Expansion of a Lepidopteran Carboxylesterase Gene Cluster Drives Xanthotoxin Detoxification in <i>Spodoptera litura</i>

In plant-insect coevolution, xanthotoxin serves as a widespread phytochemical defense against herbivores. This study aims to elucidate xanthotoxin detoxification mechanisms in the polyphagous pest <i>Spodoptera litura</i>. Genomic analyses revealed an expanded carboxylesterase (COE) cluster on chromosome 2, exhibiting xanthotoxin-inducible expression in detoxification tissues. Knockdown of ten COEs significantly increased larval sensitivity to xanthotoxin, while molecular docking confirmed stable xanthotoxin-COE binding. Heterologous expression of four COEs in <i>Escherichia coli</i> conferred bacterial tolerance to xanthotoxin, with three isoforms demonstrating direct metabolic clearance of xanthotoxin <i>in vitro</i>. Mechanistic exploration identified the transcription factors CncC and MafK as critical regulators in modulating larval xanthotoxin tolerance through the coordinated activation of COE expression. Functional validation via dual-luciferase reporter assays established direct transcriptional regulation of three COE promoters through CncC/Maf binding element interactions. Our findings systematically decode both enzymatic detoxification and upstream regulatory networks, revealing how lepidopteran pests adapt to plant chemical defenses through COE pathway specialization.