Identificació i cultiu de nanoflagel·lats heterotròfics rellevants per als sistemes marins

1. Nous Avenços en Ecologia Microbiana 2008
Identification and Culture of Relevant Marine
Heterotrophic Nanoflagellates
Avoiding Culturing Bias in HNF Isolation
J. del Campo
Department of Marine Biology and Oceanography

2. Nous Avenços en
Ecologia Microbiana
January 11th 2008
Classical vs Molecular
two different schools to study the same diversity in protists

3. Nous Avenços en
Ecologia Microbiana
January 11th 2008
Most Reported Marine HNF

4. Nous Avenços en
Ecologia Microbiana
January 11th 2008
Distribution of bicosoecids sequences
0,1
Cafeteria Cluster
Cultured vs Environmental sequences
Bicosoeca Cluster
Boroka Cluster
Unclassified Bicosoecida Cluster 1
Halocafeteria Cluster
Freshwater Clusters
Unclassified Bicosoecida Cluster 2
Caecitellus Cluster
Atlantic Ocean
Pacific Ocean
Indian Ocean
Arctic Ocean
Mediterranean Sea
Freshwater Sources
Solar Saltern
0,1
Environmental Sources
Culture Sources
Freshwater Sources

5. Nous Avenços en
Ecologia Microbiana
January 11th 2008
Probe design
Freshwater clusters Marine clusters
Solar saltern
RED: CAF01 coverage
0,1 BLUE: CET01 coverage

6. Nous Avenços en
Ecologia Microbiana
January 11th 2008
Distribution and abundance in natural samples
* All 0 counts correspond to values below 1 cel mL-1

7. Nous Avenços en
Ecologia Microbiana
January 11th 2008
Testing culturing bias
O 0% OM L 0,01% OM M 0,1% OM
Total Chrysophytes MAST Prasynophytes Bolidophytes Novel Alveolates II Cercozoa
Clones OTUs Clones OTUs Clones OTUs Clones OTUs Clones OTUs Clones OTUs Clones OTUs
O3 L5 M7
O3
21 11 16 7 1 1 2 1 - - 1 1 1 1
13 8 10 6 3 3 - - - - 1 1 1 1
L5
25 11 20 6 3 3 1 1 1 1 - - - -
7 6 4 3 3 3 - - 1 1 - - - -
M7
20 3 20 3 - - - - - - - - - -
- - - - - - - - - - - - - -
0% OM 0,01% OM 0,1% OM

8. Nous Avenços en
Ecologia Microbiana
January 11th 2008
Strategies for Cultivation of Uncultured HNF
Serial dilutions of unamended enrichments in suitable media.
Incubate the dilutions in the dark and feed with a suitable food source to
obtain a pure culture.
Identify our cultures by molecular and microscopical techniques.

9. Nous Avenços en
Ecologia Microbiana
January 11th 2008
MAST Isolation Attempt (our strategy)
HNFs
1st. Unamended Enrichments.
2nd. 24 wells cultures.
1HNF cel/well 2mL media.
3rd. Dark incubation.
4th. Direct Microscope Observation.
5th. Culture Identification.
FOOD SOURCE
1st. Unamended Enrichments.
2nd.Tangential Flow Filtration.
3rd. DAPI counts of the bacterial
concentrate.
4th. Suitable dilution in Filtered
and Sterile Aged Sea Water.

10. Nous Avenços en
Ecologia Microbiana
January 11th 2008
Where are we now?
We started with 480 dilutions with 1 “estimated” cell in each well.
From these 480 only 25 grew succesfully and were tansferred to
10mL.
10 of those 25 survived and were mantained in 50 mL cultures.
The 10 “isolates” obtained were identified by molecular means as: 4
Paraphysomonas, 2 Chlorarachnion like, 1 Novel Chrysophyceae, 1
Novel Cercozoa, 1 Novel Choanoflagellate and 1 MAST 3.
Now we need to prove if these cultures are pure or mixed. To do this
we are using diferent techniques such as Clone Libraries and
Sequencing and FISH.

11. Nous Avenços en
Ecologia Microbiana
January 11th 2008
Culture Perspectives
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.

12. Acknowledgements
Vanessa
Balagué
Dr. Fernando
Unrein
Dr. Fabrice
Not
Irene
Forn
Prof. Ramón
Massana
Raquel
Rodríguez
thanks for
your attention
Nous Avenços en
Ecologia Microbiana
January 11th 2008