An obligate microsporidian parasite modulates defense against opportunistic bacterial infection in the yellow fever mosquito, Aedes aegypti.

The ability of Aedes aegypti mosquitoes to transmit vertebrate pathogens depends on multiple factors, including the mosquitoes' life history traits, immune response, and microbiota (i.e., the microbes associated with the mosquito throughout its life). The microsporidium Edhazardia aedis is an obligate intracellular parasite that specifically infects Ae. aegypti mosquitoes and severely affects mosquito survival and other life history traits critical for pathogen transmission. In this work, we investigated how E. aedis impacts bacterial infection with Serratia marcescens in Ae. aegypti mosquitoes. We measured development, survival, and bacterial load in both larval and adult stages of mosquitoes. In larvae, E. aedis exposure was either horizontal or vertical and S. marcescens was introduced orally. Regardless of the route of transmission, E. aedis exposure resulted in significantly higher S. marcescens loads in larvae. E. aedis exposure also significantly reduced larval survival but subsequent exposure to S. marcescens had no effect. In adult females, E. aedis exposure was only horizontal and S. marcescens was introduced orally or via intrathoracic injection. In both cases, E. aedis infection significantly increased S. marcescens bacterial loads in adult female mosquitoes. In addition, females infected with E. aedis and subsequently injected with S. marcescens suffered 100% mortality which corresponded with a rapid increase in bacterial load. These findings suggest that exposure to E. aedis can influence the establishment and/or replication of other microbes in the mosquito. This has implications for understanding the ecology of mosquito immune defense and potentially disease transmission by mosquito vector species. IMPORTANCE: The microsporidium Edhazardia aedis is a parasite of the yellow fever mosquito, Aedes aegypti. This mosquito transmits multiple viruses to humans in the United States and around the world, including dengue, yellow fever, and Zika viruses. Hundreds of millions of people worldwide will become infected with one of these viruses each year. E. aedis infection significantly reduces the lifespan of Ae. aegypti and is therefore a promising novel biocontrol agent. Here, we show that when the mosquito is infected with this parasite, it is also significantly more susceptible to infection by an opportunistic bacterial pathogen, Serratia marcescens. This novel discovery suggests the mosquito's ability to control infection by other microbes is impacted by the presence of the parasite.

Sugar restriction and blood ingestion shape divergent immune defense trajectories in the mosquito Aedes aegypti.

Immune defense is comprised of (1) resistance: the ability to reduce pathogen load, and (2) tolerance: the ability to limit the disease severity induced by a given pathogen load. The study of tolerance in the field of animal immunity is fairly nascent in comparison to resistance. Consequently, studies which examine immune defense comprehensively (i.e. considering both resistance and tolerance in conjunction) are uncommon, despite their exigency in achieving a thorough understanding of immune defense. Furthermore, understanding tolerance in arthropod disease vectors is uniquely relevant, as tolerance is essential to the cyclical transmission of pathogens by arthropods. Here, we tested the effect(s) of dietary sucrose concentration and blood ingestion on resistance and tolerance to Escherichia coli infection in the yellow fever mosquito Aedes aegypti. Resistance and tolerance were measured concurrently and at multiple timepoints. We found that mosquitoes from the restricted sugar treatment displayed enhanced resistance at all timepoints post-infection compared to those from the laboratory standard sugar treatment. Blood also improved resistance, but only early post-infection. While sucrose restriction had no effect on tolerance, we show that consuming blood prior to bacterial infection ameliorates a temporal decline in tolerance that mosquitoes experience when provided with only sugar meals. Taken together, our findings indicate that different dietary components can have unique and sometimes temporally dynamic impacts on resistance and tolerance.

Bacterial communities of Aedes aegypti mosquitoes differ between crop and midgut tissues.

Microbiota studies of Aedes aegypti and other mosquitoes generally focus on the bacterial communities found in adult female midguts. However, other compartments of the digestive tract maintain communities of bacteria which remain almost entirely unstudied. For example, the Dipteran crop is a food storage organ, but few studies have looked at the microbiome of crops in mosquitoes, and only a single previous study has investigated the crop in Ae. aegypti. In this study, we used both culture-dependent and culture-independent methods to compare the bacterial communities in midguts and crops of laboratory reared Ae. aegypti. Both methods revealed a trend towards higher abundance, but also higher variability, of bacteria in the midgut than the crop. When present, bacteria from the genus Elizabethkingia (family Weeksellaceae) dominated midgut bacterial communities. In crops, we found a higher diversity of bacteria, and these communities were generally dominated by acetic acid bacteria (family Acetobacteriaceae) from the genera Tanticharoenia and Asaia. These three taxa drove significant community structure differences between the tissues. We used FAPROTAX to predict the metabolic functions of these communities and found that crop bacterial communities were significantly more likely to contain bacteria capable of methanol oxidation and methylotrophy. Both the presence of acetic acid bacteria (which commonly catabolize sugar to produce acetic acid) and the functional profile that includes methanol oxidation (which is correlated with bacteria found with natural sources like nectar) may relate to the presence of sugar, which is stored in the mosquito crop. A better understanding of what bacteria are present in the digestive tract of mosquitoes and how these communities assemble will inform how the microbiota impacts mosquito physiology and the full spectrum of functions provided by the microbiota. It may also facilitate better methods of engineering the mosquito microbiome for vector control or prevention of disease transmission.

Vector microbiota and immunity: modulating arthropod susceptibility to vertebrate pathogens.

Arthropods, including mosquitoes, sand flies, tsetse flies, and ticks are vectors of many bacterial, parasitic, and viral pathogens that cause serious disease in humans and animals. Their microbiota, that is, all microorganisms that dwell within their tissues, can impact vector immunity and susceptibility to pathogen infection. Historically, host-pathogen-microbiota interactions have not been well described, with little known about mechanism. In this review, we highlight recent advances in understanding how individual microorganisms and microbial communities interact with vectors and human pathogens, the mechanisms they utilize to achieve these effects, and the potential for exploiting these interactions to control pathogen transmission. These studies fill important knowledge gaps and further our understanding of the roles that the vector microbiota plays in pathogen transmission.

Larval Diet Abundance Influences Size and Composition of the Midgut Microbiota of Aedes aegypti Mosquitoes.

The midgut microbiota of the yellow fever mosquito Aedes aegypti impacts pathogen susceptibility and transmission by this important vector species. However, factors influencing the composition and size of the microbiome in mosquitoes are poorly understood. We investigated the impact of larval diet abundance during development on the composition and size of the larval and adult microbiota by rearing Aedes aegypti under four larval food regimens, ranging from nutrient deprivation to nutrient excess. We assessed the persistent impacts of larval diet availability on the microbiota of the larval breeding water, larval mosquitoes, and adult mosquitoes under sugar and blood fed conditions using qPCR and high-throughput 16S amplicon sequencing to determine bacterial load and microbiota composition. Bacterial loads in breeding water increased with increasing larval diet. Larvae reared with the lowest diet abundance had significantly fewer bacteria than larvae from two higher diet treatments, but not from the highest diet abundance. Adults from the lowest diet abundance treatment had significantly fewer bacteria in their midguts compared to all higher diet abundance treatments. Larval diet amount also had a significant impact on microbiota composition, primarily within larval breeding water and larvae. Increasing diet correlated with increased relative levels of Enterobacteriaceae and Flavobacteriaceae and decreased relative levels of Sphingomonadaceae. Multiple individual OTUs were significantly impacted by diet including one mapping to the genus Cedecea, which increased with higher diet amounts. This was consistent across all sample types, including sugar fed and blood fed adults. Taken together, these data suggest that availability of diet during development can cause lasting shifts in the size and composition of the microbiota in the disease vector Aedes aegypti.

Considerations for mosquito microbiome research from the Mosquito Microbiome Consortium.

In the past decade, there has been increasing interest in mosquito microbiome research, leading to large amounts of data on different mosquito species, with various underlying physiological characteristics, and from diverse geographical locations. However, guidelines and standardized methods for conducting mosquito microbiome research are lacking. To streamline methods in mosquito microbiome research and optimize data quality, reproducibility, and comparability, as well as facilitate data curation in a centralized location, we are establishing the Mosquito Microbiome Consortium, a collaborative initiative for the advancement of mosquito microbiome research. Our overall goal is to collectively work on unraveling the role of the mosquito microbiome in mosquito biology, while critically evaluating its potential for mosquito-borne disease control. This perspective serves to introduce the consortium and invite broader participation. It highlights the issues we view as most pressing to the community and proposes guidelines for conducting mosquito microbiome research. We focus on four broad areas in this piece: (1) sampling/experimental design for field, semi-field, or laboratory studies; (2) metadata collection; (3) sample processing, sequencing, and use of appropriate controls; and (4) data handling and analysis. We finally summarize current challenges and highlight future directions in mosquito microbiome research. We hope that this piece will spark discussions around this area of disease vector biology, as well as encourage careful considerations in the design and implementation of mosquito microbiome research. Video Abstract.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes.

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite's life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Inhibition of Asaia in Adult Mosquitoes Causes Male-Specific Mortality and Diverse Transcriptome Changes.

Mosquitoes can transmit many infectious diseases, such as malaria, dengue, Zika, yellow fever, and lymphatic filariasis. Current mosquito control strategies are failing to reduce the severity of outbreaks that still cause high human morbidity and mortality worldwide. Great expectations have been placed on genetic control methods. Among other methods, genetic modification of the bacteria colonizing different mosquito species and expressing anti-pathogen molecules may represent an innovative tool to combat mosquito-borne diseases. Nevertheless, this emerging approach, known as paratransgenesis, requires a detailed understanding of the mosquito microbiota and an accurate characterization of selected bacteria candidates. The acetic acid bacteria Asaia is a promising candidate for paratransgenic approaches. We have previously reported that Asaia symbionts play a beneficial role in the normal development of Anopheles mosquito larvae, but no study has yet investigated the role(s) of Asaia in adult mosquito biology. Here we report evidence on how treatment with a highly specific anti-Asaia monoclonal antibody impacts the survival and physiology of adult Anopheles stephensi mosquitoes. Our findings offer useful insight on the role of Asaia in several physiological systems of adult mosquitoes, where the influence differs between males and females.

Larval exposure to bacteria modulates arbovirus infection and immune gene expression in adult Aedes aegypti.

Here we have investigated whether bacterial challenges to larval stages of Aedes aegypti can influence the adults' immune and vector competence for dengue and Zika viruses. We show that larval exposure to live Bacillus thuringiensis Berliner and Enterobacter ludwigii can result in the modulation of virus infection at the adult stage in the absence of bacterial carry-over between the two developmental stages. We observed a significant reduction in virus infection intensity in the mosquitoes exposed to bacteria as larvae but not re-exposed as adults. The pattern of immune gene transcript regulation after bacterial exposure varied between adults, depending on whether or not they had been exposed to bacteria as larvae. Adults exposed to bacteria as larvae showed an earlier immune gene mRNA enrichment when re-exposed as adults than did adults not exposed as larvae. Bacterial exposure of larvae appears to have only modest effects on adult fitness.

Physiological and Environmental Factors Affecting the Composition of the Ejaculate in Mosquitoes and Other Insects.

In addition to transferring sperm, male mosquitoes deliver several proteins, hormones and other factors to females in their seminal fluid that inhibit remating, alter host-seeking behaviors and stimulate oviposition. Recently, bioinformatics, transcriptomics and proteomics have been used to characterize the genes transcribed in male reproductive tissues and the individual proteins that are delivered to females. Thanks to these foundational studies, we now understand the complexity of the ejaculate in several mosquito species. Building on this work, researchers have begun to identify the functions of various proteins and hormones in the male ejaculate, and how they mediate their effects on female mosquitoes. Here, we present an overview of these studies, followed by a discussion of an under-studied aspect of male reproductive physiology: the effects of biotic and abiotic factors on the composition of the ejaculate. We argue that future research in this area would improve our understanding of male reproductive biology from a physiological and ecological perspective, and that researchers may be able to leverage this information to study key components of the ejaculate. Furthermore, this work has the potential to improve mosquito control by allowing us to account for relevant factors when implementing vector control strategies involving male reproductive biology.

Hydrogen cyanide produced by the soil bacterium Chromobacterium sp. Panama contributes to mortality in Anopheles gambiae mosquito larvae.

Mosquito larvae continuously encounter microbes in their aquatic environment, which serve as food and play a critical role in successful development. In previous work, we isolated a Chromobacterium sp. (C.sp_P) with larvicidal activity from the midgut of dengue vector Aedes mosquitoes in Panama. In this study, we found a positive correlation between initial concentrations of C.sp_P and larval mortality rates, and that C.sp_P is more efficient at inducing larval mortality in a high nutrient environment. Multiple Chromobacterium species induce larval mortality with similar efficacy to C.sp_P except for C. subtsugae. We also found that a non-lethal dose of C.sp_P lengthens development time and increases mortality over multiple developmental stages, suggesting persistent effects of exposure. Additionally, we showed that larvicidal activity persists in the larval breeding water after removal of live bacteria, and that the larvicidal factor in C.sp_P-treated water is smaller than 3 kDa, heat resistant to 90 °C, and lost after vacuum centrifugation. We showed that C.sp_P produces hydrogen cyanide in culture and in larval water at concentrations sufficient to kill An. gambiae larvae, and treatment of the larval water with a cyanide antidote eliminated larvicidal activity. We conclude that a potential mechanism by which C.sp_P can induce larval mortality is via production of hydrogen cyanide.

The mosquito adulticidal Chromobacterium sp. Panama causes transgenerational impacts on fitness parameters and elicits xenobiotic gene responses.

BACKGROUND: Vector control is critical in reducing the disease burden caused by mosquitoes, and insecticides are an effective tool to control vector populations. Resistance to common insecticides is now widespread, and novel classes of insecticides are needed. In previous work, we described the mosquitocidal activity of Chromobacterium sp. Panama (C.sp_P), a bacterium found in association with mosquitoes in natural populations. In the current work, we further explored the effects of exposure to the bacterium on mosquito fitness and mosquito physiology. RESULTS: We found that C.sp_P has mosquitocidal activity against a broad range of mosquito taxa. When exposed to C.sp_P as adults, female An. gambiae suffered reduced longevity, but experienced no change in fecundity. The offspring of these females, however, had higher mortality as larvae and were slower to develop compared to offspring of control females. We also found that the mosquitocidal activity of C.sp_P was retained after removal of live cells from biofilm culture media, suggesting the bacteria secrete mosquitocidal compound(s) into the media during growth. Exposure to this cell-free C.sp_P-conditioned media caused female midgut transcriptional changes comprising detoxification, xenobiotic response, and stress response genes, suggesting the physiological response to C.sp_P is similar to that of insecticide exposure. Finally, we found that multiple members of the Chromobacterium genus had mosquitocidal activity, but this activity was highest in mosquitoes treated with C.sp_P. CONCLUSIONS: Our findings suggest that C.sp_P produces factor(s) with strong effects on mosquito longevity and fitness, which may be of interest for mosquitocide development. More generally, they indicate that further exploration of mosquito-associated and environmental microbes for novel insecticidal compounds or biocontrol agents is warranted.

Amino acid metabolic signaling influences Aedes aegypti midgut microbiome variability.

The mosquito midgut microbiota has been shown to influence vector competence for multiple human pathogens. The microbiota is highly variable in the field, and the sources of this variability are not well understood, which limits our ability to understand or predict its effects on pathogen transmission. In this work, we report significant variation in female adult midgut bacterial load between strains of A. aegypti which vary in their susceptibility to dengue virus. Composition of the midgut microbiome was similar overall between the strains, with 81-92% of reads coming from the same five bacterial families, though we did detect differences in the presence of some bacterial families including Flavobacteriaceae and Entobacteriaceae. We conducted transcriptomic analysis on the two mosquito strains that showed the greatest difference in bacterial load, and found that they differ in transcript abundance of many genes implicated in amino acid metabolism, in particular the branched chain amino acid degradation pathway. We then silenced this pathway by targeting multiple genes using RNA interference, which resulted in strain-specific bacterial proliferation, thereby eliminating the difference in midgut bacterial load between the strains. This suggests that the branched chain amino acid (BCAA) degradation pathway controls midgut bacterial load, though the mechanism underlying this remains unclear. Overall, our results indicate that amino acid metabolism can act to influence the midgut microbiota. Moreover, they suggest that genetic or physiological variation in BCAA degradation pathway activity may in part explain midgut microbiota variation in the field.

Chromobacterium Csp_P reduces malaria and dengue infection in vector mosquitoes and has entomopathogenic and in vitro anti-pathogen activities.

Plasmodium and dengue virus, the causative agents of the two most devastating vector-borne diseases, malaria and dengue, are transmitted by the two most important mosquito vectors, Anopheles gambiae and Aedes aegypti, respectively. Insect-bacteria associations have been shown to influence vector competence for human pathogens through multi-faceted actions that include the elicitation of the insect immune system, pathogen sequestration by microbes, and bacteria-produced anti-pathogenic factors. These influences make the mosquito microbiota highly interesting from a disease control perspective. Here we present a bacterium of the genus Chromobacterium (Csp_P), which was isolated from the midgut of field-caught Aedes aegypti. Csp_P can effectively colonize the mosquito midgut when introduced through an artificial nectar meal, and it also inhibits the growth of other members of the midgut microbiota. Csp_P colonization of the midgut tissue activates mosquito immune responses, and Csp_P exposure dramatically reduces the survival of both the larval and adult stages. Ingestion of Csp_P by the mosquito significantly reduces its susceptibility to Plasmodium falciparum and dengue virus infection, thereby compromising the mosquito's vector competence. This bacterium also exerts in vitro anti-Plasmodium and anti-dengue activities, which appear to be mediated through Csp_P -produced stable bioactive factors with transmission-blocking and therapeutic potential. The anti-pathogen and entomopathogenic properties of Csp_P render it a potential candidate for the development of malaria and dengue control strategies.

Reproductive status alters transcriptomic response to infection in female Drosophila melanogaster.

Mating and consequent reproduction significantly reduce the ability of female Drosophila melanogaster to defend against systemic bacterial infection. The goal of the present study was to identify genes likely to inform the mechanism of this post-mating immunosuppression. We used microarrays to contrast genome-wide transcript levels in virgin vs. mated females before and after infection. Because the immunosuppressive effect of mating is contingent on the presence of a germline in females, we repeated the entire experiment by using female mutants that do not form a germline. We found that multiple genes involved in egg production show reduced expression in response to infection, and that this reduction is stronger in virgins than it is in mated females. In germline-less females, expression of egg-production genes was predictably low and not differentially affected by infection. We also identified several immune responsive genes that are differentially induced after infection in virgins vs. mated females. Immune genes affected by mating status and egg production genes altered by infection are candidates to inform the mechanism of the trade-off between mating and immune defense.

Dietary plant phenolic improves survival of bacterial infection in Manduca sexta caterpillars.

Plant phenolics are generally thought to play significant roles in plant defense against herbivores and pathogens. Many plant taxa, including Solanaceae, are rich in phenolic compounds and some insect herbivores have been shown to acquire phenolics from their hosts to use them as protection against their natural enemies. Here we demonstrate that larvae of an insect specialist on Solanaceae, the tobacco hornworm, Manduca sexta L. (Lepidoptera: Sphingidae), acquire the plant phenolic chlorogenic acid (CA), and other caffeic acid derivatives as they feed on one of their hosts, Nicotiana attenuata L. (Solanaceae), and on artificial diet supplemented with CA. We test the hypothesis that larvae fed on CA-supplemented diet would have better resistance against bacterial infection than larvae fed on a standard CA-free diet by injecting bacteria into the hemocoel of fourth instars. Larvae fed CA-supplemented diet show significantly higher survival of infection with Enterococcus faecalis (Andrewes & Horder) Schleifer & Kilpper-Bälz, but not of infection with the more virulent Pseudomonas aeruginosa (Schroeter) Migula. Larvae fed on CA-supplemented diet possess a constitutively higher number of circulating hemocytes than larvae fed on the standard diet, but we found no other evidence of increased immune system activity, nor were larvae fed on CA-supplemented diet better able to suppress bacterial proliferation early in the infection. Thus, our data suggest an additional defensive function of CA to the direct toxic inhibition of pathogen proliferation in the gut.

Female Drosophila melanogaster suffer reduced defense against infection due to seminal fluid components.

Reduced defense against infection is commonly observed as a consequence of reproductive activity, but little is known about how post-mating immunosuppression occurs. In this work, we use Drosophila melanogaster as a model to test the role of seminal fluid components and egg production in suppressing post-mating immune defense. We also evaluate whether systemic immune system activity is altered during infection in mated females. We find that post-mating reduction in female defense depends critically on male transfer of sperm and seminal fluid proteins, including the accessory gland protein known as "sex peptide." However, the effect of these male factors is dependent on the presence of the female germline. We find that mated females have lower antimicrobial peptide gene expression than virgin females in response to systemic infection, and that this lower expression correlates with higher systemic bacterial loads. We conclude that, upon receipt of sperm and seminal fluid proteins, females experience a germline-dependent physiological shift that directly or indirectly reduces their overall ability to defend against infection, at least in part through alteration of humoral immune system activity.

Suppression of heat shock protein 27 induces long-term dormancy in human breast cancer.

The mechanisms underlying tumor dormancy have been elusive and not well characterized. We recently published an experimental model for the study of human tumor dormancy and the role of angiogenesis, and reported that the angiogenic switch was preceded by a local increase in VEGF-A and basic fibroblast growth factor. In this breast cancer xenograft model (MDA-MB-436 cells), analysis of differentially expressed genes revealed that heat shock protein 27 (HSP27) was significantly up-regulated in angiogenic cells compared with nonangiogenic cells. The effect of HSP27 down-regulation was further evaluated in cell lines, mouse models, and clinical datasets of human patients with breast cancer and melanoma. Stable down-regulation of HSP27 in angiogenic tumor cells was followed by long-term tumor dormancy in vivo. Strikingly, only 4 of 30 HSP27 knockdown xenograft tumors initiated rapid growth after day 70, in correlation with a regain of HSP27 protein expression. Significantly, no tumors escaped from dormancy without HSP27 expression. Down-regulation of HSP27 was associated with reduced endothelial cell proliferation and decreased secretion of VEGF-A, VEGF-C, and basic fibroblast growth factor. Conversely, overexpression of HSP27 in nonangiogenic cells resulted in expansive tumor growth in vivo. By clinical validation, strong HSP27 protein expression was associated with markers of aggressive tumors and decreased survival in patients with breast cancer and melanoma. An HSP27-associated gene expression signature was related to molecular subgroups and survival in breast cancer. Our findings suggest a role for HSP27 in the balance between tumor dormancy and tumor progression, mediated by tumor-vascular interactions. Targeting HSP27 might offer a useful strategy in cancer treatment.

Epoxyeicosanoids stimulate multiorgan metastasis and tumor dormancy escape in mice.

Epoxyeicosatrienoic acids (EETs) are small molecules produced by cytochrome P450 epoxygenases. They are lipid mediators that act as autocrine or paracrine factors to regulate inflammation and vascular tone. As a result, drugs that raise EET levels are in clinical trials for the treatment of hypertension and many other diseases. However, despite their pleiotropic effects on cells, little is known about the role of these epoxyeicosanoids in cancer. Here, using genetic and pharmacological manipulation of endogenous EET levels, we demonstrate that EETs are critical for primary tumor growth and metastasis in a variety of mouse models of cancer. Remarkably, we found that EETs stimulated extensive multiorgan metastasis and escape from tumor dormancy in several tumor models. This systemic metastasis was not caused by excessive primary tumor growth but depended on endothelium-derived EETs at the site of metastasis. Administration of synthetic EETs recapitulated these results, while EET antagonists suppressed tumor growth and metastasis, demonstrating in vivo that pharmacological modulation of EETs can affect cancer growth. Furthermore, inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and promoted primary tumor growth and metastasis. Thus, our data indicate a central role for EETs in tumorigenesis, offering a mechanistic link between lipid signaling and cancer and emphasizing the critical importance of considering possible effects of EET-modulating drugs on cancer.

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.