Abstract

The chemosensory protein (CSP)-mediated sequestration resistance mechanism has attracted increasing attention. This study revealed that a laboratory-selected clothianidin-resistant strain of Bradysia odoriphaga exhibited moderate cross-resistance to thiamethoxam. qPCR analysis demonstrated significant upregulation of BoCSP1 expression in the resistant strain, and its transcription could be induced by clothianidin exposure. RNAi-mediated silencing of BoCSP1 increased the susceptibility of resistant larvae to both clothianidin and thiamethoxam. Fluorescence competitive binding assays showed strong binding affinity between BoCSP1 and clothianidin/thiamethoxam, with Ki values < 3 μM. Molecular dynamics simulations and binding mode analysis further revealed that the stability of BoCSP1-clothianidin and BoCSP1-thiamethoxam complexes is primarily governed by van der Waals interactions. Computational alanine scanning (CAS) and site-directed mutagenesis identified Asn74 and Ile90 as critical residues mediating BoCSP1-clothianidin binding, while Trp101 was essential for BoCSP1-thiamethoxam interaction. Binding pocket analysis of the mutant proteins revealed that alanine substitutions disrupted hydrogen bonding networks and altered local conformational rigidity, leading to substantially reduced ligand affinity. These findings elucidate a novel CSPs-mediated resistance mechanism and provide a theoretical foundation for developing precision-engineered insecticides that exploit CSPs as unique molecular targets.