Three carboxylesterase genes are involved in spinosad resistance in Frankliniella occidentalis (Pergande).

Carboxylesterases (CarEs) are key enzymes in insect detoxification and metabolism, playing a crucial role in resistance development to various insecticides. In this study, CarE activity was significantly higher in spinosad-resistant strains of Frankliniella occidentalis (NIL-R) than in susceptible strains (Ivf03), suggesting a potential role of CarEs in spinosad resistance. RNA-seq and quantitative PCR (qPCR) validation confirmed that four CarE genes were significantly upregulated in NIL-R. RNA interference (RNAi) validated that three of these genes FoB1, FoB1L, and FoFE4 are directly involved in spinosad resistance. Notably, under low to high concentration spinosad stress, FoB1 expression was significantly upregulated, and its knockdown markedly increased susceptibility to spinosad, indicating its strong responsiveness to spinosad. FoB1L and FoFE4 also contributed to resistance, though they required higher spinosad concentrations for transcriptional activation. Tissue expression analysis showed that FoB1 was predominantly expressed in the abdomen, and its expression in the midgut was 1.92 times and 2.69 times that of FoB1L and FoFE4, respectively. Furthermore, molecular docking revealed that FoB1 exhibits a significantly higher binding affinity for spinosad than FoB1L and FoFE4. In vitro metabolic assays further confirmed that the recombinant FoB1 protein can directly metabolize approximately 47% of spinosad, providing direct evidence of its catalytic function. Taken together, these findings demonstrate that FoB1, FoB1L, and FoFE4 are involved in spinosad resistance in F. occidentalis. This study provides potential molecular targets for resistance monitoring and integrated pest management.