Strains of Uronema marinum (Scuticociliatia)  co-isolated with amoebae of the genus Neoparamoeba

Abstract

Mixed infections of histophagous ciliates and Neoparamoeba spp. Page, 1987 were diagnosed in gill tissue of farmed turbot Psetta maxima (synonym: Scophthalmus maximus) and Atlantic salmon Salmo salar during a study of amoebic gill disease. Ciliates co-isolated from lesions grossly visible on gills of both fish hosts and from 2 species of red algae Lithophyllum racemus and Palmaria palmata were characterized morphologically and by using molecular markers. Sequences of small subunit (SSU) rDNA were determined for 6 strains of ciliates isolated from hosts collected in geographically distant localities. Of these, sequences of 5 strains revealed a surprisingly high level of similarity and identified the corresponding strains with Uronema marinum Lynn et Small, 1997. Thus, the set of environmental sequences of U. marinum available in the GenBank database to date was supplemented with the first sequences of potentially histophagous strains. On the basis of SSU rDNA, the 6th strain, also isolated from affected fish gills, was identified as Aristerostoma sp. KEY WORDS: Uronema marinum · SSU rDNA · Histophagous strains Resale or republication not permitted without written consent of the publisherDis Aquat Org 89: [71][72][73][74][75][76][77] 2010 characterization (Dyková et al. 2007. Ciliates included in the present study are listed in Table 1. This set of strains contains only those co-isolated with Neoparamoeba strains from a same sample (gill tissue or organic material covering the surface of red algae). Non-nutrient 75% seawater agar (BD Bacto™ Agar) was routinely used for primary isolations. To establish the ciliates in culture, a clean population of each isolate of ciliates was selected with the aid of a light microscope and transferred (together with piece of agar) to MY75S (Malt &Yeast Extract -75% seawater) medium (catalogue of the UK National Culture Collection). The same medium was used for the cloning procedures carried out in cell wells at 20°C. For subculturing of ciliates, initially MY75S medium supplemented with autoclaved pieces of freshwater fish gill tissue was used. Later, the ciliates were fed fathead minnow Pimephales promelas (FHM) cell line cells. Culture medium used for the FHM cell line was removed from each culture flask and substituted with MY75S medium containing ciliates of the parent culture. Ciliates were subcultured weekly. Both methods of subculturing were used alternately with varying success.Morphological characterization of ciliates. Gross morphological features of living ciliates were observed with light microscopy by means of Nomarski differential interference contrast. Details of ciliature, not visible in living specimens, were studied in stained preparations and with scanning electron microscopy (SEM). For purposes of SEM, ciliates were fixed in 3% cacodylate buffered glutaraldehyde, washed in 0.1 M cacodylate buffer and postfixed with 1% osmium tetroxide. Samples were dehydrated in a graded acetone series, critical-point dried with liquid CO 2 , sputter-coated with g...

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