Only male H. anatolicum were selected for DNA extraction due to an insufficient number of female ticks. Before DNA extraction, ticks were washed in 70% ethanol and deionized water for 5 min to remove environmental contaminants in accordance with a published protocol [23 (link)]. Due to the small size of each tick, a pool of five male H. anatolicum ticks was homogenized in liquid nitrogen inside a sterile 1.5-ml microcentrifuge tube using a sterile Kimble Kontes pellet pestle (Thermo Fisher Scientific, Waltham, MA, USA). Genomic DNA was extracted from each pool using the QIAamp Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The concentration and quality of DNA were assessed using a Nano Drop ND-100 spectrophotometer (Peqlab Biotechnologie GmbH, Erlangen, Germany). DNA quality was also determined by electrophoresis in a 1% agarose gel; the bands were stained with ethidium bromide and visualized under UV light. DNA was stored at − 20 °C in the freezer until further use. Prior to sequencing, extracted DNA samples (5 DNA samples of ticks from each host) were pooled again to make one pool for each host from each location, resulting in seven DNA pools.
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