A masked gene concealed hand in glove in the forkhead protein crocodile regulates the predominant detoxification CYP6DA1 in Aphis gossypii Glover

Cytochrome P450-mediated metabolism is an important mechanism of insecticide resistance, most studies show upregulated transcript levels of P450s in resistant insect strains. Our previous studies illustrated that some upregulated P450s were associated with cyantraniliprole resistance, and it is more comprehensive to use the tissue specificity of transcriptomes to compare resistant (CyR) and susceptible (SS) strains. In this study, the expression profiles of P450s in a CyR strain compared with a SS strain in remaining carcass or midgut were investigated by RNA sequencing, and candidate genes were selected for functional study. Drosophila melanogaster bioassays suggested that ectopic overexpression of CYP4CK1, CYP6CY5, CYP6CY9, CYP6CY19, CYP6CZ1 and CYP6DA1 in flies was sufficient to confer cyantraniliprole resistance, among which CYP6DA1 was the predominant contributor to resistance (12.24-fold). RNAi suppression of CYP4CK1, CYP6CY5, CYP6CY9 and CYP6DA1 significantly increased CyR aphid sensitivity to cyantraniliprole. The CYP6DA1 promoter had two predicted binding sites for crocodile (CROC), an intron-free ORF with bidirectional transcription yielding CROC (+) and CROC (−). Y1H, RNAi and EMSA found that CROC (−) was a transcription factor directly regulating CYP6DA1 expression. In conclusion, P450 genes contribute to cyantraniliprole resistance, and the transcription factor CROC (−) regulates the expression of CYP6DA1 in A. gossypii.