The seminal odorant binding protein Obp56g is required for mating plug formation and male fertility inDrosophila melanogaster

InDrosophila melanogasterand other insects, the seminal fluid proteins (SFPs) and male sex pheromones that enter the female with sperm during mating are essential for fertility and induce profound post-mating effects on female physiology and behavior. The SFPs inD. melanogasterand other taxa include several members of the large gene family known as odorant binding proteins (Obps). Previous work inDrosophilahas shown that someObpgenes are highly expressed in the antennae and can mediate behavioral responses to odorants, potentially by binding and carrying these molecules to odorant receptors. These observations have led to the hypothesis that the seminal Obps might act as molecular carriers for pheromones or other compounds important for male fertility in the ejaculate, though functional evidence in any species is lacking. Here, we used RNAi and CRISPR/Cas9 generated mutants to test the role of the seven seminal Obps inD. melanogasterfertility and the post-mating response (PMR). We found thatObp56gis required for male fertility and the induction of the PMR, whereas the other six genes had no effect on fertility when mutated individually.Obp56gis expressed in the male’s ejaculatory bulb, an important tissue in the reproductive tract that synthesizes components of the mating plug. We found males lackingObp56gfail to form a mating plug in the mated female’s reproductive tract, leading to ejaculate loss and reduced sperm storage. We also examined the evolutionary history of these seminalObpgenes, as several studies have documented rapid evolution and turnover of SFP genes across taxa. We found extensive lability in gene copy number and evidence of positive selection acting on two genes,Obp22aandObp51a. Comparative RNAseq data from the male reproductive tract of multipleDrosophilaspecies revealed thatObp56gshows high male reproductive tract expression only in species of themelanogasterandobscuragroups, though conserved head expression in all species tested. Together, these functional and expression data suggest thatObp56gmay have been co-opted for a reproductive function over evolutionary time.