Assessing species-specific neonicotinoid toxicity using cross-species chimeric nicotinic acetylcholine receptors in aDrosophilamodel

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. The pentameric nAChR channel is made up either of five α-subunits constituting five ligand-binding sites or a mixture of two to three α and β subunits constitute two to three ligand-binding sites. Of particular note, the honey bee nAChRα8 subunit is converted into a β subunit (nAChRβ2) inDrosophila, raising the question whether this α to β conversion 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 inDrosophilaof nAChRβ2 with honey bee nAChRα8. Toxicity assessment by neonicotinoid thiamethoxam revealed significantly impaired motor functions in climbing and flight assays when comparing the α8/β2 chimeric channel to wild type or a β2 knock-out. However, both the α8/β2 chimeric channel and the β2 knock-out showed the same increased survival after neonicotinoid exposure compared to wild type flies. Combinatorial exposure to neonicotinoids also did not reveal differences. These findings highlight the critical role of nAChR subunit composition in motor control and demonstrate how subtle structural differences can profoundly impact motor function and pesticide response, offering new insights into the molecular mechanisms of neurotoxicity across species.