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Invertebrates lack the specialized immune-memory cells responsible for vertebrate-like acquired immunity. However, there is increasing evidence that past infection by the same pathogen can ‘prime’ the insect immune response, resulting in improved survival upon reinfection. The mechanisms underlying these phenomenological accounts of priming are diverse, and often not completely clear. Here, we investigated the generality, specificity and mechanistic basis of immune priming in the fruit flyDrosophila melanogasterwhen infected with the gram-negative bacterial pathogenProvidencia rettgeri. We further explore the epidemiological consequences of immune priming and find it has the potential to curtail pathogen transmission by reducing pathogen shedding. We find that priming inDrosophilais a long-lasting, pathogen-specific response, occurring in several fly genetic backgrounds and is particularly stronger in male flies. Mechanistically, we find that the enhanced survival of individuals primed with an initial non-lethal bacterial inoculum coincides with a transient decrease in bacterial loads, and that this is likely driven by the IMD-responsive antimicrobial-peptideDiptericin-Bin the fat body. Further, we show that whileDiptericinsare required as the effector of bacterial clearance, it is not solely sufficient for immune priming, and requires regulation by the peptidoglycan recognition proteinsPGRP-LB, PGRP-LCandPGRP-LE. We discuss potential explanations for the observed sex differences in priming, and discuss the epidemiological consequences of innate immune priming in invertebrates.Author summaryImmunisation using attenuated or inactivated pathogens remains one of the most successful public health practices to reduce the incidence of infectious diseases. Immunisation works because humans and other vertebrate animals have evolved an immune response capable of specific immune memory, which ensures a strong, precise, and effective response to a secondary infection. While invertebrates lack the vertebrate-like specialized immune memory cells responsible for acquired immunity, there is now substantial evidence that invertebrates are capable of immune priming, characterised by improved survival to a pathogenic infection following an initial exposure to an attenuated or inactivated pathogen. Here, we investigated the occurrence, generality, specificity and mechanistic basis of immune priming in fruit flies when infected with the gram-negative pathogenProvidencia rettgeri. We find that priming with an initial non-lethal bacterial inoculum results increased survival after a secondary lethal challenge with the same live bacterial pathogen, and that this protective response may last at least two weeks after the initial exposure, is particularly strong in male flies, and occurs in several genetic backgrounds. We also find that primed flies shed less pathogen during infection, suggesting that priming can be a useful tool to reduce pathogen transmission in insects.

Mitochondria participate in various cellular processes including energy metabolism, apoptosis, stress responses, inflammation and immunity. While mitochondrial dysfunction contributes to many diseases, mitochondrial perturbations can also be beneficial, a phenomenon coined mitohormesis. We investigated how mitochondrial variation contributes to the heterogeneity of infection outcomes and whether moderate mitochondrial dysfunction could benefit immune-challenged individuals. We took three approaches to model variation in mitochondrial oxidative phosphorylation (OXPHOS) in Drosophila melanogaster: i) inherited natural mitochondrial DNA (mtDNA) variation present in all tissues; ii) sporadic, tissue-specific silencing of OXPHOS-related genes and iii) systemic pharmacological inhibition of OXPHOS, and studied their effects on the cell-mediated innate immune response. When perturbing mitochondrial function, we detected signs of activated cellular innate immunity without infection and upon infection the perturbation caused enhanced immune response. Our data indicate that mitochondrial dysfunction can be beneficial in immune-challenged individuals when it is mild enough and does not cause complications to the host, and ultimately causes variation in infection outcomes between individuals.Author summaryMitochondria are eukaryotic cell organelles involved in various cellular processes including metabolism and cell signaling. The role of mitochondria and the variation of mitochondrial function in immune responses have been increasingly studied across various models. Mitochondrial dysfunction, originating from either the nuclear or the mitochondrial genome, is generally considered harmful for the organism. What is not yet well understood is how immune responses are affected by mitochondrial dysfunction, for example in the context of mitochondrial disease. Here, we show that in the fruit fly Drosophila melanogaster mild systemic or immune cell -specific mitochondrial perturbations enhance the innate immune response of the host to parasitoids. Furthermore, we show that due to the mitochondrial perturbations the cell-mediated immune system of the host is activated in the absence of infection and makes the immune responses more efficient upon infection. Importantly, our results demonstrate that mitochondrial genome variation is one of the underlying factors contributing to the variation in infection outcomes among individuals. Our results therefore contribute to the understanding of innate immunity, by providing information about the genetic causes underlying the variation seen in the response to infections.

Evolutionary theory predicts a late-life decline in the force of natural selection, possibly leading to late-life deregulations of the immune system. A potential outcome of such immune-deregulation is the inability to produce specific immunity against target pathogens. We tested this possibility by infecting multiple Drosophila melanogaster lines (with bacterial pathogens) across age-groups, where either individual or different combinations of Imd- and Toll-inducible antimicrobial peptides (AMPs) were deleted using CRISPR gene editing. We show a high degree of non-redundancy and pathogen-specificity of AMPs in young flies: in some cases, even a single AMP could confer complete resistance. In contrast, ageing led to a complete loss of such specificity, warranting the action of multiple AMPs across Imd- and Toll-pathways during infections. Moreover, use of diverse AMPs either had no survival benefits, or even accompanied survival costs post-infection. These features were also sexually dimorphic: females expressed a larger repertoire of AMPs than males, but extracted equivalent survival benefits. Finally, age-specific expansion of the AMP-pool was associated with downregulation of negative-regulators of the Imd-pathway and a potential damage to renal function, as features of poorly-regulated immunity, Overall, we could establish ageing as an important driver of nonspecific AMP responses, across sexes and bacterial infections.

Genetic variation for both resistance and disease tolerance has been described in a range of species infected with bacterial, viral and fungal pathogens. In Drosophila melanogaster, genetic variation in mortality following systemic Drosophila C Virus (DCV) infection has been shown to be driven by large effect polymorphisms in the viral restriction factor pastrel (pst). However, it is unclear if pst impacts variation in DCV titres (i.e. resistance), or if it also contributes to disease tolerance. We investigated systemic infection across a range of DCV challenge doses spanning nine orders of magnitude, in males and females of ten Drosophila Genetic Reference Panel (DGRP) lines carrying either a susceptible (S) or resistant (R) pst allele. Our results uncover among-line variation in fly survival, viral titers, and disease tolerance measured both as the ability to maintain survival (mortality tolerance) and reproduction (fecundity tolerance). We confirm the role of pst in resistance, as fly lines with the resistant (R) pst allele experienced lower viral titers, and we uncover novel effects of pst on host vigor, as flies carrying the R allele exhibited higher survival and fecundity even in the absence of infection. Finally, we found significant variation in the expression of the JAK-STAT ligand upd3 and the epigenetic regulator of JAK-STAT G9a. While G9a has been previously shown to mediate tolerance of DCV infection, we found no correlation between the expression of either upd3 or G9a on fly tolerance or resistance. Our work highlights the importance of both resistance and tolerance in viral defence.

Identifying how infection modifies host behaviours that determine social contact networks is important for understanding heterogeneity in infectious disease dynamics. Here, we investigate whether group social behaviour is modified during bacterial infection in Drosophila melanogaster, an established system for behavioural genetics, according to pathogen species, infectious dose, host genetic background and sex. We find that systemic infection with four different bacterial species results in a reduction in the mean pairwise distance within infected flies, and that the extent of this change depends on the infectious dose in a pathogen-specific way. In the presence of infected conspecifics, susceptible flies also tended to aggregate throughout time, but did not show any evidence of avoiding infected flies. We also observed genetic- and sex-based variation in social aggregation, with infected female flies aggregating more closely than infected males. In general, our results confirm that bacterial infection induces changes in fruit fly behaviour across a range of pathogen species, but also highlight that these effects vary between fly genetic backgrounds and can be sex-specific. We discuss possible explanations for sex differences in social aggregation and their consequences for individual variation in pathogen transmission.

Mitochondria are organelles that produce cellular energy in the form of ATP through oxidative phosphorylation, and this primary function is conserved between many taxa. Locomotion is a trait that is highly reliant on metabolic function and expected to be greatly affected by disruptions to mitochondrial performance. To this end, we aimed to examine how activity and sleep vary between Drosophila melanogaster strains with different geographic origins, how these patterns are affected by mitochondrial DNA (mtDNA) variation, and how breaking up co-evolved mito-nuclear gene combinations affect the studied activity traits. The results demonstrate that Drosophila strains from different locations differ in sleep and activity, and the extent of variation differs between sexes, females in general being more active. By comparing activity and sleep of mtDNA variants introgressed onto a common nuclear background in cytoplasmic hybrid (cybrid) strains, we establish that mtDNA variation affects both traits, sex specifically. Furthermore, by using previously published mtDNA copy number data, we detected a positive correlation between mtDNA copy number and the activity levels of the cybrid flies. Altogether, our study shows that both mtDNA variation and mitonuclear interactions affect activity and sleep patterns, highlighting the important role that both genomes play on life-history trait evolution.

The insect gut is frequently exposed to pathogenic threats and must not only clear these potential infections, but also tolerate relatively high microbe loads. In contrast to the mechanisms that eliminate pathogens, we currently know less about the mechanisms of disease tolerance. We investigated how well-described mechanisms that prevent, signal, control or repair damage during infection contribute to the phenotype of disease tolerance. We established enteric infections with the bacterial pathogen Pseudomonas entomophila in transgenic lines of Drosophila melanogaster fruit flies affecting dcy (a major component of the peritrophic matrix), upd3 (a cytokine-like molecule), irc (a negative regulator of reactive oxygen species) and egfr 1 (epithelial growth factor receptor). Flies lacking dcy experienced the highest mortality, while loss of function of either irc or upd3 reduced tolerance in both sexes. The disruption of egfr 1 resulted in a severe loss in tolerance in male flies but had no substantial effect on the ability of female flies to tolerate P. entomophila infection, despite carrying greater microbe loads than males. Together, our findings provide evidence for the role of damage limitation mechanisms in disease tolerance and highlight how sexual dimorphism in these mechanisms could generate sex differences in infection outcomes.

Disease tolerance describes a hosts ability to maintain health independently of the ability to clear microbe loads. However, we currently know little about the mechanisms that underlie disease tolerance or how known mechanisms of tissue damage signalling and repair may contribute to variation in tolerance. The Jak/Stat pathway plays a pivotal role in Drosophila humoral innate immunity, signalling tissue damage and triggering cellular renewal, making it a potential mechanism underlying the disease tolerance phenotype. Here, we show that disrupting the Jak/Stat pathway in Drosophila melanogaster alters disease tolerance during Pseudomonas entomophila systemic infection. Overall, flies with disrupted Jak/Stat show variation in survival that is not explained by variation in pathogen loads. For instance, mutations disrupting the function of ROS producing dual oxidase (duox) or the negative regulator of Jak/Stat, Socs36E render males less tolerant to systemic bacterial infection but not females. We also investigated whether the negative regulator of Jak/Stat, G9a which has previously been associated with tolerance of viral infections is also implicated in tolerance of bacterial infection. While female flies lacking G9a showed higher mortality and reduced bacterial clearance, disease tolerance did not differ between G9a mutants and the wildtype. This suggests that G9a does not affect tolerance during systemic bacterial infection as it appears to do with viral infection. Overall, our findings highlight that Jak/Stat signalling mediates disease tolerance during systemic bacterial infection and that this response differs between males and females. Our work therefore suggests that differences in Jak/Stat mediated disease tolerance may be a potential source of sexually dimorphic response to infection in Drosophila.

Disease tolerance is an infection phenotype where hosts show relatively high health despite harbouring elevated pathogen loads. Compared to the mechanisms of immune clearance our knowledge of the mechanisms underlying increased tolerance remains incomplete. Variation in the ability to reduce immunopathology may explain why some hosts can tolerate higher pathogen burdens with reduced pathology. Negative immune regulation would therefore appear to be a clear candidate for a mechanism underlying disease tolerance but this has not been tested directly for bacterial infections. Here, we examined how the negative regulation of the immune deficiency (IMD) pathway affects disease tolerance in Drosophila melanogaster when infected with the gram-negative bacterial pathogen Pseudomonas entomophila. We find that UASRNAi-mediated reduced expression of the negative regulators of IMD (pirk and caudal) severely reduced the ability to tolerate infection in both males and females across a wide range of infectious doses. While flies unable to regulate the IMD response exhibited higher expression of antimicrobial peptides and lower bacterial loads as expected, this was not accompanied by a proportional reduction in mortality. Instead, tolerance (measured as fly survival relative to its microbe load) was drastically reduced, likely due to the combination of increased immunopathology and cytotoxicity of elevated AMP expression. Our results therefore highlight that in addition to regulating an efficient pathogen clearance response, negative regulators of IMD also contribute to disease tolerance.

All genomes mutate but the consequences of the resulting deleterious mutational load are poorly understood. Helicobacter pylori lives in the human stomach, has a higher mutation rate than most bacteria and has accompanied anatomically modern humans in migrations including the out-of-Africa expansion more than 50,000 years ago. H. pylori from East Asia have accumulated at least 500 more non-synonymous mutations than African strains, which we propose is due to reduced efficacy of selection during the out-of-Africa bottleneck. H. pylori from Europe and the Middle East trace a substantially higher fraction of ancestry from modern African populations than the humans that carry them, which we find is due to at least three separate admixture events. African ancestry is elevated at positions in the genome where non-synonymous mutations are at high frequency in Asia. We propose that this is due to replacement of deleterious mutations that accumulated during the bottleneck, with the high overall African ancestry proportion due to clonal expansion of strains of African origin. We use simulations to show that a Muller9s ratchet like effect can lead to long-term segregation of deleterious mutations within bacterial populations after a bottleneck, despite high rates of homologous recombination, but that population fitness can be restored by migration of small numbers of bacteria from non-bottlenecked populations. Our results demonstrate that population bottlenecks can have long-term genomic and demographic consequences, even in species with enormous population sizes.

Ostreococcus tauri is a ubiquitous marine pico-eukaryote that is susceptible to lysis upon infection by its species specific Ostreococcus tauri viruses (OtVs). In natural populations of O. tauri, costs of resistance are usually invoked to explain the persistence or reappearance of susceptible individuals in resistant populations. Given the low costs of resistance measured in laboratory experiments with the O. tauri/OtV system to date, the question remains of why susceptible individuals persist in the wild at all. Epidemiological models of host and pathogen population dynamics are one useful approach to understand the conditions that can allow the coexistence of susceptible and resistant hosts. We used a SIR (Susceptible-Infected-Resistant) model to investigate epidemiological dynamics under different laboratory culturing regimes that are commonly used in the O.tauri/OtV system. When taking into account serial transfer (i.e. batchcycle lengths) and dilution rates as well as different resistance costs, our model predicts that no susceptible cells should be detected under any of the simulated conditions – this is consistent with laboratory findings. We thus considered an alternative model that is not used in laboratory experiments, but which incorporates one key process in natural populations: host populations are periodically re-seeded with new infective viruses. In this model, susceptible individuals re-occurred in the population, despite low costs of resistance. This suggests that periodic attack by new viruses, rather than (or in addition to) costs of resistance, may explain the high proportion of susceptible hosts in natural populations, and underlie the discrepancy between laboratory studies and observations of fresh isolates.ImportanceIn natural samples of Ostreococcus sp. and its associated viruses, susceptible hosts are common. However, in laboratory experiments, fully resistant host populations readily and irreversibly evolve. Laboratory experiments are powerful methods for studying process because they offer a stripped-down simplification of a complex system, but this simplification may be an oversimplification for some questions. For example, laboratory and field systems of marine microbes and their viruses differ in population sizes and dynamics, mixing or migration rates, and species diversity, all of which can dramatically alter process outcomes. We demonstrate the utility of using epidemiological models to explore experimental design and to understand mechanisms underlying host-virus population dynamics. We highlight that such models can be used to form strong, testable hypotheses about which key elements of natural systems need to be included in laboratory systems to make them simplified, rather than oversimplified, versions of the processes we use them to study.

Early-life conditions have profound effects on many life-history traits. In particular, early-life diet affects both juvenile development, and adult survival and reproduction. Early-life diet also has consequences for the ability of adults to withstand stressors such as starvation, temperature and desiccation. However, it is less well known how early-life diet influences the ability of adults to respond to infection. Here we test whether varying the larval diet of femaleDrosophila melanogaster(through altering protein to carbohydrate ratio, P:C) influences the long-term response to injury and infection with the bacterial pathogenPseudomonas entomophila. Given previous work manipulating adult dietary P:C, we predicted that adults from larvae raised on higher P:C diets would be more likely to survive infection and have increased reproduction, but shorter lifespans and an increased rate of ageing. For larval development, we predicted that low P:C would lead to a longer development time and lower viability. We found that early-life and lifetime egg production were highest at intermediate to high larval P:C diets, but there was no effect of larval P:C on adult survival. Larval diet had no effect on survival or reproduction post-infection. Larval development was quickest on intermediate P:C and egg-to-pupae and egg-to-adult viability were higher on higher P:C. Overall, despite larval P:C affecting several traits measured in this study, we saw no evidence that larval P:C altered the consequence of infection or injury for adult survival and early-life and lifetime reproduction. Taken together, these data suggest that larval diets appear to have a limited impact on adult response to infection.

Parental care is a key component of an organism’s reproductive strategy that is thought to trade-off with allocation towards immunity. Yet it is unclear how caring parents respond to pathogens: do infected parents reduce their amount of care as a sickness behaviour or simply from being ill, or do they prioritise their offspring by maintaining high levels of care? Here we explored the consequences of infection by the pathogenSerratia marcescenson mortality, time spent providing care, reproductive output, and expression of immune genes of female parents in the burying beetleNicrophorus vespilloides. We compared untreated control females with infected females that were inoculated with live bacteria, immune-challenged females that were inoculated with heat-killed bacteria, and injured females that were injected with buffer. We found that infected and immune-challenged females mounted an immune response and that infected females suffered increased mortality. Nevertheless, infected and immune-challenged females maintained their normal level of care and reproductive output. There was thus no evidence that infection led to either a decrease or an increase in parental care or reproductive output. Our results show that parental care, which is generally highly flexible, can remain remarkably robust and consistent despite the elevated mortality caused by infection by pathogens. Overall, these findings suggest that infected females maintain a high level of parental care; a strategy that may ensure that offspring receive the necessary amount of care but that might be detrimental to the parents’ own survival or that may even facilitate disease transmission to offspring.

Dietary restriction (DR), limiting calories or specific nutrients, extends lifespan across diverse taxa. This lifespan extension has been explained as diet-mediated changes in the trade-off between lifespan and reproduction, with survival favoured with scarce resources. Another evolutionary hypothesis suggests the selective benefit of the response is the maintenance of reproduction. This hypothesis predicts that lifespan extension is a side effect of benign laboratory conditions, where DR individuals are frailer and unable to deal with additional stressors, and thus lifespan extension should disappear under more stressful conditions. We tested this by rearing outbred female Drosophila melanogaster on 10 different protein:carbohydrate diets. Flies were either infected with a bacterial pathogen (Pseudomonas entomophila), injured or unstressed. We monitored lifespan, fecundity and ageing measures. DR extended lifespan and reduced reproduction irrespective of injury and infection. These results do not support lifespan extension under DR being a side effect of benign laboratory conditions.

Host heterogeneity in disease transmission is widespread and presents a major hurdle to predicting and minimizing pathogen spread. Using the Drosophila melanogaster model system infected with Drosophila C virus, we integrate experimental measurements of individual host heterogeneity in social aggregation, virus shedding, and disease-induced mortality into an epidemiological framework that simulates outbreaks of infectious disease. We use these simulations to calculate individual variation in disease transmission and apportion this variation to specific components of transmission: social network degree distribution, infectiousness, and infection duration. The experimentally-observed variation produces substantial differences in individual transmission potential, providing evidence for genetic and sex-specific effects on disease dynamics at a population level. Manipulating variation in social network connectivity, infectiousness, and infection duration in simulated populations reveals that these components affect disease transmission in clear and distinct ways. We consider the implications of this genetic and sex-specific variation in disease transmission and discuss implications for appropriate control methods given the relative contributions made by social aggregation, virus shedding, and infection duration to transmission in other host-pathogen systems.

Heterogeneity in disease transmission is widespread and, when not accounted for, can produce unpredictable outbreaks of infectious disease. Despite this, precisely how different sources of variation in host traits drive heterogeneity in disease transmission is poorly understood. Here we dissected the sources of variation in pathogen transmission using Drosophila melanogaster and Drosophila C Virus as a host-pathogen model system. We found that infected lifespan, viral growth, virus shedding, and viral load at death were all significantly influenced by fly genetic background, sex and female mating status. To understand how variation in each of these traits may generate heterogeneity in disease transmission, we estimated individual transmission potential by integrating data on virus shedding and lifespan alongside previously collected data on social aggregation. We found that ∼15% of between-individual heterogeneity in disease transmission was explained by a significant interaction between genetic and sex-specific variation. We also characterised the amount of variation in viral load, virus shedding, and lifespan following infection that could be explained by genetic background and sex. Amongst the determinants of individual variation in disease transmission these sources of host variation play roles of varying importance, with genetic background generally playing the largest role. Our results highlight the importance of characterising sources of variation in multiple host traits when studying disease transmission at the individual-level.

Deciding where to eat and raise offspring carries important fitness consequences for all animals, especially if foraging, feeding and reproduction increase the risk of exposure to pathogens. In insects with complete metamorphosis, foraging occurs mainly during the larval stage, while oviposition decisions are taken by adult-stage females. Selection for infection avoidance behaviours may therefore be developmentally uncoupled. Using a combination of experimental infections and behavioural choice assays, here we tested if Drosophila melanogaster fruit flies avoid potentially infectious environments at distinct developmental stages. When given conspecific fly carcasses as a food source, larval-stage flies did not discriminate between carcasses that were clean or infected with the pathogenic Drosophila C Virus (DCV), even though scavenging was a viable route of DCV transmission. Adult females however, discriminated between different oviposition sites, laying more eggs near a clean rather than an infectious carcass if they were healthy; DCV-infected females did not discriminate between the two environments. While potentially risky, laying eggs near potentially infectious carcasses was always preferred to sites containing only fly medium. Our findings suggest that infection avoidance can play an important role in how mothers provision their offspring, and underline the need to consider infection avoidance behaviours at multiple life-stages.

The ability to tolerate infection is a key component of host defence and offers potential novel therapeutic approaches for infectious diseases. To yield successful targets for therapeutic intervention, it is important that the analytical tools employed to measure disease tolerance are able to capture distinct host responses to infection. Here, we show that commonly used methods that estimate tolerance as a linear relationship may be inadequate, and that more flexible, non-linear estimates of this relationship may reveal variation in distinct components of host defence. To illustrate this, we measured the survival of Drosophila melanogaster carrying either a functional or non-functional regulator of the JAK-STAT immune pathway (G9a) when challenged with a range of concentrations of Drosophila C Virus (DCV). While classical linear model analyses indicated that G9a affected tolerance only in females, a more powerful non-linear logistic model showed that G9a mediates viral tolerance to different extents in both sexes. This analysis also revealed that G9a acts by changing the sensitivity to increasing pathogen burdens, but does not reduce the ultimate severity of infection. These results indicate that fitting non-linear models to host health-pathogen burden relationships may offer better and more detailed estimates of disease tolerance.

Viruses are major evolutionary drivers of insect immune systems. Much of our knowledge of insect immune responses derives from experimental infections using the fruit flyDrosophila melanogaster.Most experiments, however, employ lethal pathogen doses through septic injury, frequently overwhelming host physiology. While this approach has revealed a number of immune mechanisms, it is less informative about the fitness costs hosts may experience during infection in the wild. Using both systemic and oral infection routes we find that even apparently benign, sub-lethal infections with the horizontally transmitted Drosophila C Virus (DCV) can cause significant physiological and behavioral morbidity that is relevant for host fitness. We describe DCV-induced effects on fly reproductive output, digestive health, and locomotor activity, and we find that viral morbidity varies according to the concentration of pathogen inoculum, host genetic background and sex. Notably, sub-lethal DCV infection resulted in a significant increase in fly reproduction, but this effect depended on host genotype. We discuss the relevance of sub-lethal morbidity forDrosophilaecology and evolution, and more broadly, we remark on the implications of deleterious and beneficial infections for the evolution of insect immunity.

Wolbachia-mediatedprotection against viral infection has been extensively demonstrated in Drosophila and in mosquitoes that are artificially inoculatedwith D. melanogaster Wolbachia (wMel), but to date no evidence for Wolbachia-mediated antibacterial protection has been demonstrated in Drosophila.Here we show that D. melanogaster carrying wMel shows reduced mortality during enteric – but not systemic - infection with the opportunist pathogen Pseudomonas aeruginosa, and that protection is more pronounced in male flies. Wolbachia-mediated protection is associated with increased early expression of the antimicrobial peptide attacinA, followed by increased expression of a ROS detoxification gene (gstD8), and other tissue damage repair genes which together contribute to greater host resistance and disease tolerance. These results highlight that the route of infection is important for symbiont-mediated protection from infection, that Wolbachia can protect hosts by eliciting a combination of resistance and disease tolerance mechanisms, and that these effects are sexually dimorphic.

Infection avoidance behaviours are the first line of defence against pathogenic encounters. Behavioural plasticity in response to internal or external cues can therefore generate heterogeneity in infection. We tested whether Drosophila melanogaster exhibits infection avoidance behaviour during foraging, and whether this behaviour is modified by prior exposure to Drosophila C Virus (DCV) and by the risk of DCV encounter. We examined two measures of infection avoidance: (1) the motivation to feed in the presence of an infection risk and (2) the preference to feed on a clean food source over a potentially infectious source. We found no clear evidence for preference of clean food sources over potentially infectious ones. However, infection avoidance was present in female flies, which were less motivated to feed when presented with a risk of encountering DCV, but this was only the case if they had been previously exposed to this viral pathogen. We discuss the relevance of plasticity in avoidance behaviours during ecologically relevant scenarios such as foraging for host fitness and pathogen spread.

Recent insights into the human microbiome offer the hope of manipulating its composition to help fight infectious diseases. While this strategy has shown huge potential, its consequences for pathogen evolution have not been explored. Here we show that manipulating the microbiome to increase the competition that pathogens face could lead to the evolution of increased production of virulence factors that pathogens use to combat commensals, an evolutionary response that can increase total disease induced mortality in the long-term. However, if treatment with microbiome engineering is sufficiently aggressive this evolutionary response can be avoided and the pathogen eradicated. Furthermore, we show that using damage limitation therapies (e.g. anti-virulence and anti-inflammatory drugs) in combination with microbiome manipulation increases the potential for pathogen eradication. While manipulating our microbiota offers a promising alternative to antibiotics, our results show that these treatments must be designed with careful consideration of the potential evolutionary responses of target pathogens.