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Marine cnidarian-microalgal endosymbiosis is an ecologically important intracellular association. However, its underlying molecular mechanisms are essentially unknown. In light of the critical roles of host phagocytosis in intracellular fates of a variety of microbes, and the Rab small GTPases as key mediators of host-symbiont interaction, we set out to investigate the potential involvement of Aiptasia Rab proteins in the model photosynthetic endosymbiosis between the sea anemone, Aiptasia pulchella and the symbiotic dinoflagellate (commonly called zooxanthellae), Symbiodinium spp. Many Aiptasia Rab homologue-encoding cDNA fragments were first cloned through our degenerate RT-PCR and RACE reactions. Significantly, Aiptasia homologues of Rab5 and Rab7 (ApRab5 and ApRab7), two Rabs known to be critical regulators of phagosome maturation were also identified in the screen. The overall sequence identities of ApRab5 and ApRab7 to those of human Rab5C and Rab7 were very extensive, and EGFP reporter, protein fractionation, and immuno-fluorescence studies all suggested that the similarity of the Aiptasia Rabs to their human counterparts extended to the functional levels. Finally, although the phagosomes enclosing latex beads stained positive for ApRab5 and ApRab7 with kinetics characteristics of normal phagosomal maturation, the phagosomes housing zooxanthellae only stained positive for ApRab5. Furthermore, the association of ApRab5 with and the exclusion of ApRab7 from the zooxanthellae-containing phagosomes could be reversed by the heat-killed or photosynthesis-impaired symbionts. Overall, our present study has identified ApRab5 and ApRab7 as potential key regulators of the Aiptasia-Symbiodinium endosymbiosis

Cnidarians are the largest phylum of generally toxic animals, yet their toxins and venoms have not received as much scientific attention as those of many terrestrial (snakes, scorpions, spiders, etc.) and even some marine animals (i.e. cone snails). Approximately 13,000 living cnidarian species have been described by systematists. A major rationale for their study in the past, besides scientific curiousity, was to better treat victims of their envenomation. While that goal remains a high priority, it is now appreciated that the toxins of these mostly marine animals can be very useful molecular probes for the analysis of ion channels involved in electrical signaling, immune responses and other signal transduction processes of biomedical interest. For instance, anaphylaxis was discovered by Richet (1905) during experiments with sea anemone and hydrozoan tentacular extracts. Similarly, it has recently been shown that a toxin from another sea anemone is able to potently inhibit T-lymphocyte proliferation in models of certain autoimmune diseases. Thus, these natural substances continue to be of relevance for understanding and treating human diseases. In addition to introducing phylum Cnidaria (Coelenterata), we provide a short history of early (until about 1990) research on cnidarian toxins and venoms, to provide a perspective for appreciating the scientific advances of the past two decades that are summarized in the ensuing 19 papers in this special Toxicon issue.

Orientador: Vidal Haddad Júnior / Banca: Fábio Lang da Silveira / Banca: Rafael Augusto Gregati / Resumo: O presente trabalho relata a composição, abundância e distribuição dos cnidários nas diferentes estações do ano, caracterizando as espécies dominantes da comunidade nas regiões amostradas, da costa do litoral norte do Estado de São Paulo (Ubatuba 23º26' 15''S e 45º03'45''W; Caraguatatuba 23º61'39"S e 45º32'56''W; São Sebastião 23º84' 94''S e 45º24'81''W), no período compreendido entre fevereiro de 2001 a dezembro de 2002. Foram encontradas 6 espécies de cnidários potencialmente causadores de acidentes, sendo 2 cubozoários: Tamoya haplonema (Müeller, 1859) e Chiropsalmus quadrumanus (Müeller, 1859); 2 hidrozoários: Nemalecium lighti (Hargitt, 1924) e Olindias sambaquiensis (F. Müller, 1861) e 2 cifozoários: Chrysaora lactea Eschscholtz, 1829 e Lychnorhiza lucerna Haeckel, 1880. As espécies de maior abundância e freqüência na comunidade foram C. lactea e O. sambaquiensis. Estas espécies também tiveram mais representatividade em análises de abundância além de sua distribuição espacial e temporal. T. haplonema foi uma espécie de ocorrência exclusiva apenas em outono e inverno e C. lactea foi uma espécie abundante de ocorrência no ano todo, com mais representantes nas estações de outono e inverno. Outro objetivo do estudo foi o registro de acidentes causados por algumas espécies de cnidários em banhistas da região de Ubatuba, além da identificação da espécie e associação às lesões dermatológicas e à provável etiologia dos acidentes, além da correlação da quantidade dos animais com a ocorrência de acidentes registrados em meses de veraneio. Foram registrados cento e vinte e oito pacientes, e desta forma, analisados sinais e sintomas, manifestações sistêmicas e extensões das lesões no corpo da vítima, comprovou-se uma associação entre as marcas cutâneas e a provável etiologia e gravidade dos acidentes. Além disso .... / Not available / Mestre

Made available in DSpace on 2014-06-11T19:30:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-25Bitstream added on 2014-06-13T21:00:29Z : No. of bitstreams: 1 mendes_pr_me_botib.pdf: 1465265 bytes, checksum: bbb67eb612b94d3451a49d78c65fbdd4 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O presente trabalho relata a composição, abundância e distribuição dos cnidários nas diferentes estações do ano, caracterizando as espécies dominantes da comunidade nas regiões amostradas, da costa do litoral norte do Estado de São Paulo (Ubatuba 23º26’ 15’’S e 45º03’45’’W; Caraguatatuba 23º61’39”S e 45º32’56’’W; São Sebastião 23º84’ 94’’S e 45º24’81’’W), no período compreendido entre fevereiro de 2001 a dezembro de 2002. Foram encontradas 6 espécies de cnidários potencialmente causadores de acidentes, sendo 2 cubozoários: Tamoya haplonema (Müeller, 1859) e Chiropsalmus quadrumanus (Müeller, 1859); 2 hidrozoários: Nemalecium lighti (Hargitt, 1924) e Olindias sambaquiensis (F. Müller, 1861) e 2 cifozoários: Chrysaora lactea Eschscholtz, 1829 e Lychnorhiza lucerna Haeckel, 1880. As espécies de maior abundância e freqüência na comunidade foram C. lactea e O. sambaquiensis. Estas espécies também tiveram mais representatividade em análises de abundância além de sua distribuição espacial e temporal. T. haplonema foi uma espécie de ocorrência exclusiva apenas em outono e inverno e C. lactea foi uma espécie abundante de ocorrência no ano todo, com mais representantes nas estações de outono e inverno. Outro objetivo do estudo foi o registro de acidentes causados por algumas espécies de cnidários em banhistas da região de Ubatuba, além da identificação da espécie e associação às lesões dermatológicas e à provável etiologia dos acidentes, além da correlação da quantidade dos animais com a ocorrência de acidentes registrados em meses de veraneio. Foram registrados cento e vinte e oito pacientes, e desta forma, analisados sinais e sintomas, manifestações sistêmicas e extensões das lesões no corpo da vítima, comprovou-se uma associação entre as marcas cutâneas e a provável etiologia e gravidade dos acidentes. Além disso.... / Not available

Le succès évolutif des Cnidaires symbiotiques réside en grande partie dans leurs échanges trophiques établis avec les Dinoflagellés du genre Symbiodinium. Cependant, le réchauffement climatique global ainsi que les pollutions ont un impact fort sur les écosystèmes coralliens, notamment en conduisant à la rupture de la symbiose, phénomène appelé blanchissement. La compréhension des mécanismes qui régissent l’établissement, le maintien et la rupture de la symbiose est essentielle à la prévention des épisodes de blanchissement massif. Dans ce contexte, les objectifs de mon projet de thèse sont de caractériser les médiateurs chimiques de l’anémone de mer Anemonia viridis, de les localiser, et d’analyser leur réponse face à un stress. Parmi les composés caractérisés, les lipides et les bétaïnes sont les plus abondants et présentent une grande diversité. Certains sont transférés des symbiotes vers l’hôte. Des anémones de mer ont ensuite été traitées en laboratoire afin de provoquer la rupture de la symbiose et le blanchissement des individus. Une étude cinétique a été menée par une approche globale comparative identique à celle réalisée sur l’anémone symbiotique. Par ailleurs, une cartographie de l’évolution de composés clé a été réalisée par MALDI-MSI. La réponse au stress a été évaluée et a permis d’identifier des lipides de bétaïne et trois indicateurs lipidiques comme marqueurs de réponse précoce au stress. L’ensemble de ces résultats apporte de nouveaux éléments de réponse concernant le rôle des médiateurs chimiques clés dans le maintien de la symbiose, ainsi que leur influence sur sa rupture. / The ecological success of cnidarian-dinoflagellate symbiosis mainly relies on nutrient recycling. Environmental changes, such as global warming or pollution, often result to symbiosis breakdown, also called cnidarian bleaching. The understanding of mechanisms regulating the symbiosis establishment, maintenance and breakdown is essential to prevent massive bleaching phenomena. In this respect, my PhD project focused on the characterization of chemical mediators expressed in the sea anemone Anemonia viridis, their localization and their modulation by stress conditions. A comparative study was first conducted to characterize the chemical mediators and analyze their distribution within the symbiotic sea anemone. We described a great abundance and diversity of lipids in A. viridis tissues. From these results, we proposed possible transfers of FAs between the symbiotic partners. A thermal stress and a chemical stress have also been applied in laboratory-controlled conditions in order to induce symbiosis breakdown and bleaching of the sea anemones, in order to correlate A. viridis metabolome to its symbiotic status. A mapping of these metabolites has been performed by MALDI-MSI of tentacle cross-sections, as well as their evolution following stress. Some betaine lipids have thus been proposed as short-term indicators of stress. A. viridis stress response has also been evaluated with a lipidomic approach, and allowed to identify 3 lipid indicators of early stress response based on membrane fluidity markers. Overall, this study provides insight on key chemical mediators that may regulate the symbiosis maintenance, and may contribute to the symbiosis breakdown.

As climate change intensifies, the capacity of organisms to adapt to changing environments becomes increasingly relevant. Heat-induced coral bleaching –the breakdown of the symbiotic association between coral hosts and photosynthetic algae of the family Symbiodiniaceae– is rapidly degrading reefs worldwide. Hence, there is a growing interest to study symbioses that can persist in extreme conditions. The Red Sea is such a place, known as one of the hottest seas where healthy coral reef systems thrive. Here (Chapter 1), we tested the potential of symbiont manipulation as means to improve the thermal resilience of the cnidarian holobiont, particularly using heat tolerant symbiont species from the Red Sea. We used clonal lineages of the model system Aiptasia (host and symbiont), originating from different thermal environments to assess how interchanging either partner affected their short- and long-term performance under heat stress. Our findings revealed that symbioses are not only intra-specific but have also adapted to native, local environments, thus potentially limiting the acclimation capacity of symbiotic cnidarians to climate change. As such, infection with more heat resistant species, even if native, might not necessarily improve thermotolerance of the holobiont. We further investigated (Chapter 2) how environment-dependent specificity, in this case elevated temperature, affects the establishment of novel symbioses. That is, if Aiptasia hosts are, despite exhibiting a high degree of partner fidelity, capable of acquiring more thermotolerant symbionts under stress conditions. Thus, we examined the infection dynamics of multi-species symbioses under different thermal environments and assessed their performance to subsequent heat stress. We showed that temperature, more than host identity, plays a critical role in symbiont uptake and overall performance when heatchallenged. Additionally, we found that pre-exposure to high temperature plays a fundamental role in improving the response to thermal stress, yet, this can be heavily influenced by other factors like feeding. Like climate change, ocean acidification is a serious threat to corals. Yet, most research has focused on the host and little is known for the algal partner. Thus, here we studied (Chapter 3) the global transcriptomic response of an endosymbiotic dinoflagellate to long-term seawater acidification stress. Our results revealed that despite observing an enrichment of processes related to photosynthesis and carbon fixation, which might seem beneficial to the symbiont, low pH has a detrimental effect on its photo-physiology. Taken together, this dissertation provides valuable insights into the responses of symbiotic cnidarians to future climate and ocean changes.

Animal chemosensation involves several families of G protein-coupled receptors (GPCRs) and, though some of these families are well characterized in vertebrates and nematode worms, receptors have not been identified for most metazoan lineages. In this dissertation, I use a combination of bioinformatics approaches to identify candidate chemosensory receptors in three invertebrates that occupy key positions in the metazoan phylogeny. In the sea urchin Strongylocentrotus purpuratus, I uncovered 192 candidate chemosensory receptors many of which are expressed in sensory structures including pedicellariae and tube feet. In the cephalochordate Branchiostoma floridae, my survey uncovered 50 full-length and 11 partial odorant receptors (OR). No ORs were identified in the urochordate Ciona intestinalis. By exposing conserved amino acid motifs and testing the ability of those motifs to discriminate between ORs and non-OR GPCRs, I identified three OR-specific amino acid motifs that are common in cephalochordate, fish and mammalian ORs and are found in less than 1% of non-ORs from the rhodopsin-like GPCR family. To further investigate the antiquity of vertebrate ORs, I used the OR-specific motifs as probes to search for orthologs among the protein predictions from 12 invertebrates. My search uncovered a novel group of genes in the cnidarian Nematostella vectensis. Phylogenetic analysis that included representatives from the major subgroups of rhodopsin-like GPCRs showed that the cnidarian genes, the cephalochordate and vertebrate ORs, and a subset of genes S. purpuratus from my initial survey, form a monophyletic clade. The taxonomic distribution of these genes indicates that the formation of this clade began at least 700 million years ago, prior to the divergence of cnidarians and bilaterians. Furthermore, my phylogenetic analyses show that three of the four major subgroups of rhodopsin-like GPCRs existed in the ancestor of cnidarians and bilaterians. The utility of the new genes I describe here is that they can be used to identify candidate olfactory cells and organs in cnidarians, echinoderms and cephalochordates that can be tested for function. These genes also provide the raw material for surveys of other metazoans as their genomes become available. My sequence level comparison between chordates, echinoderms and cnidarians exposed several conserved amino acid positions that may be useful for understanding receptor mediated signal transduction. ORs and other rhodopsin-like GPCRs have roles in cell migration, axon guidance and neurite growth; therefore duplication and divergence in the rhodopsin-like gene family may have played a key role in the evolution of cell type diversity (including the emergence of complex nervous systems) and in the evolution of metazoan body plan diversity. / Graduate

Durant leur cycle de vie, les organismes sont exposés à des variations environnementales capables d'induire des changements physiologiques, morphologiques et comportementaux, résultant d’une plasticité phénotypique. La plasticité phénotypique est la capacité d'un génotype à générer un nouveau phénotype suite à un stress. Ici, nous avons étudié la plasticité phénotypique d’un Cnidaire symbiotique et non-calcifiant, l’anémone de mer Anemonia viridis, à de multiples niveaux de complexité structurale, in vivo et in vitro. In vivo, nous avons identifié les mécanismes sous-jacents de la plasticité phénotypique potentiellement induits par les futurs changements climatiques (acidification et réchauffement des océans). Nos résultats montrent des modifications dans l'utilisation du carbone inorganique par A. viridis exposée à une forte pCO2 lors d’un stress chronique in natura ou lors d’un stress court en conditions contrôlées. Nous avons ainsi observé une diminution des activités anhydrase carbonique, une enzyme clé des mécanismes de concentration du carbone chez les Cnidaires. Nous avons aussi démontré que l'augmentation concomittante de la température modifie la réponse observée lors d'une élévation seule de la pCO2. In vitro, nous avons établi une culture de cellules primaires viables issue de tentacules d’A. viridis en régénération. Nous avons déterminé l'origine gastrodermale des cellules cultivées et validé l'utilisation de ce nouvel outil pour l'étude de la réponse au stress au niveau cellulaire. Ce nouvel outil ouvre une multitude de perspectives pour l'étude des réponses cellulaires aux stress exogènes (changement climatique) et endogènes (contraintes dues à la symbiose) / During the course of their life cycle organisms are exposed to natural environment variations capable of inducing physiological, morphological and behaviour changes, thus a phenotypic plasticity. Phenotypic plasticity is the ability of a genotype to generate a new phenotype following exogeneous or endogeneous stress. Here, we investigated the phenotypic plasticity of the non-calcifying symbiotic cnidarian Anemonia viridis at multiple levels of structural complexity, in vivo and in vitro. In vivo, we determined the mechanisms behind the phenotypic plasticity under expected future climate change (i.e. ocean acidification and ocean warming). Our results show physiological changes in the inorganic carbon use of the sea anemone A. viridis exposed to high pCO2 during a long-term stress in natura or a short-term stress in controlled conditions. We then observed an equivalent decrease in carbonic anhydrase activity, a key enzyme of cnidarian carbon concentrating mechanisms. Also, we demonstrated that an increase in seawater temperature modified the response observed during a high pCO2 scenario. In vitro, we established a viable primary cell culture from regenerating tentacles of A. viridis. We determined the gastrodermal tissue origin of the cultivated cells and validated the use of this new tool to the in vitro study of stress response at the cellular level. The set-up of this powerful in vitro tool will open a multitude of perspectives for the study of cellular responses to exogeneous stress (as global change perturbations) and to endogeneous stress (as the symbiosis constraints experienced by symbiotic cnidarians)

In situ hybridization (ISH) has played a crucial role in developing a spatial transcriptomic understanding of emerging model organisms in the past, but advancing high-throughput RNA-sequencing (RNA-seq) technology has pushed this method into the shadows, leading to a loss of data resolution. This shift in research towards the exclusive use of RNA-seq neglects essential considerations for transcriptomic studies including the spatial and temporal expression of transcripts, available budget, experimental design needs, and validation of data. A synergy of spatiotemporal transcriptomic techniques is needed, using the bulk and unbiased analysis of RNA-seq and the visual validation and spatiotemporal resolution of ISH. Integration of this synergistic approach can improve our molecular understanding of non-model organisms and establish the background data needed for advancing research techniques. A prime example lies within an emerging model of the marine science and symbiosis fields, where I present a case study on a threatened coral reef keystone – the cnidarian-dinoflagellate symbiosis. Establishing a whole-mount ISH protocol for the emerging cnidarian model Aiptasia (sea anemone) will help future studies reveal the gene regulation underpinning the establishment, persistence, and breakdown of this complex symbiotic relationship.