Modulation of Reactive Oxygen Species (ROS)

production by hemocyte of two aquacultured bivalves,

clam and oyster, faced to pathogenic Vibrio.

Modulation of Reactive Oxygen Species (ROS)

production by hemocyte of two aquacultured bivalves,

clam and oyster, faced to pathogenic Vibrio.

Christophe LAMBERT*, Philippe SOUDANT, Marine JEGADEN,

Yannick LABREUCHE, Mouna RIFI, Jeanne MOAL and Christine PAILLARD.

UMR UMR PhysiologiePhysiologie des des MollusquesMollusques MarinsMarins

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IntroductionIntroductionIntroduction

�Question: study the interaction between bivalve hemocyte and pathogenic bacteria.

�Two Models

�Vibrio tapetis – clam Ruditapes phillipinarum

�Vibrio aestuarianus – oyster Crassostrea gigas

�Hemocyte parameter

� Reactive oxygen intermediates (ROI) and Reactive Nitrogen intermediates (RNI) production: microbicidal

��Question: study the interaction between bivalve Question: study the interaction between bivalve hemocyte hemocyte

and pathogenic bacteria. and pathogenic bacteria.

��Two Models Two Models

�Vibrio tapetis – clam Ruditapes phillipinarum

�Vibrio aestuarianus – oyster Crassostrea gigas

��HemocyteHemocyte parameterparameter

� Reactive oxygen intermediates (ROI) and Reactive Nitrogen intermediates (RNI) production: microbicidal

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Vibrio aestuarianus / Crassostrea gigasVibrio aestuarianusVibrio aestuarianus / / Crassostrea gigasCrassostrea gigas

0

0 ,2

0 ,4

0 ,6

0 ,8

1

1 ,2

1 ,4

1 ,6

100% mortality

50% mortality

01/323 02/103 SW01/32 01/64 01/14002/9202/41

Injection: 108cfu in oyster muscle

Mortality: 7 days post injection

mo

rtality

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Vibrio tapetis/ Ruditapes philippinarumVibrio tapetisVibrio tapetis/ / Ruditapes philippinarumRuditapes philippinarum

Injection: 5.107cfu in clam muscle

Mortality: 7 days post injection From G. Choquet, Phd thesis, 2004

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Phagocytosis and

ROI- RNI production

Phagocytosis andPhagocytosis and

ROIROI-- RNI productionRNI production

NO synthase NADPH oxydase

O2OH-arginine

O2-NO

NO2

NO3

H2 O2

ONOO-

peroxynitrite

H O + O

Catalase

SOD

From Paillard, 2004

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Methods: ROI- RNI production measured

by flow cytometry (FCM)

Methods: Methods: ROIROI-- RNI production measured RNI production measured

by flow by flow cytometrycytometry (FCM)(FCM)

DCFHDCFH--DADA((2’7’2’7’--dichlorofluorescin dichlorofluorescin diacetatediacetate))

hemocytehemocyte

DCFHDCFHnon fluorescentnon fluorescent

DA

PhagocytosisPhagocytosis

zymosanzymosan

PMA...PMA...

DCFDCF

ROI/ RNI: ROI/ RNI: HH22OO2 2 / O/ O22oo-- / NO/…/ NO/…

From Bass et al., 1983; Curtin et al., 2002

FL1FL1

detectiondetection

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Clam – Oyster: ROI-RNI productionClamClam –– Oyster: ROIOyster: ROI--RNI productionRNI production

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ROI-RNI production survey in the fieldROIROI--RNI production survey in the fieldRNI production survey in the field

Clam: Ruditapes philippinarumGolfe du Morbihan (Brittany)

Oyster: Crassostrea gigasAuray river (Brittany)

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In vitro effect of live Vibrio tapetis on clam ROI-RNI production

In vitroIn vitro effect of liveeffect of live Vibrio tapetisVibrio tapetis on on

clam ROIclam ROI--RNI productionRNI production

Bacteria hemocyte ratio HK: heat killedDo n

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In vitro effect of live Vibrio aestuarianus on oyster ROI-RNI production

In vitroIn vitro effect of live effect of live Vibrio aestuarianusVibrio aestuarianus on on

oyster ROIoyster ROI--RNI productionRNI production

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In vitro effect of extra cellular product (ECP) of

Vibrio aestuarianus on oyster ROI-RNI production

In vitroIn vitro effect of extra cellular product (ECP) of effect of extra cellular product (ECP) of

Vibrio aestuarianusVibrio aestuarianus on oyster ROIon oyster ROI--RNI productionRNI production

Granulocytes

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ConclusionConclusionConclusion

�Two models: two bivalves - two Vibrio species

� opposite effect on the defense system: ROI-RNI production

�Each bacteria develops its own « methods » to disturb its host

�We need more basic research to analyze the variation of the immune status of bivalve in the field.

��Two models: two bivalves Two models: two bivalves -- twotwo VibrioVibrio speciesspecies

� opposite effect on the defense system: ROI-RNI production

��Each bacteria develops its own «Each bacteria develops its own « methodsmethods » to disturb its » to disturb its

hosthost

��We need more basic research to analyze the variation of We need more basic research to analyze the variation of

the immune status of bivalve in the field. the immune status of bivalve in the field.

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Inhibition of NO-synthase and NADPH-oxidaseInhibition of NOInhibition of NO--synthasesynthase and NADPHand NADPH--oxidaseoxidase

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Hyalinocytes

Granulocytes

Agranulocytes

Granulocytes

Hyalinocytes

Agranulocytes

ROI- RNI production: Flow Cytometry analysisROIROI-- RNI production: Flow RNI production: Flow CytometryCytometry analysisanalysis

ComplexityF

luo

resc

ence

Fluorescence FL1 Oyster: Crassostrea gigasDo n

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