Drosophila female reproductive glands contribute to mating plug composition and the timing of sperm ejection

Reproductive traits that influence female remating and competitive fertilization rapidly evolve in response to sexual selection and sexual conflict. One such trait, observed across diverse animal taxa, is the formation of a structural plug inside the female reproductive tract (FRT), either during or shortly after mating. In Drosophila melanogaster, male seminal fluid forms a mating plug inside the female bursa, which has been demonstrated to influence sperm entry into storage and latency of female remating. Processing of the plug, including its eventual ejection from the female's reproductive tract, influences the competitive fertilization success of her mates and is mediated by female x male genotypic interactions. However, female contributions to plug formation and processing have received limited attention. Using developmental mutants that lack glandular FRT tissues, we reveal that these tissues are essential for mating plug ejection. We further use proteomics to demonstrate that female glandular proteins, and especially proteolytic enzymes, contribute to mating plug composition and have a widespread impact on plug formation and composition. Together, these phenotypic and molecular data identify female contributions to intersexual interactions that are a potential mechanism of post-copulatory sexual selection.