Expanded characterization and localization of male seminal fluid proteins within the female reproductive tract of the dengue vector mosquito Aedes aegypti

Abstract

Aedes aegypti mosquitoes transmit numerous viruses that impact human health. Contemporary biological control programs aim to reduce Aedes fertility despite our limited understanding of interactions between the sexes required for reproduction. During mating, males transfer seminal fluid proteins (SFPs) to females which alter their post-mating behavior, physiology and gene regulation, but the contribution of individual SFPs to fertility remains uncharacterized. In Drosophila, a small subset of SFPs localize to the sperm storage organs and oviducts or enter the hemolymph which suggests their participation in specific post-mating processes. We used mass spectrometry-based proteomics in conjunction with whole animal heavy labelling to expand the characterization of the Ae. aegypti ejaculate and identify SFPs that leave the site of insemination and localize to other female tissues. We identified 1031 ejaculate proteins, including a suite of novel SFPs. The expanded ejaculate proteome shows low conservation amongst SFPs when compared to insect model Drosophila, consistent with rapid evolutionary turnover at the genetic and proteomic levels. Further, we identify 25 SFPs that localize to the spermathecae, oviducts, and/or enter the hemolymph. This study expands our knowledge of the Ae. aegypti seminal fluid proteome and identifies candidate SFPs that may have tissue-specific, postcopulatory roles which support fertility.

Significance

Male-derived seminal fluid proteins (SFPs), transferred to females along with sperm during mating, are essential for the fertility of a mating pair. SFPs in aggregate induce several physiological and behavioral changes in mated females. Studies in the insect model Drosophila have shown that individual SFPs often participate in specific post-mating processes. In the dengue vector mosquito Aedes aegypti, 177 high confidence SFPs have been identified, but the contribution of individual SFPs in female fertility has yet not been characterized. In Drosophila, a small subset of SFPs leave the site of insemination and localize to the oviduct and sperm storage organs of the female reproductive tract or are transported to the female hemolymph, with patterns of SFP localization suggesting participation in a specific post-mating process. We used MS/MS proteomic characterization coupled with whole animal heavy labeling to expand characterization of the Ae. aegypti ejaculate proteome, increasing the number of known ejaculate proteins to 1378, including identification of 40 novel SFPs. Further, we identified 25 SFPs that leave the site of insemination and localize to the oviducts and/or spermathecae or enter the hemolymph, which can now be assessed for potential tissue-specific functions in female fertility.