The importance of fat accumulation and reserves for insect overwintering.

Winter is a challenging season for ectothermic species such as insects. In addition to thermal stress imposed by cold temperatures, food scarcity during winter can lead to starvation and energy drain. In preparation for winter, most insects accumulate lipid (fat) reserves, which are the principal source of energetic fuel during overwintering. In this review, we highlight the most recent literature on lipid metabolism in response to cold. We first discuss how lipid metabolism is affected by biotic and abiotic environmental changes in preparation for winter. We then highlight how lipid dynamics are affected during winter, including physiological and (epi)genetic mechanisms. We end our review emphasizing the importance of remaining fat reserves in spring and how climate change can negatively impact lipid metabolism and fitness.

Many parasitoids lack adult fat accumulation, despite fatty acid synthesis: A discussion of concepts and considerations for future research.

Fat reserves, specifically the accumulation of triacylglycerols, are a major energy source and play a key role for life histories. Fat accumulation is a conserved metabolic pattern across most insects, yet in most parasitoid species adults do not gain fat mass, even when nutrients are readily available and provided ad libitum. This extraordinary physiological phenotype has evolved repeatedly in phylogenetically dispersed parasitoid species. This poses a conundrum because it could lead to significant constraints on energy allocation toward key adult functions such as survival and reproduction. Recent work on the underlying genetic and biochemical mechanisms has spurred a debate on fat accumulation versus fat production, because of incongruent interpretation of results obtained using different methodologies. This debate is in part due to semantics, highlighting the need for a synthetic perspective on fat accumulation that reconciles previous debates and provides new insights and terminology. In this paper, we propose updated, unambiguous terminology for future research in the field, including "fatty acid synthesis" and "lack of adult fat accumulation", and describe the distinct metabolic pathways involved in the complex process of lipogenesis. We then discuss the benefits and drawbacks of the main methods available to measure fatty acid synthesis and adult fat accumulation. Most importantly, gravimetric/colorimetric and isotope tracking methods give complementary information, provided that they are applied with appropriate controls and interpreted correctly. We also compiled a comprehensive list of fat accumulation studies performed during the last 25 years. We present avenues for future research that combine chemistry, ecology, and evolution into an integrative approach, which we think is needed to understand the dynamics of fat accumulation in parasitoids.

The Drosophila-parasitizing wasp Leptopilina heterotoma: A comprehensive model system in ecology and evolution.

The parasitoid Leptopilina heterotoma has been used as a model system for more than 70 years, contributing greatly to diverse research areas in ecology and evolution. Here, we synthesized the large body of work on L. heterotoma with the aim to identify new research avenues that could be of interest also for researchers studying other parasitoids and insects. We start our review with a description of typical L. heterotoma characteristics, as well as that of the higher taxonomic groups to which this species belongs. We then continue discussing host suitability and immunity, foraging behaviors, as well as fat accumulation and life histories. We subsequently shift our focus towards parasitoid-parasitoid interactions, including L. heterotoma coexistence within the larger guild of Drosophila parasitoids, chemical communication, as well as mating and population structuring. We conclude our review by highlighting the assets of L. heterotoma as a model system, including its intermediate life history syndromes, the ease of observing and collecting natural hosts and wasps, as well as recent genomic advances.

Mosaic Evolution of Molecular Pathways for Sex Pheromone Communication in a Butterfly.

Unraveling the origin of molecular pathways underlying the evolution of adaptive traits is essential for understanding how new lineages emerge, including the relative contribution of conserved ancestral traits and newly evolved derived traits. Here, we investigated the evolutionary divergence of sex pheromone communication from moths (mostly nocturnal) to butterflies (mostly diurnal) that occurred ~119 million years ago. In moths, it is the females that typically emit pheromones to attract male mates, but in butterflies males emit pheromones that are used by females for mate choice. The molecular bases of sex pheromone communication are well understood in moths, but they have remained relatively unexplored in butterflies. We used a combination of transcriptomics, real time qPCR, and phylogenetics to identify genes involved in the different steps (i.e., production, regulation, and reception) of sex pheromone communication of the butterfly Bicyclus anynana. Our results show that the biosynthesis and reception of sex pheromones relies both on moth-specific gene families (reductases) and on more ancestral insect gene families (desaturases, olfactory receptors, odorant binding proteins). Interestingly, B. anynana appears to use what was believed to be the moth-specific neuropeptide Pheromone Biosynthesis Activating Neuropeptide (PBAN) for regulating sex pheromone production. Altogether, our results suggest that a mosaic pattern best explains how sex pheromone communication evolved in butterflies, with some molecular components derived from moths, and others conserved from more ancient insect ancestors. This is the first large-scale investigation of the genetic pathways underlying sex pheromone communication in a butterfly.

Pupal size as a proxy for fat content in laboratory-reared and field-collected Drosophila species.

In arthropods, larger individuals tend to have more fat reserves, but data for many taxa are still missing. For the vinegar fly Drosophila melanogaster, only few studies have provided experimental data linking body size to fat content. This is rather surprising considering the widespread use of D. melanogaster as a model system in biology. Here, we hypothesized that fat content in D. melanogaster is positively correlated with body size. To test this, we manipulated the developmental environment of D. melanogaster by decreasing food availability. We then measured pupal size and quantified fat content of laboratory-reared D. melanogaster. We subsequently measured pupal size and fat content of several field-caught Drosophila species. Starvation, crowding, and reduced nutrient content led to smaller laboratory-reared pupae that contained less fat. Pupal size was indeed found to be positively correlated with fat content. The same correlation was found for field-caught Drosophila pupae belonging to different species. As fat reserves are often strongly linked to fitness in insects, further knowledge on the relationship between body size and fat content can provide important information for studies on insect ecology and physiology.

The Evolutionary Relevance of Social Learning and Transmission in Non-Social Arthropods with a Focus on Oviposition-Related Behaviors.

Research on social learning has centered around vertebrates, but evidence is accumulating that small-brained, non-social arthropods also learn from others. Social learning can lead to social inheritance when socially acquired behaviors are transmitted to subsequent generations. Using oviposition site selection, a critical behavior for most arthropods, as an example, we first highlight the complementarities between social and classical genetic inheritance. We then discuss the relevance of studying social learning and transmission in non-social arthropods and document known cases in the literature, including examples of social learning from con- and hetero-specifics. We further highlight under which conditions social learning can be adaptive or not. We conclude that non-social arthropods and the study of oviposition behavior offer unparalleled opportunities to unravel the importance of social learning and inheritance for animal evolution.

Phenotypic plasticity explains apparent reverse evolution of fat synthesis in parasitic wasps.

Numerous cases of evolutionary trait loss and regain have been reported over the years. Here, we argue that such reverse evolution can also become apparent when trait expression is plastic in response to the environment. We tested this idea for the loss and regain of fat synthesis in parasitic wasps. We first show experimentally that the wasp Leptopilina heterotoma switches lipogenesis on in a fat-poor environment, and completely off in a fat-rich environment. Plasticity suggests that this species did not regain fat synthesis, but that it can be switched off in some environmental settings. We then compared DNA sequence variation and protein domains of several more distantly related parasitoid species thought to have lost lipogenesis, and found no evidence for non-functionality of key lipogenesis genes. This suggests that other parasitoids may also show plasticity of fat synthesis. Last, we used individual-based simulations to show that a switch for plastic expression can remain functional in the genome for thousands of generations, even if it is only used sporadically. The evolution of plasticity could thus also explain other examples of apparent reverse evolution.

Dealing with predictable and unpredictable temperatures in a climate change context: the case of parasitoids and their hosts.

The Earth's climate is changing at a rapid pace. To survive in increasingly fluctuating and unpredictable environments, species can either migrate or evolve through rapid local adaptation, plasticity and/or bet-hedging. For small ectotherm insects, like parasitoids and their hosts, phenotypic plasticity and bet-hedging could be critical strategies for population and species persistence in response to immediate, intense and unpredictable temperature changes. Here, we focus on studies evaluating phenotypic responses to variable predictable thermal conditions (for which phenotypic plasticity is favoured) and unpredictable thermal environments (for which bet-hedging is favoured), both within and between host and parasitoid generations. We then address the effects of fluctuating temperatures on host-parasitoid interactions, potential cascading effects on the food web, as well as biological control services. We conclude our review by proposing a road map for designing experiments to assess if plasticity and bet-hedging can be adaptive strategies, and to disentangle how fluctuating temperatures can affect the evolution of these two strategies in parasitoids and their hosts.

Bottom-up regulation of a tritrophic system by Beet yellows virus infection: consequences for aphid-parasitoid foraging behaviour and development.

Effects of plants on herbivores can cascade up the food web and modulate the abundance of higher trophic levels. In agro-ecosystems, plant viruses can affect the interactions between crops, crop pests, and natural enemies. Little is known, however, about the effects of viruses on higher trophic levels, including parasitoids and their ability for pest regulation. We tested the hypothesis that a plant virus affects parasitoid foraging behaviour through cascading effects on higher trophic levels. We predicted that the semi-persistent Beet yellows virus (BYV) would influence plant (Beta vulgaris) quality, as well as aphid host (Aphis fabae) quality for a parasitoid Lysiphlebus fabarum. We determined amino acid and sugar content in healthy and infected plants (first trophic level), lipid content and body size of aphids (second trophic level) fed on both plants, as well as foraging behaviour and body size of parasitoids (third trophic level) that developed on aphids fed on both plants. Our results showed that virus infection increased sugars and decreased total amino acid content in B. vulgaris. We further observed an increase in aphid size without modification in host aphid quality (i.e., lipid content), and a slight effect on parasitoid behaviour through an increased number of antennal contacts with host aphids. Although the BYV virus clearly affected the first two trophic levels, it did not affect development or emergence of parasitoids. As the parasitoid L. fabarum does not seem to be affected by the virus, we discuss the possibility of using it for the development of targeted biological control against aphids.

Sexual selection contributes to partial restoration of phenotypic robustness in a butterfly.

Phenotypic variation is the raw material for selection that is ubiquitous for most traits in natural populations, yet the processes underlying phenotypic evolution or stasis often remain unclear. Here, we report phenotypic evolution in a mutant line of the butterfly Bicyclus anynana after outcrossing with the genetically polymorphic wild type population. The comet mutation modifies two phenotypic traits known to be under sexual selection in this butterfly: the dorsal forewing eyespots and the pheromone-producing structures. The original comet mutant line was inbred and remained phenotypically stable for at least seven years, but when outcrossed to the wild type population the outcrossed comet line surprisingly recovered the wild type phenotype within 8 generations at high (27 °C), but not at low (20 °C), developmental temperatures. Male mating success experiments then revealed that outcrossed comet males with the typical comet phenotype suffered from lower mating success, while mating success of outcrossed comet males resembling wild types was partially restored. We document a fortuitous case where the addition of genetic polymorphism around a spontaneous mutation could have allowed partial restoration of phenotypic robustness. We further argue that sexual selection through mate choice is likely the driving force leading to phenotypic robustness in our system.

Variation in lipid synthesis, but genetic homogeneity, among Leptopilina parasitic wasp populations.

Lipid synthesis can have a major effect on survival and reproduction, yet most insect parasitoids fail to synthesize lipids. For parasitic wasps in the genus Leptopilina, however, studies have suggested that there is intraspecific variation in the ability for lipid synthesis. These studies were performed on only few populations, and a large-scale investigation of both lipogenic ability and population genetic structure is now needed. Here, we first examined lipogenic ability of nine Leptopilina heterotoma populations collected in 2013 and found that five of nine populations synthesized lipids. The 2013 populations could not be used to determine genetic structure; hence, we obtained another 20 populations in 2016 that were tested for lipogenic ability. Thirteen of 20 populations (all Leptopilina heterotoma) were then used to determine the level of genetic differentiation (i.e., haplotype and nucleotide diversity) by sequencing neutral mitochondrial (COI) and nuclear (ITS2) markers. None of the 2016 populations synthesized lipids, and no genetic differentiation was found. Our results did reveal a nearly twofold increase in mean wasp lipid content at emergence in populations obtained in 2016 compared to 2013. We propose that our results can be explained by plasticity in lipid synthesis, where lipogenic ability is determined by environmental factors, such as developmental temperature and/or the amount of lipids carried over from the host.

Ant-like Traits in Wingless Parasitoids Repel Attack from Wolf Spiders.

A recent study showed that a wingless parasitoid, Gelis agilis, exhibits a suite of ant-like traits that repels attack from wolf spiders. When agitated, G. agilis secreted 6-methyl-5-hepten-2-one (sulcatone), which a small number of ant species produce as an alarm/panic pheromone. Here, we tested four Gelis parasitoid species, occurring in the same food chain and microhabitats, for the presence of sulcatone and conducted two-species choice bioassays with wolf spiders to determine their degree of susceptibility to attack. All four Gelis species, including both winged and wingless species, produced sulcatone, whereas a closely related species, Acrolyta nens, and the more distantly related Cotesia glomerata, did not. In two-choice bioassays, spiders overwhelmingly rejected the wingless Gelis species, preferring A. nens and C. glomerata. However, spiders exhibited no preference for either A. nens or G. areator, both of which are winged. Wingless gelines exhibited several ant-like traits, perhaps accounting for the reluctance of spiders to attack them. On the other hand, despite producing sulcatone, the winged G. areator more closely resembles other winged cryptines like A. nens, making it harder for spiders to distinguish between these two species. C. glomerata was also preferred by spiders over A. nens, suggesting that other non-sulcatone producing cryptines nevertheless possess traits that make them less attractive as prey. Phylogenetic reconstruction of the Cryptinae reveals that G. hortensis and G. proximus are 'sister'species, with G. agilis, and G.areator in particular evolving along more distant trajectories. We discuss the possibility that wingless Gelis species have evolved a suite of ant-like traits as a form, of mimicry to repel predators on the ground.

An Evolutionary Perspective on Linoleic Acid Synthesis in Animals.

The diet of organisms generally provides a sufficient supply of energy and building materials for healthy growth and development, but should also contain essential nutrients. Species differ in their exogenous requirements, but it is not clear why some species are able to synthesize essential nutrients, while others are not. The unsaturated fatty acid, linoleic acid (LA; 18:2n-6) plays an important role in functions such as cell physiology, immunity, and reproduction, and is an essential nutrient in diverse organisms. LA is readily synthesized in bacteria, protozoa and plants, but it was long thought that all animals lacked the ability to synthesize LA de novo and thus required a dietary source of this fatty acid. Over the years, however, an increasing number of studies have shown active LA synthesis in animals, including insects, nematodes and pulmonates. Despite continued interest in LA metabolism, it has remained unclear why some organisms can synthesize LA while others cannot. Here, we review the mechanisms by which LA is synthesized and which biological functions LA supports in different organisms to answer the question why LA synthesis was lost and repeatedly gained during the evolution of distinct invertebrate groups. We propose several hypotheses and compile data from the available literature to identify which factors promote LA synthesis within a phylogenetic framework. We have not found a clear link between our proposed hypotheses and LA synthesis; therefore we suggest that LA synthesis may be facilitated through bifunctionality of desaturase enzymes or evolved through a combination of different selective pressures.

Hormetic benefits of prior anoxia exposure in buffering anoxia stress in a soil-pupating insect.

Oxygen is essential for most animals, and exposure to a complete lack of oxygen, i.e. anoxia, can result in irreparable damage to cells that can extend up to the organismal level to negatively affect performance. Although it is known that brief anoxia exposure may confer cross-tolerance to other stressors, few data exist on the biochemical and organismal consequences of repeated intermittent bouts of anoxia exposure. In nature, the Caribbean fruit fly, Anastrepha suspensa (Diptera: Tephritidae), is frequently exposed to heavy tropical rainfall while pupating in the soil, equating to multiple exposures to hypoxia or anoxia during development. Here, we tested whether prior anoxia exposures during pupal development can induce a beneficial acclimation response, and we explored the consequences of prior exposure for both whole-organism performance and correlated biochemical metrics. Pharate adults (the last developmental stage in the pupal case) were most sensitive to anoxia exposure, showing decreased survival and fertility compared with controls. These negative impacts were ameliorated by exposure to anoxia in earlier pupal developmental stages, indicating a hormetic effect of prior anoxia exposure. Anoxia exposure early in pupal development reduced the oxygen debt repaid after anoxia exposure relative to pharate adults experiencing anoxia for the first time. Lipid levels were highest in all pupal stages when exposed to prior anoxia. Prior anoxia thus benefits organismal performance and relocates resources towards lipid storage throughout pupal-adult development.

Maternal age affects offspring nutrient dynamics.

The internal physiological state of a mother can have major effects on her fitness and that of her offspring. We show that maternal effects in the parasitic wasp Eupelmus vuilleti become apparent when old mothers provision their eggs with less protein, sugar and lipid. Feeding from a host after hatching allows the offspring of old mothers to overcome initial shortages in sugars and lipids, but adult offspring of old mothers still emerged with lower protein and glycogen quantities. Reduced egg provisioning by old mothers had adverse consequences for the nutrient composition of adult female offspring, despite larval feeding from a high-quality host. Lower resource availability in adult offspring of old mothers can affect behavioural decisions, life histories and performance. Maternal effects on egg nutrient provisioning may thus affect nutrient availability and fitness of future generations in oviparous animals.

Concurrence in the ability for lipid synthesis between life stages in insects.

The ability to synthesize lipids is critical for an organism's fitness; hence, metabolic pathways, underlying lipid synthesis, tend to be highly conserved. Surprisingly, the majority of parasitoids deviate from this general metabolic model by lacking the ability to convert sugars and other carbohydrates into lipids. These insects spend the first part of their life feeding and developing in or on an arthropod host, during which they can carry over a substantial amount of lipid reserves. While many parasitoid species have been tested for lipogenic ability at the adult life stage, it has remained unclear whether parasitoid larvae can synthesize lipids. Here we investigate whether or not several insects can synthesize lipids during the larval stage using three ectoparasitic wasps (developing on the outside of the host) and the vinegar fly Drosophila melanogaster that differ in lipogenic ability in the adult life stage. Using feeding experiments and stable isotope tracing with gas chromatography/mass spectrometry, we first confirm lipogenic abilities in the adult life stage. Using topical application of stable isotopes in developing larvae, we then provide clear evidence of concurrence in lipogenic ability between larval and adult life stages in all species tested.

Honey and honey-based sugars partially affect reproductive trade-offs in parasitoids exhibiting different life-history and reproductive strategies.

Adult dietary regimes in insects may affect egg production, fecundity and ultimately fitness. This is especially relevant in parasitoid wasps where many species serve as important biological control agents of agricultural pests. Here, we tested the effect of honey and sugar diets on daily fecundity schedules, lifetime reproductive success and longevity in four species of parasitoid wasps when reared on their respective hosts. The parasitoid species were selected based on dichotomies in host usage strategies and reproductive traits. Gelis agilis and G. areator are idiobiont ecto-parasitoids that develop in non-growing hosts, feed on protein-rich host fluids to maximize reproduction as adults and produce small numbers of large eggs. Meteorus pulchricornis and Microplitis mediator are koinobiont endoparasitoids that develop inside the bodies of growing hosts, do not host-feed, and produce greater numbers of small eggs. Parasitoids were reared on diets of either pure honey (containing trace amounts of proteins), heated honey (with denatured proteins) and a honey-mimic containing sugars only. We hypothesized that the benefits of proteins in honey would enhance reproduction in the ectoparasitoids due to their high metabolic investment per egg, but not in the koinobionts. Pure honey diet resulted in higher lifetime fecundity in G. agilis compared with the honey-mimic, whereas in both koinobionts, reproductive success did not vary significantly with diet. Longevity was less affected by diet in all of the parasitoids, although there were variable trade-offs between host access and longevity in the four species. We argue that there are both trait-based and association-specific effects of supplementary nutrients in honey on reproductive investment and success in parasitoid wasps.

Multi-trait mimicry of ants by a parasitoid wasp.

Many animals avoid attack from predators through toxicity or the emission of repellent chemicals. Defensive mimicry has evolved in many species to deceive shared predators, for instance through colouration and other morphological adaptations, but mimicry hardly ever seems to involve multi-trait similarities. Here we report on a wingless parasitoid wasp that exhibits a full spectrum of traits mimicing ants and affording protection against ground-dwelling predators (wolf spiders). In body size, morphology and movement Gelis agilis (Ichneumonidae) is highly similar to the black garden ant (Lasius niger) that shares the same habitat. When threatened, G. agilis also emits a volatile chemical that is similar to an ant-produced chemical that repels spiders. In bioassays with L. niger, G. agilis, G. areator, Cotesia glomerata and Drosophila melanogaster, ants and G. agilis were virtually immune to spider attack, in contrast the other species were not. Volatile characterisation with gas chromatography-mass spectrometry identified G. agilis emissions as 6-methyl-5-hepten-2-one, a known insect defence semiochemical that acts as an alarm pheromone in ants. We argue that multi-trait mimicry, as observed in G. agilis, might be much more common among animals than currently realized.

Rising temperature reduces divergence in resource use strategies in coexisting parasitoid species.

Coexistence of species sharing the same resources is often possible if species are phylogenetically divergent in resource acquisition and allocation traits, decreasing competition between them. Developmental and life-history traits related to resource use are influenced by environmental conditions such as temperature, but thermal trait responses may differ among species. An increase in ambient temperature may, therefore, affect trait divergence within a community, and potentially species coexistence. Parasitoids are interesting models to test this hypothesis, because multiple species commonly attack the same host, and employ divergent larval and adult host use strategies. In particular, development mode (arrested or continued host growth following parasitism) has been recognized as a major organiser of parasitoid life histories. Here, we used a comparative trait-based approach to determine thermal responses of development time, body mass, egg load, metabolic rate and energy use of the coexisting Drosophila parasitoids Asobara tabida, Leptopilina heterotoma, Trichopria drosophilae and Spalangia erythromera. We compared trait values between species and development modes, and calculated trait divergence in response to temperature, using functional diversity indices. Parasitoids differed in their thermal response for dry mass, metabolic rate and lipid use throughout adult life, but only teneral lipid reserves and egg load were affected by developmental mode. Species-specific trait responses to temperature were probably determined by their adaptations in resource use (e.g. lipogenesis or ectoparasitism). Overall, trait values of parasitoid species converged at the higher temperature. Our results suggest that local effects of warming could affect host resource partitioning by reducing trait diversity in communities.

Ecological interactions drive evolutionary loss of traits.

Loss of traits can dramatically alter the fate of species. Evidence is rapidly accumulating that the prevalence of trait loss is grossly underestimated. New findings demonstrate that traits can be lost without affecting the external phenotype, provided the lost function is compensated for by species interactions. This is important because trait loss can tighten the ecological relationship between partners, affecting the maintenance of species interactions. Here, we develop a new perspective on so-called `compensated trait loss' and how this type of trait loss may affect the evolutionary dynamics between interacting organisms. We argue that: (1) the frequency of compensated trait loss is currently underestimated because it can go unnoticed as long as ecological interactions are maintained; (2) by analysing known cases of trait loss, specific factors promoting compensated trait loss can be identified and (3) genomic sequencing is a key way forwards in detecting compensated trait loss. We present a comprehensive literature survey showing that compensated trait loss is taxonomically widespread, can involve essential traits, and often occurs as replicated evolutionary events. Despite its hidden nature, compensated trait loss is important in directing evolutionary dynamics of ecological relationships and has the potential to change facultative ecological interactions into obligatory ones.