Functional and metabolic validation of CYP419A1v2 in mediating resistance of Laodelphax striatellus (Fallén) to triflumezopyrim.

Triflumezopyrim (TFM), a new type of mesoionic insecticide with good insecticidal activity, showed great value in field control of Laodelphax striatellus. However, the molecular mechanisms underlying L. striatellus resistance to TFM remain poorly understood. In the current study, a 46.67-fold resistant strain (RS31) was selected in the laboratory from a susceptible laboratory stain (SS) after 31 generations of resistance screening. Compared with the SS, the activities of cytochrome P450 monooxygenases (P450) and carboxylesterases (CarE) were significantly elevated. In addition, CYP303A1, CYP419A1v2, CarE6 and CarE12 genes were most up-regulated with 7.6-, 15.8-, 8.3-, and 16.8-folds, respectively. After silencing these four genes by RNA interference (RNAi), the mortalities of L. striatellus under TFM exposure were significantly increased compared with the dsGFP treatment, confirming the key functional roles in resistance. Furthermore, co-silencing of CYP303A1 and CYP419A1v2, CarE6 and CarE12 enhanced the susceptibility of L. striatellus to TFM, indicating a synergistic effect. The binding energies of CYP303A1, CYP419A1v2, CarE6, and CarE12 with TFM were - 6.50, -5.35, -7.41, and - 7.37 kcal/mol, respectively. The in vitro recombinant CYP419A1v2 enzyme activity was 112,270.76 mRFU/min/mg protein, with an activity ratio of 2.15 relative to GFP. The in vitro metabolic efficiency toward TFM reached 96.59%, confirming in vitro metabolic activity of CYP419A1v2. This study elucidates the metabolic resistance mechanism involving the upregulation of detoxification enzyme genes and in vitro metabolic ability, providing a scientific basis for optimizing resistance management strategies and extending the efficacy of TFM for field control of L. striatellus.