Paramecium Species Research Articles

Genome-defence small RNAs exapted for epigenetic mating-type inheritance.

In the ciliate Paramecium, transposable elements and their single-copy remnants are deleted during the development of somatic macronuclei from germline micronuclei, at each sexual generation. Deletions are targeted by scnRNAs, small RNAs produced from the germ line during meiosis that first scan the maternal macronuclear genome to identify missing sequences, and then allow the zygotic macronucleus to reproduce the same deletions. Here we show that this process accounts for the maternal inheritance of mating types in Paramecium tetraurelia, a long-standing problem in epigenetics. Mating type E depends on expression of the transmembrane protein mtA, and the default type O is determined during development by scnRNA-dependent excision of the mtA promoter. In the sibling species Paramecium septaurelia, mating type O is determined by coding-sequence deletions in a different gene, mtB, which is specifically required for mtA expression. These independently evolved mechanisms suggest frequent exaptation of the scnRNA pathway to regulate cellular genes and mediate transgenerational epigenetic inheritance of essential phenotypic polymorphisms.

Single Cell Traffic of Swimming Green Paramecia on Microchips with Micro-Flow Channels Fabricated by Micro-Casting

Members of Paramecium species are often referred to as “swimming neurons or sensory cells” applicable to micro-biorobotics or BioMEMS (biological micro-electro-mechanical systems). Paramecium bursaria known as green paramecia 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 living cells of green paramecia as micro-machines controllable under electrical, optical and magnetic signals, has a variety of engineering applications such as transportation of micro-sized particles (ingested within the cells) in the capillary systems. In the present study, we aimed to test if the swimming environment of green paramecia can be implementable on microchips. For this purpose, the series of microchips were prepared for cellular swimming platform for green paramecia through fabrication of poly(methyl methacrylate) master plates using the programmable micro-milling system followed by polydimethylsiloxane-based micro-casting. Finally, microchips equipped with optimally sized micro-flow channels for allowing the single cell traffic by swimming green paramecia were successfully prepared, and thus further studies for application of green paramecium cells in BioMEMS are encouraged.

Paramecium putrinum (Ciliophora, Protozoa): The first insight into the variation of two DNA fragments – Molecular support for the existence of cryptic species

Paramecium putrinum (Claparede & Lachmann 1858) is one of the smallest (80–140μm 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.

TheD1‐D2 region of the large subunit ribosomalDNAas barcode for ciliates

Ciliates are a major evolutionary lineage within the alveolates, which are distributed in nearly all habitats on our planet and are an essential component for ecosystem function, processes and stability. Accurate identification of these unicellular eukaryotes through, for example, microscopy or mating type reactions is reserved to few specialists. To satisfy the demand for a DNA barcode for ciliates, which meets the standard criteria for DNA barcodes defined by the Consortium for the Barcode of Life (CBOL), we here evaluated the D1-D2 region of the ribosomal DNA large subunit (LSU-rDNA). Primer universality for the phylum Ciliophora was tested in silico with available database sequences as well as in the laboratory with 73 ciliate species, which represented nine of 12 ciliate classes. Primers tested in this study were successful for all tested classes. To test the ability of the D1-D2 region to resolve conspecific and congeneric sequence divergence, 63 Paramecium strains were sampled from 24 mating species. The average conspecific D1-D2 variation was 0.18%, whereas congeneric sequence divergence averaged 4.83%. In pairwise genetic distance analyses, we identified a D1-D2 sequence divergence of <0.6% as an ideal threshold to discriminate Paramecium species. Using this definition, only 3.8% of all conspecific and 3.9% of all congeneric sequence comparisons had the potential of false assignments. Neighbour-joining analyses inferred monophyly for all taxa but for two Paramecium octaurelia strains. Here, we present a protocol for easy DNA amplification of single cells and voucher deposition. In conclusion, the presented data pinpoint the D1-D2 region as an excellent candidate for an official CBOL barcode for ciliated protists.

Genetic Differentiation of the Mitochondrial Cytochrome Oxidase c Subunit I Gene in Genus Paramecium (Protista, Ciliophora)

BackgroundThe mitochondrial cytochrome c oxidase subunit I (COI) gene is being used increasingly for evaluating inter- and intra-specific genetic diversity of ciliated protists. However, very few studies focus on assessing genetic divergence of the COI gene within individuals and how its presence might affect species identification and population structure analyses.Methodology/Principal findingsWe evaluated the genetic variation of the COI gene in five Paramecium species for a total of 147 clones derived from 21 individuals and 7 populations. We identified a total of 90 haplotypes with several individuals carrying more than one haplotype. Parsimony network and phylogenetic tree analyses revealed that intra-individual diversity had no effect in species identification and only a minor effect on population structure.ConclusionsOur results suggest that the COI gene is a suitable marker for resolving inter- and intra-specific relationships of Paramecium spp.

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.

Spliced DNA Sequences in the Paramecium Germline: Their Properties and Evolutionary Potential

Despite playing a crucial role in germline-soma differentiation, the evolutionary significance of developmentally regulated genome rearrangements (DRGRs) has received scant attention. An example of DRGR is DNA splicing, a process that removes segments of DNA interrupting genic and/or intergenic sequences. Perhaps, best known for shaping immune-system genes in vertebrates, DNA splicing plays a central role in the life of ciliated protozoa, where thousands of germline DNA segments are eliminated after sexual reproduction to regenerate a functional somatic genome. Here, we identify and chronicle the properties of 5,286 sequences that putatively undergo DNA splicing (i.e., internal eliminated sequences [IESs]) across the genomes of three closely related species of the ciliate Paramecium (P. tetraurelia, P. biaurelia, and P. sexaurelia). The study reveals that these putative IESs share several physical characteristics. Although our results are consistent with excision events being largely conserved between species, episodes of differential IES retention/excision occur, may have a recent origin, and frequently involve coding regions. Our findings indicate interconversion between somatic—often coding—DNA sequences and noncoding IESs, and provide insights into the role of DNA splicing in creating potentially functional genetic innovation.

A synthetic cadmium metallothionein gene (PMCd1syn) of Paramecium species: expression, purification and characteristics of metallothionein protein

Metallothioneins (MTs) are metal binding proteins that are rich in cysteine residues constituting 10-30% of the total protein, and in which the thiol groups bind to the metal ions. The increasing amount of metal ions in the medium have shown increased production of MTs by different organisms such as bacteria, protozoa and mammals like humans. PMCd1 is the first gene ever discovered in Paramecium, a ciliated protozoan, that could produce this MT in response to cadmium. In this study the PMCd1syn gene has been cloned in pET41a expression vector and expressed in an Escherichia coli BL21-codonplus strain for the first time. Since the gene PMCd1 amplified from Paramecium contained 10 codons, which could act as stop codons during expression in E. coli, this gene of 612 bps was synthesized to substitute these (stop) codons for the Paramecium sp. specific amino acids. For stability of the expressed protein, glutathione-S-transferase gene was fused with PMCd1syn gene and coexpressed. The cells expressing PMCd1syn demonstrated increased accumulation of cadmium. This is the first report of cadmium MT protein expressed from Paramecium species, particularly from synthetic MT gene (PMCd1syn). This fusion protein, the molecular weight of which has been confirmed to be 53.03kDa with MALDI analysis, is rich in cysteine residues, and has been shown for the first time in this ciliate to bind to and sequester Cd(2+)-ions.

Survey of Paramecium duboscqui using three markers and assessment of the molecular variability in the genus Paramecium

The genus Paramecium (phylum Ciliophora) is one of the best-known among protozoa. Nevertheless, the knowledge on the diversity and distribution of species within this genus was remarkably scarce until recent times. In the last years a constantly growing amount of data has formed, especially on the distribution of species and the characterization of molecular markers. Much effort has been made on detecting clades inside each morphospecies, which could suggest the presence of sibling species complexes as in the famous case of Paramecium aurelia. In this work we present new data on Paramecium duboscqui, one of the morphospecies that have not yet been surveyed employing DNA sequences as markers. We obtained data from nine strains sampled around the world, using the three most commonly employed markers (18S rRNA gene, ITS1-5.8S-ITS2 and COI gene sequences). Moreover, we compared our results with those already available for other Paramecium species, and performed phylogenetic analyses for the entire genus. We also expanded the knowledge on the ITS2 secondary structure and its usefulness in studies on Paramecium. Our approach, that considers the data of all the species together, highlighted some characteristic patterns as well as some ambiguities that should be further investigated.

Does high intraspecific variability of two genome fragments indicate a recent speciation process of Paramecium dodecaurelia (P. aurelia species complex, Ciliophora, Protozoa)?

Species of the Paramecium aurelia complex show different levels of intraspecific polymorphism, with P. dodecaurelia revealing a high level of intraspecific variation. Paramecium dodecaurelia strains originating from distant localities in the Palaearctic, North America (USA), and Oceania (Hawaii) were studied in terms of intraspecific differentiation and the degree of speciation. Sequences of genes encoding the ITS1–5.8S-ITS2–5’ end of LSU rDNA (1063–1097 bp) and cytochrome c oxidase subunit I mtDNA (638–644 bp) were obtained from 33 strains of P. dodecaurelia, other P. aurelia species, and another species of the genus Paramecium, with Tetrahymena sp. used as an outgroup. In phylograms, the majority of P. dodecaurelia strains from the Palaearctic appear in one cluster, while strains from Japan, Hawaii and the USA are grouped in another cluster, together with some strains from Italy and representatives of the P. aurelia species complex. Our results tend to support the hypothesis that P. dodecaurelia seems to be a polyphyletic species with several haplotypes similar to or even shared with other members of the P. aurelia species complex. However, it is still an open question whether the revealed intraspecific differences within P. dodecaurelia are the result of ongoing speciation, or perhaps they just indicate genetic differentiation within a species that has a wide geographic distribution.

Enhanced Microsphere Transport in Capillary by Conditioned Cells of Green Paramecia Used as Living Micromachines Controlled by Electric Stimuli

Some researchers have described the cells of Paramecium species as “swimming sensory cells” or “swimming neurons” applicable to micro-biorobotics and biological micro-electromechanical systems (BioMEMS). Paramecium species including green paramecia (Paramecium bursaria) migrate towards the anodic electrode when exposed to an electric field. This type of cellular movement is known as galvanotaxis. Because the ideal micromachines designed for microparticle transport must have a capacity for loading certain numbers of particles, P. bursaria was chosen as a model organism. In this study, we show enhanced microparticle transport by overcoming (i) the particle size limitation for the cell-mediated transport of microspheres of up to ca. 10 μm size (doubling the size of particles ever reported) and (ii) the limit of cellular migration distance manifested by galvanotactically stimulated cells.

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.

The impact of environmental variability and species composition on the stability of experimental microbial populations and communities

Understanding how environmental fluctuations affect the stability of populations and communities is complex, for example, because direct effects of environmental variability on populations may be modified and propagated across communities by species interactions. One way to explore and further understand these complexities is via a factorial manipulation of community composition and environmental conditions. Using laboratory based aquatic microcosms we manipulated environmental fluctuation by creating two environments; one with variable light and one with constant light. Within these environments, community composition was manipulated by constructing communities from all possible combinations of three species that vary in their reliance on light for growth (an autotroph: a diatom completely reliant on light, a heterotroph: a Paramecium species not reliant on light, and a mixotroph: a Paramecium species somewhat reliant on light). Community composition was predicted to affect populations and communities by introducing and altering competitive interactions between species and affecting the degree of niche differentiation between species. We found that population stability was predominantly influenced by an interaction between community composition and environmental variability, whereby the effect of environmental variability synergistically combined with effects of community composition to reduce population stability. Covariance of populations was determined by an interaction between community composition and environmental variability, though this did not result from the effect of niche differentiation between species. Species interactions drove correlations between population biomass and the environment which otherwise did not exist. Our results demonstrate the complex and interrelated effects of abiotic and biotic factors on population and community stability, and suggest the need to consider aspects of community composition when predicting the impact of environmental fluctuations.

ParameciumDB in 2011: new tools and new data for functional and comparative genomics of the model ciliate Paramecium tetraurelia

ParameciumDB is a community model organism database built with the GMOD toolkit to integrate the genome and biology of the ciliate Paramecium tetraurelia. Over the last four years, post-genomic data from proteome and transcriptome studies has been incorporated along with predicted orthologs in 33 species, annotations from the community and publications from the scientific literature. Available tools include BioMart for complex queries, GBrowse2 for genome browsing, the Apollo genome editor for expert curation of gene models, a Blast server, a motif finder, and a wiki for protocols, nomenclature guidelines and other documentation. In-house tools have been developed for ontology browsing and evaluation of off-target RNAi matches. Now ready for next-generation deep sequencing data and the genomes of other Paramecium species, this open-access resource is available at http://paramecium.cgm.cnrs-gif.fr.

“ Candidatus Paraholospora nucleivisitans”, an intracellular bacterium in Paramecium sexaurelia shuttles between the cytoplasm and the nucleus of its host

An intracellular bacterium was discovered in two isolates of Paramecium sexaurelia from an aquarium with tropical fish in Münster (Germany) and from a pond in the Wilhelma zoological–botanical garden, Stuttgart (Germany). The bacteria were regularly observed in the cytoplasm of the host, but on some occasions they were found in the macronucleus of the host cell. In these cases, only a few, if any, bacteria were observed remaining in the cytoplasm. The bacterium was not infectious to P. sexaurelia or other species of Paramecium and appeared to be an obligate intracellular bacterium, while bacteria-free host cells were completely viable. The fluorescence in situ hybridisation (FISH) and comparative 16SrDNA sequence analyses showed that the bacterium belonged to a new genus, and was most closely, yet quite distantly, related to Holospora obtusa. In spite of this relationship, the new bacteria differed from Holospora by at least two biological features. Whereas all Holospora species reside exclusively in the nuclei of various species of Paramecium and show a life cycle with a morphologically distinct infectious form, for the new bacterium no infectious form and no life cycle have been observed. For the new bacterium, the name Candidatus Paraholospora nucleivisitans is suggested. The host P. sexaurelia is usually known from tropical and subtropical areas and is not a species typically found in Germany and central Europe. Possibly, it had been taken to Germany with fish or plants from tropical or subtropical waters. Candidatus Paraholospora nucleivisitans may therefore be regarded as an intracellular neobacterium for Germany.

Molecular characteristics of an immobilization antigen gene of the fish-parasitic protozoanIchthyophthirius multifiliisstrain ARS-6

AbstractIchthyophthirius multi¢liis(Ich), a ciliated protozoanparasite of ¢sh, expresses surface antigens (i-anti-gens), which react with host antibodies that renderthemimmobile.Thenucleotidesequenceofani-anti-gen gene of I. multi¢liis strain ARS-6 was deduced.The predicted protein of 47493Da is comprised of460 amino acids (aa’s) arranged into ¢ve imperfectrepeats with periodic cysteine residues with thestructure: CX (19)20 CX 2 CX 16 27 CX 2 CX 20(21) CX 3 . TheN-terminal aa’s typify a signal peptide motif whilea stretch of C-terminal aa’s resemble a glycosyl^phosphatidyl^inositol (GPI)-anchor addition site.Thedegreeofdeducedi-antigenaasequenceidentityof strain ARS-6 (GenBank accession # ACH87654and # ACH95659) with other I. multi¢liis i-antigensequences present in GenBank ranges from 99% to36% identity with 52kDa i-antigens of I. multi¢liisstrain G5 (accession #s AAK94941and AAK01661respectively). Immunoblot analysis of i-antigens fol-lowing exposure of I. multi¢liis theronts to cat¢shanti-I. multi¢liis immune serum did not show anyappreciable alteration in i-antigen expression. Themechanism that regulates i-antigen expression inI.multi¢liisremainsapuzzlingquestion.Keywords: immobilization antigen, gene, nucleo-tidesequence,Ichthyophthiriusmulti¢liisIntroductionIchthyophthiriusmulti¢liis(Ich) is a hymenostomatidparasitic ciliate that causes ‘white spot’ disease inmany species of freshwater cultured and/or free liv-ing ¢sh. Fish that recover from I. multi¢liis infectionareknowntopossessbothserumandcutaneousmu-cus antibodies that are capable of immobilizing theparasite in vitro (Clark & Dickerson 1997). The anti-gens on the parasite surface that are involved in theantibody-mediatedimmobilizationarecomprisedofaclass of abundant glycosyl^phosphatidyl^inositol(GPI)-anchoredmembraneproteinsthatareaptlyde-signated as immobilization antigens (i-antigens)(Clark, Gao, Gaertig, Wang & Cheng 2001; Clark FHatanka,Umeda,YamashitaH Swennes, Findly DLee & Kim 2008).Tetrahymena and Paramecium spe-cies are free-living ciliated protists that have servedas model systems, in which i-antigens have been ex-tensively studied (Doerder 2000; Simon & Schmidt2005). Both Tetrahymena and Paramecium possess arepertoire of paralogous genes that encode alterna-tive forms of i-antigens, the expression of which isunder the in£uence of certain environmental cuessuch as temperature and medium, in which they

Micro-Particle Transporting System Using Galvanotactically Stimulated Apo-Symbiotic Cells of Paramecium bursaria

It is well known that Paramecium species including green paramecia (Paramecium bursaria) migrate towards the anode when exposed to an electric field in a medium. This type of a cellular movement is known as galvanotaxis. Our previous study revealed that an electric stimulus given to P bursaria is converted to a galvanotactic cellular movement by involvement of T-type calcium channel on the plasma membrane [Aonuma et al. (2007), Z. Naturforsch. 62c, 93-102]. This phenomenon has attracted the attention of bioengineers in the fields of biorobotics or micro-robotics in order to develop electrically controllable micromachineries. Here, we demonstrate the galvanotactic controls of the cellular migration of P bursaria in capillary tubes (diameter, 1-2 mm; length, 30-240 mm). Since the Paramecium cells take up particles of various sizes, we attempted to use the electrically stimulated cells of P bursaria as the vehicle for transportation of micro-particles in the capillary system. By using apo-symbiotic cells of P bursaria obtained after forced removal of symbiotic algae, the uptake of the particles could be maximized and visualized. Then, electrically controlled transportations of particle-filled apo-symbiotic P bursaria cells were manifested. The particles transported by electrically controlled cells (varying in size from nm to /m levels) included re-introduced green algae, fluorescence-labeled polystyrene beads, magnetic microspheres, emerald green fluorescent protein (EmGFP)-labeled cells of E. coli, Indian ink, and crystals of zeolite (hydrated aluminosilicate minerals with a micro-porous structure) and some metal oxides. Since the above demonstrations were successful, we concluded that P bursaria has a potential to be employed as one of the micro-biorobotic devices used in BioMEMS (biological micro-electro-mechanical systems).

Comparative description of macronuclear electrophoretic karyotypes of Paramecium primaurelia and Paramecium novaurelia sibling species

The macronuclear genomes of two sibling species belonging to the Paramecium aurelia complex--P. primaurelia and P. novaurelia--were analyzed by pulsed-field gel electrophoresis (PFGE). Their electrophoretic karyotypes showed a continuous spectrum of different-sized DNA molecules with a species-specific pattern of banding, which was reproducible and did not change with strain senescence. Thus, P. aurelia macronuclear PFGE profiles could be described by an approach analogous to that used for the description of metaphase chromosome banding patterns. At first, well-identifiable regions (size fractions) of a PFGE profile, which can be seen at any resolution, are determined. Then, the bands of the second order of resolution (subfractions) can be defined in some of these regions. The blocks of the first and second orders can be utilized as inner markers of the PFGE profile allowing precise comparison of different PFGE profiles. Such comparative analysis has demonstrated some marking differences in organization of the macronuclear genomes of P. primaurelia and P. novaurelia, and low level of intraspecific polymorphism. Worth noting is that the P. novaurelia strain isolated in USA was different from all other analyzed P. novaurelia strains originating from Europe. The nature of banding of P. aurelia PFGE profiles is discussed. The revealed high order and stability of the macronuclear genome organization makes possible searching for new approaches to study the mechanisms of this non-trivial genome formation and maintenance. Further PFGE analysis of the macronuclear genomes of the other Paramecium species in relation with the Paramecium phylogeny may provide insights into the directions of the evolution of the macronuclear genome in Ciliata.

Genetic Diversity in the Paramecium aurelia Species Complex

Current understanding of the population genetics of free-living unicellular eukaryotes is limited, and the amount of genetic variability in these organisms is still a matter of debate. We characterized-reproductively and genetically-worldwide samples of multiple Paramecium species belonging to a cryptic species complex, Paramecium aurelia, whose species have been shown to be reproductively isolated. We found that levels of genetic diversity both in the nucleus and in the mitochondrion are substantial within groups of reproductively compatible P. aurelia strains but drop considerably when strains are partitioned according to their phylogenetic groupings. Our study reveals the existence of discrepancies between the mating behavior of a number of P. aurelia strains and their multilocus genetic profile, a controversial finding that has major consequences for both the current methods of species assignment and the species problem in the P. aurelia complex.

Paramecium Species of the Upper and Lower Volga River Basin, Russia

The Volga, which is the largest river in Europe (3690 km long), flows from the north (Tver' region) to the south (Caspian Sea), and its extensive basin (1380 km2) includes very different biotopes. Thus, analysis ofthe occurrence of Paramecium species along this large river basin may significantly enhance our understanding of species distribution according to temperature regime, food richness and other possible factors. The present paper concerns the occurrence of species of the P. aurelia complex in the sampling areas of the Upper Volga River, and a comparison with the occurrence of species of the P. aurelia complex in the Lower Volga region. In the Upper Volga basin, P. biaurelia was the most abundant among species of the complex recorded (among P. triaurelia, P. decaurelia, P. dodecaurelia), in the Lower Volga region eight species of the complex were recorded (P. primaurelia, P. biaurelia, P. triaurelia, P. pentaurelia, P. sexaurelia, P. septaurelia, P. novaurelia, P. decaurelia).