The identification and development of probionts for use in marine fish hatcheries

Striped trumpeter (Latris lineata) is currently being researched as a new candidate for the Tasmanian aquaculture industry. Larval development is protracted and unusual in the striped trumpeter, and successful mass culture has taken over 15 years. Recent breakthroughs have been made in the successful culture of seedstock, including the use of ozonated water to reduce and control microbial communities. The subject of this thesis was the implementation of research to further improve larval and juvenile rearing success through the use of bacterial probionts. A knowledge of the microbial bacterial community of the larvae, seawater and live feeds was necessary in order to develop an understanding of what represents a healthy microbial ecology relevant for larval fish rearing success. 16S ribosomal RNA (rRNA) gene-based clone library and terminal restriction fragment length polymorphism (TRFLP) analyses were utilized to examine the microbial community associated with larvae cultured under different "greenwater" conditions. It was discovered that the larvae-associated microbial diversity was restricted but varied considerably between culture conditions. Most bacteria detected on the basis of cloned16S rRNA gene sequence data, belonged to class Alphaproteobacteria (predominantly of the Roseobacter clade), Gammaproteobacteria (genus Psychrobacter and Pseudoalteromonas) and Actinobacteria (genus Microbacterium). No association was found between larval survival and microbial community structure. Using TRFLP analysis, similar results were obtained and demonstrated that the bacterial composition was in agreement with the clone library data. It was found that the microbial community in the larvae was distinct from the bacterial community present in the surrounding water. Potential probiotic candidates were identified using antimicrobial in vitro plate testing against known pathogenic Vibrio species, with six out of 25 isolates tested selected for further testing. The 25 test bacteria were obtained in a previous experiment and were determined to be a representation of the bacterial community present in 15 days post-hatch (dph) striped trumpeter larvae. In both the rotifer and Artemia challenge trials, it was determined that Pseudoalteromonas agarivorans ST18 and Aliivibrio fischeri ST7 had the least negative effect on rotifer and Artemia survival numbers. To further assess the probiotic capability of strains ST18 and ST7 rotifer and Artemia cultures were challenged with pathogenic strain Vibrio proteolyticus V760 mixed with strains S118 or ST7. Strain ST18 was found to have a probiotic effect in that cultures containing both V760 and 5T18 were not significantly different from the controls but produced significantly better survival compared to the pathogen-only treatments. To further investigate ST18 and ST7 probiotic capability, they were added to striped trumpeter larvae cultures either directly through addition to water or through bioencapsulation in live fed rotifers. TRFLP was used to monitor the change in bacterial community and to track individual probionts by detection of unique terminal restriction fragments (TRF). The addition of strain ST7 alone was found to be disadvantageous to the culturing of striped trumpeter yolk sac larvae, while the addition of strain 5T18 and a combination of strains 5T18 with ST7 showed no significant reduction in survival. Tracking of strain ST18 using TRFLP was successful with the strain specifically detected in treatments where it was added by both bioencapsulation and direct addition