Analysis of nanoplankton community structure using flow sorting and molecular techniques.

We developed a method for the separate and simultaneous analysis of the community structure of heterotrophic nanopkankton (HNP) and autotrophic nanoplankton (ANP). This method consists of three steps. First, nanoplankton cells were concentrated using a cross-flow filtration system because cell densities in natural seawater are usually too low for genetic studies. Second, HNP and ANP were separated by flow cytometric sorting ("flow sorting") on the basis of the presence or absence of chlorophyll. Finally, the community structure was analyzed using denaturing gradient gel electrophoresis targeting 18S rRNA gene. The newly developed method was applied to the coastal surface water of Aburatsubo Inlet, Japan, in July 2008. The separation of nanoplankton into HNP and ANP was validated by phylogenetic analysis, and the trophic mode of uncultured nanoplankton was confirmed (e.g. Marine Alveolata group II [MALV II] and Marine Stramenopile clade-2 [MAST-2]). This new method involving cell concentration, flow sorting and phylogenetic analysis is a potentially powerful tool for evaluating the population dynamics and ecology of marine protozoa.