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

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January 11th 2008

Fresh

water clu

stersM

arine clu

stersSolar saltern

RED: CAF01 coverageBLUE: CET01 coverage0,1

Distribution and abundance in natural samples

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January 11th 2008

* All 0 counts correspond to values below 1 cel mL-1

Testing culturing bias

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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

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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