Frankliniella occidentalis maintenance and TSWV acquisition
A virus-free F. occidentalis colony was isolated from an indoor strain that had been kept in our laboratory for nearly 20 years. Thrips were fed with fresh bean pods and were kept in glass jars in a climate chamber at 26 ± 1℃, with 70 % relative humidity and a 16L: 8D photoperiod as described by Wan et al. (2020a). TSWV isolate TSWV-YN was maintained on Datura stramonium plants by thrips transmission. Young D. stramonium plants were mechanically inoculated with TSWV, and TSWV infection was confirmed 2 weeks later by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using a purchased kit (Agdia Incorporated. Elkhart, Indiana, USA) and following the manufacturer’s instructions. DAS-ELISA-positive plants were used for virus acquisition by newly hatched F. occidentalis nymphs (Wan et al. 2020b).The TSWV-infected plants were placed in an incubator at 22 ± 1℃ with 80 % relative humidity and a 16L: 8D photoperiod.
Obtaining male thrips with and without TSWV
The large number of male thrips used in this study were obtained from arrhenotokous parthenogenesis of virgin females. Approximately 500 adult thrips (mixed sex) were placed in a glass rearing jar and allowed to lay eggs on fresh bean pods for 48 h. The bean pods with eggs were placed in another glass jar and reared to the pupal stage with fresh bean pods provided as needed. Pupae were transferred into a 1.5 mL centrifuge tube (one pupa per tube) using a fine paint brush. The sex of the newly emerged adults was determined by microscopic examination. All newly emerged virgin females were placed in a new glass jar and fed with fresh bean pods. Newly hatched 1st-instar nymphs (< 6 h) were collected and placed in a 15-cm-diameter petri dish. Half of the nymphs were reared on TSWV-infected D. stramonium leaves, and these were regarded as the TSWV infected group or TSWV (+); the other half was reared on healthy D. stramonium leaves, and these served as the control group or TSWV (-).
Approximately 400 1-day-old males were collected from TSWV (+) and TSWV (-) groups and were rapidly frozen in liquid nitrogen after being placed in 1.5 mL RNase-free centrifuge tubes. Five cohorts were separately collected from each group as five biological replicates. For the TSWV (+) group, the TSWV acquisition rate was detected by using qRT-PCR (Wan et al. 2020b). Only cohorts with an acquisition rate > 90 % were used for subsequent proteomic assays. The samples were suspended in lysis buffer (1 % SDS, 8 M urea) that included an appropriate protease inhibitor. The concentration of protein supernatant was determined by using the Pierce Bicinchoninic acid Protein Assay Kit (Thermo, USA). The samples were dissolved in 0.5 M tetraethylammonium bromide (TEAB) (Applied Biosystems, Milan, Italy). One unit of tandem mass tag (TMT) reagent was then thawed and reconstituted in 50 µL of acetonitrile. After they were tagged for 2 h at 37℃, all samples were pooled, desalted, and vacuum-dried (Jia et al. 2018; Zhong et al. 2019). Peptides were first separated with a gradient of eluent (phase A: 5 mM ammonium hydroxide solution containing 20 % acetonitrile, pH 10; phase B: 80 % acetonitrile, pH 10) by ACQUITY Ultra Performance liquid chromatography (UPLC) (Waters, USA) with an ACQUITY UPLC BEH C18 Column (1.7 µm × 2.1 mm × 150 mm, Waters, USA). Peptides were then analyzed by online nano flow liquid chromatography tandem mass spectrometry performed on an EASY-nLC 1200 (Thermo, USA) connected to a Orbitrap Exploris 480 (Thermo, USA) through a nano-electrospray ion source (Jia et al. 2018; Zhong et al. 2019). The raw data obtained from LC/LC–MS/MS were analyzed using Proteome Discoverer (Thermo Scientific, Version 2.4) against uniprot-taxonomy-133901. unique. fasta database. The false discovery rate (FDR) of peptide identification was set as FDR ≤ 0.01. Annotation of all identified proteins was performed using GO (http://geneontology.org/) and KEGG pathways (http://www.genome. jp/kegg/).
Molecular cloning and bioinformatics analysis
Total RNA was extracted from male thrips using TRIzol (Invitrogen, Carlsbad, CA) The integrity, concentration, and purity of RNA were evaluated with a spectrophotometer (NanoDrop 2000c, MA, USA), and cDNA was synthesized using a reverse transcription kit (PrimeScript RT reagent Kit, Takara Biotech, Tokyo, Japan). Based on the transcriptome data of F. occidentalis in our laboratory, primers of FoFSCB-like were designed with Primer3plus (Table 1). PCR reactions were performed using High-Fidelity Master Mix (Beijing Kinco Biotechnology Co. Ltd.) following the manufacturer’s instructions. The 25 μL PCR reaction system contained 2 × High-Fidelity Master Mix 12.5 µL, forward and reverse primers (10 μmol/L) each 1 μL, cDNA template 1 μL, and nuclease-free water 9.5 μL. PCR reactions were performed as follows: 98℃ 2 min; 98℃ 10 s, 60℃ 15 s, 72℃ 50s, 35 cycles; 72℃ 5 min (Wan et al. 2018). The amplified PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. SignalP (http:∥www.cbs.dtu.dk/services/SignalP/) and TMHMM (http:∥www. cbs.dtu.dk/services/TMHMM/) were used to predict signal peptide and the transmembrane domains, respectively. Protein conserved domains were identified using NCBI CDD Tools (https://www.ncbi.nlm.nih.gov/cdd).PROSITE(https://prosite.expasy.org), and Pfam (Pfam: xfam.org) was used to analyze the phosphorylation sites.
The expression profile of FoFSCB-like at different developmental stages of TSWV (+) and TSWV (-) groups was determined by qRT-PCR. Approximately 100 1st-instar nymphs, 100 2nd-instar nymphs, 80 pro-pupae, 70 pupae, and 70 newly emerged male adults were collected. Total RNA extraction, cDNA synthesis, and primer design were conducted as described above. The reaction was performed in an ABI PRISM 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA) with 20 μL, containing 2 × FastFire qPCR PreMix (SYBR Green) (Tiangen Biotech Co., Ltd., Beijing, China) 10 μL, cDNA template 1 μL, forward and reverse primers (10 pmol/μL) each 0.6 μL, 50 × ROX Reference Dye (Tiangen Biotech Co., Ltd., Beijing, China) 0.4 μL, and RNase-Free ddH2O 7.4 μL. The qRT-PCR program was as follows: 95℃ 10 min (pre-denaturation); 95℃ 15 s (denaturation), 60℃ 30 s (annealing), 72℃ 30 s (extension), 40 cycles (Peng et al. 2021). Succinate dehydrogenase (SDHA) (GenBank accession no. XM026420561.1) and β-actin (GenBank accession no. GQ290644) were selected as reference genes (Cifuentes et al. 2012), and the geometric means of the Ct values were used to normalize the target gene (Vandesompele et al. 2002). The relative expression level of FoFSCB-like was calculated using the 2-ΔΔCt method (Wan et al. 2018). Four biological and technical replicates were performed for each sample.
Gene-specific dsRNA primers containing a T7 RNA polymerase promoter sequence (Table 1) were designed with the online dsRNA design tool (https://www.flyrnai.org/cgi-bin/RNAi_find_primers.pl/). The T7 RiboMAX Express RNAi kit (Promega, Madison, WI, USA) was used to synthesize dsFoFSCB-like and dsEGFP according to the manufacturer’s instructions. The integrity of the dsRNA was confirmed by 1.5 % agarose gel electrophoresis, and the dsRNA products were stored at -80°C. Because the expression of the FoFSCB-like gene was highest in pupae, pupae were used for subsequent RNAi. A nanoparticle-mediated RNAi method was performed as described by Peng et al. (2021), with some modifications. Approximately 70 pupae collected from the TSWV (-) group were placed in a disposable plastic petri dish (diameter = 3 cm). The dsRNA mixture (1 µg/µL of dsFoFSCB-like/dsEGFP and an equal volume of nanoparticles) were dripped onto pupae with a pipette. After 6 h, the treated pupae were transferred from the Petri dish to 1.5 mL centrifuge tubes for RNA extraction; control pupae were treated in the same manner except they were not exposed to the dsRNA. The interference efficiency was determined using qRT-PCR as described above. There were four biological replications for dsFoFSCB-like and dsEGFP treatments.
Approximately 40 pupae were treated with dsFoFSCB-like or dsEGFP. Newly emerged males were individually placed in a plastic cylinder (diameter = 8 cm, height = 6 cm) together with a healthy virgin female; a bean pod was provided for egg laying, and bean pods were collected and replaced every 2 days for a total of five collections. The bean pods with eggs that were collected each time were placed in a new plastic cylinder as described above and were reared in the usual way until adults emerged. The numbers of females and males were counted. At least 20 pairs (replications) were measured for each treatment.
Unless otherwise stated, all quantitative data are presented as the means ± SEM of at least three independent experiments. Differences (P < 0.05) in relative gene expression among developmental stages were determined by one-way analysis of variance (ANOVA) with Tukey’s HSD test (Graphpad Prism 8.0, San Diego, CA). Differences in relative gene expression, number of adults, and sex ratio between TSWV (+) and TSWV (-) groups, or between the dsEGFP and dsFoFSCB-like treatments, were determined using Student’s t-test (Graphpad Prism 8.0, San Diego, CA).