PROJECT SUMMARY In both mammals and invertebrates, initiation of post-mating responses in the female post-copulation is known to contribute to reproductive success. Seminal fluid proteins (Sfps) have been shown to initiate many of these responses. However, male x female interactions are underexplored as contributors to infertility. Investigating the mechanisms and evolution of male x female interactions is critical for understanding the complexities of reproduction. I will use D. melanogaster as a model system for investigating post-copulatory male x female interactions via characterization of male and female contributions to the Drosophila mating plug’s (MP) formation and ejection. In Drosophila, the MP forms in the uterus of the female via coagulation of male- ejaculated Sfps and some female reproductive tract proteins. Genetic disruption of the MP impacts reproductive outcomes by affecting sperm retention; however, little is known about the male and female proteins (and their interactions) that modulate mating plug ejection (MPE) rates either directly by contributing to MP composition/degradation or indirectly by regulating female ejection behavior. In Aims 1 & 2 I will respectively use female or male phenotypic variation in the Drosophila Genomic Reference Panel to perform a GWAS on MPE timing. I will then functionally investigate how top gene candidates mediate MPE timing. I have concluded a GWAS on female MPE timing and have so far identified 4 neuronal genes that regulate female MPE (Aim 1). Using highly tissue- and cell type- specific knockdown of gene candidate expression via RNA interference, I will more deeply characterize neuronal regulation of female MPE timing. After also identifying and characterizing male genes regulating MPE timing (Aim2) I will perform a 6x6 grid cross of males and females with disrupted function of MPE genes to investigate the presence of complex non-additive male x female MPE interactions. Finally, I will investigate the molecular evolution and function of male and female genes contributing to MP composition (Aim 3). I discovered that many male and female MP genes are closely paralogous to each other and evolving under positive selection, potentially suggestive of evolution under sexual conflict. After fully characterizing the evolution and coevolution of these male and female MP paralogs, I will investigate their sex-specific functions in MPE and MP formation. Observation of opposing functions for paralogous male and female MP genes would point to sexual conflict driven evolution caused by opposing sex-specific optimal mating strategies. Observation of complementary functions would indicate similar evolutionary pressures acting on male and female paralogs to cooperatively ensure optimal reproductive outcomes.

PROJECT NARRATIVE Male x Female interactions are necessary for reproduction, however, an understanding of the intersexual interactions mediating reproductive phenotypes is still in its nascent stages. By harnessing genetic variation from within and between Drosophila species and the genetic and genomic resources available in D. melanogaster, I will disentangle the contributions from both male and female flies to the mechanisms of mating plug formation and ejection and characterize the potential coevolution of male and female MP interaction partners. My results will lead to an understanding of the intersexual interactions that mediate reproduction and how they are shaped evolutionarily.