Insights into the differential proteome landscape of a newly isolated Paramecium multimicronucleatum in response to cadmium stress.

Employing microbial systems for the bioremediation of contaminated waters represent a potential option, however, limited understanding of the underlying mechanisms hampers the implication of microbial-mediated bioremediation. The omics tools offer a promising approach to explore the molecular basis of the bioremediation process. Here, a mass spectrometry-based quantitative proteome profiling approach was conducted to explore the differential protein levels in cadmium-treated Paramecium multimicronucleatum. The Proteome Discoverer software was used to identify and quantify differentially abundant proteins. The proteome profiling generated 7,416 peptide spectral matches, yielding 2824 total peptides, corresponding to 989 proteins. The analysis revealed that 29 proteins exhibited significant (p ≤ 0.05) differential levels, including a higher abundance of 6 proteins and reduced levels of 23 proteins in Cd2+ treated samples. These differentially abundant proteins were associated with stress response, energy metabolism, protein degradation, cell growth, and hormone processing. Briefly, a comprehensive proteome profile in response to cadmium stress of a newly isolated Paramecium has been established that will be useful in future studies identifying critical proteins involved in the bioremediation of metals in ciliates. SIGNIFICANCE: Ciliates are considered a good biological indicator of chemical pollution and relatively sensitive to heavy metal contamination. A prominent ciliate, Paramecium is a promising candidate for the bioremediation of polluted water. The proteins related to metal resistance in Paramecium species are still largely unknown and need further exploration. In order to identify and reveal the proteins related to metal resistance in Paramecia, we have reported differential protein abundance in Paramecium multimicronucleatum in response to cadmium stress. The proteins found in our study play essential roles during stress response, hormone processing, protein degradation, energy metabolism, and cell growth. It seems likely that Paramecia are not a simple sponge for metals but they could also transform them into less toxic derivatives or by detoxification by protein binding. This data will be helpful in future studies to identify critical proteins along with their detailed mechanisms involved in the bioremediation and detoxification of metal ions in Paramecium species.

Noninvasive and safe cell viability assay for Paramecium using natural pigment extracted from food.

Noninvasive, safe and cost-effective cell viability assay is important in many fields of biological research such as cell culture and counting. We examined ten typical natural pigments extracted from food to find that Monascus pigment (MP) or anthocyanin pigment (AP: purple sweet potato and purple cabbage) with Tris (Trimethylolaminomethane) works as a good indicator of viability assay for dye exclusion test (DET) of Paramecium. This was confirmed spectrally by scan-free, non-invasive absorbance spectral imaging A (x, y, λ) microscopy. We developed a new method of cell capture using a metal mesh to confine live Paramecium in a restricted space. This has the advantage that a low-cost and robust capture can be fabricated without using special equipment, compared to a conventional lab-on-a-chip. As a result, MP and AP stained dead cells as quick as methylene blue (MB), a synthetic dye conventionally used in DET within 1 min when treated with microwave and benzalkonium chloride. The natural pigments with Tris had little effect on inhibiting the growth of Paramecium, but MB killed all the cells within 1 h. MP is most useful because it allows non-invasive DET without Tris. This approach provides less invasive and safe DET.

Diclofenac sodium aqueous systems at low concentrations: Interconnection between physicochemical properties and action on hydrobionts.

Diclofenac sodium (DS) is a widely used nonsteroidal anti-inflammatory drug (NSAIDs). NSAIDs are poorly removed during standard wastewater treatment. The consequences of the presence of NSAIDs in rivers and lakes at 10-11-10-8 mol/L are not yet established; therefore, ecotoxicologists have focused their efforts on studying the effect of low-concentration NSAIDs on fish and hydrobionts, and also on predicting the potential risks to humans. Literature provides some information about the bioeffects of some NSAID solutions in low concentrations but there is no physicochemical explanation for these phenomena. Studying the physicochemical patterns of DS solutions in the low range of concentrations and establishing an interconnection between the solutions' physicochemical properties and bioeffects can provide a conceptually new and important source of information regarding the unknown effects of DS. The physicochemical properties and action of DS solutions on Ceriodaphnia affinis cladocerans, Paramecium caudatum infusoria, Chlorella vulgaris unicellular green algae, as well as on the growth of the roots of Triticum vulgare wheat seeds, were studied in the calculated concentration range of 1 × 10-3-1 × 10-18 mol/L. The relationship between these phenomena was established using the certified procedures for monitoring the toxicity of natural water and wastewater. It was shown for the first time that water solutions of DS are dispersed systems in which the dispersed phase undergoes a rearrangement with dilution, accompanied by changes in its size and properties, which affects the nonmonotonic dependences of the system's physicochemical properties and could cause nonmonotonic changes in action on hydrobionts in the low concentration range.

The Biology of General Anesthesia from Paramecium to Primate.

General anesthesia serves a critically important function in the clinical care of human patients. However, the anesthetized state has foundational implications for biology because anesthetic drugs are effective in organisms ranging from paramecia, to plants, to primates. Although unconsciousness is typically considered the cardinal feature of general anesthesia, this endpoint is only strictly applicable to a select subset of organisms that are susceptible to being anesthetized. We review the behavioral endpoints of general anesthetics across species and propose the isolation of an organism from its environment - both in terms of the afferent arm of sensation and the efferent arm of action - as a generalizable definition. We also consider the various targets and putative mechanisms of general anesthetics across biology and identify key substrates that are conserved, including cytoskeletal elements, ion channels, mitochondria, and functionally coupled electrical or neural activity. We conclude with a unifying framework related to network function and suggest that general anesthetics - from single cells to complex brains - create inefficiency and enhance modularity, leading to the dissociation of functions both within an organism and between the organism and its surroundings. Collectively, we demonstrate that general anesthesia is not restricted to the domain of modern medicine but has broad biological relevance with wide-ranging implications for a diverse array of species.

Evaluation of phenol-induced ecotoxicity in two model ciliate species: Population growth dynamics and antioxidant enzyme activity.

The application of identical exposure dosages in different species generally leads to a limited understanding of dose-response patterns because of species-specific factors. To evaluate phenol-induced ecotoxicity, antioxidant enzyme activity and population growth dynamics were compared in two model ciliates, the marine species Euplotes vannus and the freshwater species Paramecium multimicronucleatum. Dosage ranges of phenol exposure were based on tolerance limits of test ciliates as determined by their carrying capacity (K) and growth rate (r). When the exposure duration of phenol increased from 48 h to 96 h, the median effective dose (ED50) for P. multimicronucleatum decreased faster than that for E. vannus, and the ratio of the former to the latter declined from 2.75 to 0.30. When E. vannus was exposed to increasing concentrations of phenol (0-140 mg l-1), r rose initially and then dropped significantly at concentrations higher than 40 mg l-1, whereas K decreased linearly over the entire range. For P. multimicronucleatum, both r and K declined gradually over the range 0-200 mg l-1 phenol. Dose-response patterns of activities of three individual antioxidant enzymes, and the integrative index of the three enzymes, presented a biphasic (inverse U-shaped) curve at each of four durations of exposure, i.e. 12 h, 24 h, 36 h and 48 h. Cluster analyses and multidimensional scaling analyses of antioxidant enzyme activities revealed differences in the temporal succession of physiological states between the two model ciliates. In brief, combining ED50 with growth dynamic parameters is helpful for designing exposure dosages of toxicants in ecotoxicity tests.

Simulation of Paramecium Chemotaxis Exposed to Calcium Gradients.

Paramecium or other ciliates have the potential to be utilized for minimally invasive surgery systems, making internal body organs accessible. Paramecium shows interesting responses to changes in the concentration of specific ions such as K(+), Mg(2+), and Ca(2+) in the ambient fluid. Some specific responses are observed as, changes in beat pattern of cilia and swimming toward or apart from the ion source. Therefore developing a model for chemotactic motility of small organisms is necessary in order to control the directional movements of these microorganisms before testing them. In this article, we have developed a numerical model, investigating the effects of Ca(2+) on swimming trajectory of Paramecium. Results for Ca(2+)-dependent chemotactic motility show that calcium gradients are efficient actuators for controlling the Paramecium swimming trajectory. After applying a very low Ca(2+) gradient, a directional chemotaxis of swimming Paramecium is observable in this model. As a result, chemotaxis is shown to be an efficient method for controlling the propulsion of these small organisms.

Mitigation of copper toxicity by DNA oligomers in green paramecia.

Impact of transition metals which catalyze the generation of reactive oxygen species (ROS), on activation of cell death signaling in plant cells have been documented to date. Similarly in green paramecia (Paramecium bursaria), an aquatic protozoan species harboring symbiotic green algae in the cytoplasm, toxicities of various metallic ions have been documented. We have recently examined the effects of double-stranded GC-rich DNA fragments with copper-binding nature and ROS removal catalytic activity as novel plant cell-protecting agents, using the suspension-cultured tobacco cells. Here, we show that above DNA oligomers protect the cells of green paramecia from copper-induced cell death, suggesting that the phenomenon firstly observed in tobacco cells is not limited only within higher plants but it could be universally observable in wider range of organisms.

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.

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.

Inactivation of Ca2+-induced ciliary reversal by high-salt extraction in the cilia of Paramecium.

Intracellular Ca(2+) induces ciliary reversal and backward swimming in Paramecium. However, it is not known how the Ca(2+) signal controls the motor machinery to induce ciliary reversal. We found that demembranated cilia on the ciliated cortical sheets from Paramecium caudatum lost the ability to undergo ciliary reversal after brief extraction with a solution containing 0.5 M KCl. KNO(3), which is similar to KCl with respect to chaotropic effect; it had the same effect as that of KCl on ciliary response. Cyclic AMP antagonizes Ca(2+)-induced ciliary reversal. Limited trypsin digestion prevents endogenous A-kinase and cAMP-dependent phosphorylation of an outer arm dynein light chain and induces ciliary reversal. However, the trypsin digestion prior to the high-salt extraction did not affect the inhibition of Ca(2+)-induced ciliary reversal caused by the high-salt extraction. Furthermore, during the course of the high-salt extraction, some axonemal proteins were extracted from ciliary axonemes, suggesting that they may be responsible for Ca(2+)-induced ciliary reversal.

Surface interactions affect the toxicity of engineered metal oxide nanoparticles toward Paramecium.

To better understand the potential impacts of engineered metal oxide nanoparticles (NPs) in the ecosystem, we investigated the acute toxicity of seven different types of engineered metal oxide NPs against Paramecium multimicronucleatum, a ciliated protozoan, using the 48 h LC(50) (lethal concentration, 50%) test. Our results showed that the 48 h LC(50) values of these NPs to Paramecium ranged from 0.81 (Fe(2)O(3) NPs) to 9269 mg/L (Al(2)O(3) NPs); their toxicity to Paramecium increased as follows: Al(2)O(3) < TiO(2) < CeO(2) < ZnO < SiO(2) < CuO < Fe(2)O(3) NPs. On the basis of the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, interfacial interactions between NPs and cell membrane were evaluated, and the magnitude of interaction energy barrier correlated well with the 48 h LC(50) data of NPs to Paramecium; this implies that metal oxide NPs with strong association with the cell surface might induce more severe cytotoxicity in unicellular organisms.

Cylindrospermopsis raciborskii exudate-Cu complexes: impact on copper dynamics and bioavailability in an aquatic food chain.

INTRODUCTION: The increasing contamination of aquatic environments motivates studies on the interactions among natural dissolved organic matter, metals, and the biota. This investigation focused on the organic exudates of the toxic cyanobacteria Cylindrospermopsis raciborskii as a Cu carrier through a three-level aquatic trophic chain (bacteria, protozoa, and copepod). DISCUSSION: The effects of bacteria activity and growth on the metal-organic complexes were evaluated through changes in free Cu(2+) ions, total dissolved, and total particulate Cu. To be sure that the added copper would be complexed to the exudates, its complexing properties were previously determined. The cyanobacteria exudate-Cu complexes were furnished to bacteria that were further used as a food source to the protozoan Paramercium caudatum. This was then furnished as food to the copepod Mesocyclops sp. The results showed that, in general, the cyanobacterial exudates decreased Cu bioavailability and toxicity to the first trophic level (bacteria), but because the heterotrophic bacteria accumulated Cu, they were responsible for the transference for the otherwise low availability metal form. Both the bacteria and protozoan organisms accumulated Cu, but no metal accumulation was detected in the copepods.

The ciliate Paramecium shows higher motility in non-uniform chemical landscapes.

We study the motility behavior of the unicellular protozoan Paramecium tetraurelia in a microfluidic device that can be prepared with a landscape of attracting or repelling chemicals. We investigate the spatial distribution of the positions of the individuals at different time points with methods from spatial statistics and Poisson random point fields. This makes quantitative the informal notion of "uniform distribution" (or lack thereof). Our device is characterized by the absence of large systematic biases due to gravitation and fluid flow. It has the potential to be applied to the study of other aquatic chemosensitive organisms as well. This may result in better diagnostic devices for environmental pollutants.

Symbiotic Chlorella vulgaris of the ciliate Paramecium bursaria plays an important role in maintaining perialgal vacuole membrane functions.

Treatment of symbiotic alga-bearing Paramecium bursaria cells with a protein synthesis inhibitor, cycloheximide, induces synchronous swelling of all perialgal vacuoles at about 24h after treatment under a constant light condition. Subsequently, the vacuoles detach from the host cell cortex. The algae in the vacuoles are digested by the host's lysosomal fusion to the vacuoles. To elucidate the timing of algal degeneration, P. bursaria cells were treated with cycloheximide under a constant light condition. Then the cells were observed using transmission electron microscopy. Results show that algal chloroplasts and nuclei degenerated within 9h after treatment, but before the synchronous swelling of the perialgal vacuole and appearance of acid phosphatase activity in the perialgal vacuole by lysosomal fusion. Treatment with cycloheximide under a constant dark condition and treatment with chloramphenicol under a constant light condition induced neither synchronous swelling of the vacuoles nor digestion of the algae inside the vacuoles. These results demonstrate that algal proteins synthesized during photosynthesis are necessary to maintain chloroplastic and nuclear structures, and that inhibition of protein synthesis induces rapid lysis of these organelles, after which synchronous swelling of the perialgal vacuole and fusion occur with the host lysosomes.

Gamma-amino butyric acid (GABA) release in the ciliated protozoon Paramecium occurs by neuronal-like exocytosis.

Paramecium primaurelia expresses a significant amount of gamma-amino butyric acid (GABA). Paramecia possess both glutamate decarboxylase (GAD)-like and vesicular GABA transporter (vGAT)-like proteins, indicating the ability to synthesize GABA from glutamate and to transport GABA into vesicles. Using antibodies raised against mammalian GAD and vGAT, bands with an apparent molecular weight of about 67 kDa and 57 kDa were detected. The presence of these bands indicated a similarity between the proteins in Paramecium and in mammals. VAMP, syntaxin and SNAP, putative proteins of the release machinery that form the so-called SNARE complex, are present in Paramecium. Most VAMP, syntaxin and SNAP fluorescence is localized in spots that vary in size and density and are primarily distributed near the plasma membrane. Antibodies raised against mammal VAMP-3, sintaxin-1 or SNAP-25 revealed protein immunoblot bands having molecular weights consistent with those observed in mammals. Moreover, P. primaurelia spontaneously releases GABA into the environment, and this neurotransmitter release significantly increases after membrane depolarization. The depolarization-induced GABA release was strongly reduced not only in the absence of extracellular Ca(2+) but also by pre-incubation with bafilomycin A1 or with botulinum toxin C1 serotype. It can be concluded that GABA occurs in Paramecium, where it is probably stored in vesicles capable of fusion with the cell membrane; accordingly, GABA can be released from Paramecium by stimulus-induced, neuronal-like exocytotic mechanisms.

Biophysical effects of the natural product euplotin C on the Paramecium membrane.

The effect of euplotin C--a cytotoxic secondary metabolite produced by the protist ciliate Euplotes crassus--on the voltage-dependent Ca(2+) channel activity was studied in a single-celled system by analyzing the swimming behavior of Paramecium. When the intraciliary Ca(2+) concentration associated with plasma membrane depolarization increases, a reversal in the direction of ciliary beating occurs, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca(2+) influx. The present study demonstrates that the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, is longer in euplotin C-treated cells. Using selective Ca(2+) channel blockers, we demonstrate that euplotin C modulates Ca(2+) channels similar to the T- and L-types that occur in mammalian cells. Indeed, the increase of CCR duration significantly decreased when flunarizine and nimodipine-verapamil blockers were employed. Membrane fluidity measurements using a fluorescent dye, 6-lauroyl-2-dimethylaminonaphtalene (laurdan), indicated that membranes in euplotin C-treated cells are more tightly packed and ordered than membranes in control cells. Our data suggest that euplotin C enhances backward swimming in our unicellular model system by interacting with the ciliary Ca(2+) channel functions through the reduction of cell membrane fluidity.

Side-effects of fenazaquin on a cellular model of Paramecium.

Our biodiversity has long been preserved, but the main constituents of our environment have been particularly affected by the addition of molecules resulting from agricultural and industrial activities. It is well accepted that these changes may stress some species, making them more vulnerable. In this project, we determined the disruptive side-effects of a pesticide on several biochemical endpoints and the behaviour of a microorganism as the ciliate protist Paramecium sp. Here we used fenazaquin [4-(4-tert-butylphenethoxy)quinazoline] that belongs to the quinazoline class of chemicals and that is a pesticide intended to control mites and insects; its route of exposure is ingestion and dermal, and its mode of action is the disruption of the biochemistry of insect mitochondria. In our experiments with fenazaquin at 40, 60 and 80 nM, we recorded disturbances in protein and glutathione, in glutathione S-transferase, and a decrease in consumption of oxygen. The results are discussed in relation to potential risks and mechanisms of action. In addition, the data can be used as reference values in further testing with other pesticides and chemistries.

Cycloheximide induces synchronous swelling of perialgal vacuoles enclosing symbiotic Chlorella vulgaris and digestion of the algae in the ciliate Paramecium bursaria.

Cycloheximide is known to inhibit preferentially protein synthesis of symbiotic Chlorella of the ciliate Paramecium bursaria, but to hardly host protein synthesis. Treatment of algae-bearing Paramecium cells with cycloheximide induces synchronous swelling of all perialgal vacuoles that are localized immediately beneath the host's cell membrane. In this study, the space between the symbiotic algal cell wall and the perialgal vacuole membrane widened to about 25 times its normal width 24 h after treatment with cycloheximide. Then, the vacuoles detached from beneath the host's cell membrane, were condensed and stained with Gomori's solution, and the algae in the vacuoles were digested. Although this phenomenon is induced only under a fluorescent light condition, and not under a constant dark condition, this phenomenon was not induced in paramecia treated with cycloheximide in the light in the presence of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that algal proteins synthesized in the presence of algal photosynthesis serve some important function to prevent expansion of the perialgal vacuole and to maintain the ability of the perialgal vacuole membrane to protect itself from host lysosomal fusion.

Effect of the bioactive metabolite euplotin C on phagocytosis and fluid-phase endocytosis in the single-celled eukaryote Paramecium.

The effect of euplotin C -- a lipophilic bioactive metabolite produced by the ciliate Euplotes crassus -- on the kinetics of both phagocytosis of latex particles and fluid-phase uptake of dextran, was studied in the single-cell ciliate Paramecium primaurelia. The inhibition of food vacuole formation was concentration- and time-dependent (p<0.001), even if euplotin C did not completely block the phagocytosis. Following a 15 min treatment with a euplotin C (0.5 microg/ml), the latex particle uptake was inhibited up to 25%. Furthermore, the pretreatment of cells with taxol strongly counteracted euplotin C effect. The amount of extracellularly provided dextran, which is internalized exclusively by fluid-phase uptake, was quantified in cells whose phagocytic activity was blocked by trifluoperazine. The amount of the internalized dextran was about 50% of that in controls after 15 min incubation in the presence of euplotin C. Fluorescence confocal images showed that no endosomes were formed on the surface of these cells. The effect of euplotin C on the food vacuole formation and fluid-phase endocytosis is apparently mediated by a modification of microtubule network.

Infectivity of Chlorella species for the ciliate Paramecium bursaria is not based on sugar residues of their cell wall components, but on their ability to localize beneath the host cell membrane after escaping from the host digestive vacuole in the early infection process.

Paramecium bursaria cells harbor several hundred symbiotic algae in their cytoplasm. Algae-free cells can be reinfected with algae isolated from algae-bearing cells or cultivated Chlorella species through the digestive vacuoles. To determine the relationship between the infectivity of various Chlorella species and the nature of their cell wall components, algae-free P. bursaria cells were mixed with 15 strains of cultivated Chlorella species and observed for the establishment of endosymbiosis at 1 h and 3 weeks after mixing. Only 2 free-living algal strains, C. sorokiniana C-212 and C. kessleri C-531, were maintained in the host cells, whereas free-living C. sorokiniana C-43, C. kessleri C-208, C. vulgaris C-27, C. ellipsoidea C-87 and C-542, C. saccharophila C-183 and C-169, C. fusca var. vacuolata C-104 and C-28, C. zofingiensis C-111, and C. protothecoides C-150 and C-206 and the cultivated symbiotic Chlorella sp. strain C-201 derived from Spongilla fluviatilis could not be maintained. These infection-incapable strains could escape from the host digestive vacuole but failed to localize beneath the host cell membrane and were eventually digested. Labeling of their cell walls with Alexa Fluor 488-conjugated wheat germ agglutinin, GS-II, or concanavalin A, with or without pretreatment with 0.4 N NaOH, showed no relationship between their infectivity and the stainability with these lectins. Our results indicate that the infectivity of Chlorella species for P. bursaria is not based on the sugar residues on their cell wall and on the alkali-insoluble part of the cell wall components, but on their ability to localize just beneath the host cell membrane after escaping from the host digestive vacuole.