Changes In Both Trans- And Cis-Regulatory Elements Mediate Insecticide Resistance In A Lepidopteron Pest, Spodoptera Exigua

Author summaryInsect pests developing resistance to insecticides used for their control is a major problem in agriculture. Many pests including the beet armyworm, Spodoptera exigua have developed resistance to insecticides used for their control. Information on the mechanisms of resistance would help in resistance management programs. Overexpression of detoxifying enzymes were associated with insecticide resistance, but their functions and regulatory mechanisms are still unidentified. The expression levels of P450 genes between susceptible and resistant strains of S. exigua were compared and CYP321A8 was identified as the major contributor to resistance to organophosphate and pyrethroid insecticides. Further studies uncovered two independent but synergistic mechanisms; constitutive upregulation of b-Zip transcription factors and mutations in the promoter that facilitates the binding of an orphan nuclear receptor, Knirps contributing to increase in the expression of CYP321A8 and resistance to multiple insecticides in S. exigua.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.