Rearing of ticks
All stages of ticks (R. haemaphysaloides, a cattle tick from Wuhan, China) were reared in the laboratory on the ears of New Zealand white rabbits at Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Ticks were kept in a dark incubator at 27 °C and 95% humidity in the laboratory as described previously . Adult males and females were fed together to permit mating on the host.
Collection of hemolymph and dissection of tissues from ticks
Ticks were cleaned in water, dried, and glued (cyanoacrylate compound) to the bottom of a transparent Petri dish, and placed under crushed ice for 15 min. Afterward, the ice was removed, and a small incision was made along the dorsolateral margin. Exuding hemolymph (up to 30 μl) was collected in capillary tubes, and 300 μl methanol was added immediately. It was then frozen quickly by liquid nitrogen immersion and stored at −80 °C until 20E extraction and detection.
For tissue collection, after identical tick preparation, a small incision was made along the dorsolateral margin, and the dorsal surface was removed with a scalpel blade. The coelom was washed with cooled phosphate-buffered saline (PBS) and the salivary gland, fat body, gut, ovary, and synganglion were removed. These organs were then immediately separated with fine-tipped forceps under a light microscope . The sample materials were quickly frozen in liquid nitrogen and stored at −80 °C until use (for RNA and protein analysis). A section of the salivary gland (10 mg) was homogenized using an ultrasonic instrument, and 300 μl methanol was added immediately; it was then frozen quickly by liquid nitrogen immersion and stored at −80 °C until 20E extraction and detection.
Cloning the open reading frame (ORF) of the RhEcR and RhUSP genes
According to the salivary gland transcriptomes of engorged R. haemaphysaloides in our laboratory, we localized ORF regions of RhEcR and RhUSP in contig sequences (Additional file 2). Primers (Additional file 1: Table S1) were designed to clone these sequences. The amplicons with complete ORF sequences were cloned into the pET-28a vector using the ClonExpress II One Step Cloning Kit (Vazyme Biotech, Nanjing, China), and the obtained clones were sequenced. Signal peptide analysis, isoelectric point/molecular weight (PI/Mw), and nuclear localization signal (NLS)/nuclear export signal (NES) were predicted by the online software SignalP 5.0 (http://www.cbs.dtu.dk/services/SignalP/) , ExPASy (https://web.expasy.org/compute_pi/) , and NLS/NES signal (https://rostlab.org/services/nlsdb/) . Similar sequences of target genes were searched using the Smart BLAST (or Protein BLAST) server (National Center for Biotechnology Information, National Institutes of Health). The sequences of RhEcR1, 2, and 3 and RhUSP1 and 3 were aligned with the EcR/LXR and the USP/RXR of other species using Genetyx version 6 software (Genetyx, Tokyo, Japan). For phylogenetic analysis, the alignment of the sequences was performed using the CLUSTALW algorithm  and inferred using the maximum likelihood method with default settings in MEGA 6.0 software . The sequences of EcR/LXR and USP/RXR in vertebrates and invertebrates were obtained from the GenBank database (Additional file 1: Table S2).
Expression analysis of genes by quantitative real-time polymerase chain reaction (qRT-PCR)
The RhEcR and RhUSP gene expression profiles in the salivary gland, gut, fat body, ovary, and synganglion from adult female ticks who were unfed, after feeding for 3 days (Fed 3) and 5 days (Fed 5), and post-engorgement at 0 h (E 0h), 24 h (E 24h), 48 h (E 48h), 72 h (E 72h), and 96 h (E 96h) were determined by qRT-PCR using the 2−△CT analytical method. Ten female ticks per condition were analyzed. The data were normalized to elongation factor 1-alpha (EF1α). Total RNA was extracted using TRIzol reagent (15-596-018, Invitrogen, USA) and transcribed to synthesize complementary DNA (cDNA) using HiScript® III RT SuperMix (R323, Vazyme Biotech, Nanjing, China), using the following program: 42 °C for 2 min, 37 °C for 15 min, 85 °C for 5 s, and finally 16 °C for the synthesis of cDNA. The cDNA samples were analyzed by qRT-PCR using the ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme Biotech, Nanjing, China) with a QuantStudio™ 5 Real-Time PCR System (Applied Biosystems™, Waltham, MA, USA), with program parameters of 95 °C for 30 s; 40 cycles of 95 °C for 10 s and 60 °C for 30 s; and 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s.
Preparation of polyclonal antibodies (PcAb) in mice
The epitopes of RhEcR were analyzed by antibody epitope prediction using BepiPred Linear Epitope Prediction 2.0 (http://tools.iedb.org/bcell/) . The peptide sequences were selected and synthesized (GL Biochem, China) and coupled to the keyhole limpet hemocyanin (KLH) carrier protein. Antibodies against the peptides were raised in BALB/C mice by intraperitoneal immunization with 100 μg of the peptide in PBS emulsified with the same volume of Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA). Eight mice in each group, which were maintained in a specific-pathogen-free (SPF) animal area, were inoculated three times at 15-day intervals with 50 μg of peptide in PBS emulsified with the same volume of Freund’s incomplete adjuvant (Sigma-Aldrich, St. Louis, MO, USA). Seven days after the final inoculation, sera were collected and stored at −20 °C until use.
20-Hydroxyecdysone measurement using enzyme immunoassay (EIA)
The 20E levels in the salivary gland and the hemolymph were measured using an enzyme immunoassay (EIA) according to the manufacturer’s instructions (20-Hydroxyecdysone ELISA kit, #A05120.96 wells, Bertin, France). From female ticks of different feeding periods, we collected 10 mg of the salivary glands by dissection and 30 μl of hemolymph by capillary absorption during each time course.
Total protein was extracted from the salivary gland at each time point in RIPA lysis buffer containing protease inhibitor, with 1 mM phenylmethanesulfonyl fluoride (PMSF), followed by centrifugation at 16,000×g at 4 °C for 15 min. The supernatant was collected, and a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the protein concentration following the manufacturer’s instructions. For sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis, 20–50 µg of total protein per sample was applied to 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was incubated with antibodies at 4 °C overnight and then with goat anti-mouse or goat anti-rabbit conjugated with horseradish peroxidase (HRP; D20691 or A32731, Invitrogen, USA) at room temperature for 2 h. Images were captured using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA).
RNA interference for gene knockdown in ticks
Synthesis of double-stranded RNA
Double-stranded RNA (dsRNA) was synthesized using the T7 RiboMAX™ RNAi System (P1700, Promega, Madison, WI, USA) according to manufacturer’s recommendations. Primers containing T7 promoter sequences (Additional file 1: Table S1) were designed to synthesize the dsRNA. The dsRNA to RhEcR, RhUSP, and Luciferase mRNA had about 500 base pairs (bp) each. Because the R. haemaphysaloides genome sequence is not available, the siRNA specificity and potential off-targets were estimated based on similar regions of the Drosophila melanogaster ortholog gene (accession number XP_021710216) and genome sequences using the dsCheck program . Also, using BLAST, analyses were conducted using publicly available genome sequences of Ixodes persulcatus, Haemaphysalis longicornis, Dermacentor silvarum, Hyalomma asiaticum, R. sanguineus, R. microplus, and I. scapularis to confirm that the dsRNA was specific to the target genes and to reduce the possibility of off-target effects. A dsRNA targeting luciferase was used as a negative control . The quality of the dsRNA was determined by electrophoresis on 1.0% agarose gel. The dsRNA was stored at −80 °C until use.
Virgin or unfed adult female ticks (n = 80 females per group, two independent groups) were micro-injected with approximately 0.5 μl dsRNA (containing about 1 μg) to RhEcR, RhUSP, and Luciferase at the base of the fourth right leg (near the genital aperture) of the ventral surface. After microinjection, ticks were maintained in a dark incubator at 27 °C with 95% humidity for 24 h, and they were then allowed to feed on rabbit ears. Four female ticks per group were collected after 5 days of feeding for RNA extraction using TRIzol reagent (15–596-018, Invitrogen, USA), following the manufacturer’s instructions. qRT-PCR was performed to characterize gene knockdown efficiency using the 2 −△△CT analytical method. Ten female ticks per group were analyzed. The data were normalized to EF1α. The remaining ticks were allowed to feed until they detached. The tick 24-h attachment rate, engorgement rate, engorgement weight, oviposition rate, and egg-hatching rate were determined for the individual female ticks.
Histology and HE staining
The salivary glands of each groups were removed from adult female ticks by micro-dissection at different time points during blood-feeding and after engorgement. Salivary glands were fixed in 4% paraformaldehyde and embedded with paraffin. Paraffin sections were stained with hematoxylin–eosin (HE). HE staining images were obtained by light microscopy (Nikon Eclipse 80i microscopy system using a ×40 objective).
Quantitative proteomic analysis by tandem mass tag (TMT) technology
The salivary glands of normal female ticks (Fed 3, Fed 5, E 0h, and E 72h) and dsEcR-treated/dsLuciferase-treated ticks (Fed 3, Fed 5, and Fed 7) were dissected and washed with ice-cold PBS, then frozen quickly by liquid nitrogen immersion and stored at −80 °C. Each sample had 15–20 female ticks and two repeats. All the samples were treated and tagged with TMT and analyzed by Shanghai Applied Protein Technology.
Co-transfection and co-immunoprecipitation
HEK293T cells were seeded onto a six-well plate. Cells with 70–80% confluence were co-transfected with the plasmids pCMV-Flag-RhEcR1, pCMV-Flag-RhEcR2, pCMV-Flag-RhEcR3, pCMV-HA-RhUSP1, pCMV-HA-RhUSP3, GFP-RhEcR1, and mCherry-RhUSP1 using Lipofectamine™ 3000 reagent according to the manufacturer’s instructions (L3000008, Invitrogen, USA). After 24 h, the cellular supernatant was collected to detect the cytotoxicity for detection of cell death using the ToxiLight™ bioassay kit according to manufacturer’s instructions (LT07-117, Lonza, Basel, Switzerland). The transfected cells were collected and protein was extracted using a lysis buffer (50 mM Tris, 150 mM NaCl, 0.5% NP-40 (v/v), pH 7.5) containing protease inhibitor for the immunoprecipitation assay. Cell extract supernatants were incubated with anti-Flag immunomagnetic beads (B26101, Bimake, USA) and anti-HA immunomagnetic beads (B26201, Bimake, USA) according to the manufacturer’s instructions for 7–8 h at 4 °C with gentle shaking. The protein–antibody complex with immunomagnetic beads was separated magnetically and washed three times with PBST. After adding 50 μl 1× protein loading buffer and boiling for 5 min, the samples were cooled to room temperature and placed on a magnetic rack for 10 s. Finally, the protein supernatant was used for the SDS-PAGE test. The samples were subjected to SDS/PAGE (10% gel, GenScript, Nanjing, China), followed by western blot analysis with rabbit monoclonal antibodies against Flag (14793S, Cell Signaling Technology, Danvers, MA, USA) and HA (3724S, Cell Signaling Technology) to detect the target proteins. GFP-EcR1 and mCherry-USP1 were co-transfected into HEK293T cells for 24 h to detect the expression of proteins under a fluorescence microscope. We then discarded the supernatant, fixed the cells, stained the cells with DAPI for 5–10 min, and observed proteins under a fluorescence microscope.
All statistical analyses were performed using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA). One-way and two-way analysis of variance (ANOVA) for statistical differences between multiple groups was used (α = 0.05). Mean ± standard deviation (SD) values were calculated for three separate experiments, and two-tailed Student’s t-tests were used to identify significant differences (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) between groups.