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

1. Buscant els depredadors més petits de l’oceà
Aprofundint en la diversitat dels flagel·lats heterotròfics marins
Javier del Campo
Centre d’Estudis Avançats de Blanes, 18 de febrer de 2010

2. Two schools, the same diversity
Baldauf et al. Science 2003

3. Is anyone there?
Pedrós-Alió Trends in Microbiology 2006

4. Most Reported Marine HNF

5. 100%
80%
60%
40%
20%
0%
Other HNF
MALV
MAST
Choanoflagellates
Bicosoecids
Chrysophytes
HNF Clonal Abundances
Freshwater Marine

6. Culturing Bias
O3 L5 M7
No OM added Medium addition of OM
0,1%
Low addition of OM
0,01%
Uncultured
Cultured

7. The Isolation Method
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.

8. 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.

9. The isolates
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
MFI04
Uncultured marine eukaryote
CD8.18
DQ647519 98 Chrysophyceae
Chrysophyceae sp.
CCMP2296
91
MFI05
Uncultured marine eukaryote
NIF_1E11
EF526878 96 MAST3
Bolidomonas pacifica
OLI120SD
88
MFI06
Uncultured marine eukaryote
SA2_4G8
EF526931 96 Cercozoa Cryothecomonas longipes 92
MFI07
Uncultured marine eukaryote
CD8.18
DQ647519 98 Chrysophyceae
Chrysophyceae sp.
CCMP2296
91
MFI08
Uncultured marine eukaryote
AD401
DQ781330 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

10. Minorisa minuta candidatus portrait

11. CRN02 probe
Minorisa minuta candidatus family

12. Blanes Bay 2007 Abundance
4
3
2
1
0
Jan 07 Mar 07 Apr 07 May 07 Jun 07 Jul 07 Aug 07 Sep 07 Oct 07 Nov 07 Dec 07
cells/mL

13. CARD-FISH/DAPI

14. CARD-FISH/DAPI

15. 10%
9%
8%
7%
6%
5%
4%
3%
2%
1%
0%
Blanes Bay 2007 Abundance
4
3
2
1
180
160
140
120
100
80
60
40
20
0
jan mar apr may jun jul agst sept oct nov dec
0
Jan 07 Mar 07 Apr 07 May 07 Jun 07 Jul 07 Aug 07 Sep 07 Oct 07 Nov 07 Dec 07
cells/mL cells/mL
% of total HNF

16. Blanes Bay 2007 Size Distribution
100
90
80
70
60
50
40
30
20
10
0
jan mar apr may jun jul agst sept oct nov dec
1-2 μm
2-2.5 μm
>2.5 μm
cells/mL

17. Global Abundance and 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

18. 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.cat
fonamental@gmail.com
www.fonamental.cat

19. In silico Acknowledgements
Vanessa
Balagué
Dr. Fernando
Unrein
Dr. Fabrice
Not
Irene
Forn
Raquel
Rodríguez
Prof. Ramon
Massana
thanks for
your attention
Marco
Álvarez
Massimo
Pernice