CRISPR/Cas9-mediated Disruption of UGT40 Genes Increases Susceptibility to Cry1Ac Toxin and Emamectin Benzoate in Lepidoptera Noctuidae Spodoptera Litura.

UDP-glycosyltransferases (UGTs) are phase II enzymes. They catalyze sugar conjugation of endogenous and xenobiotic compounds. Through this activity, UGTs contribute to insect detoxification and physiology. Our previous work showed that disruption of either SlUGT40D20 or SlUGT40D22 increased susceptibility of Spodoptera litura larvae to Bacillus thuringiensis insecticidal proteins. Here, we used CRISPR/Cas9 mutagenesis to generate 3 additional UGT40 mutant strains: a SlUGT40F25 mutant in the wild-type background, a SlUGT40D20/D22 dual-mutant and a SlUGT40D20/F25 dual-mutant in the SlUGT40D20-KO background. Diet-overlay bioassays showed that all mutant strains were more susceptible than the WT strain to Cry1Ac. The SlUGT40D20/D22 strain had the lowest LC50, although its confidence interval overlapped that of SlUGT40D20-KO; therefore, this combined effect should be interpreted as a trend rather than statistically confirmed additivity. The SlUGT40D20/F25 strain did not differ from SlUGT40D20-KO, consistent with a nonframeshift SlUGT40F25 deletion that retained predicted transmembrane helices. Leaf-dip bioassays indicated no significant changes in responses to most chemical insecticides, except increased mortality of SlUGT40D20/D22 larvae exposed to emamectin benzoate. These findings associate the susceptibility change of S. litura to Cry1Ac and emamectin benzoate with knockout of single or double SlUGT40 genes and support a potential role of UGT40 genes in Cry1Ac tolerance and suggest compound-specific effects on insecticide susceptibility.