Intercomparison of Two Fluorescent Dyes to Visualize Parasitic Fungi (Chytridiomycota) on Phytoplankton

Fungal microparasites (here chytrids) are widely distributed and yet, they are often overlooked in aquatic environments. To facilitate the detection of microparasites, we revisited the applicability of two fungal cell wall markers, Calcofluor White (CFW) and wheat germ agglutinin (WGA), for the direct visualization of chytrid infections on phytoplankton in laboratory-maintained isolates and field-sampled communities. Using a comprehensive set of chytrid-phytoplankton model pathosystems, we verified the staining pattern on diverse morphological structures of chytrids via fluorescence microscopy. Empty sporangia were stained most effectively, followed by encysted zoospores and im-/mature sporangia, while the staining success was more variable for rhizoids, stalks, and resting spores. In a few instances, the staining was unsuccessful (mostly with WGA), presumably due to insufficient cell fixation, gelatinous cell coatings, and multilayered cell walls. CFW and WGA staining could be done in Utermohl chambers or on polycarbonate filters, but CFW staining on filters seemed less advisable due to high background fluorescence. To visualize chytrids, 1 mu g dye mL(-1) was sufficient (but 5 mu g mL(-1) are recommended). Using a dual CFW-WGA staining protocol, we detected multiple, mostly undescribed chytrids in two natural systems (freshwater and coastal), while falsely positive or negative stained cells were well detectable. As a proof-of-concept, we moreover conducted imaging flow cytometry, as a potential high-throughput technology for quantifying chytrid infections. Our guidelines and recommendations are expected to facilitate the detection of chytrid epidemics and to unveil their ecological and economical imprint in natural and engineered aquatic systems.</p

Efficient carbon and nitrogen transfer from marine diatom aggregates to colonizing bacterial groups

Bacterial degradation of sinking diatom aggregates is key for the availability of organic matter in the deep-ocean. Yet, little is known about the impact of aggregate colonization by different bacterial taxa on organic carbon and nutrient cycling within aggregates. Here, we tracked the carbon (C) and nitrogen (N) transfer from the diatom Leptocylindrus danicus to different environmental bacterial groups using a combination of C-13 and N-15 isotope incubation (incubated for 72 h), CARD-FISH and nanoSIMS single-cell analysis. Pseudoalteromonas bacterial group was the first colonizing diatom-aggregates, succeeded by the Alteromonas group. Within aggregates, diatom-attached bacteria were considerably more enriched in C-13 and N-15 than non-attached bacteria. Isotopic mass balance budget indicates that both groups showed comparable levels of diatom C in their biomass, accounting for 19 +/- 7% and 15 +/- 11%, respectively. In contrast to C, bacteria of the Alteromonas groups showed significantly higher levels of N derived from diatoms (77 +/- 28%) than Pseudoalteromonas (47 +/- 17%), suggesting a competitive advantage for Alteromonas in the N-limiting environments of the deep-sea. Our results imply that bacterial succession within diatom aggregates may largely impact taxa-specific C and N uptake, which may have important consequences for the quantity and quality of organic matter exported to the deep ocean

Intercomparison of Two Fluorescent Dyes to Visualize Parasitic Fungi (Chytridiomycota) on Phytoplankton

We revisited the applicability of two fungal cell wall markers, Calcofluor White (CFW) and wheat germ agglutinin (WGA), for the direct visualization of chytrid infections on phytoplankton in laboratory-maintained isolates and field-sampled communities. Using a comprehensive set of chytrid–phytoplankton model pathosystems, we verified the staining pattern on diverse morphological structures of chytrids via fluorescence microscopy. Empty sporangia were stained most effectively, followed by encysted zoospores and im-/mature sporangia, while the staining success was more variable for rhizoids, stalks, and resting spores. In a few instances, the staining was unsuccessful (mostly with WGA), presumably due to insufficient cell fixation, gelatinous cell coatings, and multilayered cell walls. CFW and WGA staining could be done in Utermöhl chambers or on polycarbonate filters, but CFW staining on filters seemed less advisable due to high background fluorescence. To visualize chytrids, 1 μg dye mL−1 was sufficient (but 5 μg mL−1 are recommended). Using a dual CFW–WGA staining protocol, we detected multiple, mostly undescribed chytrids in two natural systems (freshwater and coastal), while falsely positive or negative stained cells were well detectable. As a proof-of-concept, we moreover conducted imaging flow cytometry, as a potential high-throughput technology for quantifying chytrid infections. Our guidelines and recommendations are expected to facilitate the detection of chytrid epidemics and to unveil their ecological and economical imprint in natural and engineered aquatic systems. The uploaded data included complementary information for Figure 2 and Figure S6, and image analyses (data of fluorescence intensity shown in Figure 4

Marine nitrogen fixation : Cyanobacterial nitrogen fixation and the fate of new nitrogen in the Baltic Sea

Biogeochemical processes in the marine biosphere are important in global element cycling and greatly influence the gas composition of the Earth’s atmosphere. The nitrogen cycle is a key component of marine biogeochemical cycles. Nitrogen is an essential constituent of living organisms, but bioavailable nitrogen is often short in supply thus limiting primary production. The largest input of nitrogen to the marine environment is by N2-fixation, the transformation of inert N2 gas into bioavailable ammonium by a distinct group of microbes. Hence, N2-fixation bypasses nitrogen limitation and stimulates productivity in oligotrophic regions of the marine biosphere. Extensive blooms of N2-fixing cyanobacteria occur regularly during summer in the Baltic Sea. N2-fixation during these blooms adds several hundred kilotons of new nitrogen into the Baltic Proper, which is similar in magnitude to the annual nitrogen load by riverine discharge and more than twice the atmospheric nitrogen deposition in this area. N2-fixing cyanobacteria are therefore a critical constituent of nitrogen cycling in the Baltic Sea. In this thesis N2 fixation of common cyanobacteria in the Baltic Sea and the direct fate of newly fixed nitrogen in otherwise nitrogen-impoverished waters were investigated. Initially, the commonly used 15N-stable isotope assay for N2-fixation measurements was evaluated and optimized in terms of reliability and practicality (Paper I), and later applied for N2-fixation assessments (Paper II–IV). N2 fixation in surface waters of the Baltic Sea was restricted to large filamentous heterocystous cyanobacteria (Aphanizomenon sp., Nodularia spumigena, Dolichospermum spp.) and absent in smaller filamentous cyanobacteria such as Pseudanabaena sp., and unicellular and colonial picocyanobacteria (Paper II-III). Most of the N2-fixation in the Northern Baltic Proper was contributed by Aphanizomenon sp. due to its high abundance throughout the summer and similar rates of specific N2-fixation as Dolichospermum spp. and N. spumigena. Specific N2 fixation was substantially higher near the coast than in an offshore region (Paper II). Half of the fixed nitrogen was released as ammonium at the site near the coast and taken up by non-N2-fixing organisms including phototrophic and heterotrophic, prokaryotic and eukaryotic planktonic organisms. Newly fixed nitrogen was thereby rapidly turned-over in the nitrogen-depleted waters (Paper III). In colonies of N. spumigena even the potential for a complete nitrogen cycle condensed to a microcosm of a few millimeters could be demonstrated (Paper IV). Cyanobacterial colonies can therefore be hot-spots of nitrogen transformation processes potentially including nitrogen gain, recycling and loss processes. In conclusion, blooms of cyanobacteria are instrumental for productivity and CO2 sequestration in the Baltic Sea. These findings advance our understanding of biogeochemical cycles and ecosystem functioning in relation to cyanobacterial blooms in the Baltic Sea with relevance for both ecosystem-based management in the Baltic Sea, and N2-fixation and nitrogen cycling in the global ocean.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p

Denitrification and DNRA at the Baltic Sea oxic–anoxic interface : Substrate spectrum and kinetics

The dependence of denitrification and dissimilatory nitrate reduction to ammonium (DNRA) on different electron donors was tested in the nitrate-containing layer immediately below the oxic–anoxic interface (OAI) at three stations in the central anoxic basins of the Baltic Sea. Additionally, pathways and rates of fixed nitrogen transformation were investigated with 15N incubation techniques without addition of donors. Denitrification and anammox were always detected, but denitrification rates were higher than anammox rates. DNRA occurred at two sites and rates were two orders of magnitude lower than denitrification rates. Separate additions of dissolved organic carbon and sulfide stimulated rates without time lag indicating that both organotrophic and lithotrophic bacterial populations were simultaneously active and that they could carry out denitrification or DNRA. Manganese addition stimulated denitrification and DNRA at one station, but it is not clear whether this was due to a direct or indirect effect. Ammonium oxidation to nitrite was detected on one occasion. During denitrification, the production of nitrous oxide (N2O) was as important as dinitrogen (N2) production. A high ratio of N2O to N2 production at one site may be due to copper limitation, which inhibits the last denitrification step. These data demonstrate the coexistence of a range of oxidative and reductive nitrogen cycling processes at the Baltic OAI and suggest that the dominant electron donor supporting denitrification and DNRA is organic matter. Organotrophic denitrification is more important for nitrogen budgets than previously thought, but the large temporal variability in rates calls for long-term seasonal studies

Turbulence simultaneously stimulates small- and large-scale CO2 sequestration by chain-forming diatoms in the sea

Chain-forming diatoms are key CO2-fixing organisms in the ocean. Under turbulent conditions they form fast-sinking aggregates that are exported from the upper sunlit ocean to the ocean interior. A decade-old paradigm states that primary production in chain-forming diatoms is stimulated by turbulence. Yet, direct measurements of cell-specific primary production in individual field populations of chain-forming diatoms are poorly documented. Here we measured cell-specific carbon, nitrate and ammonium assimilation in two field populations of chain-forming diatoms (Skeletonema and Chaetoceros) at low-nutrient concentrations under still conditions and turbulent shear using secondary ion mass spectrometry combined with stable isotopic tracers and compared our data with those predicted by mass transfer theory. Turbulent shear significantly increases cell-specific C assimilation compared to still conditions in the cells/chains that also form fast-sinking, aggregates rich in carbon and ammonium. Thus, turbulence simultaneously stimulates small-scale biological CO2 assimilation and large-scale biogeochemical C and N cycles in the ocean

A novel measurement-based model for calculating O2 flux at interfaces in aquatic environments

Our understanding of the small-scale processes that drive global biogeochemical cycles and the Earth's climate is dependent on accurate estimations of interfacial diffusive fluxes to and from biologically-active substrates in aquatic environments. In this study, we present a novel model approach for accurate calculations of diffusive fluxes of dissolved gases, nutrients, and solutes from concentration profiles measured across the substrate-water interfaces using microsensors. The model offers a robust computational scheme for automatized determination of the interface position and enables precise calculations of the interfacial diffusive fluxes simultaneously. In contrast to other methods, the new approach is not restricted to any particular substrate geometry, does not require a priori determination of the interface position for the flux calculation, and, thus, reduces the uncertainties in calculated fluxes arising from partly subjective identification of the interface position. In addition, it is robust when applied to measured profiles containing scattered data points and insensitive to reasonable decreases of the spatial resolution of the data points. The latter feature allows for significantly reducing measurement time which is a crucial factor for in situ experiments

Active DNRA and denitrification in oxic hypereutrophic waters

Since the start of synthetic fertilizer production more than a hundred years ago, the coastal ocean has been exposed to increasing nutrient loading, which has led to eutrophication and extensive algal blooms. Such hypereutrophic waters might harbour anaerobic nitrogen (N) cycling processes due to low-oxygen microniches associated with abundant organic particles, but studies on nitrate reduction in coastal pelagic environments are scarce. Here, we report on 15N isotope-labelling experiments, metagenome, and RT-qPCR data from a large hypereutrophic lagoon indicating that dissimilatory nitrate reduction to ammonium (DNRA) and denitrification were active processes, even though the bulk water was fully oxygenated (> 224 µM O2). DNRA in the bottom water corresponded to 83 % of whole ecosystem DNRA (water + sediment), while denitrification was predominant in the sediment. Microbial taxa important for DNRA according to the metagenomic data were dominated by Bacteroidetes (genus Parabacteroides) and Proteobacteria (genus Wolinella), while denitrification was mainly associated with proteobacterial genera Pseudomonas, Achromobacter, and Brucella. The metagenomic and microscopy data suggest that these anaerobic processes were likely occurring in low-oxygen microniches related to extensive growth of filamentous cyanobacteria, including diazotrophic Dolichospermum and non-diazotrophic Planktothrix. By summing the total nitrate (NO3 −) fluxes through DNRA and denitrification, it results that DNRA retains approximately one fifth (19 %) of the fixed N that goes through the NO3 − pool. This is noteworthy as DNRA represents thus a very important recycling mechanism for fixed N, which sustains algal proliferation and leads to further enhancement of eutrophication in these endangered ecosystems