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

Microbes “run the world”

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

Rotifer culturein European marine fish hatcheries

Rotifer culture systems

random interaction of the different bacteria

++ --

M 1 2 3 4 5 6 7 M 8 9 10 11 12 13 14 MDay

DGGE fingerprinting rotifer recirculation culture

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 Artemia – seabass food chain

Gnotobiotic sea bass test system

0102030405060708090

100

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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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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

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6

0 24 48 72

Time (h)

HH

L co

ncen

trat

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(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

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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

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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

YS1 YS2

Correlation

HSP inducedbacteria

non-HSP induced bacteria

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

Yeong Yik Sung, Ronald J. Roberts & Peter Bossier

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

ACE application for Vibrio reduction in Artemia

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

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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

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evel

(mg/

g dw

t)

0.0

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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 during enrichmentincreased enrichment levels

Over 20% higher enrichment

levels!

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

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% s

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Trial 1 Trial 2

Larval survival 60 dphControl Treatment

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Trial 1 Trial 2

Total tank biomass at 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

thank you for your attention

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