PROBIOTICS AND THEIR USE IN COMMERCIALLY IMPORTANT AQUACULTURE SPECIES IN THE NORTHEASTERN UNITED STATES by JACQUELINE L. MYER A THESIS Submitted in partial fulfillment of the requirements for the degree of Master of Science in the Natural Resources Graduate Program of Delaware State University DOVER, DELAWARE December 2018 This thesis is approved by the following members of the Final Oral Review Committee: Dr. Dennis McIntosh, Committee Chairperson, Department of Agriculture, Delaware State University Dr. Dewayne Fox, Committee Member, Department of Agriculture, Delaware State University Dr. Sigrid Smith, Committee Member, Department of Agriculture, Delaware State University Dr. Eric Schott, External Committee Member, Department of Biological Sciences, University of Maryland Dr. Hal Schreier, External Committee Member, Department of Biological Sciences, University of Maryland © Jacqueline L. Myer All Rights Reserved Dedication Husband. You know what you did. i Acknowledgements I would like to express my sincere thanks to my advisor, Dr. Dennis McIntosh who was not only one of the three men who isolated the probiotics used in my thesis research, but also got funding for my thesis and helped me to achieve the work needed for this thesis. I would also thank Drs. Harold Schreier and Eric Schott, who along with Dr. McIntosh studied the probiotic strains used in this thesis. They also advised me through my thesis work and were members of my committee. I would like to thank the rest of my committee; Dr. Dewayne Fox for his help with editing, Dr. Sigrid Smith who not only helped me with the statistics for my thesis, but also helped me to better understand statistics and its necessity in my career. Additional thanks to Dr. Kevina Vulinec who was not only my first introduction to statistical science regarding natural resources, but also contributed greatly to my success in my thesis. I would also like to thank Dr. Stephen Hughes, from Cheyney University, and Mike Freeze, from Keo Fish Farms Inc., for helping me get multiple species of finfish for my experiments. None of this would have been possible without the help from all mentioned above. ii Probiotics and their use in Commercially Important Aquaculture Species in the Northeastern United States Jacqueline L. Myer Faculty Advisor: Dr. Dennis McIntosh Thesis Abstract Probiotics have shown to be beneficial to aquatic farming in aspects such as growth and development. This study, using three commercial aquaculture species, intends to ascertain if certain strains of Bacillus and Shewanella bacteria could possibly be used in aquaculture to improve finfish growth and survival, and be classified as true probiotics. Trials run at the DSU ARDF dose Nile tilapia (Oreochromis niloticus), rainbow trout (Oncorhynchus mykiss) and hybrid striped bass (Morone chrysops x M. saxatilis) with three probiotics Bacillus spp. (Iso 5 and Iso 11) and Shewanella spp. (Iso 12). Mortality was observed in three finfish trials over a 56-day trial or until one tank in the trial attained 10% survival. Ammonia, nitrite and nitrate were monitored throughout the trial. Feed conversion ratios, specific growth rates, weight gains and mortality were assessed at the end of the study. During the Nile tilapia experiment control tanks lost a total of 42 of 75 fish. ISO 12 (60/75), ISO 5 (56/75), and ISO 11 (58/75), treated tanks had statistically higher survival rates then the control tanks. Significant differences were also seen in survival during the hybrid striped bass experiment between the control tanks (16/30), ISO 12 (24/30) and ISO 11 (30/30). No significant differences were seen in survival for the rainbow trout experiment, but no treatment lost more than 10 finfish during the entirety of the experiment. iii Significant differences were observed for nitrite concentrations among tanks with probiotic treatments in the hybrid striped bass as well as with the rainbow trout experiment. ISO 5 treated tanks had a significantly lower concentration (0.107 ± 0.027 mg/l) of nitrite when compared to control tanks (0.231 ± 0.027 mg/l) during the rainbow trout experiment. Tanks treated with ISO 12 were significantly higher in nitrite concentration (0.038 ± 0.002 mg/l) than tanks treated with ISO 5 (0.030 ± 0.002 mg/l), and significantly higher than those treated with ISO 11 (0.029 ± 0.002 mg/l) during the hybrid striped bass experiment. The difference in ISO 5 and ISO 12 nitrite concentrations could be attributed to the ability of Bacillus spp. bacteria to metabolize ammonia into nitrate and nitrite, jumpstarting the nitrogen cycle. ISO 12 treated tanks very high nitrite concentration in comparison, might also be due to Shewanella spp. ability to denitrify nitrate into nitrite. If the experiment had lasted longer, perhaps an eventual decrease in nitrite would have been seen as the nitrite was converted into ammonium, a benign gaseous output. All three tested bacteria have the potential to be probiotics and used in aquaculture. Higher survival of finfish in the probiotic treatment tanks and the control tanks suggest that all three probiotics have the ability to increase survival in warm water (24 ºC) conditions, though no significant results were observed for cooler water (19 ºC). ISO 5, Bacillus cereus, and ISO 12, Shewanella spp., were both shown to have an effect on the concentrations of nitrite, but ISO 5 decreased the nitrite concentration while ISO 12 increased the concentrations when compared to ISO 5 and ISO 11, not the control. Further testing is required for ISO 12 to see if it could possibly lower the nitrite over a longer time period. Further testing is required for all three iv probiotic strains, and different systems as well as temperatures and salinities should be used to better understand the functions of these bacteria. v Table of Contents Table of Contents ........................................................................................................................... vi List of Tables ................................................................................................................................ vii List of Figures .............................................................................................................................. viii Chapter 1: Introduction ............................................................................................................... 1 Chapter 2: Literature Review ...................................................................................................... 8 Chapter 3: Materials and Meathods ......................................................................................... 11 Chapter 4: Results ...................................................................................................................... 19 Tilapia ................................................................................................................................... 19 Rainbow trout ........................................................................................................................ 26 Hybrid striped bass ............................................................................................................... 32 Chapter 5: Discussion ................................................................................................................. 38 Tilapia ................................................................................................................................... 38 Rainbow trout ........................................................................................................................ 40 Hybrid Striped Bass .............................................................................................................. 41 Probiotics .............................................................................................................................. 42 Chapter 6. Conclusions ............................................................................................................... 46 References .................................................................................................................................... 49 Appendix ...................................................................................................................................... 55 vi List of Tables Table 1-1. Value of finfish aquaculture (thousands of dollars) in the Northeast (NE) region vs. the whole United States of reporting farms in the 2012 USDA Census of Aquaculture (USDA 2013). ..................................................................................................................... 2 Table 1-2. Value in the Northeast (Table 1-1). vs. total United States value of three commercially important fish species (thousands of dollars). Data represent the farm gate value of the various species as reported in the Fisheries of the United States 2014 (NMFS 2015). .................................................................................................................................. 3 Table 1-3. Comparison of observed functions of Bacillaceae and Shewanellaceae., two families of probiotics used in aquaculture. This table indicates the genus of the probiotic tested, the host species and the observed benefit(s). ...................................................................... 5 Table 3-1. Bacteria in use for the trials detailed in this thesis. Each of the probiotics were taken from the Mummichog (Fundulus heteroclitus) in a previous effort (McIntosh et