Collection of tick specimens from turtles
Ticks infesting wild turtles were collected every month from April 2021 to March 2022. The yellow-margined box turtles (C. flavomarginata) were captured at the reservation area around the Fei-tsui Reservoir located at the Shiding District of New Taipei City in northern Taiwan (Fig. 1). In general, ticks infested on the legs, head, body and shell of the turtles were collected and cleaned by sonication in 75% ethanol solution for 5-10 min and then washed twice in sterile distilled water. All cleaned ticks were stored at -20℃ refrigerator until further analysis.
Species identification of tick specimen
Each stage of tick specimen was placed on a glass slide and photographed using a Nikon stereomicroscope (model: SMZ 1500) equipped with a fiber lamp (Chao et al. 2013). The external features of the Ambloymma ticks were recorded for species identification, as previously described (Yamaguti et al. 1971; Teng and Jiang 1991; Chao et al. 2022). In addition, molecular identification of collected ticks was also performed by targeting the internal transcribed spacer gene (ITS2), as previously described (Chao et al. 2011).
DNA extraction from tick specimens
Genomic DNA was extracted from individual tick specimens used in this study. Briefly, the individual tick specimen was homogenized in a microcentrifuge tube filled with 180-μL lysing buffer solution (DNeasy Blood &Tissue Kit, catalogue no. 69506, Qiagen, Taipei, Taiwan) and then homogenized with a TissueLyser II apparatus (catalogue no. 85300, Qiagen, Germany), instructed by the manufacturer. The homogenate was centrifuged at room temperature and the supernatant fluid was further processed by a DNeasy Tissue Kit, as instructed by the manufacturer. After filtration, the filtrated fluid was collected and the DNA concentration was determined spectrophotometrically with a DNA calculator (Epoch, Biotek, USA) and the extracted DNA is stored at -80 ℃ for further investigations.
DNA amplification by polymerase chain reaction (PCR)
DNA samples extracted from the tick specimens were used as a template for PCR amplification. A specific primer set of 3SA (50-CTAAGCGGTGGATCACTCGG-30) corresponding to the 3’ end of the 5.8S rDNA and JB9A (50-GCACTATCAAGCAACACGACTC-30) corresponding to the 5’ end of the 28S rDNA were designed to target the ITS2 rDNA gene, as described previously (Barker 1998). All PCR reagents and Taq polymerase were obtained and used as recommended by the supplier (Takara Shuzo Co., Ltd., Japan). Briefly, each 25-μl reaction mixture containing 3-μl DNA template, 1.5-μl forward and reverse primers, 2.5-μl 10X PCR buffer (Mg2+), 2-μl dNTP mixture (10 mM each), 1 unit of Taq DNA polymerase and filled-up with adequate volume of ddH2O. In contrast, adequate amounts of sterile distilled water were added for serving as a negative control. PCR amplification was performed with a thermocycler (Veriti, Applied Bioosystems, Taipei, Taiwan) and was amplified for 35 cycles with the conditions of denaturation at 95℃ for 30 s, annealing at 50℃ for 30 s, and extension at 68℃ for 2 min., as described previously (Chao et al. 2011). Thereafter, amplified DNA products were electrophoresed on 2% agarose gels in Tris-Borate-EDTA (TBE) buffer and visualized under ultraviolet (UV) light after staining with ethidium bromide. A 100-bp DNA ladder (GeneRuler, Thermo Scientific, Taiwan) was used as the standard marker for comparison. A negative control of distilled water was included in parallel with each amplification.
Sequence alignments and phylogenetic analysis
Approximately 10-μl of each selected samples with clear bands on the agarose gel was submitted for DNA sequencing (Mission Biotech Co., Ltd., Taiwan). After purification (QIAquick PCR Purification Kit, catalog No. 28104), sequencing reaction was performed with 25 cycles under the same conditions and same primer set of nested amplification by dye-deoxy terminator reaction method using the Big Dye Terminator Cycle Sequencing Kit in an ABI Prism 377-96 DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The resulting sequences were initially edited by BioEdit software (V5.3) and aligned with the CLUSTAL W software (Thompson et al. 1994). Thereafter, the aligned sequences of ITS2 genes from 7 tick strains of Taiwan were analyzed by comparing with other 9 tick strains from the different biological and geographical origin that are available from GenBank. Phylogenetic analysis was performed by neighbour-joining method to estimate the phylogeny of the entire alignment using MEGA X software package (Kumar et al. 2018). The phylogenetic tree was constructed and performed with 1000 bootstrap replications to evaluate the reliability of the construction, as described previously (Felsenstein 1985).