Characterization of Pharyngomonas kirbyi (= "Macropharyngomonas halophila" nomen nudum), a very deep-branching, obligately halophilic heterolobosean flagellate

The tetraflagellate Pharyngomonas is among the most commonly reported morphotypes of halophilic protozoa. We have established two cultures of Pharyngomonas kirbyi, SD1A and AS12B, from 300‰ and 210‰ salinity waters from the USA and Australia, respectively. 18S rRNA gene phylogenies confirm that Pharyngomonas is the same entity as 'Macropharyngomonas' (nomen nudum), and represents the deepest branch in the heterolobosean lineage. Pharyngomonas kirbyi (Strain SD1A) has flattened/discoidal cristae, and lacks conspicuous Golgi dictyosomes. It also has a heterolobosean 'double bikont' flagellar apparatus, with two right roots, each associated with an 'I' fibre and part of a rhizoplast-like complex. One right root splits shortly after its origin, and supplies most of the microtubules that support both the ventral groove, and the sub-anterior cytopharynx. Interestingly, Pharyngomonas has some potentially ancestral features not found in typical Heterolobosea, including elongated left roots associated with multilayered 'C' fibres, orthogonal basal bodies, and a spur structure that might represent a 'B' fibre homolog. Both isolates are obligate halophiles that grow best at 100-200‰ salinity and do not grow below 75‰ salinity. Pharyngomonas is therefore of considerable evolutionary importance, both as a deep-branching, plesiomorphic heterolobosean, and a borderline extreme halophile.     

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.     

A New Halophilic Heterolobosean Flagellate,Aurem hypersalina gen. n. et sp. n., Closely Related to the Pleurostomum‐Tulamoeba Clade: Implications for Adaptive Radiation of Halophilic Eukaryotes

Halophilic protozoa are independently scattered across the molecular phylogeny of eukaryotes; most of which are assigned to Heterolobosea. Here, we isolated a biflagellate from a hypersaline water of 342‰ salinity. This isolate shared several morphological features with typical halophilic heterolobosean flagellates. In addition, molecular phylogenetic trees of the 18S rRNA gene sequences clearly indicated flagellate is a heterolobosean species closely related to the halophilic Tulamoebidae. However, the flagellate was not accommodated to any described genus. Cells were ovoid‐shaped, and no amoebae were observed. The two unequal flagella beat heterodynamically. An ear‐like bulge at the margin of a cytostomal groove was observed. Flagellates could grow at 100–200‰ salinity, suggesting an obligately halophilic species. Currently, it appears that the new halophilic Aurem hypersalina forms a strong clade with Tulamoebidae, and is sister to the Tulamoebidae, indicating that this new clade is composed almost entirely of obligate halophilic taxa. Thus, A. hypersalina and the Tulamoebidae clade currently represent a unique adaptive radiation of halophilic eukaryotes.     

Biological characterization of a clinical and an environmental isolate of <Emphasis Type="Italic">Acanthamoeba polyphaga</Emphasis>: analysis of relevant parameters to decode pathogenicity

Acanthamoeba spp. consists of free-living amoebae, widespread in nature, which occasionally can cause human infections including granulomatous amoebic encephalitis and amoebic keratitis. Acanthamoeba pathogenesis is not entirely known and correlations between pathogenic potential and taxonomy are complex issues. In order to decipher the definition of a pathogenic amoeba, the objective of this work was to decipher the definition of pathogenic amoeba by characterizing two isolates of Acanthamoeba polyphaga obtained from different origins (a keratitis patient and freshwater), looking for differences among them. The clinical isolate grew faster in Peptone-yeast extract-glucose (PYG) medium, transformed more rapidly from a trophozoite to cyst and exhibited increased cytopathic effect on cultured cells. Morphological differences were also noted, since freshwater amoebae presented more acanthopodia than the clinical isolate. Moreover, actin labeling demonstrated that microfilament organization varies between isolates, with the presence of locomotory structures as lobopodia and lamellipodia in the keratitis isolate, which were less adherent on plastic. Zymography demonstrated that the keratitis isolates presented higher proteolytic activity and also were more able to invade collagen matrices. Altogether, we conclude that a group of stable physiological characteristics exist in Acanthamoeba that can be related to pathogenicity.     

Morphology and Phylogenetic Analyses of Three Novel Naegleria Isolated from Freshwaters on Jeju Island,Korea, During the Winter Period

The genus Naegleria is one of the best known heterolobosean groups, and is the causative agent of primary amoebic meningoencephalitis. This group is rarely studied in temperate regions during winter. Here, three novel Naegleria were isolated from freshwaters on Jeju Island, Korea, during winter. Two isolates were amoeboflagellates, and one of the three amoebae did not undergo enflagellation. All amoebae had eruptive pseudopodia, and the layer of refractile granules around a large nucleus. They formed a cyst with ~2 pores in the cyst stage. The amoeboflagellate form had two flagella and no division in the flagellate stage, and no cytostome. These features are very similar to typical Naegleria. Furthermore, our isolates were able to grow at > 30 °C, suggesting that they had different thermophilicity from Naegleria in polar regions. All amoebae were largely encysted at 5 or 10 °C, indicating that they were likely encysted during winter. Based on the 18S rRNA gene and the ITS1‐5.8S rRNA gene‐ITS2 sequences, the phylogenetic analyses consistently revealed that the isolates are members of the Naegleria group. However, the isolates differ from other species in both phylogenetic trees. Thus, Naegleria in cold habitats appeared to have a high degree of novelty, but their thermophilicity may be dependent on locality.     

ISOLATION AND CHARACTERIZATION OF CYANOBACTERIA POSSESSING ANTIMICROBIAL ACTIVITY FROM THE SUNDARBANS,THE WORLD’S LARGEST TIDAL MANGROVE FOREST1

Eight obligately halophilic, euryhaline cyanobacteria from intertidal soil were isolated in artificial seawater nutrients III (ASN‐III) medium. Antimicrobial activity, 16S rRNA gene sequences, phenotypic characters as well as growth and antibiosis in response to variable salinity, temperature, phosphate concentration, and pH were studied. Minimum inhibitory concentrations (MIC) of the extracts against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and multiple drug‐resistant clinical isolates ranged between 0.25 and 0.5 mg · mL⁻¹. Cytotoxicity tests showed 73%–84% human colon adenocarcinoma (HT‐29/C1) cell survival at MIC values, indicating that the extracts were nontoxic. Morphologically, six cyanobacteria were assigned to the Lyngbya‐Phormidium‐Plectonema (LPP) group B, and one each was assigned to Oscillatoria and Synechocystis genera. Glycerol, mannitol, and starch supported better photoheterotrophic growth than simpler mono‐ and disaccharides. No heterocyst formation was observed when grown under nitrogen‐starved conditions. All isolates survived 7‰ salinity, grew at minimum 32‰ salinity, and showed sustained growth throughout 32‰–82‰ salinity but matured poorly in freshwater medium supplemented with 30.0 g · L⁻¹ NaCl. Antimicrobial production occurred only at 32‰ salinity. While four of the eight isolates demonstrated sustained growth at 37°C, maximum antimicrobial activity was obtained at 25°C. All strains showed maximum growth and antimicrobial elaboration at 0.04 g · L⁻¹ phosphate. All isolates thrived at pH 9.5; six grew at pH 4.5, though antimicrobial production occurred only at pH 7.5. Molecular phylogenetic analysis based on 16S rRNA gene sequences of the filamentous isolates validated the previous taxonomic affiliations established on morphological characteristics. This is the first study of antimicrobial‐producing halophilic cyanobacteria from the mangroves.     

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.     

Testate Amoeba (Arcellinida,Euglyphida) Ecology along a Poor‐Rich Gradient in Fens of Western Poland

We investigated the relationships between testate amoebae (Arcellinida, Euglyphida), vegetation and water chemistry along environmental gradients in minerotrophic peatlands (fens) in western Poland. We hypothesized that: a) hydrochemistry significantly influences structure of testate amoeba communities, and b) testate amoeba communities are more closely correlated with the hydrochemical variables (environment) than with the vegetation data. Testate amoeba communities and vegetation from 71 sample plots were investigated together with the hydro‐chemistry and hydrology based on 16 environmental variables and vegetation composition. Testate amoeba communities revealed a distinctive poor‐rich gradient in analysed fens. Mineral‐rich habitats, which were dominated by brown mosses, were preferred by a higher number of taxa than acidic habitats, which were dominated by Sphagnum. We recorded a total of 107 testate amoebae taxa. The average species richness of testate amoebae for brown mosses was higher (20) than for Sphagnum (13). We found that testate amoebae communities were similarly correlated with vascular plants, mosses and environmental parameters. Results of direct ordination demonstrate that hydrology, pH, Mg²⁺ and sodium remain the most important environmental control for the entire data set. CCA showed that in case of brown mosses hydrology, sodium and oxygen affect testate amoeba communities significantly whereas in Sphagnum only sodium emerge as most significant determining testate amoeba assemblages. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Marinamoeba thermophila, a new marine heterolobosean amoeba growing at 50 °C

Two amoeba strains were isolated from marine sediment taken at the same place with 18 months interval from a region of the sea floor heated by extended submarine hot springs and fumaroles. These thermophilic amoebae grow at temperatures up to 50 °C. Sequences of the internal transcribed spacer demonstrated that the two strains belong to the same species and are different from any genus for which sequences are known. Phylogeny using small subunit ribosomal RNA places the amoeba in the Heterolobosea. Their closest relatives are the hypersaline flagellate Pleurostomum flabellatum and the hypersaline amoeba Tulamoeba peronaphora. The freshwater amoeboflagellate genera Naegleria and Willaertia belong to the same phylogenetic clade in the Vahlkampfiidae. The new marine species does not transform into flagellates. It forms cysts, which are round to ellipsoidal with few pores. Because of their unique place in the molecular phylogenetic tree, and because there is no morphologically identical species found in the literature, these isolates are considered to be a new species and a new genus, Marinamoeba thermophila.     

Characterization of halotolerant Bicosoecida and Placididea (Stramenopila) that are distinct from marine forms,and the phylogenetic pattern of salinity preference in heterotrophic stramenopiles

Recent culture‐based studies demonstrate the distinctiveness of the microbial eukaryote biota of very hypersaline environments. In contrast, microscopy‐based faunistic studies suggest that the biota of habitats of more moderate hypersalinity (60–150‰) overlaps substantially with that of marine environments, but this has barely been studied with modern techniques. To investigate the diversity and salinity tolerance range of these organisms, eight cultures of heterotrophic stramenopiles were established from (or from nearby) moderately hypersaline locations. These isolates represent five independent groups; Groups A, B and C are bicosoecids; Groups D and E belong to Placididea. One isolate (Group A) is a strain of the widespread marine species Cafeteria roenbergensis, and cannot grow above 100‰ salinity. The other isolates – Groups B–E – can all grow at 150–175‰ salinities and are probably moderate halophiles. Groups B–E all represent previously unsequenced species or even genera, although Group B is the sister group of the borderline extreme halophile Halocafeteria. The high level of novelty en countered suggests that moderately hypersaline environments may harbour a heterotrophic stramenopile biota distinct from that of marine environments. Interestingly, our new isolates are all most closely related to marine or halophilic forms, and our phylogenies show large clades defined by saline/non‐saline habitats within bicosoecids, placidomonads and related lineages. In particular, most freshwater/soil bicosoecids form one well‐supported clade. The sole major exception is Bicosoeca, which is intermixed with marine environmental sequences originally referred to as ‘MAST‐13’, which are from brackish water, not typical seawater. It seems that the freshwater/marine barrier has been crossed very few times in the evolutionary history of these heterotrophic stramenopile flagellates.     

Comparison of polyamine and S-adenosylmethionine contents of growing and encysted Acanthamoeba isolates

We have used High Performance Liquid Chromatography to determine metabolite characteristics of three recent isolates of Acanthamoeba which exhibit cultural characteristics consistent with those of established potential pathogens. Growing amoebae and dormant cysts of these isolates were explored in regard to their qualitative and quantitative intracellular levels of polyamine and S-adenosylmethionine metabolites. The polyamine found in the greatest concentration in the growing cells was 1,3-diaminopropane (DAP), followed by spermidine (SPD). A low level of putrescine was also found in the growing cells. These polyamines significantly decreased in concentration as the amoebae differentiated to cysts. N⁸-acetylspermidine and acetylspermine were found in both developmental stages while acetylcadaverine was found only in growing amoebae and N¹-acetylspermidine only in cysts. Acetylputrescine was present in both stages of two isolates but only in the growing amoebae of the third isolate. Spermine was not detected in any of the isolates.S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were present in growing amoebae but SAM was undetectable or barely detectable in cysts. SAH also decreased in concentration during encystation of two of the isolates to a level comparable to that of the other isolate.The developmental transition from growing amoebae to dormant cysts is characterized metabolically by a threshold adjustment in concentration of SAM, SAH and of the polyamines (esp., DAP and SPD).     

Remarkable Salinity Tolerance of Seven Species of Naked Amoebae (gymnamoebae)

The salinity tolerance of naked amoebae collected from sites ranging from ca. 0‰ to 160‰ were compared in laboratory experiments. Amoebae were collected from hypersaline ponds around the perimeter of the Salton Sea, California, where salinities averaged 160‰, and directly from the shoreline waters of the Sea where salinities were generally between 44 and 48‰. Naked amoebae were also collected from the intertidal zone of a Florida beach, a habitat subject (on occasion) to salinity fluctuations within the range 6–85‰. From these combined sites, 6 clones of amoebae were isolated for salinity tolerance experiments (2 marine beach isolates, 2 Salton Sea isolates, and 2 hypersaline pond isolates). A seventh clone, Acanthamoeba polyphaga, a common freshwater/soil amoeba, was obtained from a Culture Collection. Laboratory experiments compared the effects of gradually changing culture salinity versus no salinity acclimatization. Growth rate and culture yield were used as indices of effect. Generally, amoebae were tolerant over a wide range of salinity conditions (in terms of growth and yield) and were not markedly influenced by pre-conditioning to salinity changes throughout the experiments. Overall, the freshwater amoeba Acanthamoeba grew between 0 and 12‰, the marine clones grew in the range of 2–120‰, and the Salton Sea clones reproduced between 0 and 138 ‰. The hypersaline clones were the most resilient and grew between 0 and 270‰ salt. The survival and activity of large populations of naked amoebae in sites subject to salinity fluctuations suggest that they should be considered in future studies to better understand their, as yet, undefined ecological role.     

CYST–THECA RELATIONSHIP,LIFE CYCLE,AND EFFECTS OF TEMPERATURE AND SALINITY ON THE CYST MORPHOLOGY OF GONYAULAX BALTICA SP. NOV. (DINOPHYCEAE) FROM THE BALTIC SEA AREA1

A new species of Gonyaulax, here named Gonyaulax baltica sp. nov., has been isolated from sediment samples from the southeastern Baltic. Culture strains were established from individually isolated cysts, and cyst formation was induced in a nitrogen‐depleted medium. Although G. baltica cysts are similar to some forms attributed to Spiniferites bulloideus and the motile stage of G. baltica has affinities with G. spinifera, the combination of features of cyst and motile stage of G. baltica is unique. The culture strains were able to grow at salinity levels from 5 to 55 psu and formed cysts from 10 to 50 psu. Cultures at each salinity level were grown at 12, 16, and 20° C. Temperature‐ and salinity‐controlled morphological variability was found in the resting cysts. Central body size varied with temperature and salinity, and process length varied with salinity. Cysts that formed at extreme salinity levels displayed lower average process length than cysts formed at intermediate salinity levels, and central body length and width were lowest at higher temperature and lower salinity. Models for the relationship between central body size and temperature/salinity and process length and salinity have been developed and may be used to determine relative paleosalinity and paleotemperature levels. Our results on salinity‐dependent process length confirm earlier reports on short‐spined cysts of this species found in low salinity environments, and the model makes it possible to attempt to quantify past salinity levels.     

The Pathogenic Amoeboflagellate Naegleria fowleri: Environmental Isolations,Competitors, Ecologic Interactions,and the Flagellate-Empty Habitat Hypothesis1

From several surveys of environmental sites, the virulent human pathogen, Naegleria fowleri, was isolated from a pond in Georgia, a sewage treatment plant in Missouri, and from the Potomac and Anacostia rivers near and in Washington, D.C. Widely scattered, sparse populations seemed only a potential threat to human health at the time of sampling. The data support an estimate that the sites sampled contain 10,000 typical, low temperature, bactivorous amoebae for each heat tolerant amoeba able to grow at 45° C. Heat tolerant competitors were much more common than N. fowleri. Naegleria lovaniensis, which is heat tolerant but nonpathogenic, was isolated from and downstream from an open air thermal pollution temperature gradient. Hot piles of composting sewage sludge yielded no amoeboflagellates, many heat tolerant (45–49° C) amoebae, and one thermophilic (52° C) Acanthamoeba. Features of the methods used include two-stage incubation to increase isolation of sparse organisms and distinction of N. fowleri from almost all other amoebae on agar plates. The flagellate-empty habitat hypothesis postulates a general model in which human intervention and/ or natural events remove usual competitors and the ability to transform to a motile flagellate confers an advantage in recolonizing.     

The Effect of Different Environmental Conditions on the Viability of Naegleria fowleri Amoebae

Naegleria fowleri, a free‐living amoeba found in soil and freshwater environments, is the causative agent of Primary Amoebic Meningoencephalitis. Infection occurs when amoebae enter the nasal cavity, attach to the nasal mucosa and travel along olfactory neurons towards the olfactory bulb. Upon reaching the central nervous system, the amoebae replicate very rapidly and can cause death in 3–10 days. Little is known about the conditions in which the amoeba can survive in the environment. We have tested conditions beyond the known boundaries on the viability of amoebae by introducing them into moderate and extreme salinity, pH, and temperatures. Our data shows that although viability expectedly decreases towards each of these extreme conditions, their tolerance was much greater than anticipated, including viability in moderate salinity, a wide pH range, and temperatures higher than the previously reported 45 °C.     

Starvation and Encystment of a Soil Amoeba Hartmannella castellanii*

SYNOPSIS. The behavior of the amoeba H. castellanii was investigated in various carbon and nitrogen deficient media with a view to developing a satisfactory replacement medium for the study of encystment and excystment. Media which had been devised for other soil amoebae did not cause H. castellanii to encyst. In these media there was an efflux of material from the cells which was independent of osmolarity but which was minimized by the addition of magnesium. Maximal encystment occurred in a medium containing magnesium chloride alone. The cysts produced in the magnesium chloride replacement medium are viable and readily excyst when resuspended in the growth medium. The cysts contain cellulose, which is not present in the vegetative amoebae, and differ from the amoebae in their greater resistance to induced lysis and mechanical injury.     

Morphological Study of the Encystment and Excystment of Vermamoeba vermiformis Revealed Original Traits

Free‐living amoebae are ubiquitous protozoa commonly found in water. Among them, Acanthamoeba and Vermamoeba (formerly Hartmannella) are the most represented genera. In case of stress, such as nutrient deprivation or osmotic stress, these amoebae initiate a differentiation process, named encystment. It leads to the cyst form, which is a resistant form enabling amoebae to survive in harsh conditions and resist disinfection treatments. Encystment has been thoroughly described in Acanthamoeba but poorly in Vermamoeba. Our study was aimed to follow the encystment/excystment processes by microscopic observations. We show that encystment is quite rapid, as mature cysts were obtained in 9 h, and that cyst wall is composed of two layers. A video shows that a locomotive form is likely involved in clustering cysts together during encystment. As for Acanthamoeba, autophagy is likely active during this process. Specific vesicles, possibly involved in ribophagy, were observed within the cytoplasm. Remarkably, mitochondria rearranged around the nucleus within the cyst, suggesting high needs in energy. Unlike Acanthamoeba and Naegleria, no ostioles were observed in the cyst wall suggesting that excystment is original. During excystment, large vesicles, likely filled with hydrolases, were found in close proximity to cyst wall and digest it. Trophozoite moves inside its cyst wall before exiting during excystment. In conclusion, Vermamoeba encystment/excystment displays original trends as compare to Acanthamoeba.     

Serologic Changes Associated with the Encystment of Axenically Grown Hartmannella culbertsoni*

SYNOPSIS. Serologic reactions elicited by sonically ruptured trophic and cystic forms of Hartmannella culbertsoni were studied. The antigens of trophic amoebae reacted with their homologous rabbit antiserum showing multiple precipitin lines which could not be seen when the reacting antigens were treated with trypsin prior to application on the Ouchterlony plates. Antigens of trophic amoeba did not react with antiserum against cysts. Cyst antigens reacted with their homologous antiserum only after trypsin treatment. Antigens prepared from trophozoites excysting from cysts reacted positively with the antiserum against antigens of trophic amoebae. Antigens of trophic as well as cystic forms fixed guinea pig complement in presence of their homologous antisera. With the trophic form, this property was abolished after trypsin treatment. Non-specific complement fixation mediated by cyst antigens was abolished by treatment with cellulase. Antiserum against trophic amoebae immobilized trophozoites and, in the presence of guinea pig complement, led to their lysis.     

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.