Transcript Oceanic Phytoplankton Team

Molecular Analysis of the Genetic
Diversity and Physiological Status of
Natural Picoplanktonic Populations
RCC 371
Oceanic Phytoplankton Team
Station Biologique de Roscoff
UMR 7127 CNRS et Université Paris 6, France
Aims for BIOSOPE:
1. Mapping the Picoplankton
• Enumerate picophytoplankton cells (Prochlorococcus,
Synechococcus, picoeukaryotes) + bacteria +/- viruses
+/- photosynthetic anoxygenic bacteria (to be tested) by
flow cytometry
=> Volume needed: 10 mL all stations / all depths
+ transect (1 sample / hour)?
• Analyze the cell cycle of Prochlorococcus & Synechococcus
over L:D cycles (in combination with molecular analyses
of stress response)
=> 4 long-term stations only : Volume needed: 10 mL
every 3 h for 2-3 days, 7 depths (to be discussed)
Flow Cytometry
FACSAria
• Laser source : 488 &
635 nm
• High speed cell sorting
(> 5 103 cell./sec)
• Increased Sensitivity
• « Transportable »
(180 kg!)
FACSort
ARIA
Aims for BIOSOPE :
2. Genetic diversity
• Isolate new species & organisms representative of noncultured environmental groups
• Assess the genetic diversity of (auto- and heterotrophic)
picoeukaryotes in the euphotic hyper-oligotrophic layer
(0-200 m)
• Assess the diversity & activity of heterotrophic
picoeukaryotes over the vertical layer (0-2000 m)
• Quantify the abundance of selected taxa and identify
the dominant species/taxa
•
and more (collaborations): Synechococcus, …
Molecular Techniques
- Pre-screening of genetic diversity of picoeukaryotes by TTGE
(most stations / 6 depths)
=> Volume : 5L pre-filtered on 3 µm on-line with 0.2µm filter
- Cloning & sequencing 18S & 16S rRNA gene from natural populations
(8-10 selected stations & depths)
=> same DNA sample as for TTGE
- Quantification of the abundance of different taxa
• in situ Hybridization (FISH) using 18S & 16S rRNA probes
(8-10 selected stations at 6 depths, 1 L)
• Quantitative PCR
(most stations / 6 depths ?)
- Metagenomics ?
(Long-term stations, 2 depths, >100 L samples, in situ pumps)
Molecular
Approach
Natural sample
< 3 µm
Cultures
DNA
PCR /
Quantitative PCR
Amplification
18S/16S rRNA gene
DGGE/TTGE
Clone libraries
rRNA gene sequencing
Data banks
Phylogenetical analyses
Molecular probes
An example of DGGE analysis
DGGE: PROSOPE
Metazoa
Prasinophyceae
Cercozoa
Bacyllariophyta
Alveolate Group I
Alveolate Group II
CTD28-110M
CTD28-90M
CTD28-50M
CTD28-30M
CTD28-5M
Ref.
CTD17-110M
CTD17-95M
CTD17-80M
CTD17-55M
CTD17-25M
CTD17-5M
Ref.
CTD11-80M
CTD11-30M
D. Marie unpubl.
CTD11-3M
Ciliophora
Example of Clone Library Analysis
Oligotrophic: OLIPAC
Dictyochophyceae
Stramenopiles
Stramenopiles
Pelagophyceae
Stramenopiles
Acantharean
Ciliophora
Alveolata
Dinophyceae
Alveolata
OLI11032
OLI11019
Prymnesiophyceae
Haptophyta
Amoebophrya
Dinophyceae
Alveolata
Choanoflagellida
Prasinophyceae
Chlorophyta
Moon et al 2001
OLI11001
Alveolata
FISH: Abundance of
Prasinophyceae off
Roscoff
14 000
Eukaryotes
Micromonas
Ostreococcus
Bathycoccus
12 000
Cell/mL
10 000
8 000
6 000
4 000
2 000
0
Jul
F. Not, unpubl.
Oct
Jan
Apr
Date (2000-2001)
Jul
Oct
Available Probes for FISH (subset)
Target_class
Target_level
Eukaryotes
Supra
Autotrophic groups
Chlorophyta
Supra
Chromista
Supra
Green algae
Prasinophyceae
Clade
Prasinophyceae
Clade
Prasinophyceae
Clade
Brow n algae
Haptophyta
Division
Bolidophyceae
Class
Dictyochophyceae Class
Pelagophyceae
Clade
Eustigmatophyceae Class
Cryptophyceae
Class
Environmental
Clade
Dinophyceae
Class
Heterotrophic groups
Stramenopiles
Clade
Alveolata group II
Class
Probe_name
EUK1209
Target_group
Eukaryotes
CHLO02
NCHLO02
Chlorophyta
Most Non-Chlorophyta/Some Chlorophyta
PRAS04
PRAS01
PRAS03
Mamiellales
Pycnococcales
Prasinococcales
PRYM02
BOLI02
DICTYO01
PELA02
EUSTI01
CRYPT13
ENV01
DINO B
Haptophyta
Bolidophyceae
Dictyochophyceae
Pelagophyceae Clade
Eustigmatophyceae
Cryptophyceae
Environmental group (close to Glaucytophyceae)
Dinophyceae; some Apicomplexa
NS03
ALV01
Novel stramenopiles-clade III
Alveolata group II
Probes in yellow require development
Quantitative PCR
100
Morlaix Bay
Amorces Eucaryotes
Amorces Prasinophyte
Amorces Ostreococcus
Amorces Bathycoccus
Amorces Micromonas
10
Fluorescence normalisée
Natural sample
dilué x100
eau stérile
1
0,1
0,01
15
20
25
30
35
40
Nombre de cycles
Advantages/FISH : faster (96/3 = 32 samples at a time)
more sensitive (detection : < 0.1 ng d ’ADN)
Drawbacks/FISH : more expensive, do not visualize cell morphology
measures relative gene abundances, not cell number
=> 18S rRNA in variable number copies (2-1000 per cell)
Q-PCR: PROSOPE cruise
Fei Zhu
Available probes for Q-PCR
• Six primer sets (5’ Euk universal, 3’Group specific)
– EUK : Eukaryotes
– CHL : Chlorophyta
– MAM (=PRAS04) : Mamiellales
– MIC : Micromonas (not totally specific yet)
– BAT : Bathycoccus
– OST : Ostreococcus
Aims for BIOSOPE
3. Physiological status of natural populations
•
Expression of specific stress genes (UV stress, nutrient starvation) over a L:D
cycle every 3 h for 2-3 days)
=> at the four long-term stations only, 7 depths (subsurface,
5 m, 10 m, 20m, 30 m, 50 m, DCM (Niskin!)
+ if bottles : 5 L for DNA, 15 L for RNA
+ if in situ pumps: 20L for DNA, 80L for RNA
•
Characterization of the expression of genes of biogeochemical interest in the
picoplankton (e.g.: nifH)
=> same DNA & RNA samples
Problems to be solved with the use of in situ pumps
- Sampling need to be short (only 100 L necessary) and done as soon as
possible after a CTD cast (within an hour)
- flow cytometry needed : will be sampled at same depths with the rosette
(DCM: 2 x 10 L)
- need for RNA-free material (use of gloves to handle filters, RNA-zapping
of filter holder,…)
- need for pre-tests of RNA extraction from GF/F filters before the cruise !
Q-PCR for measuring Gene
Expression
Natural sample
RNA
cDNA
Amplification gene X
by quantitative PCR
Analysis of Gene Expression by
Q-PCR (Eilat, Sept. 2000)
0
23,8
40
23,6
60
23,4
80
23,2
60
% Cells
DCM depth (m)
70
Temperature (°C)
20
80
24,0
A
DCM depth
Temperature
C
S%
G2%
50
40
30
20
10
100
18:00
0
23,0
06:00
18:00
06:00
18:00
06:00
18:00
18:00
125
100
75
50
25
18:00
18:00
06:00
18:00
06:00
18:00
0,05
B
06:00
18:00
06:00
Time
18:00
06:00
18:00
Normalized ftsZ mRNA levels
(2-ddCT)
Prochlorococcus concentration
3
(x10 cells/mL)
150
06:00
D
0,04
0,03
0,02
0,01
0,00
18:00
06:00
18:00
06:00
18:00
06:00
18:00
Time
Holtzendorff et al. (2001)
Specific needs
Sample storage :
- Cruise start (Tahiti): 105-140 L liquid N2 to fill 3 or 4 35-liter containers
Note: Samples can be also be stored at -80°C during cruise time but fast
freeze is preferable
- Cruise end (Chili) : 50-60 kg dry ice to send all frozen samples by express mail
in a large cryobox after the cruise
Pre-cultures will be either brought back « by hand » in a
cryobox or left on board (requires light and air
conditioning left on during the transect back)
Space :
- Beside SBR container lab space, 1.5 m of bench for filtration ramps
Flow cytometer:
- Needs to be sent by plane!
Sample number (estimation)
Euk + Syn
Hetero.
Euks
Pro/Syn
Cyto
counts
Culture
TTGE &
Clone
libraries
0-200 m
FISH
0-200 m
Q-PCR
Diversity
0-200m
Clone
libraries
0-2000 m
FISH
0-2000
m
QPCR
genes
Cyto
Cell cycle
Vol. sampled (mL)
2
250
5 000
1 000
2 000
5 000
1 000
10 000
2
total station #
25
12
19
19
19
4
4
4
4
CTD # per station
1
1
1
1
1
1
1
8
8
Depth # per CTD
12
3
6
6
6
6
12
5
5
Storage container
# per Depth
1
20
1
4
1
1
2
1
1
Total sample #
300
720
114
456
114
24
96
160
160
Storage type
N2 or- 80°
Live
N2 or- 80°
-20 or -80
N2 or- 80
N2 ou- 80
-20/-80
N2 or - 80
N2 or -80
Storage containers
2 mL
cryotubes
Tubes or
flasks
2 mL
cryotubes
filter
holders
5mL
cryotubes
5mL
cryotubes
filters
5mL
cryotubes
2 mL
cryotubes
Personnel SBR Involved in BIOSOPE
1)
2)
3)
4)
5)
6)
7)
8)
9)
10)
Daniel VAULOT DR1 CNRS (leg 1)
Frédéric PARTENSKY DR2 CNRS (leg 2)
Laurence GARCZAREK CR1 CNRS (leg 1)
Laure GUILLOU CR2 CNRS
Nathalie SIMON MdC UPMC
Dominique MARIE IE CNRS (2 legs)
Florence Le GALL AI CNRS
Post-doc X (2003-2004)
PhD student Y on Diversity (2004-2007) (leg 2)
DEA or PhD student Z on Physiology (2004-2007)
Collaborations for molecular analyses
 Dave Scanlan, Warwick University : diversity Synechococcus (DNA/FISH)
+/- John Zehr, USA : nifH gene diversity (DNA/RNA)
+/- Colomban de Vargas, USA : Coccolithophorids
+/- Alex Worden : Roseobacter diversity (DNA/RNA) ?
+/- Carles Pedros-Alio : Archae diversity (DNA) ?
+/- Osvaldo Ulloa, Chili : diversity eukaryotes, anoxygenic Procs
+/- Z. Kolber, USA : anoxygenic bacteria