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

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 <i>Plutella xylostella</i> remains limited. Here, we investigated the molecular basis of high-level pyrethroid resistance in <i>P. xylostella</i>. Comparative transcriptomic analyses of a susceptible strain and two highly resistant field populations (resistance ratios >650-fold) identified <i>PxCYP6B4</i> as the most strongly overexpressed P450 (>95-fold). RNA interference-mediated knockdown of <i>PxCYP6B4</i> significantly increased larval susceptibility to pyrethroids, while heterologous expression in <i>Drosophila melanogaster</i> conferred a 5.27-fold increase in tolerance to lambda-cyhalothrin. Importantly, <i>in vitro</i> 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 <i>PxCYP6B4</i> overexpression and pyrethroid detoxification.