Differential Expression of Stress Adaptation Genes in a Diatom Ulnaria acus under Different Culture Conditions.

Diatoms are a group of unicellular eukaryotes that are essential primary producers in aquatic ecosystems. The dynamic nature of their habitat necessitates a quick and specific response to various stresses. However, the molecular mechanisms of their physiological adaptations are still underexplored. In this work, we study the response of the cosmopolitan freshwater diatom Ulnaria acus (Bacillariophyceae, Fragilariophycidae, Licmophorales, Ulnariaceae, Ulnaria) in relation to a range of stress factors, namely silica deficiency, prolonged cultivation, and interaction with an algicidal bacterium. Fluorescent staining and light microscopy were used to determine the physiological state of cells under these stresses. To explore molecular reactions, we studied the genes involved in the stress response-type III metacaspase (MC), metacaspase-like proteases (MCP), death-specific protein (DSP), delta-1-pyrroline-5-carboxylate dehydrogenase (ALDH12), and glutathione synthetase (GSHS). We have described the structure of these genes, analyzed the predicted amino acid sequences, and measured their expression dynamics in vitro using qRT-PCR. We demonstrated that the expression of UaMC1, UaMC3, and UaDSP increased during the first five days of silicon starvation. On the seventh day, it was replaced with the expression of UaMC2, UaGSHS, and UaALDH. After 45 days of culture, cells stopped growing, and the expression of UaMC1, UaMC2, UaGSHS, and UaDSP increased. Exposure to an algicidal bacterial filtrate induced a higher expression of UaMC1 and UaGSHS. Thus, we can conclude that these proteins are involved in diatoms' adaptions to environmental changes. Further, these data show that the molecular adaptation mechanisms in diatoms depend on the nature and exposure duration of a stress factor.

The effect of metabarcoding 18S rRNA region choice on diversity of microeukaryotes including phytoplankton.

Metabarcoding using high throughput sequencing of amplicons of the 18S rRNA gene is one of the widely used methods for assessing the diversity of microeukaryotes in various ecosystems. We investigated the effectiveness of the V4 and V8-V9 regions of the 18S rRNA gene by comparing the results of metabarcoding microeukaryotic communities using the DADA2 (ASV), USEARCH-UNOISE3 (ZOTU), and USEARCH-UPARSE (OTU with 97% similarity) algorithms. Both regions showed similar levels of genetic variability and taxa identification accuracy. Richness for DADA2 datasets of both regions was lower than for UNOISE3 and UPARSE datasets, which is due to more accurate error correction in amplicons. Microeukaryotic communities (autotrophs and heterotrophs) structure identified using both regions showed a significant relationship with phytoplankton (autotrophs) communities structure based on microscopy in a seasonal freshwater sample series. The strongest relationship was found between the phytoplankton species and V8-V9 ASVs produced by DADA2.

Variability of Microbial Communities in Two Long-Term Ice-Covered Freshwater Lakes in the Subarctic Region of Yakutia, Russia.

Although under-ice microbial communities are subject to a cold environment, low concentrations of nutrients, and a lack of light, they nevertheless take an active part in biogeochemical cycles. However, we still lack an understanding of how high their diversity is and how these communities are distributed during the long-term ice-cover period. Here, we assessed for the first time the composition and distribution of microbial communities during the ice-cover period in two subarctic lakes (Labynkyr and Vorota) located in the area of the lowest temperature in the Northern Hemisphere. The diversity distribution and abundance of main bacterial taxa as well as the composition of microalgae varied by time and habitat. The 16S rRNA gene sequencing method revealed, in general, a high diversity of bacterial communities where Proteobacteria (~ 45%) and Actinobacteria (~ 21%) prevailed. There were significant differences between the communities of the lakes: Chthoniobacteraceae, Moraxellaceae, and Pirellulaceae were abundant in Lake Labynkyr, while Cyanobiaceae, Oligoflexales, Ilumatobacteraceae, and Methylacidiphilaceae were more abundant in Lake Vorota. The most abundant families were evenly distributed in April, May, and June their contribution was different in different habitats. In April, Moraxellaceae and Ilumatobacteraceae were the most abundant in the water column, while Sphingomonadaceae was abundant both in water column and on the ice bottom. In May, the abundance of Comamonadaceae increased and reached the maximum in June, while Cyanobiaceae, Oxalobacteraceae, and Pirellulaceae followed. We found a correlation of the structure of bacterial communities with snow thickness, pH, Nmin concentration, and conductivity. We isolated psychrophilic heterotrophic bacteria both from dominating and minor taxa of the communities studied. This allowed for specifying their ecological function in the under-ice communities. These findings will advance our knowledge of the under-ice microbial life.

Seasonal Succession and Coherence Among Bacteria and Microeukaryotes in Lake Baikal.

Microorganisms exhibit seasonal succession governed by physicochemical factors and interspecies interactions, yet drivers of this process in different environments remain to be determined. We used high-throughput sequencing of 16S rRNA and 18S rRNA genes to study seasonal dynamics of bacterial and microeukaryotic communities at pelagic site of Lake Baikal from spring (under-ice, mixing) to autumn (direct stratification). The microbial community was subdivided into distinctive coherent clusters of operational taxonomic units (OTUs). Individual OTUs were consistently replaced during different seasonal events. The coherent clusters change their contribution to the microbial community depending on season. Changes of temperature, concentrations of silicon, and nitrates are the key factors affected the structure of microbial communities. Functional prediction revealed that some bacterial or eukaryotic taxa that switched with seasons had similar functional properties, which demonstrate their functional redundancy. We have also detected specific functional properties in different coherent clusters of bacteria or microeukaryotes, which can indicate their ability to adapt to seasonal changes of environment. Our results revealed a relationship between seasonal succession, coherency, and functional features of freshwater bacteria and microeukaryotes.

The insight into diatom diversity, ecology, and biogeography of an extreme cold ultraoligotrophic Lake Labynkyr at the Pole of Cold in the northern hemisphere.

The freshwater ultraoligotrophic Lake Labynkyr is located near the Pole of Cold in the northern hemisphere (Yakutia, Russia). The lake is covered by ice during 240 days a year. We undertook several expeditions to the lake during the ice and open water periods for sampling ice fouling, plankton and periphyton that were then analyzed by means of scanning electron microscopy. As a result, we identified a high biodiversity of diatoms-123 species and intraspecific taxa from 53 genera, among them 3 species were new for Russia and 26 taxa were new for the algal flora of Yakutia. The oligo- and xenosaprobionts and their variations dominate-71 taxa. 18 Species were evaluated as tolerant to cold oligotrophic waters, 12 occurred on the ice bottom, and 62 in the water column under ice (0-25 m). 104 taxa were found during the open water period, 70 taxa were identified in the periphyton. We showed the diatom flora of Lake Labynkyr to be unique compared with other lakes of Yakutia and to share taxa with the diatom flora of Lake Baikal. The diatoms being indicators of the global climate changes and ecological status of lakes, our data can be used as an evidence of such changes as well as to be useful studies of biogeography and history of formation of flora in Arctic and Subarctic waters.

De novo transcriptome assembly and analysis of the freshwater araphid diatom Fragilaria radians, Lake Baikal.

Diatoms are a group of eukaryotic microalgae populating almost all aquatic and wet environments. Their abundance and species diversity make these organisms significant contributors to biogeochemical cycles and important components of aquatic ecosystems. Although significant progress has been made in studies of Diatoms (Bacillariophyta) over the last two decades, since the spread of "omics" technologies, our current knowledge of the molecular processes and gene regulatory networks that facilitate environmental adaptation remain incomplete. Here, we present a transcriptome analysis of Fragilaria radians isolated from Lake Baikal. The resulting assembly contains 27,446 transcripts encoding 21,996 putative proteins. The transcriptome assembly and annotation were coupled with quantitative experiments to search for differentially expressed transcripts between (i) exponential growth phase and dark-acclimated cell cultures, and (ii) those changing expression level during the early response to light treatment in dark-acclimated cells. The availability of F. radians genome and transcriptome data provides the basis for future targeted studies of this species. Furthermore, our results extend taxonomic and environmental sampling of Bacillariophyta, opening new opportunities for comparative omics-driven surveys.

Co-occurrence patterns between phytoplankton and bacterioplankton across the pelagic zone of Lake Baikal during spring.

Phytoplankton and bacterioplankton play a key role in carbon cycling of aquatic ecosystems. In this study, we found that co-occurrence patterns between different types of phytoplankton, bacterioplankton, and environmental parameters in Lake Baikal during spring were different over the course of three consecutive years. The composition of phytoplankton and bacterial communities was investigated using microscopy and 16S rRNA gene pyrosequencing, respectively. Non-metric multidimensional scaling (NMDS) revealed a relationship between the structure of phytoplankton and bacterial communities and temperature, location, and sampling year. Associations of bacteria with diatoms, green microalgae, chrysophyte, and cryptophyte were identified using microscopy. Cluster analysis revealed similar correlation patterns between phytoplankton abundance, number of attached bacteria, ratio of bacteria per phytoplankton cell and environmental parameters. Positive and negative correlations between different species of phytoplankton, heterotrophic bacteria and environmental parameters may indicate mutualistic or competitive relationships between microorganisms and their preferences to the environment.

Correction to: Co-occurrence Networks Among Bacteria and Microbial Eukaryotes of Lake Baikal During a Spring Phytoplankton Bloom.

The original version of this article unfortunately contained mistakes in the legends of figures.

Co-occurrence Networks Among Bacteria and Microbial Eukaryotes of Lake Baikal During a Spring Phytoplankton Bloom.

The pelagic zone of Lake Baikal is an ecological niche where phytoplankton bloom causes increasing microbial abundance in spring which plays a key role in carbon turnover in the freshwater lake. Co-occurrence patterns revealed among different microbes can be applied to predict interactions between the microbes and environmental conditions in the ecosystem. We used 454 pyrosequencing of 16S rRNA and 18S rRNA genes to study bacterial and microbial eukaryotic communities and their co-occurrence patterns at the pelagic zone of Lake Baikal during a spring phytoplankton bloom. We found that microbes within one domain mostly correlated positively with each other and are highly interconnected. The highly connected taxa in co-occurrence networks were operational taxonomic units (OTUs) of Actinobacteria, Bacteroidetes, Alphaproteobacteria, and autotrophic and unclassified Eukaryota which might be analogous to microbial keystone taxa. Constrained correspondence analysis revealed the relationships of bacterial and microbial eukaryotic communities with geographical location.

The Role of Polyamine-Dependent Facilitation of Calcium Permeable AMPARs in Short-Term Synaptic Enhancement.

Depending on subunit composition AMPA receptor channels can be subdivided into two groups: GluA2-containing calcium impermeable AMPARs, and GluA2-lacking calcium permeable, AMPARs. These two groups differ in a number of biophysical properties and, most likely, in their functional role at glutamatergic synapses. GluA2-lacking channels have received a lot of attention over the last two decades mainly due to high calcium permeability, which was suggested to play a significant role in the induction of long-term synaptic plasticity in healthy tissue and neuronal death under neuropathological conditions. However, calcium permeable AMPARs possess another property that can contribute substantially to frequency dependent dynamics of synaptic efficacy. In the closed state calcium permeable AMPARs are blocked by endogenous polyamines, however, repetitive activation leads to progressive relief from the block and to the facilitation of ion flux through these channels. Polyamine-dependent facilitation of AMPARs can contribute to short-term plasticity at synapses that have high initial release probability and express calcium permeable AMPARs. During synaptic transmission activity-dependent relief from polyamine block of postsynaptic calcium-permeable AMPARs either counteracts presynaptic short-term depression in a frequency-dependent manner or, under specific stimulation conditions, induces facilitation of a synaptic response. Taking into account the fact that expression of calcium permeable AMPARs is developmentally regulated, depends on network activity and increases in diseased brain states, polyamine-dependent facilitation of calcium permeable AMPARs is an important, entirely postsynaptic mechanism of synaptic gain regulation.

A family of silicon transporter structural genes in a pennate diatom Synedra ulna subsp. danica (Kütz.) Skabitsch.

Silicon transporters (SIT) are the proteins, which capture silicic acid in the aquatic environment and direct it across the plasmalemma to the cytoplasm of diatoms. Diatoms utilize silicic acid to build species-specific ornamented exoskeletons and make a significant contribution to the global silica cycle, estimated at 240 ±40 Tmol a year. Recently SaSIT genes of the freshwater araphid pennate diatom Synedra acus subsp. radians are found to be present in the genome as a cluster of two structural genes (SaSIT-TD and SaSIT-TRI) encoding several concatenated copies of a SIT protein each. These structural genes could potentially be transformed into "mature" SIT proteins by means of posttranslational proteolytic cleavage. In the present study, we discovered three similar structural SuSIT genes in the genome of a closely related freshwater diatom Synedra ulna subsp. danica. Structural gene SuSIT1 is identical to structural gene SuSIT2, and the two are connected by a non-coding nucleotide DNA sequence. All the putative "mature" SITs contain conserved amino acid motifs, which are believed to be important in silicon transport. The data obtained suggest that the predicted "mature" SIT proteins may be the minimal units necessary for the transport of silicon is S. ulna subsp. danica. The comparative analysis of all available multi-SITs has allowed us to detect two conservative motifs YQXDXVYL and DXDID, located between the "mature" proteins. Aspartic acid-rich DXDID motif can, in our opinion, serve as a proteolysis site during the multi-SIT cleavage. The narrow distribution of the distances between CMLD and DXDID motifs can serve as additional evidence to the conservation of their function.

Bacterial community composition in the water column of the deepest freshwater Lake Baikal as determined by next-generation sequencing.

The composition of bacterial communities in Lake Baikal in different hydrological periods and at different depths (down to 1515 m) has been analyzed using pyrosequencing of the 16S rRNA gene V3 variable region. Most of the resulting 34 562 reads of the Bacteria domain have clustered into 1693 operational taxonomic units (OTUs) classified with the phyla Proteobacteria, Actinobacteria, Chloroflexi, Bacteroidetes, Firmicutes, Acidobacteria and Cyanobacteria. It has been found that their composition at the family level and relative contributions to bacterial communities distributed over the water column vary depending on hydrological period. The number of OTUs and the parameters of taxonomic richness (ACE, Chao1 indices) and diversity (Shannon and inverse Simpson index) reach the highest values in water layers. The composition of bacterial communities in these layers remains relatively constant, whereas that in surface layers differs between hydrological seasons. The dynamics of physicochemical conditions over the water column and their relative constancy in deep layers are decisive factors in shaping the pattern of bacterial communities in Lake Baikal.

The Relative Contribution of NMDARs to Excitatory Postsynaptic Currents is Controlled by Ca(2+)-Induced Inactivation.

NMDA receptors (NMDARs) are important mediators of excitatory synaptic transmission and plasticity. A hallmark of these channels is their high permeability to Ca(2+). At the same time, they are themselves inhibited by the elevation of intracellular Ca(2+) concentration. It is unclear however, whether the Ca(2+) entry associated with single NMDAR mediated synaptic events is sufficient to self-inhibit their activation. Such auto-regulation would have important effects on the dynamics of synaptic excitation in several central neuronal networks. Therefore, we studied NMDAR-mediated synaptic currents in mouse hippocampal CA1 pyramidal neurons. Postsynaptic responses to subthreshold Schaffer collateral stimulation depended strongly on the absence or presence of intracellular Ca(2+) buffers. Loading of pyramidal cells with exogenous Ca(2+) buffers increased the amplitude and decay time of NMDAR mediated EPSCs (EPSPs) and prolonged the time window for action potential (AP) generation. Our data indicate that the Ca(2+) influx mediated by unitary synaptic events is sufficient to produce detectable self-inhibition of NMDARs even at a physiological Mg(2+) concentration. Therefore, the contribution of NMDARs to synaptic excitation is strongly controlled by both previous synaptic activity as well as by the Ca(2+) buffer capacity of postsynaptic neurons.

Sub-Ice Microalgal and Bacterial Communities in Freshwater Lake Baikal, Russia.

The sub-ice environment of Lake Baikal represents a special ecotope where strongly increasing microbial biomass causes an "ice-bloom" contributing therefore to the ecosystem functioning and global element turnover under low temperature in the world's largest freshwater lake. In this work, we analyzed bacterial and microalgal communities and their succession in the sub-ice environment in March-April 2010-2012. It was found out that two dinoflagellate species (Gymnodinium baicalense var. minor and Peridinium baicalense Kisselew et Zwetkow) and four diatom species (Aulacoseira islandica, A. baicalensis, Synedra acus subsp. radians, and Synedra ulna) predominated in the microalgal communities. Interestingly, among all microalgae, the diatom A. islandica showed the highest number of physically attached bacterial cells (up to 67 ± 16 bacteria per alga). Bacterial communities analyzed with pyrosequencing of 16S rRNA gene fragments were diverse and represented by 161 genera. Phyla Proteobacteria, Verrucomicrobia, Actinobacteria, Acidobacteria, Bacteroidetes, and Cyanobacteria represented a core community independently on microalgal composition, although the relative abundance of these bacterial phyla strongly varied across sampling sites and time points; unique OTUs from other groups were rare.

Layer Specific Development of Neocortical Pyramidal to Fast Spiking Cell Synapses.

All cortical neurons are engaged in inhibitory feedback loops which ensure excitation-inhibition balance and are key elements for the development of coherent network activity. The resulting network patterns are strongly dependent on the strength and dynamic properties of these excitatory-inhibitory loops which show pronounced regional and developmental diversity. Therefore we compared the properties and postnatal maturation of two different synapses between rat neocortical pyramidal cells (layer 2/3 and layer 5, respectively) and fast spiking (FS) interneurons in the corresponding layer. At P14, both synapses showed synaptic depression upon repetitive activation. Synaptic release properties between layer 2/3 pyramidal cells and FS cells were stable from P14 to P28. In contrast, layer 5 pyramidal to FS cell connections showed a significant increase in paired pulse ratio by P28. Presynaptic calcium dynamics also changed at these synapses, including sensitivity to exogenously loaded calcium buffers and expression of presynaptic calcium channel subtypes. These results underline the large variety of properties at different, yet similar, synapses in the neocortex. They also suggest that postnatal maturation of the brain goes along with increasing differences between synaptically driven network activity in layer 5 and layer 2/3.

The structure of microbial community and degradation of diatoms in the deep near-bottom layer of Lake Baikal.

Insight into the role of bacteria in degradation of diatoms is important for understanding the factors and components of silica turnover in aquatic ecosystems. Using microscopic methods, it has been shown that the degree of diatom preservation and the numbers of diatom-associated bacteria in the surface layer of bottom sediments decrease with depth; in the near-bottom water layer, the majority of bacteria are associated with diatom cells, being located either on the cell surface or within the cell. The structure of microbial community in the near-bottom water layer has been characterized by pyrosequencing of the 16S rRNA gene, which has revealed 149 208 unique sequences. According to the results of metagenomic analysis, the community is dominated by representatives of Proteobacteria (41.9%), Actinobacteria (16%); then follow Acidobacteria (6.9%), Cyanobacteria (5%), Bacteroidetes (4.7%), Firmicutes (2.8%), Nitrospira (1.6%), and Verrucomicrobia (1%); other phylotypes account for less than 1% each. For 18.7% of the sequences, taxonomic identification has been possible only to the Bacteria domain level. Many bacteria identified to the genus level have close relatives occurring in other aquatic ecosystems and soils. The metagenome of the bacterial community from the near-bottom water layer also contains 16S rRNA gene sequences found in previously isolated bacterial strains possessing hydrolytic enzyme activity. These data show that potential degraders of diatoms occur among the vast variety of microorganisms in the near-bottom water of Lake Baikal.