CYP4C64 Directly Metabolizes Nitenpyram to IM-1-4 Conferring Resistance in the Whitefly, Bemisia tabaci

Neonicotinoid insecticides are widely used to control sap-feeding pests, but their effectiveness is increasingly limited by cytochrome P450-mediated metabolic resistance. Here, we investigated the role of CYP4C64 in nitenpyram (NIT) detoxification in Bemisia tabaci through expression analyses, bioassays, heterologous expression, and in vitro metabolism assays. CYP4C64 was functionally expressed in insect cells using a Bac-to-Bac baculovirus system. UPLC-MS analysis showed that CYP4C64 catalyzed the conversion of NIT to IM-1-4, the major metabolite identified under the experimental conditions, which exhibited substantially lower toxicity than the parent compound. Enzyme kinetic analysis further confirmed the metabolic activity of CYP4C64 toward NIT. These findings provide direct biochemical evidence that CYP4C64 contributes to nitenpyram detoxification in B. tabaci and demonstrate that the Bac-to-Bac expression system is an effective platform for functional characterization of insect P450 enzymes involved in insecticide metabolism.