OBJECTIVE 1: Determine diverse and quantitatively significant microorganisms in larval and young metamorphosed fish, feed organisms and constituents, source water and culture tanks.
Microbial sampling succeeded in meeting several of the major objectives of the project. Baseline data were collected on the occurrence and frequency of microbial populations in the different components of the culture system. Dominant and frequently occurring bacteria were identified and frozen for further study. Differences in microbial numbers and types were detected between the different components of the culture system, in particular for the algae, rotifers, and artemia used as feed organisms. For example, the data indicate that the artemia cultures favored the growth of dominant opportunistic vibrios, whereas the algal and rotifer cultures were characterized by lower overall numbers of vibrios, and a more diverse assemblage of non-vibrios. In addition, an associated increase in total vibrios was observed in the larval culture tanks when the larvae began feeding heavily on artemia. This finding is significant given that a major factor in early mortality of larval marine fish is vibriosis. During the course of this project, several unexplained incidents of mortality occurred in the larval fish during a period of time when they were feeding heavily upon artemia. Although these incidents were monitored during the course of this project, no definitive diagnoses of vibriosis could be made. However, as a result of some productive collaboration with researchers at the University of Rhode Island, a private fish diagnostics lab in Maine, and Dr. Carroll Jones of UNH Veterinary Diagnostics, two vibrios pathogenic to adult summer flounder were identified (Vibrio damsela and Vibrio carchariae). Although these organisms were not found associated with the artemia cultures, they were occasionally found associated with the tank culture water and the larval and juvenile fish. These findings provided the basis for further work.
OBJECTIVE 2: Identify probiotic microbial isolates with ecophysiological characteristics that promote competition with pathogenic bacteria and survival in larval and young fish gut microenvironments.
A major component of this study was to identify autogenous organisms that could act as probiotics. One approach towards accomplishing this is to identify organisms antagonistic towards known pathogens, but harmless to the host animal. A series of screening assays were carried out during which several strains of previously isolated and frozen bacteria from GreatBay Aquafarms were tested for their ability to inhibit the growth of the two vibrio pathogens (V. damsela and V. carchariae). A published protocol commonly used for this purpose was utilized. Each tested strain was inoculated as six discrete colonies on a nonselective agar and allowed to grow until the colonies were 2 to 3 mm in diameter. These macrocolonies were then killed with chloroform vapor, and an agar overlay containing the pathogen was poured over them. The pathogen was allowed to grow for 24 hours, after which it was checked for any growth inhibition. If the tested strain was releasing any diffusible natural antibiotic compound, it would diffuse through the agar overlay and inhibit the growth of the pathogen in the overlay. This would manifest as a clear zone of no pathogen growth around the inhibited or killed macrocolony of the tested strain.
Using the above described approach, several strains of bacteria autogenous to GreatBay Aquafarms were found capable of inhibiting the growth of the pathogenic vibrios. One strain in particular, subsequently identified as a Bacillus sp., showed a particularly strong inhibitory effect as measured by the size of the zone of inhibition. This strain was chosen for further testing.
OBJECTIVE 3: Determine the feasibility of establishing favorable microflora in early stages of summer flounder via colonization of larvae and live feed organisms by probiotic isolates.
As previously stated, the monitoring aspect of this project had indicated that the artemia were a potential source of vibrio pathogens. Artemia are filter feeding organisms, and can thus serve to bio-encapsulate microscopic particles, including bacteria, for delivery into fish digestive organs. Published studies had shown that anemia can be an efficient means of delivering either antibiotics or pathogens to larval and juvenile fish via bio-encapsulation. These findings suggested that artemia could also be used as a means of delivering potential probiotics to the fish. Several assays were conducted to assess the viability of this hypothesis. Artemia meta-nauplii were incubated for one hour in pure bacterial broth suspensions of Vibrio alginolyticus (commonly found associated with artemia cultures), the pathogens Vibrio damsela and Vibrio carchariae, and the potential probiotic Bacillus sp. No adverse effect was observed on artemia viability after one hour.
The artemia were then rinsed for one minute through a sterile mesh screen and fed to larval or juvenile fish in replicate beakers. A subsample was taken of the anemia to estimate the bacterial load. The fish were observed feeding upon the artemia, and were also subsampled for bacterial load after 3 hours, 24 hours, 48 hours, 72 hours, and 1 week. The results showed that the artemia bio-encapsulated very high bacterial titers of the vibrio and bacillus cells after one hour. In addition, the fish also showed very high levels of the bacteria three hours after feeding. These levels declined in the fish over time, but viable bacteria could still be detected 48 hours after the feeding. Surprisingly, despite the high levels of ingested and viable pathogenic vibrios in the fish, no difference in mortality was observed between tested and control fish, indicating that these vibrios were either nonpathogenic to immature fish, or were nonpathogenic when exposure was via the oral route. The mechanisms of pathogenicity and the mode of infection remain interesting and fruitful areas for further research.
In conclusion, this project succeeded in adding to the understanding of the microbial ecology of marine finfish larviculture and the relationship between indigenous microbes and fish health. Protocols were developed and used to assess the pathogenicity of microbes to larval and juvenile summer flounder, which will be applicable to other species. Artemia were identified as an excellent vector for introducing probiotic organisms into juvenile fish. Regional cooperation with other researchers was a crucial component of this project, and strengthened ties between the academic and commercial sectors. As a result of these ties, additional research in this area will continue.