Detection and functional validation of point mutations in acetylcholinesterase-1 associated with organophosphate resistance in field populations of Helicoverpa armigera

Point mutations in the acetylcholinesterase-1 gene (ace-1) have been associated with resistance to OPs in many insects. However, the presence and function of ace-1 mutations associated with OP resistance in Helicoverpa armigera (Lepidoptera: Noctuidae), a significant lepidopteran pest damaging a wide range of crops, remain largely unexplored. This study investigated resistance to the OP insecticide phoxim in 12 field populations of H. armigera from northern China in 2022, revealing low levels of resistance (2.5- to 6.7-fold). Using an amplicon sequencing approach, we screened for ace-1 mutations in 13,874 moths collected from 114 populations collected between 2006 and 2022. We found 3 amino acid substitutions (A201S, G227E, and F290V) potentially related to OP resistance. The mean frequencies of A201S, G227E, and F290V mutations were 0.0032, 0.0001, and 0.0001, respectively. To assess these mutations' role in OP resistance, we expressed wild-type and mutant AChE1 proteins in Sf9 cells. Biochemical characterization revealed a 3.1-fold and 3.3-fold increase in the I50 of chlorpyrifos-oxon for A201S and F290V mutants compared to the wild-type enzyme, correlating with a 2.9-fold and 2.7-fold decrease in the Ki value. No enzyme activity was observed in the G227E mutant, indicating that only A201S and F290V confer insensitivity to chlorpyrifos-oxon. Our study demonstrates that amplicon sequencing is an effective method for large-scale screening of resistance-associated point mutations in field populations of H. armigera and potentially other insect pests. It also identifies A201S and F290V in AChE1 as potential point mutations conferring OP resistance in field populations of H. armigera.