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.

Use of double‐stranded RNA targeting β2 divergent nicotinic acetylcholine receptor subunit to control pea aphid Acyrthosiphon pisum at larval and adult stages

“…Thus, this compensation mechanism could only take place in dsRNA‐ β 2‐treated adults because the level of expression of the β 2 subunit has reached a low threshold, whereas the dsRNA‐ β 2‐treated larvae still have a high enough expression level of this subunit to avoid triggering such a mechanism. Moreover, such a compensation mechanism has also been reported in the Colorado beetle Leptinotarsa decemlineata treated with dsRNAs targeting the Ldα1 subunit 28 and in the fruit fly Drosophila melanogaster treated with dsRNAs targeting either Dα1, Dα2 or Dα3 29 . However, in these cases, no induced mortality has been reported after RNAi treatment.…”

“…Moreover, such a compensation mechanism has also been reported in the Colorado beetle Leptinotarsa decemlineata treated with dsRNAs targeting the Ld⊍1 subunit 28 and in the fruit fly Drosophila melanogaster treated with dsRNAs targeting either D⊍1, D⊍2 or D⊍3. 29 However, in these cases, no induced mortality has been reported after RNAi treatment.…”

“…For instance, American cockroaches Periplaneta americana treated with dsRNAs targeting the α 2 subunit, 19 Colorado beetle Leptinotarsa decemlineata with dsRNAs that target Ldα1 subunit 28 or brown leafhopper Nilaparvata lugens with dsRNAs targeting the Nlα8 subunit 31 showed a significant decrease in mortality after exposure to imidacloprid. Although the use of dsRNAs targeting the D α 2 subunit in the fruit fly Drosophila melanogaster has been shown to impair the climbing ability of fruit flies, 29 it has not been shown that these dsRNAs combined with an exposure to imidacloprid decreased the fruit fly survival rate. All these studies focus solely on nAChR subunits that are highly conserved among insect species.…”

Mutation V65I in the β1 Subunit of the Nicotinic Acetylcholine Receptor Confers Neonicotinoid and Sulfoxaflor Resistance in Insects

“…laevis oocytes, which represent the first instance of recombination with the α and β subunits of agricultural pests (thrips). Previous studies have shown that altering injection ratios or coinjecting with α3, α4, and β3 can lead to the formation of nAChRs with distinct functions. , However, it is worth noting that neonicotinoids are predicted to target the α1, α2, β1, and β2 subunits of insect nAChRs . Therefore, the homozygous expression of these receptors can serve as a valuable screening tool for the discovery of novel insecticidal compounds.…”

Tissue‐specific transcriptome analyses in Drosophila provide novel insights into the mode of action of the insecticide spinosad and the function of its target, nAChRα6

“…The nAChRs are ligand-gated cation channels, conserved among insects that, once activated, lead to calcium ion (Ca 2+ ), sodium ion (Na + ), or potassium ion (K + ) flux into neurons, regulating a range of neuromotor and behavioural responses. 8 Imidacloprid targets subunits nAChR⊍1, nAChR⊍2, nAChR⊍3, nAChR⊍4, nAChR⊎1, nAChR⊎2 and nAChR⊎3, [9][10][11] whilst spinosad targets nAChR⊍6. 12,13 An imidacloprid dose of 2.5 ppm creates a long-lasting low Ca 2+ flux into neurons expressing nAChRs, further reducing their cholinergic response by more than two-fold.…”