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