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Abstract Background The influence of microbiota in ecological interactions, and in particular competition, is poorly known. We studied competition between two insect species, the invasive pest Drosophila suzukii and the model Drosophila melanogaster, whose larval ecological niches overlap in ripe, but not rotten, fruit. Results 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 triggered 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 only occurred if the competitor had its normal microbiota. Conclusions This study establishes microbiotas as potent mediators of interspecific competition and reveals a central role for context-dependent behaviors under bacterial influence. Video Abstract

An active immune response is energetically demanding and requires reallocation of nutrients to support resistance to and tolerance of infection. Insulin signaling is a critical global regulator of metabolism and whole-body homeostasis in response to nutrient availability and energetic needs, including those required for mobilization of energy in support of the immune system. In this review, we share findings that demonstrate interactions between innate immune activity and insulin signaling primarily in the insect model Drosophila melanogaster as well as other insects like Bombyx mori and Anopheles mosquitos. These studies indicate that insulin signaling and innate immune activation have reciprocal effects on each other, but that those effects vary depending on the type of pathogen, route of infection, and nutritional status of the host. Future research will be required to further understand the detailed mechanisms by which innate immunity and insulin signaling activity impact each other.

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.

Several aspects of mosquito ecology that are important for vectored disease transmission and control have been difficult to measure at epidemiologically important scales in the field. In particular, the ability to describe mosquito population structure and movement rates has been hindered by difficulty in quantifying fine-scale genetic variation among populations. The mosquito virome represents a possible avenue for quantifying population structure and movement rates across multiple spatial scales. Mosquito viromes contain a diversity of viruses, including several insect-specific viruses (ISVs) and “core” viruses that have high prevalence across populations. To date, virome studies have focused on viral discovery and have only recently begun examining viral ecology. While nonpathogenic ISVs may be of little public health relevance themselves, they provide a possible route for quantifying mosquito population structure and dynamics. For example, vertically transmitted viruses could behave as a rapidly evolving extension of the host’s genome. It should be possible to apply established analytical methods to appropriate viral phylogenies and incidence data to generate novel approaches for estimating mosquito population structure and dispersal over epidemiologically relevant timescales. By studying the virome through the lens of spatial and genomic epidemiology, it may be possible to investigate otherwise cryptic aspects of mosquito ecology. A better understanding of mosquito population structure and dynamics are key for understanding mosquito-borne disease ecology and methods based on ISVs could provide a powerful tool for informing mosquito control programs.

Abstract 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.

Abstract 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.

Genome-scale studies of population structure and high-resolution mapping of genetically complex traits both require techniques for accurately and efficiently genotyping large numbers of polymorphic sites in multiple individuals. Many high-throughput genotyping technologies require the purchase of expensive equipment or consumables and are therefore out of reach of some individual research laboratories. Conversely, less expensive technologies are often labor intensive so that the effort involved in typing large numbers of samples or polymorphic sites is prohibitive. Here we present a method of fluorescently post-labeling restriction digestion using standard dye-terminator sequencing chemistry so that RFLP and AFLP products can be visualized on an automated sequencer. This labeling method is efficient, inexpensive, easily multiplexed, and requires no unusual equipment or reagents, thus striking a balance between cost and throughput that should be appropriate for many research groups and core facilities.

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.

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.

Immune function is likely to be a critical determinant of an organism's fitness, yet most natural animal and plant populations exhibit tremendous genetic variation for immune traits. Accumulating evidence suggests that environmental heterogeneity may retard the long-term efficiency of natural selection and even maintain polymorphism, provided alternative host genotypes are favoured under different environmental conditions. ‘Environment’ in this context refers to abiotic factors such as ambient temperature or availability of nutrient resources, genetic diversity of pathogens or competing physiological demands on the host. These factors are generally controlled in laboratory experiments measuring immune performance, but variation in them is likely to be very important in the evolution of resistance to infection. Here, we review some of the literature emphasizing the complexity of natural selection on immunity. Our aim is to describe how environmental and genetic heterogeneities, often excluded from experimentation as ‘noise’, may determine the evolutionary potential of populations or the potential for interacting species to coevolve. [ABSTRACT FROM AUTHOR]

Overnutrition with dietary sugar can worsen infection outcomes in diverse organisms including insects and humans, through generally unknown mechanisms. In the present study, we show that adult Drosophila melanogaster fed high-sugar diets became more susceptible to infection by the Gram-negative bacteria Providencia rettgeri and Serratia marcescens. We found that P. rettgeri and S. marcescens proliferate more rapidly in D. melanogaster fed a high-sugar diet, resulting in increased probability of host death. D. melanogaster become hyperglycemic on the high-sugar diet, and we find evidence that the extra carbon availability may promote S. marcescens growth within the host. However, we found no evidence that increased carbon availability directly supports greater P. rettgeri growth. D. melanogaster on both diets fully induce transcription of antimicrobial peptide (AMP) genes in response to infection, but D. melanogaster provided with high-sugar diets show reduced production of AMP protein. Thus, overnutrition with dietary sugar may impair host immunity at the level of AMP translation. Our results demonstrate that dietary sugar can shape infection dynamics by impacting both host and pathogen, depending on the nutritional requirements of the pathogen and by altering the physiological capacity of the host to sustain an immune response.

AbstractLittle 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. melanogasterBeauveria bassianaB. bassianaB. bassiana

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.

Host responses to infection encompass many processes in addition to activation of the immune system, including metabolic adaptations, stress responses, tissue repair, and other reactions. The response to bacterial infection in Drosophila melanogaster has been classically described in studies that focused on the immune response elicited by a small set of largely avirulent microbes. Thus, we have surprisingly limited knowledge of responses to infection that are outside the canonical immune response, of how the response to pathogenic infection differs from that to avirulent bacteria, or even of how generic the response to various microbes is and what regulates that core response. In this study, we addressed these questions by profiling the D. melanogaster transcriptomic response to 10 bacteria that span the spectrum of virulence. We found that each bacterium triggers a unique transcriptional response, with distinct genes making up to one third of the response elicited by highly virulent bacteria. We also identified a core set of 252 genes that are differentially expressed in response to the majority of bacteria tested. Among these, we determined that the transcription factor CrebA is a novel regulator of infection tolerance. Knock-down of CrebA significantly increased mortality from microbial infection without any concomitant change in bacterial number. Upon infection, CrebA is upregulated by both the Toll and Imd pathways in the fat body, where it is required to induce the expression of secretory pathway genes. Loss of CrebA during infection triggered endoplasmic reticulum (ER) stress and activated the unfolded protein response (UPR), which contributed to infection-induced mortality. Altogether, our study reveals essential features of the response to bacterial infection and elucidates the function of a novel regulator of infection tolerance.

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.

Drosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs, we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2,000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont--which is known to be protective against virus infections in Drosophila--we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets, we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host-virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research.

Life history theory predicts that trait evolution should be constrained by competing physiological demands on an organism. Immune defense provides a classic example in which immune responses are presumed to be costly and therefore come at the expense of other traits related to fitness. One strategy for mitigating the costs of expensive traits is to render them inducible, such that the cost is paid only when the trait is utilized. In the current issue of PLOS Biology, Bajgar and colleagues elegantly demonstrate the energetic and life history cost of the immune response that Drosophila melanogaster larvae induce after infection by the parasitoid wasp Leptopilina boulardi. These authors show that infection-induced proliferation of defensive blood cells commands a diversion of dietary carbon away from somatic growth and development, with simple sugars instead being shunted to the hematopoetic organ for rapid conversion into the raw energy required for cell proliferation. This metabolic shift results in a 15% delay in the development of the infected larva and is mediated by adenosine signaling between the hematopoietic organ and the central metabolic control organ of the host fly. The adenosine signal thus allows D. melanogaster to rapidly marshal the energy needed for effective defense and to pay the cost of immunity only when infected.

Both malnutrition and undernutrition can lead to compromised immune defense in a diversity of animals, and "nutritional immunology" has been suggested as a means of understanding immunity and determining strategies for fighting infection. The genetic basis for the effects of diet on immunity, however, has been largely unknown. In the present study, we have conducted genome-wide association mapping in Drosophila melanogaster to identify the genetic basis for individual variation in resistance, and for variation in immunological sensitivity to diet (genotype-by-environment interaction, or GxE). D. melanogaster were reared for several generations on either high-glucose or low-glucose diets and then infected with Providencia rettgeri, a natural bacterial pathogen of D. melanogaster. Systemic pathogen load was measured at the peak of infection intensity, and several indicators of nutritional status were taken from uninfected flies reared on each diet. We find that dietary glucose level significantly alters the quality of immune defense, with elevated dietary glucose resulting in higher pathogen loads. The quality of immune defense is genetically variable within the sampled population, and we find genetic variation for immunological sensitivity to dietary glucose (genotype-by-diet interaction). Immune defense was genetically correlated with indicators of metabolic status in flies reared on the high-glucose diet, and we identified multiple genes that explain variation in immune defense, including several that have not been previously implicated in immune response but which are confirmed to alter pathogen load after RNAi knockdown. Our findings emphasize the importance of dietary composition to immune defense and reveal genes outside the conventional "immune system" that can be important in determining susceptibility to infection. Functional variation in these genes is segregating in a natural population, providing the substrate for evolutionary response to pathogen pressure in the context of nutritional environment.

Next-generation sequencing technologies have made it possible to address principle population genetic questions in almost any system, but high error rates associated with this data can introduce significant biases into downstream analyses, so careful consideration of experimental design and interpretation in essential in studies based on short-read sequencing. Exploration of population genetic analyses based on next-generation sequencing, has revealed some of the potential biases, but previous work has emphasized human population genetics, and further examination of parameters relevant to other systems is necessary, including when sample sizes are small and genetic variation is high. To assess experimental power to address several principal objectives of population genetic studies under these conditions, we simulated population samples under selective sweep, population growth, and population subdivision models and tested the power to recover the correct model from sequence polymorphism data inferred from 4x, 8x, and 15x short-read data. We found that estimates of population genetic differentiation and population growth parameters were systematically biased when inference was based on 4x sequencing, but biases were markedly reduced at even 8x read depth. We also found that the power to identify footprints of positive selection depends on an interaction between read depth and the strength of selection, with strong selection being recovered consistently at all read depths, but weak selection requiring deeper read depths for reliable detection. Although we have only explored a small subset of the many possible experimental designs, population genetic models and SNP calling approaches, our results reveal some general patterns and provide some assessment of what biases could be expected under similar experimental structures.

Multiple studies have shown that infection with the endosymbiotic bacterium Wolbachia pipientis confers Drosophila melanogaster and other insects with resistance to infection by RNA viruses. Studies investigating whether Wolbachia infection induces the immune system or confers protection against secondary bacterial infection have not shown any effect. These studies, however, have emphasized resistance against extracellular pathogens. Since Wolbachia lives inside the host cell, we hypothesized that Wolbachia might confer resistance to pathogens that establish infection by invading host cells. We therefore tested whether Wolbachia-infected D. melanogaster are protected against infection by the intracellular pathogenic bacteria Listeria monocytogenes and Salmonella typhimurium, as well as the extracellular pathogenic bacterium Providencia rettgeri. We evaluated the ability of flies infected with Wolbachia to suppress secondary infection by pathogenic bacteria relative to genetically matched controls that had been cured of Wolbachia by treatment with tetracycline. We found no evidence that Wolbachia alters host ability to suppress proliferation of any of the three pathogenic bacteria. Our results indicate that Wolbachia-induced antiviral protection does not result from a generalized response to intracellular pathogens.

The three-gene APL1 locus encodes essential components of the mosquito immune defense against malaria parasites. APL1 was originally identified because it lies within a mapped QTL conferring the vector mosquito Anopheles gambiae natural resistance to the human malaria parasite, Plasmodium falciparum, and APL1 genes have subsequently been shown to be involved in defense against several species of Plasmodium. Here, we examine molecular population genetic variation at the APL1 gene cluster in spatially and temporally diverse West African collections of A. gambiae. The locus is extremely polymorphic, showing evidence of adaptive evolutionary maintenance of genetic variation. We hypothesize that this variability aids in defense against genetically diverse pathogens, including Plasmodium. Variation at APL1 is highly structured across geographic and temporal subpopulations. In particular, diversity is exceptionally high during the rainy season, when malaria transmission rates are at their peak. Much less allelic diversity is observed during the dry season when mosquito population sizes and malaria transmission rates are low. APL1 diversity is weakly stratified by the polymorphic 2La chromosomal inversion but is very strongly subdivided between the M and S "molecular forms." We find evidence that a recent selective sweep has occurred at the APL1 locus in M form mosquitoes only. The independently reported observation of a similar M-form restricted sweep at the Tep1 locus, whose product physically interacts with APL1C, suggests that epistatic selection may act on these two loci causing them to sweep coordinately.

Anopheles funestus is one of the primary vectors of human malaria, which causes a million deaths each year in sub-Saharan Africa. Few scientific resources are available to facilitate studies of this mosquito species and relatively little is known about its basic biology and evolution, making development and implementation of novel disease control efforts more difficult. The An. funestus genome has not been sequenced, so in order to facilitate genome-scale experimental biology, we have sequenced the adult female transcriptome of An. funestus from a newly founded colony in Burkina Faso, West Africa, using the Illumina GAIIx next generation sequencing platform.We assembled short Illumina reads de novo using a novel approach involving iterative de novo assemblies and "target-based" contig clustering. We then selected a conservative set of 15,527 contigs through comparisons to four Dipteran transcriptomes as well as multiple functional and conserved protein domain databases. Comparison to the Anopheles gambiae immune system identified 339 contigs as putative immune genes, thus identifying a large portion of the immune system that can form the basis for subsequent studies of this important malaria vector. We identified 5,434 1:1 orthologues between An. funestus and An. gambiae and found that among these 1:1 orthologues, the protein sequence of those with putative immune function were significantly more diverged than the transcriptome as a whole. Short read alignments to the contig set revealed almost 367,000 genetic polymorphisms segregating in the An. funestus colony and demonstrated the utility of the assembled transcriptome for use in RNA-seq based measurements of gene expression.We developed a pipeline that makes de novo transcriptome sequencing possible in virtually any organism at a very reasonable cost ($6,300 in sequencing costs in our case). We anticipate that our approach could be used to develop genomic resources in a diversity of systems for which full genome sequence is currently unavailable. Our An. funestus contig set and analytical results provide a valuable resource for future studies in this non-model, but epidemiologically critical, vector insect.

It is now well established that natural populations of Drosophila melanogaster harbor substantial genetic variation associated with physiological measures of immune function. In no case, however, have intermediate measures of immune function, such as transcriptional activity of immune-related genes, been tested as mediators of phenotypic variation in immunity. In this study, we measured bacterial load sustained after infection of D. melanogaster with Serratia marcescens, Providencia rettgeri, Enterococcus faecalis, and Lactococcus lactis in a panel of 94 third-chromosome substitution lines. We also measured transcriptional levels of 329 immune-related genes eight hours after infection with E. faecalis and S. marcescens in lines from the phenotypic tails of the test panel. We genotyped the substitution lines at 137 polymorphic markers distributed across 25 genes in order to test for statistical associations among genotype, bacterial load, and transcriptional dynamics. We find that genetic polymorphisms in the pathogen recognition genes (and particularly in PGRP-LC, GNBP1, and GNBP2) are most significantly associated with variation in bacterial load. We also find that overall transcriptional induction of effector proteins is a significant predictor of bacterial load after infection with E. faecalis, and that a marker upstream of the recognition gene PGRP-SD is statistically associated with variation in both bacterial load and transcriptional induction of effector proteins. These results show that polymorphism in genes near the top of the immune system signaling cascade can have a disproportionate effect on organismal phenotype due to the amplification of minor effects through the cascade.

Natural populations of most organisms harbor substantial genetic variation for resistance to infection. The continued existence of such variation is unexpected under simple evolutionary models that either posit direct and continuous natural selection on the immune system or an evolved life history "balance" between immunity and other fitness traits in a constant environment. However, both local adaptation to heterogeneous environments and genotype-by-environment interactions can maintain genetic variation in a species. In this study, we test Drosophila melanogaster genotypes sampled from tropical Africa, temperate northeastern North America, and semi-tropical southeastern North America for resistance to bacterial infection and fecundity at three different environmental temperatures. Environmental temperature had absolute effects on all traits, but there were also marked genotype-by-environment interactions that may limit the global efficiency of natural selection on both traits. African flies performed more poorly than North American flies in both immunity and fecundity at the lowest temperature, but not at the higher temperatures, suggesting that the African population is maladapted to low temperature. In contrast, there was no evidence for clinal variation driven by thermal adaptation within North America for either trait. Resistance to infection and reproductive success were generally uncorrelated across genotypes, so this study finds no evidence for a fitness tradeoff between immunity and fecundity under the conditions tested. Both local adaptation to geographically heterogeneous environments and genotype-by-environment interactions may explain the persistence of genetic variation for resistance to infection in natural populations.

We previously identified by genetic mapping an Anopheles gambiae chromosome region with strong influence over the outcome of malaria parasite infection in nature. Candidate gene studies in the genetic interval, including functional tests using the rodent malaria parasite Plasmodium berghei, identified a novel leucine-rich repeat gene, APL1, with functional activity against P. berghei.Manual reannotation now reveals APL1 to be a family of at least 3 independently transcribed genes, APL1A, APL1B, and APL1C. Functional dissection indicates that among the three known APL1 family members, APL1C alone is responsible for host defense against P. berghei. APL1C functions within the Rel1-Cactus immune signaling pathway, which regulates APL1C transcript and protein abundance. Gene silencing of APL1C completely abolishes Rel1-mediated host protection against P. berghei, and thus the presence of APL1C is required for this protection. Further highlighting the influence of this chromosome region, allelic haplotypes at the APL1 locus are genetically associated with and have high explanatory power for the success or failure of P. berghei parasite infection.APL1C functions as a required transducer of Rel1-dependent immune signal(s) to efficiently protect mosquitoes from P. berghei infection, and allelic genetic haplotypes of the APL1 locus display distinct levels of susceptibility and resistance to P. berghei.