PROJECT SUMMARY The Drosophila female reproductive tract is morphologically and functionally complex, and as the arena where high-stakes interactions among multiple male ejaculates and female products play out, it is also likely subject to strong and conflicting selection pressures. Moreover, the reproductive tract is the initiation site for a cascade of post-mating responses in female biology, from sequestering sperm and ovipositing to changes in neural gene expression and behavior. Many of these processes may undergo sexually antagonistic selection, as outcomes affect the sexes’ divergent reproductive interests. Nevertheless, research investment in the lower female reproductive tract, including the sperm storage organs and the female accessory glands, severely lags investment in male reproductive tissues. For example, the seminal receptacle, uterus, and female accessory glands have been excluded from every major community transcriptome analysis; resources like Gal-4 drivers or marker gene labels generally do not exist for these tissues; and the function and evolution of several organs remain poorly described. These omissions limit insight into a range of important questions, including the mechanisms and evolution of female-ejaculate interactions, the extent of rapid evolution in reproductive tissues and its drivers, and the outcomes of sexual conflict. Yet these omissions also highlight an opportunity for new low-barrier, high return investigations using well-established technologies in a leading model organism, which this proposal pursues. First, single-cell sequencing of mated and unmated females from two diverged natural populations will be used to 1) characterize cell-type diversity in the female reproductive tract for the first time and 2) measure gene expression and divergence in the 5 somatic female reproductive tissues at cellular resolution (Specific Aim 1). These data will also test the hypothesis that gene expression in female reproductive tissues diverges rapidly owing to coevolving interactions with seminal fluid proteins. Next, genes that are candidates for important functional evolution will be knocked out and evaluated for reproductive effects. Specific Aim 2 tests null alleles of 7 receptors that are expressed in the seminal receptacle and show indications of rapid divergence, to test the hypothesis that these receptors mediate female post-mating responses. Specific Aim 3 uses a set of de novo genes with novel expression in the seminal receptacle to explore the mysterious process of gene birth and functional integration. Specifically, the de novo gene candidates that appear most likely to be functionally integrated based on transcript length, expression level, structural complexity, and other metrics, will be knocked out and evaluated for effects on female reproductive function.