Updated classification of the phylum Parabasalia.

The phylum Parabasalia includes very diverse single-cell organisms that nevertheless share a distinctive set of morphological traits. Most are harmless or beneficial gut symbionts of animals, but some have turned into parasites in other body compartments, the most notorious example being Trichomonas vaginalis in humans. Parabasalians have garnered attention for their nutritional symbioses with termites, their modified anaerobic mitochondria (hydrogenosomes), their character evolution, and the wholly unique features of some species. The molecular revolution confirmed the monophyly of Parabasalia, but considerably changed our view of their internal relationships, prompting a comprehensive reclassification 14 years ago. This classification has remained authoritative for many subgroups despite a greatly expanded pool of available data, but the large number of species and sequences that have since come out allow for taxonomic refinements in certain lineages, which we undertake here. We aimed to introduce as little disruption as possible but at the same time ensure that most taxa are truly monophyletic, and that the larger clades are subdivided into meaningful units. In doing so, we also highlighted correlations between the phylogeny of parabasalians and that of their hosts.

New Parabasalia symbionts Snyderella spp. and Daimonympha gen. nov. from South American Rugitermes termites and the parallel evolution of a cell with a rotating "head".

Most Parabasalia are symbionts in the hindgut of "lower" (non-Termitidae) termites, where they widely vary in morphology and degree of morphological complexity. Large and complex cells in the class Cristamonadea evolved by replicating a fundamental unit, the karyomastigont, in various ways. We describe here four new species of Calonymphidae (Cristamonadea) from Rugitermes hosts, assigned to the genus Snyderella based on diagnostic features (including the karyomastigont pattern) and molecular phylogeny. We also report a new genus of Calonymphidae, Daimonympha, from Rugitermes laticollis. Daimonympha's morphology does not match that of any known Parabasalia, and its SSU rRNA gene sequence corroborates this distinction. Daimonympha does however share a puzzling feature with a few previously described, but distantly related, Cristamonadea: a rapid, smooth, and continuous rotation of the anterior end of the cell, including the many karyomastigont nuclei. The function of this rotatory movement, the cellular mechanisms enabling it, and the way the cell deals with the consequent cell membrane shear, are all unknown. "Rotating wheel" structures are famously rare in biology, with prokaryotic flagella being the main exception; these mysterious spinning cells found only among Parabasalia are another, far less understood, example.

Fish Parasite Dinoflagellates Haidadinium ichthyophilum and Piscinoodinium Share a Recent Common Ancestor.

The dinoflagellate Haidadinium ichthyophilum Buckland-Nicks, Reimchen and Garbary 1997 is an ectoparasite of the spine-deficient, three-spine stickleback Gasterosteus aculeatus L. Reimchen 1984, a fish endemic to Rouge Lake, Haida Gwaii. Haidadinium ichthyophilum proved difficult to assign taxonomically because its morphology and complex life cycle exhibited defining characteristics of both autotrophic and heterotrophic dinoflagellates, and was tentatively assigned to the Phytodiniales. Here, we characterized a 492 bp fragment of the small subunit ribosomal RNA (SSU rRNA) from preserved H. ichthyophilum cysts. In SSU phylogeny, H. ichthyophilum branches with the fish parasites, Piscinoodinium sp., strongly supporting the inclusion of H. ichthyophilum within the Suessiales.

New Species of Spirotrichonympha from Reticulitermes and the Relationships Among Genera in Spirotrichonymphea (Parabasalia).

Spirotrichonymphea is a class of hypermastigote parabasalids defined by their spiral rows of many flagella. They are obligate hindgut symbionts of lower termites. Despite more than 100 yr of morphological and ultrastructural study, the group remains poorly characterised by molecular data and the phylogenetic positions and taxonomic validity of most genera remain in question. The genus Spirotrichonympha has been reported to inhabit several termite genera, including Reticulitermes, Coptotermes, and Hodotermopsis. The type species for this genus, Spirotrichonympha flagellata, was described from Reticulitermes lucifugus but no molecular data are yet available for this species. In this study, three new Spirotrichonympha species are described from three species of Reticulitermes. Their molecular phylogenetic position indicates that the genus is not monophyletic, as Spirotrichonympha species from Coptotermes, Paraneotermes, and Hodotermopsis branch separately. In contrast, the genus Holomastigotoides is monophyletic, as demonstrated using new sequences from Holomastigotoides species. The presence of Holomastigotoides in Prorhinotermes and the distinct phylogenetic positions of Spirotrichonympha from Reticulitermes and Coptotermes are consistent with a previously proposed symbiont fauna replacement in the ancestor of Reticulitermes.

Molecular characterization and phylogeny of four new species of the genus Trichonympha (Parabasalia, Trichonymphea) from lower termite hindguts.

Members of the genus Trichonympha are among the most well-known, recognizable and widely distributed parabasalian symbionts of lower termites and the wood-eating cockroach species of the genus Cryptocercus. Nevertheless, the species diversity of this genus is largely unknown. Molecular data have shown that the superficial morphological similarities traditionally used to identify species are inadequate, and have challenged the view that the same species of the genus Trichonympha can occur in many different host species. Ambiguities in the literature, uncertainty in identification of both symbiont and host, and incomplete samplings are limiting our understanding of the systematics, ecology and evolution of this taxon. Here we describe four closely related novel species of the genus Trichonympha collected from South American and Australian lower termites: Trichonympha hueyi sp. nov. from Rugitermes laticollis, Trichonympha deweyi sp. nov. from Glyptotermes brevicornis, Trichonympha louiei sp. nov. from Calcaritermes temnocephalus and Trichonympha webbyae sp. nov. from Rugitermes bicolor. We provide molecular barcodes to identify both the symbionts and their hosts, and infer the phylogeny of the genus Trichonympha based on small subunit rRNA gene sequences. The analysis confirms the considerable divergence of symbionts of members of the genus Cryptocercus, and shows that the two clades of the genus Trichonympha harboured by termites reflect only in part the phylogeny of their hosts.

The role of host phylogeny varies in shaping microbial diversity in the hindguts of lower termites.

The hindguts of lower termites and Cryptocercus cockroaches are home to a distinct community of archaea, bacteria, and protists (primarily parabasalids and some oxymonads). Within a host species, the composition of these hindgut communities appears relatively stable, but the evolutionary and ecological factors structuring community composition and stability are poorly understood, as are differential impacts of these factors on protists, bacteria, and archaea. We analyzed the microbial composition of parabasalids and bacteria in the hindguts of Cryptocercus punctulatus and 23 species spanning 4 families of lower termites by pyrosequencing variable regions of the small-subunit rRNA gene. Especially for the parabasalids, these data revealed undiscovered taxa and provided a phylogenetic basis for a more accurate understanding of diversity, diversification, and community composition. The composition of the parabasalid communities was found to be strongly structured by the phylogeny of their hosts, indicating the importance of historical effects, although exceptions were also identified. Particularly, spirotrichonymphids and trichonymphids likely were transferred between host lineages. In contrast, host phylogeny was not sufficient to explain the majority of bacterial community composition, but the compositions of the Bacteroidetes, Elusimicrobia, Tenericutes, Spirochaetes, and Synergistes were structured by host phylogeny perhaps due to their symbiotic associations with protists. All together, historical effects probably resulting from vertical inheritance have had a prominent role in structuring the hindgut communities, especially of the parabasalids, but dispersal and environmental acquisition have played a larger role in community composition than previously expected.

Molecular Evidence for the Polyphyly of Macrotrichomonas (Parabasalia: Cristamonadea) and a Proposal for Macrotrichomonoides n. gen.

Macrotrichomonas (Cristamonadea: Parabasalia) is an anaerobic, amitochondriate flagellate symbiont of termite hindguts. It is noteworthy for being large but not structurally complex compared with other large parabasalians, and for retaining a structure similar in appearance to the undulating membrane (UM) of small flagellates closely related to cristamonads, e.g. Tritrichomonas. Here, we have characterised the SSU rDNA from two species described as Macrotrichomonas: M. restis Kirby 1942 from Neotermes jouteli and M. lighti Connell 1932 from Paraneotermes simplicicornis. These species do not form a clade: M. lighti branches with previously characterised Macrotrichomonas sequences from Glyptotermes, while M. restis branches with the genus Metadevescovina. We examined the M. restis UM by light microscopy, scanning electron microscopy, and transmission electron microscopy, and we find common characteristics with the proximal portion of the robust recurrent flagellum of devescovinids. Altogether, we show the genus Macrotrichomonas to be polyphyletic and propose transferring M. restis to a new genus, Macrotrichomonoides. We also hypothesise that the macrotrichomonad body plan represents the ancestral state of cristamonads, from which other major forms evolved.

The phylogenetic position of Kofoidia loriculata (Parabasalia) and its implications for the evolution of the Cristamonadea.

Kofoidia loriculata is a parabasalid symbiont inhabiting the hindgut of the lower termite Paraneotermes simplicicornis. It was initially described as a lophomonad due to its apical tuft of multiple flagella that disintegrate during cell division, but its phylogenetic relationships have not been investigated using molecular evidence. From single cell isolations, we sequenced the small subunit rRNA gene and determined that K. loriculata falls within the Cristamonadea, but is unrelated to other lophomonads. This analysis further demonstrates the polyphyly of the lophomonads and the necessity to re-assess the morphological and cellular evolution of the Cristamonadea.

Toxoplasma serotype is associated with development of ocular toxoplasmosis.

BACKGROUND: Worldwide, ocular toxoplasmosis (OT) is the principal cause of posterior uveitis, a severe, life-altering disease. A Toxoplasma gondii enzyme-linked immunoassay that detects strain-specific antibodies present in serum was used to correlate serotype with disease. METHODS: Toxoplasma serotypes in consecutive serum samples from German uveitis patients with OT were compared with non-OT seropositive patients with noninfectious autoimmune posterior uveitis. OT patients were tested for association of parasite serotype with age, gender, location, clinical onset, size, visual acuity, or number of lesions (mean follow-up, 3.8 years) to determine association with recurrences. RESULTS: A novel, nonreactive (NR) serotype was detected more frequently in serum samples of OT patients (50/114, 44%) than in non-OT patients (4/56, 7%) (odds ratio, 10.0; 95% confidence interval 3.4-40.8; P < .0001). Non-OT patients were predominantly infected with Type II strains (39/56; 70%), consistent with expected frequencies in Central Europe. Among OT patients, those with NR serotypes experienced more frequent recurrences (P = .037). Polymerase chain reaction detected parasite DNA in 8/60 OT aqueous humor specimens but failed to identify Type II strain alleles. CONCLUSIONS: Toxoplasma NR and Type II serotypes predominate in German OT patients. The NR serotype is associated with OT recurrences, underscoring the value of screening for management of disease.

Trichonympha burlesquei n. sp. from Reticulitermes virginicus and evidence against a cosmopolitan distribution of Trichonympha agilis in many termite hosts.

Historically, symbiotic protists in termite hindguts have been considered to be the same species if they are morphologically similar, even if they are found in different host species. For example, the first-described hindgut and hypermastigote parabasalian, Trichonympha agilis (Leidy, 1877) has since been documented in six species of Reticulitermes, in addition to the original discovery in Reticulitermes flavipes. Here we revisit one of these, Reticulitermes virginicus, using molecular phylogenetic analysis from single-cell isolates and show that the Trichonympha in R. virginicus is distinct from isolates in the type host and describe this novel species as Trichonympha burlesquei n. sp. We also show the molecular diversity of Trichonympha from the type host R. flavipes is greater than supposed, itself probably representing more than one species. All of this is consistent with recent data suggesting a major underestimate of termite symbiont diversity.

Molecular characterization of parabasalian symbionts Coronympha clevelandii and Trichonympha subquasilla from the Hawaiian lowland tree termite Incisitermes immigrans.

An important and undervalued challenge in characterizing symbiotic protists is the accurate identification of their host species. Here, we use DNA barcoding to resolve one confusing case involving parabasalian symbionts in the hindgut of the Hawaiian lowland tree termite, Incisitermes immigrans, which is host to several parabasalians, including the type species for the genus Coronympha, C. clevelandii. We collected I. immigrans from its type locality (Hawaii), confirmed its identity by DNA barcoding, and characterized the phylogenetic position of two symbionts, C. clevelandii and Trichonympha subquasilla. These data show that previous molecular surveys of "I. immigrans" are, in fact, mainly derived from the Caribbean termite I. schwarzi, and perhaps also another related species. These results emphasize the need for host barcoding, clarify the relationship between morphologically distinct Coronympha species, and also suggest some interesting distribution patterns of nonendemic termite species and their symbionts.

Morphology and molecular phylogeny of Staurojoenina mulleri sp. nov. (Trichonymphida, Parabasalia) from the hindgut of the kalotermitid Neotermes jouteli.

Staurojoenina is a large and structurally complex genus of hypermastigont parabasalians found in the hindgut of lower termites. Although several species of Staurojoenina have been described worldwide, all Staurojoenina observed to date in different species of North American termites have been treated as the same species, S. assimilis. Here, we characterize Staurojoenina from the North American termite Neotermes jouteli using light microscopy, scanning electron microscopy, and phylogenetic analysis of small subunit ribosomal RNA, and compare it with S. assimilis from its type host, Incisitermes minor. The basic morphological characteristics of the N. jouteli symbiont, including its abundant bacterial epibionts, are similar as far as they may be compared with existing data from S. assimilis, although not consistently identical. In contrast, we find that they are extremely distantly related at the molecular level, sharing a pairwise similarity of SSU rRNA genes comparable to that seen between different genera or even families of other parabasalians. Based on their evolutionary distance and habitat in different termite genera, we consider the N. jouteli Staurojoenina to be distinct from S. assimilis, and describe a new species, Staurojoenina mulleri, in honor of the pioneering parabasalian researcher, Miklos Muller.

Molecular and morphological analysis of the family Calonymphidae with a description of Calonympha chia sp. nov., Snyderella kirbyi sp. nov., Snyderella swezyae sp. nov. and Snyderella yamini sp. nov.

Calonymphids are a group of multinucleate, multiflagellate protists belonging to the order Cristamonadida (Parabasalia) that are found exclusively in the hindgut of termites from the family Kalotermitidae. Despite their impressive morphological complexity and diversity, few species have been formally described and fewer still have been characterized at the molecular level. In this study, four novel species of calonymphids were isolated and characterized: Calonympha chia and Snyderella yamini spp. nov., from Neotermes castaneus and Calcaritermes nearcticus from Florida, USA, and Snyderella kirbyi and Snyderella swezyae, spp. nov., from Calcaritermes nigriceps and Cryptotermes cylindroceps from Colombia. Each of these species was distinguished from its congeners by residing in a distinct host and by differences at the molecular level. Phylogenetic analyses of small subunit (SSU) rDNA indicated that the genera Calonympha and Stephanonympha were probably not monophyletic, though the genus Snyderella, previously only represented by one sequence in molecular analyses, appeared with these new data to be monophyletic. This was in keeping with the traditional evolutionary view of the group in which the morphology of the genus Snyderella is considered to be derived, while that of the genus Stephanonympha is ancestral and therefore probably plesiomorphic.

Morphology and molecular phylogeny of Pseudotrichonympha hertwigi and Pseudotrichonympha paulistana (Trichonymphea, parabasalia) from neotropical rhinotermitids.

Pseudotrichonympha is a large hypermastigote parabasalian found in the hindgut of several species of rhinotermitid termites. The genus was discovered more than 100 years ago, and although over a dozen species have since been described, this represents only a small fraction of its likely diversity: the termite genera from which Pseudotrichonympha is known are all species rich, and in most cases their hindgut symbionts have not been examined. Even formally described species are mostly lacking in detailed microscopic data and/or sequence data. Using small subunit ribosomal RNA gene sequences and light and scanning electron microscopy we describe here the morphology and molecular phylogenetic position of two Pseudotrichonympha species: the type species for the genus, Pseudotrichonympha hertwigi from Coptotermes testaceus (described previously in line drawing only), and Pseudotrichonympha paulistana from Heterotermes tenuis (described previously based on light microscopy only).

Identification of an atypical strain of toxoplasma gondii as the cause of a waterborne outbreak of toxoplasmosis in Santa Isabel do Ivai, Brazil.

Multilocus DNA sequencing has identified a nonarchetypal strain of Toxoplasma gondii as the causal agent of a waterborne outbreak in Brazil in 2001. The strain, isolated from a water supply epidemiologically linked to the outbreak, was virulent to mice, and it has previously been identified as BrI. Using a serologic assay that detects strain-specific antibodies, we found that 13 (65%) of 20 individuals who were immunoglobulin (Ig) M positive during the outbreak possessed the same serotype as mice infected with the purported epidemic strain. The remaining 7 individuals, plus additional IgM-negative, IgG-positive individuals, possessed 1 of 4 novel serotypes, the most common of which matched the serotype of mice infected with strains isolated from chickens foraging near the outbreak site. The latter strains likely reflect the genetic diversity of T. gondii circulating in highly endemic regions of Brazil. The serotyping assay proved a useful tool for identification of specific individuals infected with the outbreak agent.

Characterization of new cristamonad species from kalotermitid termites including a novel genus, Runanympha.

Cristamonadea is a large class of parabasalian protists that reside in the hindguts of wood-feeding insects, where they play an essential role in the digestion of lignocellulose. This group of symbionts boasts an impressive array of complex morphological characteristics, many of which have evolved multiple times independently. However, their diversity is understudied and molecular data remain scarce. Here we describe seven new species of cristamonad symbionts from Comatermes, Calcaritermes, and Rugitermes termites from Peru and Ecuador. To classify these new species, we examined cells by light and scanning electron microscopy, sequenced the symbiont small subunit ribosomal RNA (rRNA) genes, and carried out barcoding of the mitochondrial large subunit rRNA gene of the hosts to confirm host identification. Based on these data, five of the symbionts characterized here represent new species within described genera: Devescovina sapara n. sp., Devescovina aymara n. sp., Macrotrichomonas ashaninka n. sp., Macrotrichomonas secoya n. sp., and Macrotrichomonas yanesha n. sp. Additionally, two symbionts with overall morphological characteristics similar to the poorly-studied and probably polyphyletic 'joeniid' Parabasalia are classified in a new genus Runanympha n. gen.: Runanympha illapa n. sp., and Runanympha pacha n. sp.

Specific mutations in ABCA1 have discrete effects on ABCA1 function and lipid phenotypes both in vivo and in vitro.

Mutations in ATP-binding cassette transporter A1 (ABCA1) cause Tangier disease and familial hypoalphalipoproteinemia, resulting in low to absent plasma high-density lipoprotein cholesterol levels. However, wide variations in clinical lipid phenotypes are observed in patients with mutations in ABCA1. We hypothesized that the various lipid phenotypes would be the direct result of discrete and differing effects of the mutations on ABCA1 function. To determine whether there is a correlation between the mutations and the resulting phenotypes, we generated in vitro 15 missense mutations that have been described in patients with Tangier disease and familial hypoalphalipoproteinemia. Using localization of ABCA1, its ability to induce cell surface binding of apolipoprotein A-I, and its ability to elicit efflux of cholesterol and phospholipids to apolipoprotein A-I we determined that the phenotypes of patients correlate with the severity and nature of defects in ABCA1 function.

Increased ABCA1 activity protects against atherosclerosis.

The ABC transporter ABCA1 plays a key role in the first steps of the reverse cholesterol transport pathway by mediating lipid efflux from macrophages. Previously, it was demonstrated that human ABCA1 overexpression in vivo in transgenic mice results in a mild elevation of plasma HDL levels and increased efflux of cholesterol from macrophages. In this study, we determined the effect of overexpression of ABCA1 on atherosclerosis development. Human ABCA1 transgenic mice (BAC(+)) were crossed with ApoE(-/-) mice, a strain that spontaneously develop atherosclerotic lesions. BAC(+)ApoE(-/-) mice developed dramatically smaller, less-complex lesions as compared with their ApoE(-/-) counterparts. In addition, there was increased efflux of cholesterol from macrophages isolated from the BAC(+)ApoE(-/-) mice. Although the increase in plasma HDL cholesterol levels was small, HDL particles from BAC(+)ApoE(-/-) mice were significantly better acceptors of cholesterol. Lipid analysis of HDL particles from BAC(+)ApoE(-/-) mice revealed an increase in phospholipid levels, which was correlated significantly with their ability to enhance cholesterol efflux.

Macrophage ATP-binding cassette transporter A1 overexpression inhibits atherosclerotic lesion progression in low-density lipoprotein receptor knockout mice.

BACKGROUND: ATP-binding cassette transporter A1 (ABCA1) is a key regulator of cellular cholesterol and phospholipid transport. Previously, we have shown that inactivation of macrophage ABCA1 induces atherosclerosis in low-density lipoprotein receptor knockout (LDLr-/-) mice. However, the possibly beneficial effects of specific upregulation of macrophage ABCA1 on atherogenesis are still unknown. METHODS AND RESULTS: Chimeras that specifically overexpress ABCA1 in macrophages were generated by transplantation of bone marrow from human ABCA1 bacterial artificial chromosome (BAC) transgenic mice into LDLr-/- mice. Peritoneal macrophages isolated from the ABCA1 BAC --> LDLr-/- chimeras exhibited a 60% (P=0.0006) increase in cholesterol efflux to apolipoprotein AI. To induce atherosclerosis, the mice were fed a Western-type diet containing 0.25% cholesterol and 15% fat for 9, 12, and 15 weeks, allowing analysis of effects on initial lesion development as well as advanced lesions. No significant effect of macrophage ABCA1 overexpression was observed on atherosclerotic lesion size after 9 weeks on the Western-type diet (245+/-36x10(3) microm2 in ABCA1 BAC --> LDLr-/- mice versus 210+/-20x10(3) microm2 in controls). However, after 12 weeks, the mean atherosclerotic lesion area in ABCA1 BAC --> LDLr-/- mice remained only 164+/-15x10(3) microm2 (P=0.0008) compared with 513+/-56x10(3) microm2 in controls (3.1-fold lower). Also, after 15 weeks on the diet, lesions in mice transplanted with ABCA1 overexpressing bone marrow were still 1.6-fold smaller (393+/-27x10(3) microm2 compared with 640+/-59x10(3) microm2 in control transplanted mice; P=0.0015). CONCLUSIONS: ABCA1 upregulation in macrophages inhibits the progression of atherosclerotic lesions.