Corals are cnidarian animals that build the founding structures of tropical reefs, which survival depends upon the obligate symbiotic association to photosynthetic dinoflagellate algae in the family Symbiodiniaceae. As corals are facing increasing environmental and anthropogenic stress, understanding the molecular principles governing this unique symbiotic association is crucial to predict their adaptive potential. Due to logistic, costly, and experimental difficulties of working with corals, we use the sea anemone Aiptasia (sensu Exaiptasia pallida) as a tractable model organism for the molecular study of cnidarian-algal symbiosis. A major advantage of Aiptasia is that it establishes a facultative symbiotic association with Symbiodiniaceae algae, that is, this anemone can be maintained in an aposymbiotic (symbiont-free) state, allowing for comparison of symbiotic and ‘control’ aposymbiotic processes. The main aim of this dissertation was to investigate the signaling pathways involved in the regulation of this symbiotic interaction, and in particular, phosphorylation-mediated protein signaling. Phosphorylation is indeed a major post-translational modification that mediates signal transduction within and across cells. To investigate if protein phosphorylation regulates the complex intercellular signaling that occurs between symbiotic partners, a mass spectrometry-based phosphoproteomic approach was employed. Given the novelty of this application in the field of coral reef biology, the first research chapter details the development and optimization of a protocol that allows quantification of protein phosphorylation in the sea anemone Aiptasia. This chapter includes mass spectrometric analysis in 1) data-dependent acquisition (or shotgun proteomics) for the generation of a so-called assay (spectral) library, a reference dataset that servers for 2) accurate and reproducible label-free quantification of protein phosphorylation in data-independent acquisition (DIA/SWATH-MS). In the second research chapter, the developed protocol was employed to generate a phosphopeptide assay (spectral) library for aposymbiotic and symbiotic Aiptasia, which would allow further quantification of protein phosphorylation across symbiotic conditions. We consistently quantified more than 3,000 phosphopeptides, totaling more than 1,600 phosphoproteins, across biological replicates and symbiotic conditions. Characteristic phosphoproteomic profile distinguished the two symbiotic groups and differential phosphorylation targeted biological processes that have not been previously described in the context of cnidarian-algal symbiosis, namely the phospholipase D signaling pathway and protein processing in the endoplasmic reticulum. We suggest that changes in the phosphorylation status of these signaling pathways may have a potentially relevant role in the control of an established cnidarian-algal association.

Nausithoe aurea Silveira & Morandini, 1997 é uma espécie metagenética e dióica com fecundação externa. Os oócitos são liberados continuamente (55 dias em laboratório), porém com grandes variações no número a cada dia. No desenvolvimento embrionário a clivagem, após o estágio de 8 células, passa de holoblástica e igual para pseudoespiral. A gastrulação ocorre por ingressão multipolar e inicia-se aproximadamente 24 horas após a fecundação. A estrutura histológica geral das gônadas assemelha-se a outros Scyphozoa, onde os gonócitos proliferam a partir da gastroderme, migram e diferenciam-se na mesogléia. Na gônada masculina as células germinativas formam camadas razoavelmente distintas e constituem folículos testiculares. Na gônada feminina os oócitos surgem da zona germinativa na gastroderme e apresentam um gradiente de maturação a partir deste ponto (cortes no sentido oral-aboral). Os oócitos encontram-se livres na mesogléia da gônada, sem associação com outras células. A relação espacial entre a musculatura circular, as gônadas e o sulco coronal, é uma característica a ser usada na sistemática do gênero Nausithoe Kölliker, 1853. / Nausithoe aurea Silveira & Morandini, 1997 is a metagenetic and dioecious species with external fertilization. The oocytes are released continuously (55 days in laboratory), but with great variations in the daily number. In the embryonic development the cleavage, after the 8 cells stage, changes from holoblastic and adequal to pseudospiral. The gastrulation occurs through multipolar ingression and begin 24 hours after fertilization. The general histological structure of the gonads resembles other Scyphozoa, in which the gonocytes proliferate from the gastrodermis, migrate and differentiate in the mesoglea. In the male gonad the germ cells are arranged in distinctive layers and form follicles (cysts). In the female gonad the oocytes develop from the germinative zone in the gastrodermis and present a maturing gradient from this point on (oral-aboral sections). The oocytes are free in the gonad mesoglea, without association to any cell. The spatial relation of the coronal musculature, gonads and coronal groove, is a character to be used in the systematics of the genus Nausithoe Kölliker, 1853.

Nausithoe aurea Silveira & Morandini, 1997 é uma espécie metagenética e dióica com fecundação externa. Os oócitos são liberados continuamente (55 dias em laboratório), porém com grandes variações no número a cada dia. No desenvolvimento embrionário a clivagem, após o estágio de 8 células, passa de holoblástica e igual para pseudoespiral. A gastrulação ocorre por ingressão multipolar e inicia-se aproximadamente 24 horas após a fecundação. A estrutura histológica geral das gônadas assemelha-se a outros Scyphozoa, onde os gonócitos proliferam a partir da gastroderme, migram e diferenciam-se na mesogléia. Na gônada masculina as células germinativas formam camadas razoavelmente distintas e constituem folículos testiculares. Na gônada feminina os oócitos surgem da zona germinativa na gastroderme e apresentam um gradiente de maturação a partir deste ponto (cortes no sentido oral-aboral). Os oócitos encontram-se livres na mesogléia da gônada, sem associação com outras células. A relação espacial entre a musculatura circular, as gônadas e o sulco coronal, é uma característica a ser usada na sistemática do gênero Nausithoe Kölliker, 1853. / Nausithoe aurea Silveira & Morandini, 1997 is a metagenetic and dioecious species with external fertilization. The oocytes are released continuously (55 days in laboratory), but with great variations in the daily number. In the embryonic development the cleavage, after the 8 cells stage, changes from holoblastic and adequal to pseudospiral. The gastrulation occurs through multipolar ingression and begin 24 hours after fertilization. The general histological structure of the gonads resembles other Scyphozoa, in which the gonocytes proliferate from the gastrodermis, migrate and differentiate in the mesoglea. In the male gonad the germ cells are arranged in distinctive layers and form follicles (cysts). In the female gonad the oocytes develop from the germinative zone in the gastrodermis and present a maturing gradient from this point on (oral-aboral sections). The oocytes are free in the gonad mesoglea, without association to any cell. The spatial relation of the coronal musculature, gonads and coronal groove, is a character to be used in the systematics of the genus Nausithoe Kölliker, 1853.

Coral reefs are one of the most diverse ecosystems on the planet due partially to the habitat structure provided by corals. Corals are long lived organisms that can live for hundreds of years and as a result growth of many species is very slow. As a result of this, recovery of corals from disease outbreaks is very slow and difficult and therefore the ecosystem is deteriorating rapidly. Due to this increase in disease and its detrimental effect on coral reefs, it has become imperative to study how corals respond to disease outbreaks. The response of the coral to pathogens is believed to be controlled by the innate immune system. However, the immune pathways and components of these pathways used by cnidarians to combat pathogens are still rudimentary. This work showed that C3 and heat shock protein 70 are components of the coral immune system that positively respond to disease occurrence. As disease out breaks become more frequent, the question has arisen as to whether cnidarians have homologs to of the adaptive immune system that allow them to respond more rapidly to subsequent encounters with the same bacterium. In the cnidarian model system Exaiptasia pallida, immune priming occurs up to one month after the initial sub lethal exposure to the pathogen. This transient form of priming could be the result of host energy allocation in place of establishing long term immune priming which could be too energetically costly. Cnidarians may only activate priming during summer months, when ocean temperatures and bacterial load are high. Specificity of immune priming in E. pallida requires further investigation with more bacterial pathogens. In this dissertation, one bacterial strain shows specificity while the other does not. Furthermore, the priming response involves many pathways which include pathogen recognition, inflammation, and activation of NF-κB. The discovery of immune priming in a sea anemone shows that this phenomenon evolved earlier in the tree of life than previously thought. Additionally, identification of priming in E. pallida is suggestive of its presence in corals which would allow for potential vaccinations of vulnerable corals.

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.