Disease imposes an important constraint on aquaculture and in this context vibriosis brings about massive mortalities in many commercial species, ranging from fish to shrimp, with resultant heavy monetary losses. Antibiotics are frequently used to control disease but this is expensive and entails severe negative impacts on living organisms and the environment. Therefore, an urgent need exists to develop effective prophylactic measures which will reduce antibiotic use and the environmental impact of aquaculture. During my work the application of heat shock proteins (Hsps) as an approach to disease control in aquaculture was explored, with gnotobiotic Artemia franciscana larvae as the model organism. In chapter 4, a non-lethal heat shock (NLHS) protocol for induction of endogenous Hsp70 in Artemia was optimized. Specifically, axenic larvae incubated at 28°C were heat shocked at 32, 37 and 40°C for 15, 30, 45 and 60 min, with recovery periods of 2, 6, 12 and 24 h and then tested for resistance against Vibrio campbellii and V. proteolyticus. An NLHS of 37°C for 30 min followed by a 6 h recovery period optimally enhanced resistance of Artemia larvae against pathogenic vibrios and induced Hsp70 maximally. The resulting twofold increase in survival of larvae to pathogenic Vibrio in concert with stress protein synthesis suggested that Hsp70 functions in protection. The expression of Hsp70 in Artemia larvae following exposure to abiotic stressors including a hypothermic stress from 28°C to 4°C for 1 h, a combined hypo- and hyperthermic stress with temperature reduction from 28°C to 4°C for 1 h followed by incubation at 37°C for 30 min, and several osmotic stresses with change from 30g/l to 4, 50, 100 and 150g/l for 30 min was examined in chapter 5. The effects on animal weight loss, induced thermotolerance, and resistance against V. campbellii were determined. Immunoprobing of western blots revealed a single polypeptide of approximately 70 kDa, which increased only in larvae exposed to combined hypo- and hyperthermic shock. A lower ash free dry weight, reflecting reduced growth, was detected in animals stressed at salinities of 100 and 150 g/l and from the combined hypothermic/hyperthermic treatment. Conversely, weight loss was not apparent in larvae experiencing hypothermic stress treatment and osmotic stress at 4 and 50 g/l. Thermotolerance and protection against infection by V. campbellii were significantly enhanced in larvae preconditioned with a combined hypo- and hyperthermic stress. The data support a causal link between Hsp accumulations as monitored by Hsp70 induced by abiotic stress and enhanced resistance to infection by V. campbellii, perhaps via stimulation of the Artemia immune system. In chapter 6, the protective properties of exogenous Hsps were investigated. Heat shocked E. coli strains CAG 626 and CAG 629, and transformed E. coli over-expressing the three Hsp combinations DnaK-DnaJ-GrpE and GroEL-GroES, DnaK-DnaJ-GrpE, and GroEL-GroES were fed to Artemia larvae prior to V. campbellii challenge. Heat shocking strain CAG 626 enhanced its ability to safeguard Artemia larvae from Vibrio infection by approximately twofold whereas CAG 629, which does not increase Hsp production upon heat shock, failed to provide protection. Feeding Artemia larvae with E. coli over-producing DnaK-DnaJ-GrpE significantly increased larval resistance to V. campbellii, suggesting a role for these proteins in protection, possibly via immune enhancement. In contrast, this effect was not observed in larvae fed with E. coli over-producing GroEL-GroES. To test the role of DnaK larvae were fed with E. coli over-producing only this protein and this led to a 3-fold increase in Artemia survival. The protective effects of bacterially encapsulated heat shock proteins in Artemia larvae were investigated further in chapter 7. Heated bacterial strains LVS 2 (Bacillus sp), LVS 3 (Aeromonas hydrophila), LVS 8 (Vibrio sp), GR 8 (Cytophaga sp) and GR 10 (Roseobacter sp), verified by probing of western blots to over-produce DnaK, were fed once to gnotobiotic Artemia larvae and their effects were examined in V. campbellii challenges. All heated bacterial strains were more effective than non-heated bacteria in defending gnotobiotic Artemia larvae against V. campbellii challenge. Immunoprobing of western blots and quantification by ELISA revealed that the amount of DnaK in bacteria, and their ability to enhance larval resistance to V. campbellii infection, were correlated. DnaK and/or other Hsps, improved resistance of Artemia against Vibrio infection, perhaps by immune stimulation. In conclusion, the outcome from this PhD work revealed that Artemia resistance to Vibrio infection is enhanced, perhaps by stimulation of the immune response, upon elevating endogenous Hsps by non-lethal heat shock and through administration of heat-shocked bacteria. The feeding of E. coli under an arabinose inducible promoter, also providing protection against V. campbellii, clearly placed Hsp70 as the focal point of attention. Both techniques represent efficient strategies to control Vibrio infection in Artemia and may serve as alternatives to antibiotic use in aquaculture. |
