Reproductive investment is frequently associated with compromised immune performance. In female Drosophila melanogaster, mating increases susceptibility to bacterial infections, and prior work has demonstrated that juvenile hormone (JH) signaling is necessary for this mating-induced reduction of immune performance. However, the temporal dynamics and key organ systems that mediate this JH-induced immunosuppression have not been studied. Here we show that post-mating sensitivity to bacterial infection depends on JH function spanning multiple life stages and target tissues. We find that mating induces a rapid, transient peak in JH titers and prolonged JH signaling activity. Transient treatment with exogenous JH is not sufficient to increase mortality from infection, suggesting continued JH activity is necessary for the persistent immune suppression observed after mating. Using conditional RNAi, we find that the JH receptor germ cells expressed (Gce) functions in the adipose tissue of developing pupae to potentiate post-mating sensitivity to infection in adults. In adult females, Gce appears to function in multiple tissues, including the fat body and ovary, to decrease infection resistance after mating. These results suggest that Gce function during metamorphic development sensitizes adult females to sustained JH signaling induced by mating. More broadly, our study illustrates how endocrine regulation of the interaction between development and reproduction can shape immune performance and infection outcome.

The ability to direct tissue-specific overexpression of transgenic proteins in genetically tractable organisms like Drosophila melanogaster has facilitated innumerable biological discoveries. However, transgenic proteins can themselves impact cellular and physiological processes in ways that are often ignored or poorly defined. Here we discovered that the yolk-GAL4 transgene, which directs strong expression of the yeast GAL4 transcription factor in the Drosophila fat body, induces significant physiological defects in adult female flies. We found that yolk-GAL4 disrupts adipose tissue integrity and reduces fat body lipid stores, egg production, and resistance to systemic bacterial infections. Knocking down GAL4 expression in yolk-GAL4 heterozygotes using RNAi fully suppressed each of these defects, thus confirming that the GAL4 transgene product induces these phenotypes. Comparing a panel of additional fat body driver lines, we found that GAL4 expression levels directly correlate with infection susceptibility, but not with fat levels or egg production. To determine whether other transgenic proteins can impair fat body function, we constructed new fly lines in which the yolk enhancer directs expression of either cytoplasmic or nuclear-localized mCherry, or an alternative transactivator, LexA. We found that only nuclear-localized mCherry and LexA increased infection susceptibility similarly to GAL4, suggesting that intranuclear transgenic proteins in general can curtail the fat body's induced immune response in a manner highly sensitive to transgene expression strength. Additionally, these new lines can be valuable tools for future studies. More broadly, our findings highlight the potential for transgenes to substantially impact organismal biology and emphasize the importance of rigorously characterizing genetic tools to optimally leverage model systems like Drosophila.

Bacterial infections can substantially impact host metabolic health as a result of the direct and indirect demands of sustaining an immune response and of nutrient piracy by the pathogen itself. Drosophila melanogaster and other insects that survive a sublethal bacterial infection often carry substantial pathogen burdens for the remainder of life. In this study, we asked whether these chronic infections exact metabolic costs for the host, and how these costs scale with the severity of chronic infection. We infected D. melanogaster with four bacterial species ( Providencia rettgeri, Serratia marcescens, Enterococcus faecalis and Lactococcus lactis) and assayed metabolic traits in chronically infected survivors. We found that D. melanogaster carrying chronic infections were uniformly more susceptible to starvation than uninfected controls, and that sensitivity to starvation escalated with higher chronic pathogen burden. We observed some evidence for greater depletion of triglyceride and glycogen stores in D. melanogaster carrying chronic bacterial loads, although this varied among bacterial species. Chronically infected flies exhibit sustained upregulation of the immune response, which we hypothesized might contribute to the metabolic costs. Consistent with this prediction, genetic activation of the major innate immune signaling pathways depleted metabolic stores and increased starvation sensitivity even in the absence of infection. These results demonstrate that even sublethal infections can have substantial health and fitness consequences for the hosts, arising in part from pathogen-induced immune activation, and that the consequences scale quantitatively with the severity of infection.

Endocrine signaling networks control diverse biological processes and life history traits across metazoans. In both invertebrate and vertebrate taxa, steroid hormones regulate immune system function in response to intrinsic and environmental stimuli, such as microbial infection. The mechanisms of this endocrine-immune regulation are complex and constitute an ongoing research endeavor facilitated by genetically tractable animal models. The 20-hydroxyecdysone (20E) is the major steroid hormone in arthropods, primarily studied for its essential role in mediating developmental transitions and metamorphosis; 20E also modulates innate immunity in a variety of insect taxa. This review provides an overview of our current understanding of 20E-mediated innate immune responses. The prevalence of correlations between 20E-driven developmental transitions and innate immune activation are summarized across a range of holometabolous insects. Subsequent discussion focuses on studies conducted using the extensive genetic resources available in Drosophila that have begun to reveal the mechanisms underlying 20E regulation of immunity in the contexts of both development and bacterial infection. Lastly, I propose directions for future research into 20E regulation of immunity that will advance our knowledge of how interactive endocrine networks coordinate animals' physiological responses to environmental microbes.

Symbiotic microorganisms exert multifaceted impacts on the physiology of their animal hosts. Recent discoveries have shown the gut microbiota influence host brain function and behavior, but the host and microbial molecular factors required to actuate these effects are largely unknown. To uncover molecular mechanisms that underlie the gut-microbiota-brain axis, we used Drosophila melanogaster and its bacterial microbiota as a model to identify microbiota-dependent gene expression changes in the host brain and head. Specifically, we employed RNA-seq and nanoString nCounter technology to identify Drosophila genes that exhibit altered transcript levels in fly heads upon elimination of the microbiota. The identified genes, some of which exhibited sex-specific differences, have demonstrated or inferred functional roles in the immune response, metabolism, neuronal activity, and stress resistance. Overall, this study reveals microbiota-responsive genes in the fly head, an anatomical structure not previously investigated in this context. Our results serve as a foundation for future investigations of how microbe-driven gene expression changes impact Drosophila biology.

Symbiotic microorganisms exert multifaceted impacts on the physiology of their animal hosts. Recent discoveries have shown the gut microbiota influence host brain function and behavior, but the host and microbial molecular factors required to actuate these effects are largely unknown. To uncover molecular mechanisms that underlie the gut-microbiota-brain axis, we usedDrosophila melanogasterand its bacterial microbiota as a model to identify microbiota-dependent gene expression changes in the host brain and head. Specifically, we employed RNA-seq and nanoString nCounter technology to identifyDrosophilagenes that exhibit altered transcript levels in fly heads upon elimination of the microbiota. The identified genes, some of which exhibited sex-specific differences, have demonstrated or inferred functional roles in the immune response, metabolism, neuronal activity, and stress resistance. Overall, this study reveals microbiota-responsive genes in the fly head, an anatomical structure not previously investigated in this context. Our results serve as a foundation for future investigations of how microbe-driven gene expression changes impactDrosophilabiology.

The gut microbiota impacts diverse aspects of host biology including metabolism, immunity, and behavior, but the scope of those effects and their underlying molecular mechanisms are poorly understood. To address these gaps, we used Two-dimensional Difference Gel Electrophoresis (2D-DIGE) to identify proteomic differences in male and female Drosophila heads raised with a conventional microbiota and those raised in a sterile environment (axenic). We discovered 22 microbiota-dependent protein differences, and identified a specific elevation in Alcohol Dehydrogenase (ADH) in axenic male flies. Because ADH is a key enzyme in alcohol metabolism, we asked whether physiological and behavioral responses to alcohol were altered in axenic males. Here we show that alcohol induced hyperactivity, the first response to alcohol exposure, is significantly increased in axenic males, requires ADH activity, and is modified by genetic background. While ADH activity is required, we did not detect significant microbe-dependent differences in systemic ADH activity or ethanol level. Like other animals, Drosophila exhibit a preference for ethanol consumption, and here we show significant microbiota-dependent differences in ethanol preference specifically in males. This work demonstrates that male Drosophila’s association with their microbiota affects their physiological and behavioral responses to ethanol.