Odorant-Binding Protein 18 Contributes to Tolerance-Related Responses to Flonicamid and Its Metabolite in Apis mellifera ligustica

Honey bees rely on olfaction to perceive pheromones and environmental chemicals. Odorant-binding proteins (OBPs) are key carriers in odorant recognition, while growing studies suggest that OBPs may function beyond chemosensation, particularly in response to chemical stress. In this study, OBP18 expression in the antennae of Apis mellifera ligustica was significantly upregulated after sublethal exposure to flonicamid (FLO) or its metabolite 4-(trifluoromethyl) nicotinamide (TFNA-AM). Fluorescence competitive binding assays showed that AmOBP18 exhibited high binding affinity for TFNA-AM ( K D = 0.65 ± 0.01 μM) and FLO ( K D = 1.14 ± 0.03 μM). Surface plasmon resonance (SPR) analysis confirmed these findings, with a K D value of 0.163 μM for TFNA-AM. Moreover, overexpression of AmOBP18 in Escherichia coli significantly enhanced bacterial tolerance to TFNA-AM. Silencing of AmOBP18 increased honey bee sensitivity to both FLO and TFNA-AM. We demonstrate that AmOBP18 may have functions beyond its classical olfactory role, contributing to tolerance-related responses to flonicamid and TFNA-AM