A new low cost microbiotest with the freshwater ciliate protozoan Spirostomum ambiguum

This paper reports on preliminary experiments on the controlled culturing of the freshwater ciliated protozoan Spirostomum ambiguum, as a first step in the development of a new low cost microbiotest, to complement the limited battery of standardized cost-effective toxicity tests presently available, with a representative of unicellular biota.Three strains of S. ambiguum originating from France and Poland respectively, have been grown in natural as well as in reconstituted waters, on several types of inert diets and in different temperature and light conditions.The best results in terms of survival and reproduction of the ciliates, leading to a standard recipe for stock culturing, were obtained at 20 °C, in reconstituted Volvic water and on a diet of oat flakes supplemented with dried leave powder as vegetal adjuvant, with weekly renewal of the medium and food. An average generation time of 4 days was obtained in the latter set of stock culturing conditions.     

Electron Microscopy of the Longitudinal Fibrillar Bundle and the Contractile Fibrillar System in Spirostomum ambiguum

SYNOPSIS. A study of the 'longitudinal fibrillar bundle' (LFB) and the 'contractile fibrillar system' (CFS) of a large protozoan ciliate, Spirostomum ambiguum , has been performed by means of an electron microscope. A system of sub-pellicular fibrils has been newly found and its function is discussed. Each LFB runs parallel with a longitudinal row of ciliary bases. It seems to be identical with the so-called kinetodesma. It is composed of tubular fibrils arranged in layered sheets, each of which contains 13 to 35 fibrils with the same diameter as the intra-ciliary fibrils and has a close connection to each of the ciliary bases. The CFS lies on a transitional plane between ectoplasm and endoplasm of the organism and forms a cobweb-like system of myofibrils as a whole. It stands in an intimate relationship with a characteristic vacuolar system. In a peristomial field, the fibrous structures are interrupted and somewhat thickened. A sub-pellicular system is composed of minute fibrils 20 to 26 mμ in diameter. The fibrils run parallel with each other in an antero-posterior direction, immediately beneath the inner pellicular membrane.     

Contraction of protoplasm. I. Cinematographic analysis of the anodally stimulated contraction of Spirostomum ambiguum

Electrically stimulated contraction of Spirostomum ambiguum was investigated by high speed cinematography (up to 6,000 pps). Contraction is completed in about 4 msec following a latent period of up to 30 msec. Reduction in length during contraction followed a sigmoid curve, and final length was about 50% of the original length. Contraction always started at the end of the animal directed towards the anode. When the length of each half was measured separately, it was found that the cathodal end lagged about 1 msec in all cases observed. Rate of contraction was increased when the external calcium contraction was increased, and was decreased in Ca-free and K-free solutions, but was unchanged in K-rich solutions. These results are interpreted in terms of contraction being associated with a relative increase of calcium bound to the contractile protein. The differential migration of potassium and calcium ions in an electric current would result in a temporary lowering of K⁺ at the anodal end of the animal, hence a relative rise would take place in the Ca⁺⁺ available for binding. The results of experiments using changed calcium and potassium concentrations can be explained by this hypothesis which is in general agreement with modern work on muscle contraction and relaxation.     

Macronuclear duplication in the ciliated protozoan Euplotes

The ribbon-like macronucleus of Euplotes eurystomus pinches in half amitotically at each cell division. Several hours before the actual division two lightly staining duplication bands (reorganization bands) appear at the ends of the nucleus and approach each other slowly, finally meeting near the middle. Distal to the bands, that is, in regions through which the bands have already passed, the concentration of DNA (Feulgen) and "histone" (alkaline fast green) is greater than in the central zone. These facts suggest the hypothesis that DNA-histone synthesis takes place in a sequential fashion starting at the tips of the nucleus and proceeding to the middle. That this hypothesis is correct is shown by autoradiographic and photometric observations. Tritium-labelled thymidine is incorporated only in a limited region immediately distal to the bands. The average amount of Feulgen dye bound by the nucleus rises as the duplication bands approach each other, and is double the presynthesis value by the time the bands meet. A similar rise in the alkaline fast green dye is seen in duplicating nuclei, although no completely post-synthesis values were obtained in this study. The quantitative data are consistent with the assumption that the macronucleus contains a number of DNA-histone "units," presumably chromosomes, each of which duplicates once and only once.     

Structural components of sphingophosphonolipids from the ciliated protozoan, Tetrahymena pyriformis WH-14.

1. Two sphingophosphonolipids were isolated from the lipids of the ciliated protozoan, Tetrahymena pyriformis WH-14. They were ceramide N-methyl-2-aminoethylphosphonate (CMAEP) and ceramide 2-aminoethylphosphonate (CAEP), in yields of 0.05 mg/g and 1.74 mg/g dry cells, respectively. 2. Two chromatographically distinguishable CAEP species were found, a slow-moving major component and a minor component which moved faster; the slow-moving one contained primarily hydroxy fatty acids, while in the other one nonhydroxy fatty acids were predominant. However, their long-chain base constituents were similar. 3. The major fatty acids of CAEP were 2-hydroxy acids with carbon numbers of 16 to 19, which were almost exclusively iso-types. The fatty acids of CMAEP consisted mainly of palmitic, iso-octadecanoic, and 2-hydroxy iso-heptadecanoic acids. 4. The long-chain bases were dominated by C16, C17, and C19 iso-4-sphingenine homologs.     

A pseudo-phytochelatin synthase in the ciliated protozoan Tetrahymena thermophila

Phytochelatins (PCs) and metallothioneins (MTs) are the two major heavy metal chelating peptides in eukaryotes. We report here on the identification of a biosynthetically inactive pseudo-phytochelatin synthase enzyme (TtψPCS) in the ciliate Tetrahymena thermophila, the first of this kind (pseudo-PCS) to be described in eukaryotes. TtψPCS which resembles a true PCS at the N-terminal region, while it is most divergent in its Cys-poor C-terminal region, was found to be up-regulated under cadmium stress conditions. However, only glutathione (GSH) hydrolysis products, but not PCs, could be detected in extracts from Cd-treated cells. The latter feature is reminiscent of pseudo-PCS enzymes recently identified in cyanobacteria, which are also biosynthetically inactive, but capable to hydrolyze GSH.     

The histones of the ciliated protozoan Stylonychia mytilus

Histones were extracted from pure macronuclei, micronuclei and macronucleus anlagen and from chromatin which was isolated from these different nuclear fractions. Analysis of these preparations by polyacrylamide gel electrophoresis showed differences in electrophoretic patterns between the histones of these nuclei.     

Alternative use of chromosome fragmentation sites in the ciliated protozoan Oxytricha nova.

During its life cycle, the hypotrichous ciliated protozoan Oxytricha nova transforms a copy of its micronucleus, which contains chromosome-sized DNA, into a macronucleus containing linear, gene-sized DNA molecules. A region of the micronuclear genome has been defined that gives rise to two distinct macronuclear DNA molecules during development. Through analysis of recombinant macronuclear and micronuclear clones, the generation of the two macronuclear DNA molecules was shown to be the result of alternative use of chromosome fragmentation sites. In addition, evidence was obtained that adjacent micronuclear precursors of macronuclear DNA molecules can overlap by a few base pairs. The significance of these findings in relation to developmental chromosome fragmentation is discussed.     

Actin in the ciliated protozoan Climacostomum virens: purification by DNAse I affinity chromatography, electrophoretic characterization, and immunological analysis.

The anti-actin monoclonal antibody (mab) JLA20 (Lin: Proc. Natl. Acad. Sci. U.S.A. 78:2335-2339, 1981) labels a 43 kD protein on Western blots of Climacostomum cell extracts; this protein does not react with an anti-alpha-smooth muscle actin mab (Skalli et al.: J. Cell Biol. 103:2787-2796, 1986) nor with an anti-alpha-sarcomeric actin mab (Skalli et al.: Am. J. Pathol. 130:515-531, 1988). This protein binds to DNAse I and can be purified by DNAse I affinity chromatography. The affinity-purified actin also reacts with mab JLA20. Two-dimensional gel analysis reveals that Climacostomum actin focuses as three spots which are more basic than the mammalian actin isoforms. After addition of KCl, the affinity-purified actin polymerizes into filaments as shown by electron microscopy after negative staining.     

Fine Structure of Mitochondria of Spirostomum ambiguum as Seen in Sectioned and Negatively-Stained Preparations*

SYNOPSIS. Comparisons are made between sectioned and negatively-stained mitochondria of the ciliate Spirostomum ambiguum. Particulate elements 70–80 A in diameter are associated with the surface of tubular cristae of negatively-stained and disrupted mitochondria; such particles are not seen in sectioned mitochondria fixed in various ways. As measured in sectioned material, the inner mitochondrial membrane forming the tubular cristae is about 100 A thick, while the outer mitochondrial membrane is about 50 A thick and is the more labile of the 2.     

Cultural characteristics of the marine ciliated protozoan, Uronema marinum Dujardin

The marine hymenostome ciliate Uronema marinum Dujardin was isolated from sediment from Robin Hood's Bay, Yorkshire. Experiments on the growth of U. marinum were carried out in batch cultures in which the ciliate was maintained on a diet of Vibrio sp. The duration of the lag phase varied directly with the age of the inoculum. Factorially designed experiments were used to investigate the effects of salinity and temperature on the rate of logarithmic growth of the ciliate. A response surface analysis of the data indicated that there was interaction between salinity and temperature. Regression equations predicted a minimum doubling time of 2.5 h at 32‰ and 25 °C. The cell volume of U. marinum varied during growth; it reached a maximum at the end of the lag phase and decreased during the logarithmic phase to become minimal in the stationary phase.     

Structure and expression of two temperature-specific surface proteins in the ciliated protozoan Tetrahymena thermophila.

The presence of specific proteins (known as immobilization antigens) on the surface of the ciliated protozoan Tetrahymena thermophila is under environmental regulation. There are five different classes (serotypes) of surface proteins which appear on the cell surface when T. thermophila is cultured under different conditions of temperature or incubation medium; three of these are temperature dependent. The appearance of these proteins on the cell surface is mutually exclusive. We used polyclonal antibodies raised against 30 degrees C (designated SerH3)- and 40 degrees C (designated SerT)-specific surface antigens to study their structure and expression. We showed that these surface proteins contain at least one disulfide bridge. On sodium dodecyl sulfate-denaturing polyacrylamide gels, the nonreduced 30 degrees C- and 40 degrees C-specific surface proteins migrated with molecular sizes of 69 and 36 kilodaltons, respectively. The reduced forms of the proteins migrated with molecular sizes of 58 and 30 kilodaltons, respectively. The synthesis of the surface proteins responded rapidly and with a time course similar to that of the incubation temperature. The synthesis of each surface protein was greatly reduced within 1 h and undetectable by 2 h after a shift to the temperature at which the protein is not expressed. Surface protein synthesis resumed by the end of 1 h after a shift to the temperature at which the protein is expressed. The temperature-dependent induction of these surface proteins appears to be dependent on the synthesis of new mRNA, as indicated by a sensitivity to actinomycin D. Surface protein syntheses were mutually exclusive except at a transition temperature. At 35 degrees C both surface proteins were synthesized by a cell population. These data support the potential of this system as a model for the study of the effects of environmental factors on the genetic regulation of cell surface proteins.     

Spontaneous motional activity of paramecium Spirostomum ambiguum after gamma-irradiation in a broad variety of dose as a bioassay

The spontaneous motional activity of paramecium Spirostomum ambiguum Ehrbg. has high sensitivity to the gamma-irradiation. The increase of this parameter has been observed just after irradiation in the 0.1 Gy. It is approximately on the similar level (about 35% below control) in a broad variety of doze (to 850 Gy inclusive). After that the dozes 1000-1500 Gy has been obtained, the effect became stronger. The present result has been interesting in the question of the behavior's reaction unicellular using in the indication of the radioactivity contamination of the aquatic aria. And also it is actual in the common problem of the irradiation activity on the biota.     

Distribution of a centrosomal antigen during morphogenesis in the ciliated protozoan Euplotes

Ciliates assemble basal bodies in great number at many stages of the life-cycle. In order to understand their assembly mechanisms, we screened a library of monoclonal antibodies directed against pericentriolar material. One of these antibodies, CTR210, was used previously to follow steps of this assembly process: in Paraurostyla, new basal bodies appear along a scaffold of linear structures recognized by this antibody. The very unusual behavior of this antigen deserved confirmation in other species. In the present study, we show by immunofluorescence that, in another phylogenetically very distant species, Euplotes, basal bodies are assembled in the same pathway during division. In addition, this antibody recognizes a filamentous ring located at the division furrow and linking many basal body assemblages. By cell fractionation and cytoskeletal extraction, we obtained fractions enriched in basal bodies and associated material. Such fractions still display a high complexity in protein composition. These fractions were used to characterize the main target of the antibody as a doublet of 45 kDa. These results confirm previous results in terms of functionality of the protein recognized by the antibody, but raise new questions in terms of the assignment of the recognized protein to the HSP70 family as hypothesized previously.