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Functional impact of subunit composition and compensation on Drosophila melanogaster nicotinic receptors–targets of neonicotinoids [1]

['Yuma Komori', 'Department Of Applied Biological Chemistry', 'Faculty Of Agriculture', 'Kindai University', 'Nara', 'Koichi Takayama', 'Naoki Okamoto', 'Life Science Center For Survival Dynamics', 'Tsukuba Advanced Research Alliance', 'Tara']

Date: 2023-03

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.

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.

Funding: This study was supported by KAKENHI (Grant-in-Aid for Scientific Research) from the Japan Society for the Promotion of Science (grant number 21H04718 (KM and RN), 22H02350 (MI),26250001 (HT), and 17H01378 (HT)); and the Cooperative Research Project Program of Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA Center), University of Tsukuba, Japan (grant number 202113 and 202217 (KM)).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Komori et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

To begin to address this shortfall in understanding, we first showed that the D. melanogaster Dα3 subunit co-exists with the Dα1, Dα2, Dβ1, and Dβ2 subunits in ejaculatory duct neurons of adult D. melanogaster. Co-expression in X. laevis with the Dα3 subunit increased possible nAChR subtypes from 4 to 12. All the 12 recombinant fruit fly nAChRs will be explored and their sensitivity to the transmitter acetylcholine (ACh), neonicotinoids (imidacloprid, thiacloprid, and clothianidin) and α-bungarotoxin compared. The impact of the Dα1, Dα2, and Dα3 subunits on recombinant nAChR receptor affinity is addressed. The impact of RNAi targeting particular subunits on toxicity to larvae and adult toxicity and behaviour is investigated. Finally, we studied the effects of replacing one of the α subunits in the Dα1/Dα2/Dα3/Dβ1/Dβ2 nAChRs by either the Dα4 or the Dβ3 subunit on the neonicotinoid actions to address whether such subunit compensation further expands the diversity of neonicotinoid actions in insects.

The recent finding that the transmembrane thioredoxin-related protein 3 (TMX3) enables robust functional expression of insect nAChRs in X. laevis oocytes [ 20 , 30 , 31 ] permitted a demonstration that honeybee (A. mellifera) and bumblebee (B. terrestris) α1/α8/β1 and α1/α2/α8/β1 nAChRs were more sensitive to neonicotinoids than the fruit fly (D. melanogaster) Dα1/Dβ1, Dα1/Dα2/Dβ1, Dα1/Dβ1/Dβ2, and Dα1/Dα2/Dβ1/Dβ2 nAChRs. Thiacloprid and clothianidin modulated honeybee and bumblebee nAChRs at picomolar concentrations, lower than those commonly encountered in treated fields [ 30 ]. However, the extent to which other subunits participate in the formation of native nAChRs and how that affects neonicotinoid actions is not known.

Neonicotinoid insecticides targeting insect nAChRs are effective, broad-spectrum insecticides [ 16 , 19 – 23 ]. Following the discovery of the nitromethylene heterocyclic compound nithiazine, this initial lead compound was modified extensively resulting in 3 generations of commercial neonicotinoids [ 15 ]. They exhibit higher affinity for insect over vertebrate nAChRs, thereby resulting in selective toxicity to insects [ 24 , 25 ]. Their high systemic activity in plants has permitted seed treatment which has accelerated their deployment for crop protection. However, potential adverse effects on pollinators such as honeybees, bumblebees and solitary bees are a concern [ 16 , 20 , 26 , 27 ], even though reduced numbers of pollinators and other non-target insects are not simply due to the effects of neonicotinoids [ 16 , 26 ]. Adverse effects on aquatic invertebrates and birds are also reported [ 28 , 29 ]. It is vital to understand in detail the mechanism of insect nAChR-neonicotinoid interactions but until recently that was precluded by the difficulty of heterologously expressing robust insect nAChRs.

The nicotinic acetylcholine receptors (nAChRs) are cys-loop ligand-gated cation channels playing a pivotal role in fast cholinergic neurotransmission [ 1 ]. In mammals, nAChRs underlie memory [ 2 ], learning [ 2 ], circadian rhythm [ 3 ] and immune responses [ 4 ] as well as locomotion [ 5 ] and hearing [ 6 , 7 ]. In insects, nAChRs are involved primarily in afferent synaptic transmission [ 8 ]. Roles for insect nAChRs include escape responses [ 9 ], circadian rhythm [ 10 , 11 ] and regulation of the mating-induced germline stem cell growth [ 12 ]. Hence, several classes of synthetic and natural-product based insecticides target nAChRs [ 13 – 18 ].

Results and discussion

We first examined whether the Dα3 subunit is co-expressed with Dα1, Dα2, Dβ1, and Dβ2 subunits, previously shown to be present in ejaculatory neurons of D. melanogaster [30], and if so, how such co-expression influences actions of neonicotinoids in vitro and in vivo. To analyse the expression of Dα3, we stained male ejaculatory neurons with a tyrosine decarboxylase 2 targeting antibody (anti-Tdc2) in the animals expressing GFP under control of Dα3-T2A-Gal4 and found that Dα3 is indeed expressed in the male ejaculatory neurons (Fig 1A). Another recent study has shown similar findings for the oviduct neurons in female fruit flies [12], suggesting that Dα1, Dα2, Dα3, Dβ1, and Dβ2 subunits can potentially generate diverse heteromeric nAChRs in male and female adult neurons involved in reproductive functions.

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TIFF original image Download: Fig 1. Expression of Dα3 in male ejaculatory duct neurons of D. melanogaster and characteristics of nAChRs expressed in X. laevis oocytes. (A) Dα3 is expressed in the ejaculatory ducts of male flies. Male ejaculatory ducts from Dα3-2A-Gal4>UAS-mCD8::GFP were immunostained for Tdc2 (magenta) and GFP (green). If both signals are overlapped, merged signals are shown in white. Scale bar: 25 μm. The Dα3 subunit is expressed in the neurons where Dα1, Dα2, Dβ1, and Dβ2 subunits are also expressed [30]. (B) Current responses to 100 μM ACh of nAChRs reconstructed with Dα1, Dα2, Dα3, Dβ1, and Dβ2 subunits in X. laevis oocytes. Each box plots represents 75 and 25% percentiles of data and horizonal line in each box indicates the median of data (n = 10). Whiskers indicate the range of data. The current amplitude of the ACh-induced response was compared by Kruskal-Wallis tests (*, P < 0.05; **, P < 0.01). ns: not significant. https://doi.org/10.1371/journal.pgen.1010522.g001

Given their co-localisation in certain neurons, we investigated in terms of responses to bath-applied 100 μM ACh how many kinds of robust, functional nAChRs the five subunits can reconstitute in X. laevis oocytes when co-expressed with co-factors DmRIC-3, DmUNC-50, and DmTMX3 [30]. We found that by co-expressing the Dα3 subunit, 8 more robust nAChRs (Dα3/Dβ1, Dα1/Dα3/Dβ1, Dα2/Dα3/Dβ1, Dα1/Dα2/Dα3/Dβ1, Dα3/Dβ1/Dβ2, Dα1/Dα3/Dβ1/Dβ2, Dα2/Dα3/Dβ1/Dβ2, and Dα1/Dα2/Dα3/Dβ1/Dβ2 nAChRs) were generated in addition to previously described Dα1/Dβ1, Dα1/Dα2/Dβ1, Dα1/Dβ1/Dβ2, and Dα1/Dα2/Dβ1/Dβ2 nAChRs (Fig 1B). We then determined concentration-response relationships for ACh on the 12 nAChRs (Fig A in S1 Text and Table 1). Replacing either the Dα1 or Dα2 subunit by the Dα3 subunit led to increased current amplitude of the response to 100 μM ACh. For example, the ACh response amplitudes of the Dα3/Dβ1 and Dα1/Dα3/Dβ1 nAChRs were 58.2 and 7.1-fold larger than those of Dα1/Dβ1 and Dα1/Dα2/Dβ1 nAChRs, respectively (ANOVA, P < 0.05, n = 10, Fig 1B). The affinity in terms of pEC 50 for ACh varied from 4.14 to 6.14 depending on subunit combinations (Fig 2A, Table 1, and Table A in S1 Text), indicative of 12 distinct nAChRs. Notably, the Dα1/Dα2/Dα3/Dβ1/Dβ2 nAChR is the first functional recombinant insect nAChR consisting of five different subunits. It is also the first time that all possible subunit combinations for a single insect neuron have been reported.

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TIFF original image Download: Fig 2. Concentrations-response relationships of ACh for D. melanogaster nAChRs expressed in X. laevis oocytes and actions of α-BTX on the ACh-induced responses of nAChRs. (A) Concentration-response curves of ACh and (B) heatmap representing α-BTX for the 12 nAChRs expressed in X. laevis oocytes. In (A), each data plot indicates the mean ± standard error (n = 5). In (B), high to low α-BTX sensitivity is shown in blue and white, respectively. The Dα1 subunit underpins the α-BTX sensitivity of the nAChRs tested. See Fig B in S1 Text for the ACh-induced currents measured in the absence and presence of α-BTX (Fig Ba in S1 Text) and bar graph representations of the effects of α-BTX (Fig Bb in S1 Text). https://doi.org/10.1371/journal.pgen.1010522.g002

α-Bungarotoxin (α-BTX), a peptide toxin known to block certain insect nAChRs [32], was tested on D. melanogaster nAChRs expressed in X. laevis oocytes. We found that 100 nM α-BTX effectively blocked the ACh-induced responses of nAChRs containing the Dα1 subunit (Fig 2B, and Fig B in S1 Text). In contrast, α-BTX was ineffective on nAChRs lacking the Dα1 subunit. Notably, α-BTX showed a minimal blocking effect on the Dα3/Dβ1, Dα2/Dα3/Dβ1, and Dα3/Dβ1/Dβ2 nAChRs (Fig 2B and Fig B in S1 Text). These findings accord with an earlier observation that Dα1/chicken β2 nAChR was sensitive whereas Dα2/chicken β2 nAChR was resistant to this neurotoxin [33]. Diverse pharmacology in terms of the sensitivity to α-BTX confirms that 12 distinct, robust, and functional nAChRs results from combinatorial assembly from the five subunits.

We measured agonist activity of imidacloprid, thiacloprid, and clothianidin, in terms of their pEC 50 and I max values for 8 Dα3-containing nAChRs and analysed factors determining them (Fig 3A, Fig C in S1 Text and Table 1). The pEC 50 value for each neonicotinoid relies primarily on the subunit properties (Fig 3B and Table 1). For imidacloprid, the EC 50 value for the Dα3/Dβ1 nAChR was 14.4-fold lower than that for the Dα1Dβ1 nAChR. Similarly, substituting either Dα1 or Dα2 subunit by Dα3 subunit enhanced affinity of imidacloprid and clothianidin (Dα1/Dβ1/Dβ2 nAChR (for imidacloprid, pEC 50 = 6.99, EC 50 = 102 nM; for clothianidin, pEC 50 = 6.64, EC 50 = 229 nM) vs Dα3/Dβ1/Dβ2 nAChR (for imidacloprid, pEC 50 = 8.28, EC 50 = 5.25 nM; for clothianidin, pEC 50 = 7.46, EC 50 = 34.7 nM), Fig 3B, Table 1, and Tables B and F in S1 Text for ANOVA analysis). For thiacloprid, the affinity for the Dα1/Dα3/Dβ1 nAChR (pEC 50 = 8.07, EC 50 = 8.51 nM) was higher than that for the Dα1/Dα2/Dβ1 nAChR (pEC 50 = 6.92, EC 50 = 120 nM, Fig 3B, Table 1, and Table D in S1 Text for ANOVA analysis). Inversely, the Dα2 subunit reduced the affinity of neonicotinoids (imidacloprid and clothianidin, Dα2/Dα3/Dβ1 nAChR < Dα3/Dβ1nAChR; thiacloprid, Dα2/Dα3/Dβ1 nAChR < Dα1/Dα3/Dβ1 nAChR, Tables B, D, and F in S1 Text for ANOVA analysis). Compound properties also contribute to determining the affinity as indicated by the highest pEC 50 values of thiacloprid for most of the nAChRs (Fig 3A and 3B, Table 1, Tables B, D, and F in S1 Text for ANOVA analysis). Such compound factors were more evident in the I max values (Fig 3C, Table 1, and Tables C, E, and G in S1 Text for ANOVA analysis). For all the nAChRs tested, the order of I max was clothianidin > imidacloprid > thiacloprid, similar to the efficacy order observed in the fruit fly neurons [34], which supports the utility of using the X. laevis oocytes to express nAChRs for the evaluation of neonicotinoid actions in insects.

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TIFF original image Download: Fig 3. Concentration-response relationships for agonist activity of imidacloprid, thiacloprid, and clothianidin for D. melanogaster nAChRs expressed in X. laevis oocytes and heatmap representations of the affinity and efficacy of neonicotinoids. (A) Concentration-response relationships of agonist activity of neonicotinoids for D. melanogaster nAChRs. Each data plot represents mean ± standard error (n = 5). (B) Heatmap representation of the affinity in terms of pEC 50 values for the neonicotinoids tested. (C) Heatmap representation of the efficacy in terms of I max values for the neonicotinoids tested. See Table 2 for the results of multivariate analyses of subunit and ligand factors governing variations in pEC 50 and I max values. https://doi.org/10.1371/journal.pgen.1010522.g003

To clarify the relationship of the nAChR subunits and the neonicotinoids tested with the affinity and efficacy of neonicotinoids for the 12 fruit fly nAChRs (Table 1), we quantitatively analysed the factors governing the variations in the agonist activity indices (Table 2, and Table H in S1 Text for parameter and data sets). The adjusted coefficients of Dα1 and Dα2 subunits for affinity were -0.306 and -0.754, respectively (Table 2), suggesting that both subunits reduced the neonicotinoid affinity, the Dα2 contribution being higher than the Dα1 contribution, while the coefficient of Dα3 was 0.524, indicating that the subunit enhanced affinity (Table 2). Also, the compound properties underpin the affinity (Table 2). It was impossible to elucidate the contribution of the Dβ1 subunit since it is common to all the nAChRs being an essential subunit. However, we showed previously that the R81T mutation in the Dβ1 subunit strikingly reduced the affinity and efficacy of the neonicotinoids [30], indicating its critical role in determining neonicotinoid action [16, 35, 36]. On the other hand, the I max relied mainly on the compound properties even though the values also varied with subunit composition (Fig 2C and Table 2). The highest efficacy of clothianidin probably results from hydrogen bond formation of NH of its guanidine moiety with the backbone carbonyl of the tryptophan in loop B conserved in the insect α subunits [37].

The subunit factors governing variations in neonicotinoid affinity for the various fruit fly nAChRs were derived solely from the multivariate analyses. Therefore, to confirm the results, we performed the chaid (Fig 4A) and lattice (Fig 4B) analyses of the affinity of the neonicotinoids. In the chaid analysis, the Dα1 and Dα2 subunits were negative determinants, whereas the Dα3 subunit was a positive determinant of the affinity, Dα2 being a higher contributor than Dα1 and Dα3 (Fig 4A). Mean pEC 50 of all the neonicotinoids tested for nAChRs without Dα1 and Dα2, but with Dα3 was highest (7.506), indicating that Dα3 is the most critical determinant of high sensitivity for all the neonicotinoids tested of the D. melanogaster nAChRs. In the lattice analysis (Fig 4B), Dα2 subunit was a significant negative factor for the affinity (Table I in S1 Text).

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TIFF original image Download: Fig 4. Chaid and lattice analyses of subunit factors determining the affinity of neonicoticotinoid for the D. melanogaster nAChRs. (A) Chaid analysis of subunit factors determining the affinity of neonicotinoids for the nAChRs. pEC 50 and data numbers are shown in each bracket. Abbreviations: with, w; without, wo. (B) Lattice analysis of subunit factors determining the neonicotinoid affinity for the nAChRs. https://doi.org/10.1371/journal.pgen.1010522.g004

Based on these results, we knocked down Dα1, Dα2, and Dα3 subunit genes by using a pan-neuronal Gal4 driver (elav-Gal4) and quantified the expression of genes encoding all nAChR subunits in Control (elav-Gal4>w1118) and RNAi animals in both developmental (white prepupae) and adult stages of D. melanogaster (Fig 5A). At the same time, these RNAi animals were used to examine toxicity of imidacloprid, thiacloprid, and clothianidin (Fig 5B). We here focused on Dα1, Dα2, and Dα3 subunits, because these three subunits play critical roles in determining the affinity of the neonicotinoids (Tables 1 and 2). Knockdown of Dα1, Dα2, and Dα3 differentially affected the other subunit gene expression level, depending on stage and sex (Fig 5A). During development, knockdown of each of Dα1, Dα2, and Dα3 hardly affected other subunit gene expression except for Dα2 RNAi, which significantly reduced Dα1 expression. By contrast, knockdown of Dα1 enhanced Dβ3 expression in both males and females and Dα7 expression in adult females. Knockdown of Dα2 reduced Dα1, Dα6, and Dα7 expression in both males and females, and Dβ1 expression in adult males. Furthermore, knockdown of Dα2 enhanced Dβ3 expression in adult males. On the other hand, knockdown of Dα3 reduced Dα1 expression and enhanced Dα2 expression in both males and females while enhancing Dβ3 expression in adult females (Fig 5A). These findings indicate that the subunit compensation occurs more frequently in adults than during development. Such subunit compensation can also enhance the inhibitory effect on the climbing behaviour, thereby inducing hypersensitisation to neonicotinoids.

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TIFF original image Download: Fig 5. Effects of α subunit gene RNAi on neonicotinoid toxicity in the fruit flies. (A) Relative expression of genes encoding all nAChR subunits by pan-neuronal knockdown of Dα1, Dα2, and Dα3 in white prepupae and adults of D. melanogaster. elav-Gal4>UAS-dicer2 was used to induce RNAi in all neurons. Each bar indicates the mean ± standard deviation (n = 3). Asterisk indicates that the gene expression level changes as compared with the control (elav-Gal4>w1118) (t-test, P < 0.05). (B) Toxicity of neonicotinoids by pan-neuronal knockdown of Dα1, Dα2, and Dα3 in larvae and adults of D. melanogaster. Each bar represents the mean ± standard deviation (pupariation rate assay, n = 3; adult climbing assay, control n = 20, RNAi n = 10). Asterisk indicates that the toxicity level changes compared with the control (one-way ANOVA, Bonferroni test, P < 0.05). https://doi.org/10.1371/journal.pgen.1010522.g005

Several studies investigated the effects of knocking out nAChR subunit genes in fruit flies on the toxicity of neonicotinoids to larvae or adults and showed that in almost all cases, such mutations resulted in reduced sensitivity to neonicotinoids [38–40].

Interestingly, Perry et al. showed that Dα2 knockout enhanced nitenpyram sensitivity in larvae [38]. Also, Chen et al. showed that Dα1/Dβ2 double knockouts reduced imidacloprid resistance level compared to that observed in a Dα1 or a Dα2 single gene knockout in D. melanogaster [41]. Still, the mechanism of these findings is not known. Liu et al. showed a higher Dα3 subunit contribution to the interactions with clothianidin than for other subunits tested in terms of lethal activity [40]. However, no such Dα3 preference for clothianidin was evident in our study. These findings may be attributable, at least in part, to the differences in the nAChR subunits involved in toxicity. Here we show that RNAi of Dα1 and Dα2 reduced toxicity of imidacloprid, thiacloprid, and clothianidin in larvae (Fig 5B), which is attributable to increased non-target/target nAChR ratio. However, as predicted by the multivariate analyses, RNAi of Dα2 led to hypersensitivity to imidacloprid and thiacloprid in adult males and females and to clothianidin in adult males (Fig 5B), which counsels caution in believing that reduction of drug sensitivity generally happens in response to suppressing the primary target proteins. A direct interpretation of such an observation is that Dα2 subunit is the negative factor reducing the affinity of neonicotinoids (Fig 4 and Table 2), hence reduced Dα2 gene expression results in enhanced neonicotinoid sensitivity. The qRT-PCR data (Fig 5A) revealed that in response to RNAi of Dα2, expression of genes encoding Dα5, Dα6, and Dα7 subunits, of which the Dα5 and Dα6 subunits form low imidacloprid-sensitive nAChRs [42], was reduced, offering another explanation for the enhanced toxicity of neonicotinoids. Reduced toxicity by knockdown of Dα2 and concomitant reduced Dβ1 expression was also observed in adult males (Fig 5A), which can reduce numbers of nAChRs with neonicotinoid sensitivity since the Dβ1 subunit is essential for functional expression (Fig 1 and Table 1). As such, subunit compensation in response to the knockdown of Dα1, Dα2, and Dα3 varies with developmental stages and sexes as well as the primary target of RNAi, resulting in diverse neonicotinoid actions.

These data indicate that Dα1, Dα2, and Dα3 subunits all underpin the interactions with neonicotinoids in the fruit fly. Nevertheless, a contribution of the other subunits to the neonicotinoid actions should not be underestimated because subunit compensation, which can cause replacement of subunits in nAChRs, occurs in response to RNAi of each subunit gene (Fig 5A). The t-SNE representations of the single cell gene expression data indicate co-expression of the Dα1, Dα2, Dα3, Dβ1, and Dβ2 subunits with the Dα4 subunit (Fig D in S1 Text). The Dα1, Dα2, Dα3, Dβ1, and Dβ2 subunits also co-exist with Dβ3 subunit, although such cases are limited [43]. Hence, for the first time we evaluated the effects of replacing one of the Dα1, Dα2, and Dα3 subunits in the Dα1/Dα2/Dα3/Dβ1/Dβ2 nAChRs by the Dα4 or Dβ3 subunit on the agonist activity of imidacloprid, thiacloprid, and clothianidin as well as ACh (Fig 6, Fig E in S1 Text and Table 1). Except for the substitution of the Dα3 subunit, such switching increased affinity of neonicotinoids (Fig 6, Table 1, and Table J in S1 Text). For example, pEC 50 values of imidacloprid, thiacloprid, and clothianidin for the Dα1/Dα2/Dα3/β1/Dβ2 nAChR increased from 6.47, 7.18, and 6.59 to 7.40, 7.99, and 7.11, respectively by switching the Dα1 subunit to the Dα4 subunit. On the other hand, switching the Dα1 subunit of the Dα1/Dα2/Dα3/Dβ1/Dβ2 nAChR to the Dα4 or Dβ3 subunit reduced the efficacy of imidacloprid (Dα1/Dα2/Dα3/Dβ1/Dβ2 nAChR, 0.142, Dα2/Dα3/Dα4/Dβ1/Dβ2 nAChR, 0.027; Dα2/Dα3/Dβ1/Dβ2/Dβ3 nAChR, 0.021) and thiacloprid (Dα1/Dα2/Dα3/Dβ1/Dβ2 nAChR, 0.059, Dα2/Dα3/Dα4/Dβ1/Dβ2 nAChR, 0.018; Dα2/Dα3/Dβ1/Dβ2/Dβ3 nAChR, 0.010). Similarly, replacing the Dα2 subunit of the Dα1/Dα2/Dα3/Dβ1/Dβ2 nAChR with the Dα4 subunit reduced the efficacy of imidacloprid (0.142 to 0.101) and clothianidin (0.812 to 0.425), impairing fruit fly motility in accordance with the enhanced neonicotinoid toxicity by RNAi of Dα2 (Fig 5B). By contrast, switching the Dα3 subunit to the Dα4 subunit had a minimal impact on the efficacy of imidacloprid and thiacloprid, while increasing that of clothianidin (0.812 to 1.001). Furthermore, switching the Dα3 subunit to the Dβ3 subunit reduced the efficacy of imidacloprid (0.142 to 0.080) and thiacloprid (0.059 to 0.023) while increasing that of clothianidin (0.812 to 0.974, Fig 6, Table 1 and Table J in S1 Text), explaining why targeting Dα3 had less impact on neonicotinoid toxicity than targeting Dα1 and Dα2. These results suggest that both Dα4 and Dβ3 subunits can form heteromeric nAChRs with either of Dα2/Dα3/Dβ1/Dβ2, Dα1/Dα3/Dβ1/Dβ2, and Dα1/Dα2/Dβ1/Dβ2 combinations and show unique pharmacological features in neonicotinoid actions. Also, it is conceivable that the nAChRs containing the Dα4 or Dβ3 subunit also contribute to the change of neonicotinoid toxicity in response to RNAi of Dα1, Dα2, and Dα3.

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TIFF original image Download: Fig 6. Effects of Dα1, Dα2, and Dα3 subunit substitution by Dα4 or Dβ3 subunit on agonist actions of ligands on Dα1/Dα2/Dα3/Dβ1/Dβ2 D. melanogaster nAChRs. Dα1 (A), Dα2 (B), and Dα3 (C) subunits were substituted by Dα4 or Dβ3 subunits and we compared the concentration-response curves for ACh, imidacloprid, thiacloprid, and clothianidin on the Dα1/Dα2/Dα3/Dβ1/Dβ2 nAChRs with those on the nAChRs resulting from the subunit switching. Except for the substitution of the Dα3 subunit, the subunit switching resulted in enhanced affinity of neonicotinoids. In the Dα1 and Dα2 subunit switching, Dα4 subunit addition resulted in reduced efficacy and enhanced affinity of imidacloprid and thiacloprid, whereas Dβ3 subunit addition increased the efficacy of thiacloprid. For the actions of clothianidin, Dα2 subunit switching by Dα4 or Dβ3 subunit reduced efficacy, while Dα3 subunit switching enhanced it. Each data point represents the mean ± standard error (n = 5). https://doi.org/10.1371/journal.pgen.1010522.g006

In conclusion, by studying 18 subunit combinations of subunits Dα1, Dα2, Dα3, Dα4, Dβ1, Dβ2, and Dβ3, we have found that imidacloprid, thiacloprid and clothianidin can interact with a broad range of D. melanogaster nAChRs formed not only by the Dα1, Dα2, Dα3, Dβ1, and Dβ2 subunits, but also by the Dα4 and Dβ3 subunits, which has not been described to the best of our knowledge. Although co-expression of these subunits does not necessary prove that they co-assemble to form functional nAChRs in neurons, it is clear that the three neonicotinoids exhibited diverse agonist actions on the 18 nAChRs tested, the outcome depending on both the compound as well as subunit composition. Notably, the Dα1, Dα2, Dα3, Dβ1, and Dβ2 subunits co-localise in organs underlying mating and egg laying, predicting that modulation of the nAChRs consisting of these subunits will affect the number of offspring. In future, it will be of considerable interest to test this hypothesis. If such actions are confirmed, not only for the fruit flies, but also for other insect species such as pollinators and disease vectors, this will counsel further caution in identifying target receptor subtypes simply in terms of reduced neonicotinoid sensitivity resulting only from gene disruption or suppression experiments.

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