Paramecium Species Research Articles

Induced hydrogen peroxide tolerance in Paramecium

Summary Hormesis is the phenomenon of beneficial effects induced by exposure to low doses of otherwise harmful physical or chemical agents. The present study of peroxide tolerance in two species of Paramecium provides evidence that: 1) prior exposure to peroxide can induce tolerance to higher doses of the agent; 2) the length of the recovery period after exposure is crucial for inducing cell tolerance to increased doses of the toxin in both the short-lived Paramecium tetraurelia and the long-lived Paramecium multimicronucleatum . The two species of paramecia had comparable levels of constitutive and inducible catalase levels. Two inhibitors of endogenous antioxidants were used, aminotriazole, a catalase inhibitor, and BCNU (1,3 -bis (chloroethyl)-1-nitrosourea), the antineoplastic agent carmustine, an inhibitor of glutathione peroxidase. In the presence of both inhibitors, a significant increase in cell sensitivity to peroxide was found. Since exposure to given doses of hydrogen peroxide can stimulate increased catalase, and can increase survival following oxidative challenge, the use of a hormetic strategy to increase longevity is proposed.

Cytological studies of conjugation and nuclear processes in Paramecium duboscqui Chatton & Brachon 1933

Summary Paramecium duboscqui Chatton and Brachon 1933 were collected during cold times of the year in Harbin, China. Cell growth and conjugation processes of this species were studied under various temperatures, and compared with those of P. caudatum. P. duboscqui can divide once every three days even at 5°C, whereas P. caudatum stops cell division at temperatures less than 10°C. Conjugating pair formation takes place from 5 to 20°C but not at 30°C in P. duboscqui. On the other hand, in P. caudatum, pairing is blocked at temperatures less than 10°C, but occurs at 30°C. Nuclear processes during conjugation of P. duboscqui were studied using protargol stained preparations. Changes in the micronuclei, such as 3 pregamic divisions, pronuclear exchange, synkaryon formation, and three postgamic divisions took place as were observed in other Paramecium species. After the third postgamic division, four macronuclear anlagen and four micronuclei were differentiated. Each of the four macronuclear anlagen were segregated into four daughter cells through successive two cell divisions. On the other hand, the four micronuclei divided mitotically at each cell division. The old macronucleus divided once amitotically at the time of the third postgamic division and then disappeared mostly before the first cell division.

Holospora species infecting the nuclei of Paramecium appear to belong into two groups of bacteria

Summary Ten Holospora bacteria in various Paramecium species, namely P. novaurelia, P. caudatum, P. bursaria, P. woodruffi , and P. putrinum were found to be similar concerning their morphology and life cycle. However, four species (group 1): Holospora caryophila, H. bacillata, H. curvata , and a species newly found in P. putrinum differed from the other bacteria (group 2), among them H. obtusa, H. undulata, H. elegans, H. recta , as they had a different behavior during the division of the host nucleus; in group 2 the infectious forms were collected in the connecting piece of the dividing host nucleus and were then released into the medium, whereas in group 1 the infectious forms remained distributed over the whole nucleus during its division. The bacteria of the group 1 also were not labelled by in situ hybridization using an oligonucleotide probe hybridizing with the rRNA of bacteria of group 2. The data indicate that Holospora bacteria may at least belong into two groups.

Interspecific Pair Formation Induced by Natural Mating Reaction in Paramecium

Abstract The species of Paramecium are sexually isolated primarily by the specificity of mating reaction. By this reason, it has been thought that interspecific pair formation does not occur when cells of different species are mixed. However we discovered that interspecific pair formation can be induced simply by mixing the mating reactive cells of two species. Among four species of Paramecium, P. tetraurelia, P. multimicronucleatum, P. caudatum, and P. bursaria, the interspecific pairs were observed in the former three species which belong to the “aurelia” group, but those three species did not mate with P. bursaria which belongs to the “bursaria” group. The percentage of interspecific pair formation was less than 10% in all positive cases. Macronuclear fragmentation, one of the remarkable nuclear changes in normal conjugation, was also observed in the interspecific pairs. The time course of the pair formation and macronuclear changes were similar to those of intraspecific conjugation.

Monoclonal antibody to a bacterial endonuclear symbiont Holospora cross reacts with proteins of contractile vacuole radial canals of Paramecium species.

A monoclonal antibody (mAb) IR-2-1 was raised against a 67-kDa protein purified from the macronucleus-specific bacterial symbiont Holospora obtusa of Paramecium caudatum. The mAb was found to react with two bands (31 and 67-kDa) on gels of H. obtusa. Indirect immunofluorescence microscopy showed that these antigens were distributed inside the cells. However, unexpectedly, this mAb also cross reacted with the radial arms of the contractile vacuole in P. caudatum, P. tetraurelia, P. multimicronucleatum, P. jenningsi and P. bursaria as well as with their cytoplasm. Immunoelectron microscopy showed that the antigens were located on the decorated spongiome of the radial arms. In immunoblots, mAb IR-2-1 reacted with a band of 67 kDa in all Paramecium species examined. However, no band appeared in the immunoblot of isolated macronuclei of H. obtusa-free P. caudatum and no label was seen in the nuclear matrix of the macronucleus of air-dried P. caudatum. These results suggest that the 67-kDa antigen found in H. obtusa was not imported from the host macronucleus and the same antigen in the host contractile vacuoles and cytoplasm were not derived from the symbiont. These results also showed that an epitope on the decorated spongiome of the Paramecium species is shared by its bacterial symbiont. In contrast to the decorated tubule-specific mAb, DS-1, the antigens for IR-2-1 appeared to be loosely membrane bound as they were lost in paraformaldehyde fixed and acetone permeabilized Paramecium.

Distribution and genetic variabilities of mitochondrial plasmid-like DNAs in Paramecium.

Plasmid-like DNAs were isolated from mitochondria in many stocks of Para- mecium caudatum. Fifty-two among 68 stocks examined contained unique double-stranded DNAs (designated type I) ranging from 6.0 to 6.8 kb in length, and 12 stocks contained a set of 4 kinds of DNAs (8.2, 4.1, 2.8 and 1.4 kb; type II). Five of the stocks containing the type-I DNA had an additional 8.9-kb DNA (type III). No plasmid-like DNAs were found in the remaining 4 stocks. All these DNAs are linear and closely associated with proteins which can be removed only after proteinase-K digestion, suggesting that they exist as unique DNA protein complexes in mitochondria. Restriction and Southern blot analyses revealed a large sequence divergence among both type-I and -II DNAs. Type-I DNAs were found in every lineages of mitochondrial genomic DNA (mtDNA) which were inferred from restriction analyses. Type-II DNAs were, in contrast, scattered throughout the evolutionary tree of the mtDNAs. No obvious relations were found between the distribution of the DNAs and that of syngens (mating-type groups). Type-I DNAs were also found in other species of Paramecium, P. jenningsi and P. multimicronucleatum, and type-II DNAs in P. polycaryum.

Guanine Nucleotides Modulate Calcium Currents in a Marine Paramecium

Voltage-dependent calcium channels play a critical role in many cell functions and in many cell types ranging from protozoa to vertebrates. We have shown previously that guanine nucleotides modulate the calcium action potential and the duration of backward swimming in Paramecium, both indirect measurements of calcium channel function. To determine whether guanine nucleotides to indeed alter calcium currents, the inward calcium current (ICa) in Paramecium calkinsi was studied. First, the calcium current was characterized. The magnitude of ICa increased as the extracellular calcium concentration was increased from 0.5 to 50 mmol l-1, unlike the situation in freshwater species of Paramecium where the inward calcium current magnitude is maximal when extracellular calcium levels reach 1 mmol l-1. Inorganic compounds (NiCl2 at 10 mumol l-1 and CdCl2 at 1 mmol l-1) and organic compounds (naphthalene sulfonamides, W-7 and W-12-Br at 100 and 2 mumol l-1, respectively) reduced ICa. Regardless of the holding membrane potential (from -80 to -20 mV), the threshold activation for ICa was at -10 mV and the maximum value of ICa was reached at +20 mV, suggesting that there is only one type of calcium channel in P. calkinsi. Second, we injected GTP gamma S, GTP and GDP beta S into voltage-clamped cells while monitoring calcium and/or potassium currents. GTP gamma S increased the magnitude of ICa by 42 +/- 6% (mean +/- S.D., N = 5) and the effect was irreversible, GTP increased the magnitude of ICa by 37 +/- 4% (N = 4) in a reversible manner, and GDP beta S decreased ICa by 57 +/- 8% (N = 3) irreversibly. The outward potassium currents did not change when GTP gamma S was injected into the cells. These results support the hypothesis that injection of guanine nucleotides modulates the voltage-dependent calcium channel in P. calkinsi, presumably by activating G-protein-dependent processes.

A repetitive DNA sequence in Paramecium macronuclei is related to the beta subunit of G proteins.

A repeated DNA sequence has been identified in the macronucleus of several Paramecium species. In P.tetraurelia the repeat was identified in the subtelomeric region of four randomly selected telomere clones, as well as downstream of the A type variable surface protein gene. The complete sequence of the A gene linked repeat consists of 15 tandem repeats of exactly 126 nucleotides that contain an open reading frame with significant similarity to the beta subunits of trimeric G proteins. The most striking consensus feature is the amino acid sequence DX omega WD where X is any amino acid and omega is I, L, or V spaced at precise 42 amino acids intervals. This sequence and spacing are found in G-protein beta subunits and other members of this protein motif family. Analysis of the five cloned telomeric restriction fragments showed the repeats can be found in either orientation with respect to the telomere. Poly(A) RNA transcripts containing this sequence have been identified in Paramecium tetraurelia. The conserved presence of this sequence in several species of Paramecium suggests an important physiological function, and the study of this repeat may reveal information about the evolution of this common protein motif.

Defensive function of trichocysts in Paramecium

AbstractA putative defensive function of trichocysts in Paramecium has been tested experimentally. Cells of trichocyst‐non‐discharge (tnd) mutant, artificially induced trichocyst‐deficient cells, and intact wild‐type cells were compared as prey for a carnivorous ciliate, Dileptus margaritifer. Cells of tnd mutants were eaten 9–45 times faster than wild‐type cells by the predator in P. caudatum and P. tetraurelia. Encounters between paramecia and dilepti occurred in nearly the same frequency in mutant and wild‐type cells. Paramecia with reduced numbers of trichocysts were obtained in P. caudatum, P. jenningsi, and P. tetraurelia by treating wild‐type cells with lysozyme, an inducer of trichocyst discharge. These cells were eaten faster than intact cells by the predator and the rate of predation was inversely correlated with the remained capacity of trichocyst discharge. It was concluded that the discharge of trichocysts defends these species of Paramecium against D. margaritifer. Therefore, the results of this work strongly support the hypothesis that trichocysts in Paramecium function as defensive organelles against predators. The results also support the hypothesis that the offense‐defense interaction between Dileptus and Pramecium is mediated by their extrusomes (toxicysts and trichocysts) and suggest that extrusomes in ciliates function as organelles for interspecific cell‐cell interaction.

Use of nile red as a rapid measure of lipid content in ciliates

We report the use of nile red as a rapid and inexpensive method to estimate cellular lipids in three species of Paramecium and in Tetrahymena by the direct application of the dye to living or fixed cells without extraction and purification. Qualitative estimates of the relative changes in the lipid content of cells of varying culture ages were obtained using fluorescence microscopy, while semiquantitative determinations were obtained by measuring the total fluorescence from the emission spectrum (excitation, 535 nm) of fixed cells treated with excess nile red. The relative amounts of neutral (excitation, 488 nm; emission, 540 nm) and polar (excitation, 535 nm; emission, 680 nm) lipids were approximated using fluorescence intensity at these selected spectral conditions to avoid any spill over from each other. The patterns of change with culture age in total lipids in Tetrahymena and in total, neutral and polar lipids in Paramecium obtained using nile red agreed well with published gravimetric data for these ciliates.

Karyological Studies onParamecium(Ciliophora, Protista)

SUMMARYSpecies of the Paramecium aurelia complex, characterized by inbreeding, show differences in the number of chromosomes, not only between species, but also within species. It seemed interesting to study the karyologic differentiation in Paramecium jenningsi, a taxonomic species of the genus Paramecium morphologically similar to the P. aurelia species complex, but characterized by outbreeding. Applying the method of semi-thin sections it was found that P. jenninpi strains from India, Uganda, and Madagascar differ karyologically from each other.

Circadian Rhythm of Photoaccumulation in Paramecium bursaria

Populations of Paramecium bursaria cells display a circadian rhythm of photoaccumulation. Both Chlorella-containing cells and Chlorella-free cells exhibit this rhythm. Several other species of Paramecium do not express rhythmic photoaccumulation when tested under the same conditions. In P. bursaria, photoaccumulation rhythms persist in continuous conditions (constant temperature and either continuous light or continuous darkness). The period of this rhythm is "temperature-compensated," with a Q10 of 1.10-1.12. The rhythm can be reset by pulses of light or darkness in a phase-dependent manner. Therefore, an endogenous circadian oscillator controls photoaccumulation behavior in P. bursaria.

R-body-producing bacteria.

Until 10 years ago, R bodies were known only as diagnostic features by which endosymbionts of paramecia were identified as kappa particles. They were thought to be limited to the cytoplasm of two species in the Paramecium aurelia species complex. Now, R bodies have been found in free-living bacteria and other Paramecium species. The organisms now known to form R bodies include the cytoplasmic kappa endosymbionts of P. biaurelia and P. tetraurelia, the macronuclear kappa endosymbionts of P. caudatum, Pseudomonas avenae (a free-living plant pathogen), Pseudomonas taeniospiralis (a hydrogen-oxidizing soil microorganism), Rhodospirillum centenum (a photosynthetic bacterium), and a soil bacterium, EPS-5028, which is probably a pseudomonad. R bodies themselves fall into five distinct groups, distinguished by size, the morphology of the R-body ribbons, and the unrolling behavior of wound R bodies. In recent years, the inherent difficulties in studying the organization and assembly of R bodies by the obligate endosymbiont kappa, have been alleviated by cloning and expressing genetic determinants for these R bodies (type 51) in Escherichia coli. Type 51 R-body synthesis requires three low-molecular-mass polypeptides. One of these is modified posttranslationally, giving rise to 12 polypeptide species, which are the major structural subunits of the R body. R bodies are encoded in kappa species by extrachromosomal elements. Type 51 R bodies, produced in Caedibacter taeniospiralis, are encoded by a plasmid, whereas bacteriophage genomes probably control R-body synthesis in other kappa species. However, there is no evidence that either bacteriophages or plasmids are present in P. avenae or P. taeniospiralis. No sequence homology was detected between type 51 R-body-encoding DNA and DNA from any R-body-producing species, except C. varicaedens 1038. The evolutionary relatedness of different types of R bodies remains unknown.

R-body-producing bacteria.

Until 10 years ago, R bodies were known only as diagnostic features by which endosymbionts of paramecia were identified as kappa particles. They were thought to be limited to the cytoplasm of two species in the Paramecium aurelia species complex. Now, R bodies have been found in free-living bacteria and other Paramecium species. The organisms now known to form R bodies include the cytoplasmic kappa endosymbionts of P. biaurelia and P. tetraurelia, the macronuclear kappa endosymbionts of P. caudatum, Pseudomonas avenae (a free-living plant pathogen), Pseudomonas taeniospiralis (a hydrogen-oxidizing soil microorganism), Rhodospirillum centenum (a photosynthetic bacterium), and a soil bacterium, EPS-5028, which is probably a pseudomonad. R bodies themselves fall into five distinct groups, distinguished by size, the morphology of the R-body ribbons, and the unrolling behavior of wound R bodies. In recent years, the inherent difficulties in studying the organization and assembly of R bodies by the obligate endosymbiont kappa, have been alleviated by cloning and expressing genetic determinants for these R bodies (type 51) in Escherichia coli. Type 51 R-body synthesis requires three low-molecular-mass polypeptides. One of these is modified posttranslationally, giving rise to 12 polypeptide species, which are the major structural subunits of the R body. R bodies are encoded in kappa species by extrachromosomal elements. Type 51 R bodies, produced in Caedibacter taeniospiralis, are encoded by a plasmid, whereas bacteriophage genomes probably control R-body synthesis in other kappa species. However, there is no evidence that either bacteriophages or plasmids are present in P. avenae or P. taeniospiralis. No sequence homology was detected between type 51 R-body-encoding DNA and DNA from any R-body-producing species, except C. varicaedens 1038. The evolutionary relatedness of different types of R bodies remains unknown.

Cytidine Analogues and Stomatogenic Recovery in Amicronucleate Paramecium tetraurelia and Paramecium jenningsi

ABSTRACTRemoval of the micronuclei of Paramecium tetraurelia and Paramecium jenningsi by micropipetting generates amicronucleate cell lines. These cell lines go through a period of growth depression for several dozen fissions, but they gradually recover. Amicronucleate cells in the depression period characteristically exhibit abnormal oral development, particularly reduction in the length of the buccal cavity and an abnormal pattern of the oral membranelles. To test the notion that the macronucleus is involved in the recovery of amicronucleate cell lines, DNA demethylation drugs were administered to amicronucleates in the depression period. After at least 4 fissions, the treated amicronucleates were assessed for their progress in recovery by scoring the proportion of cells with normal oral membranelles. Cvtidine analogues which demethylate cytosine specifically at the 5 position, namely 5‐azacytidine, 5‐aza‐2′‐ deoxycytidine and 5‐fluoro‐2′‐deoxycytidine. promoted recovery of the amicronucleates. Cytidine, 6‐azacytidine, 2′‐fluoro‐2′‐deoxy‐cytidine and cytosine‐β‐D‐arabinofuranoside did not. These results suggest that (i) 5‐methylcytosine is present in the macronucleus of these Paramecium species, probably in small amounts and (ii) recovery of amicronucleates involves demethylation of macronuclear DNA. This implies that in normal cells the micronuclei are involved in maintaining the macronuclear DNA in a methylated state and hence the inactivation of the macronuclear sequences that are to be employed for stomatogenic recovery. A general mechanism for the control of gene expression may therefore be employed for the regulation of specific sequences.

Predation by Didinium Nasutum: Effects of Predator and Prey Size

Experimental manipulation of the size of Didinium nasutum, a protozoan predator, feeding on large and small Paramecium species was used to examine the effects of predator and prey size on predator behavior and capture success. Didinum size is a function of prey size; large and small Didinium were obtained by conditioning predators on large or small prey species prior to experimentation. The predatory behavior of large and small predators feeding on large and small prey was monitored through an entire division cycle. Large Didinium had shorter handling times and encounter times on both sizes of prey than small Didinium. Large Didinium made more captures per division on either size of prey. Large prey required longer handling times, longer engulfing times, and were encountered more frequently than small prey for both sizes of predator. First captures were more difficult than subsequent captures for all predators. Small predators were more successful initially in capturing small prey than were large predators; first captures by large predators on small prey were particularly difficult. Encounter times, the number of encounters per capture, capture times, and handling times varied with the size of the predator and also varied within the division cycle of the predator. The effect of time within the division cycle may be due to increasing size as prey are captured and/or to adaptations to the specific prey species following initial captures. Didinium may adapt to new prey species through morphological changes in the oral apparatus, chemoreceptors, or extrusive organelles, in addition to shifts in size. That each size of Didinium had more successes per encounter on the size of prey to which it had been conditioned suggests that the size shift improves that aspect of the predatory behavior. However, other aspects (such as handling time and time to each encounter) show a clear advantage to large size. Large size also improves survival ability of Didinium under conditions of starvation (Hewett 1987). For large predators to maintain a large size on small prey would require a substantial increase in the division time. Selection forces favoring fast division rates, may impose restraints, making the size of Didinium on any prey species a compromise.

Prey Size and Survivorship in Didinium nasutum (Ciliophora: Gymnostomatida)

The ability of the ciliate predator Didinium nasutum to survive and recover from a period of starvation is affected by the previous history of the predator. The size of D. nasutum increases with increasing prey size across a range of sizes of Paramecium species. This increase in the size of the predator when feeding on larger prey contributes to the survival ability of the predator under conditions of starvation. D. nasutum that had been conditioned to larger prey (and were themselves larger) were better able to recover from starvation than were predators that had been conditioned to small prey. D. nasutum conditioned to large prey may also be more likely to form resting cysts as a response to

Artificial induction of selfing conjugation in Paramecium bursaria

AbstractIn normal conjugation of Paramecium bursaria, pairs always consist of cells of different mating types and no selfing pairs are induced with detached cilia from cells of a complementary mating type. This suggests that the initial ciliary agglutination (the mating reaction) is mating‐type specific but that the subsequent formation of conjugating pairs is not mating‐type specific as in the other species of Paramecium. The mechanism to exclude the formation of selfing pairs in normal conjugation is suggested to be the requirement of long‐lasting specific ciliary adhesion for the formation of firm pairs.

Interspecific conjugation between distantly related species of Paramecium.

Interspecific conjugation was induced artificially between the distantly related species belonging to the two different morphological groups, ‘aurelia’ and ‘bursaria’ groups, of Paramecium. Cytological observations showed that the interspecific conjugants underwent normal nuclear changes, including meiosis. However, nuclear changes of co-conjugant cells did not synchronize each other. These results suggest that conjugation in Paramecium involves non-species-specific interactions and its interactions are sufficient for nuclear activation.

The Extent of Algal and Bacterial Endosymbioses in Protozoa1,2

Long neglected has been the extensive and more or less intimate association of protozoa with a wide variety of other cells, either prokaryotic or eukaryotic in nature. Yet study of such relationships can provide important information concerning certain basic aspects of cellular evolution in general. A survey is offered here of the whole range of such symbiotic associations (i.e. with species of protozoa serving as hosts) with the purposes of drawing attention to the exciting possibilities of such research and of reviewing significant findings made to date. Because of the vastness of the overall field, examples and discussion are primarily limited to consideration of the following major studies: methanogenic bacteria in certain ciliates, bacterial endosymbionts of the large freshwater amoeba Pelomyxa palustris (itself an amazing organism from an evolutionary/phylogenetic point of view), the rod‐shaped bacteria found in Amoeba proteus, the “Greek‐letter” prokaryotes of Paramecium species, the xenosomes (sensu stricto) of the marine scuticociliate Parauronema acutum, and the diverse algal endosymbionts of similarly diverse protozoan taxa–ciliates, flagellates, radiolarians, acantharians, and foraminifera.