Phagotrophic Protists: undiscovered country Ev Barry Sherr with help from colleagues

1. Phagotrophic Protists: undiscovered countryEv & Barry Sherr (with help from colleagues)

3. Characteristics of marine phagotrophic protists Highly diverse � both phylogenetically and in terms of size Ubiquitous, biogeochemically significant as consumers of microbial biomass (bacteria and algae) and as remineralizers Complex behaviors, feeding strategies, growth patterns In situ distribution and activity still not well known

6. Themes of this talk The large: heterotrophic, athecate gymnodinoid dinoflagellates as major grazers of diatoms in the sea Linking genes and behavior: the biochemical basis of feeding behavior The small: pico-plankton sized heterotrophic flagellates: the smallest phagotrophs � a call for genomic analysis Systems biology: an approach to understanding complex biological systems (such as phagotrophic protists!)

10. Theme 1: The Large: Are athecate gymnodinoid dinoflagellates the dominant consumers of diatoms in the ocean? Armored (or thecate) heterotrophic dinoflagellates, e.g Protoperidium sp., are well known for feeding extracellularly on large sized and chain-forming diatoms

11. But, large, athecate (unarmored or naked) heterotrophic dinoflagellates in the Order Gymnodiniales � e.g. Gymnodinium and Gyrodinium spp., also can consume large diatoms and diatom chains, and are usually more abundant than are thecate heterotrophic dinoflagellates

18. Theme 2: Linking genes and behavior: genetic/ biochemical basis of feeding behavior Prior studies have shown selective grazing by phagotrophic protists based on: Prey size (e.g. for heterotrophic bacteria and a size range of phytoplankton prey) Prey species � some prey species are preferentially selected Chemical deterence, e.g. DMSP producing strains of Isochrysis are selected against compared to non-DMSP producing strains of Isochrysis (work by Gordon Wolfe and Suzanne Strom)

19. Protist feeding behavior is complex! One example: Martel, C. 2006. Prey location, recognition and ingestion by the phagotrophic marine dinoflagellate Oxyrrhis marina. J. Exp. Mar. Biol. Ecol. 335:210-220. Analysed feeding behavior of O. marina: Ingestion rates and chemosensory response to live/heat killed cells and culture filtrate for: Preferred algal food: Dunaliella primolecta �Distasteful� algal food: Isochrysis galbana

20. To measure chemosensory response I place a capillary tube containing an attractant into a microcentrifuge tube containing a suspension of protist cells. The attractant diffuses out and creates a gradient, and if the protists exhibit a chemosensory response they swim into the tube. The contents of the capillary are filtered down and put onto a microscope slide. Cell counts are completed using epifluorescence microsocopy. Protists are incubated with inhibitors prior to the start of the expirement.To measure chemosensory response I place a capillary tube containing an attractant into a microcentrifuge tube containing a suspension of protist cells. The attractant diffuses out and creates a gradient, and if the protists exhibit a chemosensory response they swim into the tube. The contents of the capillary are filtered down and put onto a microscope slide. Cell counts are completed using epifluorescence microsocopy. Protists are incubated with inhibitors prior to the start of the expirement.

24. What is the biochemical/genetic basis for selectivity of prey ingestion/chemosensory response by phagotrophic protists? That brings me to my research question: What is the biochemical basis for phagotrophy and chemosensory response in marine protists. This cartoon shows a protozoa and a bacteria. Bacteria are known to have various molecules (such as sugars and amino acids) hanging off their cell surface and eukaryotic cells are known to have membrane bound receptors which bind various signal molecules. I want to know how the interatctions between bacterial and proitstan cell surface molecules are involved in prey location, selection, and ingestion. Membrane bound receptors are a direct link to the cell�s internal machinery. Once a signal is received, a signal transduction cascade is initiated. This consists of a series of intracellular signals resulting in gene expression or some kind of physiological response resulting in a change of behavior. I am also investigating how these signal transduction pathways contribute to chemosensory response and phagocytosis of prey items.That brings me to my research question: What is the biochemical basis for phagotrophy and chemosensory response in marine protists. This cartoon shows a protozoa and a bacteria. Bacteria are known to have various molecules (such as sugars and amino acids) hanging off their cell surface and eukaryotic cells are known to have membrane bound receptors which bind various signal molecules. I want to know how the interatctions between bacterial and proitstan cell surface molecules are involved in prey location, selection, and ingestion. Membrane bound receptors are a direct link to the cell�s internal machinery. Once a signal is received, a signal transduction cascade is initiated. This consists of a series of intracellular signals resulting in gene expression or some kind of physiological response resulting in a change of behavior. I am also investigating how these signal transduction pathways contribute to chemosensory response and phagocytosis of prey items.

31. I used chemical inhibitors of g-proteins and protein kinases to evaluate the importance of signal transduction in chemosensory response and prey ingestion in protozoaI used chemical inhibitors of g-proteins and protein kinases to evaluate the importance of signal transduction in chemosensory response and prey ingestion in protozoa

32. Control test to make sure that there is not a general toxic effect of these inhibitors on protist cell health. Using flow cytometry I could then evaluate the cells for shifts in red and green fluorescence which would indicate PI and Annexin binding respectively. ****explainUsing flow cytometry I could then evaluate the cells for shifts in red and green fluorescence which would indicate PI and Annexin binding respectively. ****explain

33. Example results of cell health test : no apparent effect of inhibitors used so far Results from the programmed cell death suggest genistein and staurosporin have no affect on cell health after four hours at the concentrations used. The treatment represented by the black bar wasincubated with H2O2 to induce programmed cell death like symptoms. Control, inhibitors, DMSO. Results from the programmed cell death suggest genistein and staurosporin have no affect on cell health after four hours at the concentrations used. The treatment represented by the black bar wasincubated with H2O2 to induce programmed cell death like symptoms. Control, inhibitors, DMSO.

35. Tests of inhibitor effect on protist chemosensory response This graph shows an example of some of my data. On the X-axis is the chemosensory index. As the ciliates were incubated with higher concentrations of staurosporin the chemosensory response decreased.This graph shows an example of some of my data. On the X-axis is the chemosensory index. As the ciliates were incubated with higher concentrations of staurosporin the chemosensory response decreased.

37. Next step: exploring the genetic basis of protistan phagotrophy Can we find genes for biochemicals involved in phagotrophy in protist genomes? What is the minimum set of genes needed for a phagotrophic protist?

39. Theme 3: The small: what genes make a phagotroph? Genomic analysis of one or more of the smallest phagotrophic flagellates, i.e. do for these phagotrophs what Steve Giovannoni did with the genome of a small marine bacterium, his SAR-11 isolate This would provide a solid basis for screening the genomes other phagotrophic protists for genes similar to those found in the phagotrophic flagellate