Binding of neonicotinoid compounds to Apis mellifera chemosensory protein 3 (AmelCSP3): Insight into the molecular toxicity of compounds with both risk and potential

In recent years, honeybee populations have declined dramatically, raising serious concern due to their vital role as pollinators in maintaining global ecosystems and biodiversity. Among the various potential threats, neonicotinoid pesticides have attracted increasing attention for their sublethal toxicity to bees and the differing toxicological profiles of various neonicotinoid compounds toward non-target organisms. In this study, we investigated selective toxicity at the molecular level by examining the binding interactions between a key chemosensory protein in Apis mellifera, AmelCSP3, and three representative neonicotinoid compounds developed across different decades. The analysis was conducted using spectroscopic techniques, surface plasmon resonance, and molecular modeling. The findings reveal that clothianidin binds CSP3 most strongly, followed by thiamethoxam and then paichongding. Thermodynamic parameters derived from fluorescence analysis indicated that the binding process was spontaneous and primarily driven by hydrophobic interactions. Notably, clothianidin—a primary metabolite of thiamethoxam—demonstrated slightly stronger binding than its parent, suggesting that metabolic transformation can exacerbate non-target risk. Paichongding, a third-generation neonicotinoid, showed the highest dissociation constant (KD) and a temperature-dependent decrease in association constant (Ka), indicating it may exert weaker olfactory disruption in Apis mellifera at elevated temperatures. This study offers mechanistic insight into how specific structural features of neonicotinoids influence their binding behavior with Apis mellifera chemosensory proteins, providing molecular-level evidence for their differential non-target effects and informing the rational design of pollinator-friendly pesticides.