Corrigendum: Interactions between innate immunity and insulin signaling affect resistance to infection in insects.

[This corrects the article DOI: 10.3389/fimmu.2023.1276357.].

Facultatively ectoparasitic mites as vectors for entomopathogenic bacteria in Drosophila.

Opportunistic bacterial infections are common in insect populations but there is little information on how they are acquired or transmitted. We tested the hypothesis that Macrocheles mites can transmit systemic bacterial infections between Drosophila hosts. We found that 24% of mites acquired detectable levels of bacteria after feeding on infected flies and 87% of infected mites passed bacteria to naïve recipient flies. The probability that a mite could pass Serratia from an infected donor fly to a naïve recipient fly was 27.1%. These data demonstrate that Macrocheles mites are capable of serving as vectors of bacterial infection between insects.

Interactions between innate immunity and insulin signaling affect resistance to infection in insects.

An active immune response is energetically demanding and requires reallocation of nutrients to support resistance to and tolerance of infection. Insulin signaling is a critical global regulator of metabolism and whole-body homeostasis in response to nutrient availability and energetic needs, including those required for mobilization of energy in support of the immune system. In this review, we share findings that demonstrate interactions between innate immune activity and insulin signaling primarily in the insect model Drosophila melanogaster as well as other insects like Bombyx mori and Anopheles mosquitos. These studies indicate that insulin signaling and innate immune activation have reciprocal effects on each other, but that those effects vary depending on the type of pathogen, route of infection, and nutritional status of the host. Future research will be required to further understand the detailed mechanisms by which innate immunity and insulin signaling activity impact each other.

Microbiota-mediated competition between Drosophila species.

BACKGROUND: The influence of microbiota in ecological interactions, and in particular competition, is poorly known. We studied competition between two insect species, the invasive pest Drosophila suzukii and the model Drosophila melanogaster, whose larval ecological niches overlap in ripe, but not rotten, fruit. RESULTS: We discovered D. suzukii females prevent costly interspecific larval competition by avoiding oviposition on substrates previously visited by D. melanogaster. More precisely, D. melanogaster association with gut bacteria of the genus Lactobacillus triggered D. suzukii avoidance. However, D. suzukii avoidance behavior is condition-dependent, and D. suzukii females that themselves carry D. melanogaster bacteria stop avoiding sites visited by D. melanogaster. The adaptive significance of avoiding cues from the competitor's microbiota was revealed by experimentally reproducing in-fruit larval competition: reduced survival of D. suzukii larvae only occurred if the competitor had its normal microbiota. CONCLUSIONS: This study establishes microbiotas as potent mediators of interspecific competition and reveals a central role for context-dependent behaviors under bacterial influence. Video Abstract.

Leveraging insect-specific viruses to elucidate mosquito population structure and dynamics.

Several aspects of mosquito ecology that are important for vectored disease transmission and control have been difficult to measure at epidemiologically important scales in the field. In particular, the ability to describe mosquito population structure and movement rates has been hindered by difficulty in quantifying fine-scale genetic variation among populations. The mosquito virome represents a possible avenue for quantifying population structure and movement rates across multiple spatial scales. Mosquito viromes contain a diversity of viruses, including several insect-specific viruses (ISVs) and "core" viruses that have high prevalence across populations. To date, virome studies have focused on viral discovery and have only recently begun examining viral ecology. While nonpathogenic ISVs may be of little public health relevance themselves, they provide a possible route for quantifying mosquito population structure and dynamics. For example, vertically transmitted viruses could behave as a rapidly evolving extension of the host's genome. It should be possible to apply established analytical methods to appropriate viral phylogenies and incidence data to generate novel approaches for estimating mosquito population structure and dispersal over epidemiologically relevant timescales. By studying the virome through the lens of spatial and genomic epidemiology, it may be possible to investigate otherwise cryptic aspects of mosquito ecology. A better understanding of mosquito population structure and dynamics are key for understanding mosquito-borne disease ecology and methods based on ISVs could provide a powerful tool for informing mosquito control programs.

No evidence for trans-generational immune priming in Drosophila melanogaster.

Most organisms are under constant and repeated exposure to pathogens, leading to perpetual natural selection for more effective ways to fight-off infections. This could include the evolution of memory-based immunity to increase protection from repeatedly-encountered pathogens both within and across generations. There is mixed evidence for intra- and trans-generational priming in non-vertebrates, which lack the antibody-mediated acquired immunity characteristic of vertebrates. In this work, we tested for trans-generational immune priming in adult offspring of the fruit fly, Drosophila melanogaster, after maternal challenge with 10 different bacterial pathogens. We focused on natural opportunistic pathogens of Drosophila spanning a range of virulence from 10% to 100% host mortality. We infected mothers via septic injury and tested for enhanced resistance to infection in their adult offspring, measured as the ability to suppress bacterial proliferation and survive infection. We categorized the mothers into four classes for each bacterium tested: those that survived infection, those that succumbed to infection, sterile-injury controls, and uninjured controls. We found no evidence for trans-generational priming by any class of mother in response to any of the bacteria.

No evidence for trans-generational immune priming in Drosophila melanogaster.

Most organisms are under constant and repeated exposure to pathogens, leading to perpetual natural selection for more effective ways to fight-off infections. This could include the evolution of memory-based immunity to increase protection from repeatedly-encountered pathogens both within and across generations. There is mixed evidence for intra- and trans-generational priming in non-vertebrates, which lack the antibody-mediated acquired immunity characteristic of vertebrates. In this work, we tested for trans-generational immune priming in adult offspring of the fruit fly, Drosophila melanogaster , after maternal challenge with 10 different bacterial pathogens. We focused on natural opportunistic pathogens of Drosophila spanning a range of virulence from 10% to 100% host mortality. We infected mothers via septic injury and tested for enhanced resistance to infection in their adult offspring, measured as the ability to suppress bacterial proliferation and survive infection. We categorized the mothers into four classes for each bacterium tested: those that survived infection, those that succumbed to infection, sterile-injury controls, and uninjured controls. We found no evidence for trans-generational priming by any class of mother in response to any of the bacteria.

Infection biology: Molecular recognition of fungal spores stimulates host hygiene.

Hygienic behaviors that remove pathogens can be crucial in preventing disease. But how are such behaviors stimulated? A new study shows that Drosophila recognize proteins on the surface of Metarhizium spores as a cue to initiate grooming and spore removal.

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 single mating is sufficient to induce persistent reduction of immune defense in mated female Drosophila melanogaster.

In many species, female reproductive investment comes at a cost to immunity and resistance to infection. Mated Drosophila melanogaster females are more susceptible to bacterial infection than unmated females. Transfer of the male seminal fluid protein Sex Peptide reduces female post-mating immune defense. Sex Peptide is known to cause both short- and long-term changes to female physiology and behavior. While previous studies showed that females were less resistant to bacterial infection as soon as 2.5 h and as long as 26.5 h after mating, it is unknown whether this is a binary switch from mated to unmated state or whether females can recover to unmated levels of immunity. It is additionally unknown whether repeated mating causes progressive reduction in defense capacity. We compared the immune defense of mated females when infected at 2, 4, 7, or 10 days after mating to that of unmated females and saw no recovery of immune capacity regardless of the length of time between mating and infection. Because D. melanogaster females can mate multiply, we additionally tested whether a second mating, and therefore a second transfer of seminal fluids, caused deeper reduction in immune performance. We found that females mated either once or twice before infection survived at equal proportions, both with significantly lower probability than unmated females. We conclude that a single mating event is sufficient to persistently suppress the female immune system. Interestingly, we observed that induced levels of expression of genes encoding antimicrobial peptides (AMPs) decreased with age in both experiments, partially obscuring the effects of mating. Collectively, the data indicate that being reproductively active versus reproductively inactive are alternative binary states with respect to female D. melanogaster immunity. The establishment of a suppressed immune status in reproductively active females can inform our understanding of the regulation of immune defense and the mechanisms of physiological trade-offs.

Balancing sensitivity, risk, and immunopathology in immune regulation.

Activation of an immune response is energetically costly and excessive immune system activity can result in immunopathology, yet a slow or insufficient immune response carries the risk of pathogen establishment with consequent pathology arising from the infection. Mathematical theory and empirical data demonstrate that hosts balance the costs of immunity against the risk of infection by closely regulating immunological dynamics. An optimal immune system is rapidly and robustly deployed against a true infectious threat and rapidly deactivated once the threat has been controlled. Genetic variation in the sensitivity of an immune system, as well as in the activation and shutdown kinetics of host immune responses, can contribute to the evolution of pathogen virulence and host tolerance of infection. Improved understanding of the adaptive forces that operate on immune regulatory dynamics will clarify fundamental principles governing the evolution and maintenance of innate immune systems.

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.

The molecular architecture of Drosophila melanogaster defense against Beauveria bassiana explored through evolve and resequence and quantitative trait locus mapping.

Little is known about the genetic architecture of antifungal immunity in natural populations. Using two population genetic approaches, quantitative trait locus (QTL) mapping and evolve and resequence (E&R), we explored D. melanogaster immune defense against infection with the fungus Beauveria bassiana. The immune defense was highly variable both in the recombinant inbred lines from the Drosophila Synthetic Population Resource used for our QTL mapping and in the synthetic outbred populations used in our E&R study. Survivorship of infection improved dramatically over just 10 generations in the E&R study, and continued to increase for an additional nine generations, revealing a trade-off with uninfected longevity. Populations selected for increased defense against B. bassiana evolved cross resistance to a second, distinct B. bassiana strain but not to bacterial pathogens. The QTL mapping study revealed that sexual dimorphism in defense depends on host genotype, and the E&R study indicated that sexual dimorphism also depends on the specific pathogen to which the host is exposed. Both the QTL mapping and E&R experiments generated lists of potentially causal candidate genes, although these lists were nonoverlapping.

Drosophila Evolution over Space and Time (DEST): A New Population Genomics Resource.

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome data sets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate data sets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in >20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This data set, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental metadata. A web-based genome browser and web portal provide easy access to the SNP data set. We further provide guidelines on how to use Pool-Seq data for model-based demographic inference. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan data set. Our resource will enable population geneticists to analyze spatiotemporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.

Broad geographic sampling reveals the shared basis and environmental correlates of seasonal adaptation in Drosophila.

To advance our understanding of adaptation to temporally varying selection pressures, we identified signatures of seasonal adaptation occurring in parallel among Drosophila melanogaster populations. Specifically, we estimated allele frequencies genome-wide from flies sampled early and late in the growing season from 20 widely dispersed populations. We identified parallel seasonal allele frequency shifts across North America and Europe, demonstrating that seasonal adaptation is a general phenomenon of temperate fly populations. Seasonally fluctuating polymorphisms are enriched in large chromosomal inversions, and we find a broad concordance between seasonal and spatial allele frequency change. The direction of allele frequency change at seasonally variable polymorphisms can be predicted by weather conditions in the weeks prior to sampling, linking the environment and the genomic response to selection. Our results suggest that fluctuating selection is an important evolutionary force affecting patterns of genetic variation in Drosophila.

Host-pathogen immune feedbacks can explain widely divergent outcomes from similar infections.

A long-standing question in infection biology is why two very similar individuals, with very similar pathogen exposures, may have very different outcomes. Recent experiments have found that even isogenic Drosophila melanogaster hosts, given identical inoculations of some bacterial pathogens at suitable doses, can experience very similar initial bacteria proliferation but then diverge to either a lethal infection or a sustained chronic infection with much lower pathogen load. We hypothesized that divergent infection outcomes are a natural result of mutual negative feedbacks between pathogens and the host immune response. Here, we test this hypothesis in silico by constructing process-based dynamic models for bacterial population growth, host immune induction and the feedbacks between them, based on common mechanisms of immune system response. Mathematical analysis of a minimal conceptual model confirms our qualitative hypothesis that mutual negative feedbacks can magnify small differences among hosts into life-or-death differences in outcome. However, explaining observed features of chronic infections requires an extension of the model to include induced pathogen modifications that shield themselves from host immune responses at the cost of reduced proliferation rate. Our analysis thus generates new, testable predictions about the mechanisms underlying bimodal infection outcomes.

Antimicrobial peptides: Application informed by evolution.

Antimicrobial peptides (AMPs) are essential components of immune defenses of multicellular organisms and are currently in development as anti-infective drugs. AMPs have been classically assumed to have broad-spectrum activity and simple kinetics, but recent evidence suggests an unexpected degree of specificity and a high capacity for synergies. Deeper evaluation of the molecular evolution and population genetics of AMP genes reveals more evidence for adaptive maintenance of polymorphism in AMP genes than has previously been appreciated, as well as adaptive loss of AMP activity. AMPs exhibit pharmacodynamic properties that reduce the evolution of resistance in target microbes, and AMPs may synergize with one another and with conventional antibiotics. Both of these properties make AMPs attractive for translational applications. However, if AMPs are to be used clinically, it is crucial to understand their natural biology in order to lessen the risk of collateral harm and avoid the crisis of resistance now facing conventional antibiotics.

Nephrocytes Remove Microbiota-Derived Peptidoglycan from Systemic Circulation to Maintain Immune Homeostasis.

Preventing aberrant immune responses against the microbiota is essential for the health of the host. Microbiota-shed pathogen-associated molecular patterns translocate from the gut lumen into systemic circulation. Here, we examined the role of hemolymph (insect blood) filtration in regulating systemic responses to microbiota-derived peptidoglycan. Drosophila deficient for the transcription factor Klf15 (Klf15NN) are viable but lack nephrocytes-cells structurally and functionally homologous to the glomerular podocytes of the kidney. We found that Klf15NN flies were more resistant to infection than wild-type (WT) counterparts but exhibited a shortened lifespan. This was associated with constitutive Toll pathway activation triggered by excess peptidoglycan circulating in Klf15NN flies. In WT flies, peptidoglycan was removed from systemic circulation by nephrocytes through endocytosis and subsequent lysosomal degradation. Thus, renal filtration of microbiota-derived peptidoglycan maintains immune homeostasis in Drosophila, a function likely conserved in mammals and potentially relevant to the chronic immune activation seen in settings of impaired blood filtration.

Consequences of chronic bacterial infection in Drosophila melanogaster.

Even when successfully surviving an infection, a host often fails to eliminate a pathogen completely and may sustain substantial pathogen burden for the remainder of its life. Using systemic bacterial infection in Drosophila melanogaster, we characterize chronic infection by three bacterial species from different genera - Providencia rettgeri, Serratia marcescens, and Enterococcus faecalis-following inoculation with a range of doses. To assess the consequences of these chronic infections, we determined the expression of antimicrobial peptide genes, survival of secondary infection, and starvation resistance after one week of infection. While higher infectious doses unsurprisingly lead to higher risk of death, they also result in higher chronic bacterial loads among the survivors for all three infections. All three chronic infections caused significantly elevated expression of antimicrobial peptide genes at one week post-infection and provided generalized protection again secondary bacterial infection. Only P. rettgeri infection significantly influenced resistance to starvation, with persistently infected flies dying more quickly under starvation conditions relative to controls. These results suggest that there is potentially a generalized mechanism of protection against secondary infection, but that other impacts on host physiology may depend on the specific pathogen. We propose that chronic infections in D. melanogaster could be a valuable tool for studying tolerance of infection, including impacts on host physiology and behavior.

Sperm success and immunity.

The moment of the fertilization of an egg by a spermatozoon-the point of "sperm success"-is a key milestone in the biology of sexually reproducing species and is a fundamental requirement for offspring production. Fertilization also represents the culmination of a suite of sexually selected processes in both sexes and is commonly used as a landmark to measure reproductive success. Sperm success is heavily dependent upon interactions with other key aspects of male and female biology, with the immune system among the most important. The immune system is vital to maintaining health in both sexes; however, immune reactions can also have antagonistic effects on sperm success. The effects of immunity on sperm success are diverse, and may include trade-offs in the male between investment in the production or protection of sperm, as well as more direct, hostile, immune responses to sperm within the female, and potentially the male, reproductive tract. Here, we review current understanding of where the biology of immunity and sperm meet, and identify the gaps in our knowledge.