Studying host microbial interaction in larviculture:

the way forward

Peter Bossier

Laboratory of Aquaculture & Artemia Reference Center,Ghent University, Belgium

UGent R&D Aquaculture Consortium meeting, June 2008

How to study host-microbial interactions?

Known micro-

organisms

Host

environment

Hostsimplification

complex gnotobiotic

reality?

MC

Gnotobiotic systems

• Mammals• Zebrafish• Artemia• Brachionus• Seabass and Cod (under development)

Gnotobiotic Artemia: GART

Instar I

Instar II

Decapsulated cysts

Decapsulation

Axenic conditions

Hydrated cysts

Non-axenic conditions

20 nauplii counted for the

Experiments

Processes

• Quorum sensing• Polyhydroxybutyric acid• Yeast-bound glucan• Heat shock proteins

Quorum sensing

What is quorum sensing?

AHL

luxI

luxR

Target gene

Biofilm formation,

Bioluminescence,

Toxin production

AHL-mediated pathway in Gram-negative bacteria

N O

OH

H OH O

Quorum sensing and quenchingin the

Brachionus - Turbotfood chain

Objectives

• Study the effect of AHL on turbot larval survival

• Study the effect of an N-acyl homoserine lactone(AHL) degrading bacterial mixtures (EC5) in turbotlarviculture.

Experiment setup

• Egg disinfection: 0.005% glutaraldehyde + 50 mg l-1 rifampicin

• Temperature: 16°C

• Light intensity: 14 lux

• Mild aeration for cones, no aeration for beakers

Experiment outline

Exp Treatment

1 2 3 4 5 6

1 Control AHL addition

Rifampicin Rifampicin +AHLs

2

3

Aim: To investigate the mode of action of AHL mixture (1 mg l-1)

Survival of turbot larvae at 5 dph (Exp 1)

No AHLAHL

No rifampicin

Rifampicin0

102030405060708090

100

Surv

ival

(%)

• No negative effect of AHLs in the presence of antibiotic

• Effect of AHL probably through the stimulation of the virulence of opportunistic bacteria

Experiment outline

Exp Treatment

1 2 3 4 5 6

1 Control AHL addition

Rifampicin Rifampicin +AHLs

2 Control AHL addition

EC5 (added to water)

EC5 (added to water) + AHLs

EC5 (added to water + via rotifers)

EC5 (added to water + via rotifers) + AHLs

3 Control AHL addition

EC3 (added to water + via rotifers)

EC3 (added to water + via rotifers) + AHLs

EC5 (added to water + via rotifers)

EC5 (added to water + via rotifers) + AHLs

Aim: To compare the effect of EC3 and EC5

Survival of turbot larvae at 7 dph (Exp 3)

No ECEC3

EC5

AHL

No AHL0

102030405060708090

100

Surv

ival

(%)

EC5 can neutralize the negative effect of AHLs, while EC3 cannot

Quorum sensing in the Artemia - Macrobrachium

food chain

Effect of AHL on Macrobrachium larviculture

Survival rate on day 8

77.7c77.1c77.3c

61.5bc

48.5ab

37.3a

31323334353637383

AHL+EC5 EC5 AHL+LVS3 LVS3 AHL Control

Treatment

Sur

viva

l rat

e (%

)

Effect of AHL on Macrobrachium larviculture: LSI

Larval Stage Index (day 8)

5.0c5.2c

5.1c

4.6b4.6b4.4a

3.0

3.5

4.0

4.5

5.0

5.5

AHL+EC5 EC5 AHL+LVS3 LVS3 AHL Control

Treatment

LSI

Conclusions• QS determines virulence in vivo as demonstrated by using

qs mutant

• In Artemia disruption of AI2 is sufficient to abolish virulence

• In Brachionus disruption of AI1 and AI2 is needed

• In larviculture of turbot and macrobrachium AHL molecules have a strong negative effect on survival

• Added QS molecules can be quenched by AHL degrading MC

• Is AHL mediated virulence by prevalent opportunistic pathogenic bacteria a problem in larviculture?

Heat shock proteins

as

immunostimulants?

YS1

pblDnaK(constructed using pBAD

TOPO® vectors, InvitrogenTM, USA)

L-arabinose(0.5 mg/ml for 1 h) -

YS2

pDnaK(constructed using pBAD

TOPO® vectors, InvitrogenTM , USA)

L-arabinose(0.5 mg/ml for 4 h) DnaK

E. colistrains Plasmids Induction

Hsps encodedby plasmids

Construction of bacteria over-producing DnaK

To test the ability of E. coli overproducing DnaK to protect Artemia larvae against Vibrio infection

DnaK

DnaK expression of YS1 and YS2 on SDS-PAGE

YS1 YS2

Correlation

0

10

20

30

40

50

60

YS1 CTR YS1 ID YS2 CTR YS2 ID

Surv

ival

(%)

Run 1 Run 2

• Survival of Artemia larvae fed either induced or non-induced strainYS1 was low, results similar to those obtained with non-induced YS2

Survival after Vibrio challenge

• A significant 2 to 3-fold increase in survival occurred when larvae fedwith arabinose-induced YS2 were exposed to V. campbellii

DnaK

• DnaK accumulation correlated with enhanced survival of Artemia

• Clearly, protection against vibrio challenge is improved by DnaK,although the possibility that other Hsps have this capability is notdiscounted

0

10

20

30

40

50

60

YS1 CTR YS1 ID YS2 CTR YS2 ID

Surv

ival

(%)

Run 1 Run 2

Correlation of enhanced resistance vs. DnaK accumulation in bacteria

38°C for 30 min

A B

• Feeding HS as opposed to non-HS bacteria significantly increasedsurvival upon exposure to V. campbellii

• Protection occurred in all HS bacterial strains employed

1. Survival after Vibrio challenge

• Western blot revealed that HS increased DnaK production bybacteria

• Higher amounts of DnaK in HS bacteria correlated withenhanced ability to promote survival of Artemia larvae

Hsp70

• Quantification by ELISA demonstrated that DnaK increasedfrom 2.0 - 2.3 fold in heated bacteria

2.1 2.0 2.1 2.0 2.3

3. DnaK accumulation in bacteria

• Feeding gnotobiotic Artemia with E. coli over-producing differentprokaryotic Hsps (DnaK-DnaJ-GrpE) increased larval resistance to V.campbellii

• Immunoprobing of western blots showed that enhanced resistance toV. campbellii correlated with DnaK production in E. coli

• A definitive role for DnaK was demonstrated by feeding Artemialarvae with transformed bacteria over-producing only this protein(YS2), with survival augmented approximately 3-fold after pathogenicvibrio exposure

• Exogenous Hsps possibly trigger the Artemia innate immuneresponse, producing anti-inflammatory substances which suppressinfection

GENERAL CONCLUSIONS

• Gnotobiotic systems allow to test novel strategies for disease abatement in larviculture

• Potential treatment that deserve to be tested in non-gnotobiotic environments are

• Quorum sensing interference

• polyhyroxybutyric acid

•Yeast cell wall bound glucan

•Heat shock proteins

Slide Number 1How to study host-microbial interactions?Gnotobiotic systemsSlide Number 10ProcessesQuorum sensingSlide Number 13Quorum sensing and quenching�in the �Brachionus - Turbot �food chainSlide Number 39Slide Number 40Slide Number 41Slide Number 42Slide Number 43Slide Number 44Quorum sensing in the �Artemia - Macrobrachium �food chainEffect of AHL on Macrobrachium larvicultureEffect of AHL on Macrobrachium larviculture: LSIConclusionsSlide Number 55Slide Number 56Slide Number 57Slide Number 58Slide Number 59Slide Number 60Slide Number 61Slide Number 62Slide Number 64Slide Number 65Slide Number 75