Assessing species-specific neonicotinoid toxicity using cross-species chimeric nicotinic acetylcholine receptors in a Drosophila model

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels and the main mediators of synaptic neurotransmission in the insect brain. In insects, nAChRs are pivotal for sensory processing, cognition and motor control, and are the primary target of neonicotinoid insecticides. Neonicotinoids are potent neurotoxins, and pollinators such as honey bees are more sensitive and affected by extremely low sub-lethal doses. nAChR subtypes exist as homomers of α-subunits or heteromers composed of α and β subunits. The honey bee nAChRα8 subunit is orthologous to nAChRβ2 in Drosophila, raising the question of whether this α to β change makes flies less sensitive to neonicotinoids. To investigate species-specific aspects of neonicotinoid toxicity, we CRISPR-Cas9 engineered a cross-species chimeric nAChR subunit by swapping the ligand-binding domain in Drosophila of nAChRβ2 with honey bee nAChRα8. Phenotypic assessment revealed significantly impaired motor functions in climbing and flight assays when comparing flies carrying the α8/β2 chimeric channel to wild type or a β2 knock-out. Despite these motor deficits, both flies expressing the α8/β2 chimeric receptor and β2 knock-out flies showed significantly increased survival after exposure to neonicotinoids thiamethoxam and clothianidin, compared to wild type flies. Combinatorial exposure to different insecticides did not reveal differences. These findings highlight the critical role of nAChR subunit composition in motor control, and demonstrate how subtle structural modifications within a single nAChR subunit can profoundly impact motor function and pesticide response, offering new insights into the molecular mechanisms of neurotoxicity across species.