Cytochrome P450 and UDP‐glucosyltransferase (UGT) as phase I and phase II metabolism enzymes, respectively, play vital roles in the breakdown of endobiotics and xenobiotics. Insects can increase the expression of detoxification enzymes to cope with the stress from xenobiotics including insecticides. However, the molecular mechanisms for insecticide detoxification in Spodoptera exigua remain elusive, and the genes conferring insecticide metabolisms in this species are less well reported. In this study, 68 P450 and 32 UGT genes were identified. Phylogenetic analysis showed gene expansions in CYP3 and CYP4 clans of P450 genes and UGT33 family of this pest. P450 and UGT genes exhibited specific tissue expression patterns. Insecticide treatments in fat body cells of S. exigua revealed that the expression levels of P450 and UGT genes were significantly influenced by challenges of abamectin, lambda‐cyhalothrin, chlorantraniliprole, metaflumizone and indoxacarb. Multiple genes for detoxification were affected in expression levels after insecticide exposures. The results demonstrated that lambda‐cyhalothrin, chlorantraniliprole, metaflumizone and indoxacarb induced similar responses in the expression of P450 and UGT genes in fat body cells; eight P450 genes and four UGT genes were co‐up‐regulated significantly, and no or only a few CYP/UGT genes were down‐regulated significantly by these four insecticides. However, abamectin triggered a distinct response for P450 and UGT gene expression; more P450 and UGT genes were down‐regulated by abamectin than by the other four compounds. In conclusion, P450 and UGT genes from S. exigua were identified, and different responses to abamectin suggest a different mechanism for insecticide detoxification.

The evolution of insect resistance to insecticides is frequently associated with overexpression of one or more cytochrome P450 enzyme genes. Although overexpression of CYP450 genes is a well-known mechanism of insecticide resistance, the underlying regulatory mechanisms are poorly understood. Here we uncovered the mechanisms of overexpression of the P450 gene, CYP321A8 in a major pest insect, Spodoptera exigua that is resistant to multiple insecticides. CYP321A8 confers resistance to organophosphate (chlorpyrifos) and pyrethroid (cypermethrin and deltamethrin) insecticides in this insect. Constitutive upregulation of transcription factors CncC/Maf are partially responsible for upregulated expression of CYP321A8 in the resistant strain. Reporter gene assays and site-directed mutagenesis analyses demonstrated that CncC/Maf enhanced the expression of CYP321A8 by binding to specific sites in the promoter. Additional cis-regulatory elements resulting from a mutation in the CYP321A8 promoter in the resistant strain facilitates the binding of the orphan nuclear receptor, Knirps, and enhances the promoter activity. These results demonstrate that two independent mechanisms; overexpression of transcription factors and mutations in the promoter region resulting in a new cis-regulatory element that facilitates binding of the orphan nuclear receptor are involved in overexpression of CYP321A8 in insecticide-resistant S. exigua.