blue diatoms as a potential for sustainable shellfish ......blue diatoms as a potential for...
TRANSCRIPT
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Blue diatoms as a potential for sustainable shellfish aquaculture
By
Fiddy S. Prasetiya*, Priscilla Decottignies, Michèle Morançais, Luc A. Comeau, François Turcotte, Romain Gastineau, Iskandar, Toto Subroto, Yudi N. Ihsan, Réjean Tremblay, Bruno Cognie, Christophe Stavrakakis and Jean-Luc Mouget
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Up and down postgraduation
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Challenges for Indonesian young researchers
• Limited research funding sources
• Complexity for obtaining national grants applications
• Less funding opportunity for basic research
• Lack of transparency, unclear & long delay of funding
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Research collaboration to minimize the gap..
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• Oyster greening:
– Erratic proliferation of H. ostrearia & pigment released in oyster ponds
– Marennine fixation on oyster gills
– Added economic value
Haslea ostrearia and oyster greening
Atlantic Ocean
Marennes-Oléron
Oyster ponds in Marennes-Oléron
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Haslea ostrearia and Marennine
IMnEMn
Characteristics Type of pigment
IMn EMn
Localization Apical axis of the cell Released into the medium
Molecular weight 10.7 kDa 9.8 kDa
Marennine molecule structure?Unknown:- Pouvreau et al. (2006): Polyphenolic- Gastineau et al. (2014): Glycosidic
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Purified EMn and IMn
Pouvreau et al. (2008)
H. ostrearia in co-culturev
Skeletonema costatumChaetoceros calcitrans
Tetraselmis suecica
Prasetiya et al. (2016)
Purified EMn and IMnv
Vibrio tubiashii V. aestuarianusV. coralliilyticusV. Tasmaniensis
Gastineau et al. (2014)
.
Purified EMn and IMn
HSV-1
Gastineau et al. (2012)
Biological activities of marennine in vitro
Antiviral Antioxidant
Antibacterial Allelopathic
MarennineIMn /EMn
7
Natural antibiotic for aquaculture
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• France is the main oyster producer in Europe (87.4%)
• Pathogen infections– Disease outbreaks (OsHV-1, Vibrio aestuarianus)
– Mass mortality: juveniles (2008), adults (2013)
• Stock depletion & Economic loss
Oyster aquaculture
However…
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Assessment for utilization of marennine in shellfish aquaculture
• How the greening is occurred?
• How Haslea affect oyster’s feeding behavior?
• Consequence of greening on functional responses of bivalve?
• Prospective application of marennine in shellfish aquaculture?
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Is the greening only due to EMn in solution, or to the diatoms (IMn) consumed by oysters?
a) Control (without marennine and H. ostrearia)
b) H. ostrearia + blue water (IMn + EMn, 72h)
c) Blue water (supernatant) (EMn, 72h)
d) H. ostrearia cells (IMn, 12 weeks)
Part 1: Greening of oyster by H. ostrearia
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Persistency of greening
No de-greening after 12 weeks
Marennine fixation is persistent
Part 1: Greening of oyster by H. ostrearia
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Marennine fixation on the gills
Marennine fixation in mucocytes
Part 1: Greening of oyster by H. ostrearia
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Cognie (2001), Barille et al. (2001)
Part 2: Role of size in preingestive selection of H. ostrearia in C. gigas
Does the size really matter?
Haslea on feeding behaviour
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Photo: JD Billaud
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• Experiment A: Scanning electron microscopy (SEM)
• Experiment B: Video endoscopy directed-sampling
DT
VT
Data analysis:
- Homogenity & Normality test
- T-test (XLSTAT)
Part 2: Role of size in preingestive selection of H. ostrearia in C. gigas
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Experiment B:
Video endoscopy observation
Rejection of larger cells
Part 2: Role of size in preingestive selection of H. ostrearia in C. gigas
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Valve activity experiment (short-term effect)
Part 3: Consequences of greening by marennine on the integrative response of bivalve
Analysis of Scope for Growth
Lipids and FAs analysis (long-term effect)
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Behavioral traits
• Significant effect on mussel’s acclimation phase
• Post-acclimation effect on oyster
*
*
*
*
Part 3: Consequences of greening by marennine on the integrative response of bivalve
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Physiological traits
0.0 0.5 1.0 2.00.0
0.2
0.4
0.6
0.8A
a
a
a
a
VO
2 (
ml h
-1 g
-1)
0.0 0.5 1.0 2.00
5
10
15
20C
a
a
a,b
b
CR
(l h
-1 g
-1)
0.0 0.5 1.0 2.00
20
40
60
80
100E
a a a a
Absorp
tion r
ate
(%
)
0.0 0.5 1.0 2.00
5000
10000
15000G
a
a
a,b
b
[EMn] mg L-1
SF
G (
J h
-1 g
-1)
0.0 0.5 1.0 2.00.0
0.5
1.0
1.5B a a a
b
0.0 0.5 1.0 2.00.0
0.2
0.4
0.6
0.8D
a
a,b
a,b
b
0.0 0.5 1.0 2.00
20
40
60
80
100F a a a a
0.0 0.5 1.0 2.00
200
400
600H a
a,b
a,b
b
[EMn] mg L-1
Parameter Significancy Animal
VO2 S Oyster
CR S Mussel, oyster
AR NS Mussel, oyster
SFG S Mussel, oyster
S = SignificantNS = Non significant
*
*
**
*
*
Part 3: Consequences of greening by marennine on the integrative response of bivalve
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*
0 20
100
200
300
a
b
EMn (mg L-1)
Tota
l FA
s
0 20
50
100
150
a
a
EMn (mg L-1)
Tota
l FA
s
• *S in FAs composition (PERMANOVA)– 18:3 n-6; 20:5 n-
3 (SIMPER)
• NL, energetic reserves
• PL, membrane saturation
0 20
100
200
300
a
a
A
[EMn] (mg L-1)
Unsatu
ration index
0 20
100
200
300
a
bB
[EMn] (mg L-1)
Unsatu
ration index
• *S in UI+ 18:2 n-6, 20:3 n-3, 22:6 n-cis
- 18:3 n-6
Part 3: Consequences of greening by marennine on the integrative response of bivalve
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Physiological traits• Species specific
M. e
dulis
C. v
irgin
ica
P. m
agel
lani
cus
0
1
2
3
4B
Adult bivalve species
VO
2 (
ml O
2 h
-1 g
-1)
M. e
dulis
C. v
irgin
ica
P. m
agel
lani
cus
0
1
2
30 mg l-1_day 0
3 mg l-1_day 0
0 mg l-1_day 7
3 mg l-1_day 7
A
CR
(l h
-1 g
-1)
Part 3: Consequences of greening by marennine on the integrative response of bivalve
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Physiological traits• Age-specific
Juve
nile
Adu
lt0
1
2
3
4
5 0 mg l-1_day 0
3 mg l-1_day 0
0 mg l-1_day 7
3 mg l-1_day 7
A
CR
(l h
-1 g
-1)
Juve
nile
Adu
lt0.0
0.5
1.0
1.5
2.0
2.5B
Stage of P. magellanicus
VO
2 (
ml O
2 h
-1 g
-1)
Part 3: Consequences of greening by marennine on the integrative response of bivalve
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Conclusion and perspectives
• Haslea and its marennine (-like) pigments are promising sources of natural compounds with potential applications with probiotic and prophylactic benefits in aquaculture. Yet further studies need to be conducted..
• Perspectives: Identification on chemical structures, toxicity and to improve the cultivation of Haslea species and extraction processes for the compounds of interest, in particular when considering the production of marennine at an industrial scale.
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Related publications
Contact information:[email protected]://www.researchgate.net/profile/Fiddy_Prasetiya
Book chapter:Marennine-Like Pigments: Blue Diatom or Green Oyster Cult?
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Greening estimation: Semi-qualitative method
Quantitative method
Grounded freezed gills + Urea 8M
Heated 50°C, 24h,centrifuged 8500 rpm Spectro-UV vis on
supernatant
Part 1: Greening of oyster by H. ostrearia
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Experiment A: SEM observation3 populations differing in cell size
100 μm 10 μm
10 μm 10 μm
100 μm 100 μm
Part 2: Role of size in preingestive selection of H. ostrearia in C. gigas
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Greening estimation: Semi-qualitative Semi-qualitative vs quantitative
0 20 40 60 80 100 1500 2000 25000
2
4
6
8
10CE
HO+CE
HO
Time of exposure (h)
Gre
enin
g inte
nsity
0.00 0.05 0.10 0.15 0.200
1
2
3
4
5
R2 = 0.9642
mg EMn mg-1 wet weight
Gre
enin
g inte
nsity
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Marennine & gill interaction
Fresh gills, dissected & washed
Immersion, 3-30 kDa ultra-filtered EMn
Fixation, 10% formaldehyde, 10d
Cryostat sectionning, 40 micron
Observation under microscope
Part 1: Greening of oyster by H. ostrearia
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Greening experiment
S. costatum 30-120 103 cell mL-1 (Control)
H. ostrearia + BW, 5 mg L-1
BW, 5 mg L-1 H. ostrearia, 30-120 103 cell mL-1
BW removal, centrifugation
Greening quantification
Placed in normal medium, 12 weeks
BW: Blue water
EMn + IMn EMn IMn
Part 1: Greening of oyster by H. ostrearia
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C. gigas juveniles + EMn (5 mg/L, n=8)
C. gigas juveniles (control, n=8)
Outflow
Sampling at outflow with time T
0T
20T
40& T
60(min) for
each group
Samples were analyzed by particle counter multisizer
Analysis of CR
Marennine on CR
Part 1: Greening of oyster by H. ostrearia
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Effect of marennine on Clearance Rate (CR)
Initial response
Recovery ability
0 70
1
2
3
4
5Control
Green
a
b
aa
Days
CR
(L h
-1 g
-1)
Part 1: Greening of oyster by H. ostrearia
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• Different sizes of H. ostreariapopulations (Experiment A & B), 60 L, (3.106cells.l-1)
• 10 adult oysters wereacclimated, flow rate 10l.h-1
PF
Outflow
• Sampling each 15’, during 1h, at outflow
• Sampling of PF at the end of observation
• Fixed with Lugol & counted withbiometric microscope
Flow-through chamber system (FTC)
Part 2: Role of size in preingestive selection of H. ostrearia in C. gigas
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Behavioral traits (short-term)
Acclimation phase Post-acclimation phase
M. e
du
lisC
. vir
gin
ica
0 2 4 60
5
10
15A
Valv
e o
penin
g (°)
6 8 10 120
5
10
15B
0 2 4 60
2
4
6
8
10C
Elapsed time (h)
Valv
e o
penin
g (°)
6 8 10 120
2
4
6
8
10D
Elapsed time (h)
Part 3: Consequences of greening by marennine on the integrative response of bivalve
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Marennine fixation on the gills
Marennine fixation in mucocytes
Part 1: Greening of oyster by H. ostrearia
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Bakteri patogen kerang
Antibacterial activity in vitro
Prasetiya et al., 2018
Aktivitas Biologis Pigmen Marenin untuk Budidaya Perairan
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Kerang abalone (Pangandaran)
Kerang mutiara (Bali)Udang vanname (Pangandaran)
Larva udang (Bali)
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Biochemical traits
• NL, energetic reserves
• Significantly different in FAs composition (PERMANOVA)– 18:3 n-6; 20:5 n-3 (SIMPER)
M. edulis C. virginica
*
0 20
100
200
300
a
b
EMn (mg L-1)
Tota
l FA
s
0 20
50
100
150
a
a
EMn (mg L-1)Tota
l FA
s
Part 3: Consequences of greening by marennine on the integrative response of bivalve
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Biochemical traits
• PL, membrane saturation
0 20
100
200
300
a
a
A
[EMn] (mg L-1)
Unsatu
ration index
0 20
100
200
300
a
bB
[EMn] (mg L-1)
Unsatu
ration index
M. edulis C. virginica
Part 3: Consequences of greening by marennine on the integrative response of bivalve
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Behavioral traits
• Relationship between EMn on the gill and valve opening
0 5 10 150.00
0.05
0.10
0.15
0.20C. virginica
M. edulis
R2= 0.166
R2= -0.793
B
Valve opening (°)
0.0 0.5 1.0 1.5 2.0 2.50.00
0.05
0.10
0.15
0.20 C. virginica
M. edulis
R2 = 0.661
R2 = 0.797
A
EMn (mg L-1)
EM
n g-1
wet w
eig
ht
Part 3: Consequences of greening by marennine on the integrative response of bivalve
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Valve activity experiment (short-term effect)
(n = 32) (n = 32)
• Installation of Hall’s element sensor
• Exposed to 0, 0.5, 1 and 2 mg L-1
• Recording of valve activity (in degree)
Acclimation phase
Post-accclimation phase
• EMn quantification on the gill
Sample preparation
1L
Part 3: Consequences of greening by marennine on the integrative response of bivalve
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SFG experiment
• Exposed to 0, 0.5, 1 and 2 mg L-1
• Measurement of:• O2 consumption• Clearance rate (CR)• Absorption rate (AR)• SFG
(n = 32) (n = 32)
Sample preparation
1L
Part 3: Consequences of greening by marennine on the integrative response of bivalve
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Lipids and FAs analysis (long-term effect)
• Lipid extraction • Separation of neutral (NL) and polar lipids (PL)• Methylation• Purification• Quantification in GC MS
• Min. 100 mg of tissue wet weight (gills - digestive glands)
Statistical analysis
- One-way ANOVA,
- Multivariate analysis of variance PERMANOVA+
(n = 16) (n = 16)
Part 3: Consequences of greening by marennine on the integrative response of bivalve
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Biochemical traits
• PL, membrane saturation
• Significant correlation, UI (unsaturation index) vs PUFA+ 18:2 n-6, 20:3 n-3, 22:6 n-cis
- 18:3 n-6
*
*
*
* **
**
*
0 20 40 60
18:2 n-6
18:3 n-6
20:3 n-3
20:5 n-3
22:6 n-3
SFA
MUFA
PUFA
EFA 0 mg L-1
2 mg L-1*
*
ns
*
*
ns
ns
ns
ns
A
M. edulis
(%)
Fatty a
cid
cla
ss
0 20 40 60
18:2 n-6
18:3 n-6
20:3 n-3
20:5 n-3
22:6 n-3
SFA
MUFA
PUFA
EFA 0 mg L-1
2 mg L-1
*
*
*
*
ns
ns
ns
*
ns
B
C. virginica
(%)
Part 3: Consequences of greening by marennine on the integrative response of bivalve