Pipiserpin orchestrates mosquito reproduction through dual control of vitellogenin integrity and 20-hydroxyecdysone-directed vitellogenesis.

Mosquito-borne diseases persist as a critical global health burden, driving the demand for novel vector control approaches. Here, we identify and characterize Pipiserpin, a female-specific serine protease inhibitor (serpin) in Culex pipiens pallens, and elucidate its role in regulating reproductive fitness. Pipiserpin exhibits tissue-specific expression in the fat body and ovary, peaking post-blood meal, and features a conserved serpin fold with a reactive central loop critical for protease inhibition. RNA interference-mediated knockdown of Pipiserpin severely impaired ovarian follicular development, reducing follicle size by 7.73% and egg production by 68.10%. Mechanistically, Pipiserpin depletion destabilized vitellogenin (Vg)-a key yolk precursor-by 62.79% in the fat body and 73.01% in the ovary, despite unchanged Vg transcription. This phenotype was attributed to unchecked trypsin-mediated Vg degradation, as demonstrated by recombinant Pipiserpin's dose-dependent inhibition of trypsin activity. Crucially, exogenous 20-hydroxyecdysone (20E) supplementation restored Vg protein levels, follicular maturation and fecundity, revealing a compensatory relationship wherein the 20E-Vg hormonal pathway can functionally overcome reproductive defects caused by Pipiserpin depletion. Global transcriptomic profiling of Pipiserpin-depleted mosquitoes identified 221 differentially expressed genes, including upregulated trypsin-like proteases and metabolic pathway components, indicative of disrupted proteolytic balance and compensatory metabolic remodeling. Our findings redefine serpin functionality in arthropods, highlighting Pipiserpin's dual role in post-translational Vg stabilization and crosstalk with developmental signaling. This work positions Pipiserpin as a promising target for RNAi or small-molecule interventions to disrupt mosquito reproduction, offering a sustainable strategy for combating vector-borne diseases by precisely targeting reproduction.