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Identification of an arthropod molecular target for plant-derived natural repellents

Significance Rational control of arthropod pests is important for animal and human health as well as biodiversity preservation. As an alternative to synthetic chemical pesticides, natural repellents represent an ecological method of pest control. Through an exceptional gene library screening in Mesobuthus martensii scorpions, we here uncover a transient receptor potential ion channel as the chemosensory sensor for plant-derived repellents. Its ortholog ion channel in Drosophila melanogaster also acts as a molecular receptor of natural repellents and mediates avoidance behavior. This work thus identifies a molecular basis for arthropod chemosensing and should help update the ecological strategies for pest control while preserving biodiversity.

from Dr. Wei Yang (Zhejiang University, Hangzhou, Zhejiang, China). Drosophila NompC channel was generously provided by Dr. Wei Zhang (Tsinghua University, Beijing, China) and Zhiqiang Yan (Fudan University, Shanghai, China), and the fulllength cDNAs of 12 Drosophila TRP channels as indicated were obtained from D. melanogaster cDNA library and subcloned into the pIRES2-EGFP vector. Chimeras and site-directed mutagenesis were performed using the overlap-extension polymerase chain reaction (PCR) method as previously described (2). To express dTRP in Drosophila Schneider 2 (S2) cells, the full-length cDNA of dTRP was constructed into pAc5.1/V5-His vector. All recombinant constructs were verified by DNA sequencing.
For the analysis of expression patterns of the gene, the body of scorpion was anatomically divided into 5 segments: pedipale, prosoma, mesasoma, metasoma and ambulatory legs. Similarly, the body of Drosophila was also divided into 5 segments: proboscis, head, thorax, abdomen, and legs. Separated body parts were then treated with TriZol (Invitrogen) to obtain RNA. Afterwards, the first-strand cDNA was synthesized with a reverse transcription kit (Invitrogen). Gene expression was examined with a Bio-Rad SFX connect system using Bestar qPCR Master Mix (DBI, Germany) (SYBR Green). Data were normalized to the expression of β-Actin. Gene specific primers were shown in Supplementary Table 3.

Cell culture and expression
Scorpion sensory neurons were dissociated from ventral nerve cord (VNC) ganglia of Mesobuthus Martensii. Briefly, the adult scorpions were decapitated, and the VNC ganglia were exposed simply by removing the carapace, tergites, dorsal surface of the postabdominal rings, dorsal surface of the telson, and tissue adjacent to the central nervous system. Thereafter, three pairs of preabdominal VNC ganglia were immediately dissected and rinsed in Ca 2+ /Mg 2+ -free Hank's balanced salt solution. Ganglia were dissociated by enzymatic treatment with collagenase (Type IA, 1 mg/ml) and trypsin (type I, 0.3 mg/ml) at 30 °C for 30 min. During digestion, gentle mechanical trituration was performed every 10 min through fire-polished glass pipettes until solution become cloudy. The resulting suspension of single cells was centrifuged at 1,500 rpm for 5 minutes, and resuspended in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific, MA) containing 10% heat-inactivated fetal bovine serum (FBS), then seeded onto a poly-L-lysine pre-treated glass slides, and cultured in a humidified incubator gassed with 5% CO2. Electrophysiology experiments were performed ~4-8 h after the plating.
HEK 293T cells were grown in DMEM containing 4.5 mg/ml glucose, 10% FBS, 50 units/ml penicillin, and 50 g/ml streptomycin, and were incubated at 37°C in a humidified incubator gassed with 5% CO2. Drosophila S2 cells were kindly gifted from Dr. Xi Zhou (Wuhan Institute of Virology, Chinese Academy of Sciences). S2 cell were grown in Schneider's Insect Medium (Millipore Sigma, St. Louis, MO) supplemented with 10% heat-inactivated FBS, and were incubated at 27 °C in a precision biochemical incubator. Cells at a confluence of ~70% were transfected with the desired DNA constructs using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer's instructions, or using a standard calcium phosphate precipitation method. Transfected cells were reseeded on 12 mm round glass coverslips coated by poly-Llysine. Experiments took place ~48 h after transfection.

Electrophysiology
Conventional whole-cell and excised patch-clamp recording methods were used. Cells were voltage clamped or current clamped in the whole-cell mode using an EPC10 amplifier (HEKA, Lambrecht, Germany). Voltage commands were made from the Patchmaster program. For a subset of recordings, currents were amplified by an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA) and recorded through a BNC-2090/MIO acquisition system (National Instruments, Austin, TX) using QStudio developed by Dr. Feng Qin at the State University of New York at Buffalo. Recording pipettes were pulled from borosilicate glass capillaries (World Precision Instruments, WPI), and fire-polished to a resistance of 2-4  when filled with internal solution. Whole-cell recordings were typically sampled at 5 kHz and filtered at 1 kHz, and singlechannel recordings were sampled at 25 kHz and filtered at 10 kHz.
Electrodes were filled with (in mM): 150 CsCl, 5 EGTA, 10 HEPES, pH 7.4 adjusted with CsOH. The control bath solution contained (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 10 HEPES, pH 7.4 adjusted with NaOH. The Ca 2+ -free bath saline for whole-cell recording in HEK 293T cells consisted of (in mM): 140 NaCl, 5 KCl, 3 EGTA, and 10 HEPES, pH 7.4 adjusted with NaOH. For recordings in ventral nerve cord neurons, the bath solution consisted of (in mM): 140 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 glucose, 10 HEPES, pH 7.4 adjusted with NaOH. The bath and pipette solutions for single-channel recording was symmetrical and contained 140 NaCl, 5 KCl, 5 EGTA, and 10 HEPES, pH 7.4 (adjusted with NaOH). For cation substitution experiment, normal bath solution was used to establish a whole-cell configuration. Pipette solution contains (in mM): 140 NaCl and 10 HEPES, pH 7.4 adjusted with NaOH. After the whole-cell configuration was obtained, bath solution was replaced with specific cationic solution, and a voltage step pulse was used to measure the reversal potential. For monovalent cation X + (Na + , K + , and Cs + ), cationic solution contains (in mM): 140 XCl, 10 HEPES, pH 7.4 adjusted with XOH. For divalent cation Y 2+ (Mg 2+ , Ca 2+ , and Ba 2+ ), cationic solution contains (in mM): 110 YCl2, 10 HEPES, pH 7.4 adjusted with Y(OH)2. Isolated cells were voltage clamped and held at -60 mV before recordings. Exchange of external solutions was performed using a gravity-driven local perfusion system. As determined by the conductance tests, the solution around a patch under study was fully controlled by the application of a solution with a flow rate of 100 l/min or greater. All pharmacological experiments met this criterion. All patch-clamp recordings were made at room temperature (22-24 °C).

Intracellular Ca 2+ measurements
For intracellular Ca 2+ measurements, cells were transfected with the desired DNA constructs together with GCaMP6m (gift from Dr. Liangyi Chen at Peking University). After transfection, cells were reseeded on a poly-L-lysine pre-treated glass coverslips. The bath solution consisted of (in mM): 140 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 glucose, 10 HEPES, pH 7.4 adjusted with NaOH. In another set of experiment, fly antennas were acutely dissected in bath solution consisted of (in mM): 140 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 glucose, 10 HEPES, pH 7.15 adjusted with NaOH and fixed on a silicone (MOLYKOTE) pre-treated glass dish. Calcium imaging was performed on an inverted epifluorescence microscope (Olympus IX 73) equipped with a complete illumination system (Lambda XL, Sutter Instruments). Fluorescent images were acquired using a cool CCD camera (CoolSNAP ES2, Teledyne Photometrics) controlled by Micro-Manager 1.4 (Vale lab, UCSF) using a public 1394 digital camera driver (Carnegie Mellon University). Images at excitation of 470 nm and emission of 510 nm were taken at 0.2 or 1 Hz.

Immunostaining
HEK 293T cells transfected with desired DNA constructs were prepared for immunostaining, which took place 16-24 hours after transfection. Samples were washed twice with phosphate-buffered saline (PBS), and then were fixed with ice-cold methanol for 10 min, and thereafter blocked in PBS supplemented with 5% FBS for 30 min. Afterwards, samples were incubated with rabbit monoclonal anti-human Na + /K + -ATPase antibody at a dilution of 1:500 in 1% BSA for 1 h, and were then incubated with conjugated antibodies, the donkey anti-rabbit IgG-FITC at a dilution of 1:1000 in 1% BSA for 1 h. Then cells were seeded on glass slides with DAPI. All the procedures were performed at room temperature (22-25 °C). The outcomes were evaluated by a confocal microscopy.

Phylogenetic analysis
TRP channels of some representative species used for the analysis were shown in Supplementary Table 4. The alignments were performed using ClustalX (version 1.83), and phylogenetic trees were built with MEGA7 using Neighbor-Joining method (1,000 replicates for the bootstrapping tests).

Data analysis
Data were analyzed offline with Clampfit (Molecular Devices, Sunnyvale, CA), IGOR (Wavemetrics, Lake Oswego, OR, USA), SigmaPlot (SPSS Science, Chicago, IL) and OriginPro (OriginLab Corporation, MA, USA). For concentration dependence analysis, the modified Hill equation was used: Y = A1 + (A2 -A1) / (1 + (EC50/[toxin]) nH ), in which EC50 is the half maximal effective concentration, and nH is the Hill coefficient. Permeation ratios for monovalent cations to Na + (Px/PNa) were calculated using a modified GHK equation: PX/PNa = exp(VrevF/RT), where Vrev is the reversal potential, F is Faraday's constant, R is the universal gas constant, and T is the absolute temperature. For measurements of divalent cations' permeability (PY/PNa), the following equation was used: PY/PNa = [Na + ]i × exp(VrevF/RT) × (1+expVrevF/RT))/4[Y 2+ ]o, where the bracketed terms represent activities. Assumed activity coefficients are 0.75 for monovalents and 0.25 for divalents, following the methods established by Soledad Valera et al (3). Unless stated otherwise, the data are presented as the mean ± standard error (SEM), from a population of cells (n), with statistical significance assessed by Student's t test for two group comparison or Oneway ANOVA test with Dunnett's method and One-way ANOVA with the Tukey-Kramer test was used for comparing multiple group comparisons. Significant difference is indicated by a p value less than 0.05 (*p < 0.05, **p < 0.01, and ***p < 0.001).    Phylogenetic tree of TRP channels. Statistical confidence (bootstrap value) is indicated beside the respective branch. The tree was constructed using MEGA and ClustalX by comparing the amino acid sequences of TRP channels and was further built using Neighbor-joining method (1,000 replicates for the bootstrapping tests). sTRP1 is shaded in blue box.