Effect of Aging of a Cell Population on Lipids and Drug Resistance in Ochromonas danica*

SYNOPSIS. Stationary cultures of Ochromonas danica accumulated lipids as they aged. The bulk of the increase in fatty acids was in the unsaturates, particularly the polyunsaturates. The quantity of lipid peroxides (thiobarbituric acid-positive material) also increased with age. Aging was also associated with increase in sensitivity to inhibitory compounds. The implications of lipid accumulation for cell sensitivity are discussed.     

Ultrastructural Localization of Acid Phosphatase in Feeding Tokophrya infusionum*

A combined cytochemical and electronmicroscopic study of feeding Tokophrya revealed that it has 2 sources of acid phosphatase. One is from the prey, Tetrahymena, supplying newly formed food vacuoles with large amounts of enzyme. The other source is in Tokophrya itself, the enzyme being found in small vesicles, small dense elongate bodies surrounded by a membrane, or in residue vacuoles. It seems that the 2 former small structures contain insignificantly small amounts of phosphatase; however, large deposits of lead phosphate are present in residue vacuoles, former food vacuoles. Since Tokophrya has no cytopyge these vacuoles are not excreted. On the contrary, when feeding is resumed, they merge with food vacuoles, presumably supplying them with acid phosphatase. Whether this enzyme ultimately is derived from the prey Tetrahymena and persists undegraded in the residue vacuoles, or whether it is synthesized by Tokophrya cannot be determined from present work.     

Ultrastructural Study of Schizogony of Eimeria bovis in Cell Cultures*

SYNOPSIS. First-generation schizogony of Eimeria bovis in bovine cell culture was studied by electron microscopy. The intracellular sporozoite retained its structure for at least 6 days at which time it rounded up and lost its apical complex. Although the refractile body underwent certain morphologic changes, it was retained throughout the parasite's growth. The beginning of mitosis was marked by the formation of a cytoplasmic funnel which traversed the nucleus opening on each side toward a pair of centrioles. Subsequently, there developed an intranuclear spindle. Separation of the daughter nuclei was preceded by the formation of typical centrocones. Differentiation of merozoites was accomplished by exogenesis during the last mitotic division. A dense fiber, interpreted as a link connecting the merozoite anlage with its nucleus, extended from the developing apical complex to the nearest division pole. In the anlage, the inner membrane complex was at first composed of patches associated with pairs of subpellicular microtubules. Rhoptries appeared early in merogenesis, whereas micronemes formed at the time the merozoites detached from the residuum. The level of amylopectin, low in schizonts, rose at the beginning of merozoite formation.     

The Differentiation of Herpetomonas megaseliae: Ultrastructural Observations*

Ultrastructure of both undifferentiated (promastigote and paramastigote) and differentiated (opisthomastigote) forms of Herpetomonas megaseliae is described. There is a posterior migration of the kinetoplast at the end of the exponential growth phase. The posterior extension of the flagellar pocket precedes migration of the kinetoplast. Opisthomastigotes have an electron-translucent mitochondrial matrix in comparison with undifferentiated forms. The Golgi body changes from a stack of flattened sacs to an aggregation of vesicles. Several structures previously reported from Trypanosomatidae, e.g. subpellicular organelles, pellicular microtubules, membrane whorls, stored metabolic products, surface blebs, and an intraflagellar body are also present in H. megaseliae.     

Subcellular Distribution of Enzymes in Ochromonas malhamensis*

SYNOPSIS. The activity and distribution of 7 enzymes in Ochromonas malhamensis were studied. Subcellular organelles were separated by centrifugation at 648,000 g min to precipitate the larger particles; the resulting supernatant was centrifuged at 5,560,000 g min to separate the microsomal fraction from the supernatant. Sixty-four percent of the cytochrome oxidase (1.9.3.1 ferrocytochrome c:oxygen oxidoreductase, 81% of the catalase (1.11.1.6 hydrogen-peroxide: hydrogen-peroxide oxidoreductase) and 70% of the urate oxidase (1.7.3.3 urate:oxygen oxidoreductase) activity was associated with the larger particles, altho only 20% of the total protein was found in this fraction. Three acid hydrolases, cathepsin (3.4.4.9 cathepsin C, acid phosphatase (3.1.3.2 orthophosphoric monoesterphosphohydrolase) and acid ribonuclease (2.7.7.17 ribonucleate nucleotido-2′-transferase) were found mostly in the supernate (50-60%, yet their latency and their similar subcellular distribution indicated the presence of lysosomes. After 2.5 hr centrifugation in a sucrose density gradient (ρ= 1.08–1.25, the acid hydrolases showed a broad distribution which differed greatly from cytochrome oxidase associated with mitochondria. Catalase, which could not be separated from cytochrome oxidase by centrifuging on this gradient, had a different distribution after centrifugation on a kinetic gradient. Urate oxidase had a similar distribution to catalase and both these enzymes were latent, indicating the presence of peroxisomes.     

Ultrastructural Observations on the Development of the Microsporidian Protozoon Plistophora hyphessobryconis Schaperclaus*

SYNOPSIS. A sequence of developmental stages of Plistophora hyphessobryconis Schaperclaus, a microsporidian protozoan parasite of the muscular tissue of several species of freshwater fishes, was studied with the electron microscope. The youngest stages observed, ca. 4 × 2 μ, have a single nucleus and their plasm contains only ergastoplasmic lamellae and ribosomes. They are surrounded by a halo of lysed host tissue. They increase in volume to become large sporonts with a great number of nuclei and a thick, 2-layered membrane. Thru schizogony, a corresponding number of sporoblasts is produced within this pansporoblast membrane. Sporoblasts start to develop a thick spore membrane, and a number of smooth-membraned vesicles appear in the plasm. These vesicles fuse to make the outer membrane of the filament. Later, its inner structures originate—the axial electron-dense substance, filling the hollow lumen of the filament, and a middle, electron-transparent layer. The structure of the filament is discussed in relation to its function and with regard to the findings of other authors. The polaroplast is a laminated structure, originating possibly by transformation of endoplasmic reticulum; the polar cap forms its apical part. The cap is also lamellar; its substance reaches into the lumen of the filament for a certain distance. No micropyle was discovered in the shell; the filament is fastened to the polar cap. These observations on microsporidian development and on the structure of their spores are compared with similar data on myxosporidian species. Such a comparison speaks clearly in favor of the complete taxonomic separation of the Microsporidea from the Myxosporidea, the latter being quite different also from other sporozoa sensu lato.     

Ultrastructural and immunocytological studies on the rhizoplast in the chrysophycean alga Ochromonas danica

The rhizoplast, a striated band elongating from the flagellar basal body to the nucleus, is conspicuous in cells of Ochromonas danica Prings. In interphase cells, it runs from the basal body of the anterior flagellum to the space between the nucleus and the Golgi body. In O. danica, the rhizoplast duplicates during mitosis and the two rhizoplasts serve as mitotic poles. In the present study, we reinvestigated mitosis of O. danica using transmission electron microscopy and immunofluorescence microscopy, especially focusing on the rhizoplast. The nuclear envelope became dispersed during metaphase, and the rhizoplasts from two sets of the flagellar basal bodies functioned as the mitotic poles. Immunofluorescence microscopy using anti‐α‐tubulin, anti‐centrin and anti‐γ‐tubulin antibodies showed that centrin molecules were localized at the flagellar basal bodies, whereas γ‐tubulin molecules were detected at the rhizoplast during the whole cell cycle.     

Effect of Filtered Cultures of Flagellate Ochromonas sp. on Colony Formation in Microcystis aeruginosa

The possible effect of filtered cultures of flagellate Ochromonas sp. on colony formation in M. aeruginosa was investigated in this paper. The results show that filtered cultures of flagellates fed with M. aeruginosa could induce colony formation in M. aeruginosa. Furthermore, induction strength is clearly dependent on the concentration of flagellates and filtered cultures. However, no colonial M. aeruginosa was found in the treatments of filtered cultures of flagellates fed with Microcystis wesenbergii, filtered cultures of flagellate fed with Chlorella pyrenoidosa, and algae homogenates. This suggests that infochemicals released from flagellates fed with M. aeruginosa may be a trigger for colony formation in M. aeruginosa. The clearance rates of flagellates on algae were markedly decreased when they were cultivated with induced colonial M. aeruginosa. These indicate that colony formation in M. aeruginosa is a predator‐induced defense which could reduce predation risk from flagellates (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Ultrastructural and Morphometric Observations of Cortical Endoplasmic Reticulum in Arbacia,Spisula and Mouse Eggs*

Results of morphometric investigations indicate that Arbacia eggs possess a network of cortical endoplasmic reticulum equal in volume and surface area to that within the subcortex. The cortical endoplasmic reticulum surrounds individual cortical granules and forms associations with the plasma membrane reminiscent of junctions shared by the sarcolemma and sarcoplasmic reticulum. Mouse eggs, which also exhibit a cortical granule reaction, possess endoplasmic reticulum that is associated with cortical granules and the plasmalemma. The same relative volume of cortical endoplasmic reticulum is present in mouse eggs as in Arbacia. Significantly less cortical endoplasmic reticulum is present in Spisula eggs which do not undergo cortical granule discharge upon activation. These observations are discussed in light of the hypothesis that the cortical endoplasmic reticulum transduces the interaction of the gametes into an intracellular calcium release which initiates the cortical granule reaction and the activation of development.     

Cytochemical Changes in Aging Ochromonas; Evidence for an Alkaline Phosphatase

SYNOPSIS. Changes accompanying aging in light-grown stationary cultures of Ochromonas danica were examined cytochemically. Succinate dehydrogenase activity increased during the log phase and decreased steadily during stationary and later phases. Acid phosphatase, alkaline phosphatase and lipase activities increased during the several phases of growth, as did accumulation of lipid. These results imply loss of mitochondrial activity and a gain in lysosomal activity with aging of the cell population. Alkaline phosphatase, widely distributed in animals and believed absent from most photosynthetic organisms and bacteria, is here reported in the photosynthetic genus Ochromonas.     

Ultrastructural Observations of Experimental Naegleria Meningoencephalitis in Mice: Intranuclear Inclusions in Amebae and Host Cells*

SYNOPSIS. Primary amebic meningoencephalitis was experimentally produced in mice through intranasal instillation of pathogenic Naegleria fowleri. Experimental animals had a 64% mortality, with average time of onset of symptoms or death occurring on the 7–8th day following inoculation. Ultrastructural studies of the olfactory lobes from brains of dead (or sacrificed) animals revealed major concentrations of amebae in the perivascular regions; amebae were also seen to be under attack by host polymorphonuclear leukocytes, and in the lumina of blood vessels. Amebae in brain tissue contained 30 nm intranuclear particles arranged in clusters. In the brains of some mice, dead presumably as a result of amebic meningoencephalitis, particles and crystalloids were observed in the nuclei of degenerating cells of the central nervous system. Some alternatives are examined to explain a possible relationship between ameba intranuclear particles and mouse brain cell intranuclear inclusions.     

Studies on the Origin of the Methyl Groups of Choline in Ochromonas malhamensis*

SYNOPSIS. Five- to 6-day-old resting cells of Ochromonas malhamensis were incubated at pH 6.5 with glucose and appropriate C¹⁴ precursors of the methyl groups of phospholipid-choline. Under the experimental conditions L-methionine-C¹⁴H₃ was the most efficient source of choline-methyl groups, followed by formate-C¹⁴, formaldehyde-C¹⁴ and DL-serine-3-C¹⁴, respectively. Glycine-2-C¹⁴ was not incorporated into choline. Both L-methionine-C¹⁴H₃ and formate-C¹⁴ served as precursors for the methyl groups of monomethylethanolamine, dimethylethanolamine and choline. Addition of non-radio-active L-methionine depressed the incorporation of formate-C¹⁴ into choline-methyl groups by 50%. The results support the hypothesis that methionine can be the source of all 3 methyl groups of choline, and that formate is probably converted to the methyl group of methionine before transmethylation to choline. However, an alternate pathway from single-carbon sources cannot be excluded.     

Ultrastructural and Other Observations Which Suggest Suctorian Affinities for the Taxonomically Enigmatic Ciliate Cyathodinium*

SYNOPSIS. Two species of the taxonomically enigmatic genus Cyathodinium, C. piriforme and C. cunhai, were studied in some detail at both light and electron microscopic levels. Data obtained strongly suggest suctorian affinities for the genus, since a number of structures or features are strikingly reminiscent of similar (if not homologous) structures recently discovered in ciliates belonging to the order Suctorida. Endosprits (suctorial tentacles?) of Cyathodinium show an arrangement of microtubules not unlike that known for several suctorians, especially Acineta and Tokophrya. Haptocysts or missile-like bodies, ca. 600 mμ long, have been observed within endosprits and free in the cytoplasm; again this is reminiscent of the complex organelles recently described from several suctorian groups. Mouthlessness, coupled with the presence of a ventral depression (functioning in gathering prey at distal ends of endosprits?) and the presence of food vacuoles in the cytoplasm, further support a suctorian mode of feeding. Finally, stages in the curious life cycle of Cyathodinium suggest neoteny and a basic similarity to endogenous budding processes in certain suctorians.     

Fine Structure of the Cell Surface and Golgi Apparatus of Ochromonas*

SYNOPSIS. Ochromonas danica has an unusually flexible cell surface capable of producing projections of varying sizes and shapes: large projections, 340–360 nm long, and small projections, 50–110 nm long. These projections have been demonstrated by transmission and scanning electron microscopy; some of them may break off into the medium and be the source of extracellular membranes and vesicles reported in the cell-free O. danica growth medium. Ruthenium red stained the acid mucopolysaccharide layer just outside the cell surface as well as small blebs at the cell surface. The Golgi complex of O. danica, Ochromonas malhamensis, Ochromonas sociabilis and Ochromonas sp. produced small coated vesicles which may move toward and fuse with the plasma membrane. The role of the several vesicles is unknown but possible functions are discussed.     

Ultrastructural Observations on CTC-Induced Callose Formation in Riella helicophylla

The Ca²⁺-chelator CTC binds to a specific site on both outer surfaces of all non-meristematic cells of the unistratose thallus of Riella, known to be rich in anionic wall components and calcium, and induces there the deposition of callose. Structural changes in this region during prolonged CTC treatment have been followed by light and transmission electron microscopy. With fluorescence microscopy punctate structures can be detected after 10 min, which upon longer incubation in CTC develop into large vesicular bodies, surrounded by a circular structure. The aniline blue-derived fluorescence intensity of these structures is highest in cells of the extension growth zone. At the ultrastructural level a mosaic of numerous smooth-surfaced vesicles, presumably containing callose, initially appears subjacent to the plasma membrane. These vesicles swell and fuse with each other, forming ultimately a circular fusion profile with the plasma membrane. This complex of callose-forming vesicles is thought to develop from elements of the partially coated reticulum (PCR), based on the presence of coated vesiculation profiles on the callose vesicles and numerous aggregates of coated vesicles in their immediate vicinity. After 30 min in CTC osmiophilic particles appear around these callose vesicles and at the cytoplasmic face of mitochondria. They are later (after 60 min) deposited in the periplasmic space between wall and plasma membrane and are also released into the surrounding medium. As judged by their reaction with FeCl₃, the osmiophilic particles appear to be phenolic in nature. We propose that upon binding of CTC a local increase of cytoplasmic calcium triggers callose synthesis in PCR-like compartments beneath the plasma membrane. However it remains to be shown as to why callose is synthesized exclusively in these intracellular compartments and not at the plasma membrane.     

Phagotrophic Ingestion of a Blue-Green Alga by Ochromonas*†

Anacystis nidulans disappeared rapidly from culture in the presence of an unidentified species of Ochromonas. Disappearance was light-independent and could be induced neither by bacteria associated with, nor by soluble products released from the flagellate. Electronmicrographs of mixed cultures revealed numerous A. nidulans cells in various stages of digestion within vacuoles of Ochromonas. Evidently the disappearance of the alga from culture resulted from phagotrophy by the chrysomonad. A 2-stage digestive process is suggested whereby A. nidulans cells are initially sequestered in the posterior “leucosin” vacuole and then undergo the terminal stages of digestion and elimination in smaller, peripheral vacuoles.     

Conservation of Glycerolated Cultures of Ochromonas danica and O. malhamensis at −10 C

SYNOPSIS. Ochromonas danica in a complex natural growth medium dies at 6–10 C in 4 days; O. malhamensis in ∼2 days. O. danica grown in the medium supplemented with 4.0% glycerol survived at −10±2 C for 35 days, and with 8% glycerol 29 days. O. malhamensis lasted only to 5 days in these media supplemented with 4% glycerol. Ethylene glycol and dimethylsulfoxide were too toxic to be effective. Difficulties in freeze-preservation of certain other phagocytic cells, notably blood granulocytes having comparatively simple flexuous outer membranes, add interest to use of O. danica and O. malhamensis as test organisms for preservation methods, especially in the convenient, inexpensive -10 to -20 C range. Biphasic media with an overlay of distilled water serve for conservation at room temperature. Problems of mutational erosion of these photosynthetic phagotrophs are discussed.     

Ultrastructural Observations on Life Cycle Stages of Pneumocystis carinii

An ultrastructural study of life cycle stages of Pneumocystis carinii in infected rat lungs in situ was undertaken utilizing 8 different modes of fixation. Three of the fixatives employed gave good fixation of cysts and intracystic bodies, but for the trophic forms fixation was only fair. Both the trophic forms and intracystic bodies have nuclear pores. The mitochondria of the organism have cristac that appear lamellar. One of the fixation modes revealed a thin, electron-dense layer on the outer surface of the cell wall, a "fuzzy coat" that had not been described previously. This material appears to mediate tight adhesion of trophic forms with other trophic forms, cysts, and with pneumocytcs of the lung alveolus.