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UNIVERSITY OF SOUTHAMPTON

FACULTY OF NATURAL AND ENVIRONMENTAL SCIENCES

Ocean and Earth Science, National Oceanography Centre of Southampton

“The relationship between corals and their symbiotic dinoflagellates: Environment and Host control”

Victor Emanuel Urrutia Figueroa

Thesis for the degree of Doctor of Philosophy in Ocean and Earth Science

October 2017

UNIVERSITY OF SOUTHAMPTON

ABSTRACT

FACULTY OF NATURAL AND ENVIRONMENTAL SCIENCES

Ocean and Earth Science

Thesis for the degree of Doctor of Philosophy in Ocean and Earth Science

“THE RELATIONSHIP BETWEEN CORALS AND THEIR SYMBIOTIC DINOFLAGELLATES: ENVIRONMENT AND HOST CONTROL”

Victor Emanuel Urrutia Figueroa

Abstract

The ecological success and also the susceptibility of corals to bleaching have been attributed to their obligate relationship with Symbiodinium and its functional diversity. Deeper understanding of this symbiosis is essential to enable the existence of reef ecosystems. This thesis aimed to determine how the environment and the host exert control over the proteome of Symbiodinium and how this is related to the maintenance of symbiosis. Firstly, it was explored how the proteome related to the photoacclimation strategies of Symbiodinium, and its regulation under different light environments. An initial assessment of the proteome regulation using shotgun proteomics over Symbiodinium type C1 cultured at two experimental photon fluxes displayed differences in proteins associated with light harvesting, electron transport, carbon fixation and protein modification. Subsequent two-dimensional gel electrophoresis (2-DE) analysis between three Symbiodinium types (A1, A13 and C1) cultured at the two previous photon fluxes revealed unique proteome differences across these types and between both experimental light treatments. Database searches of the isoelectric point (pI) and the molecular weight (MW) tagged these proteins also associated with the same processes matched by previous shotgun analysis over C1 type. These results are in agreement with previous hypothesis on different photoacclimation strategies displayed among different types of Symbiodinium. These strategies comprised differential proteomic modifications in the photosynthetic unit (PSU) of the experimental types of Symbiodinium to balance ATP production and achieve homeostasis at different photon flux environments. The host control effect and regulation of the proteome and physiology of Symbiodinium ex- hospite was studied using two host release factors (HRFs) a free aminoacid mix (FAA) and the host tissue (HT) homogenate from the coral Pocillopora damicornis. To comprehend the relation between the proteome and physiology various parameters related with the photosynthate production and translocation of Symbiodinium were measured including: glucose released, excitation pressure over photosystem two (PSII), total protein, total glucose and chlorophyll a concentration in the presence and absence of the HRFs. The measured parameters showed variability among types. In general, the parameters directly related to photochemistry: Chl a and excitation pressure over PSII were controlled by photon flux and inversely correlated with each other. Protein and glucose also correlated with each other and were controlled by host factors although HT and FAA had an antagonistic effect of stimulation and inhibition of these parameters respectively. The principal component analysis (PCA) evidenced the existence of a general mechanism of glucose release which correlated with protein expression. The HRFs control over the proteome of Symbiodinium explored by SDS-PAGE analysis displayed different bands with

differential expression among types and treatments. Some of these proteins may be related with the stimulation and inhibition of the mechanism of translocation of glucose that were found. Identification of these proteins would probably clarify the physiology behind the translocation of glucose from the Symbiodinium cell to its coral host. The proteome response and physiology of Symbiodinium was assessed by the interaction of different light environments with different temperatures. Growth patterns and photochemical measurements confirmed unique acclimation processes to light and temperature conditions across the three Symbiodinium types. These acclimation strategies appeared regulated by the differential expression of proteins involved in photosynthesis, protein modification, energy metabolism and cell maintenance. This differential expression of the proteome related to these processes seemed associated with the activation of alternative electron transport pathways to balance ATP production, regulate energy metabolism and fuel the cell at the different experimental light and temperature conditions. The results obtained provide a broader view on how the differential regulation of the proteome mediates the physiological plasticity across types of Symbiodinium to photoacclimate to different light enviroments, to acclimate to high temperature and to respond to stressful conditions determining the functional diversity existing in this genus of coral symbionts.

Table of Contents

Table of Contents ...... i

Table of Tables ...... vii

Table of Figures ...... ix

Academic Thesis: Declaration Of Authorship ...... xvii

Acknowledgements ...... xix

Definitions and Abbreviations ...... xxi

Chapter 1 ...... General Introduction ...... 1 1.1 Importance of coral reef ecosystems ...... 1 1.2 The coral symbiosis ...... 2 1.3 The Symbiodinum ...... 4 1.4 Host control of symbiosis ...... 6 1.5 Breakdown of symbiosis ...... 8 1.6 Proteomics ...... 9 1.7 Thesis Hypothesis ...... 14 1.8 Thesis Objectives ...... 14

Chapter 2 ...... Photoacclimation of Symbiodinium: a proteomic approach ...... 16 2.1 Introduction ...... 17 2.2 Hypothesis ...... 18 2.3 Objective ...... 19 2.4 Materials and Methods ...... 19 2.4.1 ...... Cultures ...... 19 2.4.2 ...... Growth ...... 19 2.4.3 ...... Photochemistry ...... 20

i Table of Contents

2.4.4 ...... Proteomics ...... 21 2.4.4.1 ...... Protein extraction ...... 21 2.4.4.2 ...... Protein determination and Iso-Electro-Focusing (IEF) as 1st dimension ...... 21 2.4.4.3 ...... Equilibration ...... 22 2.4.4.4 ...... Second dimension electrophoresis 2-DE ...... 22 2.4.4.5 ...... Protein visualization by silver staining ...... 22 2.4.4.6 ...... Protein Identification ...... 22 2.4.5 ...... Statistical analyses ...... 23 2.5 Results ...... 24 2.5.1 ...... Growth ...... 24 2.5.2 ...... Photophysiology ...... 25 2.5.3 ...... Proteomics ...... 33 2.5.3.1 ...... Shotgun proteomics...... 33 2.5.3.2 ...... Two-dimensional electrophoresis (2-DE) analysis ...... 34 2.6 Discussion ...... 41 2.6.1 ...... Growth ...... 41 2.6.2 ...... Photophysiology ...... 42 2.6.3 ...... Proteomics ...... 43

ii Table of Contents

Chapter 3 ...... The coral host release factor and its effect over Symbiodinium ...... 49 3.1 Introduction ...... 50 3.2 Hypothesis ...... 52 3.3 Objective ...... 52 3.4 Materials and Methods ...... 53 3.4.1 ...... Experimental setup ...... 53 3.4.2 ...... Isolation and formulation of HRFs ...... 53 3.4.2.1 ...... i) Host tissue homogenate extraction ...... 53 3.4.2.2 ...... ii) Free Amino-acid mix formulation ...... 53 3.4.3 ...... Isolation and culture of Symbiodinium of P. damicornis ...... 54 3.4.4 ...... Cultured Clades ...... 54 3.4.5 ...... Cell quantification ...... 54 3.4.6 ...... Chlorophyll a ...... 55 3.4.7 ...... Protein ...... 55 3.4.8 ...... Glucose ...... 55 3.4.9 ...... Fast repetition rate fluorometry (FRRF) ...... 56 3.4.10 ...... Statistical analyses ...... 56 3.5 Results ...... 57 3.6 Discussion ...... 71

Chapter 4 ...... Effect of temperature on the physiology and protein expression of Symbiodinium in culture ...... 75 4.1 Introduction ...... 75

iii Table of Contents

4.2 Hypothesis ...... 78 4.3 Objectives ...... 79 4.4 Materials and Methods ...... 79 4.4.1 ...... Cultures ...... 79 4.4.2 ...... Growth ...... 80 4.4.3 ...... Photophysiology ...... 81 4.4.4 ...... Proteomics ...... 82 4.4.4.1 ...... Protein extraction ...... 82 4.4.4.2 ...... Protein determination and Iso-Electro-Focusing (IEF) as 1st dimension ...... 82 4.4.4.3 ...... Equilibration ...... 82 4.4.4.4 ...... SDS-PAGE as second dimension ...... 83 4.4.4.5 ...... Protein visualization by silver staining ...... 83 4.4.5 ...... Statistical analyses ...... 83 4.5 Results ...... 84 4.5.1 ...... Growth ...... 84 4.5.2 ...... Photophysiology ...... 86 4.5.3 ...... Proteomics ...... 96 4.6 Discussion ...... 103

Chapter 5 ...... General Discussion ...... 108 5.1 Photoacclimation metabolism ...... 108 5.2 Host control ...... 109

iv Table of Contents

5.3 Temperature and Light effect over the proteome ...... 110

Appendix A ...... 115 A.1 High resolution 2-Dimensional Electrophoresis of the protein complex mix isolated from the three experimental types (A1, A13 and C1) grown under two PFs (LL=100 and HL=350 µmol photons·m-2·s-1). 40µg of protein were loaded in 7cm (pH 3-10) and 11cm (pH 4-7) linear gradient strips for the first dimension and then resolved in 12% and 10-14.5% polyacrylamide gels for the second dimension. The more representative spots for each PF were analysed using C1 and A13 resolving gels as references for LL and HL respectively...... 115 A.2 High resolution 2-Dimensional Electrophoresis of the protein complex mix isolated from the three experimental types (A1, A13 and C1) grown under two PFs (LL=100 and HL=350 µmol photons·m-2·s-1). 40µg of protein were loaded in 11cm (pH 4-7) linear gradient strips for the first dimension and resolved in 10-14.5% polyacrylamide gels for the second dimension. The most significantly different spots between PFs were analysed...... 116

Glossary of Terms ...... 119

List of References ...... 121

Table of Tables

Table 1. Summary of proteomic studies in marine dinoflagellates from a literature search. Modified from Wang et al. (2014) to include Symbiodinium and symbiosis studies...... 12

Table 2. Specific growth rates µ (day-1), and Chl a concentration (pgcell-1) of the three experimental Symbiodinium types cultured at two photon fluxes (LL=100 and HL=350 µmol photons·m-2·s-1), (each value represents the mean value and standard deviation (±SE), obtained from three different cultures) ...... 24

Table 3. Mean values of the PSII photochemical efficiency Fv/Fm and PSII effective cross section 2 σPSII(nm ) (±SE). Each value is the average of measurements made every second day along the exponential phase of three different cultures per each type...... 30

Table 4. Maximum RCII-relative-rate of electron transfer rETRPSIImax and α of the three Symbiodinium types cultured under the two experimental PFs (LL=100 and HL=350 µmol photons·m-2·s-1). Each value is the average of three replicates...... 32

Table 5.Proteome differences displayed between C1 grown in two different photon fluxes (100 and 350 µmol photons•m2•s-1) ...... 33

Table 6 Proteome differences across the three types of Symbiodinium cultured at the two photon fluxes. The molecular weight (MW) and isoelectric point (IP) of the significantly different (Anova (p)) protein spots (Spot #) from gels choosen for identification were measured. Identities (ID) of the proteins were obtained by submitting the MW and IP of each spot in the TagIdent tool available in ExPASy bioinformatics resource portal. Databases retrieved the name (ID), access number (Access#) and the Biological Process of the proteins that corresponded with the parameters of each spot. The differences in expression across types (A1, A13 and C1) and between photon flux treatments (photon fluxes=100 and 350 µmol photons·m-2·s-1) per each spot is highlighted by up-regulated and ¯ down-regulated...... 38

Table 7. Means and standard errors of Chl a, Protein, Total Glucose, Released Glucose (pgcell-1) and excitation pressure over PSII (Q) of three cultured types (A1, A13 and C1) and Symbiodinium isolated from scleractinian coral P. damicornis (Pds) all treated with two HRFs (Host Tissue=HT and Free Amino-acid mix=FAAmix) and ASP-8A only (Control)...... 57

vii Table of Tables

Table 8. Means and standard errors ±SE (n=3) of gel lines and gel bands analysed with SDS-PAGE run with protein extracted from cells of Symbiodinium types (A1, A13 and C1) and Symbiodinium isolated from scleractinian coral P. damicornis (Pds) cultured at 350 µmol photons•m-2•s-1 and treated with two host release factors (Host Tissue=HT and Free Amino-acid mix=FAA) and ASP-8A only (Control)...... 70

Table 9. Growth parameters calculated from fit to a logistic sigmoidal model cell counts of three types of Symbiodinium cultured at two photon fluxes (350 or 100 µmol photons•m-2•s-1) and two temperatures (26 or 30ºC)...... 84

Table 10. Means and standard errors of: Chl a concentration, PSII photochemical efficiency

(Fv/Fm) and PSII absorption cross section (σPSII)...... 93

Table 11. Means and standard errors of: α, PSII maximum rate of electron transport (ETRPSIImax) and light saturation point (Ek)...... 94

Table 12. Most significantly different protein spots from 2-DE analysis of samples from cultures of three types of Symbiodinium (A1, A13 and C1) at two photon fluxes (HL=350 and LL=100) and at two temperatures (26 and 30). Gels were analysed between both temperatures (Top), between both photon fluxes and temperatures (Middle) and after subject to 33ºC of temperature stress for three consecutive days (80h, Bottom). Values displayed for significantly different spots shared between treatments after analyses.=Up- regulated...... 101

Table 13. Relevant information of protein spots identified from isoelectric point pI and molecular weight MW using Tag-Ident tool (available in Expasy site) for searches in data bases World 2DPAGE, SWISS-2DPAGE and UniPROT...... 102

viii Table of Figures Table of Figures

Figure 1. Scheme of the continuum of interactions from mutualism to parasitism between the environment, the coral host and Symbiodinium, the life-history symbiotic state characters under selection and a hypothetical example of a mutualistic and a parasitic phylotypes based on their photosynthetic contribution to the host (modified from Lesser et al. 2013)...... 4

Figure 2. Workflow of proteomic analyses used to study Symbiodinium cultures. After sample preparation, protein extraction and separation by electrophoresis, proteins were identified using mass spectrometric analyses and database search to proceed with the interpretation of experiments...... 11

Figure 3. Growth curves of the three Symbiodinium types A1, A13 and C1 cultured at two experimental PFs (LL=100 and HL=350 µmol photons·m-2·s-1) data obtained from cell counts were fitted to a sigmoidal logistic equation of the form y= α/(1+exp(-(β-x0)/γ). (Each growth curve represents three different independent cultures)...... 25

Figure 4. Chlorophyll a concentration among cultures of three types of Symbiodinium cultured at two PFs (HL=350µmol photons•m-2•s-1, white bars or LL=100 µmol photons•m-2•s-1, grey bars)...... 26

Figure 5. Chl a* specific absorption coefficient of the three Symbiodinium types cultured at the two experimental PFs (LL=100 and HL=350 µmol photons·m-2·s-1). Each spectra is the average of nine independent Chl a* calculations...... 27

Figure 6. PSII photochemical efficiency Fv/Fm between cultures of three types of Symbiodinium (A1, A13 and C1) at two photon fluxes (HL=350µmol photons•m-2•s-1 or LL=100 µmol photons•m-2•s-1) (Means ± SE, n<100)...... 28

Figure 7. PSII absorption cross section σ (nm2) between cultures of three types of Symbiodinium (A1, A13 and C1) at two photon fluxes (HL=350µmol photons•m-2•s-1 or LL=100 µmol photons•m-2•s-1) (Means ± SE, n<100)...... 29

Figure 8. PSII relative electron transfer rate rETRPSII of three Symbiodinium types cultured under two experimental PFs (100µmol photons·m-2·s-1 and 350µmol photons·m-2·s-1). Each RLC is the average of three replicates per each species and bars represent the standard error (±SE)...... 31

ix Table of Figures

Figure 9.Reference gel (A1-100µmol photons·m-2·s-1) shows analyses of protein spots. Gel highlights the 15 different spots found between types (A1, A13 and C1) and between two photon flux treatments (photon fluxes=100 and 350 µmol photons·m-2·s-1). 40µg of protein were loaded in 11cm (pH 4-7) linear gradient strips for the first dimension and then resolved in 10-14.5% polyacrylamide gels for the second dimension...... 35

Figure 10. Analysis of the differential expression after 2-DE separation of the protein spot #412 putative RuBisCo across the three cultured types at the two experimental photon fluxes (HL=350µmol photons•m-2•s-1 and LL=100µmol photons•m-2•s-1). The right bottom shows the area where the protein spot is located in the gel. The top shows a tridimensional representation of the area of the gel with the spot volume represented across the three experimental types at the two photon fluxes. The left bottom shows the mean and standard errors of the log normalised volumes (n=3) of the spot across the three experimental types at the two light photon fluxes. As shown by the normalisation values the spot was found more expressed in type A1 at both photon fluxes...... 39

Figure 11. Chl a (pgcell-1) of Symbiodinium cells cultured at two photon fluxes (HL=350 µmol photons•m-2•s-1 and LL=100 µmol photons•m-2•s-1)...... 58

Figure 12. Chl a (pgcell-1) of Symbiodinium cells was not affected by HRFs treatments (FAA=Free Amino acids and HT=Host Tissue) compared against their controls (FAAC and HTC) treated with culture media only. Highlights the effect of light over the photosynthetic pigment, higher in cultures at 100 µmol photons•m-2•s-1...... 59

Figure 13. Excitation pressure over PSII (Q) between Symbiodinium cells cultured at two photon fluxes (HL=350 µmol photons•m-2•s-1 and LL=100 µmol photons•m-2•s-1)...... 60

Figure 14. Excitation pressure over PSII (Q) of Symbiodinium cells cultured at two photon fluxes (HL=350 µmol photons•m-2•s-1 and LL=100 µmol photons•m-2•s-1), treated with two different types of host release factors and compared against their controls treated with culture media only...... 61

Figure 15. Physiological parameters related with photosynthesis, chlorophyll a and excitation pressure over PSII measurements performed on four types of Symbiodinium A1, A13, C1 and symbiont isolated from scleractinian coral Pocillopora damicornis (PD). Symbiotic algae were cultured at two photon fluxes (HL=350 µmol photons•m-2•s-1 and LL=100 µmol photons•m-2•s-1) and treated with two different types of host release factors (Free Amino-Acids=FAA, Host Tissue=HT and ASP-8A=FAAC and HTC. Al measurements

x Table of Figures

were normalized to picograms per cell (pgcell-1). a Chlorophyll a. b Excitation pressure over PSII...... 62

Figure 16. Protein (pgcell-1) measured in Symbiodinium cells cultured at two photon fluxes (HL=350 µmol photons•m-2•s-1 and LL=100 µmol photons•m-2•s-1), treated with two different types of host release factors (FAA=Free Amino Acids, HT=Host Tissue) and compared against their controls treated with culture media only...... 63

Figure 17. Total Glucose (pgcell-1) of Symbiodinium cells cultured at two photon fluxes (HL=350 µmol photons•m-2•s-1 and LL=100 µmol photons•m-2•s-1), treated with two different types of host release factors (FAA=Free Amino Acids, HT=Host Tissue) and compared against their controls treated with culture media only...... 64

Figure 18. Physiological parameters protein and total glucose measured in on four types of Symbiodinium A1, A13, C1 and the symbiont isolated from scleractinian coral Pocillopora damicornis (PD). Symbiotic algae were cultured at two photon fluxes (HL=350 µmol photons•m-2•s-1 and LL=100 µmol photons•m-2•s-1) and treated with two different types of host release factors (Free Amino-Acids=FAA, Host Tissue=HT and ASP-8A=FAAC and HTC. Al measurements were normalized to picograms per cell (pgcell-1). a Chl a. b Protein. c Total Glucose...... 65

Figure 19. Glucose released (pgcell-1) when compared Symbiodinium cells cultured at two photon fluxes (HL=350 µmol photons•m-2•s-1 and LL=100 µmol photons•m-2•s-1)...... 66

Figure 20. Glucose released (pgcell-1) of Symbiodinium cells treated with two host release factors (FAA=Free Amino Acids, HT=Host Tissue) and ASP-8A media only as controls (FAAC and HTC)...... 67

Figure 21. Glucose released (pgcell-1) measured in Symbiodinium cells cultured at two photon fluxes (HL=350 µmol photons•m-2•s-1 and LL=100 µmol photons•m-2•s-1), treated with two different types of host release factors (FAA=Free Amino Acids, HT=Host Tissue) and compared against their controls (FAAC and HTC) treated with ASP-8A media only...... 67

xi Table of Figures

Acids=FAA, Host Tissue=HT and ASP-8A=FAAC and HTC. All measurements were normalized to picograms per cell (pgcell-1)...... 68

Figure 23. Principal component analysis (PCA) of physiological parameters of Symbiodinium cultured types A1, A13, C1 and the symbiont of the scleractinian coral Pocillopora damicornis (PD). Symbiotic algae were cultured at two photon fluxes (HL=350 µmol photons•m-2•s-1 and LL=100 µmol photons•m-2•s-1) and treated with two different types of host release factors (Free Amino-Acids=FAA, Host Tissue=HT and ASP-8A=FAAC and HTC. All measurements were normalized to picograms per cell (pgcell-1)...... 69

Figure 24. a Profiles of peaks representing the bands (# 1,2,3,4,5) obtained from gel lines separated with SDS-PAGE analysis run with protein samples of A13 treated with free amino acids (A13FAA), dinoflagellate symbiont isolated from Pocillopora damicornis treated with host tissue (PDHT) and C1 treated with ASP-8A (C1Ctrl). Profiles were produced with Image J program. b Protein profiles obtained with SDS-PAGE analysis run with protein samples (40µg) of A13 treated with free amino acids (A13FAA), dinoflagellate symbiont isolated from Pocillopora damicornis treated with host tissue (PDHT) and C1 treated with ASP-8A (C1Ctrl). MW= Molecular weight in kilo Daltons. Numbers point with lines bands excised for identification analysis with MALDI-TOF...... 71

Figure 25. Growth curves of three experimental types of Symbiodinium (A1, A13 and C1) cultured at two photon fluxes (350µmol photons•m-2•s-1 or 100 µmol photons•m-2•s-1) and at two temperatures (26ºC or 30ºC). Growth curves were calculated using a sigmoidal logistic model starting from cell counts obtained with and hemocytometer. Each growth curve represents three independent cultures...... 85

Figure 26. Absorbance spectra of Chl a methanol extracts from samples of cultures of three types of Symbiodinium (A1, A13 and C1) at two photon fluxes (350µmol photons•m-2•s-1 or 100 µmol photons•m-2•s-1) and two temperatures (26ºC or 30ºC), maximum peaks of spectra were normalized to one...... 86

Figure 27. Chl a (pgcell-1) concentration between cultures at two photon fluxes (350µmol photons•m-2•s-1 or 100 µmol photons•m-2•s-1) and two temperatures (26ºC or 30ºC). 87

Figure 28. Chlorophyll a concentration among cultures of three types of Symbiodinium at two photon fluxes (350µmol photons•m-2•s-1 or 100 µmol photons•m-2•s-1) and two temperature (26ºC or 30ºC) treatments...... 87

xii Table of Figures

Figure 29. Absorption spectra normalized to Chl a absorption coefficients of three experimental types of Symbiodinium (A1, A13 and C1) acclimated at two photon fluxes (350µmol photons•m-2•s-1 or 100 µmol photons•m-2•s1) and two temperatures (26ºC or 30ºC). . 88

Figure 30. Comparison of PSII photochemical efficiency Fv/Fm between cultures at two photon fluxes (350µmol photons•m-2•s-1 or 100 µmol photons•m-2•s-1) and two temperatures (26ºC or 30ºC)...... 89

Figure 31. Comparison of PSII photochemical efficiency Fv/Fm between cultures of three types of Symbiodinium (A1, A13 and C1) at two photon fluxes (350µmol photons•m-2•s-1 or 100 µmol photons•m-2•s-1) and two temperatures (26ºC or 30ºC). (Means ± SE, n<100). 90

Figure 32. PSII absorption cross section σ (nm2) between cultures at two photon fluxes (350µmol photons•m-2•s-1 or 100 µmol photons•m-2•s-1) and two temperatures (26ºC or 30ºC). 91

2 Figure 33. Comparison of PSII absorption cross section σPSII (nm ) between cultures of three types of Symbiodinium (A1, A13 and C1) at two photon fluxes (350µmol photons•m-2•s-1 or 100 µmol photons•m-2•s-1) and two temperatures (26ºC or 30ºC). (Means ± SE, n<100). 92

Figure 34. Rapid light curves (RLCs) of three experimental types of Symbiodinium (A1, A13 and C1) acclimated at two photon fluxes (350µmol photons•m-2•s-1 or 100 µmol photons•m- 2•s-1) and two temperatures (26ºC or 30ºC). Each point represents the mean and standard errors of rETR at a given irradiance for a determined type of Symbiodinium (Means ± SE, n=100)...... 95

Figure 35. Patterns of decline of photochemical efficiency (Fv/Fm) of three experimental types of Symbiodinium (A1, A13 and C1) acclimated at two photon fluxes (350µmol photons•m- 2•s-1 or 100 µmol photons•m-2•s-1) and two temperatures (26ºC or 30ºC) after being subject to stress temperatures at 33ºC for three days...... 96

Figure 36. Reference 2D-PAGE map (A1 cultured at 100µmol photons•m-2•s-1 and 26˚C) displays all the significant different proteins (spots) found between both temperature treatments 26ºC vs30ºC (40µg of total protein were loaded in the IPG strip for the first dimension IEF, second dimension was performed using 10.5-14% polyacrylamide gels (Bio-Rad)...... 97

xiii Table of Figures

loaded in the IPG strip for the first dimension IEF, second dimension was performed using 10.5-14% polyacrylamide gels (Bio-Rad)...... 98

Figure 38. Reference 2D-PAGE map (A1 cultured at 100µmol photons•m-2•s-1 and 26˚C) displays 46 significant different proteins (spots) between photon fluxes (350µmol photons•m-2•s-1 or 100 µmol photons•m-2•s-1) and temperature treatments (26ºC or 30ºC) combined, after manual picking for removal of false positives (40µg of total protein were loaded in the IPG strip for the first dimension IEF, second dimension was performed using 10.5-14% polyacrylamide gels (Bio-Rad)...... 99

Figure 39. Reference 2D-PAGE map (C1 cultured at 100µmol photons•m-2•s-1 and 30˚C-33˚C), displays 32 significant different proteins (spots) between previous temperature treatments (26ºC or 30ºC) subject to temperature stress at 33ºC for 3 days (80h), after manual picking for removal of false positives (40µg of total protein were loaded in the IPG strip for the first dimension IEF, second dimension was performed using 10.5-14% polyacrylamide gels (Bio-Rad)...... 100

xiv Table of Figures

Academic Thesis: Declaration Of Authorship Academic Thesis: Declaration Of Authorship

I, ...... Victor Emanuel Urrutia Figueroa declare that this thesis and the work presented in it are my own and has been generated by me as the result of my own original research.

“The relationship between corals and their symbiotic dinoflagellates: Environment and Host Control” ......

......

I confirm that:

1. This work was done wholly or mainly while in candidature for a research degree at this University; 2. Where any part of this thesis has previously been submitted for a degree or any other qualification at this University or any other institution, this has been clearly stated; 3. Where I have consulted the published work of others, this is always clearly attributed; 4. Where I have quoted from the work of others, the source is always given. With the exception of such quotations, this thesis is entirely my own work; 5. I have acknowledged all main sources of help; 6. Where the thesis is based on work done by myself jointly with others, I have made clear exactly what was done by others and what I have contributed myself; 7. None of this work has been published before submission

Signed:

Date: 31 July 2018 ......

xvii

Acknowledgements

To Prof. David Smith at University of Essex for his unconditional support and invaluable advice, at every moment during development of the project. To my supervisors at University of Southampton firstly Prof. Joerg Wiedenmann for his help and support and to Prof. Tomas Bibby for his advice. To Prof. David Suggett for initial supervision of the project. To Prof. Tracy Lawson for her help at the initial phase of the project. To Dr. Metodi Metodiev for the shotgun proteomics and database identification of samples with LC/MS/MS. To the proteomics facility at the University of Bristol for the MALDI-TOF analysis. To CONACYT for the scholarship # 359280 to develop the project. To Prof. Richard Geider and Prof. Nelson Fernandez for their support and advice. To technicians Tania, Russell, Phill, Farid and John at the University of Essex for all their help and assistance during the lab work.

xix

Definitions and Abbreviations Definitions and Abbreviations

2-DE Two-dimensional Electrophoresis acpPC chlorophyll a, chlorophyll c2, peridinin Protein Complex

AET Alternative Electron Transport

CET Cyclic Electron Transport

Chl a Chlorophyll a

Chl a* Chlorophyll a specific absorption coefficient

ET Electron Transport

FAA Free Amino Acid

Fq’/Fm’ PSII effective photochemical efficiency

FRRF Fast Repetition Rate Fluorometry

Fv/Fm PSII maximum photochemical efficiency

HL photon flux of 350µmol photons•m-2•s-1

HRF Host Release Factor

LET Linear Electron Transport

LHC Light Harvesting Complex

LL photon flux of 100µmol photons•m-2•s-1

MW Molecular Weight

NPQ Non Photochemical Quenching

PCP Peridinin Chlorophyll Protein

PF Photon Flux

PFs Photon Fluxes pI Isoelectric Point

PSI Photosystem I

xxi Definitions and Abbreviations

PSII Photosystem II

PSs Photosystems

PSU Photosynthetic Unit

Q Excitation pressure over PSII

RC Reaction Centre

RCs Reaction Centres rETR relative Electron Transport Rate

SDS-PAGE Sodium dodecyl sulfate poliacrylamide gel electrophoresis

STs State Transitions

WWC Water Water Cycle

σPSII PSII maximum absorption cross section

σPSII’ PSII effective absorption cross section

xxii Chapter 1

Chapter 1 General Introduction

1.1 Importance of coral reef ecosystems

Coral reefs are one of the most important ecosystems, they offer multiple ecological goods and services including high biodiversity, species refuges, coastal protection and biogeochemical cycling (Moberg & Folke 1999). Coral reefs can mainly be found in the oligotrophic coastal seawaters of equatorial zones where they have grown to form great structures such as the Caribbean Reef Belt and the Australian Great Barrier Reef (Stanley, 2003, 2006). Along the coastal lines at these zones, human populations exploit the coral reef resources to develop based on the tourism, fisheries and other livelihoods, sometimes exceeding the reef resilience capacity (Jameson et al. 2002, D’Angelo & Wiedenmann 2014). In addition to the anthropogenic impacts on coral reefs such as overfishing and pollution; the consequences of climate change such as the increases in temperature and ocean acidification are accelerating the deterioration of these ecosystems (Sebens, 1997). It has been suggested by studies of community ecology that current conditions affecting coral reef communities are producing many irreversible changes that may only leave them suitable to deliver some of their exploitative potential (Graham et al. 2014, 2015).

After the recent bleaching events in 2016 which was suggested to be equal with the worst bleaching event on record in 1998 (Hoegh-Guldberg 1999, Hughes et al. 2017), it has become urgent to increase efforts for the conservation of coral reefs and funding for scientific research in order to improve predictions on what will be the more probable consequences on the current scenarios affecting these ecosystems (Kleypas & Hoegh-Guldberg, 2005). Some recent projections based on how increases in temperature and ocean acidification would affect coral reefs, predict that in 40 years 85% of reef locations will be altered under the Intergovernmental Panel on Climate Change (IPCC) emissions scenario (Frieler et al. 2013, Hughes et al. 2017). An estimation of the lost value of coral reef ecosystems based on climate change scenarios ranges from US$3.95 to US$23.78 billion annually (Chen et al. 2015). It has been proposed carbon budgets in some coral reefs affected by the 2016 bleaching event will still remain positive by 2030 but only if measures for conservation of the resilient factors affecting positively carbon budgets are applied (Hooidonk et al. 2014). There have been proposed many different alternatives to impede global decline of coral reef ecosystems such as assisted evolution and conservation of deep coral reefs as refuges for reseeding of coral species in shallow waters (Kleypas & Hoegh-Guldberg, 2005). A deeper understanding of coral symbiosis is crucial to aid future persistence of coral reefs.

1 Chapter 1 1.2 The coral symbiosis

Massive extensions of coral reefs exist as a result of the obligate symbiotic relationship between scleractinian corals and dinoflagellate algae of the genus Symbiodinium. This symbiosis is based on the exchange of CO2 and nutrients that the coral supply to the zooxanthellae for the translocated carbohydrates produced by photosynthesis which support calcium carbonate deposition rates of the coral (Muscatine, 1980; 1990; Trench, 1993).

In coral symbiosis, the term “holobiont” refers to the functional unit formed by the association of the algal and the animal as the main contributors to the productivity of the symbiosis (Rowan, 1998). The biology of the holobiont implies the generation of a new metabolism and although no symbiotic genes have been found a recent study suggests based on RT-PCR (real time polymerase chain reaction) that H+ATPase (proton pump ATPase) could be used as a molecular marker to distinguish between symbiotic and non-symbiotic relationships between invertebrate organisms and Symbiodinium (Davy et al. 2012, Mies et al. 2017).

The ecological success of corals has been attributed to the carbon translocation of the Symbiodinium, which in some cases can reach up to 95% of photosynthetic products to the host (Muscatine 1980, 90). In addition, the high oxygen production through photosynthesis promotes the deposition of calcium carbonate required for the construction of the reef framework (Colombo- Pallotta et al. 2010, Wijgerde et al. 2014). However, it is also known that the coral host contributes to the efficiency of its Symbiodinium population by the light scattering of the coral skeleton (Enríquez et al. 2005, Marcelino et al. 2013), the concentration of carbon (Falkowski et al. 1993), the limitation of nutrients (Dubinsky & Jokiel 1994), the induction of carbon release (Gates et al. 1995) and the stimulation of photosynthesis (Barott et al. 2015). For this reason, the differential adaptations of different types of Symbiodinium (Ziegler et al. 2015), and different species of corals (Wicks et al. 2012), to environmental variation and their specificity to constitute a stable symbiotic relationship determines the holobiont distribution (LaJeunesse 2005, LaJeunesse et al. 2014).

Given the great importance of photosynthesis to feed the holobiont it has been postulated that photoacclimation of Symbiodinium to environmental variation is a determinant for the maintenance of the symbiosis (Chang et al. 1983, Iglesias-Prieto & Trench 1994, 1997, Robison & Warner, 2006; Hennige et al. 2009). Nevertheless, it has also been postulated that the shape of the coral host and its role to provide Symbiodinium with enough CO2 for the dark reactions of photosynthesis is also determinant to upkeep the holobiont (Wooldridge 2009, 2010, 2013, 2014, Hennige et al. 2010, Oakley et al. 2014a, b, Baker et al. 2015).

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The dynamic assembly of the holobiont may range in relation to the fidelity of the Symbiodinium and the capacity of the coral host to shuffle symbionts, both traits depending on the symbiont transmission mode and the nature of the symbiosis (Stat et al. 2006, Baird et al. 2007, Baker 2001, 2003; Baker et al. 2004; Cunning & Baker 2014). Obligate symbioses with vertical transmission modes would be more restricted to shuffle symbionts than facultative symbioses with horizontal transmission modes (Buddemeier et al. 2004, Baker 2001, 2003, Stat et al. 2006). Some holobionts harbour assemblages of multiple Symbiodinium types but the vast majority are dominated by one type whilst retaining others in small abundance (Goulet, 2006, Stat et al. 2006, Leggat et al. 2007, Baird et al. 2007, Wicks et al. 2012, Kemp et al. 2014, 2015, Silva-Lima et al. 2015).

Other aspects giving shape to the holobiont under the recent climate change scenario are the debilitated immunological response of corals after a bleaching event (Tchernov et al. 2011, Pinzón et al. 2015), the frontloading of genes determining the differential response to stressful conditions between corals of the same species (Barshis et al. 2013), the reduction of carbon input for growth and calcification after infection by opportunistic types of Symbiodinium (Pettay et al. 2015) and the contributions to the symbiosis from the surrounding microbiome (Rosenberg et al. 2007, Kelly et al. 2014, Rädecker et al. 2015, Pantos et al. 2015).

The recent availability of the complete genome sequences of Symbiodinium minutum, Symbiodinium kawagutti, Symbiodinium microadriaticum and Acropora digitifera has enabled the simultaneous analysis of the symbiont and the coral host and also the study by transcriptomic assembly of some aspects representative of the holobiont (Shoguchi et al.2013, Shinzato et al. 2014 a, b).

It has been hypothesised that coral symbiosis with different species of Symbiodinium is actually representing a continuum of interaction states from parasitism to mutualism (Lesser et al. 2013). Based on the evolutionary theory of symbiosis and studies on bacterial symbiosis with eukaryotic hosts, Lesser and collaborators (2013) proposed that this continuum of interaction states would be going from corals with horizontal transfer and facultative associated (parasitic) to those where the species of algae is vertically transferred and in an obligate symbiotic state (mutualistic), (Lesser et al. 2013). They also suggest that the phenotypic differences in the photo-biological characteristics between the different clades of Symbiodinium could be modulating the evolution of the parasitic to mutualistic continuum (Lesser et al. 2013; Figure 1).

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Symbiodinium Photosynthesis

Obligate Facultative Vertical transmission Horizontal transmission e.g. Clade C e.g. Clade A ↓CO fixation ↑CO2 fixation Symbiosis 2 ↑Translocation ↓ Translocation Mutualistic Parasitic

Coral Environment Host Factors Light & T°

Modified from Lesser et al. 2013

This hypothesis still needs to be probed extensively under different experimental conditions in order to establish a model to predict the future of corals under the actual climate change scenario (Lesser et al. 2013). The dynamic complexity of coral symbiosis demands further study including not only the photophysiology but also other aspects related to the establishment and maintenance of the relationship that may influence the holobiont capacity to cope with environmental stress in the urge to persist in the future climate changing conditions (Davy et al., 2012).

1.3 The Symbiodinum

Dinoflagellates belong to the alveolate kingdom and are eukaryotic unicellular flagellates which have been classified as algae or as protozoan. They have been found to be autotrophic and heterotrophic as well as free living (Amphidinium) forming part of the marine plankton but also endosymbionts (Symbiodinium) with various groups of invertebrate including corals (Trench 1993, Iglesias-Prieto, 1996, Stat et al. 2006, Dorrel & Howe 2015).

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The use of the ITS2 (Internal transcribed spacer), (LaJeunesse & Trench, 2000, LaJeunesse 2001) and other high resolution molecular markers (LaJeunesse & Thornhill 2011, Arif et al. 2014) to identify the type of Symbiodinium harboured by a coral host has demonstrated the great diversity of phylotypes within the group composed by nine clades designated from the A to I (Blank & Trench 1985a, b, 1986, Stat et al. 2006, LaJeunesse et al. 2014).

It is acknowledged that the biogeographic distribution of Symbiodinium is largely determined by environmental parameters linked to photosynthesis including light availability and temperature (LaJeunesse et al. 2010a, 2010b, Tonk et al. 2014).

The high efficiency of Symbiodinium photosynthesis is determined by the adaptations of its photosynthetic unit (PSU) to perform in the marine environment (Iglesias-Prieto 1996, Roth 2014). The photosynthetic unit of Symbiodinium includes two specific kinds of light harvesting complexes (LHCs), the peridinin, chlorophyll a protein (PCP), (Hofmann et al. 1996, Schulte et al. 2009) and the chlorophyll a, chlorophyll c2, peridinin protein complex (acpPC) (Hiller et al. 1993, Iglesias- Prieto et al. 1993, Iglesias-Prieto 1996). Recently it’s been found that the native form of acpPC is constituted by a 64-66kDa trimer and presents sequence variations, which may be related to Symbiodinium ecophysiological adaptations (Jiang et al. 2014). The discovery of new structural proteins of the PSU of Symbiodinium, their different organization and their diversity of phylogenetic origins are raising new questions about the photobiology and the diversity of the group itself (Reynolds et al. 2008; Boldt et al. 2012, Jiang et al. 2014, Dorrell & Howe 2015, Maruyama et al. 2015, Xiang et al. 2015). High variability of the PSU is also supported by chloroplast´s genome sequences of Symbiodinium minutum and Symbiodinium C3 revealing a high genomic plasticity (Voolstra et al. 2011, Barbrook et al. 2014; Mungpakdee et al. 2014, also see Dorrel & Howe 2015).

Photoacclimation studies on the PSU of Symbiodinium measuring oxygen evolution, differences in the protein complexes and variable fluorescence have shown that different types may present differences in the organization of photosystems (PSs) in the thylakoid membrane and in their association with the LHCs in response to environmental variation (Iglesias-Prieto & Trench 1994, 1997, Warner et al. 1999, Reynolds et al. 2008, Hennige et al. 2009, Ragni et al. 2010, Krämer et al. 2012, LaJeunesse et al. 2014).

A comprehensive study has suggested that the different photoacclimation strategies between the different phylotypes of Symbiodinium are consistent with differences in cell size and cladal designation and these differences may help explain their biogeographic diversity (Suggett et al. 2015). Also, high throughput sequencing assessment in four clades of cultured Symbiodinium has revealed the shared transcriptome between them which would include the fundamental proteins and

5 Chapter 1 metabolic pathways necessary for the stability of a successful holobiont, setting the basis to study the modification of these symbiotic processes in and ex hospite (Rosic et al. 2015). Proteomic studies achieving the isolation and characterization of the symbiotic proteins shared and unshared by different clades of Symbiodinium would reveal symbiotic metabolic pathways and processes related to changes in proteins due to their post-translational and allosteric modification that are not reflected in transcriptomic studies (Leggat et al. 2011, McGinley et al. 2013).

1.4 Host control of symbiosis

The holobiont metabolism including: nutrient transfer, carbon concentration, nitrogen recycling, calcification, oxidative stress and cell communication require multiple physiological adaptations in order to keep the homeostasis of symbiosis (Weber & Medina 2012).

Evidence suggests that a coral host exerts some control over these metabolic pathways to optimize the photosynthesis of the algal symbiont and its benefits to the holobiont by the expression of different sets of proteins (e.g. carbon transporters and carbon anhydrase to deliver and concentrate inorganic carbon and make it available to the alga to be fixed (Weis et al. 1989, Leggat et al. 1999, 2002, Rodriguez-Lanetty et al., 2006, Zoccola et al. 2015, see also for other processes regulated by the coral host Yellowlees et al. 2008, Meyer & Weis, 2012).

Several studies have demonstrated the coral host exerts control over the carbon translocation from the Symbiodinium by producing unspecific host factors which stimulate or inhibit the release of photosynthetic products as in the intact symbiosis (Muscatine & Hand 1958, Muscatine & Cernichiari 1969, 1972, Trench 1971, Muscatine 1967, Kempf 1984, Sutton & Hoegh-Guldberg 1990, Gates et al. 1995, Grant et al. 2001). This induction of carbon release occurs using crude homogenate of host tissue but can be mimicked with free amino acids (especially taurine), clotrimazole and osmotic shocks (Muscatine 1967, Muscatine & Cernichiari 1969, 1972, Trench 1971, Kempf 1984, Sutton & Hoegh-Guldberg 1990, Gates et al. 1995, Wang & Douglas 1997, Ritchie et al. 1997, Suescún-Bolívar et al. 2012). In every study with a host release factor (HRF) various organic compounds were exuded from the Symbiodinium including carbohydrates, lipids and amino acids but the most abundant were glycerol or glucose (Trench 1971, Muscatine 1967, Whitehead & Douglas 2003). Apparently, glucose is the main carbon product released to the host in hospite while glycerol seems to be the major one in vitro due to experimental stress (Burriesci et al. 2012). There is evidence that the HRF is an amino acid or polypeptide with low molecular weight and an acidic isoelectric point (Grant et al. 2013). Recent findings suggest a whole set of proteins synthesized by the host may be involved in the regulation of Symbiodinium metabolic pathways to optimize the net benefits of the symbiosis (Lehnert, et al. 2014, Barott et al. 2015).

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Supporting this, the transcriptomic analysis of aposymbiotic vs symbiotic Aiptasia anemones hosting Symbiodinium showed that more than 60 proteins were associated with the nutrient transport. In particular three transcripts were found for proteins related to the transport of glucose: i) the orthologs of GLUT8, ii) the putative Na+-glucose/myo-inositol co-transporter (both localized in the outer membrane of the interspace between the coral cell and the Symbiodinium usually known as symbiosome) and iii) a specific protein of Symbiodinium transporting glucose to the symbiosome (Lehnert et al. 2014).

In one previous proteomic based study conducted also in Aiptasia, 17 different proteins were isolated from the symbiosomes, 41% of these proteins were deemed to be of animal origin and 29% of algal origin. Following functional group analysis 6% of the proteins were membrane transporters including the ABC protein membrane transporter related to ATP-dependent trans-membrane transportation of multiple organic and inorganic molecules, Hsp70 which is related to protein modification during transportation and the enzyme PDI related to transportation of lipids (Peng et al. 2010).

In a later study using symbiotic gastrodermal cells (SCGs) isolated from the coral Euphyllia glabrescens nineteen proteins of cnidarian origin were identified some of them involved with the regulation of membrane transport (Li et al. 2014). Recently, Barott et al. 2015 demonstrated how the coral host expression of a vacuole proton like ATPase (VHA) acidifies the symbiosome lumen This, enables the transport of ions and nutrients and promotes Symbiodinium photosynthesis as shown by the reduction in the oxygen evolution of the coral by inhibition of the VHA activity. This study also suggests demonstration of continued acidification of the symbiosome lumen in the dark which may help explain the nutrient-independent elongation of algal cell cycle observed in symbiosis (Barott et al. 2015).

It is probable that the expression of the VHA ATPase, the ABC protein membrane transporter, the Hsp70 in conjunction with the expression of other transport membrane proteins and carriers are involved in the nutrient transfer during symbiosis. This regulation of the supply of inorganic nutrients and the translocation of carbon products must be supplemented by proteins expressed by the host to regulate at the same time other metabolic pathways of the holobiont. This proteome mediated metabolism must define the extent of control upon the productivity of Symbiodinium and the nature and maintenance of the symbiosis.

Recent development of some models to assess the energetics of the symbiosis in terms of benefits and losses for the coral’s fitness and the maintenance of the symbiosis under stress as occurs during bleaching have proposed some predictions emphasising the importance of nutrient exchange to sustain the holobiont (Cunning et al. 2017, Raharinirina et al, 2017). In regard of this a

7 Chapter 1 proteomic study conducted on bleached and unbleached colonies of Acropora palmata showed that the bleaching process lead to the unique expression of proteins involved in growth, detoxification, endo–exophagocytosis, and catabolic processes which comprehensively are related to the nutrient exchange and energy costs of the symbiosis (Ricaurte et al. 2016). Further studies should be conducted in order to corroborate which are the most highly expressed proteins under conditions where nutrient trafficking is high vs null such as in- hospite vs ex-hospite in order to describe the molecular mechanisms by which external environmental conditions affect the interaction between the coral host and its symbionts.

1.5 Breakdown of symbiosis

Coral reefs framework is determined by the different adaptations of the holobiont to cope with the regular environmental variation, occurrence of diseases and other natural disturbances (Connel 1978, Richmond 1993, Nyström et al. 2000). The breakdown of symbiosis produced by occurrence of the phenomenon of coral bleaching has evidenced the sensitivity of the holobiont to environmental stressors such as high irradiance and high temperature (Iglesias-Prieto et al. 1992, Brown 1997, Fitt & Warner 1995, Warner et al. 1996, Jones et al. 1998, Hoegh-Guldberg 1999, Tchernov et al. 2004, Dove et al. 2006, Apprill et al. 2007).

Coral bleaching is defined as the loss of zooxanthella pigments or the expulsion of the zooxanthellae population from the coral due mainly to temperature stress. This breakdown of the symbiosis drives the holobiont into a risk of mortality unless temperature returns to an optimum to allow recovery. However, massive bleaching has been reported to occur also with extreme low temperature, nutrient loading, mechanical disturbances and infections by pathogens. In addition, massive discoloration and whitening of coral tissue have been reported independently from coral bleaching (LaJeunesse et al. 2010a, Suggett & Smith 2011, Plass-Johnson et al. 2015, Cruz et al. 2015).

A short-term exposure of a few days to extreme environmental stress (in the case of temperature stress 1-2 C° above the summer maximum) may allow the recovery of the holobiont if conditions return to an optimum but long-term disturbances of several weeks may produce damage which if crosses the threshold for recovery would produce the total breakdown of symbiosis and mortality (Goreau & MacFarlane 1990, Suggett & Smith 2011). The threshold recovery may differ in relation to the adaptations of each component of the symbiosis and their interactive contribution to the fitness of the holobiont (Rowan & Knowlton 1995, Rowan et al. 1997, Rowan 1998, 2004, Fitt et al. 2001, Furla et al. 2005, Berkelmans & Oppen 2006, Leggat et al. 2011, Howells et al. 2012, Krueger et al. 2014, Oakley et al. 2014a, b).

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Coral symbiosis is a delicate bioenergetic trade-off between partners where cell communication and nutrient transfer are the most evident of many other symbiotic metabolic pathways dedicated to optimize the physiology of the holobiont, all kinds of extreme environmental stress could potentially interrupt any of these pathways breaking down the symbiosis (Jones et al. 1998, Smith et al. 2005, Weis 2010, Wooldridge 2009, 2010, 2013, 2014). This context gives a dynamic dimension to coral symbiosis as to how Symbiodinium types and species of coral hosts may be able to adapt to climate change and generate holobionts resilient to coral bleaching, diseases and other disturbances (Baird et al. 2007, Bellantuono et al. 2011, Downs et al. 2013). In relation to this the adaptive theory of bleaching suggests that the host may be able to shuffle their algal symbiont according with the environmental conditions (Baker 2001, 2003, Baker et al. 2004, Fautin & Buddemeier, 2004, Baird et al. 2007, Cunning & Baker 2014).

Evidence suggests the possible adaptation mechanisms of the coral and the symbiont may not be fast enough or may be negative for the holobiont (Douglas 2003, Sampayo et al. 2008, 2009 Kwiatkowski et al. 2015). Some types of Symbiodinium can adapt to high temperature but others can become parasitic to the host (Barshis et al. 2010, 2013, Lesser et al. 2013, Hume et al 2015, Howells et al 2016, Pettay et al. 2015). Research suggests some species of corals affected by bleaching become immunological weakened and more susceptible to bleaching within another subsequent event (Sampayo et al. 2008, 2009, Tchernov et al. 2011, Weston et al. 2012, 2015, Kemp et al. 2014, 2015, Pinzón et al. 2015).

Although great progress has been made in understanding coral bleaching the predictions on the future of coral ecosystems under threat of this and other disturbances such as viral and bacterial diseases are still obscure. The application of proteomics in studies of the holobiont is starting to unveil the molecular mechanisms and processes involved in cell communication and metabolic pathways between the alga and the animal cells as well as define different traits between phylotypes and clades in and ex hospite (Pasaribu et al. 2015, Oakley et al. 2016, Ricaurte et al. 2016). More proteomic studies addressing the holobiont response to environmental change will help understand what forces drive the maintenance of the symbiosis.

1.6 Proteomics

Proteomics is an omic’s discovery science technology that is recently becoming a more accessible resource to study the protein expression (proteome) of cells in a determinate set of conditions (Aebersold & Watts 2000, Dunn 2014). In comparison with genomics the complexity of biological systems can be extensively approached by the inherent information of the proteome (Rabilloud et al. 2010). The dynamic expression of proteome allows comparisons in protein

9 Chapter 1 sequences, activities, structures, abundances, etc., in the same cell type at different treatments (e.g. LL vs HL, Souchelnytskyi 2005, Tyers & Mann 2003).

Different methods to separate complex mixtures of proteins from cell lysates have been developed but the most commonly used are electrophoresis and mass spectrometry (Palcy 2006). These methods present advantages and limitations and many studies use both in order to isolate and characterize the key differences in protein expression (Wang et al. 2014). Two-dimensional electrophoresis (2-DE) separates the proteins from cell lysates by their different molecular weight and isoelectric point (Herbert 1999). Mass spectrometry measures in an ion trap the mass to charge ratio m/z of ion fragments of a protein sample and generates mass-spectra. Both methods allow protein identification using search algorithms in databases. Usually proteins separated by 2DE are digested with trypsin and analysed using matrix assisted laser desorption ionization (MALDI) coupled with time of flight (TOF) mass analysers and the spectra obtained can be compared with sequence databases to identify the proteins (Gharahdaghi 1999, Gerber et al. 2003, Fenn et al. 1990, Imai & Mische1999, Figure 2).

Alternatively, the combination of liquid chromatography and mass spectrometry or LC- MS/MS shotgun proteomics can be used to analyse the proteome of cell lysates after pre- fractionation of the sample by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), (Davis et al. 2001, Figure 2). This system can generate hundreds of thousands of peptides from samples previously digested with trypsin. A caveat to this method is that the pre-fractionation of the sample (e.g. by SDS-PAGE) is required for the detection and analysis of the peptides by the collision-induced dissociation (CID) mass spectrometry technique (Michalski et al. 2011, Lee et al. 2004, Brun et al. 2009).

In spite of the efficiency of these methods for protein analyses, there is not yet a full proteome described for any species (Kim et al. 2014). The improved power of omic’s science technologies is becoming a more common resource to generate new information regarding coral symbiosis and its biological components (Pernice et al. 2015). There has recently been a lot of advances since the first omic’s studies applied to coral symbiosis (for a review see: Meyer & Weis 2012). Currently there are available full genome sequences of various species of scleractinian corals, the model organism for coral symbiosis Aiptasia, and three species of Symbiodinium (Shoguchi et al. 2013, Baumgarten 2015, Lin et al. 2015, Aranda et al. 2016,). As well there have been produced transcriptomic assemblages for the four major Symbiodinium clades symbiotic with scleractinian corals (Rosic et al. 2015, Xiang et al. 2015).

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Proteomics has been widely applied to marine dinoflagellates along many fields of study and also some proteomic research has been done related to Symbiodinium and coral symbiosis (Table 1).

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Table 1. Summary of proteomic studies in marine dinoflagellates from a literature search. Modified from Wang et al. (2014) to include Symbiodinium and symbiosis studies. Field Species Protein MS platform Database Reference separation method Symbiodinium Euphyllia glabrescens 2-DE LC–nanoESI-MS/MS NCBInr Peng et al. 2008 Stylophora pistillata 1-DE LC–MS/MS uniprot_sprot_100713 Weston et al. Euphyllia glabrescens 2-DE LC–MS/MS and LC– 2012 nanoESI-MS/MS NCBInr Clade B from Exaiptasia 1-DE MALDI-TOF MS/MS Li et al. 2014 pulchella NCBInr Pasaribu et al. 2015 Coral Bleaching Acropora microthalma 1-DE LC–MS/MS Uniprot Weston et al. Acropora palmata 2-DE MALDI-TOF MS/MS NCBInr 2015 Ricaurte et al. 2016 Cnidarian Aiptasia pulchella 1-DE LC–nanoESI-MS/MS NCBInr Peng et al. Symbiosis Aiptasia sp. SDC LC-ESI-MS/MS Swiss-Prot, Uniprot and 2010 NCBInr Oakley et al. 2016 Protein Prorocentrum triestinum 2-DE - - Chan et al. preparation 2002 and Alexandrium sp. 2-DE MALDI-TOF-MS NCBInr Wang et al. identification Alexandrium spp. and 2-DE MALDI-TOF-MS NCBInr and 2009 Scrippsiella spp de novo sequence Lee et al. 2008 Alexandrium tamarense 2-DE MALDI-TOF-MS NCBInr, Wang et al. dinoflagellate EST, 2011 and de novo sequence Cell growth Prorocentrum triestinum 2-DE MALDI-TOF-MS NCBInr, Protein data Chan et al. regulation and N-terminal bank, and Swiss-prot 2004 amino acid Sequence Alexandrium catenella 2-DE MALDI-TOF-MS NCBInr Wang et al. Lingulodinium polyedrum 2-DE LC–MS/MS NCBInr and 2012 Swiss-prot Akimoto et al. Prorocentrum donghaiense 2-D DIGE MALDI-TOF-MS NCBInr, 2004 dinoflagellate EST, Wang et al. and de novo sequence 2013

Environmental Prorocentrum triestinum 2-DE MALDI-TOF-MS NCBInr, Protein data Chan et al. stresses and N-terminal bank, and Swiss-prot 2004 amino acid Sequence

Alexandrium catenella 2-DE MALDI-TOF– NCBInr Wang et al. TOF-MS 2012 Karenia mikimotoi 1-DE MALDI-TOF– NCBInr Lei et al. 2011 TOF-MS and LC–MS/MS Alexandrium affine 2-DE MALDI-TOF-MS NCBInr and Lee et al. 2009 and LC–MS/MS de novo sequence Karenia mikimotoi 1-DE MALDI-TOF– NCBInr Lei et al. 2011 TOF-MS and LC–MS/MS Prorocentrum micans 2-DE MALDI-TOF-MS NCBInr, Swiss-Prot Shim et al. and TrEMBL 2011

Cell wall Alexandrium catenella 2-DE MALDI-TOF– NCBInr and Minge et al. and cell TOF-MS de novo-sequence 2010 surface Alexandrium catenella DH01 2-D DIGE MALDI-TOF– NCBInr and Wang et al. proteins Alexandrium affine and TOF-MS dinoflagellate EST 2011 Alexandrium tamarense 2-DE N-terminal amino Protein data bank Li et al. 2012 acid sequence and Swiss-prot

Note: 1-DE, one dimensional electrophoresis; 2-DE, two-dimensional electrophoresis; 2-D DIGE, two-dimensional fluorescence difference gel electrophoresis; EST, expressed sequence tag; NCBInr, National Center for Biotechnology Nonredundant; MALDI-TOF MS, matrix-assisted laser desorption–ionization time-of-flight mass spectrometry; MALDI-TOF–TOF MS, matrix-assisted laser desorption–ionization time-of- flight/time of flight mass spectrometry; ESI MS, electrospray ionization mass spectrometry; LC, liquid chromatography.

Proteomic work has produced some interesting results in fields regarding Symbiodinium in hospite. Isolation of pure symbiosomes from the sea anemone Aiptasia pulchella used as a model

12 Chapter 1 for coral symbiosis, allowed the identification of 17 membrane surface proteins, has clarified some of the possible mechanisms of remodelling, transport, recognition, signalling and stress control related with the maintenance of the symbiosis (Peng et al. 2010).

Proteome from gastrodermal cells of the coral Euphylia glabrescens displayed 19 proteins linked with remodelling, energy metabolism and stress response (Li et al. 2014). The Symbiodinium proteome from partially thermal bleached coral Styllophora pistillata suggested the self-mediated expulsion of the zooxanthellae and also the high susceptibility of the coral to viral disease during bleaching (Weston et al. 2012). Evidence of a self-mediated expulsion mechanism during thermal bleaching was also found in the proteome from the coral Acropora micropthalma (Weston et al. 2015).

Comparison of the proteome from endosymbiotic and cultured Symbiodinium from Exaiptasia pulchella displayed differences in transcription and translation factors besides photosynthesis and energy metabolism (Pasaribu et al. 2015) A coral bleaching model based on the proteome from bleached and unbleached Acropora palmata proposed recently that bleaching affects coral growth, detoxification, catabolism and endo-exophagocytosis and interestingly is involved in a dual function of a P700 apoprotein (Ricaurte et al. 2016).

In Aiptaisia sp. symbiosis the proteome of symbiotic cnidarian is enriched in transport and nutrient cycling proteins vs aposymbiotic organisms, which seem to produce more proteins related to oxidative stress (Oakley et al. 2016).

The advantages of proteomics over transcriptomics to characterize phenotypical differences in specific conditions or among types and subtypes produced by post-translational control could be used to asses more comprehensively unresolved hypothesis including the functional diversity of Symbiodinium in coral symbiosis and the role of host control under different environmental conditions. This thesis focuses on cultures of three types of Symbiodinium (A1, A13 and C1) which belong to two of the most widely distributed clades across corals of the world; clade A in the Caribbean and clade C in the Indopacific. The metabolism of the holobiont is supported by the photosynthesis of Symbiodinium, the different proteome and photophysiology across types compared at different environments and using different host factors could shade light on the physiological controls of the different symbiotic relationships. The main aim of this study is to understand how the environment (light and temperature) and the host (host factors) exert control over the proteome of Symbiodinium and how this is related to the physiology and maintenance of the symbiosis.

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1.7 Thesis Hypothesis

If the metabolism of the holobiont is supported by the photosynthesis of Symbiodinium then the differences in the regulation of the photophysiology and proteome across types at different environmental conditions could be the foundation of the differences in the physiology of different symbiotic relationships. Thus, the following hypotheses were tested on this thesis: H1: If the light environment drives the differential photophysiology of different types of Symbiodinium, then at different photon fluxes their photoacclimation related proteome would be differentially regulated across types revealing their diverse physiological role in the symbiosis. H2: If different host release factors stimulate or inhibit differentially the release of glucose of different Symbiodinium types then this translocation will be regulated by a differential photophysiology and proteome across different types of Symbiodinium revealing the mechanisms of glucose translocation and their differential contribution to the symbiosis. H3: If high temperature impacts photoacclimation differentially across Symbiodinium types then the acclimation to high temperature will be regulated by differences in the photophysiology and proteome across these types revealing their role in the maintenance of the diverse symbiotic relationships. H4: If different types of Symbiodinium have different tolerances to temperature stress then these differences will be regulated by differences in the photophysiology and proteome across these types revealing their role in the maintenance of the diverse symbiotic relationships.

1.8 Thesis Objectives

The overall objectives of the research presented in this thesis were as follows: Determine the mechanisms behind the regulation of the proteome and photophysiology across different types of Symbiodinium ex-hospite after their acclimation at different environmental conditions, specifically: 1. Characterise the differences in photophysiology (growth rates, Chl a concentration, absorbance and fluorescence) and the protein expression (mass spectrometry and 2-DE analyses) across three cultured types of Symbiodinium after three months acclimation at two photon fluxes (HL= 350 µmol photons•m2•s-1 and LL= 100 µmol photons•m-2•s-1).

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2. Characterise the differences in the protein expression (SDS-PAGE and MALDI- TOF analyses) and the photophysiological parameters: chlorophyll a concentration, the excitation pressure over photosystem two (PSII), total protein, total glucose and glucose released; across three cultured types of Symbiodinium acclimated at two photon fluxes (HL= 350 µmol photons•m2•s-1 and LL= 100 µmol photons•m-2•s-1), after exposure for one hour to a free amino-acid mix and a coral tissue host release factors. 3. Characterise the differences in photophysiology (growth rates, Chl a absorbance and fluorescence parameters) and the protein expression (mass spectrometry and 2- DE analyses) across three cultured types of Symbiodinium acclimated at two photon fluxes (HL= 350 µmol photons•m2•s-1 and LL= 100 µmol photons•m-2•s-1), after acclimation for ten months at two temperatures (26 ºC and 30 ºC). 4. Characterise the differences in photophysiology (Chl a fluorescence) and the protein expression (mass spectrometry and 2-DE analyses) across three cultured types of Symbiodinium acclimated at two photon fluxes (HL= 350 µmol photons•m2•s-1 and LL= 100 µmol photons•m-2•s-1) and at two temperatures (26 ºC and 30 ºC), after being subjected to constant high temperature stress at a temperature of 33 ºC for three days.

15 Chapter 2 Chapter 2 Photoacclimation of Symbiodinium: a proteomic approach

Abstract

The ecological success of coral reefs has been attributed to the obligate relationship between scleractinian corals and symbiotic dinoflagellates of the genus Symbiodinium. The high diversity of Symbiodinium demonstrated by high-resolution molecular markers has led to hypothesis over the functionality of different types to establish symbiosis with different species of corals. Evidence coming from photobiological studies investigating the functional diversity of Symbiodinium, has shown that photoacclimation occurs among types using different strategies. Although this difference in strategies seems to account for the variable persistence of symbiosis among corals this still can’t fully explain the physiology of its maintenance. Recently, omic’s science technologies are bringing new insights into the mechanisms behind the differential duration of the symbiosis. However, in spite of recent availability of full genome sequences for some species of corals and Symbiodinium, environmental experiments looking for patterns of differential expression using transcriptomic and genomic analysis suggest most proteins related with photoacclimation are controlled by posttranscriptional and posttranslational modifications. The use of proteomics is delivering some promising results to explain better the mechanisms of photoacclimation related with the symbiotic state. The present study is dedicated to the photoacclimation proteome of Symbiodinium examining three types (A1, A13 and C1) belonging to the widest distributed clades in the Caribbean (A) and in the Indopacific (C). An initial assessment of the proteome regulation using shotgun proteomics over Symbiodinium type C1 cultured at two experimental photon fluxes displayed differences in proteins associated with light harvesting, electron transport, carbon fixation and protein modification. A two- dimensional gel electrophoresis (2-DE) analysis between the three Symbiodinium types cultured at the two photon fluxes revealed unique proteome differences across these types and between both experimental light treatments. Database searches tagged these proteins also associated with the same processes matched by previous shotgun analysis over C1 type alone. These results are in agreement with previous hypothesis on different photoacclimation strategies displayed among different types of Symbiodinium. These strategies involve differential protein modifications in the PSU to achieve homeostasis at different photon flux environments resembling differential use across types of alternative electron transport pathways and photoprotection mechanisms. These findings are discussed in relation to the different photophysiologies across types of Symbiodinium to photoacclimate to different light environments and how these differences help understand the stability of the reef holobiont.

16 Chapter 2 2.1 Introduction

The mutualistic symbiosis between corals and dinoflagellates of genus Symbiodinium drives the productivity of reef building corals (Muscatine 1980, 1990). Biogeographic distribution of Symbiodinium is largely determined by environment and mainly by factors effecting photosynthesis such as light availability (LaJeunesse & Trench 2000; LaJeunesse et al. 2010b, Tonk et al. 2014). The high variability of light in the ocean has shaped the diversity of Symbiodinium enabling the differential plasticity to photoacclimate their photosynthetic machinery and establish symbiosis with many species of corals (Henninge et al. 2010, Jeans et al. 2013, Hawkins et al. 2015, Schrameyer et al. 2016).

The efficiency of Symbiodinium photosynthesis is determined by adaptations to its PSU, specific to environmental conditions experienced within marine systems (Iglesias-Prieto 1996, Roth 2014). The photosynthetic unit of Symbiodinium includes two specific kinds of light harvesting complexes (LHCs), the peridinin, chlorophyll a protein (PCP, Hofmann et al. 1996,

Schulte et al. 2009) and the chlorophyll a, chlorophyll c2, peridinin protein complex (acpPC, Hiller et al. 1993, Iglesias-Prieto et al. 1993, Iglesias-Prieto 1996). Recently it has been found that the native form of acpPC is constituted by a 64-66kDa trimer and presents sequence variations, which may be related to Symbiodinium ecophysiological adaptations and survival in different light environments (Jiang et al. 2014).

The discovery of new structural proteins of the PSU of Symbiodinium, their different organization and its diversity of phylogenetic origins are raising new questions about the photobiology and the diversity of the group itself (Reynolds et al. 2008; Boldt et al. 2012, Jiang et al. 2014, Dorrell & Howe 2015, Maruyama et al. 2015, Xiang et al. 2015). The high variability of the PSU is also supported by chloroplast´s genome sequences of Symbiodinium minutum, Symbiodinium kawagutti, Symbiodinium microadriaticum and Symbiodinium C3 revealing a high genomic plasticity (Barbrook et al. 2014; Mungpakdee et al. 2014, also see Dorrel & Howe 2015).

Research assessing photoacclimation mechanisms in the PSU with techniques such as oxygen evolution, biochemistry of protein complexes and variable fluorescence of chlorophyll a, have shown that the reaction centres among types of Symbiodinium are relatively functionally conserved, however there may be differences on their interactions with the LHCs in response to environmental variation (Iglesias-Prieto & Trench 1994, 97, Warner et al. 1999, Reynolds et al. 2008, Suggett et al. 2008, Hennige et al. 2009, Ragni et al. 2010, Krämer et al. 2012, LaJeunesse et al. 2014).

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A study by Suggett et al. (2015), suggested that the different photoacclimation strategies used by different types of Symbiodinium are consistent with cell size and cladal designation and these differences may help explain their biogeographic distribution (Suggett et al. 2015). Also, high throughput sequencing assessment of cultured Symbiodinium belonging to the four major clades that are symbiotic with scleractinian corals has revealed the shared transcriptome with the fundamental proteins and metabolic pathways necessary for the stability of a successful symbiosis, opening the question how these proteins and metabolic pathways are regulated within the holobiont (Rosic et al. 2015).

As proteomics is a useful tool to track posttranslational changes in protein expression, studies focused on isolating and characterising symbiotic proteins that are both shared and unshared by different types of Symbiodinium and also related to acclimation processes could reveal symbiotic mechanisms that are not reflected in transcriptomic studies (Leggat et al. 2011, McGinley et al. 2013, Xiang et al. 2015). One such study highlighted the implications of an apoprotein associated with the reaction centre of PSI, possibly a LHC, which has been proposed as a marker of stress in corals (Ricaurte et al. 2016). Further studies are needed that clearly define and separate between the mechanisms of host control compared to the mechanisms controlled by the symbiont only. These studies would provide a deeper understanding of how homeostasis is maintained within a holobiont that experiences fluctuating environmental conditions.

Therefore, this study is aimed at revealing the response of the Symbiodinium proteome across different light environments. Assuming Symbiodinium photosynthesis fuels the symbiosis, photoacclimation warranties sustained photosynthetic rates, translocation of carbon to the host and nutrients back for the population of symbionts. Exploring the regulation of Symbiodinium proteome during photoacclimation at different photon flux regimes can reveal important ways the external environment and/or host may control symbiont’s proteome to sustain the holobiont and also how different types of symbionts may represent more benefit as to say being more mutualistic than others for symbiosis. This may be revealed by comparison of the physiologically related proteome differences displayed between these symbionts at these different lights and host-influenced environments.

2.2 Hypothesis

If the photon flux drives the differential photophysiology of different types of Symbiodinium, then at different light environments their photoacclimation related proteome would be differentially regulated across types revealing their diverse physiological role in symbiosis

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2.3 Objective

Characterise the differences in photophysiology (growth rates, Chl a concentration, absorbance and fluorescence) and the protein expression (mass spectrometry and 2-DE analyses) across three cultured types of Symbiodinium after three months acclimation at two photon fluxes (HL= 350 µmol photons•m2•s-1 and LL= 100 µmol photons•m-2•s-1).

2.4 Materials and Methods

2.4.1 Cultures

The experiments were carried out on symbiotic dinoflagellates of the genus Symbiodinium belonging to the widest distributed clades in the Caribbean (A) and in the Indopacific (C). More precisely types A1, A13 and C1 as previously characterized by analysis of the ribosomal ITS2 sequence (LaJeunesse 2001). Type A1 was previously described as Symbiodinium microadriaticum, originally isolated from the jellyfish Cassiopeia xamachana (Freudenthal 1962). Type A13, described as Symbiodinium necroappetens was originally isolated from the sea anemone Condylactis gigantean. Finally, type C1 shares the same ITS2 with Symbiodinuim goreaui was isolated from the corallimorph Discosoma sanctithomae (Robison & Warner 2006, et al. 2015, Ragni et al. 2010, Krämer et al. 2012, Suggett et al. 2015, Yuyama et al. 2016).

Batch cultures of the three Symbiodinium types were maintained in 2L Durand flasks on 1L of ASP-8A culture media (Blank 1987) at 26° C on a 12:12 light:dark cycle in a climate controlled room under two PFs (HL= 350 µmol photons•m2•s-1 and LL= 100 µmol photons•m-2•s-1) previously measured with a 4π light sensor (Li-Cor 193SA, Lincoln, NE, USA) submerged on ASP-8A within the Durand flasks. Cultures were maintained under constant aeration and stirring to avoid the aggregation of the algal cells and its attachment to the flask’s glass walls.

2.4.2 Growth

Growth rates of the three Symbiodinium types were calculated by consecutive cell counts from 1ml aliquots of culture fixed with 10µl of a Lugol´s solution. Cell counts were made every second day using an improved haemocytometer until cultures reached the end of the exponential phase and the beginning of the stationary phase. New cultures were initiated by diluting with fresh media to reach 50000cell·ml-1 and this process was repeated at least three consecutive times per phylotype. The specific growth rate (µ, day-1) was calculated by applying the formula:

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µ=ln2/r

After fitting the cell counts to a logistic sigmoidal function of the form:

y= α/(1+exp(-(β-x0)/γ)

Were α is the horizontal asymptote or maximum growth, β is the inflection point or the half point of the growth curve and γ represents the steepness at inflection or in this case the doubling day (r in the upper formula).

2.4.3 Photochemistry

Cultures were sampled during the exponential phase of growth. Aliquots of 25-50 ml were pelleted by centrifugation for 5 min at 2500rpm and resuspended in 1ml of ASP-8A media. The absorbance of the live cells was recorded from 350-750nm using a U-3000 spectrophotometer fitted with an integration sphere to minimize the scattering (ø60, Hitachi High-Technologies, Berkshire, UK). Alternative samples were also pelleted and suspended in 1ml of 100% methanol to extract the pigments and stored at -20°C overnight. The absorbance spectra of methanol pigment extracts were recorded also from 350-750nm. The chlorophyll concentration from the methanol extracts was calculated from the equations of Ritchie (Ritchie et al. 2006). The chl a specific absorption coefficient a* was calculated from the formula:

a*= (D/ρ) ln10

Where ρ is the chl a concentration (mg·m-2) and D the optical density from 400 to 700 nm (Enríquez et al. 2005).

Fluorescence measurements to obtain the PSII maximal photochemical efficiency of dark acclimated samples (Fv/Fm), the PSII effective photochemical efficiency of samples under actinic light or the acclimation irradiance (Fq´/Fm´), PSII maximum absorption cross section of dark acclimated samples (σPSII) and PSII effective absorption cross section under actinic light or the

PSII acclimation irradiance (σPSII´) for calculations of PSII-relative-rate of electron transfer (rETR ) were made using a Fast Repetition Rate Fluorometer (Chelsea Instruments, West Molesey, UK). Samples of 3ml of culture were dark acclimatized for 30 minutes before being flashed to achieve PSII single turnover saturation with 100 flashlets of 1.1 µs applied at 1-s intervals (Suggett et al. 2003, 2004).

Rapid light curves (RLC) were made applying the same PSII single turnover saturation pulse as for dark-adapted single acquisitions. Samples were dark acclimatized for 30min and then light

20 Chapter 2 saturated every 20s intervals during a 3 min exposition to each of 12 increasing actinic lights (0, 6, 22, 55, 105, 171, 295, 505, 750, 996, 1263, 1454 and 1646 µmol photons•m-2•s-1). Calculations of the RCII-specific-rate of electron transfer were made with the formula:

rETRPSII = PPF · Fq′/Fm′ · 0.42

Where rETRPSII is the PSII-relative rate of electron transfer, PPF is the photosynthetically active photon flux density (µmol photons•m-2•s-1) and the factor 0.42 assumes a constant value of 84% of total light absorbed of which only half is transferred to PSII for photochemistry (Schreiber et al. 2012).

2.4.4 Proteomics

2.4.4.1 Protein extraction

For protein extraction, 200 ml of culture of the three experimental types of Symbiodinium at the end of the exponential phase were harvested and concentrated by centrifugation for 5 min at 2500 rpm. The protein extraction was made as described in Li et al. (2012). After Trizol reagent (1mL) was added to the cell pellet, was subsequently sonicated on ice. Then 200µL of chloroform were added to the lysate before shaking vigorously for 15 s. The mixture was allowed to stand for 5 min at room temperature before centrifugation at 12000rpm for 15 min at 4°C. The top pale-yellow to colourless layer was removed, 300µL of ethanol were added to suspend the reddish bottom layer and the mixture was centrifuged at 5000rpm for 5 min at 4°C. The supernatant was transferred to a new tube and 2 mL of isopropanol were added. Then, the mixture was stored at −20°C for at least 1 h to precipitate proteins. The mixture was then centrifuged at 14000rpm for 30 min at 4°C and the recovered pellet was briefly washed with 95% ethanol and then air dried.

2.4.4.2 Protein determination and Iso-Electro-Focusing (IEF) as 1st dimension

Protein concentration from the sample was determined by the Bradford assay (Bradford 1976). For first dimension focusing, 40ug of protein were suspended in rehydration buffer containing 8 M urea, 4% w/v CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate), 65mM DTT (dithiothreitol), 0.5 % v/v IPG (immobilized pH gradient) buffer and 2% v/v DeStreak reagent. Bio-Rad 7 and 11cm long IPG strips of linear pH gradient of 3–10 and 4-7, were passively rehydrated overnight in 125 or 200µl of rehydration buffer respectively and were covered with mineral oil to avoid evaporation and precipitation of the protein sample. IEF was performed in the following manner: 50V-125Vh, 100V-150Vh, 200V-300Vh, 500V-350Vh, 1000V-750Vh gradient mode and 4000V-16000Vh, 8000V-18000Vh hold mode.

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2.4.4.3 Equilibration

After focusing, the IPG strips were placed in a petri dish or a screw-cap tube. The IPG strips were immersed in 10 mL of equilibration buffer containing 64 mM DTT, 6 M urea, 50 mM Tris pH 8.8, 30% v/v glycerol, 2% SDS and a trace of bromophenol blue and placed on an orbital shaker for 15 min. The first equilibration buffer was decanted and the strips were immersed in 10 mL of equilibration buffer but instead of DTT with 260mM iodoacetamide and placed for another 15 min on an orbital shaker.

2.4.4.4 Second dimension electrophoresis 2-DE

The 2-DE was performed using 12% or 10-14.5% polyacrylamide gels (Bio-Rad). After equilibration, the IPG strips were rinsed 3 times with running buffer and then placed on top of the resolving gel. The top of the second-dimension gels with the strip was overlaid with 0.5% agarose in running buffer. The gel was run at 50V for 30 min and 200 V for 1 h. Proteins after 2-DE were visualized by silver staining.

2.4.4.5 Protein visualization by silver staining

The fixation with silver staining of the proteins embedded in the gel was made as described by Chan et al. (2004). Gels were fixed for 2 h initially in a solution containing 40% v/v ethanol and 10% v/v acetic acid. Gels were then sensitized in a solution containing 30% v/v ethanol, 0.2% w/v sodium thiosulfate, 6.8% w/v sodium acetate and 0.125% v/v glutaraldehyde for 30 min, followed by washing with distilled water (3 times for 5 min each). After this the gel was stained for 20 min in 0.25% w/v silver nitrate with 0.015% v/v formaldehyde before washing with distilled water again (twice for 1 min each). The gel was finally developed in 2.5% w/v sodium carbonate containing 0.0074% v/v formaldehyde. The reaction was stopped with 1.5% w/v EDTA. The Gel was scanned and the analysis of the protein spots pattern was performed with Progenesis SameSpots vers. 2.0 software (Non Linear Dynamics, Newcastle, England).

2.4.4.6 Protein Identification

Measurement of molecular weights and isoelectric points of the protein spots separated by 2-DE were made using Image J program (Schneider et al. 2012). After measurement of the molecular weights (MW) and isoelectric points (pI) of the selected proteins these two parameters were submitted for identification online using the TagIdent tool available in ExPASy bioinformatics resource portal (Artimo et al. 2012). Searches within TagIdent tool were made looking for protein sequences with cysteines in reduced form (-SH). Searches in the classification field were made using the taxa “Symbiodinium”, “Dinophyceae”, “Alveolata” or “Apicomplexa”. When

22 Chapter 2 taxonomical searches with the classification field did not match any protein the keywords “photosynthesis” or “chloroplast” were used. Each search was made amplifying or reducing the percentage range of MW and pI depending on the number of matches obtained. Finally, the protein chosen was the one that most closely matched the pI and MW measured from the gels and belonged to the most closely related taxa with Symbiodinium.

2.4.5 Statistical analyses

Fit of cell counts to the logistic sigmoidal model for the calculation of growth rates, the examination of data distribution, descriptive statistics and analysis of the variances of the data were made using R (Development Core Team 2008). To test for normality on data sets Shapiro-Wilk normality test was applied. Linear models and ANOVA tests were applied to look for significant differences among groups and interaction of factors. For homogeneity of variance Levene’s Test was performed. Post hoc Tukey test were performed to determine which groups were significantly different. Whenever data sets violated assumptions of normality, data were log, square root or arcsine transformed or if still not normally distributed or variance still not homogenous, non- parametric (Kruskal Wallis test) analyses were performed to test for significant differences between and within groups and pairwise comparisons using Conover test for multiple comparisons of independent samples was used when required to find out the significant different groups. When necessary Bonferroni adjustment for confidence intervals was applied.

23 Chapter 2 2.5 Results

2.5.1 Growth

In this study comparisons showed that the three experimental types did not present significant differences in specific growth rates (µ day-1) between the two PFs nor across types. Type A13 had the highest specific growth rate in low light (LL) while A1 exhibited the highest growth rate in high light (HL). The cell counts after fitted to a sigmoidal logistic equation showed that cultures at HL reached the stationary phase faster than cultures at LL (Figure 3).

PF Type µ(day-1) (±SE) Chla (pgcell-1) (±SE)

A1 0.18 0.04 0.35 0.05

LL A13 0.21 0.06 0.38 0.03

C1 0.19 0.05 0.64 0.1

A1 0.23 0.037 0.11 0.008

HL A13 0.17 0.019 0.1 0.01

C1 0.16 0.012 0.1 0.015

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350 µ350µmolphotonmol photons m^2 s^ m−-12s-1 100 µ100µmolphotonmol photons m^2 s^− m1 -2s-1 1500000 C1LL A13LL C1HL A1LL A13HL A1HL 1000000 1 - 1 − Cell•ml Cell...ml 500000 0

0 10 20 30 40 0 10 20 30 40 Time (days) Time(days) Time (days) Time(days)

2.5.2 Photophysiology

The three types of Symbiodinium explored in this study, exhibited significant differences in Chl a concentration (pgcell-1) when compared between photon fluxes (H(1)=12.8, p<0.001). Looking deeper into the differences across Symbiodinium types, the Chl a concentrations were significantly different (H(5)=14.4, p=0.013). Pairwise Conover’s test for multiple comparisons of independent samples showed that Chl a concentration of type C1 at LL was significantly higher than concentration of the other two types at both PFs with exception of A13 type. Types A1 and A13 at LL had a similar Chl a concentration between each other but their concentrations were significantly higher than cultures of the three types at HL. All three types were similar in Chl a concentration at HL (Table 2, Figure 4).

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0.6 ) 1 1) - − ab b 0.4 (pgcell Chl a (pg...ml a

Chl 0.2

c c c

0.0

A13HL A1HL C1HL A13LL A1LL C1LLA13.HL A1.HL C1.HL A13.LL A1.LL C1.LL

Figure 4. Chlorophyll a concentration among cultures of three types of Symbiodinium cultured at two PFs (HL=350µmol photons•m-2•s-1, white bars or LL=100 µmol photons•m-2•s-1, grey bars).

Comparisons of Chl a* specific absorption coefficients showed only cultures of type A1 at HL were significantly higher than all the other cultures at both LL and HL including also cultures of type A1 at LL clade either between light treatments and compared with the other types (H(5)=14.754, p=0.011, Figure 5).

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350 µ350µmolphotonmol photons m^2 s^− m1 -2 s- 100 µ100µmolphotonmol photons m^2 s^m−-21 s-1 1 0.06

A13 A13 0.05 A1 A1

C1 C1 ) 1 - 0.04 a 1) − hl c mg 0.03 2 (m a*(m^2 mg chla a* 0.02 0.01 0.00

450 500 550 600 650 450 500 550 600 650 Wavelength (nm)Wavelenght(nm) Wavelength (nm)Wavelenght(nm)

Comparisons of PSII photochemical efficiency Fv/Fm displayed significant differences between types and also between both PFs (H(5)=495, p<0.001). Pairwise comparisons showed that Fv/Fm in cultures of type C1 at LL was significantly higher than the Fv/FM displayed by the other two types Fv/Fm at both PFs (p<0.001). At HL A1 Fv/Fm was significantly higher compared with this parameter on the other two types at HL (p<0.001, Table 3, Figure 6). Also comparisons of PSII absorption cross section σPSII showed significant differences between types and PFs (H(5)=294, p<0.001). Pairwise comparisons of PSII absorption cross section σPSII showed a pattern among types and PFs inverse to F v/Fm. Type C1 σPSII was significantly lower than in all types at both

PFs (p<0.001). At HL A1 σPSII was significantly lower compared with this parameter on the other two types at HL (p<0.001,Table 3 Figure 7)

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0.45

0.40 Fm Fv/Fm / Fv

0.35

0.30

A13.HL A1.HL C1.HL A13.LL A1.LL C1.LL A13HL A1HL C1HL A13LL A1LL C1LL Culture Treatment

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4.0

) 3.6 2 (nm ..PSII(nm2) ..PSII(nm2) σPSII

3.2

2.8

A13.HL A1.HL C1.HL A13.LL A1.LL C1.LL A13HL A1HL C1HL A13LL A1LL C1LL Culture Treatment

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2 Table 3. Mean values of the PSII photochemical efficiency Fv/Fm and PSII effective cross section σPSII(nm ) (±SE). Each value is the average of measurements made every second day along the exponential phase of three different cultures per each type.

2 PF Type Fv/Fm (±SE) σPSII(nm ) (±SE)

A1 0.42 0.003 3.12 0.05

LL A13 0.42 0.004 3.53 0.05

C1 0.47 0.003 2.81 0.04

A1 0.35 0.003 3.83 0.07

HL A13 0.29 0.007 4.16 0.08

C1 0.31 0.005 4.13 0.09

The PSII-relative-rate of electron transfer (ETRPSII) was significantly different between the two light treatments. The three phylotypes grown in 350µmol photons·m-2·s-1 showed an increase in the

PSII electron transfer rate in comparison to 100µmol photons·m-2·s-1 (Figure 8).

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100µmolphoton m^2 s^−1 350 µ100µmolphoton350µmolphotonmol photons m^2 m^2 s^ s^− m1−1-2s- 100 µ100µmolphoton350µmolphotonmol photons m^2 m^2 s^ m− -s^21s−-11 350µmolphoton m^2 s^−1 1 100 100 A1 100 A1 A1 A1 A1 A1 A13 A13 A13 A13 A13 A13 C1 C1 C1 C1 C1 C1 80 80 80 ) 60 60 60 ru ( ETR rETR(ru) r rETR(ru) rETR(ru) 40 40 40 20 20 20 0 0 0 0 500 1000 1500 0 500 1000 1500 0 500 1000 1500 0 500 1000 1500 0 500 1000 1500 0 500 1000 1500 PAR(µmolphotonm^2s^−1) PAR(µmolphotonm2s−1) PAR(µmolphotonm^2s^−1) PAR(µPAR(µmolphotonm2smol photons − m1) -2s-1) PAR(µPAR(µmolphotonm^2s^mol photons m−1)-2s-1) PAR(µmolphotonm2s−1)

The analysis of the parameters α and rETRPSIImax showed that α did not change within and between types, as well both measures α or rETRPSIImax were not significantly different between photon flux treatments (Table 4).

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PF Type (α) (±SE) rETRPSIImax (±SE)

A1 0.18 0.01 47.5 3.3

LL A13 0.15 0.01 49 5.45

C1 0.19 0.01 41.6 1.99

A1 0.13 0.01 60.3 20.2

HL A13 0.15 0.02 63 19.2

C1 0.16 0.03 49.5 5.92

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2.5.3 Proteomics

2.5.3.1 Shotgun proteomics.

An initial assessment for proteome differences between the two photon fluxes with LC- MS/MS shotgun proteomic analyses on samples from type C1 produced a high yield of protein spectra but only 74 matched with sequences search in databases. From these proteins only 13 displayed significant differences between the two photon fluxes (Table 5).

Table 5.Proteome differences displayed between C1 grown in two different photon fluxes (100 and 350 µmol photons•m2•s-1)

Symbiodinium C1

E (µmolphotons•m-2•s-1)

350 100 t-test (p) ID Access # Biological Process

- + 0.005 PCP apoprotein Q8H1J8 Light harvesting

- + 0.01 PCP chloroplastic P51874 Light harvesting

- + 0.02 acpPC E0WEC7 Light harvesting

- + 0.005 PSII CP47 U6EFR9 Electron transport

- + 0.04 PSII CP43 U6EFN7 Electron transport

- + 0.01 RuBisCo Q41406 Carbon Fixation

+ - 0.04 RuBisCo A1L0Q2 Carbon Fixation

- + 0.03 GAPDH Q7XJJ3 Glucose metabolism

+ - 0.01 GAPDH Q76EI3 Glucose metabolism

- + 0.03 HSP90 Q0R0F5 Protein folding

- + 0.04 HSP70 Q0R0F4 ATP binding

+ - 0.01 HSP70 Q0H8V3 ATP binding

Symbiodinium type C1 proteome differences between both photon fluxes belonged to the bioprocesses of photochemistry (light harvesting and electron transport), photosynthesis, energy metabolism and housekeeping. The two light harvesting complexes known for Symbiodinium (PCP and acpPC) were more expressed in 100µmol photons•m-2•s-1 cultures (Table 5). Photosystem II core antenna proteins CP47 and CP43 were also up regulated at 100µmol photons•m-2•s-1. Proteins involved in carbon fixation RuBisCo (Ribulose 1,5-bisphosphate carboxylase oxygenase) and

33 Chapter 2 glycolysis GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) proteins were found in different isoforms and differentially expressed at both photon fluxes. Heat shock protein 90 (HSP90), a chaperone used in protein folding, was found more expressed at LL. Finally, different isoforms of Heat shock protein 70 (HSP70), which has been associated with HSP90 also involved in protein folding and stress response were found differentially expressed at both photon fluxes (Table 5).

2.5.3.2 Two-dimensional electrophoresis (2-DE) analysis

Preliminary 2-DE analysis produced a pattern of spots representing the proteome of the three types of Symbiodinium examined in this study. When gels were aligned distortion of the gels occurred most probably due to different pH of gel strips for first dimension electro-focusing used between PFs. Analysis of PFs per separate showed differences in the patterns of expression compared between LL vs HL and also some less marked differences across the three types (Appendix A, A.1).

Subsequent analysis with gel strips of same electro-focusing pH (pH=4-7), displayed 1656 spots. From these spots, after analysis looking for proteome differences across the three types of Symbiodinium, 80 significantly different spots were found (Appendix A, A.2).

After removal of false positive spots coming from stains and gel textures 31 different protein spots across types were found. Further analysis to know which proteins were different only between photon fluxes produced another 82 significantly different spots. After removal of false positives 17 different protein spots between photon fluxes were found and from these only 6 proteins matched with the former analysis across types. In total, 15 proteins were chosen for identification belonging to three different spot groups: i) The six proteins matched different with both analyses ii) Five proteins different just across types iii) and four proteins different just between photon flux treatments giving a total of 15 spots (Figure 9).

34 Chapter 2

"kDa""""""pI" 4""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""7" 120" " " " " " " " " " " " ""15"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""

After database search for protein identities of the fifteen protein spots selected, most matched with proteins involved with light harvesting, carbon fixation, electron transport, energy metabolism and protein modification and most of them were in agreement with results obtained from the former analysis made using LC-MS/MS shotgun proteomics on samples of C1 type. (Table 6).

2.5.3.2.1 i) Protein differences across types and treatments

The largest difference found between types and treatments was found in spot 710 identified as cytochrome b, a mitochondrial protein part of the respiratory chain for ATP synthesis. Type A1 presented the highest as well as the lowest expressions of this protein at LL and HL respectively. This pattern of expression was repeated on type C1. In type A13 was not differentially expressed between PFs. Spot 606 identified as Tubulin beta chain, an important protein for organization of cytoskeleton, was more expressed at HL in types C1 and A1 but not in type A13. Spot 762 matched PCP of Amphidinium carterae was significantly more expressed by A1 at LL. Protein spot 703 matched with Enolase 1 of an Apicomplexan, this protein involved in glycolysis was found in general more expressed by all types at LL and also at HL but only by A1. Spot 1231 matched with adenylate-kinase-isoenzyme 6 homolog also of an

35 Chapter 2

Apicomplexan. Alternatively this spot also matched with a chloroplastic PSII 22 kD protein, with a non-photochemical quenching function. It was more expressed in HL by type C1. Spot 385 matched with an apo-protein of PSI from dinoflagellate Amphidinium carterae. It was more expressed by A1 and C1 at LL vs HL. It was equally expressed by type A13 at both PFs (Table 6)

2.5.3.2.2 ii) Protein differences across types

In general protein spots different between types were identified as part of the electron transport chain with exception of spot 412 which matched with RuBisCo of dinoflagellate Heterocapsa triquetra (Glenodinium triquetrum). This is an essential protein for carbon fixation and also involved in photorespiration. It was found to be more expressed by type A1 at both PFs specially at HL where was 3.4 fold compared with type A13 at LL (Figure 10).

Spots 758, 820 and 699 matched with PSII protein D1 of red tide dinoflagellate Karenia mikimotoi (Gymnodinium mikimotoi) most probably as a result of the scarce information available for dinoflagellates though it can’t be ruled out these are isoforms of the same protein. Alternatively, secondary matches identified spot 758 as PSII protein CP47, spot 820 again as PSII protein D1 and 699 as PSII protein CP43, all different components of core antenna PSII reaction centre. These three proteins as well as spot 1243 identified as cytochrome b6f of dinoflagellate Amphidinium operculatum, are all associated with electron transport and were found more highly expressed in type A1 at LL (Table 6).

2.5.3.2.3 iii) Protein differences between PFs

Spot 1031 matched with chloroplastic PCP light harvesting complex of Symbiodinium and was more expressed at LL than HL. Spot 369 matched with an apo-protein of PSI from dinoflagellate Heterocapsa triquetra and it was more expressed at HL. Spot 598 matched a cytochrome c of an Apicomplexa involved in respiratory chain and was more expressed at LL.

Finally, spot 1503 matched with calmodulin involved with calcium binding and protein transport and it was more expressed at HL (Table 6).

36 Chapter 2

Chapter 2

Average%Normalised%Volumes 350µmolphotonsGm2GsI1 100µmolphotonsGm2GsI1 Spot%# Anova%(p) Fold A1 A13 C1 A1 A13 C1 IP MW ID Access# Biological%Process Ribulose)bisphosphate)carboxylase,)chloroplastic Q5ENN5 Calvin)cycle,)Carbon)dioxide)fixation,)Photorespiration,)Photosynthesis) x412 0.003 3.4 3564! 1092 1087 2967 1034" 1395 5.451 72.357 MagnesiumJchelatase)67)kDa)subunit Q9ZGE6 Chlorophyll)biosynthesis,)Photosynthesis Transcription)initiation)factor)IIF)subunit)alpha Q9SU25 Transcription,)Transcription)regulation Photosystem)II)protein)D1 Q9TJ82 Electron)transport,)Photosynthesis x758 0.004 2.6 2381 2354 1276 2952! 1245 1127" 6.012 45.619 Photosystem)II)CP47)reaction)center)protein Q0P3P8 )Transport,photosynthetic)electron)transport)in)photosystem)II NAD(P)HJquinone)oxidoreductase)subunit)H,)chloroplastic A2T389 Transport Photosystem)II)protein)D1 Q9TJ82 Photosynthetic)electron)transport)in)photosystem)II x820 0.023 2.2 8563! 7062 5061 6317 5640 3884" 5.857 41.041 Photosystem)II)protein)D1 Q46JV2 Electron)transport MagnesiumJprotoporphyrin)IX)monomethyl)ester)[oxidative])cyclase Q3AK18 )lightJindependent)chlorophyll)biosynthetic)process Cytochrome)b6 Q8WHC6 Electron)transport,)Photosynthesis x1243 0.007 3.1 1955 1247 1380 2775! 907" 927.3 6.322 22.752 Superoxide)dismutase)[Fe] Q9Y1B0 Oxidoreductase Photosystem)I)assembly)protein)Ycf3) Q4VZH4 Transport,photosynthesis Electron)transport)complex)subunit)G C6DH13 Electron)transport,)Transport,)Glycolysis Photosystem)II)protein)D1 Q9TJ82 Electron)transport,)Herbicide)resistance,)Photosynthesis, x699 0.013 6.1 506.9 392.8" 475 2390! 485.8 649.5 6.12 49.115 Photosystem)II)CP43)reaction)center)protein Q08684 )Transport,photosynthetic)electron)transport)in)photosystem)II Ribulose)bisphosphate)carboxylase)large)chain P0087 Calvin)cycle,)carbon)dioxide)fixation,)Photorespiration,)Photosynthesis Enolase Q7RA60 Glycolysis Tubulin)beta)chain Q7KQL5 Microtubule)cytoskeleton)organization §606 0.006 3.1 3578 1745 3723! 1734 1206" 1835 4.248 52.02 AcetylJcoenzyme)A)carboxylase)carboxyl)transferase)subunit)beta B5LMN4 Fatty)acid)biosynthesis,)Fatty)acid)metabolism,)Lipid)biosynthesis Trigger)factor A1SME5 Cell)cycle,)Cell)division PeridininJchlorophyll)aJbinding)protein)3 P80483 LightJharvesting)polypeptide §762 0.022 5.4 212.7 170 122" 654.2! 205.2 234.7 6.079 42.73 Photosystem)II)protein)D1 Q9TJ82 Electron)transport,)Herbicide)resistance,)Photosynthesis,)Transport Photosystem)II)D2)protein P56319 Electron)transport,)Photosynthesis,)Transport Cytochrome)b P15585 Electron)transport,)Respiratory)chain,)Transport §710 0.011 6.3 114.6" 211.8 124 725.4! 198 496.3 6.22 46.02 AlphaJ1,2)mannosyltransferase)KTR1 P27810 Protein)OJlinked)glycosylation LightJindependent)protochlorophyllide)reductase)subunit)N Q7VD37 Chlorophyll)biosynthesis,)Photosynthesis Photosystem)I)P700)chlorophyll)a)apoprotein)A2 P58383 Electron)transport,)Photosynthesis,)Transport §385 0.023 3.6 360" 772.8 601 1063 603.3 1313! 5.404 75.576 Photosystem)I)P700)chlorophyll)a)apoprotein)A1 P58309 Electron)transport,)Photosynthesis,)Transport Translation)factor)GUF1)homolog,)mitochondrial A7AQ93 Protein)biosynthesis Adenylate)kinase)isoenzyme)6)homolog Q8I236 Kinase,)Transferase §1231 0.025 3.6 9431 6639 16560! 4870 4771 4577" 4.837 22.491 Photosystem)II)22)kDa)protein,)chloroplastic P54773 nonphotochemical)quenching FibrillarinJlike)rRNA/tRNA)2'JOJmethyltransferase Q9HQG3 rRNA)processing,)tRNA)processing Enolase)1 Q9UAE6 Glycolysis §703 0.044 2.8 587.5 273.6 264" 727.8! 509.4 611.7 5.99 46.388 LightJindependent)protochlorophyllide)reductase)subunit)N Q2RWR8 LightJindependent)chlorophyll)biosynthetic)process GammaJglutamyl)phosphate)reductase A5IKB6 ADP)binding)aminoacid)biosynthesis Photosystem)I)P700)chlorophyll)a)apoprotein)A2 Q9XQV2 Electron)transport,)Photosynthesis,)Electron)transport *369 0.001 2.3 7765! 3448 4.832 80.452 Photosystem)I)P700)chlorophyll)a)apoprotein)A1 Q9XQV3 Electron)transport,)Photosynthesis,)Transport Photosystem)I)P700)chlorophyll)a)apoprotein)A2 P29255 Photosynthesis,)Transport Cytochrome)c)oxidase)subunit)1 Q4UJ69 Electron)transport,)Respiratory)chain,)Transport *598 0.034 2 619.7 1250! 5.987 53.084 Ribulose)bisphosphate)carboxylase)large)chain Q3BAN4 Respiratory)chain,)Transport,photorespiration Ribulose)bisphosphate)carboxylase)large)chain A4QJC3 Respiratory)chain,)Transport,photorespiration Calmodulin A3E4D8 Calcium)ion)binding *1503 0.037 1.7 83270! 49920 4.655 16.522 Cytochrome)b6Jf)complex)subunit)4 Q1HCL0 Electron)transport,)Photosynthesis,)Transport Photosystem)I)reaction)center)subunit)XI P37277 Photosynthesis Transcription)elongation)factor)GreA Q3J5L0 Regulation)of)DNAJtemplated)transcription,)elongation PeridininJchlorophyll)aJbinding)protein,)chloroplastic,, P51874 LightJharvesting)polypeptide *1031 0.049 2 174 353! 6.038 31.953 Dimethlysulfonioproprionate)lyase P0DN22 Dimethylpropiothetin)dethiomethylase)activity Cytochrome)f Q9BBR8 Electron)transport,)Photosynthesis,)Transport Note: !Upregulated)"Downregulated §Protein)dif)between)phylotypes)and)dif)between)treatments) xProtein)dif)only)between)phylotypes *Protein)dif)only)between)treatments

38 Chapter 2

Chapter 2

2.6 Discussion

2.6.1 Growth

Light environment is the main factor driving the physiology and bioenergetics of algae not only in the open ocean but also in the coastal oligotrophic waters where coral reefs thrive as a result of their symbiosis with Symbiodinium (Muscatine 1980, 1990). Changes in algal growth rates and photophysiological modifications are among the most important responses to variable light (Chang et al. 1983, Iglesias & Trench 1994, 97, Hennige et al. 2009). Different types of Symbiodinium have evolved different photoacclimation strategies, which allow them to adjust the pigments composition and quantity of proteins involved in photosynthesis (Iglesias & Trench 1994, 1997, Hennige et al. 2009, Sugget et al. 2015). Whether these different photoacclimation strategies are more or less advantageous for specific types may also explain their success within any environmental window including symbiosis (Iglesias-Prieto et al. 2004, Hennige et al. 2010, 2011). Growth rates in Symbiodinium as in most microalgae are mainly determined by light as the main resource available. Adaptations of the PSU of Symbiodinium enable this genus to photoacclimate and maintain a very high photosynthetic efficiency across a wide range of PFs (Iglesias-Prieto & Trench 1994, 1997, Hennige et al. 2009). In this study the three types of Symbiodinium A1, A13 and C1 exhibited a similar specific growth rate µ under the two PFs tested. Growth values at LL in this study were slightly lower than those reported in other former experiments at similar low light conditions (Hennige et al. 2009). In other studies growth rates increased at higher PFs than used here. The high PF used in this study apparently was not high enough to produce a measurable difference in growth between both light treatments (Hennige et al. 2009). Photoacclimation strategies across types of Symbiodinium also enable sustained growth even under the nutrient limitation in oligotrophic waters as well as in symbiosis when its host limits nutrient supply to the symbiosome located inside the gastrodermal cells where Symbiodinium algae are harboured (Dubinsky & Jokiel 1994, Iglesias-Prieto & Trench 1994, 1997, Rodríguez-Román & Iglesias-Prieto 2005, The present study was conducted in batch cultures and usually in these type of culture nutrients become exhausted as the culture approaches the stationary phase (Sugget et al. 2009). As shown in the fitted growth curve, as a consequence of this nutrient exhaustion maximum growth is reached faster in HL cultures of the three types. However, there were not significant differences in the specific growth rates between the two PFs. These results show that at these two PFs the three types of Symbiodinium adjusted the PSU and photoacclimate to sustain similar

41 Chapter 2 growth rates in agreement with other studies where different types of Symbiodinium maintain photochemical efficiencies long after nutrients in batch cultures are reduced (Rodríguez-Román & Iglesias-Prieto 2005, Sugget et al. 2009). Different photoacclimation strategies among Symbiodinium types should be complemented by differential regulation of other metabolic processes. These differences between types together with different mechanisms of control and adaptation of the coral host probably determine the stability of the symbiosis (Iglesias-Prieto & Trench 1994, 1997, Reynolds et al. 2008, Hennige et al. 2009, 2010, 2011).

2.6.2 Photophysiology

The three types of Symbiodinium explored showed specific photophysiological modifications in response to the two experimental PFs in agreement with known photoacclimation strategies (Iglesias & Trench 1997, Hennige et al. 2009). In the present study Chl a (pgcell-1) concentration was significantly higher in cultures of the three types growing at LL. This increase was particularly higher in C1 compared with the other two types. As well Chl a* specific absorption coefficient was incremented at HL although was only significantly higher (~2fold) in type A1 compared to LL these results are in agreement with other previous studies showing different types of Symbiodinium experiment changes in pigment concentration and Chl *a as part of photoacclimation to PF (Iglesias & Trench 1997, Hennige et al. 2009). Higher concentrations of Chl a (pgcell-1) in cultures at low PFs are indicative of larger photosystem core antennas and light harvesting complexes (LHCs) which increase the probability of photon capture and transfer to the reaction centres (RCs) (Iglesias & Trench 1994, 1997). In contrast, at high PFs core antennas and LHCs have a reduced amount of pigment to avoid photodamage of RCs as the probability of absorbing a photon increases (Iglesias & Trench 1994, 1997). It has been suggested that photoacclimation is an important trait describing the phenotypic plasticity of Symbiodinium, which has evolved to deal with light availability in order to optimize photosynthesis (Hennige et al. 2009, Suggett et al. 2009, 2015). Modifications are made in the size and amount of peripheral and inner LHCs and in the number of RCs to balance excitation and electron transport rate through the PSU. This allows the production of the carbohydrates necessary for the requirements of the algal metabolism and in-hospite to translocate a significant amount of these to the host (Iglesias & Trench 1997, Iglesias-Prieto et al. 2004, Hennige et al. 2009). Here two descriptors of changes in the PSU related to photoacclimation to PF, the PSII photochemical efficiency Fv/Fm and the PSII effective absorption cross section (σPSII) showed modification across the three types addressed. Generally, Fv/Fm was higher in the three types at LL and

42 Chapter 2 inversely the σPSII increased at HL compared to LL. It has been suggested that the frequent negative correlation between these two parameters is related to the bioenergetics of each type and is driven by nutrient availability (Suggett et al. 2009). In general, changes found in PSII relative rate of electron transport support the hypothesis that the nutrient limitation effect on growth is overweighted by light availability/utilization (Rodríguez-Román & Iglesias-Prieto 2005, Suggett et al. 2009). In HL, PSII electron transport was higher as for the three types leading to sustained growth by photosynthesis at conditions of nutrient exhaustion. This is achieved with efficient photoacclimation changes such as reducing the amount of pigment concentration to avoid photodamage but increasing the Chl a* and PSII specific absorptions to sustain a higher electron transport rate in a HL environment. Also at LL effective photoacclimation changes are achieved by increasing the amount of pigments, and reducing the Chl a* and PSII specific absorptions resulting in a higher photochemical efficiency to maintain a sustained rate of electron transport and photosynthesis in a LL environment. These modifications to photoacclimate may also occur in-hospite. The coral host amplifies the availability of light by scattering from the skeleton and when light is scarce as in mesophotic environments makes it available by florescence emitted by products of the animal cell (Enríquez et al. 2005, Marcelino et al. 2013, Bollati et al. 2017). Also, it has been suggested that the host limits the nutrient supply to the symbiont to control population densities and to increase the translocation of photosynthetic products (Falkowski et al. 1993, Dubinsky & Jokiel 1994, Yellowlees et al. 2008). As well in hospite the host can induce an elongation of the growth rates of Symbiodinium that does not occur in culture (Rodríguez-Román & Iglesias-Prieto 2005). Even though, these mechanisms of host control upon the Symbiodinium are regulated by the expression of host proteins, the photoacclimation response of Symbiodinium must be linked with other metabolic pathways under environmental and host control that can be studied with proteomics.

2.6.3 Proteomics

The protein patterns obtained in this study are consistent with previous studies examining other dinoflagellates (Chan et al. 2004). The marine dinoflagellate Alexandrium catenella cultured at 100 µmol photons•m-2•s-1 exhibits a similar 2-DE protein pattern as those shown here in the three Symbiodinium types subject to the same PF (Li et al. 2012). Also in agreement with the present study in dinoflagellate Prorocentrum triestinum different 2-DE patterns of protein expression were obtained when cultures of this species are exposed to light starvation and subsequent illumination in comparison to controls (Chan et al. 2002). Although differences between the abundance of spots between the two PFs were probably a result of the different range of pH of the resolving gels in the

43 Chapter 2 preliminary analyses, subsequent analysis with 2-DE gels of the same electro-focusing resolving pH indicated this was not the case and confirmed there were differences between PFs and across the three types subjected to similar PFs. These differences in protein patterns between the experimental types grown at the same PFs treatments showed A1 and A13 are more similar to each other than with C1. Results obtained in this study support the hypothesis that different types of Symbiodinium display differences in proteome that supplement strategies to photoacclimate to different photon flux environments. Analysis of proteome differences across clades and treatments from identification based on the isoelectric point (pI) and the molecular weight (MW) of the protein spots obtained with 2-DE gels delivered identities (IDs) of proteins with similar biological process even with searches using different tags therefore this supports interpretation of the analysis. In Symbiodinium as with other microalgae increases in the number of components and size of core antenna of PSs as well as of LHCs at low photon fluxes, have been related to a photoacclimation strategy to increase the absorption cross section and, also increase the number of photons transferred to the reaction centre (RC) for charge separation. The responses across types of Symbiodinium tested in this study show how this genus can successfully photo-acclimate to different photon fluxes implementing differential changes within their PSU as well as in metabolic pathways linked with its regulation. These changes include different protein involved in light harvesting, electron transport, carbon fixation, energy metabolism and protein modification. Most of the differentially expressed proteins across clades and between PFs are involved in electron transport (ET) in the PSU: PCP, acpPC, PSII inner antenna components (CP47, CP43), PSII protein D1, PSI apoproteins and Cytochrome b6. According to a general photo-acclimation pattern all three types up-regulated PCP at LL. In addition, results showed C1 also up regulated acpPC at this photon flux. Furthermore, PSII inner antenna components (CP47, CP43) were also more expressed at LL. This higher expression of LHCs and PSII inner antenna explains the significantly higher Chl a (pgcell-1) concentrations across types at LL. These modifications on components of the PSU are in agreement with previous research on Symbiodinium at similar experimental conditions (Iglesias & Trench 1997, Iglesias- Prieto et al. 2004, Hennige et al. 2009). Results also show that the three experimental types expressed more PSII protein D1at HL which may be indicative of a higher turnover of PSII reaction centres for charge separation to sustain electron transport, this explains the higher rETRs found at this photon flux. This differential expression of proteins may be indicative of regulation of ET determined by light. The linear electron transport (LET) may be supplemented by alternative electron transport (AET) pathways

44 Chapter 2 and interactions between chloroplastic and mitochondrial metabolic pathways to balance the proton motive force (pmf) and ATP production (Ort & Baker 2002). Previous studies have suggested types belonging to clade A photoacclimate by activation of AET pathways such as cyclic electron transport (CET), state transitions (STs) and the water water cycle (WWC), (Reynolds et al. 2008, Roberty et al. 2014). Cytochrome b6f was the most significantly different ET protein between types and it was more expressed by A1 at LL This protein mediates and balances ET between photosystems (PSs) and is involved in CET around PSI. Thus, differential expression of cytochrome b6f between types supports activation of CET and this activation of CET is crucial for the generation of pmf and ATP balance (Dumas et al. 2016). This AET pathway of electron flow around PSI has been reported to occur in members of clade A such as A1 and A13 (Reynolds et al. 2008). PSI apoproteins matched with two spots, one of them was differentially expressed across clades and was more expressed in C1 and A1 at LL compared with HL. The other PSI apoprotein was differentially expressed between both photon fluxes and was more expressed in HL. These differences in expression may also be related to the differential activation of CET around PSI. Cytochrome b6f is also involved in STs between PSI and PSII. As well, STs have been reported to occur in clade A by the migration of PCP between PSs (Reynolds et al. 2008). Also, PSII core antenna components CP43 and CP47 have been associated with AET pathways including also CET and ST. The differential expression of these proteins may be also related with the observed differences in Chl a*, σPSII absorption cross section and photochemical efficiencies between types. Previos work by Reynolds et al. (2008) using serial irradiation pulses found that type C1 harboured by Porites astreoides did not present induction of chl a fluorescence fluctuations indicative of AET (Reynolds et al. 2008). However, it can not be ruled out that type C1 may activate any form of AET pathways included photorespiration, which is considered also an O2- dependent AET (Roberty et al. 2014, Shimakawa et al. 2017). On the other hand, type C1 upregulated a PSII 22 kDa protein at 350µmol photons•m-2•s-1 probably indicative of a heat dissipation NPQ photoacclimation mechanism. AET pathways have been suggested to occur in microalgae on a general basis to keep the balance of ATP production, avoid photoinhibition as well as photoinactivation of photosynthesis and to regulate CO2 and O2 exchange between chloroplast and mitochondria (Curien et al. 2017, Shimakawa et al. 2017). In Symbiodinium has been suggested pathways of AET occurring to avoid photodamage of PSII from ROS at stress conditions (Reynolds et al. 2008, Roberty et al. 2014). Nevertheless, it is possible that these AET occur at low light intensities as reported for other groups

45 Chapter 2 of microalgae to balance ATP production (Mullineaux, et al. 2004, Munekage et al. 2004, Yamori et al. 2015). Other proteins differentially expressed were components of ET in mitochondria: Cytochrome b and Cytochrome c. These proteins involved in respiration were significantly more expressed at LL, in particular type A1expressed more cytochrome b, but it did not change in A13. The respiratory chain in dinoflagellates including genus Symbiodinium, lacks complex I. This produces a shortfall in respiratory ATP synthesis which supports the hypothesis that photosynthetic AET in the chloroplast could be balancing ATP production (Raven & Beardall, 2017). The similar growth rates found among types explored in this study may be sustained by mechanisms to balance ATP production and energy metabolism. Proteins involved in energy metabolism were also differentially expressed: RuBisCo, GAPDH, and Enolase 1. Enolase 1 is a protein involved in glycolysis and was more expressed in LL. RuBisCo was found differentially expressed with 2-DE analysis and shotgun proteomics.

RuBisCo in Symbiodinium has a low affinity for CO2 and it can be expressed for carbon fixation or as consequence of increases in photorespiration (Palmer 1996). Previous studies have suggested CET occurs in clade A at high photon fluxes as levels of expression found in RuBisCo did not correlate with electron transport rates (Brading et al. 2013). In this study, RuBisCo was more expressed in A1 at HL which, correlates with higher rETR of this type at this condition. Isoforms found for C1 were found more expressed at both lights. In type A13 its expression did not change with light. This could be produced by the dual function of RuBisCo or may indicate these two types could have activated AET (Badger et al. 2000). Shotgun analyses of type C1 displayed differences in expression of three proteins between photon fluxes that were not identified with 2-DE analyses GAPDH, HSP90 and HSP70. GAPDH protein is involved in glucose metabolism linked between mitochondria and chloroplast but as it happens with dual activation of RuBisCo its levels of expression may be due to differing pathways within respiration and glycolysis (Noguchi & Yoshida, 2008). It has an important interaction with RuBisCo and high utilization of NADPH thus it may affect photorespiration and activation of AET pathways, ROS metabolism and production of DI photorepair, also its expression levels might be related with expression of mitochondrial and chloroplasts cytochromes (Takahashi & Murata, 2006, Curien et al. 2017, Shimakawa et al. 2017). Proteins related with protein modification, transport and cell maintenance Tubulin, Calmodulin, HSP90, HSP070 were also differentially expressed. Tubulin was more expressed at HL this protein forms microtubules for organization of cytoskeleton. This may be produced by an increase in cellular protein export across organelles or by signalling between cells. It is known Symbiodinium cells tend to aggregate when in culture, and this may lead to exchange of cellular

46 Chapter 2 materials or to secretion of signalling molecules such as glycoconjugates (Markell & Wood- Charlson, 2010). Calmodulin was also found more expressed at HL, it is a protein related with growth, response to environment and protein transport (Wen et al. 2014). As mentioned before, shotgun analyses found the differential expression of HSP90 and HSP70 of type C1. Both proteins were more expressed at HL, these are highly abundant chaperones, they interact with each other, as well as cooperate with other HSPs to regulate signal transduction, they are involved in multiple cell maintenance tasks comprising membrane translocation, protein degradation, protein folding, repair and regulation of protein homeostasis in normal and stressed cells. Its high expression at HL may be due to higher cell maintenance requirements for repair and disassembling of protein complexes (Guo et al. 2015). Finally Adenylate kinase isoenzyme 6 homolog is a kinase-transferase protein involved in ATP, was more expressed by type C1 at HL. As mentioned before this protein was also identified as a PSII 22 kDa protein involved in a non-photochemical quenching (NPQ) photoacclimation mechanism. In conclusion, the differential proteome of the Symbiodinium types in this study showed how the physiological modifications to achieve photoacclimation across the genus Symbiodinium include the differential expression of proteins involved in electron transfer, carbon fixation, respiration and cell maintenance. This protein regulation implicates changes in antenna size and composition as well as adjustments in various metabolic pathways associated with the balance of energy production and utilization. The differential expression of proteins constitutive of the two photosystems (specially PSII) across the three types may be probably related with their different photoacclimation strategies, their distribution and their ability to survive in different environments. This is supported by the differential expression between photon fluxes of the two light harvesting complexes PCP and acpPC as they transfer excitation to both photosystems. As well, the differential expression of PSI reaction centre and Cytb6f across types indicates adjustments in the electron transport as this two proteins are involved in alternative electron transport pathways. These adjustments and their role in the balance of ATP and NADPH are probably related with the differential expression of RuBisCo and GAPDH involved in carbon fixation and glycolysis respectively. Respiration was also regulated as shown by the differential expression of mithocondrial proteins. All this metabolic regulation requires also the differential expression of proteins related with cell maintenance, protein modification and protein transport. This work exposes how photoacclimation adjustments in the proteome of Symbiodinium may probably be as diverse as types there are in this symbiotic genus of dinoflagellates. The full understanding of this plastic regulation in Symbiodinium may give some clues on the acclimation potential to future stressful conditions of the diversity of types and its role in the physiology of the holobiont. As well, other hypotheses regarding the actual symbiotic benefits of this functional

47 Chapter 2 diversity of Symbiodinium can be examined using the combination of the physiological and proteomic approach.

48 Chapter 3 Chapter 3 The coral host release factor and its effect over Symbiodinium

Abstract

Coral reefs thrive in oligotrophic ocean waters using the photosynthetic energy derived from their obligated symbiosis with dinoflagellates of the genus Symbiodinium. Stressful conditions due to long periods of increased ocean mean temperatures exert negative effects on the coral holobiont. This results in the loss of algal pigments or whole algal cells from the animal as a phenomenon known as coral bleaching. Variation in bleaching response across coral holobionts is well documented and in part has been attributed to the high functional diversity of Symbiodinium. However, the different host control mechanisms may well also drive the variable capacity of the holobiont to cope with changing environmental conditions. Is well documented the coral host effects carbon translocation from the algal symbiont by a host release factor (HRF). In this study, we aimed to determine and compare the proteome of three types of Symbiodinium cultured at two photon fluxes (PFs) and subjected to: a) Pocillopora damicornis tissue extracts (HT) and b) a previously characterised free amino acid mix (FAA) mimicking this coral’s HRF. As control, we also treated cultured isolates of the dominant Symbiodinium type from the coral species. In addition to the proteome, we measured the physiological parameters: i) chlorophyll a concentration, ii) the excitation pressure over photosystem two (PSII) iii) total protein, iv) total glucose and v) glucose released in the presence and absence of the HRFs. These parameters were variable among symbiont types. In general, the parameters directly related to photochemistry such as Chl a concentration and excitation pressure over PSII, were controlled by the photon flux and inversely correlated with each other. Total protein and total glucose also correlated with each other but their concentration appeared determined by the host factor treatment. As expected the HT of Pocillopora damicornis stimulated the release of glucose from its’ dominant Symbiodinium. A1 in the presence of HT and at a high PF presented elevated excitation pressure over PSII and higher concentrations of total glucose and total protein relative to controls. This maybe indicative of HF stimulation over the photosynthetic production of glucose. However, the mechanism of glucose release was not ejected in type A1 as this parameter did not significantly increase relative to controls. In A13 and C1 types cultured at a low PF the FAA inhibited the release of glucose relative to controls. A principal component analysis (PCA) revealed the existence of a general mechanism of glucose release which involves the excitation pressure over PSII, the total glucose and the total protein. SDS-PAGE protein analysis resolved different bands with differential expression among types and treatments. Id of these bands by MALDI-TOF analysis would explain at a finer resolution the mechanism of host control over Symbiodinium photosynthesis and the mechanism of translocation of carbon to the coral host.

49 Chapter 3 3.1 Introduction

Coral reef ecosystems are versatile providers of ecological services including an important contribution to bigeochemical cycles (Muscatine 1980, Muscatine & Weis, 1992, Moberg & Folke, 1999). Unfortunately, coral reefs are deteriorating and being lost rapidly (Goreau & Macfarlane, 1990). Since coral reefs thrive in oligotrophic waters, their ecological success has been attributed to the high productivity of Symbiodinium photosynthesis and its role in fueling the symbiosis (Muscatine, 1990).

The symbionts exist within the host gastrodermal cells and maximize their productivity by the host supply of inorganic nutrients, which in turn regulate symbiont driven photoassimilated carbon translocation (Enríquez et al. 2005, Davy et al. 2012). The transport of nutrients between the host and algal symbionts must be finely controlled since a disturbance of the nutrient transport cycle could jeopardize the stability of the symbiosis resulting in the expulsion of the algal cell out of the coral host (Wooldridge 2014, Lehnert et al. 2014). Although most of the mechanisms of nutrient exchange of coral symbiosis still remain to be explained, evidence suggests that the host cell exerts control over the algal cell by stimulating the release of carbon, promoting photosynthesis and perhaps also by increasing the length of cell cycle (Yellowlees et al. 2008, Barott et al. 2015).

The first evidence of control of the host over the algal symbiont arose from studies assessing the contribution of photosynthesis to coral metabolism (Muscatine & Hand 1958; Muscatine et al. 1972). These studies found symbionts isolated and exposed to a crude homogenate of host tissue, released carbohydrates in the same amount and quality as if they remained in intact symbiosis (Muscatine & Cernichiari 1969, Trench 1971c). The carbon released by the symbiont in the presence of host tissue homogenates was also found to occur in many other symbiotic relationships besides coral symbiosis including within other anthozoans (eg. anemones), bivalves (Tridacna sp.) and nudibranchia (Muscatine 1967, Kempf 1984, Sutton & Hoegh-Guldberg 1990). This induced carbon release by the symbiont was attributed to a compound synthesized by the host as it did not occur with tissue homogenates of aposymbiotic organisms (Trench 1971c, Gates et al. 1995) The stimulation of carbon release by this “host release factor” (HRF or HF) was also found to be unspecific as some experiments demonstrated the HRF of one host could induce the release of carbon on other symbiotic algae different from its own symbiont (Sutton & Hoegh-Guldberg 1990, Gates et al. 1995). Other experiments found that free amino acids, especially taurine, had a similar effect as the natural HRF obtained through coral tissue homogenation (Wang & Douglas 1997). Interestingly this kind of effect had been achieved also by using clotrimazole which is a micro-

50 Chapter 3 sporine-amino-acid like compound similar to those produced naturally by corals and which have been attributed to function as UV-protective compounds (Ritchie et al. 1997). In every study performed that examined the effects of HRF on symbiont translocation, a diversity of organic compounds including carbohydrates, lipids and amino acids have been exuded but the major carbon compound released from different species were glycerol or glucose (Muscatine 1967, Trench 1971c, Whitehead & Douglas 2003). Strong evidence suggests glucose is the major carbon product released from the symbiont in hospite while glycerol seems to be a product released in vitro due to experimental stress (Burriesci et al. 2012). Other studies have shown that osmotic shocks stimulate the release of glycerol from cultured dinoflagellates suggesting such stress could be a possible mechanism by which the coral host could be inducing the release of photosynthetic products from their symbionts (Suescún-Bolívar et al. 2012). Although the exact nature of HRF has not been determined, the most recent studies focused on isolating HRF suggest that amino acid(s) or polypeptide(s) with a low molecular weight and an acidic isoelectric point could be the dominant components (Grant et al. 2013).

Alternatively, some observations demonstrate that when symbionts are exposed to host tissue homogenate of certain corals, photosynthesis rates decrease suggesting there could be further compounds synthesized by the host that inhibit the symbiont photosynthesis; this compound has been termed Photosynthesis Inhibitory Factor (PIF) (Grant et al. 2001). It seems more likely that not only a polypeptide but a whole set of proteins synthesized by the host are involved in the regulation of Symbiodinium metabolism aimed ultimately at maximizing the net benefit of the symbiosis. Differential transcriptomic analysis of aposymbiotic and symbiotic Aiptasia anemones hosting Symbiodinium showed that more than 60 proteins were possibly associated with nutrient transport. In particular three transcripts were found for proteins related with the transport of glucose: the orthologs of GLUT8, the putative Na+-glucose/myo-inositol co-transporter both localized in the symbiosome membrane and a Symbiodinium specific protein transporting glucose to the symbiosome lumen (Lehnert et al. 2014). In one previous proteomic based study conducted also on Aiptasia, 17 different proteins were isolated from the symbiosomes, 41% of these proteins were deemed to be of animal origin and 29% of algal origin (Peng et al. 2010). Following functional group analysis, 6% of the proteins were membrane transporters included the ABC protein membrane transporter related to ATP-dependent trans-membrane transportation of multiple organic and inorganic molecules and the Hsp70 protein that is related to protein modification during transportation. Finally, the enzyme PDI related to transportation of lipids has also been found (Peng et al. 2010). Interestingly Hsp70 has been found to be highly expressed in corals subjected to light and thermal stress (Weston et al. 2015). In a later study using symbiotic

51 Chapter 3 gastrodermal cells (SCGs) isolated from the coral Euphyllia glabrescens nineteen proteins of cnidarian origin were identified, some of wich are involved with the regulation of membrane transport (Li et al. 2014). A more recent study found that the vacuole proton like ATPase (VHA) located externally to the symbiosome membrane is regulated by the gastrodemal cell, to acidify the symbiosome, to facilitate the transport of ions and nutrients and to promote photosynthesis of the Symbiodinium (Barott et al. 2015). Other proteomic studies identified differences in protein expression between cultured and freshly isolated Symbiodinium cells including five functional groups that may be related with host environment interaction (Pasaribu et al. 2015).

It seems like expression of a whole set of proteins could be involved with the host mediated control of the metabolism of Symbiodinium in hospite. Further studies should be conducted in order to corroborate this hypothesis and to describe how external environments regulate the interaction between the coral host and its symbionts. The aim of this study is to investigate the differential effect that different host release factors exert over the proteome and physiology of different types of Symbiodinium and with this understand better how the host exerts control over the Symbiodinium in order to sustain the symbiosis.

3.2 Hypothesis

If different host release factors stimulate or inhibit differentially the release of glucose of different Symbiodinium types then this translocation will be regulated by a differential photophysiology and proteome across different types of Symbiodinium revealing the mechanisms of glucose translocation and their differential contribution to the symbiosis.

3.3 Objective

Characterise the differences in the protein expression (SDS-PAGE and MALDI-TOF analyses) and the photophysiological parameters: chlorophyll a concentration, the excitation pressure over photosystem two (PSII), total protein, total glucose and glucose released; across three cultured types of Symbiodinium acclimated at two photon fluxes (HL= 350 µmol photons•m2•s-1 and LL= 100 µmol photons•m-2•s-1), after exposure for one hour to a free amino-acid mix and a coral tissue host release factors.

52 Chapter 3 3.4 Materials and Methods

3.4.1 Experimental setup

Control and experimental cultures of Symbiodinium subclades A1, A13 and C1 made in 50ml of ASP-8A (Blank 1987) media were acclimated in triplicate at two different PFs (photon flux: 100 and 350 µmol photons•m-2•s-1) at 26ºC in a photoperiod of 10:14 hours Light: Dark. Control and experimental cultures of Symbiodinium isolated from the scleractinian coral Pocillopora damicornis were acclimated (in triplicate) only at a photon flux of 350 µmol photons•m-2•s-1, at 26ºC and in a photoperiod of 10:14 hours Light: Dark. Cultures of experimental clades A1, A13 and C1 and of Symbiodinium of P. damicornis were concentrated through clumping and attaching to the bottom of the flasks and then were resuspended in 3.6ml of either Free Amino-Acid mix (FAAmix), Host Tissue (HT) or ASP-8A media (Controls) and then subject for 1 hour to HRFs under their previously acclimation irradiance. After HRFs exposure, 3ml were collected in 1.5ml eppendorf tubes, fast frozen and kept at -80ºC for protein extraction and glucose quantification and the remaining 0.6ml left were diluted ten times with ASP-8A media to 6ml which were used for FRRF measurements and quantification of cells and Chl a.

3.4.2 Isolation and formulation of HRFs

Two different types of HRFs were used for comparison with controls only with ASP-8A: i) Coral tissue homogenate extracted from various colonies of scleractinian coral species Pocillopora damicornis, ii) A free amino-acid mix (FAA) based on amino-acid composition of P. damicornis tissue previously characterized in Gates et al. (1995).

3.4.2.1 i) Host tissue homogenate extraction

For tissue extraction coral branches of P. damicornis were immersed in a solution of 3% N-Acetyl- Cysteine pH 8.2 in artificial seawater for 30 min and gently vortexed until tissue detached from the coral skeleton. Symbiodinium cells were separated from host tissue by centrifugation at 2500rpm. The supernatant with the host tissue was kept at 4ºC until used as HRF in subsequent experiments.

3.4.2.2 ii) Free Amino-acid mix formulation

Artificial FAAmix host factor was prepared based on the procedure and molar concentrations obtained by HPLC analyses in a previous study of P. damicornis HRF: Free aminoacids were

53 Chapter 3 weighted and dissolved in Nano pure water and the mix was adjusted to a final concentration of 100mM, pH was adjusted to 8.3 and salinity to 33ppt. The final mix was filtered with a 0.22µm Millipore filter and stored at 4ºC prior to use (Gates et al. 1995).

3.4.3 Isolation and culture of Symbiodinium of P. damicornis

Symbiodinium cells isolated from P. damicornis corals were washed three times with filtered seawater and cultured at a photon flux of 350µmol photons•m-2•s-1 in ASP-8A culture media with a KAS (Kanamicin, Ampicilin and Streptomycin) antibiotic mix. After pure axenic monocultures of the dominant Symbiodinium of P. damicornis were obtained the KAS antibiotic mix was no longer added to the ASP-8A culture media formulation. These Symbiodinium cultures were then used as positive controls for the HRF experiments.

3.4.4 Cultured Clades

3.4.5 Cell quantification

Measurements of the amount of Symbiodinium cells in culture and from coral colonies of P. damicornis were quantified from 1 ml aliquots from the 6ml diluted from 0.6 ml of the experimental cultures and fixed with 10µl of Lugol’s solution. Cell counts were made from10µl aliquots pipetted in the chamber of an improved Haemocytometer under the microscope.

54 Chapter 3

3.4.6 Chlorophyll a

Aliquots of 5ml from the 6ml diluted from 0.6ml samples of the experimental cultures were pelleted by centrifugation at 2500rpm, chlorophyll a extractions were made with 1 ml of 100% methanol and stored at -20ºC overnight. Absorption spectra of methanol extracts were recorded from 350-750nm and chlorophyll a was quantified using the equations of Ritchie (Ritchie, 2006).

3.4.7 Protein

For protein extraction samples of 1.5ml were defrosted from -80ºC to -20ºC and then to 4ºC and all following procedures were performed at this temperature. Samples were pelleted by centrifugation at 2500rpm. Protein were precipitated with 10% trichloroacetic acid (TCA)/Acetone (W/V), vortexed and lysed with sonication on ice, extracts were pelleted again by centrifugation at 12000rpm, then were washed twice with acetone with 0.1% dithiothreitol (DTT) and were centrifuged, vortexed and sonicated again in between each time and finally pelleted by centrifugation again. The pellet was then aired dried before being resuspended in lysis buffer (8M Urea, 2% CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate) and TM 2.8mg/ml DTT). Protein content in samples was quantified using PlusOne 2D Quant kit (GE Healthcare Life Sciences). For protein analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), (Laemmli, 1970) samples were suspended in an equal volume of sample buffer 2X (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue, 5% β-mercaptoethanol (added fresh)). Electrophoresis was performed using 10.5-14% CriterionTM precast gels at 200 V constant according to BIO-RAD. Gels were stained with Comassie Blue R- 250 and then destained. Analyses of gel bands were performed using Image J program.

3.4.8 Glucose

Measurements of concentration of glucose were made using the Glucose (GO) Assay Kit (Sigma). Cultured cells in 1.5ml aliquots were concentrated by centrifugation, supernatant was collected to measure glucose released by cells and the pellet was kept to assay total glucose concentration in the cells. Each fraction was treated as kit’s instructions for liquids and solids respectively. Protocol was adapted to use with a 96-well micro-plate reader.

55 Chapter 3

3.4.9 Fast repetition rate fluorometry (FRRF)

Fast repetition rate Chlorophyll a fluorescence measurements of Symbiodinium cells were performed in 6ml dilutions made from 0.6ml samples of experimental cultures contained in 12 ml glass test tubes fitted inside the head of a benchtop settled Fast Act Fast Repetition Rate Fluorometer (Chelsea Instruments, West Molesey, UK). Calculations of the excitation pressure over PSII (Q) of the Symbiodinium cells treated with the HRFs and controls with ASP-8A culture media, were made by using the following formula:

Q=1- (Fq´/Fm´/ Fv/Fm)

Were Fv/Fm is the PSII maximum quantum yield of dark acclimated samples and Fq´/Fm´ is the effective quantum yield at steady state of samples under actinic light or the acclimation irradiance.

3.4.10 Statistical analyses

Fit of cell counts to the logistic sigmoidal model for the calculation of growth rates, the examination of data distribution, descriptive statistics, analysis of the variances and the principal component analysis of the data were made using R Development Core Team (2008). To test for normality on data sets Shapiro-Wilk normality test was applied. Linear models and ANOVA tests were applied to look for significant differences among groups and interaction of factors. For homogeneity of variance Levene’s Test was performed. Post hoc Tukey test were performed to determine which groups were significantly different. Whenever data sets violated assumptions of normality, data were log, square root or arcsine transformed. If data were still not normally distributed or variance was still not homogenous, non-parametric (Kruskal Wallis test) analyses were performed to test for significant differences between and within groups. Post-hoc pairwise comparisons using Conover test for multiple comparisons of independent samples was used when required to find out the significant different groups. When necessary Bonferroni adjustment for confidence intervals was applied.

56 Chapter 3 3.5 Results

There was a diverse effect of treatment across the two HRFs and four types of Symbiodinium cultured at the two photon fluxes (Table 7).

The concentration of chlorophyll a (pgcell-1) was significantly different between the two photon fluxes (H (1)=35.061, p< 0.05, Figure 11) used to culture the experimental types of Symbiodinium.

Total Glucose Released Glucose Chl a (pgcell-1) Protein (pgcell-1) Q (pgcell-1) (pgcell-1) PF% HRF (µmol• Type mean %%±SE mean ±SE mean ±SE mean ±SE mean ±SE m2•s1) HT 350 A1 0.285 0.061 581 158 635 126 94 5 0.620 0.007 % A13 0.304 0.045 207 21 823 25 90 8 0.691 0.001 C1 0.306 0.013 333 44 1369 191 149 22 0.812 0.006 PdS 0.541 0.040 407 99 493 89 252 83 0.751 0.004 100 A1 0.857 0.029 374 57 1211 330 180 60 0.542 0.011 A13 0.934 0.016 346 76 1237 219 138 39 0.495 0.033 C1 0.518 0.020 221 15 1100 210 58 34 0.464 0.021 PdS E E E E E E E E E E Control 350 A1 0.222 0.005 312 88 286 73 61 6 0.422 0.063 % A13 0.429 0.041 578 100 1039 217 74 15 0.682 0.021 C1 0.177 0.027 448 60 1187 164 105 19 0.797 0.018 PdS 0.624 0.092 226 65 312 23 84 43 0.738 0.027 100 A1 0.760 0.097 213 45 1014 79 104 12 0.589 0.058 A13 0.745 0.007 485 87 1573 340 99 27 0.476 0.070 C1 0.430 0.026 176 41 1185 258 117 53 0.537 0.031 PdS E E E E E E E E E E FAAmix 350 A1 0.382 0.048 436 22 529 3 259 82 0.711 0.024 % A13 0.759 0.088 193 29 922 131 191 19 0.413 0.102 C1 0.495 0.043 198 41 494 36 149 19 0.680 0.010 PdS 0.399 0.112 197 59 511 135 93 28 0.747 0.018 100 A1 0.655 0.096 249 33 522 61 147 11 0.571 0.035 A13 0.822 0.058 253 42 597 29 100 10 0.478 0.038 C1 0.780 0.063 640 118 546 14 117 10 0.471 0.015 PdS E E E E E E E E E E Control 350 A1 0.485 0.005 467 57 425 28 167 23 0.729 0.021 % A13 0.627 0.029 254 22 773 61 179 10 0.569 0.063 C1 0.549 0.078 269 57 722 114 261 60 0.640 0.009 PdS 0.624 0.092 226 65 312 23 84 43 0.738 0.027 100 A1 0.790 0.089 240 66 605 148 180 36 0.552 0.066 A13 0.828 0.068 295 31 550 48 168 21 0.312 0.068 C1 0.859 0.010 505 86 605 61 205 33 0.493 0.024 PdS E E E E E E E E E E

57 Chapter 3 ) 1 - (pgcell a Chl

HL LL Culture Treatment

Figure 11. Chl a (pgcell-1) of Symbiodinium cells cultured at two photon fluxes (HL=350 µmol photons•m- 2•s-1 and LL=100 µmol photons•m-2•s-1).

Comparisons between the type of HRF showed that this treatment did not contributed to differences in chlorophyll a but these differences were due to the light treatment only (Figure 12).

58 Chapter 3 ) 1 - (pgcell a Chl

HLFAA HLFAAC HLHT HLHTC LLFAA LLFAAC LLHT LLHTC Culture Treatment

Excitation pressure over PSII (Q) of Symbiodinium cells was opposite compared to Chl a concentration and was significantly higher in cultures at high compared with low photon flux (ANOVA(1)=47, p<0.0001; Figure 13).

59 Chapter 3

HL LL Culture Treatment

Figure 13. Excitation pressure over PSII (Q) between Symbiodinium cells cultured at two photon fluxes (HL=350 µmol photons•m-2•s-1 and LL=100 µmol photons•m-2•s-1). Comparison between cultures of Symbiodinium cells treated for one hour with two HRFs against their respective controls in media only, showed that differences in Q were produced by the photon fluxes and not by HRF treatments (Figure 14). Comparison across Symbiodinium cell types highlights the predominant effect of photon flux on both photosynthesis related parameters Chlorophyll a and Q. Chlorophyll a concentration was lower at high photon flux (HL=350µmol photons•m-2•s-1) cultures in all types of Symbiodinium cells. Although P. damicornis symbiont (PdS) was not cultured at low photon flux, comparison with other cultured types (clades) at this photon flux shows chlorophyll a concentration of PdS was also lower when cultured at a photon flux of 350µmol photons•m-2•s-1 than chlorophyll a concentration in the other types of Symbiodinium cultured at a photon flux of 100µmol photons•m- 2•s-1 with exception of C1 cultures treated with host tissue (HT) and C1 host tissue controls (HTC) treated only with media. The only significant difference found in chlorophyll a related with HRF treatment was found in type A13 cultured at 100 µmol photons•m-2•s-1, this type of Symbiodinium displayed a significantly higher amount of Chl a after one hour treatment with host tissue (HT.LL) in comparison with controls treated with media only (HTC.LL, p=0.013, Figure 15).

60 Chapter 3

HLFAA HLFAAC HLHT HLHTC LLFAA LLFAAC LLHT LLHTC Culture Treatment

Excitation pressure over PSII was lower in all types of Symbiodinium cultured at low photon flux (LL=100µmol photons•m-2•s-1) with exception of A13 cultures treated with free amino-acid (FAA) and A1 host tissue control cultures treated only with media (HTC) (Figure 15). Also as with chlorophyll a comparison, Q couldn’t be compared directly on PDS cultures at low photon flux but as well, comparison of PdS cultured at a photon flux of 350 µmol photons•m-2•s-1 shows excitation pressure over PSII was higher than in all the other types of Symbiodinium types cultured at a photon flux of 100 µmol photons•m-2•s-1. Also similarly as happened with chlorophyll a analyses, only one type displayed a significant difference in Q related with HRF treatment but in this case was found in type A1 cultured at 350 µmol photons•m-2•s-1, this type of Symbiodinium displayed a significantly higher Q after one hour treatment with host tissue (HTHL) in comparison with controls treated with media only (HTCHL, p=0.001, Figure 15).

61 Chapter 3

a *

600 0.75 * ) 1 - 1)

− Clade

pgcell A1

( 0.50 A13

a C1 PD Chl a (pg...ml

0.25 Chlorophyll

1) Clade − A1 400 0.00 FAAHL FAACHL HTHL HTCHL FAALL FAACLL HTLL HTCLLFAA.HL FAAC.HL HT.HL HTC.HL FAA.LL FAAC.LL HT.LL HTC.LL A13 0.8 b C1 PD Chl a (pg...ml *

0.6 * Clade A1 A13 0.4 C1 PD

200

Excitation Pressure over PSII (relative) Excitation Pressure over 0.2 Excitation Pressure over PSII (relative)

0.0

FAAHL FAACHL HTHL HTCHL FAALL FAACLL HTLL HTCLLFAA.HL FAAC.HL HT.HL HTC.HL FAA.LL FAAC.LL HT.LL HTC.LL Culture Treatment

Figure 15. Physiological parameters related with photosynthesis, chlorophyll a and excitation pressure over PSII measurements performed on four types of Symbiodinium A1, A13, C1 and symbiont isolated from scleractinian coral Pocillopora damicornis (PD). Symbiotic algae were cultured at two 0 photon fluxes (HL=350 µmol photons•m-2•s-1 and LL=100 µmol photons•m-2•s-1) and treated with two different types of host release factors (Free Amino-Acids=FAA, Host Tissue=HT and ASP- 8A=FAAC and HTC. Al measurements were normalized to picograms per cell (pgcell-1). a Chlorophyll a. b Excitation pressure over PSII. HL.FAA LL.FAA HL.FAAC LL.FAAC HL.HT LL.HT HL.HTC LL.HTC

62 Chapter 3

Protein in cultured cells of Symbiodinium at both photon fluxes was not significantly different after 1 hour treatments with two types of HRFs in comparison with controls treated only with ASP-8A media (Figure 16). ) 1 - Protein (pgcell

HLFAA HLFAAC HLHT HLHTC LLFAA LLFAAC LLHT LLHTC Culture Treatment

Similarly as with protein, comparison of the effect in total glucose per cell of Symbiodinium cultured at two different photon fluxes after one hour treatments with two HRFs did not display any significant differences against controls treated only with media (Figure 17).

63 Chapter 3 ) 1 - pgcell Glucose ( Total

HLFAA HLFAAC HLHT HLHTC LLFAA LLFAAC LLHT LLHTC Culture Treatment

Comparison of protein between all types of Symbiodinium treated with two HRFs and versus controls treated only with media, at both photon fluxes, displayed significant differences in some clades treated with host tissue and their controls (H(-26)=52.9 p=0.001). Protein in Symbiodinium type A13 cultured at a photon flux of 350 µmol photons•m-2•s-1 was significantly lower in cultures treated with host tissue (HT) than controls treated only with media (HTC) (p<0.001) (Figure 18). In contrast with type A13 total protein in type A1 cultures at a photon flux of 350 µmol photons•m-2•s- 1 (p=0.047) and also at a photon flux of 100µmol photons•m-2•s-1 (p=0.024) were significantly higher in cultures treated with host tissue (HT) compared with controls treated only with media (HTC). As well as with type A1, total protein in Symbiodinium isolated from P. damicornis but only in cultures at 350 µmol photons•m-2•s-1 was significantly higher after one hour of treatment with host tissue (HT) than controls only treated with media (HTC, p=0.032; Figure 18). Comparison of total glucose between all types of Symbiodinium treated with two HRFs versus their controls treated only with media, at both photon fluxes, displayed significant differences ((H(26)=63.47 p<0.001). Only total glucose in type A1 cultured at a photon flux of 350µmol photons•m-2•s-1 was significantly higher after 1 hour treatment with host tissue (HT) compared with controls treated only with media (HTC, p=0.008; Figure 18).

64 Chapter 3

* * 600 a 600

) * 1 - 1)

− * Clade 400 A1 * A13 C1 PD

Protein (pg...ml * * * 200 Protein (pgcell

1) FAAHL FAACHL HTHL HTCHL FAALL FAACLL HTLL HTCLLFAA.HL FAAC.HL HT.HL HTC.HL FAA.LL FAAC.LL HT.LL HTC.LL Clade − A1 400 2000 b A13 ) 1 - C1 1500 PD Chl a (pg...ml 1) −

Clade A1 1000 A13 C1 PD

Glucose (pgcell * Total Glucose (pg...ml Total

500 Total *

200 0 FAAHL FAACHL HTHL HTCHL FAALL FAACLL HTLL HTCLLFAA.HL FAAC.HL HT.HL HTC.HL FAA.LL FAAC.LL HT.LL HTC.LL Culture Treatment

Figure 18. Physiological parameters protein and total glucose measured in on four types of Symbiodinium A1, A13, C1 and the symbiont isolated from scleractinian coral Pocillopora damicornis (PD). Symbiotic algae were cultured at two photon fluxes (HL=350 µmol photons•m-2•s-1 and LL=100 µmol photons•m-2•s-1) and treated with two different types of host release factors (Free Amino- Acids=FAA, Host Tissue=HT and ASP-8A=FAAC and HTC. Al measurements were normalized to picograms per cell (pgcell-1). a Chl a. b Protein. c Total Glucose.

Glucose measured in the supernatant referred from now as glucose released (GR) per cell (pgcell-1) did not differ when compared between the two culture photon fluxes (HL=350 µmol photons•m-2•s-

0 65

HL.FAA LL.FAA HL.FAAC LL.FAAC HL.HT LL.HT HL.HTC LL.HTC Chapter 3

1 and LL=100 µmol photons•m-2•s-1, Figure 19). However comparison between HRF treatments versus controls displayed significant differences (H(3)=25.53, p<0.001, Figure 20). In general cultures treated with free amino acid (FAA) were lower in glucose released than controls treated with media (FAAC, Conover’s p<0.014) and cultures treated with host tissue (HT) were higher in glucose released than controls treated only with media (HTC, p=0.022, Figure 20). Comparison of host release treatments at two photon fluxes showed significant differences (H(7)=28.08, p<0.001) only occurred, for cultures treated with FAA versus FAAC at a photon flux of 100 µmol photons•m-2•s-1 (p=0.018) and for cultures treated with HT versus HTC at 350µmol photons•m-2•s- 1(p=0.012, Fig Figure 21). Further analysis comparing glucose released between all types of Symbiodinium versus their controls showed significant differences (H(26)=48.81, p<0.004) occurred in type PDS treated with HT released significantly more glucose than controls HTC at 350µmol photons•m-2•s-1 (p=0.004) and in contrast, types C1 and A13 treated with FAA released significantly less glucose than controls FAAC at 100µmol photons•m-2•s-1 (p=0.038 and p=0.046 respectively, Figure 22). ) 1 - pgcell ( Glucose Released

HL LL Culture Treatment

Figure 19. Glucose released (pgcell-1) when compared Symbiodinium cells cultured at two photon fluxes (HL=350 µmol photons•m-2•s-1 and LL=100 µmol photons•m-2•s-1).

66 Chapter 3 ) 1 - pgcell ( Glucose Released

FAA FAAC HT HTC Culture Treatment

Figure 20. Glucose released (pgcell-1) of Symbiodinium cells treated with two host release factors (FAA=Free Amino Acids, HT=Host Tissue) and ASP-8A media only as controls (FAAC and HTC). ) 1 - pgcell ( Glucose Released

HLFAA HLFAAC HLHT HLHTC LLFAA LLFAAC LLHT LLHTC Culture Treatment

67 600

Chapter 3

1) * Clade − A1 400 300 A13 C1 ) 1 - * PD 1) Chl a (pg...ml −

pgcell 200 * Clade A1 A13 C1 PD * * * Glucose Released(pg...ml 200 100 Glucose Released (

FAAHL FAACHL HTHL HTCHL FAALL FAACLL HTLL HTCLLFAA.HL FAAC.HL HT.HL HTC.HL FAA.LL FAAC.LL HT.LL HTC.LL Culture Treatment

Figure 22. Glucose released by cells of four types of Symbiodinium A1, A13, C1 and symbiont isolated from scleractinian coral Pocillopora damicornis (PD). Symbiotic algae were cultured at two photon 0 fluxes (HL=350 µmol photons•m-2•s-1 and LL=100 µmol photons•m-2•s-1) and treated with two different types of host release factors (Free Amino-Acids=FAA, Host Tissue=HT and ASP- 8A=FAAC and HTC. All measurements were normalized to picograms per cell (pgcell-1). HL.FAA LL.FAA HL.FAAC LL.FAAC HL.HT LL.HT HL.HTC LL.HTC

Principal component analyses of physiological parameters measured in cell cultures, showed first two components explain 57.5% of the total variation observed. The third component adds 18% of total variation giving a 75.5% of total variation explained by these three components. The fourth component adds 16.7% giving a total of 92.2% of the total variation explained. Chlorophyll a is the most important variable in first component followed by Q negatively correlated with each other. The third most important element of first component is glucose released, positively correlated with Chl a. Total protein is the most important variable in the second component followed by Q as the second most important element, positively correlated with each other. Glucose released is the third most important element. In the third component, total glucose is the most important variable followed by glucose released and they are negatively correlated with each other. In fourth

68 Chapter 3 component, the most important variable is total protein, followed by Q, glucose released and total glucose, all negatively correlated with total protein (Figure 23).

Figure 23. Principal component analysis (PCA) of physiological parameters of Symbiodinium cultured types A1, A13, C1 and the symbiont of the scleractinian coral Pocillopora damicornis (PD). Symbiotic algae were cultured at two photon fluxes (HL=350 µmol photons•m-2•s-1 and LL=100 µmol photons•m-2•s-1) and treated with two different types of host release factors (Free Amino- Acids=FAA, Host Tissue=HT and ASP-8A=FAAC and HTC. All measurements were normalized to picograms per cell (pgcell-1).

Comparison over areas of lines between different protein samples of Symbiodinium types, treated with two HRFs (free amino acids (FAA) and host tissue (HT)) and ASP-8 media (Controls), showed there were not significant differences between treatments across lines of all four types.

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Line Band1 Band2 Band3 Band4 Band5 HRF Type mean ±SE mean ±SE mean ±SE mean ±SE mean ±SE mean ±SE FAA A1 48734 10568 1247 218 699 166 691 170 3440 360 2317 655 A13 53716 7909 1622 569 1844 630 1189 52 3067 485 2381 208 C1 61069 15746 1614 264 1942 156 1514 520 5018 1998 3367 1334 PdS 47130 6665 1400 128 630 126 585 102 2832 1496 2508 1387 HT A1 74167 6524 3261 1698 934 66 1331 285 2969 234 3832 931 A13 71559 3879 3458 1850 1722 621 1416 328 4502 1435 2886 583 C1 97208 4889 3522 1407 2711 602 1864 358 6860 3622 8363 1924 PdS 73538 15691 3365 1080 1498 392 1277 227 3177 2168 4901 260 Control A1 70940 20823 2150 324 1021 368 1070 495 2508 635 3447 552 A13 62239 6927 2169 765 1019 228 733 88 2735 256 3840 737 C1 71451 758 2489 1572 1760 389 1254 314 6002 3092 7056 1087 PdS 62946 4055 1556 70 778 158 722 171 3047 1295 3150 1195

A general analysis of band areas between HRFs treatments showed band 1 was significantly different between HT vs FAA (H(2)=9.79, p=0.008). More specifically after comparison between treatments and types, Band 1 was significantly different between: A1FAA vs A1HTC, C1HT, PdSHT (ps=0.05, 0.02, 0.02 respectively); PdSFAA vs C1HT (ps=0.042) and PdSHT vs PdSFAA (ps=0.042). In general Band 2 was significantly different (H(11)=26.9, p=0.035) across Symbiodinium types: PdS vs C1 and A13 (ps<0.001 and <0.05 respectively); A1 vs C1 and A13 (ps<0.001 and <0.05 respectively) and C1 vs A13 (ps=0.05). Band 3 was significantly different between HRFs (H(2)=6.88, p=0.038) HT vs HTC and FAA (ps<0.05), specifically: PdSFAA vs A1HT, A13FAA, A13HT, C1FAA, C1HT (ps=0.03, 0.04, 0.04, 0.04 and 0.004 respectively); PdSHT vs PdFAA (p=0.048); PdSHT vs C1HT (p=0.01); C1HT vs A13HTC and A1FAA (p=0.01 and 0.009 respectively). Band 4 was not significantly different between HRFs or types of Symbiodinium. In general Band 5 was significantly different between HRFs (H(2)=7.55, p=0.02), in particular FAA vs HT and HTC (ps=0.01, 0.02 respectively) and between Symbiodinium types: C1 vs A1, A13, PdS (ps=0.022, 0.015 and 0.035 respectively; Table 8, Figure 24). Band 5 also was significantly different in C1FAA vs C1HT and C1HTC (ps=0.02, 0.04 respectively; Table 8)

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A13FAA% 200#$ 120#$ 1" $85#$ 2" 3" 70#$ 60#$

$50#$

4" 40#$

PDHT% 30#$ 5"

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3.6 Discussion

Acquisition of nutrient exchange metabolism is one of the most important characteristics for the establishment and maintenance of an obligated symbiotic relationship between “the architects of the reef” scleractinian corals with their highly productive photosynthetic algae Symbiodinium (Davy et al. 2012). Light modulated by the host plus an efficient transfer of inorganic nutrients to Symbiodinium, are paid off with the translocation of photoassimilated carbon from the symbiont to its animal host (Marcelino et al. 2013, Tremblay et al. 2014). This trade must be highly regulated and controlled in order to maintain homeostasis during variable environmental conditions across different spatial scales (Cunning et al. 2015).

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Regulation of symbiosis has been studied from many different perspectives to consider mechanisms of coral host control over the Symbiodinium (Weber & Medina 2012). Some studies have suggested that the coral host can impact photosynthesis of its symbiont by reducing the amount of nutrients, limiting CO2, acidifying the symbiosome lumen or controlling translocation of carbon products (Yellowlees et al. 2008, Meyer & Weis, 2012). The present study aimed to compare the physiology and protein expression of four different types of Symbiodinium cultured at two photon fluxes after one hour treatments with two different types of HRFs versus controls treated with ASP-8A media. Results on response to treatments from measurements of parameters directly related to the photophysiology of Symbiodinium such as chlorophyll a concentration and excitation pressure over PSII (Q) were mainly affected by the light environment in all types. This is in agreement with other studies where cultures of various types of Symbiodinium at low photon fluxes display more concentration of chlorophyll a than cultures at higher photon fluxes to increase the capture of photons (Iglesias-Prieto & Trench 1997a, Hennige et al. 2009). Furthermore, other studies have shown how cultures at increased photon fluxes also display higher Q in comparison with cultures at low photon fluxes (Iglesias-Prieto et al. 2004, Suggett et al. 2015). As demonstrated by the PCA, Chl a and Q were negatively correlated (first component). This may be explained through a reduction in the amount of Chl a concentration in the external and internal light harvesting complexes acting as a form of non-photochemical quenching (NPQ) strategy to reduce the amount of excitation pressure in a high photon flux environment and thus avoid probable damage of the PSII reaction centers (Iglesias-Prieto & Trench 1997a, Iglesias-Prieto et al. 2004, Suggett et al. 2015). In this study, the significant increment on excitation pressure over PSII found on type A1 cultures at 350µmol photons•m-2•s-1, after one hour treatment with host tissue in comparison with ASP-8A treated controls, was not accompanied by a lower concentration on Chl a (pgcell-1). Distinctively type A13 cultured at 100µmol photons•m-2•s-1 were show to have a significantly higher concentration of Chl a (pgcell-1) in the presence of host tissue in comparison with its controls with media only. However, this difference did not arise with any significantly inverse effect on Q compared with media controls.

Chlorophyll a is coupled to proteins in the membrane of thylakoids of chloroplasts in the form of chlorophyll-protein-complexes such as light harvesting complexes and photosystems (Hiller et al. 1993, Iglesias-Prieto et al. 1993, Iglesias-Prieto 1996). Q is also related with the number of chlorophyll-protein-complexes and reaction centers in the photosynthetic units of chloroplasts in the cell (Suggett et al. 2015). In addition to this, other photosynthetic and non- photosynthetic proteins are expressed and regulated for many other cell processes linked with photosynthesis such as RuBisco and GAPA as well as glycolysis and respiratory mitochondrial

72 Chapter 3 proteins sensitive to photorespiration and AET or even housekeeping proteins of the HSP family (Curien et al. 2017, Shimakawa et al. 2017). Many of these proteins may be involved with mechanisms of nutrient translocation and possibly its regulation may be controlled by the host. In this study, significant differences in total protein (pgcell-1) were found in specific Symbiodinium types following one hour treatments with two types of HRFs and as compared with ASP-8A media controls. Total protein reduction in type A13 at 100µmol photons•m-2•s-1 treated with host tissue compared with controls, matched with chlorophyll a increases. It is likely that both parameters are involved in a photosynthesis-inhibitory mechanism, perhaps related to disconnection of antenna, caused by host tissue control over this type of Symbiodinium. This is supported by the observation that increases in Chl a and total protein reductions were accompanied by an evident decrease in total glucose (pgcell-1) at both photon fluxes treated with host tissue even though it was not significant. Inhibition of photosynthesis in the presence of host factors has been previously documented (Grant et al. 2001, 2013). Glucose released (pgcell-1) was significantly inhibited in this study when types C1 and A13 cultured at 350µmol photons•m-2•s-1 were treated for one hour with FAA. This free amino acid artificial host factor also seemed to inhibit the glucose released in PdS in comparison with controls and natural host tissue treatment. In PdS cultured at 350µmol photons•m-2•s-1 and with host tissue there was an increase in glucose released as well as in total protein. This result suggests protein is involved in a glucose releasing mechanism, which is also supported by the positive correlation of these two variables (also Q) within the second component of PCA.

It is well documented that glucose is released (as well as other carbon products such as amino acids and lipids) by Symbiodinium cells in the presence of HRFs similar to those used in this study (Gates et al. 1995, Burriesci et al. 2012, Grant et al. 2013). The mechanism of glucose releasement was induced in PdS in the presence of the tissue of its coral host. In ype A1 increases in total protein at both photon fluxes matched with a significant increase in Q at the higher photon flux an also with a significant increase in total glucose after an hour treatment with the host tissue treatment. In the second component of the PCA total protein, total glucose and Q were positively correlated with glucose released. Furthermore, in the third component of PCA glucose released and total glucose are the most important variables and showed a negative correlation. This analysis leads to the interpretation that although there was a significant increase in total glucose in type A1 produced by effect of the host tissue, this treatment did not induce the mechanism of glucose releasement or translocation in this type of Symbiodinium.

Previous studies on HRF isolation have suggested it is a low molecular weight amino acid or polypeptide (Grant et al. 2013). Protein analysis with SDS-PAGE of samples extracted from the

73 Chapter 3 four types of Symbiodinium following one hour of treatment with the HRFs (and appropriate culture media controls) showed significant differences in areas of protein bands that were excised for further identification with MALDI-TOF analysis (Figure 24). Of particular interest were bands 1 and 5. Band 1 showed to be significantly more expressed in the symbiont of P. damicornis treated with host tissue compared with the free amino-acid mix treatment. This band also appeared more expressed than in controls but this difference was not significant. This protein band might be involved with the mechanism of glucose releasement activated on the coral symbionts treated with their host tissue. Band 5 was significantly less expressed in C1 treated with free amino-acid mix in comparison to host tissue and control treatments. This protein band may be probably related with the inhibition of glucose releasement found in C1.

In conclusion, the parameters related with the photochemistry of the types of Symbiodinium appeared ruled by the light treatment rather than by the HRF treatment. Chl a content and excitation pressure over PSII were inversely effected by the photon flux to balance the production of ATP and NADPH for photosynthesis and other metabolic pathways. The translocation of glucose was induced in the Symbiodinium with the host tissue of its coral host P. damicornis. This HRF also appeared to stimulate photosynthesis and induce the glucose production in A1 but did not activate the mechanism of translocation in this type. The free amino-acid mix HRF inhibited the translocation of glucose in C1 and A13. Based in the distinction between the parameters influenced by the photon flux and those influenced by the HRF, it is probable that at some point between the electron transport reactions and the production of glucose, the HRF is effecting the stimulation or inhibition of glucose translocation by some protein mechanism. This mechanism of translocation of glucose seemed to be stimulated or inhibited by the differential expression of protein bands 1 and 5 respectively. The identification of these protein bands with MALDI-TOF analysis would probably clarify the mechanism of glucose release and its inhibition in the presence of these host factors. This would also give a better understanding of the control exert by the host over the physiology of the Symbiodinium and its role in the holobiont.

74 Chapter 4 Chapter 4 Effect of temperature on the physiology and protein expression of Symbiodinium in culture

Abstract

Corals build reef structures from calcium carbonate fixation by means of the photosynthetic products obtained from the symbiosis with dinoflagellates of the genus Symbiodinium. Studies suggest the different expression of photosynthetic proteins is associated with the survival of Symbiodinium within the host when environments vary. Disruption in photosynthesis of Symbiodinium caused by increased ocean temperatures is known to be the main cause for mass coral bleaching. Early studies on the short acclimation response of Symbiodinium microadriaticum to high temperature suggest photosynthetic disruption occurs above 30ºC. Recently, omic’s science technologies are delivering new insights into the mechanisms of maintenance of the symbiosis. However, studies have shown photoacclimation processes are mainly controlled by posttranslational modifications. The use of proteomics is delivering some promising results to explain the mechanisms of photoacclimation related with the symbiotic state. The present study is dedicated to dig inside the effect of temperature over the photoacclimation proteome of Symbiodinium. The physiology and proteome of three types (A1, A13 and C1) of Symbiodinium belonging to the two clades with the largest distribution in the Caribbean (clade A) and in the Indo-Pacific (clade C) were compared at two growth photon fluxes and two temperatures (100µmolm2s-1 and 350µmolm2s-1; T=26ºC and 30ºC). Furthermore, cultures of these three types acclimated at these photon fluxes and temperatures, were also compared after being subjected to three days of constant stress at 33ºC. Shotgun proteomics and 2-DE analyses displayed proteome differences in light harvesting, electron transport, carbon fixation and protein folding across types that were also differentially expressed between photon fluxes and temperature treatments as well as after being under constant stress at 33ºC. The differential proteome across types related to the processes of photoacclimation, temperature acclimation and response to stress seemed associated with the activation of alternative electron transport (AET) to balance ATP production and fuel the cell with energy at the different experimental light, temperature and stress conditions. These differences suggest that the Symbiodinium proteome for photoacclimation has an important role with stress coping hence, also with the maintenance of holobiont’s homeostasis in the reef.

4.1 Introduction

Coral reef ecosystems are endangered if the rise of ocean temperature keeps on with recent predictions based on climate change scenarios (Hooidonk et al. 2014, Kwiatkowski et al. 2015). The dynamic nature of coral symbiosis secures the stable maintenance of the holobiont’s

75 Chapter 4 physiology and is supported predominantly by the coordination of the host and symbiont metabolic interactions (Rowan, 1998).

As a general pattern the breakdown of coral symbiosis by temperature, termed bleaching is increasing in frequency and intensity (Hughes et al. 2007, 2017, 2018). Differential responses between coral species have suggested some are more susceptible or resilient to bleaching (Baird et al. 2007, Bellantuono et al. 2011, Downs et al. 2013). These variable responses raise the question whether it is a product of differential adaptations of the symbiotic alga, the coral host or the holobiont per se (Pinzón et al. 2015, Pettay et al. 2015).

The recently recognized diversity of Symbiodinium types and subtypes by the use of higher resolution molecular markers has revealed how these different genotypes can display different physiological adaptations to respond to environmental variation with some subtypes being more tolerant or sensitive than others (LaJeunesse & Trench, 2000, LaJeunesse 2001, LaJeunesse & Thornhill 2011, Arif et al. 2014, LaJeunesse et al. 2014, Tonk et al. 2014, Hume et al. 2015). This functional diversity has direct effects on the symbiotic relationship of Symbiodinium with different coral hosts in relation to the benefits and costs depending on how mutualistic or parasitic the type of Symbiodinium and how wider the host control may be interacting to determine the productivity and stability of the symbiosis (Grottoli et al. 2014, Baker et al. 2015, Pettay et al. 2015). Most studies on coral symbiosis have focused around the photophysiology of the algal symbiont and its modification with environmental variation in particular, with acclimation to different light intensities and also to different temperatures. Less research has been conducted to investigate how the modification of the photophysiology of the algal symbiont produces various effects on the physiology of the host and its response to environmental change (Iglesias-Prieto & Trench 1994, 97, Robison & Warner, 2006; Hennige et al. 2009).

Only recently, research has shown there are other biochemical and physiological pathways different than the photosynthetic metabolism of Symbiodinium that could be affected by environmental variation with cascading consequences in the symbiosis (Chen et al. 2012, Bayer et al. 2012, Lesser et al. 2013, Baker et al. 2013, 2015). Some studies suggest some subtypes of Symbiodinium are more tolerant or sensitive to the negative synergistic effect of temperature elevation together with high irradiance. This variability has been related with different rates of photo-damage and photo-repair. However, this does not explain in full the mechanisms associated with this physiological diversity (Warner et al. 1996, Robison et al. 2006, Ragni et al. 2010, Krämer et al. 2012). Previous studies on the acclimation of Symbiodinium to different photon flux densities have revealed how different types are able to modify the quantity of components and

76 Chapter 4 pigment characteristics of their photosynthetic machinery in order to optimize light utilization for photosynthesis according to different strategies (Chang et al. 1983, Iglesias-Prieto & Trench 1994, 97, Hennige et al. 2009). These acclimation strategies might also account for the different responses to temperature changes and stress (Ragni et al. 2010, Takahashi et al. 2013).

One of the first studies examined the short-term acclimation capacity to increased temperature by Symbiodinium microadriaticum suggesting that photosynthesis would decrease after a gradual rise in temperature up to 30°C as a result of changes in the lipid content of the thylakoid membranes. Other studies have demonstrated this to occur but also showed the melting point varies between different types of Symbiodinium (Iglesias-Prieto et al. 1992, Tchernov et al. 2004, Diaz-Almeyda et al. 2011). In addition, previous work has pointed to the component of the photosystem II the protein D1 as the primary site where failure of photosynthesis and consequent bleaching occurs, however this does not totally explain the variable tolerance/sensitivity of different types of Symbiodinium (Warner et al. 1996). It has been suggested the variability is conferred by different configurations of proteins and pigments for photoprotection mechanisms and alternative electron transport pathways, this may generate the different photoacclimation strategies observed among the Symbiodinium types (Robison & Warner 2006, Reynolds et al. 2008, Krämer et al. 2012, Takahashi 2008, 2009, 2013, Roberty et al. 2014). The photoacclimation mechanisms may also vary during the symbiotic state to adjust with the light microenvironment within the host. Besides, a failure in the mechanisms of host control caused by temperature stress may as well affect the mechanisms of photoacclimation of the symbiont and cause a disruption in the stability of the symbiosis. This failure may have a differential effect on the exchange of metabolites between the host and the algal symbiont, this may be in part producing the differential patterns of bleaching found in the reef (Wooldridge 2009, 2010, 2013, 2014, Hennige et al. 2010, Oakley et al. 2014a, b, Baker et al. 2015).

The variability on the thermal stress interruption of the metabolic exchange within different holobionts must involve a differential protein regulation between partners along the symbiotic range (Weber & Medina 2012, Rosic et al. 2015, Hoadley et al. 2015, Lin et al. 2015). Research focused on the differential genetic and transcriptomic response of the holobiont under temperature stress has not been able to find symbiotic patterns of protein expression to explain the variable response among host-symbiont assemblages. This suggests the response to stress mainly occur under post-translational modification of the proteins dedicated to upkeep the physiology of the holobiont (Leggat et al. 2011, Davy et al. 2012, McGinley et al. 2013, Butterfield et al. 2016).

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Due to the nature of this symbiotic relationship its study has been difficult to approach with traditional techniques (Meyer & Weis 2012). The advances in “omics” technologies allow the rapid generation of new information about the essential molecules associated with the response to bleaching and the maintenance of the symbiosis. This approach can help to understand the actual contribution of each of the partners to the physiology of the holobiont (Peng et al. 2010, Weber & Medina 2012, Meyer &Weis 2012, Lehnert et al. 2014, Weston et al. 2012, 2015, Rosic et al. 2015, Lin et al. 2015). Traditional proteomic techniques such as 2D-PAGE are being improved with the use of mass spectrometry techniques such as LC\MS-MS and MALDI-TOF to sequence and identify complex protein samples. These methods in junction with physiological approaches can reveal important information about the modifications upon biological systems such as the modifications generated in the coral symbiosis by the change in environmental variables (Li et al. 2014, Weston et al. 2012, 2015, Ricaurte et al. 2016, Oakley et al. 2016).

Climate change is acting on the holobiont diversity by challenging adaptation capacity of the partners to higher temperatures, as has been found in previous studies on extreme temperature environments (Hoadley et al. 2015, Hume et al. 2015, Howells et al. 2016). Whether these adaptations will be fast enough for the persistence of coral reefs waits to be resolved. This study is based in hypotheses that associate the different responses to temperature stress of different types of Symbiodinium with their different photophysiological strategies. The mechanisms behind these associations may be explained by the differential proteome response to temperature stress of cultures previously acclimated at different photon fluxes and different temperatures.

4.2 Hypothesis

If different types of Symbiodinium display differential acclimation to high temperature and tolerance to temperature stress then these differential responses should be regulated by their differences in photophysiology and proteome revealing their role in the maintenance of the diverse symbiotic relationships.

H1: If high temperature impacts photoacclimation differentially across Symbiodinium types then the acclimation to high temperature will be regulated by differences in the photophysiology and proteome across these types revealing their role in the maintenance of the diverse symbiotic relationships.

H2: If different types of Symbiodinium have different tolerances to temperature stress then these differences will be regulated by differences in the photophysiology and proteome

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across these types revealing their role in the maintenance of the diverse symbiotic relationships.

4.3 Objectives

O1: Characterise the differences in photophysiology (growth rates, Chl a absorbance and fluorescence parameters) and the protein expression (mass spectrometry and 2-DE analyses) across three cultured types of Symbiodinium acclimated at two photon fluxes (HL= 350 µmol photons•m2•s-1 and LL= 100 µmol photons•m-2•s-1), after acclimation for ten months at two temperatures (26 ºC and 30 ºC). O2: Characterise the differences in photophysiology (Chl a fluorescence) and the protein expression (mass spectrometry and 2-DE analyses) across three cultured types of Symbiodinium acclimated at two photon fluxes (HL= 350 µmol photons•m2•s-1 and LL= 100 µmol photons•m-2•s-1) and at two temperatures (26 ºC and 30 ºC), after being subjected to constant high temperature stress at a temperature of 33 ºC for three days.

4.4 Materials and Methods

4.4.1 Cultures

The experiments were carried out on symbiotic dinoflagellates of the genus Symbiodinium belonging to the widest distributed clades in the Caribbean (A) and in the Indopacific (C). More precisely types A1, A13 and C1 as previously characterized by analysis of the ribosomal ITS2 sequence (LaJeunesse 2001). Type A1 known to be thermo-tolerant was previously described as Symbiodinium microadriaticum, originally isolated from the jellyfish Cassiopeia xamachana (Freudenthal 1962). Type A13 is recognized as thermo-sensitive, described as Symbiodinium necroappetens was originally isolated from the sea anemone Condylactis gigantean. Finally type C1 also thermo-tolerant, shares the same ITS2 with Symbiodinuim goreaui was isolated from the corallimorph Discosoma sanctithomae (Robison & Warner 2006, et al. 2015, Ragni et al. 2010, Krämer et al. 2012, Suggett et al. 2015, Yuyama et al. 2016).

Batch cultures of the three Symbiodinium types were maintained and grown at two different temperatures, either in 2L Durand flasks on 1L of ASP-8A media (Blank 1987) at 26° C or in 3L

79 Chapter 4 water jacketed flasks also on 1L of ASP-8A media at a temperature of 30° C generated by a constant water flux from a water bath set at the experimental temperature.

Light cycle for the two experimental temperature conditions was 12:12 light: dark at two photon fluxes (PFs: high photon flux HL=350 µmol photons•m2•s-1 and low photon flux LL=100 µmol photons•m-2•s-1) as measured with a 4π light sensor (Li-Cor 193SA, Lincoln, NE, USA) submerged on ASP-8A media within the Durand flasks or the water jacketed flasks. Cultures were maintained under constant aeration and stirring to avoid air limitation and the aggregation of the algal cells and their attachment to the flasks glass walls.

For the temperature stress experiment 36 culture flasks of 500ml with 250ml of ASP-8A media with the three types of Symbiodinium previously acclimated at 26 ºC and 30 ºC (3 cultures times 3 types = 9 cultures times 2 Photon Fluxes =18 cultures times 2 Temperatures = 36 cultures in total), were set at their two previously mentioned PFs but at the stressful temperature of 33ºC. The photophysiological parameters of each culture were measured at three different times per day for 3 days and protein samples of each culture were also collected at the end of the experiment for proteomic analysis.

4.4.2 Growth

Characterization of growth rates for the three Symbiodinium types was carried out in 1ml aliquots of fresh culture fixed with 10µl of Lugol´s solution. Cell counts were made every second day using an improved haemocytometer under the microscope until cultures reached the end of the exponential phase and the beginning of the stationary phase. New cultures were initiated by dilutions using fresh media till concentrations reached densities of 50000 cell·ml-1. This process was repeated three consecutive times per type. The specific growth rate (µ, day-1) was calculated applying the following formula:

µ=ln2/r

After fitting the cell counts to a logistic sigmoidal function of the form:

y= α/(1+exp(-(β-x0)/γ)

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4.4.3 Photophysiology

Cultures were sampled along the growth curve. Aliquots of 25-50 ml were pelleted by centrifugation 5 min at 2500rpm and resuspended in 1ml of ASP-8A media. The absorbance of the live cells was recorded from 350-750nm using a U-3000 spectrophotometer fitted with an integrating sphere to minimize scattering (ø60, Hitachi High-Technologies, Berkshire, UK).

Alternative samples were also pelleted and resuspended in 1ml of 100% methanol to extract the pigments and stored at -20°C overnight. The absorbance spectra of methanol pigment extracts were recorded also from 350-750nm. The chlorophyll concentration from the methanol extracts was calculated from the equations of Ritchie (Ritchie et al. 2006). The chl a specific absorption coefficient a* was calculated from the formula:

a*= (D/ρ) ln10

Where ρ is the chl a concentration (mg·m-2) and D the optical density at 675nm (Enríquez et al. 2005).

Fluorescence measurements of the PSII maximum quantum yield of dark acclimated samples (Fv/Fm), the effective quantum yield at steady state of samples under actinic light (Fq´/Fm´), PSII cross section of dark acclimated samples (σPSII) and PSII cross section of samples under actinic light or the acclimation irradiance (σPSII´) for the calculation of the relative RCII-specific-rate of electron transfer (rETRRCII) were made using a bench-top Fast Repetition Rate Fluorometer (Chelsea Instruments, West Molesey, UK).

Samples of 3ml of culture were dark acclimatized for 30 minutes before being flashed to achieve PSII single turnover saturation with 100 flashlets of 1.1 µs applied at 1-s intervals (Suggett et al. 2003, 2004). Rapid light curves (RLC) were made applying the same PSII single turnover saturation pulse as for the dark adapted single acquisitions. Samples were dark acclimatized for 30 min and then light saturated every 20 s interval during a 3 min exposition to each of 12 increasing actinic lights (0, 6, 22, 55, 105, 171, 295, 505, 750, 996, 1263, 1454 and 1646 µmol photons•m-2•s- 1). Calculations of the RCII-relative rate of electron transfer were made with the formula:

rETRRCII = PAR · Fq′/Fm′ · 0.42

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Where rETRRCII is the RCII-relative rate of electron transfer, PAR is the photosynthetically active radiation (µmol photons•m-2•s-1) and the “ETR factor” 0.42 assuming that 84% of all light is absorbed and half is delivered to PSII for photochemistry (Szabó et al. 2014).

4.4.4 Proteomics

4.4.4.1 Protein extraction

Symbiotic dinoflagellates of the three experimental phylotypes at the end of the exponential/beginning of the stationary phase were harvested from 200 ml of fresh culture and concentrated by centrifugation for 5 min at 2500 rpm. The protein extraction was made as described by Li et al. (2012). Trizol reagent (1mL) was added to the cell pellet, followed by sonication on ice. Then 200µL of chloroform were added to the cell lysate before shaking vigorously for 15 s. The mixture was allowed to stand for 5 min at room temperature before centrifugation at 12000rpm for 15 min at 4°C. The top pale-yellow to colourless layer was removed, 300µL of ethanol were added to resuspend the reddish bottom layer and the mixture was centrifuged at 5000rpm for 5 min at 4°C. The supernatant was transferred to a new tube and 2 mL of isopropanol were added. Then, the mixture was stored at −20°C for at least 1 h to precipitate proteins. The mixture was then centrifuged at 14000rpm for 30 min at 4°C and the recovered pellet was briefly washed with 95% ethanol and then air dried.

4.4.4.2 Protein determination and Iso-Electro-Focusing (IEF) as 1st dimension

Protein concentration from the sample was determined by the Bradford assay (Bradford 1976). For the first-dimension electrophoresis, 40ug of protein were resuspended in rehydration buffer containing 8 M urea, 2 M thiourea, 2% w/v CHAPS, 65mM DTT, 0.5 % v/v IPG buffer, 2% v/v DeStreak reagent. Bio-Rad 11cm long IPG strips of linear pH gradient 4–7 were passively rehydrated overnight in 200µl of rehydration buffer covered with mineral oil to avoid evaporation and precipitation of the protein sample. Iso-Electro-Focusing (IEF) was performed in the following manner: 50V-125Vh, 100V-150Vh, 200V-300Vh, 500V-350Vh, 1000V-750Vh gradient mode and 4000V-16000Vh, 8000V-18000Vh hold mode.

4.4.4.3 Equilibration

82 Chapter 4 min. The first equilibration buffer was decanted and the strips were immersed in 10 mL of equilibration buffer DTT free and with 260mM iodoacetamide and placed for another 15 min on an orbital shaker.

4.4.4.4 SDS-PAGE as second dimension

The second dimension SDS-PAGE electrophoresis was performed using 10-14.5% polyacrylamide gels (Bio-Rad). After equilibration, the IPG strips were rinsed 3 times with running buffer and then were placed on top of the resolving gel. The top of the second-dimension gels with the strip was overlaid with 0.5% agarose in running buffer. The gel was run at 50V for 30 min and 200 V for 1 h. Proteins on the 2-DE gel were visualized by silver staining.

4.4.4.5 Protein visualization by silver staining

The fixation with silver staining of the proteins embedded in the gel was made as described in (Chen et al. 2004). Gel was fixed for 2 h initially in a solution containing 40% v/v ethanol and 10% v/v acetic acid. Then it was sensitized in a solution containing 30% v/v ethanol, 0.2% w/v sodium thiosulfate, 6.8% w/v sodium acetate and 0.125% v/v glutaraldehyde for 30 min, followed by washing with distilled water (3 times for 5 min each). After this the gel was stained for 20 min in 0.25% w/v silver nitrate with 0.015% v/v formaldehyde before washing with distilled water again (twice for 1 min each). The gel was finally developed in 2.5% w/v sodium carbonate containing 0.0074% v/v formaldehyde. The reaction was stopped with 1.5% w/v EDTA. The Gel was scanned and the analysis of the protein spots pattern was performed with Progenesis SameSpots (Non Linear Dynamics) software.

4.4.5 Statistical analyses

Fit of cell counts to the logistic sigmoidal model for the calculation of the growth rates, the examination of data distribution, descriptive statistics and analysis of the variances of the data were made using R Development Core Team (2008). To test for normality on data sets Shapiro-Wilk normality test was applied. Linear models and ANOVA tests were applied to look for significant differences among groups and interaction of factors. For homogeneity of variance Levene’s Test was performed. Post hoc Tukey test were performed to determine out which groups were significant different. Whenever data sets violated assumptions of normality, data were log, square root or arcsine transformed or if still not normally distributed or variance still not homogenous, non-parametric (Kruskal Wallis test) analyses were performed to test for significant differences

83 Chapter 4 between and within groups and pairwise comparisons using Conover test for multiple comparisons of independent samples was used when required to find out the significant different groups. When necessary Bonferroni adjustment for confidence intervals was applied.

4.5 Results

4.5.1 Growth

In this study photon fluxes (350 or 100 µmol photons•m-2•s-1) and temperature (26 or 30ºC) treatments applied to investigate the acclimation response of the three types of Symbiodinium A1, A13 and C1 did not reveal any significant differences when their growth rate parameters were compared (Table 9).

µ•day-1 doubling•day-1 divisions•day-1

˚C PF Type mean ±SE mean ±SE mean ±SE

C1 0.22 0.07 4.2 1.8 0.32 0.1

HL A13 0.26 0.1 3.5 1.1 0.37 0.14

A1 0.26 0.02 2.6 0.2 0.38 0.03 30 C1 0.30 0.06 2.5 0.6 0.44 0.08

LL A13 0.22 0.04 3.5 0.7 0.31 0.06

A1 0.21 0.01 3.3 0.1 0.30 0.01

C1 0.16 0.01 4.4 0.4 0.23 0.02

HL A13 0.17 0.02 4.1 0.4 0.25 0.03

A1 0.23 0.04 3.2 0.6 0.33 0.05 26 C1 0.19 0.05 4.4 1.3 0.27 0.07

LL A13 0.21 0.06 3.9 1.2 0.30 0.08

A1 0.18 0.04 4.0 0.7 0.27 0.05

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The fitted growth curve of the three types of Symbiodinium reached a higher maximum growth in cultures at a photon flux of 100µmol photons•m-2•s-1 and at a temperature of 26ºC with exception of type A1 which had highest maximum growth in cultures at a photon flux of 100µmol photons•m- 2•s-1 too, but at a temperature of 30ºC. Cultures of Symbiodinium type C1 at a photon flux of 350µmol photons•m-2•s-1 presented a similar growth curve at both temperatures 26 and 30ºC. Interestingly cultures of types A1 and A13 at a photon flux of 350µmol photons•m-2•s-1 and at a temperature of 30ºC displayed a higher maximum growth and a longer exponential phase than cultures at the same photon flux of 350µmol photons•m-2•s-1 but at a temperature of 26ºC (Figure 25).

26ºC 26ºC 30ºC 30ºC 26°C 30°C

A1LL A1LL A1LL A1LL A1HL A1HL A1HL A1HL A13LL A13LL A13LL A13LL A13HL A13HL A13HL A13HL C1LL C1LL C1LL C1LL 1500000 1500000 C1HL C1HL C1HL C1HL 1 - 1 1 − − 1000000 1000000 Cell•ml Cel...ml^ Cel...ml^ 500000 500000 0 0

0 010 1020 2030 3040 40 0 010 1020 2030 3040 40

Time(days)Time (days) Time(days) Time (days) Time(days) Time(days)

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4.5.2 Photophysiology

In this study, absorption spectra from methanol extracts did not present any differences on the contributions of the accessory pigments or any signs of degradation on any of the measured samples from cultures of Symbiodinium types at any photon flux or temperature treatment (Figure 26). Chl a concentration differed between photon flux regimes (H (3)=20.7, p<0.001). In general, the pairwise comparisons using Conover’s test showed Chl a concentration was significantly higher in cultures at a photon flux of 100µmol photons•m-2•s-1 and at a temperature of 26ºC than Chl a in cultures at the other photon fluxes and temperatures (p<0.001,Figure 27). Comparison of cultures across types and treatments showed chlorophyll a in cultures of type C1 at 100µmol photons•m-2•s- 1 and at a temperature of 26ºC was significantly higher than most cultures except A1 and A13 at same conditions. These two types were next in higher Chlorophyll a concentration than the rest of the cultures except cultures of A13 at low photon flux and 30ºC (Table 10, Figure 28).

26ºC 26ºC26°C 3030ºC°C 30ºC 1.0 1.0 1.0 1.0

C1HL C1HL C1HL C1HL A13HL A13HL A13HL A13HL A1HL A1HL A1HL A1HL 0.8 0.8 0.8 0.8 C1LL C1LL C1LL C1LL A13LL A13LL A13LL A13LL A1LL A1LL A1LL A1LL .) 0.6 0.6 0.6 0.6 r. u Absorbance(r.u.) Absorbance(r.u.) 0.4 0.4 0.4 0.4 Absorbance ( 0.2 0.2 0.2 0.2 0.0 0.0 0.0 0.0

400 450 400500 450550 500600 550650 600700 650 700 400 450 400500 450550 500600 550650 600700 650 700 Wavelenght(nm)Wavelength (nm) Wavelenght(nm) Wavelength (nm) Wavelenght(nm)Wavelenght(nm)

86 Chapter 4

) 1 - (pgcell a Chl

26HL 26LL 30HL 30LL Culture Treatment

Figure 27. Chl a (pgcell-1) concentration between cultures at two photon fluxes (350µmol photons•m-2•s-1 or 100 µmol photons•m-2•s-1) and two temperatures (26ºC or 30ºC).

a ) 1 - b bc (pgcell a cd Chl d d d d d d d d

A1326HL A126HL C126HL A1330HL A130HL C130HL A1326LL A126LL C126LL A1330LL A130LL C130LL Culture Treatment

87 Chapter 4

Chlorophyll a specific absorption spectra (Chl a*) showed variation between cultures of the same type and between types, at both photon fluxes and also at both temperature treatments (Figure 29).

26ºC 26ºC 30ºC 30ºC 26°C 30°C 0.08 0.08

A13HL A13HL A13HL A13HL A13LL A13LL A13LL A13LL A1HL A1HL A1HL A1HL A1LL A1LL A1LL A1LL C1HL C1HL C1HL C1HL 0.06 0.06 C1LL C1LL C1LL C1LL ) 1 - a 1) 1) − − chl 0.04 0.04 •mg 2 a*(m^2 mg chla a*(m^2 mg chla *(m a 0.02 0.02 0.00 0.00

450 500 450 550 500 600 550 650 600 650 450 500 450 550 500 600 550 650 600 650 Wavelenght(nm)WavelenghtWavelenght(nm)(nm) WavelenghtWavelenght(nm)Wavelenght(nm)(nm)

Comparisons of PSII photochemical efficiency (Fv/Fm) between cultures of three types of Symbiodinium exhibited significant differences between photon flux and temperature treatments (H(11)=843, p<0.001;). Similarly to Chl a concentration Fv/Fm was significantly higher in cultures at photon flux of 100µmol photons•m-2•s-1 and at a temperature of 26ºC than Fv/Fm in cultures at the other photon fluxes and temperatures (p<0.001, Figure 30). PSII photochemical efficiency in cultures of type C1 at a photon flux of 100µmol photons•m-2•s-1 and at a temperature of 26ºC was significantly higher (p<0.0001) than all the other cultures of types at every treatment. Cultures of type C1 at a photon flux of 100 µmol photons•m-2•s-1 and at a temperature 30ºC were significantly lower than cultures of the other two types for the same treatment (p<0.01). Cultures of type A1 were significantly higher at a photon flux of 350 µmol photons•m-2•s-1 and at a temperature

88 Chapter 4 of 26ºC than the other two types at the same conditions (p<0.0001). Cultures of three types had a similar reduction in PSII photochemical efficiency at a photon flux of 350 µmol photons•m-2•s-1 and at a temperature of 30ºC (Figure 31, Table 10) Fm / Fv

26HL 26LL 30HL 30LL Culture Treatment

Figure 30. Comparison of PSII photochemical efficiency Fv/Fm between cultures at two photon fluxes (350µmol photons•m-2•s-1 or 100 µmol photons•m-2•s-1) and two temperatures (26ºC or 30ºC).

89 Chapter 4

0.45

0.40 Fm / Fv/Fm Fv

0.35

0.30

A1326HL A126HL C126HL A1330HL A130HL C130HL A1326LL A126LL C126LL A1330LL A130LL C130LL A13.26.HL A1.26.HL C1.26.HL A13.30.HL A1.30.HL C1.30.HL A13.26.LL A1.26.LL C1.26.LL A13.30.LL A1.30.LL C1.30.LL Culture Treatment

Comparison of PSII absorption cross section (σPSII) reproduced a typical inverse relationship between Fv/Fm and σPSII values. In general, σPSII was significantly lower in cultures at a photon

-2 -1 flux of 100µmol photons•m •s and a temperature of 26ºC than σPSII of cultures at the other three conditions of photon fluxes and temperatures (Figure 32). Comparison of cultures between types and treatments showed PSII absorption cross section in cultures of type C1 at a photon flux of 100µmol photons•m-2•s-1 and at a temperature of 26ºC was significantly lower (p<0.0001) than

σPSII in all the other cultures of types at every treatment including cultures of types A1 and A13 at same condition; also at this condition, σPSII displayed by cultures of type A1 was significantly lower than σPSII in cultures of type A13. Cultures of type A1 displayed a significantly lower σPSII

90 Chapter 4 than displayed in cultures of the other two types at all the other three conditions of photon fluxes and temperatures (Figure 33, Table 10) ) 2 (nm σPSII

26HL 26LL 30HL 30LL Culture Treatment

Figure 32. PSII absorption cross section σ (nm2) between cultures at two photon fluxes (350µmol photons•m- 2•s-1 or 100 µmol photons•m-2•s-1) and two temperatures (26ºC or 30ºC).

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4.0 )

2 3.5 (nm ..PSII(nm2) σPSII

3.0

A13.26.HLA13HL26 A1.26.HL C1.26.HL A13.30.HL A1.30.HL C1.30.HL A13.26.LL A1.26.LL C1.26.LL A13.30.LL A1.30.LL C1.30.LL A1326HL A126HL C126HL A1330HL A130HL C130HL A1326LL A126LL C126LL A1330LL A130LL C130LL Culture Treatment

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Table 10. Means and standard errors of: Chl a concentration, PSII photochemical efficiency (Fv/Fm) and PSII absorption cross section (σPSII).

-1 2 PF Chl a (pgcell ) Fv/Fm σPSII(nm )

(µmol ˚C photons•m-2•s-1) Type mean ±SE mean ±SE mean ±SE

C1 0.09 0.02 0.31 0.005 3.63 0.08

350 A13 0.11 0.02 0.28 0.005 3.67 0.05

A1 0.05 0.002 0.3 0.004 3.2 0.07 30 C1 0.1 0.003 0.33 0.007 3.7 0.05

100 A13 0.17 0.06 0.33 0.005 3.8 0.06

A1 0.09 0.01 0.35 0.004 3.2 0.05

C1 0.10 0.02 0.30 0.005 4.1 0.08

350 A13 0.10 0.01 0.3 0.007 4.1 0.08

A1 0.11 0.01 0.34 0.003 3.4 0.09 26 C1 0.64 0.1 0.47 0.003 2.8 0.04

100 A13 0.38 0.03 0.42 0.004 3.5 0.05

A1 0.35 0.05 0.41 0.003 3.1 0.04

Calculations of the PSII maximum relative rate of electron transport rETRPSIImax showed a pattern of decline with reduction of photon flux from 350 to 100µmol photons•m-2s-1 and with rise in temperature from 26˚C to 30˚C. The highest rETRPSIImax values were found at 350µmol photons•m-2•s-1 and 26˚C treatment in all three experimental types. As well, the highest values of light saturation point (Ek) were found at this treatment in all three types. In contrast, the highest values of α were found at 100µmol photons•m-2•s-1 and 26ºC also in all three types (Table 11).

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Table 11. Means and standard errors of: α, PSII maximum rate of electron transport (ETRPSIImax) and light saturation point (Ek).

PSII PF α ETR max Ek (µmol °C photons•m-2•s-1) Type mean ±SE mean ±SE mean ±SE

C1 0.13 0.01 41 3.6 291 30

350 A13 0.1 0.001 33 6.1 284 17

A1 0.13 0.01 46 7.7 309 19 30 C1 0.15 0.01 31 1.8 195 7

100 A13 0.14 0.02 28 3 174 11

A1 0.15 0.01 45 6.4 267 15

C1 0.16 0.03 50 5.9 366 15

350 A13 0.15 0.02 63 19 445 43

A1 0.13 0.01 60 20 397 51 26 C1 0.19 0.01 42 2 211 6

100 A13 0.15 0.01 49 5.5 288 12

A1 0.18 0.01 48 3.3 249 9

Rapid light curves (RLCs) showed the dynamics of decline on rate of electron transport with increase in photon flux from 100 to 350µmol photons•m-2•s-1 and higher temperature from 26ºC to 30ºC (Figure 34).

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26ºC 26ºC26°C 3030ºC°C 30ºC 100 100 A1LL A1LL A1LL A1LL

A13LL A13LL A13LL A13LL

C1LL C1LL C1LL C1LL

A1HL A1HL A1HL A1HL 80 80 A13HL A13HL A13HL A13HL

C1HL C1HL C1HL C1HL 60 60 ) ru ( rETR(ru) rETR(ru) ETR r 40 40 20 20 0 0

0 0 500 5001000 10001500 1500 0 0 500 5001000 10001500 1500

PAR(µmolphotonm^2s^PAR(µPAR(µmolphotonm^2s^mol−1)photons −1) m-2s-1) PAR(µPAR(µmolphotonsm^2s^mol PAR(µmolphotonsm^2s^photons −1) m-2s-1)−1)

Figure 34. Rapid light curves (RLCs) of three experimental types of Symbiodinium (A1, A13 and C1) acclimated at two photon fluxes (350µmol photons•m-2•s-1 or 100 µmol photons•m-2•s-1) and two temperatures (26ºC or 30ºC). Each point represents the mean and standard errors of rETR at a given irradiance for a determined type of Symbiodinium (Means ± SE, n=100).

Following acclimation at the different treatments the Symbiodinium types were subject to 33ºC of temperature stress for three consecutive days (80h). Measurements of photochemical efficiencies (Fv/Fm) showed a gradual decline during exposure in all three clades acclimated to both previous photon fluxes and temperatures (Figure 35).

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26ºC 2626ºC°C 33°C 3030ºC°C 30ºC 0.5 0.5 A1HL A1HL A1HL A1HL

A1LL A1LL A1LL A1LL

A13HL A13HL A13HL A13HL

0.4 0.4 A13LL A13LL A13LL A13LL

C1HL C1HL C1HL C1HL

C1LL C1LL C1LL C1LL 0.3 0.3 Fm / Fv/Fm Fv/Fm Fv 0.2 0.2 0.1 0.1 0.0 0.0

0 020 2040 4060 6080 80 0 020 2040 4060 6080 80 Time(h) Time(h)Time(h) Time(h)Time(h) Time(h)

Figure 35. Patterns of decline of photochemical efficiency (Fv/Fm) of three experimental types of Symbiodinium (A1, A13 and C1) acclimated at two photon fluxes (350µmol photons•m-2•s-1 or 100 µmol photons•m-2•s-1) and two temperatures (26ºC or 30ºC) after being subject to stress temperatures at 33ºC for three days.

4.5.3 Proteomics

Proteomic analysis with 2-DE gels ran with protein samples extracted from cell cultures of three experimental types of Symbiodinium A1, A13 and C1 at two photon fluxes (HL=350µmol photons•m-2•s-1 and LL=100µmol photons•m-2•s-1) and at two temperatures (26ºC and 30ºC) displayed significantly different protein spots between the two temperature treatments (Figure 36).

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After manual picking for removal of false positives due to stains and gel textures 69 significantly different spots were found (Figure 37). Of these 69 spots, 8 of the most significantly different were chosen for identification in databases from measurements of their isoelectric point (pI=pH) and their apparent molecular weight (Mw=kDa) and for mass analysis with MALDI-TOF to obtain their aminoacid composition (ion peaks) and confirmation of their identities also by database search. Numbers and characteristics of the spots chosen as well as p values between treatments are displayed (Table 13). Measured isoelectric points, apparent molecular weights of spots and a posteriori information regarding their identity are displayed in table 5. After identification using the two parameters obtained from gels (pI and Mw) four of the eight protein spots were found to be important components of the electron transport chain in the thylakoid membrane of chloroplasts. Spots 667, and 431 were identified as PSII D1 and P700 apoprotein A2 respectively and were more abundant at 26ºC. Spots 868 and 1210 matched with PCP and subunit 4 of Cytochrome b6f respectively and these were more abundant at 30ºC (Table 12). The other four spots were identified as proteins involved in processes not directly related to photosynthesis such as respiration,

97 Chapter 4 cytokinesis and cell maintenance. Spot 598 was identified as subunit 1 of Cytochrome c oxidase, a mitochondrial protein, spot 610 matched with Tubulin alpha-chain, spot 957 as dymethylsulfonioproprionate lyase protein, these three spots were more expressed at 26ºC and spot 1492 identified as calmodulin was more expressed at 30ºC (Table 12).

Figure 37. Reference 2D-PAGE map (A1 cultured at 100µmol photons•m-2•s-1 and 26˚C) displays 69 significant different proteins (spots) between both temperature treatments (26ºC vs 30ºC), after manual picking for removal of false positives (40µg of total protein were loaded in the IPG strip for the first dimension IEF, second dimension was performed using 10.5-14% polyacrylamide gels (Bio-Rad).

Consequent analysis between photon fluxes and temperature treatments combined displayed 46 significantly different spots (Figure 38). Of these 46 spots 5 were found shared with previous analysis between temperature treatments (Table 12). Interestingly, of these protein spots two were photosynthesis related, PSII DI (667) and subunit 4 of Cb6f (1210) both more expressed in cultures at a photon flux of 100µmol photons•m-2•s-1 than at a photon flux of 350µmol photons•m-2•s-1. Besides these two photosynthetic chain reaction proteins the other three significantly different spots between photon fluxes were calmodulin (1492), Tubulin alpha-chain (610) and subunit 1 of Cytochrome c oxidase (598) all of them more expressed at a photon flux of 100µmol photons•m- 2•s-1. Three spots were not found significantly different between cultures at both photon fluxes in

98 Chapter 4 combination with temperature treatments: PCP (868), P700 (431) both more expressed at 30ºC and DMSP lyase more expressed at 26ºC (957).

Analyses executed over samples of cultures at 26ºC and 30ºC after exposure to temperature stress at a temperature 33ºC for 3 days (80h), delivered 32 significantly different protein spots between this two previous temperature conditions (Figure 39). Of these 32 spots 2 were found significantly different also as with previous analysis between temperature treatments and photon fluxes combined, as well as were with analysis between temperatures only (Table 12). These two spots: subunit 4 of cytochrome b6f (1210) and Tubuline alpha-chain (610) were both significantly more expressed in cultures previously set at a temperature of 26ºC compared with those previously set at a temperature of 30ºC after both were subject to temperature stress at 33ºC for 3 days (80h). Significant differences in levels of expression of Tubuline alpha chain (610) remained similar before vs after stress at 33ºC. However, in the case of subunit 4 of Cytochrome b6f (1210) levels of expression turned around after stress exposure at temperatures of 33ºC. This electron transport chain protein associated with alternative electron transport was significantly down regulated in

99 Chapter 4 cultures at 30ºC and significantly upregulated in cultures at 26ºC compared with levels of expression before exposure to temperature stress at 33ºC temperature (Table 12).

100 Chapter 4

Treatments Average Normalised Volumes Spot # Anova (p) Fold 26°C 30°C

667 0.001 1.8 8697 4856

868 0.002 1.9 3359 6285

1210 0.016 1.6 14290 22970

1492 0.028 1.3 143400 187200

598 0.03 1.9 1110 592.2

610 0.032 1.8 7198 4067

431 0.041 2.3 4852 2128

957 0.043 1.7 2080 1223

26°C- 30°C- 30°C- Spot # Anova (p) Fold HL 26°C-LL HL LL

1492 0.002 1.7 126800 160000 159600 214800

667 0.016 2.1 7841 9553 4558 5155

610 0.017 3.9 6090 8307 2144 5989

598 0.036 3.9 839.2 1380 357.3 827.1

1210 0.036 2.2 17130 11460 20560 25380

Spot # Anova (p) Fold 26°C-33°C 30°C-33°C

1210 0.025 1.6 22020 13640

610 0.05 1.7 7530 4333

101 Chapter 4

Biological pI MW Spot # ID Access # Organism Process/Function

Karenia Photosynthetic electron 6.1 47.4 667 PSII protein D1 Q9TJ82 mikimotoi transport in photosystem II

Light-harvesting 5.3 35.8 868 PCP P51874 Symbiodinium sp. polypeptide

Cytochrome b6-f Amphidinium Photosynthetic electron 4.7 20.9 1210 complex subunit 4 Q1HCL0 carterae transport chain

Karlodinium 5.2 15.6 1492 Calmodulin A3E4F9 veneficum Calcium ion binding

Cytochrome c Electron transport, 5.9 53.1 598 oxidase subunit 1 Q4UJ69 Theileria annulata Respiratory chain, Transport

Plasmodium 5.9 50.4 610 Tubulin alpha chain P14642 falciparum Microtubule-based process

Amphidinium 5.6 63.1 431 PSI P700 P58383 carterae Photosynthesis

Dimethylpropiothetin 6.2 32.1 957 DMSP lyase P0DN22 Symbiodinium sp. dethiomethylase activity

102 Chapter 4 4.6 Discussion

The high phenotypic diversity of dinoflagellates of the genus Symbiodinium has demonstrated their varied physiological traits that facilitate the establishment of symbiotic states across different invertebrate taxa as well as their ability to acclimate to different environments (Sugget et al. 2015). This functional diversity has often been claimed to drive their wide biogeographic distribution (LaJeunesse 2001). Research has demonstrated that some corals can survive thermal induced bleaching events, as a result of having established strong symbiotic relationships with some types of Symbiodinium with developed resistance to temperature elevation (Bellantuono et al. 2011, Hume et al. 2015).

Previous studies focusing on the physiological response of Symbiodinium to temperature stress have demonstrated different types of this genus display growth response mainly due to varying acclimatory capabilities to elevated temperatures (Iglesias et al. 1992, Robinson & Warner 2006, Reynolds et al. 2008, Ragni et al. 2010, Krämer et al. 2012) There is experimental evidence supporting differential production of enzymes, protein factors and other molecules among temperature sensitive vs tolerant types (Krueger et al. 2014). These molecules are mainly related to a response to stress produced by ROS production after exposure to temperature elevation (Krueger et al. 2014). Also, metabolic modification to balance ATP production from photosynthesis, account for the differential resilience of this unicellular organisms to stressful changing environments (Roberty et al. 2014, Aihara et al. 2016).

In this study, results obtained from detailed photophysiological analysis displayed type specific differences among the three types of Symbiodinium. These differences allow different types to efficiently utilize available photon sources under ambient but also elevated temperatures.

As previously shown (see Chapter 2) among the types studied, diverse photoacclimation strategies are apparent within the genus. These strategies support similar growth rates across photon flux regimes and at elevated temperature. Well known photoacclimation strategies include changes in the pigment composition and amount of the light harvesting complexes and core antennas to modulate photosystems cross-sections and energy transfer to reaction centers. These modifications regulate photochemical efficiencies and mechanisms of photoprotection and repair to respond to potential damage produced from extreme pH gradients, excess of excitation and exposure to elevated temperatures (Iglesias-Prieto & Trench 1994, 1997, Hennige et al. 2009, Suggett et al. 2015). Though these strategies may also be effective at high temperature conditions, there is a synergistic effect of high photon fluxes with temperature elevation producing a negative

103 Chapter 4 effect on photochemical efficiencies and maximum electron transport rates. Successful photoacclimation strategies to achieve a sustained growth rate, maintenance of metabolism and cell maintenance, among others, are most probably key factors to ensure survival of the holobiont during exposure to high temperatures (Iglesias et al. 1992, Takahashi et al. 2008, Ragni et al. 2010, Bellantuono et al. 2011, Hoadley et al. 2015).

Symbiodinium types cultured in this study are among the most commonly found as coral symbionts, and their distribution and dominance are in themselves signs of an effective partnership within the holobiont (Robinson & Warner 2006, Suggett et al. 2015). To sustain the holobiont, Symbiodinium must balance photosynthesis with several other metabolic pathways by regulating several important functional groups of proteins (Rosic et al. 2015, Oakley et al. 2016). The proteins involved in electron transport that drive photochemical reactions are also regulated to balance ATP by activation of AET pathways in some types of Symbiodinium (Roberty et al. 2014, Aihara et al. 2016). There is still a debate about the ability of clade A to activate AET during exposure to elevated temperatures, whether it is a mechanism of photoprotection or a stress response (Roberty et al. 2014, Aihara et al. 2016).

It has also been shown before (see Chapter 2) that proteins related to carbon fixation such as RuBisCo or to glycolysis such as GAPDH and those involved in respiration and energy metabolism are integrated with balancing proton motive force (pmf) at different photon regimes (Badger et al. 2000, Shimakawa et al. 2017). These mechanisms are used across Symbiodinium types during exposure to elevated temperature and prolonged stress conditions (Aihara et al. 2016). Results from this study have shown the proteomic response combined with decline of cellular processes caused by exposure to a constant elevated temperature (30ºC) and following acclimation to this elevation, the response to continuous thermal stress (33ºC) is different than chronic exposure to this temperature from 26ºC, suggesting pre-adaptation to high temperatures may be achievable by some types of Symbiodinium.

The significant down-regulation of P700 and D1 in cultures subjected to 30ºC compared with cultures at 26ºC can explain the observed decreases in electron transport rates found for all clades. These two proteins represent the core of reaction centers of both photosystems where separation of charge occurs (Iglesias-Prieto et al. 1993). Response to and perhaps photoacclimation to high temperature appeared to be mediated by up-regulation of PCP and Cytb6f, as these proteins are known to be involved in photoprotection and AET (Iglesias-Prieto 1996, Reynolds et al. 2008).

104 Chapter 4

PCP together with acpPC is an important light harvesting complex component in the photosynthetic machinery of Symbiodinium (Iglesias-Prieto et al. 1993, Hiller et al. 1993). It has been suggested PCP acts also as a quencher of excess of light as it is composed of the photoprotective carotenoid peridinin (Kanazawa et al. 2014). In addition, some studies have suggested this light harvesting complex moves between PSII and PSI, as occurs as a mechanism of STs in other photosynthetic organisms (Reynolds et al. 2008). In this study PCP upregulation at a temperature of 30ºC wasn’t found to be significantly different at combined comparison between photon fluxes and temperatures (Table 12) this suggests PCP might be involved in AET acting as a photoprotection mechanisms to reduce photodamage as a response to heat stress.

Similarly, at 26ºC, P700 and DMSP lyase were upregulated at both photon fluxes but significantly decreased at 30ºC demonstrating a stronger temperature effect. Lower levels of expression of DMSP lyase at a temperature of 30ºC may be a direct response to stress as DMSP is highly abundant and an important osmolyte in dinoflagellates, its production is increased in high light, and it has been suggested to play a role in scavenging ROS such as hydroxyl radical (Sunda et al. 2002).

Low expression levels of P700 at 30ºC could also be a response to avoid photodamage. This protein was recently found to be upregulated in thermally bleached corals when compared to unbleached corals. This suggest that corals with symbionts expressing less P700 at high temperature (similarly as in cultures at 30ºC in this study), would be less susceptible to bleach (Ricaurte et al. 2016). In the present study, it was not possible to fully characterise how the temperature-induced regulation of P700 varied across Symbiodinium types although it is widely accepted that A1 type is temperature tolerant (Robison &Warner, 2006, Ragni et al. 2010). It must be considered however that symbiont response may be different when in hospite as compared to in vitro and this response may depend on host control mechanisms and host driven variability in the conditions experienced by the symbiont (Bellantuono et al. 2011, Hoadley et al. 2015).

Many studies have shown the synergistic effects of light and temperature stress, particularly as it concerns photosynthetic function, and amplified photodamage (Ragni et al. 2010, Krämer et al. 2012).

Increased temperature stress under higher photon fluxes was found to occur in this study as most photosynthetic proteins were at their lowest levels of expression in cultures at a photon flux of 350µmol photons m-2s-1 particularly at 30ºC. It has been suggested that higher temperatures increase fluidity of thylakoid membranes (Tchernov et al. 2004, Díaz-Almeyda et al. 2011). Higher

105 Chapter 4 temperatures may also affect other compartments with similar bipolar permeable membranes resulting in an increased metabolic cost to maintain homeostasis. Reducing metabolic costs of the temperature stress response can be considered a general strategy of many organisms (Raharinirina et al. 2017). As shown in this study, abundance of alpha tubulin was negatively affected by increases in temperature. Levels of expression of this protein were higher in cultures at 26ºC compared with those at 30ºC. Also, levels of expression of alpha-tubulin in cultures previously at 26ºC then exposed to 33ºC where higher compared with low levels of expression of this protein in cultures previously at 30ºC then exposed to 33ºC. This differential expression depicts a cost- effective response strategy to high temperature, as alpha-tubulin protein is one of the most abundant in dinoflagellate cells, and is involved in the formation of cytoskeletal microtubules (Rosic et al. 2015). Energy is saved by reductions in cytokinesis and transport of cellular material during exposure to temperature elevation (Gagnon et al. 1996). Of note is the fact that production of this protein was significantly lower in cultures at a photon flux of 350µmol photons•m-2•s-1 and at a temperature of 30ºC suggesting a synergistic light-temperature effect. This finding also suggests that cellular levels of alpha tubuline, may be a signature of temperature stress for Symbiodinium in-hospite.

Similarly, there was a differential expression of cytochrome b6f across all experiments (e.g. upregulted at 30ºC) and may be a temperature stress response related to energy metabolism. This protein was significantly upregulated in cultures at 30ºC compared with cultures at 26ºC and was again significantly upregulated in cultures previously at 30ºC then exposed to 33ºC than cultures previously at 26ºC then exposed to 33ºC. The importance of cytochrome b6f in the temperature stress response is also supported by an evident increase in levels of expression in cultures at a temperature of 30ºC at a photon flux of 100µmol photons•m-2•s-1 compared with cultures at that same temperature of 30ºC but at a photon flux of 350µmol photons•m-2•s-1. As mentioned before cytochrome b6f is the link in electron transfer between PSI and PSII and it is involved in CET and STs, both AET have been suggested to occur in Symbiodinium to alleviate thermally induced phtodamage (Reynolds et al. 2008, Aihara et al. 2016).

Calmodulin differential expression was also observed in response to temperature stress and was significantly upregulated at 30ºC but only in those treated with a photon flux of 100µmol photons•m-2•s-1. This protein is a calcium binding protein and is used to transport other proteins specially calcium/calmodulin-dependent protein kinases and is suggested to play an important role in the establishment of symbiosis. It is highly abundant in Symbiodinium and also involved in a number of metabolic processes related to cell maintenance and cell signaling. This protein is

106 Chapter 4 involved in the physiological responses to environmental stress (Rosic et al. 2015) but within this experiment was not found to differ significantly between cultures at 26ºC compared to 30ºC when both were subsequently exposed to 33ºC. It seems highly probable that this protein was generally upregulated during the 33ºC temperature stress regardless of what temperature they were acclimated to.

Cytochrome c subunit 1 protein expression was affected by the synergistic effect of elevation in temperature and photon flux and was significantly downregulated at 30ºC at the high photon flux compared with the other three treatments. Cytochrome c subunit 1 is a mitochondrial protein involved in respiration, reducing oxygen to water to fuel metabolism with ATP production (see Chapter 2). Its upregulation at high temperature enhanced by higher light, might be related with AET occurring in the chloroplast (such as CET) as a photoprotective response to temperature stress (Aihara et al. 2016, Shimakawa et al. 2017). This is in agreement with other published findings (eg Raven and Beardall, 2017) concerning the respiratory chain of Symbiodinium, which lacks complex I ultimately resulting in a shortfall in the respiratory ATP synthesis that might be covered by AET in the chloroplast.

To conclude, this study presents a general view of some important cell processes affected by temperature stress in Symbiodinium. The main process of photosynthesis is sustained and driven by photoacclimation and other mechanisms that support growth, but are at the expense of other processes such as cytokinesis and respiration. Cell maintenance proteins and ROS scavengers also play an important role in the temperature stress response.

Although some hope lies in the power of Symbiodinium’s adaptive potential, the impacts of temperature elevation will be largely dependent on the interaction between the symbiont and coral host and at least in part, dictated by the past environmental history of the holobiont.

107 Chapter 5 Chapter 5 General Discussion

In this thesis, a proteomic approach was used, supplemented by physiological analyses to study the symbiotic relationship between corals and Symbiodinium. Specifically, the environmental and coral host control over the Symbiodinium and how these are involved in the maintenance of the symbiosis.

Photosynthesis is an important trait of the holobiont and is differentially affected by the interaction of environmental variables and the host environment. The approach used in this study was focused on Symbiodinium in culture and its photoacclimatory response to light, host factors and temperature. The proteome and photophysiology of three types of Symbiodinium were analysed. The types studied (A1, A13 and C1) belong to two (A and C) of the most widely distributed clades across corals of the world’s reefs.

The results obtained can be interpolated to the physiology of the holobiont with some caution considering that the physiology of Symbiodinium may be also influenced in hospite by other factors out of reach of this study such as the life history of the different coral hosts which obviously can have an effect on the interaction of the variables studied here and its control.

Proteomics is as a powerful platform and in spite of the recent progress made on genome and transcriptome sequences of Symbiodinium, in this study the proteome was not fully identified.

Shotgun proteomics (LC-MS/MS) analyses produced a high yield of proteins but data base searches matched only a small number of these. MALDI-TOF analyses were also unsatisfactory as the protein amount in the 2-DE spots was too small and ion-spectra only matched with trypsin digests. Identification of the proteins from the 2-DE analysis was achieved in databases using the calculated isoelectric points and molecular weights. These analyses delivered identities (IDs) of proteins with similar biological process even with searches using different tags, therefore this supports interpretation. It is also worth reporting that the two different types of proteomic analysis used (LC-MS/MS and 2-DE) and subsequent data base searches produced similar results on IDs and on the differential expression of many of these proteins

5.1 Photoacclimation metabolism

Light environment drives the photoacclimatory response of Symbiodinium in the coral host (Muscatine 1980, 1990, Szabó et al. 2014). In this study, the three types of Symbiodinium had

108 Chapter 5 similar specific growth rates at the experimental photon fluxes (PFs). The PFs used were not as different between each other and the higher PF used in this study was moderate in comparison with previous experiments (Hennige et al. 2009). However, different photoacclimatory strategies were identified across Symbiodinium types driven by adjustment of the photosynthetic unit (PSU) to photoacclimate and sustain similar growth rates. Photoacclimatory modifications included increasing the Chl a* and photosystem two (PSII) specific absorptions to sustain a higher electron transport rate in a high PF. At low PF, the increased number of pigments resulted in a higher photochemical efficiency, in agreement with previous research (Iglesias & Trench 1997, Hennige et al. 2009). Different photoacclimation strategies among Symbiodinium types are supplemented by differential regulation of other metabolic processes. Differentially expressed proteins involved in electron transport ET in the PSU (indicative of regulation of ET), may be supplemented by alternative electron transport AET pathways and by differentially expressed components of respiration in the mitochondria such as cytochrome b and cytochrome c. Energy metabolism was also modified showing light-produced differences in expression of RuBisCo, GAPDH, and Enolase 1. As well cellular maintenance was differentially modulated by regulation of Tubulin, Calmodulin and heat shock proteins (HSPs) proteins.

5.2 Host control

Nutrient exchange metabolism acquired by the holobiont during symbiosis is one of the most important characteristics for its maintenance (Davy et al. 2012). Regulation of host control over the Symbiodinium has been suggested to occur in many studies (Weber & Medina 2012). This study specifically aimed to compare four different types of Symbiodinium (A1 and A13 widely distributed among corals of the Caribbean, C1 usually found in corals of the Indo-Pacific and the isolated symbiont from Pocillopora damicornis), cultured at two photon fluxes and treated for an hour with two different types of host release factors (HRFs). The parameters most directly related to photosynthesis, chlorophyll a concentration and excitation pressure over PSII were mainly affected by the light environment in all types. These two parameters are related with the number of chlorophyll-protein-complexes and reaction centers in the PSU and were inversely correlated in the principal component analysis (PCA), (Hiller et al. 1993, Iglesias-Prieto et al. 1993, Iglesias-Prieto 1996).

Many other proteins may be involved with mechanisms of nutrient translocation and possibly its regulation may be controlled by the host (Curien et al. 2017, Shimakawa et al. 2017).

109 Chapter 5

Excitation pressure over PSII, total protein and total glucose increases matched in Symbiodinium A1 under host tissue (HT) control at a high PF. This may be involving a photosynthesis-regulatory mechanism of glucose production perhaps related with an induced disconnection of antenna.

Carbon translocation was inhibited in C1 and A13 with the free amino-acid host factor mix (FAAmix) and apparently this artificial HRF affected also the translocation mechanism in the Pocillopora damicornis symbiont (PdS). The mechanism of carbon translocation worked in PdS with HT and there was an increase in glucose released as well as in total protein. In this study, it was also shown that glucose was released in a mechanism involving protein. In A1 under HT, there was a positive correlation between the increment in total glucose and total protein but a negative correlation with the glucose released. This suggests that the production of carbon was induced by host control but the mechanism of translocation was not ejected. This may be caused by specificity of the HRF control of the HT acting with a decreased translocation effect on A1 compared with PdS. Alternatively, A1 might be less mutualistic in sharing carbon under HRF control than PdS.

There also were found different proteins expressed across the four types and HRF treatments showing that the protein pool is effectively regulated by host control. Unfortunately, analysis using MALDI-TOF did not delivered any significant value to match any protein identity in databases because of the extremely small amount of protein in the samples. However, it is possible that these proteins represent part of a specific protein mechanism of control of carbon translocation of Symbiodinium as suggested previously (Grant et al. 2013).

5.3 Temperature and Light effect over the proteome

Some species of corals can survive thermal bleaching events by establishing strong symbiotic relationships with some types of Symbiodinium resistant to high temperatures (Bellantuono et al. 2011, Hume et al. 2015). In Acropora millepora pre-acclimatization to high temperature has been shown to confer resistance to high temperature stress. This resistance is attributed to a well stablished symbiosis between both partners as is not a product of shuffling of symbionts or a microbiome mark (Bellantuono et al. 2011). The resistance to high temperatures of corals on the Persian Gulf has been attributed to their well stablished relationship with Symbiodinium thermophilum a thermally tolerant coral symbiont (Hume et al. 2015).

In this study, photophysiological analysis displayed differences across the three experimental types of Symbiodinium to efficiently utilize available photon sources under ambient but also under elevated temperatures. Evidence was found of the synergistic effect of high photon fluxes with

110 Chapter 5 temperature elevation producing a negative effect on photochemical efficiencies, electron transport rates and differences in proteome among types. Proteomic results showed that in spite of the decreases in the relative electron transport rates (rETRs) caused by exposure to a constant elevated temperature (30ºC), acclimation to this elevation occurred as differences were not significant across types nor treatments. Subsequent exposure of these temperature acclimated cultures to continuous thermal stress (33ºC) produced a different protein expression compared with that obtained by chronic stress applied in acclimated cultures from 26ºC to subsequent exposure to the same continuous thermal stress (33 ºC). This suggests pre-adaptation to high temperatures may be achievable by some types of Symbiodinium. There was observed a differential expression of PCP and Cytb6f proteins known to be involved in photoprotection and AET, which appeared to mediate photoacclimation at high temperature (Iglesias-Prieto 1996, Reynolds et al. 2008). Also DMSP lyase appeared to be regulated as part of temperature response may be playing a role in scavenging ROS in Symbiodinium as it does in other microalgae (Sunda et al. 2002).

The down regulation of P700 found at high temperature is suggested to be a mechanism to avoid photodamage in agreement with similar down regulation in corals unbleached after a high temperature event (Ricaurte et al. 2016).

The negative effect on photosynthesis caused by temperature stress and enhanced by higher photon fluxes was found to occur in this study. This was shown by low levels of expression in most of the photosynthetic proteins in cultures at high temperature and at high light (Reynolds et al. 2008, Aihara et al. 2016). This study also showed proteomic responses from the energy metabolism and cell maintenance pools in response to high stress. Proteins highly abundant in the cell such as alpha tubulin or calmodulin playing an important role in the structure of cytoskeleton and transport of cellular materials respectively, were differentially expressed under high temperature. Also, mitochondrial proteins of the respiratory chain such as cytochrome c were regulated. These proteome responses to temperature suggest a general pattern to reduce energetic costs at high temperature exposure. This pattern includes balance of ATP production in the chloroplast by activation of different forms of ET fueled by photosynthesis. Other processes included in this pattern would be the regulation of protein transport and cell maintenance.

In conclusion, the study of Symbiodinium accomplished in this thesis highlighted the high plasticity of these symbiotic algae to acclimate to different external environments not only to different lights but also to high temperature and to simulated internal host controlled environments.

111 Chapter 5

The differential protein expression across the different types and between the photon fluxes, temperatures and host factors revealed at the finest molecular level the modifications that distinguish the mechanisms of photoacclimation, temperature acclimation, stress response and glucose translocation across these types. The differential photoacclimation appeared driven by adjusting the photosynthetic unit proteins to balance the production of ATP at the different photon fluxes and achieve homeostasis.

These photoacclimation adjustments also seemed invoked to acclimate at high temperature in addition to other differential proteome adjustments to face the stressing conditions of thermal rise. This differential protein expression remained even after high temperature stress. This demonstrates the significant importance of photoacclimation plasticity to face this type of stress by the regulation of chain reaction proteins besides other important proteins dedicated to cell maintenance.

In addition, it was also shown at the protein level how photosynthesis and glucose translocation of Symbiodinium is also differentially controlled by host factor proteins in junction with the light environment. These findings demonstrate how different types of Symbiodinium have the potential to differentially adjust their proteome to achieve photoacclimation and regulate other important cell processes to adapt to different environments including high temperature and the coral cell environment. This potential adaptability of Symbiodinium may be a key factor in the subsistence of some corals enabled with mechanisms to maintain this symbiosis at the current challenging environmental conditions.

For example, tolerant symbionts to high temperature and eventually adapted to stress by proteome modifications, may be nurtured and maintained by resilient coral hosts with a lower demand for photosynthates. Similarly, settlement of corals in environments dominated by stress tolerant types of Symbiodinium may allow the survival of new stress resilient colonies if the symbiotic relationship becomes well stablished. On the other hand, laboratory manipulation and artificial selection of types with proteomes preadapted to stress may be used for the assisted evolution of holobionts resilient to stress.

The biology of the genus Symbiodinium appears as complex as vast is its diversity of types. In order to understand completely the role of this photosynthetic algae in the future of coral reefs we need to approach with higher resolution its physiology in and ex hospite. The characterization of the Symbiodinium proteome to be tested at different environmental scenarios might be one effective way to obtain the urgently needed comprehension of this endangered symbiosis.

112 Chapter 5

113

Appendix A

A.1 High resolution 2-Dimensional Electrophoresis of the protein complex mix isolated from the three experimental types (A1, A13 and C1) grown under two PFs (LL=100 and HL=350 µmol photons·m- 2·s-1). 40µg of protein were loaded in 7cm (pH 3-10) and 11cm (pH 4-7) linear gradient strips for the first dimension and then resolved in 12% and 10-14.5% polyacrylamide gels for the second dimension. The more representative spots for each PF were analysed using C1 and A13 resolving gels as references for LL and HL respectively.

115 Appendix A

A.2 High resolution 2-Dimensional Electrophoresis of the protein complex mix isolated from the three experimental types (A1, A13 and C1) grown under two PFs (LL=100 and HL=350 µmol photons·m- 2·s-1). 40µg of protein were loaded in 11cm (pH 4-7) linear gradient strips for the first dimension and resolved in 10-14.5% polyacrylamide gels for the second dimension. The most significantly different spots between PFs were analysed.

116 Appendix A

117

Glossary of Terms

Coral bleaching. Refers to coral discolouration, specifically through loss of pigmentation from corals’ symbiotic microalgae, termed zooxanthellae, but also from the coral cnidarian host tissue, as well as loss of entire zooxanthellae cells from the cnidarian tissue (Suggett & Smith 2011).

Holobiont. Coral colony consisting of the coral animal, zooxanthellae, procariotes, fungi, endolithic algae and unknown components (Rohwer et al. 2002).

Microbiome.The microbiome is the entire community of microorganisms that live in or on the body of a multicellular eukaryotic organism. Because in many cases these microorganisms can only be characterized by genetic methods, the term microbiome is often used to refer to the collective genomes present in all microorganisms in a given microbial community (Royet et al. 2011).

Photoacclimation. Phenotypic response of algae to changes in the light environment and occurs from alterations to a number of components of the photosynthetic apparatus (Hennige et al. 2009)

Proteome. The term “proteome” was coined by Marc Wilkins and introduced to the scientific community at the 1st Siena Meeting in 1994. The term proteome was originally defined as the “PROTEin complement of a genOME” but soon became to be generally accepted to mean the set of proteins expressed by a particular organelle, cell type, or tissue under a particular set of conditions (Dunn 2014).

Symbiosome. Host-derived outer membrane together with the Symbiodinium cell and the space between the two (Roth et al. 1988).

119

List of References

Aebersold, R., Hood, L. E., & Watts, J. D. (2000). Equipping scientists for the new biology. Nature Biotechnology, 18 (4), 359.

Aihara, Y., Takahashi, S., & Minagawa, J. (2016). Heat induction of cyclic electron flow around photosystem I in the symbiotic dinoflagellate Symbiodinium. Plant Physiology, 171 (1), 522-529.

Akimoto, H., Wu, C., Kinumi, T., & Ohmiya, Y. (2004). Biological rhythmicity in expressed proteins of the marine dinoflagellate Lingulodinium polyedrum demonstrated by chronological proteomics. Biochemical and Biophysical Research Communications, 315 (2), 306-312.

Anderson, N. L., Hofmann, J. P., Gemmell, A., & Taylor, J. (1984). Global approaches to quantitative analysis of gene-expression patterns observed by use of two-dimensional gel electrophoresis. Clinical Chemistry, 30 (12), 2031-2036.

Apprill, A. M., Bidigare, R. R., & Gates, R. D. (2007). Visibly healthy corals exhibit variable pigment concentrations and symbiont phenotypes. Coral Reefs, 26 (2), 387-397.

Aranda, M., Li, Y., Liew, Y. J., Baumgarten, S., Simakov, O., Wilson, M. C., ... & Ryu, T. (2016). Genomes of coral dinoflagellate symbionts highlight evolutionary adaptations conducive to a symbiotic lifestyle. Scientific Reports, 6, 39734.

Arif, C., Daniels, C., Bayer, T., Banguera-Hinestroza, E., Barbrook, A., Howe, C. J., ... & Voolstra, C. R. (2014). Assessing Symbiodinium diversity in scleractinian corals via next-generation sequencing- based genotyping of the ITS2 rDNA region. Molecular Ecology, 23 (17), 4418-4433.

Artimo, P., Jonnalagedda, M., Arnold, K., Baratin, D., Csardi, G., De Castro, E., ... & Grosdidier, A. (2012). ExPASy: SIB bioinformatics resource portal, Nucleic Acids Res, 40 (W1), W597-W603.

Badger, M. R., von Caemmerer, S., Ruuska, S., & Nakano, H. (2000). Electron flow to oxygen in higher plants and algae: rates and control of direct photoreduction (Mehler reaction) and rubisco oxygenase. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 355 (1402), 1433-1446.

Baird, A. H., Cumbo, V. R., Leggat, B., & Rodriguez-Lanetty, M. (2007). Fidelity and flexibility in coral symbioses. Marine Ecology Progress Series, 347, 307-309.

Baker, A. C. (2001). Ecosystems: reef corals bleach to survive change. Nature, 411 (6839), 765.

121 List of References

Baker, A. C. (2003). Flexibility and specificity in coral-algal symbiosis: diversity, ecology, and biogeography of Symbiodinium. Annual Review of Ecology, Evolution, and Systematics, 34 (1), 661-689.

Baker, A. C., Starger, C. J., McClanahan, T. R., & Glynn, P. W. (2004). Coral reefs: corals' adaptive response to climate change. Nature, 430 (7001), 741.

Baker, D. M., Andras, J. P., Jordán-Garza, A. G., & Fogel, M. L. (2013). Nitrate competition in a coral symbiosis varies with temperature among Symbiodinium types. The ISME journal, 7 (6), 1248-1251.

Baker, D. M., Freeman, C. J., Knowlton, N., Thacker, R. W., Kim, K., & Fogel, M. L. (2015). Productivity links morphology, symbiont specificity and bleaching in the evolution of Caribbean octocoral symbioses. The ISME journal, 9 (12), 2620.

Barbrook, A. C., Voolstra, C. R., & Howe, C. J. (2014). The chloroplast genome of a Symbiodinium sp. clade C3 isolate. Protist, 165 (1), 1-13.

Barott, K. L., Venn, A. A., Perez, S. O., Tambutté, S., & Tresguerres, M. (2015). Coral host cells acidify symbiotic algal microenvironment to promote photosynthesis. Proceedings of the National Academy of Sciences, 112(2), 607-612.

Barshis, D. J., Stillman, J. H., Gates, R. D., Toonen, R. J., Smith, L. W., & Birkeland, C. (2010). Protein expression and genetic structure of the coral Porites lobata in an environmentally extreme Samoan back reef: does host genotype limit phenotypic plasticity? Molecular Ecology, 19 (8), 1705-1720.

Barshis, D. J., Ladner, J. T., Oliver, T. A., Seneca, F. O., Traylor-Knowles, N., & Palumbi, S. R. (2013). Genomic basis for coral resilience to climate change. Proceedings of the National Academy of Sciences, 110 (4), 1387-1392.

Baumgarten, S., Simakov, O., Esherick, L. Y., Liew, Y. J., Lehnert, E. M., Michell, C. T., ... & Gough, J. (2015). The genome of Aiptasia, a sea anemone model for coral symbiosis. Proceedings of the National Academy of Sciences, 112 (38), 11893-11898.

Bayer, T., Aranda, M., Sunagawa, S., Yum, L. K., DeSalvo, M. K., Lindquist, E., ... & Medina, M. (2012). Symbiodinium transcriptomes: genome insights into the dinoflagellate symbionts of reef-building corals. PloS One, 7 (4), e35269.

Bellantuono, A. J., Hoegh-Guldberg, O., & Rodriguez-Lanetty, M. (2011). Resistance to thermal stress in corals without changes in symbiont composition. Proceedings of the Royal Society of London B: Biological Sciences, rspb20111780.

122 List of References

Berkelmans, R., & Van Oppen, M. J. (2006). The role of zooxanthellae in the thermal tolerance of corals: a ‘nugget of hope’for coral reefs in an era of climate change. Proceedings of the Royal Society of London B: Biological Sciences, 273 (1599), 2305-2312.

Blank, R. J. (1987). Cell architecture of the dinoflagellate Symbiodinium sp. inhabiting the Hawaiian stony coral Montipora verrucosa. Marine Biology, 94 (1), 143-155.

Blank, R. J. & R. K. Trench. (1985a). Symbiodinium microadriaticum: a single species? In: Proc. Fifth Int. Coral Reef Congress Tahiti, vol. 6, pp 113-117. Ed. by C. Gabrie, J. L. Toffart and B. Salvat. Papeete: Atenne du Museum National d'Histoire Naturelle et de l'Ecole Pratique des Hautes Etudes.

Blank, R. J. & R. K. Trench. (1986). Nomenclature of endosymbiotic dinoflagellates. Taxon 35, 286-294

Blank, R. J., & Trench, R. K. (1985b). Speciation and symbiotic dinoflagellates. Science, 229 (4714), 656- 658.

Boldt, L., Yellowlees, D., & Leggat, W. (2012). Hyperdiversity of genes encoding integral light-harvesting proteins in the dinoflagellate Symbiodinium sp. PLoS One, 7 (10), e47456.

Bollati, E., Plimmer, D., D’Angelo, C., & Wiedenmann, J. (2017). FRET-mediated long-range wavelength transformation by photoconvertible fluorescent proteins as an efficient mechanism to generate orange-red light in symbiotic deep-water corals. International Journal of Molecular Sciences, 18 (7), 1174.

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72 (1-2), 248-254.

Brading, P., Warner, M. E., Smith, D. J., & Suggett, D. J. (2013). Contrasting modes of inorganic carbon acquisition amongst Symbiodinium (Dinophyceae) phylotypes. New Phytologist, 200 (2), 432-442.

Brown, B. E. (1997). Coral bleaching: causes and consequences. Coral Reefs,16 (1), S129-S138.

Brun, V., Masselon, C., Garin, J., & Dupuis, A. (2009). Isotope dilution strategies for absolute quantitative proteomics. Journal of Proteomics, 72 (5), 740-749.

Buddemeier, R. W., Baker, A. C., Fautin, D. G., & Jacobs, J. R. (2004). The adaptive hypothesis of bleaching. In: Coral Health and Disease (pp. 427-444). Springer, Berlin, Heidelberg.

Burriesci, M. S., Raab, T. K., & Pringle, J. R. (2012). Evidence that glucose is the major transferred metabolite in dinoflagellate–cnidarian symbiosis. The Journal of Experimental Biology, 215 (19), 3467-3477.

123 List of References

Butterfield, E. R., Howe, C. J., & Nisbet, R. E. R. (2016). Identification of sequences encoding Symbiodinium minutum mitochondrial proteins. Genome Biology and Evolution, 8 (2), 439-445.

Chan, L. L., Hodgkiss, I. J., Wan, J. M. F., Lum, J. H. K., Mak, A. S. C., Sit, W. H., & Lo, S. C. L. (2004). Proteomic study of a model causative agent of harmful algal blooms, Prorocentrum triestinum II: the use of differentially expressed protein profiles under different growth phases and growth conditions for bloom prediction. Proteomics, 4 (10), 3214-3226.

Chan LL, Lo SCL, Hodgkiss IJ. 2002. Proteomic study of a model causative agent of harmful red tide, Prorocentrum triestinum I: optimization of sample preparation methodologies for analyzing with two-dimensional electrophoresis. Proteomics; 2:1169–86.

Chang, S. S., Prezelin, B. B., & Trench, R. K. (1983). Mechanisms of photoadaptation in three strains of the symbiotic dinoflagellate Symbiodinium microadriaticum. Marine Biology, 76 (3), 219-229.

Chen, P. Y., Chen, C. C., Chu, L., & McCarl, B. (2015). Evaluating the economic damage of climate change on global coral reefs. Global Environmental Change, 30, 12-20.

Chen, W. N., Kang, H. J., Weis, V. M., Mayfield, A. B., Jiang, P. L., Fang, L. S., & Chen, C. S. (2012). Diel rhythmicity of lipid-body formation in a coral-Symbiodinium endosymbiosis. Coral Reefs, 31 (2), 521-534.

Colombo-Pallotta, M. F., Rodríguez-Román, A., & Iglesias-Prieto, R. (2010). Calcification in bleached and unbleached Montastraea faveolata: evaluating the role of oxygen and glycerol. Coral Reefs, 29 (4), 899-907.

Cruz, I. C., Leal, M. C., Mendes, C. R., Kikuchi, R. K., Rosa, R., Soares, A. M., ... & Rocha, R. J. (2015). White but not bleached: photophysiological evidence from white Montastraea cavernosa reveals potential overestimation of coral bleaching. Marine Biology, 162 (4), 889-899.

Cunning, R., & Baker, A. C. (2014). Not just who, but how many: the importance of partner abundance in reef coral symbioses. Frontiers in Microbiology, 5, 400.

Cunning, R., Muller, E. B., Gates, R. D., & Nisbet, R. M. (2017). A dynamic bioenergetic model for coral- Symbiodinium symbioses and coral bleaching as an alternate stable state. Journal of Theoretical Biology, 431, 49-62.

Cunning, R., Vaughan, N., Gillette, P., Capo, T. R., Maté, J. L., & Baker, A. C. (2015). Dynamic regulation of partner abundance mediates response of reef coral symbioses to environmental change. Ecology, 96 (5), 1411-1420.

124 List of References

Curien, G., Flori, S., Villanova, V., Magneschi, L., Giustini, C., Forti, G., ... & Finazzi, G. (2016). The water to water cycles in microalgae. Plant and Cell Physiology, 57 (7), 1354-1363.

D’Angelo, C., & Wiedenmann, J. (2014). Impacts of nutrient enrichment on coral reefs: new perspectives and implications for coastal management and reef survival. Current Opinion in Environmental Sustainability, 7, 82-93.

Davis, M. T., Beierle, J., Bures, E. T., McGinley, M. D., Mort, J., Robinson, J. H., ... & Patterson, S. D. (2001). Automated LC–LC–MS–MS platform using binary ion-exchange and gradient reversed- phase chromatography for improved proteomic analyses. Journal of Chromatography B: Biomedical Sciences and Applications, 752 (2), 281-291.

Davy, S. K., Allemand, D., & Weis, V. M. (2012). Cell biology of cnidarian-dinoflagellate symbiosis. Microbiology and Molecular Biology Reviews, 76 (2), 229-261.

Díaz-Almeyda, E., Thomé, P. E., El Hafidi, M., & Iglesias-Prieto, R. (2011). Differential stability of photosynthetic membranes and fatty acid composition at elevated temperature in Symbiodinium. Coral Reefs, 30 (1), 217-225.

Dorrell, R. G., & Howe, C. J. (2015). Integration of plastids with their hosts: Lessons learned from dinoflagellates. Proceedings of the National Academy of Sciences, 112 (33), 10247-10254.

Douglas, A. E. (2003). Coral bleaching––how and why? Marine Pollution Bulletin, 46 (4), 385-392.

Dove, S., Ortiz, J. C., Enríquez, S., Fine, M., Fisher, P., Iglesias-Prieto, R., ... & Hoegh-Guldberg, O. (2006). Response of holosymbiont pigments from the scleractinian coral Montipora monasteriata to short- term heat stress. Limnology and Oceanography, 51 (2), 1149-1158.

Downs, C. A., McDougall, K. E., Woodley, C. M., Fauth, J. E., Richmond, R. H., Kushmaro, A., ... & Kramarsky-Winter, E. (2013). Heat-stress and light-stress induce different cellular pathologies in the symbiotic dinoflagellate during coral bleaching. PloS One, 8 (12), e77173.

Dubinsky, Z., & Jokiel, P. L. (1994). Ratio of energy and nutrient fluxes regulates symbiosis between zooxanthellae and corals. Pacific Science, 48 (3), 313-324.

Dumas, L., Chazaux, M., Peltier, G., Johnson, X., & Alric, J. (2016). Cytochrome b6f function and localization, phosphorylation state of thylakoid membrane proteins and consequences on cyclic electron flow. Photosynthesis Research, 129 (3), 307-320.

Dunn, M. J. (2014). Proteomics Comes of Age. Proteomics, 14 (1), 1-2.

125 List of References

Enríquez, S., Méndez, E. R., & Prieto, R. I. (2005). Multiple scattering on coral skeletons enhances light absorption by symbiotic algae. Limnology and Oceanography, 50 (4), 1025-1032.

Falkowski, P. G., Dubinsky, Z., Muscatine, L., & McCloskey, L. (1993). Population control in symbiotic corals. Bioscience, 43 (9), 606-611.

Fautin, D. G., & Buddemeier, R. W. (2004). Adaptive bleaching: a general phenomenon. In Coelenterate Biology 2003 (pp. 459-467). Springer Netherlands.

Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., & Whitehouse, C. M. (1990). Electrospray ionization– principles and practice. Mass Spectrometry Reviews, 9 (1), 37-70.

Fitt, W. K., & Warner, M. E. (1995). Bleaching patterns of four species of Caribbean reef corals. The Biological Bulletin, 189 (3), 298-307.

Fitt, W. K., Brown, B. E., Warner, M. E., & Dunne, R. P. (2001). Coral bleaching: interpretation of thermal tolerance limits and thermal thresholds in tropical corals. Coral Reefs, 20 (1), 51-65.

Freudenthal, H. D. (1962). Symbiodinium gen. nov. and Symbiodinium microadriaticum sp. nov., a Zooxanthella: Taxonomy, Life Cycle, and Morphology. Journal of Eukaryotic Microbiology, 9 (1), 45-52.

Frieler, K., Meinshausen, M., Golly, A., Mengel, M., Lebek, K., Donner, S. D., & Hoegh-Guldberg, O. (2013). Limiting global warming to 2 ºC is unlikely to save most coral reefs. Nature Climate Change, 3 (2), 165.

Furla, P., Allemand, D., Shick, J. M., Ferrier-Pagès, C., Richier, S., Plantivaux, A., ... & Tambutté, S. (2005). The symbiotic anthozoan: a physiological chimera between alga and animal. Integrative and Comparative Biology, 45 (4), 595-604.

Gagnon, C., White, D., Cosson, J., Huitorel, P., Eddé, B., Desbruyères, E., ... & Cibert, C. (1996). The polyglutamylated lateral chain of alpha-tubulin plays a key role in flagellar motility. Journal of Cell Science, 109 (6), 1545-1553.

Gates R.D., Hoegh-Guldberg O., McFall-Ngai M.J., Bil K.Y. & Muscatine L. (1995) Free amino acids exhibit anthozoan ‘host factor’ activity: they induce the release of photosynthate from symbiotic dinoflagellates in vitro. Proceedings of the National Academy of Sciences 92, 7430–7434.

Gerber, S. A., Rush, J., Stemman, O., Kirschner, M. W., & Gygi, S. P. (2003). Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proceedings of the National Academy of Sciences, 100 (12), 6940-6945.

126 List of References

Gharahdaghi, F., Weinberg, C. R., Meagher, D. A., Imai, B. S., & Mische, S. M. (1999). Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis: An International Journal, 20 (3), 601-605.

Goreau, T. J., & Macfarlane, A. H. (1990). Reduced growth rate of Montastrea annularis following the 1987–1988 coral-bleaching event. Coral Reefs, 8 (4), 211-215.

Goulet, T. L. (2006). Most corals may not change their symbionts. Marine Ecology Progress Series, 321, 1-7.

Graham, N. A., Cinner, J. E., Norström, A. V., & Nyström, M. (2014). Coral reefs as novel ecosystems: embracing new futures. Current Opinion in Environmental Sustainability, 7, 9-14.

Graham, N. A., Jennings, S., MacNeil, M. A., Mouillot, D., & Wilson, S. K. (2015). Predicting climate- driven regime shifts versus rebound potential in coral reefs. Nature, 518 (7537), 94-97.

Grant, A. J., Rémond, M., Withers, K. J. T., & Hinde, R. (2001). Inhibition of algal photosynthesis by a symbiotic coral. Hydrobiologia, 461 (1-3), 63-69

Grant, A., People, J., Remond, M., Frankland, S., & Hinde, R. (2013). How a host cell signalling molecule modifies carbon metabolism in symbionts of the coral Plesiastrea versipora. FEBS Journal, 280 (9), 2085-2096.

Grottoli, A. G., Warner, M. E., Levas, S. J., Aschaffenburg, M. D., Schoepf, V., McGinley, M., ... & Matsui, Y. (2014). The cumulative impact of annual coral bleaching can turn some coral species winners into losers. Global Change Biology, 20 (12), 3823-3833.

Guo, R., Youn, S. H., & Ki, J. S. (2015). Heat shock protein 70 and 90 genes in the harmful dinoflagellate Cochlodinium polykrikoides: genomic structures and transcriptional responses to environmental stresses. International Journal of Genomics, 2015.

Hawkins, T. D., Krueger, T., Wilkinson, S. P., Fisher, P. L., & Davy, S. K. (2015). Antioxidant responses to heat and light stress differ with habitat in a common reef coral. Coral Reefs, 34 (4), 1229-1241.

Hennige, S. J., Suggett, D. J., Warner, M. E., McDougall, K. E., & Smith, D. J. (2009). Photobiology of Symbiodinium revisited: bio-physical and bio-optical signatures. Coral Reefs, 28 (1), 179-195.

Hennige, S. J., McGinley, M. P., Grottoli, A. G., & Warner, M. E. (2011). Photoinhibition of Symbiodinium spp. within the reef corals Montastraea faveolata and Porites astreoides: implications for coral bleaching. Marine Biology, 158 (11), 2515-2526.

127 List of References

Hennige, S. J., Smith, D. J., Walsh, S. J., McGinley, M. P., Warner, M. E., & Suggett, D. J. (2010). Acclimation and adaptation of scleractinian coral communities along environmental gradients within an Indonesian reef system. Journal of Experimental Marine Biology and Ecology, 391 (1), 143-152.

Herbert, B. (1999). Advances in protein solubilisation for two-dimensional electrophoresis. Electrophoresis: An International Journal, 20 (4-5), 660-663.

Hiller, R. G., Wrench, P. M., Gooley, A. P., Shoebridge, G., & Breton, J. (1993). The major intrinsic light- harvesting protein of Amphidinium: Characterization and relation to other light-harvesting proteins. Photochemistry and Photobiology, 5 7(1), 125-131.

Hoadley, K. D., Pettay, D. T., Grottoli, A. G., Cai, W. J., Melman, T. F., Schoepf, V., ... & Matsui, Y. (2015).

Physiological response to elevated temperature and pCO2 varies across four Pacific coral species: understanding the unique host-symbiont response. Scientific Reports, 5, 18371.

Hoegh-Guldberg, O. (1999). Climate change, coral bleaching and the future of the world's coral reefs. Marine and Freshwater Research, 50 (8), 839-866.

Hofmann, E., Wrench, P. M., Sharples, F. P., Hiller, R. G., Welte, W., & Diederichs, K. (1996). Structural basis of light harvesting by carotenoids: peridinin-chlorophyll-protein from Amphidinium carterae. Science, 272 (5269), 1788-1791.

Hooidonk, R., Maynard, J. A., Manzello, D., & Planes, S. (2014). Opposite latitudinal gradients in projected ocean acidification and bleaching impacts on coral reefs. Global Change Biology, 20 (1), 103-112.

Howells, E. J., Abrego, D., Meyer, E., Kirk, N. L., & Burt, J. A. (2016). Host adaptation and unexpected symbiont partners enable reef-building corals to tolerate extreme temperatures. Global Change Biology, 22 (8), 2702-2714.

Howells, E. J., Beltran, V. H., Larsen, N. W., Bay, L. K., Willis, B. L., & Van Oppen, M. J. H. (2012). Coral thermal tolerance shaped by local adaptation of photosymbionts. Nature Climate Change, 2 (2), 116-120.

Hume, B. C., D'Angelo, C., Smith, E. G., Stevens, J. R., Burt, J., & Wiedenmann, J. (2015). Symbiodinium thermophilum sp. nov., a thermotolerant symbiotic alga prevalent in corals of the world's hottest sea, the Persian/Arabian Gulf. Scientific Reports, 5, 8562.

Hughes, T. P., Rodrigues, M. J., Bellwood, D. R., Ceccarelli, D., Hoegh-Guldberg, O., McCook, L., ... & Willis, B. (2007). Phase shifts, herbivory, and the resilience of coral reefs to climate change. Current Biology, 17 (4), 360-365.

128 List of References

Hughes, T. P., Barnes, M. L., Bellwood, D. R., Cinner, J. E., Cumming, G. S., Jackson, J. B., ... & Palumbi, S. R. (2017). Coral reefs in the Anthropocene. Nature, 546 (7656), 82.

Hughes, T. P., Anderson, K. D., Connolly, S. R., Heron, S. F., Kerry, J. T., Lough, J. M., ... & Claar, D. C. (2018). Spatial and temporal patterns of mass bleaching of corals in the Anthropocene. Science, 359 (6371), 80-83.

Iglesias-Prieto R (1996) Biochemical and spectroscopic properties of the Light-harvesting apparatus of dinoflagellates. In: Chaudhary BR and Agrawal SB (eds) Citology. Genetic and Molecular Biology of Algae. pp 301-322. SPB Academic Publishing, Amsterdam, The Netherlands.

Iglesias-Prieto, R., & Trench, R. K. (1997). Acclimation and adaptation to irradiance in symbiotic dinoflagellates. II. Response of chlorophyll–protein complexes to different photon-flux densities. Marine Biology, 130 (1), 23-33.

Iglesias-Prieto, R., Govind, N. S., & Trench, R. K. (1993). Isolation and characterization of three membranebound chlorophyll-protein complexes from four dinoflagellate species. Phil. Trans. R. Soc. Lond. B, 340 (1294), 381-392.

Iglesias-Prieto, R., & Trench, R. K. (1994). Acclimation and adaptation to irradiance in symbiotic dinoflagellates. I. Responses of the photosynthetic unit to changes in photon flux density. Marine Ecology Progress Series, 163-175.

Iglesias-Prieto, R., Beltran, V. H., LaJeunesse, T. C., Reyes-Bonilla, H., & Thome, P. E. (2004). Different algal symbionts explain the vertical distribution of dominant reef corals in the eastern Pacific. Proceedings of the Royal Society B: Biological Sciences, 271 (1549), 1757.

Iglesias-Prieto, R., Matta, J. L., Robins, W. A., & Trench, R. K. (1992). Photosynthetic response to elevated temperature in the symbiotic dinoflagellate Symbiodinium microadriaticum in culture. Proceedings of the national Academy of Sciences, 89 (21), 10302-10305.

Jameson, S. C., Tupper, M. H., & Ridley, J. M. (2002). The three screen doors: can marine “protected” areas be effective? Marine Pollution Bulletin, 44 (11), 1177-1183.

Jeans, J., Campbell, D. A., & Hoogenboom, M. O. (2013). Increased reliance upon photosystem II repair following acclimation to high-light by coral-dinoflagellate symbioses. Photosynthesis Research, 118 (3), 219-229.

129 List of References

Jiang, J., Zhang, H., Orf, G. S., Lu, Y., Xu, W., Harrington, L. B., ... & Blankenship, R. E. (2014). Evidence of functional trimeric chlorophyll a/c2-peridinin proteins in the dinoflagellate Symbiodinium. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1837 (11), 1904-1912.

Jones, R. J., Hoegh-Guldberg, O., Larkum, A. W., & Schreiber, U. (1998). Temperature-induced bleaching of

corals begins with impairment of the CO2 fixation mechanism in zooxanthellae. Plant, Cell & Environment, 21 (12), 1219-1230.

Kanazawa, A., Blanchard, G. J., Szabó, M., Ralph, P. J., & Kramer, D. M. (2014). The site of regulation of light capture in Symbiodinium: Does the peridinin–chlorophyll a–protein detach to regulate light capture? Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1837 (8), 1227-1234.

Kelly, L. W., Williams, G. J., Barott, K. L., Carlson, C. A., Dinsdale, E. A., Edwards, R. A., ... & Rohwer, F. (2014). Local genomic adaptation of coral reef-associated microbiomes to gradients of natural variability and anthropogenic stressors. Proceedings of the National Academy of Sciences, 111 (28), 10227-10232.

Kemp, D. W., Hernandez-Pech, X., Iglesias-Prieto, R., Fitt, W. K., & Schmidt, G. W. (2014). Community dynamics and physiology of Symbiodinium spp. before, during, and after a coral bleaching event. Limnology and Oceanography, 59 (3), 788-797.

Kemp, D. W., Thornhill, D. J., Rotjan, R. D., Iglesias-Prieto, R., Fitt, W. K., & Schmidt, G. W. (2015). Spatially distinct and regionally endemic Symbiodinium assemblages in the threatened Caribbean reef-building coral Orbicella faveolata. Coral Reefs, 34 (2), 535-547.

Kempf, S. C. (1984). Symbiosis between the zooxanthella Symbiodinium (= Gymnodinium) microadriaticum (Freudenthal) and four species of nudibranchs. The Biological Bulletin, 166 (1), 110-126.

Kim, M. S., Pinto, S. M., Getnet, D., Nirujogi, R. S., Manda, S. S., Chaerkady, R., ... & Thomas, J. K. (2014). A draft map of the human proteome. Nature, 509(7502), 575-581.

Kleypas, J. A., Buddemeier, R. W., Eakin, C. M., Gattuso, J. P., Guinotte, J., Hoegh-Guldberg, O., ... & Strong, A. E. (2005). Comment on “Coral reef calcification and climate change: the effect of ocean warming”. Geophysical Research Letters, 32 (8).

Krämer, W. E., Caamaño‐Ricken, I., Richter, C., & Bischof, K. (2012). Dynamic regulation of photoprotection determines thermal tolerance of two phylotypes of Symbiodinium clade A at two photon fluence rates. Photochemistry and Photobiology, 88 (2), 398-413.

130 List of References

Krueger, T., Becker, S., Pontasch, S., Dove, S., Hoegh‐Guldberg, O., Leggat, W., ... & Davy, S. K. (2014). Antioxidant plasticity and thermal sensitivity in four types of Symbiodinium sp. Journal of Phycology, 50 (6), 1035-1047.

Kwiatkowski, L., Cox, P., Halloran, P. R., Mumby, P. J., & Wiltshire, A. J. (2015). Coral bleaching under unconventional scenarios of climate warming and ocean acidification. Nature Climate Change, 5 (8), 777.

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227 (5259), 680.

LaJeunesse, T. C. (2001). Investigating the biodiversity, ecology, and phylogeny of endosymbiotic dinoflagellates in the genus Symbiodinium using the ITS region: in search of a “species” level marker. Journal of Phycology, 37 (5), 866-880.

LaJeunesse, T. C., & Thornhill, D. J. (2011). Improved resolution of reef-coral endosymbiont (Symbiodinium) species diversity, ecology, and evolution through psbA non-coding region genotyping. PloS One, 6 (12), e29013.

LaJeunesse, T. C. (2005). “Species” radiations of symbiotic dinoflagellates in the Atlantic and Indo-Pacific since the Miocene-Pliocene transition. Molecular Biology and Evolution, 22 (3), 570-581.

LaJeunesse, T. C., & Trench, R. K. (2000). Biogeography of two species of Symbiodinium (Freudenthal) inhabiting the intertidal sea anemone Anthopleura elegantissima (Brandt). The Biological Bulletin, 199 (2), 126-134.

LaJeunesse, T. C., Pettay, D. T., Sampayo, E. M., Phongsuwan, N., Brown, B., Obura, D. O., ... & Fitt, W. K. (2010b). Long‐standing environmental conditions, geographic isolation and host–symbiont specificity influence the relative ecological dominance and genetic diversification of coral endosymbionts in the genus Symbiodinium. Journal of Biogeography, 37 (5), 785-800.

LaJeunesse, T. C., Smith, R., Walther, M., Pinzón, J., Pettay, D. T., McGinley, M., ... & Warner, M. E. (2010a). Host–symbiont recombination versus natural selection in the response of coral– dinoflagellate symbioses to environmental disturbance. Proceedings of the Royal Society of London B: Biological Sciences, rspb20100385.

LaJeunesse, T. C., Wham, D. C., Pettay, D. T., Parkinson, J. E., Keshavmurthy, S., & Chen, C. A. (2014). Ecologically differentiated stress-tolerant endosymbionts in the dinoflagellate genus Symbiodinium (Dinophyceae) Clade D are different species. Phycologia, 53 (4), 305-319.

131 List of References

Lee, F. W. F., & Lo, S. C. L. (2008). The use of Trizol reagent (phenol/guanidine isothiocyanate) for producing high quality two-dimensional gel electrophoretograms (2-DE) of dinoflagellates. Journal of Microbiological Methods, 73 (1), 26-32.

Lee, F. W. F., Morse, D., & Lo, S. C. L. (2009). Identification of two plastid proteins in the dinoflagellate Alexandrium affine that are substantially down-regulated by nitrogen-depletion. Journal of Proteome Research, 8 (11), 5080-5092.

Lee, Y. H., Kim, M. S., Choie, W. S., Min, H. K., & Lee, S. W. (2004). Highly informative proteome analysis by combining improved N-terminal sulfonation for de novo peptide sequencing and online capillary reverse-phase liquid chromatography/tandem mass spectrometry. Proteomics, 4(6), 1684- 1694.

Leggat, W., Marendy, E. M., Baillie, B., Whitney, S. M., Ludwig, M., Badger, M. R., & Yellowlees, D. (2002). Dinoflagellate symbioses: strategies and adaptations for the acquisition and fixation of inorganic carbon. Functional Plant Biology, 29 (3), 309-322.

Leggat, W., Ainsworth, T., Bythell, J., Dove, S., Gates, R., Hoegh-Guldberg, O., ... & Yellowlees, D. (2007). The hologenome theory disregards the coral holobiont. Nature Reviews Microbiology, 5 (10).

Leggat, W., Badger, M. R., & Yellowlees, D. (1999). Evidence for an inorganic carbon-concentrating mechanism in the symbiotic dinoflagellate Symbiodinium sp. Plant Physiology, 121 (4), 1247-1255.

Leggat, W., Seneca, F., Wasmund, K., Ukani, L., Yellowlees, D., & Ainsworth, T. D. (2011). Differential responses of the coral host and their algal symbiont to thermal stress. PloS One, 6 (10), e26687.

Lehnert, E. M., Mouchka, M. E., Burriesci, M. S., Gallo, N. D., Schwarz, J. A., & Pringle, J. R. (2014). Extensive differences in gene expression between symbiotic and aposymbiotic Cnidarians. G3: Genes| Genomes| Genetics, 4 (2), 277-295.

Lei, Q. Y., & Lü, S. H. (2011). Molecular ecological responses of dinoflagellate, Karenia mikimotoi to environmental nitrate stress. Marine Pollution Bulletin, 62 (12), 2692-2699.

Lesser, M. P., Stat, M., & Gates, R. D. (2013). The endosymbiotic dinoflagellates (Symbiodinium sp.) of corals are parasites and mutualists. Coral Reefs, 32 (3), 603-611.

Li, C., Wang, D., Dong, H., Xie, Z., & Hong, H. (2012). Proteomics of a toxic dinoflagellate Alexandrium catenella DH01: Detection and identification of cell surface proteins using fluorescent labeling. Chinese Science Bulletin, 57 (25), 3320-3327.

132 List of References

Li, H. H., Huang, Z. Y., Ye, S. P., Lu, C. Y., Cheng, P. C., Chen, S. H., & Chen, C. S. (2014). Membrane Labeling of Coral Gastrodermal Cells by Biotinylation: The Proteomic Identification of Surface Proteins Involving Cnidaria-Dinoflagellate Endosymbiosis. PloS One, 9 (1), e85119.

Lin, S., Cheng, S., Song, B., Zhong, X., Lin, X., Li, W., ... & Cai, M. (2015). The Symbiodinium kawagutii genome illuminates dinoflagellate gene expression and coral symbiosis. Science, 350 (6261), 691-694.

Mammalian Gene Collection (MGC) Program Team. (2002). Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proceedings of the National Academy of Sciences, 99 (26), 16899-16903.

Marcelino, L. A., Westneat, M. W., Stoyneva, V., Henss, J., Rogers, J. D., Radosevich, A., ... & Fung, J. (2013). Modulation of light-enhancement to symbiotic algae by light-scattering in corals and evolutionary trends in bleaching. PLoS One, 8 (4), e61492.

Markell, D. A., & Wood-Charlson, E. M. (2010). Immunocytochemical evidence that symbiotic algae secrete potential recognition signal molecules in hospite. Marine Biology, 157 (5), 1105-1111.

Maruyama, S., Shoguchi, E., Satoh, N., & Minagawa, J. (2015). Diversification of the light-harvesting complex gene family via intra-and intergenic duplications in the coral symbiotic alga Symbiodinium. PLoS One, 10 (3), e0119406.

McGinley, M. P., Suggett, D. J., & Warner, M. E. (2013). Transcript patterns of chloroplast‐encoded genes in cultured Symbiodinium spp. (Dinophyceae): testing the influence of a light shift and diel periodicity. Journal of Phycology,49 (4), 709-718.

Meyer, E., & Weis, V. M. (2012). Study of cnidarian-algal symbiosis in the “omics” age. The Biological Bulletin, 223 (1), 44-65.

Michalski, A., Cox, J., & Mann, M. (2011). More than 100,000 detectable peptide species elute in single shotgun proteomics runs but the majority is inaccessible to data-dependent LC− MS/MS. Journal of Proteome Research, 10 (4), 1785-1793.

Mies, M., Voolstra, C. R., Castro, C. B., Pires, D. O., Calderon, E. N., & Sumida, P. Y. G. (2017). Expression of a symbiosis-specific gene in Symbiodinium type A1 associated with coral, nudibranch and giant clam larvae. Royal Society Open Science, 4 (5), 170253.

Minge, M. A., Shalchian-Tabrizi, K., Tørresen, O. K., Takishita, K., Probert, I., Inagaki, Y., ... & Jakobsen, K. S. (2010). A phylogenetic mosaic plastid proteome and unusual plastid-targeting signals in the green-colored dinoflagellate Lepidodinium chlorophorum. BMC Evolutionary Biology, 10 (1), 191.

133 List of References

Moberg, F., & Folke, C. (1999). Ecological goods and services of coral reef ecosystems. Ecological Economics, 29(2), 215-233.

Mullineaux, C. W., & Emlyn-Jones, D. (2004). State transitions: an example of acclimation to low-light stress. Journal of Experimental Botany, 56 (411), 389-393.

Munekage, Y., Hashimoto, M., Miyake, C., & Tomizawa, K. I. (2004). Cyclic electron flow around photosystem I is essential for photosynthesis. Nature, 429 (6991), 579.

Mungpakdee, S., Shinzato, C., Takeuchi, T., Kawashima, T., Koyanagi, R., Hisata, K., ... & Satoh, N. (2014). Massive gene transfer and extensive RNA editing of a symbiotic dinoflagellate plastid genome. Genome Biology and Evolution, 6 (6), 1408-1422.

Muscatine L. (1967) Glycerol excretion by symbiotic algae from corals and Tridacna and its control by the host. Science 156, 516–519.

Muscatine L., Pool R.R. & Cernichiari E. (1972) Some factors influencing selective release of soluble organic material by zooxanthellae from reef corals. Marine Biology 13, 298–308.

Muscatine, L. (1980). Productivity of zooxanthellae. In: Primary Productivity in the Sea. (Ed. P. G. Falkowski.) pp. 381.402. (Plenum: New York.)

Muscatine, L. (1990). The role of symbiotic algae in carbon and energy flux in reef corals. Coral Reefs 5, 1- 29.

Muscatine, L., & Hand, C. (1958). Direct evidence for the transfer of materials from symbiotic algae to the tissues of a coelenterate. Proceedings of the National Academy of Sciences, 44 (12), 1259-1263.

Muscatine, L., & Cernichiari, E. (1969). Assimilation of photosynthetic products of zooxanthellae by a reef coral. The Biological Bulletin, 137 (3), 506-523.

Muscatine, L., Pool, R. R., & Cernichiari, E. (1972). Some factors influencing selective release of soluble organic material by zooxanthellae from reef corals. Marine Biology, 13 (4), 298-308.

Muscatine, L., & Weis, V. (1992). Productivity of zooxanthellae and biogeochemical cycles. In Primary productivity and biogeochemical cycles in the sea (pp. 257-271). Springer, Boston, MA.

Noguchi, K., & Yoshida, K. (2008). Interaction between photosynthesis and respiration in illuminated leaves. Mitochondrion, 8 (1), 87-99.

Nyström, M., Folke, C., & Moberg, F. (2000). Coral reef disturbance and resilience in a human-dominated environment. Trends in Ecology & Evolution,15 (10), 413-417.

134 List of References

Oakley, C. A., Ameismeier, M. F., Peng, L., Weis, V. M., Grossman, A. R., & Davy, S. K. (2016). Symbiosis induces widespread changes in the proteome of the model cnidarian Aiptasia. Cellular Microbiology, 18 (7), 1009-1023.

Oakley, C. A., Hopkinson, B. M., & Schmidt, G. W. (2014b). Mitochondrial terminal alternative oxidase and its enhancement by thermal stress in the coral symbiont Symbiodinium. Coral Reefs, 33 (2), 543- 552.

Oakley, C. A., Schmidt, G. W., & Hopkinson, B. M. (2014a). Thermal responses of Symbiodinium photosynthetic carbon assimilation. Coral Reefs, 33 (2), 501-512.

Ort, D. R., & Baker, N. R. (2002). A photoprotective role for O2 as an alternative electron sink in photosynthesis? Current Opinion in Plant Biology, 5 (3), 193-198.

Palcy, S., &Chevet, E. (2006). Integrating forward and reverse proteomics to unravel protein function. Proteomics, 6 (20), 5467-5480.

Palmer, J. D. (1996). Rubisco surprises in dinoflagellates. The Plant Cell, 8 (3), 343.

Pantos, O., Bongaerts, P., Dennis, P. G., Tyson, G. W., & Hoegh-Guldberg, O. (2015). Habitat-specific environmental conditions primarily control the microbiomes of the coral Seriatopora hystrix. The ISME Journal, 9 (9), 1916.

Pasaribu, B., Weng, L. C., Lin, I. P., Camargo, E., Tzen, J. T., Tsai, C. H., ... & Jiang, P. L. (2015). Morphological variability and distinct protein profiles of cultured and endosymbiotic Symbiodinium cells isolated from Exaiptasia pulchella. Scientific Reports, 5, 15353.

Peng, S. E., Luo, Y. J., Huang, H. J., Lee, I. T., Hou, L. S., Chen, W. N., ... & Chen, C. S. (2008). Isolation of tissue layers in hermatypic corals by N-acetylcysteine: morphological and proteomic examinations. Coral Reefs, 27 (1), 133-142.

Peng, S. E., Wang, Y. B., Wang, L. H., Chen, W. N. U., Lu, C. Y., Fang, L. S., & Chen, C. S. (2010). Proteomic analysis of symbiosome membranes in Cnidaria–dinoflagellate endosymbiosis. Proteomics, 10 (5), 1002-1016.

Pernice, M., Dunn, S. R., Tonk, L., Dove, S., Domart-Coulon, I., Hoppe, P., ... & Meibom, A. (2015). A nanoscale secondary ion mass spectrometry study of dinoflagellate functional diversity in reef- building corals. Environmental Microbiology, 17 (10), 3570-3580.

135 List of References

Pettay, D. T., Wham, D. C., Smith, R. T., Iglesias-Prieto, R., & LaJeunesse, T. C. (2015). Microbial invasion of the Caribbean by an Indo-Pacific coral zooxanthella. Proceedings of the National Academy of Sciences, 201502283.

Pinzón, J. H., Kamel, B., Burge, C. A., Harvell, C. D., Medina, M., Weil, E., & Mydlarz, L. D. (2015). Whole transcriptome analysis reveals changes in expression of immune-related genes during and after bleaching in a reef-building coral. Royal Society Open Science, 2 (4), 140214

Plass-Johnson, J. G., Cardini, U., van Hoytema, N., Bayraktarov, E., Burghardt, I., Naumann, M. S., & Wild, C. (2015). Coral Bleaching. In: Environmental Indicators (pp. 117-146). Springer Netherlands.

R Development Core Team (2008). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org.

Rabilloud, T., Chevallet, M., Luche, S., & Lelong, C. (2010). Two-dimensional gel electrophoresis in proteomics: past, present and future. Journal of Proteomics, 73 (11), 2064-2077.

Rädecker, N., Pogoreutz, C., Voolstra, C. R., Wiedenmann, J., & Wild, C. (2015). Nitrogen cycling in corals: the key to understanding holobiont functioning? Trends in Microbiology, 23 (8), 490-497.

Ragni, M., Airs, R. L., Hennige, S. J., Suggett, D. J., Warner, M. E., & Geider, R. J. (2010). PSII photoinhibition and photorepair in Symbiodinium (Pyrrhophyta) differs between thermally tolerant and sensitive phylotypes. Mar Ecol Prog Ser, 406, 57-70.

Raharinirina, N., Brandt, G., & Merico, A. (2017). A trait-based model for describing the adaptive dynamics of coral-algae symbiosis. Frontiers in Ecology and Evolution, 5, 31.

Raven, J. A., & Beardall, J. (2017). Consequences of the genotypic loss of mitochondrial Complex I in dinoflagellates and of phenotypic regulation of Complex I content in other photosynthetic organisms. Journal of Experimental Botany, 68 (11), 2683-2692.

Reynolds, J. M., Bruns, B. U., Fitt, W. K., & Schmidt, G. W. (2008). Enhanced photoprotection pathways in symbiotic dinoflagellates of shallow-water corals and other cnidarians. Proceedings of the National Academy of Sciences, 105 (36), 13674-13678.

Ricaurte, M., Schizas, N. V., Ciborowski, P., & Boukli, N. M. (2016). Proteomic analysis of bleached and unbleached Acropora palmata, a threatened coral species of the Caribbean. Marine Pollution Bulletin, 107 (1), 224-232.

Richmond, R. H. (1993). Coral reefs: present problems and future concerns resulting from anthropogenic disturbance. American Zoologist, 33 (6), 524-536.

136 List of References

Ritchie, R. J. (2006). Consistent sets of spectrophotometric chlorophyll equations for acetone, methanol and ethanol solvents. Photosynthesis Research, 89 (1), 27-41.

Ritchie, R. J., Grant, A. J., Eltringham, K., & Hinde, R. (1997). Clotrimazole, a model compound for the host release factor of the coral Plesiastrea versipora. Functional Plant Biology, 24 (3), 283-290.

Roberty, S., Bailleul, B., Berne, N., Franck, F., & Cardol, P. (2014). PSI Mehler reaction is the main alternative photosynthetic electron pathway in Symbiodinium sp., symbiotic dinoflagellates of cnidarians. New Phytologist, 204 (1), 81-91.

Robison, J. D., & Warner, M. E. (2006). Differential impacts of photoacclimation and thermal stress on the photobiology of four different phylotypes of Symbiodinium (pyrrhophyta) 1. Journal of Phycology, 42 (3), 568-579.

Rodriguez-Lanetty, M., Phillips, W. S., & Weis, V. M. (2006). Transcriptome analysis of a cnidarian– dinoflagellate mutualism reveals complex modulation of host gene expression. Bmc Genomics, 7 (1), 23.

Rodríguez-Román, A., & Iglesias-Prieto, R. (2005). Regulation of photochemical activity in cultured symbiotic dinoflagellates under nitrate limitation and deprivation. Marine Biology, 146 (6), 1063- 1073.

Rohwer, F., Seguritan, V., Azam, F., & Knowlton, N. (2002). Diversity and distribution of coral-associated bacteria. Marine Ecology Progress Series, 243, 1-10.

Rosenberg, E., Koren, O., Reshef, L., Efrony, R., & Zilber-Rosenberg, I. (2007). The role of microorganisms in coral health, disease and evolution. Nature Reviews Microbiology, 5 (5), 355.

Rosic, N., Ling, E. Y. S., Chan, C. K. K., Lee, H. C., Kaniewska, P., Edwards, D., ... & Hoegh-Guldberg, O. (2015). Unfolding the secrets of coral–algal symbiosis. The ISME Journal, 9 (4), 844-856.

Roth, K. E., K. Jeon, and G. Stacey. (1988). Homology in endosymbiotic systems: the term "Symbiosome." In: Molecular Genetics of Plant-Microbe Interactions. pp. 220-225. R. Palacios and D. P. S. Verma, eds. APS Press, St. Paul, MN.

Roth, M. S. (2014). The engine of the reef: photobiology of the coral–algal symbiosis. Frontiers in Microbiology, 5, 422.

Rowan, R. (1998). Review—diversity and ecology of zooxanthellae on coral reefs. Journal of Phycology, 34 (3), 407-417.

Rowan, R. (2004). Coral bleaching: thermal adaptation in reef coral symbionts. Nature, 430 (7001), 742-742.

137 List of References

Rowan, R., & Knowlton, N. (1995). Intraspecific diversity and ecological zonation in coral-algal symbiosis. Proceedings of the National Academy of Sciences, 92 (7), 2850-2853.

Rowan, R., Knowlton, N., Baker, A., and Jara, J. (1997). Landscape ecology of algal symbionts creates variation in episodes of bleaching. Nature 388, 265-9.

Royet, J., Gupta, D., & Dziarski, R. (2011). Peptidoglycan recognition proteins: modulators of the microbiome and inflammation. Nature Reviews Immunology, 11 (12), 837.

Sampayo, E. M., Dove, S., & LaJeunesse, T. C. (2009). Cohesive molecular genetic data delineate species diversity in the dinoflagellate genus Symbiodinium. Molecular Ecology, 18 (3), 500-519.

Sampayo, E. M., Ridgway, T., Bongaerts, P., & Hoegh-Guldberg, O. (2008). Bleaching susceptibility and mortality of corals are determined by fine-scale differences in symbiont type. Proceedings of the National Academy of Sciences, 105 (30), 10444-10449.

Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nature Methods, 9 (7), 671-675.

Schrameyer, V., Krämer, W., Hill, R., Jeans, J., Larkum, A. W., Bischof, K., ... & Ralph, P. J. (2016). Under high light stress two Indo-Pacific coral species display differential photodamage and photorepair dynamics. Marine Biology, 163 (8), 1-13.

Schulte, T., Niedzwiedzki, D. M., Birge, R. R., Hiller, R. G., Polívka, T., Hofmann, E., & Frank, H. A. (2009). Identification of a single peridinin sensing Chl-a excitation in reconstituted PCP by crystallography and spectroscopy. Proceedings of the National Academy of Sciences, 106 (49), 20764-20769.

Sebens, K. P. (1994). Biodiversity of coral reefs: what are we losing and why? American Zoologist, 34 (1), 115-133.

Shevchenko, A., Jensen, O. N., Podtelejnikov, A. V., Sagliocco, F., Wilm, M., Vorm, O., ... & Mann, M. (1996). Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proceedings of the National Academy of Sciences, 93 (25), 14440-14445.

Shim, J., Klochkova, T. A., Han, J. W., Kim, G. H., Du Yoo, Y., & Jeong, H. J. (2011). Comparative proteomics of the mixotrophic dinoflagellate Prorocentrum micans growing in different trophic modes. Algae, 26 (1), 87.

138 List of References

Shimakawa, G., Matsuda, Y., Nakajima, K., Tamoi, M., Shigeoka, S., & Miyake, C. (2017). Diverse

strategies of O2 usage for preventing photo-oxidative damage under CO2 limitation during algal photosynthesis. Scientific Reports, 7, 41022.

Shinzato, C., Inoue, M., & Kusakabe, M. (2014a). A snapshot of a coral “holobiont”: a transcriptome assembly of the scleractinian coral, Porites, captures a wide variety of genes from both the host and symbiotic zooxanthellae. PLoS One, 9 (1), e85182.

Shinzato, C., Mungpakdee, S., Satoh, N., & Shoguchi, E. (2014b). A genomic approach to coral- dinoflagellate symbiosis: studies of Acropora digitifera and Symbiodinium minutum. Frontiers in Microbiology, 5, 336.

Shoguchi, E., Shinzato, C., Kawashima, T., Gyoja, F., Mungpakdee, S., Koyanagi, R., ... & Hamada, M. (2013). Draft assembly of the Symbiodinium minutum nuclear genome reveals dinoflagellate gene structure. Current Biology, 23 (15), 1399-1408.

Silva-Lima, A. W., Walter, J. M., Garcia, G. D., Ramires, N., Ank, G., Meirelles, P. M., ... & Thompson, F. L. (2015). Multiple Symbiodinium Strains Are Hosted by the Brazilian Endemic Corals Mussismilia spp. Microbial Ecology, 1-10.

Smith, D. J., Suggett, D. J., & Baker, N. R. (2005). Is photoinhibition of zooxanthellae photosynthesis the primary cause of thermal bleaching in corals? Global Change Biology, 11 (1), 1-11.

Souchelnytskyi, S. (2005). Bridging proteomics and systems biology: what are the roads to be traveled? Proteomics, 5 (16), 4123-4137.

Stanley Jr, G. D. (2006). Photosymbiosis and the evolution of modern coral reefs. Evolution, 1, 3.

Stanley Jr, G. D. (2003). The evolution of modern corals and their early history. Earth-Science Reviews, 60 (3-4), 195-225.

Stat, M., Carter, D., & Hoegh-Guldberg, O. (2006). The evolutionary history of Symbiodinium and scleractinian hosts—symbiosis, diversity, and the effect of climate change. Perspectives in Plant Ecology, Evolution and Systematics,8 (1), 23-43.

Suescún-Bolívar, L. P., Iglesias-Prieto, R., & Thome, P. E. (2012). Induction of glycerol synthesis and release in cultured Symbiodinium. PLoS One, 7 (10), e47182.

Suggett, D. J., Goyen, S., Evenhuis, C., Szabó, M., Pettay, D. T., Warner, M. E., & Ralph, P. J. (2015). Functional diversity of photobiological traits within the genus Symbiodinium appears to be governed by the interaction of cell size with cladal designation. New Phytologist, 208 (2), 370-381.

139 List of References

Suggett, D. J., MacIntyre, H. L., & Geider, R. J. (2004). Evaluation of biophysical and optical determinations of light absorption by photosystem II in phytoplankton. Limnology and Oceanography: Methods, 2, 316–332. http://doi.org/10.4319/lom.2004.2.316

Suggett, D. J., Moore, C. M., Hickman, A. E., & Geider, R. J. (2009). Interpretation of fast repetition rate (FRR) fluorescence: signatures of phytoplankton community structure versus physiological state. Mar. Ecol. Prog. Ser, 376 (1), 1-19.

Suggett, D. J., Oxborough, K., Baker, N. R., MacIntyre, H. L., Kana, T. M., & Geider, R. J. (2003). Fluorescence Measurements for Assessment of Photosynthetic Electron Transport in Marine Phytoplankton. European Journal of Phycology, 38 (4), 371–384. http://doi.org/10.1080/09670260310001612655

Suggett, D. J., Warner, M. E., Smith, D. J., Davey, P., Hennige, S., & Baker, N. R. (2008). Photosynthesis and production of hydrogen peroxide by Symbiodinium (Pyrrhophyta) phylotypes with different thermal tolerances. Journal of Phycology, 44 (4), 948-956.

Suggett, D. J., & Smith, D. J. (2011). Interpreting the sign of coral bleaching as friend vs. foe. Global Change Biology, 17 (1), 45-55.

Sunda, W. K. D. J., Kieber, D. J., Kiene, R. P., & Huntsman, S. (2002). An antioxidant function for DMSP and DMS in marine algae. Nature, 418 (6895), 317.

Sutton, D. C., & Hoegh-Guldberg, O. (1990). Host-zooxanthella interactions in four temperate marine invertebrate symbioses: assessment of effect of host extracts on symbionts. The Biological Bulletin, 178 (2), 175-186.

Szabó, M., Wangpraseurt, D., Tamburic, B., Larkum, A. W., Schreiber, U., Suggett, D. J., ... & Ralph, P. J. (2014). Effective light absorption and absolute electron transport rates in the coral Pocillopora damicornis. Plant Physiology and Biochemistry, 83, 159-167.

Takahashi, S., & Murata, N. (2006). Glycerate-3-phosphate, produced by CO 2 fixation in the Calvin cycle, is critical for the synthesis of the D1 protein of photosystem II. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1757 (3), 198-205.

Takahashi, S., Whitney, S. M., & Badger, M. R. (2009). Different thermal sensitivity of the repair of photodamaged photosynthetic machinery in cultured Symbiodinium species. Proceedings of the National Academy of Sciences, 106 (9), 3237-3242.

140 List of References

Takahashi, S., Whitney, S., Itoh, S., Maruyama, T., & Badger, M. (2008). Heat stress causes inhibition of the de novo synthesis of antenna proteins and photobleaching in cultured Symbiodinium. Proceedings of the National Academy of Sciences, 105 (11), 4203-4208.

Takahashi, S., Yoshioka-Nishimura, M., Nanba, D., & Badger, M. R. (2013). Thermal acclimation of the symbiotic alga Symbiodinium spp. alleviates photobleaching under heat stress. Plant Physiology, 161 (1), 477-485.

Tchernov, D., Gorbunov, M. Y., de Vargas, C., Yadav, S. N., Milligan, A. J., Häggblom, M., & Falkowski, P. G. (2004). Membrane lipids of symbiotic algae are diagnostic of sensitivity to thermal bleaching in corals. Proceedings of the National Academy of Sciences of the United States of America, 101 (37), 13531-13535.

Tchernov, D., Kvitt, H., Haramaty, L., Bibby, T. S., Gorbunov, M. Y., Rosenfeld, H., & Falkowski, P. G. (2011). Apoptosis and the selective survival of host animals following thermal bleaching in zooxanthellate corals. Proceedings of the National Academy of Sciences, 108(24), 9905-9909.

Tonk, L., Sampayo, E. M., LaJeunesse, T. C., Schrameyer, V., &Hoegh‐Guldberg, O. (2014). Symbiodinium (Dinophyceae) diversity in reef‐invertebrates along an offshore to inshore reef gradient near Lizard Island, Great Barrier Reef.Journal of Phycology, 50 (3), 552-563.

Tremblay, P., Grover, R., Maguer, J. F., Hoogenboom, M., & Ferrier-Pagès, C. (2014). Carbon translocation from symbiont to host depends on irradiance and food availability in the tropical coral Stylophora pistillata. Coral Reefs, 33 (1), 1-13.

Trench, R. K. (1971). The physiology and biochemistry of zooxanthellae symbiotic with marine coelenterates. III. The effect of homogenates of host tissues on the excretion of photosynthetic products in vitro by zooxanthellae from two marine coelenterates. Proceedings of the Royal Society of London B: Biological Sciences, 177 (1047), 251-264

Trench, R. K. (1993). Microalgal-invertebrate symbioses: a review. Endocytobiosis Cell Res, 9: 135-175

Trench, R. K. 1971c. The physiology and biochemistry of zooxanthellae symbiotic with marine coelenterates. III. The effect of homogenates of host tissues on the excretion of photosynthetic products in vitro by zooxanthellae from two marine coelenterates. Proc.R. Soc. Lond. B 177: 25 1-264.

Tyers, M., & Mann, M. (2003). From genomics to proteomics. Nature, 422 (6928), 193-197.

Voolstra, C. R., Sunagawa, S., Matz, M. V., Bayer, T., Aranda, M., Buschiazzo, E., ... & Medina, M. (2011). Rapid evolution of coral proteins responsible for interaction with the environment. PloS One, 6 (5), e20392.

141 List of References

Wang, D. Z., Zhang, H., Zhang, Y., & Zhang, S. F. (2014). Marine dinoflagellate proteomics: Current status and future perspectives. Journal of Proteomics, 105, 121-132.

Wang, D. Z., Dong, H. P., Li, C., Xie, Z. X., Lin, L., & Hong, H. S. (2011). Identification and characterization of cell wall proteins of a toxic dinoflagellate Alexandrium catenella using 2-D DIGE and MALDI TOF-TOF mass spectrometry. Evidence-Based Complementary and Alternative Medicine, 2011.

Wang, D. Z., Lin, L., Chan, L. L., & Hong, H. S. (2009). Comparative studies of four protein preparation methods for proteomic study of the dinoflagellate Alexandrium sp. using two-dimensional electrophoresis. Harmful Algae, 8 (5), 685-691.

Wang, D., Lin, L., Wang, M., Li, C., & Hong, H. (2012). Proteomic analysis of a toxic dinoflagellate Alexandrium catenella under different growth phases and conditions. Chinese Science Bulletin, 57 (25), 3328-3341.

Wang, D. Z., Zhang, Y. J., Zhang, S. F., Lin, L., & Hong, H. S. (2013). Quantitative proteomic analysis of cell cycle of the dinoflagellate Prorocentrum donghaiense (Dinophyceae). PloS One, 8 (5), e63659.

Wang, J. T., Chen, Y. Y., Tew, K. S., Meng, P. J., & Chen, C. A. (2012). Physiological and biochemical performances of menthol-induced aposymbiotic corals. PLoS One, 7(9), e46406.

Wang, J. T., & Douglas, A. E. (1997). Nutrients, signals, and photosynthate release by symbiotic algae (the impact of taurine on the dinoflagellate alga Symbiodinium from the sea anemone Aiptasia pulchella). Plant Physiology,114 (2), 631-636.

Warner, M. E., Fitt, W. K., & Schmidt, G. W. (1999). Damage to photosystem II in symbiotic dinoflagellates: a determinant of coral bleaching. Proceedings of the National Academy of Sciences, 96 (14), 8007-8012.

Warner, M. E., Fitt, W. K., & Schmidt, G. W. (1996). The effects of elevated temperature on the photosynthetic efficiency of zooxanthellae in hospite from four different species of reef coral: a novel approach. Plant, Cell & Environment, 19 (3), 291-299.

Weber, M. X., & Medina, M. (2012). The role of microalgal symbionts (Symbiodinium) in holobiont physiology. Genomic Insights into the Biology of Algae, 64, 119.

Weis, V. M., Smith, G. J., & Muscatine, L. (1989). A “CO2 supply” mechanism in zooxanthellate cnidarians: role of carbonic anhydrase. Marine Biology, 100 (2), 195-202.

Weis, V. M. (2010). The susceptibility and resilience of corals to thermal stress: adaptation, acclimatization or both? Molecular Ecology, 19 (8), 1515-1517.

142 List of References

Wen, R., Sui, Z., Bao, Z., Zhou, W., & Wang, C. (2014). Isolation and characterization of calmodulin gene of Alexandrium catenella (Dinoflagellate) and its performance in cell growth and heat stress. Journal of Ocean University of China, 13 (2), 290-296.

Weston, A. J., Dunlap, W. C., Shick, J. M., Klueter, A., Iglic, K., Vukelic, A., ... & Long, P. F. (2012). A profile of an endosymbiont-enriched fraction of the coral Stylophora pistillata reveals proteins relevant to microbial-host interactions. Molecular & Cellular Proteomics, 11 (6), M111-015487.

Weston, A. J., Dunlap, W. C., Beltran, V. H., Starcevic, A., Hranueli, D., Ward, M., & Long, P. F. (2015). Proteomics links the redox state to calcium signaling during bleaching of the scleractinian coral Acropora microphthalma on exposure to high solar irradiance and thermal stress. Molecular & Cellular Proteomics, 14 (3), 585-595.

Whitehead, L. F., & Douglas, A. E. (2003). Metabolite comparisons and the identity of nutrients translocated from symbiotic algae to an animal host. Journal of Experimental Biology, 206 (18), 3149-3157.

Wicks, L. C., Gardner, J. P. A., & Davy, S. K. (2012). Host tolerance, not symbiont tolerance, determines the distribution of coral species in relation to their environment at a Central Pacific atoll. Coral Reefs, 31 (2), 389-398.

Wijgerde, T., Silva, C. I., Scherders, V., van Bleijswijk, J., & Osinga, R. (2014). Coral calcification under daily oxygen saturation and pH dynamics reveals the important role of oxygen. Biology Open, 3 (6), 489-493.

Wooldridge, S. A. (2009). A new conceptual model for the warm-water breakdown of the coral–algae endosymbiosis. Marine and Freshwater Research, 60 (6), 483-496.

Wooldridge, S. A. (2010). Is the coral-algae symbiosis really ‘mutually beneficial’ for the partners? BioEssays, 32 (7), 615-625.

Wooldridge, S. A. (2013). Breakdown of the coral-algae symbiosis: towards formalising a linkage between warm-water bleaching thresholds and the growth rate of the intracellular zooxanthellae. Biogeosciences, 10 (3), 1647-1658.

Wooldridge, S. A. (2014). Differential thermal bleaching susceptibilities amongst coral taxa: re-posing the role of the host. Coral Reefs, 33 (1), 15-27.

Xiang, T., Nelson, W., Rodriguez, J., Tolleter, D., & Grossman, A. R. (2015). Symbiodinium transcriptome and global responses of cells to immediate changes in light intensity when grown under autotrophic or mixotrophic conditions. The Plant Journal, 82 (1), 67-80.

143

Yamori, W., Shikanai, T., & Makino, A. (2015). Photosystem I cyclic electron flow via chloroplast NADH dehydrogenase-like complex performs a physiological role for photosynthesis at low light. Scientific Reports, 5, 13908.

Yellowlees, D., Rees, T. A. V., & Leggat, W. (2008). Metabolic interactions between algal symbionts and invertebrate hosts. Plant, Cell & Environment, 31(5), 679-694. Yuyama, I., Nakamura, T., Higuchi, T., & Hidaka, M. (2016). Different stress tolerances of juveniles of the coral Acropora tenuis associated with clades C1 and D Symbiodinium. Zoological Studies, 55 (19).

Ziegler, M., Roder, C. M., Büchel, C., & Voolstra, C. R. (2015). Mesophotic coral depth acclimatization is a function of host-specific symbiont physiology. Frontiers in Marine Science, 2, 4.

Zoccola, D., Ganot, P., Bertucci, A., Caminiti-Segonds, N., Techer, N., Voolstra, C. R., ... & Tambutté, S. (2015). Bicarbonate transporters in corals point towards a key step in the evolution of cnidarian calcification. Scientific Reports, 5, 9983.

144

145