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In large groups of vertebrates and invertebrates, aggregation can affect biological characters such as gene expression, physiological, immunological and behavioral responses. The insect cuticle is covered with hydrocarbons (cuticular hydrocarbons; CHCs) which reduce dehydration and increase protection against xenobiotics. Drosophila melanogaster and D. simulans flies also use some of their CHCs as contact pheromones. In these two sibling species, males also produce the volatile pheromone 11-cis-Vaccenyl acetate (cVa). To investigate the effect of insect density on the production of CHCs and cVa we compared the level of these male pheromones in groups of different sizes. These compounds were measured in six lines acclimated for many generations in our laboratory - four wild-type and one CHC mutant D. melanogaster lines plus one D. simulans line. Increasing the group size substantially changed pheromone amounts only in the four D. melanogaster wild-type lines. To evaluate the role of laboratory acclimation in this effect, we measured density-dependent pheromonal production in 21 lines caught in nature after 1, 12 and 25 generations in the laboratory. These lines showed varied effects which rarely persisted across generations. Although increasing group size often affected pheromone production in laboratory-established and freshly-caught D. melanogaster lines, this effect was not linear, suggesting complex determinants., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Chromosome inversions are of unique importance in the evolution of genomes and species because when heterozygous with a standard arrangement chromosome, they suppress meiotic crossovers within the inversion. In Drosophila species, heterozygous inversions also cause the interchromosomal effect, whereby the presence of a heterozygous inversion induces a dramatic increase in crossover frequencies in the remainder of the genome within a single meiosis. To date, the interchromosomal effect has been studied exclusively in species that also have high frequencies of inversions in wild populations. We took advantage of a recently developed approach for generating inversions in Drosophila simulans, a species that does not have inversions in wild populations, to ask if there is an interchromosomal effect. We used the existing chromosome 3R balancer and generated a new chromosome 2L balancer to assay for the interchromosomal effect genetically and cytologically. We found no evidence of an interchromosomal effect in D. simulans. To gain insights into the underlying mechanistic reasons, we qualitatively analyzed the relationship between meiotic double-stranded break (DSB) formation and synaptonemal complex (SC) assembly. We found that the SC is assembled prior to DSB formation as in D. melanogaster; however, we show that the SC is assembled prior to localization of the oocyte determination factor Orb, whereas in D. melanogaster, SC formation does not begin until the Orb is localized. Together, our data show no evidence that heterozygous inversions in D. simulans induce an interchromosomal effect and that there are differences in the developmental programming of the early stages of meiosis., Competing Interests: Conflicts of interest The author(s) declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Wolbachia are obligate intracellular alphaproteobacteria that enhance their spreading by altering the reproductive mechanisms of several invertebrates. Among the reproductive alterations, Wolbachia also causes cytoplasmic incompatibility that leads to embryo death when infected males are crossed with uninfected females, thus selecting infected females. However, the presence of Wolbachia has important fitness costs and infected Drosophila simulans males produce less sperm than their uninfected counterparts. Such sperm suffer, indeed, of some structural alterations that hinder their proper function. We took advantage of the fact that several sperm have abnormal distal regions of the tail, in which the plasma membrane is broken and the axonemal components splayed, making the ultrastructural aspects clearly observable. We found that axoneme reduction in the distal region of the sperm does not follow a unique pattern as observed in other insects, but occurs by losing accessory tubules or peripheral doublets. The axonemal tubules contain distinct coaxial ring-like structures that are still observed after axoneme fragmentation and form large clusters of several units., (© 2024 Wiley Periodicals LLC.)

Interspecies hybrid sterility has been extensively studied, especially in the genus Drosophila . Hybrid sterility is more often found in the heterogametic (XY or ZW) sex, a trend called Haldane's rule. Although this phenomenon is pervasive, identification of a common genetic mechanism remains elusive, with modest support found for a range of potential theories. Here, we identify a single precise morphological phenotype, which we call 'needle-eye sperm', that is associated with hybrid sterility in three separate species pairs that span the Drosophila genus. The nature of the phenotype indicates a common point of meiotic failure in sterile hybrid males. We used 10 generations of backcross selection paired with whole-genome pooled sequencing to genetically map the regions underlying the needle-eye (NE) sperm phenotype. Surprisingly, the sterility phenotype was present in ~50% of males even after 10 generations of backcrossing, and only a single region of the X chromosome was associated with sterility in one direction of backcross. Owing to the common phenotype among sterile male hybrids , and the strong effect of individual loci, further exploration of these findings may identify a universal mechanism for the evolution of hybrid sterility.

Background: Reproductive isolation can result from adaptive processes (e.g., ecological speciation and mutation-order speciation) or stochastic processes such as "system drift" model. Ecological speciation predicts barriers to gene flow between populations from different environments, but not among replicate populations from the same environment. In contrast, reproductive isolation among populations independently adapted to the same/similar environment can arise from both mutation-order speciation or system drift., Results: In experimentally evolved populations adapting to a hot environment for over 100 generations, we find evidence for pre- and postmating reproductive isolation. On one hand, an altered lipid metabolism and cuticular hydrocarbon composition pointed to possible premating barriers between the ancestral and replicate evolved populations. On the other hand, the pronounced gene expression differences in male reproductive genes may underlie the postmating isolation among replicate evolved populations adapting to the same environment with the same standing genetic variation., Conclusion: Our study confirms that replicated evolution experiments provide valuable insights into the mechanisms of speciation. The rapid emergence of the premating reproductive isolation during temperature adaptation showcases incipient ecological speciation. The potential evidence of postmating reproductive isolation among replicates gave rise to two hypotheses: (1) mutation-order speciation through a common selection on early fecundity leading to an inherent inter-locus sexual conflict; (2) system drift with genetic drift along the neutral ridges., (© 2024. The Author(s).)

Animals employ different strategies to establish mating boundaries between closely related species, with sex pheromones often playing a crucial role in identifying conspecific mates. Many of these pheromones have carbon-carbon double bonds, making them vulnerable to oxidation by certain atmospheric oxidant pollutants, including ozone. Here, we investigate whether increased ozone compromises species boundaries in drosophilid flies. We show that short-term exposure to increased levels of ozone degrades pheromones of Drosophila melanogaster, D. simulans, D. mauritiana, as well as D. sechellia, and induces hybridization between some of these species. As many of the resulting hybrids are sterile, this could result in local population declines. However, hybridization between D. simulans and D. mauritiana as well as D. simulans and D. sechellia results in fertile hybrids, of which some female hybrids are even more attractive to the males of the parental species. Our experimental findings indicate that ozone pollution could potentially induce breakdown of species boundaries in insects., (© 2024. The Author(s).)

Most traits are polygenic, and the contributing loci can be identified by genome-wide association studies. The genetic basis of adaptation (adaptive architecture) is, however, difficult to characterize. Here, we propose to study the adaptive architecture of traits by monitoring the evolution of their phenotypic variance during adaptation to a new environment in well-defined laboratory conditions. Extensive computer simulations show that the evolution of phenotypic variance in a replicated experimental evolution setting can distinguish between oligogenic and polygenic adaptive architectures. We compared gene expression variance in male Drosophila simulans before and after 100 generations of adaptation to a novel hot environment. The variance change in gene expression was indistinguishable for genes with and without a significant change in mean expression after 100 generations of evolution. We suggest that the majority of adaptive gene expression evolution can be explained by a polygenic architecture. We propose that tracking the evolution of phenotypic variance across generations can provide an approach to characterize the adaptive architecture., Competing Interests: Conflict of Interest None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Background: The evolution of genomic regulatory regions plays a critical role in shaping the diversity of life. While this process is primarily sequence-dependent, the enormous complexity of biological systems complicates the understanding of the factors underlying regulation and its evolution. Here, we apply deep neural networks as a tool to investigate the sequence determinants underlying chromatin accessibility in different species and tissues of Drosophila., Results: We train hybrid convolution-attention neural networks to accurately predict ATAC-seq peaks using only local DNA sequences as input. We show that our models generalize well across substantially evolutionarily diverged species of insects, implying that the sequence determinants of accessibility are highly conserved. Using our model to examine species-specific gains in accessibility, we find evidence suggesting that these regions may be ancestrally poised for evolution. Using in silico mutagenesis, we show that accessibility can be accurately predicted from short subsequences in each example. However, in silico knock-out of these sequences does not qualitatively impair classification, implying that accessibility is mutationally robust. Subsequently, we show that accessibility is predicted to be robust to large-scale random mutation even in the absence of selection. Conversely, simulations under strong selection demonstrate that accessibility can be extremely malleable despite its robustness. Finally, we identify motifs predictive of accessibility, recovering both novel and previously known motifs., Conclusions: These results demonstrate the conservation of the sequence determinants of accessibility and the general robustness of chromatin accessibility, as well as the power of deep neural networks to explore fundamental questions in regulatory genomics and evolution., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Larval crowding is one common ecological stressor for many insect species. In Drosophila , high larval density alters multiple widely-studied phenotypes including life-history traits, morphology and behavior. Nevertheless, we still miss a holistic view of the full range of phenotypic changes and the underlying molecular mechanisms. In this study, we analyzed the adult transcriptomes of high and low larval density fly cohorts, and highlighted the molecular basis of the plastic traits. Increased cellular energy metabolism and locomotion, along with reduced reproductive investment, are key responses to high larval density. Moreover, we compared the expression changes among cohorts with different developmental delays caused by larval crowding. The majority of genes induced by larval crowding showed the strongest expression alterations in cohorts with intermediate delay. Furthermore, linear expression changes were observed in genes related to nutrition and detoxification. Comparing different high-density cohorts could provide insights into the varied responses to distinct larval crowding-induced stresses such as space competition, food degradation and waste accumulation., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

The relationship between cytoplasmic incompatibility and the obligate intracellular alphaproteobacteria Wolbachia has for a long time been reported. Although the molecular mechanisms responsible for this reproductive alteration are beginning to be understood, the effects of Wolbachia on germ cell structure and dynamics have not yet been fully investigated. We report here that the presence of Wolbachia in infected cysts of elongating spermatids is associated with major structural defects that become more evident in mature sperm. We find mitochondrial defects, an improper axoneme structure, reduced sperm numbers, and individualization failures. The large heterogeneous variety of the ultrastructural defects found in elongating spermatids and mature sperm provide the first cytological evidence for the reduced fertility associated with Wolbachia infection in Drosophila simulans males. The observed abnormalities could be the result of the mechanical stress induced by the high bacteria numbers during the process of spermatid elongation, rather than the result of the released factors affecting the proper morphogenesis of the germ cells. Moreover, high Wolbachia densities in male germ cells may not be appropriate for causing cytoplasmic incompatibility as the bacteria are harmful for spermatid differentiation, leading to abnormal sperm that is unlikely to be functional.

The recent evolutionary history of the Y chromosome in Drosophila simulans, a worldwide species of Afrotropical origin, is closely linked to that of X-linked meiotic drivers (Paris system). The spread of the Paris drivers in natural populations has elicited the selection of drive-resistant Y chromosomes. To infer the evolutionary history of the Y chromosome in relation to the Paris drive, we sequenced 21 iso-Y lines, each carrying a Y chromosome from a different location. Among them, 13 lines carry a Y chromosome that is able to counteract the effect of the drivers. Despite their very different geographical origins, all sensitive Y's are highly similar, suggesting that they share a recent common ancestor. The resistant Y chromosomes are more divergent and segregate in four distinct clusters. The phylogeny of the Y chromosome confirms that the resistant lineage predates the emergence of Paris drive. The ancestry of the resistant lineage is further supported by the examination of Y-linked sequences in the sister species of D. simulans, Drosophila sechellia and Drosophila mauritiana. We also characterized the variation in repeat content among Y chromosomes and identified multiple simple satellites associated with resistance. Altogether, the molecular polymorphism allows us to infer the demographic and evolutionary history of the Y chromosome and provides new insights on the genetic basis of resistance., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Although the biological utilities of endogenous RNAi (endo-RNAi) have been largely elusive, recent studies reveal its critical role in the non-model fruitfly Drosophila simulans to suppress selfish genes, whose unchecked activities can severely impair spermatogenesis. In particular, hairpin RNA (hpRNA) loci generate endo-siRNAs that suppress evolutionary novel, X-linked, meiotic drive loci. The consequences of deleting even a single hpRNA (Nmy) in males are profound, as such individuals are nearly incapable of siring male progeny. Here, comparative genomic analyses of D. simulans and D. melanogaster mutants of the core RNAi factor dcr-2 reveal a substantially expanded network of recently-emerged hpRNA-target interactions in the former species. The de novo hpRNA regulatory network in D. simulans provides insight into molecular strategies that underlie hpRNA emergence and their potential roles in sex chromosome conflict. In particular, our data support the existence of ongoing rapid evolution of Nmy/Dox-related networks, and recurrent targeting of testis HMG-box loci by hpRNAs. Importantly, the impact of the endo-RNAi network on gene expression flips the convention for regulatory networks, since we observe strong derepression of targets of the youngest hpRNAs, but only mild effects on the targets of the oldest hpRNAs. These data suggest that endo-RNAi are especially critical during incipient stages of intrinsic sex chromosome conflicts, and that continual cycles of distortion and resolution may contribute to speciation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Vedanayagam et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Most organismal phenotypes have a polygenic basis, which enables adaptive phenotypic responses on ecological time scales. While adaptive phenotypic changes are highly parallel in replicate populations, this does not apply to the contributing loci. In particular for small populations, the same phenotypic shift can be fueled by different sets of alleles at alternative loci (genetic redundancy). Although this phenomenon is empirically well supported, the molecular basis of the genetic redundancy is not yet understood. To fill this gap, we compared the heterogeneity of the evolutionary transcriptomic and metabolomic response in ten Drosophila simulans populations which evolved parallel high-level phenotypic changes in a novel temperature environment but used different allelic combinations of alternative loci. We showed that the metabolome evolved more parallel than the transcriptome, confirming a hierarchical organization of molecular phenotypes. Different sets of genes responded in each evolved population but led to the enrichment of similar biological functions and a consistent metabolic profile. Since even the metabolomic response was still highly heterogeneous across evolved populations, we propose that selection may operate on pathways/networks., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Understanding how species adapt to different temperatures is crucial to predict their response to global warming, and thermal performance curves (TPCs) have been employed recurrently to study this topic. Nevertheless, fundamental questions regarding how thermodynamic constraints and evolution interact to shape TPCs in lineages inhabiting different environments remain unanswered. Here, we study Drosophila simulans along a latitudinal gradient spanning 3000 km to test opposing hypotheses based on thermodynamic constrains ( hotter-is-better ) versus biochemical adaptation ( jack-of-all-temperatures ) as primary determinants of TPCs variation across populations. We compare thermal responses in metabolic rate and the egg-to-adult survival as descriptors of organismal performance and fitness, respectively, and show that different descriptors of TPCs vary in tandem with mean environmental temperatures, providing strong support to hotter-is-better . Thermodynamic constraints also resulted in a strong negative association between maximum performance and thermal breadth. Lastly, we show that descriptors of TPCs for metabolism and egg-to-adult survival are highly correlated, providing evidence of co-adaptation, and that curves for egg-to-adult survival are systematically narrower and displaced toward lower temperatures. Taken together, our results support the pervasive role of thermodynamics constraining thermal responses in Drosophila populations along a latitudinal gradient, that are only partly compensated by evolutionary adaptation.

Cases of horizontal transfer of transposable elements (TEs) between species are known for the Drosophilidae family. In the middle of the last century, the case of horizontal transfer of the P-element from the Drosophila willistoni to the D. melanogaster was described. A novel P-element invasion into the D. simulans genome from D. melanogaster occurred approximately 10 years ago. Currently, the P-element has spread across all D. melanogaster population and 30% of D. simulans populations in Europe, Africa and America. In this paper, we investigated the presence of the P-element in D. simulans lines caught in different years in three Asian populations (Tashkent, Nalchik and Sakhalin Island). We also examined the physiological characteristics (cytotype, lifespan, fecundity and locomotor activity) of D. simulans lines with and without the P-element to determine the significance of this new mobile element in the genome. The P-element was found in lines isolated from nature after 2012. The number of P-element copies per genome (two-to-three dozen according to fluorescence in situ hybridization data) was greater than in the American and comparable to the African populations. There were signs of intraspecific hybrid dysgenesis for some pairs of lines. However, in general the presence of the P-element did not adversely affect the physiological characteristics. Either adaptation to the new TE occurs very quickly, or the rate of movement of the P-element is so insignificant that its appearance in the genome remains unnoticed.

Small RNAs produced from transposable element (TE)-rich sections of the genome, termed piRNA clusters, are a crucial component in the genomic defence against selfish DNA. In animals, it is thought the invasion of a TE is stopped when a copy of the TE inserts into a piRNA cluster, triggering the production of cognate small RNAs that silence the TE. Despite this importance for TE control, little is known about the evolutionary dynamics of piRNA clusters, mostly because these repeat-rich regions are difficult to assemble and compare. Here, we establish a framework for studying the evolution of piRNA clusters quantitatively. Previously introduced quality metrics and a newly developed software for multiple alignments of repeat annotations (Manna) allow us to estimate the level of polymorphism segregating in piRNA clusters and the divergence among homologous piRNA clusters. By studying 20 conserved piRNA clusters in multiple assemblies of four Drosophila species, we show that piRNA clusters are evolving rapidly. While 70%-80% of the clusters are conserved within species, the clusters share almost no similarity between species as closely related as D. melanogaster and D. simulans. Furthermore, abundant insertions and deletions are segregating within the Drosophila species. We show that the evolution of clusters is mainly driven by large insertions of recently active TEs and smaller deletions mostly in older TEs. The effect of these forces is so rapid that homologous clusters often do not contain insertions from the same TE families., (© 2021 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Physiological responses to environmental changes play important roles in adaptive evolution. In particular, homeostatic regulatory systems that maintain constant circulating glucose levels are crucial in animals. However, variation in circulating glucose levels and the genetic effects on phenotypic variation in natural populations remain to be clarified. Here, we investigated the hemolymph glucose levels in natural populations of Drosophila melanogaster and its sibling species, D. simulans, in Japan. We quantified hemolymph glucose concentrations in third instar larvae of 27 lines for each species, which were reared on either glucose-free or glucose-rich food. In both species, genetic variation was not a major component of phenotypic variation on either glucose-free or glucose-rich food. The hemolymph glucose concentrations were much higher in D. simulans than in D. melanogaster. Genetic variance was larger in D. simulans than in D. melanogaster. The observed differences between the two species may be associated with the much more recent colonization history of D. simulans populations in Japan and/or the tolerance to environmental stresses. Our findings suggest that natural selection acting on hemolymph glucose levels in D. melanogaster is different from that in D. simulans.

Acoustic communication signals diversify even on short evolutionary time scales. To understand how the auditory system underlying acoustic communication could evolve, we conducted a systematic comparison of the early stages of the auditory neural circuit involved in song information processing between closely-related fruit-fly species. Male Drosophila melanogaster and D. simulans produce different sound signals during mating rituals, known as courtship songs. Female flies from these species selectively increase their receptivity when they hear songs with conspecific temporal patterns. Here, we firstly confirmed interspecific differences in temporal pattern preferences; D. simulans preferred pulse songs with longer intervals than D. melanogaster. Primary and secondary song-relay neurons, JO neurons and AMMC-B1 neurons, shared similar morphology and neurotransmitters between species. The temporal pattern preferences of AMMC-B1 neurons were also relatively similar between species, with slight but significant differences in their band-pass properties. Although the shift direction of the response property matched that of the behavior, these differences are not large enough to explain behavioral differences in song preferences. This study enhances our understanding of the conservation and diversification of the architecture of the early-stage neural circuit which processes acoustic communication signals., (© 2023. The Author(s).)

Transposable elements (TEs) are abundant in genomes. Their mobilization can lead to genetic variability that is useful for evolution, but can also have deleterious biological effects. Somatic mobilization (SM) has been linked to degenerative diseases, such as Alzheimer's disease and cancer. We used a Drosophila simulans strain, in which SM can be measured by counting red spots in the eyes, to investigate how chemotherapeutic agents affect expression and SM of the mariner TE. Flies were treated with Cisplatin, Dacarbazine, and Daunorubicin. After acute exposure, relative expression of mariner was quantified by RT-qPCR and oxidative stress was measured by biochemical assays. Exposure to 50 and 100 µg/mL Cisplatin increased mariner expression and ROS levels; catalase activity increased at 100 µg/mL. With chronic exposure, the number of spots also increased, indicating higher mariner SM. Dacarbazine (50 and 100 µg/mL) did not significantly alter mariner expression or mobilization or ROS levels, but decreased catalase activity (100 µg/mL). Daunorubicin (25 and 50 µM) increased mariner expression, but decreased mariner SM. ROS and catalase activity were also reduced. Our data suggest that stress factors may differentially affect the expression and SM of TEs. The increase in mariner transposase gene expression is necessary, but not sufficient for mariner SM.

Clarifying the mechanisms underlying microbial community assembly from regional microbial pools is a central issue of microbial ecology, but remains largely unexplored. Here, we investigated the gut bacterial and fungal microbiome assembly processes and potential sources in Drosophila simulans and Dicranocephalus wallichii bowringi , two wild, sympatric insect species that share a common diet of waxberry. While some convergence was observed, the diversity, composition, and network structure of the gut microbiota significantly differed between these two host species. Null model analyses revealed that stochastic processes (e.g., drift, dispersal limitation) play a principal role in determining gut microbiota from both hosts. However, the strength of each ecological process varied with the host species. Furthermore, the source-tracking analysis showed that only a minority of gut microbiota within D. simulans and D. wallichii bowringi are drawn from a regional microbial pool from waxberries, leaves, or soil. Results from function prediction implied that host species-specific gut microbiota might arise partly through host functional requirement and specific selection across host-microbiota coevolution. In conclusion, our findings uncover the importance of community assembly processes over regional microbial pools in shaping sympatric insect gut microbiome structure and function., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. iMeta published by John Wiley & Sons Australia, Ltd on behalf of iMeta Science.)

Wolbachia , a vertically transmitted endosymbiont infecting many insects, spreads rapidly through uninfected populations by a mechanism known as cytoplasmic incompatibility (CI). In CI, a paternally delivered modification of the sperm leads to chromatin defects and lethality during and after the first mitosis of embryonic development in multiple species. However, whether CI-induced defects in later stage embryos are a consequence of the first division errors or caused by independent defects remains unresolved. To address this question, we focused on ~1/3 of embryos from CI crosses in Drosophila simulans that develop apparently normally through the first and subsequent pre-blastoderm divisions before exhibiting mitotic errors during the mid-blastula transition and gastrulation. We performed single embryo PCR and whole genome sequencing to find a large percentage of these developed CI-derived embryos bypass the first division defect. Using fluorescence in situ hybridization, we find increased chromosome segregation errors in gastrulating CI-derived embryos that had avoided the first division defect. Thus, Wolbachia action in the sperm induces developmentally deferred defects that are not a consequence of the first division errors. Like the immediate defect, the delayed defect is rescued through crosses to infected females. These studies inform current models on the molecular and cellular basis of CI., Competing Interests: BW, ST, MA, GV, NL, KH, JM, WS No competing interests declared, (© 2022, Warecki, Titen et al.)

Seasonally polyphenic types have been documented in many Drosophilids, which differ significantly during thermal stress. Although Drosophila simulans is a sibling species to Drosophila melanogaster, both thrive in the temperate and tropical climates, but various climatic factors are expected to impact their distribution and abundance. As a result, D. simulans may use phenotypic plasticity to adapt to colder and drier circumstances in temperate zones, although such studies are less known. In the present study, our main aim was to find a link between adaptive plasticity and thermal tolerance in D. simulans. We characterized two morphs in D. simulans flies based on the abdominal melanization collected from the same locality and season, as this trait is highly associated with the larval developmental conditions. Our results suggested that flies reared from dark and light morph showed significant differences in the basal level of proline, carbohydrates (trehalose, glycogen), and lipids (cuticular lipids and total body lipids) within simulated seasons and morph lineages in D. simulans flies. We further showed that D. simulans reared from dark morph are better adapted to cold conditions, whereas light flies are more adapted to warm conditions. The flies, both from light and dark morph lineages, when reared at 15 °C, showed an increase in the level of total body lipids after acclimation at 0 °C but a decrease in the level of proline and carbohydrates (trehalose, glycogen). Heat acclimation increases glycogen levels in the flies from light morph lineage while decreases trehalose and proline., (Copyright © 2022. Published by Elsevier Inc.)

The popular trap model assumes that the invasions of transposable elements (TEs) in mammals and invertebrates are stopped by piRNAs that emerge after insertion of the TE into a piRNA cluster. It remains, however, still unclear which factors influence the dynamics of TE invasions. The activity of the TE (i.e., transposition rate) is one frequently discussed key factor. Here we take advantage of the temperature-dependent activity of the P-element, a widely studied eukaryotic TE, to test how TE activity affects the dynamics of a TE invasion. We monitored P-element invasion dynamics in experimental Drosophila simulans populations at hot and cold culture conditions. Despite marked differences in transposition rates, the P-element reached very similar copy numbers at both temperatures. The reduction of the insertion rate upon approaching the copy number plateau was accompanied by similar amounts of piRNAs against the P-element at both temperatures. Nevertheless, we also observed fewer P-element insertions in piRNA clusters than expected, which is not compatible with a simple trap model. The ping-pong cycle, which degrades TE transcripts, becomes typically active after the copy number plateaued. We generated a model, with few parameters, that largely captures the observed invasion dynamics. We conclude that the transposition rate has at the most only a minor influence on TE abundance, but other factors, such as paramutations or selection against TE insertions are shaping the TE composition., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Mixed-species groups describe active associations among individuals of 2 or more species at the same trophic level. Mixed-species groups are important to key ecological and evolutionary processes such as competition and predation, and research that ignores the presence of other species risks ignoring a key aspect of the environment in which social behavior is expressed and selected. Despite the defining emphasis of active formation for mixed-species groups, surprisingly little is known about the mechanisms by which mixed-species groups form. Furthermore, insects have been almost completely ignored in the study of mixed-species groups, despite their taxonomic importance and relative prominence in the study of single-species groups. Here, we measured group formation processes in Drosophila melanogaster and its sister species, Drosophila simulans. Each species was studied alone, and together, and one population of D. melanogaster was also studied both alone and with another, phenotypically distinct D. melanogaster population, in a nested-factorial design. This approach differs from typical methods of studying mixed-species groups in that we could quantitatively compare group formation between single-population, mixed-population, and mixed-species treatments. Surprisingly, we found no differences between treatments in the number, size, or composition of groups that formed, suggesting that single- and mixed-species groups form through similar mechanisms of active attraction. However, we found that mixed-species groups showed elevated interspecies male-male interactions, relative to interpopulation or intergenotype interactions in single-species groups. Our findings expand the conceptual and taxonomic study of mixed-species groups while raising new questions about the mechanisms of group formation broadly., (© The American Genetic Association. 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Shifts in trait means are widely considered as evidence for adaptive responses, but the impact on phenotypic variance remains largely unexplored. Classic quantitative genetics provides a theoretical framework to predict how selection on phenotypic mean affects the variance. In addition to this indirect effect, it is also possible that the variance of the trait is the direct target of selection, but experimentally characterized cases are rare. Here, we studied gene expression variance of Drosophila simulans males before and after 100 generations of adaptation to a novel hot laboratory environment. In each of the two independently evolved populations, the variance of 125 and 97 genes was significantly reduced. We propose that the drastic loss in environmental complexity from nature to the laboratory may have triggered selection for reduced variance. Our observation that selection could drive changes in the variance of gene expression could have important implications for studies of adaptation processes in natural and experimental populations., (© 2021 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Molecular evolutionary studies usually focus on genes with clear roles in adult fitness or on developmental genes expressed at multiple time points during the life of the organism. Here, we examine the evolutionary dynamics of Drosophila glue genes, a set of eight genes tasked with a singular primary function during a specific developmental stage: the production of glue that allows animal pupa to attach to a substrate for several days during metamorphosis. Using phenotypic assays and available data from transcriptomics, PacBio genomes, and sequence variation from global populations, we explore the selective forces acting on glue genes within the cosmopolitan Drosophila melanogaster species and its five closely related species, D. simulans, D. sechellia, D. mauritiana, D. yakuba, and D. teissieri. We observe a three-fold difference in glue adhesion between the least and the most adhesive D. melanogaster strain, indicating a strong genetic component to phenotypic variation. These eight glue genes are among the most highly expressed genes in salivary glands yet they display no notable codon bias. New copies of Sgs3 and Sgs7 are found in D. yakuba and D. teissieri with the Sgs3 coding sequence evolving rapidly after duplication in the D. yakuba branch. Multiple sites along the various glue genes appear to be constrained. Our population genetics analysis in D. melanogaster suggests signals of local adaptive evolution for Sgs3, Sgs5, and Sgs5bis and traces of selective sweeps for Sgs1, Sgs3, Sgs7, and Sgs8. Our work shows that stage-specific genes can be subjected to various dynamic evolutionary forces., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The endosymbiont bacteria of the genus Wolbachia are associated with multiple mutualistic effects on insect biology, including nutritional and antiviral properties. Members of the genus Wolbachia naturally occur in fly species of the genus Drosophila , providing an operational model host for studying how virome composition may be affected by its presence. Drosophila simulans populations can carry a variety of strains of members of the genus Wolbachia , with the w Au strain associated with strong antiviral protection under experimental conditions. We used D. simulans sampled from the Perth Hills, Western Australia, to investigate the potential virus protective effect of the w Au strain of Wolbachia on individual wild-caught flies. Our data revealed no appreciable variation in virus composition and abundance between individuals infected or uninfected with Wolbachia associated with the presence or absence of w Au. However, it remains unclear whether w Au might affect viral infection and host survival by increasing tolerance rather than inducing complete resistance. These data also provide new insights into the natural virome diversity of D. simulans . Despite the small number of individuals sampled, we identified a repertoire of RNA viruses, including nora virus, galbut virus, thika virus and La Jolla virus, that have been identified in other species of the genus Drosophila . Chaq virus-like sequences associated with galbut virus were also detected. In addition, we identified five novel viruses from the families Reoviridae , Tombusviridae , Mitoviridae and Bunyaviridae . Overall, this study highlights the complex interaction between Wolbachia and RNA virus infections and provides a baseline description of the natural virome of D. simulans .

Population genetics studies often make use of a class of nucleotide site free from selective pressures, in order to make inferences about population size changes or natural selection at other sites. If such neutral sites can be identified, they offer the opportunity to avoid any confounding effects of selection. Here, we investigate evolution at putatively neutrally evolving short intronic sites in natural populations of Drosophila melanogaster and Drosophila simulans, in order to understand the properties of spontaneous mutations and the extent of GC-biased gene conversion in these species. Use of data on the genetics of natural populations is advantageous because it integrates information from large numbers of individuals over long timescales. In agreement with direct evidence from observations of spontaneous mutations in Drosophila, we find a bias in the spectrum of mutations toward AT basepairs. In addition, we find that this bias is stronger in the D. melanogaster lineage than in the D. simulans lineage. The evidence for GC-biased gene conversion in Drosophila has been equivocal. Here, we provide evidence for a weak force favoring GC in both species, which is correlated with the GC content of introns and is stronger in D. simulans than in D. melanogaster., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

In this article, a biallelic reversible mutation model with linear and quadratic selection is analysed. The approach reconnects to one proposed by Kimura (1979), who starts from a diffusion model and derives its equilibrium distribution up to a constant. We use a boundary-mutation Moran model, which approximates a general mutation model for small effective mutation rates, and derive its equilibrium distribution for polymorphic and monomorphic variants in small to moderately sized populations. Using this model, we show that biased mutation rates and linear selection alone can cause patterns of polymorphism within and substitution rates between populations that are usually ascribed to balancing or overdominant selection. We illustrate this using a data set of short introns and fourfold degenerate sites from Drosophila simulans and Drosophila melanogaster., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Surface chemical compounds are key components of survival and reproduction in many species. Cuticular hydrocarbons (CHCs) are chemical compounds produced by all insects that are used for both desiccation resistance and chemical communication, including communication related to mating. In the species pair of Drosophila melanogaster and D. simulans , female CHCs stimulate conspecific males to mate and repel heterospecific males. While CHCs are a critical contributor to both reproductive success within a species and isolation between species, few genes underlying species variation in CHC profiles are known. Here, we use genetic mapping of the 3rd chromosome to test a suite of candidate genes for interspecies variation in CHCs. Candidate gene CG5946 was found to be involved in species differences in the production of 7,11-heptacosadiene and 7-tricosene between D. melanogaster and D. simulans . This is therefore a new candidate locus contributing to species-specific variation in the CHC profile. In the process of mapping genes for CHCs, we also identified 29 candidate genes for the reduced survival or inviability of interspecies hybrids.

Sexual selection is thought to be responsible for the rapid divergent evolution of male genitalia with several studies detecting multivariate sexual selection on genital form. However, in most cases, selection is only estimated during a single episode of selection, which provides an incomplete view of net selection on genital traits. Here, we estimate the strength and form of multivariate selection on the genitalia arch of Drosophila simulans when mating occurs in the absence of a competitor and during sperm competition, in both sperm defence and offense roles (i.e., when mating first and last). We found that the strength of sexual selection on the genital arch was strongest during noncompetitive mating and weakest during sperm offense. However, the direction of selection was similar across selection episodes with no evidence for antagonistic selection. Overall, selection was not particularly strong despite genitals clearly evolving rapidly in this species., (© 2021 The Authors. Evolution © 2021 The Society for the Study of Evolution.)

High saturated fat, sugar, and salt contents are a staple of a Western diet (WD), contributing to obesity, metabolic syndrome, and a plethora of other health risks. However, the combinatorial effects of these ingredients have not been fully evaluated. Here, using the wild-caught Drosophila simulans, we show that a diet enriched with saturated fat, sugar, and salt is more detrimental than each ingredient separately, resulting in a significantly decreased lifespan, locomotor activity, sleep, reproductive function, and mitochondrial function. These detrimental effects were more pronounced in female than in male flies. Adding regular flight exercise to flies on the WD markedly negated the adverse effects of a WD. At the molecular level, the WD significantly increased levels of triglycerides and caused mitochondrial dysfunction, while exercise counterbalanced these effects. Interestingly, fruit flies developed a preference for the WD after pre-exposure, which was averted by flight exercise. The results demonstrate that regular aerobic exercise can mitigate adverse dietary effects on fly mitochondrial function, physiology, and feeding behavior. Our data establish Drosophila simulans as a novel model of diet-exercise interaction that bears a strong similarity to the pathophysiology of obesity and eating disorders in humans., Competing Interests: The authors declare no competing financial interests., (©2020 The Authors. FASEB BioAdvances published by The Federation of American Societies for Experimental Biology.)

Within populations, adult sex ratios influence population growth and extinction risk, mating behaviours and parental care. Sex ratio adjustment can also have pronounced effects on individual fitness. Accordingly, it is important that we understand how often, and why, offspring sex ratios deviate from parity. In Drosophila melanogaster, females appear to improve their fitness by producing fewer sons when paired with older males. However, facultative sex ratio adjustment in D. melanogaster is controversial, and our understanding of how sex ratio skew affects fitness is hampered by pronounced sexual conflict in this species. Additionally, it is unclear whether maternal age or quality interacts with paternal age to influence offspring sex ratios. Here, we test whether offspring sex ratios vary as a function of maternal quality, and maternal and paternal age in Drosophila simulans, a sister species of D. melanogaster that lacks overt sexual conflict. We find that offspring sex ratios are slightly male-biased overall, but constant across the female life course, and independent of female quality, or paternal age. To really understand if, how and when females skew offspring sex ratios, we need studies linking offspring sex ratios to paternal and maternal phenotypes that are predicted to shift optimal investment in sons and daughters., (© 2020 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2020 European Society For Evolutionary Biology.)

Organisms are exposed to temperatures that vary, for example on diurnal and seasonal time scales. Thus, the ability to behaviorally and/or physiologically respond to variation in temperatures is a fundamental requirement for long-term persistence. Studies on thermal biology in ectotherms are typically performed under constant laboratory conditions, which differ markedly from the variation in temperature across time and space in nature. Here, we investigate evolutionary adaptation and environmentally induced plastic responses of Drosophila simulans to no fluctuations (constant), predictable fluctuations or unpredictable fluctuations in temperature. We whole-genome sequenced populations exposed to 20 generations of experimental evolution under the three thermal regimes and examined the proteome after short-term exposure to the same three regimes. We find that unpredictable fluctuations cause the strongest response at both genome and proteome levels. The loci showing evolutionary responses were generally unique to each thermal regime, but a minor overlap suggests either common laboratory adaptation or that some loci were involved in the adaptation to multiple thermal regimes. The evolutionary response, i.e., loci under selection, did not coincide with induced responses of the proteome. Thus, genes under selection in fluctuating thermal environments are distinct from genes important for the adaptive plastic response observed within a generation. This information is key to obtain a better understanding and prediction of the effects of future increases in both mean and variability of temperatures., (Copyright © 2020 Sørensen, Manenti, Bechsgaard, Schou, Kristensen and Loeschcke.)

Experimental evolution is becoming a popular approach to study the genomic selection response of evolving populations. Computer simulation studies suggest that the accuracy of the signature increases with the duration of the experiment. Since some assumptions of the computer simulations may be violated, it is important to scrutinize the influence of the experimental duration with real data. Here, we use a highly replicated Evolve and Resequence study in Drosophila simulans to compare the selection targets inferred at different time points. At each time point, approximately the same number of SNPs deviates from neutral expectations, but only 10% of the selected haplotype blocks identified from the full data set can be detected after 20 generations. Those haplotype blocks that emerge already after 20 generations differ from the others by being strongly selected at the beginning of the experiment and display a more parallel selection response. Consistent with previous computer simulations, our results demonstrate that only Evolve and Resequence experiments with a sufficient number of generations can characterize complex adaptive architectures., (© 2020 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Transposable elements (TEs) are the main components of genomes. However, due to their repetitive nature, they are very difficult to study using data obtained with short-read sequencing technologies. Here, we describe an efficient pipeline to accurately recover TE insertion (TEI) sites and sequences from long reads obtained by Oxford Nanopore Technology (ONT) sequencing. With this pipeline, we could precisely describe the landscapes of the most recent TEIs in wild-type strains of Drosophila melanogaster and Drosophila simulans . Their comparison suggests that this subset of TE sequences is more similar than previously thought in these two species. The chromosome assemblies obtained using this pipeline also allowed recovering piRNA cluster sequences, which was impossible using short-read sequencing. Finally, we used our pipeline to analyze ONT sequencing data from a D. melanogaster unstable line in which LTR transposition was derepressed for 73 successive generations. We could rely on single reads to identify new insertions with intact target site duplications. Moreover, the detailed analysis of TEIs in the wild-type strains and the unstable line did not support the trap model claiming that piRNA clusters are hotspots of TE insertions.

Female mate rejection acts as a major selective force within species, and can serve as a reproductive barrier between species. In spite of its critical role in fitness and reproduction, surprisingly little is known about the genetic or neural basis of variation in female mate choice. Here, we identify fruitless as a gene affecting female receptivity within Drosophila melanogaster , as well as female Drosophila simulans rejection of male D. melanogaster . Of the multiple transcripts this gene produces, by far the most widely studied is the sex-specifically spliced transcript involved in the sex determination pathway. However, we find that female rejection behaviour is affected by a non-sex-specifically spliced fruitless transcript. This is the first implication of fruitless in female behaviour, and the first behavioural role identified for a fruitless non-sex-specifically spliced transcript. We found that this locus does not influence preferences via a single sensory modality, examining courtship song, antennal pheromone perception, or perception of substrate vibrations, and we conclude that fruitless influences mate choice via the integration of multiple signals or through another sensory modality.

Transposable elements are abundant, dynamic components of the genome that affect organismal phenotypes and fitness. In Drosophila melanogaster , they have increased in abundance as the species spread out of Africa, and different populations differ in their transposable element content. However, very little is currently known about how transposable elements differ between individual genotypes, and how that relates to the population dynamics of transposable elements overall. The sister species of D. melanogaster , D. simulans , has also recently become cosmopolitan, and panels of inbred genotypes exist from cosmopolitan and African flies. Therefore, we can determine whether the differences in colonizing populations are repeated in D. simulans , what the dynamics of transposable elements are in individual genotypes, and how that compares to wild flies. After estimating copy number in cosmopolitan and African D. simulans , I find that transposable element load is higher in flies from cosmopolitan populations. In addition, transposable element load varies considerably between populations, between genotypes, but not overall between wild and inbred lines. Certain genotypes either contain active transposable elements or are more permissive of transposition and accumulate copies of particular transposable elements. Overall, it is important to quantify genotype-specific transposable element dynamics as well as population averages to understand the dynamics of transposable element accumulation over time., Competing Interests: None declared., (© 2020 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Evolve and resequencing (E&R) studies investigate the genomic responses of adaptation during experimental evolution. Because replicate populations evolve in the same controlled environment, consistent responses to selection across replicates are frequently used to identify reliable candidate regions that underlie adaptation to a new environment. However, recent work demonstrated that selection signatures can be restricted to one or a few replicate(s) only. These selection signatures frequently have weak statistical support, and given the difficulties of functional validation, additional evidence is needed before considering them as candidates for functional analysis. Here, we introduce an experimental procedure to validate candidate loci with weak or replicate-specific selection signature(s). Crossing an evolved population from a primary E&R experiment to the ancestral founder population reduces the frequency of candidate alleles that have reached a high frequency. We hypothesize that genuine selection targets will experience a repeatable frequency increase after the mixing with the ancestral founders if they are exposed to the same environment (secondary E&R experiment). Using this approach, we successfully validate two overlapping selection targets, which showed a mutually exclusive selection signature in a primary E&R experiment of Drosophila simulans adapting to a novel temperature regime. We conclude that secondary E&R experiments provide a reliable confirmation of selection signatures that either are not replicated or show only a low statistical significance in a primary E&R experiment unless epistatic interactions predominate. Such experiments are particularly helpful to prioritize candidate loci for time-consuming functional follow-up investigations., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The pervasive occurrence of sexual dimorphism demonstrates different adaptive strategies of males and females. While different reproductive strategies of the two sexes are well-characterized, very little is known about differential functional requirements of males and females in their natural habitats. Here, we study the impact environmental change on the selection response in both sexes. Exposing replicated Drosophila populations to a novel temperature regime, we demonstrate sex-specific changes in gene expression, metabolic and behavioral phenotypes in less than 100 generations. This indicates not only different functional requirements of both sexes in the new environment but also rapid sex-specific adaptation. Supported by computer simulations we propose that altered sex-biased gene regulation from standing genetic variation, rather than new mutations, is the driver of rapid sex-specific adaptation. Our discovery of environmentally driven divergent functional requirements of males and females has important implications-possibly even for gender aware medical treatments., Competing Interests: SH, AJ, VN, ML, RK, NB, EV, CS No competing interests declared, (© 2020, Hsu et al.)

When two species interbreed, the resulting hybrid offspring are often sterile, with the heterogametic (e.g. XY) hybrid usually being more severely affected. The prevailing theory for this pattern of sterility evokes divergent changes in separate lineages having maladaptive interactions when placed together in a hybrid individual, with recessive factors on the sex chromosome interacting with dominant factors on the autosomes. The effect of these interactions on gametogenesis should not be uniform across species pairs unless genetic divergence follows the same paths in different lineages or if a specific stage of gametogenesis is more susceptible to detrimental genetic interactions. Here, we perform a detailed cellular characterization of hybrid male sterility across three recently diverged species pairs of Drosophila . Across all three pairs, sterile hybrid sperm are alive but exhibit rapid nuclear de-condensation with age, with active, but non-differentiated, mitochondria. Surprisingly, all three sets of interspecies hybrids produce half of the number of sperm per round of spermatogenesis, with each sperm cell containing two tails. We identify non-disjunction failures during meiosis I as the likely cause. Thus, errors during meiosis I may be a general phenomenon underlying Drosophila male sterility, indicating either a heightened sensitivity of this spermatogenic stage to failure, or a basis to sterility other than the prevailing model.

The amount of DNA sequence variability in a genomic region is often positively correlated with its rate of crossing over (CO) [1-3]. This pattern is caused by selection acting on linked sites, which reduces genetic variability and biases the frequency distribution of segregating variants toward more rare variants than are expected without selection (skew). These effects may involve the spread of beneficial mutations (selective sweeps [SSWs]), the elimination of deleterious mutations (background selection [BGS]), or both, and are expected to be stronger with lower CO rates [1-3]. However, in a recent study of human populations, the skew was reduced in the lowest CO regions compared with regions with somewhat higher CO rates [4]. A low skew in very low CO regions, compared with theoretical predictions, is seen in the population genomic studies of Drosophila simulans described here and in other Drosophila species. Here, we propose an explanation for lower than expected skew in low CO regions, and validate it using computer simulations; explanations for higher skew with higher CO rates, as in D. simulans, will be explored elsewhere. Partially recessive, linked deleterious mutations can increase neutral variability when the product of the effective population size (N e ) and the selection coefficient against homozygous carriers of mutations (s) is ≤1, i.e., there is associative overdominance (AOD) rather than BGS [5]. AOD can operate in low CO regions, producing a lower skew than in its absence. This opens up a new perspective on how selection affects patterns of variability at linked sites., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The recent emergence and spread of X-linked segregation distorters-called "Paris" system-in the worldwide species Drosophila simulans has elicited the selection of drive-resistant Y chromosomes. Here, we investigate the evolutionary history of 386 Y chromosomes originating from 29 population samples collected over a period of 20 years, showing a wide continuum of phenotypes when tested against the Paris distorters, from high sensitivity to complete resistance (males sire ∼95% to ∼40% female progeny). Analyzing around 13 kb of Y-linked gene sequences in a representative subset of nine Y chromosomes, we identified only three polymorphic sites resulting in three haplotypes. Remarkably, one of the haplotypes is associated with resistance. This haplotype is fixed in all samples from Sub-Saharan Africa, the region of origin of the drivers. Exceptionally, with the spread of the drivers in Egypt and Morocco, we were able to record the replacement of the sensitive lineage by the resistant haplotype in real time, within only a few years. In addition, we performed in situ hybridization, using satellite DNA probes, on a subset of 21 Y chromosomes from six locations. In contrast to the low molecular polymorphism, this revealed extensive structural variation suggestive of rapid evolution, either neutral or adaptive. Moreover, our results show that intragenomic conflicts can drive astonishingly rapid replacement of Y chromosomes and suggest that the emergence of Paris segregation distorters in East Africa occurred less than half a century ago., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sensory perception modulates health and aging across taxa. Understanding the nature of relevant cues and the mechanisms underlying their action may lead to novel interventions that improve the length and quality of life. We found that in the vinegar fly, Drosophila melanogaster, exposure to dead conspecifics in the environment induced cues that were aversive to other flies, modulated physiology, and impaired longevity. The effects of exposure to dead conspecifics on aversiveness and lifespan required visual and olfactory function in the exposed flies. Furthermore, the sight of dead flies was sufficient to produce aversive cues and to induce changes in the head metabolome. Genetic and pharmacologic attenuation of serotonergic signaling eliminated the effects of exposure on aversiveness and lifespan. Our results indicate that Drosophila have an ability to perceive dead conspecifics in their environment and suggest conserved mechanistic links between neural state, health, and aging; the roots of which might be unearthed using invertebrate model systems.

We develop analytical and simulation tools for evolve-and-resequencing experiments and apply them to a new study of rapid evolution in Drosophila simulans Likelihood test statistics applied to pooled population sequencing data suggest parallel evolution of 138 SNPs across the genome. This number is reduced by orders of magnitude from previous studies (thousands or tens of thousands), owing to differences in both experimental design and statistical analysis. Whole genome simulations calibrated from Drosophila genetic data sets indicate that major features of the genome-wide response could be explained by as few as 30 loci under strong directional selection with a corresponding hitchhiking effect. Smaller effect loci are likely also responding, but are below the detection limit of the experiment. Finally, SNPs showing strong parallel evolution in the experiment are intermediate in frequency in the natural population (usually 30-70%) indicative of balancing selection in nature. These loci also exhibit elevated differentiation among natural populations of D. simulans , suggesting environmental heterogeneity as a potential balancing mechanism., (Copyright © 2019 by the Genetics Society of America.)

It is known that the effective population size (Ne) and the mutation rate (u) vary across the genome. Here, we show that ignoring this heterogeneity may lead to biased estimates of past demography. To solve the problem, we develop new methods for jointly inferring past changes in population size and detecting variation in Ne and u between loci. These methods rely on either polymorphism data alone or both polymorphism and divergence data. In addition to inferring demography, we can use the methods to study a variety of questions: 1) comparing sex chromosomes with autosomes (for finding evidence for male-driven evolution, an unequal sex ratio, or sex-biased demographic changes) and 2) analyzing multilocus data from within autosomes or sex chromosomes (for studying determinants of variability in Ne and u). Simulations suggest that the methods can provide accurate parameter estimates and have substantial statistical power for detecting difference in Ne and u. As an example, we use the methods to analyze a polymorphism data set from Drosophila simulans. We find clear evidence for rapid population expansion. The results also indicate that the autosomes have a higher mutation rate than the X chromosome and that the sex ratio is probably female-biased. The new methods have been implemented in a user-friendly package.

We have developed a Poisson random field model for estimating the distribution of selective effects of newly arisen nonsynonymous mutations that could be observed as polymorphism or divergence in samples of two related species under the assumption that the two species populations are not at mutation-selection-drift equilibrium. The model is applied to 91Drosophila genes by comparing levels of polymorphism in an African population of D. melanogaster with divergence to a reference strain of D. simulans. Based on the difference of gene expression level between testes and ovaries, the 91 genes were classified as 33 male-biased, 28 female-biased, and 30 sex-unbiased genes. Under a Bayesian framework, Markov chain Monte Carlo simulations are implemented to the model in which the distribution of selective effects is assumed to be Gaussian with a mean that may differ from one gene to the other to sample key parameters. Based on our estimates, the majority of newly-arisen nonsynonymous mutations that could contribute to polymorphism or divergence in Drosophila species are mildly deleterious with a mean scaled selection coefficient of -2.81, while almost 86% of the fixed differences between species are driven by positive selection. There are only 16.6% of the nonsynonymous mutations observed in sex-unbiased genes that are under positive selection in comparison to 30% of male-biased and 46% of female-biased genes that are beneficial. We also estimated that D. melanogaster and D. simulans may have diverged 1.72 million years ago., Competing Interests: The authors have read the journal’s policy and have the following conflicts: SZ received support from Santander Bank in the form of a salary. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Males and females share most of their genome and develop many of the same traits. However, each sex frequently has different optimal values for these shared traits, creating intralocus sexual conflict. This conflict has been observed in wild and laboratory populations of insects and affects important evolutionary processes such as sexual selection, the maintenance of genetic variation, and possibly even speciation. Given the broad impacts of intralocus conflict, accurately detecting and measuring it is important. A common way to detect intralocus sexual conflict is to calculate the intersexual genetic correlation for fitness, with negative values suggesting conflict. Here, we highlight a potential confounder of this measure-cytoplasmic incompatibility caused by the intracellular parasite Wolbachia. Infection with Wolbachia can generate negative intersexual genetic correlations for fitness in insects, suggestive of intralocus sexual conflict . This is because cytoplasmic incompatibility reduces the fitness of uninfected females mated to infected males, while uninfected males will not suffer reductions in fitness if they mate with infected females and may even be fitter than infected males. This can lead to strong negative intersexual genetic correlations for fitness, mimicking intralocus conflict. We illustrate this issue using simulations and then present Drosophila simulans data that show how reproductive incompatibilities caused by Wolbachia infection can generate signals of intralocus sexual conflict. Given that Wolbachia infection in insect populations is pervasive, but populations usually contain both infected and uninfected individuals providing scope for cytoplasmic incompatibility, this is an important consideration for sexual conflict research but one which, to date, has been largely underappreciated.

During speciation, sex chromosomes often accumulate interspecific genetic incompatibilities faster than the rest of the genome. The drive theory posits that sex chromosomes are susceptible to recurrent bouts of meiotic drive and suppression, causing the evolutionary build-up of divergent cryptic sex-linked drive systems and, incidentally, genetic incompatibilities. To assess the role of drive during speciation, we combine high-resolution genetic mapping of X-linked hybrid male sterility with population genomics analyses of divergence and recent gene flow between the fruitfly species, Drosophila mauritiana and D. simulans . Our findings reveal a high density of genetic incompatibilities and a corresponding dearth of gene flow on the X chromosome. Surprisingly, we find that a known drive element recently migrated between species and, rather than contributing to interspecific divergence, caused a strong reduction in local sequence divergence, undermining the evolution of hybrid sterility. Gene flow can therefore mediate the effects of selfish genetic elements during speciation., Competing Interests: CM, EL, KG, TR, SK, AG, JV, CM, DG, DS, DP No competing interests declared, (© 2018, Meiklejohn et al.)