Discrimination between Haemaphysalis longicornis and H. qinghaiensis based on the partial 16S rDNA and the second internal transcribed spacer (ITS-2)

Abstract

In the present study, two hard tick species, Haemaphysalis longicornis and H. qinghaiensis from North-western China were characterized genetically by the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA and partial 16S rDNA. Based on a fragment within the hypervariable region of 16S rDNA with the length of approximately 453 bp, the phylogenetic trees were constructed by Neighbor-Joining and Maximum-parsimony methods. The results indicated that the phylogenetic status of H. qinghaiensis was distant from that of H. longicornis and closer to H. flava. Furthermore, the ITS-2 rDNA was amplified by PCR and sequenced from individual ticks. The length of ITS-2 is 1,606 bp for H. longicornis and 1,162 bp for H. qinghaiensis. Although sequence variation between the immature stages of H. longicornis was 0.1-0.4%, nucleotide differences between the tested species ranged 2.1-23.2%, indicating that ITS-2 rDNA sequences are genetic markers for the differentiation of the two hard ticks in China. Hence, a PCR-linked restriction fragment length polymorphism (RFLP) approach was developed for their unequivocal differentiation based on ITS-2 rDNA, which provides the foundation for further studies on ticks in China and has implications for studying the population genetic structure of the ticks and for identification and differentiation of closely related ticks.

Characterization of Haemaphysalis flava (Acari: Ixodidae) from Qingling Subspecies of Giant Panda (Ailuropoda melanoleuca qinlingensis) in Qinling Mountains (Central China) by Morphology and Molecular Markers

“…Genetic variations between Haemaphysalis species collected from A. m. qinlingensis and H. flava were the lowest in ITS2 rDNA and mt 16S, with 2.06%–2.40% and 1.30%–4.70%, respectively. Phylogenetic relationships showed that the Haemaphysalinae were complicated and polyphyletic, which were consistent with genetic trees re-constructed by Tian et al [58]. All the Haemaphysalis collected from A. m. qinlingensis were grouped with H. flava , further confirmed that the Haemaphysalis sp.…”

Life cycle of Haemaphysalis doenitzi (Acari: Ixodidae) under laboratory conditions and its phylogeny based on mitochondrial 16S rDNA

“…Recently, mitochondrial 16S rDNA has been widely used in analyzing the phylogeny or systematic evolution of ticks (Barker and Murrell 2004;Tian et al 2011). Identification and classification of H. doenitzi based on morphology is difficult due to its small size, especially at the immature stages.…”

Haemaphysalis japonica, Haemaphysalis jezoensis and ^|^#8220;Haemaphysalis douglasi^|^#8221; (Acari: Ixodidae): Which tick is distributed in Hokkaido?

“…All of the ticks were collected by flagging vegetation during spring seasons of 2012 and 2013. Fragments of mtDNA (large subunit ribosomal RNA gene, rrnL) and nDNA (internal transcribed spacer 2, ITS2) were amplified from genomic DNA by polymerase chain reaction (PCR) using published primers (Tian et al, 2011;Barker, 1998). The genomic DNA was extracted from ticks using QIAGEN DNeasy blood and tissue kit, and the PCR was run using TaKaRa ExTaq DNA polymerase.…”