2.1 Tick Feeding and tissue collection
Adult Rhipicephalus haemaphysaloides Supino 1897 (Ixodida: Ixodidae) were collected from Wuhan in Hubei Province, China. The ticks were fed on the ears of New Zealand white rabbits (SLAC, Shanghai Institutes for Biological Science, CAS), and maintained in artificial climate incubators of the Shanghai Veterinary Research Institute . Rabbits and mice were maintained following the approved guidelines of the Institutional Animal Care and Use Committee of the Shanghai Veterinary Research Institute (IACUC approval number: SHVRI-SZ-20190726-02; SHVRI-SZ-20191024-01). Tick tissues were rapidly dissected, washed with phosphate-buffered saline (PBS, PH 7.4, with 0.14 M NaCl and 0.0027 M KCl, 0.01 M phosphate buffer; Gibco, Life Technologies), and placed in PBS or TRIzol (Invitrogen, Carlsbad, CA, USA) reagent at − 80°C.
2.2 RNA extraction and cDNA synthesis
RNA was derived from ticks dissected at different development stages (eggs, unfed and fed larvae, nymphs, and adults) and different tissues (salivary glands, midguts, and ovaries of female ticks), and preserved in TRIzol reagent. The HiScript® III RT SuperMix for qPCR (+ gDNA wiper) kit (Vazyme Biotech, Nanjing, China) was used, according to manufacturer protocol, to synthesis cDNA.
2.3 Cloning, sequence analysis, and expression of RhBcl-2 and RhBax
RhBcl-2 and RhBax primers were designed based on a comparison of the salivary gland transcriptomes of starved and engorged R. haemaphysaloides [27, 28]. A BLAST analysis of the translation products deduced from the open reading frames (ORFs) was performed. SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/)  and ExPASy (http://web.expasy.org/compute pi/)  were used for the Signal peptide analysis and isoelectric point (PI) prediction. We aligned RhBcl-2 and RhBax with the Bcl-2 and Bax protein sequences of other species using Genetyx ver. 6 (GENETYX, Tokyo, Japan). For phylogenetic analysis, the alignment of the sequences was performed using the MUSCLE algorithm  and inferred using the Maximum likelihood method with the default settings in MEGA X software . Bootstrap support values were estimated using 500 bootstrap replicates .
2.4 RT-qPCR analyses
The expression levels of RhBcl-2 and RhBax were examined in ticks from different development stages (eggs, unfed and fed larvae, nymphs, and adults) and different tissues (salivary glands, midguts, and ovaries of female ticks). After RhBcl-2 and RhBax primers (Table S1) designed using Primer Premier 5, the cDNAs of the above stages and tissues were analyzed by RT-qPCRs which were conducted using ChamQ Universal SYBR qPCR Master Mix (Vazyme) green and gene-specific (Table S1) primers with a QuantStudio 5 PCR System (Applied Biosystems, Austin, TX, USA). The RT-qPCR process was 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s, followed by analysis of the melting curve. All samples were analyzed in triplicate. The data used elongation factor-1(ELF1A, Genbank accession no. AB836665) as an internal control , and this was used to analyze the relative gene expression in each sample by the 2−△Ct method [32, 33].
2.5 Expression of recombinant RhBcl-2 or RhBax and antibody production
Specific RhBcl-2 and RhBax primers (Table S2) were designed in a pET-30a or PGEX-4T-1 vector. The RhBcl-2 and RhBax amplified-PCR products were purified and digested with BamHI, EcoRI, or XhoI (New England Biolabs, USA) and ligated into pET-28a or PGEX-4T-1(Invitrogen) using In-Fusion HD Cloning Kits (Takara Clontech, Mountain View, CA, USA) . These recombinants were transformed and expressed in Escherichia coli (E. coli) BL21 (DE3) strain (TIANGEN, Beijing, China). The strains were grown until the OD600 reached 0.8 at 37°C. After isopropyl β-D thiogalacto pyranoside (IPTG) was added to 1 mM, the protein expression was induced at 25°C for 20 h. The recombinant, containing His-tagged protein, was purified by affinity chromatography using Ni-NTA His resin (Thermo Fisher Scientific, Waltham, MA, USA) by gravity. The recombinant containing the GST-tagged protein was purified by affinity chromatography using resin and gravity.
2.6 GST-Pull Down of RhBcl-2 and RhBax
To confirm whether RhBcl-2 interacts with RhBax, 1 mM IPTG induced expression for 8 h of pET-30a-RhBax, PGEX-4T-RhBcl-2, and PGEX-4T-1 recombinant E. coli BL21. The supernatant was separated using GST agarose (Merck, Darmstadt, Germany) according to manufacturer instructions. Anti-GST antibody (Proteintech, 215 Rosemont, IL, USA) and goat anti-mouse secondary antibody (Proteintech) were used to confirm that GST/GST-RhBcl-2 was separated successfully, and anti-His antibody (Proteintech) was used to detect the target protein.
2.7 Cells and Transient Co-transfection assays
HEK 293 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco), supplemented with 8% heat-inactivated fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel) and 1% penicillin (Gibco) at 37°C .
The full-length ORF of RhBcl-2 was inserted into the p3×Flag-CMV-14 vector (MiaoLing Plasmid Sharing Platform, Wuhan, China) with Flag tag at the N-terminal with gene specific primers (Table S2). RhBax was inserted into the pcmv-HA vector using same method. The fragment of RhBax deleted BH3 domain (Rhbax- ΔBH3) and BH1 domain (Rhbax-ΔBH1) was amplified by splicing by overlap extension (SOE) of recombinant PCR with specific primers (Table S3), and they were inserted into the pcmv-HA vector . Transfection, using Lipofectamine™ 3000 Transfection Reagent (Invitrogen), was performed according to manufacturer protocol with a DNA to Lipofectamine ratio of 1: 2 w/v. The HEK 293 cells were transformed with 2.5 µg/well of plasmid or co-transfected with the equivalent amount of two different plasmids in 6-well plates .
2.8 Antibody generation and western blotting
Recombinant RhBcl-2 and RhBax were expressed and purified according to previously described protocols . Polyclonal antibodies (pcAb) against recombinant RhBcl-2 and RhBax were developed in mice. We tested the specific recognition of the recombinant protein by the antibodies using western blots. We predicted the epitopes of RhBcl-2 and RhBax online (http://www.iedb.org/), and synthesized Keyhole limpet hemocyanin (KLH)  coupled polypeptides based on the epitope amino acid sequences (RhBcl-2: GLQWNTCPPLPRPSK, RhBax: STPTHEETREE). Polypeptides were dissolved in PBS and Freund’s adjuvant (complete and incomplete; Invitrogen). Equal volumes of these solutions were then emulsified together and injected into six to eight weeks old BALC/c mice provided by the Shanghai Laboratory Animals Center (Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai, China). The mixtures were injected three times at two weeks intervals. The sera were collected on the seventh day after the third immunization.
Total proteins of different development stage ticks were extracted using Tris-buffered saline (TBS; 10 mM Tris–HCl, pH 7.5; 150 mM NaCl with 1 mM phenylmethanesulfonyl fluoride). A Bradford Protein Assay Kit (Beyotime Tiangen) was used to determine the protein concentration. Proteins were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE, 12%, Genescript, Nanjing, China) and transferred onto polyvinylidene difluoride (PDVF) membranes. After blocking, the proteins were incubated at 4°C overnight with primary antibodies. The sera anti RhBcl-2 and RhBax were used to detect RhBcl-2 and RhBax in protein extracts, and anti-tubulin (1:1000, Proteintech, Rosemont, IL, USA) primary antibody was used as a constitutive control to normalize the signal from the target protein. The membranes were rinsed five times in tris-buffered saline with Tween-20 (TBST) and incubated with a secondary antibody (goat anti-mouse, 1:5000, Cell Signaling Technology, USA) for 1 h at room temperature. The membranes were rinsed five times by TBST. Images were captured by ChemiDoc Touch (Bio-rad, Hercules, CA, USA) .
2.9 RNAi of RhBcl-2 or RhBax
The RNAi experiments were designed against RhBcl-2 and RhBax genes. For the design of RNAi primers, RhBcl-2 and RhBax sequences were screened by Primer Premier 5. Specific primers (Table S4) containing the T7 polymerase promoter sequence were used for PCR amplification. The amplicons were then purified to obtain templates for double-stranded RNA synthesis using the T7 RiboMAX Express RNAi system (Promega, Madison, WI, USA). The unrelated gene Luciferase dsRNA was synthesized using the same methods described previously and used as the negative control. Unfed female ticks (N = 20 females per group) were microinjected with approximately 1 µg of dsRNA. Control ticks were injected with unrelated Luciferase dsRNA. After dsRNA injection, female ticks were held in a humidity chamber for 1 day after which they were allowed to feed on rabbit ears. The biological parameters analyzed were attachment rate at 24 h, number of engorged ticks, death rate, and weight. RNA was used to characterize gene knockdown by real-time RT-PCR.
We interfered with the salivary gland using dsRNA in vitro. The salivary gland of female ticks fed on rabbit for 5 days was dissected and placed into complete L15 medium with 1% penicillin-streptomycin . Each well received 5 µg of dsRNA and incubated at 27°C with no CO2 for 48 h. The control group received unrelated Luciferase dsRNA. Each treatment group included the salivary glands from 10 ticks. After incubation, a pair of the salivary glands in each group was fixed in 4% paraformaldehyde for TUNEL staining or fixed in 2.5% glutaraldehyde for at least 24 h at 4°C for TEM. The remaining salivary glands were used for western blots and RNA.
2.10 TUNEL staining
Salivary glands were fixed in 4% formalin and paraffin-embedded. Sections of salivary glands were mounted on microscope slides. Tissue sections were then deparaffinized, washed in 100% ethanol, and rehydrated. Samples were washed with PBS. After antigen retrieval with 0.1% Triton X-100, the tissues were incubated for 1 h with 1:9 TdT mixed with fluorescent-labeled dUTP at 37°C, following the instructions of the Roche In Situ Cell Death Detection Kit, POD (Roche, Mannheim, Germany). After washing the Sect. 2–3 times with PBS, the sections were stained with 1 µg/mL 4’, 6’-diamidino-2-phenylindole (DAPI, Invitrogen) in distilled H2O for 20 min . After washing, the sections were mounted using Lab Vision™ PermaFluor™ (Invitrogen) medium under glass coverslips, then viewed and photographed on a Pannoramic DESK Digital Slide Scanner (3D HISTECH, Budapest, Hungary) .
2.11 Data analysis
All statistical analyses were performed using GraphPad Prism 6.0 software (Graph Pad Software Inc., San Diego, CA, USA). Mean ± standard error (SEM) values were calculated for three separate experiments, and two-tailed Student’s t tests were used to identify significant differences between groups. P < 0.05 was considered to be statistically significant.