Asymmetric diamide resistance conferred by ryanodine receptor G4946E/V mutations in Plutella xylostella

The diamondback moth, Plutella xylostella, has developed resistance to diamide insecticides, threatening global cruciferous crop production. Although convergent evolution of specific ryanodine receptor (RyR) mutations has been documented in various lepidopteran pests, functional validation within a uniform genetic background has been lacking for key agricultural species. Using CRISPR/Cas9, we generated homozygous G4946E and G4946V knockin strains in a susceptible P. xylostella background. Bioassays revealed that both mutations confer asymmetric resistance to diamide classes: extremely high resistance to the phthalic diamide flubendiamide (>1613-fold for both G4946E and G4946V) but only moderate resistance to the anthranilic diamide chlorantraniliprole (76-fold for G4946E and 87-fold for G4946V). Genetic analysis confirmed that this resistance is autosomal and incompletely recessive. By comparing these results with our previous studies, we provide a mechanistic explanation for the spatiotemporal shift of resistance alleles in the field: while G4946E provided a selective advantage during early flubendiamide use, its moderate resistance to chlorantraniliprole likely led to its displacement by I4790K, which confers high-level resistance to both diamide chemotypes. Our findings provide in vivo evidence that single G4946 substitutions are sufficient to confer substantial resistance and offer insights into the adaptive landscape and evolutionary trajectory of RyR mutations in response to changing insecticide selection pressures.