Genetically based resistance to summer mortality in …l fr e m e r Genetically based resistance to...
Transcript of Genetically based resistance to summer mortality in …l fr e m e r Genetically based resistance to...
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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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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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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
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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