larviopeningkeynotefinal - universiteit gent

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


FISH & SHELLFISH LARVAE

VALIDATION


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



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







heat shock proteins


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







heat shock proteins


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




VALIDATION


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


host‐microbe interactions


• epigenetics





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



breeding studies

• epigenetics




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



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

larviopeningkeynotefinal - universiteit gent

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