Genetic basis for the establishment of endosymbiosis in Paramecium.

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.

Rare Freshwater Ciliate Paramecium chlorelligerum Kahl, 1935 and Its Macronuclear Symbiotic Bacterium "Candidatus Holospora parva".

Ciliated protists often form symbioses with many diverse microorganisms. In particular, symbiotic associations between ciliates and green algae, as well as between ciliates and intracellular bacteria, are rather wide-spread in nature. In this study, we describe the complex symbiotic system between a very rare ciliate, Paramecium chlorelligerum, unicellular algae inhabiting its cytoplasm, and novel bacteria colonizing the host macronucleus. Paramecium chlorelligerum, previously found only twice in Germany, was retrieved from a novel location in vicinity of St. Petersburg in Russia. Species identification was based on both classical morphological methods and analysis of the small subunit rDNA. Numerous algae occupying the cytoplasm of this ciliate were identified with ultrastructural and molecular methods as representatives of the Meyerella genus, which before was not considered among symbiotic algae. In the same locality at least fifteen other species of "green" ciliates were found, thus it is indeed a biodiversity hot-spot for such protists. A novel species of bacterial symbionts living in the macronucleus of Paramecium chlorelligerum cells was morphologically and ultrastructurally investigated in detail with the description of its life cycle and infection capabilities. The new endosymbiont was molecularly characterized following the full-cycle rRNA approach. Furthermore, phylogenetic analysis confirmed that the novel bacterium is a member of Holospora genus branching basally but sharing all characteristics of the genus except inducing connecting piece formation during the infected host nucleus division. We propose the name "Candidatus Holospora parva" for this newly described species. The described complex system raises new questions on how these microorganisms evolve and interact in symbiosis.

ParTIES: a toolbox for Paramecium interspersed DNA elimination studies.

MOTIVATION: Developmental DNA elimination occurs in a wide variety of multicellular organisms, but ciliates are the only single-celled eukaryotes in which this phenomenon has been reported. Despite considerable interest in ciliates as models for DNA elimination, no standard methods for identification and characterization of the eliminated sequences are currently available. RESULTS: We present the Paramecium Toolbox for Interspersed DNA Elimination Studies (ParTIES), designed for Paramecium species, that (i) identifies eliminated sequences, (ii) measures their presence in a sequencing sample and (iii) detects rare elimination polymorphisms. AVAILABILITY AND IMPLEMENTATION: ParTIES is multi-threaded Perl software available at https://github.com/oarnaiz/ParTIES. ParTIES is distributed under the GNU General Public Licence v3.

Multiple lines of evidence shed light on the occurrence of paramecium (ciliophora, oligohymenophorea) in bromeliad tank water.

Phytotelmata are vegetal structures that hold water from the rain, and organic matter from the forest and the soil, resulting in small, compartmentalized bodies of water, which provide an essential environment for the establishment and development of many organisms. These microenvironments generally harbor endemic species, but many organisms that are found in lakes and rivers, are also present. Here, we report, for the first time, the occurrence of the ciliate genus Paramecium in the tank of the bromeliad species Aechmaea distichantha. The identification of the Paramecium species was performed based on live observations, protargol impregnation, scanning electronic microscopy, and sequencing of the 18s rRNA. The absence of Paramecium from bromeliad tank water was highlighted in several earlier investigations, and may be due to the fact that this species is unable to make cysts. The occurrence of Paramecium multimicronucleatum in our samples may be explained by the proximity between the bromeliads and the river, a potential source of the species. Further, we also believe that the counting methodology used in our study provides a more accurate analysis of the species diversity, since we investigated all samples within a maximum period of 6 h after sampling, allowing minimum loss of specimens.

Secondary structural analyses of ITS1 in Paramecium.

The nuclear ribosomal RNA gene operon is interrupted by internal transcribed spacer (ITS) 1 and ITS2. Although the secondary structure of ITS2 has been widely investigated, less is known about ITS1 and its structure. In this study, the secondary structure of ITS1 sequences for Paramecium and other ciliates was predicted. Each Paramecium ITS1 forms an open loop with three helices, A through C. Helix B was highly conserved among Paramecium, and similar helices were found in other ciliates. A phylogenetic analysis using the ITS1 sequences showed high-resolution, implying that ITS1 is a good tool for species-level analyses.

First American stand of Paramecium novaurelia and intra-specific differentiation of the species.

A stand of Paramecium novaurelia was found in Boston Massachusetts, USA, the first on this continent. Molecular studies (RAPD and sequencing ofrRNA [3'SSUrRNA-ITS1, 5' LSU rRNA] and COI mtDNA fragments) of P. novaurelia strains show intra-specific polymorphism within the species as strain clusters characterized by variable relationships.

Putative ancient microorganisms from amber nuggets.

Evolutionary microbiology studies based on the isolation of ancient DNA and/or microbial samples are scarce due to the difficulty of finding well preserved biological specimens. However, amber is a fossil resin with natural preserving properties for microbial cells and DNA. The visualization by transmission electron microscopy of different microorganism-like specimens found in amber nuggets from both the Miocene and the Cretaceous periods was accompanied by studies of ancient DNA obtained from the nuggets. After the design of specific primers based on the present sequences of both genes in Saccharomyces cerevisiae, the ancestral AGP2 sequence from the Miocene, as well as the 18S rRNA from the Cretaceous, were amplified.

Occurrence of Paramecium species in Western Siberia, Russia.

Paramecium strains collected in Central Russia, Western Siberia (the West Siberian Lowland and the Altai Mountains in the south) were studied. The presence of P. caudatum, P. bursaria, P. multimicronucleatum, P. polycaryum, and four species of the P. aurelia complex, i.e. P. primaurelia (in Omsk), P. biaurelia (in Krasnoyarsk and the Altai Mountains), P. triaurelia (in Krasnoyarsk), and P. pentaurelia (in Novosibirsk, Altai Foreland, and Altai Mountains) was revealed. P. triaurelia and P. pentaurelia were found for the first time in Asia.

Phylogenetic position of endosymbiotic green algae in Paramecium bursaria Ehrenberg from Japan.

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.

Paramecium novaurelia in the Gorgany Mts (Eastern Carpathians) in Ukraine.

In the samples collected on the territory of the recently studied region of the Eastern Carpathians, i.e. in the Gorgany Mts, the presence of P. novaurelia was recorded in the samples collected from the bog situated on the Jablonica Pass at an altitude of 100 m. This is the first record of the presence of P. novaurelia in the Gorgany Mts.

Paramecium tredecaurelia of the Paramecium aurelia complex in Israel.

The paper concerns the finding of a new habitat (Kiryat Motzkin, north of Haifa, Israel) of Paramecium tredecaurelia from the P. aurelia complex. This is only the forth known locality of the species in the world. Previously, its strains were obtained from widely separated localities: the River Seine, Paris, France; Benenitra, Madagascar, and the Cuernavaca Valley, Taxco, Mexico. The studied strain originating from Israel was identified as P. tredecaurelia on the basis of the strong (90%) conjugation between the complementary mating type of the examined clones with the appropriate standard strain 209 of P. tredecaurelia from Paris, France (restricted to odd mating type). However, the strain from Israel is restricted to the even mating type.

Paramecium aurelia species complex in a natural but newly reconstructed pond of the Botanical Garden of the Jagiellonian University in Kraków.

In a natural but newly reconstructed pond the presence of P. biaurelia was revealed. The previous studies conducted in 1992 did not reveal the occurrence of any species of the Paramecium aurelia complex here.

Locus-dependent profiles of the rescue of nonexcitable behavioral mutants during conjugation in Paramecium caudatum.

Non-excitable mutants of Paramecium caudatum, CNR (caudatum non-reversal) are unable to show the avoiding reaction due to the malfunction of voltage dependent Ca2+ channels. CNR mutants are divided into four complementation groups and are controlled by four genes at different loci: cnrA, cnrB, cnrC, and cnrD. When CNR mutants are mated with the wild type, the mutant cell temporarily expresses the wild-type phenotype (called conjugation rescue). Transfer of diffusible wild-type gene products to the mutant mate through cytoplasmic connections is thought to be the cause of this phenomenon. Excitability of mutant cells during conjugation with wild type or other mutants controlled by different loci, and immediately after pair separation was examined by K+ test solution. Restorations of excitability in CNR mutants show distinguishable characters depending upon the different loci. cnrC showed wild type character indistinguishable from wild-type cells soon after pair formation. cnrD showed clear conjugation rescue but never reached the level of wild-type cells. cnrA showed only slight conjugation rescue immediately after pair separation. cnrB never showed conjugation rescue.

Paramecium triaurelia and other species of the aurelia complex in the Kaczawskie Mts and Plateau (western Sudeten).

The present investigations revealed the occurrence of Paramecium triaurelia on the territory of the Kaczawskie Plateau, while its presence was confirmed in the Kaczawskie Mts. In Poland, the presence of this species was recorded only in the Western Sudeten, in spite of many years of investigation. As the result of the studies also the occurrence of P. biaurelia and P. novaurelia was recorded on the territory of the Kaczawskie Mts and the Kaczawskie Plateau.

Species of the Paramecium aurelia complex in Spain.

The new habitats of P. primaurelia, P. biaurelia, and P. triaurelia of the P. aurelia complex were described in Spain. P. pentaurelia was also found, for the first time in that country.

Studies on the distribution of the Paramecium aurelia species complex of Poland.

Species of the Paramecium aurelia complex occurred in the studied regions of the Carpathians and the Sudetes Mts with varying frequency. P. novaurelia appeared most often, P. biaurelia occurred less frequently but was also very characteristic, both species prevailed over P. primaurelia. P. tetraurelia and P. triaurelia were very rare.

Species of the Paramecium aurelia complex in the middle Sudeten of Czecho-Slovakia.

On the investigated territory three species of the complex were identified, namely Paramecium biaurelia, P. triaurelia, and P. novaurelia. P. novaurelia dominated over the other species with regard to the number of clones established from nature as well as to habitats.

Ciliates from a fresh water sulfuretum.

Ciliates were collected from a freshwater sulfuretum, Lake Cisó, which is part of a gypsum karstic area whose main feature is Lake Banyoles (Girona, Spain). Chromatium, Lamprocystis and Chlorobium are the major phototrophic sulfur bacteria in Lake Cisó. Blooms of a photosynthetic cryptomonad (up to 5 X 10(5) ind ml-1) were found at the metalimnion. The community of ciliates could be divided in three groups: aerobic, cosmopolitan, genera such as Stentor and Vorticella, in the epilimnion; a large population (up to 10(4) ind ml-1) of Coleps, adapted to low concentrations of both oxygen and sulfide, together with a few individuals of the equally sulfide-tolerant genus Paramecium, in the metalimnion, and anaerobic, true sulfide-loving genera such as Plagiopyla and Metopus, in the hypolimnion, where sulfide concentration was between 0.6 and 1.2 mM.