Molecular phylogenetic position of the genera Stephanonympha and Caduceia (Parabasalia) inferred from nuclear small subunit rRNA gene sequences

Nuclear small subunit (SSU) rRNA gene sequences were obtained by polymerase chain reaction from trichomonad symbionts of termites that belong to the Devescovinidae (Caduceia versatilis) and polymastigont Calonymphidae (Stephanonympha nelumbium). The unidentified SSU rRNA sequence Nk3, previously obtained from the termite Neotermes koshunensis, has also been shown to derive from a Stephanonympha sp. by in situ hybridization. These sequences were analysed in a broad phylogeny including nearly all identified parabasalid sequences available in the databases, and some as yet unidentified sequences likely deriving from the new order Cristamonadida (Devescovinidae, Calonymphidae, and hypermastigids Lophomonadida). A global phylogeny of parabasalids reveals a partial agreement between the clades identified in this work and the last classification of this phylum into four orders. However, this classification is still incongruent with our data and new taxonomic considerations are proposed. The analysis confirms the monophyly of the Cristamonadida and separates this order into two groups: the first unites nearly all the Devescovinidae including Caduceia and the Calonymphidae Coronympha and Metacoronympha, whereas the second group is composed of a few Devescovinidae, Lophomonadida, and Calonymphidae such as Stephanonympha. Caduceia is closely related to Devescovina, corroborating the marked morphological similarity between these two genera whereas Stephanonympha groups together with the Calonymphidae Snyderella and Calonympha. These data also confirm the polyphyly of the families Devescovinidae and Calonymphidae and support the arrangement of the axostyle-pelta complexes as a valuable character for taxonomic considerations within the Calonymphidae.     

Glucokinase and Fructokinase of Trichomonas vaginalis and Tritrichomonas foetus

ABSTRACT. Trichomonas vaginalis and Tritrichomonas foetus contain glucokinase and not a hexokinase of broad hexose specificity. Tritrichomonas foetus also contains a specific fructokinase which could be resolved from glucokinase by anion exchange chromatography. Native T. vaginalis glucokinase had a Mr of 76,000, and SDS-PAG electrophoresis showed two equally stained bands corresponding to Mr 40,000 and 38,000. Glucose and ATP were by far the best substrates for both trichomonad glucokinases, with Km values as low as 33–35 μM and 75–83 μM, respectively. Substrate saturation curves for these enzymes were all hyperbolic. Tritrichomonas foetus fructokinase required fructose and ATP, with Km values of 200 μM and 81 μM. None of the activities was affected by a number of potential regulatory metabolites, including glucose-6-phosphate. The only exception was AMP which in supraphysiological concentrations had an inhibitory effect on T. foetus fructokinase. In conclusion, the absence of regulation at the hexose phosphorylation step described here, as well as the presence of an easily reversible PPi: fructose-6-phosphate 1 -phosphotransferase described previously (Mertens, E., Van Schaftingen, E. & Müller, M. 1989. Mol. Biochem. Parasitol. , 37 :183–190), suggest that the rate of the 1st part of glycolysis in trichomonads is controlled only by the intracellular availability of hexoses.     

Biochemical Cytology of Trichomonad Flagellates. II. Subcellular Distribution of Oxidoreductases and Hydrolases in Monocercomonas sp.*

A primitive trichomonad, Monocercomonas sp. (strain NS-1:PRR) from Natrix sipedon, was grown axenically in Diamond's medium. Activity of NADH oxidase, malate dehydrogenase, malate dehydrogenase (decarboxylating) and of the anaerobic enzymes, pyruvate synthase and hydrogenase as well as of several hydrolases was demonstrated in homogenates. The subcellular distribution of these activities was studied by means of analytical differential and isopycnic centrifugation of homogenates prepared in 0.25 M sucrose. NADH oxidase and malate dehydrogenase are in the nonsedimentable part of the cytoplasm. Malate dehydrogenase (decarboxylating), pyruvate synthase, and hydrogenase are associated with a large particle which equilibrates at density 1.22. In its properties, this particle corresponds to the microbody-like hydrogenosomes of Tritrichomonas foetus. The 5 hydrolases studied are associated with at least 2 different particle populations: a large particle population equilibrating at densities from 1.10 to 1.16 is the exclusive location of 3 enzymes (β-galactosidase, protease and β-N-acetylglucosaminidase), 2 of which have a pH optimum close to neutrality. These particles contain part of the acid phosphatase and β-glucuronidase. Another part of these 2 enzymes is associated with a separate population of smaller granules with equilibrium densities of 1.16 to 1.18. The 2 types of hydrolase-carrying particles are also biochemically very similar to their counterparts in T. foetus.     

Detection and cultivation of intestinal trichomonads of squirrel monkeys (Saimiri sciureus)

Routine examinations of fecal samples from squirrel monkeys suggested that intestinal trichomonads might be common inhabitants of these animals. In pursuit of these observations, microscopic examination of fecal suspensions and cultures have demonstrated a 100% incidence of trichomonads in 30 arbitrarily selected animals from a colony of more than 300 housed in groups of ten. The most prominent species was Pentatrichomonas hominis. A not yet fully characterized tritrichomonad was also found on several occasions. The main obstacle in establishing individual strains in culture was the presence of bacterial and fungal flora in the samples. Nevertheless, abundant cultures were obtained from 28 animals by inoculation of fecal suspensions into tissue cultures with appropriately formulated medium and high concentration of antibiotics. In several unattended cultures maintained at room temperature, the flagellates retained motility for at least 4 months. This long survival may explain the widespread occurrence of the parasites within a confined animal community.     

Critical Analysis of Eukaryotic Phylogeny: A Case Study Based on the HSP70 Family

Trichomonads, together with diplomonads and microsporidia, emerge at the base of the eukaryotic tree, on the basis of the small subunit rRNA phylogeny. However, phylogenies based on protein sequences such as tubulin are markedly different with these protists emerging much later. We have investigated 70 kDa heat-shock protein (HSP70), which could be a reliable phylogenetic marker. In eukaryotes, HSP70s are found in cytosol, endoplasmic reticulum, and organelles (mitochondria and chloroplasts). In Trichomonas vaginalis we identified nine different HSP70-encoding genes and sequenced three nearly complete cDNAs corresponding to cytosolic, endoplasmic reticulum, and mitochondrial-type HSP70. Phylogenies of eukaryotes were reconstructed using the classical methods while varying the number of species and characters considered. Almost all the undoubtedly monophyletic groups, defined by ultrastructural characters, were recovered. However, due to the long branch attraction phenomenon, the evolutionary rates were the main factor determining the position of species, even with the use of a close outgroup, which is an important advantage of HSP70 with respect to many other markers. Numerous variable sites are peculiar to Trichomonas and probably generated the artefactual placement of this species at the base of the eukaryotes or as the sister group of fast-evolving species. The inter-phyla relationships were not well supported and were sensitive to the reconstruction method, the number of species; and the quantity of information used. This lack of resolution could be explained by the very rapid diversification of eukaryotes, likely after the mitochondrial endosymbiosis.     

Organization and composition of the striated roots supporting the Golgi apparatus,the so-called parabasal apparatus,in parabasalid flagellates

Summary— In parabasalid flagellates, trichomonads and hypermastigids, the stack of cisternae of the Golgi apparatus are supported by striated roots attached to the basal bodies of flagella forming the so-called parabasal apparatus. Monoclonal antibodies raised for several trichomonad species, Monocercomonas, Trichomonas and Tetratrichomonas, label the parabasal fibre in immunofluorescence or immunogold staining and protein bands in immunoblotting. Several antibodies cross-react between trichomonad species, and one of them labels the homologous parabasal fibre in the hypermastigids: Trichonympha, Joenia, Pseudotrichonympha and Holomastigotoides. Considering the molecular mass range of the labelled proteins (100–135 kDa) and the lack of antibody cross-reactivity with the striated root proteins (centrin, assemblin, kinetodesmal protein, ciliary root proteins of epithelial ciliated cells) of other organisms, these proteins recognized by these antibodies seem to represent a new class of protein forming striated roots. The occurrence and significance of parabasal organization in eukaryogenesis is discussed.     

THE FLAGELLAR APPARATUS OF CHILOMONAS PARAMECIUM (CRYPTOPHYCEAE) AND ITS COMPARISON WITH CERTAIN ZOOFLAGELLATES1

The major components of the internal flagellar apparatus of Chilomonas paramecium Ehr. are two large microtubular roots and a striated root paralleled by three microtubules. The two microtubular roots overlap at the basal bodies. One microtubular root follows a curved path in the anterior of the cell, and the other extends straight to the posterior passing through a groove in the nucleus. The striated root extends laterally from the basal bodies. Except that it is smaller, the posteriorly directed root bears a strong resemblance to the axostyle of oxymonads. The overall arrangement and structure of the flagellar roots is similar to the pelta, axostyle and costa of trichomonads and the pelta and axostyle of oxymonads, groups of mitochondrion-less, largely parasitic or symbiotic protozoans. An affinity between cryptomonads and oxymonads or trichomonads would have many phylogenetic implications, some of which are discussed.     

Cryopreservation and Storage of Parasitic Protozoa in Liquid Nitrogen

ABSTRACT. In 1961 I reported the first successful attempt to cryopreserve and store protozoa in liquid nitrogen. In this presentation, drawing on more than 30 years of personal experience, I discuss the factors which I consider to be most critical to the successful preservation and long term storage of several species of parasitic protozoa in liquid nitrogen. I then present my most successful protocol for cryopreservation of these parasites. Finally. I cite some longevity records for a variety of protozoa stored in liquid nitrogen vapor at the Laboratory of Parasitic Diseases. The oldest record is for a monoxenic culture of Entamoeha histolytica grown in association with Crithidia fasciculata that was frozen on January 6, 1961. Thirty-two years and 11 months later both parasites were recovered and used to initiate new culture lines.     

Evolution and Diversity of Amitochondrial Zooflagellates

ABSTRACT. The amitochondrial flagellates comprise the Archamoebae, the Metamonada (orders: Retortamonadida, Diplomonadida, Oxymonadida) and the Parabasala. Molecular rDNA sequence comparison has shown that the diplomonads are very ancient and placed at the base of the tree, but the position of the parabasalids before or after the Euglenozoa and other mitochondriate protists is not definitively determined and such molecular data are required to place the other groups. Common cytological features such as one basal body and a basal body-nucleus connector show that the mastigamoebids or Archamoebae are an evolutionary lineage. The metamonad grouping is heterogenous; the three orders have in common two pairs of basal bodies, no Golgi and could be poly- or paraphyletic. The Parabasala is an evolutionary lineage with a large set of homologous cytological characters. A biochemical study of the cytoskeletal structures such as costa, parabasal fibre, preaxostylar fibre and undulating membrane in trichomonads reveals a large diversity in the protein composition among the genera that fits with the large distance between the taxa estimated by rDNA sequencing. Molecular comparison in seven tnchomonad genera indicates that the Trichomonadinae and Tritrichomonadinae form two divergent branches, the genera Hypotrichomonas and Monocercomonas are now incorporated in the tritrichomonad branch, and Trichomitus is at the bifurcation of the two branches. This scheme is different from the classic tree but not incompatible with it.