Sex Differences in the Drosophila Immune Response: Evolution & Molecular Mechanisms

Abstract

Sex dimorphism is a common fundamental characteristic of sexually reproductive organisms, exerting influence over nearly all aspects of life history and reproduction. The pervasiveness of sexually dimorphic traits is also manifested in morphology and physiology, as well as more subtle traits such as behavior, metabolism, and immunity. Among the less conspicuous sexually dimorphic traits, sex differences in immune responses have been documented in many species, playing a foundational role in shaping host-pathogen interactions and potentially the evolution of the immune response. This dissertation aims to assess whether sexually dimorphic immune responses are conserved or have diverged across species by employing two closely related species, D. melanogaster, and D. simulans, upon infection with extracellular gram-negative bacteria, Providencia rettgeri. The results of the interspecific comparison (Part 2) pointed to common sex differences in immune gene expression, as well as species-specific differences in survival and gene expression, indicating that the sex determination pathway may commonly regulate sex differences in the expression of immune response genes. To pursue this question, I conducted experiments (Part 3) aimed at testing the hypothesis that a key central regulator of the somatic sex determination pathway, transformer, regulates immune sex dimorphism. The final part of the dissertation (Part 4) follows up on this direction of research by investigating the possible regulatory function of one of the transcription factors regulated by transformer, doublesex, which is a highly conserved gene within the sex determination pathway across diverse taxa and could be a common regulator in the context of immune response differences between sexes. To answer these questions, the studies employed survival, bacterial load, bacterial load upon death, and transcriptomic profiling. It's important to note that this dissertation study marks the first instance of an immune challenge in D. simulans, and it provides a unique comparative analysis with D. melanogaster under the same experimental conditions. In addition, this is the first attempt to understand the regulatory role of TraF and both dsx isoforms DsxF and DsxM in the context of bacterial infection and sexually dimorphic immune responses in insects. The comparative analysis of D. melanogaster and D. simulans (Part 2) found that there are sex differences in the immune response of these closely related species. Female flies generally have a stronger humoral response than male flies in both species, but the level of immune gene upregulation is higher in D. melanogaster. The study also found that IMD and Toll pathways are differentially expressed in the two species, with D. melanogaster flies showing a higher level of upregulation. Additionally, D. simulans may employ a mechanism that is different from that of D. melanogaster to activate the Toll receptors. These findings suggest that sex-based variation in the immune response is indeed conserved in both species to a certain extent. However, notable differences arise between these two closely related species of Drosophila, which may be related to differences in their life history and ecological niche. In addition to investigating the underlying molecular mechanisms, the findings of this study will inform future experiments to confirm whether the identified sexually dimorphic responses and potential molecular mechanisms identified for the response to Providencia rettgeri infections are consistent with responses to other types of immune challenges. The analysis of the tra mutation and overexpression genotypes (Part 3) identified a significant role of tra in regulating sex differences in immunity in D. melanogaster. Flies without functional TraF, either wild-type males or TraF mutant animals, have lower survival rates than wild-type females and are more similar to each other. Overexpression of tra in XY animals results in survival rates that are higher than both wild-type females and males. The analysis of up and downstream sex-differentially expressed targets of tra identified 417 genes regulated by or downstream of tra. Many of these genes are involved in the Toll and IMD signaling pathways, which are the key bacterial immune response pathways. Additionally, some sex differentially expressed genes, including several immune genes, are regulated upstream of the tra gene, indicating that they may be regulated by other branches of the sex determination pathway or as yet unidentified mechanisms. These findings suggest that the tra gene plays a crucial role in regulating immune sex dimorphism. The final study reported in this dissertation (Part 4) found that even in the absence of expression of dsx, females and males maintain sex-differential efficacy of immune responses in D. melanogaster, as revealed by survival differences. This might indicate that the majority of immune sex dimorphism that underlies survival differences is not explainable by dsx regulation. However, this study did identify several immune genes that are regulated by DsxF and DsxM, with different modes of regulation of target genes by DsxF in females and DsxM in males, which demonstrates the complexities of immune sex differences. DsxF and DsxM modulate core immune response pathway genes, including Toll, IMD, and Jak-STAT (Hop-Stat92E), in a sex-dimorphic manner. Doublesex plays a role in regulating sex dimorphic immune gene expression in Drosophila, but its role is a more complex interplay of the different isoforms DsxF and DsxM. Overall, like many other aspects of life history and reproduction, the complexity of immune sex dimorphism is intricately intertwined with hormonal, genomic, and environmental impact; as a result, these differences may not be attributable to a single mechanism, pathway, or gene alone. This study provides new insights into the molecular basis of immune sex dimorphism in Drosophila. The findings suggest sex differences in immunity are more likely regulated by tra and its target genes. As dsx is one of the target genes of tra, and isoforms of the dsx showed can also substantially explain part of the immune sex dimorphism in gene expression in Drosophila, it is certain that the sex determination pathway is regulating a major part of these differences along with other molecular mechanisms. However, differences in survival appear to be primarily regulated by tra and not by dsx; therefore, future studies are needed to investigate this less well-known branch of the sex determination pathway. The results of this dissertation also underscore the necessity of further research in this area, not only with respect to other genes of this cascade but also with tools that will reveal the dynamic changes in expression during the course of the infection. This will result in a deeper understanding of how the sex determination pathway contributes to the establishment of immune sex dimorphism.