CYP321F3 mediates metabolic resistance to methoxyfenozide in rice stem borer, Chilo suppressalis

The development of insecticide resistance in insect populations is a major challenge to sustainable agriculture and food security worldwide. Methoxyfenozide, an insect growth regulator that acts as an agonist of 20-hydroxyecdysone (20E), has severely declined in its efficacy against the rice stem borer (Chilo suppressalis), a notorious pest of rice crops in East and Southeast Asia. To date, however, the genes involved in methoxyfenozide resistance in target pests remain unclear. We conducted a long-term (seven years from 2017 to 2023) and large geographical scale (8 provinces and 45 cities in China) resistance monitoring program for methoxyfenozide in C. suppressalis. Resistance was seen to arise rapidly in this species, with >100-fold resistance being detected in nearly all the field populations after 2018. Piperonyl butoxide (PBO), an inhibitor of cytochrome P450 enzymes (P450s), significantly increased the sensitivity of resistant strains of C. suppressalis to methoxyfenozide, implicating P450s in resistance. Six P450 genes: CYP321F3, CYP6CV5, CYP9A68, CYP6AB45, CYP324A12 and CYP6SN2 were identified as highly expressed in resistant C. suppressalis by transcriptome profiling. Of these, ectopic expression of CYP321F3 in Drosophila melanogaster resulted in a 7.0-fold increase in resistance to methoxyfenozide demonstrating its causal role in resistance. Collectively, these findings provide insight into the mechanisms mediating resistance to insect growth regulators and will inform the development of future pest and resistance management strategies.