Javier del CampoCentre d’Estudis Avançats de Blanes, 18 de febrer de 2010

Buscant els depredadors més petits de l’oceàAprofundint en la diversitat dels flagel·lats heterotròfics marins

Two schools, the same diversity

Baldauf et al. Science 2003

Is anyone there?

Pedrós-Alió Trends in Microbiology 2006

Most Reported Marine HNF

0%

20%

40%

60%

80%

100%

Other HNFMALVMASTChoanoflagellatesBicosoecidsChrysophytes

HNF Clonal Abundances

Freshwater Marine

Culturing Bias

No OM added Medium addition of OM0,1%

Low addition of OM0,01%

O3 L5 M7Uncultured

Cultured

Food Source Obtention

1st. Unamended Enrichments.

2nd.Tangential Flow Filtration.

3rd. DAPI counts of the bacterial concentrate.

4th. Suitable dilution in Filtered and Sterile Aged Sea Water.

Heterotrophic Marine Nanoprotists Isolation

1st. Unamended Enrichments.

2nd. 24 wells plates cultures. 1HNF cel/well in 2mL natural bacterial community media.

3rd. Dark incubation.

4th. Direct Microscope Observation.

5th. Culture Identification.

The Isolation Method

What are we gonna do with it?

Electron Microscopy. To study the ultrastructure of the cells.

Molecular Analysis. To assign a sequence to the isolate.

Ecophysiological studies: environmental adaptation, bioenergetics,trophic preferences, etc.

Deposit the cultures in a collection and the sequence in a database.

The Results

We started with 480 dilutions with 1 “estimated” cell in each well.

From these 480 only 25 grew succesfully and were transferred to 10mL.

10 of those 25 survived and were mantained in 50 mL cultures.

Only 3 were stabilized and preserved in culture in culture.

Name Closest match CM Accession % Phylogenetic group Closest cultured match %

MFI01 Uncultured eukaryote SSRPE06 EF173002 99 Cercozoa Chlorarachnion CCMP1408 88

MFI02 Uncultured eukaryote SSRPE06 EF173002 100 Cercozoa Chlorarachnion CCMP1408 88

MFI03 Uncultured eukaryote 4DB38 AJ862465 97 Choanoflagellida Diaphanoeca grandis 90

MFI04Uncultured marine eukaryote

CD8.18DQ647519 98 Chrysophyceae

Chrysophyceae sp. CCMP2296

91

MFI05Uncultured marine eukaryote

NIF_1E11EF526878 96 MAST3

Bolidomonas pacifica OLI120SD

88

MFI06Uncultured marine eukaryote

SA2_4G8EF526931 96 Cercozoa Cryothecomonas longipes 92

MFI07Uncultured marine eukaryote

CD8.18DQ647519 98 Chrysophyceae

Chrysophyceae sp. CCMP2296

91

MFI08Uncultured marine eukaryote

AD401DQ781330 93 Bicosoecida

Pseudobodo tremulans HFCC36

93

MFI09 Developayella elegans U37107 96 Stramenopiles Developayella elegans 96

MFI10 Uncultured eukaryote SSRPE06 EF173002 100 Cercozoa Chlorarachnion CCMP1408 88

The isolates

Minorisa minuta candidatus portrait

CRN02 probe

Minorisa minuta candidatus family

Jan 07 Mar 07 Apr 07 May 07 Jun 07 Jul 07 Aug 07 Sep 07 Oct 07 Nov 07 Dec 070

1

2

3

4

Blanes Bay 2007 Abundancece

lls/m

L

CARD-FISH/DAPI

CARD-FISH/DAPI

jan mar apr may jun jul agst sept oct nov dec0

20

40

60

80

100

120

140

160

180

0%

1%

2%

3%

4%

5%

6%

7%

8%

9%

10%

Jan 07 Mar 07 Apr 07 May 07 Jun 07 Jul 07 Aug 07 Sep 07 Oct 07 Nov 07 Dec 070

1

2

3

4

Blanes Bay 2007 Abundancece

lls/m

Lce

lls/m

L

% o

f to

tal H

NF

jan mar apr may jun jul agst sept oct nov dec0

10

20

30

40

50

60

70

80

90

100

1-2 µm

2-2.5 µm

>2.5 µm

cells/

mL

Blanes Bay 2007 Size Distribution

Survey date DAPI FISH CARD-FISH HF MAST1A MAST1B MAST1C MAST2 MAST4 MAST7 Ch-l

Arctic Ocean 24/08/02 652 1 4 7 2 94 16 4  27/08/02 1003 34 12 29 7 101 170 9  29/08/02 584 2 1 7 2 48 142 9

Southern Ocean 03/12/02 1562 86 274 188 2 0 361 7  05/12/02 1668 16 57 39 4 0 313 4  11/12/02 2367 23 45 201 6 0 217 2

Indian Ocean 16/05/03 687 2 8 15 0 101 78 33  23/05/03 626 1 0 16 0 66 3 0  06/06/03 719 0 0 11 0 58 4 5

Pacific Ocean 09/05/02 - 9 24 46 10 569 357 12  15/05/02 - 0 9 0 0 30 55 0  18/05/02 - 3 121 24 5 496 523 59

Canary Islands 23/09/02 1302 0 6 19 0 160 28 54  30/09/02 814 0 4 4 0 37 2 13

Saragasso Sea 12/07/04 214 0 1 1 0 8 1 9  15/07/04 443 1 0 14 0 51 1 0  30/07/04 593 3 0 15 0 22 0 0

Survey date DAPI FISH CARD-FISH HF MAST1A MAST1B MAST1C MAST2 MAST4 MAST7 Ch-l

Arctic Ocean 24/08/02 100 0,2 0,6 1,1 0,3 14,4 2,5 0,7  27/08/02 100 3,4 1,2 2,9 0,7 10,1 16,9 0,9  29/08/02 100 0,3 0,2 1,2 0,3 8,2 24,3 1,6

Southern Ocean 03/12/02 100 5,5 17,5 12,0 0,1 0,0 23,1 0,4  05/12/02 100 1,0 3,4 2,3 0,2 0,0 18,8 0,3  11/12/02 100 1,0 1,9 8,5 0,3 0,0 9,2 0,1

Indian Ocean 16/05/03 100 0,3 1,2 2,2 0,0 14,7 11,4 4,9  23/05/03 100 0,2 0,0 2,6 0,0 10,5 0,5 0,0  06/06/03 100 0,0 0,0 1,5 0,0 8,1 0,6 0,7

Pacific Ocean 09/05/02 - - - - - - - -  15/05/02 - - - - - - - -  18/05/02 - - - - - - - -

Canary Islands 23/09/02 100 0,0 0,5 1,5 0,0 12,3 2,2 4,1  30/09/02 100 0,0 0,5 0,5 0,0 4,5 0,2 1,6

Saragasso Sea 12/07/04 100 0,0 0,5 0,5 0,0 3,7 0,5 4,1  15/07/04 100 0,2 0,0 3,2 0,0 11,5 0,2 0,0  30/07/04 100 0,5 0,0 2,5 0,0 3,7 0,0 0,0

Global Abundance and Distribution

Some ideas to bring back home

Culturing Bias in HNF exists and we must avoid it.

We need to increase our culturing efforts in order to avoid Culturing Bias.

New strategies are needed to retrieve from the environment those organisms reluctant to be cultured.

Minorisa minuta candidatus is one of the most abundant and the smallest predator of the oceans.

jdelcampo@icm.catfonamental@gmail.com

www.fonamental.cat

In silico Acknowledgements

Vanessa Balagué

Dr. FernandoUnrein

Dr. FabriceNot

IreneForn

RaquelRodríguez

Prof. Ramon Massana

thanks for

your attention

Marco Álvarez

MassimoPernice