Litopenaeus vannamei or Pacific white shrimp is the most widely farmed crustacean in the world. Shrimp are commonly fed feed containing 30-40% soybean meal or other plant-based feeds that are more economically and environmentally sustainable than animal-based feed. However, plant-based pellets are less palatable and less chemically attractive compared to animal material. Based on that, current research and practice includes the addition of specific marine animal meals in order to enhance palatability and attractability of plant-based shrimp feed. Yet, it is not sustainable or economically achievable to continue relying on marine animal meal. In the herein study, the effect of proprietary chemical mixtures designed by our research group as feed additives was examined based on their attractability and palatability in comparison to krill meal, a highly attractive and palatable supplement for shrimp feed. In palatability assays, total amount of pellets was measured before and after one-hour and three-hour periods of feeding in group-housed animals. In attractability assays, responses of shrimp were measured based on the number of probes and grabs on the source (airstone) of the stimulus being released. Each diet-set used contained different concentrations of krill meal and synthetic chemical mixtures. Results demonstrated these chemical mixtures enhance attractability and palatability of soybean based feed in L. vannamei when compared to krill meal. Furthermore, the addition of a proprietary mixture (= “premix”) improved responses in the attractability assays when compared to stimuli that did not contain the premix. Overall, results support the hypothesis that synthetic chemical mixtures can improve palatability and attractability of soybean meal based shrimp feed. This work could provide a reference for the development of synthetic chemoattractants and chemopallatants for the aquaculture of shrimp.

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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

The Pacific white leg shrimp, Litopenaeus vannamei, is a potential species for low salinity inland aquaculture. Due to several independent variables, such as species, age, size, salinity and pH, that must be taken into account, there are gaps in the literature pertaining to the toxicity of ammonia and nitrite to shrimp. This study was conducted to investigate the individual and combined effects of ammonia and nitrite on L. vannamei postlarvae (25-45 days old) at 10 ppt salinity, 28 C and a pH of 7.8. The independent variables were salinity, total ammonia as nitrogen (TAN) and nitrite-N (NO₂-N), separately and combined. The TAN experiments were conducted at 18 and 10 ppt salinity while the NO₂-N test was conducted at 10 ppt salinity. Combined TAN and NO2 tests were also conducted at 10 ppt salinity. The LC50 values for TAN at 18 ppt salinity, TAN at 10 ppt salinity, and NO2-N at 10 ppt were observed to be 42.92, 39.72 mg/L (2.26 and 2.09 mg/L unionized ammonia-N), and 153.75 mg/L, respectively. When NO₂- N was adjusted to the LOEC level and TAN concentrations were varied, synergistic effects were observed, with an LC50 calculated to be 28.2 mg/L TAN (1.49 mg/L unionized ammonia-N). However, when the ammonia level was adjusted to the LOEC and nitrite was varied, antagonistic effects were observed with an LC50 calculated to be 163.3 mg/L NO₂-N. The results suggest that further investigations into the combined effects of ammonia and nitrite at varying concentrations and lower salinities will be important in developing "standard operating procedures" for the shrimp industry. / Master of Science

Many different algal species can provide an acceptable protein ingredient, with good digestibility, for shrimp feeds. Compared to fish meal, similar protein, carbohydrate, and lipid levels can be found in select algal species. Traditional shrimp diets in aquaculture rely on fish meal and fish oil from pelagic fish fisheries. A reduction or elimination of these ingredients would reduce the dependency of shrimp aquaculture on offshore fisheries and increase economic competiveness. Biofuel production produces algal by-products of potential use to aquaculturists that might reduce or eliminate the need for fisheries products in shrimp feed. Established uses for by-products from biofuel production include fertilizer for crops, fodder for swine and poultry, and production of methane and alcohol fuels. However, using biofuel production by-products as a protein and carbohydrate source for the Pacific white shrimp, Litopenaeus vannamei, has not been investigated. Therefore, a series of feeding experiments were conducted to evaluate if the algae used to produce biofuel could be a suitable main protein source in formulated diets for L. vannamei. The feasibility of substituting biofuel algae by-product for fish meal in the juvenile L. vannamei (0.0306 ± 0.0011 g) diet was evaluated, and an adequate substitution ratio was determined. Eighteen experimental diets were evaluated using 60, 80, and 100% fish meal substitution levels. Chaetoceros calcitrans, Nannochloropsis salina, and Pavlova sp. were chosen as the algae sources as they have potentially high use in biodiesel production due to their high lipid content and each has been included in established larval shrimp aquaculture operations. Each diet varied the level of fish meal substitution (60, 80, or 100%) and either contained dried algal biomass or, alternatively, dried algal biomass with reduced lipid content to simulate algal biomass post-biodiesel production. The diets were compared, relative to their effect on weight gain in juvenile L. vannamei, to each other and to a commercially available diet (CONTROL) and a diet formulated using the ingredients used in all of the experimental diet formulations but without algal biomass (BASAL). The shrimp were held individually in 355-ml Styrofoam cups filled with 200-ml seawater with a salinity of 32 parts per thousand (ppt) salinity under a 12:12 light:dark photoperiod. Water exchange was 90% per day for six days and 100% on the seventh day when weights were taken. Each of the twenty diets was presented daily to seven replicate cups, each cup containing a single shrimp, for six weeks. Food was presented once per day to satiation, which was determined by the shrimp refusing additional feed. Each animal was weighed weekly. After six weeks, the shrimp were harvested and final weights were taken. The analysis of differences between strains, levels, and lipids indicated there was a significant difference between all of the algal-based diets and the control. Overall, significantly better growth rates were observed in the diets with less fish protein replacement. The 60% fish meal replaced diets outperformed the diets that had 80 or 100% fish meal replacement. There were no significant differences in nutritional value among the algal species. Survival rates, from an aquaculture perspective, were acceptable for all treatments (>71%). Results from these studies demonstrated that formulated diets using algal biomass from biodiesel production can be the primary protein source for L. vannamei postlarvae.

Phytic acid (PA) accounts for up to 75% of the P in soybean (Glycine max L. Merr.) seeds, but it is indigestible by mono- and agastric animals resulting in economic and environmental detriment. Soybean lines with genetically reduced PA contents have been developed using three distinct mutant alleles at the MIPS1, LPA1, and LPA2 genes resulting in up to a 75% reduction in PA. Low PA (LPA) soymeal-based feeds have been tested on several agricultural species and shown to reduce the P in the animal effluent, but they have not been tested on any aquacultural species. However, LPA soybean lines often exhibit low field emergence making them commercially inviable. The cause of this phenomenon is widely debated with possibilities ranging from increased disease pressure to decreased seedling vigor. The objectives of this research were to 1) enhance field emergence of LPA soybean varieties through pre-planting seed treatments, 2) study the impact of the LPA mutant alleles on agronomic, quality, and seed composition traits, and 3) design a low-error method for studying the effect of LPA soymeal-based feeds on aquatic animals using Pacific White Shrimp (Litoenaeus vannamei). These results describe a variety of agronomic and genetic strategies with which the low field emergence of LPA soybeans can be addressed, reveal a heretofore not reported interaction between the mips1 and lpa2 alleles to further increase the digestibility of soymeal, and a possible method for studying LPA soymeal based feed on aquacultural animals. / Master of Science

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.