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.

Methods
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=  Imax/(1+10 (logEC50-X)nH)

Where X is log[ligand (M)], EC50 is the half-maximal concentration (M), Imax is the maximum normalised response, and nH 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.

新烟碱类杀虫剂（Neonicotinoid insecticides）靶向昆虫烟碱型乙酰胆碱受体（nicotinic acetylcholine receptors, nAChRs），其对非靶标昆虫的不良影响已受到高度关注。本团队近期发现，辅助因子TMX3可使昆虫nAChRs在非洲爪蟾（Xenopus laevis）卵母细胞中实现稳定的功能性表达，并证实新烟碱类杀虫剂（吡虫啉、噻虫啉、噻虫胺）对黑腹果蝇（Drosophila melanogaster）、西方蜜蜂（Apis mellifera）及熊蜂（Bombus terrestris）的部分nAChRs具有激动剂活性，且对授粉昆虫的nAChRs作用更强。然而，nAChR家族的其他亚基仍有待探索。本研究证实，Dα3亚基与Dα1、Dα2、Dβ1及Dβ2亚基共同存在于成年黑腹果蝇的同一神经元中，仅此类细胞中潜在的nAChR亚型数量便从4种扩展至12种。Dα1与Dα2亚基的存在会降低吡虫啉、噻虫啉及噻虫胺对非洲爪蟾卵母细胞中表达的nAChRs的亲和力，而Dα3亚基则可提升该亲和力。对成虫中Dα1、Dα2或Dα3亚基进行RNA干扰（RNAi）可降低靶标亚基的表达水平，但通常会上调Dβ3的表达。此外，Dα1 RNAi可增强Dα7的表达，Dα2 RNAi会降低Dα1、Dα6及Dα7的表达，Dα3 RNAi则会降低Dα1的表达同时上调Dα2的表达。多数情况下，对Dα1或Dα2进行RNA干扰可降低幼虫对新烟碱类杀虫剂的毒性敏感性，但Dα2 RNAi会提升成虫的敏感性，这与Dα2降低亲和力的效应一致。将Dα1、Dα2及Dα3亚基中的任意一个替换为Dα4或Dβ3亚基，大多会提升新烟碱类杀虫剂的亲和力并降低其效能。因此，功能性表达研究及亚基靶向RNAi实验表明，新烟碱类杀虫剂的作用与毒性涉及多种nAChR亚基组合的整合效应，提示仅通过亚基基因修饰来解读昆虫毒性时需谨慎。

### 试剂
乙酰胆碱（ACh）与马血清购自美国MilliporeSigma公司。新烟碱类杀虫剂及盐类试剂购自日本FUJIFILM Wako Pure Chemical公司。所有试剂均未进行额外纯化便直接使用。

### 果蝇饲养与品系
所有果蝇均于25℃、12h光照/12h黑暗的循环条件下饲养。培养基为标准果蝇饲料，每升饲料包含5.5g琼脂、100g葡萄糖、40g干酵母、90g玉米粉、3mL丙酸及3.5mL 10%对羟基苯甲酸丁酯（溶于70%乙醇）。对照品系为w1118，转基因果蝇品系如下：UAS-nAChRα1 RNAi（#28688）购自Bloomington果蝇种质中心（BDSC）；UAS-nAChRα2 RNAi（#10760）、UAS-nAChRα3 RNAi（#101806）、UAS-dicer2（#60009）购自维也纳果蝇资源中心（VDRC）；UAS-mCD8::GFP（#108068）[44]购自京都果蝇种质中心。Elav-Gal4（3A3）购自Michael B. O’Connor。nAChRα3敲入2A-GAL4品系通过CRISPR/Cas9系统构建，具体方法详见下文。

#### nAChRα3敲入2A-GAL4品系的构建
通过CRISPR/Cas9介导的同源重组构建GAL4敲入黑腹果蝇品系。靶向载体设计为将T2A-GAL4与蛋白的最后一个胞内区域框内融合插入。将靶向载体与在靶位点附近切割的gRNA表达载体共同注射至母本表达Cas9蛋白的受精卵中。插入位点的侧翼序列为：5´-GAAAGAGGACTGGAAGTACGTGGCCATG/GTGCTCGATCGCCTGTTCCTGTGGATCTTCACAATAGC-3´（整合位点以斜杠标注，gRNA的20bp基因特异性序列已下划线标出）。

### 免疫荧光染色
雄性果蝇生殖系统于添加了补充成分的Grace昆虫培养基（赛默飞世尔科技，美国）中解剖，随后在室温下置于含4%多聚甲醛的Grace培养基中固定30~60min。固定后的样本用添加0.1% Triton X-100的磷酸盐缓冲液（PBS）洗涤3次。洗涤完成后，样本于室温下在封闭液（含0.1% Triton X-100与0.2%牛血清白蛋白的PBS，MilliporeSigma）中封闭1h，随后于4℃下在封闭液中与一抗共同孵育过夜。本研究使用的一抗包括小鼠抗GFP单克隆抗体（克隆号GFP-20；MilliporeSigma G6539；稀释比例1:1000）及兔抗Tdc2抗体（Abcam ab128225；稀释比例1:1000）。使用荧光素偶联二抗（Alexa Fluor 488或546，赛默飞世尔科技），以1:200的稀释比例于室温下孵育2h。洗涤完成后，所有样本均使用FluorSave试剂（MilliporeSigma）进行封片。样本通过LSM 700共聚焦显微镜（蔡司，德国）进行成像，图像使用ImageJ软件包进行处理。

### cDNA与cRNA制备
将nAChR亚基与辅助因子的cDNA克隆至pcDNA3.1(+)载体（赛默飞世尔科技）中。nAChR亚基与辅助因子的登录号如下：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）及DmTMX3（NP_648847）。使用mMESSAGE mMACHINE T7转录试剂盒（赛默飞世尔科技），按照说明书操作，以经合适限制性内切酶在cDNA 3'端酶切后的cDNA为模板制备cRNA。

### 非洲爪蟾卵母细胞cRNA表达
本研究按照1986年英国《动物（科学程序）法案》及ARRIVE 2.0指南尽可能减少非洲爪蟾的使用量。购自日本SHIMIZU Laboratory Supplies的雌性非洲爪蟾在卵母细胞摘除前用三卡因进行麻醉。卵母细胞经胶原酶处理后于无钙标准卵母细胞生理盐水（Ca2+-free SOS）中脱被膜。将浓度为0.1mg/mL的nAChR亚基与辅助因子的混合cRNA以50nL的体积注射至卵母细胞中。随后将卵母细胞置于孵育培养基（添加了丙酮酸钠、青霉素、链霉素、庆大霉素及4%马血清的SOS）中孵育3~4天，再进行电生理实验。

### 电压钳电生理实验
将每个脱被膜的非洲爪蟾卵母细胞固定于有机玻璃记录槽中，以7~10mL/min的流速灌注含0.5μM阿托品的标准卵母细胞生理盐水（SOSA）。使用两根填充2M KCl的玻璃电极刺入每个卵母细胞，将膜电位钳制在-100mV。ACh与α-银环蛇毒素（α-BTX）直接溶解于SOSA中，而新烟碱类杀虫剂的待测溶液则从DMSO储备液稀释至最终浓度。体积分数1%或更低的DMSO对新烟碱类杀虫剂或其他测试配体的反应无影响。ACh与新烟碱类杀虫剂以3min的间隔依次施加5s。α-BTX的检测方法如先前文献所述（详细信息见补充材料索引）。反应的峰值振幅通过pCLAMP软件（Molecular Devices，美国）进行测量与分析。激动剂反应数据以达到反应平台期的ACh最大反应为基准进行标准化，并使用Prism软件（GraphPad Software，美国）通过非线性回归进行拟合，拟合方程如下：
Y= Imax/(1+10 (logEC50-X)nH)
其中X为配体浓度的对数（M），EC50为半数最大效应浓度（M），Imax为标准化后的最大反应，nH为希尔系数。

### 总RNA提取与定量反转录PCR（qRT-PCR）
将动物收集至1.5mL离心管中，立即置于液氮中快速冷冻。使用TRIzol试剂（赛默飞世尔科技）按照说明书提取白色预蛹（化蛹后0小时）或成虫（羽化后3天）的总RNA。使用带有gDNA去除剂的ReverTra Ace qPCR RT Master Mix（TOYOBO）从纯化后的总RNA中合成cDNA。使用Universal SYBR Select Master Mix（Applied Biosystems）在Thermal Cycler Dice TP800系统（Takara Bio）上进行qRT-PCR。为实现mRNA的绝对定量，使用包含靶基因或rp49编码序列的质粒的系列稀释液作为标准品。计算摩尔浓度后，将靶mRNA的转录水平标准化至同一样本中的rp49水平。所用引物见表S9，检测rp49水平的引物已有文献报道。

### 化蛹率检测实验
将果蝇卵置于涂有酵母膏的葡萄汁平板上，于25℃下产卵6h。24小时后收集刚孵化的早期一龄幼虫。将幼虫（每管20只）转移至含2.0g新烟碱类杀虫剂喂食培养基的微型离心管（Sarstedt #58.487）中：每管培养基包含50mL当量的蓝色培养基粉末（Formula 4-24 Instant Drosophila Medium，Carolina，#173210）、50mL当量的酵母粉（酿酒酵母，MP Biomedicals，#903312）及100mL去离子水，其中对照组添加0.1%二甲基亚砜（DMSO；Nacalai Tesque，13407045），实验组分别添加吡虫啉（FUJIFILM Wako Chemicals，#099-03771）、噻虫啉（FUJIFILM Wako Chemicals，#205-19081）或噻虫胺（FUJIFILM Wako Chemicals，#034-22581），溶剂均为0.1% DMSO。于25℃下孵育一周后，统计每管的蛹数。

### 成虫攀爬能力检测实验
羽化后一天内收集成虫，置于正常果蝇培养基中饲养（每管不超过30只）。每日将果蝇转移至新的培养基中。羽化后2~5天，用CO2对果蝇进行短暂麻醉，按性别分离并转移至含1.0%琼脂培养基的管中进行饥饿处理（每管10只）。饥饿处理16小时后，将果蝇转移至含新烟碱类杀虫剂的培养基中，无需麻醉，继续培养6h（每管10只）。含新烟碱类杀虫剂的培养基制备方法为：将10μL稀释于DMSO中的新烟碱类杀虫剂与990μL含1%琼脂及5%蔗糖的溶液混合，每管使用该混合液。在含新烟碱类杀虫剂的培养基中培养6h后，轻轻将果蝇拍打至培养基表面，记录20s内完成攀爬的果蝇数量，使用摄像机（GZ-F270-W，JVC，日本）进行录制。使用ImageJ 1.53v软件（美国国立卫生研究院）测量果蝇在20s内的最大攀爬高度。由于培养基表面至试管顶部的高度为8cm，因此最大攀爬高度上限为8cm。

### 数据可重复性
至少两名作者独立参与数据测量，以确认结果的可重复性。对于电生理实验，每个浓度下的配体激动活性均使用至少2只青蛙的5个卵母细胞进行测定。