Callosobruchus chinensis (Coleoptera: Fabaceae) is a worldwide pest that feeds exclusively on legumes, and is the most serious pest affecting mung beans. Usually, the insect olfactory system plays a predominant role in searching for host plants and egg-laying locations. Chemosensory proteins (CSPs), are mainly responsible for transporting specific odour molecules from the environment. In this study, we found that the CSP1 gene of adult C. chinensis displayed antennae-biased expression using quantitative real-time PCR (qRT-PCR) analysis. The binding properties of 23 mung bean volatiles were then determined through several analyses of in vitro recombinant CSP1 protein, including fluorescence competitive binding assay, homology modelling, molecular docking, and site-directed mutagenesis. Fluorescence competitive binding assays showed that CchiCSP1 protein could bind to four mung bean volatiles and was most stable at pH 7.4. After site-directed mutation of three key amino acid bases (L39, V25, and Y35), their binding affinities to each ligand were significantly decreased or lost. This indicated that these three amino acid residues may be involved in the binding of CchiCSP1 to different ligands. We further used Y-tube behavioural bioassays to find that the four mung bean volatiles had a significant attraction or repulsion response in adult C. chinensis. The above findings confirm that the CchiCSP1 protein may be involved in the response of C. chinensis to mung bean volatiles and plays an important role in olfactory-related behaviours. The four active volatiles are expected to develop into new behavioural attractants or repellents in the future.
Keywords: Behavioural bioassays; Callosobruchus chinensis; Chemosensory protein; Fluorescence competitive binding assay; Mung bean volatiles; Site-directed mutagenesis.
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