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Why fine filtration?

Diversity of the eukaryotes and prokaryotes (including plastids) assemblages assessed from fine size fractionation and DGGE. Why fine filtration?. 1- Because we obtained a lot of clones that are known to belong to organisms

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Why fine filtration?

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  1. Diversity of the eukaryotes and prokaryotes (including plastids) assemblages assessed from fine size fractionation and DGGE

  2. Why fine filtration? 1- Because we obtained a lot of clones that are known to belong to organisms larger than the picoplanktonic fraction (copepods, dinoflagellates, ....) 2- Because we wanted to check whether the 0.2 - 3 µm fraction describes accurately the picoplankton. 3- To assess the size of unculturable organisms such as novel alveolates, or stramenopiles which we have found majoritary in the clone libraries.

  3. Filtering chain Pump Filters with decreasing sizes Natural sample 1- The filters must not saturate 2- But, they must have enough biomass to extract good-quality DNA 3- The lenght of the filtering chain must be as short as possible to minimize cell disruption.

  4. Barcelona Harbour (from May 2000 up to now) Parameters bimonthly sampled: Flow cytometry Phytoplankton (Uterhmol method) virus (Pulse-field-electrophoresis) Temperature Salinity Secchi disk Chl a PO4 NH4 NO2 NO3 Si Extracted DNA : >3 µm, <3 µm

  5. 28/02/2001 Cells per Litre 0.5 µm 0-20 µm > 15 µm

  6. Barcelona Harbour 28/02/2001 1 L 0.8 L CHAIN 1 Prefiltration on 10.0 µm 1.0 0.6 0.2 4 L CHAIN 2 Prefiltration on 20.0 µm 10.0 3.0 1.0 CHAIN 3 12 L 20.0 14.0 10.0

  7. PCR amplifications Eukaryotes (1F/516R-GC) 0.2- 0.6 0.6- 1.0 1.0- 10.0 3.0- 5.0 5.0- 10.0 >10.0 10.0- 14.0 14.0- 20.0 >20.0 >10.0 >20.0 - prefiltrations Chain 1 Chain 2 Chain 3 Prokaryotes (GC-358F/907R) : all fractions amplified Archaea (GC-344F/915R) : No amplifications.

  8. Results on DGGE, Eukaryotes 45% 65% 1.0- 10.0 10.0- 20.0 10.0- 14.0 14.0- 20.0 5.0- 10.0 3.0- 5.0 0.6-1.0 > 20.0 10.0 - 20.0 1.0 - 10.0

  9. Results on DGGE, Prokaryotes 40% 80% 1.0- 10.0 3.0- 5.0 5.0- 10.0 10.0- 14.0 14.0- 20.0 0.2-0.6 0.6-1.0 > 20.0

  10. Next steps • The size fractionation will be reduce in the smallest size class : • 0.2, 0.6, 1.0, 2.0, 3.0, 5.0, 10.0 ... • I am now comparing these results with other ecosystems such as • - the Blanes site (March 2001) • - the Barcelona Harbour (March 2001) • ... and perhaps deth sample from the Canyon cruise (Juny 2001) • A study of the diversity in the smallest size classes will be done by direct sequencing of the bands and/or clone library. • Calibrated the size fractionation with the “real size” of the organisms obtained from cultures.

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