| Both Bunostomum phlebotomum and Bunostomum trigonocephalum, also known as hookworms, are blood-feeding nematodes which inhabit small intestines of cattle and sheep. Adult worms attach to the mucosa of the small intestine, suck blood, and may cause anemia, rapid weight loss and mandibular edema, as well as descent the performance trait and even death with heavy infection. Ancylostomiasis is endemic worldwide and causes serious hazard to animals, resulting in considerable economic losses to the live stock industries.Previous studies showed strict host specificity for hookworms, which only infect definite hosts. However, recent studies found that some Bunostomum species can infect a variety of hosts. B. trigonocephalum not only infect sheep and goats, but also can infect cattle. This is also the case for B. phlebotomum. Therefore, it is difficult to identify these two hookworm species due to their similar morphological characters. The first part of my study was to establish a molecular method to identify B. phlebotomum and B. trigoncephalum, and to study phylogenetic relationships of these two species based on sequences of the internal transcribed spacer (ITS) ribosomal DNA (rDNA). The universal primers were designed based on published rDNA sequences of Ancylostomatidae nematodes. The total genomic DNA samples of these two species were individually extracted. The ITS rDNAs of B. phlebotomum and B. trigoncephalum were amplified, and sequenced from both sides. After analyzing restriction endonuclease sites in ITS rDNA seqeunces, the Nde I was chosen to identify the two hookworms. The ITS rDNA PCR amplicons of B. trigoncephalum were digested into two bands, while the amplicons of B. phlebotomum remain undigested. The established PCR method can effectively identify B. phlebotomum and B. trigoncephalum. Phylogenetic analyses based on the ITS sequences revealed that B. trigonocephalum and B. phlebotomum were closely related, but they represent two different species.Due to maternal inheritance, high evolutionary rates and mutation rates, mitochondrial DNA (mtDNA) sequences provide useful markers for investigating population genetic structures, systematics and phylogenetics. The second part of my study was to examine genetic variations in four mitochondrial genes of B. trigoncephalum. A total of40samples were collected from four provinces (Heilongjiang, Yunnan, Jilin, Sichuan). Four partial mt genes, namely cytochrome c oxidase subunit1(pcoxl), NADH dehydrogenase subunit1(pnadl) and4(pnad4), and cytochrome b (pcytb) were amplified and sequenced. The intra-specific sequence variations of B. trigonocephalum were0-1.9%for pcoxl,0-1.6%for pnadl and0-1.7%for pnad5, and0-2.0%for pcytb. The nucleotide variation was related mainly to changes at the third codon position, while fewer changes were detected at the first or second codon position. The inter-specific variations among Ancylostoma caninum, Ancylostoma duodenale and Necator americanus were12.1-14.2%for pcoxl,17.6-19.4%for pnadl,16.0-21.6%for pnad5and13.7-16.0%for pcytb. These results demonstrated the existence of low-level intra-specific variation in mtDNA sequences among B. trigonocephalum isolates from different geographic regions.The mitochondrial (mt) genomics represents an understudied but important field of molecular biology. Although there are many nematodes, only68complete or nearly complete mt genomes of nematodes have been sequenced. There is a paucity of information on mt genome dataset. The third part of my study was to determine sequences and structures of mt genomes of B. trigoncephalum and B. phlebotomum. PCR method was used to obtain complete mt genome sequences of B. trigoncephalum and B. phlebotomum. The phylogenetic trees were also re-constructed based on the protein-coding gene sequences of mt genomes to study the phylogenetic relationships between the B. trigoncephalum and B. phlebotomum. The complete mt genome sequences of B. trigoncephalum and B. phlebotomum were13771bp and13803bp, respectively. Both including12protein genes,2rRNA genes and22tRNA genes, lacking of ATP8gene. For B. trigoncephalum, the lengths of protein-coding gene, rRNA gene and non-coding sequence were10299bp,1654bp and324bp, respectively. The nucleotide compositions of the mt genome included27.8%,48.6%,17.0%and6.5%of A, T, G, C, and A+T contents of76.5%, and G+C of23.5%, respectively. For B. phlebotomum, the lengths of protein-coding gene, rRNA and non-coding sequences were10290bp,1655bp, and346bp, respectively. The nucleotide compositions of the mt genome included26.8%of A,50.1%of T,16.9%of G, and6.2%of C, with A+T contents of76.9%, G+C of23.1%. The mt genomes of B. trigoncephalum and B. phlebotomum were similar to prefer bases A and T, with highest for T, but lowest for C, which was consistent with other ancylostomatidae nematodes. The mt genes arrangements of two hookworms were consistent with most Strongylata nematodes and belonged to the GA7type. Phylogenetic relationships were reconstructed using the concatenated amino acid sequences of12protein-coding by three methods, namely MP, BI and NJ. The topological structures of trees were similar with different methods. Nematodes in classes Adenophorea and Secementia were located in two distinct clades, with parasites in each order clustered together. Within one order, nematodes in one Family and genus were closely related. Within the cluster of family Ancylostomatidae, the genus Bunostomum posited in one clade, and the genera Necator and Ancylostoma grouped in one clade. The B. phlebotomum isolated from Chinese yaks and that isolated from Australian dairy cattle were close related, compared with phylogenetic relationships with A. duodenale, A. caninum and N.r americanus. These results were consistent with that based on ITS rDNA, suggesting that the complete mt genome sequence was a reliable genetic marker for phylogenetic studies.In conclusion, this study firstly established PCR-RFLP method to identify B. phlebotomum and B. trigoncephalum, which provided a reference for identification of other close-related or cryptic species. Based on partial mt DNA gene sequences, the genetic variations were examined among B. trigoncephalum isolated form different hosts and geographical origins. There were low intra-specific variations but significant inter-specific differences in these mt genes. The complete mt genome sequences were obtained for B. phlebotomum and B. trigoncephalum isolated from Chinese yaks. These findings enriched information of nematode mt genome dataset. Phylogenetic trees based on mt genome sequences clearly indicated genetic relationships of B. phlebotomum, B. trigoncephalum and other nematodes, suggesting that complete mt genome sequences will be another reliable genetic marker for molecular classification, genetic variation and phylogenetic studies. This thesis provides a foundation for studying the systematics, population gentics and ecology of other hookworms and parasites. |
PDF Full Text Request