Mutation and Overexpression of CYP9A9a Confer High-Level Esfenvalerate Resistance in <i>Spodoptera exigua</i>

The beet armyworm, <i>Spodoptera exigua</i>, threatens agriculture due to its rapid evolution of insecticide resistance. CYP9A9a and CYP9A9b are close paralogous P450s, and mutant CYP9A9a (V40L/Q160H/T208A) was previously linked to cyhalothrin resistance but unvalidated <i>in vitro</i>. Here, we studied esfenvalerate resistance mediated by mutant CYP9A9a in the field-derived strain FX21 (960-fold resistance to esfenvalerate) using <i>in vitro</i> and genetic approaches. Both CYP9A9a (1500-fold) and CYP9A9b (670-fold) are overexpressed in the larval midgut. Kinetic analyses showed that CYP9A9a has a similar substrate affinity but a 2.3-fold higher catalytic turnover, yielding 1.9-fold greater metabolic efficiency than CYP9A9b. CRISPR knockout of CYP9A9b alone did not alter susceptibility, whereas dual knockout reduced resistance by 294-fold, underscoring CYP9A9a's essential role. Thus, CYP9A9a is the principal resistance determinant via qualitative mutations and quantitative overexpression, making it a key marker for monitoring pyrethroid resistance in <i>S. exigua</i>. We also address why CYP9A9b exhibits <i>in vitro</i> activity but does not confer resistance <i>in vivo</i>.