The Pacific Northwest coast is strongly influenced by the Columbia River, which is the second largest river in the U.S. The Columbia River estuary is typically classified as a detritus-driven system with much of the microbial activity being heterotrophic and associated with suspended particles derived from freshwater phytoplankton. However major heterotrophic protists in the estuary have not been identified and characterized. Molecular analysis of small subunit (SSU) rRNA gene clone libraries suggests that during April the heterotrophic flagellate Katablepharis CRE is the dominant heterotrophic protist in the Columbia River estuary. Further genetic analysis of the rRNA genes of Katablepharis CRE indicates that the long subunit (LSU) contains a 332 base pair sequence element that appears unique to Katablepharis CRE. This element is GC-rich compared to the rest of the gene (59% to 45%) and shows no significant similarity to other eukaryotic sequences in LSU sequence databases. The objective of this research was to better resolve the presence of this unique sequence element, to uncover its origin, and to assess seasonal and inter-annual trends in Katablepharis CRE distribution and abundance in the estuary through real-time PCR (RT-PCR) and fluorescent in-situ hybridization (FISH) with specific probes developed from the unique LSU element.
Results/Conclusions
To assess the presence of this unique sequence element in Katablepharis CRE and possibly other eukaryotes, PCR was performed on Columbia River estuary samples and samples from other systems (Amazon River plume, Alaskan coast, Chesapeake Bay) with a combination of primers specific for this element and general eukaryotic primers. Sequences obtained from Columbia River samples were all related to Katablepharis CRE and showed little variability, while there was no amplification from other systems, suggesting that this element might only be found in the strain of Katablepharis in the Columbia River estuary. RT-PCR and FISH were performed on estuarine samples collected throughout the year from 2007-2012. This data confirms trends observed in the SSU clone libraries, with Katablepharis CRE reaching its highest abundance during the spring and maintaining a relatively small population throughout the rest of the year. Considering its recurring population pattern and potentially high abundance, Katablepharis CRE could play an important and previously uncharacterized role in the fate of spring phytoplankton organic matter in the Columbia River system.