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DINO_DON Report Summary

Project ID: 659453
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - DINO_DON (Does vegetation derived organic nitrogen fuel algal blooms in coastal waters of the Baltic Sea?)

Reporting period: 2016-01-01 to 2017-12-31

Summary of the context and overall objectives of the project

Coastal Baltic Sea is heavily affected by eutrophication, a release of nutrient from human activities that has lead to overgrown bays, increased phytoplankton blooms and reduced water transparency. Filamentous annual macrophytes outcompeted bladder wrack (Fucus vesiculosus) and eelgrass (Zostera marina), and the common reed, Phragmites australis has increased in embayments and archipelagos of the Baltic Sea. The annual biomass of macrophytes and reed constitute a major source of organic material to be decomposed during late summer and autumn.
The phytoplankton Alexandrium ostenfeldii, a dinoflagellate, produce neurotoxins, saxitoxins and spirolides that can by contaminating seafood cause paralytic shellfish poisoning in humans, with symptoms of acute gastroenteritis, respiratory distress, muscular paralysis, and death. Alexandrium ostenfeldii is also allelopathic and releases a substance that cause cell lysis in co-occurring phytoplankton. This dinoflagellate has been present in the Baltic Sea for a long time but during the last two decades dense blooms have been regularly reported from the coast of Poland to SE Sweden and Finland. In the Föglö archipelago (Åland, Finland), annual blooms of A. ostenfeldii have been reported since 2002. This is of particular concern for the newly established and increasing mussel-farming industry in coastal water of the Baltic Sea.

The blooms typically occur when inorganic nutrient concentrations (e.g. nitrate, NO3) are low but dissolved organic nitrogen (DON) is available in high concentrations and in waters overgrown by vegetation. Could organic nutrients, released by the decaying vegetation substitute inorganic nutrients, particularly nitrogen (N), and thus favour the formation of dinoflagellate blooms at times when NO3 is limited? If Baltic Sea A. ostenfeldii is mixotrophic (able to utilize organic nutrients), a trait common among the genus Alexandrium, vegetation or phytoplankton derived DON could drive these blooms.

The overall objective of the project is to determine the importance of organic N from various forms of decaying vegetation in the nutrition of bloom forming dinoflagellates.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The project has used 15N-tracer experiments in the field, during ongoing blooms, and on isolated strains representing different phases of bloom succession to confirm and describe mixotrophic behaviour of A. ostenfeldii. The stable (non-radioactive) isotope if nitrogen (15N), enriched in various N species/substrates, can be used for uptake and preference studies. By cultivating vegetation on 15N-nitrate and collecting released DON, an enriched 15N-DON substrate can be produced and used in uptake studies with A. ostenfeldii. Both macro- (i.e. bladder wrack, eel grass, reed) and micro-vegetation (other phytoplankton) has been used to produces various 15N-DON substrate for uptake experiments.
Single strain experiments were performed to describe N uptake and preference in A. ostenfeldii using substrate of nitrate, ammonium, urea and amino acids enriched to 99% with the stable isotope 15N. To distinguish bacterial uptake and transformation of N substrates from that of the dinoflagellate, an antibiotic treatment were used as control. For the visualization of DON uptake by single cells we used second ion mass spectrometry (SIMS) to analyze uptake of nitrate, amino acids and 15N-DON from co-occurring microvegetation (cryptophyte Rhinomonas nottbecki).
Nitrogen uptake rate and preference experiments were performed in the field in on-going A. ostenfeldii blooms during two field seasons (July-August) in Föglö archipelago (Åland, Finland). Extensive background measurements were done for a more detailed description of bloom conditions. To describe mixotrophic activity in A. ostenfeldii during bloom succession a model system with 25 strains isolated from 5 separate bloom phases was used for 15N-DON incubations. Simultaneous measurement of lytic activity in the strains will provide a more detailed picture of how lytic activity facilitates N uptake during an A. ostenfeldii bloom. A species distribution model based on A. ostenfeldii observations and background data was constructed to examine a possible relationship between A. ostenfeldii occurrences and DON-rich coastal habitats. The model was built using the Maxent package and presence-only data.

Our findings support the concept that dense blooms of A. ostenfeldii utilize DON as N source and that it’s allelopathic activity (release of lytic substances) facilitates N uptake. 15N-tracer experiments in the field and on isolated strains shows high uptake rates of organic N forms, regardless if bacteria were inhibited and a preference for amino acids to other tested N species. We were able to analyse 15N incorporation on a cellular level using second iron mass spectrometry (SIMS) and visualized direct uptake of DON by A. ostenfeldii from a co-occurring cryptophyte (small phytoplankton). The species distribution model based on observations of A. ostenfeldii in the Baltic Sea showed that vegetation cover together phosphorus, distance to shore, and temperature are important explanatory factors. In conclusion we show that nitrogen supply and utilization mechanisms are an important part of understanding bloom expansion of A. ostenfeldii.

The results of the project have been presented at international scientific conferences, at several department lectures (Finnish environment institute (SYKE) Helsinki, Finland; Biology and Environmental Science, Linnæus university, Kalmar, Sweden; Ecology, University of Lund, Sweden) and to the public at numerous events. Publications aimed for peer-reviewed scientific journals are in preparation.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The project is the first to confirm DON uptake and mixotrophy in A. ostenfeldii from the Baltic Sea and shows that A. ostenfeldii prefer organic forms of nitrogen over inorganic. Using second iron mass spectrometry (SIMS) we were able to analyze DON uptake on single cell level and visualize direct uptake of DON from a co-occurring microvegetation, a cryptophyte lysed by toxins released from A. ostenfeldii. This is the first study showing the direct coupling between A. ostenfeldii blooms and vegetation derived DON. Eutrophication and consequent increase in vegetation and phytoplankton blooms in coastal water are of importance to the initiation and maintenance of A. ostenfeldii blooms by providing a source of nitrogen. Measures against eutrophication are therefor important in the work to manage new and confirmed bloom sites of A. ostenfeldii. The species distribution model of A. ostenfeldii in Finnish waters, produced by this project is unique for the Baltic Sea. The vegetation cover together with factors such as depth and temperature identified substantial areas of shallow coastal waters (Baltic proper, FI) as potential bloom sites for A. ostenfeldii. Toxins produced by A. ostenfeldii accumulate in shellfish, such as blue mussels, currently cultivated in the Baltic Sea for animal feed production. The results of this project have implication for coastal management and where to intensify eutrophication- and restoration measures.

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