VU Research Portal

Parasitism and the Evolutionary Loss of Lipogenesis Visser, B.

2012

document version Publisher's PDF, also known as Version of record

Link to publication in VU Research Portal

citation for published version (APA) Visser, B. (2012). Parasitism and the Evolutionary Loss of Lipogenesis. Ipskamp B.V.

General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.

• Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ?

Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

E-mail address: [email protected]

Download date: 06. Oct. 2021 Parasitism and the Evolutionary Loss of Lipogenesis Cover: Symbiose mensuur tussen natuur en cultuur. Cover design: Leendert Verboom Lay-out: Bertanne Visser Printing: Ipskamp Drukkers B.V., Enschede

Thesis 2012-1 of the Department of Ecological Science VU University Amsterdam, the Netherlands

This research was supported by the Netherlands Organisation for Scientiﬁc Research (NWO, Nederlandse organisatie voor Wetenschappelijk Onderzoek), grant nr. 816-03-013. isbn xxx

VRIJE UNIVERSITEIT

Parasitism and the Evolutionary Loss of Lipogenesis

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad Doctor aan de Vrije Universiteit Amsterdam, op gezag van de rector magnificus prof.dr. L.M. Bouter, in het openbaar te verdedigen ten overstaan van de promotiecommissie van de faculteit der Aard- en Levenswetenschappen op woensdag 25 januari 2012 om 13.45 uur in de aula van de universiteit, De Boelelaan 1105

door

Bertanne Visser

geboren te Delft

promotor: prof.dr. J. Ellers

Voor Adriana Visser-Verboom en Hugo Kok

Contents

Contents 1 1 General introduction 3 2 Lack of lipogenesis in parasitoids: A review of physiological mechanisms and evolutionary implications 15 3 Loss of lipid synthesis as an evolutionary consequence of a parasitic lifestyle 29 4 Can host manipulation drive the evolutionary loss of traits in parasitoids? 49 5 Host exploitation efficiency in a gall wasp community 61 6 Lack of transcription of the key gene in lipid synthesis, fatty acid synthase, reflects loss of lipogenesis in adult parasitic wasps 75 7 Discriminating between energetic content and dietary composition as an explanation for dietary restriction effects 101 8 Effects of a lipid-rich diet on adult parasitoid income resources and survival 115 9 Synthesis 125 Bibliography 141 Summary 179 Samenvatting 183 Acknowledgements 189 Curriculum vitae 195 Publications 197 Affilliation of committee members 199 Affiliation of co-authors 201

Chapter 1

General introduction

A brief history of evolutionary theory

Nowadays some scientists might say that the continued reference to the original theories of evolution has become a cliché and that many theoretical concepts pertaining to evolution have undergone far-reaching conceptual revisions. I agree that science in general should progress toward novel or extended theories and researchers in evolutionary biology should be critical to concepts in evolutionary theory to elucidate the natural world. However, in essence, science can only progress through building on the foundation of knowledge acquired throughout the history of science. Evolutionary biology still relies heavily on the fundamental theories incepted by Alfred Russell Wallace in his seminal paper ‘On the tendency of varieties to depart indefinitely from the original type’ (1858) and Charles Darwin’s book ‘On the origin of species by means of natural selection, or the preservation of favoured races in the struggle for life’ (1859). While highly controversial at that time and contradicting contemporary views on inheritance and observed variation between organisms, their pivotal work explains the process of descent with modification and natural selection acting on existing herita- ble variation within populations as the main driving force behind adaptive evolution. With the foundation of evolutionary theory in place, another major contribution to evolutionary theory was fuelled by the rediscovery of Mendel’s laws of genetics in 1900. Up to that point in time the mechanisms of inheritance had remained elusive and its eventual discovery was initially regarded contradictory to theory on the process of natural selection. Rediscovery of Mendel’s laws paved the way for numerous scientists, such as Ronald Fisher (1930), John Haldane (1932) and Sewall Wright (1932), to develop the field of population genetics, concerned with studying changes in allele propor- tions within populations through the processes of natural selection, genetic drift, mutation and gene flow. Within the following decades several other prominent scientists significantly contributed to the development of evolutionary theory, in which their considerations reached beyond the realm of genetics to include fields such as ecology, paleontology and botany, as

3 Chapter 1 well as the concept of speciation (Dobzhansky, 1937; Mayr, 1942; Rench, 1959; Simpson, 1944; Stebbins, 1950). These advances in evolutionary biology were collectively termed the Modern Synthesis (MS) by Julian Huxley (1942). Amongst others, multidisciplinarity of the MS led to a conceptual framework addressing i) the key importance of genetic diversity within populations; ii) the phenotype as a means for natural selection to act upon within variable environments; and iii) the relationship between micro- and macro-evolutionary changes. Over the last couple of years further extensions of the MS have been propagated (Pigliucci, 2007; Pigliucci & Müller, 2010). Based on a theory originally proposed by Popper (Platnick & Rosen, 1987), these authors and others have argued that evolutionary theory began as a theory of form that through the inception of the MS led to a theory of genes, but that there is a current need to update the initial theory of form (Pigliucci, 2007). This expansion of the MS is referred to as the Extended Evolutionary Synthesis (EES). The EES conceptualizes evolutionary theory through the addition of numerous fields in biology. As summarized by Pigliucci (2007), extensions of the MS should embrace developmental biology, a field of research that was completely left out of the original MS, even though developmental biology was already an established field of research at the time the MS was formulated. Another major advancement revolves around incorporation of ecological theory. Despite the inclusion of ecology into the MS, the ecological settings that bring about evolutionary changes have remained largely unexplored (Maynard Smith & Szathmáry, 1995). Through technological advances during the last few decades, biology has witnessed the rise of other important fields, namely that of genomics, proteomics and metabolomics. These fields provide us with ever-increasing insights to move away from the black box principle that has been a frequent necessity in the concep- tion and extensions of evolutionary theory. Moreover, several important concepts employed in current research in evolutionary biology were not incorporated in the MS, such as phenotypic plasticity (a norm of reaction or a variable phenotype of a single genotype across environments) and epi- genetic inheritance (changes in gene expression without alterations in the underlying DNA sequences that can be passed on to the next generation). Evolutionary theory has seen major advancements and will certainly un- dergo further extensions in the future. A major advancement formulated in the MS pertains to the importance of genetic variation and its translation into the phenotype for natural selection to act upon. Undeniably, these aspects are of key importance to fuel evolutionary changes. It is becoming

4 General introduction increasingly clear, however, that variables other than standing genetic variation significantly contribute to evolutionary change. In this regard, one important concept is the evolution of novel traits, which is tackled by the EES. Yet the reciprocal process of trait loss continues to be regarded as an inevitable consequence of evolution and with it the notion that it only has a minor contribution to evolutionary change. It has been an area of research that, therefore, received considerably less attention. This thesis focuses on the loss of traits and why and how trait loss occurs during the course of evolution. I will first describe the importance of trait acquisition, how trait acquisition is linked to trait loss and current concepts and theories regarding the loss of traits. I will then outline the importance of ecological conditions and in particular nutrition to trait dynamics and fitness (average contribution of a genotype to the next generation), and last I will introduce the model system employed in the work described in this thesis.

Trait acquisition, loss and evolutionary mechanisms

Over the last few decades a key objective in evolutionary biology has been to unravel why novel traits arise, which mechanisms underlie trait acquisition and the ways in which novel traits contribute to an individual’s fitness. The most obvious examples of trait acquisition concern that of evolutionary innovations or novelties (Müller & Wagner, 1991; Shubin et al., 2009). Ac- cording to Pigliucci (2008), an evolutionary novelty should be regarded as the evolutionary gain of new traits or traits composed of a combination of pre-existing traits performing a new function within the ecological setting of the lineage in which it arose. For instance consider the morphological innovation of helmets in hemipteran treehoppers used for camouflage (Moczek, 2011; Prud’homme et al., 2011) and beetle horns employed during com- bat (Moczek, 2009). Behavioural novelties have also been described, such as the famous example of great tits opening milk bottles (Hawkins, 1950). While the MS provides us with answers on how variations in traits segre- gate throughout populations, it has not addressed the imperative question of how evolutionary novelties arise (Pigliucci, 2008). Up to now, this gap of knowledge in evolutionary theory has and continues to be filled mainly by research within the field of evo-devo (evolution and development). First, I should point out that from a mechanistic point of view, trait acquisition can still be tightly linked to genetic variation, since new traits can arise by employing pre-existing traits, although this is not necessarily the case. Genetic variation does not inevitably pertain simply to standing ge-

5 Chapter 1 netic variation that translates into a phenotype, as it can further involve genetic (or alternative) pathways that are currently unemployed in phenotypic expression, also referred to as cryptic genetic variation (Moczek, 2007). Referring back to the previous example of helmets in treehoppers, this novelty arose through employment of the genetic pathway originally involved in insect wing development in a place on their body where expression for wing development is normally silenced and that in turn is used to form this new structure (Moczek, 2011; Prud’homme et al., 2011). While most of these studies focus on how novel traits arise through developmental architecture, a crucial link that is missing in this context is why novel traits arise. In other words, how environmental variation interacts with genotypes and developmental pathways to drive evolution (Odling-Smee, 2010). There are many different ways in which developmental pathways, genotype and environment can interact. The genotype by environment interaction is a well-known concept that pertains to environmental sensitivity in terms of how different phenotypes arise through differential responses of several genotypes to a particular environmental change. This concept is tightly linked to canalization, in which expression of genetic or developmental pathways is shielded under certain environmental conditions to produce a single phenotype, hence decreasing phenotypic variability (Waddington, 1942). An example of canalization can be found in the developmental processes underlying halter formation in dipterans, in which the development of wings is repressed, whilst halters are formed that serve as a balancing organ (Gibson & Hogness, 1996; Hornstein & Shomron, 2006; Ronshaugen et al., 2005). A related theory is that of phenotypic plasticity, in which a range of phenotypes can be generated from one genotype under varying environmental conditions. For example, seasonality affects the extent of eyespot formation in polyphenic butterflies in response to changes in ambient temperature (Beldade & Brakefield, 2002). Whilst steady progression is made in identifying the importance of these mechanisms in trait acquisition, it remains challenging to establish how these mechanisms contribute to evolutionary trait dynamics. When considering the progression of research on evolutionary novelties and the importance of cryptic genetic variation in evolution, it is remarkable that the reciprocal process, i.e. trait loss, has received considerably less attention. A trait can be considered lost when its effect on the phenotype is silenced under certain environmental conditions that would normally warrant phenotypic trait expression in the ancestral lineage, hence cryptic genetic variation plays a key role in both trait loss and acquisition. Trait

6 General introduction loss typically occurs when traits are released from selection or when bearing a trait negatively affects the phenotype leading to purifying selection against the trait (Lahti et al., 2009). Similar to evolutionary innovation, phenotypic regression has been observed in a wide array of traits, including traits affecting morphology, behaviour and physiology. For instance, in some beetle species males have evolved extensive horns to fight off competitors, but these structures have been lost secondarily in females (Moczek, 2005). An example of behavioural regression can be found in weaver birds, which evolved egg rejection behaviour towards heterospecific eggs when broods of these birds were heavily cuckooed by other birds (Cruz, 1989). However, once a new population was founded in an environment without cuckooing birds, egg rejection behaviours were lost. While numerous cases of trait loss through neutrality or purifying selection have been identified (Fong et al., 1995; Lahti et al., 2009; Porter & Crandall, 2003; Regal, 1977), research on the underlying mechanisms of trait loss is only just beginning. We have yet to discover a unifying theory explaining why trait loss occurs, since some cases of trait loss are due to trait redundancy, whilst other cases reveal re-employment of previously unused traits to exert a novel function. Moreover, we still need to uncover how trait loss contributes to evolutionary trait dynamics within an ecological framework. The concept of trait loss through environmental compensation deals with the loss of traits in ecological interactions. It addresses how trait loss occurs when biotic interactions with the environment provide resources that render the trait obsolete, even though the function is still essential for survival, growth and reproduction of the organism (Visser et al., 2010). Instead, the phenotype is maintained by interaction partners or the environment rather than through investment of the organism itself, leading to a loss of traits that would normally be considered indispensable. With trait loss through environmental compensation typically overlooked and many examples remaining to be discovered, phenotypic regression of indispensable traits has mainly been observed in organisms that are involved in intricate symbiotic relationships with other organisms. In these cases, one symbiotic partner facilitates an essential phenotype, leaving the trait in its recipient prone to phenotypic decay through neutrality or the impos- ing costs on fitness. Extreme examples of trait loss through environmental compensation can be found in endosymbiotic bacteria. These bacteria live inside a host that facilitates numerous beneficial traits, and has led to extensive genome reductions in these organisms (Blanc et al., 2007; Burke & Moran, 2011; Dale & Moran, 2006; Hershberg et al., 2007; Moran, 2002).

7 Chapter 1

Vice versa, endosymbiotic bacteria in aphids were shown to supply the host with amino acids, which has led to the loss of amino acid biosynthesis in aphids (Douglas, 1998; Oliver et al., 2010; Pérez-Brocal et al., 2006). As a result, both partners benefit from an exchange of trait phenotypes, but their dependence on the symbiotic partner increases considerably through the loss of these traits. Typically, the loss of indispensable traits through symbiotic interactions tightens the dependence of organisms and might be an important aspect in preventing interacting partners from splitting up. Other routes to compensated trait loss occur when the environment provides essential dietary components to organisms, which is followed by a phenotypic loss in the organism that benefits from this dietary supply. Such an example of trait loss through dietary compensation can be found in animals, including humans, which have lost the ability to synthesize vitamin C de novo. Consumption of food sources containing high levels of vitamin C provides a sufficient supply of this resource and has led to non-functionality of the gene underlying vitamin C synthesis (Chatterjee, 1973; Ohta & Nishikimi, 1999). The dietary requirement in animals that have lost active vitamin C synthesis remains, but the detrimental effects can only be observed when the diet is devoid of vitamin C. Such dietary shifts have occurred numerous times during human history, for instance in 15th to 18th century sailors void of sufficient dietary sources containing vitamin C leading them to suffer greatly from scurvy. Identification of more cases of compensated trait loss and the mechanisms underlying such losses will shed light on how traits are lost and how ecological interactions contribute to evolutionary trait dynamics.

Nutrient acquisition, storage and life history strategies

Resource acquisition, composition, timing of investment, allocation deci- sions and environmental conditions all play an important role in determining an individual’s fitness. The acquisition of appropriate nutrients from the environment is critical for many organisms, yet resource availability and acquisition of optimal resources might be constrained by the lifestyle of an organism within the ecological conditions it faces during life (Strand & Casas, 2008). Whilst the first challenge in nutrient acquisition is that of finding suitable food sources, a second problem is that the nutritional composition of a food source is typically suboptimal for meeting the energetic demands of an organism. For instance because the environment might only facilitate suboptimal ratios of certain required nutrients (Behmer, 2009) or

8 General introduction because changes in environmental conditions during life might prevent or complicate either food location or acquisition that can detrimentally affect fitness (Coley et al., 2006; Mattson, 1980). Once nutrients have successfully been obtained allocation of nutrients typically shows a trade-off between investment into survival, growth and reproduction, as the energy acquired can only be spent once (Boggs, 1992; Stearns, 1989). The trade-off between survival and reproduction is thus further complicated by the relative ease with which organisms can acquire different types of nutrients and the constraints in redistributing resources for subsequent investment. For instance, certain butterflies readily utilize available food sources during adult life for reproduction, whilst capital reserves obtained as larvae are accessed only when these resources are not available in the adult diet (Boggs, 1997). One mechanism by which organisms have adapted to cope with environmental variability in abundance and quality of food sources is to store excessively available nutrients for use at a later time. This strategy provides a buffer against unfavourable environmental conditions that could otherwise severely affect fitness. Energy storage is an important physiological mechanism adopted by all animals that involves metabolic processing of dietary nutrients into metabolites that can be stored for a longer time and accessed later during life. At times when food is abundantly available, dietary metabolites such as sugars and other carbohydrates can be converted for storage into glycogen and lipids (Arrese & Soulages, 2010). Whilst glycogen is a short-term storage reserve that can be accessed when glucose levels are low, lipid reserves comprise the bulk form in which excess energy is stored and lipid catabolism releases most energy compared to other metabolites (Arrese & Soulages, 2010). Moreover, lipid breakdown releases substantial amounts of water that can be used to resist desiccation. A clear example of the requirement to increase storage reserves occurs at times when food might not be available for longer periods of time, for instance during diapause (Hahn & Denlinger, 2007). Accumulation of storage reserves in the form of lipids is thus an important mechanism by which organisms can prepare themselves to face unfavourable environmental conditions.

The parasitoid lifestyle

Parasitoids are parasitic insects that lay their eggs on or inside an arthropod host (Godfray, 1994). Initially parasitoids were referred to as parasites, yet this term does not accurately describe the parasitoid lifestyle. True parasites typically invade one or several hosts during their entire lifetime and

9 Chapter 1 although hosts might endure severe consequences following infection, it is often not lethal and reproduction is usually not impeded. In contrast, parasitoids differ from true parasites mainly because only part of their lives is spent in association with a host (the larval stage) and ultimately the host is killed before it can reproduce. The parasitoid lifestyle has evolved numerous times in insects, with representative species found within the orders Strepsiptera (twisted-winged parasites), Neuroptera (net-winged insects), Trichoptera (caddisflies), Lepidoptera (moths and butterflies), Coleoptera (beetles), Diptera (flies) and Hymenoptera (bees, ants and wasps) (Quicke, 1997). Most parasitoid species are found within Hymenoptera (77%) and in this order the parasitoid lifestyle has evolved once in the common ancestor (Eggleton & Belshaw, 1992; Quicke, 1997). The remainder of parasitoids is predominantly found within Coleoptera and Diptera (22%) with only a small fraction represented within the other orders (1%). Within Coleoptera and Diptera the parasitoid lifestyle is estimated to have evolved at least 14 and 21 times, respectively (Eggleton & Belshaw, 1992; Quicke, 1997). From parasitic hymenopterans numerous other lifestyles have evolved, such as phytophagy (seed-feeders, gall wasps, inquilines, fig wasps) and predation (Quicke, 1997). The diversity in parasitoid lifestyles makes them excellent study systems for questions pertaining to evolutionary biology, ecology, behavioural biology and physiology. Competing hypotheses exist concerning the evolution of the parasitoid lifestyle within the common ancestor of parasitic Hymenoptera (Godfray, 1994; Quicke, 1997). One frequently adopted theory involves the shift from a wood-feeding species to consuming coleopteran larvae developing within the same niche. Another hypothesis poses that symbiotic fungi deposited inside wood during egg-laying provided the required nutrient conversion for digestion by the ancestral insect. These fungi oviposited by conspecifics were subsequently exploited, eventually leading to a shift from feeding on fungi to the insects feeding on them (Eggleton & Belshaw, 1992). What is clear, however, is that the parasitoid lifestyle has seen extensive radiations and an incredible diversity in strategies have been adopted by parasitic Hy- menoptera. Differentiation between these strategies can be based on several traits. First, parasitoids can deposit their eggs either inside or onto a host for development (Pennacchio & Strand, 2006; Strand, 2000) and although parasitoids typically only attack one lifestage of their arthropod hosts, parasitoid taxa attacking each lifestage (eggs, larvae, pupae and adults) are found (Pennacchio & Strand, 2006). Second, parasitoids may only deposit a single egg per host, or multiple eggs are deposited to develop within one

10 General introduction host (Mayhew, 1998). Third, different life history strategies have evolved in parasitoids, in which some parasitoid species will emerge with a full complement of eggs (pro-ovigenic), whilst others will start egg maturation after emergence (synovigenic), although a range of intermediary strategies can also be adopted (Jervis & Ferns, 2004). A fourth means of categorizing parasitoid traits is through the interactions with their hosts (Pennacchio & Strand, 2006; Jervis & Ferns, 2011). Using this scheme divides the parasitoid lifestyle into two main categories: Idiobionts that arrest the host’s development and koinobionts that allow the host to continue feeding and growing whilst the parasitoid larva is developing (Pennacchio & Strand, 2006). Co-evolution between host and parasitoid has led to intriguing adaptations by both partners to cope with the constraints posed by their interaction. One extraordinary example is the ability of several parasitoid species to alter their host’s behaviour to their own benefit (Brodeur & Vet, 1994; Grosman et al., 2008; Seyahooei et al., 2009; Tanaka & Ohsaki, 2006, 2009). One example are wasps that feed and develop inside their host, yet prior to pupation they form cocoon clusters that are bound by a silken substance, whilst the host remains alive up to several days after parasitoids have pu- pated. Hosts are known to strengthen the silk web that clusters the cocoons and to fight off hyperparasitoids that seek to oviposit on the primary parasitoids. Not only can parasitoids alter their host’s behaviour, extensive physiological manipulation is also known to occur frequently. Since parasitoids can only develop on a single host, it is critical for parasitoids to carry over sufficient nutrients to meet their own energetic requirements for development, survival and reproduction. For instance, parasitoids can allow their host to maintain growing and feeding (koinobionts), while suppressing the host’s immune defences (Fellowes & Godfray, 2000; Pennacchio & Strand, 2006). Moreover, nutrient levels can be elevated, for instance through the action of venom or other parasitoid-associated substances that are either injected during oviposition or exerted by the developing larvae to increase sugar, protein and lipid levels (Bischof & Ortel, 1996; Caccia et al., 2005; Consoli & Vinson, 2004; Coudron et al., 1997, 1998; Nakamatsu et al., 2001; Rivers & Denlinger, 1994). Venom of the parasitoid Nasonia vitripennis released during oviposition leads to rapid increases in fat body lipid levels of the host that are subsequently taken over by her progeny (Rivers and Den- linger, 1995). It is this ability of parasitoids to increase nutrient levels of the host that distinguishes them from predacious species, which are unable to maximize nutrient resources once a prey has been captured.

11 Chapter 1

Parasitism and the evolutionary loss of lipogenesis

The basic principle in evolutionary theory concerns genetic variation that is translated into a phenotype on which natural selection acts to fuel adaptive evolution. However, evolutionary changes through environmental changes can also lead to the acquisition of novel traits or to the loss of traits that are dispensable in the newly posed environmental conditions. When considering trait loss the environment can further amplify the rate with which traits are lost when conditions render a trait dispensable and can even occur in traits that are still indispensable for an organism’s fitness. The evolutionary loss of a trait can thus also involve traits that are of crucial importance to fitness, such as those involved in nutrient storage. Lipids are the most comprehensive form in which nutrients can be stored and are of importance to traits affecting fitness, such as survival and reproduction. At the onset of the work presented in this thesis numerous parasitic insects had been found to lack accumulation of lipids used as reserves (Casas et al., 2003; Ellers, 1996; Fadamiro et al., 2005; Giron & Casas, 2003; Lee et al., 2004a; Olson et al., 2000; Rivero & West, 2002). These studies showed that under conditions that would normally induce lipid synthesis these species lacked phenotypic expression, suggesting this trait has been lost during the course of evolution. These observations fuelled the formulation of several questions regarding the loss of this trait in parasitic insects: i) Has co-evolution between host and parasitoid led to the evolutionary loss of lipogenesis in these insects?; ii) Which mechanisms underlie the loss of this essential metabolic trait in parasitoids?; And iii) How does dietary intake of nutrients affect life histories of organisms lacking lipogenesis? These questions are tackled in the following chapters of this thesis: Chapter 2 aims at providing a synthesis of literature regarding physiological mechanisms and evolutionary implications of the lack of lipid synthesis in parasitoids. One aspect of particular importance to the lack of lipogenesis is hypothesized to lie within the ability of parasitoids to manipulate their host into increasing lipid reserves. Redundancy of larval lipogenesis is expected, since costly conversion of nutrients into lipid reserves can be avoided and is thought to have led to a similar phenotype in the adult life- stage. This chapter further outlines prospects for future research that are for the largest part dealt with in the remainder of this thesis. The link between parasitism and the evolutionary loss of lipogenesis is central in this thesis. In chapter 3 I test the hypothesis of correlated evolution between these two traits through a comparative analysis using 94 insect

12 General introduction species. Indeed, the evolution of the parasitic lifestyle in insects is concurrent with that of lack of lipogenesis and evolved in parallel in parasitoids of 3 different insect orders. Phylogenetic analyses further reveals that numerous parasitoids re-evolved lipogenesis and shows that these species typically adopt a large host range, i.e. generalists. This study further introduces a new concept in evolutionary biology pertaining to the loss of indispensable traits through environmental compensation. The re-evolution of lipogenesis in generalist parasitoids is directly linked to the prediction that extensive host manipulation by parasitoids underlies the loss of this trait. There are, however, some exceptions to this rule, in which generalist parasitoids have lost lipogenesis similar to their specialist relatives. In chapter 4 I examine host manipulative abilities of the parasitoid Pachycrepoideus vindemmiae in relation to its ability to synthesize lipids. Results show that this generalist lacking lipogenesis further deviates from expectations in lacking the ability to manipulate its host. Hence, in some species factors other than manipulation might play an important role in the loss of this trait. The parasitoid lifestyle has led to the evolution of many other lifestyles, one of which is phytophagy. Quite similar to parasitoids, these gall wasps are able to manipulate their host plant in a manner that substantially increases resource availability during development. In chapter 5 I explore the efficiency with which gall wasps, inquiline and associated parasitoids within this community are able to carry over nutrients from their host and how these species further modify these nutrients, in particular their fatty acid composition, to meet their metabolic requirements. I show that both gall wasp and the majority of associated insects are highly efficient in carrying over nutrients from their host and that whilst some of these species readily incorporate the composition of fatty acids from their host, others further modify their fatty acid composition. The lack of lipogenesis thus pertains only to synthesis of fatty acids and accumulation of lipid reserves and not to the ability to elongate and desaturate fatty acids. A major question regarding trait loss revolves around the molecular mechanisms that underlie the loss of traits. In chapter 6 gene transcription patterns of 28 key nutrient metabolic genes are studied in response to sugar-feeding in the parasitoid Nasonia vitripennis and compared to the transcriptional profile of D. melanogaster, a species that actively synthesizes lipids. This study shows that the transcriptional profile of N. vitripennis deviates severely from that D. melanogaster. The critical gene involved in the biosynthesis of fatty acids, fatty acid synthase, lacks a transcriptional

13 Chapter 1 response. This gene would normally be transcribed at a high rate in response to dietary sugars in order to facilitate fatty acid synthesis, therefore lack of transcription of this gene explains the lack of lipogenesis in parasitic insects at the mechanistic level. Nutrient acquisition is particularly important in organisms that are metabolically compromised, such as parasitoids. Chapter 7 tests whether parasitoids respond to variation in sugar intake in a similar manner to that observed in other organisms, in which a reduced caloric intake should lead to an extension of longevity without compromising reproductive output. Although it was expected that parasitoids would benefit from a high caloric intake, due to their inability to accumulate lipids as adults, this study shows that there is an optimal level of caloric intake that maximizes longevity. Caloric values beyond the optimum decrease longevity, hence sugar sources with a high caloric value are detrimental to survival. In agro-ecosystems sugar sources are typically highly limited or absent. In chapter 8 I test whether the inclusion of lipids in the diet of adult parasitoids leads to an increase in lipid reserves and an increase in survival. This study shows that lipid reserves are higher or maintained at a high levels for a longer duration through provision of a lipid source in the diet, yet survival is detrimentally affected, conforming to our findings in chapter 7. Dietary composition and caloric value are clearly important in determining success of such an approach and it is suggested that further optimization of a lipid-rich diet could potentially benefit parasitoid reproduction and survival that would be useful in particular for parasitoids that are employed as natural enemies in agro-ecosystems. At the completion of this thesis, several major questions regarding the lack of lipogenesis in parasitic insect have been answered, yet other aspects have remained unexplored or unresolved. Chapter 9 provides a synthesis of this thesis and particularly focuses on new hypotheses and research questions resulting from this thesis that warrant further study.

14 Chapter 2

Lack of lipogenesis in parasitoids: A review of physiological mechanisms and evolutionary implications

Bertanne Visser and Jacintha Ellers Journal of Insect Physiology 54, 2008, 1315-1322

Abstract The ability of organisms to adapt to fluctuating food conditions is essential for their survival and reproduction. Accumulating energy reserves, such as lipids, in anticipation of harsh conditions, will reduce negative effects of a low food supply. For Hymenoptera and Diptera, several parasitoid species lack adult lipogenesis, and are unable to store excess energy in the form of lipid reserves. The aim of this review is to provide a synthesis of current knowledge regarding the inability to accumulate lipids in parasitoids, leading to new insights and prospects for further research. We will emphasize physiological mechanisms underlying lack of lipogenesis, the evolution of this adaptation in parasitoids and its biological implications with regard to life history traits. We suggest the occurrence of lack of lipogenesis in parasitoids to be dependent on the extent of host exploitation through metabolic manipulation. Currently available data shows lack of lipogenesis to have evolved independently at least twice, in parasitic Hymenoptera and Diptera. The underlying genetic mechanism, however, remains to be solved. Furthermore, due to the inability to replenish adult fat reserves, parasitoids are severely constrained in resource allocation strategies, in particular the trade-off between survival and reproduction.

15 Chapter 2

Introduction

To survive periods of food scarcity, organisms have developed strategies to anticipate these unfavourable environmental conditions. By accumulating energy reserves during periods of food abundance they are able to meet their energetic requirements for survival and reproduction when food is scarce or not available. Energy reserves can be stored in the form of carbohydrates, for example glycogen and other sugars, but those only provide a short-term energy supply as the storage capacity for those compounds is limited. A large, long-term, energy source is provided by the lipid reserves, which usually can contain enough energy to last several days to weeks without food. Accumulation of lipid reserves is stimulated by a diet rich in sugars and other carbohydrates (Zinke et al., 2002) or by the expectation of poor nutritional conditions, such as during diapause. During diapause the uptake of additional nutrients is usually restricted or absent and lipids have been found to increase considerably prior to diapause, for instance in the Mediterranean tiger moth, Cymbalophora pudica and the cabbage armyworm, Mamestra brassicae (Ding, 2003; Kostal et al., 1998), as well as other insects (Hahn & Denlinger, 2007). The storage of energy reserves in periods of food abundance is typically among the most conserved metabolic responses throughout the animal kingdom. One obvious exception to this rule are species that are not capable of feeding, for instance when adults posses non-functional or no mouthparts. These species do not consume or accumulate any external resource as adults, but instead have to rely on nutrients and energy reserves obtained during the juvenile stages of life. Adult starvation has been shown to accelerate depletion of larval reserves and reduce longevity, fecundity and dispersal in several insect species (Eijs et al., 1998; Gianoli et al., 2007; Shirai, 2006). As a consequence, adult lifespan of non-feeding insects is usually extremely limited and this lifestyle should be favoured only when no ﬁtness beneﬁts can be gained by an increase in nutritional resources through feeding. Remarkably, several parasitoid species have also been shown to lack pos- sibilities to accumulate adult energy supplies (Table 2.1). Although these species are capable of feeding and can utilize dietary nutrients to meet short-term energy demands, they appear to be unable to convert excess carbohydrates to long-term storage in the form of lipids (Casas et al., 2003; Ellers, 1996; Fadamiro et al., 2005; Lee et al., 2004a; Olson et al., 2000; Rivero & West, 2002). Feeding on sugar-rich substrates, such as honey or nectar, does not result in an increase in adult lipid reserves in these species.

16 Physiological mechanisms and evolutionary implications

The physiological mechanisms underlying this inability to store excess energy in parasitoids, as well as its evolution, have not yet been resolved. The diversity of life histories represented within this group of parasitoids makes it hard to see the adaptive value of this exceptional physiology. Parasitoids, however, are unusual because of their parasitic larval lifestyle, during which the developing larvae can manipulate host metabolism to increase its dietary quality. We propose that the intimate host-parasitoid interaction is one of the physiological processes driving the inability of adult parasitoids to accumulate lipids. In this review, we will evaluate empirical evidence on the presence and lack of lipogenesis in species within the class Insecta, and discuss novel hypotheses regarding its evolution. The underlying physiological and genetic mechanisms involved in insect metabolism are reviewed, and we evaluate which pathways are potentially relevant to lack of lipogenesis in parasitoids. The physiological interactions between host and developing parasitoid are examined, including their ability to manipulate host resource availability. We, furthermore, set forth the possible implications of lack of lipogenesis for important life history traits.

Lack of lipogenesis in adult parasitoids

Only a decade ago, it was ﬁrst noticed that a parasitoid species did not conform to the general metabolic model. In a study by Ellers (1996), teneral lipid levels of adult parasitoids were compared with lipid levels present after feeding on a sugar-rich diet (Figure 2.1). The lipid content of female Asobara tabida was highest at emergence and declined monotonically with age, despite continuous ad libitum feeding. In starved females, lipid reserves declined even more quickly with age, indicating that access to carbohydrates slowed down the rate of lipid depletion. Hence, lack of lipid accumulation was not due to an inability to consume or digest the food, but to the inability to convert excess carbohydrates to long-term lipid storage. Several other parasitoid species have since also been shown to lack lipogenesis, including Leptopilina heterotoma (Eijs et al., 1998), Macrocentrus grandii (Olson et al., 2000), Nasonia vitripennis (Rivero & West, 2002), Eupelmus vuilletti (Giron & Casas, 2003), Venturia canescens (Casas et al., 2003), and Diadegma insulare (Lee et al., 2004a). These parasitoid species come from a variety of superfamilies within the parasitic Hymenoptera, which demonstrates the widespread occurrence of lack of lipogenesis among adult parasitoid species.

17 Chapter 2

Table 2.1: Presence and lack of lipogenesis within the class Insecta.

Family Species Lipogenesis Parasitic References Y/N Y/N Order Odonata Calopterygidae Calopteryx atrata Y N Matsubara et al. (2005) Calopterygidae Calopteryx maculata Y N Marden & Waage (1990) Order Hemiptera Cicadidae Magicicada septendecim Y N Hoback et al. (1999) Order Orthoptera Acrididae Schistocerca gregaria Y N Chegwidden and Spencer (1996) Acrididae Schistocerca americana Y N Hahn (2005) Gryllidae Gryllus bimaculatus Y N Lorenz (2001) Gryllidae Gryllus ﬁrmus Y N Zhao & Zera (2001) Order Neuroptera Nemopteridae Lertha sheppardi Y N Cakmak et al. (2007b) Order Coleoptera Curculionidae Smicronyx fulvus Y N Vick & Charlet (1996) Curculionidae Smicronyx sordidus Y N Vick & Charlet (1996) Tenebrionidae Tenebrio molitor Y N Khebbeb et al. (1997) Gyrinidae Gyrinus opacus Y N Svensson (2005) Order Diptera Culicidae Aedes aegypti Y N Ziegler & Ibrahim (2001) Culicidae Aedes sollicitans Y N van Handel (1965) Culicidae Aedes cantans Y N Renshaw et al. (1995) Culicidae Aedes punctor Y N Renshaw et al. (1995) Culicidae Culex tarsalis Y N Gray & Bradley (2003) Cuterebridae Cuterebra austeni N Y Kemp & Alcock (2003) Scathophagidae Scathophaga stercoraria Y N Otronen (1995) Drosophilidae Drosophila melanogaster Y N Geer et al. (1985) Tephritidae Anastrepha serpentina Y N Jacome et al. (1995) Tephritidae Ceratitis capitata Y N Warburg & Yuval (1996) Phoridae Pseudacteon tricuspis N Y Fadamiro et al. (2005) Order Lepidoptera Saturniidae Hyalophora cecropia Y N Chino & Gilbert (1965) Bombycidae Bombyx mori Y N Horie et al. (1968) Noctuidae Spodoptera exempta Y N Lee et al. (2004b) Sphingidae Manduca sexta Y N Fernando-Warnakulasuriya et al., (1988) Nymphalidae Aglais urticae Y N Pullin (1987) Nymphalidae Inachis io Y N Pullin (1987) Nymphalidae Danaus plexippus Y N Brown and Chippendale (1974), Gibo & McCurdy (1993) Order Hymenoptera Ichneumonidae Venturia canescens N Y Casas et al. (2003) Ichneumonidae Pimpla turionellae N Y Ortel (1991) Ichneumonidae Diadegma insulare N Y Lee et al. (2004a) Braconidae Asobara tabida N Y Ellers (1996) Braconidae Asobara rufescens N Y Ellers, unpublished Braconidae Macrocentrus grandii N Y Olson et al. (2000) Figitidae Leptopilina heterotoma N Y Eijs et al. (1998) Pteromalidae Nasonia vitripennis N Y Rivero & West (2002) Eupelmidae Eupelmus vuilletti N Y Giron & Casas (2003) Pompilidae Hemipepsis ustulata Y N Kemp & Alcock (2003) Sphecidae Philanthus triangulum Y N Strohm (2000) Vespidae Vespula vulgaris Y N Harris & Beggs (1995) Vespidae Mischocyttarus Y N Markiewicz and mastigophorus O’Donnell (2001) Formicidae Leptothorax albipennis Y N Blanchard et al. (2000) Formicidae Camponotus festinatus Y N Rosell & Wheeler (1995) Apidae Bombus terrestris Y N Pereboom (2001) 18 Physiological mechanisms and evolutionary implications

0.05 no food 0.04 food

0.03

0.02

fat fat reserves (mg) 0.01

0.00 0 5 10 15 20 25 30 35 40 45 50 age (days)

Figure 2.1: Decline in lipid reserves (in mg) throughout adult life (in days) in honey fed and starved females of Asobara tabida. Graph reproduced with permission of BRILL.

Theoretically, the observed lack of lipid accumulation could be due to an enhanced lipid breakdown, which would remain unnoticed using the experimental design of Ellers (1996). Using radioactively labelled glucose, Giron & Casas (2003) were able to track the fate of incoming nutrients obtained through feeding in the parasitic wasp Eupelmus vuilletti, and test if the radioactive signal remained conﬁned to carbohydrates and proteins or was transformed to lipids. Their approach demonstrated a rapid increase in body sugar and body glycogen levels in glucose-fed females. This is consistent with the rise in the level of radioactivity in the extraction phase containing total sugars, and showed that the glucose was indeed digested. More importantly, however, lipid levels never exceeded teneral levels, and the amount of radioactivity measured in the hydrophobic phase remained very low. These trace experiments conﬁrm the results obtained with feeding experiments: no evidence of de novo lipid synthesis has been found in parasitoids under food conditions that would normally induce lipogenesis in other insects (Zinke et al., 2002). To identify a possible proximate cause of this atypical metabolic behaviour we need to consider more closely the variety of physiological pathways involved in lipogenesis. The primary synthetic pathway in lipogenesis is fatty acid synthesis, which converts the glucose derivative pyruvate to the long-chain palmitic acid (Garrett & Grisham, 1999). The synthesis of palmitic acid is catalysed by fatty acid synthase (FAS), a 250–270 kD multifunctional polypeptide. A central intermediate in fatty acid synthesis is acetyl coenzyme A (CoA), which is partly carboxylated to malonyl-CoA by

19 Chapter 2 the rate-limiting enzyme acetyl-CoA carboxylase. FAS performs the condensation of acetyl-CoA and malonyl-CoA, using NADPH as a reducing equivalent to produce the 16-carbon saturated fatty acid palmitate. The rate of fatty acid synthesis is highly dependent on nutritional conditions. High levels of dietary polyunsaturated fatty acids decrease lipogenesis by suppressing gene expression of fatty acid synthase (Jump et al., 1994). Con- versely, a diet rich in carbohydrate stimulates lipogenesis because glucose can be glycolytically converted to acetyl-CoA, which promotes fatty acid synthesis. In Drosophila larvae, ingestion of glucose will induce the expression of a number of genes involved in lipogenesis, such as acetyl-CoA carboxylase, acetyl-CoA citrate lyase, and Zwischenfernment (Zinke et al., 2002). The cascade of genes involved in lipogenesis makes it difficult to infer which genes are causing lack of lipogenesis in parasitoids without further molecular genetic experiments. It is clear that FAS has a central role in fatty acid synthesis, which makes it a prime candidate to explain lack of lipogenesis. A mutation in a key position of the fas gene, or in one of the other genes encoding enzymes involved in fatty acid synthesis could render it ineffective in synthesizing fatty acids and disable the entire lipid synthesis. Even though it may seem unlikely that a key enzyme like FAS would become dysfunctional or dis- appear from the genome, recently an example of such evolutionary change was documented in Malassezia globosa, a fungus associated with the skin diseases dandruff and seborrheic dermatitis in humans (Xu et al., 2007). Whole-genome analysis showed the absence of a gene encoding fatty acid synthase, explaining M. globosa’s dependence on external lipids for growth. No studies have been undertaken on fed adult parasitoids to compare gene expression profiles to other insects that are capable of lipogenesis nor is it known if fatty acid synthesis genes are lacking. Recent completion of the genome of the parasitoid Nasonia vitripennis (Nasonia Genome Project) has revealed a homolog to the fatty acid synthase gene of Drosophila, however, its functionality remains to be tested. Further genome analysis should enable the identification of evolutionary changes causing lack of lipogenesis, taking into account structural or regulatory changes in FAS and the other enzymes involved in lipogenesis. The second part of the lipogenesis pathway is modification of saturated long-chain fatty acids to form more complex fatty acids, which are used for the synthesis of various cellular lipids such as phospholipids, triacylglycerols, and cholesterol esters or as precursors in the synthesis of pheromones, waxes, and eicosanoids (Stanley, 2006). Several of the latter compounds are

20 Physiological mechanisms and evolutionary implications essential to organisms and it is highly unlikely that production of these fatty acid derivatives is affected by lack of lipogenesis. The same holds true for the production and modification of phospholipids, which are an essential part of cell membranes for all organisms. Elongation and unsaturation of phospholipids are part of the homeoviscous adaptation of membranes to changing temperature (Hazel, 1995). Observations indicate that these functions are not altered in parasitoids, which suggests that elongases and desaturases are functioning normally. The final step in the lipogenesis pathway is the storage of fatty acids in the insect fat body in the form of triacylglycerols, which consist of three fatty acids and one glycerol molecule. In Drosophila, fat storage is regulated by a number of antagonistic enzymes including lipid storage droplet protein LSD2 (Teixeira et al., 2003), and the lipase Brummer BMM (Grönke et al., 2005). Because the mechanisms guiding fat storage are conserved across taxa, parasitoid homologues are expected, but have not yet been identified. Further scrutiny of the Nasonia genome could offer useful insight into such homologues in parasitoids. In summary, the evidence suggests that adult parasitoids do not synthesize fatty acids de novo. Analogous to other taxa, this inability could possibly be due to a mutation of a key enzyme in the fatty acid synthesis pathway, but this possibility remains to be examined further. The implications of such an evolutionary scenario are far-reaching; for instance, it would implicate that parasitoids are completely dependent on larval lipid resources as adults. It, furthermore, raises the question as to how larvae accumulate these lipid resources, if key enzymes are rendered dysfunctional. Is endogenous lipogenesis lacking in larvae as well, so that they are entirely dependent on exogenous lipogenesis? An important constraint is the con- finement of larvae to a single host. It may, therefore, be crucial to take into account host-parasitoid interactions with regard to nutrient dynamics of both host and parasitoid to understand how adequate lipid levels can be obtained during larval life.

Larval lipid-accumulation strategies

Parasitoids have evolved a complex array of nutritional, physiological, and behavioral interactions with their host (Pennacchio & Strand, 2006). Be- cause the growth and survival of parasitoid larvae is largely dependent on host quality, parasitoids have to ensure a suitable environment for their developing oﬀspring by manipulating their host’s development. For instance, koinobionts, allowing host growth and development, can alter host hormone

21 Chapter 2 titers to influence the feeding habit and growth of their host (Alleyne & Beckage, 1997; Schafellner et al., 2007). Similarly, idiobionts, which arrest host development, are capable of increasing the duration of paralysis prior to death, allowing for a longer time period and optimal use of host resources (Rivers & Denlinger, 1995). Parasitoids are also capable of manipulating the physiology of their hosts to increase the nutritional composition, which will be our focus in the next section to answer the question of larval lipid accumulation. Parasitism has been found to induce changes in the amount of amino acids, proteins, pyruvate and carbohydrates within the host in both endo and ectoparasitoids (Bischof & Ortel, 1996; Caccia et al., 2005; Consoli & Vinson, 2004; Coudron et al., 1997; Nakamatsu et al., 2001; Rivers & Denlinger, 1994). The effects of parasitization on host lipids include an increase in whole body lipid content (Rivers & Denlinger, 1994, 1995), an enhanced metabolisation of fat body triacylglycerols (Nakamatsu, 2003; Nakamatsu & Tanaka, 2004), and a higher level of free fatty acids in the hemolymph. The effects of host manipulation depend on the stage of the developing parasitoid larva, because usually in the early stages the larva mainly consumes host hemolymph, while later the larva feeds directly on the host’s fat body (Nakamatsu et al., 2002; Salvador & Consoli, 2008). These changes in the nutritional content of the host are brought about by a variety of mechanisms such as teratocytes, venom, and associated mutualistic viruses. Teratocytes are cells derived from the dissociation of the embryonic membrane of parasitoid species of the families Braconidae, Scelionidae, and Platygastridae (Dahlman, 1991), and of the Chalcidoidea (Pedata et al., 2003). Cells resembling teratocytes have also been found in the family Ichneumonidae (Rouleux-Bonnin et al., 1999). Teratocytes play an important role in nutritional exploitation by parasitoid larvae because they attach themselves to the host’s fat body and contribute to its disruption (Suzuki & Tanaka, 2007). Several proteins have been identified, including a teratocyte-specific carboxylesterase, assumed to be involved in the hydrolysis of host lipids (Gopalapillai et al., 2005), a fatty acid binding protein putatively involved in transport of host fatty acids to the developing parasitoid larva (Falabella et al., 2005), and two collagenases which may attack the collagen sheath surrounding the fat body to permit selective release of fat body cells (Qin et al., 2000). In addition to the effects of teratocytes, the parasitoid larva itself is also capable of bringing about physiological changes, for instance in hormone and lipid levels, aiding its own development (Beckage & Gelman, 2004; Rivers & Denlinger, 1995).

22 Physiological mechanisms and evolutionary implications

Maternal substances, which are transferred along with the egg during oviposition, provide an additional way of host exploitation. These include diﬀerent types of viruses such as polyDNAviruses, non-polyDNAviruses, and virus-like-particles, which are found most frequently in ichneumonoid and braconid wasps (Lawrence, 2005), as well as venom. Parasitoid venom contains various proteins that may disrupt the host’s fat body, such as matrix metalloproteinase present in venom of Euplectrus separatae, causing lysis of cells and release of lipid particles from the fat body (Nakamatsu & Tanaka, 2004). Also, envenomation leads to increased lipid levels in the host’s fat body and hemolymph (Rivers & Denlinger, 1995).

The multitude of mechanisms employed to manipulate host metabolism ensures an abundance of lipid resources during development, providing parasitoid larvae with a unique opportunity to consume host lipids instead of synthesizing them de novo. Manipulation and consumption of host lipids probably provides a selective advantage for parasitoid larvae, because de novo lipid synthesis is energetically expensive. As a consequence, larval lipogenesis is hypothesized not to occur in parasitoids, and, therefore, to be evolutionary redundant. The hypothesis of direct assimilation of host lipids is supported by the fact that several parasitoid species have similar lipid compositions as their host, although this is not true for all species (Thomp- son & Barlow, 1974). Obviously, elongation and unsaturation of host lipids can alter lipid composition after uptake by the parasitoid. Experiments using radioactively marked glucose could be used to look into larval capa- bilities of lipogenesis, similar to the study done by Giron & Casas (2003).

Does this mean that parasitoid larvae lack the ability to synthesize lipids de novo? Little conclusive evidence exists, and the few available studies are based on experiments with artificial diets. Thompson (1979) found that larvae of Exeristes roborator developed into adulthood on a fat-free artificial diet, suggesting larvae are to some extent capable of de novo synthesis of lipids. In contrast, larvae of Itoplectis conquisitor did not complete development successfully on a fat-free artificial diet (Yazgan, 1972). So far, no species have been tested at both the larval and adult life stage. It seems unlikely, however, that uncoupling of this trait occurs during metamorphosis, and we would thus expect a lack in larval lipogenesis to be concurrent with that in adults. This would suggest not all parasitoids are incapable of de novo synthesis of lipids, since larval lipogenesis has been shown to occur in E. roborator (Thompson, 1979).

23 Chapter 2

The evolutionary loss of lipogenesis in parasitoids

How can an essential metabolic trait such as lipogenesis have been lost during the course of evolution? Evolutionary theory predicts that a trait can only be lost if there is a selective advantage for individuals without the trait, for example because maintaining the trait bears an energetic cost. Alter- natively, traits may be lost if the trait is no longer under natural selection and accumulated mutations do not affect fitness. These two mechanisms may not necessarily exclude one another, however, in that an energetic cost could accelerate the loss of an unnecessary trait. The most well-known example of evolutionary loss of function is the loss of ascorbic acid (vitamin C) synthesis in humans, higher primates, guinea pigs, and fruit bats (Chat- terjee, 1973; Ohta & Nishikimi, 1999). The inability to synthesize ascorbic acid is due to inactivation of L-gulono-γ-lactone oxidase (GLO), the enzyme that catalyzes the terminal step of L-ascorbic acid synthesis. The mutation leading to the loss of such an essential gene was, however, neutral and not lethal because these animals have a high dietary intake of vitamin C. Without further selective pressure the human non-functional GLO gene has accumulated a large number of mutations since it ceased to function approximately 40–50 million years ago (Nishikimi et al., 1994). The inability to synthesize ascorbic acid was lost a second time independently in guinea pigs 20–25 million years ago (Nishikimi et al., 1992), which demonstrates the relative ease with which this trait is lost. Similar to vitamin C production, lack of lipogenesis may have evolved in an environment rich in lipids, which would have made lipogenesis evolutionary redundant. The opportunity for parasites and parasitoids to exploit and manipulate their host’s resources provides them with a lipid-rich environment and could have made lipogenesis a selectively neutral trait prone to mutation accumulation. This hypothesis provides us with a strong prediction as to which species can and cannot synthesis lipids de novo. We only expect species that feed on lipid rich resources and have a continuous availability of lipid supplies to have lost the ability to synthesize lipids during the course of evolution. To our knowledge, these conditions are only met by parasitic species that are able to manipulate their host’s resources to a sufficient extent. All other species, which consume resources poor in lipids, or are prone to experience periods of lipid scarcity during life should rely on synthesis of lipids to prevent starvation. The hypothesis of exploitation of host lipid reserves has empirical support in parasitic bacteria (Fraser et al., 1995; Mushegian & Koonin, 1996), parasitic fungi (Katinka et al., 2001; Xu

24 Physiological mechanisms and evolutionary implications et al., 2007), and parasitic nematodes (Kohler & Voight, 1988). The parasitic lifestyle of these species ensures an abundance of lipid resources from the host, which has made de novo lipogenesis unnecessary. In all three taxa, this has resulted in the lack of genes encoding fatty acid synthase, uptake of host-derived fatty acids, and a limitation to condensation of exogenous fatty acids with glycerol to produce triacylglycerols. The parasitic fungus M. globosa even possesses a unique set of genes encoding lipases and other hydrolases, which enhance the breakdown of host skin lipids (Xu et al., 2007). In insects, the hypothesis could explain why only parasitoid species seem to forego the ability to accumulate lipid reserves as adults. To substantiate our hypothesis we have looked at the phylogenetic distribution of the cases of lack of lipogenesis that have been reported so far. An extensive literature study across the class Insecta has shown a total of 35 species are able to produce lipids and 11 species showing lack of lipogenesis (Table 2.1). All insects that so far have been proven to be capable of synthesizing lipids as adults are non-parasitic and distributed over all the major superfamilies within the insects. In contrast, the species with lack of lipogenesis are confined to the dipteran and hymenopteran parasitoids. Currently available data on the distribution of lack of lipogenesis provide strong support for the release of selection hypothesis, because lack of lipogenesis is only found in parasitoids. Obviously, a full test of the hypothesis would require testing more parasitoid species, preferably those that are phylogenetically independent. Within the insects, parasitoids occur in a number of taxa including Hymenoptera, Diptera, Coleoptera, and even Neuroptera, allowing identification of at least four evolutionary independent transitions. Moreover, an important suborder for testing lack of lipogenesis are the sawflies or Sym- phyta, which comprises the most basal lineage within the Hymenoptera, in which parasitism is thought to have originated. Two additional predictions can be derived from our hypothesis. First, because of the independence of the evolutionary events leading to lack of lipogenesis in the different taxa, it is not expected that the same accumulated mutations are causing lack of lipogenesis. Second, mutation accumulation is a runaway process that is not easily reversed (Siddall, 1993). A reversion to a fully functional lipid synthesis requires retracing of numerous evolutionary events and is thought to be highly unlikely. Therefore, we expect to find lack of lipogenesis to be evolutionary conserved within genera or families, so that even in those species that use plant hosts as a secondary adaptation, such as gall wasps and fig wasps, may lack lipogenesis.

25 Chapter 2

One argument against the evolutionary redundancy hypothesis is the complex life cycle of parasitoids, which are parasitic during larval development but free-living as adults. Lipogenesis may have become redundant in the larval stages but adult parasitoids may experience severe disadvantages of lack of lipogenesis, such as a reduced lifespan and fecundity. Evolutionary loss of lipogenesis could have occurred only in short-lived species that do not beneﬁt from feeding, for example strongly pro-ovigenic species, in which eggs have matured prior to eclosion. Our hypothesis also does not exclude the possibility that some species may have evolved alternative pathways or behaviours that do allow them to counter the disadvantages of lack of lipogenesis by obtaining some extra lipids as adults, especially in long-lived species.

Ecological implications of lacking lipogenesis

The inability of adult parasitoids to synthesize fat limits the amount of lipids available during adult life. Adult parasitoids cannot obtain a sufficient amount of lipids through dietary intake, since their diet consists mainly out of nectar, honeydew, and occasionally hemolymph of their host, all of which contain carbohydrates and proteins, but hardly any lipids (Eijs et al., 1998; Giron et al., 2002; Giron & Casas, 2003). One class of lipids, the sterols, present in small quantities in host hemolymph, have been found to be of importance for egg viability in the parasitoid Eupelmus vuilletti (Mondy et al., 2006). These lipids, however, cannot be used as a long- term energy source comparable to triacylglycerols. Teneral lipid reserves, therefore, affect many interrelated life history traits, such as survival and fecundity. It has been shown that fat reserves are positively correlated to body size, longevity, and fecundity in parasitoids (Colinet et al., 2007; Eijs & Van Alphen, 1999; Ellers et al., 1998). It can, therefore, be expected that there are strong selection pressures optimizing the size of the teneral lipid reserves, which, in turn, shape developmental traits such as host use efficiency, development time, and body size. In species entering diapause, for instance, an increase in diapause length has been shown to decrease the amount of teneral lipid reserves (Ellers & Van Alphen, 2002). In species lacking lipogenesis, however, survival and reproductive success are determined by both length of the season and the amount of lipids obtained prior to pupation. Similarly, when different host species can be parasitized, host choice could have severe effects on fitness, since different host species might also differ in their nutritional value to the parasitoid, for instance when the

26 Physiological mechanisms and evolutionary implications parasitoid is more successful in manipulating host physiology in one host species compared to another, as has been shown for Nasonia vitripennis (Rivers & Denlinger, 1995). A major challenge for parasitoids is to optimally allocate their limited resources either to eggs or survival. Resource allocation is dependent on the timing of egg maturation, either before eclosion (proovigeny) or after eclosion (synovigeny), on oosorption ability and on host availability (Jervis et al., 2008; Olson et al., 2000). In general, parasitoids appear to be time limited rather than egg limited (Ellers et al., 1998, 2000b; Rosenheim, 1996; Sevenster et al., 1998), so that lifetime reproductive success is limited by fat reserves, rather than egg load in the majority of wasp species (Ellers et al., 1998). In species lacking oosorption, allocation of lipids into reproduction will increase the number of eggs and results in decreased plasticity (Ellers & Van Alphen, 1997). The inability of parasitoids to accumulate fat reserves as adults implies they are severely constrained in resource allocation strategies, in particular the trade-off between survival and reproduction. According to Boggs’ model (1981) on nutrient dynamics and resource allocation in insects, the amount of nutrients that can be obtained during the adult life stages determines allocation to reserves at hatching. In addition, biochemical research has shown that insects maintain separate nutrient pools for essential and non-essential nutrients, which fuel different functions, and, therefore, not only the amount of nutrients is of importance, but also which specific nutrients can be obtained (O’Brien et al., 2000). If we would apply this model to lipogenesis in parasitoids, it would be expected long-lived species to be capable of lipogenesis and to eclose with relatively few lipids, opposed to short-lived species which have accumulated a sufficient amount of nutrients to sustain throughout the adult life stage.

Conclusions

We have provided an overview of current knowledge on lack of lipogenesis and its eﬀect on several aspects of parasitoid biology. Parasitoids are prone to lose the capability of lipid synthesis because of their ability to exploit host nutritional resources, but it is unsure if all parasitoids lack lipogenesis and what the exact physiology is. Future research should follow several leads, including the identiﬁcation of additional parasitoid species within major superfamilies of Hymenoptera, Diptera, and Coleoptera with lack of lipogenesis, which will allow us to determine how many times lack of lipogenesis has evolved independently within these groups and if it is present in

27 Chapter 2 all parasitoid lifestyles. Furthermore, the underlying physiological and genetic pathways that are involved in lack of lipogenesis in parasitoids should be identiﬁed, both in larva and adult parasitoids.

Acknowledgements

We are grateful to Jacques van Alphen and Gerard Driessen for stimulating discussions and to Nico van Straalen and two anonymous referees for helpful comments on earlier drafts of the manuscript. BV was supported by the Netherlands Organisation for Scientiﬁc Research (NWO), ALW-grant no. 816.01.013. JE was supported by NWO, VIDI-grant no. 864.03.003.

28 Chapter 3

Loss of lipid synthesis as an evolutionary consequence of a parasitic lifestyle

Bertanne Visser, Cécile Le Lann, Frank J. den Blanken, Jeﬀrey A. Harvey, Jacques J.M. van Alphen and Jacintha Ellers Proceedings of the National Academy of Sciences of the United States of America 107, 2010, 8677-8682

Abstract Evolutionary loss of traits can result from negative selection on a specific phenotype, or if the trait is selectively neutral, because the phenotype associated with the trait has become redundant. Even essential traits may be lost, however, if the resulting phenotypic deficiencies can be compensated for by the environment or a symbiotic partner. Here, we demonstrated that loss of an essential metabolic trait in parasitic wasps has evolved through environmental compensation. We tested 24 species for the ability to synthesize lipids de novo and collected additional data from the literature. We found the majority of adult parasitoid species to be incapable of synthesizing lipids and phylogenetic analyses showed that the evolution of lack of lipogenesis is concurrent with that of parasitism in insects. Exploitive host manipulation, in which the host is forced to synthesize lipids to the benefit of the parasitoid, presumably facilitates loss of lipogenesis through environmental compensation. Lipogenesis re-evolved in a small number of parasitoid species, particularly host generalists. The wide range of host species in which generalists are able to develop may impede effective host manipulation and could have resulted in regaining of lipogenic ability in generalist parasitoids. As trait loss through environmental compensation is unnoticed at the phenotypic level, it may be more common than currently anticipated, especially in species involved in intricate symbiotic relationships with other species.

29 Chapter 3

Introduction

Evolutionary changes are frequently associated with the acquisition of novel traits, but loss of traits can also curb the course of evolution (Fong et al., 1995; Porter & Crandall, 2003). Trait loss can be the result of negative selection for a specific phenotype, for example to reduce costs associated with the trait (Lahti et al., 2009). Alternatively, a trait may become selectively neutral due to ecological or evolutionary shifts which render the phenotype associated with the trait redundant. Well-known examples are the loss of eyes in cave-dwelling organisms (Poulson & White, 1969) and the loss of wing function in birds and insects (Roff, 1994). In these examples, respectively vision and ability for flight have become redundant, resulting in mutation accumulation and loss of function. The evolutionary loss of a trait, however, is not inevitably accompanied by a loss of the phenotype associated with that trait. If the loss of function is compensated by environmental or biotic factors the phenotype will be maintained. For instance, several plant species have lost the ability for photosynthesis (Krause, 2008) although the photosynthetic requirements are met through exploitation of other plants. Similarly, despite the loss of vitamin C production in humans, vitamin C is still an essential vitamin for human health but it is provided through dietary intake (Chatterjee, 1973; Ohta & Nishikimi, 1999). Environmental compensation can therefore release traits from selection because the phenotype is not affected by loss of such traits. Moreover, if a trait is energetically costly environmental compensation can even result in selection against expression of this trait, because reduced expression will save energetic expenditure. Environmental compensation of trait loss is frequently observed in species that are involved in symbiotic, co-evolutionary relationships, such as occurs in instances of mutualism or parasitism. The supply of essential resources by a symbiotic partner makes the production of such resources superfluous in the receiving organism, and renders the genes involved prone to mutation accumulation. As a consequence, co-evolution may lead to loss of genes that is unnoticed at the phenotypic level. To illustrate this point, many obligate endosymbionts such as Buchnera and Baumannia have undergone massive genome reduction, although which genes are lost depends on their specific host (Dale & Moran, 2006; Wu et al., 2006). Other relevant examples include the parasitic fungi, which have lost several genes involved in metabolism (Xu et al., 2007). Trait loss or a reduction in trait functioning is only expected to evolve in long-term, stable co-evolutionary physiological

30 The evolutionary loss of lipogenesis relationships. However, the relative frequent occurrence of such tight inter- specific interactions implies that environmental compensation for trait loss may be much more prevalent than currently appreciated (Cairney, 2000; Houck & O’Connor, 1991). Here we unravel a case of parallel evolution of compensated trait loss in insect parasitoids. The exceptional lifestyle of parasitoids provides unique opportunities to study environmental compensation for trait loss (Godfray, 1994). Parasitoids develop in or on arthropod hosts during the larval stage; the larvae are therefore completely dependent on their host for nutrient acquisition (Jervis et al., 2008). Various ways have evolved in which parasitoid larvae can manipulate their host’s physiology to increase nutrient availability, including host exploitation for lipids (Rivers & Denlinger, 1994; Vinson & Iwantsch, 1980). For example, parasitism by the hymenopteran Euplec- trus separatae results in a release of fat particles from the host’s fat body and an increase in hemolymph free fatty acids of the host (Nakamatsu & Tanaka, 2004). Direct uptake of lipids from the host tissue is highly advan- tageous for parasitoid larvae because they can avoid substantial metabolic costs that are associated with lipogenesis (Garrett & Grisham, 1999). In addition, it has been shown that some parasitoid species are unable to accumulate lipids as adults due to the absence of de novo lipid synthesis (Giron & Casas, 2003; Visser & Ellers, 2008), even under conditions that would induce enhanced lipogenesis in other animals (Zinke et al., 2002). We propose that the loss of lipogenesis is an evolutionary consequence of the parasitoid lifestyle because parasitism facilitates redundancy of traits that are involved in lipid production. So far lack of adult lipid accumulation has been found in two parasitic dipterans and nine closely related parasitic hymenopterans (Visser & Ellers, 2008). To demonstrate phylogenetic congruence between loss of lipogenesis and a parasitoid lifestyle, data is needed from multiple independent phylogenetic groups. We used a two-pronged approach to obtain these data. First, we performed an ex- haustive survey of the literature and acquired data on lipogenic ability of 70 species. Second, we tested an additional 24 species for their lipogenic ability by conducting feeding experiments and physiological measurements. The final dataset included almost 30 parasitoid species from three different orders which enabled us to answer the question if the evolution of parasitism results in loss of lipogenic ability in insects. Furthermore, we tested for correlated evolution between lipogenic ability and key parasitoid traits associated with the parasitoid lifestyle.

31 Chapter 3

Materials and Methods

Insects

All insects were obtained from existing laboratory cultures or ﬁeld col- lections, either as larvae and pupae or inside their host. The following species were obtained from existing cultures: C. glomerata, P. puparum, A. nens, L. nana, and G. agilis (NIOO-KNAW, the Netherlands), C. rubecula (University of Wageningen, the Netherlands), L. boulardi (University of Leiden, the Netherlands). A. ervi and A.abdominalis (Koppert BV, the Netherlands), A. rosae (University of Bielefeld, Germany), P. vindemmiae (University of Lyon 1, France), A. bilineata, A. rhopalosiphi, A. picipes, L. heterotoma, A. tabida, S. erythromera and T. drosophilae (University of Rennes 1, France) and G. nephantidis and G. legneri (University of Not- tingham, United Kingdom). Galls containing D. rosae, O. mediator and P. brandtii were collected at several sites near Wassenaar (the Netherlands) in September and October 2007. Porcellio scaber, hosts of P. maculata were collected at several sites in the provinces of North and South Holland (the Netherlands) during January, February and March 2008. All insects were kept at 20◦C, RH 75% and L:D 12:12, except A. ervi, and P. puparum, which were kept at RH 50% and C. glomerata, G. agilis, L. nana, A. nens, L. boulardi, P. vindemmiae and C. rubecula, which were kept at 23◦C, RH 75% and L:D 12:12. Galls containing D. rosae, O. mediator and P. brandtii were kept subsequently at 20◦C, 10◦C, 5◦C, 10◦C and 20◦C until emergence to mimic temperature conditions in their natural environment. Vials and galls were inspected daily for newly emerged females.

Feeding experiments

To determine lipogenic ability, newly emerged females were randomly assigned to two treatments: emergence, in which females were frozen directly after emergence, or fed, in which individuals were allowed to feed on honey ad libitum for approximately half their average lifespan. During the experiments only adult females were used and provided with water on cotton wool in addition to honey. An exception is A. bilineata, for which sex could not be determined and both sexes were used in experiments. A. bilineata was, furthermore, allowed to feed on Drosophila food medium, as this species is predacious rather than nectivorous. Medium consisted of 20 g agar, 50 g saccharose, 35 g yeast, 9 g kalmus (10 parts acidum tartaricum, 4 parts ammonium sulphate, 3 parts potassium phosphate and 1 part magnesium

32 The evolutionary loss of lipogenesis sulphate) and 10 mL nipagin (100 g 4–methyl hydroxyl benzoate per liter ethanol) per liter water. At the end of the experiment females were frozen at –20◦C until further processing. Fat content was determined based on the method by David et al. (1975). Whole insects were freeze-dried for 2 days, after which dry weight was determined. Individuals were subsequently placed in a glass tube containing 4 mL of ether. After 24 hours, ether was removed and insects were washed with fresh ether. Insects were freeze-dried for 2 days after ether extraction and dry weight determined again. For A. bilineata, P. maculata, D. rosae, O. mediator, P. brandtii and A. rosae freeze-drying was extended to 4 days and the quantity of ether was 8mL because of their increased cuticle toughness and larger size. Fat content was calculated by subtracting dry weight after ether extraction from dry weight before ether extraction, and then converted to the percentage of lipids to correct for diﬀerences in body size among individuals.

Reconstruction of phylogenetic relationships

Relationships between insect orders within the Hemimetabola are based on morphological data according to Kristensen (1999) and the phylogeny of the endopterygotes was completed with molecular data on relationships between Holometabola, as reported in Castro and Dowton (2005). New insights suggest an alternative placement of Hymenoptera within the Holo- metabola, but vary considerably between studies (Savard et al., 2006; Tim- mermans et al., 2008; Wiegmann et al., 2009). The phylogeny used in this study has been constructed with regard to the more conventional placement of Hymenoptera as a sister group of Mecopterida (Diptera + Lepi- doptera). However, alternative placement of Hymenoptera does not affect our findings. Inference of phylogenetic relationships at the superfamily, family and genus level has been made using available morphological and molecular data (Text 3.1). The enormous diversity of hymenopteran parasitoids poses difficulties in determining phylogenetic relationships, mainly at the superfamily level. Consensus between phylogenies based on morphology and molecular data has not yet been reached satisfyingly, and relationships among hymenopteran superfamilies are thus regarded as polytomous in this study. Within Hymenoptera, data on lipogenic ability has been obtained for parasitoids of the superfamilies Proctotrupoidea, Cynipoidea and Chalcidoidea (Proctotrupomorpha), Ichneumonoidea and Chrysidoidea (Aculeata) (Buckner & Hagen, 2003; Cripps et al., 1986; Visser & Ellers,

33 Chapter 3

2008). As the analysis does not allow polytomies, three alternative consensus trees were used for phylogenetic testing within Hymenoptera. The three trees assumed Ichneumonoidea and Aculeata, Proctotrupomorpha and Ac- uleata, or Ichneumonoidea and Proctotrupomorpha, to be sister groups, respectively.

Statistical analysis

Scatterplots and regression lines of dry weight before ether extraction compared to dry weight after ether extraction were used to find potential outliers in the data. Outliers were only removed if they deviated from a 99% confidence interval of the linear regression line. Normality of the data was determined by looking at the error structure of the residuals of the data. Non-normal data was transformed to normality using a log or cube root transformation. Treatments showing normally distributed data were compared using independent samples T-test if variances were equal and Welch’s T-test if variances were unequal. If data was not normally distributed, the non-parametric Kruskal-Wallis test was used. Statistical analyses were done using R project version 2.9. Phylogenetic tests were performed with each of the three consensus trees using all 94 insect species as a target clade. Lipogenic ability and parasitic lifestyle were scored as presence/absence characters. To test for correlations between parasitic lifestyle (independent character) and lipogenic ability (dependent character), ancestral states were reconstructed using parsimony for the three consensus trees. Concentrated Changes Tests (Maddison & Mad- dison, 2003) using minstate and maxstate simulations with a sample size of 10000 were carried out using MacClade 4.0.8. Correlated evolution between parasitoid traits (independent characters) and lipogenic ability (dependent character) was tested using a phylogeny consisting only of parasitic Hymenoptera. Data on the presence or absence of parasitoid traits was obtained from the literature and each character was scored binomially i.e. ectoparasitoid or endoparasitoid (0/1), idiobiont or koinobiont (0/1), other host stage attacked or larval host stage attacked (0/1), pupal host stage attacked or other host stage attacked (0/1), other host stage attacked or adult host stage attacked (0/1), solitary or gregarious (0/1) primary or facultative hyperparasitoid and hyperparasitoid (0/1), non host-feeding or host-feeding (0/1), proovigenic or synovigenic (0/1) and specialist or generalist (0/1). As clear definitions of generalism and specialism are lacking for hymenopteran parasitoids, we used two criteria based on

34 The evolutionary loss of lipogenesis

Stireman (2005): The ﬁrst criterion assumes generalists to attack 5 or more host families and specialists 4 or fewer host families. The second criterion assumes generalists to attack 10 or more host species and specialists 9 or fewer host species. When reconstructing generalism and specialism, the ancestral state of the clade comprising A. nens, L. nana, G. agilis and P. turionellae was equivocal. This has been resolved to generalism being the ancestral state of this clade as the majority of species within the subfam- ily Cryptinae are known to be generalists (Laurenne et al., 2008; Shwarz & Shaw, 2000). Ancestral states were reconstructed using parsimony and analyses carried out using Concentrated Changes Tests with exact count. For several trait states the sample sizes were low. A power analysis showed that even if maximal concurrence between trait state and lipogenic ability was assumed, a lack of signiﬁcance was found; hence for these traits the power of the analyses was too low and they were excluded from the analyses. These trait states included endoparasitism, koinobiosis, larval host stage attacked, solitary, primary, synovigenic and specialism. Concen- trated Changes Tests were not performed if fewer than 2 gains occurred on branches of the trait state in question, which was the case for adult host stage attacked, gregarious and pro-ovigenic trait states.

Results

Feeding experiments and lipogenic ability

The lipogenic ability of species was determined by comparing the lipid levels of individuals at emergence and after several days of feeding. Of the 24 species tested, 18 were demonstrated to lack lipid accumulation in a situa- tion of excess food. The staphylinid beetle Aleochara bilineata (Coleoptera) and the rhinophorid fly Paykullia maculata (Diptera) showed a significant decrease in lipid levels between newly emerged individuals and fully fed individuals, which is indicative of lack of lipogenesis (Figure 3.1; Table 3.2). Furthermore, nearly all hymenopteran parasitoids tested lacked lipid accumulation. Individuals in the feeding treatments either had decreased lipid levels or failed to increase their lipid levels, despite the fact that they had access to a surplus of carbohydrates. Only five parasitoid species proved an exception to this pattern: Gelis agilis, Lysibia nana, Acrolyta nens, Pteromalus puparum and Leptopilina heterotoma were shown to significantly increase their lipid levels during their lifetimes when fed. The only non-parasitoid species in our feeding experiment was the symphytan

35 Chapter 3

Difference in mean % fat between emerged and fed individuals Order/Superfamily Species -30-20 -10 0 10 20 30 Coleoptera Aleochara bilineata *** Diptera Paykullia maculata *** Hymenoptera Tenthredinoidea Athalia rosae ***

Ichneumonoidea Gelis agilis *** Acrolyta nens *** Lysibia nana *** Aphidius picipes ns Aphidius ervi ns Aphidius rhopalosiphi ns Cotesia glomerata * Cotesia rubecula *** Orthopelma mediator *** Asobara tabida ***

Cynipoidea Leptopilina heterotoma *** Periclistus brandtii ns Leptopilina boulardi *** Diplolepis rosae ***

Chalcidoidea Pteromalus puparum *** Aphelinus abdominalis ns Pachycrepoideus vindemmiae ** Spalangia erythromera ***

Proctotrupoidea Trichopria drosophilae ***

Chrysidoidea Goniozus legneri *** Goniozus nephantidis ***

Figure 3.1: Difference in mean lipid content (%) between emerged and fed individuals as measured in the feeding experiments. Asterisks indicate significant differences between emerged and fed individuals: *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. Sample sizes for each treatment and species are listed in Table 3.2.

Athalia rosae, belonging to the most basal lineage within Hymenoptera. This species signiﬁcantly increased its lipid levels when fed (Figure 3.1; Table 3.2).

Evolution of lack of lipid accumulation in relation to parasitism

Our literature survey and feeding experiments resulted in data on the lipogenic ability of 94 species. 26 species exhibited a lack of lipid accumulation, including one coleopteran parasitoid, three dipteran parasitoids and 20 hymenopteran parasitoids. All other, non-parasitoid hymenopteran species showed an increase in lipid levels when fed, with the exception of the gall wasp Diplolepis rosae and its inquiline Periclistus brandtii (Figure

36 The evolutionary loss of lipogenesis

3.1, Table 3.2). Phylogenetic analysis of lipogenic ability is complicated by uncertainties in the hymenopteran phylogeny (Dowton & Austin, 2001; Sharkey, 2007). We, therefore, consider three possible consensus trees, but the conclusions of our analyses are qualitatively the same for all trees. The parasitoid lifestyle can be inferred to have evolved independently four times and lost twice when lack of lipogenesis is considered the ancestral state at the root of the tree (for the consensus trees in which Ichneumonoidea and Aculeata, as well as Proctotrupomorpha and Aculeata are considered sister groups). The evolution of lack of lipid accumulation is concurrent with that of parasitism at least one time in Coleoptera, two times in Diptera and one time in Hymenoptera, demonstrating lack of lipid accumulation has evolved four times independently in parasitoid lineages throughout the insects. Lack of lipogenesis is signiﬁcantly more likely to originate on parasitic than on non-parasitic branches in the phylogeny (Concentrated Changes Test, p<0.001). One of the consensus trees, in which Ichneumonoidea and Proctotrupomorpha are considered sister groups, shows the ancestral state at the root of the hymenopteran tree to be equivocal. The ancestral state could be either non-parasitic or parasitic and lipogenic ability to be absent or present (Figure 3.2). We compared all possible combinations of character evolution and found lack of lipogenesis to have evolved concurrently with parasitism 3, 4, or 5 times. Each comparison showed concurrent evolution between lack of lipogenesis and parasitism to be signiﬁcant (Concentrated Changes Test, p<0.05 for each comparison using Benjamini and Hochberg’s (1995) False Discovery Rate as a correction for multiple testing).

Evolution of lipogenesis in relation to parasitoid traits

Not all parasitoid species showed a lack of lipogenic ability. Within the parasitic Hymenoptera five parasitoid species were found to be capable of lipogenesis (Figure 3.1, Table 3.2), showing an exception to the general pattern. Phylogenetic reconstruction of lipogenic ability in insects suggested the ability for lipogenesis in these five species to be a secondarily derived character rather than an ancestral trait, suggesting they have regained lipogenic ability (Figure 3.2). To identify possible selection pressures that may have caused a reversal to lipogenic ability, we analyzed the conditional probability of reversed lipogenesis for several parasitoid traits. For each of the consensus trees, as shown in Table 3.1, no significant relationship was found with developmental characters (mode of parasitism, host stage attacked, host developmental arrest and facultative hyperparasitism), nor

37 Chapter 3

Lepisma sacharina Thysanura Ephemerella walkeri Ephemeroptera Calopteryx atrata Calopteryx maculata Odonata Hyponeura sp. Bradynotes obesa Melanoplus sanguinipes Schistocerca gregaria Schistocerca americana Cratypedes neglectus Camnula punctata Gryllus bimaculatus Orthoptera Gryllus firmus Acheta domesticus Leucophaea maderae Nauphoeta cinerea Symploce capitata Periplaneta japonica Periplaneta fuliginosa Acroneuria sp. Plecoptera Forficula auricularia Dermaptera Myzus cerasi Prociphilis fraxinifolly Planococcus citri Bemesia argentifolii Hemiptera Magacicada septendecim Oncopeltus fasciatus Lygaeus kalmii Chrysopa carnea Lertha sheppardi Neuroptera Gyrinus opacus Aleochara bilineata Dermestus maculatus Smicronyx fulvus Coleoptera Smicronyx sordidus Hippodamia convergens Tenebrio molitor Aedes sollicitans Aedes punctor Aedes cantans Aedes aegypti Culex tarsalis Pseudacteon tricuspis Cuterebra austeni Diptera Paykullia maculata Scathophaga stercoraria Ceratitis capitata Anastrepha serpentina Drosophila melanogaster Aglais urticae Inachis io Danaus plexippus Bombyx mori Hyalophora cecropia Manduca sexta Lepidoptera Trichoplusia ni Spodoptera exempta Galleria mellonella Athalia rosae Tenthredinoidea Acrolyta nens Lysibia nana Gelis agilis Pimpla turionellae Orthopelma mediator Venturia canescens Diadegma insulare Aphidius ervi Aphidius rhopalosiphi Ichneumonoidea Aphidius picipes Asobara tabida Macrocentrus grandii Cotesia glomerata Hymenoptera Cotesia rubecula Leptopilina boulardi Leptopilina heterotoma Periclistus brandtii Cynipoidea Diplolepis rosae Trichopria drosophilae Proctotrupoidea Aphelinus abdominalis Spalangia erythromera Eupelmus vuilletti Chalcidoidea Pachycrepoideus vindemmiae Pteromalus puparum Nasonia vitripennis Goniozus nephantidis Chrysidoidea Goniozus legneri Philanthus triangulum Osmia lignaria Apoidea Lipogenesis Bombus terrestris Lack of lipogenesis Hemipepsis ustulata Equivocal Leptothorax albipennis Gain of parasitic lifestyle Camponotus festinatus Vespoidea Loss of parasitic lifestyle Vespula vulgaris Mischocyttarus mastigophorus

Figure 3.2: Phylogeny based on morphological and molecular data, showing inferred gains (black x) and losses (white x) in parasitic lifestyle. Character tracing for lipogenic ability is shown, in which dark gray branches refer to an ability to accumulate lipids, whereas light gray branches refer to a lack of lipid accumulation in adults. In this phylogeny, Ichneumonoidea and Proctotrupomorpha are considered sister groups.

38 The evolutionary loss of lipogenesis

Table 3.1: Presence and lack of lipogenesis within the class Insecta.

Ichneumonoidea and Proctotrupomorpha as sister groups Lack of lipogenesis Lipogenesis ancestral ancestral Traits Gains p-value Gains Losses p-value Developmental characters Ectoparasitic 2 0.295 2 1 0.138 Pupal host stage attacked 2 0.207 2 0 *0.027 Idiobiont 2 0.404 2 2 (or 1) 0.270 (0.175) Facultative hyper and hyper 2 0.097 2 0 *0.015 Host range Generalism (>5 host families) 2 0.068 2 0 *0.012 Generalism (>10 species) 3 *0.005 3 0 *<0.001 Adult feeding Non host-feeding 2 0.537 2 0 0.066 Host-feeding 2 0.366 7 2 2 0.412 Asterisks indicate signiﬁcant diﬀerences after correction for multiple testing using Ben- jamini and Hochberg’s False Discovery Rate.

with adult diet (host-feeding behavior) when lack of lipogenesis is the ancestral state at the root of the tree. Reversals from lack of lipogenesis to lipogenesis are not significantly associated with these parasitoid traits. When Ichneumonoidea and Proctotrupomorpha are considered sister groups, lipogenesis could have been the ancestral state at the root of the tree. In that case, similar results are obtained, except for pupal host stage attacked and facultative hyperparasitism, which are significantly correlated with the ability for lipogenesis (Table 3.1). In contrast, reversals to lipogenesis were concentrated to lineages with a generalist host species range i.e. that attacked 10 or more host species (Figure 3.3) when either lack of lipogenesis or lipogenesis are considered the ancestral state. Parasitoids attacking many different host species seem to be capable of lipogenesis, opposed to more specialist parasitoids, attacking relatively few hosts that lack lipogenesis. In addition to defining generalists by the number of host species they parasitize, generalists can also be defined by the number of host families they attack (i.e. 5 or more host families). We found a similar correlation between species adopting a wide host family range and lipogenesis (Table 3.1) when lipogenesis is considered ancestral. However, when lack of lipogenesis is considered the ancestral state, reversals to lipogenesis are not significantly concentrated to hymenopteran parasitoid lineages adopting a wide host family range.

39 Chapter 3

Acrolyta nens Lysibia nana Gelis agilis Pimpla turionellae Orthopelma mediator Venturia canescens Diadegma insulare Aphidius ervi Aphidius rhopalosiphi Aphidius picipes Asobara tabida Macrocentrus grandii Cotesia glomerata Cotesia rubecula Leptopilina boulardi Leptopilina heterotoma Trichopria drosophilae Aphelinus abdominalis Spalangia erythromera Eupelmus vuilletti Pachycrepoideus vindemmiae Pteromalus puparum Nasonia vitripennis Goniozus nephantidis Specialism Goniozus legneri Generalism (>10 host species) Gain in lipogenic ability

Figure 3.3: Phylogeny showing inferred character states for host range (specialist/generalist) for hymenopteran parasitoids, in which gains (white x) in lipogenic ability are shown. In this phylogeny, Ichneumonoidea and Proctotrupomorpha are regarded as sister groups.

Discussion

Our phylogenetic study has yielded two key results. The ﬁrst result is the evidence of a concurrent loss of lipogenesis with the evolution of parasitism in insects. Lack of lipogenesis has evolved repeatedly and independently in four insect lineages with a parasitic lifestyle. The notion that lack of lipid accumulation is restricted to parasitic species is substantiated by the presence of full lipogenic abilities in Symphyta, a sister group of Hymenoptera, and non-parasitic aculeates. Parallel evolution of compensated trait loss in insect parasitoids is possibly produced by the tight interspeciﬁc interactions between host and parasitoid and, to hypothesize, by the physiological manipulation that allows loss of lipogenic function without burdening the energetic state of the parasitoid larvae or emerging adults. The second key result reveals that lipogenic ability can re-evolve in species adopting large

40 The evolutionary loss of lipogenesis host ranges, i.e. host species generalists. Insuﬃcient host specialization prevents the ﬁne-tuned physiological matching that is needed for host manipulation and optimal exploitation of the host, thus requiring the need to synthesize lipids.

Lack of lipogenic ability and adult performance

As a preface to discussing the adaptive evolution of lack of lipogenesis, we start by considering alternative explanations for the lack of lipid accumulation in well-fed parasitoids. Lipid synthesis is generally regarded as an involuntary dose-dependent physiological process when there is a surplus of carbohydrates available in the diet (Garrett & Grisham, 1999). Neverthe- less, the net increase in lipid reserves hinges on the rate at which lipids are metabolized. If lipids are broken down at an equal or higher rate than they are produced, then the effect on lipid accumulation would be similar to the pattern observed in our feeding experiments. Even though we deem such a scenario unlikely due to the low energetic efficiency this would entail, we cannot exclude this possibility because we did not measure the rate of lipogenesis and lipid breakdown directly. Our case is corroborated, however, by a radiotracer study in the parasitoid Eupelmus vuilletti, which showed that ingestion of radioactively labeled glucose was not incorporated into lipid reserves, excluding the possibility of de novo lipogenesis (Giron & Casas, 2003). Additional studies on other parasitoid species that lack adult lipid accumulation are needed to verify the generality of this finding. Another assumption that requires consideration is the ability of species to ingest carbohydrate-rich food sources. Evolutionary reduction of the mouthparts or digestive organs limits nutrient uptake and hence restrains lipid accumulation. The species used in the feeding experiments have been shown to utilize the source of carbohydrates presented (Text 3.1, B. Visser, pers. obs.), with the exception of the gall wasp Diplolepis rosae (Randolph, 2005) and the parasitic fly Cuterebra austeni (Kemp & Alcock, 2003), which possess fully developed mouthparts, but have not been shown to ingest food. From these findings, we conclude that the lack of lipid accumulation can clearly occur with full feeding abilities. Lack of lipogenic ability in adults may appear to be highly disadvantageous, hence challenging the assumption of selective neutrality that is required for the evolution of trait loss. However, the necessity of acquiring additional lipid reserves as adults can be evaded if sufficient resources are carried over from the larval stage to sustain lipid use during adult life

41 Chapter 3

(Colasurdo et al., 2009). From this perspective, additional lipid synthesis as an adult is even unwanted because the production of lipids from carbohydrates is less efficient than the direct metabolism of carbohydrates, due to energetic conversion costs. Indeed, for some species that lack lipogenesis, capital lipid reserves can mount to more than 30 percent. Parasitoids can also economize on lipid use if acute energy requirements can be met by regular ingestion of sugars through feeding on nectar, honeydew, or carbohydrate-rich oviposition substrates (Eijs et al., 1998). For example, in the parasitoid Venturia canescens nectar feeding occurs frequently in the field, postponing the moment of lipid depletion despite relatively low capital lipid levels (Casas et al., 2003). Adult females also face a high demand for lipids from egg production (Ellers & Van Alphen, 1997); hence we expected a lack of lipogenesis to favor pro-ovigenic reproductive strategies, in which a large complement of eggs is already mature at emergence (Jervis & Ferns, 2004; Jervis et al., 2008). Alternatively, host-feeding may have evolved to provide an income resource of lipids for egg maturation (Giron et al., 2004), as it is known that host hemolymph contains non-trivial, albeit low, amounts of lipids (Beenakkers et al., 1985). However, our results showed that neither of these reproductive traits was significantly associated with lipogenic ability. It has been suggested that most parasitoids are time-limited, i.e. energy resources are depleted before all eggs have been deposited (Ellers et al., 2000a; Rosenheim, 1996). Presumably, such allocation strategies helped parasitoids to overcome the constraints of lacking lipogenic ability as adults.

Larval exploitation and host range

Adaptation by parasitoids to their host has resulted in highly speciﬁc mechanisms to increase lipid levels and optimize host exploitation (Nakamatsu & Tanaka, 2004; Rivers & Denlinger, 1995; Suzuki & Tanaka, 2007). In agreement with the specialized nature of host-parasitoid interactions, our phylogenetic analysis indicates breadth of host range is a key trait in the evolution of lipogenic abilities. Lack of lipogenesis is predominantly found in specialist parasitoids, whereas parasitoids adopting large host ranges have re-evolved lipogenic ability. Larval lipogenesis may be no longer redundant in these generalist species because the physiological manipulation of diﬀerent host species is expected to be problematic, leading to poor host exploitation and lower capital lipid reserves in adults. Nonetheless, some generalist parasitoids were found incapable of syn-

42 The evolutionary loss of lipogenesis thesizing lipids as adults, such as Nasonia vitripennis and Pachycrepoideus vindemmiae. In N. vitripennis, it has been shown that although this species can develop on over 60 different host species, it has a preference for sar- cophagous flies (Desjardins et al., 2010). From this perspective, it is a specialist because the injection of venom during oviposition can only increase lipid levels in Sarcophaga species (Rivers & Denlinger, 1995). A certain level of specialism, in which parasitoids have adapted to specific hosts in order to manipulate their physiology, is thus required to obtain sufficient resources during development. It is this unique ability to manipulate their hosts that distinguishes parasitoids from predacious species, and confines the evolution of lack of lipogenesis to parasitoids. Predators can maximize energetic gain by increasing the number of prey or selecting more nutritious prey, but they are unable to attain higher resource availability once a prey has been captured. A special case of host manipulation and redundancy of lipogenesis may be found in gall wasps. Gall wasps have evolved phytophagy secondarily within Hymenoptera (Grimaldi & Engels, 2005; Quicke, 1997). Similar to parasitoid-host interactions, they are able to manipulate their host plant’s physiology to increase lipid levels in cells surrounding the developing larva (Harper et al., 2004). Indeed, these species also have intimate physiological relationships with their host plants and these intricate co-evolutionary relationships have thus resulted in loss of lipid accumulation as adults.

Toward further understanding of the evolutionary loss of traits

The evolutionary loss of lipogenesis in parasitic insects is one of few examples of repeated trait loss through environmental compensation (Chatterjee, 1973; Ohta & Nishikimi, 1999; Krause, 2008). This study demonstrates that compensated trait loss of physiological traits can be masked by parasitoid- host interactions. We found that species with superficially similar developmental strategies can vary considerably in physiological dependence on their host. Closer examination of other stable co-evolutionary relationships can be expected to yield more such examples. Parallel evolution of traits might be the result of quite different underlying genetic mechanisms. For example, similar coat patterns and pigmentation in mice species has been shown to have evolved through different genetic mechanisms (Steiner et al., 2009). Similar to the evolution of novel traits, parallel trait loss can also be expected to occur through various genetic mechanisms. Further genome analysis is needed to assess if specific

43 Chapter 3 genes have been preferred targets of mutation accumulation. A prime candidate to explain lack of lipogenesis would be fatty acid synthase (FAS) which has a central role in the production of lipids. FAS has previously been documented to be absent in the genome of a parasitic fungus that is dependent on external lipids for growth (Xu et al., 2007). Recent completion of the genome of the parasitoid N. vitripennis (Werren et al., 2010) has revealed a homolog to the FAS gene of Drosophila, although its functionality remains to be tested. A fundamental question regarding the evolutionary loss of traits is whether physiological abilities that have been lost can be regained. Indeed, a striking ﬁnding is that lipogenesis has re-evolved three times within Hymenoptera, a fact which challenges several widespread views regarding trait loss. Trait loss is usually thought to involve one-way directional evolution, in which a lost trait does not re-evolve (Bull & Charnov, 1985). However, exceptions to this general rule have been found (Bely & Sikes, 2010; Cruickshank & Paterson, 2006), and lipogenic ability may be one of them. We propose that after the loss of lipogenic ability, much of the lipogenic pathway may remain intact, perhaps due to pleiotropic eﬀects on other physiological pathways. Conversion of carbohydrates to triglycerides employs key enzymes that are functional in other physiological processes, such as pyruvate metabolism, citrate cycle, and biosynthesis of secondary metabolites (Kanehisa & Goto, 2000). The lipogenesis pathway may be protected from severe degradation due to shared components with essential metabolic pathways, hence enabling a fully functional lipid synthesis to re-evolve. It is essential to identify the underlying genetic and physiological mechanisms involved to understand the evolutionary dynamics of the loss of this trait.

Acknowledgements

We would like to thank Caroline Müller, Anne-Marie Cortesero, Ian Hardy, Femmie Kraaijeveld, Roland Allemand and Hans Smid for providing us with insects. Eric Kok, Mirte Fritz and Louis Boumans we would like to thank for their efforts in obtaining exceptionally large amounts of rose galls and woodlice necessary to provide sufficient material for testing. We are grateful to Donald Quicke and Mark Dowton for their information on hymenopteran phylogenetic relationships, Dan Hahn for his help in finding laboratory cultures of parasitoids and two anonymous referees for their comments on the earlier draft of the manuscript. BV was funded by the

44 The evolutionary loss of lipogenesis

Netherlands Organisation for Scientiﬁc Research (NWO) ALW grant no. 816.01.013.

45 Chapter 3

Text 3.1 Additional references

Papers on the ability to ingest carbohydrate-rich food sources in species used in feeding experiments

Azzouz et al. (2004); Chen et al. (2005); Ellers (1996); Hogervorst et al. (2007); Jervis (2007); Lee et al. (2004a); Olson et al. (2000); Özalp & Emre (2001); Randolph (2005); Rivero & West (2002); Schneider et al. (2001)

Inference of phylogenetic relationships

Brothers (1999); Campbell et al. (2000); Castro & Dowton (2006); Dohlen & Moran (1995); Dowton & Austin (2001); Flook et al. (1999); Han & Ro (2005); Harbach (2007); Hunt et al. (2007); Kristensen et al. (2007); Laurenne et al. (2008); Quicke et al. (2000); Reinert et al. (2008); Wheeler et al. (2001); Yeates et al. (2007); Zwick (2008)

46 The evolutionary loss of lipogenesis

Table 3.2: Mean percentage fat in adults after emergence and after feeding.

Species Mean % fat n Mean % fat n χ2 or p-value at emergence after feeding t-value† (± 1 s.e.m.) (± 1 s.e.m.) Order Coleoptera Aleochara bilineata 39.31 (14.86) 7 12.29 (5.02) 6 7.196 <0.001

Order Diptera Paykullia maculata 26.37 (11.78) 5 15.43 (5.83) 7 9.335 <0.001

Order Hymenoptera; Symphyta Athalia rosae 11.51 (4.07) 8 21.37 (8.08) 7 -6.018 <0.001

Order Hymenoptera; Apocrita Superfamily Ichneumonoidea Gelis agilis 3.61 (0.72) 25 25.19 (4.94) 26 -14.138 <0.001 Acrolyta nens 8.12 (1.66) 24 22.46 (6.00) 14 -7.050 <0.001 Lysibia nana 7.17 (1.46) 24 21.36 (5.03) 18 -6.919 <0.001 Aphidius picipes 4.71 (1.18) 16 6.95 (2.10) 11 -1.381 0.180 Aphidius ervi 12.09 (2.37) 26 12.93 (2.19) 35 -1.186 0.244 Aphidius rhopalosiphi 6.61 (1.77) 14 7.34 (1.73) 18 -0.429 0.671 Cotesia glomerata 14.44 (2.55) 32 12.68 (2.64) 23 2.313 0.029 Cotesia rubecula 11.49 (2.79) 17 4.31 (1.04) 17 8.971 <0.001 Orthopelma mediator 17.26 (3.38) 26 8.79 (1.72) 26 19.292 <0.001 Asobara tabida 20.48 (4.58) 20 9.98 (2.29) 19 5.130 <0.001

Superfamily Cynipoidea Leptopilina heterotoma 22.80 (4.98) 21 29.38 (7.35) 16 -4.539 <0.001 Periclistus brandtii 28.35 (7.58) 14 30.78 (8.54) 13 -0.616 0.544 Leptopilina boulardi 25.56 (5.22) 24 12.92 (3.45) 14 10.852 <0.001 Diplolepis rosae 31.79 (5.53) 33 13.30 (3.23) 17 16.376 <0.001

Superfamily Chalcidoidea Pteromalus puparum 8.52 (1.48) 33 13.87 (2.62) 28 -5.303 <0.001 Aphelinus abdominalis 19.15 (4.51) 18 23.86 (5.79) 17 -1.210 0.235 Pachycrepoideus 15.72 (3.61) 19 10.72 (2.68) 16 7.922 0.005 vindemmiae Spalangia erythromera 26.70 (7.14) 14 14.00 (4.04) 12 12.233 <0.001

Superfamily Proctotrupoidea Trichopria drosophilae 21.85 (3.86) 32 15.56 (2.84) 30 34.834 <0.001 Aculeata;

Superfamily Chrysidoidea Goniozus legneri 34.80 (10.49) 11 27.79 (9.83) 8 4.031 <0.001 Goniozus nephantidis 43.28 (9.68) 20 34.77 (7.77) 20 4.027 <0.001

† χ2 is shown for the results of Kruskal-Wallis tests; t-value is shown for the results of t-tests and Welch’s t-tests.

Chapter 4

Can host manipulation drive the evolutionary loss of traits in parasitoids?

Bertanne Visser, Menno Voogt and Jacintha Ellers Submitted

Abstract Storing excess energy in the form of lipid reserves is a universal trait adopted by animals to cope with unfavourable environmental conditions. An exception to this general pattern was found in parasitoids, in which the majority of species have lost their lipogenic ability. As larvae, parasitoids maintain an intimate physiological interaction with their host. Extensive manipulation of the host’s physiology by the developing parasitoid is thought to have made lipogenesis redundant in larvae and has been suggested causal to losing lipogenesis. However, an explicit test of host manipulation ability in relation to lipogenic ability is lacking so far. Here, we examine the relation between host manipulation and lack of lipogenesis in the generalist parasitoid Pachycrepoideus vindemmiae attacking the dipteran host Drosophila melanogaster as a primary parasitoid and the parasitoids Asobara tabida and Leptopilina heterotoma as a facultative hyperparsitoid. Through quantification of lipid reserves of pupae and freshly emerged adults, we were able to determine that P. vindemmiae did not conform to our expectations. We found that P. vindemmiae did not increase lipid reserves of these three different host species through physiological manipulation. Our data suggest that P. vindemmiae is limited by the amount of resources it can carry over from its hosts. Lipid levels obtained from the host therefore suffice in providing P. vindemmiae with the resources needed to fuel its free-living adult life, while lacking lipogenesis. Host manipulation remains a prime candidate to explain the evolutionary loss of lipogenesis in the majority of parasitoids, yet for some species, like P. vindemmiae, mechanisms other than host manipulation drive the evolutionary loss of this trait.

49 Chapter 4

Introduction

Evolutionary theory predicts that redundant or unused traits are released from selection, and that organisms harboring energetically costly traits should experience negative selection pressures that potentially lead to vestigialization, regression or loss of traits (Lahti et al., 2009). Phenotypic regression of traits is particularly important for evolutionary dynamics when traits affect an organism’s survival or reproductive effort. For instance, several plant species have abolished their sexual mode of reproduction when certain environmental conditions favored clonal propagation (Dorken et al., 2004; Eckert, 2001). Recently however, several cases of trait loss have been described in which the lost trait is still essential for successful development, growth, and reproduction of the organism (Suen et al., 2011; Visser et al., 2010). For example, several species of parasitic plants have been found to lack photosynthetic abilities necessary for generating energy, yet their photosynthetic requirements are met by exploiting other plants with active photosynthesis (Krause, 2008). Loss of indispensable traits can occur when the resulting deficiencies are provided for by a symbiotic partner. This process is referred to as environmental compensation, as the environment provides certain resources or functions compensating the phenotypic requirement of the organism (Visser et al., 2010). Environmental compensation can thus either release traits from selection or cause the remaining selection pressures to be negative, even though phenotypic function of the trait is maintained when the trait is lost. Tight co-evolutionary interactions between parasitic insects and their hosts have led to the loss of an essential trait in nutrient metabolism (Visser et al., 2010). These insects attack other arthropod hosts to complete larval development, while they are free-living as adults (Godfray, 1994). Sev- eral parasitic wasp species were shown to lack lipid accumulation during their adult life-stage (Casas et al., 2003; Ellers, 1996; Fadamiro et al., 2005; Giron & Casas, 2003; Lee et al., 2004a; Olson et al., 2000; Visser & Ellers, 2008; Visser et al., 2010). Lipids are the most comprehensive form in which excess energy obtained through feeding can be stored for use at a later time and lipid reserves typically contribute largely to survival and reproduction at times when environmental conditions are unfavorable (Eijs et al., 1998; Ellers et al., 1998; Ellers & Van Alphen, 1997). Lack of lipogenesis is hypothesized to result from the parasitic larval lifestyle adopted by these insects, in which costly conversion of nutrients into lipid reserves can be avoided because these nutrients are provided by the host (Visser & Ellers,

50 Host manipulation

2008). Moreover, many parasitic wasps, or parasitoids, have evolved elaborate mechanisms to manipulate their host’s metabolism into increasing its lipid content (Beckage et al., 1997; Nakamatsu, 2003; Nakamatsu & Tanaka, 2004; Rivers & Denlinger, 1994, 1995; Suzuki & Tanaka, 2007). Parasitoid larvae thus ensure sufficiently elevated host lipid levels for direct consumption to satisfy their needs in the adult life-stage when they actively seek for new hosts. Although the loss of lipogenesis predominates among parasitic wasp species, the loss of this trait is not universal for all parasitoids. Com- parative analysis of parasitic wasp lineages revealed that lipogenesis has re- evolved in several generalist species, each of which adopt a large host range of ten or more different host species (Visser et al., 2010). In these species, females rapidly accumulate lipid reserves during their adult life when provided with dietary carbohydrates. Active host manipulation is hypothesized to be restricted to more specialized parasitoids that attack only one or few host species, as physiological alterations require specific adaptations to the host environment (Asgari & Rivers, 2010; Le Ralec et al., 2011; Vinson & Iwantsch, 1980). An exception is the parasitic wasp Nasonia vitripennis, an extreme generalist that has the potential to develop on over 60 different fly species (either under field or laboratory conditions) and the adults of which do not accumulate lipids during life (Blanchot, 1994; Rivero & West, 2002; Whiting, 1967), yet these wasps are only able to manipulate and increase host lipid levels when developing on their preferred hosts within the genus Sarcophaga (Desjardins et al., 2010; Rivers & Denlinger, 1994). This parasitoid has thus specialized to some degree on the limited number of host species that it typically encounters in the field. Clearly, a large host range is not necessarily indicative of lipogenesis in adult parasitoids, yet an ability to manipulate host physiology in more specialized parasitoids possibly explains the loss of lipogenesis in parasitic insects. However, an explicit test of this hypothesis is lacking so far. In this study, we have looked at the relation between host species, manipulative ability and lack of lipogenesis in the parasitoid Pachycrepoideus vindemmiae. P. vindemmiae is a facultative hyperparsitoid that can parasitize over 60 different host species (either in the field or in the laboratory)(Carton et al., 1986; Visser et al., 2010; Wang & Messing, 2004). We, therefore, suspected P. vindemmiae to be more specialized on hosts it frequently encounters in the field, leading to a certain degree of specialization in terms of host manipulative abilities. We tested this by allowing P. vindemmiae to parasitize its common dipteran host Drosophila melanogaster

51 Chapter 4 as a primary parasitoid, and the parasitoids Leptopilina heterotoma and Asobara tabida as a facultative hyperparasitoid. Of these larval parasitoids, A. tabida is more specialized and lacks lipogenesis, whereas L. heterotoma is a generalist that synthesizes lipid as an adult. We tested whether P. vindemmiae was able to manipulate the physiology of its hosts, leading to increased lipid levels in parasitized pupae and adult parasitoids. We compared the amount of lipid reserves of the parasitoid species and the unparasitized host at two time points during development: in the pupal and in the adult stage. We relate our ﬁndings to the lipogenic ability of this species to determine if host manipulation can be the major force driving the evolutionary loss of lipogenesis in parasitoids adopting a wide host range.

Materials and methods

Study organisms

The host ﬂy D. melanogaster was obtained from an existing laboratory culture at the University of Leiden, the Netherlands. Primary parasitoids A. tabida and L. heterotoma were obtained from laboratory cultures at the Uni- versity of Rennes 1, France and the generalist facultative hyperparasitoid P. vindemmiae was obtained from a laboratory culture at the University of Lyon 1, France. All insects were maintained at a temperature of 20◦C, a relative humidity of 75% and a 16:8 light:dark regime. To determine the ability of P. vindemmiae to manipulate its hosts, we measured lipid reserve levels at speciﬁed times during the pupal stage and after metamorphosis in adults of host(s) and parasitoids. For all treatments, approximately 100 females of D. melanogaster were allowed to oviposit during 1 day on 40mL of agar medium containing 20g agarose, 9g kalmus (10 parts of acidum tartaricum, 4 parts ammonium sulphate, 1 part magnesium sulphate and 3 parts potassium phosphate), 10mL nipagin (100g 4-methyl hydroxyl benzoate per liter ethanol), 50g sucrose and 35g yeast per liter water. For the control treatments containing D. melanogaster only, experimental jars were prepared that each contained approximately 200 3-day old second instar larvae that were placed on 25mL of medium containing 20g agarose, 9g kalmus, 5mL nipagin and 4mL proprionic acid per liter water covered by thin layer of liquid yeast until pupae were collected 9 days after hatching. For parasitized treatments, D. melanogaster larvae underwent the treatment as decribed previously for unparasitized pupae, except that approximately 200 second-instar larvae per jar were available during 1 day

52 Host manipulation for oviposition by two mated females of either A.tabida or L. heterotoma. For P. vindemmiae 2 mated females per jar were allowed to oviposit during 1 day on approximately 50 9-day old D. melanogaster pupae or on pupae containing either of the two primary parasitoids. The developmental stages used for hyperparasitism of primary parasitoids by P. vindemmiae were based on optimal parasitism rates. Van Alphen and Thunissen (1982) describe optimal parasitism rates for P. vindemmiae developing on L. heterotoma and A. tabida. For L. heterotoma timing of oviposition was deduced from data obtained at 25◦C. Therefore a conversion factor of 1.59 (Zwaan et al., 1992) was used to deduce the optimal time for parasitism. Hence, P. vindemmiae was allowed to oviposit on developing L. heterotoma after 13 and 21 days of development and on developing A.tabida after 17 days of development, in which the physiological status of A. tabida at 17 days is similar to that of the longer development time of L. heterotoma at 21 days. To verify that optimal developmental stages were reached at the time of oviposition, at least 5 parasitized pupae per treatment were visually inspected under a microscope. For all species jars were inspected daily for emerged adults that were collected and immediately frozen at -20◦C until further processing.

Experimental set-up

To determine the ability of P. vindemmiae to manipulate the physiology of its hosts, we measured lipid levels of unparasitized D. melanogaster pupae, pupae of D. melanogaster parasitized by the primary parasitoids A. tabida and L. heterotoma (at two time points) and freshly emerged adults of all species, including treatments with hyperparasitism by P. vindemmiae on each of the primary parasitoids and on D. melanogaster. We used both males and females in adult treatments as sex cannot be determined at the pupal stage of D. melanogaster or developing parasitoids. Lipid levels were determined using ether extraction of triglycerides based on the method described by David et al. (1975). Samples containing pupae and adults were freeze-dried for 2 days, after which dry weight was determined. Samples were placed in a vial containing 4mL of ether that was removed after 24 hours. Samples were rinsed with fresh ether and subsequently freeze-dried during 2 days, after which dry weight was determined again. The amount of lipids was calculated by subtracting dry weight after ether extraction from dry weight before ether extraction.

53 Chapter 4

Statistical analyses

Scatterplots and regression lines of dry weight before ether extraction were compared to dry weight after ether extraction to find potential outliers in the data. Following Visser et al. (2010), outliers were only removed if they deviated from a 99% confidence interval of the linear regression line. Data was inspected for normality and homogeneity of variances. Error structures that deviated from a normal distribution were cube root transformed to normality. For two datasets there was significant heterogeneity of variances. We therefore used ANOVA to test for differences between treatments followed by a Tukey test when variances were equal and Dunnett’s T3 test to determine differences between groups assuming unequal variances and sample sizes (Dunnett, 1980).

Results

Pupal lipid reserves of D. melanogaster and primary parasitoids

Lipid reserves of D. melanogaster pupae were 0.523 mg (±0.045 mg, 1SE; n=46; Figure 4.1A) after 9 days of development, whereas lipid reserves of D. melanogaster pupae containing L. heterotoma after 13 days of development were 0.103 mg (±0.007 mg, 1SE; n=19) which declined to 0.058mg (±0.004 mg, 1SE; n=21) on day 21. Of the primary parasitoids, A. tabida pupae contained the highest lipid levels of 0.347 mg (± 0.023 mg, 1SE; n=60). The differnece in lipid reserves of D. melanogaster pupae and pupae of primary parasitoids was significant between treatments (Figure 4.1A; F3,145 = 33.305, P < 0.001). D. melanogaster pupae had significantly higher lipid levels compared to pupae containing parasitoids (Dunnett’s T3: P < 0.01 for all comparisons). A. tabida developing on D. melanogaster had significantly higher lipid levels when compared to L. heterotoma at 13 and 21 days of development (Dunnett’s T3: P < 0.001), whereas the pupal stage after 13 days of development of L. heterotoma had significantly higher lipid levels when compared to 21 days of development (Dunnett’s T3: P < 0.001).

Adult lipid reserves of D. melanogaster, primary parasitoids and P. vindemmiae as facultative hyperparasitoid

Adult D. melanogaster emerged with an average of 0.029 mg (±0.003, 1SE; n=50) lipids, whereas the primary parasitoids L. heterotoma and A. tabida emerged with reserve levels of 0.020 mg (±0.002, 1SE; n=34) and 0.043

54 Host manipulation

A 0,6 a

60 0,5 50

(mg) d

0,4 (%)

40 fat fat 0,3 30 0,2 b 20

Amount c 0,1 fat Amount 10 0 0 D. L. heterotoma L. heterotoma A. tabida D. L. heterotoma L. heterotoma A. tabida melanogaster day 13 day 21 melanogaster day 13 day 21 Species Species 35

B 0,06

b 30 0,05 (%) 25 (mg) 0,04

ab fat 20 fat fat 0,03 a 15 0,02 10 c

0,01 Amount 5 Amount 0 0 D. L. heterotoma A. tabida P. vindemmiae D. L. heterotoma A. tabida P. vindemmiae melanogaster melanogaster Species Species

C 0,025 b 25

0,02 b 20 (%) 0,015 15

0,01 a 10 a

0,005 fat Amount 5 Amount (mg) fat Amount 0 0 D. L. heterotoma L. heterotoma A. tabida D. L. heterotoma L. heterotoma A. tabida melanogaster day 13 day 21 melanogaster day 13 day 21 Species Species

Figure 4.1: Mean amount of fat in mg ±1SE (left column) and in percentage ±1SE (right column) in pupae (A), adults (B) and P. vindemmiae parasitizing D. melanogaster and primary parasitoids (C).

mg (±0.005, 1SE; n=35), respectively. When P. vindemmiae developed as a primary parasitoid on D. melanogaster it contained 0.006 mg (±0.0007, 1SE; n=39) lipids. The comparison of adult lipid levels of D. melanogaster with primary parasitoids and P. vindemmiae parasitizing as primary parasitoid showed a significant difference between species (Figure 4.1B; F3,157 = 33.309; P < 0.001). We found that lipid levels of adult D. melanogaster were not significantly different from those of L. heterotoma and A. tabida adults (Dunnett’s T3: P = 0.380 and P = 0.214, respectively) and higher than that of adult P. vindemmiae (Dunnett’s T3: P < 0.001). Lipid levels of adult A. tabida were higher than that of adult L. heterotoma (Dunnett’s T3: P < 0.01) and adult P. vindemmiae (Dunnett’s T3: P < 0.001), and L. heterotoma lipid levels were significantly higher than that of P. vindemmiae (Dunnett’s T3: P < 0.001). When P. vindemmiae acted as a secondary parasitoid on L. heterotoma, lipid levels at emergence were 0.006 mg (±0.001, 1SE; n=6; Figure 4.1C) and 0.020 mg (±0.003, 1SE; n=7), when developing on parasitized pupae aged 13 and 21 days, respectively. When A. tabida was used as a host, P.

55 Chapter 4 vindemmiae emerged with reserve levels of 0.018 mg (±0.0008, 1SE; n=38). Lipid resources obtained by P. vindemmiae from hosts when it acted as a primary parasitoid or as a hyperparasitoid differed significantly between treatments (Figure 4.1C; F3,89 = 44.889, P < 0.001). P. vindemmiae obtained most lipid resources when it parasitized L. heterotoma after 21 days of development and A. tabida: lipid levels were significantly higher when compared to those of P. vindemmiae developing on D.melanogaster as primary parasitoid (Tukey: P < 0.001 for both comparisons). P. vindemmiae parasitizing A. tabida did not differ in lipid content from individuals developing on L. heterotoma after 21 days of development (Tukey: P = 0.831). We further found no differences in lipid levels between P. vindemmiae on D. melanogaster and P. vindemmiae developing on L. heterotoma after 13 days of development (Tukey: P = 0.989). Lipid levels were significantly higher in P. vindemmiae parasitizing L. heterotoma after 21 days of development and after developing on A. tabida when compared to L. heterotoma after 13 days of development (Tukey: P < 0.001 for both comparisons).

Discussion

This study focused on the relationship between the ability to manipulate host physiology and the evolutionary loss of lipogenesis in parasitoids, in which host manipulation is expected to drive the loss of this trait. Our results showed that the parasitoid L. heterotoma was least eﬃcient in acquiring lipid resources from its host D. melanogaster during its development. After 13 and 21 days of development, pupal lipid levels were lower when compared to 9-day old pupae of the host. A further decrease in lipid reserves during development suggests that L. heterotoma was not increasing its lipid reserves by synthesizing lipids as a larva, even though this species has been shown to actively synthesize lipids in its adult life-stage (Visser et al., 2010). We found no evidence of L. heterotoma manipulating D. melanogaster to increase its lipid levels. A lack of manipulative abilities of this parasitoid is consistent with active lipogenic abilities in adults and conforms to our expectation that active adult lipogenesis foregoes the need to manipulate the host to increase larval parasitoid resources. The parasitoid A. tabida was most eﬃcient in utilizing its host during development. Although we found lower lipid reserves in developing parasitoids compared to host pupae, lipid reserves remained at a high level considering reserve levels of host pupae were determined 8 days prior to sampling of parasitized pupae. Moreover, 66% of the initial fat amount was

56 Host manipulation still available to the parasitoid at this time. Although the high reserve levels of A. tabida might reflect an extraordinary ability to carry over reserves from its host, these high levels could also reflect the ability of this parasitoid to actively manipulate D. melanogaster into increasing its lipid levels. Af- ter moulting into the pupal stage, D. melanogaster acquired capital lipid reserves of nearly 50% its initial dry weight, consistent with findings in other studies (Djawdan et al., 1997; Simmons & Bradley, 1997). Although other insect species are known to acquire even higher lipid levels (Arrese & Soulages, 2010), a further increase in lipid reserves might be constrained by larval size, in which maximum lipid levels have been attained in the fat body of the host. It remains unclear whether A. tabida manipulated lipid levels of D. melanogaster, but at the least this parasitoid was highly efficient in carrying over lipid reserves from its host. Similar findings have been obtained for other parasitoids, in which efficiency in utilizing host resources was shown to be over 75 or even 90% depending on trophic level (Harvey et al., 2009, 2011). In comparison to other animals that typically exploit resources with an efficiency ranging from 20 to 40% some parasitoids have an exceptional ability to efficiently carry over host resources (Howell & Fisher, 1977). A key result of this study is the finding that the extreme generalist parasitoid P. vindemmiae did not manipulate its host’s resources, which is contrary to our predictions, since this species does not accumulate lipids in its adult life-stage. However, hyperparasitism of the parasitoids A. tabida and L. heterotoma on day 21 did lead to higher lipid reserves than could be attained from the hosts D. melanogaster or L. heterotoma (on day 13). This reflects the efficiency of A. tabida in obtaining high lipid levels from D. melanogaster and a high efficiency of P. vindemmiae to acquire resources from both A. tabida and L. heterotoma after 21 days of development. Sim- ilarity in lipid reserves obtained from D. melanogaster and L. heterotoma after 13 days of development could be due to the size of L. heterotoma at this stage of development. At this point in development the parasitoid larva is relatively small and D. melanogaster tissues predominate at this stage, hence similar amounts of lipid reserves can be carried over from the host (van Alphen & Thunnissen, 1982). Hyperparasitism has further been shown to bring a high cost to P. vindemmiae. In a study by Grandgirard et al. (2002) it was shown that successful parasitism, mortality, development time, sex ratio and adult size were negatively affected when P. vindemmiae hyperparasitized A. tabida. In all cases, these parameters were all negatively affected when P. vindemmiae hyperparasitized A. tabida, i.e.

57 Chapter 4 parasitism success and size decreased, whereas development time and mortality increased, whilst sex ratios were more male-bias. Even though we did not test for these parameters, these costs likely pertain to hyperparasitism by P. vindemmiae on L. heterotoma in our study, in which only small sample sizes were obtained. However, total mass of P. vindemmiae developing on L. heterotoma and A. tabida was higher when compared to development on D. melanogaster and the number of P. vindemmiae adults obtained from development on A. tabida did not suggest such a fitness cost when this species was used as a host. As predicted by the ‘Jack of all trades, master of none’ hypothesis, generalist parasitoids are expected to be less efficient in utilizing host resources in comparison to specialists (Egas et al., 2005; Levins, 1962). Overall P. vindemmiae attained the lowest lipid levels from its host when compared to the other parasitoids, in line with expectations inferred from its extensive host range. However, with regard to the lack of lipogenesis in P. vindemmiae, a degree of specialization was expected for this parasitoid, similar to findings in the extreme generalist N. vitripennis which has specialized on its preferred hosts within the genus Sarcophaga. D. melanogaster is a common host for P. vindemmiae (Carton et al., 1986), and this species has been shown to favour D. melanogaster for oviposition over hyperpar- sitizing A. tabida and L. heterotoma (van Alphen & Thunnissen, 1982). However, naive females do not seem to prefer the relatively small host D. melanogaster over the larger host Musca domestica (Morris & Fellowes, 2002) and acceptance rates for oviposition were shown to be higher on the larger host Delia radicum (Goubault et al., 2004), yet P. vindemmiae does prefer D. melanogaster over three other dipterans (Wang & Messing, 2004). Even if P. vindemmiae has specialized to some degree on D. melanogaster, this was not reflected by an ability to manipulate its host’s physiology to increase resources. Another explanation for the lack of host manipulation and lipogenesis in P. vindemmiae is that this species might attain sufficient reserve levels for its survival and reproduction that foregoes the need to adapt to their host through physiological manipulation. During oviposition P. vindemmiae in- jects venom into the host that not only arrests host development, but also detrimentally affects other parasitoids developing inside the pupa (Wang & Messing, 2004). Moreover, once P. vindemmiae has exploited a patch it actively defends a patch from potential intra-specific competitors through fighting (Goubault, 2005). Even though the pupal parasitoid P. vindemmiae arrives later in a patch than the parasitoids A. tabida and L. heterotoma,

58 Host manipulation active defence against competitors and facultative hyperparasitism seem to provide a successful strategy for this parasitoid. Furthermore, in a study by Wang and Messing (2004) P. vindemmiae was shown to only partially consume large tephritid hosts and thus P. vindemmiae might be constrained by the extent it can physically consume host tissues. Although their study also showed that P. vindemmiae completely consumes D. melanogaster pupae, it is plausible that the higher lipid levels of primary parasitoids can only be consumed up to a certain level. This is consistent with the finding that lipid reserves are similar between P. vindemmiae developing on L. heterotoma (on day 21) and A. tabida. Following these observations, lipid exploitation is limiting yet sufficient for P. vindemmiae to find and parasitize hosts during its adult life without a need to synthesize lipids. In this species, the cost of converting carbohydrates obtained in the diet likely exceeds the benefits as fitness can be optimized regardless of lacking lipogenesis. The reciprocal perspective on the expected relationship between host range and lipogenesis would predict that P. vindemmiae can increase its lipid reserves, which is not supported by our findings. In a study by Le Lann et al. (2011) the specialist parasitoids Aphidius rhopalosiphi and A. picipes did not increase lipid levels in the adult stage, yet these parasitoids were found to lack manipulative abilities. Although manipulative capabili- ties are still expected to significantly contribute to the evolutionary loss of lipogenesis in parasitoids, in some systems other factors might contribute to the loss of this trait. Constraints in resource consumption or an overall sufficiency in resource levels obtained during larval feeding might render lipogenesis unnecessary for attaining a higher fitness. For example, some insects have entirely lost their feeding abilities as adults, as capital nutrient reserves provide sufficient resources for maintaining adult survival and reproduction (Gagné, 1994; Irwin & Lee Jr, 2000). We conclude that host manipulation ability of parasitoids has likely facilitated the evolutionary loss of lipogenesis in the majority of parasitoids (Visser & Ellers, 2008), yet in this study host manipulation was found not to be the driving force behind the evolutionary loss of lipogenesis in P. vindemmiae.

Acknowledgements

We would like to thank Cécile Le Lann, Femmie Kraaijeveld and Roland Allemand for providing insects and Gerard Driessen for providing helpful comments on earlier drafts of the manuscript. BV was supported by the

59 Chapter 4

Netherlands Organisation for Scientiﬁc Research (NWO), ALW-grant no. 816-01-013.

60 Chapter 5

Host exploitation eﬃciency in a gall wasp community

Bertanne Visser, Coby van Dooremalen, Alba Vazquez-Ruiz and Jacintha Ellers Submitted

Abstract Acquiring sufficient nutrients during developmental stages is essential for insects to allocate resources into survival and reproduction during the adult stage. The nutritional composition of food sources is, however, typically suboptimal for meeting the energetic demands of insects. Therefore, insects have evolved numerous mechanisms to overcome the constraints in nutrient levels and composition posed upon them. Acquiring sufficient resources is particularly important in insects that are unable to synthesize certain nutrient types de novo, as is the case in parasitoid species, many of which do not synthesize lipid reserves during adult life. Parasitoids therefore depend completely on the lipid reserves they acquire during larval development. In this study, we investigate host exploitation and lipid dynamics in a hymenopteran gall wasp community. Using the gall wasp Diplolepis rosae (Hymenoptera: Cynipidae) and associated parasitoids, we measured efficiency of host conversion and lipid exploitation between trophic levels, the ability for lipid synthesis and changes in fatty acid compositions over trophic levels. Our results showed that the majority of species within the rose gall community are highly efficient in exploiting resources with the percentage of dry body mass attained from the host ranging between 61 and 70% and that of lipid reserves between 53 and 70%. We further found that all species lacked lipid synthesis in their adult life-stage. With the exception of the parasitoid Or- thopelma mediator at the third trophic level, which was shown to alter its fatty acid composition, all species showed a high similarity in fatty acid composition to host plant and gall wasp. Our results suggest gall wasps and associated parasitoids are highly efficient in acquiring resources from their hosts, particularly lipid reserves. The manipulative nature of the galling lifestyle in terms of nutrient quality could explain why these systems are typically excessively exploited by species higher in the trophic cascade.

61 Chapter 5

Introduction

Phytophagy is one of the most common host exploitation strategies employed by insects (Janz et al., 2006; Lawton, 1983). As a consequence plant diversity has contributed greatly to the radiation of insects using plants as their main food source (Jaenike, 1990). Although plants provide a large pool of resources to sustain exploitation by phytophagous insects, many species face highly challenging conditions to sustain growth and survival on their host plants. For instance, many plants contain only low levels of nitrogen, or have physical and chemical defence barriers that can severely reduce exploitation success of phytophagous insects (Coley et al., 2006). Moreover, phytophagous insects are in turn excessively exploited by other organisms, such as predatory insects and insect parasitoids. Due to their intermediary position in the food web, phytophagous insects are of crucial importance in relaying the energy flow up to higher trophic levels (Futuyma & Agrawal, 2009) and an important question is whether nutrient concentrations increase through the nutrient flow over trophic levels. The suboptimal ratio of nutrients available in lower trophic levels requires insects to selectively attain certain resources over others (Behmer, 2009). For example, grasshopper nymphs that were offered a diet with varying ratios of protein to carbohydrate were shown to simultaneously regulate and balance protein and carbohydrate levels through consuming different meal numbers from diets diferring in nutritional composition (Chambers et al., 1995). Nutrient composition can further be adjusted once food has been acquired (Behmer, 2009). Such physiological processes following ingestion include selective absorption, differential egestion and altered metabolic processing (Anderson et al., 2005; Frost et al., 2005). A combination of these strategies has been found in grasshoppers: high levels of ingested protein led to increased egression, whereas high carbohydrate levels led to enhanced respiration rates (Zanotto et al., 1993, 1997). The elaborate mechanisms that insects have evolved to cope with unfavourable nutrient compositions allows them to successfully exploit their host plants. Acquiring sufficient quantities of nutrients during the larval stage is especially important for essential nutritional components, i.e. nutrients that cannot be produced de novo. For parasitoids, lipids form such an essential resource as parasitoids generally lack the ability to synthesize lipids de novo (Visser & Ellers, 2008; Visser et al., 2010). Lipid reserves are an important energy source to survive periods of food scarcity and typically all organisms readily synthesize lipids when excess carbohydrates are available in the diet.

62 Host exploitation eﬃciency

Parasitoids, however, form an exception to this general rule (Visser & Ellers, 2008). Parasitoids develop in or on other arthropods as larvae, but are free- living as adults (Godfray, 1994). Since the diet of adult parasitoids contains no or only few lipids, the main part of the necessary lipid reserves should be collected during the larval stage (Eijs et al., 1998; Giron et al., 2002; Giron & Casas, 2003). However, larval development is restricted to a single host, so contrary to predacious insects they cannot compensate for unfavourable nutrient ratios through selective feeding on several hosts. To deal with the dietary constraints posed by developing on a single host, parasitoid larvae have evolved intricate mechanisms to increase resource availability of the host through physiological manipulation (Nakamatsu, 2003; Nakamatsu & Tanaka, 2004; Rivers & Denlinger, 1994). Further- more, parasitoids are highly efficient in host exploitation, which is essential when dietary resources are limited. For example, the facultative hyperparasitoid Gelis agilis maintained 90% of the body mass of its host Cotesia glomerata when parasitizing at the fourth trophic level, and 75% at the fifth trophic level (Harvey et al., 2009). Moreover, specific fatty acids are required for different metabolic functions, hence acquiring high levels of certain fatty acids over others could be an important strategy to sustain specific metabolic functions in the adult stage. Similarly, butterflies acquire essential amino acids during larval development, while renewing nonessen- tial amino acids during their adult stage (O’Brien et al., 2002). The number of trophic levels that can be sustained within an insect community might therefore not necessarily be constrained by the amount of lipids carried over from the host, but can also be determined by the fatty acid composition of species at the base of the trophic cascade and the efficiency with which these fatty acids are carried over from the host. In this study, we look at host exploitation in a gall wasp community. Galls commonly sustain a large insect community with several parasitoid species attacking the gall inhabitants. Gall formation is initiated by the gall inducer or its progeny to increase nutrient availability and protection from predators and parasitoids (Hartley, 1998; Hartley & Lawton, 1992; Price & Pschorn-Walcher, 1988; Price et al., 1987). Gall wasps have evolved secondarily from a parasitic ancestor within Hymenoptera, and similar to their close parasitic relatives, gall wasps can substantially alter their host’s physiology in terms of nutrient composition. Gall induction leads to redirection of the plant’s physiology and growth (Shorthouse et al., 2005). For instance, a study by Harper et al. (2004) showed that galls induced by cynipid wasps attacking oak trees formed lipid-rich nutritive tissues lining the inner gall

63 Chapter 5 chamber for larval feeding by the insect. This suggests that the ability of gall wasps to manipulate and substantially increase their host’s lipid levels has evolved in response to similar nutritional constraints as parasitoids, i.e. development within a single gall. The question is how this has affected host exploitation efficiency of higher trophic levels such as parasitoids and hyperparasitoids We use the gall wasp Diplolepis rosae (Hymenoptera: Cynipidae), its associated inquiline Periclistus brandtii (Hymenoptera: Cynipidae) and parasitoids Orthopelma mediator (Hymenoptera: Ichneumonidae), Ptero- malus bedeguaris (Hymenoptera: Pteromalidae) and Torymus bedeguaris (Hymenoptera: Torymidae) to measure efficiency of host conversion and lipid exploitation between trophic levels, the ability for lipid synthesis, and changes in fatty acid composition over trophic levels. We predict a high conversion efficiency of body mass in each of the wasp species because they are confined to a single host. We expect all wasp species to lack lipogenic ability, as has been found in general for parasitoids, and to have a high exploitation efficiency of host lipids. Host manipulation to increase lipid reserves is not expected, however, since the hosts themselves are incapable of lipogenesis. Fatty acid composition is predicted to show similarity between trophic levels, which would be indirect evidence of direct consumption of host lipid reserves.

Materials and methods

Insects

The cynipid wasp Diplolepis rosae induces gall formation on plants of the Rosaceae family. D. rosae and its inquiline Periclistus brandtii are the main inhabitants of these galls and are attacked by several parasitoids, including Orthopelma mediator, Pteromalus bedeguaris and Torymus bedeguaris (Fig- ure 5.1). The parasitoid O. mediator mainly parasitizes gall-maker and has been found to only parasitize 2.8% of P. brandtii hosts (Randolph, 2005). P. bedeguaris can act as a primary parasitoid on D. rosae and as a secondary hyperparasitoid on both O. mediator and T. bedeguaris (Randolph, 2005). T. bedeguaris is able to parasitize gall-maker and occasionally the primary parasitoid O. mediator (Randolph, 2005). Galls containing D. rosae, its inquiline P. brandtii and parasitoids were collected near Wassenaar, the Netherlands and Lyon, France in September 2007 and 2008. Until wasp emergence, galls were subsequently placed at 20◦, 10◦, 5◦, 10◦ and 20◦ to

64 Host exploitation eﬃciency

Torymus bedeguaris Periclistus brandtii

Diplolepis rosae Rosa sp.

Pteromalus bedeguaris Orthopelma mediator

Figure 5.1: Overview of interactions within the gall community. Photograph courtesy of Robin Williams. mimic natural conditions during autumn, winter and spring. Prior to emergence, galls were individually placed in glass jars with foam stoppers at a temperature of 20◦, relative humidity of 75% and a photoperiod of 12:12 L:D.

Experiments

Emergence was monitored daily and freshly emerged females were placed singly in small jars containing moist cotton wool. Experiments were designed to determine feeding ability, lipogenic ability and lipid compositions of galls and wasps. To test feeding ability, freshly emerged females were randomly assigned to two treatments: starvation or honey-feeding for four days. To test lipogenic ability, females were either frozen at -20◦C or allowed to feed on honey for fourteen days. Fat amount was measured for single individuals of D. rosae, P. brandtii, O. mediator and P. bedeguaris using ether extraction as described in Visser et al. (2010). Low parasitism rates of T. bedeguaris led to insuﬃcient sample sizes to test feeding and lipogenic

65 Chapter 5 ability. To assess fatty acid composition freshly emerged individuals were frozen at -20◦C. Each sample contained three pooled individuals, or a single rose gall. The number of replicates for each species was: D. rosae n=6; P. brandtii n=3; O. mediator n=5; P. bedeguaris n=3; T. bedeguaris n=2; and galls n=3. Diﬀerences in sample sizes are reﬂected by the proportional parasitism levels between species.

Sample preparation for GC-FID

Samples were prepared following van Dooremalen et al. (2009). Fatty acids were extracted from the samples using dichloromethane/methanol (2:1 v/v). After vial headspace was flushed with nitrogen gas, 0.3 µg of C19:0 internal standard (Fluka) was added to each sample. Samples were saponified in a methanolized sodium hydroxide solution (45g NaOH, 150mL CH3OH, 150 mL milli-Q H2O) at 70◦C for 90 minutes (Chamberlain et al., 2004). Saponi- fication was followed by acid methanolysis in methanolized HCl (325mL 6.0N HCl, 275mL CH3OH) at 80±1◦C for 10 minutes. Methylated fatty acids were extracted using hexane/methyl tertiary butyl ether (1:1 v/v). This solution was dried with nitrogen gas and methylated fatty acids were dissolved in hexane and stored at -80◦C until further processing. Fatty acid composition analysis was performed using GC-FID equipped with a standard split/splitless injector (Agilent technologies 6890, Santa Clara CA, USA) and a polar BPX70 column (SGE International, 60m x 0.25mm i.d., df 0.25µm). Samples were injected (1 µL-aliquot) in the pulsed splitless mode. The temperature-programmed oven was set to 70◦ for two minutes, which then increased by 20◦C/min to 150◦C, continuing with a gradient of 15◦C/min to 250◦C during 10 minutes.

Analyses

Feeding experiments The amount of fat of each individual was calculated by subtracting dry weight after ether extraction from dry weight before ether extraction. Data were inspected for potential outliers prior to analysis as described in Visser et al. (2010). Normality of error structures was inspected and heterogeneity of variances tested using Levene’s test. Non-normal data were cube root transformed to normality. Datasets assuming normal distributions were compared using T-tests if variances were equal and Welch’s T-tests were used if variances were unequal. Statistical analyses were performed using R Project 2.12.

66 Host exploitation eﬃciency

Table 5.1: Dry weight and mean amount of fat at emergence for all species and eﬃciency of host exploitation on diﬀerent hosts.

Species Dry weight Mean amount of fat Eﬃciency on D. rosae Eﬃciency on O. mediator (mg ±1SE) (mg ±1SE) (body mass) (fat amount) (body mass) (fat amount) D. rosae 1.077 (0.099) 0.280 (0.029) - - - - P. brandtii 0.754 (0.110) 0.196 (0.041) 70 70 - - O. mediator 0.712 (0.048) 0.118 (0.016) 66 67 - - P. bedeguaris 0,437 (0.106) 0.099 (0.026) 41 35 61 53

Lipid composition data Data handling and analysis was done as described in van Dooremalen et al. (2010). Fatty acids were identified based on comparison of retention times between samples and a Supelco standard (Supelco 37 Component FAME Mix). Fatty acid concentrations below the detection limit were excluded from further analysis, i.e. C20:0 (22% missing values). All peak areas were log-ratio transformed (Nash et al., 2008) and multivariate statistics performed to explore changes in fatty acid composition. We used Principle Component Analysis (PCA) to obtain two principle components (PC1, PC2) for each sample using PAST software (Hammer et al., 2001). PC1 and PC2 scores were correlated to individual fatty acids using Pearson’s correlation coefficient to determine the contribution of each fatty acid to differences between species. Subsequently, we performed ANOVA and post-hoc Bonferroni correction to assess the effect of species on fatty acid compositions.

Results

Host exploitation eﬃciency

All parasitoids use D. rosae as the main host for development and the inquiline P. brandtii develops on similar resources as the gall wasp. P. brandtii was less efficient than D. rosae in attaining resources from the gall; both dry body mass and fat amount of P. brandtii were 70% of that found for D. rosae (Table 5.1). The primary parasitoid O. mediator obtained a total dry mass of 66% and fat reserves of 67% that of the host D. rosae. P. bedeguaris was least efficient in exploiting the gall wasp’s resources with a total dry body mass at 41% and 35% of D. rosae’s fat reserves. However, P. bedeguaris is also able to develop on O. mediator and T. bedeguaris and exploitation efficiency when O. mediator is assumed to be the host was 61% for dry body mass and 53% for fat reserves.

67 Chapter 5

0,35 NS ** *** NS

0,3 Starved 0,25 Fed 4 days

0,2

0,15

0,1 Amount of mg) (in fat Amount 0,05

0 D. rosae P. brandtii O. mediator P. bedeguaris

Figure 5.2: Mean amount of fat (±1SE) for starved (dark gray bars) and 4-day fed (light gray bars) females. NS = Not signiﬁcant; ** P < 0.01; *** P < 0.001.

Feeding ability

The amount of fat of the gall-inducer D. rosae was 0.284 mg (±0.028, 1SE) after starving for four days and 0.284 mg (±0.021, 1SE) after feeding for a similar duration. After four days fat levels did not differ significantly (Figure 5.2; t = 0.316, P = 0.753, n = 104), either suggesting this species did not economize on lipid-use when sugar sources were available or that it was unable to ingest food. Similarly, P. bedeguaris fat reserves were 0.050 mg (±0.016, 1SE) and 0.052 mg (±0.011, 1SE) after four days of starvation and feeding, respectively, without significant differences between treatments (Figure 5.2; t = 0.090, P = 0.930, n = 10). Fat reserves of the inquiline P. brandtii were 0.155 mg (±0.027, 1SE) after four days of starvation and 0.278 mg (±0.041, 1SE) after feeding, and were significantly differerent between the two treatments (Figure 5.2; t = 2.808, P < 0.01, n = 44). O. mediator fat amount was 0.052 mg (±0.009, 1SE) after starvation and 0.093 mg (±0.010, 1SE) after feeding. Fed females had significantly higher lipid levels compared to starved females (Figure 5.2; t = 3.889, P < 0.001, n = 84). Both P. brandtii and O. mediator are thus able to economize on their lipid reserves when food is available and capable of ingesting and utilizing dietary food sources as an adult.

68 Host exploitation eﬃciency

0,35 *** NS *** NS

0,3 Emergence 0,25 Fed 14 days

0,2

0,15

0,1 Amount of mg) (in fat Amount 0,05

0 D. rosae P. brandtii O. mediator P. bedeguaris

Figure 5.3: Mean amount of fat (±1SE) for females at emergence (dark gray bars) and after 14 days of feeding (light gray bars). NS = Not signiﬁcant; *** P < 0.001.

Lipogenic ability

None of the species increased their lipid reserves after fourteen days of feeding when compared to levels at emergence. D. rosae emerged with the highest fat amount (0.280 mg ±0.029, 1SE), while P. bedeguaris emerged with the lowest fat reserves (0.099 ±0.026, 1SE). Two species showed a significant decrease in fat reserves after 14 days of feeding: D. rosae (Figure 5.3; t = 8.8, P < 0.001, n = 35) and O. mediator (Figure 5.3; Welch’s t = 4.166, P < 0.001, n = 37). In the two other species fat reserves remained stable after feeding: in P. brandtii and P. bedeguaris there was no significant difference between the fat reserves at emergence and after feeding (Figure 5.3; P. brandtii: Welch’s t = 0.280, P = 0.787, n = 9; P. bedeguaris: t = 0.151, P = 0.884, n = 9). None of the species were shown to increase lipid reserves after feeding, hence all species within the gall wasp community lack lipogenesis as adults.

Fatty acid composition over trophic levels

The lipid fraction contained six saturated fatty acids (SFAs) ranging in carbon length between 12 and 20 C-atoms, two mono-unsaturated fatty acids (MUFAs), and three polyunsaturated fatty acids (PUFAs). The most abundant fatty acids were the SFA palmitic acid (C16:0), the MUFA oleic acid (C18:1n9c) and the two PUFAs linoleic and linolenic acid (C18:2n6c, C18:3n3)(Table 5.2).

69 Chapter 5 D. rosae P. brandtii O. mediator P. bedeguaris T. bedeguaris PC1scores PC2scores Species lation coefficient lation coefficient SFA 30,453 9,273 9,550 18,111 9,801 11,168 C12:0C14:0C15:0C16:0C16:1 0,741C17:0 -0,285C18:0 0,000 0,147 0,198 -0,546 0,514 -0,425 0,009 0,641 0,048 -0,399 0,799 0,001 -0,860 0,066 0,000 -0,816 0,000 -0,590 0,607 0,000 1,010 0,170 0,004 0,126 0,379 0,426 0,450 19,510 0,522 0,521 0,048 0,041 6,422 0,684 0,013 0,055 0,195 0,607 0,363 8,340 0,044 4,949 0,162 0,017 2,000 0,429 0,385 16,624 0,080 0,281 4,027 0,062 0,373 0,170 0,134 4,652 0,053 0,828 0,022 0,474 0,187 0,518 6,503 0,044 4,347 0,386 0,247 4,164 PUFA 42,194 53,499 47,152 36,375 51,226 48,946 MUFA 26,514 36,955 43,133 45,412 38,774 39,639 C18:3n3C20:4n6 -0,840 -0,777 0,000 0,000 0,449 0,408 0,036 0,059 10,610 30,698 0,206 0,082 23,850 0,147 25,271 0,103 23,712 25,759 0,172 0,172 C18:1n9cC18:2n6c -0,782 0,295 0,000 0,182 0,437 0,680 0,042 0,000 25,830 36,593 31,378 22,720 42,749 23,155 45,040 11,001 38,300 27,341 39,253 23,015 Fatty acid Pearson’s corre- P-value Pearson’s corre- P-value Rose gall Table 5.2: Pearson correlationproportion coefficients of of the each single single fatty fatty acids acid on of PC1 the and total PC2 extracted with fatty associated acids P-values. is For each given. species the mean

70 Host exploitation eﬃciency

Rose gall D. rosae P. brandtii O. mediator P. bedeguaris T. bedeguaris 1,500 a PC1 PC2 1,000 z z z b 0,500 b

0,000 b y

PCscores b -0,500 xy

-1,000 x c

-1,500

Figure 5.4: Principle component scores of PC 1 (mean ±1SE; dark gray bars) and PC2 (mean ±1SE; light gray bars) for all species. Different letters represent significant differences at the α = 0.05 level.

To compare the proportional abundance of these 11 fatty acids among trophic levels, we used principal component analysis of the variation in fatty acid composition. Two principal components were extracted. PC1 explained 46.4% of the variation, with high positive loadings of C12:0 and C18:0 and high negative loadings of C18:1n9c,C18:3n3 and C20:4n6. PC1 scores were signiﬁcantly higher for the rose galls than for all of the wasp species

(Figure 5.4; F5,16 = 21.68, P < 0.001). The largest difference was observed between the rose galls and the parasitoid O. mediator and this species also had significantly lower PC1 scores than the other wasp species. On average, the rose galls showed higher levels of C18:0 and C20:4n6 and lower levels of C18:1n9c and C18:3n3 than the wasp species (Table 5.2). The second principal component explained 30.3% of the eigenvalue variation. PC2 was positively correlated to the PUFA C18:2n6c, while it had negative loadings of the SFAs C14:0,C15:0 and C16:0. The rose galls differed significantly in PC2 scores from all wasp species, with the exception of O. mediator (Figure 5.4; F5,16 = 12, P < 0.001). The rose galls and O. mediator contained up to 2.5 and 4.2 times more C16:0 than the other wasps. In addition, the rose galls contained the highest levels of C18:2n6c when compared to the wasp species, while O. mediator contained the lowest levels of this fatty acid (Table 5.2).

Discussion

Galling has evolved numerous times throughout the insects and this strategy of host exploitation shows a high similarity to that of parasitoids that

71 Chapter 5 utilize only a single host during development. This study investigated host exploitation efficiency in a rose gall community in terms of acquiring body mass and lipid reserves from the host, the ability to synthesize lipids as adults and changes in fatty acid composition between trophic levels. The gall wasp D. rosae was shown to obtain the highest dry body mass from development on a single rose gall. All wasp species were shown to have lower body masses, yet they were still highly efficient in carrying over resources with a range of dry weights from 61 to 70% that of their host, with the exception of P. bedeguaris developing on D. rosae that attained a dry weight of 41%. High conversion efficiencies have been observed in other parasitoids, for instance, the parasitoid Venturia canescens showed an overall conversion efficiency of host resources of 60% (Howell & Fisher, 1977). Another study on parasitoids further revealed that even higher efficiencies were found up to the fifth trophic level (Harvey et al., 2009, 2011), even though exploitation efficiency is typically much lower in other animals, ranging between 20 to 40% (Howell & Fisher, 1977). The adaptation of gall wasps to developing on a single host has thus resulted in high exploitation efficiencies, similar to that observed in parasitoids. We found two wasp species were unable to conserve on lipid-use when food was available during four days, suggesting that these species do not feed at all or sugar sources are not used to delay the need to burn their lipid reserves. The gall wasp D. rosae does not economize on its reserves, conforming to previous observations that suggest this species refrains from feeding as an adult. Hence this species does not benefit from the provision of sugar sources (Randolph, 2005). In a similar manner, our results suggest that the parasitoid P. bedeguaris refrains from feeding or utilizing food. Regardless of feeding ability, we found none of the wasps were increasing their lipid reserves, hence all species tested within this community lack de novo lipid synthesis and do not increase lipid reserve levels during life. Across trophic levels the absolute amount of fat decreases, although similar to our observations of dry body mass, conversion efficiencies of lipid reserves are relatively high compared to other animals, ranging from 61 to 70% in our wasp species. One potential consequence of decreasing fat reserves is that body size decreases higher in the trophic cascade. Dry weight decreases with increasing trophic level, however, we did not measure body size directly and body size could potentially decrease at higher trophic levels. Another consequence of decreasing fat reserves higher in the trophic cascade could be that lipid levels are at the threshold of sustaining sufficient allocation of reserves into survival and reproduction in the adult stage. At

72 Host exploitation eﬃciency some point, lipid reserves might be too low and it could be expected that this constraint might lead to a switch in lipogenic strategy towards active lipogenesis in adults, as has been observed in other parasitoids (Visser et al., 2010). Fatty acid compositions were similar between all wasp species, with the exception of O. mediator. O. mediator distinguishes itself from the other species by the high level of palmitic acid (C16:0) and low levels of linoleic acid (C18:2n6c). Palmitic acid is produced during fatty acid synthesis, and high levels suggest an active lipogenesis pathway. However, our results from the feeding experiment showed that O. mediator does not accumulate lipids when fed, therefore the high level of palmitic acid is either due to direct uptake from its host, or through modiﬁcation of other fatty acids, such as

C18:1n9c and C18:3n3. Since the level of palmitic acid is lower in the host D. rosae, low levels of linoleic acid (C18:2n6c) suggest that O.mediator takes over this fatty acid from its host that is further saturated and reduced to form palmitic acid. Although parasitoids that actively synthesize fatty acids have been shown to elongate and desaturate fatty acids (Barlow & Bracken, 1971; Thompson & Barlow, 1972), this is the first report on the ability of a parasitoid lacking lipid synthesis to alter its fatty acid composition. We found, furthermore, that all wasp species had substantially higher levels of oleic (C18:1n9c) and linolenic acid (C18:3n3) when compared to the rose gall. High levels of these fatty acids found in all wasps could be due to an increase in fatty acid levels within the nutritive tissue of the gall that is consumed by the larvae (Tooker & De Moraes, 2009). These nutritive tissues line the inner part of the gall chamber in which the larvae develop, thus nutrient levels tend to vary throughout the gall, in which only nutritive cells are consumed by galler and inquiline (Harper et al., 2004). Our analysis of fatty acid composition within the gall might therefore reflect the composition in the outer layer of the galling tissue or only partially that of the nutritive cells, since wasps are expected to have consumed the majority of nutritive cells at the time of sampling. Another explanation for high levels of oleic and linolenic acid in all species could be that all wasps alter their fatty acid composition. Biosyn- thesis of linoleic and linolenic acid has been observed in some insect species (Cripps et al., 1986), yet synthesis of this essential fatty acid has not yet been reported for any hymenopteran. This leads to the expectation that the observed distribution of oleic and linolenic acid in comparison to other fatty acids within wasps reflects the composition within nutritive tissue of the gall, rather than modification of fatty acids, hence it is likely that lipid

73 Chapter 5 reserves are directly carried over from the host. Other studies have shown either that the fatty acid composition is reflected by the diet, such as observed in the common jellyfish Aurelia aurita (Fukuda & Naganuma, 2001) or that the fatty acid composition can be modified higher in the trophic cascade, as has been observed in a terrestrial predator-prey system (Cakmak et al., 2007a). Our results further indicate that levels of arachidonic acid

(C20:4n6) increase over trophic levels, an important fatty acid for egg laying and immunity, as this fatty acid forms the precursor of eicosanoids (Stan- ley, 2006), suggesting that this fatty acid is acquired through elongation and desaturation of other fatty acids. In our system, the majority of fatty acids are carried over from the host and the overall amount of lipid reserves is efficiently carried over from the host, but decreases at higher trophic levels, suggesting the amount of lipids acquired from the host rather than fatty acid composition might determine exploitation potential higher in the trophic cascade. High quality resources maintained through nutrient regulation of the plant makes galling insects prime candidates for exploitation by other insects at higher trophic levels. Nutrient concentrations are expected to increase with trophic level and could potentially aid in attaining high resource levels, as is the case for parasitoids with high exploitation efficiencies. A high efficiency in resource exploitation could potentially increase food web complexity, particularly the length of the food chains, although it remains to be tested how exploitation efficiency and certain nutrient pools are of importance in determining the length of trophic cascades. Future studies should reveal how the nutrient flow over trophic levels is affected in galling insects and their communities and determine the potential of these systems to sustain a larger number of trophic levels that are typically observed in food webs.

Acknowledgements

We would like to thank Eric Kok for his help during field collection, Cécile Le Lann and Jeff Harvey for discussion and helpful comments on earlier drafts of the manuscript and Robin Williams for providing us with pictures of the gall community. BV was supported by the Netherlands Organisation for Scientific Research (NWO), ALW-grant no. 816-01-013.

74 Chapter 6

Lack of transcription of the key gene in lipid synthesis, fatty acid synthase, reﬂects loss of lipogenesis in adult parasitic wasps

Bertanne Visser, Dick Roelofs, Daniel A. Hahn, Peter E.A. Teal, Janine Mariën and Jacintha Ellers Submitted Abstract Loss of redundant morphological, behavioral or physiological traits is a common process contributing to evolutionary dynamics, but studies linking the evolutionary loss of redundant traits to the molecular decay in the genome are rare. The majority of parasitic insects do not accumulate lipid reserves during the adult life-stage. It is hypothesized that host manipulation during larval development renders lipid synthesis redundant. Manipulation of the host’s physiology allows parasitoids to acquire lipids directly from their hosts, leaving metabolic pathways involved in parasitoid lipid synthesis prone to phenotypic regression. Here, we study transcriptional changes associated with loss of lipogenesis in the parasitic wasp Nasonia vitripennis. We first confirmed the lack of lipogenesis in N. vitripennis by showing a reduction in lipid reserves despite ingestion of dietary sugar, and a lack of incorporation of isotopic labels into lipid reserves when fed deuterated sugar solution. Second, we investigated transcriptional patterns of 28 genes involved in major nutrient metabolic pathways in short- and long-term sugar-fed females relative to starved females of N. vitripennis. Numerous genes involved in carbohydrate metabolism had a lower transcription in fed than in starved females. Sugar-feeding did not induce transcription of fatty acid synthase (fas), a critical gene involved in the lipid biosynthesis pathway. We further compare our findings to gene transcription of orthologous genes in Drosophila melanogaster, a species that actively synthesizes lipids. Our results reveal that N. vitripennis gene transcription involved in sugar and lipid pathways deviates severely from that of D. melanogaster, mainly through unresponsiveness of fas to dietary sugar in N. vitripennis. This study is the first to identify major changes in gene transcription that underlie the loss of lipogenesis in parasitic insects and provides new insights into the molecular mechanisms underlying trait loss.

75 Chapter 6

Introduction

Phenotypic degradation and loss of morphological, behavioral or physiological traits are common processes contributing to evolutionary trait dynamics (Fong et al., 1995; Porter & Crandall, 2003). Trait loss typically occurs when a trait is selected against or when redundancy renders a trait selectively neutral (Lahti et al., 2009). For example, sexually selected male ornamental traits that were once maintained by sexual selection can be lost after release from selection through environmental changes (Endler, 1983; Saetre et al., 1997; Wiens, 2001). The molecular mechanisms responsible for trait loss include mutation accumulation in the gene underlying a trait, distortions of gene regulatory mechanisms, and deletion of genes or partial genome losses (Dale & Moran, 2006; Maughan et al., 2007). Trait losses are regularly discovered when the phenotype is severely affected, but trait loss frequently has minor effects on the phenotype due to redundancy of the trait (Visser et al., 2010). The increasing availability of genome sequence information should allow for a more precise evaluation of the mechanisms underlying trait loss. Parasitic insects are rapidly becoming model systems to study the evolutionary and ecological consequences of trait loss. Numerous studies have demonstrated that different species of parasitic wasps and flies have lost the ability to synthesize lipids de novo in their adult life-stage (Casas et al., 2003; Ellers, 1996; Fadamiro & Heimpel, 2001; Giron & Casas, 2003; Lee et al., 2004a; Olson et al., 2000; Rivero & West, 2002; Visser & Ellers, 2008). Although parasitic insect species are capable of utilizing dietary carbohydrates to meet immediate energy demands (Eijs et al., 1998; Jervis et al., 2008), the conversion of such carbohydrates to long-term storage in the form of lipids is impaired. It has been suggested that de novo lipid synthesis has become redundant in parasitic insects because host manipulation results in increased lipid levels in the host that are subsequently taken up by the parasitoid (Visser & Ellers, 2008). The molecular mechanism underlying lack of lipogenesis has not yet been resolved, but phylogenetic analysis revealed that loss of this essential metabolic trait has evolved independently in parasitic insects of three different orders (Visser et al., 2010). The recurrent loss of lipogenesis is remarkable since major metabolic pathways are typically highly conserved across taxa (Arrese & Soulages, 2010; Grönke et al., 2005; Turkish & Sturley, 2009). Given the recent completion of the full genome sequence of the parasitic wasp Nasonia vitripennis (Werren et al., 2010), this species offers an excellent opportunity to study regulatory and

76 Lipogenesis lost through lack of transcription structural genetic changes underlying trait loss in parasitic insects. N. vitripennis feeds on nectar and host haemolymph during its free- living adult life, but does not convert these carbohydrate-rich food sources into fatty acids and triglycerides for lipid storage (Rivero & West, 2002). The conversion of glucose to triglycerides involves three different pathways, each of which is a candidate for harboring genetic changes causing the evolutionary loss of lipogenesis (Figure 6.1). Ingestion of glucose first activates the glycolytic pathway that produces pyruvate from glucose (Scrutton & Utter, 1968). Second, through several enzymatic steps pyruvate is then converted into acetyl coenzyme A (acetyl-CoA), an important intermediary metabolite in many metabolic processes. To synthesize fatty acids de novo acetyl-CoA is then carboxylated to malonyl CoA by acetyl-CoA carboxylase (ACC), a substrate used by the multi-domain enzyme fatty acid synthase (FAS) to form fatty acids through a multi-step process (Wakil, 1989). Thirdly, these fatty acids are the raw materials used in the formation of more complex glycerolipids, such as membrane and storage lipids. A prime candidate gene to explain the loss of lipid synthesis is fatty acid synthase (fas), a highly conserved gene, the functioning of which is essential for synthesis of palmitic acid. Palmitic acid serves as the precursor for various lipid types, such as short- and other long-chain fatty acids used in glycerolipid synthesis. Absence of fas has been associated with the evolutionary loss of lipogenesis in the parasitic fungus Malassezia globosa (Xu et al., 2007). Deficiencies of another gene involved in fatty acid synthesis, acetyl-CoA carboxylase (acc), result in severely reduced lipid levels in mice, or even lethal effects during embryonic development (Abu-Elheiga et al., 2001, 2005), signifying the crucial importance of a functional lipogenesis pathway in animals. Finally, lack of lipid accumulation may be due to disruption of triglyceride synthesis, as was found in mice with a null mutation and rearrangement of their lipin-1 gene (Csaki & Reue, 2010). Mutations in lipin-1 inhibit phosphatidate phosphatase activity, an essential enzyme in the formation of diglycerides prior to triglyceride synthesis (Carman & Han, 2006). Lack of lipogenesis in N. vitripennis could thus result from reduced or inhibited functioning of one or several genes within either fatty acid or triglyceride synthesis pathways. Here, we aim to unravel the transcriptional profile associated with lack of lipogenesis in the parasitic wasp N. vitripennis. First, we confirm the phenotypic observation that adult wasps do not accumulate lipids by comparing lipid levels in fed and starved wasps at several time points during adult life. We further confirmed that adult wasps do not synthesize storage

77 Chapter 6

Glycolysis/Gluconeogenesis

Glucose Pentose-phosphate pathway Glycogen synthesis

D-6-P-Glucono-1,5-Lactone -D-Glucose-6P -D-Glucose-1P g6pd ugpase pgi

NADPH -D-Fructose-6P UDP-glucose pfk Glycerolipid metabolism Glycogen Glycerone-P -D-Fructose-1,6-P2

gpdh gpat D-Glyceraldehyde-3P Acylglycerol-3P Glycerol-3P

agpat gk

1,3-P2 Glycerate Diacylglycerol-1P Glycerol

pl-d 3-P-D glycerate Phosphatidylcholine pp pgm

2-Phosphoglycerate Phosphatidylethanolamine eno Diacylglycerol TCA cycle Phosphoenolpyruvate pepck dgat tgl Fatty acids pyk Oxaloacetate Triacylglycerol pc Pyruvate

Acetyl coA metabolism cs Hydroxyethyl TPP atpcl Citrate

Acetyldihydrolipoamide

dlat

Acetyl CoA Acetate Fatty acid synthesis accoas

acc

Malonyl CoA

Glycerolipid metabolism fas

fas scd Palmitate Palmitoyl ACP Octadecanoyl ACP Oleoyl ACP

lcfacs

Palmitoyl coA

Figure 6.1: Key nutrient metabolic pathways involved in lipids synthesis. Genes from pathways other than carbohydrate, fatty acid and glycerolipid metabolism include AMP activating protein kinase (ampk), cGMP-dependent protein kinase (pkg), and lipid storage droplet-2 (lsd2 ). For a list explaining abbreviations see tables 6.2 to 6.4

78 Lipogenesis lost through lack of transcription lipids by feeding females isotopically labeled solution of sugar in deuterated water showing no incorporation of isotopes in lipid reserves by GC-MS. Second, we compared gene transcription patterns in metabolic pathways of importance for lipid synthesis, as induced after short-term (2 to 8 hours) and long-term (1 to 3 days) sugar-feeding or starvation. Using quantitative RT- PCR assays, we examine the transcription of 28 key genes contributing to lipid synthesis, including genes involved in carbohydrate metabolism, fatty acid and glycerolipid metabolism. To relate our findings to observations in a species with fully functional lipogenic abilities, we compare our findings to gene transcription patterns of orthologous genes in the fruit fly D. melanogaster using data of a previously published microarray experiment (Zinke et al., 2002) comparing sugar-fed with starved D. melanogaster. This is the first study to assess the deviations in transcriptional profile associated with the lack of lipid synthesis.

Materials and methods

Lipogenic ability at the phenotypic level

Strain AsymC of N. vitripennis was obtained from an existing laboratory culture at the University of Rochester, NY, USA (van den Assem & Jach- mann, 1999). Insects were kept at a temperature of 25◦C, RH 75% and a photoperiod of 16:8 L:D. For experiments 6 females were allowed to oviposit on 6 pupae of the ﬂesh ﬂy Sarcophaga bullata (Diptera: Sarcophagi- dae)(Carolina Biological Supply Company). After emergence from host pupae individual females were randomly assigned to treatment tubes. To test whether female N. vitripennis lack lipogenesis, lipid levels were measured in four treatments: 1) at emergence, 2) after three days of starvation with access to water on cotton wool, and after 3) three and 4) seven days of feeding on a 10% (w/v) sucrose solution. For 8 to 18 females per treatment, fat content was determined following the method of David et al. (1975). Fe- males were dried for 5 days at 70◦C after which dry weight was determined. Females were subsequently placed individually in a glass tube containing 4mL of ether. After 24 hours ether was removed and females washed with fresh ether. Insects were dried for 5 days at 70◦C after ether extraction and dry weight determined again. To trace the fate of isotopic labels, 5 females per treatment were fed with a 10% (w/v) sucrose solution in water as a control or sucrose solution with added deuterium oxide (Sigma-Aldrich) at 50% (v/v) of total water

79 Chapter 6 added. After 7 days females were frozen at -20◦C until further processing. To compare our findings with a species that accumulates lipids as adults, we used Apis mellifera as a positive control. Freshly emerged A. mellifera were collected from an existing colony at CMAVE, Gainesville, FL, USA. Insects were kept at a temperature of 20◦C, a relative humidity of 40% and in complete darkness. Treatments were similar to those described for N. vitripennis, but bees were frozen at -20◦C until further processing after 4 days of feeding. Lipids were extracted and fractionated for A. mellifera females following the method described by Wessels & Hahn (2010). For N. vitripennis neutral and polar fractions were separated after application of 4mL of chloroform and 3mL of methanol, respectively. For GC-MS analyses only neutral lipid fractions containing triglycerides were used. To prepare samples for GC-MS, 10µL of a 1µg/µl solution of heptade- canoic acid in methylene chloride (Sigma) was added to lipid fractions as an internal standard, after which samples were dried under a stream of nitrogen. 100µL methanolic HCl (Supelco) was added and heated for 15 minutes at 65◦C. Methanolic HCl converts all fatty acids, including free fatty acids, di- and triglycerides, within the sample into methylesters. After cooling at room temperature 1mL of pentane was added and the vial vortexed for 1 minute prior to centrifugation for 8 minutes at 18,000 g. The pentane layer was removed for analysis. Routine chemical analyses were conducted using chemical ionization mass spectroscopy (CIMS, isobutane reagent gas) with an Agilent 5975C® MS interfaced to Agilent 7890A® gas chromatograph (GC). The GC was equipped with a cool-on-column injector fitted with a 10cm length of 0.5mm (id) deactivated fused silica tubing which was in turn connected to 1m x 0.25mm (id) length of deactivated fused silica tubing as a retention gap. The retention gap was connected to a 30m x 0.25mm (id, 0.25µm Coating thickness) DB5MS® analytical column. The conditions of chromatography were: Initial oven and injector temperature = 30◦C, 5 min; oven and injector temperatures increased at 10◦C/min; final temperature = 225◦C. We also obtained total ion spectra (60-500amu). Electron impact spectra (60-300amu) were obtained using an Agilent 5975B® instrument interfaced to a 7890 GC® equipped as above except that the analytical column used was a 30m x 0.25mm DB1MS® (id, 0.25µm Coating thickness). For analyses we compared fragmentation patterns and retention times with those of authentic standards. The base peak for straight chain methylesters using CI-MS with isobutane as reagent gas is the result of addition of a pro- ton to the ester resulting in a m+1 fragment. Thus although m for methyl palmitate (C16:0) is m/z=270 the parent ion is m/z=271. We took this

80 Lipogenesis lost through lack of transcription adduct effect into account when assessing mass label incorporation into fatty acids by the insects. Thus, for an addition of 1 deuteron to methyl palmitate we used abundance of m/z= 272, for m+2 we used m/z=273 and so forth. We also analyzed selected samples by electron impact mass spectroscopy to confirm identities of the methylesters. For these studies we used an Agilent 5975B® instrument interfaced to a 7890 GC® equipped as above except that the analytical column used was a 30m x 0.25 mm (id, 0.25 µm Coating thickness) DB1MS®. As in CI-MS studies retention times and fragmentations patterns were used to confirm identities of natural esters.

Statistical analysis of experiments testing lipogenic ability at the phenotypic level

The amount of lipid per female was calculated by subtracting dry weight after ether extraction from dry weight before ether extraction. We used the percentage of lipids to correct for diﬀerences in body size. Normality was inspected using the error structure of the data and homogeneity of variances determined using Levene’s test. Data was log-transformed to normality and equal variances. To compare treatments, we used ANOVA followed by a Tukey test to correct for multiple testing. To analyze isotopic labeling data, we divided the m/z abundance by the C17 internal standard for that sample and calculated the amount of methyl palmitate in ng for each sample. For N. vitripennis abundances of m+4 to m+6 were too low to accurately detect with sample amounts below 0,05 ng/sample. Normality was inspected using the error structure of the data and homogeneity of variances was determined using Levene’s test. We performed T-tests to compare the deuterated sugar water treatment with the sugar water control when variances were equal and Welch’s T-test if variances were unequal. All statistical analyses were done using SPSS 14.0.

Gene transcription experiment

Strain AsymC of the parasitic wasp N. vitripennis (Hymenoptera: Pteroma- lidae) was obtained from a laboratory culture at the University of Gronin- gen, the Netherlands. Insects were kept at a temperature of 25◦C, RH 75% and a photoperiod of 16:8 L:D. For the experiments 20 to 40 females were allowed to oviposit during 24 to 48 hours on 20 pupae of the blowﬂy Cal- liphora sp. (Diptera: Calliphoridae) in glass jars sealed with foam stoppers. Jars were inspected daily between 9 and 11am for newly emerged individuals. Emerged females were randomly assigned to treatments. Females in

81 Chapter 6 starvation treatments were allowed to feed on water only presented on a piece of wet cotton wool; females in feeding treatments were allowed access to water on cotton wool and honey ad libitum applied to the foam stoppers. We applied two feeding treatments: 1) the short-term treatment, in which emerged females were starved for 24 hours and subsequently either starved or fed for a short-term 2, 4, 6 or 8 hours; and 2) the long-term treatment, in which newly emerged females were immediately starved or fed for 1, 2 or 3 days. Ten females per treatment were snap-frozen in liquid nitrogen and stored at -80◦C for further qRT-PCR analysis. Each treatment consisted of three biological replicates. RNA was isolated using the SV Total RNA isolation system (Promega) according to the manufacturer’s protocol. Successful isolation was confirmed by visual inspection of ribosomal RNA on a 1% agarose gel and RNA quantities were determined using a nanodrop ND-1000 spectropho- tometer (Nanodrop Technologies). Nanodrop 260-280nm and 260-230nm ratios were inspected to assess protein and organic salt contamination. Po- tential DNA contamination was tested using 1µL of RNA and a PCR with Taq-polymerase, using the primer set of phosphoeolpyruvate carboxykinase (pepck), of which the product was run on a 2% agarose gel. Total RNA quantities of clean samples ranged between 50 and 200 ng/µl and were further diluted to a concentration of 50 ng/µl for each sample. cDNA synthesis was done using the M-MLV Reverse Transcriptase system (Promega). cDNA was diluted 8x and stored at -20◦C until further processing. Relevant gene functions were obtained by searching KEGG (Kanehisa & Goto, 2000) and orthologs of N. vitripennis for metabolic genes of interest retrieved from GenBank. Primers for candidate and reference genes were designed using the program Primer Express 1.5 (Applied Biosystems). Program settings were according to Roelofs et al. (2006). GenBank accession numbers, primer sequences and efficiencies are listed in supplementary table S6.1. In order to determine PCR efficiency, standard curves were obtained in triplicate for the qRT-PCR primer set with fourfold dilutions of a reference batch of cDNA (Pfaffl, 2001). For each qRT-PCR reaction a total volume of 20µl was used consisting of 2µl cDNA template, 10µl SYBR Green (SensiMix™ SYBR No-ROX kit, Bioline), 1µl of forward and reverse primer (20 pmol, Eurofins MWG Operon) and 7µLH2O. qPCR cycling was performed on a DNA Engine Opticon 1 (Biorad) with three replicates per sample. Cycling settings were programmed according to Roelofs et al. (2006). Reference genes were selected using a pilot dataset consisting of a subset of 8 treatments. The pilot included 5 potential reference genes: elon-

82 Lipogenesis lost through lack of transcription gation factor 1 alpha (ef1a), ribosomal protein 49 (rp49 ) (Loehlin et al., 2010), ubiquitin conjugating enzyme (ubc), alpha tubulin (at) and V-type ATPase (atpase), and 3 target genes: pyruvate kinase (pyk), fatty acid synthase (fas) and diacylglycerol o-acyltransferase (dgat). We used the geNorm analysis application as available in the software package GeNex Light to se- lect the most suitable reference genes (MultiD Analyses AB, 2008). Stable reference genes in our pilot experiment were ef1a and rp49.

Statistical analysis of qRT-PCR data

Opticon Monitor 3 software (Biorad) was used to calculate Cycle threshold (Ct) values. The cycle threshold was set at 0.03 at the beginning of the exponential phase of the curve for all assays. Ct values of 3 technical replicates were averaged if the standard error percentage did not exceed 20%. If a standard error percentage exceeded 20% all curves were inspected and the deviating curve removed. For all assays, averages of at least two technical replicate Ct values were used. Ct values were corrected for primer efficiency and normalized based on the formula described by Simon (2003) and using the geometric mean of the two reference genes (Vandesompele et al., 2002). For both short- and long-term treatments, we performed two-way ANOVA. Except for atpcl within the long-term feeding treatment, we did not find any significant interaction between treatments (fed vs. starved) and time, nor did we find an effect of time as main effect. Consequently, we performed one-way ANOVAs for all genes per time series. Normality was inspected using the error structure of the data and homogeneity of variances was determined using Levene’s test. Non-normal data or data with unequal variances were log2 transformed. If log2 transformation did not improve normality or variance the non-parametric Mann-Whitney U test was applied.

Statistical analysis of D. melanogaster microarray data

To compare transcription patterns observed in N. vitripennis with a species capable of lipogenesis, we used D. melanogaster microarray data from Zinke et al. (2002). In this study the authors performed a microarray study of sugar-fed and starved larvae at 3 time points: 1 hour, 4 hours and 12 hours. We selected D. melanogaster orthologs of our candidate genes from Aﬃmetrix datasets by comparing amino acid sequences of N. vitripennis to that of D. melanogaster using the BLASTP search in KEGG and selected the closest matching amino acid sequence of D. melanogaster as orthologs

83 Chapter 6 with highest similarity to our candidate genes. We found D. melanogaster orthologs with similar annotations for all our candidate genes. From the Affymetrix datasets, we used the ‘Average difference’ for calculations. This value represents the average intensity of different probe sets for one gene after global normalization and scaling. We performed similar analyses on these data as described previously for N. vitripennis gene transcription data.

Results and Discussion

Lack of lipogenesis, isotope tracing and gene transcription in N. vitripennis

N. vitripennis females emerged with an average of 16.3% (±1.1%, 1SE) lipids. After sugar feeding, lipid levels declined to 7.6% (±0.7%, 1SE) and 6.9% (±0.8%, 1SE) after 3 and 7 days, respectively (Figure 6.2). The lowest lipid levels were found in starved females with 4.8% (±0.5%, 1SE) lipids. Reserve levels were significantly different between treatments (F3,53 = 25.728; n = 57, P < 0.001), with significantly lower lipid levels after starvation, 3 days, and 7 days of sugar feeding compared to females at emergence (Tukey: P < 0.01 for all comparisons), thus confirming the lack of lipogenesis in N. vitripennis (Rivero & West, 2002). We further found that females fed sugar for 3 days had significantly higher lipid levels when compared to females that were starved for a similar duration (Tukey: P = 0.017), suggesting that N. vitripennis females successfully ingested food, leading to a reduced rate of lipid expenditure when sugar was available. No increase in the quantity of isotope labels was detected in palmitic acid, an abundant representative fatty acid in the neutral lipid fraction of N. vitripennis when females were fed sugar and deuterated water compared to females on a sugar control lacking isotopic labels (Table 6.1). An identical labeled isotope diet did lead to significantly increased levels of isotopes incorporated in the storage lipids in the non-parasitic hymenopteran Apis mellifera, which is known to readily synthesize fatty acids de novo (Table 6.1). These findings provide strong evidence that N. vitripennis does not synthesize fatty acids when fed sugars, neither to accumulate lipids nor to balance an increased catabolism of fatty acids. The gene transcription assays showed that 10 out of 28 candidate genes in nutrient metabolic pathways were differentially transcribed after short- term feeding compared to starvation (Figure 6.3a; Table 6.2 and 6.3). In the short-term treatment, 1-day old starved females were either fed with

84 Lipogenesis lost through lack of transcription

8 Lipid levels (%) levels Lipid 6

0 Emergence Starved 3 days Fed 3 days Fed 7 days

Figure 6.2: N. vitripennis phenotypic response to food. Mean % lipids (±1SE) for N. vitripennis females at emergence, three days of starvation and three and seven days after sugar feeding.

Table 6.1: Results of isotope tracing into the lipid fraction through synthesis of palmitate (C16:0)

Nasonia vitripennis n=5 per treatment Water Deuterated water Added deuterons Mean ng/sample (±1SE) Mean ng/sample (±1SE) t-value p-value m+1 210.047 (26.368) 187.707 (39.535) 0.470 0.651 m+2 22.453 (2.730) 21.559 (4.410) 0.172 0.867 m+3 1.527 (0.427) 1.946 (0.392) -0.721 0.491 m+4 - - - - m+5 - - - - m+6 - - - - Apis mellifera n=5 per treatment Water Deuterated water Added deuterons Mean ng/sample (±1SE) Mean ng/sample (±1SE) t-value p-value m+1 936.773 (50.396) 1165.238 (215.928) -1.030 0.333 m+2 103.344 (5.580) 167.777 (25.980) -2.425 0.042 m+3 11.076 (2.519) 33.478 (2.775) -5.977 < 0.001 m+4 0.108 (0.047) 0.882 (0.074) -8.807 < 0.001 m+5 0.260 (0.056) 1.217 (0.491) -1.937a 0.123a m+6 0.611 (0.057) 0.825 (0.375) 0.102 0.588 aIndicates the result of Welch’s T-test.

85 Chapter 6 honey for 2, 4, 6, or 8 hours or kept without food for the same length of time. No significant effect of time was detected, nor did we find any significant interaction between treatments (fed vs. starved) and time, hence in the analysis we combined the samples from different time points. Numerous genes involved in carbohydrate metabolism had lower transcript abundance in fed than in starved females. These genes included phosphofructo kinase (pfk), phosphoglucose isomerase (pgi) and phosphoglucose mutase (pgm), located at the beginning and end of the glycolytic pathway. Furthermore, the gene UDP-glucose phosphorylase (ugpase) involved in glycogen synthesis had lower transcript abundance in fed compared to starved females. Two genes involved in the glycolytic pathway and tricarboxylic acid cycle (TCA cycle) also had a higher transcription in starved females, phosphoenolpyruvate carboxykinase (pepck) and citrate synthase (cs). Pepck is an important enzyme in glucose homeostasis, while cs performs the first step of the TCA cycle (Figure 6.1). Our finding that the majority of genes involved in carbohydrate metabolism and TCA cycle were transcribed at a higher rate in starved females is consistent with the response to starvation observed in other animals (Duplus & Forest, 2002; Kersten, 2001). First, starvation causes an acute shortage of glucose that typically leads to an activation of the gluconeogenesis pathway, increasing the availability of glucose. Starvation is known to induce phosphorylation of pyruvate kinase which inhibits its activity and prevents phosphoenolpyruvate to be converted to pyruvate. The increased transcription of pepck, which catalyzes the reverse reaction, is the rate limiting step for converting pyruvate to glucose in a series of gluconeogenesis reactions (Reshef et al., 2003) (Figure 6.1). Second, starvation requires catabolism of triglycerides to increase the availability of free fatty acids for use in other metabolic pathways and for β-oxidation to release energy. While pepck plays a pivotal role in gluconeogenesis, this gene is further involved in glyceroneogenesis, an alternative pathway that produces glycerides from pyruvate by using an alternative precursor than glucose. Glyceroneogenesis allows re-esterification of fatty acids during lipolysis, thereby restraining loss of free fatty acids (Reshef et al., 1970, 2003). An increase in levels of unsaturated and long-chain fatty acids can, furthermore, act as signals involved in gene regulation, for instance by stimulating transcription of pepck (Duplus & Forest, 2002).

86 Lipogenesis lost through lack of transcription

A 4,5

3,5

2,5

1,5 -log10 -log10 p-value

0,5

0 -2 -1,5 -1 -0,5 0 0,5 1 1,5 2 Fold Change

B 4,5

3,5

2,5

-log10 -log10 p-value 1,5

0,5

0 -2 -1,5 -1 -0,5 0 0,5 1 1,5 2 Fold Change

C 4,5

3,5

2,5

1,5 -log10 -log10 p-value

0,5

0 -4 -3 -2 -1 0 1 2 3 4 Fold Change

Figure 6.3: Gene transcription in relation to significance. Volcano plots showing fold changes calculated using the log2 ratio of the Mean Normalized Expressions (MNE) of fed vs. starved females (x-axis) of each gene against the level of significance (-log10 P- value, y-axis) for N. vitripennis after short-term feeding (A), long-term feeding (B) and Drosophila short-term sugar-feeding (C). Grey points refer to non-significant changes in gene transcript abundance between fed and starved females; black points refer to significant differences in gene transcription between fed and starved females. 87 Chapter 6 ) ) ) ) ) ) ) ) ) ) ) ) ) ) 5 5 4 5 5 4 4 4 4 4 6 5 4 4 − − − − − − − − − − − − − − e e e e e e e e e e e e e e (2.2 (5.1 (1.2 (8.6 (2.1 (3.3 (1.9 4 4 4 4 4 5 4 − − − − − − − e e e e e e e )) 0.006 (9.8 ) 0.004 (3.2 1.6 )) 0.003 (3.4 3.4 )) 0.006 (5.4 6.8 ) 0.001 (1.8 ) 4.5 )) 0.001 (5.1 1.3 ) 1.9 ) 0.005 (0.002) ) 9.1 ) 0.002 (3.5 4 4 5 4 5 4 4 4 5 4 5 6 4 4 4 − − − − − − − − − − − − − − − e e e e e e e e e e e e e e e (1.2 (2.0 (7.7 (3.4 (3.1 (5.0 (2.5 4 4 4 4 4 5 4 − − − − − − − e e e e e e e ) 1.7 )) 0.005 (5.3 4.8 ) 0.008 (8.7 ) 0.001 (1.2 ) 4.1 )) 0.001 (1.4 1.2 ) 6.9 ) 2.6 ) 9.7 5 4 5 4 4 4 4 5 5 6 4 − − − − − − − − − − − e e e e e e e e e e e (3.7 (5.5 (1.2 (3.6 (4.8 4 4 4 4 5 − − − − − e e e e e ) 0.005 (4.5 ) 0.011 (7.6 ) 0.002 (3.2 ) 0.003 (2.0 ) 1.9 ) 5.1 ) 0.001 (6.4 ) 8.6 ) 2.0 ) 0.002 (2.7 ) 3.7 gene transcription assays. 4 4 4 4 5 5 5 5 5 4 6 − − − − − − − − − − − e e e e e e e e e e e (2.0 (4.4 (5.2 (2.1 (4.4 4 4 4 4 5 − − − − − 0.014 (0.001)0.006 (0.001) 0.015 (0.002) 0.005 (0.001) 0.011 (7.9 0.005 (4.8 0.197 (0.022) 0.175 (0.008) 0.142 (0.012) 0.134 (0.010) 0.234 (0.016)0.032 (0.004) 0.2470.006 (0.015) (0.001) 0.028 (0.004) 0.206 0.004 (0.015) (0.001) 0.025 (0.002) 0.003 (3.1 0.135 (0.014) 0.017 (0.002) 0.089 (0.006)0.053 (0.005) 0.1560.028 (0.008) (0.002) 0.0790.042 (0.007) (0.006) 0.083 0.0530.465 (0.007) (0.003) (0.046) 0.052 0.0630.028 (0.005) (0.008) (0.003) 0.078 0.024 0.5880.029 (0.006) (0.003) (0.039) (0.007) 0.066 0.048 0.0440.105 (0.007) (0.014) (0.004) (0.078) 0.024 0.407 0.0890.005 (0.002) (0.029) (0.009) (0.001) 0.036 0.026 0.056 (0.004) (0.003) (0.007) 0.360 0.043 0.0070.044 (0.043) (0.027) (0.001) (0.005) 0.018 0.0590.010 (0.002) (0.003) (0.002) 0.003 (9.1 0.157 0.048 (0.041) (0.003) 0.038 0.009 (0.007) (0.002) 0.039 (0.004) 0.009 (0.002) 0.037 (0.004) 0.005 (0.001) e e e e e 0.004 (4.6 0.008 (7.3 0.001 (1.4 0.002 (1.1 0.001 (8.7 0.002 (2.4 1. 3.9 4.3 1.7 4.5 N. vitripennis 1SE) of ± pp tgl pgk gpdh agpat dgat gpat pld ampk lsd2 lcfacs gk g6pd acc fas Treatment Short-term (n=24)pgi pfk pgm eno pyk Long-termugpase (n=18) pepck atpcl pc dlat accoas cs scd Phosphatidate phosphatase Triacylglycerol lipase cGMP-dependent protein kinase 3-phophate acyltransferase Diacylglycerol o-acyltransferase Glycerol-3P o-acyltransferase Phospholipase d AMP-activated protein kinase Lipid storage droplet 2 Glycerol kinase Long-chain fatty acyl-CoA synthethase Acetyl-CoA carboxylase Fatty acid synthase 6-phophofructo-2-kinase Phosphoglycerate mutase Enolase Pyruvate kinase UDP-glucose pyrophosphorylase ATP citrate lyase Pyruvate carboxylase subunit A component of pyruvate dehydrogenase Acetyl-CoA synthethase Citrate synthase Stearoyl CoA desaturase Gene Abbreviation Fed Starved Fed Starved Table 6.2: Mean normalised expression ( GlycerolipidsOther 1-acyl-sn-glycerol- pathways Glycerolipid metabolism Glycerol Glycerol-3P dehydrogenase Pentose Phosphate pathwayFatty acid metabolism Glucose-6P dehydrogenase N. vitripennis TCA cycleAcetyl-CoA Phosphoenolpyruvate carboxykinase Dihydrolipoamide acetyltransferase Carbohydrate metabolism Glycolysis/Gluconeogenesis Glucose-6P isomerase

88 Lipogenesis lost through lack of transcription ↓ ↑ ↑ ↑ * * * * 86 0.013 0.007 18 0.153 0.28 0.605 3.0150.008 0.104 0.93 0.029 0.867 0.318 0.582 0.199 0.662 16.656 0.002 ↓ ↓ ↓ ↓ ↓ ↑ ↓ ↓ ↓ ↓ * * * * * * * * * 27 0.016 4935 0.295 0.05648 7.863 0.014 0.268 4.526 0.052 0.282 0.603 0.46 0.505 0.094 0.764 0.57 0.4590.63 0.436 40 0.368 3.786 0.072 3.4994.219 0.075 0.0532.047 0.003 0.891 0.957 0.1670.258 0.361 0.617 1.104 0.311 2.668 0.125 0.6653.9551.155 0.424 0.0614.33 0.295 0.001 6.5340.349 0.023 0.932 1.083 0.561 0.351 0.198 0.316 1.177 11.739 0.661 0.004 2.661 0.29 11.955 0.004 0.118 25 2.387 0.491 0.145 2.8979.809 0.103 0.005 0.006 0.942 48.565 < 0.001 11.64439.931 0.003 < 0.001 10.61840.397 < 0.004* 0.001 23.167 < 0.001 17.066 < 0.001 gene transcription assays. Treatment Short-term (n=24)pgi Long-term (n=18) pfk pgm eno pyk ugpase atpcl pc dlat accoas cs pepck g6pd acc fas scd lcfacs gpdh gk agpat dgat gpat pp pld tgl ampk pgk lsd2 N. vitripennis refers to genes that are significantly down regulated in fed compared to starved ↓ Gene Abbreviation Test statistic P-value Test statistic P-value 6-phophofructo-2-kinase Phosphoglycerate mutase Enolase Pyruvate kinase UDP-glucose pyrophosphorylase component of pyruvate dehydrogenase Citrate synthase ATP citrate lyase Pyruvate carboxylase subunit A Acetyl-CoA synthethase Acetyl-CoA carboxylase Fatty acid synthase Stearoyl CoA desaturase Long-chain fatty acyl-CoA synthethase Phosphatidate phosphatase Triacylglycerol lipase Glycerol kinase 3-phophate acyltransferase Diacylglycerol o-acyltransferase Glycerol-3P o-acyltransferase Phospholipase d AMP-activated protein kinase cGMP-dependent protein kinase Lipid storage droplet 2 Table 6.3: Results of statistical analyses of N. vitripennis Carbohydrate metabolism Glycolysis/Gluconeogenesis Glucose-6P isomerase Acetyl-CoA Dihydrolipoamide acetyltransferase TCA cycle Phosphoenolpyruvate carboxykinase Pentose Phosphate pathway Glucose-6P dehydrogenase Fatty acid metabolism Glycerolipid metabolism Glycerol Glycerol-3P dehydrogenase Glycerolipids 1-acyl-sn-glycerol- Other pathways denotes genes that are significantly up regulated in fed compared to starved females. ↑ indicates differences are significant after correction for multiple testing; * females;

89 Chapter 6

Catabolism of lipids for use in glyceroneogenesis to maintain cell membrane lipids or energy release further requires genes involved in lipolysis, such as long-chain fatty acyl CoA synthethase (lcfacs). We found higher transcript abundance of lcfacs during starvation, suggesting that aside from re-use of fatty acids through the glyceroneogenesis pathway, fatty acids are broken down during β-oxidation releasing acetyl-CoA for use in gluconeogenesis. Also, cGMP-dependent protein kinase (pkg) transcription was higher in starved females. This gene is involved in phosphorylation of protein substrates which stimulates lipolysis (Holm et al., 2000). Fur- thermore, increased transcription of pkg has been associated with starvation and increased food searching behavior in several species of insects, including other hymenopterans like honeybees and ants (Kaun & Sokolowski, 2009). Many animals initially increase food searching behavior under starvation conditions, and our observations of increased transcript abundance for glycolysis/gluconeogenesis and TCA-cycle genes is consistent with an increase in catabolism that could be due to increased food searching behavior in starved wasps. Our results for N. vitripennis females under starvation thus conform to ﬁndings in other animal species, in which gluconeogenesis is activated to increase glucose levels and free fatty acids are burned or re-used for the formation of other lipid types. Consistent with our biochemical data, our estimates of transcript abundance of lipid synthetic genes suggest that lipogenesis is not occurring in sugar-fed wasps. Glucose can activate lipogenic genes in three ways (Ker- sten, 2001). First, glucose itself stimulates activation of lipogenic genes as it is the substrate for glycolysis that leads to the formation of the precursor for fatty acid synthesis, acetyl-CoA. Second, the uptake of glucose increases the transcriptional activity of sterol regulatory element binding protein (SREBP) that regulates genes involved in cholesterol and fatty acid metabolism. Third, glucose ingestion stimulates the release of insulin, stimulating SREBP mRNA expression that activates lipogenic genes. We found no eﬀect of sugar feeding on the transcription levels for any of the lipogenic genes, including fatty acid synthase (fas), the enzyme of which performs the majority of steps involved in fatty acid synthesis and for which an active gene transcription is crucial for lipogenesis. Regardless of its nutritional status, only trace levels of fas transcripts were found in N. vitripennis, consistent with the lack of active lipid synthesis in N. vitripennis. We further found that glucose-6P dehydrogenase (g6pd) of the pentose- phosphate pathway had a lower transcript abundance in fed compared to starved wasps. G6pd produces the reducing agent of nicotinamide adenine

90 Lipogenesis lost through lack of transcription dinucleotide phosphate (NADP), NADPH, and should typically increase after feeding to generate higher NADPH levels for use in fatty acid synthesis (Salati & Amir-Ahmady, 2001). Availability of NADPH is essential for fatty acid synthesis, yet a lacking response of g6pd at the transcriptional level suggests N. vitripennis did not respond to glucose by increasing the production of NADPH through the pentose-phosphate pathway. The only gene that showed increased transcription levels after short-term sugar- feeding was ATP citrate lyase (atpcl), which catalyzes the release of cytosolic acetyl-CoA for fatty acid synthesis, hence the metabolic fate of this precursor remains unclear. Long-term feeding led to differences in transcript abundance for 4 out of 28 genes when comparing fed to starved N. vitripennis females (Figure 6.3b, Table 6.2 and 6.3). In the long-term treatment, newly emerged N. vitripennis females were either fed with honey or starved for 1, 2 or 3 days. No differences in transcription levels were detected among days; therefore we combined the samples from different time points in the analysis. As in the short-term feeding treatment, no induction of fas transcription was observed after feeding and constitutive fas gene transcription was reduced to trace levels. Again this is consistent with fatty acid synthesis being impaired in N. vitripennis. Two of the four differentially transcribed genes were also observed in short-term fed females, i.e. phosphoenolpyruvate carboxykinase (pepck) had lower transcript abundance in sugar-fed wasps, while ATP citrate lyase (atpcl) had higher transcript abundance under fed conditions (Figure 6.1). Two genes that did not respond in the short-term fed treatment had greater transcript abundance after long-term feeding: glycerol-3P dehydrogenase (gpdh), and 1-acyl-sn-glycerol-3P acyltransferase (agpat). The former is essential for glycerol synthesis, an important component of di- and triglycerides, the latter catalyzes the reaction that converts acylglycerol-3P to diacylglycerol-1P for use in glycerolipids. The response of these genes leads to a redistribution of lipids, for example, phospholipids may still be synthesized from triglyceride stores and be used to maintain the integrity of cell membranes.

Lipid regulation in parasitic and non-parasitic insects

To compare our ﬁndings in the parasitic wasp N. vitripennis with a species that rapidly increases lipid levels after sugar-feeding, we used data from a microarray study on D. melanogaster, in which gene transcription of sugar-

91 Chapter 6 fed and starved larvae was compared (Geer et al., 1985; Zinke et al., 2002). We searched for D. melanogaster orthologs of our candidate genes by comparing amino acid sequences of N. vitripennis to those of D. melanogaster from Affimetrix datasets using the BLASTP search in KEGG and selected the closest matching amino acid sequence of D. melanogaster as orthologs with highest similarity to our candidate genes. D. melanogaster showed differential gene transcription in 4 out of the 28 genes: namely fas, accoas, atpcl and gpdh, all of which were up regulated after sugar-feeding (Figure 6.3c; Table 6.4). The most notable change in gene transcription patterns between D. melanogaster and N. vitripennis was a significant increase in the transcription of fas in Drosophila (Figure 6.1). As expected, D. melanogaster actively synthesizes fatty acids when it has access to sugar, a crucially different response from that observed in N. vitripennis, in which fatty acid synthesis is lacking. A second gene that responded differently to sugar-feeding in D. melanogaster compared to N. vitripennis was acetyl-CoA synthethase (accoas). Consistent with an increase in fatty acid synthesis, D. melanogaster accoas showed higher transcript abundance after short-term feeding, suggesting that acetyl-CoA is released for use in the fatty acid synthesis pathway. Furthermore, transcription of atpcl and gpdh was induced by feeding in D. melanogaster, similar to what was observed in N. vitripennis. In contrast, the reduced transcription of genes involved in carbohydrate metabolism, as found in sugar-fed N. vitripennis, was not observed in D. melanogaster. This suggests that lack of lipogenesis is associated with a concerted down regulation of genes involved in various metabolic pathways. In addition to the comparison of gene transcription of single genes, we inspected overall transcription levels of all candidate genes within species. Overall levels of gene transcription among D. melanogaster genes were higher (Figure 6.4). This holds true in particular for genes involved in carbohydrate metabolism and fas. In contrast, transcription levels in N. vitripennis show very low transcript abundances that approach the qRT-PCR detection limit for numerous candidate genes used in our assays.

92 Lipogenesis lost through lack of transcription ↑ ↑ ↑ ↑ microarray data. D. melanogaster 259 (78) 94 (31) 8 0.109 790 (266)396 (582) 686 (156) 1159 (665) 0.114 0.743 0.747 0.408 3490 (360)1328 4552 (335) (412)1103 (490) 1054 (409)2438 1351 (707) (607) 3.764177 1891 0.081 (1395) (409) 27456 0.269 (10136) 0.1014646 0.616 (561)4673 0.758 (800) 0.448 5436 (607) 6229 0.518 (889) 4 0.025 0.913 1.693 0.362 0.222 9058 (2054) 5241 (2135)5365 (1002) 5918 (741) 1.66 0.227 0.197 0.666 8747 (2570) 1209 (530) 98.229 0.006* -2187 (2384) -3172 (1680) 0.114 0.743 20832 (3868) 17902 (2648) 0.391 0.546 48769 (7955)36836 30140 (8241) (2484) 22337 (3481)15687 (2131) 13401 (1175) 4 11 0.025 32970 (3900) 0.262 24118 (3081) 0.88242558 (8999) 23355 (3865) 0.37 36256 (6357) 3.18333016 39039 (3547) (3949) 15938 0.105 (3437)44168 (6923) 12971 (4613) 8 0.138 0.109 0.718 14.064 2 0.004* 0.01 78401 (10642)95847 64106 (13595) (3179) 55262 (3840)53924 (14219) 6197165804 (10158) (16766) 17394 (3397) 8.253 8 0.017 0.212 0.109 84625 (17548) 0.655 20363 (4529) 0 0.004* 0 0.004* Treatment Short-term (n=12)pgi Short-term (n=12) pfk pgm eno pyk ugpase pepck atpcl pc dlat accoas cs g6pd acc fas scd lcfacs gpdh gk agpat dgat gpat pp pld tgl pgk lsd2 ampk 1SE) and results of statistical analysis of ± Gene abbreviation Fed Starved Test statistic p-value 6-phophofructo-2-kinase Phosphoglycerate mutase Pyruvate carboxylase subunit A component of pyruvate dehydrogenase Acetyl-CoA synthethase Citrate synthase Fatty acid synthase Stearoyl CoA desaturase Enolase Pyruvate kinase UDP-glucose pyrophosphorylase ATP citrate lyase Long-chain fatty acyl-CoA synthethase Glycerol kinase Phosphatidate phosphatase Triacylglycerol lipase 3-phophate acyltransferase Diacylglycerol o-acyltransferase Glycerol-3P o-acyltransferase Phospholipase d cGMP-dependent protein kinase Lipid storage droplet 2 Table 6.4: Average intensity ( Carbohydrate metabolism Glycolysis/Gluconeogenesis Glucose-6P isomerase TCA cycleAcetyl-CoAPentose Phosphate pathwayFatty acid Phosphoenolpyruvate metabolism carboxykinase Glucose-6P dehydrogenase Dihydrolipoamide acetyltransferase Acetyl-CoA carboxylase Glycerolipid metabolism Glycerol Glycerol-3P dehydrogenase GlycerolipidsOther 1-acyl-sn-glycerol- pathways AMP-activated protein kinase

93 Chapter 6

Evolutionary changes in lipogenic regulatory mechanisms

Lack of lipogenesis is unlikely to be due to mutation accumulation in the structural part of the gene or truncation of fas from the genome. Fas is present in N. vitripennis and a comparison of the FAS amino acid sequence with D. melanogaster and the related hymenopteran, A. mellifera, reveals no irregularities such as an increased number of stop-codons (e-values were <0.001 when amino acid sequences were compared between species). This suggests that no mutations have accumulated in the structural part of the fas gene of N. vitripennis. Amino acid sequence comparison furthermore suggests that transcription of fas leads to the formation of a fully functional enzyme, although enzyme assays are needed to confirm FAS functionality. Alternatively, impaired enzyme functioning could be due to other factors that optimize enzyme activity, such as sufficient availability of enzymatic co-factors. In Saccharomyces cerevisiae, the lack of sufficient iron substrates acting as a co-factor for enzymes involved in lipid synthesis prohibits their functioning and leads to an impaired triglyceride synthesis (Shakoury-Elizeh et al., 2010). Moreover, the rate of metabolomic responses typically depends only partly on gene transcription and can also be affected by substrate or product levels and the physiological status within certain tissues (Iizuka et al., 2004; ter Kuile & Westerhoff, 2001). Despite the lack of induced gene transcription of fas in fed compared to starved females, we detected very low fas transcript levels under both conditions, leading us to question why lipogenesis is not active in N. vitripennis even though we recover fas transcripts in our gene transcription assays. First, transcript persistence of fas in N. vitripennis could be the result of directed gene transcription, initiated from the fas promoter, albeit at very low levels. In this case, transcription might not be sufficient to generate substantial amounts of enzyme necessary for a fully functional fatty acid synthesis pathway. Second, low transcript levels of fas might be the result of the physical positioning of the gene. In D. melanogaster, highly transcribed genes are known to affect the rate of transcription of genes in close proximity (Spellman & Rubin, 2002). The majority of genes neigh- boring fas in N. vitripennis have various functions, including an ATPase AAA family protein, a splicing factor, a purine nucleoside phosphorylase and a ubiquitin activating protein. Read-through of RNA polymerase actively transcribing genes nearby fas could have resulted in the leaky low transcript levels that we observe in our presented qPCR assays. Another plausible explanation for unresponsiveness of fas to food in N.

94 Lipogenesis lost through lack of transcription

Fatty acid Carbohydrate metabolism metabolism Glycerolipid metabolism Other A 1 0,95 0,9 0,85 0,8 0,75 0,7 0,65 0,6 0,55 0,5 0,45 0,4 0,35

Relative expression Relative 0,3 0,25 0,2 0,15 0,1 0,05 0

Gene

B 1 0,95 0,9 0,85 0,8 0,75 0,7 0,65 0,6 0,55 0,5 0,45 0,4 0,35

Relative expression Relative 0,3 0,25 0,2 0,15 0,1 0,05 0 -0,05 -0,1

Gene

Figure 6.4: Overall transcription levels in N. vitripennis and D. melanogaster. Relative transcript levels of candidate genes as a ratio to the highest transcribed gene, pyruvate kinase (pyk), in N. vitripennis (A) and D.melanogaster (B).

95 Chapter 6 vitripennis is regulatory inhibition of gene transcription, either through mutation accumulation in promoter regions or mutations in genes underlying transcription factors that are involved in lipid synthesis. Non-coding promoter regions are not under purifying selection and typically the rate of mutation accumulation is increased in these regions (Stone & Wray, 2001; Wray et al., 2003). Computational studies show the incredible rate with which evolutionary changes in transcription initiation binding sites can occur, both in terms of loss and acquisition of novel transcriptional regulatory sequences through local point mutation, recombination and transposition. In rats, two promoter regions of fas have been discovered that contribute to fatty acid synthesis (Rufo et al., 2001). A relatively small promoter region that increases fas promoter activity 2-3 fold in response to insulin, and a larger promoter region located over 6,000 base-pairs upstream of fas, which contains a carbohydrate responsive element that increases activity of the fas promoter 20-30 fold when glucose is available. Mutations accumulated in the promoter region of fas could have resulted in the occurrence of transcriptional repressor elements, which in turn silence fas and impair lipogenic ability in parasitoids. Alternatively, mutations in genes encoding transcription factors may prohibit lipogenesis. For example, mutant D. melanogaster larvae that lack the homolog of sterol regulatory element binding protein (SREBP), an essential transcription factor for cholesterol and unsaturated fatty acid synthesis, exhibited pronounced growth defects and died prematurely due to improper gene regulation (Kunte et al., 2006). Identification and testing the functionality of genes underlying essential transcription factors for successful functioning of fas in N. vitripennis could reveal if the unresponsiveness of fas is due to mutation in genes encoding essential transcription factors. Irrespective of the exact molecular mechanism that has led to transcriptional unresponsiveness of fas to sugars, a lack of transcription of fas would be expected to lead in time to associated accumulation of mutations in the coding part of the gene. Lack of lipogenesis is thought to have originated in the basal parasitic Hymenoptera, with the first parasitic groups appearing in the fossil record during the early Jurassic (180-200 million years ago) (Quicke, 1997; Visser et al., 2010). Unused genes should be expected to have accumulated mutations, even when conservative mutation rates are considered. The fact that such degradation of the fas gene has not occurred in N. vitripennis is remarkable and could only be explained if there was still a selective advantage to maintain functionality of fas, although the nature of such benefit would remain speculative. The absence of mutation

96 Lipogenesis lost through lack of transcription accumulation in fas is consistent with the relative ease with which lipogenic ability re-evolves in parasitic wasps (Visser et al., 2010). Re-gaining of lipogenesis has been observed at least 3 times in the hymenopteran phylogeny, mainly in species that are expected to lack active physiological manipulation of their hosts, but regaining of lipogenesis has also been reported between different populations of the same species under varying environmental conditions (Moiroux et al., 2010). Contrary to current theory, these observations imply that loss of complex features can readily be reversed, reflecting findings from other recent phylogenetic studies on evolutionary reversals (Bely & Sikes, 2010).

Conclusion

The importance of trait loss in shaping evolutionary dynamics is often neglected. Moreover, only a limited number of studies have addressed loss of redundant traits at both the phenotypic and genetic level. Our phenotypic experiments and gene transcription study on loss of lipogenesis in the parasitic wasp N. vitripennis showed that adults do not actively synthesize fatty acids and that this essential metabolic trait has been lost through unresponsiveness of the gene fatty acid synthase (fas) to sugar. Unresponsiveness of fas, a key gene for fatty acid synthesis, eliminates an important link in nutrient metabolic pathways that prohibits lipogenesis from taking place in N. vitripennis. Gene transcription patterns in D. melanogaster are in sharp contrast with those observed in N. vitripennis and further highlights the diﬀerences in nutrient metabolism between a species that is actively synthesizing lipids and N. vitripennis, which lacks lipogenesis in its adult life-stage. The loss of lipogenesis in parasitic wasps is hypothesized to result either from low gene transcription or from distorted gene regulation of fas. Future studies on signaling pathways, genomic architecture and genetics underlying trait loss will increase our understanding of the mechanisms that are responsible for observations at the phenotypic level and will aid in understanding the process of trait loss from an evolutionary perspective. Moreover, mechanisms underlying major changes in traits, whether involved in the acquisition of novel traits or in trait losses, might result from similar processes that aﬀect trait dynamics.

97 Chapter 6

Acknowledgements

We would like to thank Bart Pannebakker, Jack Werren and Amanda Avery for providing insects and Thomas Blankers for his help during experiments. We are grateful to Rinaldo Bertossa, David Loehlin, Christopher Desjardins, Ben Nota, Tjalf de Boer and Elaine van Ommen Kloeke for discussion, Ingo Zinke for providing data, Zoltán Bochdanovits for advice on the statistical analyses and Nico van Straalen for helpful comments on earlier drafts of the manuscript.

98 Lipogenesis lost through lack of transcription 2 2 3’) Eﬃciency R → 3’) Reverse primer (5’ → Accession no. NM_001170906 ATTCCAAAAACCGAGTCACCGXM_001604192 CGTCAACACGACTCGTCTGAAGXM_001602598 CTGTCCGAGAAAGGCAAAAGC TGAACATTGCATCACGAGCC GTCAGGAATGTCATAGTGAGCCGXM_001607492 CGTGACTGAGAATGGACTCTTCCXM_001600601 GATTTGTCATTGTCCCTGAGCTC TCTGGTCAACTGAGAGGTATGGG GCCTGTTATCTGCGCTACTCAGA 1.8XM_001606149 GCTCCATCAAGAATAGCATTAGCC CAACAGAACGATGGTTTCAAGC 1.9XM_001600177 1.9 1.7 1.7 ACAACCAGGTCAACGACGATCT CTCTGGCAATCTTTGAATCTGGXM_001604270 AAACCCCTTGTTGCATGGTG 0.985 AGCATCATCCAAGTCTTCACAGCXM_001600169 ACCAAATCGGTGCTCCTATGC 0.998 XM_001606511 0.986 1.9 0.993 0.993 AAAAGAATGTGACGATCGGTCG 1.9XM_001599602 AGTTTCAAGGTTCGAGTGGGC ATATACCAGCCGAATCCTGAGC GCAGAAAGAACAACAAGAGCTGC CGAGCCGTCATCTTTGTAGTCCXM_001606354 AGACCAAGGGCCAAATGTACG GGATGCTTTATCGACCTCTCGTXM_001600277 1.9 0.998 ACATCTCAAAGCACTCTTCACCG 0.999 XM_001606924 1.8 1.8 TTGGCGAATCTGAGACTGAGG CCCAGTGTTTTTCTGATGTCTGC GCATTTCTAACGACAGCGGATACXM_001605009 AGTACGACTCGAACCTAATCACACC 1.8 ACTCCACTCGCACAATTGAGC 1.8XM_001599615 0.977 2 TGGTGCCACAGAGCTTACAAAGXM_001605905 0.992 0.982 1.9 CGAGTGGCTCATCAGCTATATGG TTTGAGGGATGTGAGCCAGAG ACTCGTACAGGTGATTCTGGAAGG TCAGGCAGTAGTGCAGTTCACC 0.999 2.1 0.995 XM_001607887 TGGTGCTGGTTTTGTTGACG 0.992 XM_001603084 1 AACAGATATGGCCCGCTCATC 1.9 1.9XM_001603785 TCTGTACTTCGCCACATTCGG 0.999 XM_001602238 CGTCGACAAACTTGATCATTTCC TGTTCTGCTCCTTTCCTCACG TTAAGACCTCGGCAGTATTGGCXM_001599807 AAATCGCACCACCTATCTCAGC 0.999 0.998 GAATTGACGGTACTTTGGGCC 1.7XM_001605407 AGAACCAACACCGCTACATCG TGAGAAGCCTGAAATCCAGTCC 2.1XM_001606674 GCTACTCTTTTCGTGACCTGAGAAG GTCCCGAAAACATCAAAGTTCTG 2.1XM_001607962 GGAATGCGACGCTTTGATCTAC ATAGAGTAAGCGCAGTGGGTGC GAAGGGAAGGACAGCCTGAGTT 1.8XM_001599824 0.993 1.9 TTTCAGTCCTAGCAATGAGGCC ACCCACCACAGAGATGTTTCTTG 0.972 XM_001603499 AGGTGGAAGTGTCGAGAGATGG 0.982 2 1.9 TTGGGTAGGAGTAGTGCTCAGAGAGNM_001159980 2 ATTCTCAGCACCCAGTACGGAG 1.8 GTCCTCTTGCAGTTGTAAAGAATGG 0.994 2XM_001605557 0.999 CTGGTCACTTGATCTACAAATGCG GACCCAGGCATACTTGAAGGAAC GTGGAAAATAGTGATCGAGAAGCCLoehlin et al. 1.7 0.999 (2010) 1.9 0.987 0.995 0.98 XM_001607335 CTTCCGCAAAGTCCTTGTTC GAATCTTTCCTGCCCCAGTTG 0.991 XM_001606820 AACTCTACTGCCTGGAGCATGG 0.993 XM_001604635 0.999 GAAAGACATCAACGAACTCACCG CCTCCGGCTTGTGAAGATACAT CGCTGAAGAAGGTGTTGAAGCT AACTCCATGGGCAATTTCTG GGTTGAATTCCCAGGCTATGG 1.7 1.9 1.8 1.7 0.959 0.999 0.988 0.991 pfk pgm eno pyk ugpase atpcl pc accoas cs fas scd lcfacs gk dgat gpat pp pld tgl pgk lsd2 rp49 ubc at atpase pgi pepck dlat g6pd acc gpdh agpat ampk ef-1a Table 6.5: Candidate genes, abbeviations, GenBank accession numbers, forward and reverse primer sequence, qPCR eﬃciency and R Carbohydrate metabolism Glycolysis/Gluconeogenesis TCA cycle Acetyl-CoA Pentose Phosphate pathway Fatty acid metabolism Glycerolipid metabolism Glycerol Glycerolipids Other pathways Reference genes Candidate genes Abbr. GenBank Forward primer (5’

Chapter 7

Discriminating between energetic content and dietary composition as an explanation for dietary restriction eﬀects

Jacintha Ellers, Bas Ruhé and Bertanne Visser Journal of Insect Physiology 57, 2011, 1670-1676

Abstract A reduction in dietary calories has been shown to prolong life span in a wide variety of taxa, but there has been much debate about confounding factors such as nutritional composition of the diet, or reallocation of nutrients from reduced reproduction. To disentangle the contribution of these different mechanisms to extension of life span, we study the effect of caloric restriction on longevity and fecundity in two species of sugar-feeding parasitoid wasps. They have a simple diet that consists of carbohydrates only, and they do not resorb eggs, which rules out the proposed alternative explanations for beneficial effects of caloric restriction. In addition, parasitoids are unable to biosynthesize lipids from sugars, adding to the importance of income calories from carbohydrates. Two caloric restriction treatments were applied: First, dietary dilution to investigate the effect of carbohydrate concentration in the diet; and second, intermittent feeding to examine the effect of feeding frequency on longevity and fecundity. Only the dietary dilution treatment showed an effect of caloric restriction with the highest longevity recorded at 80% sucrose (w/v). No effect of dietary regime was found on fecundity. We also measured the weight increase of the parasitoids after feeding to obtain an estimate of consumption. A constant quantity of the sugar solution was consumed in all dietary dilution treatments, hence caloric intake was proportional with sucrose concentrations. Although the present study does not disqualify the relevance of nutrient composition in other species, our data unequivocally support the caloric restriction hypothesis and invalidate alternative explanations.

101 Chapter 7

Introduction

It has become increasingly clear over the last decade that dietary restriction (DR) without malnutrition prolongs life. Reduced nutrient availability increases life span and ameliorates age-related diseases in a wide array of species (Colman et al., 2009; Nemoto & Finkel, 2004; Weindruch & Wal- ford, 1988). Modest restriction of dietary intake was first applied in rats (McCay et al., 1935), but its effects have been found to hold for other mammals (Colman et al., 2009), invertebrates (Kaeberlein et al., 2007; Partridge et al., 2005) and micro-organisms (Skinner & Lin, 2010), with the exception of some studies finding no detrimental effect of nutrient-rich diets (Bauer- feind et al., 2009; Cooper et al., 2004; Molleman et al., 2009). Although the effect DR seems phylogenetically well-conserved, it is less clear if common principles and mechanisms underlie the life-extending effects of DR. The relevance of understnading the costs and benefits of dietary composition is highlighted by the fact that obesity has been increasing rapidly in the western world and the interest in the potential life-extending effects of DR in humans is growing. The mechanisms responsible for the beneficial effects of DR have remained elusive, but proposed mechanisms can be broadly divided into three categories. First, the caloric restriction hypothesis assumes that a reduction in energetic content of the food has a life extending effect, because of reduced oxidative stress (Harman, 1956; Sohal & Weindruch, 1996). With lower available energy, metabolic rate will slow down and reduce free radical formation. This widely held view emerged from early work on DR, but has recently been challenged by studies that suggest specific nutrients are involved in aging and life span. Hence, a second proposed mechanism focuses on the composition of diet rather than its caloric content. Dietary components may act independently on signalling pathways such as the insulin pathway (Kimura et al., 1997) or the TOR pathway which determine life span in Drosophila (Kapahi et al., 2004). Also, deficiencies of specific nutrients may lead to excessive consumption of others. Thirdly, DR often reduces reproductive output, which may increase life span because of the trade-off between reproduction and longevity. The relative importance of the direct effects of nutrient composition versus the indirect effects of reallocation of nutrients, has remained largely unaddressed in DR protocols (Bass et al., 2007). To disentangle the contribution of different mechanisms on extension of life span, it is indispensable to extend the study of DR to invertebrates,

102 Dietary restriction in sugar-feeding insects which are more amenable to experimental manipulation. Invertebrates offer the opportunity to reveal more subtle effects of diet because of their relative fast life cycle and possibility of larger sample sizes. The recent surge in studies using Drosophila has made important contributions to the understanding of life span extension, such as a detailed evaluation of the effect of dietary protein to carbohydrate ratios versus energetic content (Lee et al., 2008; Simpson & Raubenheimer, 2007) and the role of olfaction and odorant receptors in life span regulation (Libert et al., 2007). Invertebrates offer a wider range of experimental manipulations than mammals because dietary complexity is more variable among species (Slan- sky & Rodriguez, 1989). Elaborate diets are found in predatory arthropods, and life span in these species will probably be strongly determined by dietary composition and nutritional ratios (Hvam & Toft, 2005; Toft, 1999). On the other hand, several invertebrate species have relatively simple diets that contain no proteins or lipids and consist of carbohydrates only. For example, adult diets may exist only of nectar consumed from flowers or extrafloral nectaries (Wäckers, 2001). Many of these species are nearly pro- ovigenic, meaning that they emerge with the majority of their eggs ready for oviposition (Jervis et al., 2001). If they do not resorb eggs during adult life, reallocation of nutrients from reproduction will play only a minor role in life span extension. Such species offer a yet unexploited opportunity to discriminate between the role of energetic content and dietary composition in DR. In this study, we investigate the effect of caloric restriction on longevity and fecundity in two parasitoid species, Asobara tabida and Trichopria drosophilae. During the larval stage, these parasitoids develop in Drosphila larvae or pupae respectively, and kill their host to complete development. However, the adult diet of both species is simple and consists only of sugar- rich substances (Eijs et al., 1998). Adult feeding is of particular importance in parasitoid species because the majority of parasitoids are unable to accumulate lipids, even when a surplus of food is available (Ellers, 1996; Giron & Casas, 2003; Visser & Ellers, 2008). A comparative study showed that the parasitic lifestyle had led to the evolutionary loss of this essential metabolic trait (Visser et al., 2010), which means that in order to save irreplaceable lipid reserves, they need permanent access to food sources. Indeed, carbohydrate feeding lowers lipid depletion rates and extends longevity in A. tabida and other species (Heping et al., 2008), and under natural conditions parasitoids feed frequently on carbohydrates (Desouhant et al., 2010). Due to the continuous dependence on adult feeding, we expect no benefits of

103 Chapter 7 caloric restriction on longevity in these two parasitoid species. We test this hypothesis using a two-pronged approach to restrict calorie intake: First, a dietary diluation experiment to investigate the eﬀect of carbohydrate concentration in the diet; and second, an intermittent feeding treatment to examine the eﬀect of feeding frequency on longevity and fecundity of A. tabida and T. drosophilae.

Materials and Methods

Insects

Drosophila melanogaster and Drosophila subobscura hosts were kept at 20◦C, RH 75% and a 12:12h L:D regime on food medium containing 20 g agar, 50 g sucrose, 35 g yeast, 9 g kalmus (10 parts acidum tartaricum, 4 parts ammonium sulphate, 3 parts potassium phosphate and 1 part magnesium sulphate) and 10 mL nipagin (100 g 4-methyl hydroxyl benzoate per liter ethanol) per liter water. For the rearing of Asobara tabida 100-200 two-day old larvae of D. subobscura were collected and subsequently placed in pots with wasp medium containing 20g agar, 9g kalmus, 5mL nipagin and 4mL proprionic acid per liter water, covered by a thin layer of liquid yeast. Two mated females were placed with the larvae for parasitization and allowed to feed on honey. All experimental pots were kept at 23◦C, RH 75% and a 16:8 L:D regime for further parasitoid development. Success- fully parasitized pupae were transferred individually into vials containing moist cotton wool. For the rearing of Trichopria drosophilae 30 one-day old pupae of D. melanogaster were collected and subsequently placed in pots containing wasp medium. Conditions for parasitization and development were similar to those described for A. tabida, with the exception that pots and vials were kept at a 12:12h L:D regime.

Feeding regimes and longevity, egg load and body size measurements

After emergence, adult female wasps were kept singly in a vial containing wet cotton wool and were randomly assigned to treatments. For dilution treatments, sucrose was diluted with water to the following concentrations in weight per volume (w/v): 0%, 20% (20 grams sucrose in 100 mL water), 40%, 60%, 80% or 100%, provided ad libitum on a small piece of paraﬁlm containing a droplet of the appropriate dilution. For intermittent feeding treatments, adult female wasps were allowed to feed on a 40% w/v concentration of sucrose and water (cf Hogervorst et al 2007) placed on a piece

104 Dietary restriction in sugar-feeding insects of paraﬁlm for the duration of 2 hours. Adult females were presented with water or with food once, weekly, twice weekly, daily or ad libitum. Dilu- tions were refreshed every week to prevent fungal growth. For longevity measurements all vials were inspected daily for deaths and subsequently frozen at -18◦C. Body size was determined by placing a single adult female under a microscope (Leica DC 200; 500x) and measuring hind tibia length. After size had been determined the adult female was placed in a droplet of demineralised water, dissected and the number of eggs counted.

Food uptake experiments

Food uptake in the dilution treatment was determined for all six sucrose concentrations. Freshly emerged females of A. tabida and T. drosophilae were randomly assigned to feed on one of the concentrations during 2 hours, after which individuals were frozen at -18◦C. Food uptake was determined by measuring the wet weight of 5 pooled individuals on a microbalance (Metler Toledo), with 8 replicates per concentration per species.

Statistical analysis

Longevity data were checked for normality and homogeneity of variances. General Linear Models were used to test for an eﬀect of treatment on egg load and longevity for each species. Tibia length was included in these analyses as a covariable. Posthoc comparisons were carried out using a Tukey test. Data on food uptake were subjected to Analysis of Variance, followed by a Tukey test. Statistical analyses were done using SPSS version 17.

Results

Dilution treatment

Dietary dilution had a significant influence on longevity of both parasitoid species. For A. tabida the shortest longevity was observed at 0% sucrose (6.72 days ± 1.14, 1SE) and the highest longevity at 80% sucrose (30.93 days ± 2.97, 1SE; Figure 7.1A). When fed the highest concentration of sucrose (100%), longevity of A. tabida was significantly lower than when fed 60% (P < 0.026) or 80% (P < 0.001) sucrose. T. drosophilae also had the shortest longevity when fed 0% sucrose (11.21 days ± 0.61, 1SE) and the highest longevity when fed 80% sucrose (86.25 days ± 3.72, 1SE; Figure 7.1B).

105 Chapter 7

Table 7.1: Mean body size and egg load of two parasitoid species subjected to caloric restriction through dietary dilution and intermittent feeding.

A. tabida T. drosophilae Dietary dilution Treatment Mean tibia length Mean egg load Mean tibia length Mean egg load (mm ±1SE) (±1SE) (mm ±1SE) (±1SE) 0% 0.980a (0.021) 146.9a (8.64) 0.786a (0.014) 80.00a (4.01) 20% 0.985a (0.018) 139.6a (18.19) 0.778a (0.016) 74.62a (2.22) 40% 0.993a (0.032) 138.00a (23.67) 0.767a (0.012) 75.44a (5.17) 60% 0.969a (0.038) 145.20a (9.56) 0.771a (0.017) 75.36a (4.75) 80% 0.995a (0.029) 173.00a (38.25) 0.760a (0.020) 65.20a (6.12) 100% 0.998a (0.025) 173.20a (19.23) 0.760a (0.018) 65.11a (7.38)

Intermittent feeding No food 0.916a (0.023) 154.00a (10.15) 0.708a (0.029) 68.43a (4.24) Once 0.956a (0.024) 149.89a (14.60) 0.759ab (0.018) 79.88a (3.62) Weekly 0.978a (0.024) 172.83a (11.21) 0.750ab (0.016) 70.88a (3.65) Twice weekly 0.954a (0.018) 167.63a (10.44) 0.756ab (0.018) 78.31a (3.73) Daily 0.964a (0.025) 112.06b (10.92) 0.803b (0.024) 81.81a (9.07) Permanent 1.009a (0.018) 150.42a (13.21) 0.750ab (0.021) 77.33a (6.79)

Different letters represent differences significant at α=0.05 level.

Similar to A. tabida, T. drosophilae had a significantly reduced longevity when fed 100% sucrose compared to 80% (Tukey test: P = 0.049). Body size was not a significant variable in explaining variation in longevity among treatments in either of the two species (A. tabida: P = 0.89; T. drosophilae: P = 0.80, Table 7.1). The average egg load of A. tabida (151.3 ± 6.91, 1SE) was higher than that of T. drosophilae (73.17 ± 2.02SE), but for neither of the two species did sucrose concentration affect egg load (A. tabida:F5,46 = 0.728, P = 0.606; T. drosophilae:F5,58 = 1.565, P = 0.184, Table 7.1).

Intermittent feeding

Intermittent feeding had a significant influence on the longevity of both parasitoid species. In A. tabida there was a clear dichotomy between feeding treatments: average longevity was uniformly high when females were fed daily or had permanent access to food (Tukey: P = 0.506, Figure 7.2A), while both treatments differed significantly from treatments with less frequent feeding (Tukey: all pairwise comparisons P < 0.001). When females were fed twice a week or less frequently, longevity was consistently low. In fact, there was no significant difference in longevity between the starvation treatment and being fed twice a week (P = 0.477). Egg load did not differ among treatments except for the daily feeding treatment which had a lower

106 Dietary restriction in sugar-feeding insects

A d

cd bc bc b

0% 20% 40% 60% 80% 100%

B c bc bc b b

0% 20% 40% 60% 80% 100%

Figure 7.1: Average longevity (days ±1SE) of A) A. tabida and B) T. drosophilae when exposed to dietary dilution treatments with different sucrose concentrations. Different letters represent differences significant at α = 0.05 level.

107 Chapter 7 egg load than the other treatments (Tukey: all comparisons P<0.05, Table 7.1). Egg load and tibia length contributed significantly to the variation in longevity (egg load: F=4.66, P=0.034; tibia length: F = 5.71, P = 0.019). Long-lived individuals were larger and had a higher egg load compared to shorter-lived females. In T. drosophilae the highest average longevities were also observed in the daily and permanent treatment (Figure 7.2B). The two most frequent feeding treatments differed significantly from the other treatments (all comparisons P<0.001), but not from each other (P = 1.00). Tibia length was significant as covariable in the model (F = 4.34, P = 0.04) with larger females living longer. Egg load was not a significant covariable in the model (F = 1.40, P = 0.425) and egg load did not differ among treatments (F = 0.36, P = 0.88, Table 7.1).

Feeding experiments

To ascertain that the differences in longevity were not due to the quantity of food imbibed, we measured the weight gain of females after feeding for two hours. In A. tabida there were no differences among sucrose concentrations in the weighed gained by females after feeding (Figure 7.3A), indicative of similar feeding activity. Only in the water treatment (0% sucrose concentration) did females gain less weight than in the 100% sucrose treatment (P = 0.014). Also in T. drosophilae, all feeding treatments yielded similar weight gain after two hours (all comparisons P > 0.05), indicating that there were no differences in food uptake among females in different treatments (Figure 7.3B).

Discussion

Dietary restriction has been thought to increase longevity due to reduced calorie intake, but there is uncertainty about the cause of the life-extending effects. Confounding effects include an unsuitable ratio of macronutrients in the richest diets and re-allocation of resources from reproduction to survival under restricted diet (Simpson & Raubenheimer, 2007). The parasitoid species investigated in this study have a simple adult diet consisting of carbohydrates only and lack egg resorption (Eijs et al., 1998), which rules out the proposed alternative explanations for beneficial effects of DR. The dilution treatments we applied showed a clear decrease in longevity for A. tabida and T. drosophilae at the highest sucrose concentration without

108 Dietary restriction in sugar-feeding insects

A b b

a a a a

No food Once Weekly 2x /week Daily Permanent

B c c

ab ab a

No food Once Weekly 2x /week Daily Permanent

Figure 7.2: Average longevity (days ± 1SE) of A) A. tabida and B) T. drosophilae when exposed to an intermittent feeding regime with different feeding frequencies of 40% w/v sucrose solution. Different letters represent differences significant at α = 0.05 level.

109 Chapter 7

A 2 b b b b b 1.5 a

1 imbibed 0.5

0 0 20% 40% 60% 80% 100%

B 2

a a a a a 1.5 a

1 (mg) Amount of sucrose solution Amount of sucrose 0.5

0 0 20% 40% 60% 80% 100%

Feeding treatment

Figure 7.3: Average food intake (mg ± 1SE) of A) A. tabida and B) T. drosophilae when fed different sucrose concentrations during 2 hours. Values represent the gain in wet weights of five pooled individuals. Different letters represent differences significant at α = 0.05 level.

110 Dietary restriction in sugar-feeding insects any concomitant effect on fecundity. This finding strongly supports the hypothesis that high nutritional content can be detrimental, and that the reduction in energy intake is a key element of the dietary restriction effect (Masoro, 2005). In contrast, we did not find evidence for a beneficial effect of caloric restriction in the intermittent feeding treatment. The feeding frequency was an important determinant of longevity, especially with frequencies of two feedings per week or more, but no decrease in longevity was detected when sucrose solution was available permanently compared to daily or twice weekly. A possible explanation for the lack of effect of caloric restriction is the relatively low sucrose concentration in the intermittent feeding experiment (40% w/v) compared to the highest concentration in the dilution experiment (100% w/v). Even if a 40% sucrose solution is available permanently it does not lead to a caloric restriction effect in either of the two species (Figure 1). Repeating the experiment with higher energetic content in the food could reveal an effect of DR. Alternatively, several studies have demonstrated the capability of organisms to compensate for food-deprived periods by overeating when food is available again (Anson et al., 2003; Cerqueira & Kowaltowski, 2010). Compensatory feeding can produce comparable overall food intake or growth rates compared to continuous feeding (Inness & Metcalfe, 2008), so that no life span extension is to be expected. One of the problems that have hampered a thorough investigation of the mechanisms underlying DR is the lack of information on how much the animals actually eat (Carvalho et al., 2005). In the current experiment we measured the weight increase of the parasitoids after feeding to obtain an estimate of consumption. The data showed that a constant quantity of the sugar solution was consumed in all dietary dilution treatments; only the water control was consumed to a significantly lesser extent. Therefore, the amount of calories consumed was directly proportional with the concentration of sucrose in the food. The lack of differential intake suggests that the parasitoids are constrained by the volume of their stomach, which may prevent a compensatory feeding strategy in the low concentration treatments. In contrast, in D. melanogaster, individuals were found to adjust the volume they ingested to account for differences in the concentrations of nutrients (Carvalho et al., 2005; Lee et al., 2008). If A. tabida and T. drosophilae would have a comparable strategy to Drosophila, a decreased intake at higher concentrations would have been expected. More importantly, the similarity in quantity imbibed between the two highest concentrations validates the assumption that the energy intake is higher in the highest

111 Chapter 7 sucrose concentrations. A second confounding factor in the interpretation of many DR experiments is the lack of distinction between reduced energy content and changed nutrient composition (Grandison et al., 2009; Lee et al., 2008). Re- cent techniques in nutrient geometry have been successfully used to show that carbohydrate:protein ratio is responsible for life span reduction in D. melanogaster and other species (Fanson et al., 2009), and that detrimental effects can easily be re-mediated by addition of specific amino acids, for example (Grandison et al., 2009). An alternative approach to avoid interaction between the effect of nutrient composition and energy content is to choose study organisms with a diet containing only a single component. Carbohydrates are the only dietary component for non-host feeding parasitoid species (Jervis et al., 2008), as they feed on honeydew, (extra) floral nectary secretions, fruit juice or fruit pulp (Eijs et al., 1998; Sivinski et al., 2006; Hein & Dorn, 2008). The principle sugars present in these food sources are glucose, fructose and sucrose, which have been shown to have the greatest beneficial effects on parasitoid longevity compared to other, less prevalent sugar sources (Hogervorst et al., 2007; Wäckers, 2001). Also, variation in the proportion of glucose, fructose and sucrose in the diet does not affect its profitability (Tompkins et al., 2010), despite earlier sugges- tions (Vattala et al., 2006). The DR effect we found in A. tabida and T. drosophilae occurred when provided with different concentrations of sucrose solutions, therefore nutrient composition should not be of any influence on our results. A third mechanism to account for the increased life span under restricted dietary conditions is a reallocation of nutrients from reproduction to somatic maintenance, which is thought to be an adaptive response to aid survival of food shortages in nature (Holliday, 1989; Williams, 1966). Indeed, DR lowers fecundity in several taxa (Chapman & Partridge, 1996; Klass, 1977); for example, in the mosquito Aedes aegypti a restricted number of blood meals significantly extended longevity at the cost of producing fewer eggs (Joy et al., 2010). In A. tabida and T. drosophilae as well as many other parasitoids species, oogenesis and vitellogenesis starts well before female eclosion, so that a major proportion of the eggs is mature at female emergence (Jervis et al., 2001). Due to the early timing of egg development, the only possibility for reallocation of nutrients during adult life is through egg resorption, which is known to occur in periods of nutritive stress in certain species (Asplen & Byrne, 2006; Bodin et al., 2007; Wakefield et al., 2010). However, no evidence of egg resorption was found in any of our dietary

112 Dietary restriction in sugar-feeding insects treatments as the egg load of females was independent of sugar concentration or feeding frequency. Reallocation of nutrients therefore does not explain the responses to DR found in this study. The detrimental effects of a high calorie intake put parasitoids in a particularly difficult evolutionary dilemma. Due to the inability to convert carbohydrates into lipids (Visser et al., 2010), a continuous intake of carbohydrates is necessary in order to economize on capital lipid reserves and maintain egg production potential (Ellers & Van Alphen, 1997; Desouhant et al., 2010; Giron & Casas, 2003). On the other hand, the present results prove a high sugar consumption to be disadvantageous for longevity, and suggest selection may exist against overeating. A crucial factor is the quality of sugar sources in the field, both with regard to the type and concentration of sugars as well as the availability of food sources. However, sugar concentrations in fruit pulp or concentrated honeydew may well be high enough to induce unfavourable effects. Recently, there has been a surge of papers convincingly demonstrating the importance of nutritional balance in the diet in order to obtain maximal life span (Fanson et al., 2009; Grandison et al., 2009; Lee et al., 2008). Although the present study does not disqualify the relevance of nutrient composition in other species, our data unequivocally support the caloric restriction hypothesis and invalidate alternative explanations. A reduced energetic content is thought to be associated with a reduction in metabolic rate that leads to decreased ROS production (Hunt et al., 2006; Sohal & Weindruch, 1996). A next step in disentangling the mechanisms responsible in life span extension in parasitoids would be to investigate the association between caloric restriction and resting metabolic rate, as has been found in stick insects (Roark & Bjorndal, 2009). In addition, measuring several indicators of oxidative stress under a full and restricted diet could resolve the role of reactive oxygen species in life span (Monaghan et al., 2009).

Acknowledgements

This study was supported by the Netherlands Organisation for Scientiﬁc Research (NWO) with an ALW-grant no. 816-01-013 to support B. Visser.

113

Chapter 8

Eﬀects of a lipid-rich diet on adult parasitoid income resources and survival

Bertanne Visser and Jacintha Ellers In press in Biological Control

Abstract Effectiveness of parasitoids as natural enemies in agro-ecosystems depends on key traits such as fecundity and longevity. Energy sources allocated into survival and reproduction can be mobilized from capital stores acquired during larval feeding, or from income resources through adult feeding. Adult parasitoids have a restricted diet consisting solely of carbohydrates and most species do not replenish lipid levels after emergence from their host. Here, we have adopted a novel approach that could improve pest elimination by parasitoids, in which we added different ratios of olive oil to the food of adults to reveal the potential of a lipid-rich diet to increase lipid levels and allocation into survival. Our results show that males of the parasitoid Cotesia glomerata had higher lipid levels when fed a diet containing a ratio of 90:10 percent honey to olive oil when compared to males at emergence and males fed honey-only. For females, lipid levels at emergence were similar to those of females fed a diet supplemented with a ratio of 75:25 percent honey to olive oil, yet in both of these treatments lipid levels were significantly higher when compared to females fed honey-only. This suggests that females on a lipid-containing diet economized on their lipid use. In contrast, survival of males and females was negatively affected by the addition of olive oil and no differences in survival were found when wasps were fed 1 day on various ratios of honey to olive oil compared to honey-only. Our results show that the addition of a lipid source can increase or maintain nutrient availability and further research into lipid supplementation could minimize detrimental effects in order to increase the effectiveness of parasitoids in certain agro-ecosystems.

115 Chapter 8

Introduction

Parasitoids are widely used as natural enemies of pest species in agro- ecosystems, but their effectiveness depends on life history traits such as longevity and fecundity (Jervis, 2007). Resource allocation to these key traits depends on the nutritional content of the host that the parasitoid larva developed on, as well as on the environmental conditions the adult parasitoid encounters (Jervis et al., 2008), particularly the availability of income food sources, such as nectar, fruit juices, honeydew, and haemolymph (Eijs et al., 1998; Godfray, 1994). Dietary concentrations of different types of carbohydrates, such as sugars (Wäckers, 2001), honeydew (Lee et al., 2004a) and nectar (Winkler et al., 2006) have been shown to drastically increase parasitoid fecundity and longevity under laboratory conditions (Jervis et al., 1996; Wäckers et al., 2005). For example, in a laboratory study Witting-Bissinger and colleagues (2008) have shown that longevity of two parasitoid species Trichogramma exiguum and Cotesia congregata increased 8.5 fold when wasps had access to floral nectar. For T. exiguum fecundity was shown to increase 2.3-6.3 folds when provided with nectar, depending on plant species. However, carbohydrates are typically limited in agro-ecosystems, possibly putting constraints on pest elimination efficiency in agro-ecosystems (Heimpel & Jervis, 2005; Wäckers, 2003). To increase parasitoid effectiveness in eliminating pests, research has focused on the provisioning of carbohydrates in the field. Approaches to increase carbohydrate availability in the field include spraying of sugar- rich food sources and the introduction of flowering plants (Landis et al., 2000). Indeed, increased carbohydrate availability positively affects parasitoid effectiveness under field conditions. A study by Cappuccino and colleagues (1999) showed that sugar provisioning by means of spraying resulted in higher levels of parasitism by the parasitoid Elachertus cacoeciae on its host Choristoneura fumiferana in treated plots, when compared to untreated control plots and plots where understory herbs had been removed. Similar findings have been obtained in other studies (Baggen, 1998; Evans et al., 2010). Although sugar-rich food sources positively affect survival and reproduction, the benefits for parasitoids are limited compared to those for other insect species. In general, insects exploit available sugar resources not only for acute energy needs, but also convert them into fatty acids and triglycerides to serve as a long-term energy source. Most parasitoid species, however, do not accumulate lipids as adults, even when they have access to carbohy-

116 Lipid-feeding in parasitoids drates (Giron & Casas, 2003; Visser & Ellers, 2008; Visser et al., 2010). This means that although the rate of lipid-use can be reduced through carbohydrate feeding, lipid reserves are not replenished during life (Ellers, 1996). As a consequence, parasitoids need continuous access to carbohydrates in order to preserve their irreplaceable lipid stores and to achieve fitness effects of carbohydrate feeding. On the contrary, a diet that would increase parasitoid lipid reserves would only have to be accessed intermittently to have long-term fitness benefits, as is the case in other insect species (Jacome et al., 1995; Leahy & Andow, 1994). Many insects use dietary lipids during life and take up lipids from the gut for storage, egg production or catabolism to release energy (Turunen, 1979; Grillo et al., 2007). Even though the diet of parasitoids typically contains few lipids, uptake of more complex lipids through host feeding is essential for egg production in some species (Mondy et al., 2006). Here, we explored the potential of a lipid-rich food source to increase nutrient availability and associated life history traits in parasitoids. We fed adults of the parasitoid Cotesia glomerata, a species that does not accumulate lipids as an adult (Visser et al., 2010), with various mixtures of honey and olive oil. Compared to other oils, such as palm or sunflower oil, olive oil shows highest similarity in the composition of fatty acids to Hymenoptera and Lepidoptera, especially in the ratio of the predominant fatty acids, palmitic, oleic and linoleic acid (Belitz et al., 2009; Ramirez- Tortosa et al., 2006; Thompson, 1973). We tested if this novel approach of provisioning a lipid-rich dietary substrate increased overall lipid content and longevity of adult C. glomerata.

Materials and methods

Species

Parasitized cocoons containing C. glomerata were obtained from an existing laboratory culture at the University of Wageningen, the Netherlands. C. glomerata is a parasitoid that attacks caterpillars of Pieris sp., a pest species found on plants of the cabbage family Brassicaceae. Coccoons were kept in small pots at 23◦C at a 12:12 light:dark regime and a relative humidity of 75%.

117 Chapter 8

Experimental set-up

Pots were inspected daily for newly emerged individuals. After emergence 20 males and 20 females per treatment were isolated and either frozen directly at -20◦C or placed in a vial containing wet cotton wool. Twenty males and females per treatment were presented with different ratios of mixed honey and olive oil (v/v) on a piece of parafilm; 100% honey (100:0), 90% honey and 10% olive oil (90:10) or 75% honey and 25% olive oil (75:25). After four days of continuous exposure, surviving individuals were frozen and stored at -20◦C for lipid content analyses. Lipid levels were determined using the following method: individuals were freeze-dried for 2 days, after which dry weight was determined to the nearest µg on a microbalance (Mettler Toledo UMT2). Individuals were subsequently placed in a vial containing 4 mL of ether to extract triglycerides. After 24 hours, ether was removed and individuals were washed with fresh ether. Individuals were freeze-dried for 2 days after ether extraction and dry weight was determined again. Twenty-five to 30 males and females were used per treatment in longevity experiments. After emergence from host pupae, individuals were isolated and allowed to feed for 1 day on a ratio of 100:0 or a ratio of 75:25 honey to olive oil. In addition, in three treatments parasitoids were allowed to feed continuously on a ratio of 100:0, 90:10 or 75:25 honey to olive oil. Individuals were monitored every morning for survival.

Statistics

Data on males and females were analyzed separately. Lipid content was determined by subtracting dry weight after ether extraction from dry weight before ether extraction, which was then converted to the percentage of lipids to correct for diﬀerences in body size. Normality of the data was inspected by looking at the error structures of the residuals of the data. Non-normal data was either log or cube root transformed to normality. Treatments were compared using an ANOVA followed by a Tukey test. Longevity data of males and females were analyzed separately. Using survival analysis, various models were compared to describe survival data and a model assuming a Weibull distribution of the data was used as this model showed the best ﬁt. All statistical analyses were performed using R project version 2.9 (R Development Core Team, 2009).

118 Lipid-feeding in parasitoids

Results

Lipid levels

Adult diet had a signiﬁcant eﬀect on lipid levels in C. glomerata males

(F3,45 = 3.715, P = 0.018). Dietary intake of a ratio of 90:10 honey to olive oil resulted in a significant increase in male lipid content compared to males that had recently emerged (Tukey: P = 0.025, n = 26) and males fed honey-only (Tukey: P = 0.047, n = 24), but a ratio of 90:10 did not differ from that of 75:25 honey to olive oil (Tukey: P = 0.856, n = 11; Figure 1A). No significant differences were found when comparing recently emerged males with those fed on a ratio of 75:25 (Tukey: P = 0.295, n= 25) and honey-only (Tukey: P = 0.989, n = 38) or a ratio of 75:25 with honey-only (Tukey: P = 0.412, n = 23). For females, dietary treatment also had a significant effect on lipid content (F3,40 = 8.336, P < 0.001). Dietary intake of honey-only resulted in a significant decrease in lipid levels when compared to females fed on a ratio of 75:25 (Tukey: P < 0.001, n = 23) and freshly emerged females (Tukey: P = 0.001, n = 23; Figure 1B). Lipid levels of females fed 90:10 honey to olive oil did not differ significantly from recently emerged females (Tukey: P = 0.438, n = 21) and those fed 75:25 honey to olive oil (Tukey, P = 0.272, n = 21) and honey-only (Tukey: P = 0.081, n = 22).

Longevity

Males fed one day on a ratio of 75:25 honey to olive oil and honey-only lived on average 2.3 and 2.7 days, whereas females lived on average 3.3 and 3.6 days on those dietary treatments, respectively. Males and females that were fed one day with a ratio of 75:25 honey to olive oil showed no extended longevity compared to those fed on honey-only (males: P = 0.350, n = 58; females: P = 0.577, n = 46; figure 8.1A). Continuous feeding on a ratio of 75:25, 90:10 and honey-only resulted in an average lifespan of 2.9, 2.4 and 12.6 days respectively for males and 7.6, 6.5 and 18.9 for females. When fed continuously on honey-only, males and females lived significantly longer compared to those fed on 75:25 and 90:10 honey to olive oil (males: P < 0.001, n = 61; females: P < 0.001, n = 73; figure 8.1B).

119 Chapter 8

A 30 ab 25 b 20 15 a a 10

5 Lipid level (in %) level (in Lipid 0 Emergence 100:0 90:10 75:25 Treatment

B 30 25 a 20 a ab 15 b 10

Lipid level (in %) (in level Lipid 5 0 Emergence 100:0 90:10 75:25 Treatment

Figure 8.1: Percentage of lipids for male (A) and female (B) C. glomerata at emergence and after feeding for 4 days on different ratios of honey to olive oil. Different letters represent significant differences at α = 0.05.

Discussion

Our results show that parasitoids can ingest lipids when allowed to feed on a food source containing honey supplemented with olive oil. The uptake of lipids in adult males resulted in an increase in lipid levels; in females the inclusion of olive oil in the diet resulted in a lower rate of decrease of lipid reserves compared to females that were fed on honey-only. If fed on a diet containing only carbohydrates, females need to utilize their capital lipid stores, obtained during larval feeding, for allocation into survival and reproduction. Females fed on honey that was supplemented with olive oil can economize on their lipid use, because a source of lipids is provided in the food. Therefore, their lipid levels may remain stable or decrease at a slower rate. We found no evidence that dietary lipids can be used to extend longevity. Our data show that one day of feeding on honey supplemented with olive oil did not result in increased lifespan. Moreover, longevity was negatively

120 Lipid-feeding in parasitoids

Figure 8.2: Figure 2: Kaplan-Meier curves of survivorship in days for C. glomerata females fed for 1 day (A) and fed continuously (B) on diﬀerent ratios of honey to olive oil (Kaplan-Meier curves for males were similar to those for females).

121 Chapter 8 aﬀected by the addition of olive oil when parasitoids were allowed to feed on the mixture ad libitum during their lives. This would suggest that olive oil becomes toxic to C. glomerata, even after only a few days of feeding. We expected olive oil to be a suitable lipid substrate, because the main component consists of triglycerides with a total fatty acid composition that shows most similarity to that of insects in comparison to other oils (Belitz et al., 2009; Ramirez-Tortosa et al., 2006; Thompson, 1973). Although lipid compositions are comparable, relatively large quantities of oleic acid are present in olive oil (between 68 to 81.5 percent) (Belitz et al., 2009; Ramirez-Tortosa et al., 2006), whilst Hymenoptera, in particular, have on average 45.8 percent oleic acid (Thompson, 1973). Aside from the relative large quantity of oleic acid, olive oil contains several other compounds, such as hydrocarbons, sterols, non-glyceride esters, and alcohols (Ramirez- Tortosa et al., 2006). Mismatch in lipid ratios or the presence of other compounds in olive oil could be responsible for the observed toxicity in parasitoids when fed a lipid-rich diet continuously. An important question that remains to be answered is whether parasitoids are capable of using lipids provided in their diet for allocation of resources into reproduction. Other insects have been shown to readily incorporate dietary lipids in their fat body (McGuire & Gussin, 1967) and to subsequently use these lipid stores for allocation into eggs (Ziegler & Ibrahim, 2001). Inclusion of lipids in the diet of C. glomerata could increase egg size or number; however, C. glomerata fecundity is very high compared to most other parasitoids. Newly emerged females usually contain over 450 eggs, and those fed on honey for a week contain more than 950 eggs (Sato, 1975). It is unclear if capital lipid reserves are limiting lifetime fecundity, or if fecundity is limited by other factors such as egg maturation rate. Our novel approach to increase lipid reserves in parasitoids was only partly successful. We have shown that C. glomerata can successfully ingest lipids during adult life, and employ these to increase lipid reserves or economize on the rate of lipid use. One possibility to avoid toxicity of ingested lipids would be to solely add palmitic acid to the diet. Palmitic acid is the end-product of the lipogenesis pathway, which can be modiﬁed into other fatty acids by elongation or desaturation (Garrett & Grisham, 1999). In a study using the cockroach Periplaneta americana labeled palmitic acid was readily ingested and subsequently absorbed and redistributed as diglycerides in the haemolymph of the insect (Chino & Downer, 1979). Even though parasitoids do not increase their lipid reserves, they are likely capa-

122 Lipid-feeding in parasitoids ble of modifying palmitic acid, for instance for the formation of phospholipids that are an important part of the cell membrane and as precursors for the production of pheromones, sterols and eicosanoids (Stanley-Samuelson et al., 1988). Experiments in which more complex fatty acids are added, such as certain sterols or triglycerides, could also prove valuable as sterols are important for oogenesis (Mondy et al., 2006) and triglycerides are the bulk form in which lipids are stored in the insect fat body.

Acknowledgements

We are grateful to Eric Kok for his help during experiments. We would like to thank Gerard Driessen and Cécile Le Lann for helpful comments on earlier drafts of the manuscript. BV was supported by the Netherlands Organisation for Scientiﬁc Research (NWO), ALW-grant no. 816-01-013.

123

Chapter 9

Synthesis

A key objective in evolutionary biology is to unravel how and why organisms change over time to adapt to environmental conditions and how traits within populations have propagated to define a population or species into their current forms. This thesis has focused on the loss of an essential metabolic trait in parasitoids, which mechanisms underlie trait loss in this group of organisms and how the environment, and in particular nutrition, affects parasitoid traits. In this final chapter I will summarize the main findings of this thesis. I will further discuss the prevalence of lack of lipogenesis in other organisms and the implications of lacking lipid synthesis for our understanding of parasitoid physiology. Host manipulative abilities and the lack of larval lipid synthesis will be discussed, followed by the importance of nutrition in evolutionary processes. The final part of this synthesis deals with the mechanisms that underlie trait loss, the re-evolution of lost traits and the role of trait loss in evolutionary theory. Throughout this synthesis I will highlight potential research avenues to be pursued based on the work presented in this thesis, as in science elucidating the answers to some questions should ultimately lead to the development of perhaps even more new questions.

Loss of lipid synthesis through environmental compensation and underlying physiological pathways

Over the last two decades research on parasitoids revealed that numerous parasitic insects lack lipid accumulation in their adult life-stage (Ellers, 1996; Giron et al., 2002)(Chapter 2 and references therein). A pertinent question that arose was the extent to which lack of lipogenesis occurred within insects and if the evolutionary loss of this trait was restricted to parasitic insect lineages. Using a comparative approach, phylogenetic analysis of lipogenic ability of 94 insect species revealed that within Diptera, Coleoptera and Hymenoptera, lack of lipogenesis is conﬁned to parasitic lineages (Chapter 3). A lack of an essential metabolic trait, such as lipid synthesis, is quite rare and only few other organisms have been found to lack this metabolic trait. One example is the parasitic fungus Malassezia

125 Chapter 9 globosa (Xu et al., 2007). This fungus lives on the human scalp or on the skin of other warm-blooded animals where it feeds on lipids provided by the host. A lack of lipogenesis further occurs in another fungus (Katinka et al., 2001), bacteria (Fraser et al., 1995; Mushegian & Koonin, 1996) and several parasitic flatworms that do not synthesize de novo palmitate, yet they are able to modify their phospholipids that are of particular importance to the formation of cell membranes (Brouwers et al., 1997; Kohler & Voight, 1988; Tielens & Hellemon, 2006). In all three cases, the environment (in this case the host) facilitates these organisms with sufficient lipid levels, rendering their own lipid synthesis pathway prone to regression. A lack of lipogenesis is further only found in animals that are unable to eat, in which nutrients acquired during early development suffice in maintaining the required reproductive output (Gagné, 1994; Irwin & Lee Jr, 2000; Randolph, 2005). However, in organisms for which lipogenesis is still an essential phenotypic requirement, lipogenesis has been lost during the course of evolution due to the close association with their hosts, signifying that organisms involved in intricate symbiotic relationships are especially prone to lose otherwise indispensable traits. A lack of lipid synthesis was previously thought to occur in the parasitic protist, Trypanosoma brucei, responsible for causing human sleeping disease (Lee et al., 2007; Smith & Butikofer, 2010; Stephens et al., 2007). It is an obligate parasite of mammals, transmitted through Tsetse flies as the intermediary host. For more than 30 years the mammalian form of this parasite was thought unable to synthesize fatty acids de novo, after studies had shown radioactively labelled acetate, the intermediary precursor for fatty acid synthesis, was not recovered from their lipid fraction. However, this parasite was shown to exploit an alternative pathway for fatty acid synthesis, in which fatty acids are synthesized de novo by elongases, instead of the conventional pathway through the action of fatty acid synthase (FAS) that is typically employed by animals and fungi (Type I, (Smith, 1994)) and bacteria and plants (Type II, (Rock & Jackowski, 2002)). As the name suggests, in most organisms elongases elongate the product of the fatty acid biosynthesis pathway, palmitate, that is generated through the enzymatic function of fatty acid synthase (FAS), into longer chain fatty acids. How- ever, in T. brucei four separate elongases are used to form the bulk of fatty acids. Each of these four elongases is attached to the membrane of the endoplasmic reticulum where precursors are converted first to capric acid (C10:0) and myristic acid (C14:0), of which the latter is subsequently elongated by a third elongase into oleic acid (C18:0). The last elongase uses host lipid

126 Synthesis

substrates to elongate the polyunsaturated arachidonic acid C20:4 to C22:4 that are used as precursors for more complex lipid types. Clearly, in some organisms enzymes other than FAS, that are involved in lipid synthesis or other biochemical processes, can be used in the biosynthesis of fatty acids. A recent comparison of 56 genome sequences of organisms ranging from bacteria and fungi to animals showed that elongase and desaturase (that introduce a double bond into fatty acids) genes and enzymes are highly variable even between closely related organisms, in which enzyme substrate specificity can show a remarkable divergence (Hashimoto et al., 2008). Elon- gation and desaturation typically occur through enzymes associated with the membrane of the endoplasmic reticulum (ER), whilst in animals the majority of fatty acids are synthesized in the cell’s cytosol. A study using the house fly Musca domestica revealed a novel function of FAS, in which this enzyme was found in association with the ER (recovered as partial ER vesicles called microsomes after chemical analyses) and capable of synthesizing both fatty acids and methyl-fatty acids that are used as precursors for pheromone production (Gu et al., 1997). Cytosolic and microsomal FAS were shown to differ in their amino acid composition and could possibly explain the divergent affinity of both enzymes for producing either fatty acids or methyl-fatty acids. It remained unclear from this study, however, whether differences between these enzymes are caused by two different genes or post-transcriptional changes. Clearly, metabolic pathways that were once considered highly conserved are now commonly found to have changed in numerous organisms to enhance their fitness (Eanes, 2011; Ginger et al., 2010) and perhaps some parasitoid species have evolved novel ways to deal with lacking lipid synthesis. Radiotracer and isotopic labelling studies in parasitoids have shown that neither glucose nor deuterium oxide were incorporated in the lipid fraction, demonstrating the lack of conventional lipid synthesis at a deeper biochemical level in at least two parasitoid species (Giron & Casas, 2003) (Chapter 6). These studies refute the possibility that declining lipid reserves observed in most feeding experiments is due to stable lipid loss, in which lipids are burned at a higher rate than at which they are synthesized. As described in chapter 2, lack of lipogenesis is expected to encompass only the fatty acid synthesis pathway producing the 16-carbon fatty acid palmitate. Further modification of this fatty acid requires desaturation and elongation into long-chain fatty acids that are important components of cell membranes, precursors for eicosanoids (signalling molecules) and other more complex lipid types (Stanley, 2006). Whilst earlier studies reported on the ability of

127 Chapter 9 numerous parasitoids to modify their fatty acid composition into deviating from that of the host, their lipogenic ability as adults remains to be tested (Barlow & Bracken, 1971; Jones et al., 1982; Thompson, 1979). In Chap- ter 5 I have shown that at least one parasitoid lacking lipid synthesis was able to adjust its fatty acid composition by modifying levels of linoleic and palmitic acid. An isotopic labelling study using N. vitripennis further revealed that deuterium labels were not recovered in saturated palmitic acid

(C16:0), stearic acid (C18:0) and the unsaturated linoleic acid (C18:2), suggesting these fatty acids are not synthesized. In contrast, deuterons were recovered from the mono-unsaturated oleic acid (C18:1) fraction (Box 1). One explanation for these findings is that palmitic acid (C16:0) is first desaturated into palmitoleic acid (C16:1), which is then elongated to oleic acid (C18:1), the most abundant fatty acid found in Hymenoptera (Thompson, 1973). A similar mechanism of elongation is found in parasitic helminths lacking lipid synthesis: No fatty acids are synthesized (C16:0), yet C18:1 is elongated to form (C20:1) (Brouwers et al., 1997). Hence in parasitoids, modification of lipid compositions through elongation and desaturation remains unaffected, even though fatty acids are not synthesized de novo.

Host manipulation ability and lack of larval lipid synthesis

A prime hypothesis explaining the loss of lipogenesis in parasitoids is their ability to extensively manipulate host physiology to increase resource availability. It is thus not only the eﬃciency with which parasitoids can carry over nutritional resources from the host, but also manipulation that is thought to play a key role in regression of the fatty acid synthesis pathway (Chapters 2, 4 and 5). In chapter 2, we hypothesized that excessive availability of lipids induced by physiological manipulation of the host renders parasitoids prone to losing their lipogenic ability. As a consequence, adults are constrained in the allocation of their resources by their teneral lipid reserves as trait non-functionality extends to the adult life-stage. Numerous parasitoids lacking lipid synthesis have been found to manipulate their host’s physiology, such as N. vitripennis (Rivers & Denlinger, 1994), Aphidius ervi (Caccia et al., 2005; Falabella et al., 2005) and Cote- sia rubecula (Harvey et al., 1999). Chapter 4 of this thesis showed that a Drosophila parasitoid, Leptopilina heterotoma, that actively synthesizes lipids as an adult, does not seem to manipulate its host’s physiology, whilst the parasitoid Asobara tabida potentially forces the host into increasing lipid availability. In contrast, the parasitoid Pachycrepoideus vindemmiae was

128 Synthesis

Box 1 Elongation of fatty acids in the parasitoid Nasonia vitripennis This experiment aimed at determining if labeled isotopes were incorporated in the lipid fraction of parasitoids and in particular whether or not the lack of lipid synthesis in parasitoids is due to stable lipid loss. We fed the parasitoid Nasonia vitripennis and the closely related aculeate Apis mellifera either deuterated sugar solution or a sugar water control and measured incorporation of deuterons into various fatty acids using GC-MS (methods are described in Chapter 6).

Our results showed that N. vitripennis lacking lipid synthesis in feeding experiments did not incorporate deuterons in palmitic acid (C16:0), the end-product of the lipogenesis pathway (Chapter 6; Table 9.1; Figure 9.1). We further found no incorporation in stearic acid (C18:0) and linoleic acid (C18:2) nor that deuterons were incorporated into oleic acid (C18:1), the most dominant fatty acid in Hymenoptera. These results suggest that palmitic acid is first desaturated to stearic acid and then elongated to oleic acid, hence the ability to elongate and desaturate fatty acids has not been lost in parasitoids that lack de novo lipid synthesis.

ns 250 ns water deuterium

200

150

ng/sample 100 * ns 50

0 C16:0 C18:0 C18:1 C18:2 Figure 9.1: Average amount of incorporation of 3 deuterons (ng/sample ± 1SE)

Table 9.1 N. vitripennis: Isotope tracing into the lipid fraction m+1 m+2 m+3

C16:0 Sugar water 210.047 (26.268) 22.453 (2.731) 1.527 (0.428) Deuterated water 187.707 (39.535) 21.559 (4.410) 1.946 (0.392)

C18:0 Sugar water 155.343 (12.773) 18.367 (1.557) 1.725 (0.158) Deuterated water 164.672 (36.552) 20.257 (4.490) 1.979 (0.417)

C18:1 Sugar water 20.194 (6.257) 5.130 (1.574)* 0.727 (0.209)* Deuterated water 44.063 (8.339) 11.875 (2.105)* 2.168 (0.383)*

C18:2 Sugar water 12.981 (1.950) 5.212 (1.138) 0.806 (0.198) Deuterated water 13.525 (2.645) 5.766 (1.445) 1.019 (0.370) * p-value < 0.05

129 Chapter 9

As a positive control we tracked deuteron incorporation in the aculeate A. mellifera. This species actively synthesizes lipids, which is reflected by the incorporation of deuterons in

palmitic and stearic acid (C16:0 and C18:0)(Figure 9.2; Table 9.2). Oleic acid C18:1 did not contain significant amounts of deuterons, nor did linoleic acid (C18:2), suggesting C18:1 is obtained through reduction of longer fatty acids, whereas linoleic acid is not synthesized, conforming to findings in other insects and N. vitripennis.

60 ns

50 ***

30 ***

ng/sample 20 ns 10

0 C16:0 C18:0 C18:1 C18:2 Figure 9.2: Average amount of incorporation of 3 deuterons (ng/sample ± 1SE)

Table 9.2 A. mellifera: Isotope tracing into the lipid fraction m+1 m+2 m+3

C16:0 Sugar water 936.773 (50.396) 103.344 (5.580)* 11.076 (2.519)*** Deuterated water 1165.238 (215.928) 167.777 (25.980)* 33.4783 (2.775)***

C18:0 Sugar water 781.455 (57.793) 92.350 (6.607) 8.155 (0.740)*** Deuterated water 960.175 (123.155) 132.880 (16.884) 18.392 (2.477)***

C18:1 Sugar water 987.311 (166.688) 162.728 (25.893) 19.704 (3.038) Deuterated water 1568.737 (447.713) 285.623 (69.169) 43.668 (9.645)

C18:2 Sugar water 43.326 (4.254) 15.679 (3.401) 2.659 (0.754) Deuterated water 51.652 (11.377) 19.364 (5.889) 3.215 (1.026)

* p-value < 0.05; *** p-value <0.001

130 Synthesis found unable to manipulate its host, yet this parasitoid lacks lipogenesis as an adult, demonstrating that for some species mechanisms other than host manipulation might underlie the lack of lipid synthesis or that the ability to manipulate host physiology has been lost in this species. Although loss of lipid synthesis through host manipulation remains an important hypothesis explaining the extensive occurrence of lack of lipogenesis within parasitic Hymenoptera, more studies on host manipulative abilities are necessary to confirm the correlation between manipulative abilities and the lack of lipid synthesis. Large differences in teneral lipid reserves are observed between species differing in their lipogenic abilities, in which species lacking synthesis emerge with reserves over 30%, whilst lipogenic species emerge with considerably lower reserves between 5 and 10% (Chapter 3). These observations further suggest that whether or not the host is manipulated to increase resources, it is essential to carry over sufficient lipid reserves in order to render the lipogenesis pathway prone to regression. The second part of this hypothesis concerns the lack of larval lipogenesis. Most studies so far, including this thesis, have only measured lack of adult lipogenesis, while theory predicts lack of lipogenesis in larvae. It is during the larval stage that lipogenesis is redundant or even costly as conversion of sugar sources into lipid reserves can be avoided by carrying over resources from the host. The benefit for larvae to lack lipid synthesis is, however, traded off by the disadvantages for adults, as their diet mainly includes sugar sources that contain only few or no lipids at all (Eijs et al., 1998; Giron & Casas, 2003; Giron et al., 2002). As discussed in chapter 2 of this thesis, for only few parasitoid species larvae have been tested for their lipogenic ability and larval lipogenesis was shown to vary between species, however, adult lipogenic abilities have not been tested for these species (Thompson, 1979; Yazgan, 1972). These studies have used artificial rearing media lacking lipid sources to determine developmental nutrient requirements. Although this method can be used in species for which artificial media have been developed, preferably this hypothesis would be tested under more natural conditions. Such an approach is however complicated by the difficulty with which host and parasitoid metabolic activities can be partitioned. The availability of the genome sequence of the parasitoid Naso- nia vitripennis might prove a valuable resource to elucidate larval lipogenic abilities (Werren et al., 2010). By evaluating transcription levels of key genes in the lipid synthesis pathway throughout larval developmental stages, we should be able to reveal larval lipogenic ability. Developmental stage- specific responses in gene transcription have been observed in Drosophila

131 Chapter 9 buzzatii, in which either larvae or adults selected for increased responses to induced heat, showed a stage-speciﬁc manner of expressing Heat Shock Protein 70 (hsp70 ) at a higher rate that is uncoupled from expression in the other developmental stage (Loeschcke & Krebs, 1996). Although intu- itively it would be expected that activity of metabolic pathways underlying lipogenesis is uniform in all developmental stages, at this point in time, we have yet to discover whether or not lipogenesis is uncoupled in larval and adult stages.

The eﬀects of nutrition on life history, trait acquisition and loss

Nutrition plays a pivotal role in determining an organism’s fitness through its inherent link with traits such as reproduction and survival (Boggs, 1981, 1992; Ellers & Van Alphen, 1997; O’Brien et al., 2004). The constraints posed by fluctuating environmental conditions on resource acquisition are expected to be particularly important in animals that do not feed or in parasitic wasps that do not accumulate lipids as adults. Hence parasitoids are expected to benefit greatly from acquiring additional food sources during the adult life-stage to conserve on the rate of lipid use. In chapter 7, we have explored the effect of caloric restriction on fecundity and longevity, showing that two parasitoids respond to a restricted caloric intake similar to many other animals by extending longevity while maintaining a stable egg load. This is the first study revealing an effect of caloric restriction in parasitoids and further showed that sugar sources accessed intermittently during two hours on a daily basis are sufficient to sustain a similar investment in survival and reproduction compared to an ad libitum food availability. Whilst under natural conditions sugar sources might be readily available, the environmental conditions within agro-ecosystems typically lack sufficient food sources to sustain the dietary needs of parasitoids. In chapter 8, I have explored the potential of dietary lipid intake by adult parasitoids to allevi- ate the negative effects associated with lacking food availability and lipid synthesis. In conjunction with findings of chapter 7, inclusion of lipids in the adult diet ultimately resulted in a decreased longevity either due to toxicity, or because a high calorie intake negatively affects survival. More studies are required to find an optimal balance between dietary lipid and sugar sources to increase survival and reproductive output of parasitoids employed as natural enemies. Many organisms are able to deal effectively with suboptimal dietary conditions through increasing resource exploitation efficiency (chapter 5) or by

132 Synthesis adapting to restrictions in food sources. Moreover, resource availability can substantially affect evolutionary changes (van Noordwijk & de Jong, 1986; Sgro & Hoffmann, 2004). For instance, analysis of the honey bee genome revealed numerous novel genes and gene functions have evolved in response to their diet, consisting mainly out of nectar and pollen (Kunieda et al., 2006). Another example is adaptation of the moth Plutella xylostella to counter the negative effects of a high-calorie diet through reducing lipid synthesis, whilst a low calorie diet led to an increase in propensity for lipid synthesis (Warbrick-Smith et al., 2006). Trait loss through dietary environmental compensation, as described in the introduction of this thesis and chapter 3, further highlight the ability of organisms to adapt to their diet. One of the few studies that directly linked nutritional resources to the mechanisms underlying trait loss was done by Hall and Colgrave (2008). In their study, experimental evolution was used to determine the effect of varying carbon availability on trait loss in the bacterium Pseudomonas fluorescens. P. fluorescens loses unnecessary swimming motility, yet varying carbon concentrations are important determinants of the rate and mechanisms that underlie the loss of this trait: low carbon availability revealed a negative genetic correlation between swimming motility and fitness, whilst effects at higher carbon levels were neutral. Clearly, nutritional conditions do not only affect life histories and associated trade-offs, but at a longer time scale can lead to the evolutionary loss of traits.

Mechanisms underlying lack of lipogenesis and trait loss

Trait loss has frequently been observed in a diverse range of organisms, yet the mechanisms that underlie trait loss have received considerably less attention (Maughan et al., 2007). The most obvious mechanism underlying the loss of a trait is through loss of certain genes or partial genome reductions. For instance, lack of lipogenesis in the parasitic fungus Malassezia globosa results from loss of the gene fatty acid synthase from its genome (Xu et al., 2007), whereas extensive genome reductions have been described mainly for endosymbiotic bacteria (Blanc et al., 2007; Dale & Moran, 2006; Hershberg et al., 2007). In a comparison of genome sequences from 5 vertebrates and 5 insects, it was revealed that orthologous gene losses have occurred frequently, in which hundreds of genes were lost from the genome entirely (Wyder et al., 2007). Moreover, the rate of gene loss is consistent with rates of molecular evolution, in which insects showed an 8-fold higher propensity to lose orthologous genes when compared to vertebrates.

133 Chapter 9

Another mechanism underlying trait loss comprises mutation accumulation within the coding region of a gene. The loss of vitamin C biosynthesis in humans and several other animals has resulted from a mutation in the coding region of the gene underlying vitamin C synthesis (Chatterjee, 1973; Ohta & Nishikimi, 1999). Similarly, the loss of sporulation in the bacterium Bacillus subtilis was found to be due to mutation accumulation (Maughan et al., 2007). The consequences of mutation accumulation in coding regions of a gene compromises functionality and is typically restricted to genes maintaining one function only (Prud’homme et al., 2007). Although gene loss and mutation accumulation can be directly linked to trait loss, the loss of a trait does not necessarily involve coding regions, complete gene losses or genome reductions. Trait loss can also result from less severe genomic changes, in which underlying pathways remain present but are rendered non-functional through modification of transcriptional regulation of trait expression. Hormones responding to metabolite levels typically occupy an important function in regulating gene transcription. An important hormone involved in sugar and lipid metabolism in vertebrates is insulin. Typically, insulin levels increase in response to increasing sugar availability activating transcription factors and consequently gene transcription (Murray-Rust et al., 1992; Le Roith et al., 1980; Ebbering et al., 1989). Although most research on the activity of insulin has been carried out in vertebrates, recent studies revealed a similar function for insulin in insects. In D. melanogaster insulin was found to be directly involved in fat metabolism, showing that fat storage increases when insulin levels increase (DiAngelo & Birnbaum, 2009). Recent studies further revealed that insulin signalling is affected by the common insect endosymbiont Wolbachia pipientis, in which removal of the endosymbiont leads to decreased insulin signalling, highlighting an important role for this endosymbiont in affecting regulation of fat metabolism in some insects (Ikeya et al., 2009). Phenotypic regression can further result from alteration in gene transcription through gene regulation by transcription factors. Changes in the regulatory region occur most frequently for genes with pleiotropic functions (one gene influences several traits), as pleiotropy poses constraints on mutations in the coding region (Prud’homme et al., 2007), i.e. mutation accumulation or gene loss also render the pleiotropic phenotype non-functional. Examples of trait loss through altered gene regulation have been found in Drosophila, in which parallel inactivation of regulatory elements has resulted in loss of pigmentation (Jeong et al., 2006; Prud’homme et al., 2006). In chapter 6 I studied the molecular mechanism underlying the loss of lipo-

134 Synthesis genesis in parasitic insects and showed that the gene transcription profile of Nasonia vitripennis deviates severely from that of Drosophila melanogaster, a species that actively synthesizes lipids. A lack of transcription in the key gene underlying fatty acid biosynthesis, fatty acid synthase (fas), explains the loss of this trait in parasitoids. Inspection of the amino acid sequence of FAS further suggests that no mutations have accumulated in the coding region of the gene, thus lack of transcription is most likely due to alterations in regulatory mechanisms or post-transcriptional interference. The rate of evolutionary change in regulatory elements is quite extraordinary and can lead to complicated multi-component systems that affect regulation of a single gene (Stone & Wray, 2001; Wray et al., 2003). In Drosophila it has been shown that three different enhancers (short DNA sequences that stimulate gene transcription) all differentially affect the gene underlying trichome pattern (McGregor et al., 2007). Future studies should aim at identifying the deviating nature of regulatory mechanisms that underlie the lack of lipogenesis in parasitoids; however, identification of regulatory involvement might not be straightforward for several reasons. First, because gene regulation is facilitated by numerous transcription factors that can differentially affect a trait (Wray, 2007); second, because gene regulatory mechanisms can be highly variable even between closely related organisms (Ronald et al., 2005); and third, because transcriptional activity can even differ between allelic variants (Ruvkun et al., 1991). Yet another mechanism potentially involved in trait loss concerns post- transcriptional processes. MicroRNAs are small strands of RNA that affect functionality of complementary strands of messenger RNA (mRNA) that are in turn translated into amino acid sequences for enzyme formation (Ambros, 2004). At least 100 miRNA genes have been found in the genomes of D. melanogaster and Caenorhabditis elegans (Ambros, 2004). Although the function of only few miRNAs has been characterized and information regarding trait loss through the action of microRNAs is lacking, in Drosophila a miRNA was found to affect lipid storage (Xu et al., 2003). When miRNAs were knocked out, flies showed elevated levels of di- and triglycerides, whereas an increase in miRNA levels led to the oppos- ing effect, in which di- and triglyceride levels decreased in comparison to wild-type flies. These findings suggest that miRNA regulation of gene expression is dose-dependent in flies. Whereas the rate of evolutionary change is expected to be higher when comparing transcription factors to structural alterations in coding regions, miRNAs have so far only been shown to have a repressive effect and are expected to evolve at an even faster evolutionary

135 Chapter 9 rate. Moreover, even though similar transcription factors can exert different effects within and between organisms, transcription factor families have remained highly conserved (Chen & Rajewsky, 2007; Hsia & McGinnis, 2003). Although some miRNAs also seem highly conserved, new miRNAs are formed more readily, some which are estimated to affect hundreds of different genes (Rajewsky, 2006). miRNAs have further been shown to not just affect mRNA translation, but also mRNA levels itself and interference of these miRNAs might be an alternative explanation for low transcription levels observed for numerous genes in N. vitripennis and an important mechanism involved in trait loss in general (Lim et al., 2005).

Re-evolution of lost traits and evolutionary trait dynamics

A surprising finding of our phylogenetic study (Chapter 3) was that lipogenic ability has re-evolved on three separate occasions within the parasitic Hymenoptera. This finding is in direct contrast with conventional views that highlight the improbability of regaining lost traits. The first person to describe the concept of irreversible evolution was Louis Dollo, stating that organisms cannot partially or fully return to a previous ancestral state (Collin & Miglietta, 2008). These views were re-formulated by Simpson (1953) stating that complex characters cannot re-evolve because phylogenetic observations suggest complex trait do not re-evolve (birds that have not regained teeth or snakes that have not regained legs) and because unused characters should be prone to mutation accumulation in the genes underlying an unnecessary trait, making reversion to a functional state unlikely (Collin & Miglietta, 2008). In recent years, several studies have found conflicting evidence regarding irreversible evolution showing that certain traits do re-evolve (reviewed in Collin & Miglietta (2008); Porter & Cran- dall (2003); Teotónio & Rose (2001)). An important notion with respect to Dollo’s law on the re-occurrence of lost traits is that these laws pertain to original state reversion that assume similar genes or genetic pathways underlie the reacquired trait when compared to the genetic architecture underlying the ancestral trait. With that concept in mind, estimation of time to loss-of-function of unused genes showed that gene function of unused characters can be maintained in the genome for up to 8 million years (Lynch & Conery, 2000; Marshall et al., 1994). Such long latency in gene degradation indicates that re-evolution of traits by genes with a lost function may be less constrained than previously thought, even on considerable evolutionary time scales.

136 Synthesis

Traits can be considered lost when their effect on the phenotype is silenced under certain environmental conditions that would normally warrant phenotypic trait expression in the ancestral lineage. Given this def- inition of trait loss, re-evolution of traits does not necessarily involve the re-acquisition of traits from ancestral genes with similar functions or reversal of mutation accumulation through counteracting beneficial mutations. Pleiotropy can be an important mechanism to retain genes within the genome even though one of its functions has lost its usefulness under current environmental conditions as selection might still act in favour of other gene functions (Collin & Miglietta, 2008). This could be an important mechanism by which genes underlying lipid synthesis have remained intact in the parasitoid genome. Re-evolution of traits could potentially even occur more easily when other genetic pathways can be co-opted. Other mechanisms that could provide a basis for evolution in reverse are gene duplication (gene duplicates are typically long-lived within the genome), alternative splicing (that affect mRNA transcript variance), evolution of alternative transcription start sites (to re-use a previously silenced gene) and post-translational enzyme structure modification (proteolytic cleavage or attachment of modifiers to one or more amino acids) (Lynch & Con- ery, 2000; Mann & Jensen, 2003; Meyer & Schartl, 1999), all of which have the potential for fuelling evolution in reverse concerning the re-evolution of lipogenesis in parasitoids. Evolutionary changes are brought about by differences in reproductive success between individuals harbouring a certain trait set. Whilst consensus exists between disciplines on the importance of trait acquisition, the effect of trait loss on evolutionary trait dynamics is grossly underestimated. However, in yeast 85% of the genome has been found dispensable without affecting viability and some trait losses within this species can exert beneficial effects on fitness under certain environmental conditions when compared to strains with functional genes (Goebl & Petes, 1986). The notion that trait loss leads to diminished genetic variation and specialization to certain environmental conditions is directly related to the view that trait loss might not be as important in evolutionary trait dynamics as is trait acquisition. As proposed by Olson (1999), it is the persistence of loss-of- function genes within the genome that could provide a large base for facilitating evolutionary changes through re-use of lost genes (cryptic genetic variation, Figure 9.1). One assumption of critical importance to this idea is that environmental conditions should not remain stable for prolonged periods of time. However, environmental conditions typically fluctuate not just

137 Chapter 9

Phenotype

Genetic variation translated to phenotype Trait loss

Trait acquisition

Cryptic genetic variation

Figure 9.1: Components of variation of importance to trait acquisition and loss.

through abiotic factors (such as temperature or humidity) but also through an organisms interactions within the environment that lead to alterations in environmental conditions for the organisms itself and its oﬀspring (i.e. niche construction theory (Odling-Smee, 1988; Odling-Smee et al., 2003; Laland & Sterelny, 2006). Olson (1999) further proposes that cycles of genetic loss of function are likely followed by re-use composing an important mechanism for molecular evolution and consequently evolutionary change.

Evolutionary theory and the loss of traits

It falls within the legacy of the Modern Synthesis (MS) that the overall majority of research is concerned with standing genetic variation to explain evolutionary changes. Without disregarding the importance of these classic views on evolution, the genomic era has and will reveal other mechanisms that affect evolutionary change that extend beyond the effects of standing genetic variation. Trait acquisition, and in particular evolutionary novelties. are already dealt with in the Extended Evolutionary Synthesis (EES), but trait loss still occupies only a small portion of research in evolutionary biology and its importance still tends to be ignored. As is becoming increasingly clear, trait loss significantly contributes to the part of genetic variation that remains hidden from selection. Although this might appear trivial, shifts in environmental conditions can lead to re-employment of

138 Synthesis cryptic genetic variance, the acquisition of novel traits and re-evolution of lost traits that are once again available for selection to act upon (Figure 9.1). Elucidating the range of mechanisms that underlie trait loss and the effects of trait dynamics will be an important area of research to explain evolutionary changes. With regard to evolutionary theory it is further particularly interesting to note that ecological theory still plays only a minor role in evolutionary biology research. As is exemplified by the concept of trait loss through environmental compensation, ecology plays a key role in affecting evolutionary changes. Hence by incorporating trait loss and ecology into current evolutionary theory, we can move forward to a framework explaining all aspects of importance to evolution.

139

Bibliography

Abu-Elheiga, L., Matzuk, M.M., Abo-Hashema, K. & Wakil, S.J. 2001. Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl coA carboxylase 2. Science 291: 2613–2616. Abu-Elheiga, L., Matzuk, M.M., Kordari, P., Oh, W., Shaikenov, T., Gu, Z. & Wakil, S.J. 2005. Mutant mice lacking acetyl-CoA carboxylase 1 are embryonically lethal. Proceedings of the National Academy of Sciences of the United States of America 102: 12011–12016. Alleyne, M. & Beckage, N.E. 1997. Parasitism-induced effects on host growth and metabolic efficiency in tobacco hornworm larvae parasitized by Cotesia congregata. Journal of Insect Physiology 43: 407–424. Ambros, V. 2004. The functions of animal microRNAs. Nature 431: 350– 355. Anderson, T.R., Hessen, D.O., Elser, J.J. & Urabe, J. 2005. Metabolic stoiciometry and the fate of excess carbon and nutrients in consumers. The American Naturalist 165: 1–15. Anson, R.M., Guo, Z., de Cabo, R., Iyun, T., Rios, M., Hagepanos, A., Ingram, D.K., Lane, M.A. & Mattson, M.P. 2003. Intermittent fasting dissociates beneficial effects of dietary restriction on glucose metabolism and neuronal resistance to injury from calorie intake. Proceedings of the National Academy of Sciences 100: 6216–6220. Arrese, E.L. & Soulages, J.L. 2010. Insect fat body: Energy, metabolism, and regulation. Annual Review of Entomology 55: 207–225. Asgari, S. & Rivers, D.B. 2010. Venom proteins from endoparasitoid wasps and their role in host-parasite interactions. Annual Review of Entomology 56: 313–335. Asplen, M.K. & Byrne, D.N. 2006. Quantification and ultrastructure of oosorption in Eretmocerus eremicus (Hymenoptera: Aphelinidae). Jour- nal of Morphology 267: 1066–1074. Azzouz, H., Giordanengo, P., Wäckers, F.L. & Kaiser, L. 2004. Effects of feeding frequency and sugar concentration on behavior and longevity of the adult aphid parasitoid: Aphidius ervi (Haliday) (Hymenoptera: Braconidae). Biological Control 31: 445–452. Baggen, L.R. 1998. The influence of food on Copidosoma koehleri (Hy- menoptera: Encyrtidae), and the use of flowering plants as a habitat management tool to enhance biological control of potato moth, Phthori-

141 maea operculella (Lepidoptera: Gelechiidae). Biological Control 11: 9– 17. Barlow, J.S. & Bracken, G.K. 1971. Incorporation of Na-1-14C-acetate into the fatty acids of two insect parasites (Hymenoptera) reared on different hosts. Canadian Journal of Zoology 49: 1297–1300. Bass, T.M., Grandison, R.C., Wong, R., Martinez, P., Partridge, L. & Piper, M.D.W. 2007. Optimization of dietary restriction protocols in Drosophila. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 62: 1071–1081. Bauerfeind, S.S., Perlick, J.E.C. & Fischer, K. 2009. Disentangling environmental effects on adult life span in a butterfly across the metamorphic boundary. Experimental Gerontology 44: 805–811. Beckage, N.E. & Gelman, D.B. 2004. Wasp parasitoid disruption of host development: Implications for new biologically based strategies for insect control. Annual Review of Entomology 49: 299–330. Beckage, N.E., Gelman, D.B., Chappell, M. & Alleyne, M. 1997. Effects of parasitism by the braconid wasp Cotesia congregata on metabolic rate in host larvae of the tobacco hornworm, Manduca sexta. Journal of Insect Physiology 43: 143–154. Beenakkers, A.M., van der Horst, D.J. & van Marrewijk, W.J. 1985. Insect lipids and lipoproteins, and their role in physiological processes. Progress in Lipid Research 24: 19–67. Behmer, S.T. 2009. Insect herbivore nutrient regulation. Annual Review of Entomology 54: 165–187. Beldade, P. & Brakefield, P.M. 2002. The genetics and evo-devo of butterfly wing patterns. Nature Reviews Genetics 3: 442–452. Belitz, H.D., Grosch, W. & Schieberle, P. 2009. Edible fats and oils. In: Food Chemistry, pp. 640–669. Springer-Verslag, Berlin. Bely, A.E. & Sikes, J.M. 2010. Latent regeneration abilities persist following recent evolutionary loss in asexual annelids. Proceedings of the National Academy of Sciences of the United States of America 107: 1464–1469. Benjamini, Y. & Hochberg, Y. 1995. Controlling the false discovery rate: A practical and powerful approach to multiple testing. Journal of the Royal Statical Society 57: 289–300. Bischof, C. & Ortel, J. 1996. The effects of parasitism by Glyptapanteles liparidis (Braconidae: Hymenoptera) on the hemolymph and total body

142 Bibliography

composition of gypsy moth larvae (Lymantria dispar, Lymantriidae: Lep- idoptera). Parasitology Research 82: 687–692. Blanc, G., Ogata, H., Robert, C., Audic, S., Suhre, K., Vestris, G., Claverie, J.M. & Raoult, D. 2007. Reductive genome evolution from the mother of Rickettsia. PLoS Genetics 3: e14. Blanchard, G.B., Orledge, G.M., Reynolds, S.E. & Franks, N.R. 2000. Divi- sion of labour and seasonality in the ant Leptothorax albipennis: worker corpulence and its influence on behaviour. Animal Behaviour 59: 723– 738. Blanchot, P. 1994. Inventaire des parasitoides de mouches synanthropes recenses en France. EPHE Biologie et Evolution des Insectes 7: 111–119. Bodin, A., Jaloux, B., Mandon, N., Vannier, F., Delbecque, J.P., Monge, J.P. & Mondy, N. 2007. Host-induced ecdysteroids in the stop-and-go oogenesis in a synovigenic parasitoid wasp. Archives of Insect Biochemistry and Physiology 65: 103–111. Boggs, C.L. 1981. Nutritional and life-history determinants of resource allocation in holometabolous insects. The American Naturalist 117: 692– 709. Boggs, C.L. 1992. Resource allocation: Exploring connections between foraging and life history. Functional Ecology 6: 508–518. Boggs, C.L. 1997. Reproductive allocation from reserves and income in butterfly species with differing adult diets. Ecology 78: 181–191. Brodeur, J. & Vet, L.E.M. 1994. Usurpation of host behaviour by a parasitic wasp. Animal Behaviour 48: 187–192. Brothers, D. 1999. Phylogeny and evolution of wasps, ants and bees (Hy- menoptera, Chrysidoidea, Vespoidea and Apoidea). Zoologica Scripta 28: 233–250. Brouwers, J.F.H.M., Smeenk, I.M.B., van Golde, L.M.G. & Tielens, A.G.M. 1997. The incorporation, modification and turnover of fatty acids in adult Schistosoma mansoni. Molecular and Biochemical Parasitology 88: 175– 185. Brown, J.J. & Chippendale, G.M. 1974. Migration of the Monarch butterfly Danaus plexippus: Energy sources. Journal of Insect Physiology 20: 1117–1130. Buckner, J.S. & Hagen, M.M. 2003. Triacylglycerol and phospholipid fatty acids of the silverleaf whitefly: Composition and biosynthesis. Archives

143 of Insect Biochemistry and Physiology 53: 66–79. Bull, J.J. & Charnov, E.L. 1985. On irreversible evolution. Evolution 39: 1149–1155. Burke, G.R. & Moran, N.A. 2011. Massive genomic decay in Serratia sym- biotica, a recently evolved symbiont of aphids. Genome Biology and Evo- lution 3: 195–208. Caccia, S., Leonardi, M.G., Casartelli, M., Grimaldi, A., de Eguileor, M., Pennacchio, F. & Giordana, B. 2005. Nutrient absorption by Aphidius ervi larvae. Journal of Insect Physiology 51: 1183–92. Cairney, J.W. 2000. Evolution of mycorrhiza systems. Naturwissenschaften 87: 467–75. Cakmak, O., Bashan, M. & Bolu, H. 2007a. The fatty acid compositions of predator Piocoris luridus (Heteroptera: Lygaeidae) and its host Monoste- ria unicostata (Heteroptera: Tingidae) reared on almond. Insect Science 14: 461–466. Cakmak, O., Bashan, M. & Satar, A. 2007b. Total lipid and fatty acid compositions of Lertha sheppardi (Neuroptera: Nemopteridae) during its main life stages. Biologia 62: 774–780. Campbell, B., Heraty, J., Rasplus, J.Y., Chan, K., Steffen-Campbell, J. & Babcock, C. 2000. Molecular systematics of the Chalcidoidea using 28S-D2 rDNA. In: Hymenoptera Evolution, Biodiversity and Biological Control (A. Austin & M. Dowton, eds), pp. 59–73. CSIRO, Canberra. Cappuccino, N., Houle, M.J. & Stein, J. 1999. The influence of understory nectar resources on parasitism of the spruce budworm Choristoneura fumiferana in the field. Agricultural and Forest Entomology 1: 33–36. Carman, G.M. & Han, G.S. 2006. Roles of phosphatidate phosphatase enzymes in lipid metabolism. Trends in Biochemical Sciences 31: 694– 699. Carton, Y., Boulétreau, M., van Alphen, J.J.M. & van Lenteren, J.C. 1986. The Drosophila parasitic wasps. In: The genetics and biology of Drosophila, pp. 347–394. Cambridge University Press, Cambridge. Carvalho, G.B., Kapahi, P. & Benzer, S. 2005. Compensatory ingestion upon dietary restriction in Drosophila melanogaster. Nature methods 2: 813–815. Casas, J., Driessen, G., Mandon, N., Wielaard, S., Desouhant, E., van Alphen, J.J.M., Lapchin, L., Rivero, A., Christides, J.P. & Bernstein, C.

144 Bibliography

2003. Energy dynamics in a parasitoid foraging in the wild. Journal of Animal Ecology 72: 691–697. Castro, L. & Dowton, M. 2006. Molecular analyses of the Apocrita (Insecta: Hymenoptera) suggest that the Chalcidoidea are sister to the diaprioid complex. Invertebrate Systematics 20: 603–614. Castro, L.R. & Dowton, M. 2005. The position of the Hymenoptera within the Holometabola as inferred from the mitochondrial genome of Perga condei (Hymenoptera: Symphyta: Pergidae). Molecular Phylogenetics and Evolution 34: 469–479. Cerqueira, F.M. & Kowaltowski, A.J. 2010. Commonly adopted caloric restriction protocols often involve malnutrition. Ageing Research Reviews 9: 424–430. Chamberlain, P.M., Bull, I.D., Black, H.I., Ineson, P. & Evershed, R.P. 2004. Lipid content and carbon assimilation in Collembola: Implications for the use of compound-specific carbon isotope analysis in animal dietary studies. Oecologia 139: 325–335. Chambers, P., Simpson, S.J. & Raubenheimer, D. 1995. Behavioural mechanisms of nutrient balancing in Locusta migratoria nymphs. Animal Be- haviour 50: 1513–1523. Chapman, T. & Partridge, L. 1996. Female fitness in Drosophila melanogaster: An interaction between the effect of nutrition and of en- counter rate with males. Proceedings of the Royal Society B: Biological Sciences 263: 755–759. Chatterjee, I.B. 1973. Evolution and the biosynthesis of ascorbic acid. Sci- ence 182: 1271–1272. Chegwidden, W. & Spencer, I.M. 1996. Carbonic anhydrase provides bicar- bonate for de novo lipogenesis in the locust. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 115: 247– 254. Chen, K. & Rajewsky, N. 2007. The evolution of gene regulation by transcription factors and microRNAs. Nature Reviews Genetics 8: 93–103. Chen, L., Onagbola, E.O. & Fadamiro, H.Y. 2005. Effect of temperature, sugar availability, gender, mating, and size on the longevity of phorid fly Pseudacteon tricuspis (Diptera: Phoridae). Environmental Entomology 34: 246–255. Chino, H. & Downer, R.G.H. 1979. The role of diacylglycerol in absorption

145 of dietary glyceride in the American cockroach, Periplaneta americana L. Insect Biochemistry 9: 379–382. Chino, H. & Gilbert, L.I. 1965. Studies on the interconversion of carbohydrate and fatty acid in Hyalophora cecropia. Journal of Insect Physiology 11: 287–295. Colasurdo, N., Gélinas, Y. & Despland, E. 2009. Larval nutrition affects life history traits in a capital breeding moth. Journal of Experimental Biology 212: 1794–1800. Coley, P.D., Bateman, M.L. & Kursar, T.A. 2006. The effects of plant quality on caterpillar growth and defense against natural enemies. Oikos 115: 219–228. Colinet, H., Boivin, G. & Hance, T. 2007. Manipulation of parasitoid size using the temperature-size rule: Fitness consequences. Oecologia 152: 425–433. Collin, R. & Miglietta, M.P. 2008. Reversing opinions on Dollo’s law. Trends in Ecology & Evolution 23: 602–609. Colman, R.J., Anderson, R.M., Johnson, S.C., Kastman, E.K., Kosmatka, K.J., Beasley, T.M., Allison, D.B., Cruzen, C., Simmons, H.A., Kemnitz, J.W. & Weindruch, R. 2009. Caloric restriction delays disease onset and mortality in rhesus monkeys. Science 325: 201–204. Consoli, F.L. & Vinson, S.B. 2004. Host regulation and the embryonic development of the endoparasitoid Toxoneuron nigriceps (Hymenoptera: Braconidae). Comparative Biochemistry and Physiology Part B: Bio- chemistry & Molecular Biology 137: 463–473. Cooper, T.M., Mockett, R.J., Sohal, B.H., Sohal, R.S. & Orr, W.C. 2004. Effect of caloric restriction on life span of the housefly, Musca domestica. The FASEB Journal 18: 1591–1593. Coudron, T.A., Brandt, S.L. & Raqib, A. 1997. Comparison of the response of Heliothis virescens to parasitism by Euplectrus comstockii and Euplectrus plathypenae. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 116: 197–202. Coudron, T.A., Raqib, A., Brandt, S.L. & Wright, M.K. 1998. Comparison of the hemolymph proteins in permissive and non-permissive hosts of Euplectrus comstockii. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 120: 349–357. Cripps, C., Blomquist, G.J. & de Renobales, M. 1986. De novo biosynthesis

146 Bibliography

of linoleic acid in insects. Biochimica et Biophysica Acta 876: 572–580. Cruickshank, R.H. & Paterson, A.M. 2006. The great escape: Do parasites break Dollo’s law? Trends in Parasitology 22: 509–515. Cruz, A. 1989. The decline of an adaptation in the absence of a presumed selection pressure. Evolution 43: 55–62. Csaki, L.S. & Reue, K. 2010. Lipins: Multifunctional lipid metabolism proteins. Annual Review of Nutrition 30: 257–272. Dahlman, D.L. 1991. Teratocytes and host-parasitoid interactions. Biolog- ical Control 1: 118–126. Dale, C. & Moran, N.A. 2006. Molecular interactions between bacterial symbionts and their hosts. Cell 126: 453–465. Darwin, C. 1859. On the origin of species by means of natural selection or the preservation of favoured races in the struggle for life. John Murray, London. David, J., Cohet, Y. & Gouillet, P. 1975. Physiology of starvation and use of reserves in Drosophila melanogaster adults. Archives de Zoologie Experimentale et Generale 116: 579–590. Desjardins, C.A., Perfectti, F., Bartos, J.D., Enders, L.S. & Werren, J.H. 2010. The genetic basis of interspecies host preference diﬀerences in the model parasitoid Nasonia. Heredity 104: 270–277. Desouhant, E., Lucchetta, P., Giron, D. & Bernstein, C. 2010. Feeding activity pattern in a parasitic wasp when foraging in the ﬁeld. Ecological Research 25: 419–428. DiAngelo, J.R. & Birnbaum, M.J. 2009. Regulation of fat cell mass by insulin in Drosophila melanogaster. Molecular and Cellular Biology 29: 6341–52. Ding, L. 2003. Physiological and biochemical changes in summer and winter diapause and non-diapause pupae of the cabbage armyworm, Mamestra brassicae L. during long-term cold acclimation. Journal of Insect Physi- ology 49: 1153–1159. Djawdan, M., Rose, M.R. & Bradley, T.J. 1997. Does selection for stress resistance lower metabolic rate? Ecology 78: 828–837. Dobzhansky, T. 1937. Genetics and the origin of species. Colombia Uni- versity Press, New York. Dohlen, C.D. & Moran, N.A. 1995. Molecular phylogeny of the homoptera:

147 A paraphyletic taxon. Journal of Molecular Evolution 41: 211–223. Dorken, M.E., Neville, K.J. & Eckert, C.G. 2004. Evolutionary vestigialization of sex in a clonal plant: Selection versus neutral mutation in geographically peripheral populations. Proceedings of the Royal Society B: Biological Sciences 271: 2375–2380. Douglas, A.E. 1998. Insect-microbial symbioses: Aphids and their symbiotic bacteria Buchnera. Annual Review of Entomology 43: 17–37. Dowton, M. & Austin, A.D. 2001. Simultaneous analysis of 16S, 28S, COI and morphology in the Hymenoptera: Apocrita - evolutionary transitions among parasitic wasps. Biological Journal of the Linnean Society 74: 87– 111. Dunnett, C.W. 1980. Multiple comparisons in the unequal pairwise variance case. Journal of the American Statistical Association 75: 796– 800. Duplus, E. & Forest, C. 2002. Is there a single mechanism for fatty acid regulation of gene transcription? Biochemical Pharmacology 64: 893– 901. Eanes, W.F. 2011. Molecular population genetics and selection in the glycolytic pathway. The Journal of Experimental Biology 214: 165–171. Ebbering, R.H.M., Smit, A.B. & Van Minnen, J. 1989. The insulin family: Evolution of structure and function in vertebrates and invertebrates. Biological Bulletin 177: 176–182. Eckert, C.G. 2001. The loss of sex in clonal plants. Evolutionary Ecology 15: 501–520. Egas, M., Sabelis, M. & Dieckmann, U. 2005. Evolution of specialization and ecological character displacement of herbivores along a gradient of plant quality. Evolution 59: 507–520. Eggleton, P. & Belshaw, R. 1992. Insect parasitoids: An evolutionary overview. Philosophical Transactions of the Royal Society B: Biological Sciences 337: 1–20. Eijs, I.E.M., Ellers, J. & van Duinen, G.J. 1998. Feeding strategies in drosophilid parasitoids: The impact of natural food resources on energy reserves in females. Ecological Entomology 23: 133–138. Eijs, I.E.M. & Van Alphen, J.J.M. 1999. Life history correlations: Why are hymenopteran parasitoids an exception? Ecology Letters 2: 27–35. Ellers, J. 1996. Fat and eggs: An alternative method to measure the trade-

148 Bibliography

off between survival and reproduction in insect parasitoids. Netherlands Journal of Zoology 3: 227–235. Ellers, J., Driessen, G. & Sevenster, J.G. 2000a. The shape of the trade-off function between egg production and life span in the parasitoid Asobara tabida. Netherlands Journal of Zoology 50: 29–36. Ellers, J., Sevenster, J.G. & Driessen, G. 2000b. Egg load evolution in parasitoids. The American Naturalist 156: 650–665. Ellers, J., Van Alphen, J.J.J.M. & Sevenster, J.G. 1998. A field study of size-fitness relationships in the parasitoid Asobara tabida. Journal of Animal Ecology 67: 318–324. Ellers, J. & Van Alphen, J.J.M. 1997. Life history evolution in Asobara tabida: Plasticity in allocation of fat reserves to survival and reproduction. Journal of Evolutionary Biology 10: 771–785. Ellers, J. & Van Alphen, J.J.M. 2002. A trade-off between diapause duration and fitness in female parasitoids. Ecological Entomology 27: 279–284. Endler, J.A. 1983. Natural and sexual selection on color patterns in poeciliid fishes. Environmental Biology of Fishes 9: 173–190. Evans, E.W., Anderson, M.R. & Bowling, P.D. 2010. Targeted sugar provision promotes parasitism of the cereal leaf beetle Oulema melanopus. Agricultural and Forest Entomology 12: 41–47. Fadamiro, H.Y., Chen, L.I., Onagbola, E.O. & Graham, L. 2005. Lifespan and patterns of accumulation and mobilization of nutrients in the sugar- fed phorid fly, Pseudacteon tricuspis. Physiological Entomology 30: 212– 224. Fadamiro, H.Y. & Heimpel, G.E. 2001. Effects of partial sugar deprivation on lifespan and carbohydrate mobilization in the parasitoid Macrocentrus grandii (Hymenoptera: Braconidae). Annals of the Entomological Society of America 94: 909–916. Falabella, P., Perugino, G., Caccialupi, P., Riviello, L., Varricchio, P., Tranfaglia, A., Rossi, M., Malva, C., Graziani, F., Moracci, M. & Pennac- chio, F. 2005. A novel fatty acid binding protein produced by teratocytes of the aphid parasitoid Aphidius ervi. Insect Molecular Biology 14: 195– 205. Fanson, B.G., Weldon, C.W., Pérez-Staples, D., Simpson, S.J. & Taylor, P.W. 2009. Nutrients, not caloric restriction, extend lifespan in Queens- land fruit flies (Bactrocera tryoni). Aging Cell 8: 514–523.

149 Fellowes, M.D.E. & Godfray, H.C.J. 2000. The evolutionary ecology of resistance to parasitoids by Drosophila. Heredity 84: 1–8. Fernando-Warnakulasuriya, G., Tsuchida, K. & Wells, M. 1988. Eﬀect of dietary lipid content on lipid transport and storage during larval development of Manduca sexta. Insect Biochemistry 18: 211–214. Fisher, R.A. 1930. The genetical theory of natural selection. Clarendon Press, Oxford. Flook, P.K., Klee, S. & Rowell, C.H.F. 1999. Combined molecular phylogenetic analysis of the Orthoptera (Arthropoda, Insecta) and implications for their higher systematics. Systematic Biology 48: 233–253. Fong, D.W., Kane, T.C. & Culver, D.C. 1995. Vestigialization and loss of nonfunctional characters. Annual Review of Ecology and Systematics 26: 249–268. Fraser, C.M., Gocayne, J.D., White, O., Adams, M.D., Clayton, R.A., Fleischmann, R.D., Bult, C.J., Kerlavage, A.R., Sutton, G., Kelley, J.M., Fritchman, J.L., Weidman, J.F., Small, K.V., Sandusky, M., Fuhrmann, J., Nguyen, D., Utterback, T.R., Saudek, D.M., Phillips, C.A., Merrick, J.M., Tomb, J.F., Dougherty, B.A., Bott, K.F., Hu, P.C., Lucier, T.S., Peterson, S.N., Smith, H.O., Clyde A. Hutchison III, C.A. & Venter, J.C. 1995. The minimal gene complement of Mycoplasma genitalium. Science 270: 397–404. Frost, P.C., Evans-White, M.A., Finkel, Z.V., Jensen, T.C. & Matzek, V. 2005. Are you what you eat? Physiological constraints on organismal stoichiometry in an elementally imbalanced world. Oikos 109: 18–28. Fukuda, Y. & Naganuma, T. 2001. Potential dietary eﬀects on the fatty acid composition of the common jelly Aurelia aurita. Marine Biology 138: 1029–1035. Futuyma, D.J. & Agrawal, A.A. 2009. Macroevolution and the biological diversity of plants and herbivores. Proceedings of the National Academy of Sciences 106: 18054–18061. Gagné, R.J. 1994. The gall midges of the neotropical region. Cornell Uni- versity Press, Ithaca, NY, USA. Garrett, R.H. & Grisham, C.M. 1999. Lipid biosynthesis. In: Biochemistry (S. Kiselica, L. Boyle Riley & S. Fitz-Hugh, eds), pp. 802–851. Saunders College Publishing, Orlando. Geer, B.W., Langevin, M.L. & McKechnie, S.W. 1985. Dietary ethanol

150 Bibliography

and lipid synthesis in Drosophila melanogaster. Biochemical Genetics 23: 607–622. Gianoli, E., Suárez, L.H., Gonzáles, W.L., González-Teuber, M. & Acuña Rodríguez, I.S. 2007. Host-associated variation in sexual size dimorphism and fitness effects of adult feeding in a bruchid beetle. Entomologia Ex- perimentalis et Applicata 122: 233–237. Gibo, D.L. & McCurdy, J.A. 1993. Lipid accumulation by migrating monarch butterflies (Danaus plexippus L.). Canadian Journal of Zoology 71: 76–82. Gibson, G. & Hogness, D.S. 1996. Effect of polymorphisms in the Drosophila regulatory gene Ultrabithorax on homeotic stability. Science 271: 200– 203. Ginger, M.L., Fritz-Laylin, L.K., Fulton, C., Cande, W.Z. & Dawson, S.C. 2010. Intermediary metabolism in protists: A sequence-based view of facultative anaerobic metabolism in evolutionarily diverse eukaryotes. Pro- tist 161: 642–671. Giron, D. & Casas, J. 2003. Lipogenesis in an adult parasitic wasp. Journal of Insect Physiology 49: 141–147. Giron, D., Pincebourde, S. & Casas, J. 2004. Lifetime gains of host-feeding in a synovigenic parasitic wasp. Physiological Entomology 29: 436–442. Giron, D., Rivero, A., Mandon, N., Darrouzet, E. & Casas, J. 2002. The physiology of host feeding in parasitic wasps: Implications for survival. Functional Ecology 16: 750–757. Godfray, H.C.J. 1994. Parasitoids: Behavioural and evolutionary ecology. Princeton University Press, West Sussex. Goebl, M.G. & Petes, T.D. 1986. Most of the yeast genomic sequences are not essential for cell growth and division. Cell 46: 983–992. Gopalapillai, R., Kadono-Okuda, K. & Okuda, T. 2005. Molecular cloning and analysis of a novel teratocyte-specific carboxylesterase from the parasitic wasp, Dinocampus coccinellae. Insect Biochemistry and Molecular Biology 35: 1171–1180. Goubault, M. 2005. Patch exploitation strategies of parasitic wasps under intraspecific competition. Behavioral Ecology 16: 693–701. Goubault, M., Fourrier, J., Krespi, L., Poinsot, D. & Cortesero, A. 2004. Selection strategies of parasitized hosts in a generalist parasitoid depend on patch quality but also on host size. Journal of Insect Behavior 17:

151 99–113. Grandgirard, J., Poinsot, D., Krespi, L., Nénon, J.P. & Cortesero, A.M. 2002. Costs of secondary parasitism in the facultative hyperparasitoid Pachycrepoideus dubius: Does host size matter? Entomologia Experi- mentalis et Applicata 103: 239–248. Grandison, R.C., Piper, M.D.W. & Partridge, L. 2009. Amino-acid imbal- ance explains extension of lifespan by dietary restriction in Drosophila. Nature 462: 1061–1064. Gray, E.M. & Bradley, T.J. 2003. Metabolic rate in female Culex tarsalis (Diptera: Culicidae): Age, size, activity, and feeding effects. Journal of Medical Entomology 40: 903–911. Grillo, L.A.M., Majerowicz, D. & Gondim, K.C. 2007. Lipid metabolism in Rhodnius prolixus (Hemiptera: Reduviidae): Role of a midgut triacylglycerol-lipase. Insect Biochemistry and Molecular Biology 37: 579–588. Grimaldi, D. & Engels, M. 2005. Evolution of insects. Cambridge Univ Press, New York. Grönke, S., Mildner, A., Fellert, S., Tennagels, N., Petry, S., Müller, G., Jäckle, H. & Kühnlein, R.P. 2005. Brummer lipase is an evolutionary conserved fat storage regulator in Drosophila. Cell Metabolism 1: 323– 330. Grosman, A.H., Janssen, A., de Brito, E.F., Cordeiro, E.G., Colares, F., Fonseca, J.O., Lima, E.R., Pallini, A. & Sabelis, M.W. 2008. Parasitoid increases survival of its pupae by inducing hosts to fight predators. PLoS ONE 3: e2276. Gu, P., Welch, W.H., Guo, L., Schegg, K.M. & Blomquist, G.J. 1997. Char- acterization of a novel microsomal fatty acid synthetase (FAS) compared to a cytosolic FAS in the housefly, Musca domestica. Comparative Bio- chemistry and Physiology Part B: Biochemistry & Molecular Biology 118: 447–456. Hahn, D. 2005. Larval nutrition affects lipid storage and growth, but not protein or carbohydrate storage in newly eclosed adults of the grasshopper Schistocerca americana. Journal of Insect Physiology 51: 1210–1219. Hahn, D.A. & Denlinger, D.L. 2007. Meeting the energetic demands of insect diapause: Nutrient storage and utilization. Journal of Insect Physi- ology 53: 760–73.

152 Bibliography

Haldane, J.B.S. 1932. The time of action of genes, and its bearing on some evolutionary problems. American Naturalist 55: 5–24. Hall, A.R. & Colegrave, N. 2008. Decay of unused characters by selection and drift. Journal of Evolutionary Biology 21: 610–617. Hammer, O., Harper, D.A.T. & Ryan, P.D. 2001. Paleontological statistics software package for education and data analysis. Han, H.Y. & Ro, K.E. 2005. Molecular phylogeny of the superfamily Tephri- toidea (Insecta: Diptera): New evidence from the mitochondrial 12S, 16S, and COII genes. Molecular Phylogenetics and Evolution 34: 416–430. Harbach, R.E. 2007. The Culicidae (Diptera): A review of taxonomy, classification and phylogeny. Zootaxa 1668: 591–638. Harman, D. 1956. Aging: A theory based on free radical and radiation chemistry. Journal of gerontology 11: 298–300. Harper, L.J., Schonrogge, K., Lim, K.Y., Francis, P. & Lichtenstein, C.P. 2004. Cynipid galls: Insect-induced modifications of plant development create novel plant organs. Plant, Cell and Environment 27: 327–335. Harris, R.J. & Beggs, J.R. 1995. Variation in the quality of Vespula vulgaris L. queens (Hymenoptera: Vespidae) and its significance in wasp population dynamics. New Zealand Journal of Zoology 22: 131–142. Hartley, S.E. 1998. The chemical composition of plant galls: Are levels of nutrients and secondary compounds controlled by the gall-former? Oe- cologia 113: 492–501. Hartley, S.E. & Lawton, J.H. 1992. Host-plant manipulation by gall-insects: A test of the nutrition hypothesis. Journal of Animal Ecology 61: 113– 119. Harvey, J., Jervis, M., Gols, R., Jiang, N. & Vet, L. 1999. Development of the parasitoid, Cotesia rubecula (Hymenoptera: Braconidae) in Pieris rapae and Pieris brassicae (Lepidoptera: Pieridae): Evidence for host regulation. Journal of Insect Physiology 45: 173–182. Harvey, J., Wagenaar, R. & Gols, R. 2011. Differing host exploitation efficiencies in two hyperparasitoids: When is a ’match made in heaven’? Journal of Insect Behavior 24: 282–292. Harvey, J.A., Wagenaar, R. & Bezemer, T.M. 2009. Interactions to the fifth trophic level: Secondary and tertiary parasitoid wasps show extraordinary efficiency in utilizing host resources. The Journal of Animal Ecology 78: 686–692.

153 Hashimoto, K., Yoshizawa, A.C., Okuda, S., Kuma, K., Goto, S. & Kane- hisa, M. 2008. The repertoire of desaturases and elongases reveals fatty acid variations in 56 eukaryotic genomes. Journal of lipid research 49: 183–91. Hawkins, T.H. 1950. Opening of milk bottles by birds. Nature 165: 435– 436. Hazel, J.R. 1995. Thermal adaptation in biological membranes: Is homeoviscous adaptation the explanation? Annual Review of Physiology 57: 19–42. Heimpel, G. & Jervis, M. 2005. Does floral nectar improve biological control by parasitoids? In: Plant-provided food for carnivorous insects: A protective mutualism and its applications, pp. 267–304. Cambridge University Press, Cambridge (MA). Hein, S. & Dorn, S. 2008. The parasitoid of a fruit moth caterpillar utilizes fruit components as nutrient source to increase its longevity and fertility. Biological Control 44: 341–348. Heping, W., Ling, M. & Baoping, L. 2008. Effects of feeding frequency and sugar concentrations on lifetime reproductive success of Meteorus pulchricornis (Hymenoptera: Braconidae). Biological Control 45: 353– 359. Hershberg, R., Tang, H. & Petrov, D. 2007. Reduced selection leads to accelerated gene loss in Shigella. Genome Biology 8: R164. Hoback, W.W., Rana, R.L. & Stanley, D.W. 1999. Fatty acid compositions of phospholipids and triacylglycerols of selected tissues, and fatty acid biosynthesis in adult periodical cicadas, Magicicada septendecim. Com- parative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 122: 355–362. Hogervorst, P.A.M., Wäckers, F.L. & Romeis, J. 2007. Effects of honeydew sugar composition on the longevity of Aphidius ervi. Entomologia Experimentalis et Applicata 122: 223–232. Holliday, R. 1989. Food, reproduction and longevity: Is the extended lifespan of calorie-restricted animals an evolutionary adaptation. Bioessays 10: 125–127. Holm, C., Osterlund, O., Laurell, H. & Contreras, J.A. 2000. Molecular mechanisms regulating hormone-sensitive lipase and lipolysis. Annual Review of Nutrition 20: 365–93.

154 Bibliography

Horie, Y., Nakasone, S. & Ito, T. 1968. The conversion of 14C- carbohydrates into CO2 and lipid by the silkworm, Bombyx mori. Journal of Insect Physiology 14: 971–981. Hornstein, E. & Shomron, N. 2006. Canalization of development by mi- croRNAs. Nature Reviews Genetics 38: S20–S24. Houck, M.A. & O’Connor, B.M. 1991. Ecological and evolutionary significance of phoresy in the Astigmata. Annual Review of Entomology 36: 611–636. Howell, J. & Fisher, R.C. 1977. Food conversion efficiency of a parasitic wasp, Nemeritis canescens. Ecological Entomology 2: 143–151. Hsia, C.C. & McGinnis, W. 2003. Evolution of transcription factor function. Current Opinion in Genetics & Development 13: 199–206. Hunt, N.D., Hyun, D.H., Allard, J.S., Minor, R.K., Mattson, M.P., Ingram, D.K. & de Cabo, R. 2006. Bioenergetics of aging and calorie restriction. Ageing Research Reviews 5: 125–143. Hunt, T., Bergsten, J., Levkanicova, Z., Papadopoulou, A., John, O.S., Wild, R., Hammond, P.M., Ahrens, D., Balke, M. & Caterino, M.S. 2007. A comprehensive phylogeny of beetles reveals the evolutionary origins of a superradiation. Science 318: 1913–1916. Huxley, J.S. 1942. Evolution: The modern synthesis. Allen & Ulwin, Lon- don. Hvam, A. & Toft, S. 2005. Effects of prey quality on the life history of a harvestman. Journal of Arachnology 33: 582–590. Iizuka, K., Bruick, R.K., Liang, G., Horton, J.D. & Uyeda, K. 2004. De- ficiency of carbohydrate response element-binding protein (ChREBP) reduces lipogenesis as well as glycolysis. Proceedings of the National Academy of Sciences of the United States of America 101: 7281–7286. Ikeya, T., Broughton, S., Alic, N., Grandison, R. & Partridge, L. 2009. The endosymbiont Wolbachia increases insulin/IGF-like signalling in Drosophila. Proceedings of the Royal Society B: Biological Sciences 276: 3799–3807. Inness, C.L.W. & Metcalfe, N.B. 2008. The impact of dietary restriction, intermittent feeding and compensatory growth on reproductive investment and lifespan in a short-lived fish. Proceedings of the Royal Society B: Biological Sciences 275: 1703–1708. Irwin, J.T. & Lee Jr, R.E. 2000. Mild winter temperatures reduce sur-

155 vival and potential fecundity of the goldenrod gall fly, Eurosta solidaginis (Diptera: Tephritidae). Journal of Insect Physiology 46: 655–661. Jacome, I., Aluja, M., Liedo, P. & Nestel, D. 1995. The influence of adult diet and age on lipid reserves in the tropical fruit fly Anastrepha serpentina (Diptera: Tephritidae). Journal of Insect Physiology 41: 1079– 1086. Jaenike, J. 1990. Host specialization in phytophagous insects. Annual Review of Ecology and Systematics 21: 243–273. Janz, N., Nylin, S. & Wahlberg, N. 2006. Diversity begets diversity: Host expansions and the diversification of plant-feeding insects. BMC Evolu- tionary Biology 6: 4. Jeong, S., Rokas, A. & Carroll, S.B. 2006. Regulation of body pigmentation by the Abdominal-B Hox protein and its gain and loss in Drosophila evolution. Cell 125: 1387–1399. Jervis, M. 2007. Insects as natural enemies: A practical perspective. Springer, Dordrecht. Jervis, M. & Ferns, P. 2011. Towards a general perspective on life-history evolution and diversification in parasitoid wasps. Biological Journal of the Linnean Society 104: 443–461. Jervis, M., Kidd, N. & Heimpel, G. 1996. Parasitoid adult feeding behaviour and biocontrol - A review. Biocontrol News and Information 17: 11N– 26N. Jervis, M.A., Ellers, J. & Harvey, J.A. 2008. Resource acquisition, allocation, and utilization in parasitoid reproductive strategies. Annual Review of Entomology 53: 361–385. Jervis, M.A. & Ferns, P.N. 2004. The timing of egg maturation in insects: Ovigeny index and initial egg load as measures of fitness and of resource allocation. Oikos 107: 449–461. Jervis, M.A., Heimpel, G.E., Ferns, P.N., Harvey, J.A. & Kidd, N.A.C. 2001. Life-history strategies in parasitoid wasps: A comparative analysis of ‘ovigeny’. Journal of Animal Ecology 70: 442–458. Jones, D., Barlow, J.S. & Thompson, S.N. 1982. Exeristes, Itoplectis, Aphaereta, Brachymeria, and Hyposoter species: In vitro glyceride synthesis and regulation of fatty acid composition. Experimental Parasitology 54: 340–351. Joy, T.K., Arik, A.J., Corby-Harris, V., Johnson, A.A. & Riehle, M.A.

156 Bibliography

2010. The impact of larval and adult dietary restriction on lifespan, reproduction and growth in the mosquito Aedes aegypti. Experimental Gerontology 45: 685–690. Jump, D.B., Clarke, S.D., Thelen, A. & Liimatta, M. 1994. Coordinate regulation of glycolytic and lipogenic gene expression by polyunsaturated fatty acids. Journal of Lipid Research 35: 1076–1084. Kaeberlein, M., Burtner, C.R. & Kennedy, B.K. 2007. Recent developments in yeast aging. PLoS Genet 3: e84. Kanehisa, M. & Goto, S. 2000. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Research 28: 27–30. Kapahi, P., Zid, B.M., Harper, T., Koslover, D., Sapin, V. & Benzer, S. 2004. Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway. Current Biology 14: 885–890. Katinka, M.D., Duprat, S., Cornillot, G.E., Méténier, F., Thomarat, G., Prensier, V., Barbe, E., Peyretaillade, P., Brottier, P., Wincker, F., Del- bac, H., Alaoui, E., Peyret, P., Saurin, W., Manolo, G., Weissenbach, J. & Vivarès, C.P. 2001. Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi. Nature 414: 450–453. Kaun, K.R. & Sokolowski, M.B. 2009. cGMP-dependent protein kinase: Linking foraging to energy homeostasis. Genome 52: 1–7. Kemp, D.J. & Alcock, J. 2003. Lifetime resource utilization, flight physiology, and the evolution of contest competition in territorial insects. The American Naturalist 162: 290–301. Kersten, S. 2001. Mechanisms of nutritional and hormonal regulation of lipogenesis. EMBO Reports 2: 282–286. Khebbeb, M.E.H., Delachambre, J. & Soltani, N. 1997. Lipid metabolism during the sexual maturation of the mealworm (Tenebrio molitor): Effect of ingested diflubenzuron. Pesticide Biochemistry and Physiology 58: 209–217. Kimura, K.D., Tissenbaum, H.A., Liu, Y. & Ruvkun, G. 1997. daf-2, an Insulin Receptor-Like Gene that regulates longevity and diapause in Caenorhabditis elegans. Science 277: 942–946. Klass, M.R. 1977. Aging in the nematode Caenorhabditis elegans: Major biological and environmental factors influencing life span. Mechanisms of Ageing and Development 6: 413–429. Kohler, P. & Voight, K.P. 1988. Nutrition and metabolism. In: Parasitology

157 in Focus (H. Mehlhorn, ed), pp. 412–453. New York. Kostal, V., Sula, J. & Simek, P. 1998. Physiology of drought tolerance and cold hardiness of the Mediterranean tiger moth Cymbalophora pudica during summer diapause. Journal of Insect Physiology 44: 165–173. Krause, K. 2008. From chloroplasts to ‘cryptic’ plastids: Evolution of plas- tid genomes in parasitic plants. Current Genetics 54: 111–121. Kristensen, N.P. 1999. Phylogeny of endopterygote insects, the most successful lineage of living organisms. European Journal of Entomology 96: 237–253. Kristensen, N.P., Scoble, M.J. & Karsholt, O. 2007. Lepidoptera phylogeny and systematics: The state of inventorying moth and butterﬂy diversity. Zootaxa 1668: 699–747. Kunieda, T., Fujiyuki, T., Kucharski, R., Foret, S., Ament, S.A., Toth, A.L., Ohashi, K., Takeuchi, H., Kamikouchi, A., Kage, E., Morioka, M., Beye, M., Kubo, T., Robinson, G.E. & Maleszka, R. 2006. Carbohydrate metabolism genes and pathways in insects: Insights from the honey bee genome. Insect Molecular Biology 15: 563–576. Kunte, A.S., Matthews, K.A. & Rawson, R.B. 2006. Fatty acid auxotrophy in Drosophila larvae lacking SREBP. Cell Metabolism 3: 439–448. Lahti, D.C., Johnson, N.A., Ajie, B.C., Otto, S.P., Hendry, A.P., Blumstein, D.T., Coss, R.G., Donohue, K. & Foster, S.A. 2009. Relaxed selection in the wild. Trends in Ecology & Evolution 24: 487–496. Laland, K.N. & Sterelny, K. 2006. Seven reasons (not) to neglect niche construction. Evolution 60: 1751–1762. Landis, D.a., Wratten, S.D. & Gurr, G.M. 2000. Habitat management to conserve natural enemies of arthropod pests in agriculture. Annual Review of Entomology 45: 175–201. Laurenne, N., Karatolos, N. & Quicke, D.L.J. 2008. Hammering homoplasy: Multiple gains and losses of vibrational sounding in cryptine wasps (In- secta: Hymenoptera: Ichneumonidae). Biological Journal of the Linnean Society 96: 82–102. Lawrence, P.O. 2005. Non-poly-DNA viruses, their parasitic wasps, and hosts. Journal of Insect Physiology 51: 99–101. Lawton, J.H. 1983. Plant architecture and the diversity of phytophagous insects. Annual Review of Entomology 28: 23–39.

158 Bibliography

Le Lann, C., Visser, B., Baaren, J., Alphen, J.J.M. & Ellers, J. 2011. Comparing resource exploitation and allocation of two closely related aphid parasitoids sharing the same host. Evolutionary Ecology doi:10. 1007/s10682-011-9498-2. Le Ralec, A., Ribule, A., Barragan, A. & Outreman, Y. 2011. Host range limitation caused by incomplete host regulation in an aphid parasitoid. Journal of Insect Physiology 57: 363–371. Le Roith, D., Shiloach, J., Roth, J. & Lesniak, M.A. 1980. Evolutionary origins of vertebrate hormones: Substances similar to mammalian insulins are native to unicellular eukaryotes. Proceedings of the National Academy of Sciences of the United States of America 77: 6184–6188. Leahy, T.C. & Andow, D.A. 1994. Egg weight, fecundity and longevity are increased by adult feeding in Ostrinia nubilalis (Lepidoptera: Pyralidae). Annals of the Entomological Society of America 87: 342–349. Lee, J.C., Heimpel, G.E. & Leibee, G.L. 2004a. Comparing floral nectar and aphid honeydew diets on the longevity and nutrient levels of a parasitoid wasp. Entomologia Experimentalis et Applicata 111: 189–199. Lee, K.P., Simpson, S.J., Clissold, F.J., Brooks, R., Ballard, J.W.O., Taylor, P.W., Soran, N. & Raubenheimer, D. 2008. Lifespan and reproduction in Drosophila: New insights from nutritional geometry. Proceedings of the National Academy of Sciences 105: 2498–2503. Lee, K.P., Simpson, S.J. & Raubenheimer, D. 2004b. A comparison of nutrient regulation between solitarious and gregarious phases of the specialist caterpillar, Spodoptera exempta (Walker). Journal of Insect Physiology 50: 1171–1180. Lee, S.H., Stephens, J.L. & Englund, P.T. 2007. A fatty-acid synthesis mechanism specialized for parasitism. Nature Reviews Microbiology 5: 287–297. Levins, R. 1962. Theory of fitness in a heterogeneous environment. I. The fitness set and adaptive function. American Naturalist 96: 361–373. Libert, S., Zwiener, J., Chu, X., VanVoorhies, W., Roman, G. & Pletcher, S.D. 2007. Regulation of Drosophila life span by olfaction and food- derived odors. Science 315: 1133–1137. Lim, L.P., Lau, N.C., Garrett-Engele, P., Grimson, A., Schelter, J.M., Cas- tle, J., Bartel, D.P., Linsley, P.S. & Johnson, J.M. 2005. Microarray analysis shows that some microRNAs downregulate large numbers of tar-

159 get mRNAs. Nature 433: 769–773. Loehlin, D.W., Oliveira, D.C.S.G., Edwards, R., Giebel, J.D., Clark, M.E., Cattani, M.V., van de Zande, L., Verhulst, E.C., Beukeboom, L.W., Muñoz Torres, M. & Werren, J.H. 2010. Non-coding changes cause sex- specific wing size differences between closely related species of Nasonia. PLoS genetics 6: e1000821. Loeschcke, V. & Krebs, R.A. 1996. Selection for heat-shock resistance in larval and in adult Drosophila buzzatii: Comparing direct and indirect responses. Evolution 50: 2354–2359. Lorenz, M.W. 2001. Synthesis of lipids in the fat body of Gryllus bimaculatus: Age-dependency and regulation by adipokinetic hormone. Archives of Insect Biochemistry and Physiology 47: 198–214. Lynch, M. & Conery, J.S. 2000. The evolutionary fate and consequences of duplicate genes. Science 290: 1151–1155. Maddison, W.P. & Maddison, D.R. 2003. MacClade 4: Analysis of phylogenetic evolution. Mann, M. & Jensen, O.N. 2003. Proteomic analysis of post-translational modifications. Nature biotechnology 21: 255–261. Marden, J.H. & Waage, J.K. 1990. Escalated damselfly territorial contests are energetic wars of attrition. Animal Behaviour 39: 954–959. Markiewicz, D. & O’Donnell, S. 2001. Social dominance, task performance and nutrition: Implications for reproduction in eusocial wasps. Journal of Comparative Physiology A: Sensory, Neural, and Behavioral Physiology 187: 327–333. Marshall, C.R., Raff, E.C. & Raff, R.A. 1994. Dollo’s law and the death and resurrection of genes. Proceedings of the National Academy of Sciences 91: 12283–12287. Masoro, E.J. 2005. Overview of caloric restriction and ageing. Mechanisms of Ageing and Development 126: 913–922. Matsubara, K., Tojo, S. & Suzuki, N. 2005. Age-related changes in flight muscle mass, lipid reserves and flight capacity during adult maturation in males of the territorial damselfly Calopteryx atrata (Odonata: Caloptery- gidae). Zoological Science 22: 587–592. Mattson, W.J. 1980. Herbivory in relation to plant nitrogen-content. An- nual Review of Ecology and Systematics 11: 119–161.

160 Bibliography

Maughan, H., Masel, J., Birky, C.W. & Nicholson, W.L. 2007. The roles of mutation accumulation and selection in loss of sporulation in experimental populations of Bacillus subtilis. Genetics 177: 937–48. Mayhew, P.J. 1998. The evolution of gregariousness in parasitoid wasps. Proceedings of the Royal Society of London B: Biological Sciences 265: 383–389. Maynard Smith, J. & Szathmáry, E. 1995. The major transitions in evol- tuion. W.H. Freeman, Oxford. Mayr, E. 1942. Systematics and the origin of species. Colombia University Press, New York. McCay, C.M., Rowell, M.F. & Maynard, L.A. 1935. The effect of retarded growth upon the length of life span and upon the ultimate body size. Journal of Nutrition 10: 63–79. McGregor, A.P., Orgogozo, V., Delon, I., Zanet, J., Srinivasan, D.G., Payre, F. & Stern, D.L. 2007. Morphological evolution through multiple cis- regulatory mutations at a single gene. Nature 448: 587–590. McGuire, J.L. & Gussin, A.E.S. 1967. Effect of diet on the lipid composition of the cockroach Blaberus discoidalis. Comparative Biochemistry and Physiology 22: 427–433. Meyer, A. & Schartl, M. 1999. Gene and genome duplications in vertebrates: The one-to-four (-to-eight in fish) rule and the evolution of novel gene functions. Current Opinion in Cell Biology 11: 699–704. Moczek, A.P. 2005. The evolution and development of novel traits, or how beetles got their horns. BioScience 55: 937–951. Moczek, A.P. 2007. Developmental capacitance, genetic accommodation, and adaptive evolution. Evolution & Development 9: 299–305. Moczek, A.P. 2009. Differential recruitment of limb patterning genes during development and diversification of beetle horns. Proceedings of the National Academy of Sciences of the United States of America 106: 8992– 8997. Moczek, A.P. 2011. The origins of novelty. Nature 473: 34–35. Moiroux, J., Le Lann, C., Seyahooei, M.A., Vernon, P., Pierre, J.S., Van Baaren, J. & Van Alphen, J.J.M. 2010. Local adaptations of life-history traits of a Drosophila parasitoid, Leptopilina boulardi: Does climate drive evolution? Ecological Entomology 35: 727–736.

161 Molleman, F., Ding, J., Boggs, C.L., Carey, J.R. & Arlet, M.E. 2009. Does dietary restriction reduce life span in male fruit-feeding butterflies? Ex- perimental Gerontology 44: 601–606. Monaghan, P., Metcalfe, N.B. & Torres, R. 2009. Oxidative stress as a mediator of life history trade-offs: Mechanisms, measurements and interpretation. Ecology Letters 12: 75–92. Mondy, N., Corio-Costet, M.F., Bodin, A., Mandon, N., Vannier, F. & Monge, J.P. 2006. Importance of sterols acquired through host feeding in synovigenic parasitoid oogenesis. Journal of Insect Physiology 52: 897– 904. Moran, N.A. 2002. Microbial minimalism: Genome reduction in bacterial pathogens. Cell 108: 583–586. Morris, R.J. & Fellowes, M.D.E. 2002. Learning and natal host influence host preference, handling time and sex allocation behaviour in a pupal parasitoid. Behavioral Ecology and Sociobiology 51: 386–393. Müller, G.B. & Wagner, G.P. 1991. Novelty in evolution: Restructuring the concept. Annual Review of Ecology and Systematics 22: 229–256. MultiD Analyses AB 2008. GenEx Light Software. Murray-Rust, J., McLeod, A.N., Blundell, T.L. & Wood, S.P. 1992. Struc- ture and evolution of insulins: Implications for receptor binding. BioEs- says 14: 325–331. Mushegian, A.R. & Koonin, E.V. 1996. A minimal gene set for cellular life derived by comparison of complete bacterial genomes. Proceedings of the National Academy of Sciences of the United States of America 93: 10268–10273. Nakamatsu, Y. 2003. Venom of ectoparasitoid, Euplectrus sp. near plathypenae (Hymenoptera: Eulophidae) regulates the physiological state of Pseudaletia separata (Lepidoptera: Noctuidae) host as a food resource. Journal of Insect Physiology 49: 149–159. Nakamatsu, Y., Fujii, S. & Tanaka, T. 2002. Larvae of an endoparasitoid, Cotesia kariyai (Hymenoptera: Braconidae), feed on the host fat body directly in the second stadium with the help of teratocytes. Journal of Insect Physiology 48: 1041–1052. Nakamatsu, Y., Gyotoku, Y. & Tanaka, T. 2001. The endoparasitoid Cote- sia kariyai (Ck) regulates the growth and metabolic efficiency of Pseu- daletia separata larvae by venom and Ck polydnavirus. Journal of Insect

162 Bibliography

Physiology 47: 573–584. Nakamatsu, Y. & Tanaka, T. 2004. Venom of Euplectrus separatae causes hyperlipidemia by lysis of host fat body cells. Journal of Insect Physiology 50: 267–275. Nash, D.R., Als, T.D., Maile, R., Jones, G.R. & Boomsma, J.J. 2008. A mosaic of chemical coevolution in a large blue butterﬂy. Science 319: 88–90. Nemoto, S. & Finkel, T. 2004. Ageing and the mystery at Arles. Nature 429: 149–152. Nishikimi, M., Fukuyama, R., Minoshima, S., Shimizu, N. & Yagi, K. 1994. Cloning and chromosomal mapping of the human nonfunctional gene for L-gulono-gamma-lactone oxidase, the enzyme for L-ascorbic acid biosynthesis missing in man. Journal of Biological Chemistry 269: 13685– 13688. Nishikimi, M., Kawai, T. & Yagi, K. 1992. Guinea pigs possess a highly mutated gene for L-gulono-gamma-lactone oxidase, the key enzyme for L-ascorbic acid biosynthesis missing in this species. Journal of Biological Chemistry 267: 21967–21972. O’Brien, D.M., Boggs, C.L. & Fogel, M.L. 2004. Making eggs from nectar: The role of life history and dietary carbon turnover in butterﬂy reproductive resource allocation. Oikos 105: 279–291. O’Brien, D.M., Fogel, M.L. & Boggs, C.L. 2002. Renewable and nonre- newable resources: Amino acid turnover and allocation to reproduction in Lepidoptera. Proceedings of the National Academy of Sciences of the United States of America 99: 4413–4418. O’Brien, D.M., Schrag, D.P. & del Rio, C.M. 2000. Allocation to reproduction in a hawkmoth: A quantitative analysis using stable carbon isotopes. Ecology 81: 2822–2831. Odling-Smee, F., Laland, K. & Feldman, M. 2003. Niche construction: The neglected process in evolution. Princeton University Press, Princeton (NJ). Odling-Smee, F.J. 1988. Niche-constructing phenotypes. In: In the role of behavior (H. Plotkin, ed), pp. 73–132. The MIT Press, Cambridge (MA). Odling-Smee, F.J. 2010. Niche inheritance. In: Evolution the extended synthesis (M. Pigliucci & G.B. Müller, eds), pp. 175–208. The MIT Press, Cambridge (MA).

163 Ohta, Y. & Nishikimi, M. 1999. Random nucleotide substitutions in pri- mate nonfunctional gene for L-gulono-gamma-lactone oxidase, the missing enzyme in L-ascorbic acid biosynthesis. Biochimica et Biophysica Acta 1472: 408–411. Oliver, K.M., Degnan, P.H., Burke, G.R. & Moran, N.A. 2010. Facultative symbionts in aphids and the horizontal transfer of ecologically important traits. Annual Review of Entomology 55: 247–266. Olson, D., Fadamiro, H., Lundgren, J.G. & Heimpel, G.E. 2000. Effects of sugar feeding on carbohydrate and lipid metabolism in a parasitoid wasp. Physiological Entomology 25: 17–26. Olson, M.V. 1999. When less is more: Gene loss as an engine of evolutionary change. American journal of human genetics 64: 18–23. Ortel, J. 1991. Effects of lead and cadmium on chemical composition and total water content of the pupal parasitoid, Pimpla turionellae. Ento- mologia Experimentalis et Applicata 59: 93–100. Otronen, M. 1995. Energy reserves and mating success in males of the yellow dung fly Scatophaga stercoraria. Functional Ecology 9: 683–688. Özalp, P. & Emre, I. 2001. The effects of carbohydrates upon the survival and reproduction of adult female Pimpla turionellae L.(Hym., Ichneu- monidae). Journal of Applied Entomology 125: 177–180. Partridge, L., Piper, M.D.W. & Mair, W. 2005. Dietary restriction in Drosophila. Mechanisms of Ageing and Development 126: 938–950. Pedata, P.A., Garonna, A.P., Zabatta, A., Zeppa, P., Romani, R. & Isidoro, N. 2003. Development and morphology of teratocytes in Encarsia berlesei and Encarsia citrina: First record for Chalcidoidea. Journal of Insect Physiology 49: 1063–1071. Pennacchio, F. & Strand, M.R. 2006. Evolution of developmental strategies in parasitic hymenoptera. Annual Review of Entomology 51: 233–58. Pereboom, J.J.M. 2001. Size dimorphism in bumblebees: A result of caste specific differences in fat body metabolism? Netherlands Journal of Zoology 51: 323–333. Pérez-Brocal, V., Gil, R., Ramos, S., Lamelas, A., Postigo, M., Michelena, J.M., Silva, F.J., Moya, A. & Latorre, A. 2006. A small microbial genome: The end of a long symbiotic relationship? Science 314: 312–3. Pfaffl, M.W. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research 29: 16–21.

164 Bibliography

Pigliucci, M. 2007. Do we need an extended evolutionary synthesis? Evo- lution 61: 2743–2749. Pigliucci, M. 2008. What, if anything, is an evolutionary novelty. Philosophy of Science 75: 887–898. Pigliucci, M. & Müller, G.B. 2010. Elements of an extended evolutionary synthesis. In: Evolution The extended synthesis (M. Pigliucci & G. Muller, eds), pp. 3–17. The MIT Press, Cambridge (MA). Platnick, N.I. & Rosen, D.E. 1987. Popper and evolutionary novelties. History and philosophy of the life sciences 9: 5–16. Porter, M.L. & Crandall, K.A. 2003. Lost along the way: The signiﬁcance of evolution in reverse. Trends in Ecology & Evolution 18: 541–547. Poulson, T.L. & White, W.B. 1969. The cave environment. Science 165: 971–981. Price, P.W., Fernandes, G.W. & Waring, G.L. 1987. Adaptive nature of insect galls. Environmental Entomology 16: 15–24. Price, P.W. & Pschorn-Walcher, H. 1988. Are galling insects better protected against parasitoids than exposed feeders? A test using tenthre- dinid sawﬂies. Ecological Entomology 13: 195–205. Prud’homme, B., Gompel, N. & Carroll, S.B. 2007. Emerging principles of regulatory evolution. Proceedings of the National Academy of Sciences 104: 8605–8612. Prud’homme, B., Gompel, N., Rokas, A., Kassner, V.A., Williams, T.M., Yeh, S.D., True, J.R. & Carroll, S.B. 2006. Repeated morphological evolution through cis-regulatory changes in a pleiotropic gene. Nature 440: 1050–1053. Prud’homme, B., Minervino, C., Hocine, M., Cande, J.D., Aouane, A., Dufour, H.D., Kassner, V.A. & Gompel, N. 2011. Body plan innovation in treehoppers through the evolution of an extra wing-like appendage. Nature 473: 83–86. Pullin, A.S. 1987. Adult feeding time, lipid accumulation, and overwintering in Aglais urticae and Inachis io (Lepidoptera: Nymphalidae). Journal of Zoology 211: 631–641. Qin, Q., Gong, H. & Ding, T. 2000. Two collagenases are secreted by teratocytes from Microplitis mediator (Hymenoptera: Braconidae) cultured in vitro. Journal of Invertebrate Pathology 76: 79–80.

165 Quicke, D.L.J. 1997. Parasitic wasps. Chapman & Hall, London. Quicke, D.L.J., Fitton, M.G., Notton, D.G., Broad, G.R. & Dolphin, K. 2000. Phylogenty of the subfamilies of Ichneumonidae (Hymenoptera): A simultaneous molecular and morphological analysis. In: Hymenoptera Evolution, Biodiversity and Biological Control (A. Austin & M. Dowton, eds), pp. 74–83. R Development Core Team 2009. R: A language and environment for statistical computing. Rajewsky, N. 2006. microRNA target predictions in animals. Nature Ge- netics 38: S8–S13. Ramirez-Tortosa, M.C., Granados, S., Quiles, J.L. & Yaqoob, P. 2006. Chemical composition, types and characteristics of olive oil. In: Olive Oil and Health, pp. 45–61. CABI, Wallingford. Randolph, S. 2005. The natural history of the rose bedguar gall and its insect community. The British Plant Gall Society, Suffolk, UK. Regal, P.J. 1977. Evolutionary loss of useless features: Is it molecular noise suppression? The American Naturalist 111: 123–133. Reinert, J.F., Harbach, R.E. & Kitching, I.J. 2008. Phylogeny and classification of Ochlerotatus and allied taxa (Diptera: Culicidae: Aedini) based on morphological data from all life stages. Zoological Journal of the Linnean Society 153: 29–114. Rench, B. 1959. Evolution above the species level. Colombia University Press, New York. Renshaw, M., Silver, J.B. & Service, M.W. 1995. Differential lipid reserves influence host-seeking behaviour in the mosquitoes Aedes cantans and Aedes punctor. Medical and Veterinary Entomology 9: 381–387. Reshef, L., Hanson, R.W. & Ballard, F.J. 1970. A possible physiological role for glyceroneogenesis in rat adipose tissue. Journal of Biological Chemistry 245: 5979–5984. Reshef, L., Olswang, Y., Cassuto, H., Blum, B., Croniger, C.M., Kalhan, S.C., Tilghman, S.M. & Hanson, R.W. 2003. Glyceroneogenesis and the triglyceride/fatty acid cycle. The Journal of Biological Chemistry 278: 30413–30416. Rivero, A. & West, S.A. 2002. The physiological costs of being small in a parasitic wasp. Evolutionary Ecology Research 4: 407–420.

166 Bibliography

Rivers, D.B. & Denlinger, D.L. 1994. Redirection of metabolism in the flesh fly, Sarcophaga bullata, following envenomation by the ectoparasitoid Nasonia vitripennis and correlation of metabolic effects with the diapause status of the host. Journal of Insect Physiology 40: 207–215. Rivers, D.B. & Denlinger, D.L. 1995. Venom-induced alterations in fly lipid metabolism and its impact on larval development of the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae). Journal of Invertebrate Pathology 66: 104–110. Roark, A.M. & Bjorndal, K.A. 2009. Metabolic rate depression is induced by caloric restriction and correlates with rate of development and lifespan in a parthenogenetic insect. Experimental Gerontology 44: 413–419. Rock, C.O. & Jackowski, S. 2002. Forty years of bacterial fatty acid synthesis. Biochemical and Biophysical Research Communications 292: 1155– 1166. Roelofs, D., Overhein, L., de Boer, M.E., Janssens, T.K.S. & van Straalen, N.M. 2006. Additive genetic variation of transcriptional regulation: Met- allothionein expression in the soil insect Orchesella cincta. Heredity 96: 85–92. Roff, D.A. 1994. The evolution of flightlessness: Is history important? Evolutionary Ecology 8: 639–657. Ronald, J., Brem, R.B., Whittle, J. & Kruglyak, L. 2005. Local regulatory variation in Saccharomyces cerevisiae. PLoS genetics 1: e25. Ronshaugen, M., Biemar, F., Piel, J., Levine, M. & Lai, E.C. 2005. The Drosophila microRNA iab-4 causes a dominant homeotic transformation of halteres to wings. Genes & Development 19: 2947–2952. Rosell, R.C. & Wheeler, D.E. 1995. Storage function and ultrastructure of the adult fat body in workers of the ant Camponotus festinatus (Buckley) (Hymenoptera: Formicidae). International Journal of Insect Morphology and Embryology 24: 413–426. Rosenheim, J.A. 1996. An evolutionary argument for egg limitation. Evo- lution 50: 2089–2094. Rouleux-Bonnin, F., Renault, S., Rabouille, A., Periquet, G. & Bigot, Y. 1999. Free serosal cells originating from the embryo of the wasp Diadro- mus pulchellus in the pupal body of parasitized leek-moth, Acrolepiosis assectella. Are these cells teratocyte-like? Journal of Insect Physiology 45: 479–484.

167 Rufo, C., Teran-Garcia, M., Nakamura, M.T., Koo, S.H., Towle, H.C. & Clarke, S.D. 2001. Involvement of a unique carbohydrate-responsive factor in the glucose regulation of rat liver fatty-acid synthase gene transcription. The Journal of Biological Chemistry 276: 21969–21975. Ruvkun, G., Wightman, B., Bürglin, T. & Arasu, P. 1991. Dominant gain- of-function mutations that lead to misregulation of the C. elegans het- erochronic gene lin-14, and the evolutionary implications of dominant mutations in pattern-formation genes. Development Supplement 1: 47– 54. Saetre, G.P., Moum, T., Bures, S., Kral, M., Adamjan, M. & Moreno, J. 1997. A sexually selected character displacement in flycatchers reinforces premating isolation. Nature 387: 1995–1998. Salati, L.M. & Amir-Ahmady, B. 2001. Dietary regulation of expression of glucose-6-phosphate dehydrogenase. Annual Review of Nutrition 21: 121–40. Salvador, G. & Consoli, F.L. 2008. Development and morphology of teratocytes in Encarsia berlesei and Encarsia citrina: First record for Chal- cidoidea. Biological Control 45: 103–110. Sato, Y. 1975. Rearing Apanteles glomeratus L. on the larvae of Pieris rapae crucivora Boisduval fed on an artificial diet. Kontyu 43: 242–249. Savard, J., Tautz, D. & Lercher, M. 2006. Genome-wide acceleration of protein evolution in flies (Diptera). BMC Evolutionary Biology 6: 7. Schafellner, C., Marktl, R.C. & Schopf, A. 2007. Inhibition of juvenile hormone esterase activity in Lymantria dispar (Lepidoptera, Lymantri- idae) larvae parasitized by Glyptapanteles liparidis. (Hymenoptera, Bra- conidae). Journal of Insect Physiology 53: 858–68. Schneider, M.V., Gimenez, B., Driessen, G. & Van Alphen, J.J.M. 2001. Longevity in sexual and parthenogenetic Venturia canescens (Hy- menoptera). Entomologia Experimentalis et Applicata 12: 37–40. Scrutton, M.C. & Utter, M.F. 1968. The regulation of glycolysis and gluconeogenesis in animal tissues. Annual Review of Biochemistry 37: 249– 302. Sevenster, J.G., Ellers, J. & Driessen, G. 1998. An evolutionary argument for time limitation. Evolution 52: 1241–1244. Seyahooei, M.A., Kraaijeveld-Smit, F.J.L., Kraaijeveld, K., Crooijmans, J.B.M., Van Dooren, T.J.M. & Van Alphen, J.J.M. 2009. Closely related

168 Bibliography

parasitoids induce different pupation and foraging responses in Drosophila larvae. Oikos 118: 1148–1157. Sgro, C.M. & Hoffmann, A.A. 2004. Genetic correlations, tradeoffs and environmental variation. Heredity 93: 241–248. Shakoury-Elizeh, M., Protchenko, O., Berger, A., Cox, J., Gable, K., Dunn, T.M., Prinz, W.A., Bard, M. & Philpott, C.C. 2010. Metabolic response to iron deficiency in Saccharomyces cerevisiae. Journal of Biological Chemistry 285: 14823–14833. Sharkey, M.J. 2007. Phylogeny and classification of Hymenoptera. Zootaxa 1668: 521–548. Shirai, Y. 2006. Flight activity, reproduction, and adult nutrition of the beet webworm, Spoladea recurvalis (Lepidoptera: Pyralidae). Applied Entomology and Zoology 41: 405–414. Shorthouse, J.D., Leggo, J., Sliva, M. & Lalonde, R. 2005. Has egg location influenced the radiation of (Hymenoptera: Cynipidae) gall wasps on wild roses? Basic and Applied Ecology 6: 423–434. Shubin, N., Tabin, C. & Carroll, S. 2009. Deep homology and the origins of evolutionary novelty. Nature 457: 818–823. Shwarz, M. & Shaw, M.R. 2000. Western palearctic Cryptinae (Hy- menoptera: Ichneumonidae) in the national museums of Scotland, with nomenclatural changes, taxonomic notes, rearing records and special reference to the British check list. Part 3. Tribe Phygadeuontini, subtribes Chiroticin. Entomologist’s Gazette 51: 147–186. Siddall, M.E. 1993. Phylogeny and the reversibility of parasitism. Evolution 47: 308–313. Simmons, F.H. & Bradley, T.J. 1997. An analysis of resource allocation in response to dietary yeast in Drosophila melanogaster. Journal of insect physiology 43: 779–788. Simon, P. 2003. Q-Gene: Processing quantitative real-time RT-PCR data. Bioinformatics 19: 1439–1440. Simpson, G.G. 1944. Tempo and mode in evolution. Colombia University Press, New York. Simpson, S.J. & Raubenheimer, D. 2007. Caloric restriction and aging revisited: The need for a geometric analysis of the nutritional bases of aging. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 62: 707–713.

169 Sivinski, J., Aluja, M. & Holler, T. 2006. Food sources for adult Diachas- mimorpha longicaudata, a parasitoid of tephritid fruit flies: Effects on longevity and fecundity. Entomologia Experimentalis et Applicata 118: 193–202. Skinner, C. & Lin, S.J. 2010. Effects of calorie restriction on life span of microorganisms. Applied Microbiology and Biotechnology 88: 817–828. Slansky, F. & Rodriguez, J.G. 1989. Effects of calorie restriction on life span of microorganisms. Wiley, New York. Smith, S. 1994. The animal fatty acid synthase: One gene, one polypeptide, seven enzymes. The FASEB Journal 8: 1248–1259. Smith, T.K. & Butikofer, P. 2010. Lipid metabolism in Trypanosoma brucei. Molecular and Biochemical Parasitology 172: 66–79. Sohal, R.S. & Weindruch, R. 1996. Oxidative stress, caloric restriction, and aging. Science 273: 59–63. Spellman, P. & Rubin, G. 2002. Evidence for large domains of similarly expressed genes in the Drosophila genome. Journal of Biology 1: 5. Stanley, D.W. 2006. Prostaglandins and other eicosanoids in insects: Bio- logical significance. Annual Review of Entomology 51: 25–44. Stanley-Samuelson, D.W., Jurenka, R.A., Cripps, C., Blomquist, G.J. & de Renobales, M. 1988. Fatty acids in insects: Composition, metabolism, and biological significance. Archives of Insect Biochemistry and Physiol- ogy 9: 1–33. Stearns, S.C. 1989. Trade-offs in life-history evolution. Functional Ecology 3: 259–268. Stebbins, G.L. 1950. Variation and evolution in plants. Colombia University Press, New York. Steiner, C.C., Römpler, H., Boettger, L.M., Schöneberg, T. & Hoekstra, H.E. 2009. The genetic basis of phenotypic convergence in beach mice: Similar pigment patterns but different genes. Molecular Biology and Evo- lution 26: 35–45. Stephens, J.L., Lee, S.H., Paul, K.S. & Englund, P.T. 2007. Mitochon- drial fatty acid synthesis in Trypanosoma brucei. Journal of Biological Chemistry 282: 4427–4436. Stireman, J.O. 2005. The evolution of generalization? Parasitoid flies and the perils of inferring host range evolution from phylogenies. Journal of

170 Bibliography

Evolutionary Biology 18: 325–36. Stone, J.R. & Wray, G.A. 2001. Rapid evolution of cis-regulatory sequences via local point mutations. Molecular Biology and Evolution 18: 1764–70. Strand, M. 2000. Developmental traits and life history evolution in parasitoids. In: Parasitoid population biology (M.E. Hochberg & A.R. Ives, eds), pp. 139–162. Princeton University Press, Princeton, NJ. Strand, M.R. & Casas, J. 2008. Parasitoid and host nutritional physiology in behavioral ecology. In: Behavioral Ecology of Insect Parasitoids (E. Wajnberg, C. Bernstein & J.J. van Alphen, eds), pp. 113–128. Black- well Publishing, London. Strohm, E. 2000. Factors affecting body size and fat content in a digger wasp. Oecologia 123: 184–191. Suen, G., Teiling, C., Li, L., Holt, C., Abouheif, E., Bornberg-Bauer, E., Bouffard, P., Caldera, E.J., Cash, E., Cavanaugh, A., Denas, O., El- haik, E., Favé, M.J., Gadau, J., Gibson, J.D., Graur, D., Grubbs, K.J., Hagen, D.E., Harkins, T.T., Helmkampf, M., Hu, H., Johnson, B.R., Kim, J., Marsh, S.E., Moeller, J.a., Muñoz Torres, M.C., Murphy, M.C., Naughton, M.C., Nigam, S., Overson, R., Rajakumar, R., Reese, J.T., Scott, J.J., Smith, C.R., Tao, S., Tsutsui, N.D., Viljakainen, L., Wissler, L., Yandell, M.D., Zimmer, F., Taylor, J., Slater, S.C., Clifton, S.W., Warren, W.C., Elsik, C.G., Smith, C.D., Weinstock, G.M., Gerardo, N.M. & Currie, C.R. 2011. The genome sequence of the leaf-cutter ant Atta cephalotes reveals insights into its obligate symbiotic lifestyle. PLoS Genetics 7: e1002007. Suzuki, M. & Tanaka, T. 2007. Development of Meteorus pulchricornis and regulation of its noctuid host, Pseudaletia separata. Journal of Insect Physiology 53: 1072–8. Svensson, B.W. 2005. Seasonal fat content fluctuations in a gyrinid beetle: Comparison between an arctic and a temperate zone population (Coleoptera: Gyrinidae). Aquatic Insects 27: 11–19. Tanaka, S. & Ohsaki, N. 2006. Behavioral manipulation of host caterpillars by the primary parasitoid wasp Cotesia glomerata (L.) to construct de- fensive webs against hyperparasitism. Ecological Research 21: 570–577. Tanaka, S. & Ohsaki, N. 2009. Does manipulation by the parasitoid wasp Cotesia glomerata (L.) cause attachment behaviour of host caterpillars on cocoon clusters? Ethology 115: 781–789.

171 Teixeira, L., Rabouille, C., Rorth, P., Ephrussi, A. & Vanzo, N.F. 2003. Drosophila Perilipin/ADRP homologue Lsd2 regulates lipid metabolism. Mechanisms of Development 120: 1071–1081. Teotónio, H. & Rose, M.R. 2001. Perspective: Reverse evolution. Evolution 55: 653–660. ter Kuile, B.H. & Westerhoff, H.V. 2001. Transcriptome meets metabolome: Hierarchical and metabolic regulation of the glycolytic pathway. FEBS letters 500: 169–171. Thompson, S. 1973. A review and comparative characterization of the fatty acid compositions of seven insect orders. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry 45: 467–482. Thompson, S. 1979. The effects of dietary carbohydrates on larval development and lipogenesis in the parasite, Exeristes roborator (Frabri- cius)(Hymenoptera: Ichneumonidae). Journal of Parasitology 65: 849– 854. Thompson, S.N. & Barlow, J. 1974. Fatty acid composition of parasitic Hymenoptera. Annals of the Entomological Society of America 67: 627– 632. Thompson, S.N. & Barlow, J.S. 1972. Synthesis of fatty acids by the parasite Exeristes comstockii (Hymenop.) and two hosts, Galleria mellonella (Lep.) and Lucilia sericata (Dip.). Canadian Journal of Zoology 50: 1105–1110. Tielens, A.G.M. & Hellemon, J.J. 2006. Unusual aspects of metabolism in flatworm parasites. In: Parasitic flatworms: Molecular biology, biochemistry, immunology and physiology (A. Maule & N. Marks, eds). CABI, Wallingford. Timmermans, M.J.T.N., Roelofs, D., Mariën, J. & van Straalen, N.M. 2008. Revealing pancrustacean relationships: Phylogenetic analysis of ribosomal protein genes places Collembola (springtails) in a monophyletic Hexapoda and reinforces the discrepancy between mitochondrial and nuclear DNA markers. BMC Evolutionary Biology 8: 83. Toft, S. 1999. Prey choice and spider fitness. Journal of Arachnology 27: 301–307. Tompkins, J.M.L., Wratten, S.D. & Wäckers, F.L. 2010. Nectar to improve parasitoid fitness in biological control: Does the sucrose:hexose ratio matter? Basic and Applied Ecology 11: 264–271.

172 Bibliography

Tooker, J.F. & De Moraes, C.M. 2009. A gall-inducing caterpillar species increases essential fatty acid content of its host plant without concomitant increases in phytohormone levels. Molecular plant-microbe interactions 22: 551–559. Turkish, A.R. & Sturley, S.L. 2009. The genetics of neutral lipid biosynthesis: An evolutionary perspective. American Journal of Physiology. Endocrinology and Metabolism 297: E19–E27. Turunen, S. 1979. Digestion and absorption of lipids in insects. Comparative Biochemistry and Physiology Part A: Physiology 63: 455–460. van Alphen, J.J.M. & Thunnissen, I. 1982. Host selection and sex allocation by Pachycrepoideus vindemiae Rondani (Pteromalidae) as a facultative hyperparasitoid of Asobara tabida Nees (Braconidae; Alysiinae) and Leptopilina heterotoma (Cynipoidea; Eucoilidae). Netherlands Journal of Zoology 33: 497–514. van den Assem, J. & Jachmann, F. 1999. Changes in male perseverance in courtship and female readiness to mate in a strain of the parasitic wasp Nasonia vitripennis over a period of 20+ years. Netherlands Journal of Zoology 49: 125–137. van Dooremalen, C. & Ellers, J. 2010. A moderate change in temperature induces changes in fatty acid composition of storage and membrane lipids in a soil arthropod. Journal of Insect Physiology 56: 178–184. van Dooremalen, C., Pel, R. & Ellers, J. 2009. Maximized PUFA measurements improve insight in changes in fatty acid composition in response to temperature. Archives of Insect Biochemistry and Physiology 72: 88–104. van Handel, E. 1965. The obese mosquito. The Journal of Physiology 181: 478–486. van Noordwijk, A.J. & de Jong, G. 1986. Acquisition and allocation of resources: Their influence on variation in life history tactics. The American Naturalist 128: 137–142. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A. & Speleman, F. 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3: Research0034–Research0034.11. Vattala, H., Wratten, S., Phillips, C. & Wäckers, F.L. 2006. The influence of flower morphology and nectar quality on the longevity of a parasitoid biological control agent. Biological Control 39: 179–185.

173 Vick, B.A. & Charlet, L.D. 1996. Comparative fatty acid composition and metabolism in larvae of the red and of the gray sunflower seed weevils (Coleoptera: Curculionidae). Journal of the Kansas Entomological Soci- ety 69: 160–176. Vinson, S.B. & Iwantsch, G.F. 1980. Host suitability for insect parasitoids. Annual Review of Entomology 25: 397–419. Visser, B. & Ellers, J. 2008. Lack of lipogenesis in parasitoids: A review of physiological mechanisms and evolutionary implications. Journal of Insect Physiology 54: 1315–1322. Visser, B., Le Lann, C., Den Blanken, F.J., Harvey, J.A., Van Alphen, J.J.M. & Ellers, J. 2010. Loss of lipid synthesis as an evolutionary consequence of a parasitic lifestyle. Proceedings of the National Academy of Sciences of the United States of America 107: 8677–8682. Wäckers, F.L. 2001. The influence of flower morphology and nectar quality on the longevity of a parasitoid biological control agent. Journal of Insect Physiology 47: 1077–1084. Wäckers, F.L. 2003. The parasitoids ‘need for sweets’: Sugars in mass rearing and biological control. CABI, Wallingford. Wäckers, F.L., van Rijn, P. & Bruin, J. 2005. Plant-provided food for carnivorous insects: A protective mutualism and its application. Cambridge University Press, Cambridge (MA). Waddington, C.H. 1942. Canalization of development and the inheritance of acquired characters. Nature 150: 563–565. Wakefield, M.E., Bell, H.A. & Gatehouse, A.M.R. 2010. Longevity and fecundity of Eulophus pennicornis, an ectoparasitoid of the tomato moth Lacanobia oleracea, is affected by nutritional state and diet quality. Agri- cultural and Forest Entomology 12: 19–27. Wakil, S.J. 1989. Fatty acid synthase, a proficient multifunctional enzyme. Biochemistry 28: 4523–4530. Wallace, A.R. 1858. On the tendency of varieties to depart indefinitely from the original type. Proceedings of the Linnean Society of London 3: 53–62. Wang, X.G. & Messing, R.H. 2004. The ectoparasitic pupal parasitoid, Pachycrepoideus vindemmiae (Hymenoptera: Pteromalidae), attacks other primary tephritid fruit fly parasitoids: Host expansion and potential non-target impact. Biological Control 31: 227–236. Warbrick-Smith, J., Behmer, S.T., Lee, K.P., Raubenheimer, D. & Simpson,

174 Bibliography

S.J. 2006. Evolving resistance to obesity in an insect. Proceedings of the National Academy of Sciences of the United States of America 103: 14045–14049. Warburg, M.S. & Yuval, B. 1996. Effects of diet and activity on lipid levels of adult Mediterranean fruit flies. Physiological Entomology 21: 151–158. Weindruch, R. & Walford, R.L. 1988. The retardation of aging and disease by dietery restriction. C. C. Thomas, Springfield, IL. Werren, J.H., Richards, S., Desjardins, C.A., Niehuis, O., Gadau, J., Col- bourne, J.K., Beukeboom, L.W., Desplan, C., Elsik, C.G., Grimmelikhui- jzen, C.J.P., Kitts, P., Lynch, J.A., Murphy, T., Oliveira, D.C.S.G., Smith, C.D., Van De Zande, L., Worley, K.C., Zdobnov, E.M., Aerts, M., Albert, S., Anaya, V.H., Anzola, J.M., Barchuk, A.R., Behura, S.K., Bera, A.N., Berenbaum, M.R., Bertossa, R.C., Bitondi, M.M.G., Borden- stein, S.R., Bork, P., Bornberg-Bauer, E., Brunain, M., Cazzamali, G., Chaboub, L., Chacko, J., Chavez, D., Childers, C.P., Choi, J.H., Clark, M.E., Claudianos, C., Clinton, R.A., Cree, A.G., Cristino, A.S., Dang, P.M., Darby, A.C., De Graaf, D.C., Devreese, B., Dinh, H.H., Edwards, R., Elango, N., Elhaik, E., Ermolaeva, O., Evans, J.D., Foret, S., Fowler, G.R., Gerlach, D., Gibson, J.D., Gilbert, D.G., Graur, D., Gründer, S., Hagen, D.E., Han, Y., Hauser, F., Hultmark, D., Hunter, H.C., Hurst, G.D.D., Jhangian, S.N., Jiang, H., Johnson, R.M., Jones, A.K., Junier, T., Kadowaki, T., Kamping, A., Kapustin, Y., Kechavarzi, B., Kim, J., Kim, J., Kiryutin, B., Koevoets, T., Kovar, C.L., Kriventseva, E.V., Kucharski, R., Lee, H., Lee, S.L., Lees, K., Lewis, L.R., Loehlin, D.W., Logsdon, J.M., Lopez, J.A., Lozado, R.J., Maglott, D., Maleszka, R., Mayampurath, A., Mazur, D.J., McClure, M.A., Moore, A.D., Morgan, M.B., Muller, J., Munoz-Torres, M.C., Muzny, D.M., Nazareth, L.V., Neupert, S., Nguyen, N.B., Nunes, F.M.F., Oakeshott, J.G., Okwuonu, G.O., Pannebakker, B.A., Pejaver, V.R., Peng, Z., Pratt, S.C., Predel, R., Pu, L.L., Ranson, H., Raychoudhury, R., Rechtsteiner, A., Reese, J.T., Reid, J.G., Riddle, M., Robertson, H.M., Romero-Severson, J., Rosen- berg, M., Sackton, T.B., Sattelle, D.B., Schlüns, H., Schmitt, T., Schnei- der, M., Schüler, A., Schurko, A.M., Shuker, D.M., Simões, Z.L.P., Sinha, S., Smith, Z., Solovyev, V., Souvorov, A., Springauf, A., Stafflinger, E., Stage, D.E., Stanke, M., Tanaka, Y., Telschow, A., Trent, C., Vattathil, S., Verhulst, E.C., Viljakainen, L., Wanner, K.W., Waterhouse, R.M., Whitfield, J.B., Wilkes, T.E., Williamson, M., Willis, J.H., Wolschin, F., Wyder, S., Yamada, T., Yi, S.V., Zecher, C.N., Zhang, L. & Gibbs, R.A.

175 2010. Functional and evolutionary insights from the genomes of three parasitoid Nasonia species. Science 327: 343–348. Wessels, F.J. & Hahn, D.A. 2010. Carbon 13 discrimination during lipid biosynthesis varies with dietary concentration of stable isotopes: Impli- cations for stable isotope analyses. Functional Ecology 24: 1017–1022. Wheeler, W.C., Whiting, M., Wheeler, Q.D. & Carpenter, J.M. 2001. The phylogeny of the extant hexapod orders. Cladistics 17: 113–169. Whiting, A.R. 1967. The biology of the parasitic wasp Mormoniella vitripennis [=Nasonia brevicornis](Walker). Quarterly Review of Biology 42: 333–406. Wiegmann, B., Trautwein, M., Kim, J.W., Cassel, B., Bertone, M., Win- terton, S. & Yeates, D. 2009. Single-copy nuclear genes resolve the phylogeny of the holometabolous insects. BMC Biology 7: 34. doi: 10.1186/1741-7007-7-34. Wiens, J.J. 2001. Widespread loss of sexually selected traits: How the peacock lost its spots. Trends in Ecology & Evolution 16: 517–523. Williams, G.C. 1966. Adaptation and natural selection. Princeton Univer- sity Press, Princeton (NJ). Winkler, K., Wäckers, F.L., Bukovinszkinekiss, G. & van Lenteren, J.C. 2006. Sugar resources are vital for Diadegma semiclausum fecundity under field conditions. Basic and Applied Ecology 7: 133–140. Witting-bissinger, B., Orr, D. & Linker, H. 2008. Effects of floral resources on fitness of the parasitoids Trichogramma exiguum (Hymenoptera: Tri- chogrammatidae) and Cotesia congregata (Hymenoptera: Braconidae). Biological Control 47: 180–186. Wray, G.A. 2007. The evolutionary significance of cis-regulatory mutations. Nature Reviews Genetics 8: 206–216. Wray, G.A., Hahn, M.W., Abouheif, E., Balhoff, J.P., Pizer, M., Rockman, M.V. & Romano, L.A. 2003. The evolution of transcriptional regulation in eukaryotes. Molecular Biology and Evolution 20: 1377–1419. Wright, S. 1932. The roles of mutation, inbreeding, crossbreeding and selection in evolution. Proceedings of the Sixth International Congress of Genetics 1: 356–366. Wu, D., Daugherty, S.C., Van Aken, S.E., Pai, G.H., Watkins, K.L., Khouri, H., Tallon, L.J., Zaborsky, J.M., Dunbar, H.E., Tran, P.L., Moran, N.A. & Eisen, J.A. 2006. Metabolic complementarity and genomics of the dual

176 Bibliography

bacterial symbiosis of sharpshooters. PLoS Biology 4: e188. Wyder, S., Kriventseva, E., Schroder, R., Kadowaki, T. & Zdobnov, E. 2007. Quantification of ortholog losses in insects and vertebrates. Genome Biology 8: R242. Xu, J., Saunders, C.W., Hu, P., Grant, R.A., Boekhout, T., Kuramae, E.E., Kronstad, J.W., DeAngelis, Y.M., Reeder, N.L., Johnstone, K.R., Leland, M., Fieno, A.M., Begley, W.M., Sun, Y., Lacey, M.P., Chaud- hary, T., Keough, T., Chu, L., Sears, R., Yuan, B. & Dawson, T.L. 2007. Dandruff-associated Malassezia genomes reveal convergent and divergent virulence traits shared with plant and human fungal pathogens. Proceed- ings of the National Academy of Sciences of the United States of America 104: 18730–18735. Xu, P., Vernooy, S.Y., Guo, M. & Hay, B.A. 2003. The Drosophila mi- croRNA Mir-14 suppresses cell death and is required for normal fat metabolism. Current Biology 13: 790–795. Yazgan, S. 1972. A chemically defined synthetic diet and larval nutritional requirements of the endoparasitoid Itoplectis conquisitor (Hymenoptera). Journal of Insect Physiology 18: 2123–2141. Yeates, D.K., Wiegmann, B.M., Courtney, G.W., Meier, M., Lambkin, C. & Pape, T. 2007. Phylogeny and systematics of Diptera: Two decades of progress and prospects. Zootaxa 1668: 565–590. Zanotto, F.P., Gouveia, S., Simpson, S.J. & Calder, D. 1997. Nutritional homeostasis in locusts: Is there a mechanism for increased energy expenditure during carbohydrate overfeeding? Journal of Experimental Biology 200: 2437–2448. Zanotto, F.P., Simpson, S.J. & Raubenheimer, D. 1993. The regulation of growth by locusts through post-ingestive compensation for variation in the levels of dietary protein and carbohydrate. Physiological Entomology 18: 425–434. Zhao, Z. & Zera, A. 2001. Enzymological and radiotracer studies of lipid metabolism in the flight-capable and flightless morphs of the wing- polymorphic cricket, Gryllus firmus. Journal of Insect Physiology 47: 1337–1347. Ziegler, R. & Ibrahim, M.M. 2001. Formation of lipid reserves in fat body and eggs of the yellow fever mosquito, Aedes aegypti. Journal of Insect Physiology 47: 623–627.

177 Zinke, I., Schütz, C.S., Katzenberger, J.D., Bauer, M. & Pankratz, M.J. 2002. Nutrient control of gene expression in Drosophila: Microarray analysis of starvation and sugar-dependent response. The EMBO Journal 21: 6162–6173. Zwaan, B.J., Bijlsma, R. & Hoekstra, R.F. 1992. On the developmental theory of ageing. II. The eﬀect of developmental temperature on longevity in relation to adult body size in D. melanogaster. Heredity 68: 123–130. Zwick, A. 2008. Molecular phylogeny of Anthelidae and other bombycoid taxa (Lepidoptera: Bombycoidea). Systematic Entomology 33: 190–209.

178 Summary

An important process contributing to evolutionary changes is trait loss. This field of research has, however, received little attention and its importance is typically overlooked. A trait can be considered lost when phenotypic expression is silenced under certain environmental conditions that warranted trait expression in the ancestral lineage. Trait loss can occur when a trait remains unused or when selection acts against it. The loss of traits can be accelerated when the environment compensates for a phenotypic function, in which a resource or function is provided by the environment, leaving the trait in the receiving organism prone to phenotypic degradation. Environmental compensation of traits can be realized in systems when the diet facilitates an essential resource or when an interacting partner provides a certain phenotypic function. Ecological interactions with the biotic environment are thus expected to play a key role in evolutionary trait dynamics. Particularly important cases of trait loss pertain to traits that involve the loss of certain nutrient metabolic functions. Nutrition plays a key role in determining fitness through its inherent link to survival, growth and reproduction. An example in which an indispensable trait has been lost was found in parasitoids that lack lipid synthesis in their adult life-stage. These insects feed and develop on other arthropods during their larval stages, yet they are free-living as adults to search for new hosts. Lipogenesis has been considered a highly conserved pathway that is uniformly adopted by all animals to synthesize fatty acids used in the formation of triglycerides to store energy. Nutrient storage is particularly important when an animal is faced with unfavourable environmental conditions, in which food might not be available to meet direct energetic demands. Co-evolution between parasitoid and host was expected to fuel the loss of lipogenesis, since valuable resources in terms of lipid reserves are provided by the host during development. The parasitoid readily carries over these nutrients during development, compensating for this trait at the phenotypic level and rendering pathways underlying lipid synthesis prone to degradation. The first aspect of this hypothesis relates to the link between this metabolic aberration in larvae and adults: lack of lipogenesis is expected to be favoured in larvae that directly benefit from the host’s lipid supply, yet adults are constrained by the level of teneral lipid levels obtained from the host. The second aspect involves the ability of parasitoids to alter physiological processes in their

179 hosts, in which nutrient resources, including lipids, are increased through interference by the parasitoid. Host manipulation is thus expected to play an important role in the evolutionary loss of lipogenesis, because sufficient nutrient levels are required to sustain metabolic functions in the adult life- stage. Three pertinent questions regarding the evolutionary loss of lipogenesis and its relation to the parasitic lifestyle were tackled in this thesis: i) Has co-evolution between host and parasitoid led to the evolutionary loss of lipogenesis? ii) Which mechanisms underlie the loss of this essential metabolic trait in parasitoids? iii) How does dietary intake of nutrients affect life histories of organisms lacking lipogenesis? To answer the first question the link between parasitism and lack of lipogenesis was tested using phylogenetic analyses. 24 parasitoids were tested for their lipogenic ability and data on lipogenic ability of 70 other insect species obtained from the literature. The evolutionary loss of lipogenesis was found to have occurred in parallel in three different insect orders, all adopting the parasitoid lifestyle. This study further showed that lipogenesis had re-evolved in three parasitoid lineages that were characterized by their broad host range, i.e. generalists. Environmental compensation of lipid reserves by the host has led to the evolutionary loss of lipogenesis in parasitoids, yet this trait seems to re- evolve with relative ease, particularly in species that are expected to lack host manipulation to increase the host’s lipid resources. Parasitoids that are specialized on one or only few hosts typically lack lipid synthesis, presumably because host manipulation substantially increases lipid resources of the host. Alternatively, in generalists adopting a large host range host manipulation is likely prohibited leading to a lower level of resources that can be taken over from the host. There are, however, exceptions to this general pattern, in which generalists have been found to lack lipogenesis. In one of these species, Nasonia vitripennis, host manipulation does occur, in which lipid levels are increased, hence this species is specialized at least to some extent on a number of hosts it prefers to use for oviposition. Another parasitoid adopting a large host range and lacking lipogenesis is Pachycrepoideus vindemmiae. By determining lipid reserves of host and parasitoid this species was shown to lack host manipulation, hence the loss of lipogenesis is not inevitably related to host manipulation ability. At least in this species, mechanisms other than manipulation have led to the lack of lipogenesis, such as sufficiency in teneral reserves to maintain fitness or because the ability to manipulate the host has been lost.

180 Summary

One assumption regarding the lack of lipogenesis is that it involves only a lack of fatty acid synthesis, hence elongation and desaturation of fatty acids remains possible for metabolising other lipid types. By determining efficiency of host exploitation in a gall wasp community, it was found that at least one parasitoid species was able to adjust certain fatty acids ratios. This work further showed that all of the species tested within this community lacked lipid synthesis and that the majority of species showed an extraordinary efficiency in carrying over resources from their host. This is the first study to show that a parasitoid can adjust its fatty acid composition despite lack of lipogenesis. While more cases of trait loss are currently being discovered, the mechanisms underlying the loss of traits have largely remained elusive. Study- ing transcriptional changes in response to food in the parasitoid Nasonia vitripennis, it was found that the transcriptional profile of this paraistoid severely deviates from that of Drosophila melanogaster, a species that actively synthesizes lipids. A lack of transcription of the key gene in fatty acid synthesis, fatty acid synthase (fas), explains the lack of lipogenesis in parasitoids. Lack of lipogenesis is not due to mutation in the coding region of this gene, because inspection of the amino acid sequence revealed no irregularities when compared to sequences of species that synthesize lipids. Lack of lipogenesis likely results from alterations in gene regulation, either through its response to hormones, non-functionality of transcription factors or deviations in other regulatory mechanisms involved in activating genes within the fatty acid synthesis pathway. The acquisition of sufficient nutrient reserves is particularly important in organisms that are metabolically compromised, such as parasitoids. It can therefore be expected that nutrient sources with high caloric values are favoured over lower quality resources. In most organisms, however, a negative correlation has been found between a high-calorie diet and lifespan. In that sense, parasitoids lacking lipid synthesis are expected to differ from this general pattern, since consuming food sources with a higher caloric value should reduce the demand for limited lipid reserves and therefore correlate positively with life history traits. Using a two-pronged approach to discover the relationship between calorie intake, longevity and fecundity, two parasitoid species were subjected either to various dietary sugar dilutions or intermittent sugar-feeding. Dietary dilutions led to a typical caloric restriction effect, in which longevity decreased at higher sugar concentrations, while fecundity remained stable. When sugar sources were accessed intermittently, no effect of calorie restriction was found. These findings suggest

181 that, contrary to expectations, parasitoids do not benefit from feeding on a high calorie diet. Parasitoids are frequently employed as natural enemies to fight pests in agro-ecosystems, but sugar sources are typically scarce within those environmental settings. Because sugar sources are scarce parasitoids can potentially benefit from feeding on a lipid-rich substrate as adults to increase their lipid reserves. Inclusion of a lipid-substrate in the diet of the parasitoid Cotesia glomerata led to an increase in lipid reserves or levels were maintained at a high level for a longer period of time. However, a lipid- rich diet significantly decreased longevity, either due to toxicity or because caloric values detrimentally affected longevity. Even though this experiment was only partly successful, it could be worthwhile to further explore optimal ratios for lipid provisioning to increase lipid resources benefiting allocation into longevity and reproduction. A wide variety of traits have been lost during the course of evolution. It remains unclear however why trait loss occurs and how trait loss contributes to evolutionary trait dynamics within an ecological framework. What is clear is that phenotypic decay substantially adds to the component of genetic variation that remains hidden from selection and cryptic genetic variation plays a key role in both trait acquisition and re-evolution of lost traits. Therefore, trait loss plays an important role in facilitating the molecular resources that can be reacquired to extend the part of genetic variation expressed to the phenotype followed by selection and evolutionary change. Once we get more insight into the variety of mechanisms underlying trait loss we can entangle why and how trait loss contributes to evolutionary trait dynamics and reveal the importance of trait loss in evolutionary processes.

182 Samenvatting

Het verlies van eigenschappen is een belangrijk proces dat bijdraagt aan evolutionaire veranderingen van organismen. Desondanks heeft onderzoek op dit gebied relatief weinig aandacht gekregen en wordt het belang van verloren eigenschappen vaak over het hoofd gezien. Een eigenschap kan als verloren worden beschouwd als er, onder bepaalde omgevingscondities, geen expressie optreedt van het fenotype ondanks dat deze eigenschap wel tot expressie kwam in de gemeenschappelijke voorouder. Zo’n situatie kan ontstaan als eigenschappen ongebruikt blijven of als selectie tegen een eigenschap optreedt met een negatief effect op de fitness. Het proces kan versneld worden als de omgeving voor een bepaalde fenotypische functie compenseert. Hierbij voorziet de omgeving, in plaats van het organisme zelf, in een voedingsstof of functie met een degradatie van het fenotype als gevolg. Compensatie van eigenschappen door de omgeving kan bijvoorbeeld tot stand komen in systemen waarbij het dieet voorziet in een essentiële stof of wanneer een interactiepartner een bepaalde fenotypische functie uitvoert. Ecologische interacties met de omgeving spelen daarom een aanzienlijke rol in de evolutionaire dynamiek tussen eigenschappen. Belangrijke voorbeelden van verloren eigenschappen betreffen eigenschappen betrokken bij de stofwisseling. Voeding is van grote invloed op de fitness, met name door het inherente verband met overleving, groei en voort- planting. Het verlies van een essentieel kenmerk dat betrokken is bij de stofwisseling van voedingsstoffen komt voor in parasitoïden die geen vet- voorraden aanleggen als adulten. Deze insecten voeden en ontwikkelen zich op of in andere geleedpotigen tijdens het larvale stadium, maar zijn vrij le- vend als adulten om nieuwe gastheren te zoeken. Het aanmaken van vetten is een geconserveerde eigenschap die universeel door organismen gebruikt wordt om vetzuren te produceren voor opslag in vetreserves. De opslag van voedingsstoffen is vooral belangrijk wanneer organismen geconfronteerd worden met ongunstige omgevingscondities, waarbij voedsel wellicht niet of maar in beperkte hoeveelheid aanwezig is om te voorzien in de directe ener- giebehoefte. De parasitoïde neemt vetten over van de gastheer tijdens de larvale ontwikkeling, waarbij het fenotype van dit kenmerk wordt gecom- penseerd door de omgeving. Dit heeft tot gevolg dat fenotypische degradatie optreedt in belangrijke eigenschappen betrokken bij de vetaanmaak. Co- evolutie tussen gastheer en parasitoïde lijkt daarom ten grondslag te liggen aan het verlies van de vetaanmaak, omdat kostbare stoffen in de vorm van

183 vetreserves worden aangemaakt door de gastheer. Het eerste aspect met betrekking tot deze hypothese berust op het verband tussen deze metabo- lische afwijking in larven en adulten: Het is aannemelijk dat een gebrek aan vetaanmaak voordelig is in het larvale stadium, terwijl adulten worden beperkt door de hoeveelheid vetreserves die beschikbaar zijn na de meta- morfose naar het adulte stadium. Het tweede aspect heeft betrekking op het vermogen van parasitoïden om de fysiologie van de gastheer te beïnvloeden, waarbij de hoeveelheid voedingsstoﬀen kan worden verhoogd. Gastheer- manipulatie lijkt daarom een belangrijke rol te spelen bij het evolutionaire verlies van de vetaanmaak. Drie vragen met betrekking tot het evolutionair verlies van de vetaanmaak worden in dit proefschrift behandeld: i) Heeft co-evolutie tussen gastheer en parasitoïde geleid tot het evolutionaire verlies van de vetaanmaak? ii) Welke mechanismen liggen ten grondslag aan het verlies van deze essen- tiële eigenschap in parasitoïden? iii) Hoe beïnvloedt het dieet belangrijke levensloopstrategieën in organismen die geen vetten aanmaken? Om een antwoord te vinden op de eerste vraag is er gekeken naar het verband tussen de parasitaire levensstijl en het verlies van vetaanmaak door middel van een fylogenetische analyse. Hierbij werd de vetaanmaak van 24 parasitoïden onderzocht en data voor 70 andere insecten verkregen uit de literatuur. De vetaanmaak is tijdens de evolutie verloren gegaan in drie verschillende orden binnen de insecten die elk een parasitaire levensstijl hebben aangenomen. Naast deze bevinding heeft dit onderzoek laten zien dat de vetaanmaak he- revolueerde in drie groepen sluipwespen die gekarakteriseerd worden door de brede verscheidenheid aan gastheren die zij parasiteren, ofwel generalisten. Compensatie van vetreserves door de omgeving heeft geleidt tot het evolutionaire verlies van de vetaanmaak in de meeste parasitoïden. Des- ondanks lijkt het dat deze eigenschap gemakkelijk herevolueert, met name in soorten waarbij gastheermanipulatie (en daarmee het verhogen van de vetreserves van de gastheer) onwaarschijnlijk lijkt. Gespecialiseerde parasitoïden leggen eitjes in maar één of enkele gast- heersoorten en hebben over het algemeen een gebrek aan lipogenese, omdat gastheermanipulatie de vetreserves van de gastheer substantieel kan laten toenemen. Daarentegen wordt voor generalisten verwacht dat de grote verscheidenheid aan potentiële gastheren fysiologische manipulatie bemoeilijkt. Generalisten kunnen daarom een relatief lagere vethoeveelheid overnemen van de gastheer waardoor een functionele vetaanmaak genoodzaakt blijft. Er zijn echter uitzonderingen gevonden waarbij generalisten geen vetreserves aanleggen. Één van deze soorten, Nasonia vitripennis, is wel in staat

184 Samenvatting tot gastheermanipulatie en is daarmee dan ook tot op zekere hoogte gespe- cialiseerd op enkele gastheren. Een andere parasitoïde soort met een grote verscheidenheid aan gastheren en een gebrek aan vetaanmaak is Pachycre- poideus vindemmiae. Door vethoeveelheden van gastheer en parasitoïde te bepalen is gebleken dat deze soort zijn gastheer niet manipuleert, wat leid tot de conclusie dat in sommige parasitoïden andere mechanismen dan gastheermanipulatie ten grondslag liggen aan het verlies van de vetaanmaak. Potentiële verklaringen voor deze bevindingen kunnen zijn dat reeds toe- reikende vethoeveelheden verkregen worden tijdens de larvale ontwikkeling wat manipulatie van de gastheer onnodig maakt. Anderzijds bestaat de mogelijkheid dat gastheermanipulatie verloren is gegaan tijdens de evolutie van deze soort. Een aanname met betrekking tot het verlies van lipogenese was dat het verlies van deze eigenschap alleen betrekking heeft op het aanmaken van vetzuren, waarbij het verlengen of oververzadigd maken van vetzuren nog tot de mogelijkheden behoort om verschillende typen vetmoleculen aan te maken. Door te kijken naar de efficiëntie waarmee gastheren geëxploiteerd kunnen worden binnen een galwespengemeenschap werd gevonden dat één van de geteste parasitoïde soorten in staat was de ratio van bepaalde typen vetzuren aan te passen. Dit wijst erop dat deze soort in staat is tot het verlengen en overzadigd maken van vetzuren. Deze studie heeft verder onthuld dat geen van de soorten binnen de galwespengemeenschap vetten aanmaken en dat er een zeer hoge efficiëntie is waarmee voedingsstoffen kunnen worden overgenomen van de gastheer. Dit onderzoek is daarmee het eerste dat laat zien dat een parasitoïde in staat is de vetzuurcompositie aan te passen, ook al is de vetaanmaak verloren gegaan. Ondanks het feit dat er momenteel veel nieuwe voorbeelden worden gevonden waarbij eigenschappen verloren gaan door compensatie door de omgeving zijn de onderliggende mechanismen onduidelijk gebleven. Door naar veranderingen in gentranscriptie te kijken in gevoerde en gehongerde vrouwtjes van de sluipwesp Nasonia vitripennis werd gevonden dat trans- criptiepatronen van deze wesp aanzienlijk afwijken van die geobserveerd bij de vlieg Drosophila melanogaster, een soort die wel vetten aanmaakt. Een zeer lage transcriptie van het belangrijkste gen betrokken bij de vetaanmaak, fatty acid synthase fas, verklaard waarom parasitoïden geen vet aanmaken. Er zijn twee mogelijke oorzaken voor het gebrek aan vetaanmaak: Mutaties zijn geaccumuleerd in het coderende gedeelte van dit gen of een verandering in genregulatie ligt ten grondslag aan de lage transcriptie. Het werd niet aannemelijk bevonden dat de lage transcriptie van fas het

185 gevolg was van geaccumuleerde mutaties, omdat er geen onregelmatigheden werden gevonden in de aminozuursequentie wanneer deze vergeleken werd met andere insecten die wel vetten aanmaken. Het is daarom aannemelijk dat het gebrek aan vetaanmaak veroorzaakt wordt door veranderingen in genregulatie, bijvoorbeeld door de invloed van hormonen, non-functionele transcriptiefactoren of andere veranderingen in de regulatie van genen betrokken bij vetmetabolisme. Het verkrijgen van voldoende reserves is met name belangrijk in organismen die metabolisch gecompromitteerd zijn, zoals parasitoïden. Het kan daarom verwacht worden dat voedingsstoffen met een hoge calorische waarde een gunstig effect hebben op fitness-gerelateerde eigenschappen. Desondanks wordt er in de meeste organismen een negatief effect gevonden tussen een dieet met een hoge calorische kwaliteit en levensduur. Parasito- ïden die geen vet aanmaken worden verwacht af te wijken van dit patroon, omdat het opnemen van voedsel met een hogere calorische waarde juist voordelig zou moeten zijn met betrekking tot belangrijke fitness-gerelateerde eigenschappen. Gebruik makend van een tweedelige opzet werd onderzocht hoe twee sluipwespen reageerden op voedsel van variërende calorische waarde met betrekking tot de levensduur en de aanmaak van eieren: verschillende suikerconcentraties werden aangeboden of de toegankelijkheid tot voedsel werd gevarieerd. Daarbij werd aangetoond dat hoge suikerconcentraties een negatief effect hebben op de levensduur, terwijl de aanmaak van eieren stabiel bleef. Variatie in de frequentie waarmee deze soorten toegang kregen tot suikerbronnen liet dit effect niet zien. Deze bevindingen geven aan dat, in tegenstelling tot de verwachting, parasitoïden geen voor- deel ondervinden van het consumeren van voedselbronnen met een hogere calorische waarde. Parasitoïden worden steeds vaker gebruikt als natuurlijke vijanden tegen schadelijke insecten in agro-ecosystemen. Het kan daarom economisch voordelig zijn om parasitoïden gebruikt in de biologische bestrijding te voorzien van een adult dieet waarin een vetcomponent aanwezig is om de vetreserves en mogelijk de fitness te verhogen. Toevoeging van een vetcomponent in het dieet van de sluipwesp Cotesia glomerata leidde tot een verhoging van de vetreserves of reserves werden voor langere tijd op een hoog niveau gehou- den. Echter, deze vetcomponent had een nadelige invloed op de levensduur. Mogelijk was deze vetcomponent in de huidige concentratie toxisch. Ander- zijds kan het zijn dat de calorische waarde van de vetcomponent te hoog was. Het is daarom essentieel een geschikte vetcomponent te vinden om vetreserves en fitness te verhogen voor parasitoïden die gebruikt worden in

186 Samenvatting de biologische bestrijding. Er is grote variatie in het aantal eigenschappen dat verloren is gegaan gedurende de evolutie, waarbij het onduidelijk blijft waarom bepaalde eigenschappen verloren gaan en hoe dit verlies bijdraagt aan de evolutionaire dynamiek tussen eigenschappen in een ecologische context. Wat wel duidelijk is is dat fenotypische degradatie substantieel bijdraagt aan de component van de genetische variatie die verscholen blijft voor selectie, waarbij deze cryptische genetische variatie een belangrijke rol speelt in zowel het ontstaan van nieuwe eigenschappen als het verlies daarvan. Om deze re- den speelt het verlies van eigenschappen dan ook een belangrijke rol in de voorziening van het moleculaire substraat dat potentieel gebruikt kan worden om staande genetische variatie te vergroten. Zodra eigenschappen herevolueren en deel uit gaan maken van de genetische variatie die vertaald wordt naar het fenotype is deze eigenschap weer beschikbaar voor selectie en evolutionaire veranderingen. Zodra we meer inzicht krijgen in de verscheidenheid aan mechanismen die ten grondslag liggen aan het verlies van eigenschappen kunnen we ontrafelen waarom en hoe verloren eigenschappen bijdragen aan de evolutionaire dynamiek en de grote rol die verloren eigenschappen in evolutionaire processen spelen.

187

Acknowledgements

I had a great time working at the Animal Ecology department. I would therefore like to thank all my colleagues that have supported me during these four years: if it had been through a long pep talk, some chit chat in the hallways, or a friendly smile. Thank you also for all those times you did not ﬁnish the coﬀee before me. I miss all of you already.

There are also some people that I would like to thank personally for their support during these four years:

Jacintha Ellers, ik denk dat ik een van de meest gelukkige AIO’s ben geweest met jou als begeleider. Bedankt voor alles wat je me geleerd hebt en de manier waarop je mij gestimuleerd hebt werk neer te zetten dat het mogelijk maakt door te gaan in de wetenschap. Voor dit project hebben we beide het uiterste gegeven en daarbij ontzettend mooie resultaten behaald. We zijn samen een heel sterk team en ik denk dat onze professionele relatie een uitzonderlijke was. Onze meningen waren vaak overeenkomstig, desondanks was er ook de mogelijkheid om open en eerlijk te zijn over hoe we vonden dat de dingen moesten gaan. Je hebt me de ruimte gegeven verder te groeien als wetenschapper en me geholpen op alle momenten waar ik niet zonder mijn begeleider kon. Jij bent een uitzonderlijke wetenschapper en het was een eer om met je samen te werken. Blijf je AIO’s en post-docs stimuleren en draag je kennis over waar dat kan, zoals je dat bij mij hebt gedaan, want ik weet dat ik me geen betere begeleider had kunnen wensen. Bedankt voor alles.

Cécile Le Lann, merci pour ton obstination à rejoindre notre équipe il y a déjà 3 ans. Merci pour toutes les fois où tu as passé du temps à relire mes manuscrits (et de nombreuses fois il y eut). Merci de m’avoir supportée de quelque manière qu’elle soit, et de continuer à me supporter. Il y a 3 ans, j’étais très excitée à l’idée d’avoir enﬁn une copine de parasitoïde au labo, et je n’imaginais pas trouver une meilleure amie que toi. Heureusement, tu t’es sentie comme chez toi dans notre labo et tu y es revenue pour un plus long moment, dont j’ai apprécié chaque instant. Merci pour tous tes con- seils, qui ont fait de moi une meilleure scientiﬁque et aussi très important: merci d’être une de mes amies les plus proches (et je sais que tu ressens la même chose, depuis que tu as déménagé à moins de 150 mètres de chez

189 moi: ma porte sera toujours ouverte pour toi).

Daniel Hahn, thank you for having me in your lab and for all the things that you have taught me during that short period. You have always regarded me as a fellow scientist and not as a student. It meant a great deal to me to have all your trust and confidence. Thank you for taking care of me and Eric and for giving me the best time imaginable working in your lab. I would also like to take the opportunity to thank my fellow lab members Giancarlo Lopez, Greg Ragland and Frank Wessels for their scientific discussions and the great time that I had with them in the lab. Giancarlo, thanks for putting up with me taking over the lab and for not throwing liquid nitrogen in my face. Also, you are now officially my P-NAS buddy: deal with it! It has been a pleasure working with all of you.

My collaborators, Daniel Hahn, Peter Teal, Dick Roelofs, Jacques van Alp- hen, Jeﬀrey Harvey, Ken Kraaijeveld, Cécile Le Lann, Coby van Doorema- len, Barbara Reumer, Toby Kiers and Cameron Currie. Thanks for all that you have taught me and for the opportunity to work with you and I hope our collaborations will continue.

My students, Frank den Blanken, Bas Ruhé, Arjan van der Linden, Alba Vazquez-Ruiz, Thomas Blankers, Menno Voogt en Jamie Jenner. Thank you for the hard work you have put into your projects and for teaching me how to be a better teacher.

The thesis committee members Jetske de Boer, David Giron, Toby Kiers, Louise Vet and Bregje Wertheim I would like to thank for the time and eﬀort they have invested in evaluating my thesis.

My fellow teachers, Matty Berg, Hans Cornelissen, Jurgen van Hal and An- dré Dias. Thanks for everything that you have taught me while teaching the course ’Ecologie, Mens en Natuur’. I had a great time working together with all of you teaching the students. I have very much enjoyed our time in the ﬁeld with the students and all the fun we’ve had while teaching. Obrigado.

The ’klein beraad’ members, Jacintha Ellers, Matty Berg, Gerard Dries- sen, Toby Kiers, Roel Pel, Cécile Le Lann, Maartje Liefting, Coby van Dooremalen, Elaine van Ommen Kloeke, Erik Verbruggen, Marie Duhamel

190 Acknowledgements and André Dias. Thank you all for the numerous scientiﬁc discussions that I found very constructive.

Nico van Straalen, bedankt voor je steun tijdens mijn promotieonderzoek. Ik heb het naar mijn zin gehad als medewerker van de afdeling Dierecologie onder jouw leiding. Bedankt ook dat je in je column aandacht hebt besteed aan mijn onderzoek.

De twee mensen die het allemaal mogelijk maken, Janine Mariën en Rudo Verweij. Bedankt voor het beantwoorden van al mijn vragen en voor het mogelijk maken van mijn onderzoek. Ik heb veel van jullie geleerd en zonder jullie hulp had ik mijn praktische werk niet kunnen uitvoeren.

My roommates, Jeroen Hoﬀer, Roel Pel, Muriel de Boer, Elferra Swart and Yifu Pei I would like to thank you for putting up with me these four years. You were all great roommates and I enjoyed all the conversations and fun we had in our beautiful room.

David Giron et Jérôme Casas, merci à vous deux pour tout le temps passé et les eﬀorts que vous avez consacrés à notre demande de subventions. J’espère que cela nous permettra d’avoir l’opportunité de travailler ensemble, que ce soit maintenant ou dans un avenir proche.

Jeﬀrey Harvey, thank you for giving me the opportunity to work in your lab. I would have been lost had I not had the opportunity to work at the NIOO. Thank you for the support that you are giving me and for the opportunity to learn from you. I would also like to thank Helen Snaas and Vincent de Vries for their hard work in the lab together with me and for making my time at the NIOO so much fun.

My bbf’s, Elaine van Ommen Kloeke and Elferra Swart. It is you that have kept me sane when times were hard. Thank you for forcing me to take a break every once in a while. Elaine, ik heb nog nooit zo snel een klik met iemand gehad als met jou. Gelijk na onze ontmoeting was al duidelijk dat wij vriendinnen zouden worden en je bent voor mij een geweldige collega geweest. Superelf, thank you for all the times you sang religious songs to me in the molecular lab. Although perhaps a bit disturbing, I found your voice very soothing while working. Thanks also for all the dirty jokes you send me while I was in Florida. You have been a great colleague and friend to

191 me and I hope that both you and Elaine know that you can always count on me for help, science-related issues or whatever else you can come up with. Lastly, thank you both for wanting to be my paranimfs during the defence, I could not have wished for two better sidekicks.

Monday mornings have never been such a challenge (and strangely enough I am not talking about the Monday Morning Meetings): Vielen dank Daniel Giesen für Ihre deutsche Ansatz in der Turnhalle. Thanks also to Elferra Swart and Cécile Le Lann for going through all this together with me on all those early Monday mornings: Dhanyavaad et merci beaucoup.

Mijn vrienden, Sjoerd de Vlieger, Miranda van Barneveld, Léonie van Da- len, Mirte Fritz, Johan Straver, Anna Raap, Erica van Maren, Martijn van Manneveld, Inke van der Sluijs, Kees Hofker, Charlotte Lindeyer, Henk van Goor en Barbara Reumer. Bedankt voor alle leuke dingen die we samen gedaan hebben en voor jullie hulp als ik weer eens stoppen moest maken of veldwerk moest doen. Mijn sociale leven heeft in ieder geval niet geleden onder mijn promotieonderzoek. Ik heb genoten van al onze etentjes, uitjes en vakanties. Bedankt.

Leendert Verboom, heel erg bedankt voor het ontwerpen van de mooist mogelijke kaft voor mijn proefschrift. Niemand anders had een mooiere of relevantere kaft kunnen maken.

Peter en Ego Visser. Als er iemand is die weet hoe je moet motiveren dan ben jij het wel pap. Je hebt me altijd gesteund en gezorgd dat ik bleef door- zetten om het hoogst mogelijke te bereiken. Bedankt voor alle discussies en wijze raad. Eeg, bedankt voor het aanhoren van al mijn verhalen en de gezelligheid bij onze wekelijkse etentjes in huize Visser. Binnenkort vertrek ik naar het buitenland en ik weet nu al dat ik jullie enorm ga missen. Ik beloof elke week te skypen, maar deze keer komen jullie er niet onderuit: Waar ik ook ben, jullie moeten langskomen!

Saskia (muti) Kok-de Jongh, Paul Kok en Dorijn Otto, bedankt voor jullie steun en de gezelligheid in huize Kok. Toen mijn promotieonderzoek begon was het voor ons allemaal een roerige tijd door het overlijden van Huug. Vooral voor jou Sas was het een vreselijke periode, maar je hebt je er ge- weldig doorheen geslagen en nog steeds. Jullie, en ook Huug, hebben me altijd gesteund en me het gevoel gegeven dat ik in huize Kok in mijn eigen

192 Acknowledgements huis was.

Eric Kok, naast misschien een aantal van mijn collega’s is er niemand die zo nauw betrokken is geweest bij mijn promotieonderzoek als jij. Los van alle momenten dat we samen in het weekend naar de VU gingen om te werken aan experimenten (en dat waren er echt heel erg veel) heb jij me altijd gesteund. Bedankt voor alle tijd en moeite die je in mijn onderzoek en de afronding van dit proefschrift hebt gestopt. Ik had het simpelweg niet kunnen doen zonder jouw hulp en steun. Bedankt voor alles.

Adriana Visser-Verboom en Hugo Kok. Mijn moeder en schoonvader hebben mijn tijd op de VU niet meer mee kunnen maken, maar zij hebben mij de steun en het doorzettingsvermogen gegeven om te beginnen aan dit project en het goed af te ronden. Bedankt voor alles.

193

Curriculum vitae

1984 13th of October born in Delft

1996-2001 Zandvliet College, The Hague

2001-2002 Propaedeutic degree in Biology and Medical Laboratory Sci- ences, Hogeschool Leiden, Leiden

2002-2005 BSc Biologie, University of Leiden, Leiden

Literature survey at the department of Behavioural Biology (University of Leiden) under supervision of Dr. R. Lachlan on: The theory of mind in animals.

Internship at the department of Animal Ecology (University of Leiden) under supervision of Dr. I. van der Sluijs and Prof. Dr. J. van Alphen on: The genetic mechanism underlying co- evolution between mate choice and coloration in Lake Victoria cichlids.

2005-2007 MSc Evolutionary and Ecological Science, University of Lei- den

Literature survey at the department of Evolutionary Biology (University of Leiden) under supervision of Dr. P. Beldade and Prof. Dr. B. Zwaan on: Genetics of sexual behaviour.

Internship at the department of Evolutionary Biology (Uni- versity of Leiden) under supervision of Dr. P. Beldade and Prof. Dr. B.J. Zwaan on: Genetics of sex-reversed courtship behaviours in the butterﬂy Bicyclus anynana.

Internship at the Center for Ecology and Conservation (Uni- versity of Exeter, Cornwall Campus, UK) under supervision of Dr. Z. Lewis, Prof. Dr. N. Wedell and Prof. Dr. B. Zwaan on: Genetics underlying life history traits in the moth Plodia interpuctella.

195 2007-2011 PhD in Evolutionary Ecology, VU University Amsterdam

Doctoral internship at the department of Entomology and Ne- matology (University of Florida, USA) under supervision of Dr. D. Hahn on: Stable lipid loss in parasitoids.

Presentation of results on 10 national and international con- ferences.

Supervision of 5 BSc and 2 MSc students.

196 Publications

In peer-reviewed journals

Visser, B. & Ellers, J. Eﬀects of a lipid-rich diet on adult parasitoid income resources and survival. In press in Biological Control.

Le Lann, C., Visser, B., van Baaren, J., Van Alphen, J.J.M. & Ellers, J. Comparing resource exploitation and allocation of two closely related aphid parasitoids sharing the same host. Published online in Evolutionary Ecology. DOI: 10.1007/s10682-011-9498-2.

Ellers, J., Ruhé, B. & Visser, B. 2011. Discriminating between energetic content and dietary composition as an explanation for dietary restriction eﬀects. Journal of Insect Physiology 57: 1670-1676.

Visser, B., Le Lann, C., den Blanken, F.J., Harvey, J.A., Van Alphen, J.J.M. & Ellers, J., 2010. Loss of lipid synthesis as an evolutionary consequence of a parasitic lifestyle. Proceedings of the National Academy of Sciences of the United States of America 107: 8677-8682.

Visser, B. & Ellers, J., 2008. Lack of lipogenesis in parasitoids: A review of physiological mechanisms and ecological implications. Journal of Insect Physiology 54: 1315-1322.

Submitted manuscripts

Visser, B., Roelofs, D. Hahn, D.A., Teal, P.E.A., Mariën. J. & Ellers, J. Lack of transcription of the key gene in lipid synthesis, fatty acid synthase, reﬂects loss of lipogenesis in adult parastic wasps. Encouraged to resubmit to Molecular Ecology.

Visser, B., Voogt, M. & Ellers, J. Can manipulation of host physiology drive the loss of traits in parasitoids? Submitted to Journal of Evolutionary Biology.

Visser, B., van Dooremalen, C., Vazquez-Ruiz, A. & Ellers, J. Host exploitation over trophic levels in a gall wasp community. Submitted to Oikos.

197 Manuscripts in preparation

Ellers, J., Kiers. T.E., Currie, C.R. & Visser, B. Evolutionary loss of traits in ecological interactions. Pre-submission accepted by Ecology Letters. Reumer, B., Visser, B., Ellers, J., van Alphen, J.J.M. & Kraaijeveld, K. Diversity of life history traits among populations of the wasp Tetrastichus coeruleus infected with Wolbachia. Le Lann, C., Visser, B., Moiroux, J., van Baaren, J., van Alphen, J.J.M. & Ellers, J. Thermal reaction norms of Drosophila parasitoids attacking diﬀerent host stages.

198 Aﬃlliation of committee members

Promotor Prof. Dr. Jacintha Ellers Section Animal Ecology, Department of Ecological Science, Amsterdam Global Change Institute, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, the Netherlands

Committee members Dr. J. G. de Boer Evolutionary Genetics, Centre for Ecological and Evolutionary Studies, University of Groningen, 9700 CC Groningen, the Netherlands

Dr. D. Giron Institut de Recherche sur la Biologie de l’Insecte, Faculté des Sciences et Techniques, Université François-Rabelais, Parc Grandmont, 37200 Tours, France

Dr. E.T. Kiers Section Animal Ecology, Department of Ecological Science, Amsterdam Global Change Institute, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, the Netherlands

Prof. Dr. L.E.M. Vet Department of Terrestrial Ecology, Netherlands Institute of Ecology, Droeven- daalsesteeg 10, 6708 PB Wageningen, the Netherlands

Dr. B. Wertheim Evolutionary Genetics, Centre for Ecological and Evolutionary Studies, University of Groningen, 9700 CC Groningen, the Netherlands

199

Aﬃliation of co-authors

Jacintha Ellers, Cécile Le Lann, Frank J. den Blanken, Menno Voogt, Alba Vazquez-Ruiz, Dick Roelofs, Janine Mariën and Bas Ruhé Section Animal Ecology, Department of Ecological Science, Amsterdam Global Change Institute, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, the Netherlands

Jeﬀrey A. Harvey Department of Terrestrial Ecology, Netherlands Institute of Ecology, Droeven- daalsesteeg 10, 6708 PB Wageningen, the Netherlands

Jacques J.M. van Alphen IBED, University of Amsterdam, P.O. Box 94248, 1090 GE Amsterdam, The Netherlands/Netherlands Centre for Biodiversity Naturalis, P.O. Box 9517, 2300 RA Leiden, the Netherlands

Coby van Dooremalen Bees wur, Plant Research international, Wageningen University, Droeven- daalsesteeg 1, 6708 PB Wageningen, the Netherlands

Daniel A. Hahn Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611-0620, USA

Peter E.A. Teal Chemistry Research Unit, CMAVE-USDA-ARS, 1700 SW 23 Dr., Gainesville, FL 32608, USA

201