Analysis And Tentative Application Of Molecular Markers Of Piroplasma And Hard Tick

Piroplasma included Babesia and Theileria, is protozoon that usuallyparasitizes in erythrocytes, lymphyblast, and macrophage. Hard tick,transmitted vector of piroplasma and belonged to Acari, Ixodida, Ixodidae, areobligate ectoparasites, and seem to be second in importance only tomosquitoes as vectors of human and animal diseases. All species areexclusively hematophagous in all feeding stages to host from reptile, birds tomammal (excepted fish). The babesiosis, theileriosis, and some tick-bornediseases cause a huge loss to the livestock industry and increase the risk ofpublic health. However, there are many proplems of systematization andphylogenetic relationships about piroplasma and their host. In this study, wefocus on some samples that isolated and collected in China, and somesequences from GenBank to research the molecular taxonomy, phylogeny,and coevolution of them. The major results are presented as following:1. Compare the phylogenetic relationships of Babesia spp. using large subunitribosomal RNA, i.e.,28S rRNA, and the united28S+18S rRNA sequence fragments from eleven isolates of Babesia spp. collected in China. Due tosequence length and variability, the28S rRNA gene contained moreinformation than the18S rRNA gene and could be used to elucidate thephlyogenetic relationships of Babesia motasi, Babesia major, and Babesiabovis. Thus,28S rRNA is another candidate marker that can be used for thephylogenetic analysis of Babesia spp. However, the united fragment (28S+18S) analysis provided better supported phylogenetic relationships than singlegenes for Babesia spp. in China.2. The ribosomal large-subunit RNA gene sequences of thirteen ChineseTheileria stocks infective to cattle and sheep were sequenced. The segmentswere from3,031bp to3,061bp in length. We also selected the D2–D3regionfrom the complete28S rRNA for phylogenetic analysis. The putativestructures of the D2–D3region of the28S rRNA gene were predicted. Ouranalyses of28S rRNA gene sequences revealed:28S rRNA was applicable asa phylogenetic marker to the analysis of relationships among the Theileria spp.in ruminants; the D2–D3region as a short segment can play same role as thewhole28S rRNA sequence in the phylogenetic analysis of Theileria and may be a ideal DNA barcode.3. The abilities of eight DNA regions including18S rRNA,28S rRNA,internal transcribed spacer (ITS) regions, COI genes, and their partialsegments (18SD,28SD, ITS1, ITS2) have been compared as candidates ofDNA barcodes for piroplasma. In total,484sequences of piroplasma werecollected from this study and GenBank. The eight proposed DNA regionswere evaluated according to the criterion of Consortium for the Barcode ofLife (CBOL). From this evaluation, ITS2had100%PCR amplificationefficiency, an ideal sequence length, the largest gap between the intra-andinter-specific divergence,98%identification efficiency at the genus level, and92%at the species level. Thus, we propose that ITS2is the most ideal DNAbarcode based on the current database for piroplasma.4. We collected165sequences from60species, covering seven genera of hardticks. According to DNA barcoding approach, the5’ region of themitochondrial Cytochrome c Oxidase subunit1(COI) region (586bp) hasbeen used to investigate intra-and interspecific variations and the neighborjoining (NJ) method for developing phylogenetic trees have been used for barcoding utility assessment. As a result, by comparing the distance of intra-and interspecies,49samples were shown to have overlapping barcodes. Fromthe phylogenetic analysis,85%of the species were identified correctly fromtheir DNA barcodes. The remaining nine species were not monophyletic intheir COI sequences because they shared barcode sequences with closelyrelated species. On deeper analysis of these species, using single molecularmarker, presence of cryptic species, and mistakes of submitted data toGenBank maybe important reasons for above results. Therefore, further workis required to delineate species boundaries and to develop a more completeunderstanding of hard tick diversity over a larger scale.5. To examine coevolutionary relationships between piroplasma and hard tick,we conducted phylogenetic analyses and calculated the molecular clock ofboth group based on COI sequences. As a result, the origin date of piroplasma(about56Mya) is later than their host (hard tick about86Mya). This idcatedthe diversification of piroplasma was not caused by codivergence along withtheir hosts. From analyses of evolution of piroplasma lineages and their hostlineages and their association, we can say the piroplasma with high host specificity and was main driving force for the host switch events. However,cospeciation as one of factor also played important role in the complicatedprocess of host-parasite coevolution.