Identification and functional analysis of <i>CYP9A32</i>, a cytochrome P450 gene associated with emamectin benzoate resistance in <i>Spodoptera frugiperda</i>

The fall armyworm (Spodoptera frugiperda, J. E. Smith) is a highly invasive pest causing significant damage to global crops and has developed resistance to multiple insecticides. Cytochrome P450 monooxygenases (P450s) are crucial for insecticide detoxification and resistance. However, the roles of P450 genes in mediating resistance to emamectin benzoate in S. frugiperda are unclear. This study examined the expression of 15 P450 genes in susceptible and emamectin benzoate-resistant populations. CYP9A32 was highly expressed in resistant populations (EBRS, FZJRS, and FHKRS) with resistance ratios of 32.7-, 28.7-, and 48.9-fold, respectively. P450 activity was significantly higher in these resistant populations than in the susceptible population (SS), with increases of 3.52-, 7.23-, and 6.12-fold. In contrast, no significant differences were observed in glutathione S-transferase and esterase activities. RNA interference (RNAi) of CYP9A32 increased susceptibility to emamectin benzoate and reduced P450 activity by 38.74%, 26.38%, and 21.03% in SS, EBRS, and FZJRS populations, respectively. Mortality rates of 3rd instar larvae treated with emamectin benzoate at median lethal concentration dosage increased significantly due to CYP9A32 RNAi, with increases of 92.98%, 64.06%, 51.61%, and 65.52% for SS, EBRS, FZJRS, and FHKRS populations. Conversely, transgenic Drosophila melanogaster overexpressing CYP9A32 showed enhanced tolerance to emamectin benzoate. Homology modeling and molecular docking analyses revealed tight binding of CYP9A32 to emamectin benzoate. These findings demonstrate that CYP9A32 upregulation contributes to emamectin benzoate resistance in S. frugiperda. This study enhances our understanding of P450-mediated resistance mechanisms and provides insights for improving insecticide resistance management in pest control programs.