Unveiling the functional contribution of GSTe16 to pyrethroid detoxification in Spodoptera litura

Glutathione S-transferases (GSTs) play pivotal roles in insect metabolic adaptation to xenobiotic challenges; however, the mechanistic basis of GST-mediated insecticide detoxification, particularly the interaction between GSTs and pyrethroids remains poorly characterized. This study demonstrates that exposure to three pyrethroids (β-cypermethrin, λ-cyhalothrin, and fenvalerate) induces significant elevation in GST activity in the tobacco cutworm Spodoptera litura. The synergistic effects of the GST-specific inhibitor diethyl maleate dramatically potentiated pyrethroid toxicity, indicating a critical role of GST-mediated detoxification. Transcriptional profiling revealed selective induction of GSTe11 and GSTe16 under pyrethroid challenge, with RNA interference-mediated GSTe16 knockdown substantially increasing larval susceptibility. In vivo validation through CRISPR/Cas9 mutagenesis and transgenic Drosophila melanogaster models established GSTe16 as a critical determinant of pyrethroid detoxification. In vitro analyses uncovered the bifunctional capacity of GSTe16: direct metabolic processing of pyrethroids via conjugation and secondary antioxidant defense through reactive oxygen species neutralization. Molecular docking and site-directed mutagenesis identified Arg111 and Asn122 as substrate-specificity determinants in the binding and catalytic subsites, with catalytic mutants retaining full antioxidant activity. This functional specialization reflects evolutionary adaptation of GST architecture, coordinating xenobiotic metabolism with oxidative stress responses. Collectively, these results establish an evolutionary-driven functional compartmentalization within GST architecture, proposing a dual-defense model that synergizes pyrethroid metabolism with oxidative stress resilience.