Poly(Ethylene Oxide), a New Slowing Agent for Protozoa

SYNOPSIS The water-soluble, viscoelastic resin Polyox® (WSR 301), a poly(ethylene oxide) of high molecular weight (4 million) is introduced as a new slowing agent for protozoa. Generally, as the kinetic viscosity of the resin increased from 0.25% to 1% (w/v), the swimming velocity of Euglena gracilis, Didinium nasutum, Paramecium aurelia, Blepharisma undulans , and Prorodon platyodon decreased. The 1.0% solution had the highest viscosity and decreased velocity more effectively than 1.0% methyl cellulose and Protoslo® solutions. The Polyox solutions differed from those of methyl cellulose and Protoslo by having, in addition to viscous drag, an elastic recoil that pulled the protozoa backwards when their swimming efforts stopped. The toxicity of these slowing agents was determined using 10 P. aurelia /test slide preparation. Paramecium numbers decreased in 1.0% methyl cellulose and Protoslo to nearly zero by 24 hr; in Polyox, not only were most of these ciliates alive after 24 hr, but many survived for 96 hr and divisions occurred in 0.25% and 0.50% solutions.     

Swimming velocity of Paramecium under the conditions of weightlessness

During the 6 min-lasting "free-fall conditions" (4 x 10(-6) g) of the parabolic flight of a sounding rocket Paramecium aurelia cells showed an increase of 7.5 % in their mean swimming velocity. A detailed analysis revealed that the kinetic response was transient: after 3 min the velocity decreased to the speed of the former horizontal swimming at 1 g. Control experiments simulating the influence of vibration and hypergravity during launch of the rocket lead to the conclusion that the increase of the velocity during the parabolic flight was exclusively induced by the transition to 0 g. An increased velocity was also observed under the condition of simulated weightlessness on a fast-rotating clinostat microscope.     

Defective ion regulation in a class of membrane-excitation mutants in Paramecium.

The "paranoiac" mutants of Paramecium aurelia show prolonged backward swimming in solutions containing Na+, unlike wild-type paramecia, which jerk back and forth in Na+ solutions. The paranoiac mutants in Na+ solutions also show large losses of cellular K+ and large influxes of Na+. Three different paranoiac mutants all show similar defects in ion regulation but to different degrees. Wild-type Paramecium, in contrast, shows no Na+ -dependent loss of cellular K+ and a much smaller Na+ influx. In K+ -containing solutions, there is no difference between wild-type and paranoiac paramecia with respect to their cellular K+ content. The Na+ influx, the K+ loss, and the duration of backward swimming are all proportional to the extracellular Na+ concentration. Electrophysiologically, the backward swimming of the paranoiac mutants corresponds to a prolonged depolarization of the membrane potential, while the backward jerks of wild-type Paramecium correspond to a series of transient depolarizations. We propose that the large Na+ influxes and the large K+ effluxes in paranoiacs occur during the periods of backward swimming, while the membrane is depolarized.     

Chemical Homologies Between Protozoa

SYNOPSIS. Nucleic acid hybridisation involving DNA samples was used to study relationships between various protozoa. The most hybridisation or nucleic acid homology was always in the homologous reaction between 2 DNA samples from the same source. It was concluded that all the protozoa tested have nucleotide sequences or genes in common with Paramecium aurelia. The ciliates could be placed in a sequence of decreasing homology relative to Paramecium: Tetrahymena, Colpidium, Stentor, Didinium, Dileptus, Blepharisma. Actinosphaerium had fewer sequences than any of the ciliates and the flagellate Euglena had the fewest sequences in common. The bacterium Aerobacter had none. Similar relationships were inferred from competitive hybridisation experiments; these relationships were also in general mirrored by morphologic relationships and overall G + C base compositions which ranged from 46% for Euglena to 32% for P. aurelia. These experiments, it is hoped, will contribute to studies on origins of the metazoa.     

Responses of Paramecium to Temperature Change

SYNOPSIS. The effect of temperature on the swimming velocity of Paramecium was investigated. When paramecia cultured at 25 C were transferred to various temperatures, their swimming velocity was increased immediately and then decreased exponentially with time to a new steady velocity. The relaxation time was about 1 min, independent of the new temperature. At a constant temperature the steady velocity was inversely proportional to viscosity. The velocity acceleration was observed when the sudden temperature change was larger than ± 1 C. Its magnitude became constant when the temperature change was greater than several degrees. The steady velocity as a function of temperature had a sharp maximum at the culture temperature and decreased on both sides of this temperature. Incubation of paramecia at 30 C for several hr after cultivation at 25 C shifted the maximum temperature of the steady velocity to 30 C. The temperature at which paramecia gathered in a temperature gradient cell correlated closely with the temperature of the maximum steady velocity.     

Orientation of paramecium swimming in a static magnetic field: Dependence on membrane lipid fluidity

We studied the swimming orientation of the ciliated protozoan Paramecium aurelia in a static magnetic field (0.78 T). P. aurelia is a complex of species termed syngens, whose cell morphology appears similar on microscopic examination. In the magnetic field, the cells of some syngens gradually changed their swimming orientation so that they were swimming perpendicular or parallel to the magnetic field, although such sensitivity to magnetic fields differs between syngens. When the temperature of the cell suspension was raised, the magnetic sensitivity of the cells was decreased. On the other hand, when the cells were cultured beforehand at a high temperature, their magnetic sensitivity was increased. These results raise the possibility that membrane lipid fluidity, which is inversely proportional to the membrane lipid order, contributes to the magnetic orientation of syngens. In this study, measurements of membrane lipid fluidity obtained using fluorescence image analysis with the lipophilic dye, laurdan (6-lauroyl-2-dimethylaminonaphtalene), showed that the degree of membrane lipid fluidity was correlated with the differences in magnetic orientation between syngens. That is, the syngens with decreased membrane fluidity showed an increased degree of magnetic orientation. Therefore, the membrane lipid order is a key factor in the magnetic orientation of Paramecium swimming.     

A computer method for assessing the average rate of movement of Paramecium caudatum by a cross-sectional light beam

The possibility of using the motility of Paramecium caudatum for computer biotesting of toxic substances was studied. The method is based on measuring the number and duration of impulses that arise when protozoa chaotically moving in a layer 0.5 mm thick cross a red light beam (1 mm in diameter). It was found that, in the presence of Cu2+ ions and menadione bisulfite, a compound having prooxidant properties, the number of impulses decreased with a concurrent increase in the duration of impulses. The high correlation between the number of impulses and the reciprocal of impulse duration indicates that the motility of paramecium by the action of Cu2+ ions and menadione slow down, and even an immobilization of some of them occurs. The mean swimming velocity of protozoa was reduced by half within 20 min by the action of Cu2+ at a concentration of 90 microM (6 mg/l of Cu2+ ions) and within 80 min by the action of menadione bisulfite at a concentration of 50 microM. It was shown that the slowing down of motility, which indicates the presence of a toxicant in the medium, is a faster physiological reaction than cell death.     

西藏温泉两种中国新记录纤毛虫第一双小核草履虫和明布雷斯四膜虫的形态学和系统发育学研究

研究利用活体观察、蛋白银和氨银染色技术对采自西藏温泉的寡膜纲咽膜类纤毛虫第一双小核草履虫(Paramecium primaurelia)和膜口类纤毛虫明布雷斯四膜虫(Tetrahymena mimbres)进行了形态学研究, 首次描述了这两种纤毛虫细胞核器的形态和位置、纤毛图式和口膜的排布模式; 并且测定了SSU rDNA和COXⅠ标记基因序列, 基于这两种基因的系统发育树均支持P. primaurelia聚在草履虫P. aurelia复合种中, T. mimbres聚在四膜虫T. borealis类群中。两种纤毛虫均为中国新记录种, 且首次在高原温泉中发现, 不仅为西藏地区温泉原生动物生物资源的发掘提供新的方法和思路, 也为原生动物环境适应性研究提供基础资料。     

Magnetic field influence on paramecium motility

The influence of a moderately intense static magnetic field on movement patterns of free swimming Paramecium was studied. When exposed to fields of 0.126 T, these ciliated protozoa exhibited significant reduction in velocity as well as a disorganization of movement pattern. It is suggested that these findings may be explained on the basis of alteration in function of ion specific channels within the cell membrane.     

Diaminobenzidine reactivity of mitochondria and peroxisomes in Tetrahymena and in wild-type and cytochrome oxidase-deficient Paramecium.

Reactivity of mitochondria and peroxisomes to diaminobenzidine was investigated in Tetrahymena pyriformis and in wild-type and cytochrome oxidase-deficient Paramecium aurelia. Wild-type and cytochrome oxidase-deficient Paramecium gave positive mitochondrial reactions in the absence of added H2O2, and the deposits were enhanced by the addition of H2O2, whereas Tetrahymena gave positive mitochondrial reactions only upon addition of H2O2. These results are discussed in the light of the current ideas concerning the mechanism of staining by diaminobenzidine. Peroxisome-like organelles which react positively to diaminobenzidine, the reaction being partially inhibited by aminotriazole, were identified in both protozoa.     

Comparison of the evolutionary distances among syngens and sibling species of Paramecium

The morphospecies of the genus Paramecium have several mating type groups, so-called syngens, composed of cells of complementary mating types. The Paramecium aurelia complex is composed of 15 sibling species assigned to the species from the syngen. To increase our understanding of the evolutionary relationships among syngen and sibling species of the genus Paramecium, we investigated the gene sequences of cytosol-type hsp70 from 7 syngens of Paramecium caudatum and 15 sibling species of P. aurelia. Molecular phylogenetic trees indicated that the P. aurelia complex could be divided into four lineages and separated into each sibling species. However, we did not find any obvious genetic distance among syngens of P. caudatum, and they could only be separated into two closely related groups. These results indicated that the concept of syngens in P. caudatum differs quite markedly from that of the P. aurelia complex. In addition, we also discuss the relationships among these species and other species, Paramecium jenningsi and Paramecium multimicronucleatum, which were once classified as varieties of P. aurelia.     

SOME PHYSIOLOGICAL ACTIONS OF CYANIDES

1. The physiological actions of HCN and its salts appear to be due (a) to the ease with which HCN molecules penetrate living cells and then ionizing, exert their influence by means of H ions and CN ions; (b) to the weakness of HCN as an acid, which permits at neutrality or at even slight alkalinity the presence of a considerable amount of free HCN molecules in the presence of their salts; (c) to specific effects occasioned by its chemical activity. 2. The order of resistance of various protozoans to HCN resembles that of the same protozoa to CO₂ and to H₂S, but is the reverse of their resistance to mineral acids. 3. HCN in acid, neutral, or slightly alkaline media produces intracellular acidity because of the rapid penetration of HCN molecules into the cell. 4. HCN acts specifically on certain species of protozoa, the order of resistance of Paramecia being Paramecium caudatum > Paramecium mullimicronucleatum > Paramecium aurelia > Paramecium bursaria.     

The role of cortical orientation in the control of the direction of ciliary beat in Paramecium

The swimming behavior of many ciliate protozoans depends on graded changes in the direction of the ciliary effective stroke in response to depolarizing stimuli (i.e., the avoiding reaction of Paramecium). We investigated the problem of whether the directional response of cilia with a variable plane of beat is related to the polarity of the cell as a whole or to the orientation of the cortical structures themselves. To do this, we used a stock of Paramecium aurelia with part of the cortex reversed 180 degrees. We determined the relation of the orientation of the kineties (ciliary rows) to the direction of beat in these mosaic paramecia by cinemicrography of particle movements near living cells and by scanning electron microscopy of instantaneously fixed material. We found that the cilia of the inverted rows always beat in the direction opposite to that of normally oriented cilia during both forward and backward swimming. In addition, metachronal waves of ciliary coordination were present on the inverted patch, travelling in the direction opposite to those on the normal cortex. The reference point for the directional response of Paramecium cilia to stimuli thus resides within the cilia or their immediate cortical surroundings.     

Identification of Specific Antibodies Prepared Against Purified Immobilization Antigens of Paramecium aurelia

SYNOPSIS The identity of antibodies prepared against preparations of isolated immobilization antigen protein of Paramecium aurelia has been studied. Antisera were fractionated by ammonium sulfate precipitation, sucrose gradient centrifugation, and ion exchange chromatography on DEAE cellulose. The different fractions were assayed for the presence of specific antibodies by the immobilization test, and the globulins were identified by immuno-electrophoresis. It is concluded that in the sera studied in this investigation, specific antibodies are excusively of the class Ig G.     

A Method for the Isolation of Macronuclei from Paramecium aurelia

SYNOPSIS. A method is described for the isolation of macronuclei from Paramecium aurelia. It has also been successfully employed with Didinium nasutum. The yield is up to 50% with P. aurelia. Some results on the DNA, RNA and protein contents are given. The isolated macronuclei of P. aurelia are able to incorporate ATP and UTP into acid-insoluble material, a process which is probably RNA synthesis mediated by RNA polymerase. The macronuclei were thus highly purified, and retained a measure of biosynthetic activity.     

A two-locus molecular characterization of Paramecium calkinsi

Paramecium calkinsi (Ciliophora, Protozoa) is a euryhaline species which was first identified in freshwater habitats, but subsequently several strains were also collected from brackish water. It is characterized by clockwise spiral swimming movement and the general morphology of the "bursaria type." The present paper is the first molecular characterization of P. calkinsi strains recently collected in distant regions in Russia using ITS1-5.8S- ITS2-5'LSU rDNA (1100bp) and COI (620bp) mtDNA sequenced gene fragments. For comparison, our molecular analysis includes P. bursaria, exhibiting a similar "bursaria morphotype" as well as species representing the "aurelia type," i.e., P. caudatum, P. multimicronucleatum, P. jenningsi, and P. schewiakoffi, and some species of the P. aurelia species complex (P. primaurelia, P. tetraurelia, P. sexaurelia, and P. tredecaurelia). We also use data from GenBank concerning other species in the genus Paramecium and Tetrahymena (which used as an outgroup). The division of the genus Paramecium into four subgenera (proposed by Fokin et al. 2004) is clearly presented by the trees. There is a clear separation between P. calkinsi strains collected from different regions (races). Consequently, given the molecular distances between them, it seems that these races may represent different syngens within the species.     

Cytochrome b sequence data suggest rapid speciation within the Paramecium aurelia species complex

We investigated mitochondrial Cytochrome b sequences from all 15 members of the enigmatic Paramecium aurelia species complex (Ciliophora). The analysis revealed high genetic distances between the different P. aurelia species (6.1-19.8%) and a largely unresolved, star-like phylogenetic tree. This result strongly supports a rapid radiation in the evolutionary history of this species complex and it correlates well with the hypothesis that the extant species diversity may have originated from the neutral consequences of a whole genome duplication in the common ancestor of P. aurelia.     

Relationships of new sibling species of Paramecium jenningsi based on sequences of the histone H4 gene fragment

Paramecium jenningsi Diller & Earl, 1958 belongs to the "aurelia" subgroup of the genus, together with Paramecium caudatum, Paramecium multimicronucleatum, Paramecium schewiakoffi and species of the Paramecium aurelia complex. The original assumption that the morphospecies P. jenningsi was a single genetic species was questioned because a comparison of genome analyses suggested the possibility that this morphospecies contained two sibling species. To refine understanding of relationships between the strains of P. jenningsi, a molecular phylogenetic analysis was conducted using H4 gene sequences. Some polymorphic sites were found among the compared sequences, and specific patterns of single nucleotide polymorphism (SNP) markers characterize two groups of strains of P. jenningsi. Phylogenetic trees constructed by different methods identified two clearly different groups (from Japan and mainland Asia) whatever the method used. The sequences of the H4 gene analyzed in the present study are closely related, and provide a good subject for phylogenetic analysis. The presence of two isolated groups of strains in the P. jenningsi group can reveal the evolutionary relationship between them; it confirms the presence of two sibling species among the known strains of P. jenningsi, and the close relationships between them and species of the P. aurelia complex.     

Negative Geotactic Behavior of Paramecium Caudatum is Completely Described by the Mechanism of Buoyancy-Oriented Upward Swimming

This paper presents evidence that the negative geotactic behavior of Paramecium caudatum takes place by the mechanism of buoyancy-oriented upward swimming. Photographs of swimming pathways of the organisms were completely described by two dynamic equations for the translational motion of the center of gravity of the organism's body and for the rotational motion of the organism's body about its center of gravity, where the rotational torque is induced by a slight difference in position between the center of gravity and the center of buoyancy. It now seems unlikely that complicated mechanisms such as the statocyst mechanism and the gravity-propulsion mechanism, which have been proposed by many investigators, need be considered for other protozoa since preliminary observation and analysis of other ciliates such as Paramecium multimicronucleatum, Paramecium tetraurelia, and Tetrahymena pyriformis also strongly suggested that their negative geotaxis is due to buoyancy-oriented upward swimming.     

Histochemical Use of Borohydrides as Aldehyde Blocking Reagents

Complete blocking of the Schiff reaction applied after HIO₄ oxidation is attained by 1%, 0.5% or 0.2% NaBH₄ in 1% Na₂HPO₄ with a 1 min exposure, 0.1% NaBH₄ requires 2 min. KBH₄ was also completely effective in the same solvent at 1, 0.5 and 0.25% in 1 min. The solutions deteriorate on standing, so that 0.5% NaBH₄ is effective in 1 min at 7 hr but 5 min is needed at 24 hr and is ineffective at 36 hr; 1% KBH₄ requires 4 min at 24 hr when fresh, and showed deterioration in 2-4 days. Saturated (0.4%) isopropanol and 1% pyridine solutions required 10-15 min when fresh and showed deterioration in 2-4 days. No satisfactory results were obtained with 80% or absolute dioxan or with methyl cellosolve in times of an hour or less; even a 24 hr exposure was ineffective with 0.2% in 80% dioxan.