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This report considers the geographic and taxonomic spread of non-constitutive mixoplankton (NCM). NCM are mixoplankton (protists that are both phototrophic and phagotrophic) by virtue of acquired phototrophy; they acquire their potential for photosynthesis from their prey. NCM are quantitatively important members of the protistan communities in Arctic, temperate and Subtropical waters. Generally, ciliates were the quantitatively dominant NCM across climate zones, with NCM dinoflagellates and amoebic were of less importance.

A guide for the sampling and analysis of mixoplankton in natural environments is provided that offers guidelines to assist students and research scientists initiating studies of mixoplankton in natural waters. The guide contains methods on how to sample, preserve and analyse mixoplankton abundance and diversity directly from natural environments. As mixoplankton are fully integrated constituents of the protist plankton community, many of the sampling strategies and techniques described in this guide are applicable also to phytoplankton and protozooplankton. Accordingly, methods cover traditional methods but also evolving new molecular techniques developed for applications to field and discrete studies of plankton diversity.

MixITiN Project Report D3.8.-- 69 pages, 19 figures, 7 tables, Despite the supposed relevance of mixoplankton (Stoecker et al. 2017, Flynn et al. 2019) we still lack standardized techniques to measure their grazing impact in the field. This is mostly because of the difficulty in separating grazers and prey (similar sizes and/or pigments). To date, most approaches to estimate mixoplankton grazing are based on extrapolating to the field the rates obtained in the laboratory (e.g. Yih et al. 2004). For instance, to estimate grazing on protists it is common to measure the disappearance of prey during 24h incubations under strictly controlled conditions. Given the impossibility to obtain data for all the species present at sea on a given time, and to predict the particular interactions between groups, the approach is, if not completely erroneous, at least very limited and inaccurate. Moreover, the fact that some species, like Prymnesium parvum or Karlodinium armiger, release allelopathic compounds (e.g. Shilo & Aschner 1953, Rasmussen et al. 2017) that may cause immobilization and lysis of potential prey may introduce further complications to the interpretation of the result. [...], Project MixITiN has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 766327

International audience; Arranging organisms into functional groups aids ecological research by grouping organisms (irrespective of phylogenetic origin) that interact with environmental factors in similar ways. Planktonic protists traditionally have been split between photoautotrophic “phytoplankton” and phagotrophic “microzooplankton”. However, there is a growing recognition of the importance of mixotrophy in euphotic aquatic systems, where many protists often combine photoautotrophic and phagotrophic modes of nutrition. Such organisms do not align with the traditional dichotomy of phytoplankton and microzooplankton. To reflect this understanding, we propose a new functional grouping of planktonic protists in an eco-physiological context: (i) phagoheterotrophs lacking phototrophic capacity, (ii) photoautotrophs lacking phagotrophic capacity, (iii) constitutive mixotrophs (CMs) as phagotrophs with an inherent capacity for phototrophy, and (iv) non-constitutive mixotrophs (NCMs) that acquire their phototrophic capacity by ingesting specific (SNCM) or general non-specific (GNCM) prey. For the first time, we incorporate these functional groups within a foodweb structure and show, using model outputs, that there is scope for significant changes in trophic dynamics depending on the protist functional type description. Accordingly, to better reflect the role of mixotrophy, we recommend that as important tools for explanatory and predictive research, aquatic food-web and biogeochemical models need to redefine the protist groups within their frameworks.

International audience; Upon phosphorus (P) deficiency, marine phytoplankton reduce their requirements for P by replacing membrane phospholipids with alternative non-phosphorus lipids. It was very recently demonstrated that a SAR11 isolate also shares this capability when phosphate starved in culture. Yet, the extent to which this process occurs in other marine heterotrophic bacteria and in the natural environment is unknown. Here, we demonstrate that the substitution of membrane phospholipids for a variety of non-phosphorus lipids is a conserved response to P deficiency among phylogenetically diverse marine heterotrophic bacteria, including members of the Alphaproteobacteria and Flavobacteria. By deletion mutagenesis and complementation in the model marine bacterium Phaeobacter sp. MED193 and heterologous expression in recombinant Escherichia coli, we confirm the roles of a phospholipase C (PlcP) and a glycosyltransferase in lipid remodelling. Analyses of the Global Ocean Sampling and Tara Oceans metagenome data sets demonstrate that PlcP is particularly abundant in areas characterized by low phosphate concentrations. Furthermore, we show that lipid remodelling occurs seasonally and responds to changing nutrient conditions in natural microbial communities from the Mediterranean Sea. Together, our results point to the key role of lipid substitution as an adaptive strategy enabling heterotrophic bacteria to thrive in the vast P-depleted areas of the ocean.

International audience; Upon phosphorus (P) deficiency, marine phytoplankton reduce their requirements for P by replacing membrane phospholipids with alternative non-phosphorus lipids. It was very recently demonstrated that a SAR11 isolate also shares this capability when phosphate starved in culture. Yet, the extent to which this process occurs in other marine heterotrophic bacteria and in the natural environment is unknown. Here, we demonstrate that the substitution of membrane phospholipids for a variety of non-phosphorus lipids is a conserved response to P deficiency among phylogenetically diverse marine heterotrophic bacteria, including members of the Alphaproteobacteria and Flavobacteria. By deletion mutagenesis and complementation in the model marine bacterium Phaeobacter sp. MED193 and heterologous expression in recombinant Escherichia coli, we confirm the roles of a phospholipase C (PlcP) and a glycosyltransferase in lipid remodelling. Analyses of the Global Ocean Sampling and Tara Oceans metagenome data sets demonstrate that PlcP is particularly abundant in areas characterized by low phosphate concentrations. Furthermore, we show that lipid remodelling occurs seasonally and responds to changing nutrient conditions in natural microbial communities from the Mediterranean Sea. Together, our results point to the key role of lipid substitution as an adaptive strategy enabling heterotrophic bacteria to thrive in the vast P-depleted areas of the ocean.

Aquatic Sciences Meeting, Aquatic Sciences: Global And Regional Perspectives - North Meets South, 22-27 February 2015, Granada, Spain, Phosphorus (P) is an essential nutrient for life. The replacement of membrane phospholipids with non-phosphorus lipids constitutes an important strategy to allow growth on phosphate-limited environments. In the ocean, this strategy has been reported in phytoplankton, but not in heterotrophic bacteria. A phospholipase C was recently identified to be required for lipid remodelling in a soil bacterium during P-limitation. In this study we show this enzyme is widespread in bacterial taxa and frequently found in ocean metagenomes. In SAR11 and other proteobacterial isolates, this gene is frequently neighbour to a glycosyltransferase that could be involved in glycolipid biosynthesis. Experiments with marine isolates showed up-regulation of transcription of both the phospholipase C and the glycosyltransferase upon P-starvation. These results suggest that marine heterotrophic bacteria may substitute phospholipids for glycolipids to reduce their P-demand. Lipid measurements in environmental samples confirmed that indeed there is an important enrichment in glycolipids in the heterotrophic bacterial fraction under P-depleted conditions. Hence, lipid remodelling seems to be a widespread mechanism among marine microbes for surviving in the vast oligotrophic areas of the ocean

20 pages, 8 figures, 1 table, The use of inorganic phosphate (Pi) and dissolved organic phosphorus (DOP) by different bacterial groups was studied in experimental mesocosms of P-starved eastern Mediterranean waters in the absence (control mesocosms) and presence of additional Pi (P-amended mesocosms). The low Pi turnover times in the control mesocosms and the increase in heterotrophic prokaryotic abundance and production upon Pi addition confirmed that the bacterial community was originally P-limited. The bacterioplankton groups taking up Pi and DOP were identified by means of microautoradiography combined with catalysed reporter deposition fluorescence in situ hybridization. Incubations with leucine were also performed for comparative purposes. All the probe-identified groups showed a high percentage of cells taking up Pi and DOP in the control, P-limited, mesocosms throughout the experiment. However, in response to Pi addition two contrasting scenarios in Pi use were observed: (i) on day 1 of the experiment Pi addition caused a clear reduction in the percentage of SAR11 cells taking up Pi, whereas Gammaproteobacteria, Roseobacter and Bacteroidetes showed similar percentages to the ones in the control mesocosms and (ii) on day 4 of the experiment, probably when the bacterial community had fully responded to the P input, all the probe-identified groups showed low percentages of cells taking up the substrate as compared with the control mesocosms. These differences are likely related to different P requirements among the bacterial groups and point out to the existence of two contrasting strategies in P use, The mesocosm experiment in the Cretacosmos facility was arranged in cooperation by project Nutritunnel financed by the Research Council of Norway and the MESOAQUA EU network. The processing of the samples was supported by the grants FOSMICRO (CTM2009-07679-E), STORM (CTM2009-09352/MAR), SUMMER (CTM2008-03309/MAR) and MALASPINA (CSD2008 – 00077) funded by the Spanish Ministry of Science and Innovation. We thank Irene Forn and Clara Ruiz for their help with the CARDFISH and MARFISH techniques, Iordanis Magiopoulos for performing the flow cytometer analyses and Tatiana Tsagaraki for performing the inorganic nutrient analyses. Marta Sebastián was supported by the Spanish Ministry of Science and Innovation through a ‘Juan de la Cierva’ award

Mediterranean fish farming has grown exponentially during the last 20 years. Although there is little evidence of the impact on the trophy status around fish farms, there are concerns that the release of solute wastes from aquaculture might affect larger scales in the ecosystem by changing the nutrient load. After combining information from various sources on waste production and on nutrient loads, it was concluded that the overall N and P waste from fish farms in the Mediterranean represents less than 5% of the total annual anthropogenic discharge, and the overall annual increase in P and N pools in the Mediterranean, under a production rate of 150000 tons, is less than 0.01%. The proportion of fish farming discharged nutrients was slightly higher in the eastern Mediterranean. A simple model was used to assess the long-term effects of nutrients released from various sources taking into account the water renewal rate in the Mediterranean. We conclude that, in the long term, fish farm waste could cause a 1% increase in nutrient concentrations in contrast to other anthropogenic activities which might double the Mediterranean nutrient pool., La piscicultura en el Mediterráneo ha crecido exponencialmente durante los ultimos 20 años. Aunque hay pocas evidencias del impacto sobre el estado trófico alrededor de las granjas de peces, existe la preocupación de que la liberación de residuos solubles procedentes de la acuicultura pueda afectar al ecosistema, de manera notable, a través de cambios en la carga de nutrientes. Después de combinar información de varias fuentes sobre la producción de residuos cargados de nutrientes, se concluyó que el N y el P totales liberados en las granjas de peces en el Mediterráneo, representan menos del 5% de la descarga anthropogénica anual total y el aumento anual del contenido en P y de N en el mediterráneo, bajo una tasa de producción de 150000 toneladas, es menor de 0.01%. La proporción de la descarga de nutrientes procedentes de la piscicultura fue ligeramente mayor en el Mediterráneo Oriental. Se utilizó un modelo sencillo para determinar los efectos, a largo plazo, de la liberación de nutrientes desde varias fuentes considerando la tasa de renovación del agua en el Mediterráneo. Se concluyó que, a largo plazo, las granjas de peces podrían dar lugar a un aumento del 1% de las concentraciones de nutrientes, en contraste con otras actividades antropogénicas que podrían doblar el contenido de nutrientes del Mediterráneo.

A manual for the isolation and maintenance of mixoplankton is provided that offer guidelines to assist students and scientists initiating studies of mixoplankton. The manual contains specific information on: Facilities required, including temperature regulated rooms or cabinets, instruments and consumables Growth media and aseptic precautions General methods and recommendation for isolation of cells from nature and subsequent establishment of cell cultures Specific recommendation for the isolation and culture of different mixoplankton functional groups

A manual for the isolation and maintenance of mixoplankton is provided that offer guidelines to assist students and scientists initiating studies of mixoplankton. The manual contains specific information on: Facilities required, including temperature regulated rooms or cabinets, instruments and consumables Growth media and aseptic precautions General methods and recommendation for isolation of cells from nature and subsequent establishment of cell cultures Specific recommendation for the isolation and culture of different mixoplankton functional groups

A guide for the sampling and analysis of mixoplankton in natural environments is provided that offers guidelines to assist students and research scientists initiating studies of mixoplankton in natural waters. The guide contains methods on how to sample, preserve and analyse mixoplankton abundance and diversity directly from natural environments. As mixoplankton are fully integrated constituents of the protist plankton community, many of the sampling strategies and techniques described in this guide are applicable also to phytoplankton and protozooplankton. Accordingly, methods cover traditional methods but also evolving new molecular techniques developed for applications to field and discrete studies of plankton diversity.

This report considers the geographic and taxonomic spread of non-constitutive mixoplankton (NCM). NCM are mixoplankton (protists that are both phototrophic and phagotrophic) by virtue of acquired phototrophy; they acquire their potential for photosynthesis from their prey. NCM are quantitatively important members of the protistan communities in Arctic, temperate and Subtropical waters. Generally, ciliates were the quantitatively dominant NCM across climate zones, with NCM dinoflagellates and amoebic were of less importance.