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

Abstract

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 Dα3 subunit co-exists with Dα1, Dα2, Dβ1, and Dβ2 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 Dα1 and Dα2 subunits reduced the affinity of imidacloprid, thiacloprid, and clothianidin for nAChRs expressed in Xenopus laevis oocytes, whereas the Dα3 subunit enhanced it. RNAi targeting Dα1, Dα2 or Dα3 in adults reduced expression of targeted subunits but commonly enhanced Dβ3 expression. Also, Dα1 RNAi enhanced Dα7 expression, Dα2 RNAi reduced Dα1, Dα6, and Dα7 expression and Dα3 RNAi reduced Dα1 expression while enhancing Dα2 expression, respectively. In most cases, RNAi treatment of either Dα1 or Dα2 reduced neonicotinoid toxicity in larvae, but Dα2 RNAi enhanced neonicotinoid sensitivity in adults reflecting the affinity-reducing effect of Dα2. Substituting each of Dα1, Dα2, and Dα3 subunits by Dα4 or Dβ3 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. Author summary In this paper, we show that the Drosophila melanogaster nicotinic acetylcholine receptor (nAChR) Dα3 subunit is co-expressed in ejaculatory duct neurons with Dα1, Dα2, Dβ1, and Dβ2 subunits. All 5 subunits combine to form 12 functional nAChRs in Xenopus laevis oocytes. The functional expression of 18 nAChRs generated from combinations of subunits Dα1−4 and Dβ1−3 are also reported. Dα1 and Dα2 reduced the affinity of D. melanogaster heteromeric nAChRs for imidacloprid, thiacloprid, and clothianidin, whereas Dα3 enhanced it. RNAi of Dα1, Dα2 or Dα3 in adult flies reduced expression of the targeted subunits but commonly enhanced Dβ3 expression; other subunits were also affected in some cases. RNAi targeting either Dα1 or Dα2 reduced neonicotinoid toxicity in larvae but targeting Dα2 led to hyper-neonicotinoid sensitivity in adults consistent with the affinity-reducing effect on neonicotinoids of Dα2. Since RNAi induced subunit compensation was detected, each of Dα1, Dα2, and Dα3 subunits was substituted by Dα4 or Dβ3 subunit. Such subunit compensation mostly increased neonicotinoid affinity and reduced efficacy, impairing the climbing ability of the flies. These results are important because they indicate that neonicotinoid action and toxicity involve the integrated actions of multiple nAChR subunit combinations and counsel caution in interpreting neonicotinoid actions in terms of reduced toxicity.