Diseases affecting shrimp contribute to billions of dollars of economic loss yearly to the aquaculture industry. Recently, one of the primary causative agents of disease has been Vibrio parahaemolyticus; in 2009, a new strain causing Early Mortality Syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp emerged. Shrimp losses attributed to pathogens can be greatly reduced through probiotic use, which are known to act as natural immune enhancers and promote pathogen resistance. However, research on probiotic treatment against EMS disease is lacking. The overall project goal was to improve intensive shrimp production through direct application of probiotics in aquaculture feeds. The value of probiotics for the shrimp industry was evaluated by (1) reducing severity or mortality of V. parahaemolyticus disease in shrimp, (2) qPCR confirmation of Bacillus spore germination in shrimp gut, and (3) probiotic effectiveness evaluation for improving disease resistance. The virulence of several Vibrio spp. strains was examined and it was concluded the V. parahaemolyticus strain identified as the causative agent of EMS was the most lethal; EMS-infected shrimp exhibited 100% mortality within 36-hours of feed inoculation. The number of bacterial cells added to feed directly correlated with pathogenicity and only cells, not filtrate, was capable of causing death. Probiotic strains were evaluated and it was concluded that probiotic strains A, A/B blend, and B were the best candidates for promoting disease resistance against EMS. This research will provide the shrimp farming industry with information vital to developing a means for reducing economic loss from Vibrio-infected shrimp. / Master of Science in Life Sciences

Early Mortality Syndrome (EMS) is a widespread bacterial infection of shrimp, attributed to pathogenic Vibrio parahaemolyticus strains (VP-EMS). This disease threatens aquaculture production and global food security. A valuable and alternative approach to using antibiotics for pathogen control, is the practice of incorporating direct-fed microbes (DFM) or probiotics. In order to evaluate the hypothesis that probiotics (specific strains of Bacillus subtilis spores) are able to provide shrimp, Litopenaeus vannamei, protection to the EMS disease, a pathogen growth model, disease challenge model, and probiotic feed coating methodologies were developed and refined, allowing independent shrimp probiotic trials to be piloted. A single probiotic strain of Bacillus subtilis: O14VRQ and a blend of Bacillus subtilis strains: Plus10, were evaluated as feed additives or as water additions, for their efficacy. Accordingly, two independent trials were conducted in which shrimp were fed daily with a probiotic-coated feed for seven days, before a challenge with VP-EMS. Each trial consisted of a negative control (no VP-EMS exposure, no probiotic) and positive control (VP-EMS exposure, no probiotic), with five additional probiotic treatment groups, which were fed and exposed to VP-EMS in the same manner as the positive control. Shrimp were observed for clinical signs of disease after the initial exposure and were continuously exposed every 24 hours until 50% of the population remained in the positive control treatment. Both probiotics studied were shown to significantly (p < 0.05) improve shrimp survival. Overall the data presented in this work demonstrates that probiotic prophylaxis is reliant upon probiotic dose, regardless of application. / Master of Science in Life Sciences / Aquaculture is one of the fastest growing agricultural sectors in the world allowing it to greatly contribute to global food security. Seafood products are known for their excellent health benefits, providing good sources of protein, fatty acids, and vitamins. However, the animals raised in this industry, like in many facets of animal agriculture, are susceptible to disease. Diseases can be costly to treat and if no treatment exists, can be detrimental to farms, especially to highly valued species such as shrimp. Traditionally, many diseases have been treated with antibiotics, however this can promote the growth of antibiotic resistant bacteria, which is a public health concern especially when involving animals fit for human consumption. An alternative to this approach is administering probiotics or beneficial bacteria to these animals. When incorporated with feed or applied to water, these beneficial bacteria can prevent diseases and help promote the growth of healthy animals. Two novel probiotics were fed to shrimp, before exposing them to the bacteria, Vibrio parahaemolyticus, which causes Early Mortality Syndrome, and is responsible for annual shrimp losses of more than $1 billion USD. Signs of this disease and survival were observed to assess if this probiotic could provide protection against this bacterium. Results from these studies show that these probiotics were capable of offering protection to shrimp when they were fed or introduced into tank water in high concentrations. Such probiotic applications could have beneficial effects on intensive shrimp aquaculture and help prevent this disease.

Recruitment failure of lake trout (Salvelinus namaycush) in the Great Lakes has been attributed in part to the consumption of alewife (Alosa pseudoharengus) by adult lake trout, leading to Thiamine Deficiency Complex (TDC) and early mortality in fry. The current understanding of thiamine deficiency in lake trout fry is based on information from culture and hatchery settings, which do not represent conditions fry experience in the wild and may influence the occurrence of TDC. In the wild, lake trout fry have access to zooplankton immediately following hatching; previous studies found that wild fry begin feeding before complete yolk-sac absorption. However, hatchery-raised fry are not provided with food until after yolk-sac absorption, long after the development of TDC. Zooplankton are a potential source of dietary thiamine for wild fry in the early life stages that has not previously been considered in the occurrence of thiamine deficiency. We postulated that wild-hatched fry could mitigate thiamine deficiency through early feeding on natural prey. Specifically, we hypothesized 1) feeding should increase thiamine concentrations relative to unfed fry and 2) feeding should increase survival relative to unfed fry. Feeding experiments were conducted on lake trout fry reared from eggs collected from Lake Champlain in 2014 and Cayuga Lake in 2015. A fully crossed experimental design was used to determine the effect of early feeding by lake trout fry in thiamine replete and thiamine deplete treatments before and after feeding. Overall, thiamine concentrations and survival did not significantly differ between fed and unfed fry. Thiamine concentrations increased from egg stage to hatching in both years, suggesting a potential source of thiamine, which had not previously been considered, was available to the lake trout eggs during development.

Prebiotics and probiotics, proposed alternatives to antibiotics in shrimp aquaculture, are reported to improve growth parameters, promote disease resistance, and influence the gut microbial community. This study aimed to investigate the influence of prebiotic- or probiotic-coated feed and/or exposure to the Early Mortality Syndrome-causing strain of Vibrio parahaemolyticus ( VP-EMS) on the mid and hindgut microbiome of Pacific white shrimp (Litopenaeus vannamei). A monoculture probiotic strain of Bacillus subtilis spores: O14VRQ, and a prebiotic product of cultured Saccharomyces cerevisiae cell walls: MOS, were administered to shrimp as feed additives for 14 days, before a pathogen challenge to VP-EMS. Based on previous efforts, animals in this study were fed experimental diets for 14 days to allow ample amount of time for the prebiotic to be metabolized by health-promoting bacteria and for the probiotic spores to germinate. The pathogen challenge consisted of negative disease control (no VP-EMS exposure, commercial feed), positive disease control (VP-EMS exposure, commercial feed) and two treatment groups, probiotic (VP-EMS exposure) and prebiotic (VP-EMS exposure). DNA extraction, 16S rRNA gene amplicon sequencing, polymerase chain reaction (PCR), and sequencing were utilized to create an overview of the mid and hindgut microbial composition. No significant differences in survival were shown between experimental diets following exposure to sublethal levels of VP-EMS. Bioinformatic analyses revealed no distinct shifts in the mid and hindgut microbiome of shrimp across experimental diets and time points. Results of this data revealed that dominant members of the intestinal microbiome, Proteobacteria, Actinobacteriota, Bacteroidota, Verrucomicrobiota, Flavobacteriaceae, Demequinaceae, Vibrionaceae, Shewanellaceae, Rhodobacteriaceae, and Rubritaleaceae were relatively stable across treatments and time points. Sequencing methods such as metagenomics or metatranscriptomics should be utilized for a higher microbiome resolution. Furthermore, the use of quantitative polymerase chain reaction to quantify ingested probiotic spores, prebiotic-associated bacteria, and VP-EMS is recommended. / Master of Science in Life Sciences / Shrimp aquaculture is one of the fastest-growing aquaculture sectors that provides another supply of feed that save wild fish populations. The use of prebiotics and probiotics are reported to improve growth, provide disease protection, influence the gut community, improve the immune system, and serve as substitutes to antibiotics. In this study, the effect of probiotic-, or prebiotic-coated diets and/or exposure to the Early Mortality Syndrome-causing Vibrio parahaemolyticus (VP-EMS) strain on the gut communty of shrimp (Litopenaeus vannamei) was investigated. DNA extraction, 16S rRNA gene amplification, and sequencing were utilized to identify the microbes in the intestines of shrimp. Based on previous studies, animals in this study were fed experimental diets for 14 days to allow enough time for prebiotic-associated bacteria and probiotic spores to multiply within the intestines. Exposure to a sublethal level of VP-EMS did not significantly affect shrimp survival between treatments. Analyses showed no noticeable differences in the intestinal microbial communities between treatments and time points. This research provided initial proof of what microbes occupy the mid and hindgut microbiome. A higher resolution sequencing method is recommended to gain a better understanding of the microbes and their roles in the intestines. The use of quantitative polymerase chain reaction is warranted to evaluate the amount of ingested probiotic spores, prebiotic-associated bacteria, and VP-EMS.