Complete genome sequence of Pseudomonas entomophila strain TVIN-A01.

The complete genome sequence of Pseudomonas entomophila strain TVIN-A01 is reported in this manuscript. It is a commonly used laboratory strain of Pseudomonas entomophila known to infect insects and, hence, often studied in host-pathogen interactions. Oxford Nanopore sequencing and Illumina sequencing were performed to assemble the genome completely.

Intraspecific genetic variation in host vigour, viral load and disease tolerance during Drosophila C virus infection.

Genetic variation for resistance and disease tolerance has been described in a range of species. In Drosophila melanogaster, genetic variation in mortality following systemic Drosophila C virus (DCV) infection is driven by large-effect polymorphisms in the restriction factor pastrel (pst). However, it is unclear if pst contributes to disease tolerance. We investigated systemic DCV challenges spanning nine orders of magnitude, in males and females of 10 Drosophila Genetic Reference Panel lines carrying either a susceptible (S) or resistant (R) pst allele. We find among-line variation in fly survival, viral load and disease tolerance measured both as the ability to maintain survival (mortality tolerance) and reproduction (fecundity tolerance). We further uncover novel effects of pst on host vigour, as flies carrying the R allele exhibited higher survival and fecundity even in the absence of infection. Finally, we found significant genetic variation in the expression of the JAK-STAT ligand upd3 and the epigenetic regulator of JAK-STAT G9a. However, 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.

Inherent constraints on a polyfunctional tissue lead to a reproduction-immunity tradeoff.

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

A robust method to isolate Drosophila fat body nuclei for transcriptomic analysis.

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

Experimental evolution reveals sex-specific dominance for surviving bacterial infection in laboratory populations of Drosophila melanogaster.

Males and females are subjected to distinct kinds of selection pressures, often leading to the evolution of sex-specific genetic architecture, an example being sex-specific dominance. Sex-specific dominance reversals (SSDRs), where alleles at sexually antagonistic loci are at least partially dominant in the sex they benefit, have been documented in Atlantic salmon, rainbow trout, and seed beetles. Another interesting feature of many sexually reproducing organisms is the asymmetric inheritance pattern of X chromosomes, which often leads to distinct evolutionary outcomes on X chromosomes compared to autosomes. Examples include the higher efficacy of sexually concordant selection on X chromosomes, and X chromosomes being more conducive to the maintenance of sexually antagonistic polymorphisms under certain conditions. Immunocompetence is a trait that has been extensively investigated for sexual dimorphism with growing evidence for sex-specific or sexually antagonistic variation. X chromosomes have been shown to harbor substantial immunity-related genetic variation in the fruit fly, Drosophila melanogaster. Here, using interpopulation crosses and cytogenetic cloning, we investigated sex-specific dominance and the role of the X chromosome in improved postinfection survivorship of laboratory populations of D. melanogaster selected against pathogenic challenge by Pseudomonas entomophila. We could not detect any contribution of the X chromosome to the evolved immunocompetence of our selected populations, as well as to within-population variation in immunocompetence. However, we found strong evidence of sex-specific dominance related to surviving bacterial infection. Our results indicate that alleles that confer a survival advantage to the selected populations are, on average, partially dominant in females but partially recessive in males. This could also imply an SSDR for overall fitness, given the putative evidence for sexually antagonistic selection affecting immunocompetence in Drosophila melanogaster. We also highlight sex-specific dominance as a potential mechanism of sex differences in immunocompetence, with population-level sex differences primarily driven by sex differences in heterozygotes.

Absence of reproduction-immunity trade-off in male Drosophila melanogaster evolving under differential sexual selection.

BACKGROUND: The theory of trade-off suggests that limited resources should lead to trade-off in resource intensive traits such as, immunity related and sexually selected traits in males. Alternatively, sexual exaggerations can also act as an honest indicator of underlying immunocompetence, leading to positive correlations between these traits. Evidences in support of either hypothesis in invertebrates are equivocal. Whereas several studies have addressed this question, few have used naturally occurring pathogens and realized post infection realized immunity (e.g., survivorship) to assay the fitness correlations between these two sets of traits. RESULTS: Adopting an experimental evolution approach, we evolved replicate populations of Drosophila melanogaster under high and low sexual selection regimes for over a hundred generations and found the following in virgin and mated males in three separate assays: a.Post infection survivorship against two natural pathogens - Pseudomonas entomophila (Pe) and Staphylococcus succinus (Ss): Mated males survived better against Pe, but were no different than virgins against Ss.b.Bacterial clearance ability against a third natural pathogen Providencia rettgeri (Pr): Mated males had significantly lower CFUs than virgins. However, sexual selection history had no effect on realized immunity of either virgin or mated males. CONCLUSION: We show that while mating can affect realized immunity in a pathogen specific way, sexual selection did not affect the same. The results highlight that complex polygenic traits such as immunity and reproductive traits not necessarily evolve following a binary trade-off model. We also stress the importance natural pathogens while studying sexual selection-immunity correlations.

Evolution of the metabolome in response to selection for increased immunity in populations of Drosophila melanogaster.

We used NMR-based metabolomics to test two hypotheses-(i) there will be evolved differences in the metabolome of selected and control populations even under un-infected conditions and (ii) post infection, the metabolomes of the selected and control populations will respond differently. We selected replicate populations of Drosophila melanogaster for increased survivorship (I) against a gram-negative pathogen. We subjected the selected (I) and their control populations (S) to three different treatments: (1) infected with heat-killed bacteria (i), (2) sham infected (s), and (3) untreated (u). We performed 1D and 2D NMR experiments to identify the metabolic differences. Multivariate analysis of the metabolic profiles of the untreated (Iu and Su) flies yielded higher concentrations of lipids, organic acids, sugars, amino acids, NAD and AMP in the Iu treatment as compared to the Su treatment, showing that even in the absence of infection, the metabolome of the I and S regimes was different. In the S and I regimes, post infection/injury, concentration of metabolites directly or indirectly associated with energy related pathways (lipids, organic acids, sugars) declined while the concentration of metabolites that are probably associated with immune response (amino acids) increased. However, in most cases, the I regime flies had a higher concentration of such metabolites even under un-infected conditions. The change in the metabolite concentration upon infection/injury was not always comparable between I and S regimes (in case of lactate, alanine, leucine, lysine, threonine) indicating that the I and S regimes had evolved to respond differentially to infection and to injury.

Nonlinear disease tolerance curves reveal distinct components of host responses to viral infection.

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 should be complemented with more flexible, nonlinear estimates of this relationship which may reveal variation in distinct components such as host vigour, sensitivity to increases in pathogen loads, and the severity of the infection. 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 nonlinear 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 disease. These results indicate that fitting nonlinear models to host health-pathogen burden relationships may offer better and more detailed estimates of disease tolerance.

The route of infection determines Wolbachia antibacterial protection in Drosophila.

Bacterial symbionts are widespread among metazoans and provide a range of beneficial functions. Wolbachia-mediated protection against viral infection has been extensively demonstrated in Drosophila. In mosquitoes that are artificially transinfected with Drosophila melanogaster Wolbachia (wMel), protection from both viral and bacterial infections has been demonstrated. However, no evidence for Wolbachia-mediated antibacterial protection has been demonstrated in Drosophila to date. Here, we show that the route of infection is key for Wolbachia-mediated antibacterial protection. Drosophila melanogaster carrying Wolbachia showed reduced mortality during enteric-but not systemic-infection with the opportunist pathogen Pseudomonas aeruginosaWolbachia-mediated protection was more pronounced in male flies and is associated with increased early expression of the antimicrobial peptide Attacin A, and also increased expression of a reactive oxygen species detoxification gene (Gst D8). 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. We discuss the importance of using ecologically relevant routes of infection to gain a better understanding of symbiont-mediated protection.

No apparent cost of evolved immune response in Drosophila melanogaster.

Maintenance and deployment of the immune system are costly and are hence predicted to trade-off with other resource-demanding traits, such as reproduction. We subjected this longstanding idea to test using laboratory experimental evolution approach. In the present study, replicate populations of Drosophila melanogaster were subjected to three selection regimes-I (Infection with Pseudomonas entomophila), S (Sham-infection with MgSO4 ), and U (Unhandled Control). After 30 generations of selection flies from the I regime had evolved better survivorship upon infection with P. entomophila compared to flies from U and S regimes. However, contrary to expectations and previous reports, we did not find any evidence of trade-offs between immunity and other life history related traits, such as longevity, fecundity, egg hatchability, or development time. After 45 generations of selection, the selection was relaxed for a set of populations. Even after 15 generations, the postinfection survivorship of populations under relaxed selection regime did not decline. We speculate that either there is a negligible cost to the evolved immune response or that trade-offs occur on traits such as reproductive behavior or other immune mechanisms that we have not investigated in this study. Our research suggests that at least under certain conditions, life-history trade-offs might play little role in maintaining variation in immunity.

Evolution of mate-harm, longevity and behaviour in male fruit flies subjected to different levels of interlocus conflict.

BACKGROUND: Interlocus conflict predicts (a) evolution of traits, beneficial to males but detrimental to females and (b) evolution of aging and life-span under the influence of the cost of bearing these traits. However, there are very few empirical investigations shedding light on these predictions. Those that do address these issues, mostly reported response of male reproductive traits or the lack of it and do not address the life-history consequence of such evolution. Here, we test both the above mentioned predictions using experimental evolution on replicate populations of Drosophila melanogaster. We present responses observed after >45 generations of altered levels of interlocus conflict (generated by varying the operational sex ratio). RESULTS: Males from the male biased (high conflict, M-regime) regime evolved higher spontaneous locomotor activity and courtship frequency. Females exposed to these males were found to have higher mortality rate. Males from the female biased regime (low conflict, F-regime) did not evolve altered courtship frequency and activity. However, progeny production of females continuously exposed to F-males was significantly higher than the progeny production of females exposed to M-males indicating that the F-males are relatively benign towards their mates. We found that males from male biased regime lived shorter compared to males from the female biased regime. CONCLUSION: F-males (evolving under lower levels of sexual conflict) evolved decreased mate harming ability indicating the cost of maintenance of the suit of traits that cause mate-harm. The M-males (evolving under higher levels sexual conflict) caused higher female mortality indicating that they had evolved increased mate harming ability, possibly as a by product of increased reproduction related activity. There was a correlated evolution of life-history of the M and F-males. M-regime males lived shorter compared to the males from F-regime, possibly due to the cost of investing more in reproductive traits. In combination, these results suggest that male reproductive traits and life-history traits can evolve in response to the altered levels of interlocus sexual conflict.

Sexual activity increases resistance against Pseudomonas entomophila in male Drosophila melanogaster.

BACKGROUND: Maintenance and deployment cost of immunity is high, therefore, it is expected to trade-off with other high cost traits like sexual activity. Previous studies with Drosophila melanogaster show that male's ability to clear bacteria decreases with increase in sexual activity. We subjected this idea to test using two pathogens (Pseudomonas entomophila and Staphylococcus succinus) and three different populations of Drosophila melanogaster. RESULTS: We found that sexual activity enhanced male survivorship in a pathogen specific manner. Sexually active males show higher resistance than virgins upon infection with Pseudomonas entomophila. Interestingly, the beneficial effects of sexual activity increased with time of co-habitation with females and declined when access to females was restricted. We observed no change in male survivorship upon experimentally varying the number of sexual interactions. CONCLUSION: Our results show that the sexual activity-immunity trade-off in males cannot be generalised. The trade-off is potentially mediated through complex interactions between the host, pathogen and the environment experienced by the host.

Sperm competitive ability evolves in response to experimental alteration of operational sex ratio.

In naturally polygamous organisms such as Drosophila, sperm competitive ability is one of the most important components of male fitness and is expected to evolve in response to varying degrees of male-male competition. Several studies have documented the existence of ample genetic variation in sperm competitive ability of males. However, many experimental evolution studies have found sperm competitive ability to be unresponsive to selection. Even direct selection for increased sperm competitive ability has failed to yield any measurable changes. Here we report the evolution of sperm competitive ability (sperm defense-P1, offense-P2) in a set of replicate populations of Drosophila melanogaster subjected to altered levels of male-male competition (generated by varying the operational sex ratio) for 55-60 generations. Males from populations with female-biased operational sex ratio evolved reduced P1 and P2, without any measurable change in the male reproductive behavior. Males in the male-biased regime evolved increased P1, but there was no significant change in P2. Increase in P1 was associated with an increase in copulation duration, possibly indicating greater ejaculate investment by these males. This study is one of the few to provide empirical evidence for the evolution of sperm competitive ability of males under different levels of male-male competition.