Inorganic Carbon and Nitrogen Utilization in Mixotrophic Ciliates Donald M
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University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 5-11-2013 Inorganic Carbon and Nitrogen Utilization in Mixotrophic Ciliates Donald M. Schoener University of Connecticut, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Schoener, Donald M., "Inorganic Carbon and Nitrogen Utilization in Mixotrophic Ciliates" (2013). Doctoral Dissertations. 90. https://opencommons.uconn.edu/dissertations/90 Inorganic Carbon and Nitrogen Utilization in Mixotrophic Ciliates Donald Matthew Schoener University of Connecticut 2013 Mixotrophy is a common nutritional strategy that uses both heterotrophy and photosynthesis. Kleptoplastidic mixotrophs do not make their own plastids but acquire them from their algal prey. Before we can add mixotrophs to standard ecological models we need to understand how much each nutritional mode contributes to mixotrophic growth, and how this balance may be influenced by plastid acquisition, retention, and turnover. In order to examine the role of captured chloroplasts in the metabolism of the oligotrich ciliate Strombidium rassoulzadegani . I evaluated the uptake and retention of chloroplasts, the ability of two different algae to supply functional chloroplasts, and the photosynthetic uptake of inorganic carbon by the chloroplasts once inside the ciliate. In addition, the ability of the ciliate to take up inorganic forms of nitrogen and its role as net mineralizer or utilizer of inorganic nitrogen was examined, using stable isotope tracers of N in nitrate and ammonium. I compared mixotrophic ingestion and inorganic uptake to that of the heterotrophic ciliate Strombidinopsis . The kleptoplastidic ciliate had higher growth efficiency (GGE) on a chlorophyte diet than a cryptophyte diet. When compared to the heterotroph, the mixotrophic ciliate had improbably high GGEs at low algal food concentrations. However, mixotrophic ingestion did not saturate as the food concentration increased as it did with the heterotroph, suggesting that the mixotroph continues to consume algae at a high rate in order to maintain a fresh supply of chloroplasts. Mixotrophic inorganic carbon uptake did not change with algal food concentration, but its importance to the ciliate’s carbon budget did increase. Although there was measurable inorganic carbon uptake due to still-active algae in food vacuoles, it did not contribute significantly to the growth of the heterotrophic ciliate. In terms of nitrogen, mixotrophic and heterotrophic ciliates had similar GGEs with the mixotroph’s being slightly higher. Furthermore, ammonium uptake was slightly higher in the mixotroph. Inorganic nitrogen uptake did not contribute significantly to the nitrogen budget of either the heterotrophic or mixotrophic ciliate, and nitrogen GGEs were always less than one. Kleptoplastidic ciliates like S. rassoulzadegani may survive algal scarcity by ensuring that they have the freshest and most suitable plastids they can at any given time. Like the heterotrophs, kleptoplastidic ciliates are net remineralizers of nitrogen, and thus not competing with algae for this resource. However, they may outcompete heterotrophs due to higher growth efficiency and high ingestion rates. ii Inorganic Carbon and Nitrogen Utilization in Mixotrophic Ciliates Donald Matthew Schoener BS Temple University 2002 MS Temple University 2005 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2013 Copyright by Donald Matthew Schoener 2013 iv APPROVAL PAGE Doctor of Philosophy Dissertation Inorganic Carbon and Nitrogen Utilization in Mixotrophic Ciliates Presented by Donald Matthew Schoener, BS. MS. Major Advisor____________________________________________________________ George B. McManus Associate Advisor_________________________________________________________ Hans G. Dam Associate Advisor_________________________________________________________ Pieter Visscher Associate Advisor_________________________________________________________ Diane Stoecker Associate Advisor_________________________________________________________ Craig Tobias University of Connecticut 2013 v Acknowledgments No one truly does anything on their own, there are too many people who have helped me to thank here, but several standout in my mind. First, I need to thank my wife June Park for her support and patience. I thank my advisor George McManus for his gentle approach and strong advocacy. To associate advisors Hans Dam, Pieter Visscher, Craig Tobias and Diane Stoecker, thank you for your invaluable advice. Thank you to the staff of the marine science department, especially Claudia Keorting, Dennis Arbige, Pat Evans, and Deb Schuler. These dedicated professionals set up instrumentation, fix incubators, order supplies and open the doors (figurative and literal) that make the work presented in this dissertation possible. My friends and labmates, Jill Tomaras, Barbara Costas, Willysthssa Pierre-Louis, Chelsea Roy, Amanda Liefeld, Michal Tian, Chris Perkins, and Joanna York, for their help and support with numerous experiments. A special thanks to my good friends from writers anonymous (WA), Kim Gallagher, Lauren Stefaniak, and Rita Kuo for all the peer pressure. Finally I would like to thank my family, my father Louis, may Aunt Kathy and my brothers. They have always encouraged me to pursue my interests wherever they might lead me. Funding for this work was provided by a National Science Foundation grant to GBM (OCE0751818) and the University of Connecticut. vi Table of Contents……………………………………….…………………………………………………….…..Page Chapter 1 1 Mixotrophy: who does it, what do they get out of it, and why is it important? 1 Chapter 2 13 Plastid Retention, Use, and Replacement in a Kleptoplastidic Ciliate 13 Abstract: 14 Introduction: 15 Materials and Methods: 17 Cultures: 17 Plastid Replacement: 18 Plastid Retention during starvation: 18 Ciliate Mortality When Starved: 19 Growth and Grazing Rates: 20 Gross Growth Efficiency 22 Feeding selectivity 22 Results: 23 Cultures: 23 Plastid Replacement: 24 Plastid Retention during starvation: 26 Ciliate Mortality When Starved: 27 Growth and Grazing 29 Gross Growth Efficiency 32 Feeding selectivity 33 Discussion: 34 Chapter 3 40 Mixotrophic Carbon Uptake: Comparison of Grazing and Direct Inorganic Uptake in a Kleptoplastidic Ciliate 40 Abstract 41 Introduction 42 Methods 43 Cultures 43 Growth, ingestion and GGE 45 Inorganic carbon uptake 47 Pulse chase 48 Results 49 Cultures 49 Growth, ingestion and GGE 49 Total Growth Efficiency 55 Pulse chase 57 Discussion 59 vii Chapter 4 65 Comparison of Grazing and Inorganic Nitrogen Uptake in a Mixotrophic and a Heterotrophic Ciliate: Is the Kleptoplastidic ciliate Strombidium rassoulzadegani a Source or Sink of Inorganic Nitrogen? 65 Abstract 66 Introduction 67 Materials and Methods 68 Cultures 68 Growth and Grazing: 69 Inhibition experiments 72 Ammonium Addition 72 Statistics 73 Results 74 Cultures 74 Growth, Grazing and GGE 75 15 N Uptake in Strombidinopsis sp. 84 Discussion 88 Chapter 5 94 Summary, Discussion and Future Directions 94 Summary and Discussion 95 Future Directions 98 References 101 viii List of Equations………………………………………….……………………………………………….…..…..Page Eq. 2.1 Ingestion calculations ......................................................................................... 20 Eq. 2.2 Michaelis-Menten equation ............................................................................... 22 Eq. 4.1 Inorganic nitrogen uptake rate ........................................................................... 72 ix List of Tables………..………….……………………………………………………………………………………..Page Table 2.1 Fluorescence area, a proxy for chlorophyll content, in cell squashes of Strombidium rassoulzadegani when starved after being acclimated on a diet of either Tetraselmis or Rhodomonas . ....................................................................... 27 Table 2.2 Result of extra sum of squares F-test comparing growth and grazing rates of S. rassoulzadegani on algal diets of Tetraselmis chui and Rhodomonas lens . ............ 32 Table 3.1 Parameter estimates for Michaelis-Menten curve fits in terms of carbon ...... 53 Table 3.2 Specific inorganic uptake rates for various ciliates species. 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Table 4.1 Algal and ciliate cell size, carbon and nitrogen content .................................. 62 Table 4.2 Parameter estimates for Michaelis-Menten curve fits in terms of nitrogen.... 79 x List of Figures……………………………………………………………………….………………………………...Page Figure 2.1 Strombidium rassoulzadegani replaces Rhodomonas-derived plastids with Tetrasemis-dirved plastids within 48 hours of being switched from a diet consisting of Rhodomonas to a diet of only Tetraselmis. ........................................................ 25 Figure 2.2 Strombidium rassoulzadegani retains some Tetrasemis-dirved plastids 48 hours after being switched from a diet consisting of Tetraselmis to only consisting of only Rhodomonas. .......................................................................................... 25 Figure 2.3 Changes in abundance of Strombidium rassoulzadegani when starved after being fed a diet of either Tetraselmis or Rhodomonas . ........................................ 28 Figure 2.4 Specific growth rate ( µµµ) vs. algal food concentration expressed as µgC L -1. ... 30 Figure 2.5 Specific ingestion rate (IR) vs. algal food concentration