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Genetically based resistance tosummer mortality in the Pacificoyster (Crassostrea gigas) and

initial related physiological characteristics .

Genetically based resistance toGenetically based resistance tosummer mortality in the Pacificsummer mortality in the Pacificoyster (oyster (CrassostreaCrassostrea gigasgigas) and ) and

initial related physiological initial related physiological characteristicscharacteristics ..

JF Samain, L.Degremont, P.Boudry, P.Soletchnik, S.Pouvreau, M.Ropert, E.Bedier, J.Haure, J.Moal, K.Costil, C.Lambert, V.Boulo, JL.Nicolas, F.Le Roux, T.Renault, T.Burgeot, C.Bacher, J.Knoery as representatives of Morest partners

Styli 2003 Npumea

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er…Mortalities

ReportedReported in in JapanJapan as as soonsoon as 1940, in as 1940, in NorthNorth AmericaAmerica1950 1950 andand in France 1990in France 1990

30 30 àà 60% 60% dependingdepending on areason areasA large A large diversitydiversity of sitesof sites

DistributedDistributed in patches, in patches,

Are Are associatedassociated withwith summersummer temperaturestemperatures andandreproductive reproductive periodperiodMostlyMostly juvenilesjuveniles, but , but alsoalso 11--2 2 yearyear oldold oystersoystersNot Not totallytotally explainedexplained by a single by a single pathogenpathogen (virus or (virus or

vibriovibrio) )

Styli 2003 Npumea

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GeneticsAge

PhysiologyDefenceNutrition

HOST PATHOGEN

ENVIRONMENT

Genetics

VirulenceNutrition

Temperature, Salinity, O2 , Trophic conditions, Stress and Pollution

1rst 1rst statementstatement : : SummerSummer mortalitiesmortalities : a : a multifactorialmultifactorial systemsystem

MOREST : a cooperative project

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er11--Temperature 19Temperature 19°°CC

44-- PhytoPhyto levellevel

66--Opportunistic Opportunistic PathogensPathogens

Mortality Date

33--output output fromfromwatershedswatersheds A

mpl

ifica

tion

Environment

22--ReproductionReproduction

77--geneticsgenetics

55--StressStressand

and

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11-- GeneticGenetic effecteffect : CharacterizationCharacterization in 3 in 3 fieldfieldareas in Franceareas in France

Marennes-Oléron

Rivière d’Auray

Baie des Veys

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25 27 32 35 39 41 43 48 CN2

7

8

9

10

11

12

P2

CN2

0

10

20

30

40

50

60

70

80

90

100

Mortality rate (%)

Female

Male

1- Genetic effect : First generation2001

11-- GeneticGenetic effecteffect : : FirstFirst generationgeneration20012001

BDV

Ronce

La Trinité

Juveniles

Control : wild spat

3 x 15 = 45 3 x 15 = 45 familiesfamilies on on

3 sites3 sites

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Susceptible: 43 %Resistants : 7 %Control 2N: 24%Control 3N: 7%

Wild spat (15%)

Auray site

0

10

20

30

40

50

60

70

80

90

Z L P F C AD

AB

M J X U W T Q CNA

CNF

AC

R D S I Y H A N K

Mor

talit

és (%

)

Genetic effect : divergent generation G2

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erGenetic effect : Field performancesNovember 03 :GeneticGenetic effecteffect : Field performances: Field performancesNovemberNovember 0303 ::

G3SD (site Auray) :G3SD (site Auray) :

LivestockLivestock MortalityMortalitySusceptiblesSusceptibles 72,5 % 72,5 % ResistantsResistants 27,1 %27,1 %Control 2NControl 2N 47,8 %47,8 %

GrowthGrowth14,7 g14,7 g12,9 g12,9 g15,7 g15,7 g

YieldYield0,8 %.j0,8 %.j--11

3,3 %.j3,3 %.j--11

1,8 %.j1,8 %.j--11

- Positive response to selection.- High realized heritability.-- No No effecteffect ofof selectionselection on on growthgrowth performance.performance.-- SignificantSignificant positive positive effecteffect ofof selectionselection on on yieldyield..

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Sensitive

Resistant

R R andand S S oysteroyster comparisoncomparison

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0

25

50

75

100

Gon

ad o

ccup

atio

n (%

)

Sens itive familyResistant family

APR. MAY JUNE JULY AUGUS T S EPT. O CT. NOV. DEC

*

*

*

*

*

*

*

*

*

*

0%

10%

20%

30%

May-1 May-31 June-30 July 30 August 29

Cum

ulat

edm

orta

lity

(%)

-- TheThe reproductive reproductive strategystrategy appearedappeared differentdifferent for R for R andand S. S.

-- S S hadhad a a higherhigher reproductive effortreproductive effort

-- R R hadhad a total a total spawningspawning contrarycontrary to Sto S

-- MortalityMortality affectedaffected SS

1- Reproduction: R and S quantitative histology

11-- Reproduction: R Reproduction: R andand S quantitative S quantitative histologyhistology

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Phyto Repro

2- Nutrition : food level and reproduction22-- Nutrition : Nutrition : foodfood levellevel andand reproductionreproduction

CN1

CN3

CN2 ?

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0

20

40

60

80

Gon

advo

lum

e/to

tal f

lesh

vol.

(%)

R

S

April May June July August0

20

40

60

80

Gon

advo

lum

e/to

tal f

lesh

volu

me

(%)

R

S

April May June July August

CN1 CN3*

- Reproduction intensity increased with food level

- R and S had similar reproductive intensity in low foodconcentration

- S had a higher reproductive intensity than R at highfood concentration and demonstrated partial spawnings

FoodFood levellevel andand reproduction reproduction intensityintensity

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Phyto Repro Energy Infection

3- R and S energy balance?33-- R R andand S S energyenergy balance?balance?

? ?

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Consumption = f(food level) Consumption = f(food level) No differences between phenotypes R and SNo differences between phenotypes R and S

0

20

40

60

80

100

120

Con

sum

ptio

nra

te (

J.h-1

.g-1

)

CN1RCN3RCN1SCN3S

May June July August

CN3

CN1

ConsumptionConsumption

S

R

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0

20

40

60

80A

bsor

ptio

n ef

ficie

ncy

(%)

RCN1SCN1RCN3SCN3

May June July August

* CN3

CN1

Absorption efficiencyAbsorption efficiency

-- AE decreased in July (19AE decreased in July (19°°C) and with food levelC) and with food level

-- No differences between R and S (except S in June)No differences between R and S (except S in June)

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0,5

0,55

0,6

0,65

0,7

0,75

0,8

0,85

0,9

15-avr 12-m ai 10-juin 17-juil 14-août 10-sept

AEC

CN3 RCN3 SCN1 RCN1 S

Adenylic Energy charge

Storage consumptionStorageStorage consumptionconsumption

R and S :

- No significant difference in glycogen utilisation

- Nor in Adenylic Energy Charge (AEC)

0

5

10

15

20

25

30

35

April May June July Aug. Sept.

% c

arbo

hydr

ate

/ DW

CN1 RCN1 SCN3 RCN3 S

Glycogen (in mg glucose)

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-- Respiration S > Respiration RRespiration S > Respiration R-- Oxygen consumption in CN3 > Oxygen consumption in CN1 Oxygen consumption in CN3 > Oxygen consumption in CN1

0

2

4

6

8

10

Oxy

gen

cons

umpt

ion

rate

(J.

h-1.g

-1)

CN1RCN3RCN1SCN3S

May June July August

**

*

*

Oxygen consumptionOxygen consumption

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0

5

10

15

20M

orta

lity

cum

ulat

ive

rate

(%)

RCN1RCN3SCN1SCN3

May June July August

Scope for growthScope for growthSFG (J.h-1.g-1) CN1R CN1S CN3R CN3S

JUNE 10.1 15.3 25.3 16.0

JULY 5.9 7.6 1.6 -0.7

AUGUST 12.0 15.1 13.3 11.0

R CN1

S CN1

S CN3

R CN3

R CN1

S CN1

S CN3

R CN3

R CN1

S CN1

S CN3

R CN3

- SFG decreased in July more at high food level than at lower one

- SFG small differences between R and S, but significant mortality

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GLUCOSE

Glucose 6Phosphatase ? Hexokinase

Glycogène synthétase

G6PdesH2 PGMPENTOSES GG LL UU CC OO SS EE 66 -- PP G1P GLYCOGÈNE

Glycogène phosphorylase

PHOSPHOENOLPYRUVATE

PEPCK PK

PYRUVATE ACIDES AMINESLACTATE

NADPH

LIPIDES AcetylcoAacides gras synthetase

Krebbs cycle

Gene expression for Glucose 6P metabolic pathways

- Higher expression of genes leading to Glucose 6P in R than in S oysters during the critical period

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er4- Stress :

Effect of pesticides and hypoxia

44-- Stress :Stress :

EffectEffect of pesticides of pesticides andand hypoxiahypoxia

Resistant "R"

control

PesticidesNormoxia

J0 J1 J3 J71.8

1.9

2.0

2.1

2.2

2.3

2.4

2.5

2.6

2.7

2.8

2.9

3.0

Log

(Cat

alas

e ac

tivity

in µ

mol/

min

/mg

of p

rote

in)

Hypoxia

J0 J1 J3 J7

Susceptible "S"

Control

Pestic idesNormoxia

J0 J1 J3 J71.8

1.9

2.0

2.1

2.2

2.3

2.4

2.5

2.6

2.7

2.8

2.9

3.0

Log

(Cat

alas

e ac

tivity

in µ

mol

/min

/mg

of p

rote

in)

Hypoxia

J0 J1 J3 J7

* *

*

- Catalase activity was lower in S oysters

- Only R oysters reacted to pesticides and oxygen availability

- In general S oysters were not able to react to stress

Resistant R Susceptible S

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Repro hemocyte Infection

5- Are defence affected :

Hemocyte activities?

55-- Are Are defencedefence affectedaffected : :

HemocyteHemocyte activitiesactivities??

?

Energy

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erHemocyte response

- Reactive Oxygen Species ROSHemocyteHemocyte responseresponse

-- ReactiveReactive OxygenOxygen SpeciesSpecies ROSROS

Hyalinocytes

0

50

100

150

200

April

May

June

July

AugustSep

tember

RO

S pr

oduc

tion

(arb

itrar

y un

it)

R

S

Granulocytes

0

50

100

150

200

250

300

April

May

June

July

AugustSep

tember

RO

S pr

oduc

tion

(arb

itrar

y un

it)

R

S

- S had a significant higher ROS levels than R beforemortality

- No difference between CN1 and CN3

lfrem

erHyalinocytes and phagocytosisHyalinocytesHyalinocytes andand phagocytosisphagocytosis

0

5

10

15

20

25

April May June July August September

Phag

ocyt

osis

(% a

ctiv

e ce

lls)

R

SHyalinocytes

0,E+00

1,E+05

2,E+05

3,E+05

4,E+05

5,E+05

6,E+05

7,E+05

April May June July August September

conc

entr

atio

n (c

ell p

er m

L) R

S

* *Phagocytosis

Hyalinocytes

- Hyalinocytes concentration and phagocytosis increased more in S than in R before mortality

Question : were S oysters infected before R ones?

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Concentration Bacterienne dans l'hemolymphe

1,E+03

1,E+04

1,E+05

1,E+06

14m

a

05ju

10ju

16ju

27ju

17-ju

il

10-s

ept

Dates de prelevements

Con

cent

ratio

n ba

ct/m

l

R15R70S15S703N15

BacteriaBacteria in in haemolymphhaemolymphSpawning

-- BacteriaBacteria concentration in concentration in hemolymphhemolymph increasedincreased duringduringspawningspawning periodperiod afterafter mortalitymortality withoutwithout detrimentaldetrimental effecteffect..

44-- Field infection Field infection processprocess : : bacteriabacteria andand spawningspawning

Mortality

-- WhatWhat about partial about partial spawningspawning??

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R and S oysters had mainly a difference in reproductive intensity and spawning strategy

However, S and R oysters demonstrated similar energy balance problems at the end of gametogenesis, that cannot explainobserved differences in R and S mortality rates.

Preliminary conclusions :

One month before mortality :

S oysters had partial spawnings

Absorption efficiency decreased drastically in S before R oysters

S oysters had a lower expression of genes controling G6P

S oysters could not react to stress

hemocyte characteristics suggested a higher infection level of S oysters compared to R ones.

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In relation to partial spawning of S oysters onemonth before mortality allowing their infection by opportunistic pathogens

The genetic origin of the R and S difference in reproductive strategy, in glucose mobilisation, absorption and in defence, is studied using molecular tools as SSH and micro-array technologies.

A difference in infection period and intensity ofS oysters by opportunistics pathogens

Inducing or associated to a limited capacity of S oysterto provide glucose 6P to metabolism, to absorb nutriments and to react to environmental stresses and infections.

Possible interpretation :

Styli 2003 Npumea

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erPartnership

PhysiologyLaboratoire Conchylicole de Poitou-Charentes (LCPC), IFREMER,

La TrembladeLaboratoire Conchylicole de Bretagne (LCB), IFREMER, La

Trinité sur mer Laboratoire Conchylicole des Pays de la Loire (LCPL), IFREMER,

BouinLaboratoire Conchylicole de Normandie (LCN), IFREMER,Port en

BessinCentre de Recherche en Ecologie Marine et Aquaculture

(CREMA), CNRS-IFREMER, L’HoumeauLaboratoire de Physiologie des Invertébrés (LPI), IFREMER,

PlouzanéLaboratoire de Biologie et Biotechnologies Marines (LBBM),

Université de CaenStation de Biologie Marine, Muséum National d’Histoire Naturelle,

Concarneau

PathologyLaboratoire de Génétique et

Pathologie (LGP), IFREMER, La Tremblade

Laboratoire de Biologie et d’Environnement Marins (LBEM), Université de la Rochelle

EcotoxicologyDEL/PC, IFREMER, Nantes(en attente 2002)Laboratoire des sciences de

l'environnement marin (LEMAR), Université de Bretagne Occidentale ; Institut Universitaire Européen de la Mer, Plouzané

ProfessionStructures régionales

(SMIDAP, CREAA, SMEL, CEPRALMAR)

Ecloseurs-nurseursProducteurs

GeneticsLaboratoire de Génétique et Pathologie

(LGP), IFREMER, La TrembladeSyndicat des Sélectionneurs Avicoles et

Aquacoles Français (SYSAAF), RennesLe Centre Régional d’Expérimentation et

d’Application Aquacole (CREAA), Le Château d’Oléron

ImmunologyDéfense et Résistance chez les

Invertebrés Marins (DRIM), CNRS-IFREMER, Université Montpellier 2

Laboratoire des sciences de l'environnement marin (LEMAR), Université de Bretagne Occidentale; Institut Universitaire Européen de la Mer, Plouzané

Environnement DEL-RA réseaux et

modélisation

15 labs and 70 contributors

Manythank

s to all

of the

MOREST communi

ty