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.

Chemicals: ACh and horse serum were purchased from MilliporeSigma (USA). The neonicotinoids and salts were purchased from FUJIFILM Wako Pure Chemical (Japan). These reagents were used without further purification.

Flies: All flies were raised at 25°C under 12 hours/12 hours light/dark cycle. The animals were reared on standard fly food containing 5.5 g agar, 100 g glucose, 40 g dry yeast, 90 g cornflour, 3 mL propionic acid, and 3.5 mL 10% butyl p-hydroxybenzoate (in 70% ethanol) per liter. The control strain was w¹¹¹⁸, and transgenic flies are as follows: UAS-nAChRα1 RNAi (#28688) was obtained from the Bloomington Drosophila Stock Center (BDSC); UAS-nAChRα2 RNAi (#10760), UAS- nAChRα3 RNAi (#101806); UAS-dicer2 (#60009) were obtained from the Vienna Drosophila Resource Center (VDRC); and UAS-mCD8::GFP (#108068) [44] was obtained from Kyoto Stock Center. Elav-Gal4 (3A3) was obtained from Michael B. O’Connor. nAChRα3-knock-in 2A-GAL4 was generated by the CRISPR/Cas9 system as described in detail below.

Generation of nAChRα3-knock-in T2A-GAL4 strain: The GAL4 knock-in D. melanogaster flies were generated by CRISPR/Cas9-mediated homologous recombination. A targeting vector was designed such that the T2A-GAL4 is inserted in frame with the last intracellular region of the protein. The targeting vector and a gRNA expression vector that cuts near the target site were co-injected into fertilized eggs maternally expressing Cas9 protein. The flanking sequences of the insertion are: 5´-GAAAGAGGACTGGAAGTACGTGGCCATG/GTGCTCGATCGCCTGTTCCTGTGGATCTTCACAATAGC-3´ (The site of integration is indicated by a slash, The 20-bp gene-specific sequence of the gRNA is underlined.)

Immunostaining: Male Drosophila reproductive systems were dissected in Grace’s Insect Medium, supplemented (Thermo Fisher Scientific, USA), and fixed in 4% paraformaldehyde in Grace’s medium for 30-60 min at room temperature (RT). The fixed samples were washed three times in phosphate-buffered saline (PBS) supplemented with 0.1% Triton X-100. After washing, samples were blocked in the blocking solution (PBS with 0.1% Triton X-100 and 0.2% bovine serum albumin, MilliporeSigma) for 1 h at RT and then incubated with a primary antibody in the blocking solution at 4°C overnight. The primary antibodies used in this study were mouse anti-GFP monoclonal antibody (clone GFP-20; MilliporeSigma G6539; 1:1000) and rabbit anti-Tdc2 antibody (Abcam ab128225; 1:1000). Fluorophore (Alexa Fluor 488 or 546)-conjugated secondary antibodies (Thermo Fisher Scientific) were used at a 1:200 dilution and incubated for 2 h at RT in the blocking solution. After washing, all samples were mounted in FluorSave reagent (MilliporeSigma).  Samples were visualized on an LSM 700 confocal microscope (Zeiss, Germany). Images were processed using the ImageJ package.

cDNAs and cRNAs: cDNAs of the nAChR subunits and co-factors were cloned into pcDNA3.1 (+) vector (Thermo Fisher Scientific). The accession numbers of the nAChR subunits and cofactors are as follows: Dα1 (NP_524481), Dα2 (NP_524482), Dα3 (NP_525079), Dα4 (CAB77445), Dβ1 (NP_523927), Dβ2 (NP_524483), Dβ3 (NP_525098), DmRIC-3 (CAP16647), DmUNC-50 (NP_649813), and DmTMX3 (NP_648847). cRNAs were prepared using the mMESSAGE mMACHINE T7 Transcription Kit (Thermo Fisher Scientific) according to the manual with the cDNA template which was cut with appropriate restriction enzymes at the 3’ end of the cDNA.

cRNA expression in Xenopus laevis oocytes: We minimized the use of X. laevis according to the U.K. Animals (Scientific Procedures) Act, 1986, and the ARRIVE 2.0. Female X. laevis purchased from SHIMIZU Laboratory Supplies (Japan) were anaesthetized with tricaine prior to oocyte excision. Oocytes were defolliculated after collagenase treatment in Ca²⁺-free standard oocyte saline (Ca²⁺-free SOS). cRNAs of the nAChR subunits and co-factors mixed at a concentration of 0.1 mg/mL was injected into oocytes at a volume of 50 nL. Then the oocytes were incubated in the incubation medium (SOS supplemented with sodium pyruvate, penicillin, streptomycin, gentamycin, and 4% horse serum) for 3−4 days prior to electrophysiology.

Voltage-clamp electrophysiology: Each defolliculated X. laevis oocyte was secured in a Perspex recording chamber and perfused with the standard oocyte saline (SOS) containing 0.5 µM atropine (SOSA) at a flow rate of 7−10 mL/min. Two glass electrodes filled with 2 M KCl were impaled into each oocyte and the membrane potential was clamped at -100 mV. ACh and α-BTX were dissolved directly in SOSA, while test solutions of the neonicotinoids were diluted to the final concentration from DMSO stock solutions. DMSO at 1% (v/v) or lower had no effect on the responses to neonicotinoids or other ligands tested. ACh and neonicotinoids were applied for 5 s successively at 3 min intervals. α-BTX was tested as previously described in the literature (See SI Index for details). The peak amplitude of the response was measured by and analysed by pCLAMP (Molecular Devices, USA). The agonist response data were normalised to the maximum response to ACh at concentrations at which the response amplitude attained plateau and fitted by non-linear regression using Prism (GraphPad Software, USA), according to the following equation.

Y=  I_max/(1+10 ^{(logEC50-X)n_H})

Where X is log[ligand (M)], EC₅₀ is the half-maximal concentration (M), I_max is the maximum normalised response, and n_H is the Hill coefficient.

Total RNA extraction and quantitative reverse transcription (qRT)-PCR: Animals were collected in 1.5 ml tubes and immediately flash-frozen in liquid nitrogen. Total RNA from white prepupa (0 hours after puparium formation) or adults (3 days after eclosion) was extracted using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. cDNA was generated from purified total RNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). qRT-PCR was performed on the Thermal Cycler Dice TP800 system (Takara Bio) using Universal SYBR Select Master Mix (Applied Biosystems). For absolute quantification of mRNAs, serial dilutions of plasmids containing coding sequences of the target genes or rp49 were used for standards. After the molar amounts were calculated, transcript levels of the target mRNA were normalised to rp49 levels in the same samples. The primers used are listed in Table S9. The primers to detect rp49 levels are reported before.

Pupariation rate assay: Eggs were laid on grape juice plates with yeast paste at 25°C for 6 h. After 24 h, early (just hatched) first instar larvae were collected. Larvae (20 larvae/vial) were transferred into a mini-vial (Sarstedt #58.487) with 2.0 g of neonicotinoid feeding assay food: 50 mL eq. of blue food powder (Formula 4-24 Instant Drosophila Medium, Carolina, #173210), 50 mL eq. of yeast powder (Brewer’s yeast, MP Biomedicals, #903312), and 100 mL dH2O containing 0.1% dimethyl sulfoxide (DMSO; Nacalai Tesque, 13407045) (for control), imidacloprid (FUJIFILM Wako Chemicals, #099-03771), thiacloprid (FUJIFILM Wako Chemicals, #205-19081), clothianidin (FUJIFILM Wako Chemicals, #034-22581), in 0.1% DMSO. After a week of incubation at 25°C, pupal numbers were scored in each vial.

Adult climbing assay: Adult flies were collected within a day following eclosion and placed in normal fly food (less than 30 flies/vial). Flies were transferred daily to new fly food. 2-5 days after eclosion, flies were briefly anesthetized with CO₂, and the sexes were separated and sorted into fly vials containing 1.0% agar food for starvation (10 flies/vial). After 16 h starvation, flies were transferred to vials containing neonicotinoid-containing food without anesthesia and cultured for 6 h (10 flies/vial). Neonicotinoid-containing foods were prepared by mixing 10 µL of diluted neonicotinoids dissolved in DMSO with 990 µL of a solution containing 1% agar and 5% sucrose for each vial. After 6 h cultured in vials containing neonicotinoid-containing food, flies were gently tapped down to the surface of the food, and flies that climbed within 20 s after tapping were recorded by a video camera (GZ-F270-W, JVC, Japan). The maximum climbing heights of the flies within 20 s after tapping were measured using ImageJ1.53v (National Institute of Health, USAf). Since the height from the surface of the food to the vial top is 8 cm, the maximum climbing height is 8 cm.

Reproducibility of data: At least two authors participated independently in measuring data to confirm the reproducibility of the results. For electrophysiology, five oocytes from at least two frogs were used to determine the agonist activity of each ligand at each concentration.

All data tables were made using Microsoft Excel.