From Field Overexpression to Biochemical Function: PxCYP6B4-Mediated Hydroxylation Confers Pyrethroid Resistance in Plutella xylostella

The rapid evolution of insecticide resistance threatens global food security, with cytochrome P450-mediated detoxification representing a key adaptive mechanism in insects. However, direct biochemical evidence linking specific P450s to pyrethroid metabolism in Plutella xylostella remains limited. Here, we investigated the molecular basis of high-level pyrethroid resistance in P. xylostella . Comparative transcriptomic analyses of a susceptible strain and two highly resistant field populations (resistance ratios >650-fold) identified PxCYP6B4 as the most strongly overexpressed P450 (>95-fold). RNA interference-mediated knockdown of PxCYP6B4 significantly increased larval susceptibility to pyrethroids, while heterologous expression in Drosophila melanogaster conferred a 5.27-fold increase in tolerance to lambda-cyhalothrin. Importantly, in vitro assays using recombinant PxCYP6B4 expressed in Sf9 cells, combined with LC–MS/MS analysis, demonstrated direct hydroxylation of lambda-cyhalothrin to 4′-hydroxy-lambda-cyhalothrin. These results establish a direct molecular and biochemical link between PxCYP6B4 overexpression and pyrethroid detoxification.