Justine MARCHAND

Assistant Professor
EA 2160 Sea, Molecule, Health University of Maine justine.marchand@univ-lemans.fr

Genetic adaptation p and responses to environmental stress in marine organisms

Understand genetic adaptation and estimate effects of environmental stress on marine organisms and populations Create potential tools to evaluate coastal degradation and their ability for restoration

Fields:
Molecular biology Gene characterization Polymorphism mRNA expression (QPCR) toward Transcriptomic siRNA

Model species : Fish Bivalves Crustaceans C t Microalgae

Cerastoderma glaucum Cerastoderma edule Platichthys flesus

Bythograea thermydron

C Cyanagraea praedator d

Carcinus maenas Phaeodactylum tricornutum

Segonzacia mesatlantica

Experimental stress : Pesticides P i id Metals Effluents Endocrine disruptors Light

Field studies : Hydrothermal y vents Estuaries of the Atlantic Coast Sites from the Golf of Gabs (TunisiaGenera)

Different levels of study

day life several generations

MOLECULE CELLULE
Physiological regulation

INDIVIDUAL
Phenotype modifications

POPULATION
Population genetic and dynamic

COMMUNITY
Species diversity

TOXICOLOGICAL signification

Integrated approach

ECOLOGICAL signification

day

life

several generations

MOLECULE CELLULE
Physiological regulation

INDIVIDUAL
Phenotype modifications

POPULATION
Population genetic and dynamic

COMMUNITY
Species diversity diversity

Genetic polymorphism ( ll (allozyme, DNA)

Ca t Can the e polymorphism poly o p s associated assoc ated to particular pa t cula loc loci have ave a functional role in contaminated systems ? Can p particular g genetic variants be associated to resistance or sensibility to stress ?

DNA polymorphism Enzymatic y polymorphism p y p PGM (PhosphoGlucoMutase)

PGI (PhosphoGlucose Isomerase) MPI (Mannose Phosphate Isomerase) IDH (Isocitrate DesHydrogenase)
General metabolism glycolysis
exon 1
(96) (200)

exon 2
(140)

(97)

exon 3

(80) (79) (117) (95) (101) (86) (3)

exon 4

exon 5

exon 6

SSCP
( (Single l Strand d Conformation f Polymorphism l h )

AAT(Aspartate AminoTransferase)

Allle 110 Allle 100 Alll 85 Allle

Profil dlectrophorse enzymatique (PGM)

GAPDH BHMT PGDS P53 CK

gnotypes

sequencing i Identification of the different alleles

Sampling sites

5 estuaries sampled
Ster of Lesconil Seine Loire Gironde Vilaine reference site Diffuse contamination ++++ Diffuse contamination ++ Heavy metals +++ pesticides +++

 Weak heterozygosity in contaminated systems/reference Genetic differentiation (Fst) reference/contaminated systems Higher g allelic frequency q y of p particular alleles in contaminated systems/"reference" Phosphoglucomutase p53 BHMT PGDS

selective pressure ? direct/indirect ? Allele associated to "resistance" in contaminated systems ?

day

life

several generations

MOLECULE CELLULE
Physiological regulation

INDIVIDUAL
Phenotype modifications

POPULATION
Population genetic and dynamic

COMMUNITY
Species diversity diversity

Physiological parameters Fecundity Condition index Chromosomal damage (flow cytometry) Growth rate (otolith)

Growth rate
Ster Loire GirondeVilaineSeine Ster

Condition index
Ster L i Loire Gironde Vilaine Seine

fecundity
Loire Gironde Seine

In contaminated sites :

of parameters linked to fitness

Energetic cost linked to exposition to contaminants ?

Genotoxicity : chromosomal damage

Loire Ster Gironde Vilaine Vilaine Seine

No clear tendancy

Selective pressure acting on individuals able to maintain DNA integrity (protection and/or reparation) ?

Coupling genotypes-physiological parameters

Differential physiological performances between individuals carrying different genotypes ?

longueur g

Htrozygotie

Loire

Coupling genotypes-contaminant bioacumulation

Differential bioaccumulations between individuals carrying different genotypes ?

Loire

Integrated approach

Individual responses
Coupling genotypes-phenotypes
Allelic variants Better ability to maintain DNA integrity in contaminated systems

Population responses

PGM-85 Energy production P53-A Genome integrity BHMT3-A BHMT3 A PGDS-A metabolism detoxification resistant alleles (selection ?) Higher frequency in contaminated systems

Metabolism orientated toward survival

PGM-85 PGM 85

day

life

several generations

MOLECULE CELLULE ELL LE

Physiological regulation

INDIVIDUAL
Phenotype modifications

POPULATION
Population genetic and dynamic

PEUPLEMENT
Species diversity

Particular proteins or modifications of gene expression

Important components of organisms acclimatization and/or adaptation to environmental changes Usually precede manifestations of strong functioning alterations markers of early stress

 Gene characterization Global approach

- Subtractive Suppressive Hybridization (SSH) for identification - Gene expression (QPCR) for validation

A priori approach
- Oxidative stress (SOD, catalase, GPx) - Metallothioneins - MultiXenobiotic Resistance (MXR) - Heat Shock Proteins - Endocrine disruptors (Vitellogenin, Estrogen Receptor)

Example : flounder exposition to pesticides

~ 270 sequences of genes potentially regulated by pesticides on flounders were identified

Up regulated genes Up-regulated

NADH dehydrogenase Elongin c

Down regulated genes Down-regulated

GAPDH Insulin-like growth factor

ADI

EST Energetic metabolism General metabolism

EST

ADI

Cytochrome c oxydase ATP synthase Hemoglobin C Cysteine i proteinase i Transferrine

Immune system Proteins of regulation P t i of Proteins f structure t t EST Proteins of transport Ribosomal proteins EST (Expressed Sequence Tags) EST

Glyphosate

Glyphosate

Creatine kinase Prostaglandine D synthase B t i Betaine h homocystine ti methyltransferase th lt f Carboxylestrase RACK

ENERGY PRODUCTION DETOXIFICATION CELLULAR PERTURBATIONS AND TUMOR DEVELOPMENT

Validation of the regulation in EXPERIMENTAL CONDITIONS

Cytochrome c oxydase

Up-regulated U Upl d liver

on 14 different genes on 3 tissues (liver, muscle, gills)

Up-regulated Upliver
reference ADI Glyphosate

Relative R expr ression

Elongin C

Validation Val dat on of the regulat regulation on Up-regulated Upli liver

BHMT

Tissu-dependant p expression p
liver = target of the regulation

Time-dependant p expression p

Explorations in FIELD
VILAINE estuary pesticides juveniles Gene expression femelles males

Cytochrome c oxydase

ATP synthase

WINTER

SUMMER

VILAINE (pesticides) and STER (reference)

BHMT

Results in accordance with those obtained in experimental conditions

Relative expre ession

WINTER

SUMMER

WINTER SUMMER

WINTER SUMMER

Project n6 of GENERA The use of two bivalves Ruditapes p decussatus and Cerastoderma glaucum in pollution biomonitoring: effects of metals and endocrine disruptors on the stress response and reproduction based on biomarkers
N National ti lM Museum of f Natural N t l Hi History t (F (France) ) Le Mans University (France) Sfax University (Tunisia)

G lf of Gulf fG Gabs b
Increasing industrial ad urban activities SIAP Phosphate treatment plant (phosphogypse) High contamination by heavy metals In particular Cd

Biomarkers studied
Metallothioneins
able to bind both physiological and xenobiotic heavy metals (high cysteine content)

MultiXenobiotic Resistant Protein (Pgp-1)

transmembrane protein transporter that acts by actively transporting toxic compounds out of the cell

Heat Heat-Shock Shock Proteins 70

ubiquitous and universal cytosolic chaperones, involved in the folding/refolding of newly synthesized and damaged proteins

Cytochrome oxidase I
last enzyme of the respiratory electron transport chain of mitochondria

 Superoxide dismutases (MnSOD and CuZnSOD) Catalase

antioxidant enzymes that limit reactions brought about by endogenous and exogenous reactive oxygen species. Minimize cell oxidative damage

Biological model
Model species : The cockle Cerastoderma glaucum

Mollusca bivalvia Cardiidea Sedentary and filter feeding Lives in the superficial sediment Suitable for field and laboratory experiments common in Mediterranean Sea validated as a bioindicator organism reflecting the pollution state of the Tunisian Coast

1st step: cDNA sequencing of the biomarkers

Target genes
MXR CuZnSOD MnSOD Catalase HSP70 COI MT

Housekeeping genes
actin -tubulin -tubulin EF1a

Genbank accession N

Analyses performed
Stress on stress tests Each sampling time: Condition index Cadmium contents Gene expressions
T Target t genes
MXR CuZnSOD MnSOD Catalase HSP70 COI MT

One individual CORRELATIONS

H Housekeeping k i genes
actin -tubulin -tubulin EF1a

Experimental exposure to CdCl2

C t lt Control tank k C1 tank = 50 g/L C2 tank = 5 mg/L

10

15

18 days

0.5

200 samples /tank

Plateform presentation on Wednesday 10h

Control

CdC1

CdC2

Control

CdC1

CdC2

Correlations: Principal Component Analysis

+ time of exposure (p<0.02)

COI: + C1 time of exposure

Correlations: Projections of individuals

Experimental exposure to effluents

Control l F1 F2

Ellouza Sidi Mansour Gargour SIAP

1/2

days

Field sampling

Ellouza Sidi Mansour Gargour SIAP

Poster session

The regulation of Carbon metabolism in marine microalgae

EffectsoflightstressonmRNAexpressionofenzymesoftheCarbon metabolisminPhaeodactylumtricornutum

30mol/(m2.s):lowlightintensity 300mol/(m2.s):optimumlightintensity 1000mol/(m2.s) s):highlightintensity

300/1000
xylulose-5-P y

Cytosol
RPE ribulose-5-P ribulose 5P PGDH gluconate-6-P PGL
ribulose-5-P ribulose 5P
RPE RPI PRK

Chloroplaste
2-phosphoglycolate O2
Rubisco PGP

glycolate
GDH1

ribulose-1-5-bisP CO2

glycolate
GK ????

glyoxylate MLS

xylulose-5-P TKL fructose-6-P

ribose-5-P

3-phosphoglycerate
PGK 1-3-bisP-glycerate

glycerate

isocitrate
PGAM 2-phosphoglycerate

malate

TKL

6-P-gluconolactone GPDH D-glucose-6-P GPI fructose-6-P

FBP PFK

Sedoheptulose-7-P p TKL TKL TAL erythrose-4-P

GAPDH

Cycle glyoxylate succinate ICL

glyceraldehyde-3-P TPI enolase dihydroxyacetone-P

Acetyl CoA

fructose-1-6-bisP GPI glucose-6-P PGM

FBA PYC2 HCO3 Lipides

OAA PPDK pyruvate PK PEP AroA AroB EPSP Shikimate

O idation Oxidation Acides gras

glucose-1-P

Peroxisome

erythrose-4-P erythrose 4P

TAL

PEPC1

PEP+ HCO3

OAA

sedoheptulose-7-P PEP SBP sedoheptulose-1-7-bisP AL dihydroxyacetone-P dihydroxyacetone P TPI FBA PFK fructose-1-6-bisP FBP

PK

glycerate

glycolate
GDH2

pyruvate
HCO 3 pyruvate
PK

HPR

enolase? 2-phosphoglycerate PGAM? 3-P-glycerate PGK 1,3-bisP-glycerate GAPDH glyceraldehyde-3-P

hydroxypyruvate
SPT-AGT PYC1 CO2 NH3

glyoxylate glycine
SPT-AGT

serine

PEP
Enolase

GDC-SHMT

2-phosphoglycerate
PGAM

PEPCK

CO2 OAA
MDH

3-phosphoglycerate
PGK

PEPC2

citrate

1,3-biphosphoglycerate
GAPDH

malate
ME1

TCA

PGM GPI fructose-6-P glucose-6-P glucose-1-P

glyceraldehyde 3-phosphate

CO2

succinate

Mitochondrie

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