identificació i cultiu de nanoflagel·lats heterotròfics rellevants per als sistemes marins
TRANSCRIPT
J. del Campo
Avoiding Culturing Bias in HNF Isolation
Nous Avenços en Ecologia Microbiana 2008
Department of Marine Biology and Oceanography
Identification and Culture of Relevant Marine Heterotrophic Nanoflagellates
Classical vs Moleculartwo different schools to study the same diversity in protists
Nous Avenços en Ecologia Microbiana
January 11th 2008
Most Reported Marine HNF
Nous Avenços en Ecologia Microbiana
January 11th 2008
Nous Avenços en Ecologia Microbiana
January 11th 2008
Distribution of bicosoecids sequences
0,1
Cafeteria Cluster
Bicosoeca Cluster
Boroka Cluster
Unclassified Bicosoecida Cluster 1
Halocafeteria Cluster
Freshwater Clusters
Unclassified Bicosoecida Cluster 2
Caecitellus Cluster
Atlantic OceanPacific OceanIndian OceanArctic OceanMediterranean SeaFreshwater SourcesSolar Saltern 0,1
Environmental SourcesCulture SourcesFreshwater Sources
Cultured vs Environmental sequences
Probe design
Nous Avenços en Ecologia Microbiana
January 11th 2008
Fresh
water clu
stersM
arine clu
stersSolar saltern
RED: CAF01 coverageBLUE: CET01 coverage0,1
Distribution and abundance in natural samples
Nous Avenços en Ecologia Microbiana
January 11th 2008
* All 0 counts correspond to values below 1 cel mL-1
Testing culturing bias
Nous Avenços en Ecologia Microbiana
January 11th 2008
O3 L5 M7
Total Chrysophytes MAST Prasynophytes Bolidophytes Novel Alveolates II CercozoaClones OTUs Clones OTUs Clones OTUs Clones OTUs Clones OTUs Clones OTUs Clones OTUs
O321 11 16 7 1 1 2 1 - - 1 1 1 113 8 10 6 3 3 - - - - 1 1 1 1
L525 11 20 6 3 3 1 1 1 1 - - - -7 6 4 3 3 3 - - 1 1 - - - -
M720 3 20 3 - - - - - - - - - -- - - - - - - - - - - - - -
0% OM 0,01% OM 0,1% OM
O 0% OM L 0,01% OM M 0,1% OM
Strategies for Cultivation of Uncultured HNF
Nous Avenços en Ecologia Microbiana
January 11th 2008
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.
MAST Isolation Attempt (our strategy)
Nous Avenços en Ecologia Microbiana
January 11th 2008
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.
Where are we now?
Nous Avenços en Ecologia Microbiana
January 11th 2008
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.
Culture Perspectives
Nous Avenços en Ecologia Microbiana
January 11th 2008
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.
Acknowledgements
Vanessa Balagué
Dr. FernandoUnrein
Dr. FabriceNot
IreneForn
RaquelRodríguez
Prof. Ramón Massana
thanks for
your attention
Nous Avenços en Ecologia Microbiana
January 11th 2008