New Approach to the Ultrastructure of the Capillitium in the Order Trichiales (Myxomycetes, Amoebozoa) and its Phylogenetic Implications.

Myxomycetes constitute one of the major lineages within the supergroup Amoebozoa. At the end of their life cycles, most myxomycetes produce spore-bearing fruiting bodies, in which additional structures develop, like the capillitium, a system of sterile filaments intermingled with the spores. The capillitium is a relevant structure in the taxonomy of the order Trichiales, the target group in this study. However, the introduction of molecular phylogenies in Myxomycetes systematics is challenging our comprehension of this structure. We studied the capillitium of 25 species representing nine Trichiales genera, with both scanning and transmission electron microscopy. In this order, the capillitium showed higher diversity than so far recognized. Thus, we distinguished and described five capillitium types and two subtypes based on the presence or absence of a lumen and the wall ultrastructure. These types followed the evolutionary history reported in recent phylogenies, although not all of them defined monophyletic groups. Besides, the spiral ornamentation, which most taxonomists considered to have appeared once, occurred in three different capillitium types. The ultrastructural approaches in Myxomycetes systematics enable the reconsideration of their morphological features in the new phylogenetic scenario.

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.

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.

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.

Stygamoeba cauta n. sp. (Amoebozoa, Discosea) - a new brackish-water species from Nivå Bay (Baltic Sea, The Sound).

Several evolutionary lineages of Amoebozoa are characterized by unusual morphological and ultrastructural features that impede resolving of their position in the phylogenetic tree. Among them is the genus Stygamoeba, not yet reliably placed on the phylogenetic tree even by a phylogenomic analysis. Only two species of Stygamoeba are known at present, and molecular data exists on one species only. Here, we present a description of the mesohaline species Stygamoeba cauta n. sp. isolated from the bottom sediments of Nivå Bay (Baltic Sea, The Sound). This stick-like, flattened amoeba morphologically resembles the previously described species Stygamoeba regulataSmirnov, 1996. However, the molecular analysis based on the 18S rRNA gene sequences and differences in cell behavior and pattern of locomotion provide strong support for establishing a new species.

Description of Armaparvus languidus n. gen. n. sp. Confirms Ultrastructural Unity of Cutosea (Amoebozoa, Evosea).

The American Type Culture Collection (ATCC) PRA-29 isolate has a publicly available transcriptome, which has led to its inclusion in recent phylogenomic analyses. The ATCC PRA-29 isolate was originally identified and deposited as "Pessonella sp." This taxon branches robustly within the recently discovered clade Cutosea, very distantly related to the clade in which the genus Pessonella is believed to branch based on morphological data. Using detailed light and electron microscopy, we studied the morphology and ultrastructure of ATCC PRA-29 as well as other cutosean amoebae to better elucidate the morphological affinity of ATCC PRA-29 to other amoebozoans. Here, we show that ATCC PRA-29 was misidentified by the original depositor as Pessonella and name it Armaparvus languidus n. gen. n. sp. We show that a cell coat of microscales separated from the cell membrane is a unique trait found in all known cutosean amoebae. As Cutosea represents a clade at the deepest bifurcation in the amoebozoan group Evosea and because this clade is currently taxon-poor, but likely represents a major understudied group it will be important to isolate and describe more cutosean amoebae in the future.

In vitro gill cell monolayer successfully reproduces in vivo Atlantic salmon host responses to Neoparamoeba perurans infection.

An in vitro model to study the host response to Neoparamoeba perurans, the causative agent of amoebic gill disease (AGD), was evaluated. The rainbow trout gill derived cell line, RTgill-W1, was seeded onto permeable cell culture supports and maintained asymmetrically with apical seawater. Cells were inoculated with either a passage attenuated or a recent wild clone of N. perurans. Amoebae, loaded with phagocytosed fluorescent beads, were observed associated with host cells within 20 min post inoculation (pi). By 6 h small foci of cytopathic effect appeared and at 72 h cytolysis was observed, with total disruption of the cell monolayer at 96 h pi. Due to cell monolayer disruption, the platform could not support proliferation of amoebae, which showed a 3-log reduction in parasite 18S rRNA mRNA after 72 h (106 copies at 1 h to 103 at 72 h pi). SEM observations showed amoebae-like cells with either short pseudopodia and a malleiform shape, or, long pseudopodia embedded within the gill cells and erosion of the cell monolayer. To study the host immune response, inoculated gill cells were harvested from triplicate inserts at 0, 1, 3, 6, 24 and 48 h pi, and expression of 12 genes involved in the Atlantic salmon response to AGD was compared between infected and uninfected cells and between amoebic clones. Both clones induced similar host inmate immune responses, with the up-regulation of proinflammatory cytokine IL1ß, complement C3 and cell receptor MHC-1. The Th2 pathway was up-regulated, with increased gene expression of the transcription factor GATA3, and Th2 cytokines IL10, IL6 and IL4/13A. PCNA and AG-2 were also up-regulated. The wild clone induced significantly higher up-regulation of IL1ß, MHC-1, PCNA, lysozyme and IL10 than the attenuated clone for at least some exposure times, but AG-2 gene expression was higher in cells inoculated with the attenuated one. A principal component analysis showed that AG-2 and IL10 were key genes in the in vitro host response to N. perurans. This in vitro model has proved to be a promising tool to study host responses to amoebae and may therefore reduce the requirement for in vivo studies when evaluating alternative therapeutants to AGD control.

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.

Phylogenomics of Thecamoebida (Discosea, Amoebozoa) with the Description of Stratorugosa tubuloviscum gen. nov. sp. nov., a Freshwater Amoeba with a Perinuclear MTOC.

Thecamoebida Smirnov and Cavalier-Smith, 2011 (Discosea, Amoebozoa) has been molecularly understudied. The group until recently consisted of three genera containing species that live in terrestrial or aquatic environments. Here, we describe a fourth genus, Stratorugosa tubuloviscum gen. nov. sp. nov., which was isolated from a freshwater Amoeba proteus Ward's Science culture. Although this species most closely morphologically resembles a large, rugose Thecamoeba, S. tubuloviscum gen. nov. sp. nov. can be differentiated from Thecamoeba spp. by the following: 1) the presence of definitive finger-like (lobate-like) subpseudopodia extending at both the anterior and lateral parts of the cell during locomotion; 2) a peculiar locomotive mechanism with two sections, frontal and back, of the cells moving in a pulling and piggyback movement, respectively; 3) the presence of fibrillar cytoplasmic microtubules (MTs) organized by a prominent, perinuclear microtubule-organizing center (MTOC). A phylogenomic analysis of 511 genes assembled from transcriptomic data showed that this new genus was highly supported as sister to Stenamoeba. Despite the variance in gross morphology, Stenamoeba and S. tubuloviscum gen nov. sp. nov. both have MTOCs unlike two Thecamoeba spp., which display dot-like cytoplasmic MTs and lack an MTOC.

More amoebae from the deep-sea: Two new marine species of Vexillifera (Amoebozoa, Dactylopodida) with notes on taxonomy of the genus.

Two marine members of the genus Vexillifera Schaeffer, 1926 (Amoebozoa, Dactylopodida) are described. Vexillifera abyssalis n. sp. originates from an abyssal sample of the Western Atlantic 4.5 km deep, which is the first unambiguous record of a deep-sea Vexillifera. The second species, V. kereti n. sp. was isolated from the soft bottom sediments of the White Sea (depth 106 m). An analysis of available data on the genus Vexillifera shows that it comprises many different species, yet they are very unevenly studied. The majority of species have only been described using light microscopy, and their phylogenetic relationships with other amoebae are unclear. However, available small-subunit (SSU) rRNA gene sequences of Vexillifera spp. form a robust, yet very heterogeneous clade in the phylogenetic tree. These species demonstrate a wide range of morphological and ultrastructural characters and originate from diverse habitats, suggesting that Vexillifera may need to be subdivided into several genera in the future. In addition to the described species, we sequenced the COI gene of original CCAP strains of Vexillifera bacillipedes, V. minutissima and Pseudoparamoeba pagei, thereby performing a phylogenetic reconstruction of the Dactylopodida based on a decent taxonomic sampling.

Clydonella sawyeri n. sp. (Amoebozoa, Vannellida): Morphological and molecular study and a re-definition of the genus Clydonella Sawyer, 1975.

We isolated and described a brackish-water amoeba, Clydonella sawyeri n. sp. (Amoebozoa, Vannellida), from the littoral habitat in Kandalaksha Bay (The White Sea, northwestern Russia). Morphology of this amoeba corresponds to the initially proposed diagnosis of the genus Clydonella Sawyer, 1975, although it is sufficiently different from other described species of this genus to warrant a distinct species designation. Phylogenetic analysis based on the small-subunit rRNA gene shows that this species is closely related to the two previously studied ATCC strains identified as Clydonella sp., for which only ultrastructural and molecular data were published. Cytochrome oxidase subunit 1 (COI) gene sequence of the studied species was for the first time obtained for Clydonella. Analysis of this marker shows that this genus belongs to Vannellida and is separated from the other vannellids sampled to date. Phylogenetic analysis of the concatenated SSU rRNA and COI genes dataset yields the best resolved position of Clydonella compared to both markers when analysed separately. Based on the data presented we finally link light microscopic, ultrastructural and molecular data in a description of a single strain, which allows a refinement of the diagnosis of the genus Clydonella.

Sappinia sp. (Amoebozoa: Thecamoebida) and Rosculus sp. (SAR: Cercozoa) Isolated From King Penguin Guano Collected in the Subantarctic (South Georgia, Salisbury Plain) and their Coexistence in Culture.

Two amoeboid organisms of the genera Sappinia Dangeard, 1896 and Rosculus Hawes, 1963 were identified in a sample containing king penguin guano. This sample, collected in the Subantarctic, enlarges the list of fecal habitats known for the presence of coprophilic amoebae. The two organisms were co-isolated and subcultured for over 6 mo, with continuous efforts being invested to separate each one from the mixed culture. In the mixed culture, Rosculus cells were fast growing, tolerated changes in culturing conditions, formed cysts, and evidently were attracted by Sappinia trophozoites. The separation of the Rosculus strain was accomplished, whereas the Sappinia strain remained intermixed with inseparable Rosculus cells. Sappinia cell populations were sensitive to changes in culturing conditions; they improved with reduction of Rosculus cells in the mixed culture. Thick-walled cysts, reportedly formed by Sappinia species, were not seen. The ultrastructure of both organisms was congruent with the currently accepted generic characteristics; however, some details were remarkable at the species level. Combined with the results of phylogenetic analyses, our findings indicate that the ultrastructure of the glycocalyx and the presence/absence of the Golgi apparatus in differential diagnoses of Sappinia species require a critical re-evaluation.

Vannella pentlandii n. sp., (Amoebozoa, Discosea, Vannellida) a small, cyst-forming soil amoeba.

We describe a new species of cyst-producing soil amoeba Vannella pentlandii from course pasture in the Pentland Hills, Scotland. Analysis of the 18S rDNA gene reveals that it belongs to the sub-group within the genus, presently composed of V. placida, V. epipetala and V. fimicola (the PEF group). This group share features such as longitudinal folds/ridges on the lamella (the anterior hyaline region of the trophozoite), stubby floating forms and cyst production. While each PEF species contain cyst producing strains, not all strains within these species do so. V. fimicola produces cysts on stalks leading to its former classification as a slime mould, however no such stalks were evident in the V. pentlandii, instead groups of cysts become piled on top of each other forming clumps. The encysting amoebae crawl toward each other, pushing some off the surface to form these mounds. The V. pentlandii trophozoites are of typical size for the genus but the cysts at 6.9 µm in diameter, are the smallest so far described in genus Vannella. Other cyst producing species are found in various branches within the Vannella phylogenetic tree, probably meaning that this ability was ancestral but lost in many branches (particularly in marine species), and perhaps re-gained in others.

Description of Flamella daurica n. sp., with notes on the phylogeny of the genus Flamella and related taxa.

A new species of naked amoebae, Flamella daurica (Amoebozoa, Conosa, Variosea), from the Russian Far East is studied and described. In addition, we isolated a strain, identified as Flamella aegyptia from Lake Ontario in Canada. Its SSU rRNA gene sequence is almost identical with that of the type strain, except for the presence of an intron in the SSU sequence. The phylogenetic analysis of a variosean alignment after inclusion of a number of environmental sequences found in GenBank confirmed that the species Talaepolella tubasferens Lahr 2012 is closely related to the Flamella clade but probably represents an independent genus and includes no less than two different species. This conclusion is supported by considerable differences between Talaepolella and Flamella in morphology and life history. Talaepolella tubasferens is an evolutionary important species demonstrating the transition from compact flamellian to expanded acramoebian morphology. The genus Flamella now includes twelve named species, which makes it one of the most species-rich genera of naked lobose amoebae.

Mycamoeba gemmipara nov. gen., nov. sp., the First Cultured Member of the Environmental Dermamoebidae Clade LKM74 and its Unusual Life Cycle.

Since the first environmental DNA surveys, entire groups of sequences called "environmental clades" did not have any cultured representative. LKM74 is an amoebozoan clade affiliated to Dermamoebidae, whose presence is pervasively reported in soil and freshwater. We obtained an isolate from soil that we assigned to LKM74 by molecular phylogeny, close related to freshwater clones. We described Mycamoeba gemmipara based on observations made with light- and transmission electron microscopy. It is an extremely small amoeba with typical lingulate shape. Unlike other Dermamoebidae, it lacked ornamentation on its cell membrane, and condensed chromatin formed characteristic patterns in the nucleus. M. gemmipara displayed a unique life cycle: trophozoites formed walled coccoid stages which grew through successive buddings and developed into branched structures holding cysts. These structures, measuring hundreds of micrometres, are built as the exclusive product of osmotrophic feeding. To demonstrate that M. gemmipara is a genuine soil inhabitant, we screened its presence in an environmental soil DNA diversity survey performed on an experimental setup where pig cadavers were left to decompose in soils to follow changes in eukaryotic communities. Mycamoeba gemmipara was present in all samples, although related reads were uncommon underneath the cadaver.

Copromyxa laresi n. sp. (Amoebozoa: Tubulinea) and Transfer of Cashia limacoides (Page, 1967) to Copromyxa Zopf, 1885.

Five amoeboid organisms of different origin (isolated from fish organs, soil and digestive tract of earthworm) that shared light microscopical and ultrastructural features including type and arrangement of mitochondrial cristae were subjected to phylogenetic analyses based on sequences of SSU rDNA and protein coding genes (actin, cytochrome oxidase I, and eukaryotic elongation factor 2). The reconstruction of multigene phylogeny of the strains studied (i) revealed that they belong to the same single-genus Copromyxa clade; (ii) strongly supported position of Copromyxa cantabrigiensis (syn. Hartmannella cantabrigiensis) within the genus; (iii) together with comparisons of light and electron microscopy data justified reclassification of Cashia limacoides (syn. Vexillifera expectata) to Copromyxa limacoides n. comb., and (iv) justified description of a new species, Copromyxa laresi n. sp.

Environmental Free-Living Amoebae Isolated from Soil in Khon Kaen, Thailand, Antagonize Burkholderia pseudomallei.

Presence of Burkholderia pseudomallei in soil and water is correlated with endemicity of melioidosis in Southeast Asia and northern Australia. Several biological and physico-chemical factors have been shown to influence persistence of B. pseudomallei in the environment of endemic areas. This study was the first to evaluate the interaction of B. pseudomallei with soil amoebae isolated from B. pseudomallei-positive soil site in Khon Kaen, Thailand. Four species of amoebae, Paravahlkampfia ustiana, Acanthamoeba sp., Naegleria pagei, and isolate A-ST39-E1, were isolated, cultured and identified based on morphology, movement and 18S rRNA gene sequence. Co-cultivation combined with a kanamycin-protection assay of B. pseudomallei with these amoebae at MOI 20 at 30°C were evaluated during 0-6 h using the plate count technique on Ashdown's agar. The fate of intracellular B. pseudomallei in these amoebae was also monitored by confocal laser scanning microscopy (CLSM) observation of the CellTracker™ Orange-B. pseudomallei stained cells. The results demonstrated the ability of P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 to graze B. pseudomallei. However, the number of internalized B. pseudomallei substantially decreased and the bacterial cells disappeared during the observation period, suggesting they had been digested. We found that B. pseudomallei promoted the growth of Acanthamoeba sp. and isolate A-ST39-E1 in co-cultures at MOI 100 at 30°C, 24 h. These findings indicated that P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 may prey upon B. pseudomallei rather than representing potential environmental reservoirs in which the bacteria can persist.

Pseudoparamoeba microlepis n. sp., Korotnevella fousta n. sp. (Amoebozoa, Dactylopodida), with notes on the evolution of scales among dactylopodid amoebae.

The present paper describes two new species of lobose amoebae belonging to the family Paramoebidae within the order Dactylopodida: Pseudoparamoeba microlepis n. sp. and Korotnevella fousta n. sp. Among them, P. microlepis formally fits the diagnosis of the genus Korotnevella, because it has scales and lacks a parasome. At the same time its 18S rDNA gene sequence robustly groups with Pseudoparamoeba pagei and never branches among those of Korotnevella spp. Korotnevella fousta in 18S rDNA tree groups among other species of the genus Korotnevella. It has uniform scales with an elliptical basal plate and spine arising from its central part and surrounded by an "inverted skirt" structure. These findings show that the current differentiation between the genera Korotnevella and Pseudoparamoeba based on the presence of scales is not entirely valid. At the moment only molecular data can reliably differentiate these two genera.

New data on the ultrastructure of Paradermamoeba levis (Amoebozoa, Discosea, Dermamoebida): Cytoplasmic MTOCs are found among Dermamoebida.

In this detailed electron microscopic study of Paradermamoeba levis Smirnov et Goodkov 1994 (phylum Amoebozoa, class Discosea, subclass Longamoebia, order Dermamoebida, family Dermamoebidae) based on numerous fixations and studies of a large number of cells we provide the first comprehensive description of the ultrastructure of this species. P. levis possesses cytoplasmic microtubule-organizing centres (MTOCs) associated with dictyosomes of the Golgi complex. This finding adds evidence to our earlier suggestion that the presence of cytoplasmic MTOCs is a synapomorphy of the phylogenetic lineages forming the subclass Longamoebia. The so-called "supernumerary nucleus" of P. levis noted in the initial description was found to be not an individual structure but an outgrowth of the cell nucleus containing its own nucleolus. Enigmatic trichocyst-like bodies were noted in all studied strains, originating from different geographic locations. This proves that these bodies are integral parts of the cell structure, not an occasional property of the type strain. P. levis is now reliably recorded from several European locations (North-Western Russia, Croatia, Switzerland, UK) and Far East of Russia.

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.