Vannella primoblina n. sp. – an unusual species of the genus Vannella (Amoebozoa,Discosea, Vannellida) with pronounced dorsal ridges and folds

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.     

Phylogeny, evolution, and taxonomy of vannellid amoebae

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.     

Dermamoeba algensis n. sp. (Amoebozoa, Dermamoebidae): an algivorous lobose amoeba with complex cell coat and unusual feeding mode

The genus Dermamoeba unifies oblong, flattened amoebae of lingulate morphotype, possessing a thick multilayered cell coat. It includes two species, D. granifera and D. minor. In this paper we describe a third species of this genus, D. algensis n. sp. This species is algivorous; engulfing a large algal cell, it destroys part of the cell coat liberating the plasma membrane, which forms the food vacuole. Thus the glycocalyx never appears inside the phagosome. This observation confirms that some of the thick-coated amoebae may use this way to avoid energetically costly digestion of their own glycocalyx. Studies of the physiology of this organism show that it feeds most actively at a temperature of 22-25 °C. Below and above this temperature the feeding intensity drastically decreases. The new species can survive NaCl concentrations up to 5%, which roughly corresponds to 50 ppt salinity. Accordingly, D. algensis has a wide range of salinity tolerance.     

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.     

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.     

Description of a new species of marine amoeba Korotnevella mutabilis n. sp. (Amoebozoa,Dactylopodida)

Korotnevella (Amoebozoa, Dactylopodida) is a genus of naked lobose amoebae with a dactylopodial morphotype. The cell membrane of these amoebae is covered with a monolayer of scales. The structure and size of scales are considered as species-specific features. Here, we describe a new marine species, Korotnevella mutabilis n. sp., isolated from the bottom sediment sample of Nivå Bay (Baltic Sea, The Sound) and studied with light and electron microscopy as well as with molecular phylogenetic analysis. This species has a number of morphological similarities with Korotnevella monacantholepis, such as size of the cell, L/B ratio, the nucleus structure and the type of a biotope from which both species were isolated. At the same time, Korotnevella mutabilis n. sp. differs from K. monacantholepis in the structure of basket-shaped scales: Korotnevella mutabilis n. sp. has an enclosed hammock-shaped latticework basket and up to two spines while K. monacantholepis has an opened two-row latticework basket and never has two spines. According to molecular phylogenetic analyses based on the sequences of the mitochondrial COI gene, Korotnevella mutabilis n. sp. is a distinct species, highly divergent from other Korotnevella species.     

Vannella samoroda n. sp. (Amoebozoa) — First member of the genus from a continental saline habitat placed in a molecular tree

Vannella samoroda n. sp. (Amoebozoa, Vannellida) was isolated from the mouth of the Malaya Samoroda river flowing into Elton, the largest European hypersaline lake (Russia). Among all rivers of the area, it has the highest salt content (ca. 110‰). Amoebae maintained in seawater medium with ca. 77‰ salts concentration had a set of morphological characters typical of Vannella spp.: rounded, fan-shaped, or spatulate locomotive form, floating form with bent, blunt-ended hyaline pseudopodia, and a cell coat consisting of regularly packed palisade elements and scarce simple filaments. Phylogenetic analyses based on SSU rRNA and cytochrome C oxidase subunit 1 genes show that the amoeba is most closely related to Vannella ebro Smirnov, 2001, but represents a distinct species. The clade of V. ebro and V. samoroda branches among marine species of Vannella. The studied species is the first member of the genus Vannella from a continental saline habitat described using molecular data. Interestingly, it has a broad range of salinity tolerance: cells reproduce above 18‰, while survival of a few cells regularly occurs even in highly diluted Prescott and James medium. The normal culture restores itself when PJ medium is substituted with 77‰ seawater medium even after months of experimental incubation.     

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.     

Cunea russae n. sp. (Amoebozoa,Dactylopodida), another cryptic species of Cunea Kudryavtsev and Pawlowski, 2015, inhabits a continental brackish-water biotope

The genus Cunea Kudryavtsev and Pawlowski, 2015 (Amoebozoa, Dactylopodida) was initially described from the oceanic benthos: C. profundata, from over 5 km depth in the Atlantic Ocean, and C. thuwala from the Red Sea benthos at ca. 60 m depth. Both species are identical to each other in morphology (including cell coat ultrastructure), but differ significantly in the gene sequence data, including barcoding loci of small subunit ribosomal RNA and cytochrome oxidase subunit 1 gene, as well as actin. This paper describes the third species of Cunea, C. russae n. sp. isolated from a brackish water habitat without a direct connection to the ocean, a small spring of brackish water (19‰) emerging from a 246 m deep hole in the earth. This species is morphologically identical to the previous two amoebae, but differs from them significantly in the gene sequence data and ecological preferences. In particular, this species has the broadest salinity tolerance range, being able to reproduce well already at 2.5‰. It is also capable of resisting cold temperatures, like C. profundata. The data obtained suggest that the genus Cunea may comprise a significant taxonomic diversity represented by morphologically identical, but quickly diverging species with significant ecological plasticity.     

Thecamoeba foliovenanda n. sp. (Amoebozoa,Discosea, Thecamoebida) – One more case of sibling species among amoebae of the genus Thecamoeba

Until recently it seemed that amoebae of the genus Thecamoeba can be reliably identified using light-microscopic characters, like the size and shape of the cell, a characteristic pattern of folds and ridges and structure of the nucleus. However, recent molecular studies show the presence of sibling species that can be reliably distinguished based on the gene sequence data only. Here we describe a new terrestrial species, Thecamoeba foliovenanda n. sp. This species is almost identical with Thecamoeba similis in light-microscopy, which has minor differences in the ultrastructure but considerable differences in the SSU rRNA gene sequence. We investigated the light-microscopic data, as well as transmission electron-microscopic images and videorecords on the type strain of T. similis 1583/8 and performed its comparison with the new species. This study further supports the conclusion that species of the genus Thecamoeba nowadays require gene sequencing for reliable identification and species distinction.     

Pellitidae n. fam. (Lobosea, Gymnamoebia) – a new family, accommodating two amoebae with an unusual cell coat and an original mode of locomotion, Pellita catalonica n.g., n.sp. and Pellita digitata comb. nov

Two species of amoebae, the marine Pellita catalonica n. g., n. sp. discovered in the Ebro Delta (Spain) and Nivå Bay (Denmark) and the freshwater Pellita digitata n. comb., previously known from the UK and Switzerland and now found in North-Western Russia, have a very thick (0.5–0.8 μm) cell coat consisting of a fuzzy fibrous basal layer, covered with the dense layer of complex pentagonal glycostyles. The cell coat is integrated with the cell membrane and entirely envelops the amoeba, like a typical glycocalyx. For purposes of locomotion and phagocytosis the cell produces short papilliform subpseudopodia that protrude through the cell coat and are covered solely by the cell membrane. In order to accommodate these unusual organisms we established a new family, Pellitidae n. fam., within the subclass Gymnamoebia sensu Page, 1987, order Euamoebida.     

Thecamoeba cosmophorea n. sp. (Amoebozoa,Discosea, Thecamoebida) — An example of sibling species within the genus Thecamoeba

A new species Thecamoeba cosmophorea n. sp. (Amoebozoa, Discosea, Thecamoebida) was isolated from leaf litter collected in the surroundings of Saint-Petersburg (Russia). This species resembles T. quadrilineata in light-microscopic morphology, but has certain morphological differences and significantly differs in 18S rRNA gene sequence. We performed a direct comparison of this newly isolated species with the Thecamoeba strain isolated from leaf litter in East Siberia (Russia) and identified as T. quadrilineata both at the morphological and the molecular level. There is no type strain of T. quadrilineata, and the type material on this species is represented with the stained slide by F.C. Page that he designated as neotype in 1977. The sequence of the 18S rRNA gene of this species deposited in the GenBank belongs to the isolate identified as T. quadrilineata by Rolf Michel in 1998 and deposited as CCAP 1583/10 strain. Hence we cannot be entirely sure that morphological and molecular data on T. quadrilineata belong to the same amoebae species. The use of molecular data for reliable species differentiation is getting obligate even within the amoebae genus Thecamoeba, which until recently was believed to be among few genera of naked lobose amoebae allowing morphological identification of species.     

A diversity of amoebae colonise the gills of farmed Atlantic salmon (Salmo salar) with amoebic gill disease (AGD)

Neoparamoeba perurans is the aetiological agent of amoebic gill disease (AGD) in salmonids, however multiple other amoeba species colonise the gills and their role in AGD is unknown. Taxonomic assessments of these accompanying amoebae on AGD-affected salmon have previously been based on gross morphology alone. The aim of the present study was to document the diversity of amoebae colonising the gills of AGD-affected farmed Atlantic salmon using a combination of morphological and sequence-based taxonomic methods. Amoebae were characterised morphologically via light microscopy and transmission electron microscopy, and by phylogenetic analyses based on the 18S rRNA gene and cytochrome oxidase subunit I (COI) gene. In addition to N. perurans, 11 other amoebozoans were isolated from the gills, and were classified within the genera Neoparamoeba, Paramoeba, Vexillifera, Pseudoparamoeba, Vannella and Nolandella. In some cases, such as Paramoeba eilhardi, this is the first time this species has been isolated from the gills of teleost fish. Furthermore, sequencing of both the 18S rRNA and COI gene revealed significant genetic variation within genera. We highlight that there is a far greater diversity of amoebae colonising AGD-affected gills than previously established.     

Primary and Secondary Structure of the Small-Subunit Ribosomal RNA of the Naked, Marine Amoeba Vannella anglica: Phylogenetic Implications

The primary and secondary structure of the small-subunit ribosomal RNA (ssrRNA) gene from the naked, marine amoeba, Vannella anglica (subclass Gymnamoebia), was determined. The ssrRNA is 1962 nucleotides in length, with a low G+C content of 37.1%. The ssrRNA is composed of several uncommon secondary structure features including helix E8-1, which may be a useful target for rRNA probes for the direct identification of isolates in mixed culture. Phylogenetic analysis of sequence data showed that V. anglica branched prior to the rapid diversification of the eukaryotes. It did not associate with the other naked, lobose amoebae represented by Acanthamoeba and Hartmannella, indicating that Vannella represents a separate amoeboid lineage and the subclass Gymnamoebia is polyphyletic. Received: 9 July 1998 / Accepted: 16 November 1998     

Two new small-subunit ribosomal RNA gene lineages within the subclass gymnamoebia

Phylogenetic analysis of small-subunit ribosomal RNA gene sequences for gymnamoebae of the families Vexilliferidae, Paramoebidae, and Vannellidae identified two distinct lineages that are supported by gross morphological characters. This analysis indicates that paramoebids and vexilliferids are part of one lineage and that vannellids belong to another. A shared morphological character unique to the paramoebid/vexilliferid lineage members is the presence of dactylopodiate subpseudopodia. However, cell surface structures, normally used for taxonomic discrimination, range from simple hair-like filaments without any apparent organization (Neoparamoeba), to hexagonal glycostyles (Vexillifera) or more elaborate surface scales (Korotnevella). Taxa within the vannellid lineage all lack subpseudopodia and appear flabellate, spatulate or linguiform while in locomotion. Cell surface structures of taxa within the vannellid lineage range from filaments organized into hexagonal arrays (Lingulamoeba, Platyamoeba) to pentagonal glycostyles (Clydonella, Vannella). Vannellid lineage members of the genera Clydonella and Lingulamoeba were studied at the level of electron microscopy. Unique cell surface features validate these as genera distinct from Vannella and Platyamoeba. Genetic and ultrastructural data are used to discuss the phylogenetic interrelationships for the taxa studied.     

Pseudoparamoeba garorimi n. sp., with Notes on Species Distinctions within the Genus

A new marine species of naked lobose amoebae Pseudoparamoeba garorimi n. sp. (Amoebozoa, Dactylopodida) isolated from intertidal marine sediments of Garorim Bay, Korea was studied with light and transmission electron microscopy. This species has a typical set of morphological characters for a genus including the shape of the locomotive form, type of subpseudopodia and the tendency to form the single long waving pseudopodium in locomotion. Furthermore, it has the same cell surface structures as were described for the type species, Pseudoparamoeba pagei: blister‐like glycostyles with hexagonal base and dome‐shaped apex; besides, cell surface bears hair‐like outgrowths. The new species described here lacks clear morphological distinctions from the two other Pseudoparamoeba species, but has considerable differences in the 18S rDNA and COX1 gene sequences. Phylogenetic analysis based on 18S rDNA placed P. garorimi n. sp. at the base of the Pseudoparamoeba clade with high PP/BS support. The level of COX1 sequence divergence was 22% between P. garorimi n. sp. and P. pagei and 25% between P. garorimi n. sp. and P. microlepis. Pseudoparamoeba species are hardly distinguishable by morphology alone, but display clear differences in 18S rDNA and COX1 gene sequences.     

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.