The Drosophila melanogaster post-mating response (PMR) is a well-characterized suite of changes that occur after mating, accompanied by a flux in gene expression. Given the significant male contribution to the PMR, as well as increasing understanding of the role of microRNAs (miRNAs) in regulating gene expression, we began our work by determining what role, if any, male miRNAs play in the female PMR. We confirmed the presence of conserved seminal miRNAs in male ejaculate and tested whether interrupting miRNA processing in the male reproductive tract affects the PMR. While largely initiated and influenced by male molecules, we also explore the potential influence of female-endogenous microRNAs (miRNAs) on the PMR in this work. In performing a genetic screen of miRNA mutants, we identified several miRNAs that either increase or decrease the magnitude and duration of the PMR, as measured by assays of fecundity and remating latency. Finally, we developed and deployed four parallel pipelines to reanalyze an existing dataset of two female D. melanogaster tissue types before and after mating. In comparing our study with the previous analysis of this dataset, we find our results to be more stringent, though we do identify a number of significant genes not found before. We also found variation among our own separate experiments, with gene-to-transcript isoform number and index building playing important roles in outcome. We identified a set of genes found by our pipeline that were not identified by the previous study and proposed potential roles for these genes in post-mating biology. Together, this work proposes the possibility of both male and female miRNA affecting the gene expression underlying the PMR and presents a critique of current RNA-seq analysis techniques and presents multiple workflow adjustments that can increase the sensitivity, specificity, and stringency of differential gene expression studies.
