Growth of uncultured heterotrophic flagellates in unamended seawater incubations

Unamended dark incubations of 3 mu m-filtered seawater were prepared with surface samples from the Norwegian Sea, the NW Mediterranean and the Indian Ocean. Except for the very oligotrophic Indian Ocean samples, this simple treatment promoted the growth of heterotrophic flagellates, with rates (0.3 to 1.2 d(-1)) typical of natural assemblages. Marine stramenopile (MAST)-1 and MAST-4 cell counts, obtained by fluorescence in SitU hybridization (FISH), increased in most incubations. These yet uncultured protists were first detected in molecular surveys of marine picoplankton and have been recently shown to be globally distributed heterotrophic flagellates. Three incubations from the Norwegian Sea were studied in more detail by denaturing gradient gel electrophoresis (DGGE), 18S rRNA gene clone libraries and FISH counts for 5 MAST groups. Protist diversity changed gradually during the incubation, but the DGGE bands selected were already present at the beginning of the incubation. Clone libraries from the peaks in abundance of heterotrophic flagellates were dominated by MAST sequences. FISH counts revealed MAST-1B to be a very successful organism in the 3 incubations, accounting for 15 to 30%, of heterotrophic flagellates after 6 to 8 d; MAST-1A and -1C cells were also abundant. MAST-4 cells peaked before the other groups and MAST-2 was the least represented. We concluded that unamended seawater incubations can select for heterotrophic flagellates abundant in situ but not yet isolated in pure culture. Therefore, they allow investigation of the growth requirements and dynamics of these uncultured protists, and provide promising preliminary stages for their isolation.