Expanded UDP-glycosyltransferase gene clusters underlie insecticide detoxification in Spodoptera frugiperda

As Phase II detoxification enzymes, UDP-glycosyltransferases (UGTs) catalyze the conjugation of sugar moieties to various hydrophobic xenobiotics, thereby increasing their solubility and facilitating excretion. Certain UGT gene families are often massively expanded as clusters in insect genomes, yet whether such expansions confer a preadaptive advantage for insecticide detoxification remains unclear. In this study, we employed three previously generated Spodoptera frugiperda knockout strains, in which three UGT gene clusters-comprising ten and four UGT33 genes and fifteen UGT40 genes-were deleted using CRISPR-Cas9. By comparing the insecticide susceptibility of these knockout strains to that of the background strain across six insecticides with distinct modes of action, we found that the UGT40 gene cluster contributes to tolerance to emamectin benzoate and lambda-cyhalothrin. Further functional assays using transgenic Drosophila melanogaster lines overexpressing each of the 15 UGT40 genes identified five genes involved in insecticide detoxification, including four associated with emamectin benzoate (UGT40R14, UGT40F19, UGT40M10, and UGT40L10) and one (UGT40R17) with lambda-cyhalothrin. Computational modelling of protein structure and ligand binding analyses suggested that, in the case of the UGTs associated with emamectin benzoate tolerance, the main component of the insecticide, emamectin B1a, binds in a position where the key interactions are with the N-terminal domain, an orientation with the sugar acceptor and metabolic activity. Together, our results provide a systematic dissection of UGT gene cluster function in insecticide detoxification and highlight UGTs as an underappreciated but important component of metabolic resistance in agricultural pests.