Aquatic animals, such as fish and shellfish, provide important economic and nutritional benefits for human society. Due to overexploitation of natural fish sources through traditional wild-caught fisheries, aquaculture (generally described as fish farming or culturing) has grown into an economically important industry. A major focus area for the aquaculture field is related to sustainability by ensuring the health and welfare of the aquatic animals. Communities of microorganisms inhabiting the various niches of a given host comprise its microbiome and provide several key health benefits. The microbiome impacts nutrient acquisition, gut homeostasis, protection against pathogens, and immune system modulation. Therefore, much attention has been placed on studying how various culturing conditions and host factors impact the microbiomes of aquatic animals.
Here, multiple studies were conducted to elucidate the impacts of various parameters on the microbiomes of rainbow trout, steelhead trout, and Nile tilapia, including dietary supplementation, administration of probiotics and animal age. Though there is a significant correlation between the diet fed to fish and their microbiome communities, small dietary changes such as the inclusion of a dried and lysed yeast product, acting as a protein source alternative to unsustainable fishmeal did not significantly alter the intestinal adherent microbiome of rainbow trout. Moreover, an optimal percentage of yeast replacement that did not negatively impact weight gain for the aquaculture-raised fish was identified, suggesting its efficacy for the industry. Similarly, the intestinal adherent microbiomes of steelhead trout were not significantly altered by diet supplementation with a Bacillus subtilis probiotic. The total microbiome of steelhead trout (mucosa combined with digesta) was instead significantly changed when they were only fed the probiotic additive at an early stage of intestinal development. This change in the microbiome of steelhead trout correlated with a significant increase in weight gain compared to fish only fed the probiotic during later stages of intestinal development. These findings also corroborate previous observations wherein the intestinal microbiome of fish varies during their developmental stages but then stabilizes over time.
Determining the core set of bacteria present in fish microbiomes, independent of treatment variables, is another important factor when considering attempts to manipulate the microbiome. To that end, a literature review was conducted in which the phyla Firmicutes, Proteobacteria and, to a lesser extent, Actinobacteria, Bacteroides, and Tenericutes were identified as likely members of the rainbow trout core microbiome. Bacterial families identified as part of the core phyla included Lactobacilliaceae that are commonly used as probiotics and Mycoplasmataceae that lack cell walls. Preventing dysbiosis of the rainbow trout microbiomes will be crucial to ensuring the health of the fish hosts and increasing longevity and profitability of the aquaculture industry.
Another important aquaculture-raised species is the Eastern oyster. This animal is critical for the ecological health of the Chesapeake Bay, and it is also an important source of revenue. A significant portion of the revenue flow is the harvest and sale of live oysters for consumption. Unfortunately, consumption of raw or undercooked oysters is the most common route of infection by the human pathogen Vibrio parahaemolyticus (VP) as oysters are a natural reservoir for VP. This bacterium is responsible for a debilitating acute gastroenteritis with potential to cause fatal septicemia. Despite efforts to mitigate infection by this CDC-reportable pathogen, cases continue to increase. The understudied host-microbe relationship between the Eastern oyster and VP has been implicated as a path to research for potential future therapeutics. A novel culturing system for oysters was created using fermentation jars within a BSL-2 ready biosafety cabinet. Using this system, the effect of harvest season was tested against the inoculation efficiency of VP. It was found that higher native Vibrio levels within the oysters were present during the summer compared to the winter. Moreover, addition of the bacteriostatic antibiotic chloramphenicol (Cm) enabled a higher inoculation efficiency by VP during both the summer and winter compared to oysters not exposed to the antibiotic. During the winter, exposure to Cm led to the highest inoculation efficiency (~100%). These findings confirm the importance of the existing microbial communities against exogenous inoculation. Therefore, a year-long study was conducted to investigate the microbiome of oysters during each season. This pan-microbiome study identified a significant impact of harvest season on the microbiome structure. An increased diversity, including higher levels of Cyanobacteriaceae, was observed during the summer. Whereas an increase in Arcobacteriaceae was observed during the winter. Bacteria that persisted throughout the year included Mycoplamataceae and Spirochaeteacae; these families may represent potential members of the Eastern oyster core microbiome.
Further work is needed to study the localization patterns of VP within oysters. Such work includes further optimization of immunohistochemistry (IHC) and intracellular colonization assay methods under development here. Collectively, studies of the oyster-microbe interactions will help improve aquaculture methods and identify mitigation targets to reduce VP-related clinical infections. / Doctor of Philosophy / Fish and shellfish provide important economic and nutritional benefits for human society across the globe. Unfortunately, over-fishing of traditional sources of fish and shellfish has led to a reduced supply for world markets, even as the human population increases. Aquaculture, or fish farming, has been around for centuries, but its role in society has significantly increased in the past 50 years. It currently provides about half of fish and other aquatic products on the market today. To better maintain and increase the sustainability and profitability of this industry, more focus is being placed on the health of the fish. The microbiome is the collection of communities of microorganisms, including bacteria, fungi, and archaea, that inhabit various environments including animal hosts. The majority of this dissertation focuses on the impact of factors like diet and age on the microbiomes of aquaculture-raised animals, especially fish. Dietary changes such as the addition of dried yeast-products had a significant impact on fish health but not on the microbiome communities. However, a common probiotic, Bacillus subtilis, did significantly increase not only the growth rate of trout but it also significantly altered the total intestinal microbiome found in the feces and the intestinal mucosal layer. Moreover, it was found that early exposure of the animals to the probiotic had enhanced benefits even though the microbiome appeared to stabilize over time as the fish developed. Maintaining or improving the microbiomes of fish, paying close attention to the microbes that exist as part of a core group of bacteria always present, is vital to ensuring fish health and understanding vertebrate host-microbe relationships. Thus, an analysis of the core microbiome of trout was performed.
The final set of projects within this dissertation focused on the relationship between the Eastern oyster, a mollusk native to the Chesapeake Bay, and the bacterial human pathogen Vibrio parahaemolyticus (VP). VP is the leading cause of seafood-borne acute gastroenteritis worldwide, and efforts are needed to mitigate the increasing rate of human infections. Therefore, a simple system using fermentation jars within the laboratory biosafety cabinet was designed to enable safe culture of oysters that were exposed to VP under experimentally controlled conditions. Oysters harvested during the summer naturally harbored higher amounts of native Vibrio organisms in contrast to the winter oysters that harbored much lower levels. A separate microbiome analysis revealed large shifts in the oyster microbiome between summer and winter, although some microbes were continually present. The lower levels of existing Vibrio species detected in winter oysters may have allowed for the higher efficiency of inoculation of winter animals by VP. In fact, these winter animals had Vibrio microbiomes that were completely dominated by the inoculated strain which will enable future work to observe the pattern by which VP localizes, or colonizes, the oysters. Ultimately, these efforts may lead to the development of future disease mitigation strategies against VP.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/109607 |
| Date | 07 April 2022 |
| Creators | Hines, Ian Samuel |
| Contributors | Biological Sciences, Stevens, Ann M., Aylward, Frank O., Kuhn, David D., Popham, David L. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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