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.     

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.     

Correlation Between Loss of A Mg2+ Conductance and an Adaptation Defect In A Mutant of Paramecium Tetraurelia

Paramecium tetraurelia responds to chronic KCl-induced depolarization by swimming backward, but the ciliate recovers within seconds and then undergoes a prolonged adaptation period during which sensitivity to external stimuli is altered radically. We examined the role of Mg2+ in this phenomenon, prompted by finding that mutations in the eccentric-A gene both suppressed a Mg(2+)-specific conductance and prevented adaptation. Adaptation of the wild type proceeded normally when extracellular Mg2+ was varied from 0-20 mM, however, suggesting that channel-mediated Mg2+ fluxes were not involved. In seeking alternative explanations for the eccentric mutant phenotype, we ascertained that there was an osmotic component to adaptation but that K(+)-induced depolarization was the primary stimulus. We also noted that wild-type and eccentric mutant cells depolarized by equivalent amounts in KCl, suggesting that the genetic lesion must lie downstream of membrane-potential change. We also examined whether the adaptation-induced behavioral changes and, indeed, the defect in eccentric might be explained in terms of Mg2+ and Na+ efflux during behavioral testing, but experimental observations failed to support this notion. Finally, we consider the possibility that eccentric gene mutation prevents adaptation by interfering with intracellular free Mg2+ homeostasis in Paramecium.     

Membrane potential responses of paramecium caudatum to external Na+

The membrane potential responses of Paramecium caudatum to Na+ ions were examined to understand the mechanisms underlying the sensation of external inorganic ions in the ciliate by comparing the responses of the wild type and the behavioral mutant. Wild-type cells exhibited initial continuous backward swimming followed by repeated transient backward swimming in the Na+-containing test solution. A wild-type cell impaled by a microelectrode produced initial action potentials and a sustained depolarization to an application of the test solution. The prolonged depolarization, the depolarizing afterpotential, took place subsequently after stimulation. The ciliary reversal of the cell was closely associated with the depolarizing responses. When the application of the test solution was prolonged, the wild-type cell produced sustained depolarization overlapped by repeated transient depolarization. A behavioral mutant defective in the Ca2+ channel, CNR (caudatum non reversal), produced a sustained depolarization but no action potential or depolarizing afterpotential. The mutant cell responded to prolonged stimulation with sustained depolarization overlapped by transient depolarization, although it did not show backward swimming. The results suggest that Paramecium shows at least two kinds of membrane potential responses to Na+ ions: a depolarizing afterpotential mediating initial backward swimming and repeated transient depolarization responsible for the repeated transient backward swimming.     

Effect of SERCA Pump Inhibitors on Chemoresponses in Paramecium

ABSTRACT. Inhibitors of SERCA (sarcoplasmic/endoplasmic reticulum Ca²⁺-dependent ATPase) calcium pumps were used to investigate the involvement of internal Ca²⁺ stores in the GTP response in Paramecium . External application of these inhibitors was found to dramatically alter the typical behavioral and electrophysiological responses of Paramecium to extracellular chemical stimulation. In particular, 2.5-di-tert-butylhydroquinone (BHQ) strongly inhibited the backward swimming response of paramecia to externally applied GTP, though it did not inhibit the associated whirling response. BHQ also prolonged the normally brief electrophysiological response of these cells to GTP. BHQ completely blocked the behavioral and electrophysiological responses of Paramecium to extracellular Ba²⁺, but had no measurable effect on the behavioral or electrophysiological responses of these cells to another depolarizing stimulus, elevated external K⁺ concentration. These results suggest the involvement of nonciliary Ca²⁺ ions in the GTP and Ba²⁺ responses.     

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.     

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.     

Response kinetics of tethered bacteria to stepwise changes in nutrient concentration

We examined the changes in swimming behaviour of the bacterium Rhodobacter sphaeroides in response to stepwise changes in a nutrient (propionate), following the pre-stimulus motion, the initial response and the adaptation to the sustained concentration of the chemical. This was carried out by tethering motile cells by their flagella to glass slides and following the rotational behaviour of their cell bodies in response to the nutrient change. Computerised motion analysis was used to analyse the behaviour. Distributions of run and stop times were obtained from rotation data for tethered cells. Exponential and Weibull fits for these distributions, and variability in individual responses are discussed. In terms of parameters derived from the run and stop time distributions, we compare the responses to stepwise changes in the nutrient concentration and the long-term behaviour of 84 cells under 12 propionate concentration levels from 1 nM to 25 mM. We discuss traditional assumptions for the random walk approximation to bacterial swimming and compare them with the observed R. sphaeroides motile behaviour.     

Diffusion Coefficient of Paramecium as a Function of Temperature1

The motion of Paramecium caudatum has been investigated at various temperatures by measuring the transient behavior of spatial distribution in the diffusion process of organisms that, by electric stimulus, are initially gathered at a single place in the glass culture cell. The spatial distribution through the course of diffusion has a nearly Gaussian profile. Dispersion was obtained at 1 sec intervals and increased linearly with time. The time dependence of the dispersion gave a diffusion coefficient for the random motion of the organisms. The results show that the diffusion coefficient has a maximum at the temperature at which the paramecia were cultivated.     

The Relationship between Stimulus Intensity and Oriented Phototactic Response (Topotaxis) in Chlamydomonas

Two techniques have been used to study the quantitative relationship between stimulus intensity and oriented phototactic response (topotaxis) in Chlamydomonas. The net response of a cell population was monitored photometrically and was recorded continuously against time. The responses of individual cells were observed through a microscope and their swimming tracks were recorded on film. The net response of the population is positive at low stimulus intensity and negative at high intensity. The direction of response can be reversed within two seconds by raising or lowering the intensity. The intensity-response curve for phototaxis is similar to the dose-response curve for phototropism. The net response has no distinct threshold; it increases linearly with log intensity; then it decreases and finally becomes negative. The individual-cell studies reveal that the intensity-dependent increase in net topotactic response is due primarily to an increase in the number of cells responding and in the directness of their swimming path. As stimulus intensity is raised, the swimming path becomes increasingly well-aligned with the stimulus beam, whether net response is positive throughout the intensity range tested, negative throughout that range, or changing from positive to negative. Changes in swimming rate do not contribute significantly to the intensity-dependent changes in net response. Swimming rate shows virtually no change throughout the intensity range of positive topotaxis and shows only a small increase in the negative range. However, a transient decrease in swimming rate (stop response) is often observed at the onset of stimulation. The implications of these results for the orientation mechanism are discussed.     

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.     

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.     

Behavioral Mutants in PARAMECIUM CAUDATUM

Mutants of Paramecium caudatum with abnormal swimming behavior or responses to cations were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Some of the mutants, like pawn in P. tetraurelia, cannot swim backward and are called CNR. Seven independently obtained CNR mutants belonged to three complementation groups, designated as cnrA, cnrB and cnrC. Some characteristics of double homo- and heterozygotes were compared with single homo- and heterozygotes. Other behavioral mutants shown to have a genic basis included K⁺-sensitive, temperature-shock behavioral and slow swimmer. All those mutants except for slow swimmer had lesions in the membrane because Triton-extracted models of them show almost the same swimming behavior as wild type.     

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.     

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.     

Limits of Feedback Control in Bacterial Chemotaxis

Inputs to signaling pathways can have complex statistics that depend on the environment and on the behavioral response to previous stimuli. Such behavioral feedback is particularly important in navigation. Successful navigation relies on proper coupling between sensors, which gather information during motion, and actuators, which control behavior. Because reorientation conditions future inputs, behavioral feedback can place sensors and actuators in an operational regime different from the resting state. How then can organisms maintain proper information transfer through the pathway while navigating diverse environments? In bacterial chemotaxis, robust performance is often attributed to the zero integral feedback control of the sensor, which guarantees that activity returns to resting state when the input remains constant. While this property provides sensitivity over a wide range of signal intensities, it remains unclear how other parameters such as adaptation rate and adapted activity affect chemotactic performance, especially when considering that the swimming behavior of the cell determines the input signal. We examine this issue using analytical models and simulations that incorporate recent experimental evidences about behavioral feedback and flagellar motor adaptation. By focusing on how sensory information carried by the response regulator is best utilized by the motor, we identify an operational regime that maximizes drift velocity along chemical concentration gradients for a wide range of environments and sensor adaptation rates. This optimal regime is outside the dynamic range of the motor response, but maximizes the contrast between run duration up and down gradients. In steep gradients, the feedback from chemotactic drift can push the system through a bifurcation. This creates a non-chemotactic state that traps cells unless the motor is allowed to adapt. Although motor adaptation helps, we find that as the strength of the feedback increases individual phenotypes cannot maintain the optimal operational regime in all environments, suggesting that diversity could be beneficial.     

Intracellular stimulation of sensory cells elicits swimming activity in the medicinal leech

Intracellular stimulation of each of three different types of mechanoreceptors, the T, P and N cells, evokes swimming behavior in leech preparations. Stimulation of an individual N cell or P cell evoked swimming in 75% and 53% respectively, of the preparations tested. Stimulation of an individual T cell was ineffective in eliciting swimming; however, simultaneous stimulation of two T cells evoked swimming in 59% of our preparations. Stimulation of mechanosensory neurons elicited swimming activity for a limited number of trials; i.e. the response habituated. The number of swim episodes evoked before habituation to criterion did not differ significantly for the different types of mechanoreceptors. The duration of swim episodes declined significantly over the course of N cell stimulation. The tendency for swim length to decline with repeated stimulation was present as well for swim episodes elicited by P or T cell stimulation. Swim initiation recovered spontaneously following habituation resulting from T cell stimulation. Spontaneous recovery following N cell stimulation was not demonstrated. However, N cell stimulation evoked swimming again after DP nerve shock or to a limited extent, after cell 204 stimulation. Spontaneous recovery of swim initiation to P cell stimulation was not investigated. A previous study detailed habituation of swimming activity to mechanical stimulation of the body wall (Debski and Friesen 1985). Only the T cells are activated significantly by this stimulus. Stimulation of sensory receptors other than mechanoreceptors was not effective in eliciting swimming in our preparation. We conclude that T cells mediate swim initiation elicited by stroking of the body wall and that the cessation of swimming to this stimulus is not due to sensory adaptation.     

Motility response of Rhodobacter sphaeroides to chemotactic stimulation.

Tethered rotating cells of Rhodobacter sphaeroides varied widely in their stopping frequency; 45% of cells showed no stops of longer than 1 s, whereas others showed stops of up to several seconds. Individual cells alternated between stops and rotation at a fairly constant rate, without continuous variation. Addition of the chemoattractant propionate to free-swimming cells of R. sphaeroides increased the mean population swimming speed from 15 to 23 microns s-1. After correction for nonmotile cells, the percentage swimming at less than 5 microns s-1 dropped from approximately 22 to 8, whereas the percentage swimming at greater than 50 microns s-1 increased from 6 to 15. However, cells already swimming did not swim faster after propionate addition; the increase in the mean population speed after propionate addition was caused by an increase in the mean run length between stops from 25 to 101 microns. The increased run length was the result of a drop in both the stopping frequency and the length of a stop. Addition of propionate over the range of 10 microM to 1 mM decreased the stopping frequency; this decrease was almost entirely blocked by benzoate, a competitive inhibitor of propionate transport. The chemoattractants acetate and potassium had the same effect as propionate on the distribution of stopping frequency, which demonstrated that this is a general behavioral response to chemotactic stimulation. Adaptation to propionate stimulation was slow and very variable, cultures frequently showing little adaptation over 30 min. This characteristic may be the result of the lack of a highly specific chemosensory system in R. sphaeroides.     

Conspecific and heterospecific alarm substance induces behavioral responses in piau fish Leporinus piau

In ostariophysan fish, the detection of alarm substance released from the skin of a conspecific or a sympatric heterospecific may elicit alarm reactions or antipredator behavioral responses. In this study, experiments were performed to characterize and quantify the behavioral response threshold of Leporinus piau, both individually and in schools, to growing dilutions of conspecific (CAS) and heterospecific skin extract (HAS). The predominant behavioral response to CAS stock stimulation was biphasic for fish held individually, with a brief initial period of rapid swimming followed by a longer period of immobility or reduced swimming activity. As the dilution of skin extract was increased, the occurrence and magnitude of the biphasic alarm response tended to decrease, replaced by a slowing of locomotion. Slowing was the most common antipredator behavior, observed in 62.5% of animals submitted to HAS stimulation. School cohesion, measured as proximity of fish to the center of the school, and swimming activity near the water surface significantly increased after exposure to CAS when compared with the control group exposed to distilled water. Histological analysis of the epidermis revealed the presence of Ostariophysi-like club cells. The presence of these cells and the behavioral responses to conspecific and heterospecific skin extract stimulation suggest the existence of a pheromone alarm system in L. piau similar to that in Ostariophysi, lending further support for the neural processing of chemosensory information in tropical freshwater fish.     

Ca2+ oscillations mediated by exogenous GTP in Paramecium cells: assessment of possible Ca2+ sources

We applied exogenous guanosine trisphosphate, GTP, to Paramecium tetraurelia cells injected with Fura Red for analysing changes of free intracellular Ca(2+) concentrations, [Ca(2+)](i), during periodic back-/forward swimming thus induced. Strain ginA (non-responsive to GTP) shows no Ca(2+) signal upon GTP application. In strain nd6 (normal Ca(2+) signalling) an oscillating [Ca(2+)](i) response with a prominent first peak occurs upon GTP stimulation, but none after mock-stimulation or after 15 min adaptation to GTP. While this is in agreement with previous electrophysiological analyses, we now try to identify more clearly the source(s) of Ca(2+). Stimulation of nd6 cells, after depletion of Ca(2+) from their cortical stores (alveolar sacs), shows the same Ca(2+) oscillation pattern but with reduced amplitudes, and a normal behavioural response is observed. Stimulation with GTP, supplemented with the Ca(2+) chelator BAPTA, results in loss of the first prominent Ca(2+) peak, in reduction of the following Ca(2+) amplitudes, and in the absence of any behavioural response. Both these observations strongly suggest that for the initiation of GTP-mediated back-/forward swimming Ca(2+) from the extracellular medium is needed. For the maintenance of the Ca(2+) oscillations a considerable fraction must come from internal stores, probably other than alveolar sacs, rather likely from the endoplasmic reticulum.