bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effects of Supraoptimal Temperatures on Population Growth and Cortical Patterning in Tetrahymena pyriformis and Tetrahymena thermophila: A Comparison
Authors: FRANKEL JOSEPH , NELSEN E. MARLO
Published: July 2005

ABSTRACT. In this investigation, we compare the multiplication rates and morphogenetic responses of the two most studied Tetrahymena species, T. pyriformis and T. thermophila, at supraoptimal temperatures. Although the upper temperature limits differ greatly in the two species, the pattern of growth responses to high temperature is for the most part similar, with some differences in detail. The transient recovery of cell division at the highest temperature that allows cell division, characteristic of T. pyriformis, is observed in a less distinct form in T. thermophila. Moreover, there is a remarkable difference in developmental response, with drastic abnormalities in patterning of oral structures during the transient recovery of cell division in T. pyriformis, and far more limited abnormalities under similar conditions in T. thermophila. The abnormalities result from spatial disorder in the alignment and orientation of basal body pairs within the early oral primordium, followed by failures in the realignment that normally occurs as oral structures (membranelles and undulating membrane) mature. Both the initial spatial disorder and the failures in realignment are far more severe in T. pyriformis than in T. thermophila.
Diversity and Phylogeny of Insect Trypanosomatids Based on Small Subunit rRNA Genes: Polyphyly of Leptomonas and Blastocrithidia
Authors: MERZLYAK EKATERINA , YURCHENKO VYACHESLAV , KOLESNIKOV ALEXANDER A. , ALEXANDROV KIRILL , PODLIPAEV SERGEI A. , MASLOV DMITRI A.
Published: July 2005

ABSTRACT. With the aim of further investigating phylogenetic relationships in insect trypanosomatids, we have determined the sequences of small subunit rRNA genes from ten isolates, which were originally classified as Leptomonas, Blaslocrithidia, and Wallaceina based on their morphology in the hosts. The inferred maximum likelihood, parsimony, and distance trees indicate that the Leptomonas and Blastocrithidia are polyphyletic, and confirm the polyphyly of Herpetomonas and Crithidia. Blaslocrithidia triatoma and Leptomonas collosoma were among the earliest branching lineages among the insect trypanosomatids, while most other isolates were found within a closely related terminal clade, which also included Crithidia fasciculata. This analysis has clearly demonstrated that the morphological classification system of insect trypanosomatids does not always reflect their genetic affinities warranting its revision in the future.
Experimental Pathogenicity of a Presumed Monoxenous Trypanosomatid Isolated from Humans in a Murine Model
Authors: GARIN YVES J. F. , SULAHIAN ANNIE , MÉNECEUR PASCALE , PRATLONG FRANCINE , PRINA ERIC , GANGNEUX JEAN‐PIERRE , DEDET JEAN‐PIERRE , DEROUIN FRANCIS
Published: July 2005

ABSTRACT. Two strains of a presumed lower trypanosomatid isolated from immunocompetent and HIV‐infected humans in French West Indies were investigated in vitro and in vivo in a murine experimental model. The ability of parasites to grow in vitro in bone marrow‐derived macrophages and their virulence in vivo were assessed. For in vivo infection, two groups of BALB/c mice were inoculated either by the subcutaneous or intravenous route with 107 promastigotes at day 0. Infection was monitored by measuring parasite load in liver, spleen, foot pad, popliteal, and mesenteric lymph nodes and brain from day 7 to day 150 post‐infection using a microtitration technique. Parasites multiplied in mouse macrophages in vitro. In vivo, both strains proved infective to mice and capable of visceralization and dissemination in the popliteal and mesenteric lymph nodes, liver, spleen, and even brain. Both strains elicited a strong humoral response against trypanosomatid antigen in mice, which cross‐reacted with Leishmania antigen. Contrasting with the straightforward dissemination of parasites, the infection was strikingly well tolerated by the murine host with no clinical signs and minimal tissue changes around parasitized macrophage infiltrates.
Enzymatic Characterization of Phospholipase D of Protozoan Tetrahymena Cells
Authors: WANG SHULIN , BANNO YOSHIKO , NAKASHIMA SHIGERU , NOZAWA YOSHINORI
Published: July 2005

ABSTRACT. Phospholipase D (PLD), which is present in plant, bacterial, and mammalian cells, has been proposed to be involved in a number of cellular processes including transmembrane signaling and membrane deterioration. We demonstrated the existence of evolutionally related PLD activity in the unicellular eukaryotic protozoan Tetrahymena. The partial characterization of this enzyme showed that PLD in Tetrahymena cells was a neutral phospholipase, which catalyzed both transphosphatidylation and hydrolysis reactions. The activity was markedly stimulated by phosphatidylinositol 4, 5‐bisphosphate (PIP2) but was insensitive to phorbol 12‐myristate 13‐acetate (PMA) and guanosine 5′–3‐O‐(thio)triphosphate (GTPγS), suggesting that it is a PIPrdependent PLD and that protein kinase C (PKC) and GTP‐binding proteins are not implicated in the regulation of this enzyme. For its maximal activity Ca2+ was not required. This enzyme was also capable of hydrolyzing phosphatidylcholine (PC) but not phosphatidylethanolamine (PE), implying that PC was a preferred substrate. Subcellular fractionation showed that PLD‐like activity localized mainly to the membrane fraction, especially microsomes. As an initial step to explore the functions of PLD in Tetrahymena, the PLD‐like activity was determined during the different culture phases, and it was found to be significantly and transiently elevated in the early logarithmic phase, indicating its possible role in the development of Tetrahymena.
Reconstruction of the Flagellar Apparatus in Ploeotia costata (Euglenozoa) and its Relationship to Other Euglenoid Flagellar Apparatuses
Authors: LINTON ERIC W. , TRIEMER RICHARD E.
Published: July 2005

ABSTRACT. The flagellar apparatus of Ploeotia costata Farmer and Triemer was reconstructed using serial sectioning and TEM. The flagellar apparatus is similar to other euglenoids having two flagella arising from basal bodies connected by a striated fiber, and three asymmetrically arranged roots. The flagella emerge subapically from between the two ventral pellicle strips. The dorsal flagellum is 1/2 the body length and actively pulls the cell, while the ventral flagellum is twice the body length and drags along the substrate surface. The ventral and dorsal roots are on the opposite sides of their respective basal bodies, while the intermediate root is associated with the ventral flagellum on the side closest to the dorsal basal body. The dorsal root lines the dorsal side of the reservoir and after giving rise to the dorsal band lines the right side of the reservoir/canal. The ventral and intermediate roots join at the reservoir forming the intermediate‐ventral root, which lines the left and ventral sides of the reservoir/canal. There was no evidence of a microtubule‐rcinforced pocket in P. costata. Comparisons with Ploeotia vitrea, Lentomonas applanatum, and related flagellar apparatuses led to the conclusion that the basic euglenoid flagellar structure is symplesiomorphic but with enough variation to be taxonomically diagnostic.
Relationships of Microsporidian Genera, with Emphasis on the Polysporous Genera, Revealed by Sequences of the Largest Subunit of RNA Polymerase II (RPB1)
Authors: CHENEY SARAH A. , LAFRANCHI‐TRISTEM NATHALIE J. , BOURGES DOROTHEE , CANNING ELIZABETH U.
Published: July 2005

ABSTRACT. Molecular data have proved useful as an alternative to morphological data in showing the relationships of genera within the phylum Microsporidia, but until now have been available only for ribosomal genes. In previous studies protein‐coding genes of microsporidia have been used only to assess their position in the evolution of eukaryotes. For the first time we report on the use of a protein‐coding gene, the A‐G region of the largest subunit of RNA polymerase II (RPB1) from 14 mainly polysporous species, to generate an alternative phylogeny for microsporidia. Using the amino acid sequences, the genera and species fell into the same main groupings as had been obtained with 16S rDNA sequences, but the RPB1 data provided better resolution within these groups. The results supported the pairings of Trachipleistophora hominis with Vavraia culicis and Pleistophora hippoglossoideos with Pleistophora typicalis. They also confirmed that the genus Pleistophora is not monophyletic and that it will be necessary to transfer Pleistophora ovariae and Pleistophora mirandellae into one or more other genera, as has already been effected for Pleistophora anguillarum.
Recombinant CD40 Ligand Administration Does Not Decrease Intensity of Pneumocystis carinii Infection in Scid Mice
Authors: VESTERENG VIBEKE H. , KOVACS JOSEPH A.
Published: July 2005

SUMMARY: X‐linked Hyper IgM Syndrome (HIM) is a rare congenital immunodeficiency recently demonstrated to be caused by a mutation in the gene encoding CO40 ligand. These patients are susceptible to Pneumocystis carinii pneumonia, which implies an important role for CD40L in host defense against P. carinii. In this study we undertook to investigate whether treatment of P. carinii infected scid mice with murine recombinant CD40 ligand trimer (muCD40L) for 21 days would facilitate clearance of the organisms. We found no significant difference in organism burden in treated compared to control animals. Therefore in this model treatment with muCD40L alone is ineffective in clearing P. carinii infection.
Development of an RT‐PCR on the Heat Shock Protein 70 Gene for Viability Detection of Pneumocystis carinii f. sp. hominis in Patients with Pneumocystosis and in Air Sample
Authors: LATOUCHE SOPHIE , TOTET ANNE , LACUBE PHILIPPE , BOLOGNINI JOSIANE , NEVEZ GILLES , ROUX ATRICIA
Published: July 2005

SUMMARY. To test the viability of Pneumocystis carinii f. sp. hominis, an RT‐PCR assay that employs specific primers from the Heat Shock Protein 70 gene was developed. Using this method, the viability of P.c. hominis in bronchoalveolar lavage fluids from patients developing PCP and in the environment of PCP patients was established.
Active Transport of L‐Proline in Trypanosoma cruzi
Authors: SILBER ARIEL M. , TONELLI RENATA R. , MARTINELLI MARCELA , COLLI WALTER , ALVES MARIA JúLIA M.
Published: July 2005

ABSTRACT. L‐proline is the main energy source in insect vector stages of most trypanosomatids, including Trypanosoma cruzi epimastigotes. This is the first biochemical description of two active proline transporter systems in T. cruzi. Uptake of this amino acid occurred by a low affinity system B and a high affinity system A. System B consistently appeared more specific than System A when excess competing amino acids were used in transport inhibition assays. Furthermore, the high affinity system is 70% inhibited by L‐tryptophan, but the low affinity system is not. Both systems were found to be insensitive to the intracellular proline concentration and D‐proline did not inhibit L‐proline uptake showing that both systems are stereospecific. Both systems were Na+ and K+ independant but dependant on energy since ATP depletion impairs L‐proline uptake. The combined action of carbonyl cyanide p‐trifluoromethoxyphenyl hydrazone (FCCP) and oligomycin, and the dependence of activity on pH, further differentiated between the two systems leading to the conclusion that the high affinity system is a H+ gradient‐dependant transporter whereas the low affinity system depends directly on ATP.
Phylogenetic Position of Licnophora, Lechriopyla, and Schizocaryum, Three Unusual Ciliates (Phylum Ciliophora) Endosymbiotic in Echinoderms (Phylum Echinodermata)
Authors: LYNN DENIS H. , STRÜDER‐KYPKE MICHAELA
Published: July 2005

ABSTRACT. Various echinoderms are colonized by species from several classes of the Phylum Ciliophora, indicating that the eachinoderm “habitat” has been invaded independently on numerous occasions throughout evolutionary history. Two “echinoderm” cliates whose phylogenetic positions have been problematic are Licnophora macfarlandiStevens, 1901 and Schizocaryum dogieli poljansky and Golikova, 1957. Licnophora macfarlandi is an endosymboint of the repirato;ry trees of holothuroids, and S. dogieli is found in the esophahus of echinoids. A third species, Lechriopyla mystax Lynch, 1930, is plagiopylid ciliate found in the intestine of echino‐ids. Host echinoderms were collected near the Friday Harbor Laboratories, San Juan Island, WA. Specimens of S. dogieli and L. mystax were obtained from the eso[phgue and tintestine, repectively, of the sea urchin Strongylocentrothus pallidus. Specimens of L. Macfarlandi were collected from the fluid obtained from the respiratory trees of Parastichopus califormicus. Using small subunit ribosomal RNA (SSrRNA) sequences of these three ciliates and a global alignement of SSrRNA sequnes of other ciliates, we estabilished the following. 1) Licnophora is a spirotrich ciliate, clearly related to the hypotrichs and stichotrichs; this is corrovorated by its possession of macronuclear replication bands. 2) Lechriopyla is the sister genus to Plagiopyla and is a number of the Class Plagiopylea, which was predicted based on its cytology. 3) Schizocaryum clusters in the Class Oligohymenophorea and is most closely related to the scuticociliates; there are currently no morphologica features known to relate Schizocaryum to the scuticociliates.
Identification and Phylogenetic Analysis of Morphologically Similar Naked Amoebae Using Small Subunit Ribosomal RNA
Authors: SIMS GARY P. , AITKEN ROBERT , ROGERSON ANDREW
Published: July 2005

ABSTRACT
Fan‐shaped, naked amoebae are commonly encountered in samples from freshwater and marine habitats suggesting that they are an important component of the microbial food web. However, there are considerable problems in both detecting these amoebae and identifying them, given their morphological similarity. In this study we used restriction analysis and partial sequence analysis of the small‐subunit 18S ribosomal RNA gene to examine the phylogenetic relationships between nine “fan‐shaped”Vannella and Platyamoeba species. The molecular phylogeny showed that the marine Vannella and Platyamoeba isolates are closely related, whereas the freshwater isolates are disparate. Thus, the current reliance on the fine structure of the cell coat (glycocalyx) used to separate these genera is not justified. The study also highlights sequence elements that might be targeted by fluorescent probes for the direct detection of these amoebae in field samples. The molecular data were also used to aid the identification of three unknown fan‐shaped isolates. All three unknowns resembled Vannella or Platyamoeba. However, one of the strains (a small < 10 μm, benthic, fan‐shaped amoeba) probably represents a new genus.
Myxobolus desaequalis n. sp. (Myxozoa, Myxosporea), Parasite of the Amazonian Freshwater Fish, Apteronotus albifrons (Teleostei, Apteronotidae)
Authors: AZEVEDO CARLOS , CORRAL LAURA , MATOS EDILSON
Published: July 2005

ABSTRACT. Myxobolus desaequalis n. sp. is described from the gill lamellae of the freshwater fish Apteronotus albifrons, collected in the Amazon River, near the city of Salvaterra, Brazil. Large spherical plasmodia filled with disporic pansporoblasts and spores were observed. Ellipsoidal to pyriform spores are 18.3 μm length × 11.2 μm width × 4.4 μm thickness. The anterior end of the spores contain two extremely unequal pyriform polar capsules measuring: (larger): 11.2 μm length, 4.9 μm width, and an isofilar polar filament with 11 to 12 turns obliquely to the longitudinal axis; (smaller): 4.6 μm length, 2.8 μm width, and an isofilar polar filament with 4 to 5 turns, obliquely to the longitudinal axis.
Azadirachtin Disrupts Formation of Organised Microtubule Arrays during Microgametogenesis of Plasmodium berghei
Authors: BILLKER OLIVER , SHAW MICHAEL K. , JONES IAN W. , LEY STEVEN V. , (LUNTZ) A. JENNIFER MORDUE , SINDEN ROBERT E.
Published: July 2005

ABSTRACT. Transmission of malaria parasites from vertebrate blood to the mosquito vector depends critically on the differentiation of the gametocytes into gametes. This occurs in response to environmental stimuli encountered by the parasite in the mosquito bloodmeal. Male gametogenesis involves three rounds of DNA replication and endomitosis, and the assembly de novo of 8 motile axonemes. Azadirachtin, a plant limnoid and insecticide with an unkown mode of action, specifically inhibits the release of motile gametes from activated microgametocytes but does not inhibit growth and replication of asexual blood stages. We have combined confocal laser scanning microscopy and transmission electron microscopy to examine the effect of azadirachtin on the complex reorganisation of the microtubule cytoskeleton during gametogenesis in Plasmodium berghei. Neither the replication of the genome nor the ability of tubulin monomers to assemble into microtubules upon gametocyte activation were prevented by azadirachtin. However, the drug interfered with the formation of mitotic spindles and with the assembly of microtubules into typical axonemes. Our observations suggest that azadarachtin specifically disrupts the patterning of microtubules into more complex structures, such as mitotic spindles and axonemes.
The Vacuolar‐ATPase of Paramecium multimicronucleatum: Gene Structure of the B Subunit and the Dynamics of the V‐ATPase‐rich Osmoregulatory Membranes
Authors: Fok Agnes K. , Yamauchi Kiyoshi , Ishihara Akinori , Aihara Marilynn S. , Ishida Masaki , Allen Richard D.
Published: July 2005

ABSTRACT. Previous studies have shown that the vacuolar‐ATPase (V‐ATPase) of the contractile vacuole complexes (CVCs) in Paramecium multimicronucleatum is necessary for fluid segregation and osmoregulation. In the current study, immunofluorescence showed that the development of a new CVC begins with the formation of a new pore around which the collecting canals form. The decorated membranes are then deposited around the newly formed collecting canals. Quick‐freeze deep‐etch techniques reveal that six 10‐nm‐wide V‐ATPase V, sectors, tightly packed into a 20 × 30‐nm rectangle, form two rows of these compacted sectors that helically wrap around the cytosolic side of decorated membrane tubules. During new CVC formation, packing of decorated tubules around mature CVCs was temporarily disrupted so that some of these decorated tubules became transformed into decorated vesicles. Freeze‐fracturing of these decorated vesicles revealed a highly pitted E‐face and a particulate P‐face. The V‐ATPase was purified for the first time in any ciliated protozoan and shown to contain, as in other cells, the V1subunits A to E, and four 14–20 kDa polypeptides. The B subunit was cloned and found to be encoded by one gene containing four short introns. This subunit has 510 amino acid residues with a predicted molecular weight of 56.8 kDa, a value similar to B subunits of other organisms. Except for the N‐ and C‐termini, it has a 75% sequence identity with other B subunits, suggesting that the B subunits in Paramecium, like other species, have been conserved and that the entire surface of this subunit may be important in interacting with other subunits.
Entamoeba histolytica Schaudinn, 1903 and Entamoeba dispar Brumpt, 1925: Differences in their Cell Surfaces and in the Bacteria‐containing Vacuoles
Authors: PIMENTA PAULO F. P. , DIAMOND LOUIS S. , MIRELMAN DAVID
Published: July 2005

ABSTRACT. Entamoeba histolytica Schaudinn, 1903 and Entamoeba dispar Brumpt, 1925 are two of eight species of Entamoeba that sometimes inhabit the human colon. The former is an invasive organism capable of causing life‐threatening intestinal and extraintestinal disease; the latter appears not to be invasive. Because the two species, when viewed by light microscopy appear morphologically similar, they were long regarded as a single species. However, recent biochemical, immunological, and genetic studies provided convincing evidence that they belong to separate species. Our ultrastructural studies revealed distinct differences in at least two features of the trophozoites. 1) The cell surfaces of the trophozoites of each species differ with regard to structures exposed on the surface, and the distribution and arrangement of intra‐membranous proteins. 2) The phagocytosis of bacteria differs in respect to the formation of the phagocytic vacuoles. Loose vacuoles containing several bacteria were seen in E. histolytica whereas tight vacuoles containing a single bacterium were observed in E. dispar. Furthermore, bacteria were found only within vacuoles in E. histolytica; in E. dispar, bacteria were found within vacuoles and some were found free in the cytoplasm.
Morphological, Small Subunit rRNA, and Physiological Characterization of Trimyema minutum (Kahl, 1931), an Anaerobic Ciliate from Submarine Hydrothermal Vents Growing from 28°C to 52°C
Authors: BAUMGARTNER MANUELA , STETTER KARL O. , FOISSNER WLHELM
Published: July 2005

ABSTRACT. A thermophilic strain of Trimyema minutum was isolated from the hydrothermally heated sea floor at Vulcano Island (Italy) and cultivated monoxenically on Marinobaaer sp. and Methanococcus thermolithotrophicus. It can be propagated strictly an aerobically and is sensitive to oxygen: if exposed to air at 48°C all cells die within 60 min. It grows from 0.45–7.2% (w/v) salt and at pH 6.0–8.0. The isolate is the most extreme thermophilic ciliate which ever has been cultivated, exhibiting an optimal growth temperature of 48°C (doubling time 6 h). Growth occurs between 28°C and 52°C. Trimyema minutum is redescribed using live observation and silver impregnation. Its morphology and the small subunit ribosomal RNA sequence is distinctly different from that of T. compressum, but morphology is highly similar to that of T. shoalsiaNerad et al. 1995, which is thus probably a junior synonym of T. minutum. To stabilize the bewildering species taxonomy in Trimyema. we suggest to recognize our population as a neotype of T. minutum.
“Novel Cytoskeletal Proteins in Protists”: Introductory Remarks1
Authors: FLEURY‐AUBUSSON ANNE
Published: July 2005

ABSTRACT. Protists provide the opportunity to integrate analyses from a low (molecular) to a high (organism) level of complexity within a broad evolutionary framework. The perpectives they offer in the cytoskeletal field are discussed with respect to emerging concepts of cellular biology.
Novel Cytoskeletal Proteins in the Cortex of Tetrahymena1
Authors: HONTS JERRY E. , WILLIAMS NORMAN E.
Published: July 2005

ABSTRACT. An important unsolved problem lies in the mechanisms that determinhd overall size, shape, and the localization of subcellular structures in eukaryotic cells. The membrane skeleton must play a central role in these processes in many cell types, and the ciliate membrane skeleton, or epiplasm, offers favorable opportunities for exploring the molecular determinants of cortical organization. Among the ciliates, Tetrahymena is well suited for the application of a wide range of molecular and cellular approaches. Progress has been made in the identification and sequencing of genes and proteins that encode epiplasmic and cortical proteins. The amino acid sequences of these proteins suggest that they define new classes of cytoskeletal proteins, distinct from the articulin and epiplasmin proteins. We will also discuss recent in vivo and in vitro studies of the regulation of assembly of these cortical proteins. This will include information regarding the down‐regulation of epiplasmic proteins during cleavage, their topographic regulation in the cell cycle, and the results of in vitro assembly and binding studies of the epiplasmic C protein.
Occurrence of Articulins and Epiplasmins in Protists1
Authors: Huttenlauch Irm , Stick Reimer
Published: July 2005

ABSTRACT. The cortex of ciliates, dinoflagellates, and euglenoids comprises a unique structure called the epiplasm, implicated in pattern‐forming processes of the cell cortex and in maintaining cell shape. Articulins, a novel class of cytoskeletal proteins, are major constituents of the epiplasm in the flagellate Euglena gracilis and the ciliate Pseudomicrothorax dubius. The hallmark of articulins is a core domain of repetitive motifs of alternating valine and proline residues, the VPV‐motif. The VPV‐motif repeats are 12 residues long. Positively and negatively charged residues segregate in register with valine and proline positions. The VPV‐motif is unique to articulins. The terminal domains flanking the core are generally hydrophobic and contain a series of hexa‐ or heptapeptide repeats rich in glycine and hydrophobic residues. Using molecular and immunological tools we show that articulins are also present in the dinofla‐gellate Amphidinium carterae and the ciliates Paramecium tetraurelia and Paramecium caudatum, Tetrahymena pyrifarmis, and Euplotes aediculatus. Our analysis further shows that epiplasmins, a group of epiplasmic proteins first characterized in Paramecium, are also present in all these species. Moreover, we present evidence that epiplasmins and articulins represent two distinct classes of cytoskeletal proteins.
Ultrastructure, Eneystment and Cyst Wall Composition of the Resting Cyst of the Peritrich Ciliate Opisthonecta henneguyi
Authors: CALVO PURIFICACIÓN , FERNANDEZ‐ALISEDA M. CARMEN , GARRIDO JOSE , TORRES ANTONIO
Published: July 2005

ABSTRACT. The cyst wall of Opisthonecta henneguyi has been studied ultrastructurally and cytochemically by light and electron microscopy, as well as by chemical and electrophoretic analyses, to examine the structure of the cyst wall and its composition. The cyst wall consists of four morphologically distinct layers. The ectocyst is a thin dense layer. The mesocyst is the thickest layer and is composed of a compact material. The endocyst is a thin layer like the ectocyst, but less dense. The granular layer varies in thickness and is composed of a granular material. In the resting cyst, kinetosomes of both oral apparatus and trochal band as well as the myoneme system are maintained, and only cilia are resorbed. The sugars present in the cyst wall are predominantly N‐acetylglucosamine (90%) and glucose (10%). The niesocyst is composed of chitin, and the endocyst includes glycoproteins and acid mucopolysaccharides. During secretion of the cyst wall, the endocyst and granular layer are secreted from precursors synthesized “de novo”. No cytoplasmic precursors of ectocyst and mesocyst have been detected.
Freeze‐Fracture and Cytoehemical Studies on the in Vitro Cyst Form of Reptilian Blastocystis pythoni
Authors: YOSHIKAWA HISAO , NAGASHIMA MINAKO , MORIMOTO KEIKO , YAMANOUTI YASUKO , YAP EU HIAN , SINGH MULKIT
Published: July 2005

ABSTRACT. After cultured cysts are osmotically shocked by treating with distilled water, there is an exponential increase in the cyst form of Blastocystis pythoni; this was demonstrated by an immunofluorescence antibody assay against the culture organisms. In 11‐day‐old cultures of B. pythoni, 68.8% of the organisms (= 2.2 × 108 cysts/ml) were in the cyst form. Examination of thin sections of cysts revealed many similarities to the cyst forms of Blastocystis obtained from fecal samples in previous investigations. Freeze‐fracture images of the plasma membrane of non‐cyst cells also revealed a similar distribution of the intramembrane particles (IMP) when compared to non‐cysts of B. hominis, while the plasma membrane of the cyst form showed practically no IMP. The size and morphology of particle‐rich small depressions and smooth small protrusions observed on the P face and E face of non‐cyst cells, respectively, were similar to endocytic sites reported for B. hominis. In the present study glycogen was cytochemically demonstrated at the ultrastructural level by an alkaline bismuth staining method in both cyst and non‐cyst cells.
A Multidisciplinary Approach to Describe Protists: Redescriptions of Novistrombidium testaceum Anigstein 1914 and Strombidium inclinatumMontagnes, Taylor, and Lynn 1990 (Ciliophora, Oligotrichia)
Authors: MODEO LETIZIA , PETRONI GIULIO , ROSATI GIOVANNA , MONTAGNES DAVID J. S.
Published: July 2005

ABSTRACT. We combined behavioral, morphological (live, stained, scanning and transmission electron‐microscope), and molecular data to redescribe two common, intertidal oligotrich ciliates, Novistrombidium testaceum and Strombidium inclinatum. Both species were collected from a rocky shore near Leghorn, Ligurian Sea. A literature review revealed four morphotypes of Novistrombidium testaceum that differ in subtle ways, including oral morphology. These differences may be diagnostic, but we do not consider them sufficient to distinguish different taxa. Although other studies have synonymised Strombidium inclinatum and S. sulcatum, based on oral structures, size, and nuclear structure, there are morphological distinctions between them. In particular, the present study supports a lack of anterior protuberance in both live and preserved S. inclinatum, while S. sulcatum possesses a protuberance. The 18S rDNA molecular data, in accordance with morphological and ultrastructural observations, indicate that the Strombidiida (Oligotrichia) constitute a well‐supported clade. The separation of the genera within this clade, even between Novistrombidium and Strombidium, remains unresolved, and the analysis of more species is required. Finally, we recommend that when possible, ecologists, morphological taxon‐omists, and molecular biologists combine their expertise to provide comprehensive taxonomic descriptions.
Analysis of Genes of Mitochondrial Origin in the Genus Entamoeba
Authors: BAKATSELOU CHRISTINA , BESTE DANY , KADRI AYODEJI O. , SOMANATH SUSHELA , CLARK C. GRAHAM
Published: July 2005

ABSTRACT. The amitochondriate protistan parasite Entamoeba histolytica has lost most mitochondria functions secondarily but has retained a reduced organelle of mitochondrial origin, the mitosome. We here investigate the presence, origins, and expression in other species of Entamoeba of three genes of mitochondrial origin—pyridine nucleotide transhydrogenase and the mitochondrial‐type chaperonins cpn60 and hsp70. The genes appear to be present in all species and specifically related, confirming that the E. histolytica mitosomal genes were not acquired recently by lateral transfer from another organism. Detection of expression was not possible in all cases under the culture conditions used, but several genes were induced during recovery from exposure to a heat shock. This includes the transhydrogenase, which to our knowledge has not been shown previously to be a heat‐shock protein.
First Report of Three Kudoa Species from Eastern Australia: Kudoa thyrsites from Mahi mahi (Coryphaena hippurus), Kudoa amamiensis and Kudoa minithyrsites n. sp. from Sweeper (Pempheris ypsilychnus)
Authors: WHIPPS CHRISTOPHER M. , ADLARD ROBERT D. , BRYANT MAL S. , LESTER ROBERT J.G. , FINDLAV VANESSA , KENT MICHAEL L.
Published: July 2005

ABSTRACT. Fish species around the world are parasitized by myxozoans of the genus Kudoa, several of which infect and cause damage of commercial importance. In particular, Kudoa thyrsites and Kudoa amamiensis infect certain cultured fish species causing damage to muscle tissue, making the fish unmarketable. Kudoa thyrsites has a broad host and geographic range infecting over 35 different fish species worldwide, while K. amamiensis has only been reported from a few species in Japanese waters. Through morphological and molecular analyses we have confirmed the presence of both of these parasites in eastern Australian waters. In addition, a novel Kudoa species was identified, having stellate spores, with one polar capsule larger than the other three. The SSU rDNA sequence of this parasite was 1.5% different from K. thyrsites and is an outlier from K. thyrsites representatives in a phylogenetic analysis. Furthermore, the spores of this parasite are distinctly smaller than those of K. thyrsites, and thus it is described as Kudoa minithyrsites n. sp. Although the potential effects of K. minithyrsites n. sp. on its fish hosts are unknown, both K. thyrsites and K. amamiensis are associated with flesh quality problems in some cultured species and may be potential threats to an expanding aquaculture industry in Australia.
Locomotion and Phenotypic Transformation of the Amoeboflagellate Naegleria gruberi at the Water‐Air Interface
Authors: PRESTON TERENCE M. , KING CONRAD A.
Published: July 2005

ABSTRACT. The protozoon Naegleria gruberi is able to carry out amoeboid locomotion at the water—air interface in a manner indistinguishable from that exhibited on solid substrata with the production of focal contacts and associated filopodia. The speed of locomotion at this interface can be modulated by changes in electrolyte concentrations; these speed changes are identical to those observed at a water‐glass interface. The nature of the water—air interface is discussed leading to the hypothesis that surface tension alone could provide suitable properties for the adhesion and translocation of amoebae at this interface without necessitating specific, absorbed molecules. The temporary swimming flagellate stage of Naegleria is able to dock at the interface, make stable adhesions to it, and revert to the amoeboid phenotype. Conversely, amoebae resident at the water‐air interface can transform to swimming flagellates and escape into the bulk liquid phase. We report the presence of Naegleria amoebae in the surface microlayers of natural ponds; thus, in freshwater bodies there may be active shuttling of Naegleria amoebae from the benthos to the surface microlayers by means of the non‐feeding, swimming flagellate phenotype. The public health implication of this behaviour in the case of the pathogenic relative, Naegleria fowleri, is discussed.
Feeding by the Heterotrophic Dinoflagellate Oxyrrhis marina on the Red‐Tide Raphidophyte Heterosigma akashiwo: a Potential Biological Method to Control Red Tides Using Mass‐Cultured Grazers
Authors: JEONG HAE JIN , KIM JAE SEONG , YOO YEONG DU , KIM SEONG TAEK , KIM TAE HOON , PARK MYUNG GIL , LEE CHANG HOON , SEONG KYEONG AH , RANG NAM SEON , SHIM JAE HYUNG
Published: July 2005

ABSTRACT. As part of the development of a method to control the outbreak and persistence of red tides using mass‐cultured heterotrophic protist grazers, we measured the growth and ingestion rates of cultured Oxyrrhis marina (a heterotrophic dinoflagellate) on cultured Heterosigma akashiwo (a raphidophyte) in bottles in the laboratory and in mesocosms (ca. 60 liter) in nature, and those of the cultured grazer on natural populations of the red‐tide organism in mesocosms set up in nature. In the bottle incubation, specific growth rates of O. marina increased rapidly with increasing concentration of cultured prey up to ca. 950 ng C ml−1 (equivalent to 9,500 cells ml−1), but were saturated at higher concentrations. Maximum specific growth rate (μmax), KGR (prey concentration sustaining 0.5 μmax) and threshold prey concentration of O. marina on H. akashiwo were 1.43 d−1, 104 ng C ml−1, and 8.0 ng C ml−1, respectively. Maximum ingestion and clearance rates of O. marina were 1.27 ng C grazer−1 d−1 and 0.3 μ1 grazer−1 h−1, respectively. Cultured O. marina grew well effectively reducing cultured and natural populations of H. akashiwo down to a very low concentration within 3 d in the mesocosms. The growth and ingestion rates of cultured O. marina on natural populations of H. akashiwo in the mesocosms were 39% and 40%, respectively, of those calculated based on the results from the bottle incubation in the laboratory, while growth and ingestion rates of cultured O. marina on cultured H. akashiwo in the mesocosms were 55% and 36%, respectively. Calculated grazing impact by O. marina on natural populations of H. akashiwo suggests that O. marina cultured on a large scale could be used for controlling red tides by H. akashiwo near aquaculture farms that are located in small ponds, lagoons, semi‐enclosed bays, and large land‐aqua tanks to which fresh seawater should be frequently supplied.
Glycogen Phosphorylase Sequences from the Amitochondriate Protists, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica and Giardia intestinalis1
Authors: WU GANG , MÜLLER MIKLÖS
Published: July 2005

ABSTRACT. Glycogen phosphorylase genes or messages from four amitochondriate eukaryotes, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica (two genes) and Giardia intestinalis, have been isolated and sequenced. The sequences of the amitochondriate protist enzymes appear to share a most recent common ancestor. The clade containing these sequences is closest to that of another protist, the slime mold (Dictyostelium discoideum), and is more closely related to fungal and plant phosphorylases than to mammalian and eubacterial homologs. Structure‐based amino acid alignment shows conservation of the residues and domains involved in catalysis and allosteric regulation by glucose 6‐phosphate but high divergence at domains involved in phosphorylation‐dependent regulation and AMP binding in fungi and animals. Protist phosphorylases, as their prokaryotic and plant counterparts, are probably not regulated by phosphorylation.
Isolation of a ThermotolerantParavahlkampfiasp. from Lizard Intestine: Biology and Molecular Identification
Authors: SCHUSTER FREDERICK L. , DE JONCKHEERE JOHAN F. , MOURA HERCULES , SRIRAM RAMA , GARNER MICHAEL M. , VISVESVARA GOVINDA S.
Published: July 2005

ABSTRACT.An amoeba was isolated from the intestines of several moribund pink‐tongued skinks (lizards),Hemisphaeriodon ger‐rardiUnusual features of this isolate were its ability to grow at temperatures of ≥ 37 °C, and its inability to useEscherichia colias a food source or to grow axenically on a variety of enriched culture media suitable for other soil amoeba isolates. Growth was abundant, however, on tissue culture cells, with amoebae clearing cell monolayers in ∼48 h at 37 °C. Trophozoites had a vahlkampfiid‐like morphology, moving by means of an anterior eruptive pseudopod. Cysts, round to slightly ovoid and lacking exit pores, were formed in culture. Tests for enflagellation of trophic amoebae were negative. Indirect immunofluorescence staining was negative forNaegleria fowleriandWillaertiasp. The isolate was sensitive to azithromycin, but not to amphotericin B, pentamidine isethionate, fluconazole, 5‐fluorocytosine, and sulfadiazine. Phylogenetic analysis based on the PCR‐amplified small subunit ribosomal DNA, identified the organism asParavahlkampfia ustiana, an amoeba not previously isolated from either poikilothermic or homeothermic hosts. No evidence of pathology was seen in stained sections of lizard intestine, suggesting that the ameba was part of the normal fauna of the lizard gut. Its diet in the lizard intestine is unknown and the organism may have unusual growth requirements. Thus,P. ustianajoins other soil amoebae that have been isolated from mammals, amphibia, fish, and reptiles, which have the potential of becoming opportunistic pathogens.
Extrusomes in Ciliates: Diversification, Distribution, and Phylogenetic Implications
Authors: ROSATI GIOVANNA , MODEO LETIZIA
Published: July 2005

ABSTRACT. Exocytosis is, in all likelihood, an important communication method among microbes. Ciliates are highly differentiated and specialized micro‐organisms for which versatile and/or sophisticated exocytotic organelles may represent important adaptive tools. Thus, in ciliates, we find a broad range of different extrusomes, i.e ejectable membrane‐bound organelles. Structurally simple extrusomes, like mucocysts and cortical granules, are widespread in different taxa within the phylum. They play the roles in each case required for the ecological needs of the organisms. Then, we find a number of more elaborate extrusomes, whose distribution within the phylum is more limited, and in some way related to phylogenetic affinities. Herein we provide a survey of literature and our data on selected extrusomes in ciliates. Their morphology, distribution, and possible function are discussed. The possible phylogenetic implications of their diversity are considered.
Molecular Characterization of Nuclear Small Subunit (ISS)‐rDNA Pseudogenes in a Symbiotic Dinoflagellate (Symbiodinium, Dinophyta)
Authors: SANTOS SCOTT R. , KINZIE ROBERT A. , SAKAI KAZUHIKO , COFFROTH MARY ALICE
Published: July 2005

ABSTRACT. For the dinoflagellates, an important group of single‐cell protists, some nuclear rDNA phylogenetic studies have reported the discovery of rDNA pseudogenes. However, it is unknown if these aberrant molecules are confined to free‐living taxa or occur in other members of the group. We have cultured a strain of symbiotic dinoflagellate, belonging to the genus Symbiodinium, which produces three distinct amplicons following PCR for nuclear small subunit (18S) rDNA genes. These amplicons contribute to a unique restriction fragment length polymorphism pattern diagnostic for this particular strain. Sequence analyses revealed that the largest amplicon was the expected region of 18S‐rDNA, while the two smaller amplicons are Symbiodinium nuclear 18S‐rDNA genes that contain single long tracts of nucleotide deletions. Reverse transcription (RT)‐PCR experiments did not detect RNA transcripts of these latter genes, suggesting that these molecules represent the first report of nuclear 18S‐rDNA pseudogenes from the genome of Symbiodinium. As in the free‐living dinoflagellates, nuclear rDNA pseudogenes are effective indicators of unique Symbiodinium strains. Furthermore, the evolutionary pattern of dinoflagellate nuclear rDNA pseudogenes appears to be unique among organisms studied to date, and future studies of these unusual molecules will provide insight on the cellular biology and genomic evolution of these protists.
Cryptosporidium Species and Genotypes in HIV‐Positive Patients in Lima, Peru
Authors: Cama Vitaliano A. , Bern Caryn , Sulaiman I.M. , Gilman Robert H. , Ticona Eduardo , Vivar Aldo , Kawai Vivian , Vargas Daniel , Zhou Ling , Xiao Lihua
Published: July 2005

ABSTRACT: Cryptosporidium parasites from a cross‐sectional study conducted in two national hospitals in Lima, Peru were genetically characterized to deteimine the diversity of Cryptosporidium spp. in HIV‐positive people. A total of 2,672 patients participated in this study and provided 13,937 specimens. Cryptosporidium oocysts were detected by microscopy in 354 (13.3%) of the patients. Analysis of 951 Cryptosporidium‐ positive specimens from 300 patients using a small subunit rRNA‐based PCR‐RFLP tool identified 6 genotypes; Cryptosporidium hominis was the species most frequently detected (67.5%), followed by C. meleagridis (12.6%) and C. parvum (11.3%). Cryptosporidium canis (4.0%), C. felis (3.3%), and Cryptosporidium pig genotype (0.5%) were also found. These findings indicate that C. hominis is the predominant species in Peruvian HIV‐positive persons, and that zoonotic Cryptosporidium spp. account for about 30% of cryptosporidiosis in these patients.
Characterization of a Cryptosporidium parvum Gene Encoding a Protein with Homology to Long Chain Fatty Acid Synthetase
Authors: Camero Leonardo , Shulaw William P. , Xiao Lihua
Published: July 2005

ABSTRACT: We describe here the cloning, sequencing, and characterization of a novel Cryptosporidium parvum gene, encoding a protein with significant homology to the long‐chain fatty acyl‐CoA synthetase (LCFA, EC 6.2.13). The gene has an open reading frame of 2,301 bp, coding for a 766 amino acid polypeptide, and with an estimated MW of 86.1 kDa. By indirect immunofluorescence assay, monoclonal antibodies C3CE7 and ESD labeled the anterior pole of fixed C. parvum sporozoites and developmental stages in C. parvum‐infected cultures at 24, 48, and 72 h post‐infection. These monoclonal antibodies inhibited more than 3.5% of parasite growth in vitro. The effect of triacsin C, a potent selective inhibitor of LCFA synthetase, on parasite growth was assessed in cell culture; complete inhibition of parasite growth at 2.5 ug/inl was obtained with little evidence of drug‐associated cytotoxicity. These results suggest that the fatty acyl‐CoA synthetase may be a useful target in the development of selective inhibitors and immunologic interventions against C. parvum
Molecular Epidemiology of Cryptosporidiosis in Children in Malawi
Authors: PENG MICHAEL M. , MESHNICK STEVE R. , CUNLIFFE NIGEL A. , THINDWA BENSON D.M. , HART C. ANTHONY , BROADHEAD ROBIN L. , XIAO LIHUA
Published: July 2005

ABSTRACT: Few studies have examined the molecular epidemiology of cryptosporidiosis in developing countries. In this study, DNA of 69 microscopy‐positive human fecal samples collected from Malawi were examined by multilocus genetic analyses. From 43, 27 and 28 of the samples, the SSU rRNA, 70 kDa heat shock protein (HSP70) and 60 kDa glycoprotein (GP60) genes, respectively, were successfully PCR‐amplified. Restriction analysis of the SSU PCR products showed that 41 of the 43 PCR‐positive samples had C. hominis and 2 had C. parvum. Sequence analysis of the HSP70 and GP60 gene contirmed the species identification by SSU rRNA PCR‐RFLP analysis, but also revealed high intraspecific variations. Altogether, six HSP70 subtypes and six GP60 subtypes (belonging to lour subtype alleles) of C. hominis were found. Linkage diseyuilibrum analysis of the two genetic loci showed possible intraspecitic recombination. Thus, cryptosporidiosis in the study area was largely caused by anthroponotic transmission. The high intraspecitic variation and existence of genetic recombination were probably results of high transmission of cryptosporidiosis in this area.
Loading of Peptide on to MHC Molecule is Not Essential for Clearance of Cryptosporidium pawum
Authors: PONNURAJ ESTHER M. , SANAPAREDDY NINA , HAYWARD ANTHONY R.
Published: July 2005

ABSTRACT: Transgenic and knockout mice usefully model the mechanisms that result in the clearance of Cryptosporidium parvum from the gut. CD4+ cells, cells expressing MHC class II, and CD154/CD40 interactions are essential. Unexpectedly, AND RAG‐/‐ and DO11.10 RAG‐/‐ mice with single specificities of T cells successfully clear Cryptosporidium infection. Clearance is accompanied by activation of CD4+ cells in the MLN. The ability of T cells bearing receptors for apparently irrelevant and non‐cross reactive antigens to activate and to clear infection is surprising. The requirement for class II MHC expression for Cryptosporidium clearance raises the alternative possibilities that (a) class II MHC is required to present a peptide that is loaded as a consequence of infection or (b) that the cytokine environment engendered by a Cryptosporidium infection allows affinity for self MHC to activate nahe T cells. In order to test the hypothesis that peptide loading is necessary, we used ApEα‐/‐Ii‐/‐ mice that express a hybrid IA‐IE MHC molecule. They also carry a transgene that makes an Ea peptide while disruption of their invariant chain blocks the loading of a foreign peptide on to their MHC class II molecules. After oral gavage, the course of infection was followed by ELISA. CD4+ cells in the MLN of these mice were activated to express CD69 and the infection was cleared. We conclude that the loading of a Cryptosporidium or other infection‐dependent peptide onto the MHC class II molecules of APCs is not necessary for clearance of Cryptosporidium. Instead the TcR affinity for self‐MHC must suffice for T cell activation in the cytokine environment resulting from infection