An electrophysiological and kinematic model of Paramecium, the "swimming neuron".

Paramecium is a large unicellular organism that swims in fresh water using cilia. When stimulated by various means (mechanically, chemically, optically, thermally), it often swims backward then turns and swims forward again in a new direction: this is called the avoiding reaction. This reaction is triggered by a calcium-based action potential. For this reason, several authors have called Paramecium the "swimming neuron". Here we present an empirically constrained model of its action potential based on electrophysiology experiments on live immobilized paramecia, together with simultaneous measurement of ciliary beating using particle image velocimetry. Using these measurements and additional behavioral measurements of free swimming, we extend the electrophysiological model by coupling calcium concentration to kinematic parameters, turning it into a swimming model. In this way, we obtain a model of autonomously behaving Paramecium. Finally, we demonstrate how the modeled organism interacts with an environment, can follow gradients and display collective behavior. This work provides a modeling basis for investigating the physiological basis of autonomous behavior of Paramecium in ecological environments.

Anoctamin-like protein 1 regulates repolarization in Paramecium behavioral responses.

Paramecium exhibits responsive behavior to environmental changes, moving either closer to or further away from stimuli. Electrophysiological experiments have revealed that these behavioral responses are controlled by membrane potentials. Anoctamin, a Ca2+-activated Cl- channel, is involved in the regulation of membrane potential in mammals. However, it remains uncertain whether Cl- channels like anoctamin regulate Paramecium behavior. Herein, replacement of external Cl- ions with acetate ion and application of Cl- channel blocker niflumic acid (NFA, 0.1 µM) increased spontaneous avoiding reactions (sARs). Hence, we hypothesized that anoctamin is involved in the stabilization of membrane potential fluctuation. Paramecium cells in which the anoctamin-like protein 1 gene was knocked down displayed frequent sARs in the culture medium without external stimulation. Treatment of anoctamin-like protein 1-knockdown cells with the Ca2+ chelator BAPTA or Ca-channel blocker nicardipine reversed the increase in sARs. Electrophysiological experiments revealed extension of membrane depolarization when positive currents were applied to anoctamin-like protein 1-knockdown cells. We concluded that anoctamin-like protein 1 works as a Cl-channel and stabilizes the membrane potential oscillation, reducing sARs.

Micromanipulation in Paramecium: From non-mendelian inheritance to the outlook for versatile micromachines.

This review addresses nine areas of knowledge revealed by micromanipulations performed with Paramecium. Microinjection has shown that sexual maturation and senescence of Paramecium caudatum is a programmed process conducted by a specific gene and its product protein. In Paramecium tetraurelia, autogamy was revealed to depend on the number of DNA syntheses rather than the number of cell divisions in clonal aging. The cytoplasmic complementarity test established that microinjection of wild-type cytoplasm can correct genetic defects of mutants. The concept of complementarity together with protein chemistry revealed compounds that control membrane excitability. In non-Mendelian inheritance, noncoding small RNAs made from the parental micronucleus regulate the rearrangement of the progeny's macronuclear DNA. The macronucleus has the potential to be used as a factory for genetic engineering. The development and differentiation of progeny's nuclei in mating pairs are controlled by the parental macronucleus. The chemical reaction processes associated with exocytosis have been revealed by microinjection of various enzymes and antibodies. Using the fusion gene of histone H2B and yellow-fluorescence protein, it was revealed that the fusion gene-mRNA is transferred between cells during mating. Experiments with endosymbiotic bacteria and the host shed light on the conditions needed to establish sustainable symbiotic relationships.

Ion channels of cilia: Paramecium as a model.

Holotrichous ciliates, like Paramecium, swim through their aqueous environment by beating their many cilia. They can alter swimming speed and direction, which seems to have mesmerized early microscopists of the 1600s. We know from extensive and elegant physiological studies and generation of mutants that these cells can be considered little swimming neurons because their ciliary beating is under bioelectric control of ion channels in the cilia. This chapter will focus on the ionic control of swimming behavior by ciliary ion channels, primarily in the holotrichous ciliate Paramecium. Voltage-gated and calcium-activated channels for calcium, magnesium, sodium, and potassium are regulated in a closely orchestrated manner that allows cilia to bend and propel the cell forward or backward. Sensory input that generates receptor potentials feeds into the control of this channel activity and allows the cell to turn or speed up. This in turn helps the cell to avoid predators or toxic conditions. While the focus is on P. tetraurelia and P. caudatum, the principles of ciliary ion channel activity and control are easily extendable to other ciliates and protists. The high conservation of channel and ion pump structures also extends the lessons from Paramecium to higher organisms.

Membrane traffic and Ca2+ signals in ciliates.

A Paramecium cell has as many types of membrane interactions as mammalian cells, as established with monoclonal antibodies by R. Allen and A. Fok. Since then, we have identified key players, such as SNARE proteins, Ca2+ -regulating proteins, including Ca2+ -channels, Ca2+ -pumps, Ca2+ -binding proteins of different affinity, etc., at the molecular level, probed their function and localized them at the light and electron microscopy level. SNARE proteins, in conjunction with a synaptotagmin-like Ca2+ -sensor protein, mediate membrane fusion. This interaction is additionally regulated by monomeric GTPases whose spectrum in Tetrahymena and Paramecium has been established by A. Turkewitz. As known from mammalian cells, GTPases are activated on membranes in conjunction with lumenal acidification by an H+ -ATPase. For these complex molecules, we found in Paramecium an unsurpassed number of 17 a-subunit paralogs which connect the polymeric head and basis part, V1 and V0. (This multitude may reflect different local functional requirements.) Together with plasmalemmal Ca2+ -influx channels, locally enriched intracellular InsP3 -type (InsP3 R, mainly in osmoregulatory system) and ryanodine receptor-like Ca2+ -release channels (ryanodine receptor-like proteins, RyR-LP), this complexity mediates Ca2+ signals for most flexible local membrane-to-membrane interactions. As we found, the latter channel types miss a substantial portion of the N-terminal part. Caffeine and 4-chloro-meta-cresol (the agent used to probe mutations of RyRs in man during surgery in malignant insomnia patients) initiate trichocyst exocytosis by activating Ca2+ -release channels type CRC-IV in the peripheral part of alveolar sacs. This is superimposed by Ca2+ -influx, that is, a mechanism called "store-operated Ca2+ -entry" (SOCE). For the majority of key players, we have mapped paralogs throughout the Paramecium cell, with features in common or at variance in the different organelles participating in vesicle trafficking. Local values of free Ca2+ -concentration, [Ca2+ ]i , and their change, for example, upon exocytosis stimulation, have been registered by flurochromes and chelator effects. In parallel, we have registered release of Ca2+ from alveolar sacs by quenched-flow analysis combined with cryofixation and X-ray microanalysis.

Polarization grating based on diffraction phase microscopy for quantitative phase imaging of paramecia.

This study presents a polarization grating based diffraction phase microscopy (PG-DPM) and its application in bio-imaging. Compared with traditional diffraction phase microscopy (DPM) of which the fringe contrast is sample-dependent, the fringe contrast of PG-DPM is adjustable by changing the polarization of the illumination beam. Moreover, PG-DPM has been applied to real-time phase imaging of live paramecia for the first time. The study reveals that paramecium has self-helical forward motion characteristics, or more specifically, 77% clockwise and 23% anti-clockwise rotation when moving forward. We can envisage that PG-DPM will be applied to many different fields.

Roles of Adenylate Cyclases in Ciliary Responses of Paramecium to Mechanical Stimulation.

Paramecium shows rapid forward swimming due to increased beat frequency of cilia in normal (forward swimming) direction in response to various kinds of stimuli applied to the cell surface that cause K+ -outflow accompanied by a membrane hyperpolarization. Some adenylate cyclases are known to be functional K+ channels in the membrane. Using gene-specific knockdown methods, we examined nine paralogues of adenylate cyclases in P. tetraurelia to ascertain whether and how they are involved in the mechanical stimulus-induced hyperpolarization-coupled acceleration of forward swimming. Results demonstrated that knockdown of the adenylate cyclase 1 (ac1)-gene and 2 (ac2)-gene inhibited the acceleration of forward swimming in response to mechanical stimulation of the cell, whereas that spared the acceleration response to external application of 8-Br-cAMP and dilution of extracellular [K+ ] induced hyperpolarization. Electrophysiological examination of the knockdown cells revealed that the hyperpolarization-activated inward K+ current is smaller than that of a normal cell. Our results suggest that AC1 and AC2 are involved in the mechanical stimulus-induced acceleration of ciliary beat in Paramecium.

Phenomena of synchronized response in biosystems and the possible mechanism.

Phenomena of synchronized response is common among organs, tissues and cells in biosystems. We have analyzed and discussed three examples of synchronization in biosystems, including the direction-changing movement of paramecia, the prey behavior of flytraps, and the simultaneous discharge of electric eels. These phenomena and discussions support an electrical communication mechanism that in biosystems, the electrical signals are mainly soliton-like electromagnetic pulses, which are generated by the transient transmembrane ionic current through the ion channels and propagate along the dielectric membrane-based softmaterial waveguide network to complete synchronized responses. This transmission model implies that a uniform electrical communication mechanism might have been naturally developed in biosystem.

Trichocysts-Paramecium's Projectile-like Secretory Organelles: Reappraisal of their Biogenesis, Composition, Intracellular Transport, and Possible Functions.

This review summarizes biogenesis, composition, intracellular transport, and possible functions of trichocysts. Trichocyst release by Paramecium is the fastest dense core-secretory vesicle exocytosis known. This is enabled by the crystalline nature of the trichocyst "body" whose matrix proteins (tmp), upon contact with extracellular Ca2+ , undergo explosive recrystallization that propagates cooperatively throughout the organelle. Membrane fusion during stimulated trichocyst exocytosis involves Ca2+ mobilization from alveolar sacs and tightly coupled store-operated Ca2+ -influx, initiated by activation of ryanodine receptor-like Ca2+ -release channels. Particularly, aminoethyldextran perfectly mimics a physiological function of trichocysts, i.e. defense against predators, by vigorous, local trichocyst discharge. The tmp's contained in the main "body" of a trichocyst are arranged in a defined pattern, resulting in crossstriation, whose period expands upon expulsion. The second part of a trichocyst, the "tip", contains secretory lectins which diffuse upon discharge. Repulsion from predators may not be the only function of trichocysts. We consider ciliary reversal accompanying stimulated trichocyst exocytosis (also in mutants devoid of depolarization-activated Ca2+ channels) a second, automatically superimposed defense mechanism. A third defensive mechanism may be effectuated by the secretory lectins of the trichocyst tip; they may inhibit toxicyst exocytosis in Dileptus by crosslinking surface proteins (an effect mimicked in Paramecium by antibodies against cell surface components). Some of the proteins, body and tip, are glycosylated as visualized by binding of exogenous lectins. This reflects the biogenetic pathway, from the endoplasmic reticulum via the Golgi apparatus, which is also supported by details from molecular biology. There are fragile links connecting the matrix of a trichocyst with its membrane; these may signal the filling state, full or empty, before and after tmp release upon exocytosis, respectively. This is supported by experimentally produced "frustrated exocytosis", i.e. membrane fusion without contents release, followed by membrane resealing and entry in a new cycle of reattachment for stimulated exocytosis. There are some more puzzles to be solved: Considering the absence of any detectable Ca2+ and of acidity in the organelle, what causes the striking effects of silencing the genes of some specific Ca2+ -release channels and of subunits of the H+ -ATPase? What determines the inherent polarity of a trichocyst? What precisely causes the inability of trichocyst mutants to dock at the cell membrane? Many details now call for further experimental work to unravel more secrets about these fascinating organelles.

Maintenance of algal endosymbionts in Paramecium bursaria: a simple model based on population dynamics.

Algal endosymbiosis is widely distributed in eukaryotes including many protists and metazoans, and plays important roles in aquatic ecosystems, combining phagotrophy and phototrophy. To maintain a stable symbiotic relationship, endosymbiont population size in the host must be properly regulated and maintained at a constant level; however, the mechanisms underlying the maintenance of algal endosymbionts are still largely unknown. Here we investigate the population dynamics of the unicellular ciliate Paramecium bursaria and its Chlorella-like algal endosymbiont under various experimental conditions in a simple culture system. Our results suggest that endosymbiont population size in P. bursaria was not regulated by active processes such as cell division coupling between the two organisms, or partitioning of the endosymbionts at host cell division. Regardless, endosymbiont population size was eventually adjusted to a nearly constant level once cells were grown with light and nutrients. To explain this apparent regulation of population size, we propose a simple mechanism based on the different growth properties (specifically the nutrient requirements) of the two organisms, and based from this develop a mathematical model to describe the population dynamics of host and endosymbiont. The proposed mechanism and model may provide a basis for understanding the maintenance of algal endosymbionts.

Disentangling the Taxonomy of Rickettsiales and Description of Two Novel Symbionts ("Candidatus Bealeia paramacronuclearis" and "Candidatus Fokinia cryptica") Sharing the Cytoplasm of the Ciliate Protist Paramecium biaurelia.

In the past 10 years, the number of endosymbionts described within the bacterial order Rickettsiales has constantly grown. Since 2006, 18 novel Rickettsiales genera inhabiting protists, such as ciliates and amoebae, have been described. In this work, we characterize two novel bacterial endosymbionts from Paramecium collected near Bloomington, IN. Both endosymbiotic species inhabit the cytoplasm of the same host. The Gram-negative bacterium "Candidatus Bealeia paramacronuclearis" occurs in clumps and is frequently associated with the host macronucleus. With its electron-dense cytoplasm and a distinct halo surrounding the cell, it is easily distinguishable from the second smaller symbiont, "Candidatus Fokinia cryptica," whose cytoplasm is electron lucid, lacks a halo, and is always surrounded by a symbiontophorous vacuole. For molecular characterization, the small-subunit rRNA genes were sequenced and used for taxonomic assignment as well as the design of species-specific oligonucleotide probes. Phylogenetic analyses revealed that "Candidatus Bealeia paramacronuclearis" clusters with the so-called "basal" Rickettsiales, and "Candidatus Fokinia cryptica" belongs to "Candidatus Midichloriaceae." We obtained tree topologies showing a separation of Rickettsiales into at least two groups: one represented by the families Rickettsiaceae, Anaplasmataceae, and "Candidatus Midichloriaceae" (RAM clade), and the other represented by "basal Rickettsiales," including "Candidatus Bealeia paramacronuclearis." Therefore, and in accordance with recent publications, we propose to limit the order Rickettsiales to the RAM clade and to raise "basal Rickettsiales" to an independent order, Holosporales ord. nov., inside Alphaproteobacteria, which presently includes four family-level clades. Additionally, we define the family "Candidatus Hepatincolaceae" and redefine the family Holosporaceae IMPORTANCE: In this paper, we provide the characterization of two novel bacterial symbionts inhabiting the same Paramecium host (Ciliophora, Alveolata). Both symbionts belong to "traditional" Rickettsiales, one representing a new species of the genus "Candidatus Fokinia" ("Candidatus Midichloriaceae"), and the other representing a new genus of a "basal" Rickettsiales According to newly characterized sequences and to a critical revision of recent literature, we propose a taxonomic reorganization of "traditional" Rickettsiales that we split into two orders: Rickettsiales sensu stricto and Holosporales ord. nov. This work represents a critical revision, including new records of a group of symbionts frequently occurring in protists and whose biodiversity is still largely underestimated.

Host control and nutrient trading in a photosynthetic symbiosis.

Photosymbiosis is one of the most important evolutionary trajectories, resulting in the chloroplast and the subsequent development of all complex photosynthetic organisms. The ciliate Paramecium bursaria and the alga Chlorella have a well established and well studied light dependent endosymbiotic relationship. Despite its prominence, there remain many unanswered questions regarding the exact mechanisms of the photosymbiosis. Of particular interest is how a host maintains and manages its symbiont load in response to the allocation of nutrients between itself and its symbionts. Here we construct a detailed mathematical model, parameterised from the literature, that explicitly incorporates nutrient trading within a deterministic model of both partners. The model demonstrates how the symbiotic relationship can manifest as parasitism of the host by the symbionts, mutualism, wherein both partners benefit, or exploitation of the symbionts by the hosts. We show that the precise nature of the photosymbiosis is determined by both environmental conditions (how much light is available for photosynthesis) and the level of control a host has over its symbiont load. Our model provides a framework within which it is possible to pose detailed questions regarding the evolutionary behaviour of this important example of an established light dependent endosymbiosis; we focus on one question in particular, namely the evolution of host control, and show using an adaptive dynamics approach that a moderate level of host control may evolve provided the associated costs are not prohibitive.

Did Gause Have a Yeast Infection?

We planned to develop predator-prey models using Paramecium and yeast, but they have not been empirically examined since work by Gause in the 1930s. Therefore, we evaluated if Paramecium aurelia ingests and grows on eight yeasts. Recognising that it ingested yeasts but could not grow, we assessed if it might grow on other yeasts, by empirically parameterising a predator-prey model that relies on ingestion, not growth. Simulations were compared to P. aurelia-yeast time-series data, from Gause. We hypothesised that if the model simulated predator-prey dynamics that mimicked the original data, then possibly P. aurelia could grow on yeast; simulations did not mimic the original data. Reviewing works by Gause exposed two issues: experiments were undoubtedly contaminated with bacteria, allowing growth on bacteria, not yeast; and the population cycle data cannot be considered a self-sustaining time series, as they were manipulated by adding yeast and ciliates. We conclude that past and future work should not rely on this system, for either empirical or theoretical evaluations. Finally, although we show that P. aurelia, P. caudatum, Euplotes patella, and Blepharisma sp. cannot grow on yeast, Tetrahymena pyriformis and Colpidium striatum can; these may provide models to explore predator-prey dynamics.

The Evolutionary Relationships between Endosymbiotic Green Algae of Paramecium bursaria Syngens Originating from Different Geographical Locations.

Paramecium bursaria (Ehrenberg 1831), a freshwater ciliate, typically harbors hundreds of green algal symbionts inside the cell. The aim of present study was the molecular identification of newly analyzed P. bursaria symbionts. The second aspect of the present survey was testing a hypothesis whether endosymbionts prefer the specified syngen of the host, and the specified geographical distribution. Ten strains of endosymbionts isolated from strains of P. bursaria originating from different geographical locations were studied. We analyzed for the first time, both the fragment of plastid genome containing 3'rpl36-5' infA genes and a fragment of a nuclear gene encoding large subunit ribosomal RNA (LSU rDNA). The analysis of the LSU rDNA sequences showed the existence of 3 haplotypes and the haplotype diversity of 0.733, and 8 haplotypes for the 3'rpl36-5' infA gene fragment and haplotype diversity of 0.956. The endosymbionts isolated from P. bursaria strains were identified as Chlorella vulgaris, Ch. variabilis and Micractinium conductrix. There was no correlation between the syngen of P. bursaria and the species of endosymbiont.

Disturbance-mediated colonization-extinction dynamics in experimental protist metacommunities.

Colonization-extinction dynamics and species sorting among habitats deter- mine the distribution of species within metacommunities. Theory suggests that disturbances reduce the importance of species sorting and enhance spatial patterning and stochastic effects, however this has not yet been experimentally shown. We examined how extinctions in a heterogeneous landscape of patches affects the influence of environmental, spatial, and stochastic factors on community composition in a simple two-species, two-habitat, protist metacommunity where each species dominates in a different habitat type. We imposed four different levels of random extinctions on local patches and monitored changes in the metacommunity through time. We found that near-steady state patterns of community variability developed relatively rapidly (within nine colonization-extinction cycles) and that increased extinction rate produced altered patterns of community regulation by reducing environmental control and increasing spatial and stochastic effects. Our results indicate a possible explanation for the combination of environmental, spatial and stochastic effects observed in natural metacommunities.

L-shaped prey isocline in the Gause predator-prey experiments with a prey refuge.

Predator and prey isoclines are estimated from data on yeast-protist population dynamics (Gause et al., 1936). Regression analysis shows that the prey isocline is best fitted by an L-shaped function that has a vertical and a horizontal part. The predator isocline is vertical. This shape of isoclines corresponds with the Lotka-Volterra and the Rosenzweig-MacArthur predator-prey models that assume a prey refuge. These results further support the idea that a prey refuge changes the prey isocline of predator-prey models from a horizontal to an L-shaped curve. Such a shape of the prey isocline effectively bounds amplitude of predator-prey oscillations, thus promotes species coexistence.

Use of a novel cell adhesion method and digital measurement to show stimulus-dependent variation in somatic and oral ciliary beat frequency in Paramecium.

When Paramecium encounters positive stimuli, the membrane hyperpolarizes and ciliary beat frequency increases. We adapted an established immobilization protocol using a biological adhesive and a novel digital analysis system to quantify beat frequency in immobilized Paramecium. Cells showed low mortality and demonstrated beat frequencies consistent with previous studies. Chemoattractant molecules, reduction in external potassium, and posterior stimulation all increased somatic beat frequency. In all cases, the oral groove cilia maintained a higher beat frequency than mid-body cilia, but only oral cilia from cells stimulated with chemoattactants showed an increase from basal levels.

Trapping of swimming microorganisms at lower surfaces by increasing buoyancy.

Models suggest that mechanical interactions alone can trap swimming microorganisms at surfaces. Testing them requires a method for varying the mechanical interactions. We tuned contact forces between Paramecia and surfaces in situ by varying their buoyancy with nonuniform magnetic fields. Remarkably, increasing their buoyancy can lead to ∼100% trapping at lower surfaces. A model of Paramecia in surface contact passively responding to external torques quantitatively accounts for the data implying that interactions with a planar surface do not engage their mechanosensing network and illuminating how their trapping differs from other smaller microorganisms.

Pharmacological characterization of NMDA-like receptors in the single-celled organism Paramecium primaurelia.

Paramecium primaurelia is a unicellular eukaryote that moves in freshwater by ciliary beating and responds to environmental stimuli by altering motile behaviour. The movements of the cilia are controlled by the electrical changes of the cell membrane: when the intraciliary Ca(2+) concentration associated with plasma membrane depolarization increases, the ciliary beating reverses its direction, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca(2+) influx. Here, we evaluated the effects due to the activation or blockade of N-methyl-d-aspartic acid (NMDA) receptors on swimming behaviour in Paramecium. Paramecia normally swim forward, drawing almost linear tracks. We observed that the simultaneous administration of NMDA and glycine induced a partial ciliary reversal (PaCR) leading to a continuous spiral-like swim. Furthermore, the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, was longer in NMDA+glycine-treated cells. NMDA action required the presence of Ca(2+), as the normal forward swimming was restored when the ion was omitted from the extracellular milieu. The PaCR and the enhancement of CCR duration significantly decreased when the antagonists of the glutamate site D-AP5 or CGS19755, the NMDA channel blocker MK-801 or the glycine site antagonist DCKA was added. The action of NMDA+glycine was also abolished by Zn(2+) or ifenprodil, the GluN2A and the GluN2B NMDA-containing subunit blockers, respectively. Searches of the Paramecium genome database currently available indicate that the NMDA-like receptor with ligand-binding characteristics of an NMDA receptor-like complex, purified from rat brain synaptic membranes and found in some metazoan genomes, is also present in Paramecium. These results provide evidence that functional NMDA receptors similar to those typical of mammalian neuronal cells are present in the single-celled organism Paramecium and thus suggest that the glutamatergic NMDA system is a phylogenetically old behaviour-controlling mechanism.

Finding the ciliary beating pattern with optimal efficiency.

We introduce a measure for energetic efficiency of biological cilia acting individually or collectively and numerically determine the optimal beating patterns according to this criterion. Maximizing the efficiency of a single cilium leads to curly, often symmetric, and somewhat counterintuitive patterns. However, when looking at a densely ciliated surface, the optimal patterns become remarkably similar to what is observed in microorganisms like Paramecium. The optimal beating pattern then consists of a fast effective stroke and a slow sweeping recovery stroke. Metachronal coordination is essential for efficient pumping and the highest efficiency is achieved with antiplectic waves. Efficiency also increases with an increasing density of cilia up to the point where crowding becomes a problem. We finally relate the pumping efficiency of cilia to the swimming efficiency of a spherical microorganism and show that the experimentally estimated efficiency of Paramecium is surprisingly close to the theoretically possible optimum.