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Simplifying Methods for in Vitro Metamorphosis of Glochidia

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Simplifying Methods for in Vitro Metamorphosis of Glochidia View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Missouri State University: BearWorks BearWorks MSU Graduate Theses Summer 2017 Simplifying Methods for in Vitro Metamorphosis of Glochidia Morgan A. Kern [email protected] As with any intellectual project, the content and views expressed in this thesis may be considered objectionable by some readers. However, this student-scholar’s work has been judged to have academic value by the student’s thesis committee members trained in the discipline. The content and views expressed in this thesis are those of the student-scholar and are not endorsed by Missouri State University, its Graduate College, or its employees. Follow this and additional works at: https://bearworks.missouristate.edu/theses Part of the Biology Commons Recommended Citation Kern, Morgan A., "Simplifying Methods for in Vitro Metamorphosis of Glochidia" (2017). MSU Graduate Theses. 3132. https://bearworks.missouristate.edu/theses/3132 This article or document was made available through BearWorks, the institutional repository of Missouri State University. The work contained in it may be protected by copyright and require permission of the copyright holder for reuse or redistribution. For more information, please contact [email protected]. SIMPLIFYING METHODS FOR IN VITRO METAMORPHOSIS OF GLOCHIDIA A Masters Thesis Presented to The Graduate College of Missouri State University TEMPLATE In Partial Fulfillment Of the Requirements for the Degree Master of Science, Biology By Morgan A. Kern August 2017 Copyright 2017 by Morgan Ashley Kern ii SIMPLIFYING METHODS FOR IN VITRO METAMORPHOSIS OF GLOCHIDIA Biology Missouri State University, August 2017 Master of Science Morgan A. Kern ABSTRACT Captive culture of Unionoid mussels is complicated by the parasitic larval stage, which normally requires a host fish for metamorphosis. Alternatively, some mussel species can metamorphose in vitro, i.e. in an artificial medium in Petri dishes. Most workers have used 5% CO2 atmosphere and bicarbonate to stabilize pH, requiring a specialized incubator. In the present study, in vitro metamorphosis success of Anodonta oregonensis and other species were higher or similar in air than in 1%, or 5% CO2. The nutritional role of the medium was tested by substituting physiological saline without nutrients at varying intervals before metamorphosis was complete. Pyganodon grandis metamorphosed without external nutrition during more than half of the incubation period, suggesting that development, once triggered, can continue largely on internal reserves. Post-metamorphic growth rates of P. grandis from medium, from saline, and from host fish were similar. Previous studies indicate that species which grow substantially during metamorphosis are unsuccessful in vitro. It was hypothesized that higher nutrient use by these species might result in local diffusion-limited depletion of the growth medium, which might be alleviated by circulation. However, initial attempts to metamorphose Leptodea fragilis glochidia in media circulated by a slow rocker system were unsuccessful. KEYWORDS: Freshwater mussels, in vitro, development, metamorphosis, Unionoid This abstract is approved as to form and content _______________________________ M. Christopher Barnhart, PhD Chairperson, Advisory Committee Missouri State University iii SIMPLIFYING METHODS FOR IN VITRO METAMORPHOSIS OF GLOCHIDIA By Morgan A. Kern A Masters Thesis Submitted to the Graduate College Of Missouri State University In Partial Fulfillment of the Requirements For the Degree of Master of Science, Biology August, 2017 Approved: _______________________________________ M. Christopher Barnhart, PhD _______________________________________ Ryan S. Udan, PhD _______________________________________ Dan Beckman, PhD _______________________________________ Julie Masterson, PhD: Dean, Graduate College In the interest of academic freedom and the principle of free speech, approval of this thesis indicates the format is acceptable and meets the academic criteria for the discipline as determined by the faculty that constitute the thesis committee. The content and views expressed in this thesis are those of the student- scholar and are not endorsed by Missouri State University, its Graduate College, or its employees. iv ACKNOWLEDGEMENTS I would like to express my deepest gratitude and respect for my research advisor, Dr. Chris Barnhart. I hope that a small part of his passion for natural history and endless curiosity has rubbed off on me. I have learned so much from him through this process and hope to continue to learn from him in the years to come. My sincere thanks must also go to the members of my thesis committee: Dr. Udan and Dr. Beckman. They generously gave their time to offer me valuable advice and comments that have improved my work. They have both been patient with me, as deadlines seemed to zoom past. There is no way to express how much it has meant to me to be a member of the Mussel Lab, which has been like a second family. These brilliant friends and colleagues have inspired me during the short time we worked together: Kendell Loyd, Kathryn Martin, Bryce Maynard, Amy Maynard, and all the other current and former Mussel Lab grad students and visitors that I know. I am most grateful to Beth Glidewell for the friendship, assistance, and advice she has given me. It has been invaluable in this process. Thanks to Amy, Beth, Bryce and anyone else I roped into helping count juveniles or collect mussels. I must also offer special thanks to Jordan Murray. Without her dedication and attention to detail as an undergraduate worker, I would have never slept. Kendell, working with you has made teaching bearable and I hope you really do start a blog for your life lessons. Finally, I would like to thank my parents, Kim and Ken Kern and the rest of my family for the endless love and support you have offered me. You have made the distance from home easier by never turning down a chance to visit or call. I consider myself truly fortunate to have all of you in my life! v TABLE OF CONTENTS Introduction ..........................................................................................................................1 Life History of Freshwater Mussels .........................................................................1 Glochidia Development ...........................................................................................3 Propagation ..............................................................................................................5 Metamorphosis In Vivo on Fish Hosts. ........................................................6 In Vitro Metamorphosis. ..............................................................................8 Goal and Objectives. ..............................................................................................11 Methods .............................................................................................................................13 Mussel Collection and Care ...................................................................................13 Media Preparation ..................................................................................................14 Loading Culture Dishes ........................................................................................15 Judging Survival, Metamorphosis and Growth .....................................................16 Experiments ...........................................................................................................17 Results ...............................................................................................................................22 Discussion ..........................................................................................................................24 CO2, pH, and Medium Formulations ....................................................................24 Optimizing In Vitro Incubation ..............................................................................26 Developmental Triggers and Nutrition during Development ................................27 Quality of In Vitro vs In Vivo Juveniles .................................................................28 Effect of Agitation of Culture Medium on L. fragilis ............................................28 Measuring Success of In Vitro Culture ................................................................29 References ..........................................................................................................................31 Appendices .........................................................................................................................60 Appendix A. In Vitro Culture Medium Composition ............................................60 Appendix B. Lebovitz’s Medium Components .....................................................61 Appendix C. Components of M199 ......................................................................62 Appendix D. Fish Saline Composition ..................................................................63 vi LIST OF TABLES Table 1. Species and collection localities of mussels used for experiments. .....................37 Table 2. Effect of CO2 #1 (LIV).. .......................................................................................37 Table 3. Effect of CO2 #1 (LW).. ........................................................................................38
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