Testis-specific serine/threonine kinase 3 regulates the size of sperm reservoir in Anopheles stephensi.

Mosquito reproductive biology is an underexplored area with potential for developing novel vector control strategies. In this study, we investigated the role of the testis-specific serine/threonine-protein kinase (tssk) family, an essential regulator of spermiogenesis in mammals, in mosquitoes. We identified tssk homologues, As_tssk3 and Aea_tssk1, in Anopheles stephensi and Aedes aegypti, respectively and analyzed their expression across different developmental stages. Functional characterization of As_tssk3 was performed by RNAi mediated gene silencing in An. stephensi. The efficiency of As_tssk3 knockdown was analyzed using qRT-PCR, and the testes of knockdown and control males were compared for phenotypic changes. Additionally, the effect of As_tssk3 knockdown on the reproductive potential of male mosquitoes was analyzed via mating analysis. Further, in-silico studies were conducted to identify interactions between As_TSSK3 and the homologues of known Drosophila TSSK targets involved in the process of spermatogenesis, in Anopheles. As_tssk3 and Aea_tssk1 were found to express in a male-specific manner throughout development. Efficient knockdown in the expression of As_tssk3 gene was observed upon feeding An. stephensi larvae with target dsRNA. The knockdown of As_tssk3 led to a reduced sperm reservoir in adult males. As_TSSK3 was found to interact with the Nucleosome Assembly Protein1 like-4 (NAP1like-4), a putative transcription factor in our in-silico analysis. We hypothesize that As_TSSK3 might regulate the transcription of the protamine gene via the NAP protein. The study presented here is the first report of the characterization of tssk homologues in mosquitoes' and demonstrates a possible role of As_tssk3 in maintaining sperm stores.