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.     

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.     

Comparison of two aposymbiotic ciliate clones of Climacostomum virens by their ability for reinfection

The ability of two aposymbiotic (algae-free) subclones of the same green clone of C. virens to establish a stable symbiotic association with Chlorella sp. has been studied by light and electron microscopy. Alga-free subclone No. 1 was obtained from the original green clone by a long-term cultivation in darkness, while subclone No. 2 originated from one cell that spontaneously lost the algae and was found among normal green cells during daily inspection. For infection, algae isolated from ciliates with chlorellae of parental clone of C. virens were used. 5-10 minutes after feeding with Chlorella, specimens of both subclones show numerous algae mostly inside food vacuoles, but some rare algae (3-4 per cell) may occur in individual perialgal vacuoles. Later on, the number of symbiotic chlorellae in ciliates of subclone No. 1 increased, and a stable symbiotic association was reestablished. Unlike, in specimens of subclone No. 2 all newly ingested algae were seen digested within food vacuoles. Within 24-28 h all the ciliates investigated appeared free of algae. However, obviously stable symbiotic ciliate-algae systems in this subclone were obtained after improving the microinjection technique. Injection of algae into alga-free ciliates resulted in maintenance of intact chlorellae in these ciliates. The algae were seen to be located individually within perialgal vacuoles, being presumably protected against host lytic enzyme attack. The endosymbiont population in ciliates was established from as many as 3-5 originally injected algae. The number of symbiotic chlorellae increased steadily reaching the value equal to that in the parental clone 28-30 days after the start of experiment.     

Infection of algae-free Climacostomum virens with symbiotic Chlorella sp. isolated from algae-containing C. virens

The ciliate Climacostomum virens normally contains algae as symbionts in its cytoplasm and retains them over many generations. An aposymbiotic strain of C. virens which cannot re-establish a new symbiotic association by ingestion of algae derived from green Climacostomum was recently isolated in our laboratory. Results of infection experiments showed that all newly ingested, potentially symbiotic algae were digested in food vacuoles. To clarify whether these ciliates have completely lost their ability to sustain symbiosis with algae or whether this ability can eventually be re-established, infection experiments were performed using a microinjection technique. We have achieved successful infection of algae-free Climacostomum using this method. The endosymbiont population was established in ciliates from as few as 3-5 injected algae, which have retained an intact perialgal vacuole membrane around them. To our knowledge, this is the first evidence of successful infection of aposymbiotic ciliates with algae by microinjection.     

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.     

Comparative Proteomics of Symbiotic and Aposymbiotic Juvenile Soft Corals

The symbiotic association between corals and photosynthetic unicellular algae is of great importance in coral reef ecosystems. The study of symbiotic relationships is multidisciplinary and involves research in phylogeny, physiology, biochemistry, and ecology. An intriguing phase in each symbiotic relationship is its initiation, in which the partners interact for the first time. The examination of this phase in coral–algae symbiosis from a molecular point of view is still at an early stage. In the present study we used 2-dimensional polyacrylamide gel electrophoresis to compare patterns of proteins synthesized in symbiotic and aposymbiotic primary polyps of the Red Sea soft coral Heteroxenia fuscescens. This is the first work to search for symbiosis-specific proteins during the natural onset of symbiosis in early host ontogeny. The protein profiles reveal changes in the host soft coral proteome through development, but surprisingly virtually no changes in the host proteome as a function of symbiotic state.     

Timing of perialgal vacuole membrane differentiation from digestive vacuole membrane in infection of symbiotic algae Chlorella vulgaris of the ciliate Paramecium bursaria

Each symbiotic Chlorella of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole to protect from lysosomal fusion. To understand the timing of differentiation of the perialgal vacuole from the host digestive vacuole, algae-free P. bursaria cells were fed symbiotic C. vulgaris cells for 1.5min, washed, chased and fixed at various times after mixing. Acid phosphatase activity in the vacuoles enclosing the algae was detected by Gomori's staining. This activity appeared in 3-min-old vacuoles, and all algae-containing vacuoles demonstrated activity at 30min. Algal escape from these digestive vacuoles began at 30min by budding of the digestive vacuole membrane into the cytoplasm. In the budded membrane, each alga was surrounded by a Gomori's thin positive staining layer. The vacuoles containing a single algal cell moved quickly to and attached just beneath the host cell surface. Such vacuoles were Gomori's staining negative, indicating that the perialgal vacuole membrane differentiates soon after the algal escape from the host digestive vacuole. This is the first report demonstrating the timing of differentiation of the perialgal vacuole membrane during infection of P. bursaria with symbiotic Chlorella.     

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.     

Symbiosis between hydra and chlorella: Molecular phylogenetic analysis and experimental study provide insight into its origin and evolution

Although many physiological studies have been reported on the symbiosis between hydra and green algae, very little information from a molecular phylogenetic aspect of symbiosis is available. In order to understand the origin and evolution of symbiosis between the two organisms, we compared the phylogenetic relationships among symbiotic green algae with the phylogenetic relationships among host hydra strains. To do so, we reconstructed molecular phylogenetic trees of several strains of symbiotic chlorella harbored in the endodermal epithelial cells of viridissima group hydra strains and investigated their congruence with the molecular phylogenetic trees of the host hydra strains. To examine the species specificity between the host and the symbiont with respect to the genetic distance, we also tried to introduce chlorella strains into two aposymbiotic strains of viridissima group hydra in which symbiotic chlorella had been eliminated in advance. We discussed the origin and history of symbiosis between hydra and green algae based on the analysis.     

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.     

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.     

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.     

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.     

Aposymbiotic Plesiastrea versipora continues to produce cell-signalling molecules that regulate the carbon metabolism of symbiotic algae

The scleractinian coral Plesiastrea versipora produces cell signals that regulate the carbon metabolism of its symbiotic algae. Host release factor (HRF) stimulates the release of photosynthate, and photosynthesis inhibiting factor (PIF) partially inhibits carbon fixation in freshly isolated symbiotic algae. Naturally occurring aposymbiotic specimens of P. versipora are rare in Port Jackson, Australia, but one that was collected contained HRF and PIF. Artificially produced aposymbiotic corals of P. versipora that had been kept in the dark for up to 23 months continued to produce both HRF and PIF in the absence of photosynthetically active algae. Aposymbiotic P. versipora from which most of the tissue had been removed, regenerated when they were kept in the dark and fed; the regenerated tissue also contained HRF and PIF. These results suggest that the presence of symbiotic algae is not required for the production of HRF and PIF in P. versipora. We suggest that these cell signals may have evolved in response to symbiosis with Symbiodinium sp. but are now always expressed in the coral P. versipora.     

The Hydra viridis/Chlorella symbiosis. Growth and sexual differentiation in polyps without symbionts

To investigate interactions between the basal metazoan Hydra viridis and its symbiotic Chlorella algae, we generated aposymbiotic hydra lacking algae and compared them to symbiotic ones with regard to growth and sexual differentiation. Under standard feeding conditions aposymbiotic polyps proliferated similarly to symbiotic polyps. Under moderate and low feeding conditions asexual growth was reduced in polyps lacking algae, indicating that the symbionts supply nutrients to their hosts. In addition, the Chlorella symbionts had a strong influence on the sexual reproduction of Hydra viridis: in most cases female gonads were produced only when symbiotic algae were present. Spermatogenesis proceeded similarly in symbiotic and aposymbiotic polyps. Since during oogenesis symbionts are actively transferred from endodermal epithelial cells to the ectodermal oocytes, this oogenesis promoting role could indicate that the symbionts are critically involved in the control of sexual differentiation in green hydra.     

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.     

Value of the Hydra model system for studying symbiosis

Green Hydra is used as a classical example for explaining symbiosis in schools as well as an excellent research model. Indeed the cosmopolitan green Hydra (Hydra viridissima) provides a potent experimental framework to investigate the symbiotic relationships between a complex eumetazoan organism and a unicellular photoautotrophic green algae named Chlorella. Chlorella populates a single somatic cell type, the gastrodermal myoepithelial cells (also named digestive cells) and the oocyte at the time of sexual reproduction. This symbiotic relationship is stable, well-determined and provides biological advantages to the algal symbionts, but also to green Hydra over the related non-symbiotic Hydra i.e. brown hydra. These advantages likely result from the bidirectional flow of metabolites between the host and the symbiont. Moreover genetic flow through horizontal gene transfer might also participate in the establishment of these selective advantages. However, these relationships between the host and the symbionts may be more complex. Thus, Jolley and Smith showed that the reproductive rate of the algae increases dramatically outside of Hydra cells, although this endosymbiont isolation is debated. Recently it became possible to keep different species of endosymbionts isolated from green Hydra in stable and permanent cultures and compare them to free-living Chlorella species. Future studies testing metabolic relationships and genetic flow should help elucidate the mechanisms that support the maintenance of symbiosis in a eumetazoan species.     

Delay in migration of symbiotic algae in Hydra viridis by inhibitors of microtubule protein polymerization

An English strain of the fresh water symbiotic coelenterate Hydra viridis was experimentally "bleached" of its Chlorella algae and maintained indefinitely by feeding. The algal symbiosis could be re-established by injecting other symbiotic algae into aposymbionts. Although algal uptake and recognition were not affected by microtubule protein polymerization inhibitors, these compounds i.e., podophyllotoxin, beta-peltatin and vinblastine had delaying effects on the migration of the algae through the host digestive cells. Picropodophyllotoxin did not delay migration. The rates, the reversibility and the sensitivity of algal migration to low concentrations of drugs known to bind tubulin suggests the symbionts migrate somehow via labile polymerization of host hydra tubulin into microtubules.     

Colchicine, nocodazole and trifluralin: different effects of microtubule polymerization inhibitors on the uptake and migration of endosymbiotic algae in Hydra viridis

Hydra viridis (= Chlorohydra viridissima) the freshwater coelenterate, is symbiotic. Each individual animal harbours Chlorella sp. in its endodermal cells. The symbiosis may be disestablished, the partners grown independently, and then re-established experimentally. The most effective method to produce alga-free hydra was developed by Pardy (1976). In this study algae from homogenized H. viridis, English (= European) strain, were either used directly or first grown in pure culture and then injected to re-establish the symbiosis. The uptake of algae grown in culture was compared with that of algae released directly from fresh hydra. Fewer cultured algae were taken up. Four strains were tested: English, Carolina, Frome, and Jubilee. Each takes up a characteristic number of Chlorella when injected in excess. Colchicine, nocodazole and trifluralin, were tested for their effects on either uptake or migration. Colchicine had no effect. Nocodazole and trifluralin reduced both the number of algae taken up and the rate at which they were transported to the distal region of the hydra digestive cell. The effects of these drugs tended to be proportional to the concentration between 10(-8) and 10(-5) M.     

Identification of an algal carbon fixation-enhancing factor extracted from Paramecium bursaria

The green ciliate Paramecium bursaria contains several hundred symbiotic Chlorella species. We previously reported that symbiotic algal carbon fixation is enhanced by P. bursaria extracts and that the enhancing factor is a heat-stable, low-molecular-weight, water-soluble compound. To identify the factor, further experiments were carried out. The enhancing activity remained even when organic compounds in the extract were completely combusted at 700 degrees C, suggesting that the factor is an inorganic substance. Measurement of the major cations, K+, Ca2+, and Mg2+, by an electrode and titration of the extract resulted in concentrations of 0.90 mM, 0.55 mM, and 0.21 mM, respectively. To evaluate the effect of these cations, a mixture of the cations at the measured concentrations was prepared, and symbiotic algal carbon fixation was measured in the solution. The results demonstrated that the fixation was enhanced to the same extent as with the P. bursaria extract, and thus this mixture of K+, Ca2+, and Mg2+ was concluded to be the carbon fixation-enhancing factor. There was no effect of the cation mixture on free-living C. vulgaris. Comparison of the cation concentrations of nonsymbiotic and symbiotic Paramecium extracts revealed that the concentrations of K+ and Mg2+ in nonsymbiotic Paramecium extracts were too low to enhance symbiotic algal carbon fixation, suggesting that symbiotic P. bursaria provide suitable cation conditions for photosynthesis to its symbiotic Chlorella.