The molecular stress response in the Indo-Pacific model scleractinian coral, Acropora millepora

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(1)This file is part of the following reference:. Seneca, François (2010) The molecular stress response in the Indo-Pacific model scleractinian coral, Acropora millepora. PhD thesis, James Cook University. Access to this file is available from: http://eprints.jcu.edu.au/15336.

(2) The Molecular Stress Response in the Indo-Pacific Model Scleractinian Coral, Acropora millepora. Thesis submitted by François Seneca (B.Sc. Zoology, University of Hawai‘i) September 2010. for the degree of Doctor of Philosophy in the School of Pharmacy and Molecular Sciences James Cook University.

(3) STATEMENT ON THE CONTRIBUTION OF OTHERS. Funding for the research within this thesis was obtained from the Australian Research Council (ARC), James Cook University (JCU), and the Australian Institute of Marine Science (AIMS). The Government of Monaco (Principality of Monaco) and the School of Pharmacy and Molecular Sciences (JCU) provided stipend support. A supervisory team consisting of Professor David Miller (JCU), and Dr Madeleine van Oppen (AIMS) provided intellectual and editorial support throughout the thesis. Dr Eldon Ball (Australian National University) provided intellectual and editorial support for Chapter 2 and access to facilities for the microarray experiment in Chapter 3. Dr Sylvain Forêt (ARC Centre of Excellence for Coral Reef Studies, JCU) provided statistical, intellectual and editorial support for Chapter 2, 3, and 4. Dr Nicolas Goffard (ANU) provided intellectual support in bioinformatics for Chapter 3. Dr Lauretta Grasso (JCU) provided technical support for the microarray experiment in Chapter 3. Dr Carolyn Smith-Keune (JCU) provided intellectual and editorial support as well as the samples used in Chapter 3. Erin Noonan, Thea Jacob, Dr David Abrego and Eneour Puill-Stephan provided assistance in the laboratory. Allison Palley, and Thomas Stevens provided assistance in the field. Patricia Warner provided editorial support throughout the thesis..

(4) Acknowledgements I always knew that I would have to work in contact with the ocean to be happy with my job. The end of this thesis feels like I have accomplished my dream. I became somewhat of a marine biologist. I am just looking at marine living things a bit more closely than most people do. The molecular side of my studies was unexpected. My unquenchable curiosity took me there. My PhD was a bumpy road, but I can still say that I am passionate about what I am doing. I hope this will stay in me as long as possible.. I would like to thank my supervisors, David and Madeleine, for giving me the opportunity to accomplish this PhD. David, thanks for allowing me the freedom to do my work when and where I wanted, and showing positive support every time I came to see you. Madeleine, your enthusiasm had a real boosting effect on my work. Thanks for always finding some time to listen and giving me the opportunity to use the AIMS facilities. Working at AIMS will remain a highlight of my PhD memories. Thank you, Dr. D aka. Danielle DeJong, for making me feel welcome and guiding my first steps in the DM lab. A special thanks to the ANU team, Eldon, David and Lauretta (at the time). Although the science was pretty stressful, I had a great time working with you in Canberra. Eldon, you are an inspiration for your passion and hard work, thanks for letting me come over and use your lab. Sylvain, merci pour ton aide avec les stats pour les experiences de qRTPCR et microarray.. J‘aimerais aussi remercier ma famille, Sylvaine, Jean-Charles et lolo pour ne jamais avoir manquer de demander quand est ce que j‘aurais fini ma these! Mam, je sais que ca a ete long et t‘as cause beaucoup de stress. Mais, tu vois, j‘y suis arrive! Pap, je sais que tu n‘as pas toujours compris mes choix, mais tu as toujours ete la quand j‘ai eu besoin de toi. Ce PhD est aussi le votre..

(5) Françoise, Vincent et Camille, je n‘aurais jamais pu etre la ou j‘en suis sans votre support. Merci.. Ninou, t‘es de la bombe! Je suis tellement reconnaissant pour ton aide et ton temps. Merci mon pote. JB, merci poulet pour les stats de derniere minutes! I would like to thank all the people and friends who helped me in the lab, Erin, Thea, Lesa, Lauretta, Julie, David, et tonton aka. Eneour.. And finally, Patricia Warner, thank you ma fiancée, for your love and support during those past two years. This is happening because of you. You gave me the strength and motivation to finish it. I am so looking forward to our future in science and in life together..

(6) Publications resulting from the research in this thesis. Seneca F, Forêt S, Ball E, Smith-Keune C, Miller D J and van Oppen M. (2009) Patterns of gene expression in a scleractinian coral undergoing natural bleaching. Marine Biotechnology: 10.1007/s10126-009-9247-5.. Forêt S#, Seneca F#, de Jong D, Bieller A, Hemmrich G, Augustin R, Hayward DC, Ball EE, Bosch TCG, Agata K, Hassel M and Miller DJ (Accepted) Phylogenomics reveals an anomalous distribution of USP genes in metazoans. Molecular Biology and Evolution. #These authors contributed equally..

(7) i. ABSTRACT A better understanding of the molecular coral bleaching response to environmental stressors is essential to determine how global climate change and water quality degradation affect corals. A central part of an individual‘s response to biotic or abiotic stressor exposure is the regulation of genes within the cells. The expression pattern of particular genes, which play key roles in the fate of cells under stress, can indicate the level and source of intra-cellular disturbances.. The study of gene. expression, or transcriptomics, can therefore be used to diagnose the health of an organism or population. The research reported here has for goal to improve our current knowledge of the coral transcriptomic stress response by: (1) providing optimized methods for the measurement of gene expression during coral bleaching in the field (Chapter 2); (2) exploring the changes in gene expression during bleaching and subsequent recovery period (Chapter 3); and, (3) describing a novel coral gene family potentially involved in the stress response (Chapter 4). Coral bleaching is a major threat to coral reefs worldwide and is predicted to intensify with increasing global temperature. The present study represents the first investigation of gene expression in an Indo-Pacific coral species undergoing natural bleaching which involved the loss of algal symbionts. Quantitative real-time PCR (qRTPCR) experiments were conducted to select and evaluate coral internal control genes (ICGs), and to investigate selected coral genes of interest (GOIs) for changes in gene expression in nine colonies of the scleractinian coral Acropora millepora undergoing bleaching at Magnetic Island, Great Barrier Reef (GBR), Australia. Among the six ICGs tested, glyceraldehyde 3-phosphate dehydrogenase and the ribosomal protein genes S7 and L9 exhibited the most constant expression levels between samples.

(8) ii. from healthy looking colonies and samples from the same colonies when severely bleached a year later. These ICGs were therefore utilized for normalization of expression data for seven selected GOIs. Of the seven GOIs, homologs of catalase, Ctype lectin and chromoprotein genes were significantly up-regulated as a result of bleaching by factors of 1.81, 1.46 and 1.61 (linear mixed models analysis of variance, p < 0.05), respectively. I present these genes as potential key coral bleaching response genes. In contrast, three genes, including one putative ICG, showed highly variable levels of expression between coral colonies. Potential variation in microhabitat, gene function unrelated to the stress response, and individualized stress responses may influence such differences between colonies, and need to be better understood when designing and interpreting future studies of gene expression in natural coral populations. In the next experiment, I used an extensive coral cDNA microarray assay to conduct a novel investigation of transcriptomic changes in colonies undergoing a natural summer bleaching event in the field environment. Four colonies of the IndoPacific model reef-building coral, Acropora millepora, were sampled in situ during a bleaching event and the subsequent recovery period on the GBR, in summer 2000-01. Significant change in the expression of hundreds of genes representing approximately 14 different processes/mechanisms shows that the natural bleaching stress response is fundamentally a general rearrangement involving almost all aspects of the cellular machinery. I identified a large number of genes, additional to those published in prior laboratory experiments, that are involved in previously identified cellular processes as well as new processes and gene groups unique to this study, such as exo- and endocytic pathway, defense and inflammation response, protein/cell degradation and death, cell cycle and division, and DNA/protein repair. Surprisingly, the relationship between the level of stress and changes in expression of hallmarks genes of the stress response, such.

(9) iii. as HSPs and antioxidants, remains equivocal.. However, the detection of key. transcription factors, calcium binding, cytoskeleton and extracellular matrix protein genes provide further information about the characteristics of natural coral bleaching. Moreover, my results from a natural bleaching event enable comparisons to laboratory studies, which both enhance our understanding of the genes and processes implicated in the stress response as well as suggest the limitations and benefits of both approaches. In the last chapter, I describe a novel coral stress related gene family, the Universal Stress Proteins (USP). Members of the USP family were identified in bacteria in the context of the stress response, and the USP-like domain occurs in a phylogenetically diverse range of prokaryotes, fungi, protists and plants. Here, I report that members of the USP family also occur in the animal kingdom, but that their distribution follows an unusual pattern. USP genes are present in urochordates as well as all Cnidaria and Lophotrochozoa examined, but are not present in any ecdysozoans or non-urochordate deuterostomes. The vast majority of the metazoan USPs are short, single domain proteins, and phylogenetically distinct from the prokaryotic, plant, protist and fungal members of the protein family. Phylogenetic analyses imply that one or a few USP loci were present in the common metazoan ancestor, and have undergone independent expansions in some lineages but have been lost from others. Most of the metazoan USP genes contain introns, the position of one of which is conserved across the Metazoa and possibly also with some of the plant sequences. By contrast, most (22 of 24) of the Hydra genes encoding USPs are atypical in that they are intronless and these clustered together in phylogenetic analyses, whereas the remaining Hydra sequences contain introns and seem to have counterparts in other cnidarians, urochordates and lophotrochozoans. Expression patterns were determined for several cnidarian USPs, including two genes belonging to the intronless clade, and these imply.

(10) iv. a diversity of functions. The apparent paradox of implied diversity of roles despite high overall levels of similarity parallels the situation in bacteria. I hypothesize that the absence of USP genes in ecdysozoans and most deuterostomes may be a consequence of functional redundancy or specialization in taxon-specific roles, for example USP genes have been recently detected in bleaching experiments and may play an important role in the coral stress response. This calls attention to an exciting novel coral gene family for future research. This thesis provides future research aiming to explain the molecular stress response of reef-building corals with relevant information about the genes involved in the natural coral bleaching response. With greater understanding of the molecular effect of particular stressors and/or stress events on corals, we can better comprehend the expected changes to corals under global climate change, improve our forecasting of coral reef deterioration, and prioritize management of the most serious threats..

(11) v. CONTENTS i. Abstract. viii. List of Tables. x. List of Figures. Chapter 1.0. Background and General Introduction. 1. 1.1. Corals under threat. 1. 1.2. Background on coral biology. 3. 1.3. Coral bleaching and the molecular stress response. 4. 1.3.1 1.3.2 1.3.3 1.3.4 1.3.5. Coral bleaching definition General bleaching response Molecular stress response and understanding symbiosis Cellular mechanisms of bleaching Quest for coral health biomarker. 4 4 6 10 11. 1.4. Challenges in coral molecular ecology. 14. 1.5. Implications for management on coral reefs. 15. 1.6. Thesis objectives. 16. Chapter 2.0 Patterns of gene expression in a scleractinian coral undergoing natural bleaching. 19. 2.1. Introduction. 20. 2.2. Materials and methods. 23. 2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6. 2.1 2.1.1 2.1.2. 2.2. Sample collection Bleaching condition: Symbiodinium cell counts and chlorophyll content Total RNA extraction ICGs and GOIs selection Two step qRTPCR Statistical analysis. Results. 29. Changes in Symbiodinium densities, chlorophyll a content and water temperature29 ICGs, differentially expressed genes and inter-colony variability 30. Discussion. 2.2.1 ICGs for coral bleaching 2.2.2 Regulation of GOIs during bleaching 2.2.2.1 AmCatalase 2.2.2.2 AmChromoprotein 2.2.2.3 AmC-type lectin 2.2.3 Intercolony variability in levels of gene expression in a natural population. 2.3. 23 23 24 25 27 28. Conclusion. 33 34 36 37 38 39 40. 41.

(12) vi. Chapter 3.0 Changes in gene expression in the reef-building coral Acropora millepora during a natural bleaching episode and subsequent recovery period in the field 43 3.1. Introduction. 44. 3.2. Materials and methods. 47. 3.2.1 Sample collection 3.2.2 Physiological parameters: Symbiodinium cell counts, chlorophyll a, and protein content 48 3.2.3 RNA extraction and microarray hybridisation 3.2.4 EST analysis and microarray description 3.2.5 Experimental design 3.2.6 Normalization 3.2.7 Background correction and between array normalization 3.2.8 Detecting differential expression 3.2.9 Bioinformatic analysis of differentially expressed genes 3.2.9.1 Annotation and functional classification 3.2.9.2 Hierarchical clustering. 3.3. Results. 3.3.1 Onset of bleaching 3.3.1.1 Environmental changes 3.3.1.2 Physiological changes 3.3.1.3 Changes in coral gene expression levels 3.3.2 Recovery phase 3.3.2.1 Environmental changes 3.3.2.2 Physiological changes 3.3.2.3 Changes in coral gene expression levels. 3.4 3.4.1. Discussion. 47. 49 50 50 52 52 53 54 54 54. 55 56 56 59 60 69 69 70 70. 74. Coral gene expression in relation to time, increased temperature and symbiont loss 74 3.4.2 Molecular functions and cellular processes in relation to coral bleaching 75 3.4.2.1 System perturbation 78 3.4.2.1.1 HSPs and chaperones 78 3.4.2.1.2 Immunity and inflammation 80 3.4.2.1.3 Oxidative stress protection and detoxification 82 3.4.2.2 System maintenance 84 3.4.2.2.1 Calcium homeostasis 84 3.4.2.2.2 Cytoskeleton and cell-cell adhesion 85 3.4.2.2.3 Extra-cellular matrix 85 3.4.2.2.4 Protein degradation and cell death 86 3.4.2.2.5 Metabolism 86 3.4.2.3 System regulation 87 3.4.2.3.1 Transcription 87 3.4.2.3.2 Protein synthesis 87 3.4.2.3.3 Exocytic and endocytic pathways 88 3.4.2.4 Unknown genes 89 3.4.3 Comparison with laboratory bleaching microarray study 93 3.4.4 Hypothetical Molecular Coral Bleaching Model 94 3.4.5 Interpretation of the molecular functions and cellular processes in relation to recovery from bleaching 98 3.4.6 Improvements and directions for future studies 100 3.4.7 Candidate selection for the development of a coral bleaching response assay (CBRA) 101.

(13) vii. 3.5. Conclusion. Chapter 4.0 metazoans. 103. Phylogenomics reveals an anomalous distribution of USP genes in 105. 4.1. Introduction. 106. 4.2. Materials and methods. 110. 4.2.1 4.2.2 4.2.3. 4.3. Sequences Phylogenetics In-situ hybridisation. 112. Results. 4.3.1 4.3.2 4.3.3 4.3.4. 110 111 111. The phylogenetic distribution of USP genes General characteristics of the metazoan USP proteins Phylogenetic analysis Heterogeneity of USP expression patterns. 112 114 114 117. 4.4. Discussion. 120. 4.5. Conclusion. 124. Chapter 5.0. General discussion, major findings, and future research. 126. Reef flat dominated by Acropora millepora, Miall Is., Keppels, AUS. 126. 5.1. General Discussion. 127. 5.2. Major findings of this thesis. 132. 5.3. The future. 133. References. 135. Appendices. 152.

(14) viii. LIST OF TABLES Table 2.1. Regulation of seven GOIs (based on qRTPCR) in nine field colonies of A. millepora undergoing bleaching. The same nine colonies were sampled exactly one year apart and were healthy-looking at the first and severely bleached at the second sampling time point. The fold changes of gene expression are shown with the P-values for those genes that were significantly differentially expressed. The E-value corresponds to the best match of a Tblastx search in the nucleotide collection (nr/nt). 32 Table 3.1. Symbiont losses and gains over the experimental period calculated as total algal cell content relative to the initial colony condition on the 21 November 2001 (time point 1) for the onset of bleaching and 8 February 2002 (time point 5) for the recovery phase. Blue and red values correspond to loss and gain, respectively; time intervals (days) shown in parentheses. 60 Table 3.2. Annotated differentially expressed genes from the onset of bleaching microarray experiment (n=4 colonies) focusing on the change in gene expression between four successive time points in A. millepora colonies undergoing bleaching in the field. Genes are grouped according to the categories represented in DeSalvo et al. (2008), as well as additional newly defined categories for the purposes of these results. Fold changes of gene expression and adjusted P values are shown for differentially expressed genes, and genes detected more than once are highlighted in grey. Putative molecular functions are identified from Uniprot keywords and pathways, KEGG pathways, GO molecular functions, biological processes, and cellular components in addition to literature search deduced categories. Gene ID‘s are directly usable and uniquely linked to sequences from the A. millepora EST collection publicly available from the GenBank database (except for ID starting with D). GenBank accession numbers for early life stage (gene ID starting with A-B-C) differentially expressed genes presented in the following table are accessible as electronic supplementary material on demand. Asterisks (*) denote the genes in common with the DeSalvo et al. (2008) experiments. 62 Table 3.3. Annotated differentially expressed genes grouped by similar expression profiles during onset of bleaching in four A. millepora colonies; clusters derived from the analysis in Figure 3.5. The best match ID from the Entrez PROTEIN database and corresponding E-values shown in parenthesis. Genes highlighted grey significantly represent a certain gene group or cellular process. The array column indicates in which comparison genes were found differentially expressed. 65 Table 3.4. Differentially expressed genes in comparisons performed for the microarray experiment on A. millepora colonies (n=4) recovering from bleaching in the field.. 71. Table 3.5. Annotated differentially expressed genes from the recovery phase microarray experiment focusing on the change in gene expression between four successive time points in A. millepora colonies (n=4) regaining healthy symbiont densities. Genes are grouped into a subset of the categories presented in Table 3.2. Fold changes of gene expression and adjusted p values are shown for differentially expressed genes. Genes detected more than once are highlighted in grey. 71 Table 3.6. The transcriptomic data on coral HSPs and chaperones in response to elevated temperature.. 79. Table 3.7. Current knowledge of coral lectins.. 81. Table 3.8. Transcriptomic data on the oxidative stress response in corals exposed to elevated temperature. 83 Table 3.9. Coral genes with no record in the literature on cnidarian research, but regarded as very relevant candidates for future studies on coral stress response. 89 Table 3.10. Summary comparing the 3 coral bleaching microarray studies at the time of this thesis. 93.

(15) ix. Table 3.11. Log fold change of the seven differentially expressed genes in common to the DeSalvo et al. (2008) experiments and the onset of bleaching experiment in the present study. Question mark denotes a very low e-value for the best tBlastx hit using the EST sequence in parenthesis. 94 Table 3.12. Candidate coral bleaching biomarker genes in common between the bleaching and recovery periods as well as genes detected at the beginning and the end of the recovery. 101 Supplementary Table S.1. Primers used for the genes under investigation in the qRTPCR experiment. The best Tblastx match and E value are shown for the EST sequence corresponding to the indicated accession number. The Symbiodinium-specific PCNA primers were designed on the same sequences used in Boldt et al. (2009). 152 Supplementary Table S.2. Ranking of the candidate ICGs according to their M and CV values (Hellemans et al. 2007) calculated between healthy-looking and severely bleached samples across the nine colonies used in qRTPCR experiment. 152 Supplementary Table S.3. MIQE checklist (Bustin et al. 2009).. 153.

(16) x. LIST OF FIGURES Fig. 1.1. Photosynthetic electron transport chain of the thylakoid membrane (reprint based on (Taiz and Zeiger 1998). 8 Fig. 2.1. Comparison of (A) the mean Symbiodinium cell densities and (B) chlorophyll a concentrations in samples used in the qRTPCR experiment (n = 9) at two time points, 24th of January 2001 (2001) and 24th of January 2002 (2002). *paired two-tailed t-test, P < 0.05. The percentages of degenerate Symbiodinium cells are indicated. 30 Fig. 2.2. Box plots comparing the log-normalized relative quantity (log (NRQ)) for nine Acropora millepora colonies in healthy-looking (2001) and severely bleached (2002) condition for: (A) three GOIs, AmCat, AmCTL and AmCh, and (B) three ICGs, rpS7, GAPDH and rpL9. *linear mixed models analysis of variance, P < 0.05 (• outliers,  superposed outliers). 31 Fig. 2.3. Log (NRQ) data for three genes showing great inter-colony variability in expression among the nine colonies. *linear mixed models analysis of variance, P < 0.05 (qRTPCR reaction triplicates for one colony are depicted by the same symbol). 33 Fig. 3.1. Loop design used to compare change in gene expression between 4 time points and replicated 51 Fig. 3.2. Reprinted by permission from Macmillan Publishers Ltd: The ISME Journal (Bourne et al.), copyright (2008). Temperature trends for Nelly Bay reef during the total sampling period. (A) Daily average water temperature on Nelly Bay reef flat from October 2000 to January 2003, and (B) from 1 November 2000 to 31 April 2001: daily average (—) and daily temperature range (grey area), 1990-2000 daily average with 14 d smoothing function applied (—), sampling times () and bleaching first observed (). Note that only 19 sampling times shown as temperature data not available beyond January 2003 55 Fig. 3.3. Adapted by permission from Macmillan Publishers Ltd: The ISME Journal (Bourne et al.), copyright (2008). A) Colony distribution according to visual health assessment categories (N=normal; 58 Fig. 3.4. Summary of the differentially expressed ESTs detected in the onset of bleaching microarray experiment (n=4 colonies) partitioned into three groups: A) ESTs appearing strictly in comparison #1; B) ESTs shared by both comparisons #1 and #4; and, C) ESTs exclusively in comparison #4. Each group is further divided into the number of up and down regulated genes (clustered ESTs) and the ranges for log fold change is indicated for each group. Different ESTs belonging to the same gene, but only differentially expressed in one or the other comparison, are also included in the intersection B. 61 Fig. 3.5. Cluster analysis based on the expression profiles (heat maps) of the 156 genes found differentially expressed (p < 0.05) in comparisons #1 and #4 of the onset of bleaching experiment. Annotated genes show unique expression profiles. Genes of each cluster are detailed in Table 3.3. 65 Fig. 3.6. Cluster analysis based on the expression profiles (heat maps) of the 590 genes found differentially expressed (p < 0.05) in comparison #8 of the recovery phase experiment. Clusters labeled with the functional category found significantly over-represented (adjusted p-values < 2.58e-5) and the representative percentage of genes when applicable, or alternatively a list of genes with relevance to coral bleaching response. 73 Fig. 3.7. A hypothetical molecular model of the Acropora millepora natural bleaching response.96 Fig. 4.1. The USP domain. This alignment includes all the Hydra magnipapillata sequences and the bacterial 1MJH. The two Hydra sequences indicated in red are those lacking introns. The secondary structure is shown above the alignment and the residues involved in ATP binding are indicated by a ―*‖ under the alignment. The color coding of residues is based on that used in the.

(17) xi. Jalview implementation of ClustalX (see www.jalview.org/help/help.html), as follows: blue, A, I, L, M, F, W, V, C; red, R, K; green, N, Q, S, T; pink, C; magenta, E, D; orange, G; cyan, H, Y; yellow, P. Thresholds for color coding were optimized to clearly delineate the boundaries of secondary structure features. 110 Fig. 4.2. Phylogenomic distribution of USP genes. For each clade, the name of a representative species is indicated and colored in red if the genome of that species encodes USPs and in black if it does not. The number of USP genes found in each genome is given to the right of the species name. Branches where losses of the entire USP family have occurred are highlighted by red dots. The Cnidaria are highlighted in blue, the Lophotrochozoa in purple, the Ecdysozoa in green and the Deuterostomia in brown. Abbreviations: LUCA last universal common ancestor; LECA last eukaryotic common ancestor. 113 Fig. 4.3. Phylogenetic analysis of animal USP protein sequences. The analyses were based on the complete USP complements of the animals with fully sequenced genomes (Fig 4.1) plus the coral Acropora millepora. The tree shown is the result of Bayesian analysis, with posterior probabilities of the 115 Fig. 4.4. Expression patterns of Hydra and Acropora USP genes. (A) Expression of Hmag01 in Hydra magnipapillata. This gene is expressed throughout the trunk endoderm, but transcripts are absent from the tentacle. (B) In Hydra magnipapillata Hmag05 is strongly expressed in an endodermal stripe across the base of the polyp. (C) Expression of Hmag10/teba1 in Hydra vulgaris. This gene is strongly expressed in the endoderm at the tentacle base. Note that H. vulgaris and H. magnipapillata are closely related sister taxa (Hemmrich et al. 2007), and the (H. magnipapillata) Hmag10 and (H. vulgaris) teba1 proteins are identical. (D) Expression of the Acropora millepora USP gene Amil10. (D1) This gene is first expressed 119 Supplementary Fig. S.1. Consistent difference between average quantification cycles of healthy (dark grey) and bleached (light grey) samples in nine colonies, for the best performing ICGs: GAPDH, rpL9 and S7 154 Supplementary Fig. S.2. Maximum likelihood tree of the USP domain sequences in animals. The numbers on the nodes indicate their support by a SH-like likelihood ratio test. 155 Supplementary Fig. S.3. Maximum likelihood tree of the USP domain sequences in animals. The numbers on the nodes indicate their bootstrap support. 156 Supplementary Fig. S.4. Bayesian tree of the USP domain sequences in all species. The numbers on each node represent the posterior probability. 157 Supplementary Fig. S.5. Maximum likelihood tree of the USP domain sequences in all species. The numbers on the nodes indicate their support by a SH-like likelihood ratio test. 158 Supplementary Fig. S.6. Maximum likelihood tree of the USP domain sequences in all species. The numbers on the nodes indicate their bootstrap support. 159 Supplementary Fig. S.7. Likelihood mapping assessing the monophyly of animal USPs. 79.1% of 10,000 random quartets are consistent with metazoan (a) and non-metazoan sequences (b) grouping separately. 160 Supplementary Fig. S.8. Multiple sequences alignment of USP domains. Vertical bars indicate an intron between two amino acid positions, left slanted bars indicate that a codon has 1 nucleotide in the 5' exon and 2 nucleotides in the 3' exon, right slanted bars indicate that a codon has 2 nucleotide in the 5' exon and 1 nucleotides in the 3' exon. The boxes above the alignment denote the approximate position of alpha helices (pink) and beta strands (blue) in the secondary structure of 1MJH. Asterisks under the alignment highlight the position of residues involved in ATP binding in 1MJH. The arrow under the alignment points at the position of the intron conserved between plants and animals. 162.

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