Haemaphysalis flava, a kind of ticks belonging to the family of Ixodidae, is geographically ubiquitous in the Far East and many countries in Asia (Yan and Cheng 2015). In China, except for Tibet, Qinghai and Xinjiang, it can be found in the rest provinces, especially in southern and eastern China (Yan and Cheng 2015). H. flava is able to survive in vacuum condition under scanning electron microscopes, comparable to tardigrades (trivial name “water bear”), an ecdysozoa known for resistance to various extreme environments (Ishigaki et al., 2012). It attacks human, cattle, goats, dogs, giant pandas and hedgehogs etc., causing lesions, dermatitis, anemia and even death in hosts, and carries specific pathogenic microorganisms like encephalitis virus (Sungjin et al. 2010), Francisella tularensis (Ozawa et al., 1982), Rickettsiae (Noh et al., 2017), Coxiella, Pseudomonas, Ehrlichia, Escherichia, Acinetobacter, Citrobacter, Cupriavidus, Staphylococcus and Wolbachia (Duan and Cheng, 2016) etc. The extensive distribution, and vectors of a variety of pathogens, as well as a wide range of hosts, make it of specific importance for public health.

It is generally believed that the most promising way for control of tick infections is to develop vaccines against ticks. To screen possible candidates for vaccines development, we previous conducted the sialotranscriptome and midgut transcriptome of H. flava using next-generation sequencing (NGS) (Xu et al., 2015a, b). A bunch of genes involved in feeding and digestion were identified, like genes encoding cysteine protease, longipain, subolesin, calreticulin, metalloproteases, serine protease inhibitor, enolase and AV422 (Xu et al., 2015a, b) etc. Specifically, the expression patterns of SubA and Enolase, as well as possible functions, were further characterized in H. flava in our lab (Liu et al., 2016; Xu et al., 2016).

HSP, cellular chaperones expressed both under normal physiological situations and stresses, are involved in the folding and transportation of the newly synthesized peptides. HSP can be divided into 6 major subsets based on their molecular weights, including HSP100, HSP83–90, HSP70, HSP60, HSP40 and small HSP family etc. (Wang et al., 2013) HSP70 are the most widely studied ones, and currently can be further divided into 4 members according to their localizations in cells, namely Hsp70, Hsc70, Hsp75 and glucose-regulated protein 78 (GRP78) (Bausero et al., 2005). The cytosolic localized HSP70 members includes Hsc70 and Hsp70, the former being constitutive expressed while the latter being heat-inducible (Daugaard et al., 2007; Kabani and Martineau, 2008). Accumulated evidences are supporting a role of HSP70 family in the differentiation, virulence adaption, resistance and protection from oxidative damage of parasites (Polla, 1991). We previously found that Hsc70 was a protein of high abundance using saliva and midguts content proteomes of H. flava (unpublished data). The DNA fractions encoding Hsc70 (Contig2727) were also identified in libraries of salivary glands and midguts transcriptomes (Xu et al., 2015a, b).

So far the full-length and expression patterns of Hsc70 have been studied in turtles, frogs, shrimps and other invertebrates etc., but not extensively in ticks. We speculated that Hf-Hsc70 would also be conserved, as seen in other species, and its transcript levels would be varied in different organs and life phases, but would be stable under different ambient temperatures. In the present study, for the first time, the full length of Hf-Hsc70 were cloned by RACE, and its expression patters under different circumstances were determined by RT-PCR.

Materials and methods

Ticks

H. flava were collected, identified and cultured in Hunan Agricultural University, Changsha, China. Fasted H. flava (adult ticks) were inoculated onto 4 hedgehogs, 40 ticks (20 males, 20 females) each. H. flava then were harvested at half-engorged and full-engorged state, respectively. Procedures involving hedgehogs in the present study were approved and overseen by the Hunan Agricultural University Institutional Animal Care and Use Committee (No.43321503).

Primers

5′-primers and 3′-primers for RACE were synthesized based on Contig2727 in the transcriptome library of salivary glands in H. flava (NCBI-GSE67247). Primers for RT-PCR were synthesized based on hypervariable regions of Hf-Hsc70. All primers were synthesized and sequenced by Sangon (Shanghai).

Total RNA extraction

Ticks were sterically observed under stereomicroscope after surfaces were cleaned, and sterilized by 75% ethanol. Ticks were dissected to isolate salivary glands and midguts. Dissected tick organs, as well as whole ticks were well grinded in liquid nitrogen, and were extracted for RNA by an EasyPure RNA Kit (Transgen Biotech, Beijing). The A260 to A280 ratio of RNA extracts was measured by a NanoDrop ND-2000 (Thermo Scientific Waltham, MA), and the RNA extracts was further quantified by an Agilent Bioanalyzer 2100 (Agilent, CA).

Rapid amplification of cDNA ends (RACE) for Hf-Hsc70

The 3′ and 5′ RACE were applied to Contig2727 to clone the full-length of Hf-Hsc70 using a 3′-Full RACE Core Set with PrimeScript™ Rtase (Takara, Dalian) and a SMARTer™ RACE cDNA Amplification Kit (Takara, Dalian). Gene-specific primers (GSP) for 3′-RACE and 5′-RACE were listed in Table 1.

Table 1 Primers for RACE and RT-PCR
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The PCR conditions and follow-up operations were similar to our previous study (Liu et al., 2016). First-strand cDNA was reversely transcripted from 1.5 μL of RNA using PrimeScript Reverse Transcriptase from the kit. For the 3′ RACE, the first round of amplification was accomplished with the synthesized cDNA as templates, and 3’R-GSP1 and 3’R-outerP as primers through 22 cycles. The PCR programs were: initial denaturation at 94 °C for 3 min, denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 45 s, and a final extension at 72 °C for 10 min. A 1.0 μL of PCR products from the first amplification was used to perform the second round of amplification with 3’R-GSP2 and 3’R-innerP as primers through 32 cycles. The PCR programs were: initial denaturation at 94 °C for 3 min, denaturation at 94 °C for 30 s, annealing at 65 °C for 30 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 10 min.

The outer PCR for 5′-RACE was conducted with 5’R-GSP1 and 5’R-outerP as primers through 32 cycles. The PCR programs were initial denaturation at 94 °C for 3 min, denaturation at 94 °C for 30 s, annealing at 64 °C for 30 s, extension at 70 °C for 30 s, and a final extension at 70 °C for 10 min. The inner PCR for 5′-RACE was conducted with 5’R-GSP2 and 5’R-innerP as primers through 32 cycles. PCR programs were the same to the outer PCR except annealing at 60 °C instead of 64 °C.

The full-length of Hf-Hsc70 was obtained by reassembly of the 3′ end and 5′ end of PCR products and Contig2727. The encoding protein sequence and homology of Hsc70 was analyzed by software DNA (version 2.5.1). The protein structure and motifs were analyzed by InterPro scan (http://www.ebi.ac.uk/interpro/search/sequence-search). A phylogenetic tree of Hf-Hsc70 was also generated using software MEGA 5.0 based on available mRNA sequences of typical selected species in Genbank.

Expression pattern of Hf-Hsc70 in different organs, life phases and ambient temperatures

40 female adult H. flava, half engorged, were randomly divided into 5 groups. 10 ticks in control group were placed at room temperature (25 °C) for 3 h. Other 30 ticks were placed at 23 °C, 28 °C, 33 °C and 38 °C for 3 h (n = 7 or 8). Total RNA extracts from different organs and whole ticks in different life phases were achieved as described previously.

cDNA synthesis was accomplished using 2 μL total RNA according to the manual of PrimeScript™ RT reagent Kit with gDNA Eraser (Transgen Biotech, Beijing). A 20 μL reaction system of RT-PCR with 2 μL of cDNA templates was constructed by following the manual of SYBR® Premix Ex Taq™ (Takara, Dalian). The program for RT-PCR was as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 31 s, 95 °C for 15 s, and a final 60 °C for 1 min and 95 °C for 15 s. Expression levels of Hf-Hsc70 was estimated by the 2-△△Ct method. Normalized expression of Hf-Hsc70 was compared among different organs, life phases and ambient temperatures using Student’s t test.

Results

Total RNA extraction

The A260 to A280 ratios were 1.98 and 2.00 for full-engorged ticks by spectrophotometer, as shown in Fig. 1. It revealed that the extracted total RNA were of sound quality.

Fig. 1
figure 1

Electrophoresis of total RNA from H. flava in different life phases. 1 the fasted adult ticks, 2 the larvae, 3 the female nymphs, 4 the female engorged adult ticks, M Markers

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RACE for Contig2727

The amplified fragments with 3’R-GSP1, 3’R-outerP, 3’R-GSP2 and 3’R-innerP as primers were 250 bp in length (Fig. 2). After gene sequence alignment, it was found that 180 bp was lost from 3’end of Contig2727 (Fig. 4).

Fig. 2
figure 2

Amplification of Hf-Hsc70 by 3’RACE and the 3’end sequence of Hf-Hsc70. (a) Amplification of Hf-Hsc70 by 3’RACE (b) The 3’end sequence of Hf-Hsc70

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Similarly, the amplified fragments with 5’R-GSP1, 5’R-outerP, 5’R-GSP2 and 5’R-innerP as primers were 750 bp in length (Fig. 3). It was found that 562 bp was lost from 5’end of Contig2727 (Fig. 4).

Fig. 3
figure 3

Amplification of Hf-Hsc70 by 5’RACE and the 5’end sequence of Hf-Hsc70. (a) Amplification of Hf-Hsc70 by 5’RACE (b) The 5’end sequence of Hf-Hsc70

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Fig. 4
figure 4

Comparison of Contig2727 from H. flava with Hsc70 from I. scapularis. The amino acid sequences in gray indicate detected peptide fractions using proteomics

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After alignment, it was revealed that the full-length of Hf-Hsc70 was 2363 bp, and contained an ORF of 1965 bp.

Sequence and phylogenetic analysis of full length of Hf-Hsc70

The full-length of Hf-Hsc70 was 2363 bp, with an ORF at 1965 bp in length. The encoded protein was 648 aa in length, with molecular weight at 71.09 kDa and pI at 5.25.

The complete amino acid sequence could be separated into three separate regions: ATPase domain (1 aa-385 aa), the peptide-binding domain (386 aa-543 aa) and C-terminal domain (538 aa-648 aa) (Fig. 5). Three signature sequences of HSP70 was detected, and that was IDLGTTyS (9 aa-16 aa), IFDLGGGTfdvSIL (197 aa-210 aa), and IvLvGGsTRIPkIqK (334 aa-348 aa). It was also found that 2 tetrapeptide motifs (GGMP and GGFP), the putative bipartite nuclear location signal (KK and RRLRT), a potential non-organelle eukaryotic consensus motif (RARFEEL), the extreme C-terminal domain (SGPTIEEVD) motif and 2 glycosylation sites (NKSI and NVSA) were included in the protein structure (Fig. 5). Based on those structural characteristics, it could be concluded that the protein encoded was actually Hsc70.

Fig. 5
figure 5

The predicted structure of Hsc70 encoded by Hf-Hsc70. The protein structure and motifs were analyzed by InterPro scan (http://www.ebi.ac.uk/interpro/search/sequence-search). The characteristic HSP70 family signatures are underlined; the putative ATP/GTP binding site, localization signals, glycosylation domains and GGXP motifs are shown in gray

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As shown by both Blast P and gene alignment, Hsc70 was highly homologous with the identity of the protein in vertebrates like Pisces, Amphibian, Reptilian, Aves and mammals over 89.9%, and it was also highly homologous in invertebrates like Crustacea, Arachnoidea, and insect, with the identity over 90.33%. Notably, the identity among H. flava, Ixodes scapularis and Ixodes ricinus was over 99.02% (Fig. 6). It could be concluded that Hf-Hsc70 was an evolutionarily conserved gene.

Fig. 6
figure 6

Phylogenetic analysis of Hsc70 genes from representative species. A phylogenetic tree of Hsc70 was constructed by software MEGA 5.0 based on available mRNA sequences of typical species in Genbank. The accession numbers of Hsc70 sequences from multiple species are shown after their names

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Expression patterns of Hf-Hsc70 in different life phases, organs and ambient temperatures

Expression levels of Hf-Hsc70 in different organs were in the order of salivary glands of the female half-engorged > midguts of the female half-engorged > salivary glands of the female full-engorged > midguts of the female full-engorged. Specifically, the relative expression level of Hf-Hsc70 in salivary glands was steadily higher than that in midguts (p < 0.05) regardless of feeding status (Fig. 7 A). Expression patterns of Hf-Hsc70 in different life stages were in the order of female half engorged adult ticks > female full engorged adult ticks > male adult ticks > female fully engorged nymphs > female larvae (Fig. 7 B). Hsc70 expression was highest at 33 °C, and lowest at 38 °C, but not significantly different (Fig. 7 C).

Fig. 7
figure 7

The expression of Hf-Hsc70 in different organs (A), life phases (B) and ambient temperatures (C) by RT-PCR. Hf-Hsc70 expression in different organs, life phases and ambient temperatures was compared by Student’s t-test. Data not sharing a common letter indicated there was a significant difference (p < 0.05)

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Discussion

Our previous data from saliva and midgut content proteome in H. flava by LC/MS/MS revealed that 8 peptide fragments, namely ARFEELNADLFR, DAGTIAGLNVLR, IINEPTAAAIAYGLDK, SINPDEAVAYGAAVQAAILIGDK, STAGDTHLGGEDFDNR, TTPSYVAFTDTER, WLDTNQLADKEEYEHR and VEIIANDQGNR, were labeled as HSP70 unique peptide fragments of I. scapularis with total 69 specific peptides detected (unpublished data). 16 unigenes were annotated as Hsp70 fragments of I. scapularis by searching in libraries of salivary glands and midguts transcriptomes in H. flava (GSE67247). Among them, contig2727 was 1629 bp in length, and showed an 86.40% identity with Hsp70 of I. scapularis (XM_002407088.1) and a 97.42% identity of Hsc70 (GI:241,153,675), but lacking VEIIANDQGNR in amino terminal and VCNPIITKLYQDGGMPAGGFPGAGAPGGAGAAPGAGAGSGPTIEEVD in carboxyl terminal. We concluded that contig2727 was an encoding gene fraction of Hsc70, but was lack of the 3′ end and 5′ end.

Of the 6 HSP families, HSP70 had the most members, and all members showed different bioactivity and different expression patterns (Bolhassani and Rafati, 2008). In the current study, full length of Hf-Hsc70 was cloned by 3′-RACE and 5′-RACE based on the contig2727. It was found that the sequence obtained from RACE had 3 signature sequences of HSP70 family in the putative amino acids sequences, 2 characteristic motifs of Hsc70 (GGMP, SGPTIEEVD). Hence we claimed the sequence to be Hsc70 (Bairoch, 1991; Rensing and Maier, 1994).

The phylogenetic analysis further indicated clearly the gene cloned was a constitutive HSP70 form other than a stress inducible form. Hf-Hsc70 showed close relationships with Hsc70 sequences of other typical selected vertebrates and invertebrates, especially with that of I. scapularis (GI:241,153,675) and I. ricinus (GI:442,751,305). However, Hf-Hsc70 was not clustered with the sequence of H. longicornis (GI:169,809,131) to form a distinct clade. Only an identity of 64.6% was found between those two sequences (Tian et al., 2011). The author claimed that the Hsp70, as well as that in I. scapularis both belonged to ER-localized HSP70 proteins (Tian et al., 2011), not Hsc70.

Our data revealed that Hf-Hsc70 transcription was not significantly up-regulated after 3-h heat stress, indicating the protein encoded was not inducible by the 3-h heat stress. The observation was consistent with the data in frogs (Simoncelli et al., 2010), pleurodeles (Delelis-Fanien et al., 1997) and zebrafish etc. However, there were also data showing that Hsc70 in some invertebrates and vertebrates, like moths (Sonoda et al., 2006), clams (Wu et al., 2014), shrimps (Lo et al., 2004), endoparasitoids (Wang et al., 2008) and turbots (Wang et al., 2013) etc., had an up-regulation pattern upon heat stress. One explanation for this was that possibly Hsc70 was only account for the acquisition of tolerance to the beginning of heat stress, and other members of HSP70 would be induced and play a part in the long term heat exposure, like seen in Pteromalus puparum and Drosophila melanogaster (Li et al., 2012; Wang et al., 2008).

Expression pattern of Hf-Hsc70 in different life phases showed that its expression was associated positively with the amount of blood ticks sucked. Salivary glands and midguts were both capable to express Hsc70 and secreted it into saliva and midgut contents as confirmed by transcriptomics and proteomics. Those observations suggested that Hsc70 was likely to play a role in blood digestion, more likely, to play a part in anticoagulant or facilitation of the uptake of blood by somatic cells of ticks. Hsc70 depletion by RNAi knockdown in Rhodnius prolixus was lethal 40 d after dsRNA injection, and those challenged insects died even earlier within 14–20 d after blood-feeding (Paim et al., 2016). It was suggested that Hsc70 knockdown bugs had impaired signaling pathways involved in blood processing and digestion. This observation was consistent with our assumptions. Hsc70 was expressed in platelets, and was involved in the regulation of platelet adhesion by binding to protein phosphatase 1, and thus was functional in blood coagulation (Polanowska-Grabowska and Gear, 2000; Polanowska-Grabowska et al., 1997). However, more detailed molecular mechanism of Hsc70 on blood coagulation/anticoagulation awaits further investigations. Thus screening of peptides antigens based on the variable region of Hsc70 would be of significance to develop vaccines for the control of tick infestations. However, those assumption needs to be confirmed in further.

In conclusion, the current study cloned the full length of Hf-Hsc70 and analyze its expression patterns under different circumstances. Hf-Hsc70 was a conserved gene and belonging to a constitutive form of HSP70. Expression patterns of Hf-Hsc70 in different life phases and organs suggested a possible role in blood feeding. Further work is needed to explore the functional roles of Hsc70 in the blood feeding process of ticks and make it a competent candidate for the development of vaccines against ticks.