Functional characterization of CYP321A8 and CYP6AB60 in diamide insecticides resistance in a field population of Spodoptera litura

Spodoptera litura , a globally distributed agricultural pest, has developed increasing resistance to the diamide insecticides commonly used for its control. In this study, bioassays and synergist bioassays were performed to determine the resistance ratios and synergism ratios of chlorantraniliprole and cyantraniliprole between the Wuhan (WH) field population and the laboratory-sensitive (SS) population. The results indicated that cytochrome P450 monooxygenases (P450s) play important roles in the resistance of S. litura to both insecticides. Subsequently, transcriptome sequencing was conducted to screen for P450s potentially involved in detoxification pathways, and six candidate genes ( CYP321A10 , CYP321A15 , CYP321B1 , CYP321A8 , CYP6B38 , and CYP6AB60 ) were identified for further study. Quantitative PCR revealed that the relative expression levels of all six P450s were significantly higher in the WH population than in the SS population, and that all six genes were markedly overexpressed in the midgut compared with the fat bodies and Malpighian tubules. Furthermore, transgenic Drosophila lines ectopically overexpressing each of these six P450s were generated. Bioassay results demonstrated that, compared with the control lines (UAS-P450s), the P450-overexpressing lines (Act5C > UAS-P450s or Esg > UAS-P450s) exhibited enhanced tolerance to both chlorantraniliprole and cyantraniliprole. Additionally, in vitro metabolism assays indicated that both CYP321A8 and CYP6AB60 could directly metabolize chlorantraniliprole and cyantraniliprole into their corresponding metabolites. Molecular docking results further showed that these two P450s exhibit favorable binding interactions with the two diamide insecticides. In conclusion, these findings indicate that upregulated expression of the six P450s contributes to enhanced detoxification capacity of S. litura against both diamide insecticides, with CYP321A8 and CYP6AB60 in particular being capable of directly metabolizing these compounds. These findings may have significant potential for application in resistance monitoring and the development of sustainable pest management strategies.