1

Reproductive Strategies in Parasitic

by

Ian Charles Wrighton Hardy

A thesis submitted for the degree of Doctor of Philosophy of the University of London and for the Diploma of Imperial College

Department of Biology and Centre for Population Biology, Imperial College at Silwood Park, Ascot, Berkshire, SL5 7PY, U.K.

1991

(Submitted November 1990) 2

Abstract

This thesis investigates the evolutionary ecology of reproduction by wasps.

In haplodiploid populations some females are constrained to produce sons only, theor­ etically, the optimal progeny sex ratio of unconstrained females may be influenced. Prevalences of constrained females are assessed in of D ro so p h ila and from the literature. Constrained oviposition is generally rare, however, in some species constrained females are sufficiently common to affect unconstrained female’s sex ratios.

Goniozus nephantidis females remain with their broods until the offspring pupate. G. nephantidis competes for hosts with conspecific and non-conspecific parasitoids. The costs of remaining seem at least partially offset by the prevention of oviposition by competing parasitoids.

To predict clutch size, the relationship to the p e r c a p ita fitness of offspring must be known and also the parental trade-off between present and future reproduction. Since trade-offs are assumed unimportant in G. nephantidis clutch fitness should be maximised, this is achieved at the ’Lack clutch size’. Females adjust clutch size to host size. Manipulation of clutch size on standard hosts shows that developmental mortality is unaffected by clutch size, but larger females emerge from smaller clutches and have greater longevity and fecundity. Lack clutch sizes are calculated using longevity and fecundity as estimators of offspring fitness. Calculated clutch sizes are larger than those produced by the parasitoids. Disparities are attributed to incorrect assumptions about the importance of trade-offs or to inadequate measurement of offspring fitness (laboratory-based approaches are unable to assess all components of fitness).

Precise sex allocation is theoretically advantageous over binomial sex allocation, since the number of mated females is maximised when brood sex ratio variances are reduced. G. nephantidis sex ratios are compared with those expected under binomial sex allocation. Precise sex ratios are produced at most brood sizes, although precision is limited, probably by factors such as pre-adult mortality. To Esther, to John and to the rest of my family. 4

Table of Contents

Abstract ...... 2 Table of Contents ...... 4 List of Figures ...... 7 List of T ab les...... 8 Acknowledgements ...... 9

Chapter One Introduction...... 10 1.1 Behavioural ecology and reproductive strategies ...... 10 1.2 Parasitoids, parasitic diversity and the Bethylidae ...... 11 1.3 Parasitoids and evolutionary stu d ies ...... 13 1.3.1 Maternal Care ...... 13 1.3.2 Clutch size ...... 14 1.3.3 Sex ratio ...... 16 1.4 Statistical an alysis...... 21

Chapter Two Constrained Sex Allocation in ...... 23 2.1 Abstract ...... 23 2.2 Introduction...... 24 2.3 Constrained Oviposition by D ro so p h ila Parasitoids...... 30 2.3.1 Biology of D r o s o p h ila parasitoids ...... 30 2.3.2 Methods ...... 31 2.3.3 R esults...... 32 2.4 Literature R ev iew ...... 34 2.5 Discussion ...... 39 2.5.1 D r o s o p h ila parasitoids ...... 39 2.5.2 General prevalence of constrained oviposition...... 39

Chapter 3 Brood guarding in the bethylid wasp nephantidis ...... 42 3.1 Abstract ...... 42 3.2 Introduction...... 43 3.2.1 Biology of G. nephantidis...... 43 3.3 M ethods...... 44 3.3.1 A nim als...... 44 5

3.3.2 Experimental procedures ...... 45 3.4 Results ...... 46 3.4.1 Control ...... 46 3.4.2 Brood survivorships ...... 47 3.4.3 Presence or absence of the initial female and intruder oviposition .. 48 3.4.4 Survival of the initial female's brood and intruder oviposition ...... 49 3.5 Discussion ...... 52

Chapter 4 Clutch size in Goniozus nephantidis ...... 55 4.1 Abstract ...... 55 4.2 Introduction ...... 56 4.2.1 Biology of G. nephantidis ...... 57 4.3 Methods...... 58 4.3.1 ...... 58 4.3.2 Clutch size and host size ...... 59 4.3.3 Clutch size manipulations ...... 59 4.3~4 The effects of the mother remaining with her clutch ...... 59 4.3.5 Reproductive female's longevity and fecundity ...... 60 4.3.6 Female longevity without reproduction ...... 60 4.3.7 Statistical analysis ...... 60 4.4 Results ...... 61 4.4.1 Clutch size and host size ...... 61 4.4.2 Clutch size manipulations ...... 64 4.4.3 The effects of mother remaining with her clutch ...... 67 4.4.4 Influence of adult size on fitness ...... 68 4.4.4.1 Female fecundity ...... 68 4.4.4.2 Reproductive female's longevity ...... 70 4.4.4.3 Female longevity without reproduction ...... 72 4.5 Discussion ...... 73

Chapter 5 Precise Sex Ratios in Goniozus nephantidis ...... 82 5.1 Abstract ...... 82 5.2 Introduction ...... 83 5.2.1 Evidence for precise sex ratios ...... 84 5.2.2 The biology of Goniozus nephantidis ...... 93 6

5.2.3 Primary sex ratio...... 94 5.2.4 Male insemination capacity ...... 95 5.3 Materials and Methods ...... 96 5.3.1 A nim als...... 96 5.3.2 Assessment of primary sex ratio...... 96 5.3.3 Male insemination capacity ...... 96 5.3.4 Analysis of G. nephantidis sex ratio strategy ...... 97 5.4 R esu lts...... 98 5.4.1 Primary sex ratio...... 98 5.4.2 Male insemination capacity ...... 98 5.4.3 Precise sex ratio ...... 99 5.5 Discussion ...... 102 5.5.1 Primary sex ratio...... 102 5.5.2 Male insemination capacity ...... 102 5.5.3 Precise sex ratio ...... 103

Chapter Six Discussion ...... 109 6.1 Constrained sex allocation in Hymenoptera ...... 109 6.2 Brood guarding in Goniozus nephantidis...... 110 6.3 Clutch size in Goniozus nephantidis...... 112 6.4 Precise sex ratios in Goniozus nephantidis ...... 113 6.5 C onclusions...... 114

References 115 7

List of Figures

Fig. 1.1 A female G. nephantidis guarding her larvae ...... 14 Fig. 1.2 Per captita fitness and the Lack clutch s iz e ...... 15 Fig. 1.3 Sex ratio and number of foundresses ...... 18 Fig. 1.4 Binomial distributions of sex ratios ...... 20 Fig. 2.1 Sex ratio reponses of unconstrained females...... 28 Fig. 2.2 Numbers of parasitoids caught during the season ...... 33 Fig. 2.3 Distribution of percentage constrained females...... 38 Fig. 4.1 Clutch size and host weight ...... 62 Fig. 4.2 Cube of female thoracic length and host w eig h t...... 63 Fig. 4.3 Survival and numbers of hatched eggs ...... 65 Fig. 4.4 Female size and numbers of hatched eggs ...... 66 Fig. 4.5 Male size and numbers of hatched e g g s ...... 67 Fig. 4.6 Fecundity and size of females ...... 69 Fig. 4.7 Size and longevity of reproducing fem ales ...... 70 Fig. 4.8 Cohort survival for reproducing females ...... 71 Fig. 4.9 Size and longevity for non-reproducing females ...... 72 Fig. 4.10 Cohort survival for non-reproducting fem ales...... 73 Fig. 4.11 Clutch size and fitness (reproductive longevity) ...... 77 Fig. 4.12 Clutch size and fitness (non-reproductive longevity) ...... 78 Fig. 4.13 Clutch size and fitness (fecundity)...... 79 8

List of Tables

Table 2.1 Estimates of constraints in D ro so p h lia parasitoids ...... 34 Table 2.2 Prevalence of constrained oviposition ...... 36 Table 3.1 Control egg to larval survival ...... 47 Table 3.2 Survival o f G. nephantidis in different situations ...... 48 Table 3.3 Intmder laying with or without mother ...... 49 Table 3.4 Survival of clutch and intmder la yin g...... 50 Table 3.5 Mean number of intmder offspring per h o s t...... 51 Table 4.1 Probabilities of survival in G. nephantidis...... 62 Table 5.1 Evidence for variance in brood sex ratios ...... 84 Table 5.2 Evidence for sequences of sex allocation...... 89 Table 5.3 A test of binomial sex ratio in G. nephantidis...... 99 Table 5.4 Binomial sex ratios in broods with low m ortality...... 101 9

Acknowlegements

I thank Charles Godfray for his excellent supervision of my studies at Silwood Park. Some of the work presented in this thesis has been in collaboration with Charles Godfray (Chapters 2 & 4), Tim Blackburn (Chapter 3), Nigel ’Griff’ Griffiths (Chapter 4) and James Cook (Chapter 5). All deserve thanks for their valuable contributions. Hefin Jones (fy hoff post-doc) provided discussion and support in innumerable ways.

In addition those named above, I have had helpful discussions with most people at Silwood, but especially Simon Gates, Clare Towner, Brad Hawkins, Jeremy Field, Mike Hochberg, Trev Williams, Guy Forrester, Carlos ’el Enano’ Garcfa-Saez de Nanclares, Solange Brault, Niall Broekhuizen and Aris Lopez-Avila. For statistical advice, particular thanks are due to Mark Rees, Charles Godfray and Mick Crawley. For technical, admi- nistratitive and library assistance I thank many people at Silwood, notably Dave, Ena, Ed, Ken, Anthea, Karen, Richard, Len, Frank, Paddy, Barbara, Jackie, Anne, Pauline, Diana and Mike.

My future collegues at the University of Leiden have been very hospitable, I thank them for this and for some very useful discussions, especially Jaques van Alphen, Marianne van Dijken, Marcel Visser and Patsy Haccou (die ook de rauwe haring verzorgde). For helpful discussion, I should also like to thank the following, from various institutions, B. Shorrocks, W.D. Hamilton, J. Weiner, A.R. Ives, A.D. le Masurier, M. Begon, P.A.C.R. Perera, R. Singh, J. Rosenheim and all the people whose unpublished data or ideas I have quoted. I also thank Jack Cohen for being generally stimulating, and NERC for funding my studies.

The course of my studies has been eased by many friends in many places, I thank them all for this. Finally, I am grateful to my family for being very supportive in numerous ways. Introduction 10

Chapter One

Introduction

This thesis presents an investigation into the evolution and ecology of several aspects of reproduction in parasitic wasps. This chapter provides a general introduction to the biology and behavioural ecology of parasitic wasps, and explains terminology and stat­ istical methods used throughout the thesis. The central chapters of the thesis (Chapters 2, 3, 4 & 5) contain empirical work, each of which may be read independently, but their themes are interrelated. Chapter 2 investigates the sex ratio consequences of virgin reproduction in parasitic wasps, and examines evidence for this in parasitoids of D r o s o ­ p h ila . Chapters 3, 4 and 5 each examine different aspects of reproduction by G o n io zu s nephantidis\ parental investment, clutch size and sex ratio respectively. Finally, Chapter 6 reviews the preceding chapters and contains suggestions for further investigations. 1.1 Behavioural ecology and reproductive strategies

Behavioural ecology is the study of how behaviour is influenced by natural selection in relation to ecological conditions. The Darwinian assumption is made that, given a set of alternative traits, natural selection will favour the trait that leads to the maximum fitness for it s bearer. Fitness may be defined as the organism’s genetic contribution to future generations. The reproductive strategy of an individual organism is the way that it allocates resources to reproduction in the attempt to maximise fitness. For a simple example, an organism might produce many offspring and invest a small amount of resource (such as nutrition or protection) in each, alternatively it could produce fewer offspring with a larger p e r c a p ita investment, in each case allocating the same total resources to reproduction, but in very different ways (Smith & Fretwell 1974). The strategy which is adopted as the result of natural selection depends largely on the environment of the organism. For parasitic wasps the reproductive strategy may be the result of making a series of evolutionary "decisions", for instance whether or not to parasitize a host once it has been encountered, and if so, how many eggs to lay and the sex of the offspring (Chamov & Skinner 1984). Introduction 11

Optimization models have been employed to understand how organisms are adapted to their environment. To examine adaptation, optimization models must partition organisms into traits and the environment into problems. It is assumed that the isolation of characters is valid and that interactions between characters are secondary, representing constraints on the adaptation of each character separately. It is also assumed that all aspects of the organism are adaptive (Lewontin 1978). A problem arising from the latter assumption is that if functional hypotheses fail to predict behaviour correctly, they can always be modified by the a d h oc proposal of additional functions which act as constraints on the adaptation of first. Thus, given sufficient ingenuity, adaptive explanations can always be found for traits and the assumption is irrefutable. If this assumption is dropped, however, then traits which are difficult to explain can simply be declared non-adaptive, thus allowing the explanation of only those traits that fit the mode of explanation. The theory of adaptation has thus been criticized because it is considered to be untestable (Gould & Lewontin 1979, Lewontin 1978). Maynard Smith (1978a) considers that such criticisms are invalid since the theory of adaptation itself is not under test, but a specific set of hypotheses in a particular model. If a hypothesis is falsified as a complete explanation, but has some success (i.e. qualitative agreement) Maynard Smith considers that it is appropriate to modify the hypothesis. What is not justifiable, however, is to modify the hypothesis and to claim that the model is confirmed by observation (Maynard Smith 1978a).

1.2 Parasitoids, parasitic wasp diversity and the Bethylidae

The vast majority of species are (May 1986), and a large proportion of these are entomophagous. Entomophagous insects may be roughly categorized as parasitic or predatory, although there is no sharp demarcation between the two (Clausen 1940). Many parasitic entomophagous insects kill the host during their development. A more exact term for these parasitic insects is "parasitoids", since aspects of their lifestyle are shared with both predators and "true" parasites. Parasitoids occur amongst the Hymenoptera, Diptera, Lepidoptera, Strepsiptera and Coleoptera (Clausen 1940), but most parasitoid species are Hymenoptera. The terms "parasitoid" and "parasitic wasp" are used synonymously throughout this thesis to refer to these Hymenoptera.

Individual parasitoids attack and lay eggs (oviposit) on or in a host, usually paralysing the host with an injection of venom in the process. On hatching, the parasitoid larvae feed on the host, which is eventually killed. Parasitoids are free living as adults. species differ widely in their developmental habits. For instance, endoparasites Introduction 12

develop inside the host and ectoparasites develop externally on the host. Solitary parasitoids develop singly, one parasitoid offspring emerging from each host, while gregarious parasitoids develop in groups. Parasitoids also vary in the type of host they attack; primary parasitoids are parasitic on free living hosts and secondary- or hyper-parasitoids are parasitic on a primary parasitoid itself. A few species, tertiary parasitoids, are parasitoids of secondary parasitoids. Some parasitoid species are able to develop on another parasitoid or on the free living host, these are facultative hyper- parasitoids. Obligate hyperparasitoids are unable to develop as primary parasitoids.

The stage in the host life cycle attacked by the parasitoid also varies between species; egg, larval, pupal and adult parasitoids attack host eggs, larvae, pupae and adults respectively. Egg, larval and pupal parasitoids are all relatively common but parasitoids of adult insects are less common. The host stage at the completion of the parasitoid development may not be the same as at oviposition, for example an egg-pupal parasitoid oviposits into the egg of the host species and the adult parasitoid emerges from the pupa of the host. Likewise, egg-larval and larval-pupal parasitoids are found. The parasitoid may change it’s relationship to the host during development, for instance some initially endoparasitic larval-pupal parasitoid species feed ectoparasitically after the pupation of the host (e.g. some of the parasitoids of D r o s o p h ila, Carton e t a l. 1986). Monophagous parasitoids are restricted to a single host species, while oligophagous and polyphagous parasitoids are able to develop in range of host species.

The majority of this thesis is concerned with the reproductive strategies of G o n io zu s nephantidis, a bethylid wasp. The family Bethlidae is reviewed by Clausen (1940) and Griffiths (1990). The family Bethlidae is thought to have originated from a primitive Aculeate group (Gordh e t al. 1983), but shows many advanced characteristics making bethylids functionally equivalent to the . The members of the family Bethylidae almost exclusively develop on the larvae of Lepidoptera and Coleoptera. Their hosts are mostly stored products pests or pests of agricultural plants, usually living in some kind of enclosed space, such as a tunnel, a rolled leaf or a leaf mine. Bethylids are, with few exceptions, beneficial to man and have been used in biological control programs, some with success (Greathead 1986, Murphy & Moore 1990). Typically females subdue the host by repeated stinging resulting in complete or partial paralysis. All bethylids are ectoparasites, and the majority develop gregariously, usually with extensive inbreeding. Male and female aptery (winglessness) occurs in some bethylid species, though not in G. Introduction 13

nephantidis. Bethylids exhibit a tendency towards communal life, with maternal care of the brood in some species. In each of the chapters dealing with reproductive strategies in G. nephantidis aspects of it s biology specifically relating to that chapter are reviewed.

1.3 Parasitoids and evolutionary studies

Parasitoids can be viewed as key organisms fortesting and extending natural selection theory (Chamov & Skinner 1985). The association between behaviour and fitness in parasitoids is often much clearer than in many organisms. For example, in species where adults do not feed upon the host tissues, parasitoids forage directly for reproductive opportunities, rather than for some other quantity (such as energy) indirectly related to the production of offspring. This close association between observable behaviour and measurable fitness has made parasitoids ideal for testing evolutionary theories. Con­ veniently, parasitoids are also small and easy to manipulate experimentally, although their small size may make field studies difficult. Developments in the understanding of evolutionary processes gained from the study of parasitoids, are not confined in their implications only to parasitoids, despite the existence of phenomena which generally do not occur in other types of organisms (e.g. superparasitism). 1.3.1 Maternal Care

Females of most parasitoid species complete their investment of resources in off­ spring at oviposition, but in a few, mothers remain with their broods. Amongst the Bethylidae this behaviour is relatively common. It has been generally considered that remaining with the brood constitutes some form of maternal care (Griffiths 1990). Maternal care may significantly reduce the number of offspring a mother is able to p r o d u c e in her lifetime, but if it is more profitable, in terms of the number of su rv iv in g offspring, than alternative strategies then such a trait will have been selected. It has not been well understood how the presence of the mother is beneficial to developing offspring. An investigation of how the function of maternal care is fulfilled in G. nephantidis is presented in Chapter 3. Figure 1.1 shows a female G. nephantidis guarding her larvae developing on Corcyra cephalonica. Introduction 14

Fig. 1.1 An adult female G. nephantidis guarding her larvae developing on Corcyra cephalonica.

1.3.2 Clutch size

An important component of a parasitoid’s reproductive strategy is the number of eggs, or clutch size, that is laid on the host. The study of the evolution of clutch sizes began when Lack (1947) suggested that the clutch size laid by birds should maximise the number of fledgling young. More recently the theory of clutch size evolution has been applied to invertebrates, particularly gregarious parasitoids (e.g. Begon & Parker 1986, Chamov & Skinner 1984,1985, Godfray 1987a, 1987b, Godfray & Ives 1988, Ives 1989, Iwasa e t al. 1984, Mangel 1987, Parker & Courtney 1984, Parker & Begon 1986, Skinner 1985, Waage 1986, Waage & Godfray 1985). Introduction 15

The host constitutes the only food source for developing parasitoids until they reach adulthood and this resource is divided roughly equally between developing individuals. The amount of resource available to each wasp, and it’s ultimate fitness, are thus strongly influenced by the size of the clutch produced by it’s mother. In the absence of trade-offs between present and future reproductive success, parasitoids are predicted to produce the clutch size that maximises brood productivity: the "Lack clutch size" (Chamov & Skinner 1984). Normally, the marginal increase in fitness for a parent decreases as eggs are added to achieve the Lack clutch size, and beyond this clutch size, total brood fitness declines (Fig. 1.2).

CO CO cCD -f —» Ll

1 2 3 4 5 6 7 Clutch size Fig. 1.2 The relationship between clutch size and offspring fitness. Individual fitness (filled bars only) declines as clutch size increases. The total fitness of offspring in the clutch is the individual fitness multiplied by the clutch size (stacked bars). The clutch size at which the total fitness is at a maximum is the Lack clutch size, in this case 4 eggs. Introduction 16

The reduction of marginal increase in fitness leads to a number of potential trade-offs between present and future reproductive success resulting in selection for clutches smaller than the Lack clutch size (Skinner 1985). For example, if the reproductive success of the animal is limited by time, it may not be worth spending time completing the Lack clutch size when that time could more profitably be spent searching for fresh hosts (Chamov & Skinner 1984, Parker & Courtney 1984, Skinner 1985). If eggs are limiting, using eggs in the completion of the Lack clutch size might be an inferior strategy to starting new clutches (Parker & Courtney 1984, Waage & Godfray 1985). A number of other possible trade-offs are discussed by Godfray (1987a).

Although many empirical studies have found relationships between parasitoid clutch size and offspring characteristics such as juvenile mortality, body size, fecundity and longevity (Chapter 4), relatively few quantitative predictions of clutch size have been attempted. These, based on the relationship between p e r c a p ita fitness and clutch size, have predicted Lack clutch sizes which are greater than the clutch sizes naturally laid by the parasitoid (Chamov & Skinner 1984, 1985, Waage 1986, Waage & Godfray 1985). Predicted clutch sizes may be greater than the observed clutch sizes either due to the inadequate estimation of individual fitness or to the importance of trade-offs between present and future reproduction. Only Dijkstra (1986) attempted to predict clutch size in a species for which there is an expectation for the unimportance of trade-offs between present and future reproduction. Using different offspring characters as estimators of fitness Dijkstra predicted Lack clutch sizes both above and below the observed clutch size. Chapter 4 presents an investigation of the consequences of clutch size in G. nephantidis, and an attempt to predict it’s clutch size.

1.3.3 Sex ratio

Another important component of the reproductive strategy of parasitic wasps is the sex of their offspring. In arrhenotokous (haplodiploid) parasitoids fertilized eggs develop into females and unfertilized eggs develop into males. Control of the sex of offspring is achieved by controlling the fertilization of eggs (e.g. Cole 1981, van Dijken & Waage 1987, Flanders 1956, Suzuki e t al. 1984). In general, the major factors leading to biased sex ratios in parasitoids seem to be understood. It has been possible to verify detailed quantitative predictions, making this a very productive field of evolutionary biology (re­ views in Chamov 1982, Frank 1983, King 1987, Waage 1986, Waage & Godfray 1985, Werren 1987). Introduction 17

Darwin considered the problem of the evolution of sex ratios, but left the solution for the future (May & Seger 1985). It was Fisher (1930) who first explained why the equilibrium sex ratio of most species is equal numbers of males and females. Briefly, Fisher’s argument is that in a population with a biased sex ratio, offspring belonging to the rarer sex have better mating prospects than those belonging to the more common sex. Parents with a genetic tendency to produce more of the rarer sex than the more common sex, thus attain a higher than average number of grandchildren. The tendency to produce the rare sex becomes more widespread in the population, and thus the sex ratio bias decreases. The advantage associated with the production of the rare sex decreases with reduction in the extent of sex ratio bias. Frequency dependant selection thus returns sex ratio biases to equality. These arguments apply equally to biases in favour of either sex.

Strictly, Fisher’s theory applies to the investment ratio required to produce the sexes, rather than to ratio of the numbers of each sex. His arguments also apply to the primary sex ratio and are unaffected by differential mortality between the sexes (Leigh 1970), provided that this is uncorrelated with sex-ratio genotypes (Hamilton 1967). A greater developmental mortality in one sex is exactly compensated for by the increased repro­ ductive success enjoyed by the survivors of that sex.

Although Fisher’s prediction of equal investment ratios explaintfhe sex ratio of many species, deviations from sex ratio equality are common amongst parasitoids (e.g. King 1987, Waage 1986). While Fisher’s prediction assumes random-mating (panmixis) many species of parasitic wasps do not conform to these conditions. Deviations from equal investment ratios were first attributed to non-random mating by Hamilton (1967). Hamilton considered extreme inbreeding amongst diploid animals; each patch being colonised by ’n’ females, their progeny mating randomly with each other before dispersal. In such cases the optimal sex ratio for mothers to produce becomes biased towards the sex that competes least for a limiting resource. Classically, if mating opportunities with females are the limiting resource, then the optimal sex ratio should be female biased to reduce the competition for mates amongst males.

Hamilton (1967) predicted that the optimal strategy is to produce a sex ratio with the proportion of males equal to (n-l)/2n. Hamilton’s original model was later extended to show that for haplodiploid animals, such as the Hymenoptera, the optimal sex ratio (proportion males) is given by (n-l)(2n-l)/n(4n-l) (Hamilton 1979, Suzuki & Iwasa 1980, Introduction 18

Taylor & Bulmer 1980). Differences between the models are due to asymmetries in relatedness between parents and offspring and also between siblings, existing in haplo-diploid but not diplo-diploid species (Fig. 1.3).

Number of foundresses(n) Fig. 1.3 The relationship between optimal sex ratio and the number of females (foundresses) repro­ ducing on a patch. The upper curve is the sex ratio for diplo-diploid animals, the lower curve is for haplo-diploid animals. Offspring in a patch are assumed to mate randomly amongst themselves prior to dispersal.

For large values of ’n ’, the optimal sex ratio is 0.5, as predicted by Fisher’s (1930) theory. When only one female colonises a patch, n= 1, the models predict that the optimum sex ratio strategy is to produce entirely female offspring. This is interpreted as that the number of sons should be sufficient only to fertilize the full compliment of daughters in the patch (Hamilton 1967). When n=l, the sex ratio produced by these models reduces the level of competition amongst males in the patch to the minimum, hence this is known as the theory of Local Mate Competition, or LMC (Hamilton 1967). Under such conditions Introduction 19

a mother’s fitness is assumed to be the number of mated daughters produced (Green e t al. 1982, Hamilton 1967, Hard 1971). Since males are capable of inseminating more than one female, the optimal sex ratio is female biased.

The sub-division of the mating population in patchy environments that leads to LMC also results in increased inbreeding. There had been uncertainty as to whether female biased sex ratios found under LMC were responses only to competition for matings between siblings, or whether biases were also responses to inbreeding itself. In haplodiploid species inbreeding leads to an increased mother-daughter relatedness, but mother-son relatedness remains the same under any conditions. This asymmetry in relatedness can lead to female biased sex ratios independently o f LMC. Herre (1985) showed theoretically that sex ratio biases should reflect both the level of sibling competition and the average level of inbreeding. For example, with a given number of foundresses (females colonising each patch) and at a given level of sibling competition, more highly inbred species should show greater sex ratio biases. Herre also provided empirical evidence strongly supporting his theoretical predictions, by using natural variation, both within and between species, in the number of foundresses colonising each fig and examining the sex ratios produced.

LMC theory only predicts the bias of the overall sex ratio produced by inbreeding parasitoids, but it is the sex ratio within the brood which affects the numbers of mated females produced and hence the mother’s fitness. Hard (1971) suggested that, to ensure the maximum average number of mated females produced in each brood, the overall sex ratio should be greater than that predicted by LMC models. Hard assumed that sex was allocated to eggs randomly. If sex allocation is random the probability of each egg being male is equal to the overall sex ratio (proportion males) produced by the mother. Under random sex allocation the frequency distribution of brood sex ratios will have random (binomial) variance (Fig. 1.4). Thus, not all broods contain the optimal offspring sex ratio and to maximise the numbers of mated females produced the overall sex ratio must be greater than that predicted by LMC theory. Introduction 2 0

O c CD 3 c r 0 ) ul

n-----r 9 10 Number of males in clutch

Fig. 1.4 Examples of binomial frequency distributions of sex ratios. Local mate competition theory (Fig 1.3) predicts the overall sex ratio mothers should produce. If, forexample, the sex ratio is predicted as 0.1 (proportion males) then under binomial sex allocation, for clutches of ten eggs, the most frequently occurring number of males in a clutch is 1. Clutches containing between zero and ten (inclusive) males will also be produced, with the relative frequencies shown by the black bars. If mating occurs between siblings, females developing in clutches containing no males will not be mated. To reduce the number of unmated females produced under binomial sex allocation the sex ratio should thus be less female biased than predicted by LMC theory. For example, an increase in the sex ratio to 0.2 gives the sex ratio frequency distribution shown by the grey bars. The formula which generates these patterns is, Frequency = r'" • (1 - m )' 'r • ( c! / r! ( r! ( c - r )!), where r is the sex ratio, m is the number of males in a clutch of size c.

Green e t al. (1982) showed, however, that the sex of offspring should be allocated with precise control. If sex is allocated to eggs precisely (non-randomly) the brood sex ratio variance is less than under binomial sex allocation and the number of mated females produced can be maximised without increasing the sex ratio overall. An examination of the sex ratio strategy of G. nephantidis is presented in Chapter 5.

Fisher’s (1930) theory assumed a homogeneous environment, this assumption can be violated by non-random mating (see above) and also by relative qualitative differences between hosts. If such qualitative differences occur, the sex allocation strategy of parasitoids may be altered if fitness of male and female offspring is affected differentially by the heterogeneity. Chamov (1979) and Chamov e t a l. (1981) showed that such conditions Introduction 21

lead to selection on mothers to place the sex that benefits proportionally more greatly, in the better quality hosts. This phenomenon is termed conditional sex expression, or CSE. Empirical support for CSE has been found in several species of parasitoids and other species of Hymenoptera (Chamov e t al. 1981).

Another factor that may lead to biased sex ratios in haplodiploid animals is the influence of oviposition by females constrained in their sex allocation possibilities. Until recently the consequences of reproduction by constrained females had not been explored. Although not of comparable importance to LMC or CSE, oviposition by constrained females can influence population sex ratios and the individual sex ratios produced by mated females (Godfray 1990), empirical evidence for this is examined in Chapter 2.

1.4 Statistical analysis

Most of the analysis presented in this thesis was performed using generalised linear modelling techniques (Aitkin e ta l. 1989,McCullagh&Nelder 1983,Healy 1988) available in the GLIM statistical package (GLIM 3.77, Numerical Algorithms Group, Oxford, 1985).

Generalised linear models are fitted by maximum likelihood methods (the parameter estimates adopted are those which make the observed data the most probable). Generalised linear models assume independent (or uncorrelated) observations. It is possible to use data with non-normal error variances distributions (e.g. Poisson, gamma, or binomial) without transformation since the important properties of generalised linear models depend not on normality, but on the assumption of constant variance. Thus, the parameter estimates do not depend upon the exact distributional form, but only on the variance to mean relationship and the independence of observations. This is fortunate since it is often not possible to be confident that the assumed distributional form is correct.

Fitting models summarizes patterns by replacing a set of data by a set of values derived from a model with a relatively small number of parameters. Given N observations, there can be N parameters. The null model is the simplest, having only one parameter (the overall mean), and consigns all variation in the data to the random component. The full model fits a parameter for every data point and thus matches the data exactly, consigning all variation to systematic components and none to the random component.

Full models achieve no reduction in complexity (because ragged data is not replaced by a simple theoretical pattern) but give a baseline for measuring the discrepancy between models with intermediate numbers of parameters and the data. These more simple models Introduction 22

are chosen by the inclusion of only those parameters accounting for a significant amount of the discrepancy between the model and the data. The contribution of a parameter to the overall explanation of discrepancy (or "goodness of fit") is quantified by the "deviance", this is the logarithm of the ratio of two likelihoods. The analysis of deviance examines the effects of parameters and their interactions. Constrained sex allocation 23

Chapter Two

Constrained Sex Allocation in Hymenoptera

2.1 Abstract

Sex ratio theory has assumed that females can produce offspring of both sexes. Some females in haplodiploid populations are only able to produce sons (constrained sex allo­ cation), the causes of this are reviewed.

It has been suggested that the presence of such females influences the optimal progeny sex ratio produced by unconstrained females. The relevance of these ideas to sex ratios in the natural populations is largely untested.

The frequencies of constrained oviposition in three, effectively panmictic, D r o s o p h ila parasitoid species are low or zero. Constrained, ovipositing females were distinguishable by the absence of sperm in the spermatheca.

Data from the literature that allow an estimate of the frequency of constrained females indicate that constrained oviposition is generally rare. There are some species, however, in which constrained females are sufficiently common for there to be selection for an observable sex ratio bias by unconstrained females. Constrained sex allocation 24

2.2 Introduction

The study of sex ratios is one of the most successful areas of evolutionary biology (reviews in Chamov 1982, Frank 1983, King 1987, Waage 1986, Waage & Godfray 1984, Werren 1987). Many of the empirical tests of sex ratio theory have been performed using haplodiploid organisms, in particular the Hymenoptera. Haplodiploid females normally have control over the sex of their offspring via the control of egg fertilization (e.g. Flanders 1956, Gerber & Klostermeyer 1970, Wilkes 1965); fertilized (diploid) eggs develop into females whereas unfertilized (haploid) eggs develop into males. This control permits the evolution of sex ratios adapted to local conditions (e.g. Colwell 1981, Frank 1985,1986, Godfray 1986, Hamilton 1967, Nunney 1985, Nunney & Luck 1988, Taylor 1981, Taylor & Bulmer 1980). Most sex ratio theory assumes that all females in the population are capable of the production of both sons and daughters. However, some females in haplodiploid populations are only able to produce offspring of one sex. Such females are referred to here as showing constrained sex allocation.

The causes of constrained sex allocation in the parasitic wasps are reviewed by Godfray (1990) and are classified as: V irg in ity: Unmated females are able to produce, but are unable to fertilize their eggs and are therefore constrained to lay only males. In many species of parasitic wasps, virgin females oviposit readily, at least in the laboratory (e.g Clausen 1940, Prince 1976, Singh & Sinha 1982, Sundarmurthy & Santhanakrishnan 1978), though in some species virgins may be more reticent to reproduce (Antolin 1989, Balfour Browne 1922, McColloch & Yuasa 1915, Tagawa 1987). There is probably no physiological impediment to virgin oviposition in most haplodiploid insects. Pseudo-virginity: This is a category of virginity which could be also termed "pseuo-mated" or "pseudo-inseminated" (Mathews 1975). Various factors may leave the female unwilling to copulate but without sperm, thus constraining her to only produce male offspring. These factors include interrupted coitus before insemination (van den Assem 1969, 1970, Raw & O’Toole 1979), lack of coitus during some initial critical time period (Crandell 1939, Drea e t a l. 1972, Stary 1970, Wiackowski 1962) or the act of (virgin) oviposition (Subba Rao & Shama 1962, Veri 1942). In some species virgin females are willing to copulate after oviposition (e.g. Askew & Ruse 1974, Javahery 1967), in some cases with their own sons (Balfour Browne 1922). Constrained sex allocation 25

S p erm d e p le tio n: The spermatheca can only store a finite number of sperm and once these are used only male eggs can be laid, unless the female mates again (Gordh 1976). Sperm depletion has been found to occur in many Hymenoptera (e.g. Gordh 1976, Gordh e t al. 1983, Pandey e ta l . 1983a, Rotary & Gerling 1973, Schlinger & Hall 1960,1961, Sekhar 1957, Tepedino & Torchio 1989, Chapter 4). The probability of sperm depletion increases if the male has run short of sperm (for instance after a large number of copulations (but see Wilkes 1965)) but is still willing to mate (e.g. Gordh & DeBach 1976, Laing & Cal- tagirone 1969, Nadel & Luck 1985, Simmonds 1953, Wilson 1961). If no sperm is transferred during coitus, a condition akin to pseudo-virginity will result unless the female mates with other males. Females may circumvent this problem by rejecting males with exhausted sperm supplies (Singh & Sinha 1980), though how females might assess the sperm supply of a male is unclear. Post-copulation constraints:

1) Enforced male production: For a period of time following mating a female may only be able to lay male eggs (van den Assem 1977, van den Assem & Feuth-de Bruijn 1977, Flanders & Oatman 1982, Genieys 1942, Javahery 1967, Mackauer 1976, Pandey e t a l. 1983b, Sandlan 1979). Sperm are initially unusable, possibly because it takes time for sperm to reach the spermatheca (Wilkes 1965) or because secretions in the spermatheca must activate the sperm (Flanders 1956) or be dissipated (van den Assem 1977) before sperm are usable. 2) Enforced fem ale production: In some species belonging to the family (Hymenoptera: Chalcidoidea), once females have mated they may be constrained to produce daughters only suggesting that females cannot control egg fertilization. This constraint has been reported by Colgan & Taylor (1981), Flanders (1936) and D. Gerling (pers. comm, to H.C.J. Godfray). Walter (1983) has, however, shown these reports to be generally mistaken, but believes that this constraint may occur in some species. If this constraint does exist the population sex ratio will be determined by the dynamics of mating (Godfray & Waage 1990). A slight variation on both of the above phenomena is that newly mated females are constrained to produce sons only, but after a time become constrained to produce daughters only (Mackauer 1976). Too many matings: If matings are too frequent then females may only be able to produce male progeny; possibly because the spermducts of the female become blocked with too many ejaculates (e.g. Flanders 1946, M.R. Strand pers. comm, to Godfray 1990) Constrained sex allocation 26

Constraints determined by oviposition opportunities: In the majority of the Aphelinidae, males and females develop in different kinds of hosts. Unconstrained females which have the choice of hosts suitable for the production of either sex should preferrentially oviposit into the relatively less common host. Thus the production of either only males or only females can be enforced (further details are given by Godfray 1990, Godfray & Waage 1990). Some of the consequences of constrained sex allocation leading to enforced male production have recently been explored (Bryan 1983, Godfray & Grafen 1988, Godfray 1990, Godfray & Hardy in p r e s s, Wellings 1988). Constraints have been considered to be disadvantageous to a female (e.g. Mathews 1975). Consider a panmictic haplodiploid population of unconstrained females where the costs of the production of sons and daughters are equal and where the sex ratio is 0.5 (proportion males) as predicted by Fisher’s (1930) principle of equal allocation. As the population is at sex ratio equilibrium, a mother anticipates the same fitness benefits from the production of either a son or a daughter. A rare gene for constrained (e.g. virgin) oviposition will not be selected against, since sons are equal in value to daughters. Indeed, as time spent waiting to be mated is time spent lost in reproduction, the rare gene is likely to be at an advantage. As the trait spreads, the population sex ratio will become male biased and this will lead to selection on unconstrained females to produce a female biased sex ratio such that the overall population sex ratio returns to equality (Fig. 2.1). Thus, sex ratio considerations will not limit the spread of constrained oviposition. The fraction of unconstrained females in the population will probably be determined by the ease with which males can locate and mate newly emerged females.

If this ease depends on biotic and abiotic influences, the fractions of virgin females are likely to vary from generation to generation (Godfray 1990). Females may thus adjust their progeny sex ratio to ambient levels of constraints. However, a prerequisite is that females are able to assess levels of constraints, perhaps by correlating environmental factors and virginity, or by using more direct evidence about local levels of virginity; the period before being approached by a male or the number of males attempting courtship, might imply the frequency of virginity in a population (Godfray & Hardy in p r e s s ). There is some evidence that parasitoid wasps produce more females after a long delay before mating (Hoelscher & Vinson 1971, Rotary & Gerling 1973), though the interpretation of these results is difficult due to other age-dependent sex ratio effects. The opposite result: sex ratios should be more male biased after a delay before mating, was predicted for species w ith o ut virgin oviposition by Werren and Chamov (1978) who suggested that delays before Constrained sex allocation 27

mating indicate transitory shortages of males. Females should thus produce more sons to capitalise on this shortage. For this strategy to be selected, the shortage of males must continue until the mother’s progeny reach sexual maturity. Species with short development times and long adult reproductive periods are most likely to fit the m odel’s assumptions (Godfray & Hardy in p r e s s ).

The above simple depiction of the spread of virgin oviposition is complicated if there are (1) other benefits to mating in addition to the acquisition of sperm; (2) costs to mating or (3) if unconstrained females do not alter their sex ratio as the population sex ratio becomes male biased (Godfray 1990, Godfray & Hardy in p r e s s ). Relaxing the assumption of panmixis also affects the conclusions. Hamilton (1967) demonstrated that frequent mating between siblings leads to the evolution of female biased sex ratios through a process he called Local Mate Competition (LMC) (Chapters 1 & 5). Initial attempts to incorporate constrained oviposition into LMC models (Godfray 1990) suggest that constrained ovi­ position will be strongly selected against since females that cannot produce daughters cannot take advantage of the population structure. The presence of constrained females will lead to little change in the optimal progeny sex ratio produced by unconstrained females (Fig. 2.1). Godfray’s (1990) model assumes diploid genetics for mathematical simplicity. Such models have not been developed further due to the complicated algebra involved in modelling the distribution of virgins across patches, and to difficulties of incorporating haplodiploid genetics, which affect the levels of inbreeding and influence the optimal sex ratio of unconstrained females (Godfray 1990, Godfray & Hardy in p r e s s ). Constrained sex allocation 28

Proportion constrained females

Fig. 2.1 The sex ratio produced by unconstrained females in response to the proportion of constrained females. Unconstrained females in panmictic populations should adjust the sex ratio of their progeny in response to the proportion of constrained females. Unconstrained females in inbreeding species should make little adjustment to their progeny sex ratio in response to the presence of constrained females (from God fray 1990).

Generally, female parasitoids are selective in their choice of mates, and males are relatively more promiscuous (van den Assem 1986). Perhaps females of species where there is little or no disadvantage to virgin oviposition are especially particular in their willingness to mate and in their mate choice. Unfortunately, very little is known about mate choice in haplodiploids (an exception is provided by Grant e t a l. (1974, 1980) and by White & Grant (1977) for N. vitripennis, a species with marked LMC). In many species, males locate mates by the sex pheromones produced by females. Pheromones are often complex molecules which are frequently metabolically expensive to produce and broadcast. Sex pheromones should thus be less common, and cheaper to produce, in haplodiploid insects in comparison with similar diplodiploid insects (Godfray & Hardy in p r e s s ). Pheromones are widespread in haplodiploid insects though a careful comparison of their distribution and nature has not been made. Constrained sex allocation 29

Virginity and the evolution of eusociality

Many eusocial animals are Hymenopterans, these may be predisposed to eusociality by their biology and their haplodiploid genetics may be incidental (Chamov 1978). Alternatively, the asymmetries in relatedness of siblings of different sexes in haplodiploid insects may have influenced the evolution of eusociality (e.g. Hamilton 1964a, 1964b). Sisters are more closely related to each other than mothers are to daughters, this may have led to the evolution of sibling caring by females (one of the main components of euso­ ciality). Sibling caring by females is advantageous if a randomly chosen sibling is more likely to be a sister than would be predicted from the overall population sex ratio. This occurs under certain situations were generations partially overlap (Grafen 1987a, Seger 1983) but can also occur if there is virgin oviposition (Godffay & Grafen 1988). The presence of virgin, ovipositing females leads to the population sex ratio becoming more male biased than the sex ratio of the progeny of mated females. Since females only occur in the progeny of mated parents, a randomly chosen sibling will be more likely to be a sister than would be predicted from the population sex ratio. This effect occurs whether or not mated females alter their progeny sex ratio in response to the presence of unmated females.

Although constrained reproduction has often been recognised as a laboratory phe­ nomenon, a complication to theoretical models (e.g. Hard & Brown 1970) and used as a tool in investigating parasitoid biology (e.g. Hardy & Blackburn in p r e s s , Chapter 3); the relevance of speculations on the consequences of constrained reproduction to field sex ratios remains largely untested. Here an attempt to estimate the frequency of constrained oviposition in field populations of several parasitoids of D r o s o p h ila is made. Evidence from the literature for the importance of constrained oviposition is also critically reviewed. Constrained sex allocation 30

2.3 Constrained Oviposition by D ro so p h ila Parasitoids

Before describing the field experiments, the biology of D r o s o p h ila parasitoids is briefly discussed.

2.3.1 Biology of D ro so p h ila parasitoids

The biology of the parasitoids attacking Drosophila spp. has been reviewed by Carton e t al. (1986). Three species which attack D ro so p h ila breeding in fermenting fruit (es­ pecially D. obscura, D. subobscura and D. melanogaster) were studied: Leptopilina het- erotoma Thompson (: Eucoilidae); Asobara tabida Nees von Esenbeck (: Alysiinae) and Tanycarpapunctata van Achterberg (Braconidae: Alysiinae). These species are solitary endoparasitoids ovipositing into D ro so p h ila larvae; the parasitoids remain as eggs or first instar larvae until the host pupates when they complete their development and themselves pupate within the puparium of the host.

Males emerge before females and mating takes place soon after female emergence. Males exhibit elaborate and specific courtship behaviour. Females only mate once in their lifetime, while males are polygynous (Carton e t al. 1986 and references therein). Estimates of the sex ratios of L. heterotoma and A . ta b id a have been made by rearing immature specimens collected from the field, in the laboratory. Carton e t a l. (1986) record sex ratios at emergence of 43/83 males (%2, N.S.) for L. heterotoma, and 78/195 males (%2 p<0.01) for A . ta b id a . These sex ratio estimates may be biased by differential mortality. However J.J.M. van Alphen(pers. comm.) reports that female biased sex ratios are common in field collections of both L. heterotoma and A . ta b id a and that in laboratory experiments females alter their sex ratio in response to the number of conspecifics ovipositing in the patch, as predicted by LMC theory.

D ro so p h ila typically breed in specialized resources such as rotting fruit and vegetable matter (Shorrocks 1982). These are usually ephemeral and scattered, so adult parasitoids do not usually emerge in a patch where hosts suitable for oviposition are available. Thus female parasitoids must disperse to find new oviposition sites. Females locate suitable habitats by olfaction (Vet e t a l. 1984, Vet & van der Hoeven 1984, Vet & van Opzeeland 1985) . Males do not disperse: only females are attracted to the host habitat (Carton e t al. 1986) .

In the laboratory, fed D r o s o p h ila parasitoids of both sexes, live for approximately one month. Longevity is greater in L. heterotoma (Carton e t a l. 1986). Longevity of femaleL. h e te ro to ma and A. ta b id a is shortened by continuous oviposition in the laboratory Constrained sex allocation 31

and is considered to be not more than two weeks in the field (Baker 1979). A. tabida, L. h etero to ma and T. punctata are all provigenic (emerge as adults with a complete com­ pliment of eggs) and are probably able to lay over 300 eggs (van Lenteren 1976, Baker 1979).

2.3.2 Methods

Fieldwork was carried out at Silwood Park, Berkshire, U.K. during May-September 1989. Parasitoids were collected as they searched for hosts in jars of D r o s o p h ila media. Following Baker (1979), the media for the baits were made by macerating apple, banana, or swede and adding 1cm3 of a solution of baker’s yeast (250g Litre'1) for each 200g of medium. Standard-sized jam jars were about half filled with medium. The mouths of the jars of bait were covered with 2mm plastic mesh to exclude unwanted organisms such as birds, squirrels and social wasps, but to allow free passage to D r o s o p h ila and their parasitoids. Collections were made in mature woodland or at the edge of woodland next to grassy areas and also in an apple orchard, at a total of eight sites. The baits were hung horizontally (to keep out rainwater) in groups from the branches of trees at about 1.5m from the ground. Each week one fresh jar of each type of medium was hung at each site. Each site was visited approximately every day throughout the study season. When a site was visited, any parasitoids then in the jars were caught with a pooter.

This study was an assessment of the proportion of reproducing females which are constrained. It was not possible to enure that parasitoids emerging from hosts within the bait media were actively host searching or ovipositing. These parasitoids had thus to be excluded from the collections. Baker (1979) reports the minimum developmental time for British D ro so p h ila parasitoids as 17 days. Female D ro so p h ila are not attracted to baits for a few days until these have reached a certain stage of fermentation (Vet & van Opzeeland 1985). It also takes time for the D ro so p h ila offspring to develop to a stage suitable for parasitisation (e.g. van Lenteren & Bakker 1975, van Alphen & Drijver 1982). Therefore, to prevent parasitoids emerging in the bait media while these were still in the field, each jar was collected 21 days after being hung at a site.

Captured parasitoids were identified in the laboratory. Whether a female was con­ strained by lack of sperm was detected by examination of the content of the spermatheca. The wasps were dissected in Insect Ringers and the spermatheca located under a binocular microscope (magnification up to x40). The spermathecae are lightly pigmented, those of L. h etero to ma are flask shaped whereas those of A. ta b id a and T. punctata are spherical. Constrained sex allocation 32

The presence or absence of sperm within the spermatheca was determined under the higher magnification of a compound microscope (up to xlOOO). While individual sperm could not be distinguished, living sperm appeared as a writhing mass inside the spermatheca. On occasion, motionless sperm were found, probably killed during the dissection. However, distinguishing between empty speimathecae and those containing dead sperm was not difficult.

Confidence limits on the proportion of constrained females in the population were estimated by assuming that mating is a Bernoulli trial leading to a binomial distribution. Likelihood-based confidence limits were calculated using the GLIM macro ‘BINREL’ (Aitken e t a l. 1989). The calculated standard errors are probably underestimates as there is likely to be between-individual variability in the probability of a female finding a mate.

2.3.3 Results

Eight species of D ro so p h ila parasitoid were found over the course of the study though only three species (L. heterotoma, A. tabida & T. punctata) were sufficiently common that estimates of the prevalence of constrained females could be made. Fig. 2.2 shows how the catches of these three species varied over the study period. The captures of all three species reached a maximum in June, and had declined to zero by mid-September: the sampling thus covered the majority of the flight period.

Of 58 dissected female A . ta b id a none were found to be sperm-less, while 6 out of 97 T. punctata and 2 out of 119 L. heterotoma were without sperm (Table 2.1). Constrained sex allocation 33

May June July August Fig 2.2 Numbers of parasitoids caught during the season (weekly averages of parasitoids caught per day). Parasitoids were dissected from the beginning of June. Solid line, L. heterotoma; dashed line, T. pu n c­ ta ta ; dotted line, A . tabida. Constrained sex allocation 34

Table 2.1 Estimates of the prevalence of constrained females in three species of D r o s o p h ila parasitoid.

Species Numbers Number with Proportion 95% confi­ dissected empty with empty dence spermatheca spermatheca limits Asobara tabida 58 0 0 0, 0.033

Tanycarpa punctata 97 6 0.062 0.014, 0.121

Leptopilina 119 2 0.017 0.003,0.051 h etero to m a

N o male L. heterotoma or A . ta h id a were captured at the baits and only one male T. p u n c ta ta . Females were often seen ovipositing and offspring of all three species were reared from the media in the laboratory after the baits were collected. It therefore seems certain that the females were captured whilst searching for oviposition rather than mating opportunities. Lack of sperm after dispersal is expected to be permanent since males do not disperse (Carton e t a l. 1986), and females searching for oviposition opportunities will not encounter emerging males since oviposition sites are ephemeral (Shorrocks 1982).

Sperm-less oviposition is thus rare or absent in these D r o s o p h ila parasitoids. Before discussing this finding, other estimates of the prevalence of constrained oviposition from the literature are reviewed.

2.4 Literature Review

Few studies have deliberately set out to estimate the proportion of constrained females in natural populations, though sometimes this can be inferred from data collected for other purposes. All identified studies from which this estimate can be made are collected in Table 2.2. Some studies reporting levels of virginity have been anectdotal (Balfour Browne 1922) or do not exclude the possibility of other factors giving the apperance of constraints (Raw & O’Toole 1979) these are excluded from consideration.

Nearly all the species for which data are available are parasitoid wasps, the exceptions being aculeate Hymenoptera in the family . The method by which the estimates are obtained is given in the second column of Table 2.2 and are further defined below. Constrained sex allocation 35

(1) Spermatheca dissection: Some estimates were made by dissection of the spermatheca. It is important to collect females for dissection while they are in the act of oviposition though in cases, where males do not disperse away from the natal site (e.g. wingless fig wasps) this is not essential (dissection and non-dispersing males).

(2) Gregarious broods: Many haplodiploid species lay many eggs into a host and their larvae develop together gregariously. Frequently, the majority of broods have approxi­ mately equal or female-biased sex ratios with a few broods consisting of all males. It is likely that these all-male broods are produced by virgin (or otherwise constrained) females and that the frequency of all-male broods is a rough guide to the frequency of constrained females in the population.

(3) Non-dispersing males: In some species of fig wasp males never leave the fig in which they develop. If a female develops in a fig with no males, or leaves a fig unmated, there is no prospect of mating. Dissection of the fig just prior to wasp emergence can determine the sexual composition of the wasps in each fmit, the proportion of females developing in figs which contain no adult males can then be calculated. This method assumes that all females developing in figs with at least one male are mated.

(4) Laboratory oviposition: Wild caught females can be allowed to oviposit in the lab­ oratory to test their ability to produce daughters. However, sufficient progeny from each female are required to eliminate statistical error, and care is needed to ensure that hosts suitable for daughter production are provided. Constrained sex allocation 36

Table 2.2 Estimates of the prevalence of constrained oviposition in field popula­ tions of haplodiploid insects (all Hymenoptera).

Species Method Propor­ Total Reference (Family) (see text) tion examined con­ strained

Ceratosolen dentifer Non-dispersing 0.020 100 Godfray (1988) () males and dissection

Philotrypesis sp. Non-dispersing 0.020 100 Godfray (1988) () males and dissection

Apocryptophagus sp. Non-dispersing 0.040 1212t Godfray (1988) (Torymidae) males

Apocyrta mega Non-dispersing 0.230 154f Godfray (1988) (Torymidae) males

Osmia lignaria Gregarious broods 0.294 17 T epedino & T orchio p ro p in q u a (1982)* (Megachilidae)

Osmia bruneri Gregarious broods 0.125 16 Frohlich & (Megachilidae) Tepedino (1986)*

Apanteles glomeratus Gregarious broods 0.119 535 Tagawa (1987) (Braconidae) (see Godfray 1990)

Apanteles glomeratus Gregarious broods 0.000 18 M.R. Shaw (Braconidae) {pers. comm.)

Apanteles glomeratus Gregarious broods 0.190 63 le Masurier (1987a) (Braconidae)

Apanteles abjectus Gregarious broods 0.182 11 M.R. Shaw (Braconidae) {pers. comm.)

Apanteles bignelii Gregarious broods 0.063 16 M.R. Shaw (Braconidae) {pers. comm.)

Apanteles fulvipes Gregarious broods 0.000 39 M.R. Shaw (Braconidae) {pers. comm.)

Apanteles melitaerum Gregarious broods 0.111 9 M.R. Shaw (Braconidae) {pers. comm.)

Apanteles spurius Gregarious broods 0.000 13 M.R. Shaw (Braconidae) {pers. comm.) Constrained sex allocation 37

A p a n te le s Gregarious broods 0.000 24 M.R. Shaw zyg a en a ru m {pers. comm.) (Braconidae)

(Apanteles) flavipes Gregarious broods 0.050 20 A.I. Mohyuddin (Braconidae) {pers. comm.)

Asobara tabida Laboratory 0.000 46 J.M. Cook (Braconidae) oviposition {pers. comm.)

Asobara tabida Dissection 0.000 58 This study (Braconidae)

T a n y c a rp a p u n c ta ta Dissection 0.062 97 This study (Braconidae)

Leptopilina Dissection 0.017 119 This study h etero to m a (Eucoilidae)

H a b ro c y tu s sp. Dissection 0.032 124 J. Wilson & H.C.J. () Godfray {u n p u b .) (see Godfray 1990)

E n ca rsia Dissection 0.000 48 M.S. Hunter pergandiella {pers. comm.) (Aphelinidae)

Trioxys indicus Laboratory 0.080 199 Singh & Sinha (Aphidiidae) oviposition (1980)

Trioxys indicus Laboratory 0.000 47 R. Singh (Aphidiidae) oviposition {pers. comm.)

Lysiphlebia mirzai Laboratory 0.000 78 R. Singh (Aphidiidae) oviposition {pers. comm.)

Lysiphlebia delhiensis Laboratory 0.000 112 R. Singh (Aphidiidae) oviposition {pers. comm.)

fTotal females examined from 100 ( Apocryptophagus) and 46 (A p o cryp ta) figs. "Data from greenhouse populations. ______Constrained sex allocation 38

9

Percentage constrained females

Fig 2.3 Distribution of percentage constrained females. Estimates from Table 2.2 are rounded to the nearest integer value. A single mean is taken for estimates of percentage constraints in T. indicus and for A . ta b id a , but the estimates for A. glomeratus are treated as separate values (see text).

Fig. 2.3 is a histogram of the distribution of percentage virginity in all studies shown in Table 2.2. Caution should be exercised in drawing conclusions from Fig 2.3 as it is a compilation of very heterogeneous studies. Nevertheless, two conclusions can be drawn: firstly, the majority of studies indicate low (<10%) frequencies of constrained oviposition in natural populations, but secondly, in some populations constrained females can be quite common and are probably a significant factor affecting observed sex ratios. Constrained sex allocation 39

2.5 Discussion

2.5.1 D ro so p h ila parasitoids

In the D ro s o p h ila parasitoid populations examined sperm-less females were absent or rare. Virginity, pseudo-virginity and sperm depletion cannot be differentiated as con­ straints in this study since females in all these conditions are sperm-less. Sperm depletion is probably an unlikely cause of lack of sperm under natural conditions, since males will probably ensure that the female has sufficient sperm for her lifetime needs. Any females constrained by too many matings and post-copulation constraints cannot be distingushed from unconstrained females in this study, thus the proportion of constrained females may be underestimated.

Fruit-feeding D r o s o p h ila inhabit temporary habitats but may be quite common in each resource patch. A number of hosts and parasitoids may thus develop together and as mating takes place around the pupation site prior to female dispersal, there may be a high probability of mating. In addition, if van Alphen’s observation that female biased sex ratios are often found by collections from the field applies to these populations, females may be selected to delay dispersal from their natal patch until they are mated. Thus, aspects of the biology of D ro s o p h ila parasitoids may lead to a high likelihood of females being located by males.

2.5.2 General prevalence of constrained oviposition

The studies reported in Table 2.2 are not a random selection of haplodiploid insects: for example fig wasps and the genus A p a n te le s are grossly over-represented. It is thus difficult to draw conclusions from any pattern of constrained oviposition. However, the hypothesis that constrained oviposition is less common in species with gregarious broods was tested. The rationale behind this is that gregarious broods are frequently associated with sibling mating (LMC) which leads to a female-biased sex ratio (Hamilton 1967). Constrained oviposition should be much more disadvantageous in a non-panmictic population with a female biassed sex ratio, since constrained females cannot take advantage of the population stmcture (see Introduction). In fact, levels of virginity are significantly higher in gregarious species than in solitary species (Gregarious, n=16, mean=9.0%; Solitary, n=8, mean=2.4%; non-parametric two sample test, p<0.05). Without further data it is difficult to explain this result. It is possible that in gregarious species, where most mating takes place amongst siblings, virgin females occur when all males in a brood die. Constrained sex allocation 40

Ovipositing females will then experience conflicting selection pressures to lay a greater proportion of females, to capitalise on population structure, but also more males, to avoid the risk of leaving daughters unfertilized (Hard 1971). Alternatively, the association of gregariousness with local mate competition may be an over-simplification: for example, there is evidence that the solitary D ro so p h ila parasitoids may experience local mate competition.

Available evidence suggests that the amount of constrained oviposition can differ greatly between species. For three species there is some data on within-species variations in virginity. Three independent estimates for different A. glomeratus populations give a wide variation in result (0-19%), while estimates on the same population of T. in d icu s in four consecutive seasons averaged 8% but varied between 6% and 14%, a fifth estimate for this same population in a later year found no females constrained to produce only males. Two independant estimates for a population of A . ta b id a in consecutive years found no constrained females. In the analysis the data for A. g lo m e ra tu swas treated as distinct data points since the estimates came from geographically very distant populations, though the r. in d icu s and A. ta b id a results were treated as single data points for each species, since each referred to a single population.

At what level does the presence of constrained, ovipositing females become important to empirical studies of sex ratio? This is an essentially statistical question as the answer depends on the accuracy with which sex ratios are measured. Suppose that unconstrained females adjust their sex ratio to compensate for the presence of constrained females. Sex ratio measurements made on unconstrained females (e.g. mated in the laboratory) would reveal a female-biased sex ratio instead of the expected sex ratio of equality. The optimal sex ratio, r, is related to the proportion of constrained and ovipositing females, x, by the relationship r=(0.5)(l-2x)/(l-;t) (Godfray 1990, Fig. 2.1). Whether a significantly biased sex ratio is detected depends both on the magnitude of r and the size of the sample population, n. Applying the normal approximation to the binomial theorem one can calculate that with a sample of 25 individuals, only sex ratios smaller than 0.3044 will be significantly different from 0.5; with a sample of 100 individuals, this minimum significant sex ratio rises to 0.402 and, with a sample size of 500 individuals, to 0.456. Sex ratios of 0.304, 0.402 and 0.456 are predicted if the proportion of constrained females are 0.282, 0.164 and 0.081 respectively. These rough calculations suggest that quite large sample sizes will be needed to detect the effects of constrained females if these females constitute Constrained sex allocation 41

less than about 10% of the population. 29% (7 out of 24) of the species listed (Table 2.2), exceed 10% constrained females indicating that at least in some species constrained ovi- position may be important enough to influence observed sex ratios. Brood guarding 42

Chapter 3

Brood guarding in the bethylid wasp G oniozus nephantidis

3.1 Abstract

Female Goniozus nephantidis Muesebeck remain with their broods until the offspring pupate. This behaviour is unusual amongst the parasitoid Hymenoptera and will only have evolved if the consequent reduction in fecundity is outweighed by fitness returns.

G. nephantidis competes for hosts with conspecific and non-conspecific parasitoids. The effectiveness of G. nephantidis at deterring superparasitism and multiparasitism is tested. Brood survivorships were compared when G. nephantidis and Bracon hebetor intrude on hosts with the mother present and absent and with offspring at different developmental stages.

Laying by the intruder reduced brood survivorship. Guarding reduced oviposition on unparasitized hosts by intruding females, and prevented superparasitism of hosts with egg broods. Hosts with larval broods were rarely superparasitized. Multiparasitism was common except on hosts with guarded larval broods, but even here survivorship was reduced.

Competitive asymmetries determined the outcome of contests for possession of host resources.

The costs of remaining seem at least partially offset by the prevention of oviposition by competing parasitoids. Brood guarding 43

3.2 Introduction

Typically, female parasitoid Hymenoptera simply provide their eggs with sufficient food resources for their development to adulthood (Doutt 1973). Females of some species increase their investment in each brood by remaining with the clutch until the offspring reach an advanced stage of development. Such females have a reduced reproductive potential if clutches can be laid more frequently than this behaviour allows. Remaining with broods will only evolve when the costs of the behaviour, in terms of lost reproductive opportunities, are outweighed by it s benefits (Steams 1989, Tallamy & Denno 1981, Tallamy & Wood 1986). Such benefits may include protection of offspring against abiotic (Ichikawa 1988) or biotic factors (Morse 1988, Nafiis & Schreiner 1988, Tallamy & Denno 1981).

Female Goniozus nephantidis Muesebeck (Hymenoptera: Bethylidae) remain with their broods following oviposition. This behaviour is unusual amongst parasitic wasps, but occurs in a number of other bethylid species (Griffiths & Godfray 1988). This study examines some of the factors that may favour this time investment. First, the biology of this wasp and it s environment is reviewed.

3.2.1 Biology of G. nephantidis

G o n io zu s(.Parasierola) nephantidis is a gregarious primary larval ectoparasitoid of Opisina arenosella Walker ( Nephantis serinopa Meyrick) (Lepidoptera: Oecophoridae), a coconut defoliator in the Indian sub-continent. A clutch of, usually, 10-15 eggs (Cock & Perera 1987, Chapter 4) is laid approximately one day after paralysing the host. Usually, the eggs are laid onto the dorsal or lateral surfaces of the host. A female G. nephantidis will stay with her brood until the offspring leave the host to pupate (Antony & Kurian 1960, Cock & Perera 1987, Remadevi e t al. 1981), 4-5 days after oviposition (Perera 1987, RamachandraRao&Cherian 1927, Remadevi e ta l. 1981 ,pers. obs.), but is physiologically capable of laying clutches at more frequent intervals than this behaviour allows. If a female is removed from her clutch and presented with a fresh host, a complete new clutch can be laid within the period that she would otherwise have been guarding ( pers. obs). However, reproductive opportunities are normally rare since O. arenosella is an outbreak pest which usually has very low population levels (Cock & Perera 1987, Perera 1987, Perera e t al. 1988). Brood guarding 44

The host, O. arenosella, has many pathogens, predators and parasitoids (Cock & Perera 1987, Dharmaraju 1952, 1962, Jayaratnam 1941a) which may also affect G. nephantidis broods. The female could be protecting broods against these factors. G. nephantidis will "clean" it s eggs with it s mandibles (pers. obs.) and so may ward off attack by fungal pathogens (Cock & Perera 1987, Ramachandra Rao & Cherian 1927). However, in laboratory conditions, the presence or absence of the mother makes no sig­ nificant difference to the survival of G. nephantidis eggs (Chapter 4, see also Doutt 1973). Hence, the behaviour is not explained by protection of the brood from pathogens, although this may be more important in nature.

The predatory mite Pyremotes venticosus Newport (Acarina) will destroy broods of G. nephantidis (Ramachandra Rao & Cherian 1927, Cock & Perera 1987). Remaining with the brood may not protect offspring from these predators, however, since stored product mites (.Acarus siro L.) can destroy clutches in laboratory cultures with the mother present {p e r s. o b s.). Since G. nephantidis is small (3-5mm), remaining with the brood may also provide little protection against larger generalist predators such as (Hymenoptera: Formicidae), spiders (Araneae) and crows (Aves: Corvidae) (Perera 1987).

Conspecific parasitoids can act as superparasitoids (conspecific individuals which oviposit on the same host). There are several species of other larval parasitoid competing for O. arenosella (Cock & Perera 1987) which are potential multiparasitoids (individuals of different parasitoid species which oviposit on the same host). G. nephantidis broods also have several species of hyperparasitoids (parasitoids of other parasitoids), which can destroy the clutch (Perera 1987).

As there is already some evidence that remaining with the brood does not protect against pathogens or predators, this study tested the effectiveness of G. nephantidis mothers in guarding their broods against potential superparasitoids and multiparasitoids. The aim was to ascertain whether defence of the brood against these competitors is a function of remaining with the brood after oviposition.

3.3 Methods 3.3.1 Animals

The competitor species used in these experiments was the gregarious larval ecto- parasitoid Bracon hebetor {sensu Lato) (Hymenoptera: Braconidae) (the of the braconids is poorly understood; the B ra c o n species used in this study is B . h e b e to r Say or Brood guarding 45

a closely related species). B . h e b e to r is a natural parasitoid of O. arenosella (Perera 1987), and therefore a potential multiparasitoid (and possible hyperparasitoid, (Dharmaraju 1952)) of G. nephantidis. In common with other braconids, B . h e b e to r does not remain with it s brood after oviposition, although behaviour similar to that of G. nephantidis is known in one braconid species (Beeson & Chatterjee 1935). B . h e b e to r lays a clutch of 8-12 eggs on the ventral surface of the host. The period from oviposition to eclosion is 7-9 days (Ramachandra Rao e t a l. 1950).

Since their natural host O. arenosella is difficult to culture in the laboratory, G. nephantidis and B . h e b e to r were reared on an alternative host, the stored product pest Corcyra cephalonica Stainton (Lepidoptera: Pyralidae). All parasitoid cultures and experiments were performed at 30°C, 70% r.h. with a 16L:8D photoperiod. The parasitoid stocks originated from cultures at the Coconut Research Institute, Lunuwila, Sri Lanka.

3.3.2 Experimental procedures

An adult female G. nephantidis with no prior oviposition experience, was placed in a glass vial with a single host larva weighing 30-40mg. Once this female had paralysed the host, the host was transferred to the centre of a plastic petri dish (5cm. diameter). The female was either moved with the host and allowed to settle after this disturbance, or removed from the experiment. A female intruder of either G. nephantidis or B . h e b e to r was introduced into the petri dish. Intruders had no prior oviposition experience. Con- specific intruders were not siblings of the initial female, but were of a similar age. This procedure was repeated with initial females that had laid a clutch of eggs, and again with initial females whose clutches had developed to the larval stage. Both initial female and intruder were removed from the petri dish once the brood had pupated.

When the host was paralysed (before a clutch was laid), and both the initial female and a conspecific intruder were present, the initial female used was a virgin, produced by rearing pupae in isolation. The Hymenoptera have haplodiploid sex determination; thus, unmated G. nephantidis females can produce offspring, but only males (Sundaramurthy & Santhanakrishnan 1978). Otherwise, there are no differences in clutch sizes or brood survivorships between mated and unmated mothers (Chapter 4). Therefore, it would be certain that any female offspring were the progeny of the intruder. Had not the initial female been a virgin, inference as to the parentage of female offspring would have been impossible. Thus, twelve different experimental combinations were created, in which the initial female was either present or absent, her brood was at one of three developmental Brood guarding 46

stages and the intruder was a female belonging to either of two species. Broods were observed at intervals until their development was complete. Brood survivorships were assessed by counting the numbers of offspring at each of the developmental stages; egg, larvae, pupae and adult.

When the initial female and a conspecific intruder were both present, each was marked with enamel paint for identification. When a conspecific intruder was introduced with the initial females brood at the egg stage, eggs were marked, without being removed from the host, with an aqueous solution of Eosin stain. This was to distinguish the initial clutch from any subsequently laid eggs. Marking the eggs, however, may affect their probability of survival. To test the effect of marking eggs on brood survivorship, six controls were performed. Clutches of eggs were either marked or unmarked, mothers were marked, unmarked or removed. The brood survivorships were recorded as above.

Analysis was performed using the GLIM general linear modelling program (Aitkin e ta l . 1989, McCullagh & Nelder 1983, Healy 1988, Chapter 1). For brood survivorship analysis binomial error variances were assumed, since survivorship values are proportions.

3.4 Results

3.4.1 Control

There was no significant difference between the egg to larval survivorships (pro­ portion hatching) of control broods guarded by unmarked females and control broods with no guarding female (t = 1.92, d.f. = 42, p > 0.05) (Table 3.1). Marking the female significantly reduced egg to larval survivorship compared to situations in which the female was unmarked or absent (t = 2.76, d.f. = 63, p < 0.01) (Table 3.1). However, analysis of the residuals showed that this difference was due to the effect of two replicates with much lower survivorship than the rest, reducing the survivorship of the doubly marked control group. With these replicates omitted, the difference between the survivorships disappeared (t = 1.67, d.f. = 61 , p > 0.05). There was no difference between the survivorships of marked and unmarked eggs (t = 1.02, d.f. = 63, p > 0.05). The mean survivorship for control broods did not differ significantly from the equivalent value calculated from data collected during investigations of G. nephantidis clutch size (Chapter 4) (7.32% egg to adult, 81% larva to adult) (t = 0.77, d.f. = 61,p > 0.05). Since the sample size used to obtain this survivorship value was much greater than for this control, the Chapter 4 survivorship values were taken as the "normal" values in the subsequent analysis. Brood guarding 47

Table 3.1 Mean percentage egg to larval survivorship of control group clutches with different combinations of marked eggs and adults. The number of replicates is shown in brackets.

Eggs marked Eggs unmarked

Mother absent 81.2(10) 75.0(11)

Mother present and marked 67.2(11) 82.2(11)

Mother present and unmarked 89.4(11) 81.2(11)

3.4.2 Brood survivorships

In the presence of a conspecific intruder, the survivorship of broods at the egg stage was significantly lower when the initial female (the mother) was absent than when she was present (t = 6.83, d.f. = 29, p < 0.001) (Table 3.2). However, when a conspecific intruder was introduced to the brood at the larval stage, the presence or absence of the mother made no difference to brood survivorship (t = 0.347, d.f. = 18,p > 0.1). Conspecific intruders have no effect on the survivorship of larval broods, compared to normal survi­ vorship, whether the mother was present (t = 1.74, d.f. = 105, p > 0.05) or absent (t = 1.23, d.f. = 105, p > 0.05). Conspecific intruders have no effect on the survivorship, compared to normal survivorship, of any broods guarded by the mother (Eggs, t = 0.145, d.f. = 102, p > 0.05: Larvae, t = 1.74, d.f. = 105, p > 0.05).

The survivorship of G. nephantidis broods was always very low when B . h e b e to r was the intruder (Table 3.2). However, the presence of the mother significantly increased the survivorship of larval broods (mean = 42%, 95% Confidence Intervals 31.5%-52.9%) compared to that of unguarded larvae (mean 0%, 95% C.I. 0%-2.5% calculated directly from the likelihood surface, see Aitkin e t a l. (1989), pi 12-118). There was no significant difference between the survivorships of guarded and unguarded eggs in the presence of B. h e b e to r (t = 2.08, d.f. = 18, p > 0.05). Under equivalent conditions of mother presence and brood development, the brood survivorship was always significantly lower with B. h e b e to r as the intruder compared to G. nephantidis (Table 3.2). Brood guarding 48

Table 3.2 Comparison of the mean percentage egg or larval to adult survivorship for the initial female’s brood with different intruder species in the presence or absence of the initial female. The number of replicates is shown in brackets.

Intruder species

Initial Initial female’s female G. nephantidis B. hebetor t d.f. P brood

Egg Present 74.2 (10) 8.3 (10) 7.96 18 <.001

Absent 27.0 (25) 1.1 (10) 4.34 33 <.001

Larval Present 89.3(11) 42.0 (10) 5.71 19 <.001

Absent 87.5(11) 0* (10) - - -

* Standard errors cannot be assigned to zero in a binomial distribution; hence t cannot be calculated. However the 95% confidence intervals of these survivorships do not overlap, indicating that they are significantly different at the 5% level (see text).

3.4.3 Presence or absence of the initial female and intruder oviposition

When a conspecific intruder was introduced with the host paralysed by the initial female but before the initial female had laid eggs, and with the initial female present, at least 30% of intruding females oviposited on these hosts (Table 3.3). If the initial female had been removed, significantly more (90%) of the conspecific intruders laid (Table 3.3). In the same situations but w ith#, h eb eto r as the intruder, B . h e b e to r females laid eggs on 25% of paralysed hosts with the initial female present, and on 90% when the initial female had been removed (Table 3.3). Brood guarding 49

Table 3.3 Comparison of the proportion of experimental replicates in which the intruder laid in a host in the presence or absence of the initial female.

Frequency of laying by intruder

Intruder Brood Initial Initial species stage female female d.f. p (2-tailed) present absent

G. nephantidis Para. 6/20 9/10 1 0.002* © o o H - r Eggt 0/10 13/21 1

Larval 0/10 1/10 1 >0.05

B . h e b e to r Para. 2/8 9/10 1 0.005*

Egg 10/11 9/10 1 >0.05

Larvalf 3/10 10/10 1 0.003 p values obtained using Fisher’s exact test, except * from Pearson’s X2. t occasions when the presence of the initial female increases the survivorship of her brood in the presence of an intruder.

3.4.4 Survivorship of the initial female’s brood and intruder oviposi- tion

Table 3.4 shows the survivorship of the initial female ’ s brood when the intruder either laid or did not lay for both intruder species, irrespective of the presence or absence of the initial female. Comparing these; when the intruder did not lay, the mean survivorship of initial female’s broods did not significantly differ from "normal" (Chapter 4), except when the B . h eb eto r intruded with the brood at the egg stage. In the exception the G. nephantidis brood survivorship was significantly lower than normal. When the intruders of either species laid, the mean survivorships of the initial female ’ s broods were always significantly lower than the normal. Brood guarding 50

Table 3.4 Comparison of the survivorships of initial female’s broods when the intruder either does or does not lay a clutch. Survivorships in the presence and absence of initial females, are combined.

Percentage survivorship when intruder

Intruder species Brood Lays Doesn’t Lay t d.f. P stage

G. nephantidis Egg 0.75 74.52* 5.85 29 <0.001

Larval 20.00 90.73* 3.18 18 <0.01

B . h e b e to r Egg 5.20 0 0.35 18 >0.1

Larval 4.72 70.96* 6.44 16 <0.001

All survivorships except those marked * are significantly lower than normal laboratory survivorships calculated in Chapter 4.

When the presence of the initial female (the mother) had a significant effect on brood survival (Table 3.2), her presence also significantly reduced the probability of the intruder laying (Table 3.3). Conversely, when her presence had no effect on brood survival (Table 3.2), it also had no effect on the probability of the intruder laying (Table 3.3). If the B. h e b e to r intruder laid eggs, then the presence of the initial female made no difference to the survivorship of either egg (survivorship when mother present 0%, absent 1.4%, t = 0.24, d.f. = 12, p > 0.1) or larval (present 22.7%, absent 0%, t = 0.26, d.f. = 11, p > 0.1) broods (N.B. this 0% is treated as 0.01% by GLIM, since standard errors cannot otherwise be assigned to zero, see also * Table 3.2). Therefore, significant differences in survivorship between guarded and unguarded broods were always a consequence of the mother reducing the probability of the intruder laying.

The poor survivorship of unguarded eggs with a conspecific intruder (Table 3.2) was due to their destmction by the intrader. The intruding G. nephantidis female was observed to commit ovicide by eating unguarded eggs. B . h e b e to r was not o b s e r v e d to commit ovicide but the zero survivorship of G. nephantidis eggs when B . h e b e to r did not lay (Table 3.4) indicates that ovicide was committed. The normal survivorships of larval broods Brood guarding 51

when the B . h e b e to r did not lay indicates that larvicide was not committed, at least in isolation from oviposition. G. nephantidis brood survivorships were also reduced when B . h e b e to r laid; to discover if the detrimental effect of multiparasitism was due to ovicide or larvicide by the adult B. hebetor, or to B . h e b e to r larvae being superior competitors a manipulation experiment was performed. Eight B . h e b e to r eggs were placed onto ten natural sized clutches of G. nephantidis eggs using fine seekers, in the absence of adults of either species. The broods were allowed to develop to adulthood. The resultant mean survivorship of the G. nephantidis broods was 44%, significantly lower than the normal survivorship of 73.1 % (t = 5.48, d.f. = 81, p < 0.001), but higher than the 1.1 % survivorship when the adult B . h e b e to r was also present (t = 4.27, d.f. = 18, p < 0.001).

The reproductive success of intruders of both species on guarded and unguarded broods was the opposite of the success of the initial female (Tables 3.2 & 3.5). When the mother’s brood was successful, the intruder had poor success, and vice versa. Under identical conditions of guarding, and brood stage, B . h e b e to r intruders always had greater reproductive success than conspecific intruders (Table 3.5). Of the 14 occasions when a conspecific intruder superparasitized, 13 occurred within 24 hours of intrusion. O f the 43 instances when a non-conspecific intruder laid, 40 occurred within 24 hours of intrusion.

Table 3.5 Mean number of intruder offspring per host.

Intruder G.nephantidis B . he b e to r Species

Initial female Present Absent Present Absent

Paralysed host * 5.3 (10) 1.88 (8) 8.6(10)

Egg stage brood 0(10) 4.5 (22) 3.7(10) 6.6(10)

Larval stage brood 0(10) 0.2 (10) 0.4(10) 9.3(10)

The number of replicates is shown in brackets ^impossible to infer parentage of offspring Brood guarding 52

3.5 Discussion

By remaining with her brood, a female G. nephantidis guards against both super­ parasitism and multiparasitism. This has benefits in terms of brood survivorship. Guarding is always effective against conspecific intruders, which never superparasitize when the mother is present. However, the mother only needs to guard eggs against conspecifics, since superparasitism or destruction of unguarded larval broods rarely occurs. Conversely, brood guarding against B . h e b e to r is only effective at the larval stage, although the survivorship of guarded larvae is below normal. However, there is clearly a benefit to guarding larvae because unguarded larvae are always killed. Thus, the benefits of brood guarding change as the brood develops.

One condition favouring host guarding is that the chances of finding unparasitized hosts are low. The number of hosts naturally encountered by a female G. nephantidis over a lifetime is, however, unknown (Chapter 4). As the probability of a female parasitoid finding more than one host decreases, so the proportion of her reproductive success represented by each clutch increases. Hence, when hosts are rare, a female will be selected to maximize her fitness gain from each host encountered, for example by laying the Lack clutch size (Godfray 1987a, Chapter 4) and by host guarding (sen su patch guarding, van Alphen & Visser 1990). Furthermore, facultative hyperparasitism, multiparasitism and superparasitism are all most likely to occur when hosts are rare (Godfray 1987a). In this case, the benefits of guarding are greatest when the opportunity costs are lowest.

Competition for possession of the host can be interpreted using a game theory approach. As the brood develops and uses host resources, the reproductive potential of the host to a conspecific intruder decreases, whereas the value of the brood to the mother increases (since the current brood becomes more valuable relative to future broods, n ot because of the mothers increasing investment, Dawkins & Carlisle 1976, Trivers 1972). Hence, there is an increasing asymmetry in resource value to the mother and the intruder (Maynard Smith & Parker 1976). When this asymmetry favours the mother it is not expected that the intruder will gain possession of the host (Parker 1974).

When a paralysed host is guarded before a brood has been laid, there is no competitive resource value asymmetry, since the host is of equal value to both intruder and mother in terms of future fitness returns. Consequently, the outcome of contests for possession of the host may be decided by the relative "resource holding potentials" of the competitors. These may be related to the resource (e.g. "ownership status") or to some factor uncorrelated with the resource (e.g. fighting ability) (Hammerstein 1981, Maynard Smith & Parker Brood guarding 53

1976). Conspecific intruders laid clutches on 30% of guarded paralysed hosts, whereas they never laid on guarded hosts with broods. Further, the incidence of intruder oviposition may have been underestimated, since any all-male broods laid by the intruder would have been counted as laid by the virgin guarding mother. That intruders sometimes gained possession of the guarded paralysed host, but conspecific superparasitism of guarded broods never occurred, supports this interpretation.

When conspecific intruders superparasitized unguarded hosts with broods of eggs, they first consumed the initial female’s eggs, and then laid a clutch of their own {con tra Jayaratnam 1941b, see also Remadevi e t al. 1978). Such behaviour is also known in other bethylids (Goertzen& Doutt 1975, Malyshev 1968, Venkatraman & Chacko 1961) and other parasitoid families (e.g. van Alphen & Visser 1990, Beeson & Chatterjee 1935). Strand and Godfray (1989) predicted conditions when ovicide in conjunction with superparasitism would occur; notably, when the rate of host encounter is low, the proportion of hosts parasitized is high, and the time taken to kill eggs is short. The biology of G. nephantidis conforms to these conditions, so it is perhaps not surprising that females perform ovicide.

It is unclear why intruding female G. nephantidis did not kill unguarded larvae and replace them with their own clutches. It is surprising that a female may be unable to kill larvae, although there is some indirect evidence for this. On the one occasion when a conspecific intruder did superparasitize a larval clutch, two of the four offspring produced were from the original clutch, showing that at least part of the clutch escaped larvicide. The bethylids G. p la ty n o ta e(Ashm.) and Scleroderma macrogaster (Ashm.) also kill eggs but not larvae (Goertzen & Doutt 1975, Wheeler 1928), although G. m a ra sm i (Kurian) will kill both eggs and small larvae (Venkatraman & Chacko 1961). Perhaps larvicide does not occur because it is too time consuming (Strand & Godfray 1989), or simply because once the members of the clutch have developed to larvae the host resources are too depleted for superparasitsm to be profitable.

B . h e b e to r is sometimes described as a facultative hyperparasitoid of the parasitoids of O. arenosella (Cock & Perera 1987, Dharmaraju 1962); this may account for it s higher reproductive success on larval G. nephantidis broods. Thus, the benefits of guarding against multiparasitoids and facultative hyperparasitoids may be quantitatively similar. Doutt (1973) considered that protection against hyperparasitism was the main reason for brood guarding by the bethylid Perisierola bicarinata (Brues). Unfortunately, no definite facultative hyperparasitoid was available to test this for G. nephantidis. Brood guarding 54

When B . h e b e to r intruders did not lay, the survivorship of G. nephantidis broods of eggs, but not of larvae, were reduced. B . h e b e to r intruders thus probably committed ovicide but not larvicide, in isolation to oviposition. This is not surprising since B . h e b e to r Say kills eggs of conspecific clutches by puncturing them with it s ovipositor (Strand & Godfray 1989). When B . h eb eto r intruders laid the survivorship of G. nephantidis clutches was always reduced. This may have been due to the destruction of clutches by the adult B. h e b e to r intruders or to the B . h e b e to r clutch winning the competition for resources: perhaps because the developmental period of B . h e b e to r is shorter than that of G. nephantidis (Cock & Perera 1987, Benson 1973). Similarly, competition for resources by B . h e b e to r causes increased mortality amongst developing G. m a ra sm i (Venkatraman & Chacko 1961). The manipulation of B . h eb eto r eggs onto a host with a G. nephantidis clutch demonstrated that brood competition could only cause about half of the mortality observed when an adult B . h e b e to r was present. It should be noted that the manipulated B . h e b e to r clutch was an arbitrarily chosen constant size. Thus, although it was within the clutch size range laid by intruding B . h e b e to r females, the manipulated clutch size may have differed from the natural mean, and so may have had a quantitatively different, but qualitatively similar effect on G. nephantidis brood survivorship. Therefore, the adult B . h e b e to r accounts for most G. nephantidis brood mortality, although a role for larval competition cannot be ruled out.

There is no measurement of natural intrusion frequencies or exact host availability so a quantitative cost/benefit analysis of guarding cannot be provided. The more limited conclusion is drawn that the fitness costs of remaining with the brood after oviposition are at least partially offset by the prevention of and of oviposition by other parasitoids. Clutch size 55

Chapter 4

Clutch size in Goniozus nephantidis

4.1 Abstract

Clutch size is an important component of the reproductive strategy of animals which lay eggs in discrete batches. In order to predict clutch size it s relationship to the p e r c a p ita fitness of offspring must be known and also the parental trade-off between present and future reproductive success.

The importance of trade-offs is usually difficult to assess, but there is a strong a p r io r i case for their unimportance in the bethylid wasp Goniozus nephantidis. Theory suggests that in the absence of trade-offs the clutch fitness should be maximised, this is achieved at the ’Lack clutch size’.

G. nephantidis females adjust the size of their clutches to the host size. Manipulation of clutch size on standard sized hosts shows that offspring developmental mortality is unaffected by clutch size, but larger females emerge from smaller clutches. The longevity of reproducing females and their fecundity are both positively correlated with their body size.

The Lack clutch size is calculated using each of longevity and fecundity as estimators of offspring fitness. The calculated Lack clutch sizes are notably larger than the clutch size produced by the parasitoid.

Disparities between observed and predicted clutch sizes could be attributed to incorrect assumptions about the importance of trade-offs or to inadequate measurement of offspring fitness. The latter is likely since a laboratory-based approach is unable to assess all components of adult offspring fitness. In a species ideal for such studies the fitness consequences of clutch size would be resticted to effects on offspring developmental mortality rather than to their adult fitness. Clutch size 56

4.2 Introduction

Clutch size is an important component of the reproductive strategy of those animals which lay eggs in discrete clutches. The evolution of clutch size in birds has been studied since Lack’s (1947) suggestion that the clutch size laid should maximise the number of fledgling young. More recently, there has been interest in the evolution of clutch size in invertebrates, particularly insects (e.g. Begon & Parker 1986, Chamov & Skinner 1984, 1985, Godfray 1987a, 1987b, Godfray & Ives 1988, Ives 1989, Iwasa e ta l . 1984, Mangel 1987, Parker & Begon 1986, Parker & Courtney 1984, Skinner 1985, Smith & Lessells 1985, Stamp 1980, Waage 1986, Waage & Godfray 1985). Much of this interest has applied to gregarious parasitic wasps, these lay clutches of eggs into or onto the body of their hosts. In many species, the host resources constitute the only food source for developing members of the brood.

As eggs are added to a clutch the fitness of individuals in the clutch is affected. Amongst parasitic wasps the p e r c a p ita fitness of clutch members usually declines monotonically as clutch size increases due to competition between siblings for limiting resources (Godfray 1987a). The total fitness of the clutch is simply the number of indi­ viduals in the brood multiplied by the p e r c a p ita fitness. The clutch size that maximises total fitness has become known as the ’Lack clutch size’ (Chamov & Skinner 1984, Waage & Godfray 1985, Fig. 1.2), beyond this clutch size the total fitness declines. Clutch size theory suggests that in the absence of trade-offs between present and future reproductive success, the clutch fitness should be maximised and that the Lack clutch size should thus be laid (Godfray 1987a). However, when trade-offs exist between present and future reproductive success, clutches smaller than the Lack clutch size may be selected. For example, if the reproductive success of the mother is limited by time, or eggs, using the limiting resource to complete the Lack clutch size might be an inferior strategy to using time to search for new clutch sites (Chamov & Skinner 1984, Parker & Courtney 1984, Skinner 1985) or eggs to start new clutches at fresh sites (Parker & Courtney 1984, Waage & Godfray 1985).

In order to predict clutch size in parasitic wasps, it is necessary to know the rela­ tionship between clutch size and the p e r c a p ita fitness of the offspring and also the parental trade-off between present and future reproductive success. The importance of parental trade-offs between present and future reproductive success is difficult to quantify since this usually involves the measurement of components of lifetime reproductive success for individual wasps in the field. Some components of offspring fitness are more readily Clutch size 57

measurable, such as survival to adulthood. However, there may also be effects of clutch size on the fitness of emergent adult progeny (Waage & Ng 1984, Chamov & Skinner 1984, 1985), e.g. clutch size may influence adult size which in turn may affect longevity and fecundity. The importance of these effects may be difficult to measure in the field.

The relatively few empirical studies of the relationship between p e r c a p ita fitness and clutch size (Godffay 1987a) have used either of two approaches. Firstly, natural variation in clutch size can be used to assess the fitness of individuals developing in different sized clutches (Godffay 1987a). However, for proper comparison of brood fitness ovi- position sites must be of identical quality; this is difficult to establish (Godfray 1987a). Secondly, the clutch size can be altered by manipulation and the fitness of the progeny established (Dijksra 1986, Taylor 1988). A problem with this approach is that adaptations unrelated to the selective forces which influenced the evolution of the clutch size may m ain tain the clutch size (Chew & Robins 1984, Godfray 1987a, 1987b). Such adaptations limit the power of the manipulative approach.

This chapter is an investigation of clutch size strategy in the gregarious parasitoid, Goniozus nephantidis. Firstly, the clutch size response to hosts of different qualities is examined. Secondly manipulations of clutch size on standard quality hosts explore the consequences of clutch size on two components of offspring fitness, pre-adult mortality and adult size. The effects of adult size on two further components of offspring fitness, their adult longevity and fecundity, are investigated. There is a strong a p r io r i case for the unimportance of trade-offs between present and future reproductive success in G. nephantidis. Maximising lifetime fitness is thus equivalent to maximising brood fitness and the attainment of the Lack clutch size is predicted. The Lack clutch size is calculated and compared to the clutch size produced by the parasitoid. Before explaining the experimental methods the biology of G. nephantidis is briefly described.

4.2.1 Biology of G. nephantidis

Goniozus (Perisierola) nephantidisMuGsebeck (Hymenoptera: Bethylidae) is a small wasp, c.0.5cm in length. It is a parasitoid of Opisina arenosella Walker (= N ep h a n tis se rin o p a Meyrick) (Lepidoptera: Oecophoridae), a coconut defoliator occurring in the Indian sub-continent. The female G. nephantidis lays a clutch of eggs onto the paralysed host about a day after paralysing it. The elongate eggs are laid onto the dorsal or lateral surfaces of the host larvae and orientated parallel to it’s long axis. On hatching, the parasitoid larvae feed ectoparasitically, pupating around the remains of the host several Clutch size 58

days later (Perera 1987, Ramachandra Rao & Cherian 1927, Remadevi e t al. 1981). On eclosion, the adult progeny mate amongst themselves prior to dispersal. The sex ratio is female biased, probably as a result of Local Mate Competition (Hamilton 1967).

Unlike most parasitoids, though in common with a number of other Bethylidae (Griffiths & Godfray 1988), the female remains with her developing brood until the off­ spring pupate (Antony & Kurian 1960, Cock & Perera 1987, Remadevi e t a l. 1981), probably to guard the brood against the detrimental actions of other parasitoids (Hardy & Blackburn in p r e s s, Chapter 3). The parent wasp thus makes a large time investment in each clutch. Females are physiologically capable of laying clutches at more frequent intervals than this time investment allows (Remadevi e t al. 1978, pers. obs.). Thus, the fecundity of G. nephantidis is limited not by egg supplies, but by the time invested in producing and guarding clutches. The reproductive success of the wasp is thus likely to be limited by it’s opportunities to produce clutches, consequently trade-offs between present and future reproductive success are expected to be absent or weak. Maximising brood fitness is thus equivalent to maximising lifetime fitness and the optimal clutch size is predicted to be the Lack clutch size.

4.3 Methods 4.3.1 Animals

The G. nephantidis stock originated from the Coconut Research Institute, Lunuwila, Sri Lanka where it had been cultured for more than ten years on Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae). This stock had been intermittently supplemented by wild caught G. nephantidis.

During culturing and experiments G. nephantidis were reared at 30°C, 70% r.h. and 16L:8D photoperiod. G. nephantidis plus one C. cephalonica host were housed in 2.5cm by 7.5cm glass vials stoppered by gauze and cotton wool. Food for the adult wasp was provided by making a small streak of 50% honey solution in each vial. C. cephalonica was cultured in darkness at 30°C, 70% humidity, on a medium composed of wheat bran, com meal, yeast and glycerol. Clutch size 59

4.3.2 Clutch size and host size

128 female G. nephantidis were each allowed one experience of oviposition on a host larva, and were then separately presented with a host of known head capsule width, length and weight. The number of offspring at each developmental stage (egg, larvae, pupae, adult) were counted. The mother was removed when the brood pupated to prevent confusion with her progeny. Pre-adult mortality was separated into four components; egg mortality, larval mortality, mortality during pupation and pupal mortality. Upon eclosion the sexes of the offspring were recorded, and their thoracic length measured using a bin­ ocular microscope with a calibrated eye piece graticule.

4.3.3 Clutch size manipulations

Female G.nephantidis were individually presented with hosts weighing 30-40mg. When a clutch of eggs had been laid, the host was removed from the mother and placed between two plasticine mounds (to steady the host) in a small plastic Petri dish. All eggs were then removed from the host using fine seekers. Abnormal clutch sizes were then produced by replacing the eggs and adding eggs from other clutches. No adult G. nephantidis were present with the clutch. 127 manipulated clutches were created and the numbers of eggs, larvae, pupae and adults were counted as the brood developed. Adult progeny were sexed and measured as above. Since host weight ranged between 30 and 40mg (mean 34.9mg) the influence of host weight was examined when studying the effects of the manipulated clutch size on progeny size and survival.

4.3.4 The effects of the mother remaining with her clutch

To test whether the removal of the mother influenced pre-adult mortality in the manipulation experiment, mothers were removed from 40 unmanipulated clutches. The pre-adult mortality and adult size of progeny from these clutches was compared to those of the 128 clutches in which the mother was allowed to remain with her brood (Section 4.3.2). Clutch size 60

4.3.5 Reproductive female’s longevity and fecundity

On eclosion, 76 females of a range of sizes (0.05cm-0.13cm) from the broods created in the manipulation experiment (Section 4.3.3) were each presented with a host weighing 30-40mg. The numbers of eggs, larvae and pupae were counted as the broods developed and the resulting adult progeny were measured and sexed. Upon brood pupation, mothers were removed and presented with another host. This process was repeated until the female died, and the length of her adult life recorded. Hence, a record of the size and longevity of each female was obtained along with the number, pre-adult mortality, size and sex of her progeny.

4.3.6 Female longevity without reproduction

A wide range of female sizes w as created by clutch size manipulation or by carefully removing the late instar larvae from the host (thus reducing the amount of food available to the developing parasitoid). On eclosion, 205 females were placed in individual vials and fed once with a streak of 50% honey solution. 87 females were treated similarly but were not fed. Females were not provided with hosts. When each wasp died it was measured and the length of it’s adult life recorded.

4.3.7 Statistical analysis

Data were analysed using generalised linear modelling techniques (McCullagh & Nelder 1983, Aitkin e t a l. 1989, Chapter 1) allowing the analysis of covariance and the use of data with binomial or Poisson error variances without transformation. In analyses with non-normal error variances, there is no exact theory for the distribution of the deviances and a chi-squared approximation is used with conservative significance levels. When poisson error variances are assumed, the logarithm of the response variable is assumed to be a linear function of the explanatory variable(s) In the case of binomial errors, the ’log odds’ of the response variable (i.e. the ’logit’) is assumed to be the linear function (all logarithms are logj. In the analysis of data using poisson or binomial error distributions, a heterogeneity factor (H.F.) is used to adjust the standard errors in cases where the residual deviance is substantially larger than the residual degrees of freedom, indicating overdispersion (McCullagh & Nelder 1983). Box-Cox plots (Box & Cox 1964) are used to select suitable transformations for analysis of data with normal error dis­ tributions. Clutch size 61

In some cases the percentage deviance explained is given: this is the percentage reduction in the total deviance achieved by fitting the model, and is an assessment of the explanatory power of the model. When the error variances are normally distributed, variance and deviance are equivalent, i2 is the percentage variance explained. In reporting the statistics, the degrees of freedom are sometimes considerably less than the number of experimental replicates. This is due to cases where missing values of a particular parameter effectively reduce the number of replicates during consideration o f that parameter and it’s interactions.

4.4 Results 4.4.1 Clutch size and host size

G. nephantidis adjusts it’s clutch size in response to the size of the host, this rela­ tionship is shown in Fig. 4.1. The fitted regression is highly significant (F(,>75) = 81.25, p < 0.001) and explains 52% of the total variance in clutch size. Some of the clutches consisted exclusively of males indicating that the mothers were unmated. There was, however, no statistical difference in the relationship between clutch size and host weight for virgin and mated females (F(1 >75) = 0.12, p > 0.05). Whether host length or host head width were better predictors of clutch size was explored. Both measures were highly correlated with host weight (Length; F(1127) = 21.06, p < 0.01 :Width; F (U27) = 38.1, p < 0.01) and with clutch size (Length; F (1 ;75) = 79.05, p <0.01: Width F(175) = 65.64, p < 0.01), explaining 51.3% and 46.7% of the variance in clutch size respectively. The mean naturally laid clutch size on the host weight range selected for the clutch size manipulation (Section 4.4.3) was 9.26 (S.E. = 8.51/10.10) (Fig. 4.1).

The smaller number of eggs laid on smaller hosts reduces the competition for resources within the clutch. However, even after this compensatory behaviour by the mother, offspring survival and eventual adult size may still be related to host size. Survival was thus regressed on both host weight and also on host weight divided by the number of eggs that hatched, a crude measure of the magnitude of competition, survival was not affected by either of these (Host weight; X2 = 2.56, d.f. = 71 ,p > 0.05: Host weight divided by number of hatching eggs; X2 = 2.87, d.f. = 68, p > 0.05). There was no significant effect of clutch size on offspring survival (X2 = 3.55, d.f. = 71, p > 0.05). There was also no significant difference between survival in broods exclusively containing males and in mixed broods (containing mostly females) (X2 = 0.54, d.f. =71, p > 0.05). Clutch size 62

Fig. 4.1 The relationship between clutch size and host weight. The fitted line is the regression equation y = 0.125x + 4.95.

The overall probability of an egg surviving to adulthood, and through each of the developmental stages is shown in Table 4.1. There was no significant relationship between any of these sub-mortalities and clutch size, host weight or host weight divided by clutch size.

Table 4.1 Probabilities of survival in G. nephantidis

Probability S.E. H.F.

Overall survival 0.732 0.027 2.9

Egg survival 0.907 0.016 2.0

Larval survival 0.938 0.016 3.5

Survival during pupation 0.953 0.011 2.4

Pupal survival 0.924 0.012 1.8 Clutch size 63

A plot of the cube of female thoracic length against host weight is shown in Fig. 4.2 (Box-Cox plots suggest a fourth power transformation of female thoracic length, but a third power transformation was chosen since host weight is likely to influence parasitoid weight, which will be related to the cube of parasitoid length). The average size of females from a brood was significantly related to host weight (F(U6) = 26.47, p < 0.01, r2 = 50.4%) and less significantly to clutch size (F(1>26) = 7.42, p < 0.05, r2 = 2 2 .0 Adding the effect of clutch size does not increase the significance of the host weight regression (F(1<25) = 0.16, p < 0.05). Male size (after cubic transformation) was not significantly related to host weight (F(159) = 3.16, p > 0.05, r2 = 5.1%), as for female size, adding the effect of clutch size does not increase the significance of the regression.

Fig. 4.2 The relationship between the cube of female thoracic length and host weight. The mothers had been allowed to oviposit naturally on hosts. The fitted line is the regression equation y = 1.667x + 92.94.

It was not possible to assess the sex ratio at oviposition (Chapter 5) but the sexual composition of the adults that survived in each brood was recorded. Excluding broods composed solely of males since these were probably produced by virgin females; the Clutch size 64

maximum number of males recorded in a brood was two, and the overall sex ratio (pro­ portion males) was 0.113 (S.E. = 0.017, H.F. = 0.71). The low value of the heterogeneity factor indicates less than binomial variance in the sex ratio (Chapter 5). There is no evidence of any change in sex ratio with increasing brood size (logistic regression, X?w./.)~0*35,p > 0.05) this results in a significant increase in the number of males with brood size (log linear regression Xfw/)~11.61,p~0.001).

Thus female wasps lay larger clutches on larger hosts. Survival is not affected by clutch size or host size. Female wasps developing on large hosts are greater in size than those developing on small hosts. There was no effect of host size on male size. Brood sex ratios are very female biased, with less than binomial variance and are not affected by clutch size.

4.4.2 Clutch size manipulations

The probability of survival through the egg stage in the broods where clutch size had been manipulated was 0.428 (S.E. = 0.016, H.F. = 3.38). Egg survival was unaffected by clutch size (X2 = 0.09, d.f. = 119, p > 0.05) and host weight (X2 = 2.92, d.f. = 118, p > 0.05) (logistic regressions). The lower survival of eggs in manipulated broods compared to eggs in unmanipulated broods suggests that the process of manipulation itself was responsible for the increased mortality. The probability of survival through the larval period was 0.944 (S.E. = 0.010, H.F. = 3.08) which was not significantly different from the larval survival unmanipulated broods (z = 0.64 (normal approximation),/? ~ 0.75). As for eggs, larval survival was unaffected by clutch size (X2 = 0.57, d.f. = 119 , p > 0.05) and host weight (X2 = 0.85, d.f. = 118 ,p > 0.05) (logistic regressions). It therefore appears that the manipulations caused a marked reduction in egg survival but had no effect on larval survival. Hence, the clutch size is taken as the number of hatched eggs in the assessment of the consequences of clutch size on offspring fitness.

The overall probability of survival through pupation was 0.883 (S.E. = 0.014, H.F. = 2.363). However, survival significantly declined with increasing manipulated brood size although the strength of the relationship was weak (f,18 ~ 2.80, p < 0.01, deviance explained = 7.6%). Examination of the residuals from the regression suggested that this relationship was caused by a few large clutches with high mortality. There was no rela­ tionship between pupation mortality and host weight (X2 = 0.04, d.f. = 119, p >0.05). The Clutch size 65

average probability of survival through the pupal stage was 0.829 (S.E. = 0.019, H.F. = 3.063), there was no relationship between pupal survival and brood size (X2 =6.8, d.f. =110,p > 0.05) or host weight (X2 =6.15, d.f. = 110, p >0.05).

The overall probability of survival from hatching to the emergence of the adult was 0.712 (S.E. = 0.022, H.F. = 3.063) and this was not related to either the number of eggs hatched (X2 = 0.92, d.f. = 112, p >0.05, Fig. 4.3) or to host weight (X2 = 6.88, d.f. = 109, p > 0.05) (logistic regressions).

1 - i

■ ■ "

0.8

13 ■o 0.6 03

C Z3 "as 0.4 >

if) 0.2

0 -I 0 10 20 30 40 Clutch size (number of eggs hatched)

Fig. 4.3 The relationship between the number of eggs that hatched and their survival to adulthood. There was no relationship between survival and the number of hatched eggs.

Female size was strongly influenced by clutch size (Fig. 4.4) and this relationship is described by the regression

[Average female size] = -0.506[Clutch size] + 55.28 (4.1) Clutch size 66

F(Uo2) = 74.57,p < 0.001, r2 = 42.3% (Fig. 4.4) (Box-Cox plots showed very little advantage to transforming female size). In addition to the effect of clutch size, host size had a sig­ nificant effect on female size (F(U01) = 17.3, p < 0.001, r2 = 50.9%. However, adding sex ratio to the clutch size regression had no discernible influence (F(U01) = 0.026, p > 0.05, r2 = 1.5x10'^. Box-Cox plots showed no advantage to transforming male size.

Fig. 4.4 The relationship between the average size of a female G. nephantidis emerging from a brood and the number of hatched eggs. The fitted regression is Equation 4.1.

Male size was also influenced by clutch size (Fig. 4.5) (F(157) = 13.04, p < 0.01, r2 = 21.2%), but this effect was much weaker than for female size. Adding either the effect of host weight or of sex ratio did not increase the explanatory power of the regression. Clutch size 67

Fig. 4.5 The relationship between the average size of a male G. nephantidis emerging from a brood and the number of hatched eggs. The fitted line is the regression y = -0.326x + 45.62.

To conclude, the experimental manipulations markedly lowered egg survival though not larval survival. Survival was unaffected by clutch size. Adult female size was strongly affected by clutch size, adult male size was also influenced though less strongly.

4.4.3 The effects of mother remaining with her clutch

The effect of maternal presence on survival was not significant for the immature stages (X2(ldf x -1.53, p ~ 0.12). The presence or absence of the mother did not affect the size of her adult progeny (Females; F{lJ9) = 0.43, p > 0.05, r2 = 0.55| Males; F(1|27) = 2.32, p > 0.05, r2 = 1.8 !?• Clutch size 68

4.4.4 Influence of adult size on fitness

Two components of adult female fitness were examined; longevity (both with and without reproduction) and fecundity. The fitness of males is not assessed in this study. Since mating takes place within the brood, a single male does not have to compete for mates. For a number of other parasitoids, it has been argued that male fitness is much less dependent on body size than female fitness (van den Assem et al. 1989), and clutch size theory has been confined to female offspring factors only (Chamov & Skinner 1984, 1985). 4.4.4.1 Female fecundity

The total number of progeny produced over the female’s lifetime was strongly influenced by her size (Fig. 4.6). Examination of the residuals from this standard regression showed that females which produced no offspring tended to be outliers or to have a large influence on the regression equation. Thus the analysis was modified to examine first the effect of clutch size on the probability that a female has progeny (binary regression) and second, the influence of size on the number of progeny produced by those females that had at least some offspring (standard regression).

The probability of producing at least some offspring increased with size (X$ld f )~9.l6,p < 0.01, deviance explained = 17.1%) and was described by the equation

Probability of ______1______(4.2) _ reproducing. 1 + exp f-T 0.19size- 7.23611 where ’size’ is the size of the female. Given that a female reproduces, the number of 2. offspring she produces is influenced by her size (F(158) = 23.77, p < 0.01, r =29.1% ) and was described by the equation.

Number of some = 2.976[size]-99.62 (4.3) _ progeny reproduction where ’size’ again refers to female size.

There was no significant relationship between the number of female progeny a mother produced and her size (excluding virgin females) (F(132) = 1.39, p > 0.05, ^ =4.17%) Clutch size 69

Fig. 4.6 The total number of progeny produced by different sized female G. nephantidis. The fitted regression is the relatioship between female size and her fecundity (given that she reproduces) (Equation 4.3). Clutch size 70

4.4.4.2 Reproductive female’s longevity

The effect of body size on the longevity of reproducing females was investigated, Fig. 4.7 shows the data. Fig. 4.8 shows the cohort survival curve for the 76 females studied. A Weibull distribution (Cox & Oats 1984) was fitted to the data which, though providing a reasonable fit, underestimated the mortality of young adults. To investigate whether this discrepancy was due to female size a second Weibull distribution was fitted in which the logarithm of the distribution’s rate parameter, X, was a linear function of female size (a standard technique of survival analysis, e.g. Aitkin e t al. 1989, p.281). Incorporation of female size significantly improved the fit of the distribution (Xjw./.)~20.04, p < 0.001). The mean of the Weibull distribution is proportional to \fk , and the dependence of average longevity on female size can be written

Average 1 (4.4) .longevity. exp{-0.115[size] -1.76} where ’size’ again refers to female size.

80 -i

70 -

60 -

CO aJ so TD

. t l 40 - > CD O)c o 30 - © CtJ 20 E 0 10

30 40 50 60 Female size (50 units = 1 mm)

Fig. 4.7 The longevity of reproductive females against their size. Clutch size 71

Fig. 4.8 Cohort survival for reproductive females. The fitted curve is the Weibull distribution without the effects of female size (Proportionsurviving =exp[-0.00136(0' 805], where t is the time in days) showing the extreme age depen- dancy of female survival. Clutch size 72

4.4.4.3 Female longevity without reproduction

The effect of body size on the longevity of females which did not reproduce was investigated, Fig. 4.9 shows this relationship.

Female size (50 units = 1 mm)

Fig. 4.9 The relationship between size and longevity of non-reproducing females. The data for fed and unfed females are lumped since feeding has no effect on longevity (see text). The fitted line is the regression y = -0.279x+40.94. Larger females live, on average, for less time than smaller females, this relationship is significant at the 5% level (FiX >234) = 5.7, 0.05< p > 0.01, i* = 2.3$.

As for reproducing females, a Weibull distribution was fitted to the cohort survival curve (Fig. 4.10), though providing a reasonable fit a descrepancy remained. To investigate whether this discrepancy was due to female size more Weibull distributions were fitted in which the logarithm of the distributions’ rate parameters, X, were linear functions of female size, or of whether the females were fed or unfed. Incorporation of female size significantly improved the fit of the distribution (Xfw/)~13.9,p > 0.01). Adding the effects of feeding did not significantly improve the fit of the distribution (Xfw>/-)~0.3,p > 0.05), nor did the effect of feeding alone f > 0.05). Clutch size 73

The mean of the Weibull distribution is proportional to 1/A,, and the dependence of average longevity on female size can be written

Average ______1______(4.5) .longevity. exp{0.037[size] -9.162} where ’size’ refers to female size.

Fig. 4.10 Cohort survival for non*reproductive females. The fitted curve is the Weibull distribution without the effects of female size (Proportion surviving = exp[-0.000907(r)2052], where t is the time in days) showing the extreme age dependancy of female survival.

4.5 Discussion

Clutch size theory predicts that clutch size should vary with the total amount of resource available to the developing offspring (e.g Skinner 1985). Consistent with this prediction, the unmanipulated experiment revealed that G. nephantidis females adjust the size of their clutches to changes in host size. Most theoretical studies (e.g. Iwasa e t al. Clutch size 74

1984, Waage & Godfray 1985) have assumed that, for a fixed amount o f resource, the p e r c a p ita fitness of offspring declines as the clutch size increases. One component of offspring fitness is survival until adulthood, however, in G. nephantidis there is little effect of clutch size on offspring survival. Another component of offspring fitness is reproductive success when adult; longevity and fecundity. In G. nephantidis larger females emerge from smaller clutches. Fecundity is positively correlated with adult female size. The longevity of females that reproduce (but not of those that do not) is also positively correlated with size. Thus, for females which reproduce, the p e r c a p ita fitness consequences of increased clutch size are qualitatively consistent with theoretical predictions.

Empirical studies of other parasitoid species have obtained similar results. Clutch size has been found to vary in cases where the host size varies interspecifically (e.g. Klomp & Teerink 1962, le Masurier 1987b, Takagi 1986), larger clutches being laid on larger host species. Within one host species parasitoids are commonly observed to lay larger clutches on larger hosts (e.g. Dijkstra 1986, Klomp & Teerink 1967,1978, Luck e ta l. 1982, Luck & Podoler 1985, Neser 1973, Takagi 1986).

In G. nephantidis clutch size has little effect on offspring survival. In other species, however, offspring survival declines with increasing clutch size (Benson 1973, Klomp & Teerink 1978, Takagi 1986, Taylor 1988, Waage & Godfray 1985). Consistent with the results from G. nephantidis, negative correlations between clutch size and offspring body size have been found in some other parasitoid species (e.g. Benson 1973, Bouletreau 1971, 1974, Klomp & Teerink 1978, Pyomila 1977). In contrast, Chacko (1964) found that the adult size of B ra c o n g e le c h ia eoffspring was not affected by clutch size, however longevity and fecundity were both negatively correlated with clutch size. The finding that in G. nephantidis clutch size had both little effect on offspring survival and a marked effect on adult wasp size, is consistent with some other parasitoid species (Klomp & Teerink 1967, Chamov & Skinner 1984, Taylor 1988). The positive correlations of adult size with longevity and fecundity found in G. nephantidis are also found in other species (e.g. Benson 1973, Takagi 1985, Waage & Ng 1984).

Although the association between clutch size and host size has frequently been observed, quantitative estimates of the precision of the mother’s clutch size response have rarely been made. The fitness consequences of non-precise clutch size allocation have been explored theoretically (Godfray & Ives 1988). In G. nephantidis about half the Clutch size 75

variance in clutch size is explained by host weight although, even after the mother had adjusted her clutch size to the size of the host, G. nephantidis offspring fitness was influenced by host weight.

Few quantitative predictions of clutch size have been attempted. Using offspring fecundity as a measure of fitness, Chamov & Skinner (1984, 1985) calculated the Lack clutch size for Nasonia vitripennis and Trichogramma embryophagum and found that the calculated Lack clutch size was greater than the clutch size laid by the parasitoids. Similar results have also been found in other parasitoid species using the probability of offspring survival to adulthood as a measure of fitness (Waage 1986, Waage & Godfray 1985). Explanations for the observed clutch size being smaller than the Lack clutch size have postulated the presence of trade-offs between present and future reproductive success: if maximising fitness per host is not equivalent to maximising lifetime fitness, then the optimal clutch size is less than the Lack clutch size (Chamov & Skinner 1984,1985, Iwasa e t a l. 1984, Godfray 1987a, Parker & Courtney 1984, Skinner 1985, Waage 1986, Waage & Godfray 1985). Alternatively, the discrepancy between observed and predicted clutch sizes may be due to inadequate estimation of individual fitness (Chamov & Skinner 1984, Godfray 1987a, Waage 1986).

With no evaluation of the extent of trade-offs between present and future repro­ duction, calculations of the Lack clutch size provide a theoretical clutch size which should not be exceeded, but cannot predict the optimal clutch size if such trade-offs are present. For some species a p r io r ipredictions can be made that trade-offs between present and future reproduction are unimportant, in these species the Lack clutch size is predicted to be optimal. One such species is the eulophid wasp Colpoclypeus florus, an ectoparasite of leafrolling tortricid moth larvae. The number of hosts parasitised by C. f lo r u s females is low (2-3) and a large amount of time is invested in the parasitization of each host. Thus, in an attempt to predict the optimum clutch size in C . flo r u s , Dijkstra (1986) assumed that maximising fitness per clutch is equivalent to maximising lifetime fitness. Dijkstra found that the chief effect of clutch size manipulation was on the size of the adult offspring: on hosts of the same size, larger clutches gave rise to smaller females. Adult size affected female longevity (in the absence of hosts) and fecundity, at least in the laboratory. Dijkstra reasoned that if the wasp was strongly host limited, the correct measure of a mother’s fitness would be the total potential longevity of all her daughters, since this would maximise the number of hosts her daughters could parasitize. However, if the wasp was not limited by hosts but by egg supply, the mother should lay a clutch size that resulted in the maximum total fecundity of her daughters. Dijkstra calculated the Lack clutch size for each of these Clutch size 76

assumptions. He found that if the mother was truly host limited, clutch sizes greater than those observed should be laid whereas if she were tmly egg limited, smaller clutch sizes than those observed should be produced.

The biology of G. nephantidis suggests that trade-offs between present and future reproduction are unimportant. Thus, there is an a p r io r iprediction that G. nephantidis should produce the Lack clutch size. If G. nephantidis is host limited then the correct measure of a mother’s fitness would be the total longevity of her female offspring. Two separate estimates of female longevity were made, one for longevity without reproduction and the other for longevity with reproduction (Section 4.3). The longevity of a reproductive G. nephantidis female from a particular sized clutch can be calculated by combining equations 4.1 and 4.4. The total reproductive longevity of the clutch is therefore a female’s reproductive longevity multiplied by the number of females in the clutch. The sex ratio has less than binomial variance (Section 4.4.1, Chapter 5) and the modal number of males per clutch is one. Thus, to a simple approximation, the number of females in the clutch is the clutch size minus one. The relationship between total reproductive female longevity and clutch size is shown in Fig. 4.11, the Lack clutch size according to this calculation is 18.19 eggs. Clutch size 77

Clutch size

Fig. 4.11 The theoretical relationship between the clutch size produced by a female and her fitness. Fitness is defined as the total longevity of her (reproductive) daughters. The Lack clutch size (where total longevity of daughters is at a maximum) is 18.19 eggs.

Similarly, the longevity of a non-reproductive female from a particular sized clutch can be calculated by combining equations 4.1 and 4.5. The total non-reproductive longevity of females in the clutch is the individual longevity multiplied by the clutch size minus one. The relationship between total non-reproductive longevity and clutch size is shown in Fig. 4.12. The relationship shows the fitness of a females increasing exponentially with increase in clutch size. This result is obtained because the longevity of these females does not increase with their size (Section 4.4.4.3, Fig. 4.9). The p e r c a p ita "fitness" of offspring in the clutch thus does not decline as clutch size increases. The rationale behind measurement of female longevity without reproduction was to totally separate longevity from fecundity. This was to approximate to a more natural situation in which females produce at maximum a few clutches with, possibly, long periods between eclosion and host location and between successive hosts. Tbe absence of a decline in p e r ca p ita longevity Clutch size 78

as clutch size increases implies that the longevity of females which do not reproduce is not a component of fitness, this prevents the use of this measurement for calculation of the Lack clutch size.

Clutch size

Fig. 4.12 The theoretical relationship between the clutch size produced by a female and her fitness. Fitness is defined as the total longevity of her (non-reproductive) daughters. There is a negative correlation between a daughter’s size and her (non-reproductive) longevity (Fig 4.9). If non-reproductive female longevity is assumed to be related to fitness the clutch size which theoretically maximizes offspring longevity is thus infinite (see text).

If G. nephantidis is not host limited, but reproductive success is limited by egg supplies, the appropriate measure of a mothers fitness is the total fecundity of her female offspring. The fecundity of a female developing in a particular sized clutch can be calculated by combining equations 4.1, 4.2 and 4.3. The total fecundity of the members of the clutch is the individual fecundity multiplied by the clutch size minus one. The relationship between total female offspring fecundity and clutch size is shown in Fig. 4.13, the Lack clutch size according to this calculation is 18.31 eggs. Clutch size 79

Fig. 4.13 The theoretical relationship between the clutch size produced by a female and her fitness. Fitness is defined as the total fecundity of her daughters (or number of grandchildren produced). The Lack clutch size (where total fecundity of daughters is at a maximum) is 18.31 eggs.

The Lack clutch size calculated using each of the measurements of reproductive female fitness are very similar. Both these are notably larger than the mean naturally laid clutch size at the host weight used in the manipulation experiment (9.26 eggs; Section 4.4). The disparities between observed and predicted clutch sizes in G. nephantidis could be attributed to clutch size theory being incorrect, to incorrect assumptions about the importance of trade-offs between present and future reproduction or to the inadequate measurement of offspring fitness.

If trade-offs between present and future reproduction are important then the optimal clutch size would be less than the Lack solution. The importance of trade-offs depends on the influence of clutch size on future reproduction. Clutch size could influence future reproduction by affecting the mother’s mortality. The mother’s action in guarding the developing brood may expose her to extra risk of mortality, for example while defending it from intruders (Hardy & Blackburn in p r e s s, Chapter 3), this may reduce the probability Clutch size 80

of the mother surviving to lay subsequent clutches. If this extra mortality were a function of her clutch size a mother may be selected to produce smaller clutch sizes. However, it is unlikely that this risk should be clutch size dependant.

A mother may be selected to produce clutch sizes below the Lack solution if either the time or the eggs required to produce the clutch are limiting. It is considered that each of these will not affect future reproduction in G. nephantidis since the time invested by G. nephantidis guarding each clutch is independent of clutch size since broods of different sizes develop at the same rate. Furthermore, the time taken to lay a clutch is a small fraction of the total time a mother spends with her brood. The egg supply of G. nephantidis is probably not limiting since in the laboratory some females can produce over a hundred eggs during their life. The average number of clutches naturally produced by G. nephantidis is unknown. Although some mothers produced up to eleven clutches, once they had produced four or five, they became sperm depleted and produced exclusively male off­ spring in subsequent clutches. This may imply a maximum of around five clutches under natural conditions. Furthermore, the natural host, O. arenosella, normally occurs at low population densities, though during population outbreaks the local host density can be high (Cock & Perera 1987, Perera 1987, Perera e t a l. 1988). Since hosts are usually widely dispersed it is expected that search time is relatively large in G. nephantidis. Both the large amount of time spent with and between clutches support the assumption of host availability limiting reproductive success.

In summary, the assumption of the unimportance of trade-offs between present and future reproduction is not indisputable, since the number of clutches produced naturally \< \ by G. nephantidis is unknown. A variety of evidence suggests, however, thatG. nephantidis these trade-offs are probably unimportant or absent.

An alternative explanation for the disparities between observed and predicted clutch sizes is that the measurement of offspring fitness was inadequate. This is likely if offspring fitness depends on factors other than longevity or fecundity (e.g. host searching ability or brood guarding ability) or if the measurement of juvenile survival, fecundity or longevity in the laboratory is not representative of the natural situation. The experiments were able to identify clearly the effects of clutch size on juvenile survival and adult progeny size (note that clutch-size independent larval mortality, which may vary between the field and laboratory, does not affect the optimal clutch size prediction). The importance of female size to fitness may have been underestimated if size affects the female’s ability to guard her broods from intruders (Chapter 3), the effects of size on this ability are not known Clutch size 81

(Chapter 6). A female’s ability to find hosts also may depend her size, there is no measurement of this in G. nephantidis. This problem is common to other studies which have estimated parasitoid fitness (van den Assem e t al. 1984, Chamov & Skinner 1984, Dijkstra 1986). If larger females are better at finding hosts than small females, perhaps because they live longer and thus have more time in which to search (but see Section 4.4.43), then the Lack solution will be less than the calculated value. It could also be argued that smaller females may be better at searching for hosts. This may be true if hosts are widely dispersed, since small females may be better at dispersal, perhaps due to lower wing loads (amount of adult dry weight per unit wing length) (Takagi 1985). If smaller females are more successful in host location than larger females then the Lack clutch size would be larger than the calculated value. If the effects of female size on host searching ability were known then this consideration could be added to the predictive equations. When calculating fitness consequences it is best to have as complete a measure of fitness as possible. Shapes of fitness relationships may differ greatly with different partial measures used and thus combining partial measures gives a better measure of fitness (Skinner 1985). However, a d hoc factors should not be "added to the model until it fits" since this will negate the value of any test. Factors added must be are natural and general considerations, which may explain a discrepancy (Chamov & Skinner 1984).

In conclusion, some of the relationships between clutch size and offspring fitness have been determined in a parasitoid for which there is an a p r io r iexpectation that the optimal clutch size is the Lack clutch size. The measured fitness relationships have been combined to provide simple calculations of the Lack clutch size. Disparities between the observed and the predicted clutch size are found, possibly due to inadequate ability to assess offspring fitness using a laboratory-based approach. The inability to find agreement between observed and predicted clutch sizes in this simple system raises questions about the possibilities of testing clutch size theory. T o avoid the problems of potentially important but unmeasurable influences of the natural environment on adult life, it would be ideal to investigate clutch size in a species with a similar biology to G. nephantidis, but in which the penalties of exceeding the optimal clutch size were restricted to the survival of offspring rather than affecting adult fitness. Precise sex ratios 8 2

Chapter 5

Precise Sex Ratios in Goniozus nephantidis

5.1 Abstract

Amongst inbreeding parasitic wasps precise sex allocation is theoretically advantageous over the alternative of binomial sex allocation, since it maximises the number of mated females produced by reducing variance in brood sex ratios.

Evidence from inbreeding parasitoids mostly supports the theory of precise sex ratios.

The life history of Goniozus nephantidis leads to the prediction of precise sex allocation. The sex ratio strategy of G. nephantidis is investigated by comparison of observed sex ratios with those expected under binomial sex allocation. Attempts are made to test assumptions of the equivalency of primary and secondary sex ratios and also of male mating ability.

G. nephantidis produces precise sex ratios (with less than binomial variance) at most brood sizes, although precision is limited, probably by factors such as pre-adult mortality. Precise sex ratios 83

5.2 Introduction

Fisher (1930) first explained why the equilibrium sex ratio of most animal species is equal numbers of males and females (Chapter 1). Deviations from sex ratios of equality are, however, common amongst parasitoids (e.g. King 1987, Waage 1986). Fisher’s theory assumes panmixis (random mating), and the life histories of many parasitoids violate this assumption. Biased sex ratios were first attributed to non-random mating by Hamilton (1967) who predicted the optimal sex ratio a mother should produce to reduce competition formating opportunities amongst her offspring (Chapter 1). Hamilton’s theory has become known as the theory of local mate competition, or LMC. LMC theory (Hamilton 1967, 1979, Taylor & Bulmer 1980, Suzuki & Iwasa 1980, Chapter 1) only predicts the overall sex ratio produced by mothers; the variance in brood sex ratios is not considered (Figs. 1.2, 1.4). Since siblings mate with each other, the sex ratios within broods affect the number of mated daughters a mother produces, a measure of her fitness (Green e t a l. 1982, Hamilton 1967, Hard 1971). Mothers are thus expected to maximise the average number of mated daughters produced in each brood.

The Hymenoptera have haplodiploid sex determination: unfertilized (haploid) eggs develop into males and fertilized (diploid) eggs develop into females. Sperm stored in mated females’ spermathecae is released to fertilize eggs (e.g. Flanders 1956, Gerber & Klostermeyer 1970, Wilkes 1965). The allocation of progeny sex ratios is thus potentially under maternal control. How then should mothers maximise the numbers of mated females produced in each brood? If sex is allocated to eggs randomly (binomially) then the probability of each egg being male is equal to the overall sex ratio (proportion males) produced by the mother. Under these conditions the frequency distribution of brood sex ratios will have binomial variance. Hence, some broods will not contain the optimal (overall) offspring sex ratio, and these will give rise to fewer mated females than an equal sized brood containing the optimal number of males (Fig. 1.4). The lower number of mated females produced will either be due to the brood containing insufficient males to mate with all the females or to a greater than optimal number of males, with a consequent reduction in the number of females in the brood. In extreme cases, when broods contain exclusively males or females, no mated females will be produced. Members of such broods are assumed to have extremely low fitness (Chapter 2). To maximise the number of mated females produced under binomial sex allocation, the overall sex ratio must be greater than predicted by LMC theory (Cornell 1988, Hard 1971). Precise sex ratios 84

An alternative to increasing the sex ratio to increase the numbers of mated daughters, is to decrease the variance in brood sex ratios. This can be achieved by replacing the random (binomial) allocation of sex to each egg by non-random sex allocation, fewer broods thus contain non-optimal numbers of males. This is termed precise sex allocation (Green 1980, Green e t al. 1982), precise control of the sex ratio of offspring in each brood is theoretically advantageous over the binomial alternative.

This chapter examines the brood sex ratios produced by Goniozus nephantidis, a parasitic wasp which lays eggs in discrete clutches. G. nephantidis siblings mate amongst themselves prior to dispersal from the natal patch. In agreement with the predictions of LMC theory, the overall sex ratio of G. nephantidis is female biased (proportion males = 0.113, S.E. = 0.017, H.F. = 0.71, Chapter 4). The biology of G. nephantidis suggests that mothers should allocate sex to eggs precisely. The low value of the heterogeneity factor indicates less than binomial variance in the sex ratios of broods. Other than this, the sex ratios within G. nephantidis broods have not been examined. The biology of G. nephantidis and evidence for precise control of sex ratios in other Hymenoptera are first reviewed. 5.2.1 Evidence for precise sex ratios

Evidence for variance in brood sex ratios in parasitic wasps is collected in Table 5.1. Precise sex ratio theory predicts that the variance in sex ratio of broods should be less than binomial in inbreeding species.

Table 5.1 Estimates of variance in parasitic wasp brood sex ratios.

Species Method and estimate Reference

Goniozus (gallicola) Variance in brood (secondary) sex ratios Green 1980, g o rd h iEvans compared to binomial models. Green e t al. (Hymenoptera: Variance is significantly less than binomial 1982 Bethylidae) at most brood sizes.

Goniozus emigratus Variance in brood (secondary) sex ratios Green e t al. Rohwer compared to precise and binomial models. 1982 (Hymenoptera: Sex ratio not precise, but shows less than Bethylidae) binomial variance. Precise sex ratios 85

L a e liu s p e d a tu s Say Methods not given. Mertins (Hymenoptera: One male per brood (3-4 eggs) in ten out of pers. comm. Bethylidae) eleven cases. to Green e t a l. 1982

Laelius utilis Cockrell Methods not givenf. Mertins (Hymenoptera: Almost always one male in a brood. 1985 Bethylidae)

Eulophus larvarum L. Variance in brood secondary sex ratios Godfray & (Hymenoptera: examined. In the spring generation (with Shaw 1987 ) LMC) sex ratio has binomial variance. In the summer generation (with less LMC and many single sex broods) sex ratio has greater than binomial variance.

Achrysochariodes cillia Examined sex ratios of broods with no Askew & Walker mortality1. Males and females in separate Ruse 1974, (Hymenoptera: broods, both sometimes gregarious. Sex see also Eulophidae) ratio has greater than binomial variance. Bryan 1983

Achrysochariodes latreil- Examined sex ratios of broods with no lei Curtis mortality*. Males and females in separate (Hymenoptera: broods. Females sometimes gregarious, Eulophidae) males solitary. Sex ratio has greater than binomial variance.

Achrysochariodes n iv eip e s Thompson (Hymenoptera: Eulophidae)

Achrysochariodes brutus Walker (Hymenoptera: Eulophidae) Precise sex ratios 86

Gryon atriscapus Gahan Frequency of males in egg masses com­ Waage (Hymenoptera: pared to binomial distribution. 1982a Scelionidae) Variance is significantly less than binomial.

Telenomus nitidulus Frequency of males in egg masses com­ Thomson pared to binomial distribution. (Hymenoptera: Variance is significantly greater than Scelionidae) binomial.

Trissolcus reticulatus Frequency of broods containing one male Remaudiere (Asolcus simoni spp. compared to Poisson distribution. & S k a f1963 reticulatus) Deluchi Differences are significant, males are not (Hymenoptera: distributed randomly. Scelionidae)

Asolcus mitsukurii Ash- Nine, twelve-egg host masses presented. Hokyo e t al. mead One male and eleven females emerged from 1966 (Hymenoptera: each. Scelionidae)

Asolcus waloffae sp.n Methods not given*. Javahery (Hymenoptera: At least one male per egg batch. 1967 Scelionidae)

Asolcus silwoodensis sp.n Methods not given1. (Hymenoptera: Normally at least one male per egg batch. Scelionidae)

Asolcus clavatchii sp.n Methods not given1. (Hymenoptera: Sex ratio precisely determined. Scelionidae) Precise sex ratios 87

Trichogramma kalkae Frequencies of brood secondary sex ratios Feijen & Schulten & Feijen compared to binomial distribution. Schulten (Hymenoptera: Variance is significantly less than binomial. 1981 T richogrammatidie)

Trichogramma pinneyi Schulten & Feijen (Hymenoptera: Trichogrammatidie)

Trichogrammatoidea sim- m o n d si Nagraja (Hymenoptera: T richogrammatidie)

Nasonia vitripennis Superparasite’s brood sex ratio have greater Orzack 1986 Walker variance than those of first parasite. (Hymenoptera: Precise control is either not possible or Pteromalidae) selection for superparasite sex ratio is weak (sex ratio genotype may be important).

Asobara persimilis Papp Variance in broods’ secondary sex ratios Owen 1983 (Hymenoptera: compared to binomial model. Variance is Braconidae) just significantly greater than binomial (out- breeding is probably normal in nature).

Pachysomoides stupidus Variance in brood secondary sex ratios Pickering Cresson compared to binomial model. Variance in 1980 (Hymenoptera: brood sex ratio is significantly greater than ) binomial*.

Caraphractus cinctus Methods not given*. Jackson Walker One thrust of the ovipositor usually lays 1958 (Hymenoptera: one male and two female eggs. Mymaridae)

fNot quantitative, sample sizes not given. ^Outbreeding species with overall sex ratio of 1:1, and fairly high developmental mortality. Precise sex ratios 88

The species studied are unevenly distributed among taxonomic groups, for example the Eulophidae and Scelionidae are over-represented. Some of the evidence for brood sex ratio variances is not quantified and constitutes anecdotal evidence only. Most species listed in Table 5.1 provide evidence that sex is not allocated binomially (with a given probability, resulting in a given overall sex ratio) to eggs. However, sex ratio variances are found both greater and less than binomial. Less than binomial sex ratio variances imply precise control of sex allocation. Greater than binomial variances in brood sex ratios are not evidence for precise sex ratios, but do imply a tendency to produce broods containing only one sex of offspring. An explanation for this is that the survival of individuals in broods containing only siblings of the same sex is better than that of individuals in mixed sex broods (Godfray & Shaw 1987). Thus, provided that the opportunities for outbreeding are not scarce, the oviposition of single sex broods will be favoured over binomial sex allocation. Green e t al. (1982) found that the sex ratio was not precise in the bethylid G. e m ig ra tu s, but that the number of males per brood had less than binomial variance. This is possibly attributable to incorrect assumptions about the optimal number of males per brood at larger brood sizes. Green e t a l. assumed that one male is sufficient to inseminate all the females in the brood but provided no measurement of the limits of the mating ability of males.

How might females produce precise sex ratios? Some parasitoids have been shown to lay batches of eggs with the sexes deposited in a particular sequence. Such non-random sequences of sex allocation provide evidence for the non-random allocation of sex to each egg, and give clues to the mechanisms involved (e.g. Putters & van den Assem 1985). Evidence for non-random sequences in sex allocation during ovipositional bouts is col­ lected in Table 5.2. Precise sex ratios 89

Table 5.2 Evidence for sequences of sex allocation in gregarious parasitic wasps and solitary parasitoids laying in discrete patches of hosts

Species Source of evidence Reference

Nesolynx albiclavus Sequence assessed by observation of ovi- Putters & Kerrich position and removal of female during ovi- van den (Hymenoptera: position bout. Assem 1985 Eulophidae) Male egg laid sixth in the sequence, then not until 10-20 eggs later (dependent upon time intervals).

Colpoclypeus florus Sequence assessed by observation followed Dijkstra Walker by cytology. 1986 (Hymenoptera: Usually male eggs are laid last in the clutch. Eulophidae)

Anisopteromalus calandre Sequence assessed by observation of ovi- Waage Howard position and removal of female during ovi- 1982b (Hymenoptera: position bout. Pteromalidae) Male eggs are laid late in a bout.

Trichogramma Sequence assessed by observation of order Waage & Ng e v a n e sc e n s Westwood of host acceptance and sex ratio of subse­ 1984, Waage (Hymenoptera: quent broods. & Lane 1984 T richogrammatidie) Male eggs produced early in the sequence and at intervals thereafter.

Sequence assessed by observation of differ­ van Dijken ences in abdominal movements. & Waage Male eggs laid early (usually second) in the 1987 bout.

Sequence assessed by removal of female Flanders during oviposition bout. 1935, 1956 First egg laid is invariably female. When clutch size is three, first egg is female, sec­ ond usually female, third is male. Precise sex ratios 90

Trichogramma chilonis Sequence assessed by observation of differ­ Suzuki e t al. Ishii ences in abdominal movements. 1984 (Hymenoptera: Male eggs usually laid second in the Trichogrammatidie) sequence, then about every eighth egg after­ wards.

Trichogramma kalkae Examination of single egg and single sex Feijen & Schulten & Feijen clutches. Shulten (Hymenoptera: Sequences begin with a female egg. 1981 T richogrammatidie)

Sex ratio frequency data compared to binomial and fixed-squence models. Closer fit to fixed-sequence models.

Trichogramma pinneyi Examination of single egg and single sex Schulten & Feijen clutches. (Hymenoptera: No definite sequence-starting sex. T richogrammatidie)

Sex ratio frequency data compared to binomial and fixed-squence models. Closer fit to fixed-sequence models.

Trichogrammatiodea Sex ratio frequency data compared to sim m o n d si Nagraja binomial and fixed-squence models. (Hymenoptera: Closer fit to fixed-sequence models. T richogrammatidie)

Gryon atriscapus Gahan Sequence assessed by observation of order Waage (Hymenoptera: of host acceptance, and sex of emerging 1982a Scelionidae) offspring. Males laid early in the bout and sometimes, in large egg masses, towards the end.

Telenomus remus Nixon Sequence assessed by observation of order van Welzen (Hymenoptera: of host acceptance, and sex of emerging & Waage Scelionidae) offspring. 1987 Males laid early in the bout. Precise sex ratios 91

Asolcus basalis Woll. Sequence assessed by observation of order Cumber (Hymenoptera: of host acceptance, and sex of emerging 1964 Scelionidae) offspring. Males laid generally early in the bout, but no fixed sequence of sex allocation.

Asolcus mitsukurii Varied size of host egg masses. Hokyo e t al. Ashmead When only one host, male egg always laid, 1966 (Hymenoptera: thus male eggs first in bout. Scelionidae)

Asolcus waloffae sp.n Sequence assessment method not given1. Javahery (Hymenoptera: Tendency to produce males early and late in 1967 Scelionidae) the oviposition bout, though occasionally produced in the middle.

Asolcus silwoodenis sp.n Sequence assessment method not given*. (Hymenoptera: Tendency to produce males early and late in Scelionidae) the oviposition bout.

Trissolcus reticulatus Sequence assessed by observation of order Safavi 1968 (Asolcus simoni spp. of host acceptance. reticulatus) Deluchi Male egg (usually) laid first in oviposition (Hymenoptera: bout. Scelionidae)

Phanurus beneficiens Sequence assessment method not given1. Okada & Zehnter Male eggs laid early and late in oviposition Maki 1921 (Hymenoptera: bout. Scelionidae)

L a e liu s p e d a tu s Say Sequence assessment method not given. Mertins (Hymenoptera: The last (temporally and spatially) egg laid 1980 Bethylidae) is malef. Typically one male per brood.

Laelius udlis Cockrell Sequences assessed by eggs being laid in Mertins (Hymenoptera: different positions'!-. 1985 Bethylidae) Almost always last egg in brood is male. Precise sex ratios 92

Cephalonomia tarsalis Sequence assessment method not given1. Powell 1938 Ash. Clutch size is two, female egg then male (Hymenoptera: egg laid. Bethylidae)

Heterospilus prosopidis Sequence assessed by observation of ovi- Waage Vier position and removal of female during ovi- 1982b (Hymenoptera: position bout. Braconidae) Males laid early in a bout.

Microbracon hebetor Say Sequence assessment method not given1. Flanders (Hymenoptera: Last eggs laid in clutch are usually male. 1956 Braconidae)

Aphytis melinus DeBach Sequence assessed by position of eggs Abdelrah- (Hymenoptera: which indicates sex**. man 1974 Aphelinidae) Male eggs laid after females.

*Abdelrahman’s (1974) correlation of sex Luck e t al. and position is not absolute, but is sup­ 1982 ported. Dorsally laid eggs are usually female and ventral eggs are usually male. When two eggs are laid, usually the first is laid dorsally and the second ventrally.

Copidosoma truncatellum Sequence assessed by examination of egg Leiby 1926 Dalman cell nuclei prior to cleavage. (Hymenoptera: When two eggs deposited in a host the sec­ Encytidae) ond is male in about 80% of instances

fNot quantitative, sample sizes not given. Precise sex ratios 93

There are insufficient samples for a detailed comparative study of patterns of sequence types within and between taxonomic groups. Studies of sex allocation sequences have not been distributed evenly across the parasitoid taxa (for example the Scelionidae are grossly over-represented). Sequences of sex allocation do seem to differ between taxonomic groups, the studied scelionid and trichogrammatid species lay male eggs early in the oviposition sequence (and also later in larger broods), whereas the Bethylidae, Ptero- malidae and Eulophidae lay male eggs later in the sequence. Both early and late male-laying sequences are found amongst the Braconidae.

There is (Tables 5.1 & 5.2) evidence from many species of parasitic wasps, for non-random, if not precise, control of sex allocation to eggs. Non-random sex allocation is taxonomically widespread (the studied species belong to various taxonomic groups within the Parasitica and ) and may create either greater than binomial variance in brood sex ratios or less than binomial variance (precise sex ratios). Precise sex ratios may be created by the allocation of sex to eggs in various non-random sequences during the oviposition bout.

5.2.2 The biology of Goniozus nephantidis

Goniozus nephantidis Muesebeck (Hymenoptera: Bethylidae) is a gregarious, parasitoid of the larvae of Opisina arenosella Walker (= Nephantis serinopa Meyrick) (Lepidoptera: Oecophoridae), a coconut defoliator occurring in the Indian sub-continent (Cock & Perera 1987, Dharmaraju & Pradhan 1977). The host is paralysed by the injection of venom through the wasp’s ovipositor (Ramachandra Rao & Cherian 1927, Remadevi e ta l. 1978) and a clutch of eggs is laid approximately 1-3 days later (Dharmaraju & Pradhan 1977, Jayaratnam 1941b). The clutch size depends upon the size of the host (Chapter 4). Unlike most parasitoids, though in common with a number of other bethylid species (Griffiths & Godfray 1988), the female remains with her developing brood until the off­ spring leave the host to pupate (Antony & Kurian 1960, Cock & Perera 1987, Remadevi e t a l. 1981) probably to guard the clutch against the actions of other parasitoids (Hardy & Blackburn in p r e s s, Chapter 3). Pupation takes place 4-5 days after oviposition (Perera 1987, Ramachandra Rao & Cherian 1927, Remadevi e t a l. 1981).

Approximately 74% of eggs survive development to adulthood (Chapter 4). At the end of pupal development the adult offspring may remain inactive within the cocoon for up to three days (Ramachandra Rao & Cherian 1927, Remadevi e ta l. 1981). Broods hatch approximately synchronously, although males generally emerge first and mate with sisters Precise sex ratios 94

before these have left their cocoons (Remadevi e t a l. 1981) or within approximately a day of their emergence (Jayaratnam 1941b, Ramachandra Rao & Cherian 1927). The popu­ lation sex ratio (proportion males) has been reported to be between 0.1 and 0.4 (Antony & Kurian 1960, Jayaratnam 1941b, Remadevi e t a l. 1981, Chapter 4). Males copulate many times but females are thought to copulate only once under natural conditions (Jayaratnam 1941b, c o n tra Remadevi e t al. 1981). In the laboratory virgin females survive long enough to mate with their sons (J.M. Cook pers. comm.), the female producing daughters in later broods. Other species of G o n io zu sare also normally found to copulate only once, but may successfully re-mate after sperm-depletion of the female (Gordh 1976). There is a period of relative inactivity of adults before dispersal in other bethylid species (Kearns 1934), mating may take place during this period. This may also occur in G. nephantidis. Nothing is known of dispersal from the natal site by G. nephantidis. Females must leave to forage for reproductive opportunities. Males possess apparently functional wings, and thus m a y disperse in search of mating opportunities with non-sibling females. There do not appear to be detrimental effects of inbreeding on the sex ratio (e.g. production of unviable diploid males, A.R. Whiting 1925, P.W. Whiting 1943) under continued inbreeding (J.M. Cook pers. comm.).

G. nephantidis is apparently naturally monophagous on O. arenosella (Cock & Perera 1987), although O. arenosella itself is not confined to coconut palm (Nirula 1956, Perera 1987). The density of hosts on coconut palm is usually low, though occasionally there are host population outbreaks when local densities can be high (Cock & Perera 1987, Perera 1987, Perera e t al. 1988). Oviposition opportunities for female G. nephantidis are thought to be usually very limited (Cock & Perera 1987) as a large amount of time is invested by the female in remaining with each brood and hosts are usually widely dispersed.

Goniozus nephantidis appears to conform closely to the biofacies of extreme inbreeding described by Hamilton (1967), Local Mate Competition (LMC) theory predicts that only sufficient males will be produced to fertilize the female offspring in the clutch. Precise sex ratio theory (Green e t al. 1982) predicts that the frequency distribution of brood sex ratios will have less than binomial variance.

5.2.3 Primary sex ratio

Green e t al. (1982) assumed that examination of the brood (secondary) sex ratio is equivalent to examination of the primary sex ratio. To test this assumption, both the Precise sex ratios 95

primary and secondary sex ratios must be known. This is possible if there is no devel­ opmental mortality amongst members of a clutch. In some species, the sex of the egg may be indicated by differences in the position (Abdelrahman 1974, Flanders 1950, Gerber & Klostermeyer 1970, Luck e t a l. 1982, Mertins 1985, Walter 1983) or morphology (Tooke 1955) of male and female eggs. In some species differences in the oviposition behaviour of the mother indicate the sex of an egg (Cole 1981, van Dijken & Waage 1987, Gerber & Klostermeyer 1970, Suzuki e t al. 1984). Hence, in some species both the primary and secondary brood sex ratio can be assessed directly. In other species, such as G. nephantidis, the primary sex ratio cannot be assessed directly. Separate estimates of primary and secondary sex ratios must be made. Since males are haploid and females are diploid the sex of a hymenopteran egg can be identified by the chromosome compliment of it’s cells; this can be established by cytological staining.

Cytological staining techniques have been used to determine the primary sex ratio of several species of parasitic wasps (Dijkstra 1986, M.J. van Dijken unpublished, van Heemert 1974). The chromosome content of an egg is assessed by staining the nucleus of the egg cells during the metaphase of mitotic division, when the chromosomes are maximally contracted and possible to count. Somatic cells in a developing egg at first undergo synchronous mitotic division. Later egg cells divide asynchronously, thus at any time there are some cells at each stage of mitosis. In some species, chromosomes in somatic cells replicate without cell division, producing somatic polyploidy (Aubert 1954, 1959). Once the somatic cells become polyploid it is difficult to infer whether they began as haploid or diploid, consequently the sex of the egg cannot be established. As the number of cells increases their size decreases and it becomes progressively more difficult to obtain single cell layer squashes (M.J. van Dijken unpublished). Thus, the best stage of cell division for egg cytology is when the cells are dividing asynchronously, before too many small cells are present and before the onset of somatic polyploidy.

5.2.4 Male insemination capacity

In addition to assuming equivalency of primary and secondary sex ratios, the analysis of Green e t al. (1982) assumed that a single male is sufficient to inseminate all female siblings, and hence that "one male per brood" is optimal. The number of females that a male can inseminate is investigated in G. nephantidis to estimate the optimal number of males for each brood size. Precise sex ratios 96

5.3 Materials and Methods

5.3.1 Animals

The G. nephantidis stock originates from the Coconut Research Institute, Lunuwila, Sri Lanka. This species has been cultured for over ten years on an alternative host, C o rc y ra cephalonica Stainton (Lepidoptera: Pyralidae). G. nephantidis stock had been intermit­ tently augmented by wild material. During culturing and experiments G. nephantidis was reared at 30°C, 70% r.h., which is optimal (Dharmaraju & Pradhan 1977), and 16L:8D photoperiod. C. cephalonica, was reared in darkness at 30°C, 70% r.h., on a medium composed of wheat bran, com meal, yeast and glycerol.

5.3.2 Assessment of primary sex ratio

Cytological staining techniques were used in an attempt to establish the primary sex ratio of broods laid by G. nephantidis. The fixative and stain, 2% lacto acetic orcine, LAO (a chromosome specific dye), was made by dissolving l.Og natural orcine in a mixture of 10ml lactic acid (85% pure), 25ml glacial acetic acid and 15ml distilled water. The resulting solution was gently boiled in a reflux cooler for one hour, slowly cooled and then filtered (MJ. van Dijken unpublished, Dijkstra 1986, van Heemert 1974)

Cells were stained by placing the egg on a microscope slide and adding a drop of LAO. 25 minutes later the egg was squashed under a coverslip which was then sealed with nail varnish. After 24 hours at 20°C the tissue was inspected under a compound microscope (magnification xlOOO).

5.3.3 Male insemination capacity

In G. nephantidis siblings emerge approximately, but not exactly synchronously, it is thus unknown how much time males have in which to copulate with sisters in nature (section 5.2.2). Fifteen males were individually allowed access to females as these emerged "naturally", four having access to females for 24 hours after m a le emergence, and eleven males having access to females for 12 hours after the last female had emerged. Four males were individually presented with groups of females simultaneously, and were allowed to remain with these for 24 hours. After removal from contact with a male, each female was presented with a host and allowed to oviposit. The resulting broods were reared to maturity, and the number of females which produced broods containing daughters recorded. Precise sex ratios 97

5.3.4 Analysis of G. nephantidis sex ratio strategy

Data from other studies of G. nephantidis (Chapter 4, J.M. Cook unpublished data) were combined to provide sex ratios for 875 broods. For many of these the clutch size, survivorship and sex ratio were known. Each brood’s position in the mother’s ovipositional history was known, for example whether the brood was the first, second or third etc. brood the mother had laid.

Under investigation were the sex ratio decisions made by ovipositing G. nephantidis; thus, broods produced by females identifiably constrained in their sex allocation decisions (virgin females and sperm-depleted females, Chapter 2) were excluded from the investi­ gation. Virgin females were identified as females which, while information was collected from them, produced no adult female progeny. Sperm-depleted females were identified as those which, after having produced daughters, later produced male offspring exclusively (Gordh 1976, Gordh & Hawkins 1981). Sperm-depletion may initially occur during the oviposition of a clutch, thus the brood sex ratios of females which suffered sperm-depletion was examined. If the brood preceding the first exclusively male brood contained an unusually high proportion of males, it was considered that the sex ratio of this brood had also been affected by sperm-depletion. Eight such broods were excluded from further analysis.

The remained 595 broods produced by females without identifiable constraints; of these, 36 broods exclusively contained male offspring. The sex ratio of these broods may be due to hidden constraints (e.g. temporary malfunction of the spermatheca, Flanders 1956) or may be the product of deliberate sex allocation strategy by unconstrained females. It is almost certain that exclusively male broods are not the product of binomial sex allocation. Given the overall highly female biased sex ratio, the probability of producing a full brood of males is very small (Fig. 1.4). Thus, if exclusively male broods do arise, this is evidence for non-binomial sex allocation. The data from these exclusively male broods were initially excluded from the analysis, with the caveat that they should be considered as possibly valid. A large data set from 559 broods remained with which to examine the sex ratio strategy of G. nephantidis.

The analysis of Green (1980) and Green e t a l. (1982) was followed to examine sex ratio strategy in G. nephantidis. The assumption was made that one male is sufficient to successfully mate all his sisters, hence "one male per brood" is assumed to be optimal (section 5.4.2). It was also assumed that pre-adult mortality does not affect the brood sex ratio (i.e. that brood sex ratios are equivalent to primary sex ratios) (section 5.4.1). For Precise sex ratios 98

each brood size, the number of broods containing one male was compared with the expected number if sex were determined binomially. If the distribution of the number of males in broods of a given size is binomial, the probability, P, of exactly one male in a brood of size N is given by

5.1

where X = number o f males in the brood, N = brood size and p = probability that each egg will be unfertilized (male). This probability will be greatest when P = 1 /N. In this case P(X=1) = (1-1 / N f ' 1, and this value is named a.

The null hypothesis, that X is binomially distributed, is tested against the hypothesis that sex ratios are more precisely determined. If n = frequency of broods of size N and k = the number of broods of size N having exactly one male, then, under the null hypothesis, k is a random variable having a binomial distribution with parameters n and P when P < a. The probability of observing as many as k (or more) broods with exactly one male will be no larger than

5.2

5.4 Results 5.4.1 Primary sex ratio

Despite repeated attempts and variations in the above protocol, no clear preparations were obtained, thus the primary sex ratio could not be assessed.

5.4.2 Male insemination capacity

The number of replicates from these estimates of male insemination capacity are insufficient for statistical analysis. Without more data only speculative interpretations of patterns can be made. When females were presented simultaneously, male mating ability was low, the maximum number of females inseminated was 8, but the mean (3.25) was considerably lower, and in no case were all the presented females inseminated. With the natural presentation of females, and a fixed maximum time of 24 hours with females, the maximum number of females inseminated was 12, mean 6.75. In no case were all the presented females inseminated. With a fixed minimum time of 12 hours in contact with each female, in all but one replicate males inseminated all of their sisters. In the remaining Precise sex ratios 99

replicate, all but one female was inseminated. The maximum number of sisters inseminated was 9, mean 7.5. The number of inseminated females was limited by the brood size. Larger clutch sizes did not occur, so an estimate of the maximum insemination capacity o f males cannot be made.

5.4.3 Precise sex ratio

Table 5.3 shows, for each brood size, the upper bound on the probability of at least the observed number of one-male broods being produced by binomial sex determination.

T ab le 5.3 A test of binomial sex ratio in G nephantidis

Brood size Frequency Number of one- Upper bound on the prob- male broods ability of observing k or N n k more one-male broods

1 5 0 2 7 2 0.937 3 14 2 0.996 4 14 1 0.995 5 27 7 0.966 6 27 6 0.985 7 51 28 0.019* 8 89 48 0.355 x 10'2" 9 57 35 0.507 x 10'3" 10 95 62 1.550 x 10'7‘* 11 65 44 0.2 x 10-4** 12 62 49 7.8 x 10n** 13 22 15 0.432 x 10'2" 14 12 8 0.043* 15 8 4 0.361 16 1 0 t 17 3 2 t

* Significant at the 5% level ** Significant at the 1% level 1 Differences from binomial distribution not detectable at 1% or 5% significance levels, due to small frequency. Total broods: 559 Precise sex ratios 100

For broods of between 7 and 15 adults (inclusive) there were significantly too many one-male broods for the sex ratio distribution to be binomial. Thus, at these brood sizes, G. nephantidis produces a more precise sex ratio than if the sex of each egg were determined binomially, in agreement with theoretical predictions (Green 1980, Green e ta l. 1982). For the largest two brood sizes (16 and 17) any differences from a binomial distribution would not be detectable, because of the small sample sizes (frequencies). At brood sizes 2-6 (inclusive) and 15 the variance in sex ratios produced by G. nephantidis are not significantly different from binomial. Although it is mathematically possible to detect significant differences from binomial sex ratios at each of these brood sizes occurring with these frequencies, the power of the test to detect differences is low when frequencies are small. At small frequencies very high proportions of broods must contain only one male for differences to be significant, whereas with greater frequencies the proportions of one male broods need not be so large. Thus, this lack of significant difference from binomial variance is very probably due to low sample sizes.

The larger the developmental mortality, the less strongly the secondary sex ratio reflects the primary sex ratio and thus the sex allocation strategy. To remove any effects of developmental mortality, the broods with known complete survival were examined separately. The data set was consequently reduced in size and at all but one brood size the frequencies were too small to allow detection of precise sex ratios. At the remaining brood size, 10, there was no significant difference in the observed number of one-male broods from expected under binomial sex allocation. This contrasts with the precise result obtained at this brood size when all data are combined (Table 5.3). This is, again, very probably due to the low frequencies of broods in this reduced data set. To try to avoid the problem of small sample sizes, but still reduce possible effects of developmental mortality, broods with a known mortality of less than 20% were examined (Table 5.4). Precise sex ratios 101

Table 5.4 A test of binomial sex ratio in G. nephantidis broods with less than 20% mortality

Brood size Frequency Number of one- Upper bound on the prob- male broods ability of observing k or N n k more one-male broods

4 1 0 t 5 2 1 t 6 1 0 t 7 5 4 0.084 8 8 6 0.045* 9 4 2 0.507 10 11 6 0.219 11 12 6 0.297 12 5 5 0.834 x 10‘2‘" 13 5 4 0.746 14 2 1 t 15 2 1 t 16 0 0 17 1 1 t

* Significant at the 5% level ** Significant at the 1% level f Differences from binomial distribution not detectable at 1% or 5% significance levels, due to small frequency. Total broods: 59

The numbers of one-male broods are significantly more precise than expected under a binomial distribution at brood sizes of 8 and 12, many fewer than when all the data are combined (Table 5.3). At brood sizes of 7, 9-11 and 13, sex ratios are not significantly different from the binomial expectation, precise sex ratios were found at all these brood sizes when all data were combined. This pattern is again probably due to the lower frequencies in this reduced data set. For broods containing 6 or less, and 14 or more offspring there are insufficient frequencies to make detection of differences from the binomial distribution possible. Precise sex ratios 102

In summary, at the middle range of brood sizes (probably the most common in the natural range) G. nephantidis produces precise sex ratios. At the extremes of the clutch size range sex ratios are not significantly different from binomial, but the lack of signifi­ cance is probably due to the small data set at these brood sizes. Investigation of effects of developmental mortality is hampered by the necessarily reduced data set.

5.5 Discussion

5.5.1 Primary sex ratio

The cytological technique for assessing the primary sex ratio produced by G. nephantidis has not yet been made to work. Consequently, it is not possible to test whether the brood sex ratio differed from the primary sex ratio. The technique may not have worked for a number of reasons, it may be that the eggs were not squashed at the best developmental stage to show nuclei in metaphase of mitosis. The age of the egg at which this stage occurs is likely to depend upon the species and on environmental conditions, such as temperature. This best stage may not have been found for G. nephantidis. The Hymenoptera have notoriously small chromosomes (Aubert 1959) which make application of chromosome staining techniques difficult.

5.5.2 Male insemination capacity

There are insufficient data for an adequate understanding of the mating abilities of male G. nephantidis. Males were found to be capable of inseminating up to twelve females. Simultaneous presentation of females to the male reduced the estimate of his mean insemination potential in comparison to "natural" presentation. Simultaneous presentation with females may lead to high copulation frequencies for males. High copulation fre­ quencies may cause temporary sperm depletion in males (Nadel & Luck 1985), such that a female receives no sperm during copulation. If G. nephantidis females refuse subsequent courtship, such females would remain effectively virgin (a condition termed "pseudovir­ ginity" by van den Assem 1969, Chapter 2). Simple observational experiments could test this hypothesis.

The asynchronous presentation of females leads to increased estimates of mean male insemination ability compared to those obtained from simultaneous presentation. These estimates seem to depend on the time period over which males had contact with females. Precise sex ratios 103

The more time the males had in contact with females, the greater the estimate of insemi­ nation potential. Males given at least 12 hours in contact with each female were capable of inseminating all their sisters. Unfortunately no clutches containing more than nine females were produced, so it remains uncertain whether males are capable of fertilizing all sisters in larger clutches before dispersal.

Obviously, more data are needed to quantify mean male insemination capacity, and the dependence of mating capacity on factors such as male size. Two general conclusions can be made from these initial attempts. Firstly, the choice of protocol is of the utmost importance in attempts to estimate mating ability of male gregarious parasitoids. Secondly, the assumption that one male is sufficient to fertilize all his sisters in a brood has not been strictly tested, but it seems that there is no reason to reject the assumption without further investigation. This assumption is made in the analysis of G. nephantidis sex ratios.

5.5.3 Precise sex ratio

G. nephantidis exhibits precise sex allocation at some brood sizes, which were generally the most commonly produced. Precise sex ratios were not found at all brood sizes. Although the possibility that this is a biological phenomenon cannot be eliminated, this result is very probably an artifact of small frequencies of some brood sizes. Small sample size (frequency) is a more important limiting factor in the analysis at small brood sizes. For smaller broods, the proportion of one male broods must be relatively higher than in larger broods for differences from binomial sex ratios to be significant. Precise sex ratios will not be found in cases where they exist biologically but are statistically indistinguishable from binomially produced sex ratios.

At the largest brood sizes sex ratios were not significantly different from binomial, perhaps due to small frequencies, but also perhaps because the assumption of one male per brood may be invalid at large (greater than 14) brood sizes. The validity of this assumption depends on the limits of male ability to fertilize sisters. A precise strategy allocating more than one male per brood will not be revealed under this test. Additionally, G. nephantidis may not have the ability to allocate sex precisely when laying large and small broods, especially if these are relatively uncommon in nature. For the same overall sex ratio, binomial variance is, however, greatest among smaller broods and thus the advantage of precise sex allocation is also greatest (Green e t a l. 1982). Precise sex ratios 104

For the most common brood sizes, sex ratios with less than binomial variance were produced by G. nepharttidis. However, brood sex ratios are not perfectly precise (not all broods contained only one male, T able 5.3). If brood sizes with less than binomial variance in the sex ratio are examined separately, the proportion of broods containing exactly one male is generally between a third and a half. This seems a surprisingly low degree of precision. Furthermore, 26% of this group of broods do not contain any males at all. Since G. nephantidis is assumed to be highly inbreeding, females from such broods are expected to have very small probabilities of finding a mate, and thus extremely low fitness (Hardy & Godfray 1990, Chapter 2).

Factors which limit the degree of sex ratio precision attained in G. nephantidis must influence the primary sex ratio or cause differences between primary and secondary sex ratios.

The primary sex ratio of G. nephantidis is not perfectly precise since broods without mortality did not all contain exactly one male (although most, 14/19, did). Precision in the primary sex ratio could be limited by the control of egg fertilization or by the mother’s ability to assess the optimal sex ratio. The use of C. cephalonica as a host rather than O. a re n o se lla may have altered the degree of precision in the primary sex ratio. C. cephalonica may be on average smaller than a O. arenosella. If parasitoids fail to adjust their clutch size d e c isio n s fully to the C. cephalonica size distribution, and if male eggs tend to be deposited later in the clutch (Section 5.2.1), then more broods with fewer than optimal number of males will be produced. There has been no comparison of size distributions of O. arenosella and C. cephalonica. G. nephantidis clutch size is correlated with C. cephalonica size, although not perfectly (Chapter 4). Male eggs are laid at the end of clutch oviposition in some bethylid species (Mertins 1980,1985, Powell 1938, Table 5.2), but the sequence of sex allocation is not known for G. nephantidis. The possibility that clutch size decisions limit sex ratio precision in G. nephantidis thus cannot be excluded. Evidence contrary to this explanation is provided by Ramachandra Rao & Cherian (1927) who reared G. nephantidis on O. arenosella and found that 42% (5/12) of broods consisted entirely of females. The number of broods examined by Ramachandra Rao & Cherian is much smaller than that analysed here, but the similar pattern of their data does indicate that exclusively female broods are not an artifact of culturing G. nephantidis on C. cephalonica. It seems that other factors may be limiting precision in the brood sex ratio of G. nephantidis. Precise sex ratios 105

Differences between primary and secondary sex ratios are caused by developmental mortality. The advantage of precise over binomial sex allocation is least when the developmental mortality is highest (Green e t al. 1982). The mean mortality during development from egg to adult in G. nephantidis is 26.8% (probability of survival from egg to adult = 0.732, S.E. = 0.027, Heterogeneity Factor = 2.9, Chapter 4). How could this affect the degree of sex ratio precision? If one male egg is laid in each brood, 26.8% of broods are expected to contain no adult male since 26.8% of male eggs will die. Amongst the brood sizes at which precise sex ratios are found (Table 5.3) the percentage of broods exclusively containing female offspring is 26%, almost exactly the same value. Devel­ opmental mortality may thus greatly account for the degree of brood sex ratio precision achieved by G. nephantidis. This simple explanation assumes that the primary sex ratio strategy is not affected by developmental mortality, that offspring survive independently of their siblings and that there is no differential mortality between the sexes.

Green e t al. (1982) showed that under precise sex allocation the primary sex ratio should increase with the probability of developmental mortality. The increased numbers of males thus ensures that the average number of mated females in a brood is maximised. Hence, if a mother is to take into account developmental mortality she must make a more complicated decision when solving for the sex ratio that maximises her number of mated daughters. An analogous situation exists in the theory of superparasitism in which a female adjusts her clutch size (Parker & Courtney 1984, Strand & Godfray 1989) and sex ratio (Suzuki & Iwasa 1980, Werren 1980, Wylie 1976) on the basis of the probability of superparasitism. More than one male egg is thus expected in G. nephantidis clutches at oviposition, and hence few broods with no adult males should occur. The occurrence of so many exclusively female broods in G. nephantidis is thus not fully explained by the overall mortality.

The high value of the heterogeneity factor implies that individual survival is not independent of siblings in G. nephantidis. Thus, the assumption that offspring survive independently of their siblings is probably incorrect. How does this affect the optimal primary sex ratio? For the same overall mortality, the compensatory increase in primary sex ratio should be less if offspring survival is not independent of sibling survival than if it is. This is because the sex ratio is irrelevant if all members of a brood die (Green e t al. 1982). The remaining broods will suffer relatively little mortality and members will enjoy greater than the average individual survival. The primary sex ratio is only important to these latter broods and thus will not be very different from the optimal primary sex ratio when mortality is absent from all broods. Precise sex ratios 106

It is assumed above that there is no differential mortality between the sexes. The developmental mortality of haploid males may be higher than that of diploid females since deleterious mutations (recessive) are always unmasked in the haploid state, but only in homozygous diploids (Hard & Brown 1970, Manning & Jenkins 1980, Smith & Shaw 1980, White 1973). Although there is no direct evidence of this in G. nephantidis, the mortality of all male broods is not different from that of "mixed" broods containing mostly females (Chapter 4).

If differential mortality occurs how would it affect the sex ratio strategy of parasitoids producing precise sex ratios? Sex ratio theory (Fisher 1930) has assumed that a differential mortality between the sexes will not affect the primary sex ratio since a greater develop­ mental mortality one sex is compensated for by the increased reproductive success of the survivors of that sex (Chapter 1). This will be correct for panmictic species but in an inbreeding parasitoid with a highly biased sex ratio there may be insufficient survivors of a sex to enjoy enhanced reproductive success; for example, if the only male in a brood dies there is no remaining male to enjoy enhanced reproduction. Females in the brood will remain unmated and will have extremely low fitness (Chapter 2). In such cases a mother should thus increase the primary sex ratio she produces to compensate for a greater mortality amongst males than females, similar to the production of higher sex ratios with higher non-differential mortality.

The factors discussed so far have all potentially reduced the degree of sex ratio precision attained. Cannibalism or combat amongst males may reduce sex ratio variance independently of the mother’s sex ratio strategy (Green e ta l. 1982). There is no evidence of cannibalism or post-eclosion male combat in G. nephantidis (with the rare exception of larvae feeding on siblings rather than on the host, pers. obs.).

The total number of broods examined (559) was large in comparison to previous studies of other species (e.g. Green e t al. (1982) examined 185 broods of G . g o rd h iand 74 of G. emigratus). How do the brood sex ratios of G. nephantidis compare with those in other parasitoids? Developmental mortality of G o n io zu s g o rd h was i measured by Gordh (1976), but the value was not reported. In Green (1980) and Green e t a l .' s (1982) analysis of Gordh’s data, the mortality is only qualitatively reported as "low". Full comparison of the sex ratio strategies of G. nephantidis and G. g o rd h iis difficult with no exact estimate of mortality in G. g o rd h i. As in G. nephantidis, G. gordhi brood sex ratios were not Precise sex ratios 107

perfectly precise. At each brood size, there are proportionally more one male broods in G. g o rd h icompared to G. nephantidis. In the species with (apparently) lower develop­ mental mortality, the brood sex ratios are thus more precise.

In G. nephantidis many (32% overall) broods contain exclusively female offspring. It has been assumed that females which do not mate with male siblings have effectively zero fitness, and thus that the brood sex ratio is an important determinant of the mother’s fitness. How valid are these assumptions? Unmated females could produce sons and mate with these (Balfour-Browne 1922) thus avoiding the penalties of constrained sex allocation (Chapter 2). These females are, however, expected to have low fitness due to the time required to produce mates. Alternatively, unmated females could mate with non-sibling males. The probability of outbreeding is unknown in G. nephantidis. Outbreeding requires that males disperse from the natal site. Wingless males are often associated with inbreeding (Hamilton 1967, 1979), but G. nephantidis males possess functional wings: perhaps out- breeding is less rare than assumed. Since O. arenosella populations are usually at low densities, but occasionally have locally high density outbreaks (Cock & Perera 1987, Perera 1987, Perera e t a l. 1988), outbreeding opportunities for G. nephantidis could be usually low, but greater during periods of high host density. It is not known whether mated G. nephantidis females employ a sex ratio strategy which alters sex ratios in accordance to local host density.

Thelytoky (diploid parthenogenesis) abandons the need to produce mates for females and thus avoids problems associated with the production of optimal sex ratios. It has been proposed that thelytoky should replace arrhenotoky (haploid parthenogenesis) as an extreme response to selection for the production of highly female biased sex ratios (Cornell 1988). Thelytoky may arise by mutation from haplodiploid forms (Cuellar 1977, Suo- malaineinen 1962, White 1954) or due to the influence of extrachromosomal factors, such as microorganisms (Hurst e t a l. 1990, Stouthamer e t a l. 1990). The adoption of thelytoky is the abandonment of sexual for asexual reproduction, the relative advantages of which are reviewed by Bell (1982), Maynard Smith (1978b) and Williams (1975).

Though thelytoky appears in nearly all major parasitoid families (Clausen 1940) it has not become established throughout groups (Suomalaineinen 1962, White 1954). There is contradictory evidence that G. nephantidis is not sometimes thelytokous; Sundaramurthy & Santhanakrishnan (1978) and J.M. Cook {p e r s. co m m .) found that virgin females only produce male offspring, whereas Antony & Kurian (1960) report that unmated females produce both male and female offspring. Most other bethylid species appear to lack Precise sex ratios 108

thelytoky, although there has also been contradictory evidence for it’s occurrence in some (Gordh 1976). If G. nephantidis is not thelytokous then females in broods containing no males, can be assumed to have fitness related to their (apparently low) outbreeding opportunities.

In summary, Green e t al. (1982) showed theoretically that precise sex allocation results in larger average numbers of mated females produced per brood than under binomial sex allocation. G. nephantidis produces sex ratios mostly in agreement with these pre­ dictions. Even with precise sex allocation many broods did not contain males. Until there are better data for the primary sex ratio, the relative importance of developmental mortality and other factors which limit precision in G. nephantidis brood sex ratios, cannot be evaluated. Discussion 109

Chapter Six

Discussion

Specific discussions have been presented in the individual chapters. This chapter is intended only as a short review of the experimental work presented in the thesis and for suggestions for future investigations. 6.1 Constrained sex allocation in Hymenoptera

Constrained sex allocation in three species of D ro so p h ila parasitoids was absent or rare (Chapter 2). The available evidence suggests that, amongst the Hymenoptera, con­ strained sex allocation is generally rare. There are, however, some species in which constrained oviposition is quite common. Amongst the Hymenoptera for which there is evidence of the prevalence of constraints some taxa are over-represented. Interpretation of patterns is difficult without a phylogenetic analysis considering the number of separate evolutionary transitions involved. Investigation of constrained oviposition in more species is required before there will be sufficient data for a comparative approach. One possibility for investigation of prevalence of constraints are A m m o p h ila species; these aculiate wasps carry their hosts to a nest, it can thus be clear whether captured females are reproducing.

A prediction was made that constrained sex allocation should be less common in species with gregarious broods. In making this prediction, the assumption was made that species with gregarious broods are associated with inbreeding and thus these species would be subject to LMC. For such species the penalties of producing only one sex of offspring are expected to be high (Chapters 2 & 5). However, the available evidence (Chapter 2) does not support this prediction. Interestingly, the species with the highest percentages of constraints are the fig wasp, Apocyrta mega, (with high levels of inbreeding) and some of the gregariously developing braconids (Chapter 2). The assumed correlation of gregarious offspring development and LMC may be a oversimplification. There have been few studies of the prevalence of outbreeding amongst gregarious parasitoids. Tagawa and Kitano (1981) examined the mating behaviour of Apanteles glomeratus as offspring eclosed in the field, 60% of the observed mating was between siblings. Some (5/19) females dispersed Discussion 110

before mating, whether these females remained virgins or mated later is unknown. Levels of inbreeding in gregarious species may thus be lower than assumed. Similarly, inbreeding among solitary parasitoids may be more common than assumed (e.g. some parasitoids of D r o s o p h ila, Chapter 2, Nadel & Luck 1985).

The optimal sex ratio for a gregarious parasitoid to produce is less female biased when she superparasitizes than when she parasitises a fresh host (Suzuki & Iwasa 1980, Werren 1980). The difference between the sex ratios produced by constrained females and unconstrained females is thus least when these superparasitise. Hence, in gregarious species subject to LMC, constrained females may be more willing to superparasitise than unconstrained females. Superparasitism may reduce the penalties of the constraint and provide mates for the offspring of constrained females.

6.2 Brood guarding in Goniozus nephantidis

The possible reasons why G. nephantidis females remain with hosts following ovi- position were investigated in Chapter 3. Remaining with the brood incurs loss of repro­ ductive opportunities, but it seems that the cost of this is offset by benefits to the offspring, via the prevention, by the mother, of detrimental actions of other parasitoids. There is still much to be discovered about G. nephantidis. The behavioural interactions between mother and intruder have not been fully investigated. Initial attempts (I.C.W. Hardy & T.M. Blackburn unpublished data) examined the behaviour of the mother and the intruder in the first 30 minutes after the introduction of the intruder. It was expected that in situations where the presence of the mother made a difference to brood survival, this would be reflected in the behaviour of the parasitoids: perhaps mothers deter oviposition and ovicide by intruders by aggressively driving them away from the developing brood. However, the behaviour of the parasitoids was not consistent with these expectations. Aggression by mothers towards B . h e b e to r made no difference to the probability of the intruder laying eggs (equivalent analysis for conspecific intruders could not be performed because they never laid when the mother was present). Similarly, the positions of mother and intruder relative to the host were generally unrelated to the probability of the intruder laying. So, unfortunately, these behavioural observations gave no clue as to how contests for resources might be decided.

Fighting between initial females and intruders was rarely observed. Aggressive interactions usually consisted of the guarding G. nephantidis opening it s mandibles and adopting an apparently threatening posture towards an intruder and, on occasion, chasing Discussion 111

the intruder away. Fighting can, however, be induced by allowing two G. nephantidis, separated by a barrier, to lay clutches simultaneously on either end of the same host. On removal of the barrier, the two mothers discover each other and a short intense fight often ensues. The "loser" is driven away, the "victor" remains with the broods. The result is the same whether the broods are at the egg stage or have been allowed to develop to the larval stage when the barrier is removed. The victor has not been observed to kill the loser’s brood. The same behaviour is observed if separate guarded hosts are placed in close proximity either side of the barrier, which is then removed as above. This is technically more convenient since the host does not need to be squashed by the barrier: squashing the host can cause it’s death, and females are more likely to oviposit when presented with an unsquashed host.

These pilot "simultaneous oviposition" experiments have not yet been quantified, but are reported here as part of a suggestion for further work. An interpretation of competition for the host based on game theory was suggested in Chapter 3. This could be extended to investigate the nature of the asymmetries between females. Conspecific intruders never laid on guarded hosts with broods (Chapter 3), G. nephantidis intruders may thus follow simple mles based on asymmetries in "ownership status", such as "if owner is present then retreat". Such simple rules would prevent escalated conflicts (fighting). Similar situations occur in other invertebrate intruder/occupier conflicts (e.g. Crespi 1986, Davies 1978,Marden e ta l. 1990,Waage 1988). Grafen( 1987b)has, however, pointed out that rules based on asymmetries which create consistent losers cannot be used to settle contests in which the outcome represents a major part of the fitness of the con­ testants. Consistent losers will not respect such rules since if the chances of reproduction are rare then the intruder has nothing to loose and much to gain. Applying this to G. nephantidis; that conspecific intruders never laid on guarded hosts with broods implies that the probability of finding a fresh host is sufficient to make avoidance of escalation worthwhile for the intruder. Simultaneous oviposition experiments remove the asymmetry in "ownership status" of the contestants, the importance of other factors such as female size, clutch size, host weight or brood development stage on the outcome of contests could thus be investigated. It is not expected that these experiments would represent natural situations, but that they may elucidate underlying features of contest in G. nephantidis.

Although conspecific intruders never laid on guarded hosts with broods, they sometimes gained possession of paralysed hosts guarded by the initial female (Chapter 3). Perhaps, in these cases, the superior "fighting ability" of the intruder overcame any advantages of "ownership" the initial female may have had. This may occur because the Discussion 112

resource value asymmetry between initial female and intruder is less before oviposition by the initial female than once a clutch has been laid (Maynard Smith & Parker 1976, Chapter 3). It seems likely that fighting abilities are related to the size of the female. Investigation of how the outcome of contests for a paralysed host depend on the sizes of the contestant females may clarify the components of the "resource holding potential" (Hammerstein 1981, Maynard Smith & Parker 1976) and further elucidate the importance of female size in G. nephantidis (Chapter 4).

In nature, there are other parasitoid species which may interact with G. nephantidis, these interactions have not yet been investigated. One important, but unknown, quantity is the scarcity of oviposition opportunities for G. nephantidis. Unfortunately, it is much easier to ask questions of the field biology of G. nephantidis than to provide answers.

6.3 Clutch size in Goniozus nephantidis

There is a strong prediction that G. nephantidis produces the Lack clutch size, this maximises the total fitness of offspring developing in the clutch. Chapter 4 examined the clutch size of G. nephantidis and found that developmental mortality was not affected by clutch size, but offspring size was reduced in larger broods. Female size affects both fecundity and longevity. These measurements of fitness were combined to provide calculation of the Lack solution. The calculated values were larger than the observed clutch size. Possible explanations are that the measurements of fitness were incomplete or that the parasitoid is not producing the Lack solution. The prediction of the attainment of the Lack solution relies on the assumption that there are no trade-offs between present and future reproduction. The biology of G. nephantidis implies that this assumption is correct but without better information about the natural history of this wasp it is not possible to be certain. Although, trade-offs are potentially unaffected by the number of hosts a female parasitises in nature, this quantity may be important and is unknown.

The more likely cause of the disparities between prediction and observation is that fitness cannot be adequately measured in the laboratory. If the advantages of large size are greater for females than estimates of fecundity or longevity predict, then the Lack clutch size will be less than the calculated value and thus closer to the observed value. Perhaps female size is an important influence in the mothers success guarding broods against intruders (Section 6.2 & Chapter 3) or perhaps the ability of females to search for hosts is improved by larger size. Neither of these relationships have been investigated. It Discussion 113

should also be noted that there may be advantages to small size which have not been discovered, such advantages will increase the value of the calculated Lack clutch size, and also the discrepancy between observation and prediction.

The natural history of G. nephantidis is much more complicated than can be repre­ sented in the laboratory, however that life-history of this species is relatively simple in comparison with other animals. Prediction of the Lack clutch size would have been simplified if the penalties of increased clutch size had been manifest in brood survival rather than via the effects of size on adult fitness. The ideal choice of species to test sex ratio theory would be one with a similar biology to that of G. nephantidis, but in which the penalties of exceeding the optimal clutch size were restricted to the survival of offspring rather than affecting adult fitness. However, there is no way of knowing how clutch size will affect offspring fitness until investigations are carried out! Another type of species for which there are a p r io r iexpectations for the absence of trade-offs, and which may be amenable fortesting clutch size theory, are polyembryonic Hymenoptera (Godfray 1987a). In these the clutch size decision is made by the offspring not by the parent. The parent lays one or a few eggs which divide asexually to produce groups of identical siblings, trade-offs thus should be absent and the Lack clutch size is predicted.

6.4 Precise sex ratios in Goniozus nephantidis

Inbreeding parasitoids are expected to achieve less than binomial variance in the sex ratios of their broods (Green e t al. 1982). Chapter 5 showed that the sex ratios produced by G. nephantidis mostly match this expectation. Despite non-binomial sex allocation, not all broods contained the (assumed) optimal sex ratio. The penalties (Chapter 2) of non-optimal sex ratios are thought to be high in G. nephantidis since the number of mated females produced is strongly influenced by the brood sex ratio. It is unknown why G. nephantidis sex ratios are not more precise.

In extreme cases broods contained no males; the proportion of broods containing no males was the same as the probability of developmental mortality. Perhaps, in G. nephantidis, there is no adjustment of primary sex ratio to compensate for developmental mortality ( c o n tra theoretical expectation, Green e t al. 1982). Similar proportions of exclusively female broods are obtained if G. nephantidis is reared on it’s natural host (Ramachandra Rao & Cherian 1927) indicating that this phenomenon is not due to the use of C. cephalonica as the host. Discussion 114

Despite a large data set, there are insufficient data from broods with no (or very low) mortality to examine primary sex ratios directly. Cytological techniques, which could be used to assess primary sex ratios, have not yet been successful with G. nephantidis eggs. Before a better understanding of the sex ratio strategy of G. nephantidis can be reached, the mating ability of males and the outbreeding possibilities of both sexes need to be quantified.

6.5 Conclusions

In general, results from studying G. nephantidis agree, at least qualitatively, with theoretical predictions and with empirical work on other parasitoid species. Females remain with their broods to look after their developing offspring, the sex ratios they produce are precise, and the fitness of their offspring is affected by the clutch size they produce (even if all aspects of this may not have been examined). In other Hymenoptera the phenomenon of virgin oviposition has been explored and it’s potential importance identified.

Despite qualitative agreement between predictions and observations, quantitative discrepancies remain (Chapter 4 particularly). How important are these discrepancies to the understanding of parasitoid reproduction? Assessing the significance of discrepancies between prediction and observation brings it s own problems (Parker & Maynard Smith 1990). If differences are not significant the important factors in the evolution of the behaviour may have been identified. If discrepancies are significant, how should investigations of reproductive strategies proceed? The assumptions of the theory should be re-examined and the theory modified. In this thesis, a large part of the explanation of discrepancies has been that too little is known of the importance of factors in the parasitoid’s natural environment. If these were examined and included in predictive theories, and the new predictions tested, this would provide an iterative approach to understanding the important evolutionary factors affecting the parasitoid’s behaviour (Parker & Maynard Smith 1990, Chapter 1). In other words, disparities between prediction and observation have indicated factors which may reduce these disparities when incorporated into theories, and thus which factors should next be investigated. 115

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