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URI [dataset] UNIVERSITY OF SOUTHAMPTON FACULTY OF NATURAL AND ENVIRONMENTAL SCIENCES School of Ocean and Earth Science Fluorescence on Coral Reefs by Elena Bollati Thesis for the degree of Doctor of Philosophy April 2018 A Claudia e Gabriella, Che mi hanno insegnato ad imparare E a non temere le alghe ballerine To Claudia and Gabriella, Who taught me to learn And not to fear the dancing seaweeds UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF NATURAL AND ENVIRONMENTAL SCIENCES Ocean and Earth Sciences Thesis for the degree of Doctor of Philosophy FLUORESCENCE ON CORAL REEFS by Elena Bollati Coral reefs are highly biodiverse ecosystems and provide vital resources for the human population. Due to increasing pressure by climate change and local stressors, these ecosystems currently face a global crisis. The development of tools to monitor how coral reefs respond to environmental change is a key aspect of the conservation of their biodiversity and resources. A number of reef organisms produce fluorescent molecules, including photosynthetic pigments and green fluorescent protein (GFP)-like pigments found in Anthozoa. These pigments are responsive to environmental conditions and can be optically monitored in vivo, making them a promising tool to investigate organism- and community-level processes on coral reefs. In this thesis, the fundamental principles and technological developments necessary for the application of fluorescence as a biomarker are explored. First, the mechanisms regulating coral fluorescence are considered for two functionally and biochemically distinct groups of GFP-like proteins. In mesophotic and depth-generalist symbiotic corals, incomplete light-driven maturation of the red GFP-like protein pool is shown to determine the spectrum of fluorescence emission. A role of this mechanism in adaptation to the reduced mesophotic light spectrum is discussed. In corals from shallow water environments, enhancement of internal light fluxes due to reduced absorption by symbiont pigments during bleaching is shown to induce expression of GFP- like proteins. High-level expression of these pigments in bleached tissue is shown to promote recovery of the symbiotic algae complement after a stress event. Second, a novel approach to fluorescence imaging for coral reef surveys is presented. The method enables automatic classification of reef benthic organisms based on the intensity of fluorescent signal in different excitation and emission bands. These findings demonstrate the potential of fluorescence as an in vivo marker for physiological and ecological studies of coral reef organisms, contributing to ongoing efforts to monitor and preserve the health of these ecosystems. i Table of Contents Table of Contents ................................................................................................ iii Table of Tables .................................................................................................... vii Table of Figures .................................................................................................... ix Academic Thesis: Declaration Of Authorship ................................................... xi AcKnowledgements ............................................................................................ xiii Definitions and Abbreviations ............................................................................ xv Chapter 1 Introduction .................................................................................... 1 1.1 BacKground ......................................................................................................... 1 1.1.1 Coral reefs in the Anthropocene .................................................................... 1 1.1.2 Biomarkers of change .................................................................................... 3 1.1.3 Fluorescence as a biomarker ........................................................................ 4 1.2 Thesis overview .................................................................................................. 6 1.2.1 Thesis aims .................................................................................................... 6 1.2.2 Thesis structure ............................................................................................. 8 Chapter 2 Fluorescent proteins and chromoproteins in reef corals ......... 11 2.1 Fluorescence of living organisms ................................................................... 11 2.2 Biochemistry of coral fluorescent proteins .................................................... 12 2.2.1 GFPs ............................................................................................................ 13 2.2.2 CFPs ............................................................................................................ 14 2.2.3 RFPs ............................................................................................................ 14 2.2.4 pcRFPs ........................................................................................................ 15 2.2.5 CPs .............................................................................................................. 15 2.3 GFP-like proteins in reef-building corals ........................................................ 16 2.3.1 Distribution of GFP-like proteins in coral tissue ........................................... 16 2.3.2 Regulation of coral GFP-like proteins .......................................................... 17 2.3.3 Functions of coral GFP-like proteins ............................................................ 19 2.4 Corals and light ................................................................................................. 20 2.5 Conclusion ......................................................................................................... 21 Chapter 3 FRET-mediated long-range wavelength transformation by photoconvertible fluorescent proteins as an efficient mechanism to generate orange-red light in symbiotic deep water corals .............................................. 23 3.1 Abstract .............................................................................................................. 23 iii 3.2 Introduction ....................................................................................................... 23 3.3 Results ............................................................................................................... 26 3.3.1 Effects of light quality on photoconvertible red fluorescent proteins (pcRFPs) 26 3.3.2 Photoconversion along a simulated depth-irradiance gradient .................... 32 3.3.3 Wavelength transfer by Förster Resonance Energy Transfer (FRET) ......... 34 3.3.4 Efficiency of FRET-mediated wavelength transfer ....................................... 36 3.4 Discussion ......................................................................................................... 37 3.4.1 Effects of spectral quality on photoconversion of pcRFPs ........................... 37 3.4.2 Wavelength transformation via FRET .......................................................... 38 3.5 Materials and methods ..................................................................................... 41 3.5.1 Sample culture and aquarium set up ........................................................... 41 3.5.2 Live colony fluorescence ............................................................................. 42 3.5.3 Protein expression and purification .............................................................. 42 3.5.4 Photoconversion and spectroscopy of purified proteins .............................. 42 3.5.5 Photoconversion potential of light sources .................................................. 43 3.5.6 Estimation of FRET in live colonies ............................................................. 44 3.5.7 Evaluation of wavelength conversion in vitro ............................................... 45 3.6 Supplementary Figures .................................................................................... 46 3.7 Supplementary Tables ...................................................................................... 46 Chapter 4 Bleaching corals express bright colours to facilitate recovery49 4.1 Abstract .............................................................................................................. 49 4.2 Main text ............................................................................................................. 49 4.3 Materials and