Tick Identification and Molecular Analysis

Ticks were examined with a Keyence VHX-900F microscope (Itasca, IL, USA) and identified to species level using morphological keys (Filippova 1977 ; Pérez-Eid 2007 ; Hornok et al. 2014 (link)). In cases of questionable specimens, those were objected to molecular analysis. DNA was extracted using the QIAamp mini DNA extraction kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The 16S rRNA gene was amplified by PCR and Sanger sequenced according to Halos et al. (2004 (link)), using the tick-specific primer pair TQ16S + 1F (5’-CTGCTCAATGATTTTTTAAATTGCTGTGG-3’) and TQ16S-2R (5’-ACGCTGTTATCCCTAGAG-3’) of Black and Piesman (1994 (link)). Sequences were edited and primers trimmed using the Geneious Prime 2022.2.1 software (https://www.geneious.com). A BLASTN search (Zhang et al. 2000 (link)) in GenBank was performed to molecularly annotate the individual 16S rDNA sequences. 16S rDNA sequence data can be found in GenBank and the supplementary material.