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.     

A two-locus molecular characterization of Paramecium calkinsi

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 (1100bp) and COI (620bp) 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.     

ARDRA and RAPD-fingerprinting reject the sibling species concept for the ciliate Paramecium caudatum (Ciliophora, Protoctista)

Morphologically indistinguishable sibling species also known as syngens are a characteristic taxonomic feature of the ciliate genus Paramecium . This has been convincingly demonstrated for the P. aurelia species complex. For a long time this feature has also been assumed for P. caudatum . Classical morphology based techniques of taxonomic analysis are often inefficient to study sibling specie. We therefore investigated 14 P. caudatum strains of seven supposedly different syngens using random amplified polymorphic DNA (RAPD)-fingerprinting and amplified ribosomal DNA restriction analyses (ARDRA, Riboprinting). The RAPD patterns revealed by five different random primers were similar between the different strains of the same syngen (similarity index ranging from 73 to 91%) and also between strains of supposedly different syngens (similarity index ranging from 67 to 91%). The amplified 18S rRNA-fragments of supposedly different syngens, as well as the restriction patterns of these fragments digested by five different endonucleases, were identical for all investigated P. caudatum stains. Consequently we reject the sibling species hypothesis for P. caudatum . According to our molecular analysis, P. caudatum is not a species complex, but just one single species.     

Comparison of the evolutionary distances among syngens and sibling species of Paramecium

The morphospecies of the genus Paramecium have several mating type groups, so-called syngens, composed of cells of complementary mating types. The Paramecium aurelia complex is composed of 15 sibling species assigned to the species from the syngen. To increase our understanding of the evolutionary relationships among syngen and sibling species of the genus Paramecium, we investigated the gene sequences of cytosol-type hsp70 from 7 syngens of Paramecium caudatum and 15 sibling species of P. aurelia. Molecular phylogenetic trees indicated that the P. aurelia complex could be divided into four lineages and separated into each sibling species. However, we did not find any obvious genetic distance among syngens of P. caudatum, and they could only be separated into two closely related groups. These results indicated that the concept of syngens in P. caudatum differs quite markedly from that of the P. aurelia complex. In addition, we also discuss the relationships among these species and other species, Paramecium jenningsi and Paramecium multimicronucleatum, which were once classified as varieties of P. aurelia.     

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.     

Initial steps of infection of the ciliate Paramecium with bacteria Holospora sp

New light and electron microscope data on the initial steps of endocytobiosis establishment between the ciliate Paramecium and specific intranuclear bacteria Holospora are provided. At the cytoplasmic step of infection bacteria of all Holospora species are found in a vesicle originating from the membrane of the host cell phagosome. The association between host cell microfilaments and the bacterium bearing vesicle may suggest a possible involvement of the ciliate cytoskeleton in the transportation of bacteria to the host cell nucleus. The authors subdivide the process of infection into 6 steps. Some strains of P. caudatum never take up infectious Holospora bacteria in the course of phagocytosis.     

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.     

Parasitic infection reduces dispersal of ciliate host

Parasitic infection can modify host mobility and consequently their dispersal capacity. We experimentally investigated this idea using the ciliate Paramecium caudatum and its bacterial parasite Holospora undulata. We compared the short-distance dispersal of infected and uninfected populations in interconnected microcosms. Infection reduced the proportion of hosts dispersing, with levels differing among host clones. Host populations with higher densities showed lower dispersal, possibly owing to social aggregation behaviour. Parasite isolates that depleted host populations most had the lowest impact on host dispersal. Parasite-induced modification of dispersal may have consequences for the spatial distribution of disease, host and parasite genetic population structure, and coevolution.     

Obtaining and characterization of “Holospora curviuscula” and Holospora obtusa, bacterial symbionts of the macronuclei of Paramecium bursaria and Paramecium caudatum

The symbionts of the macronuclei of Paramecium bursaria and P. caudatum, “Holospora curviuscula” 02AZ16 and H. obtusa 88Ti, respectively, were obtained and investigated. The 16S rDNA nucleotide sequences of “Holospora curviuscula” were obtained for the first time. The differences in 16S rDNA (3.4%) suggest their classification within the genus Holospora. Molecular phylogenetic analysis of the symbionts revealed that these intranuclear symbionts of the ciliates belonged to the order Rickettsiales, forming within a compact cluster of related species.     

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.     

Effects of antibiotics on the early infection process of a macronuclear endosymbiotic bacterium Holospora obtusa of Paramecium caudatum

We examined the effects of antibiotics involved in bacterial DNA, RNA and protein synthesis and host protein synthesis on the early infection process of the bacterium Holospora obtusa, a macronucleus-specific symbiont of the ciliate Paramecium caudatum. Infection of the host macronucleus by the bacterium was not inhibited by mitomycin C, rifampicin and chloramphenicol. However, ingestion of the bacterium into the host digestive vacuoles and escape of the bacterium from the vacuoles to the host cytoplasm were significantly arrested with emetine. The results suggest that newly synthesized host proteins play an important role in the early infection process.     

Phylogenetic relationships of the genus Paramecium inferred from small subunit rRNA gene sequences

The genus Paramecium includes species that are well known and very common in freshwater environments. Species of Paramecium are morphologically divided into two distinct groups: the "bursaria" subgroup (foot-shaped) and the "aurelia" subgroup (cigar-shaped). Their placement within the class Oligohymenophorea has been supported by the analysis of the small subunit rRNA gene sequence of P. tetraurelia. To confirm the stability of this placement and to resolve relationships within the genus, small subunit rRNA gene sequences of P. bursaria, P. calkinsi, P. duboscqui, P. jenningsi, P. nephridiatum, P. primaurelia, and P. polycaryum were determined and aligned. Trees constructed using distance-matrix, maximum-likelihood, and maximum-parsimony methods all depicted the genus as a monophyletic group, clustering with the other oligohymenophorean taxa. Within the Paramecium clade, P. bursaria branches basal to the other species, although the remaining species of the morphologically defined "bursaria" subgroup do not group with P. bursaria, nor do they form a monophyletic subgroup. However, the species of the "aurelia" subgroup are closely related and strongly supported as a monophyletic group.     

Paramecium caudatum acquires heat-shock resistance in ciliary movement by infection with the endonuclear symbiotic bacterium Holospora obtusa

Holospora obtusa is a macronucleus-specific bacterium of the ciliate Paramecium caudatum. Three types of P. caudatum cells (H. obtusa-free cells, cells bearing the reproductive form of H. obtusa and cells bearing the predominantly infectious form of H. obtusa) cultured at 25 degrees C were transferred to 4, 10, 25, 35 and 40 degrees C and their swimming velocities were measured by taking photomicrographs with two-second exposures. The H. obtusa-free cells almost ceased swimming at both 4 and 40 degrees C, while cells bearing the reproductive form and those bearing the predominantly infectious form actively swam even at these temperatures. These results show that the host cell can acquire heat-shock resistance when infected by H. obtusa in the macronucleus. This is the first evidence to show that the endonuclear symbiont Holospora contributes to maintain the ciliary movement of the host even at temperatures unsuitable for the host growth.     

Induction of Conjugation by Methyl Cellulose in Paramecium

ABSTRACT An improved method has been developed for the induction of selfing conjugation in Paramecium. Methyl cellulose induces selfing conjugation simply and efficiently in all species examined. Induction of conjugation by methyl cellulose was characterized in P. caudatum , where it occurred only in sexually mature, mating reactive cells. Conjugation produced sexual offspring and the time course of nuclear processes was substantially the same as that in natural conjugation between cells of complementary mating types. The method is useful for genetic studies in a wide variety of Paramecium species including P. caudatum, P. tetraurelia, P. multimicronucleatum and P. bursaria.     

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.     

Inter- and intrasyngenic variations of the hemoglobin components in Paramecium caudatum.

The ciliated protozoan Paramecium contains hemoglobin in heterogeneous monomeric forms. In particular, Paramecium caudatum is characterized by the presence of a major component called Hb10 and a basic component named bHb. We found that in P. caudatum both of these hemoglobin components show some variation according to stock. The types and distributions of these hemoglobin components were examined on 16 stocks in five different syngens and one stock in an unidentified syngen using high performance liquid chromatography. The results indicate that in a variety of stocks the major component, Hb10, was divided into three types, A, B or A + B, and that the basic hemoglobin component was composed of a combination of two or three variants out of four possible, i.e. bHb 1, bHb 2, bHb 3 and bHb 4. Neither the Hb10 types nor the bHb variants, however, could be used to distinguish syngen in P. caudatum, since all of the Hb10 types and bHb variants were widely distributed over syngens and identical profiles appeared to some stocks in different syngens.     

Cryopreservation of Holospora undulata, a bacterial parasite of the ciliate Paramecium caudatum

We investigated cryopreservation of horizontal transmission stages of Holospora undulata, a micronucleus-specific bacterial parasite of Paramecium caudatum. Unlike in previous studies on related Holospora species, protocols using glycerol as cryoprotectant failed entirely. In contrast, freezing with dimethyl sulfoxide (Me2SO) conserved infectiousness of nearly all replicate inocula, although infection success was considerably lower than that of fresh inocula. Infection probability was enhanced by increasing the Me2SO concentration from 5 to 10%, and by freezing at -196 degrees C rather than -80 degrees C. Prolonged storage of up to 3 months had no significant effect on the viability of the inocula.     

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.     

The endosymbiotic bacterium Holospora obtusa enhances heat-shock gene expression of the host Paramecium caudatum

The bacterium Holospora obtusa is a macronuclear-specific symbiont of the ciliate Paramecium caudatum. H. obtusa-bearing paramecia could survive even after the cells were quickly heated from 25 degrees C to 35 degrees C. To determine whether infection with H. obtusa confers heat shock resistance on its host, we isolated genes homologous to the heat shock protein genes hsp60 and hsp70 from P. caudatum. The deduced amino acid sequences of both cDNAs were highly homologous to hsp family sequences from other eukaryotes. Competitive PCR showed that H. obtusa-free paramecia expressed only trace amounts of hsp60 and hsp70 mRNA at 25 degrees C, but that expression of hsp70 was enhanced immediately after the cells were transferred to 35 degrees C. H. obtusa-bearing paramecia expressed high levels of hsp7O mRNA even at 25 degrees C and the level was further enhanced when the cells were incubated at 35 degrees C. In contrast, the expression pattern of hsp60 mRNA was the same in H. obtusa-bearing as in H. obtusa-free paramecia. These results indicate that infection with its endosymbiont can confer a heat-shock resistant nature on its host cells.     

Inter- and Intrasyngenic Variations of the Hemoglobin Components in Paramecium caudatum

ABSTRACT The ciliated protozoan Paramecium contains hemoglobin in heterogeneous monomeric forms. In particular, Paramecium caudatum is characterized by the presence of a major component called Hb10 and a basic component named bHb. We found that in P. caudatum both of these hemoglobin components show some variation according to stock. The types and distributions of these hemoglobin components were examined on 16 stocks in five different syngens and one stock in an unidentified syngen using high performance liquid chromatography. The results indicate that in a variety of stocks the major component, Hb10, was divided into three types, A, B or A + B, and that the basic hemoglobin component was composed of a combination of two or three variants out of four possible, i.e. bHb 1, bHb 2, bHb 3 and bHb 4. Neither the Hb10 types nor the bHb variants, however, could be used to distinguish syngen in P. caudatum , since all of the Hb10 types and bHb variants were widely distributed over syngens and identical profiles appeared to some stocks in different syngens.