Estimating Protistan Diversity Using High-Throughput Sequencing.

Sequencing hypervariable regions from the 18S rRNA gene is commonly employed to characterize protistan biodiversity, yet there are concerns that short reads do not provide the same taxonomic resolution as full-length sequences. A total of 7,432 full-length sequences were used to perform an in silico analysis of how sequences of various lengths and target regions impact downstream ecological interpretations. Sequences that were longer than 400 nucleotides and included the V4 hypervariable region generated results similar to those derived from full-length 18S rRNA gene sequences. Present high-throughput sequencing capabilities are approaching protistan diversity estimation comparable to whole gene sequences.

Investigating microbial eukaryotic diversity from a global census: insights from a comparison of pyrotag and full-length sequences of 18S rRNA genes.

Next-generation DNA sequencing (NGS) approaches are rapidly surpassing Sanger sequencing for characterizing the diversity of natural microbial communities. Despite this rapid transition, few comparisons exist between Sanger sequences and the generally much shorter reads of NGS. Operational taxonomic units (OTUs) derived from full-length (Sanger sequencing) and pyrotag (454 sequencing of the V9 hypervariable region) sequences of 18S rRNA genes from 10 global samples were analyzed in order to compare the resulting protistan community structures and species richness. Pyrotag OTUs called at 98% sequence similarity yielded numbers of OTUs that were similar overall to those for full-length sequences when the latter were called at 97% similarity. Singleton OTUs strongly influenced estimates of species richness but not the higher-level taxonomic composition of the community. The pyrotag and full-length sequence data sets had slightly different taxonomic compositions of rhizarians, stramenopiles, cryptophytes, and haptophytes, but the two data sets had similarly high compositions of alveolates. Pyrotag-based OTUs were often derived from sequences that mapped to multiple full-length OTUs at 100% similarity. Thus, pyrotags sequenced from a single hypervariable region might not be appropriate for establishing protistan species-level OTUs. However, nonmetric multidimensional scaling plots constructed with the two data sets yielded similar clusters, indicating that beta diversity analysis results were similar for the Sanger and NGS sequences. Short pyrotag sequences can provide holistic assessments of protistan communities, although care must be taken in interpreting the results. The longer reads (>500 bp) that are now becoming available through NGS should provide powerful tools for assessing the diversity of microbial eukaryotic assemblages.

Photoresponse in the heterotrophic marine dinoflagellate Oxyrrhis marina.

Expressed rhodopsins were detected by proteomic analysis in an investigation of potential signal receptors in the cell membrane of the marine heterotrophic dinoflagellate Oxyrrhis marina (CCMP604). We inferred these to be sensory rhodopsins, a type of G-protein-coupled receptor trans-membrane signaling molecule. Because phototactic behavior based on sensory rhodopsins has been reported in other protists, we investigated the photosensory response of O. marina. This dinoflagellate exhibited strongest positive phototaxis at low levels (2-3 µE/m(2)/s) of white light when the cells were previously light adapted and well fed. Positive phototaxis was also found for blue (450 nm), green (525 nm), and red (680 nm) wavelengths. In a further test, O. marina showed significantly greater phototaxis toward concentrated algal food illuminated by blue light to stimulate red chlorophyll-a autofluorescence in the prey, compared with using bleached algae as prey. Concentration of a cytoplasmic downstream messenger molecule, cyclic adenosine monophosphate, a component of the signaling pathway of G-protein-coupled receptor molecules, rapidly increased in O. marina cells after exposure to white light. In addition, treatment with hydroxylamine, a rhodopsin signaling inhibitor, significantly decreased their phototactic response. Our results demonstrate that a heterotrophic marine dinoflagellate can orient to light based on rhodopsins present in the outer cell membrane and may be able to use photosensory response to detect algal prey based on chlorophyll autofluorescence.

Using inhibitors to investigate the involvement of cell signaling in predation by marine phagotrophic protists.

Phagotrophic protists are major consumers of microbial biomass in aquatic ecosystems. However, biochemical mechanisms underlying prey recognition and phagocytosis by protists are not well understood. We investigated the potential roles of cell signaling mechanisms in chemosensory response to prey, and in capture of prey cells, by a marine ciliate (Uronema sp.) and a heterotrophic dinoflagellate (Oxyrrhis marina). Inhibition of protein kinase signal transduction biomolecules caused a decrease in both chemosensory response and predation. Inhibition of G-protein coupled receptor signaling pathways significantly decreased chemosensory response but had no effect on prey ingestion. Inhibitor compounds did not appear to affect general cell health, but had a targeted effect. These results support the idea that cell signaling pathways known in other eukaryotic organisms are involved in feeding behavior of free-living protists.

Significance of predation by protists in aquatic microbial food webs.

Predation in aquatic microbial food webs is dominated by phagotrophic protists, yet these microorganisms are still understudied compared to bacteria and phytoplankton. In pelagic ecosystems, predaceous protists are ubiquitous, range in size from 2 gm flagellates to > 100 microm ciliates and dinoflagellates, and exhibit a wide array of feeding strategies. Their trophic states run the gamut from strictly phagotrophic, to mixotrophic: partly autotrophic and partly phagotrophic, to primarily autotrophic but capable of phagotrophy. Protists are a major source of mortality for both heterotrophic and autotrophic bacteria. They compete with herbivorous meso- and macro-zooplankton for all size classes of phytoplankton. Protist grazing may affect the rate of organic sinking flux from the euphotic zone. Protist excretions are an important source of remineralized nutrients, and of colloidal and dissolved trace metals such as iron, in aquatic systems. Work on predation by protists is being facilitated by methodological advances, e.g., molecular genetic analysis of protistan diversity and application of flow cytometry to study population growth and feeding rates. Examples of new research areas are studies of impact of protistan predation on the community structure of prey assemblages and of chemical communication between predator and prey in microbial food webs.