Transcript title of presentation

MICROBIAL MANAGEMENT IN FISH AND
SHELLFISH LARVICULTURE:
FROM GNOTOBIOTIC EXPERIMENTS TO
APPLICATIONS
NPC mtg Feb 2012
Conceptual framework
Immuno
stimulant
eggs
larvae
MCe
MCs
MCe
MCs
Modulators
composition
Modulators of
activity
nutrition
juveniles
MCe
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
MCs
slide 2 of 39
Experimental approach:
• Gnotobiotic systems
– Artemia
– Brachionus
– Seabass
• Non-gnotobiotic verification
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 3 of 39
How to study host-microbial interactions?
Host
simplification
MC
Host
environment
reality?
complex
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
Known
microorganisms
gnotobiotic
slide 4 of 39
Gnotobiotic Artemia: GART
Decapsulation
Instar I
Decapsulated cysts
20-50 nauplii
counted for the
Experiments
Hydrated cysts
Non-axenic conditions
Instar II
Axenic conditions
Gnotobiotic challenge: add Vibrio
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 5 of 39
Gnotobiotic Artemia –seabass food chain
DAH1
DAH3
DAH7
Vibrio anguillarum
Artemia
DAH14
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
At 16°C
slide 6 of 39
blank
120
110
100
90
80
70
60
50
40
30
20
10
0
Virulent
Vibrio anguillarum
Strain HI610
challenge
0
1
2
3
4
5
6
7
8
serovar O2a
9
10
11
12
time (day)
120
100
Avirulent
Vibrio anguillarum
Strain 43
serovar 01
survival (%)
survival (%)
Gnotobiotic Artemia –seabass food chain
80
60
40
20
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
time (day)
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 7 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 Bossier
Ghent University Aquaculture Research Consortium
slide 8 of 39
Yeast cell wall-bound glucan
as
immunostimulant?
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 9 of 39
Yeast mutants with altered cell wall composition
Mannoproteins
WT-
exp YNB
–
exp YNB
43%
16%
55%
75%
2%
9%
mnn9
 (1,6) glucans
 (1,3) glucans
Chitin
Plasma
membrane
(adapted from EUROCELLWALL project from EU)
 Since -glucans are well-known immunostimulants,
possibly the mnn9 yeast acts as immunostimulant,
allowing Artemia to be protected against pathogens
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 10 of 39
Artemia survival in GART
90
80
70
untreated+VC
60
treated+VC
50
40
30
20
10
0
WT
mnn9 mnn6
fks1
knr4
kre6
gas1
chs3
LVS3
yeast strains
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 11 of 39
Non-gnotobiotic: mussel larvae
Survival of 2 week
Survival old larvae
80
100%
70
0%
Algae
60%
40%
Mnn9 yeast
60
%
50
40
30
20
10
14
28
49
39
50
100%
90/10
80/20
70/30
60/40
0
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 12 of 39
Heat shock proteins
as
immunostimulants?
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 13 of 39
DnaK (HSP70) overexpression:
E. coli strain YS2
DnaK
WB
DnaK
YS1: control strain, no DnaK overproduction
YS2: positive strain, DnaK overproduction by arabinose induction
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 14 of 39
Enhanced resistance by DnaK feeding in a Vibrio
challenge
60
Run 1
Run 2
Survival (%)
50
40
30
20
10
0
YS1 CTR
YS1 ID
YS2 CTR
YS2 ID
DnaK
• 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
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 15 of 39
Priming
(g)HOST
MiC’s, heat shock proteins (DnaK),..
HOST + MAMPs
HOST + MAMPs + pathogen
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
CONCLUSIONS HSPs
• Exogenous HSPs feeding possibly triggers
the Artemia innate immune response,
producing
anti-inflammatory
activity
(Phenoloxidase activity) which suppresses
infection
• Sofar,
no
confirmation
gnotobiotic conditions
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
under
non-
slide 18 of 39
POLY-β-HYDROXYBUTYRATE (PHB)
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 19 of 39
POLY-β-HYDROXYBUTYRATE (PHB)
• Linear polymer of β-hydroxybutyric acid
O
HO
O
O
O
O
OH
n
103-106
Could PHB also be used to protect
Artemia from luminescent vibriosis?
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 20 of 39
SHORT-CHAIN FATTY ACIDS
• 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)
H+
fatty acid
H+
H+
proton pump
H+
bacterium
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 21 of 39
PHB UPTAKE BY ARTEMIA
• Starved nauplii without feed or with PHB particles
No PHB
PHB
Light
microscopy
The PHB particles are ingested by the
nauplii
Fluorescence
microscopy
(Nile Blue)
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
(bar = 250 µm)
slide 22 of 39
EFFECT ON STARVED ARTEMIA
• Sterile Artemia nauplii: no feed added
Longer
or only PHB particles (1 g/l) at survival
day 0with
Artemia survival (%)
120
PHB particles
No feed
PHB
100
The nauplii
can obtain
energy from
the particles
80
60
40
20
0
0
1
2
Time (days)
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
3
The particles
must be
(partially)
degraded
in
slide 23 of 39
the gut
PHB: non-gnotobiotic
An Artemia – Macrobrachium
food chain example
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 24 of 39
Macrobrachium larval survival feeding on
PHB enriched Artemia nauplii
100
90
80
78
82B
a
65A
70
Survival (%)
PHB
Control
89b
50
60
2
50
40
24
30
1
20
10
0
Day 5
Day 10
Day 15
Rearing time
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 25 of 39
PHB combined with a classical HUFA
enrichment
90
a
A
80
b
b
70
Survival (%)
x
B B
60
X
c
50
y
40
y
C
30
Y
Y
z
20
Z
10
0
Day 10
• No PHB, no Hufa
Day 15
Day 20
Day 28
Rearing time
• No PHB, Hufa
• PHB, no Hufa
• PHB, Hufa
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 26 of 39
CONCLUSIONS PHB
• PHB particles protect Artemia from luminescent
vibriosis
• Positive effects in the aquaculture food chain:
but further verification is needed.
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 27 of 39
Quorum sensing
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 28 of 39
What is Quorum Sensing (QS)?
• 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
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 29 of 39
What type of processes are under the control of QS?
• Many bacterial behaviors are regulated by
quorum sensing
– luminescence
– Symbiosis
– Virulence
– Antibiotic production
– Biofilm formation
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 30 of 39
QS molecules: acyl homoserine lactones (AHL)
N-Heptanoyl-DL-homoserine
N-Hexanol-DL-homoserine
lactone (C7-HSL)
lactone (C6-HSL)
N-Butyryl-DL-homoserine
N-Octanoyl-DL-homoserine
lactone (C4-HSL)
lactone (C8-HSL)
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 31 of 39
What is Quorum sensing (QS)?
Can we demonstrate that quorum
sensing is important in microbial
interference with (larval) stage of
aquatic animals?
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 32 of 39
Quorum sensing: non-gnotobiotic
An Artemia – Macrobrachium
food chain example
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 33 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 Bossier
Ghent University Aquaculture Research Consortium
slide 34 of 39
Effect of AHL and EC5D on
Macrobrachium larviculture:
survival
Survival rate on day 8
77.3c
83
Survival rate (%)
73
63
77.7c
77.1c
61.5bc
48.5ab
53
37.3a
43
33
23
13
3
AHL+EC5
EC5
•daily AHL addition of 1 mg/l
AHL+LVS3
LVS3
AHL
Control
Treatment
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 35 of 39
Effect of AHL and EC5D on
Macrobrachium larviculture: LSI
Larval Stage Index (day 8)
5.5
c
5.1
5.2c
5.0c
5.0
4.6b
4.6b
LVS3
AHL
LSI
a
4.4
4.5
4.0
3.5
3.0
AHL+EC5
EC5
•daily AHL addition of 1 mg/l
AHL+LVS3
Control
Treatment
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 36 of 39
ENZYMATIC AHL INACTIVATION
AHL degradation by pure Bacillus strains isolated
from shrimp (LT3, LT12) and sea bass (LCDR16)
6
5
[HHL] (mg/L)
•
4
Control
3
LT3
LT12
2
LCDR16
1
0
0
3
6
Time (h)
9
12
ENZYMATIC AHL INACTIVATION
Use of signal-degrading bacteria as probionts, e.g.
in Macrobrachium larvae:
a
50
a
40
Survival (%)
•
30
b
20
c
10
0
C–
C+
D+
Treatment
Is this effect by AHL degradation?
L+
Quorum 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 Bossier
Ghent University Aquaculture Research Consortium
slide 39 of 39
General conclusions
Feeds,
Feeds
eg PHB
Quorum sensing
analysis
Pathogenic
bacteria
Probiotic bacteria
Immunostimulants
Antimicrobial
Peptides
Gnotobiotic
Artemiamodel
test
Artemia
system
Performance
Quantitative
analysis of
the bacterial
community
Host gene
expression
analysis
Biochemical
analysis
e.g. antimicrobial substances
Fish and shellfish
larvae validation
Marker
genes
LARVI09 Keynote on microbial management – Peter Bossier
Ghent University Aquaculture Research Consortium
slide 40 of 39