Morphological and molecular divergence of Rhipicephalus turanicus tick from Albania and China

Rhipicephalus turanicus is an important tick species potentially carrying tick-borne pathogens. Several tick species have obvious subspecies divergence. However few studies aimed to examine the existence of divergence within R. turanicus. Therefore, a detailed morphological and molecular analysis was conducted for comparing R. turanicus from the Mediterranean Basin (represented by Albania) and Central Asia (Northwestern China). Altogether 315 adult ticks of R. turanicus (103 from Albania and 212 from China) were morphologically and molecularly analysed. DNA samples were used for mitochondrial 16S rRNA and cox1 gene sequences analysis. In addition, as potentially genetic markers, three fragments including partial nad1-16S rRNA, nad2-cox1, cox1-tRNA-Lys, were designed and then phylogenetically analyzed. Based on detailed morphological observations, only basis capituli length:width ratio (females), the length, the width and the length:width ratio of the scutum (males) had differences between R. turanicus from China and Albania. Gene divergences of 16S rRNA, cox1, partial nad1-16S rRNA, nad2-cox1 and cox1-tRNA-Lys from China and Albania ticks were 3.53–4.84, 3.57–4.92, 3.57–4.07, 3.57–4.39 and 3.18–4.69%, respectively. The evaluated five genetic markers revealed two phylogenetic branches in R. turanicus. Obvious differences exist within R. turanicus based on morphological and genetic analysis. Three newly designed genetic markers (partial nad1-16S rRNA, nad2-cox1 and cox1-tRNA-Lys) in this study may be suitable genetic tools for identification and analysis in R. turanicus. Subspecies analysis of R. turanicus from other regions of the world should be initiated in the future.


Introduction
Rhipicephalus turanicus is widely distributed in the Mediterranean sub-region, Africa and Asia, infesting a rich variety of domestic and wild hosts such as sheep, cattle, horses, dogs, cats, and Corsican hares, occasionally even human beings (Chochlakis et al. 2014;Dantas-Torres et al. 2013). It is a potential or competent vector of several categories of tick-borne pathogens such as Rickettsia spp. (Germanakis et al. 2013) among various hosts and negatively impacts agricultural economies globally. While several tick species have valid subspecies around the world, as exemplified by R. evertsi (Guglielmone et al. 2014) and Haemaphysalis erinacei (Hornok et al. 2016), few studies aimed to examine the existence of distinction within R. turanicus, despite the known uncertainty in its taxonomy (Dantas-Torres et al. 2013).
Cox1 and 16S rRNA genes are well established barcoding genes for the molecular identification and phylogenetic analyses of ticks. Intraspecies sequence divergence within R. turanicus from eastern and central parts of China has been investigated (Lv et al. 2014). Intraspecies genetic variation in the 16S rRNA and cytochrome c oxidase subunit I (cox1) target region have been recorded concerning R. turanicus in Yining County, Xinjiang Uygur Autonomous Region (XUAR) , where R. turanicus was considered one of the dominant tick species .
Here, based on complete mitochondrial sequence analysis of the members of Rhipicephalus sanguineus complex (with at least 17 species), 3 mitochondrial hypervariable region fragments, namely, partial nad1-16S rRNA gene, partial nad2-cox1 gene and partial cox1-tRNA-Lys gene, together with Cox1 and 16S rRNA fragments and detailed morphological study were analysed to investigate divergence of R. turanicus originated from Albania and Northwestern China.

Morphological analysis
Ticks were first identified as R. turanicus according to their morphological characteristics based on the work of Walker et al. (2000) and Filippova (1997). Pictures were made and measurements were performed with a Keyence VHX-5000 digital microscope (Osaka, Japan). The same parameters were measured and compared in the case of males (92 from China, 53 from Albania) and females (120 from China, 50 from Albania), except for the peritreme process (used only for females). Morphological study included anteriolateral setae (palp article II), anterior setae (palpal article III), inner length (palpal articles II ? III), outer length (palpal articles II ? III), palpal width (between palpal articles II/III), basis capituli length, basis capituli width, ratio basis capituli length:width, scutum median length, scutum width, ratio scutum length:width, peritreme median length, peritreme width, peritreme process width and peritreme process median length. Structures of females measured are illustrated in Additional Figure 1. Measurements of ticks were compared with Student t test. Mean values were considered significantly different if P \ 0.05 (Additional Table 3).

Molecular analysis
Total genomic DNA of 315 R. turanicus (212 from China and 103 from Albania) was extracted from individual specimens using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China).
All ticks were first evaluated based on 16S rDNA and cox1 genetic fragments (Black and Piesman 1994;Chen et al. 2014). Moreover, 13 complete mitochondrial genomes of Rhipicephalus spp. available in GenBank were used to analyze their conservative motifs by DNAMAN 6.0 software (Additional Table 2). Three pairs of primers aimed to hypervariable region fragments were designed by Primer Premier 5.0 software, and their cycling conditions were shown in Additional File 1. The 654 bp fragment of N1 (partial nad1-16S rRNA gene), 595 bp fragment of N2 (partial nad2-cox1 gene), and 780 bp fragment of C1 (partial cox1-tRNA-Lys gene) were used as novel genetic markers to analyze divergence of R. turanicus. Data analysis methods are described in references (Dantas-Torres et al. 2013;Lv et al. 2014). PCR products were purified using the TIANgel Midi Purification Kit (NGEN, Beijing, China), and sequenced by Sangon Biotech (Shanghai, China). The automatic MEGA model selection method (analysis: Maximum Likelihood model selection, substitution type: nucleotide) was applied to choose the appropriate model for phylogenetic analyses. The dataset was resampled 1000 times to generate bootstrap values. Phylogenetic analyses were conducted with the Maximum Likelihood method by using MEGA v.6.0. Outgroups of phylogenetic trees were selected from GenBank. The 75 nucleotide sequences (16S rDNA: KY583065-KY583081; cox1: KY606287-KY606303; N1: KY620098-KY620114; N2: KY626023-KY626039; and C1: KY996824-KY996840) were deposited in the GenBank database.

Results and discussion
Concerning evaluated morphological parameters, only the ratio basis capituli length:width was significantly different between R. turanicus females from China and Albania (P = 0.022). Three parameters were significantly different between R. turanicus males Exp Appl Acarol (2017) 73:493-499 495 from China and Albania, i.e. the length, the width and the length: width ratio of the scutum (P = 0.0125, 0.0018 and 0.0027, respectively) (Additional Table 3). The observed differences between one parameter of female and three parameters of male R. turanicus collected in China and Albania might be partialy interpreted as geographically related intraspecific morphological variations. Similar differences were noted by Filippova (1997) when comparing R. turanicus from four geographical regions in Central Asia (Dagestan, Badaj-Tugaj, Taskent, Sumbar). Sequencing data confirmed the morphological results on the basis of BLASTn analysis of 16S rRNA and cox1 gene, as these had 98.83-99.56 and 98.71-99.88% homology with the GenBank Italian and American sequences of R. turanicus. Based on 16S rDNA sequence data and Italian R. turanicus reference sequence (accession number: KC243864) (Dantas-Torres et al. 2013), 315 R. turanicus ticks indicated two different lineages of R. turanicus originated from from northwestern China and Albania, respectively (Fig. 1). The analysis of sequence divergences showed that the 16S rRNA sequences from Chinese R. turanicus were 0-1.32% whereas from Albania they were 0-0.44%. Interestingly, the sequence divergences between R. turanicus from Albania and China were 3.53-4.84%. In addition, based on cox1 sequence data, two significantly different lineages also appeared in R. turanicus ticks (Fig. 2). The analysis of sequence divergences showed that the cox1 sequences from Chinese inners, Albania inners and Chinese-Albania interface were 0-0.47, 0-1.42 and 3.57-4.92%, respectively. The latter value might inspire us whether subspecies within it or species complex (Dantas-Torres et al. 2013). 16S rRNA gene phylogenetic trees showed two branches were presented in R. turanicus. All R. turanicus sequences of northwestern China clustered into the same group, which contains 16S rRNA gene  (Fig. 1). Similarly, based on cox1 phylogenetic analysis, R. turanicus sequences of northwestern China were in the same phylogenetic group, which included sequences from Israel (KF219719, KF219747, KF219750 and KF251021) and Iraq (KM235717-KM235719). Meanwhile, together with KC243921 (from Italy reference sequence) (Dantas-Torres et al. 2013), KC243913 (from Greece) and AF132841 (from Australia), the sequences originating from Albania clustered into another branch (Fig. 2).
Three pairs of newly primers aimed to hypervariable mitochondrial region fragments were designed based on 13 complete mitochondrial genomes of Rhipicephalus spp. available in GenBank, which are superior to 16S rRNA and cox1 gene owing to their widely coverage than 16S rRNA and cox1 gene. When analysing three novel genetic markers (N1, N2 and C1), two different lineages also appeared respectively in 315 R. turanicus ticks (Fig. 3). The sequence divergences showed that the N1, N2 and C1 sequences from Chinese inners were 0-0.46, 0-1.01 and 0-0.87%, respectively. The divergences from Albania inners were 0-1.22, 0-1.27 and 0-1.67%, while the data from Chinese-Albania interface was 3.57-4.07, 3.57-4.39 and 3.18-4.69%, respectively. Our three genetic markers of concatenated phylogenetic analysis also support two branches in R. turanicus, thus these three genetic markers may be suitable genetic tools for distinction identification analyses (Fig. 3). More ticks originated from other part of the world should be molecularly analyzed by 3 newly markers for the divergence of R. turanicus.
In this study, R. turanicus originated from northwestern China were collected in semidesert regions, neighbored to Takla makan desert or Gurbantunggut desert. Interestingly, as for geographical characteristics, Afghanistan, Kyrgyzstan and Israel belong to semi-desert Overall, morphological, molecular data divergence and phylogenetic trees based on five mitochondrial genetic markers showed that two genetic branches might exist within R. turanicus. Phylogenetic analyses generated trees that segregated our tick sequences together with sequences in GenBank, into two distinct clades: one is represented by Central Asia region; the second clade is from the European region, which is in line with former observations by Dantas-Torres on R. sanguineus sensu lato from the New and Old worlds (Dantas-Torres et al. 2013). Herein, our findings suggest that R. turanicus samples evaluted here from two geographically distant regions showed distinctly divergence. In the future, more efforts are needed to study this, based on a larger number of R. turanicus specimens collected throughout its vast geographical range around the world.

Conclusion
The present study showed the existence of morphological and molecular divergence among R. turanicus (from Albania and China), which needs to be further analysed by larger scale sampling of this species in the Palaearctic. Three novel genetic markers (N1, N2 and C1), in addition to 16S rRNA and cox1 gene, may be suitable genetic tools of R. turanicus phylogenetic analysis for distinction delineation.