Abstract
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.
Highlights
Insects often develop resistance to insecticides that are used for their control
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
Bioassays revealed that the P450 inhibitor, piperonyl butoxide (PBO) significantly enhanced the toxicity of chlorpyrifos, cypermethrin, and deltamethrin in the resistant strain (S1 Table)
Summary
Insects often develop resistance to insecticides that are used for their control. This microevolutionary change in response to environmental challenges constitutes a constant battle between insects and humans [1,2,3]. Cytochrome P450 (P450) enzymes that are capable of metabolizing synthetic insecticides and plant compounds including toxins play a major role in metabolic resistance [4]. Overexpression of genes coding for P450s has been shown to associate with resistance to insecticides in a wide range of insect species [5,6,7,8]. Overexpression of CYP6G1 confers resistance to the insecticides DDT and imidacloprid in Drosophila melanogaster [9]
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