The closest unicellular relatives of animals.

Molecular phylogenies support a common ancestry between animals (Metazoa) and Fungi, but the evolutionary descent of the Metazoa from single-celled eukaryotes (protists) and the nature and taxonomic affiliation of these ancestral protists remain elusive. We addressed this question by sequencing complete mitochondrial genomes from taxonomically diverse protists to generate a large body of molecular data for phylogenetic analyses. Trees inferred from multiple concatenated mitochondrial protein sequences demonstrate that animals are specifically affiliated with two morphologically dissimilar unicellular protist taxa: Monosiga brevicollis (Choanoflagellata), a flagellate, and Amoebidium parasiticum (Ichthyosporea), a fungus-like organism. Statistical evaluation of competing evolutionary hypotheses confirms beyond a doubt that Choanoflagellata and multicellular animals share a close sister group relationship, originally proposed more than a century ago on morphological grounds. For the first time, our trees convincingly resolve the currently controversial phylogenetic position of the Ichthyosporea, which the trees place basal to Choanoflagellata and Metazoa but after the divergence of Fungi. Considering these results, we propose the new taxonomic group Holozoa, comprising Ichthyosporea, Choanoflagellata, and Metazoa. Our findings provide insight into the nature of the animal ancestor and have broad implications for our understanding of the evolutionary transition from unicellular protists to multicellular animals.

The nucleariid amoebae: more protists at the animal-fungal boundary.

Nucleariid amoebae are naked amoebae, generally characterized by a spherical or sometimes flattened body with radiating filopodia. Most species preferentially consume algal prey or cyanobacteria. Phylogenetic analyses of the small-subunit rRNA coding regions from four nucleariid amoebae place these species near the origin of the animal-fungal divergence, together with the choanoflagellate-Corallochytrium and the ichthyosporean clades. The species Nuclearia delicatula, N. moebiusi, and N. simplex form a monophyletic group, while ATCC 30864, tentatively but possibly incorrectly assigned to Nuclearia sp., represents a separate line of descent. These nucleariids are unrelated to the lineage containing the testate filose amoebae (Testaceafilosia). Our findings expand the morphological and phylogenetic diversity of protists at the animal-fungal divergence.

Korotnevella hemistylolepis n. sp. and Korotnevella monacantholepis n. sp. (Paramoebidae), two new scale-covered mesohaline amoebae.

Two new species of Korotnevella Goodkov, 1988, Korotnevella hemistylolepis n. sp. and Korotnevella monacantholepis n. sp., are described from mesohaline ecosystems. The amoebae are characterized on the basis of light and electron microscopy with special emphasis on the structure of the basket scales, which have species-specific architecture. The two new species are the second and third ones recovered from environments other than freshwater. In terms of scale morphology they most closely resemble a freshwater species, Korotnevella bulla (Schaeffer, 1926) Goodkov, 1988. Two genus names, Dactylamoeba Korotnev, 1880 and Korotnevella Goodkov, 1988, are in current use. The latter name is preferred, pending rediscovery and characterization of Dactylamoeba elongata Korotnev, 1880, the type species of the genus. Korotnevella species can be divided into three groups on the basis of scale morphology, suggesting that the genus may not be monophyletic. A key to species is provided.

A molecular reassessment of the Leptomyxid amoebae.

Leptomyxid amoebae encompass a diverse assemblage of amoeboid protists that have been implicated as encephalitis-causing agents. This characteristic is attributed to recent studies identifying new members of the Leptomyxidae, in particular, Balamuthia mandrillaris, that cause the disease. Their morphologies range from limax to plasmodial, as well as reticulated and polyaxial. Although systematic studies have identified B. mandrillaris as a new member of the Leptomyxidae, its precise placement within the leptomyxids is uncertain. To further assess the taxonomic placement of Balamuthia among the leptomyxid amoebae and to determine whether the members of the Leptomyxida form a monophyletic assemblage, we have sequenced 16S-like rRNA genes from representatives of three leptomyxid families. Our phylogenetic analyses revealed that current members of the order Leptomyxida do not constitute a monophyletic assemblage. Our analyses clearly show that Gephyramoeba, as well as Balamuthia do not belong in the order Leptomyxida. We highlight where molecular data give differing insights than taxonomic schemes based on traditional characters.

Ultrastructure of Trimastix pyriformis (Klebs) Bernard et al.: similarities of Trimastix species with retortamonad and jakobid flagellates.

Trimastix pyriformis (Klebs 1893) Bernard et al. 1999, is a quadriflagellate, free-living, bacterivorous heterotrophic nanoflagellate from anoxic freshwaters that lacks mitochondria. Monoprotist cultures of this species contained naked trophic cells with anterior flagellar insertion and a conspicuous ventral groove. Bacteria were ingested at the posterior end of the ventral groove, but there was no persistent cytopharyngeal complex. The posterior flagellum resided in this groove, and bore two prominent vanes. A Golgi body (dictyosome) was present adjacent to the flagellar insertion. The kinetid consisted of four basal bodies, four microtubular roots, and associated fibers and bands. Duplicated kinetids, each with four basal bodies and microtubular root templates, appeared at the poles of the open mitotic spindle. Trimastix pyriformis is distinguishable from other Trimastix species on the basis of external morphology, kinetid architecture and the distribution of endomembranes. Trimastix species are most similar to jakobid flagellates, especially Malawimonas jakobiformis, and to species of the retortamonad genus Chilomastix. Retortamonads may have evolved from a Trimastix-like ancestor through loss of "canonical" (easily seen with electron microscopy) endomembrane systems and elaboration of cytoskeletal elements associated with the cytostome/cytopharynx complex.

Effects of low temperatures on viability of Cryptosporidium parvum oocysts.

Microcentrifuge tubes containing 8 x 10(6) purified oocysts of Cryptosporidium parvum suspended in 400 microliters of deionized water were stored at 5 degrees C for 168 h or frozen at -10, -15, -20, and -70 degrees C for 1 h to 168 h and then thawed at room temperature (21 degrees C). Fifty microliters containing 10(6) oocysts was administered to each of five to seven neonatal BALB/c mice by gastric intubation. Segments of ileum, cecum, and colon were taken for histology from each mouse 72 or 96 h later. Freeze-thawed oocysts were considered viable and infectious only when developmental-stage C. parvum organisms were found microscopically in the tissue sections. Developmental-stage parasites were not found in tissues from any mice that received oocysts frozen at -70 degrees C for 1, 8, or 24 h. All mice that received oocysts frozen at -20 degrees C for 1, 3, and 5 h had developmental-stage C. parvum; one of 6 mice that received oocysts frozen at -20 degrees C for 8 h had a few developmental-stage parasites; mice that received oocysts frozen at -20 degrees C for 24 and 168 h had no parasites. All mice that received oocysts frozen at -15 degrees C for 8 and 24 h had developmental-stage parasites; mice that received oocysts frozen at -15 degrees C for 168 h had no parasites. All mice that received oocysts frozen at -10 degrees C for 8, 24, and 168 h and those that received oocysts stored at 5 degrees C for 168 h had developmental-stage parasites. These findings demonstrate for the first time that oocysts of C. parvum in water can retain viability and infectivity after freezing and that oocysts survive longer at higher freezing temperatures.

Acanthamoeba pearcei n. sp. (Protozoa: Amoebida) from sewage contaminated sediments.

Seabottom sediments from a discontinued Philadelphia-Camden 40-Mile ocean sewage disposal site were cultured for cyst-forming free-living amoebae. Barge delivered wastes were discharged at the site from 1973 until 1980 when the site was closed. One station at the southeast margin of the site was sampled at a depth of approximately 50 m, twice in 1978 and once in 1982, 1983 and 1984. Sediment from the 1978 collection yielded Acanthamoeba polyphaga, Vahlkampfia sp., and an unknown amoeba with stellate endocysts similar to those of A. astronyxis. Trophozoites and cysts of the isolate were typical of those described for the genus Acanthamoeba. Biochemical tests employing enzyme electrophoresis and morphological studies on live and stained specimens showed that the isolate was distinct from other well-described species within the family Acanthamoebidae Sawyer & Griffin, 1975.

Phylogeny of trichomonads inferred from small-subunit rRNA sequences.

Small subunit (16S-like) ribosomal RNA sequences were obtained from representatives of all four families constituting the order Trichomonadida. Comparative sequence analysis revealed that the Trichomonadida are a monophyletic lineage and a deep branch of the eukaryotic tree. Relative to the early divergent eukaryotic assemblages the branching pattern within the Trichomonadida is very shallow. This pattern suggests the Trichomonadida radiated recently, perhaps in conjunction with their animal hosts. From a morphological perspective the Devescovinidae and Calonymphidae are considered more derived than the Monocercomonadidae and Trichomonadidae. Molecular trees inferred by distance, parsimony and likelihood techniques consistently show the Devescovinidae and Calonymphidae are the earliest diverging lineages within the Trichomonadida, however bootstrap values do not strongly support a particular branching order. In an analysis of all known 16S-like ribosomal RNA sequences, the Trichomonadida share most recent common ancestry with unidentified protists from the hindgut of the termite Reticulitermes flavipes. The position of two putative free-living trichomonads in the tree is indicative of derivation from symbionts rather than direct descent from some free-living ancestral trichomonad.

The unusually long small subunit ribosomal RNA of Phreatamoeba balamuthi.

The small subunit ribosomal RNA (rRNA) of the anaerobic amoeba Phreatamoeba balamuthi is the longest 16S-like rRNA sequenced to date. Secondary structure analysis suggests that the additional sequence is incorporated in canonical eukaryotic expansion regions and is not due to the presence of introns. Reverse transcriptase sequencing of total RNA extracts confirmed that two uncommonly long expansion regions are present in native P. balamuthi 16S-like rRNA. Primary sequence comparison and similar secondary structure indicate a 61 base stem and loop repeat within an expansion region; a mechanism whereby the repeat may have been incorporated is presented. P. balamuthi provides further evidence that 16S-like rRNA length does not correlate with phylogenetic position.

Small subunit ribosomal RNA+ of Hexamita inflata and the quest for the first branch in the eukaryotic tree.

A phylogenetic analysis of the small subunit ribosomal RNA (16S-like rRNA) coding region from Hexamita inflata demonstrates that parasitism alone cannot explain early diverging eukaryotic lineages. Parasitic and free-living diplomonads, as well as trichomonads and microsporidia, diverge at the base of the eukaryotic tree. The relative branching order of diplomonads, trichomonads and microsporidia is influenced by outlying prokaryotic taxa with different G+C compositions in their rRNA coding regions. The high G+C prokaryotes position Giardia lamblia at the base of the eukaryotic tree but split diplomonads into a paraphyletic group. When the outlying groups are restricted to rRNAs with nominal G+C compositions, diplomonads form a monophyletic group that diverged after the microsporidia and trichomonads. This unstable branching pattern correlates with unusual nucleotide compositions in the rRNAs of G. lamblia (75% G+C) and Vairimorpha necatrix (35% G+C). In contrast, the 51% G+C composition of the H. inflata rRNA is typical of other eukaryotic rRNAs. Its divergence after trichomonads is strongly supported by bootstrap replicates in distance analyses that do not include G. lamblia. Because of a low G+C composition in its rRNA coding region, the phylogenetic placement of V. necatrix is uncertain and the identity of the deepest branching eukaryotic lineage is ambiguous.

Reclinomonas americana N. G., N. Sp., a new freshwater heterotrophic flagellate.

A new heterotrophic flagellate has been discovered from sites in Maryland, Michigan and Wyoming. The flagellate resides within a lorica constructed of a meshwork of intertwined fibrils with the outer surface invested with nail-shaped spines. The organism "reclines" within the lorica with its ventral aspect directed upward, and has two heterodynamic flagella, neither of which bears mastigonemes. One flagellum is directed upward and the other is arched over the ventral aspect of the body. Ingestion of bacteria takes place at the left posterior half of the cell. The organism is anchored to the lorica on the right posterior side by a series of regularly spaced cytoplasmic bridges and at the left anterior of the cell by a cytoplasmic appendage similar to the "languette cytoplasmique" found in some bicosoecids. The right side of the cell is raised into a flattened lip with the outer margin reinforced by a ribbon of microtubules. The new flagellate has mitochondria with tubular cristae and lacks a Golgi. A new genus is created to accommodate both the new flagellate described herein and Histiona campanula Penard. A new family is proposed to include the new genus and Histiona.

Studies on cryopreservation of Cryptosporidium parvum.

Neonatal BALB/c mice received oocysts or sporozoites of Cryptosporidium parvum pretreated by a variety of cryopreservation protocols. Histologic sections of infected and control mice were examined to determine if pretreated organisms established infection in the intestine. Sporozoites were inoculated rectally, oocysts orally. Freshly excysted sporozoites were frozen in Hanks' balanced salt solution (HBSS) containing dimethylsulfoxide (DMSO) or glycerol at concentrations of 5%, 10%, or 15% at cooling rates of -1 C and -10 C per min. Other sporozoites were frozen to -70 C in the absence of cryoprotectant without controlled reduction of temperature, others placed in HBSS with 10% DMSO but not subjected to freezing, whereas others were placed in vitrification media containing 5.5 M propylene glycol, 6.5 M glycerol, or 8 M ethylene glycol for 1 min before resuspension in fresh HBSS and inoculation into mice. Intact oocysts were frozen without controlled reduction of temperature directly to -70 C in HBSS containing no cryoprotectant or in HBSS that contained 10% DMSO. Others were cooled at -0.3 C per min from 4 C to -70 C in HBSS with 5% or 10% DMSO. Still others were cooled at a rate of -1 C per min until reaching -40 C and then cooled at -10 C per min until reaching -70 C in HBSS with 7.5% DMSO. Oocysts and sporozoites not exposed to cryoprotectants were inoculated into mice orally and rectally, respectively, for control purposes. Only unfrozen oocysts and sporozoites not exposed to cryoprotectant, and some of the unfrozen oocysts and sporozoites exposed to 10% DMSO, successfully established infections in mice.

Ultrastructure of Amastigomonas bermudensis ATCC 50234 sp. nov.: A new heterotrophic marine flagellate.

The ultrastructure of a new marine heterotrophic flagellate is described. The cell is dorso-ventrally flattened and displays a steady gliding forward movement. A longitudinal groove whose lips are appressed to the substrate runs along the ventral aspect of the cell. The two heterodynamic flagella originate from the proximal end of a cytoplasmic sheath that enfolds most of the length of the anterior flagellum. The lips of this sheath are continuous with the margins of the ventral groove. The posterior trailing flagellum is held closely appressed to the cell body on one side of the ventral groove. Three bands of microtubules extend posteriorly into the cell body and are associated with the kinetosomes. The part of the surface membrane extending over the cell's dorsal and lateral aspect is five-layered, giving the semblance of two fused cell membranes. There is a fibrillar layer underneath these membranes. There is also a fibrous network whose arrays are oriented in different directions within the cytoplasm. Mitochondria have tubular cristae. Based on a comparison with previously described species of Amastigomonas, we establish the species, A. bermudensis n.sp. We further conclude that Thecamonas is a junior synonym of Amastigomonas and move the three nominal species to the latter genus.

Characterization of an axenic strain of Hartmannella vermiformis obtained from an investigation of nosocomial legionellosis.

A free-living amoeba identified as Hartmannella vermiformis was isolated from a water sample obtained during an investigation of nosocomial legionellosis. Hartmannella vermiformis is known to support the intracellular multiplication of Legionella pneumophila. This strain of H. vermiformis, designated CDC-19, was cloned and established in axenic culture to develop a model for the study of the pathogenicity of legionellae. Isoenzyme patterns of axenically-cultivated strain CDC-19 were compared with two strains of H. vermiformis derived from the type strain, one axenic (ATCC 50236) and the other grown in the presence of bacteria (ATCC 30966). Enzyme patterns suggested that all three strains are assignable to the species H. vermiformis. Axenic H. vermiformis strain CDC-19 has been deposited with the American Type Culture Collection (ATCC 50237) and should prove useful in the study of protozoan-bacterial interaction.

Partial purification and characterization of Naegleria fowleri beta-glucosidase.

Naegleria fowleri cells, grown axenically, contain high levels of beta-D-glucosidase which catalyzes the hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside (4MUGlc) (Km, 0.9 mM), octyl-beta-D-glucoside (Km, 0.17 mM), and p-nitrophenyl-beta-D-glucopyranoside at relative rates of 1.00, 2.88, and 1.16, respectively (substrate concentration, 3.0 mM). When the amebae are subjected to freeze-thawing, sonication, and centrifugation (100,000 g, 1 h), 85% of the beta-glucosidase activity appears in the supernatant fraction. The beta-glucosidase was purified 40-fold (34% yield) using a combination of chromatographic steps involving DE-52 cellulose, concanavalin A-Sepharose, and hydroxylapatite followed by isoelectric focusing. The predominant soluble beta-D-galactosidase activity in the Naegleria extract copurifies with the beta-D-glucosidase; the two activities have the same isoelectric point (pI, 6.9), similar heat stabilities, are both inhibited by lactobionic acid (Ki, 0.40 mM), and exhibit optima at pH 4.5, indicating that they are probably the same enzyme. The Naegleria beta-D-glucosidase has an apparent molecular weight of 66,000, a Stokes radius of 25 A, and a sedimentation coefficient of 4.2S. The beta-glucosidase is not inhibited by conduritol beta-epoxide or galactosylsphingosine but is completely inhibited by 1.25 mM bromo conduritol beta-epoxide. The latter compound, when present in the growth medium, inhibits the growth of the organism and profoundly alters its ultrastructure, the main effect being the apparent inhibition of cytokinesis and the generation of multinucleate cells. The issue of the role of the beta-glucosidase in the metabolism of the ameba and its possible role in pathogenic mechanisms are discussed.

Demonstration of various acid hydrolases and preliminary characterization of acid phosphatase in Naegleria fowleri.

Extracts of the pathogenic ameba Naegleria fowleri, prepared by freeze-thawing and sonication, were analyzed for their content of various hydrolytic enzymes that have acid pH optima. The organism is rich in acid phosphatase activity as well as a variety of glycosidases which include beta-glucosidase, beta-galactosidase, beta-fucosidase, alpha-mannosidase, hexosaminidase, arylsulfatase A, and beta-glucuronidase. The crude extract contained only negligible levels of sphingomyelinase, neuraminidase, or arylsulfatase B. All of the hydrolases exhibited higher activity at pH 5.5 than at 7.0, indicating that they are truly "acid" hydrolases. In general, after centrifugation (100,000 g, 1 h), except for arylsulfatase B, more than half of the activity of each of the various hydrolases was recovered in the supernatant fraction. The acid phosphatase in the high-speed supernatant was purified 45-fold (32% yield) by chromatography on QAE-Sephadex and Sephadex G-200 and shown to have the following properties: pH optima, 5.5; Km (4-methylumbelliferyl phosphate), 0.60 mM; molecular weight (estimated by gel filtration chromatography), 92,000; inhibited by heteropolymolybdate complexes but not by L(+) sodium tartrate (0.5 mM) or sodium fluoride (0.5 mM). In addition, unlike the tartrate-resistant acid phosphatase of Leishmania donovani, the major acid phosphatase of N. fowleri is less than 5% as effective in inhibiting superoxide anion production by f-Met-Leu-Phe-stimulated human neutrophils. The finding of high levels of a number of acid hydrolases in Naegleria fowleri raises several questions that merit further study: Do the hydrolases perform a housekeeping function in this single cell eukaryote or do they play some role in the pathogenic process that ensues when the organism infects a suitable host?

A molecular approach to the phylogeny of Acanthamoeba.

Isoenzyme electrophoresis of three different enzyme systems was used to compare 71 strains assigned to the 15 currently recognized species of Acanthamoeba. A phylogenetic (cladistic) analysis of the zymograms indicated an arrangement of strains in 15 distinguishable lineages, but not all corresponding to current taxonomic assignments. Five of the groups corresponded to the recognized species A. castellanii, A. culbertsoni, A. griffini, A. lenticulata and A. royreba. But none of these groups consisted of only strains which had been previously assigned to each respective species. The type-equivalent strains for two species, A. hatchetti and A. tubiashi, were not closely aligned to any other strain and thus are considered to be monotypic. Strains of A. triangularis, A. astronyxis and A. palestinensis occurred together in a single group suggesting possible synonymy; however, on morphologic criteria, the strains assigned to these species are readily distinguishable. Strains assigned to A. polyphaga and A. rhysodes were interspersed throughout the other species groups. The strains of these two species were either misidentified or the species could not be recognized. Two groups previously not recognized as unique formed distinctive clusters which could be considered as new species. The analysis also made it possible to place strains which had previously been identified only to genus into species complexes. These results therefore suggest that previous criteria which have been used to classify Acanthamoeba are not adequate for fully resolving taxa at the species level.