Research On Molecular Phylogeny Of E. Hortense

Echinostoma hortense belongs to Platyhelminthes, Trematoda, Digenea, Echinostomata,Echinostomatidae, Echinostoma. Adult worms of E. hortensis live in the small intestine of mammals such as rat, dog, cat and pig. Although the majority of people mildly infected by echinotomes have no obvious symptoms, severe infections may have anorexia, lower extremity edema, anemia, weight loss, dysplasia, or combination with other diseases and even to death.Besides, E. hortensis is a zoonotic pathogen and can live in human. The E. hortensis are mainly distributed in Korea, Japan, China and Southeast Asia.In recent years, researches mainly focused on the epidemiology, diagnosis, treatment and prevention. The complete mitochondrial genomics of E. hortensis has not been reported in any literature except on cox1 and nad1.Genomic DNA of E. hortensis was extracted from parasites isolated from individual host using SDS-proteinase K. The first part of the study was to amplify the completed mitochondrial genome of E. hortensis and to further analyze their evolutionary relationship through phylogenic trees based on 12 protein-coding genes. The second part was to obtain 5 E. hortensis ribosomal sequences by molecular biology method and to analyze each part of 5 ribosomal sequences, such as gene size and arrangement. The phylogenic trees were created to compare with other trematoda, and their phylogenetic relationships were examined based on the small sub-unit(18 S)ribosomal DNA and the internal transcribed spacer 1(ITS1).The results showed that the complete mitochondrial genome of E. hortensis was 14,994 bp in length, including 22 tRNA genes, 12 protein-coding genes, 2 rrnA genes and 1 noncoding region.The lengths of protein-coding gene, rrnA gene and non-coding sequence were 10,047 bp, 1,732 bp and 1,553 bp, respectively. The mitochondrial nucleotide is composed with T(41.69%), G(24.79%), A(21.36%) and C(12.16%); their A+T contents is 63.05%. The mitochondrial genome of E. hortensis was similar to prefer A and T bases, which was consistent with other trematodes. The arrangement of 12 protein-coding genes, 2 rrnA and 22 tRNA genes in E.hortensis belongs to GO4 type. Phylogenetic relationships were reconstructed based on the amino acid sequences of 12 protein-coding genes using BI, MP and ML methods. The results of topological structure trees were similar with each other, which were divided into two branches,Clinostomoidea mesh flukes(Clinostomum complanatum) independent evolutionary branch,Opisthorchiata, Echinostomata and Plagiorchiata orders for another branch. E. hortensis was separated into the order Echinostomata according to morphological classification. These results indicate that 12 protein-coding genes can be used as a genetic Marker for studying the systematics, phylogenetics of Digenea.The five rDNA sequences are 6,810 bp in length, which contain rDNA(28 S, 18 S and 5.8 S)and ITS1-2. There is no difference in length among all the examined 18 S, ITS1, 5.8S, ITS2 and28 S rDNA sequences, which were 1,981 bp, 444 bp, 162 bp, 440 bp and 3,783 bp, respectively.The intra-specific variations within E. hortensis were 0~0.1% for 18 S, 0% for 5.8 S-28 S,0~0.20% for ITS1-ITS2. Phylogenetic relationships among members of trematodes were constructed based on 18 S rDNA and ITS1 using BI, MP and ML; their topological structures of trees were similar with each other. The results showed that E. hortensis was grouped into the order Echinostomata according with morphological classification. These results indicate that 18 S rDNA and ITS1 were useful for studying the systematics, phylogenetics of Digenea.Isthmiophora hortensis and E. hortensis is in the same branch; E. hortensis and I. hortensis is a synonym.In this study, mitochondrial genome were firstly obtained for E. hortensis isolated from dog,which enriched the trematode mitochondrial genome data. Based on mitochondrial 12protein-coding genes, ribosomal 18 S and ITS1 sequences, phylogenetic trees were constructed to discuss the evolutionary relationships with other trematode. This study enriches the trematode mitochondrial genome, ribosomal gene data, for further study of spine trematode classification,system, and round nursery spine fluke of origin laid the foundation.