Importance of CYP6ER1 Was Different among Neonicotinoids in Their Susceptibility in Nilaparvata lugens

CYP6ER1 overexpression is a prevalent mechanism for neonicotinoid resistance in Nilaparvata lugens . Except for imidacloprid, the metabolism of other neonicotinoids by CYP6ER1 lacked direct evidence. In this study, a CYP6ER1 knockout strain ( CYP6ER1 –/– ) was constructed using the CRISPR/Cas9 strategy. The CYP6ER1 –/– strain showed much higher susceptibility to imidacloprid and thiacloprid with an SI (sensitivity index, LC 50 of WT/LC 50 of CYP6ER1 –/– ) of over 100, which was 10–30 for four neonicotinoids (acetamiprid, nitenpyram, clothianidin, and dinotefuran) and less than 5 for flupyradifurone and sulfoxaflor. Recombinant CYP6ER1 showed the highest activity to metabolize imidacloprid and thiacloprid and moderate activity for the other four neonicotinoids. Main metabolite identification and oxidation site prediction revealed that CYP6ER1 activities were insecticide structure-dependent. The most potential oxidation site of imidacloprid and thiacloprid was located in the five-membered heterocycle with hydroxylation activity. For the other four neonicotinoids, the potential site was within the ring opening of a five-membered heterocycle, indicating N -desmethyl activity.