Paramecium Species Research Articles

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.

Respiratory inhibition by cyanide and salicylhydroxamic acid on the three species of Paramecium in stationary growth phase

1. 1. Paramecia in stationary growth phase consumed oxygen at a constant rate, exhibiting 21.5 nM/min/mg protein of the cells at 27°C. 2. 2. Respiratory inhibition by 1 mM KCN was significantly high in the following order: P. caudatum (96%) > P. multimicronucleatum (87%) > P. tetraurelia (77%). 3. 3. In the presence of cyanide (CN), 1 mM salicylhydroxamic acid (SHAM) suppressed a large part of the CN-insensitive respiration in the cells, and the cumulative inhibition by CN and SHAM reached approx. 95% in all of the Paramecium species examined. SHAM in the absence of CN caused no detectable change in the respiration of the cells. 4. 4. Oxygen affinity for the CN-sensitive respiration was far stronger than that for the CN-insensitive respiration in the whole cells of Paramecium.

Paramecium mitochondrial genes. I. Small subunit rRNA gene sequence and microevolution.

The sequences of the small subunit mitochondrial rRNA genes from two divergent species of Paramecium (primaurelia and tetraurelia) were determined. The gene lies near the center of the linear mitochondrial genome, on the same strand as are all other currently identified genes. The sequences generally resemble their counterparts found in cytoplasmic, procaryotic, and other mitochondrial sources. The rDNA gene boundaries were located by nuclease S1 protection. Small subunit rDNA spans about 1680 nucleotides, including an extraneous 83-base pair sequence very near the 3' end which is unique to Paramecium mitochondria. This "insert" occurs at the apex of the highly variable in length penultimate helix, according to proposed models for small subunit rRNA secondary structure. A discontinuity occurs in isolated rRNA near the start of the insert, resulting in a stable 13 S RNA species and a small segment containing the remaining 3' portion of the gene. The overall rRNA gene sequence was 94% conserved between the two species, and the nucleotide differences consisted of 53% transitions, 37% transversions, and 9% insertions plus deletions. These substitutions were somewhat clustered, and the two most divergent regions coincided with the gene boundaries. The sequence was aligned with Escherichia coli 16 S rRNA for direct comparison of sequence and structure.

Paramecium mitochondrial genes. II. Large subunit rRNA gene sequence and microevolution.

The sequences of the small subunit mitochondrial rRNA genes from two divergent species of Paramecium (primaurelia and tetraurelia) were determined. The gene lies near the center of the linear mitochondrial genome, on the same strand as are all other currently identified genes. The sequences generally resemble their counterparts found in cytoplasmic, procaryotic, and other mitochondrial sources. The rDNA gene boundaries were located by nuclease S1 protection. Small subunit rDNA spans about 1680 nucleotides, including an extraneous 83-base pair sequence very near the 3' end which is unique to Paramecium mitochondria. This insert occurs at the apex of the highly variable in length penultimate helix, according to proposed models for small subunit rRNA secondary structure. A discontinuity occurs in isolated rRNA near the start of the insert, resulting in a stable 13 S RNA species and a small segment containing the remaining 3' portion of the gene. The overall rRNA gene sequence was 94% conserved between the two species, and the nucleotide differences consisted of 53% transitions, 37% transversions, and 9% insertions plus deletions. These substitutions were somewhat clustered, and the two most divergent regions coincided with the gene boundaries. The sequence was aligned with Escherichia coli 16 S rRNA for direct comparison of sequence and structure.

HIGH LEVELS OF PHENOTYPIC VARIABILITY OF METAL AND TEMPERATURE TOLERANCE IN PARAMECIUM.

The selection of resistant individuals by a concentration of a chemical sufficient to kill sensitive individuals is the most easily understood and easily demonstrated type of natural selection. Yet at a polluted habitat it is clear that species differ in their capacity to respond to stress (e.g., Gartside and McNeilly, 1974). Are differences in tolerance between species and within species the product of previous interactions with environmental stress or do they have non-selective origins? How much phenotypic variation in tolerance is there within species and on what scale are populations spatially or temporally differentiated? What is the genetic basis of tolerance? We have a long-standing interest in these environmentally-important evolutionary problems, particularly in how the answers to these questions are affected by breeding systems (Nyberg, 1974). In this report we will describe the extent of phenotypic variation in tolerance to eight heavy metals and high temperature in two species of Paramecium. The two species, Paramecium primaurelia and P. triaurelia, are cryptic species in the inbreeding P. aurelia complex (Sonneborn, 1975). No gene flow is possible between these species, but they are morphologically similar and occur together (Landis, 1981), though P. primaurelia has a maximum abundance in lower latitudes than P. triaurelia (Sonneborn, 1957). All the species of the P. aurelia complex are considered to be inbreeders, because all stocks have the capacity to undergo an autogamous fertilization which results in a completely homozygous diploid. Within this inbreeding complex, P. primaurelia and P. triaurelia have similar breeding systems (Sonneborn, 1957). The interval from conjugation to autogamy has recently been estimated to be 49 fissions in P. triaurelia (Nyberg, 1979) and 44 fissions in P. primaurelia (Nyberg, 1982). We chose inbreeding species because Nyberg (1974) had proposed, and found some support for, the hypothesis that phenotypic variation in tolerance should be greater among stocks of inbreeding species than among stocks of outbreeding species. In P. primaurelia 26 stocks from 14 locations were studied, and in P. triaurelia 10 stocks from seven locations. All but one of the stocks were collected in 1975 and 1976. Some of the stocks were collected from a location known to be polluted by heavy metals, Sudbury, Ontario. The 48 h median tolerance limit (MTL) to cadmium, chromium (III), cobalt, copper, manganese (II), mercury, nickel, zinc, and high temperature has been determined for each stock. Heavy metals are a class of compounds whose environmental importance and evolutionary impact has been well documented, especially in plants (Antonovics et al., 1971). Correlations of tolerances to pairs of metals have been calculated to determine whether or not tolerances to different metals are independent. The variation among the stocks within each species has been divided into variation among locations and variation within a location. Our basic outlook is that tolerance limits are the result of selection on tolerance. We imagine that brief, highly localized episodes of selection for resistance are followed by longer periods of weak selection for sensitives (at most locales), followed by even longer periods

Comparison of the Esterases and Acid Phosphatases in Paramecium multimicronucleatum, Syngens 1–5, P. jenningsi, P. caudatum, and the P. aurelia Complex1

ABSTRACT. Forty‐eight stocks in Paramecium jenningsi, syngens 1–5 of P. multimicronucleatum, P. caudatum, P. primaurelia, P. biaurelia, and P. tetraurelia were grown axenically and tested for their esterases and acid phosphatases using starch gel electrophoresis. The five esterases and the acid phosphatases previously characterized in species of the P. aurelia complex were also found in P. jenningsi, and three to four of the esterases and the acid phosphatases were found in the P. multimicronucleatum species complex and in P. caudatum. Additional subtypes were observed for each of the enzyme phenotypes in these new (though here unnamed) species of Paramecium. Two of the new acid phosphatase subtypes, which depart radically in mobility and in pattern, were found in syngen 3 of P. multimicronucleatum and in P. caudatum. Except for syngens 1 and 5 in P. multimicronucleatum, the degree of similarity between syngens 1, 5 and 2, 3, and 4 appears to be very low—perhaps even lower than that seen for species in the aurelia complex. More realistically, the syngens of P. multimicronucleatum should be considered as separate species although they are not here given separate taxonomic names. Limited sharing of subtypes occurred between species in different species complexes. This observation suggests that the molecular distances between species complexes may be even greater than between species within a complex.

Immunological crossreactivity of Paramecium surface antigens: conformational differences account for cell surface specificity.

Surface antigens (i-Ags) G and D of Paramecium primaurelia, reported earlier to be completely unrelated on the basis of peptide mapping and immunological tests, were compared and found to be similar in molecular weight (Mr 235,000), isoelectric point (ca. 4.5), and amino acid composition. They are tightly folded proteins with more than 100 intramolecular disulfide bonds. Native i-Ags and their CNBr fragments were blotted onto nitrocellulose and labeled with a battery of homologous and heterologous antibodies fractionated on native and reduced i-Ag immunoadsorbents. It was found the i-Ags retained part of their antigenic structure upon cleavage and reduction. Analysis of labeling revealed that the two proteins share many of their antigenic sites, though they are completely distinct in their native conformation. Immunofluorescence and immunoadsorption on fixed cells confirmed these results. Antigenic crossreactivity also was found with surface antigens of a ciliate outside the Paramecium species complex. It is concluded that surface specificity of paramecia is due, in this case, to the expression of related proteins in different conformations.

Cytoplasmic streaming inParamecium

Certain species ofParamecium demonstrate rotational cytoplasmic streaming, in which most cytoplasmic particles and organelles flow along permanent route, in a constant direction. By means of novel methods of immobilization, observation and recording, some dynamic properties of cytoplasmic streaming have been described. It was found that the velocity profiles of coaxial layers of cytoplasm have a (parabolic) paraboidal shape and the mean output of cytoplasm flow in different examined zones of streaming is constant. As the consequence of randomly distributed elementary propulsion units within the cytoplasm, particles, which serve as markers of movement, exhibit movements of a saltatory nature; this form of movement is seen inParamecium streaming only in cases of error due to polarization of the saltating particles. Interaction of actin filaments and myosin is likely to occur under specific conditions in microcompartments of cytoplasm where local solations are generated eventually leading to contractions which might propagate on gelated neighbouring areas. Places of elementary contractions are scattered. Therefore the motile effect appears as streaming. Rotational cytoplasmic streaming inParamecium may serve as a convenient model for the study of the dynamics and function of cytoplasmic motility.

A protein called immaturin controlling sexual immaturity in Paramecium.

As in many metazoans, clones of some species of Paramecium have, after conjugation, a period of immaturity during which the cells cannot mate. The duration of immaturity is measured by the number of cell generations, which remains fairly constant, although duration in time varies with rate of cell reproduction. Genic involvement is shown by mutants with reduced periods of immaturity. In three different groups of Paramecium species, the cytoplasm of immature cells apparently contains the same substance which represses mating activity when injected into sexually mature cells. The immaturity-inducing substance seems to be absent from sexually mature cells, as brei made not only from mature cells in the stationary phase (mating-reactive cells), but also from those in the log phase (mating-non-reactive cells), does not repress mating activity when injected into mature cells. Variations in the amount of the substance during immaturity suggest that it controls the duration of the period. We have isolated and partially characterized the substance-a single protein called immaturin. The activity of immaturin is dose dependent and associated with a heat-labile protein of molecular weight (MW) 10,000.

The Effect of Prey Size on the Functional and Numerical Responses of a Protozoan Predator to Its Prey

The functional response of Didinium nasutum to variation in density of three Paramecium species was found to decrease with increasing prey size. The attack coefficient is similar on small and intermediate—sized Paramecium and declines sharply on the largest prey species. Handling time increases with increasing prey size. Actual division rates of the predator are highest, however, on the intermediate—sized prey and lowest on the smallest prey species. This suggest that there is an optimal Paramecium size at which Didinium division rates are highest. The density of prey and the size of prey were also found to have a significant effect on the volume of the Didinium cells. It is suggested that there may be contrasting advantages to Didinium of being of as large a size as possible and of dividing as quickly as possible.

Prey‐Dependent Cell Size In A Protozoan Predator*

SYNOPSIS. the cell size of Didinium nasutum was found to be dependent on the size of the Paramecium species available as prey. Didinium feeding on P. tetraurelia averaged 5.6 × 105μm3. the cell volume of Didinium increased with increasing prey size for the 5 prey species tested, to 9.1 × 105μm3 for Didinium feeding on P. caudatum. Didinium nearing a cell division ranged in size from 8.6 × 105μm3 on P. tetraurelia to 12.9 × 105μm3 on P. caudatum. the range in cell volume is such that Didinium feeding on P. caudatum are larger than the size at which Didinium divide when feeding on P. tetraurelia. This morphologic plasticity in cell volume allows Didinium to exploit a wide size range of Paramecium species as prey. It is proposed that the size of a Didinium may have profound effects on its ability to encounter and capture prey of different sizes.

Electrophysiological responses of Didinium nasutum to Paramecium capture and mechanical stimulation

Didinium nasutum is a carnivorous ciliate with a preference for feeding on species of Paramecium. Accidental contact between a Paramecium and the proboscis of a hungry Didinium (Fig. 1a) causes instantaneous discharge of extrusive organelles from the proboscis. If the strike is successful, some of these organelles penetrate the Paramecium, which immediately stops swimming and discharges many trichocysts, mostly from the struck side (Fig. 1b). Cytoplasmic streaming pulls the discharged organelles inward, the captured Paramecium reaches the proboscis, and then the Didinium swallows the prey, opening its proboscis1–10 (Fig. 1c and d). On the other hand, a hungry Didinium does not discharge extrusive organelles when it collides with a glass wall. Instead it shows an avoiding reaction. When a Paramecium collides with the proboscis of a fed Didinium, the proboscis similarly does not discharge extrusive organelles, and the fed Didinium makes an avoiding response. We found that a hungry Didinium held steady by two glass capillary microneedles performed normal capture and ingestion in a solution containing Paramecium caudatum. We describe here an electrophysiological investigation of Didinium nasutum in collision with P. caudatum or with a glass rod while held still by glass capillary microneedles.

A scanning electron-microscopic study of the local degeneration of cilia during sexual reproduction in Paramecium.

The location and extent of local degeneration of cilia during sexual reproduction of Paramecium was studied using scanning electron microscopy to examine cells undergoing conjugation and autogamy. At some time during the mating reaction, but prior to conjugant pair formation, ciliary degeneration begins at the antero-ventral tip of cells and proceeds posteriorly along the suture. In the anterior part of the cell, degeneration occurs on both sides of the suture, but in the posterior part it is restricted to the right side of the suture. In 5 species of Paramecium examined, degeneration occurred in nearly the same region. No degeneration of cilia is observed in natural autogamy of P. tetraurelia, whereas in chemically induced autogamy of P. caudatum degeneration occurs as in ordinary conjugation. Conjugant pairs never expose any deciliated cell surface except at the postero-ventral tip. The maximum extent of ciliary degeneration is best seen in the chemically induced autogamous cells: 7 kinetics (rows of unit teritories) at the anterior-left, 4 kinetics at the anterior-right, 10 or more kinetics at the posterior-right and the right wall of the vestibule of the mouth. Before complete disappearance of the cilia, many short cilia are observed. This suggests that ciliary degeneration is due to resorption. Degeneration extends more rapidly in cells with stronger mating reactivity. The relations between mating reactivity, ciliary degeneration and nuclear activation are discussed.

Effect of Acetate on Esterase C Activity During the Growth Cycle of Paramecium*

SYNOPSIS Enhanced esterase C activity could be demonstrated by starch gel electrophoresis in various stocks of Paramecium spp. (P. primaurelia stocks 90 and 540, P. biaurelia stock 93, P. tetraurelia stock 29. P. pentaurelia stock 87, P. octaurelia stocks 31 and 300, and P. multimicronucleatum species 3, stock 8 MO) grown in Adaptation Medium. This esterase, however, was barely detectable when they were cultivated in Axenic Medium. Addition of trypticase to Adaptation Medium resulted in reduction of esterase C in the ciliates. This effect is ascribable to Na acetate present in trypticase. Since esterase C increased with the decrease in acetate concentration (as estimated by gas‐liquid chromatography) during growth of Paramecium, acetate appears to be utilized by the cells. Sensitivity of esterase C to acetate occurs in all 6 species of Paramecium examined. Different stocks within a species may have different levels of sensitivity; in one case this is genetically determined. The results emphasize the importance of controlling and manipulating growth conditions for the assessment of inter‐ and intraspecies variations in the isozymes of Paramecium.

The evolutionary significance of ultrastructural variations in the micronuclear spindle apparatus in the genus Paramecium

Ultrastructural variations of micronuclear mitosis in three species of Paramecium have been reviewed for the purpose of hypothesizing possible evolutionary relationships. P. bursaria is considered to be the most primitive species because it has complex kinetochore-equivalents termed microlamellae which attach to microfilaments. P. multimicronucleatum is considered more advanced since it has typical kinetochores which intercalate between the kinetochores and polar microfilaments. P. aurelia is considered the most advanced member of the genus, and has typical diffuse or well defined kinetochores and chromosomal microtubules while it lacks obvious meshworks of microfilaments. A hypothetical model of chromosome movement in the genus involves the interaction of microtubules and microfilaments.

Genetics of chemical induction of conjugation in Paramecium aurella.

Certain stocks of P. aurelia, syngen 8, could not be induced to conjugate in a solution (KCl + acriflavine + calcium-poor conditions) which was effective in inducing conjugation in other species of Paramecium as well as in other stocks of syngen 8. Both stocks could conjugate by interaction with cells of complementary mating type. Breeding analysis shows that each of the two stocks is homozygous for a recessive gene that blocks induction of conjugation by the KCl-acriflavine solution. These two genes are neither allelic nor linked. Analyses of the phenotypes of the two uninducibles and the wild type were carried out by attempting to induce mating in cells of a single mating type by exposing them to detached mating-reactive cilia from cells of complementary mating type and to the KCl-acriflavine solution, either sequentially or simultaneously. The results confirm the conclusions of others that there is at least one unique step in chemical induction not shared with induction by interaction of complementary mating types. But the results also indicate that there is more than one unique step in chemical induction and that the effects of the two genes described here operate during different periods of the hour required for chemical induction.

Intrinsic Rates of Increase, Saturation Densities, and Competitive Ability. I. An Experiment with Paramecium

In a set of experiments with pure and mixed cultures of two species of Paramecium "aurelia," it was found that there was no consistent relationship between r and K and competitive success, and that simple environmental changes affect competitive ability much less than they affect either r or K. Competition was manifest as interference rather than exploitation. It is suggested that the endosymbioses of Paramecium with bacterial species may provide some species of Paramecium with a mechanism of interference, which may play an important role in determining the relative abundance of protozoan species in natural communities.

The Relationship between Species Diversity and Stability: An Experimental Approach with Protozoa and Bacteria

Small experimental communities of bacteria and Protozoa were designed to test the widely held hypothesis that higher species diversity brings about greater stability. Three species of bacteria, three species of Paramecium and two species of protozoan predators, Didinium and Woodruffia, were used. The communities were maintained by regular additions of the appropriate combinations of species of bacteria. Stability was measured as persistence of all species and as a tendency to maintain evenness of the species abundance distribution. The measures were in essential agreement. Stability at the Paramecium trophic level was increased by increasing diversity at the bacterium level, but three species of Paramecium were less stable than two. An important finding was that one pair of Paramecium species consistently showed greater stability without the third species than with it. This finding indicates that there were significant second—order effects, with two species having an interaction that was detrimental to the third species. We conclude that much more experimental and observational work is necessary before the nature of any functional relationship between diversity and stability can be claimed with confidence.

Induction of conjugation by chemical agents in Paramecium.

AbstractInduction of conjugation by chemical agents was studied in nine species of Paramecium. KCl + acriflavine + Ca‐poor conditions were effective in inducing conjugation in P. caudatum, P. multimicronucleatum, P. aurelia, P. jenningsi and probably in P. trichium. KCL + Ca‐poor conditions were effective in P. caudatum, P. traunsteineri and probably in P. trichium. KCl + acriflavine + Ca‐poor conditions were also effective in inducing interspecific conjugation. Interaction between complementary mating types is not involved in the chemical induction of conjugation, but otherwise chemically‐induced conjugation is, in at least some pairs, essentially identical to normal conjugation induced by the interaction between complementary mating types. The mechanism of induction of conjugation and applications of chemical induction of conjugation to genetic studies are discussed.

Effects of metabolic inhibitors on Paramecium aurelia during the cell generation cycle

The ability of a number of metabolic inhibitors (namely, p- dl-fluorophenylalanine, puromycin, chloramphenicol, 5-fluoro-2-deoxyuridine, actinomycin D, triparanol citrate, and 2,4-dinitrophenol) to interfere with cell division has been studied in the species Paramecium aurelia. First, the lowest concentrations sufficient to prevent further division in newly-divided paramecia continuously exposed to the compound were found. Then the effects of exposure to these concentrations for 30 and/or 60 min were tested throughout the interval between two successive divisions, and assessed in terms of prolongation of the cell generation cycle. In all cases the most characteristic feature was an abrupt change from long to short prolongations, which occurred with small increases in the age of the cells, about an hour prior to completion of division. In a discussion on the possible mechanism underlying this response, comparison has been drawn with the parallel behaviour of Tetrahymena pyriformis.