Population Genentic Structures Of Taenia Multiceps And Taenia Hydatigena In China Based On Mitochondrial Genes

Taenia multiceps and Taenia hydatigena were the most commom Taenia cestodes. Both cestodes(at their adult stage) usually inhabit the small intestine of dogs and other canids (e.g., wolfs, foxs and jackals), and widely distributed in all over the world. Coenurus cerebralis, the metacestode or larvae of T. multiceps, is frequently found in the nervous system, including brain and spinal cord in various herbivores(e.g., sheep, goats and cattle) and occasionally humans, can cause severe coenurosis. Cysticercus tenuicollis, the metacestode or larvae of T. hydatigena, is usually found on the liver surface, omentum and mesentery of swine, goats and sheep, and cause C. tenicollis cysticercosis. For the two cestodes, the relevant researhes on the molecular taxonomy are vacant and limited in our country. Until now there has been no information available on the population genetic diversity of the two Taenia cestodes. In order to learn more about the population genetic structure and evolution history of T. multiceps and T. hydatigena, in the present study, population genetic structures of these two cestodes were carried out, and the main research work were as follows:1. Population genetic diversity of T. multiceps from different regions in Sichuan based on mitochondrial coxl and Cyt b geneTo study the population genetic diversity and phylogentic relationship among T. multiceps, the complete sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) and cytochrome b(Cyt b) genes of 20 Coenurus isolates from goats (11 were isolated from brain, and 9 were isolated from intramuscular) in Sichuan were analyzed. The results showed that the complete lengths of mitochondrial gene sequenced in this study were 1 623 bp for cox1 gene and 1 068 bp for Cyt b gene. Variable sites were 59 and 9, respectively. The sequence divergence between the cox1 sequences of Coenurus isolated from Sichuan ranged from 0.1% to 2.5%, while differences in Cyt b sequences ranged from 0.1% to 0.7%.17 haplotypes and 6 haplotypes were detected in cox1 and Cyt b, with the global haplotype diversity being 0.968±0.033 (cox1) and 0.737±0.068 (Cyt b). Additionally, the nucleotide diversities were 0.00598±0.00133 for coxl and 0.00306±0.00079 for Cyt b, and the average genetic distances were 0.006 for coxl and 0.003 for Cyt b, respectively. The phylogenetic trees inferred from both coxl and Cyt b showed that all 20 Coenurus isolates from Sichuan were the species T. multiceps. However, the phylogenetic tree based on the coxl gene showed that a closer relationship between Sichuan isolates and Iran and Turkey isolates than between Sichuan and Gansu, Guangzhou and Italy isolates. The results indicated that coxl and Cyt b gene were suitable markers for detecting genetic diversity of T. multiceps, and that there was a low diversity and geographic variation observed in T. multiceps. Moreover, the diversity of T. multiceps based on Cyt b gene was lower than coxl gene.2. Population genetic diversity of T. multiceps from different regions in Sichuan based on mitochondrial nadl and nad4 geneIn order to further investigate genetic structure and the phylogenetic relationship of T. multiceps from different regions in Sichuan, the partial sequences of mitochondrial nadl and nad4 genes of 20 isolates of cerebral and non-cerebral Coenurus in goats were analyzed. The results showed that the lengths of mitochondrial genes sequenced in this study were 753 bp for pnadl gene and 801 bp for pnad4 gene, and there were 2 variable sites in pnadl sequences, and 8 variable sites in pnad4 sequences. The sequence divergences of pnadl and pnad4 were 0～0.3% and 0～0.8%, respectively.3 haplotypes and 6 haplotypes were detected in pnadl and pnad4, with the global haplotype diversity being 0.574±0.055(pnadl) and 0.742±0.074 (pnad4). The nucleotide diversity were 0.00082±0.00053 for pnadl and 0.00288±0.001 for pnad4, and the average genetic distances were 0.002 for pnadl and 0.003 for pnad4, respectively. The phylogenetic trees showed that all T. multiceps isolates clustered together. The phylogenetic tree based on the pnad4 gene showed that all isolates from Sichuan clustered into three subgroups, and the cerebral and non-cerebral specimens grouped together, indicating that all isolates were species T. multiceps. The results suggested that the diversity of T. multiceps from Sichuan were low, particularly when with using the gene pnadl for analysis. Whether pnadl and pnad4 gene are suitable markers for detecting genetic diversity of T. multiceps are need to further research, although they were suitable markers for molecular taxonomy of T. multiceps.3. Population genetic structure analysis of T. hydatigena from Sichuan and Yunnan provinces based on mitochondrial cox2 geneIn order to explore the population genetic structure and phylogenetic relationship of T. hydatigena from Sichuan and Yunnan provinces, the partial fragments of mitochondrial gene cox2 of C. tenuicollis were amplified by PCR, and the sequences were then used to population genetic structure analysis. The results showed that the lengths of pcox2 sequence in 33 isolates were 523 bp, and no indels,33 variable sites and 19 haplotypes were detected in these sequences. The sequence divergence was 0.2%～2.9%, and the global haplotype diversity, nucleotide diversity and average genetic distance were 0.932±0.032,0.01097±0.00271 and 0.011, respectively. AMOVA analysis revealed that the major genetic variation was observed within populations, was 96.18%, but no significance was found. In the NJ tree, all 33 isolates from 7 different regions in the Sichuan and Yunnan provinces were grouped into a single cluster, and had not any directly correlation with geography and host. The results indicated that the diversity of T. hydatigena based on cox2 gene was high, and the isolates from different regions appeared to exhibit disordered distribution patterns, and the genetic differentiation among populations was not obvious.4. Population genetic structure analysis of T. hydatigena from Sichuan and Yunnan provinces based on mitochondrial Cyt b geneTo explore the genetic population structure and phylogenetic relationship of T. hydatigena,33 isolates of T. hydatigena from seven different regions in Sichuan and Yunnan provinces were analyzed based on the complete sequences of mitochondrial Cyt b gene. NJ tree and MP trees of haplotypes were constructed using the MEGA and PAUP software, respectively. The results showed that the complete sequence of Cyt b gene of C. tenuicollis was 1068 bp, and there were 67 variable sites and 22 haplotypes were detected in these sequences. Sequence divergence between haplotypes was 0.1%～2.8%, and the global haplotype diversity, nucleotide diversity and average genetic distance were 0.951±0.027,0.01055±0.00189 and 0.012, respectively. AMOVA analysis revealed that a major genetic variation observed within populations, was 98.48%. The phylogenetic trees of haplotypes based on Cyt b gene showed that all the haplotypes of seven populations were formed a single cluster that could be divided into three subgroups. There was no significant structural pattern, had no obvious geography cluster or host cluster found in all populations. The results indicated that Cyt b gene was suitable marker for studying genetic population structure of T. hydatigena, and that the diversity of T. hydatigena from Sichuan and Yunnan province was high, but the genetic differentiation among populations was not obvious.5. Population genetic structure analysis of T. hydatigena from Sichuan and Yunan provinces based on mitochondrial nadl gene and coxl geneThe partial sequences of mitochondrial nadl gene (pnad1) and the complete sequences of the cox1 gene of 24 C.tenuicollis samples isolated from five different regions in Sichuan and Yunnan province were analyzed to reveal the population genetic structure and phylogenetic relationship between these isolates and other Taenia cestodes studied. The results showed that the partial sequences of nad1 gene and complete sequences of coxl gene of C. tenuicollis were 780 bp and 16 20 bp, respectively. There were 43 and 86 variable sites and 19 and 6 haplotypes were detected in pnad1 and cox1, respectively.The global haplotype diversities were 0.978±0.019 for pnadl and 0.995±0.016 for coxl, and the nucleotide diversities were 0.008640.00223 for pnadl and 0.00685±0.00149 for coxl. Interestingly, the average genetic distances were both 0.007. The diversities based on the two genes were both low, but the diversity based on pnadl was higher than coxl gene. The major genetic variations occurred within populations in AMOVA analysis based on the two genes, but the genetic differentiation among populations was not obvious. The phylogenetic trees showed that all the C. tenuicollis isolates formed a single cluster that could be further divided into three subgroups, and there was no obvious geography cluster or host cluster. Taken together, the gene nad1 was more suitable to study population genetic structure of T. hydatigena.6. Studies on population genetic structure of T. hydatigena from Sichuan, Yunan and Qinghai provinces based on mitochondrial nad4 geneIn order to explore the relationship among different populations of T. hydatigena, the partial sequences of mitochondrial nad4 gene (pnad4) of 38 C.tenuicollis samples isolated from eight different cities in Sichuan, Yunnan and Qinghai provinces were analyzed to reveal the population genetic structure. As for the 801 bp pnad4 sequences of 38 individuals, there was no indels,31 haplotypes and 63 polymorphic sites (7.87%) were detected, including 38 parsimony informative sites. The sequence divergence between haplotypes was 0.1%～4.0%, and the global haplotype diversity, nucleotide diversity and average genetic distance were 0.989±0.009,0.01333±0.00236 and 0.014, respectively. It showed a high diversity in T. hydatigena. The results of population dynamic analysis supported that T. hydatigena have undergone population expansion in the past. The results of the AMOVA showed that the percentage of variation within populations (55.29%) was more than that among populations (44.71%), suggesting that a major genetic variation occurred within populations. The Fst value was 0.44709, indicating that the genetic differentiation among populations was significant. The phylogenetic tree of haplotypes based on nad4 gene showed that all the 31 haplotypes were clearly divided into two different branchs (Ⅰ and Ⅱ), and some individuals from different populations clustered together across Ⅰ and Ⅱ branchs, and the populations did not form significant structural pattern. The results showed that there was no obvious population structure of T. hydatigena from Sichuan, Yunnan and Qinghai provinces.7. A PCR method established for detecting T. multiceps egg DNATo establish a suitable PCR method for identifying T. multiceps egg DNA and providing a tool for epidemiological survey of T, multiceps infection.5 experimental dogs were separately infected with one fresh cyst of C.cerebralis by oral. After 45 day, each infected dog discharged of gravid proglottides, and each dog was then anatomical examination and collected the adult tapeworms and dog feces. DNA was directly extracted from infected dog feces, subsequently a mitochondrial nad5 gene fragments were amplified by PCR for developing a PCR diagnostic method for T. multiceps infection. Its specificity, sensitivity, and manifestation in field application were estimated. All amplified fragments were analyzed by sequencing. The fragments were 433 bp, with 99.5% similarity with two nad5 gene fragments of T. multiceps in GenBank(nos:GQ228818 and FJ495086). The PCR positive rate was 100% of the 5 adult tapeworms, while T. hydatigena and T. pisiformis were not detected during this procedure. The PCR was capable of detecting 10 eggs of T. multiceps. In the field application, five of six taeniid egg-positive feces were positive by PCR diagnostic method. This PCR method should have wide application value. The results indicated that the PCR diagnostic method to T. multicep developed in this study was successfully established and it showed a good specificity and sensitivity.