Collaborative Research: Combining rRNA Probes and Cell Cycle Analysis to Investigate In Situ Growth Rates of Eucaryotic Phytoplankton

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The goal of the project is to assess both species diversity and the distribution of growth rates among eukaryotic phytoplankton populations under natural conditions. Relatively little is known about phytoplankton species composition in any particular sample of seawater, let alone the growth rates of those species. The wide range of growth rates and responses to environmental conditions observed for phytoplankton has obvious implications for species succession and the efficiency of the biological puinp. The investigators will focus on eukaryotic phytoplankton because these cells are responsible for the bulk of production in many regions, including most marine phytoplankton blooms. They will employ a three-tiered approach that combines: 1) high-throughput DNA sequencing and fragment analyses to allow us to determine species diversity and to design species-specific rRNA-targeted probes for ecologically relevant organisms; 2) a newly emerging class of molecular phylogenetic probes known as peptide nucleic acids (PNAs), which are highly sensitive and relatively easy to design and use; and 3) flow cytometric cell cycle analyses of PNA-labeled cells to determine intrinsic growth rates of targeted species.Since the division cycles of most phytoplankton are phased to the daily light:dark cycle, the intrinsic growth rate of a population can be determined by monitoring DNA cell cycle distributions over a diel period. If the flow cytometric signature (pigment fluorescence/light scatter characteristics) of the organism in question is sufficiently distinctive (as with the prokaryotic picoplankter Prochlorococcus), DNA distributions (measured with a fluorescent stain) in natural samples can provide growth rates at the species level. Direct flow cytometric analyses of eukaryotic phytoplankton cell cycling are not practical because multiple species in a given sample typically have similar morphology and flow cytometric signatures, but different amounts of DNA or cell division timing. Therefore, the investigators will selectively analyze cells of particular species by using fluorescentlylabelled PNA rRNA probes. Target species will be chosen from among the phytoplankton present at diel sampling sites in coastal and open ocean waters. They will extract DNA from phytoplankton purified either by flow cytometric cell sorting or size fractionation and amplify eukaryotic rDNA using PCR. A combination of terminal restriction fraction length polymorphism (T-RFLP) analysis and the sequencing of 18S clone libraries generated from selected sites will be used to select species of interest for further study. Specific rRNA probes to selected species will then be designed. The invetigators will use fluorescently-labeled PNA probes, in conjunction with DNA staining and dual-beam flow cytometry, to obtain cell cycle (and thus growth rate information for target species). In addition to speciesTm growth rates, these analyses will provide new perspectives on the distribution and diversity of eukaryotic phytoplankton in the sea and will improve our capabilities to address a variety of oceanographic questions on ecologically relevant scales.