Transcriptional Signaling Cascade Orchestrates <i>CYP6AE48</i> Overexpression to Drive λ-Cyhalothrin Detoxification in <i>Spodoptera litura</i>

Lambda-cyhalothrin (λ-cyhalothrin) is a broad-spectrum pyrethroid against lepidopteran pests, yet its efficacy is increasingly threatened by insect resistance. Although <i>CYP6AE48</i> overexpression has been implicated in λ-cyhalothrin detoxification in <i>Spodoptera litura</i>, the regulatory mechanism remains unclear. Here, CRISPR/Cas9-generated <i>CYP6AE48</i> knockout increased larval susceptibility to λ-cyhalothrin, confirming its essential role in detoxification. λ-Cyhalothrin exposure triggered the expression of several transcription factors, including <i>CncC</i>, <i>MafK</i>, <i>EcR</i>, <i>USP</i>, <i>AhR</i>, and <i>ARNT</i>. Their knockdown suppressed <i>CYP6AE48</i> expression, thereby compromising larval tolerance. Dual-luciferase reporter assay demonstrated that these transcription factors regulated <i>CYP6AE48</i> transcription by binding to the specific <i>cis</i>-acting elements. Moreover, protein kinases AKT, ERK, JNK, and RSK2 were implicated as upstream regulators, orchestrating transcription factor expression and subsequent CYP6AE48-mediated detoxification. These findings reveal a multilayered regulatory circuitry that activates <i>CYP6AE48</i> expression to facilitate λ-cyhalothrin detoxification, providing novel insights into the evolutionary adaptation of detoxification mechanisms and highlighting potential molecular targets for future pest management strategies.