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Application of DNA barcodes in the genetic diversity of hard ticks (Acari: Ixodidae) in Kazakhstan
1Abstract
Forty-five tick species have been recorded in Kazakhstan. However, their genetic diversity and evolutionary relationships, particularly when compared to ticks in neighbouring countries, remain unclear. In the present study, 148 mitochondrial cytochrome c oxidase subunit I (COI) sequence data from our laboratory and NCBI (National Center for Biotechnology Information; https://www.ncbi.nlm.nih.gov/) data were used to address this knowledge gap. Phylogenetic analyses showed that i) Hyalomma anatolicum anatolicum (Koch, 1844) ticks from Jambyl Oblast (southeastern Kazakhstan) and Gansu Province (northwestern China) constituted a newly deviated clade; and ii) Dermacentor reticulatus (Fabricius, 1974) ticks from South Kazakhstan Oblast were closer to those in Romania and Turkey. The network diagram of haplotypes showed that i) the H-1 and H-2 haplotypes of Dermacentor marginatus (Sulzer, 1776) ticks from Zhetisu and Almaty were all newly evolved; and ii) the H-3 haplotypes of Haemaphysalis erinacei (Pavesi, 1884) from Almaty Oblast and Xinjiang Uygur Autonomous Region (northwestern China) were evolved from the H-1 haplotype from Italy. In the future, more COI data from different tick species, especially from Kazakhstan and neighbouring countries, should be employed in the field of tick DNA barcoding.
Supplementary Information
The online version contains supplementary material available at 10.1007/s10493-023-00893-1.
Introduction
Hard ticks are ectoparasites of terrestrial vertebrates that require a different host for each developmental stage, namely the larval, nymph, and adult stages (Leal et al. 2020). Host activity, such as the seasonal migration of migratory birds and the international livestock trade, plays an important role in tick dissemination (Tsao et al. 2021).
Kazakhstan, with an area of 2,724,900 square kilometers (Yang et al. 2002), is the ninth-largest country in the world, and is adjacent to Russia, Turkmenistan, Uzbekistan and China. To date, 45 tick species, belonging to seven genera including Ixodes, Hyalomma, Dermacentor, Rhipicephalus, Haemaphysalis, Argas and Ornithodoros, have been reported in Kazakhstan (Perfilyeva et al. 2020).
Mitochondrial genes, such as 12 S rDNA, 16 S rDNA, cytochrome c oxidase subunit I (COI), and COII, are the most common molecular markers used to identify tick species (Murrell et al. 2000; Leo et al. 2010). The mitochondrial COI, as the standard for DNA barcoding, has played an important role in intra-/inter-species identification and the study of genetic diversity (Hebert et al. 2003). However, the biology of ticks plays a decisive role in the epidemic characteristics of tick-borne diseases in different regions, and limited studies have been conducted in Kazakhstan using DNA barcodes to analyze the genetic diversity and evolutionary relationships of ticks, especially compared to its neighbouring countries. To address this knowledge gap, this study conducted phylogenetic analyses based on COI sequences to explore intra-/inter-species tick evolution in central Asia.
Materials and methods
Tick sampling and morphological identification
Under the cooperation agreement between Shihezi University and Kazakh National Agrarian University, a total of 13,095 nymphs and adult ticks were collected in Kazakhstan during 2016–2023. Parasitizing ticks were collected from the entire body of each animal, including camel (Camelus bactrianus), cattle (Bos taurus), chicken (Gallus gallus f. domestica), dog (Canis lupus familiaris), hedgehog (Hemiechinus auritus), horse (Equus ferus caballus), and sheep (Ovis aries) (Horak et al. 2011; Wang et al. 2015). Off-host ticks were collected using the dragging-flagging method and by directly capturing ticks from the ground (Tsunoda et al. 2004; Zhang et al. 2016). The sampling information is shown in Table 1. All ticks were placed in tubes with 75% ethanol, stored at − 20 °C and morphologically identified according to standard taxonomic keys (Walker et al. 2003).
Table 1
Tick collection data during 2016–2023 in Kazakhstan
| Year | Oblast | Host | Hard/ soft tick | Number |
|---|---|---|---|---|
| 2016–2019 | Almaty | Cattle, sheep, hedgehog, off-host | Hard tick | 596 |
| 2018–2019 | East Kazakhstan | Cattle, sheep | Hard tick | 1625 |
| 2018–2019 | Jambyl | Cattle, sheep | Hard tick | 752 |
| 2018–2019, 2023 | Kyzylorda | Camel, cattle, sheep, | Hard tick | 4693 |
| 2018–2019, 2023 | South Kazakhstan | Cattle, chicken, sheep, dog, off-host | Hard tick | 3886 |
| 2018–2019 | South Kazakhstan | Chicken, off-host | Soft tick | 1472 |
| 2023 | Zhetisu | Cattle | Hard tick | 71 |
| Total | 13,095 |
DNA extraction, polymerase chain reaction (PCR) amplification, and sequencing
After morphological identification, 464 representative tick specimens, with three to 10 ticks for each tick species obtained at each sampling site, were used to analyze the genetic diversity. DNA was extracted from representative ticks using TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’ s instructions. About 710-bp fragments of the COI genes were amplified via PCR. The primers and PCR cycling conditions are shown in Appendix Table 1. The newly generated sequences of COI were manually edited, aligned, and compared to the reference GenBank (National Center for Biotechnology Information, NCBI) sequences using the nucleotide BLASTN program (https://blast.ncbi.nlm.nih.gov) (Hornok et al. 2016). A total of 38 COI sequences of tick samples were deposited in the NCBI GenBank (accession No. MN907836, MN907834, MN907826, MN907832, MN964336, MN907838, MN964340, MN907848, MT079206, MN689420, MN689425, MN689410, MN907846, MN841463, MN689436, MN689429, MN689434, MN907845, MN689404, MN964337, MN964342, MN821375, MN964341, MN907835, MN853164, MN853163, OR533607, OR533660 and OR533790-OR533799).
Sequence analyses
The data obtained in the laboratory were combined with the NCBI (https://www.ncbi.nlm.nih.gov/) data retrieved on March 27, 2023. The above data were resampled 1,000 times to generate bootstrap values. Phylogenetic relationships were inferred using the maximum likelihood (ML) method. Evolutionary analyses were conducted in MEGA X (shown in Appendix Fig. 1). The genetic diversity was estimated using the haplotype (h), haplotype diversity (Hd) and nucleotide diversity (Pi) indices with the DNAsp ver. 5.10.01 program. Median-joining (MJ) networks were generated with the Network ver. 10.2.0 software to display the configuration of haplotypes (Wetjen et al. 2020).
Results and discussion
Morphological and molecular identification confirmed that seven hard tick species were identified, namely Dermacentor marginatus, Dermacentor reticulatus, Rhipicephalus sanguineus (Latreille, 1806), Rhipicephalus turanicus (Pomerantzev, 1940), Hyalomma anatolicum, Hyalomma asiaticum asiaticum (Schulze et Schlottke, 1929) and Hyalomma scupense (Schulze, 1918). Photographs of them in turn are shown in Fig. 1.
The analysis of the COI phylogenetic tree showed that i) Hy. anatolicum ticks (accession Nos. MN841463 and MH459382) from Jambyl Oblast and Gansu Province (northwestern China) constituted a newly deviated clade, while the corresponding species from Khorasan-e Razavi Province (northeastern Iran) (KP219867) and Turkey (MT230046), were ancestor populations; ii) D. reticulatus ticks (OR533790) from South Kazakhstan Oblast were more closely related to those in Romania (KT87452) and Turkey (KT877453); and iii) D. marginatus ticks from Kazakhstan had two clades. One clade (MN907848) from Almaty Oblast was closer to that (FN394327) from Balint (Romania), and another clade (MN907836) from East Kazakhstan Oblast was closer to that (MN517831) from Xinjiang Uygur Autonomous Region (XUAR, northwestern China). Previously, H. erinacei was classified into three subspecies, including H. e. erinacei (distribution in North Africa and southern Europe, in particular in Spain, Italy and the western Balkans) (Tovornik and Cerný 1974), H. e. taurica (distributed in the Middle East and the eastern Balkans) (Zlatanova 1991) and H. e. turanica (distributed in Central Asia) (Hornok et al. 2016). In this study, H. erinacei (accession No. MN841464) from Almaty Oblast was clustered into H. e. turanica, together with the corresponding sequence (KU880621) from XUAR (northwestern China), rather than those from Europe, including Tokat Province (Turkey) (KX901844), Romania (KU885986), and Italy (KX237631). The COI BLAST analysis showed that the sequence from Kazakhstan shared 100% identity with that from China (KU880621), but had only 95.13%, 95.10% and 94.81% identities with those from Turkey (KX901844), Romania (KU885986) and Italy (KX237631), respectively. This finding was consistent with the analysis of the 16 S rDNA phylogenetic tree (Hornok et al. 2016).
The network diagram based on the COI in the haplotypes was shown as follows. Firstly, D. marginatus ticks were highly divergent, and nine haplotypes were found. The haplotype diversity was 0.9394, while the nucleotide diversity was 0.02101. The H-3 haplotype was the most dominant. The H-1 and H-2 haplotypes from Zhetisu Oblast and Almaty Oblast, the H-6 haplotype from XUAR (northwestern China), the H-7 haplotype from Iran, the H-8 haplotype from Turkey and the H-9 haplotype from Germany were all newly evolved. More interestingly, D. marginatus ticks (belonging to the H-3 and 6 haplotypes) in Russia and XUAR (northwestern China), respectively, were evolved from the H-5 haplotype from South Kazakhstan Oblast (shown in Fig. 2). This result might be related to the close geographical distance between southeast Kazakhstan, northwestern China and the far east of Russia. Secondly, H. erinacei harbored four haplotypes. The haplotype diversity was 0.8095, and the nucleotide diversity was 0.02972. The H-3 haplotypes from Almaty Oblast (MN841464) and XUAR (northwestern China) (KU880621 and MT890494) were evolved from the H-1 haplotype from Italy (KX237631) (shown in Fig. 3). Although there is limited COI data available around the world, the ancestor of H. erinacei may have originated in Europe and developed morphological and molecular differences with crustal movement and host migration.
Central Asia covers about 4 million km2, exhibits many shared characteristics in terms of climate, wildlife hosts and geographical habitats. In total, 148 COI sequences were involved in the current study. In future work, more COI data from different tick species, especially data from Kazakhstan and its neighbouring countries, will be employed. This will allow for the more systematic exploration of intra-/inter-species tick evolution in central Asia.
Conclusions
This article describes the genetic diversity and evolutionary relationships of hard ticks in Kazakhstan based on the analysis of COI data. Hy. anatolicum ticks from Jambyl Oblast and Gansu Province (northwestern China) constituted a new population. D. reticulatus ticks from South Kazakhstan Oblast were closer to those in Romania and Turkey. The H-1 and H-2 haplotypes of D. marginatus ticks from Zhetisu Oblast and Almaty Oblast were newly evolved. The H-3 haplotypes of H. erinacei from Almaty Oblast and XUAR (northwestern China) were evolved from the H-1 haplotype from Italy.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Acknowledgements
We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
Author contributions
MHY and YZW conceived and designed the study and critically revised the manuscript. All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by ZWZ, WXZ, SWW, WBT, XBL, KK, LGM, WRLHZHZH, GL, MHY and YZW. The first draft of the manuscript was written by ZWZ and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding
This work was supported by National Key Research and Development Program (2022YFC2304004), National Natural Science Foundation of China (Grant numbers 82260399 and 81960379), Natural Science Key Project of Xinjiang Uygur Autonomous Region (2022B03014) and High-Level Talent Initiative Foundation of Shihezi University (RCZK202369 and KX019303/0305).
Data availability
The datasets generated during the current study are available in the GenBank repository, http://www.ncbi.nlm.nih.gov/genbank/.
Declarations
This study was approved by theimal Ethics Committee of Shihezi University (Approval No. AECSU2023-28).
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Ziwei Zheng, Weixin Zeng, Suwen Wang, Wenbo Tan and Xiaobo Lu contributed equally to this work.
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