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Cotton plants accumulate phytotoxins, such as gossypol and related sesquiterpene aldehydes, to resist insect herbivores. The survival of insects exposed to toxic secondary metabolites depends on the detoxification metabolism mediated by limited groups of cytochrome P450. Gossypol has an antibiotic effect on Aphis gossypii, and as the concentrations of gossypol were increased in the present study, the mortality of cotton aphids increased from 4 to 28%. The fecundity of the cotton aphids exposed to gossypol was also significantly reduced compared with the control. The transcriptional levels of CYP6DA2 in cotton aphids were significantly induced when exposed to gossypol, and knockdown of the CYP6DA2 transcripts by RNA interference (RNAi) significantly increased the toxicity of gossypol to cotton aphids. To further understand the gossypol regulatory cascade, the 5'-flanking promoter sequences of CYP6DA2 were isolated with a genome walker, and the promoter was very active and was inducible by gossypol. Co-transfection of the cap 'n' collar isoform C (CncC) and CYP6DA2 promoters dramatically increased the expression of CYP6DA2, and suppression of the CncC transcripts by RNAi significantly decreased the expression levels of CYP6DA2, and significantly increased the toxicity of gossypol to cotton aphids. Thus, the transcriptional regulation of CYP6DA2 involved the transcriptional factor CncC.

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The cotton aphid, Aphis gossypii, is one of the most economically important agricultural pests worldwide as it is polyphagous and resistant to many classes of insecticides. Overexpression of the cytochrome P450 monooxygenase (P450) CYP6DA2 has previously been found to be associated with gossypol and spirotetramat tolerance in the cotton aphid. In the present study, the elements located in the promoter region (-357:-343; -250:-241; -113:-104) of CYP6DA2 were shown to control promoter activity, and gossypol induction was observed. We hypothesized that the expression of CYP6DA2 is subject to transcriptional regulation. To investigate the underlying mechanism, we assessed two transcription factors, aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT), and found that the abundance of AhR was highly correlated with CYP6DA2 abundance. RNA interference of AhR or ARNT significantly decreased the levels of the target gene as well as those of its counterpart, and both dramatically repressed CYP6DA2 expression. Cotransfection of the ARNT, AhR, or AhR plus ARNT and CYP6DA2 promoter constructs elevated CYP6DA2 promoter activity, with the AhR plus ARNT cotransfection being the most effective. Thus, these elements located in the promoter were responsible for CYP6DA2 transcription, and CYP6DA2 expression was regulated by the transcription factors AhR and ARNT.

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Cyantraniliprole targets the ryanodine receptor and shows cross-spectrum activity against a broad range of chewing and sucking pests. In this study, a cyantraniliprole-resistant cotton aphid strain (CyR) developed resistance 17.30-fold higher than that of a susceptible (SS) strain. Bioassay results indicated that CyR developed increased cross-resistance to cyfluthrin, α-cypermethrin, imidacloprid, and acephate. In CyR, piperonyl butoxide synergistically increased the toxicity of cyantraniliprole, α-cypermethrin, and cyfluthrin. The cytochrome P450 activities in the CyR strain were significantly higher than those in the SS strain. The mRNA expression of CYP6CY7, CYP6CY12, CYP6CY21, CYP6CZ1, CYP6DA1, and CYP6DC1 in the CYP3 clade, and CYP380C6, CYP380C12, CYP380C44, CYP4CJ1, and CYP4CJ5 in the CYP4 clade, was significantly higher in CyR than in SS. The depletion of the most abundant CYP380C6 transcript by RNAi also significantly increased the sensitivity of CyR to cyantraniliprole. Transgenic expression of CYP380C6, CYP6CY7, CYP6CY21, and CYP4CJ1 in Drosophila melanogaster suggested that the expression of CYP380C6 and CYP4CJ1 was sufficient to confer cyantraniliprole resistance, with CYP380C6 being the most effective, and that CYP380C6, CYP6CY7, and CYP6CY21 were related to α-cypermethrin cross-resistance. These results indicate the involvement of P450 genes in cyantraniliprole resistance and pyrethroid cross-resistance and provide an overall view of the metabolic factors involved in resistance development.