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) nAChRs 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. Therefore,
functional expression studies and RNAi targeting of subunits show that
neonicotinoid action and toxicity involve the integrated actions of
multiple nAChR subunit combinations, counseling caution in interpreting
toxicity to insects by subunit gene modification alone.