Regulation of sex differences in innate immunity by sex-determining gene transformer in Drosophila melanogaster

Sex dimorphism is one of the key features of dioecious organisms, which shapes almost all the aspects of life history traits from early development to cessation of life. However, the specific regulatory differences between males and females that underlie sex dimorphism are underexplored for most quantitative traits, including immune sex dimorphism. Complex patterns of variation, for example, with respect to numbers and types of contributing alleles, dynamic changes in gene expression, and context dependency, have created challenges in understanding the mechanistic basis of sex differences in immunity. To investigate the regulatory basis of sex dimorphism in immunity, we focused on the sex determination hierarchy master switch, transformer ( tra ), in Drosophila melanogaste r. Two different perturbations of sex determination were examined, pseudomales ( tra- mutant) and pseudofemales ( tra- overexpression). Changes in gene expression patterns in response to the infection of Providencia rettgeri , an ex tra cellular gram-negative bacterium, were examined for each of these perturbations and for controls with wild-type sex determination phenotypes. To the best of our knowledge, this is the first study to report the immune response to bacterial infection in any insects with mutant or overexpressed tra genotypes. Survival and bacterial load were first characterized to assess the overall impact of tra on sex differences in immunity. To examine the regulatory role of the tra gene, sex differentially regulated genes were identified in comparisons of wound control to bacterial infected flies for males, females, and tra- mutant or tra- overexpression animals. The survival assay showed that in the absence of the female-specific isoform of tra (Tra ^F ), tra- mutant XX animals showed significantly lower survival than wild-type females, and their survival rate aligned with males. The overexpression of Tra ^F in the XY animals resulted in a surprising outcome, much higher survival of these animals relative to both males and females. The DEG analysis of genes with a significant interaction between sex and infection status identified 235 and 417 genes regulated upstream and downstream of tra in the sex hierarchy pathway, respectively. Interestingly, the GO enrichment analysis found 49 bacterial infection response-related biological processes enriched among genes regulated downstream of tra , including the Toll signaling pathway, and no enrichment for immune response-related categories among genes regulated upstream of tra . Further analysis of the genes and regulators of the Toll signaling pathway identified a significant regulatory role of tra or its downstream targets in signal detection, transduction, cellular response, and regulators of the pathway. Moreover, several genes regulated downstream of tra can regulate IMD pathways via the transcription factor NF-kB-Relish and some of its regulators, such as Diap2/IAP2 and Charon. Overall, the findings highlighted a strong potential role of tra to establish immune sex dimorphism in Drosophila.