Larvae of the brine shrimp Artemia franciscana serve as important feed in fish and shellfish larviculture; however, they are subject to bacterial diseases that devastate entire populations and consequently hinder their use in aquaculture. Exposure to abiotic stress was shown previously to shield Artemia larvae against infection by pathogenic Vibrio, with the results suggesting a mechanistic role for heat shock protein 70. In the current report, combined hypothermic/hyperthermic shock followed by recovery at ambient temperature induced Hsp70 synthesis in Artemia larvae. Thermotolerance was also increased as was protection against infection by Vibrio campbellii, the latter indicated by reduced mortality and lower bacterial load in challenge tests. Resistance to Vibrio improved in the face of declining body mass as demonstrated by measurement of ash-free dry weight. Hypothermic stress only and acute osmotic insult did not promote Hsp70 expression and thermotolerance in Artemia larvae nor was resistance to Vibrio challenge augmented. The data support a causal link between Hsp70 accumulation induced by abiotic stress and enhanced resistance to infection by V. campbellii, perhaps via stimulation of the Artemia immune system. This possibility is now under investigation, and the work may reveal fundamental properties of crustacean immunity. Additionally, the findings are important in aquaculture where development of procedures to prevent bacterial infection of feed stock such as Artemia larvae is a priority.

Heat shock proteins (Hsps) are robustly induced by diverse stressors that denature proteins. In addition to stress resistance, Hsps are involved in the folding of nascent proteins, plant and animal development, aging, environmental adaptation and the immune response, demonstrating the fundamental importance of these proteins to cell survival. Heat shock proteins are induced in aquatic organisms by perturbations of temperature and salinity, environmental contaminants, handling, hormones and biotic stressors. Exposure to sublethal stress may enhance tolerance to a subsequent stress, a process termed induced thermotolerance, and provide protection to stressors other than the initial stress, known as cross-tolerance. In the present review, we briefly describe the established approaches that are used to control disease during aquaculture. This is followed by documentation of Hsp induction after exposure to stressors commonly encountered by aquatic organisms. Induced thermotolerance, cross-tolerance and immune enhancement by Hsps are also considered. Although physiological stress is known to decrease disease resistance it is now becoming clear that stress-induced Hsps enhance the tolerance of aquatic organisms to disease. Potential applications for Hsps in the commercial production of fish, crustaceans and molluscs are indicated, an issue of significance when the importance of aquaculture in feeding the world's population is realized

Among their numerous physiological effects, heat shock proteins (Hsps) are potent immunomodulators, a characteristic reflecting their potential as therapeutic agents and which led to their application in combating infection. As an example, the up-regulation of endogenous Hsp70 in the branchiopod crustacean Artemia franciscana (Kellogg) is concurrent with shielding against bacterial infection. To better understand this protective mechanism, gnotobiotic Artemia were fed with Escherichia coli treated to over-produce different prokaryotic Hsps. This was shown to increase larval resistance to experimental Vibrio campbellii exposure. Immunoprobing of Western blots showed that the enhanced resistance to V. campbellii correlated with DnaK production in E coli. A definitive role for DnaK was then demonstrated by feeding Artemia larvae with transformed bacteria over-producing only this protein, although other Hsps such as DnaJ and grpE also provided tolerance against Vibrio infection. Feeding of bacteria synthesizing selected Hsps is therefore suggested as an alternative to antibiotic use as a means of enhancing resistance of Artemia larvae to bacterial infection, which may have potential applications in aquaculture.

SUMMARYEncysted embryos (cysts) of the brine shrimp Artemia undergo diapause, a state of profound dormancy and enhanced stress tolerance. Upon exposure to the appropriate physical stimulus diapause terminates and embryos resume development. The regulation of diapause termination and post-diapause development is poorly understood at the molecular level, prompting this study on the capacity of hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO) to control these processes. Exposure to H 2 O 2 and NO, the latter generated by the use of three NO generators, promoted cyst development, emergence and hatching, effects nullified by catalase and the NO scavenger 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). The maximal effect of NO and H 2 O 2 on cyst development was achieved by 4 h of exposure to either chemical. NO was effective at a lower concentration than H 2 O 2 but more cysts developed in response to H 2 O 2 . Promotion of development varied with incubation conditions, indicating for the first time a population of Artemia cysts potentially arrested in post-diapause and whose development was activated by either H 2 O 2 or NO. A second cyst sub-population, refractory to hatching after prolonged incubation, was considered to be in diapause, a condition broken by H 2 O 2 but not NO. These observations provide clues to the molecular mechanisms of diapause termination and development in Artemia, while enhancing the organism's value in aquaculture by affording a greater understanding of its growth and physiology.

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Feeding of bacterially encapsulated heat shock proteins (Hsps) to invertebrates is a novel way to limit Vibrio infection. As an example, ingestion of Escherichia coli overproducing prokaryotic Hsps significantly improves survival of gnotobiotically cultured Artemia larvae upon challenge with pathogenic Vibrio campbellii. The relationship between Hsp accumulation and enhanced resistance to infection may involve DnaK, the prokaryotic equivalent to Hsp70, a major molecular chaperone in eukaryotic cells. In support of this proposal, heat-stressed bacterial strains LVS 2 (Bacillus sp.), LVS 3 (Aeromonas hydrophila), LVS 8 (Vibrio sp.), GR 8 (Cytophaga sp.), and GR 10 (Roseobacter sp.) were shown in this work to be more effective than nonheated bacteria in protecting gnotobiotic Artemia larvae against V. campbellii challenge. Immunoprobing of Western blots and quantification by enzymelinked immunosorbent assay revealed that the amount of DnaK in bacteria and their ability to enhance larval resistance to infection by V. campbellii are correlated. Although the function of DnaK is uncertain, it may improve tolerance to V. campbellii via immune stimulation, a possibility of significance from a fundamental perspective and also because it could be applied in aquaculture, a major method of food production.

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