Molecular Basis of Behaviorally Active Terpenoid Volatile Recognition by Odorant-Binding Proteins in Tomicus pilifer

Tomicus pilifer is an important wood-boring forest pest in China, and its host localization and intraspecific communication rely on the perception of volatile chemical cues. However, the molecular mechanisms underlying odor recognition in this species remain largely unknown. In this study, we systematically investigated the behaviorally active volatiles present in the hindgut and feces of T. pilifer and elucidated the roles of odorant-binding proteins (OBPs) in their recognition. Gas chromatography–mass spectrometry (GC–MS) identified eight volatile compounds common to both hindgut and fecal samples. Among them, five terpenoid compounds, α-pinene, 3-carene, D-limonene, camphene, and β-myrcene, elicited significant electroantennogram (EAG) responses and induced positive behavioral attraction in adults. Based on antennal transcriptome data, phylogenetic relationships with functionally characterized homologous OBPs, preliminary molecular docking analyses, and tissue-specific expression patterns, three candidate OBPs (TpilOBP5, TpilOBP16, and TpilOBP29) were selected from 51 identified TpilOBP genes and subsequently expressed as recombinant proteins. Fluorescence competitive binding assays demonstrated that all three OBPs bound to the five behaviorally active terpenoid volatiles, with TpilOBP29 exhibiting the broadest ligand-binding spectrum and the highest binding affinity. Molecular docking and interaction analyses further revealed that the binding pocket of TpilOBP29 forms a continuous hydrophobic core composed of multiple conserved hydrophobic residues, which cooperatively stabilizes ligand binding through hydrophobic interactions, π–alkyl interactions, and van der Waals forces, thereby conferring broad-spectrum and high-efficiency odorant recognition. These findings provide new insights into the molecular mechanisms underlying the recognition of key behaviorally active terpenoid volatiles in T. pilifer, identify TpilOBP29 as a key mediator of odor recognition, and provide a potential molecular target for the development of environmentally friendly semiochemical-based behavioral management strategies against bark beetle pests.