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.     

Orientation of paramecium swimming in a DC magnetic field

We found that a ciliated protozoan, Paramecium, swam perpendicular to a static (DC) magnetic field (0.68 T). The swimming orientation was similar even when the ionic current through the cell membrane disappeared after saponin treatment. To determine the diamagnetic anisotropy of intracellular organs, macronuclei, cilia, and secretory vesicles, trichocysts, were selectively isolated. Both cilia and trichocysts tended to align their long axis parallel to the magnetic field (0.78 T). Paramecium mutants that lack trichocysts also swam perpendicular to the magnetic field, although the proportion fraction was smaller than the normal population. Since large numbers of cilia and trichocysts are arranged at right angles to the long axis of the cell, the diamagnetic anisotropies of cilia and trichocysts cause the long axis of the cell to align perpendicular to the magnetic field. In contrast to the DC magnetic field, an alternative (AC) magnetic field (60 Hz, 0.65 T) had almost no effect on the swimming orientation of Paramecium.     

Identification of Paramecium bursaria syngens through molecular markers--comparative analysis of three loci in the nuclear and mitochondrial DNA

This is the first attempt to resolve the phylogenetic relationship between different syngens of Paramecium bursaria and to investigate at a molecular level the intraspecific differentiation of strains originating from very distant geographical locations. Herein we introduce a new collection of five P. bursaria syngens maintained at St Petersburg State University, as the international collection of syngens was lost in the 1960s. To analyze the degree of speciation within Paramecium bursaria, we examined 26 strains belonging to five different syngens from distant and geographically isolated localities using rDNA (ITS1-5.8S-ITS2-5'LSU) fragments, mitochondrial cytochrome c oxidase subunit I (COI), and H4 gene fragments. It was shown that P. bursaria strains of the same syngens cluster together in all three inferred molecular phylogenies. The genetic diversity among the studied P. bursaria strains based on rDNA sequences was rather low. The COI divergence of Paramecium bursaria was also definitely lower than that observed in the Paramecium aurelia complex. The nucleotide sequences of the H4 gene analyzed in the present study indicate the extent of genetic differences between the syngens of Paramecium bursaria. Our study demonstrates the diagnostic value of molecular markers, which are important tools in the identification of Paramecium bursaria syngens.     

Electrokaryotypes of macronuclei of several Paramecium species

A comparative study of macronuclear DNA molecules from the following Paramecium species: the P. aurelia complex, P. caudatum, P. bursaria, P. putrinum and P. multimicronucleatum was performed using pulsed-field gel electrophoresis. The electrophoretic pattern was constant and unique for each species, and is referred to herein as its electrokaryotype. Large differences were observed between Paramecium species according to the range and major size of macronuclear DNA fragments, while different strains of the same species, even belonging to different syngens, were characterized by the same electrokaryotype. In this respect sibling species from the P. aurelia complex are as similar as syngens in other Paramecium species, but are unlike conventional species. The principles and value of electrokaryotype analysis for application to ciliates are discussed.     

Effects of an extremely low frequency electromagnetic field on the cell division rate and plasma membrane of Paramecium tetraurelia

The eukaryotic protozoan, Paramecium, was examined as a model for effects of pulsated electromagnetic fields (PEMF) on cells. A 72-Hz PEMF similar to fields employed clinically increased cell division rates in Paramecium by 8.5%. Two calcium transport mutants of these organisms showed differential responses to the same field. Verapamil, a calcium channel blocker, abolished any effect of PEMFs on cell division rates. A fluorescent probe that is thought to sense changes in membrane potential also manifested an altered response in the PEMF-exposed cells whereas a fluorescent lipid bilayer fluidity probe produced evidence of decreased membrane fluidity in the exposed cells. An effect of PEMFs on ion transport mediated by either a direct or indirect effect on the cell membrane is suggested by these studies.     

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.     

Evidence for a correlation between swimming velocity and membrane fluidity of Tetrahymena cells

The influence of the physical state of the membrane on the swimming behaviour of Tetrahymena pyriformis was studied in cells with lipid-modified membranes. When the growth temperature of Tetrahymena cells was increased from 15 degrees C to 34 degrees C or decreased from 39 degrees C to 15 degrees C, their swimming velocity changed gradually in a similar to the adaptive change in membrane lipid composition. Therefore, such adaptive changes in swimming velocity were not observed during short exposures to a different environment. Tetrahymena cells adapted to 34 degrees C swam at 570 microns/s. On incubation at 15 degrees C these cells swam at 100 microns/s. When the temperature was increased to 34 degrees C after a 90-min incubation at 15 degrees C, the initial velocity was immediately recovered. On replacement of tetrahymanol with ergosterol, the swimming velocity of 34 degrees C-grown cells decreased to 210 microns/s, and the cells ceased to move when the temperature was decreased to 15 degrees C. To investigate the influence of the physical state of the membrane on the swimming velocity, total phospholipids were prepared from Tetrahymena cells grown under these different conditions. The fluidities of liposomes of these phospholipid were measured using stearate spin probe. The membrane fluidity of the cells cooled to 15 degrees C increased gradually during incubation at 15 degrees C. On the other hand, the fluidity of the heated cell decreased during incubation at 34 degrees C. Replacement of tetrahymanol with ergosterol decreased the membrane fluidity markedly. Consequently, a good correlation was observed between swimming velocity and membrane fluidity; as the membrane fluidity increased, the swimming velocity increased linearly up to 600 microns/s. These results provide evidence for the regulation of the swimming behaviour by physical properties of the membrane.     

Temperature-dependent changes in the swimming behaviour of Tetrahymena pyriformis-NT1 and their interrelationships with electrophysiology and the state of membrane lipids

The swimming velocity and the amplitude of the helical swimming path of T. pyriformis-NT1 cells grown at 20 degrees C (Tg 20 degrees C) and 38 degrees C (Tg 38 degrees C) were monitored between 0 and 40 degrees C in the presence and absence of electric fields. Within physiological limits the swimming velocity increased and the amplitude decreased as temperature was raised. The temperature profiles of these properties were not linear, and showed discontinuities at different temperatures for the different cultures. The break points in Arrhenius plots of the resting potential, regenerative spike magnitude, repolarization time, swimming velocity and swimming amplitude are tabulated and compared. The initial breakpoints upon cooling were clustered about the breakpoints in fluorescence polarization of D.P.H. in extracted phospholipids, and around the transition temperatures estimated from the literature for the pellicular membrane of these cells. The average of the initial breakpoints on cooling was 22.9 degrees C for Tg 38 degrees C cells and 13.7 degrees C for Tg 20 degrees C cells, a shift of 9.2 degrees C. Unlike Paramecium there is no depolarizing receptor potential in Tetrahymena upon warming. It is suggested that this may be the basis of a behavioural difference between Tetrahymena and Paramecium--namely that in Tetrahymena maximum swimming velocity occurs above growth temperature whereas in Paramecium the two points coincide. Swimming velocity and resting potential were correlated with membrane fluidity within physiological limits, but for other parameters the relationship with fluidity was more complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Evidence for a correlation between swimming velocity and membrane fluidity of Tetrahymena cells

The influence of the physical state of the membrane on the swimming behaviour of Tetrahymena pyriformis was studied in cells with lipid-modified membranes. When the growth temperature of Tetrahymena cells was increased from 15°C to 34°C or decreased from 39°C to 15°C, their swimming velocity changed gradually in a similar to the adaptive change in membrane lipid composition. Therefore, such adaptive changes in swimming velocity were not observed during short exposures to a different environment. Tetrahymena cells adapted to 34°C swam at 570 μm/s. On incubation at 15°C these cells swam at 100 μm/s. When the temperature was increased to 34°C after a 90-min incubation at 15°C, the initial velocity was immediately recovered. On replacement of tetrahymanol with ergosterol, the swimming velocity of 34°C-grown cells decreased to 210 μm/s, and the cells ceased to move when the temperature was decreased to 15°C. To investigate the influence of the physical state of the membrane on the swimming velocity, total phospholipids were prepared from Tetrahymena cells grown under these different conditions. The fluidities of liposomes of these phospholipid were measured using stearate spin probe. The membrane fluidity of the cells cooled to 15°C increased gradually during incubation at 15°C. On the other hand, the fluidity of the heated cell decreased during incubation at 34°C. Replacement of tetrahymanol with ergosterol decreased the membrane fluidity markedly. Consequently, a good correlation was observed between swimming velocity and membrane fluidity; as the membrane fluidity increased, the swimming velocity increased linearly up to 600 μm/s. These results provide evidence for the regulation of the swimming behaviour by physical properties of the membrane.     

Effect of a 60 Hz magnetic field on the behavior of Paramecium

Paramecium multimicronucleatum was used as a model cell to study the effects of 60 Hz magnetic fields on swimming behavior. When exposed to a vertical field of 0.6 T, the cells accumulated at the upper end of the cuvette. An analysis of the swimming behavior revealed that the exposure to the field increased the number of cells swimming upwards maximally at 1 min after onset of the exposure. This effect of the magnetic field was transient, disappearing within a few minutes during the exposure. It is suggested that the magnetic field may amplify to a large extent the negative gravitaxis of Paramecium. Effects of an induced electric field on the swimming behavior are also discussed.     

Poly(ethylene oxide), a new slowing agent for protozoa.

The water-soluble, viscoelastic resin Polyox WSR 301), a poly(ethylene oxide) of high molecular weight (approximately 4 million) is introduces 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, Didnium 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 these ciliates alive after 24 hr, but many survived for 96 hr and divisions occurred in 0.25% and 0.50% solutions.     

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.     

Does Paramecium sense gravity?

In order to get an insight into the cellular mechanisms for the integration of the effects of gravity, we investigated the gravitactic behaviour in Paramecium. There are two main categories for the model of the mechanism of gravitaxis; one is derived on the basis of the mechanistic properties of the cell (physical model) and the other of the physiological properties including cellular gravireception (physiological model). In this review article, we criticized the physical models and introduced a new physiological model. Physical models postulated so far can be divided into two; one explaining the negative gravitactic orientation of the cell in terms of the static torque generated by the structural properties of the cell (gravity-buoyancy model by Verworn, 1889 and drag-gravity model by Roberts, 1970), and the other explaining it in terms of the dynamic torque generated by the helical swimming of the cell (propulsion-gravity model by Winet and Jahn, 1974 and lifting-force model by Nowakowska and Grebecki, 1977). Among those we excluded the possibility of dynamic-torque models because of their incorrect theoretical assumptions. According to the passive orientation of Ni(2+)-immobilized cells, the physical effect of the static torque should be inevitable for the gravitactic orientation. Downward orientation of the immobilized cells in the course of floating up in the hyper-density medium demonstrated the gravitactic orientation is not resulted by the nonuniform distribution of cellular mass (gravity-buoyancy model) but by the fore-aft asymmetry of the cell (drag-gravity model). A new model explaining the gravitactic behaviour is derived on the basis of the cellular gravity sensation through mechanoreceptor channels of the cell membrane. Paramecium is known to have depolarizing receptor channels in the anterior and hyperpolarizing receptors in the posterior of the cell. The uneven distribution of the receptor may lead to the bidirectional changes of the membrane potential by the selective deformation of the anterior and posterior cell membrane responding to the orientation of the cell in the gravity field; i.e. negative- and positive-going shift of the potential due to the upward and downward orientation, respectively. The orientation dependent changes in membrane potential with respect to gravity, in combination with the close coupling of the membrane potential and the ciliary locomotor activity, may allow the changes in swimming direction along with those in the helical nature of the swimming path; upward shift of axis of helix by decreasing the pitch angle due to hyperpolarization in the upward-orienting cell, and also the upward shift by increasing the pitch angle due to depolarization in the downward-orienting cell. Computer simulation of the model demonstrated that the cell can swim upward along the "super-helical" trajectory consisting of a small helix winding helically an axis parallel to the gravity vector, after which the model was named as "Super-helix model". Three-dimensional recording of the trajectories of the swimming cells demonstrated that about a quarter of the cell population drew super-helical trajectory under the unbounded, thermal convection-free conditions. In addition, quantitative analysis of the orientation rate of the swimming cell indicated that gravity-dependent orientation of the swimming trajectory could not be explained solely by the physical static torque but complementarily by the physiological mechanism as proposed in the super-helix model.     

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.     

Catalase activity in Paramecium aurelia]

The catalase activity of Paramecium aurelia was determined by the procedure of Sinha after bacteria elimination from culture medium. A significant level of catalase activity was shown, higher than in other cell kinds. The role of catalase activity in Paramecium sensitivity to low doses of ionizing radiations is discuted.     

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.     

ARDRA and RAPD-fingerprinting reject the sibling species concept for the ciliate Paramecium caudatum (Ciliophora, Protoctista)

Morphologically indistinguishable sibling species also known as syngens are a characteristic taxonomic feature of the ciliate genus Paramecium . This has been convincingly demonstrated for the P. aurelia species complex. For a long time this feature has also been assumed for P. caudatum . Classical morphology based techniques of taxonomic analysis are often inefficient to study sibling specie. We therefore investigated 14 P. caudatum strains of seven supposedly different syngens using random amplified polymorphic DNA (RAPD)-fingerprinting and amplified ribosomal DNA restriction analyses (ARDRA, Riboprinting). The RAPD patterns revealed by five different random primers were similar between the different strains of the same syngen (similarity index ranging from 73 to 91%) and also between strains of supposedly different syngens (similarity index ranging from 67 to 91%). The amplified 18S rRNA-fragments of supposedly different syngens, as well as the restriction patterns of these fragments digested by five different endonucleases, were identical for all investigated P. caudatum stains. Consequently we reject the sibling species hypothesis for P. caudatum . According to our molecular analysis, P. caudatum is not a species complex, but just one single species.     

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.