Three Chemosensory Proteins Contribute to Chlorfluazuron Tolerance in <i>Bradysia odoriphaga</i>

Chemosensory proteins (CSPs), a class of small soluble proteins, have recently been implicated in insecticide detoxification. In this study, we observed that the transcription levels of three CSP genes (<i>BoCSP2</i>, <i>BoCSP4</i>, and <i>BoCSP5</i>) in <i>Bradysia odoriphaga</i> were significantly increased after exposure to chlorfluazuron. Silencing <i>BoCSP2</i>, <i>BoCSP4</i>, or <i>BoCSP5</i> markedly enhanced the larval susceptibility to chlorfluazuron. Fluorescence competitive binding experiments revealed high-affinity interactions between these BoCSPs and chlorfluazuron (<i>K</i>_<i>i</i> < 1.0 μM). Molecular docking and molecular dynamics analysis revealed the dominance of van der Waals and electrostatic interactions in ligand binding and elucidated the binding modes and stability of BoCSP-chlorfluazuron complexes. Alanine scanning mutagenesis further pinpointed key amino acid residues essential for maintaining binding affinity. These findings suggest that BoCSPs confer the tolerance of larvae to chlorfluazuron through high binding affinity that sequesters the insecticide, reducing its bioavailability and subsequent toxicity. The study provides new insights into the detoxification mechanisms of insects to chlorfluazuron and offers valuable references for future insecticide development and pest management strategies.