Knockout of two uridine diphosphate-glycosyltransferase genes increases the susceptibility of Spodoptera litura to Bacillus thuringiensis toxins

Uridine diphosphate-glycosyltransferases (UGTs) catalyze sugar conjugation of endogenous and exogenous molecules in insects. In this study, 45 putative UGT genes in 11 families were identified from the genome of S. litura. Exposure to Bt toxins in 5th-instar larvae of the WT strain led to a significant upregulation of midgut UGT40 expression, particularly of SlUGT40D20, SlUGT40D22, and SlUGT40F25. This upregulation was not observed following exposure to chemical pesticides. Knockout of the UGT genes SlUGT40D20 and SlUGT40D22 in S. litura (mutant strains SlUGT40D20-KO and SlUGT40D22-KO) via CRISPR/Cas9-mediated mutagenesis increased susceptibility of S. litura to Bacillus thuringiensis (Bt) insecticidal proteins. However, in comparison with the wild-type (WT) strain, the mutants did not change susceptibility to chemical pesticides. Observations of 5th-instar larval midgut by electron microscopy revealed severe damage to the midgut epithelium caused by Cry1Ac toxin at 10 μg/g in the SlUGT40D20-KO strain compared to the WT. SDS-PAGE and LC MS/MS analyses identified a specific protein band corresponding to putative proteoglycans in the peritrophic matrix of the WT strain, which was absent in the SlUGT40D20-KO strain. Our study suggests an inverse correlation between expression of some UGTs and the susceptibility of S. litura larvae to some Bt toxins.