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Paramecium spp. is types of free-living protists that live in freshwater environments. They are ciliates with high motility and phagocytosis and have been used to analyze cell motility and as a host model for pathogens. Besides such biological characteristics, apart from the usual morphological and genetic classification of species, the existence of taxonomies (such as syngens) and mating types related to Paramecium’s unique reproduction is known. In this study, we attempted to develop a simple method to identify Paramecium strains, which are difficult to distinguish morphologically, using random amplified polymorphic DNA (RAPD) analysis. Consequently, we can observe strain-specific band patterns. We also confirm that the presence of endosymbiotic Chlorella cells affects the band pattern of P. bursaria. Furthermore, the results of the RAPD analysis using several P. caudatum strains with different syngens show that it is possible to detect a band specific to a certain syngen. By improving the reaction conditions and random primers, based on the results of this study, RAPD analysis can be applied to the identification of Paramecium strains and their syngen confirmation tests.

The genus Stentor is a relatively well-known ciliate owing to its lucid trumpet shape. Stentor pyriformis represents a green, short, and fat Stentor, but it is a little-known species. We investigated 124 ponds and wetlands in Japan and confirmed the presence of S. pyriformis at 23 locations. All these ponds were noticeably oligotrophic. With the improvement of oligotrophic culture conditions, we succeeded in long-term cultivation of three strains of S. pyriformis. The cytoplasm of S. piriformis contains a large number of 1–3 μm refractive granules that turn brown by Lugol’s staining. The granules also show a typical Maltese-cross pattern by polarization microscopy, strongly suggesting that the granules are made of amylopectin-rich starch. By analyzing the algal rDNA, it was found that all S. pyriformis symbionts investigated in this study were Chlorella variabilis. This species is known as the symbiont of Paramecium bursaria and is physiologically specialized for endosymbiosis. Genetic discrepancies between C. variabilis of S. pyriformis and P. bursaria may indicate that algal sharing was an old incident. Having symbiotic algae and storing carbohydrate granules in the cytoplasm is considered a powerful strategy for this ciliate to withstand oligotrophic and cold winter environments in highland bogs.

Francisella novicida is a facultative intracellular pathogen and the causative agent of tularemia. Although cases of infection caused by exposure to contaminated water have been reported, its natural host and ecology in the environment remain unclear. In this study, we investigated in vitro the possibility that Paramecium bursaria may be a useful tool as a protist host model of F. novicida. Experimental infection with F. novicida resulted in a stable intracellular relationship within P. bursaria. This symbiotic intracellular relationship was not observed in experimental infections with other Francisella species and Legionella pneumophila. We found that F. novicida showed similar behaviour to that of the eukaryotic endosymbiont of P. bursaria, the green algae Chlorella, in the internalization process. In addition, stable intracellular localization of F. novicida was possible only when Chlorella was not present. Although we investigated the type VI secretion system of F. novicida as a candidate for the bacterial factor, we found that it was not involved in the establishment of an intracellular relationship with P. bursaria. These results suggested that P. bursaria is potentially a protist host model for F. novicida and may be a useful tool for understanding the relationship between protist hosts and their symbionts.

Ciliates are a diverse protistan group and many consist of cryptic species complexes whose members may be restricted to particular biogeographic locations. Mitochondrial genes, characterized by a high resolution for closely related species, were applied to identify new species and to distinguish closely related morphospecies. In the current study, we analyzed 132 sequences of COI mtDNA fragments obtained from P. bursaria species collected worldwide. The results allowed, for the first time, to generate a network of COI haplotypes and demonstrate the relationships between P. bursaria strains, as well as to confirm the existence of five reproductively isolated haplogroups. The P. bursaria haplogroups identified in the present study correspond to previously reported syngens (R1, R2, R3, R4, and R5), thus we decided to propose the following binominal names for each of them: P. primabursaria, P. bibursaria, P. tribursaria, P. tetrabursaria, and P. pentabursaria, respectively. The phylogeographic distribution of P. bursaria species showed that P. primabursaria and P. bibursaria were strictly Eurasian, except for two South Australian P. bibursaria strains. P. tribursaria was found mainly in Eastern Asia, in two stands in Europe and in North America. In turn, P. tetrabursaria was restricted to the USA territory, whereas P. pentabursaria was found in two European localities.

The protist (mostly single-celled organisms), Paramecium bursaria, forms an intracellular symbiotic relationship with the single-celled algae, Chlorella variabilis, where P. bursaria provides nutrients (i.e., Ca2+, Mg2+, and K+), carbon dioxide for photosynthesis and protection from viruses, while C. variabilis provides oxygen, carbon fixation, and nutrients. Key to this successful relationship is the perialgal vacuole (PV) membrane, which surrounds C. variabilis and protects it from digestion by P. bursaria. The membrane is fragile and difficult to analyze using conventional methods therefore very little is known about the molecular composition. We used the OrbiSIMS, a new high-resolution mass spectrometer with subcellular resolution imaging, to study the compartmentalization of endosymbionts and elucidate biomolecular interactions between the host and endosymbiont. Ions from the region of interest, close to C. variabilis, and specific to the target samples containing PVs were found based on the chemical mapping and masses of the ions. We show chemical localizations of oligosaccharides in close proximity of C. variabilis endosymbionts in P. bursaria. These oligosaccharides are detected in host-endosymbiont samples containing PV membrane-bound algae and absent in free-living algae and digestive vacuole (DV) membrane-bound algae in P. bursaria.

The single-celled ciliate Paramecium bursaria is an indispensable model for investigating endosymbiosis between protists and green-algal symbionts. To elucidate the mechanism of this type of endosymbiosis, we combined PacBio and Illumina sequencing to assemble a high-quality and near-complete macronuclear genome of P. bursaria. The genomic characteristics and phylogenetic analyses indicate that P. bursaria is the basal clade of the Paramecium genus. Through comparative genomic analyses with its close relatives, we found that P. bursaria encodes more genes related to nitrogen metabolism and mineral absorption, but encodes fewer genes involved in oxygen binding and N-glycan biosynthesis. A comparison of the transcriptomic profiles between P. bursaria with and without endosymbiotic Chlorella showed differential expression of a wide range of metabolic genes. We selected 32 most differentially expressed genes to perform RNA interference experiment in P. bursaria, and found that P. bursaria can regulate the abundance of their symbionts through glutamine supply. This study provides novel insights into Paramecium evolution and will extend our knowledge of the molecular mechanism for the induction of endosymbiosis between P. bursaria and green algae.

Paramecium bursaria (Ehrenberg 1831) is a ciliate species living in a symbiotic relationship with green algae. The aim of the study was to identify green algal symbionts of P. bursaria originating from distant geographical locations and to answer the question of whether the occurrence of endosymbiont taxa was correlated with a specific ciliate syngen (sexually separated sibling group). In a comparative analysis, we investigated 43 P. bursaria symbiont strains based on molecular features. Three DNA fragments were sequenced: two from the nuclear genomes—a fragment of the ITS1-5.8S rDNA-ITS2 region and a fragment of the gene encoding large subunit ribosomal RNA (28S rDNA), as well as a fragment of the plastid genome comprising the 3′rpl36-5′infA genes. The analysis of two ribosomal sequences showed the presence of 29 haplotypes (haplotype diversity Hd = 0.98736 for ITS1-5.8S rDNA-ITS2 and Hd = 0.908 for 28S rDNA) in the former two regions, and 36 haplotypes in the 3′rpl36-5′infA gene fragment (Hd = 0.984). The following symbiotic strains were identified: Chlorella vulgaris, Chlorella variabilis, Chlorella sorokiniana and Micractinium conductrix. We rejected the hypotheses concerning (i) the correlation between P. bursaria syngen and symbiotic species, and (ii) the relationship between symbiotic species and geographic distribution.

Metabolic symbiosis is a form of symbiosis in which organisms exchange metabolites, typically for mutual benefit. For example, acquired phototrophs like Paramecium bursaria obtain photosynthate from endosymbiotic green algae called Chlorella. In addition to facilitating the persistence of P. bursaria by providing a carbon source that supplements P. bursaria’s heterotrophic digestion of bacteria, symbiotic Chlorella may impact competitive interactions between P. bursaria and other bacterivores, with cascading effects on community composition and overall diversity. Here, we tested the effects of metabolic symbiosis on coexistence by assessing the impacts of acquired phototrophy on priority effects, or the effect of species arrival order on species interactions, between P. bursaria and its competitor Colpidium. Our results suggest light-dependent priority effects. The acquired phototroph benefited from metabolic symbiosis during sequential arrival of each organism in competition, and led to increased growth of late-arriving Colpidium. These findings demonstrate that understanding the consequences of priority effects for species coexistence requires consideration of metabolic symbiosis.

Forty-seven species in 25 genera of gall midges (Diptera: Cecidomyiidae: Cecidomyiinae) known from Indonesia are reviewed. Available historic types were examined, and taxa are revised. Leefmansiella Kolesik & Gagné gen. nov. is erected for Trishormomyia pandani Felt. Actilasioptera falcaria (Felt) is found to be a senior synonym of Actilasioptera tumidifolium Gagné. Apoasphondylia orientalis Felt is transferred to Bruggmanniella Tavares, new combination; Asphondylia bursaria Felt is changed to Bruggmanniella, new combination; Contarinia eragrostidis Felt to Stenodiplosis Reuter, new combination; the Australian Contarinia brevipalpis Harris to Stenodiplosis, new combination; Dasyneura tetrastigma Felt to Gephyraulus Rübsaamen, new combination; and the Philippine Itonida paederiae Felt to Eucalyptodiplosis Kolesik, new combination. For each species, type data, a description, the basic biology and the known geographical distribution are given. A key to Cecidomyiidae genera based on adults, with references to described species, is provided. Species of economic importance, as pests of cultivated plants or biological control agents of weeds and pest arthropods, are listed.

International audience; Digestion-resistant bacteria (DRB) refer to the ecological bacterial group that can be ingested, but not digested by protistan grazers, thus forming a specific type of bacteria-protist association. To test the hypothesis that the environment affects the assembly of DRB in protists, a mixotrophic ciliate, Paramecium bursaria, and a heterotrophic ciliate, Euplotes vannus, were reared at different temperatures, light conditions, and concentration gradients of antibiotic oxytetracycline and heavy metals. Community profiling indicated that the composition of DRB in both species varied significantly across the manipulated conditions, except for in P. bursaria under light/dark treatments. Clone library analysis of bacterial 16S rRNA genes showed that DRB were diverse. Pseudomonas became more abundant during the warmer treatment of P. bursaria, whereas the dominance of Pseudoalteromonas weakened and Vibrio became more abundant in E. vannus at a higher temperature. During the treatment of diel light:dark cycles, Aestuariibacter and Alteromonas were selected for in E. vannus but not Pseudoalteromonas, which was highly represented in the all-light and alldark treatments. In contrast, P. bursaria consistently hosted Nevskia, Curvibacter, and Asticcacaulis under all light conditions. There were many bacterial species co-resistant to oxytetracycline and to protistan digestion, in which Sphingomonas, Alteromonas, Aestuariibacter, Puniceicoccaceae (Verrucomicrobia), Pseudomonas, and Sulfitobacter were frequently abundant. Flectobacillus and Aestuariibacter were major lead-resistant bacteria associated with the studied protists. Acinetobacter and Hydrogenophaga were abundant in the P. bursaria treated with a high dose of mercury. Aestuariibacter was found as a dominant group of DRB in E. vannus across all cadmium treatments. In summary, this study demonstrates for the first time that environmental stress selects for bacterial populations associated with protists and that there are diverse bacterial species that not only are resistant to pollution stresses but can also survive protistan predation. This work highlights that bacteria-protists associations need to be taken into account in understanding ecological and environmental issues, such as resilience of bacterial community and function, microbial co-occurrence, and quantity and distribution of antibiotic resistant bacteria and genes.

The &lsquo, green&rsquo, ciliate Paramecium bursaria lives in mutualistic symbiosis with green algae belonging to the species Chlorella variabilis or Micractinium conductrix. We analysed the diversity of algal endosymbionts and their P. bursaria hosts in nine strains from geographically diverse origins. Therefore, their phylogenies using different molecular markers were inferred. The green paramecia belong to different syngens of P. bursaria. The intracellular algae were assigned to Chl. variabilis, M. conductrix or, surprisingly, Choricystis parasitica. This usually free-living alga co-occurs with M. conductrix in the host&rsquo, s cytoplasm. Addressing the potential status of Chor. parasitica as second additional endosymbiont, we determined if it is capable of symbiosis establishment and replication within a host cell. Symbiont-free P. bursaria were generated by cycloheximid treatment. Those aposymbiotic P. bursaria were used for experimental infections to investigate the symbiosis specificity not only between P. bursaria and Chor. parasitica but including also Chl. variabilis and M. conductrix. For each algae we observed the uptake and incorporation in individual perialgal vacuoles. These host-symbiont associations are stable since more than five months. Thus, Chor. parasitica and P. bursaria can form an intimate and long-term interaction. This study provides new insights into the diversity of P. bursaria algal symbionts.

The endosymbiosis between Paramecium bursaria and Chlorella spp. is mutualistic. Symbiotic algae localize beneath the host Paramecium cell cortex compete for their attachment sites with preexisting organelle trichocysts. To examine the relationship between P. bursaria trichocysts and their symbiotic algae, algae-bearing or alga-free P. bursaria were starved for several days and the changes in the number of Chlorella sp. and presence or absence of trichocysts were evaluated. We conducted an indirect immunofluorescence microscopy with an anti-trichocyst monoclonal antibody against P. bursaria cells. Indirect immunofluorescence microscopy demonstrated that under starvation and darkness conditions, the immunofluorescence of trichocysts in alga-free P. bursaria decreased much faster than that in the normal algae-bearing P. bursaria. In the latter case, our observations proposed the possibility that the nutrition obtained from symbiotic algal digestion may promote trichocysts synthesis. This algal digestion mechanism may permit host P. bursaria cells to survive for a longer time under starvation condition. To the best of our knowledge, this may be a new benefit that host P. bursaria gain from harboring symbiotic algae.

Many chloroviruses replicate in endosymbiotic zoochlorellae that are protected from infection by their symbiotic host. To reach the high virus concentrations that often occur in natural systems, a mechanism is needed to release zoochlorellae from their hosts. We demonstrate that the ciliate predator Didinium nasutum foraging on zoochlorellae-bearing Paramecium bursaria can release live zoochlorellae from the ruptured prey cell that can then be infected by chloroviruses. The catalysis process is very effective, yielding roughly 95% of the theoretical infectious virus yield as determined by sonication of P. bursaria. Chlorovirus activation is more effective with smaller Didinia, as larger Didinia typically consume entire P. bursaria cells without rupturing them, precluding the release of zoochlorellae. We also show that the timing of Chlorovirus growth is tightly linked to the predator-prey cycle between Didinium and Paramecium, with the most rapid increase in chloroviruses temporally linked to the peak foraging rate of Didinium, supporting the idea that predator-prey cycles can drive cycles of Chlorovirus abundance.

Chloroviruses are large viruses that replicate in chlorella-like green algae and normally exist as mutualistic endosymbionts (referred to as zoochlorellae) in protists such as Paramecium bursaria. Chlorovirus populations rise and fall in indigenous waters through time, however, the factors involved in these virus fluctuations are still under investigation. Chloroviruses attach to the surface of P. bursaria but cannot infect their zoochlorellae hosts because the viruses cannot reach the zoochlorellae as long as they are in the symbiotic phase. Predators of P. bursaria, such as copepods and didinia, can bring chloroviruses into contact with zoochlorellae by disrupting the paramecia, which results in an increase in virus titers in microcosm experiments. Here, we report that another predator of P. bursaria, Bursaria truncatella, can also increase chlorovirus titers. After two days of foraging on P. bursaria, B. truncatella increased infectious chlorovirus abundance about 20 times above the controls. Shorter term foraging (3 h) resulted in a small increase of chlorovirus titers over the controls and more foraging generated more chloroviruses. Considering that B. truncatella does not release viable zoochlorellae either during foraging or through fecal pellets, where zoochlorellae could be infected by chlorovirus, we suggest a third pathway of predator virus catalysis. By engulfing the entire protist and digesting it slowly, virus replication can occur within the predator and some of the virus is passed out through a waste vacuole. These results provide additional support for the hypothesis that predators of P. bursaria are important drivers of chlorovirus population sizes and dynamics.

The amoeba, Mayorella viridis contains several hundred symbiotic green algae in its cytoplasm. Transmission electron microscopy revealed strong resemblance between symbiotic algae from M. viridis the symbiotic Chlorella sp. in the perialgal vacuoles of Paramecium bursaria and other ciliates. Although it is thought that the M. viridis and symbiotic algae could be model organisms for studying endosymbiosis between protists and green algae, few cell biological observations of the endosymbiosis between M. viridis and their symbiotic algae have been published. In this study, we characterized the specificity of endosymbiotic relationships between green algae and their hosts. Initially, we established stable cultures of M. viridis in KCM medium by feeding with Chlorogonium capillatum. Microscopic analyses showed that chloroplasts of symbiotic algae in M. viridis occupy approximately half of the algal cells, whereas those in P. bursaria occupy entire algal cells. The symbiotic algae in P. bursaria contain several small spherical vacuoles. The labeling of actin filaments using Acti-stain™ 488 Fluorescent Phalloidin revealed no relationship between host actin filaments and symbiotic algal localization, although the host mitochondria were localized around symbiotic algae. Symbiotic algae from M. viridis could infect algae-free P. bursaria but could not support P. bursaria growth without feeding, whereas the original symbiotic algae of P. bursaria supported its growth without feeding. These data indicated the specificity of endosymbiotic algae relationships in M. viridis and P. bursaria.

The unicellular green alga Chlorella variabilis NC64A is an endosymbiont of the ciliate Paramecium bursaria. The host's control, including the transfer of biochemical substrates from P. bursaria to C. variabilis, is involved in symbiotic relationships. C. variabilis NC64A that had been re-infected to P. bursaria for more than 1 year and isolated from the host showed higher chlorophyll levels compared to those in free-living cells. Unlike the host, the expression of C. variabilis NC64A heat shock 70 kDa protein was independent of establishment of endosymbiosis. In symbiotic cells, the levels of PII signal transduction protein (CvPII) that coordinate the central C/N anabolic metabolism were slightly higher than those in free-living cells. Furthermore, the environmental cues (light and host food bacteria availability) affected the abundance of CvPII, suggesting that synthesis of the protein was influenced by the host. Moreover, arginine concentrations in the symbiotic algae of P. bursaria were also controlled by the host's nutritional conditions. Together, our results imply that signal substrates and/or products of metabolism in host cells might act as messengers mediating the regulation of key events in symbiont cells.

Chlorella variabilis strain NC64A is an intracellular photobiont of the ciliate Paramecium bursaria. NC64A was isolated from P. bursaria nearly 50 years ago and was thereafter cultivated outside the host. This study was undertaken to detect changes in its infectivity to P. bursaria and its auxotrophy for growth outside the host induced during long-term cultivation. NC64A can grow in Modified Bold's Basal Medium but not in C medium, whereas another symbiotic Chlorella variabilis strain, 1N, that was recently isolated from the host grew in C medium but not in Modified Bold's Basal Medium. With regards infectivity, NC64A in the logarithmic phase of growth showed low infectivity to alga-removed P. bursaria cells, whereas those in the early stationary phase showed high infectivity of about 30%. Those in the decay phase of growth showed no infectivity. Results show that NC64A has infectivity, but the infection rate depends on their culture age in the growth curve. Furthermore, NC64A that had been re-infected to P. bursaria for more than one year and isolated from the host showed a nearly 100% infection rate, which indicates that NC64A can recover its infectivity by re-infection to P. bursaria., Summary: This study was undertaken to detect changes in infectivity induced during long-term cultivation of Chlorella variabilis to alga-free Paramecium bursaria, and its auxotrophy for growth outside the host.

Although some Paramecium species are suitable research objects in many areas of life sciences, the biodiversity structure of other species is almost unknown. In the current survey, we present a molecular analysis of 60 Cypriostomum strains, which for the first time allows for the study of intra- and interspecific relationships within that subgenus, as well as the assessment of the biogeography patterns of its morphospecies. Analysis of COI mtDNA variation revealed three main clades (separated from each other by approximately 130 nucleotide substitutions), each one with internal sub-clusters (differing by 30 to 70 substitutions - a similar range found between P. aurelia cryptic species and P. bursaria syngens). The first clade is represented exclusively by P. polycaryum; the second one includes only four strains identified as P. calkinsi. The third cluster seems to be paraphyletic, as it includes P. nephridiatum, P. woodruffi, and Eucandidatus P. hungarianum. Some strains, previously identified as P. calkinsi, had COI sequences identical or very similar to P. nephridiatum ones. Morphological reinvestigation of several such strains revealed common morphological features with P. nephridiatum. The paper contains new information concerning speciation within particular species, i.e. existence of cryptic species within P. polycaryum (three) and in P. nephridiatum (six).

Paramecium bursaria is composed of five syngens that are morphologically indistinguishable but sexually isolated. The aim of the present study was to confirm by molecular methods (analyses of mitochondrial COI) the identification of P. bursaria syngens originating from different geographical locations. Phylograms constructed using both the neighbor-joining and maximum-likelihood methods based on a comparison of 34 sequences of P. bursaria strains and P. multimicronucleatum, P. caudatum and P.calkinsi strains used as outgroups revealed five clusters which correspond to results obtained previously by mating reaction. Our analysis shows the existence of 24 haplotypes for the COI gene sequence in the studied strains. The interspecies haplotype diversity was Hd = 0.967. We confirmed genetic differentiation between strains of P. bursaria and the occurrence of a correlation between geographical distribution and the correspondent syngen.

We used cells of the yeast Saccharomyces cerevisiae expressing green fluorescent protein (GFP) as fluorescently labelled prey to assess the phagocytic activities of the mixotrophic ciliate Paramecium bursaria, which harbours symbiotic Chlorella-like algae. Because of different fluorescence spectra of GFP and algal chlorophyll, ingested GFP-expressing yeast cells can be distinguished from endosymbiotic algal cells and directly counted in individual P. bursaria cells using fluorescence microscopy. By using GFP-expressing yeast cells, we found that P. bursaria altered ingestion activities under different physiological conditions, such as different growth phases or the presence/absence of endosymbionts. Use of GFP-expressing yeast cells allowed us to estimate the digestion rates of live prey of the ciliate. In contrast to the ingestion activities, the digestion rate within food vacuoles was not affected by the presence of endosymbionts, consistent with previous findings that food and perialgal vacuoles are spatially and functionally separated in P. bursaria. Thus, GFP-expressing yeast may provide a valuable tool to assess both ingestion and digestion activities of ciliates that feed on eukaryotic organisms.

Paramecium putrinum (Claparede & Lachmann 1858) is one of the smallest (80–140 μm long) species of the genus Paramecium . Although it commonly occurs in freshwater reservoirs, no molecular studies of P. putrinum have been conducted to date. Herein we present an assessment of molecular variation in 27 strains collected from widely separated populations by using two selected DNA fragments (ITS1-5.8S-ITS2-5′LSU rDNA and COI mtDNA). Both the trees and haplotype networks reconstructed for both genome fragments show that the studied strains of P. putrinum form five main haplogroups. The mean distance between the studied strains is p -distance = 0.007/0.068 (rDNA/ COI ) and exhibits similar variability as that between P. bursaria syngens . Based on these data, one could hypothesize that the clusters revealed in the present study may correspond to previously reported syngens and that there are at least five cryptic species within P. putrinum .

Paramecium bursaria is an unicellular organism that lives widely in fresh water environments such as rivers and ponds. Recent studies have suggested that in vivo cellular robotics using the cells of P. bursaria as micro-machines controllable under electrical and optical stimuli, has a variety of engineering applications such as transport of micro-sized particles in the capillary systems. The present study aimed to test if the swimming cells of P. bursaria, implementable in capillaries or on chips, are applicable for detection of metal ions. For model assays, rare earth elements (REEs) were chosen as target chemicals. In P. bursaria, LC50values for REE ions ranged between 2.0 and 62.7 µM. Among them, Sc was shown to be most toxic. In addition to the lethal impacts of REE ions, most of REE ions at sub-lethal concentrations at around 10 - 30 µM, showed inhibitory action against the motility of the cells during the electrically forced motility known as galvanotaxisis. In conclusion, in the non-lethal ranges of REE concentration, swimming cells of P. bursaria report the presence of REE ions, by lowering the motility.

Bursaria cayzerae I.Telford & L.M.Copel. (Pittosporaceae), endemic to north-eastern New South Wales, is described. Its distribution is mapped, and habitat and conservation status discussed. A table comparing distinguishing attributes of the new species to those of B. longisepala and B. spinosa is provided. The keys to species in NSW FloraOnline and the Flora of New South Wales are modified to accomodate the new species.

Chlorella spp. and ciliate Paramecium bursaria share a mutual symbiosis. However, both alga-removed P. bursaria and isolated symbiotic algae can grow independently. Additionally, mixing them experimentally can cause algal reinfection through host phagocytosis. Although the symbiotic algal localization beneath the host cell cortex is a prerequisite phenomenon for maintenance of the relationship of their endosymbiosis, how and where the algae locate beneath the host cell cortex remains unknown. To elucidate this phenomenon, algal distribution patterns during algal removal and reinfection were observed. During algal removal, algae at the host anterior cortex were easier to remove than at the posterior and ventral or dorsal cortex areas. During algal reinfection, the algae after separation from the host digestive vacuoles tended to localize beneath the host ventral or dorsal cortex more readily than that at other cortices. Algae that reinfected trichocyst-removed paramecia didn’t show this localization. Trichocyst-discharge experiments clarified that trichocysts of the anterior cortex are difficult to remove. In 14 strains of P. bursaria, some of the paramecia lacked their symbiotic algae at the anterior cortex. These observations demonstrate that symbiotic algae of P. bursaria are difficult to localize at the anterior cortex and that they are easy to remove from the area.

Paramecium bursaria (Ehrenberg 1831), a freshwater ciliate, typically harbors hundreds of green algal symbionts inside the cell. The aim of present study was the molecular identification of newly analyzed P. bursaria symbionts. The second aspect of the present survey was testing a hypothesis whether endosymbionts prefer the specified syngen of the host, and the specified geographical distribution. Ten strains of endosymbionts isolated from strains of P. bursaria originating from different geographical locations were studied. We analyzed for the first time, both the fragment of plastid genome containing 3'rpl36-5' infA genes and a fragment of a nuclear gene encoding large subunit ribosomal RNA (LSU rDNA). The analysis of the LSU rDNA sequences showed the existence of 3 haplotypes and the haplotype diversity of 0.733, and 8 haplotypes for the 3'rpl36-5' infA gene fragment and haplotype diversity of 0.956. The endosymbionts isolated from P. bursaria strains were identified as Chlorella vulgaris, Ch. variabilis and Micractinium conductrix. There was no correlation between the syngen of P. bursaria and the species of endosymbiont.

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.

We redescribe Paramecium chlorelligerum, a forgotten species, which Kahl (Tierwelt Dtl., 1935, 30:651) briefly but precisely described in the addendum to his ciliate monographs as a Paramecium with symbiotic green algae. The redescription is based on classical morphological methods and the analysis of the small subunit (SSU) rDNA. Morphologically, P. chlorelligerum differs from P. (C.) bursaria, the second green species in the genus, by having a special swimming shape, the length of the caudal cilia, the size of the micronucleus, the size of the symbiotic algae, the contractile vacuoles (with collecting vesicles vs. collecting canals), and the number of excretory pores/contractile vacuole (1 vs. 2-3). The molecular investigations show that P. chlorelligerum forms a distinct branch distant from the P. (Chloroparamecium) bursaria clade. Thus, we classify P. chlorelligerum in a new subgenus: Paramecium (Viridoparamecium) chlorelligerum. The symbiotic alga belongs to the little-known genus Meyerella, as yet recorded only from the plankton of a North American lake.

The association of ciliate Paramecium bursaria with symbiotic Chlorella sp. is a mutualistic symbiosis. However, both the alga-free paramecia and symbiotic algae can still grow independently and can be reinfected experimentally by mixing them. Effects of the host's nutritional conditions against the symbiotic algal cell division and density were examined during early reinfection. Transmission electron microscopy revealed that algal cell division starts 24 h after mixing with alga-free P. bursaria, and that the algal mother cell wall is discarded from the perialgal vacuole membrane, which encloses symbiotic alga. Labelling of the mother cell wall with Calcofluor White Stain, a cell-wall-specific fluorochrome, was used to show whether alga had divided or not. Pulse labelling of alga-free P. bursaria cells with Calcofluor White Stain-stained algae with or without food bacteria for P. bursaria revealed that the fluorescence of Calcofluor White Stain in P. bursaria with bacteria disappeared within 3 days after mixing, significantly faster than without bacteria. Similar results were obtained both under constant light and dark conditions. This report is the first describing that the cell division and density of symbiotic algae of P. bursaria are controlled by the host's nutritional conditions during early infection.

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.

Paramecium calkinsi (Ciliophora, Protozoa) is a euryhaline species which was first identified in freshwater habitats, but subsequently several strains were also collected from brackish water. It is characterized by clockwise spiral swimming movement and the general morphology of the “bursaria type.” The present paper is the first molecular characterization of P. calkinsi strains recently collected in distant regions in Russia using ITS1-5.8S- ITS2-5′LSU rDNA (1100 bp) and COI (620 bp) mtDNA sequenced gene fragments. For comparison, our molecular analysis includes P. bursaria, exhibiting a similar “bursaria morphotype” as well as species representing the “aurelia type,” i.e., P. caudatum, P. multimicronucleatum, P. jenningsi, and P. schewiakoffi, and some species of the P. aurelia species complex (P. primaurelia, P. tetraurelia, P. sexaurelia, and P. tredecaurelia). We also use data from GenBank concerning other species in the genus Paramecium and Tetrahymena (which used as an outgroup). The division of the genus Paramecium into four subgenera (proposed by Fokin et al. 2004) is clearly presented by the trees. There is a clear separation between P. calkinsi strains collected from different regions (races). Consequently, given the molecular distances between them, it seems that these races may represent different syngens within the species.

The green ciliate, Paramecium bursaria, has evolved a mutualistic relationship with endosymbiotic green algae (photobionts). Under culture conditions, photobionts are usually unified (to be single species) within each P. bursaria strain. In most cases, the algal partners are restricted to either Chlorella variabilis or Micractinium reisseri (Chlorellaceae, Trebouxiophyceae). Both species are characterized by particular physiology and atypical group I intron insertions, although they are morphologically indistinguishable from each other or from other Chlorella-related species. Both algae are exclusive species that are viable only within P. bursaria cells, and therefore their symbiotic relationship can be considered persistent. In a few cases, the other algal species have been reported as P. bursaria photobionts. Namely, P. bursaria have occasionally replaced their photobiont partner. This paper introduces some P. bursaria strains that maintain more than one species of algae for a long period. This situation prompts speculations about flexibility of host-photo-biont relationships, how P. bursaria replaced these photobionts, and the infection theory of the group I introns.

The behavior of Paramecium sp. (Paramecium bursaria) in aqueous solutions containing Sr and Pb was investigated to determine the role of protozoa in the migration of radionuclides in the environment. Precultured living cells of P. bursaria were exposed to aqueous solutions containing 0.01 or 0.05 mM Sr or Pb at pH 7 for 24 h. For comparison, pre-killed cells were treated with the metal solutions in the same way. Two-dimensional elemental mappings of cells were obtained by micro-PIXE. Aquatic species of Sr and Pb were analyzed by size exclusion chromatography (SEC) coupled online to ultraviolet (UV) spectroscopy and inductivity coupled plasma mass spectroscopy (ICP-MS). The amounts of Sr adsorbed or taken up by the cells surviving for 24 h and adsorbed on pre-killed cells were below the detection limit. Cells of P. bursaria adsorbed or took up a fraction of Pb. The Pb adsorbed or taken up by the cells surviving for 24 h in the Pb solution was barely detectable, while the Pb adsorbed on pre-killed cells was clearly mappable. These findings suggest that living cells of P. bursaria have functions that reduce adsorption or uptake of Pb on the cells. Quantitative and SEC–UV–ICP-MS analyses of the Sr and Pb in aqueous phases showed no clear evidences that living cells of P. bursaria alter the chemical form of Sr or Pb remaining in the aqueous phases after the cell-solution contact.

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) Verifications experimentales de la theorie mathematique de la lutte pour la vie. Actualites Scientifiques et Industrielles, 277]. He showed the coexistence of Paramecium caudatum with a potentially allelopathic species, Paramecium bursaria. less thanbrgreater than less thanbrgreater than2. 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. less thanbrgreater than less thanbrgreater than3. 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. less thanbrgreater than less thanbrgreater than4. 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. bursarias 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. less thanbrgreater than less thanbrgreater than5. Our results expose the hidden diversity of mechanisms that underlie competitive interactions. They thus support Gausess 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.

Nilova, M. & Oskolski, A.A.: Comparative bark anatomy of Bursaria, Hymenosporum and Pittosporum (Pittosporaceae) — Plant Div. Evol. 128: 491–500. 2010. — ISSN 1869-6155. Bark anatomy in 3 species of Bursaria, 9 species of Pittosporum, and in the single species of monotypic genus Hymenosporum (Pittosporaceae) was examined. The members of these three genera resemble Araliaceae, Myodocarpaceae and Apiaceae in the occurrence of axial secretory canals in cortex and secondary phloem, the pattern of alternating zones in secondary phloem, and the absence of fibres in this tissue. We therefore confirm a relationship between Pittosporaceae and other Apiales (van Tieghem 1884, Dahlgren 1989, Takhtajan 1997, Plunkett et al. 1996, 2004) rather than its traditional placement into Rosales (Cronquist 1981). Hymenosporum differs markedly from Bursaria and Pittosporum in the presence of primary phloem fibres, in the cortical (vs subepidermal) initiation of the periderm and in the occurrence of numerous (more than 25) sieve areas on compound sieve plates. These features confirm the isolated position of Hymenosporum within Pittosporaceae, as suggested both by traditional taxonomy and gross morphology (Cayzer et al. 2000) and by molecular phylogenetics (Chandler et al. 2007).

SUMMARY Symbiotic algae of the ciliate Paramecium bursaria (Ehrenberg) Focker are key species in the fields of virology and molecular evolutionary biology as well as in the biology of symbiotic relationships. These symbiotic algae were once identified as Zoochlorella conductrix Brandt by the Dutch microbiologist, Beijerinck 120 years ago. However, after many twists and turns, the algae are today treated as nameless organisms. Recent molecular analyses have revealed several different algal partners depending on P. bursaria strains, but nearly all P. bursaria contains a symbiont belonging to either the so-called ‘American’ or ‘European’ group. The absence of proper names for these algae is beginning to provoke ill effects in the above-mentioned study areas. In the present study, we confirmed the genetic autonomy of the ‘American’ and ‘European’ groups and described the symbionts as Chlorella variabilis Shihira et Krauss and Micractinium reisseri Hoshina, Iwataki et Imamura sp. nov., respectively (Chlorellaceae, Trebouxiophyceae).

Ten strains of Paramecium bursaria and also P. caudatum, P. multimicronucleatum, P. tetraurelia strains (as outgroups) were characterized by using Random Amplified Polymorphic DNA (RAPD), Amplified Ribosomal DNA Restriction Analysis (ARDRA) and sequencing of the non-coding ribosomal internal transcribed spacer (ITS) regions. RAPD analysis revealed that all Paramecium bursaria strains possessed characteristic band patterns; there was a correlation between the degree of differentiation of DNA revealed by RAPD-fingerprinting and the geographic origin of a particular strain. ARDRA riboprinting (using a fragment of SSU-LSU rDNA, about 3085 bp) with restriction enzymes DraI, EcoRV, HhaI, HindIII, MspI, PstI distinguished groups of P. bursaria strains with characteristic band patterns originating from different sites. Comparison of the 550 bp ITS 1-5.8S-ITS2 fragment showed differentiation (0.9%) of the P. bursaria strains as three main groups of strains connected by site of origin in the constructed tree.

We assessed the photoprotective role of symbiotic Chlorella in the ciliate Paramecium bursaria by comparing their sensitivity to UV radiation (UVR) with Chlorella-reduced and Chlorella-free (aposymbiotic) cell lines of the same species. Aposymbiotic P. bursaria had significantly higher mortality than the symbiotic cell lines when exposed to UVR. To elucidate the protection mechanism, we assessed the algal distribution within the ciliate using thin-sections and transmission electron microscopy and estimated the screening factor by Chlorella based on an optical model. These analyses evidenced a substantial screening factor ranging, from 59.2% to 93.2% (320nm) for regular algal distribution. This screening efficiency reached up to approximately 100% when Chlorella algae were dislocated to the posterior region of the ciliate. The dislocation was observed in symbiotic ciliates only under exposure to UV plus photosynthetically active radiation (PAR) or to high PAR levels. Moreover, under exposure to UVB radiation and high PAR, symbiotic P. bursaria aggregated into dense spots. This behavior could represent an efficient avoidance strategy not yet described for ciliates. Analyses of the intact symbiosis and their algal symbionts for UV-screening compounds (mycosporine-like amino acids and sporopollenin) proved negative. Overall, our results show that photoprotection in this ciliate symbiosis represents an additional advantage to the hitherto postulated nutritional benefits.

Little is known about the relationship between fire regimes and plant diversity in Australia's temperate grassy woodlands. The effect of fire frequency on shrubs in grassy woodland remnants across Western Sydney's Cumberland Plain was examined. Shrub species richness and composition were compared in sites that had experienced a high, moderate or low frequency of fire over the previous 20 years. Nine sites were surveyed, three in each fire frequency category; most sites, including all low-fire-frequency sites, had burnt 9–36 months prior to sampling. Fire frequency had a profound effect on the composition and structure of the shrub layer. Per cent frequency and density of the prickly shrub Bursaria spinosa (Pittosporaceae) was considerably higher in low-fire-frequency sites than where fires had occurred at least once a decade. In sites where fire had been absent for decades prior to a recent fire, this species dominated the landscape, while elsewhere it occurred as clumps in a grassy matrix. Per cent frequency of other native shrubs, particularly obligate seeders, was greatest at moderate fire frequencies. Exotic shrubs were recorded most often where fire had been rare. While ordination clearly separated out the low-fire-frequency sites, complete separation between high- and moderate-fire-frequency blocks was not achieved. The increase in Bursaria in the absence of fire mirrors the encroachment of woody plants into a range of grassy ecosystems around the world. The sensitivity of obligate seeder species, many of them short-lived legumes with fire-cued seeds, to both very frequent and very infrequent fire shows the vulnerability of these species to extreme fire regimes, despite the safeguards conferred by hard-seededness. Competition from Bursaria, as well as loss of viable seed in the soil, may have contributed to the low frequency of these species after a long inter-fire interval.

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.

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.

Summary 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.

Paramecium bursaria is composed of a "host" ciliate and a "symbiont" green alga. Based upon physiology, DNA hybridization and virus infection, two types of symbionts, called "American" type and "European" type, have been reported to date. Here, we determined the 18S rDNA and internal transcribed spacer 2 (ITS2) regions for both "American" and "European" types. Sequence features clearly separated into two lineages; NC64A (USA), Syngen 2-3 (USA), Cs2 (Chinese), MRBG1 (Australian), and Japanese strains belong to the "American", whereas PB-SW1 (German) and CCAP 1660/11 (British) strains belong to the "European". In "American" 18S rDNA, three introns were inserted in the same positions as for previously described Japanese symbionts. In "European" 18S rDNA, a single intron occurred in a different position than in the "American". Between the types, sequence differences were seven or eight nucleotides (0.39 %) in the 18S rDNA exon, and more than 48 nucleotides (19.2 %) in ITS2 regions. We subsequently sequenced the host 18S rDNA. As a result, two groups: Cs2, MRBG1, and Japanese strains, and PB-SW1 and CCAP 1660/11 strains, were separated (with 23 substitutions and 4 insertions or deletions between the groups). The congruent separations between hosts and symbionts may imply that the type of symbiont depends on the host type.

Cells of the green paramecium, Paramecium bursaria, contain several hundred endosymbiont cells. The properties of the symbionts are considered to vary depending on the collection site of the host. Difficulties in achieving axenic cells and maintenance of axenic strains for long periods have been reported for symbiotic algae from P. bursaria isolated in Japan. To establish axenic algal strains from such Japanese P. bursaria, symbionts were isolated carefully, and isolated axenic strains were grown on an agar medium containing organic nitrogen compounds. Symbiotic algal strains were obtained from three Japanese P. bursaria strains and their axenicity was confirmed by DAPI staining, cultural tests of bacterial contamination, and DGGE-PCR. These axenic strains have been maintained for over 2 years. Utilization of carbohydrates and nitrogen compounds by symbionts was examined. Monosaccharides (glucose and fructose) increased the growth of the symbiont but disaccharides (maltose and sucrose) did not. Japanese axenic symbionts were able to use ammonia and amino acids, but not nitrate or nitrite. While potent nitrite reductase activity was stimulated by nitrate induction, nitrate reductase activity was not. Nitrate utilization of Japanese symbionts differed from that reported for European and American symbionts.

The effects of hexavalent chromium on the cell growth of green paramecium, Paramecium bursaria (P. bursaria), were investigated in this study. Two strains (MB-1 and F1 generation) of P. bursaria were incubated in lettuce media supplemented with different concentrations of potassium dichromate under LL (24 hr light), LD (12 hr light : 12 hr dark) and DD (24 hr dark) conditions, and the IC50 values were obtained. The IC50 7-day value showed that the toxicity of potassium dichromate was light-sensitive in both strains of P. bursaria. The results of the toxic effect of chromium on the cell shape of P. bursaria (BWK-4) showed that the body ratio of P. bursaria increased, even if the cells were incubated for 24 hr with 0.5 µM potassium dichromate solution, indicating that the cell shape of P. bursaria is very sensitive to potassium dichromate. The average amount of chromium accumulated in green paramecium ranged from 1.72 to 15.5 pg Cr/cell in a time- and concentration-dependent manner. This finding indicates the possibility of the use of P. bursaria as a biomonitor and bioaccumulator for chromium contaminants in aquatic environments. The experiment with electrical stimuli into the culture of P. bursaria indicated that cells accumulated at the negative electrode, suggesting that P. bursaria carried a positive charge. Thus, because the positive charge does not cause any significant absorption of chromium into the cells, P. bursaria may develop a variety of mechanisms for chromium accumulation in the cell. Further research studies are required to elucidate the mechanism of chromium uptake in P. bursaria.

The triple symbiotic system (TSS): P. bursaria-Chlorella-Chlorovirus, was studied. In Eurasia we found only 2 forms TSS, named N, northern and S, southern ecotypes. Each ecotype manifested at 32°C ts (N)-or tr (S)-phenotypes. In northeren parts of P. bursaria areals, from Karelia up to Kamchatka, near Baikal and in Armenia highlands, we find only ts-viruses, in Central Asia - only tr-types. Two types of genome characters were shown by PCR of 18 S RNA-genes. According all this characters populations of zoochlorella in P. bursaria - 2 clones of obligate symbionts

Endosymbiotic green algae of Japanese Paramecium bursaria were phylogenetically analyzed based on DNA sequences from the ribosomal DNA operon (18S rDNA, ITS1, 5.8S rDNA, and ITS2). Phylogenetic trees constructed using 18S rDNA sequences showed that the symbionts belong to the Chlorella sensu stricto (Trebouxiophyceae) group. They are genetically closer to the C. vulgaris Beijerinck group than to C. kessleri Fott et Nováková as proposed previously. Branching order in C. vulgaris group was unresolved in 18S rDNA trees. Compared heterogeneities of 18S rDNA, ITS1, 5.8S r, and ITS2 among symbionts and two Chlorella species, indicated that the ITS2 region (and probably also ITS1) is better able to resolve phylogenetic problems in such closely related taxa. All six symbiotic sequences obtained here (approximately 4000-bp sequences of 18S rDNA, ITS1, 5.8S rDNA, and ITS2) were completely identical in each, strongly suggesting a common origin.

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.

Paramecium bursaria contains hundreds of algae in its cytoplasm as symbionts and retains them over many generations. Recently, aposymbiotic cells of P. bursaria were collected from a pond. To know whether the natural aposymbiotic strain (Ysa2) lost ability to associate with algae, infection experiments were performed using symbiotic algae isolated from green paramecia. Results showed that Ysa2 establish symbiotic association in the same way as artificially obtained aposymbiotic cells (control). However, clustered algae appeared in some Ysa2 cells after infection. Algal clusters differ in number and size from cell to cell. Isolation line culture was performed to pursue the fate of cells having algal clusters. Within several cell divisions, some descendants retained large clusters and such cells were sometimes misshapen, ceasing proliferation and eventually dying. At cell division of cluster-bearing cells, unequal distribution of clustered algae occurred because many algal clusters tend to locate in the posterior of the host cell. Most extremely, all algae were distributed to the posterior daughter cell, whereas the anterior daughter cell contained no algae. This is one possible mechanism of production of Ysa2 in nature. From these results, we conclude that Ysa2 can make symbiotic association with potentially symbiotic algae, but they have some incompatibilities with the algae.

Along with algae as producers in ecosystems and industrial applications, some microalgae existing in special ecological niches through endosymbiosis with other organisms represent fascinating examples of biological evolution. Although reproducing endosymbiosis experimentally is difficult in many situations, endosymbiosis of several ongoing types is possible. Endosymbiosis in Paramecium bursaria is a particularly excellent model. Although many studies of P. bursaria have specifically examined infection processes such as the host recognition of symbionts, coordination of host-symbiont division, which has been explored for eukaryotic organelles, is worth pursuing. Evaluating the cell (life) cycle of algae is crucially important for algal applications. Flow cytometry (FCM) has been used to study cell cycles of several eukaryotic cells including microalgae. Microscopy, however, has been used mainly to study endosymbiosis, as with P. bursaria, because of their larger size than suitable cells for FCM with hydrodynamic focusing. Vast amounts of time have been expended for microscopic analysis. This review presents an approach using capillary FCM to elucidate the endosymbiosis of P. bursaria. Results reveal that endosymbiotic algae of P. bursaria finely adjust their cell cycle schedule with their comfortable host and show that a coincident endosymbiont–host life cycle is virtually assured in their endosymbiosis.