Molecular response of the Pacific oyster Crassostrea gigas to experimental hypoxia

Application of biomarkers on estuarian populations along the Atlantic coast of France

Molecular response of the Pacific oyster Molecular response of the Pacific oyster CrassostreaCrassostrea gigasgigas to experimental hypoxia to experimental hypoxia

Application of biomarkers on Application of biomarkers on estuarianestuarian populations populations along the Atlantic coast of Francealong the Atlantic coast of France

Elise David1, Arnaud Tanguy2, Adeline Hilaire1, Jean Laroche1,

and Dario Moraga1

1 Laboratoire des Sciences de l'Environnement Marin (LEMAR), UMR-CNRS 6539, Institut UniversitaireEuropéen de la Mer, Université de Bretagne Occidentale, Place Nicolas Copernic, 29280, Plouzané, France

2 Laboratoire Adaptation et Diversité en Milieu Marin, UMR-CNRS 7127, Station Biologique de Roscoff, B.P. 74, 29682 Roscoff cedex, France

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Introduction

Context:

Chemical pollution

Hydrocarbons, pesticides, heavy metals, …

Hypoxia

Nutrient inputs

Eutrophication

STRESS for organisms

RESPONSE of the organisms

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Introduction

• privileged place for anthropogenic contaminants producingactivities establishment (harbours, industry, etc.)

• high influence of catchment basins discharging pesticides, nutrients and other contaminants

• high biological interest (transition environment)

ESTUARIES

Need of evaluation of environmental stress on estuarian populations

toxicity and risk evaluation

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Introduction

Integration levels of stress response:

gene

cell

organism

population

functional groups

ecosystems

gene expression, genotoxicity, immunotoxicity

physiology

genetic polymorphism, life history traits

ecology

ALTERED GENE EXPRESSION = EARLY, FAST and SENSITIVEmeans of stress response detection

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Objectives:

� improve knowledge on molecular and physiological mecanisms of toleranceand adaptation to hypoxic stress

� develop new genetic biomarkers of environmental stress for evaluation ofenvironmental toxicity

Introduction

Approach :

1 Identification of genes implicated in hypoxia response

Realization of SSH (suppression subtractive hybridization) libraries after7-10 days and 24 days of hypoxia exposure : 30% of oxygen saturation

2 Study of expression kinetic of genes potentially regulated by hypoxia

after 3, 7, 10, 14, 17, 21 and 24 days of hypoxia exposure by semi-quantitative RT-PCR

3 Test and comparison of expression of environmental stress regulatedgenes on natural populations from contaminated estuariesby semi-quantitative RT-PCR

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Pacific oyster Crassostrea gigas:

- Filter feeder bivalve mollusc with large distribution in Atlantic French estuaries

- High economical interest

- Facility in sampling and laboratorymaintenance

- Model species in international programs

Biological model

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SSH method (Suppression Subtractive Hybridization)

Control oysters

Exposedoysters

Comparison of expressed genes

Up-regulated genes Down-regulated genes

7-10 and 24 days of exposure

Identification of genes potentially up- and down-regulated in hypoxiaexposure without previous knowledge of the genome

Digestive gland GillsMantle

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Results of SSH

Up-regulated genesafter 7-10 days

Up-regulated genesafter 24 days

Down-regulated genesafter 7-10 days

Down-regulated genesafter 24 days

Cytoskeleton, structure, matrix

Respiratory chain

Nucleic acids regulation

Detoxification

Amino-acids metabolism

Energetic metabolism

Protein regulation

Cellular communication, immune system,

receptor

Stress protein

Reproduction

Lipids metabolism

Development, differenciation

Ribosomal protein

Unknown functions

ESTs

615 sequences potentially regulated by hypoxia in C. gigas after 7-10 days and 24 days of exposure

Carbonic anhydraseGlutathione peroxidase

HSP70

SODHSP70

Delta-9 desaturaseMetallothionein

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0

2

4

6

8

10

12

14

0 3 7 10 14 17 21 24

Time of exposure (days)

Relative Expression (digestive gland)

Activation of carbonicanhydrase gene expression at7 days and after 21 days ofhypoxia exposure

Results of experimental hypoxia

exposed oysters

control oysters

0

0,5

1

1,5

2

2,5

3

3,5

4

0 3 7 10 14 17 21 24

Delta-9 desaturase

Carbonic anhydrase

Activation of delta-9 desaturase gene expression after 10-17 days of hypoxiaexposure

Same patterns of response in the 3 tissues

** *

*

*

*

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Toward a multi-stress approach

Considering expression of genes regulated in different environmental stress

?

HypoxiaHeavy metals

Hydrocarbons

Pesticides

Low salinity

Temperature changes

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• Belon estuary

• Vilaine estuary

• Loire estuary

• Gironde estuary

« reference estuary »

pesticides contamination +++

diffuse contamination ++

metal contamination +++

Multi-stress estuaries alongAtlantic French coast

Expression in estuarian natural populations

Natural populations sampled in january(reproductive rest period)

Belon

No data on oxygen saturation

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Results of expression in estuarian populations

0,0

0,5

1,0

1,5

2,0

2,5

Gironde Loire Vilaine Belon

Control at intermediate levelHigh expression in Vilaine

Higher level in Vilaine = Hypoxicstress ?

Expression down-regulated in experimental hydrocarbon exposure

--> low level in Gironde

Relative Expression

Delta-9 desaturase

*

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

Gironde Loire Vilaine Belon

Lower in contaminated estuariesthan in the reference

Low level due to chemicalcontaminants ?

Other influencing parameter ?Relative Expression

Carbonic anhydrase

***

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• 615 partial sequences of cDNA identified, corresponding to hypoxia potentiallyregulated genes under experimental conditions in C. gigas, involved in major physiological functions

• experimental hypoxia --> response in delta-9 desaturase and carbonicanhydrase gene expressionnatural estuarian environment --> more complex

• Concerns on significativity of gene expression as indicator of environmentalstress:

– Confusing effects between stressors

– What ecological signification of such a parameter considered separatly ?

Conclusions

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1. Comparison with summer samples (reproduction period)

2. Micro-arrays constructed with sequences obtained under experimentalexposure to: hydrocarbon

pesticideshypoxialow salinityand to come differential temperatures

--> comparison of expression patterns of hundreds of genes in the different estuaries

3. At last, study polymorphism of genes involved in environmental stress response (candidate genes)

--> study the possible link between expression level andgenotype for a same gene

--> relation between phenotype (expression, other physiologicalparameters) and genotype

Prospects

--> more integrated vision of adaptative strategies

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