Functional Validation of GmGSTs2 inthe Resistance to Abamectin in the Oriental Fruit Moth, Grapholita molesta (Lepidoptera: Tortricidae)

Abamectin has been used for decades as an insecticide and acaricide in arthropod pest management. However, there is no direct evidence from CRISPR/Cas9 studies confirming the involvement of GSTs in insect resistance to abamectin. The oriental fruit moth, Grapholita molesta, is a destructive pest of fruit trees worldwide. The role of GSTs in the oriental fruit moth remains unclear. In this study, an abamectin-resistant strain (AB-R) was derived from a susceptible laboratory strain (AB-S) of G. molesta. Synergist bioassays showed that the GST inhibitor diethyl maleate (DEM) significantly increased abamectin toxicity in AB-R. Biochemical assays indicated that glutathione S-transferase (GST) activity in AB-R was 1.63-fold higher than in AB-S. Among 25 GST genes examined, GmGSTs2 showed the largest expression difference between AB-R and AB-S and was expressed across developmental stages and body parts. Recombinant GmGSTs2 significantly reduced the effective quantity of abamectin in vitro. CRISPR/Cas9 knockout of GmGSTs2 in both genetic backgrounds increased susceptibility to abamectin and significantly affected the development and survival of G. molesta. The transgenic Drosophila melanogaster strain expressing GmGSTs2 showed an LC₅₀ of 74.12 mg L^–1 (34.59–126.63) versus 25.48 mg L^–1 (12.28–39.82) in W¹¹¹⁸ controls, indicating a 2.91-fold difference. Together, synergism assays, enzyme activity measurements, in vitro metabolism, CRISPR knockout in both resistant and susceptible backgrounds, and a heterologous in vivo assay identify GmGSTs2 as a key metabolic driver of abamectin resistance in G. molesta, providing a practical target for resistance management.