Plasmodium structure of Intoshia linei (Orthonectida).

Orthonectids are enigmatic parasitic bilaterians whose exact position on the phylogenetic tree is still uncertain. Despite ongoing debate about their phylogenetic position, the parasitic stage of orthonectids known as "plasmodium" remains underexplored. There is still no consensus on the origin of the plasmodium: whether it is an altered host cell or a parasitic organism that develops in the host extracellular environment. To determine the origin of the orthonectid parasitic stage, we studied in detail the fine structure of the Intoshia linei orthonectid plasmodium using a variety of morphological methods. The orthonectid plasmodium is a shapeless multinucleated organism separated from host tissues by a double membrane envelope. Besides numerous nuclei, its cytoplasm contains organelles typical for other bilaterians, reproductive cells, and maturing sexual specimens. Reproductive cells, as well as developing orthonectid males and females, are covered by an additional membrane. The plasmodium forms protrusions directed to the surface of the host body and used by mature individuals for egress from the host. The obtained results indicate that the orthonectid plasmodium is an extracellular parasite. A possible mechanism for its formation might involve spreading parasitic larva cells across the host tissues with subsequent generation of a cell-within-cell complex. The cytoplasm of the plasmodium originates from the outer cell, which undergoes multiple nuclear divisions without cytokinesis, while the inner cell divides, giving rise to reproductive cells and embryos. The term "plasmodium" should be avoided and the term "orthonectid plasmodium" could be temporarily used instead.

Endothelial Cell Behavior and Nitric Oxide Production on a-C:H:SiOx-Coated Ti-6Al-4V Substrate.

This paper focuses on the surface modification of the Ti-6Al-4V alloy substrate via a-C:H:SiOx coating deposition. Research results concern the a-C:H:SiOx coating structure, investigated using transmission electron microscopy and in vitro endothelization to study the coating. Based on the analysis of the atomic radial distribution function, a model is proposed for the atomic short-range order structure of the a-C:H:SiOx coating, and chemical bonds (C-O, C-C, Si-C, Si-O, and Si-Si) are identified. It is shown that the a-C:H:SiOx coating does not possess prolonged cytotoxicity in relation to EA.hy926 endothelial cells. In vitro investigations showed that the adhesion, cell number, and nitric oxide production by EA.hy926 endothelial cells on the a-C:H:SiOx-coated Ti-6Al-4V substrate are significantly lower than those on the uncoated surface. The findings suggest that the a-C:H:SiOx coating can reduce the risk of endothelial cell hyperproliferation on implants and medical devices, including mechanical prosthetic heart valves, endovascular stents, and mechanical circulatory support devices.

Study of the Embryonic Toxicity of TiO2 and ZrO2 Nanoparticles.

Currently, the widespread use of TiO2 and ZrO2 nanoparticles (NPs) in various industries poses a risk in terms of their potential toxicity. A number of experimental studies provide evidence of the toxic effect of TiO2 and ZrO2 NPs on biological objects. In order to supplement the level of knowledge and assess the risks of toxicity and danger of TiO2 and ZrO2 NPs, we decided to conduct a comprehensive experiment to study the embryonic toxicity of TiO2 and ZrO2 NPs in pregnant rats. For the experiment, mongrel white rats during pregnancy received aqueous dispersions of powders of TiO2 and ZrO2 NPs at a dose of 100 mg/kg/day. To characterize the effect of TiO2 and ZrO2 NPs on females and the postnatal ontogenesis of offspring, a complex of physiological and biochemical research methods was used. The results of the experiment showed that TiO2 NPs as ZrO2 NPs (100 mg/kg per os) cause few shifts of similar orientation in the maternal body. Neither TiO2 NPs nor ZrO2 NPs have an embryonic and teratogenic effect on the offspring in utero, but both modify its postnatal development.

Evolutionary relationships of Metchnikovella dogieli Paskerova et al., 2016 (Microsporidia: Metchnikovellidae) revealed by multigene phylogenetic analysis.

The species Metchnikovella dogieli (Paskerova et al. Protistology 10:148-157, 2016) belongs to one of the early diverging microsporidian groups, the metchnikovellids (Microsporidia: Metchnikovellidae). In relation to typical ('core') microsporidia, this group is considered primitive. The spores of metchnikovellids have no classical polar sac-anchoring disk complex, no coiled polar tube, no posterior vacuole, and no polaroplast. Instead, they possess a short thick manubrium that expands into a manubrial cistern. These organisms are hyperparasites; they infect gregarines that parasitise marine invertebrates. M. dogieli is a parasite of the archigregarine Selenidium pygospionis (Paskerova et al. Protist 169:826-852, 2018), which parasitises the polychaete Pygospio elegans. This species was discovered in samples collected in the silt littoral zone at the coast of the White Sea, North-West Russia, and was described based on light microscopy. No molecular data are available for this species, and the publicly accessible genomic data for metchnikovellids are limited to two species: M. incurvata Caullery & Mesnil, 1914 and Amphiamblys sp. WSBS2006. In the present study, we applied single-cell genomics methods with whole-genome amplification to perform next-generation sequencing of M. dogieli genomic DNA. We performed a phylogenetic analysis based on the SSU rRNA gene and reconstructed a multigene phylogeny using a concatenated alignment that included 46 conserved single-copy protein domains. The analyses recovered a fully supported clade of metchnikovellids as a basal group to the core microsporidia. Two members of the genus Metchnikovella did not form a clade in our tree. This may indicate that this genus is paraphyletic and requires revision.

Polychaos centronucleolus n. sp. - a new terrestrial species of the genus Polychaos (Amoebozoa, Tubulinea) with nontypical nuclear structure.

A new species of the "proteus-type" naked amoebae (large cells with discrete tubular pseudopodia) was isolated from tree bark sample of a birch tree in the surrounding of Kislovodsk town, Russia and named Polychaos centronucleolus n. sp. (Amoebozoa, Tubulinea). Amoebae of this species have a filamentous cell coat and a nucleus with a central compact nucleolus. This type of nucleolar organization has not been previously known for the genus Polychaos. A sequence of the 18S rRNA gene of this strain was obtained using whole genome amplification of DNA from the single amoeba cell, followed by NGS sequencing. The analysis of molecular data robustly groups this species with Polychaos annulatum within the family Hartmannellidae. Our results, together with the results of our previous studies, show that the taxonomic assignment of "proteus-type" amoebae species is becoming increasingly complex, and the taxonomic characters that can be used to classify these organisms are becoming more shadowed.

Extreme Genome and Nervous System Streamlining in the Invertebrate Parasite Intoshia variabili.

Orthonectida is a small, rare, and in many aspects enigmatic group of organisms with a unique life cycle and a highly simplified adult free-living stage parasitizing various marine invertebrates [1, 2]. Phylogenetic relationships of Orthonectida have remained controversial for a long time. According to recent data, they are close to Annelida, specifically to Clitellata [3-5]. Several studies have shown that parasitism can not only lead to a dramatic reduction of the body plan and morphological structures but also affect organisms at the genomic level [6, 7]. Comparative studies of parasites and closely related non-parasitic species could clarify the genome reduction degree and evolution of parasitism. Here, we report on the morphology, genome structure, and content of the smallest known Orthonectida species Intoshia variabili, inhabiting the flatworm Graffiellus croceus. This orthonectid with an extremely simplified nervous system demonstrates the smallest known genome (15.3 Mbp) and one of the lowest reported so far gene numbers (5,120 protein-coding genes) among metazoans. The genome is extremely compact, due to a significant reduction of gene number, intergenic regions, intron length, and repetitive elements. The small genome size is probably a result of extreme genome reduction due to their parasitic lifestyle, as well as of simplification and miniaturization of the free-living stages. Our data could provide further insights into the evolution of parasitism and could help to define a minimal bilaterian gene set.

Molecular Phylogeny of Polychaos annulatum (Amoebozoa, Tubulinea, Euamoebida) Shows that Genus Polychaos Belongs to the Family Hartmannellidae.

We have obtained a sequence of the 18S rRNA gene of the species Polychaos annulatum (Penard 1902) Smirnov et Goodkov 1998 using the isolation of a single nucleus from an amoeba cell. Attempts to amplify the 18S rRNA gene from the DNA of this species by conventional PCR were not successful, so we applied the whole genome amplification of the nuclear DNA followed by NGS sequencing. The 18S rRNA gene was found among the resulting contigs. The analysis unexpectedly shows that P. annulatum robustly groups within the family Hartmannellidae, but not Amoebidae. This finding warrants revision of the basic morphological criteria used to classify Euamoebida families and show that "proteus-type" amoebae may belong to other families rather than Amoebidae. This makes taxonomic assignments of such species more complex and the borders between Euamoebida families more nuanced. It is getting evident that molecular data are necessary to clarify the position of species even in this most "classical" order of naked lobose amoebae.

A Comparative Characterization of the Mitochondrial Genomes of Paramoeba aparasomata and Neoparamoeba pemaquidensis (Amoebozoa, Paramoebidae).

Marine amebae of the genus Paramoeba (Amoebozoa, Dactylopodida) normally contain a eukaryotic endosymbiont known as Perkinsela-like organism (PLO). This is one of the characters to distinguish the genera Neoparamoeba and Paramoeba from other Dactylopodida. It is known that the PLO may be lost, but PLO-free strains of paramoebians were never available for molecular studies. Recently, we have described the first species of the genus Paramoeba which has no parasome-Paramoeba aparasomata. In this study, we present a mitochondrial genome of this species, compare it with that of Neoparamoeba pemaquidensis, and analyze the evolutionary dynamics of gene sequences and gene order rearrangements between these species. The mitochondrial genome of P. aparasomata is 46,254 bp long and contains a set of 31 protein-coding genes, 19 tRNAs, two rRNA genes, and 7 open reading frames. Our results suggest that these two mitochondrial genomes within the genus Paramoeba have rather similar organization and gene order, base composition, codon usage, the composition and structure of noncoding, and overlapping regions.

Paramoeba aparasomata n. sp., a symbiont-free species, and its relative Paramoeba karteshi n. sp. (Amoebozoa, Dactylopodida).

Two brackish water amoebae have been isolated and studied from the benthic biotopes of the Chupa Inlet (Kandalaksha Bay, northwestern Russia). Both strains can be identified as new species of the genus Paramoeba (Amoebozoa, Dactylopodida, Paramoebidae) based on light microscopical characters, structure of microscales on the cell surface and molecular evidence based on the analyses of two genes, nuclear SSU rRNA and mitochondrial cytochrome c oxidase subunit 1 (COI). Paramoeba aparasomata n. sp. is of particular interest because this amoeba is permanently lacking a symbiotic Perkinsela-like organism (PLO) present in other species of Paramoeba and Neoparamoeba. The results obtained show that scaly dactylopodial amoebae lacking PLO are not necessarily members of Korotnevella. In particular, we suggest that Korotnevella nivo Smirnov, 1997, with microscales very similar to those of Paramoeba eilhardi and the species studied here in structure, may be in fact a member of Paramoeba. Molecular data on K. nivo have to be obtained and analysed to test this hypothesis. Based on our new results we emend the diagnosis of the genus Paramoeba to make it more fit to the current phylogenetic conception.

The complete mitochondrial genome of Paravannella minima (Amoebozoa, Discosea, Vannellida).

We present a complete sequence and describe the organization of the mitochondrial genome of the amoeba Paravannella minima (Amoebooza, Discosea, Vannellida). This tiny species represents a branch at the base of Vannellida tree, to the moment being its earliest-branching lineage. The circular mitochondrial DNA of this species has 53,464 bp in length and contains 30 protein-coding genes, 2 ribosomal RNAs, 23 transfer RNAs, and 15 open reading frames. This genome is significantly longer and contains more protein-coding genes than any yet sequenced mitochondrial genome of vannellid amoebae. Unlike the previously sequenced mitochondrial genomes of Vannellida, which should be translated using the "Table 4" (the mold, protozoan, and coelenterate mitochondrial code), that of P. minima can be properly translated using the universal genetic code.

Mitochondrial Genome of Vannella croatica (Amoebozoa, Discosea, Vannellida).

Mitochondrial genome sequence of Vannella croatica (Amoebozoa, Discosea, Vannellida) was obtained using pulse-field gel electrophoretic isolation of the circular mitochondrial DNA, followed by the next-generation sequencing. The mitochondrial DNA of this species has the length of 28,933 bp and contains 12 protein-coding genes, two ribosomal RNAs, and 16 transfer RNAs. Vannella croatica mitochondrial genome is relatively short compared to other known amoebozoan mitochondrial genomes but is rather gene-rich and contains significant number of open reading frames.

The complete mitochondrial genome of Vannella simplex (Amoebozoa, Discosea, Vannellida).

Vannella simplex (Amoebozoa, Discosea, Vannellida) is one of the commonest freshwater free-living lobose amoebae, known from many locations worldwide. In the present study, we describe the complete mitochondrial genome of this species. The circular mitochondrial DNA of V. simplex has 34,145öbp in length and contains 27 protein-coding genes, 2 ribosomal RNAs, 16 transfer RNAs and 4 open reading frames. Mitochondiral genome of V. simplex is one of the most gene compact due to overlapping genes and reduced intergenic space. It has much in common with its closest relative, mitochondrial genome of V. croatica GenBank number MF508648. In the same time, both of them show considerable differences in length and in gene order from the next close relative - that of Neoparamoeba pemaquidensis KX611830 (deposited as Paramoeba) and even more - from other sequenced amoebozoan mitochondrial genomes. The present study confirms the opinion that the level of synteny between the mitochondrial genomes across the entire Amoebozoa clade is low. More or less considerable similarity yet was found only between members of the same clade of the genera or family level, but hardly - among more distant lineages.

Phylogeny and Systematics of Leptomyxid Amoebae (Amoebozoa, Tubulinea, Leptomyxida).

We describe four new species of Flabellula, Leptomyxa and Rhizamoeba and publish new SSU rRNA gene and actin gene sequences of leptomyxids. Using these data we provide the most comprehensive SSU phylogeny of leptomyxids to date. Based on the analyses of morphological data and results of the SSU rRNA gene phylogeny we suggest changes in the systematics of the order Leptomyxida (Amoebozoa: Lobosa: Tubulinea). We propose to merge the genera Flabellula and Paraflabellula (the genus Flabellula remains valid by priority rule). The genus Rhizamoeba is evidently polyphyletic in all phylogenetic trees; we suggest retaining the generic name Rhizamoeba for the group unifying R. saxonica, R.matisi n. sp. and R. polyura, the latter remains the type species of the genus Rhizamoeba. Based on molecular and morphological evidence we move all remaining Rhizamoeba species to the genus Leptomyxa. New family Rhizamoebidae is established here in order to avoid paraphyly of the family Leptomyxidae. With the suggested changes both molecular and morphological systems of the order Leptomyxida are now fully congruent to each other.

Energy of the Isolated Metastable Iron-Nickel FCC Nanocluster with a Carbon Atom in the Tetragonal Interstice.

The energy of the isolated iron-nickel nanocluster was calculated by molecular mechanics method using Lennard-Jones potential. The cluster included a carbon atom that drifted from an inside octahedral interstice to a tetrahedral interstice in [Formula: see text] direction and after that in <222> direction to the surface. In addition, one of 14 iron atoms was replaced by a nickel atom, the position of which was changing during simulation.The energy of the nanocluster was estimated at the different interatomic distances. As a result of simulation, the optimal interatomic distances of Fe-Ni-C nanocluster was chosen for the simulation, in which height of the potential barrier was maximal and face-centered cubic (FCC) nanocluster was the most stable.It is shown that there were three main positions of a nickel atom that significantly affected nanocluster's energy.The calculation results indicated that position of the carbon atom in the octahedral interstice was more energetically favorable than tetrahedral interstice in the case of FCC nanocluster. On the other side, the potential barrier was smaller in the direction [Formula: see text] than in the direction <022>.This indicates that there are two ways for carbon atom to drift to the surface of the nanocluster.

Lineage-Specific and Highly Derived Gene Sequences Among Amoebozoa, Revealed by the Comparative Analysis of Transcriptomes from Twelve Amoebozoan Species.

Amoebozoa represent a difficult group for traditional morphology-based taxonomy. Molecular approaches, such as gene sequencing and DNA barcoding have greatly enhanced our knowledge of the diversity of these organisms. However, metagenomic studies of Amoebozoa still did not provide as impressive results as they did among some other groups of protists. In environmental DNA surveys done on fragments of SSU rDNA gene and other traditional DNA barcodes, Amoebozoa genes normally constitute a minor part of the total gene diversity and represent only the most abundant lineages. A potential way to resolve this problem is the usage of DNA barcodes based on genes, which are unique or highly derived in this group of organisms. In the present study, we attempted to find such genes and gene families with a low level of paralogy, potentially appropriate as Amoebozoa-specific DNA barcodes. For this we re-assembled transcriptomes of 12 amoebozoan species available from the public databases and performed gene annotation and identification of orthologous genes. In our analysis Amoebozoa-specific and highly derived sequences formed 53,182 clusters of orthologs, containing from 2 to 299 proteins each. Some of these genes may be a potential target for DNA barcoding of Amoebozoa.

Genetic structure of a morphological species within the amoeba genus Korotnevella (Amoebozoa: Discosea), revealed by the analysis of two genes.

Amoebae of the genus Korotnevella are covered with scales, the structure of which is believed to be species-specific and allows distinguishing species reliably at the morphological level. We studied members of this genus in order to assess the genetic structure of the local populations of amoebae. For the present study we isolated nine freshwater strains of Korotnevella, belonging to three species, from two locations in North-Western Russia. In order to obtain data on the population structure of these amoebae, we identified all isolates based on the light-microscopic morphology and scale structure and investigated both inter-strain and intra-strain polymorphism of Cox I and 18S rRNA genes. Results show that both genes provide congruent patterns of population structure. The Cox I gene appears to be more reliable DNA barcode while the 18S rRNA gene shows an interesting pattern of polymorphism, which may represent phylotypes of amoebae. Local population of amoebae in every studied species consists of a number of genetic lineages (phylotypes), some shared between the populations while others are unique to a local habitat.

Morphology and phylogeny of Vannella croatica n. sp. (Amoebozoa, Discosea, Vannellida).

We isolated and described a new species of freshwater vannellid amoeba from Krka natural reserve in Croatia--Vannella croatica n. sp. This species has certain morphological differences from all known vannellids and differs at the level of SSU sequence. It resembles in size and morphology Vannella lata; to facilitate direct comparison we publish images of V. lata CCAP 1589/12 strain (type strain, which is now lost) taken in 1999. Vannela croatica feeds on bacteria and can be easily grown in large amount in relatively pure culture and thus is suitable for molecular and biochemical studies requiring large amounts of material.

Viable Species of Flamella (Amoebozoa: Variosea) Isolated from Ancient Arctic Permafrost Sediments.

Six viable strains of amoebae belonging to the genus Flamella (Amoebozoa, Variosea) were isolated from permafrost sediments sampled in the Russian Arctic region. Two of them are from late Pleistocene permafrost in North-East Siberia, and four--from Holocene and late Pleistocene in North-West Siberia. Light- and electron-microscopic study and molecular phylogeny show that these isolates represent two new species belonging to the genus Flamella. Both species are cyst-forming. This is a remarkable case of high resistance of protozoan cysts, allowing them to survive and recover an amoebae population after a very long, geologically significant period of rest; a "snapshot" of evolution in time. This study directly shows for the first time that amoeba cysts can be conserved not only for years and decades but for many thousand years and then recover, contributing to the formation of an active microbial community. We propose to name the new species as Flamella pleistocenica n.sp. and Flamella beringiania n.sp. Phylogenetic analysis shows that the genus Flamella is a robust and potentially species-rich group of Variosea.

Change of Energy of the Cubic Subnanocluster of Iron Under Influence of Interstitial and Substitutional Atoms.

Energy change of an iron face-centred cubic subnanocluster was evaluated using molecular mechanics method depending on the position of a carbon interstitial atom and substitutional atoms of nickel. Calculations of all possible positions of impurity atoms show that the energy change of the system are discrete and at certain positions of the atoms are close to continuous.In terms of energy, when all impurity atoms are on the same edge of an atomic cluster, their positions are more advantageous. The presence of nickel atoms on the edge of a cubic cluster resulted in decrease of potential barrier for a carbon atom and decrease in energy in the whole cluster. A similar drift of a carbon atom from central octahedral interstitial site to the surface in the direction <011> occurred under the influence of surface factors.Such configuration corresponds to decreasing symmetry and increasing the number of possible energy states of a subnanocluster, and it corresponds to the condition of spontaneous crystallization process in an isolated system.Taking into account accidental positions of the nickel atom in the iron cluster, such behaviour of the carbon atom can explain the mechanism of growth of a new phase and formation of new clusters in the presence of other kind of atoms because of surface influence.