Introduction

Ticks are hematophagous arthropods transmitting several harmful pathogens of human and animal, like viruses, Rickettsia, bacteria, and protozoa (Shpynov et al. 2001; Whitehouse 2004; Shayan et al. 2007). Several studies dialing with the characterization of Ticks occurring in Iran were performed (Delpy 1936, 1938; Abbasian 1961; Mazlum 1971; Hoogstral and Valdez 1980; Rahbari 1995; Telmadarraiy et al. 2004; Nabian et al. 2007). The last information about the tick fauna in Iran revealed that 20 tick species were identified as Hyalomma anatolicum anatolicum, Hyalomma marginatum, Hyalomma detritum, Hyalomma aegptium, Hyalomma schultzei, Hyalomma dromedarii, Hyalomma asiaticum asiaticum, Hyalomma anatolicum excavatum, Haemaphysalis punctata, Haemaphysalis Parva, Haemaphysalis concinna, Haemaphysalis choldokovsky, Ixodes ricinus, Rhipicephalus sanguineus, Rhipicephalus Bursa, Rhipicephalus Turanicus, Boophilus annulatus, Dermacentor niveus, Dermacentor marginatus, and Ornithodoros lahorensis (Rahbari et al. 2007). For the identification and speciation of ticks, the identification key of Arthur (1960), Kaiser and Hoogstraal (1963), and Walker et al. (2003) were normally used in Iran. Such identification keys are based in the first line on the morphological features and characteristics of tick genera and in the second line are extended on the morphological features and characteristics of species. Although these identification keys are well prepared, in some cases, due to the strong overlapping characteristics between species like D. marginatus and D. niveus, these accompanied with serious problems. Currently, Nabian et al. (2008) reported the occurrence of three species of Dermacentor (D. marginatus, D. niveous, and Dermacentor raskamensis) in Iran. They specified D. marginatus and D. niveus as separate species. Some other investigators reported also about D. marginatus and D. niveus as separate species (Telmadarraiy et al. 2004; Rahbari et al. 2007; Nabian et al. 2008). Interestingly, some other investigators (Estrada-Pena and Estrada-Pena 1991; Roman and Sicrat 1957) believed that D. niveus is synonyms of D. marginatus. Since D. marginatus and D. niveus have been intermittently used synonymously and there is no a generally agreement with the specification of Dermacentor species, we used the molecular-based techniques to answer this problem. For this aim, the nucleotide sequence of ITS-2 region of ribosomal RNA gene were determined by both species and compared with each other.

Materials and methods

Samples and species determination

D. marginatus and D. niveus were collected from different area of Iran and preserved in 70% ethanol until used (50 male ticks from each species). The specification was carried out on the basis of morphological characteristic keys recommended by Arthur (1960) and Estrada-Pena and Estrada-Pena (1991) and the male ticks were used for genetical analysis. The preparation of salivary glands was performed according to Brown and Askenase (1986).

Extraction of DNA from salivary gland of ticks

Deoxyribonucleic acid (DNA) was extracted using a DNA isolation kit (MBST, Iran) according to the manufacturer’s instructions. Briefly, salivary gland was first lysed in 180 μl lysis buffer, and the proteins were degraded with 20 μl proteinase K for 10 min at 55°C. After addition of 360 μl binding buffer and incubation for 10 min at 70°C, 270 μl ethanol (100%) was added to the solution, and after vortexing, the complete volume was transferred to the MBST column. The MBST column was first centrifuged and then washed twice with 500 μl washing buffer. Finally, DNA was eluted from the carrier with 50 μl elution buffer.

PCR amplification

Approximately 100–500 ng DNA was used for the polymerase chain reaction (PCR) analysis. The PCR was performed on 100 μl total volume including one-time PCR buffer; 2.5 U Taq Polymerase (Cina gene, Iran); 2 μl of each primer (P1/P2, P3/P4, 20 mM, Cina gene); 200 μM of each deoxyadenosine triphosphate, deoxythymidine triphosphate, deoxycytidine triphosphate, and deoxyguanosine triphosphate (Fermenta); and 1.5 mM MgCl2 in automated Thermocycler (Eppendorf, Germany) with the following program: 5 min incubation at 95°C to denature double-strand DNA, 36 cycles of 45 s at 58°C (annealing step), 45 s at 72°C (extension step), and 45 s at 94°C (denaturing step). Finally, PCR was completed with the additional extension step for 10 min. The PCR products were analyzed on 1.8% agarose gel in 0.5× TBE buffer (5× TBE buffer, 54 g Tris base, 27.5 g acide boric, and 20 ml 0.5 M EDTA (pH 8.0) in 1 l H2O) and visualized using ethidium bromide and an UV illuminator (Table 1).

Table 1 The nucleotide sequence of primers, corresponding accession no of genes used for designing of primers and the PCR product size were listed in the table
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The complete ITS-2 nucleotide sequence was amplified in two steps. In the first step the upstream region of the ITS-2 was amplified using forward primer derived from the nucleotide sequence 1 to 20 of 5.8 S rRNA gene (P1, accession no. AF199114 ) and the reverse primer derived from nucleotide sequence 690 to 708 of ITS-2 region (P2). In the second step the downstream region of ITS-2 was amplified using forward primer derived from upstream of the first reverse primer from nucleotide 628 to 647 (P3, accession no. AF199114 ) and the reverse primer derived from nucleotide 1,091 to 1,110 of 5` region of 28 S rRNA (P4, accession no. AF199114 ) to have the PCR product with the overlapping region with the first PCR product. The primers are listed in Table 2.

Table 2 The main differentiation characters between D. marginatus and D. niveus are given
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Sequencing of PCR products

The PCR products were sequenced after they were extracted from agarose gel or after purification of PCR products. Twenty microliters of PCR product was run on a 1.5% agarose in 0.5% TBE buffer. After visualization of the positive band using ethidium bromide under UV, the PCR product was extracted from the gel using DNA extraction kit from agarose gel (MBST, Iran) according to the manufacturer’s instructions. Briefly, the PCR product was cut from the gel under UV control and dissolved in 340 μl binding buffer for 5 min at 60°C. After the addition of 255 μl ethanol (96%) to the sample, the mixture was applied to the spin column and centrifuged for 1 min at 8,000×g. The column was washed twice with washing buffer, and the adsorbed DNA was eluted from the column using 20 μl elution buffer.

PCR product was purified from the salts and proteins using PCR purification kit (MBST, Iran). Briefly, 200 μl binding buffer was added to 100 μl PCR product solution. After adding 150 μl ethanol (96%) to the sample, the mixture was applied into the column. The column was washed twice with washing buffer, and PCR product was eluted from the column using 100 μl elution buffer. The sequencing was performed from both sites of each PCR products by Kawsar Biotech Company in Iran basis on Sanger method (1977).

Results and discussion

D. marginatus and D. niveus were specified according to the main specific characters such as dimensions of basis capituli, the basis color of scutum, and the second segment of palp. Basis capituli of D. marginatus is like D. niveus which is rectangular. This was dorsally one and a half times as broad and as long in D. marginatus as well as in D. niveus. These parameters were consistent in 50 ticks from each D. marginatus and D. niveus species. In contrast Estrada-Pena and Estrada-Pena (1991) have measured the dorsal basic capituli for D. marginatus 0.5–0.9 times as broad and as long (excluding cornua) and they have characterized D. niveus provided by Arthur as Dermacentor species conspecific with D. marginatus lacking the produced dorsal palpal spur and having a darker dorsal pattern than those of the paratypic series of niveus collected at Iran. They have also analyzed three males collected from Morocco displaying a palpal shape typical for D. niveus, with pronounced spur at the dorsal side of the second palpal segment and a dark color of scutum. In accordance with our results Arthur (1960) described the basis capituli of D. niveus one and a half times as broad and as long (including cornua).

The dorsal scutum of D. marginatus is like D. niveus which is ornamented and multishape, but its basic color in D. marginatus is enamel white, whereas the corresponding areas in D. niveus is enamel yellow-white. Arthur (1960) described the base color of dorsal scutum of D. niveus as brown, enamel yellow-white such as we observed in our samples. According to the scutum pattern, our observation confirmed the description of Estrada-Pena and Estrada-Pena (1991). They described the scutal pattern of D. marginatus as very variable, with spots in the scutum generally with the inner surface of each spot mostly studded with base color pattern. Furthermore, we observed that the two central dorsal spots are organized approximately triangular with the apical angle directed antrolateral in D. marginatus whereas these two spots are vice versa in D. niveus (Fig. 1). The second segment of palp in D. marginatus had small or strong postero-median spur. In contrast, the second segment of palp in D. niveus had strong pronounced spur at the dorsal side (Fig. 1).

Fig. 1
figure 1

Basis capituli of D. marginatus is like D. niveus rectangular and is dorsally one and half times as broad as long in both species. The central dorsal spots are organized approximately triangular with the apical angle directed antrolateral in D. marginatus whereas these two spots are vice versa in D. niveus

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After speciation of these two species, DNA was extracted from salivary gland of each tick separately. DNA was then amplified using specific primers to amplify the complete ITS-2 region in two steps. In the first step of PCR, PCR product of approximately 708 bp in length was amplified using primers P1/P2 and in the second step PCR product of approximately 482 bp in length was amplified using primers P3/P4. The PCR products were purified or extracted from the gel and sequenced from both sites of the DNA. The nucleotide sequence of both PCR products had overlapping region and could be adapted (Fig. 2). The complete nucleotide sequence of D. marginatus and D. niveus consist of 1,099 bp and 1,100 bp respectively. The alignment of these two sequences with each other was performed by BLAST software and showed 99% nucleotide homology. The both nucleotide sequences distinguished only in 5 bp scattered in the whole sequences. The alignment of both sequences with the nucleotide sequence registered in the DataBank showed 98% homology to the nucleotide sequence of D. marginatus (Accession no S83081.1), 87% with Dermacentor reticulates (accession no. S83080.1). Nucleotide sequence of ITS-2 region of D. marginatus had 85% homology to the D. andersoni, whereas D. niveus had 86% homology to this sequence (accession no. EU520395.1). Zahler et al. (1995) showed that the nucleotide sequence of ITS-2 region of D. reticulates had 87.8% homology to D. marginatus and D. andersoni was 79.4% and 79.5% identical to D. reticulates and D. marginatus, respectively. They analyzed the nucleotide sequence of ITS-2 region of D. reticulates individuals from different geographic origins and determined a 99.6% homology between the individuals and described it as intra-specific differences. In their study the differences in nucleotide sequence of more than 10% was determined as interspecific identity, which can be understood as not suggestive for common gene pool. ITS-2 sequence was already used for determination of validity of species status of Ixodes dammini (Wesson et al. 1993), or for differentiation of Rhipicepalus spp. as well (Barker 1998). Since the nucleotide sequence of ITS-2 region of D. marginatus in Iran has 98% sequence homology to the other in GenBank registered ITS-2 sequences of D. marginatus, and the morphological characteristics between both examined species showed minimal differences, therefore we believe that the D. marginatus and D. niveus could belong to the same species and 1% differences in nucleotide sequence of ITS-2 region between these two species can be understood as an intra-species polymorphism. It seems that the ITS-2 nucleotide sequence can be used as a sufficient molecular marker for phylogenetical analysis of Dermacentor spp.

Fig. 2
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Alignment of complete nucleotide sequence of ITS-2 regions of rRNA genes from Iranian D. niveus (D. n Iran, GenBank accession no. GQ144706), Iranian D. marginatus (D. m Iran, GenBank accession no. GQ144707), D. marginatus (D. m GenBank accession no. S83081.1), D. reticulatus (D. r GenBank accession no. S83080.1), and D. andersoni (D. a GenBank accession no. EU520395.1) using CLUSTAL format alignment by MAFFT

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Rahbari et al. (2007) and Nabian et al. (2008) determined the tick fauna in Iran and could find D. marginatus and D. niveus mostly in the mountainous areas. Interestingly, they could find D. niveus in North-East of Iran (Khorassan province) whereas D. marginatus was more collected in North-West of Iran. Taken these results with our results together, it could be speculated that ecological environments could have direct influence on some morphological characters of ticks, which must be studied in future.