Introduction

Ticks, as obligate hematophagous ectoparasites, play a significant role in the transmission of various pathogens, such as viruses, protozoa, fungi, bacteria and helminths, affecting humans and animals health worldwide (Silaghi et al. 2016; Benelli et al. 2016). The tick Haemaphysalis flava is widely prevalent in many countries and regions and feeds on blood from many wild and domestic animals (Andoh et al. 2013; Lu et al. 2013). This tick transmits various disease agents (Francisella tularensis, Rickettsia and Ehrlichia) and causes significant vector-borne diseases (encephalitis, lyme borreliosis and spotted fever), posing a huge threat to the health of both humans and animals (Xu et al. 2016).

Mitochondrial (mt) DNA sequences have been proven useful and reliable genetic markers due to their maternal inheritance, fast rate of evolutionary change and relatively conserved genome structures than nuclear ribosomal genome (Blouin 2002). mtDNA sequences can provide valuable markers for investigating population genetic structures, systematics and phylogenetics of ticks. For example, mt 16S ribosomal RNA gene sequences have been shown as useful genetic markers for the identification and differentiation of Dermacentor nuttalli (Kulakova et al. 2014). mt cox1 and 12S ribosomal RNA gene sequences are useful genetic markers for studying intra-specific variation of Rhipicephalus appendiculatus (Kanduma et al. 2016). mt cox1 gene sequences are new and useful markers for studying the population genetic variations in Ixodes holocyclus (Song et al. 2011). mt 16S ribosomal RNA and cox1 gene sequences are useful markers for studying of the genetic polymorphism of I. persulcatus and I. pavlovskyi tick populations (Livanova et al. 2015). Although genetic variation in a number of ticks have been studied, there is a paucity of information on sequence variation among populations of H. flava of socio-economic significance.

The objectives of the present study were to examine genetic variation in three mtDNA genes, namely cytochrome c oxidase subunits 1 (cox1) and NADH dehydrogenase subunits 1 and 4 (nad1 and nad4), among H. flava isolates from wild hedgehogs in China. Based on the combined sequences of these three mtDNA regions, phylogenetic relationships of H. flava with other two Haemaphysalis species were also re-constructed.

Materials and methods

Parasites and DNA extraction

All adult ticks of H. flava (n = 20) were obtained from wild hedgehogs in Henan and Hunan provinces of China (Table 1). These samples were fixed in 70% (v/v) ethanol and stored at −20 °C until use. Total genomic DNA was extracted from individual samples using sodium SDS/proteinase K treatment, followed by spin column purification (TIANamp Genomic DNA Purification System, TIANGEN) and eluted into 50 µL H2O according to the manufacturer’s recommendations.

Table 1 Geographical origins in China of Haemaphysalis flava samples used in the present study, as well as their GenBank accession numbers for sequences of partial mitochondrial cytochrome c oxidase subunit 1 gene (pcox1), and NADH dehydrogenase subunits 1 and 4 genes (pnad1 and pnad4)
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Enzymatic amplification and sequencing

The primer sets (Table 2) for amplifying mt cox1, nad1 and nad4 were designed based on well-conserved mt sequences of H. flava (NC_005292) (Shao et al. 2004). PCR reactions (25 μL) were performed in 3.0 μL of MgCl2 (25 mM), 0.25 μL of each primer (50 pmol/μL), 2.5 μL 10 × rTaq buffer (100 mM Tris–HCl and 500 mM KCl), 2 μL of dNTP Mixture (2.5 mM each), 0.25 μL of rTaq (5 U/μL) DNA polymerase (TaKaRa Biotechnology, Dalian, China) and 2 µL of DNA sample in a thermocycler (Biometra, Göttingen, German). The cycling conditions were: 94 °C for 5 min (initial denaturation), followed by 35 cycles of 94 °C for 30 s (denaturation), 53–56 °C for 30 s (annealing), 72 °C for 1 min (extension) and then 72 °C for 5 min (final extension). Negative control (without DNA template) was included in each amplification run. Each amplicon (5 μL) was examined by 1% (w/v) agarose gel electrophoresis to validate amplification efficiency. PCR products were sent to BGI-Shenzhen (Shenzhen, China) for sequencing from both directions.

Table 2 Sequences of primers used to amplify a portion of the cytochrome c oxidase subunit 1 gene (pcox1), NADH dehydrogenase subunit 1 and 4 genes (pnad1 and pnad4) from Haemaphysalis flava
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Sequences analysis and reconstruction of phylogenetic relationships

Sequences of the three mt genes were separately aligned using the software Clustal X 1.83 (Thompson et al. 1997). The level of sequence differences (D) among H. flava isolates were calculated by pairwise comparisons using the formula D = 1−(M/L), where M is the number of alignment positions at which the two sequences have a base in common, and L is the total number of alignment positions over which the two sequences are compared (Chilton et al. 1995). The haplotypes, nucleotide diversity (Pi) and haplotype diversity (Hd) of each gene were determined using the DnaSP 5.0 program (Librado and Rozas 2009).

The combined sequences of pcox1, pnad1 and pnad4 of all tick samples in this study were used for phylogenetic analyses. Maximum likelihood (ML) was used for phylogenetic re-constructions. ML analyses were performed using PhyML 3.0 (Guindon et al. 2010), and the GTR + I model with its parameter for the concatenated dataset was determined for the ML analysis using JModeltest (Posada 2008) based on the Akaike information criterion (AIC). Bootstrap support (BS) for ML trees was calculated using 100 bootstrap replicates. To study the phylogenetic relationships with other Haemaphysalis species, H. formosensis (NC_020334) (Burger et al. 2013), H. parva (NC_020335) (Burger et al. 2013) and H. flava (NC_005292) (Shao et al. 2004) were considered into the present study, with Bothriocroton concolor (NC_017756) as the outgroup. Phylograms were drawn using the Tree View program version 1.65 (Page 1996).

Results and discussion

Genetic variation is widespread in tick populations, and the accurate analysis of genetic variation in ticks has important implications for studying epidemiology, population genetics and biology of ticks. Advances in molecular genetics and bioinformatic methods are providing unique opportunities to explore the biology of ticks. mtDNA markers are receiving more attention for this purpose than nuclear DNA (e.g., internal transcribed spacer) due to high mutation rate (Blouin 2002). However, given the different evolutionary rates occurring in different gene regions of tick mtDNA and the high degree of conservation of the cox1 gene (Guo et al. 2016). Therefore, in the present study, three mt markers (cox1, nad1 and nad4) were combined to further explore the genetic variation of the tick H. flava.

Amplicons of pcox1, pnad1 and pnad4 (approximately 900, 330 and 670 bp, respectively) were amplified individually and subjected to agarose gel electrophoresis. For each mtDNA region, in no case was product amplified from no DNA sample or host DNA control (not shown). The sequences of pcox1, pnad1 and pnad4 were 849, 285 and 626 bp in size, respectively. There were no difference in the lengths of any of the three pcox1, pnad1 and pnad4 sequences among H. flava from different geographic origins. These sequences have been deposited in the GenBank database (Table 1). The A + T contents of the sequences were 60.6–62.6% (pcox1), 56.3–60.6% (pnad1) and 67–73.6% (pnad4), respectively. Five haplotypes, nucleotide diversity (Pi = 0.00092) and haplotype diversity (Hd = 0.653) were determined among pcox1 sequences. Two haplotypes, nucleotide diversity (Pi = 0.00118) and haplotype diversity (Hd = 0.337) were determined among pnad1 sequences. Three haplotypes, nucleotide diversity (Pi = 0.00046) and haplotype diversity (Hd = 0.279) were determined among pnad4 sequences. The intra-specific sequence variations among different populations of H. flava isolates were 0–0.4% for pcox1, 0–0.4% for pnad1 and 0–0.3% for pnad4, while the inter-specific sequence differences among members of the Haemaphysalis were significantly higher, being 2.5–14.3%, 13.6–24.8% and 14.8–19% for pcox1, pnad1 and pnad4. These studies have clearly indicated that mt cox1, nad1 and nad4 gene sequences provide reliable genetic markers for specific identification and differentiation of H. flava. These results were consistent with that of previous studies (Chitimia et al. 2010; Mangold et al. 1998).

Many studies have demonstrated that mtDNA sequences are valuable genetic markers for phylogenetic studies of different groups of parasites, including ticks (Song et al. 2011; Liu et al. 2013; Burger et al. 2014). In the present study, phylogenetic analyses of the combined sequences of pcox1, pnad1 and pnad4 among 20 individual H. flava isolates from China, 3 other ticks including B. concolor as outgroup, using ML is shown in Fig. 1. In this tree, the H. flava form monophyletic group with high statistical support (BS = 100), and all the H. flava isolates were more closely related to H. formosensis than to H. parva. Our results were consistent with those of previous studies (Burger et al. 2013; Williams-Newkirk et al. 2015).

Fig. 1
figure 1

Phylogenetic relationship among Haemaphysalis flava isolates in China with other Haemaphysalis species inferred by maximum likelihood analyses using the combined dataset (cox1 + nad1 + nad4), with Bothriocroton concolor (NC_017756) as outgroup

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Our study presents the first attempt to characterize genetic variation in mt cox1, nad1 and nad4 genes of H. flava isolates from wild hedgehogs in China, but we believe it is still necessary to carry out more experimental research. Future studies could (1) examine population structure using larger number of samples from different hosts and geographical locations, (2) employ other more variable molecular markers, (3) detailed morphological re-description of these ticks H. flava.

In conclusion, sequence variations among H. flava isolates from different geographical localities in China were revealed by sequence analyses of mt cox1, nad1 and nad4 genes. The molecular approach employed provides a powerful tool for elucidating the epidemiology, genetics and biology of H. flava in China and elsewhere in Asia.