Updates on subgenus Ixodes in the Mediterranean region: validity of Ixodes festai Rondelli, 1926, reinstatement of Ixodes tatei Arthur, 1959, and a new species closely related to Ixodes gibbosus Nuttall, 1916
Description
Raw data in FASTA format for 16S and COX1 sequences of Ixodes acuminatus, Ixodes eldaricus, Ixodes festai, Ixodes gibbosus, Ixodes paragibbosus and Ixodes tatei.
Files
Steps to reproduce
In this study, 21 Ixodes spp. tick specimens were analyzed morphologically and molecularly. These samples were collected from birds, rodents or from the vegetation between 2013-2024 in the Mediterranean region (Israel, Turkey) and the southern part of central-eastern Europe (Hungary, Romania). The ticks were stored in 96% ethanol at -4 C until usage. Tick species were morphologically identified using the best available keys according to their species (for all Ixodes species: Estrada-Peña et al., 2017a; for I. eldaricus: Filippova, 1974; for I. festai: Gilot and Perez, 1936, Contini et al., 2011; for I. tatei: Arthur, 1959 and 1968; for I. gibbosus: Nuttall, 1916; Saratsiotis, 1970). Pictures were made with a Keyence VHX-5000 digital microscope (Osaka, Japan). DNA was extracted with the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany), including an overnight digestion in tissue lysis buffer and Proteinase K at 56 oC. PCR amplification of an approx. Molecular-phylogenetic relationships were analyzed based on two mitochondrial genetic markers. First, amplification of an approx. 710-bp-long fragment of the cytochrome c oxidase subunit I (cox1) barcoding gene was attempted with the primer pairs LCO1490 (5’- GGT CAA CAA ATC ATA AAG ATA TTG G -3’) and HCO2198 (5’ - TAA ACT TCA GGG TGA CCA AAA AAT CA- 3’). Samples not yielding PCR product in this test were analyzed further with other primer pairs designed to amplify 680- to 850-bp-long fragments of the same region of this gene (Table 2). As the second genetic marker, an approx. 460-bp-long fragment of the 16S rDNA gene of Ixodidae was also amplified with the primers 16S+1 (5’-CTG CTC AAT GAT TTT TTA AAT TGC TGT GG-3’) and 16S-1 (5’-CCG GTC TGA ACT CAG ATC AAG T-3’). Cycling conditions of these PCRs are summarized in Table 2. PCR reaction components included 5 µl of extracted DNA were added to 20 µl of reaction mixture containing 1 U HotStar Taq Plus DNA Polymerase (5U/µl) (QIAGEN, Hilden, Germany), 0.5 µl dNTP Mix (10mM), 0.5 µl of each primer (50µM), 2.5 µl of 10x Coral Load PCR buffer (15mM MgCl2 included) and 15.8 µl PCR water. PCR products were visualized in 1.5% agarose gel. Purification and sequencing of the PCR products were done by Eurofins Biomi Ltd. (Gödöllő, Hungary). Quality control and trimming of sequences were performed with the BioEdit program. Obtained sequences were compared to GenBank data by the nucleotide BLASTN program (https://blast.ncbi.nlm.nih.gov). New sequences were submitted to GenBank under the following accession numbers (cox1 gene: PV274492-PV274501, 16S rRNA gene: PV277790-PV277808). For the phylogenetic analyses, only sequences with high (98-100%) coverage to sequences from this study were retrieved. Phylogenetic trees were constructed with the Neighbor-Joining method, p-distance model in MEGA11, and sequence datasets were resampled 1,000 times to generate bootstrap values.