Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue...

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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

Transcript of Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue...

Page 1: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 2: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

Up and down postgraduation

Page 3: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,
Page 4: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 5: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

Research collaboration to minimize the gap..

Page 6: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,
Page 7: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

• 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

Page 8: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 9: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 10: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

• 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…

Page 11: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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?

Page 12: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 13: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

Persistency of greening

No de-greening after 12 weeks

Marennine fixation is persistent

Part 1: Greening of oyster by H. ostrearia

Page 14: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

Marennine fixation on the gills

Marennine fixation in mucocytes

Part 1: Greening of oyster by H. ostrearia

Page 15: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 16: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

Photo: JD Billaud

Page 17: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

• 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

Page 18: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

Experiment B:

Video endoscopy observation

Rejection of larger cells

Part 2: Role of size in preingestive selection of H. ostrearia in C. gigas

Page 19: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,
Page 20: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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)

Page 21: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 22: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 23: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

*

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

Page 24: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 25: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 26: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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.

Page 27: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,
Page 28: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,
Page 29: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

Related publications

Contact information:[email protected]://www.researchgate.net/profile/Fiddy_Prasetiya

Book chapter:Marennine-Like Pigments: Blue Diatom or Green Oyster Cult?

Page 30: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 31: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 32: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 33: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 34: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 35: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 36: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 37: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

• 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

Page 38: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 39: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

Marennine fixation on the gills

Marennine fixation in mucocytes

Part 1: Greening of oyster by H. ostrearia

Page 40: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

Bakteri patogen kerang

Antibacterial activity in vitro

Prasetiya et al., 2018

Aktivitas Biologis Pigmen Marenin untuk Budidaya Perairan

Page 41: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

Kerang abalone (Pangandaran)

Kerang mutiara (Bali)Udang vanname (Pangandaran)

Larva udang (Bali)

Page 42: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 43: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 44: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 45: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 46: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 47: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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

Page 48: Blue diatoms as a potential for sustainable shellfish aquaculture Semba Prasetiya.pdf · Blue diatoms as a potential for sustainable shellfish aquaculture By Fiddy S. Prasetiya*,

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