Phylogenetic Analysis Of Mitochondrial RrnL And Nad5Gene And Expression Profile Of MicroRNAs Of Taenia Pisiformis

Taenia pisiformis is a taeniid tapeworm, which resides in small intestine of dogs, wolves and foxes. Cysticercus pisiformis is the larva of T. pisiformis, which ordinarily resides in the liver, mesentery and greater omentum of intermediate hosts including rabbits, hares and some rodents. Infection may occur when the definite hosts, such as canines, are fed with infected intermediates. About one month after infection,C. pisiformis will develop to the adult (T. pisiformis). T. pisiformis distributes globally and causes significant social-economic losses in rabbits and dog breeding, especially in developing countries which normally pay no attention to the detection and treatment of cysticercus and Taenia carriers.The present studies involved two parts, the first part of the thesis was to examine sequence variation in the mitochondrial large subunit ribosomal rRNA(rrnL) and NADH dehydrogenase subunit5(nad5) gene among T. pisiformis isolates from Gansu and Henan provinces and to study its phylogenetic relationships with other cestodes using rrnLand nad5sequences. The partial rrnL (prrnL) and nad5(pnad5) gene sequences were amplified from each T. pisiformis sample (C. pisiformis) by polymerase chain reaction (PCR), and prrnL and pnad5sequences were aligned using the ClustalX1.81. Maximum parsimony (MP) tree was constructed using the software PAUP4.0Beta10. The lengths of prrnL and pnad5sequences were847-848bp and602bp, respectively. Sequence variation in rrnL among the examined T. pisiformis samples were0.12-0.35%, and0-0.5%for nad5, whereas inter-specific differences among taeniid cestodes were significantly higher than16.6%for rrnL, and higher than14.3%for nad5. Phylogenetic analysis showed that all the T. pisiformis isolates clustered in the same clade. It is concluded that prrnL and pnad5 sequences can be used as genetic markers for the identification and differentiation of taeniid cestodes.The second part of the thesis was to characterize and compare the global miRNA profiles of larvae and adults of T. pisiformis using next-generation sequencing technology and real-time quantitative PCR. A total of5and7miRNA candidates were identified from12.29and10.58million raw sequencing reads in adults and larvae, respectively, with5of them known in S. japonicum and/or S. mansoni. Among which, some miRNAs derived from genes located at different scaffolds of the reference genome, such as miR-466i in cysticercus, which located in two different scaffolds named as SJC_S000096and SJC_S019419. Five miRNA candidates were found to express in the both stages, and two others were found in larvae only. No specific miRNA candidate was found in adults. Targets of the five common and the two specific miRNAs were successfully predicated and analyzed from the17401mRNA and EST non-redundant sequences of the closely related species, S. japonicum. To our knowledge, this is the first report of miRNAs in larval and adult T. pisiformis.In conclusion, after cloning and phylogenetic analysis of mitochondrial rrnL and nad5gene, we proved that rrnL and nad5sequences can be used as genetic markers for the identification and differentiation of taeniid cestodes. We investigated and compared the miRNA profiles of larval and adult T. pisiformis by HiSeq deep sequencing approach and real-time quantitative PCR, which provided fundamental information and novel resources for the further understanding of the parasite and for the development of control strategies and agents against the disease it caused.