PCR AMPLIFICATION, SEQUENCING AND ANALYSIS OF 18S rDNA OF|BALANTIDIUM CTENOPHARYNGODONI INHABITING GRASS CARP

Balantidium ctenopharyngodoni, an obligatory intestinal symbiont and possibly opportunistic parasite ofgrass carp (Ctenopharyngodon idella), was first discovered and named by Chen (1955). It mainly inhabits the cream-likeluminal contents and spaces between mucosal folds of the hindgut. In the present study, twenty two or three-summergrass carp were bought from Wuhan Fruit-Lake Market in March 2009 and transported alive to the laboratory for furtherexamination. All fish samples were dissected and the intestines were cut to collect the luminal contents into Petri dishesfor examination. Then 0.65% saline solution was added to the contents and waited for a few minutes to allow B. ctenopharyngodoniswim free of luminal contents. The ciliates were collected with Pasteur micropipette and washed twice indistilled water. The parasite gDNA was isolated by proteinase K digestion overnight at 37℃, followed by phenol-chloroform protein extraction and ethanol precipitation. Its 18S rDNA was then sequenced, which contains 1638base pairs. Phylogenetic trees were also constructed by the neighbour joining method, the maximum parsimony methodand the Bayesian method, respectively. The results indicated that the subclass Trichostomatia was monophyletic andcomposed of the order Entodiniomorphida, Vestibuliferida and Australian clade (100% Bay; 100% MP; 100% NJ). Theoder Entodiniomorphida (100% Bay; 98% MP; 93% NJ) and Australian clade (100% Bay; 97% MP; 99% NJ) showedtheir monophyletic while the order Vestibuliferida showed its paraphyly. As to the phylogenetic position, B. ctenopharyngodonifirst clustered with the Australian clade with higher bootstrap values (100% Bay; 100% MP; 100% NJ).Then they formed a sister clade to “Entodiniomorphida+ Vestibuliferida” (100% Bay; 85% MP; 72% NJ). B. coli, as anisolated taxon, was joined together with “Australian clade + B. ctenopharyngodoni” and “Entodiniomorphida + Vestibuliferida(part)” to form the subclass Trichostomatia (100% Bay; 100% MP; 100% NJ). It also suggested that the genusBalantidium should be recognized as a higher taxon.