The effect of diatom secondary
metabolites on other
phytoplankton
Raffaella Casotti
François Ribalet
Sabina Mazza
100
80
80
60
40
20
0
0.0 0.5 1.0 1.5 2.0
Copepod
% Relative cell numbers
100
% Cleavage
% Egg hatching
Effect on model animals
60
40
20
0
0.0 0.5 1.0 1.5 2.0
Sea urchin
125
100
75
50
25
0
0
5
10
human
15
20
Aims
• Test the effect of different aldehydes on
different phytoplankton species
•
•
•
•
Diatoms/non-diatoms
Describe the cellular effects and localization
Investigate the action as infochemicals
Modulation by environmental factors (light, nutrients)
• Bloom dynamics in situ
Phytoplankton models
• Thalassiosira
weissflogii
Phaeodactylum tricornutum
• Skeletonema
marinoi
Flow Cytometry
PMT
4
Flow cell
PMT
Dichroic
Filters
3
PMT
2
Bandpass
Filters
PMT
1
Laser
Fluorescence Activated
Cell Sorting
488 nm laser
FALS Sensor
Fluorescence detector
Charged Plates
Single cells sorted
into test tubes
-
+
Aldehydes tested
Decadienal (A3) (SIGMA)
Decatrienal (A1) (synthesized)
Octadienal (SIGMA)
Heptadienal (SIGMA)
dose-dependent effect
160000
0.00
0.18
0.50
1.00
5.00
10.00
concentration (cell ml-1 )
140000
120000
100000
80000
60000
40000
20000
0
0
Casotti et al. 2005
12 24 36 48 60 72 84 96 108 120
time (h)
Time-dependent
cells/ml
160000
600
♦ control
□ 1.00
120000
1h
400
80000
200
a
0
0
24
48
72
96
120 144 168 192
160000
120000
80000
40000
c
0
0
24
48
72
96
120 144 168 192
160000
120000
80000
40000
e
0
0
24
48
72
96
Green fluorescence (r. u.)
12 h
Cell concentration (cell cm-3)
40000
6h
Metabolic activity (FDA)
0
0
24
48
72
96
120
144
168
192
0
24
48
72
96
120
144
168
192
0
24
48
72
96
120
144
168
192
0
24
48
72
96
120
144
168
192
600
400
200
0
600
400
200
0
120 144 168 192
600
200000
160000
400
120000
24 h
80000
200
g
40000
0
0
0
Casotti et al. 2005
24
48
72
96
120 144 168 192
time (h)
time (h)
Effects on metabolic activity
Fv/Fm
Metabolic activity (FDA)
a.u.
300
A3-1
A3-5
control
200
100
0
0
1
2
3
4
5
6
9
24
time (hours from addition)
a.u.
400
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
A3-1
A3-5
control
0
1
2
3
4
5
6
24
time (hours from addition)
• Photosynthesis is affected slower than
esterase activity
9
Cell cycle
control
2629
2341
G1
G1
1314
657
0
0
128
GREEN
192
0
256
1436
128
GREEN
192
256
64
128
GREEN
192
256
192
256
1709
854
0
64
128
GREEN
192
0
256
1779
0
2677
889
G2 34%
G2 (34%)
444
0
G1
G1
2007
Counts
G1
G1
1334
Counts
64
G1
G1
2563
718
Casotti et al. 2005
0
3418
Counts
Counts
64
G1
2154
0
1170
585
2872
0
G1
G1
1755
Counts
1971
Counts
+ A3
64
128
GREEN
1338
G2
5%
G2 (5%)
669
192
256
0
0
DNA
64
128
GREEN
Subpopulations
319
dead
239
Counts
vital
159
dying
79
0
10
0
10
1
10
2
SYTOX
Casotti et al. 2005
10
3
10
4
Active process of cell death
Morfologia
Casotti et al. 2005
a
b
c
d
TW
b values
y = a exp(bconc ) + c
FDA
cell ml-1
red fluor.
SX+
Dt/(Dt+Dd)
Fv/Fm
A3 conc
2.97
1.37
0.88
0.48
0.42
0.27
200
175
150
0.18 A3
125
100
75
control
50
25
0
0
12
24
36
48
60
72
84
100
% dead cells
75
0
0+2
50
0.1
0.1+2
25
0
0
10
20
30
40
50
time (h from 1st DECA addition)
60
• Mediators of cell death (ROS or NO)
• Comparative analyses on other
phytoplankton (F. Ribalet)
MODEL SPECIES
f/2 medium, under Light:Dark 12:12 at 150 µE.mol-1.s-1 at 17°C in environmental chamber
SPECIES
CLASS
GROWTH RATE (d-1)
Amphidinium carterae (AC
(AC))
Dinoflagellate
0.43 ± 0.02
Dunaliella tertiolecta (DT
(DT))
Chlorophyceae
0.69 ± 0.09
Isochrysis galbana (IG
(IG))
Prymnesiophyceae
0.65 ± 0.03
Micromonas pusilla (MP
(MP))
Prasinophyceae
0.84 ± 0.05
Skeletonema costatum (SC
(SC))
Bacillariophyceae
0.85 ± 0.08
Tetraselmis suecica (TS
(TS))
Prasinophyceae
0.96 ± 0.07
AX
I
N
E
C
50 mL of exponentially growing
cultures (10 000 cells / mL)
exposed to different concentrations
of PUA (from 0.1 to 4 mg / L).
For each treatment, compilation of data from 2 series of experiments
in triplicats
Ribalet 2005
Statistical Analysis
2.5
Decadienal
Octadienal
Heptadienal
EC50 Value (mg/L)
2.0
1.5
1.0
0.5
SC
TS
D
T
A
C
M
P
IG
0.0
Figure 3: 24h-EC50 values for decadienal, octadienal and heptadienal for the different species.
Data are means ± 95% IC (n=3).
Ribalet 2005
Ribalet 2005
(Wichard et al., 2005)
In situ work
(more questions than answers)
• Study of a diatom bloom: Skeletonema
costatum in the North Adriatic Sea
Venice lagoon
Po delta
Croatia
Aldehydes can be effectors of a bloom at sea?
• Mastication by grazers
• Autolysis
• Endogenous (ciclo cellulare)
• Exogenous (virus, bacteria, UV, mixing,
nutrients)
Tentative approach for in situ
studies
• Characterize the Skeletonema bloom dynamics
•
•
(growth rates, interactions with other
phytoplankton)
Estimate lysis rates (FDA method)
Estimate apoptotic cells
•
•
•
Relate to effects on copepods
Presence of aldehydes dissolved?
Enhancement due to environmental factors?
0.045
20000
100
0.024
cell ml
-1
% TUNEL+
75
10000
50
25
0
0
0
20
40
julian day
60
80
0
20
40
julian day
60
80
TUNEL