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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 deregulations is the inability to produce specific immunity against target pathogens. We tested this possibility by infecting multiple

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.

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.

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 deregulations 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. However, ageing led to drastic reductions in such specificity to target pathogens, warranting the action of multiple AMPs across Imd and Toll pathways. Moreover, use of diverse AMPs either lacked survival benefits or even accompanied survival costs post-infection. These features were also sexually dimorphic: females required a larger repertoire of AMPs than males but extracted equivalent survival benefits. Finally, age-specific expansion of the AMP-repertoire was accompanied with ageing-induced downregulation of negative-regulators of the Imd pathway and damage to renal function post-infection, as features of poorly regulated immunity. Overall, we could highlight the potentially non-adaptive role of ageing in producing less-specific AMP responses, across sexes and pathogens.

Mitochondria are organelles that produce cellular energy in the form of ATP through oxidative phosphorylation, and this primary function is conserved among 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. Our results demonstrate that Drosophila strains from different locations differ in sleep and activity, and that females are generally more active than males. By comparing activity and sleep of mtDNA variants introgressed onto a common nuclear background in cytoplasmic hybrid (cybrid) strains, we were able to quantify the among-line variance attributable to mitochondrial DNA, and we establish that mtDNA variation affects both activity and sleep, in a sex-specific manner. Altogether our study highlights the important role that mitochondrial genome variation plays on organismal physiology and behaviour.

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 fruit flies ( Drosophila melanogaster ) according to pathogen species, infectious dose, host genetic background and sex. In one experiment, we find that systemic infection with four different bacterial species results in a reduction in the mean pairwise distance within infected female flies, and that the extent of this change depends on pathogen species. However, susceptible flies did not show any evidence of avoidance in the presence of infected flies. In a separate experiment, we observed genetic- and sex-based variation in social aggregation within infected, same-sex groups, 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 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.

The Gram-negative bacterium

Campylobacter coli and C. jejuni, the causing agents of campylobacteriosis, are described to be undergoing introgression events, i.e., the transference of genetic material between different species, with some isolates sharing almost a quarter of its genome. The participation of phages in introgression events and consequent impact on host ecology and evolution remain elusive. Three distinct prophages, named C. jejuni integrated elements 1, 2, and 4 (CJIE1, CJIE2, and CJIE4), are described in C. jejuni. Here, we identified two unreported prophages, Campylobacter coli integrated elements 1 and 2 (CCIE1 and CCIE2 prophages), which are C. coli homologues of CJIE1 and CJIE2, respectively. No induction was achieved for both prophages. Conversely, induction assays on CJIE1 and CJIE2 point towards the inducibility of these prophages. CCIE2-, CJIE1-, and CJIE4-like prophages were identified in a Campylobacter spp. population of 840 genomes, and phylogenetic analysis revealed clustering in three major groups: CJIE1-CCIE1, CJIE2-CCIE2, and CJIE4, clearly segregating prophages from C. jejuni and C. coli, but not from human- and nonhuman-derived isolates, corroborating the flowing between animals and humans in the agricultural context. Punctual bacteriophage host-jumps were observed in the context of C. jejuni and C. coli, and although random chance cannot be fully discarded, these observations seem to implicate prophages in evolutionary introgression events that are modulating the hybridization of C. jejuni and C. coli species., F.F.V. is funded by Fundação para a Ciência e a Tecnologia (FCT) through an Assistant Researcher grant CEECIND/03023/2017, and a project grant (PTDC/BTM-SAL/28978/2017) that supported this work. The work is partially supported by National funds from FCT, projects UIDB/04138/2020 and UIDP/04138/2020. Campylobacter strains were sequenced under the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020—Operational Programme for Competitiveness and Internationalization (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by FCT. This work was also supported by Fundos FEDER through the Programa Operacional Factores de Competitividade—COMPETE and by Fundos Nacionais through the FCT within the scope of the project UID/BIM/00009/2019 (Centre for Toxicogenomics and Human Health-ToxOmics).

Erratum in: Nat Commun. 2023 Mar 20;14(1):1539. doi: 10.1038/s41467-023-37302-5. Helicobacter pylori lives in the human stomach and has a population structure resembling that of its host. However, H. pylori fromEurope and the Middle East trace substantially more ancestry from modern African populations than the humans that carry them. Here, we use a collection of Afro-Eurasian H. pylori genomes to show that this African ancestry is due to at least three distinct admixture events. H. pylori from East Asia, which have undergone little admixture, have accumulated many more non-synonymous mutations than African strains. European and Middle Eastern bacteria have elevated African ancestry at the sites of these mutations, implying selection to remove them during admixture. Simulations show that population fitness can be restored after bottlenecks bymigration and subsequent admixture of small numbers of bacteria from non-bottlenecked populations. We conclude that recent spread of African DNA has been driven by deleterious mutations accumulated during the original out-of-Africa bottleneck. This work was supported by Sequencing Grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (221S0002, 18KK0266, 19H03473, 21H00346 and 22H02871) to Y.Y. F.F.V. is financed by FCT through Assistant Researcher grant CEECIND/03023/2017 and a project grant PTDC/BTM-TEC/3238/ 2020. I.K. studentship was funded by the National Strategic Reference Framework Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014-2020, project No. MIS5002486) and sequencing of strains was supported by the InfeNeutra Project (NSRF 2007-2013, project no. MIS450598) of the Ministry of Culture and Edu- cation, Greece. K.T. and the sequencing of KI isolates was supported by Erik Philip-Sörensen Foundation grant G2016-08, and Swedish Society for Medical research (SSMF). All primary bioinformatics and parts of the comparative genomics were performed on resources provided by Swedish National Infrastructure for Computing (SNIC) through Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX) under projects snic2018-8-24 and uppstore2017270. Work by S.S. was supported by the German Research Foundation (DFG, project number 158 989 968–SFB 900/A1) and by the Bavarian Ministry of Sci- ence and the Arts in the framework of the Bavarian Research Network “New Strategies Against Multi-Resistant Pathogens by Means of Digital Networking—bayresq.net”. D.F. was supported by Shanghai Municipal Science and Technology Major Project No. 2019SHZDZX02. info:eu-repo/semantics/publishedVersion

Early-life conditions have profound effects on many life-history traits, where 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 environmental challenges such as starvation, temperature and desiccation. However, it is less well known how early-life diet influences the consequences of infection in adults. Here we test whether varying the larval diet of female Drosophila melanogaster (through altering protein to carbohydrate ratio, P:C) influences the long-term consequences of injury and infection with the bacterial pathogen Pseudomonas entomophila. Given previous work manipulating adult dietary P:C, we predicted that adults from larvae raised on higher P:C diets would have increased reproduction, but shorter lifespans and an increased rate of ageing, and that the lowest larval P:C diets would be particularly detrimental for adult survival in infected individuals. 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 this was independent of injury and infection. There was no effect of larval P:C on adult survival. Larval development was quickest on intermediate P:C and egg-to-pupae and egg-to-adult viability were slightly higher on higher P:C. Overall, despite larval P:C affecting several measured traits, we saw no evidence that larval P:C altered the consequence of infection or injury for adult survival or early-life and lifetime reproduction. Taken together, these data suggest that larval diets appear to have a limited impact on the adult life history consequences of infection.