1. Detection of Protistan Genomes in the Environment Rebecca J. Gast Woods Hole Oceanographic Institution

2. Identification…. Detection ….. Enumeration……. ………Of marine protists

3. Identification of cells Morphology -Most useful for larger protists -Motility of cells also diagnostic Sequences -Small subunit ribosomal RNA gene -Phylogenetically/taxonomically informative

4. Detection of organisms Indirect - detection without visual confirmation of cell -Sequences -Proteins -Lipids Direct -Microscopy -In situ probe hybridization

5. Oligonucleotide probes Different levels of specificity - Extensive sequence databases are an important resource for designing probes. -Kingdom (archea, bacteria, eukaryote) -Group (methanotroph, prymnesiophyte) -Individual organism (E. coli, Paraphysomonas) Hybridization -Southern blots, dot blots -Arrays -Whole cell - In situ (whole cell) hybridization is often considered the “gold standard” because it provides visual confirmation of a cell.

6. Reverse Dot Blot Hybridization (aka arrays) Detection and identification of acanthamoeba ribotypes in environmental samples –Ribotypes recovered by enrichment culture are potentially not representative of the natural sample –New ribotypes can be detected with this method

7. Fluorescence in situ hybridization Litaker et al., 2002. Journal of Phycology, 38 (3):442-463.

8. Still difficult to address Abundance -Quantitative analyses require the ability to retain and recover cells. Monitoring over scales relevant to the microbial system -Spatial - there may be significant changes over very small distances -Temporal - community changes may occur rapidly in response to physical or chemical perturbation.

9. New Technology Automated water samplers New probe types -Peptide nucleic acid (PNA) New instrumentation -ESP -In situ flow cytometers

10. What is a PNA? Behave like nucleic acid, but lack the negatively charged backbone. PNA probes are desirable for in situ hybridization shorter probes can be used (increased % mismatch) lower salt concentration (increased specificity) better cell penetration

11. Flow cytometric detection of hybridized cells

12. In situ hybridization chamber Increase spatial sampling capability Couple to flow cytometer Quantitative

13. Collaborators and support Robert Olson Heidi Sosik Mark Dennett Alexi Shalapyonok Ken Doherty WHOI Green Technology Award NSF Biocomplexity IDEA