Li_2026_Insect.Biochem.Mol.Biol_189_104518

Pyrethroid resistance in Lepidopteran pests poses a serious threat to sustainable pest management. Although carboxylesterases (COEs) contribute to metabolic resistance, their precise functional roles and regulation remain obscure. Here, a lambda-cyhalothrin-resistant strain of the tobacco cutworm Spodoptera litura (93.29-fold resistance) showed significantly increased COE activity. Synergism assays using triphenyl phosphate confirmed COE involvement, increasing lambda-cyhalothrin toxicity with synergistic ratios of 2.26 and 2.33 in susceptible and resistant strains. Three COE genes (COE052, COE054, and COE066) were markedly overexpressed in the resistant strain, with midgut expression upregulated by 140.63-, 102.60-, and 99.98-fold, respectively. RNA interference revealed that COE052 and COE066 are essential for resistance, while COE054 knockdown had no effect, likely due to compensatory overexpression of the other two COEs. Heterologous expression in bacteria demonstrated that all three COEs confer increased lambda-cyhalothrin tolerance by 3.13-, 2.26-, and 3.29-fold and exhibit antioxidant activity. In vitro metabolism assays confirmed their capacity to degrade lambda-cyhalothrin, with COE066 displaying the highest metabolic efficiency (47.20%). Molecular docking and mutagenesis identified Asn446 and His447 in COE066 as key residues for lambda-cyhalothrin binding, metabolism, and antioxidant function. His447 was particularly critical: its mutation decreased binding free energy from -34.96 to 3.28 kcal mol(-1), reduced metabolic efficiency from 51.10% to 14.39%, and significantly impaired antioxidant activity. Furthermore, the transcription factor HNF4 was found to regulate the expression of all three COEs, establishing an HNF4-COE regulatory axis underlying lambda-cyhalothrin resistance. This study elucidates the mechanism of COE-mediated pyrethroid resistance and highlights potential targets for resistance management.