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.

Dactylomonas gen. nov., a Novel Lineage of Heterolobosean Flagellates with Unique Ultrastructure, Closely Related to the Amoeba Selenaion koniopes Park, De Jonckheere & Simpson, 2012.

We report the discovery of a new genus of heterolobosean flagellates, Dactylomonas gen. nov., with two species, D. venusta sp. nov. and D. crassa sp. nov. Phylogenetic analysis of the SSU rRNA gene showed that Dactylomonas is closely related to the amoeba Selenaion, the deepest-branching lineage of Tetramitia. Dactylomonads possess two flagella, and ultrastructural studies revealed an unexpected organization of the flagellar apparatus, which resembled Pharyngomonada (the second lineage of Heterolobosea) instead of Tetramitia: basal bodies were orthogonal to each other and a putative root R1 was present in the mastigont. On the other hand, Dactylomonas displayed several features uncommon in Heterolobosea: a microtubular corset, a distinctive rostrum supported by the main part of the right microtubular root, a finger-like projection on the proximal part of the recurrent flagellum, and absence of a ventral groove. In addition, Dactylomonas is anaerobic and seems to have lost mitochondrial cristae. Dactylomonas and Selenaion are accommodated in the family Selenaionidae fam. nov. and order Selenionida ord. nov. The taxonomy of Tetramitia is partially revised, and the family Neovahlkampfiidae fam. nov. is established.

Fine structure of Leptomyxa ambigua n. sp. CCAP 1546/2 strain, formerly known as "Rhizamoeba flabellata" (Amoebozoa, Tubulinea, Leptomyxida).

The species Leptomyxa flabellata was described by Goodey in 1915 and re-isolated by Pussard and Pons in 1976. It seems that it was never seen (or never recognized) again since that time. The strain designated as "Leptomyxa flabellata CCAP 1546/2" was studied by Cann in 1984, however the quality of the electron microscopic images of that time was poor. Based on the cyst structure and size characters, Page in 1988 suggested that this strain is not co-specific with Goodey's Leptomyxa flabellata, but represents a species 'Ripidomyxa' australiensis Chakraborty and Pussard, 1985, nowadays known as Rhizamoeba australiensis. In the present paper light- and electron-microscopic images of CCAP 1546/2 strain, which is now lost, are provided. Based on the morphological evidences it is suggested to establish it in a rank of a new species, Leptomyxa ambigua n. sp. Neither "true" L. flabellata Goodey, 1915 nor original R. australiensis Chakraborty et Pussard, 1985 are nowadays represented in the culture collections, and no original type material is available on both these species.

Leptomyxa valladaresi n. sp. (Amoebozoa, Tubulinea, Leptomyxida), from Mount Teide, Tenerife, Spain.

Leptomyxa valladaresi was isolated from soil in a pine forest on the southern flank of Mt Teide in Tenerife, Spain. It feeds on bacteria and on a range of other amoebae, and it was possible to establish bi-axenic cultures with L. valladaresi and Acanthamoeba. It is easily propagated on a E. coli also. 18S rDNA gene sequence analysis suggests that it is most closely related to Leptomyxa variabilis, however this amoeba differs in important detail. L. valladaresi is primarily mononucleate whereas L. variabilis is multinucleate. L. valladaresi is a larger amoeba and although the cysts are similar in size, there is no sign of the pore-like structures described in L. variabilis cysts. L. valladaresi can adopt a rapid monopodal and tubular morphology similar to that described for L. neglecta and Rhizamoeba matisi, and is never reticulated as larger L. variabilis individuals tend to be. The mean generation time was found to be 18 h, in line with amoebae of this size. Like other members of the genus, L. valladaresi is reported to harbour intracellular, presumably endosymbiotic bacteria, and a Delftia sp has been identified by 16S PCR a bacterium which is also known to grow within Acanthamoeba. The availability of this easily cultured species will help to characterize of this little studied genus and family and their relationship with bacteria, both prey and symbionts.

Origin and diversity of testate amoebae shell composition: Example of Bullinularia indica living in Sphagnum capillifolium.

Testate amoebae are free-living shelled protists that build a wide range of shells with various sizes, shapes, and compositions. Recent studies showed that xenosomic testate amoebae shells could be indicators of atmospheric particulate matter (PM) deposition. However, no study has yet been conducted to assess the intra-specific mineral, organic, and biologic grain diversity of a single xenosomic species in a natural undisturbed environment. This study aims at providing new information about grain selection to develop the potential use of xenosomic testate amoebae shells as bioindicators of the multiple-origin mineral/organic diversity of their proximal environment. To fulfil these objectives, we analysed the shell content of 38 Bullinularia indica individuals, a single xenosomic testate amoeba species living in Sphagnum capillifolium, by scanning electron microscope (SEM) coupled with X-ray spectroscopy. The shells exhibited high diversities of mineral, organic, and biomineral grains, which confirms their capability to recycle xenosomes. Mineral grain diversity and size of B. indica matched those of the atmospheric natural mineral PM deposited in the peatbog. Calculation of grain size sorting revealed a discrete selection of grains agglutinated by B. indica. These results are a first step towards understanding the mechanisms of particle selection by xenosomic testate amoebae in natural conditions.

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.

Regeneration of test in testate amoebae of the genus Arcella (Tubulinea, Arcellinida).

Although the presence of test is the most fundamental synapomorphy of arcellinid lobose amoebae, the significance of tests in maintenance of the biological functions of these organisms is still largely unclear. This paper demonstrates the effect of the artificial test removal on the behaviour, ultrastructure and reproduction of the testate lobose amoebae belonging to the two species of the genus Arcella. The studied cells can survive after artificial removal of their test, and many specimens are capable of building a test de novo. We investigated this process and found that test-free cells of Arcella in culture remained alive for weeks; they are able to move and feed, but never undergo normal cell division accompanied by mitosis. Test-free cells can restore the test in three different ways: (1) building almost normal test; (2) building a very small single-chambered test; (3) building a test which consisted of several small chambers attached to each other. Although newly constructed tests are abnormal, cells restore shape, size and other characteristic features of the test in the next one or two generations of descendants. The results obtained suggest that the test may be critically important for completing a life cycle of an amoeba.

Eight species in the Nebela collaris complex: Nebela gimlii (Arcellinida, Hyalospheniidae), a new species described from a Swiss raised bog.

We describe here a new species of sphagnicolous testate amoeba found abundantly in the forested part of the Le Cachot peatland (Jura Mountains, Neuchâtel, Switzerland) based on microscopical observations (LM, SEM). The new species, called Nebela gimlii was placed in a phylogenetic tree based on mitochondrial cytochrome oxidase sequences (COI), and branched robustly within the N. collaris complex next to the morphologically similar N. guttata and N. tincta. It is however genetically clearly distinct from these two species, and differs morphologically from them by its smaller size and stouter shape of the shell. This new species completes the phylogeny of the Nebela collaris species complex, with now eight species described, mostly from peatlands and acidic forest litter, and further demonstrates the existence of an unknown diversity within testate amoebae. Improving the taxonomy of testate amoebae in peatlands and clarifying the ecology of newly discovered species should make these organisms even more valuable as bioindicator and for palaeoecological reconstruction.

Morphological and molecular diversification of Asian endemic Difflugia tuberspinifera (Amoebozoa, Arcellinida): a case of fast morphological evolution in protists?

Planktonic arcellinid testate amoebae exhibit a broad-range of morphological variability but it is currently unclear to what extent this variability represents phenotypic plasticity or if it is genetically determined. We investigated the morphology and phylogenetic relationships of three endemic east-asian Difflugia taxa 1) the vase-shaped D. mulanensis, 2) and a spinose and a spineless morphotypes of D. tuberspinifera using scanning electron microscopy and two ribosomal genetic markers (SSU rDNA and ITS sequences). Our phylogenetic analyses shows that all three taxa are genetically distinct and closely related to D. achlora and Netzelia oviformis. The genetic variations between the spineless and spinose morphotypes of D. tuberspinifera were low at the SSU rRNA level (0.4%), but ten times higher at the ITS level (4.5-6%). Our data suggest that the two forms of D. tuberspinifera are sufficiently differentiated in terms of morphology and genetic characteristics to constitute two separate entities and that the presence of spines does not result from phenotypic plasticity due to environmental selective pressure. However further observational and experimental data are needed to determine if these two forms constitute different biological species.

Ptolemeba n. gen., a novel genus of hartmannellid amoebae (Tubulinea, Amoebozoa); with an emphasis on the taxonomy of Saccamoeba.

Hartmannellid amoebae are an unnatural assemblage of amoeboid organisms that are morphologically difficult to discern from one another. In molecular phylogenetic trees of the nuclear-encoded small subunit rDNA, they occupy at least five lineages within Tubulinea, a well-supported clade in Amoebozoa. The polyphyletic nature of the hartmannellids has led to many taxonomic problems, in particular paraphyletic genera. Recent taxonomic revisions have alleviated some of the problems. However, the genus Saccamoeba is paraphyletic and is still in need of revision as it currently occupies two distinct lineages. Here, we report a new clade on the tree of Tubulinea, which we infer represents a novel genus that we name Ptolemeba n. gen. This genus subsumes a clade of hartmannellid amoebae that were previously considered in the genus Saccamoeba, but whose mitochondrial morphology is distinct from Saccamoeba. In accordance with previous research, we formalize the clade as distinct from Saccamoeba. Transmission electron microscopy of our isolates illustrate that both molecularly discrete species can be further differentiated by their unique mitochondrial cristal morphology.

Evidence for two different morphotypes of Difflugia tuberspinifera from China.

Difflugia is a morphologically diverse genus of testate amoebae, which are common components of freshwater ecosystems. We observed a new morphotype similar to Difflugia tuberspinifera but without spine in four Xiamen reservoirs, Fujian Province, southeast China. We investigated its morphology and biometry using light and scanning electron microscopy. The linear discriminant analysis and principal component analysis of biometric characters revealed that the spiny and spineless forms of D. tuberspinifera differed only in the presence or absence of spine. Shell height, shell diameter, aperture diameter and collar height did not differ significantly between the two morphotypes. The number of conical spines varies from 0 to 8. However, the distribution of spine numbers showed two main modes at 0 (spineless form 45.1% of individuals) and 4-6 (38.9%), suggesting the possible existence of two genetically distinct lineages. Spines may have ecological and evolutionary significance. Our results suggested that the spiny and spineless morphotypes of D. tuberspinifera represent either a single variable taxon with different ecotypes or sibling species. Further morphological studies on clonal variations and molecular approaches are needed to clarify if the spineless morph represents an independent species or not.

Three-dimensional morphological and mineralogical characterization of testate amebae.

Testate amebae are unicellular shelled protozoa commonly used as indicators in ecological and paleoecological studies. We explored the potential application of three-dimensional (3D) X-ray micro-tomography used in addition to 2D techniques (environmental scanning electron microscopy, electron probe micro-analysis, and cathodoluminescence) for detailed characterization of agglutinated shells of protozoa. We analyzed four specimens of the aquatic testate ameba Difflugia oblonga (Arcellinida), to test whether size distribution and mineral composition of shell grains diverged from sediment size distribution and mineralogical composition. From the 3D images, the geometry of the specimens (size and mass) and of the individual grains forming the specimen (grain size distribution and volume) were calculated. Based on combined chemical, mineralogical, and morphological analyses we show that D. oblonga is able to selectively pick up the small size fraction of the sediment with a preference for low-density silicates close to quartz density (~2.65). The maximum size of the grains matches the size of the pseudostome (shell aperture), suggesting the existence of a physical limit to grain size used for building the shell. This study illustrates the potential of this combined approach to characterize agglutinated shells of protozoa. This data can be useful for detailed morphological studies with applications in taxonomy and ecology.

Occurrence of the lobose testate amoeba Pseudonebela africana (Amoebozoa, Arcellinida) in the Brazilian "cerrado".

Flagship species are defined as microbial eukaryote species with characteristic morphologies and restricted geographic distributions. These are proposed as ideal systems to elucidate patterns of geographical distribution in microbial eukaryotes. Here we present new records of the putative flagship species Pseudonebela africana, a lobose testate amoeba (Arcellinida) characterized by a cross or clover-shaped aperture and geographic distribution previously believed to be restricted to Africa. We have sampled P. africana from 5 separate ponds in the Central and Southwest Brazillian "cerrado", and characterized individuals both by light and electron microscopy. We provide a brief description to facilitate further studies on this poorly understood taxon, and show that light microscopy is sufficient for identification, an important feature for ecological and biogeographical studies.

Saccamoeba lacustris, sp. nov. (Amoebozoa: Lobosea: Hartmannellidae), a new lobose amoeba, parasitized by the novel chlamydia 'Candidatus Metachlamydia lacustris' (Chlamydiae: Parachlamydiaceae).

An amoeba isolated from an aquatic biotope, identified morphologically as Saccamoeba limax, was found harbouring mutualistic rod-shaped gram-negative bacteria. During their cultivation on agar plates, a coinfection also by lysis-inducing chlamydia-like organisms was found in some subpopulations of that amoeba. .Here we provide a molecular-based identification of both the amoeba host and the two bacterial endosymbionts. Analysis of the 18S rRNA gene revealed that this strain is the sister-group to Glaeseria, for which we proposed the name Saccamoeba lacustris. The rod-shaped endosymbiont was identified as a member of Variovorax paradoxus group (Comamonadaceae, Beta-Proteobacteria). No growth on bacteriological agars was recorded, hence this symbiont might be strictly intracellular. The chlamydia-like parasite was unable to infect Acanthamoeba and other amoebae in coculture, showing high host specificity. Phylogenetic analysis based on the 16S rDNA indicated that it is a new member of the family Parachlamydiaceae (order Chlamydiales), for which we proposed the name 'Candidatus Metachlamydia lacustris'.

Description and phylogenetic relationships of Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. (Amoebozoa, Arcellinida).

Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. were isolated and described from dry epiphytic moss. The morphology and ultrastructure of both species clearly demonstrate that they belong to the genus Spumochlamys (family Microchlamyiidae). They differ from its only described member, Spumochlamys iliensis (as well as from species of Microchlamys), in the relief of the dorsal surface of the test, revealed by scanning electron microscopy, which can represent a good characteristic for species identification. They also differ in the structure of the dorsal part of the test wall (especially S. perforata). Small subunit ribosomal DNA-based molecular phylogenetic analyses show that Spumochlamys is a deeply branching lineage of the Arcellinida, without any close affinities. Actin gene sequence analysis places this genus within the Tubulinea, close to two other arcellinid lineages but without forming a monophyletic group with them. These data together strongly suggest that the lack of resolution in the arcellinid molecular phylogenies is due to serious undersampling of taxa, a limited number of sequence data, and high divergence rates in most of the species.

Balamuthia mandrillaris: staining properties of cysts and trophozoites and the effect of 2,6-dichlorobenzonitrile and calcofluor white on encystment.

Here, we determined the staining properties of Balamuthia mandrillaris cysts, and assessed the effect of 2, 6-dichlorobenzonitrile (DCB), a cellulose synthesis inhibitor, and calcofluor white, a brightening agent, on its encystment. Periodic acid-Schiff reagent stained the inner wall intensely and middle and outer walls weakly suggesting that the cyst wall of B. mandrillaris may contain glycans. Furthermore, cysts, but not trophozoites, fluoresced when stained with calcofluor white. Calcofluor white and DCB, a cellulose synthesis inhibitor, inhibited B. mandrillaris encystment. This is the first report suggesting possible glycan biosynthesis in B. mandrillaris encystment, and this pathwaymay provide a potentially useful drug target and help improve treatment.

Pentagonia zhangduensis nov. spec. (Lobosea, Arcellinida), a new freshwater species from China.

The morphology of a new testate amoeba Pentagonia zhangduensis nov. spec. was investigated using light and scanning electron microscopy. The new species was discovered in the sediments of Lake Zhangdu, Hubei Province, China. The low coefficients of variation and normal size frequency distribution suggest that P. zhangduensis is a size-monomorphic species. P. zhangduensis differs from the one other species in this genus (P. maroccana), by its larger size and its quadrangular cross section with two parallel longitudinal ridges on each of the two flattened sides of the test.

Correct identification of species makes the amoebozoan rRNA tree congruent with morphology for the order Leptomyxida Page 1987; with description of Acramoeba dendroida n. g., n. sp., originally misidentified as 'Gephyramoeba sp.'.

Morphological identification of protists remains an expert task, especially for little known and poorly described species. Culture collections normally accept organisms under the name provided by depositors and are not responsible for identification. Uncritical acceptance of these names by molecular phylogeneticists may result in serious errors of interpretation of phylogenetic trees based on DNA sequences, making them appear more incongruent with morphology than they really are. Several cases of misidentification in a major culture collection have recently been reported. Here we provide evidence for misidentifications of two more gymnamoebae. The first concerns "Gephyramoeba sp." ATCC 50654; it is not Gephyramoeba, a leptomyxid with lobose pseudopods, but a hitherto undescribed branching amoeba with fine, filamentous subpseudopods named here Acramoeba dendroida gen. et sp. nov. We also sequenced 18S rRNA of Page's strain of Rhizamoeba saxonica (CCAP 1570/2) and show that it is the most deeply branching leptomyxid and is not phylogenetically close to 'Rhizamoeba saxonica' ATCC 50742, which was misidentified. Correcting these misidentifications improves the congruence between morphological diversity of Amoebozoa and their rRNA-based phylogenies, both for Leptomyxida and for the Acramoeba part of the tree. On morphological grounds we transfer Gephyramoebidae from Varipodida back to Leptomyxida and remove Flamella from Leptomyxida; sequences are needed to confirm these two revisions.

Amoeba at attention: phylogenetic affinity of Sappinia pedata.

The genus Sappinia, a taxon of free-living amoebae with trophozoites that typically have two closely appressed nuclei, contains two named species, Sappinia pedata, the type species, and S. diploidea. The amoebae of both species are essentially identical according to the literature. The two species are distinguished by S. pedata having a standing amoeba stage, incorrectly interpreted as a cyst, and S. diploidea having sessile, bicellular cysts. Using four isolates of S. pedata collected from around the world, we present detailed light micrographic illustrations of all stages of its life cycle. We confirm that the standing amoeba lacks a cell wall. In two isolates of S. pedata, there are bicellular cysts indistinguishable from those of S. diploidea. Using sequence data from the nuclear small subunit ribosomal RNA gene, we conclude that S. pedata and the published neotype of S. diploidea are congeneric but not conspecific. The genus branches within Thecamoebidae. Sequencing of the actin gene confirms the inclusion of Sappinia in Thecamoebidae. Resolving the taxonomy of Sappinia is gaining importance because it has recently been attributed as an opportunistic human pathogen.

Mechanism of intrusion of a microspordian-like organism into the nucleus of host amoebae (Vannella sp.) isolated from a keratitis patient.

Free-living amoebae (FLA) occur ubiquitously in many aquatic habitats and humid soils as well as in "artificial" water samples. In addition to their role as pathogens, FLA are known to serve as natural hosts and vehicles of transmission for various intracellular organisms. An otherwise healthy 24-year-old female patient presented with keratitis in her inflamed left eye. She was a contact lens wearer and had no history of corneal trauma. No acanthamoebae could be determined by culture methods. A Vannella strain (called VanAun0) isolated from corneal scrapings showed intracellular aggregating organisms. Within 1-2 days, the host amoebae ruptured, and numerous coccoid organisms (called Kaun1) were released. We succeeded in detecting the mechanisms of infection and intrusion of this eukaryotic organism, growing within the nucleus of the FLA, by light and electron microscopy. It could be shown that the spores at the cell membrane of strain KAun1 resemble Microsporidia and were taken up into the Amoeba by phagocytosis after adhesion of the spores and food cup formation (infective phase). The spores were transported into the cytoplasm of the vannellae in food vacuoles. Phase contrast microscopy revealed early stages of the parasites moving through the cytoplasm into the nucleus of the host amoeba. Electron microscopy showed the proliferation of polymorphic stages within the karyoplasm. The life cycle of these microsporidian-like organisms ended up with a sporogenic phase in which a terminal differentiation took place and numerous spores were released by rupture of the host cell wall. With the rupture of the host amoeba's cell membrane, the cycle started again from the beginning, the released infectious spores being ingested by other host amoebae. In particular, the morphology of the organelles made visible by electron microscopy finally allowed us to classify the endocytobionts as a microsporidan-like organism. Infection of Vannella sp. with the microsporidia-like organism strain KAun1 is a suitable model for studying the host-parasite relations of organisms using their hosts as so-called Trojan horses.