Complete mitochondrial genome and phylogenetic analysis of Ixodes acutitarsus (Acari: Ixodidae)

Abstract Ixodes acutitarsus is regarded as the largest Ixodes tick around the world. I. acutitarsus is capable to transmit a wide range of animal and human pathogens. This research pioneered sequencing of the complete mitochondrial genome of I. acutitarsus. With a length of 14,475 bp, the complete mitochondrial genome encodes 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and one replication-initiating region. The phylogenetic relationship was established using the Maximum-likelihood method to indicate that I. acutitarsus and the others of the genus Ixodes fit into the same branch, which confirms the inclusion of I. acutitarsus in the genus Ixodes. The complete mitogenome of sequenced I. acutitarsus provides molecular evidence for the taxonomic status and phylogenetic position of several Ixodes species.

The obligate hematophagous arthropod Ixodes acutitarsus (Karsch, 1880) (Acari: Ixodidae) is widely distributed in many Asian countries and regions, including Southwest China, Nepal, Taiwan, Burma, Japan, and India (Daniel 1979). As the principal transmission vector, I. acutitarsus acts as the reservoir of various tick-transmitted pathogens that include a virus, bacteria, and protozoa (Chao and Shih 2012). However, there remains a considerable gap in molecular epidemiology and genetics owing to the lack of appropriate genetic markers for these ticks (Shao et al. 2005). Up to now, there is only a single 'unverified' (and lacking accurate genetic annotation information) mitochondrial sequence of I. acutitarsus, which is a linear sequence with a size of 14,484 bp.
Herein, the first complete mitochondrial genome of I. acutitarsus was sequenced, annotated, and verified. Adult ticks (three male and three females) were collected in February 2021 from High Li Gong Shan, Fugong county-level town, Nujiang Lisu Autonomous Prefecture, northwest Yunnan Province, China (26 34 0 N, 98 48 0 E). Species identification was performed by Professor Chunhong Du according to the exact morphological characteristics . One male and one female were treated as voucher specimens and the remaining ones were used for DNA extraction. Then, the collected specimens were stored in the Parasitological Museum, Dali University (Yunnan, China) under the collection number: DLUP2102_11-12 (Url: http://www.dali.edu.cn/jcyxy/xkpt/jcyx syjxzx/6431.htm, Contact person: Xing Yang, yang08220013@ 163.com) ). The circle genomics DNA was isolated using the standard CTAB technique and then preserved in 75% ethanol at À20 C. The complete mitochondrial genome was sequenced on the Illumina NovaSeq platform (Shanghai Personal Biotechnology Co, Ltd, Shanghai, China), assembled using A5-miseq software (Coil et al. 2015), and annotated using the MITOS web server (http://mitos.bioinf.uni-leipzig.de/) (Bernt et al. 2013).
An alignment of published complete mitogenomes of Ixodidae species was constructed with the 13 PCGs together with Limulus polyphemus (NC_003057) as an outgroup. A phylogenetic tree was then obtained applying a Maximumlikelihood analysis implemented in MEGA7.0 software ( Figure  1) (Kumar et al. 2016).
The phylogenetic tree show two phylogroups: Metastriata and Prostriata (Cummings et al. 1995). The first branch includes species of seven genera, namely, Rhipicephalus, Hyalomma, Rhipicentor, Dermacentor, Amblyomma, Archaeocroton, Haemaphysalis. The second branch includes only the species of the genus Ixodes. Ixodes acutitarsus clusters within the Ixodes clade. Besides, the species showed a closer relationship with I. hexagonus, followed by I. simplex, I. vespertilionis, I. ricinus, I. persulcatus, and I. pavlovskyi. The sequences have only 61.01% similarity to unconfirmed sequences, but more than 90% similarity to confirmed partial sequences. The sequences as measured in this study were found to be identical to some of the published I. acutitarsus genes in NCBI, despite a low-level resemblance to the unconfirmed complete sequence data. In conclusion, the new complete mitochondrial genome of I. acutitarsus is an important resource to enhance future phylogenetic studies within Ixodidae. (Tao et al. 2014).

Ethical approval
This study was approved by the Administration Committee of Experimental Animals, Dali University, Yunnan Province, China.

Author contributions
XYL conceived the study and wrote the manuscript. DDJ and CHD carried out the experiments and analyzed the data. CHR, JQY and YHF contributed to the collection of I. acutitarsus and discussions, XY is responsible for the interpretation of experimental data, critical revision of important knowledge content and final approval of the version to be published.

Disclosure statement
No potential conflict of interest was reported by the author(s).