The Complex Contributions of Genetics and Nutrition to Immunity in Drosophila melanogaster

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.     

Genetic Dissection of Anopheles gambiae Gut Epithelial Responses to Serratia marcescens

Genetic variation in the mosquito Anopheles gambiae profoundly influences its ability to transmit malaria. Mosquito gut bacteria are shown to influence the outcome of infections with Plasmodium parasites and are also thought to exert a strong drive on genetic variation through natural selection; however, a link between antibacterial effects and genetic variation is yet to emerge. Here, we combined SNP genotyping and expression profiling with phenotypic analyses of candidate genes by RNAi-mediated silencing and 454 pyrosequencing to investigate this intricate biological system. We identified 138 An. gambiae genes to be genetically associated with the outcome of Serratia marcescens infection, including the peptidoglycan recognition receptor PGRPLC that triggers activation of the antibacterial IMD/REL2 pathway and the epidermal growth factor receptor EGFR. Silencing of three genes encoding type III fibronectin domain proteins (FN3Ds) increased the Serratia load and altered the gut microbiota composition in favor of Enterobacteriaceae. These data suggest that natural genetic variation in immune-related genes can shape the bacterial population structure of the mosquito gut with high specificity. Importantly, FN3D2 encodes a homolog of the hypervariable pattern recognition receptor Dscam, suggesting that pathogen-specific recognition may involve a broader family of immune factors. Additionally, we showed that silencing the gene encoding the gustatory receptor Gr9 that is also associated with the Serratia infection phenotype drastically increased Serratia levels. The Gr9 antibacterial activity appears to be related to mosquito feeding behavior and to mostly rely on changes of neuropeptide F expression, together suggesting a behavioral immune response following Serratia infection. Our findings reveal that the mosquito response to oral Serratia infection comprises both an epithelial and a behavioral immune component.     

Elaboration, Diversification and Regulation of the Sir1 Family of Silencing Proteins in Saccharomyces

This study quantifies the effects of naturally occurring X-linked variation on immune response in Drosophila melanogaster to assess associations between immunity genotypes and innate immune response. We constructed a set of 168 X-chromosomal extraction lines, incorporating X chromosomes from a natural population into co-isogenic autosomal backgrounds, and genotyped the lines at 88 SNPs in 20 X-linked immune genes. We find that genetic variation in many of the genes is associated with immune response phenotypes, including bacterial load and immune gene expression. Many of the associations act in a sex-specific or sexually antagonistic manner, supporting the theory that with the selective pressures facing genes on the X chromosome, sexually antagonistic variation may be more easily maintained.THE deep evolutionary conservation of many specific genes in innate immunity underscores the potent forces of natural selection maintaining this vital function. While it is widely accepted as the ancestral form of immune response, its role in the activation of adaptive immune response further motivates investigation into variation in its function (Medzhitov and Janeway 1997). Drosophila has been used as a valuable model organism to identify and characterize functions of the components of innate immune pathways as well as the evolutionary patterns present among the genes comprising these pathways (reviewed in Brennan and Anderson 2004; Irving et al. 2004; Ferrandon et al. 2007). The humoral response, resulting in the production of antimicrobial peptides in response to bacterial or fungal infection, relies mainly on Toll and imd signal transduction pathways, both of which are highly homologous to pathways in mammalian immunity (reviewed in Kimbrell and Beutler 2001). The cellular component, on the other hand, incorporates phagocytic engulfment as well as melanization and encapsulation of infecting particles. While less well defined in the Drosophila model, portions of other systems also appear to affect the effectiveness of immune response, including JAK/STAT and JNK signaling pathways, hematopoesis, and iron metabolism.Population genetic analysis can be used to determine whether sequence polymorphism and divergence patterns among Drosophila genes in innate immune pathways are consistent with signatures of selection acting within and between species of flies. If, for example, the innate immune pathways are involved in an evolutionary “arms race” with pathogenic organisms, genes in these pathways would be expected to show signs of positive selection driven by evolutionary pressure to counter virulence mechanisms of invading microbes. When signs of selection (as inferred from sequence comparisons within Drosophila simulans populations and between D. simulans and D. melanogaster) in immune genes and nonimmune genes were evaluated, immune genes as a group were found to have higher K_A/K_S ratios than nonimmune genes, providing evidence for elevated adaptive evolution (Schlenke and Begun 2003). Since receptor, effector, and signaling proteins function in different portions of the immune response pathways, these may be exposed to differing levels of contact with invading microbes and may display nonuniform levels of functional redundancy or pleiotropy. Thus, genes from different functional groups may be exposed to distinct selective pressures. Antimicrobial peptides, which might be expected to encounter unique selective pressures due to their direct interactions with invading microbes, have shown little sign of positive selection, bearing low levels of amino acid divergence (Clark and Wang 1997; Date et al. 1998; Ramos-Onsins and Aguadé 1998; Lazzaro and Clark 2003). Furthermore, sequence analyses of immune-related receptors have shown evidence for purifying selection in peptidoglycan recognition proteins (PGRPs), while others, including some scavenger receptors (SRs), appear to be rapidly evolving under pressures consistent with positive selection (Jiggins and Hurst 2003; Lazzaro 2005). On a deeper evolutionary timescale, sequence comparisons between immune genes in multiple Drosophila species (based on full-genome sequence data) have shown striking differences among functional groups of immune genes, with recognition molecules showing much more positive selection than either signaling or effector genes (Sackton et al. 2007).Beyond using sequence data and the analysis of polymorphism and divergence to infer levels and modes of selection that have previously acted on immune genes (either individually or in functional groups), other studies have investigated correlations between autosomal variation in genotype and immune response phenotype in natural populations of Drosophila (Lazzaro et al. 2004, 2006). These experiments tested associations between naturally occurring genetic variation in immune-related genes and postinfection bacterial load. In these studies, genetic variation in many of the immune genes was found to associate significantly with one or more of the bacterial load phenotypes. Specifically, polymorphisms in autosomal genes encoding recognition and signaling proteins (but not antimicrobial peptides) associate consistently with bacterial load phenotypes, suggesting that not all functional classes of immune-related genes harbor equally influential genetic variation.The focus of this study is X-linked immune genes, which may be under unique regulatory and selective pressures simply because they are hemizygous in males, are dosage compensated, and face elevated influence of random genetic drift due to their smaller effective population size. As a consequence, the X chromosome should favor the more rapid fixation of beneficial recessive alleles and more rapid loss of harmful recessive alleles compared to the autosomes (Charlesworth et al. 1987; Singh et al. 2008). Thus, with different selective pressures compared to autosomal genes, X-linked immunity genes are expected to bear different standing levels of variation, and segregating polymorphisms in these genes may have different impacts on phenotype.Different exposures of X-linked genes to selection in males and females can also contribute to sexual dimorphism. Rice (1984) suggested that X-linked sexually antagonistic alleles may more freely influence sexually dimorphic traits than can those on autosomes. In fact, the X chromosome appears to favor the maintenance of sexually antagonistic variation (Gibson et al. 2002); if a given allele is slightly deleterious in one sex, it may be maintained in the population by being beneficial to the other sex. Immune-related genes may be particularly prone to bearing sexual dimorphism in Drosophila, since males and females have been shown to have different evolutionary optima for energetic expenditure on immune response, and thus their respective immune responses may differ on the basis of conditions such as food or reproductive resource availability (McKean and Nunney 2001, 2005). If sexually antagonistic traits are responsible for some of the observed sexual dimorphism, variation in X-linked genes could contribute to phenotypic differences, and so X-linked variation in immune genes could face unique selective pressures.In this report we investigate the standing levels of variation in X-linked immune genes in natural populations of D. melanogaster and quantify the impacts of that variation on immune response phenotypes. We genotyped 168 lines at single-nucleotide polymorphisms (SNPs) across 20 X-linked immunity loci and quantified postinfection bacterial load and immune gene expression phenotypes. We found significant variation across the lines for bacterial load after infection, and we were able to identify polymorphisms in immune-related genes that associate with immune response phenotypes individually and in interacting pairs of SNPs. Additionally, some of the genetic variation was found to associate with a sex difference in immune competence, with alleles acting in either a sex-specific or a sexually antagonistic manner. This provides evidence for X-linked genetic variation in immune-related loci associating with both phenotypic variation among lines and sex differences in these phenotypes.     

Female and male genetic contributions to post-mating immune defence in female Drosophila melanogaster

Post-mating reduction in immune defence is common in female insects, and a trade-off between mating and immunity could affect the evolution of immunity. In this work, we tested the capacity of virgin and mated female Drosophila melanogaster to defend against infection by four bacterial pathogens. We found that female D. melanogaster suffer post-mating immunosuppression in a pathogen-dependent manner. The effect of mating was seen after infection with two bacterial pathogens (Providencia rettgeri and Providencia alcalifaciens), though not after infection with two other bacteria (Enterococcus faecalis and Pseudomonas aeruginosa). We then asked whether the evolution of post-mating immunosuppression is primarily a ‘female’ or ‘male’ trait by assaying for genetic variation among females for the degree of post-mating immune suppression they experience and among males for the level of post-mating immunosuppression they elicit in their mates. We also assayed for an interaction between male and female genotypes to test the specific hypothesis that the evolution of a trade-off between mating and immune defence in females might be being driven by sexual conflict. We found that females, but not males, harbour significant genetic variation for post-mating immunosuppression, and we did not detect an interaction between female and male genotypes. We thus conclude that post-mating immune depression is predominantly a ‘female’ trait, and find no evidence that it is evolving under sexual conflict.     

Rapid recruitment of innate immunity regulates variation of intracellular pathogen resistance in Drosophila

Genetic variation in susceptibility to pathogens is a central concern both to medicine and agriculture and to the evolution of animals. Here, we have investigated the link between such natural genetic variation and the immune response in wild-type Drosophila melanogaster, a major model organism for immunological research. We found that within nine wild-type strains, different Drosophila genotypes show wide-ranging variation in their ability to survive infection from the pathogenic bacteria Listeria monocytogenes. Canton-S, a resistant strain, showed increased capacity to induce stronger innate immune activities (antimicrobial peptides (AMPs), phenol oxidase activity, and phagocytosis) compared to the susceptible strain (white) at early time points during bacterial infection. Moreover, PGRP-LE-induced innate immune activation immediately after infection greatly improves survival of the susceptible strain strongly suggesting a mechanism behind the natural genetic variation of these two strains. Taken together we provide the first experimental evidence to suggest that differences in innate immune activity at early time points during infection likely mediates infection susceptibility in Drosophila.     

Sick of mating: Sexual transmission of a pathogenic bacterium in Drosophila melanogaster

Internal fertilization protects gametes from inhospitable environments and ensures sufficient proximity for gamete union. However, close contact between individuals during mating also increases the risk of pathogen transfer. We developed an approach to transfer the entomopathogenic bacterium Serratia marcescens from males to females during courtship and mating in Drosophila melanogaster. We then examined the frequency of contamination and bacterial loads of females copulating with males for varying durations, showing that while courtship is sufficient for bacterial transmission, mating significantly increases the bacterial load received in a time-independent manner. S. marcescens transmission from contaminated males during mating was sufficient to establish rapid, systemic infection and death in mated females.     

Polymorphisms in Anopheles gambiae Immune Genes Associated with Natural Resistance to Plasmodium falciparum

Many genes involved in the immune response of Anopheles gambiae, the main malaria vector in Africa, have been identified, but whether naturally occurring polymorphisms in these genes underlie variation in resistance to the human malaria parasite, Plasmodium falciparum, is currently unknown. Here we carried out a candidate gene association study to identify single nucleotide polymorphisms (SNPs) associated with natural resistance to P. falciparum. A. gambiae M form mosquitoes from Cameroon were experimentally challenged with three local wild P. falciparum isolates. Statistical associations were assessed between 157 SNPs selected from a set of 67 A. gambiae immune-related genes and the level of infection. Isolate-specific associations were accounted for by including the effect of the isolate in the analysis. Five SNPs were significantly associated to the infection phenotype, located within or upstream of AgMDL1, CEC1, Sp PPO activate, Sp SNAKElike, and TOLL6. Low overall and local linkage disequilibrium indicated high specificity in the loci found. Association between infection phenotype and two SNPs was isolate-specific, providing the first evidence of vector genotype by parasite isolate interactions at the molecular level. Four SNPs were associated to either oocyst presence or load, indicating that the genetic basis of infection prevalence and intensity may differ. The validity of the approach was verified by confirming the functional role of Sp SNAKElike in gene silencing assays. These results strongly support the role of genetic variation within or near these five A. gambiae immune genes, in concert with other genes, in natural resistance to P. falciparum. They emphasize the need to distinguish between infection prevalence and intensity and to account for the genetic specificity of vector-parasite interactions in dissecting the genetic basis of Anopheles resistance to human malaria.     

The Toll pathway underlies host sexual dimorphism in resistance to both Gram-negative and Gram-positive bacteria in mated <Emphasis Type="Italic">Drosophila</Emphasis>

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.

     

Stage-Specific Effects of Candidate Heterochronic Genes on Variation in Developmental Time along an Altitudinal Cline of Drosophila melanogaster

Background

Previously, we have shown there is clinal variation for egg-to-adult developmental time along geographic gradients in Drosophila melanogaster. Further, we also have identified mutations in genes involved in metabolic and neurogenic pathways that affect development time (heterochronic genes). However, we do not know whether these loci affect variation in developmental time in natural populations.

Methodology/Principal Findings

Here, we constructed second chromosome substitution lines from natural populations of Drosophila melanogaster from an altitudinal cline, and measured egg-adult development time for each line. We found not only a large amount of genetic variation for developmental time, but also positive associations of the development time with thermal amplitude and altitude. We performed genetic complementation tests using substitution lines with the longest and shortest developmental times and heterochronic mutations. We identified segregating variation for neurogenic and metabolic genes that largely affected the duration of the larval stages but had no impact on the timing of metamorphosis.

Conclusions/Significance

Altitudinal clinal variation in developmental time for natural chromosome substitution lines provides a unique opportunity to dissect the response of heterochronic genes to environmental gradients. Ontogenetic stage-specific variation in invected, mastermind, cricklet and CG14591 may affect natural variation in development time and thermal evolution.     

Genetic Architecture of Abdominal Pigmentation in Drosophila melanogaster

Pigmentation varies within and between species and is often adaptive. The amount of pigmentation on the abdomen of Drosophila melanogaster is a relatively simple morphological trait, which serves as a model for mapping the genetic basis of variation in complex phenotypes. Here, we assessed natural variation in female abdominal pigmentation in 175 sequenced inbred lines of the Drosophila melanogaster Genetic Reference Panel, derived from the Raleigh, NC population. We quantified the proportion of melanization on the two most posterior abdominal segments, tergites 5 and 6 (T5, T6). We found significant genetic variation in the proportion of melanization and high broad-sense heritabilities for each tergite. Genome-wide association studies identified over 150 DNA variants associated with the proportion of melanization on T5 (84), T6 (34), and the difference between T5 and T6 (35). Several of the top variants associated with variation in pigmentation are in tan, ebony, and bric-a-brac1, genes known to affect D. melanogaster abdominal pigmentation. Mutational analyses and targeted RNAi-knockdown showed that 17 out of 28 (61%) novel candidate genes implicated by the genome-wide association study affected abdominal pigmentation. Several of these genes are involved in developmental and regulatory pathways, chitin production, cuticle structure, and vesicle formation and transport. These findings show that genetic variation may affect multiple steps in pathways involved in tergite development and melanization. Variation in these novel candidates may serve as targets for adaptive evolution and sexual selection in D. melanogaster.     

Secreted Bacterial Effectors and Host-Produced Eiger/TNF Drive Death in a Salmonella-Infected Fruit Fly

Death by infection is often as much due to the host's reaction as it is to the direct result of microbial action. Here we identify genes in both the host and microbe that are involved in the pathogenesis of infection and disease in Drosophila melanogaster challenged with Salmonella enterica serovartyphimurium (S. typhimurium). We demonstrate that wild-type S. typhimurium causes a lethal systemic infection when injected into the hemocoel of D. melanogaster. Deletion of the gene encoding the secreted bacterial effector Salmonella leucine-rich (PslrP) changes an acute and lethal infection to one that is persistent and less deadly. We propose a model in which Salmonella secreted effectors stimulate the fly and thus cause an immune response that is damaging both to the bacteria and, subsequently, to the host. In support of this model, we show that mutations in the fly gene eiger, a TNF homolog, delay the lethality of Salmonella infection. These results suggest that S. typhimurium-infected flies die from a condition that resembles TNF-induced metabolic collapse in vertebrates. This idea provides us with a new model to study shock-like biology in a genetically manipulable host. In addition, it allows us to study the difference in pathways followed by a microbe when producing an acute or persistent infection.