Download - Buscant els depredadors més petits de l’oceà
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.
[email protected]@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