Fish meal replacement with soybean meal in yellow perch (Perca flavescens) diets: responses of nutritional programming on growth, transcriptome and isoflavone accumulation DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Megan Marie Kemski Graduate Program in Food Science and Technology The Ohio State University 2018 Dissertation Committee Dr. Macdonald Wick, Advisor Dr. Konrad Dabrowski, Co-Advisor Dr. Chad Rappleye Dr. Yael Vodovotz 1 Copyrighted by Megan Marie Kemski 2018 2 Abstract One of the most economical and sustainable ways to reduce the consumption of wild fish resources, is to replace fish meal protein in aquafeeds with plant-based proteins. Aquaculturists, however, have found that they are not able to entirely replace fish meal with plant-based proteins in diets due to amino acid profile differences and possible anti- nutritional factors present, which have caused fish to have hindered growth, survival and reproductive quality. Thus, more research needs to be done for plant-based proteins to be fully integrated into fish diets that ultimately allow for optimal fish growth and health. The current study addresses a significant gap in the literature by focusing on fish meal replacement in yellow perch (Perca flavescens). The central hypothesis of this project is that there is a phenotype-diet interaction that occurs through nutritionally programming fish to a soybean meal-based diet. Nutritional programming is described as early dietary events that occur during critical developmental windows that can result in permanent changes later in life such as, growth potential health and metabolic status. To test this hypothesis, a series of experiments were designed. In the first experiment (Chapter 2), yellow perch were nutritionally programmed over 4 phases; growth performance (growth, survival and specific growth rate (SGR) was measured, and subsequently, the fish were reproduced to determine if soybean meal in the diet affected reproductive quality. In the ii second experiment (Chapter 3), the nutritionally programmed adult yellow perch from the previous study were reproduced annually over the course of three years (2015, 2016 and 2017) to determine if the offspring had improved growth performance when given a fish meal (FM) or soybean meal-based (SBM) diet as their first formulated feed. The aim of this project was to determine if possible parental inheritance of nutritional programming occurred, and if it had an effect on the growth of offspring when they were fed a SBM- based diet. It was determined that parental inheritance and initial offspring diets, independent of one another, had a significant effect on percent weight gain on the progeny. In Chapter 4, a more mechanistic approach was taken, in which RNA- sequencing (RNA-seq) was done to determine transcriptional differences in the mid- intestine of juvenile yellow perch, after being fed either a FM or SBM-based diet as their first formulated feed. In this experiment, the goal was to examine the gene expression differences between juveniles when fed FM and SBM-based diets as their first formulated fed. RNA-seq analysis revealed that nine of the genes up-regulated in the SBM-fed fish were directly involved in the cholesterol biosynthesis pathway, and these fish were found to have similar cholesterol levels compared to the FM-fed fish. Because higher inclusion levels of soybean meal in aquafeeds is needed for more sustainable aquaculture production, and ultimately as a food product, it was important to also examine yellow perch after being fed a SBM-based diet from the consumer’s perspective. In Chapter 5, the soybean isoflavone (phytoestrogen) content within the soybean meal-based diets was analyzed, along with the possible accumulation in the yellow perch fillets. Studies of isoflavone accumulation are limited in fish, and this was iii the first such study in yellow perch. It is hypothesized that by feeding yellow perch diets with a high concentration of SBM, isoflavone accumulation will occur within the fillet (muscle tissue) of these fish in a dose dependent manner. This is important from a consumer’s perspective because isoflavones have been shown to play roles as antioxidants, and have exhibited anticancer, and anti-inflammatory activities within mammals. However, they are also structurally similar to estrogen, and can cause agonistic/antagonistic estrogenic effects. Results of this study discovered that isoflavone accumulation occurred within the muscle tissue of yellow perch at low concentrations (ng isoflavones/g muscle tissue), and was significantly effected by the first feed given to fish as juveniles. Interestingly, there was no effect of sex of fish, weight or dose dependent response seen on isoflavone accumulation in the muscle tissue. Overall, these outcomes of the presented studies contributed to the knowledge of how nutritional programming can be used to raise carnivorous fish on diets with high levels soybean meal-based protein without compromise to survival, growth or reproductive quality. Transcriptomic results revealed specific alterations to the metabolism of these fish after being fed a SBM-based diet, which will allow for better diet formulation and a possible explanation of why depressed growth is seen when these fish are juveniles. Finally, the findings that isoflavone accumulation was only affected by initial dietary history are important from a consumer’s perspective, as these fish do not seem to have an increase in accumulation after prolonged exposure to soybean meal. iv Dedication To Jim and my family who have supported me, believed in me, and encouraged me. v Acknowledgments I have been lucky to work with some amazing people during my time here at The Ohio State University, and would not have been able to accomplish my degree without the help and support of the following; To my advisor Dr. Macdonald Wick, words cannot express the gratitude that I have for your guidance, knowledge, patience and support throughout the years. Without your backing and encouragement, this degree would not have been possible. You have helped me grow as a scientist and as a person, and for that, I thank you. Thank you to Dr. Konrad Dabrowski, for teaching and sharing your limitless knowledge about fish with me. Through your training and patience I have gained so much, and you have made this entire project possible, for which I am truly grateful. Thank you to Dr. Chad Rappleye, you have provided knowledge, guidance and tremendous encouragement as I grew from a lab tech to a PhD graduate. Without your help, I would have never been able to navigate the RNAseq data, and I am appreciative of your help and support over the years. Thank you to Dr. Yael Vodovotz, for your support, trust and providing a new and exciting opportunity for me to advance. You brought a unique perspective to my project, and allowed my passion for food science to thrive. vi In addition, thank you to Dr. Bruno who has contributed scientific intellect and allowed me to work in his lab, and his post doc, Dr. Priyankar Dey, who spent many hours teaching me and helping me with protocols. I also could not have made it through my time without my awesome lab members who have also become friends, and are the only other people that really understood the process. To Mackenzie Miller, John Grayson, Kevin Fisher, Dr. Thomas Delomas, Kristen Towne and the other members of the aquaculture lab past and present, thank you for your continuous help and support. I could not have completed this project without your many hours of assistance, and it always helped to have some humor and fun along the way! There are also those that have helped me over the years and I am also so grateful for all of your patience and assistance, but also your friendship. Thank you to Dr. Stephanie Hutsko and Dr. Jackie Griffin, Tim Vasquez, Kelsey Morris, and to the many others that I have had the pleasure to get to know during my time as a graduate student. Finally, thank you to my family especially my mother, who has been my strength and rock for the past 5 years, supporting me through all of the highs and lows, and never stopped encouraging me along the way. To my father, who provided love and motivation. And to my brother who has provided true friendship and advice, as well as being good distraction from the stress. Above all, thank you to Jim, who has been by my side during this entire journey, supporting me throughout. Thank you for your continuous patience, understanding, encouragement and love. I could not have done it without you! vii Vita 2003-2008 B.A. Zoology (Biology), Miami University, Oxford, OH 2008-2011 Research Assistant, Kao Corporation, Cincinnati, OH 2011-2013 Research Associate, Department of Microbiology, The Ohio State University, Columbus, OH 2013-Present Graduate Research Associate, Department of Food Science and Technology, The Ohio State University, Columbus, OH Publications Kemski, M., Wick, M., Dabrowski, K. (2018). Effect of Nutritional Programming on Growth, Reproduction and Embryonic Development in Yellow Perch (Perca flavescens) fed Soybean Meal-Based Diets. Aquaculture. In press. Miller, M., Kemski, M., Grayson, J., Towne, K., Dabrowski, K. (2018). Yellow Perch sperm motility, cryopreservation and viability of resulting larvae/juveniles. North American Journal of Aquaculture. 80(1), 3-12. Kemski, M. M., Stevens, B., & Rappleye, C. A. (2013). Spectrum of T-DNA integrations for insertional mutagenesis of Histoplasma capsulatum. Fungal Biology, 117(1), 41-51 Edwards J.A., Kemski M.M.,Rappleye C.A. (2013). Identification of an aminothiazole with antifungal activity against intracellular Histoplasma capsulatum. Antimicrobial Agents and Chemotherapy, 57(9), 4349-4 viii Edwards J.A., Kemski M.M., Rappleye C.A., Chen C., Hu J.,Mitchell T.K. (2013). Histoplasma yeast and mycelial transcriptomes reveal pathogenic-phase and lineage-specific gene expression profiles.