Coordinated sequestration and transcriptional regulation drive CSP2 ‐mediated chlorpyrifos resistance in Nilaparvata lugens

BACKGROUND: Insecticide resistance in the brown planthopper, Nilaparvata lugens, poses a major threat to rice production. Beyond metabolic detoxification, sequestration by chemosensory proteins (CSPs) has emerged as a vital resistance mechanism. Our earlier work identified CSP2 as a contributor to chlorpyrifos resistance. However, the molecular basis of its insecticide binding and the transcriptional regulation governing its overexpression remained elusive. RESULTS: = 5.40 ± 0.27 μm). Integrated molecular docking, site-directed mutagenesis, and fluorescence competitive binding assays identified distinct binding sites. Lys20 and Asp51 were essential for chlorpyrifos recognition, whereas Asp25 was critical for interacting with chlorpyrifos-oxon. Mutations in these residues severely impaired the protective function of CSP2. Furthermore, we uncovered a key transcriptional mechanism: the transcription factor FoxO acts as a transcriptional repressor of CSP2. Dual-luciferase reporter assays showed FoxO suppresses promoter activity by 34.31%, and RNA interference knockdown of FoxO led to significant upregulation of CSP2 expression and decreased susceptibility of N. lugens to chlorpyrifos. This derepression, resulting from FoxO downregulation in the resistant strain, underpins CSP2 overexpression. CONCLUSION: Our findings reveal a sophisticated, multi-layered resistance strategy in N. lugens. CSP2 provides a frontline, sequestration-based defense that complements the previously characterized CYP6BD12-inhibition mechanism of CSP15. The functional differentiation between these CSPs, coupled with the novel FoxO-mediated regulatory pathway for CSP2, illustrates a complex adaptive network. © 2026 Society of Chemical Industry.