Application of DNA barcodes in the genetic diversity of hard ticks (Acari: Ixodidae) in Kazakhstan

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 ninthlargest 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.

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).

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

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).
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 Rus- sia 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 km 2 , 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.
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Table 1
(Hornok et al. 2016) during 2016-2023 in Kazakhstan .DNA was extracted from representative ticks using TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) according to the manufacturer' s instructions.About 710bp fragments of the COI genes were amplified via PCR.The primers and PCR cycling conditions are shown in Appendix Table1.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)(Hornoketal. 2016).A total of 38 COI sequences of tick samples diversity