Identification of a serine protease involved in spinosad degradation in the yellow fever mosquito, Aedes aegypti.

Spinosad is a widely used insecticide effective in controlling Aedes aegypti populations, but the molecular mechanisms underlying resistance remain poorly understood. This study explores the role of a serine protease, AeaSP (AAEL002624), in the potential detoxification ability of spinosad. Our results showed the crude protein of Ae. aegypti degraded approximately 48% of spinosad in vitro within 1 h; based on our previous research, AeaSP was believed to be potentially involved in the degradation of spinosad. Subsequently, AeaSP was recombinantly expressed in vitro, and its enzymatic activity was tested using BAEE as a substrate, with a Michaelis constant (KM) of 0.88 mmol/L. Spatiotemporal expression profiles revealed that AeaSP expression peaked in third instar larvae and thoraxes. In vitro assays showed that AeaSP degraded approximately 63% of spinosad (500 ng/mL) within 6 h. RNAi knockdown of AeaSP significantly increased larval mortality under spinosad exposure and raised spinosad residue levels in larvae by 37% under 0.15 μg/mL spinosad. Our findings suggest AeaSP may play a critical role in detoxifying spinosad in Ae. aegypti and serve as a target for improving spinosad efficacy and mosquito control strategies.