Taxonomic And Phylogenetic Study Of Fish Trypanosomes

The genus Trypanosoma of flagellated parasitic protozoa is found worldwide in all classes of vertebrates. In natural condition, both marine and freshwater fish commonly carry trypanosomes, which are transmitted by bloodsucking aquatic leeches. Although ubiquitous, the taxonomy and phylogenetic relationships of fish trypanosomes are uncertain. The present dissertation chose Chinese fish trypanosomes as main research objection. The use of SSU rRNA gene sequences in combination with current morphological data greatly promotes the correct identification of Chinese fish trypanosomes and study the taxonomy and phylogenetic relationship within fish trypanosomes. In addition, in order to gain purified trypanosomes, the methods of isolation of fish trypanosomes were discussed in this paper. The main results and conclusions are summarized as follows:1. The blood of Monopterus albus with trypanosomes was treated with 51% Percoll reagent and centrifugation at 2.9×10~3 rpm and 15 min is best for removing the most blood cells and recovery of trypanosome. To gain purified trypanosomes, the collected suspension contained trypanosomes were through DEAE-cellulose column. This technique is sensitive and has a potential diagnostic value and may provide reliable data for the study of epidemiology of Trypanosoma monopteri.2. It was first time that the recombinant aerolysin protein was used to isolate fish trypanosomes from blood. Purified trypanosomes were alive for 3-4 days in PGSS buffer at 4℃, which was equivalent with untreated trypanosomes. The result indicated recombinant aerolysin protein was harmless to trypanosomes. 3. The trypanosomes from the blood of Ophiocephalus argus and Odontobutis obscura were used for light microscopy investigations and studied morphology using 13 morphometric characterizations. By compared with related species, these trypanosomes were tentatively identified as Trypanosoma ophlcephali Chen, 1964. Infection with trypanosomes of Odontobutis obscura was recorded for the first time.4. The trypanosomes from the blood of Siniperca chuatsi from Niushan Lake were used for light microscopy investigations. These trypanosomes had great similarity in morphology with Trypanosoma siniperca described by the former researcher and tentatively identified as T. siniperca. At the same time, T. siniperca was redescribed using 13 morphometric characterizations and compared with related species. In addition, SSU rDNA sequences were compared between T. siniperca and related species, and the position of T. siniperca was shown in the phylogenetic analysis.5. Generally, the trypanosomes from the blood of Pseudobagras fulvidraco and Cyprinus carpio were all considered to be Trypanosoma pseudobagri. Morphological analyses indicated the trypanosomes from Niushan Lake were three forms: one from Cyprinus carpio was T. sp Carpio and two from P. fulvidraco were T. sp Pseudobagri and T. sp, respectively. Following the current standard, the size and morphometric characteristics of these trypanosomes show significant differences among the 3 forms. T. sp Carpio and T. siniperca had great similarity in not only morphological characteristics but 18S rDNA sequence. Moreover, in phylogenetic analysis they clustered together and formed sister species. Therefore, T. sp Carpio and T. siniperca probably are the same species. T. sp Pseudobagri was first observed. Due to high sequence similarity (99.8%), T. sp Pseudobagri and Marv might be the same genotypes. The position of T. sp was placed at the bottom of the freshwater fish trypanosome group and became a single isolate. The 18S rRNA gene sequence of T. sp differs from those of freshwater fish trypanosomes by 3.5-5.8%. T. sp might be tentatively identified as T. pseudobagri. Results mentioned above show trypanosomes were not strictly specific to the hosts from which they were recovered. 6. The fine structure of T. pseudobagri was observed with transmission electron microscopy. The pellicle of trypanosome was a unit membrane with characteristic 3-layered structure, which was covered by a well-developed glycoprotein surface coat. The structure and attachment of the flagellum and the subpellicular microtubules show the usual structural and organizational features. Kinetoplast, mitochondria, nucleus and vacuoles, which occur in trypanosomidae, were observed in electron micrographs. The significance of similarities and differences of these fine structures is uncertain in terms of the evolutionary relationship of trypanosomes.7. Phylogenetic analysis was conducted by using 18S rRNA gene sequences, which contained 7 sequences of fish trypanosomes from China. The results indicated that all fish trypanosomes fell in a single clade, which freshwater fish trypanosomes was separate from trypanosomes of marine fish and formed sister group. Trypanosomes from China were nested within the fish clade and further subdivided into at least 2 subgroups.