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.
