The synergism between metabolic and target-site resistance enhances the intensity of resistance to pyrethroids in Spodoptera exigua

The widespread application of insecticides imposes intense selective pressure on pest populations, driving the evolution of high-level resistance and leading to frequent control failures of pest. Insecticide resistance is primarily mediated through two primary mechanisms: target-site insensitivity and enhanced metabolic detoxification. However, the potential interactions and synergistic effects between these mechanisms remain largely unexplored. In this study, we demonstrate a striking cooperative interaction between these two major resistance mechanisms in a field-derived strain of Spodoptera exigua exhibiting extreme resistance (631-fold) to the pyrethroid insecticide lambda-cyhalothrin. Through genetic mapping and linkage analysis, we identified that this resistance phenotype is conferred by the combined effects of overexpression of the P450 CYP9A9 (two copies: CYP9A9a and CYP9A9b) and a target-site mutation (L1014F, kdr) in the voltage-gated sodium channel. Using an introgression approach, we generated two near-isogenic strains: WH-kdr, carrying only the target-site resistance allele (6.2-fold resistance), and WH-CYP9A, harboring only the metabolic resistance genes (79-fold resistance), both compared to the susceptible WH-S strain. CRISPR/Cas9-mediated knockout of both CYP9A9 copies in the QP19 strain dramatically reduced resistance from 631-fold to 19-fold, while transgenic expression of the CYP9A9a variant (containing three amino acid substitutions) from QP19 strain in Helicoverpa armigera conferred 39-fold resistance to lambda-cyhalothrin. These findings provide compelling evidence that target-site resistance can significantly potentiate metabolic resistance, resulting in substantially higher resistance levels than either mechanism alone in S. exigua. These findings enhance the understanding of higher level resistance mechanisms mediated by interactions between resistance genes and provide theoretical basis for devising management strategies of insecticide resistance.