Introducing the Laboratory of Aquaculture & Artemia Reference Center, Ghent University

Centre of Excellence for
Aquaculture
Research & Development
Peter Bossier, Laboratory for Aquaculture
& Artemia Reference Center
January, 2014

of …
1970: Start research on Artemia
culturing biology at
Ghent University
(Patrick Sorgeloos)
1978: Artemia Reference Center
established upon suggestion
of the FAO
HOW IT ALL BEGAN…
2013: • 2 professors + 4 post docs
• 5 senior scientists
• > 20 PhD (6 Chinese)
• 7 lab technicians + 2 secr staff
• since 1991 >350 MSc alumni from 50 countries
• since 1983 >65 PhD alumni from 21 countries

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Sustainable aquaculture: the “learning curve”
from an empirical approach to a knowledge-based industry
Husbandry
techniques
Nutritional
research
Microbial
management
Breeding
Shifting bottlenecks for the aquaculture industry:
– yields and rentability very variable, still too unpredictable
– limited knowledge of the biology of the cultured species
– limited automation & system control

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 yields & quality 
 disease 
 sustainability
 food security
Aquaculture requires a multidisciplinary approach

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Key partners in Aquaculture UGent
FACULTY OF
BIOSCIENCE ENGINEERING
• Animal Production - Aquaculture
• Biochemical and Microbial Technology
• Food Chemistry and Human Nutrition
• Applied Ecology and Environmental Biology
• Applied Analytical & Physical Chemistry
• Agriculture Economics
• Crop Protection
• Organic Chemistry FACULTY OF SCIENCES
• Biochemistry, Physiology and Microbiology
• Marine biology
• Evolutionary Morphology of Vertebrates
• Vertebrate Morphology & Developmental Biology
• Protistology & Aquatic Ecology
• Molecular Genetics
FACULTY OF
VETERINARY MEDICINE
• Pathology, Bacteriology and Poultry
Diseases
• Virology, Parasitology & Immunology
• Morphology
3 Faculties
12 professors
10 research groups
 100 researchers
 1 business developer

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OUR MISSION
 Perform and foster multidisciplinary
research in aquaculture
 Provide research-based education in
aquaculture
 Valorise our expertise for the sector
and society

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Disease control
Commercial
competitiveness
Microbial
management
Nutrition
Zootechnical
aspects
Diverse & longstanding expertise in
fish & shellfish larviculture
Egg quality &
broodstock
management

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MICROBIAL MANAGEMENT
How  by steering host-microbial interactions
– Stimulating the host’s immune response
•Immunostimulants: yeast cell wall-bound glucan
•Heat shock proteins: upregulate immune system
– Influencing microbial numbers or microbial activity
•Polyhydroxybutyric acid
•Quorum sensing
Goal  Health management
– Improve larval survival
– Alternative techniques for
disease prevention

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HOW TO STUDY
LARVAL HOST-MICROBIAL INTERACTIONS?
HOST
ENVIRONMENT
COMPLEX GNOTOBIOTIC
MICROBIAL
COMMUNITY
HOST
KNOWN MICROBIAL
COMMUNITY

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Experimental approach:
 Gnotobiotic systems
– Artemia
– Brachionus
– Seabass
– Cod (NTNU, Norway; O. Vadstein)
– Tilapia (under development)
– mussel larvae (see Aaron Plovie)
 Non-gnotobiotic verification

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Gnotobiotic 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 Vibrio harveyi

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Immune-priming in Artemia
12
(g)HOST
HOST + MAMPs
HOST + MAMPs + pathogen
Immunostimulants,
microbes, compounds

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Priming by glucans
13
(g)Artemia
HOST + glucan
HOST + glucan + V. harveyi
Glucan from different
YEAST strains

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Phenoloxidase expression
48 hours after challenge

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Priming by DnaK (HSP70)
15
(g)Artemia
HOST + MAMPs
HOST + MAMPs + pathogen
HSP70

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Priming by HSP70
step 1: arabinose –induced HSP production in E. coli
YS1 (-): E coli not overproducing DnaK; YS1 (+): E coli not overproducing
DnaK(arabinose)
YS2 (-): E .coli not overproducing DnaK; YS2(+): E coli overproducing DnaK
by arabinose induction
0
10
20
30
40
50
60
YS1 CTR YS1 ID YS2 CTR YS2 ID
Survival(%)
Run 1 Run 2
WB

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Priming by DnaK
Step 2: Prophenoloxidase Gene
Expression
after Vibrio challenge

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• Cell-to-cell communication in bacteria
• Gene regulation mechanism: activity based
on presence of extracellular signal
molecules ≈ hormones
• Wide range of phenotypes are under QS
control
 Virulence factors
QUORUM SENSING (QS)
Quorum
sensing
Flagellar motility
Metalloprotease
Type III secretion
Siderophore
Phospholipase
Chitinase
+
-
Flagellar
motility
Metalloprotease

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• Quorum sensing regulates virulence
in various aquatic host-pathogen systems
• Quorum sensing-disrupting agents can be recruited
from the aquatic environment
• Signal-degrading bacteria isolated from healthy fish, shrimp and algal
cultures
• Algae (macro & micro) producing antagonists
and protect aquatic animals from infection
QUORUM SENSING (QS)

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• Non-toxic synthetic flavouring substance that is
widely used in food (GRAS)
• Long time known for antibacterial properties
• Subinhibitory concentrations  QS disruption
• Mechanism = furanones
CINNAMALDEHYDE
Cinnamaldehyde
Brackman et al. (2008) BMC Microbiol. 8: 149

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CINNAMALDEHYDE
0
20
40
60
80
100
Survival(%)
0
20
40
60
80
100
Survival(%)
0
20
40
60
80
100
Survival(%)
• Effective in different host-microbe systems
Brine shrimp –
V. harveyi
Macrobrachium
– V. harveyi
Burbot –
A. hydrophila
Crustaceans: 10-100 µM Fish: 0.01 µM

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• Bacterial inactivation (degradation) by 2 enzymes:
1) AHL lactonase: opens ring; e.g. Bacillus
2) AHL acylase: removes acyl chain; e.g. Variovorax
ENZYMATIC AHL INACTIVATION
R
NH
O
O
O
R OH
O
NH2
O
O
R
NH
O
OH
O
OH
AHL
fatty acid homoserine lactone
+
N-acyl homoserine
acylase
lactonase
Defoirdt et al. (2004) Aquaculture 240: 69-88

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• Enrichment of AHL degrading bacteria in medium
containing AHL as
• Sole C source (e.g. add NH4
+ as N source)
• Sole N source (e.g. add glycerol as C source)
• Sole C and N source
• Isolation of pure strains from the mixed culture by
spread-plating and picking of single colonies
O
O
NH
O

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• AHL degradation by Bacillus strains isolated from
shrimp (LT3, LT12) and sea bass (LCDR16)
0
1
2
3
4
5
6
0 3 6 9 12
[HHL](mg/L)
Time (h)
Control
LT3
LT12
LCDR16
R
NH
O
O
O
R
NH
O
OH
O
OH
lactonase
Defoirdt et al. (2011) Aquaculture 311: 258-260

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0
10
20
30
40
50
60
Survival(%)
• Use of signal-degrading bacteria as probionts, e.g.
in Macrobrachium larvae:
Nhan et al. (2011) J. Appl. Microbiol. 109: 1007-1016

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• Linear polymer of β-hydroxybutyric acid
POLY-β-HYDROXYBUTYRATE (PHB)
OH
O
O
O
O
O
OH
n
103-106

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H+H+
H+
H+
H+
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

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• Artemia nauplii challenged with Vibrio campbellii
• PHB particles added to culture water at start of test
0
20
40
60
80
100
Control
Vibrio
Vibrio + PHB(10)
Vibrio + PHB(100)
Vibrio + PHB(1000)
Artemiasurvival(%)
Significantly
increased
survival at
100 mg/l PHB
or more
1000 mg/l PHB:
complete protectionPHB effectively protects Artemia from
luminescent vibriosis
EFFECT in GART: Artemia survival

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0
20
40
60
80
100
Control
Vibrio
Vibrio + PHB2 (32% PHB)
Vibrio + PHB2 (2% PHB)
Artemiasurvival(%)
• Brachymonas strain PHB2 isolated from PHB-
accumulating enrichment culture
• Fed-batch enrichment with PHB → 32% on VSS
• Strain PHB2 added at 107 cells/ml (~ 10 mg/l PHB)
complete
protection
Effect is due
to PHB
accumulated
by the strain
PHB containing bacteria protect
shrimp from luminescent vibriosis
PHB-ACCUMULATING BACTERIA

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24
1
65A
78a
50
2
82B
89b
0
10
20
30
40
50
60
70
80
90
100
Day 5 Day 10 Day 15
Rearing time
Survival(%)
Control PHB
Macrobrachium larval survival feeding on
PHB enriched Artemia nauplii

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a A
x
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
Survival(%)
PHB combined with a classical HUFA
enrichment
• No PHB, no Hufa
• PHB, no Hufa
• No PHB, Hufa
• PHB, Hufa

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European sea bass larvae (Dicentrarchus labrax)
Nile Tilapia (Oreochromis niloticus)
GNOTOBIOTIC FISH LARVAE CULTURE
AS A MODEL SYSTEM

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Bone development/deformities
Development of the digestive tract
AS A MODEL SYSTEM

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Effect/mode of action of probiotic bacteria on survival of fish larvae
Virulence factors of different wild-type/mutant strains of fish
pathogens
AS A MODEL SYSTEM

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 Effect of host tissue to pathogen virulence
AS A MODEL SYSTEM

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 Fish gene-expression in relation to pathogen infection
AS A MODEL SYSTEM

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NUTRITIONAL RESEARCH
Live food production
– Artemia:
• culturing biology,
• natural occurrence,
• production techniques,
• strain characterization,
• nutritional value enrichment;
• automation in production & cleaning
techniques
– Rotifer culture and enrichment
– Micro-algae
– Special focus on production, nutritional
manipulation (lipids, vitamin C and E)

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GENOMICS
 Genome sequencing of Artemia
 Proof-of-concept as a model
organism for Crustaceans

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Environmental monitoring & design of
integrated concepts
 Biofloc technology
– Nitrogen recovery
– Waste to feed
 Biopolymers
– Compound poly--hydroxybutyrate (PHB)
– Disease control and growth promotion

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AQUATIC VETERINARY MEDICINE
 White Spot Syndrome Virus (WSSV) in penaied shrimp
 Edwardsiella & Flavobacterium causing fish diseases

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Research on WSSV in shrimp and prawn
1. Development of a reproducible WSSV challenge
procedure by intramuscular and oral routes for
testing products to control WSSV disease
a) Production of a WSSV stock and in vivo titration in SPF
Litopenaeus vannamei
b) Determination of the minimal dose able to cause
infection and disease in all the inoculated shrimps by
each of the routes tested
2. Testing effects of products (e.g. the antiviral
cidofovir and Spirulina) and environmental
circumstances (e.g. high temperature)

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3. Study of WSSV pathogenesis by oral inoculation
using low dose and high dose at 27°C in SPF
Litopenaeus vannamei
4. Comparison of WSSV-isolates  challenge
model with low and high virulent isolates
5. Effect of molt stage on WSSV infection
6. Resistance of Macrobrachium prawn to WSSV

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7. Development and use of shrimp haemocyte
culture systems (in attachment and in
suspension) for testing of immunostimulants

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RESEARCH OUTPUT
Published A1 papers Citations

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• to deliver researchers able
to perform and design research in
various aquaculture fields;
• to deliver experts who can draw and
implement strategies for future
development in the aquaculture
industry;
MSc in Aquaculture
• to form key persons who can act as a nucleus in their local
environment through dissemination and teaching their acquired
knowledge;
• to deliver academically trained staff for the aquaculture ndustry
objectives:

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MSc in Aquaculture
GENERAL COURSES
Applied Statistics
Physiology of Aquatic Organisms
Biology of Fishes
Aquatic Ecology
Microbial Ecology and Environmental Sanitation
SPECIFIC COURSES
Technology of Fishery Products
Algae Culture
Aquatic Farm Management Training
Mollusc and Crustacean Culture
Aquaculture and the Environment
Marine Fish Larviculture
General Aspects of Aquaculture
Aquaculture Nutrition
Management in the Aquaculture Industry
Aquaculture Genetics
Diseases in Aquaculture
Freshwater Fish Culture Techniques
knowledge &
application
of knowledge
Generic skills
• judgement forming
• communication
• learning skills

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IMAQUA
O.4. Specific character of the programme
• focus exclusively on aquaculture
• gradient of increasingly specialised and research-
sustained courses
across the 2 year-programme
• aims at acquisition of existing scholarly knowledge and
competency to apply, integrate and expand it
• research-embedded, not research-based
• daily management by ARC, but course framework
provided by UGent Aquaculture R&D consortium
• focus on maximal internationalisation (even expected to
further increase)

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GQS 1. Targeted outcome level
Strengths
• only master of its kind in Flanders (so no definition for discipline-
specific LOs)
• desciption of LOs very much in line with UGent competency model
• course framework provided by UGent Aquaculture Consortium,
supported by business developer
• strong international links (in Europe and beyond)
Weaknesses
• some more emphasis on scholarly competencies in compulsory
programme is advisable (presently ‘solved’ by advocating
‘Project’ and ‘Internship’
• diversity of intake students: quality assurance during selection
• new aquaculture challenges (immunology, microbial ecology...) to be
reflected in the curriculum
Future prospect
joint curricula with Can Tho University and Stellenbosch University

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GQS 2. Educational process
Strengths
• research-embedded multidisciplinary programme with highly
international dimension, permanent courseware update and preparing
for highest level in aquaculture R&D, industry and policy-making (cfr
alumni profiles)
• closely embedded in/coached by multidisciplinary and international
team of ARC personnel
• facilities: excellent library, state-of-the-art equipment for
microbiological and molecular research
Weaknesses
• high study load and uncertain funding (e.g. for thesis work abroad)
• high pressure of students on (limited) ARC staff
• scattered location of lecture rooms; limited facilities for thesis work
and practicum rooms
Future prospect
• joint curricula (CTU and SU)
• development of new courses (e.g. Aquaculture immunology)
• more hands-on experience: promote Internship

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GQS 3. Outcome level achieved
Strengths
• consistence in teaching and evaluation tools thanks to closely
interacting ARC-lecturers
• overall good scores for thesis work thanks to good procedures and
fair guidance
• overall successful transfer of knowledge & skills: most students
graduate within standard learning path
• alumni esteem IMAQUA as preparing them well for their career
Weaknesses
• heavy focus on written examinations
• limited insight of non-ARC lecturers in each other’s evaluation tools
• limited insight of lecturers in various assignments given to students
• quality assurance of evaluation system = permanent point of concern
Future prospects
• future re-positioning of curriculum and re-definition of graduate’s
profile will have major effect on evaluation tools
• overall platform of IMAQUA lecturers (meeting e.g. annually), may
increase quality and coherence of programme and evaulation

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• European Aquaculture Technology & Innovation Platform
- EATIP (founding member)
• ASEM Aquaculture Platform (coordinator)
• AquaTNET Thematic Network for European Higher
Aquaculture Education (partner, former coordinator)
• AquaExcel: Aquaculture Infrastructures for Excellence in
European Fish Research (partner)
• Aquacultuur Vlaanderen (secretariat)
• …

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ASEM Aquaculture Platform
Patrick Sorgeloos - Coordinator
Jean Dhont - Secretary
Ghent University (Belgium)
THE BRIDGE BETWEEN THE ASIAN AND
EUROPEAN AQUACULTURE SECTOR
ASEM Aquaculture
Platform

The ASEM Aquaculture Platform: a bridge between European and Asian aquaculture sector
slide 54 of 19
ASEM Aquaculture
Platform
• Reconcile concerns for seafood quality and safety
• Identify joint research, education and business
opportunities
WHAT IS IT ?

THEMATIC NETWORK
Promoting innovation and a European dimension
through Lifelong Learning in the field of Aquaculture,
Fisheries and Aquatic Resources Management
82 partners in 26 countries
45 Universities or HE Institutions
19 Research organisations
5 Associations, networks or platforms
5 Commercial or consultancy organisations
8 Others

LLP – Erasmus – Erasmus Network
OUTCOMES
• European MSc database /webportal
• Mobility portal
• Portal of dedicated PhD courses
• Teaching innovation guide
• Generic skills: Identification of
training gaps & recommendations
• Framework for sharing digital
teaching material
• Much more at www.aquatnet.com

The ASEM Aquaculture Platform: a bridge between European and Asian aquaculture sector
slide 57 of 19
thank you for your attention

Introducing the Laboratory of Aquaculture & Artemia Reference Center, Ghent University

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