Proteases and protease inhibitors have been identified in the ejaculates of animal taxa ranging from invertebrates to\nmammals and form a major protein class among Drosophila melanogaster seminal fluid proteins (SFPs). Other than a single\nprotease cascade in mammals that regulates seminal clot liquefaction, no proteolytic cascades (i.e. pathways with at least\ntwo proteases acting in sequence) have been identified in seminal fluids. In Drosophila, SFPs are transferred to females\nduring mating and, together with sperm, are necessary for the many post-mating responses elicited in females. Though\nseveral SFPs are proteolytically cleaved either during or after mating, virtually nothing is known about the proteases\ninvolved in these cleavage events or the physiological consequences of proteolytic activity in the seminal fluid on the\nfemale. Here, we present evidence that a protease cascade acts in the seminal fluid of Drosophila during and after mating.\nUsing RNAi to knock down expression of the SFP CG10586, a predicted serine protease, we show that it acts upstream of the\nSFP CG11864, a predicted astacin protease, to process SFPs involved in ovulation and sperm entry into storage. We also\nshow that knockdown of CG10586 leads to lower levels of egg laying, higher rates of sexual receptivity to subsequent\nmales, and abnormal sperm usage patterns, processes that are independent of CG11864. The long-term phenotypes of\nfemales mated to CG10586 knockdown males are similar to those of females that fail to store sex peptide, an important\nelicitor of long-term post-mating responses, and indicate a role for CG10586 in regulating sex peptide. These results point to\nan important role for proteolysis among insect SFPs and suggest that protease cascades may be a mechanism for precise\ntemporal regulation of multiple post-mating responses in females.
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