The dinoflagellate genus Symbiodinium is a popular research topic in the coral reef molecular biology field. Primarily because these organisms serve as the coral holobiont’s primary source of energy, carrying out photosynthesis, and providing hydrocarbons to the coral host. Previous studies have shown the difficulty of isolating Symbiodinium as well as the inherent problems in trying to quantify the diversity of this genus and to qualify the distinct reactions of different Symbiodinium sp. to changing environmental conditions. The main goals of this study are: (1) to detail the relationship between the genetic classification of the organism and its physiology in regard to photosynthesis with a number of established Symbiodinium cultures; and (2) to isolate Symbiodinium from coral of the central Red Sea. To evaluate the photosynthetic physiology of Symbiodinium, a microsensor was used to measure oxygen concentrations along with a phytoplankton analyzer system that used pulse-amplitude-modulation (Phyto-PAM) to measure fluorescence. In order to identify the particular clade that the isolates belonged to, denaturing gradient gel electrophoresis (PCR-DGGE) was used to identify Symbiodinium based on their internal transcribed spacer 2 (ITS2) region. These techniques helped us to achieve our goals in the following ways: Symbiodinium sp. from a culture collection were classified to the subclade level; species-specific and clade-specific photosynthetic profiles were generated; and a Symbiodinium sp. was isolated from the central Red Sea. This study provided preliminary correlation between the photosynthetic difference and Symbiodinium genetic classification; showed the probable existence of a self-protection system inside the Symbiodinium cells by comparing the difference between the initial oxygen production at the beginning of each light step and the oxygen production after light adaptation; and confirmed the possibility of the isolation of Symbiodinium.

Animal and plant genomes produce numerous small RNAs (smRNAs) that regulate gene expression post-transcriptionally affecting metabolism, development, and epigenetic inheritance. In order to characterize the repertoire of endogenous microRNAs and potential gene targets, we conducted smRNA and mRNA expression profiling over nine experimental treatments of cultures from the dinoflagellate Symbiodinium sp. A1, a photosynthetic symbiont of scleractinian corals. We identified a total of 75 novel smRNAs in Symbiodinum sp. A1 that share stringent key features with functional microRNAs from other model organisms. A subset of 38 smRNAs was predicted independently over all nine treatments and their putative gene targets were identified. We found 3,187 animal-like target sites in the 3’UTRs of 12,858 mRNAs and 53 plantlike target sites in 51,917 genes. Furthermore, we identified the core RNAi protein machinery in Symbiodinium. Integration of smRNA and mRNA expression profiling identified a variety of processes that could be under microRNA control, e.g. regulation of translation, DNA modification, and chromatin silencing. Given that Symbiodinium seems to have a paucity of transcription factors and differentially expressed genes, identification and characterization of its smRNA repertoire establishes the possibility of a range of gene regulatory mechanisms in dinoflagellates acting post-transcriptionally.

Broad-scale population genetics can reveal population structure across an organism’s entire range, which can enable us to determine the most efficient population-wide management strategy depending on levels of connectivity. Genetic variation and differences in genetic diversity on small-scales have been reported in anemones, but nothing is known about their broad-scale population structure, including that of “host” anemone species, which are increasingly being targeted in the aquarium trade. In this study, microsatellite markers were used as a tool to determine the population structure of a sessile, host anemone species, Heteractis magnifica, across the Indo-Pacific region. In addition, two rDNA markers were used to identify Symbiodinium from the samples, and phylogenetic analyses were used to measure diversity and geographic distribution of Symbiodinium across the region. Significant population structure was identified in H. magnifica across the Indo-Pacific, with at least three genetic breaks, possibly the result of factors such as geographic distance, geographic isolation and environmental variation. Symbiodinium associations were also affected by environmental variation and supported the geographic isolation of some regions. These results suggests that management of H. magnifica must be implemented on a local scale, due to the lack of connectivity between clusters. This study also provides further evidence for the combined effects of geographic distance and environmental distance in explaining genetic variance.

Cette thèse confidentielle public/privé porte sur l’étude des sources biologiques de la palytoxine (PlTX) dont l’identification est nécessaire afin de développer un protocole industriel standardisé permettant d’obtenir des quantités importantes de toxine pour réaliser diverses études. Premièrement, l’activité anticancéreuse in vitro de la PlTX a été évaluée sur des lignées cellulaires cancéreuses humaines et murines. La cytotoxicité de la PlTX est 106 fois plus importante sur les lignées cancéreuses par rapport aux lignées non cancéreuses témoins. La PlTX utilisée a été purifiée à partir d’une espèce encore non décrite de Palythoa, qui a été caractérisée phylogénétiquement et morphologiquement et qui fournit les quantités de PlTX pures les plus élevées à ce jour (2,8 ± 0,45 mg/g de matière fraîche). La localisation de la PlTX dans les tissus de cette espèce a été décrite par des techniques innovantes d’imagerie par spectrométrie de masse MALDI. Deuxièmement, une étude phylogénétique et une quantification de la PlTX par HPLC chez 29 échantillons de Zoantharia a été réalisée. La spectrométrie de masse ESI a permis de donner la structure chimique du type de PlTX purifiée à partir de ces échantillons. La dernière partie présente une étude préliminaire sur la culture ex hospite des endosymbiotes unicellulaires de Palythoa spp. appartenant au genre Symbiodinium afin de tester leur implication dans la synthèse de PlTX. En conclusion, il ressort que la PlTX présente un effet anticancéreux très prometteur. Le moyen le plus efficace actuellement pour obtenir de la PlTX en quantité importante est de l’extraire à partir d’une espèce de Palythoa caractérisée grâce à ce travail. / The work presented in this manuscript is the outcome of a confidential public/private collaboration. It concerns the study of biological sources of PlTX. Their identification is required to develop an industrial-standard protocol in order to obtain sufficient quantity of toxin to perform several studies. In the first part, the in vitro anti-cancer activity of PlTX was evaluated upon several human and murine cancerous cell lines. The cytotoxic activity of PlTX was 106-times more powerful against cancerous lines compared to non-cancerous control lines. The PlTX used in this work was purified from an undescribed species of Palythoa which was characterized based on phylogenetic and morphological analyses. This species produces the highest PlTX levels ever recorded (2.8 ± 0.45 mg/g fresh matter). PlTX localization in tissues of this Palythoa species was also described by innovative MALDI mass spectrometry imaging techniques. The second part presents a phylogenetic study of 29 samples of Zoantharia and quantification of PlTX by HPLC. The ESI-mass spectrometry allowed to characterize the chemical structure of the type of PlTX purified from these samples. The last part of this thesis constitutes a preliminary study on the ex hospite culture of unicellular endosymbionts belonging to the genus Symbiodinium. Ensosymbiont cells were isolated from Palythoa species containing high PlTX concentrations in order to test their implication in PlTX synthesis. In conclusion, PLTX has a very promising anticancer activity, and the best way to obtain large quantities of PlTX is the extraction from a Palythoa species characterized in this work.

Les coraux vivent en association symbiotique avec des algues dinoflagellées du genre Symbiodinium, distinguées en différents clades (A à I). Un même hôte peut abriter seul ou plusieurs de ces clades, a priori dépendant d’un contexte environnemental, suggérant qu’ils possèdent des propriétés physiologiques distinctes. Ainsi, les capacités de flexibilité et d’acquisition de clades pourraient être une issue pour la survie de l’holobionte face à divers stress. Dans le cadre de cette thèse, ces capacités ont été explorées chez plusieurs espèces coralliennes, décrites avec différentes flexibilités et sensibilités aux conditions environnementales. La diversité et la dynamique des communautés de Symbiodinium associées aux coraux ont été estimées à l’aide de la technique de PCR en temps réel et évaluées dans différents contextes environnementaux: naturels, lors d’un suivi spatio-temporel de 18 mois à Moorea, ou contrôlés, lors d'une incubation à différentes pCO2. Les coraux ont montré la possibilité d’héberger jusqu’à 3 ou 4 clades distincts, reconsidérant la notion de spécialiste (i.e. symbionte unique). Néanmoins, propre à l’hôte corallien, il a été mis en évidence la régulation de ‘signatures symbiotiques’ caractérisées par des profils particuliers d’association de clades, stables et durables. Des modifications sporadiques de ces profils, par switching et shuffling ont put être observées, indépendamment d’un contexte environnemental donné, suggérant la dynamique comme un mécanisme constitutif chez les coraux. Néanmoins, en cas de d’apparition d’épizooties de Vibrio spp. et d’une maladie corallienne il est suggéré que le clade D participe activement au ‘fitness’ de leur hôte. / Corals live in symbiosis with dinoflagellate algae of the genus Symbiodinium, divided into 9 clades (A to I). The same host can harbour one or more of these clades depending on the environmental context. This lead to the suggestion that each clade has its unique physiological property. Therefore the capacity of flexibility and acquisition of clades may be an issue for the survival of the holobiont against various stresses. In this thesis, these capacities have been explored in several coral species, described with various flexibilities and sensitivities facing different environmental conditions. The diversity and dynamics of Symbiodinium communities associated to corals were estimated by using the real-time PCR technique, and evaluated in different environmental contexts : natural, in an 18-month spatio-temporal survey around Moorea, or controlled, upon incubation with different pCO2. Some corals showed the ability to host up to 3 or 4 distinct clades, leading to a reconsideration of the specialist-concept (i.e. host associated with one symbiont type). Nevertheless, depending on the coral host, ‘symbiotic signature’ regulations have been revealed. Latter are characterized by specific clade combination profils, which are stable and sustainable. In addition, sporadic changes by switching and shuffling, were observed in a given profil, regardless of a given environmental context, suggesting the dynamics as an constitutive mechanism in corals. However, records of coral disease together with Vibrio spp outbreaks, suggested an active participation of clade D for the ‘fitness’ of the coral host.

Dans le contexte du réchauffement climatique, les récifs coralliens subissent des stress thermiques de plus en plus fréquents et intenses. Dans le but de mieux comprendre les mécanismes de la thermo­tolérance des coraux, j’ai développé une approche intégrative sur l’holobionte corallien (méta­organisme composé de l’hôte corallien, son algue symbiotique etson microbiote). Pour cela, j’ai réalisé une expérience de stress thermique écologiquement réaliste sur une espèce de corail, Pocillopora damicornis. Cette espèce étant présente dans l’ensemble de l’Indo­Pacifique, j’ai pu comparer la réponse de deux populations dont la thermotolérance est différente puisqu’elles sont soumises à des régimes thermiques contrastés. J’ai analysé, pour chacune d’entre­elles, la réponse de l’hôte corallien (par RNAseq), ainsi que la structure et les changements au niveau des microbiotes algaux et bactériens (par métabarcoding). Les résultats obtenus montrent qu’alors que la structure du microbiote n’est pas influencée par le stress, le corail y répond de façon très différente selon la population étudiée. La population issue d’un environnement plus fluctuant met en place une réponse plus efficace et plus plastique, probablement grâce à l’intervention de mécanismes épigénétiques. Une autre étude réalisée sur différentes populations de P. damicornis dans le cadre de cette thèse montre que la composition du microbiote est influencée par le génome de l’hôte ainsi que par le régime thermique. Un des clades de l’algue symbiotique connu pour améliorer la thermo­ tolérance de l’hôte corallien semble plus sensible aux basses températures que les autres. / In the context of global warming, coral reefs are experiencing thermal stresses which are becoming more frequent and intense. In order to get a better understanding of the mechanisms of coral thermo­tolerance, I developed an integrative approach on the coral holobiont (meta­ organism composed of the coral host, its symbiotic algae and microbiota). For this, I performed an ecologically realistic thermal stress experiment on a coral species, Pocillopora damicornis. This species is widespread in the Indo­Pacific area. I compared the response of two populations whose thermotolerance is different since they are subjected to contrasting thermal regimes. I analyzed, for each of them, the response of the coral host (by RNAseq), as well as the structure and changes in the algal and bacterial microbiota (by metabarcoding). The results show that,while the structure of the microbiota is not influenced by stress, coral responds very differently depending on the population studied. The population from a more fluctuating environment displays a more effective and more plastic response, probably thanks to the involvement of epigenetic mechanisms. Another study carried out on different populations of P. damicornisshowed that the composition of the microbiota is influenced by the host genome and the thermal regime. One of the clades of the symbiotic algae, known to improve the heat­tolerance of the coral host, appears more sensitive to low temperatures than the others.

Caribbean coral reefs are in crisis. Degradation of living coral and fish assemblages has accelerated during the past half century, with a suite of anthropogenic drivers –from local fishing pressure to unprecedented global scale climate change– implicated. Accompanying these losses is the physical disintegration of the three-dimensional calcium carbonate reef structure. Flattening of reefs, synonymous with loss of ecosystem function and provision of services, is caused by an imbalance in the carbonate budget: a trade-off between carbonate production and consolidation by calcifying organisms (principally coral-algal symbioses) and framework breakdown by bioeroding organisms and storms. This thesis focuses on expanding our understanding of two functionally critical issues that strongly influence Caribbean coral reef community composition and dynamics, and which look likely to have a key bearing on the future state of reefs in the region: coral photosynthetic endosymbionts, and carbonate budgets. The former exert an important role in the production of the coral carbonate framework, whilst the latter reflect the dynamics of reef carbonate production and erosion. In the first part of the thesis, existing information on rates of carbonate production and erosion on Caribbean reefs is utilised to construct a detailed theoretical carbonate budget model. The model is used to chart historic changes in Caribbean carbonate budgets, tracking reef flattening across time and identifying key ecological drivers of these changes. This “eco-geomorphic” model is then coupled with state-of-the-art climate and ecological models, to project reef processes to the end of the century, asking the question ‘at what point will Caribbean reefs shift to net erosional regimes?’. The models are also used to explore the efficacy of local management and climate mitigation in altering the negative trajectory of reefs under projected warming and ocean acidification. In the second part of the thesis, 632 corals from across the wider Caribbean are screened, to construct the largest recorded baseline of symbiont biogeography for the region’s key remaining reef framework builder, Montastraea annularis. Spatial patterns of symbiont diversity are explored in terms of environmental, geographic and genetic factors, contributing to the growing body of work currently in the early stages of cataloguing symbiont diversity and its ecological significance. Although carbonate budget models forecast a bleak outlook for the Caribbean, detection of widespread low-level prevalence of thermally-tolerant endosymbionts in M. annularis provides a weak ‘nugget of hope’ for potential coral acclimation. Combined local management and aggressive mitigative action on carbon emissions are pre-requisites for maintenance of functioning reefs into the next century. Coral reef conservation efforts can be improved if we fully appreciate the contributions of all reef components –not just the enigmatic ones– to healthy reef functioning.

The pathway of seawater ammonium assimilation and influence of light on amino acid synthesis remain unresolved in cnidarian symbioses. Labeled ammonium (10 μM 15NH4Cl) in seawater was used to trace the pathway of the incorporation into amino acids in host tissue, Zoanthus sp., and zooxanthellae, Symbiodinium microadriaticum. Freshly isolated zooxanthellae were exposed to 20 μM 15NH4Cl with coral homogenate to evaluate the role of host factors on amino acid synthesis. High performance liquid chromatography and mass spectrometry was used to measure percent labeling and concentrations of amino acids. In zooxanthellae, ammonium was assimilated into glutamine likely via glutamine synthetase and into glutamate via glutamine 2-oxoglutarate amidotransferase. Interrupting photosynthesis with DCMU did not inhibit glutamine and tryptophan synthesis however reduced the 15N-enrichment of glutamate, aspartate, and ornithine in zooxanthellae, as well as arginine, ornithine, and lysine in host tissue. Coral homogenate had little effect on the 15N-enrichment of glutamine, aspartate, and alanine in freshly isolated zooxanthellae. Evidence is presented to support the uptake of ammonium ions and data shows that glutamine and not glutamate is translocated to the coral host.

Coral reefs constitute some of the most prolific and diverse ecosystems on our planet, but also among the most threatened. This thesis investigates the effects of environmental stressors on corals’ metabolism and symbiont diversity. Paper I shows that the coral Turbinaria mesenterina withstood a single stressor while a combination of two stressors (decreased salinity and increased seawater temperature) lead to decreased metabolism. Increased seawater temperature in combination with two stressors (enhanced nutrients and decreased salinity) lead to rapid mortality of all specimens. Paper II shows that chronic stress in combination with increased seawater temperature affects coral species differently. Porites lutea did not show any difference in response to temperature increase, regardless of environmental disturbance history, while Galaxea fascicularis’ metabolism was negatively affected in chronically disturbed corals but not in corals from less disturbed areas. The main explanation for the difference in response between the two species is different compositions of endosymbionts as found in paper III. P. lutea only harboured the symbiont C15, regardless of environment, whilst D1a dominated the nearshore G. fascicularis and C1 dominated offshore corals. In paper IV there was a clear inshore-offshore pattern of D1a along the whole coast of Vietnam, where D1a dominated inshore. In contrast, the five symbionts belonging to group C displayed a strong latitudinal gradient, with diversity increasing from north to south. The coral host showed higher diversity offshore than inshore. The thesis emphasizes the importance of improving water quality (paper I and II) and protecting marginal areas since tolerant coral hosts and symbionts can be found there (paper III and IV), as well as safeguarding areas with high symbiont diversity (paper IV) to increase the ability of corals to withstand future environmental changes. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

No description available.