Investigation of the contribution of RyR target-site mutations in diamide resistance by CRISPR/Cas9 genome modification in Drosophila.

Diamide insecticides are used widely against lepidopteran pests, acting as potent activators of insect Ryanodine Receptors (RyRs) and thus inducing muscle contraction and eventually death. However, resistant phenotypes have recently evolved in the field, associated with the emergence of target site resistance mutations (G4946E/V and I4790M). We investigated the frequency of the mutations found in a resistant population of Tuta absoluta from Greece (G4946V ~79% and I4790M ~21%) and the associated diamide resistance profile: there are very high levels of resistance against chlorantraniliprole (9329-fold) and flubendiamide (4969-fold), but moderate levels against cyantraniliprole (191-fold). To further investigate functionally the contribution of each mutation in the resistant phenotype, we used CRISPR/Cas9 to generate genome modified Drosophila carrying alternative allele combinations, and performed toxicity bioassays against all three diamides. Genome modified flies bearing the G4946V mutation exhibited high resistance ratios to flubendiamide (91.3-fold) and chlorantraniliprole (194.7-fold) when compared to cyantraniliprole (5.4-fold). Flies naturally wildtype for the I4790M mutation were moderately resistant to flubendiamide (15.3-fold) but significantly less resistant to chlorantraniliprole (7.5-fold), and cyantraniliprole (2.3-fold). These findings provide in vivo functional genetic confirmation for the role and relative contribution of RyR mutations in diamide resistance and suggest that the mutations confer subtle differences on the relative binding affinities of the three diamides at an overlapping binding site on the RyR protein.