Mutation and Overexpression of CYP9A9a Confer High-Level Esfenvalerate Resistance in Spodoptera exigua

The beet armyworm, Spodoptera exigua, 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 in vitro. Here, we studied esfenvalerate resistance mediated by mutant CYP9A9a in the field-derived strain FX21 (960-fold resistance to esfenvalerate) using in vitro 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 S. exigua. We also address why CYP9A9b exhibits in vitro activity but does not confer resistance in vivo.