The Pennsylvania State University The Graduate School Eberly College of Science UNDERSTANDING TEMPERATURE ACCLIMATION IN SYMBIODINIUM: AN INTERDISCIPLINARY APPROACH A Dissertation in Biology by Erika Díaz-Almeyda © 2016 Erika Díaz-Almeyda Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2016 The dissertation of Erika Díaz-Almeyda was reviewed and approved* by the following: Monica Medina Associate Professor of Biology The Pennsylvania State University Dissertation Advisor Charles R. Fisher Professor and Distinguished Senior Scholar of Biology The Pennsylvania State University Chair of Committee Todd C. LaJeunesse Associate Professor of Biology The Pennsylvania State University Mark Guiltinan Professor of Plant Molecular Biology and Adjunct Professor of Biology The Pennsylvania State University Tracy Langkilde Professor of Biology The Pennsylvania State University Department Head of Biology *Signatures are on file in the Graduate School ii Abstract I quantified the thermotolerance in 11 cultures from different populations of five species of Symbiodinium clade A. we grew cultures at 26°C and 32°C over 18 days, measuring growth and photochemical efficiency (Fv/Fm). Thermotolerance was not restricted to a single species but it was widespread across species and cultures, showing a gradient from susceptible to tolerant. All cultures at 32°C decreased growth and Fv/Fm. To test the synergistic effect of temperature and light, we cultured three strains (tolerant, intermediate, and susceptible) in five different light intensities at 26°C and 32°C. Strains surviving stressful light and temperature exhibited less growth and quicker damage by light. To investigate the mechanisms behind thermoacclimation, we cultured S. microadriaticum (CassKB8) with intermediate thermotolerance at 26°C and 32°C. Gene expression was explored first using cDNA microarrays before (day -2), and during acclimation (day 6 and 16). Differentially expressed genes (DEG) due to increased temperature were time dependent. DEG on day 16 were likely a result of the start of the stationary phase in culture. Similarly, RNA-Seq data (day 5 and 7) suggest temporal variation in gene expression with major changes in heat-shock proteins and chaperones. Retrotransposons were highly expressed on day 7, indicating high stress during thermal exposure. Adaptation to higher temperatures is not restricted to a single clade or species but it is widespread within species. However, acclimating to higher temperatures compromises health and increases chaperone activity. iii Table of Contents List of Figures ......................................................................................................................... vii List of Tables ............................................................................................................................. x Acknowledgments ................................................................................................................ xii Chapter 1. Introduction ......................................................................................................... 1 Symbiodinium diversity .................................................................................................................. 2 Symbiodinium gene expression changes under high temperature conditions. ........... 4 Symbiodinium as a model organism to understand the effects of climate change ..... 6 Table ..................................................................................................................................................... 8 References ........................................................................................................................................... 9 Chapter 2. Within and among species variation in thermotolerance and photoacclimation in Symbiodinium dinoflagellates .................................................. 17 Abstract .............................................................................................................................................. 17 Introduction ..................................................................................................................................... 17 Methods ............................................................................................................................................. 20 Symbiodinium cultures: growth conditions ...................................................................................... 20 High temperature acclimation: experimental design ................................................................... 21 High temperature acclimation and photoacclimation: experimental design .................... 21 Photochemical Efficiency .......................................................................................................................... 22 Population Growth ...................................................................................................................................... 22 Statistical analysis ........................................................................................................................................ 22 Results ................................................................................................................................................ 23 Extensive variation in temperature sensitivity within and among species ........................ 23 The ability to acclimate to high temperature is not species specific ..................................... 24 High temperature acclimation has a cost ......................................................................................... 24 The effect of light and temperature in photochemistry and growth ..................................... 25 Discussion ......................................................................................................................................... 26 Widespread thermotolerance variation within species from clade A lineage .................. 26 Combined effect of light and temperature in physiological acclimation ............................. 28 Conclusions ....................................................................................................................................... 28 iv Tables ................................................................................................................................................. 30 Figures ................................................................................................................................................ 33 Supplementary Figures ................................................................................................................ 38 References ......................................................................................................................................... 41 Chapter 3. Temporal transcriptomic response of Symbiodinium microadriaticum (CassKB8) under high temperature acclimation ..................... 47 Introduction ..................................................................................................................................... 47 Materials & Methods ...................................................................................................................... 50 Symbiodinium growth and experimental design ............................................................................ 50 Physiological measurements .................................................................................................................. 50 Microarray experiment .............................................................................................................................. 51 RNAseq experiment .................................................................................................................................... 52 Results ................................................................................................................................................ 54 Symbiodinium microadriaticum acclimates to high temperature. .......................................... 54 RNA-Seq libraries quality and assembly ............................................................................................ 55 Differential expression is higher over time than between temperatures ........................... 55 Functions differentially expressed at high temperature during multiple time points .. 56 Time-dependent gene expression ......................................................................................................... 58 Photosynthetic functions under thermal acclimation .................................................................. 59 Nitrogen metabolism .................................................................................................................................. 60 Chaperone and heat-shock proteins express time and temperature specific ................... 60 Other important DEG .................................................................................................................................. 61 Discussion ......................................................................................................................................... 61 Physiological acclimation to high temperature has a cost in growth. ................................... 61 Differential expression is higher with time rather than with high temperature ............. 62 Thermal acclimation affects