Stenamoeba polymorpha, a New Species Isolated from Domesticated Horse Equus ferus caballus.

A new species of lobosean amoeba, Stenamoeba polymorpha n. sp., was isolated from the diarrheic stool of a domesticated horse in Great Falls Virginia, U.S. It shares characteristics with the five other described Stenamoeba species. However, electron microscopy revealed S. polymorpha has a substantially thickened cell surface lamina. Under light microscopy, the amoebae had a dynamic polymorphic appearance because hyaloplasm readily formed and resorbed subpseudopodia from any peripheral region of the cell. While in locomotion, the amoebae produced subpseudopodia that led and alternated the direction of movement with an apparent zigzag path. Sometimes, stationary amoebae had a vertical cell posture that was substantially taller than wide or long. The new species description is also supported by small subunit ribosomal ribonucleic acid gene analyses using phylogenies with both broad and narrow taxon sampling, pairwise nucleotide comparisons, and in-silico secondary structure predictions. The latter suggested Stenamoeba spp. discriminatory motifs in variable region eight. Stenamoeba polymorpha n. sp. is the first of its genus to be recovered from mammal feces, the first to yield a potential group one intron and the second described from North America. Here, the phylogenies suggest Stenamoeba limacina is this new species' nearest known relative.

Characterization of a new pathogenic Acanthamoeba Species, A. byersi n. sp., isolated from a human with fatal amoebic encephalitis.

Acanthamoeba spp. are free-living amoebae that are ubiquitous in natural environments. They can cause cutaneous, nasopharyngeal, and disseminated infection, leading to granulomatous amebic encephalitis (GAE) in immunocompromised individuals. In addition, they can cause amoebic keratitis in contact lens wearers. Acanthamoeba GAE is almost always fatal because of difficulty and delay in diagnosis and lack of optimal antimicrobial therapy. Here, we report the description of an unusual strain isolated from skin and brain of a GAE patient. The amoebae displayed large trophozoites and star-shaped cysts, characteristics for acanthamoebas belonging to morphology Group 1. However, its unique morphology and growth characteristics differentiated this new strain from other Group 1 species. DNA sequence analysis, secondary structure prediction, and phylogenetic analysis of the 18S rRNA gene confirmed that this new strain belonged to Group 1, but that it was distinct from the other sequence types within that group. Thus, we hereby propose the establishment of a new species, Acanthamoeba byersi n. sp. as well as a new sequence type, T18, for this new strain. To our knowledge, this is the first report of a Group 1 Acanthamoeba that is indisputably pathogenic in humans.

A description of a new "Amoebozoan" isolated from the American lobster, Homarus americanus.

Our knowledge of the diversity of amoeboid protists is rapidly expanding as new and old habitats are more fully explored. In 2003, while investigating the cause of an amoeboid disease afflicting lobsters on the East Coast, samples were examined for the presence of amoebae from the carapace washings of the American lobster, Homarus americanus. During this survey a unique community of gymnamoebae was discovered. Among the new taxa discovered was a small Thecamoeba-like organism with a single posteriorly directed pseudopodium. Although resembling Parvamoeba rugata, this amoeba displayed distinctive morphology from that isolate or any other amoebozoan. Phylogenetic analysis shows this amoeba is distantly related to the Thecamoebidae. In this paper we describe the unique morphology of a second species of Parvamoeba and discuss its phylogenetic position with respect to the "Amoebozoa."

A multigene analysis of Corallomyxa tenera sp. nov. suggests its membership in a clade that includes Gromia, Haplosporidia and Foraminifera.

We combine a morphological description with a multigene analysis to assess the phylogenetic placement of a poorly known amoeboid taxon Corallomyxa within the eukaryotic tree of life. A detailed morphological analysis including transmission electron microscopy and light microscopy of Corallomyxa sp. ATCC 50975 demonstrates that this isolate is a new species, herein designated, Corallomyxa tenera sp. nov. This species possesses features of the genus, such as a multinucleate, reticulate plasmodium with localized bidirectional streaming and occasional formation of surface buds, but is differentially characterized from other species by its delicate appearance, short duration of the anastomosing reticulate network and production of round smooth-walled cysts. The new species also lacks some features found in some Corallomyxa species, including cytoplasmic condensation and an electron dense "chromocenter". A Bayesian analysis of four concatenated genes (SSU-rDNA, actin, alpha- and beta-tubulin) from a wide diversity of eukaryotes places the new species together with taxa placed in the putative supergroup 'Rhizaria'. All molecular loci refute the traditional placement of Corallomyxa within the supergroup 'Amoebozoa', which includes other Mycetozoidea and Lobosea. Maximum likelihood and Bayesian analyses of the two well-sampled genes, SSU-rDNA and actin, with increased taxon sampling of 'Rhizaria' show a close affinity of Corallomyxa with Foraminifera, Gromia and, for SSU-rDNA, Haplosporidia. We further identify a novel stem, herein designated E23-13-1, in the predicted SSU-rDNA secondary structure that supports this relationship. A hypothesis is presented for the evolution of morphological and molecular synapomorphies in a clade containing Gromia, Corallomyxa, Foraminifera and Haplosporidia.

Monopylocystis visvesvarai n. gen., n. sp. and Sawyeria marylandensis n. gen., n. sp.: two new amitochondrial heterolobosean amoebae from anoxic environments.

Two new species of heterolobosean amoebae from anoxic environments, Monopylocystis visvesvarai and Sawyeria marylandensis, are described on the basis of light microscopy, electron microscopy, and their phylogenetic affiliation based on analyses of nuclear small-subunit ribosomal RNA gene sequences. Both species lack mitochondria but have organelles provisionally interpreted as hydrogenosomes, and neither can tolerate aerobic conditions. As their conditions of culture do not exclude all oxygen, they may be microaerophiles rather than strict anaerobes. Both species have unusual nucleolar morphologies. Monopylocystis visvesvarai, from a marine sediment, has nucleolar material distributed around the nuclear periphery. It is the first non-aerobic heterolobosean protist for which a cyst is known; the cyst is unmineralized and unornamented except for a single, raised, plugged pore. Sawyeria marylandensis, from an iron-rich freshwater stream, has nucleolar material distributed in one or two parietal masses, which persist during mitosis. In phylogenetic analyses of small-subunit rRNA gene sequences, Monopylocystis visvesvarai, Sawyeria marylandensis and Psalteriomonas lanterna converge to form a single clade of non-aerobic (anaerobic/microaerophilic) heteroloboseans.

Subulatomonas tetraspora nov. gen. nov. sp. is a member of a previously unrecognized major clade of eukaryotes.

While a large number of aerobic free-living protists have been described within the last decade, the number of new anaerobic or microaerophilic microbial eukaryotic taxa has lagged behind. Here we describe a microaerophilic genus and species of amoeboflagellate isolated from a near-shore marine site off the coast at Plymouth, Massachusetts: Subulatomonas tetraspora nov. gen. nov. sp. This taxon is closely related to Breviata anathema based on both microscopical features and phylogenetic analyses of sequences of three genes: SSU-rDNA, actin, and alpha-tubulin. However, Subulatomonas tetraspora nov. gen. nov. sp. and B. anathema are morphologically distinctive, differ by 14.9% at their SSU-rDNA locus, and were isolated from marine and 'slightly brackish' environments, respectively. Phylogenetic analyses of these two taxa plus closely related sequences from environmental surveys provide support for a novel clade of eukaryotes that is distinct from the major clades including the Opisthokonta, Excavata, Amoebozoa and 'SAR' (Stramenopile, Alveolate, Rhizaria).

Phylogenetic placement of diverse amoebae inferred from multigene analyses and assessment of clade stability within 'Amoebozoa' upon removal of varying rate classes of SSU-rDNA.

Placing amoeboid lineages on the eukaryotic tree of life is difficult due to the paucity of comparable morphological characters and the limited molecular data available for many groups. This situation has led to the lumping of distantly related lineages into large inclusive groups, such as Sarcodina, that do not reflect evolutionary relationships. Previous analyses of molecular markers with limited taxon sampling reveal members of Sarcodina are scattered in five of the six proposed supergroups. We have used multigene analyses to place seven diverse amoeboid lineages-two Nolandella spp., Rhizamoeba sp., Pessonella sp., Arcella hemisphaerica, Arachnula sp. and Trichosphaerium sp.-on the eukaryotic tree of life. Bayesian analysis of the concatenated data of the four genes sequenced (SSU-rDNA, actin, alpha-tubulin and beta-tubulin), including diverse representatives of eukaryotes, indicates that all seven taxa group within the 'Amoebozoa' supergroup. We further performed separate analyses of the well-sampled SSU-rDNA and actin genes using Bayesian and Maximum Likelihood analyses to assess the positions of our newly characterized taxa. In the case of SSU-rDNA, we performed extensive analyses with removal of the fastest rates classes to evaluate the stability and resolution of various taxonomic hypotheses within 'Amoebozoa'. Five of our seven amoeboid lineages fall within well-supported clades that are corroborated by morphology. In contrast, the positions of Arachnula sp. and Trichosphaerium sp. in the SSU-rDNA gene trees are unstable and vary by analyses. Placement of these taxa will require additional data from slowly evolving genes combined with taxon-rich phylogenetic analyses. Finally, the analyses without the fastest rate classes demonstrate that SSU-rDNA has a limited signal for deep relationships within the 'Amoebozoa'.

Vannella epipetala n. sp. isolated from the leaf surface of Spondias mombin (Anacardiaceae) growing in the dry forest of Costa Rica.

As part of a Microbial Observatory of Caterpillars located in the Area de Conservacíon Guanacaste (ACG) in northwestern Costa Rica, we isolated a novel species of the genus Vannella associated with the food of the caterpillars of the saturniid moth Rothschildia lebeau, namely the leaves of the dry forest deciduous tree Spondias mombin (Anacardiaceae). The new species can be distinguished from other described species of the genus by the presence of a plasmalemma coated with a thickened, osmiophilic lamina containing glycostyles, and by its unusual habitat, the leaf surfaces or phylosphere of S. mombin. We further established the novelty of our isolate by sequencing its nuclear small-subunit (SSU) rRNA gene and inferring its phylogenetic position among all other currently sequenced members of the genera Vannella and Platyamoeba. Our results reveal that our isolate shares most recent common ancestry with three strains of Platyamoeba placida, the type species of the genus Platyamoeba. Despite this placement, the isolate clearly possesses glycostyles that are the hallmark of the genus Vannella. In addition to the cultured isolate, we also present a closely related sequence from a SSU rRNA gene clone library constructed from a DNA extract of leaf-wash of S. mombin with sterile water.

Etiology of ulcerative lesions of Atlantic menhaden (Brevoortia tyrannus) from James River, Virginia.

We observed ulcerative lesions on live Atlantic menhaden, Brevoortia tyrannus, during ichthyofaunal sampling in the tidal James River in October 1999 (near Jamestown, VA, USA). Other synoptically collected fishes exhibited no signs of lesions or pre-ulcerative tissues. Live fish were classified as unremarkable (no dermal anomalies), pre-ulcerative (integument intact with boil-like swelling), and ulcerative (severe focal lesions). Specimens were analyzed for bacteria, fungi, and pathogenic protozoans including amphizoic amoebae, Pfiesteria piscicida, and Kudoa sp. No Pfiesteria were detected in any tissue specimen. All B. tyrannus examined, including tissues from unremarkable fish, tested positive for presence of the known fish parasite Kudoa. Only ulcerative lesions were also colonized by bacteria, fungi, and amphizoic amoebae. The absence of bacteria, fungi, and protozoans from unremarkable and pre-ulcerative fish suggests that association of other potential pathogens with B. tyrannus ulcers was due to secondary colonization following lesion formation as a result of Kudoa infection.

The new higher level classification of eukaryotes with emphasis on the taxonomy of protists.

This revision of the classification of unicellular eukaryotes updates that of Levine et al. (1980) for the protozoa and expands it to include other protists. Whereas the previous revision was primarily to incorporate the results of ultrastructural studies, this revision incorporates results from both ultrastructural research since 1980 and molecular phylogenetic studies. We propose a scheme that is based on nameless ranked systematics. The vocabulary of the taxonomy is updated, particularly to clarify the naming of groups that have been repositioned. We recognize six clusters of eukaryotes that may represent the basic groupings similar to traditional "kingdoms." The multicellular lineages emerged from within monophyletic protist lineages: animals and fungi from Opisthokonta, plants from Archaeplastida, and brown algae from Stramenopiles.

Platyamoeba nucleolilateralis n. sp. from the Chesapeake Bay Region.

Members of the genus Platyamoeba are among the most common of the free-living brackish and marine amoebae; yet, to date only twelve species have been documented in the literature and only a limited number of habitats have been sampled globally. During the course of a systematic survey of salt-marsh amoebae along the east coast of the United States, a new species of Platyamoeba was discovered in sediment samples obtained from a salt marsh at Assateague Island, VA. The species can be distinguished from all other described species within the genus by the presence of a nucleus with a single parietal nucleolus and a floating form with long tapering pseudopods. Its shape varies from flabellate to spatulate as described for species of Platyamoeba and Vannella. The fine structure of the glycocalyx, however, is characteristic of Platyamoeba.

Identification of amoebae implicated in the life cycle of Pfiesteria and Pfiesteria-like dinoflagellates.

This study was undertaken to assess whether amoebae commonly found in mesohaline environments are in fact stages in the life cycles of Pfiesteria and Pfiesteria-like dinoflagellates. Primary isolations of amoebae and dinoflagellates were made from water and sediment samples from five tributaries of the Chesapeake Bay. Additional amoebae were also cloned from bioassay aquaria where fish mortality was attributed to Pfiesteria. Electron microscopy and small subunit (SSU) rRNA gene sequence analysis of these isolates clearly demonstrated that the commonly depicted amoeboid form of Pfiisteria is very likely a species of Korotnevella and is unrelated to Pfiesteria or Pfiesteria-like dinoflagellates. We have determined that the Pfiesteria and Pfiesteria-like dinoflagellates examined in this study undergo a typical homothallic life cycle without amoeboid stages. Furthermore, we have demonstrated that cloned amoebae sharing morphological characteristics described for stages in the life cycle of Pfiesteria do not transform into dinozoites. The strict clonal isolation and cultivation techniques used in this study substantially support the conclusion that the amoebae and some of the flagellates depicted in the life cycle of Pfiesteria are environmental contaminants of the Pfiesteria culture system and that the Ambush Predator Hypothesis needs to be rigorously reevaluated.

Two new small-subunit ribosomal RNA gene lineages within the subclass gymnamoebia.

Phylogenetic analysis of small-subunit ribosomal RNA gene sequences for gymnamoebae of the families Vexilliferidae, Paramoebidae, and Vannellidae identified two distinct lineages that are supported by gross morphological characters. This analysis indicates that paramoebids and vexilliferids are part of one lineage and that vannellids belong to another. A shared morphological character unique to the paramoebid/vexilliferid lineage members is the presence of dactylopodiate subpseudopodia. However, cell surface structures, normally used for taxonomic discrimination, range from simple hair-like filaments without any apparent organization (Neoparamoeba), to hexagonal glycostyles (Vexillifera) or more elaborate surface scales (Korotnevella). Taxa within the vannellid lineage all lack subpseudopodia and appear flabellate, spatulate or linguiform while in locomotion. Cell surface structures of taxa within the vannellid lineage range from filaments organized into hexagonal arrays (Lingulamoeba, Platyamoeba) to pentagonal glycostyles (Clydonella, Vannella). Vannellid lineage members of the genera Clydonella and Lingulamoeba were studied at the level of electron microscopy. Unique cell surface features validate these as genera distinct from Vannella and Platyamoeba. Genetic and ultrastructural data are used to discuss the phylogenetic interrelationships for the taxa studied.