Discontinuous genetic variation among mesophilic Naegleria isolates: further evidence that N. gruberi is not a single species.

Naegleria isolates which are currently placed in the type species N. gruberi display great genetic, physiological and morphological heterogeneity. There are two possible interpretations of the nature of this species--that N. gruberi is a species complex or that it is a single continuously variable species. To distinguish between these alternatives, allelic states were determined for 33 loci in 74 new isolates selected to represent wide geographic sources and diverse temperature limits for growth. The results were compared with data for culture collection strains of N. gruberi and other species in the genus. The isolates formed a discontinuous series of clusters, separated by genetic distances similar to those separating the better-characterised taxa N. fowleri, N. lovaniensis, N. jadini, N. australiensis australiensis and N. australiensis italica. Culture collection strains assigned to N. gruberi fell into six distinct clusters, while other clusters were not represented by reference strains. The data are most consistent with the interpretation that N. gruberi is a group of several distinct species, each equivalent to the recently described species in the genus. Naegleria andersoni andersoni and N. andersoni jamiesoni also formed two distinct clusters, equivalent to species. Characteristics temperature limits for growth show that the mesophilic species are ecological as well as genetic entities.     

Discontinuous Genetic Variation Among Mesophilic Naegleria Isolates: Further Evidence that N. gruberi Is Not a Single Species

Naegleria isolates which are currently placed in the type species N. gruberi display great genetic, physiological and morphological heterogeneity. There are two possible interpretations of the nature of this species–that N. gruberi is a species complex or that it is a single continuously variable species. To distinguish between these alternatives, allelic states were determined for 33 loci in 74 new isolates selected to represent wide geographic sources and diverse temperature limits for growth. The results were compared with data for culture collection strains of N. gruberi and other species in the genus. The isolates formed a discontinuous series of clusters, separated by genetic distances similar to those separating the better-characterised taxa N. fowleri, N. lovaniensis, N. jadini, N. australiensis australiensis and N. australiensis italica . Culture collection strains assigned to N. gruberi fell into six distinct clusters, while other clusters were not represented by reference strains. The data are most consistent with the interpretation that N. gruberi is a group of several distinct species, each equivalent to the recently described species in the genus. Naegleria andersoni andersoni and N. andersoni jamiesoni also formed two distinct clusters, equivalent to species. Characteristic temperature limits for growth show that the mesophilic species are ecological as well as genetic entities.     

The ant-parasitic genus Rhipipalloidea Girault (Hymenoptera: Eucharitidae), with a description of a new species

The genus Rhipipalloidea Girault (1934 ), from the Australian region is revised. Rhipipalloidea gruberi Girault (1940 ) is synonymised with R. mira Girault 1934 . A new species, R. madangensis , is described from Papua New Guinea. This species is separated from R. mira from Australia by the distinct striation on the head, and ramose, 12-segmented antennae of the females. Males and a host for the genus are described for the first time.     

Evaluation of the Indirect Fluorescent-Antibody Technique for Identification of Naegleria Species

The indirect fluorescent-antibody technique was used to assess a rapid method for identification of amoebae belonging to the genus Naegleria. Thirty-eight Naegleria and eight other limax amoeba strains were examined by using one N. gruberi and two N. fowleri antisera. All pathogenic Naegleriae, most of which originated from fatal cases of primary amoebic meningo-encephalitis, were identified as belonging to the fowleri species. Most of the N. gruberi strains showed irregular fluorescence. Other limax amoebae, such as Vahlkampfia, Acanthamoeba, Hartmannella, and Schizopyrenus sp. gave negative responses with the prepared antisera. The indirect fluorescent-antibody technique allows the identification of N. fowleri in a mixed culture of both N. fowleri and N. gruberi strains. Twenty-two Naegleria isolated from a suspected stream, other surface waters, and muddy soil could be excluded from the fowleri species with the indirect fluorescent-antibody technique. The results obtained demonstrate that this immunological technique is a valid method for the rapid identification of N. fowleri trophozoites.     

Restriction endonuclease analysis of mitochondrial DNA as an aid in the taxonomy of Naegleria and Vahlkampfia

Using restriction enzyme analysis, mitochondrial DNA fragment patterns from seven strains of pathogenic and nonpathogenic Naegleria and one strain of Vahlkampfia were compared to estimate nucleotide sequence divergence. Significantly high levels of estimated genetic variation between strains of N. gruberi, N. fowleri, and N. jadini support the current taxonomic level of the individual Naegleria species and suggest a distinct phylogeny for each group. Naegleria lovaniensis, strain TS, was shown to have significant nucleotide sequence homology with N. gruberi, strain EGs, suggesting that the two groups share a close taxonomic relationship. The pathogenic strain MB-41 of N. fowleri exhibited distinct genetic divergence from the highly homologous, pathogenic strain Nf66 and the drug-cured strain 6088. Morphologically distinct strains EGs and 1518/la of N. gruberi exhibited significantly large sequence divergence consistent with a more distant taxonomic relationship. Amoebae from the genus Vahlkampfia expressed genetic similarity with strains of N. gruberi.     

Phylogeny of the Pluteaceae (Agaricales, Basidiomycota): taxonomy and character evolution

The phylogeny of the genera traditionally classified in the family Pluteaceae (Agaricales, Basidiomycota) was investigated using molecular data from nuclear ribosomal genes (nSSU, ITS, nLSU) and consequences for taxonomy and character evolution were evaluated. The genus Volvariella is polyphyletic, as most of its representatives fall outside the Pluteoid clade and shows affinities to some hygrophoroid genera (Camarophyllus, Cantharocybe). Volvariella gloiocephala and allies are placed in a different clade, which represents the sister group of Pluteus, and a new generic name, Volvopluteus, is proposed to accommodate these taxa. Characters such as basidiospore size and pileipellis structure can be used to separate Pluteus, Volvariella and Volvopluteus. The genus Pluteus is monophyletic and includes species with partial veil traditionally classified in the genus Chamaeota. The evolution of morphological features used in the infrageneric taxonomy of the genus, such as metuloid cystidia and pileipellis structure, was analyzed. Agreement between the molecular phylogeny and morphological subdivision of Pluteus is, generally speaking, good, though some rearrangements are necessary: (i) species with non-metuloid pleurocystidia and pileipellis as a cutis are placed either in sect. Celluloderma, together with the species characterized by a hymenidermal pipeipellis, or in sect. Pluteus, with the metuloid bearing species; (ii) subdivision of sect. Celluloderma according to the presence/absence of cystidioid elements in the pileipellis is not supported by molecular data.     

Molecular definition and the ubiquity of species in the genus Naegleria

To investigate the variability within species of the genus Naegleria, the ITS1,5.8S and ITS2 rDNA were sequenced of several strains of N. lovaniensis and its Western Australian variants, N. australiensis, N. fowleri, N. andersoni, N. jamiesoni, N. tihangensis, N. pringsheimi, N. pagei, N. gruberi sensu lato and a Naegleria lineage that lost a group I intron from the SSUrDNA twintron. As a result, it is possible to define a molecular species within the Naegleria genus. In addition, one strain of each different allozyme cluster was sequenced to investigate whether they belong to described species or should be treated as distinct new species. This leads to the proposal of eleven new species. The sequencing results from those Naegleria spp. of which several strains are available indicate that these species are ubiquitous. The only exception might be the species represented by the WA variants. However, there are still many Naegleria spp. for which only one strain has been isolated, hence, it is important that the search for more isolates should be continued worldwide.     

Learamoeba waccamawensis, N. G., N. Sp. (Heterolobosea: Vahlkampfiidae), a New Temperature-Tolerant Cyst-Forming Soil Amoeba

Soil from a roadside ditch at Lake Waccamaw, North Carolina, was tested for cyst-forming free-living amoebae, and water from the same ditch was tested for fecal coliform bacteria. Soil samples incubated at room temperature (21-23° C) yielded Acanthamoeba polyphaga, Amoeba sp., Hyperamaeba sp., Mayorella penardi. Naegleria gruberi, Naegleria minor , and unidentified ciliates, testaceans and slime molds. Incubation at 37-39° C yielded Acanthamoeba jacobsi, Platyamoeba schaefferi , and a new amoeba to be described herein. Fecal contamination of the ditch was confirmed by a direct membrane filtration technique that yielded a mean of 1,410 + 134 bacteria/ ml. The new amoeba is assigned to the class Heterolobosea Page & Blanton, 1985 on the basis of promitotic nuclear division, and a flagellated stage with a cytostome as seen in the genus Tetramitus. Amoebae studied in hanging drop preparations were flattened and irregular as described for the genus Stachyamoeba in the family Gruberellidae but changed to a lobose cylindrical form as described for species of Vahlkampfia in the family Vahlkampfiidae. A new genus and species, Learamoeba waccamawensis , is proposed for the amoeba described herein.     

Naegleria-Like Cysts In Cretaceous Amber From Central Kansas

ABSTRACT. Fossil protist cysts are reported from the mid-Cretaceous amber of Ellsworth County, Kansas, which is rich in terrestrial microfossils but contains no known macrofossils. On the basis of their distinctive morphology, the cysts can be referred to the genus Naegleria (Schizopyrenida); they most closely resemble cysts of the living species Naegleria gruberi. This is the first known fossil record for this group of amoebas. the current phylogenetic position and paleoecological role of Naegleria are discussed in relation to this find; it provides direct confirmation of morphological stasis in this group, which had previously been inferred from rRNA sequence divergence data.,     

Occurrence of Naegleria and Acanthamoeba in aquaria

Samples from 24 aquaria were incubated at 28, 37, and 45 degrees C for the isolation of Naegleria and Acanthamoeba. Naegleria was the predominant genus (60.9%), whereas Acanthamoeba represented 15.5% of the isolates. No pathogenic N. fowleri was identified, although a high number of strains were closely related to this species. One isolate (Aq/9/1/45D) was compared with an aquarium isolate (PPMFB-6) from Australia. The Belgian isolate was found to be more related to N. fowleri, whereas the Australian isolate was closer to N. gruberi.     

rRNA genes of Naegleria gruberi are carried exclusively on a 14-kilobase-pair plasmid.

An extrachromosomal DNA was discovered in Naegleria gruberi. The 3,000 to 5,000 copies per cell of this 14-kilobase-pair circular plasmid carry all the 18S, 28S, and 5.8S rRNA genes. The presence of the ribosomal DNA of an organism exclusively on a circular extrachromosomal element is without precedent, and Naegleria is only the third eucaryotic genus in which a nuclear plasmid DNA has been found.     

Occurrence of Naegleria and Acanthamoeba in aquaria.

Samples from 24 aquaria were incubated at 28, 37, and 45 degrees C for the isolation of Naegleria and Acanthamoeba. Naegleria was the predominant genus (60.9%), whereas Acanthamoeba represented 15.5% of the isolates. No pathogenic N. fowleri was identified, although a high number of strains were closely related to this species. One isolate (Aq/9/1/45D) was compared with an aquarium isolate (PPMFB-6) from Australia. The Belgian isolate was found to be more related to N. fowleri, whereas the Australian isolate was closer to N. gruberi.     

Analysis of the 5.8S rRNA gene and the internal transcribed spacers in Naegleria spp. and in N. fowleri

Internal transcribed spacers (ITS) and the 5.8S ribosomal gene of 21 Naegleria fowleri strains and eight other species including Naegleria gruberi were sequenced. The results showed that this region can help differentiate between and within species. The phylogeny of Naegleria spp. deduced from the ITS and the 5.8S gene produced four major lineages, fowleri-lovaniensis, galeacystis-italica-clarki-gruberi-australiensis, andersoni-jamiesoni, and pussardi, that fit perfectly with those inferred from the 18S rRNA gene analysis. The N. gruberi isolate, NG260, was closely related to Naegleria pussardi. The other N. gruberi isolates branched together with Naegleria australiensis in another lineage. The ITS and 5.8S results for N. fowleri were congruent with those previously deduced by RAPD analysis. The phylogenetic analysis inferred from ITS and RAPD data revealed two major groups. The French Cattenom and Chooz and South Pacific strains constituted the first group. The second group encompassed the strains corresponding to the Euro-American and Widespread RAPD variants and shared the same substitution in the 5.8S gene. In addition, it was possible to define species specific primers in ITS regions to rapidly identify N. fowleri.     

Genetic Variations in the Internal Transcribed Spacer and Mitochondrial Small Subunit rRNA Gene of Naegleria spp.

ABSTRACT: Naegleria spp. are widely distributed free-living amebas, but one species in the genus, N. fowleri , causes acute fulminant primary amebic meningoencephalitis in humans and other animals. Thus, it is important to differentiate N. fowleri from the rest in the genus of Naegleria , and to develop tools for the detection of intra-specific genetic variations. In this study, one isolate each of N. australiensis, N. gruberi, N. jadini , and N. lovaniensis and 22 isolates of N. fowleri were characterized at the internal transcribed spacers (ITS) and mitochondrial small subunit rRNA (mtSSU rRNA) gene. The mtSSU rRNA primers designed amplified DNA of all isolates, with distinct sequences obtained from all species examined. In contrast, the ITS primers only amplified DNA from N. lovaniensis and N. fowleri , with minor sequence differences between the two. Three genotypes of N. fowleri were found among the isolates analyzed in both the mtSSU rRNA gene and ITS. The extent of sequence variation was greater in the mtSSU rRNA gene, but the ITS had the advantage of length polymorphism. These data should be useful in the development of molecular tools for rapid species differentiation and genotyping of Naegleria spp.     

Circular ribosomal RNA genes are a general feature of schizopyrenid amoebae

We have recently shown that the ribosomal RNA genes of the amoebo-flagellate Naegleria gruberi Schardinger, 1899, strain NEG-M are carried exclusively on a 14 kilobasepair plasmid. To explore the distribution of this unique gene arrangement, we have examined another strain of N. gruberi and four other species from the order Schizopyrenida. All have this unusual gene arrangement although the size of the plasmid varies widely. Species groups based on morphological criteria do not agree with those resulting from comparison of plasmid restriction enzyme patterns.     

Circular Ribosomal RNA Genes Are a General Feature of Schizopyrenid Amoebae

ABSTRACT We have recently shown that the ribosomal RNA genes of the amoebo-flagellate Naegleria gruberi Schardinger, 1899, strain NEG-M are carried exclusively on a 14 kilobasepair plasmid. To explore the distribution of this unique gene arrangement, we have examined another strain of N. gruberi and four other species from the order Schizopyrenida. All have this unusual gene arrangement although the size of the plasmid varies widely. Species groups based on morphological criteria do not agree with those resulting from comparison of plasmid restriction enzyme patterns.     

Restriction Endonuclease Analysis of Mitochondrial DNA as an Aid in the Taxonomy of Naegleria and Vahlkampfia1

ABSTRACT Using restriction enzyme analysis, mitochondrial DNA fragment patterns from seven strains of pathogenic and nonpathogenic Naegleria and one strain of Vahlkampfia were compared to estimate nucleotide sequence divergence. Significantly high levels of estimated genetic variation between strains of N. gruberi, N. fowleri, and N. jadini support the current taxonomic level of the individual Naegleria species and suggest a distinct phylogeny for each group. Naegleria lovaniensis, strain TS, was shown to have significant nucleotide sequence homology with N. gruberi, strain EGs, suggesting that the two groups share a close taxonomic relationship. The pathogenic strain MB-41 of N. fowleri exhibited distinct genetic divergence from the highly homologous, pathogenic strain Nf66 and the drug-cured strain 6088. Morphologically distinct strains EGs and 1518/la of N. gruberi exhibited significantly large sequence divergence consistent with a more distant taxonomic relationship. Amoebae from the genus Vahlkampfia expressed genetic similarity with strains of N. gruberi.     

Bottled Mineral Waters Polluted by Protozoa in Mexico

A survey of protozoa polluting bottled mineral water in Mexico was carried out using samples obtained from the three best-selling brands of bottled mineral water in the country. The organisms were concentrated through filtration procedures and subsequently cultured in sterile media. The cultures were observed over four weeks, with identification to the level of genus and species. Most commonly found were the amoebae Naegleria gruberi, Acanthamoeba astronyxis, and Vahlkampfia vahlkampfi (trophic as well as cystic stages) plus one flagellate, Bodomorpha minima. No ciliates were detected. The public health importance of the findings is obvious, since some strains of Naegleria and Acanthamoeba have the potential to cause human disease that may lead to death.     

Naegleria fowleri: functional expression of the Nfa1 protein in transfected Naegleria gruberi by promoter modification

To establish a transient transfection system in a Naegleria, we constructed three nfa1-pEGFP-N1 vectors by the promoter replacement and insertion of a nfa1 gene and transfected the DNAs into Naegleria gruberi using a lipid reagent. The transfection efficiency and usefulness of the three modified vectors were estimated by identifying the expressions of the EGFP and Nfa1 protein from N. gruberi. After transfection, the Nfa1 protein was functionally expressed on pseudopodia of N. gruberi. The strong GFP fluorescence was observed in N. gruberi transfected with the actin-nfa1-pEGFP-N1 vector, of which the CMV promoter region in the expression vector was replaced with the actin 5' UTR region. Additionally, when transgenic N. gruberi trophozoites were co-cultured with CHO target cells, the Nfa1 protein was also located on cytoplasm and pseudopodia, especially on a food cup that was formed in contact with target cells as it shown in pathogenic N. fowleri.