Silica-scaled heterotrophic protists: Rotosphaerida, Thaumatomonadida and Centroplasthelida in Arctic waters of Russia.

Maintaining high levels of biological diversity in various ecosystems is necessary for stable functioning of the Earth's biosphere. The article describes diversity and ecology of heterotrophic siliceous protists - rotosphaerids, colourless free-living thaumatomonad flagellates, and centrohelid heliozoans - in Arctic waters located of Asian Russia. Samples were collected in the mouths of the Olenyok, Yana, Indigirka, Kolyma Rivers - and small freshwater ponds near Settlement of Tiksi in Yakutia. Based on electron microscopy, 35 centrohelids (Heliozoa), 11 rotosphaerid species as well as four thaumatomonad flagellate species were found in the region. Seven species were recorded in Russia for the first time: Rabdiaster multicosta, Rabdiaster reticulata, Turriplaca denticulata, Choanocystis cf. cordiformis parvula, Raineriophrys pteromorphos, Pseudoraphidocystis glutinosa and Pseudoraphidiophrys formosa. For 43 species, the study area is the most northern location they were described. Morphological details of scales are discussed for selected species, particularly for widespread species of rotosphaerids - Pinaciophora fluviatilis. The literature on hydrochemical conditions was analyzed to find taxa with high sensitivity towards environmental changes. Such species could be further used for monitoring plankton, recovering evolutionary changes, and reconstructing past environments.

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.

Molecular Evolution of Tubulins in Diatoms.

Microtubules are formed by α- and ß-tubulin heterodimers nucleated with γ-tubulin. Tubulins are conserved eukaryotic proteins. Previously, it was shown that microtubules are involved in diatom silica frustule morphogenesis. Diatom frustules are varied, and their morphology is species-specific. Despite the attractiveness of the problem of elucidating the molecular mechanisms of genetically programmed morphogenesis, the structure and evolution of diatom tubulins have not been studied previously. Based on available genomic and transcriptome data, we analyzed the phylogeny of the predicted amino acid sequences of diatom α-, ß- and γ-tubulins and identified five groups for α-tubulins, six for ß-tubulins and four for γ-tubulins. We identified characteristic amino acids of each of these groups and also analyzed possible posttranslational modification sites of diatom tubulins. According to our results, we assumed what changes occurred in the diatom tubulin structures during their evolution. We also identified which tubulin groups are inherent in large diatom taxa. The similarity between the evolution of diatom tubulins and the evolution of diatoms suggests that molecular changes in α-, ß- and γ-tubulins could be one of the factors in the formation of a high morphological diversity of diatoms.

Silica-scaled heterotrophic protists Rotosphaerida, Thaumatomonadida, and Centroplasthelida in the large continuous ecosystem connecting Lake Baikal to the Kara Sea.

Heterotrophic protists Rotosphaerida, Thaumatomonadida, and centrohelid heliozoans are among the less studied silicified protists in terms of their biogeography and ecology. These organisms inhabit fresh and brackish water, and leave behind siliceous structural elements after death that are species-specific and amenable to electron microscopic analysis. This paper is the first to present data on species richness and taxonomic structure of silica-scaled heterotrophic protists-rotosphaerids, colorless free-living thaumatomonad flagellates and centrohelid heliozoans-in the large continuous water system of Siberia connecting Lake Baikal to the Kara Sea. In the study area, electron microscopy revealed 21 centrohelid heliozoan species from the genera Raphidiophrys (1), Acanthocystis (7), Choanocystis (3), Raineriophrys (2), Raphidocystis (6), and Pterocystis (2), seven rotosphaerid species from the genera Pinaciophora (3), Turriplaca (2), Rabdiophrys (1), and Pompholyxophrys (1), and one thaumatomonad flagellate species Thaumatomastix. Two species of rotosphaerids, Rabdiophrys cf. anulifera and Pinaciophora tridentata, and two species of centrohelid heliozoans, Acanthocystis cf. tubata, and A. cf. cornuta, were found in the waters of Russia for the first time. The most widespread species in fresh water from Lake Baikal to the Lower Yenissei River were Pinaciophora fluviatilis and Raineriophrys cf. fortesca. These species disappeared from the protist assemblages of the Yenissei gulf of the Kara Sea due to higher salinity, with only three species, Acanthocystis cf. mylnikovi (at 3.12‰ salinity) and A. pectinata and Raphidocystis sp. 2 (at 8‰ salinity), found in the area. The sensitivity of silica-scaled heterotrophic protists to changes in habitat parameters suggests that these microeukaryotes could be new indicator organisms.

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.

Early structural and functional changes in Baikal Sculpin gills exposed to suspended soot microparticles in experiment.

The toxic influence of soot microparticles on terrestrial organisms has been well studied, although there is scarce data on how microparticles could affect hydrobionts. We performed a first-ever study of the short-term (5 days) impact of furnace soot (0.005 g/L) on the structural and functional features of gill cells in the Baikal Sculpin species Paracottus knerii, Dybowski, 1874. The soot samples used in the experiment were composed of small (10-100 nm) particles and larger (up to 20 µm) aggregates. The dominant fractions of the polycyclic aromatic hydrocarbons of these microparticles were phenanthrene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzofluoranthenes, benzopyrenes, indeno[1,2,3-c,d]pyrenes, and benzo[ghi]perylene. Trace element analysis of the soot detected the presence of C, S, Si, Al, Ca, K, Mg, P, and Fe. The gill condition was assessed with electron scanning, transmission, and laser confocal microscopy. Soot induces degenerative changes in the macrostructure and surface of secondary lamellae and increases mucus production in fish gills. A decrease in mitochondrial activity, an increase in reactive oxygen species production, and an increase in the frequency of programmed cell death in gill epithelium were observed under the influence of soot. In chloride cells, an induction of macroautophagy was detected. In general, the changes in fish gills after the short-term influence of soot microparticles indicate the stress of respiratory and osmotic regulation systems in fish. The data obtained are important for forming a coherent picture of the impact of soot on hydrobionts and for developing bioindication methods for evaluating the risks of their influence on aquatic ecosystems.

Phylogeny and structural peculiarities of the EB proteins of diatoms.

The end-binding proteins are a family of microtubule-associated proteins; this family belongs to plus-end-tracking proteins (+TIPs) that regulate microtubule growth and stabilisation. Although the genes encoding EB proteins are found in all eukaryotic genomes, most studies of them have centred on one or another taxonomic group, without a broad comparative analysis. Here, we present a first phylogenetic analysis and a comparative analysis of domain structures of diatom EB proteins in comparison with other phyla of Chromista, red and green algae, as well as model organisms A. thaliana and H. sapiens. Phylogenetically, diatom EB proteins are separated into six clades, generally corresponding to the phylogeny of their respective organisms. The domain structure of this family is highly variable, but the CH and EBH domains responsible for binding tubulin and other MAPs are mostly conserved. Homologous modelling of the F. cylindrus EB protein shows that conserved motifs of the CH domain are positioned on the protein surface, which is necessary for their functioning. We hypothesise that high variance of the diatom C-terminal domain is caused by previously unknown interactions with a CAP-GLY motif of dynactin subunit p150. Our findings contribute to wider possibilities for further investigations of the cytoskeleton in diatoms.

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.

Frustule morphogenesis of raphid pennate diatom Encyonema ventricosum (Agardh) Grunow.

Diatoms stand out among other microalgae due to the high diversity of species-specific silica frustules whose components (valves and girdle bands) are formed within the cell in special organelles called silica deposition vesicles (SDVs). Research on cell structure and morphogenesis of frustule elements in diatoms of different taxonomic groups has been carried out since the 1950s but is still relevant today. Here, cytological features and valve morphogenesis in the freshwater raphid pennate diatom Encyonema ventricosum (Agardh) Grunow have been studied using light and transmission electron microscopy of cleaned frustules and ultrathin sections of cells, and scanning electron and atomic force microscopy of the frustule surface. Data have been obtained on chloroplast structure: the pyrenoid is spherical, penetrated by a lamella (a stack of two thylakoids); the girdle lamella consists of several short lamellae. The basic stages of frustule morphogenesis characteristic of raphid pennate diatoms have been traced, with the presence of cytoskeletal elements near SDVs being observed throughout this process. Degradation of the plasmalemma and silicalemma is shown to take place when the newly formed valve is released into the space between sister cells. The role of vesicular transport and exocytosis in the gliding of pennate diatoms is discussed.

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.

Evolutionary history of the chitin synthases of eukaryotes.

Chitin synthases are widespread among eukaryotes and known to have a complex evolutionary history in some of the groups. We have reconstructed the chitin synthase phylogeny using the most taxonomically comprehensive dataset currently available and have shown the presence of independently formed paralogous groups in oomycetes, ciliates, fungi, and all diatoms except raphid pennates. There were also two cases of horizontal gene transfer (HGT): transfer from fungus to early diatoms gave rise to diatom paralogous group, while transfer from raphid pennate diatom to Acantamoeba ancestor is, to our knowledge, restricted to a single gene in amoeba. Early evolution of chitin synthases is heavily obscured by paralogy, and further sequencing effort is necessary.

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.

Changes in the micro- and nanostructure of siliceous valves in the diatom Synedra acus under the effect of colchicine treatment at different stages of the cell cycle.

The important role of the cytoskeleton in the morphogenesis of siliceous frustule components, which are synthesized within the diatom cells, has been revealed due to experiments with microtubule inhibitors. It has been shown that colchicine entering the diatom cell inhibits polymerization of tubulin, the main protein of microtubules, thereby disrupting the normal processes of biogenic silica deposition and daughter valve morphogenesis. In this study, experiments with a synchronized culture of the pennate diatom Synedra acus have been performed to determine the timing and duration of the formation of various valve components and analyze the effect of colchicine at a subtoxic concentration on the structure of daughter valves at different stages of their morphogenesis. Electron microscopic analysis has revealed several types of micro- and nanoscale anomalies in daughter valve morphology, with their frequency varying depending on the time of colchicine treatment. Laser scanning microscopy of preparations vitally stained with Tubulin Tracker Green has shown that polymerized tubulin at early stages of valve morphogenesis is localized along the periphery of the developing valve. This is evidence for an important role of microtubules in the horizontal growth of the valve at the stage when its general structural pattern is established, including its shape and arrangement of basic micro- and nanostructures. Treatment with a microtubule inhibitor at a certain stage of valve morphogenesis makes it possible to obtain new forms with a specific structure of siliceous components that hold promise for use in nanotechnologies.

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.

Specific features of mandible structure and elemental composition in the polyphagous amphipod Acanthogammarus grewingkii endemic to Lake Baikal.

BACKGROUND: In crustaceans, several mechanisms provide for the mechanical strength of the cuticular "tools" (dactyli, claws, jaws), which serve to catch and crush food objects. Studies on the mandibles of the endemic Baikal amphipod Acanthogammarus grewingkii by means of electron microscopy and elemental analysis have revealed specific structural features of these mouthparts. METHODOLOGY: The fine structure of the mandible has been studied by means of SEM, TEM, and AFM; methods used to analyze its elemental and phase composition include XEPMA, XPS, SEM-EDS analysis, and XRD. CONCLUSION: Functional adaptations of the mandible in A. grewingkii provide for the optimum combination of mechanical hardness and fracture resistance, which is achieved due to a complex structure and composition of its cutting parts. Teeth of the mandible are covered by a thin layer of silica (10-20 µm). Their epicuticle is characterized by a high density, consists of three layers, and increases in thickness toward the tooth apex. The epicuticle is enriched with Br, while the concentrations of Ca and P reach the peak values in the softer internal tissues of the teeth. These data broaden the view of the diversity of adaptation mechanisms providing for the strengthening of cuticular "tools" in crustaceans.

Complete sequence of the mitochondrial genome of a diatom alga Synedra acus and comparative analysis of diatom mitochondrial genomes.

The first two mitochondrial genomes of marine diatoms were previously reported for the centric Thalassiosira pseudonana and the raphid pennate Phaeodactylum tricornutum. As part of a genomic project, we sequenced the complete mitochondrial genome of the freshwater araphid pennate diatom Synedra acus. This 46,657 bp mtDNA encodes 2 rRNAs, 24 tRNAs, and 33 proteins. The mtDNA of S. acus contains three group II introns, two inserted into the cox1 gene and containing ORFs, and one inserted into the rnl gene and lacking an ORF. The compact gene organization contrasts with the presence of a 4.9-kb-long intergenic region, which contains repeat sequences. Comparison of the three sequenced mtDNAs showed that these three genomes carry similar gene pools, but the positions of some genes are rearranged. Phylogenetic analysis performed with a fragment of the cox1 gene of diatoms and other heterokonts produced a tree that is similar to that derived from 18S RNA genes. The introns of mtDNA in the diatoms seem to be polyphyletic. This study demonstrates that pyrosequencing is an efficient method for complete sequencing of mitochondrial genomes from diatoms, and may soon give valuable information about the molecular phylogeny of this outstanding group of unicellular organisms.

Silicon nanotechnologies of pigmented heterokonts.

Many pigmented heterokonts are able to synthesize elements of their cell walls (the frustules) of dense biogenic silica. These include diatom algae, which occupy a significant place in the biosphere. The siliceous frustules of diatoms have species-specific patterns of surface structures between 10 and a few hundred nanometers. The present review considers possible mechanisms of uptake of silicic acid from the aquatic environment, its transport across the plasmalemma, and intracellular transport and deposition of silica inside the specialized Silica Deposition Vesicle (SDV) where elements of the new frustule are formed. It is proposed that a complex of silicic acid with positively charged proteins silaffins and polypropylamines remains a homogeneous solution during the intracellular transport to SDV, where biogenic silica precipitates. The high density of the deposited biogenic silica may be due to removal of water from the SDV by aquaporins followed by syneresis--a process during which pore water is expelled from the network of the contracting gel. The pattern of aquaporins in the silicalemma, the membrane embracing the SDV, can determine the pattern of species-specific siliceous nanostructures.