Functional analysis of inducible choline/carboxylesterase CCE01 associated with fenpropathrin and abamectin...

The two-spotted spider mite, Tetranychus urticae, represents a globally significant and highly destructive polyphagous pest, notorious for its capacity to develop severe resistance to numerous acaricides. Our previous investigative work identified a specific esterase gene, designated TuCCE01, which was consistently found to be over-expressed in multiple resistant mite populations. This study systematically investigated the functional role of the esterase gene TuCCE01 in metabolizing acaricides. Quantitative real-time PCR revealed that TuCCE01 expression in a susceptible strain was significantly induced by sublethal doses of fenpropathrin and abamectin, showing 3.6-fold and 3.8-fold increases after 24 h, respectively. RNA interference-mediated knockdown of TuCCE01 increased mite susceptibility to both acaricides. Critically, CRISPR/Cas9-mediated gene editing was successfully employed to generate homozygous TuCCE01 knockout mutant lines. Bioassays demonstrated that this specific knockout strain exhibited significantly increased sensitivity to both fenpropathrin and abamectin, with LC₅₀ values markedly reduced. Conversely, transgenic Drosophila melanogaster overexpressing TuCCE01 exhibited enhanced tolerance. Importantly, heterologous expression and in vitro incubation assays confirmed that recombinant TuCCE01 could effectively deplete both acaricides. Homology modeling and molecular docking demonstrated that both acaricides bind to the substrate-binding pocket of TuCCE01, indicating direct enzymatic metabolism, which was consistent with the metabolic assays. This study enhances our understanding of CCE-mediated acaricide acute detoxification and provides a candidate molecular marker for future resistance monitoring.