Referenced Transcriptomics Identifies a Core Set of Cytochrome P450 Genes Driving Broad-Spectrum Insecticide Detoxification in Phthonandria atrilineata

Phthonandria atrilineata, also known as the mulberry looper, is a major defoliator of mulberry trees. This feeding behavior directly affects the growth of the trees and reduces the quality and yield of mulberry leaves for its use in sericulture. Despite its importance the molecular basis of its resistance to insecticides remains poorly understood. Therefore, this study aimed to comprehensively characterize the cytochrome P450 monooxygenases (P450s) gene family in P. atrilineata and identify key effectors responsible for responses to diverse chemical stressors. We integrated genome-wide re-annotation, phylogenetic analysis, and comparative transcriptomics following exposure to five chemically distinct insecticides. We identified a high-confidence set of 70 P450 genes, dominated by the CYP6 and CYP4 families, whose expansion was driven by tandem gene duplication. Transcriptomic analysis revealed a powerful yet highly selective “elite-driven” response, wherein a small subset of P450s was strongly induced by multiple insecticides. Random Forest and Support Vector Machine (SVM) models converged with differential expression data to pinpoint a core trio of P450s as primary drivers of detoxification: two generalists, CYP6(09521) and CYP6(04876), responsive to all compounds, and one potent specialist, CYP4(04803), exhibiting massive induction to a specific subset of insecticides. Our findings uncover a complex, energy-efficient metabolic strategy in P. atrilineata and identify pivotal P450 genes for broad-spectrum detoxification. These genes represent high-priority targets for developing molecular diagnostic tools for resistance monitoring and informing scientifically guided insecticide rotation strategies.