larvi th fish & shellfish larviculture symposium ghent university, belgium 2 5 september2013

Transcription

1 larvi th fish & shellfish larviculture symposium ghent university, belgium 2 5 september2013 ARTEMIA AS MODEL ORGANISM IN LARVICULTURE RESEARCH Patrick Sorgeloos Conductor of the UGent Aquaculture Orchestra

2 1960s State University of Ghent Faculty of Medicine Laboratory of Anatomy brine shrimp Artemia as model organism in embryology research Julien Fautrez Lieve Criel

3 1969 State University of Ghent Faculty of Sciences Laboratory of Ecology start research on Artemia culturing biology Patrick Sorgeloos Guido Persoone

4 Duke University Marine Laboratory Beaufort, North Carolina USA Artemia as food C.G. Bookhout & John D. Costlow

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6 SouthEast Asian Development Center Herminio Rabanal study stays at SEAFDEC

7 decapsulated cysts umbrella stage instar I nauplius TECHNIQUE enriched metanauplii

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10 larvi 91EGG QUALITY fish & crustacean larviculture symposium broodstock nutrition august 27-30, 1991 gent, NUTRITION belgium COMMERCIAL COMPETITIVENESS LARVICULTURE OF FISH & SHELLFISH DISEASE CONTROL ZOOTECHNICAL ASPECTS MICROBIAL CONDITIONS probionts / pathogens

11 ADVANTAGES OF GREEN WATER VERSUS CLEAR WATER TECHNIQUE water quality conditioning pre/probiotic more hypotheses than proofs black box approach need for new research approach

12 NEW APPROACH IN THE STUDY OF HOST MICROBE INTERACTIONS host host environment microbial community known microbial community COMPLEX GNOTOBIOTIC

13 Gnotobiotic culture of Artemia axenic hatching

14 Gnotobiotic culture of Artemia axenic hatching axenic food Harvest micro algae / yeast / bacteria Day 0 Evaluation criteria survival length total biomass immune parameters Day 3 Pathogen (Vibrio campbellii)

15 Development of innovative microbial management systems pre/probiotics immunostimulants gnotobiotic Artemia system performance: survival growth qual/quant analysis of bacterial composition host gene expression analysis & identification marker genes biochemical analyses FISH & SHELLFISH LARVAE VALIDATION

16 ARTEMIA AS MODEL SYSTEM IN LARVICULTURE RESEARCH host microbe interactions breeding studies epigenetics nutrition studies bioflocs

17 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

18 Quorum Sensing (QS) bacteria sense and respond to environmental changes and to each other through extracellular signal molecules hormones in higher organisms AHA!

19 Presence of QS signal molecules affects gene expression f.ex. virulence factors (biofilm formation, toxin secretion, etc.) H H N OH O HAI 1 O O HO O O B - OH AI 2 O OH OH OH O CAI 1 LuxP LuxN LuxQ LuxS CqsS LuxM CqsA srna s + Hfq LuxR LuxO LuxU σ 54 Promoter of target genes vibrios

20 new concept: QS disruption to control bacterial infections verification with Vibrio harveyi QS mutants effect on Artemia survival H OH O O H N HAI 1 O HO OH B - O O O AI 2 OH OH OH O CAI 1 LuxP LuxN LuxQ LuxS CqsS LuxM LuxU CqsA srna s + Hfq LuxR LuxO σ 54 Promoter of target genes

21 QS disruption to control bacterial infections use of QS inhibitors (e.g. plant extracts) degradation of QS signals by other bacteria Survival (%) Survival (%) Survival (%) Artemia Vibrio harveyi Macrobrachium Vibrio harveyi Burbot Aeromonas hydrophila Crustaceans: µm Fish: 0.01 µm

22 QS disruption to control bacterial infections use of QS inhibitors (e.g. plant extracts) degradation of QS signals by other bacteria f.ex. Bacillus strains isolated from aquatic organisms 6 signal molecule concentration [HHL] (mg/l) Signal signal molecule degradation Control LT3 LT12 LCDR Time (h)

23 QS disruption to control bacterial infections use of QS inhibitors (e.g. plant extracts) degradation of QS signals by other bacteria use of signal degrading probionts in Macrobrachium larviculture Survival (%)

24 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

25 Poly β hydroxybutyrate PHB as a bio control strategy in larviculture PHB = bacterial storage compound 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?

26 PHB as dietary ingredient for Artemia challenge test with pathogenic Vibrio campbellii significant increase of survival at 100 mg/l and 1000 mg/l PHB

27 Application of PHB in fish and shellfish larviculture Improved parameters: growth performance larval survival infection resistance

28 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

29 Heat shock proteins (Hsps) Hsp highly conserved proteins, available in all living cells Induced after exposure to stressors (heat, cold, O 2 deprivation, radicals, disease etc) Inside the cell, act as molecular chaperones assist in protein biogenesis and degradation Extracellular Hsps serve as danger signals and modulate both innate and adaptive immune responses

30 Hsps effects in Artemia Vibrio challenge test Survival after Vibrio challenge

31 Hsps effects in Artemia Vibrio challenge test Endogenous Hsp accumulation Survival after Vibrio challenge Hsp70 Hsp70 Correlation exists between enhanced protection and Hsp70 accumulation

32 new concept: use of Hsp inducing compounds heat shock is not an ideal way to enhance Hsps in aquaculture less traumatic approaches are needed to manipulate Hsps expression can compound(s) extracted from plants induce Hsp70 in aquaculture animals? can they confer protection against stress and disease?

33 Protective effect of Hsp inducing compounds against Vibrio harveyi Survival after Vibrio challenge 2-fold Hspi concentration

34 Protective effect of Hsp inducing compounds against Vibrio harveyi Survival after Vibrio challenge 2-fold Induction of Hsp70 Marker Hela cells control Hspi Hspi concentration

35 Validation with common carp Cyprinus carpio Hsp inducing plant extract protects against lethal ammonia toxicity treated = exposure to 1 h LD50 (5.92 mg L 1 NH 3 )

36 Development of innovative microbial management systems pre/probiotics immunostimulants gnotobiotic Artemia system performance: survival growth qual/quant analysis of bacterial composition host gene expression analysis & identification marker genes biochemical analyses FISH & SHELLFISH LARVAE VALIDATION

37 The Artemia genome so far 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) 37

38 The Artemia genome today Satellites 1% introns 6% exons 3% Unclassified repeats 31% intergenic region 43% DNA elements 5% LTR elements 1% LINEs 10% 23,860 coding genes identified

39 ARTEMIA AS MODEL SYSTEM IN LARVICULTURE RESEARCH host microbe interactions breeding studies epigenetics nutrition studies bioflocs

40 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

41 lethal heat shock T C iso thermic conditions TF1. CF1

42 lethal heat shock T C iso thermic conditions TF1 TF2. CF1 CF2 TF12 CF12

43 Allele shift detection in relevant genes by whole genome re sequencing selected population Whole genome sequencing control population Hsp70

44 ARTEMIA AS MODEL SYSTEM IN LARVICULTURE RESEARCH host microbe interactions breeding studies epigenetics nutrition studies bioflocs

45 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)

46 Parthenogenetic Artemia strain (clonal population) daily non lethal heat shock T iso thermic conditions C TF1 CF1

47 Parthenogenetic Artemia strain (clonal population) daily non lethal heat shock T iso thermic conditions C TF1 TF2 TF3 I S O T H E R M I C CF1 CF2 CF3

48 Proportion of the survived animals 1 0,8 0,6 0,4 0,2 0 *** TF1 *** CF1 Thermo-tolerance test (common garden experiment) *** *** *** *** *** *** Time (h) TF1

49 Proportion of the survived animals 1 0,8 0,6 0,4 0,2 0 *** TF1 *** CF1 Thermo-tolerance test (common garden experiment) *** *** *** *** *** *** Time (h) TF1 Proportion of the survived animals 1 0,8 0,6 0,4 0,2 0 *** *** CF2 *** *** TF2 CF2 *** *** *** *** ** ** Time (h) TF2 Proportion of the survived animals 1 0,8 0,6 0,4 0,2 0 CF3 * TF3 CF3 * * * * * * Time (h) TF3

50 Opportunities for application of epigenetics in aquaculture epigenetics Pittman et al., 2013

51 ARTEMIA AS MODEL SYSTEM IN LARVICULTURE RESEARCH host microbe interactions breeding studies epigenetics nutrition studies bioflocs Artemia as a model for microbe based feeding

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53 Industry yields & quality disease Morphological development Society sustainability food security Life cycle analysis Aquatic veterinary medicine Environmental monitoring Nutritional research Microbial management Genomics

54 Faculty of Bioscience Engineering Animal Production - Patrick Sorgeloos and Peter Bossier Biochemical and Microbial Technology Nico Boon and Tom Van de Wiele Ecotoxicology and Environmental Sanitation Colin Janssen Environmental 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

55 Algeria Bangladesh Belgium Brazil Chili China Colombia Ecuador Egypt Greece Indonesia Iran Morocco

56 Thank you slide layout courtesy Jean Dhont

57 Thank you slide layout courtesy Jean Dhont

58 Thank you slide layout courtesy Jean Dhont

59 Thank you slide layout courtesy Jean Dhont