Effect of Exposure Period and Algal Concentration on the Frequency of Infection of Aposymbiotic Ciliates by Symbiotic Algae from Paramecium bursaria

The effect of exposure period and concentration of algae on the frequency of infection of aposymbiotic ciliates by algae obtained from the same clone of Paramecium bursaria syngen 2, was studied. The frequency of infection was roughly proportional to the algal concentration and to the exposure time of ciliates to algae. The relationship of algal concentration to infection frequency closely fitted the Poisson distribution curve for N = 1, suggesting that the minimum number of algae required to infect a single ciliate is 1. However, the data also strongly suggested that the average number of algae required to initiate infection of an average ciliate was ? 1,000. Three possible resolutions of this situation are: (a) the selection by the ciliate of a rare infective variant from a heterogeneous population: (b) the rare escape of an alga from digestion by the ciliate; and (c) the requirement for a large number of algae-ciliate contacts to induce susceptibility in the ciliate. Splitting the exposure of ciliates to algae into 2 periods of 0.5 h, separated by 5 h in the absence of algae, produced a much higher frequency of infection than a single l-h exposure, supporting the suggestion that the large number of algae is required to induce susceptibility in the ciliate which can then be infected by as few as a single algal cell.     

Transfer of Photosynthetically Produced Carbohydrate from Endosymbiotic Chlorellae to Paramecium bursaria*

Paramecium bursaria (Ehrenberg) and an endozoic zoochlorella Chlorella conductrix (Brandt) live in a symbiotic relationship. Uptake of NaH¹⁴CO₃ was studied to determine if carbohydrate products of photosynthesis are transferred to the host paramecium. Paramecium bursaria containing the algal symbionts took up NaH¹⁴CO₃ but those without the algal symbionts did not. Radioactive maltose, glucose, fructose and malate were identified from the ethanolic extract of paramecia. Transfer of materials from Paramecium to Chlorella and the transfer of other materials from Chlorella to Paramecium, led to the conclusion that this is a mutualistic relationship, both organisms benefiting from the relationship.     

Correlation of Sugar Release and Concanavalin A Agglutinability with Infectivity of Symbiotic Algae from Paramecium bursaria for Aposymbiotic P. bursaria

Synopsis.
Eighteen strains of algae, including 17 formerly symbiotic with Paramecium bursaria , were tested for capacity to release sugar. Detectable amounts of sugar were found in the supernatant fluids from 10 strains, including 6 strains infective for aposymbiotic P. bursaria syngen 2. The other 4 sugar-releasing strains were noninfective and released ˜26–46 g sugar/mg dry cell weight compared to ˜90–175 g sugar/mg dry cell weight for infective strains. This relationship of infectivity with capacity to release sugar supplements data that indicate a relationship of infectivity with resistance to Con A agglutination. The correlation is completed if we assume that resistance to Con A agglutination and capacity for sugar release must both be present in an algal strain for infectivity. The data thus strongly suggest that these 2 characteristics must be present for infectivity by any algal strains for aposymbiotic P. bursaria syngen 2.     

The Spatial and Temporal Distribution of Paramecium bursaria in the Littoral Zone1

The spatial and seasonal distribution of Paramecium bursaria in two small Indiana ponds was studied using a sampling grid. Very small (5.0 ml) samples were taken so that the individual microhabitats could be studied. The results were evaluated in comparison to the data collected for the P. aurelia complex collected in the same manner and at the same sites. It was found that P. bursaria exist in a clumped distribution, but that the distribution was not very different from random. Paramecium bursaria also exist at the surface and at the mud-water interface. Temperature does not seem to play a statistically significant role in determining population size. The breeding system of P. bursaria is optimized for an outbreeding population of low density. In comparison, the species of the P. aurelia complex exist in a very clumped distribution, are found only at the mud-water interface, and are inbreeders. The evolutionary strategies of the two types of paramecia are discussed.     

Life Cycle of Paramecium bursaria Syngen 1 in Nature

ABSTRACT. Studies were completed on the natural population density of Paramecium bursaria syngen 1 and on the life cycle stages to which the individuals belonged. Green paramecia were collected from two streams once every 20 days for over one year: 413 individuals on 26 collection dates in Mikumarikyo stream and 83 individuals on 23 collection dates in Momijidani-gawa stream. Individuals in nature did not maintain at a steady density but fluctuated greatly depending on the month. It seems that conjugation occurred from April to June in the Mikumarikyo stream and from May to June in the Momijidani-gawa stream. The appearance of individuals with mating ability might be related closely to increasing population so that sexual reproduction probably occurred near the peak of the population density. The 413 individuals from Mikumarikyo stream were examined to determine their position within the life cycle; 309 (74%) were immature, 55 (13%) were adolescent, and 49 (12%) were mature. No senile individuals were observed. The fraction of individuals with mating ability was generally less than 30% at any collection. Four mating types were observed occurring with about equal frequencies in mature individuals. The results show the frequencies of the recessive genes for mating types (a and b) are higher than for dominant genes (A and B). Of 83 individuals from Momijidani-gawa stream, 44 (52%) were immature, 21 (25%) were adolescent, and 18 (21%) were mature. Again, no senile individuals were observed. Because only two mating types were found, II and III (genotypes aaB- and aabb), it seems possible that the dominant gene A was rare or absent in the Momijidani-gawa population.     

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.     

Photosynthetic Shutdown in Chlorella NC64A Associated with the Infection Cycle of Paramecium bursaria Chlorella Virus-1

The effects of the algal virus Paramecium bursaria Chlorella virus-1 on the photosynthetic physiology of its host, Chlorella NC64A, was studied by observing changes in Chl fluorescence quenching and O2 exchange. Metabolic changes were calibrated against electron microscopic analysis of the morphological changes that occur during the infection cycle. It takes approximately 10 h from attachment of the virus to final lysis of the host cell, so a complete infection cycle can be observed continuously in one experiment. During the early stages of the infection cycle many rapid changes occurred in the host cell's metabolism and these were reflected in changes of photosynthetic and respiratory rates. The dramatic inhibition of photosynthesis in Chlorella NC64A cells by P. bursaria Chlorella virus-1 has facilitated the use of fluorescence quenching as an accurate measure of the first phase of viral infection (attachment and penetration of the host cell) and the extent to which a population of host cells is infected. Effects of temperature and cation requirement of the infection cycle are described. The relevance of our observations to the events observed during viral infection of higher plants is discussed.     

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.     

DNA amounts in the nuclei of Paramecium bursaria

DNA amounts in the macronuclei and micronuclei of syngen 4 of Paramecium bursaria were determined. There was a wide distribution of values for the amount of DNA in the macronucleus. This distribution remained wide, but moved to lower values as the animals were starved. The micro-nuclear DNA values were constant under all culture conditions, at a value of 7.5 × 10^–12 g per nucleus. This gave an estimate of the haploid genome size, 2.25 × 10¹² daltons, which is approximately ten times that obtained by DNA renaturation studies.This work was supported by a Commonwealth Scholarship to the author. Thanks are due to the Medical Research Council for funds to purchase the Vickers densitometer.     

The metabolic interactions between Paramecium bursaria Ehrbg. and Chlorella spec. in the Paramecium bursaria-symbiosis

In an organism (strain C 1/2 from Dr. P. R. Hayes, Leeds) regarded as a typical representative of the genus Flavobacterium, flexirubin-type pigments have been identified. The Flavobacterium pigments contain structural elements of both, the pigments of the genus Flexibacter and the pigments of the genus Cytophaga. As flexirubin-type pigments seem to have a rather restricted distribution among bacteria, and have formerly proved to be useful chemosystematic markers for the flexibacteria, this new observation may indicate that there is a relatively close phylogenetic relationship between this type of flavobacteria and the Cytophaga-Flexibacter group.     

Digestion of Added Homologous Algae by Chlorella-Bearing Paramecium bursaria

SYNOPSIS. Endosymbiotic algae from Paramecium bursaria when added to the culture medium are ingested by Chlorella -bearing P. bursaria at a rate of 2,000 algae/organism/day. That the ingested algae are digested and assimilated by the ciliates is suggested by the more rapid growth of Paramecium when algae are added to the medium ( G = 40 hr with algae compared to 190 hr without). The digestion by the ciliates of exogenous algae contrasts with the survival of these algae under normal growth conditions. It is suggested that the protection of the endogenous algae is related to their location in peripheral perialgal vacuoles.     

Effect of symbiotic algae on the photoaccumulation capacity of cells of the ciliate Paramecium bursaria

The character of the effect produced by symbiotic algae on photodependent behavior of their host ciliates, Paramecium bursaria, was determined. Partially alga-freed paramecia showed a reliable increase in the rates of photoaccumulation. The photoaccumulation rate gradually decreased with a further decrease in the number of zoochlorellae. Once the chlorophyll content fell down to 20-25 mu/l, the ciliates lost their capacity for photoaccululation. A mathematical model of photoaccumulation has been constructed.     

Infection of Alga-Free Paramecium bursaria with Strains of Chlorella, Scenedesmus, and a Yeast

SYNOPSIS. Twenty-one different stocks of Paramecittm bursaria , belonging t o 4 separate varieties (syngens), whose endosymbiotic chorellae had been removed, were tested for reinfection by several strains of Chorella , some previously isolated from P. bursaria , and others free-living. In addition, infection of P. bursaria by a single strain of the green alga Scenedesmus sp., and an unidentified strain of yeast was attempted. Most combinations involving Chlorella yielded infected paramecia, and all those with Scenedesmus or the yeast did so. The failures with Chlorella were attributed to low infectibility of the stocks of Paramecium concerned, rather than to inability of the Chlorella to survive inside the paramecia. Little evidence was found that the strains of P. bursaria differed genetically in ability to maintain the symbiotic organisms.     

Relations Between Photobehavior and Ionically Stimulated Swimming Behavior in Paramecium bursaria1

ABSTRACT. Cells of Paramecium bursaria, which harbor a symbiotic alga of the genus Chlorella, reverse the effective beat of their cilia and swim backward when stimulated in either of two ways. Ionic stimulation is introduction of cells into a solution high in K⁺ while step-down photostimulation is a sharp reduction in the intensity of light falling on the culture. Much is known about the mechanisms of ionic stimulation of ciliary reversal, but little is known about step-down photobehavior. Inhibitors of ionically stimulated ciliary reversal were applied to cells undergoing step-down photobehavior; Ca-channel inhibitors, neomycin and W-7, inhibit both behaviors. Activation of Ca-channels in the ciliary membrane is involved in step-down photobehavior, suggesting that the algae may alter the Paramecium membrane to make it more excitable.     

Multiple origins of the symbioses in Paramecium bursaria

Many organisms have symbioses with photosynthetic algae as typified by corals, clams, lichens, and some protozoa. Paramecium bursaria contains green algal symbionts and this unicellular ciliate is a textbook example used for microscopic observation in junior high school science projects. We have determined molecular phylogenies for the green algal symbionts. The symbiotic algae are the main constituent of the Paramecium cytoplasm, and we have recognized a total of four species, of which two were newly discovered in the present study. One should be regarded genetically as Chlorella vulgaris, and it belongs phylogenetically to the Chlorella clade (Chlorellaceae, Trebouxiophyceae) as well as "American" and "European" groups, which we previously introduced. Their genetic dissimilarities are 0.50-0.83% in 18S rDNA comparisons, but those of the internal transcribed spacer 2 (ITS2) reach an unambiguous level (22.6-26.6%). These dissimilarities suggest that they are equivalent to discrete species derived from multiple origins as paramecian symbionts. Another newcomer was clearly separated from the Chlorellaceae, and this alga clustered with Coccomyxa spp. in ITS2 analyses. These symbiotic relations indicate multiple origins of symbionts.