larvi 20136th fish & shellfish larviculture symposium
ghent university, belgium2‐5 september 2013
ARTEMIA AS MODEL ORGANISMIN LARVICULTURE RESEARCH
Patrick Sorgeloos Conductor of the UGent Aquaculture Orchestra
1960sState University of Ghent ‐ Faculty of Medicine ‐ Laboratory of Anatomy
Julien Fautrez Lieve Criel
brine shrimp Artemia as model organism in embryology research
1969 State University of Ghent ‐ Faculty of Sciences ‐ Laboratory of Ecology
Guido PersoonePatrick Sorgeloos
start research on Artemia culturing biology
Duke University Marine LaboratoryBeaufort, North Carolina‐USA
C.G. Bookhout &John D. Costlow
Artemia as food
1976‐78 study stays at SEAFDEC
Herminio Rabanal
SouthEast Asian Development Center
instar I nauplius
enriched metanauplii
umbrella stage
TECHNIQUE
decapsulated cysts
LARVICULTUREOF
FISH & SHELLFISH DISEASE CONTROL
COMMERCIAL COMPETITIVENESSNUTRITION
EGG QUALITY broodstock nutrition
ZOOTECHNICAL ASPECTS MICROBIAL CONDITIONS
probionts / pathogens
larvi ‘91fish & crustacean larviculture symposiumaugust 27-30, 1991gent, belgium
ADVANTAGES OF GREEN‐WATER VERSUSCLEAR‐WATER TECHNIQUE
water quality conditioning
more hypotheses than proofs black box approach – need for new research approach
pre/probiotic pre/probiotic
host
environment
COMPLEX GNOTOBIOTIC
microbialcommunity
NEW APPROACH IN THE STUDY OF HOST‐MICROBE INTERACTIONS
host
known microbial community
Gnotobiotic culture of Artemia
axenic hatching
Day 3
micro algae / yeast / bacteriaDay 0
Harvestaxenic food
Gnotobiotic culture of Artemia
• survival• length• total biomass• immune parameters
Pathogen (Vibrio campbellii)
axenic hatching
Evaluation criteria
biochemical analyses
host‐gene expression analysis
&identificationmarker genes
qual/quant analysis of bacterial
composition
performance:‐ survival‐ growth
gnotobioticArtemiasystem
gnotobioticArtemiasystem
FISH & SHELLFISH LARVAE
VALIDATION
FISH & SHELLFISH LARVAE
VALIDATION
pre/probiotics immunostimulants
Development of innovative microbial management systems
ARTEMIA AS MODEL SYSTEM IN LARVICULTURE RESEARCH
• host‐microbe interactions
• breeding studies
• epigenetics
• nutrition studies – bioflocs
ARTEMIA AS MODEL SYSTEM IN LARVICULTURE RESEARCH
• host‐microbe interactions
Influencing microbial numbers or activity
quorum sensing / quorum quenching
Poly‐β‐hydroxybutyrate
Stimulating the host’s immune response
heat shock proteins
yeast cell wall‐bound glucan
bacteria sense and respond to environmental changes
and to each other through extracellular
signal molecules ≈ hormones in higher organisms
Quorum Sensing (QS)
AHA!
Presence of QS signal molecules affects gene expressionf.ex. virulence factors (biofilm formation, toxin secretion, etc.)
LuxSLuxQ
LuxP
LuxM
LuxN CqsS
CqsA
LuxU
LuxO
LuxR
sRNA’s + Hfq
σ54
HAI‐1AI‐2
OH
O
CAI‐1
Promoter of target genes
OOB
-
OOH
OH
OH OH
vibrios
N O
OH
H OH O
verification with Vibrio harveyi QS mutants ‐ effect on Artemia survival
LuxSLuxQ
LuxP
LuxM
LuxN CqsS
CqsA
LuxU
LuxO
LuxR
sRNA’s + Hfq
σ54
HAI‐1AI‐2 OH
O
CAI‐1
Promoter of target genes
OOB
-
OOH
OH
OH OH
N O
OH
H OH O
new concept: QS‐disruption to control bacterial infections
use of QS inhibitors (e.g. plant extracts) degradation of QS signals by other bacteria
0
20
40
60
80
100
Surv
ival
(%)
0
20
40
60
80
100
Surv
ival
(%)
0
20
40
60
80
100
Surv
ival
(%)
ArtemiaVibrio harveyi
MacrobrachiumVibrio harveyi
Burbot Aeromonas hydrophila
Crustaceans: 10‐100 µM Fish: 0.01 µM
QS‐disruption to control bacterial infections
Signal Signal
0
1
2
3
4
5
6
0 3 6 9 12
[HHL] (m
g/L)
Time (h)
Control
LT3
LT12
LCDR16
QS‐disruption to control bacterial infections use of QS inhibitors (e.g. plant extracts)
degradation of QS signals by other bacteriaf.ex. Bacillus strains isolated from aquatic organisms
signal m
olecule concen
tration
signal molecule degradation
0
10
20
30
40
50
60Survival (%
)
QS‐disruption to control bacterial infections use of QS inhibitors (e.g. plant extracts)
degradation of QS signals by other bacteriause of signal‐degrading probionts in Macrobrachium larviculture
ARTEMIA AS MODEL SYSTEM IN LARVICULTURE RESEARCH
• host‐microbe interactions
Influencing microbial numbers or activity
quorum sensing / quorum quenching
Poly‐β‐hydroxybutyrate
Stimulating the host’s immune response
heat shock proteins
yeast cell wall‐bound glucan
Poly‐β‐hydroxybutyrate PHB as a bio‐control strategy in larviculture
3‐hydroxybutyric acid
Short chain fatty acids (SCFA) and organic acids: bacteriostatic and bacteriocidal effects on pathogens influence invasion capacity of pathogens beneficial effects on host intestinal cells
Does PHB have a protective effect against Vibrio infection?
PHB = bacterial storage compound
PHB as dietary ingredient for Artemiachallenge test with pathogenic Vibrio campbellii
significant increase of survival at 100 mg/L and 1000 mg/L PHB
Improved parameters:‐ growth performance‐ larval survival‐ infection resistance
Application of PHB in fish and shellfish larvicultureApplication of PHB in fish and shellfish larviculture
ARTEMIA AS MODEL SYSTEM IN LARVICULTURE RESEARCH
• host‐microbe interactions
Influencing microbial numbers or activity
quorum sensing / quorum quenching
Poly‐β‐hydroxybutyrate
Stimulating the host’s immune response
heat shock proteins
yeast cell wall‐bound glucan
highly conserved proteins, available in all living cells Induced after exposure to stressors (heat, cold,
O2 deprivation, radicals, disease etc)
Inside the cell, act as molecular chaperones –assist in protein biogenesis and degradation
Extracellular Hsps serve as danger signalsand modulate both innate and adaptive immuneresponses
Heat shock proteins (Hsps)HspHsp
Survival after Vibrio challenge
Hsps effects in Artemia ‐ Vibrio challenge test
Endogenous Hsp accumulation Survival after Vibrio challenge
Hsp70
Hsp70
Correlation exists between enhanced protection and Hsp70accumulation
Hsps effects in Artemia ‐ Vibrio challenge test
heat shock is not an ideal way to enhance Hsps inaquaculture
less traumatic approaches are needed to manipulate Hsps expression
can compound(s) extractedfrom plants induce Hsp70 inaquaculture animals?
can they confer protectionagainst stress and disease ?
new concept: use of Hsp‐inducing compounds
Survival after Vibrio challenge
2-fold
Protective effect of Hsp‐inducing compounds against Vibrio harveyi
‐‐‐‐‐‐‐‐‐ Hspi concentration‐‐‐‐‐‐‐‐‐‐
Induction of Hsp70Survival after Vibrio challenge100
75
Marker Hela cells control Hspi
2-fold
Protective effect of Hsp‐inducing compounds against Vibrio harveyi
‐‐‐‐‐‐‐‐‐ Hspi concentration‐‐‐‐‐‐‐‐‐‐
treated = exposure to 1‐h LD50 (5.92 mg L−1 NH3)
Validation with common carp Cyprinus carpio
Hsp‐inducing plant extract protects against lethal ammonia toxicity
Development of innovative microbial management systems
pre/probiotics
biochemical analyses
host‐gene expression analysis
&identificationmarker genes
qual/quant analysis of bacterial
composition
performance:‐ survival‐ growth
immunostimulants
gnotobioticArtemiasystem
gnotobioticArtemiasystem
FISH & SHELLFISH LARVAE
VALIDATION
FISH & SHELLFISH LARVAE
VALIDATION
• mtDNA sequenced(Valverde et al., 1994)
• 2n = 42 (A. persimilis 44)(Abreu‐Grobois, 1987; Badaracco et al., 1987)
• Genome size estimation ~ 1 Gb (De Vos et al., 2013)
• AFLP‐based linkage map with 433 AFLP markers
(De Vos et al., 2013)
The Artemia genome so far
37
introns6% exons
3%
intergenic region43%
LINEs10%
LTR elements1%
DNA elements5%
Unclassified repeats31%
Satellites1%
The Artemia genome today
23,860 coding genes identified
ARTEMIA AS MODEL SYSTEM IN LARVICULTURE RESEARCH
host‐microbe interactions
• breeding studies
• epigenetics
• nutrition studies – bioflocs
ARTEMIA AS MODEL SYSTEM IN LARVICULTURE RESEARCH
host‐microbe interactions
• breeding studies
Artemia: suitable tool as
gene discovery platform
for crustaceans ?
1st test case:
breeding for thermo‐tolerance
..TF1 CF1
lethal heat shock T C iso‐thermic conditions
..TF1 CF1
TF2 CF2
TF12 CF12
lethal heat shock T C iso‐thermic conditions
Whole genome sequencing
Hsp70
selected population
control population
Allele shift detection in relevant genesby whole genome re‐sequencing
ARTEMIA AS MODEL SYSTEM IN LARVICULTURE RESEARCH
host‐microbe interactions
breeding studies
• epigenetics
• nutrition studies – bioflocs
ARTEMIA AS MODEL SYSTEM IN LARVICULTURE RESEARCH
host‐microbe interactions
breeding studies
• epigenetics
heritable modification of phenotypes
without modification at genotype level
(modification of DNA/histones)
(clonal population)Parthenogenetic Artemia strain
TF1 CF1
daily non‐lethal heat shock T iso‐thermic conditions C
(clonal population)Parthenogenetic Artemia strain
TF1 CF1
TF2 CF2
TF3 CF3
ISO
THERMIC
daily non‐lethal heat shock T iso‐thermic conditions C
Thermo-tolerance test (common garden experiment)
******
*** *** *** ****** ***
0
0,2
0,4
0,6
0,8
1
0 3 6 9 12 15 18 21 24 27
Prop
ortio
n of th
e survived
animals
Time (h)
TF1
CF1CF1
TF1
*
* ** * * *
0
0,2
0,4
0,6
0,8
1
0 3 6 9 12 15 18 21 24 27
Prop
ortio
n of th
e survived
animals
Time (h)
TF3CF3
Thermo-tolerance test (common garden experiment)
******
*** *** *** ****** ***
0
0,2
0,4
0,6
0,8
1
0 3 6 9 12 15 18 21 24 27
Prop
ortio
n of th
e survived
animals
Time (h)
TF1
CF1
*** ***
*** ***
*** *** ******
** **
0
0,2
0,4
0,6
0,8
1
0 3 6 9 12 15 18 21 24 27
Prop
ortio
n of th
e survived
animals
Time (h)
TF2
CF2CF1
TF1 TF2
CF2
CF3
TF3
epigenetics
Pittman et al., 2013
Opportunities for application of epigenetics in aquaculture
ARTEMIA AS MODEL SYSTEM IN LARVICULTURE RESEARCH
host‐microbe interactions
breeding studies
epigenetics
• nutrition studies ‐ bioflocs
Artemia as a model for
microbe‐based feeding
Morphologicaldevelopment
Aquatic veterinarymedicine
Nutritionalresearch
GenomicsMicrobial
management
Environmentalmonitoring
Life cycle analysis
Industry yields & quality disease
Industry yields & quality disease
Society• sustainability • food security
Society• sustainability • food security
Faculty of Bioscience Engineering Animal Production - Patrick Sorgeloos and Peter Bossier Biochemical and Microbial Technology – Nico Boon and Tom Van de WieleEcotoxicology and Environmental Sanitation – Colin JanssenEnvironmental Sustainability Assessment – Jo Dewulf
Faculty of Veterinary Medicine Morphology – Wim Van den Broeck and Annemie Decostere Virology, Parasitology and Immunology – Hans Nauwynck
Faculty of Sciences Biology – Dominique Adriaens and Magda Vincx Molecular Genetics – Marnik Vuylsteke, Yves Van de Peer and Dirk Inzé
AlgeriaBangladeshBelgiumBrazilChiliChinaColombiaEcuadorEgyptGreeceIndonesiaIranMorocco
Thank you
www.aquaculture.ugent.beslide layout courtesy Jean Dhont
Thank you
www.aquaculture.ugent.beslide layout courtesy Jean Dhont
Thank you
www.aquaculture.ugent.beslide layout courtesy Jean Dhont
Thank you
www.aquaculture.ugent.beslide layout courtesy Jean Dhont
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