A Light and Electron Microscopical Study of a New,Polymorphic Free-Living Amoeba,Phreatamoeba balamuthi n. g., n. sp.1

ABSTRACT. A new amoeba, isolated from well water in Gambia, West Africa, is described and named Phreatamoeba balamuthi n. g., n. sp. Requiring anaerobic conditions for growth, it is easily cultured monoxenically with Escherichia coli or axenically in complex, undefined organic media. Three phenotypes have been observed in the life cycle: an amoeba, a flagellate, and a cyst. The amoeba moves by monopodia, is predominantly multinucleate, and varies from 11 to 160 μm in length. The flagellate has a single flagellum and is from 6 to 50 μm long. The cyst is surrounded by a resistant wall that lacks pores and ranges from 9 to 18 μm in diameter. The transformation from amoeba to flagellate can be induced nutritionally, the exact inducing factor(s) being unknown. Sexual reproduction has not been observed.     

Entamoeba bubalus, n.sp., from Carabao

SUMMARY. Twelve carabao in the Philippines were found to be lightly infected with intestinal amoebae. Trophic forms (12 microns in diameter) of this protozoan possessed a definite ectoplasm and a homogeneous endoplasm. They were found only in stained preparations. The nucleus was similar to that of the cyst. All unconcentrated fecal smears contained at least a few cysts (8 microns in diameter). In these forms the cytoplasm usually contained a large vacuole and one or more irregular chromatoidal bodies. The nucleus (2.6 microns in diameter) possessed a pronounced, deeply staining, uniform peripheral ring and a large irregular endosome. There was no periendosomal ring. This amoeba is designated as Entamoeba bubalus , n.sp.     

Characterization of a Deep‐Branching Heterolobosean,Pharyngomonas turkanaensis n. sp., Isolated from a Non‐Hypersaline Habitat,and Ultrastructural Comparison of Cysts and Amoebae Among Pharyngomonas Strains

An unusual heterolobosean amoeba, isolate LO, was isolated recently from a sample with a salinity of ~4‰, from Lake Turkana in East Africa. 18S rDNA phylogenies confirm that isolate LO branches among halophilic amoeboflagellates assigned to Pharyngomonas. We examined the ultrastructure of the amoeba and cyst stages of isolate LO, as well as the amoebae and cysts of Pharyngomonas kirbyi (isolates AS12B and SD1A). The amoebae of all three isolates lacked discrete dictyosomes and had discoidal/flattened mitochondrial cristae, but the mitochondria were not enrobed by rough endoplasmic reticulum. The cysts of all three isolates showed a thick, bipartite cyst wall, and lacked cyst pores. The cysts of isolate LO were distinct in that the ectocyst was very loose‐fitting, and could contain “crypts”. No flagellate form of isolate LO has been observed to date, and a salinity‐for‐growth experiment showed that isolate LO can grow at 15–100‰ salinity, indicating that it is halotolerant. By contrast, other studied Pharyngomonas isolates are amoeboflagellates and true halophiles. Therefore, we propose isolate LO as a new species, Pharyngomonas turkanaensis n. sp. It is possible that P. turkanaensis descended from halophilic ancestors, and represents a secondary reestablishment of a physiology adapted for moderate salinity.     

Platyamoeba stenopodia. n. g., n. sp., a Freshwater Amoeba*

SYNOPSIS. Platyamoeba stenopodia n. g., n. sp., isolated from a lake in Alabama, is an amoeba with flattened form, usually more than 2.5 × as long as broad, the anterior half hyaline and the posterior half granular, with a single vesiculate nucleus at the posterior edge of the hyaline region. It forms a transitory floating stage with slender, hyaline pseudopods. Its cyst is spherical, uninucleate, with a smooth inner wall and a thinner, closely applied outer wall, which is often slightly wrinkled. Length of locomotive amoeba 15–36 μ diameter of cyst 7–11.5 μ Reproduction by binary fission with mesomitotic nuclear division, the nuclear membrane disappearing in prophase. The genus Platyamoeba is erected for amoebae including this organism; the type species is P. placida.     

Characterization of Tulamoeba bucina n. sp., an Extremely Halotolerant Amoeboflagellate Heterolobosean Belonging to the Tulamoeba–Pleurostomum Clade (Tulamoebidae n. fam.)

Most protozoans that have been cultivated recently from high salinity waters appear to be obligate halophiles. Phylogenetic analyses indicate that these species mostly represent independent lineages. Here, we report the cultivation, morphological characterization, and phylogenetic analysis of two strains (XLG1 and HLM‐8) of a new extremely halotolerant heterolobosean amoeboflagellate. This species is closely related to the obligate halophiles Tulamoeba peronaphora and Pleurostomum flabellatum, and more specifically to the former. Like Tulamoeba, the new species has a monopodial limax amoeba stage, however, its cyst stage lacks an intrusive pore plug. The flagellate stage bears a combination of a planar spiral feeding apparatus and unequal heterodynamic flagella that discriminates it from described Pleurostomum species. Strain XLG1 grows at salinities from 35‰ to 225‰. This degree of halotolerance is uncommon in protozoa, as most species showing growth in seawater are unable to grow at 200‰ salinity. The unrelatedness of most halophilic protozoa suggested that independent colonization of the hypersaline environment is more common than speciation within it. However, this study supports the idea that the Tulamoeba–Pleurostomum clade underwent an adaptive radiation within the hypersaline environment. A new species Tulamoeba bucina n. sp. is described, with Tulamoebidae n. fam. proposed for the Tulamoeba–Pleurostomum clade.     

A New Heterolobosean Amoeboflagellate,Tetramitus dokdoensis n. sp., Isolated from a Freshwater Pond on Dokdo Island in the East Sea,Korea

The genus Tetramitus is a representative amoeboflagellate group within the Heterolobosea, and currently contains over a dozen species. Here, a new heterolobosean amoeboflagellate was isolated from a freshwater pond on Dokdo Island, Korea. The amoebae have eruptive pseudopodia, no uroidal filament, and a nucleus with a central nucleolus. The length and width of the amoebae are 15.5–28.0 μm and 5.4–12.6 μm, respectively. The flagellates are conical, with 4 flagella of equal length (~10 μm). There is a discrete rostrum in the subapical region of the flagellate form. The cyst has thin endo‐ and ectocyst layers and no cyst pores. The amoeba shows slow movement at 37 °C, but does not move at 42 °C under a light microscope. Phylogenies of the 18S rRNA gene and the ITS1‐5.8S rRNA gene‐ITS2 sequence show that the strain belongs to a subclade of Tetramitus that includes Tetramitus rostratus, Tetramitus waccamawensis and Tetramitus entericus, amongst others. Nonetheless, the strain is distinct from other species in both molecular phylogenetic trees. Thus the strain isolated from the Dokdo Island is proposed as a novel species, Tetramitus dokdoensis n. sp.     

Scale Formation in an Amoeba, Cochliopodium sp.

During excystment of an amoeba, Cochliopodium sp., scale formation was examined with light and electron microscopy. This amoeba was covered with scales. When the amoeba encysted, the scales remained on the external surface of the cyst wall. Soon after the induction of excystment the Golgi complex began to develop. Many vesicles were extruded from it and changed into vacuoles. Scales were observed first in the vacuole adjacent to the Golgi complex and later in inside the cyst wall. When the amoeba excysted it had been coated by the newly formed scales. It is suggested that the scale formation is dependent on the activity of the Golgi complex.     

Adelphamoeba galeacystis n. g., n. sp., an Amoeboflagellate Isolated from Soil*

SYNOPSIS. An amoeboflagellate isolated from common soil is described. The amoeboid stage is typically limax and contains a well differentiated uroid region. The flagellate has 2 flagella, which emerge anteriorly and are equal in length. It has a ventral cytostome near the anterior border. The cyst is helmet-shaped and without opercula. Polar masses are present during nuclear division.     

Characterization of Selenaion koniopes n. gen., n. sp., an Amoeba that Represents a New Major Lineage within Heterolobosea,Isolated from the Wieliczka Salt Mine

A new heterolobosean amoeba, Selenaion koniopes n. gen., n. sp., was isolated from 73‰ saline water in the Wieliczka salt mine, Poland. The amoeba had eruptive pseudopodia, a prominent uroid, and a nucleus without central nucleolus. Cysts had multiple crater‐like pore plugs. No flagellates were observed. Transmission electron microscopy revealed several typical heterolobosean features: flattened mitochondrial cristae, mitochondria associated with endoplasmic reticulum, and an absence of obvious Golgi dictyosomes. Two types of larger and smaller granules were sometimes abundant in the cytoplasm—these may be involved in cyst formation. Mature cysts had a fibrous endocyst that could be thick, plus an ectocyst that was covered with small granules. Pore plugs had a flattened dome shape, were bipartite, and penetrated only the endocyst. Phylogenies based on the 18S rRNA gene and the presence of 18S rRNA helix 17_1 strongly confirmed assignment to Heterolobosea. The organism was not closely related to any described genus, and instead formed the deepest branch within the Heterolobosea clade after Pharyngomonas, with support for this deep‐branching position being moderate (i.e. maximum likelihood bootstrap support—67%; posterior probability—0.98). Cells grew at 15–150‰ salinity. Thus, S. koniopes is a halotolerant, probably moderately halophilic heterolobosean, with a potentially pivotal evolutionary position within this large eukaryote group.     

Isolation of a vahlkampfiid amoeba from a contact lens: Tetramitus ovis (Schmidt, 1913) n. comb.

A vahlkampfiid amoeba has been isolated from a contact lens. Based on 5.8S rDNA sequence analysis the strain belongs to the genus Tetramitus. The internal transcribed spacer (ITS1 and ITS2) sequences differ by 3 and 9 bp, respectively, from T. lobospinosus. The morphology of the cyst does not correspond to T. lobospinosus, but is identical to that published for Vahlkampfia ovis at the beginning of the last century. There is no reference strain of V. ovis to investigate using molecular techniques. Therefore, we propose that the strain under study should be considered to represent the neotype strain of V. ovis, now classified as T. ovis (Schmidt, 1913) n. comb.     

A new heterolobosean amoeba Solumitrus palustris n. g., n. sp. isolated from freshwater marsh soil

During the course of research on the bacterial feeding behavior and resistance of amoebae to virulent pathogens, we isolated a new strain of amoeba from organic rich soil at the margin of freshwater swamp in the northeastern United States. Light microscopic morphology is characteristically heterolobosean, resembling vahlkampfiids, including a broadened, limax shape, and eruptive locomotion, but occasionally becoming more contracted and less elongated with lateral or anterior bulges and somewhat branching sparse, uroidal filaments. Electron microscopic evidence, including mitochondria with flattened cristae surrounded by rough endoplasmic reticulum, further indicates a heterolobosean affinity. The solitary nucleus contains a centrally located nucleolus. Cysts are rounded with occasionally an eccentrically located nucleus. The cyst walls are relatively thin, becoming crenated, and loosely enclosing the cyst when mature. Molecular genetic evidence places this isolate among the Heterolobosea, branching most closely in a clade including Allovahlkampfia spelaea and previously isolated, un-named strains of soil amoebae. Based on differentiated features, including morphology of the uroid, cyst wall structure, and molecular genetic evidence that distinguish it from A. spelaea, a new genus and species, Solumitrus palustris, is proposed for this new heterolobosean.     

Ultrastructure and Description of a Fungus-Feeding Amoeba,Trichamoeba mycophaga n. sp. (Amoebidae,Amoebea), from Australia1

ABSTRACT. An amoeba isolated from a wheatfield and a forest soil in Australia has been identified as Trichamoeba mycophaga n. sp. Trophozoites of this amoeba are palmate to elongate and measure 45–136 μm in length and 25–94 μm in width. Amoebae in continuous locomotion may be limax with a villous-bulb uroid. Both the lobose pseudopodia and the advancing margin of a limax trophozoite bear an ectoplasmic crescent. The plasma membrane is coated with an electron-dense amorphous layer ca. 100 nm thick. Endoplasm is granular with elongate to bipyramidal crystals and contains bacterial endosymbionts. Trophozoites have a single, spherical to oval nucleus, 4–10 μm in diameter, which contains a centrally located, spherical to oval nucleolus, 2.8–5.0 μm in diameter. The nucleoplasm contains aggregations of filaments distributed radially within the nuclear membrane. Cysts are 21–60 μm in diameter, with ecto- and endocyst walls separated by an amorphous layer.     

Paravahlkampfia francinae n. sp. Masquerading as an Agent of Primary Amoebic Meningoencephalitis

ABSTRACT. Paravahlkampfia francinae n. sp., a new species of the free-living amoeba genus Paravahlkampfia , designated as CDC:V595, was isolated from the cerebrospinal fluid of a patient with headache, sore throat, and vomiting, typical symptoms of primary amoebic meningoencephalitis (PAM) caused by Naegleria fowleri . The isolate grew at 33 °C, 37 °C, 40 °C, and 42 °C and destroyed mammalian cell cultures. However, it did not kill young mice upon intranasal inoculation. P. francinae does not produce flagellates and does not grow on agar plates coated with Gram-negative bacteria such as Escherichia coli , the usual food source of Paravahlkampfia ustiana , the type species of the genus. The trophozoite at light microscopy exhibited eruptive locomotion and possessed a single vesicular nucleus. Ultrastructurally, the trophozoites had numerous mitochondria with discoidal cristae but did not have a Golgi apparatus. The trophozoites differentiated into cysts after consuming most of the monolayer. The cyst had an inner well-differentiated endocyst and an outer thin, wrinkled, and wavy ectocyst with no pores. During excystation trophozoites ruptured the cyst wall and emerged from the cysts. A unique feature seen in the cysts was the presence of bacterial endosymbionts, both in the endoplasm and within the cyst wall. Full-length sequencing analysis of the 18S and 5.8S RNA genes of P. francinae showed that they were distinct from those of other Paravahlkampfia species. The patient recovered within a few days indicating that some of the previously reported cases of PAM that survived may have been due to P. francinae .     

Spumochlamys iliensis n.g. n. sp. (Testacealobosia, Microchlamyiidae) from Central Asia, with notes on the diversity of Microchlamys-like testate amoebae

Spumochlamys iliensis n. g., n. sp. was isolated from a mineral pond in an arid, semi-desert region in Kazakhstan (Central Asia). This amoeba is covered with a plate-shaped, flexible organic spongious test. The thin membranous margin of this test extends ventrally to surround a flexible ventral aperture. In its morphological features and behaviour this amoeba is very similar to Microchlamys patella (Claparède and Lachmann, 1859) Cockerell, 1911, but differs from this species and from M. sylvatica Golemansky, Skarlato and Todorov, 1987 in the lack of an additional membrane separating the cell body from the test, a feature that can only be detected using electron microscopy. The presence of this membrane is considered to be a principal characteristic of the genus Microchlamys and family Microchlamyiidae; however, this conclusion was reached from the study of only two species. The data presented show a higher diversity of Microchlamys-like testate amoebae; we therefore suggest that the described species should be included in the family Microchlamyiidae with emendation of its diagnosis, but that a separate genus within this family should be established to accommodate it.     

Cultivation and Lyophilization of Mastigina sp.

SYNOPSIS Mastigina sp. is an amoeboid flagellate isolated from pine frass collected in the Guadarrama Mountains in Spain. It feeds on bacteria and yeasts. It prefers yeasts that produce extracellular polysaccharides, and the 2 species that have been used predominantly for cultivation of the flagellate are Pachysolen tannophilus and Hansenula holstii. Mastigina sp. is easily isolated in axenic culture and grows abundantly therein. Its locomotive form, averaging 27 μ in length, resembles that of a limax amoeba, with a vesiculate nucleus at the anterior end. Cells are capable of simultaneous movement by pseudopodia and flagella. It develops rapidly on dead or living yeast cells in shaken cultures and the trophozoites may convert quantitatively to cysts. The cysts remain viable for long periods of time in refrigerated suspensions and in the lyophilized state. They are spherical or ovoid and smooth-walled cysts; the trophozoite emerges from them by breaking the wall.     

Monocercomonas molae n. sp., a Flagellate from the Sunfish Mola mola*

SYNOPSIS. Monocercomonas molae from the hindgut of the sunfish Mola mola is described. The host was taken from southern California coastal waters in October, 1964. The body of the flagellate is 8.0 × 10.7 microns. A single basal granule complex gives rise to 4 flagella, one of which is recurrent. The axostyle is relatively stout, with argentophilic granules, and possesses a periaxostylar ring. The capitulum of the axostyle continues into the sickle-shaped pelta, and the parabasal body is rod-shaped or lobed and roughly triangular in cross-section.