Functional Validation of <i>GmGSTs2</i> in the Resistance to Abamectin in the Oriental Fruit Moth, <i>Grapholita molesta</i> (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, <i>Grapholita molesta</i>, 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 <i>G. molesta</i>. 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, <i>GmGSTs2</i> 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 <i>in vitro</i>. CRISPR/Cas9 knockout of <i>GmGSTs2</i> in both genetic backgrounds increased susceptibility to abamectin and significantly affected the development and survival of <i>G. molesta</i>. The transgenic <i>Drosophila melanogaster</i> 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 <i>W</i>^<i>1118</i> controls, indicating a 2.91-fold difference. Together, synergism assays, enzyme activity measurements, <i>in vitro</i> metabolism, CRISPR knockout in both resistant and susceptible backgrounds, and a heterologous <i>in vivo</i> assay identify GmGSTs2 as a key metabolic driver of abamectin resistance in <i>G. molesta</i>, providing a practical target for resistance management.