UGent Aquaculture R&D Consortium

1. UGent Aquaculture R&D Consortium MICROBIAL MANAGEMENT IN FISH AND SHELLFISH LARVICULTURE: FROM GNOTOBIOTIC EXPERIMENTS TO APPLICATIONS NPC mtg Feb 2012

2. Conceptual framework Immuno stimulant eggs MCe MCs Modulators composition larvae MCe MCs Modulators of activity nutrition MCe MCs juveniles

3. Gnotobiotic systems Artemia Brachionus Seabass Non-gnotobiotic verification Experimental approach:

4. How to study host-microbial interactions? Host Host simplification MC environment Known micro-organisms reality? complex gnotobiotic

5. GnotobioticArtemia: GART Decapsulation Instar I Decapsulated cysts 20-50 nauplii counted for the Experiments Hydrated cysts Instar II Axenic conditions Non-axenic conditions Gnotobiotic challenge: add Vibrio

6. Gnotobiotic Artemia –seabass food chain DAH1 DAH3 DAH7 Vibrio anguillarum Artemia DAH14 At 16°C

7. Gnotobiotic Artemia –seabass food chain blank Virulent Vibrio anguillarum Strain HI610 serovar O2a challenge Avirulent Vibrio anguillarum Strain 43 serovar 01

8. 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

9. Yeast cell wall-bound glucan as immunostimulant?

10. WT- exp YNB mnn9– exp YNB 43% 16% Mannoproteins  (1,6) glucans 75% 150 nm 55% CELL WALL  (1,3) glucans 9% 2% Chitin Plasma membrane (adapted from EUROCELLWALL project from EU) Yeast mutants with altered cell wall composition • Since -glucansare well-known immunostimulants, possibly the mnn9 yeast acts as immunostimulant, allowing Artemia to be protected against pathogens

11. 90 80 70 untreated+VC 60 treated+VC 50 40 30 20 10 0 WT mnn9 mnn6 fks1 knr4 kre6 gas1 chs3 LVS3 yeast strains Artemia survival in GART Survival (%)(day 6)

12. Non-gnotobiotic: mussel larvae Survival of 2 week old larvae Algae 100% 60% 40% 0% Mnn9 yeast

13. Heat shock proteins as immunostimulants?

14. DnaK (HSP70) overexpression: E. coli strain YS2 DnaK DnaK WB YS1: control strain, no DnaK overproduction YS2: positive strain, DnaK overproduction by arabinose induction

15. Enhanced resistance by DnaK feeding in a Vibrio challenge DnaK • 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 DnaK overproducing YS2 were exposed to V. campbellii

16. Priming (g)HOST MiC’s, heat shock proteins (DnaK),.. HOST + MAMPs HOST + MAMPs + pathogen

17. Priming the innate immune system:PO expression in Artemia as modulated by DnaK and Vibrio YS2(-): E coli not overproducing DnaK (HSP70 homolog) YS2(+): E coli overproducing DnaK (HSP70 homolog) VC: Vibrio campbellii challenge with a delay of 6 h

18. CONCLUSIONS HSPs • Exogenous HSPs feeding possibly triggers the Artemia innate immune response, producing anti-inflammatory activity (Phenoloxidase activity) which suppresses infection • Sofar, no confirmation under non-gnotobiotic conditions

19. POLY-β-HYDROXYBUTYRATE (PHB)

20. 103-106 POLY-β-HYDROXYBUTYRATE (PHB) • Linear polymer of β-hydroxybutyric acid Could PHB also be used to protect Artemia from luminescent vibriosis?

21. Energy H+ H+ H+ H+ H+ 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 pH optimal • Effect is pH-dependent (lower pH → higher effect) fatty acid proton pump bacterium

22. PHB UPTAKE BY ARTEMIA • Starved nauplii without feed or with PHB particles No PHB PHB Light microscopy The PHB particles are ingested by the nauplii Fluorescence microscopy (Nile Blue) (bar = 250 µm)

23. EFFECT ON STARVED ARTEMIA • Sterile Artemia nauplii: no feed added or only PHB particles (1 g/l) at day 0 Longer survival with PHB particles The nauplii can obtain energy from the particles The particles must be (partially) degraded in the gut

24. An Artemia – Macrobrachiumfood chain example PHB: non-gnotobiotic

25. Macrobrachium larval survival feeding on PHB enriched Artemia nauplii

26. PHB combined with a classical HUFA enrichment • No PHB, no Hufa • No PHB, Hufa • PHB, no Hufa • PHB, Hufa

27. CONCLUSIONS PHB • PHB particles protect Artemia from luminescent vibriosis • Positive effects in the aquaculture food chain: but further verification is needed.

28. Quorum sensing

29. 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 What is Quorum Sensing (QS)?

30. Many bacterial behaviors are regulated by quorum sensing luminescence Symbiosis Virulence Antibiotic production Biofilm formation What type of processes are under the control of QS?

31. N-Hexanol-DL-homoserine lactone (C6-HSL) N-Heptanoyl-DL-homoserine lactone (C7-HSL) N-Butyryl-DL-homoserine lactone (C4-HSL) N-Octanoyl-DL-homoserine lactone (C8-HSL) QS molecules: acyl homoserine lactones (AHL)

32. Can we demonstrate that quorum sensing is important in microbial interference with (larval) stage of aquatic animals? What is Quorum sensing (QS)?

33. An Artemia – Macrobrachiumfood chain example Quorum sensing: non-gnotobiotic

34. Effect of AHL mixture in Artemia – Macrobrachiumfood chain • Survival of Macrobrachium larvae on day 7 post-hatch, (mean ± SD, n = 6). • daily AHL addition of 1 mg/l

35. Effect of AHL and EC5D on Macrobrachium larviculture:survival • daily AHL addition of 1 mg/l

36. Effect of AHL and EC5D on Macrobrachium larviculture: LSI • daily AHL addition of 1 mg/l

37. ENZYMATIC AHL INACTIVATION • AHL degradation by pure Bacillus strains isolated from shrimp (LT3, LT12) and sea bass (LCDR16)

38. ENZYMATIC AHL INACTIVATION • Use of signal-degrading bacteria as probionts, e.g. in Macrobrachium larvae: V. harveyi + AHL degraders Untreated V. harveyi AHL degraders Is this effect by AHL degradation?

39. 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

40. General conclusions Feeds, eg PHB Feeds Pathogenic Pathogenic Quorum sensing Quorum sensing Antimicrobial Antimicrobial Probiotic bacteria Probiotic bacteria Immunostimulants Immunostimulants bacteria bacteria analysis analysis Peptides Peptides Gnotobiotic Gnotobiotic Gnotobiotic Gnotobiotic Artemia Artemia test test Artemia Artemia model model system system system system Quantitative Quantitative Biochemical Biochemical Host gene Host gene Performance Performance Fish and shellfish Fish and shellfish Fish and shellfish Fish and shellfish analysis of analysis of analysis analysis expression expression larvae validation larvae validation larvae validation larvae validation the bacterial the bacterial analysis analysis e.g. e.g. antimicrobial substances antimicrobial substances community community Marker Marker genes genes