Microbial communities associated with gastrointestinal tract of animals play a critical role in gut development, digestion and resistance to disease, thus the prospect of altering these communities beneficially by using probiotics is attractive. In terrestrial animals, the gut provides a stable, moist habitat in an otherwise moisture-limited environment, thus microbial communities tend to be very stable. In contrast, farmed aquatic animals reside within an environment that can support microbes in high densities, and as many marine animals drink continuously for osmoregulation, they are subjected to potential re-inoculation. Consequently, little is known of the stability of gut microbial communities in marine shrimp or whether it is possible to establish beneficial bacteria in the gut. The aims of this thesis were therefore to examine the midgut microbial community associated with farmed black tiger shrimp, Penaeus monodon, and to investigate whether the introduction of potentially probiotic Bacillus could alter the species diversity or abundance of the present microbes. Using culture methods it was found that B. pumilus was able to transfer between animals via the water column and persisted in the midgut for at least 7 days, while B. subtilis was only recovered from animals directly fed the bacteria and persisted for less than 24 h in the midgut. V. parahaemolyticus, a known shrimp pathogen,remained in the tanks it was originally found in, and did not transfer via the water column to other tanks and is therefore tightly associated with its host. A bacterium with apparent probiotic qualities was isolated from control animals in the above study and identified as a strain of B. pumilus. Its safety for food animal use was confirmed due to the absence of B. cereus toxin genes, and the isolate’s pH and salt tolerances were investigated. Moreover, the isolate was highly inhibitory to crustacean pathogens in the family Vibrionaceae. Methods to investigate the gut microbiota using the full cycle 16S rRNA methodology were optimized. Fluorescence in situ hybridization (FISH) probes designed specifically targeting B. pumilus, B. subtilis and B. licheniformis, commercially available probiotics, were validated for specificity and optimal hybridization conditions. For FISH analysis of bacteria in situ in histological sections of shrimp midgut trunks, fixation times in 4 % paraformaldehyde wereoptimizedfor bacterial RNA retention whilst maintaining tissue integrity. Due to the broad range of autofluorescence in the shrimp tissue, spectral imaging is required to adequately differentiate between host tissue and multiple bacterial probes. The richness and diversity of the midgut microbiota of animals treated with the novel strain of B. pumiluswere analyzed using 16S rRNA gene clone libraries and FISH analysis of histological sections. It was confirmed that B. pumilus can enter the midgut via top-coated feed and through water inoculation. In the tanks that were treated with B. pumilus the proportion of Vibrio sp. in the microbial community decreased, however, only in the systems in which B. pumilus was recovered from the shrimp midgut did the proportion of pathogenic Vibrio species decrease. The application of the B. pumilus caused a shift in the shrimp midgut microbiota, but the community returned to its initial diversity over time. The midgut microbiota of P. monodon is relatively stable but can be adjusted using probiotics. The transience or residence of the probiotics is strain-specific and should be tested for any new strains before determining optimum application protocols. The methods designed in this study are applicable to future research in this field.
| Identifer | oai:union.ndltd.org:ADTP/279247 |
| Creators | Jessica Hill |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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