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.     

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.     

Effects of static magnetic fields on growth of Paramecium caudatum

Little is known about the influence of magnetic fields on growth of primitive eukaryotes such as the ciliate Paramecium. The latter are known to exhibit interesting characteristics such as electrotaxis, gravitaxis, and membrane excitability not commonly encountered in higher organisms. This preliminary study reports the effects of static magnetic fields on growth of Paramecium caudatum. The microorganisms were either permanently or 24 h on-and-off exposed to North and South polarity magnetic fields of average field gradient 4.3 T/m, for a period of 96 h. The growth rate and lag phase of all exposed populations were not significantly different from control ones exposed to normal geomagnetic field (P > .05). However, a significant negative shift in t(max) (time taken for maximum growth) of 10.5%-12.2% and a significant decrease (P < .05) in population size of 10.2%-15.1% during the 96 h of experimental conditions were recorded for exposed populations compared to control. Our results suggest that magnetic fields, irrespective of polarity and exposure period reduce Paramecium growth by triggering early senescence of the population. The mechanisms underlying the small changes in population growth are unknown at this level, but various hypotheses have been suggested, including disorganization of swimming patterns resulting from (i) changes in cell membrane electric potential due to high speed movement through a gradient magnetic field and (ii) thermodynamic effect of anisotropic magnetic energies on cell membrane components affecting functioning of calcium channels. Altered swimming movements could in turn affect highly orchestrated processes such as conjugation, essential for survival of the organisms during development of adverse environmental conditions as thought to occur in the closed culture system used in this study.     

An automated assay for quantifying the swimming behavior of Paramecium and its use to study cation responses

Paramecium tetraurelia is a ciliated protist that alters its swimming behavior in response to various stimuli. Like the sensory responses of many organisms, these responses in Paramecium show adaptation to continued stimulation. For quantitative studies of the initial response to stimulation, and of the time course of adaptation, we have developed a computerized motion analysis assay that can detect deviations from the normal swimming pattern in a population of cells. The motion of an average of ten cells was quantified during periods ranging from 15 to 60 seconds, with a time resolution of 1/15 seconds. During normal forward swimming, the maximum deviation from a straight-line path was less than 17 degrees. Path deviations above this threshold value were defined as changes in swimming direction. The percentage of total path time that cells spent deviating from forward swimming was defined as percent directional changes (PDC). This parameter was used to construct dose-response curves for the behavioral effects of various externally added cations known to induce behavioral changes and also to show the time course of adaptation to a depolarizing K+ stimulus. This assay is a valuable tool for studies of chemoeffectors or mutations that alter the swimming behavior of Paramecium and may also be applicable to other motile organisms.     

Regulation of ciliary motility by membrane potential in Paramecium: a role for cyclic AMP

The membrane potential of Paramecium controls the frequency and direction of the ciliary beat, thus determining the cell's swimming behavior. Stimuli that hyperpolarize the membrane potential increase the ciliary beat frequency and therefore increase forward swimming speed. We have observed that 1) drugs that elevate intracellular cyclic AMP increased swimming speed 2-3-fold, 2) hyperpolarizing the membrane potential by manipulation of extracellular cations (e.g., K+) induced both a transient increase in, and a higher sustained level of cyclic AMP compared to the control, and 3) the swimming speed of detergent-permeabilized cells in MgATP was stimulated 2-fold by the addition of cyclic AMP. Our results suggest that the membrane potential can regulate intracellular cAMP in Paramecium and that control of swimming speed by membrane potential may in part be mediated by cAMP.     

Behavior of free-swimming cells under various accelerations

The different steps of the gravity signal-transduction chain on the cellular level are not identified. In our experiments performed up to now we mainly stressed our attention on the last step, the response of the cells. Swimming behavior is a suitable indicator for the physiological status of a Paramecium cell. Depending on membrane potential and/or concentrations of Ca++, cGMP and cAMP the beating direction and the beating velocity of the cilia are influenced in a characteristical way leading to a changed swimming activity of the cell. The behavior of Paramecium is influenced by various stimuli from their environment. Previous studies have demonstrated that under controlled conditions Paramecium shows a clear gravity-dependent behavior resulting in negative gravitaxis and gravikinesis (speed regulation in dependence of gravity). By changing the orienting stimulus (gravity) we expected changes of the swimming behavior. Additional experiments were performed using pawn mutant d4-500r. Due to defective Ca(2+)-channels the membrane of this mutant cannot depolarize. As a consequence d4-500r cannot perform phobic responses and swim backwards. Comparative experiments are also performed with the ciliate Loxodes striatus. In contrast to Paramecium this ciliate possesses statocyst-like organelles--the Müller Organelles.     

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.     

Aligning Paramecium caudatum with static magnetic fields

As they negotiate their environs, unicellular organisms adjust their swimming in response to various physical fields such as temperature, chemical gradients, and electric fields. Because of the weak magnetic properties of most biological materials, however, they do not respond to the earth's magnetic field (5 x 10(-5) Tesla) except in rare cases. Here, we show that the trajectories of Paramecium caudatum align with intense static magnetic fields >3 Tesla. Otherwise straight trajectories curve in magnetic fields and eventually orient parallel or antiparallel to the applied field direction. Neutrally buoyant immobilized paramecia also align with their long axis in the direction of the field. We model this magneto-orientation as a strictly passive, nonphysiological response to a magnetic torque exerted on the diamagnetically anisotropic components of the paramecia. We have determined the average net anisotropy of the diamagnetic susceptibility, Deltachi(p), of a whole Paramecium: Deltachi(p) = (6.7+/- 0.7) x 10(-23) m(3). We show how the measured Deltachi(p) compares to the anisotropy of the diamagnetic susceptibilities of the components in the cell. We suggest that magnetic fields can be exploited as a novel, noninvasive, quantitative means to manipulate swimming populations of unicellular organisms.     

A comparison of the polycation receptors of Paramecium tetraurelia and Tetrahymena thermophila

Chemorepellents are compounds that cause ciliated protozoans to reorient their swimming direction. A number of chemorepellents have been studied in the ciliated protozoans, Paramecium and Tetrahymena. Chemorepellents, such as polycations, cause the organism to exhibit "avoidance behavior," a swimming behavior characterized by jerky movements and other deviations from normal forward swimming, which result from ciliary reversal. One well-characterized chemorepellent pathway in Tetrahymena is that of the proposed polycation receptor that is activated by lysozyme and pituitary adenylate cyclase activating polypeptide (PACAP). In this study, we compare the response of Paramecium to the chemorepellents lysozyme, vasoactive intestinal peptide (VIP), and PACAP to the previously studied polycation response in Tetrahymena. Our results indicate that lysozyme, VIP, and PACAP are all chemorepellents in Paramecium, just as they are in Tetrahymena. However, the signaling pathways involved appear to be different. While previous pharmacological characterization indicates that G-proteins are involved in polycation signaling in Tetrahymena, we present evidence that similar reception in Paramecium involves activation of a tyrosine kinase pathway in order for lysozyme avoidance to occur. Polycation responses of both organisms are inhibited by neomycin sulfate. While PACAP is the most effective of the three chemorepellents in Tetrahymena, lysozyme is the most effective chemorepellent in Paramecium.     

Temperature-Sensitive Behavior of Paramecium caudatum

SYNOPSIS. The effect of temperature on the behavior of swimming cells of Paramecium caudatum has been investigated by photographic analyses of their tracks in uniform temperature, in temperature gradient, or in temperature changing with time. When the cells were placed in the temperature gradient, the frequency of discontinuous directional changes of cells swimming toward the optimal temperature, the temperature of the culture, was much lower than that of the cells swimming in the opposite direction. This difference in the frequency of directional changes explained the observed accumulation of the cells at - the optimal temperature. When the temperature was suddenly changed toward the optimum, a transient decrease of the frequency of directional changes was observed and when the temperature was changed in the reverse direction, a transient increase of the frequency was noted. This transient response to the temperature change was the origin of the dependence of the frequency of directional changes on the swimming direction in the temperature gradient. Finally, the relation between the magnitude of the transient response and the rate of the temperature change was derived.     

磁场作用对小鼠抗应激能力的影响

目的 :探讨磁场作用对小鼠抗应激能力的影响。方法 :对磁场处理 30分钟和 1 5分钟的两组实验组与非磁场处理的正常对照组进行游泳耐疲劳运动时间的比较。结果 :游泳耐疲劳运动实验表明 30分钟磁场处理组与正常对照组比较 ,动物游泳耐疲劳运动时间延长 ,且具有显著性差异 (p <0 .0 5 ) ;1 5分钟磁场处理组与正常对照组比较 ,动物游泳耐疲劳运动时间无明显差异 (p >0 .0 5 ) ;30分钟磁场处理组与 1 5分钟磁场处理组比较 ,动物游泳耐疲劳运动时间延长 ,且具有显著性差异 (p <0 .0 5 )。结论 :在一段时间( 30天 )内 ,每天给予小鼠 30分钟的磁场处理明显提高了小鼠的抗应激能力 ,而磁场处理 1 5分钟对小鼠的抗应激能力不产生影响。     

Okadaic acid, an inhibitor of protein phosphatase 1 in Paramecium, causes sustained Ca2(+)-dependent backward swimming in response to depolarizing stimuli.

Backward swimming is a stereotypic behavioural response of Paramecium. It is triggered by depolarizing stimuli, which open calcium channels in the excitable ciliary membrane. The influx of Ca2+ causes the reversal of ciliary beat and initiates backward swimming. Here, we demonstrate that the protein phosphatase inhibitor okadaic acid does not affect the normal forward swimming pattern of Paramecium, but greatly extends the duration of backward swimming as initiated by depolarization caused by a rise in extracellular K+. Chelation of external Ca2+ results in an immediate resumption of forward swimming. The results suggest that the voltage-operated calcium channel is inactivated by a dephosphorylation event, and that okadaic acid blocks this dephosphorylation without any effect on the motile apparatus of the cilia. In addition, Paramecium is unique among eukaryotic cells, in that okadaic acid inhibits just one protein phosphatase, namely a type 1 enzyme, 75% of which is tightly associated with the excitable ciliary membrane. The type 2A protein phosphatases in Paramecium are unaffected by okadaic acid. The results indicate that protein phosphatase 1 is the enzyme responsible for the dephosphorylation and closure of the calcium channel in Paramecium.     

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.     

G-protein modulators alter the swimming behavior and calcium influx of Paramecium tetraurelia

To assess the potential role of G-proteins in chemokinesis, Paramecium tetraurelia was pre-incubated with the G-protein modulator pertussis toxin. Pertussis toxin pretreatment significantly reduced Paramecium chemoattraction to sodium acetate and ammonium chloride in T-maze behavioral assays and depressed the frequency of avoidance reactions, indicating that heterotrimeric G-proteins may be involved with the motility response. To determine whether G-proteins exert their effect via the ciliary voltage-sensitive calcium channel, we examined responses of P. tetraurelia to the potent voltage-sensitive calcium channel agonist, deltamethrin. Pertussis toxin preincubation significantly reduced the toxic effects of deltamethrin exposure as determined by survival under depolarizing conditions and reduced the duration of backward swimming episodes in behavioral bioassays. Furthermore, non-hydrolyzable analogs of guanine nucleotides altered deltamethrin-stimulated calcium influx via calcium channels in isolated ciliary vesicles. Heterotrimeric G-protein subunits were subsequently detected in ciliary vesicles of P. tetraurelia by antibodies produced against Galpha and Gbeta subunits, and by 32P-ADP-ribosylation, indicating that proteins of the appropriate molecular weight are the target of pertussis toxin in these vesicles. These findings provide additional evidence that heterotrimeric G-proteins are associated with ciliary vesicles and that they play a role in the modulation of swimming behavior and the toxic action of deltamethrin in Paramecium.     

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)     

Effect of Extracellular pH on Motility and K+-Induced Ciliary Reversal in Paramecium caudatum1

ABSTRACT. Paramecium caudatum, reared on bacterized hay infusions at pH 6.5 to 6.9, were washed into various buffered solutions containing 0.016 mM CaCl₂ and a pH of 3.5 to 10.4. Solutions of pH 4.5 to 9.5 support strong swimming of the cells for at least 24 h. At pH values acid to the culture medium, cells show an increasing frequency of spontaneous ciliary reversal episodes (“avoiding reactions”). Uninterrupted forward swimming is usually observed over the pH range of 7.1 to 8.5, and above pH 8.5, forward motion is interrupted by circular swimming. For all pH values tested, transfer of cells to a more acidic test solution than the solutions into which they were washed (adaptation solution) usually induced short duration, periodic ciliary reversal behavior. With transfer to a more alkaline test solution than the adaptation solution, the cells shift from forward left spiralling motion to forward right spiralling motion. With decreasing pH, the cells show progressively less sensitivity to KC1 stimulation, and at pH values of less than 5.0, cells fail to show significant ciliary reversal response to any KC1 concentration tested (1 - 128 mM). At alkaline pH values and higher KC1 concentrations, the cells show very pronounced ciliary reversal behaviors but usually fail to regain forward swimming behavior.     

Propulsive force of Paramecium as revealed by the video centrifuge microscope

Using the video centrifuge microscope we constructed, we observed the behavior of Paramecium cells in a solution of graded densities under centrifugal acceleration. Beyond 300g, they not only gather in the zone where the density is closest to theirs, but also orient themselves with their longitudinal axis parallel to the direction of centrifugation turning their anterior ends toward either centripetal or centrifugal direction. Since all of them retain still active swimming capacity, it is possible to calculate their propulsive force from the difference in density between theirs (1.04 g cm-3) and that of the upper or lower layer which they can reach. The propulsive force of single Paramecium cells thus obtained was calculated to be about 7 x 10(-4) dyn.     

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.     

Rhythmicity of swimming activity in an axenic population of Paramecium multimicronucleatum

A unicellular organism, Paramecium, exhibits circadian rhythm activities in many physiological phenomena, i.e., mating reactivity, photoaccumulation in Paramecium bursaria and mating type reversals in Paramecium multimicronucleatum. In this study, we used an image-processing system to analyze swimming activity in a population of Paramecium multimicronucleatum cultured axenically under 12 h-light/12 h-dark cycles (LD 1212). Swimming behavior was recorded both under LD 1212 and constant darkness and images tracing the tracks of Paramecium were produced every 4 min. Swimming activity was represented by the occupied area by the tracks relative to the total observed area. It is high during daytime and low at night and exhibits a freerunning rhythm in constant darkness. Furthermore, criteria for two major components of swimming behavior, straight and circle swimming, were established and analyzed. The results indicate that swimming behavior alters depending on the time of day: straight swimming increased during the day and circling was dominant around dusk both under LD 1212 and constant darkness.Abbreviations ZT Zeitgeber Time - LD 1212 12h- light/12h-dark cycles - TF transversing frequency     

Orientation of Paramecium under the conditions of weightlessness

A cell culture of Paramecium with a precise negative gravitaxis was exposed to 4 x l0(-6) g during a parabolic flight of a sounding rocket for 6 min. Computer image analysis revealed that without gravity stimulus the individual swimming paths remained straight. In addition, three reactions could be distinguished. For about 30 s, paramecia maintained the swimming direction they had before onset of low gravity. During the next 20 s, an approximate reversal of the swimming direction occurred. This period was followed by the expected random swimming pattern. Similar behavior was observed under the condition of simulated weightlessness on a fast-rotating clinostat. Control experiments on the ground under hyper-gravity on a low-speed centrifuge microscope and on a vibration test facility proved that the observed effects were caused exclusively by the reduction of gravity.