Funded projects

Temporal trends of planktonic viruses in an oligotrophic coastal system (DIVAS) Summary Viruses are key players in the functioning of microbial food webs, affect all biogeochemical cycles, can manipulate microbial metabolisms, and each infection has the potential to introduce new genetic information into the cell or progeny viruses, thereby driving genetic remodeling of both host and viral populations. Hence, viruses are considered the largest reservoir of genetic diversity. In recent years, the improvement in methods for counting viral abundance and the use of molecular tools to study their diversity have allowed progress in understanding the ecology and spatial distribution of viruses in the ocean. However, there is a lack of knowledge about the temporal patterns of viral abundance, diversity and the impact on their hosts (virus-host interactions) in the sea. Indeed, to perform these studies, it is necessary the availability of time series where these variables are considered together with other biotic and abiotic environmental parameters. Since 2001, there has been a continuous monthly monitoring of microbial abundances, diversity and biogeochemical functions in the Blanes Bay Microbial Observatory (BBMO, Catalan coast), constituting an ideal time-series for the present proposal. The main goal of the DIVAS project is to detect, within the context of the BBMO time series, the existence of recurrent seasonal and interannual patterns in viral abundance, viral diversity and virus-host interactions. Furthermore, we will investigate how these temporal trends are shaped by biotic (abundance of viruses, prokaryotes and protists, together with their diversity and activity) and abiotic parameters (light, temperature, salinity and nutrients), some of them indicators of climate change. We will also identify specific viruses infecting dominant prokaryotic and picoeukaryotic species using single cell genomics. The relative abundance over time of specific viruses will be investigated in environmental sequencing datasets (metaviromes and metagenomes). The metaviromes (<0.2 μm size fraction) are expected to have free viruses, mostly bacteriophages, while the metagenomes (0.2-3 μm) would include larger free viruses (mostly infecting eukaryotes) and viruses within bacterial and picoeukaryotic cells. Finally, we will evaluate the temporal links of planktonic viruses with abiotic and biotic parameters, how these affect and shape viral abundance community structure and virus-host interactions, and whether there is a hint relating viral ecology and climate change.

Marine eukaryotic protists offer a huge but currently underexploited reservoir of metabolic pathways with biotechnological potential. Given their unique adaptations through symbiosis, endosymbiosis and organelle acquisition, the ecofunctionalities of protists present a hitherto untapped source to discover novel metabolic pathways and bioactivities whilst bearing a high chance of discovering different activities compared to those identified in marine sources, such as bacteria. The scientific approach and rationale sets PROMiSE apart from many previous scientific initiatives exploiting the biotechnological potential of marine bacteria. The PROMiSE experimental workflow enables this by employing a comprehensive set of Omics methods. This approach spans the encoded metabolic potential to identify biosynthetic gene clusters which in turn guide the targeted metabolite profiling, merged with discovery-based metabolomics. The goal is to target identified candidate compound classes and their pathway-related metabolites and conjugations dereplicated from the Omics information. By linking these methods back to the source cell through single cell Omic methods, PROMiSE offers a unique way to recognize functional gene clusters and to understand how metabolism is partitioned across ecosystems. This is important to unravel how the identified pathways work in nature, and by extension, how they can be expressed and utilized for technological adaptations relevant to a human health and biotechnology market. The vertically integrated extraction and analyses procedure within PROMiSE are supported by a comprehensive array of cutting-edge in vitro and in vivo bioassays for reliably assessing biological activities by High-Content profiling and antibacterial screening. Analytical chemistry, including high resolution mass spectroscopy and nuclear magnetic resonance spetroscopy approaches, will be used to elucidate compounds found in the bioactive fractions, which will tie back the molecular data to identify relevant enzymes, pathways, and compounds.

Environmental microbial surveys have revealed a remarkable diversity of microeukaryotic life in most ecosystems, the majority of which had previously escaped detection. From an ecological point of view this work highlighted our ignorance of critical microbial players in natural environmental processes, including primary production, biogeochemical cycling and trophic interactions such as parasitism and grazing. Consequently, our understanding of community function is partial, limiting our ability to study environmental change. While, from an evolutionary perspective, we are missing major components of the Tree of Life giving rise to a fragmented understanding of how major cellular functions have evolved. Single cell genomics (SCG), including single cell transcriptomics, is an emerging technology that has the potential to retrieve genomic information from individual uncultured microbes recovered directly from natural environments and promises to provide new tools to investigate microeukaryotes in unparalleled detail. The aim of this ITN is therefore to train a new generation of scientists with the highest expertise in SCG, from the initial stages of cell sorting to genome sequencing and gene annotation, to the full exploitation of the data obtained. Such progress will allow the European research community for the first time to address critical ecological and evolutionary questions. SINGEK will drive training through research by both local and network-wide activities, secondments, and workshops, and by establishing an environment that extends far beyond each partner team. This training environment will also provide the transferable skills essential for successful career development. This network of well connected and highly qualified scientists with expertise in eukaryotic SCG will be ready to implement this technology beyond ecology and evolution to other fields such as biomedicine or biotechnology driving innovation across the EU.

The perception that marine microbial life is extremely diverse is well grounded based upon molecular diversity surveys and the existence of a wide variety of cultured forms. This also applies to the unpigmented smallest eukaryotes in planktonic systems, the heterotrophic flagellates (HFs), which form a diverse collection of tiny flagellated cells that are important agents in prokaryotic mortality through grazing and crucial in nutrient remineralization. HFs came to light a few decades ago, when they were included in marine food webs models as the trophic link between prokaryotes and larger protists like ciliates or dinoflagellates. However, HF cells have received relatively little attention in oceanographic efforts developed up to now, due to two main factors: (1) the lack of automatized counting procedures since they are not easily quantifiable by flow cytometry and (2) a particularly severe culturing bias, by which they seem to be composed by a myriad of novel and uncultured species. As a result, further research is needed to explore which are the dominant species forming HF assemblages, and go beyond the only fragmented information currently available based in scattered molecular surveys. The ALLFLAGS project aims to take advantage of new analytic tools and recent extensive sampling datasets to perform, for the first time, a global study to identify the dominant HF species and better define their ecological relevance in the marine environment. As novel tools, we will develop an automatized microscopy routine for counting HF cells and fully exploit the potential of High-Throughput Sequencing (HTS), together with new bioinformatic developments for diversity studies. Afterwards, we will apply these tools to quantify HF assemblages and identify the dominant species in recently available sampling surveys. In particular, we will process the dataset obtained in a global sampling effort done in the major oceans during the Malaspina expedition, and the samples from a long-term temporal survey performed at the Blanes Bay Microbial Observatory (BBMO). Once detected the dominant HF species in these two extensive surveys, we will undertake a detailed analysis of their distribution and abundance, including the definition of their genetic variability related to the environmental context, and the establishment of their relative activity measuring specific grazing rates. Finally, we will perform comparative genomics to identify the gene basis for ecological adaptation of selected dominant HF species using already published genomes and those obtained in novel metagenomes constructed here. This analysis will be based on the detection of gene families in all genomes and placing a particular focus on viral signatures as proxies of mortality susceptibility. The use of this genomic-based approach to its full potential will allow a better understanding of HFs role in the global marine ecosystem and biogeochemical cycles, as well as to elucidate the question of whether or not cultured and uncultured HF species can be differentiated based on their gene content. The confluence of HTS surveys and extensive datasets opens the possibility for the first time to develop a global assessment of HF diversity, the identification of the dominant HF species, and the search for their ecological niche.

REMEI es un proyecto diseñado para comprender el potencial funcional de las comunidades microbianas y los factores que lo regulan. Combinaremos (a) la secuenciación del contenido genómico de una comunidad procariota costera (metagenoma, metaG) en coordinación con una descripción detallada de los ciclos biogeoquímicos en el ambiente de estudio, y (b) una serie de experimentos diseñados para entender los factores biológicos que regulan la presencia de cada función procariota. En (a) se espera entender cuáles son los factores oceanográficos y de estacionalidad que regulan la presencia de los diferentes genes y en (b), con la eliminación experimental controlada de la presión de depredación, los efectos de los virus, o la limitación por nutrientes y por carbono, esperamos entender cómo estos diferentes factores biológicos contribuyen a la regulación. Nuestros datos proporcionarán a) un catálogo completo de los genes del océano costero en el Mediterráneo noroccidental (a añadir a los análisis de una sola vez obtenidos mediante iniciativas como el Ocean Sampling Day o TaraOceans, b) la información obtenida experimentalmente ayudará en la interpretación de los numerosos otros estudios publicados recientemente de metagenomas marinos, o que están en curso, y que son trabajos primordialmente descriptivos. Nuestro objetivo es la generación de una base de datos de los genes asociados a una lista de atributos (por ejemplo, un gen pufM particular aparece en este momento particular del año y con estas condiciones oceánicas, y su aparición es reprimida, por ejemplo, por depredadores), Análisis filogenéticos de las muestras (de los fragmentos de 16S extraídos de los metaG) podría permitir la asociación de genes filogenéticamente no informativos a los probables organismos a los que pertenecen y con los análisis de metaG, en teoría sin sesgo, tendremos la oportunidad de estimar el crecimiento verdadero y las tasas de crecimiento brutas de procariotas a nivel de OTU, y también de genes funcionales. Sin embargo, también vamos a aislar procariotas con medios de cultivo innovadores que podrían servir como base para la comprensión de los vínculos entre la filogenia y la función y también vamos a analizar algunos SCG (genomas de células individuales) de organismos separados por citometría de flujo. Las fortalezas y originalidad del proyecto REMEI provienen del uso combinado de los metagenomas de referencia con la descripción estacional de los principales ciclos biogeoquímicos; y, sobre todo, ii) del uso de manipulaciones experimentales para determinar el papel de los virus, los depredadores y la limitación de nutrientes sobre cada una de las unidades taxonómicas de procariotas presentes en la comunidad (a través de los marcadores de 16S rDNA), y para cada uno de los genes identificados en la conjunto de la comunidad.

Biological communities are systems (ecosystems) composed of many interacting parts (species). Macro ecologists have advanced substantially our understanding of ecosystems involving mainly animals and plants, generating models where species and their ecological interactions are understood as networks, which have specific characteristics and architectures that can influence ecosystem functioning. In comparison, our knowledge of microbial interaction networks is rudimentary, and in most community studies, microbes are still pooled by their function (e.g. grazers), thus missing their species-specific interactions. This represents a major knowledge gap, as microbes are key players in almost all ecosystems, particularly in the oceans, and without comprehending their interactions we cannot increase our understanding of the functioning of the biosphere, which is particularly needed in a context of global change. The reason for the current state-of-affairs is that understanding microbial interactions (and diversity) has proven to be highly challenging. However, recent technological advance in Single-Cell genomics, High-Throughput sequencing, and High-Performance computing makes now feasible to capture the most important microbes of a given community and determine their ecological interactions. Therefore, in this project we propose to focus in a model marine microbial community aiming to a) predict important ecological interactions using association networks, b) test whether predicted interactions exist and determine interaction type (e.g. symbiosis, predation, parasitism) using Single Cell genomics, c) test if other predicted interactions can be considered as metabolic cooperation using metagenomics and metatranscriptomics and d), determine the ubiquity of selected significant interactions in the global ocean using existing molecular data from large marine expeditions (Malaspina and Tara Oceans). Overall, the main results of this project will be: a) identification of the most important ecological interactions within the studied microbial community in a temporal context, b) determination of core metabolic interactions c) assembly of a comprehensive network for this model marine site, analyzing its main characteristics and d) identification of the ubiquity of core microbial interactions in the global ocean. Overall, the knowledge produced by this project will move forward the research frontier in microbial ecology by determining pairwise species-specific ecological interactions.

Life on Earth is mostly microscopic. The advent of molecular tools in the last decades has completely transformed our world-view of biodiversity, and now we know that the contribution of visible life to biodiversity is indeed small. Furthermore, microbes have tremendous metabolic diversity and they are the engines that drive Earths’s biogeochemical cycles. Given that the marine environment is the largest ecosystem on Earth, the ecological function of bacteria inhabiting this system is essential for life in our planet. Bacterial communities are composed by few dominant species and a large number of species represented only by a few individuals. These rare species were until recently invisible, yet they constitute a vast repository of functional and phylogenetic diversity. For this reason the study of the mechanisms that allow the maintenance of the “rare biosphere” and how they contribute to the functioning of microbially dominated ecosystems has become the focus of intense research in the last years. Rare bacteria were originally thought to be dead or dormant, but it is increasingly believed they have important ecological roles such as stabilizing ecosystem processes after disturbance or maintaining critical biogeochemical functions. In this proposal we will combine flow cytometry, high-throughput sequencing, and “omics” techniques with experimental microbiology to provide an integrative view of the physiological status of rare bacteria and their potential contribution to ecosystem functioning.

Oceanic life plays a major role in global biogeochemical cycles and the oceans act as sink for atmospheric CO2. How an ecosystem influences the carbon cycle depends on its balance between primary production and respiration. Primary producers convert inorganic carbon into their biomass, thereby removing CO2 from the atmosphere. Conversely, consumers take up organic material to fuel their respiration and release CO2 back into the environment. The two key processes in this cycle, primary production and consumption of organic material, are usually considered as being performed by different groups of organisms, autotrophs and heterotrophs. However, some organisms, the so-called mixotrophs can perform both of these processes simultaneously and hence, can have opposing effects on the carbon cycle depending on their balance between autotrophic and heterotrophic nutrition. Mixotrophic eukaryotes have recently been found as both abundant and important for key ecosystem processes in the ocean. However, the basic biology of mixotrophs is not well understood, particularly the environmental drivers that influence their nutritional balance. The goal of this project is to gain a mechanistic understanding of the role mixotrophs play in the marine carbon cycle. To achieve this, mixotrophs will be studied at all relevant organizational levels, ranging from gene expression patterns at the molecular level to the diversity and activity patterns on the ecosystem scale. In particular (i) a molecular study using transcriptome and proteome analysis will elucidate the metabolic integration of their nutritional pathways, (ii) their contribution to biogeochemical cycles will be quantified by NanoSIMS technology, and (iii) ecological factors controlling their distribution and diversity in the ocean will be assessed. This multidisciplinary approach will provide the foundation necessary for linking molecular processes to the contribution of mixotrophs to the marine carbon cycle.

Coral reefs are home to the greatest density of species in shallow marine waters, including unusually diverse microbial communities. However, almost everything we know about reef microbial communities is confined to prokaryotes and viruses: next to nothing is known about the microbial eukaryotes, with the exception of the symbiont dinoflagellate Symbiodinium. Coral reefs are like most other marine environments, where protists are consistently overlooked. Chromera and Vitrella are two reef-associated algae assumed to have a symbiotic relationship similar to that of Symbiodinium. As photosynthetic relatives of apicomplexan parasites, they held answers to long-debated questions about plastid evolution, but equally important questions about their functional relationship to corals and the reef community have hardly been asked. It has been observed that bacterial sequence surveys are heavily ‘contaminated’ with eukaryotic plastid sequences. The apicomplexanrelated lineages (ARLs) are the richest source of still-unidentified plastid diversity, and virtually all of this diversity is restricted to coral reefs. We know they exist, but have no direct information on their biology or role in ecosystems whatsoever. ARL-V is the most common lineage, but we cannot even say whether the organisms are photosynthetic symbionts or intracellular parasites. To address these questions, an ecological approach was needed.

The Role of Viruses in Structuring the Marine Environment in the Red Sea The Red Sea is a largel underexplored ocean and often the most basic information is missing. It is the world's northernmost tropical sea featuring distinct environmental conditions along its latitudinal spread that give rise to a multitude of distinct ecosystems, e.g. extensive shallow-water coral reefs, deep-sea hydrothermal brine pools, cold seeps, and deep-sea corals. To acquire missing knowledge that is critical for a basic understanding of the functioning of Red Sea ecosystems, the RSRC proposed two broad themes in its 5-year funding request as well as a third applied theme: a) Biodiversity and resilience of Red Sea ecosystems b) Nutrients origin and fate in the Red Sea c) Environmental protection and sustainable use. These research themes hold diverse and current areas of research efforts in the RSRC, but did not incorporate the study of viruses in the Red Sea. Viruses are by far the most abundant organisms in the oceans that influence the composition of marine communities and are a major force behind biogeochemical cycles. Further, viruses are reservoirs of genetic diversity in the marine environment and have the potential to introduce new genetic information into an organisms, therefore, playing a role in adaptive evolution and resilience of organisms to environmental changes. In summary, viruses are hugely important in structuring the marine environment, but are so far unaddressed in research efforts in the RSRC.

Most of the biodiversity in oceans is constituted by microbes which dominate the biomass and have key roles in ecosystem functioning and biogeochemical cycling. Recent culture-independent studies of marine planktonic protists have unveiled a large diversity at all phylogenetic scale, notably among the heterotrophic microeukaryotes compartment (2-5 µm cell size), and the existence of novel groups. Among these novel uncultured lineages, Marine Stramenopiles groups (MASTs) are essentially composed of small flagellated eukaryotes (<3 µm) widely distributed in marine systems. These flagellated organisms are recognized to play essential roles for the functioning of marine ecosystems as they channel large amounts of organic carbon to the upper trophic levels and control the population sizes of bacteria and archaea. During the last decade, the microbial diversity and the distribution of the twelve lineages which composed this new stramenopiles are already started, however, little information on their quantitative importance and activity in ecosystems functioning are already acquired. Here, we proposed to i) evaluate the dynamics of the diversity and abundance of these uncultured flagellates in marine environments and ii) investigate their ecological significance in oceans through the study of their grazing activity and the analysis of their general genomic structure. In this proposal, the dynamics, genomics and role of six constitutive lineages of these stramenopiles (MAST-1, -2, -3, -4, -7 and -8) will be studied by the use of new state-of-art techniques (i.e. 454- and Illumina pyrosequencing, Whole Genome Amplification after flow cytometry cell-sorting) and several standard ones (i.e. grazing experiments, TSA-FISH), applied together on an unique set of environmental samples from a circumglobal navigation providing samples with a wide geographical coverage and from a temporal survey based on a coastal oligotrophic station.

The new concept for the Microbial Carbon Pump (MCP) postulates mechanisms by which refractory organic matter is produced in the oceans through microbial activities, and the role that this refractory pool plays in carbon sequestration in marine systems. This postulate evokes the need to increase our understanding of organic matter transformation, particularly for the processes of organic carbon degradation. In this context, key questions about the drivers that govern these processes have been posed and require scientific answers. DO-RE-MI will examine the biological and biogeochemical mechanisms that hinder total DOC remineralisation in marine systems.

We will apply a multidisciplinary approach to the design of experiments, the field program and the analysis of the results. Our approach builds upon the strengths of the team members and collaborators who have varied expertise and can bring diverse methodological techniques and tools, and ideas originating in their different disciplines - microbial ecology, biogeochemistry, and physical oceanography. With these varied methodological strategies we will work to achieve the following objectives:

1) To quantify DOM remineralization in contrasting marine systems, 2) To test the effects of nutrient availability and microbial metabolic capability on the efficiency of DOM degradation, 3) To experimentally test whether DOC concentration and DOM chemical diversity can explain the limits of DOM degradation in the deep ocean and 4) to evaluate the role of different bacterial community structures in the degradation processes.

Bacteria (prokaryotes) are a relevant component of the ocean’s food networks; they are responsible for 30% of the primary biomass production (fixing CO2) and 95% of the respiration of the ocean. They are accounting with 1029 cells in the global ocean representing a major player of the microbial world but only few thousands of species has been described so far. Although the concept of bacterial species is continuously transformed and under debate after a decade of comparative genomics, there is an accepted view of bacteria as a pan-genome. This bacterial concept is described by a \"core genome\" containing genes present in all strains, and a \"flexible or adaptive genome\" integrating those genes shared by few or unique to single strains usually constrained in genomic islands. New sequencing technologies have allowed to sequence many genomes from the same species to get a deeper resolution refereed as ‘Pan-Genomics’. This new concept has been mostly restricted to cultured microorganisms and mostly to pathogenic bacteria but the majority of bacteria in the ocean are uncultured and most importantly, are embedded in a network of plankton organisms (microbial loop) that range from viruses to zooplankton. Surprisingly the genetic complexity and interactions among these microorganisms remain largely un-investigated. The objectives of this proposal are: 1) Sequence ecologically relevant and uncultured bacterial genomes through Single Cell Genomics (SCG). 2) Describe the pool of core and adaptive genes of these uncultured genomes 3) Develop a new concept for bacterial “species” (or “units”) in the ocean for uncultured genomes, referred here as microbial ocean pangenomes by SCG. In addition, we will investigate (i) the distribution and genetic interaction of these bacterial ocean Pangenomes with different microbial plankton components (from protist to viruses) and (ii) the evolutionary mechanisms driving speciation. Taking into account that horizontal gene transfer (HGT) is a main diversification mechanism that occurs among distinct bacterial phylogenetic groups and based on the coexistence of bacterial with eukaryotic protist and bacteriophages, the evolutionary processes underlying the genomic patterns and structure within microbial ocean pangenomes may be expanded across domains of life. To successfully develop this proposal Single Amplified Genomes (SAGs) of uncultured and ecologically relevant bacterial genomes of the photic and deep ocean would be analyzed coupled with their corresponding Metagenomes and correlated to other quantitative and oceanographic data collection. Expected outcomes of this proposal are: (i) to develop a new concept of bacterial “unit” refereed as microbial ocean pangenomes in the ocean by SCG, (ii) to provide reference genomes for a meaningful interpretation of metagenome and metratranscriptome at population and community level and (iii) a unique framework to correlate microbial ocean pangenomes with eco-physicochemical parameters and other fractions of the microbial food web. The results of this proposal will shed light on the bacterial species concept, biogeography, ecological functioning and evolutionary mechanisms driving bacterial diversity in the oceans.

BioMarKs integrates 8 EU research institutes and 30 EU experts in eukaryotic microbial taxonomy and evolution, marine biology and ecology, genomics and molecular biology, bioinformatics, as well as marine economy and policy, to assess the taxonomic depth, environmental significance, human health and economical implications of arguably the least explored biodiversity compartment in the biosphere: the unicellular eukaryotes or protists.
Marine protists typically live in huge populations with rapid turnover. They may build complex (in)organic skeletal structures which profoundly impact biogeochemical cycles and climate; they have complex genomes with thousands of genes producing molecules which influence marine ecosystem functioning, human health and economy, and which represent outstanding potential for future green energies, pharmaceutics and cosmetics. Based on phenotypic data, marine protists comprise <200k “species”. However, exploration of ribosomal (r) DNA clone libraries over the last decade has revealed ever-increasing biodiversity in both novel lineages and groups which were believed to be species-poor.
BioMarKs will reassess coastal marine protist biodiversity using massive rDNA sequencing integrated into a network of taxonomic expertise and comprehensive contextual phenotypic and environmental metadata. 454-pyrosequencing technology permits acquisition of several hundred thousand sequence tags in a single run, providing the prospect, for the first time, of conducting nearly-exhaustive surveys of microbial diversity and population dynamics. In collaboration with GENOSCOPE (France), we propose to use 45X 454-runs (18 to 45 million sequence-tags) to assess protist biodiversity at 3 depths (subsurface, deep-chlorophyll maximum, surface sediment) in 9 EU coastal water sites from Spitzbergen to the Black Sea.
This general strategy will be used to (i) establish a baseline of protist biodiversity in EU coastal waters, (ii) measure biodiversity change in marine protist communities facing ocean acidification, (iii) evaluate the impact of ballast water and pollution on marine protist biodiversity. In addition to significantly enhancing our basic knowledge of eukaryote biodiversity and ecology (Who? How Many? When? Where? Why?), BioMarKs will provide baseline data and new methods for future surveys of marine biodiversity change and for evaluation of its ecological and economic cost.

In microbial ecology, the biomass, activity and ecological role of a given microbial assemblage is often addressed as a bulk property, obviating the fact that these assemblages are formed by distinct species with diverse evolutionary histories, cellular traits and metabolic capacities. The main reason for this is that microorganisms are morphologically similar and often impossible to identify without molecular tools, which use in microbial ecology is relatively recent. One of such microbial assemblages in aquatic systems are the "heterotrophic flagellates", a consortium of tiny unpigmented eukaryotic cells that play critical roles as microbial grazers (controlling prokaryotic abundance) and nutrient remineralizers. The biomass and grazing impact of heterotrophic flagellates in marine systems is relatively well established, but their phylogenetic and functional diversity is still poorly known. Therefore, the natural following step in the research of this microbial consortium is moving on from their bulk properties to the investigation of the different component populations. In this project we propose to study the population ecology of three model species of heterotrophic flagellates that represent different scenarios with respect to culturability and dominance in the marine plankton. These model taxa are Cafeteria roenbergensis (culturable and low in situ abundance), MAST-4 (unculturable and high in situ abundance), and Minorisa minuta , recently isolated in our laboratory (culturable and moderate in situ abundance). Our approach consists in a sequence of steps. First, we will study the physiological properties of these model organisms, such as growth and grazing rates, numerical and functional responses, and prey selectivity. Second, we will develop molecular tools (FISH and q-PCR) to study their distribution and abundance in the marine environment, taking advantage of the Malaspina cruise (which provides samples with a wide geographical coverage) and the Blanes Bay Microbial Observatory (a well-characterized temporal sampling site). Third, we will select a reduced number of samples to study the population genetic structure of one of the taxa (MAST-4) using molecular markers and high-throughput pyrosequencing. Fourth, we will aim at isolating specific viruses for the two cultured taxa and study viral genomics, estimate viral abundances in the sea and infer the viral impact on natural populations of flagellates. Fifth, we will take the preliminary steps in genome projects of the cultured species. The results generated will shed light into the processes leading to the adaptation and success of each taxa to the marine environment and will serve to start understanding heterotrophic flagellates in nature. Focusing on the population ecology of a few model species provides a unique possibility of going one step further in disentangling the structure and function of microbial assemblages in nature.

Organic carbon (OC) in the marine coastal pelagic environment originates from a variety of sources, some are autochthonous such as primary production, and some are allochthonous, such as sediment resuspension, river and groundwater run-off, and atmospheric deposition. It is well known that ecosystem metabolism depends, to a large extent, on the origins of the organic C processed in the coastal ocean. The relevance of different OC sources to coastal function, however, derive not only from the magnitude, but also from OC structure. OC from different sources appears in different “structure” or “architecture” (dissolved, particulate, in different types of particles…). Unappreciated until recently, the structure of the OC has an impact on the ecology of the coastal environment, by generating microenvironments where numerous biogeochemical processes take place. Microenvironmental structure also promotes biological diversity by increasing the number of niches available for microbial growth. The origin of the OC in the coastal ocean, and its inherent structures, control system-wide biogeochemical cycling and ecology. STORM will study this physical and microbial structure by, first describing the relative importance of sources of OC understudied in the marine environment, such as atmospheric deposition and resuspension. Sampling will be done at two coastal Mediterranean stations that are well known (Blanes Bay Microbial Observatory) or that have in situ instrumentation (Barcelona Littoral Station) to evaluate OC dynamics. We will systematically analyze the structure of the OC pool with a suite of microscopical and molecular biology approaches to describe the seasonality in the structure of the dominant OC pool, the microbes associated with this structure, and the microbial hotspots from a biogeochemical point of view. Microbial function will be investigated by means of qPCR and genetic libraries of functional genes relevant in the cycling of P, N, C, and in light use. Other components of STORM will focus on the fluxes between types of OC, and on environmental factors affecting structure and fluxes. STORM will examine the factors and processes that control microbial structuring of organic matter in seawater and how structure and microbes interact physically in the processes that determine OC fate. Biogeochemical C cycling in the coastal ocean is largely driven by microbes, and C cycling has profound effects on the final fate of both coastal primary production and C inputs. Furthermore, C cycling is expected to vary as a consequence of Global Climate Change. Gaining of knowledge about the structure and dynamical cycling of OC will facilitate forecasting and modelling of future biogeochemical scenarios.

Esta acción rentabilizaría el esfuerzo que se viene invirtiendo en la serie oceánica de l’Estartit. La serie cuenta con datos de temperatura desde 1973, y constituye la serie oceánica más larga e ininterrumpida del Mediterráneo Noroccidental. Desde el año 2008, investigadores del proyecto ACDC muestrean agua para analizar pH, alcalinidad y carbono inorgánico. En esta propuesta se solicita una pequeña ayuda económica para estudiar el compartimento de carbono orgánico y poder examinar con más precisión la capacidad tamponadora de las aguas mediterráneas así como la variabilidad de los microorganismos en relación con la tendencia a la acidificación y calentamiento.

Gracias a los microorganismos planctónicos que viven en los primeros 200 m de la superficie del mar, el 50% de la producción global de CO2 a la Tierra es absorbida por el océano. De este porcentaje, un 30% es absorbido por microorganismos de origen bacteriano. Si se tiene en cuenta que las bacterias marinas son además responsables del 95% de la respiración del océano, está claro que estos microorganismos son imprescindibles para mantener en equilibrio de la atmósfera del planeta.

Entre 2009 y 2012 se lleva a cabo la expedición científica TARA OCEANS a bordo del TARA, un velero de 36 metros de longitud, que permitirá a mas de un centenar de científicos explorar la diversidad microbiana en los océanos de todo el planeta y su sensibilidad al cambio climático, un cambio que está sucediendo a una velocidad 100 veces más rápida del que se haya documentado previamente por estudios geológicos.

Esta expedición científica, de tres años de duración, es una oportunidad única de circunnavegación global de los océanos en el cual científicos expertos en biología, ecología, oceanografía, física, genómica y bioinformática tendrán la oportunidad de analizar los organismos que viven en el plancton marino como un ecosistema único, desde larvas de peces hasta microorganismos invisibles al ojo humano (como protistas, bacterias, arqueas y virus) y su relación con el ambiente.

El proyecto TANIT explorará el “mundo bacteriano” estudiando la biogeografía y función de las bacterias marinas en los diferentes océanos del planeta, integrando técnicas de ecología molecular y de genómica. Está previsto recolectar muestras en casi 400 puntos alrededor del planeta y aplicar técnicas genómicas (estudio de metagenomas y metatranscriptomas) para explorar la diversidad genética y funcional de estas comunidades microbianas, para evaluar y posteriormente modelar su adaptación a condiciones climáticas futuras.

ENLACES:

Expedición TARA OCEANS

Las bacterias aeróbicas anoxigénicas fototróficas (AAPs) constituyen un grupo de microorganismos mixotróficos, cuya relevancia en el océano no está del todo clara. Estos organismos contienen bacterioclorofila a (BChla) y aunque no son capaces de fijar CO2, pueden utilizar la luz para generar ATP lo que les aporta una ventaja ecológica frente a otros heterótrofos cuando la disponibilidad de C orgánico es limitada. Por ello, cuando inicialmente se descubrieron se pensó que éstas serian abundantes en ambientes oligotróficos, dónde de hecho pueden representar hasta el 10-20% del total de procariotas. Sin embargo, más tarde se vió que las AAPs también pueden ser abundantes en ambientes eutróficos por lo que no está muy claro cúal es su papel ecológico. Con esta Acción Complementaria hemos obtenido financiación para participar en un proyecto de circunnavegación global del océano (Tara-Oceans) dónde convertiremos un fluorómetro convencional en uno ultrasensible en el infrarojo que permite detectar de forma rápida y sencilla la presencia de AAPs. Este instrumento será utilizado también en el proyecto de circunnavegación Malaspina. Tara-Oceans durante tres años, y Malaspina en 7 meses recorrerán las principales provincias oceánicas. Estas dos campañas oceanográficas nos brindan una oportunidad única de estudiar la dinámica de las AAPs a escala global, en ambientes de distinto nivel trófico nunca estudiados anteriormente, lo que sin duda aportará información de gran valor para entender su papel ecológico en el océano y su impacto en el ciclo del C.

El objetivo de la presente propuesta es financiar el proyecto Bacteriomics englobado dentro de la campaña de circumnavegación global TARA Oceans que colectará muestras en las 375 estaciones previstas durante los 3 años que perdurará la expedición (2009-2012). Bacteriomics se engloba dentro del consorcio de procariotas de TARA Oceans (TANIT) el cual coordino y que explorará la diversidad, biogeografía y función de las comunidades bacterianas a una escala global. Este proyecto se coordinará y se desarrollará en el departamento de Biología Marina y Oceanografía en el Instituto Ciencias del Mar (ICM) uno de los centros del CSIC y pioneros en las investigaciones marinas. Como coordinadora de TANIT, nuestro laboratorio en el ICM ha sido designado como sede oficial del banco de muestras de TANIT de toda la campaña de Tara Oceans. En este proyecto, Bacteriomics, se presenta dos objetivos principales: (i) preservar todas las muestras de TARA Oceans de forma adecuada ya que se dispondrá de unas 10,000 muestras en distintos formatos y desarrollar una base de datos específica para dicha campaña y (ii) analizar la biogeografía de las bacterias marinas a través de técnicas de secuenciación masiva y de ecología microbiana de muestras ya colectadas durante el primer año de expedición (2009) procedentes del Mar Mediterráneo, Mar Rojo, Mar Arábico y Océano Indico. En esta Acción Complementaria se solicita la compra de un arcón congelador imprescindible para preservar este legado y el análisis de dos placas de pirosecuenciación para realizar un análisis preliminar de muestras de TANIT de TARA Oceans. Estos análisis preliminares serán de vital importancia para preservar parte del material para futuras generaciones y para realizar posteriormente el metagenoma/metatranscriptoma de estaciones piloto de TARA Oceans en coordinación con Genoscope a un coste mínimo. Esta propuesta complementa a los proyectos del Plan Nacional GEMMA (CTM2007-63753-C02-01) y MICRODIVERSITY (CGL2008-00762/BOS) .

Microbes dominate the biomass of oceans, having vital roles in the functioning of ecosystems and the biogeochemical cycling. Yet, little is known about the total extent and structuring patterns of their genetic diversity, especially at the species and population levels. Pointing into this research direction, this proposal has two general aims: (a) to go deeper into assessing the extent of picoeukaryotic (2-5μm cell size) diversity in marine environments and (b) to investigate genetic patterns at the species and population levels. The chosen lineage for this study is the MArine STramenopiles -4 (MAST-4), a recently discovered group of heterotrophic picoeukaryotes that can be relative abundant (and therefore important) in marine environments. Two novel molecular techniques are considered for this proposal: (1) Pyrosequencing and (2) Single-Cell-Whole-Genome-Amplification. The use of these new techniques and several standard ones, together with the availability of a unique marine environmental DNA set of samples from a variety of geographic locations around the world, will allow a strong assessment of the extent of the MAST-4 diversity, with the potential to unveil new lineages as well as genetic structuring patterns at the species and population levels. The genetic patterns found at different evolutionary hierarchies will be correlated with environmental data in order get insight into ecological adaptation and evolutionary diversification.

Microbial diversity is a crucial element in our understanding of the evolution and ecology of natural environments but also an important resource for biotechnology and medical research. Molecular and genomic approaches not only have pointed out the vast diversity within microbial communities, but also have revealed the predominance of high level of sequence microheterogeneity “microdiversity” within co-existing bacterioplankton. However, the relevance, extension, functional differentiation and mechanisms responsible for such microdiversity patterns are barely known. For a better understanding of function and speciation in microbial populations in the environment more studies focusing on co-existing bacterial genomes within bacterial populations are needed at different temporal and space scales.
The major goal of this proposal is to shed light on the role and mechanisms of maintenance for microdiversity patterns in environmental microbial populations by exploring the genomic microdiversity of two specific marine bacterial populations (Bacteroidetes y Pseudanabaena) and the evolutionary mechanisms by which this microdiversity is generated and preserved. The innovation of the present project is based on the way to analyze the genomic microdiversity at different ecological contexts by exploring the microdiversity of two bacterial populations that exhibit abundances and different ecological strategies at three levels of resolution: (i) we will explore the genomic microdiversity from two bacterial populations at a global level (i) we will investigate the inherent microdiversity in a specific ecotype and the divergence between two different ecotypes and finally (iii) we will sequence two individual genomes that belong to same ecotype.
Without this knowledge, population biology, biogeography or environmental microbial genomics (metagenomics) cannot be rigorously explored.

Las bacterias aeróbicas anoxigénicas fototróficas (AAPs) constituyen un grupo de microorganismos mixotróficos, cuya relevancia en el océano no está del todo clara. Estos organismos contienen bacterioclorofila a (BChla) y aunque no son capaces de fijar CO2, pueden utilizar la luz para generar ATP lo que les aporta una ventaja ecológica frente a otros heterótrofos cuando la disponibilidad de C orgánico es limitada. Por ello, cuando inicialmente se descubrieron se pensó que éstas serian abundantes en ambientes oligotróficos, dónde de hecho pueden representar hasta el 10-20% del total de procariotas. Sin embargo, más tarde se vió que las AAPs también pueden ser abundantes en ambientes eutróficos por lo que no está muy claro cúal es su papel ecológico. Con esta Acción Complementaria hemos obtenido financiación para participar en un proyecto de circunnavegación global del océano (Tara-Oceans) dónde convertiremos un fluorómetro convencional en uno ultrasensible en el infrarojo que permite detectar de forma rápida y sencilla la presencia de AAPs. Este instrumento será utilizado también en el proyecto de circunnavegación Malaspina. Tara-Oceans durante tres años, y Malaspina en 7 meses recorrerán las principales provincias oceánicas. Estas dos campañas oceanográficas nos brindan una oportunidad única de estudiar la dinámica de las AAPs a escala global, en ambientes de distinto nivel trófico nunca estudiados anteriormente, lo que sin duda aportará información de gran valor para entender su papel ecológico en el océano y su impacto en el ciclo del C. Summary: Aerobic anoxygenic phototrophic bacteria (AAPs) are a group of mixotrophic organisms, which role in the ocean is still unclear. These organisms contain bacteriochlorophyll a (BChla), and, although they are not capable of fixing CO2, they can generate ATP using light energy, which could give them an ecological advantage under carbon limiting conditions. For that reason, it was first postulated that they would be very abundant in the oligotrophic ocean, where they can in fact make up to 10-20% of the total of prokaryotes. However, later on the AAPs were found to be also abundant in eutrophic environments, and therefore their ecological role is still controversial. This project has provided funding to participate in the global ocean expedition Tara-Oceans. We will upgrade a standard fluorometer into an ultra-sensitive infrared fluorometer that can easily register the signal of AAP bacteria. The instrument will be used in Tara-Oceans but also in the Malaspina expedition, which will sail the main provinces of the world’s oceans. These two circumnavigation cruises will allow us to study the dynamics of AAPs at a global scale, in environments of different trophic level never studied before. The data obtained will definitely contribute significantly to understand which is the ecological role of AAPs in the ocean and their impact in the carbon cycling.

Los virus se consideran los componentes más abundantes del plancton y son los responsables de un gran porcentaje de la mortalidad de microorganismos en sistemas marinos, con consecuencias para los flujos de carbono a través de las redes tróficas microbianas. Sabemos que la infección vírica está propiciada por la densidad de la presa, asumiéndose que hay una gran especificidad entre virus y huéspedes. Pero aún se conoce muy poco cual es el espectro de infección de diferentes virus sobre diferentes cepas de microorganismos, y como este afecta al papel ecológico que juegan los virus dentro de las comunidades microbianas. Los objetivos de este proyecto son: (1) Caracterizar los diferentes virus que infecten a bacterias que siempre están presentes en una zona costera del Mediterráneo (Roseobacter, alfa-proteobacteria) y a diferentes especies de una alga tóxica del genero Alexandrium spp; (2) Identificación y aislamiento de eucariotas heterotróficos y el consiguiente aislamiento de virus; (3) espectros de infección de los virus aislados sobre las distintas cepas de un mismo grupo de microorganismos; (4) variabilidad anual de comunidades naturales Roseobacter al ser infectadas por diferentes virus aisladas. Al mismo tiempo se determinará la variabilidad anual de comunidades naturales de virus y bacterias. En este proyecto se sigue una aproximación interdisciplinar en la que se examinarán aspectos sobre la ecología de los virus, utilizando técnicas convencionales de microbiología clásica y algunos de los métodos más prometedores dentro del campo de la biología molecular.

La luz solar facilita la mayor parte de la vida sobre la Tierra, también de los océanos, donde inicia la cadena trófica mediante la fotosíntesis del fitoplancton. Sin embargo, esta luz contiene también energía en longitudes de onda que son dañinas para los seres vivos. En los ecosistemas marinos la mayor parte de la materia y la energía se canaliza a través de las bacterias, que son los principales organismos encargados del reciclaje de la materia orgánica. La luz UV afecta directamente a éste bacterioplancton, pero también provoca alteraciones en la materia orgánica y los nutrientes alterando su biodegradabilidad y su composición. Por lo tanto, cambios en la concentración y en la composición tanto del bacterioplancton como de la materia orgánica ocasionarán cambios en el flujo de materia orgánica y energía y por tanto, en la productividad de los ecosistemas marinos. El objetivo del proyecto es determinar los efectos de la radiación solar sobre la actividad bacteriana y la utilización de la materia orgánica por parte de las bacterias marinas. Para ello, se llevarán a cabo experimentos en el laboratorio para evaluar los efectos directos sobre el bacterioplancton y los efectos a través de la materia orgánica.

Recent molecular studies based on sequencing rDNA directly retrieved from the marine plankton have revealed the existence of eukaryotic microorganisms never seen before. A significant number of the sequences retrieved affiliated within the stramenopiles, a very heterogeneous group that includes from diatoms to fungi-like cells, but were not similar to any known organism and were then named “novel stramenopiles”. The main objective of the project ESTRAMAR is the phylogenetic and ecological characterization of this novel group. First, we will analyze in detail the phylogenetic relationships among novel stramenopiles and other organisms, since we know this is a very diverse and complex group. Then, we will design phylogenetic probes against the most important subgroups that will allow visualizing the organisms in natural samples by FISH (Fluorescent In Situ Hybridization). Using FISH and the new probes, we will study the abundance and distribution of novel stramenopiles in samples obtained in a coastal seasonal study and in oceanographic cruises of opportunity. Finally, we will perform experiments to assess the ecological role of this novel group. Some novel stramenopiles are small heterotrophic flagellates that consume bacteria. We will develop a method to estimate the ability and rates of bacterivory by each different subgroup of novel stramenopiles. Moreover, we will also assess whether some subgroups are able to incorporate dissolved organic matter. The results expected during the project ESTRAMAR will contribute significantly to understand this novel group of microorganisms and will have clear implications for biodiversity studies and the understanding of the structure and function of marine food webs.

This project addresses the key issue of the relationship between diversity and ecological function in the uncultured heterotrophic eukaryotic microbes dominant in marine planktonic food webs. On one hand, heterotrophic flagellates is a functional group wit h accepted central roles in marine ecosystems (grazing on picoplankton, nutrient remineralization) and studies so far have treated them as a black box, formed mostly by small and unidentified cells. On the other hand, several recent molecular studies based on environmental rDNA sequences have revealed the existence of unknown eukaryotic microorganisms. A significant number of these sequences were found to be affiliated to the stramenopile taxon, a very heterogeneous assemblage of protists, but were not similar to any known group. Those were named MAST (Marine Stramenopiles), and some of them are heterotrophic and bacterivorous flagellates. Even though scientists start to have an idea of the distribution and phylogenetic relationships within these MAST groups, their role and significance in marine microbial food webs remains largely unknown. In the proposed research project, we will use a combination of innovative methodologies within a multi-disciplinary oceanographic context to open the heterotrophic flagellates' black box and investigate the ways MAST organisms interact with the other components of the natural microbial assemblage, particularly with photosynthetic picoplankton, which are major primary producers in the oceans. The host research team is one of the most experienced European teams in that field and the structured training program is designed to provide broad experience in the application of cutting-edge techniques to investigate major cross disciplinary issues in modern microbial ecology and oceanography. The training objectives are directly motivated by the applicant's ambition to acquire complementary skills in order to develop an independent high level research career in Europe.