The Effect of Habitat on Insect Movements: Experimental Evidence from Wild-Caught Butterflies.

There is broad evidence that the main driver of the ongoing biodiversity crisis is land-use change, which reduces and fragments habitats. The consequence of habitat fragmentation on behavioural responses of fitness-related traits in insects have been so far understudied. In herbivorous insects, oviposition-related behaviours determine access to larval food, and the fate of the next generation. We present a pilot study to assess differences in behaviours related to movement and oviposition in Limenitis camilla butterflies from Wallonia (Belgium), one of the most fragmented regions in Europe. We first quantified variation in functional habitat connectivity across Wallonia and found that fragmented habitats had more abundant, but less evenly distributed host plants of L. camilla. Secondly, we quantified the behaviours of field-caught L. camilla females originating from habitats with contrasted landscape connectivity in an outdoor experimental setting. We found differences in behaviours related to flight investment: butterflies from fragmented woodlands spent more time in departing flight, which we associated with dispersal, than butterflies from homogenous woodlands. Although results from this study should be interpreted with caution given the limited sample size, they provide valuable insights for the advancement of behavioural research that aims to assess the effects of global changes on insects.

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.

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.

Polyphenism of visual and chemical secondary sexually-selected wing traits in the butterfly Bicyclus anynana: How different is the intermediate phenotype?

Polyphenism is a type of phenotypic plasticity supposedly adaptive to drastic and recurrent changes in the environment such as seasonal alternation in temperate and tropical regions. The butterfly Bicyclus anynana shows polyphenism with well-described wet and dry seasonal forms in sub-Saharan Africa, displaying striking morphological, physiological and behavioural differences in response to higher or lower developmental temperatures. During the seasonal transition in the wild, the intermediate phenotype co-occurs with wet and dry phenotypes. In this study, we aimed to characterize the secondary sexually-selected wing traits of the intermediate form to infer its potential fitness compared to wet and dry phenotypes. Among the previously described wing morphological traits, we first showed that the area of the fifth eyespot on the ventral hindwing is the most discriminant trait to identify wet, dry and intermediate phenotypes in both sexes. Second, we characterized the intermediate form for two secondary sexually-selected wing traits: the area and UV reflectance of the dorsal forewing pupil and the composition of the male sex pheromone. We showed that values of these two traits are often between those of the wet and dry phenotypes. Third, we observed increasing male sex pheromone production in ageing dry and wet phenotypes. Our results contrast with previous reports of values for sexually-selected traits in wet and dry seasonal forms, which might be explained by differences in rearing conditions or sample size effects among studies. Wet, dry and intermediate phenotypes display redundant sexually dimorphic traits, including sexually-selected traits that can inform about their developmental temperature in sexual interactions.

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.

Adaptive learning in non-social insects: from theory to field work, and back.

We review the evidence that learning affects fitness in non-social insects. Early accounts date back from the 1970s and were based on field-based observational and experimental work, yet exploration of the ways in which various forms of learning increase fitness remains limited in non-social insects. We highlight the concerns that arise when artificial laboratory settings, which do not take the ecology of the species into account, are used to estimate fitness benefits of learning. We argue that ecologically-relevant experimental designs are most useful to provide fitness estimates of learning, that is, designs that include: firstly, offspring of wild-caught animals producing newly established stocks under relevant breeding conditions, combined with common-garden and reciprocal transplant experiments; secondly, the spatio-temporal dynamics of key ecological resources; and thirdly, the natural behaviours of the animals while searching for, and probing, resources. Finally, we provide guidelines for the study of fitness-learning relationships in an eco-evolutionary framework.

Differentiation in putative male sex pheromone components across and within populations of the African butterfly Bicyclus anynana as a potential driver of reproductive isolation.

Sexual traits are often the most divergent characters among closely related species, suggesting an important role of sexual traits in speciation. However, to prove this, we need to show that sexual trait differences accumulate before or during the speciation process, rather than being a consequence of it. Here, we contrast patterns of divergence among putative male sex pheromone (pMSP) composition and the genetic structure inferred from variation in the mitochondrial cytochrome oxidase 1 and nuclear CAD loci in the African butterfly Bicyclus anynana (Butler, 1879) to determine whether the evolution of "pheromonal dialects" occurs before or after the differentiation process. We observed differences in abundance of some shared pMSP components as well as differences in the composition of the pMSP among B. anynana populations. In addition, B. anynana individuals from Kenya displayed differences in the pMSP composition within a single population that appeared not associated with genetic differences. These differences in pMSP composition both between and within B. anynana populations were as large as those found between different Bicyclus species. Our results suggest that "pheromonal dialects" evolved within and among populations of B. anynana and may therefore act as precursors of an ongoing speciation process.

Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression profile of the target candidate genes.

The scent of inbreeding: a male sex pheromone betrays inbred males.

Inbreeding depression results from mating among genetically related individuals and impairs reproductive success. The decrease in male mating success is usually attributed to an impact on multiple fitness-related traits that reduce the general condition of inbred males. Here, we find that the production of the male sex pheromone is reduced significantly by inbreeding in the butterfly Bicyclus anynana. Other traits indicative of the general condition, including flight performance, are also negatively affected in male butterflies by inbreeding. Yet, we unambiguously show that only the production of male pheromones affects mating success. Thus, this pheromone signal informs females about the inbreeding status of their mating partners. We also identify the specific chemical component (hexadecanal) probably responsible for the decrease in male mating success. Our results advocate giving increased attention to olfactory communication as a major causal factor of mate-choice decisions and sexual selection.

Cracking the olfactory code of a butterfly: the scent of ageing.

Although olfaction is a primary mode of communication, its importance in sexual selection remains understudied. Here, using the butterfly Bicyclus anynana, we address all the parameters of importance to sexual selection for a male olfactory signal. We show that variation in the male sex pheromone composition indicates male identity and male age. Courting males of different ages display small absolute (c. 200 ng) but large relative (100%) change of one specific pheromone component (hexadecanal) which, unlike the other components, showed no heritability. Females prefer to mate with mid-aged over younger males and the pheromone composition is sufficient to determine this preference. Surprisingly refined information is thus present in the male olfactory signal and is used for sexual selection. Our data also reveal that there may be no 'lek paradox' to resolve once the precise signal of importance to females is identified, as hexadecanal is, as expected, depleted in additive genetic variation.

The male sex pheromone of the butterfly Bicyclus anynana: towards an evolutionary analysis.

BACKGROUND: Female sex pheromones attracting mating partners over long distances are a major determinant of reproductive isolation and speciation in Lepidoptera. Males can also produce sex pheromones but their study, particularly in butterflies, has received little attention. A detailed comparison of sex pheromones in male butterflies with those of female moths would reveal patterns of conservation versus novelty in the associated behaviours, biosynthetic pathways, compounds, scent-releasing structures and receiving systems. Here we assess whether the African butterfly Bicyclus anynana, for which genetic, genomic, phylogenetic, ecological and ethological tools are available, represents a relevant model to contribute to such comparative studies. METHODOLOGY/PRINCIPAL FINDINGS: Using a multidisciplinary approach, we determined the chemical composition of the male sex pheromone (MSP) in the African butterfly B. anynana, and demonstrated its behavioural activity. First, we identified three compounds forming the presumptive MSP, namely (Z)-9-tetradecenol (Z9-14:OH), hexadecanal (16:Ald ) and 6,10,14-trimethylpentadecan-2-ol (6,10,14-trime-15-2-ol), and produced by the male secondary sexual structures, the androconia. Second, we described the male courtship sequence and found that males with artificially reduced amounts of MSP have a reduced mating success in semi-field conditions. Finally, we could restore the mating success of these males by perfuming them with the synthetic MSP. CONCLUSIONS/SIGNIFICANCE: This study provides one of the first integrative analyses of a MSP in butterflies. The toolkit it has developed will enable the investigation of the type of information about male quality that is conveyed by the MSP in intraspecific communication. Interestingly, the chemical structure of B. anynana MSP is similar to some sex pheromones of female moths making a direct comparison of pheromone biosynthesis between male butterflies and female moths relevant to future research. Such a comparison will in turn contribute to understanding the evolution of sex pheromone production and reception in butterflies.

Parasites: proxies for host genealogy and ecology?

Genetic information is used extensively to reconstruct the evolutionary and demographic history of organisms. Recently, it has been suggested that genetic information from some parasites can complement genetic data from their hosts. This approach relies upon the hypothesis that such parasites share a common history with their host. In some cases, parasites provide an additional source of information because parasite data can better reconstruct the common history. Here, we discuss which parasite traits are important in determining their usefulness for analysing host history. The key is the matching of the traits of the parasite (e.g. effective population size, generation time, mutation rate and level of host specificity) with the timescales (phylogenetic, phylogeographic and demographic) that are relevant to the issues of concern in host history.