R&D Innovations in Microbial Management in Fish and Shellfish Larviculture
Patrick Sorgeloos, Peter Bossier & co-workersUGent Aquaculture R&D Consortium
Ghent University, Belgium
Patrick Lavens, Geert Rombaut & co-workersINVE Technologies SA, Dendermonde, Belgium
2011 International Symposium on Grouper Culture November 8-11, 2011, Pingtung, Taiwan
Priorities for future aquaculture Priorities for future aquaculture
resulting in new concepts & products for a sustainable aquaculture
from empiricial farming towards
a knowledge-based bio-industry
1. Complete independence from natural stocks through DOMESTICATION
2.2. Improved / more costImproved / more cost‐‐effective effective SEEDSEED PRODUCTION
3.3. Better targeted Better targeted SPECIES SELECTIONSPECIES SELECTION
4.4. Development of more efficient stocks through Development of more efficient stocks through SELECTIVE BREEDING SELECTIVE BREEDING
5.5. More More MICROBIAL MANAGEMENT MICROBIAL MANAGEMENT for more sustainable production for more sustainable production
6.6. Better understanding of Better understanding of IMMUNE SYSTEMS IMMUNE SYSTEMS in vertebrates and in vertebrates and invertebratesinvertebrates
7.7. More More INTEGRATED PRODUCTION SYSTEMS INTEGRATED PRODUCTION SYSTEMS for plant and animal farmingfor plant and animal farming
8.8. COASTAL AND OFFCOASTAL AND OFF‐‐SHORE FARMSSHORE FARMS of food and energyof food and energy
9.9. Full independence from fisheries stocks for Full independence from fisheries stocks for LIPID AND PROTEIN LIPID AND PROTEIN INGREDIENTS INGREDIENTS in aquatic feeds in aquatic feeds
10.10. More attention for More attention for INTEGRATIONINTEGRATION of restocking activities with of restocking activities with FISHERIESFISHERIESmanagementmanagement
11.11. SOCIETAL LEVERAGE:SOCIETAL LEVERAGE:
•• multimulti‐‐stakeholder interactionstakeholder interaction
•• International cooperation on a winInternational cooperation on a win‐‐win basiswin basis
1. Complete independence from natural stocks through DOMESTICATION
2. Improved/more cost‐effective seed production3.3. Better targeted Better targeted SPECIES SELECTIONSPECIES SELECTION
4.4. Development of more efficient stocks through Development of more efficient stocks through SELECTIVE BREEDINGSELECTIVE BREEDING
5.5. More microbial management for more More microbial management for more sustainable production sustainable production
6.6. Better understanding of immune systems in Better understanding of immune systems in vertebrates and invertebratesvertebrates and invertebrates
7.7. More More INTEGRATED PRODUCTION SYSTEMS INTEGRATED PRODUCTION SYSTEMS for plant and animal farmingfor plant and animal farming
8.8. COASTAL AND OFFCOASTAL AND OFF‐‐SHORE FARMSSHORE FARMS of food and energyof food and energy
9.9. Full independence from fisheries stocks for Full independence from fisheries stocks for LIPID AND PROTEIN INGREDIENTS LIPID AND PROTEIN INGREDIENTS iiaquatic feeds aquatic feeds
10.10. More attention for More attention for INTEGRATIONINTEGRATION of restocking activities with of restocking activities with FISHERIESFISHERIESmanagementmanagement
11.11. SOCIETAL LEVERAGE:SOCIETAL LEVERAGE:
Improved / more costImproved / more cost‐‐effective and qualitative effective and qualitative SEEDSEED PRODUCTIONPRODUCTION
example:sea bass/bream larviculture in the Mediterranean
• annual production of 1 billion fry
• market value of 15 Euro cents a piece
• average survival 20 % by day 60
• unpredictable survival = critical bottleneck for future cost efficiency and sustainability of the industry
• microbial interference considered to be the main culprit
• no selected breeds available yet
Improved / more costImproved / more cost‐‐effective and qualitative effective and qualitative SEEDSEED PRODUCTIONPRODUCTION
LIVE FOOD USED IN LARVICULTURE
of fish & shellfish species
microalgae rotifers brine shrimp
Present applications are based on trial & error approach - limited microbial quality control - much room for optimisation
Effects of probionts: more hypotheses than proofs
Production of inhibitory compoundsCompetition for chemicals or energy change microbial community?Competition for adhesion sites
Improvement of water quality lower bacterial load ?
Supply of extra nutrients in the digestive tract ?
Stimulate immune system or other critical functions at first feeding ?
2 – 5 % PHB induces significant weight increase
Dietary effect of PHB addition to the diet of sea bass larvae
Sea bass growth
polyhydroxybutyrate
pH of sea bass intestine
Dietary effect of PHB addition to the diet of sea bass larvae
production of 3production of 3--HB HB or other SCFA in the or other SCFA in the
gut?gut?
change in microbial change in microbial communitycommunity
higher uptake of PHB higher uptake of PHB results in lower intestinal results in lower intestinal
pHpH
New experimental approach:
• Gnotobiotic systems– Artemia
– Brachionus
– Seabass
• Non‐gnotobiotic verification / validation
Gnotobiotic sea bass test system
0102030405060708090
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time (day)
surv
ival
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)
bacteria free
control
Effect of light stress on survival of xenic sea bass larvae
Axenic sea bass larvae are not sensitive to light stress
Gnotobiotic sea bass test system
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controllight
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light
Faculty of Bioscience EngineeringAnimal Production - P. Sorgeloos and P. Bossier Biochemical and Microbial Technology – W. Verstraete and N. Boon
Faculty of Veterinary MedicineMorphology – W. Van den Broeck and A. Decostere Pathology, Bacteriology and Poultry Diseases – F. Pasmans Virology, Parasitology and Immunology – H. Nauwynck
Faculty of SciencesBiochemistry, Physiology and Microbiology – P. Vandamme and P. De VosBiology – D. Adriaens and W. Vyverman Molecular Genetics – D. Inzé, Frank Van Breusegem
Development of innovative microbial management systems
Quantitative analysis of
the bacterial community
Qual/Quant analysis of
the bacterial composition
Biochemical analysis
Biochemical analysis
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 Artemia test
system
Gnotobiotic Artemia test
system
Quorum sensing analysis Probiotic bacteria Pathogenic
bacteria
Feeds
Antimicrobial PeptidesImmunostimulantsQuorum sensing Probiotic bacteria Pathogenic
bacteria
Micro Algae /Feeds
Antimicrobial peptidesImmunostimulants
PerformancePerformance
Artemiasystem
Gnotobioticmodel
systems
Gnotobiotic
Heat-shock proteins
2. Improved / more cost2. Improved / more cost‐‐effective effective SEEDSEED PRODUCTIONPRODUCTION
Larval Gonotobiotic Systems
with - invertebrates (Artemia)- vertebrates (seabass)
Head start: gnotobiotic systems, microbial community fingerprinting, transciptome profiling
Objectives: explore established model systems to study effects of- host genomic make-up- innate immunity- virulence factors
Major outcomes: - annotated Artemia genome
- microbial community functionality
- host pathogen interplay at molecular and immunological level
• Stimulating the host’s immune response- heatshock proteins- yeast cell wall-bound components
• Influencing microbial numbers or activity - quorum-sensing interference– intestinal pH modulation (polyhydroxybutyric acid)
Examples of steering hostExamples of steering host--microbial interactionsmicrobial interactions
• Before: bacteria = separate entities
• Now: bacteria sense and respond to environment and to each other• Extracellular signal molecules• ≈ hormones in higher organisms
QUORUM SENSING
AHA!
AHL chemicals(Acyl Homoserine Lactone)
Communication Between Bacteria = Quorum Sensingg in Vibrio harveyi
When quorum of bacteria is present a threshold concentration of the AHL chemicals is reached. They bind to the sensor protein, which then turns on the lux control box and activates the lux and virulence genes
sensor protein
Synthesis of RNA Enzymes for light production and virulence factorssensor
proteins
Macrobrachium non-gnotobiotic food chain
Treatments Survival LSI
Control 70.0 ± 4.2b 5.3 ± 0.4b
AHL 1ppm 49.2 ± 2.6a 4.9 ± 0.3a
Effect of 1ppm AHL on growth & survival of giant freshwater prawn larvae
Baruah et al. (2009) Aquaculture 288: 233-238
Larval Stage Index (growth)
= disruption of quorum sensingf ex thru inactivation of signal molecules
QUORUM QUENCHING
Synthase
Receptor
Target genes
Signal
0
1
2
3
4
5
6
0 24 48 72
Time (h)
HH
L co
ncen
trat
ion
(mg/
L)
Positive control (pME 6863)
Negative control (pME 6000)
EC5(D)
EC5(S)Q
EC5(S)QHS
EC5(S)N
EC5(S)NHS
AHL degradation by different Enrichment Cultures ECs
SIGNIFICANCE FOR AQUACULTURE
• Use of signal-degrading bacteria as probionts, e.g. in turbot larvae
0
20
40
60
80
100
120
no AHL + AHL (1 ppm)
Treatment
Surv
ival
(%)
Control
+ AHL degraders
Tinh et al. (2008) Aquaculture 285: 52-62
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
Surv
ival
rate
(%)
daily addition of 1 mg/l AHL mixture
SIGNIFICANCE FOR AQUACULTURE
• Effect on survival of Macrobrachium larvae
• Micro algae are important in aquaculture:• Feed• Green water: mechanism???
• Interactions between algae and bacteria• Growth inhibition• Interference with activity: QS disruption?
METABOLITES OF MICRO ALGAE
Chlamydomonas Chlorella
• Supernatants of axenic freshwater micro algae inhibit QS in reporters • C. violaceum CV026 + 1 mg/L HHL• E. coli JB523 + 1 µg/L OHHL
• QS inhibitory compounds identity still unknown
METABOLITES OF MICRO ALGAE
0
10
20
30
40
50
60
70
Chlamydomonasreinhardtii
Chlorella vulgaris Chlorella emersonii Chlorellasaccharophila
% Q
S in
hibi
tion
CV026JB523
• Stimulating the host’s immune response- heatshock proteins- yeast cell wall-bound components
• Influencing microbial numbers or activity - quorum-sensing interference– intestinal pH modulation (polyhydroxybutyric acid)
Examples of steering hostExamples of steering host--microbial interactionsmicrobial interactions
What are Heat Shock Proteins? What are Heat Shock Proteins? Intracellular Intracellular
Sets of proteins synthesized constitutively in cells of all living organisms (
Induced after Induced after exposure to heat exposure to heat stress stress (cold, O2 deprivation, radicals, disease and etc)
Functions: Protein chaperones and Protein chaperones and cell maintenancecell maintenance
Extracellular (Hsp60/70/90) Extracellular (Hsp60/70/90)
Functions: Play significant role in Play significant role in innate and adaptive immunity (immune innate and adaptive immunity (immune systemsystem))
Necrosis / Absence of Necrosis
- involve in TLR2 and TLR4 signaling -transduce inflammatory danger signal to immune cells
- via MHC-peptide I/II complex - Antigen presenting
- secretory pathway: induce monocytes and macrophages to produce of NO synthase, NO, TNF-α, IL-1β, IL-6
Endogenous HSP70 accumulationEndogenous HSP70 accumulation
HS treatments
(OC)
24h Survival (%)
A B
Non-HS 36 ± 4a 38 ± 6a
HS 32 65 ± 2b 63 ± 5b
HS 37 70 ± 7b 71 ± 2b
HS 40 68 ± 8b 63 ± 1b
Survival after Vibrio challengeSurvival after Vibrio challenge
p70
Hsp70
vsvs
Larvae accumulating endogenous Hsp70 have higher resistance Larvae accumulating endogenous Hsp70 have higher resistance against pathogensagainst pathogens
Enhanced challenge resistance following HSP feedingEnhanced challenge resistance following HSP feeding
• 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 HSP overproducing E. coli (YS2) were challenged with V. campbellii
HSP 70
Benefits: Reduced bacterial load in rotifers and surrounding water, strong Vibriosuppression
Resulting in:• Improved rearing environment for larval fish• More reliable production of fish larvae
SURE application for Vibrio reduction in rotifers
Vibrio count on TCBS medium
Test results (average over 40 tests)
Bacterial presentce in culture water
012345678
Control Treatment
LOG
CFU
/ml
Marine Agar TCBS
Bacterial presence in rotifers
0
1
2
3
4
5
Control TreatmentLO
G C
FU /r
otife
r
Marine Agar TCBS
Sanocare® ACE during hatchingBacterial load
Effect of different concentrations of Sanocare® ACE on the bacterial development in the hatching medium of EG
Artemia cysts after 24h incubation
6-7 logunit reduction!
Sanocare ACE during enrichmentHigher enrichment levels
Enrichment levels of EPA, DHA and total HUFA obtained after enrichment with Easy DHA Selco for 24h
in the absence and presence of Sanocare® ACE
0
10
20
30
40
50
60
70
80
600 ppm EASY DHA 750 ppm EASY DHA 600 ppm EASY DHA 750 ppm EASY DHA
no Sanocare® ACE no Sanocare® ACE 600 µl Sanocare® ACE 750 µl Sanocare® ACE
300 npl/ml 600 npl/ml 300 npl/ml 600 npl/ml
Enr
ichm
ent l
evel
(mg/
g dw
t)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
DH
A/EP
A ra
tio
EPA 20:5(n-3) DHA 22:6(n-3) HUFA n-3 >= 20:3 DHA/EPA
Figure 1 Microscopic view of the head of an enriched Artemia harvested after 24H enrichment without using Sanocare® ACE
Figure 2 Microscopic view of the head of an enriched Artemia harvested after 24H enrichment using Sanocare® ACE
Sanocare SURE / Sanocare ACE – Fish trial
Fish tests were performed on Gilthead Seabream (Sparus aurata) in the production unit at Maricoltura di Rosignano Solvay
– Hatched larvae were stocked at ±150 larvae/ L – Salinity: 38ppt.– Temperature: 19±1°C– using a semi-closed system
Effect on performance of seabream larvae was determined in
2 consecutive trials (no replicates).
0
5
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% s
urvi
val
Trial 1 Trial 2
Larval survival 60 dphControl Treatment
disease free
disease resistant
certified seed
develop and apply new microbial managements systems develop and apply new microbial managements systems ‐‐resulting in improved / more costresulting in improved / more cost‐‐effective seed productioneffective seed production
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