Two odorant receptors regulate 1-octen-3-ol induced oviposition behavior in the oriental fruit fly
Insect rearingWT B. dorsalis were collected from Haikou, Hainan province, China, in 2008. They were maintained at the Key Laboratory of Entomology and Pest Control Engineering in Chongqing at 27 ± 1 °C, 70 ± 5% relative humidity, with a 14-h photoperiod. Adult flies were reared on an artificial diet containing honey, sugar, yeast powder, and vitamin C. Newly hatched larvae were transferred to an artificial diet containing corn and wheat germ flour, yeast powder, agar, sugar, sorbic acid, linoleic acid, and filter paper.Behavioral assaysDouble trap lure assays were set up to compare the olfactory preferences of gravid and virgin females in a 20 × 20 × 20 cm transparent cage with evenly distributed holes (diameter = 1.5 mm) on the side walls. The traps were refitted from inverted 50-mL centrifuge tubes and were placed along the diagonal of the cage. The top of each trap was pierced with a 1-mL pipette tip, which was shortened to ensure flies could access the trap from the pipette. For the olfactory preference assay with mango, one trap was loaded with 60 mg mango flesh and the other trap with 20 μL MO in the cap of a 200-μL PCR tube. For the olfactory preference assay with 1-octen-3-ol (≥98%, sigma, USA), one trap was loaded with 20 μL 10% (v/v) 1-octen-3-ol diluted in MO, and the other with 20 μL MO. A cotton ball soaked in water was placed at the center of the cage to provide water for the flies. Groups of 30 female flies were introduced into the cage for each experiment, and each experiment was repeated to provide eight biological replicates. All experiments commenced at 10 am to ensure circadian consistency. The number of flies in each trap was counted every 2 h for 24 h. We compared the preferences of 3-day-old immature females, 15-day-old virgin females, and 15-day-old mated females. The olfactory preference index was calculated using the following formula41: (number of flies in mango/odorant trap – number of flies in control trap)/total number of flies.Oviposition behavior was monitored in a 10 × 10 × 10 cm transparent cage with evenly distributed holes on the side walls as above. A 9-cm Petri dish filled with 1% agar was served as an oviposition substrate, and the mango flesh, 10% (v/v) 1-octen-3-ol or MO were added at opposite edges of the dish. We tested the preference of flies for different substrates: (1) ~60 mg of mango flesh on one edge and 20 μL of MO on the other; (2) 20 μL of 1-octen-3-ol on one edge and 20 μL of MO on the other; (3) ~60 mg mango flesh on one edge and 20 μL of 1-octen-3-ol on the other; and (4) ~60 mg mango flesh plus 20 μL 1-octen-3-ol on one side and ~60 mg of mango flesh plus 20 μL MO on the other. The agar disc was covered in a pierced plastic wrap to mimic fruit skin, encouraging flies extend their ovipositor into the plastic wrap to lay eggs. The agar disc was placed at the center of the cage, and we introduced eight 15-day-old gravid females. Two Sony FDR-AX40 cameras recorded the behavior of the flies for 24 h, one fixed above the cage to record the tracks and the other placed in front of the cage to record the oviposition behavior. Based on the results from double traps luring assays, a 3 h duration (6–9 h) of the videos was selected to analyze the tracks and spent time of all flies in observed area (the surface of Petri dish). The videos were analyzed using EthoVision XT v16 (Noldus Information Technology) to determine the total time of all flies spent on each side in seconds and the total distance of movement in centimeters, and the tracks were visualized in the form of heat maps17. The number of eggs laid by the eight flies in each experiment was counted under a CNOPTEC stereomicroscope, and each experimental group comprised 7–16 replicates.Annotation of B. dorsalis OR genesD. melanogaster amino acid sequences downloaded from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) were used as BLASTP queries against the B. dorsalis amino acid database with an identity cut-off of 30%. The candidate OR genes were compared with deep transcriptome data from B. dorsalis antennae42, maxillary palps and proboscis, and other tissues.Cloning of candidate B. dorsalis OR genesHigh-fidelity PrimerSTAR Max DNA polymerase (TaKaRa, Dalian, China) was used to amplify the full open reading frame of BdorOR genes by nested PCR using primers (Supplementary Table 2) designed according to B. dorsalis genome data. Each 25-μL reaction comprised 12.5 μL 2 × PrimerSTAR Max Premix (TaKaRa), 10.5 μL ultrapure water, 1 μL of each primer (10 μM), and 1 μL of the cDNA template. An initial denaturation step at 98 °C for 2 min was followed by 35 cycles of 10 s at 98 °C, 15 s at 55 °C and 90 s at 72 °C, and a final extension step of 10 min at 72 °C. Purified PCR products were transferred to the vector pGEM-T Easy (Promega, Madison, WI) for sequencing (BGI, Beijing, China).Transcriptional profilingTotal RNA was extracted from (i) male and female antennae, maxillary palps, head cuticle (without antenna, maxillary palps, and proboscis), proboscis, legs, wings and ovipositors, and (ii) from the heads of 15-day-old virgin and mated females using TRIzol reagent (Invitrogen, Carlsbad, CA). Genomic DNA was eliminated with RNase-free DNase I (Promega) and first-strand cDNA was synthesized from 1 µg total RNA using the PrimeScript RT reagent kit (TaKaRa). Standard curves were used to evaluate primer efficiency (Supplementary Table 3) with fivefold serial dilutions of cDNA. Quantitative real-time PCR (qRT-PCR) was carried out using a CFX Connect Real-Time System (Bio-Rad, Hercules, CA) in a total reaction volume of 10 µL containing 5 μL SYBR Supermix (Novoprotein, Shanghai, China), 3.9 μL nuclease-free water, 0.5 μL cDNA (~200 ng/μL) and 0.3 μL of the forward and reverse primers (10 μM). We used α-tubulin (GenBank: GU269902) and ribosomal protein S3 (GenBank: XM_011212815) as internal reference genes. Four biological replicates were prepared for each experiment. Relative expression levels were determined using the 2−∆∆Ct method43, and data were analyzed using SPSS v20.0 (IBM).Two-electrode voltage clamp electrophysiological recordingsVerified PCR products representing candidate B. dorsalis OR genes and BdorOrco were transferred to vector pT7Ts for expression in oocytes. The plasmids were linearized for the synthesis of cRNAs using the mMESSAGE mMACHINE T7 Kit (Invitrogen, Lithuania). The purified cRNA was diluted to 2 µg/µL and ~60 ng cRNA was injected into X. laevis oocytes. The oocytes were pre-treated with 1.5 mg/mL collagenase I (GIBCO, Carlsbad, CA) in washing buffer (96 mM NaCl, 5 mM MgCl2, 2 mM KCl, 5 mM HEPES, pH 7.6) for 30–40 min at room temperature before injection. After incubation for 2 days at 18 °C in Ringer’s solution (96 mM NaCl, 5 mM MgCl2, 2 mM KCl, 5 mM HEPES, 0.8 mM CaCl2), the oocytes were exposed to different concentrations of 1-octen-3-ol diluted in Ringer’s solution from a 1 M stock in DMSO. Odorant-induced whole-cell inward currents were recorded from injected oocytes using a two-electrode voltage clamp and an OC-725C amplifier (Warner Instruments, Hamden, CT) at a holding potential of –80 mV. The signal was processed using a low-pass filter at 50 Hz and digitized at 1 kHz. Oocytes injected with nuclease-free water served as a negative control. Data were acquired using a Digidata 1550 A device (Warner Instruments, Hamden, CT) and analyzed using pCLAMP10.5 software (Axon Instruments Inc., Union City, CA).Calcium imaging assayVerified PCR products representing candidate B. dorsalis OR genes and BdorOrco were transferred to vector pcDNA3.1(+) along with an mCherry tag that confers red fluorescence to confirm transfection. High-quality plasmid DNA was prepared using the Qiagen plasmid MIDIprep kit (QIAgen, Düsseldorf, Germany). The B. dorsalis OR and BdorOrco plasmids were co-transfected into HEK 293 cell using TransIT-LT1 transfection reagent (Mirus Bio LLC, Japan) in 96-well plates. The fluorescent dye Fluo-4 AM (Invitrogen) was prepared as a 1 mM stock in DMSO and diluted to 2.5 μM in Hanks’ balanced salt solution (HBSS, Invitrogen, Lithuania) to serve as a calcium indicator. The cell culture medium was removed 24–30 h after transfection and cells were rinsed three times with HBSS before adding Fluo 4-AM and incubating the cells for 1 h in the dark. After three rinses in HBSS, 99 μL of fresh HBSS was added to each well before testing in the dark with 1 μL of diluted 1-octen-3-ol. Fluorescent images were acquired on a laser scanning confocal microscope (Zeiss, Germany). Fluo 4-AM was excited at 488 nm and mCherry at 555 nm. The relative change in fluorescence (ΔF/F0) was used to represent the change in Ca2+, where F0 is the baseline fluorescence and ΔF is the difference between the peak fluorescence induced by 1-octen-3-ol stimulation and the baseline. The healthy and successfully transfected cells (red when excited at 555 nm) were used for analysis. The final concentration of 10−4 M was initially used to screen corresponding ORs, and then to determine the response of screened ORs to stimulation with different concentrations of 1-octen-3-ol. Each concentration of 1-octen-3-ol was tested in triplicate. Concentration–response curves were prepared using GraphPad Prism v8.0 (GraphPad Software).Genome editingThe exon sequences of BdorOR7a-6 and BdorOR13a were predicted using the high-quality B. dorsalis genome assembly. Each gRNA sequence was 20 nucleotides in length plus NGG as the protospacer adjacent motif (PAM). The potential for off-target mutations was evaluated by using CasOT to screen the B. dorsalis genome sequence. Each gRNA was synthesized using the GeneArt Precision gRNA Synthesis Kit (Invitrogen) and purified using the GeneArt gRNA Clean-up Kit (Invitrogen). Embryos were microinjected as previously described20. Purified gRNA and Cas9 protein from the GeneArt Platinum Cas9 Nuclease Kit (Invitrogen) were mixed and diluted to final concentrations of 600 and 500 ng/µL, respectively. Fresh eggs (laid within 20 min) were collected and exposed to 1% sodium hypochlorite for 90 s to soften the chorion. The eggs were fixed on glass slides and injected with the mix of gRNA and Cas9 protein at the posterior pole using an IM-300 device (Narishige, Tokyo, Japan) and needles prepared using a Model P-97 micropipette puller (Sutter Instrument Co, Novato, CA). Eggs were injected with nuclease-free water as a negative control. Injection was completed within 2 h. The injected embryos were cultured in a 27 °C incubator and mortality was recorded during subsequent development.G0 mutants were screened as previously described20. G0 adult survivors were individually backcrossed to WT flies (single pair) to collect G1 offspring. Genomic DNA was extracted from G0 individuals after oviposition using the DNeasy Blood & Tissue Kit (Qiagen). The region surrounding each gRNA target was amplified by PCR using the extracted DNA as a template, the specific primers listed in Supplementary Table 2, and 2 × Taq PCR MasterMix (Biomed, Beijing, China). PCR products were analyzed by capillary electrophoresis using the QIAxcel DNA High Resolution Kit (Qiagen). PCR products differing from the WT alleles were purified and transferred to the vector pGEM-T Easy for sequencing. To confirm the mutation was inherited, genomic DNA was also extracted from one mesothoracic leg of G1 flies using InstaGene Matrix (Bio-Rad, Hercules, CA) and was analyzed as above. To avoid potential off-target mutations, heterozygous G1 mutants were backcrossed to WT flies more than 10 generations before self-crossing to generate homozygous mutant flies.Electroantennogram (EAG) recordingThe antennal responses of 15-day-old B. dorsalis adults to 1-octen-3-ol were determined by EAG recording (Syntech, the Netherlands) as previously reported20. Briefly, antennae were fixed to two electrodes using Spectra 360 electrode gel (Parker, Fairfield, NJ, USA). The signal response was amplified using an IDAC4 device and collected using EAG-2000 software (Syntech). Before each experiment, 1-octen-3-ol and other three volatiles (ethyl tiglate, ethyl acetate, ethyl butyrate) were diluted to 10%, 1% and 0.1% (v/v) with MO to serve as the electrophysiological stimulus, and MO was used as a negative control. A constant air flow (100 mL/min) was produced using a controller (Syntech) to stimulate the antenna. We then placed 10 µL of each dilution or MO onto a piece of filter paper (5 × 1 cm), and the negative control (MO) was applied before and after the diluted odorants to calibrate the response signal. The EAG responses at each concentration were recorded for 15–20 antennae, and each concentration was recorded twice. Each test lasted 1 s, and the interval between tests was 30 s. EAG response data from WT and mutant flies for the diluted odorants were analyzed using Student’s t test with SPSS v20.0.Molecular docking and site-directed mutagenesisThe three dimensional-structures of BdorOR7a-6 and BdorOR13a were modeled using AlphaFold 2.044. The quality and rationality of each protein structure was evaluated online using a PROCHECK Ramachandran plot in SAVES 6.0 (https://saves.mbi.ucla.edu/). AutoDock Vina 1.1.2 was used for docking analysis, and the receptor protein structure and ligand molecular structure were pre-treated using AutoDock 4.2.6. The docking parameters were set according to the protein structure and active sites, and the optimal docking model was selected based on affinity (kcal/mol). Docking models were imported into Pymol and Discovery Studio 2016 Client for analysis and image processing. Based on the molecular docking data, three residues (Asn86 in OR7a-6, Asp320, and Lys323 in OR13a) were replaced with alanine by site-directed mutagenesis45 using the primers listed in Supplementary Table 2. Calcium imaging assays and molecular docking of mutated proteins were then carried out as described above.Statistics reproducibilityAll of the olfactory preference assays, oviposition bioassays, expression profiles analysis, EAG recording assays were analyzed using Student’s t-test (*p More
