Sustained Post-Mating Response in Drosophila melanogaster Requires Multiple Seminal Fluid Proteins

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Abstract
Successful reproduction is critical to pass genes to the next generation. Seminal proteins contribute to important reproductive processes that lead to fertilization in species ranging from insects to mammals. In Drosophila, the male's accessory gland is a source of seminal fluid proteins that affect the reproductive output of males and females by altering female post-mating behavior and physiology. Protein classes found in the seminal fluid of Drosophila are similar to those of other organisms, including mammals. By using RNA interference (RNAi) to knock down levels of individual accessory gland proteins (Acps), we investigated the role of 25 Acps in mediating three post-mating female responses: egg production, receptivity to remating and storage of sperm. We detected roles for five Acps in these post-mating responses. CG33943 is required for full stimulation of egg production on the first day after mating. Four other Acps (CG1652, CG1656, CG17575, and CG9997) appear to modulate the long-term response, which is the maintenance of post-mating behavior and physiological changes. The long-term post-mating response requires presence of sperm in storage and, until now, had been known to require only a single Acp. Here, we discovered several novel Acps together are required which together are required for sustained egg production, reduction in receptivity to remating of the mated female and for promotion of stored sperm release from the seminal receptacle. Our results also show that members of conserved protein classes found in seminal plasma from insects to mammals are essential for important reproductive processes. In sexually reproducing organisms, sperm enter the female in combination with seminal proteins that are critical for fertility. These proteins can activate sperm or enhance sperm storage within the female, and can improve the chance that sperm will fertilize eggs. Understanding the action of seminal proteins has potential utility in insect pest control and in the diagnosis of certain human infertilities. However, the precise function of very few seminal proteins is known. To address this, we knocked down the levels of 25 seminal proteins individually in male fruit flies, and tested the males' abilities to modulate egg production, sperm storage/release, or behavior of their mates. We found five seminal proteins that are necessary to elevate offspring production in mated females. Four of these proteins are needed for efficient release of sperm from storage to fertilize eggs, a function that had not been previously assigned to any seminal protein. All four are in biochemical classes that are conserved in seminal fluid from insects to humans, suggesting they may play similar sperm-related roles in other animals. In addition to assigning functions to particular seminal proteins, our results suggest that fruit flies can serve as a model with which to dissect the functions of conserved protein classes in seminal fluid.