epidemicconsiderationsin shellfishrestoration effect of injection … pm/icsr05... · 2006. 2....
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Effect of in vivo pesticide exposure and injection of bacteria on immune gene expression in the Pacific oyster,
Crassostrea gigas
Béatrice Gagnaire1, Tristan Renault1, Hélène Thomas-Guyon2
and Thierry Burgeot3
ICSR 2005 – Pathology andepidemic considerations in
shellfish restoration
1IFREMER, Laboratoire de Génétique et Pathologie, La Tremblade, France2Université de La Rochelle, Laboratoire de Biologie et Environnement
Marins, France3IFREMER, Département Polluants chimiques, Nantes, France
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French oyster production
Marteilia
refringens
Bonamia
ostreae
Iridovirus
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Pollutants, diseases and oyster farming Rearing areas = estuary areas receiving pollutants
– Industry : heavy metals, PCBs
– Boats : PAHs
– Agriculture : pesticides
Crassostrea gigas
– Sedentary
– filter-feeding
Presence of infectious agents in C. gigas
– Rearing conditions
– No vaccination
Effects of pollutants on oyster defence capacities?
Relations between pollutants and susceptibility to diseases?Do n
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In vivo pesticide exposure
7 days : control (C) and pesticides (P) (3 experiments)
Mixture of 8 pesticides:
– Alachlor, metolachlor, atrazine, diuron, glyphosate,
terbutylazine, fosteyl aluminium, carbaryl
– Environmental concentrations (0.05 to 0.8 µg.L-1)
Flow cytometry: measurements of hemocyte
parameters (mortality, esterase, ROS, phagocytosis)Do n
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Results
Cell mortality: few variations
ROS and esterase positive cells : P<C after 3 days only
in the first experiment
Phagocytosis :
– P<C after 7 days of pesticide exposure
– Reproducible results for the 3 experiments
Decrease of phagocytosis activity due to pesticide
mixtureDo n
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Bacteria injection
After 7 days of pesticide exposure, injection on C and P of ASW or bacteria
– mixture of 2 Vibrio isolated from mortality events (2.107
or 4.107 bacteria/oyster)
Oyster mortality measurements
RNA extraction from hemocytes after 24 h post-injection
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1. Receptor binding 2. Transduction3. Cytoskeleton
modification
4. Post-phagocytosis degradations and
cellular protection mechanisms
Ficolin Vav-3 protein Ankyrin Isocitrate deshydrogenase
Galectin 4 Importin-α Cofilin pro-Cathepsin L
LBP/BPI protein ECSIT Filamin TIMP
LPS/ß-1,3-glucan protein DOCK 180 protein Rho protein SOD
c-Src kinase Lysozyme
Defensin
Defensin
Expression of genes involved in defencemechanisms (phagocytosis)
RT
Real-time PCR (Icycler, BioRad)
Comparison to a reference gene (elongation factor)
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0
10
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0 20 40 60 80
Temps (heures)
Po
urc
en
tag
e d
e m
ort
alité
T EDMAS
P EDMAS
T 2.10^7
P 2.10^7
**
**
*
*
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0 20 40 60 80
Temps (heures)
Po
urc
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tag
e d
e m
ort
alité
T EDMAS
P EDMAS
T 2.10^7
P 2.10^7
**
**
*
*
Time (hours)
Mo
rtality
(perc
en
tag
e)
T ASW
P ASW
0
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40
50
0 20 40 60 80
Temps (heures)
Po
urc
en
tag
e d
e m
ort
alité
T EDMAS
P EDMAS
T 2.10^7
P 2.10^7
**
**
*
*
0
10
20
30
40
50
0 20 40 60 80
Temps (heures)
Po
urc
en
tag
e d
e m
ort
alité
T EDMAS
P EDMAS
T 2.10^7
P 2.10^7
**
**
*
*
Time (hours)
Mo
rtality
(perc
en
tag
e)
T ASW
P ASW
C
C
Comparison between C+Vibrio and P+Vibrio Oyster mortality after injection
Higher mortality for contaminated oysters after
injection of 2.107 CFU only
2.107 CFU/animal 4.107 CFU/animal
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0 5 10 15 20 25Temps (heures)
Mo
rta
lité
(p
ou
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nta
ge
)
T EDMAS
P EDMAS
T 4.10^7
P 4.10^7
*
**
0
10
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40
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60
0 5 10 15 20 25Temps (heures)
Mo
rta
lité
(p
ou
rce
nta
ge
)
T EDMAS
P EDMAS
T 4.10^7
P 4.10^7
*
**
Time (hours)
Mo
rtality
(perc
en
tag
e)
T ASW
P ASW
0
10
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30
40
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60
0 5 10 15 20 25Temps (heures)
Mo
rta
lité
(p
ou
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nta
ge
)
T EDMAS
P EDMAS
T 4.10^7
P 4.10^7
*
**
0
10
20
30
40
50
60
0 5 10 15 20 25Temps (heures)
Mo
rta
lité
(p
ou
rce
nta
ge
)
T EDMAS
P EDMAS
T 4.10^7
P 4.10^7
*
**
Time (hours)
Mo
rtality
(perc
en
tag
e)
T ASW
P ASW
0
10
20
30
40
50
60
0 5 10 15 20 25Temps (heures)
Mo
rta
lité
(p
ou
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nta
ge
)
T EDMAS
P EDMAS
T 4.10^7
P 4.10^7
*
**
0
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40
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0 5 10 15 20 25Temps (heures)
Mo
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(p
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T EDMAS
P EDMAS
T 4.10^7
P 4.10^7
*
**
Time (hours)
Mo
rtality
(perc
en
tag
e)
T ASW
P ASW
C
C
P = CP > C
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Comparison between C+Vibrio and P+Vibrio Phagocytosis gene expression at 24 h post-injection (P vs C):
Up-regulation of genes with pesticides, whatever thedose of bacteria may be
2.107 CFU/animal 4.107 CFU/animal
Re
lative
exp
ressio
n o
ftr
an
scrip
ts(p
esticid
es +
2
.10
7 C
FU
)
0
2
4
6
8
10
12
14
16
18Id
h
Pro
CL
TIM
P
SO
D
Lysozy
me
Défe
nsin
e
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*
0
1
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Idh
Pro
CL
TIM
P
SO
D
Lysozy
me
Défe
nsin
e
*
*
*
*
***
Re
lative
exp
ressio
n o
ftr
an
scrip
ts(p
esticid
es +
4
.10
7 C
FU
)
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minate
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t auth
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ation
Discussion & Conclusion
Pesticides act on phagocytosis at cellular level and
may disrupt the immune response to an infection
Decrease of phagocytosis after a 7 day-contamination period:
– susceptibility of oysters to environmental contaminants
Oyster mortality higher for P only with 2.107
– 4.107: too elevated concentration to see differences
Up-regulation of genes at 24 h post-injection of Vibrio in P
compared to C:
– dysfunction of host immune response: harmful inflammation
Same gene response for both Vibrio concentrations:
– No difference for mortality, but response to disease
BUT only one experiment: need to confirm these results
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Thank you
for your
attention ! !
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