A Genome of Temperate Enterococcus Bacteriophage Placed in a Space of Pooled Viral Dark Matter Sequences.

In the human gut, temperate bacteriophages interact with bacteria through predation and horizontal gene transfer. Relying on taxonomic data, metagenomic studies have associated shifts in phage abundance with a number of human diseases. The temperate bacteriophage VEsP-1 with siphovirus morphology was isolated from a sample of river water using Enterococcus faecalis as a host. Starting from the whole genome sequence of VEsP-1, we retrieved related phage genomes in blastp searches of the tail protein and large terminase sequences, and blastn searches of the whole genome sequences, with matches compiled from several different databases, and visualized a part of viral dark matter sequence space. The genome network and phylogenomic analyses resulted in the proposal of a novel genus "Vespunovirus", consisting of temperate, mainly metagenomic phages infecting Enterococcus spp.

The Roles of Mosquitoes in the Circulation of Monoxenous Trypanosomatids in Temperate Climates.

Monoxenous (insect-restricted) trypanosomatids are highly diverse and abundant in nature. While many papers focus on the taxonomy and distribution of these parasites, studies on their biology are still scarce. In particular, this concerns trypanosomatids inhabiting the ubiquitous mosquitoes. To shed light on the circulation of monoxenous trypanosomatids with the participation of mosquitoes, we performed a multifaceted study combining the examination of naturally- and experimentally-infected insects using light and electron microscopy and molecular identification of parasites. Our examination of overwintering mosquitoes (genera Culex and Culiseta) revealed that their guts contained living trypanosomatids, which can be spread during the next season. Experimental infections with Crithidia spp. demonstrated that imagines represent permissive hosts, while larvae are resistant to these parasites. We argue that for the parasites with wide specificity, mosquitoes act as facultative hosts. Other trypanosomatids may have specific adaptations for vertical transmission in these insects at the expense of their potential to infect a wider range of hosts and, consequently, abundance in nature.

Morphology and spicules elemental composition of Marophrys nikolaevi spec. nov. (Haptista: Centroplasthelida).

Marophrys is a genus of spicules-bearing centrohelids belonging to Heterophrys-like organisms (HLO's). Here Marophrys nikolaevi spec. nov. is described. Four strains were isolated from brackish waters (16-22 ppt) of the Tuzlukkol' River, the Tuzluchnoe Lake (South Urals, Russia) and the Black Sea. All the strains were characterised with light microscopy and electron microscopic study of the whole mount preparations. Molecular phylogenetic analysis has put SSU rDNA sequences, obtained for all strains, inside Marophryidae clade sister to M. marina. The organisms have a cell diameter of 4-11 µm and are surrounded with organic spicules of two types. Short (0.6-0.7 µm) and thin (0.01-0.02 µm) mostly tangentially oriented spicules form a lax sheath, surrounding the cell. Longer (3-6 µm) and thicker (0.04-0.05 µm) spicules are embedded in this sheath and are radially or obliquely oriented. Energy-dispersive X-ray analysis has shown that the spicules are purely organic. The taxonomy of marine Heterophrys-like organisms is discussed.

On the phylogenetic position of Raphidocystis pallida with some notes on its life cycle.

Raphidocystis pallida, a centrohelid heliozoan with unusually shaped tubular siliceous scales, was reisolated from Jamor river, Portugal, and studied with the use of light and electron microscopy. In enriched cultures, the cells were naked, devoid of siliceous external skeleton with the exception of several scales present in one cell. Instead, such cells were covered with a layer of rod-shaped bacteria. In clonal cultures, the cells gradually acquired siliceous coverings typical for this species and retained them in next generations. Phylogenetic position of R. pallida was clarified with SSU rDNA-based molecular phylogenetics, and its placement within the genus Raphidocystis despite unusual coverings structure was confirmed. The implications of phylogenetic placement of R. pallida and possible origins of the previously undescribed naked form were discussed.

Development of two species of the Trypanosoma theileri complex in tabanids.

BACKGROUND: Trypanosoma theileri species complex includes parasites of Bovidae (cattle, sheep, goat, etc.) and Cervidae (deer) transmitted mainly by Tabanidae (horse flies and deerflies) and keds (Hippoboscidae). While morphological discrimination of species is challenging, two big clades, TthI and TthII, each containing parasites isolated from bovids and cervids, have been identified phylogenetically. To date, the development in the vector has been studied in detail only for the ked-transmitted sheep parasite T. melophagium (TthII), while the fate of trypanosomes in tabanids was described only briefly by light microscopy. METHODS: We collected infected tabanids of various species and identified trypanosomes by molecular phylogenetic analysis. The morphology and development of trypanosomes was studied using the combination of statistical analyses as well as light and electron microscopy. RESULTS: Two trypanosome species belonging to both TthI and TthII clades of the T. theileri complex were identified. The phylogenetic position of these two trypanosomes suggests that they parasitize deer. Both species were indiscernible by morphology in the vector and showed the same development in its intestine. In contrast to the previously described development of T. melophagium, both trypanosomes of tabanids only transiently infected midgut and settled mainly in the ileum, while pylorus and rectum were neglected. Meanwhile, the flagellates developing in the tabanid ileum (pyriform epimastigotes and metacyclic trypomastigotes) showed similarities to the corresponding stages in T. melophagium by morphology, mode of attachment to the host cuticle and formation of the fibrillar matrix surrounding the mass of developing parasites. In addition, for the first time to our knowledge we documented extraintestinal stages in these trypanosomes, located in the space between the epithelium and circular muscles. CONCLUSIONS: The development of different species of flagellates of the T. theileri complex in their insect vectors shows many similarities, which can be explained not only by their common origin, but also the same transmission mode, i.e. contamination of the oral mucosa with the gut content released after squashing the insect either by tongue or teeth. The observed differences (concerning primarily the distribution of developmental stages in the intestine) are associated rather with the identity of vectors than the phylogenetic position of parasites.

Phylogenetic position and morphology of Raphidiophrys elongata sp. nov. (Haptista: Centroplasthelida) with notes on cyst wall structure and evolution.

The majority of centrohelids bear external coverings consisting of organic spicules or siliceous scales. Cyst coverings are usually reinforced with additional layers of modified scales. The cyst wall of Raphidiophrys heterophryoidea has an unusual and complex structure. It consists of three different types of scales and includes the mosaic scale layer not known in other centrohelids. During excystment, the cyst wall fragments along the sutures of the mosaic layer. For other Raphidiophrys species, cyst coverings are not studied. The present paper describes a new Raphidiophrys species, R. elongata, belonging to the NC7 environmental clade. Trophozoites bore thin plate scales with reduced upper plate. Under starvation, cysts emerged in clonal cultures. Cyst coverings of R. elongata and R. heterophryoidea were studied in comparison with the use of FIB-SEM. Cyst wall of R. elongata was significantly thinner than in R. heterophryoidea and was formed with 3-5 layers of uniform overlapping scales. No mosaic scale layer was present. During excystment, trophozoite exited cyst shell through random fissure. Possible evolutionary events and driving forces behind the complication of cyst wall within Raphidiophrys were discussed.

Clypifer cribrifer gen. nov., sp. nov. (Clypiferidae fam. nov., Pterocystida, Centroplasthelida), with notes on evolution of centrohelid siliceous coverings.

A new family, genus and species of centrohelid heliozoans, Clypifer cribrifer gen. nov., sp. nov. (Clypiferidae fam. nov.), from the Gulf of Aqaba (Israel) was studied with light and electron microscopy and SSU rRNA gene sequencing. Clypifer cribrifer has only one type of scales, partially running up the sides of the axopodia. Plate scales [0.8-2.3 (av. 1.5)×0.6-1.8 (av. 1.2) µm] are flat, elliptical or circular, fenestrated with holes of irregular shape and have a marginal rim and a very short axial rib. The cell diameter is 3.9-9.6 (av. 6.0) µm. Molecular phylogenetic analysis robustly places C. cribrifer in the C4 clade for which the new family Clypiferidae is proposed here. This position is confirmed with the short sequences in the panacanthocystid increased regions. The morphology of the new genus has similarities to the genus Raphidocystis. The probability that another Clypifer species was described under a different name in the centrohelid literature is discussed. Clypiferidae represent the second lineage of Pterocystida, which are characterized by the presence of only tangentially oriented plate scales of one type. Possible ways of evolution of the centrohelid siliceous coverings are also discussed.

The long-time orphan protist Meringosphaera mediterranea Lohmann, 1902 [1903] is a centrohelid heliozoan.

Meringosphaera is an enigmatic marine protist without clear phylogenetic affiliation, but it has long been suggested to be a chrysophyte-related autotroph. Microscopy-based reports indicate that it has a worldwide distribution, but no sequence data exist so far. We obtained the first 18S rDNA sequence for M. mediterranea (identified using light and electron microscopy) from the west coast of Sweden. Observations of living cells revealed granulated axopodia and up to 6 globular photosynthesizing bodies about 2 µm in diameter, the nature of which requires further investigation. The ultrastructure of barbed undulating spine scales and patternless plate scales with a central thickening is in agreement with previous reports. Molecular phylogenetic analysis placed M. mediterranea inside the NC5 environmental clade of Centroplasthelida (Haptista) along with additional environmental sequences, together closely related to Choanocystidae. This placement is supported by similar scales in Meringosphaera and Choanocystidae. We searched the Tara Oceans 18S V9 metabarcoding dataset, which revealed four OTUs with 94.8%-98.2% similarity, with oceanic distribution similar to that based on morphological observations. The current taxonomic position and species composition of the genus are discussed. The planktonic lifestyle of M. mediterranea contradicts the view of some authors that centrohelids enter the plankton only temporarily.

The smallest known heliozoans are the Erebor lineage (nom. clad. n.) inside Microheliella maris (Eukaryota, Diaphoretickes), with the amendation of M. maris diagnosis and description of Berkeleyaesol magnus gen. nov., comb. nov. (Eukaryota, incertae sedis).

A new strain of planktonic heliozoans (ZI172) belonging to the genus Microheliella (the sister group of Cryptista in Diaphoretickes), closely related to the only one known strain of Microheliella maris (CCAP 1945/1), was studied with light microscopy and SSU rRNA gene sequencing. Morphometric data obtained from 127 cells and based on 254 measurements showed that this strain represents the smallest heliozoan (1.66-3.42 µm, av. 2.56 µm) in diameter known to date and one of the smallest free-living eukaryotes. We also did morphometry for strain CCAP 1945/1. Its cell body size is 3.20-6.47 µm (av. 4.15 µm; n=141; m=282). The secondary structures of hairpin 15 of the SSU rRNA molecules were reconstructed for ZI172 and CCAP 1945/1 and they were compared The possible biochemical explanation for the smaller size of the ZI172 strain, which is smaller than the CCAP 1945/1 strain, is discussed, including all published electron micrographs of CCAP 1945/1. The necessary taxonomic work is also carried out. The diagnosis of Microheliella maris is amended and the new infraspecific clade Erebor is described to include ZI172. The measurements and systematics of the enigmatic heliozoan 'Raphidiophrys' magna O'Donoghue 1922 (non 1921; the biggest known heliozoan) are also discussed and it is transferred to the new genus Berkeleyaesol.

Phenotypic masquerade: Polymorphism in the life cycle of the centrohelid heliozoan Raphidiophrys heterophryoidea (Haptista: Centroplasthelida).

The life cycle of the centrohelid heliozoan Raphidiophrys heterophryoidea Zlatogursky, 2012 was studied with light and electron microscopy in clonal cultures from the type locality. The alternation of two types of trophozoites, having contrastingly different morphology, was observed. Type 1 trophozoites morphology matched the original description. Type 2 trophozoites tended to form colonies usually of 6-8 individuals, connected with cytoplasmic bridges and their cell size was noticeably bigger, namely 43-45 µm compared to 24.5 µm on average in type 1 trophozoites. Some colonies were forming stalks composed of three or four axopodia covered with scales. Spicules were lacking completely, while plate-scales differed from those of type 1 trophozoites: they had oblong-elliptical shape, larger (5.9-14.1 × 2.4-5.8 µm) size, non-branching septa always reaching scale centre, solid upper plate. The conspecificity of the two trophozoite types was confirmed with the comparison of SSU rDNA gene sequence data. Both types of trophozoites were capable of encystment and excysted individuals always were type 1 trophozoites. A new type of cyst-scales (cup-scales) was described. Transitions between cysts and the two trophozoites types were documented. The diagnosis of R. heterophryoidea was improved accordingly. The possible functions, driving forces, and taxonomic consequences of the polymorphism were discussed.

Pinjata ruminata gen. et sp. n.-A New Member of Centrohelid Family Yogsothothidae (Haptista: Centroplasthelida) from the Brackish River.

A new genus and species of centrohelid heliozoan Pinjata ruminata from the Tuzlukkol' River (Orenburg Region of Russia) and Gor'koe Lake (Chelyabinsk Region of Russia) is studied with light- and electron microscopy. Pinjata ruminata has two types of plate scales, partially running up the sides of the axopodia. Inner plate scales (3.2-4.9 × 1.5-2.6 µm) are flat, ovate-oblong and have a broad axial thickening and a thin electron-dense border. Outer plate scales (4.2-6.7 × 1.5-3.0 µm) are concave, elongated, of irregular shape, often curved, and broadened towards one end. Roundish depressions are forming two rows on both sides of the narrow axial thickening. The cells are attached to the substratum. Molecular phylogenetic analysis based on the SSU rDNA robustly placed P. ruminata in the family Yogsothothidae. This position is confirmed with the presence of five panacanthocystid increase regions. The morphology of the new genus is in a good accordance with diagnosis of the family. The status of a genus "Heteroraphidiophrys" is discussed. Other potential findings of Pinjata from literature are analyzed. Pinjata represents the third lineage of centrohelids, characterized with the presence of only tangentially oriented plate scales. The halophilic nature of Yogsothothidae is suggested.

Yogsothoth knorrus gen. n., sp. n. and Y. carteri sp. n. (Yogsothothidae fam. n., Haptista, Centroplasthelida), with Notes on Evolution and Systematics of Centrohelids.

Two closely related new species of centrohelid heliozoans with unusual morphology were studied with light and electron microscopy. Sequences of the 18S rRNA gene were also obtained and secondary structure of 18S rRNA molecule reconstructed. The cells, covered with inner siliceous plate scales formed colonies. The entire colony was surrounded with a thick layer of external scales. Inner scales were tabulate and had a patternless surface, except for the presence of an axial rib. Outer scales had a boat-like (Yogsothoth knorrus gen. nov., sp. nov.) or pot-like (Yogsothoth carteri sp. nov.) shape with an axial rib and numerous conical papillae on the scale surface. Analysis of 18S rRNA gene sequences robustly placed the new taxa within centrohelids, but not in any existing family. Scaled Yogsothoth represents a genetically divergent closest outgroup of Acanthocystida, branching after the supposedly primary non-scaled Marophrys, and together with acanthocystids, forming the novel taxon Panacanthocystida. Reconstruction of presumptive 18S rRNA secondary structure reveals interspecific differences in expansion segments 7 and 9 of Yogsothoth. Analysis of 18S rRNA secondary structure of other centrohelids allowed identification of length increases characteristic for Panacanthocystida location and reconstruction of 18S rRNA elongation in the course of the evolution of this group.

On the phylogenetic position of the genus Raphidocystis (Haptista: Centroplasthelida) with notes on the dimorphism in centrohelid life cycle.

Centrohelid heliozoan Raphidocystis glabra was reisolated for the first time after initial description and put into a clonal culture. Its correct identification was confirmed by scanning and transmission electron microscopy of scales. The first light microscopy data from the living cells were obtained. Phylogenetic analysis of its position using 18S rDNA sequences was also performed. This species branches inside of the Polyplacocystis clade, being closely related to the strain HLO4. The latter was isolated from the same sample with R. glabra and has spicules, not siliceous scales, unlike all the other members of this clade. Using another strain isolated from this sample the co-specificity of HLO4 and R. glabra was demonstrated, and the presence of spicule-bearing stages in the life cycle of Raphidocystis was shown. Polyplacocystis revealed to be paraphyletic with Raphidocystis embedded in it. Moreover, representatives of both genera share the similar monolayered plate scales with hollow inflected margin. Thus, a family-level name Raphidocystidae for the whole clade was proposed and all the Polyplacocystis species were transferred to Raphidocystis which name has a priority. The evolution of centrohelid coverings in the light of new findings was discussed, as well as the possible wide distribution of dimorphism in centrohelid life cycles.