Functional analysis of P450s in afidopyropen detoxification and their regulation by microRNAs in Aphis craccivora

Throughout their evolutionary history, insects have consistently encountered xenobiotic challenges, including insecticides, and have evolved sophisticated metabolic detoxification systems to ensure survival. Afidopyropen is a key insecticide used for controlling Aphis craccivora. However, the rapid evolution of resistance compromises its field efficacy, and the molecular mechanisms responsible remain not fully understood. This study employed an integrated approach combining transcriptomics, functional validation, and regulatory analysis to systematically elucidate the detoxification network of A. craccivora in response to afidopyropen. Transcriptome analysis revealed a specific enrichment of cytochrome P450 monooxygenase (P450) genes in the midgut and fat body of afidopyropen-exposed A. craccivora. Functional investigations identified CYP6YC1 and CYP6CY52 as core detoxification genes. Both inhibition of overall P450 enzyme activity and RNA interference-mediated silencing of these two genes significantly restored aphid susceptibility to the insecticide. Further research uncovered a crucial regulatory layer involving two novel miRNAs, PC-5p-117250_8 and PC-3p-120485_7. These miRNAs directly target the coding sequences of CYP6YC1 and CYP6CY52, respectively. Afidopyropen exposure significantly downregulated these miRNAs, while exogenous delivery of their corresponding agomirs effectively suppressed target P450 gene expression and enhanced susceptibility to the insecticide. This study demonstrates that afidopyropen detoxification in A. craccivora is driven by the synergistic action of the specific overexpression of CYP6YC1 and CYP6CY52, fine-tuned by the post-transcriptional regulation mediated by PC-5p-117250_8 and PC-3p-120485_7. These findings enhance our understanding of the adaptive mechanisms underlying insecticide resistance and provide a theoretical basis for targeted resistance management and the discovery of novel pest control targets.