MICROBIAL MANAGEMENT IN FISH AND SHELLFISH LARVICULTURE:
FROM GNOTOBIOTIC EXPERIMENTS TO APPLICATIONS
MICROBIAL MANAGEMENT IN FISH AND SHELLFISH LARVICULTURE:
FROM GNOTOBIOTIC EXPERIMENTS TO APPLICATIONS
NPC mtg Feb 2012
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 2 of 39
Conceptual framework
eggs
MCe MCs
juveniles MCe MCs
MCe MCs
larvae
Immuno
stimulant
nutrition
Modulators
composition
Modulators of activity
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
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Experimental approach:
• Gnotobiotic systems– Artemia– Brachionus– Seabass
• Non-gnotobiotic verification
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 4 of 39
Known micro-
organisms
Host
environment
Hostsimplificati
on
complex gnotobiotic
reality?
MC
How to study host-microbial interactions?
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 5 of 39
Gnotobiotic Artemia: GARTGnotobiotic Artemia: GART
Instar I
Instar II
Decapsulated cysts
Decapsulation
Axenic conditions
Hydrated cysts
Non-axenic conditions
20-50 nauplii counted for the
Experiments
Gnotobiotic challenge: add VibrioGnotobiotic challenge: add Vibrio
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 6 of 39
Gnotobiotic Artemia –seabass food chainDAH1
DAH3
DAH14
Artemia
Vibrio anguillarum
DAH7
At 16°C
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
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0102030405060708090
100110120
0 1 2 3 4 5 6 7 8 9 10 11 12
time (day)
surv
ival
(%
)Gnotobiotic Artemia –seabass food
chain
Virulent
Vibrio anguillarum
Strain HI610
serovar O2a
Avirulent
Vibrio anguillarum
Strain 43
serovar 010
20
40
60
80
100
120
0 1 2 3 4 5 6 7 8 9 10 11 12 13
time (day)
surv
ival
(%
)
blank
challenge
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 8 of 39
Steering host-microbial interactions
• Stimulating the host’s immune response– yeast cell wall-bound glucan– heat shock proteins
• Influencing microbial numbers or activity – polyhydroxybutyric acid– quorum sensing
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 9 of 39
Yeast cell wall-bound glucanas
immunostimulant?
Yeast cell wall-bound glucanas
immunostimulant?
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 10 of 39
MannoproteinsMannoproteins
(1,6) glucans(1,6) glucans
(1,3) glucans(1,3) glucans
ChitinChitin
1616%%
7575%%
9%9%
mnn9mnn9 – exp – exp YNBYNB
2%
55%
43%
WTWT- - exp YNBexp YNB
(adapted from EUROCELLWALL project from EU)
Plasma membranePlasma membrane
Since -glucans-glucans are well-known immunostimulants, possibly the mnn9 yeast acts as immunostimulant, allowing Artemia to be protected against pathogens
Yeast mutants with altered cell wall composition
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 11 of 39
Su
rviv
al (
%)(
da y
6)
Artemia survival in GART
0
10
20
30
40
50
60
70
80
90
WT mnn9 mnn6 fks1 knr4 kre6 gas1 chs3 LVS3
yeast strains
untreated+VC
treated+VC
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 12 of 39
Non-gnotobiotic: mussel larvae
Survival
14 28 49 39 500
10
20
30
40
50
60
70
80
100% 90/10 80/20 70/30 60/40
%
Survival of 2 week old larvae
Algae100%
60%
Mnn9 yeast0% 40%
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 13 of 39
Heat shock proteins as
immunostimulants?
Heat shock proteins as
immunostimulants?
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 14 of 39
DnaK
DnaK (HSP70) overexpression:
E. coli strain YS2
YS1: control strain, no DnaK overproduction
YS2: positive strain, DnaK overproduction by arabinose induction
DnaKWB
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 15 of 39
Enhanced resistance by DnaK feeding in a Enhanced resistance by DnaK feeding in a Vibrio challengeVibrio challenge
• Survival of Artemia larvae fed either induced or non-induced negative control strain YS1 was low.
• Survival of non-induced YS2 strains as in negative control
• A significant increase in survival in larvae fed with arabinose-induced DnaK overproducing YS2 were exposed to V. campbellii
DnaK
Priming
(g)HOST
HOST + MAMPs
HOST + MAMPs + pathogen
MiC’s, heat shock proteins (DnaK),..
16
Priming the innate immune system:PO expression in Artemia as modulated by DnaK
and Vibrio
YS2(-): E coli not overproducing DnaK (HSP70 homolog)YS2(+): E coli overproducing DnaK (HSP70 homolog)VC: Vibrio campbellii challenge with a delay of 6 h
17
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 18 of 39
• Exogenous HSPs feeding possibly triggers the Artemia innate immune response, producing anti-inflammatory activity (Phenoloxidase activity) which suppresses infection
• Sofar, no confirmation under non-gnotobiotic conditions
CONCLUSIONS HSPsCONCLUSIONS HSPs
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 19 of 39
POLY-β-HYDROXYBUTYRATE (PHB) POLY-β-HYDROXYBUTYRATE (PHB)
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 20 of 39
• Linear polymer of β-hydroxybutyric acid
POLY-β-HYDROXYBUTYRATE (PHB)
OH
O
O
O
O
O
OH
n103-106
Could PHB also be used to protect Artemia from luminescent vibriosis?
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 21 of 39
H+H+H+
H+
H+
Energy
bacterium
proton pump
fatty acid
• Short-chain fatty acids (SCFA): formic, acetic, propionic, butyric and valeric acid
• Known to inhibit growth of enteric bacteria (Salmonella, Klebsiella, Escherichia coli)
– Acidification of cytoplasm
– Energy needed to keep internal pH optimal
– Effect is pH-dependent (lower pH → higher effect)
SHORT-CHAIN FATTY ACIDS
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 22 of 39
• Starved nauplii without feed or with PHB particles
No PHB PHB
Light microscopy
Fluorescence microscopy (Nile Blue)
(bar = 250 µm)
The PHB particles are ingested by the nauplii
PHB UPTAKE BY ARTEMIA
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 23 of 39
0
20
40
60
80
100
120
0 1 2 3
Time (days)
Art
em
ia s
urv
ival (%
) No feedPHB
• Sterile Artemia nauplii: no feed added or only PHB particles (1 g/l) at day 0
Longer survival with PHB particles
The nauplii can obtain
energy from the particles
The particles must be
(partially) degraded in
the gut
EFFECT ON STARVED ARTEMIA
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 24 of 39
An Artemia – Macrobrachium food chain example
PHB: non-gnotobiotic
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
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241
65A
78a
502
82B
89b
0
10
20
30
40
50
60
70
80
90
100
Day 5 Day 10 Day 15
Rearing time
Su
rviv
al (
%)
Control PHB
Macrobrachium larval survival feeding on PHB enriched Artemia nauplii
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 26 of 39
a Ax
X
b
B
y
Y
b
B
y
Y
c
C
z
Z
0
10
20
30
40
50
60
70
80
90
Day 10 Day 15 Day 20 Day 28
Rearing time
Su
rviv
al (
%)
PHB combined with a classical HUFA enrichment
• No PHB, no Hufa
• PHB, no Hufa
• No PHB, Hufa
• PHB, Hufa
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 27 of 39
• PHB particles protect Artemia from luminescent vibriosis
• Positive effects in the aquaculture food chain: but further verification is needed.
CONCLUSIONS PHBCONCLUSIONS PHB
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 28 of 39
Quorum sensingQuorum sensing
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 29 of 39
• QS: a mechanism by which bacteria regulate gene
expression in response to their population density by
producing, releasing and detecting small signal molecules
(quorum sensing molecules) (Fuqua et al., 1997).
• QS: process of bacterial cell-to-cell
communication/conversation with signal molecules
What is Quorum Sensing (QS)?What is Quorum Sensing (QS)?
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 30 of 39
• Many bacterial behaviors are regulated by quorum sensing
– luminescence– Symbiosis– Virulence– Antibiotic production– Biofilm formation
What type of processes are under the control of QS?What type of processes are under the control of QS?
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 31 of 39
QS molecules: acyl homoserine lactones (AHL)QS molecules: acyl homoserine lactones (AHL)
N-Butyryl-DL-homoserine
lactone (C4-HSL)
N-Hexanol-DL-homoserine
lactone (C6-HSL)
N-Heptanoyl-DL-homoserine
lactone (C7-HSL)
N-Octanoyl-DL-homoserine
lactone (C8-HSL)
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 32 of 39
Can we demonstrate that quorum
sensing is important in microbial
interference with (larval) stage of
aquatic animals?
What is Quorum sensing (QS)?What is Quorum sensing (QS)?
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 33 of 39
An Artemia – Macrobrachium food chain example
Quorum sensing: non-gnotobiotic
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 34 of 39
Effect of AHL mixture in Artemia – Macrobrachium
food chain
Treatments Survival LSI
Control 70.0± 4.2b 5.3 ± 0.4b
AHLmix1 49.2 ± 2.6a 4.8 ± 0.3a
1
• Survival of Macrobrachium larvae on day 7 post-hatch, (mean ± SD, n = 6).
• daily AHL addition of 1 mg/l
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 35 of 39
Effect of AHL and EC5D on Macrobrachium larviculture:
survivalSurvival rate on day 8
77.7c77.1c77.3c
61.5bc
48.5ab
37.3a
3
13
23
33
43
53
63
73
83
AHL+EC5 EC5 AHL+LVS3 LVS3 AHL Control
Treatment
Su
rviv
al r
ate
(%)
•daily AHL addition of 1 mg/l
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 36 of 39
Effect of AHL and EC5D on Macrobrachium larviculture: LSI
Larval Stage Index (day 8)
5.0c5.2c
5.1c
4.6b4.6b
4.4a
3.0
3.5
4.0
4.5
5.0
5.5
AHL+EC5 EC5 AHL+LVS3 LVS3 AHL Control
Treatment
LS
I
•daily AHL addition of 1 mg/l
• AHL degradation by pure Bacillus strains isolated from shrimp (LT3, LT12) and sea bass (LCDR16)
ENZYMATIC AHL INACTIVATION
c
b
aa
0
10
20
30
40
50
C– C+ D+ L+
Treatment
Su
rviv
al (
%)
• Use of signal-degrading bacteria as probionts, e.g. in Macrobrachium larvae:
ENZYMATIC AHL INACTIVATION
Untreated
V. harve
yi
AHL degraders
V. harve
yi + A
HL
degraders
Is this effect by AHL degradation?
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 39 of 39
Quorum sensing: conclusionsQuorum sensing: conclusions
•QS is important in host-microbial interactions in the aquatic environment
•Data on in vivo QS molecule concentration are mostly lacking, necessary to further substantiate QS importance for an aquaculture setting
LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium
slide 40 of 39
General conclusionsGeneral conclusions
Quantitative analysis of
the bacterial community
Quantitative analysis of
the bacterial community
Biochemical analysis
e.g.antimicrobial substances
Biochemical analysis
e.g.antimicrobial substances
Host gene expression analysis
Marker genes
Host gene expression analysis
Marker genes
Fish and shellfish Fish and shellfish larvae validationlarvae validationFish and shellfish Fish and shellfish larvae validationlarvae validation
Gnotobiotic Artemiatest
system
Gnotobiotic Artemiatest
system
Quorum sensing analysis Probiotic bacteria
Pathogenic bacteria
Feeds
Antimicrobial PeptidesImmunostimulantsQuorum sensing
analysis Probiotic bacteriaPathogenic
bacteria
Feeds, eg PHB
Antimicrobial PeptidesImmunostimulants
PerformancePerformance
Artemiamodelsystem
GnotobioticArtemiamodel
system
Gnotobiotic