Expansion of a Lepidopteran Carboxylesterase Gene Cluster Drives Xanthotoxin Detoxification in Spodoptera litura

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 Spodoptera litura. 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 Escherichia coli conferred bacterial tolerance to xanthotoxin, with three isoforms demonstrating direct metabolic clearance of xanthotoxin in vitro. 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.