Quantification and distinction of aplastidic and plastidic marine nanoplankton by fluorescence in situ hybridization

We developed a protocol for the microscopic detection of nanoplankton by fluorescence in situ hybridization (FISH) with 18S rDNA targeting oligonucleotide probes in combination with tyramide signal amplification (TSA). The use of tyramides labeled with a UV-excitable fluorochrome allowed simultaneous identification and classification of the hybridized organisms according to their trophic modes. The protocol was initially validated on pure cultures and was subsequently compared with a standard technique for the enumeration of protists in a time series from North Sea waters. Cell counts with the new protocol were significantly higher for both aplastidic and plastidic nanoplankton, mainly due to superior detection of cells between 2 and 5 µm. FISH-TSA with specific probes for Pedinellales and a group of novel stramenopiles revealed that these lineages of bacterivores were not abundant in coastal North Sea surface waters at the time of investigation. In combination with specific 18S rRNA targeted oligonucleotide probes the new protocol may provide a valuable tool for a simultaneous rapid analysis of the identity and trophic mode of nanoplankton in environmental samples