1.4.2 The status of Macrobrachium rosenbergii culture
1.4.3 Macrobrachium rosenbergii culture practices
1.4.4 Disease in M. rosenbergii aquaculture
1.5 The brine shrimp Artemia
1.5.1 Biology and ecology of Artemia
1.5.2 The role of Artemia in aquaculture
1.5.2.1 The supply and demand of cysts
1.5.2.2 Artemia as live food
1.5.2.3 Risks associated with the use of Artemia as live food in aquaculture
1.5.2.4 Artemia as a model test organism
1.6 Measures to control diseases in aquaculture
1.6.1 Water control
1.6.2 Immunostimulation and vaccination
1.6.3 Quorum sensing interference
1.6.4 Probiotics and prebiotics
1.6.5 Alcaligenes spp. and Bacillus spp. as probiotics
1.7 Poly-β-hydroxybutyrate as antimicrobial agent in aquaculture
1.7.1 The group of polyhydroxyalkanoates
1.7.2 The metabolism of polyhydroxyalkanoates
1.7.3 The production and cost of polyhydroxyalkanoates
1.7.4 The degradation of polyhydroxyalkanoates
1.7.5 The potential of poly-β-hydroxybutyrate as an antimicrobial agent for aquaculture application
1.7.6 Obstacles for the application of PHB in aquaculture
1.8 Thesis objectives and outline
CHAPTER 2
SECTION 1
2.1.1 Introduction
2.1.2 Materials and methods
2.1.2.1 Origin of Macrobrachium prawn larvae
2.1.2.2 Experimental feed preparation
2.1.2.2.1 Axenic hatching of Artemia franciscana
2.1.2.2.2 PHB-accumulated Alcaligenes eutrophus and PHB particles preparation
2.1.2.2.3 Enrichment of axenic Artemia nauplii with PHB-accumulated A. eutrophus and crystalline PHB particles
2.1.2.3 Experimental design
2.1.2.4 Analyses
2.1.2.4.1 Measurement of PHB content in enriched Artemia nauplii
2.1.2.4.2 Measurement of PHB content in enriched and purged Artemia nauplii
2.1.2.4.3 M. rosenbergii larval survival
2.1.2.4.4 Larval stage index (LSI)
2.1.2.4.5 Bacteria in the gut of M. rosenbergii larvae
2.1.2.4.6 Statistics
2.1.3 Results
2.1.3.1 PHB content in Artemia nauplii
2.1.3.2 Experiment 1: survival and growth test
2.1.3.2.1 Effect of feeding PHB enriched Artemia nauplii on survival and growth of M. rosenbergii larvae
2.1.3.2.2 Effect of feeding PHB enriched Artemia nauplii on the gut microbiota of M. rosenbergii larvae
2.1.3.3 Experiment 2: Challenge test
2.1.3.3.1 Effect of feeding PHB enriched Artemia nauplii on the survival of M. rosenbergii larvae challenged with V. harveyi BB120
2.1.3.3.2 Effect of feeding PHB enriched Artemia nauplii on the number of vibrios in the gut of M. rosenbergii larvae challenged with V. harveyi BB120
2.1.4 Discussion
SECTION 2
2.2.1 Introduction
2.2.2 Materials and methods
2.2.2.1 Origin of Macrobrachium prawn larvae
2.2.2.2 Axenic hatching of Artemia franciscana
2.2.2.3 Seed preparation of Alcaligenes eutrophus
2.2.2.4 A. eutrophus H16 preparation for feeding and PHB measurement
2.2.2.5 Enrichment of axenic Artemia nauplii with A. eutrophus as feed for M. rosenbergii larvae
2.2.2.6 Experimental design
2.2.2.7 Analyses
2.2.2.7.1 Cell dry weight (CDW)
2.2.2.7.2 Measurement of PHB content in A. eutrophus and enriched Artemia nauplii
2.2.2.7.3 Artemia nauplii and M. rosenbergii larval survival
2.2.2.7.4 Bacteria in the gut of M. rosenbergii larvae
2.2.2.8 Statistics
2.2.3 Results
2.2.4 Discussion
CHAPTER 3
3.1 Introduction
3.2 Materials and methods
3.2.1 Axenic cysts of Artemia franciscana
3.2.2 Bacillus sp. LT12 preparation
3.2.3 Experimental design
3.2.4 Hatching success of Artemia cysts in experiment 1
3.2.5 Carbon and nitrogen analyses of AHMA samples from experiment 1
3.2.5.1 Glycerol
3.2.5.2 Glycogen
3.2.5.3 Trehalose
3.2.5.4 Total organic carbon (TOC)
3.2.5.5 Total nitrogen (TN)
3.2.6 OD of Bacillus LT12 in experiment 2
3.2.7 Verification of axenity during hatching in experiment 1 and experiment 2
3.2.8 Statistics
3.3 Results
3.3.1 Hatching success
3.3.2 Glycerol, glycogen and trehalose content in the AHMA of Artemia
3.3.3 Total organic carbon content in the hatching medium of Artemia
3.3.4 Total nitrogen content in the hatching medium of Artemia
3.3.5 Growth of Bacillus sp. LT12 in axenic hatching medium of Artemia (Experiment 2)
3.4 Discussion
CHAPTER 4
4.1 Introduction
4.2 Materials and methods
4.2.1 Source of Bacillus strains and growth conditions
4.2.2 Axenic hatching medium and sterile nauplii of Artemia franciscana
4.2.3 Preparation of Bacillus strains for the experiments
4.2.4 Experimental design
4.2.5 Analysis
4.2.5.1 Survival and total length of Artemia nauplii
4.2.5.2 Cell dry weight (CDW) of Bacillus sp. LT12
4.2.5.3 Measurement of PHB content in Bacillus sp. LT12
4.2.5.4 Statistics
4.3 Results
4.3.1 Experiment 1 (Selecting Bacillus strains)
4.3.1.1 Survival of Artemia nauplii fed the different Bacillus strains as a sole food
4.3.1.2 Total length (TL) of Artemia nauplii
4.3.1.3 PHB content of Bacillus sp. LT3 and LT12 cultured in LB (12 g/L salinity)
4.3.2 Experiment 2 (In vivo challenge tests)
4.3.2.1 PHB content of Bacillus sp. LT12 cultured in LB, AHMA16 and AHMA20 medium
4.3.2.2 Effect of feeding Bacillus sp. LT12 cultured in LB, AHMA16 or AHMA20 medium on the survival of Artemia nauplii in in vivo challenge test with Vibrio campbellii LMG21363
4.4 Discussion
CHAPTER 5
5.1 Introduction
5.2 Materials and methods
5.2.1 Origin of Macrobrachium prawn larvae and nursing conditions
5.2.2 Experimental live food preparation
5.2.3 Experimental design
5.2.4 Larval rearing procedure and challenge test
5.2.5 Analyses
5.2.5.1 Cell density of Bacillus LT12
5.2.5.2 Cell dry weight (CDW) of Bacillus LT12
5.2.5.3 Measurement of PHB content in Bacillus LT12 and Artemia nauplii
5.2.5.4 M. rosenbergii larval survival
5.2.5.5 TCBS plate counts of bacteria in the gut of M. rosenbergii larvae
5.2.5.6 Statistics
5.3 Results
5.3.1 Experiment 1
5.3.2 Experiment 2
5.4 Discussion
CHAPTER 6
6.1 General discussion
6.1.1 The importance of the poly-β-hydroxybutyrate form for application at the larval stage
6.1.2 Reuse of Artemia hatching medium to culture PHB-accumulating bacteria
6.1.3 The economics and sustainability of reusing Artemia hatching medium for the production of PHB accumulating bacteria
6.1.4 The proposed model for application of amorphous poly-β-hydroxybutyrate on crustacean larviculture in the future