Coral reef ecosystems are in rapid decline due to global and local anthropogenic factors. Being among the most diverse ecosystems on Earth, a loss will decrease species diversity, and remove food source for people along the coast. The coral together with its symbionts (i.e. Symbiodinium, bacteria, and other microorganisms) is called the ‘coral holobiont’. The coral host offers its associated symbionts suitable habitats and nutrients, while Symbiodinium and coral-associated bacteria provide the host with photosynthates and vital nutrients. Association of corals with certain types of Symbiodinium and bacteria confer coral stress tolerance, and lack or loss of these symbionts coincides with diseased or bleached corals. However, a detailed understanding of the coral holobiont diversity and structure in regard to diseases and health states or across global scales is missing. This dissertation addressed coral-associated symbiont diversity, specifically of Symbiodinium and bacteria, in various coral species from different geographic locations and different health states. The main aims were (1) to expand the scope of existing technologies, (2) to establish a standardized framework to facilitate comparison of symbiont assemblages over coral species and sites, (3) to assess Symbiodinium diversity in the Arabian Seas, and (4) to elucidate whether coral health states have conserved bacterial footprints. In summary, a next generation sequencing pipeline for Symbiodinium diversity typing of the ITS2 marker is developed and applied to describe Symbiodinium diversity in corals around the Arabian Peninsula. The data show that corals in the Arabian Seas are dominated by a single Symbiodinium type, but harbor a rich variety of types in low abundant. Further, association with different Symbiodinium types is structured according to geographic locations. In addition, the application of 16S rRNA gene microarrays to investigate how differences in microbiome structure relate to differences in health and disease demonstrate that coral species share common microbial footprints in phenotypically similar diseases that are conserved between regional seas. Moreover, corals harbor bacteria that are species-specific and distinct from the diseased microbial footprints. The existence of conserved coral disease microbiomes allows for cataloging diseases based on bacterial assemblage over coral species boundaries and will greatly facilitate future comparative analyses.

Looking at the metabolite profile of an organism provides insights into the metabolomic state of a cell and hence also into pathways employed. Little is known about the metabolites produced by corals and their algal symbionts. In particular, corals from the central Red Sea are understudied, but interesting study objects, as they live in one of the warmest and most saline environments and can provide clues as to the adjustment of corals to environmental change. In this study, we applied gas chromatography – mass spectrometry (GC–MS) metabolite profiling to analyze the metabolic profile of four coral species and their associated symbionts: Fungia granulosa, Acropora hemprichii, Porites lutea, and Pocillopora verrucosa. We identified and quantified 102 compounds among primary and secondary metabolites across all samples. F. granulosa and its symbiont showed a total of 59 metabolites which were similar to the 51 displayed by P. verrucosa. P. lutea and A. hemprichii both harbored 40 compounds in conjunction with their respective isolated algae. Comparing across species, 28 metabolites were exclusively present in algae, while 38 were exclusive to corals. A principal component and cluster analyses revealed that metabolite profiles clustered between corals and algae, but each species harbored a distinct catalog of metabolites. The major classes of compounds were carbohydrates and amino acids. Taken together, this study provides a first description of metabolites of Red Sea corals and their associated symbionts. As expected, the metabolites of coral hosts differ from their algal symbionts, but each host and algal species harbor a unique set of metabolites. This corroborates that host-symbiont species pairs display a fine-tuned complementary metabolism that provide insights into the specific nature of the symbiosis. Our analysis also revealed aquatic pollutants, which suggests that metabolite profiling might be used for monitoring pollution levels and assessing environmental impact.

Symbiosis has been an important factor in evolution, and continues to drive speciation and allows organisms to fill new ecological niches. Symbiotic relationships in which both partners benefit from the association, or mutualisms, are ubiquitous in both terrestrial and aquatic ecosystems. Many of the symbionts in these associations are photosynthetic algae or cyanobacteria that fix carbon through photosynthesis and translocate a portion of this energy to their hosts. Host organisms utilize this fixed carbon for a variety of physiological processes, including growth and development, thus, photosynthetically-fixed carbon is vital for many hosts. The following chapters will describe carbon fixation and translocation in two algal symbioses: the freshwater association between the alga Oophila and the eggs of Ambystoma maculatum salmanders, and the relationship between the dinoflagellate Symbiodinium and marine zoanthids. These chapters will discuss carbon flux in symbiosis, and reveal some of the ways in which environmental factors alter photosynthesis in algal mutualisms. / Biology

L’endosymbiose trophique établie entre un hôte Cnidaire et ses symbiotes Dinoflagellés photosynthétiques est à l’origine du succès évolutif des écosystèmes coralliens. Les symbiotes sont internalisés par un mécanisme de phagocytose et maintenus dans les cellules du gastroderme de l'hôte. La symbiose est régie par un dialogue moléculaire intime entre les deux partenaires, interrompu lors de perturbations environnementales ou anthropiques, responsables du déclin mondial des récifs coralliens. Les objectifs de mon projet de recherche sont de définir les acteurs moléculaires localisés à l’interface symbiotique chez l’anémone de mer, Anemonia viridis. Premièrement, nous avons étudié les mécanismes cellulaires impliqués dans différents types de rupture de la symbiose et mis en évidence des phénomènes d’apoptose, nécrose et symbiophagie. Parallèlement, nous avons caractérisé chez l’anémone les gènes npc1 et npc2, impliqués chez les vertébrés dans le transport endosomal de stérols, et dont l’expression est modulée par l’état symbiotique. Nous avons pu montrer que le gène npc2d est issus d’une duplication et vraisemblablement d’une sub-fonctionnalisation et que les protéines NPC1 et NPC2 sont exprimées au voisinage des symbiotes. Nous proposons donc que la protéine NPC2-d soit utilisée comme marqueur de l’état de santé des Anthozoaires symbiotiques et que la protéine NPC1 soit un marqueur de la membrane périsymbiotique. Nous avons également développé un protocole afin d’identifier les protéines associées à l’interface symbiotique entre les deux partenaires. A terme, les cibles identifiées permettront une meilleure compréhension des mécanismes qui régulent la relation symbiotique. / The trophic endosymbiosis interaction between a cnidarian host and its photosynthetic dinoflagellatessymbionts form the basis of coral reef ecosystems. Cnidarians host their symbionts in gastrodermis cells, in a phagocytosis-derived vacuole. Establishment and maintenance of the symbiotic interaction depend on an intimate molecular communication between the two partners. However, environmental and/or anthropogenic disturbances can lead to the breakdown of the symbiotic association, which is responsible for the worldwide decline of coral reefs. The main objectives of my research project are to improve the knowledge regarding symbiosis maintenance and disruption mechanisms, but also to define the molecular key players involved at the symbiotic interface in the sea anemone, Anemonia viridis. First, we have described the cellular mechanisms involved in the different types of symbiosis breackdown. Meanwhile, the characterization of npc1 and npc2 genes (involved in endosomal sterol transport), showed a duplication and a sub-functionalization of the npc2d gene. Both NPC1 and NPC2 proteins are expressed around symbionts. We therefore suggest that the duplicated protein NPC2-d is a biomarker of symbiosis health and that NPC1 protein is a marker of the perisymbiotic membrane. We then developed a protocol to characterize the proteome of the symbiotic interface between the two symbiotic partners. The newly-identified symbiotic key players will increase the general knowledge on the symbiotic interaction and its regulation during both stable and bleaching conditions.

Symbiotic dinoflagellates in the genus Symbiodinium are critical to the success of scleractinian reef corals in shallow tropical seas. These symbionts are commonly isolated from hosts and cultured separately in liquid media (f/2 or ASP8a), but initial isolations can be prone to abundant contaminants that can persist long-term in culture. To help remove these contaminants, I developed a solid growth substrate composed of 1% f/2 medium in agar, supplemented with a variety of antibiotics, to help isolate individual clones and establish new ?axenic? cultures. I found that an antibiotic cocktail of kanamycin (50 µg/mL), ampicillin (100 µg/mL) and streptomycin (50 µg/mL) was the most effective at eliminating visual signs of contamination without apparent harm to a variety of Symbiodinium in culture. Photophysiological measurements of Symbiodinium grown on f/2 agar plates, taken using an Imaging Pulse Amplitude Modulated (I-PAM) fluorometer, were comparable with those grown in liquid f/2, both with and without antibiotics. Eight types of Symbiodinium in clades A-D grown on f/2 agar plates at low irradiance (19-46 µmol photons m-2 s-1) were exposed to higher irradiance conditions (50-90 µmol photons m-2s-1) for 13 days and their photosynthetic efficiencies (Fv/Fm) were compared using the I-PAM. There were significant differences among and within clades, except for two types in clade C (C2 and C3) which did not perform differently from eachother. All types showed reduced Fv/Fm after 12 days in higher light. Type D1a showed high mortality after 13 days of higher light stress. Finally, preliminary work to fluorescently label Symbiodinium determined that available vital stains were not generally practical for symbiosis studies. Attempts to transform Symbiodinium with a variety of plasmids containing fluorescent reporters and/or genes for antibiotic resistance were not successful, but did provide a baseline for future work. In summary, Symbiodinium cultures grown on solid substrates supplemented with antibiotics are useful for: (1) isolating individual cells or clones for subsequent applications and establishing and maintaining ?axenic? cultures that are free of observable contaminants; (2) directly comparing the photophysiology of different cultures using an I-PAM fluorometer; (3) quantifying cells on agar plates using the I-PAM and (4) selecting possible transgenic symbionts for symbiosis studies.

Coral reefs belong to the most diverse and the most threatened ecosystems on earth. Anthropogenic stressors and climate change have led to mortalities at levels unprecedented in modern times. The aims of this thesis are to investigate aspects of the corals’ ability to reproduce, disperse, adapt and survive. Papers I-III study reproduction in a common soft coral species, Sarcophyton elegans, with previously unknown reproductive modes. Paper IV investigates genetic distribution of coral-symbiont associations in Galaxea fascicularis focusing on adaptation to the environment along the coastline of Vietnam. Sarcophyton  elegans is a gonochoric broadcast spawner with a 1:1 sex ratio. Reproduction is strictly size dependent. Oogenesis takes 19-24 months, with a new cycle commencing every year. Spermatogenesis takes 10-12 months. The majority of gametes were released during the annual austral mass spawning event after full moon in November, but spawning also occur between August and February. The polyps at the outer edge of the colonies released their gametes first, followed by polyps situated closer to the center during subsequent months. Colonies upstream in the prevailing current spawn earlier than those downstream. The colonies were arranged in clusters of alternating males and females, which spawned simultaneously and were of the same genotype. Fission and buddying is a common mode to expand locally. Additionally, females undergoing fission divided into the most fecund size classes. The G. fascicularis and their associated symbionts were not genetically coupled to each other but to environmental factors. The host displayed an inshore-offshore zonation, with higher diversity offshore. The D1a symbiont exhibited an inshore- offshore zonation. In contrast; the 5 different C symbiont types showed a latitudinal distribution gradient, which shifted in dominance north to south. The study highlights the importance of protecting resilient coral and algal genotypes in stressed areas and the need to understand reproductive modes for coral conservation. / Các rạn san hô là một trong những hệ sinh thái có tính đa dạng và bị đe dọa cao nhất trên trái đất. Các áp lực từ con người và nhiệt độ nước biển tăng (SSTs) đã gây ra hiện tượng “tẩy trắng” gây chết san hô ở mức độ cao chưa từng thấy trong thời điểm hiện tại. Mục tiêu của nghiên cứu này là tìm hiểu khả năng của san hô trong thích nghi, phân tán và sống sót nhằm duy trì quần thể. Bài báo số II-III là những nghiên cứu đầu tiên về đặc điểm sinh sản của loài san hô mềm phổ biến, Sarcophyton elegan tại Australia. Bài báo số IV nghiên cứu về phân bố nguồn gen của tảo cộng sinh trong loài san hô Galaxea fascicularis, tập trung vào sự thích nghi với môi trường dọc theo vùng biển Việt Nam, khu vực bị ô nhiễm từ lục địa. Sarcophyton elegans được biết với đặc điểm sinh sản cả vô tính và hữu tính. Loài này là loài sinh sản bằng cách phân tán trứng, với tỷ lệ giới tính là 1:1 và sự sinh sản hữu tính bị khống chế nghiêm ngặt bởi kích cỡ của tập đoàn (Bài báo II, phần phương pháp của Bài báo I). Quá trình tạo trứng kéo dài từ 19 đến 24 tháng với chu kỳ sinh sản lặp lại hàng năm, và sự sinh tinh kéo dài từ 10 đến 12 tháng. Phần lớn giao tử được giải phóng trong một thời gian ngắn sau ngày trăng tròn của tháng 11, nhưng giao tử vẫn được giải phóng trong ngày trăng tròn của các tháng từ tháng 8 đến tháng 1 năm sau. Các polyp autozooid nằm phía ngoài của tập đoàn giải phóng giao tử trước, sau đó là các polyp nằm gần lõi trong các tháng tiếp theo. Các tập đoàn ngược lên trong dòng chảy thịnh hành đẻ trứng sớm hơn các tập đoàn xuôi dòng khoảng một tháng (Bài báo II). Các tập đoàn được sắp xếp thành từng đám từ 7 đến hàng trăm tập đoàn trong mỗi nhóm, bao gồm cả đực và cái. Các tập đoàn trong cùng một nhóm sinh sản cùng một thời điểm. (Bài báo II) và mỗi nhóm có cùng một kiểu di truyền (Bài báo III) có đầy đủ 13 (có thể là 22) kiểu di truyền  khác nhau. Sự phân đôi và kết đôi phụ thuộc hoàn toàn vào kích thước và có lẽ là phương thức mở rộng phổ biến nhất. Sự phân đôi phải mất 2 năm hoặc hơn mới hoàn thành. Thêm vào đó, con cái trải qua quá trình phân đôi thành kích cỡ có khả năng sinh sản cao nhất (Bài báo III). Có 6 nhóm haplotypes (mtDNA) của loài G. fascicularis và tảo cộng sinh Symbiodinium (ITS2 rDNA) không đóng cặp với nhau nhưng lại gắn với các yếu tố môi trường, có thể như kết quả của phương thức sinh sản của vật chủ (Bài báo IV). Vật chủ có sự phân vùng rõ rệt giữa gần bờ và xa bờ, với sự đa dạng cao hơn hẳn của các rạn xa bờ so với các rạn gần bờ, khu vực thường xuyên bị độ đục, ô nhiễm và lắng đọng trầm tích tác động. Tảo cộng sinh Symbiodinium D1a ITS2 điểm hình của sự phân vùng gần bờ và xa bờ. Ngược lại, 5 loại C khác lại có sự phân vùng theo vĩ tuyến, với sự tăng lên rõ rệt theo chiều Bắc-Nam, cùng với sự ổn định SST và sự tăng lên của các SST. Nghiên cứu này đã chỉ rõ tầm quan trọng trong bảo vệ các loài san hô và tảo biển bản địa tại các khu vực bị đe dọa (Bài báo IV) và sự cần thiết phải hiểu các phương thức sinh sản (Bài báo II-III) và các thông số môi trường trong việc xác định mức độ đa dạn sinh học và sự hấp thụ của sinh vật cộng sinh trong san hô cứng và san hô mềm. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Many members of the Phylum Cnidaria are mutualistic with unicellular dinoflagellates belonging to the genus Symbiodinium. Corals are the most widely recognized example of these associations due to their key ecological importance in coral reef ecosystems where they serve as the structural and trophic foundation of these rich ecosystems. Coral reefs are severely threatened by human activities worldwide and are at great risk from global climate change, in particular the increase in seasurface temperatures. Detailed knowledge of how corals respond to stress is scarce. The most serious and immediate response of corals to environmental stress is a process referred to as coral bleaching (a.k.a. cnidarian bleaching). Nevertheless, the cellular and molecular processes by which elevated temperatures elicit the bleaching response are poorly understood. This dissertation deals with this important question by describing two mediators of cnidarian bleaching in the model symbiotic tropical sea anemone Aiptasia pallida (Verril), namely nitric oxide and cyclophilin. After an introduction to the topic of cnidarian-algal symbioses and cnidarian bleaching (Chapter 1), I present results from a study describing the involvement of nitric oxide (NO) in the anemone A. pallida (Chapter 2). Elevated temperature as well as oxidative stress induces production of NO and exposure of A. pallida to NO induces bleaching at non-stressful temperatures. Co-incubation with an NO scavenger suppresses bleaching. I propose that the host up-regulates NO production in response to elevated oxidative stress and that this situation leads to cytotoxicity and bleaching. Chapter 3 examines the role of cyclophilin from A. pallida in the regulation of the symbiosis. Cyclophilins belong to a highly conserved family peptydyl-prolyl cistrans isomerases (PPIases). Incubation of A. pallida with cyclosporin A (CsA), a potent inhibitor of cyclophilin resulted in bleaching and a decrease in tolerance to elevated temperatures. Protein extracts from A. pallida exhibited CsA-sensitive PPIase activity. Laser scanning confocal microscopy using superoxide and nitric oxide-sensitive fluorescent dyes on live A. pallida revealed that CsA strongly induced the production reactive oxygen species as well as NO. We tested weather the CsAsensitive isomerase activity is important for maintaining the activity of the antioxidant enzyme superoxide dismutase (SOD). SOD activity of protein extracts was not affected by pre-incubation with CsA in vitro. In Chapter 4 I review what is known about the molecular and cellular mechanisms of bleaching and describe a model of bleaching based on the results presented herein as well as studies of non-cnidarian models. / Graduation date: 2007

Les dinoflagellés sont une famille d'eucaryotes unicellulaires trouvés dans les écosystèmes marins et d'eau douce et sont d'importants producteurs primaires. Ils sont réputés pour plusieurs comportements distinctifs, notamment la formation de proliférations d'algues nuisibles appelées « marées rouges », l'émission de bioluminescence dans l'océan et leur contribution à la formation de récifs coralliens. Leur structure génomique est inhabituelle avec de grandes quantités d'ADN et des chromosomes condensés en permanence à toutes les étapes du cycle cellulaire. L’ADN est sans nucléosome et se trouve dans une structure de cristaux liquides. Plusieurs gènes sont codés dans de multiples répétitions situées dans des réseaux en tandem produisant des protéines pratiquement identiques sans aucun élément conservé détecté dans les régions présumées promotrices en amont de la séquence codante. Ces caractéristiques uniques rendent difficile à comprendre comment les cellules régulent l'expression des gènes. Cette thèse examine l’hypothèse que la régulation de transcription est difficile et peu utilisée chez les dinoflagellés. Les dinoflagellés présentent une rareté des facteurs de transcription, les protéines du domaine de choc froid (CSP) représentant la majorité des protéines de liaison à l'ADN potentielles dans le transcriptome de Lingulodinium polyedra et le génome de Symbiodinium kawagutii. Le potentiel des CSP de dinoflagellés à agir en tant que facteurs de transcription spécifiques à la séquence a été testé en utilisant des tests de déplacement de mobilité électrophorétique. Ces études ont révélé que quatre CSP différentes ont montré une préférence pour l'ARN par rapport à l'ADN simple et double brin. Une deuxième approche a examiné le ciblage de la séquence spécifique par des tests de sélection et de liaison d'amplification, et cela n'a révélé aucun motif consensus détectable dans la liaison à l'ADN. Nous concluons que les CSP dinoflagellés sont plus susceptibles de fonctionner comme des protéines de liaison à l'ARN que comme des facteurs de transcription. Il a été rapporté que l'expression de nombreux gènes chez plusieurs espèces de dinoflagellés était régulée par l'exposition à la lumière. Cela a été testé pour trois gènes, dont l'expression régulée par la lumière chez l'espèce formant des récifs Symbiodinium kawagutii. La régulation de ces gènes a été rapportée dans la littérature suggérant la possibilité d’identifier les éléments régulateurs dans le promoteur. Cependant, l'analyse par transfert de Northern n'a pas pu valider le modèle d'expression de ces trois gènes chez S. kawagutii. De plus, le séquençage d'ARN à haut débit a confirmé que ces trois gènes n'étaient pas induits par la lumière. Au total, seuls sept gènes ont été exprimés de manière différentielle à l'aube et au crépuscule en utilisant RNA-Seq, et tous étaient de moindre abondance à la fin de la période de lumière sur un 12: 12 cycle L: D. Trois des sept ont également été examinés en utilisant une analyse qPCR, et seule deux des trois ont pu être confirmés comme étant altérés, mais avec une différence de facteur inférieure à celle observée avec RNA-Seq. Nous en concluons qu'il y a peu de régulation lumineuse de l'expression génique dans cette espèce dinoflagellé. Dans l’ensemble, les études décrites ici appuient l’hypothèse que les dinoflagellés ont un moins grande dépendance sur la régulation transcriptionnelle que d’autres organismes. / Dinoflagellates are a large family of unicellular eukaryotes found in marine and freshwater ecosystems and are important primary producers in marine ecosystem. They are famous for several distinctive behaviors including forming harmful algal blooms called “red tides”, emission of bioluminescence in the ocean, and contributing to the formation of coral reefs. They have an unusual genome structure with large amounts of DNA and permanently condensed chromosomes throughout all stages of the cell cycle. The chromatin lacks observable nucleosomes and has a liquid crystal structure. Some genes are encoded in multiple repeats located in tandem arrays producing virtually identical proteins without any known conserved elements detected in the upstream promoter regions or intergenic spacers. These unique features make it difficult to understand how gene expression is regulated. This thesis describes two experimental tests for the hypothesis that transcriptional regulation is difficult and is not the primary means of regulating gene expression in dinoflagellates. Dinoflagellates show a paucity of transcription factors, and of these, cold shock domain proteins (CSPs) account for the majority of potential DNA binding proteins in the transcriptome. Here, the potential of dinoflagellate CSPs from free-living Lingulodinium polyedra and reef-forming Symbiodinium kawagutii (recently renamed to Fugacium kawagutii) to act as sequence specific transcription factors was tested. These studies using four different CSPs showed a preference for RNA over both single and double stranded DNA using electrophoretic mobility shift assays (EMSA). A second approach, testing for specific sequence binding by three cycles of selection and amplification binding (SAAB) did not enrich any consensus motif for any of the four proteins. We conclude dinoflagellate CSPs are more likely to function as RNA binding proteins than as transcription factors. Expression of many genes in many dinoflagellate species has been reported to be regulated by light. This was tested for three genes whose expression was reported to be light-regulated in Symbiodinium kawagutii. The availability of a genome sequence for this species suggested that it might be possible to identify potential regulatory elements in the promoter of these genes. However, Northern blot analysis was unable to confirm differential expression of these three genes over a 24 hour light-dark cycle. Furthermore, RNA-Seq of samples taken at the end of the day and night also indicated these three genes were not light-induced. In total, only seven genes were found to be differentially expressed at dawn and dusk using RNA-Seq in triplicate with a false discovery rate (FDR) of 0.1. All were of lower abundance at the end of the light period on a 12:12 L:D cycle suggesting possible repression by light. Three of these seven, picked at random, were examined using qPCR analysis. Only two of the three had lower abundance at the end of the day by this technique, and the fold difference was less than what was observed with RNA-Seq. We conclude from this that there is little light regulation of gene expression in this dinoflagellate species. Taken together, the studies described here support the hypothesis that dinoflagellates do not rely on regulation of genes at the transcriptional level to the same extent as other organisms.