A New Thermophilic Heterolobosean Amoeba, Fumarolamoeba ceborucoi, gen. nov., sp. nov., Isolated Near a Fumarole at a Volcano in Mexico

Headnote

Summary. An amoeba was isolated from a soil sample collected at the edge of a fumarole of the volcano Ceboruco in the state of Nayarit, Mexico. The trophozoites of this new isolate have eruptive pseudopodes and do not transform into flagellates. The strain forms cysts that have a double wall. This thermophilic amoeba grows at temperatures up to 500C. Molecular phylogenetic analysis of the small subunit ribosomal DNA (SSU rDNA) places the amoeba into the Heterolobosea. The closest relatives are Paravahlkampfia spp. Like some other heterolobosean species, this new isolate has a group I intron in the SSU rDNA. Because of its position in the molecular phylogenetic tree, and because there is no species found in the literature with similar morphological and physiological characteristics, this isolate is described as a new genus and a new species, Fumarolamoeba ceborucoi gen. nov, sp. nov.

Key words: Group I intron, Heterolobosea, new genus, new species, SSU rDNA, thermophilic.

INTRODUCTION

The Heterolobosea are amoebae, which move with eruptive pseudopodia, have intranuclear promitosis, have mitochondrial cristae which are flattened, often disc-like, and in which stacked Golgi bodies are absent (Page and Blanton 1985). In the family Vahlkampfiidae within this class, the genus Vahlkampfia included all species in which the amoebae do not transform into flagellates (Page 1988). When it was discovered that one Vahlkampfia sp. did transform into flagellates it was transferred to a newly established genus, Paratetramitus (Darbyshire et al. 1976). Later, the small subunit ribosomal DNA (SSU rDNA) sequences proved that this new genus Paratetramitus actually belongs to the genus Tetramitus (Brown and De Jonckheere 1999). The molecular analyses also demonstrated that only two of the morphologically denned Vahlkampfia spp. are related closely enough to belong to the genus Vahlkampfia. All the others, except two, were found to belong to the genus Tetramitus. The two unrelated species were placed into newly established genera, Paravahlkampfia and Neovahlkampfia, respectively. As such the freshwater species Vahlkampfia ustiona became known as Paravahlkampfia ustiona and the marine species V. damariscottae became Neovahlkampfia damariscottae (Brown and De Jonckheere 1999). These conclusions were confirmed by the sequences of the internal transcribed spacers (ITS), including the 5.8S rDNA (De Jonckheere and Brown 2005).

Since that time no other Neovahlkampfia sp. and only two other Paravahlkampfia spp. have been described, P. lenta (Brown and De Jonckheere 2004) and P. francinae (Visvesvara et al. 2009). An isolate from the intestine of a lizard showed to have SSU rDNA sequences similar to P. ustiona but the differences were considered too small (0.2%) to describe the strain as a different species (Schuster et al. 2003). Another Paravahlkampfia strain was isolated from the eye of a keratitis patient but there was not enough information on this isolate to consider it as a different species (Ozkoc et al. 2008). In databases there are also different sequences (AY082995, AY394431, DQ388521) from uncultured amoebae, which show a close relationship to the sequences of the genus Paravahlkampfia, from samples taken from the acidic 'river of fire' in Spain (Zettler et al. 2002), from an acid mine drainage in the USA (Baker et al. 2004) and an unknown origin (Shutt and Gray, unpublished), respectively.

Recently, the new genus Allovahlkampfia was created for an isolate from a cave (Walochnik and Mulec 2009). In the phylogenetic tree this new genus forms a separate branch with some unidentified heterolobosean isolates near the Paravahlkampfia and Neovahlkampfia branches.

We here report the isolation of a heterolobosean amoeba, which belongs to a branch separate from the Paravahlkampfia, while the Neovahlkampfia and Allovahlkampfia branches are found to be totally unrelated to the former two. This new isolate from a fumarole near the volcano Ceboruco in Mexico is described herein as a new species and new genus, Fumarolamoeba ceborucoi.

MATERIALS AND METHODS

Soil samples showing algal growth were collected on December 6, 2008 at the edge of five different fumaroles of the volcano Ceboruco (21°125' N; 104°508' W), in the state of Nayarit, Mexico. The temperature of the vapor escaping from the fumarole has been recorded previously to be 83°C (Taran et al. 2002). The samples were transported at ambient temperature to the laboratory in Belgium and processed for isolation of amoebae 9 days later.

Soil samples were incubated on a solid medium (1.5% nonnutrient agar (NNA) spread with a lawn of Escherichia coli) and in a liquid medium (Page amoeba saline (PAS) with a suspension of E. coli) (Page 1988). The incubation temperature was 44°C.

Strains FUMI and FUM4 were isolated from two samples incubated in the liquid medium PAS supplemented with E. coli. AU further tests were performed with strain FUMI, as both strains appeared to be identical: they had the same ITS sequences (see results). Growth was tested at different temperatures at up to 52°C in the liquid PAS medium supplemented with E. coli.

DNA extraction, amplification of the SSU rRNA gene by PCR, and sequencing were carried out as described previously (Murase et al. 2010). The ITS, including the 5.8S rDNA, sequences were obtained as described by De Jonckheere and Brown (2005). The SSU rRNA sequence of strain FUMI was placed into an alignment of selected heterolobosean SSU rRNA sequences obtained from the on-line comprehensive ribosomal RNA database Silva (<http:// www.arb-silva.de/>http://www.arb-silva.de/; Pruesse et al. 2007). The SSU RNA sequence from strain FUMI, and of other heterolobosean sequences not available at the Silva database, were manually aligned to this set. The final dataset for tree construction comprised 39 other heterolobosean taxa (accession numbers): Paravahlkampfia ustiona (AJ224890), P. francinae (FJ169185), Paravahlkampfia sp. strain 113 (CDC: V453) (AJ550994), Paravahlkampfia sp. strain LA (DQ388521), Neovahlkampfia damariscottae (AJ224891), Allovahlkampfia spelaea (EU696948), Tetramitus aberdonicus (AJ224888), T. jugosus (M9805), T. entericus (AJ224889), T. rostratus (M98051), T. lobospinosus (M98052), T. thermacidophilus (AJ621575), Vahlkampfia avara (AJ 22488), V. inornata (AJ22488), Naegleria fowleri (U80059), N. andersoni (U80057), Willaertia magna (X93221, X93223 and AY2663 15), Pflabellatum (DQ979962), Tulamoeba peronaphora (FJ222603), Acrasis rosea (AFOl 1458), A. helenhemmesae (GU437220), Heteramoeba clara (AFOl 1460), 'Plaesiobystra hypersalinicd' (AFOl 1459), Psalteriomonas lanterna (X94430), Sawyeria marylandensis (AF43935 1), Monopylocystis visvesvarai (AFOl 1463), Stachyamoeba sp. ATCC 50324 (AFOl 1461), Percolomonas cosmopolitus (AF5 19443 and AFOl 1464), Stephanopogon minuta (AB365646), 'Macropharyngomonas halophila' (AFO 11 46 5), Marinamoeba thermophila (FM244741), Vrihiamoeba italica (AB5 13360), Oramoeba thermophila (FN668558), the environmental heterolobosean isolates OSA (DQ388520), AND9 (AY965861) and AND12 (AY965862), and two uncultured heterolobosean clones, RT5in38 (AY082995) and WIM43 (AMI 14803). Names with quotation marks indicate that these names are not formally described. The sequences of the Euglenozoa Euglena gracilis (AY029409) and Trypanoplasma borreli (AY028454) were used as outgroups. A total of 645 of unambiguously aligned sites common to all sequences was retained for phylogenetic analysis. This alignment is available upon request. Phylogenetic trees were inferred by distance matrix neighbor-joining as implemented in ClustalX version 2 (Thompson et al. 1997), maximum likelihood (Felsenstein 198 1) as implemented by the program PhyML version 2.4.5 (Guindon and Gascuel 2003), and by Bayesian analysis using MrBAYES version 3.1.2 (Huelsenbeck and Ronquist 2001). The general time reversible (GTR) model of evolution (Tavaré 1986) was selected as the best model from 28 using the ModelFind program (http://www.hiv.lanl.gov/ content/sequence/ findmodel/findmodel.html). The optimal parameters for the GTR model were estimated using the PhyML program. This model extended with gamma-shaped rate variation with four rate categories was used for both maximum likelihood (100 bootstrap samplings in PhyML) and Baysian analysis (MrBayes). To estimate Bayesian posterior probabilities, Markov Chain Monte Carlo (MCMC) chains were run for 500,000 generations until convergence and sampled every 100 generations (burn-in: 1,000 generations).

A percentage identity matrix was obtained with alignments in ClustalX for genera closely related to the new isolate under investigation, for the SSU rDNA sequences (Table 1) and for the ITS, including the 5.8S rDNA, sequences (Table 2).

RESULTS

Amoebae were isolated from two out of the five samples incubated in liquid medium, not from those incubated on the agar medium. A free-swimming form was also observed in the cultures, which turned out to be a contaminating ciliate. The amoebae were made free of the contaminating ciliate by culturing on agar medium. On the agar medium the ciliate could not grow while the amoebae formed cysts. These cysts were transferred back to liquid medium, and as such the culture was free of the contaminating ciliate.

The amoeba grows at up to 500C in liquid medium, but not at 520C. At 51 0C it does not multiply but just survives. At room temperature (22 0C) the amoebae survive and keep moving. When tested at 44° C the strain does not grow on agar (NNE), and the amoebae rapidly encyst, except if PAS is added on top of the agar. The trophozoites rarely show the limax locomotion, but are mostly rounded, forming eruptive pseudopodes in all directions, rather than moving unidirectionally (Fig. Ia). The pseudopodes are continuously eruptive, whereby the hyaloplasm bulges outwards and spills around the periphery of the cell. No typical uroid or uroidal filaments are observed. In young cultures most trophozoites have two nuclei, but sometimes up to six nuclei are observed (Fig. Ic, d, e). As the culture ages the number of nuclei diminishes to one per cell (Fig. Ib). The mean length and width of the amoeba grown on agar covered with PAS is 26.0 and 13.8 µp?, ranging from 21.0 to 36.6 µp? and 7.5 to 20.5 µp?, respectively.

The amoeba does not transform into a flagellate stage but it forms cysts. The cysts have no pores but a double cyst wall, with the outer wall detached from the inner wall (Fig. 2). Cysts with two nuclei are frequently observed. The diameter of the cysts varies from 5.5 to 11.0 µp? (mean 6.2 µp?).

The SSU rDNA sequence is 2,371 bp long and contains a group I intron of 478 bp long (EBI accession N0 FR719836). In a phylogenetic tree of the SSU rDNA sequence strain FUMI was found to belong to a branch separate from the Paravahlkampfia, Neovahlkampfia and Allovahlkampfia clusters (Fig. 3). Its sequence clusters with the sequence of the uncultured heterolobosean clone RT5in38.

The SSU rDNA sequence of strain FUMI shows 81% identity (Table 1) with P. ustiona, P. francinae (Visvesvara et al. 2009) and strain LA, an environmental isolate stated as being a Paravahlkampfia sp. in the database (DQ388521), and 75% identity to A. spelaea (Walochnik and Mulec 2009).

The length of the ITS 1 is 133 bp, the 5.8S rDNA 154 bp, and the ITS2 125 bp (EBI accession N0 FR719837) for both isolated strains, FUMI and FUM4, and the sequences are identical. Therefore, all experiments were only performed with strain FUMI. There is very low % identity in the ITS sequence of strain FUMI, including the 5.8S rDNA, with the Paravahlkampfia spp. and A. spelaea (Table 2).

DISCUSSION

Phylogenetic analysis based on SSU rDNA sequences shows that strain FUMI, together with clone RT5in38, form a clade which lies outside the clade formed by an environmental heterolobosean isolate LA and the Paravahlkampfia spp. Both clades are totally unrelated to the Neovahlkampfia and Allovahlkampfia branches (Fig. 3). While the SSU rDNA sequence of P. ustiona is 100% identical to that oí P. francinae, strain FUMI shares 81% residues with the former two and 76%o with clone RT5in38 (Table 1). A percentage of 8 lis too low for strain FUMI to belong to the genus Paravahlkampfia, while the sequence of the LA isolate is 98% identical to the Paravahlkampfia spp. Thus the LA strain probably belongs to the genus Paravahlkampfia. The SSU rDNA sequence of .P lenta is not available, only the ITS, including the 5.8S rDNA, sequence has been determined (Brown and De Jonckheere 2004). The ITS, including the 5.8S rDNA, sequence differences (Table 2) of the Paravahlkampfia spp. are large enough (25, 28 and 32%, respectively) to consider them as separate species within the genus. The differences of strain FUMI with the Paravahlkampfia spp. is almost 60%, which is very similar to that of A. cavaea with the Paravahlkampfia spp. However, this sequence of strain FUMI differs also with that of A. cavaea by 42%. Together with the SSU rDNA sequence analysis this is further evidence that strain FUMI belongs to another genus. The SSU rDNA of strain OSA (DQ388520; Shutt and Gray, unpublished) is 97% identical with that of A. speiaea, and only 90% identical to the SSU rDNA sequence of strain AND12 (Lara et al. 2007). Therefore, the former sequence probably belongs to the genus Allovahlkampfia, while strain AND12 should be considered a separate genus or species. It is interesting to note that the only strain (AND9) mentioned in the literature to be a close relative to N. damariscottae (Lara et al. 2007) also has only 72% identity in the SSU rDNA (Table 1) with the latter and, therefore, does not belong to the Neovahlkampfia genus. This is supported by the fact that N. damariscottae is a marine organism (Table 3) while strain AND9 was isolated from soil. In the Heterolobosea all strains of a genus are either freshwater or marine isolates (Fig. 3).

The morphology of the FUMI amoebae seems to be similar to that oí Paravahlkampfia spp. by the fact that it forms hemispherical bulges in all directions (Brown and De Jonckheere 2004), while the limax form is infrequently observed, although in other Paravahlkampfia reports the monopodal-type of locomotion seems to be more the norm (Schuster et al. 2003, Visvesvara et al. 2009). Uroidal filaments are common in Paravahlkampfia spp., but we did not observe them in strain FUMI. The biggest difference might seem to be the presence of multinucleated trophozoites, but it has been reported that species of several vahlkampfiid genera have a strong tendency to supernumerary nuclei (Page 1988). The cysts appear to be similar to those of Paravahlkampfia spp. by the fact that the outer cyst wall often is detached from the inner wall. None of the Paravahlkampfia spp. grows at a temperature higher than 42°C (Table 3), while strain FUMI was isolated at 44°C and grows at a temperature up to 500C.

In Allovahlkampfia the outer wall is closely attached to the endocyst, and the trophozoites are mostly monopodial and show prominent, very long uroid filaments (Walochnik and Mulec 2009). Also in N. damariscottae uroidal filaments seem to be common, but this marine species does not form cysts (Page 1983). None of the mentioned genera, and strain FUMI, have pores in the cysts. Nothing is known on the morphology of clone RT5in38 as the sequence was obtained from an environmental sample, without culturing the amoeba (Zettler et al. 2002). But the low % identity (Table 1) of the SSU rDNA sequences of clone RT5in38 with strain FUMI precludes that they could belong to the same genus.

The morphology supports the conclusion from the molecular data that strain FUMI belongs to a new species and new genus, Fumarolamoeba ceborucoi, gen. nov, sp. no v. It is also obvious that some of the sequences of Heterolobosea reported unnamed in the databases do belong to novel genera, and the organisms, if isolated (e.g. AND9 and AND 12), should be investigated in detail to give them a proper scientific name.

DIAGNOSIS

Fumarolamoeba ceborucoi, gen. nov., sp. nov.

Most trophozoites are rounded displaying markedly eruptive pseudopodes in all directions. In young cultures most trophozoites have two nuclei, but up to six can be observed. This number diminishes to one nucleus per cell as the culture ages. The cysts lack pores and in most cysts the outer cyst wall is detached from the inner wall. No flagellates are observed. The maximum growth temperature is 5O0C. The organism does grow in liquid medium but not on agar, except if the latter is covered with liquid.

The species has a unique ITS, including 5.8S rDNA, sequence which allows it to be identified from other Vahlkampfiidae, especially from Paravahlkampfia spp.

which seems to be the closest related genus.

Observed habitat: soil near a fumarole at the volcano Ceboruco in Nayarit, Mexico.

Etymology: Fumarolamoeba ceborucoi, gen. nov., sp. nov. is named to denote the origin of the type strain, the fiimaroles at the Ceboruco volcano in Nayarit, Mexico.

Acknowledgements. We thank MdL. Lara Alcaraz (Sonora, Mexico) for technical support during sampling and A. Tsyganov (University of Antwerp) for help in obtaining micrographs of the cysts.

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Received on 10* November, 20 10; revised on 22nd December, 20 10; accepted on 22nd December, 2010