Showing papers in "Journal of Eukaryotic Microbiology in 1985"

Occurrence of a new microsporidan: Enterocytozoon bieneusi n.g., n. sp., in the enterocytes of a human patient with AIDS.

Abstract: A new microsporidium is reported infesting the enterocytes of a Haitian patients with AIDS. The stages observed were diplokaryotic cells, sporogonial plasmodia, unikaryotic sporoblasts, and spores. Neither a sporophorous vesicle (pansporoblastic membrane) nor parasitophorous vacuole were differentiated around the developmental stages, which were in direct contact with the host cell cytoplasm. The polar tube (5-6 coils) was differentiated before fission of the sporogonial plasmodium. The mature spores measured 1.5 micron X 0.5 micron. The spore wall was very thin as the endospore was absent or poorly differentiated. The organism is named Enterocytozoon bieneusi n. g., n. sp. and is assigned to the suborder Apansporoblastina.

Protozoa in Planktonic Food Webs1,2

Abstract: Protozoa are now being recognized as important members of planktonic food webs. This is due to the inclusion of microbial links in our paradigm of trophic relationships. Heterotrophic microflagellates and ciliates are major grazers of bacteria. They can stimulate production through nutrient recycling and can transform microbial production into larger particles, which are then available for macroconsumers. In this paper we add new groups, the small (< 20 μm) ciliates and myxotrophic flagellates, to the planktonic food web.

Nutritional Requirements of Plasmodium falciparum in Culture. I. Exogenously Supplied Dialyzable Components Necessary for Continuous Growth

Abstract: Continuous cultivation of Plasmodium falciparum presently requires the nutritionally complex medium, RPMI 1640. A basal medium of KCl, NaCl, Na2HPO4, Ca(NO3)2, MgSO4, glucose, reduced glutathione, HEPES buffer, hypoxanthine, phenol red (in RPMI 1640 concentrations), and 10% (v/v) exhaustively dialyzed pooled human serum was used to determine which vitamins and amino acids had to be exogenously supplied for continuous cultivation. Supplementation of basal medium with calcium pantothenate, cystine, glutamate, glutamine, isoleucine, methionine, proline, and tyrosine was necessary for continuous growth. This semi-defined minimal medium supported continuous growth of four isolates of P. falciparum at rates slightly less than those obtained with RPMI 1640. Adding any other vitamin or amino acid did not improve growth. Incorporation of several non-essential amino acids, particularly phenylalanine and leucine, into proteins was markedly enhanced in the minimal medium compared to RPMI 1640.

Functional Groups in the Protozoa: Roles in Differing Ecosystems1,2

Abstract: Feeding habits of freshwater protozoa were used to group species into functional, trophic groups. Community structure in differing ecosystems was examined in relation to the number of species occumng in the functional group categories. Six wetland ecosystems and a large river ecosystem were studied. Changes in community structure during the colonization of artificial substrates were also examined. Changes during colonization were studied in a mesotrophic lake, in low-order streams, and in laboratory micro- ecosystems. In the latter case, the response of colonizing communities to a heavy metal toxicant was studied. All communities studied were dominated by bactivorous-detritivorous species and, to a lesser extent, by photosynthetic species. The chief functional role of substrate-associated protozoans appears to be the processing of dead organic matter and its associated bacterial flora. Functional groups utilizing resources other than detntal or mineral nutrients (saprotrophs, algivores, omnivores, and predators) were always minor community components. Colonizing communities were often dominated by photosynthetic species during early colonization stages but were again dominated by bactivorous-detntivorous species at species equilibrium. Low levels of toxicant (Cd) reduced numbers of both photosynthetic and bactivorous-detritivorous species. Higher toxicant levels virtually eliminated photosynthetic species and reduced bacterial detritivores by over one-half. Roles of protozoan species in ecosystems are closely tied to the processing of detritus and the recycling of mineral nutrients. Enumeration of individuals in functional categories is proposed as a simplified method for studying the abundance and activity of protozoa in ecosystems. Examination of changes in functional group composition and the relationship of functional group abundances to rates of carbon processing are suggested for studies of the importance of protozoa to the flow of energy and materials in ecosystems. OST ecosystems are not fragile, random associations of M species but are composed of species associations with multiple redundancies that act to maintain system function even when a few or many component parts may be changing or miss- ing. Species patterns in aquatic ecosystems often appear ran- dom, and the probability of collecting a given taxon at a given site is frequently not great. Understanding large species arrays

Protozoa as Hosts for Endosymbioses and the Conversion of Symbionts into Organelles1,2

Abstract: . Protozoa may be thought of as preadapted to serve as hosts for cellular endosymbionts by virtue of their widespread ability to take up particles by endocytosis. The absence of the cell wall so characteristic of plants and fungi and the commonly large size of most protozoa are additional factors favoring protozoan cells for endosymbioses. The conversion of symbiont into a cellular organelle (e.g. a mitochondrion or chloroplast) is more complicated, especially since the latter do not code for all of their own proteins. Thus, such conversions are held to be rare. Among protozoa, numerous foraminifera appear to have characteristics making them very favorable as hosts for certain algae. Such adaptations, both physiological and morphological in nature, are discussed. Also discussed in this paper are the ways by which (present-day) chloroplasts and mitochondria may have been derived from early endosymbionts: a single ancestral cyanobacterium, in the first case, and a single ancestral purple-nonsulfur bacterium, in the second. Mechanisms for insertion of proteins into and across the organellar membranes had to be evolved for all genes transferred from the symbionts into the host nucleus.

The mitochondrion of Plasmodium falciparum visualized by rhodamine 123 fluorescence.

Abstract: Rhodamine 123 (Rh 123) has been used to probe the functional status of the mitochondrion present within the asexual, intraerythrocytic stages of the malarial parasite Plasmodium falciparum. This cationic fluorescent dye accumulates specifically in negatively charged cellular compartments, such as mitochondria. Using epifluorescence microscopy the development of what appears to be a single mitochondrion has been followed through the intraerythrocytic cycle. Mitochondrial development progresses from a fine thread-like organelle that becomes longer and eventually branched. Each daughter merozoite receives a branch or piece of the parent organelle. Cytoplasmic Rh 123 accumulation was also observed, indicating that there exists a transmembrane potential across the outer plasma and parasitophorous vacuolar membranes of the parasite. The effects of uncouplers (protonophores), ionophores, and inhibitors were examined by monitoring Rh 123 accumulation and retention. Our results demonstrate that the mitochondrion of P. falciparum actively maintains a high transmembrane potential, the function of which is as yet undefined.

Identification of the meiotic division of malarial parasites.

Abstract: Zygotes of Plasmodium berghei were cultured 15–25 h in vitro to yield mature infective ookinetes. Samples taken in the first 5 h of culture were examined by electron microscopy. Meiotic figures were detected in the nuclei of the zygotes. Threadlike leptotene chromatids (chromosomes) condensed from attachment plaques on the nuclear envelope; chromatid pairing followed (zygotene), with synaptonemal complexes subsequently appearing (pachytene). These complexes persisted into metaphase but dissociated when the chromatids rapidly decondensed during anaphase. At telophase of the first meiotic division the kinetochores were retracted toward two small spindle complexes, which were found at widely separated poles in the nuclear envelope. The observations are consistent with a haploid genome of 8–10 chromosomes.

PKX, the causative agent of proliferative kidney disease (PKD) in Pacific salmonid fishes and its affinities with the Myxozoa.

Abstract: Proliferative kidney disease (PKD), caused by an unclassified protozoan (PKX), is reported from Pacific salmon, Oncorhynchus tshawytscha (Walbaum) and O. kisutch (Walbaum), and steelhead trout, Salmo gairdneri Richardson, held at the Mad River Hatchery in California, USA. The cumulative mortality attributed to the disease was 95, 13, and 18% respectively. The mortalities were greatest at mean water temperatures of 12-14°C during July 1983. The ultrastructure of the PKX organism and its associated pathology during clinical disease in all three species were consistent with those of the parasite in rainbow trout (Salmo gairdneri) as described in European outbreaks. Significant mortalities did not occur after August, at which time the parasite could no longer be detected in the salmon species. The steelhead continued to exhibit parasites in the kidney interstitium and epithelium and lumens of the tubules. Myxosporidan trophozoites and developing spores were also observed in the lumens of the kidney tubules of these fish. Although a mixed infection with another parasite may have occurred, evidence suggests that the myxosporidans are later stages of PKX. They were only observed in fish exposed to water with the infective stage and were particularly prominent in recovering fish. The PKX organism is similar to UBO, an unclassified protozoan of carp suspected to be an early stage of Sphaerospora renicola Dykova & Lom. Both parasites infect the blood and kidney, divide by endogeny, and are released by disintegration of the primary mother cell. The intraluminal myxosporean forms show similarities to Sphaerospora spp. in that they are monosporous and sporoblasts are formed within pseudoplasmodia. It is possible that PKX migrates to the lumen of the kidney tubule and subsequently sporulates. If the myxosporean forms are later stages of PKX, then it would belong to the phlyum Myxozoa.

Structural bases of the cytolytic mechanisms of Entamoeba histolytica

Abstract: The cellular bases of the powerful cytolytic activity of the human protozoan parasite Entamoeba histolytica were explored by studying the effect of the virulent strain HM1:IMSS on epithelial monolayers of MDCK cells using a combination of time-lapse microcinematography and transmission and scanning electron microscopy. Early alterations of the epithelial cell membranes were detected by measuring changes in the transepithelial electrical resistance of MDCK monolayers mounted in Ussing chambers. The aggressive mechanism of E. histolytica trophozoites was found to be a complex, multifactorial phenomenon that included hit-and-run damage to the plasma membrane of effector cells mediated through contact, phagocytosis of lysed or apparently intact, but detached, MDCK cells, and intracellular degradation of ingested cells. Following contact with amebas, the epithelial monolayers showed a pronounced lowering of transepithelial resistance, opening of tight junctions, distortion of microvilli, surface blebbing, and the presence of minute focal discontinuities in the plasma membrane. There was no evidence of amebic exocytosis, membrane fusion, or junction formation between the parasite and host plasma membranes. Although modifications in the epithelial cell membranes usually preceded lysis, the cytolytic activity of the parasite did not exclusively involve damage to the plasma membrane of the cultured host cells but also was mediated by avid phagocytosis, the displacement and separation of neighboring cells by means of pseudopodial activity, and the "pinching-off" of the peripheral cytoplasm of epithelial cells.

The biochemistry and functional morphology of the Entamoeba.

Abstract: Article de synthese sur ce genre concernant l'organisation infracellulaire, le metabolisme et les aspects moleculaires des relations hote parasite

Paramoeba invadens n.sp. (Amoebida, Paramoebidae), a pathogenic amoeba from the sea urchin, Strongylocentrotus droebachiensis, in Eastern Canada

Abstract: Paramoeba invadens n. sp. is described from sea urchin tissues and from culture. Amoebae are 20–40 μm in length with an irregular hyaline region producing short subpseudopodia. One parasome per cell is present. The parasome is bipolar with basophilic, Feulgen-positive poles and a Feulgen-negative median segment. The fine structure of the parasome resembles those in other species of Paramoeba and the surface of the amoeba is plain with no hairs or scales. Amoebae dislodged from tissues often adopt a semifloating form; floating forms have been seen in culture. Amoebae circulate within lumina of the water vascular system and can migrate readily into or out of tissues.

RNA and protein synthesis during meiotic prophase in Tetrahymena thermophila.

Abstract: Tetrahymena is one of the few organisms from which large amounts of precisely staged meiotic material can be obtained. We took advantage of this fact to monitor RNA and protein synthesis during meiosis. The rate of total protein synthesis as well as the synthesis of the majority of heavily labeled conjugation-specific polypeptides (monitored by high resolution two-dimensional gel electrophoresis) was maximal during meiotic prophase. We therefore cloned cDNAs corresponding to genes active during this time. The mRNA levels of three conjugation-specific genes (pC1, pC2, and pC7) and one conjugation-induced gene (pC3) were followed by using the corresponding labeled cDNAs to probe RNA isolated from different times during mating that was also followed cytologically. Synthesis of the conjugation-specific mRNAs was maximal just prior to maximum crescent stage (pachytene). Evidence is presented for transcription by the normally inactive micronucleus just prior to the maximum crescent stage, confirming an earlier report. The significance of these results is discussed.

Phagocytized Intracellular Microsporidian Blocks Phagosome Acidification and Phagosome‐Lysosome Fusion

Abstract: This study examines the relationship between phagosome acidification and phagosome-lysosome fusion events using phagocytized Glugea hertwigi spores. The incidence of lysosome fusion with Glugea spores in phagosomes of mouse peritoneal macrophages and of Tetrahymena was monitored using colloidal gold and acridine orange as labels for secondary lysosomes. Over 80% of the Glugea phagosomes remained segregated from the labeled compartments in macrophages after 60 min; this inhibition of fusion was still evident after 4 h. In Tetrahymena, Glugea spores also showed a high capacity to block fusion with secondary lysosomes (67%); however, spores coated with cationized ferritin showed an 80% fusion rate with labeled acidic compartments (i.e. lysosomes) after 60 min with both Tetrahymena and macrophages. The pH of phagosome compartments was monitored by measuring the emissions of fluorescein isothiocyanate (FITC)-labeled Glugea ingested by Tetrahymena. Tetrahymena phagosomes with FITC-Glugea did not acidify within the first hour after phagocytosis; however, phagosomes with cationized ferritin-labeled Glugea underwent acidification during this time period. This acidification took place although the capability of the host cells' lysosomes to fuse was blocked by pretreatment with poly-D-glutamic acid. The cationized ferritin bound to Glugea spores was uncoupled from the spore wall prior to fusion with colloidal gold-labeled compartments. In vitro testing showed that ferritin dissociation requires an acid pH, indicating that phagosomes acidify prior to lysosome fusion.

Ichthyophthirius multifiliis (Ciliophora) Invasion of Gill Epithelium1

Abstract: The sequence of morphologic events associated with Ichthyophthirius rnultifliis invasion of gill epithelium began in the theront with differentiation of secretory mucocysts and the perforatorium. After escaping from the cyst the theront, which stained intensely with Mallory' stain, sought a host. As it approached the host epithelium, the contents of the mucocysts were discharged, enveloping the ciliate in sticky material, which made initial contact with the host epithelium. Rapid penetration by the theront caused disruption of one or more host cells and resulted in a focal necrosis associated with the anterior margin of the ciliate. Within five minutes postexposure, the parasite completed its invasion of the epithelial layer and stained less intensely. The remnants of host cells disrupted during its entry surrounded the trophont until they were ingested by the parasite. Within 40 min postexposure, synthetic activity of the parasite appeared to increase as evidenced by an abundance of organelles, particularly ribosomes and crystalline mucocysts. At this point, the overlying host epithelium appeared normal.

Symposium on “Protozoan Ecology”: The Role of Protozoa in Litters and Soils1

Abstract: Soil is the focus of organic matter turnover in terrestrial ecosystems and is an interstitial mosaic of microsites composed of particle aggregates and pore spaces, where transformation, decomposition, mineralization, and humification of organic matter takes place. Microorganisms and animals are scattered discontinuously in these microsites. Microarthropods and larger fauna increase the rate and amount of mineralization by comminution of organic matter and by redistribution of microsites through movements of earthworms and large arthropods; however, mineralization and return of nutrients to plants occurs in the community of bacteria, fungi, protozoa, and nematodes living in the water films covering aggregates and filling pore spaces. Protozoa, especially small amoebae, are important bacterial grazers because they can enter tiny spaces unavailable to nematodes. The latter graze bacteria, fungi, and protozoa. Protozoan and nematode predation increase the amounts of soluble nutrients and decrease the competitive abilities of bacteria, thus making these nutrients more available to plants. Protozoa enhance nutrient recycling out of proportion to their biomass.

Nutritional requirements of Plasmodium falciparum in culture. III. Further observations on essential nutrients and antimetabolites.

Abstract: In a semi-defined minimal medium for cultivation of Plasmodium falciparum, ribose, mannose, fructose, galactose, and maltose could not replace glucose. Hypoxanthine was the preferred purine source for the parasite over adenine, guanine, inosine, adenosine and guanosine although all supported growth equally. Inhibitors of nucleoside uptake had low potency in killing the parasites but depressed incorporation of [3H]adenosine more than [3H]hypoxanthine. Glutamate could not be replaced by 5-oxoproline, indicating that the gamma-glutamyl transferase pathway for amino acid uptake is probably not found in this organism. Adenine, nicotinamide, and orotic acid could not supplement glutamine-deficient medium. The pyridoxine antagonists isoniazid and 4-deoxypyridoxine were reversed by amino acid supplementation, suggesting that transaminases may be targets of these drugs. Orotic acid, but not glutathione or its amino acid components, partially reversed the effects of 8-methylamino-8-desmethyl riboflavin. Thus, the flavin enzyme, dihydroorotic acid dehydrogenase, but not glutathione reductase, appears to be a target of this riboflavin antagonist. Five biotin antagonists had no significant activity. The choline antagonist 2-(tert-butylamino)ethanol and thiamin uptake inhibitors had nonspecific inhibitory effects, which were not reversed by the respective target vitamin. Buthionine sulfoximine and methionine sulfoximine, inhibitors of glutathione synthesis, had significant oxygen-dependent toxicity. Six sulfonamides showed marked variation in potency and efficacy. Sulfathiazole and sulfadoxine were reversed differentially by p-aminobenzoic acid, folic acid, and folinic acid. Folinic acid was more effective than folic acid at reversing the toxicity of the dihydrofolate reductase inhibitors aminopterin and pyrimethamine; p-amino-benzoic acid had no effect.

Stage-dependent toxicity of N-acetyl-glucosamine to Plasmodium falciparum.

Abstract: The effects of N-acetyl-glucosamine on growth of synchronized cultures of Plasmodium falciparum were assessed by morphological observations and by measurement of parasite incorporation of 3H-hypoxanthine. Inhibition of 3H-hypoxanthine incorporation was more marked during the later stages of the erythrocytic cycle. At concentrations of the sugar below 20 mM, however, the deleterious effects were mainly a result of failure of released merozoites to invade erythrocytes, rather than a failure of schizonts to mature or release merozoites. These results are compatible with the hypothesis that a lectin-like substance on the merozoite interacts with a surface glycoprotein on the red cell and that sugar residues on this glycoprotein may be involved in this recognition.

Biochemical identification and phylogenetic relationships in free-living amoebas of the genus Naegleria.

Abstract: Using isoelectric focusing, the zymograms of 23 pathogenic and nonpathogenic Naegleria strains were studied for the activity of 16 enzymes. Certain enzymes (lactate dehydrogenase, L-threonine dehydrogenase, superoxide dismutase, acid phosphatase, malic enzyme, and leucine aminopeptidase) proved particularly useful from a practical point of view as they allow easy and reliable identification of pathogenic N. fowleri and N. australiensis as well as nonpathogenic N. lovaniensis strains. Genetic interpretation of these zymograms gave estimates of genetic distances that largely confirmed the taxonomic position of the Naegleria species. In addition, the genetic data suggest that there are two main phylogenetic groups in the genus Naegleria.

Morphological, Biometric, and Morphogenetic Comparison of Two Closely Related Species, Stylonychia vorax and S. pustulata (Ciliophora: Oxytrichidae)1

Abstract: Differences in the morphology of Stylonychia vorax Stokes, 1885 and S. pustulata (Muller, 1786) Ehrenberg, 1838 recognizable in vivo are the shape, the ventral cirral pattern, the caudal cirri, and the mode of moving. The dorsal-bristle complexes are distinguishable by the length of dorsal kinety four and the spaces among the pairs of basal bodies. When the ranges of variation of different populations and clones are compared by biometric analyses, S. vorax shows a relatively stable cortical pattern whereas in S. pustulata the cortical elements are regulated depending on the size of the body and the number of adoral membranelles. In S. vorax morphogenesis begins with a proliferation of basal bodies close to the transverse cirri. In contrast, in S. pustulata, the oral primordium appears de novo between the left marginal row and the postoral cirri. All other morphogenetic events are the same for both species. In proters and opisthes the six anlagen of the frontal-ventral-transverse cirri are of different origin and evolve independently. Three anlagen of the opisthe separate from the oral primordium, two originate from the right, and one from the left postoral cirrus. Three anlagen of the proter evolve from the posteriormost cirrus in the frontal area, one from the parental undulating membranes, one from the buccal cirrus, and one from the cirrus below the buccal cirrus. The anlagen one to six generate one, three, three, three, four, and four cirri. The characteristic arrangement of the undulating membranes and the participation of only two postoral cirri in the formation of primordia provide features that distinguish between the often confused genera Oxytricha and Stylonychia.

Vertical distribution of potentially pathogenic free-living amoebae in freshwater lakes.

Abstract: The vertical distribution of thermotolerant (37 degrees C and 45 degrees C) free-living amoebae (FLA) in warm monomictic lakes was determined in relation to the onset of thermal stratification and associated physical and chemical changes. The position of abiotic or biotic particulate layers in the water column was located by using a submersible horizontal beam transmissometer that measures attenuance, or the absorption and scattering of light by particulates in the water column. During mixis, the vertical distribution of amoebae was sporadic with significant numbers of FLA only occurring in clay layers caused by runoff after heavy rains. With the onset of thermal stratification in the lakes, phytoplankton layers began to form. Few amoebae were isolated from layers containing flagellated phytoplankton; however, significant (P less than 0.005) numbers of FLA were isolated from two particulate layers dominated by the filamentous blue-green algae Aphanizomenon and Lyngbya, respectively. By late June, a persistent detrital or decomposition layer formed in the lower metalimnion, as well as a hypolimnetic iron layer where the Fe2+ state was predominant. In this midsummer period, 13 Naegleria fowleri were isolated, with three from the detrital layer and seven from the iron layer. The presence of attenuation zones was found to be the best indicator of the vertical distribution of FLA in the water column, and such layers represent an important, previously undescribed habitat for potentially pathogenic FLA.

The Extent of Algal and Bacterial Endosymbioses in Protozoa1,2

Abstract: Long neglected has been the extensive and more or less intimate association of protozoa with a wide variety of other cells, either prokaryotic or eukaryotic in nature. Yet study of such relationships can provide important information concerning certain basic aspects of cellular evolution in general. A survey is offered here of the whole range of such symbiotic associations (i.e. with species of protozoa serving as hosts) with the purposes of drawing attention to the exciting possibilities of such research and of reviewing significant findings made to date. Because of the vastness of the overall field, examples and discussion are primarily limited to consideration of the following major studies: methanogenic bacteria in certain ciliates, bacterial endosymbionts of the large freshwater amoeba Pelomyxa palustris (itself an amazing organism from an evolutionary/phylogenetic point of view), the rod-shaped bacteria found in Amoeba proteus, the “Greek-letter” prokaryotes of Paramecium species, the xenosomes (sensu stricto) of the marine scuticociliate Parauronema acutum, and the diverse algal endosymbionts of similarly diverse protozoan taxa–ciliates, flagellates, radiolarians, acantharians, and foraminifera.

Ultrastructure of Cryptosporidium parvum oocysts and excysting sporozoites as revealed by high resolution scanning electron microscopy.

Abstract: Cryptosporidium parvum oocysts isolated from calf feces were examined by scanning electron microscopy during excystation. Intact C. parvum oocysts were spheroid to ellipsoid, approximately equal to 3.5 X 4.0 micron, with length : width ratio = 1.17. The oocyst wall had a single suture at one pole, which spanned 1/3 to 1/2 the circumference of the oocyst. During excystation the suture dissolved, resulting in a slit-like opening, which the sporozoites used to exit the oocyst. Sporozoites were 3.8 X 0.6 micron and had a rough outer surface.

Invasion and early development of Sarcocystis muris (Apicomplexa, Sarcocystidae) in tissue cultures.

Abstract: Ultrastructural observations on the invasion and early development of merozoites (bradyzoites) of Sarcocystis muris in Madin-Darby canine kidney (MDCK) cells are presented. Invading merozoites cause the host cell plasmalemma to invaginate; they form a membrane junction (moving junction) and move into the host cell where they are enclosed in a primary parasitophorous vacuole (PV). Within 30-45 min after becoming intracellular, merozoites begin to vacate the newly established primary PV and move, forming a new membrane junction, into a secondary PV. Simultaneously with the movement of the parasite, the contents of dense granules in the apical part of the merozoites are shed by exocytosis into the lumen of the developing secondary PV. A lamella of the endoplasmic reticulum of the host cell becomes attached to the PV membrane, forming a PV limited by three host cell membranes.

Chemosensory responses in Tetrahymena: the involvement of peptides and other signal substances

Abstract: Starved Tetrahymena thermophila (or cells growing in Holz's defined medium) are attracted by a chemosensory response to complex peptide mixtures as proteose peptone, yeast extract, and extracts of blood platelets containing platelet-derived growth factor. Starved cells are also significantly attracted by mixtures of amino acids and of nucleosides of Holz's defined medium; however, no individual amino acid or nucleoside could be identified as the major chemo-attractant. The positive chemosensory response can be abolished by cycloheximide (CHX) but is insensitive to actinomycin D and puromycin, possibly indicating that de novo RNA and protein synthesis are not necessary for a change in chemosensory behavior. Three mutants resistant to CHX show normal response in the presence of the drug. The possible role of peptides as naturally occurring food signals of Tetrahymena is discussed.

Chemotaxis by Entamoeba histolytica.

Abstract: A micropore membrane procedure to assay taxis by Entamoeba histolytica is described and the results of studies of responses to a variety of soluble substances, bacteria, an rat colon washings using this procedure are reported Trophozoites migrated in blind well chambers through 8-micron pore size polycarbonate membranes but not nitrocellulose membranes up to 12 micron pore size Amoebae were attracted toward fresh axenic culture medium (TYI-S), an enzymatic hydrolysate of casein (Trypticase), and a partially purified preparation of N-acetylneuraminic acid from egg mucin, but not purified N-acetylneuraminate or a variety of other low molecular weight metabolites The response was verified as chemotaxis by checkerboard analysis Amoebae migrated most dramatically toward suspensions of all of seven bacterial species tested, including motile and non-motile, gram-negative and gram-positive rods and cocci This response was diminished when the bacteria concentration gradient was eliminated The response to bacteria culture filtrates was less than 10% of that to bacterial suspensions A response to clarified washings from the rat colon was detected; this was diminished but not eliminated by filter sterilization of the washings We concluded that some soluble molecules, possibly of intermediate molecular size, whole bacteria, and both soluble and particulate components of the rat colon provide tactic stimuli for E histolytica Scanning electron micrographs of trophozoites migrating towards attractants through membranes showed narrow, extended pseudopodia entering the membrane pores, and enlarging spheres exiting as the cells proceeded through

On the Organization and Affinities of the Amoeba, Pompholyxophrys punicea Archer, Based on Ultrastructural Examination of Individual Cells from Wild Material

Abstract: The genus Pompholyxophrys includes amoebae which have a spherical body, fine radiating pseudopodia, and a layer of adhering siliceous “perles.” These organisms are normally regarded as a type of heliozoon. Ultrastructural examination of P. punicea reveals that those characters associated with well characterized heliozoa, such as microtubular axonemes and extrusomes, are lacking. The species has much in common with nucleariid filose amoebae with which it, and the related genus Pinaciophora, are regarded as having affinities. The species P. punicea is rare, and this study was made possible by the application of techniques developed for the ultrastructural examination of single cells. The assessment of protistan diversity and interrelationships relies heavily on the use of ultrastructural characters. Although techniques that are based on the examination of a small number of individual cells have limitations, they do allow rare organisms to be included in the re-evaluation of protistan systematics.

Rhoptry secretion of membranous whorls by Plasmodium berghei sporozoites

Abstract: Electron microscopy of sporozoites of the rodent malaria parasite, Plasmodium berghei, reveals electron-dense multilaminate membranous whorls within components of the rhoptry-microneme complex after fixation with tannic acid in conjunction with glutaraldehyde. This multilaminate material, which has a dark line to dark line periodicity of approximately 5 nm, appears to be secreted from the sporozoite since it is also found adhering to the sporozoite's external surface. The material may function in sporozoite gliding motility and in invasion of host cells.

Nutritional requirements of Plasmodium falciparum in culture. II. Effects of antimetabolites in a semi-defined medium.

Abstract: A semi-defined minimal medium, in which pantothenic acid is the only vitamin, was used to culture Plasmodium falciparum for the analysis of antimetabolite drugs. Analogs of riboflavin, nicotinamide, pyridoxine, and thiamin inhibited the growth of this parasite; for each drug, effects were much more pronounced after 96 h of exposure compared to 48 h. The most potent drug examined was 8-methylamino-8-desmethyl riboflavin (ICso value approximately 1.0 x M at 96 h). Avidin, a protein which complexes and thus inactivates biotin, did not affect parasite viability. Other antimalarial drugs, including chloroquine and quinine derivatives and antibiotics, were equipotent in the minimal medium and in RPMI 1640. Four strains of P. fakiparum showed only minor differences in sensitivity to these antimetabolites. The use of prolonged drug exposure times and a vitamin-depleted medium allowed the preliminary characterization of antimalarial antimetabolites in vitro. ECHNIQUES for the continuous cultivation of Plasmo- T dium falciparum (42) have facilitated investigations of par- asite biology. A greater understanding of the biochemistry of this organism is essential for the rational development of new drugs for malaria; containment of drug resistance (6, 24, 46) may require novel therapeutic agents. The recently developed minimal medium for cultivation of P. fakiparum (9) can be used to investigate the role of water- soluble vitamins in parasite development and multiplication. Although vitamins such as biotin, nicotinamide, pyridoxine, riboflavin, and thiamin are not exogenous requirements for con- tinuous growth (9), inhibition of survival by specific antime- tabolites could more clearly define their importance and offer insight into parasite metabolism. In addition, such antimetabo- lites are known to have prominent anticoccidial effects; these parasites are taxonomically closely related to Plasmodium spp., but few of these drugs have been examined in P. falciparum. These drugs represent a class of compounds which may be of importance as prototype antimalarials.

The Cytology of Sheep Rumen Ciliates. II. Ultrastructure of Eudiplodinium maggii1

Abstract: The anterior adoral zone of syncilia (AZS) of Eudiplodinium maggii is mounted on an extrusible peristome within a vestibulum. The peristome contains cytopharyngeal components derived from the infraciliature. These components include a crescent-shaped palisade of nematodesmata, two types of sub-membrane cytopharyngeal ribbons, and an ensheathing fibrous layer enclosing a phagoplasmic zone containing the other components. A convoluted esophagus is continuous with and extends from the posterior of the cytopharynx adjacent to the macronucleus. A posterior cytoproct has specialized cytoplasm around it and associated myoneme-like elements. The skeletal plate is composed of finely granular platelets and lies under the cortex ventral to the macronucleus. The endoplasm is separated from the ectoplasm by a fibrous boundary layer. The cortex has an external glycocalyx, a membranous layer, epiplasm, and microtubular and microfilament layers. The AZS infraciliature is of the usual cntodiniomorph type, kinetosomes linked by a sub-kinetosomal rod and with associated bifurcated kinetodesma, postciliary and transverse microtubules-the latter extending into the cytopharynx—nematodesmata, and a fibrous reticulum. A possible vestigial, somatic infraciliature consisting of short, barren kinetosomes with associated basal and cortex-directed microtubules and a periodic incomplete fiber, is found subcortically throughout the cell.

Fine structure of exoerythrocytic merozoite formation of Plasmodium berghei in rat liver.

Abstract: The fine structure of exoerythrocytic merogony of Plasmodium berghei was studied after perfusion-fixation of rat livers from 51 h post-inoculation onwards. Meroblast formation was effected by clefts originating from the parasite plasmalemma and by fusion of vacuoles with each other. Invaginations at the periphery resulted in labyrinthine structures providing the parasites with an enormous increase in surface area, which might facilitate exchange of metabolites. When the parasitophorous vacuole membrane collapsed, the newly formed merozoites were lying free in the hepatocytic cytoplasm, which degenerated until the merozoites were sticking together by a stroma, obviously a remnant of the host hepatocyte. Groups of merozoites, still kept together by the spongy stroma, were subsequently released in the bloodstream. At 53 h most of the developmental stages leading to the release of merozoites could be found and thereafter parasite numbers decreased while large granulomas became apparent.