Functional impact of subunit composition and compensation on Drosophila melanogaster nicotinic receptors-targets of neonicotinoids

Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs) and their adverse effects on non-target insects are of serious concern. We recently found that cofactor TMX3 enables robust functional expression of insect nAChRs in Xenopus laevis oocytes and showed that neonicotinoids (imidacloprid, thiacloprid, and clothianidin) exhibited agonist actions on some nAChRs of the fruit fly (Drosophila melanogaster), honeybee (Apis mellifera) and bumblebee (Bombus terrestris) with more potent actions on the pollinator nAChRs. However, other subunits from the nAChR family remain to be explored. We show that the D alpha 3 subunit co-exists with D alpha 1, D alpha 2, D beta 1, and D beta 2 subunits in the same neurons of adult D. melanogaster, thereby expanding the possible nAChR subtypes in these cells alone from 4 to 12. The presence of D alpha 1 and D alpha 2 subunits reduced the affinity of imidacloprid, thiacloprid, and clothianidin for nAChRs expressed in Xenopus laevis oocytes, whereas the D alpha 3 subunit enhanced it. RNAi targeting D alpha 1, D alpha 2 or D alpha 3 in adults reduced expression of targeted subunits but commonly enhanced D beta 3 expression. Also, D alpha 1 RNAi enhanced D alpha 7 expression, D alpha 2 RNAi reduced D alpha 1, D alpha 6, and D alpha 7 expression and D alpha 3 RNAi reduced D alpha 1 expression while enhancing D alpha 2 expression, respectively. In most cases, RNAi treatment of either D alpha 1 or D alpha 2 reduced neonicotinoid toxicity in larvae, but D alpha 2 RNAi enhanced neonicotinoid sensitivity in adults reflecting the affinity-reducing effect of D alpha 2. Substituting each of D alpha 1, D alpha 2, and D alpha 3 subunits by D alpha 4 or D beta 3 subunit mostly increased neonicotinoid affinity and reduced efficacy. These results are important because they indicate that neonicotinoid actions involve the integrated activity of multiple nAChR subunit combinations and counsel caution in interpreting neonicotinoid actions simply in terms of toxicity.

Automated summary

Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs) and their adverse effects on non-target insects are of serious concern. Other subunits from the nAChR family remain to be explored. The study found that neonicotinoids exhibited agonist actions on nAChRs of fruit flies, honeybees, and bumblebees, with more potent actions on the pollinator nAChRs.