Stressor-induced ecdysis and thecate cyst formation in the armoured dinoflagellates Prorocentrum cordatum.

Ecdysis, the process of extensive cell covering rearrangement, represents a remarkable physiological trait of dinoflagellates. It is involved in the regulation of the population and bloom dynamics of these microorganisms, since it is required for the formation of their thin-walled cysts. This study presents laboratory data on ecdysis in Prorocentrum cordatum, a harmful dinoflagellate species of high environmental significance. We studied external stressors triggering this process and changes in the cell ultrastructure accompanying it. Our experiments showed that mass ecdysis and formation of cysts in P. cordatum could be induced by centrifugation, temperature decrease, changes in salinity, and treatment by 2,6-dichlorobenzonitrile, whereas temperature increase, changes in pH and treatment by tetracycline did not have this effect. Obtained cysts of P. cordatum did not contain the pellicular layer and were formed in the end of the first stage of this process, i.e. removal of the plasma membrane and the outer amphiesmal vesicle membrane, whereas its second stage, removal of theca, represented excystment. Based on our findings, we conclude that such cysts can be attributed to thecate cysts and suggest P. cordatum as a promising model organism for the investigation of cellular and molecular aspects of ecdysis in dinoflagellates.

The Uncoupled Assimilation of Carbon and Nitrogen from Urea and Glycine by the Bloom-forming Dinoflagellate Prorocentrum minimum.

The spread of harmful dinoflagellate blooms has been linked to the increasing availability of nitrogen, including its dissolved organic forms. The relationships between organic and inorganic nutrient uptake by dinoflagellates are not completely understood; moreover, it is not clear whether organic substances are used exclusively as nitrogen or also as carbon sources. We used laboratory culture experiments to investigate the concurrent uptake of glycine and nitrate by Prorocentrum minimum and estimate a role of two widespread organic substrates, glycine and urea, as carbon sources. Glycine uptake exceeded the uptake of nitrate when both nutrients were present in equal nitrogen amounts. Carbon of urea and glycine constituted only 0.4% and 1.3% of the total carbon uptake by cells, respectively, and this amount of carbon was disproportionately small compared to nitrogen taken up from the same organic substrates indicating uncoupling of organic carbon and nitrogen assimilation. We suggest that the observed uncoupling of organic nitrogen and carbon assimilation is a result of urea and glycine metabolic processing by urease and the glycine decarboxylation complex. We argue that such uncoupling reduces the net dissolved inorganic carbon (DIC) removal by dinoflagellates since the acquisition of nitrogen from urea and glycine leads to DIC release.

Diversity and evolution of four-domain voltage-gated cation channels of eukaryotes and their ancestral functional determinants.

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, Cavß-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 Cavß-binding.

Superposition of Individual Activities: Urea-Mediated Suppression of Nitrate Uptake in the Dinoflagellate Prorocentrum minimum Revealed at the Population and Single-Cell Levels.

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.

Obtaining spheroplasts of armored dinoflagellates and first single-channel recordings of their ion channels using patch-clamping.

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.

Nitrogen isotope effects induced by anammox bacteria.

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 (N2) 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 N2 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 N2 (+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 (NOx) pool is depleted in (15)N relative to NOx. This net N isotope effect by anammox is superimposed on the N isotope fractionation by the co-occurring reduction of nitrate to nitrite in suboxic waters, possibly enhancing the overall N isotope effect for N loss from oxygen minimum zones.