Wild adult H. doenitzi ticks were collected from vegetation by flag-dragging in Cangxi county (31°37′-32°10′N, 105°43′-106°28′E), Sichuan province. They were placed on the ears of New Zealand white rabbits glued with cloth bags for feeding. Those in non-parasitic stages are kept in an incubator at 26 ± 1°C and 75 ± 3% relative humidity under a 16/8-ligh/dark photoperiod. All experiments in this study were administrated with the permission of the Animal Ethics Committee of the Hebei Normal University (Protocol Number: IACUC-156027).

A number of unfed adult ticks with total body weights ranging from 50 to 80 mg were randomly selected and extracted for RNA according to the instructions of the TransZol™ Up Plus RNA Kit (TransGen Biotech, China). A cDNA library was synthesized according to Easy Script^® first-strand cDNA Synthesis SuperMix (TransGen Biotech, China) and stored at −80°C.

Based on the transcriptome library of H. doenitzi deposited on GenBank (accession number: MZ981733), specific primers (forward primer 5′-CAGCGAAATGGCTTCTG-3′ and reverse primer 5′-TCAGTATTTGCAGGCGAAC-3′) were designed using Primer Premier 5.0 (Premier Biosoft International, United States). The PCR program was as follows: 94°C for 10 min, followed by 28 cycles each at 94°C for 30 s, 56°C for 30 s, 72°C for 1 min, and then a final elongation at 72°C for 10 min. The target PCR product was isolated by agarose gel electrophoresis (1%) and purified with gel extraction kits. Subsequently, the purified PCR product was inserted into the pMD^®19-T Simple Vector (BGI, China) for sequencing. The confirmed sequence was analyzed by BLAST on NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignments were performed using the DNAMAN software (http://www.lynnon.com). The analysis selected H. longicornis (BAH02683.1), H. flava (ANA67892.1), R. microplus (XP_037274758.1), D. variabilis (ACF35510.1), and I. scapularis (XP_029835135.2). Then, the molecular weight (MW) and theoretical isoelectric point (pI) were predicted using Compute PI/Mw (http://web.expasy.org/compute_pi/). Signal peptides of the sequence were predicted based on SignalP (http://www.cbs.dtu.dk/services/SignalP/).

A total of 120 partially fed female adult ticks were used to collect hemolymph, salivary glands, ovaries and midguts, and eggs (incubated for 10 days); larvae (10 days after hatching), nymphs (10 days after molting), and adults (10 days after molting) were used to determine different life stages (unfed) and different tissue expression patterns of Doenitin-1. Total RNA was extracted as described above, and specific primers (qRT-PCR forward primer 5′-ATGGCTTCTGTCGGCTTCC-3′ and qRT-PCR reverse primer 5′-TTAGAGTCTCGAAGTTGTTCTCGTT-3′) were used. β-actin was used as a reference gene (β-actin forward primer 5′-CGTTCCTGGGTATGGAATCG-3′ and β-actin reverse primer 5′-TCCACGTCGCACTTCATG-3′) (27). Then, the SPSS 16.0 software for Windows (SPSS Inc, USA) was used to conduct a one-way ANOVA for qRT-PCR results (28).

The fragment was inserted into pET-32a with the Not I and BamH I restriction sites and then transformed into Escherichia coli (TransGen Biotech, China) cells and cultured overnight at 37°C. We added IPTG until the final concentration was 0.2 mM, and E. coli was induced for 6 h at 200 r/min at 37°C. The bacteria were centrifuged at 12,000 × g for 10 min, and the resultant supernatant was filtered with a 0.4-μm filter.

Recombinant proteins were purified using a ProteinIso™ Ni-NTA Resin kit (TransGen Biotech, China). The sample was added to the chromatography column filled with Ni-NTA for affinity for 30 min. Then, it was eluted with different imidazole concentrations (20, 50 100, 200, 250, and 500 mM) to get the recombinant protein in the gravity affinity chromatography. Finally, after elution with a 3-ml elution buffer (0.025 M NaCl, 0.05 M NaHPO₄, and 0.02 M imidazole, pH = 7.4), the chromatography column was sealed with 3 ml anhydrous ethanol.

Preliminary purified samples were lyophilized and then diluted to a suitable concentration with ddH₂O. Subsequently, dialysis membranes with a molecular weight cutoff of 8–14 kDa (Solarbio, China) and an ultrafiltration centrifugal tube (3 kDa) were used to further purify the eluted recombinant protein. Aliquots of a 20-μl sample (1 mg/ml) was boiled for 5 min and then separated by SDS-PAGE (12%). The concentration of the purified protein was determined using a BCA protein detection kit (Transgenic, China). The purified recombinant protein was identified by LC-MS/MS using a M-Class nanoACQUITY ultra performance liquid chromatography (UPLC) (Waters, USA) and a Q Exactive HF mass spectrometer (Thermo Fisher Scientific).

Healthy adult human venous blood (2 ml) was collected in anticoagulant tubes containing sodium (KWS, China) and centrifuged at 4,000 r/min for 5 min to separate the plasma. The activated partial thromboplastin time (APTT) kit and the prothrombin time (PT) kit were purchased from Sunbio (China). The plasma and reagents were incubated at 37°C for 3 min. First, we used an ACLTOP 700 coagulation analyzer (Werfen, Spain) to test APTT and PT as the group of control. The results were recorded, and three normal samples were selected (PT normal value: 12–16 s; normal APTT: 24–36 s). Second, the recombinant protein was diluted at different concentrations of 0, 3.5, 7, 14, and 28 μM. Then, the groups were added separately to normal plasma samples, and the APTT and PT results were recorded after re-mixing. The experiment was repeated thrice.

After mixing 80 μl fibrinogen solution and 80 μl rDoenitin-1 with different concentrations (0, 0.5, 1, 1.5, 2, and 2.5 μM), the mixtures were mixed and placed at a 37°C constant temperature water bath and incubated for 2 min. Then, 80 μl thrombin (Sigma, China) was added, and the absorbance of the mixture was immediately determined at 650 nm every 12 s with a UV spectrophotometer and monitored for 30 min. The experiment was repeated thrice.

rDoenitin-1 was mixed with three serine peptidases (thrombin, trypsin, and cathepsin G; Sigma, China). According to Assumpção et al. (29), corresponding chromogenic substrates were added to detect the residual activity of the peptidase. The buffers selected for the reaction of thrombin, trypsin, and cathepsin G with recombinant proteins were 50 mM Tris-HCl (pH 8), 150 mM NaCl, 20 mM CaCl₂, and 0.01% Triton X-100. Boc-ASP-Pro-Arg-AMC (24.2 μM), Boc-GLN-Ala-Arg-AMC (250 μM), and SUC-Ala-Ala-Pro-Phe-AMC (250 μM) (Njpeptide, China) were selected as the chromogenic substrate for thrombin (27 nM), trypsin (10 nM) and cathepsin G (10 nM), respectively, and the concentration was 24.2 μM when the substrate was dissolved in a buffer under dark conditions. Dissolved thrombin at a concentration of 27 nM was used. The recombinant protein was diluted at a concentration gradient (32, 16, 8, 4, 2, 1, and 0.5 nM for thrombin; 200, 100, 50, 25, 12.5, and 6.25 nM for trypsin and cathepsin G) and no recombinant protein at 0 nM. Serine peptidase (20 μl) and recombinant protein (20 μl) with different concentration gradients were placed into an incubator and incubated at 37°C for 30 min (40-μl buffer was added into the control well). On a 96-well black ELISA plate (Corning, United States), a 60-μl buffer was added to each well, followed by the addition of a 100-μl color developing substrate under dark conditions. The microplate was then put into a microplate meter; the excitation wavelength was set at 355 nm and the emission wavelength at 460 nm to measure the OD value of each well. The experimental group OD value was A_sample, the recombinant protein concentration at 0 nM was A_black, and the control well OD value was A_control. Residual peptidase activity (%) = (A_sample-A_control)/A_black × 100% (30). The experiment was repeated thrice.

The healthy adult venous blood (6 ml) was mixed with 6 ml normal saline and centrifuged for 4 min at 3,000 × g, and the supernatant discarded. An appropriate amount of normal saline was added and centrifuged for 4 min at 3, 000 × g, and the supernatant was discarded. We repeated the previous step until the supernatant of the liquid changes from red to transparent after centrifugation. We added 9 ml normal saline to suspend the packed red blood cells and added the following solution into a 1.5-ml EP tube according to the following system: positive control group (absorbance value is A): 20 μl 1%Triton X-100 and hematocyte suspension 980 μl; negative control group (absorbance value is B): 20 μl normal saline and hematocyte suspension 980 μl; experimental group (absorbance value is C): 20 μl recombinant protein and hematocyte suspension 980 μl. The reagents were placed in a 37°C constant temperature water bath and incubated for 1 h; 300 μl of the supernatant was collected after centrifugation at 3,000 × g for 3 min. The absorbance value of each group was determined with a UV spectrophotometer at 540 nm. Three replicates were performed for each group.

Three repeated tests were performed to calculate the hemolysis rate of rDoenitin-1 according to the following formula: hemolysis rate =[(C–B)/(A–B)] × 100%.

The versatile functions of Doenitin-1 during feeding and reproduction of H. doenitzi were explored using RNAi. Briefly, the primers including the T7 promoter sequence at the 5′-end of both primers (Doenitin-1 primer: 5′-TAATACGACTCACTATAGGAACTGGCTGCAAGCCAG-3′ and 5′- TCAGTATTTGCAGGCGAAC-3′; 5′-AACTGGCTGCAAGCCAG-3′ and 5′- TAATACGACTCACTATAGGTCAGTATTTGCAGGCGAAC-3′; GFP primer: 5′-TAATACGACTCACTATAGGGACGTAAACGGCCACAAGT-3′ and 5′- GCTTCTCGTTGGGGTCTTT-3′; 5′-GACGTAAACGGCCACAAGT-3′ and 5′- TAATACGACTCACTATAGGGCTTCTCGTTGGGGTCTTT-3′) were used to synthesize dsRNA. Unfed female and male ticks with uniform body size were selected, and the interference group was injected with Doenitin-1 dsRNA, while the control group was injected with GFP dsRNA. About 0.6 μl dsRNA (2,400 ng) was injected into the fourth basal segment of the abdomen of each female tick under a microscope using a beveled Hamilton's needle (10 μl). The injected ticks were placed in an incubator and observed for 24 h. After 24 h of observation, dead or weak ticks were removed, and ticks in the interference group and the control group were put into ear bags with a ratio of 20 female ticks to 15 male ticks per rabbit ear. Twenty-four hours later, the number of feeding ticks was checked to calculate the rate of attachment rate. After 4 days, 2–3 partially fed female ticks were removed from each group to determine the relative expression of the genes by qPCR. The experimental data were analyzed according to the 2^−ΔΔCt algorithm, and the RQ value of each sample was calculated according to the formula and analyzed by biostatistics with the SPSS software. The time, body weight, and engorgement rate were recorded after the ticks were collected. Engorged female ticks were placed in an incubator, and the oviposition rate and hatching rate of eggs were recorded.

A statistical analysis was performed using SPSS 13.0 (SPSS Inc., Chicago, IL). Data organization and analysis were performed with Graphpad Prism 8.0.1. The statistical method were one-way ANOVA and Student's t test. Whether there is a significant difference between different samples is indicated by different letters. All the data were expressed as mean ± standard deviation (SD) or percentage. P < 0.05 was considered statistically significant.

The open reading frame (ORF) of Doenitin-1 (accession number: MZ981733) is 426 bp and encodes 141 amino acids (Figure 1). It is predicted that Doenitin-1 is 15.8 kDa, and that the theoretical isoelectric point (pI) is 4.76. It is presumed that Doenitin-1 contains a signal peptide of 15 amino acids and two Kunitz domains. Identity-based BLAST searches showed that 90% identity was found with H. longicornis (BAH02683.1), 83% identity with H. flava (ANA67892.1), 66% identity with R. microplus (Q8WPI2.1), 63% identity with Dermacentor variabilis (ACF35510.1), and 59% identity with Ixodes scapularis (XP_002434145.1). The multiple sequence alignment is shown in Figure 2.

The qRT-PCR results showed that Doenitin-1 was expressed in all tissues (partially fed) and stages (unfed) (Figure 3). By one-way ANOVA, the expression level of Doenitin-1 in the midgut was significantly higher than in other tissues (P < 0.05) (Figure 3A), and the highest expression level was found in unfed adult ticks (Figure 3B).

The recombinant protein of Doenitin-1 was successfully expressed through a prokaryotic expression system, and the molecular weight of the fusion protein was about 35 kDa (immature protein 15.8 kDa and label protein 19 kDa). The optimum elution concentration of imidazole for preliminary purification was 250 mM. After further purification by dialysis and concentration by ultrafiltration, the SDS-PAGE showed that purified rDoentitin-1 was free of impurities (Figure 4). The mass spectrometric data were searched in the UniProt protein database contain rDoenitin protein sequence using Proteome Discoverer 2.2 (Thermo Fisher Scientific). After enzymatic digestion, the peptides of the recombinant protein identified by LC-MS/MS correctly matched with the rDoenitin-1 protein (Table 2), which implied the E. coli prokaryotic expression system we constructed correctly expressed the rDoenitin protein of of the tick H. doenitzi. The normal APTT coagulation was 28.37 ± 1.47 s without rDoenitin-1. APTT coagulation was significantly prolonged after the addition of different concentrations of rDoenitin-1 (P < 0.05). When the concentration was 28 μM, APTT coagulation was extended to 77.8 ± 5.66 s (Figure 5A). However, PT coagulation was not significantly prolonged (Figure 5B). These results indicated that rDoenitin-1 can inhibit the intrinsic coagulation pathway but has no obvious effect on the extrinsic coagulation pathway.

Fibrinogen coagulation time was 2.03 ± 0.68 min without rDoenitin-1, and fibrinogen coagulation occurred almost immediately. When the concentration of rDoenitin-1 was 2.5 μM, fibrinogen solidification time was extended to 29.67 ± 2.08 min (Figure 6A). The results showed that coagulation time was increased significantly with increase in rDoenitin-1 protein concentration, indicating that the inhibition of fibrinogen clot formation was gradually enhanced.

When rDoenitin-1 concentration was 0.5 nM, the residual peptidase activity of thrombin was 91.67%. When rDoenitin-1 concentration was 32 nM, the residual peptidase activity of thrombin was 4.85% (Figure 6B). The IC50 for thrombin was 6.546 nM (R² = 0.9677). It can be seen that thrombin residual peptidase activity was gradually decreased with the increase in recombinant protein concentration, indicating inhibition of chromogenic substrate hydrolysis. When rDoenitin-1 concentration was 6.25 nM, the residual peptidase activity of cathepsin G was 77.39%. When rDoenitin-1 concentration was 200 nM, the residual peptidase activity of cathepsin G was 12.68 ± 5% (Figure 6C). The IC50 for cathepsin G was 30.01 nM (R² = 0.9259). It showed that with increase in recombinant protein concentration, the residual peptidase activity of cathepsin G was gradually decreased, and no significant correlation was found between protein concentration and residual peptidase activity. There was no significant change in the residual peptidase activity of trypsin in the results (Figure 6D), indicating that rDoenitin-1 has no significant inhibitory effect on trypsin. The hemolytic rate of rDoenitin-1 was 4.25% (<5%), indicating that rDoenitin-1 had no significant hemolytic activity in healthy adult blood cells (Table 3).

The qPCR results showed that the relative expression level of Doenitin-1 in the control group was 2.93 times higher than that of the interference group, indicating that the expression of Doenitin-1 was reduced by 65.7% (Figure 7). The engorgement weight and engorgement rate of the female ticks were significantly reduced after Doenitin-1 interference (P < 0.05). Compared with the GFP group, the egg hatchability was decreased significantly (P < 0.05), the egg hatchability of the GFP group was 85.36%, while it was only 46.67% in the experimental group. There were no significant changes in feeding time and 24-h attachment rate (P > 0.05) (Table 4). This result would suggest that Doenitin-1 is not directly involved in the feeding site but may play a role in keeping the blood meal in liquid form in the gut.