| Paramphistomum cervi, Dicrocoelium chinensis and Dicrocoelium dendriticum are threeparasites of ruminant animals, P. cervi belongs to Echinostomata, Paramphistomatidae, while D.chinensis and D. dendriticum belong to Plagiorchiata, Dicrocoeliidae. All of these threetrematodes infection can cause serious trematodiasis, named Paramphistomosis andDicrocoeliosis, attention should be paid to these trematode because they can also parasitize inthe liver bile duct and gallbladder of human, lead to disease, it has been reported humaninfections in America, Korea, Japan country. In order to better control these trematodes,further understand should be taken to learn their genetic characteristics, then to study theirecology, genetic model, classification and the drug resistance. Hence, it is great significanceof this research in studying molecular taxonology, population genetics of these trematodes,specificity diagnosising and controlling of these diseases, protecting human health and publichealth.In the first section, the ribosomal DNA internal transcribed spacer (ITS) between the28Sand18S ribosomal RNA genes was sequenced and characterized for P. cervi, D. chinensis andD. dendriticum populations from different endemic regions. The length of the ITS rDNA ofthree trematodes was854bp～868bp,955bp～960bp and1140bp～1144bp, theintraspecific sequence variations was0～2.475%,0～2.5%and0～0.62%, respectively.Phylogenetic analyses was operated by Bayes based on the concatenated sequences of theITS–2sequence. In the tree, all the same kind specises grouped together with high statisticalsupport, D. chinensis and D. dendriticum were more closely to Paragonimus westermani thanto schistosoma japonicum, clonorchis sinensis and opisthorchis felineus, more distantrelationship with Fasciola hepatica, furthest with P. cervi.The objectives in the second section were to examine the polymorphic analysis in thetwo part regions of mitochondrial DNA (mtDNA), named cytochrome coxidase subunit1(cox1) and NADH dehydrogenase subunit1(nad1) within the species about P. cervi and D.chinensis, The length of pcox1and pnad1for P.cervi were399bp and532bp, respectively.The intra–specific sequence variations among the examined P.cervi samples were0～15.4%for pcox1and0～8.4%for pnad1, whereas the inter–specific sequence differences among the members of the examined trematodes were significantly higher, being21.8～27.3%for pcox1and25.6～32.4%for pnad1. The sequences of pcox1and pnad1for Dicrocoelium chinensiswere358and458bp in length, respectively. The intraspecific sequence variations among theexamined D. chinensis samples were0～0.84%for pcox1and0～0.87%for pnad1, whereasthe inter–specific sequence differences among the members of the examined trematodes were22.9～29.3%for pcox1and33.6～41.5%for pnad1. Phylogenetic analyses was operated byBayes based on the concatenated sequences of the cox1+nad1sequence, showed that D.chinensis and S. japonicum were more closely related than to P. westermani, C. sinensis andO. felineus, furthest distant relationship with F. hepatica. P. cervi was more closely relatedwith C. sinensis and O. felineus than to P. westermani and F. hepatica, furthest distantrelationship with S. japonicum. Phylogenetic tree show P. cervi samples may containedcryptic species.In the thrid section, we sequenced the complete mitochondrial DNA (mtDNA) sequencesof P. cervi, D. chinensis and D. dendriticum. The mt genome sequence length was14,014bp,14910bp and14884bp in size, contained36genes, including12protein-coding genes,2ribosomal RNA genes and22transfer RNA genes, all genes are transcribed in the samedirection, which is consistent with F. hepatica and P. westermani. Phylogenetic analyses wereoperated by Bayes using concatenated amino acid sequences of the12protein–coding geneswith the flukes had been reported, respectively. Resuits demonstrates that P. cervi is closer toF. hepatica, P. westermani and is far away from Schistosoma spp., D.chinensis and D.dendriticum are closer to O. felineus and C. sinensis, and is far away from Schistosoma spp..The present study gained the ITS rDNA sequence of P. cervi, D. chinensis and D.dendriticum, and sequenceed the mitochondrial partial genes (pcox1, pnad1) and completegenomes for the first time. Results comprehensively reflects the genetic variability andpopulation structure of P. cervi, D. chinensis and D. dendriticum in mainland China, thecomparison of the result shows that internal transcribed spacer (ITS) and mitochondrialgenome methods in studying genetic variation and population structure is accurate. Thesedata filled the gaps of knowledge about these tremotades and provide molecular tools for therapid and specific detection of them, and have implications for the prevention and control ofP. cervi, D. chinensis and D. dendriticum in different regions of China. |
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