An experimental test of the symbiosis specificity between the ciliate Paramecium bursaria and strains of the unicellular green alga Chlorella

The ciliate Paramecium bursaria living in mutualistic relationship with the unicellular green alga Chlorella is known to be easily infected by various potential symbionts/parasites such as bacteria, yeasts and other algae. Permanent symbiosis, however, seems to be restricted to Chlorella taxa. To test the specificity of this association, we designed infection experiments with two aposymbiotic P. bursaria strains and Chlorella symbionts isolated from four Paramecium strains, seven other ciliate hosts and two Hydra strains, as well as three free-living Chlorella species. Paramecium bursaria established stable symbioses with all tested Chlorella symbionts of ciliates, but never with symbiotic Chlorella of Hydra viridissima or with free-living Chlorella. Furthermore, we tested the infection specificity of P. bursaria with a 1:1:1 mixture of three compatible Chlorella strains, including the native symbiont, and then identified the strain of the newly established symbiosis by sequencing the internal transcribed spacer region 1 of the 18S rRNA gene. The results indicated that P. bursaria established symbiosis with its native symbiont. We conclude that despite clear preferences for their native Chlorella, the host-symbiont relationship in P. bursaria is flexible.     

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.     

A green Paramecium strain with abnormal growth of symbiotic algae

Some hundred cells of Chlorella-like green algae are naturally enclosed within the cytoplasm of a single cell of green paramecia (Paramecium bursaria). Therefore, P. bursaria serves as an experimental model for studying the nature of endo-symbiosis made up through chemical communication between the symbiotic partners. For studying the mechanism of symbiotic regulations, the materials showing successful symbiosis are widely used. Apart from such successful model materials, some models for symbiotic distortion would be of great interest in order to understand the nature of successful symbiosis. Here, we describe a case of unsuccessful symbiosis causing unregulated growth of algae inside the hosting ciliates. Recently, we have screened some cell lines, from the mass of P. bursaria cells survived after paraquat treatment. The resultant cell lines (designated as KMZ series) show novel and unusual morphological features with heavily darker green colour distinguishable from the original pale green-coloured paramecia. In this type of isolates, endo-symbiotic algae are restricted within one or two dense spherical structures located at the center of the host cells' cytoplasm. Interestingly, this isolate maintains the host cells' circadian mating response which is known as an alga-dependent behaviour in the host cells. In contrast, we discuss that KMZ lacks the host-dependent regulation of algal growth, thus the algal complex often over-grows obviously exceeding the original size of the normal hosting ciliates. Additionally, possible use of this isolate as a novel model for symbiotic cell-to-cell communication is discussed.     

Correlation of Infectivity and Concanavalin A Agglutinability of Algae Exsymbiotic from Paramecium bursaria

SYNOPSIS. Eighteen strains of algae, including 17 exsymbiotic from Paramecium bursaria , were tested for infectivity for P. bursaria , syngen 2 aposymbiotes, and Concanavalin A (Con A) agglutinability. All 6 infective algal strains were relatively resistant to agglutination by Con A, suggesting that algal surface characteristics are correlated with infectivity. Among the noninfective strains, high and low agglutinability were about equally represented, indicating that the Con A titer alone is not a sufficient indicator of infectivity. It is suggested that noninfective algal strains are the progeny of mutations occurring within the endozoic population and fortuitously selected by the external culture medium.     

Characteristics of the digestive vacuole membrane of the alga-bearing ciliate Paramecium bursaria

Cells of the ciliate Paramecium bursaria harbor symbiotic Chlorella spp. in their cytoplasm. To establish endosymbiosis with alga-free P. bursaria, symbiotic algae must leave the digestive vacuole (DV) to appear in the cytoplasm by budding of the DV membrane. This budding was induced not only by intact algae but also by boiled or fixed algae. However, this budding was not induced when food bacteria or India ink were ingested into the DVs. These results raise the possibility that P. bursaria can recognize sizes of the contents in the DVs. To elucidate this possibility, microbeads with various diameters were mixed with alga-free P. bursaria and traced their fate. Microbeads with 0.20μm diameter did not induce budding of the DVs. Microbeads with 0.80μm diameter produced DVs of 5-10μm diameter at 3min after mixing; then the DVs fragmented and became vacuoles of 2-5μm diameter until 3h after mixing. Each microbead with a diameter larger than 3.00μm induced budding similarly to symbiotic Chlorella. These observations reveal that induction of DV budding depends on the size of the contents in the DVs. Dynasore, a dynamin inhibitor, greatly inhibited DV budding, suggesting that dynamin might be involved in DV budding.     

Endosymbionts in paramecium

Paramecium species are extremely valuable organisms to enable experiments for the reestablishment of endosymbiosis. This is investigated in two different systems, the first with Paramecium caudatum and the endonuclear symbiotic bacterium Holospora species. Although most endosymbiotic bacteria cannot grow outside the host cell as a result of their reduced genome size, Holospora species can maintain their infectivity for a limited time. We found that an 89-kDa periplasmic protein has an important function for Holospora's invasion into the target nucleus, and that Holospora alters the host gene expression; the host thereby acquires resistance against various stresses. The second system is the symbiosis between P. bursaria and symbiotic Chlorella. Alga-free P. bursaria and the algae retain the ability to grow without a partner. Consequently, endosymbiosis between the aposymbiotic host cells and the symbiotic algae can be reestablished easily by mixing them. We now found four checkpoints for the reestablishment of the endosymbiosis between P. bursaria and the algae. The findings in the two systems provide excellent opportunities for us to elucidate not only infection processes but also to assess the associations leading to eukaryotic cell evolution. This paper summarizes recent progresses on reestablishment of the primary and the secondary endosymbiosis in Paramecium.     

Ingredients for protist coexistence: competition, endosymbiosis and a pinch of biochemical interactions

1. The interaction between mutualism, facilitation or interference and exploitation competition is of major interest as it may govern species coexistence. However, the interplay of these mechanisms has received little attention. This issue dates back to Gause, who experimentally explored competition using protists as a model [Gause, G.F. (1935) Vérifications expérimentales de la théorie mathématique de la lutte pour la vie. Actualités Scientifiques et Industrielles, 277]. He showed the coexistence of Paramecium caudatum with a potentially allelopathic species, Paramecium bursaria. 2. Paramecium bursaria hosts the green algae Chlorella vulgaris. Therefore, P. bursaria may benefit from carbohydrates synthesised by the algae. Studying endosymbiosis with P. bursaria is possible as it can be freed of its endosymbiont. In addition, C. vulgaris is known to produce allelochemicals, and P. bursaria may benefit also from allelopathic compounds. 3. We designed an experiment to separate the effects of resource exploitation, endosymbiosis and allelopathy and to assess their relative importance for the coexistence of P. bursaria with a competitor that exploits the same resource, bacteria. The experiment was repeated with two competitors, Colpidium striatum or Tetrahymena pyriformis. 4. Results show that the presence of the endosymbiont enables the coexistence of competitors, while its loss leads to competitive exclusion. These results are in agreement with predictions based on resource equilibrium density of monocultures (R*) supporting the idea that P. bursaria's endosymbiont is a resource provider for its host. When P. bursaria and T. pyriformis coexist, the density of the latter shows large variation that match the effects of culture medium of P. bursaria. Our experiment suggests these effects are because of biochemicals produced in P. bursaria culture. 5. Our results expose the hidden diversity of mechanisms that underlie competitive interactions. They thus support Gauses's speculation (1935) that allelopathic effects might have been involved in his competition experiments. We discuss how a species engaged both in competition for a resource and in costly interference such as allelopathy may counterbalance these costs with a resource-provider endosymbiont.     

Association of Paramecium bursaria Chlorella viruses with Paramecium bursaria cells: ultrastructural studies

Paramecium bursaria Chlorella viruses were observed by applying transmission electron microscopy in the native symbiotic system Paramecium bursaria (Ciliophora, Oligohymenophorea) and the green algae Chlorella (Chlorellaceae, Trebouxiophyceae). Virus particles were abundant and localized in the ciliary pits of the cortex and in the buccal cavity of P. bursaria. This was shown for two types of the symbiotic systems associated with two types of Chlorella viruses - Pbi or NC64A. A novel quantitative stereological approach was applied to test whether virus particles were distributed randomly on the Paramecium surface or preferentially occupied certain zones. The ability of the virus to form an association with the ciliate was investigated experimentally; virus particles were mixed with P. bursaria or with symbiont-free species P. caudatum. Our results confirmed that in the freshwater ecosystems two types of P. bursaria -Chlorella symbiotic systems exist, those without Chlorella viruses and those associated with a large amount of the viruses. The fate of Chlorella virus particles at the Paramecium surface was determined based on obtained statistical data and taking into account ciliate feeding currents and cortical reorganization during cell division. A life cycle of the viruses in the complete symbiotic system is proposed.     

Electrokaryotypes of macronuclei of several Paramecium species

A comparative study of macronuclear DNA molecules from the following Paramecium species: the P. aurelia complex, P. caudatum, P. bursaria, P. putrinum and P. multimicronucleatum was performed using pulsed-field gel electrophoresis. The electrophoretic pattern was constant and unique for each species, and is referred to herein as its electrokaryotype. Large differences were observed between Paramecium species according to the range and major size of macronuclear DNA fragments, while different strains of the same species, even belonging to different syngens, were characterized by the same electrokaryotype. In this respect sibling species from the P. aurelia complex are as similar as syngens in other Paramecium species, but are unlike conventional species. The principles and value of electrokaryotype analysis for application to ciliates are discussed.     

Arrest of cytoplasmic streaming induces algal proliferation in green paramecia

A green ciliate Paramecium bursaria, bearing several hundreds of endosymbiotic algae, demonstrates rotational microtubule-based cytoplasmic streaming, in which cytoplasmic granules and endosymbiotic algae flow in a constant direction. However, its physiological significance is still unknown. We investigated physiological roles of cytoplasmic streaming in P. bursaria through host cell cycle using video-microscopy. Here, we found that cytoplasmic streaming was arrested in dividing green paramecia and the endosymbiotic algae proliferated only during the arrest of cytoplasmic streaming. Interestingly, arrest of cytoplasmic streaming with pressure or a microtubule drug also induced proliferation of endosymbiotic algae independently of host cell cycle. Thus, cytoplasmic streaming may control the algal proliferation in P. bursaria. Furthermore, confocal microscopic observation revealed that a division septum was formed in the constricted area of a dividing paramecium, producing arrest of cytoplasmic streaming. This is a first report to suggest that cytoplasmic streaming controls proliferation of eukaryotic cells.     

Identification of Chlorella viruses in Paramecium bursaria clones by pulse-field electrophoresis

The ciliates Paramecium bursaria contain endosymbiotic green algae Chlorella spp. in their cytoplasm. The algae isolated from P. bursaria are sensitive to large DNA-containing viruses of the family Phycodnaviridae. The type virus of this family is PBCV-1 (Paramecium bursaria Chlorella virus). Investigation of the total DNA of P. bursaria clones by pulse-field electrophoresis (PEGE) revealed a pronounced band on PEGE profiles of some P. bursaria clones; the band was formed by DNA molecules of approx. 300 kb. This band probably contained the DNA of Chlorella virus. Two approaches were used in the present work to confirm this hypothesis. Microbiological tests were used to scan a collection of P. bursaria clones for specific types of viruses; the 300-kb band was revealed only in the PEGE profiles of virus-containing clones. Blot hybridization of P. bursaria total DNA separated by pulse-field electrophoresis with the virus-specific probe revealed that the band under study was formed by the DNA of a Chlorella virus. Paramecium clones were shown to contain approx. 10⁵ copies of nonintegrated viral DNA.     

Growth kinetics of algal populations exsymbiotic from Paramecium bursaria by flow cytometry measurements.

BACKGROUND: The ciliate Paramecium bursaria normally exists as a green paramecium system because each animal cell carries several hundred, unicellular, green, algal cells in its cytoplasm. One of the remarkable and poorly understood pecularities of this system is the steady state in the number of algae per protozoan cell. A major point in the study of mechanisms governing the persistence of symbiont numbers is adequate understanding of the algal life cycle. METHODS: Asynchronously growing cell populations of several algal strains (SA-1, SA-3, and SA-9) exsymbiotic from P. bursaria were characterized by flow cytometry. Algal endogenous chlorophyll and DNA contents were monitored to analyze cell growth kinetics at logarithmic and stationary culture phases. Cell sorting visualized the morphology of algae corresponding to the hyperhaploid (2C and 4C) DNA peaks. RESULTS: Cell-division cycle-dependent changes in chlorophyll and DNA content distributions were most dramatic in logarithmically growing algal populations (an increase in the number of S-phase cells and cells with more chlorophyll), which are thought to be associated with accelerated DNA and chlorophyll metabolism in log-phase algal cultures. Upon reaching the stationary phase of growth, algal populations distinctly showed, in addition to one haploid (1C) DNA peak, two hyperhaploid peaks (2C and 4C) corresponding mainly to cells with two and four nuclei, respectively. CONCLUSIONS: Growth characteristics of algae exsymbiotic from P. bursaria monitored by flow cytometry provide valuable information for the analysis of the algal life cycle, which is important for understanding the regulation mechanisms of symbiont numbers.     

Identification of Paramecium bursaria syngens through molecular markers--comparative analysis of three loci in the nuclear and mitochondrial DNA

This is the first attempt to resolve the phylogenetic relationship between different syngens of Paramecium bursaria and to investigate at a molecular level the intraspecific differentiation of strains originating from very distant geographical locations. Herein we introduce a new collection of five P. bursaria syngens maintained at St Petersburg State University, as the international collection of syngens was lost in the 1960s. To analyze the degree of speciation within Paramecium bursaria, we examined 26 strains belonging to five different syngens from distant and geographically isolated localities using rDNA (ITS1-5.8S-ITS2-5'LSU) fragments, mitochondrial cytochrome c oxidase subunit I (COI), and H4 gene fragments. It was shown that P. bursaria strains of the same syngens cluster together in all three inferred molecular phylogenies. The genetic diversity among the studied P. bursaria strains based on rDNA sequences was rather low. The COI divergence of Paramecium bursaria was also definitely lower than that observed in the Paramecium aurelia complex. The nucleotide sequences of the H4 gene analyzed in the present study indicate the extent of genetic differences between the syngens of Paramecium bursaria. Our study demonstrates the diagnostic value of molecular markers, which are important tools in the identification of Paramecium bursaria syngens.     

Photo-oxidative stress in symbiotic and aposymbiotic strains of the ciliate Paramecium bursaria.

We tested the hypothesis that photo-oxidative stress is greater in symbiotic representatives of the freshwater ciliate Paramecium bursaria than in aposymbiotic (i.e., without Chlorella) ones. The level of oxidative stress was determined by assessing reactive oxygen species (ROS) with two fluorescent probes (hydroethidine and dihydrorhodamine123) by flow cytometry in exponential and stationary growth phases of both strains. Photo-oxidative stress was assessed in the laboratory after exposure of the ciliates to photosynthetically active radiation (PAR: 400-700 nm) and PAR+ultraviolet radiation (UVR: 280-400 nm). Additionally, both strains were screened for their antioxidant defenses by measuring the activity of the enzymes catalase, superoxide dismutase (SOD), and glutathione reductase. The results showed that aposymbiotic ciliates had higher levels of PAR-induced oxidative stress than symbiotic ones. Significant differences in PAR-induced oxidative stress were also found in both strains when comparing exponential and stationary growth phases with generally higher values in the former. After exposure to UVR, aposymbiotic ciliates in the stationary phase had the highest levels of ROS despite an increase in SOD activity. By contrast, exposure to UVR decreased catalase activity in both strains. Overall, our results suggest that in this ciliate symbiosis, the presence of symbionts minimizes photo-oxidative stress. This work represents the first assessment of photo-oxidative stress in an algal-ciliate mutualistic symbiosis.     

Genetics of the relationship between the ciliate Paramecium bursaria and its symbiotic algae

Abstract. Paramecium bursaria , a freshwater protozoan, typically harbors hundreds of symbiotic algae ( Chlorella sp.) in its cytoplasm. The relationship between host paramecia and symbiotic algae is stable and mutually beneficial in natural environments. We recently collected an aposymbiotic strain of P. bursaria . Infection experiments revealed that the natural aposymbiotic strain (Ysa2) showed unstable symbiosis with Chlorella sp. The algae aggregated at the posterior region of the host, resulting in aposymbiotic cell production after cell division. Cross-breeding analyses were performed to determine the heritability of the aposymbiotic condition. In crosses of Ysa2 with symbiotic strains of P. bursaria , F1 progeny were able to form stable symbioses with Chlorella sp. However, unstable symbiosis, resembling Ysa2 infection, occurred in some F2 progeny of sibling crosses between symbiotic F1 clones. Infection experiments using aposymbiotic F2 cells showed that these F2 subclones have limited ability to reestablish the symbiosis. These results indicate that the maintenance of stable symbiosis is genetically controlled and heritable, and that Ysa2 is a mutant lacking the mechanisms to establish stable symbiosis with Chlorella sp.     

Mycosporines from freshwater yeasts: a trophic cul-de-sac?

Mycosporine-like amino-acids (MAAs) are found in aquatic bacteria, algae, and animals. A related compound, the mycosporine-glutaminol-glucoside (myc-glu-glu), has recently been reported in freshwater yeasts. Although animals depend on other organisms as their source of MAAs, they can efficiently accumulate them in their tissues. In this work we assessed the potential transfer of the yeast mycosporine myc-glu-glu from the diet into the copepod Boeckella antiqua and the ciliate Paramecium bursaria. For this purpose, we performed experiments to study the feeding of B. antiqua and P. bursaria on the yeast Rhodotorula minuta and their ability to bioaccumulate myc-glu-glu. Bioaccumulation of myc-glu-glu in B. antiqua was assessed through long-term factorial experiments manipulating the diet (Chlamydomonas reinhardii and C. reinhardii + yeasts) and radiation exposure (PAR and PAR + UVR). Shorter term experiments were designed in the case of P. bursaria. The composition and concentration of MAAs in the diet and in the consumers were determined by HPLC analyses. Our results showed that even though both consumers ingested yeast cells, they were unable to accumulate myc-glu-glu. Moreover, when exposed to conditions that stimulated the accumulation of photoprotective compounds (i.e. UVR exposure), an increase in MAAs concentration occurred in copepods fed C. reinhardii plus yeasts as well as in those fed only C. reinhardii. This suggests that the copepods were able to modify their tissue concentrations of MAAs in response to environmental clues but also that the contribution of yeast mycosporines to total MAAs concentration was negligible.     

Method for the simultaneous establishment of many axenic cultures of Paramecium

A method is described for the simultaneous treatment of 42 (or more) stocks of Paramecium, and their adaptation to growth in axenic culture. Samples of dense cultures of these ciliates growing with Enterobacter aerogenes are rendered bacteria-free by migration through 2 sets of tubes containing Adaptation Medium (Peters' salts solution, stigmaterol, vitamins, and autoclaved E. aerogenes). The 2nd set of tubes contains Adaptation Medium plus antibiotics. Bacteria-free samples containing approximately 100 animals are then transferred to test tubes containing Adaptation Medium without antibiotics. This medium also serves as a growth medium. It supports indefinite growth of all Paramecium stocks tested. After adaptation to this medium, the ciliattes can be grown in the axenic medium developed by Soldo, Godoy & van Wagtendonk. On a single trial at least half of the stocks can be expected to produce axenic cultures within 5 to 10 days by these procedures. The method has been applied successfully to several of the species of the Paramecium aurelia complex, to all syngens of Paramecium multimicronucleatum, to several stocks of Paramecium jenningsi, and to 1 stock of Paramecium caudatum and Paramecium calkinsi. A modification of the method also works for Didinium nasutum.     

Flow cytometry as a strategy to study the endosymbiosis of algae in Paramecium bursaria

BACKGROUND: The stable symbiotic association between Paramecium bursaria and algae is of interest to study such mechanisms in biology as recognition, specificity, infection, and regulation. The combination of algae-free strains of P. bursaria, which have been recently established by treating their stocks of green paramecia with herbicide paraquat (Hosoya et al.: Zool Sci 12: 807-810, 1995), with the cloned symbiotic algae isolated from P. bursaria (Nishihara et al.: Protoplasma 203: 91-99, 1998), provides an excellent clue to gain fundamental understanding of these phenomena. METHODS: Flow cytometry and light microscopy have been employed to characterize the algal cells after they have been released from the paramecia by ultrasonic treatment. Algal optical properties such as light scattering and endogenous chlorophyll fluorescence intensity have been monitored for symbiotic and free-living strains, and strains at stages of interaction with a host. RESULTS: Neither algal morphology nor chlorophyll content has been found to be altered by sonication of green paramecia. This fact allows to interpret in adequate degree changes in the optical properties of symbiont that just has been released from the association with a host (decreased forward light scatter and chlorophyll fluorescence signals). Optical characterization of both symbiotic and free-living algal strains with respect to their ability to establish symbioses with P. bursaria showed that chlorophyll content per cell volume seems to be a valuable factor for predicting a favorable symbiotic relationship between P. bursaria and algae. CONCLUSIONS: Flow cytometry combined with algae-free paramecia and cloned symbiotic algae identifies algal populations that may be recognized by host cells for the establishment of symbioses.     

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.     

Green paramecia as an evolutionary winner of oxidative symbiosis: a hypothesis and supportive data

A single cell of the green paramecia (Paramecium bursaria) harbors several hundreds of endo-symbiotic Chlorella-like algae in its cytoplasm. Removal of algae from the host organism and re-association of ex-symbiotic host paramecia with ex-symbiotic algae can be experimentally demonstrated in the laboratory. However, the mechanism precisely governing the alga-protozoan association is not fully understood, and the origin of symbiosis in the evolutionary view has not been given. Here, we propose the possible biochemical models (models 1 and 2) explaining the co-evolution between Paramecium species and algal symbionts by pointing out that algal photosynthesis in the host paramecia plays a dual role providing the energy source and the risk of oxidative damage to the host. Model 1 lays stress on the correlation between the (re)greening ability of the paramecia and the tolerance to oxidative stress whereas model 2 emphasizes the cause of evolutionary selection leading to the emergence of Paramecium species tolerant against reactive oxygen species.