Isolation, identification and in vitro culture of three species scuticociliatid ciliates from different hosts
GAO Yanqi, WANG Sen, LIU Juan, DANG Huifeng, LI Ruijun*
College of Fisheries and Life Science, Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea, Ministry of Agriculture and Rural Affairs，Dalian Key Laboratory of Marine Animal Disease Control and Prevention, Dalian Ocean University, Dalian 116023, China
Abstract: In order to further identify and culture the scuticociliatid ciliates causing the body surface ulceration of representatively farming marine fish and echinoderms, scuticociliatid ciliates were isolated from body surface of the scuticociliatosis sea cucumber Apostichopusjaponicus, tiger puffer Takifugurubripes and turbot Scophthalmusmaximus from October 2016 to May 2017 in Dalian, Liaoning Province. The morphological characteristics of the three scuticociliatid ciliates species were investigated by in vivo observation, formaldehyde fixation samples, scanning electron microscopy observation, acetic acid magenta staining and methyl green petuline staining. The 18S rDNA gene of the three scuticociliatid ciliates was sequenced by PCR, compared with homologous sequence and analyzed by phylogenetic tree. The three scuticociliatid ciliates were cultured in L-15 medium, MEM medium or RPMI1640 medium for 7 days at 20 ℃ under the same conditions to screen the optimal culture medium by comparing the population density. Then the population densities of the three scuticociliatid ciliates were comparatively cultured in the L-15 medium under different temperatures(10, 15, 20, and 30 ℃), salinities (13, 20, 30, and 40)and pH values (4.2, 5.2, 7.2, and 9.2) for 7 days to probe into the effects of environmental factors on the population densities of the three scuticociliatid ciliates. The results showed that the ciliates isolated from sea cucumber and tiger puffer were similar to Uronema in morphology and 18S rDNA sequence, with as high as 99% of consistency and the maximal confidence value of 100% clustered into the phylogenetic tree. The scutellaria isolated from turbot were very similar to Pseudomonas in morphology, and as high as 99% of the consistency in 18S rDNA sequence with Pseudomonas, and the maximal confidence value of 100% in the phylogenetic tree. The best culture of the three scuticociliatid ciliates was observed under the conditions of L-15 medium, temperature of 20 ℃, salinity of 30 and pH 7.2. The findings provide the references for isolation and identification of scuticociliatid ciliates from the skin ulceration of marine animals and the culture conditions in vitro for development of the whole parasite vaccine.
高延奇, 王森, 刘娟, 党慧凤, 黎睿君. 3种宿主源盾纤毛虫的分离鉴定及体外培养研究[J]. 大连海洋大学学报, 2021, 36(2): 260-267.
GAO Yanqi, WANG Sen, LIU Juan, DANG Huifeng, LI Ruijun. Isolation, identification and in vitro culture of three species scuticociliatid ciliates from different hosts. Journal of Dalian Ocean University, 2021, 36(2): 260-267.
2021, Vol. 36 
