Differentiation Of Dermacentor Silvarum And D.nuttalli By Pcr-rflp And Studies Complete Mitochondrial Genome Sequences Of D.silvarum

Ticks are obligate blood-sucking ectoparasites, which mainly parasitize on the body surface of domestic(such as cattle, sheep, goats and horses) and wild animals, and also attacks human. Ticks can carry and transmit a number of pathogens, including bacteria, viruses and parasites. Furthermore, ticks bite can lead to direct parasitic damages due to their paralyses and toxicoses, irritation and allergy, which not only causes enormous economic loss in husbandry production but also is a threat to human health.Dermacentor silvarum and D. nuttalli are common in northern China and their similar morphology results in the difficulty for identification the two kind of ticks. The first part of the study was to establish PCR-RFLP method to distinguish D. silvarum from D. nuttalli based on the sequences of internal transcribed spacer 2(ITS2) in ribosomal DNA(r DNA). The total genomic DNA was extracted from each worm of the two species, then designed primers to amplify ITS2, and their PCR products were sequenced in both directions. PCR-RFLP method was carried out to identify D. silvarum and D. nuttalli through Spe I existed in their ITS2 r DNA seqeunces. The results showed no Spe I site in ITS2 sequence of D. silvarum but one in D. nuttalli, which suggested that PCR-RFLP based on ITS2 sequences can distinguish D. silvarum from D. nuttalli.The previous reports about Dermacentor mainly focused on their morphology, life cycles, biological characteristics, epidemiology and transmitted tick-borne diseases; very little is known about their mitochondrial genome expect cox1 and rrn L. So the second part of the study was to amplify and analyze the complete mitochondrial genome of D. silvarum. The results showed that the complete mitochondrial genome of D. silvarum is 14,945 bp in length, which contains 37 genes including 13 protein-coding genes(cox1-3, nad1-6, nad4 L, cytb, atp6 and atp8), 2 ribosomal RNA genes and 22 transfer RNA genes. The nucleotide is composed of A(38.89%), T(39.89%), G(8.94%) and C(12.29%), and their A+T contents is 78.78%. The mitochondrial gene arrangements in D. silvarum is consistent with that of Metastriata. The mt DNA of D. silvarum contained a variable repeat region consisting of a “similar to nad1” motif that was repeated three times. To determine the phylogenetic relationships of D. silvarum, phylogenetic trees were constructed based on the amino acid sequences of 13 mitochondrial protein-coding genes using BI, ML and MP methods. The results showed that the topologies of the three phylogenetic trees were similar.and the examined hard ticks formed two large branches, namely Metastriata and Prostriata. And the trees revealed Dermacentor, Rhipicephalus spp. and Bothriocroton formed independent branches, while Amblyomma and Haemaphysalis became complex branches. In Metastriate branch, D. silvarum and D. nitens gathered together with high statistical support(BP=100%; PP=1) and D. silvarum is closer to D. nitens campared to Rhipicephalus spp..This study firstly established PCR-RFLP method to identify D. silvarum and D. nuttalli, which provided a reference for identification of the two ticks. Meanwhile, the mitochondrial genome of D. silvarum was determined in the present study for the first time, and compared with genetic component. All these results not only enriched mitochondrial DNA resources, but also laid the foundation for studying tick classification and the evolutionary relationships.