Search

Most organisms are under constant and repeated exposure to pathogens, leading to perpetual natural selection for more effective ways to fight-off infections. This could include the evolution of memory-based immunity to increase protection from repeatedly-encountered pathogens both within and across generations. There is mixed evidence for intra- and trans-generational priming in non-vertebrates, which lack the antibody-mediated acquired immunity characteristic of vertebrates. In this work, we tested for trans-generational immune priming in adult offspring of the fruit fly,Drosophila melanogaster, after maternal challenge with 10 different bacterial pathogens. We focused on natural opportunistic pathogens ofDrosophilaspanning a range of virulence from 10% to 100% host mortality. We infected mothers via septic injury and tested for enhanced resistance to infection in their adult offspring, measured as the ability to suppress bacterial proliferation and survive infection. We categorized the mothers into four classes for each bacterium tested: those that survived infection, those that succumbed to infection, sterile-injury controls, and uninjured controls. We found no evidence for trans-generational priming by any class of mother in response to any of the bacteria.

In many species, female reproductive investment comes at a cost to immunity and resistance to infection. Mated Drosophila melanogaster females are more susceptible to bacterial infection than unmated females. Transfer of the male seminal fluid protein Sex Peptide reduces female post-mating immune defense. Sex Peptide is known to cause both short- and long-term changes to female physiology and behavior. While previous studies showed that females were less resistant to bacterial infection as soon as 2.5 h and as long as 26.5 h after mating, it is unknown whether this is a binary switch from mated to unmated state or whether females can recover to unmated levels of immunity. It is additionally unknown whether repeated mating causes progressive reduction in defense capacity. We compared the immune defense of mated females when infected at 2, 4, 7, or 10 days after mating to that of unmated females and saw no recovery of immune capacity regardless of the length of time between mating and infection. Because D. melanogaster females can mate multiply, we additionally tested whether a second mating, and therefore a second transfer of seminal fluids, caused deeper reduction in immune performance. We found that females mated either once or twice before infection survived at equal proportions, both with significantly lower probability than unmated females. We conclude that a single mating event is sufficient to persistently suppress the female immune system. Interestingly, we observed that induced levels of expression of genes encoding antimicrobial peptides (AMPs) decreased with age in both experiments, partially obscuring the effects of mating. Collectively, the data indicate that being reproductively active versus reproductively inactive are alternative binary states with respect to female D. melanogaster immunity. The establishment of a suppressed immune status in reproductively active females can inform our understanding of the regulation of immune defense and the mechanisms of physiological trade-offs.

Background Single tissues can have multiple functions, which can result in constraints, impaired function, and tradeoffs. The insect fat body performs remarkably diverse functions including metabolic control, reproductive provisioning, and systemic immune responses. How polyfunctional tissues simultaneously execute multiple distinct physiological functions is generally unknown. Immunity and reproduction are observed to trade off in many organisms but the mechanistic basis for this tradeoff is also typically not known. Here we investigate constraints and trade-offs in the polyfunctional insect fat body. Results Using single-nucleus sequencing, we determined that the Drosophila melanogaster fat body executes diverse basal functions with heterogenous cellular subpopulations. The size and identity of these subpopulations are remarkably stable between virgin and mated flies, as well as before and after infection. However, as an emergency function, the immune response engages the entire tissue and all cellular subpopulations produce induce expression of defense genes. We found that reproductively active females who were given bacterial infection exhibited signatures of ER stress and impaired capacity to synthesize new protein in response to infection, including decreased capacity to produce antimicrobial peptides. Transient provision of a reversible translation inhibitor to mated females prior to infection rescued general protein synthesis, specific production of antimicrobial peptides, and survival of infection. Conclusions The commonly observed tradeoff between reproduction and immunity appears to be driven, in D. melanogaster, by a failure of the fat body to be able to handle simultaneous protein translation demands of reproductive provisioning and immune defense. We suggest that inherent cellular limitations in tissues that perform multiple functions may provide a general explanation for the wide prevalence of physiological and evolutionary tradeoffs.

Gene expression profiles are typically described at the level of the tissue or, often in Drosophila, at the level of the whole organism. Collapsing the gene expression of entire tissues into single measures averages over potentially important heterogeneity among the cells that make up that tissue. The advent of single-cell RNA-sequencing technology (sc-RNAseq) allows transcriptomic evaluation of the individual cells that make up a tissue. However, sc-RNAseq requires a high-quality suspension of viable cells or nuclei, and cell dissociation methods that yield healthy cells and nuclei are still lacking for many important tissues. The insect fat body is a polyfunctional tissue responsible for diverse physiological processes and therefore is an important target for sc-RNAseq. The Drosophila adult fat body consists of fragile cells that are difficult to dissociate while maintaining cell viability. As an alternative, we developed a method to isolate single fat body nuclei for RNA-seq. Our isolation method is largely free of mitochondrial contamination and yields higher capture of transcripts per nucleus compared to other nuclei preparation methods. Our method works well for single-cell nuclei sequencing and can potentially be implemented for bulk RNA-seq.

Activation of an immune response is energetically costly and excessive immune system activity can result in immunopathology, yet a slow or insufficient immune response carries the risk of pathogen establishment with consequent pathology arising from the infection. Mathematical theory and empirical data demonstrate that hosts balance the costs of immunity against the risk of infection by closely regulating immunological dynamics. An optimal immune system is rapidly and robustly deployed against a true infectious threat and rapidly deactivated once the threat has been controlled. Genetic variation in the sensitivity of an immune system, as well as in the activation and shutdown kinetics of host immune responses, can contribute to the evolution of pathogen virulence and host tolerance of infection. Improved understanding of the adaptive forces that operate on immune regulatory dynamics will clarify fundamental principles governing the evolution and maintenance of innate immune systems.

Little is known about the genetic architecture of antifungal immunity in natural populations. Using two population genetic approaches, quantitative trait locus (QTL) mapping and evolve and resequence (E&R), we explored D. melanogaster immune defense against infection with the fungus Beauveria bassiana. The immune defense was highly variable both in the recombinant inbred lines from the Drosophila Synthetic Population Resource used for our QTL mapping and in the synthetic outbred populations used in our E&R study. Survivorship of infection improved dramatically over just 10 generations in the E&R study, and continued to increase for an additional nine generations, revealing a trade-off with uninfected longevity. Populations selected for increased defense against B. bassiana evolved cross resistance to a second, distinct B. bassiana strain but not to bacterial pathogens. The QTL mapping study revealed that sexual dimorphism in defense depends on host genotype, and the E&R study indicated that sexual dimorphism also depends on the specific pathogen to which the host is exposed. Both the QTL mapping and E&R experiments generated lists of potentially causal candidate genes, although these lists were nonoverlapping.

A long-standing question in infection biology is why two very similar individuals, with very similar pathogen exposures, may have very different outcomes. Recent experiments have found that even isogenic Drosophila melanogaster hosts, given identical inoculations of some bacterial pathogens at suitable doses, can experience very similar initial bacteria proliferation but then diverge to either a lethal infection or a sustained chronic infection with much lower pathogen load. We hypothesized that divergent infection outcomes are a natural result of mutual negative feedbacks between pathogens and the host immune response. Here, we test this hypothesis in silico by constructing process-based dynamic models for bacterial population growth, host immune induction and the feedbacks between them, based on common mechanisms of immune system response. Mathematical analysis of a minimal conceptual model confirms our qualitative hypothesis that mutual negative feedbacks can magnify small differences among hosts into life-or-death differences in outcome. However, explaining observed features of chronic infections requires an extension of the model to include induced pathogen modifications that shield themselves from host immune responses at the cost of reduced proliferation rate. Our analysis thus generates new, testable predictions about the mechanisms underlying bimodal infection outcomes.

Many of the molecular mechanisms for antifungal immunity inDrosophila melanogasterhave been defined, but relatively little is known about the genetic basis for variation in antifungal immunity in natural populations. Using two population genetic approaches, Quantitative Trait Locus (QTL) Mapping and Evolve and Resequence (E&R), we explored the genetics underlyingD. melanogasterimmune defense against infection with the fungusBeauveria bassiana. Immune defense was highly variable both in the recombinant inbred lines from the Drosophila Synthetic Population Resource used for our QTL Mapping and in the synthetic outbred populations used in our E&R study. Survivorship of infection improved dramatically over just 10 generations in the E&R study, and continued to increase for an additional 9 generations, revealing a trade-off with uninfected longevity. Populations selected for increased defense againstB. bassianaevolved cross resistance to a second, distinctB. bassianastrain but not to bacterial pathogens. The QTL mapping study revealed that sexual dimorphism in defense depends on host genotype, and the E&R study indicated that dimorphism also depends on the specific pathogen to which the host is exposed. Both the QTL Mapping and E&R experiments generated lists of potentially causal candidate genes, although these lists were non-overlapping.

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome datasets from natural populations of this species have been published over the last 20 years. A major challenge is the integration of these disparate datasets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution and population structure of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 population samples from over 100 locations in >20 countries on four continents based on a combination of 121 unpublished and 150 previously published genomic datasets. Several of these locations have been sampled at different seasons across multiple years. This dataset, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental meta-data. A web-based genome browser and web portal provide easy access to the SNP dataset. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan dataset. Our resource will enable population geneticists to analyze spatio-temporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.

The use of one tissue for multiple purposes can result in constraints, impaired function, and tradeoffs. The insect fat body performs remarkably diverse functions including metabolic control, reproductive provisioning, and systemic immune responses. Immunity and reproduction are observed to trade off in many organisms, although the mechanistic basis for the tradeoff is generally unknown. More generally, how do polyfunctional tissues simultaneously execute multiple distinct physiological functions? Using single-nucleus sequencing, we determined theDrosophila melanogasterfat body executes diverse basal functions with heterogenous cellular subpopulations. However, as an emergency function, the immune response engages the entire tissue. We found that reproductively active females exhibit impaired capacity to produce new protein in response to infection, resulting in the reproduction-immunity tradeoff. We suggest that such inherent internal limitations may provide a general explanation for the wide prevalence of physiological and evolutionary tradeoffs.

A longstanding question in infection biology is why two very similar individuals, with very similar pathogen exposures, may have very different outcomes. Recent experiments have found that even isogenic Drosophila melanogaster hosts, given identical inoculations of some bacterial pathogens at suitable doses, can experience very similar initial bacteria proliferation but then diverge to either a lethal infection or a sustained chronic infection with much lower pathogen load. We hypothesized that divergent infection outcomes are a natural result of mutual negative feedbacks between pathogens and the host immune response. Here we test this hypothesis in silico by constructing process-based dynamic models for bacterial population growth, host immune induction, and the feedbacks between them, based on common mechanisms of immune system response. Mathematical analysis of a minimal conceptual model confirms our qualitative hypothesis that mutual negative feedbacks can magnify small differences among hosts into life-or-death differences in outcome. However, explaining observed features of chronic infections requires an extension of the model to include induced pathogen modifications that shield themselves from host immune responses at the cost of reduced proliferation rate. Our analysis thus generates new, testable predictions about the mechanisms underlying bimodal infection outcomes.

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome datasets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate datasets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in > 20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This dataset, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental meta-data. A web-based genome browser and web portal provide easy access to the SNP dataset. We further provide guidelines on how to use Pool-Seq data for model-based demographic inference. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan dataset. Our resource will enable population geneticists to analyze spatio-temporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail., DrosEU is funded by a Special Topic Networks (STN) grant from the European Society for Evolutionary Biology (ESEB). MK (M. Kapun) was supported by the Austrian Science Foundation (grant no. FWF P32275); JG by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (H2020-ERC-2014-CoG-647900) and by the Spanish Ministry of Science and Innovation (BFU-2011-24397); TF by the Swiss National Science Foundation (SNSF grants PP00P3_133641, PP00P3_165836, and 31003A_182262) and a Mercator Fellowship from the German Research Foundation (DFG), held as a EvoPAD Visiting Professor at the Institute for Evolution and Biodiversity, University of Münster; AOB by the National Institutes of Health (R35 GM119686); MK (M. Kankare) by Academy of Finland grant 322980; VL by Danish Natural Science Research Council (FNU) grant 4002-00113B; FS Deutsche Forschungsgemeinschaft (DFG) grant STA1154/4-1, Project 408908608; JP by the Deutsche Forschungsgemeinschaft Projects 274388701 and 347368302; AU by FPI fellowship (BES-2012-052999); ET Israel Science Foundation (ISF) grant 1737/17; MSV, MSR and MJ by a grant from the Ministry of Education, Science and Technological Development of the Republic of Serbia (451-03-68/2020-14/200178); AP, KE and MT by a grant from the Ministry of Education, Science and Technological Development of the Republic of Serbia (451-03-68/2020-14/200007); and TM NSERC grant RGPIN-2018-05551.

To advance our understanding of adaptation to temporally varying selection pressures, we identified signatures of seasonal adaptation occurring in parallel among Drosophila melanogaster populations. Specifically, we estimated allele frequencies genome-wide from flies sampled early and late in the growing season from 20 widely dispersed populations. We identified parallel seasonal allele frequency shifts across North America and Europe, demonstrating that seasonal adaptation is a general phenomenon of temperate fly populations. Seasonally fluctuating polymorphisms are enriched in large chromosomal inversions, and we find a broad concordance between seasonal and spatial allele frequency change. The direction of allele frequency change at seasonally variable polymorphisms can be predicted by weather conditions in the weeks prior to sampling, linking the environment and the genomic response to selection. Our results suggest that fluctuating selection is an important evolutionary force affecting patterns of genetic variation in Drosophila.

Gene expression profiles are typically described at the level of the tissue or, often in Drosophila, at the level of the whole organism. Collapsing the gene expression of entire tissues into single measures averages over potentially important heterogeneity among the cells that make up that tissue. The advent of single-cell RNA-sequencing technology (sc-RNAseq) allows transcriptomic evaluation of the individual cells that make up a tissue. However, sc-RNAseq requires a high-quality suspension of viable cells or nuclei, and cell dissociation methods that yield healthy cells and nuclei are still lacking for many important tissues. The insect fat body is a polyfunctional tissue responsible for diverse physiological processes and therefore is an important target for sc-RNAseq. The Drosophila adult fat body consists of fragile cells that are difficult to dissociate while maintaining cell viability. As an alternative, we developed a method to isolate single fat body nuclei for RNA-seq. Our isolation method is largely free of mitochondrial contamination and yields higher capture of transcripts per nucleus compared to other nuclei preparation methods. Our method works well for single cell nuclei sequencing and potentially can be implemented for bulk RNA-seq.

Species that share resources often avoid competition with context-dependent behaviors. This is the case for the invasive insect pest Drosophila suzukii, whose larval ecological niche overlaps with that of Drosophila melanogaster in ripe, but not rotten, fruit. We discovered D. suzukii females prevent costly interspecific larval competition by avoiding oviposition on substrates previously visited by D. melanogaster. More precisely, D. melanogaster association with gut bacteria of the genus Lactobacillus triggers D. suzukii avoidance. However, D. suzukii avoidance behavior is condition-dependent, and D. suzukii females that themselves carry D. melanogaster bacteria stop avoiding sites visited by D. melanogaster. The adaptive significance of avoiding cues from the competitor’s microbiota was revealed by experimentally reproducing in-fruit larval competition: reduced survival of D. suzukii larvae was dependent on the presence of gut bacteria in the competitor. This study unveils a new role for the symbiotic microbiota and plastic behaviors in mediating interspecific competition.

In many animal species, females and males differ in physiology, lifespan, and immune function. The magnitude and direction of the sexual dimorphism in immune function varies greatly and the genetic and mechanistic bases for this dimorphism are often unknown. Here we show that Drosophila melanogaster females are more likely than males to die from infection with several strains of the fungal entomopathogen Beauveria bassiana. The sexual dimorphism is not exclusively due to barrier defenses and persists when flies are inoculated by injection as well as by surface exposure. Loss of function mutations of Toll pathway genes remove the dimorphism in survivorship. Surprisingly, loss of function mutation of relish, a gene in the Imd pathway, also removes the dimorphism, but the dimorphism persists in flies carrying other Imd pathway mutations. The robust sexual dimorphism in D. melanogaster survival to B. bassiana presents opportunities to further dissect its mechanistic details, with applications for biological control of insect vectors of human disease and insect crop pests.

Defence against pathogenic infection can take two forms: resistance and tolerance. Resistance is the ability of the host to limit a pathogen burden, whereas tolerance is the ability to limit the negative consequences of infection at a given level of infection intensity. Evolutionarily, a tolerance strategy that is independent of resistance could allow the host to avoid mounting a costly immune response and, theoretically, to avoid a co-evolutionary arms race between pathogen virulence and host resistance. Biomedically, understanding the mechanisms of tolerance and how they relate to resistance could potentially yield treatment strategies that focus on health improvement instead of pathogen elimination. To understand the impact of tolerance on host defence and identify genetic variants that determine host tolerance, we defined genetic variation in tolerance as the residual deviation from a binomial regression of fitness under infection against infection intensity. We then performed a genomewide association study to map the genetic basis of variation in resistance to and tolerance of infection by the bacterium Providencia rettgeri. We found a positive genetic correlation between resistance and tolerance, and we demonstrated that the level of resistance is highly predictive of tolerance. We identified 30 loci that predict tolerance, many of which are in genes involved in the regulation of immunity and metabolism. We used RNAi to confirm that a subset of mapped genes have a role in defence, including putative wound repair genes grainy head and debris buster. Our results indicate that tolerance is not an independent strategy from resistance, but that defence arises from a collection of physiological processes intertwined with canonical immunity and resistance.

Even when successfully surviving an infection, a host often fails to eliminate a pathogen completely and may sustain substantial pathogen burden for the remainder of its life. Using systemic bacterial infection in Drosophila melanogaster, we characterize chronic infection by three bacterial species from different genera - Providencia rettgeri, Serratia marcescens, and Enterococcus faecalis-following inoculation with a range of doses. To assess the consequences of these chronic infections, we determined the expression of antimicrobial peptide genes, survival of secondary infection, and starvation resistance after one week of infection. While higher infectious doses unsurprisingly lead to higher risk of death, they also result in higher chronic bacterial loads among the survivors for all three infections. All three chronic infections caused significantly elevated expression of antimicrobial peptide genes at one week post-infection and provided generalized protection again secondary bacterial infection. Only P. rettgeri infection significantly influenced resistance to starvation, with persistently infected flies dying more quickly under starvation conditions relative to controls. These results suggest that there is potentially a generalized mechanism of protection against secondary infection, but that other impacts on host physiology may depend on the specific pathogen. We propose that chronic infections in D. melanogaster could be a valuable tool for studying tolerance of infection, including impacts on host physiology and behavior.

The moment of the fertilization of an egg by a spermatozoon—the point of “sperm success”—is a key milestone in the biology of sexually reproducing species and is a fundamental requirement for offspring production. Fertilization also represents the culmination of a suite of sexually selected processes in both sexes and is commonly used as a landmark to measure reproductive success. Sperm success is heavily dependent upon interactions with other key aspects of male and female biology, with the immune system among the most important. The immune system is vital to maintaining health in both sexes; however, immune reactions can also have antagonistic effects on sperm success. The effects of immunity on sperm success are diverse, and may include trade-offs in the male between investment in the production or protection of sperm, as well as more direct, hostile, immune responses to sperm within the female, and potentially the male, reproductive tract. Here, we review current understanding of where the biology of immunity and sperm meet, and identify the gaps in our knowledge.

Genes of the immune system often evolve rapidly and adaptively, presumably driven by antagonistic interactions with pathogens [1–4]. Those genes encoding secreted antimicrobial peptides (AMPs), however, have failed to exhibit conventional signatures of strong adaptive evolution, especially in arthropods (e.g., [5, 6]) and often segregate for null alleles and gene deletions [3, 4, 7, 8]. Furthermore, quantitative genetic studies have failed to associate naturally occurring polymorphism in AMP genes with variation in resistance to infection [9–11]. Both the lack of signatures of positive selection in AMPs and lack of association between genotype and immune phenotypes have yielded an interpretation that AMP genes evolve under relaxed evolutionary constraint, with enough functional redundancy that variation in, or even loss of, any particular peptide would have little effect on overall resistance [12, 13]. In stark contrast to the current paradigm, we identified a naturally occurring amino acid polymorphism in the antimicrobial peptide, Diptericin, that is highly predictive of resistance to bacterial infection in Drosophila melanogaster [13]. The identical amino acid polymorphism arose in parallel in the sister species D. simulans, by independent mutation with equivalent phenotypic effect. Convergent substitutions to arginine at the same amino acid residue have evolved at least five times across the Drosophila genus. We hypothesize that the alternative alleles are maintained by balancing selection through context-dependent or fluctuating selection. This pattern of evolution appears to be common in antimicrobial peptides, but is invisible to conventional screens for adaptive evolution that are predicated on elevated rates of amino acid divergence.

Species compositions of gut microbiomes impact host health [1–3], but the processes determining these compositions are largely unknown. An unexplained observation is that gut species composition varies widely between individuals but is largely stable over time within individuals [4, 5]. Stochastic factors during establishment may drive these alternative stable states (colonized versus non-colonized) [6, 7], which can influence susceptibility to pathogens such as Clostridium difficile. Here we sought to quantify and model the dose-response, dynamics, and stability of bacterial colonization in the fruit fly (Drosophila melanogaster) gut. Our precise, high-throughput technique revealed stable between-host variation in colonization when individual germ-free flies were fed their own natural commensals (including the probiotic Lactobacillus plantarum). Some flies were colonized while others remained germ-free even at extremely high bacterial doses. Thus, alternative stable states of colonization exist even in this low complexity model of host-microbe interactions. These alternative states are driven by a fundamental asymmetry between the inoculum population and the stably colonized population that is mediated by spatial localization and a population bottleneck, which makes stochastic effects important by lowering the effective population size. Prior colonization with other bacteria reduced the chances of subsequent colonization, thus increasing the stability of higher diversity guts. Therefore, stable gut diversity may be driven by inherently stochastic processes, which has important implications for combatting infectious diseases and for stably establishing probiotics in the gut.

BACKGROUND: Antimicrobial peptides (AMPs) are small proteins with potent antibacterial, antiviral, and antifungal activity. AMPs are ubiquitous among multicellular eukaryotes, with most plant and animal species expressing dozens of distinct AMP genes in epithelial tissues and in response to infection. The diversity and potency of AMPs make them attractive candidates for translational application, and several are already in clinical trials. However, if AMPs are to be used effectively and sustainably, it will be imperative to understand their natural biology and evolution in order to lessen the risk of collateral harm and avoid the resistance crisis currently facing conventional antibiotics. ADVANCES: For most of the past 25 years, the prevailing wisdom has been that AMPs are generally nonspecific and functionally redundant—largely interchangeable provided that they were produced quickly enough to a threshold that could contain infection. Support for this model was drawn from molecular evolutionary observations that AMP genes are rapidly duplicated and pseudogenized within and between species, often with little evolution at the level of the primary amino acid sequence. Furthermore, it was believed that the biochemical simplicity of AMPs reflected fundamentally irresistible modes of action, including permeabilization of the cell envelope through the formation of open pores, which was assumed to largely prevent bacterial evolution of resistance. New evidence within the past 5 years, however, has begun to overturn that model. We now know that AMPs can exhibit remarkable levels of specificity and that some of the evolutionary degradation of AMP gene families may be adaptive. We are learning that genetic variability in AMPs, even at the level of single amino acids, can dramatically alter resistance to infection. There are now multiple documentations of convergent evolution of identical amino acid variants between species and of shared allelic diversity between species. It is increasingly clear that AMPs are highly functionally diversified and that they play roles in varied biological processes, including the regulation of symbiotic communities. It is also becoming apparent that bacteria can evolve resistance to AMPs, although the pharmacodynamics and mechanisms of killing of AMPs are much more favorable than those of conventional antibiotics for the prevention of resistance evolution. OUTLOOK: AMPs hold considerable promise for translational applications, but developing their potential will require more sophisticated foundational understanding. AMPs function synergistically in vivo, and emerging evidence indicates that their activities in biological contexts may not be fully captured with classical in vitro assays. Further development of mathematical approaches to study synergies will be required, especially for higher-order interactions, in order to rationally develop cocktails that have high efficacy at low concentrations. Synergies between AMPs and conventional antibiotics should be exploited to rescue drugs that are currently lost to resistance. AMPs should be mined from all domains of life: Although more than 3100 naturally occurring AMPs have been described from taxa representing the breadth of life on earth, almost 40% of AMPs under clinical trial are of human origin. This is potentially risky because any evolved resistance to those AMPs may result in collateral resistance to endogenous human immunity. The biochemical properties and pharmacodynamics of AMPs make them far more refractory to resistance evolution than conventional antibiotics, but care should still be taken to deploy them responsibly. Translational use of AMPs in clinical and other applied settings will be greatly enhanced by understanding how specific AMPs function in their natural contexts and how their evolutionary history may predict their future utility. If we combine the insights from the evolutionary diversification of the AMPs, their activity in the context of synergistic cocktails, and our growing understanding of how to limit resistance evolution, we may avoid repeating the mistakes that have resulted in the current crisis of antibiotic resistance.

Hosts and pathogens impose coevolutionary pressure on each other as pathogens strive to establish themselves and hosts seek to suppress infection. RNA interference (RNAi) is a mechanism by which cells repress viruses and transposable elements, thereby serving as a form of immune defense. Previous studies have shown that antiviral RNAi genes evolve extraordinarily quickly in the fruit fly Drosophila melanogaster, suggesting that they may adaptively coevolve with viruses and transposable elements. An article by Palmer and colleagues extends this observation to nematodes and multiple insects. Their article can be combined with this Primer to demonstrate the use of comparative genomics and molecular evolutionary analyses in the measurement of natural selection. Related article in GENETICS: Palmer, W. H., J. D. Hadfield, and D. J. Obbard, 2018 RNA-Interference pathways display high rates of adaptive protein evolution in multiple invertebrates. Genetics 208: 1585–1599.

To advance our understanding of adaptation to temporally varying selection pressures, we identified signatures of seasonal adaptation occurring in parallel amongDrosophila melanogasterpopulations. Specifically, we estimated allele frequencies genome-wide from flies sampled early and late in the growing season from 20 widely dispersed populations. We identified parallel seasonal allele frequency shifts across North America and Europe, demonstrating that seasonal adaptation is a general phenomenon of temperate fly populations. Seasonally fluctuating polymorphisms are enriched in large chromosomal inversions and we find a broad concordance between seasonal and spatial allele frequency change. The direction of allele frequency change at seasonally variable polymorphisms can be predicted by weather conditions in the weeks prior to sampling, linking the environment and the genomic response to selection. Our results suggest that fluctuating selection is an important evolutionary force affecting patterns of genetic variation inDrosophila.

Autophagy and phagocytosis are cellular immune mechanisms for internalization and elimination of intracellular and extracellular pathogens. Some pathogens have evolved the ability to inhibit or manipulate these processes, raising the prospect of adaptive reciprocal co-evolution by the host. We performed population genetic analyses on phagocytosis and autophagy genes in Drosophila melanogaster and D. simulans to test for molecular evolutionary signatures of immune adaptation. We found that phagocytosis and autophagy genes as a whole exhibited an elevated level of haplotype homozygosity in both species. In addition, we detected signatures of recent selection, notably in the Atg14 and Ykt6 genes in D. melanogaster and a pattern of elevated sequence divergence in the genderblind (gb) gene on the D. simulans lineage. These results suggest that the evolution of the host cellular immune system as a whole may be shaped by a dynamic conflict between Drosophila and its pathogens even without pervasive evidence of strong adaptive evolution at the individual gene level.

International audience; Speciation as a process remains a central focus of evolutionary biology, but our understanding of the genomic architecture and prevalence of speciation in the face of gene flow remains incomplete. The Anopheles gambiae species complex of malaria mosquitoes is a radiation of ecologically diverse taxa. This complex is well-suited for testing for evidence of a speciation continuum and genomic barriers to introgression because its members exhibit partially overlapping geographic distributions as well as varying levels of divergence and reproductive isolation. We sequenced 20 genomes from wild A. gambiae s.s., Anopheles coluzzii, Anopheles arabiensis, and compared these with 12 genomes from the "GOUNDRY" subgroup of A. gambiae s.l. Amidst a backdrop of strong reproductive isolation, we find strong evidence for a speciation continuum with introgression of autosomal chromosomal regions among species and subgroups. The X chromosome, however, is strongly differentiated among all taxa, pointing to a disproportionately large effect of X chromosome genes in driving speciation among anophelines. Strikingly, we find that autosomal introgression has occurred from contemporary hybridization between A. gambiae and A. arabiensis despite strong divergence ( approximately 5x higher than autosomal divergence) and isolation on the X chromosome. In addition to the X, we find strong evidence that lowly recombining autosomal regions, especially pericentromeric regions, serve as barriers to introgression secondarily to the X. We show that speciation with gene flow results in genomic mosaicism of divergence and introgression. Such a reticulate gene pool connecting vector taxa across the speciation continuum has important implications for malaria control efforts.

Individuals are genetically variable for the way in which they process nutrients and in the effects of dietary content on reproductive success, immunity, and development. Here, we surveyed genetic variation for nutrient stores (glucose, glycogen, glycerol, protein, triglycerides, and wet weight) in the Drosophila Genetic Reference Panel (DGRP) after rearing the flies on either a low-glucose or high-glucose diet. We found significant genetic variation for these nutritional phenotypes and identified candidate genes that underlie that variation using genome-wide associations. In addition, we found several significant correlations between the nutritional phenotypes measured in this study and other previously published phenotypes, such as starvation stress resistance, oxidative stress sensitivity, and endoplasmic reticulum stress, which reinforce the notion that these lines can be used to robustly measure related phenotypes across distinct laboratories.

Background Host sexual dimorphism is being increasingly recognized to generate strong differences in the outcome of infectious disease, but the mechanisms underlying immunological differences between males and females remain poorly characterized. Here, we used Drosophila melanogaster to assess and dissect sexual dimorphism in the innate response to systemic bacterial infection. Results We demonstrated sexual dimorphism in susceptibility to infection by a broad spectrum of Gram-positive and Gram-negative bacteria. We found that both virgin and mated females are more susceptible than mated males to most, but not all, infections. We investigated in more detail the lower resistance of females to infection with Providencia rettgeri, a Gram-negative bacterium that naturally infects D. melanogaster. We found that females have a higher number of phagocytes than males and that ablation of hemocytes does not eliminate the dimorphism in resistance to P. rettgeri, so the observed dimorphism does not stem from differences in the cellular response. The Imd pathway is critical for the production of antimicrobial peptides in response to Gram-negative bacteria, but mutants for Imd signaling continued to exhibit dimorphism even though both sexes showed strongly reduced resistance. Instead, we found that the Toll pathway is responsible for the dimorphism in resistance. The Toll pathway is dimorphic in genome-wide constitutive gene expression and in induced response to infection. Toll signaling is dimorphic in both constitutive signaling and in induced activation in response to P. rettgeri infection. The dimorphism in pathway activation can be specifically attributed to Persephone-mediated immune stimulation, by which the Toll pathway is triggered in response to pathogen-derived virulence factors. We additionally found that, in absence of Toll signaling, males become more susceptible than females to the Gram-positive Enterococcus faecalis. This reversal in susceptibility between male and female Toll pathway mutants compared to wildtype hosts highlights the key role of the Toll pathway in D. melanogaster sexual dimorphism in resistance to infection. Conclusion Altogether, our data demonstrate that Toll pathway activity differs between male and female D. melanogaster in response to bacterial infection, thus identifying innate immune signaling as a determinant of sexual immune dimorphism. Electronic supplementary material The online version of this article (doi:10.1186/s12915-017-0466-3) contains supplementary material, which is available to authorized users.

The role of microbial secondary metabolites in the ecology of the organisms that produce them remains poorly understood. Variation in aflatoxin production by Aspergillus flavus is maintained by balancing selection, but the ecological function and impact on fungal fitness of this compound are unknown. We hypothesize that balancing selection for aflatoxin production in A. flavus is driven by interaction with insects. To test this, we competed naturally occurring aflatoxigenic and non-aflatoxigenic fungal isolates against Drosophila larvae on medium containing 0–1750 ppb aflatoxin, using quantitative PCR to quantify A. flavus DNA as a proxy for fungal fitness. The addition of aflatoxin across this range resulted in a 26-fold increase in fungal fitness. With no added toxin, aflatoxigenic isolates caused higher mortality of Drosophila larvae and had slightly higher fitness than non-aflatoxigenic isolates. Additionally, aflatoxin production increased an average of 1.5-fold in the presence of a single larva and nearly threefold when the fungus was mechanically damaged. We argue that the role of aflatoxin in protection from fungivory is inextricably linked to its role in interference competition. Our results, to our knowledge, provide the first clear evidence of a fitness advantage conferred to A. flavus by aflatoxin when interacting with insects.

A central problem in infection biology is understanding why two individuals exposed to identical infections have different outcomes. We have developed an experimental model where genetically identical, co-housed Drosophila given identical systemic infections experience different outcomes, with some individuals succumbing to acute infection while others control the pathogen as an asymptomatic persistent infection. We found that differences in bacterial burden at the time of death did not explain the two outcomes of infection. Inter-individual variation in survival stems from variation in within-host bacterial growth, which is determined by the immune response. We developed a model that captures bacterial growth dynamics and identifies key factors that predict the infection outcome: the rate of bacterial proliferation and the time required for the host to establish an effective immunological control. Our results provide a framework for studying the individual host-pathogen parameters governing the progression of infection and lead ultimately to life or death.

Understanding the rate of evolutionary change and the genetic architecture that facilitates rapid adaptation is a current challenge in evolutionary biology. Comparative studies show that genes with immune function are among the most rapidly evolving genes in a range of taxa. Here, we use immune defense in natural populations of D. melanogaster to understand the rate of evolution in natural populations and the genetics underlying the rapid change. We probed the immune system using the natural pathogens Enterococcus faecalis and Providencia rettgeri to measure post-infection survival and bacterial load of wild D. melanogaster populations collected across seasonal time along a latitudinal transect on the eastern North America (Massachusetts, Pennsylvania, and Virginia). There are pronounced and repeatable changes in the immune response over approximately 10 generations between the spring and fall populations with a significant but less distinct difference among geographic locations. Genes with known immune function are not enriched among alleles that cycle with seasonal time, but the immune function of a subset of seasonally cycling alleles in immune genes was tested using reconstructed outbred populations. We find that flies containing seasonal alleles in Thioester-containing protein 3 (Tep3) have different functional responses to infection and that epistatic interactions among seasonal Tep3 and Drosomycin-like 6 (Dro6) alleles produce the immune phenotypes observed in natural populations. This rapid, cyclic response to seasonal environmental pressure broadens our understanding of the complex ecological and genetic interactions determining the evolution of immune defense in natural populations.

Summary Hormonal signaling provides metazoans with the ability to regulate development, growth, metabolism, immune defense, and reproduction in response to internal and external stimuli. The use of hormones as central regulators of physiology makes them prime candidates for mediating allocation of resources to competing biological functions (i.e., hormonal pleiotropy) [1]. In animals, reproductive effort often results in weaker immune responses (e.g., [2–4]), and this reduction is sometimes linked to hormone signaling (see [5–7]). In the fruit fly, Drosophila melanogaster , mating and the receipt of male seminal fluid proteins results in reduced resistance to a systemic bacterial infection [8, 9]. Here, we evaluate whether the immunosuppressive effect of reproduction in female D. melanogaster is attributable to the endocrine signal juvenile hormone (JH), which promotes the development of oocytes and the synthesis and deposition of yolk protein [10, 11]. Previous work has implicated JH as immunosuppressive [12, 13], and the male seminal fluid protein Sex Peptide (SP) activates JH biosynthesis in female D. melanogaster after mating [14]. We find that transfer of SP activates synthesis of JH in the mated female, which in turn suppresses resistance to infection through the receptor germ cell expressed ( gce ). We find that mated females are more likely to die from infection, suffer higher pathogen burdens, and are less able to induce their immune responses. All of these deficiencies are rescued when JH signaling is blocked. We argue that hormonal signaling is important for regulating immune system activity and, more generally, for governing trade-offs between physiological processes.

Preventing aberrant immune responses against the microbiota is essential for the health of the host. Microbiota-shed pathogen-associated molecular patterns translocate from the gut lumen into systemic circulation. Here, we examined the role of hemolymph (insect blood) filtration in regulating systemic responses to microbiota-derived peptidoglycan. Drosophila deficient for the transcription factor Klf15 (Klf15NN) are viable but lack nephrocytes-cells structurally and functionally homologous to the glomerular podocytes of the kidney. We found that Klf15NN flies were more resistant to infection than wild-type (WT) counterparts but exhibited a shortened lifespan. This was associated with constitutive Toll pathway activation triggered by excess peptidoglycan circulating in Klf15NN flies. In WT flies, peptidoglycan was removed from systemic circulation by nephrocytes through endocytosis and subsequent lysosomal degradation. Thus, renal filtration of microbiota-derived peptidoglycan maintains immune homeostasis in Drosophila, a function likely conserved in mammals and potentially relevant to the chronic immune activation seen in settings of impaired blood filtration.

International audience; Human malaria causes nearly a million deaths in sub-Saharan Africa each year. The evolution of drug-resistance in the parasite and insecticide resistance in the mosquito vector has complicated control measures and made the need for new control strategies more urgent. Anopheles gambiae s.s. is one of the primary vectors of human malaria in Africa, and parasite-transmission-blocking vaccines targeting Anopheles proteins have been proposed as a possible strategy to control the spread of the disease. However, the success of these hypothetical technologies would depend on the successful ability to broadly target mosquito populations that may be genetically heterogeneous. Understanding the evolutionary pressures shaping genetic variation among candidate target molecules offers a first step towards evaluating the prospects of successfully deploying such technologies. We studied the population genetics of genes encoding two candidate target proteins, the salivary gland protein saglin and the basal lamina structural protein laminin, in wild populations of the M and S molecular forms of A. gambiae in Mali. Through analysis of intraspecific genetic variation and interspecific comparisons, we found no evidence of positive natural selection at the genes encoding these proteins. On the contrary, we found evidence for particularly strong purifying selection at the laminin gene. These results provide insight into the patterns of genetic diversity of saglin and laminin, and we discuss these findings in relation to the potential development of these molecules as vaccine targets.

International audience; The recent discovery of a previously unknown genetic subgroup of Anopheles gambiae sensu lato underscores our incomplete understanding of complexities of vector population demographics in Anopheles. This subgroup, named GOUNDRY, does not rest indoors as adults and is highly susceptible to Plasmodium infection in the laboratory. Initial description of GOUNDRY suggested it differed from other known Anopheles taxa in surprising and sometimes contradictory ways, raising a number of questions about its age, population size and relationship to known subgroups. To address these questions, we sequenced the complete genomes of 12 wild-caught GOUNDRY specimens and compared these genomes to a panel of Anopheles genomes. We show that GOUNDRY is most closely related to Anopheles coluzzii, and the timing of cladogenesis is not recent, substantially predating the advent of agriculture. We find a large region of the X chromosome that has swept to fixation in GOUNDRY within the last 100 years, which may be an inversion that serves as a partial barrier to contemporary gene flow. Interestingly, we show that GOUNDRY has a history of inbreeding that is significantly associated with susceptibility to Plasmodium infection in the laboratory. Our results illuminate the genomic evolution of one of probably several cryptic, ecologically specialized subgroups of Anopheles and provide a potent example of how vector population dynamics may complicate efforts to control or eradicate malaria.