenicity of the entomopathogen Meturhizium anisopliae. Bitter eggs (with cucurbitacins) survive exposure to conidia concentrations differing by several orders of magnitude while the hatching rate of non-bitter eggs, which were obtained by feeding females with a cucurbitacin-free diet, is greatly reduced and is related to conidia dosage (Tallamy et al., 1998). Cantharidin, the terpenoid anhydride produced by beetles in the families Meloidae and Oedemeridae, exhibits strong in vitro activity against fungi (Trichophyfon and Microsporum species). Meloid males biosynthesize cantharidin and transfer large amounts during copulation which are bestowed upon the eggs.

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As these are deposited in the soil, it has been argued that the protection of developing embryos from entomopathogenic fungi appears to be the toxin’s main function (Blum, 1996). However, there is no evidence available that proves this directly (compare Chapter 3). Another defensive compound, the pyrrolizidine alkaloid monocrotaline sequestered by the arctiid moth Utetheisu ornutrix, was tested against two entomopathogenic fungi (Beuuveriu bussiunu and Puecilornyces lilacinus). Both monocrotaline-containing and monocrotaline-free eggs proved to be vulnerable to the fungal infections. Furthermore, the free base and the N-oxide (the latter also tested against Meturhiziurn unisopliue and Puecilornycesfurnosoroseus) had no in vitro inhibiting effect on the fungi (Storey et al., 1991). The presence of antimicrobialsubstanceson the egg surfaceis a further constraint for the success of pathogens, in addition to moisture levels, temperature, and UV light (Vega et al., 1997).The first example for antibacterial coating of insect eggs has been reported in the Mediterranean fruit fly, Cerutitis cupitutu: accessory reproductive glands in sexually mature females produce three antibacterial peptides, called ceratotoxin A-C (Marchini et al., 1993; Rosetto et al., 1996). At oviposition, the accessory gland secretion is spread onto the eggs where it is clustered in droplets that contain the antibacterial ceratotoxins (Marchini et al., 1997). After hatching of the larvae, the progeny themselvesare able to acquire chemical protection by sequestering plant natural products from their diet. Thus, they do not rely on maternal supply. Sequestration of antifungal plant compounds has been observed, for example, in larvae of the cucumber beetle Diubroticu undecirnpunctutu howurdi, which acquire cucurbitacins in addition to the maternal endowment of the eggs already mentioned above (Tallamyet al., 1998).Inoculation with entomopathogens per 0s may be inhibited even without actual sequestration of a toxic compound. In case of ingestion of the pathogen together with fungicidecontaining foliage, germination of conidia is suppressed as they get in contact with the plant toxin inside the insect’s gut (Costa and Gaugler, 1989b).The pathogen will be removed from the gut without infection of the host. Susceptibility of the Colorado potato beetle, Leptinotursu decernlineutu, larvae towards Beuuveriu bussiunu was reduced when larvae consumed plants containing glycoalkaloids, although they only tolerate but do not sequester these compounds (Costa and Gaugler, 1989a).

6.4

Attraction of Gravid Females towards Oviposition Sites by Cues of Microorganisms

Besides pathogenic or mutualistic relationships, microbial assemblages have been shown to attract certain insect species. Insect oviposition is mediated by various chemical cues which are used by females to identify the appropriate site. In herbivorous species plant volatiles attract females and non-volatile chemical cues of the host plant may elicit recognition and oviposition (Chapter 7).Oviposition

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Attraction of Gravid Females towards Oviposition Sites by Cues of Microorganisms

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behaviour of numerous carnivorous insects is known to be affected by chemicals released from their prey and their prey’s host plant (Chapter 10, 11).However, some insects have been found to be attracted by chemical cues that are not emitted by the host plant itself but originate from microorganisms (Dicke, 1996, and references therein). The chemical composition of the attractive blends has not yet been characterized. In particular, oviposition by several dipteran species has been shown to be influenced by the presence of microorganismsat the oviposition site. For example, host finding and oviposition has been studied in detail in several anthomyiid fly species of the genus Delia. In the cabbage root fly, Delia radicum, oviposition into the soil depends both on quality of nearby host plants and on a substrate with moist particles beneath a dry surface.Volatile chemicalsignals from the root-substrate complex containing microorganisms that are associated with plant decomposition stimulate oviposition (KoStfil et al., 2000). In the seedcorn maggot, Delia platura, specific bacteria stimulating oviposition have been identified: Flavobacterium sp., Erwinia herbicola, and Xanthomonas campestris contribute to the stimulating effectsof moist soil and decaying organic matter with other bacteria being less stimulatory. Odours proved to be the attractive factor because females preferred inoculated substrates even in the absence of direct contact (Hough-Goldstein and Bassler, 1988). Microbial activity on or near the plant has been suggested as a main factor for oviposition in the onion fly, Delia antiqua, as it may influence the volatiles produced (Ellis et al., 1979). Infested onions show short range attraction of females due to by-products of microbial metabolism (Judd and Borden, 1992). Production of these volatile metabolites during plant decomposition is based on a complex interaction of different environmental factors. However, distinct bacterial strains give reproducible headspace profiles on certain substrates, which differ in their attractancy for gravid females. For example, Erwinia carotovora cultured on onion is more attractive than Klebsiella pneumoniae on the same substrate or E. carotovora on potato (Hausmann and Miller, 1989). The compounds identified so far are n-dipropyl disulfide, a predominant onion secondary metabolite, 2-phenylethanol and pentanoic acid. The synthetic chemicals are, however, unable to simulate the effect of the natural blend (Dindonis and Miller, 1981; Hausmann and Miller, 1989). Another case of microbial attractancy has been reported from the apple maggot fly, Rhagoletis pomonella. This tephritid fly is known to be attracted by olfactory lures like protein hydrolysate that may mimic bacterial odours. Several bacterial isolates were visited more often by females than by males (MacCollomet al., 1992). Gravid females search for bacteria and application of antibiotics to the fruit surface greatly reduces infestation (MacCollom et al., 1994).Emission of attractive odours is limited to members of the Enterobacteriaceaeand certain strains of Enterobacter agglomerans have been shown to be most attractive (Lauzon et al., 1998).As the bacteria are found not only at oviposition sites but also in the alimentary tract of

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R. pomonella, this relationship might have an almost symbiotic quality when the bacteria, after colonizationof the gut, may be transferred from a contaminated site to other fruit that are in turn rendered more attractive for the flies, increasing the probability of another colonization. Indeed, attractancy of microbial odours appears to be a rather widespread phenomenon in Diptera and is not only well-known in Drosophila flies (e.g.Barker and Starmer, 2000). Agar plates inoculated with decomposing onion microorganisms were found to be even more attractive to the house fly, Fannia canicularis, than to the onion fly, Delia antiqua, mentioned above (Dindonis and Miller, 1981). Furthermore, gravid female mosquitoes (Culex quinquejasciatus and C. tarsalis) are attracted by fermented infusions of dried bulrushes, Schoenoplectus acutus, which also stimulate oviposition (Du and Millar, 1999)(compareChapter 10).The presence of bacteria may indicate valuable protein sources and thus affect foragingbehaviour of flies, as has been suggested for Rhagoletis pornonella (MacCollom et al., 1994). Microbial presence and odour has not only been shown to affect foraging and oviposition of dipteran species. Microbial odour is also known to attract sap beetles (Nitidulidae).These beetles transmit fungi among their host plants, e.g. the diseasecausing Fusarium verticillioides (= F. moniliforme, Hyphomycetes) in corn. Several strains analysed of F. verticillioides consistently produced a volatile blend of five alcohols (most abundantly ethanol), acetaldehyde, and ethyl acetate which in wind-tunnel bioassays attracted the nitidulid Carpophilus humeralis (Bartelt and Wicklow, 1999). A remarkable example of microbial oviposition stimulation is known from a specialized siricid woodwasp, Xeris spectrum. Siricid and xiphydriid woodwasps are known to kill trees as their larvae bore tunnels in the wood where they live in symbiosis with basidiomycete fungi. Adult females possess mycangia filled with fungal spores that are lacking in the males. During oviposition the trunk is inoculated with the fungi that assist the offspringin utilizing the nutrient-deficient wood (Kajimura, 2000). Whereas other siricids live in symbiosis with specific Amylostereum fungi, Xeris spectrum femalescarry no fungal spores and deposit their eggs into logs already inoculated by fungus-carrying females of other species. Lack of a specific Amylostereum fungus may account for the extended host range and wider distribution of X . spectrum (Fukuda and Hijii, 1997).

6.5

Beneficial Effects of Microorganisms for Eggs and Progeny

In most tight associations of microorganismsand insects the host benefits from the metabolic activities of its accompanying symbionts. The main exception is the infection of insects with Wolbachia, where such beneficial effects are unknown (see Section 6.3.1).

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6.5.1

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Beneficial Effects of Microorganisms for Eggs and Progeny

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Nutritional Interactions

Although the egg stage is an important link for vertical transmission, the positive effect of mutualistic relationships is postponed to other, often larval stages. In ectosymbioticrelationshipslike that between woodwasps and basidiomycete fungi, the larva’s digestion of plant material is aided by the symbiosis. Presumably, Amylostereum fungi render the host tree more assimilable as they are able to decompose polysaccharides, like cellulose, and l i p n (Fukuda and Hijii, 1997). Other xylophagous larvae like cerambycid beetles host their endosymbionts for similar purposes (Campbell, 1989). Whether it is an ecto- or an endosymbiotic relationship, the larvae depend on symbionts to make use of their diet. The mycetocyte endosymbiont of aphids, Buchneru uphidicolu, which belongs to the +Proteobacteria, is particularly well analysed with regard to its nutritional interactions with the host. The bacteria supply the aphids with essential amino acids that are deficient in their phloem sap diet. For methionine, both biosynthesis and transfer to the host tissues have been shown experimentally. Other amino acids probably provided by Buchneru are tryptophan and aromatic amino acids. Nitrogen recycling may occur, but is not quantitatively important and aphids are nutritionally independent in case of lipid biosynthesis (Douglas, 1998). The physiological evaluation of Buchneru’s role was complicatedby the fact that neither the host nor the endosymbiont can be reproduced independently. An elegant solution of this problem is the sequencing of the complete genome of Buchneru sp. APS, a strain harboured by the pea aphid, Acyrthosiphon pisum. That strain was shown to have genes for the biosynthesis of amino acids essential for the host, including isoleucine and the pyruvate family (valineand leucine)that share several biosynthetic enzymes. On the other hand, genes for non-essential amino acids are almost completely lacking. Thus, Buchneru needs such non-essential amino acids (glutamate and aspartate) as precursors for biosynthesis of essential amino acids (known as nitrogen recycling from physiological experiments). These mutually dependent pathways allow a much reduced genome size of the obligate endosymbiont (Shigenobu et al., 2000). Vitamins have been suggested as microbial metabolites in a number of endosymbiotic associations. Although vitamins are not generally supplied by Buchneru in aphids (Douglas, 1998), young Acyrthosiphon pisum aphids could be shown to need provision of riboflavin (vitaminB2complex)by Buchneru (Nakabachi and Ishikawa, 1999).In the brown-winged green bug, Pluutiu stuli, carotin (vitamin A,) and vitamin E were detected only in the gastric caeca, where the symbionts occupy mycetocytes (Abe et al., 1995).Tsetse flies (Glossinu sp.) may be provided with B-group vitamins by their endosymbiotic Wigglesworthiu. While awaiting the sequencing of the Wigglesworthiu genome, the application of gene arrays from the related Escherichiu coli preliminarily confirmed the presence of biosynthetic enzymes necessary to produce the B-vitamins (Aksoy et al., 1997; Akman and Aksoy, 2001). The presence of the intracellular endosymbiont of the rice weevil, Sitophilus oryzae, which belongs to the y3-Proteobacteria, decreases larval developmental time.

162

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Panthotenic acid and riboflavin, two vitamins of the B, complex, are supplied by this bacterium (Heddi et al., 1999). Nutritional interactions between host and symbiont could also be important during egg production and embryo development in some cases. Endosymbionts present in the ovarioles of the German cockroach, BlutteZZu gerrnunicu, are in close contact with microvilli of the oolemma throughout ovarian development. This tight association and their long stay in the ovarioles may indicate that they are involved in oogenesis. Possibly they supply nutrients (Sacchiet al., 1988).Furthermore, they may be involved in embryo development as they invade and lyse some of the yolk granules, allowing specialized vitellophages to protrude towards the yolk (Giorgi and Nordin, 1994; Sacchi et al., 1996).

6.5.2

Defensive Substances

In addition to substances of nutritive value, endosymbionts might produce compounds that are advantageous for defense against pathogens or carnivores. Microorganisms associated with eggs may produce antibiotics which prevent the larvae hatching from such eggs from pathogen infection. For example, the brown-winged green bug larvae, Pluutiu stdi, die from opportunistic infections caused by fungi and bacteria when deprived of endosymbionts and hatching from sterilized eggs (Abe et al., 1995). Endosymbiotic microorganisms associated with eggs of a herbivorousinsect have also been shown to produce antibiotics that inhibit growth of a plant pathogen.

Figure 6-2 Structures of a n antibiotic, andrimid (a, Fredenhagen et al., 1987),and a haemolymph toxin, pederin (b, Kocienski et al., 1996),that are available to insects due to

endosymbioticbacteria.

Concludina Remarks

The antibioticcomponent andrimid (Figure6-2) was isolated from the culture broth of symbionts obtained from brown planthopper eggs, Nilupurvuta lugens. This compound exhibited strong specific activity against the white blight pathogen of rice plants, Xunthornonas curnpestris pv. oryzue (Fredenhagen et al., 1987).It has been suggested that the planthoppers might be protected against destruction by pathogens invading the host plant (Ishikawa, 1989). However, it is unknown whether the compound is only produced in vitro or is indeed supplied to the planthopper. Insect substances with defensive effects against antagonists are generally assumed to be either products synthesized de novo by the insect or derived from the host plant (Chapter 3,4). However, pederin (Fig. 6-2), a defensive compound against wolf spiders, is accumulated by staphylinid beetle females of the genus Puederus (e.g.P.fuscipes, P. ripurius)only if they possess their specificendosymbionts. These are called (+)-females. (-)-Females lacking endosymbionts lay eggs without toxin (compare Section 6.2.4).Larvae descended from both types of female as well as the males cannot biosynthesize the compound (Kellner, 1998,1999).If (-)-larvae are attacked by wolf spiders they are at risk of being eaten while their counterparts that had received maternal pederin are unpalatable and survive the attack (Kellner and Dettner, 1996). The (+)-larvae are thus chemically protected against wolf spider attacks by a metabolite provided by their endosymbiont-harbouring mother. The endosymbionts, which belong to the y-Proteobacteria, are transmitted via the surfaces of (+)-eggs laden with the product and enable only the resulting females to biosynthesize the toxin (Kellner, 1999,2001,2002).

6.6

Concluding Remarks

In the first half of the 20th century analysis of the insects’ relationship to microorganisms was largely confined to phenomenological descriptions of the interactions and histological studies of symbionts’ localization. Today molecular biology techniques such as PCR (polymerase chain reaction), gene sequencing, and in situ hybridization help to identify the microorganisms.For example, these techniques led to the discovery that pseudococcids actually contain two endosymbionts. Previously, they had been regarded as monosymbiotic although the symbionts were observed to exhibit morphological variations that could only be interpreted as transformations (Fukatsu and Nikoh, 2000).Such modern studies on endosymbiont localization have, however, been performed only in a few insect species (e.g.Fukatsu and Nikoh, 1998; Fukatsu et al., 1998; von Dohlen et al., 2001). Furthermore, the differentiation between symbiosis and pathogenesis that has been stressed traditionally appears to be of low importance when considering the molecular mechanisms regulating infection of insects by symbiotic and pathogenic microorganisms.With the exception of the different consequences for the insect, no general principles could be found justifying this distinction (Hentschel et al., 2000). For example, both pathogens and endosymbionts rely on type I11 secretion systems for infection of insect cells. There may be thus a parasitism-mutualism

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continuum enabling horizontally transmitted parasites to evolve into mutualistic endosymbionts that are transmitted vertically (Dale et al., 2001). Such an in-depth analysis of the molecular mechanisms is only feasible through isolation and maintenance of maternally transmitted insect endosymbionts. This was successful for the first time in Sodulis glossinidius, using co-culture with insect cells (Dale and Maudlin, 1999; Dale et al., 2001). Every stage in the relationship of insects and microorganismscan now be followed more precisely than with previous morphological and culture techniques. This includes both transient gut bacteria that may be found only a short time after ingestion together with the food and obligate endosymbionts that have lost a whole set of genes normally essential for bacterial life. The continuing advance and widespread availability of molecular biology techniques provide a set of methods to the entomologist by which new insights into the fascinating association of insects and microorganisms could be gained in the near future.

6.7

Acknowledgements

I would like to thank Prof. M. Hilker for inviting me to write this chapter, Prof. K. Dettner for entrusting me with the topic, an anonymous reviewer for comments, and S. Kellner for her support.

6.8

References

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Che moecoIogy of Egg Deposition

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Chapter 7 Plant Chemical Cues Important for Egg Deposition by Herbivorous Insects Erich Stadler

Table of Contents 7.1 7.2 7.2.1 7.2.2 7.2.2.1 7.2.2.2 7.2.2.3 7.2.3 7.2.4 7.2.5 7.2.6 7.3 7.3.1 7.3.2 7.3.3 7.4 7.4.1 7.4.2 7.4.3 7.4.4 7.4.5 7.4.6 7.4.7 7.4.8 7.4.9 7.5 7.5.1 7.5.2 7.5.3 7.5.4 7.6 7.7 7.8

Introduction “Insect Active” Compounds in Plants Different Plant Parts Leaf Surface Surface Waxes Trichomes Boundary Layer Plant Interior Variability within the Plant Species Variability between the Plant Species Distribution of Oviposition-Relevant Chemicals in the Plant Kingdom Characteristics of “Insect Active” Plant Compounds Primary versus Secondary Metabolites Volatiles and Non-Volatiles Polarity Insects’ Responses t o Plant Chemicals Methods of Investigation Orientation by Ovipositing Females t o Host Plant Host Selection Behaviour “Host Plant Search Image”: The Essence of Multi-Component Mixtures Interactions between Plant Compounds Specialization Prior Experience and Learning Host Selection Related t o Different Stages Oviposition Related t o Performance of the Progeny Oviposition and Chemoreception Chemosensory Coding in General Coding of Quality Coding of Quantity Independent of Quality Perception of Mixtures Concluding Remarks Acknowledgements References

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Abstract

Plant chemicals are essential cues that mediate egg deposition by herbivorous insects. The leaf surface waxes play an important role for host plant selection by ovipositing females of different species. But it is not yet clear if they are perceived chemically or mechanically. Behaviourally active compounds that guide gravid females include volatiles and non-volatiles with varying polarity. Volatiles acting as long range attractants during search for oviposition sites are the exceptionrather than the rule. Compounds with host plant specific distribution as well as more generally occurringplant allelochemicalsand primary metabolites have been found to influence oviposition.Mixtures of compounds are common and may also contain substances that alone or at higher concentration inhibit oviposition as repellents or deterrents. Important stimulants may occur at extremely low concentrationsand the receptor neurons perceiving them have been found to be extremely sensitive. Different insects ovipositing on the same host plant are stimulated often not by the same mixtures of compounds. Oligophagous insects may react to specific combinations of plant compounds of their different host plants, indicating an ability to recognise plant specific "search or "recognition images". Examples of plant compounds influencing oviposition by generalist (polyphagous) herbivores are fewer and probably act mainly as repellents or deterrents. Unexpectedly, receptor neurons for these substances have been found to be very specific and sensitive. Oviposition may be influenced by prior experience. Learning of associated cues can cause changes in preference and, in addition to visual cues, plant volatiles have been found to be also important in this respect.

7.1

Introduction

During the last 10 years several reviews have appeared that treat different aspects of the role of plant chemicals for oviposition by herbivorous insects. Stadler and Roessingh (1991)and Eigenbrode (1996)reviewed the influence and the interaction of insects with the plant leaf surface. Stadler (1994)surveyed the chemoreceptors that mediate the perception of plant compounds leading to oviposition.An excellent review on host plant selection by herbivorous insects has been published by Bernays and Chapman (1994).The book "Insect-Plant Biology, from Physiology to Evolution" by Schoonhoven et al. (1998) is a great addition because it adds the broader overview of the relations between insects and plants. The vast majority of herbivorous insects are specialists.This is an important fact that influences the insect's efficiency and success to a large extent. Bernays (2001) has reviewed this aspect recently and focused on the role of the nervous system in allowing control of the efficient behavioural responses. The specialist insects of Cruciferae and Liliaceae have attracted a lot of attention. Many of these herbivores and their parasites react to sulphur-containing plant metabolites- a topic reviewed recently by Stadler (2000).

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”Insect Active” Compounds in Plants

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,,,,,,,

,

In recent years it has become apparent that plants are no “immobile”partners in herbivore - plant relationships. Induced responses and their effects on herbivorous insects have recently been reviewed in a book by Karban and Baldwin (1997)and in two symposia volumes by Chadwick and Goode (1999)and Agrawal et al. (2000). Oviposition behaviour of specific groups of insects and the role of plant compounds have also been treated in special publications. Honda (1995) concentrated on Lepidoptera in general, Chew and Renwick (1995)on the chemical ecology of host plant choice in Pieris butterflies, Feeny (1995) on the evolution of host plant utilization by swallowtail butterflies, and Nishida (1995) on the oviposition stimulants of swallowtail butterflies. The evolution of insect-plant associations and the role of sensory perception were recently surveyed by Menken and Roessingh (1998). Plant chemicals can first of all influence the behaviour of insects via their sensory organs for volatiles and non-volatile plant compounds. The most recent reviews of olfaction has been published by Hansson (1999) and about contact chemoreception as related to host plant selection by Schoonhoven et al. (1998). Oviposition is not only influenced by plant chemicals but also by compounds produced by insects of the same or other species. Important examples are male bark beetles that attract females with pheromones (Byers, 1995). In most of these species only one sex begins the attack and releases a species-specific blend of chemicals composing an aggregation pheromone (in Ips and Pityogenes species the males begin to gnaw the entrance hole). Males may choose the host plants and may influence the host choice of their females also via volatile pheromones, but also via marking the plants, as shown recently for the cabbage root fly by De Jong and Stadler (2001) (compare Chapter 9). Another case are some tephritid fruit flies in which the males guard and mark host fruit, and this has been shown by Papaj et al. (1996)to have an effect on oviposition by the walnut fruit fly, Rhugoletis boycei. Plant compounds are in general the most important sensory cues mediating oviposition in herbivorous insects. But depending on the insect, mechanical and visual characters can play a significant role, too (Prokopy, 1986). Several reports can be found in the literature. An example was provided by the recent study of Degen and Stadler (1996)who compared the reaction of three herbivorous flies to the same set of surrogate leaf shapes and found that each species showed a more or less pronounced preference for the shapes resembling their natural host plant leaves.

7.2

173.

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“Insect Active” Compounds in Plants

7.2.1 Different Plant Parts All plant parts may be attacked by herbivorous insects and may be the source of oviposition stimulants or inhibitors. This is best known for the aerial parts: leaves, barks, and fruits. But the roots, mostly not accessible to the females, may contain much higher concentrations of oviposition stimulants than the aerial parts. For

174

Plant Chemical Cues Important for Egg Deposition by Herbivorous Insects . . ...................... .................................... .............................. .................................................................. . .............. .. .............. .................. . ........

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example, de Jong et al. (2000) found that roots of rutabaga contain orders of magnitude more of the stimulant CIF ("cabbage identificationfactor"; see 7.4.4. for details)than the leaves. Similarly,the concentration of the most active glucosinolates (indolyl and benzyl) are much higher in the roots of Brassica rapa and B. carnpestris (Carlson et al., 1987)and B. oleracea than in their leaves (Rosa and Rodrigues, 1998). The roots might release compounds via exudates into the soil or may produce (together with microorganisms?)volatiles that could be detected by the herbivore insects above the ground, as suggested by experiments performed by de Jong and Stadler (1999).

7.2.2

leaf Surface

7.2.2.1 Surface Waxes

The leaf surface and its waxes play an important role in host plant selection by ovipositingfemales of different species. Ovipositing females have no direct access to the plant interior during their approach. Only after contact many, but not all, species may gain access to the interior of the plant. Most leaves are covered with different layers of waxes, as described by Jetter et al. (2000).Stimulatingcompounds acting as volatiles are part of or must pass these wax layers. The interaction of attracting or stimulating compounds with the waxes is not well understood and it is still open whether chemical or mechanical properties of surface waxes explain the observed synergistic effects (seebelow). Most plant compounds that have been shown to influence oviposition were isolated from total leaf extracts. But even if the leaf surfaces were extracted with special care, no guarantee can be given that the plant interior is not at least partially extracted too. However, as recently demonstrated by Jetter et al. (2000), new techniques have been developed that will allow a more precise extraction of layers of the wax in the future. Leaf surfaces form an environment suitable for many different organisms. Recently, Mercier and Lindow (2000) showed that the population size of the bacterium Pseudomonas fluorescens is limited by the abundance of nutrients in the form of carbon sources leached from inside onto the leaf surface. Ovipositing females of Ostrinia nubilalis have been shown to be influenced by both sugars (fructose)(Derridj et al., 1996)and inhibitory compounds. The latter are produced by yeast (Sporobolornyces roseus) and possibly other microorganisms that can establish themselveson the leaf surface too (Martinet al., 1993).The authors suggested several possible mechanisms that might explain the reduced oviposition: 1)The yeast cells might take up carbohydrates that have been shown to induce oviposition. 2) The yeast cells might excrete polyamines that are transformed amino acids and are known to reduce oviposition. 3) The dense colonies of yeast cells might even inhibit contact of the ovipositor with the leaf surface. In addition it should be kept in mind that eggs of herbivorous insects are associated with many microorganisms that can influence their lives in many ways, as pointed out by Kellner (Chapter 6).

"Insect Active" Compounds in Plants

7.2.2.2 Trichomes

The leaves of many plants are covered with trichomes that may mechanically and chemically affect insects that try to establish contact for feeding or oviposition. As an example, Haddad and Hicks (2000) found that leaf pubescence (leaf surface trichomes) of Sussufius albidum negatively influences the growth and survivorship of the butterfly Pupilio troilus (Lepidoptera: Papilionidae). In accordance, females preferred to oviposit and caterpillarspreferred to feed on glabrous leaves. But other herbivores have been found that are actually stimulated by compounds contained in trichomes. Peterson et al. (1994) found that the pickleworm moth (Diuphuniu nitidulis, Lep., Pyralidae) is attracted and stimulated by 14 volatile compounds occurring in leaf trichomes of yellow squash (Cucurbitu pepo). Except for germacreneD, no single compound was attractive alone, but only in the mixture. The total mixture of the highly volatile fractionswas as attractive as volatiles emanating from whole intact leaves and corresponded with oviposition. Of special interest is also that two of the compounds present in the extract, R-(+)- and (S)-(-)-1.imonene, were repellent and reduced oviposition when tested alone. Other examples have been presented by Jackson et al. (1986) who isolated and identified duvane diterpenes from trichomes of green tobacco that stimulated oviposition in the tobacco budworm moth (Heliothis virescens). Yet another noctuid moth, Helicoverpu zed, was found by Coates et al. (1988)to be stimulated by a-santalenoic and endop-bergamotenic acids occurring in the trichomes of wild tomato. Inhibitory effects of trichome compounds have been found to occur in various plants, too. An example are the acylsugars of wild tomato Lycopersicon pennellii leaves that alter settling and reduce oviposition by Bemisiu urgentifolii (Homoptera: Aleyrodidae) (Lied1et al., 1995). 7.2.2.3 Boundary Layer

As discussed by Stadler (1986) the boundary layer above the leaves might contain in addition to a higher humidity a higher concentration of volatiles emanating from the leaf surface and its waxes and possibly also through the stomata. Insects that "inspect" the plant leaves before host acceptance and oviposition might be able to perceive these compounds. To our knowledge no attempt has been made yet to investigate this assumption even though the behaviour of some insects is suggestive (seebelow). The concentrations of isothiocyanates,an example of plant specific crucifer volatiles, in the headspace above undamaged crucifer plants are extremely low (Finch, 1978; Tollsten and Bergstrom, 1988). It is therefore not surprising that Finch and Skinner (1982) found no signs of a long-distance orientation (about 100 m) in the cabbage root fly, Delia rudicum, to host plant patches (Brussicu olerucea). However, as shown by several authors (reviewed in Nottingham, 1988)odour-modulated anemotaxis to isothiocyanatesdoes exist and orientation to host plant odour occurs in the field probably over distances of up to about 5 m (Finch and Skinner, 1982).

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Plant Chemical Cues Important for Egg Deposition by Herbivorous Insects

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7.2.3

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Plant Interior

It is difficult to understand how compounds occurring exclusively in the plant interior can influence ovipositing females. Roessingh et al. (1992) found that leaf surface extracts contained about one to two orders of magnitude less glucosinolates than the total leaf (the surface or the total of leaves with same weight extracted). But the surface extraction technique used was certainly not selective enough to exclude contamination by compounds occurring inside the leaves. This indicates that the surface may contain even smaller amounts. Thus, the relationship of stimulatory plant compounds inside and outside the leaves still needs further investigation. Some females have the capacity to damage the surface and detect compounds from inside. Classicalexamplesare beetles that bite into the leaf and feed before they oviposit. Butterflies, such as the monarch butterfly, drum on the leaf surface and might get access to inside the leaf, too. An example is the study by Haribal and Renwick (1998) who found that females can rupture the plant surface using the spines on the mesothorax leg. As shown by Baur et al. (1998), compounds released on the surface could be detected with contact chemoreceptor sensilla on the antennae and tarsi. Other insects that gain access to the interior of the plant are fruit flies that use their ovipositor not only for the final act of egg laying but apparently also to test the fruit quality inside (seebelow).Also sawflies(Tenthredinidae)and galling insects (Cynipoidea)lay their eggs into the leaf tissue. Craig et al. (1988)found that wound compounds of the host plant Sulix Zusiolepis function as oviposition deterrent in the stem-galling sawfly Euuru hiolepis. Roininen et al. (1999) showed that the phenol glucosides 2’-O-acetylsalicortin of the natural host (Sulix pentundra) and tremulacin of the foreign SuZix host plants are potent probing and oviposition stimulants for this sawfly. Thus, it seems that the stimulatory phenolic glucosides are present on the twig surface and the inhibitory compounds are present in the plant tissue and appear at the surface only through scars. Other authors also indicated that compounds from inside the leaf might be inhibitory. This might also explain the negative results obtained with certain host-plant extracts. They could contain compounds from within the leaves that are normally not exposed to the ovipositinginsect. We found such indications in host plant (celeriac)extracts tested in the carrot fly. Surrogate leaves treated with total steam distillate leaf extracts were stimulatory only after they had been exposed to air, depending on the concentration, for one to two weeks (Stadler,1971/72).The most likely explanation was that inhibitory compounds were decomposed or evaporated during this time. The plant vacuole represents, according to Matile (1990),the most conspicuous storage compartment of secondary metabolites in leaves. The author listed 20 different compounds, many of them glyco- or glucosides, that have been shown to be present in the vacuoles of different plants. Several of these compounds are known to stimulate or inhibit oviposition and feeding in herbivores. Prominent examples of such secondary metabolites are the glucosinolates sinigrin, gluconasturtiin and glucobrassicin.In the meantime Kelly et al. (1998)have shown

“InsectActive” CornDoundsin Plants

that sinigrin,at least in the seedlingsof Brussicujunceu, partitions with storageprotein also. The biosynthesis, degradation and transport of glucosinolates has recently been reviewed by Chen and Andreasson (2001). The enzyme necessary for the cleavage of glucosinolates, the myrosinase, is stored in different compartments. Thus, the reaction of myrosinase with the substrate and the production of volatile isothiocyanatesonly occur after leaf damage. This is confirmed indirectly by Finch (1978) and Tollsten and Bergstrom (1988), who found that the natural release of the volatiles of undisturbed plants is very low. The low concentrations probably also limit the possibility for insects to locate crucifer host plants over a longer distance. Latex and secretory resin canals are also sourcesof allelochemicals.Xie and Isman (1992)and Farrell et al. (1991) showed that these plant secretory organs play a role in plant defence against feeding insects. It seems likely that components of such resins influence oviposition, too, as seems to be the case for bark beetles (Byers, 1995).Phloem also contains secondary plant compounds (example glucosinolates: Brudenell et al., 1999; quinolizidine alkaloids: Wink and Witte, 1991). It can thus be assumed that the settling and sucking behaviour of aphids and indirectly also their oviposition (larviposition)will be affected by phloem components. Dorschner (1993) showed in elegant experiments that honeydew collected from aphids and added to artificial diets does affect the two tested aphids, Phorodon hurnuli and Myzus persicue. The aphids grew and reproduced only on diets that contained honeydew (probablycontaining secondary metabolites)obtained from their respective hosts.

7.2.4

Variability within the Plant Species

Plants vary in metabolites due to their genetics and the environment. Female herbivores are known to react to plant variability and often they choose plants allowing an optimal growth of their offspring (larvae emerging from the eggs) (e.g. Craig et al., 1989,2000; Degen et al., 1999a).Variability in allelochemicalsis known to exist between leaves of the same plant, between different growth stages, between plant individuals and between plant species of the same genus. For example, oviposition by the butterfly Iunoniu coeniu (Nymphalidae) is stimulated by the iridoid glycosides of its host plants (Plantaginaceae).Klockars et al. (1993)showed that old leaves contained almost none of the stimulants, whereas young leaves, which are preferred by the females, contain up to 9% of their dry weight. De Moares et al. (2001)found that tobacco plants release herbivore-induced volatiles during both night and day. But several volatiles were released exclusively at night and were found to be highly repellent to female moths of Heliothis virescens. “Daytime volatiles” were significantly less repellent than nocturnal volatiles. Udagayri and Mason (1995) compared leaf extracts of different parts of the same plant and of different plants as oviposition stimulants for the European corn borer, Ostriniu nubilulis. The authors found that plant phenology affects chemically mediated oviposition response resembling the response of the moths in the field. Anderson and Alborn (1999)found yet another moth, Spodopteru littorulis, responding to plant

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Plant Chemical Cues Important for Egg Deposition by Herbivorous Insects 178 ................................ .................................... .............................. ............. . .................... ............... . .................................................................................

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age related changes. The females of this species lay more eggs on damaged versus undamaged cotton plants with 5 leaves. In larger plants with 10 leaves the preference is reversed and the undamaged plants receive more eggs. The environment of the plant, especially light intensity, C 0 2concentration and soil nutrients, does have an influence on plant compounds. Stange (1997)concluded that sensory organs detecting C 0 2 are common in herbivorous moths and butterflies. As the C 0 2 gradients in the vicinity of plants depend on their physiological condition, C 0 2 could provide a sensory cue for the suitability of the plant. The author found indeed that the gas concentration did affect oviposition by the moth Cuctoblustis cuctorurn on its host, the plant Opuntiu strictu. Another indication that the photosyntheticactivity of plants can be perceived by herbivorous insects was recently found by Langan et al. (2001).The authors studied in the field the egg-laying preferences of Pieris rupue and related it to photosynthesis or transpiration of the chosen host plants. They examined the relationships among host choice, plant gas exchange activity, and plant size and found that the postalighting preferences were related to plant height and photosynthetic rate. The question how these butterflies detect the plant photosynthetic rate remains to be investigated but in this case transpiration seems not to be involved. Plant nutrient effects, either in relation to secondary metabolites or not, on ovipositing females have rarely been tested. Wolfson (1980) showed that the available soil nutrients may change the attractiveness of Brussicu nigva plants to the small white butterfly, Pieris rupue. Leaf water content (and not the content of secondary plant compounds!) was implicated as a phenotypic characteristic associated with oviposition preference. Plants colonized by other organisms of the same or different species might be more or less attractive for ovipositingfemales. Host plants already infested by larvae or adults of Oreinu cuculiue were more attractive than undamaged plants (Kalberer et al., 2001b).Also artificially damaged host plants (Adenostyles ulliuriue) were more attractive for these beetles as long as the damage was not more than half an hour old. In aphids, Pettersson et al. (1998) found that apterae of Aphis cruccivoru responded with positive anemotaxis to air passed over plants with small groups of the same species but negatively to bigger groups of individuals. Further, alatae were repelled by groups of alatae, independent of their number, showing some type of interaction between pheromones and host plant odour. In the case of Heliothis virescens, de Moares et al. (2001) found that the moths clearly preferred undamaged host plants. As reviewed by Karban and Baldwin (1997),damage or stress does enhance or stimulate defensive mechanisms in plants in the form of an increased biosynthesis of allelochemicals or the formation of new compounds. One of the plant stress hormones involved in the mobilization of these mechanisms in response to herbivory is jasmonic acid and its derivatives.Intuitively the induction or increase of defensive compounds should decrease the propensity of female herbivores to lay eggs. This does not need to be so in every case. Stanjek et al. (1997)found that treatment of celery leaves with jasmonic acid stimulated the biosynthesis of

"Insect Active" Compounds in Plants

furanocoumarins that are known oviposition stimulants for the carrot fly (Stadler and Buser, 1984; Stadler, 1986). The females responded with an increased oviposition to the leaf surface extracts from induced celery leaves. Similarly, phytoalexinsinduced in crucifersin response to attackby different microorganisms have been found by Baur et al. (1998)to be oviposition stimulants for the cabbage root fly.

7.2.5

Variability between the Plant Species

For several oligophagous insects the importance of the chemical cues of different host plant species have been compared. A recent example is the monarch butterfly which in nature is faced with host plants containing a large variety of flavonol glycosides that have been shown to be oviposition and probably feeding (for the larvae) stimulants (Haribal and Renwick, 1998). The authors suggested that the geographical pattern of distribution of these stimulants may have affected the evolution of host recognition. Feeny and colleagues (Feeny, 1995) compared not only different host plants of the same butterfly species but identified also the oviposition stimulants (hydroxycinnamicacid derivatives) of different swallowtail butterflies. The recent identification of oviposition stimulants for the zebra butterfly, Eurytides marcellus, (Haribal and Feeny, 1998) adds yet another facet and allows predictions about the oviposition cues of ancestors of the subfamily Papilioninae. Further examples are the identification of oviposition stimulants present in different host plant species of the carrot fly by Degen and Stadler (1998) and Degen et al. (1999a), and the cabbage root fly by Griffiths et al. (2001) and Stadler et al. (2002).

7.2.6

Distribution of Oviposition-Relevant Chemicals in the Plant Kingdom

A review of the literature about compounds influencing oviposition (and larviposition) revealed about 50 examples of such insect plant relationships. Only in 6 of these publications had the identified active compounds not yet been identified before by phytochemists as typical components of the plant families in question (Hegnauer, 1962-1989). Hegnauer's extensive compendium of phytochemicals is based mainly on plant compounds that were identified with a pharmaceutical application in mind. On the other hand our knowledge about insect active plant compounds is probably biased, too, because many studies were done with readily available compounds. But despite these biases several conclusions can be made (referencesin Stadler 1992,1994and further references mentioned in this chapter): (1) Primary metabolites that occur in most plants are far less important for plant recognition than the secondary metabolites. Without secondary plant compounds ovipositing females seem unable to locate and choose their host plants. (2) Oligophagous and monophagous insects often use "typical"plant compounds of their host plants. A typical example is the carrot fly (Figures 7-1,7-2,7-3).

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Plant Chemical Cues Important for Egg Deposition by Herbivorous Insects

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2000000

~

1000000

eugenoI

100000 10000 1000 100

10 1

6 gle Carrot leaf surface mix Activity of single compounds

ODI: Oviposition discrimination index: - i- 8o [(Treatment-Control)/(Treatrnent+Control)~lOO

- 1: 70

-1 60 -F 50

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"Insect Active" Compounds in Plants

Figure 7-2 Contents (log scale)of oviposition stimulants for the carrot fly in different host plants: The plants have been ranked according to the preference (A = highest, B = medium, C = lowest)in oviposition choice experiments(datafrom Degen et al., 1999a).Three different

patterns could be discerned that are characterized by dominance of propenylbenzenesand polyacetylenes (l), or furanocoumarinsand polyacetylenes (2),or mainly furanocoumarins (3). The flies seem to choose all the three patterns about equally, indicating that widely different host-plant patterns can be acceptable. (3) Not all insects associated with the same groups of plants make use of the same compounds. As an example, Brooks et al. (1996)extracted the known oviposition stimulants (flavonoid glycosides, chlorogenic acid) for Pupilio polyxenes from the leaf surface of wild carrots and studied their variability. Not unexpectedly the extraction method replicating best the host plant ranking with real leaves was different than that for the carrot fly (Degen et al., 1999a).This fly reacts to completely different compounds that also occur in carrots. An exception seems to be the big group of crucifer insects. Fifteen or more species belonging to 5 orders have been shown to respond to glucosinolatesor their volatile metabolites (isothiocyanates).However, even these insects have preferences for certain glucosinolates, as pointed out for two Pieris species by Huang and Renwick (1994),and the activity of specific glucosinolates varies between species. (4) Not all typical host plant compounds (from a chemosystematic point of view) have to be attractants and stimulants in a specific insect. They may be neutral, being not perceived and possibly excreted or detoxified with special mechanisms. They may also be deterrents of oviposition with the result that plant parts or plant individuals are avoided. An example is the Colorado potato

181

Figure7-3 Distribution of oviposition stimulants ofthe carrot fly in the plant orders arranged as a cross section through the plant kingdom (Hijwegen, 1979).In orders filled with one or more (overlaid) pattern(s) all plants investigated contain the respective group(s) of compounds. Circular patterned spots within the orders indicate that only some plants were found t o contain the particular group of compounds. The three groups of stimulants do occurtogether only in the Araliales enclosingthe host-plants (Umbelliferae). Distribution of compoundsderived from data of Hegnauer (1962-1996).

Y

"Insect Active" Compounds in Plants

beetle, Leptinotursu decemlineutu, that has chemoreceptor neurons sensitive to host plant specific glycoalkaloids (Mitchell and Harrison, 1985) that are inhibitory for feeding (Schreiber,1958;Yencho et al., 2000)and presumably also for subsequent oviposition.Recently, Dickens et al. (2001)managed to identify blends of potato compounds that attract the Colorado potato beetle, and these stimulants are indeed not related to the alkaloids. Another example is the monarch butterfly, Dunuus plexippus. Zalucki et al. (1990) found post-alighting rejection of plants (Asclepius species) by females with low or high cardiac glycoside levels. Further, Zalucki et al. (2001)found that cardiac glycosides and other as yet undetermined plant factors all have a negative effect on first-instar larval survival. Thus, female butterflies avoiding the highest levels of cardenolides increase the survival of their progeny. Some cardenolides, below a damaging level for the caterpillars, are however beneficial because these compounds offer an effective defence against predators (Malcolm, 1995). (5) Mixtures and rarely single compounds: Single compounds at natural concentrations have been found to have a stimulatory effect. This is the case in the females of the noctuid moth Helicoverpu (Heliathis)urmigeru that are attracted in a wind tunnel to components of steam distillates of a host plant (Cujunus cujun) (Hartlieb and Rembold, 1996). One isolated and identified sesquiterpene, abulnesene, was found to increase the approaches and another, a-humulene, the contacts with the odour sources, by as much as the mixture of six isolated sesquiterpenes. The mixture in the original proportions of the individual compounds was as attractive as the distillate itself. Further examples are the

10mM KCI

0.01

0.1

1

10

100

I000

CIF 1 (nglml)

Figure 7-4 CIF dose-response curve generated by the nerve impulses of the sensitive neuron in the C-sensillum of the cabbage root fly Delia radicum (adaptedfrom Hurter et al., 1999). The response threshold was estimated to be about 10-11M. This neuron is different from

another neuron inthesamesensillumsensitivetoglucosinolates(Stadleretal., unpublished).

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Plant Chemical Cues Important for Egg Deposition by Herbivorous Insects

cabbage root fly reacting to the CIFs (see 7.4.4) and the carrot fly that is stimulated by falcarindiol (Stadlerand Buser, 1984; Stadler, 1986).But even in these insects, mixtures of several plant specific compounds are more active andor might explain the preference for some host plants much better (Figure7-1).For insects, highly important stimulants or components of mixtures can occur at extremely low concentrations. A good example is the cabbage root fly with its extremely sensitive receptor neurons for CIF (Figure 7-4) (Hurter et al., 1999). (6) Inhibitory compounds seem to dominate host plant selection of less specific insects such as polyphagous and some oligophagous insects. Bernays and Chapman (1994) concluded that host plant range is ultimately defined by the occurrence of deterrent compounds in non-hosts. This conclusion seems still to hold true in the reaction of feeding and ovipositing females in contact with potential host plants. But, recent investigations of ovipositing females of polyphagous species gave evidence that plant specific compounds may attract these females as well. Rojas (1999a) studied the polyphagous noctuid moth Murnestru brussicue and found allylisothiocyanate,a typical volatile of one of the possible host plants, to be stimulating upwind flight and landing. This observation is remarkable because mated femalesflew upwind more often than virgins or males. The generally occurring green leaf volatiles, (E)-2-hexenaland (Zj-3-hexenylacetate, elicited upwind flight and landing too. These results are in accordance with the observation of the author that females were attracted landed and laid more often on mechanically damaged than undamaged cabbage plants (Rojas, 1999b).The reaction of this polyphagous species to the plant specific allylisothiocyanateis unexpected. But it is in fact not new, since Chapman (1977)and Le Berre and Tira (1977)showed that polyphagous insects can be stimulated by specific plant allelochemicals,too. Recently Anderson et al. (1996)and Jonsson and Anderson (1999)investigated the olfactory receptor neurons of the antennae of two polyphagous noctuid moths (Agrotis segeturn, Spodopteru littoralis). The authors found in both moths receptor neurons highly sensitive and also very specific for components of volatiles of damaged and undamaged plants. Polyphagousmoths are no exception in this regard. Hansson et al. (1999)identified in the antennae of the polyphagous Japanese scarabbeetle, Phylloperthu diversu, olfactory receptor neurones that are highly specific and sensitive to some individual green leaf volatiles ((Z)-3-hexenyl acetate, (E)-2hexenal, (Z)-3-hexenol).Thus, it seems that polyphagous insects can respond during orientation to specific as well as non-specific host plant compounds. However, an important part of the discriminationbetween plants will take place in contact, as has been recently shown for Murnestru brussicueby Rojas et al. (2000), and in this phase deterrents that may be also plant specific play a crucial role. These studies seem to indicate that our view of host plant selection of polyphagous insects is still incomplete and needs more studies.

.......................,,,,..,,,,,,.......,.........................

.. ,

Characteristics of “Insect..................................... Active” Plant Compounds 185 .., .,......................... . ..................................................... .

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7.3

Characteristics of “Insect Active” Plant Compounds

7.3.1

Primary versus Secondary Metabolites

As pointed out, a vast majority of ovipositing females seem to use plant secondary metabolites as cues to locate and select suitable plants. But the importance of primary metabolites for oviposition cannot be excluded in every case. As mentioned above (see Section 7.2.2), females of Ostrinia nubilalis use primary metabolites: In this species, Derridj et al. (1992) found that oviposition is influenced by carbohydrates, especially fructose on the leaf surface. Electrophysiological recordings from their contact chemoreceptor sensilla on the ovipositor (Roessingh, Baur and Stadler, unpubl.) showed that they contain a sugar receptor neuron sensitive to fructose, but not to glucose. This unusual sensitivity matches well the observed correlation between the susceptibility of corn cultivars, the preference for plant growth stages and the content of fructose. This does of course not contradict the findings that terpenoids (Binder et al., 1995)and leaf volatiles with waxconstituents (Udayagiri and Mason, 1995,1997)have attractive and/or stimulatory properties. Eisemann and Rice (1985)found that Ducus tryoniis stimulated by p-D(-)fructose to lay significantly more eggs in an agar substrate of fruit surrogates. The threshold for the detection of fructose was 4 mM whereas, remarkably, sucrose and glucose were not stimulating at all. CaC1, was found to be deterrent, whereas NaCl had no effect. The Caribbean fruit fly, Anastrepha suspensa, however, was found by Szentesi et al. (1979)to be not sensitive to sucrose but inhibited by secondary plant compounds such as naringin and quinine. Crnjaret al. (1989)succeededin recording reactions from neurones in the contact chemoreceptor and olfactory sensilla on the ovipositor of another fruit fly, the apple maggot fly, Rhugoletis pornonella. This indicates that fruit flies can detect compounds from the fruit interior and that primary metabolites, mainly sugars, and inhibitory secondary metabolites play a role in the final acceptance or rejection of the fruit. In general it seems tempting to believe that primary metabolites influence mainly the distribution of the eggs within the same plant species or between different plant parts.

7.3.2

Volatiles and Non-Volatiles

The so far identified attractants and/or stimulants include volatiles and nonvolatiles.In some insects, the cabbage root fly is an example, non-volatilestimulants seem to be far more important than volatile compounds. However, also in this insect, volatileswere found to influence synergisticallystimulationby non-volatiles (de Jong and Stadler, 1999).In other insects, such as the carrot fly, electroantennogram recordings revealed that all the stimulating compounds seem to be detected with the antennae (Stadler,unpubl.). This is rather surprising because some of the stimulants, for example, the polyacetylene falcarindiol, have a very low vapour pressure and we did not succeed in showing an olfactory orientation, either in the lab or in the field, to it. We assume that these compounds as well as the furano-

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coumarins are perceived only at short distance in the boundary layer of the leaf surface.Other host plant compounds, the propenylbenzenes (methyl iso-eugenol, asarone), act on a larger scale and attract flies also to traps in the field (Guerin et al., 1983).

7.3.3

Polarity

Ovipositionstimulants may vary largely in polarity. In the case of the glucosinolates, the glucosides acting as contact stimulants on the leaf surface are, as all glucosides are, very polar and water-soluble. The aglucones of the glucosinolates, the isothiocyanates,are apolar, very volatile and have synergisticeffects as attractants/ stimulants for most herbivorous insects associated with crucifers. Many other insect plant relationships are mediated by polar, non-volatile compounds as well as by non-polar volatiles.

7.4

Insects' Responses to Plant Chemicals

7.4.1 Methods of Investigation Despite the progress in phytochemistry and analytical techniques, the isolation and identification of oviposition stimulants is still a challenge. This is particularly true for non-volatile, polar compounds. But, as pointed out, many very important stimulants are not volatile and it may well be that our present knowledge about plant chemicals influencing oviposition is biased toward volatiles. One of the cornerstones of any isolation is the bioassay guiding the fractionation of extracts that often requires study of different surrogates and extracting methods first. Oviposition bioassays vary widely in the methodology used. First of all it has to be decided which phase in the sequence of behavioural steps will be considered, and this will influence the chosen design of the bioassay as well as the chosen extraction and fraction procedure. No assumptions about the volatility of the compounds involved can be made when oviposition behaviour is observed in contact with a leaf or surrogate. For large butterflies "hand-held individual assays have proven to be most useful (see cited references about swallowtail butterflies; e.g. 7.4.5). The test of individual females has, as pointed out by Singer (1986) and Singer and Lee (2000), important advantages such as the control of effects due to individual preferences and the influence of prior experience, age, nutrition and egg maturation. Jallow and Zalucki (1995)used this approach also for a moth and showed that it can generate reliable data. The obvious disadvantage of individual female bioassays is the additional time required to perform the test, and this is the more limiting the more fractions have to be tested. This is the reason that often only the end result of oviposition behaviour, i.e. the number of eggs laid, has been used as a measure of activity. Problems with "disappearing" activity of plant extracts on insect oviposition during extract purification are well known. One of the reasons might be a loss due

Insects' Responsesto Plant Chemicals

to purely chemical problems caused by destruction or permanent binding to column material. Another cause may be the fact that individual fractions or compounds might at natural concentrations not be active when tested singly because individual components might synergize each other (Figure7-1).A possible remedy is the recombination of fractions after the individual fractions have been bioassayed. This procedure has been used with success in the cited studies of papilionid butterflies (see e.g. 7.2.5).

7.4.2

Orientation by Ovipositing Females to Host Plant

In insects such as beetles and many Homoptera which feed as adults on the host plants, no clear discrimination can be made between orientation to feeding or oviposition sites. Often the plant may be a source of nutrition needed for egg maturation and at the same time provide an oviposition site. On the other hand attraction or anemotaxis to volatiles by female insects might not be directly related to oviposition but rather reflect orientation to food sources like flowers or honeydew. Long range orientation to host plant volatiles that would be comparable to the olfactory modulated anemotaxis of males in the response to female pheromones, probably rarely if ever existsin nature. In the case of the onion fly, Judd and Borden (1989)found that dipropyl-disulphides, typical host plant compounds, can attract female flies over a distance of 100 m. This record value might have occurred because relatively high (50 mg) and efficient odour sources were used. But even this is surprising because Harris et al. (1987) found that the same compound (n-propyl disulphide) inhibits oviposition in a wind tunnel above release rates of 10 ndsec. The other extreme about distances of olfactory Orientation is the Colorado potato beetle, Leptinotarsa decernlineuta. Jermy et al. (1988)showed that walking beetles can discriminate between two plant species (potato, a host plant and cabbage, a nonhost plant) in the field only at distances of less than 60 cm. In the field the chrysomelid beetle Oreinu cuculiue displayed a directed, olfactory orientation to its host plant. Kalberer (2001) found that marked beetles landed randomly and walked upwind to their host plants over a distance of 3.2 m up to 15 m. Experiments in a wind tunnel confirmed that beetles orientated to olfactory cues when locating plants for feeding and for larviposition.This study also showed that the type of habitat might determine the maximal distance allowing anemotactic orientation. The authors made their observations on a flat plane devoid of higher vegetation ideal for anemotaxis in a small walking insect. The distance travelled to the host plant under the influence of volatiles might indeed be more dependent on other factors than odour concentration. Prokopy et al. (1983) showed convincinglythat the cabbage root fly uses mainly visual cues to find its host plants, mainly crucifers. This does not exclude anemotactic orientation, as proven in the wind tunnel by Nottingham (1988)and several other authors.

187

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7.4.3

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Host Selection Behaviour

Host plant selection proceeds, depending on the species, through a sequence of different behavioural phases. All behavioural patterns may be influenced by plant chemical cues. Prior to landing, many butterflies are known to flutter with their wings in a hovering flight. This might allow the females to get a visual impression with a higher resolution. After landing, several butterflies species are known to briefly drum the leaf surface (Terofal,1965).Other insects, like many flies, perform exploratory runs on the plant surface before continuing the behavioural sequence leading to actual oviposition on or near the plant. During these runs and drumming with the tarsi, possibly many sensory structures, also on other parts of the body, like the proboscis, palps and antennae, touch the plant. The fluttering might produce an air stream over the antennae that could also contain volatiles from the boundary layer of the leaves. Ablation experiments and investigations of the chemosensory sensilla of the appendages have given evidence that in these behavioural phases multiple organs are involved in the perception of different plant compounds (reviewed by Stadler, 1994).

7.4.4

“Host Plant Search Image”: The Essence of Multi-Component Mixtures

The term search image was probably first used by Luuk Tinbergen and later extended by Atema et al. (1980)to the “chemical search image” of yellowfin tuna in the marine environment. The “host plant search images” used by ovipositing female insects include, as stated in the introduction, visual and mechanical properties of host plants. In order to show that this concept is valid, all the major physical and chemical characters of a plant eliciting oviposition need to be identified. This is accomplished if it can be shown that the ovipositing female will accept a surrogate plant as often as a natural plant. It would be even better if the choice between a whole range of different host plants could be reproduced with surrogates. Kanno and Harris (2000) came close to this aim with their study of the host selection behaviour of the Hessian fly. These authors established the ranking of six grasses for ovipositing females and then ran choice tests with plant models that incorporated physical and/or chemical features of the six grasses. The effects of the physical features of the models turned out to be at least as important as the effects of the chemical features (1-octacosanaland 6-methoxy-2-benzoxazolinone; Morris et al., 2000). In tests with surrogate models treated with extracts of the six grasses, egg counts were similar to egg counts on real plants, proving that the major components of the host plant search image had been identified. As shown by Roessingh and Stadler (1990)and confirmed by Degen and Stadler (1996),the cabbage root fly, Deliu rudicum, is also sensitive to physical characters of the host plant, albeit to a lesser degree than other flies. We recently compared the preference behaviour of this fly for leaf surface extracts of different plants differing in their susceptibility to larval attack. We found that the observed hierarchy in

insects' Responsesto Plant Chemicals

189

oviposition ranking was related to the content of the extracts of known oviposition stimulants, the glucosinolates and the recently isolated and identified "CIF" (cabbage identification factors; 1,2-dihydro-3-thia-4,lO,lOb-triaza-cyclopenta[.a.] fluorene-1-carboxylic acid; Figure 7-4)compounds. We also found a partial correlation between the quantity of benzyl and indolyl glucosinolates (Griffiths et al., 2001). However, some of the plant extracts containing stimulants received very few eggs and additional experimentsrevealed that inhibitorycompounds also occur in the extracts of some plants (Stadler et al., 2002). Thus, these analytical and behavioural data support the hypothesis that inhibitory compounds may change the host plant search image so much that it will no longer be acceptable. The identification of plant compounds stimulating feeding or oviposition on host plants raised the question whether quantifying the stimulating compounds could be used to predict the susceptibilityof specific varieties. Nielsen et al. (2001) investigated this question using flea beetles (Phyllotretu spp.) and compared the feeding response on wild Arubidopsis thulium with that on plants bio-technologically engineered to contain four-fold more p-hydroxybenzyl-glucosinolate(sinalbin)in the leaves. Surprisingly, the beetles did not discriminate between transgenic and wild-type plants, despite the fact that sinalbin is a known feeding stimulant. The authors reviewed published studies on the effect of different glucosinolate levels within the same plant on feeding and oviposition of crucifer insects. They concluded that the effect of glucosinolates on adapted insects often does not depend on the concentration of the glucosinolates, even if these compounds when tested on their own have been shown to be stimulants. An explanation may be that specific host plant compounds might act in adapted specialist insects as sign (yeslno),token or flag stimuli without having a concentration dependent effects on behaviour. This is in line with the observation of Blau et al. (1978)that in adapted specialist insects such as Pieris rupae glucosinolates show no or little dosage-dependent effects on growth. An alternative hypothesis was put forward by Nielsen et al. (2001).They suggested that the effect of glucosinolates in such insects also depends on interaction with other compounds in the plant. It remains open whether one or the other or even both hypotheses might be true.

7.4.5

Interactions between Plant Compounds

Active plant components show their effect mostly in concert with numerous other ones. This is evident but often forgotten when single compounds are tested that influence ovipositionbehaviour. Thus, a full appreciation of the role of a compound can only be obtained if other compounds present in the host plant(s) are tested in mixtures, too. An indication of the importance of mixtures is that plant compounds of which a stimulatory activity was found at unnaturally high concentrations are at their natural concentration not stimulatory at all. Synergism of individual plant compounds seems to occur frequently, as was shown for oviposition by the carrot fly (Stadlerand Buser, 1984;Stadler,unpubl.; Figure 7-1)and swallowtailbutterflies (Sachdev-Gupta et al., 1993,1999; Nishida, 1994; Carter et al., 1998).This effect

190 Plant Chemical Cues Important for Egg Deposition by Herbivorous Insects . ........................ . ............................................................................................... .................................................................... ................. ............................................

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holds true also for other butterflies, like the pierid butterfly Colius erute studied by Honda et al. (1997) or the danaid butterfly ldeopsis similis that is stimulated by a synergistic mixture of five phenanthro-indolizidine alkaloids (Honda et al., 2001). In the case of the cabbage root fly, Delia rudicum, we also found a synergism between volatile and non-volatile compounds that might originatefrom different plant parts (de Jong and Stadler, 1999). The interaction between allelochemicals on the leaf surface with the epicuticular waxes also seems to be very common (Eigenbrode, 1996; Spencer, 1996). By mechanically removing the epicuticular wax of the host plant, Tunucetum vulgure, Miiller and Hiker (2001)found out that waxes of the lower leaf surface contain crucial information for oviposition in the chrysomelid beetle Cussidu stigmuticu. It remains open whether this is a purely "chemical" interaction or a mechanical effect of the waxes might be involved, as suggested by results from the cabbage root fly (Roessingh and Stadler, 1990). Synergism and the effect of mixtures are not limited to stimulatory compounds. Jermy (1983)found that the effect of individual deterrents was potentiated by the inhibitory effect of other feeding deterrents. This holds true also for oviposition as shown by Jermy and Szentesi (1978).More recently Peterson et al. (1994)presented an interesting example of trichome originating compounds that interact when combined. Huang and Renwick (1994) confirmed earlier studies showing that glucosinolates are major oviposition stimulants for the pierid butterflies Pieris rupae and P. napi oleruceu. These authors noticed that several plants containing copious amounts of these stimulants were rejected as host plants. Subsequently, Renwick and his collaborators isolated and identified different allelochemicalcompounds that inhibit oviposition and can explain the negative selection by the butterflies (Renwick, 1996). This is yet another example of inhibitory compounds having a significant influence on host plant selection.Jermyand Szentesi(1978)also reviewed the older literature giving evidence that the distribution of deterring or inhibiting compounds strongly determine host plant selection by ovipositing females.

7.4.6

Specialization

Different insect species attacking the same host plant may perceive completely different spectra of compounds or mixtures. This indicates that associations with a specific host plant evolved differently. Some oligophagous insects are stimulated by different combinations of plant compounds showing an ability to recognize specific host plant "search or "recognition" images (Figure 7-2).As pointed out by Bernays (2001),specialization has certain advantages, including the fact that it requires chemosensoryinput from fewer compounds perceived with higher sensitivity (loss of neuron number, narrower sensitivity spectrum, increased sensitivity). The reaction to non-hosts could be lost and the sensitivity to general stimulants as well. But the sensitivity to broad-band repellentddeterrents might be higher. Polyphagous species might also be a collectionof individualseach with a different preference and thus not fundamentally different from specialist species in their

Insects’ Responsesto Plant Chemicals

host selection behaviour. Jallow and Zalucki (1995,1996)studied the polyphagous species Helicoverpa armigera and found no differences between populations but observed significant variance between individual females within the populations. This was expressed in a reversed ranking of host plants. The inter-individual differences were found to be under genetic control and heritable. Janz and Nylin (1997)presented an interesting comparison of populations of the same species with a different specialization. The authors found that females of more specialized populations of Polygonia c-album discriminated against bad quality host plants (Urtica dioica) whereas the generalists lay equally on nettles of differing quality. This is in accordance with the hypothesis of Bernays (1996)that generalists are less efficient in discriminating between host plants because of their limitations in sensory processing. As pointed out by different authors and summarized and discussed by Bernays (2001),real polyphagy has some benefits, too, and generalist species seem to have evolved special mechanisms to limit the disadvantages of a generalized food-choice. She points to the more sophisticated nervous systems, plasticity of behaviour such as learning (of course also dependent on a more complex central nervous system), and larvae that are mobile enough to avoid toxicity of host plants initially chosen by their mothers by switching plants (food mixing).

7.4.7

Prior Experience and learning

Past experience can influence oviposition behaviour and the reaction to plant stimuli in different ways. Prior contact with plants can influence oogenesis, and this has a dramatic effect on the reactions of females. In the walnut fly, Rhagoletis juglandis, even the presence of surrogate fruit strongly enhanced production of eggs in the first maturation cycle (Lachmann and Papaj, 2001). Host plant compounds influence oogenesis very strongly in many other insects as well, for example the onion fly Delia antiqua (Harris and Miller, 1988).Most likely associative learning during alightingor selection of an oviposition site and/or actual oviposition may change the subsequent preference for specificplants. Szentesi and Jermy (1990) and Papaj and Lewis (1993)reviewed this subject and concluded that experience induced changes of oviposition preference has been demonstrated in only a few herbivorous insects. In this context the ”Hopkins’host selection principle” (Hopkins, 1917)has to be mentioned. It states that oviposition choice is influencedby prior feeding experience of the larvae. Many authors have tested this principle in many different insects with negative results. However, recently, Anderson et al. (1995) found that Spodoptera littoralis females prefer the food they have been feeding on as larvae for oviposition. This acquired preference was lost after amputation of the antenna. This is an indication that a changed sensitivity of the antenna1 chemoreceptors or the processing of olfactory information in the CNS is involved. The question remains why so many other insects failed to show a similar effect (discussedin van Emden et al., 1996).Recently, Fox and Savalli (2000) added yet another insect, a

191

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bruchid beetle, to the growing list of examples showing that a priori the “Hopkins’ host selection principle” cannot be discounted and that the ”legacy hypothesis” (Corbet, 1985) has to be considered to hold at least for some herbivorous insects (compare Chapter 13).

7.4.8

Host Selection Related to Different Stages

Genetic determination of behavioural and physiologicalreactions may be different in larvae and in adults, as has been shown for example in Yponorneutu moths studied by Roessingh et al. (2000).The authors investigated feeding reactions of the larvae and the ovipositionin the same species. Dulcitol, a sugar alcohol typical for the host plants, stimulated feedingin the larvae but had no effect on oviposition. Another indication for differences in genetic determination of host selection behaviour of larvae and adults is the range of plants accepted by both stages. The potential host range of larvae is usually wider than the range chosen by females. This aspect was discussed by Janz and Nylin (1997) and is further elaborated in Chapter 13. Special cases are aphids that are polymorphic and have different summer and winter host plants for feeding and oviposition. For the bird-cherry oat aphid, Rhopalosiphum pudi, Quiroz and Niemeyer (1998) have shown that wheat and oat volatiles are attractive for the aphid colonizing cereals in the spring. Interestingly, during this phase the winter host plant component methyl salicylate released from Prunus pudus reduced colonization of the summer host (cereals)(larvi-position,no oviposition)(Petterssonet al., 1994).Sandstrom and Pettersson (2000)showed that winter host plant specificity is controlled mainly by the preference of the females re-immigrating (gynoparae) to the winter host, P. pudus, in autumn and there is little doubt that olfaction plays a role during this phase, too (Pettersson, 1993).

7.4.9

Oviposition Related to Performance of the Progeny

Larval performance, digestion, excretion or detoxification is strongly influenced by the secondary plant compounds encountered in the host plant. In many insects the females’ oviposition preference correlated well with subsequent larval performance. An example is the carrot fly, Psilu rosue. Degen et al. (199913) showed that the females preferred leaves of plants that produced on their roots more pupae from the same number of eggs. Craig et al. (1989)studying a shoot-gallingsawfly, also found a strong relationship between oviposition preference and larval performance. But there are exceptions to this rule. Many other insects have been found in which the female makes wrong or even fatal decisions. Courtney et al. (1990) concluded ”mother doesn’t know best” and Larsson et al. (1995) found a high oviposition rate of the gall midge Dusineuru rnurgznerntorquenson Sulix virninulis genotypes that are unsuitable for offspring survival. An explanation for such “errors” might be that during evolution the species may not have been exposed to these unsuitable plants (compare Chapter 13).

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Oviposition and Chemoreception

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Herbivorous insects can sequester allelochemicals and use them actively or passively via aposematic colouration for their own defence against parasites and/or predators. The question arises whether the same or related compounds are also used by the female insect to localize and identify the host plants. Indeed, this has been reported in several insects.A recent example is the papilionid butterfly Battus philenor that has long been known to be stimulated and protected by the alkaloid aristolochic acid and has now been shown by Sime et al. (2000) to sequester this compound from its host plant, Aristolochia rnacrophyllurn, in eggs, larval integument, larval osmeteria, pupa and adults (compare Chapter 3).

7.5

Oviposition and Chemoreception

7.5.1

Chemosensory Coding in General

As worded by Dethier (1982),one of the founding leaders in this field: “The sensing and encoding characteristics of the peripheral sensory system determine what information is made available to the central nervous system.” The information relevant for herbivorous insects must be decoded in respect of 1)modality, 2) type of compounds and 3) the concentration(s)of the perceived compounds. The inputs of the two modalities, volatiles and non-volatiles, are probably separated by the type of sensilla stimulated. Most olfactory sensilla are located on the antennae and palpae and their output is connected via the axons with the olfactory glomeruli in the central nervous system (referencesin Hansson, 1999).The axons of the contact chemoreceptor sensilla have separate targets in the CNS (Mitchellet al., 1999)and lack discrete centres (discussionin Chapman, 1999).However, the two modalities seem not always to be clearly separated, since Stadler and Hanson (1975)identified a classical contact-chemoreceptor sensillum that contains one or several receptor neurones sensitive to plant volatiles (Stadler, 1984).

7.5.2

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Coding of Quality

The type of a compound can be detected either by one specificor several less specific receptor neurones. If one important plant compound (stimulant or deterrent) is detected by a receptor neurone that reacts exclusively to this compound this type of coding is termed “labelled coding” (for detailed discussion see Schoonhoven et al., 1998).An example is the tarsal receptor neuron sensitive to CIF compounds (= cabbage identification factors; see 7.4.4. for details; Hurter et al., 1999; de Jong et al., 2000; Figure 7-4). Receptor neurons for these compounds might be expected to be highly selective and very sensitive. This was found to be indeed the case (Roessingh et al., 1997; Hurter et al., 1999)and the high sensitivitywith a threshold of about 10-11 M matches the extremely low concentration of these compounds in the leaf surface. Intensity is coded in the frequency of nerve impulses generated by the sensory receptor cell and relayed via the axon to the central nervous system. The behavioural reactions released by the motor neurones in the central nervous

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Figure 7-5 Reactionsof two receptor neurons in the tarsal contact-chemoreceptor sensilla of three Pieris butterflies t o stimulation with different glucosinolates (Du et al., 1995; Stadler et al., 1995).The two receptor cells showed in all three species a different structure-activity relationship. Thus, not onlythe quantity ofthe stimulating compounds but also their quality is coded by these receptor neurons. The response patterns of the two closely related Pieris napi species were more similar t o each other than t o Pieris rapae. But, Du e t al. (1995) also found significant differences between these two allopatric species, as indicated by stars in the columns.

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Oviposition and Chemoreception

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system in response to the nervous impulses originating in the receptor neurones, may be very fast. In the case of the salt sensitive contact chemoreceptor neurone of the blowfly Dethier (1968)found that 14nerve impulses (spikes)suffice to release a behaviour reaction (retractionof proboscis) after less than 100 ms of contact with a 200 mM NaCl solution. As pointed out by Schoonhoven et al. (1998)the number of identified secondary plant compounds exceeds now 100,000. This number shows that herbivorous animals cannot have so many receptor neurones specialized for all possibly encountered compounds. Dethier (1971)summarized this situation in the title “A surfeit of stimuli: a paucity of receptors”.As the survey by Chapman (1982)shows the number of receptor neurones in some herbivorous, especially polyphagous, insects may be remarkably high. But since many of the neurons have identical or similar sensitivity spectra the theoretical possibility for labelled coding of thousands of compounds is very slight and such coding would overwhelm the CNS with information. Since not all compounds are important they do not all need to be perceived, but the coding mechanisms need to filter out the important message. The solution to the existence of many important stimuli and limited numbers of receptor neurones seem to be present in across fibre (neuron) coding. This coding mechanism requires fewer, but less specialized receptor neurons. The same type of compound may be detected by different receptor neurons but with a differing specificity.If the range of compounds detected (sensitivityspectrum)differs among the receptor neurons, each compound will produce a specific pattern of reaction in theses neurons (Figure 7-5).

7.5.3

195

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Coding of Quantity Independent of Quality

The question arises how across fibre coding handles different concentrations of compounds. Differently concentrated compounds will only generate a compound specific pattern if the dose response curves are similar and neither saturation nor sub-threshold concentrations are tested. Most likely in nature these conditions are not always met and thus coding and behavioural responses to compounds and to mixtures will, as pointed out by Dethier (1973), not easily be predictable. But despite these complications and conditions, across fibre coding seems to be important in ovipositing females. An example is the oviposition response of Pieris r a p e to different glucosinolates. Du et al. (1995)and Stadler et al. (1995)found that both P. napi and P. r a p e have contact chemoreceptor sensilla on the prothorax legs. Each sensillum contains two receptor neurones that are sensitive to glucosinolates.The sensitivity of the receptor neurons varies between compounds, which means that the most active and least active compounds are not identical for the two cells (Figure 7-5). Stadler et al. (1995) found that this simple pattern is constant between 0.1 and 1.0 mg/ml of the 10 glucosinolates tested. Furthermore, these authors established that differencesin activitybetween the two cells correlated best with the stimulatory activity of the same compounds in oviposition experiments. One of the receptor neurons in the same tarsal sensillum was also

196 . . ,

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found to react to cardenolides that have been found by Huang and Renwick (1994) to inhibit oviposition. This observation added an inhibitory element to the coding by these receptor neurons that needs to be considered if we want to understand the coding of the tarsal neurons. This example shows that indeed across fibre coding functions and presents a meaningful code in the output of the sensory neurons. However, the decoding mechanism of the CNS is still a blackbox that awaitsinvestigation.

7.5.4

Perception of Mixtures

The importance of compound mixtures and sensory coding is also apparent in our recent comparison of the oviposition preferences of two root flies for different host plants. Baur et al. (1996)compared the oviposition preference of Delia rudicurn and D.floralis for four Brussicu cultivars with major oviposition stimulants occurring in leaf surface extracts.Of these genotypes we compared both the oviposition response and the electrophysiological recordings from respective receptor neurons to leaf surface fractions containing glucosinolates or CIF (cabbage identification factor: Hurter et al., 1999; de Jong et al., 2000). The ovipositionpreference of both fly species correlated nicely with the activity of the tarsal receptor neuron for CIF (see 7.4.4. for details of CIF).Thus, the behavioural preferences of the females for the different plant genotypes could be explained by the activity of this receptor neuron. This indicates a case of “labelled line (fibre)” coding. These promising results were recently extended to eighteen different host plants of the cabbage root fly, D. rudicurn. Griffiths et al. (2001) found a clear relationship between the leaf surface contents of indolyl- and benzyl glucosinolates and oviposition preference. There was no correlationto the quantities of the other functional groups of glucosinolates, which are less effective stimulants for the fly and its tarsal receptors. Stadler et al. (2002), using the same collection of plants, analysed the CIF contents of theses extracts as well. The correlation between oviposition and the activity of the CIF receptor neurons was only apparent for a subset of plants. Several of the other plant extracts produced substantial CIF activity, but were not very stimulatory in the oviposition tests. A closer investigation revealed that these plants contain inhibitory compounds that can explain the reaction of the flies. These results seem to indicate that sensory coding is more complex and that in this species also, “across fibre coding” is the likely sensory coding mechanism involved in host plant perception. However, a more definite conclusion about coding has to await more detailed analysis of the host plant compounds as well as the sensory physiology of the various receptor organs involved. This conclusion applies also to other herbivores and their sensory physiology mediating host plant selection.

7.6

Concluding Remarks

If we want to understand the evolution of oviposition by herbivorous insects, and insect plant relationships in general, we need detailed data about the plant cues, mainly the plant compounds that mediate these relationships. When the essential

References

compounds and the mixture components are known and available,this will provide a basis for the study of the insect sensory physiology. The present and future possibility to analyse and modify the genetics of the plant as well as that of the insects will open up new ways to test our conclusionsmore rigorously in the sense that cause and effect can be scrutinized. The first promising steps in this direction have been taken by Nielsen et al. (2001).Given the number of herbivorous insects, a concentration on some insects will be necessary. The comparative study of related groups of herbivores varying in host plant specializationhas been favoured by the late Vince Dethier (Dethier and Crnjar, 1982) and was applied successfully to the study of candidate codes in the gustatory system of caterpillars.There is no reason why such a research tactic should not also be successful in future studies of plant cues important for oviposition.

7.7

Acknowledgements

I thank Drs. Peter Anderson, Reg Chapman, Frank Hanson, Monika Hilker and Louis Schoonhoven for improvements of the contents.This research was supported by grant # 31-52409.97(31-65'016.01)of the SchweizerischerNationalfonds.

7.8

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Rosa, EAS, Rodrigues, PMF. 1998. The effect of light and temperature on glucosinolate concentration in the leaves and roots of cabbage seedlings. J. Sci. Food Agric. 78: 208-212. Sachdev-Gupta, K, Feeny, P, Carter, M. 1993. Oviposition stimulants for the pipevine swallowtail butterfly, Battus philenor, from an Aristolochia host plant: synergism between inositols, aristolochic acids and monogalactosyl diglyceride. Chemoecol. 4: 19-28. Sandstrom,J, Pettersson, J. 2000. Winter host plant specializationin a host-alternatingaphid. J. Insect Behav. 13: 815-825. Schoonhoven, LM, Jermy, T, van Loon, JJA. 1998. Insect-Plant Biology. Chapman and Hall, London. Schreiber, K. 1958. Inhaltsstoffe der Solanaceen und ihre Bedeutung fur die Kartoffelkaferresistenz. Entomol. Exp. Appl. 1:28-37. Sime, KR, Feeny, PP, Haribal, MM. 2000. Sequestration of aristolochic acids by the pipevine swallowtail, Battus philenor (L. ): evidence and ecological implications. Chemoecol. 10: 169-1 78. Simmonds, MSJ. 2001. Importance of flavonoids in insect-plant interactions: feeding and oviposition. Phytochemistry. 56: 245-252. Singer,MC. 1986.The definition and measurement of ovipositionpreference in plant-feeding insects. In: Miller, JR, Miller, TA (eds.)Insect-Plant Interactions.Pp. 65-94. Springer,New York. Singer, MC, Lee, JR. 2000. Discrimination within and between host species by a butterfly: implications for design of preference experiments. Ecol. Lett. 3: 101-105. Spencer, JL. 1996. Waxes enhance Plutella xylostella oviposition in response to sinigrin and cabbage homogenates. Entomol. Exp. Appl. 81: 165-173. Stadler,E. 1971/1972.h e r die Orientierung und das Wirtswahlverhalten der Mohrenfliege, Psila rosae F. (Diptera: Psilidae) I. Larven. Z . Angew. Entomol. 69: 425438. Stadler, E. 1984. Contact chemoreception. In: Bell, WJ, Carde, RT (eds.) Chemical Ecology of Insects. Pp. 3-35. Chapman and Hall, London. Stadler, E. 1986. Oviposition and feeding stimuli in leaf surface waxes. In: Juniper, BE, Southwood, TRE (eds. ) Insects and the Plant Surface. Pp. 105-121. Edward Arnold, London. Stadler, E. 1992. Behavioral responses of insects to plant secondary compounds. In: Berenbaum, MR, Rosenthal, GA (eds. ) Herbivores: Their Interaction with Secondary Plant Metabolites, 2nd edn, Volume 11. Pp. 45-88. Academic Press, New York. Stadler, E. 1994. Oviposition behavior of insects influenced by chemoreceptors. In: Kurihara, K, Suzuki, N, Ogawa, H. (eds. ) Olfaction and TasteXI. Pp. 821-826. Springer-Verlag,Tokyo. Stadler, E. 2000. Secondary sulfur metabolites influencing herbivorous insects. In: Brunold, C, Rennenberg, H, de Kok, LJ, Stulen, I, Davidian, JC (eds.) Sulfur Nutrition and Sulfur Assimilation in Higher Plants: Molecular, Biochemical and Physiological Aspects. Pp. 187-202. Paul Haupt, Bern. Stadler, E, Baur, R, De Jong, R. 2002. Sensory basis of host-plant selection: in search of the "fingerprints" related to oviposition of the cabbage root fly. Acta Biol. Hung. 52: in press. Stadler, E, Buser, H-R. 1984. Defense chemicals in leaf surface wax synergistically stimulate oviposition by a phytophagous insect. Experientia. 40: 1157-1159. Stadler, E, Hanson, FE. 1975. Olfactory capabilities of the "gustatory" chemoreceptors of the tobacco hornworm larvae. J. Comp. Physiol. A. 104: 97-102. Stadler, E, Renwick, JAA, Radke, CD, Sachdev-Gupta, K. 1995. Ovipositional and sensory responses of tarsal sensilla of Pieris rapae (Lep. , Pieridae) to stimulating glucosinolates and deterring cardenolides. Physiol. Entomol. 20: 175-187. Stadler, E, Roessingh, P. 1991. Perception of surface chemicals by feeding and ovipositing insects. In: Szentesi, A, Jermy, T (eds. ) Proc. 7th Int. Symp. Insect-Plant Relationships, Symposia Biologica Hungarica. 39: 71-86. Stange, G. 1997. Effects of changes in atmospheric carbon dioxide on the location of hosts by the moth, Cactoblastis cactorum. Oecologia. 110: 539-545. Stanjek, V, Herhaus, Chr, Ritgen, U, Boland, W, Stadler, E. 1997. Changes in the leaf surface chemistry of Apium graueolens (Apiaceae) stimulated by jasmonic acid and perceived by a specialist insect. Helv. Chim. Acta. 80: 1408-1420. Szentesi, A, Greany, PG, Chambers, DL. 1979. Oviposition behavior of laboratory-reared and wild Caribbean fruit flies (Anastrepha suspensa; Diptera: Tephritidae): I. Selected chemical influences. Entomol. Exp. Appl. 26: 227-238.

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Plant Chemical Cues Important for Egg Deposition by Herbivorous Insects Szentesi, A, Jermy, T. 1990. The role of experience in host plant choice by phytophagous insects. In: Bernays, EA (ed. ) Insect-Plant Interactions. Vol. 2. Pp. 39-74. CRC Press, Boca Raton. Terofal, F. 1965. Zum Problem der Wirtsspezifitat bei Pieriden (Lep. ). Unter besonderer Berucksichtigung der einheimischen Arten Pieris brussicue L. ,P. nupi L. und P. rupue L. Mitt. Munch. Entomol. Ges. 55: 1-76. Tollsten, L, Bergstrom, G. 1988. Headspace volatiles of whole plants and macerated plant parts of Brussicu and Sinupis. Phytochem. 27: 2073-2077. Udayagiri, S, Mason, CE. 1995. Host plant constituents as oviposition stimulants for a generalist herbivore: European corn borer. Entomol. Exp. Appl. 76: 59-65. Udayagiri, S, Mason, CE. 1997. Epicuticular wax chemicalsin Zea mays influence oviposition in Ostrinia nubilulis. J. Chem. Ecol. 23: 1675-1687. Van Emden, HF, Sponagl, B, Baker, T, Ganguly, S, Douloumpaka, S. 1996. Hopkins’ “host selection principle”, another nail in its coffin. Physiol. Entomol. 21: 325-328. Vernon, RS, Judd, GJR, Borden, JH, Pierce, HDJr, Oehlschlager, AC. 1981. Attraction of Hylemyu untiquu (Meigen) (Diptera: Anthomyiidae) in the field to host-produced oviposition stimulants and their nonhost analogues. Can. J. Zool. 59: 872-881. Wegener, R, Schulz, S, Meiners, T, Hadwich, K, Hilker, M. 2001. Analysis of volatiles induced by oviposition of elm leaf beetle Xunthogulerucu luteolu on Ulmus minor. J Chem Ecol. 27: 499-5 15. Wiklund, C. 1981. Generalist vs. specialist oviposition behaviour in Pupilio muchuon (Lepidoptera) and functional aspects on the hierarchy of oviposition preferences. Oikos 36: 163-170. Wink, M, Witte, L. 1991. Storage of quinolizidine alkaloids in Macrosiphum ulbifrons and Aphis genistue (Homoptera: Aphididae). Entomol. Gener. 15: 237-254. Wolfson, JL. 1980. Oviposition response of Pieris rupue to environmentally induced variation in Brussicu nigru. Entomol. Exp. Appl. 2 7 223-232. Xie, Y, Isman, MB. 1992. Antifeedant and growth inhibitory effects of tall oil and derivatives against the variegated cutworm, Peridromu suuciu Hubner (Lepidoptera: Noctuidae). Can. Entomol. 124: 861-869. Yencho, GC, Kowalski, SP, Kennedy, GG, Sanford, LL. 2000. Segregation of leptine glycoalkaloids and resistance to Colorado potato beetle (Leptinotursu decemlineutu (Say)) in F2 Solunum tuberosum (4x) x S-chacoense (4x) potato progenies. Am. J. Potato Res. 77: 167-178. Zalucki, MP, Brower, LP, Alonso-M, A. 2001. Detrimental effects of latex and cardiac glycosides on survival and growth of first-instarmonarch butterfly larvae Dunaus plexippus feeding on the sandhill milkweed Asclepius humistrutu. Ecol. Entomol. 26: 212-224. Zalucki, MP, Brower, LP, Malcolm, SB. 1990. Oviposition by Dunuus plexippus in relation to cardenolide content of three Asclepius species in the southeastern U.S.A. Ecol. Entomol. 15: 231-240.

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Chapter 8 The Plant’s Response towards Insect Egg Deposition Monika Hilker, Odette Rohfritsch and Torsten Meiners

Table of Contents Introduction Plant Tissue: Changes in Response towards Oviposition Plant Tissue: Changes in Response towards Oviposition by Non-Galling Insects 8.2.1.1 Formation of Neoplasms 8.2.1.2 Hypersensitive Responses 8.2.2 Plant Tissue: Changes in Response towards Oviposition by Gall Insects 8.2.2.1 Galls Induced by Hymenoptera 8.2.2.2.Galls Induced by Coleoptera 8.3 Plant Surface Chemicals: Changes in Response towards Oviposition 8.4 Plant Volatiles: Changes in Response towards Oviposition 8.4.1 Local and Systemic Plant Response towards Oviposition by Emission of Vol at iIes 8.4.2 Eliciting Mechanisms of Induction of Plant Volatiles by Oviposition 8.4.3 Role of Jasmonic Acid in Induction of Plant Volatiles by Oviposition 8.4.4 Chemistry of Released Plant Volatiles Induced by Oviposition 8.5 Concluding Remarks Acknowledgements 8.6 8.7 References

8.1 8.2 8.2.1

Abstract

Plant responses towards insect egg depositions range from changes in plant tissue to alterations of the plant’s volatile bouquet. Such responses may result either in defence against herbivores or even in their protection. Defensive tissue changes in plants caused by egg depositions are known as formation of neoplasms and as hypersensitive response. Tissue changes that positively affect herbivores are induced by egg depositions of several gall forming insects. These manipulate plants to produce nutritive and protective tissues for the progeny. Indirect plant defence responses towards egg deposition do not affect the eggs or hatching larvae directly, but employ the third trophic level. Changes in the plants’ volatile emission induced by insect egg deposition are known to attract egg

206

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The Plant’s Response towards Insect Egg Deposition ... .....................................................

.. .

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

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

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.

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

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

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

parasitoids and may thus defend the plant against herbivores. The diversity of plant responses towards egg depositionsis reflected by various elicitorsand eliciting mechanisms. Compounds from the egg chorion or glandular secretions associated with oviposition may act as elicitor. For some plant responses, egg deposition is associated with wounding of plant tissue by e.g. the ovipositor valves of the egg laying female or its mouthparts. Thus, elicitors may also be located at the female’s ovipositor or mouth. This chapter reviews the various ways plants react to egg depositions, the still scarce knowledge about the different mechanisms eliciting these responses, and their different ecological effects. The studies considered in this review indicate a high complexity of insect - plant interactions that can be initiated as soon as insect eggs touch the plant.

8.1

Introduction

When we study oviposition of a herbivorous insect on a plant, the insect is usually considered to play the active part, while the plant is attributed the passive role of being selected and being accepted for oviposition (see Chapter 7). Even though oviposition is the very first step taken by numerous herbivorous insects to exploit a plant, the active role of the plant under herbivorous attack has mostly been studied only when larvae have already hatched and started feeding. Feeding activity of the larvae is well known to induce direct and indirect defence responses of a plant (Price, 1986; Dicke and Sabelis, 1988; Sabelis et al., 1999).Direct plant responses directly affect the herbivore by reducing plant quality due to e.g. production of toxins like those targeting the nervous system or inhibiting digestion (Edwards and Wratten, 1987; Baldwin, 1994; Duffey and Stout, 1996). Indirect defence responses towards feeding herbivores employ interactions with the third trophic level (Price et al., 1980). They draw on predators and parasitoids to use their potential to attack and kill the threatening herbivores. Various studies have shown that the feeding activity of herbivores can induce a change in the volatile pattern in plants so that the emitted plant odour becomes attractive to natural enemies of the herbivores (e.g. Tumlinson et al., 1993; Dicke, 1994; Turlings et al., 1995). However, there is growing evidence that a plant may not only respond towards feeding by herbivores, but already to the first sign of future attack, towards the oviposition.Also, the defensive plant responses towards oviposition may be direct or indirect. Oviposition-induced direct responses may affect the egg, the hatching larvae or the gravid female. Oviposition-induced indirect responses are able to act indirectlyagainst the herbivoresby employing the third trophic level and attracting egg parasitoids (see Table 8-1). In this chapter, we are going to provide an overview of our knowledge about plant responses towards oviposition by insects. We will first address plant tissue changes in response towards egg deposition (see Section 8.2). Such plant tissue changes may directly affect the herbivore and reduce the number of viable eggs

"Cry for help" Diprion pini in the "pine system"

Pinus sylvestris

Ulmus minor

Xanthogaleruca luteola

"Cry for help" in the "elm system"

Indirect

Brassica oleracea

Pieris brassicae

Oviposit ion deter rence

egg cement

production and emission of synornones

production and emission of synomones

parasitization of oviduct eggs by Chrysono- secretion tomyia ruforum

parasitization of oviduct eggs by Oomyzus secretion gallerucae

Timing

plant hybrids

unknown

species specific for plant and herbivore

unknown

24-72 h

24-72 h

24 h

5-7 d

24h

plant 24h individuals

Specfficity

isolation, extracts from genetic desiccation, eggs, bruchins lines of isolated from plant hatching larva cannot bite beetles through neoplasm, egg drop, enhanced predator pressure

detachment, egg cement egg drop, or egg shell enhanced predator pressure

isolation, desiccation

Elicitor/ location of elicitor

change in surface deterrence of egg batches chemistry further oviposition

Bruchus Pisum increased cell pisorum sativum and division activity, Callosobruchus Lathyrus spp. neoplasrna maculatus formation

necrotic zone

necrotic zone

Proximateeffect Final effect of oviposiion on herbivore on plant

Neoplasma formation

Solanum tuberosum

Hypersensitive Leptinotarsa response decemlineata

Host plant

Brassica nigra

Ovipositing herbivore

Direct Hypersensitive Pieris rapae response Pieris napi

Category of defence

Hilker et al., 2002

Meiners and Hilker, 1997,2000 Meiners et al., 2000

Blaakmeer e t al., 1994a

Doss et al., 1995 Doss et al., 2000 Oliver et al., 2000

Balbyshev and Lorenzen, 1997

Shapiro and De Vay, 1987

Reference

Table 8-1 Overview of direct and indirect plant defence responses towards insect egg deposition. Timing: time after egg deposition when the effect has been observed

208 The Plant’s Responsetowards Insect Egg Deposition ...................... ... . . ................... . ... ....................... .. .. . . ................. .. ............... ... ........... . ,.,

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. ... .

by different means, e.g. by egg drop due to the formation of neoplasm (see Section 8.2.1.1)or by desiccation of the eggs due to a hypersensitive response (see Section 8.2.1.2).These direct defensive plant responses are restricted to the tissue onto which the eggs are laid and to the immediately adjacent tissue. Not only plant tissue changes but also changes in plant surface chemicals in response to egg deposition may directly defend the plant against the herbivore. In Section 8.3, we consider a study which shows that change in plant surface chemicals in response towards egg deposition directly affects the herbivore by reducing plant acceptance of further oviposition after the first eggs have been laid on the plant. A very sophisticated way of response towards egg deposition indirectly affecting the herbivore has been shown in plants that start to release volatiles that attract egg parasitoids when eggs are laid upon them. In Section 8.4 we outline studies on this induction of volatiles by oviposition,which is not restricted to the plant tissue with eggs but extends also to adjacent leaves without eggs. While ovipositionof several herbivorous insects has been shown to induce plant defensive responses, gall insects succeeded in exploiting plant responses for their purposes. They manipulate plants in such a way that the plant tissue responds towards their ovipositional activity or larval feeding activity by forming galls, which protect and nourish the gall insect. Counteradaptations of plants against ovipositionof galling insects have hardly been studied up to now (see Section8.2.2). Likewise, further studies are needed to elucidate whether non-galling herbivorous insects may take advantage of oviposition induced plant responses. Special emphasis will be given in this chapter to the mechanisms eliciting the responses wherever any knowledge is available.As far as known, the physiological, chemical, and molecular mechanisms of the plant responses towards insect oviposition will be outlined.

8.2

Plant Tissue: Changes in Response towards Oviposition

Herbivorous insect egg deposition can evoke tissue changes in plants with two different effects on the eggs or hatching larvae: (1)Tissue changes for plant defence aim at killing eggs or isolating hatching larvae from nutritive tissue (see Section 8.2.1). (2) Tissue changes that positively affect herbivores are induced by egg depositions by several gall forming insects (see Section 8.2.2).

8.2.1

Plant Tissue: Changes in Response towards Oviposition by Non-Calling Insects

Plants under herbivore attack may change their tissue physiologically and/or morphologically and reduce its suitability in response to both feeding activity and oviposition. Feeding induced tissue changes are known to result in enhanced

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

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

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

,

Plant Tissue: Changes in Responsetowards Oviposition

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

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

209

.....,,......................................... ................................................... ..........................................

formation of defensive structures like glands, thorns or trichomes (e.g., Young, 1987; Bauer et al., 1991; Agrawal and Karban, 2000). Defensive tissue changes in plants caused by egg depositions are known as formation of neoplasm and as hypersensitive response.

8.2.1.1 Formation of Neoplasms Neoplasms arise when cell division is stimulated in non-meristematic areas. Tumour-like growth of the cells leads to the development of undifferentiated tissue, the so-called neoplasm. Recently neoplasm formation has been shown to occur in specific genetic lines of pea (Pisurn sativurn L.) in response to oviposition of the pea weevil (Bruchus pisorurn L.) and the cowpea weevil (Callosobruchus rnaculatus) (Doss et al., 2000). These beetles are not really weevils (Curculionidae), but belong to the Bruchidae. They lay their eggs singly on the surface of pea pods. Usually, the larva leaves the egg just at that site where it is attached to the pod and thus directly enters the inside of the pod to feed upon the seeds inside without being exposed to any danger from the outside. Pods of the pea lines with the socalled N p allele (Neoplastic Pod allele) respond by increased cell division at the site of egg attachment. The new cells form a small protuberance consisting of undifferentiated tissue with the egg on its top (Figure 8-la). This protuberance may protect the pea pod in two ways: (1)the protuberance with the egg on it may be detached from the pod prior to larval hatching; (2) the hatching larva leaves the egg and needs to crawl down the neoplastic bump on the outside to penetrate the pod at a site with healthy tissue. Thus, the larva hatching from an egg on a neoplasm is exposed to carnivores and desiccation when looking for its way into the pod.

Figure 8-1 Neoplasma formation by cell division of undifferentiated tissue on pea pod in response to (a) bruchid egg or (b) bruchins.

.

210

The Plant's Response towards Insect Egg Deposition

\ OH

\ OH

I

OH

d

OH

Figure 8-2 Bruchins A-D. Bruchin A is (Z)-9-docosene-1,22-diol, 1-(3-hydroxypropanoate) ester, bruchin B is in addition to bruchin A also esterified at C-22with 3-hydroxypropanoic ester, bruchin C is (Z)-9-tetracosene-l,24-diol,-1,24-(3-hydroxypropanoate)diester, a n d bruchin D is (Z)-9,17-tetracosadiene-1,24-(3-hydroxypropanoate)diester (Oliveret al., 2000).

Field trials with Np/Np pea lines revealed that the pea weevil infests pods of these peas significantlyless than pods of other pea lines without this high neoplastic activity (Dosset al., 2000). Pea pods of Lathyrus tingitanus and L. sativus also respond towards oviposition of the pea weevil B. pisorurn by formation of neoplasms. In these Lathyrus pod neoplasms, the hatching larvae just died in the callus tissue because they did not manage to penetrate in the pods from there (Annis and OKeeffe, 1984). Chemical analyses of the components initiating the neoplastic growth in P. sativurn by stimulation of mitogenesis revealed the so-called bruchins, i.e. longchain a,w-monounsaturated Cz-diols and a,w- mono- and diunsaturated C,-diols, monoesterified or dieesterified with 3-hydroxypropanoicacid (Figure 8-2) (Oliver et al., 2000). Synthetic samples of the bruchins B, C, and D induced very clear neoplasm formation within a week at concentrations of as little as 1fmol (about 5 pg) when applied to a P. sativurn pod of a responsive genetic line (Figure 8-lb). The bruchins are thought to be products of fatty acid biosynthesis or metabolism. They have been identified from extracts of adults of C. rnaculatus and B. pisorurn. Also, extracts of pea weevil eggs and accompanyingfluid have been shown to have mitogenetic activity potential (Doss et al., 1995). Studies on the specificity of the bruchins have revealed that these components show specific activity only on pod tissue of the pea (P. sativurn). They do not cause

Plant Tissue. Changes in Response towards Oviposition

mitogenesis on leaves or stems of the pea. Also, pods of Lathyrus tingitanus are induced to form neoplasmswhen bruchin B is applied. Severalpods of other legume species are known to be able to form neoplasms in response to egg deposition of bruchids. However, it has not been proved yet whether bruchins are the stimulating factors in these species (Doss et al., 2000). First structure-activity studies on the bruchins and closely related components indicate that the unsaturation in the diol chain is not required, whereas the 3-hydroxypropanoate function seems to be crucial (Doss et al., 2000). The function of bruchins in the bruchid life cycle is unknown. Extracts of sexually mature female pea weevils have been shown to have much higher neoplasm-inducing activity than extracts of males and immature females (Doss et al., 1995). However, in the cowpea weevil, extracts of newly emerged males and females revealed similar activities (Doss et al., 2000). 8.2.1.2 Hypersensitive Responses

Hypersensitive responses were first described in the context of plant defence against microbial disease (Ward, 1902; Stakman, 1915; Klement and Goodman, 1967). A hypersensitive response is made visible by a rapid, locally restricted necrosis of cells at the infection site. This form of induced defence has been shown to act against phytopathogenic viruses, bacteria, fungi, and nematodes (Klement & Goodman, 1967; Wood, 1982; Moerschbacher and Reisener, 1997; Sticher et al., 1997; Somssich and Hahlbrock, 1998; van Loon et al., 1998; Kombrink and Schmelzer, 2001).The necrosis of cells is generally thought to kill the invader or at least to inhibit its growth. Fernandez (1990, 1998) describes several examples of formation of locally restricted necrotic cells in the response of plants towards feeding activity of mites and gall insects.Feeding of the gall mite Aceria cludophthirus causes a hypersensitive response in Solanum dulcamara. Depending on the attacking mite species it can trigger either induced resistance or induced susceptibilityin the leaves (Westphal et al., 1991, 1992). Hypersensitive responses as a defensive reaction of the plant towards the gall insects will be addressed below (see Section 8.2.2).Fernandez also considers the necrosis of tissue surrounding bark beetle galleries to be a hypersensitiveresponse. Similarly,he mentions the death of woody tissue damaged by oviposition of Sirex females (Siricidae)in the context of hypersensitive response. However, only two studies are available which mention hypersensitive response of plant leaves towards insect egg deposition, i.e. the formation of necrotic leaf tissue in response to eggs. Shapiro and de Vay (1987)were the first to show that insect eggs can trigger a hypersensitive response in a plant. When eggs of Pieris brussicue and P. nupi are laid on leaves of mustard, Brussicu n i p , necrotic zones appear at the site where the eggs are lying within 24 hours after deposition. The necrotic zone leads to desiccation of the eggs within 3 days. The elicitor of this response is supposed to be located in the egg cement that glues the eggs to the leaves. Eggs themselves, after detachment and reattachment with a brush, were not active. No bacteria or fungi associated with the eggs were found to elicit the necrosis. A hypersensitive

211

212

The Plant’s Response towards Insect Egg Deposition

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

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

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

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

..

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

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

Figure 8-3 Hyper-

sensitive response in a clone of a hybrid of Solanurn spp. as response to egg deposition of the Colorado potato beetle Leptinotarsa decemlineata. Part of a

leaf with egg mass on nedrocic tissue.

response appears only in certain individuals of the plant population, indicating genetic differences in the ability of Brussicu plants to employ this defence mechanism. Balbyshev and Lorenzen (1997) also found hypersensitive response of a clone of a Solanurn hybrid, when the Colorado potato beetle Leptinotursu decernlineutu lays its egg masses onto the leaves. The leaf tissue around the egg mass becomes brownish and necrotic within a zone of about 2 cm (Figure 8-3), disintegrates, detaches, and thus, ”drops” the egg mass from the leaf. The elicitor of this hypersensitive response is unknown. Since no bacteria were isolated from rinsates of egg masses from the Colorado potato beetle, this induction of a hypersensitive response is probably not due to bacterial microorganisms. While the hypersensitive response of Brussicu n i p towards eggs of Pieris spp. increased egg mortality by desiccation (Shapiro and de Vay, 1987), dropped eggs of the Colorado potato beetle were more robust and did not suffer increased mortality due to water loss. However, in the field, egg masses and hatching larvae on the ground are exposed to high predator pressure by e.g. spiders and ground beetles. Field data revealed that only276 of the L. decernlineutu larvae which hatched from egg masses on the ground near the plant succeeded in colonizing the plant again. If the egg masses on the ground were more than 15 cm away, none of the larvae were found to reach the plant again (Balbyshev and Lorenzen, 1997). Abiotic conditions like shading of the plants sigruficantlyaffect the hypersensitive response towards egg depositions (Shapiro and de Vay, 1987; Balbyshev and Lorenzen, 1997).Shaded plants did not display as strong responses as plants fully exposed to normal daylight. Light is also known to act as a modulator of the hypersensitive response of plants towards bacterial disease (Lozana and Sequira, 1970). High temperature may suppress the hypersensitive response of plants towards fungal disease (e.g. Silverman, 1959).However, the impact of temperature

Plant Tissue: Changes in Response towards Oviposition

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

,

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

.

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

213

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

on the hypersensitive response of a plant towards egg deposition has not been investigated yet. No studies on the mechanisms of plant hypersensitive responses towards insect egg deposition are available up to now. In contrast, phytopathologists have intensively studied hypersensitive responses towards plant pathogens. Invasion of pathogens into plant cells is known to evoke a series of rapid, initial responses, as (a)changes in inorganicion fluxes across the plasma membrane, (b) accumulation of reactive oxygen intermediates (oxidative burst), (c) changes in the phosphorylation status of various proteins with signalling functions, and (d) local gene activation encoding proteins that are either directly or indirectly inhibitory towards the invading pathogens (Kombrinkand Schmelzer, 2001; and references therein). The oxidative burst, which may originate in the activity of a plasma membrane located NADPH-dependent oxidase complex and a cell wall peroxidase, is controlled by induction of antioxidant enzymes. A biphasic accumulation of reactive oxygen intermediates has been observed in several systems in response to bacterial and fungal elicitors. The first peak of accumulation is considered nonspecific, while the second one is dependent on the pathogen race attacking the cells. The oxidative burst leads to the death of a plant cell (autotoxicity),to direct inhibition of the pathogen, and plays a role as a signal for induction of cellular protectants and defence responses in adjacent cells. The cells undergoing a hypersensitive response reorganize their cytoskeletonarchitecture and synthesize cell wall phenolics and callose. The hypersensitive response of a plant towards a pathogen is not necessary for resistance. Plants may also display resistance against pathogens without any hypersensitive response when attacked (Kombrink and Schmelzer, 2001; and references therein). In spite of numerous microscopic studies of cells displaying hypersensitive response, no consistent morphological features could be defined to distinguish death of plant cells due to hypersensitive response from cell death caused by other factors. The hypersensitive responses described towards egg depositions look very similar to the ones towards plant pathogens. But unequivocal proof is lacking whether the plants responding towards egg deposition with formation of necrotic cells indeed show the same typical cytological,physiologicaland molecular changes as plant cells do that respond hypersensitively towards pathogens. A parallel between the plants responding towards insect egg deposition by formation of necrotic cells and neoplasms on the one hand and the plants responding towards pathogens on the other hand is their specificity.Only certain plant varieties or certain individuals within a population show these responses (Table8-1).The high specificityof the plant-pathogen interaction has been described by the gene-for-gene model which postulates that pathogen and plant have corresponding genes which are responsiblefor the outcome of the interaction (Flor, 1971). While several pathogen elicitors are known to initiate hypersensitive responses in plants ( e g Cervone et al., 1997),nothing is known about the elicitors associated with insect egg deposition.

214

The Plant’s Response towards Insect Egg Deposition

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8.2.2

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

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

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

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Plant Tissue: Changes in Response towards Oviposition by Call Insects

Gall insects may induce the plant to grow a gall by oviposition and feeding. This subchapter outlines the different responses of plants towards gall insects, especially towards gall insect oviposition. Even though several studies have analysed the biochemical changes in plant tissue under attack from gall insects (Bronner, 1992; Bayer, 1994), knowledge about the chemistry of the elicitors and the molecular mechanisms of these plant responses is scarce. However, possible elicitors will be discussed on the basis of the available studies and observations (Hori, 1992). A gall or “cecidium”is usually defined as the atypical grown structure of a plant that provides food and shelter for the developingimmature stages of the gallinsect. The atypical, but limited growth of plant tissue elicited by gall insects is due to cell enlargement, mitosis and often change in the morphogenesis of the induced cells. Most galls are considered to be limited neoplasms in contrast to the unlimited growth of cancerous tumours. Some galls are just conglomerations of e.g. parenchymaticcells, while others show a high degree of differentiation.The process of gall formation involves striking biochemical changes in the tissue attacked by the gall insect. Since galls often act as sinks for water and nutrients, they also change the physiology of the tissue surrounding the gall (Shorthouseand Rohfritsch,1992). The relationships between plants and gall inducing insects are usually very specific, suggesting tight coevolutionary processes. Even though the galls provide nutritive tissue and shelter for the gall insect, decades ago Zweigelt (1931)asserted that a plant produces a gall not primarily for the benefit of the gall inducer, but to isolate the gall insect. While the gall insect benefits from this “isolating”gall tissue, other induced plant defences are known to kill the gall insect. Fernandes (1990; 1998)describes resistance responses associated with cell necrosis in plants around the feeding site of gall midge larvae. The necrotic tissue prevents normal development of the gall insect and its access to nutrients. Also lignification may be an efficient mode of defence of a plant against a gall insect. Rey (1976)observed that eggs of the cynipid Biorrhiu puZZidu laid into the roots of oak may become encircled by lignified cells which first appear at the posterior egg pole. Within a period of two weeks the eggs are totally encircled by lignified sheath and many of them die. Gall inducers are found in all insect orders and the host plants belong to nearly all branches of the plant kingdom. Most gall inducing insects are highly host and organ specific. Among the dipteran gall insects, gall midges (Cecidomyiidae) constitute a large group of gall inducers and they represent the most general gall type. In this group, oviposition and eggs have no effect on the plant tissue, but the larva and its activity induces the formation of the gall. This so-called cecidomyiid model (Rohfritsch, 1992) is followed by numerous other gall inducers such as Homoptera, Hemiptera, Thysanoptera, most Lepidoptera, and Acarina. In contrast, all galls induced by Hymenoptera start at oviposition. Nevertheless, the activity of the larva may also play a role as is outlined below for the cynipids and chalcidoids.

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Plant Tissue: Changes in Response towards Oviposition 215 ................................. ........................................... .. . . .............................. . .

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Table 8-2 Overview of oviposition induced mechanisms of gall growth

Gall insect

Hymenoptera/in general

Hymenoptera Cy n i pidae : Diplolepis Hymenoptera Chalcidoidea

Hymenoptera Tenthredinidae Coleoptera Curculionidae: Ceutorhynchus

Elicitingof gall growth by ovipositional wounding: -physical process of wounding -chitin and chitosan of ovipositor valves?

other oviposition associated factors larval feeding activity ovipositional wounding (see Hym./in general) components of the egg larval feeding activity ovipositional wounding (see Hym./in general) components of the egg larval feeding activity ovipositional wounding (see Hym./in general) componentsof the accessory glandular secretions larval activity ovipositional wounding (see Hym./in general) the eggs components associated with the eggs?

Table 8-2 summarizes the knowledge about the elicitors or eliciting mechanisms of galls induced by oviposition of the different taxa. The following section considers“true” galls (cecidia)that ”host”the gallinginsect during its entire development. However, so-called “procecidia” are also known which confine only the egg stage of an insect but decay as soon as the larva hatches (Mani, 1964). Such procecidia occur, for example, when the tenthredinid sawfly Arge enoides deposits its eggs on leaves of Rosa. The egg deposition induces the growth of small pustules from which the hatching larvae escape (Mani, 1964). Another example is provided by Heliozela stanneella (Lepidoptera, Heliozelidae). The female induces procecidial growth on oak stems by ovipositional wounding and the fluid covering the eggs (Prota, 1963).In contrast to the neoplasms described above (see Section 8.2.1.1), these procecidia embed and thus maybe protect the eggs. 8.2.2.1 Galls Induced by Hymenoptera The hymenopterous gall inducers have evolved a sophisticated ovipositional wounding behaviour and specialized chitinous ovipositors.The physical wounding process per se may initiate gall growth by disturbing the tissue architecture, but chitinous components of the ovipositor valves may also play an important role together with the egg. Chitin, a polymer of p-1,4 N-acetyl-D-glucosamine,and chitosan, a deacetylated derivative of chitin, are considered to be elicitorsin plant defence responses (Hahn et al., 1993; Cervone et al., 1997). The plant receptor for chitin oligomersis assumed to play a role in detecting chitin-containing organisms, e.g. pathogenic fungi and arthropods (Boller, 1995). Chito oligomers and so-called nod-factors (lipo-

216

The Plant’s Responsetowards Insect Egg Deposition

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chitooligomers)stimulate the activity of chitinase isoenzymes (Xie et al., 1999) in soybean roots, and both induce cell division and formation of a plastem in roots of legumes (Spaink, 1996).The chitinous cheliceral stylets of the mite Eriophyies cludophtirus have been shown to be involved in the induction of galls in susceptible varieties of Solunum dulcumura,while it induces a hypersensitiveresponse in resistant plants (Bronner et al., 1988, 1989; Westphal et al., 1996).Nod factors have been suggested to be involved in formation of galls (Schonrogge et al., 1998).Similarly, chitin and chitosan may also act as components, which elicit the initial processes of gall growth.

Call Wasps (Cynipidae) The phytophagous gall wasps either induce galls in various plants or live as inquilines in galls induced by other species. Different organs of the plants are used for gall induction, such as roots, leaves, buds, and several parts of the flower. Plant tissue induced by cynipids to form galls experiences a high degree of redifferentiation.Cynipid galls often appear as new organs with a complex external structure, but the most prominent structural characteristic of these galls is the presence of concentriclayers of differentiated cells around the larval chamber. The innermost layer is composed of the well-characterizednutritive tissue cells, common to all cynipid galls (Bronner,1992).Gall initiationby cynipidsis thought to be driven by several factors, including wounding of the tissues with the ovipositor, activity of the egg, and activity of the larva (Rohfritsch, 1992). The highly specific shape and structure and epidermal outgrowths of the mature gall are initially induced by the act of oviposition in those cynipid species whose larvae stay at the site of oviposition (Folliot, 1964). The oviposition of several Diplolepis species has been studied in detail (Magnus, 1914; Meyer, 1957; Bronner, 1985) (Figure 8-4a). Diplolepis females have been observed to fix their eggs by piercing a single epidermal cell of the plant tissue and to “anchor” the egg in this cell with ovipositional fluid from collateral glands (Figure 8-4b, d). The fluid may also serve as interface between the egg chorion and the plant tissue. The egg itself together with the wound is the most important component for the initiation of cynipid galls. The cells around the posterior pole of the egg are activated in such a way that proteosynthesis and RNA synthesis are greatly increased (large nucleus and nucleolus, a fragmented vacuole, a cytoplasm enriched with ribosomes).If phenolics are present in the vacuole they are fragmented. This first stage of gall growth appears within 18 h of oviposition. About 1 day after egg deposition, plant cell hypertrophy occurs, but only 2-3 cell layers beneath the eggshell are involved in the case of Diplolepis rosaefolii (Figure 8-4c). Cell divisions begin 2 days after egg deposition (Figure 8-4e) and cell growth gives rise to a pad of tissue supporting the egg: the gall plastem. About 3 days after egg deposition, plant cells of the pad in close contact with the eggshell begin to show lyses. A lytic cavity begins to form (Figure8-4f). The eggs are known to exert a proteolytic, cellulolytic and pectinolytic activity in vitro (Rey, 1976; Bronner, 1977). Bronner (1985) demonstrated that the lytic enzymes of the eggs

Plant Tissue: Chanaes in Response towards Oviposition

Figure 8-4 Development of a cynipid gall in a Rosa bud. (a) Oviposition of the gall wasp Diplolepisbicolorin a bud of Rosaacicularis (Shorthouse,1974).Bar: 1mm. (b) Eggs of Diplolepis rosaefolii fixed on the abaxial epidermis of Rosa virginiana along secondary veins. The ovipositional fluid surroundingthe distal pole ofthe egg is visible (star) (Leblanc and Lacroix, 2001). Bar: 100pm. (c) Mature galls of D. bicolor on R. acicularis (Shorthouse, 1974).Bar: 1 cm. (d)A higher magnification of b. Bar: 10pm. (e) Initiation of D. rosaefoliigall. Cross section of a leaflet of R. virginiana showing the point of attachment of an egg (E) on the abaxial epidermal layer. Earlycellularchanges are observed in 2 or 3 cell layers beneath the insertion of the egg (arrows). The cells have a dense cytoplasm, a large nucleus and nucleolus, the vacuoles are reduced in size. Cells divide. Of: ovipositional fluid, E: egg. (f) Later modifications observed 3 days after oviposition. Beneaththe attachment side of the egg a pad oftissue has formed, a number of cells beneath the insertion side (arrows) becomevacuolated,cell content progressivelydisappears, the lytic process has started. A t the periphery of the lytic area, cells have a dense cytoplasm and multiply, Of: ovipositional fluid. E: Egg. Bar: 10pm.

217

218 The Plant’s Responsetowards Insect Egg Deposition ......................... ................................................. .. . .. ............................................... ......................

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are excreted by the whole egg chorion. Bronner observed that the chorion has the lytic activity from the very beginning of the oviposition and that the eggs keep their lytic activity even after washing. This indicates that the enzymes are not part of the ovipositional secretion and that the embryo does not secrete them. During embryological development the eggs also exert a polarizing effect on the newly formed plant tissues. This effect is most pronounced at the distal or posterior egg pole. The cells lining the lytic cavity formed by egg enzymes grow and divide in such a way that they nearly obliterate the wound. A few cells highly stimulated to proteosynthesis appear at the bottom of the cavity beneath the zone of contact with the eggshell. Thus the larval cavity is lined with a newly formed tissue called the lysenchym.Cell lyses and regeneration are the two complementarymechanisms induced by the egg enzymes that form this larval cavity (Magnus, 1914; Bronner, 1977; Meyer, 1987; Rey, 1992; Rohfritsch, 1992; Leblanc and Lacroix, 2001). When the larva hatches from the egg, it feeds within the cavity. The larval activity stimulates the plant tissue to provide nutritive cells and sheltering tissue. Hori (1992)reviews possible gall elicitorsin the larval saliva that promote growth of the gall during larval feeding. Little is known about the chemistry of the gall elicitors associated with the cynipid egg that initiate the gall growth.

Chalcid Wasps (Chalcidoidea) Chalcidoidea and Cynipidae are considered to be sister groups (Roskam, 1992). Little is known about the galls induced by chalcid wasps, namely members of the Eurytomidae and Perilampidae. Chalcid females deposit their eggs deeply within plant tissues preferably in the vicinity of the vascular cambium, using a long and sharp ovipositor (Magnus, 1914). The eggs are usually deposited in a row with each oviposition wound and egg being separated by a few millimetres. Both the wounding process and components from the eggs stimulate cell growth. A pad of tissue - the so-called plastem - is formed around the chalcid egg similar to that described for the cynipid egg. However, the chalcid egg does not have the lytic activity and the polarizing effect of cynipid eggs (Magnus, 1914; West and Shorthouse, 1989).When the larva has hatched, its feeding activity may further promote gall growth.

Sawflies (Tenthredinidae) Most Tenthredinidae are external leaf feeders or leaf miners; only a few induce galls and nearly all of these are restricted to leaves of SuZix and PopuZus (Smith, 1970)(Figure8-5a).The gall inducers belong mainly to the genus Pontuniu and Euuru. Galls induced by sawflies are relatively unstructured; the cell layers of the gall keep the characteristics of leaf or shoot tissues (Figure 8-5b). Thus, plant cells of galls induced by sawflies do not show the high degree of differentiation of cell layers known for cynipid galls. With the saw-like ovipositor valves the female saws a pocket-shaped slot stretching out tangentially to the leaf blade. Along with the egg that is inserted adjacent to the vascular tissue, the female introduces a substance

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Plant Tissue: Changes in Response towards Oviposition

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Figure8-5 Leaf gallsof sawflyfontuniuproximuonSulixtriundru (Rey,1992).(a) Maturegalls, collected mid-summer. Bar: 0.5 cm. (b) Cross section of a gall 60 h after oviposition. Cells in every layer have divided several times by periclinal mitosis. E: egg; le: lower epidermis; ue: upper epidermis;vr: median vascular region. Bar: 50pm.

which is secreted from the accessory glands and bathes the eggs (Magnus, 1914; Rey, 1992). About 3 days after egg deposition, the tissue starts to form a protuberance. About 6-7 days after oviposition, the typical bean galls are apparent in Pontania proxima (Leitch, 1994). Growth of sawfly galls is initiated during oviposition. The wounding process provides the stimulus during oviposition and/or the secretion of accessory glands coating the eggs. The host tissues react to the wounding and the ovipositionalfluid by cell division and cell expansion in all leaf cell layers. The wounding process per se could induce gall growth by disturbing the leaf blade architecture. Normal leaf blade morphogenesis results from an inhibitory effect that upper half and lower half of the leaf lamina exert on each other to avoid supernumerary cell layers (Mbdard, 1988).Since the wounding of the leaf by sawfly oviposition separates the two parts of the leaf lamina, the oviposition wound and fluid may allow supernumerary cell layers to grow on both sides of the lamina. Chemical analyses of the secretion of the accessory glands revealed that this ovipositional fluid is rich in both nucleic acids and protein (Hovanitz,1959).McCalla et al. (1962)identified uric acid, two adenine derivatives, glutamic acid, and possibly uridine. Leitch (1994) showed that the amounts of 4 iso-pentenyl adenine type cytokinins increased significantly in leaf tissue induced by gall insect oviposition within a period of 4 to 6 days. These cytokinins may stimulate the cell divisions observed during growth of sawfly galls. Leitch suggests that precursors of these cytokinins may be present in the ovipositional fluid. This suggestion is supported by the detection of the adenine derivatives identified by McCalla et al. (1962).The vascular tissue adjacent to the sawfly galls is also a known source of cytokinins (Leitch, 1994). However, a study by Higton and Mabberley (1994)indicates that the cytokinins might promote the sawfly gall growth, but doubt that these components induce the galls. Higton and Mabberley (1.c.) wounded leaves of the willow Salixfragilis using a microscalpel in a similar way as the sawfly female of P. proxima does. They

220

The Plant’s Response towards Insect Egg Deposition

introduced an ultrafiltrate of the fluid of the accessory glands into this wound. Their results show that components of the accessory glandular secretion with less than 3 kDa and an UV absorption peak at 247 nm are able to produce a typical gall of P. proximu when applied into a leaf wound. 8.2.2.2 Galls Induced by Coleoptera

Most of the coleopterous gall inducers belong to the family Curculionidae (Meyer, 1987).In this group, egg deposition and not larval feeding is thought to induce the growth of the gall. The female wounds the tissues (e.g. roots, young stems, legume pods) with its head and mouthparts to prepare a small channel into which the egg is introduced. In the curculionid Ceuforhynchus nupi living on Brussicu nupus L., a membranous material (exochorion according to LePape and Bronner, 1987, a secretion of the oviduct bathing the eggs according to Deubert, 1955)is known to coat the egg laid into the pith of the stem. This material forms a close pouch around the egg and has been suggested to protect the egg from desiccation (Deubert, 1955). About 10 h after egg deposition, plant cells around the egg are activated, the nuclei enlarge and 1 day later cells divide to form a nodule of small, metabolically active cells around the egg. Whether the exochorion has the inducing effect on the plant cell division is unknown. Cells of the pith surrounding this nodule start to degenerate. This lytic process observed around the egg nodule is of the same kind as that observed in the pith of normal plants. However, the eggshell has no lytic activity. According to Deubert (1955) and to LePape and Bronner (1987) the egg induces translocation of water and nutrients from the plant tissue to the nodule. This redistribution of nutrients and water might cause the degeneration of the surrounding cells.

8.3

Plant Surface Chemicals: Changes in Response towards Oviposition

While tissue changes as a response to oviposition are apparent at first glance, other plant responses are not. Egg depositions of Pieris brussicue on cabbage leaves (Brussicu oleruceu) induces the plant to produce oviposition deterrents which reduce the number of subsequent egg depositions of this butterfly at that site (Blaakmeer et al., 1994a).It has long been known that the butterfly avoids egg depositions at those leaves that already carry conspecific eggs. However, this effect has been attributed to oviposition deterrents associated with the eggs, since methanolic and watery egg rinsing applied to cabbage leaves also reduced the number of egg depositions (Rothschild and Schoonhoven, 1977).From these egg washes, the socalled miriamides, which are three novel avenanthramide alkaloids, have been identified as active oviposition deterrents (Blaakmeer et al., 1994b). However, Blaakmeer and co-authorsfound in a further study that cabbage leaves keep their oviposition deterring activity even after removal of the Pieris eggs (Blaakmeer et al., 1994a).They did not find any miriamides left on cabbage leaves from which the eggs had been removed. Thus they concluded that plant surface

Plant Volatiles: Chanzes in Response towards Oviposition

chemicalsmust have changed in response towards the egg deposition.And indeed, they were able to isolate oviposition deterrents from cabbage leaves from which the eggs had been removed by dipping them into methanol. In the discussion of their results the authors mention that cabbage leaves adjacent to those with eggs were also found to receive less eggs than control leaves from a plant that never had carried any Pieris eggs (Blaakmeeret al., 1994a).Thus the oviposition of Pieris brassicae induces both locally and systemicallya change in plant surface chemicals, which results in reduced acceptance of these plants for further oviposition. Judd and Borden (1992) have reported an example of plant surface chemical changes induced by oviposition leading to aggregations of egg depositions. Since this plant response does not reduce herbivore attack, we do not consider it to be a defensive reaction of the plant. Egg washes and eggs from the onion maggot fly Delia antiqua (Meigen) transferred to onion slices (Allium cepa) enhanced the oviposition rate of conspecific females. Neither eggs alone nor egg washes that were processed through a bacterial filter and added to the onion slices resulted in an enhanced ovipositionrate. But microorganisms associated with the egg surface infesting the host after oviposition were found to be responsible for producing metabolic by-products that attract females of D. antiqua and lead to aggregated oviposition. Further chemicals such as host-derived alkyl sulphides and a femalederived aggregation pheromone additionally mediate the host selection process in D. antiqua (Vernon et al., 1977; Judd and Borden, 1992). The herbivore’s benefits from aggregated oviposition on bacteria-infestedonions are not clear yet, but it is suggested that infested onions may facilitate larval attack or enhance the availability of certain nutrients to larvae (for a discussion see Judd and Borden, 1992).Several microbial species are known to enhance oviposition in D. antiqua when transferred to onion tissue (Hausmann and Miller, 1989; Hough et al., 1981),stressing the potential role of microorganisms for the chemical ecology of insect oviposition. The role of microorganisms for insect eggs and progeny is addressed in Chapter 6.

8.4

Plant Volatiles: Changes in Response towards Oviposition

Feeding activity of herbivores is well known to induce qualitativeand quantitative changes in the plant’s volatile emissions. These changes in volatiles have been shown to be specific for the plant and the herbivore species (e.g. De Moraes et al., 1998; Turlings et al., 1998a; but see Rose et al., 1998) and even for the feeding life stage of the herbivore (e.g. Takabayashi et al., 1995).Predators and parasitoids of the feeding stages of the herbivorous arthropod are known to be attracted by volatiles emitted from plants with feeding damage (e.g. Tumlinson et al., 1993; Dicke, 1994; Turlings et al., 1995). Reduction of the number of herbivores caused by the attracted predators and parasitoids may benefit the plant. The attraction of predators and parasitoids by feeding induced volatiles (= synomones) has been interpreted as a “cry for help” from the plant addressed to the third trophic level

221

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The Plant’s Responsetowards Insect Egg Deposition

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(Dicke, 1994). Feeding induced release of volatiles can reduce the number of herbivore eggs on the plant in two ways: (1)by reducing oviposition by gravid females on these plants, and (b)by attracting predators that kill the eggs (evidence is given by De Moraes et al., 2001, and Kessler and Baldwin, 2001). Not only may feeding by a herbivorous arthropod induce the release of plant volatiles; even oviposition of a herbivorous insect has been shown to induce the plant to emit volatiles that are attractive to an egg parasitoid. A comparison of induction by feeding damage and insect ovipositionis given by Hilker and Meiners (2002).Both an angiosperm tree, the field elm Ulrnus minor, and a gymnosperm tree, the pine Pinus sylvestris, have recently been shown to respond to oviposition of herbivorous insects by a change in volatiles that attract egg parasitoids specializing in these herbivores (Table 8-3). Oviposition of the elm leaf beetle Xunthogulerucu luteolu Muller (Coleoptera, Chrysomelidae) induces leaves of the field elm to emit volatiles that are attractive towards the egg parasitoid Oornyzus gullerucue (Hymenoptera, Eulophidae) (here referred to as the “elm system”). Artificial damage to elm leaves and feeding of the adult elm leaf beetles is not relevant for host searching of 0.gullerucue (Meinersand Hilker, 1997,2000).Odour from pine twigs of Pinus sylvestris that carry egg masses of Diprion pini (Hymenoptera, Diprionidae) attract the eulophid egg parasitoid Chrysonotornyiu ruforurn (here referred to as the “pine system”).Volatiles from pine twigs that have experienced mechanical damage are not attractive to C. ruforum (Hilker et al., 2002). In the elm system it has been shown that the elicitation process is highly specific (Table8-3). While eggs of the elm leaf beetle induce field elm leaves to emit volatiles attractive to egg parasitoids, eggs of Gulerucu tunuceti (closely related to X . luteola) have no such effect. Moreover, oviposition of the elm leaf beetle on twigs of the mountain elm (U.glubru) does not induce volatiles attractive to the egg parasitoids (Meiners et al., 2000). A plant that attracts egg parasitoids in response to egg deposition of herbivores starts its defence against the larvae prior to any feeding damage. While plants, which have attracted parasitoids in response to larval feeding activity will have to bear herbivory of parasitized larvae for some time, parasitized eggs will cause no future damage. Therefore, if plants are part of a trophic system where egg parasitoids of a severe herbivorous attacker can exhibit strong control of the herbivore, plants are expected to benefit from employing such an indirect defence mechanism of chemically “calling for help” towards egg parasitoids. Table 8-3 Overview of knowledge about mechanismsof indirect plant defence responses towards insect egg deposition

Mechanism

Elm

Pine System

Induction of synomones by oviposition Specificity: high Spatial scale: local and systemic induction Elicitor: oviduct secretion Wound signal: response inducible byjasmonic acid

+

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Plant Volatiles: Changes in Response towards Oviposition

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The plants that are induced by feeding to emit volatiles attractive towards antagonists of the feeding herbivores are mainly herbaceous plants. In contrast, up to now, the oviposition-inducedplants emitting synomones are trees. However, especially short-living, annual plants should benefit from release of ovipositioninduced synomones. Due to their short life time and much lower biomass, feeding damage in a herbaceous plant can be much more costly in terms of fitness than in a tree. Thus, we expect that insect egg deposition may also induce herbaceousplants to release volatiles attractive towards antagonists of eggs of the herbivores.

8.4.1

local and Systemic Plant Response towards Oviposition by Emission of Volatiles

Feeding of herbivores has been shown to induce the plant to change its volatile pattern not only locally at the site of feeding, but also in undamaged parts of the plant adjacent to the feeding damaged parts (e.g., Dicke, 1994; Rose et al., 1996; Du et al., 1998).Such a systemically induced response may have advantages over a localized response for very mobile predators or parasitoids (Zangerl, 1999).It might increase the amounts of volatiles released and thus enhance attraction. Also, oviposition of insects may locally and systemically induce the emission of volatiles that attract the egg parasitoids (Table 8-3). The elm U. minor has been shown to emit the volatiles attractive to the egg parasitoid 0.gallerucae both from leaves carryingeggs of the elm leaf beetle and from egg-freeleaves of a twig adjacent to the egg-laden twig (Meiners and Hilker, 2000). The pine P. sylvesfris is able to release volatiles that attract the egg parasitoid C. ruforum from an egg-free part of a pine twig adjacent to pine needles laden with eggs of the sawfly D. pini (Hilker et al., 2002). Egg parasitoids do not use oviposition induced plant volatiles only for host location; host kairomones may also be important (Vinson, 1988; Godfray, 1994).In the case of the egg parasitoid of the elm leaf beetle, faeces of the beetles have been shown to contain contact cues (as well as volatile kairomones) that increase the searching activity of the parasitoid (Meiners and Hilker, 1997). In the case of the egg parasitoid of the pine sawfly, sex pheromones of the sawfly are attractive towards the egg parasitoid (Hilker et al., 2000). However, up to now, we do not know whether or how these infochemicals interact.

8.4.2

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Eliciting Mechanisms of Induction of Plant Volatiles by Oviposition

Induction of plant volatiles by feeding insects are known to be elicited by orally released components of the feeding larvae (e.g. Mattiacci et al., 1994; Alborn et al., 1997; Felton and Eichenseer, 1999; Halitschke et al., 2001). Our knowledge about the elicitor of oviposition induced synomones in the “elm” and ”pine” system will be outlined in this section (Table 8-3). The gravid elm leaf beetle female X. lufeola removes plant tissue from the lower

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surface of a leaf by just scratching the surface with its mouthparts. Afterwards the female glues its eggs to the scratched surface with a secretion from the oviduct. This removal of plant tissue by the female prior to oviposition does not in itself induce the emission of synomones attractive to the egg parasitoids (Meiners and Hilker, 2000). When freshly laid eggs are removed from leaves and transferred into artificially scratched grooves on the lower surface of other previously egg-free elm leaves, these leaves with transferred eggs emit volatiles that attract the egg parasitoid. Thus, we studied whether the elicitor is located on or associated with the egg. Freshly laid elm leaf beetle eggs are always coated with oviduct secretion, which glues the eggs onto the leaf surface.Artificialscratchingof the elm leaf surface and subsequent applicationof the oviduct secretion (obtained from gravid females) leads to the induction of the synomones (Meiners and Hilker, 2000). Thus, the elicitor is located in the oviduct secretion coating the eggs. Although the epidermal damage done to the leaf surface by the female elm leaf beetle prior to oviposition cannot induce the emission of synomones, it is necessary for the induction. Application of oviduct secretion onto the undamaged leaf surface does not induce the release of the attractive volatiles (Meiners and Hilker, 2000). Probably the epidermal damage brings the elicitor from the oviduct secretion in contact with the damaged plant cells. It cannot be ruled out that the female applies an additional elicitor to the leaf through the mouthparts when scratching the grooves for egg deposition. But this elicitor would not be necessary for the synomone induction, since mimicking the wounding with a scalpel and adding oviduct secretion also induces the elm leaves to emit synomones (as described above). Furthermore, elm leaves damaged by feeding elm leaf beetles but carrying no eggs do not emit the volatiles that attract the egg parasitoid (Meinersand Hilker, 1997). The elicitor of induced synomones caused by oviposition of pine sawfly D. pini is also located in the oviduct secretion. Females of D. pini slit the pine needle with the ovipositor to lay their eggs in the wound, embedding them in oviduct secretion. This secretion, when transferred onto needles slit with a scalpel, has been shown to elicit the release of synomonesthat attract the egg parasitoid C. ruforum. Covering secretion that is applied to the eggs at the end of oviposition does not elicit attractive volatiles in pine needles (Hilkeret al., 2002). In contrast to the elm leaf beetle, females of D. pini do not feed upon the pine needles. Thus, an additional elicitor associated with female feeding can be ruled out in the pine system. The chemical nature of the elicitors in the oviduct secretion of X . luteolu and D. pini is unknown. In contrast, elicitors of plant synomones have been identified from regurgitate of feeding herbivores. In Pieris brussicue larvae, P-glucosidase is orally released and possibly hydrolyses stored glycosides in the plant cell, thus leading to the release of the volatile aglyca or to the release of aglyca that induce next steps of the signal transduction cascade (Mattiacciet al., 1995).Another elicitor has been identified in larvae of Spodopteru e x i p a as 17-hydroxylated linolenic acid conjugated with glutamine (Alborn et al., 1997, 2000; Turlings et al., 2000). This compound has been named volicitin and induces de nozm synthesis of volatile

Plant Volatiles: Changes in ResDonse towards OviDosition

compounds in corn and potato rather than causing their release from storage (Par6 and Tumlinson, 1997, 1998).Furthermore, the regurgitate of Munducu sextu also contains conjugates of fatty acids and amino acids that have been shown to elicit the release of volatiles emitted by feeding damaged tobacco plants (Baldwin et al., 2001; Halitschke et al., 2001).

8.4.3

Role of Jasmonic Acid in Induction of Plant Volatiles by Oviposition

Jasmonicacid and its precursors are known to act as mediators and wound signals in the feeding induced plant response (Koch et al., 1999; and references therein). Jasmonic acid is produced via the octadecanoid pathway from linolenic acid (Karban and Baldwin, 1997; and references therein). Application of jasmonic acid onto the plant was shown to mediate the de nova production and release of volatiles (Hopke et al., 1994; Boland et al., 1992,1995).When jasmonic acid was added to the water supplying elm twigs for a period of three days, the leaves released volatiles that attracted the egg parasitoid (Meiners and Hilker, 2000). The same result was obtained when pine twigs were supplied with jasmonic acid (Hilker et al., 2002) (Table 8-3). These results indicate that jasmonic acid is invoIved in mediating the oviposition-induced release of plant volatiles.

8.4.4

Chemistry of Released Plant Volatiles Induced by Oviposition

Plant odours released in response to herbivory exceed those of undamaged plants by far in number and amount of emitted substances (Dicke and Sabelis, 1988; Turlings et al., 1990; Hopke et al., 1994).Freshly damaged plants release a blend of "green leaf" volatiles. These six carbon alcohols, aldehydes, and acetates emanating from destroyed plant cells (Loughrin et al., 1994; Turlings et al., 1998b) are products of the lipoxygenase pathway, which involvesthe oxidationof linolenic acid. Instead of branching then to formation of jasmonic acid (see Section 8.4.3), this pathway leads to degradation of the oxidation product into a CI2-acidand (Z)3-hexenal. The latter component may be transformed into its esterified, reduced, or rearranged derivatives (Par6and Tumlinson, 1996,1997).Additionally, constitutively present defensive compounds ( e g , terpenoids in glands) can be released immediately with the green leaf volatiles (Loughrin et al., 1994). While their emission ceases not long after the feeding damage comes to an end, plants start the production and release of terpenoids. Two biosyntheticpathways for terpenoid production are known: the mevalonate-dependent pathway and the mevalonateindependent, so-called l-deoxy-D-xylulose-5-phosphat pathway (Lichtenthaler, 1998; Pie1 et al., 1998; Zeidler et al., 1998).Other induced compounds like methyl salicylate and indole emanate from the third known pathway involved in induction processes, the shikimic acidtryptophan pathway (Par6 and Tumlinson, 1997). Plant odours released in response to oviposition are currently being studied. First results are available on the elm system (Wegener et al., 2001). While intact

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

.

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

.

................................................................,,.,................ . ................... ......................................... . ................ ........................

.

elm leaves emit only small amounts of volatiles consisting of terpenes (monoterpenes, a homoterpene, and sesquiterpenes), herbivore treated elm leaves emit more than 40 compounds, most of them being terpenoids (with (€,€)-a-farnesene, P-caryophyllene and (€)-4,8-dimethyl-1,3,7-nonatriene as major compounds). Elm leaves adjacent to those carrying eggs emit fewer compounds than the locally oviposition induced leaves. Comparative headspace analyses of elm leaves with eggs and of feeding damaged leaves revealed similarities, but also differences in the blend of volatiles. When treated with jasmonic acid, elm leaves emit a blend which consists almost exclusively of terpenoids. A pentane fraction of jasmonic acid treated elm leaves containing terpenoid hydrocarbons such as (€)-P-ocimene,(E)-4,8-dimethyl-1,3,7nonatriene, P-caryophyllene, a-humulene, germacrene D, (Z,E) and (€,€)-alphafarnesene was attractive to the parasitoids. This indicates that jasmonic acid stimulates the production of terpenoid hydrocarbons, which might convey the information about egg deposition to the parasitoid. The volatile patterns of oviposition induced elm leaves and jasmonic acid treated ones differed (Wegener et al., 2001). This latter result is paralleled by results of comparative studies of the volatiles emitted by feeding induced and jasmonic acid treated plants. Gols et al. (1999) and Dicke et al. (1999) showed that the plant volatile pattern induced by application of jasmonic acid is similar to, but not the same as the one induced by feeding of the herbivore. However, the treatment of plants with jasmonic acid through the cut stem does not precisely mimic herbivore action. Therefore the actual role of jasmonic acid in inducing volatiles remains to be elucidated.

8.5

Concluding Remarks

The plant's defensive responses towards feeding activity of herbivores have been intensively studied for numerous years (Dicke and van Loon, 2000; Walling, 2000, and referencestherein; Baldwin et al., 2001).But for severalplants is has been shown that they do not need to wait until they are damaged by feeding: they respond already to the initial attack of a herbivore, towards the egg deposition. Plants may isolate and "try to get rid of" eggs of herbivores by hypersensitive response or growing of neoplasms. They may avoid further egg depositions by changing their surface chemistry after having received the first egg batch or they may even attract egg parasitoids or egg predators when they carry eggs. When we look at the few cases of ovipositioninduced defence known in plants, we see a high specificity of response. Not every species of a plant genus responds towards contact with insect eggs and even not every individual of one plant species responds to ovipositionwith the formation of neoplasms or hypersensitive response (see Section 8.2.1). There is also specificity on the side of the eliciting egg, showing specificity for the herbivore species (Meiners et al., 2000). Furthermore, a variance of the eliciting capacity of herbivore eggs within a species cannot be excluded. Females that succeed in laying eggs without eliciting direct or indirect plant defensive responses would benefit largely from circumventing the plant defence

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

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Acknowledgements

against their progeny. The specificity of the plants’ responses towards oviposition parallels the high specificity of known plant responses towards feeding (Dicke, 1999). Our knowledge about the chemicaland molecular processes induced in the plant by egg deposition is slight. Jasmonic acid, which is well known as a wound signal in feeding induced systems, has been shown to be also involved in induction of synomones by oviposition (see Section 8.4.3). However, since feeding damaged plants do not emit the volatiles that attract egg parasitoids, further or other signals must be necessary to trigger plant synomone induction by oviposition. Symbiotic microorganisms might play a crucial role in the production of the elicitor in the regurgitate of feeding herbivores (Spitelleret al., 2000). It will be a highly interesting task of future studies to answer the question whether microorganisms associated with the eggs are important for eliciting the induction of plant responses by oviposition (compare Chapter 6). Up to now, no studies have tried to analyse the costs and benefits of oviposition induced responses. The physiological costs of oviposition induced responses face the benefits of reduction of the number of viable eggs and thus, of the avoidance of future feeding damage. In addition to these physiological costs, possible counteractions of the herbivores need to be considered. While the gall insects succeed in using the plant response towards oviposition and feeding for their own purposes, hardly anything is known about counteractions of other herbivorous insects towards ovipositioninduced plant responses. For example, we do not know whether gravid insect females avoid plants that have shown hypersensitive response or growth of neoplasms. We have just started to analyse how the gravid females respond towards volatiles from oviposition induced plants (compare Section 8.4, response of gravid females to feeding induced volatiles). The volatiles released by oviposition induced plants might indicate to the herbivore female that her future progeny will suffer a higher risk of predation or competition at this oviposition site. Since so little is known about the herbivore’s counteractiontowards oviposition induced plant responses, the estimation of the benefit of the plant from these responses needs further studies. We have just realized that plants can respond to insect oviposition even by addressing the third trophic with oviposition induced volatiles. May the studies described in this chapter have enough “elicitingsubstance” to induce a cascade of physiological, chemical, molecular and evolutionary studies on the induction of plant responses towards insect oviposition from both the plant’s and the insect’s perspective.

8.6

227.

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Acknowledgements

Many thanks are due to Robert P. Doss and James E. Oliver for providing Figure 8-1, and to Nikolay F. Balbyshev and James H. Lorenzen for providing the photo to draw Figure 8-3. We are also very grateful to Anurag Agrawal for his critical comments on an earlier version of this manuscript.

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Shorthouse, JD, Rohfritsch, 0.1992. Biology of Insect lnduced Galls. Oxford University Press, Oxford. Silverman, W. 1959. The effect of variations in temperature on necrosis associated with infection type 2 uredia of the wheat stem fungus. Phytopathology 49: 827-830. Smith, EL. 1970. Biosystematics and morphology of Symhyta. 11. Biology of gall-making nematine sawflies in the California region. Ann. Entomol. SOC.h e r . 63: 36-51. Spaink, HP. 1996. Regulation of plant morphogenesis by lipo-chitin oligosaccharides. Crit. Rev. Plant Science 15: 559-582. Somssich, IE, Hahlbrock, K. 1998. Pathogen defense in plants - a paradigm of biological complexity. Trends in Plant Science 3: 8690. Spiteller, D, Dettner, K, Boland,W. 2000. Gut bacteria may be involved in interactions between plants, herbivores and their predators: microbial biosynthesis of N-acylglutamine surfactants as elicitors of plant volatiles. Biol. Chem. 381: 755-762. Sticher, L, Mauch-Mani, B, Metreaux, JP. 1997. Systemic aquired resistance. Ann. Rev. Phytopathol. 35: 235-270. Stakman, EC. 1915.Relation between Pucciniagraminis and plants highlyresistant to its attack. J. Agric. Res. 4: 193-299. Takabayashi, J, Takahashi, S, Dicke, M, Posthumus, M. 1995. Developmental stage of herbivore Pseudaletia separata affects production of herbivore-induced synomoneby corn plants. J. Chem. Ecol. 21: 273-287. Tumlinson, JH, Turlings, TCJ, Lewis, WJ. 1993. Semiochemicallymediated foraging behavior in beneficial parasitic insects. Arch. Insect Biochem. Phys. 22: 385-391. Turlings, TCJ, Alborn, HT, Loughrin, JH, Tumlinson, JH. 2000. Volicitin, an elicitor of maize volatiles in oral secretion of Spodoptera exigua: Isolation and bioactivity. J. Chem. Ecol. 26: 189-202. Turlings, TCJ, Bernasconi, M, Bertossa, R, Bigler, F, Caloz, G, Dorn, S. 1998a. The induction of volatile emissions in maize by three herbivore species with different feeding habits: possible consequences for their natural enemies. Biol. Control 11: 12Z129. Turlings, TCJ, Lengwiler, UB, Bernasconi, ML, Wechsler, D. 1998b. Timing of induced volatile emissions in maize seedlings. Planta 207: 146152 Turlings, TCJ, Loughrin, JH, McCall, PJ, Rose, USR, Lewis, WJ, Tumlinson, JH. 1995. How caterpillar-damaged plants protect themselves by attracting parasitic wasps. Proc. Natl. Acad. Sci. USA 92: 41694174. Turlings, TCJ,Tumlinson, JH, Lewis, WJ. 1990. Exploitationof herbivore-induced plant odors by host seeking parasitic wasps. Science 250: 1251-1253. Van Loon, LC, Bakker, PAHM, Pieterse, CMJ. 1998. Systemic resistance induced by rhizosphere bacteria. Ann. Rev. Phytopathol. 36: 453-483. Vernon, RS, Borden, JH, Pierce, HD, Jr, Oehlschlaeger, AC. 1977. Host selection by Hylemya antiqua: Laboratory bioassay and methods of obtaining host volatiles. J. Chem. Ecol. 3: 359-368. Vinson, SB. 1988. Comparison of host characteristics that elicit host recognition behaviour of parasitic Hymenoptera. Pp. 285-291. In: Gupta, VK (ed). Advances in Parasitic Hymenoptera Research. E.J. Brill, Leiden. Walling,LL. 2000. The myriad plant responses to herbivores.J. Plant Growth Regul. 19:195-216. Ward, HM. 1902. On the relations between host and parasite in bromes and their brown rust, Puccinia dispersa (Erikss.).Ann. Bot. 16: 233-315. Wegener, R, Schulz, S, Meiners, T, Hadwich, K, Hilker, M. 2001. Analyses of volatiles induced by oviposition of the elm leaf beetle Xanthogaleruca luteola on Ulmus minor. J. Chem. Ecol. 27: 499-515 West, A, Shorthouse JD. 1989.Initiation and development of the stem gall induced by Hemadas nubilipennis (Hymenoptera: Pteromalidae) on lowbush blueberry, Vacciniumangustifolium (Ericaceae).Can. J. Bot. 67: 2187-2198. Westphal, E, Dreger, F, Bronner, R. 1991. Induced resistance in Solanum dulcamara triggered by the gall mite Aceria cladophthirus (Acari: Eriophyoidea). Exp. Appl. Acar. 12: 111-118. Westphal, E, Perrot-Minnot, MJ, Kreiter, S, Gutierrez, J. 1992. Hypersensitive reaction of Solanum dulcamara to the gall mite Aceria cladophthirus causes an increased susceptibility to Tetranychus urticae. Exp. Appl. Acar. 15: 15-26.

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Chapter 9 Oviposition Pheromones in Herbivorous and Carnivorous Insects Peter Anderson

Table of Contents Introduction 9.1 9.2 Oviposition Deterring Pheromones (= ODPs) 9.2.1 ODPs Deposited by Females 9.2.1.1 Behaviour during Release of ODP 9.2.1.2 Factors Influencing the Activity of ODP 9.2.1.3 ODP Production Site in Females 9.2.1.4 Duration of ODP Activity after Release 9.2.1.5 Effects of Presence of ODP: Competitive Advantage of Larvae 9.2.2 ODPs Associated with Eggs 9.2.3 ODPs from Larvae 9.2.3.1 Origin: Exocrine Glands and Integument 9.2.3.2 Origin: Larval Faeces 9.2.3.3 Origin: Larval Regurgitate Oviposition Stimulating Pheromones 9.3 Chemical Structure of Oviposition Pheromones 9.4 Detection of Oviposition Pheromones 9.5 lntraspecific Effects of ODPs on Behaviour other than Oviposition 9.6 lnterspecific Effects 9.7 9.7.1 Response of Competitors towards ODPs 9.7.2 Response of Carnivores towards ODPs of their Prey Applications 9.8 Concluding Remarks 9.9 9.10 Acknowledgements References 9.11 Abstract In many insects the choice of oviposition site is affected by pheromones, which may function either as deterrents or as stimulants. Oviposition deterring pheromones have been found in about 100 insect species, while oviposition stimulating pheromones have been detected in only a few. The pheromones are produced by the ovipositing female or by conspecific larvae co-occurring with

236 Oviposition Pheromones in Herbivorous and Carnivorous Insects ................................. . . . . . ...................... . . .................. .. ............................................................ ..................................... .... ................. ..................

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

gravid females. Insect females release the glandular or faecal pheromone either directly with the egg or mark the oviposition substrate adjacent to the egg. Larvae emit pheromones affecting ovipositionof conspecificfemales from exocrine glands, faeces, or regurgitate. The chemical structure of oviposition pheromones is known only for a few species. There is a large variation in the compound structure and both volatile and non-volatile compounds occur. Adult females detect oviposition pheromones by odour receptors on the antennae, as well as by contact chemoreceptors on tarsi, mouthparts and antennae. These pheromones released by females or larvae may intraspecifically also influence behaviours other than oviposition.Competitorsand carnivoresare known to exploit ovipositiondeterring pheromones interspecifically. Successful field applications using oviposition deterring pheromones have been made in one system.

9.1

Introduction

Cues originating from conspecific insects can be important for females choosing a suitable oviposition site. Ovipositionstimulatingpheromones may indicate suitable oviposition sites to other females, whereas oviposition deterring pheromones may provide the information that the site is already occupied. Sites occupied by conspecificsmay become overcrowded if further progeny are deposited there. Thus, it is advantageous to avoid sites marked with such pheromones if better alternatives are available. Oviposition stimulating and deterring pheromones of herbivorous and most carnivorous insects are presented in this chapter, while oviposition pheromones of blood feeding insects are presented in Chapter 10. Reviews on pheromones released by herbivorous insects during ovipositionand resulting in deterrence of further oviposition at the pheromone-marked site have been written by Prokopy (1981a,b), Roitbergand Prokopy (1987),and Papaj (1994). The presence and role of oviposition pheromones have been included in reviews on parasitic wasp behaviour by van Lenteren (1981) and Kainoh (1999). Recently, Nufio and Papaj (2001) reviewed host marking behaviour in herbivorous insects and parasitoids. This review makes an overview of oviposition pheromones in herbivorous and carnivorous insects, including predatory insects. I will address the behaviour, detection, production sites and chemical structure of the oviposition pheromones emitted by females and larvae as well as factors that affect the activity of the pheromones. Additionally,other intra- and interspecificfunctionsof the oviposition pheromones will be considered. The evolutionary ecology of the oviposition marking pheromones is presented in Chapter 12.

9.2

Oviposition Deterring Pheromones (= ODPs)

Females and larvae of many species (Tables 9-1 and 9-2) are known to release components that deter oviposition of conspecifics. Thus, these components have

Oviposition Deterring Pheromones (= ODPs)

Table 9-1 Herbivorous insects where oviposition deterring pheromones have been reported and the developmental stage that produces the pheromone

OlEDER

Pamity specl= COLEOPTERA Anobiidae Lasioderma serricorne Bruchidae Acanthoscelides obtectus Callosobruchus chinensis C. maculatus C. rhodesianus C. subinnotatus Zabrotes subfashiatus Cerambycidae Monochamus alternatus Chrysomelidae Castrophysa viridula Phaedon cochleariae Phratora vitellinae Phyllodecta vulgatissima Curculionidae Anthonomus grandis Ceutorhynchus assimilis C.floralis DIPTERA Agromyzidae Agromyza frontella Anthomyiidae Hylemya spp. Strobilomyia neanthracina Muscidae Antherigona soccata Tephritidae Anastrepha fraterculus A. ludens A. suspensa Bactrocera (Dacus)jarvisi B. oleae B. tryoni Ceratitis capitata Chaerorellia australis Orellia rufcauda Paraceratitella eurycephala Rhagoletis alternata R. basiola R. berberis R. cerasi R. cingulata R. completa R. conversa R. cornivora R. fausta

adult

Kohno e t al.. 1986

adult female female? female female female

Szentesi, 1981 Oshima et al., 1973 Giga and Smith, 1985 Giga and Smith, 1985 Mbata, 1992 Umeya, 1966 (in Prokopy, 1981a)

female

Ambutsu and Togashi, 2000

larvae larvae larvae larvae

Hilker, 1989 Hilker, 1989 Hilker, 1989 Hilker and Weitzel, 1991

larvae female female

Stansly and Cate, 1984 Kozlowski et al., 1983. Kozlowski, 1989

female

McNeil and Quiring, 1983

eggs female

Zimmerman, 1979 Quiring et al., 1998

eggs

Raina, 1981

female female female larvae larvae larvae female/larvae female female female female female female female female female female female female

Prokopy et al., 1982a Papaj and Aluja, 1993 Prokopy et al., 1977 Fitt, 1984 Girolami et al., 1981 Fitt, 1984 Prokopy et al., 1978 Pittara and Katsoyannos, 1990 Lalonde and Roitberg, 1992 Fitt, 1981 Bauer, 1986 Averill and Prokopy, 1981 Mayes and Roitberg, 1986 Katsoyannos, 1975 Prokopy et al., 1976 Cirio et al., 1972 (in Prokopy, 198lb) Frias et al., 1984 Prokopy et al., 1976 Prokopy, 1975

237

238

....

Oviposition Pheromones in Herbivorous and Carnivorous Insects

Table 9-1Continues

R. indifferens R. juglandis R. mendax R. pomonella R. tabellaria R. zephyria Thephritis bardanae

HY ME NOPTE RA Diprionidae Diprion pini Neodiprionfulviceps

female female female female female female female

Prokopy et at., 1976 Nufio and Papaj, 2001 Prokopy et al., 1976 Prokopy, 1972 Prokopy et al., 1976 Averill and Prokopy, 1982 Straw,1989

larvae eggs/female

Hilker and Weitzel, 1991 Tisdale and Wagner, 1991

female

Kouloussis and Katsoyannos, 1991

eggs

Rothschild and Schoonhoven, 1977

fema le/eggs larvae larvae

Brantjes,1976 Hilker, 1985 Renwick and Radke, 1980

eggs eggs eggs

Dempster,1992 Rothschild and Schoonhoven, 1977 Rothschild and Schoonhoven, 1977

female

Huth and Pellmyr, 1999

larvae larvae larvae larvae eggs larvae

Mudd and Corbet, 1973 Mudd and Corbet, 1973 Corbet, 1973 Dittrick et al., 1983 Thiery and Le Quere, 1991 Mudd and Corbet, 1973

eggs

Gabel and Thiery, 1992

Eurytomidae Eurytoma amygdali

LE PI DOPTE RA

Danaidae Danaus plexippus

Noctuidae Hadena bicruris Spodoptera littoralis Trichoplusia ni

Pieridae Anthocharis cardamines Pieris brassicae P. rapae

Prodoxidae Tegeticulayuccasella

Pyralidae Ephestia cautella E. elutella E. kuehniella Ostrinia nubilalis Plodia interpundella

Tort ricidae Lobesia botrana

been named oviposition deterring pheromones (Prokopy, 1972).However, when oviposition sites without presence of such pheromones are limited, females have been found to lay eggs even in the presence of these pheromones (e.g. Roitberg and Prokopy, 1983;van Alphen and Visser, 1990).Furthermore, these pheromones have been found not only to deter oviposition, but also to affect intraspecifically behaviours other than oviposition (see Section 9.6).For these reasons, some authors have suggested using the name host marking pheromones instead of oviposition deterring pheromone (Averill and Prokopy, 1988; Roitberg and Mangel, 1988; Aluja and Boller, 1992). When analysing the selective factors that might have favoured the evolution of such pheromones released at oviposition, not only the

Oviposition Deterring Pheromones (= ODPs)

Table 9-2 Carnivorous insects where oviposition deterring pheromones have been reported and the developmental stage that produces the pheromone

COLEOPTERA Coccinellidae Adalia bipunctata Coccinella septempunctata Cryptolaemus montrouzieri

larvae larvae larvae

Doumbia e t al., 1998 Doumbia e t al., 1998 Merlin et al., 1996

DIPTERA Cecidomyiidae Aphidoletes aphidimyza

larvae

Ruzicka and Havelka, 1998

female female

Chow and Mackauer, 1986 Hofsvang, 1988

female

Kainoh, 1999

female female female female female female

Kainoh, 1999 Vet e t al., 1984 Vet e t al., 1984 Kainoh, 1999 Kainoh, 1999 Kainoh, 1999

female

Kainoh, 1999

female

van Dijken et al., 1992

female female

Cardenghi et al., 1994 van Alphen, 1980

female female female female female female female

Kainoh, 1999 Price, 1970 Price, 1970 Kainoh, 1999 Kainoh, 1999 Kainoh, 1999 Price, 1970

female

Kainoh, 1999

female female female female

Conti et al., 1997 van Baaren et al., 1994 van Baaren and Boivin, 1998 Kainoh, 1999

female female female

Hoffmeister and Roitberg, 1997 Kainoh, 1999 Kainoh, 1999

HYMENOPTERA Aphidiidae Ephedrus californicus E. cerasicola Bethylidae Cephalonomia stephanoides Braconidae Ascogaster reticulatus Asobara rufescens A. tabida Cardiochiles nigriceps Dapsilarthra rujiventris Orgilus lepidus Cynipidae Pseudeucoila bochei Encyrtidae Epidinocarsis lopezi Eulophidae Edovum puttleri Tetrastichus asparagi lchneumonidae Campoletis perdistinctus Endasys subclavatus Mastrus aciculatus Nemeritis canescens Phaeogenes cynarae Pleolophus basizonus P. indistictus Megaspilidae Dendrocerus carpenteri Myrmaridae Anaphes iole A. sordidatus A. victus Caraphractus cinctus Pteromalidae Halticoptera laevigata Muscidifurax zaraptor Nasonia vitripennis

239

240 Oviposition Pheromones in Herbivorous and Carnivorous Insects . . . . .. ......................... ..................... ......................................... .. ............................ .................. . ................... ....................

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

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

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

Table 9-2 (Continued)

ORDER

Scelionidae Eumicrosoma blissae Telenomusfariai T. heliothidis T. sphingis T. triptus Trichograrnrnatidae Trichogramma evanescens NEUROPTERA Chrysopidae Chrysopa carnea C. commata C. oculata C. perla

female female female female female

Sadoyama, 1998 Kainoh, 1999 Strand and Vinson, 1983 Rabb and Bradley, 1970 Higushi and Suzuki, 1996

female

Kainoh, 1999

larvae larvae larvae larvae

Ruzicka, 1998 Ruzicka, 1998 Ruzicka, 1994 Ruzicka, 1996

deterrent effects needs consideration, but also other functions of the ODPs (see Chapter 12).However, since I will focus on the ovipositiondeterrent effect of these compounds, I will use the term ODP throughout this chapter. The ODP can be produced by the adult female or by the larvae. The female produced pheromone may be associated directly with the eggs or be deposited by the female near the oviposition site in connection with oviposition. ODPs have been found in about 100 species from five insect orders (Tables 9-1 and 9-2).

9.2.1 ODPs Deposited by Females 9.2.1.1 Behaviour during Release of ODP Behaviour of females during release of ODPs has been studied in detail in some dipteran species of the family Tephritidae (fruit flies). Many species of this taxon oviposit in fruits, in which the larvae feed and develop until maturity (Prokopy, 1981a).Many fruits offer limited resources and can only support a limited number of larvae. Ovipositing females in several species of tephritid flies respond to a female-produced ODP by avoiding already infested fruits (Table 9-1). The oviposition behaviour of the European cherry fruit fly, Rhugoletis cerusi, is typical for fruit flies. Katsoyannos (1975) divides the oviposition behaviour of R. cerusi on cherry fruits into three phases: Preoviposition (or exploitatory)phase. The period from the arrival of the female on the cherry to the first boring with the ovipositor.After arrival the female exhibits zigzag movements over the surface searching for a suitable oviposition site. Oviposition phase. Includes all oviposition attempts and true ovipositions. When a suitable oviposition site is found, the female penetrates the fruit slun with the ovipositor and deposits an egg under the fruit skin.

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Oviposition Deterring Pheromones (= ODPs)

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. .

241.

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Figure 9-1Afemale Rhagoletis fly marking a fruit with ODP after oviposition.The wavy line shows the ODP trail on the fruit.

Postoviposition phase. Immediately after oviposition the female drags her extended ovipositor over the surface of the cherry and deposits an ODP. Several full circles around the cherry fruit are made (Figure 9-1). A similar behaviour has been observed in R. pornonella and several other fruit fly species (Prokopy,1972; Prokopy, 1981b).A female arrivingat a fruit starts the normal searching behaviour of the preoviposition phase. If she encounters an ODP trail she interrupts the searching and leaves the fruit. The ODP greatly reduces the probability that a female will oviposit a second time in a fruit that she has already visited, as well as deters other conspecific females from ovipositingin fruit already infested by other females (Roitberg and Mangel, 1988). The marking behaviour of tephritid flies can be affected by repeated contact with ODP. In Anastreph ludens the presence of earlier markings on a fruit stimulated subsequent females to deposit longer trails of ODP after oviposition (Papaj and Aluja, 1993),while females of R. pornonella deposited the same amount of pheromone on already marked fruit as on unmarked (Averilland Prokopy, 1988).Roitberg and Prokopy (1981) reported that tephritid females needed previous experience of pheromone marking to be able to discriminate between ODP-marked and unmarked host fruits. One day of experience of the ODP was enough to elicit avoidance of a marked fruit. Experienced flies retained their ability to recognize the ODP up to at least four days of deprivation of pheromone experience (Roitberg and Prokopy, 1981). Behaviour of release of ODPs in beetles has especiallybeen examined in weevils that use seeds and buds as oviposition sites. In the boll weevil, Anthonornus grandis, the female plugs the oviposition punctures with frass that deters other females from oviposition at that site (Stansly and Cate, 1984). In Callosobruchus species, studies on the origin of the oviposition deterrent component are somewhat

242

Oviposition Pheromones in Herbivorous and Carnivorous Insects . ............................ ......................... . .................... .................,.. ............................................................

....................................................,.........................

,

,

,

,

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contradictory (see Ignacimuthu et al., 2000, for detailed references). For example, Messina and Renwick (1985)provide evidence that C. rnaculatusfemales mark seeds with a pheromone after oviposition, while Credland and Wright (1990) showed deterrent effects by undamaged seeds treated with extracts from glass beads exposed to either female or male C. rnaculatus. Females of the cerambycid beetle Monochamus alternutus gnaw a hole into the bark, insert the ovipositor into the hole and lay an egg inside. The hole is closed by deposition of a jelly, and after that the female brushes the entrance of the hole with the abdominal tip. An ODP is deposited with either the jelly or the abdominal rubbing at the oviposition site (Anbutsu and Togashi, 2000). Female parasitic wasps mark hosts in several ways (van Lenteren, 1981). The marking pheromone may be deposited inside the host (internal marker) or on the surface of the host (external marker). As found in several herbivorous insects, females of some parasitic wasps may also mark an area around the oviposition site with a trail of pheromone (van Lenteren, 1981).The different types of markers may be used in combination. Females of the aphid parasitoid Ephedrus cerasicola use both an external and an internal marker (Hofsvang, 1988).The external marker, detected by the antennae, had a short lifetime and was active only a few hours. Later a short probing with the ovipositor, to get in contact with the internal marker, was needed for the female to discriminate between parasitized and unparasitized hosts (Hofsvang, 1988). As for herbivorous insects, parasitoid females spend less time in areas marked with a trail pheromone than in unvisited areas (e.g.van Dijken et al., 1992; Bernstein and Driessen, 1996). Another way to more efficiently discriminate between parasitized and unparasitized hosts has been found in Anaphes victus (van Baaren and Boivin, 1998). The females learn to associate an external pheromone with an internal pheromone. After encountering a few parasitized hosts and having probed them with the ovipositor, subsequent parasitized hosts encountered are rejected after only antenna1 drumming (van Baaren and Boivin, 1998). 9.2.1.2 Factors Influencing the Activity of ODP

Numerous biotic and abiotic factors may affect female behaviour when releasing and responding to ODPs. Tanaka (1991,2000) found that realized heritability for the responsiveness of females of C. rnaculatus towards ODP is one order of magnitude smaller than the phenotypic repeatabilityof responsiveness.According to Tanaka (ZOOO), individual-specificnon-genetic effects rather than genetic effects explain a major part of the individual variation. A large individual quantitative variation of ODP deposited was found in females of R. pomonella. The amount of pheromone deposited was mainly dependent on the time spent dragging the ovipositor after oviposition. Females laid longer pheromone trails on large fruits than on small ones. On large fruits more pheromone is necessary for oviposition deterrence than on small ones (Averill and Prokopy, 1987; 1988). On the other hand, females of Anastrepha fvaterculus showed higher motivation to drag the ovipositor over a small fruit than over a large fruit. Each

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

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

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Oviposition Deterring Pheromones (= ODPs)

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

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.

.. .

dragging period was also longer on small fruits (Prokopy et al., 1982a).The risk for competition can be higher in a small fruit than in a large one and may explain the increased marking activity on small fruits. In addition to pheromone quantity, several other biological and physical factors have been examined for their effects on the activity of the ODP in fruit flies, such as fly size and age, host size, temperature and humidity (Prokopy et al., 1982a; Averill and Prokopy, 1988; Papaj and Aluja, 1993; Mbata and Ramaswamy, 1995). Female egg load may also affect the response to ODP. Females with low egg load of the parasitic wasp, Dendrocerus curpenferi, avoided patches that had been previously marked by themselves, while females with high egg load avoided patches visited by conspecificfemales, but not by themselves (Holler and Hormann, 1993). 9.2.1.3 ODP Production Site in Females

In most species where ODPs have been demonstrated, the production site of the pheromone is not known. In most of the investigated species the ODP released by females is produced in the abdomen and is deposited with release of faeces during ovipositor dragging or emitted from glandular tissue associated with the ovipositor. An exception from this pattern is found in the spruce cone fly, Strobilornyiu neunthrucinu, where the ODP is produced in the head or in the thorax of female flies and is deposited by the mouthparts (Quiring et al., 1998). The ODP of R. pornonella is produced by tissue located in the midgut and is secreted into the gut content of the flies (Prokopy et al., 1982b).It is released through the anus together with the faeces during ovipositor-dragging.After ovipositionfemales of the cabbage seed weevil, Ceutorhynchus ussirnilis, brush pods of oilseed rape with their abdomen and deposit an ODP (Kozlowski et al., 1983). The pheromone is produced by glandular tissue on the 7th urotergite of the abdomen and the "brush that applies the ODP on the pod is situated on the 8th urotergite. During the pod marking behaviour the "brush comes in contact with the glandular tissue during extension and retraction of the 8th tergite and collects the ODP that is transferred to the pod (Ferguson et al., 1999a). 9.2.1.4 Duration of ODP Activity after Release

The time of persistence of ODPs released by females at the oviposition sites varies greatlybetween species.For example, the lifetime of the ODP deposited by females of C. ussirnilisis only 1-2 hours (Ferguson and Williams, 1991).Such a short lifetime is mainly used by the female to avoid oviposition in the same pod that she has already visited, while it gives only a limited protection against oviposition of other conspecific females (Ferguson, pers. comm.). In contrast, the activity of the ODP of R. cerusi lasts for at least 12 days (Katsoyannos, 1975). 9.2.1.5 Effects of Presence of ODP: Competitive Advantage of larvae

Several studies considered the competitiveadvantage of larvae hatching from eggs that had been deposited at sites marked with pheromone. For example, when

243.

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244

Oviposition Pheromones in Herbivorous and Carnivorous Insects

b 1000

1

n

750 U

E

.f 500 -3 2 250 a Q

A sing

1st 2nd

sing

1st 2nd

Figure 9-2 (a) Per cent larval mortality of Agromyzafrontella and (b) pupal weight of a larva developing singly (sing)in a leaflet or in competition with two different-aged (24 hrs) larvae (1st and 2nd larvae in a leaflet).Data from Quiring and McNeil, 1984a.

deposition of ODP by R. pornonella after egglaying on a fruit deters subsequent oviposition at this site for 3 4 days, this time is sufficient to give the first deposited egg and its hatching larva a large competitive advantage over subsequently deposited eggs (Prokopy, 1981b). The effect of competition on the first hatching and subsequent larvae has also been studied in detail in the alfalfa blotch leafminer, Agrornyza frontella. After oviposition a female of A.fvontella drags her ovipositor on the underside of an alfalfa leaflet and deposits a water soluble ODP (McNeil and Quiring, 1983). The pheromone is active for at least 24 hours after deposition under field conditions (Quiring and McNeil, 1984b).Larvae of the leafminer develop in leaflets of alfalfa and are confined to the leaflet where they hatch. Quiring and McNeil(1984b)tested if 24 hours would be sufficient for the first larva to gain a competitive advantage over later merginglarvae. The experimentsshowed that larvae from later deposited eggs suffered much higher mortality than the first larva in a leaflet (Figure 9-2). Additionally, for the surviving larvae the mean pupal weight was much higher for the first larva than for later ones (Quiring and McNeil, 1984b).Females with lower pupal weight produce a less effective ODP as adults, probably due to a lower quantity of the pheromone (Quiring and McNeil, 1984~). The authors suggested that the females obtained the precursors of the ODP from alfalfa and that a lower intake of food by smaller females reduced their ability to produce pheromone. Alfalfa blotch leaf miner larvae in the same leaflet engaged in both interference and exploitation competition (Quiring and McNeil, 1984a).First and second instar larvae are cannibalistic and smaller larvae are often eaten by larger conspecifics.

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Oviposition Deterring Pheromones (= ODPs)

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Third instar larvae are not cannibalistic, and avoid aggressive encounters. Sixtyfive percent of the total consumption occurs during the third instar and the first larva reaching this stage in a leaflet has a great advantage in the scramble for resources (Quiring and McNeil, 1984a).These results show that 24 h active life of the ODP gives the first larvae of A. frontella in a leaflet a great advantage in the competition for resources.

9.2.2

245

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ODPs Associated with Eggs

This category differs from the previous in that the ODP is associated directly with the eggs. The pheromone may be present on the surface of the eggs or in the “glue” that attaches the eggs to the oviposition substrate. The “egg glue” may be produced in glandular tissue of the oviduct or in special glands associated with the female reproductive organs (compare Chapter 2). Females of the dipteran genus Hylemyu lay their eggs in flowers of several plants. The deposition of an ODP after oviposition varies between different plant species. On the perennial herb Polemoniumfoliosissimun, females attach an egg to the inside of a sepal in a flower bud and at the same time deposit an ODP associated with eggs (Zimmerman, 1979). Later arriving females attempting to oviposit in the flower bud detect the ODP and leave without ovipositing. However, on a second host plant, lpomopsis aggregutu, no evidence for deposition of ODP was recorded. Experiments showed that whether a female chooses to deposit an ODP or not depended on the host plant. Thus, the difference in behaviour was not due to two different types of females in the population preferring different host plants (Zimmerman, 1982).Also interactions between the pheromone and the host plant could be ruled out as an explanation for the variable oviposition behaviour. Egg mortality is much higher on 1. uggregutu than on P. foliosissirnun. Normally, only one larva will survive and develop in each flower even if several eggs are deposited. Thus, the risk for competitionis low in 1.uggregutu flowers and the effect of a marking pheromone is limited.An intriguing question though is why the flies use 1.aggregutu at all when mortality is so high on this host plant (Zimmerman, 1980). Females of both the European grape wine moth, Lobesiu botrunu, and of the European corn borer, Ostriniu nubilalis, avoid ovipositing on substrates treated with an extract of eggs washed in methanol (Thi6ry and Le Qubr6,1991; Gabel and Thi6ry, 1992).Increasing the dose of the extract also increased the deterrent effect. A reduction in the number of eggs a female lays has also been recorded in L. botrunu when egg wash was present at oviposition sites (Gabel and Thibry, 1996). In two species of cabbage butterflies, Pieris brussicue and P. rupue, the presence of egg batches on a cabbage leaf deters females from oviposition on a leaf (Rothschild and Schoonhoven, 1977).The oviposition deterring effect of deposited eggs on cabbage leaves persists for at least 14 days (Schoonhoven et al., 1981).Active nonvolatile compounds on the eggs were extracted with water or methanol. When sprayed on cabbage leaves without eggs, the extract deterred oviposition (Behan and Schoonhoven,1978;Klijnstra, 1986;Blaakmeer et al., 1994a).Cabbagebutterflies

246 .

Oviposition Pheromones in Herbivorous and Carnivorous Insects

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lay their eggs on the underside of cabbage leaves. Prior to oviposition a female examines only the upper surface of the leaf. She deposits her eggs on the lower surface of a leaf, by curving her abdomen over the leaf edge so that it comes in contact with the underside of the leaf. However, subsequent females attempting to oviposit on a leaf with eggs abandoned the leaf after examining only the upper side of the leaf and without coming in contact with the egg batch. No presence of the deterrent can be traced on the upper side of the leaves and it was concluded that the deterring compounds were not produced by the female, but by the plant in a response to deposition of eggs (Blaakmeer et al., 1994b) (see Chapter 8). The solitary wasp Epidinocarsis lopezi spent as much time searching a suitable patch with hosts if it had been visited earlier by the wasp itself or by conspecifics (van Dijken et al., 1992).However, a female superparasitized fewer hosts previously parasitized by herself than hosts that had been already parasitized by conspecifics. This indicates that there is an individual-specificmarker in or on the host associated with the egg.

9.2.3 ODPs from Larvae Larvae co-occurring with conspecific gravid females are also known to produce ODPs. The presence of developing larvae indicates to the female that the risk of competition for food is high at that site. There is also a risk for cannibalism, where larger larvae feed on eggs or small larvae. Thus, it is advantageous for females to avoid patches where larvae already are present when choosing an oviposition site. 9.2.3.1 Origin: Exocrine Glands and Integument

When larvae of the Mediterranean meal moth, Ephestia kuehniella, meet head to head, small droplets of secretion are emitted from their mandibular glands and deposited on the substrate (Corbet, 1971).Medium to high concentrations of the secretion are deterrent to ovipositingfemales (Corbet, 1973; Anderson and Lofqvist, 1996).At high larval densities very few eggs are deposited even if females have no other alternative (Corbet, 1973).The mandibular glands of the related species, E. cautella, E. elutella and Plodia interpunctella contain similarcompounds to those found in E. kuehniella and Mudd and Corbet (1973) suggested that they elicit similar behaviours in these species. However, oviposition experimentswith P. interpunctella did not show any deterrent effect of the larval secretion on conspecific females (Anderson and Lofqvist, 1996). Several chrysomelid larvae release exocrine glandular secretion that not only deters generalist predators such as ants, but also conspecificadults. Egg deposition by Gastrophysa viridula, Phaedon cochleariae,and Phratora vitellinae were deterred by the secretion of conspecific larvae (Hilker, 1989).Also several carnivorous larvae have been shown to release ODPs. In the ladybird beetle Cryptolaemus montrouzieri the ovipositiondeterrent was associated with the abundant wax filamentscovering the larval body. Larvae may lose the wax filaments and thus release tracks of oviposition deterrents on the substrate. Also larvae of the ladybird species Adulia

Oviposition Deterring Pheromones (= ODPs)

bipunctata and Coccinella septempunctata are known to release tracks that deter oviposition by conspecifics (Doumbia et al., 1998).Furthermore, larvae of several Chrysopa species (Ruzicka, 1994, 1996, 1998) and larvae of the cecidomyiid fly Aphidoletes aphidimyza (Ruzicka and Havelka, 1998) release components on the oviposition substrate that deter oviposition by conspecific females. The determination of the precise site of production of the pheromones in these latter mentioned carnivorous species needs further study. 9.2.3.2 Origin: larval Faeces Larval faeces have been shown to deter oviposition in several insect species (Renwick and Radke, 1980; Dittrick et al., 1983; Hilker, 1985).In most species, the chemicals with oviposition deterrent activity have not been isolated and identified from the larval faeces. Thus, in these cases it remains unknown whether the oviposition deterrent compounds in the faeces are indeed a pheromone (i.e.larval produced components) or whether plant components highly concentrated in the faeces or microbial products elicit the oviposition deterrent effect (see e.g. Thibout, et al., 1995).In the noctuid moth Trichoplusia ni, for example, both larval faeces and suspensions of macerated host plant leaf tissue have been shown to deter oviposition (Renwick and Radke, 1980,1981). In contrast, in the Egyptian cotton leafworm, Spodoptera littoralis, larval frass was shown to deter oviposition,whereas a suspension of macerated cotton plant leaves did not affect egglaying behaviour (Hilker, 1985).Chemical analyses of larval frass of S. littoralis revealed that a mixture of six compounds has oviposition deterring activity (Klein et al., 1990; Anderson et al., 1993).

9.2.3.3 Origin: larval Regurgitate Larvae of the sawfly Diprion pini release a droplet of foregut content when disturbed. This regurgitate is well known in closely related diprionids to repel generalist predators (Eisner et al., 1974). However, larvae of D. pini also release regurgitate when disturbed by co-occurringconspecific females. The regurgitate repels females and deters them from oviposition (Hilker and Weitzel, 1991).In contrast, laboratory experiments revealed that regurgitate released by larvae of Neodiprion sertifer does not deter conspecifics from oviposition (Bliimke and Anderbrant, 1997). This difference may be explained by the different life cycles of D. pini and N.sertifer. While D. pini females may search for oviposition sites when conspecificlarvae are still present, larvae and gravid females of N. sertifer do not co-occur in nature. The volatile components of the larval regurgitate of larvae of D. pini represent typical terpenoids of the host plant, Pinus spp. This is not a surprising result since larvae store resin components of the needles in their foregut pouches. However, the quantitative composition of monoterpenes in the regurgitate of D. pini larvae differ from the quantitative pattern of monoterpenes of the needles upon which larvae had fed (Weitzel, 1991).The quantitative pattern of monoterpenes seems to be important for the oviposition deterring activity, since regurgitate significantly deters oviposition, whereas needle resin does not (Weitzel and Hilker, 1993).

247

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Oviposition Pheromones in Herbivorous and Carnivorous Insects

9.3

Oviposition Stimulating Pheromones

Gregarious phase desert locusts, Schis tocercu greguriu, are attracted to and deposit eggs at sites where other females have oviposited even though other suitable areas are available. After oviposition the female covers the hole where she has deposited her eggs with a plug of foam (froth) that attracts other ovipositing females of S. greguviu (Saini et al., 1995).Volatiles emitted from the froth were shown to attract females about to oviposit. In addition, females touched the sand around an oviposition site with their antennae and palpi, indicating that non-volatile compounds from the froth also may be important during oviposition. Extracts using solvents of different polaritiessuggested that both polar and non-polar compounds are active in attracting the ovipositing female (Saini et al., 1995). Another example of oviposition stimulating pheromones was found in the two moths E. kuehniellu and P. interpunctellu, in which secretion from the mandibular glands of conspecific larvae stimulated oviposition (Corbet, 1973; Anderson and Lofqvist, 1996).Low densities of larvae in the substrate stimulated oviposition in E. kuehniellu, while a wide range of larval densities stimulated oviposition in P. interpunctellu (Anderson and Lofqvist, 1996). Aggregation pheromones are known for numerous herbivorousand carnivorous insects (e.g.Tillmann et al., 1999; Wertheim, 2001; and references therein). A major benefit of using aggregation pheromones may be aggregated oviposition when eggs and the progeny take advantage of development in aggregations (Wertheim, 2001; Chapter 13).Numerous non-social insects show aggregation behaviour and aggregated egg depositions. Prokopy and Roitberg (2001)review the diversity of types of costs and benefits of individuals who decide to aggregate and join conspecifics. Aggregation pheromones of bloodsucking Hemiptera and Diptera and their importance for oviposition of these insects are addressed in detail in Chapter 10.

9.4

Chemical Structure of Oviposition Pheromones

In most species the oviposition pheromones studied show very low volatility and long persistence. Surprisingly many of the ODPs are water soluble, making them vulnerable to unfavourable weather conditions. In a few species, volatile compounds acting as oviposition pheromones have been found (Hilker,1989;Anderson et al., 1993; Rai et al., 1997).In many cases the structure of the active compounds is unknown. The oviposition pheromones identified so far show a high diversity in chemical structure (Figure 9-3). The ODP of R. cerusi was the first one to be identified. Hurter et al. (1987)isolated the pheromone from the female’s faeces and identified it as N[15(P-glucopyranosyl)-oxy-8-hydroxypalmitoyl]-taurine (Figure 9-3a). The molecule has four different stereoisomers, as it has two chiral centres at the C-8 and C-15 positions (Hurter et al., 1987).After the four different stereoisomerswere synthesized (Ernst

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

.............................,,,,, ,, ,,...............

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

Chemical Structure of Oviposition Pheromones

................... . ,,,.,,,..........

H

a

OH

H

I

0

0

\d’OH II 0

HO

d

0

II HjC - 0 - C

H0’ -0

i

249.

....,,,,...............,,,,..,........... . ...................................................................................

a

O

C OCH3 H 3

Figure 9-3 Examples of chemical structures of oviposition pheromones. The insect species producing the pheromone is indicated in brackets after the structure name. (a) 15R(Pgl ucopyranosyl)-oxy-8R-hydroxypalmitoyll-ta urine (Rhagoletis cerasi),(b) and (c)a-serricorone ([2S,3R, 1‘S]-2,3-dihydro-3,5-dimethyl-2-ethyl-6-[l’-methyl-2’-oxobutyl]-4~-pyran-4-one) and p-serricorone(Lasioderma serricorne), (d) methyl stearate (Ostrinia nubilalis),(e) 2-(lo’€)10’-hexadecenoylcyclohexane-1,3-dione (Ephestia kuehniella), (f) nerolidol (Spodoptera littoralis), (g) thymol (5. littoralis), (h) acetophenone (Schistocerca gregaria), (i) veratrole (S. gregaria).

250

Oviposition Pheromones in Herbivorous and Carnivorous Insects

and Wagner, 1989),it could be shown that a racemic mixture of two isomers (8R, 15s and 8S, 15R isomers) deterred oviposition (Boller and Aluja, 1992). Females of the cigarette beetle, Lasioderma serricorne, are deterred by a female produced ODP (Kohno et al., 1986). From adult female body extract, the two '-methylepimers a-serricorone([2S,3R,1 'S]-2,3-dihydro-3,5-dimethyl-2-ethyl-6-[1 2 '-oxobutyl]-4H-pyran-4-one)and p-serricorone (corresponding 1-R '-epimer) (Figure 9-3b and 9-3c), were identified (Imai et al., 1990). The two compounds epimerize easily, but through careful treatment during the isolation procedure it was shown that both epimers are present in the body of the beetle. Both epimers also showed similar oviposition deterring activity. An ODP associated with the eggs has been extracted with methylene chloride in L. botrana (Gabel and ThiQy, 1996). Two groups of compounds affected oviposition behaviour in different ways. Three fatty acids (palmitic, palmitoleic and oleic acid) were involved in the selection of oviposition site, while two esters (methyl oleate and methyl palmitate) reduced the number of eggs laid (Gabeland Thikry, 1996). Methyl esters of fatty acids were also found in the deterring egg wash of 0. nubilalis (Figure 9-3d) (Thiery and Le QuQ6,1991). In P. brassicae three aventhramide alkaloids were first identified as the active compounds in the glue of egg batches that elicited the ovipositiondeterring effect (Blaakmeer et al., 1994a). However, it was later shown that these compounds are not directly responsible for the avoidance of occupied leaves (Blaakmeer et al., 1994b). ODPs released by larvae have been identified from the mandibular gland secretionsof E. kuehniella: 16different P-triketones were isolated from the secretion and suggested to deter female oviposition (Mudd, 1978, 1983). However, the behavioural effect of these compounds has not been established. The content of P-triketones in larval secretion of E. kuehniella and the related species, E. cautella, E. elutella and P. interpunctella, showed large similarities, but some interspecific differences were found (Figure 9-3e) (Nemoto et al., 1987).Behavioural tests also indicated that the larval secretions affecting oviposition of E. kuehniella and P. interpunctella were similar, but not identical (Anderson and Lofqvist, 1996).In the ladybird beetle A. bipunctata, the ODP released by the larvae contains a mixture of alkanes, of which n-pentacosane is the major component (Hemptinne et al., 2001). The major volatile components of the glandular secretions of the chrysomelid larvae deterring oviposition of conspecifics (see Section 9.2.3.1) are well known as typical defensive components in these species. Gastrophysa viridula and Phaedon cochleuriae produce the cyclopentanoid monoterpene chrysomelidial and (epi)chrysomelidial,respectively (Pasteels et al., 1982),while the willow leaf beetle Phratora vitellinae secretes salicylaldehyde as major component (Pasteels et al., 1983). Both salicylaldehyde (Hilker, 1989) and (epi)chrysomelidial (Gross and Hilker, 1995)have been shown to act as oviposition deterrents against conspecific females. Faeces of larvae of S. littoralis feeding upon cotton plant leaves emit a large number of volatile compounds (Klein et al., 1990).From the complex mixture six compounds, benzaldehyde and the five terpenoids, carvacrol, eugenol, nerolidol

Detection of Oviposition Pheromones

(Figure 9-3f), phytol and thymol (Figure 9-3g), were isolated and found to be sufficient to elicit oviposition deterrence in conspecific females (Anderson et al., 1993).Each of these six components is important for oviposition deterrence. If one of the compounds was excluded from the mixture, no deterrent effect was found (Anderson et al., 1993). When considering the oviposition stimulating pheromones, both volatile and non-volatile compounds are probably important for the attraction of gregarious S. greguriu females to sites already containingeggs (Sainiet al., 1995).The two volatile compounds, acetophenone and veratrole (Figure 9-3h and 9-3i), identified from the froth plug deposited after oviposition, were found to attract ovipositing females (Rai et al., 1997). From sand near oviposition sites, three compounds affecting oviposition (Z)-6-octen-2-one,(E,E)-3,5-octadien-2-one and (E,Z)-3,5-octadien-2one, were identified using gas chromatography-electroantennographicdetection (Torto et al., 1999). The non-volatile compounds from froth or sand affecting oviposition have yet to be identified.

9.5

Detection of Oviposition Pheromones

Sensilla with receptor neurons responding to chemical stimuli are found on many different parts of the insect body. Volatile compounds are detected by receptor neurons found in several different sensillum types on the antennae and are very rarely found on other parts of the insect body (Hansson, 1995). Contact chemoreceptors for non-volatile compounds are found in sensilla on mouthparts, tarsi, ovipositor and antennae (Stadler, 1984).Parasitic wasps use sensilla on the antennae to detect external markers on the host (van Lenteren, 1981).For detection of internal markers in the hosts contact chemosensilla on the ovipositor are used. It is also possible that contact chemoreceptors on tarsi may be involved in the detection of the pheromones (van Lenteren, 1981). In R. pornonella the ODP is detected by sensilla on the fore tarsi (Prokopy and Spatcher, 1977). Using an electrophysiological tip-recording technique, several types of contact chemoreceptors on the fore-tarsi were mapped for their response to the ODP (Crnjar and Prokopy, 1982).The longest type of sensilla, the D-hairs, responded strongly to an ODP extract, while no responses were recorded from the shorter types of sensilla. Also in R. cerusi, receptor neurons responding to a crude pheromone extract were found in the D-hairs of the tarsi (Figure9-4) (Stadler and Katsoyannos, 1979). The D-hairs contain 4 chemosensory neurons and only one of the neurons responded to the pheromone (Stadler et al., 1994).The receptor neurons in young male flies were more sensitive to pheromone than those of females, indicating that deterring oviposition is not the only function of the pheromone (Stadler et al., 1994).Structure-activity experiments showed that all four moieties of the molecule, taurine, palmitic acid, C(8)-hydroxyl group and glucose (C15)were important for the activity. Of the four structural isomers of the pheromone (N{15R,S[~-glucopyranosyl]-oxy-8RS-hydroxypalmitoyl)-taurine), the 8R, 15R and 8S, 15R isomers showed the highest activity in the electrophysiological

251

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Oviposition Pheromones in Herbivorous and Carnivorous Insects

Figure 9-4 Scanning electron micrograph showing the distal three segments of a prothorax tarsus of Rhagoletis cerasi. The tips of the visible contact chemoreceptor D-sensilla have been circled. They contain a receptor neuron sensitive to the oviposition deterring pheromone produced by mature female flies. Note that the identical D-sensilla of the 2nd segment are not in the picture.

recordings. These two isomers were equally active and showed a mean threshold for response at about 2 x 10-10M. The two 155isomers were approximately 13times less active. The purified pheromone contained a racemic mixture of the two 15R isomers. However, no synergism was found at the receptor level between the two 15R isomers, as the individual isomers were as active as a racemic mixture (1:l) (Stadler et al., 1994). In the weevil C. assirnilis the ODP is detected by contact chemoreceptors on the antennae (Ferguson et al., 1999b).However, it seems that the antennae are not in contact with the surface of the plant, and thus do not contact the pheromone, before the behaviour to abandon a marked pod is taken. This indicates that the pheromone has some volatility and that it may be perceived by the contact chemoreceptors at a distance (ca. 1mm) (Ferguson et al., 1999b). The oviposition deterring compounds identified from larval frass of S. littoralis are detected by receptor cells on the antennae (Hilker and Klein, 1989). Electrophysiological single-cell recordings on trichoid sensilla on the antennae revealed an abundant receptor cell type responding equally well to three of the compounds of the deterring mixture, carvacrol, eugenol and thymol. Neurons responding to only one of the deterring compounds were found for carvacrol, eugenol, nerolidol and phytol (Anderson et al., 1993; Anderson et al., 1995). Intracellularrecordings of interneurons in the antennal lobe of S. littoralis showed that responses to oviposition deterring compounds were found in both local interneurons, that are confined to the antennal lobe, and projection neurons, that connect the antennal lobe with other parts of the brain (Anton and Hansson, 1994).

Interspecific Effects

Neurons responding to single compounds as well as neurons responding to several of the deterrents were found. On the antennae of S. gregaria neurons specificallyresponding to the oviposition stimulating pheromone compounds, veratrole or acetophenone, were found in trichod andor basiconic sensilla (Hansson et al., 1996; Ochieng’ et al., 1999),and in intracellular recordings on neurons in the antennal lobe (Ignell et al., 1998). Generalist interneurons, responding both to oviposition pheromones and to plant compounds or to other pheromone compounds, were found in both S. littoralis and in S. gregaria (Anton and Hansson, 1994; Ignell et al., 1998).Thus, in these two species the information on oviposition pheromones is integrated with other olfactory input already in the antennal lobe.

9.6

lntraspecific Effects of ODPs on Behaviour other than Oviposition

Compounds acting as ovipositionpheromones may intraspecificallyalso influence other behaviour than oviposition. The pattern of movement of insects can be affected by the presence of oviposition pheromones. Three examples of increased dispersal have been found. The wandering time of E. kuehniella at the end of the larval development was increased by high concentrations of larval secretion in the food substrate of the larvae (Corbet, 1971).In large field case experiments both A. frontella (Quiring and McNeil, 1987)and R. pornonella (Roitberget al., 1982)dispersed from the patch when encountering only ODP-markedleaves and fruit but remained in the patch for the duration of the experiment when encountering unmarked fruit. Female R. pornonella encountering a high rate of ODP-marked fruit displayed long distance flight more frequently, and flew on average longer distances, than did females encountering uninfested fruit (Roitberget al., 1984).Male R. pornonella were on the other hand arrested by ODP at a site and stayed longer on sites with ODP than on sites without. It is possible that the pheromone indicates to males that females are present in the area (Prokopy and Bush, 1972). In the beetle L. serricorne, one of the compounds found to deter oviposition is also a minor component in the sex pheromone of females and thus has a bifunctional role (Imai et al., 1990).

9.7

Interspecific Effects

9.7.1 Response of Competitorstowards ODPs For species competing for the same resources, it would be advantageous to detect the pheromones of their competitors. Several species of herbivorous (Prokopy, 1981b; Giga and Smith, 1985; Sakai et al., 1986; Hilker, 1989; Schoonhoven et al., 1990; Anderson and Lofqvist, 1996)and carnivorous insects (Price, 1970; Vet et al., 1984; van Baaren et al., 1994; Ruzicka, 1996) have been shown to respond to oviposition deterring pheromones from closely related species. Thus, ODPs may

253

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Oviposition Pheromones in Herbivorous and Carnivorous Insects

not only serve intraspecificallyas pheromone, but also interspecificallyas allomones to deter competitors. Prokopy (1981b) showed that several fruit flies within the same species group showed cross-recognitionof the marking pheromone, while no cross-recognition was found between fruit fly speciesbelonging to different groups (Prokopy, 1975). Also in parasitic wasps of the genus Asoburu closely related species responded to each other’s ODP, while more distantly related species did not (Vet et al., 1984). The level of response of two species to each other’s ODP may, however, be different. Females of R. pornonella were equally deterred by the marking pheromone of R. zephyriu as by their own pheromone, while females of R. zephyriu showed a much weaker response to the pheromone of R. pornonella than to their own pheromone (Averilland Prokopy, 1981).An asymmetry in response towards ODP has also been found between C. muculutus and C. rhodesiunus, where the latter species is deterred by heterospecific ODP, while the former species is not (Giga and Smith, 1985).A similar pattern was found in the two stored product pests E. kuehniellu and P. interpunctellu, which occur sympatrically.Females of both speciesresponded equally well to heterospecific as to conspecific larval secretion, when compared to an uninfested substrate (Anderson and Lofqvist, 1996). However, when comparing conspecific and heterospecific secretion, females of E. kuehniellu oviposited on substrates with heterospecific secretion,while femalesof P. interpunctellu deposited equal amounts of eggs on the two substrates (Anderson and Lofqvist, 1996). Different interspecific competitive abilities may explain the differencesin response between the related species. For example, when occurring together C. muculutus eliminates C. rhodesiunus through intense larval competition (Giga and Smith, 1985).

9.7.2

Response of Carnivores towards ODPs of their Prey

There are several examples where parasitic wasps use the ODP as a kairomone to locate host larvae (Prokopyand Webster, 1978; Mossadegh, 1980; Mudd and Corbet, 1982; Strand et al., 1989; Roitberg and Lalonde, 1991; Hoffmeister and Gienapp, 1999; Kumazaki et al., 2000). The mandibular glandular secretion from larvae of E. kuehniellu and P. interpunctellu is used by at least two species of parasitic wasps to locate hosts (Mossadegh, 1980; Mudd and Corbet, 1982; Strand et al., 1989).Single P-triketones from the secretion elicited arrestment, antennation and probing response in the ectoparasitoid Brucon hebutor when present in a patch, but no difference in behaviour was found to the different p-triketones from the larval secretion (Strand et al., 1989). The parasitoid Nerneritis cunescens, on the other hand, showed a different number of ovipositional movements to different P-triketones (Mudd and Corbet, 1982; Mudd et al., 1984).When deposited linearly the P-ketones elicited a trail-following response in B. hebutor (Strand et al., 1989). No such trail-following was found in Opius lectus, a parasitoid of eggs and small larvae of R. pornonella (Prokopy and Webster, 1978). The ODP of R. pornonella

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Concluding Remarks

retained 0. lectus on the fruit and stimulated antenna1 tapping. Presence of pheromone on a fruit reinforced an intensified searching behaviour, rather than attracting the parasitoid to its host. The behaviour of a second parasitoid, 0.allocus, was not influenced by the pheromone (Prokopy and Webster, 1978). These parasitoids attack 3rd instar R. pornonella larvae, which are not present in the fruit until at least 13 days after oviposition. In some species, the ODP also acts as an allomone against antagonists. The oviposition deterring effect shown for the exocrine secretion of some chrysomelid larvae (Hilker, 1989)is not the only function of these secretions. The secretions of these speciesare primarily known for their ability to defend larvae against predators (Pasteels et al., 1988).Additionally, they have been shown to act as antimicrobial agents (Gross et al., 1998).Also the oviposition deterring larval regurgitate of the sawfly D. pini functions primarily as a defence against predators.

9.8

Applications

Soon after the discovery of an ODP in the tephritid fly R. cerusi, encouraging attempts were made to use ODPs to protect cherry trees against fruit fly attack (Katsoyannos and Boller, 1976, 1980). A crude female ODP was collected in the laboratory and applied on oviposition susceptible cherries in the field. The application resulted in a reduction of infestationof 63-90% (Katsoyannosand Boller, 1976,1980).After the identification of the ODP of R. cerasi, synthetic pheromone was applied on cherry trees in the field to examine their potential for control of the fruit fly (Aluja and Boller, 1992). Over the years the field applications have followed the same principle methods, with a first application just before the oviposition starts and a second application a week later (Boller and Hurter, 1998). Spraying entire trees or the bottom half of trees with synthetic pheromone reduced the infestation to a low level (Aluja and Boller, 1992).The authors suggest that the ideal pheromone deployment would be to exclusively spray the cherry clusters approximately every 7 days during the susceptiblestage of the cherries. Every 10th tree in the orchard should be left unsprayed to avoid adaptation to the pheromone. The flies should be able to recover the sensitivity of their receptor neurons to the ODP when resting on untreated trees (Aluja and Boller, 1992). Applying this method, field experiments were conducted in 1990-1994 under different meteorological conditions. The efficacy of the ODP was over 90% during years with unfavourable weather conditions, and 99.9 and 100%in years with favourable weather (Boller and Hurter, 1998).

9.9

255

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Concluding Remarks

It is now 30 years since more systematicinvestigations of oviposition pheromones were initiated. Until now oviposition pheromones have been found in about 100 herbivorous and carnivorous insect species. The deposition of ODPs at oviposition

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Oviposition Pheromones in Herbivorous and Carnivorous Insects

sites, which offers limited food to the progeny, may be widespread among both herbivorous and carnivorous insects. The origin of the compounds affecting oviposition behaviour may not always be clear.Future studies should consider that egg deposition or larval feeding might induce the host to change its chemicalsso that gravid females will be deterred from this site. For example, in P. brassicae and in Callosobruchus ssp. it has been shown that oviposition sites (cabbageleaves and seeds, respectively) keep the oviposition deterrent activity after removal of previously laid eggs (Blaakmeer et al., 1994a; Mbata and Ramaswamy, 1995; Ignacimuthu et al., 2000). Numerous studies have shown that larval feeding or oviposition on a plant may induce a plant to release volatiles that affect insect behaviour (e.g. Karban and Baldwin, 1997; Dicke and Van Loon, 2000, and Chapter 8). Thus, it is possible that oviposition deterrents thought to be associated with eggs or feeding larvae are produced by the plant in response to the egg deposition or feeding larvae. The field experiments on R. cerasi are very promising and show the potential of ODPs in crop protection. Applied experiments with oviposition pheromones in other species are desired. However, the identification of the active compounds is difficult, probably due to their low volatility and, thus, due to the high amounts needed for analyses. For deeper understanding of the role of oviposition pheromones and for exploration of their possibilities for applied use, more efforts need to be put into identification of the active pheromone.

9.10 Acknowledgements I am very grateful to Drs. J. Agrell, T. Hoffmeister and J. Steidle for valuable comments on earlier versions of the manuscript. I also thank Dr. Erich Stadler for kindly providing me with the SEM-micrographof a Rhagoletis cerasi tarsus.

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Chapter 10 Chemoecology of Oviposition in Insects of Medical and Veterinary Importance Philip J. McCall

Table of Contents 10.1 10.2 10.2.1 10.2.2 10.2.3 10.3 10.3.1 10.3.2 10.3.3 10.4 10.4.1 10.4.2 10.5 10.6 10.7 10.8 10.9

Introduction Oviposition Attraction Pheromones Aquatic Environment - Mosquitoes and Blackflies Terrestrial Environment - Sandflies, Tsetse Flies and Bugs On the Host Oviposition Attraction Allelochemicals and their Interaction with Pheromones and Other Sensory Information Aquatic Terrestrial On the Host Oviposition Repellents Cues from Plants Predator and Parasite-AssociatedCues The Influence of Experience on Responses t o Oviposition Cues Responses t o Plant Mimics and Plant Allelochemicals Concluding Remarks Acknowledgements References

Abstract

The insects that cause or transmit human and animal disease constitute a wide range of taxa sharing a similar feeding habit, but differing in most other aspects of their life histories, including oviposition.This chapter mainly deals with oviposition or larviposition behaviour within the Diptera, since the major groups that exhibit such behaviour and that constitute pests of major medical or veterinary importance are true flies. Aggregation behaviour in bloodsucking Hemiptera is discussed briefly.Bloodfeeding groups generally oviposit at sites remote from their vertebrate hosts: mosquitoes (Culicidae) and blackflies (Simuliidae) oviposit in aquatic environments, and sandflies (Psychodidae), tsetse flies (Glossinidae),bedbugs (Cimicidae) and kissing bugs (Reduviidae) oviposit in terrestrial environments. Facultative or obligatory carnivorous Diptera, where the immature stages can be

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saprotrophic or parasitic on the same animals on which the adult flies feed, also locate and select oviposition or larviposition sites using chemical cues. These are the warble flies, bot flies (Oestridae), blowflies, screwworms (Calliphoridae) and flesh flies (Sarcophagidae). Knowledge of the chemoecology of oviposition in these insects lags behind that on herbivorousand parasitic insects and attractant compounds have been identified in only a few cases. A number of plant species have evolved to mimic animal odour cues to attract carnivorous fly species as pollinators. Because of the importance of these insects as vectors of human and animal disease, interest in this field has been driven primarily by a search for chemicallybaited traps for monitoring and control. Odour baited oviposition traps are currently used in the surveillance of a number of important pest species. Odour baited trapping for the control of pest populations may be feasible for certain species.

10.1 Introduction The intimate association between insects and humans has forced us to speculate on how these pests locate and select the individuals they choose to land on. How they locate and select their oviposition sites has for many been a secondaryquestion. Generally, our knowledge of oviposition behaviour in these groups lags behind that of herbivorous or parasitic insects, but has improved markedly over the past twenty five years, with greatest progress made in dismantling the olfactory mechanisms involved in oviposition site location and selection within the past ten to fifteen years. While different taxonomic groups of haematophagous and carnivorous (species that feed on living or dead vertebrate tissue) insects utilize generally similar visual and olfactory cues to find hosts, their ovipositionbehaviours may not be unrelated to the feeding habit and a wide range of natural histories exists. In the haematophagous groups, oviposition takes place remote from and often unrelated to the location of the host, in terrestrial and aquatic sites. The calyptrate Diptera (blowflies and screwworms) on the other hand may oviposit or larviposit in the same vertebrate carcasses or living individuals on which the adult flies also feed. In some cases very little is known of oviposition behaviour in nature, and our knowledge derives primarily, as is the case with the Psychodidae, almost entirely from the laboratory. Conversely, studies on the Simuliidae, which are notoriously difficult to colonize, have been carried out in field laboratoriesusing wild populations of flies. This chapter deals primarily with Diptera, since in the field of medical and veterinary entomology, the true flies are without doubt the most important insects. The role of aggregation pheromones in bloodsucking Hemiptera is discussed briefly (Section 10.3.1). Oviposition behaviour in those insect pests that typically oviposit either within the nest or burrow of the host or on the host itself (Siphonaptera, fleas), or that live permanently on the host (Anoplura,lice), are not

Introduction

Table 10-1 Checklist of the families of Diptera and Hemiptera discussed in this chapter, the diseases they transmit or cause, a brief outline of the method of oviposition or larviposition and the habitat in which the larvae develop

laxon DIPTERA Culicidae (Mosquitoes) subfamily Anophelinae subfamily Culicinae

Vectors or causative agents of

Simuliidae (Blackflies)

Malaria, lymphatic filariasis, o'nyong nyong virus Lymphatic filariasis, yellow fever, dengue, encephalitis arboviruses (e.g. Japanese encephalitis, West Nile virus) Onchocerciasis (river blindness), mansonellosis

Psychodidae (Sandflies) Glossinidae (Tsetse flies)

Leishmaniasis, bartonellosis, sandfly fever Trypanosomiasis in humans (sleeping sickness) and animals.

Oestridae (Warble and Botflies) Calliphoridae (Blowflies, Screwworms)

Myiasis

HEMIPTERA Cimicidae Biting nuisance, can cause (Bedbugs) anaemia (Hepatitis B ?) Reduviidae Chagas disease subfamily Triatominae (Kissingor cone-nose bugs)

Oviposition behaviour and lawal habitat Eggs laid on water surface, on damp substrates close to water or adhered to floating vegetation; standing water; sites range from animal footprints, discarded cans and tyres to large mangrove swamps and rice paddies. Eggs typically laid on trailing or submerged vegetation and stones, or dropped into flowing water. Eggs laid in cracks and holes in soil, floors, masonry, leaf litter. Larviporous: egg develops in utero, single larva deposited in sandy soil under bushes, trees, animal burrows. Females deposit eggs or larvae in wounds or body orifices, or onto surfaces with which the host will eventually contact

Eggs deposited in cracks and crevices of buildings and furniture Eggs deposited in cracks and crevices in mud-walled buildings thatched roofs, animal burrows, and palm trees.

discussed. A list of the families discussed, the diseases they transmit a n d a brief summary of their oviposition behaviour a n d larval habitat are given in Table 101. As vectors o f h u m a n or animal disease or as the causative agents of myiasis (defined by Zumpt [1965] as the infestation of live human and vertebrate animals with

dipterous larvae, which, at least for a certain period,feed on the host's dead or living tissue, liquid body substances or ingested food), the importance of these insect groups is incalculable. A n n u a l deaths from malaria alone probably exceed 1.5 m i l l i o n persons worldwide, with African children most affected. Acute diseases like yellow fever, dengue a n d dengue haemorrhagic fever are increasing their range, w h i l e recent epidemics o f leishmaniasis a n d h u m a n trypanosomiasis demonstrate that m a n y h u m a n vector-borne diseases remains as serious as ever. Chronic diseases like

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lymphatic filariasis and onchocerciasis continue to affect hundreds of millions worldwide. Emerging zoonotic diseases like the mosquito-borne West Nile and Rift Valley fevers, and many of the diseases already mentioned, are increasing their range and the risk of infection to an ever-increasing world population. Consequently, much research has concentrated on the more important pest species with a view to the development of odour baited traps for surveillanceand control. Most studies are applied in nature, with an emphasis on characterizingbehaviours and identifying the compounds that mediate them, rather than developing hypotheses regarding the evolutionary origins and advantages of such behaviour.

10.2 Oviposition Attraction Pheromones The range of ovipositionbehaviour exhibited by the insect groups under discussion here is very wide in nature, and the only common feature is their dependence on vertebrate hosts for either bloodfeeding or for egg deposition. Coincidentally perhaps, egg aggregation appears to be common to many. The species that exhibit aggregation of eggs and immature stages breed in relatively large, potentially unrestricted food environments in which the advantages of a large population outweigh the disadvantages of larval competition or infection. Different strateges exist suggesting that the nature and advantages of aggregation may be complex. For example, staggered development and emergence of larvae may reduce the possibility of competition, while deposition of portions of an egg batch in different aggregations may reduce the risk of infection introduced by another female or consumption of a single aggregation by a large predator. There may be an optimal size to an aggregation of conspecifics or congenerics, after which the egg mass becomes less attractive. A summary of details of the oviposition and larviposition pheromones identified from haematophagous and carnivorous Diptera is given in Table 10-2.

10.2.1 Aquatic Environment - Mosquitoes and Blackflies The two major groups of bloodfeeding Diptera with aquatic immature stages are the mosquitoes(Culicidae)and the blackflies (Simuliidae).Mosquitoes deposit their eggs either directly onto the surface of, or on dry ground close to, pools of standing water where the larvae and pupae will develop. Blackflies oviposit on trailing vegetation or partially submerged rocks in running water. A vast amount has been written about mosquito oviposition, with most studies concentrating on the major vector genera, Culex, Aedes and Anopheles (Bentley and Day 1989; Clements, 1999). Many Culex sp. females lay their eggs in clusters called rafts on the water surface (Figure 10-1). Early work focussing on the rafts as a potential source of attraction for gravid female Culex spp. showed that 1.3-diacylglycerolswashed from the eggs elicited preferential oviposition (Starratt and Osgood, 1972,1973).Later, the apical droplet, a drop of fluid extruded from the posterior pole of each egg prior to darkening of the endochorion (Christophers, 1945), was shown to attract gravid

Oviposition Attraction Pheromones

Table 10-2 Oviposition/larvipositionl aggregation pheromones identified in haemato-

phagous and carnivorous insects Family and species Origin Culicidae Culex quinquefasciatus egg apical droplets larvae

Aedes aegypti

Campound

Rderences

erythro-6-acetoxy-S- Laurence and Pickett, hexadecanolide 1982,1985 heneicosane Mendki et al., 2000

Psychodidae Lutzomyia longipalpis

dodecanoic acid

Dougherty and Hamilton, 1997

unidentified

McCaII et al., 1997a,b and unpublished

Simuliidae Simulium damnosum 5.1.

(two methylbranched unsaturated hydrocarbons)

data

Glossinidae Clossina morsitans morsitans Clossina morsitans centralis

larval anal exudate larval anal exudate

n-pentadecanel

Saini et al., 1996

n-dodecanel

Saini et al., 1996

female cuticular lipids

unidentified

Emmens, 1981

Calliphoridae Lucilia cuprina

1 Major components with

(branched saturated C26 hydrocarbon)

biological activity

C. quinquefasciatus to oviposit in laboratory bioassays (Bruno and Laurence, 1979). The active substance, subsequently identified, synthesized and shown to have biological activity comprised a single compound, erythro-6-acetoxy-5-hexadecanolide (Laurence and Pickett, 1982,1985)(Table 10-2).The oviposition aggregation pheromone in C. quinquefasciatus was the first pheromone identified in any species of medical or veterinary importance. The pheromone has an orthokinetic (change in speed) but no klinokinetic (change in the rate or magnitude of direction) effect on incoming female mosquitoes, and in addition to its aggregation characteristics, it may also stimulate oviposition (Pile et al., 1991, 1993). The pheromone is attractive at distances up to 10 m (Otieno et al., 1988),but does not appear to be attractive in the absence of those allelochemicals that are indicative of high organic quality at the site (see Section 10.3). This pheromone is genus rather than species-specific, eliciting similar responses from C. tarsalis and C. pipiens molestus as well as C. quinquefasciatus (Bruno and Laurence, 1979). Considering the breeding preferences of Culex spp., this is not surprising. Many species (notably C. quinquefasciatus, the cosmotropical urban mosquito) preferentially lay eggs in organically rich or foetid water, ranging from ditches and pools to storm drains, septic tanks and pit latrines. Such environments are very high in nutrient levels, likely not to be food-limited and so capable of supporting large numbers of individuals. Moreover, large aggregations of larvae

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Figure 10-1Oviposition and larviposition in the haernatophagous vectors of human and animal diseases in which aggregationpheromones have been demonstrated. (a) Aggregation of the eggs ofthe neotropicalsandfly Lutzomyia longipalpis on an artificial substrate, showing egg aggregation close to the edge of the container on the right. (b) Vertical view of egg rafts of the cosrnotropical mosquito Culex quinquefasciatus with the apical droplet, from where the pheromone is released,visibleon each egg. (c) Largeaggregation ofeggsofthe Palaearctic blackflySimuliumornatumon a single leaf surface. (d) FemaleAfrotropicaltsetsefly, Clossina sp., depositing her fully-developed single larva, which releases the aggregation pheromone in i t s anal exudate.

Oviposition Attraction Pheromones

would help prevent the formation of the surface scum that could hinder access for breathing, in addition to the advantages conferred by large group sizes in relation to predation (McCalland Cameron, 1995).Since the pheromone released from the eggs reaches maximum after about 24 hours, Pickett and Woodcock (1996) have postulated that these mature eggs also signal the safety and durability of the breeding site to incoming gravid females. Unlike Culex, many Aedes mosquitoes breed in smaller and less eutrophic environments, such as tree holes, temporary ground pools or in manmade containers,like flower vases, discarded cans or rubber tyres. Characteristically,Aedes deposit their eggs singly on moist surfaces that are likely to become submerged. The eggs of most species can withstand desiccation throughout long dry seasons. This ability appears to be related to the resistance to water loss as viability is reduced in hotter drier conditions (Clements, 1992). A variety of studies have suggested that Aedes avoid (Chadee et al., 1990) or prefer (Soman and Reuben, 1970; Gubler, 1971; Kalpage and Brust, 1973; McDaniel et al., 1976,1979; Trimble and Wellington, 1980; Maire, 1984; Yap et al., 1995; AUan and Mine, 1998) sites where pre-existing eggs of congenerics have been laid or sites holding water in which congenerics previously occurred, suggesting a repellent or attractive effect respectively. Both observations may be true. Zahiri and Rau (1998)found that the attraction of Ae. aegypti to water in which congeneric larvae had lived, increased as that biomass of larvae increased, peaked and then declined. Comparable responses occurred as the volume of water decreased or the number of larvae increased, suggesting a feedback mechanism that results in the maintenance of larval populations at an optimal level. Starved larvae also rendered water unattractive to gravid female Ae. aegypti (Zahiri et al., 1997a), suggesting that an attractant was produced in the larval environment only under optimal conditions (see Section 10.4.2).Responses may vary seasonally, with the presence of larvae being repellent early in the season, when they represent a source of competition, but attractive later in the season when they indicate habitat quality to females searching for suitable sites to lay eggs that will enter diapause (Edgerlyet al., 1998). Evidence suggests that these attractants are chemical in nature, but whether they are all of mosquito origin, or by-products of bacterial metabolism of mosquito faeces, remains to be determined. A pheromone, heneicosane, has been identified in both larval conditioned water and larval cuticle extracts and appears to be at least partly responsible for the attraction (Mendkiet al., 2000) (Table10-2).Whilst the advantage to the detectors of an attractant seems clear, the advantage of producing an aggregation pheromone is unclear. Chadee et al. (1990)found that femaleAe. aegypti would preferentially avoid laying eggs in the presence of their own eggs, when given a choice between their own and those of conspecifics, suggesting a degree of kin recognition not previously seen in mosquitoes.Sherratt et al. (1999)however, found no evidence for this ability in the tree-hole breeding Toxorhynchites and Trichoprosopon. Anopheles mosquitoes typically breed in ground pools, with some species found in tree holes. These mosquitoes do not appear to exhibit aggregation but some

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evidence suggests that the presence of mosquito larvae in a breeding pool may be repellent (McCrae, 1984). Blackflies lay their eggs in or close to running water. Different species prefer lotic environments varying from gentle flows within the detritus on forest floors to white water rapids within large rivers. Blackflies may locate the suitable stretch of water for oviposition visually and anemotactically (Crosskey, 1990). Within these water stretches, aggregation of eggs in large masses is common (Crosskey, 1990; McCall, 1995) (Figure 10-1). Females have been observed laying eggs communally in a manner which over a period of a few hours can lead to deposits of thousands of eggs on a single substrate. In the Afrotropical species complex, Simulium damnosum d., this behaviour is mediated by a volatile pheromone emitted from freshly laid eggs (McCall, 1995). The pheromone has both attractant and stimulant properties (McCall et al., 1994; Wilson et al., 2000) and comprises two methyl-branched saturated hydrocarbons with molecular weights of 292 and 294 (McCall,unpublished data) (Table 10-2).The pheromone is released for only about 12 hours after oviposition, corresponding to the nocturnal period between crepuscular oviposition activity and dawn, when further oviposition may also occur. Since eggs of this speciesmay have progressed some way through embryonation within this time, attraction of further oviposition would be detrimental as new layers of eggs could inhibit development (Kyorku and Raybould, 1987; McCall, 1995). In an insect that oviposits in streams that may fluctuate in depth within a matter of hours, communal oviposition aggregation at single sites may ensure that a large egg mass is heavy enough to remain submerged if the water level drops, and retain water within a large gelatinous mass if left above the water level (McCall, 1995;McCall and Cameron, 1995).Inner (the first laid) egg layers will be protected by the outer layers, if the egg mass remains out of water for a long period. Aggregation would reduce the effect of predation by increasing the chance of survival of at least some eggs or larvae from each female, should predation (by Odonata, Ephemeroptera, Plecoptera or Trichoptera) be significant in the larval environment (McCall, 1995).However, there is a risk of suffocation, since after a certain period of time, bottom layers will not hatch (Kyorku and Raybould, 1987), and a single female with a microsporidian or fungal infection might infect the offspring of every female in the egg mass. Depositing portions of each egg batch in different aggregations (Weltonand Bass, 1980)may reduce these disadvantages. As in Culex mosquitoes, blackfly aggregation pheromones are also common to sympatric congenerics.All members of the S. damnosum complex examined to date have been found to share the two compounds that constitute the pheromone (McCallet al., 1997a,b). Communal oviposition by more than one species has also been observed (Imhof and Smith, 1979; Hywel-Jones and Ladle, 1986).

10.2.2 Terrestrial Environment - Sandflies, Tsetse Flies and Bugs The immature stages of two families of medically important Diptera, the sandflies (Psychodidae)and the tsetse flies (Glossinidae),develop in terrestrial environments.

Oviposition Attraction Pheromones

Both families include species that produce oviposition (sandflies)or larviposition (tsetse) aggregation pheromones. Sandflies oviposit in rough substrata that are rich in organic material on which the larvae feed, and are attracted to the odours and physical qualities of those sites (Figure 10-1and Section 10.3.2).Females of the South American speciesLutzomyia Iongipalpisproduce a pheromone in the accessory glands that is passed on to the eggs as they are laid, and that both attracts and stimulates other gravid females to oviposit at the same site (El Naiem and Ward, 1991; Dougherty et al., 1992, 1994). The pheromone has been identified as dodecanoic acid (Dougherty and Hamilton, 1997) (Table 10-2). Tsetse females deposit their large single larva in soil at the base of vegetation (Figure 10-1). The fully mature larva burrows into the soil and pupariates within about 2 hrs. In Glossina morsitans morsitans and G. m. centralis, aggregation is mediated by a pheromone in the anal exudate of the larva (Nash et al., 1976;Leonard and Saini, 1993).The major components of the pheromone are n-pentadecane and n-dodecane in G. rn. morsitans and G. m. centralis, respectively (Saini et al., 1996) (Table 10-2). Interestingly, while females prefer to oviposit in wet areas, gravid tsetse may be most responsive to the pheromone when the differential between soil moisture and ambient humidity is large (Leonardand Saini, 1993).Aggregations of puparia, often found in such specific locations during the dry season in contrast to the more dispersed distribution during the wet season (Nash, 1969). Two families of Heteropteran bugs bloodfeed on vertebrates: the Cimicidae or bedbugs and the Triatominae (familyReduviidae) called kissing bugs or cone-nose bugs. Two species of bedbugs, Cimex lectularius and C. hemipterus rest and breed in cracks and crevices of walls and furniture in human habitations, emerging at night to feed on their sleeping hosts. The odour associated with infestations of bedbugs is often likened to the spice coriander, a name deriving from korus, the Greek word for "bug". C. lectularius produces volatile alarm and assembly pheromones to which both adults and nymphs respond (Levinson and Bar-Ilan, 1971; Levinson et al., 1974). The alarm pheromone is emitted by the metasternal scent glands and mainly comprises trans-oct-2-en-1-a1 and trans-hex-Zen-1-al. The assembly pheromone has not been characterized.Although not solely involved in oviposition, the assembly pheromone may, by maintaining aggregations of all stages of bedbugs, facilitate aggregation of eggs and immatures. Triatomine bugs occur worldwide but are of major medical importance in South and Central America where they feed on humans and transmit Trypanosoma cruzi, the causative agent of Chagas disease (Table 10-1). Rather like bedbugs, the most important vectors inhabit the cracks and crevices in walls and the thatched roofs of human or animal dwellings, though some species will also inhabit trees and animal burrows and nests. Triatoma infesfans exit shelters to actively defecate at the entry point, thus marking the site for other bugs that prefer marked refuges (Lorenzo and Lazzari, 1996).Bug faeces contain an aggregation or assembly pheromone (Schofield and Patterson, 1977; Cruz-Lopez et al., 1993), possibly ammonia (Taneja and Guerin, 1997)which appears to act interspecifically (LorenzoFigueiras and Lazzari, 1998a). Another assembly odour is deposited by walking insects (Lorenzo Figueiras and

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Lazzari, 1998b).Oviposition occurs within these assemblies, where all stages rest when not hostseeking. Proposed advantages of such assemblies, including the increased likelihood of uptake of essential symbionts by coprophagic neonates (Taneja and Guerin, 1997; Lorenzo Figueiras and Lazzari, 1998a), have not been investigated.

10.2.3 On the Host Host-derived olfactory cues appear to be more important than pheromones in host location by the sheep blowflies Luciliu sericutu and L. cuprina (Ashworth and Wall, 1995)and probably by other myiasis-causing species (Table10-1and Section 10.3.3). However, there is good evidencethat L. cuprinu produces an oviposition pheromone that leads to group oviposition at single wounds on the host (Barton Browne et al., 1969). This putative aggregation pheromone seems to be more attractive to females than males and activity is associated with a relatively non-volatile nonpolar branched saturated C26 hydrocarbon isolated from female cuticular lipids (Emmens, 1981) (Table 10-2).

10.3 Oviposition Attraction Allelochemicals and their

Interaction with Pheromones and Other Sensory Information As in other insects, the location and selection of suitable oviposition sites by bloodfeeders and carnivores is achieved through the combination of vision and olfaction. A range of olfactory cues involved have been identified for mosquitoes, blackflies and sandflies, although in most cases demonstration and identification of semiochemical activity has been limited to the laboratory.

10.3.1 Aquatic Many mosquito species oviposit in organically rich waters in which rich bacterial broths produce a complex blend of volatile chemicals that provide potential olfactory cues. Darkor coloured waters, presumably indicativeof such high organic content, are often more attractive (Bates, 1940; Lund, 1942; Field and Matsui, 1965; Beehler et al., 1993; Dhileepan, 1997).Clements has recently compiled a detailed and comprehensive review of the allelochemicals known to attract gravid mosquitoes (Clements,1999; Table 40-5, pp. 569-570). A wide range of compounds, encompassing saturated and unsaturated carboxylic acids, ketones, phenols and indoles, have all been shown to elicit responses either by bioassay or electroantennography. A few have been investigated in some detail. Gravid female Culex spp. are attracted to fermented grass infusions (Millar et al., 1992; Isoe et al., 1995) and water enriched variously with alfalfa, rabbit or bovine faeces or chicken feed (Reisen and Meyer, 1990; Beehler and Mulla, 1995; Rodcharoen et al., 1997).A number of attractants have been isolated from fermented grass infusions and of these, skatole (3-methyl-indole)has consistently proven to

Oviposition Attraction Allelochemicals and their Interaction with Pheromones

be the most attractive in laboratory studies (Millar et al., 1992; Blackwell et al., 1993; Beehler et al., 1994).In the field, bowls treated with skatole at 1 V o r lVgremained attractive to gravid females over a seven-day period (Mboera et al., 2000a). Other species are attracted to odours associated with their specific environments. Aedes triseriatus oviposit in tree-holes and respond to infusions of decaying wood and certain synthetic analogues associated with these broths. P-cresol(4-methyl-indole) is an effective attractant for this mosquito (Bentley et al., 1979).P-cresol was also identified from grass infusions and is weakly attractive to Cx.quinquefasciatus. It is likely that many volatile compounds will be common to fermentations of different media. The compounds or other cues that confer specificity are unknown. Mosquito species that breed in significantly less organically rich environments and that are rarely if ever found in the sites described above, will also respond to olfactory cues indicative of larval nutrients. In an early study on anophelines, Lund (1942) observed that Anopheles quadrirnaculatus preferred water that contained algae. Rejmankovaet al. (1996)suggested that A. albirnanus may locate cyanobacteria mats for ovipositionby orienting to the high C 0 2emitted by such sites (seeChapter 6 for further information on microorganisms involved in insect egg deposition). Extracts of water from A. garnbiae breeding sites contained skatole, indole, rn-cresol and 4-methylcyclohexanol, all of which elicited electroantennographic responses in A. garnbiae by electrophysiology (Blackwell and Johnson, ZOOO), though their importance in nature remains to be determined. The container breeding Aedes albopictus is attracted to ovipositin water with hay infusions (Allanand Kline, 1995). Ae. aegypti showed some responses to phenol and 4-ethylphenol in the same study. The larvae of Mansonia mosquitoes are dependent on certain aquatic macrophytes for respiration and although certain plant species are preferred (Viswamet al., 1989) and aggregation of egg masses occurs at single sites, the attractants remain unknown (Lounibosand DeWald, 1989; Clements, 1999).An interestingobservation by Tyagi et al. (1981)found that Ae. aegypti and to a lesser extent C. quinquefasciatus were attracted to containersbaited with scavenger snail (Lyrnnaeaacurninata)excreta. Predatory mosquito species also respond to the same environmental cues as their prey. Thus the tree-hole breeding Toxorhynchites rnoctezurna and T. arnboinensis respond to p-cresol, rn-cresol and o-cresol (Collins and Blackwell, 1998), cues to which potential prey such as tree-hole breeding species like Aedes triseriatus also respond (Bentleyet al., 1979; Bentley et al., 1981).C. tigripes, another larval predator, was collected in oviposition traps baited with water from a pit where its prey C. quinquefasciatus was breeding (Mboera et al., 1999).Interestingly, C. tigripes was not caught in bowls baited with the Culex ovipositionaggregation pheromone (Mboera et al., 1999),suggesting that although a number of species within the genus respond to it (see Section 10.2.1), in this case the predator does not share an aggregation pheromone with its prey. In C. quinquefasciatus the interaction of the oviposition aggregation pheromone with site derived odours has an additive effect (Mordue et al., 1992; Millar et al., 1994).Thus, the whole mosquito oviposition site environment may be assessed for its potential as a suitable larval environment by gravid mosquitoes. Certain

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compounds, like skatole and p-cresol, are indicators of site quality to a range of mosquito species with differing breeding site preferences, though response thresholds may differ. In addition to the odours from the site and the presence of oviposition aggregationpheromone, site quality may be gauged further by its colour and size, presence of predators and/or parasites (Section 10.4.2). Blackflies (Diptera: Simuliidae)preferentially oviposit on a variety of substrates in clean running waters that would not present odour cues like those described for mosquitoes. Many species are known to prefer pale substrates for oviposition (Golini and Davies, 1987; Elsen and Hkbrard, 1977; Walsh, 1984), a strategy that suggeststhey select more secure living vegetation over the darker colours associated with dead vegetation. Blackfly egg masses gradually change from white to dark brown as they age, and as the aggregation pheromone released from the eggs also wanes, rendering the eggs less attractive to incoming flies as the time of hatching approaches. Apart from the aggregation pheromone in Simulium damnosum s.1. (Section 10.2.1),no odour cues from any other source have been identified.

10.3.2 Terrestrial Surprisingly little is known of the life history of sandflies despite their wide distribution and their importance as Leishmaniasis vectors. Little is known of oviposition activity in nature, and most information has come from laboratory studies, in particular on the neotropical species Lutzomyia longipalpis. This species prefers to deposit its eggs in cracks, crevices and edges within an oviposition substrate, where the moist environment is organically richer (El Naiem and Ward, 1992a) (Figure 10-1). Gravid females are further attracted by the oviposition aggregationpheromone released from conspecificeggs (Section 10.2.1).L. longipalpis also orient to odours emitted by extracts of rabbit food and faeces, hay and chicken faeces, indicative of a nutrient-rich larval environment (ElNaiem and Ward, 1992b; Dougherty et al., 1993; 1995).Hexanal alone and 2-methyl-2-butanolwith hexanal were attractive in bioassay, while electrophysiologyindicated that R( +)-a-pinene, R( +)-P-pinene, S(-)-a-pinene, S(-)-P-pinene,a-terpinene, benzaldehyde may also be important (Dougherty et al., 1995).

10.3.3 On the Host Responses to semiochemicals are an important part of the suite of behaviours (visual, olfactory, tactile and thermal) resulting in blowflies landing on their vertebrate hosts to lay eggs (see Hall, 1995).The resulting pathological condition of the host, invasion of tissue by fly larvae, is termed myiasis (definition, Section 10.1). Research in the field has been reviewed by Ashworth and Wall (1994), Spradbery (1994)and Hall (1995). Adult Oestridae (Diptera) do not feed but function simply as reproductive vehicles for the relatively long parasitic larval stage. No olfactory cues have yet been identified for those species that either oviposit in the nests or burrows of their hosts, or for species like Dermatobia hominis that lay their eggs on other "porter

Oviposition Attraction Allelochemicals and their Interaction with Pheromones

flies” (Hall, 1995).D. hominis effectivelyutilizes the host location ability of the porter fly species (such as day-flyingmosquitoes,a range of other fly species or even ticks), to carry the parasite’s eggs with them when they visit the host to feed. The developed larva within the egg hatches in response to host temperature, and enters the host skin. The calliphorid fly Cordylobiu unthropophugu, the ”Tumbu”, “Pulse” or ”Mango” fly also parasitizes the skin of man, dogs and other animals. Females lay eggs on ground contaminated with host urine or faeces or possibly other unidentified odours, and also on clothing when placed on the ground or even hung on a washing line (Hall and Smith, 1993).Hatched larvae burrow into the host’s skin when activated by body temperature. Many other oestrid species oviposit or deposit larvae directly onto their host, usually one or a few preferred host species, and usually with great accuracy onto or within a particular part of the host’s body. Oestrus ovis for example, always deposits live first stage larvae within the nostrils of its hosts (sheep, goats, equines, etc.), rarely (in error, since the larvae will not develop) within the eyes of humans. While the non-specific host attractants C 0 2and 1-octen-3-01have been shown to attract a number of species, colour and shape are also very important (Hall, 1995) possibly permitting species recognition by the gravid female flies. Specific odour cues have not yet been identified. The most important species within the Calliphoridae and Sarcophagidae cause traumatic myiasis in vertebrates (Table 10-1). Some species, the New World screwworm Cochliomyiu hominivorux, the Old World screwworm Chrysomyu bezziunu and the flesh fly, Wohlfahrtiu mugnifica, are obligate parasites of living animals and oviposit (larviposit in the case of Wohlfahrtiu) in fresh wounds or body orifices. Other species like Luciliu sericutu and L. cuprinu are primary facultative parasites capable of initiating myiasis on a host (called “strike” in sheep), though they will also deposit eggs in carrion. Aggregation of larvae within wounds has long been observed in myiasis. Fenton et al. (1999) suggest that the greater the number of larvae feeding within a wound, the greater the external digestion through larval enzyme secretion, resulting in greater feeding efficiency for each larva. The odours emitted from putrefying tissue are highly attractive to primary facultative myiasis species. Luciliu spp. are attracted to sheep fleece by a variety of factors. Strike can also be associated with a condition called fleece rot, a superficial dermatitis caused by Pseudomonus ueruginosu and a chronic dermatitis (Dermatophilosis) caused by Dermutophilus congolensis, although natural skin flora may also be involved (Ashworthand Wall, 1994).Bacterial infectionsresult in the production of sulphur-containing compounds that are highly attractive to gravid Luciliu spp. Activation, long-distance orientation and landing occur in response to the sulphur rich volatiles, while oviposition is elicited by ammonia-rich compounds (Ashworth and Wall, 1994). A synthetic attractant, LucilureB, developed for L. cuprinu in Australia consists of 49% butyric acid, 42% mercaptoethanol, 9% indole presented in one bottle and 20% Na2Sin water in another (to generate H,S), and has been shown to be effective in the field (Hall, 1995) (Table 10-3). Luciliu spp. respond to oviposition site odours when protein-starved or gravid

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Table 10-3 Olfactory stimuli attractingflyspecies that cause traumatic myiasis in domestic mammals and humans (Table after Hall [1995] with the permission ofthe Liverpool School

of Tropical Medicine] Species and behaviour

Stimuli

Lucilia sericata/ cuprina

Put refact ive, sul p h u r-rich volat iles, (e.g.H,S, ethanethiol,dimethyl disulphide)from: (1)Bacterial decomposition products of cystine component of wool (flystruck sheep > sound wet sheep > sound dry sheep) ( 2 ) Sulphurous decomposition products of carrion (especially when enhanced by Na,S) Oviposition Ammonia-rich compounds ( e g ammonia, ammonium carbonate used as baits)and indole. Cochliomyia hominivorax/ Decomposing I iver; bacteria I ly inoculated blood Chrysomya bezziana "Sworm I u re" Attraction [iso-butyl alcohol, n-butyric acid, n-valeric acid, acetic acid, dimethyl disulphide, phenol, p-cresol, indole, benzoic acid (plus acetone in early formulations)] Attraction + oviposition Wounded host (natural wound fluid), especially if wounds a re infested; blood. Wohlfahrtia magnifica Carrion for feeding,carrion with H,S and Na,S; Attraction abscess and dematitis exudates from sheep Attraction + larviposition wounded hosts for feeding and larviposition (wounded host > sound host). Attraction

(Ashworth and Wall, 1995)and will feed and oviposit on infected hosts. However, necrotic tissue sites on the host are less attractive to obligate parasites like Cochliomyiu hominivorux, Chrysomyu bezziunu and Wohlfuhrtiu mugnificu, which feed but do not oviposit at such sites, preferring to oviposit at fresh wounds. C. hominivorux are attracted to wounds on healthy animals by the odour from the serosanguineous exudate, particularly if already infested with conspecific larvae (Hall, 1995). A synthetic bait for screwworms was developed in the 1970s, and improved some years later (Mackleyand Brown, 1984).This bait, now called Swormlure-4, consists of ten compounds (Table 10-3) identified from decomposing animal wounds. Although routinely used as a bait for traps in the field, it may not yet include the best attractants for these species: sex ratios and ages of flies caught on baited traps differ from those caught on wounded animals with fewer gravid females caught with the synthetic lures (Hall, 1995; Hall et al., 1995).Larval-infested wounds are most attractive to C. horninivorux (Hammack and Holt, 1983)through the volatiles produced by Proteus and Providenciu bacteria (Bromel et al., 1983). Using electrophysiology,Cork (1994)identified 25 non-sulphur-containing compounds that elicited responses from C. hominivorux. Hall (1995)reported certain (unnamed) formulations of these compounds to be more effective than Swormlure-4 in the field. In addition, W. rnugnificu is often found in body openings, particularly the genital orifices of sheep of both sexes. No cues involved in this behaviour have been identified. A summary of the host-derived stimuli involved in the attraction of myiasis-causing flies to their hosts is given in Table 10-3.

Oviposition Repellents

10.4 Oviposition Repellents 10.4.1 Cues from Plants A wide range of plants contains compounds with oviposition deterring or suppressing activity for mosquitoes. The Neem tree, Azadirachta indica (Meliaceae) has been well studied in this respect, and in addition to the larvicidal and ovicidal effects noted by many studies, it also appears to inhibit or suppress oviposition in mosquitoes (Dhar et al., 1996; Mulla and Su, 1999).The water fern Azolla imbricata grows in mats on rice fields and can completely cover the water surface, preventing mosquito larvae from reaching the surface to breathe. Azolla pinnata has been found to inhibit oviposition of both Culex quinquefasciatus and Anopheles culicijacies in the laboratory (Reuben et al., 1990).Similarly, C. pipiens pipiens avoid ovipositing in sites with duckweed, Lemna minor (Lemnaceae),which has larvicidal properties (Eid et al., 1992). The rhizomatous herb lmperata cylindrica (Gramineae) deters oviposition in C. quinquefasciatus (Mohsen et al., 1995).The role of chemical cues in these cases has not been determined.

10.4.2 Predator and Parasite-Associated Cues The effect upon oviposition preference of gravid female mosquitoes and the presence of larvae in the potential oviposition site has been discussed in Section 10.2.1. An additional level of discriminatory ability appears to exist in these mosquitoes. Entomopathogenic digenean Plagiorchis sp. (Trematoda: Plagiorchiidae) are intestinal parasites of birds and mammals that release their eggs in the host’s faeces, ending up in water where the eggs are consumed by and develop within lymnaeid snails (the first intermediate host). Here the parasites emerge as cercariae (an active infective stage) that penetrate the cuticle of various insects (the second intermediate host). The resulting metacercariae (inactive infective stage) in the insect host infect the definitive (vertebrate) host when the insect is ingested. Parasitized Ae. aegypti larvae exhibit significant behavioural changes, increasing their chances of ingestion by a predator (Webber et al., 1987). Gravid Ae. aegypti females avoid sites where such parasitized larvae occur or water in which they have been reared (Lowenberger and Rau, 1994), with the degree of repellency increasingas the intensity of infections increases (Zahiriet al., 199%).The repellency is not species-specific (Zahiri et al., 1997c) and repellent activity of the water is retained even after boiling, antibiotic treatment or filtration (Lowenbergerand Rau, 1994), suggesting a stable semiochemical. These authors suggest that moribund larvae increase their inclusive fitness by reducing oviposition by kin in infected sites, a likely possibility in a mosquito species that does not disperse widely. The repellent effect induced by the stresses of starving, overcrowding or parasitization is similar, and may be associated with depletion of nutrients in all cases (Zahiri et al., 1998). Avoidance of potential oviposition sites in which potential predators or competitors already exist by detecting their presence prior to ovipositionis a highly

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efficient strategy. Ovipositing Musca domestica, houseflies, avoid sites with high densities of the black soldier fly Hermetia illucens (Diptera: Stratiomyidae), a behaviour that may be chemically mediated (Bradley and Sheppard, 1984).Culex longiareolata mosquitoes avoid laying eggs in habitats that harbour nymphs of the dragonfly Anax imperator (Odonata: Aeshnidae) (Stav et al., 1999, 2000), the backswimmer Notonecta maculata (Hemiptera: Notonectidae) (Blaustein, 1998)and green toad tadpoles Bufo viridis (Blaustein and Kotler, 1993).Aedes taeniorhynchus avoid sites containing fish (Ritchie and Laidlaw-Bell, 1994) and Culex spp. avoid sites with notonectids (Chesson, 1984). This may be mediated by chemicals associated with the predatorkompetitor. Petranka and Fakhoury (1991)found that both the mosquito Anopheles punctipennis and the phantom midge Chuoborusalbatus (Diptera: Chaoboridae) avoided ovipositing in pools into which waters from fish or amphibians diffused. While both species avoided the predatory fish, only A. punctipennis avoided the tadpoles; A. punctipennis competes with tadpoles for the same food resources, but C. albatus does not. Interestingly,Stav et al. (2000)reported that while Culex longiareolataavoided sites with free swimmingAnax nymphs, they do not appear to perceive a predation risk when the dragonfly nymphs were caged. In contrast, Torres-Estrada et al. (2001) found that Aedes aegypti females preferred to lay eggs in water that currently or previously contained the copepod Mesocyclops longisetus, (possibly in response to the copepod-derived terpenes in the water) despite the efficiency of the copepod predator.

10.5 The Influence of Experience on Responses to Oviposition Cues Although the ability of many parasitic Hymenoptera or herbivorous insects to improve oviposition site location through experience is well known (Papaj and Lewis, 1993),similar abilitiesin haematophagous insects had not been demonstrated until very recently. Charlwood et al. (1988) found evidence that Anopheles farauti in Papua New Guinea had a memorized "home range" within which it moved efficiently from oviposition sites to bloodmeal sources. Renshaw et al. (1994) reported similar behaviour in European Aedes cantans. McCall et al. (2001) showed that after bloodfeeding, Anopheles arabiensis females flew over 400m to their oviposition sites before returning to the same house in which they had previously successfully obtained the bloodmeal. Visual memory is likely to be important in such behaviour but chemical cues may also have a role. McCall and Eaton (2001) found that Culex quinquefasciatus mosquitoes reared in water containing skatole at a level normally repellent to ovipositing females, preferred to oviposit in water containing that compound rather than in water with an otherwise attractive odour compound. This induced preference was not inherited. Conditioning with other cues remains to be investigated. Experimentally, Reisen (1975) observed that Anopheles stephensi reared at very high densities preferentially avoided water in which larvae had been reared at high densities when selecting oviposition sites, unlike individuals reared at normal densities. Conversely, Hunter and Jain (2000)

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found that members of the Sirnulium venustumlverecundum species complex in North America did not exhibit oviposition or natal site fidelity.

10.6 Responses to Plant Mimics and Plant Allelochemicals Certain plants are highly attractive to a variety of bloodfeeding and carnivorous insects. Both males and females of many bloodfeeding insect species feed on plant sugars for flight energy and although relatively little is known of this activity, many species orient to specificodours and colours associated with preferred plant species. Bowen (1992) identified specific receptors in mosquitoes that responded to plantassociated terpenes, and both anophelines and culicines have long been known to frequent certain plants (see Proctor and Yeo, 1973). Many bloodfeeding and carnivorous insects orient to dark colours when hostseeking, but choose lighter colours when searching for oviposition sites ( e g ,vegetation) or sugar meals (flowers or plants). Examination of the parous condition of flies caught visiting plants would help to determine why they were choosing a particular plant. Although separation of this sugar-feeding habit from other activities on plants is often difficult, certain plants do in fact emit chemical signals that are attractive to gravid saprotrophic flies, that act as pollinators for the plant. Such plants may also mimic the colours that are attractive to gravid host-seeking blowflies. Wiens (1978)considered such sapromyophily to be the best example of chemical mimicry. Females of Luciliu cuprinu visit the flowers of Stupeliu fluvirostris (Asclepiadaceae) (see Ashworth and Wall, 1994), which are coloured red and release a foetid putrefying meat odour. While pollinating the flower, the gravid flies will even lay eggs within the plant and may obtain nectar, but their larvae will not survive. Luciliu, Surcophugu and Culliphorusp. visit and disperse spores of the stinkhorn fungus Phallus irnpudicus, which emits a putrefactive odour (Smith, 1956). This odour blend includes hydrogen sulphide, phenol, 2-phenylethanol, and indole (Stowe, 1988) which are known attractants for blowflies (Ashworth and Wall, 1994; Hall, 1995) and also occur in Cochliornyiu horninivorux larval wound fluids (Cork, 1994). The inflorescences of many Araceae produce volatiles that include skatole, indole, trimethylamine and ammonia (Dafni, 1984).Ammonia and indole stimulate oviposition in Luciliu sp. (Ashworth and Wall, 1994). Production of C 0 2 and putrefaction odours by Arum and Aristolochiu (and the production of heat by Arum to facilitate dissemination of the odours) attract a variety of coprophagous and saprophagous insects but responses of those insect species of interest in this chapter are not known. Whether gravid females orient to the plant mimics to lay eggs is not always clear. A number of orchid species also smell strongly of rotting flesh but the compositions of their odour blends (Kaiser, 1993)do not include compounds known to be attractive to gravid bloodfeeding or carnivorous insects. Interestingly, some species like Druculu chestertonii emit l-octen-3-01 (Kaiser, 1993), a known attractant for many insect species that seek mammal hosts for bloodmeals or oviposition. l-octen-3-01has also been identified in Cochliornyiu horninivorux larval wound fluid (Cork, 1994).

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10.7 Concluding Remarks Whilst many aspects of oviposition behaviour and the untested hypotheses regarding the evolutionary advantages they confer to the species discussed are extremely interesting and certainly warrant further investigation, it is the practical uses these attractants might have that interest most researchers in this field. Odourbaited traps show great promise as surveillance tools for certain pest species. Ovitraps are commonly used for monitoring populations of Aedes, Culex and other mosquitoes of public health importance (Fay and Eliason, 1966; Surgeoner and Helson, 1978; Service, 1993; Rawlins et al., 1998). Many studies have shown that odour (either pheromones or site-derived cues) can enhance mosquito ovitraps to improve their sampling efficiency (Reiter et al., 1991; Otieno et al., 1988; Trexler et al., 1998; Mboera et al., 2000b), and simple modifications may convert a sampling tool into a lethal trap for control purposes (Cheng et al., 1982; Zeichner and Perich, 1999).Screwworm populations can be monitored either through the use of feeding stations baited with meat, or with synthetic odour (Swormlure-4) baited traps (Parker and Welch, 1991,1992;Spradbery, 1993; Hall, 1995).There are good reasons to expect that odour-baited traps may be capable of controlling or suppressing populations of myiasis-causing blowflies (Torr and Hall, 1992; Green et al., 1993; Ashworth and Wall, 1994; Hall, 1995). Visually attractive oviposition traps were used to a limited extent during control of the SimuIium dumnosum complex of onchocerciasis vectors in West Africa (Bellec, 1976) and the possibility of baiting such traps with aggregation pheromone to develop an early-warning surveillance trap for this migratory vector has been proposed (McCall, 1995). Surveillance of haematophagous or carnivorous insects is crucial to detect immigration of potential pest species, to determine population size and distribution, to sample vector species for pathogen prevalences, to provide data for the evaluation of transmission indices and to evaluate progress of control or eradication programmes. Characterization of the odours and other parameters determining oviposition site selection by pests could aid in the development of new sampling devices or improve existing traps. This information might also allow accurate prediction of geographical areas where they are most likely to breed, by identifying vegetation, soil, associated animal species or other local parameters that affect distribution of vector breeding. In combination with improved sampling at ground level, this would improve the accuracy of the remote sensing techniques now being developed for applications in arthropod surveillance and control worldwide.

10.8 Acknowledgements I am grateful to those individuals who provided information, reprints or advice on specific topics within this field: Martin Hall, Monika Hilker, Richard Ward, Mark Klowden, Alan Robinson, John Pickett, and Nigel French. Thanks also to Marcel Hommel who digitally prepared the photographs for Figure 10-1, Lisa Bluett for secretarialassistance and Victoria Warbrick for assistance and tolerance throughout.

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10.9 References Allan, SA, Kline, DL. 1995. Evaluation of organic infusions and synthetic compounds mediating oviposition in Aedes albopictus and Aedes aegypti (Diptera: Culicidae). J. Chem. EcoI. 21: 1847-1860. Allan, SA, Kline, DL. 1998. Larval rearing water and preexisting eggs influence oviposition by Aedes aegypti and Ae. albopictus (Diptera: Culicidae). J. Med. Entomol. 35: 943-947. Ashworth, JR, Wall, R. 1994. Responses of the sheep blowflies Lucilia sericata and L. cuprina to odour and the development of semiochemical baits. Med. Vet. Entomol. 8: 303-309. Ashworth, JR, Wall, R. 1995. Effects of ovarian development and protein deprivation of the activity and locomotor responses of the blowfly, Lucilia sericata to liver odour. Physiol. Entomol. 20: 281- 285. Barton Browne, L, Bartell, RJ, Shorey, HH. 1969. Pheromone mediated behaviour leading to group oviposition in the blowfly Lucilia cuprina. J. Insect Physiol. 15: 1003-1014. Bates, M. 1940. Oviposition experiments with anopheline mosquitoes. Am. J. Trop. Med. 20: 569-583. Beehler, JW, Millar, JG, Mulla, MS. 1993.Synergism between chemical attractants and visual cues influencing oviposition of the mosquito, Culex quinquefasciatus (Diptera: Culicidae). J. Chem. Ecol. 19: 635-644. Beehler, JW, Millar,JG, Mulla, MS. 1994. Field evaluation of synthetic compounds mediating oviposition in Culex mosquitoes (Diptera: Culicidae). J. Chem. Ecol. 20: 281- 291. Beehler, JW, Mulla, MS. 1995. Effects of organic enrichment on temporal distribution and abundance of culicine egg rafts. J. Am. Mosq. Control Assoc. 11: 167-171. Bellec, C. 1976. Captures dadultes de Simulium damnosum Theobald, 1903 (Diptera: Simuliidae) a I'aide de plaques daluminium en Afrique de I'Ouest. Cah. ORSTOM, Ser Entomol. Med. Parasitol. 14: 209- 217. Bentley, MD, Day, JF. 1989.Chemical ecology and behavioral aspects of mosquito oviposition. Ann. Rev. Entomol. 34: 401421. Bentley, MD, McDaniel, IN, Yatagai, M, Lee, H-P, Maynard, R. 1979.p-cresol: an oviposition attractant of Aedes triseriatus. Environ. Entomol. 8: 206-209. Bentley, MD, McDaniel, IN, Yatagai, M, Lee, H-P, Maynard, R. 1981. Oviposition attractants and stimulants of Aedes triseriatus Say (Diptera: Culicidae). Environ. Entomol.. 10: 186189. Blackwell, A, Johnson, SN. 2000. Electrophysiological investigation of larval water and potential oviposition chemo-attractants for Anopheles gambiae S . S . Ann. Trop. Med. Parasitol. 94: 389- 398. Blackwell,A, Mordue (Luntz)AJ, Hansson, BS, Wadhams, LJ, Pickett, JA. 1993.A behavioural and electrophysiological study of oviposition cues for Culex quinquefasciatus. Physiol. Entomol. 18: 343-348. Blaustein, L. 1998. Influence of the predatory backswimmer, Notonecta maculata, on invertebrate community structure. Ecol. Entomol. 23: 246-252. Blaustein, L, Kotler, BP. 1993. Oviposition habitat selection by the mosquito, Culiseta longiareolata: effects of conspecifics, food and green frog tadpoles. Ecol. Entomol. 18: 104-108. Bowen, MF. 1992. Terpene-sensitive receptors in female Culex pipiens mosquitoes electrophysiology and behavior. J. Insect Physiol. 38: 759- 764. Bradley, SW, Sheppard, DC. 1984. House fly oviposition by larvae of Hermetia illucens, the black soldier fly. J. Chem. Ecol. 10: 853- 859. Bromel, M, Duh, FM, Erdmann, GR, Hammack, L, Gassner, G. 1983. Bacteria associated with the screwworm fly Cochliomyia hominivorux (Coquerel) and their metabolites. Endocytobiol. 2: 791- 800. Bruno, DW, Laurence, BR. 1979. The influence of the apical droplet of Culex egg rafts on oviposition of Culex pipiens fatigans (Diptera: Culicidae). J. Med. Entomol. 6: 300-305. Chadee, DD, Corbet, PS, Greenwood, JJD. 1990. Egg-laying yellow fever mosquitoes avoid sites containing eggs laid by themselves or by conspecifics. Entomol. Exp. Appl. 57: 29.5298. Charlwood, JD, Graves, I'M, de C. Marshall, TF. 1988. Evidence for a "memorized home

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Chapter 11 Chemoecology of Parasitoid and Predator Oviposition Behaviour Johannes L. M. Steidle and Joop J. A. van Loon

Table of Contents 11.1 Introduction 11.1.1 Parasitoids 11.1.1.1Habitat Location

11.1.1.2 Host Location 11.1.1.3 Host Recognition 11.1.1.4 Host Acceptance 11.1.2 Predators: Oviposition Site Location and Acceptance 11.2 Theories on the Use of lnfochemicals for Location of Oviposition Sites by Natural Enemies 11.2.1 The Reliability-Detectability Problem 11.2.2 The Variable Response Model 11.2.3 Dietary Specialization and lnfochemical Use in Natural Enemies 11.2.4 lnfochemical Use in Koinobiont and ldiobiont Larval Parasitoids 11.2.5 Plant-Entomophage Mutualism 11.3 Concluding Remarks 11.4 Acknowledgements 11.5 References

Abstract

Host foraging by insect parasitoids consists of several behavioural phases that mostly end up with oviposition in a host. These phases have been termed host habitat location, host location, host recognition, and host acceptance. In contrast, many adult predators forage for prey either to acquire nutrition or to oviposit in the prey’s vicinity, preventing a clear distinction of different phases in oviposition behaviour. In parasitoids and predators foraging is often guided by chemical cues, the origin of which has been examined for many parasitoid species but only for few predator species. The cues may originate from the hosvprey, from the food plants of the host/prey, from con- or heterospecifics or from associated organisms. The exact identity of the cues has been established in a few dozen cases so far. Several theories have been proposed aiming to explain the use of cues (1) originating from either the host/prey or its food plant; (2)of either general or specificoccurrence;

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(3) of either innate or learned nature. Important common concepts in these theories refer to the reliability and detectability of the cues, the constraints in the specificity of information transmitted via these cues and the relationship between the degree of dietary specialization of the entomophagous insect and characteristics of the cues used. We evaluate these theories in view of the current literature.

11.1 Introduction In this chapter, we discuss the chemoecology of oviposition in parasitoids and predators, which share their carnivorous feeding habits but differ in many other respects. Both are obligatory biotrophs and are largely dependent on living arthropod tissues to retrieve nutrients. Parasitoids are parasitic insects that in the majority of cases oviposit in or on so-called host organisms, which eventually will die as a result of this. Predators feed on their prey organisms and thereby kill them soon after feeding has commenced and do not oviposit in or on their prey, but often in the prey’s vicinity. Parasitoid larvae hatch and feed in or on a host whereas predator eggs may hatch remote from prey. The larval stage of parasitoids typically is the carnivorous life stage. Adults generally limit feeding to plant-derived sugar sources such as (extra)floralnectar or honeydew, although host feeding,i.e. feeding of haemolymph, could also be observed for quite a few, mostly idiobiont species (Quicke, 1997). Conversely, only early larval instars of predators with piercingsucking mouthparts may limit their feeding to plant saps whereas later larval instars and adults are carnivorous, but several predatory species can temporarily switch to plant-derived liquid food or pollen during times when arthropod food is not available (van Rijn and Tanigoshi, 1999; Lemos et al., 2001).Thus, host foraging in parasitoids mostly ends up with oviposition in a host, whereas prey foraging by many adult predators may or may not result in oviposition. The elaborate behavioural sequence leading to the acceptance of a host for ovipositionin parasitoids is a prominent feature of their biology and has most likely been a key factor for the impressive diversification this insect group displays (Quicke, 1997). In contrast, detailed sequences have not been documented for predators, neither for the location of oviposition sites nor for prey finding. Both parasitoids and predators often use chemical cues during the search for oviposition sites and several theories have been proposed with respect to the origin of these cues, their general or specific occurrence and their innate or learned nature. This chapter focuses on interspecifically relevant chemical cues (allelochemicals) that affect host foraging and ovipositon of predators and parasitoids, i.e. natural enemies that kill their hosts or prey species. The role of pheromones, i.e. intraspecifically acting infochemicals in oviposition of natural enemies is covered by Anderson (Chapter 9). The chemoecology of oviposition behaviour of parasitic insects, i.e. insects that do not kill their host, is reviewed by McCall (Chapter 10).

Introduction

Our chapter also considers the behavioural mechanisms that are activated by the allelochemicals mentioned, and will evaluate the respective theories in view of the current literature for both, parasitoids and predators. Although some of these theories have been originally proposed for parasitoids only, we will discuss them for predators as well, in some cases referring to data on prey foraging. We hypothesize that similar selective forces should work on oviposition and prey foraging behaviour of predators (see below). Probably the most important differences are that (1)predators generally are more polyphagous than parasitoids and that (2) much more is known about parasitoids.

11.1.1 Parasitoids A considerable amount of information has accumulated on cues utilized by parasitoids during foraging for hosts. Several reviews have succinctly summarized this body of literature, which is still growing (Vinson, 1976,1981,1985,1991,1998; Weseloh, 1981; Vet and Dicke, 1992; Turlings et al., 1993; Godfray, 1994; Vet et al., 1995; Powell, 1999).The sequence of events leading to successful parasitism has been subdivided into a number of behavioural phases, largely for reasons of conceptual clarity: (host) habitat location, host location, host recognition, host acceptance and host suitability (e.g. Vinson, 1976,1998). Host searchingmay start immediatelyupon eclosion or, in the case of synovigenic species, only after a preoviposition period has passed. During searching, female parasitoids which have not yet encountered a host respond to those chemical cues to which they have genetically programmed chemosensory sensitivity. On the eclosion site itself, or in its vicinity, females may perceive chemical cues derived from the host which they exploited as a larva. Such chemosensory experiences early in adult life may cause "imprinting" of the female, which may affect subsequent sensitivityto cues that elicit innate responses and may affect preference for larval hosts (HQard et al., 1988; Turlings et al., 1993). According to present knowledge, host experience of the parasitoid larva has no impact on the behaviour of the adult parasitoid (van Emden et al., 1996). Parasitoid females are known to use different sensory modalities during the process of host selectionbehaviour (Figure11-1).During host habitat location,visual and olfactory cues are used to generate a taxis or kinesis type of oriented locomotory responses. Thus, the parasitoid achieves contact with the substrate in or on which the host is located, e.g. a leaf in the case of parasitoids attacking folivorous insect herbivores. Now the full range of sensory modalities (visual, mechanosensory, olfactory and gustatory) is most likely utilized in attempts to locate the host. Once the host has been located by contact, mechanosensory and in particular gustatory cues are important for host recognition. In many cases, a potential host will try to escape or perform defensive movements (Gross, 1993) and is often seen to emit defensive exudates. Host acceptance, which expresses itself as the actual deposition of one or more eggs, is triggered by gustatory cues in particular (Weseloh, 1981; Vinson, 1991, 1998). Finally, host suitability refers to physiological interactions

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Figure 11-1Generalized sequence of host and prey searching and selection behaviour of parasitoids and predators. Left column: behavioural phase or event, boxes indicate transition between two phases. Middle column: common behavioural elements occurring within a behavioural phase. Right column: sensory modality by which the major stimuli affectingthe corresponding behaviour (middle column) are perceived is indicated by filled circles. Open circle: suggested or probable.. Prey contact is not necessary in all predator species for oviposition t o occur.

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between the immature parasitoid stages and the host and occurs only after the decision to oviposit has been made by the adult parasitoid.Therefore host suitability is not within the scope of this chapter. 11.1.1.1Habitat location

Upon eclosion the female parasitoid emerges in a certain habitat, which is often identical to the hosts’ habitat. Several abiotic environmental factors are assumed to affect the behavioural decision to either stay in or leave the habitat in which the adult emerged. These include light intensity, temperature, humidity, and wind speed (Vinson, 1991).Empirical studies so far are available only for humidity (e.g. Geden, 1999).Biotic factors such as the availability of food sources, e.g. (extra)floral nectar or honeydew, and refuges as provided by structural characteristics of the vegetation promote staying in the eclosion habitat. The presence of intra- and interspecific competitors or chemical oviposition marks released by them (Kainoh, 1999; see also Chapter 9 and 12) as well as predators may promote dispersal. During habitat location parasitoids may also use volatile attractants and arrestants (reviewed by Weseloh, 1981; Vinson, 1991).These cues are produced by the (micro-)habitat of the host. Often they are plant-derived, or more generally food-derived cues and are not originating directly from the host. Microorganisms have been shown to play a role in the production of odours (Thibout et al., 1993) (compare Chapter 6). It has been argued that in the initial phases of foraging, parasitoids respond to host-associated cues rather than to cues actually produced by the host itself (Vinson, 1991). It is assumed that oriented responses towards these cues can occur at a distance of at least several meters (Vinson, 1998),although few explicit attempts have been made to quantify their action radius. At a greater distance searching is most likely random. Rutledge (1996)listed 28 systems for which chemicals used for habitat location had been identified in olfactometersor wind-tunnels. However, attraction to single compounds, although demonstrating that a compound has a kairomonal effect, gives only preliminary information on the role of such a compound as part of the complex blend actually encountered under natural foraging conditions. Many species of parasitoids exploit larval plant-feeding insects as hosts. Volatile blends released by plants not infested by herbivores generally comprise several dozens of volatile compounds, which are emitted in small amounts (Visser,1986; Knudsen et al., 1993; Schoonhoven et al., 1998). An additional level of complexity is added because plant volatile blend changes as a result of herbivore feeding damage, both qualitatively and quantitatively (Turlings et al., 1995; Dicke, 1999a). Volatile blends collected from herbivoredamaged plants are commonly composed of cu. 20 to over 200 compounds ( e g Turlingset al., 1995; Dicke, 1999a;van Loon and Dicke, 2001).Due to the complexity, the minimally effective volatile blend is known only for two parasitoid wasp species [Cotesiurnurginiventris (Hymenoptera:Braconidae)and Aphidius emi (Hymenoptera: Aphidiidae)],although it has not been resolved whether all components are actually

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utilized (Turlings et al., 1991; Du et al., 1998).We will address the phenomenon of herbivore-induced volatile production more in detail in Section 11.2.1 and 11.2.5. The intraspecific sexual chemical communication system of many host insects employs readily volatilized compounds known as sex pheromones, which provide direct information for the parasitoid on the presence of the adult host. Host sex pheromones and also aggregation pheromones have been shown to act as kairomones for several species of egg parasitoids (reviewed by Powell, 1999) and larval parasitoids (Wiskerke et al., 1993; Wertheim et al., submitted). 11.1.1.2 Host Location

Within the habitat in which eclosion took place, or after having reached the hosts’ habitat, the female will generally have to search over at least some distance for hosts (= oviposition sites) within that habitat. The female may use volatile chemical cues during her search for a host (Figure 11-1).To what extent such volatiles provide directional information strongly depends on the complexity of the environment. For parasitoids of larval herbivorous insects, vegetational diversity is the major factor affecting complexity of volatile chemical blends (Sheehan, 1986).Diversity of herbivorous insects, hosts and non-hosts, on the same plant may also affect parasitoid foraging to the extent that at a distance volatile information is indistinct and time is spent landing and searching on leaves where no suitable hosts are present (Vos et al., 2001). Once arrived in the vicinity of the host, e.g. after landing on the host’s substrate, additional volatile cues with only moderate volatility as well as taste cues may become available that act as searching stimulants. In many species the female parasitoid in this phase brings her antennae in contact with the substrate (antennation) while walking around. She may also alternate bouts of walking with bouts of antennation while standing still and showing ortho- and klinokinetic responses ( e g Vet and Papaj 1992; Khandakar and Jahan, 1993),which effectively retain the female in an area or patch (area-restricted search). Both volatile and non-volatile chemicalsare perceived by olfactory and gustatory receptors, respectively, present on the antennae. These cues involved in host location behaviour may be derived from the host’s food (Horikoshi et al., 1997; TakAcs et al., 1997)and from the host itself, such as the hosts’ silk (Mattiacciand Dicke, 1995a; van Baarlen et al., 1996),oral secretions (e.g. Corbet, 1971), chemical trails (e.g. Howard and Flinn, 1990), faeces ( e g Meiners and Hilker, 1997; Steidle and Scholler, 1997), honeydew (e.g. Grasswitz, 1998), defensive substances (Mattiacciet al., 1993; Al Abassi et al., 2001), and pheromones (Powell, 1999).However, only in a few cases have the molecular structures been identified. Until 1994, cues had been identified for 12 systems (Rutledge, 1996). More recent examples are provided by Glinwood et al. (1999), Dutton et al. (2000), Hilker et al. (ZOOO), Kumazaki et al. (ZOOO), and Ruther and Steidle (2000). 11.1.1.3 Host Recognition

Area-restricted search may at some point bring the female parasitoid physically into contact with a host or its covering, e.g. the host cocoon, a gall, or a seed in

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Figure 11-2Host recognition and host acceptance behaviours preceding oviposition by three species of parasitoids: Cotesia glomerata (le Masurier, 1990; Steidle, pers. obs.), Lariophagus distinguendus (Steidle, 2000; Steidle, unpubl.), and Oomyzus galerucivorus (Meiners et al., 1997).

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internal seed feeding hosts. Now the parasitoid has to recognize the host species. In this phase, physical cues (reviewed by Schmidt, 1991),gustatory cues (Ohara et al., 1996; Hare et al., 1997; Rostas et al., 1998; Steidle and Ruther, 2000), or a combination of both may have a major impact. These cues either originate from the host itself, from its covering, or from host by-products like silkand faeces. They are perceived with the antennae drumming (antennation, drumming) on the surface, the abdominal tip (tapping) and/or the ovipositor, which is equipped with mechanosensory and contact-chemosensory receptors, probing and drilling into the potential host or its covering (Quicke, 1997).After the decision to accept the host has been made, the parasitoid may either proceed with oviposition on the outside (ectoparasitoids)or inside the host (endoparasitoids) (Figure 11-2).

11.1.1.4 Host Acceptance Acceptance,expressed as the actual deposition of an egg, may be based on chemical cues from the hosts’ products, its cuticle or may ensue only after drilling with the ovipositor and evaluating the hosts’ internal chemical composition, mostly that of the haemolymph (e.g.Cooperband and Vinson, 2000).For ectoparasitoidsit is likely that cues from the haemolymph are perceived either during stinging the host for paralysis or during host feeding, both occurring prior to oviposition (Steidle, unpublished). Rutledge (1996) listed 16 systems for which chemicals eliciting drilling or ovipositionhad been identified until 1994.These cues are predominantly alkanes, amino acids or derivativesand (as yet incompletelycharacterized)proteins, fatty acid and cholesterol derivatives and high concentrations of inorganic ions. Host discrimination, i.e. the rejection of an already parasitized host, is achieved by using external or internal marking pheromones that have been applied on the hosts during previous ovipositions.Accordingly,discrimination occurs at the level of host recognition or host acceptance. In a recent review, Kainoh (1999) presents a list of parasitoid species for which the presence of marking pheromones has been demonstrated. Anderson (Chapter 9) gives some more recent examples.

11.1.2 Predators: Oviposition Site location and Acceptance Theoretically, the behavioural sequence leading to oviposition in parasitoids also applies to predators. However, when predators are attracted to prey it is unclear whether they are searching for prey or for oviposition sites. Behavioursbelonging to ovipositionsite location can only be identified when oviposition is also observed, making the distinction between cues for oviposition site location and oviposition site acceptance difficult at present. Therefore, no distinction is made here between different phases leading to oviposition by predators. Compared to the body of information on parasitoids, much less information is available on those chemical cues that guide predators during location and acceptance of oviposition sites. The fact that some predators prefer specific plant species for oviposition (Ballal and Singh, 1999)or specific parts of plants (Sanford, 1964; Richards and Harper, 1978; Richards and Schmidt, 1996) indicates that

Introduction

chemical cues might be involved. For the mind bug Mucrolophus culiginosus, it has been demonstrated that more eggs are laid on substrate treated with plant extract as compared to moist substrate (Constant et al., 1996). Most is known about predator taxa that predominantly prey on aphids and other Homoptera: Syrphidae (Diptera), Chrysopidae (Neuroptera), and Coccinellidae (Coleoptera). For all of these specialist and generalist predators, honeydew is generally an oviposition stimulant. Sadeghi and Gilbert (2000) summarized oviposition behaviour of 14 species of Syrphidae. Several species respond with oviposition to volatiles of aphid-produced honeydew alone, whereas in others, the aphid prey themselves are needed for oviposition to occur. Aphid cornicle secretions were implied as providing necessary gustatory cues for oviposition in Eupeodes (Metusyrphus)corollue and Ischiodon scutelluris. In several species, more eggs were laid in the vicinity of higher aphid densities, e.g. larger colony sizes. The syrphids M . corollue and E. bulteutus obviously adjust the number of eggs deposited to the number of aphids present based on chemical host cues (Shonouda, 1996; Scholz and Poehling, 2000). However, the impact of physical cues cannot be excluded. Specificityin selectionbehaviour, seen as preference for oviposition, was found in several species, e.g. in the marmalade hoverfly Episyrphus bulteatus. In Table 11-1, additional references on studies investigating chemical cues guiding Table 11-1 Chemical oviposition cues used by predators

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Episyrphus balteatus specialist on (Syrphidae) Acyrthosiphon pisum

aphid extracts Bargen et al., 1998 (HzO, pentane, methanol) and honeydew aphids and Scholz and Poehling,

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tryptophan attracted by indole acetaldehyde and other breakdown products of tryptophan aphid odour and/or honeydew

Hagen et al., 1976 van Emden and Hagen,

leaf extract (ethanol) of lnula viscosa water-solublecues from conspecific and heterospecific eggs fresh snail faeces

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1976

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(Phytoseiidae) Pherbellia cinerella

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Coupland, 1996

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oviposition of syrphids as well as of other predator groups have been summarized. In only few cases has the identity of the chemical cues stimulating oviposition of predators been established. For the neuropteran Chrysopa carnea, breakdown products of the amino acid tryptophan, especially indole acetaldehyde, have been identified as the key stimulant in honeydew (van Emden and Hagen, 1976).Other amino acids were not active. A mixture of long chain hydrocarbons (C23-C28) of limited volatility were shown to be preferred for oviposition by the syrphid Metasyrphus corollae (Shonouda et al., 1998). In the specialist predatory hoverfly Parasyrphus melanderi, salicylaldehyde is a strong oviposition stimulant. Salicylaldehyde is a defensive compound of larvae of the leaf beetle Chrysornela aeneicollis, the prey of P.rnelanderi (Rank and Smiley, 1994). Also only very little detailed information is available on the use of chemical cues for the rejection of oviposition sites. First evidence has been obtained recently demonstrating that, similarly to parasitoids, predators use oviposition deterrent pheromones to avoid competition. Chapter 9 summarizes studies on oviposition deterring pheromones in herbivorous and carnivorous insects. For example, the C25 hydrocarbon n-pentacosane, present in the tracks of conspecific larvae, was identified as the major compound of a mixture of alkanes exerting an oviposition deterrent effect on the two spot ladybird beetle, Adalia bipunctata, (Hemptinne et al., 2001). Other examples of oviposition deterrency in response to con- or heterospecific eggs have been documented for predators (e.g. Faraji et al., 2000; Scholz and Poehling, 2000), but the active chemicals have not yet been identified.

11.2 Theories on the Use of lnfochemicals for Location of Oviposition Sites by Natural Enemies The facts presented so far demonstrate that the behaviour of natural enemies during the search for oviposition sites is strongly affected by chemical cues. To identify general rules, hypotheses have been proposed that predict whether the chemical cues used (1)originate from either the host/prey or its environment; (2)are of either general or specific occurrence; (3) stimulate either innate or learned reactions (Vet et al., 1990; Vet et al., 1991; Vet and Dicke, 1992; Vet et al., 1995; Vinson, 1998; Dicke, 1999a).Even though these concepts are frequently cited in empirical studies, only few of those are explicitly designed to test them. Furthermore, most studies deal with parasitoids and only few with predators. Thus, to stimulate more hypothesis-driven research in the field of infochemical use by natural enemies, the most influential theories will be presented and discussed here. To counterbalance the existing experimental bias towards parasitoids, in some cases data on prey location by predators have also been included. Regardless if searched by parasitoids for oviposition or by predators for feeding or oviposition, selection pressure on the host or prey organism to remain undetected is strong, which will have similar consequences for the availability of cues and both parasitoids and predators have to deal with a variety of different cues in either

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case. Therefore, theories originally developed for parasitoids are expected to be valid for predators as well and therefore are discussed below for natural enemies in general.

11.2.1 The Reliability-Detectability Problem As stated above, the majority of parasitoids display a high degree of specialization in host selection, whereas this is not observed to quite the same extent in prey selection by predators. In a seminal review, Vet and coworkers (Vet et al., 1991; Vet and Dicke, 1992) (see below) provided a conceptual framework for understanding the ecology and evolution of infochemicaluse by explicitly invoking the multitrophic context in which parasitoid-host and predator-prey interactions evolved. Since then, many authors have referred to this review to put their results in perspective. Central in their reasoning is the reliability-detectability problem faced by carnivores when searching for herbivores. Although cues provided by the herbivore itself (second trophic level) are reliable indicators of herbivore presence, their detectability is low due to their small biomass relative to that of the plants they are feeding on, especially at larger distances (see Section 11.1.1.1). Conversely, plant-produced volatile infochemicals(firsttrophic level)are supposed to be available in larger quantities, especially when plants are damaged by herbivores, and thereby better detectable, but do not necessarily convey reliable information. Low detectabilityof hosvprey-derived stimuliis hypothesized to impose a major constraint on the evolution of host location capacity and searching efficiency of carnivores. Vet and Dicke (1992)discussthree solutions to overcome this constraint. (1)One solution for egg and larval parasitoids is to use infochemicals produced from the adult stage (sex, aggregation or oviposition deterring pheromones) to locate eggs or larvae (see Section 11.1.1.1). (2) A second solution is to use volatile infochemicals derived from the hosts’ food, especially when this is a living plant. As mentioned above, feeding by herbivorous hosts results in a considerable increase in the amounts of volatiles released by the host plant as well as to quantitative and in several cases qualitative changes in blend composition (Turlings et al., 1995; Dicke, 1999a). Such blends have been termed herbivore-inducedsynomones (HIS).Many cases have been documented in which parasitoids and predators have been shown to discriminate between volatile blends released by intact plants and plants damaged by herbivores, and HIS are to be considered as a frequently used solution to the reliability-detectability problem (reviewed by Dicke, 1999b). Discussion about the general significance of the reliability-detectability paradigm has later focussed on the degree of specificity conveyed by information derived from the first trophic level (Dicke, 1999c; Vet, 1999; Dicke and van Loon, 2000) thereby linking the concepts reliability and specificity. For several tritrophic systems,parasitoids and predators are known to discriminate between volatile blends released by plants damaged by closely related

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herbivores or between early and late instars of the same herbivore species, either innately or after experience (reviewedby Dicke, 1999a,c; Vet, 1999).The chemical basis for this discrimination capacity has been notoriously difficult to pinpoint at the level of individual blend compounds (see Section 11.1.1.1). (3) A third solution that has been proposed for the reliability-detectability problem is to associatively link detectable cues with reliable, host-derived cues through associative learning. Once a host has been encountered and successful oviposition has resulted in parasitization of the host, host habitat preference may change as a result of associative learning. The capacity for associative learning is known to be particularly well developed in parasitoids belonging to widely different taxa (reviewed by Vet et al., 1995) and has been shown to improve the discriminationcapacity of female parasitoids (Vinsonand Williams, 1991; Vet et al., 1998). In contrast to the substantial body of information on learning in parasitoids, few studies addressed learning in predatory arthropods ( e g Dicke et al, 1990; Drukker et al., 2000) and these dealt with prey foraging rather than oviposition behaviour. The reliability-detectability paradigm assumes that reliability and detectability of cues are not compatible with each other. Vinson (1998) has argued that this is not necessarily true. In those cases in which parasitoids utilize sex pheromones as host location cues, a positive correlation exists: the most reliable cues are also the most detectable ones and vice versa. Host location guided by any infochemicals that are crucial to the sexual communication system of the host or prey is predicted to be a stable strategy, as the host is likely under strong selection pressure not to change the chemistry of its sexual communication. Vet and Dicke (1992)agree on this, but it must be considered that the detectability will be low in another way, i.e. in time because pheromone emission will be temporally restricted so as to avoid the costs of eavesdropping enemies.The chemicaldetour and herbivore-inducedsynomones is another case for which reliability and detectability do not need to be in conflict. For several cases in which plant-produced volatile synomones are induced by herbivore feeding, a specific and thus reliable message on the herbivore species present is transmitted (Dicke, 1999c)- still this specificity seems to be less clear than for pheromones according to current data. The reliability-detectability paradigm has been quite influential in parasitoid and predator chemoecology. It has provided a conceptual framework for understanding the evolutionary forces shaping the chemosensory-basedsearching strategies of carnivorous insects and the phenotypic plasticity that is observed in these strategies. Many authors have referred to the paradigm without explicitly testing the predictions resulting from it. Quantifying the detectability of cues can be achieved in relatively straightforward bioassays, quantifying reliability is more complicated when knowledge about the nature of those chemical cues that are actually utilized for host or prey acceptance is lacking, which is the case in most studies.

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11.2.2 The Variable Response Model The variable response model was developed by Vet et al. (1990,1995)to address the response plasticity of parasitoids towards chemical cues. Central to the model is the idea that each chemical stimulus perceivable by a parasitoid has its own specific response potential. Depending on the behavioural response elicited, different parameters and concomitant units are used for quantification, e.g. for arresting chemicals as allocation time in the odour field of an olfactometer or as walking speed for the response to volatile stimuli eliciting orientation. According to their response potential all stimuli can be ranked from stimuli with high response potential to stimuliwith low response potential (Figure11-3).The response potential is not fixed but variable to a certain degree, depending on the parasitoids experience, its physiological state, and its genotype. Several predictions result from this general framework, the most important ones being presented below. So far, few studies have explicitly tested these predictions but evidence for or against some of the predictions can nevertheless be found in the literature. Major ideas of this model are: (1) Chemical cues can be ranked according to their response potential

A ranking order of stimuli is frequently found in parasitoid studies (e.g. Potting et al., 1995;Du et al., 1996;Steidle and Scholler, 1997;Steidle,2000). This phenomenon is not restricted to parasitoids but is also found in other insects. For example, when honeybees are confronted with mixtures of different syntheticvolatiles, a hierarchy among the components of the mixture can be established and some compounds

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Figure 11-3Variable response model. After Vet et al. (1990).

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are more easily learned than others (Laloi et al., 1999). As suggested by Bernays and Wcislo (1994), the ultimate reason for the ranking might be to improve information processing in a complex environment by focussing on some stimuli only. (2) Stimuli with high innate response potential are more reliable to indicate host or prey presence than stimuli with low potential As discussed above for the reliability-detectability concept, stimuli can be assumed to differ with respect to their reliability. It is reasonable to assume that the most reliable stimuli have the highest response potential. These stimuli should be most important for foraging and a strong innate response towards them should be favoured by natural selection. It is assumed that host-derived stimuli have a high response potential because they are directly associated with the presence of a host. Plant cues, on the other hand, are believed to have low reliability and therefore have only low innate response potential. To test this idea, it would be necessary not only to assess the response potential of chemical cues but also to estimate the reliability of cues associated with a host. As mentioned above, this is more complicated as it requires detailed information on the hosts’ biology. For example faeces, one of the most common host-derived cues, are reliable only as long as the hosts are in their vicinity. To our knowledge, there are no studies available that actually measured reliability. Therefore, the idea that stimuli with high innate response potential are more reliable than stimuli with low potential remains untested so far.

(3) The variability of the response to a stimulus depends on its response potential It is assumed that the variability of the response between individual parasitoids of the same species is inversely related to its response potential, i.e. the variability is low towards stimuli with a high response potential and high towards stimuli with a low response potential. This assumption is supported by many studies. It is a common phenomenon that parasitoids are less likely to be distracted in experiments (e.g. by the observer) from strong stimuli than from weak ones. Additional to the confirmingstudies cited by Vet et al. (1990,1995),data from several other studies examining the response of parasitoids towards chemical stimuli have been collected to test this prediction (Figure 11-4). Data were analysed for a correlation between response potential, expressed as mean response, and variability, expressed as coefficientof variation (Sokaland Rohlf, 1981).In agreement with the prediction, the Spearman’s rank correlation was negative, although not always significant, in all 9 studies. (4) Experience can change the response potential of stimuli A changing response towards stimuli is an inevitable consequence of associative learning (for definitions on learning see Vet et al., 1995). Thereby learning can increase the response towards an experienced stimulus by positive associative learning or sensitizationbut it also can decreasethe response by negative associative learning or habituation (Eisenstein and Reep, 1989).However, this change does

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Mean Response Figure 11-4Correlation between response potential and variability of the response towards chemical cues. For 9 studies taken from the literature, the response potential of chemical cues (meanof response)was analysed for significant correlation (p = level of probability)with the variability of the response (coefficient of variation; Sokal a n d Rohlf, 1981). 1 (0) Agelopoulosand Keller(1994):p=0.000;2(O)-Borgesetal.(1999):p=0.334;3(A)-Mattiacci and Dicke (1995a): p = 0.024; 4 (A)- Cortesero et al. (1995): p = 0.072; 5 (0) - Steidle and Scholler(1997):p=0.049; 6(H)-Steidleet al.(2001b):p=O.O23;7(O)-Steidleet aL(2001a): p = 0.000; 8 (+) - Meiners and Hilker (1997):p = 0.005; 9 (V)-Meiners and Hilker (2000): p = 0.025. Numbers at the beginning and end of each graph indicate the number of the study it belongs to.

not refer only to the learned stimulus. When stimuli are arranged in a ranking order, changes in the response potential of one specific stimulus also changes the ranking of the response to other stimuli. The increase of response potential towards one stimulus (or one set of stimuli),in comparison to others, becomes obvious when parasitoids learn to prefer stimuli from one host complex over stimuli from other complexes. Learning of preferences has been found many times in parasitoids (e.g. Bjorksten and Hoffmann 1998; Geervliet et al., 1998; Steidle et al., 2001a). Oviposition experience with one host complex reinforces the reaction to stimuli from this host complex and causes a preference over stimuli from other complexes. In conclusion, the variable response model seems to be a useful tool to predict the variability of responses towards chemical cues depending on experience in parasitoids. Apart from the idea that stimuli with high response potential are more reliable than low response stimuli, all predictions seem to be well supported by empirical data. However, studies are still needed to examine whether the predictions hold not only for parasitoids but also for predators, for stimuli associated with both oviposition sites and prey.

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11.2.3 Dietary Specializationand lnfochemical Use in Natural Enemies This theory was published by Vet and Dicke (1992)and has received wide attention since then. It predicts that the origin of infochemicals eliciting an innate response as compared to that of infochemicals that elicit a learned response by a foraging carnivore will depend on the dietary specializationof the carnivore and that of its hodprey. According to the degree of specialization, Vet and Dicke distinguish between the following systems with increasing generalism: Systems with specialist carnivores of specialist hosts/prey that feed on one plant species only. For these natural enemies the correct decision to oviposit into or next to specific hosts is crucial for the offsprings’ survival, probably even more so than for specialist herbivores where hatched larvae might be able to find the correct host plant themselves. Systems of carnivores with several specialist host/prey species that all feed on the same host plant. Systems of carnivores with one hodprey species feeding on several host plants. Generalist systems with many host/prey species all feeding on several host plants. These categories represent extremes and the authors acknowledge that many systems will be intermediates. Several predictions on infochemical use are made for these systems, some of which are presented below and evaluated in view of the current literature. Especially the first two predictions have been explicitly tested in empirical studies. (1) lnnate use of specific cues by specialists and innate use of general cues by generalists Upon emergence, natural enemies have no foraging experience yet and therefore have to rely exclusivelyon cues that are innately attractive or cues that are acquired during development, most likely by imprinting before or shortly after emergence of the adult (e.g. van Emden et al., 1996). For specialist natural enemies, it is assumed that innately attractive cues are specific for the respective host/prey species or their host plants. Generalists, on the other hand, should innately respond to general cues that are associated with all potential host/prey species. The innate use of specific cues by generalists is considered unlikely because of physiological constraints caused by the diversity of specific cues expected from several hosts or prey species (Vet and Dicke, 1992).It is conceivable that these constraints exist (a) on the level of the chemosensory system,because only a limited number of specific chemoreceptors sensitive to specific infochemicals can be present, andor (b) on the level of information processed in the central nervous system (e.g.Dukas, 1998a). Some studies have been conducted to test the assumption that the specificityof behaviourally active chemical cues corresponds to the host range by comparing the behaviour of related natural enemies that differ in host range. These studies revealed that parasitoids with a broader host range seem to use more general cues than more specialized parasitoids (Hedlund et al., 1996; Vet et al., 1993; Rose et al., 1998; Bruni et al., 2000). However, in other studies, no or only minor differences were found (Geervliet et al., 1996; Cortesero et al., 1997).In electrophysiological

Theories on the Use of Infochemicalsfor Location of Oviposition Sites bv Natural Enemies

studies with olfactory antenna1 receptors of predatory bugs, a slightly higher sensitivity was found towards prey-induced plant compounds for the specialist Perillus bioculatus than for the generalist Podisus maculiventris (Dickens,1999).Thus, the studies available do not allow a general conclusion. This might be due to the circumstance that for the species compared the differences in host range were too small. Almost none of the generalists tested conformed to the definition used for herbivores (e.g. Bernays and Chapman 1994), i.e. that generalists feed on hosts from different families. In most cases, hosts were closely related and belonging to one family only. Alternatively to this comparative approach, the predictions mentioned above on the innate use of cues by generalists and specialists can be tested by evaluating studies where the response of single specialist or generalist species towards general or specificcues has been examined.To categorize chemicalcues as general or specific in this case, the relevant cues have to be identified and information on their general presence in other potential hostlprey systems is required. Furthermore, the experience of the test organisms must be controlled to ascertain that the reaction was innate. Although many studies do not meet these conditions, the innate use of specific foraging stimuli, both from the preyhost and from its food plant for specialist carnivores, is well established in the literature (e.g. Coupland, 1996; Colazza et al., 1997; Shonouda et al., 1998; Meiners et al., 2000). Likewise,some studies demonstrate for generalistparasitoidsan innate response to general cues common to several host systems (e.g. Nemoto et al., 1987; Auger et al., 1989; Steidle et al., 2001a), thus also supporting the hypothesis. In contrast to the prediction, however, an innate reaction to specific hostlprey cues by generalists was also found in some studies (Noldus et al., 1991; Teerling et al., 1993; Steidle et al., 2001c; Reddy et al., 2002). These innate responses could be the "ghost of monophagy", i.e. behavioural relicts from monophagous ancestors of the extant species, which evolved into generalists. Alternatively, it cannot be excluded that the physiological constraints mentioned above might be less severe and that at least generalists with a smaller host range are in fact able to innately use specific information from different preyhost species. Thus, the innate use of specific cues by specialists and the innate use of general cues by generalists seem to be well supported by empirical studies. However, it is presently unclear whether the innate use of specific cues by generalists is a more general phenomenon or whether it occurs only in some generalists where it is inherited from monophagous ancestors. To differentiate between these two potential explanations, it would be important to know if generalists innately react to specific cues from one or from several hostlprey species. (2) Learning of infochemicals is expected in generalist carnivores and not in specialists This prediction is based on the widely accepted idea that for animals in general behavioural plasticity due to learning is advantageous for speciesliving in a variable environment whereas fixed behaviour is favoured under conditions of no environmental variation (e.g. Dukas, 1998b). Therefore, learning behaviour is

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expected in generalists where hostlprey species available might vary within and between generations but not in specialists without host/prey variation. For parasitoids, during the last years several studies have addressed the question whether generalistshave the capacity to learn whereas specialistslack this capacity. The results are contradictory. In accordance with the hypothesis, learning ability was demonstrated in almost every generalist parasitoid for which it was examined but was absent in some specialists (Potting et al., 1997; Geervliet et al., 1998). However, learning was also found in specialists (see Vet et al., 1995and references therein). A possible explanation for these findings is provided by the idea that learning might be a plesiomorphicproblem-solvingstrategy and the ability to learn is present in all species regardless of specializationbut not used for all tasks (Papaj, 1993).It might therefore be more important to know how the ability to learn is used by species that differ with respect to specialization than to look at the ability per se (Vet et al., 1995). (3) No infochemical use in extreme generalists Withincreasing diet breadth, the use of specific cues mentioned above is envisaged to be increasingly constrained. Furthermore, when potential hostlprey species are widely distributed, extreme generalist natural enemies should not focus (by using chemical cues) on only some of them, as this will reduce the encounter rates below a level that can be achieved by pure random search. Thus, it is concluded that the use of chemical cues by extreme generalists is unlikely. Empirical evidence for this idea is contradictory. The absence of infochemical use for oviposition site selection and the use of very general physical cues was demonstrated for polyphagous natural enemies such as the tachinid fly Drino inconspicua (Dippeland Hilker, 1998),the predatory bugAnthocoris nemorum (Parker, 1981),Trichograrnrna spp. (Schmidt and Smith, 1985)and Pirnpla instigator (Henaut and Guerdoux, 1982).Several studies with extreme generalist predators, however, demonstrated the use of chemical foraging cues for the solitary hunting ant Cataglyphisfortis (Wolf and Wehner, 2000), the trail followingMyrmicu rubra (Muller and Hilker, 1999), two yellowjacket wasps (Aldrich et al., 1986; Hendrichs et al., 1994), different carabid beetles (Chiverton, 1988; Kielty et al., 1996; Trefhs et al., 2001), and a spider (Kaspi, 2000). Although these predators used chemical cues for prey finding and not for oviposition, these results indicate that the use of infochemicals for foragingby extreme generalists might be much more widespread than expected but has not yet been sufficiently examined. Thus, it remains to be elucidated which rules apply for the infochemical use in extreme generalists, i.e. whether infochemical use is a common phenomenon and whether the reaction towards the infochemicals is innate or learned. In conclusion, the hypothesis on dietary specialization and infochemical use provides a valid general framework for the prediction of behaviour during foraging for hosts and oviposition sites in natural enemies. It is reasonable to assume that dietary specializationhas a significantimpact on the use of infochemicalsby natural enemies. However, although the hypothesis has been frequently cited within the

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last ten years after being published, several important aspects are still poorly examined.Especiallywith respect to generalistnatural enemies,many more studies are needed to elucidate the relationship between infochemical use and dietary breadth.

11.2.4 lnfochemicalUse in Koinobiont and ldiobiont larval Parasitoids The theories discussed above focus on the infochemical use by parasitoids and other natural enemies depending on the origin of infochemicals, the dietary specialization, and the behavioural plasticity. Vinson (1998) drew attention to the fact that for infochemical use in parasitoids the life history strategy might also be important, namely whether parasitoids are idiobionts or koinobionts. Numerous differences in traits can be associated with idiobiont or koinobiont life strategies (Askew and Shaw, 1986; Quicke, 1997) and it is reasonable to assume that infochemical use is also affected. By definition, koinobionts are parasitoids whose hosts continue to grow and develop after ovipositionhas taken place, whereas hosts of idiobionts are paralysed and stop their development (Haeselbarth 1979; Askew and Shaw, 1986).In larval parasitoids, it is expected that koinobiont parasitoids should prefer to oviposit in younger hosts to avoid competition with other parasitoids. Because hosts continue to grow, this preference does not result in a limited resource for the developing parasitoid. Idiobionts,on the other hand, should prefer to oviposit into older larvae to obtain larger resources for their offspring. According to Vinson (1998),this difference in host age preference should result in differences in infochemical use. Koinobionts should preferably respond to cues associated with young larvae, e.g. plant cues that are released immediately after infestation. Idiobionts should use chemical cues linked to older larvae, e.g. specific blends of plant cues released later during infestation, volatiles from the host itself, or from associated organisms. Few studies so far have examined infochemical use for the assessment of host quality. Nevertheless, it has been demonstrated for koinobionts that the location of preferred younger host larvae by Cotesia spp. can be guided by cues from the host and by host stage specific volatile blends from the host plant (Agelopoulos et al., 1995; Mattiacci and Dicke, 1995a,b; Takabayashi et al., 1995).For koinobionts preferring older larval stages, host recognition can be based on the increased amount of chemical cues present in older stages (Hare et al., 1993; Steidle and Fischer, 2000).

11.2.5 Plant-Entomophage Mutualism An increasing number of studies demonstrate that plants actively release chemicals to manipulate the behaviour of insects. As described above, several species of parasitoids and predators have been shown to use plant-derived chemicals for the location of herbivores feeding on these plants, either to use the herbivores as hosts for oviposition or as prey for feeding. In Chapter 10, those studies are outlined

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which show that plants mimic the odour of breeding sites of blowflies to attract gravid fly females for pollination. Chapter 8 summarizes studies on the orientation of egg parasitoids which are attracted towards plant odour induced by oviposition of the host species. The phenomenon that herbivore feeding damage induces plants to release volatile cues that are exploited by natural enemies of the feeding stage of the herbivores has been termed induced indirect plant defence (Dicke, 1999a). Potentially both the plant and the natural enemy benefit: predators kill herbivore prey, thus causing an immediate ending of herbivore feeding damage. For parasitoids, several distinctions must be made: that between koinobionts and idiobionts and between solitary and gregarious species. Solitary koinobionts, in almost all cases documented, induce a drastic reduction of feeding rates by the host (Guillot and Vinson, 1973; van Loon et al., ZOOO), whereas for gregarious koinobionts the effect they exert on host feeding rate seems to depend on the size of egg clutches laid. For all the categories of entomophages mentioned, the utilization of chemosensory information on host or prey presence as provided by plants is generally believed to increase their foraging efficiency, as it allows for oriented locomotion which is likely to save time and energy ( e g Vinson, 1998; Dicke and Vet, 1999). When both partners in an informational interaction benefit (via synomones) there is potential for mutualism to evolve (Takabayashi and Dicke, 1996; Vinson, 1999; see also Vet, 1999). Very few experimental studies explicitly addressed the quantification of both plant and entomophagous fitness under natural conditions of information transfer and utilization (van der Meijden and Klinkhamer, 2000). A recent report demonstrated the extent to which solitarykoinobiont parasitoid ovipositionresults in feeding reduction by the host which subsequently benefits plant fitness (van Loon et al., 2000). However, at present it is not possible to generalize on the occurrence of mutualism in plant-entomophagous informational interactions. The complexity of the infochemical-mediated networks existing in multitrophic interactions is only at the onset of being unravelled (Dicke and van Loon, 2000).

11.3 Concluding Remarks It is well established that chemical cues play an important role in host foraging and, thus, the oviposition behaviour of natural enemies. Much more information is available on parasitoids than on predators. Several theories have been put forward on the behavioural ecology of infochemical use in natural enemies. They provide predictions on the origin of the cues and the variability of the response towards these cues depending on the ecology of the natural enemy and its host/prey. Several of these predictions seem to hold in the light of the present knowledge. However, there are still important gaps in our knowledge. To elaborate existing theories further, the predictions should be explicitly tested, rather than being taken as a framework in which results should fit as is common practice. Especially for predators and generalist parasitoids more studies are required.

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Results from empirical studies and theories developed from these studies are not only of interest for basic research but are important when natural enemies are used for the biological control of pest organisms. By applying artificial chemical cues or by using plant varieties that produce increased amounts of herbivore induced synomones, natural enemies might be attracted to the habitat of pest species.Knowledge on the chemical nature of host recognition and host acceptance cues might be important for mass propagation of natural enemies, to stimulate oviposition in artificial substrates. When important foraging cues have to be learned it might be necessary to provide natural enemies with experience before release. Finally, when exotic natural enemies are considered as potential agents in classical biological control, knowledge on their foraging cues might be important for the assessment of potential non-target effects. Thus, when looking at the still growing efforts worldwide to use biological instead of chemical methods to control agricultural pests, the study of infochemical use in natural enemies should be even more important in the future as it was in the past.

11.4 Ac knowIedgement s We are very grateful to Monika Hilker, Eric Wajnberg and Louise Vet for critical comments on an earlier version of this manuscript.

11.5 References Agelopoulos, NG, Dicke, M, Posthumus, MA. 1995. Role of volatile infochemicals emitted by feces of larvae in host-searching behavior of parasitoid Cotesiu rubeculu (Hymenoptera: Braconidae): a behavioral and chemical study. J. Chem. Ecol. 21: 1789-1811. Agelopoulos, NG, Keller, MA. 1994. Plant-natural enemy associationin the tritrophic system, Cotesiu rubeculu-Pieris rupue-Brassicaceae (Cruciferae):I. Sourcesof infochemicals. J. Chem. Ecol. 20: 1725-1734. A1 Abassi, S, Birkett, MA, Pettersson, J, Pickett, JA, Wadhams, LJ, Woodcock, CM. 2001. Response of the ladybird parasitoid Dinocumpus coccinellue to toxic alkaloids from the seven-spot ladybird, Coccinella septempunctutu. J. Chem. Ecol. 27: 33-43. Aldrich, JR, Lusby, WR, Kochansky, JP. 1986. Identification of a new predaceous stink bug pheromone and its attractiveness to the eastern yellowjacket. Experientia 42: 583-585. Askew, RR, Shaw, MR. 1986.Parasitoid communities: Their size, structure and development. In: Waage, JK, Greathead, D. (eds.) Insect Purusitoids. Pp. 225-264. Academic Press, New York. Auger, J, Lecomte, C, Paris, J, Thibout, E. 1989.Identification of leek-moth and diamondbackmoth frass volatiles that stimulate parasitoid, Diudromus pulchellus. J. Chem. Ecol. 15: 1391-1398. van Baarlen, P, Topping, CJ, Sunderland, KD. 1996.Host location by Gelisfestinuns, an eggsac parasitoid of the linyphiid spider Erigone utru. Entomol. Exp. Appl. 81: 155-163. Ballal, CR, Singh, SP. 1999. Host plant-mediated orientational and ovipositional behavior of three species of chrysopids (Neuroptera: Chrysopidae). Biol. Contr. 16: 47-53. Bargen, H, Saudhof, K, Poehling, HM. 1998. Prey finding by larvae and adult females of Episyrphus bulteutus. Entomol. Exp. Appl. 8 7 245-254. Bernays, EA, Chapman, RF. 1994.Host-Plant Selection by Phytophugous Insects. Chapman and Hall, New York. Bernays, EA, Wcislo, W. 1994. Sensory capabilities, information processing and resource specialization. Quat. Rev. Biol. 69: 187-204.

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Bjorksten, TA, Hoffmann, AA. 1998. Persistence of experience effects in the parasitoid Trichogramma nr. brassicae. Ecol. Entomol. 23: 110-117. Bruni, R, Sant’-Ana,J, Aldrich, JR, Bin, F. 2000. Influence of host pheromone on egg parasitism by scelionid wasps: Comparison of phoretic and nonphoretic parasitoids. J. Insect Behav. 13: 165-174. Borges, M, Costa, MLM, Sujii, ER, Cavalcanti, MDG, Redigolo, GF, Resck, IS, Vilela, EF. 1999. Semiochemical and physical stimuli involved in host recognition by Telenomus podisi (Hymenoptera: Scelionidae)toward Euschistus heros (Heteroptera: Pentatomidae). Phys. Entomol. 24: 227-233. Chiverton, PA. 1988. Searching behaviour and cereal aphid consumption by Bembidion lampros and Pterostichus cupreus, in relation to temperature and prey density. Entomol. Exp. Appl. 47: 173-182. Colazza, S, Rosi, MC, Clemente, A. 1997. Response of egg parasitoid Telenomus busseolae to sex pheromone of Sesamia nonagrioides. J. Chem. Ecol. 23: 2437-2444. Constant, B, Grenier, S, Bonnot, G. 1996. Artificial substrate for egg laying and embryonic development by the predatory bug Macrolophus caliginosus (Heteroptera: Miridae). Biol. Control. 7: 140-147. Cooperband, MF, Vinson, SB. 2000. Host-acceptance requirements of Melittobia digitata (Hymenoptera: Eulophidae), a parasitoid of mud dauber wasps. Biol. Control. 17: 23-28. Corbet, SA. 1971.Mandibular gland secretion of larvae of the flour moth, Anagasta kuehniella, contains an epideictic pheromone and elicits oviposition movements in a hymenopteran parasite. Nature 232: 481484. Cortesero, AM, De Moraes, CM, Stapel, JO, Tumlinson, JH, Lewis, WJ. 1997. Comparisons and contrasts in host-foraging strategies of two larval parasitoids with different degrees of host specificity. J. Chem. Ecol. 23: 1589-1606. Coupland, JB. 1996. Influence of snail feces and mucus on oviposition and larval behavior of Pherbellia cinerella (Diptera: Sciomyzidae).J. Chem. Ecol. 22: 183-189. Dicke, M. 1999a. Evolution of induced indirect defense of plants. In: Tollrian, R, Harvell, CD (eds.) The Ecology and Evolution of Inducible Defenses. Pp. 6 H 8 . Princeton University Press, New Jersey. Dicke, M. 1999b. Are herbivore-induced plant volatiles reliable indicators of herbivore density to foraging carnivorous arthropods? Entomol. Exp. Appl. 92: 131-142. Dicke, M. 1999c. Specificity of herbivore-induced plant defences. In: Chadwick, DJ, Goode, J (eds.) Insect-Plant lnteractions and Induced Plant Defence. Pp. 43-59. Novartis Foundation Symposium 223. Wiley, Chicester. Dicke, M, van der Maas, KJ, Takabayashi, J, Vet, LEM. 1990. Learning affects response to volatile allelochemicalsby predatory mites. Proc. Exp. Appl. Entomol. N.E.V. 1: 31-36. Dicke, M, van Loon, JJA. 2000. Multitrophic effects of herbivore-induced plant volatiles in an evolutionary context. Entomol. Exp. Appl. 97: 237-249. Dicke, M, Vet, LEM. 1999. Plant-carnivore interactions: evolutionary and ecological consequences for plant, herbivore and carnivore. In: Olff, H, Brown, VK, Drent, RH (eds.) Herbivores: Between Plants and Predators. Pp. 483-520. Blackwell Science, Oxford. Dickens, JC. 1999. Predator-prey interactions: olfactory adaptations of generalist and specialist predators. Agric. Forest Entomol. 1: 47-54. Dippel, C, Hilker, M. 1998.Effects of physical and chemical signals on host foraging behavior of Drino inconspicua (Diptera: Tachinidae), a generalist parasitoid. Environ. Entomol. 27: 682487. Drukker, B, Bruin, J, Sabelis, MW. 2000. Anthocorid predators learn to associate herbivoreinduced plant volatiles with presence or absence of prey. Phys. Ent. 25: 260-265. Du, YJ, Poppy, GM, Powell, W. 1996. Relative importance of semiochemicals from first and second trophic levels in host foraging behavior of Aphidius ervi. J. Chem. Ecol. 22: 1591-1605. Du, YJ, Poppy, GM, Powell, W, Pickett,JA, Wadhams, LJ, Woodcock, CM. 1998.Identification of semiochemicals released during aphid feeding that attract parasitoid Aphidius ervi. J. Chem. Ecol. 24: 1355-1368. Dukas, R. 1998a. Constraints on information processing and their effects on behaviour. In: Dukas, R (ed.) Cognitive Ecology. Pp. 89-127. University of Chicago Press, Chicago.

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Chapter 12

Evolutionsry Ecology of Oviposition Marking Pheromones Thomas 5. Hoffmeister and Bernard D. Roitberg

Table of Contents 12.1 12.2 12.2.1 12.2.2 12.2.3 12.2.4 12.3

Introduction Four Major Questions on the Use of Oviposition Marking Pheromones Why Use Oviposition Marking Pheromones? Where Should Insects Deposit their Mark? When Should an Insect Mark i t s Oviposition Site? What Information Should Oviposition Marking Pheromones Convey? How Might Natural Selection Shape the Use and Chemistry of Oviposition Marking Pheromones? 12.3.1 Should Marking Be Circumstance-Dependent? 12.3.2 What Is the Optimal Persistence of an Oviposition Mark? 12.3.3 Should Marking Pheromones Be Variable or Invariant among Individuals? 12.3.3.1 The Evolution of Mark Variability under Enemy Attack 12.3.3.2 A Population Model on the Evolution of Mark Variability 12.3.3.3 The Evolution of Mark Variability without Enemy Attack 12.4 Concluding Remarks 12.5 Acknowledgements 12.6 References Appendix Abstract

In this chapter we review and synthesize literature on oviposition marking pheromones from a functional perspective. This allows us to use an evolutionary approach towards communication in a multitrophic context. It leads us to pose a series of questions. We start with the question what information oviposition marking pheromones might convey. Taking the perspective of a marking individual, we ask why natural selection would favour individuals that mark their ovipositionsites and what information should be contained in the mark. This leads to the discussion on the costs and benefits of oviposition marks and measures of relative fitness. We further ask where an insect should apply its mark, whether internal or external marks should be employed.Another question regards the timing of mark deposition, i.e. whether an insect should mark before, coincident with, or

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subsequent to oviposition. A second complex of questions regards how natural selection might shape the use and chemistry of existing marking pheromones. We review concepts on the optimal persistence of marking pheromones and the circumstancesunder which marking should occur,including the physiologicalstate of the marker, the presence of predators, and the presence or absence of competitors. Subsequently, we depart from our synthetic review to develop novel concepts for the evolution of mark variability and individual-specific marks. Using gametheoretic tools we develop conceptual theory for exploring the frequency dependent relationship among individual markers, conspecifics, and natural enemies.We find that mark variability or even individual-specificmarks can evolve only under a limited set of conditions. In summary, we find an enormous complexity associated with the evolution of use and chemistry of oviposition marking pheromone systems. As a result, simple generalizations and predictions are not easy to make and usually require precise parameterization of the ecologicalcontext.

12.1 Introduction Oviposition marking pheromones make up a subset of marking pheromones that have been described for a variety of taxa, from trail marking pheromones in parasitoids (Price, 1970) to territorial marks in wolves (Lewis and Murray, 1993). They have been found in a large number of herbivorous and entomophagous insects (see Chapter 9; van Lenteren, 1981; Roitberg and Prokopy, 1987; Godfray, 1994; Kainoh, 1999; Nufio and Papaj, 2001). Almost as numerous as the taxa that use oviposition marks are the questions related to marking pheromones. In this chapter we focus on herbivorous and parasitoid insects that use discrete resources for offspringproduction (Price, 1977).Here we ask why such insects apply pheromones that mark their host plant or host insect, where and when the mark is applied, how marks are deposited, and what the individual and populationlevel repercussions are. Throughout this chapter we try to answer some of these questions by taking an evolutionary perspective. In particular, we assume that evolution proceeds primarily via natural selection. Whenever an evolutionary biologist posits a particular evolutionary process to have occurred, the following conditions have to be met (see Endler, 1986):Firstly, the trait of interest must have varied amongst individuals. For example, animals might vary in the amount of marking pheromone applied per ovipositionor in the pheromone blend. Secondly, those differences among individuals must have a genetic component and be at least partially heritable. Thirdly, there has to be a consistent relationship between trait value and fitness. For example, there might exist some fitness cost to female moths that produce a sex pheromone blend which does not optimize male response (Lofstedt, 1990).Our analysis in what follows mostly focuses on the relationship between trait value and fitness or what is often called a functional approach (Williams, 1966), although we acknowledge that a full consideration of the evolutionary ecology of marking pheromones involves all three conditions.

Introduction

Even though it is true that a functional approach has its share of critics (Gould and Lewontin, 1979),it is obvious that a purely causal understanding of marking pheromones could severely limit their application. Consider, for example, that we might try to use a marking pheromone to deter a frugivorous insect from attacking a commercial fruit. It is well known that the “deterring pheromone” will not deter individual insects that are deprived of unmarked oviposition sites (Roitberg and Prokopy, 1983;van Alphen and Visser, 1990)as would occur in a pheromone-treated orchard system. There are two ways to view this problem. On the one hand, using a purely causal approach, the lack of deterrence may be interpreted as a failure in an insect’s response to the pheromone (seevan Lenteren, 1981 for alternativecausal explanations). By contrast, a functional approach anticipates conditions under which a lack of deterrence is an adaptive response to the scarcity of oviposition sites. This would allow us to develop counter-measures to take advantage of the frugivore flexibility, such as using untreated trap-trees (Roitbergand Angerillt, 1986; E. F. Boller, personal communication). Through our functional approach we examine ovipositionmarking pheromones as a communication device within and among individuals. Following Wiley (1994), we define communication as any alteration in a receiver produced by a signaller by means of a signal. We define a signal as any pattern of energy or matter produced by one individual (the signaller)and altering some property of another (the receiver) (see Nufio and Papaj, 2001, for an alternative interpretation). Consequently, signals differ from cues that may also provide information about the presence of competitorsto the offspringof a foraging insect, like the plant-produced substances that convey information about present egg clutches to foraging female pierid butterflies (Nufioand Papaj, 2001).Here, we will restrict our discussionto the former. A special feature of the communication system we are discussing here is the fact that oviposition marking pheromones signal information about past action rather than present action. The function of an oviposition marking pheromone is thus very different from that of a sex pheromone that is released and announces an emitter’s current location. Moreover, it is the case that marking pheromones frequently function as intra-individual communication devices and provide information about previous exploitation of an oviposition site by the signaller to the signaller, though they may also function as conspecificor heterospecific signals. Another key feature of oviposition marking pheromones is that, unlike classic territorial pheromones that confer information on current user’s presence, such pheromones typically signal the presence of non-signallingoffspringand thus have cross-generational effects. Consistent with our communication-based approach we will continue to call these pheromones oviposition marking pheromones or host marking pheromones (HMPs), although they are frequently called oviposition deterring pheromones (ODPs)(see Chapter 9) and recently have been named marking pheromones (MPs) (Nufio and Papaj, 2001). While MPs may comprise territorial marks, trail marks, and marks left on exploited and unexploited sites, the terms we use convey the notion that such a pheromone is released into or onto or near a larval resource

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either subsequent to or coincident with oviposition in order to inform the recipient that oviposition has occurred at that site. In contrast, usage of the term ODP conveys the notion that such a pheromone is released into or onto or near a larval resource to prevent the recipient from ovipositing at that site. The latter involves the intentional manipulation of the receiver’s behaviour. As we will show, it is advantageous to consider these pheromones from both the releaser’s and the recipient’s perspective, and in so doing, evaluate these compounds in an unbiased manner.

12.2 Four Major Questions on the Use of Oviposition Marking Pheromones Marking pheromones potentially could convey information on a number of aspects relating to the oviposition site, including the time since oviposition, the identity of the marker, the quality of the oviposition site, and the density of competitors. Clearly, different costs and benefits are associated with these different components. Thus, here we ask why insects should provide marks at all, whether specific locations and specific times of marking are favoured and what information content natural selection will favour. Recall our argument that marking pheromones function as a communication device. Thus, the appropriate manner in which to address this question is by the methodology of communication theory, a subdiscipline of behavioural ecology (Dawkins and Krebs, 1978; Krebs and Dawkins, 1984; Grafen and Johnstone, 1993; Johnstone, 1997).We agree with the notion that signalscan only evolve if they benefit the sender of the signal. Recalling our definition of communication, we define a signal as information that is overtly sent from the sender to a receiver. We can envision at least two routes by which a sender of a signalmight benefit. Benefits can accrue in situations of mutual interests of signallers and recipients through the transmission of reliable signals, or, when there is a conflict of interest, through the manipulation of the receiver. Following, we will explore potential routes by which signallers might benefit.

12.2.1 Why Use Oviposition Marking Pheromones? In the case of oviposition marking pheromones, there are two different classes of intended receivers, the signaller itself and conspecifics.We will first consider cases of mutual interests. Oddly, when a marking animal deposits a signal for itself, there is mutuality between that individual’s current interests as a signallerand its future interests as a receiver. If the marking individual were to encounter its own mark, it can make an informed “decision” to reject that site and possibly avoid superparasitizing that resource (note, when we say decision we do not suggest any cognitive process but rather that individuals respond in particular ways to particular situations). Self-superparasitism is generally thought to be especially costly for solitary parasitic insects because a single offspring will emerge regardless of the number of ovipositions by an individual (Godfray, 1994; but see Visser, 1993 for

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cases under which self-superparasitism could pay). As such, eggs and time are wasted when females self-superparasitize.The same problem also holds for insects that lay multi-egg clutches, so long as superparasitism results in competition between the offspring for limited host resources. Opportunities for selfsuperparasitism abound for insects that search for patchily distributed hosts and thus concentrate their search in patches (Bell, 1991).Thus, individuals that mark exploited sites receive direct benefits by reducing the probability of selfsuperparasitism and subsequent severe sibling competition. By contrast, if the recipient of the signal is a conspecific or a heterospecific, there is potential for conflict regarding reproduction at a particular oviposition site. This conflict is resolved via a reliable signal (Grafenand Johnstone, 1993)that announces that the marked site is already occupied by offspring that will compete for possession of the site. The sender only benefits if receivers of the signal respond with reduced propensity for oviposition, in cases where the resident larva is not the guaranteed winner. A benefit to the recipient arises from the information about the presence of a competitor which allows it to make a decision whether or not to reuse this site for oviposition. As noted above, due to the potential for larval competition, already parasitized sites generally are poorer resources than are unexploited resources. As such, recipients frequently reject marked sites though the conditions for adaptive conspecific superparasitism are not nearly so restricted as they are for self-superparasitism (van Alphen and Visser, 1990). If the fitness return from the reduced chances of offspring survival outweighs the fitness return from site rejection and search for an alternative oviposition site, a female should reuse the site and oviposit. Thus, acceptance of marked sites will critically depend upon the survival chances of the second offspring in a contest for the site. Consequently, by marking hosts, markers potentially increase the fitness of conspecifics. This leads to the question as to why females should engage in what at first appears to be altruistic behaviour. Here again, two functional explanations are possible. First, so long as the marker gains more from marking than do conspecifics, she will receivegreaterrelativefitness (Roitbergand Mangel, 1988).This will be the case when re-encounter rates with self-parasitized sites are high as noted above. Thus, information for conspecifics would be a by-product of intra-individual signal evolution. Second, so long as there is some chance that the second individual to enter the oviposition site will win larval competition, it pays the first females to inform conspecifics of the initial oviposition, thus saving its offspring from potentially fatal competition and likewise the conspecific’s offspring. Thus, in this second case, oviposition marking pheromones function as reliable signals where both parties benefit (Roitbergand Mangel, 1988; Grafen and Johnstone, 1993). In summary, deployment of HMPs should be adaptive whenever the likelihood of re-encountering exploited sites is high, exploited sites cannot be recognized as such (e.g. site recognition without marks; Roitberg and Prokopy, 1984; van Giessen et al., 1993; Hoffmeister and Gienapp, 2001), and the potential cost of competition for the first larva at a site outweighs any physical or physiological costs associated

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with the host marking. Thus, based on our fitnessarguments alone, we don’t expect to find HMPs in all species of insects. Further, even within a species it may not always be adaptive to mark hosts nor should marks necessarilyendure. We consider such possibilities in Sections 12.3.1 and 12.3.2, respectively.

12.2.2 Where Should Insects Deposit their Mark? Here, it is essential to note that in many insects the time that can be allocated to foraging for oviposition sites is limited, and efficient use of the limited time is of major importance for an insect’s lifetime reproductive success (Sevenster et al., 1998).Since oviposition marking pheromones evolved primarily as a communication device within and among individuals to inform recipients about occupied sites, and since area concentrated search is common (Bell, 1991) and leads to frequent encounters with one’s own marks, natural selection should favour deposition of marks that leads to rapid recognition. Thus, the question arises, where should marking insects deposit their mark. A first concern is the time costs associated with mark assessment. Although this behaviour is rarely studied directly,published rejection times at marked sites allow us to infer that it can vary from a few seconds in aphid and drosophilid parasitoids (Hofsvang,1988; Volkl and Mackauer, 1990; Haccou et al., 1991)to minutes in some tephritid flies (Aluja and Boller, 1992).Regardless of the absolute value, time spent assessing marks necessarily detracts from time that could be spent exploitingother hosts (Holling, 1959). This is particularly true for insects with relatively short oviposition times and unlimited egg supplies. Under such conditions, oviposition marks may even not evolve. A second concern is the risk-to-life that may be associated with mark assessment. Since most oviposition marks are contact pheromones, they must, by definition, be assessed at the resource site, meaning that mark assessors cannot easily circumvent assessment costs. For example, approaching parasitoids risk being kicked and injured by both healthy and already-parasitized aphids (Dixon, 1998). In addition, most insects are at risk to predation when foraging for hosts (Lima and Dill, 1990).This contrasts with the situation faced by recipients of sex pheromones, wherein assessments can often be made from safe sites. Although this question of where to deposit the mark applies to herbivorous as well as parasitoid insects, this is an especially intriguing question for endoparasitic hymenopteran parasitoids where eggs are laid into the body of a host insect and where a variety of marking modes has been described. Here, marking pheromones might either be injected internally into the host haemolymph (e.g. Greany and Oatman, 1972; Rogers, 1972; van Lenteren, 1976; van Alphen and Nell, 1982) or applied externally onto the host (e.g. Salt, 1937; Rabb and Bradley, 1970; Chow and Mackauer, 1986), or they may be applied internally as well as externally (e.g. Harrison et al., 1985; van Baaren and Boivin, 1998b).If the host is concealed in some substrate, the mark may also be placed externally on the host-harbouring substrate, which might either be marked locally at the ovipositionpuncture (e.g. Hoffmeister,

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Four Major Questions on the Use of Oviposition Marking Pheromones 325 ...... . ........................................................... .............................................. . ................. .. ............... . . . . .......... ... ...................... .

2000) or globally on the entire surface of the substrate (e.g.Hoffmeister and Roitberg, 1997; Chow and Mackauer, 1999). External markers are most efficient for rapid recognition. Thus, we ask whether such interspecific variation in marking mode represents adaptive responses to costs and benefits that vary with host distribution and search behaviour of markers. Costs to foraging insects will accrue from the time needed to apply the mark and the time needed to assess the mark upon encounters with marked sites. While internal marks may be applied concomitant with oviposition, the application of external marks always requires extra time, sometimes even more than the entire behavioural sequence of ovipositor insertion, oviposition, and ovipositor withdrawal (e.g. Hoffmeister, 2000). Thus, with respect to the limited searching time of foraging parasitoids, external marks should be more costly than internal marks. In contrast, internal marks should be more costly in terms of the time needed for assessment. This is especially true when host detection within the substrate is difficult (e.g. Hoffmeister and Roitberg, 1997).Under such conditions, external marks might be favoured even if they lead to incomplete exploitation of hosts within the marked substrate (Hoffmeister and Roitberg, 1997). In general, we predict that external marks will evolve whenever re-encounters with self-marked sites are sufficiently frequent to offset the costs of increased marking time through time savings from faster mark recognition (Figure 12-1).

100

Time savings through recognition of external mark (s) Figure 12-1 Equal-fitness surface for external and internal markers as a function of the time savings through recognition of external marks, the extra time needed for applying external marks, and the re-encounter rate with marked relative to unmarked oviposition sites. The region abovetheequal-fitnesssurfacewouldfavourexternalmarkerswhilethe region below the equal-fitness surface would favour internal markers.

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Moreover, internal marks may provide little benefit under some conditions, because their effectiveness depends upon diffusion through the host. When parasitoids reencounter hosts shortly after parasitizing them, incomplete diffusion of the marking pheromone can lead to maladaptive superparasitism due to the lack of recognizing a previous oviposition (Rogers, 1972; van Alphen and Nell, 1982). Finally, if parasitoids mark the substrate harbouring the host, should they apply local or global marks? We predict that local marks should only be used whenever the host is immobile and can thus be identified through a local mark (e.g. Hoffmeister, 2000). In contrast, whenever the host can move away from the attack site, the entire structure harbouring the host should be marked (e.g. Hoffmeister and Roitberg, 1997; Holler et al., 1994), at least if this does not interfere with attacking further hosts within the same structure (cf.Vet and Dicke, 1992; discussion of the reliability-detectability problem).

12.2.3 When Should an Insect Mark i t s Oviposition Site? Here it is important to distinguish between marking oviposition sites and use of marks when foraging per se. Marking need not be restricted to the former. For example, trail marks are commonly used by parasitoids to identify already searched areas and to alter the searching effort accordingly (Price, 1970; Galis and van Alphen, 1981; van Dijken et al., 1990; Holler and Hormann, 1993). Between oviposition marks and trail marks, however, we note an asymmetry. Oviposition marks may be used to make patch-leaving decisions (e.g. van Lenteren, 1991; Hemerik et al., 1993; Hoffmeister and Roitberg, 1997; but see van Alphen, 1993for a different viewpoint), whereas trail marks are not known to directly affect oviposition decisions. So, when should an animal mark its oviposition site? By definition, marks should only be applied after the individual has committed itself to oviposition. Thus, in general, the mark itself should be applied coincident with or subsequent to oviposition. For external marks it has been shown that they are applied following oviposition (Rabb and Bradley, 1970; Strand and Vinson, 1983; Hoffmeister, 2000) whereas for internal marks the exact timing of the marking behaviour is less clear. If the marking chemical has a dual purpose (e.g. if it also functions in host paralysis or necrosis or modifies host development (Greany and Oatman, 1972; Strand, 1986; Holler et al., 1994) it may be deposited prior to oviposition.

12.2.4 What Information Should Oviposition Marking Pheromones Convey? Oviposition marking pheromones function as a communication device with a temporal component, i.e. they reside at the marked site and signal a past event. As such, marks can undergo changes over time through degradation (Section 12.3.2) or chemical reaction, thus potentially changing the information content. Thus, potentially, marks could, most simply, convey information about the presence

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of a potential competitor to the offspring of the mark-assessing insect, but they could also convey information about the identity of the marking individual, the age of the mark and thus the age of the potentially competing offspring, and the number of potential competitors. However, we expect that natural selection will favour marks that maximize the benefit of the signaller, i.e. the individual that provided the mark. Potentially, there is a conflict of interest between signaller and recipient of the mark, if the recipient is a con- or heterospecific. Therefore, we will examine benefits from the perspective of a recipient and a signaller and discuss what kind of information marks should convey. We will first discuss what kind of reliable information marks should convey, and then ask whether the evolution of deceptive marks is likely. Imagine a female insect foraging in a patch that contains potential oviposition sites. Further, imagine that she encounters a site that carriesa marking pheromone. What information could that forager extract from such a mark and what action might she take based upon that information? A functional perspective points to two critical decisions, acceptance of the oviposition site and emigration from the patch. We can think of three pieces of reliable mark-based information that could impact recipient decisions. (1)The presence of a mark signals that an oviposition has occurred at the focal site. Of course, that need not guarantee that the site is still occupied if for example, the previous occupant has been killed by a predator. Frequently, however, a mark will signal a high chance that the site is still occupiedby a living competitor. How should a female respond? Natural selection should favour contextdependent host exploitation behaviour. For example, when encounters with marked sites occur at a high rate, females should either emigrate from such patches (Roitberg et al., 1984; van Lenteren, 1991; Nelson and Roitberg, 1995; but see van Alphen, 1993for a counter argument) or superparasitize (Roitberg and Prokopy, 1983; van Alphen and Visser, 1990). The optimal response will depend upon life history attributes and how they trade off (Charnov and Skinner, 1985). (2) An oviposition mark may confer the identity of the marker (Section12.3.3).How should a female use such information to determine her oviposition behaviour? If she recognizes the mark as her own, and laying a second egg does not increase the overall chance of producing a surviving offspring, she should always reject the site (Visser, 1993).On the other hand, if she can be sure that the mark is not her own, superparasitism would be favoured under a broader range of conditions. This is because females can now expect a higher fitness return than if that site potentially contained her own offspring. (3) The mark might confer information on the time since the original oviposition took place. This could occur either through differential degradation of pheromone components (Holler et al., 1991)or production of metabolites. This time element can provide information on the likelihood that the second larva in would win a mortal competition. In most systems of contest competition it is the older larva that wins (Chow and Mackauer, 1986)and in many cases the

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age differentialcorrelateswith likelihood of winning (Strand and Godfray, 1989; Visser, 1993).The same logic applies to insects engaging in scrambleor exploitation competition, even though competition is not necessarily mortal. Since this asymmetry usually varies with the time lag between ovipositions, recipients should benefit from precise information about the time lag since deposition of the mark. Further, because the sender does not benefit from such information in the mark, regardless of whether the sender itself or a conspecific is the recipient, we predict that marks should evolve that are devoid of information regarding competitor asymmetries (for a different view, see Holler et al., 1991). Finally, we now consider cases of deceptive signals. There are two ways in which an oviposition marking pheromone can be used in a deceptive context. Animals might either mark sites which do not contain offspring or refrain from marking sites containing offspring. The former would be spiteful behaviour against conspecifics or predators. Expending time and energy to harm others (spite) is rarely if ever adaptive (Schaffer,1988; Gadagkar, 1993).Moreover, spiteful markers would often hurt themselves as recipients of their own deceitful marks. According to the definition by Dawkins & Krebs (1978),oviposition-markingpheromones can be considered manipulative, since they potentially modify the behaviour of the recipient. Because of the evolution of oviposition marking pheromones as an intraindividual communication device, the receiver should never be a victim of its own manipulation to its detriment. Finally, as discussed in the introduction, manipulation via an oviposition deterrent does not benefit the receiver and is therefore evolutionary unstable, because it would be susceptible to non-responding mutants. The above logic can as well be applied to the failure to mark as a spiteful behaviour. This should hold true so long as females encounter oviposition sites that they themselves exploited more often than oviposition sites that were exploited by a conspecific (Roitberg and Mangel, 1988).

12.3 How Might Natural Selection Shape the Use and

Chemistry of Oviposition Marking Pheromones? Up to here, we have considered particular characteristics of oviposition marking pheromones and the costs and benefits associated with their use. Of necessity, our discussion has focused on specific trait values whereas the aforementioned traits may vary considerably within individuals, among individuals, or over time. Thus, in the following, we will discuss phenotypic plasticity in the use of oviposition marking pheromones within individuals and Darwinian selection that leads to change of mark detectability over time and variance in the chemical composition of marks among individuals, respectively. We will point out that in this context, it is essential to acknowledge the multitrophic context in which oviposition marking pheromones operate, and that frequently, frequency dependent selection acts upon marking pheromones.

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12.3.1 Should Marking be Circumstance-Dependent? In Section 12.2, we argued that marking pheromones function to inform the marking individuals and others regarding occupation of the site. However, if marking has both benefits and costs, and we suggest that it does, we can ask if marking should be universal or circumstance-dependent. In the jargon of the evolutionary biologist, we no longer treat the trait to oviposition mark as a fixed trait (think of proboscis extension reflex in flies [Dethier, 1962]),but rather as a reaction norm wherein phenotypic expression for a given genotype depends upon environment (Stearns, 1992).Alternatively, from a more mechanistic perspective, think of the quality and magnitude of marking behaviour as a plastic response to a suite of cues both positive and negative (cf. the rolling fulcrum of Miller and Strickler, 1984). Will natural selection favour circumstance-dependent marking? We believe that the following three scenarios are worth considering. The first scenario considers the presence of mark-exploiting natural enemies. A precondition for this form of plasticity in the ovipositing animal is recognition of the presence of the aforementioned natural enemies. In a number of cases this has been demonstrated (Dicke and Grostal, 2001 and references therein). We predict that recognition of this cost (i.e. increased risk to offspring)could offset the benefits (i.e. reduced larval competition) and thus should favour a reduction in both marking tendency and magnitude of mark. We know of no cases where the aforementioned plasticity has been documented. The second scenario is the presence of competitors. A precondition for this form of plasticity in the ovipositing animal is recognition of conspecifics. Evidence for recognition of the presence of competitors through direct or indirect cues is widespread in conspecifics (e.g. Hoffmeister et al., 2000; Prokopy, 1981a; Strand and Godfray, 1989; Visser et al., 1990; Visser et al., 1992; see also Chapter 9) and even found in heterospecifics (e.g. McBrien and Mackauer, 1990). There is no unambiguous prediction we can make, because the benefits of any marking strategy will depend upon the number and frequency of alternative strategies and the response by the receivers. In Sections 12.3.2 and 12.3.3we take a closer look at some of these situations. Finally, a physiological cost of mark production can alter the marking behaviour if it outweighs the benefits from marking. This could happen if the production and/or release of pheromone compete with other physiological processes in the animal.For example, in the apple maggot fly, deposition of the marking pheromone depends upon the excretion of water in faeces as a carrier (Prokopy, 1981b).

12.3.2 What Is the Optimal Persistence of an Oviposition Mark? From the few data available on the persistence of oviposition marks, a tremendous variation is already obvious. Among species, marks can remain active and perceivable from a few hours to more than a week (Quiring and McNeil, 1984; Harrison et al., 1985; Turlingset al., 1985; Sugimoto et al., 1986; Averill and Prokopy,

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1987; Ferguson and Williams, 1991; Holler et al., 1991; Micha et al., 1992; Nelson and Roitberg, 1993; Gauthier et al., 1996).We thus may ask how natural selection operates upon the persistence of marking pheromones. Recalling the possible benefits and costs of marks, four parameters should affect the optimal persistence of marks. First and foremost is the spatial search of markers. With more spatially restricted searching, a foraging insect will re-encounter sites that contain its own offspring for a greater duration and at a higher frequency. This will select for unequivocal recognition across extended time periods to enable the avoidance of sibling competition. Second, the degree of competition with conspecifics for oviposition sites should affect mark persistence. Since the response to conspecific marks follows a reaction norm (Stearns, 1992) rather than a fixed response, predictions for optimal persistence of marks will be difficult to make without an explicit parameterization of the ecological scenario, e.g. the degree of competition for resources. Third, physiological costs of pheromone synthesis should be important, assuming that pheromones with a longer persistence are more costly to produce. Fourth, if natural enemies exploit the marking pheromone to facilitate host location (see Section 12.3.3), marking pheromones with reduced persistence should be favoured by natural selection. To date, data are unavailable for a comparative approach that could explain the degree to which the observed variation in the persistence of oviposition marking pheromones can be explained by ecological and phylogenetic effects.Recalling the interactivenature of HMPs, a game theory approach (seeHoffmeister and Roitberg,

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Figure 12-2Optimal half-life of oviposition marking pheromones as a function of the degree ofcompetitionforoviposition sites,forarea restricted search (a)and random search (b),when (1)insects pay no cost to marking (dotted line), (2) marking behaviour has some physiological cost (dash-dotted line), (3) when natural enemies at low density exploit the ovposition marking pheromone (dashed line), and (4)when natural enemies at high density exploit the oviposition marking pheromone (solid line). Note the log-scaled nature of the ordinate. For details, see Hoffmeister and Roitberg, (1998).

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1998 for details) suggests that all four parameters in concert strongly affect the optimal persistence of oviposition marks (Figure 12-2). Area-concentrated search in general leads to higher persistence than random search for oviposition sites. Interestingly, competition with conspecifics for oviposition sites and physiological costs of mark synthesis affect optimal mark persistence only under random search. Here, increased mark persistence is favoured with increasing competition for oviposition sites. While the benefit of persistent marks offsets small physiological costs under area-concentrated search, physiological costs select for reduced mark persistence under random search.As expected, natural enemies strongly affect mark persistence. Under high rates of mortality by mark-exploiting natural enemies, the optimal persistence of oviposition marks can be reduced to a half-life of less than a day. Thus, depending on ecological circumstances, our game theory predicts a range of mark persistence across two magnitudes, consistent with that found in natural systems.

12.3.3 Should Marking Pheromones Be Variable or Invariant

among Individuals? Thus far we have undertaken a functional approach to understand the evolution of marking pheromones. This approach assumes at equilibrium that optimal trait values will either go to fixation (Travis, 1989) or reach some evolutionary stable state of marking variants (Maynard-Smith, 1982). Here we look at one aspect of marking pheromones, i.e. the chemical composition of the pheromone blend, and ask whether a single blend will dominate or whether the composition of marking pheromone components will vary between individuals. We previously argued that the major benefit in the evolution of oviposition marking behaviour comes from the intra-individual communication regarding occupied sites that facilitates avoidance of unrecognized self-superparasitism. As soon as marking becomes a common behavioural strategy in a population of animals, however, markers benefit not only from self-recognition,but also from conspecific recognition and avoidance of sites that contain a marker’s offspring (Roitberg and Mangel, 1988).Conspecific oviposition avoidance requires that marks can be recognized not only by the producing animal, but also by conspecifics. Thus, there should be a selective advantage for individuals of a population to use a marking chemical that is identical among individuals. Are marking pheromones invariant? In fact, there is indirect evidence for variation of marks within populations in that some parasitoids have been shown to distinguish between own and conspecific parasitized hosts (Volkl and Mackauer, 1990; van Dijken et al., 1992; Danyk and Mackauer, 1993; Marris et al., 1996; van Baaren and Boivin, 1998a). While no information is available for the marking pheromones that facilitated this discrimination, evidence from the sex-pheromone composition of insects clearly shows abundant heritable variation at the species and individual level ( e g Collins and Carde, 1985; Hansson et al., 1987; Priesner and Baltensweiler, 1987; Hefetz and Graur, 1988; Lofstedt, 1990).

332 Evolutionary Ecology of Oviposition Marking Pheromones ................................................................................................................................................ .. . . .............. .............

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While it is clearly adaptive to be able to recognize conspecifically marked sites, the ability to distinguish between own and conspecific marks should also be beneficial. When unoccupied oviposition sites become scarce, it may be adaptive to accept sites occupied by conspecific offspring for oviposition (van Alphen and Visser, 1990). Yet, while in conspecific superparasitism the fitness increment a female gains from an oviposition is usually identical with the survival chances of the second egg in the host, in self-superparasitized hosts fitness increments for the female are usually zero, since all fitness increments gained from survival chances of the second egg have to be subtracted from the fitness gains accrued from the first oviposition (but see Visser, 1993 for conditions under which self-superparasitism pays off). Clearly, variation in marking pheromones among individuals can impact parasitism decisions and associated fitness. In this section we will explore if and under what conditions evolution can favour inter-individual variation in marking chemicals. There are two means by which such variation could arise. Firstly, mutations could lead to alteration of compounds or changes in the relative amount of different components of a pheromone blend. Secondly, differencescould arise via the use of substancesthat function as additional pheromone components that differ between individuals, e.g. polar hydrocarbon differences as is known in social Hymenoptera (Espelie et al., 1994; Tsuji and Liu, 1999) or amino acids and terpenoids in social desert isopods (Schildknecht and Linsenmair, 1988). In the latter case, these differences could be genetically or environmentallydetermined. We will consider each of these evolutionarypathways in turn. As we have shown in Section 12.3.1, costs and benefits associated with the deposition of a marking pheromone depend upon the response of recipients, which may either be the marker, itself, a conspecific, or a natural enemy. Any variation as noted above can alter the relative costs andor benefits for the mutant marker via interactions with all three recipient classes. A further necessary complication is the means of recognition of the deviant mark that can either be innate or learned. This recognition could occur in any or all of the three recipients. Below, we will deal with the repercussions of the different possibilities. Whenever costs and benefits of a strategy depend upon the action of others, evaluation necessitates a game theory approach (Maynard-Smith,1982).Obviously, this condition applies to marking pheromones. Thus, we follow the classic game theory approach and ask under what conditions a mutant marker might invade a population of animals that uses identical oviposition marking pheromones. Invasion by a deviant marker (the mutant) is a necessary condition for the origin of variance in marking pheromones. However, for the maintenance of variation, no single successful invader should go to fixation. Otherwise, variation returns to zero, even though the mean population mark has changed. When evaluating the potential for deviant marks to invade, it is essential to acknowledge that an individual that carries a mutation for a deviant marking pheromone will not necessarily have the ability to recognize its own marking pheromone with certainty. Full recognition of the deviant mark might depend on

How Might Natural Selection Shape the Use and Chemistry of Oviposition Marking Pheromones?

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Table 12-1 Scenarios for the evolution and recognition of deviant marks. Column 2 refers to the mutation in the recognition system, column 3 to 5 refer to recognition abilities in the focal individual emitting the mutant mark, conspecifics emitting normal marks, and natural enemies of the markers, respectively. The first digit in columns 3 to 5 refers to the recognition of the mutant mark, the second digit to the recognition of the normal mark; 1 =full recognition, 0 = impaired recognition;* indicates a bearer of the mutation noted in column 2. A generalized recognition indicatesthat animals carrying that recognition mutation can fully recognize normal and deviant marks. A specific recognition, in contrast, indicatesfull recognition of one type of mark and partial recognition of the other. In the imprinting column, a 0 indicates innate response to marks, while a 1 indicates that focal and conspecific imprint on their own mark, while the natural enemy imprints on the mark it first encounters Conspecific Natural enemy Imprinting Scenario Mark recognition Focal 1 2 3 4 5 6 7 8 9 10 11 12 13 14

No

Generalized Generalized Generalized Specific Specific Specific No

Generalized Generalized Generalized Specific Specific Specific

0;l 1;1* 0;l 0;l l;o* 0;l 0;l 0;l 1;1* 0;l 0;l

0;l 0;l 1;1* 0;l 0;l l;o* 0;l 0;l 0;l 1;1* 0;l

l;o

0;l

*

0;l 0;l

0;1* 0;l

0;l 0;l 0;l 1;1* 0;l 0;l l;o 0;l 0;l 0;l

1;1* 0;l 0;l 0;1or l;O *

333.

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

0 0 0 0 0 0 0 1 1 1 1 1 1 1

a mutation in the recognition system of the animal. Thus, the problem is analogous to the invasion of a marker population by a non-marker (see Roitberg and Mangel, 1988),but differs in the respect that the deviant mark provides at least some benefit even if the mark is recognized only with some uncertainty. Moreover, an individual with a deviant mark is not necessarily able to recognize this mark as her "own" mark, but just as a different mark. Thus, producing a deviant mark does not necessarily allow the recognition of self vs. conspecific marked sites. Mutations in the chemical composition and the recognition system can occur in a variety of ways that are listed as different scenarios in Table 12-1. A full analysis of these different scenariosis beyond the scope of this chapter, thus at this point we will briefly explain what we believe are the most important scenarios and will analyse one of the more simple of these scenarios to demonstrate how individual variation in oviposition marking pheromones can be studied. 12.3.3.1 The Evolution of Mark Variability under Enemy Attack Imagine a population of insects that is comprised of individuals all of whom employ the identical marking pheromone and have the innate ability to fully recognize it. Further imagine that some developmental stage of these insects is attacked by a species of natural enemy that exploits the marking pheromone to

334 Evolutionary Ecology of Oviposition Marking Pheromones .. ..... ............................ ,. .. ..................... ,. ...,..................... . .................... . ................... .............................................................................. ................. , ,,

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,,,,,

, ,,

,

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.

locate its victims. Now imagine a “mutant” arises that deviates from the norm in that the marking pheromone deviates slightly in the chemical structure or composition of pheromone components from the rest of the population which we will call”norma1s”.We can now ask whether this mutant will increase in frequency in the population and if so, will go to fixation or reach a stable equilibrium, or whether it will be selected against and be driven to extinction. That depends upon at least 3 factors: (1)the cost to the mutant from not fully recognizing her own signal and possibly depositing eggs at sites where her offspring already exists (the cost would be from sibling competition); (2) the costs from competition between the mutant’s offspring and those from normals based upon the same impaired recognition as discussed above; (3) the benefit to the mutant if natural enemies were less likely to attack offspring when their ability to recognize the mutant’s offspring is impaired due to the structure of the mutant signal. Recall that the payoffs should be frequency dependent. For example, when mutants are rare they will less often encounter mutant marks and vice versa. We can think of at least 7 scenarios through which the above mentioned changes in the marking pheromone system could originate (Table 12-1, Imprinting 0). In the simplest scenario there is no coincident mutation in the pheromone recognition system and thus the mutant, normals, and natural enemies will all suffer from reduced recognition of the deviant mutant mark. Other, more complicated scenarios include simultaneous or subsequent mutations in the recognition system of either the mutant marker, a normal marker, or a natural enemy. These scenarios could be further subdivided into two classes, where recognition in the mutant recognizer is either generalized,and thus the deviant mark of the mutant marker and the normal mark are both recognized without impairment, or specialized, wherein the ability to fully recognize the mutant mark impairs the ability to recognize the normal mark. We can imagine another broad class of 7 scenarios where recognition of the mark is not innate, but imprinted (Table 12-1, Imprinting 1). In contrast to the innate recognition abilities discussed above, here recognition ability is dependent on historical events, i.e. which kinds of marks are first encountered. Furthermore, in these scenarios exploitation through natural enemies now becomes frequency dependent, plus there is a potential for self- and non-self-recognition of marks.

12.3.3.2 A Population Level Model of the Evolution of Mark Variability As an example, we now introduce a population level model for solitary herbivorous or parasitoid insects. Our objective is to determine the likelihood that a mutant marker can invade a population of normal markers in our simplest scenario from Section 12.3.3.1, i.e. with no mutant recognizers (and thus impaired mark recognition) and the simplistic assumption that mark recognition always leads to site rejection. In order to do this, we track the production of offspring of mutants and normals within and across generations until the evolutionary stable strategy (ESS) is established. At the beginning of each generation we assume that a fixed number of oviposition sites exists that are all equally accessible, uninfested, and unmarked. Over time, some of these sites are visited by normals and mutants. If

How Might Natural Selection Shape the Use and Chemistrv of Oviposition Marking Pheromones?

Table 12-2 Definition of oviposition sites as a function of oviposition and marking by

mutant and normal markers Oviposltionsite class

Definition

uninfested and unmarked sites sites infested and marked by normals sites infested and marked by mutants sites marked by mutants and normals and inhabited by mutants following survival from offspring competition sites marked by mutants and normals and inhabited by normals following survival from offspring competition Figure 12-3Transition pathways by which normal (n) and mutant (m)

markers convert empty oviposition sites (No)into sites that will produce normal (Nl, N4)or mutant (N2,N3) offspring and carry normal (Nl), mutant (N2),or both normal and mutant (N3,N4)marks. Here we make the simplifying assumption that upon recognition of a mark, a site is always rejected, and oviposition occurs only if no mark is detected. no mark is detected at such a site, the visitor oviposits and marks the site. If a site is already marked and the mark is recognized, the visitor refrains from ovipositing and marking. If an existing mark is not recognized, superparasitism and subsequent mortal competition between the inhabitants of the site will ensue. These actions create five different classes of oviposition sites (Table 12-2). The number of sites per class that exist at any given point of time is described by a model that consists of five difference equations (E12.1 to E12.5), which depend on the number of transitions (T) (see Appendix) between the aforementioned different states of sites (Figure 12-3):

4 t4 N , ( t + A r ) = N , ( t ) + 4 N n . N ,b 4 N z , N , )

Nn (t + At>= Nn (t>-

N,,N,

N,.N,

E12.1 E12.2 E12.3 E12.4

For example, in equation E6.1 the number of empty sites declines as sites are converted from uninfested to infested, normal-marked and infested, mutantmarked. Likewise, there is only one way to increase the number of N4-sites and that is for a normal to not recognize a mutant mark, oviposit and mark at this site and have its offspring win the competition.

335

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At the end of each generation, we expose the offspring within sites to natural enemies that respond to the marking pheromone on the site. Survivors from these attacks are summed up across sites for each of the different marker types. Those sums give us the frequencies for normal and mutant markers as follows:

where a p d = attack rate of predators and api = attack rate of predator independent of pheromone recognition

From these equations, we calculate and define fitness as the relative increase or decrease in frequency. Recall our postulate that high re-encounter rates with previously marked sites favour high recognition abilities. To explore this effect, our model contains a term that simulates re-encounter rates, which are caused by different strengths of area-concentratedsearch (Appendix)(Vinson,1977;Roitberg, 1985; Bell, 1991). Figure 12-4 shows the conditions for mutant invasion with respect to three important variables, which are (1) the probability of the mutant marks being recognized, (2)the aforementionedre-encounter term, and (3)the baseline offspring mortality caused by natural enemies. We present these results as an equal-fitness surface.Figure 12-4can be read as follows: along the equal-fitnesssurface, mutants and normals do equally well; above the equal-fitness surface are conditions that favour invasion by the mutant, whereas conditions in the space below the equalfitness surface do not allow invasion of the mutant marker. First, note that there are conditions under which a mutant marker can invade. However, a deeper look suggests some interesting complexities.For example, the impaired recognition of mutant marks has multiple, even offsetting effects. With decreasing recognition, mutants get penalized for self-superparasitism,but benefit from decreased exposure to natural enemies. Generally, with increasing recognition, higher levels of enemyinduced mortality are needed to favour mutant markers to compensate for the smaller differencebetween normal and mutant marks. Similarly,high re-encounter rates disfavour mutants and so we find that increasing levels of natural enemy attacks are needed to favour mutants as re-encounter rates increase. These interactions give the non-linear surface apparent in the Figure 12-4. Figure 12-4shows conditions for mutant invasion but it does not imply whether either normals or mutants go to fixation or coexist at an evolutionary stable equilibrium. In fact, in this simple scenario, there are no conditions for stable coexistence.Mutants either go to fixation or go extinct.Under conditionsthat favour mutants, an important question is whether the marking systemperseis evolutionary unstable, i.e. invadable by non-markers. One way to approach this question is to

.

How Might Natural Selection Shape the Use and Chernistryof Oviposition Marking Pheromones?

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Re-encounter term 8 Probability of mutant mark being recognized Figure 12-4 Equal-fitness surface for mutant genotypes that produce deviant marks with impaired recognition by the marker, conspecifics, and natural enemies, as a function of the probability of the mutant mark being recognized, likelihood of oviposition sites being reencountered,and the baseline level of enemy-inducedmortality.The region abovetheequalfitness surface would favour mutant markers over normal markers while the region below the equal-fitnesssurface would favour normal markers.

introduce a non-marking mutant and test whether this non-marker can spread in the population of mutant markers. Returning to the suite of scenariospresented in Table 12-1,we may briefly discuss which of these scenarios most likely allow the invasion of deviant marks and/or the coexistence of two or more types of marks. Scenarios 2 and 9 will allow the invasion of a deviant mark under the broadest set of conditions, because mutants pay no cost to impaired recognition but benefit from reduced mortality by natural enemies. By contrast, they receive no benefits but suffer extra mortality in scenarios 4 and 7. Regardless of the above, we can only find one scenario that will likely allow for coexistence and thus for the evolution of intraspecificvariance in marking pheromones (scenario 14).With negatively frequency dependent selection through imprinting natural enemies, mutants will be disproportionately more frequently attacked the more common they become. 12.3.3.3 The Evolution of Mark Variability without Enemy Attack

Up to this point we have demonstrated that natural enemies can facilitate the evolution of deviant marks and, under restricted conditions, mark variability. An important question is whether, and if so under what conditions, variation in marks would be favoured in the absence of mark-exploiting enemies. We have

338

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

demonstrated that the only way to allow the invasion of a mutant marker with impaired recognition is if the benefit from reduced enemy attacks outweighs the costs of increased self and conspecific superparasitism. The only other condition we can think of requires that the mutant marker can distinguish between self and conspecifically marked sites. This is not the same as scenario 2 and 5 in Table 12-1, where full recognition of own and conspecific marks is achieved. This does not necessarily lead to self versus non-self recognition. The adaptive nature of selfrecognition can only be understood in the context of marks providing information to animals that can adjust their oviposition decisions to differential benefits from ovipositing into sites where offspring competes either with sibs or non-related conspecifics. Recall, we constrain our argument to the general case that selfsuperparasitism of sites leads to no increment in fitness, while conspecific superparasitism generally provides limited fitness gains. We can think of no situation where the recognition of self-produced marks would not be beneficial, though the gains under some circumstances might be marginal. The existence of self-recognition has only been shown in a few systems (e.g. Volk and Mackauer, 1990; van Dijken et al., 1992; Danyk and Mackauer, 1993; Marris et al., 1996; van Baaren and Boivin, 1998a), though it may be very common. Recall that there are two means to generate individual variation in marks. The first is via point mutations affecting the pheromone blend; the second is via additional highly variable substances to the blend. In contrast to point mutations, there are no restrictions for the maintenance of mark variation if it is produced by the addition of substances to the pheromone blend. Thus, the critical feature of this category is the incorporation of the recognition of inherent variation in marking components. So long as additional variable substances in the marking complex are not recognized, they are, by definition, not components of the pheromone blend. We could imagine four scenarios for an evolutionary response to variation via the addition of substances (Table 12-3). In scenarios 1 and 3, there is innate vs. learned recognition of the main blend, but the ability for recognizing additional Table 12-3 Scenarios for the evolution and recognition of deviant marks without markexploiting natural enemies. Column 2 refers to the mutation in the recognition system, column 3 to 4 refer to recognition abilities in the focal individual emitting the mark

containing individual-specific information,conspecifics emitting normal marks, and natural enemies of the markers, respectively. The first digit in columns 3 to 4 refers to the recognition of the additional component to the mark, the second digit to the recognition of the normal mark; 1=full recognition, 0 = impaired recognition;* indicates a bearer of the mutation noted in column 2. A generalized recognition indicates that animals carrying that recognition mutation can fully recognize the normal pheromone blend plus additional individual-specificsubstances in the marks. In the imprinting column, a 0 indicates innate response to marks, while a 1indicates that focal and conspecific imprint on their own mark. Scenario

Mark Recognition Focal

Conspecific

Imprinting

1 2

No

Generalized

0;l 0;l 0;l 0;l

0 0 1 1

3

No

4

Generalized

0;l 1;1* 0;l 1;1*

Concluding Remarks

substances is lacking. In such a case, the presence of additional substances is evolutionarily neutral and a communication system employing these substances will not evolve. In scenario 2, innate recognition would just allow an animal to recognize differences in marks, yet no benefit accrues from such ability. In contrast, in scenario 4, which allows for learned recognition, we would expect the mutant recognizer to go to fixation, but counter-intuitively, that would allow variation to persist. In summary, we find a narrow set of scenarios that could lead to the maintenance of variation in marking pheromones over evolutionary time. What these scenarios tell us is that ecological circumstances are at least as important as are physiological or biochemical parameters for the origin and maintenance of mark variation.

12.4 Concluding Remarks It is now 20 years since two groundbreaking papers were written, dealing with the evolution and ecology of marking pheromone deposition (Prokopy, 1981a) and the flexible responses to these compounds (van Lenteren, 1981). Since that time an enormous amount of information has been gathered on the use of marking pheromones in plant parasitic and entomophagous insects (see Chapter 9; Godfray, 1994; Nufio and Papaj, 2001). Concomitant with this has been an explosion in the growth of communication theory (Dawkins and Krebs, 1978; Krebs and Dawkins, 1984; Wiley, 1994; Johnstone, 1997) which spurred conceptual advances on the evolutionary ecology of marking pheromones (Roitberg and Mangel, 1988; Hoffmeister and Roitberg, 1998).In this chapter, we reaffirmed this approach and have extended it to new areas, in particular to the question of the origin and maintenance of intraspecific variability in marking pheromone systems. Our work has discovered an enormous complexity associated with marking systems that make simple generalizations and predictions nearly impossible. However, we have demonstrated that with the appropriate approach, this complexity informs more than impedes us. Taking into account frequency and context dependent processes provides a guiding light for understanding the huge amount of variability (not noise!) found in oviposition marking systems in nature. Clearly, we have only scratched the surface of some of the areas of marking pheromone research that still warrant investigation. Currently, we have very limited knowledge about the variation in the chemical composition of oviposition marking pheromone blends within species, and even among species. To our knowledge, no comparative analysis with full phylogenetic weighting has as yet been applied to uncover ecological correlates associated with oviposition marking pheromones (cf. Nufio and Papaj, 2001). For example, our predictions from game theory analysis of HMP persistence requires testing of this sort, in order to explain the tremendous variation that can be found. Furthermore, there is still ongoing debate to what extent marking pheromones are innately recognized or learned (van Alphen and van Dijken, 1988). It is our hope that we have provided a firm theoretical foundation for studying aspects of this fascinating phenomenon.

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12.5 Acknowledgements We thankNSERC Canada, DAAD Germany, and the University of Kiel for financial support of this work. We are grateful to K.E. Linsenmair for valuable suggestions and Marcel Dicke, Ron Prokopy, Cesar Nufio, and an anonymous reviewer for comments on earlier versions of this chapter.

12.6 References Aluja, M, Boller, EF. 1992. Host marking pheromone of Rhagoletis cerasi: foraging behavior in response to synthetic pheromonal isomers. J. Chem. Ecol. 18: 1299-1311. Averill, AL, Prokopy, RJ. 1987. Residual activity of oviposition-deterring pheromone in Rhugoletis pomonella (Diptera: Tephritidae) and female response to infested fruit. J. Chem. Ecol. 13: 167-177. Bell, WJ. 1991. Searching Behuuiour: The Behuuioural Ecology of Finding Resources. Chapman and Hall, London. Charnov, EL, Skinner, SW. 1985. Complementary approaches to the understanding of parasitoid oviposition decisions. Environ. Entomol. 14: 383-391. Chow, A, Mackauer, M. 1999. Marking the package or its contents: Host discrimination and acceptance in the ectoparasitoid Dendrocerus carpenteri (Hymenoptera: Megaspilidae). Can. Entomol. 131: 495-505. Chow, FJ, Mackauer, M. 1986. Host discrimination and larval competition in the aphid parasite Ephedrus culifornicus. Entomol. Exp. Appl. 41: 243-254. Collins, RD, Carde, RT. 1985.Variation and heritability of aspects of pheromone production in the pinkbollworm moth, Pectinophoragossypiella (Lepidoptera: Gelechiidae).Ann. Am. Entomol. SOC.78: 229-234. Collins, RD, Card6, RT. 1989. Heritable variation in pheromone response of the pink bollworm, Pectonophoragossypiella (Lepidoptera:Gelechiidae).J. Chem. Ecol. 15:2647-2659. Danyk, TP, Mackauer, M. 1993. Discrimination between self- and conspecific-parasitized hosts in the aphid parasitoid Praon pequodorum Viereck (Hymenoptera: Aphidiidae). Can. Ent. 125: 957-964. Dawkins, R, Krebs, JR. 1978. Animal signals: information or manipulation? In: Krebs, JR, Davies, NB (eds.) Behauiourul Ecology. A n Evolutionary Approach, 1 edn. Pp. 282-309 Blackwell Scientific, Oxford. Dethier, VG. 1962. To Know a Fly. Holden-Day, San Francisco. Dicke, M, Grostal, P. 2001. Chemical detection of natural enemies by arthropods: An ecological perspective. Annu. Rev. Ecol. Syst. 32: 1-23. Dixon, AFG. 1998. Aphid Ecology, 2 edn. Chapmann and Hall, London. Endler, JA. 1986. Natural Selection in the Wild. Princeton University Press, Princeton. Espelie, KE, Gamboa, GJ, Grudzien, TA, Bura, EA. 1994.Cuticular hydrocarbons of the paper wasp, Polistesfuscutus: a search for recognition pheromones. J. Chem. Ecol. 20: 1677-1687. Ferguson, AW, Williams, IH. 1991. Deposition and longevity of oviposition-deterring pheromone in the cabbage seed weevil. Physiol. Entomol. 16: 27-33. Gadagkar, R. 1993. Can animals be spiteful? Trends in Ecology and Evolution 8: 232-234. Galis, F, van Alphen, JJM. 1981. Patch time allocation and search intensity of Asoburu tabida Nees (Braconidea), a larval parasitoid of Drosophila. Neth. J. Zool. 31: 596-611. Gauthier, N, Monge, JP, Huignard, J. 1996. Superparasitism and host discrimination in the solitary ectoparasitoid Dinarmus busalis. Entomol. Exp. Appl. 79: 91-99. Godfray,HCJ.1994.Purasitoids. Behavioral and Euolutionay Ecology. Princeton University Press, Princeton. Gould, SJ, Lewontin, RC. 1979. The spandrels of St.Marco and the Panglossian paradigm: A critique of the adaptationist programme. Proc. R. SOC.Lond. B 205: 581-598. Grafen, A, Johnstone, RA.1993. Why we need ESS signalling theory. Philosoph. Transact. Royal SOC.London B - Biol. Sciences 340: 245-250.

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ADDendix

Appendix Transition equations for population level evolution of deviant marks via point mutations in the pheromone - scenario 1(Table 12-1) In this scenario, a mutant mark arises by point mutation in the pheromone blend. Neither the focal individual producing the deviant mark nor conspecifics have complete recognition of this mark. Assuming that full recognition leads to rejection of marked sites and that partial recognition of sites marked with the deviant mark leads to rejection of marked sites with some probability (Y,) and oviposition into the site with probability (1 - Y,,), we can derive transitions between the 5 different states of sites, NO through N4, introduced in the population model in Section 12.3.3.2, i.e., the identity of the individual developing in the site and the kind of mark the site carries. To derive these transition equations, we assume the following. 1) All individuals within a population have a fixed amount of time T available during their lifetime. During that time T, mutually exclusive activities may be performed, i.e. searching for hosts, ovipositing into and marking hosts, and rejecting hosts. 2) Normals (n) and mutants (m) have a constant rate of search (a), 3) they are not egg-limited, 4) they search independently from one another, 5) there is a constant amount of time required to oviposit and mark a site (T,,),and (6) there is a constant amount of time required to reject a site where a mark was recognized (TJ. In addition to the 5 constants, we assume that animals display an area-concentrated random search pattern that leads to the increasing probability of re-encountering previously visited sites (ri(t)) as a function of a shape parameter (0) and the number of previous per individual. ovipositions (nj(t))

EA12.2 EA12.3 Given the assumptions above, we can characterize the transitions by a modified Holling disc equation (Holling, 1959).The essential feature of the Holling equation is that due to the mutual exclusivity of the aforementioned activities the time available for the search of oviposition sites declines with increasing time spent in handling oviposition sites. Thus, when the density of sites increases, exploitation of sites (N,) increases in a non-linear (decelerating) fashion, because increasing amounts of time are spent in handling oviposition sites:

EA12.4 In our model, this logic remains intact. However, we recognize the existence of different types of sites and consequently, time spent handling each of those types

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of sites. Whereas in the Holling model, all sites encountered are equal and are never rejected, we here assume rejection of marked sites as long as they are recognized as being marked. Thus, handling time in our denominator contains terms for 1) acceptance of hosts (i.e. ovipositing and marking) (TJ and rejection of hosts (Tr). Moreover, our denominator contains terms for the different types of sites, and the different types of handling time that can apply to these sites. For example, acceptance of a site can only occur when the site is either unmarked or when a mark is not detected. Likewise, rejection of a site can only occur when the site is marked and the mark is recognized. Also note that the denominator in each equation is divided into two processes, due to the re-encounter function that we introduced. The first considers random encounters with different kinds of sites at a global proportional abundance at probability (1 - -yi(t)), the second considers random encounters with different kinds of previously attacked sites (ni)at a local proportional abundance at probability (ri(t)). Furthermore, the number of each type of sites available in the habitat changes over time. While at t = 0 all sites are uninfested with eggs and do not contain marks (No),they may be turned into N l , N2, N3, or N4 sites over time, while the overall number of sites remains constant. Thus: EA12.5

The number of transitions from uninfested and unmarked sites to sites infested and marked by normals in the time interval At is:

The number of transitions from uninfested and unmarked sites to sites infested and marked by mutants in the time interval At is:

EA12.7

i

The number of transitions from sites infested and marked by mutants to sites marked by mutants and normals and inhabited by mutants following survival from offspring competition (sl) in the time interval At is:

EA12.8

Amendix

The number of transitions from sites infested and marked by mutants to sites marked by mutants and normals and inhabited by normals following survival from offspring competition in the time interval At is:

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Chapter 1 3 EvoIut iona ry Ecology of Oviposition Strategies Niklas Janz

Table of Contents 13.1 Introduction 13.2 Phylogenetic Patterns 13.3 Oviposition Site Selection 13.3.1 Searching, Finding and Accepting 13.3.2 Maternal Care 13.3.2.1 Quality versus Quantity 13.3.2.2 Egg Protection 13.3.3 The Pros and Cons of Laying Egg Clutches 13.3.4 Genetics, Plasticity and Learning 13.4 Specialization 13.4.1 Internal versus External Factors 13.4.2 The Cost of Information 13.5 Preference-PerformanceCorrelations 13.5.1 Why is the Correlation not Always Perfect? 13.5.1.1 Optima lity Hypotheses 13.5.1.2 Constraint Hypotheses 13.6 Concluding Remarks 13.7 Acknowledgements 13.8 References Abstract

Finding and choosing a good site for oviposition is a challenging task for females of herbivorous insects, and their decisions have far-reaching and profound consequences for the life history of the offspring. One of the most prominent features of host plant preference is that the range of host plants accepted for oviposition is often very narrow. The reason for this widespread specialization is a question that has puzzled researchers for many years, and even though interesting progress has been made, it still waits for a completely satisfying answer. The oviposition strategy of an insect is a complex trade-off between many, sometimes contradictory factors, including host plant range, clutch size, host quality, the difficulty of finding hosts of sufficient quality, the chances of finding even better

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hosts, predation risks on the female and her offspring, larval mobility and hostfinding capability, microclimate, etc. Thus, although the female’s prime goal may be to choose an oviposition site that maximizes offspring survival and performance the outcome is not necessarily a perfect match between female host plant preference and larval performance.

13.1 Introduction Most insect larvae are small and relatively immobile, so the ovipositing female often single-handedly makes the choice of larval food. This sets the stage for an interesting but complex problem. While choosing oviposition site, the female not only has to evaluate the suitability of the host as larval food, she must also take mortality risks into account, due to microhabitat and enemies. In addition, she must choose a strategy that maximizes search efficiency and host encounter rates, and she must decide on how long she should spend searching for the best host before accepting alternatives. Of course, she must also at all times ensure that she herself survives to lay her full complement of eggs. In short, the process of choosing oviposition sites must be a daunting task for a small insect. As there are so many variables involved, and so many insect species, it is understandable that the evolutionary solutions to this task are manifold. It is the objective of this chapter to outline some of the generalizations that nevertheless can be made, as well as some of the important exceptions. The subject of the chapter is very broad, and there will be some biases. A small fraction of the amazingly diverse group of insects stands for a lion’s share of the available studies. The groups that have received most attention are typically conspicuous and easy to spot and follow or have a large economic impact, e.g. as agricultural pests. As most of these groups are herbivorous, there will be a bias toward plant-feeding insects as well.

13.2 Phylogenetic Patterns Comparative methods offer an increasingly rich set of tools to investigate the largescale patterns behind insect oviposition strategies. Phylogenetic methods can be used to test for broad correlations between characters, investigate alternative historical scenarios, as well as to test specific hypotheses about historical sequences of events. The main limiting factor has been the poor availability of robust phylogenies with adequate resolution for the involved lineages. However, such phylogenies are slowly emerging, and as a consequence, so are studies using comparative methods to investigate the relationship between insects and hosts. Much of this work has been devoted to the questions of specialization, of conservatism in host associations and the role of host plant chemistry in determining the patterns of host use. The large and interesting issue of host specialization will be treated under its own heading below.

Phylogenetic Patterns

One of the earliest non-cladistic attempts at a broad comparative study of host use was Ehrlich and Raven’s (1964) seminal study on the coevolution between butterflies and plants. The main conclusions from this paper were that most butterfly-plant associations are relatively specialized and that there is a general tendency for related butterflies to be associated with related plants. They also proposed a coevolutionaryexplanation for these patterns based on plant secondary metabolites and escape and radiation of the associated lineages. A more recent reanalysis of the butterfly-plant system using more robust phylogenetic methods confirmed the general patterns of high specializationand conservatismin the group, but concluded that these patterns could not have been caused by the coevolutionary process that Ehrlich and Raven proposed (Janz and Nylin, 1998).Coevolution may still be an important process in insect-plant interactions, but it cannot be the cause of these large scale patterns. Even though the influence of host plant chemistry on the patterns of host use is often substantial (Becerra, 1997; Wahlberg, 2001), other factors may also have significant effects, such as habitat choice (Anderson, 1993),plant growth form (Janz and Nylin, 1998; Beccaloni and Symons, 2000) and diapause pattern (Carey, 1994). Predation has also been shown to affect oviposition patterns in many species (Bernays, 1989; Stamp and Bowers, 1993; Eigenbrode et al., 1995) and could potentially affect phylogenetic patterns of host use (but see Keese, 1997). A complicating factor for comparative studies of host use is to distinguish ovipositionpatterns from larval feeding patterns, which can often be quite different (see below). Host use in many herbivorous insects is really two characters, only partially correlated. At the same time, this complication offers an opportunity to understand how larval feeding capacities influence the oviposition strategies of the female. Following that reasoning, the complication has been turned into an advantage in a series of interesting studies on the history of host associations of the beetle genus Ophruellu (Futuyma et al., 1993; Futuyma et al., 1994; Funk et al., 1995; Futuyma et al., 1995; Keese, 1998).By screening the species in the genus for genetic variation in host use traits they have been able to demonstrate a connection between present oviposition patterns and historical host use. Genetic variation for larval feeding and survival was typically found on plants that are used as hosts by close relatives (e.g. Futuyma et al., 1995). Present oviposition patterns and past colonizationof novel hosts have thus been constrained by genetic variance for host use, which itself is influenced by historical host use (e.g. Funk et al., 1995). Using a somewhat similar approach, Janz et al. (2001) showed that this phenomenon is not unique for phyllophagous beetles. Larval survival on nonhosts in the butterfly tribe Nymphalini was biased towards plants that are used by other members of this group. In fact, almost all members of the tribe had some capacity to feed and survive on stinging nettles, the ancestral host plant of the tribe. Phylogenetic optimizations also showed a remarkably high number of colonizations and losses within the Nymphalini, certainly not conforming to the conservative pattern of host use found at higher levels in this same system. As a limited set of plant families was responsible for a majority of these changes in host

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use, it was concluded that there is non-independence in these patterns of host associations (Janzet al., 2001).Clearly,there is a historical component in the present patterns of host use, suggesting a complex interaction between "hidden" larval feeding capacities, oviposition preferences and host plant availability and suitability. Phylogenetic studies on different levels of resolution and studies on preference variation within and between conspecific populations have reinforced this somewhat puzzling paradox regarding the flexibility of oviposition preference hierarchies. The Papilio machaon species complex of butterflies is associated with the same general set of plants (mainlyApiaceae, but also Asteraceae and occasionally Rutaceae), but differ in ranking and degree of specialization. Thompson (1998) showed how modest modificationsin oviposition preference within a geographic mosaic of populations have resulted in the present pattern of host use among species in this group. Almost all studies that have investigated oviposition preference variation using individually controlled experiments have found significant individual variation within populations (Tabashnik et al., 1981; Rausher, 1983; Singer, 1983; Stanton and Cook, 1983; Thompson, 1988c; Janz et al., 1994; Bossart and Scriber, 1999; Sadeghi and Gilbert, 1999). Detailed studies on variation in oviposition preference in the butterfly E u p h y d y u s editha have also revealed evidence for considerabledifferencesin ovipositionpreference between populations in close geographical proximity (e.g. Singer et al., 1989; Radtkey and Singer, 1995; Singer and Thomas, 1996), painting a picture of oviposition preference as a relatively flexible trait allowing for rapid evolution of host associations. How does this conform to the conservative patterns found at higher levels?Janz and Nylin (2001) offered an explanation for this paradox, suggesting that the conservative patterns on higher levels are caused by the fact that many changes in host associations tend to go back and forth between the same plants, masking a good deal of the dynamics at lower levels and on an ecological scale. Thus, oviposition preference may be ecologically and evolutionarily flexible and opportunistic, but only within certain constraints, set by the ability to feed and survive on the plants. This also suggests that, with enough knowledge of a system, we should be able to make predictions about future colonizations and host shifts, which could be of high importance for pest management and control.

13.3 Oviposition Site Selection The process whereby an ovipositing insect approaches a potential host plant and decides whether to lay an egg on it or not involves several behavioural phases, and engages to varying degrees all insect senses. These phases include the choice of habitat, the approaching of a plant from a distance, the decision to land on the potential host or not, the decision to ovipositor not, and, when applicable, the size of the egg batch (e.g. Jones, 1991; Bernays and Chapman, 1994; Schoonhoven et al., 1998).It is necessary to emphasize at an early stage that the relative importance of the various host finding phases and the senses used in each phase will vary

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considerablybetween species. Perhaps the most important behavioural distinction lies between pre- and post-alighting decisions, i.e. the decisionsleading the female to approach and land on a plant and the decisions to oviposit on the plant after encountering it. These terms refer to fundamentally different behavioural processes; the first involves searching for a potential host, the other involves assessment of host quality leading to acceptance or rejection of the host. Potential hosts will usually vary in quality. Preference is generally expressed as a hierarchical ranking of acceptablehosts. Wiklund (1975,1981)first drew attention to the hierarchical nature of host plant ranges. Female Papilio machaon butterflies were more likely to oviposit on some plants, but will accept lower ranked plants to a lesser degree, especiallyif the higher-ranked plants are not available. The degree to which an ovipositing insect discriminates against plants lower down in the hierarchy is referred to as its specificityor degree of specialization.Wiklund (1981) proposed a dual function for the preference hierarchy: to ensure that most eggs are oviposited on the optimal host plant as long as it is present, and to make possible the deposition of eggs on sub-optimal plants when the optimal host plant is not present. The hierarchical nature of host preference has been a fundamental key to understanding the evolutionary ecology of oviposition strategies (Singer, 1982; Singer, 1983; Courtney et al., 1989; Singer et al., 1992; Thompson, 1993; Singer, 2001).

13.3.1 Searching, Finding and Accepting The first task facing a female insect about to oviposit is to find a suitable patch in which to search for hosts, i.e. where suitable hosts are relatively abundant. It is clear that ovipositing insects do have an ability to aggregate in patches with high local host availability (Wiklund and Ahrberg, 1978; Stanton, 1982; Thomas and Singer, 1987).Sometimes habitat choice can even overshadow the choice of actual host species (Kibota and Courtney, 1991). Both olfactory and visual cues can be important at this stage, as in the next: the location of a potential host from a distance (Bernays and Chapman, 1994; Schoonhoven et al., 1998).However, the extent to which insects actually search for their hosts differs among species. While some species use visual search images of shape, size or spectral quality to identify host plants from a distance (Prokopy and Owens, 1983),others use olfactory cues to direct movement towards the source of the odour (Willis and Arbas, 1991; Meiners et al., 2000). Quite often the visual and olfactory stimuli must interact to cause arousal and directed movement in the insect (Schoonhoven et al., 1998). However, some herbivorous insects that use small, inconspicuous, or otherwise "unapparent" plants as hosts can show a more or less random flight behaviour within a patch of high host abundance, landing on hosts and non-hosts roughly according to their abundance (Wiklund, 1977; Stanton, 1982;Parmesan et al., 1995). After alighting on a potential host, the female must decide whether to oviposit or not. While previous decisions may involve search images, learning and other methods to maximize host encounter rates, the post alighting decisionsare probably

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more focused on evaluating the quality of this particular host in relation to other potential hosts (Papaj and Rausher, 1987). Furthermore, while the pre-alighting phases mainly involve visual and/or olfactory cues, the post-alighting phase is probably more based on contact chemoreception, although olfaction and vision may still play a role (Bernaysand Chapman, 1994).Some species, such as parasitoids and fruit or seed feeders, ovipositinto hosts or host structures that can only support a very limited number of larvae. These species may also use cues that do not originate from the host to discriminate against already occupied hosts, such as the presence of conspecific larval frass (e.g. Hilker, 1985; Hilker and Klein, 1989) or deterrent pheromone markings from other females (see Chapter 9). As the insect at least in part evaluates different types of cues during the different phases, preference trials could potentially produce divergent results, depending on what behavioural phase or phases the trial is designed to investigate. Most oviposition trials are based on one of two methods: either they use simultaneous choice trials to pickup a combined effect of the pre- and post-alighting phases ( e g Wiklund, 1974; Wiklund, 1975; Thompson, 1993; Janz and Nylin, 1997),or they use sequential trials to measure only the post-alighting decisions (e.g. Singer, 1982; Singer, 1986; Singer and Lee, 2000). Both these methods have their strengths and weaknesses, but it is troublesome that so few attempts have been made to compare their outcomes (cf. Janz, 1999; Janz and Thompson, 2002).

13.3.2

Maternal Care

13.3.2.1 Quality versus Quantity The fitness of an ovipositing female is determined by the product of her realized fecundity and the mean fitness of her offspring. The tremendous variation in egg size among insects indicates that the resource invested per individual offspring is quite variable (Fox and Czesak, 2000). Wiklund and Persson (1983) pointed out that the time spent by a female searching for good sites for oviposition can be seen as a form of maternal care that counterbalances the lower oviposition rate. By spending more time searching for a good oviposition site, she assures a higher fitness for her offspring.The female may increase her oviposition rate by including lower-ranked hosts in her repertoire, but the eggs laid on these plants will have a lower chance of surviving and reproducing. Thus, host plant specificity can be seen as a form of maternal care in itself. At the other endpoint we have species where the choice of host plant has been almost completely displaced from the female to the larvae. In these cases, the first instar larvae have developed adaptations, such as ballooning, that allow them to search and find host plants by themselves (e.g. Marques et al., 1994; Tammaru et al., 1995). This opens for a potential conflict between the mother and her offspring: they may not agree on what is a suitable host plant or oviposition site (Roitberg and Mangel, 1993; Weisser et al., 1994; Nylin et al., 1996; Scheirs et al., 2000).The tradeoff will be most pronounced in situations where the best larval host plant is rare. In such cases females may search for a poorer plant that is more abundant, as long

oviposition Site Selection

as this increases her oviposition rate more than the loss in larval fitness (cf.Bernays and Chapman, 1994).Thus, even when the ranking of hosts by ovipositing females and larvae is in close agreement, there could be a potential trans-generational conflict in the degree of specialization on the “best” hosts (Nylin and Janz, 1996) (compare also Section 13.3.4 and Section 13.5).

13.3.2.2 Egg Protection In addition to the search for suitable oviposition sites, the type of protection the female provides for the offspring can vary substantially, from purely behavioural to physical or chemical protection. Perhaps the most common (and presumably the least costly) form of egg protection is camouflage.Most eggs have some degree of crypsis on the normal oviposition sites. Many species also oviposit on structures of the host where the egg is more difficult for a predator to see, such as the leaf edges. In some species the eggs (or scales covering the eggs) are instead brightly coloured. This is typically interpreted as aposematic signalling of unpalatability in the form of chemical or physical protection (Rothschild, 1992; Floater, 1998). Physical protection may be provided behaviourally and morphologically. The female may physically protect the offspring by guarding the eggs or young larvae (e.g. Trivers, 1972; Mappes and Kaitala, 1994; Talamy, 1999).The golden egg bug, Phyllornorphu luciniutu, has an even more refined strategy: The females protect their eggs by ovipositing on the backs of conspecific males and other females, where the eggs gain protection from their bearers (Kaitala and Axen, 2000; Kaitala et al., 2000; Miettinen and Kaitala, 2000).Physical protection by morphologicalstructures may be provided by formation of tough eggshells and other structures that make the eggs more difficult to handle for predators (e.g. Floater, 1998; Gerson et al., 1998).For example, covering the eggs with faeces may make them less accessible towards predators (Arakaki, 1988; Damman and Cappuccino, 1991). The diversity of chemicalprotection of eggs is enormous, and is comprehensively treated in Chapters 3 to 5. In some species the protective chemicals are transferred to the ovipositing female via the spermatophore as a nuptial gift at mating (Dussourd et al., 1991; Conner et al., 2000),which adds another layer of evolutionary complexity to the issue (compare Chapter 4). Evolutionary questions on the endowment of eggs with protective chemicals will be considered below (Section 13.3.3)in context with egg clustering and aposematism. In conclusion, egg protection can be seen as a form of maternal care, whereby the ovipositing female increases the chances of survival for her offspring by providing some kind of defence for the eggs, which would otherwise be more exposed to enemies. If also the father is able to invest in egg protection, it may be the female’s choice to mate preferably with those males that invest most (e.g. Zeh and Smith, 1985; Tallamy, 2000; and see Chapter 4). In most cases, egg protection probably carries a substantial cost, whether the protection is chemical, physical or behavioural. Thus, a female that invests heavily in egg defence will not be able to lay as many eggs as a female that does not protect her eggs. Hence, the investment in egg defence is very similar to the trade-off between quality and quantity of

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offspring discussed in the previous section. Egg protection should thus be correlated with degree of specialization, especially in cases where the protective function is more or less tied to a specific host.

13.3.3 The Pros and Cons of Laying Egg Clutches One of the most striking aspects of insect oviposition strategies is the variation in clutch size. While females of some species always lay their eggs singly, other species lay several hundred eggs per batch. Parasitoid systems are especially well suited for these questions,because the resource to be exploited is particularlywell defined, but many of the problems apply to herbivorous insects as well. Much effort has gone into trylng to understand the causes of this variation,but the answer is elusive. There are about as many theories as there are researchers. Many researchers have found a positive correlation between clutch size and the size or local density of the food resource (Pilson and Rausher, 1988; Vasconcellosneto and Monteiro, 1993; Lemasurier, 1994; Fox et al., 1996; Zaviezo and Mills, 2000). Egg clustering for a time-constrained ovipositing female may be advantageous if it reduces search time (Courtney, 1984).Under some circumstances it can also decrease the egg desiccation risk (Clark and Faeth, 1998).For species that lay the eggs openly on the host (like many herbivorous insects),it has been suggested that the production of toxic or otherwise protected eggs and larvae (who often feed communally on the host) will favour the evolution of egg clustering (Stamp, 1980). Communal feeding can in itself increase larval survival, either by allowing them to more efficiently utibze the food resource (Crowe,1995),or by predator deterrence (Gamberaleand Tullberg, 1996;Gamberale and Tullberg,1998).Finally, competition over limited resources has been suggested to reduce clutch size (Vasconcellosneto and Monteiro, 1993; Visser, 1996; Visser and Rosenheim, 1998). Part of the reason why clutch size variation has proven so hard to explain is probably because clutch size is part of a tightly co-evolved trait complex, where causal relationships can be complicated to tease apart. The relationship between unpalatability of eggs and larvae, clutch size, and aposematic coloration can serve as an example of this, and of how a combination of experimental, phylogenetic and theoretical studies can be used to disentangle even closely intertwined characters like this. As mentioned above, toxic or otherwise unpalatable eggs and larvae have been suggested to favour the evolution of egg clustering. Or is it egg clustering that favours the evolution of unpalatable eggs and larvae?Unpalatability is often coupled with aposematic signalling, adding yet another layer of complexity to the picture. Using phylogenetic techniques, Sillen-Tullberg(1988)showed that aposematic coloration typically preceded group living in butterfly larvae, rejecting the hypothesis aposematism must evolve by kin selection (Harvey, 1983).Instead, aposematism seemed to favour the evolution of egg clustering (and larval gregariousness). Experimental and theoretical studies have confirmed this by demonstrating that aposematic (and distasteful) larvae often survive predator attacks (Wiklund and Jarvi, 1982; Tullberg et al., 2000), and that larval aggregations

Ovioosition Site Selection

will increase the effect of the aversive signal (Gamberale and Tullberg, 1996; Gamberale and Tullberg, 1998).

13.3.4 Genetics, Plasticity and Learning The factors determining oviposition preference are still relatively poorly known, and this is an interesting field that deserves much more attention in the future. A number of studies have demonstrated a high heritability of oviposition preference, indicating a strong genetic component in host choice (e.g. Tabashnik et al., 1981; Singer et al., 1988; Carriere and Roitberg, 1995; Barker and Starmer, 1999).Several studies have also reported a large effect of major genes on host plant use (Guldemond, 1990; Sheck and Gould, 1993; de Jong et al., 2000; Craig et al., 2001). Furthermore, there is evidence for sex linkage of oviposition preference, especially among the Lepidoptera where a substantial part of the species and population differentiating variation appears to be concentrated in the paternally inherited X chromosome (Thompson, 1988b; Scriber et al., 1991; Scriber, 1994; Janz, 1998,2002). These observations indicate that oviposition preference can sometimes be part of large co-adapted gene complexes, which may play an important role in population and species differentiation (Charlesworth et al., 1987; Hagen and Scriber, 1989; Scriber, 1994; Sperling, 1994; Hagen and Scriber, 1995). Perhaps more surprising is the common observation, across several taxonomic groups, that oviposition preference and larval performance are under different genetic control. This was suggested in early studies on butterflies and weevils (Wiklund, 1974; Wasserman and Futuyma, 1981),and has later been confirmed in as diverse organisms as papilionid butterflies (Thompson, 1988b; Thompson et al., 1990), nymphalid butterflies Uanz, 1998; Janz, 2002), chrysomelid beetles (Keese, 1996),and aphids (Guldemond, 1990)(comparealso Section 13.3.2.1and Section 13.5). As it must be very important for the female to oviposit on plants that the larvae can feed most efficiently on, why is not a good correlation assured by linkage or pleiotropy? Part of the answer may lie in the asymmetrical relationship between the traits. While ovipositions on plants that the larvae cannot survive on are very costly for the female, there is not necessarily a high cost associated with the capacity to feed on a wider range of plants than the female normally oviposits on. On the contrary, this would probably be adaptive, as females sometimes make oviposition mistakes (see below) and the original food plant can be depleted. Different genetic determination of preference and performance also permits a larger flexibility in host plant selection, allowing the female to base her choice on other criteria than larval performance. Such flexibility in host choice can be achieved by phenotypic plasticity in preference, including learning. Plasticity in oviposition preference appears to be very common. For example, many studies have shown changes in preference caused by the insect's motivational state, as defined by egg load or time since last oviposition (Singeret al., 1992; Prokopy et al., 1994b;Ueno, 1999; Agnew and Singer, 2000). Adjustment of preference based on physiological state allows the female to

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oviposit on the best hosts when they are readily available, but to accept poorer hosts after prolonged times without oviposition. Learning has been demonstrated from a number of taxa of ovipositing insects and appears to be an important means of preference modification in many species (Papaj and Prokopy, 1989; Papaj and Lewis, 1992). While motivational changes typically affect post-alighting decisions, i.e. the acceptance of a host after finding it (e.g. Prokopy et al., 1994b), most examples of learning by ovipositing females involve the host searching stage (Rausher, 1978; Prokopy et al., 1994a; Allard and Papaj, 1996).The most common objective of learning in insect oviposition is to avoid wasting time on preferred hosts that are rare. Thus, although the underlying mechanisms may be different, both these types of plastic adjustment of preference allows the female to maintain a rigid, genetically determined rank order of hosts, with retained flexibility in the face of uncertainty. The role of adult learning in oviposition preference is well established as a fairly common phenomenon (Papaj and Lewis, 1992). From time to time, it has been suggested that oviposition preference can also be affected by exposure of immature stages to different hosts. According to this hypothesis, host plant exposure (feeding) results in an ”imprinting” on the larva that remains through metamorphosis. The imprinting then results in an elevated preference in the ovipositing female for the larval host plant. This idea originated in the first part of the last century (Hopkins, 1917; Craighead, 1921)and has come to be known as the “Hopkins’ host selection principle” or pre-adult experience. The hypothesis requires a mechanism for transferring information of larval experience through metamorphosis to the adult female. Corbet (1985) proposed that chemical cues originating from the larval food could persist within the pupa to affect the emerging adult insect (the “chemicallegacy hypothesis”).The transfer of memory information through metamorphosis appears possible in principle (Elamrani et al., 1991; Tully et al., 1994),but its relevance for host preference has yet to be convincingly demonstrated (Veltman and Corbet, 1991; Barron and Corbet, 1999). Several studies have tried to experimentally test the Hopkins host selection principle. The overwhelming majority of these studies have failed to find empirical support for it (Wiklund, 1974; Tabashnik et al., 1981; Williams, 1983; van Emden et al., 1996; Rojas and Wyatt, 1999; Solarz and Newan, 2001). Moreover, early adult learning in species that pupate on or in the host (e.g. Cortesero and Monge, 1994) can easily be confused with pre-adult learning. Nevertheless, a few studies have indicated that information on larval feeding experience might indeed have passed to the ovipositing female and affected host choice (e.g. Anderson et al., 1995; Bossart and Scriber, 1999).Thus, it cannot be entirely ruled out that this process might play a role under certain circumstances, and it may be worth investigating what those circumstances are. From the scattered studies made thus far, we can conclude that a vast majority of the observed preference variance in nature has a genetic background, often modified by plasticity and adult learning. However, the relative importance of these

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factors across insect taxa remains to be determined. The same can be said about the contribution of sex chromosomes vs. autosomes and of major genes vs. genes with quantitative effects to the observed preference variation within and among individuals and populations, and across species.

13.4 Specialization Host plant range is a prominent and much debated feature of the interaction between herbivorous insects and plants. In most insects, the ovipositing female determines the feeding site for her offspring. Thus, the behavioural processes of egg deposition probably play a large role in host range evolution. A vast majority of insects are relative specialists, i.e. they oviposit on (or in) and feed on a very restricted number of host species (Janzen, 1988; Futuyma, 1991; Bernays and Chapman, 1994; Thompson, 1994; Schoonhoven et al., 1998). Considering the obvious advantages of being able to use a wider set of resources, this bias towards specializationis puzzling and calls for a general explanation (e.g. Futuyma and Moreno, 1988).Although present across most insect taxa, the pattern is not universally true; a few groups contain mostly generalists,such as Orthoptera (Bernaysand Minkenberg, 1997),and the groups that are dominated by specialists typically include polyphagous exceptions (Janz and Nylin, 1998; Janz et al., 2001). If there is such a strong push towards specialization,why is the trend not universal? There is a need to explain not only the rule of high specialization, but also the exceptions of polyphagy (Janz, 1999). Scriber (1973)demonstrated a latitudinal trend among papilionid butterflies in degree of specialization: Papilionidae species tend to have wider host ranges in temperate than in tropical areas. In a more recent study, comparing Papilionidea (excludingHesperiidae)in South-EastAsia and the western Palearctic,Fiedler (1998) found no corresponding increase in host plant range in the temperate regions. In fact, the pattern was reversed for one group of lycaenid butterflies. Thus, if there is a relationship between host range and latitude, it is not likely to be a simplistic increase or decrease. Moreover, as Fiedler (1998) pointed out, taxonomic idiosyncrasies can render it difficult to draw generalized conclusions. However, the relationship between latitude and host plant range need not be static and uniform. There are more subtle indications of a connection between parts of the temperate regions and increased host plant ranges. Nylin (1988),and Scriber and Lederhouse (1992)hypothesized that specialization in many temperate species will depend on voltinism patterns (see also Hodkinson, 1997). The number of generations a species can fit into the period with favourable temperatures will depend on latitude. The length of the season will force the females to use different oviposition strategies. In regions where it is just about possible to have a partial second generation, there will be strong selection to use plants that can sustain fast larval development. On the other hand, in some regions there will never be enough time for a second generation and selection to focus on the "fastest" host plants will be relaxed, allowing for other plants to be incorporated into the repertoire.

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A long-standing issue is whether host specialization is a directional process leading to an evolutionary dead end (see Thompson, 1994). While it seems clear that host specificity is not irreversible, recent phylogenetic studies have shown that there can exist a trend toward increasing specialization, at least within certain lineages (Wiegmann et al., 1993; Kelley and Farrell, 1998).Yet, other studies have shown that the trend is not universal (Thompson, 1998); some groups even show a reversed trend (Janz et al., 2001; Termonia et al., 2001). The most important conclusion from these studies is probably that host range evolution is very dynamic, with repeated host range expansions followed by re-specialization. The trend for any particular group depends on the evolutionary phase the group happens to be in at the time-slice under investigation (Janz et al., 2001).

13.4.1 Internal vs. External Factors The factors influencing insect diet width are often divided into chemical and ecological aspects. However, just as most chemical factors act through ecological interactions, many of the ecological interactions involve chemical stimuli (Feeny, 1992). Chemistry and ecology are thus closely intertwined. A more accurate terminology would perhaps be to distinguish between internal and external factors, where internal factors refer to features intrinsic to the insect-plant system, such as plant chemistry, herbivore behaviour and physiology, and plant and herbivore genetics. External effects then include all extrinsic factors acting on the insect-plant interaction, such as ecological interactions with other organisms (Table 13-1). There is little doubt that the chemical constitution of the plants is of importance for their suitability as hosts for herbivorous insects (see Chapter 7). It has long been Table 13-1 Factors influencing evolution of oviposition behaviour of herbivorous insects (A) INTERNAL FACTORS OF PLANT-HERBIVORE INTERACTIONS

Plant factors Plant genome and phenotype Plant physiology Chemical cues Physical cues; plant architecture Plant density Plant distribution

Herbivore factors Female: -Genome and phenotype - Physiology (e.g.age, egg load) -Search time; fitness Offspring (eggs,larvae, pupae): -Performance of offspring on host chosen by the female (phenotype,plasticity and selection)

(B) EXTERNALFACTORS OF PLANT-HERBIVORE INTERACTIONS

Abiotic conditions (soil,temperature, humidity, etc.) - effects on herbivores -effects on plants Competitors - of herbivores -of plants Enemies of herbivores -effects on females -effects on offspring (eggs,larvae, pupae)

Specialization

known that the insects use chemical cues when identifying plants for oviposition (Dethier, 1941; Fraenkel, 1959; Feeny, 1992). Similarly, it is obvious that growth and survival of larvae are affected by plant chemistry (e.g. Thorsteinson, 1960; Scriber, 1988; Zangerl and Berenbaum, 1993).Early models of coevolution and host tracking had a strong emphasis on plant chemistry (eg. Ehrlich and Raven, 1964; Feeny, 1975; Berenbaum, 1983; Jermy, 1984) and a recent phylogenetic study on melitaeine butterflies and their hosts also showed that at least these species do track certain aspects of host plant chemistry (Wahlberg, 2001). The most commonly used explanation for host plant specificity is the difficulty for the feeding larvae to cope with the diverse chemistry of angiosperm plants (e.g. Schultz, 1988; Feeny, 1990; Feeny, 1992; Zangerl and Berenbaum, 1993; Dobler et al., 1996; Becerra, 1997).The reasoning is that the specific adaptations needed to handle the specific chemicals in one plant (or set of plants) will interfere with the capacity to efficiently utilize other plants, with a different set of secondary compounds. Much effort has gone into demonstrating such a trade-off in feeding efficiency across plant species and results are mixed. Most studies have presented disappointing results (e.g.Scriber and Feeny, 1979;Futuyma and Wasserman, 1981; Futuyma et al., 1993; Carriere and Roitberg, 1994; Fox and Caldwell, 1994; Futuyma et al., 1994; Thompson, 1996; Camara, 1997; Keese, 1998),but a few studies have found evidence for it (Via, 1991; Mackenzie, 1996; Traxler and Joern, 1999).The difficultieswith demonstrating such trade-offshave led many authors to shift focus towards other explanations for the widespread host specificity. Bernays and Graham (1988) argued that the role of plant chemistry in insectplant interactions has been overemphasized and that generalist predators instead play a dominating role in the evolution of host range. This ignited an intense discussion on the role of plant chemistry as opposed to external factors such as enemies (see Strong, 1988).However, there is no need to polarize, there is too much evidence on the importance of both internal and external factors for any of them to be ignored. Indeed, plant chemistry and predation on the herbivorous insects (as well as other factors such as plant abundance) probably often interact as causes of specialization.When an insect feeds on a plant with a chemical composition it handles poorly, the most direct consequence is that it grows slower. Slower growth may imply that the insect is exposed to enemies for a longer time and it is therefore more likely to die from predation (Feeny, 1976; Courtney, 1988; Bernays, 2001). However, the empirical evidence for this slow-growth-high-mortalityhypothesis is somewhat equivocal (Williams, 1999).

13.4.2 The Cost of Information At least a couple of circumstances suggest that explanations for host specificity should be searched for primarily in female oviposition behaviour, rather than in larval growth and survival.First, in most plant-feedinginsects, the choice of feeding location for the larvae is more or less exclusively made by the ovipositing female. In any case, the female choice precedes any additional larval choices. Furthermore,

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females are often more restricted than larvae in their acceptance of host species (Wiklund, 1975; Smiley, 1978; Roininen and Tahvanainen, 1989; Penz and Araujo, 1990),which indicates that it may be fruitful to search for the primary restrictions on host specificityin the host searching strategies of egg-laying females. One such theory that has recently gained renewed attention focuses on the role of neural limitations in host range evolution ( e g Bernays and Wcislo, 1994; Bernays, 2001). Levins and MacArthur (1969) first drew attention to the fact that evaluating host plant quality will be increasingly difficult with increasing host range. Although some researchers returned to this idea during the following decades (Courtney, 1983; Futuyma, 1983), the idea did not really catch on until a few years ago with several both theoretical (e.g. Fox and Lalonde, 1993; Larsson and Ekbom, 1995; Bernays, 1996; Dall and Cuthill, 1997; Holmgren and Getz, 2000; Shelton, 2000) and empirical studies (e.g. Nylin and Janz, 1993; Janz and Nylin, 1997; Bernays and Funk, 1999; Nylin et al., 2000; Bernays and Bright, 2001). Discriminatingand evaluating several host species can be very challenging (Fox and Lalonde, 1993; Nylin and Janz, 1993; Larsson and Ekbom, 1995; Nylin et al., 2000). The outcome will largely depend on the cost of acquiring the relevant information to tell the host apart from non-hosts (Kotlerand Mitchell, 1995).Insects searching for oviposition sites must locate and identify an appropriate host species, host individual, and often a suitable part of the host, against a very complex background of competing stimuli, both physical and chemical. Oviposition on a host that cannot support larval growth and survival, and failure to oviposit on a host that can, will be very costly. In a complex sensory world, adoption of highcontrast signals would be expected to ensure rapid and appropriate responses (Bernaysand Wcislo, 1994).However, each host in an insect's repertoire will carry its own identification problems. They will contain different chemicals, different concentrations and mixtures of similar chemicals, as well as specific mortality risks not only by toxic effects,but alsoby e.g. desiccationand predation. It is quite possible that the same chemical cue will have different implications for different hosts. Individual variation between conspecificplant individuals will further complicate the problem (Shelton, 2000; Singer and Lee, 2000). Thus, with increasing host range, it will be increasingly difficult to find the unambiguous noise-free signals that are necessary to make correct decisions fast. An insect that incorporates several host types into its repertoire will need to allocate more of its neural capacities towards discriminating and evaluating alternative host plants and will have to pay by a reduced efficiency of neural processing. The costs associated with this need to identify and assess several host plant species can be of two types: the insect will have to pay with either a longer decision time or a higher error rate (Bernays, 1998). This hypothesis might go a long way towards a general explanation for the predominance of specialized oviposition strategies,especially since empirical support comes from such different insect groups as butterflies, grasshoppers and aphids (with indirect support from even more groups [Bernays, 20011). Relating the neural constraint hypothesis to the discussion of internal and

Specialization

Climate (length of season) phenology Plant chemistry

Host abundance and

Enemy load Predictability Female life-span

J Metabolic efficiency Search efficiency Life-cyclefit

Fecundity Risk spreading Diet mixing

Figure 13-1“The balance of specialization”: a visualization of host range evolution,showing the major fitness advantagesof specialized and generalized oviposition strategies.Therelative weightofthesefitnessadvantageswill depend onthestateand importanceoftheecological factors listed above.

external factors, Bernays (2001)argues that the main problem that plant chemical diversity causes for ovipositing insects is not to detoxify or metabolize these compounds, but to evolve ways to detect distinctive signals that allow fast and accurate identification of each particular plant chemotype. Failure to do so will result in higher error rates or in prolonged decision times, which will both have serious fitness consequences. Prolonged decision times will reduce vigilance against predators, by forcing the insect to direct more attention towards host identification and evaluation. With this view, it is largely the interaction between internal and external factors, mediated by insect physiology and behaviour, which makes it difficult to evolve and maintain wide host ranges (Bernays, 2001). To summarize, host range evolution, the range of plants actually used by females for oviposition, can be visualized as a balance between various fitness correlates (Figure 13-1).If we picture a “balance of specialization”,fitness advantages such as increased fecundity, risk spreading and diet mixing (when applicable) will be in the generalist scale. In the specialist scale we find increased efficiency in host use, including metabolic efficiency as well as efficiency in host finding and identification. Specialization will also allow a better fit of the insect’slife cycle to

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that of its host, and increased enemy protection in the form of host-specific defence features. A number of ecological factors will then determine how much weight should be put in each of these scales, and the balance will tip over towards increased or decreased specialization. A highly specialized species, with a life cycle that is tightly co-adapted to its host, will not easily become a generalist. Still, if local conditions were to change, the road of generalization will always be open.

13.5 Preference-Performance Correlations In most ovipositing insects, the site chosen by the female at oviposition determines to a high degree where the emerging larva will live and feed. It is therefore a reasonable assumption that there should be a close match between the hosts that femalesprefer to oviposit on (or in) and the hosts where larval fitnesswill be highest. This presumed correlation between female oviposition preference and offspring performance has been the focus of much research for several decades (Thompson, 1988a)(compare also Section 13.3.2.1and Section 13.3.4). Many studies have demonstrated a good correspondence between preference and performance (eg. Via, 1986; Singer et al., 1988; Craig et al., 1989; Siemens et al., 1991; Hanks et al., 1993; Nylin and Janz, 1993; Barker and Maczka, 1996). However, there appears to be an equally large number of studies where a correlation could not be found (e.g. Rausher, 1979; Penz and Araujo, 1990; Valladares and Lawton, 1991; Fox and Eisenbach, 1992; Burstein and Wool, 1993; Fox, 1993; Underwood, 1994; Larsson et al., 1995).

13.5.1 Why is the Correlation not Always Perfect? It may appear peculiar that so many studies have reported a poor correlation between preference and performance, but there are several possible explanations for a weak relationship, thoroughly reviewed by Thompson (1988a)and Thompson and Pellmyr (1991).Generally,these explanations fall into one of two main groups, summarized in Table 13-2. (1)Optimality hypotheses: the females are indeed making the correct choices to maximize offspring success, but they base their choices on one or several parameters that have not been measured, or (2) Constraint hypotheses: the females are not optimal in their prediction of offspring performance, due to rapid changes in the environment or to physiological or phylogenetic constraints. Some of these explanations have been touched upon above (see Sections 13.3.2.1 and 13.3.4),while some will be dealt with more comprehensively below.

.13.5.1.1 Optimality Hypotheses The most obvious reason for a poor correlation between preference and performance is that the study failed to identify and include factors that influence offspring success. Factors that are external to the insect-plant relationship in a

Preference-Performance Correlations

Table 13-2 Categorization of hypotheses to explain poor correlations between oviposition

preference and offspring performance Type of hypothesis

Optimal ity-based

Constraint-based

Description

External factors, such as predation, are more important than the direct effects of the host. The study failed to identify the relevant performance measure. Adult survival and/or performance in adult feeders are more important than offspring performance. Identification problems: Confusion of host and non-host. There has not been enough time to adjust preference and performance on a newly colonized host.

narrow sense, such as local environmental conditions, intra- or interspecific competition, and predation or parasitism, are typically difficult to study under laboratory conditions, and hence are often not included in experimental studies of preference and performance. There are many indications that such external factors can have a strong influence on oviposition preference (Rausher, 1979; Bernays and Graham, 1988; Bernays, 1989; Fox and Eisenbach, 1992; Ryoo and Chun, 1993; Bernays, 1997; Bigger and Fox, 1997; Bjorkman et al., 1997; Camara, 1997).Are laboratory-based preference-performance studies of no use then? Not necessarily: the problem of not including factors in the experiments that could potentially be of high importance can be turned into an advantage. By being able to experimentally separate these “internal” and ”external” factors, it is sometimes possible to evaluate their relative importance in a particular system, provided that the researcher has been able to include the relevant “internal” performance measurements for the system. Unfortunately, this is not as straightforward as it may seem. As mentioned, larval performance is a composite term for a variety of measurable fitness components, such as development time, growth rate, pupal weight and survival. For practical reasons, most studies investigating the relationship between oviposition preference and offspring performance only measure one of these components. In an ideal world, one should take all parts of the insect’s life cycle into account when trying to understand the host plant choices of the females. A good host plant for the larva is not necessarily good for the egg, the pupa or the emerging adult butterfly (Reavey and Lawton, 1991). Moreover, the different performance components affecting e.g. the larval stage need not be correlated among themselves (Thompson, 1988a; Nylin et al., 1996).In reality, of course, we should expect female host plant preference to be correlated with total fitness and not with just any performance measurement. Total fitness of the ovipositing female also includes factors that act on the female herself, such as predator pressure and the cost of searching for a suitable host (Courtney, 1983; Futuyma, 1983; Stanton, 1984; Underwood, 1994; Janz and Nylin, 1997; Nylin et al., 2000; Bernays, 2001), as well as adult performance in insects that feed on host plants as adults (Scheirs et al., 2000).

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A series of studies involving the polyphagous butterfly Polygoniu c-album and two commonly used host plants, Urticu dioicu and Sulix cupreu, can serve as an example of how complex the relationship between female preference and offspring fitness can be, even without”externa1factors”.Larvae of P. c-album reached a larger size on Sulix than on Urticu, and the larger size resulted in a higher fecundity. On the other hand, larvae grew faster and had higher survival on Urticu (Janz et al., 1994). The larval host plant also had a significant effect on the choice of developmental pathway. Larvae reared on Urticu were more likely to opt for direct development (Wedell et al., 1997),which could potentially have profound fitness consequences (cf. Hunter and McNeil, 1997). Moreover, males reared on Urticu allocated more protein to their abdomen and reached higher protein contents in their spermatophores (Wedell, N., Janz, N. and Nylin, S., unpubl.). Radioactive labelling showed that the nitrogen in spermatophores is used to produce eggs, so that females mated with males reared on Urticu can spend less of their own resources to produce eggs and they also lived longer (Wedell, 1996).Host plant preference is thus a complex trade-off between a large numbers of factors and the outcome of the trade-off is likely to vary on a spatial as well as a temporal scale. Even if practical constraints will not allow more than one or a few performance measurements to be included in a study, it is important to “know your system” enough to make the most relevant choice of performance components to investigate (Nylin et al., 1996). 13.5.1.2 Constraint Hypotheses

Various constraints acting on the ovipositing female may interfere with her ability to choose the best possible site for the development of her offspring. Some cases, where the female deposits her eggs on plants that are lethal for the larvae, are hard to explain in other ways. Such oviposition mistakes have been documented in several species (e.g. Chew, 1977; Feldman and Haber, 1998), and will be more common when the preferred host is easily confused with a non-host that provides the ovipositing female with similar cues. Larsson and Ekbom (1995)suggested that these mistakes could often be the first step towards the colonization of a new host. As mentioned above, the oviposition error rate can be expected to increase with increasinghost range (Janzand Nylin, 1997) and with increasing similaritybetween hosts (Fox and Lalonde, 1993; Nylin et al., ZOOO), suggesting an interesting link between polyphagy and host shifts. Another, but related, reason for poor preference-performance correlations is that the host is so newly colonized that the insect has not had time to adjust ovipositionpreference after offspringperformance (Camara, 1997).

13.6 Concluding Remarks It should be clear by now that the behavioural processes involved in host finding and choice of oviposition sites, as well as their causes and consequences, can be exceedingly complex.Several decades of research has advanced our understanding

References

of these processes tremendously, to the extent that some broad generalizations can be made. Examples include the general patterns of specializationand host range evolution, the sequence of behavioural processes involved in host finding and acceptance, and the types of cues involved in this process. Nevertheless, many challengesremain. Much of our knowledge is stillbased on a relatively small sample of insect species, with a heavy bias toward those that are easy to study and work with in the laboratory and field. In a strict sense, generalizations are always wrong, and we need better knowledge of taxon-specific idiosyncrasies to be able to make better generalizations as well as to sharpen our specific hypotheses. The various features of insect oviposition, such as specificity, clutch size, preferenceperformancecorrelations,search efficiency,defence systems, etc., are probably parts of tightly coadapted complexes, and disentangling the causal relationshipsbetween them has proven to be very challenging. It has become increasingly clear that an understanding of these relationships calls for a multidisciplinary approach. Much insight has been gathered in the fields of behavioural ecology, chemical ecology, population ecology, physiology and systematics, but the communication between the fields has not always been perfect. I believe that future progress in this field will call for more cooperation and synergy between these fields.

13.7 Acknowledgements I wish to thank the editors of this volume along with an anonymous reviewer for helpful comments on the manuscript.

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,,,,

,,,

.................,,,,,...............,, ,,,,.,................

,,,,

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

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

.

an eruptive herbivore, Disonycha pluriligata (Coleoptera: Chrysomelidae). Environ. Entomol. 23: 887-892. Meiners, T, Westerhaus, C, Hilker, M. 2000. Specificity of chemical cues used by a specialist egg parasitoid during host location. Entomol. Exp. Appl. 95: 151-159. Miettinen, M, Kaitala, A. 2000. Copulation is not a prerequisite to male reception of eggs in the golden egg bug Phyllomorpha laciniata (Coreidae; Heteroptera). J. Insect Behav. 13: 731-740. Nylin, S. 1988.Host plant specializationand seasonality in a polyphagous butterfly, Polygonia c-album (Nymphalidae). Oikos 53: 381-386. Nylin, S, Bergstrom, A, Janz, N. 2000. Butterfly host plant choice in the face of possible confusion. J. Insect Behav. 13: 469482. Nylin, S, Janz, N. 1993. Oviposition preference and larval performance in Polygoniu c-album (Lepidoptera:Nymphalidae):the choicebetween bad and worse. Ecol. Entomol.18:394-398. Nylin, S, Janz, N. 1996. Host plant preferences in the comma butterfly (Polygonia c-album): Do parents and offspring agree? Ecoscience 3: 285-289. Nylin, S, Janz, N, Wedell, N. 1996. Oviposition plant preference and offspring performance in the comma butterfly: Correlations and conflicts. Entomol. Exp. Appl. 80: 141-144. Papaj, DR, Lewis, AC 1992. lnsect Learning. Ecological and Evolutionary Perspectives. Chapman & Hall, New York Papaj, DR, Prokopy, RJ. 1989. Ecological and evolutionary aspects of learning in phytophagous insects. Annu. Rev. Entomol. 34: 315-350. Papaj, DR, Rausher, MD. 1987. Components of conspecific host discrimination behavior in the butterfly Battus philenor. Ecology 68: 245-253. Parmesan, C, Singer, MC, Harris, I. 1995. Absence of adaptive learning from the oviposition foraging behaviour of a checkerspot butterfly. Anim. Behav. 50: 161-175. Penz, CM, Araujo, AM. 1990. Interaction between Papilio hectorides (Papilionidae) and four host plants (Piperaceae) in a southern Brazilian population. J. Res. Lepid. 29: 161-171. Pilson, D, Rausher, MD. 1988. Clutch size adjustment by a swallowtail butterfly. Nature 333: 361-363. Prokopy, RJ, Bergweiler, C, Galarza, L, Schwerin, J. 1994a.Prior experience affects the visual ability of Rhugoletis pomonellu flies (Diptera, Tephritidae) to find host fruit. J. Insect Behav. 7: 663-677. Prokopy, RJ, Owens, ED. 1983.Visual detection of plants by herbivorous insects. Ann. Rev. Entomol. 28: 337-364. Prokopy, RJ, Roitberg, BD, Vargas, RI. 1994b.Effects of egg load on finding and acceptance of host fruit in Ceratis capitata flies. Physiol. Entomol. 19: 124-132. Radtkey, RR, Singer, MC. 1995. Repeated reversals of host-preference evolution in a specialist insect herbivore. Evolution 49: 351-359. Rausher, MD. 1978. Search image for leaf shape in a butterfly. Science 200: 1071-1073. Rausher, MD. 1979. Larval habitat suitability and oviposition preference in three related butterflies. Ecology 60: 505511. Rausher, MD. 1983. Conditioning and genetic variation as causes of individual variation in the oviposition behaviour of the tortoise beetle, Deloyalaguttata. Anim. Behav. 31: 74S747. Reavey, D, Lawton, JH. 1991. Larval contribution to fitness in leaf-eating insects. In: Bailey, WJ, Ridsdill-Smith, J (eds.) Reproductive Behaviour of Insects: lndividuals and Populutions. Pp. 293-329. Chapman & Hall, London. Roininen, H, Tahvanainen, J. 1989. Host selection and larval performance of two willowfeeding sawflies. Ecology 70: 129-136. Roitberg, BD, Mangel, M. 1993. Parent-offspring conflict and life-history consequences in herbivorous insects. Am. Nat. 142: 443-456. Rojas, JC, Wyatt, TD. 1999.The role of pre- and post-imaginal experience in the host- finding and oviposition behaviour of the cabbage moth. Physiol. Entomol. 24: 83-89. Rothschild, M. 1992. Egg protection by the Atala hairstreak butterfly (Eumaeus atalaflorida). Phytochemistry 31: 1959-1960. Ryoo, MI, Chun, YS. 1993. Oviposition behavior of Callosobruchus chinensis (Coleoptera; Bruchidae) and weevil population growth -effects of larval parasitism and competition. Environ. Entomol. 22: 1009-1015.

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.

\

1

1

I

375

376 Evolutionary Ecology of Oviposition Strategies ...... .......................................................... ................................ .... ..................... ... ...................... ... . ................ .............. . . ............ . . ................. ................................................................................ Williams, KS. 1983. The coevolution of Euphydryus chulcedonu and their larval host plants. 111. Oviposition behavior and host plant quality. Oecologia 56: 336-340. Willis, MA, Arbas, EA. 1991. Odor-modulated upwind flight of the sphinx moth, Munducu sextu L. J. Comp. Physiol. B. 169: 427-440. Zangerl, AR, Berenbaum, MR. 1993.Plant chemistry, insect adaptations to plant chemistry, and host plant utilization patterns. Ecology 74: 47-54. Zaviezo, T, Mills, N. 2000. Factors influencing the evolution of clutch size in a gregarious insect parasitoid. J. Anim. Ecol. 69: 1047-1057. Zeh, DW, Smith, RL. 1985. Paternal investment by terrestrial arthropods. Am. Zool. 25: 785-805.

377

Subject Index Page numbers in bold type refer to figures and/or tables.

A accessory gland 98,108,219,273 accessory gland protein 108 accessory reproductive gland (= ARG) 38 -, development 39 -,female ARG 40 -,male ARG 39 -, structure 39 acetophenone 249,251 adaline 71 adalinine 71 aeropyle 12 alkaloid 65,71,74,79 -,transfer 79 allylisothiocyanate 81,184 ammonia 278 ammonium carbonate 278 amyrin 73,74 andrimid 162,163 andropin 108 anthraquinone 65,69 anthrone 65,69 antibacterial activity 128 antibacterial peptide 54,65,66 antibiotic 128,150 antimicrobial capacity 106 antimicrobial factor 107 antimicrobial peptide 108 antiseptic chemical 127 aposematism 356 aristolochic acid 74,77 asarone 180,186 avenanthramide alkaloid 220 B

bactericide 128 benzaldehyde 250,276 P-bergamotenic acid 175 bergapten 180 betaine 65,72 biological control XIX blood 278

brood 118 -,cannibalism 139 -,chambers 121 -,defence 129 -,diseases 122 -,female 136 -,male 136 -,parasitism 120 -, protection 120 bruchin 210 bulnesene 183 butyric acid 277 butyrolactone 105 C cabbage identification factor 189 CaC1, 185 cajin 72f calcium oxalate 48 cannibalism 81,107,132,139,154 cantharidin 65,67,73,74,157 -, advertisement 98 -, transmission 96 cantharidiphile 97,99 cardenolide 65,69,74,77,196 cardiac glycoside 183 carminic acid 69 p-carotene 68 carrion 277 carvacrol 250 p-caryophyllene 226 cellular defence 118 cement gland 42 ceratotoxin 54,66,108,158 chagas disease 267 chemical legacy hypothesis 192,358 chemoreception 354 -,contact 173 -,olfaction 173 chemoreceptor 47,191 -, contact 193,195,251 -, olfactory 193 chlorogenic acid 181

378

Subject Index

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

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

choline ester 73 chorion 8,19 -,crystalline layer 5 -, endochorion 5 -, exochorion 5 -, formation 22 -, melanization 29 -, proteins 24f, 28 - -,crosslinking 25 -,waxlayer 5 chorionic layer 8 chrysomelidial 250 CIF 184 cluster -,eggs 63 COZ 178,275,277,281 coding -,across fibre 196 -,,,labelled line (fibre)" 196 -,quality 195 -,quantity 195 coevolution 351 collateral gland 216 -,calcium oxalate 48 -,female 41f -, P-glucosidase 48 -,male 39 -,morphology 39 -, phenoloxidase 48 -,protein 48 -,protocatechuic acid glucoside 48 -, spumaline 49 -,tubules 39 ,,colleterial" gland 49 competition 132,244,334f competitor 253,329 coremata l O l f , 104 courtship 103,104,105 o,p-cresol 275 crystalline layer 8,25 cucurbitacin 74,75,105f, 157 cuticular hydrocarbon 82 cyanogenic component 65,70,74,78 cyanogenic glucoside 78 cyanoglycoside 70 cycasin 74,80 cytochrome P-450 79 cytoplasmic incompatibility 155

D danaidone 104 defensive components -,autogenously produced - -,onto the eggs 64 - -,within the eggs 67 -,extrinsic origin 73

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

I-deoxy-D-xylulose-5-phosphat pathway 225 dermatitis 277 diet breadth 308 dimethyl disulphide 278 dipropyl-disulphide 159,187 n-dodecane 273 dodecanoic acid 53,273 Dufour's gland 43,134,137 dulcitol 192

E p-ecdysone 40,51 ecdysteroid 29,42,52 egg -,adhesives 49 -, aggregation 268,271 -, cannibalism 81, 107,132,154 - -, social insect 130 -, cement 49,54,211 -, chemical protectants 50 -, clustering 356 -,clutch size 356 -,cocoon 49 -, covering 47,64 --,faeces 62 --, hairs 62 - -, plant material 73 --,scales 62 - -,secretion 62 -, defences 93 -,enemies 92 -, formation 13 -, lytic activity 218,220 -, mortality 212 -,odour 138 -,polarity 16 -, protection 355 -,size 354 -,stalks 66 -,toxicity 63 -,toxins 50 -, viability 157 eggshell 19,355 -,assembly 24,26 -,composition 24 -,formation 19 -,hardening - -,quinone tanning 28 -,layers 5,26 -,morphogenesis 19,26 -,peroxidase 25,28 -,physiological functions 9 ejaculatory duct 43 elicitor 213ff, 224ff -,feeding induced synomones

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

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

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

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

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

-, conjugates of fatty acids and amino acids 225 - -,P-glucosidase 224 - -,volicitin 224 -, hypersensitive responses 213 -,neoplastic growth - -, bruchins 210 -, oviposition induced synomones 223 -, plant gall 214 embryo 62 embryogenesis 6,12,19 encapsulation 67 endochorion 6,8 endosymbiont 72,15lf, 161 erythro-6-acetoxy-5-hexa-decanolide269 escape and radiation 351 ethanethiol 278 4-ethylphenol 275 eugenol 250 evolutionary stable strategy 334 exochorion 8,220 experience 191,241,280 -

F faeces 62,247,277,296,304,355 falcarindiol 180,185 a-farnesene 226 fatbody 18 fecundity 157 fecundity enhancing substances (= FES) 43 -, accessory gland protein 44 -tJH 44 -, matrone 44 -, myotropin 45 -,prostaglandin synthetase 44 -, sex peptide 44 ,,femme fatale" 72,78,93 filariasis 268 fitness 223,323f, 332,363 fitness cost 320 flavonoid 128 flavonoid glycoside 181 flavonol glycoside 179 fleece rot 277 follicle -,cell 14,16,20 -, maturation 14 -,polarity 16 fungal infection 79 fungi 156 fungicide 128 furanocoumarin 179,181,185 G gall -, differentiation 214

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

...........

Subject Index

379.

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

-,elicitor 218 - -, accessory glandular secretion 220 - -, chitin 215 - -,chito oligomers 215 - -,cytokinins 219 - -,nod-factors 215 - -,ovipositional fluid 219 -,inducers 214 -,initiators 50 -,necrotic tissue 214 gall insect 214 game theory 320,332 generalist 191,306,308,359 genetic determination 192 germcell 13 germacrene D 226 germarium 14,16 gift 92,100,106,110 glucobrassicin 176 gluconasturtiin 176 glucosinolate 81,174,176,181,189,195 glycoalkaloid 158,183 green leaf volatile 184,225 H habituation 304 hair 62, 64 HCN 78 heneicosane 271 9-hentriacontene 132 Zheptanone 125 heritability 357 hexanal 184,276 (E)-Zhexenal 184 trans-hex-2-en-1-a1 273 (Z)-3-hexenal 225 (Z)-3-hexenol 184 (Z)-3-hexenylacetate 184 histamine 65,73 honey 128 honeydew l77,295f, 299 Hopkins' host selection principle 191,358 hormonal vitellogenesis regulation 30 hormone balance 29 H,S 278 a-humulene 183,226 hydrogen sulphide 281 hydroxydanaidal l O l f 10-hydroxy-2-decenoic acid 128 hygienic behaviour 122 -,,,licking" 66 hypersensitive response 211f I immune response 50f, 67 indole 225,277,278,281 induction plant responses by oviposition

380

Subject Index

-, change plant surface chemicals 221

-, elicitor 211 -,galls 214 -, hypersensitive response 211f - -,elicitors 213 - -, oxidative burst 213 -, jasmonic acid 225 -, neoplasms 209 -, octadecanoid pathway 225 -, tissue changes 208 -, volatiles 221 insemination 47 interneuron 252 iridoid glycoside 177 isopentyl acetate 125 isothiocyanate 175 isoxazolinone derivate 65 isoxazolinone glucoside 71

microbial attractancy 159f microbial disease 62 microorganism 47,54,129,150,159,174, 221,275,295 -, defensive substances 162 -, nutritional interactions 161 micropyle 10,153 micropyle apparatus 9,lO miriamide 66,220 monocrotaline 103,158 morphogenic factor 23 motor neurone 193 mustard oil 74,81 mycetocyte 151f mycetome 151f myiasis 267,277 myrosinase 177

N

J

jasmonic acid 178,225 JH 40,42,44,51f juvenile hormone (JH) 29,40,42,44,51f

K kin selection 356

L larval hatching 12 larval performance 192,365 larviposition 177 latex 177 learning 191,302,304,307,358 leishmaniasis 267 lignification 214 limonene 129,175 linolenic acid 225 lucibufagin 74,78,93 Lucilurea 277 M malaria 267 mate choice 101f maternal care XVIII, 354f mercaptoethanol 277 6-methoxy-Zbenzoxazolinone 188 2-methyl-Zbutanol 276 2-met hyl-3-butene-2-01 126 N-3-methylbutylacetamide 126 4-methylcyclohexanol 275 methyl-iso-eugenol 180 methyl oleate 250 methyl palmitate 124,250 N-methylquinolinium 2-carboxylate 95 methyl salicylate 225 methyl stearate 249 cis-Zmethyl-6-undecyl piperidine 132

NaCl 185 naringin 185 NazS 277,278 natural selection 320 nematode infection 76 neoplasm 210 nerolidol 249,250 nest defence 119 neural constraint hypothesis 362 (E)-4,8-dimethyl-1,3,7-nonatriene 226

0 (E)-P-ocimene 226 1-octacosanal 188 octadecanoid pathway 225 (E,E)-3,5-octadien-2-one 251 (E,Z)-3,5-octadien-2-one 251 1-octen-3-01 277,281 trans-oct-Zen-1-a1 273 (Z)-6-octen-2-one 251 oleic acid 65,66,69,250 onchocerciasis 267,268 oocyte 16,18,22 -,chorion 5 -, development 109 -, maturation 108 -, vitelline membrane 5 oogenesis 4,14,29,191 ootheca 49 -,formation 48 orientation -,anemotactic 187 -, olfactory 187 osthol 180 ovariole 14 -, meroistic 13 -,panoistic 13 ovary 40

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

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

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

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

-, calyx fluid 51

-, habitat location 295

oviduct -, lateral 41,45 -, secretion 224 - -, elicitor 53 oviposition -, aggregated 221 -, aggregation pheromone 275 -, aquatic environment 268 -,bioassays 186 -, deterrent 176,220 -, deterring pheromone 53,236,321 - -, activity 242 - -, advantage of larvae 243 - -, applications 255 - -, associated with eggs 245 - -, carnivorous insects 239 - -, cross-recognition 254 - -, duration of activity 243 - -, from larvae 246 - -, herbivorous insects 237 - -, interspecific effects 253 - -, intraspecific effects 253 - -,production site 243 -, discrimination index 180 -, fluid 216,219 -, induced plant volatiles 223 -, inhibitors 173 -, mark assessment 324 -, marking pheromone 320,328 -, microbial stimulation 160 -, patterns 351 -, persistence of marks 329 -, pheromones - -,chemical structure 248 -, preference 192,196,279,352,357 - -,constraint hypotheses 364 - -, optimality hypotheses 364 -, repellents 279 -, site 172, 274,279,280,292,298, 308,334, 350 -, site selection 352 -, stimulants l73,176f, 179,182,186,189, 300 -, stimulating pheromone 53,236,248 -, stimulation 43,269 -, strategies 350 -,wound 218 -, wounding 215 ovipositor 151 ovulation 43

-, host acceptance 293,298 -, host discrimination 298

P palatability XV parasite 47,120 parasitoid 49f, 62,92,206,292 -, foraging 293

Subject Index

381 ..,.

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

-, host location 296 -, host recognition 293,296 -,host searching 293

-, host suitability 293 -, idiobionts 309 -, koinobionts 309

-, self-superparasitism 322 -, superparasitism 332 parental care XVIII paternal care XVIII paternity 38,43,106 pathogen 62,92,120,150,155f pederin 65,72,153,162,163 n-pentacosane 250,300 n-pentadecane 273 pentanoic acid 159 peroxidase 25,28 phenol 281 phenolglycoside 76 phenolic glucoside 176 phenoloxidase 48 phenotypic plasticity 302,328,357 2-phenylethanol 159,281 pheromone -, aggregation 248,266,271ff, 276 -, alarm 125,273 -,brood 133 -, cannibalism 139 -,courtship 79,102,105 -,eggs 272f -, host marking 321 -,oviposition 235f -, oviposition deterring 236 -,sex 223 physical barrier 81 physical protective device 62 phytoalexin 179 phytol 251 phytosterol 70 a-pinene 129 pinene 276 plant -, abundance 361 -, cell hypertrophy 216 -, chemotype 363 -, counteradaptations 208 -, defence responses 206,207 -, defensive mechanisms 178 -, density 360 -, distribution 360 -,genome 360 -,height 178 -, host selection 188 -, induced responses 206ff

,

382

Subject Index

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

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

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

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

-, inhibitory compounds 184 -, leaf pubescence 175 -,leaf surface 174,188 -,mimics 281 -,necrosis 211,214 -, nonvolatiles 185 -,nutrient 178 -,pathogens 213 -,phenotype 360 -,photosynthetic rate 178 -,primary metabolites 179,185 -,quality 362 -,secondary compounds 192 -, secondary metabolites 178,185,351 -,specialization 190 -, specificity 361 -, surface 220 -, tissue changes 208 -,toxins 73 -, trichome 175,190 -, volatiles 174,185 - -, feeding induced 221 - -, herbivore-induced 177 - -, oviposition induced 222 plastem 218 plastron -, respiration 12 polyacetylene 181 polydnavirus 50f, 67 precopulatory interaction 101 predation XV, 272 predator 47,49,62,92,120,206,212,279, 292,298,355 projection neuron 252 propenylbenzene 181 propolis 125,128 prothoracicotropic hormone (PTTH) 52 pro-toxin 70,79 pterostilbene 128 puddling 109f pyrrolizidine alkaloids (PAS) 79,100

Q

quinine 185

R receptivity-inhibiting substances (= "3)

-, sex peptide 46 -, site of action 46 receptor 307 receptor neuron -,CIF compounds 193 -,coding 193 -, olfactory 184 -,sugar 185 -, sensitivity 195 recognition

...........

-,brood 120 reflex bleeding 71 refractoriness 46 reliability-detectability problem 301,326 resin 122,125,177 rolling fulcrum 329 royal jelly 128

S salicin 74,76 salicortin 76 salicylaldehyde 76,250,300 sarmentosin 78 scales 62 scent organ 105 search image 188 sensitivity spectrum 195 sensitization 304 sensory processing 191 sequestration 18,73,77-80 semcorone 249,250 sex linkage 357 sex peptide 109 silk 49,296 sinalbin 189 sinigrin 176 skatole 274,280 slow-growth-high-mortality hypothesis 361 social insect 118f -, alarm pheromones 125 -, anarchistic workers 139 -,brood recognition 132 -,burrow plugs 122 -, camouflage 121 -, caste determination 132 -, chemical barriers 124 -, chemical defence 124 -, entrance turrets 122 -,grooming 123 -,hygienic behaviour 122 -,interspecific defence 121 -,kin conflicts 130 -,male production 136 -, mechanical defence 121 -, nest site 121 -,oophagy 133 -, parthenogenic laying 134 -, queen-queen conflict 132 -, queen-worker conflict 134 -, relative relatedness asymmetry 135 -,sex of brood 136 -,worker policing 138 -,worker-worker conflict 133 sodium 110 specialist 306,359 specialization 306,350,359

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

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

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

sperm

venom

-, capacitation 38

-, ectoparasitoid 50 -, endoparasitoid 51 -, proteins 51

-,entry 38 -, storage 109 spermatophore 47,105f spumaline 49 strike 277 Swormlure-4 278 symbiont 150 -, transmission 151,153 synomone 53,221,224,301,310 -, herbivore-induced 302 -,oviposition-induced 223

-, social Hymenoptera 127 veratrole 249,251 vitamin 161 vitelline membrane 5,24ff -,formation 22 vitellogenesis 109 -,autosynthetic 17 -,heterosynthetic 17 vitellogenin 17,19 -,receptors 30

-,activation 38 -, competition 47

T taurine 248,249,251 terpene 226 a-terpinene 276 testes 43 thymol 249,251 trade-off 355 P-triketone 250 triterpene 105 triterpenoid 73,75 trypanosomiasis 267 tryptophan 299 U urine 277 usurpation -,nest 130

V vaginal pocket 151 variable response model 303

W wax 122,125,128 -, chorion layers 22 -,eggs 5 -,leaf surface 174,190 -, ,,toxin label" 63 wound fluid 278,281

x xanthotoxin 180

Y yeast 174 yolk -, protein receptors 19 -, proteins 19 - -,cochineal yolk protein 18 - -, microvitellogenin 18 - -,paravitellogenin 18 - -,vitellins 17 -, sequestration 18 -,spheres 19

383 ................................

Subject Index

...................,.................................

384

Taxonomic Index Page numbers in bold type refer to figures and/or tables.

1.Animals, Bacteria, Fungi A

Acalyrnma uittatum 75 Acanthoscelides obtectus 237 Acheta domesticus 44,157 Acraeinae 65,71 Acrididae 23,39 Acridoidea 42,48 Acyphas 65 Acyrthosiphon pisum 161 Adalia bipunctata 71,81,239,246,300 Aedes 268,271 -,aegypti 17ff, 24f, 44,269 -, taeniorhynchus 280 -, triseriatus 275 Aeshnidae 280 Agabus guttatus 82 Agelastica alni 49,63 Agromyzafrontella 237,244 Agromyzidae 237 Agrotis segetum 184 Aleyrodidae 175 Allocontarina sorghicola 23 Anastrepha -,fraterculus 237,242 -,ludens 241 -, suspensa 185 Andrenidae 128 Anobiidae 237 Anopheles 268,271 -,arabiensis 280 -,quadrimaculatus 275 Anophelinae 267 Anoplura 266 ant 120,127,130,132 Antheraea polyphemus 6,10,19,26 Anthicidae 74,99 Anthocoris nemorum 308 Anthomyiidae 237 Anthonomus grandis 237,241 Anthophoridae 128

aphid 177,357 Aphidiidae 239 Aphidius erui 295 Aphidoletes aphidimyza 247 Aphis -,cracciuora 178 -,jacobaeae 80 -, nerii 78 Apiomerus flauiuentris 95 Apis 138 --,cerana 123 -, mellifera 47,133 apple maggot fly 159,185 Apterygota 9 Arctia caja 65, 73 arctiid 81 Arctiidae 65,74,79 arctiid moth 102 Ascogaster reticulatus 239 Atala hairstreak 80 Atrophaneura alcinous 74,77,81

B Bacillus sphaericus 155 bacteria l23,155f, 278 bark beetle 177 Battus philenor 193 Beauveria bassiana 155,158 bedbug 267,273 bee 120,130,132 Belonogaster 121 Bemisia argentifolii 156,175 Bethylidae 239 blackfly 267,272,274,276 Blattella germanica 152,162 blowfly 266,267,274 bollweevil 241 Bombus -, hypnorum 130,137 -,terrestris 137 Bombyx mori 8,19,20,26 Borborphilus primitiua 12 Bracon hebator 254

Taxonomic index

Braconidae 50f, 239 Bruchidae 237

Bruchus

-, pisorum 207,209 Bucknera 161 C cabbage root fly 159,175,179,184,188,190 Cactoblastis cactorum 178 Caenocoris 78 Calliphora 281 -, erythrocephala 18 calliphorid 277 Calliphoridae 267,269 Callosobruchus 241 -, chinensis 237 -, maculatus 207,209

Campoletis -, perdistinctus 239 -, sonorensis 52 Camponotus 122 Carausius rnorosus 10 Cardiochiles nigriceps 52,239 Caribbean fruit fly 185 Carpophilus humeralis 160 Cassida 151 -, stigmatica 190 Cassidinae 49

Cataglyphis fortis 308 Cecidomyiidae 239 centipede 95 Ceraeochysa smithi 95 Cerambycidae 237 cerambycid beetle 151,242 Ceratitis capitata 8,10, 12, 18f, 25,28,54, 65,108,158,237 Ceratoma arcuata 76 Ceratopogonidae 74 Ceutorhynchus 215 -,assirnilis 237,243 -,nupi 220 chalcidoid 214 Chalcidoidea 215,218 Chaoboridae 280 Chaoborus albatus 280 Chironomidae 49 Chrysolina 50 Chrysolinina 65,70 Chrysomela 71,74,76 -,aeneicollis 300 chrysomelid 357 Chrysomelidae 49f, 65,74,237 Chrysomelinae 65 Chrysomya bezziana 278 Chrysonotomyia ruforum 54,222 Chysopa 78,247 -, carnea 240,299

chrysopid 93,100 Chrysopidae 65,240,299 cigarette beetle 250 Cimex lectularius 273 Cimicidae 267,273 Clytra 63 Coccidae 65

Coccinella

-,septempunctata 71,80,239,247,299 -,undecimpunctata 78 coccinellid 71,79 Coccinellidae 65,239,299 Cochliomyia hominiuorax 277,278 cockroach 39,48,95 Coleoptera 8,41,43,49f, 97 Colias erate 190 Collembola 22 Colletidae 128 Colorado potato beetle 183,187 cone-nose bug 273 Coptosoma scutellatum 153 Coptotermes 122 Cordylobia anthropophaga 277 Cotesia 309 -,marginiuentris 295 cowpea weevil 209 Creatonotos 102 -, transiens 100 Crematogaster deformis 129 cricket 157 Cryptotermes 122 cucumber beetle 105,157 Culex 160,268,271,274 -,quinquefasciatus 269,280 -,tarsalis 46 Culicidae 267,268 Culicinae 267 curculionid 152,220 Curculionidae 209,215,237 cynipid 214,216 Cynipidae 215,218,239 Cynipoidea 176 D

Dactylopius confusus 18,65,69 Dacus -, oleae 12,18f, 25,26,151 -,tryoni 185 Danaidae 238 Danainae 74,79,103 danaines 104

Danaus

-,gilippus 103 -,plexippus 74,77,183 Delia

-,antiqua 159, 191 -, platura 159

385

386 Taxonomic Index . . ... .. ............................ .................................

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

..

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

-, radicum 159, 175,188,190, 196 Dendrocerus carpenteri 243 Dermaptera 39 Diabrotica 105f

-, undecimpunctata howardi 157f -, uirgifera uirgifera 75 Diabroticina 74 diabroticite beetle 75 Diaphania nitidalis 175 Dinoponera quadriceps 137 Diplolepis 215,216 Diprionidae 238 Diprion pini 54,207,237,247 Diptera 8,17,39,43,49,97 Dolichouespula -, saxonica 137 Donacia 151 -, semicuprea 153 Drino inconspicua 308 Drosophila 14,19,46,160 -,fasciculisetae 10 -, melanogaster 10,14,20,26,40,43,108 -, mimica 10 -, setosimentum 10 -, uirilis 6,14,20,24, 26 Drosophilidae 23

F fire ant 129 firefly 72,78,93 flea 266 flea beetle 189 fleshfly 277 Formica 126,140 -, exsecta 136 -, rufa 137 fruit fly 240

Fusarium uerticillioides 160 G

Galeruca 69

-, tanaceti 222 Galerucella 69

-, lineola 63

Galerucinae 65,69 Galerucini 69 gall midge 192 garden tiger moth 73 Gastrophysa 71 -, cyanea 65,68 -, uiridula 63,237,246 Glossina 161 -,austeni 23 -, rnorsitans 153, 269

-, morsitans morsitans 273 E Eciton 121 Edouum puttleri 239

Glossinidae 267,269,272 Gomphocerus rufus 46

Graptopsaltria nigrofuscata 17

empidid fly 92 Encyrtidae 239 Enocyla pusilla 49

grasshopper 45 gypsymoth 23

Enterobacter agglomerans 159 Ephedrus cerasicola 242

H

Ephemeroptera 39,41,49

Halictidae 128 Heliconius 65, 71

Ephestia -, cautella 238 -, kuehniella 246 Epicauta 67 -,funebris 97 Episyrphus balteatus 299 Eriophyies cladophtirus 216 Escherichia coli 161 Eulophidae 222,239

Eulophus pennicornis 51 Eumaeus atala 80 Euphydryas editha 352 Euproctis 65 European corn borer 177,245 Eu rytides marcellus 178 Eurytoma amygdali 6,10,22f, 25 Eurytomidae 218,238 Euura 218 Exochomus 4-pustulatus 71 Eyprepocnemis plorans 9,lO

Habrobraconjuglandis 8 He1icouerpa

-, armigera 183,191 -, zea 45,175 Heliothis uirescens 52,175,177f Heliozelidae 215 Hemiptera 43,49f, 97 Hemisphaerota cyanea 95 Hermetia illucens 280 Hesperiidae 359 Hessian fly 188 Heteroptera 41,77 Homoptera 17,77 honey bee 125,128,133,139 housefly 280 Hyalophora cecropia 8,13,18,45 Hydrogaleruca 69 Hydrophilidae 42 Hylemya 237,245 Hylesia 66

...

Taxonomic Index

Hylobius abietis 152 Hymenoptera 8,42,49f, 97,118,140 I Ichneumonidae 50f, 239 ldeopsis similis 190 lphiseius degenerans 299 Ips 173 Ithomiinae 74,79,104

J

Junonia coenia 177 K kissing bug 273 Korscheltellus lupulinus 22 L Lacanobia oleracea 51 lacewings 78 Lampyridae 65,72,74 Lariophagus distinguendus 297 Lasioderma serricorne 250 Lasioglossum -, malachurum 130 -, zephyrum 135 Lathyrus tingitanus 210 Lepidoptera 8,43,49f, 64 Leptinotarsa decemlineata 6,20,23,25,156, 158,183,187,207,212 Leptothorax -, aceruorum 133 -, longispinosus 72 Leptura rubra 151 lice 266 Linaeidea 71 Linepithema humile 136 Lobesia botrana 238,245 Lochmaea 69 locust 45,248 Locusta migratoria 17f, 44 Lucilia -, cuprina 269,281 -, sericata 274,277,278 Lutzomyia longipalpis 269,273,276 lycaenid 80 Lycaenidae 74 lygaeid 78 Lygaeidae 74 Lymantriidae 65 Lytta 67 -, nuttalli 40,49 -, uesicatoria 96 M Macrolophus caliginosus 299 Mamestra brassicae 184

Manduca sexta 18,45,225 mantids 48 mealybug 152 Mecoptera 49,92 medfly 54,108 Mediterranean fruit fly 158 Megaspilidae 239 Melanoplus sanguinipes 40,44,156 Meloe 67 Meloidae 65,67,157 meloid beetle 96 Messor pergandei 132 Metarhiziurn anisopliae 106,156ff Miridae 299 Mischocyttarus 121 monarch butterfly 70,77,104,176,183 Monochamus alternatus 237,242 mosquito 160,267,274 Musca domestica 18,43,280 Muscidae 237 Myrmaridae 239 Myrmica -,rubra 308 -, tahoensis 135 Myrmiciinae 129 Myzus persicae 177 N Nasonia vitripennis 8 Nasutitermes 126,129 Neodiprion -,fuluiceps 238 -,sertifer 247 Neopyrochroa flabellata 98 Nepidae 12 Neuroptera 49 Nitidulidae 160 noctuid 184 Noctuidae 238 notonectid 280 nymphalid 78,155,357 Nymphalidae 65,74,177 Nymphalini 351

0 oataphid 192 Odonata 39,49 Oedemeridae 65,67,97,157 Oestridae 267,276 Oestrus ouis 277 olive fruit fly 151,153 Oncopeltus fasciatus 74,78 onionfly 191 onion maggot fly 221 Oomyzus gallerucae 53,222 Ophraella 351 Opius lectus 254

387

388

Taxonomic Index

Oreina 80

-,cacaliae 178,187 -, elongata 74 Orthoptera 9,41,43,47,50,77 Ostrinia nubilalis 174, 177,245

P

Paecilomyces lilacinus 156, 158 Paederus 65,72,82,153,163 paper wasp 121

Papilio -,machaon 352f -, polyxenes 181 -, troilus 175 papilionid 78,81,193,357 Papilionidae 74,77,175 Papilionidea 359 Papilioninae 179 Parabolybia 121 Parasyrphus melanderi 300 Parischnogaster nigricans 124 Parnassius phoebus 78 Paropsis atomaria 65,70 pea 161 pea weevil 209 pentatomid 151 Perilampidae 218 Perillus bioculatus 307 Periplaneta americana 39 Phaedon 71 -, cochleariae 237,246 Phaedonia 71 Pherbellia cinerella 299 Photinus 72,78,93 Photuris 65,72,74,78,93 Phratora 71 -, vitellinae 74, 76,237,246 Phthiraptera 49 Phyllomorpha laciniata 355 Phyllopertha diversa 184 Phyllotreta 189 Phytoseiidae 299 Pieridae 74,238 pierid butterfly 154 Pieris 80, 181, 212 -, brassicae 53,74,207,211,220,224,245 -,nupi 207 -, rapae l78,189f, 194,195,207 -, rapae crucivora 154 Pimpla instigator 308 pinesawfly 54 Pityogenes 173 Plagiodera 71 Plebeia 122 Plecoptera 39,41,49 Plodia interpunctella 238,246 Podisus maculiventris 307

Poecilocerus bufonius 7 4 7 7 Poecilocerus pictus 77 Pogonomyrmex 122f Polistes 121,133 -,fuscatus 124 Polybia 119, 126 Polygonia c-album 191, 366 Ponerinae 129 ponerine ant 137 Pontania 218 -, proxima 219 Porcris geryon 70 Prodoxidae 238 pseudococcid 152 Psila rosae 180,192 Psocoptera 41 Psychodidae 266,267,269,272 Pteromalidae 239 Pyralidae 238 Pyrgomorphidae 74 pyrochroid 99 Pyrochroidae 74 pyrochroid beetle 98 Pyrrhalta 69 -, viburni 50,73,74

R Reduviidae 267 reduviid bug 95 Reticulitermes 123

Rhagoletis

-,alternata 237 -, boycei 173 -,cerasi 8,10,240 -,juglandis 191 -,pomonella 159f, 185 Rhodnius prolixus 42,45 Rhopalosiphum padi 192 Rickettsia 155 Ropalidia 121 S sandfly 267,272,274 Sarcophaga 281 Saturniidae 65 sawfly 176 scarab beettle 184 Scelionidae 240 Schistocerca gregaria 248 Schizotus pectinicornis 99 Sciomyzidae 299 screwworm 266,267,277 Serratia marcescens 156 silkmoth 23

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

Simuliidae 266,267,268,276 Simulium damnosum 269,272,276 Siphonaptera 266 Sirex 211 siricid 160 Siricidae 211 Sitophilus oryzae 161 Solenopsis 129 -, invicta 132 Sphecomyrma 129 Spodoptera -,exigua 224 -, littoralis 177,184,191,247 staphylinid 72,163 Staphylinidae 65 staphylinid beetle 153 Stratiomyidae 280 Strobilomyia neanthracina 243 sunflower beetle 50 swallowtail butterfly 77,186,189 Syrphidae 299

T Telenomus fariai 240 Teleogryllus commodus 44 Tenthredinidae 176,215,218 Tephritidae 23,237,240 tephritid fly 255 tephritid fruit fly 173 termite 119,120,127 Tetragona 122 Tetragonisca 122 Tetrastichus asparagi 239 Tetrodontophora bielanensis 22 Thaumetopoea 66 Thaumetopoeidae 65 Thysanura 39,41 Timarcha 69 tobacco budworm moth 175 Tortricidae 238 Toxorhynchites 271 -, moctezuma 275 Trematoda 279 Triatoma infestans 273 Triatominae 267,273 Trichogramma 308 -,manescens 240 Trichogrammatidae 240 Trichophaga tapetzella 6 Trichoplusia ni 247 Trichoprosopon 271 Trichoptera 49 Trigona 122 tsetse fly 42,161,267,272 U Utetheisa 81,102,107 -,ornatrix 50,100

Taxonomic fndex

389

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

V Vespa crabro 126,137 Vespula 119 -, pennsylvanica 129 -, squamosa 126 W walnut fly 191 walnut fruit fly 173 wasp 119,120,132 weevil 209 whitefly 156 Wigglesworthia 161 -,glossinidia 153 Wohlfahrtia magnifica 277,278 Wolbachia 150,152,155,160

X Xanthogaleruca 69 -,luteola 53,207 Xanthomonas campestris 163 Xeris spectrum 160 Y

Yponomeuta 192 Z

Zygaena 70,78 -, trifolii 73 Zygaenidae 65,74 Zygogramma exclamationis 50

2. Plants A

Adenostyles alliariae 178 alfalfa 244 Allium cepa 221 Apiaceae 352 Apium graveolens 181 Arabidopsis thaliana 189 Araceae 281 Aristolochia 77,281 -,macrophyllum 193 Aristolochiaceae 77 Arum 281 Asclepiadaceae 69,77,281 Asclepias spp. 77,183 Asteraceae 71,79,352 Azadirachta indica 279 Azolla imbricata 279

390

Taxonomic index

B Boraginaceae 79 Brassica 196 -,juncea 177 -,napus 220 -,nigra 178,207,211f -,oleracea 175,207,220 -,rapa 174 C cabbage 53,184,187,220,245 Calotropis gigantea 77 carrot 180,181 celery 178 cherry 2.40 Conium maculatum 181 cotton 250 Crotalaria spp. 100 -,spectabilis 103 crucifer 175,186,189 Cruciferae 80 Cucurbita pep0 175 Cucurbitaceae 75,105

D

Daucus carota 181 Deliaantiqm 221 Dracula chestertonii 281

E Eucalyptus 70

F Fabaceae 79

G

Galium spp. 69

H Heracleum sphondylium 181

L hthyrus spp. 207 leguminous plant 100 Lemna minor 279 Lotus corniculatus 70 Lycopersicon pennellii 175 M milkweed 77 mustard 211

onion 221

Opuntia stricta 178 orchid 281 Orchidaceae 79

P Passifloraceae 71

Pastinaca sativa 181 pea 209

Petroselinum crispum 181 pine 222 Pinus 247

-, sylvestris

54,207,222

Pisum sativum 207,209 Plantaginaceae 177 poplar 76 Populus 218 potato 187 Prunus padus 192

R Rosa acicularis 217 Rutaceae 352

s Salicaceae 76 Salix 176,218 -,caprea 366 -,pugilk 219 -,viminalis 192

Sedum stenopetalum 78 Senecio 80 -,jacobea 71 Solanaceae 105

Solanum 212 -,dulcamara 211,216 -, tuberosum 207 Stapelia flavirostris 281

T Tanacetum vulgare 190 tomato 175

U Ulmus minor 53,207,222 Umbelhferae 182 Urtica dioica 191,366

V Viburnum spp. 50,73

N Neemtree 279

W

Nerium oleander 78

wheat 192 willow 76,250

0

oat 192 oleander 78