Nitrogen (N) isotope ratios ( 15 N/ 14 N) provide integrative constraints on the N inventory of the modern ocean. Anaerobic ammonium oxidation (anammox), which converts ammonium and nitrite to dinitrogen gas (N 2 ) and nitrate, is an important fixed N sink in marine ecosystems. We studied the so far unknown N isotope effects of anammox in batch culture experiments. Anammox preferentially removes 14 N from the ammonium pool with an isotope effect of +23.5‰ to +29.1‰, depending on factors controlling reversibility. The N isotope effects during the conversion of nitrite to N 2 and nitrate are (i) inverse kinetic N isotope fractionation associated with the oxidation of nitrite to nitrate (−31.1 ± 3.9‰), (ii) normal kinetic N isotope fractionation during the reduction of nitrite to N 2 (+16.0 ± 4.5‰), and (iii) an equilibrium N isotope effect between nitrate and nitrite (−60.5 ± 1.0‰), induced when anammox is exposed to environmental stress, leading to the superposition of N isotope exchange effects upon kinetic N isotope fractionation. Our findings indicate that anammox may be responsible for the unresolved large N isotope offsets between nitrate and nitrite in oceanic oxygen minimum zones. Irrespective of the extent of N isotope exchange between nitrate and nitrite, N removed from the combined nitrite and nitrate (NO x ) pool is depleted in 15 N relative to NO x . This net N isotope effect by anammox is superimposed on the N isotope fractionation by the cooccurring reduction of nitrate to nitrite in suboxic waters, possibly enhancing the overall N isotope effect for N loss from oxygen minimum zones.T he nitrogen (N) isotope effect associated with N loss pathways allows the isotopic tracing of N transformations in ocean waters and provides critical constraints on the global marine N budget (1-5). Culture studies have shown that heterotrophic denitrification exhibits a large N isotope effect (e)* of up to +30‰ (6-9). For decades, heterotrophic denitrification was the only known N loss pathway in the ocean (10). Consequently, strong enrichment of 15 N in residual nitrite and nitrate (NO x ) from oxygen-deficient waters, as for example in the Eastern Tropical North Pacific and the Arabian Sea, was fully attributed to water column denitrification with an N isotope effect around +25‰ (2, 11). Recent studies have highlighted the significance of anaerobic ammonium oxidation (anammox) for regional N fluxes (12, 13), with possible ramifications regarding the global N balance (14, 15). However, N isotope effects associated with the anammox metabolism were unknown and therefore their potential impacts on the distribution of oceanic N isotopes could not be addressed. This lack of knowledge severely hampers the N-isotope-based assessment of the relative importance of watercolumn N loss compared with sedimentary N loss, the most poorly constrained flux in the marine combined nitrogen budget (16). ResultsWe used the only available highly enriched (>98%) culture of anammox single cells (Kuenenia stuttgartiensis) (17, 18) to investi...

Dinoflagellates readily use diverse inorganic and organic compounds as nitrogen sources, which is advantageous in eutrophied coastal areas exposed to high loads of anthropogenic nutrients, e.g., urea, one of the most abundant organic nitrogen substrates in seawater. Cell-to-cell variability in nutritional physiology can further enhance the diversity of metabolic strategies among dinoflagellates of the same species, but it has not been studied in free-living microalgae. We applied stable isotope tracers, isotope ratio mass spectrometry and nanoscale secondary ion mass spectrometry (NanoSIMS) to investigate the response of cultured nitrate-acclimated dinoflagellates Prorocentrum minimum to a sudden input of urea and the effect of urea on the concurrent nitrate uptake at the population and single-cell levels. We demonstrate that inputs of urea lead to suppression of nitrate uptake by P. minimum, and urea uptake exceeds the concurrent uptake of nitrate. Individual dinoflagellate cells within a population display significant heterogeneity in the rates of nutrient uptake and extent of the urea-mediated inhibition of the nitrate uptake, thus forming several groups characterized by different modes of nutrition. We conclude that urea originating from sporadic sources is rapidly utilized by dinoflagellates and can be used in biosynthesis or stored intracellularly depending on the nutrient status; therefore, sudden urea inputs can represent one of the factors triggering or supporting harmful algal blooms. Significant physiological heterogeneity revealed at the single-cell level is likely to play a role in alleviation of intra-population competition for resources and can affect the dynamics of phytoplankton populations and their maintenance in natural environments.

Four-domain voltage-gated cation channels (FVCCs) represent a large family of pseudo-tetrameric ion channels which includes voltage-gated calcium (Cav) and sodium (Nav) channels, as well as their homologues. These transmembrane proteins are involved in a wide range of physiological processes, such as membrane excitability, rhythmical activity, intracellular signalling, etc. Information about actual diversity and phylogenetic relationships of FVCCs across the eukaryotic tree of life is scarce. We for the first time performed a taxonomically broad phylogenetic analysis of 277 FVCC sequences from a variety of eukaryotes and showed that many groups of eukaryotic organisms have their own clades of FVCCs. Moreover, the number of FVCC lineages in several groups of unicellular eukaryotes is comparable to that in animals. Based on the primary structure of FVCC sequences, we characterised their functional determinants (selectivity filter, voltage sensor, Nav-like inactivation gates, Cavβ-interaction motif, and calmodulin-binding region) and mapped them on the obtained phylogeny. This allowed uncovering of lineage-specific structural gains and losses in the course of FVCC evolution and identification of ancient structural features of these channels. Our results indicate that the ancestral FVCC was voltage-sensitive, possessed a Cav-like selectivity filter, Nav-like inactivation gates, calmodulin-binding motifs and did not bear the structure for Cavβ-binding.

Ion channels are tightly involved in various aspects of cell physiology, including cell signaling, proliferation, motility, endo- and exo-cytosis. They may be involved in toxin production and release by marine dinoflagellates, as well as harmful algal bloom proliferation. So far, the patch-clamp technique, which is the most powerful method to study the activity of ion channels, has not been applied to dinoflagellate cells, due to their complex cellulose-containing cell coverings. In this paper, we describe a new approach to overcome this problem, based on the preparation of spheroplasts from armored bloom-forming dinoflagellate Prorocentrum minimum. We treated the cells of P. minimum with a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), and found out that it could also induce ecdysis and arrest cell shape maintenance in these microalgae. Treatment with 100–250 µM DCB led to an acceptable 10% yield of P. minimum spheroplasts and was independent of the incubation time in the range of 1–5 days. We show that such spheroplasts are suitable for patch-clamping in the cell-attached mode and can form 1–10 GOhm patch contact with a glass micropipette, allowing recording of ion channel activity. The first single-channel recordings of dinoflagellate ion channels are presented.

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