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Background: Selection acts on the phenotype, yet only the genotype is inherited. While both the phenotypic and genotypic response to short-term selection can be measured, the link between these is a major unsolved problem in evolutionary biology, in particular for complex behavioural phenotypes., Results: Here we characterize the genomic and the transcriptomic basis of associative learning ability in the parasitic wasp Nasonia vitripennis and use gene network analysis to link the two. We artificially selected for improved associative learning ability in four independent pairs of lines and identified signatures of selection across the genome. Allele frequency diverged consistently between the selected and control lines in 118 single nucleotide polymorphisms (SNPs), clustering in 51 distinct genomic regions containing 128 genes. The majority of SNPs were found in regulatory regions, suggesting a potential role for gene expression evolution. We therefore sequenced the transcriptomes of selected and control lines and identified 36 consistently differentially expressed transcripts with large changes in expression. None of the differentially expressed genes also showed sequence divergence as a result of selection. Instead, gene network analysis showed many of the genes with consistent allele frequency differences and all of the differentially expressed genes to cluster in a single co-expression network. At a functional level, both genomic and transcriptomic analyses implicated members of gene networks known to be involved in neural plasticity and cognitive processes., Conclusions: Taken together, our results reveal how specific cognitive abilities can readily respond to selection via a complex interplay between regulatory and sequence evolution.

The mechanisms allowing the widespread invasive pest Drosophila suzukii to survive from early spring until the availability of the first fruit crops are still unclear. Seasonal biology and population dynamics of D. suzukii were investigated in order to better understand the contribution of the early spring hosts to the infestation of the first fruit crops of the season. We identified hosts available to D. suzukii in early spring and assessed their suitability for the pest oviposition and reproductive success under field and laboratory conditions. The natural infestation rate of one of these hosts, Aucuba japonica , was assessed over springtime and the morphology of the flies that emerged from infested A. japonica fruits was characterized under field conditions. Then, these findings were correlated with long-term monitoring data on seasonal reproductive biology and morphology of the pest, using a cumulative degree-days (DD) analysis. Field sampling revealed that overwintered D. suzukii females were physiologically able to lay eggs at 87 DD which coincided with the detection of the first infested early spring hosts. The latter were continuously and increasingly infested by D. suzukii eggs in nature from early spring until the end of May, in particular Aucuba japonica . Individuals emerged from most of these hosts were characterized by a poor fitness and a rather low success of emergence. In the field, only few summer morphs emerged from naturally infested A. japonica fruits around the end of May-beginning of June. However, field monitoring in orchards revealed that D. suzukii individuals consisted solely of winter morphs until mid-June. These observations indicate that overwintered D. suzukii females are the predominant source for the infestations in the first available fruit crops of the season. We discuss these findings in the context of possible pest control strategies.

The development of biological control methods for exotic invasive pest species has become more challenging during the last decade. Compared to indigenous natural enemies, species from the pest area of origin are often more efficient due to their long coevolutionary history with the pest. The import of these well-adapted exotic species, however, has become restricted under the Nagoya Protocol on Access and Benefit Sharing, reducing the number of available biocontrol candidates. Finding new agents and ways to improve important traits for control agents ("biocontrol traits") is therefore of crucial importance. Here, we demonstrate the potential of a surprisingly under-rated method for improvement of biocontrol: the exploitation of intraspecific variation in biocontrol traits, for example, by selective breeding. We propose a four-step approach to investigate the potential of this method: investigation of the amount of (a) inter- and (b) intraspecific variation for biocontrol traits, (c) determination of the environmental and genetic factors shaping this variation, and (d) exploitation of this variation in breeding programs. We illustrate this approach with a case study on parasitoids of Drosophila suzukii , a highly invasive pest species in Europe and North America. We review all known parasitoids of D. suzukii and find large variation among and within species in their ability to kill this fly. We then consider which genetic and environmental factors shape the interaction between D. suzukii and its parasitoids to explain this variation. Insight into the causes of variation informs us on how and to what extent candidate agents can be improved. Moreover, it aids in predicting the effectiveness of the agent upon release and provides insight into the selective forces that are limiting the adaptation of indigenous species to the new pest. We use this knowledge to give future research directions for the development of selective breeding methods for biocontrol agents.

Chemical warfare including insecticidal secondary metabolites is a well-known strategy for environmental microbes to monopolize a food source. Insects in turn have evolved behavioural and physiological defences to eradicate or neutralize the harmful microorganisms. We studied the defensive repertoire of insects in this interference competition by combining behavioural and developmental assays with whole-transcriptome time-series analysis. Confrontation with the toxic filamentous fungus Aspergillus nidulans severely reduced the survival of Drosophila melanogaster larvae. Nonetheless, the larvae did not behaviourally avoid the fungus, but aggregated at it. Confrontation with fungi strongly affected larval gene expression, including many genes involved in detoxification (e.g., CYP, GST and UGT genes) and the formation of the insect cuticle (e.g., Tweedle genes). The most strongly upregulated genes were several members of the insect-specific gene family Osiris, and CHK-kinase-like domains were over-represented. Immune responses were not activated, reflecting the competitive rather than pathogenic nature of the antagonistic interaction. While internal microbes are widely acknowledged as important, our study emphasizes the underappreciated role of environmental microbes as fierce competitors., (© 2017 John Wiley & Sons Ltd.)

Background: Parasitoid resistance in Drosophila varies considerably, among and within species. An immune response, lamellocyte-mediated encapsulation, evolved in a subclade of Drosophila and was subsequently lost in at least one species within this subclade. While the mechanisms of resistance are fairly well documented in D. melanogaster, much less is known for closely related species. Here, we studied the inter- and intra-species variation in gene expression after parasitoid attack in Drosophila. We used RNA-seq after parasitization of four closely related Drosophila species of the melanogaster subgroup and replicated lines of D. melanogaster experimentally selected for increased resistance to gain insights into short- and long-term evolutionary changes., Results: We found a core set of genes that are consistently up-regulated after parasitoid attack in the species and lines tested, regardless of their level of resistance. Another set of genes showed no up-regulation or expression in D. sechellia, the species unable to raise an immune response against parasitoids. This set consists largely of genes that are lineage-restricted to the melanogaster subgroup. Artificially selected lines did not show significant differences in gene expression with respect to non-selected lines in their responses to parasitoid attack, but several genes showed differential exon usage., Conclusions: We showed substantial similarities, but also notable differences, in the transcriptional responses to parasitoid attack among four closely related Drosophila species. In contrast, within D. melanogaster, the responses were remarkably similar. We confirmed that in the short-term, selection does not act on a pre-activation of the immune response. Instead it may target alternative mechanisms such as differential exon usage. In the long-term, we found support for the hypothesis that the ability to immunologically resist parasitoid attack is contingent on new genes that are restricted to the melanogaster subgroup.

In Drosophila, diet is considered a prominent factor shaping the associated bacterial community. However, the host population background (e.g. genotype, geographical origin and founder effects) is a factor that may also exert a significant influence and is often overlooked. To test for population background effects, we characterized the bacterial communities in larvae of six genetically differentiated and geographically distant D. melanogaster lines collected from natural populations across Europe. The diet for these six lines had been identical for ca. 50 generations, thus any differences in the composition of the microbiome originates from the host populations. We also investigated whether induced shifts in the microbiome-in this case by controlled antibiotic administration-alters the hosts' resistance to parasitism. Our data revealed a clear signature of population background on the diversity and composition of D. melanogaster microbiome that differed across lines, even after hosts had been maintained at the same diet and laboratory conditions for over 4 years. In particular, the number of bacterial OTUs per line ranged from 8 to 39 OTUs. Each line harboured 2 to 28 unique OTUs, and OTUs that were highly abundant in some lines were entirely missing in others. Moreover, we found that the response to antibiotic treatment differed among the lines and significantly altered the host resistance to the parasitoid Asobara tabida in one of the six lines. Wolbachia, a widespread intracellular endosymbiont associated with parasitoid resistance, was lacking in this line, suggesting that other components of the Drosophila microbiome caused a change in host resistance. Collectively, our results revealed that lines that originate from different population backgrounds show significant differences in the established Drosophila microbiome, outpacing the long-term effect of diet. Perturbations on these naturally assembled microbiomes to some degree influenced the hosts' resistance against natural parasites., Competing Interests: The authors have declared that no competing interests exist.

Background: Organisms have evolved a variety of defence mechanisms against natural enemies, which are typically used at the expense of other life history components. Induced defence mechanisms impose minor costs when pathogens are absent, but mounting an induced response can be time-consuming. Therefore, to ensure timely protection, organisms may partly rely on constitutive defence despite its sustained cost that renders it less economical. Existing theoretical models addressing the optimal combination of constitutive versus induced defence focus solely on host adaptation and ignore the fact that the efficacy of protection depends on genotype-specific host-parasite interactions. Here, we develop a signal-transduction network model inspired by the invertebrate innate immune system, in order to address the effect of parasite coevolution on the optimal combination of constitutive and induced defence., Results: Our analysis reveals that coevolution of parasites with specific immune components shifts the host's optimal allocation from induced towards constitutive immunity. This effect is dependent upon whether receptors (for detection) or effectors (for elimination) are subjected to parasite counter-evolution. A parasite population subjected to a specific immune receptor can evolve heightened genetic diversity, which makes parasite detection more difficult for the hosts. We show that this coevolutionary feedback renders the induced immune response less efficient, forcing the hosts to invest more heavily in constitutive immunity. Parasites diversify to escape elimination by a specific effector too. However, this diversification does not alter the optimal balance between constitutive and induced defence: the reliance on constitutive defence is promoted by the receptor's inability to detect, but not the effectors' inability to eliminate parasites. If effectors are useless, hosts simply adapt to tolerate, rather than to invest in any defence against parasites. These contrasting results indicate that evolutionary feedback between host and parasite populations is a key factor shaping the selection regime for immune networks facing antagonistic coevolution., Conclusion: Parasite coevolution against specific immune defence alters the prediction of the optimal use of defence, and the effect of parasite coevolution varies between different immune components.

Complex traits are manifestations of intricate gene interaction networks. Evolution of complex traits revolves around the genetic variation in such networks. Genomics has increased our ability to investigate the complex gene interaction networks, and characterize the extent of genetic variation in these networks. Immunity is a complex trait, for which the ecological drivers and molecular networks are fairly well understood in Drosophila. By characterizing the natural variation in immunity, and mapping how the genome changes during the evolution of immunity in Drosophila, we can integrate our knowledge on the complex genetic architecture of traits and the molecular basis of evolutionary processes.

Background: Haplodiploidy, where females develop from diploid, fertilized eggs and males from haploid, unfertilized eggs, is abundant in some insect lineages. Some species in these lineages reproduce by thelytoky that is caused by infection with endosymbionts: infected females lay haploid eggs that undergo diploidization and develop into females, while males are very rare or absent. It is generally assumed that in thelytokous wasps, endosymbionts merely diploidize the unfertilized eggs, which would then trigger female development., Results: We found that females in the parasitoid wasp Asobara japonica infected with thelytoky-inducing Wolbachia produce 0.7-1.2% male offspring. Seven to 39% of these males are diploid, indicating that diploidization and female development can be uncoupled in A. japonica. Wolbachia titer in adults was correlated with their ploidy and sex: diploids carried much higher Wolbachia titers than haploids, and diploid females carried more Wolbachia than diploid males. Data from introgression lines indicated that the development of diploid individuals into males instead of females is not caused by malfunction-mutations in the host genome but that diploid males are most likely produced when the endosymbiont fails to activate the female sex determination pathway. Our data therefore support a two-step mechanism by which endosymbionts induce thelytoky in A. japonica: diploidization of the unfertilized egg is followed by feminization, whereby each step correlates with a threshold of endosymbiont titer during wasp development., Conclusions: Our new model of endosymbiont-induced thelytoky overthrows the view that certain sex determination mechanisms constrain the evolution of endosymbiont-induced thelytoky in hymenopteran insects. Endosymbionts can cause parthenogenesis through feminization, even in groups in which endosymbiont-diploidized eggs would develop into males following the hosts' sex determination mechanism. In addition, our model broadens our understanding of the mechanisms by which endosymbionts induce thelytoky to enhance their transmission to the next generation. Importantly, it also provides a novel window to study the yet-poorly known haplodiploid sex determination mechanisms in haplodiploid insects.

In this study, we characterize changes in the genome during a swift evolutionary adaptation, by combining experimental selection with high-throughput sequencing. We imposed strong experimental selection on an ecologically relevant trait, parasitoid resistance in Drosophila melanogaster against Asobara tabida. Replicated selection lines rapidly evolved towards enhanced immunity. Larval survival after parasitization increased twofold after just five generations of selection. Whole-genome sequencing revealed that the fast and strong selection response in innate immunity produced multiple, highly localized genomic changes. We identified narrow genomic regions carrying a significant signature of selection, which were present across all chromosomes and covered in total less than 5% of the whole D. melanogaster genome. We identified segregating sites with highly significant changes in frequency between control and selection lines that fell within these narrow 'selected regions'. These segregating sites were associated with 42 genes that constitute possible targets of selection. A region on chromosome 2R was highly enriched in significant segregating sites and may be of major effect on parasitoid defence. The high genetic variability and small linkage blocks in our base population are likely responsible for allowing this complex trait to evolve without causing widespread erosive effects in the genome, even under such a fast and strong selective regime.

Understanding the genomic basis of evolutionary adaptation requires insight into the molecular basis underlying phenotypic variation. However, even changes in molecular pathways associated with extreme variation, gains and losses of specific phenotypes, remain largely uncharacterized. Here, we investigate the large interspecific differences in the ability to survive infection by parasitoids across 11 Drosophila species and identify genomic changes associated with gains and losses of parasitoid resistance. We show that a cellular immune defense, encapsulation, and the production of a specialized blood cell, lamellocytes, are restricted to a sublineage of Drosophila, but that encapsulation is absent in one species of this sublineage, Drosophila sechellia. Our comparative analyses of hemopoiesis pathway genes and of genes differentially expressed during the encapsulation response revealed that hemopoiesis-associated genes are highly conserved and present in all species independently of their resistance. In contrast, 11 genes that are differentially expressed during the response to parasitoids are novel genes, specific to the Drosophila sublineage capable of lamellocyte-mediated encapsulation. These novel genes, which are predominantly expressed in hemocytes, arose via duplications, whereby five of them also showed signatures of positive selection, as expected if they were recruited for new functions. Three of these novel genes further showed large-scale and presumably loss-of-function sequence changes in D. sechellia, consistent with the loss of resistance in this species. In combination, these convergent lines of evidence suggest that co-option of duplicated genes in existing pathways and subsequent neofunctionalization are likely to have contributed to the evolution of the lamellocyte-mediated encapsulation in Drosophila.

An attractive way to improve our understanding of sex determination evolution is to study the underlying mechanisms in closely related species and in a phylogenetic perspective. Hymenopterans are well suited owing to the diverse sex determination mechanisms, including different types of Complementary Sex Determination (CSD) and maternal control sex determination. We investigated different types of CSD in four species within the braconid wasp genus Asobara that exhibit diverse life-history traits. Nine to thirteen generations of inbreeding were monitored for diploid male production, brood size, offspring sex ratio, and pupal mortality as indicators for CSD. In addition, simulation models were developed to compare these observations to predicted patterns for multilocus CSD with up to ten loci. The inbreeding regime did not result in diploid male production, decreased brood sizes, substantially increased offspring sex ratios nor in increased pupal mortality. The simulations further allowed us to reject CSD with up to ten loci, which is a strong refutation of the multilocus CSD model. We discuss how the absence of CSD can be reconciled with the variation in life-history traits among Asobara species, and the ramifications for the phylogenetic distribution of sex determination mechanisms in the Hymenoptera.

As a measure of parasitoid resistance, hemocyte load and encapsulation ability were measured in lines collected from natural populations of Drosophila melanogaster in Europe. Results show large geographic variation in resistance against the parasitoid wasp Asobara tabida among the field lines, but there was no clear correlation between resistance and total hemocyte load, neither before nor after parasitization. This was in contrast to the patterns that had been found in a comparison among species of Drosophila, where total hemocyte counts were positively correlated to encapsulation rates. This suggests that the mechanisms underlying between-species variation in parasitoid resistance do not extend to the natural variation that exists within a species. Although hemocyte counts did not correspond to encapsulation ability within D. melanogaster, the ratios of lamellocytes and crystal cells were very similar in lines with successful encapsulation responses. Apart from variation in the hemocytic response of the different hemocyte types, within-species variation was also observed for accurate targeting of the foreign body by the hemocytes. These results are discussed in the context of possible causes of variation in immune functions among natural populations., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Polyploidy is rarer in animals than in plants. Why? Since Muller's observation in 1925, many hypotheses have been proposed and tested, but none were able to completely explain this intriguing fact. New genomic technologies enable the study of whole genomes to explain the constraints on or consequences of polyploidization, rather than focusing on specific genes or life history characteristics. Here, we review a selection of old and recent literature on polyploidy in animals, with emphasis on the consequences of polyploidization for gene expression patterns and genomic network interactions. We propose a conceptual model to contrast various scenarios for changes in genomic networks, which may serve as a framework to explain the different evolutionary dynamics of polyploidy in animals and plants. We also present new insights of genetic sex determination in animals and our emerging understanding of how animal sex determination systems may hamper or enable polyploidization, including some recent data on haplodiploids. We discuss the role of polyploidy in evolution and ecology, using a gene regulation perspective, and conclude with a synopsis regarding the effects of whole genome duplications on the balance of genomic networks. See also the sister articles focusing on plants by Ashman et al. and Madlung and Wendel in this themed issue., (Copyright © 2013 S. Karger AG, Basel.)

Seminal fluid proteins (Sfps) alter female behaviour and physiology and can mediate sexual conflict. In Drosophila melanogaster, a single Sfp, the sex peptide (SP), triggers remarkable post-mating responses in females, including altered fecundity, feeding, immunity and sexual receptivity. These effects can favour the evolutionary interests of males while generating costs in females. We tested the hypothesis that SP is an upstream master-regulator able to induce diverse phenotypes through efficient induction of widespread transcriptional changes in females. We profiled mRNA responses to SP in adult female abdomen (Abd) and head+thorax (HT) tissues using microarrays at 3 and 6 h following mating. SP elicited a rich, subtle signature of temporally and spatially controlled mRNAs. There were significant alterations to genes linked to egg development, early embryogenesis, immunity, nutrient sensing, behaviour and, unexpectedly, phototransduction. There was substantially more variation in the direction of differential expression across time points in the HT versus Abd. The results support the idea that SP is an important regulator of gene expression in females. The expression of many genes in one sex can therefore be under the influence of a regulator expressed in the other. This could influence the extent of sexual conflict both within and between loci.

Clinal variation in quantitative traits is widespread, but its genetic basis awaits identification. Drosophila melanogaster shows adaptive, clinal variation in traits such as body size along latitudinal gradients on multiple continents. To investigate genome wide transcription differentiation between North and South that might contribute to the clinal phenotypic variation, we compared RNA expression patterns during development of D. melanogaster from tropical northern and temperate southern populations using whole genome tiling arrays. We found that genes that were differentially expressed between the cline ends were generally associated with metabolism and growth, and experimental alteration of expression of a sample of them generally resulted in altered body size in the predicted direction, sometimes significantly so. We further identified the serpent (srp) transcription factor binding sites to be enriched near genes up-regulated in expression in the south. Analysis of clinal populations revealed a significant cline in the expression level of srp. Experimental over-expression of srp increased body size, as predicted from its clinal expression pattern, suggesting that it may be involved in regulating adaptive clinal variation in Drosophila. This study identified a handful of genes that contributed to clinal phenotypic variation through altered gene expression level, yet misexpression of individual gene led to modest body size change.

Individual hosts normally respond to parasite attack by launching an acute immune response (a phenotypic plastic response), while host populations can respond in the longer term by evolving higher level of defence against parasites. Little is known about the genetics of the evolved response: the identity and number of genes involved and whether it involves a pre-activation of the regulatory systems governing the plastic response. We explored these questions by surveying transcriptional changes in a Drosophila melanogaster strain artificially selected for resistance against the hymenopteran endoparasitoid Asobara tabida. Using micro-arrays, we profiled gene expression at seven time points during development (from the egg to the second instar larva) and found a large number of genes (almost 900) with altered expression levels. Bioinformatic analysis showed that some were involved in immunity or defence-associated functions but many were not. Previously, we had defined a set of genes whose level of expression changed after parasitoid attack and a comparison with the present set showed a significant though comparatively small overlap. This suggests that the evolutionary response to parasitism is not a simple pre-activation of the plastic, acute response. We also found overlap in the genes involved in the evolutionary response to parasitism and to other biotic and abiotic stressors, perhaps suggesting a 'module' of genes involved in a generalized stress response as has been found in other organisms., (© 2010 Blackwell Publishing Ltd.)

Escherichia coli DNA photolyase and cryptochrome 1 isolated from Vibrio cholerae, a member of the CRY-DASH family, are directly compared using a variety of experimental methods including UV-vis and Raman spectroscopy, reduction potential measurements, and isothermal titration calorimetry. The semiquinone form of the cryptochrome has an absorption spectrum that is red-shifted from that of the photolyase, but the Raman spectrum indicates that the FAD binding pocket is similar to that of photolyase. The FADH(-)/FADH* reduction potential of the cryptochrome is significantly higher than that of the photolyase at 164 mV vs NHE, but it also increases upon substrate binding (to 195 mV vs NHE), an increase similar to what is observed in photolyase. The FADH(-)/FADH* reduction potential for both proteins was found to be insensitive to ATP binding. Isothermal titration calorimetry found that photolyase binds tighter to substrate (K(A) approximately 10(5) M(-1) for photolyase and approximately 10(4) M(-1) for cryptochrome 1), and the binding constants for both proteins were slightly sensitive to oxidation state. Based upon this work, it appears that this cryptochrome has significant spectroscopic and electrochemical similarities to CPD photolyase. The thermodynamic cycle of the enzymatic repair in the context of this work is discussed.

Measurement of food intake in the fruit fly Drosophila melanogaster is often necessary for studies of behaviour, nutrition and drug administration. There is no reliable and agreed method for measuring food intake of flies in undisturbed, steady state, and normal culture conditions. We report such a method, based on measurement of feeding frequency by proboscis-extension, validated by short-term measurements of food dye intake. We used the method to demonstrate that (a) female flies feed more frequently than males, (b) flies feed more often when housed in larger groups and (c) fly feeding varies at different times of the day. We also show that alterations in food intake are not induced by dietary restriction or by a null mutation of the fly insulin receptor substrate chico. In contrast, mutation of takeout increases food intake by increasing feeding frequency while mutation of ovo(D) increases food intake by increasing the volume of food consumed per proboscis-extension. This approach provides a practical and reliable method for quantification of food intake in Drosophila under normal, undisturbed culture conditions.

We assessed the extent to which traits related to ejaculate investment have evolved in lines of Drosophila melanogaster that had an evolutionary history of maintenance at biased sex ratios. Measures of ejaculate investment were made in males that had been maintained at male-biased (MB) and female-biased (FB) adult sex ratios, in which levels of sperm competition were high and low, respectively. Theory predicts that when the risk of sperm competition is high and mating opportunities are rare (as they are for males in the MB populations), males should increase investment in their few matings. We therefore predicted that males from the MB lines would (1) exhibit increased investment in their first mating opportunities and (2) deplete their ejaculates at a faster rate when mating multiply, in comparison to FB males. To investigate these predictions we measured the single mating productivity of males from three replicates each of MB and FB lines mated to five wild-type virgin females in succession. In contrast to the first prediction, there was no evidence for differences in productivity between MB and FB line males in their first matings. The second prediction was upheld: mates of MB and FB males suffered increasingly reduced productivity with successive matings, but the decline was significantly more pronounced for MB than for FB males. There was a significant reduction in the size of the accessory glands and testes of males from the MB and FB regimes after five successive matings. However, the accessory glands, but not testes, of MB males became depleted at a significantly faster rate than those of FB males. The results show that male reproductive traits evolved in response to the level of sperm competition and suggest that the ability to maintain fertility over successive matings is associated with the rate of ejaculate, and particularly accessory gland, depletion.

Aggregation pheromones are used by fruit flies of the genus Drosophila to assemble on breeding substrates, where they feed, mate and oviposit communally. These pheromones consist of species-specific blends of chemicals. Here, using a phylogenetic framework, we examine how differences among species in these pheromone blends have evolved. Theoretical predictions, genetic evidence, and previous empirical analysis of bark beetle species, suggest that aggregation pheromones do not evolve gradually, but via major, saltational shifts in chemical composition. Using pheromone data for 28 species of Drosophila we show that, unlike with bark beetles, the distribution of chemical components among species is highly congruent with their phylogeny, with closely related species being more similar in their pheromone blends than are distantly related species. This pattern is also strong within the melanogaster species group, but less so within the virilis species group. Our analysis strongly suggests that the aggregation pheromones of Drosophila exhibit a gradual, not saltational, mode of evolution. We propose that these findings reflect the function of the pheromones in the ecology of Drosophila, which does not hinge on species specificity of aggregation pheromones as signals.

Background: Parasitoids are insect parasites whose larvae develop in the bodies of other insects. The main immune defense against parasitoids is encapsulation of the foreign body by blood cells, which subsequently often melanize. The capsule sequesters and kills the parasite. The molecular processes involved are still poorly understood, especially compared with insect humoral immunity., Results: We explored the transcriptional response to parasitoid attack in Drosophila larvae at nine time points following parasitism, hybridizing five biologic replicates per time point to whole-genome microarrays for both parasitized and control larvae. We found significantly different expression profiles for 159 probe sets (representing genes), and we classified them into 16 clusters based on patterns of co-expression. A series of functional annotations were nonrandomly associated with different clusters, including several involving immunity and related functions. We also identified nonrandom associations of transcription factor binding sites for three main regulators of innate immune responses (GATA/srp-like, NF-kappaB/Rel-like and Stat), as well as a novel putative binding site for an unknown transcription factor. The appearance or absence of candidate genes previously associated with insect immunity in our differentially expressed gene set was surveyed., Conclusion: Most genes that exhibited altered expression following parasitoid attack differed from those induced during antimicrobial immune responses, and had not previously been associated with defense. Applying bioinformatic techniques contributed toward a description of the encapsulation response as an integrated system, identifying putative regulators of co-expressed and functionally related genes. Genome-wide studies such as ours are a powerful first approach to investigating novel genes involved in invertebrate immunity.

Although the use of aggregation pheromones has been reported for hundreds of nonsocial arthropod species, the evolutionary ecological aspects of this behavior have received little attention. Despite the elaborate literature on mechanisms, robust data on costs and benefits of aggregation pheromones are scant. Existing literature indicates that, in contrast to the diversity of mechanisms, the ecological conditions in which aggregation pheromones are used are more alike. This points to a few general categories for costs and benefits of aggregation pheromones, and these are discussed. We subsequently review interspecific interactions that may be affected by the use of aggregation pheromones. We encounter a strikingly frequent association of aggregation pheromones with fungi and microorganisms and address cross-attraction by competitor species and exploitation by natural enemies. We show that aggregative behavior by individuals through the use of pheromones can profoundly affect ecological interactions and advocate further evolutionary and ecological investigations of pheromone-mediated aggregation.