bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrastructural Responses in Field‐Bleached and Experimentally Stressed Amphistegina gibbosa (Class Foraminifera)
Authors: TALGE HELEN K. , HALLOCK PAMELA
Published: July 2005

ABSTRACT. Amphistegina are the most common foraminifers with algal endosymbionts found on reefs and carbonate shelves worldwide. Like zooxanthellate corals and other reef organisms with algal symbionts, Amphistegina respond to photoxidative stress by bleaching. This paper documents Ultrastructural changes that occur during bleaching under field and laboratory conditions. Nine chambers from the outer whorl of each of 22 normal‐appearing and 11 partly bleached specimens of Amphistegina gibbosa, which were collected from Conch Reef, Florida, USA, were examined using transmission electron microscopy. The condition and numbers of algal symbionts, as well as the cell area occupied by 10 other intracellular structures of the host, were quantified. Normal‐appearing specimens averaged three times more viable symbionts and less than a fourth as many deteriorating symbionts as partly bleached specimens. Foraminifers experimentally exposed to visible light intensities a 13 μmole photon m−2 s−1 for 35 d were statistically similar to partly bleached field specimens in the number and condition of symbionts, and in chamber area occupied by the evaluated host structures. Exposure to 32 °C water temperature at 6–8 μmole photon m−2 s−1 for 28 d induced symbiont loss but did not degrade host endoplasm.
Molecular Phylogeny and Surface Morphology of Colpodella edax (Alveolata): Insights into the Phagotrophic Ancestry of Apicomplexans
Authors: LEANDER BRIAN S. , KUVARDINA OLGA N. , ALESHIN VLADIMIR V. , MYLNIKOV ALEXANDER P. , KEELING PATRICK J.
Published: July 2005

ABSTRACT. The molecular phylogeny of colpodellids provides a framework for inferences about the earliest stages in apicomplexan evolution and the characteristics of the last common ancestor of apicomplexans and dinoflagellates. We extended this research by presenting phylogenetic analyses of small subunit rRNA gene sequences from Colpodella edax and three unidentified eukaryotes published from molecular phylogenetic surveys of anoxic environments. Phylogenetic analyses consistently showed C. edax and the environmental sequences nested within a colpodellid clade, which formed the sister group to (eu)apicomplexans. We also presented surface details of C. edax using scanning electron microscopy in order to supplement previous ultrastructural investigations of this species using transmission electron microscopy and to provide morphological context for interpreting environmental sequences. The microscopical data confirmed a sparse distribution of micropores, an amphiesma consisting of small polygonal alveoli, flagellar hairs on the anterior flagellum, and a rostrum molded by the underlying (open‐sided) conoid. Three flagella were present in some individuals, a peculiar feature also found in the microgametes of some apicomplexans.
Protein Binding to Expanded Telomere Repeats in Tetrahymena thermophila
Authors: MCGUIRE JENNIFER M. , GANA JOYCE ACHE , PETCHERSKAIA MARINA , KIRK KAREN E.
Published: July 2005

ABSTRACT. The ends of eukaryotic chromosomes are protected by DNA‐protein structures called telomeres. Telomeric DNA is highly conserved, usually consisting of long tracts of a repeating G‐rich sequence. Tetrahymena thermophila telomeric DNA consists of alternating blocks of GGGG and TT sequences (i.e. a G4T2 repeat sequence). We examined the relative importance of the guanine and thymine elements of the repeat sequence in promoting in vitro binding by T. thermophila proteins. We identified single‐ and, for the first time, double‐stranded telomere binding activities from a crude T. thermophila protein extract and tested the binding of these activities to altered telomere repeat sequences. All deletions or substitutions made to the guanine element virtually abolished binding, indicating that four G's are essential for recognition by the binding activity. However, G's alone are not sufficient for efficient binding, as elimination of the thymine element dramatically reduced binding. By contrast, substantial expansion of the thymine element was well tolerated, even though one such change, G4T4, is lethal in vivo. We tested up to a four‐fold expansion of the thymine element and found that highly efficient binding was still achieved. These results suggest a minimal recognition sequence for T. thermophila proteins, with the T element providing an important spacer between essential G elements. demonstrate some flexibility in the binding of many telomere proteins, because the variant sequences tested often contain more than one base change, the minimal changes that permit or disrupt binding cannot be deduced.
G‐Protein Modulators Alter the Swimming Behavior and Calcium Influx of Paramecium tetraurelia
Authors: DE ONDARZA JOSÉ , SYMINGTON STEVEN B. , VAN HOUTEN JUDITH L. , CLARK J. MARSHALL
Published: July 2005

ABSTRACT. To assess the potential role of G‐proteins in chemokinesis, Paramecium tetraurelia was pre‐incubated with the G‐protein modulator pertussis toxin. Pertussis toxin pretreatment significantly reduced Paramecium chemoattraction to sodium acetate and ammonium chloride in T‐maze behavioral assays and depressed the frequency of avoidance reactions, indicating that heterotrimeric G‐proteins may be involved with the motility response. To determine whether G‐proteins exert their effect via the ciliary voltage‐sensitive calcium channel, we examined responses of P. tetraurelia to the potent voltage‐sensitive calcium channel agonist, deltamethrin. Pertussis toxin preincubation significantly reduced the toxic effects of deltamethrin exposure as determined by survival under depolarizing conditions and reduced the duration of backward swimming episodes in behavioral bioassays. Furthermore, non‐hydrolyzable analogs of guanine nucleotides altered deltamethrin‐stimulated calcium influx via calcium channels in isolated ciliary vesicles. Heterotrimeric G‐protein subunits were subsequently detected in ciliary vesicles of P. tetraurelia by antibodies produced against Ga and Gβ subunits, and by 32P‐ADP‐ribosylation, indicating that proteins of the appropriate molecular weight are the target of pertussis toxin in these vesicles. These findings provide additional evidence that heterotrimeric G‐proteins are associated with ciliary vesicles and that they play a role in the modulation of swimming behavior and the toxic action of deltamethrin in Paramecium.
Interstitial Ciliates: Benthic Microaerophiles or Planktonic Anaerobes?
Authors: HAYWARD BRETT H. , DROSTEB RITA , EPSTEIN SLAVA S.
Published: July 2005

ABSTRACT. We observed marine benthic interstitial ciliates Geleia sp. and Tracheloraphis sp. inhabiting the water column of a chemically stratified salt pond. This habitat is uncharacteristic for interstitial ciliates, yet they displayed active and abundant planktonic populations (up to 800 and 250 cells/liter, respectively) and a well‐defined pattern of vertical distribution. Completely absent from the oxygenated epilimnion, they first appeared at the oxic/anoxic interface and were present throughout the anoxic hypolimnion. The data could not be explained by a passive removal (e.g. by currents) of these ciliates from their conventional habitat (soft sediments) to water column. The results suggest that 1) these ciliates favored an anoxic environment, and 2) they switched to a planktonic lifestyle as appropriate conditions (seasonal anoxia) developed in the water column. This sharply contrasts the classic view of these ciliates as specifically benthic and aerobic (albeit microaerophilic) organisms. We hypothesize that Geleia sp. and Tracheloraphis sp. can readily grow in either water column or benthos, but are typically found in sediments simply because they contain their preferred (anoxic) niche.
Description of a Marine Peritrichous Ciliate, Pseudovorticella sinensis n. sp. (Ciliophora, Peritrichia) from China
Authors: JI DAODE , SONG WEIBO , AL‐RASHEID KHALED A. S.
Published: July 2005

ABSTRACT. A new marine peritrich ciliate, Pseudovorticella sinensis n. sp. was isolated from a shrimp‐farming pond in the littoral area of Qingdao, China. The morphology, infraciliature, and silverline system were studied based on living and silver‐impregnated specimens. This species is characterized by (1) an elongated bell‐shaped body that measures 50–60 × 35‐45 μm in vivo, (2) one large, ventrally located contractile vacuole, and (3) a pellicle covered by a layer of transparent, cortical vesicles. The number of transverse silverlines from the peristomial area to the aboral ciliary wreath is 26–32, and from the aboral ciliary wreath to the scopula is 12–15. The stalk measures about 160–250 μm long × 5–6 μm wide. The spasmoneme has one row of conspicuous thecoplasmic granules, which are about 0.8 μm in diameter.
Role of Gregarine Parasite Ascogregarina culicis (Apicomplexa: Lecudinidae) in the Maintenance of Chikungunya Virus in Vector Mosquito
Authors: MOURYA D. T. , SINGH D. K. , YADAV P. , GOKHALE M. D. , BARDE P. V. , NARAYAN N. B. , THAKARE J. P. , MISHRA A. C. , SHOUCHE Y. S.
Published: July 2005

ABSTRACT. Ascogregarina culicis and Ascogregarina taiwanensis are common gregarine parasites of Aedes aegypti and Aedes albopictus mosquitoes, respectively. These mosquito species are also known to transmit dengue and Chikungunya viruses. The sporo‐zoites of these parasites invade the midgut epithelial cells and develop intracellularly and extracellularly in the gut to complete their life cycles. The midgut is also the primary site for virus replication in the vector mosquitoes. Therefore, studies were carried out with a view to determine the possible role of these gregarines in the vertical transmission of dengue and Chikungunya viruses from larval to adult stage. Experiments were performed by exposing first instar mosquito larvae to suspensions containing parasite oocysts and viruses. Since Ascogregarina sporozoites invade the midgut of first instar larvae, the vertical transmission was determined by feeding the uninfected first instar larvae on the freshly prepared homogenates from mosquitoes, which were dually infected with viruses and the parasite oocysts. Similarly, the role of protozoan parasites in the vertical transmission of viruses was determined by exposing fresh first instar larvae to the dried pellets of homogenates prepared from the mosquitoes dually infected with viruses and the parasite oocysts. Direct vertical transmission and the vertical transmission of CHIK virus through the oocyst of the parasites were observed in the case of Ae. aegypti mosquitoes. It is suggested that As. culicis may have an important role in the maintenance of CHIK virus during the inter‐epidemic period.
Expression of GFP‐Actin Leads to Failure of Nuclear Elongation and Cytokinesis in Tetrahymena thermophila
Authors: HOSEIN ROLAND E. , WILLIAMS SELWYN A. , HAYE KESTER , GAVIN R. H.
Published: July 2005

ABSTRACT. Green fluorescent protein (GFP)‐tagged actin was used to investigate the distribution and function of actin in Tetrahymena. A strain that expresses both GFP‐actin and endogenous actin was developed by transformation of Tetrahymena thermophila with a ribosomal DNA‐based replicative vector. Confocal microscopy of living cells and immunogold electron microscopy confirmed localization of GFP‐actin to basal bodies and the contractile ring. Incorporation of the fusion protein into these and other actin‐related structures correlated with severe impairment of macronuclear elongation and cytokinesis. At 30 °C macronuclear elongation failed to occur in 25% of the transformants despite completion of micronuclear division. At 20 °C macronuclear elongation failed to occur in 2% of the population. Arrest of cytokinesis coincided with failure of macronuclear elongation. Arrested cells developed into homopolar doublets with two sets of oral structures. This study indicates a requirement for actin in nuclear elongation and cytokinesis. Although GFP‐actin can interfere with the functioning of actin‐containing structures, the GFP‐actin transformant strain can be used to monitor actin distribution and dynamics and is therefore an important new tool for further studies of Tetrahymena actin.
Leptomonas wallacei Shows Distinct Morphology and Surface Carbohydrates Composition Along the Intestinal Tract of Its Host Oncopeltus fasciatus (Hemiptera: Lygaeidae) and in Axenic Culture
Authors: ROMEIRO ALEXANDRE , SARAIVA ELVIRA M. B. , DE SOUZA WANDERLEY , ATTIAS MÁRCIA
Published: July 2005

ABSTRACT. Leptomonas wallacei is a monoxenic trypanosomatid that colonizes the digestive tract of the phytophagous hemipteran Oncopeltus fasciatus. This infection was specific and took place exclusively in midgut intestinal ventricles V3 and V4, and in the hindgut. Abundances of parasites in the hindgut were 54% less than those in the hindgut. Parasites in the hindgut were more slender and had a longer flagellum than those from the hindgut, which were rounded, with a shorter flagellum. Moreover, hindgut forms expressed sugar residues on the cell surface, recognized by the lectins from Griffonia simplicifolia‐I (α‐galactose, α‐N‐acetyl‐galactosamine) and Helix pomatia (N‐acetyl‐galactosamine); those sugar residues were not present in protozoa from the midgut. In culture, parasites were morphologically similar to midgut forms, but differed from them because they did not express sugar residues that bind to lectin (β‐galactose(l‐S) N‐acetyl‐galactosamine) from Arachis hypogaea.
An Improved Description of Strombidium sulcatum Claparéde & Lachmann, 1859 (Ciliophora) from Slides of Fauré‐Fremiet, and a Designation of Type Material
Authors: GRANDA ANGÉLICA P. , MONTAGNES DAVID J. S.
Published: July 2005

ABSTRACT. Strombidium sulcatum, the type species of the genus, is examined using silver‐stained material made by Faure‐Fremiet in the 1950s and 1960s. We found that the stained specimens possessed a prominent protuberance, as previously described; the number of anterior polykinetids (APk) differed between the accounts (15 this study, 12 previous reports); although previously illustrated, no contractile vacuole was observed; and the macronuclear shape, position, and size differed from those previously reported. Our work provides the first detailed re‐analysis of the silver‐stained material of S. sulcatum. We use data on S. sulcatum to distinguish it from a morphologically similar species, Strombidium inclinatum. Type material for S. sulcatum has been deposited in the Natural History Museum, London.
Caspase‐like Activity is Required for Programmed Nuclear Elimination during Conjugation in Tetrahymena
Authors: EJERCITO MYLEE , WOLFE JASON
Published: July 2005

ABSTRACT. During conjugation in the binucleate ciliate, Tetrahymena thermophila, the old macronucleus is eliminated as new macronuclei and micronuclei are ontogenetically derived from the zygote nucleus. The mechanism of programmed nuclear elimination in ciliates may be related to the mechanism of apoptosis in higher organisms since its chromatin undergoes major condensation, its DNA is digested into nucleosome‐sized fragments, and it stains positively for TUNEL. The present study explores whether caspases are involved in programmed macronuclear degradation in Tetrahymena. We show here that caspase‐like activity is detectable using two specific colorimetric substrates, and that the activity is reduced with specific caspase inhibitors. In addition, using the fluorigenic substrate PhiPhiLux, active caspase‐like activity is detected in living cells, localized to cytoplasmic vesicles; activity is not detected in pre‐ or post‐condensed macronuclei. Finally, three different inhibitors of caspase activity cause a block to macronuclear chromatin condensation and elimination. Therefore, a caspase‐like enzyme activity is necessary for regulating macronuclear elimination in Tetrahymena. These data support the possibility that macronuclear elimination is related, evolutionarily, to regulated cell death in multicellular organisms.
Observations on the Life Stages of Sphaerothecum destruens n. g., n. sp., a Mesomycetozoean Fish Pathogen Formally Referred to as the Rosette Agent
Authors: ARKUSH KRISTEN D. , MENDOZA LEONEL , ADKISON MARK A. , HEDRICK RONALD P.
Published: July 2005

ABSTRACT. The rosette agent is an obligate intracellular parasite that causes morbidity and mortality in salmonid fish. In laboratory cultures, the spore stage (2–6 uμm diam.) replicates in a salmonid cell line by sequential asexual division, giving rise to daughter cells. If infected cell cultures are transferred to distilled water, the spore stage undergoes internal division to give rise to at least 5 cells each of which develops into a uniflagellated zoospore with a body of approximately 2 μm and a flagellum approximately 10 μm long. Zoosporulation does not occur in cell culture medium alone, artificial seawater, or phosphate‐buffered saline. This parasite is currently classified as a member of the Class Mesomycetozoea (formerly Ichthyosporea) based on phylogenetic analyses of the small subunit ribosomal DNA of three different isolates from fish. Given these new morphological observations combined with the available molecular phylogenetic data on other mesomycetozoeans, we propose to classify the rosette agent as Sphaerothecum destruens, n. g., n. sp. This new genus has unique features including (1) intracellular development of spore stages in various organs eliciting a host granulomatous response; and (2) the differentiation of mature spores into multiple, flagellated zoospores. Taken together, these characteristics clearly distinguish it from the closely related genera Dermocystidium and Rhinosporidium.
A Marine Dinoflagellate, Amphidinium eilatiensis n. sp., from the Benthos of a Mariculture Sedimentation Pond in Eilat, Israel
Authors: LEE JOHN J. , OLEA RAUL , CEVASCO MEGAN , POCHON XAVIER , CORREIA MARIA , SHPIGEL MUKI , PAWLOWSKI JAN
Published: July 2005

ABSTRACT. A species of Amphidinium bloomed in a mariculture sedimentation pond that was used to grow bivalves near the Gulf of Eilat, Israel. Its overall length averaged 13 μm, the hypocone was 11 μm, and its width was 8μm. It has a ventral ridge. The sulcus begins at the longitudinal flagellar pore and does not project forward in the apex toward the transverse flagellar pore and left margin of the cingulum. The sulcus is a very shallow groove that projects variably about a third of the body length toward the antapex. The cingulum is a deep groove as it circles the cell from the left ventral side to the dorsal side and then becomes very shallow on the right ventral side as it arches posterior toward the longitudinal flagellar pore. Using a modified method for studying dinoflagellate chromosomes in the SEM, we observed 31 chromosomes. The plastid is dorsal and peripheral with 6 ventrally projecting peripheral digital lobes that wrap around the sides of the ventral and posterior nucleus. Amphidinium eilatiensis n. sp. is morphologically closest to Amphidinium carterae and Amphidinium rhynchocephalum, but it does not have the obvious thecal plates or polygonal units described for the former species. Instead, it has a series of spicules, bumps, and ridges on its surface. It differs from A. rhynchocephalum by two morphological characters: surface morphology and gross plastid architecture.

The amplified fragments of the rDNA from A. eilatiensis n. sp. isolated from 2 separate sedimentation ponds in Eilat include the 3′‐end of the SSU rDNA (about 100 nt), the whole ITS region (ITS1 + 5. 8S + ITS2) and the 5′‐end of the LSU rDNA (about 900 nts). The total length of the sequences ranged from 1,460 nt. (A. eilatiensis isolate #1) to 1,461 nts. (A. eilatiensis isolate #2). The latter sequences are identical, the difference in length being due to three insertions. Amphidinium eilatiensis is genetically more closely related to A. carterae than to A. klebsii, with respectively 2. 36% and 6. 93% of sequence divergence.
New Contributions to Two Heterotrichous Ciliates, Folliculina simplex (Dons, 1917), Condylostoma curvaBurkovsky, 1970 and One Licnophorid, Licnophora lyngbycolaFauré‐Fremiet, 1937 (Protozoa, Ciliophora): Descriptions of Morphology and Infraciliature
Authors: SONG WEIBO , WARREN ALAN , JI DAODE , WANG MEI , AL‐RASHEID KHALED A. S.
Published: July 2005

ABSTRACT. The living morphology and infraciliature of two heterotrichous and one licnophorid ciliates from the north China seas, i.e. Folliculina simplex (Dons 1917), Condylostoma curvaBurkovsky, 1970, and Licnophora lyngbycolaFauré‐Fremiet, 1937, were investigated using protargol impregnation and in vivo observations. Updated diagnoses and redescriptions of each species are provided. The improved diagnosis of Folliculina simplex is as follows: blue‐greyish to blue‐greenish Folliculina with broad and unequal‐sized peristomial lobes; adoral zone of membranelles comprises about 400 membranelles and forms two‐and‐half turns within buccal cavity; paroral membrane extends entire length of adoral zone of membranelles; about 50 somatic kineties in mid‐body; single ovoid to ellipsoid macronucleus and several micronuclei; lorica vase‐shaped, about 150‐250 μm long, often with jelly‐like covering and inconspicuous neck; marine habitat. Improved diagnosis for Licnophora lyngbycola: marine Licnophora with cup‐shaped body and glabrous adhesive disc with which the organism attaches to the filaments of the colourless cyanobacterium Lyngbya; in vivo 80–130 μm long; about 140 adoral membranelles, one micronucleus and a single C‐shaped, moniliform macronucleus consisting of about 14 nodules; two specialized dorsal kineties; five peripheral rings on adhesive disc, the 2nd, 3rd, and 5th (outermost) which are incomplete. Based on the Qingdao population, an improved diagnosis for Condylostoma curva is supplied: yellow‐brownish Condylostoma with oval to elongated body shape; about 150–350 μm long in vivo; about 68–108 membranelles in adoral zone of membranelles, 4–8 frontal cirri, 22–32 somatic kineties; macronucleus moniliform with 5–13 nodules; with cortical granules; marine habitat.
Morphological and Phylogenetic Studies of Marine Myxobolus spp. from Mullet in Ichkeul Lake, Tunisia
Authors: BAHRI SIHEM , ANDREE KARL B. , HEDRICK RONALD P.
Published: July 2005

ABSTRACT. Six species of the genus Myxobolus (Myxozoa) from the marine environment were collected from two species of mullet (Mugil cephalus and Liza ramada) in Ichkeul Lake, Tunisia. Four of these species were described previously (Myxobolus bizerti, Myxobolus ichkeulensis, Myxobolus spinacurvatura, and Myxobolus episquamalis) and two (Myxobolus exiguus and Myxobolus muelleri) are redescribed. The small subunit ribosomal (18S rDNA) sequences of these six myxozoans were obtained and compared to traditional criteria used in the identification and taxonomy of myxozoan species (such as spore morphology, host specificity, and tissue tropism). A distance analysis of 1,600–1,700 base pairs of the 18S rDNA of the six species indicates that they formed a monophyletic group separate from Myxobolus spp. found as parasites of freshwater fish. The sequence analyses also confirm that these morphologically different Myxobolus spp. that infect mullet represent different species. Lastly, M. exiguus and M. muelleri, which were found in the same host, exhibit clear differences in spore morphology but sequencing of two different regions of the 18S rDNA show they are closely related. These results demonstrate the utility of DNA sequence data in providing more detailed relationships among the Myxobolus species based upon existing morphological taxonomic approaches. We suggest that future descriptions of Myxobolus spp. provide both careful spore descriptions as part of the traditional criteria but also 18S rDNA sequence data that will aid in situations where morphological details may be absent or misleading.
RpoA: A Useful Gene for Phylogenetic Analysis in Diatoms
Authors: FOX MARTIN G. , SORHANNUS ULF M.
Published: July 2005

ABSTRACT. The aim of this study was to compare the usefulness of two chloroplast‐encoded genes (rpoA and rbcL) and the nuclearencoded small subunit (SSU) ribosomal RNA for reconstructing phylogenetic relationships among diatoms at lower taxonomic levels. To this end, the rpoA and rbcL genes for selected centric and pennate diatoms were sequenced. The new rpoA and rbcL sequences, and an existing nuclear‐encoded SSUrRNA data set, were subjected to weighted/unweighted parsimony, maximum likelihood, minimumevolution, and Bayesian analyses. All of the tree‐building methods employed showed, based on the support values, that the rpoA genewas the most useful, relative to the rbcL and SSUrRNA genes, in determining phylogenetic relationships among the sampled diatoms.The support values for the relationships among the pennate lineages were, in many instances, greater in the rpoA trees than in the SSUrRNA trees. These results suggest that rpoA might be of value in determining phylogenetic relationships among pennate lineages.
Redescription of the Sarcocysts of Sarcocystis rileyi (Apicomplexa: Sarcocystidae)
Authors: DUBEY J. P. , CAWTHORN R. J. , SPEER C. A. , WOBESER G. A.
Published: July 2005

ABSTRACT. The intermediate hosts for Sarcocystis rileyi (Stiles 1893) Minchin 1903 are ducks (Anas spp.), and the striped skunk (Mephitis mephitis) is its definitive host. The structure of sarcocysts from an experimentally infected shoveler duck (Anas cylpeata) fed sporocysts from an experimentally‐infected M. mephitis was studied and compared with type specimens from a naturally infected duck. The experimentally infected duck was killed 154 d after feeding sporocysts. By light microscopy the sarcocyst wall was 3–5 μm thick with indistinct villar protrusions. Ultrastructurally, the sarcocyst wall was a type‐23 cyst wall with anastomosing villar protrusions that were up to 7.5 μm long. The villar projections contained filamentous structures. The bradyzoites were 12–14 μm long. Structurally, the sarcocyst from the naturally infected and experimentally infected ducks appeared similar.
Small Subunit Ribosomal DNA Suggests that the Xenophyophorean Syringammina corbicula1 is a Foraminiferan
Authors: PAWLOWSKI JAN , HOLZMANN MARIA , FAHRNI JOSE , RICHARDSON SUSAN L.
Published: July 2005

ABSTRACT. Xenophyophorea are giant deep‐sea rhizopodial protists of enigmatic origins. Although species were described as Foraminifera or sponges in the early literature, the xenophyophoreans are currently classified either as a class of Rhizopoda or an independent phylum. To establish the phylogenetic position of Xenophyophorea, we analysed the small subunit (SSU) rRNA gene sequence of Syringammina corbicula Richardson, a newly described xenophyophorean species from the Cape Verde Plateau. The SSUrDNA analyses showed that S. corbicula is closely related to Rhizammina algaeformis, a tubular deep‐sea foraminiferan. Both species branch within a group of monothalamous (single‐chambered) Foraminifera, which include also such agglutinated genera as Toxisarcon, Rhabdammina, and Saccammina, and the organic‐walled genera Gloiogullmia and Cylindrogullmia. Our results are congruent with observations of similar cytoplasmic organisation in Rhizammina and Syringammina. Thus, the Xenophyophorea appear to be a highly specialised group of deep‐sea Foraminifera.
Genetic Variations in the Internal Transcribed Spacer and Mitochondrial Small Subunit rRNA Gene of Naegleria spp.
Authors: ZHOU LING , SRIRAM RAMA , VISVESVARA GOVINDA S. , XIAO LIHUA
Published: July 2005

ABSTRACT: Naegleria spp. are widely distributed free‐living amebas, but one species in the genus, N. fowleri, causes acute fulminant primary amebic meningoencephalitis in humans and other animals. Thus, it is important to differentiate N. fowleri from the rest in the genus of Naegleria, and to develop tools for the detection of intra‐specific genetic variations. In this study, one isolate each of N. australiensis, N. gruberi, N. jadini, and N. lovaniensis and 22 isolates of N. fowleri were characterized at the internal transcribed spacers (ITS) and mitochondrial small subunit rRNA (mtSSU rRNA) gene. The mtSSU rRNA primers designed amplified DNA of all isolates, with distinct sequences obtained from all species examined. In contrast, the ITS primers only amplified DNA from N. lovaniensis and N. fowleri, with minor sequence differences between the two. Three genotypes of N. fowleri were found among the isolates analyzed in both the mtSSU rRNA gene and ITS. The extent of sequence variation was greater in the mtSSU rRNA gene, but the ITS had the advantage of length polymorphism. These data should be useful in the development of molecular tools for rapid species differentiation and genotyping of Naegleria spp.
An Evaluation of Molecular Diagnostic Tools for the Detection and Differentiation of Human‐Pathogenic Cryptosporidium spp.
Authors: Jiang Jianlin , Xiao Lihua
Published: July 2005

ABSTRACT: The performance of 10 commonly used genotyping tools in the detection and differentiation of 7 human‐pathogenic Cryptosporidium spp. (C. hominis, C. parvum, C. meleagridis, C. felis, C. canis, C. muris and Cryptosporidium pig genotype I) was evaluated. All 3 SU rRNA gene‐based tools could amplify the DNA of 7 Cryptosporidium spp. efficiently. However, the tools based on the antigens TRAP‐C1, TRAP‐C2 and COWP genes, the housekeeping genes HSP70 and DHFR, or a genomic sequence, failed to detect the DNA of C. felis, C. canis, Cryptosporidium pig genotype I, and C. metris. With the exception of 1 tool based on the TRAP‐C2 gene, the PCR‐RFLP or the PCR sequencing tools evaluated in this study could differentiate C. hominis, C. parvum and C. meleagridis from each other, and 2 SSU rRNA genebased tools could differentiate all 7 Cryptosporidium spp. Thus, a thorough understanding of the strength and weakness of each technique is needed when using molecular diagnostic tool in epidemiological investigations of human cryptosporidiosis.
Identification of a New Microsporidian Parasite Related to Vittaforma corneae in HIV‐Positive and HIV‐Negative Patients from Portugal
Authors: SULAIMAN IRSHAD M. , MATOS OLGA , LOBO MARIA L. , XIAO LIHUA
Published: July 2005

ABSTRACT. Fecal samples from 22 HIV‐positive and 3 HIV‐negative patients from Portugal with symptomatic diarrhea were diagnosed positive for microsporidia by microscopy, with most parasites detected significantly bigger than Enterocytozoon bieneusi and Encephalitozoon spp. Sequence characterization of the small subunit (SSU) rRNA gene identified a microsporidian parasite with 96% homology to two published Vittaforma corneae sequences. Phylogenetic analysis confirmed the genetic relatedness of this new microsporidian parasite to Vittaforma corneae as well as Cystosporogenes operophterae. Results of the study demonstrate the presence of a new human‐pathogenic microsporidian species, which is responsible for significant number of infections in HIV‐positive and HIV‐negative patients in Portugal.
Immunocompetent Infants as a Human Reservoir for Pneumocystis jirovecii: Rapid Screening by Non‐Invasive Sampling and Real‐Time PCR at the Mitochondria1 Large Subunit rRNA Gene
Authors: TOTET ANNE , MELIANI; LEILA , LACUBE; PHILIPPE , PAUTARD; JEAN CLAUDE , RACCURT CHRISTIAN , ROUX PATRICIA , NEVEZ GILLES
Published: July 2005

ABSTRACT. We tested a real‐time PCR assay targeting the Pneumocystis jirovecii mitochondria1 large subunit rRNA gene on 240 archival nasopharyngeal aspirates from non‐immunosuppressed infants. The sensitivity of this assay appeared close to that of a conventional nested‐PCR assay targeting the same locus. Because of its one‐step procedure, and its sensitivity and rapidity, the real‐time PCR assay is particularly suitable for screening individuals parasitized by P. jirovecii within large populations.
Multilocus Genotyping of Pneumocystis jirovecii in Patients Developing Diverse Forms of Parasitism: Implication for a Wide Human Reservoir for the Fungus
Authors: TOTET ANNE , LATOUCHE SOPHIE , DUWAT HYACINTHE , MAGOIS ELINE , LACUBE PHILIPPE , PAUTARD JEAN‐CLAUDE , SCHMIT JEAN‐LUC , JOUNIEAUX VINCENT , ROUX PATRICIA , RACCURT CHRISTIAN , NEVEZ GILLES
Published: July 2005

ABSTRACT. Pneumocystis jirovecii ITS and DHPS genotypes were identified in 3 patient groups developing diverse forms of P. jirovecii infections: 13 patients with Pneumocystis pneumonia, 8 patients merely colonized by the fungus, and 19 immunocompetent infants with bronchiolitis developing mild P. jirovecii infection. Common P. jirovecii genotypes were found in the 3 patient groups, suggesting that common sources of P. jirovecii were involved in the fungus acquisition, and that transmission cycles of P. jirovecii infections in these patient groups are not independent. Parasitized patients, whatever the form of parasitism they present, may he part of a common reservoir for P. jirovecii.
Isospora belli Infection: Observation of Unicellular Cysts in Mesenteric Lymphoid Tissues of a Brazilian Patient with AIDS and Animal Inoculation
Authors: Frenkel Jacob K. , Silva Marcia Benedita De Oliveira , Saldanha Joao , De Silva Mario Leon , Filho V. Dalmo Correia , Barata Cristina Hueb , Lages Eliane , Ramirez Luis Eduardo , Prata Aluizio
Published: July 2005

ABSTRACT. We describe the finding of unizoic cysts of Isospora helli in lymphoid tissues of a Brazilian patient with AIDS, and discuss the possibilities of their drug resistance, they being the cause of relapses, and of being an indication for the existence of intermediary or paratenic animal hosts.
Cell Death Mechanisms in the Human Opportunistic Pathogen Candida albicans
Authors: Lemar Katey M. , Müller Carsten T. , Plummer Susan , Lloyd David
Published: July 2005

ABSTRACT. Difficulties arising during chemotherapy of Candida albicans necessitate novel chemotherapeutic strategies. Garlic extract and two of its constituents, diallyl disulphide and allyl alcohol, are potentially useful anti‐candidal agents. Flow Cytometry has been used to measure the population distributions of apoptotic/necrotic cell death using annexin V‐FITC/propidium iodide and oxidative stress dichlorodihydrofluorescein. Candicidal mechanisms may be due to programmed cell death induced by oxidative stress, mediated by the generation of reactive oxygen species or alternatively by the depletion of cellular thiols, which normally act as redox buffer systems for defence. We suggest that mechanisms that these anti‐candidal agents have in common is the triggering some of the characteristics of apoptotic cell death.
Sporulation and Survival of Toxoplasma gondii Oocysts in Seawater
Authors: Lindsay David. S. , Collins Marina V. , Mitchell Sheila M. , Cole Rebecca A. , Flick George J. , Wetch Carly N. , Lindquist Alan , Dubey J. P.
Published: July 2005

ABSTRACT. We have been collaborating since 1992 in studies on southern sea otters (Enhdyra lutris nereis) as part of a program to define factors, which may be responsible for limiting the growth of the southern sea otter population. We previously demonstrated Toxoplasma gondii in sea otiers. We postulated that cat feces containing oocysts could be entering the marine environment through storm run‐off or through municipal sewage since cat feces are often disposed down toilets by cat owners. The present study examined the sporulation of T. gondii oocysts in seawater and the survival of sporulated oocysts in seawater. Unsporulated oocysts were placed in 1.5 ppt artificial seawater, 32 ppt artificial seawater or 2% sulfuric acid (positive control) at 24 C in an incubator. Samples were examined daily for 3 days and development monitored by counting 100 oocysts from each sample. From 75 to 80% of the oocysts were sporulated by 3 days post‐inoculation under all treatment conditions. Groups of 2 mice were fed 10,000 oocysts each from each of the 3 treatment groups. All inoculated mice developed toxoplasmosis indicating that oocysts were capable of sporulating in seawater. Survival of sporulated oocysts was examined by placing sporulated T. gondii oocysts in 15 ppt seawater at room temperature 22–24 C (RT) or in a refrigerator kept at 4 C. Mice fed oocysts that had been stored at 4C or RT for 6 months became infected. These results indicate that T. gondii oocysts can sporulate and remain viable in seawater for several months.
Mode of Action of Ponazuril Against Toxoplasma gondii Tachyzoites in Cell Culture
Authors: Mitchell Sheila M. , Zajac Anne M. , Davis Wendell L. , Lindsay David S.
Published: July 2005

ABSTRACT.Toxoplasma gondiiis an important apicomplexan parasite of humans and other warm‐blooded animals. Ponazuril is a triazine anticoccidial recently approved for use in horses in the United States. We investigated the inode of action of ponazuril against developing RH strainT. gondiitachyzoites in African green monkey kidney cells. Host cells were infected with2.0 × 105tachyzoites and treated with 5 μg/ml ponazuril. Cultures were fixed and examined by transmission electron microscopy 3 days after treatment. Ponazuril interfered with normal parasite division. This led to the presence of multinucleate schizonts stages. Up to six tachyzoites were observed partially budded from the surface of these schizonts. Large vacuoles developed in these schizonts and they eventually degenerated.
Emergence of Zoonotic Canine Leishmaniasis in the United States: Isolation and Immunohistochemical Detection of Leishmania infantum from Foxhounds from Virginia.
Authors: ROSYPAL ALEXA C. , TROY GREGORY C. , ZAJAC ANNE M. , DUNCAN ROBERT B. , WAKI KAYOKO , CHANG K.‐P. , LINDSAY DAVID S.
Published: July 2005

ABSTRACT. Previously considered an exotic disease, canine leishmaniasis caused by Leishmania infantum has recently been detected within the foxhound population in the United States and parts of Canada. Leishmania infantum is the etiologic agent of visceral leishmaniasis in many areas of the world and dogs are considered a major reservoir host for human Leishmania infections. Human visceral leishmaniasis has recently emerged as an opportunistic infection among individuals co‐infected with HIV/AIDS and in persons taking immunosuppressive drugs. We report the isolation of L. infantum from 3 naturally infected foxhounds from Virginia by culture of popliteal lymph node and bone marrow, and the development of an immunohistochemical test to detect the parasite in tissues.
Introductory Remarks: Bacterial Endosymbionts or Pathogens of Free‐Living Amebae1
Authors: MARCIANO‐CABRAL FRANCINE
Published: July 2005

Free‐living amebae are ubiquitous in the environment and can be isolated from a variety of habitats including water, soil, air, hospital water systems, dental units, contact lens cases, and cooling towers. The interaction of amebae with other microorganisms in their environment is varied. Bacteria are a major food source for free‐living amebae. However, some bacteria have established a stable symbiotic relationship with amebae. Recent reports indicate an association of amebae with intracellular bacterial pathogens. Such amebae may serve as reservoirs for maintaining and dispersing pathogenic bacteria in the environment or as vectors of bacterial disease in humans.
Genetic and Physiological Interactions in the Amoeba‐Bacteria Symbiosis1
Authors: JEON KWANG W.
Published: July 2005

Amoebae of the xD strain of Amoeba proteus that arose from the D strain by spontaneous infection of Legionella‐like X‐bacteria are now dependent on their symbionts for survival. Each xD amoeba contains about 42,000 symbionts within symbiosomes, and established xD amoebae die if their symbionts are removed. Thus, harmful infective bacteria changed into necessary cell components. As a result of harboring X‐bacteria. xD amoebae exhibit various physiological and genetic characteristics that are different from those of symbiont‐free D amoebae. One of the recent findings is that bacterial symbionts control the expression of a host's house‐keeping gene. Thus, the expression of the normal amoeba sams gene (sams1) encoding one form of S‐adenosylmethionine synthetase is switched to that of sams2 by endosymbiotic X‐bacteria. Possible mechanisms for the switching of sams genes brought about by endosymbionts and its significance are discussed.
Bacterial Endosymbionts of Free‐living Amoebae1
Authors: HORN MATTHIAS , WAGNER MICHAEL
Published: July 2005

The occurrence of bacterial endosymbionts in free‐living amoebae has been known for decades, but their obligate intracellular lifestyle hampered their identification. Application of the full cycle rRNA approach, including 16S rRNA gene sequencing and fluorescence in‐situ hybridization with 16S rRNA‐targeted oligonucleotide probes, assigned the symbionts of Acanthamoeba spp. and Hartmannella sp. to five different evolutionary lineages within the Proteobacteria, the Bacteroidetes, and the Chlamydiae, respectively. Some of these bacterial symbionts are most closely related to bacterial pathogens of humans, and it has been suggested that they should be considered potential emerging pathogens. Complete genome sequence analysis of a chlamydia‐related symbiont of Acanthamoeba sp. showed that this endosymbiont uses similar mechanisms for interaction with its eukaryotic host cell as do the well‐known bacterial pathogens of humans. Furthermore, phylogenetic analysis suggested that these mechanisms have been evolved by the ancestor of these amoeba symbionts in interplay with ancient unicellular eukaryotes.
Modulation of a “CD59‐like” Protein in Naegleria fowleri Amebae by Bacteria1
Authors: FRITZINGER ANGELA E. , MARCIANO‐CABRAL FRANCINE
Published: July 2005

Found in soil and freshwater habitats, Naegleria fowleri are free‐living amebae that cause a fatal disease in humans called Primary Amebic Meningoencephalitis. In the natural environment, amebae feed on bacteria. In the infected host, the amebae lyse and ingest nerve tissue. Recently, we have established that N. fowleri expresses a “CD59‐like” surface protein, but the function of this protein in the ameba has not been elucidated. In mammalian cells, CD59 is a complement‐regulatory protein that inhibits complement‐mediated lysis of cells expressing this protein. In the present study, expression of the “CD59‐like” protein in response to bacteria and bacterial toxins was investigated by Western immunoblot analysis. Co‐culture of N. fowleri with log phase Escherichia coli or Pseu‐domonas aeruginosa resulted in differential expression of the “CD59‐like” protein. Co‐cultures of amebae and bacteria were examined by electron microscopy. The results of our study implicate a possible protective role of the “CD59‐like” protein in response to bacterial predators and bacterial toxins, because amebae remained intact after co‐culture with bacteria.
Plastid‐Targeting Peptides from the Chlorarachniophyte Bigelowiella natans
Authors: ROGERS MATTHEW B. , ARCHIBALD JOHN M. , FIELD MATTHEW A. , LI CATHERINE , STRIEPEN BORIS , KEELING PATRICK J.
Published: July 2005

Chlorarachniophytes are marine amoeboflagellate protists that have acquired their plastid (chloroplast) through secondary endosymbiosis with a green alga. Like other algae, most of the proteins necessary for plastid function are encoded in the nuclear genome of the secondary host. These proteins are targeted to the organelle using a bipartite leader sequence consisting of a signal peptide (allowing entry in to the endomembrane system) and a chloroplast transit peptide (for transport across the chloroplast envelope mem branes). We have examined the leader sequences from 45 full‐length predicted plastid‐targeted proteins from the Chlorarachniophyte Bigelowiella natans with the goal of understanding important features of these sequences and possible conserved motifs. The chemical characteristics of these sequences were compared with a set of 10 B. natans endomembrane‐targeted proteins and 38 cytosolic or nuclear proteins, which show that the signal peptides are similar to those of most other eukaryotes, while the transit peptides differ from those of other algae in some characteristics. Consistent with this, the leader sequence from one B. natans protein was tested for function in the apicomplexan parasite, Toxoplasma gondii, and shown to direct the secretion of the protein.
Assessing the Effectiveness of Coding and Non‐coding Regions in Antisense Ribosome Inhibition of Gene Expression in Tetrahymena
Authors: JACOBS MARY ELLEN , CORTEZZO DONNAMARIE E. , KLOBUTCHER LAWRENCE A.
Published: July 2005

In Tetrahymena thermophila, an “antisense ribosome” technology has been developed for inhibiting gene expression and generating novel mutants. Short segments of genes are inserted in antisense orientation into an rDNA vector in a region corresponding to an external loop of the folded rRNA. DNA segments derived from the 5′‐ends of genes have proven most effective in reducing cognate gene expression. To investigate the efficacy of other genie regions, we generated Tetrahymena cell lines with antisense ribosome constructs containing 100‐bp DNA segments derived from the 5′‐ends, 3′‐ends, and internal coding regions of two non‐essential genes, granule lattice protein 1 and macronuclear histone H1. The 5′‐ and 3′‐end constructs inhibited gene expression, but antisense ribosomes derived exclusively from coding regions had little effect.
Identification of Amoebae Implicated in the Life Cycle of Pfiesteria and Pfiestena‐Like Dinoflagellates
Authors: PEGLAR MICHAEL T. , NERAD THOMAS A , ANDERSON O. ROGER , GILLEVET PATRICK M.
Published: July 2005

This study was undertaken to assess whether amoebae commonly found in mesohaline environments are in fact stages in the life cycles of Pfiesteria and Pfiesteria‐like dinoflagellates. Primary isolations of amoebae and dinoflagellates were made from water and sediment samples from five tributaries of the Chesapeake Bay. Additional amoebae were also cloned from bioassay aquaria where fish mortality was attributed to Pfiesteria. Electron microscopy and small subunit (SSU) rRNA gene sequence analysis of these isolates clearly demonstrated that the commonly depicted amoeboid form of Pfiesteria is very likely a species of Korotnevella and is unrelated to Pfiesteria or Pfiesteria‐like dinoflagellates. We have determined that the Pfiesteria and Pfiesteria‐like dinoflagellates examined in this study undergo a typical homothallic life cycle without amoeboid stages. Furthermore, we have demonstrated that cloned amoebae sharing morphological characteristics described for stages in the life cycle of Pfiesteria do not transform into dino7.oites. The strict clonal isolation and cultivation techniques used in this study substantially support the conclusion that the amoebae and some of the flagellates depicted in the life cycle of Pfiesteria are environmental contaminants of the Pfiesteria culture system and that the Ambush Predator Hypothesis needs to be rigorously reevaluated.