Elsevier

Protist

Volume 160, Issue 2, May 2009, Pages 265-283
Protist

ORIGINAL PAPER
Ultrastructure and Molecular Phylogeny of two Heterolobosean Amoebae, Euplaesiobystra hypersalinica gen. et sp. nov. and Tulamoeba peronaphora gen. et sp. nov., Isolated from an Extremely Hypersaline Habitat

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We isolated two amoebae, Tulamoeba peronaphora gen. et sp. nov. and Euplaesiobystra hypersalinica gen. et sp. nov. from the high salinity waters (293–300‰ salinity) of a Korean solar saltern. These new species show features typical of Heterolobosea – a limax form with eruptive pseudopodial formation, flattened/discoidal mitochondrial cristae, cysts with plugged pores, and no discrete, stacked dictyosomes. 18S rRNA gene phylogenies place both species within the Heterolobosea. Tulamoeba peronaphora appears to lack a flagellate phase, and has one cyst pore that penetrates the cyst wall. In 18S rRNA gene trees, Tulamoeba peronaphora is specifically related to Pleurostomum flabellatum, an extreme halophile that is observed only as a flagellate. Its next closest relatives are Naegleria and Willaertia. Euplaesiobystra hypersalinica has 2–4 cyst pores in the ectocyst wall (only), and has a bi-flagellated flagellate phase with no obvious cytostome. Its closest described relative is Heteramoeba clara, which is marine, has a cytostome, lacks cyst pores, and has a different nucleolus organization. The Euplaesiobystra hypersalinica 18S rRNA gene is 99.5% identical to a sequence accessed under the nomen nudum ‘Plaesiobystra hypersalinica’ – we consider them the same species. Tulamoeba peronaphora grows at 75–250‰ salinity, while E. hypersalinica grows at 100–300‰ (at least) salinity. Both amoebae seem to be ‘extreme halophiles’, and their ancestors invaded high salinity environments independently of each other. These results provide more evidence that there is a substantial ecological and phylogenetic diversity of heterotrophic eukaryotes capable of growing in very high salinity environments, and these ecosystems may be more complex than usually assumed.

Introduction

Extremely hypersaline habitats (>300‰ salinity) are widely, but sparsely distributed across Earth (Javor 1989). Microorganisms known to inhabit these environments are mainly prokaryotes (e.g. Haloarchaea and Salinibacter, Antón et al. 2000; Guixa-Boixareu et al. 1996) and the autotrophic eukaryote Dunaliella (Javor 1989). Heterotrophic eukaryotes are often regarded as absent from high salinity waters (Guixa-Boixareu et al. 1996; Pedrós-Alió et al. 2000), implying that these ecosystems may lack typical predators. This view, however, sits uncomfortably with several anecdotal/faunistic accounts of various heterotrophic protists living in saturated or near-saturated brines (Hauer and Rogerson 2005a; Kirby 1932; Namyslowski 1913; Patterson and Simpson 1996; Post et al. 1983; Ruinen, 1938a, Ruinen, 1938b; Volcani 1945). Recently Park et al. (2003) demonstrated that heterotrophic eukaryotes, specifically small flagellates, were causing significant prokaryote mortality in a Korean saltern with a salinity of >300‰. Two extremely halophilic, bacterivorous heterotrophic flagellates – Halocafeteria seosinensis and Pleurostomum flabellatum – were subsequently isolated from this environment (Park et al., 2006a, Park et al., 2007). On the basis of ultrastructural characteristics and molecular phylogeny, Halocafeteria seosinensis and Pleurostomum flabellatum were identified as belonging to Bicosoecida and Heterolobosea, respectively (Park et al., 2006a, Park et al., 2007). Both species actively grow in media of >300‰ salinity, and Pleurostomum flabellatum actually seems to grow optimally at this salinity level (Park et al. 2007).

It seems unlikely, however, that flagellates are the only ecologically relevant heterotrophic eukaryotes in extremely hypersaline habitats. Prokaryotic mortality caused by heterotrophic flagellates was estimated to be 25–85% in >300‰ salinity waters (Park et al. 2003). Considering that viral lysis was estimated to be responsible for <5% of total prokaryotic losses in high salinity water (Guixa-Boixareu et al. 1996; Pedrós-Alió et al. 2000), other factors, perhaps other predatory or parasitic organisms, could be contributing significantly to prokaryotic mortality. In other aquatic environments, ciliates and amoebae consume prokaryotes. Recently, Cho et al. (2008) reported the isolation and culture of an extremely halophilic ciliate that ingests both prokaryotes and Dunaliella sp. and through morphological and molecular phylogenetic studies identified this organism as a new species, Trimyema koreanum, in the class Plagiopylea (Cho et al. 2008). However, we are unaware of any comparable polyphasic studies of amoebae that have been cultured from extremely hypersaline habitats.

There are several reports of bacterivorous amoebae of various kinds in moderately hypersaline or very hypersaline environments (e.g. Hamburger 1905; Hauer and Rogerson 2005a; Hauer et al. 2001; Post et al. 1983; Rogerson and Hauer 2002; Ruinen and Baas Becking 1938; Volcani 1943), collectively representing dozens of distinguishable species (Hauer and Rogerson 2005b; Rogerson and Hauer 2002). Ruinen and Baas Becking (1938) summarized previous studies and recorded new observations of amoebae from saline habitats. Included along with polypodial lobose amoebae and forms with filose pseudopodia are accounts of two nominal species of limax amoebae with eruptive pseudopodia that likely represent heteroloboseans. Volcani (1943) reported that an amoeba isolated from the Dead Sea at >300‰ salinity grew optimally at 150–180‰ salinity and tolerated >330‰ salinity. Volcani's amoeba could transform into a flagellate with two flagella, and may therefore also have been a heterolobosean. Post et al. (1983) observed amoebae that they identified as members of the heterolobosean genera Heteramoeba and Naegleria, plus three other unclassified species, in samples with salinities of 170–200‰, with the ‘Naegleria sp.’ observed in cultures up to saturation. More recently, Rogerson and Hauer (2002) isolated several amoebae identified by light microscopy as Heterolobosea (e.g. Vahlkampfia) from samples of ∼160‰ salinity from a pond at the margin of the hypersaline Salton Sea, California. Furthermore, an 18S rRNA gene sequence that clearly branches within the Heterolobosea has been accessed to Genbank under the name ‘Plaesiobystra hypersalinica’ and appears under this name in several published phylogenies (Nikolaev et al. 2004; Park et al. 2007). There are no published morphological data, nor a formal description of this genus or species, but the organism is an amoeba that was isolated from ∼140‰ salinity water (T.A. Nerad, pers. comm.). Thus, there is good evidence that a variety of amoebae exist in hypersaline environments (sensu lato), and that many of them might be members of Heterolobosea.

Here, we investigate the light microscopical and ultrastructural characteristics, and 18S rRNA gene sequences, of two amoebae that have been isolated from extremely high salinity waters (293–300‰ salinity), and maintained as monoprotistan cultures. Both are bacterivorous, produce cysts with plugged pores and can grow in very hypersaline media. Our study demonstrates that both amoebae belong to the Heterolobosea, but are not closely related to each other. One might never been observed before in detail, and is described as Tulamoeba peronaphora gen. et sp. nov. Its closest known relative is the extremely halophilic flagellate Pleurostomum flabellatum. The other amoeba (which also has a flagellate phase) is 99.5% identical to ‘Plaesiobystra hypersalinica’ in 18S rRNA gene sequence. Given the nomenclatural unavailability of the name ‘Plaesiobystra’ this second species is described as Euplaesiobystra hypersalinica gen. et sp. nov.

Section snippets

Light Microscopy

The length and width of the actively moving trophozoites (amoebae) were 6–17 μm (mean±SD of 8.8±2.3 μm, n=30) and 2–6 μm (mean±SD of 4.1±0.9 μm, n=30), respectively. The average ratio of length to width of the trophozoites was 2.2 (range 1.4–3.4). The trophozoites generally had a monopodial limax appearance (Fig. 1A–D). Pseudopodial progression was markedly eruptive. Some trophozoites occasionally showed a long hyaloplasm (up to half of total cell length) when feeding on prokaryotes (Fig. 1E). Fine

Identity and Taxonomy

Tulamoeba peronaphora and Euplaesibystra hypersalinica clearly share numerous characteristics with previously examined heterolobosean amoebae (Page 1988; Page and Blanton 1985; Patterson et al. 2000). Both T. peronaphora and E. hypersalinica are limax amoebae with eruptive pseudopodial formation. The average ratio of length to width is <3. Their mitochondria have flattened/discoidal cristae encircled, at least partly, by rough endoplasmic reticulum. Discrete stacked dictyosomes were observed

Tulamoeba n. gen.

Heterolobosean limax amoeba without known flagellate phase, but forming cysts. Cyst with a simple cyst wall (no ectocyst) and with a single plugged pore. A single nucleus with a central nucleolus. Restricted to hypersaline habitats. Type species Tulamoeba peronaphora n. sp.

Tulamoeba peronaphora n. sp.

Trophozoites: 6–17 μm (average: 8.8 μm) and 2–6 μm (average: 4.1 μm) in length and width, respectively, during locomotion; average L/B ratio=2.2; sometimes with fine uroidal filaments; a single nucleus with a single nucleolus.

Methods

Isolation and cultivation: Tulamoeba peronaphora (isolate A1) and Euplaesiobystra hypersalinica (isolate A2) were isolated from 300‰ and 293‰ salinity waters collected in May 2002 and June 2001 respectively, from a solar saltern located at Seosin on the west coast of Korea (37° 09′ 36″N, 126° 40′ 44″E). The saltern commenced operation around 1950 and was composed of 72 salt ponds, with a total area of about 50,000 m2 when full (Park et al. 2003). The average water depth of the ponds was about 10 

Acknowledgements

The authors thank A. Heiss (Dalhousie University) for assistance with scanning electron microscopy, and H. Tarrant (University of Newcastle, Australia) for advice on ancient Greek. The present study was supported by NSERC grant 298366-04 to AGBS. Computational resources were funded by the ‘‘Prokaryotic Genome Evolution and Diversity’’ Genome Atlantic/Genome Canada large-scale project. JSP is supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, No.

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