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Metchnikovellids are a deep-branching group of microsporidia, parasites of gregarines inhabiting the alimentary tract of polychaetes and some other invertebrates. The diversity and phylogeny of these hyperparasites remain poorly studied. Modern descriptions and molecular data are still lacking for many species. The results of a light microscopy study and molecular data for Metchnikovella spiralis Sokolova et al., 2014, a hyperparasite of the eugregarine Polyrhabdina sp., isolated from the polychaete Pygospio elegans, were obtained. The original description of M. spiralis was based primarily on the analysis of stained preparations and transmission electron microscopy images. Here, the species description was complemented with the results of in vivo observations and phylogenetic analysis based on the SSU rRNA gene. It was shown that in this species, free sporogony precedes sac-bound sporogony, as it occurs in the life cycle of most other metchnikovellids. Spore sacs are entwined with spirally wound cords, and possess only one polar plug. Phylogenetic analyses did not group M. spiralis with M. incurvata, another metchnikovellid from the same gregarine species, but placed it as a sister branch to Amphiacantha. The paraphyletic nature of the genus Metchnikovella was discussed. The taxonomic summary for M. spiralis was emended.

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.

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.

Amoebae of the order Vannellida (Amoebozoa, Discosea) have a fairly recognizable spatulate, fan-shaped or semi-circular outlines and a wide area of frontal hyaloplasm. They can be easily distinguished from the other groups of lobose amoebae even by light microscopy. The dorsal side of these amoebae is usually smooth and rarely bears ridges or folds, which are never numerous or regular. We have isolated an unusual species of vannellid amoebae, called Vannella primoblina n. sp. from a terrestrial substrate. It has well-developed dorsal relief consisting of regularly appearing folds and ridges. This amoeba superficially resembles members of the genus Thecamoeba. However, molecular analysis showed that this strain belongs to the genus Vannella. This finding indicates that dorsal folds may also be a characteristic of some species of vannellid amoebae and probably are a functional detail of the cell morphology rather than an apomorphy of Thecamoebida lineage. Overall outlines of the cell and the presence of the expanded frontal hyaline area remains the most reliable characters used to differentiate vannellid amoebae from other gymnamoebae lineages.

International audience; Abstract Metchnikovellids are highly specialized hyperparasites, which infect and reproduce inside gregarines (Apicomplexa) inhabiting marine invertebrates. Their phylogenetic affiliation was under constant discussion until recently, when analysis of the first near-complete metchnikovellid genome, that of Amphiamblys sp., placed it in a basal position with respect to most Microsporidia. Microsporidia are a highly diversified lineage of extremely reduced parasites related to Rozellida (Rozellosporidia = Rozellomycota = Cryptomycota) within the Holomycota clade of Opisthokonta. By sequencing DNA from a single-isolated infected gregarine cell we obtained an almost complete genome of a second metchnikovellid species, and the first one of a taxonomically described and well-documented species, Metchnikovella incurvata. Our phylogenomic analyses show that, despite being considerably divergent from each other, M. incurvata forms a monophyletic group with Amphiamplys sp., and confirm that metchnikovellids are one of the deep branches of Microsporidia. Comparative genomic analysis demonstrates that, like most Microsporidia, metchnikovellids lack mitochondrial genes involved in energy transduction and are thus incapable of synthesizing their own ATP via mitochondrial oxidative phosphorylation. They also lack the horizontally acquired ATP transporters widespread in most Microsporidia. We hypothesize that a family of mitochondrial carrier proteins evolved to transport ATP from the host into the metchnikovellid cell. We observe the progressive reduction of genes involved in DNA repair pathways along the evolutionary path of Microsporidia, which might explain, at least partly, the extremely high evolutionary rate of the most derived species. Our data also suggest that genome reduction and acquisition of novel genes co-occurred during the adaptation of Microsporidia to their hosts.

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.

Morphological identification of naked lobose amoebae has always been a problem, hence the development of reliable molecular tools for species distinction is a priority for amoebae systematics. Previous studies based on SSU rDNA sequences provided a backbone for the phylogeny of Amoebozoa but were of little help for the species distinctions in this group. On one hand, the SSU rDNA sequences were rather conserved between closely related species; on the other hand, the intra-strain polymorphism of the SSU gene obscured species identification. In the present study, a 3' fragment of the SSU, a complete ITS1-5.8S-ITS2 block and a 5' fragment of COI gene were cloned and sequenced for six Vannella morphospecies, of which V. simplex was represented by six different isolates. SSU rDNA and ITS were found to be inappropriate for species differentiation, while distinctive and homogenous COI sequences were obtained for each well-defined morphospecies. Moreover, a number of distinct COI genotypes have been identified among V. simplex isolates. This suggests that COI may be a good candidate for DNA barcoding of amoebae, but further studies are necessary to confirm the accurateness of the COI gene as a barcode in other gymnamoebae, and to understand the taxonomic meaning of COI variations.

A new species of Leptomyxida, named Rhizamoeba neglecta was found during studies of the amoeba fauna of the inner Lake Leshevoe located at Valamo archipelago (The Lake Ladoga, North-Western Russia). Light-microscopical and ultrastructural studies indicated that it represents a new species of Leptomyxida. The partial 18S rDNA sequence of this amoeba is very similar to that of Leptomyxa reticulata.. These organisms, however, are very different in LM morphology and biology. Organisms assigned to the genus Rhizamoeba do not form a single clade in the 18S rDNA tree. This may indicate that the genus is an artificial grouping or that a number of studied strains were misidentified. The phylogeny and the systematics of leptomyxids require further investigation.

Lobose amoebae are abundant free-living protists and important pathogenic agents, yet their evolutionary history and position in the universal tree of life are poorly known. Molecular data for lobose amoebae are limited to a few species, and all phylogenetic studies published so far lacked representatives of many of their taxonomic groups. Here we analyse actin and small-subunit ribosomal RNA (SSU rRNA) gene sequences of a broad taxon sampling of naked, lobose amoebae. Our results support the existence of a monophyletic Amoebozoa clade, which comprises all lobose amoebae examined so far, as well as the amitochondriate pelobionts and entamoebids, and the slime molds. Both actin and SSU rRNA phylogenies distinguish two well-defined clades of amoebae, the “Gymnamoebia sensu stricto” and the Archamoebae (pelobionts+entamoebids), and one weakly supported and ill-resolved group comprising some naked, lobose amoebae and the Mycetozoa.

We sequenced 18S rRNA genes from 21 vannellid amoebae (Amoebozoa; Vannellidae), including nearly all available type cultures, and performed a comprehensive phylogenetic analysis for 57 Vannellidae sequences. The results show that species of Vannella and Platyamoeba are completely mixed and do not form distinct clades. Several very closely related species pairs exist, each with a Vannella and a Platyamoeba species differing in only a few nucleotides. Therefore, presence (Vannella) or absence (Platyamoeba) of glycostyles in the cell surface coat is an invalid generic distinction; the genera must be merged. As Vannella has priority, we formally transferred Platyamoeba species into Vannella, except for the non-vannellid P. stenopodia, here renamed Stenamoeba stenopodia gen. n. comb. n. and transferred to the family Thecamoebidae. Our trees show that Vannella glycostyles were probably easily and repeatedly evolutionarily lost. We have established a new genus Ripella, with distinct morphology and sequence signatures for Vannella platypodia and morphologically similar species that form a clearly separate clade, very distant from other Vannellidae. Vannellids form four well-separated single-genus clades: Vannella sensu stricto, Ripella, Clydonella, and Lingulamoeba. Species of the revised genus Vannella comprise four closely related, well-supported subclades: one marine and three freshwater. Here, we provide an illustrated checklist for all 40 known Vannellidae species.