Investigation of prey ingestion and digestion in the ciliate predator Tetrahymena pyriformis and the impact on growth Oliver Ga Tae Wong A thesis submitted to Lancaster University in fulfilment of the requirements for the degree MSc (research) Biomedical Science (by research) September 2017 Declaration This dissertation is entirely my own work and has not been submitted in full or in part for the award of a higher degree at any other educational institution. No sections of this thesis have been published. Word count: 35,320 words I Acknowledgements I would like to thank all the people who contributed in some way to the work described in this thesis. First and foremost, I thank my academic advisor and supervisor Dr Jackie Parry for her support and patience, and also her contribution towards helping me developing the work and ideas for this dissertation. My thanks also extends to all that have supported me during my lab time, notably Janice Drinkall. I would also like to thank all that are close to me. But most importantly my parents, David and Anita, for their unlimited support, and Louise for her love and patience. Thank you! II Table of Contents Declaration ............................................................................................................................. I Acknowledgements ............................................................................................................... II Table of Contents ................................................................................................................. III List of Figures ..................................................................................................................... VII List of Tables ....................................................................................................................... IX Abstract................................................................................................................................. 1 Chapter 1. Introduction ...................................................................................................... 3 1.1. Protists .................................................................................................................... 3 1.1.1 Amoebae ......................................................................................................... 4 1.1.2 Flagellates ....................................................................................................... 5 1.1.3 Ciliates ............................................................................................................. 6 1.2 Ecological significance ............................................................................................ 8 1.2.1 Microbial loop................................................................................................... 8 1.2.2 Role in pathogenesis ..................................................................................... 10 1.3 Phagocytosis ........................................................................................................ 13 1.3.1 Phagocytes in the immune system ................................................................. 13 1.3.1.1 Opsonised phagocytosis ......................................................................... 14 1.3.1.2 Non-opsonised phagocytosis .................................................................. 16 1.3.2 Phagocytosis in protists ................................................................................. 18 1.3.3 Factors that affect phagocytosis..................................................................... 20 1.4 Digestion ............................................................................................................... 23 1.4.1 Vacuole passage time .................................................................................... 23 1.4.2 Digestive vacuole (phagosome) maturation ................................................... 24 1.4.2.1 Acidification and condensation (Phase-II) ............................................... 25 1.4.2.2 Lysosomal fusion and digestion (Phase-III) ............................................. 27 1.4.2.3 Defecation competent (Phase-IV) ........................................................... 30 1.4.2.4 Membrane recycling................................................................................ 31 1.5 The result of phagocytosis .................................................................................... 32 1.6 Aims ..................................................................................................................... 33 Chapter 2. Materials and methods ................................................................................... 34 2.1. Preparation and maintenance of organisms/FLMs ................................................ 34 2.1.1 Fluorescently-labelled microspheres (FLMs) .................................................. 34 2.1.2 Routine maintenance of heterotrophic bacteria .............................................. 34 2.1.3 Transforming bacteria to express the Red Fluorescing Protein (RFP) ............ 36 2.1.4 Heat-killed-DTAF-stained heterotrophic bacteria (‘Dead’) .............................. 37 2.1.5 Live autotrophic bacteria (‘Pico’) .................................................................... 39 III 2.1.6 The ciliate Tetrahymena pyriformis ................................................................ 39 2.2. Counting cells/FLMs ............................................................................................. 40 2.2.1 Counting non-fluorescent bacteria ................................................................. 40 2.2.2 Counting fluorescent prey .............................................................................. 40 2.2.3 Counting Tetrahymena pyriformis .................................................................. 41 2.2.4 Counting prey within Tetrahymena pyriformis digestive vacuoles................... 41 2.3. Particle sizing ........................................................................................................ 42 2.3.1 FLM volumes ................................................................................................. 42 2.3.2 Bacterial biovolumes (Pico, RFP-bacteria and DTAF-stained bacteria) .......... 42 2.4. Ingestion-only experiments ................................................................................... 43 2.4.1 Ingestion at 2x107 particles/ml ....................................................................... 43 2.4.2 Ingestion at different bacterial concentrations ................................................ 43 2.5. Pulse-chase experiments – ingestion and digestion .............................................. 44 2.5.1 Experimental protocol .................................................................................... 44 2.5.2 Parameter determination ............................................................................... 44 2.5.2.1 Vacuole passage time (VPT) .................................................................. 44 2.5.2.2 Ingestion and egestion rates ................................................................... 45 2.5.2.3 Digestion rate and digestion period ......................................................... 46 2.6. Growth experiments .............................................................................................. 47 2.7. Statistics ............................................................................................................... 48 2.7.1 T-test ............................................................................................................. 48 2.7.2 Error .............................................................................................................. 48 Chapter 3. Results........................................................................................................... 49 3.1. Red Fluorescent Protein (RFP) transformations .................................................... 49 3.2. Digestive vacuole processing using pulse-chase experiments .............................. 51 3.3. Phase 1: Ingestion ................................................................................................ 53 3.3.1 Effect of prey biovolume on digestive vacuole formation and content ............ 53 3.3.2 Effect of prey state on digestive vacuole formation and content ..................... 56 3.3.2.1 General overview of between-prey state analysis ................................... 56 3.3.2.2 Effect of bacterial strain within each prey state ....................................... 57 3.3.2.2.1 FLMs ................................................................................................... 57 3.3.2.2.2 Picos ................................................................................................... 60 3.3.2.2.3 Live bacteria ........................................................................................ 61 3.3.2.2.4 Dead bacteria ...................................................................................... 63 3.3.2.3 Effect of bacterial concentration within each prey state ........................... 67 3.3.2.4 Effect of time on digestive vacuole formation and content ....................... 71 3.4. Phase 2: Prey digestion ........................................................................................ 74 3.4.1 Digestive period ............................................................................................