Eucalyptus volatiles recognition mechanism of chemosensory protein 5 in <i>Endoclita vietnamensis</i> (Lepidoptera: Hepialidae)

Endoclita vietnamensis (Buchsbaum & Grehan) (Lepidoptera: Hepialidae) is a major stem-boring pest of eucalyptus. Host location in this species is critically dependent on the larval olfactory recognition of eucalyptus volatiles. However, the volatile recognition mechanism of E. vietnamensis remains unclear. To provide a molecular case study on how a key chemosensory protein (CSP) facilitates this critical ecological behavior, we selected the highly expressed CSP5 of E. vietnamensis (EvieCSP5). We used AlphaFold3 to predict and verify its 3-dimensional structure, which exhibits a typical 6-α-helix bundle forming a cup-like structure, with a central hydrophobic cavity formed by helices α1, α3, α5, and α6 that serves as the ligand-binding site. Phylogenetic analysis further showed that EvieCSP5 clusters with known lepidopteran CSPs involved in host recognition. Molecular docking screened 19 eucalyptus volatiles with high binding affinity to EvieCSP5, and fluorescence competitive binding assays confirmed that m-xylene and dibutyl ether exhibit the strongest binding capacity. Interaction analysis revealed that multiple high-affinity ligands (eg. 2-phenyl-2-propanol, camphene, m-xylene) are primarily anchored by hydrophobic interactions with key residues (ILE90, VAL36, LYS87/94, and ALA91) within the central hydrophobic cavity. This study demonstrated the broad-spectrum binding of eucalyptus volatiles by EvieCSP5 and systematically characterized its recognition mechanism of multi-residue cooperative recognition through hydrophobic interactions. Our findings not only elucidate the molecular basis for host selection in this pest but also provide a detailed example supporting the broader functional plasticity of the CSP family in insects, offering a mechanistic foundation and a key molecular target for developing volatile-based pest control.