As one of the main lepidopteran pests in Chinese tea plantations, Ectropis obliqua Warren (tea geometrids) can severely decrease yields of tea products. The olfactory system of the adult tea geometrid plays a significant role in seeking behaviors, influencing their search for food, mating partners, and even spawning grounds. In this study, a general odorant-binding protein (OBP) gene, EoblGOBP2, was identified in the antennae of E. obliqua using reverse transcription quantification PCR (RT-qPCR). Results showed that EoblGOBP2 was more highly expressed in the antennae of males than in females relative to other tissues. The recombinant EoblGOBP2 protein was prepared in Escherichia coli and then purified through affinity chromatography. Ligand-binding assays showed that EoblGOBP2 had a strong binding affinity for some carbonyl-containing tea leaf volatiles (e.g., (E)-2-hexenal, methyl salicylate, and acetophenone). Electrophysiological tests confirmed that the male moths were more sensitive to these candidate tea plant volatiles than the female moths. Immunolocalization results indicated that EoblGOBP2 was regionally confined to the sensilla trichoid type-II in the male antennae. These results indicate that EoblGOBP2 may be primarily involved in the olfactory activity of male E. obliqua moths, influencing their ability to sense tea leaf volatiles. This study provides a new perspective of insect GOBPs and implies that olfactory function can be used to prevent and control the tea geometrid.

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Nowadays the excessive usage of neonicotinoid insecticides always results in residues in Chinese tea fields. It is not clear whether the insecticide residue at the sublethal level influences the physiological processes of tea pests. Here, we provide evidence of interaction between the neonicotinoid imidacloprid and a general odorant-binding protein, EoblGOBP2, from the tea geometrid moth, Ectropis obliqua. The interacting process was demonstrated through multiple fluorescence spectra, UV absorption spectra, circular dichroism (CD) spectra, molecular docking, etc. The binding mode was determined to be static (from 300 to 310 K) and dynamic quenching (from 290 to 300 K). The binding distance was calculated to be 6.9 nm on the basis of FRET theory. According to the thermodynamic analysis, the process was mainly driven by enthalpy (ΔH < 0), and hydrogen bond and van der Waals interactions were the main driving forces in the static and dynamic binding cases, respectively. Moreover, synchronous fluorescence spectra and CD spectra analysis showed stretching of the EoblGOBP2 peptide chains with a decreasing α-helix when imidacloprid was added. Molecular docking was applied and predicted that two hydrogen bonds were formed between imidacloprid and Arg110 in the mature peptide of EoblGOBP2. Moreover, when the absolute amounts of EoblGOBP2 in the moth antennae were measured and calculated by using real-time PCR, it was estimated that imidacloprid at sublethal level (about 0.233 and 0.175 ng/male and female moth antennae, respectively) inhibited the binding of a tea volatile, E-2-hexenal, to EoblGOBP2 at about half. This study indicates that neonicotinoid insecticide at sublethal level may still affect the olfactory cognition of the tea geometrid moth to volatile compounds from tea leaves.