PROJECT SUMMARY In all animals with internal fertilization, hundreds of seminal fluid proteins (SFPs) are transferred to females during mating. These SFPs have canonically been primarily thought of as a set of male proteins that advance male interests during post-mating processes, including by manipulatively influencing female behavior and reproductive investment. From this perspective, SFPs are thought to be an important substrate of interlocus sexual conflict against which females must continually evolve defensive countermeasures, which has been hypothesized to explain observations that SFPs evolve rapidly. Using single-nucleus RNAseq of the Drosophila melanogaster female reproductive tract (uterus, oviduct, glands, and sperm storage organs), I discovered that females express >40% of the 292 known D. melanogaster SFPs. Moreover, multiple female reproductive cell types specialize in producing SFPs prior to mating. For example, in a cell type in the seminal receptacle, SFP genes comprise 30% of mRNAs in the average sequenced nucleus (up to 65% of mRNAs in some sequenced nuclei). This result demands a re-evaluation of SFP biology that integrates the function and possible selection on the female-contributed protein fraction, which I take on in this proposal by asking: 1) What are the functions of female-expressed SFPs, i.e. shared reproductive secreted proteins? Do the female- and male-contributed protein fractions have the same function? And 2) How do shared secreted reproductive proteins evolve? Do the sex-shared secreted proteins exhibit different evolutionary modalities than the sex-limited SFPs? Do evolutionary and functional patterns in these genes better support a model of cooperation or sexual antagonism? Importantly, for the sex-shared proteins, any potential sexual conflict would have to be intra-locus conflict, with implications for the predicted duration and outcome of the conflict. I address these knowledge gaps using cutting-edge genome engineering methods, specifically gene knock-out and scarless CRISPR insertion of transgenic nanobody-epitope tagged proteins, to allow both immunofluorescent visualization and functional characterization. A second set of experiments uses comparative genomics and transcriptomics in a phylogenetic framework to determine the history of the proteins, the persistence of their expression by each sex, their evolutionary rate, and the evidence for either directional or stabilizing selection on the protein sequences. Because the cell types in D. melanogaster females that specialize in SFP production belong to the sperm storage organs, the proposed work will additionally test whether convergently evolved gains of sperm storage in a third female organ, observed among select non-melanogaster Drosophila species, are associated with gains of SFP expression in additional cell types. In total, the proposed work will provide a missing perspective on a famous set of genes in a premier genetic model organism. Simultaneo...