MICROBIAL MANAGEMENT IN FISH AND SHELLFISH LARVICULTURE FROM

MICROBIAL MANAGEMENT IN FISH AND SHELLFISH LARVICULTURE: FROM GNOTOBIOTIC EXPERIMENTS TO APPLICATIONS NPC mtg Feb 2012

Conceptual framework Immuno stimulant eggs larvae MCs MCe MCs Modulators composition Modulators of activity nutrition juveniles MCe LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium MCs slide 2 of 39

Experimental approach: • Gnotobiotic systems – Artemia – Brachionus – Seabass • Non-gnotobiotic verification LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 3 of 39

How to study host-microbial interactions? Host simplification MC Host environment reality? complex LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium Known microorganisms gnotobiotic slide 4 of 39

Gnotobiotic Artemia: GART Decapsulation Instar I Decapsulated cysts 20 -50 nauplii counted for the Experiments Hydrated cysts Non-axenic conditions Instar II Axenic conditions Gnotobiotic challenge: add Vibrio LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 5 of 39

Gnotobiotic Artemia –seabass food chain DAH 1 DAH 3 DAH 7 Vibrio anguillarum Artemia DAH 14 LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium At 16°C slide 6 of 39

Gnotobiotic Artemia –seabass food chain blank Virulent Vibrio anguillarum Strain HI 610 challenge serovar O 2 a Avirulent Vibrio anguillarum Strain 43 serovar 01 LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 7 of 39

Steering host-microbial interactions • Stimulating the host’s immune response – yeast cell wall-bound glucan – heat shock proteins • Influencing microbial numbers or activity – polyhydroxybutyric acid – quorum sensing LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 8 of 39

Yeast cell wall-bound glucan as immunostimulant? LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 9 of 39

Yeast mutants with altered cell wall composition WT- exp YNB Mannoproteins 43% mnn 9 – exp YNB 16% (1, 6) glucans 55% 75% 2% 9% (1, 3) glucans Chitin Plasma membrane (adapted from EUROCELLWALL project from EU) ü Since -glucans are well-known immunostimulants, possibly the mnn 9 yeast acts as immunostimulant, allowing Artemia to be protected against pathogens LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 10 of 39

Artemia survival in GART 90 Survival (%)(day 6) 80 70 untreated+VC 60 treated+VC 50 40 30 20 10 0 WT mnn 9 mnn 6 fks 1 knr 4 kre 6 gas 1 chs 3 LVS 3 yeast strains LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 11 of 39

Non-gnotobiotic: mussel larvae Survival of 2 week old larvae 100% 0% Algae Mnn 9 yeast LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium 60% 40% slide 12 of 39

Heat shock proteins as immunostimulants? LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 13 of 39

Dna. K (HSP 70) overexpression: E. coli strain YS 2 Dna. K WB Dna. K YS 1: control strain, no Dna. K overproduction YS 2: positive strain, Dna. K overproduction by arabinose induction LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 14 of 39

Enhanced resistance by Dna. K feeding in a Vibrio challenge 60 Run 1 Run 2 Survival (%) 50 40 30 20 10 0 YS 1 CTR YS 1 ID YS 2 CTR YS 2 ID Dna. K • Survival of Artemia larvae fed either induced or non-induced negative control strain YS 1 was low. • Survival of non-induced YS 2 strains as in negative control • A significant increase in survival in larvae fed with arabinose-induced Dna. K overproducing YS 2 were exposed to V. campbellii LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 15 of 39

Priming (g)HOST Mi. C’s, heat shock proteins (Dna. K), . . HOST + MAMPs + pathogen 16

Priming the innate immune system: PO expression in Artemia as modulated by Dna. K and Vibrio YS 2(-): E coli not overproducing Dna. K (HSP 70 homolog) YS 2(+): E coli overproducing Dna. K (HSP 70 homolog) VC: Vibrio campbellii challenge with a delay of 6 h 17

CONCLUSIONS HSPs • Exogenous HSPs feeding possibly triggers the Artemia innate immune response, producing anti-inflammatory activity (Phenoloxidase activity) which suppresses infection • Sofar, no confirmation gnotobiotic conditions LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium under non- slide 18 of 39

POLY-β-HYDROXYBUTYRATE (PHB) LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 19 of 39

POLY-β-HYDROXYBUTYRATE (PHB) • Linear polymer of β-hydroxybutyric acid 103 -106 Could PHB also be used to protect Artemia from luminescent vibriosis? LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 20 of 39

SHORT-CHAIN FATTY ACIDS • 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 p. H optimal – Effect is p. H-dependent (lower p. H → higher effect) H+ fatty acid H+ H+ proton pump y H+ Energ bacterium LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 21 of 39

PHB UPTAKE BY ARTEMIA • Starved nauplii without feed or with PHB particles No PHB Light microscopy The PHB particles are ingested by the nauplii Fluorescence microscopy (Nile Blue) LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium (bar = 250 µm) slide 22 of 39

EFFECT ON STARVED ARTEMIA • Sterile Artemia nauplii: no feed added or Longer only PHB particles (1 g/l) at day 0 with survival PHB particles The nauplii can obtain energy from the particles LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium The particles must be (partially) degraded in slide 23 of 39 the gut

PHB: non-gnotobiotic An Artemia – Macrobrachium food chain example LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 24 of 39

Macrobrachium larval survival feeding on PHB enriched Artemia nauplii LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 25 of 39

PHB combined with a classical HUFA enrichment • No PHB, no Hufa • No PHB, Hufa • PHB, no Hufa • PHB, Hufa LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 26 of 39

CONCLUSIONS PHB • PHB particles protect Artemia from luminescent vibriosis • Positive effects in the aquaculture food chain: but further verification is needed. LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 27 of 39

Quorum sensing LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 28 of 39

What is Quorum Sensing (QS)? • QS: a mechanism by which bacteria regulate gene expression in response to their population density by producing, releasing and detecting small signal molecules (quorum sensing molecules) (Fuqua et al. , 1997). • QS: process of bacterial cell-to-cell communication/conversation with signal molecules LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 29 of 39

What type of processes are under the control of QS? • Many bacterial behaviors are regulated by quorum sensing – luminescence – Symbiosis – Virulence – Antibiotic production – Biofilm formation LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 30 of 39

QS molecules: acyl homoserine lactones (AHL) N-Heptanoyl-DL-homoserine N-Hexanol-DL-homoserine lactone (C 7 -HSL) lactone (C 6 -HSL) N-Butyryl-DL-homoserine N-Octanoyl-DL-homoserine lactone (C 4 -HSL) lactone (C 8 -HSL) LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 31 of 39

What is Quorum sensing (QS)? Can we demonstrate that quorum sensing is important in microbial interference with (larval) stage of aquatic animals? LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 32 of 39

Quorum sensing: non-gnotobiotic An Artemia – Macrobrachium food chain example LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 33 of 39

Effect of AHL mixture in Artemia – Macrobrachium food chain • Survival of Macrobrachium larvae on day 7 post-hatch, (mean ± SD, n = 6). • daily AHL addition of 1 mg/l LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 34 of 39

Effect of AHL and EC 5 D on Macrobrachium larviculture: survival • daily AHL addition of 1 mg/l LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 35 of 39

Effect of AHL and EC 5 D on Macrobrachium larviculture: LSI • daily AHL addition of 1 mg/l LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 36 of 39

ENZYMATIC AHL INACTIVATION AHL degradation by pure Bacillus strains isolated from shrimp (LT 3, LT 12) and sea bass (LCDR 16) 6 5 [HHL] (mg/L) • 4 Control 3 LT 12 2 LCDR 16 1 0 0 3 6 Time (h) 9 12

ENZYMATIC AHL INACTIVATION • Use of signal-degrading bacteria as probionts, e. g. in Macrobrachium larvae: re t Un ed t a i s ey v r er d a a h V. L H A gr e d V. L AH rs i + ade y ve egr r ha d Is this effect by AHL degradation?

Quorum sensing: conclusions • QS is important in host-microbial interactions in the aquatic environment • Data on in vivo QS molecule concentration are mostly lacking, necessary to further substantiate QS importance for an aquaculture setting LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 39 of 39

General conclusions Feeds, Feeds eg PHB Quorum sensing analysis Pathogenic bacteria Probiotic bacteria Immunostimulants Antimicrobial Peptides Gnotobiotic Artemiatest Artemia model system Performance Quantitative analysis of the bacterial community Host gene expression analysis Biochemical analysis e. g. antimicrobial substances Fish and shellfish larvae validation Marker genes LARVI 09 Keynote on microbial management – Peter Bossier Ghent University Aquaculture Research Consortium slide 40 of 39