Wide ecological niches ensure frequent harmful dinoflagellate blooms.

Harmful algal blooms (HABs) and their consequences cause multiple devastating effects in various freshwater, brackish and marine ecosystems. However, HAB species at moderate population densities have positive ecological roles as primary producers of organic matter and food for zooplankton and fish. They also enhance benthic-pelagic coupling and participate in the biogeochemical cycles. The consequences of HABs are transported across the conventional environmental boundaries by numerous cascade effects in the food webs and beyond. Meanwhile, forecasts of bloom events are still limited, largely because of scarcity of reliable information on ecological niches of the bloom-forming algae. To fill up this knowledge gap, this study focused on dinoflagellates, a diverse group of mostly photosynthesizing protists (unicellular eukaryotes) capable of mixotrophy, since they play a key role in primary production and formation of blooms in marine and brackish waters worldwide. In this study, ecological niches of 17 abundant bloom-forming dinoflagellate species from coastal regions of the southern Baltic Sea were identified for the first time. It was hypothesized that wider ecological niches ensure more frequent dinoflagellate blooms compared to the species with narrower niches. This hypothesis was verified using the long-term (44 years) database on phytoplankton abundance and physical-chemical characteristics of the environment. It were analyzed 4534 datasets collected from 1972 to 2016. Fourteen abiotic parameters (water temperature, salinity, Secchi depth, pH, Chl a, and concentration of basic nutrients) were considered as ecological niche dimensions. The Principal Component Analysis presented the dissolved inorganic nitrogen, total nitrogen, Chl a, and temperature as principal niche dimensions of dinoflagellates. The algal bloom criteria were refined. It was for the first time proved statistically that HAB frequency of dinoflagellate species robustly correlated with the width of their ecological niches.

The first arctic strain of Rhizochromulina: Morphology, ultrastructure, and position in the evolutionary tree of the order Rhizochromulinales (Heterokontophyta, Dictyochophyceae).

Rhizochromulina marina is a unicellular amoeboid alga capable of forming flagellate cells; it is a single validly named species in the genus. Besides, there are numerous environmental sequences and undescribed strains designated as Rhizochromulina sp. or R. marina. The biogeography of the genus is understudied: rhizochromulines from the Indian, Southern, and Arctic Oceans are unknown. Here, we present the description of Rhizochromulina sp. B44, which was for the first time isolated from an arctic habitat. Biofilms of this microalga grow at the bottom of a culture vessel, where neighbouring amoeboid cells form associations through a common network of pseudopodia, i.e. meroplasmodia. Pseudopodia branch, anastomose mainly during meroplasmodia formation, and are supported by microtubules that arise from the perinuclear zone. Actin filaments are localized in the cytoplasm and can be revealed only near the bases of pseudopodia. We succeeded in inducing the transformation of amoeboid cells into flagellates using a prolonged agitation of cultures. Morphological and molecular analyses revealed that the studied strain is most closely related to the type strain of R. marina. At the same time, 18S rDNA sequences of early branching-off rhizochromulinids differ significantly from Rhizochromulina sp. B44, suggesting a high divergence at the genus level.

Phosphorus deficiency induces sexual reproduction in the dinoflagellate Prorocentrum cordatum.

Nitrogen (N) and phosphorus (P) are essential elements whose availability promotes successful growth of phytoplankton and governs aquatic primary productivity. In this study, we investigated the effect of N and/or P deficiency on the sexual reproduction of Prorocentrum cordatum, the dinoflagellate with the haplontic life cycle which causes harmful algal blooms worldwide. In P. cordatum cultures, N and the combined N and P deficiency led to the arrest of the cell cycle in the G0/G1 phases and attenuation of cell culture growth. We observed, that P, but not N deficiency triggered the transition in the life cycle of P. cordatum from vegetative to the sexual stage. This resulted in a sharp increase in percentage of cells with relative nuclear DNA content 2C (zygotes) and the appearance of cells with relative nuclear DNA content 4C (dividing zygotes). Subsequent supplementation with phosphate stimulated meiosis and led to a noticeable increase in the 4C cell number (dividing zygotes). Additionally, we performed transcriptomic data analysis and identified putative phosphate transporters and enzymes involved in the phosphate uptake and regulation of its metabolism by P. cordatum. These include high- and low-affinity inorganic phosphate transporters, atypical alkaline phosphatase, purple acid phosphatases and SPX domain-containing proteins.

Putative Meiotic Toolkit in the Dinoflagellate Prorocentrum cordatum: Additional Evidence for Sexual Process from Transcriptome.

Prorocentrum cordatum (Ostenfeld) Dodge-is a planktonic armored dinoflagellate that is a bloom-forming, potentially toxic cosmopolitan species. The transition from vegetative reproduction to the sexual process has been recently shown for this organism. Here, we present the results of transcriptomic data analysis that uncovered one syngamy-associated and 16 meiosis-associated proteins in P. cordatum. We also detected an amino acid sequence homologous to bacterial MutS2 protein. The MutS2 presence and origin in dinoflagellates are discussed for the first time.

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.

Diversity of voltage-gated potassium channels and cyclic nucleotide-binding domain-containing channels in eukaryotes.

Voltage-gated potassium channels (Kv) and cyclic nucleotide-binding domain-containing cation channels HCN, CNG, and KCNH are the evolutionarily related families of ion channels in animals. Their homologues were found in several lineages of eukaryotes and prokaryotes; however, the actual phylogenetic and structural diversity of these ion channels remains unclear. In this work, we present a taxonomically broad investigation of evolutionary relationships and structural diversity of Kv, HCN, CNG, and KCNH and their homologues in eukaryotes focusing on channels from different protistan groups. We demonstrate that both groups of channels consist of a more significant number of lineages than it was shown before, and these lineages can be grouped in two clusters termed Kv-like channels and CNBD-channels. Moreover, we, for the first time, report the unusual two-repeat tandem Kv-like channels and CNBD-channels in several eukaryotic groups, i.e. dinoflagellates, oomycetes, and chlorarachniophytes. Our findings reveal still underappreciated phylogenetic and structural diversity of eukaryotic ion channel lineages.

Life Cycle Stages and Evidence of Sexual Reproduction in the Marine Dinoflagellate Prorocentrum minimum (Dinophyceae, Prorocentrales).

Prorocentrum minimum is a potentially toxic marine dinoflagellate that often forms massive blooms in estuarine and coastal sea waters. In this study, the life cycle of P. minimum was investigated and sexual reproduction in culture was described for the first time. Morphology of the mitotic stages was revised and several distinguishing features from sexual steps were described. The sexual reproductive stages were observed in the stationary culture and compared with a well-studied closely related species, Prorocentrum micans. Prorocentrum minimum has a haplontic life cycle and homothallic sexual process. The gametes were isogamous and morphologically indistinguishable from the vegetative cells. Unlike P. micans, P. minimum isogametes fused, but did not conjugate, partially reorganizing their cell coverings. Newly formed planozygotes were distinguished by their irregular shape and a large asymmetrically located nucleus. No long-term resting cyst stages (hypnozygotes) were documented. The late planozygotes underwent meiosis and formed tetrads of cells. The second meiotic division could be delayed or arrested in one of the daughter nuclei leading to formation of trinucleate cells with three pairs of flagella. So, similar to P. micans, P. minimum may have two possible scenarios of sexual division: (a) formation of a four-cell stage through two successive divisions or (b) asynchronous divisions of the zygote. Changes in the DNA content were confirmed by quantitative image cytometry.

Chaos theory discloses triggers and drivers of plankton dynamics in stable environment.

Despite the enticing discoveries of chaos in nature, triggers and drivers of this phenomenon remain a classical enigma which needs irrefutable empirical evidence. Here we analyze results of the yearlong replicated mesocosm experiment with multi-species plankton community that allowed revealing signs of chaos at different trophic levels in strictly controlled abiotic environment. In mesocosms without external stressors, we observed the "paradox of chaos" when biotic interactions (internal drivers) were acting as generators of internal abiotic triggers of complex plankton dynamics. Chaos was registered as episodes that vanished unpredictably or were substituted by complex behaviour of other candidates when longer time series were considered. Remarkably, episodes of chaos were detected even in the most abiotically stable conditions. We developed the Integral Chaos Indicator to validate the results of the Lyapunov exponent analysis. These findings are essential for modelling and forecasting behaviour of a variety of natural and other global systems.

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.

Ecological niche partitioning of the invasive dinoflagellate Prorocentrum minimum and its native congeners in the Baltic Sea.

This study analyses three decades of the peculiar bloom-formation history of the potentially toxic invasive planktonic dinoflagellates Prorocentrum minimum (Pavillard) Schiller in the SW Baltic Sea. We tested a research hypothesis that the unexpectedly long delay (nearly two decades) in population development of P. minimum prior to its first bloom was caused by competition with one or several closely related native dinoflagellate species due to ecological niche partitioning which hampered the spread and bloom-forming potential of the invader. We applied the ecological niche concept to a large, long-term phytoplankton database and analysed the invasion history and population dynamics of P. minimum in the SW Baltic Sea coastal waters using the data on phytoplankton composition, abundance and biomass. The ecological niche dimensions of P. minimum and its congener P. balticum were identified as the optimum environmental conditions for the species during the bloom events based on water temperature, salinity, pH, concentration of nutrients (PO43-; total phosphorus, TP; total nitrogen, TN; SiO44-), TN/TP-ratio and habitat type. The data on spatial distribution and ecological niche dimensions of P. minimum have contributed to the development of the "protistan species maximum concept". High microplankton diversity at critical salinities in the Baltic Sea may be considered as a possible reason for the significant niche overlap and strong competitive interactions among congeners leading to prolonged delay in population growth of P. minimum preceding its first bloom in the highly variable brackishwater environment.

The size of DNA molecules and chromatin organization in the macronucleus of the ciliate Didinium nasutum (Ciliophora).

Pulsed-field gel electrophoresis (PFGE) was applied to analyze the molecular karyotype of the ciliate Didinium nasutum. The data obtained indicate that D. nasutum belongs to the ciliate species with subchromosomal macronuclear genome organization. No short "gene-sized" DNA molecules were detected. Macronuclear DNAs formed a continuous spectrum from 50 kbp to approximately 1,000 kbp in size with a peak plateau between 250 and 400 kbp. The macronuclear DNA molecules were packed into chromatin bodies of 80-265 nm in size. Comparison of the PFGE and electron microscopic data shows that most if not all chromatin bodies contain more than one DNA molecule.

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.

Quantitative analysis of nucleolar chromatin distribution in the complex convoluted nucleoli of Didinium nasutum (Ciliophora).

We have earlier shown that the typical Didinium nasutum nucleolus is a complex convoluted branched domain, comprising a dense fibrillar component located at the periphery of the nucleolus and a granular component located in the central part. Here our main interest was to study quantitatively the spatial distribution of nucleolar chromatin structures in these convoluted nucleoli. There are no "classical" fibrillar centers in D.nasutum nucleoli. The spatial distribution of nucleolar chromatin bodies, which play the role of nucleolar organizers in the macronucleus of D.nasutum, was studied using 3D reconstructions based on serial ultrathin sections. The relative number of nucleolar chromatin bodies was determined in macronuclei of recently fed, starved D.nasutum cells and in resting cysts. This parameter is shown to correlate with the activity of the nucleolus. However, the relative number of nucleolar chromatin bodies in different regions of the same convoluted nucleolus is approximately the same. This finding suggests equal activity in different parts of the nucleolar domain and indicates the existence of some molecular mechanism enabling it to synchronize this activity in D. nasutum nucleoli. Our data show that D. nasutum nucleoli display bipartite structure. All nucleolar chromatin bodies are shown to be located outside of nucleoli, at the periphery of the fibrillar component.

Quantitative analysis of nucleolar chromatin distribution in the complex convoluted nucleoli of Didinium nasutum (Ciliophora)

We have earlier shown that the typical Didinium nasutum nucleolus is a complex convoluted branched domain, comprising a dense fibrillar component located at the periphery of the nucleolus and a granular component located in the central part. Here our main interest was to study quantitatively the spatial distribution of nucleolar chromatin structures in these convoluted nucleoli. There are no "classical" fibrillar centers in D.nasutum nucleoli. The spatial distribution of nucleolar chromatin bodies, which play the role of nucleolar organizers in the macronucleus of D.nasutum, was studied using 3D reconstructions based on serial ultrathin sections. The relative number of nucleolar chromatin bodies was determined in macronuclei of recently fed, starved D.nasutum cells and in resting cysts. This parameter is shown to correlate with the activity of the nucleolus. However, the relative number of nucleolar chromatin bodies in different regions of the same convoluted nucleolus is approximately the same. This finding suggests equal activity in different parts of the nucleolar domain and indicates the existence of some molecular mechanism enabling it to synchronize this activity in D. nasutum nucleoli. Our data show that D. nasutum nucleoli display bipartite structure. All nucleolar chromatin bodies are shown to be located outside of nucleoli, at the periphery of the fibrillar component.

Macroalgal diversity along the Baltic Sea salinity gradient challenges Remane's species-minimum concept.

Remane's species-minimum concept, which states that the lowest number of taxa occurs at the horohalinicum (5-8psu), was tested by investigating macroalgal diversity on hard substrates along the natural salinity gradient in the Baltic Sea. Field data on species occurrence and abundance were collected by SCUBA diving along 10 transects of the Finnish, Swedish and German coasts, covering a salinity range from 3.9 to 27psu. Macroalgal species numbers declined steadily with salinity, decreasing until 7.2psu was reached, but in the horohalinicum, a marked reduction of species number and a change in diversity were indicated by the Shannon index and evenness values. The non-linear decrease in macroalgal diversity at 5-8psu and the lack of increase in species numbers at salinities below 5psu imply a restricted applicability of Remane's species-minimum concept to macroalgae.

Emerging microsporidian infections in Russian HIV-infected patients.

Microsporidia were identified in stool specimens by histochemistry and PCR of 30 (18.9%) of 159 HIV-infected patients presenting to the S. P. Botkin Memorial Clinical Hospital of Infectious Diseases, St. Petersburg, Russia. The higher prevalence of Encephalitozoon intestinalis, in 21 (12.8%) patients, than of Enterocytozoon bieneusi, in 2 patients (1.2%), was unexpected. Encephalitozoon cuniculi was detected in three patients: one with strain I and two with strain II. Encephalitozoon hellem was detected in one patient, and two patients were identified as being infected by Microsporidium species. One patient was infected with both E. intestinalis and E. cuniculi. In two patients, the microsporidian species were not identifiable. No statistically significant differences in gender, age, and stage of AIDS were observed between the microsporidian-positive and -negative HIV-infected patients. HIV-infected patients diagnosed with microsporidian infection, however, were significantly more likely to exhibit ≤ 100 CD4(+) T cells/µl blood (20/30 patients [67%]; odds ratio [OR], 3.150; 95% confidence interval [CI(95)], 1.280 to 7.750; P = 0.0116) and weight loss of >10% of the baseline (19/30 patients [63%]; odds ratio, 2.995; CI(95), 1.100 to 8.158; P = 0.0352) than HIV-infected patients not diagnosed with microsporidian infection. In summary, this is the first report describing the diagnosis of microsporidian infection of HIV-infected patients in Russia and the first detection of E. cuniculi strain II in a human.

Analogs of the Golgi complex in microsporidia: structure and avesicular mechanisms of function.

Microsporidia are obligatory intracellular parasites, most species of which live in the host cell cytosol. They synthesize and then transport secretory proteins from the endoplasmic reticulum to the plasma membrane for formation of the spore wall and the polar tube for cell invasion. However, microsporidia do not have a typical Golgi complex. Here, using quick-freezing cryosubstitution and chemical fixation, we demonstrate that the Golgi analogs of the microsporidia Paranosema (Antonospora) grylli and Paranosema locustae appear as 300-nm networks of thin (25- to 40-nm diameter), branching or varicose tubules that display histochemical features of a Golgi, but that do not have vesicles. Vesicles are not formed even if membrane fusion is inhibited. These tubular networks are connected to the endoplasmic reticulum, the plasma membrane and the forming polar tube, and are positive for Sec13, gammaCOP and analogs of giantin and GM130. The spore-wall and polar-tube proteins are transported from the endoplasmic reticulum to the target membranes through these tubular networks, within which they undergo concentration and glycosylation. We suggest that the intracellular transport of secreted proteins in microsporidia occurs by a progression mechanism that does not involve the participation of vesicles generated by coat proteins I and II.