New insights on the ecology of free-living heterotrophic nanoflagellates based on the use of molecular biological approaches

Abstract

Nanoplanktonic protists comprise a diverse assemblage of flagellate species responsible for various trophic processes in marine and freshwater ecosystems. Current methods for identifying small protists do not readily permit identification and enumeration of nanoplanktonic flagellates in cultures or field samples. The aim of this study was to apply molecular biological techniques for identification and enumeration of nanoflagellate taxa in water samples. Restriction fragment length polymorphism (RFLP) analysis of small subunit ribosomal DNA (SSU rONA) amplified by the polymerase chain reaction (PCR) differentiated pure cultures of heterotrophic nanoflagellates according to established taxonomic classification at the generic and species level. In addition, RFLP analysis of amplified SSU rONA permitted discrimination of polymorphic forms among species of flagellates from the genus Paraphysomonas. A fluorescent in situ hybridization method that uses rRNA-targeted oligonucleotide probes for counting protists from cultures and environmental water samples was developed. Preserved cells were intensely labeled with multiple eukaryote-specific oligonucleotide probes end-labeled with biotin and detected by fluorescein (FITC)-avidin. This probe hybridization method gave estimates of nanoplankton abundances that were often more representative of natural abundances than estimates obtained by commonly employed fluorochrome stains. The geographical distribution and seasonal abundance of the cosmopolitan heterotrophic flagellate, Paraphysomonas imperforata was examined in coastal waters using species-specific oligonucleotide probes. P. imperforata was found to occur at extremely low abundances in coastal environments, constituting ≤1% of the TNAN. However, P. imperforata often dominated the nanoplankton (up to 98% of TNAN) when water samples were enriched with bacteria. P. imperforata appears to be an opportunistic species capable of growing rapidly to high abundances when prey are abundant. Water temperature, small differences in the absolute abundance of P. imperforata, and nanoplankton grazers appeared to play a role in determining P. imperforata dominance. Results from this study also suggest that enrichment cultivation or perhaps incubations in general can select for nanoflagellates such as P. imperforata that may not be representative of abundant oceanic species.