Coral reefs thrive in nutrient-deficient environments yet function among the most productive ecosystems on Earth as a consequence of the symbiosis between coral hosts and their symbiotic zooxanthellae. The symbiotic unit (holobiont) can utilize both inorganic and organic sources of nutrients for the accumulation of carbon and nitrogen required for metabolism, growth, and reproduction. An iterative model was created to describe the flux of carbon and nitrogen between a host and its algae. The model design is based on a previously published conceptual model of algal symbioses; functions and values of input parameters are based on published studies of the coral species Stylophora pistillata. The model is designed to simulate responses of the coral, zooxanthellae and the holobiont to different environmental variables, either one at a time or changing simultaneously. Simulations presented are for default values based on previously published data for S. pistillata adapted to high-light (shallow-euphotic) and low-light (deep-euphotic) environments, and for single-variable manipulations of rates of a) host feeding, b) photosynthesis, and c) dissolved inorganic nitrogen (DIN) uptake. Simulations examining feeding rates between 0% and 6.5% of host biomass indicate that biomass of both high-light and low-light adapted holobionts increase exponentially with increased feeding, with benefit to the high-light holobiont ~8 times greater than to the low-light holobiont. Increasing rates of photosynthesis illustrated that a low-light holobiont is carbon limited, is primarily dependent upon host feeding, and can benefit from a small increase in photosynthesis rate. Simulations examining rates of DIN input indicate that the high-light holobiont functions optimally when inorganic nitrogen input is very low. Increase in DIN up to 0.5% resulted in benefit to the holobiont, but more resulted in unrealistically excessive growth by the zooxanthellae until a function to maintain a fixed range for the host-zooxanthellae biomass ration function was included in the model. Simulations for the low-light holobiont did not indicate any benefit from DIN input. The model was originally designed using a spreadsheet-based program which frequently became overloaded when testing multiple variables. Modification of the model in software better designed for modeling is recommended for future work.

Les diatomées jouent un rôle primordial dans l'écologie de la planète, car elles sont responsables du 20-40% de la productivite mondial d’oxygène. Elles figurent parmi les organismes à fort potentiel biotechnologique pour des applications biocarburant. Les diatomées sont des organismes symbiotiques issus de la fusion d'un ancêtre hétérotrophe avec une ou plusieurs micro-algues photosynthétiques. Grace à cette histoire évolutive complexe, les diatomées ont un métabolisme très flexible. Comme la plus part des microalgues elles peuvent utiliser la photosynthèse pour leur croissance, mais aussi la mixotrophie, i.e. la capacité de croître en présence de lumière et d’une source de carbone réduit. L'utilisation simultanée de la photosynthèse et de la respiration peut augmenter la productivité de la biomasse des microalgues et réduire ainsi le coût de leur exploitation industrielle. Dans cette thèse j’ai étudié le mécanisme et les conséquences du métabolisme mixotrophique chez la diatomée modèle Phaeodactylum tricornutum. J’ai contribué à étudier le mécanisme moléculaire à la base des interactions énérgétiques entre chloroplaste et mitochondrie. Dans ce travail, nous avons démontré que le NADPH généré dans le chloroplaste est exporté vers la mitochondrie pour générer de l’ATP requis pour la fixation du CO2 dans le chloroplaste. Cette interaction entre les deux organites cellulaires augmente la croissance de diatomées, et suggère que l'utilisation simultanée d’une source de carbone et de l'énergie lumineuse (mixotrophie) devrait augmenter la productivité de la biomasse chez les diatomées. Cette hypothèse a été testée dans la deuxième partie de ma thèse, où j’ai etudié les conséquences de la mixotrophie sur le métabolisme de Phaeodactylum. Grace à une approche métabolomique, transcriptomique, lipidomiques et de physiologie j’ai contribué à éclaircir les principales voies métaboliques (métabolisme centrale, métabolisme des lipides, métabolisme des polymères de réserve) concerné la mixotrophie. Dans la dernière partie de ce travail j’ai optimisé les conditions de culture et la composition du milieu afin d’améliorer la productivité en croissance mixotrophe chez Phaeodactylum. Ce résultat a été validé dans des photobioréacteurs à l'échelle labo pour tester le potentiel de l'exploitation industrielle de cet organisme. / Diatoms are photosynthetic organisms with a strong influence on the global biogeochemistry. Moreover, they are extremely interesting as potential feedstocks for the production of high-value molecules and biofuel. They are endosymbiotic organisms originated by the fusion of a heterotrophic ancestor with one or more photosynthetic microalgae. This has led to an extremely flexible cell metabolism. Like other microalgae, diatoms are able to grow in the presence of both light and of a reduced carbon source. The simultaneous use of photosynthesis and respiration can increase biomass productivity and reduce the energy cost of the industrial exploitation of diatoms.In this project, the mechanism and the consequences of mixotrophic metabolism have been studied in the model diatom Phaeodactylum tricornutum. In the first part, I have studied the molecular mechanism governing the interactions between chloroplast and mitochondrion. We have demonstrated that the NADPH generated in the plastid is exported to the mitochondrion to generate additional ATP, which, once back to the plastid, is used for carbon fixation. Overall, this work shows that the interaction between these two organelles increases carbon fixation and growth in diatoms. We hence suggest that the simultaneous use of carbon and light energy sources (i.e. mixotrophy) should enhance biomass productivity in diatoms. This hypothesis has been tested in the second part of my thesis, where I focused on the consequences of mixotrophy on metabolism. By combining metabolomic, transcriptomic, lipidomic and physiology approaches, I have contributed to elucidate the main pathways targeted by mixotrophy (central carbon, lipid and storage carbon metabolism). In the last part of this work, I have worked on improving the culture conditions and medium composition to boost microalgal productivity by mixotrophy. These conditions have been scaled-up in lab scale photobioreactors, revealing the industrial exploitation potential of Phaeodactylum.

Much of the world’s aquatic food webs and nutritional relationships have been blurred by the ever-increasing evidence that many phytoplankton are not exclusively heterotrophic or autotrophic, but instead mixotrophic. Mixotrophy is a continuum of different energy and carbon-acquisition mechanisms utilizing both autotrophy and heterotrophy which distorts the concept of single trophic tier modality. This makes mixotrophs flexible to adapt to environmental pressures and is becoming more the rule than the exception in many aquatic ecosystems. One unique environmental setting where mixotrophy could be highly beneficial to food web stability is in seasonally occurring ephemeral pools – aka vernal pools. Mid-Atlantic vernal pools are biodiverse biogeochemical hotspots and critical breeding habitats for a diverse number of endemic taxa including many endangered amphibian species. Vernal pools are not permanent standing bodies of water and have fluxes in hydrology, temperatures, nutrients, and irradiance to name a few. These extremes make vernal pools an ideal setting for mixotrophic phytoplankton, however it’s never been investigated. Our survey found mixotrophy in every vernal pool sampled, as well as elevated grazing rates in pools experiencing nontypical seasonal conditions. From these small-scale forest pools to the world’s oceans mixotrophy is a widespread nutritional strategy. The Southern Ocean is essential for powering worldwide ocean circulation, regional biogeochemical cycles, and global climate. One of the major hurdles with understanding mixotrophy is identifying the phytoplankton capable of shifting nutritional strategies. While many Southern Ocean plankters have been properly identified as mixotrophic, one such keystone species has gone mislabeled until now. Phaeocystis antarctica is a well-studied Haptophyte algae that plays major roles in the global carbon and sulfur cycles. This species has been historically labeled as an obligate phototroph, but contradictory survives the long dark Antarctic winter without any known evidence of encystment. We suspect that this highly abundant species is in fact mixotrophic, capable of phagocytosis to supplement the irradiance shortcomings of the Antarctic dark. We experimented with varying degrees of light and nutrient limitations to determine possible triggers for P. antarctica grazing. Our results showed P. antarctica ingesting in every treatment, but its highest grazing rates corresponded with limitations to its primary photosynthetic mode. Apart from the newly realized complexity P. antarctica brings to the Southern Ocean food web, it is an environment that suffers from microplastic pollution that can impede these mixotrophic species. Mismanaged plastic waste around the world, especially microfiber discharge from laundered synthetic textiles, escape into the natural environment, and eventually concentrate in the oceans. The Southern Ocean can become disproportionately polluted in regions due to microfibers becoming sequestered once crossing the Antarctic circumpolar current and even becoming trapped in sea-ice formations. While it is easy to see the devastation plastic waste has on megafauna (i.e. turtles, fish, birds, and whales), its microscopic devastation is less obvious. Plastic waste comes in many forms and one less researched form is buoyant polyester microfibers <1mm that interact with colony forming algae. We utilized different concentrations of polyester microfibers and mixing speeds to determine if microfiber interactions with colony formations increases or decreases overall colony buoyancy. Smaller concentrations of polyester microfibers can impart a positively buoyant effect onto P. antarctica colonies regardless of mixing speed, however larger concentrations negatively affected colony buoyancy regardless of mixing speed. / Biology

Environmental heterogeneity in both space and time has significant repercussions for community structure and ecosystem processes. Dimictic lakes provide examples of vertically structured ecosystems that oscillate between stable and mixed thermal layers on a seasonal basis. Vertical patterns in abiotic conditions vary during both states, but with differing degrees of variation. For example, during summer thermal stratification there is high spatial heterogeneity in temperature, nutrients, dissolved oxygen and photosynthetically active radiation. The breakdown of stratification and subsequent mixing of the water column in fall greatly reduces the stability of the water column to a vertical gradient in light. Nutrients and biomass that were otherwise constrained to the depths are also suspended, leading to a boom in productivity. Freshwater lakes are teeming with microbial diversity that responds to the dynamic environment in a seemingly predictable manner. Although such patterns have been well studied for nanoplanktonic phototrophic and heterotrophic populations, less work has been done to integrate the influence of mixotrophic nutrition to the protistan assemblage. Phagotrophy by phytoplankton increases the complexity of nutrient and energy flow due to their dual functioning as producers and consumers. The role of mixotrophs in freshwater planktonic communities also varies depending on the relative balance between taxon-specific utilization of carbon and energy sources that ranges widely between phototrophy and heterotrophy. Therefore, the role of mixotrophy in the microbial food web is difficult to predict because functional types of mixotrophs along a gradient of nutritional strategies contribute differently to nutrient cycling and carbon sequestration. The overall objective of this work was to advance existing knowledge of the abundance and activity of phagotrophy phytoplankton in lacustrine systems. The incorporation of mixotrophy into the microbial food web requires the complement of physiological studies in culture (as described in chapter 2) and quantification of activity (including abundance and bacterivory) in relation to strict phototrophs and heterotrophs in situ (as described in chapter 3 and 4). Information on the physiological ecology of mixotrophic protists is crucial to understanding their role in planktonic food webs and influence on the dynamic microbial community structure in lake ecosystems. An understanding of the ecological functioning of lakes has ultimate consequences for management of water resources, particularly in the face of global climate change. / Biology

Polar regions impose harsh conditions, including low temperatures, and prolonged periods of darkness on resident microbial communities. Despite these challenges, the conditions in these environments can also create opportunities for organisms utilizing combined trophic strategies (Mixotrophy). Only a limited number of studies have identified mixotrophic behavior in polar microbial eukaryotes, and even fewer studies have quantified the response of mixotrophs to likely environmental drivers of trophic behavior (light and nutrients). The goal of this work is to provide an identification of mixotrophic behavior and elucidate of some of the factors that influence algae isolated from polar environments. First, a study of the Arctic prasinophyte, Micromonas pusilla is presented in the first species-specific identification of mixotrophy in a eukaryotic phytoflagellate of this size class. M. pusilla grazed on bacteria under all experimental conditions, responding to nutrient limitation with increased rates of bacterivory. M. pusilla also showed evidence of prey selection. In contrast to the phagotrophic response, photosynthetic production was decreased under low-nutrient conditions. In an additional study of microbial eukaryotes from the Antarctic environment, identification of phagotrophy in photosynthetic nanoflagellates representing multiple evolutionary lineages: Cryptophyceae (Geminigera cryophila) and Prasinophyceae (Pyramimonas tychotreta and Mantoniella antarctica), showed that mixotrophy is more widespread in the Southern ocean that previously thought. G. cryophila and M. antarctica increased ingestions in dark treatments, but did not respond to difference in nutrient concentrations. In contrast, no significant grazing activity was observed in P. tychotreta under high nutrient conditions. When nutrients were reduced, ingestion of bacteria by P. tychotreta was observed and grazing increased in dark as compared to illuminated treatments. Finally, through a series of experimental assays, the competitive advantages of mixotrophic flagellates as opposed to monotrophic specialists were evaluated, using organisms isolated from the Southern Ocean. In these experiments, G. cryophila is emerged as a dominant competitor against two solely autotrophic diatoms (Fragilaria sp. and Fragilariopsis sp.). In contrast, P. tychotreta was outcompeted by the solely heterotrophic chrysophyte Paraphysomonas antarctica. These results show that mixotrophic ability can confer advantages to organisms in some cases, while in other interactions the cost associated with maintenance of multiple trophic strategies results in competitive exclusion by a specialist. These results present novel identification as well as rigorous investigation of mixotrophic behaviors in phototrophic flagellates from both polar (Arctic and Antarctic) environments representing two evolutionary lineages. This work provides a significant contribution to our understanding of the versatile nature of the physiology and trophic ecology of microbial eukaryotic organisms occupying polar marine ecosystems. / Biology

BACKGROUND: Organelle retention is a form of mixotrophy that allows organisms to reap metabolic benefits similar to those of photoautotrophs through capture of algal prey and sequestration of their plastids. Mesodinium rubrum is an abundant and broadly distributed photosynthetic marine ciliate that steals organelles from cryptophyte algae, such as Geminigera cryophila. M. rubrum is unique from most other acquired phototrophs because it also steals a functional nucleus that facilitates genetic control of sequestered plastids and other organelles. We analyzed changes in G. cryophila nuclear gene expression and transcript abundance after its incorporation into the cellular architecture of M. rubrum as an initial step towards understanding this complex system. METHODS: We compared Illumina-generated transcriptomes of the cryptophyte Geminigera cryophila as a free-living cell and as a sequestered nucleus in M. rubrum to identify changes in protein abundance and gene expression. After KEGG annotation, proteins were clustered by functional categories, which were evaluated for over- or under-representation in the sequestered nucleus. Similarly, coding sequences were grouped by KEGG categories/ pathways, which were then evaluated for over- or under-expression via read count strategies. RESULTS: At the time of sampling, the global transcriptome of M. rubrum was dominated (~58-62 %) by transcription from its stolen nucleus. A comparison of transcriptomes from free-living G. cryophila cells to those of the sequestered nucleus revealed a decrease in gene expression and transcript abundance for most functional protein categories within the ciliate. However, genes coding for proteins involved in photosynthesis, oxidative stress reduction, and several other metabolic pathways revealed striking exceptions to this general decline. CONCLUSIONS: Major changes in G. cryophila transcript expression after sequestration by M. rubrum and the ciliate's success as a photoautotroph imply some level of control or gene regulation by the ciliate and at the very least reflect a degree of coordination between host and foreign organelles. Intriguingly, cryptophyte genes involved in protein transport are significantly under-expressed in M. rubrum, implicating a role for the ciliate's endomembrane system in targeting cryptophyte proteins to plastid complexes. Collectively, this initial portrait of an acquired transcriptome within a dynamic and ecologically successful ciliate highlights the remarkable cellular and metabolic chimerism of this system.

Os recifes de coral estão entre os ambientes marinhos mais produtivos e ricos em biodiversidade. Esta biodiversidade está em parte associada a complexas estruturas formadas por corais escleractíneos. Apesar da importância ecológica, social e econômica dos recifes de corais, eles são expostos a várias ameaças relacionadas às atividades humanas. Dentre os impactos antrópicos em recifes, o branqueamento, ou perda de zooxantelas, é o mais notável e é diretamente relacionado à mortalidade dos corais. Por possuírem uma associação simbiótica com essas zooxantelas, alguns corais escleractíneos são considerados mixotróficos, caracterizados por modos de alimentação autotrófico (através de simbiose com o dinoflagelado Symbiodinium) e heterotrófico (predação sobre zooplâncton). Alguns estudos comprovam que corais com maior capacidade de alimentação heterotrófica são mais resistentes ao branqueamento e, consequentemente, às alterações climáticas. A fim de analisar se o coral escleractíneo Mussismilia hispida, é capaz de alternar seu modo nutricional entre predominante autotrófico e predominante heterotróficos, dezoito colônias foram amostradas ao longo de um ano. Marcadores Tróficos de Ácidos Graxos (FATM, na sigla em inglês) foram utilizados para determinar a fonte nutricional de carbono em tecido de corais. A concentração de células de Symbiodinium e a temperatura local também foram avaliadas. Branqueamento foi observado nos meses mais quentes do ano, quando a concentração de Symbiodinium diminuiu, voltando a aumentar nos meses mais frios. O marcador para dieta heterotrófica CGA (C20: 1&#969;9) foi encontrado em amostras de zooplâncton de toda a área de estudo. Em laboratório, colônias sem acesso a zooplâncton apresentaram perda significativa deste marcador após 10 dias. Amostras de colônias naturalmente branqueadas não apresentaram nenhum vestígio dos marcadores de autotrofia SDA (18: 4&#969;3) e DPA (22: 5&#969;3), mas continham tanto CGA e DHA (22: 6&#969;3). Isso confirmou que SDA e DPA são marcadores autotróficos viáveis e CGA é um marcador de heterotrofia. FATM relacionados com autotrofia apresentaram padrão semelhante ao observado para as concentrações de Symbiodinium e foram positivamente correlacionados com a densidade numérica de simbiontes e negativamente com a temperatura. Para explorar os dados de concentração dos FATM, o Índice Trófico de Corais foi desenvolvido para exibir as alternâncias entre modos nutricionais. Mussismilia hispida de fato alterna entre predominância de modo nutritivo ao longo do ano, sendo mais heterotrófica em períodos mais quentes e em condições climáticas adversas, porem na maior parte do ano é predominantemente autotrófica. A validação dos ácidos graxos marcadores tróficos específicos como referência para autotrofia e heterotrofia em corais abre perspectivas para novos estudos em ecologia trófica bêntica em recifes de coral. Este trabalho também inclui o primeiro monitoramento de um ano do comportamento alimentar em um coral hermatípico no Atlântico Sul e o acompanhamento de um evento de branqueamento. / Coral reefs are among the most productive and biodiverse marine environments. This remarkable biodiversity is partly associated to the complex structures formed by scleractinian corals. Despite the ecological, social and economic importance of coral reefs, they are constantly exposed to several threats mainly related to human activities. Climate changes are one of the most notable impacts of human activity related to coral mortality, mainly due to coral bleaching. Some scleractinian corals are proved to be mixotrophs, displaying both autotrophic (through Symbiodinium) and heterotrophic (predation on zooplankton) nutrition modes. Many studies emphasize that corals with greater capability of heterotrophic feeding are more resilient to bleaching and consequently to climate change. In order to analyze whether the endemic scleractinian coral Mussismilia hispida is capable of shifting from predominant autotrophic and predominant heterotrophic in Ubatuba-SP, 18 colonies were sampled monthly for 12 months. The Fatty Acid Trophic Markers (FATM) approach was used to determine the source of carbon on coral tissues. Symbiodinium cell density and local seawater temperature were also assessed. A mild bleaching was observed showing a decrease in Symbiodinium numerical density during warmer months, but increasing in colder months. Reference samples validated the relation between all selected FATM and its corresponding nutritional mode. The heterotrophic feeding marker CGA (C20:1&#969;9) was found in zooplankton samples collected throughout the study area. Laboratory starved colonies (no access to zooplankton) lost any trace of this marker after 10. Samples from naturally bleached colonies presented no traces of the autotrophic feeding markers SDA (18:4&#969;3) and DPA (22:5&#969;3), but contained both CGA (C20:1&#969;9) and DHA (22:6&#969;3). These results confirmed that the FATM analyzed where reliable trophic markers. Autotrophic FATM presented a pattern similar to that observed for Symbiodinium concentration in M. hispida tissues and were positively correlated with the symbiont and negatively with temperature. The Coral Trophic Index showed that M. hispida undergoes shifts in nutritional modes along the year, being more heterotrophic in adverse conditions. The validation of specific FATM as proxies for autotrophic and heterotrophic feeding in corals opens new perspectives for further studies in benthic trophic ecology in coral reefs. This work also presents the first yearlong monitoring of the feeding behavior in a hermatypic coral in the South Atlantic and the monitoring of a mild bleaching event.

Os recifes de coral estão entre os ambientes marinhos mais produtivos e ricos em biodiversidade. Esta biodiversidade está em parte associada a complexas estruturas formadas por corais escleractíneos. Apesar da importância ecológica, social e econômica dos recifes de corais, eles são expostos a várias ameaças relacionadas às atividades humanas. Dentre os impactos antrópicos em recifes, o branqueamento, ou perda de zooxantelas, é o mais notável e é diretamente relacionado à mortalidade dos corais. Por possuírem uma associação simbiótica com essas zooxantelas, alguns corais escleractíneos são considerados mixotróficos, caracterizados por modos de alimentação autotrófico (através de simbiose com o dinoflagelado Symbiodinium) e heterotrófico (predação sobre zooplâncton). Alguns estudos comprovam que corais com maior capacidade de alimentação heterotrófica são mais resistentes ao branqueamento e, consequentemente, às alterações climáticas. A fim de analisar se o coral escleractíneo Mussismilia hispida, é capaz de alternar seu modo nutricional entre predominante autotrófico e predominante heterotróficos, dezoito colônias foram amostradas ao longo de um ano. Marcadores Tróficos de Ácidos Graxos (FATM, na sigla em inglês) foram utilizados para determinar a fonte nutricional de carbono em tecido de corais. A concentração de células de Symbiodinium e a temperatura local também foram avaliadas. Branqueamento foi observado nos meses mais quentes do ano, quando a concentração de Symbiodinium diminuiu, voltando a aumentar nos meses mais frios. O marcador para dieta heterotrófica CGA (C20: 1&#969;9) foi encontrado em amostras de zooplâncton de toda a área de estudo. Em laboratório, colônias sem acesso a zooplâncton apresentaram perda significativa deste marcador após 10 dias. Amostras de colônias naturalmente branqueadas não apresentaram nenhum vestígio dos marcadores de autotrofia SDA (18: 4&#969;3) e DPA (22: 5&#969;3), mas continham tanto CGA e DHA (22: 6&#969;3). Isso confirmou que SDA e DPA são marcadores autotróficos viáveis e CGA é um marcador de heterotrofia. FATM relacionados com autotrofia apresentaram padrão semelhante ao observado para as concentrações de Symbiodinium e foram positivamente correlacionados com a densidade numérica de simbiontes e negativamente com a temperatura. Para explorar os dados de concentração dos FATM, o Índice Trófico de Corais foi desenvolvido para exibir as alternâncias entre modos nutricionais. Mussismilia hispida de fato alterna entre predominância de modo nutritivo ao longo do ano, sendo mais heterotrófica em períodos mais quentes e em condições climáticas adversas, porem na maior parte do ano é predominantemente autotrófica. A validação dos ácidos graxos marcadores tróficos específicos como referência para autotrofia e heterotrofia em corais abre perspectivas para novos estudos em ecologia trófica bêntica em recifes de coral. Este trabalho também inclui o primeiro monitoramento de um ano do comportamento alimentar em um coral hermatípico no Atlântico Sul e o acompanhamento de um evento de branqueamento. / Coral reefs are among the most productive and biodiverse marine environments. This remarkable biodiversity is partly associated to the complex structures formed by scleractinian corals. Despite the ecological, social and economic importance of coral reefs, they are constantly exposed to several threats mainly related to human activities. Climate changes are one of the most notable impacts of human activity related to coral mortality, mainly due to coral bleaching. Some scleractinian corals are proved to be mixotrophs, displaying both autotrophic (through Symbiodinium) and heterotrophic (predation on zooplankton) nutrition modes. Many studies emphasize that corals with greater capability of heterotrophic feeding are more resilient to bleaching and consequently to climate change. In order to analyze whether the endemic scleractinian coral Mussismilia hispida is capable of shifting from predominant autotrophic and predominant heterotrophic in Ubatuba-SP, 18 colonies were sampled monthly for 12 months. The Fatty Acid Trophic Markers (FATM) approach was used to determine the source of carbon on coral tissues. Symbiodinium cell density and local seawater temperature were also assessed. A mild bleaching was observed showing a decrease in Symbiodinium numerical density during warmer months, but increasing in colder months. Reference samples validated the relation between all selected FATM and its corresponding nutritional mode. The heterotrophic feeding marker CGA (C20:1&#969;9) was found in zooplankton samples collected throughout the study area. Laboratory starved colonies (no access to zooplankton) lost any trace of this marker after 10. Samples from naturally bleached colonies presented no traces of the autotrophic feeding markers SDA (18:4&#969;3) and DPA (22:5&#969;3), but contained both CGA (C20:1&#969;9) and DHA (22:6&#969;3). These results confirmed that the FATM analyzed where reliable trophic markers. Autotrophic FATM presented a pattern similar to that observed for Symbiodinium concentration in M. hispida tissues and were positively correlated with the symbiont and negatively with temperature. The Coral Trophic Index showed that M. hispida undergoes shifts in nutritional modes along the year, being more heterotrophic in adverse conditions. The validation of specific FATM as proxies for autotrophic and heterotrophic feeding in corals opens new perspectives for further studies in benthic trophic ecology in coral reefs. This work also presents the first yearlong monitoring of the feeding behavior in a hermatypic coral in the South Atlantic and the monitoring of a mild bleaching event.

Les espèces protistes ont été traditionnellement classifiées comme des plantes ou des animaux en raison de l’absence ou présence des chloroplastes. L’état actuel de la connaissance indique qu’un grand nombre d’espèces protistes portent des chloroplastes mais que physiologiquement elles sont capables d’utiliser l’autotrophie (photosynthèse) ou l’hétérotrophie pour se nourrir. La combinaison de ces deux modes trophiques par une même cellule est nommée mixotrophie. Chez les protistes l’hétérotrophie peut s’effectuer soit par la consommation des particules par phagocytose, e.g. des proies bactériennes, ou bien par l’absorption des composants organiques dissouts, i.e. osmotrophie. La mixotrophie est de plus en plus décrit chez les protistes dans tous les habitats aquatiques. Les écologistes du plancton constatent la récurrence de la mixotrophie chez les formes traditionnelles « phyto»plancton et micro »zoo »plancton. Cependant, identifier et quantifier la mixotrophie reste toujours un défi méthodologique. Dans cette étude nous nous sommes intéressés à la mixotrophie chez les espèces phytoplanctoniques marines, en particulier à leur nutrition phagotrophique de proies bactériennes. Nous avons testé des techniques modernes afin d’identifier la mixotrophie dans des cellules phytoplanctoniques. La technique cytogénétique d’hybridation in situ Card-FISH en utilisant de sondes d’ARN ribosomique 16S a été effectuée suivant des protocoles existant pour des bactéries et des protistes. Cette technique s’est avérée être un outil précieux pour visualiser des groupes phylogénétiques bactériens en association avec le phytoplancton à l’aide de la microscopie à épifluorescence, sans avoir besoin d'un isolement préalable des cellules ou des interférences avec l'association microbienne. Cependant, la méthode a échoué pour visualiser mixotrophie chez le phytoplancton car la sonde eubactérienne générale(EUB338) combine une large gamme d'espèces phytoplanctoniques, ce qui rend impossible de discriminer les signaux fluorescents provenant de tissus bactérienne ou phytoplanctonique. Le contexte de ces études est le phytoplancton et les bactéries hétérotrophes lesquels constituent des principaux concurrents pour les nutriments inorganiques dissouts. Dans le cas où la croissance bactérienne est limitée par le carbone, l'augmentation de la concentration de carbone organique dissous(DOC) renforce la croissance bactérienne et la consommation de nutriments dissous et ainsi affecte négativement la croissance du phytoplancton autotrophe. Cependant, les consommateurs de bactéries, i.e.phytoflagellés mixotrophes, peuvent être favorisés dans de telles situations car la hausse de DOC donne lieu à l'abondance plus élevé des proies bactériennes.En outre, nos résultats indiquent un potentiel effet positif de la température sur le mode de nutrition hétérotrophe de l’espèce, ainsi qu’une croissante contribution des espèces mixotrophes au sein des communautés de phytoplancton dans des conditions des hautes températures des eaux de surface de la mer. / Protist species were traditionally classified morphologically as either „plants“ or „animals“, based on the absence or presence of chloroplasts. State of science is that a high number of protist species carrychloroplasts but are nutritionally able to employ both autotrophy (photosynthesis) and heterotrophywithin a single cell. This combination of autotrophic and heterotrophic mode of nutrition within a single species is named mixotrophy. In protists, heterotrophy can be realized either by the uptake of food particles (e.g. bacterial prey) through phagocytosis or by the uptake of dissolved organic compounds (i.e.osmotrophy). Mixotrophy is globally and increasingly described in protists from all types of aquatic habitats. Plankton ecologists nowadays assess mixotrophy among the traditionally typified “phyto”plankton and mikro”zoo”plankton species as regularity. Nevertheless, detection and quantification of mixotrophy is still a methodological challenge. In this study, we focused on mixotrophy in marine phytoplankton species and put emphasis on its phagotrophic nutrition from heterotrophic bacterial prey. State of the art methodology was tested to visualize mixotrophy in single phytoplankton cells. Catalyzedreported deposition-fluorescence in situ hybridization (Card-FISH), using 16S ribosomal RNA probes,was employed based on existing protocols for bacteria and protists. The method proved to be a valuable tool to visualise bacterial phylogenetic groups in association with phytoplankton by epifluorescence microscopy without need for prior isolation of cells or interference with the microbial association.However, the method failed to visualize mixotrophy in phytoplankton since the general eubacterial probe(EUB338) hybridised a broad range of phytoplankton species making it impossible to discriminate fluorescent signals originating from bacterial or phytoplankton tissue. Background of these studies is phytoplankton and heterotrophic bacteria being major competitors for dissolved inorganic nutrients. In case that bacterial growth is carbon limited, increasing concentrations of degradable dissolved organic carbon (DOC) enhance bacterial growth and consumption of dissolved nutrients and there by negatively affect autotrophic phytoplankton growth. Bacteria consuming mixotrophic phytoflagellates, however, may gain in importance in such situations since DOC provokes higher bacterial prey supply.In addition, our results indicate a potential positive effect of temperature on O. minima´s heterotrophic nutrition mode, and indicate a potential increasing contribution of mixotrophic species to phytoplankton communities under increasing sea surface water temperatures.

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No. of bitstreams: 1 TeseAEM.pdf: 3699711 bytes, checksum: 3b2fdf705faaa9314eeacec123aa76fd (MD5) Previous issue date: 2017-06-09 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / In aquatic environments, phytoplankton consists mostly of photosynthetic microorganisms that serve as the basis of food chains. However, besides photoautotrophy, it is widely reported in the literature that many microalgae can take up dissolved organic matter present in the environment concomitantly with the photosynthesis, a metabolic pathway known as mixotrophy. Little is known about the ecophysiology of mixotrophy in microalgae, and almost all studies are focused on the use of this metabolic pathway to increase the production of algal biomass and stimulate the production of specific biomolecules. Another important issue, considering the current anthropic activity, is that most of the contaminants eliminated in aquatic environments, such as metals and nanoparticles, affect the phytoplankton. However, so far, no ecotoxicological study involving mixotrophic metabolism was found in the literature. To better understand mixotrophy in microalgae, this work chose the chlorophycean freshwater Chlorella sorokiniana as test organism. We divided the study into two parts: the first focused on the physiological/biotechnological interest through the study of growth, photosynthetic parameters, changes in cellular volume, and production of biomolecules (proteins, carbohydrates and lipids); the second part focused on the ecotoxicological effects of cadmium (Cd) and titanium dioxide nanoparticles (NPs-TiO2). To stimulate mixotrophy, glucose (1.0 g.L-1 or 5 x 10-3 mol.L-1) was used as the organic carbon source. The results showed that during mixotrophy, the microalga C. sorokiniana presented higher population growth and production of biomolecules, such as chlorophyll a and lipids, when compared to photoautotrophic cultures. It was also observed that the photosynthetic parameters were affected by mixotrophy, although they did not interfere in the growth of the microalga, and that the presence of bacteria in the cultures acted as a stimulant factor in the production of algal biomass. Regarding the ecotoxicological effects of contaminants, microalgae in mixotrophy were more resistant to both Cd and NPs-TiO2 than those in photoautotrophy, but with changes in the biochemical composition what can affected the energy transfer in the environment. In general, we can conclude that mixotrophy should be considered in studies with phytoplankton, since aquatic environments present a myriad of organic carbon that can be used by these microorganisms. As general conclusions, we can mention that organic carbon acted as an extra source of structural carbon and energy for microalgae, not necessarily relying solely on photosynthesis to survive, so stimulating the growth and production of biomolecules of biotechnological interest, and increased cellular viability in environments contaminated with metals and nanoparticles. This study is a contribution to the understanding of mixotrophy and photoautotrophy metabolisms in a freshwater Chlorophyta with biotechnological potential. / Nos ambientes aquáticos, o fitoplâncton é formado basicamente de microrganismos fotossintetizantes que servem como base das cadeias alimentares. Entretanto, além da fotoautotrofia, é vastamente citado na literatura que muitas microalgas alimentam-se de matéria orgânica dissolvida presente no ambiente concomitantemente à realização da fotossíntese, uma via metabólica conhecida como mixotrofia. Sabe-se pouco sobre a ecofisiologia em metabolismo mixotrófico nas microalgas, sendo os estudos, em sua quase totalidade, voltados ao uso dessa via metabólica para aumentar a produção de biomassa algal e estimular a produção de biomoléculas específicas. Outra questão importante, considerando a atividade antrópica atual, é que a maioria dos contaminantes eliminados nos ambientes aquáticos, como metais e nanopartículas, são estudados em fitoplâncton sob metabolismo fotoautotrófico, não sendo encontrados trabalhos ecotoxicológicos envolvendo o metabolismo mixotrófico na literatura. Para entender melhor o metabolismo algal em mixotrofia, este trabalho escolheu a microalga Chlorophyta de água doce Chlorella sorokiniana como organismo-teste. Para melhor organizá-lo, foi dividido em duas partes: a primeira focou no interesse fisiológico/biotecnológico através do estudo do crescimento, parâmetros fotossintéticos, volume celular, e produção de biomoléculas (proteínas, carboidratos e lipídeos); a segunda parte focou nos efeitos ecotoxicológicos de cádmio (Cd) e de nanopartículas de dióxido de titânio (NPs-TiO2). Para estimular a mixotrofia, glicose (1.0 g.L-1 ou 5 x 10-3 mol.L-1) foi utilizada como fonte de carbono orgânico. Os resultados mostraram que durante a mixotrofia, a microalga C. sorokiniana apresentou maiores crescimento populacional e produção de biomoléculas, como clorofila a e lipídeos, quando comparada com as culturas em fotoautotrofia. Também foi observado que os parâmetros fotossintéticos foram afetados em mixotrofia, porém não interferindo no crescimento da microalga, e que a presença de bactérias pode ter atuado como fator estimulante na produção de biomassa algal. Em relação aos efeitos ecotoxicológicos dos contaminantes, as microalgas em mixotrofia foram mais resistentes tanto ao Cd quanto às NPs-TiO2 do que em fotoautotrofia, porém com mudanças na composição bioquímica, podendo afetar a transferência de energia nos ecossistemas aquáticos. De modo geral, podemos concluir que a mixotrofia deve ser considerada em estudos com fitoplâncton, visto que os ambientes aquáticos apresentam uma miríade de fontes de carbono orgânico para esses microrganismos. Na mixotrofia, o carbono orgânico funciona como uma fonte extra de carbono estrutural e de energia para as microalgas, não dependendo obrigatoriamente somente da fotossíntese para sobreviver, estimulando o crescimento e produção de biomoléculas de interesse biotecnológico, além de aumentar a viabilidade celular em ambientes contaminados tanto com Cd quanto com NPs-TiO2. Este estudo é uma contribuição ao entendimento dos metabolismos mixotróficos e fotoautotróficos em uma Chlorophyta de água doce com potencial biotecnológico. / CNPq: 302175/2015-6 / FAPESP: 2014/15894-0