Paramecium (Oligohymenophorea, Ciliophora) diversity in Thailand sheds light on the genus biogeography and reveals new phylogenetic lineages.

Paramecium (Ciliophora, Oligohymenophorea) is a good model to study ciliate biogeography. Extensive sampling mainly in northern hemisphere has led to 16 valid morphological species description thus far. However, a majority of hard-to-reach regions, including South East Asia, are underinvestigated. Our study combined traditional morphological and molecular approaches to reveal the biodiversity of Paramecium in Thailand from more than 110 samples collected in 10 provinces. Representatives of seven morphological species were identified from our collection, including the rare species, such as P. gigas and P. jenningsi. Additionally, we detected five different sibling species of the P. aurelia complex, described a new cryptic species P. hiwatashii n. sp. phylogenetically related to P. caudatum, and discovered a potentially new genetic species of the P. bursaria species complex. We also documented a variety of bacterial cytoplasmic symbionts from at least nine monoclonal cultures of Paramecium.

Structural maintenance of chromosomes (SMC) proteins are required for DNA elimination in Paramecium.

Chromosome (SMC) proteins are a large family of ATPases that play important roles in the organization and dynamics of chromatin. They are central regulators of chromosome dynamics and the core component of condensin. DNA elimination during zygotic somatic genome development is a characteristic feature of ciliated protozoa such as Paramecium This process occurs after meiosis, mitosis, karyogamy, and another mitosis, which result in the formation of a new germline and somatic nuclei. The series of nuclear divisions implies an important role of SMC proteins in Paramecium sexual development. The relationship between DNA elimination and SMC has not yet been described. Here, we applied RNA interference, genome sequencing, mRNA sequencing, immunofluorescence, and mass spectrometry to investigate the roles of SMC components in DNA elimination. Our results show that SMC4-2 is required for genome rearrangement, whereas SMC4-1 is not. Functional diversification of SMC4 in Paramecium led to a formation of two paralogues where SMC4-2 acquired a novel, development-specific function and differs from SMC4-1. Moreover, our study suggests a competitive relationship between these two proteins.

Paramecium Genetics, Genomics, and Evolution.

The ciliate genus Paramecium served as one of the first model systems in microbial eukaryotic genetics, contributing much to the early understanding of phenomena as diverse as genome rearrangement, cryptic speciation, cytoplasmic inheritance, and endosymbiosis, as well as more recently to the evolution of mating types, introns, and roles of small RNAs in DNA processing. Substantial progress has recently been made in the area of comparative and population genomics. Paramecium species combine some of the lowest known mutation rates with some of the largest known effective populations, along with likely very high recombination rates, thereby harboring a population-genetic environment that promotes an exceptionally efficient capacity for selection. As a consequence, the genomes are extraordinarily streamlined, with very small intergenic regions combined with small numbers of tiny introns. The subject of the bulk of Paramecium research, the ancient Paramecium aurelia species complex, is descended from two whole-genome duplication events that retain high degrees of synteny, thereby providing an exceptional platform for studying the fates of duplicate genes. Despite having a common ancestor dating to several hundred million years ago, the known descendant species are morphologically indistinguishable, raising significant questions about the common view that gene duplications lead to the origins of evolutionary novelties.

Dynamics of Gene Loss following Ancient Whole-Genome Duplication in the Cryptic Paramecium Complex.

Whole-genome duplications (WGDs) have shaped the gene repertoire of many eukaryotic lineages. The redundancy created by WGDs typically results in a phase of massive gene loss. However, some WGD-derived paralogs are maintained over long evolutionary periods, and the relative contributions of different selective pressures to their maintenance are still debated. Previous studies have revealed a history of three successive WGDs in the lineage of the ciliate Paramecium tetraurelia and two of its sister species from the Paramecium aurelia complex. Here, we report the genome sequence and analysis of 10 additional P. aurelia species and 1 additional out group, revealing aspects of post-WGD evolution in 13 species sharing a common ancestral WGD. Contrary to the morphological radiation of vertebrates that putatively followed two WGD events, members of the cryptic P. aurelia complex have remained morphologically indistinguishable after hundreds of millions of years. Biases in gene retention compatible with dosage constraints appear to play a major role opposing post-WGD gene loss across all 13 species. In addition, post-WGD gene loss has been slower in Paramecium than in other species having experienced genome duplication, suggesting that the selective pressures against post-WGD gene loss are especially strong in Paramecium. A near complete lack of recent single-gene duplications in Paramecium provides additional evidence for strong selective pressures against gene dosage changes. This exceptional data set of 13 species sharing an ancestral WGD and 2 closely related out group species will be a useful resource for future studies on Paramecium as a major model organism in the evolutionary cell biology.

Comparative transcriptome and antioxidant biomarker response reveal molecular mechanisms to cope with zinc ion exposure in the unicellular eukaryote Paramecium.

The development of industry has resulted in excessive environmental zinc exposure which has caused various health problems in a wide range of organisms including humans. The mechanisms by which aquatic microorganisms respond to environmental zinc stress are still poorly understood. Paramecium, a well-known ciliated protozoan and a popular cell model in heavy metal stress response studies, was chosen as the test unicellular eukaryotic organism in the present research. In this work, Paramecium cf. multimicronucleatum cells were exposed in different levels of zinc ion (0.1 and 1.0 mg/L) for different periods of exposure (1 and 4 days), and then analyzed population growth, transcriptomic profiles and physiological changes in antioxidant enzymes to explore the toxicity and detoxification mechanisms during the zinc stress response. Results demonstrated that long-term zinc exposure could have restrained population growth in ciliates, however, the response mechanism to zinc exposure in ciliates is likely to show a dosage-dependent and time-dependent manner. The differentially expressed genes (DEGs) were identified the characters by high-throughput sequencing, which remarkably enriched in the phagosome, indicating that the phagosome pathway might mediate the uptake of zinc, while the pathways of ABC transporters and Na+/K+-transporting ATPase contributed to the efflux transport of excessive zinc ions and the maintenance of osmotic balance, respectively. The accumulation of zinc ions triggered a series of adverse effects, including damage to DNA and proteins, disturbance of mitochondrial function, and oxidative stress. In addition, we found that gene expression changed significantly for metal ion binding, energy metabolism, and oxidation-reduction processes. RT-qPCR of ten genes involved in important biological functions further validated the results of the transcriptome analysis. We also continuously monitored changes in activity of four antioxidant enzymes (SOD, CAT, POD and GSH-PX), all of which peaked on day 4 in cells subjected to zinc stress. Collectively, our results indicate that excessive environmental zinc exposure initially causes damage to cellular structure and function and then initiates detoxification mechanisms to maintain homeostasis in P. cf. multimicronucleatum cells.

Single-cell transcriptome reveals cell division-regulated hub genes in the unicellular eukaryote Paramecium.

The transition from growth to division during the cell cycle encompasses numerous conserved processes such as large-scale DNA replication and protein synthesis. In ciliate cells, asexual cell division is accompanied by additional cellular changes including amitotic nuclear division, extensive ciliogenesis, and trichocyst replication. However, the molecular mechanisms underlying these processes remain elusive. In this study, we present single-cell gene expression profiles of Paramecium cf. multimicronucleatum cells undergoing cell division. Our results reveal that the most up-regulated genes in dividing cells compared to growing cells are associated with 1) cell cycle signaling pathways including transcription, DNA replication, chromosome segregation and protein degradation; 2) microtubule proteins and tubulin glycylases which are essential for ciliogenesis, nuclei separation and structural differentiation signaling; and 3) trichocyst matrix proteins involved in trichocyst synthesis and reproduction. Furthermore, weighted gene co-expression network analysis identified hub genes that may play crucial roles during cell division. Our findings provide insights into cell cycle regulators, microtubules and trichocyst matrix proteins that may exert influence on this process in ciliates.

Paramecium: RNA sequence-structure phylogenetics.

Organisms classified as members of the genus Paramecium belong to the best-known group of single-celled eukaryotes. Nevertheless, the phylogeny within the genus Paramecium has been discussed and revisited in recent decades and remains partly unresolved. By applying an RNA sequence-structure approach, we attempt to increase accuracy and robustness of phylogenetic trees. For each individual 18S and internal transcribed spacer 2 (ITS2) sequence, a putative secondary structure was predicted through homology modelling. While searching for a structural template, we found, in contrast to the available literature, that the ITS2 molecule consists of three helices in members of the genus Paramecium and four helices in members of the genus Tetrahymena. Two sequencestructure neighbor-joining overall trees were reconstructed with (1) more than 400 taxa (ITS2) and (2) more than 200 taxa (18S). For smaller subsets, neighbor-joining, maximum-parsimony, and maximum-likelihood analyses were executed using sequence-structure information simultaneously. Based on a combined data set (ITS2+18S rDNA) a well-supported tree was reconstructed with bootstrap values over 50 in at least one of the applied analyses. Our results are in general agreement with those published in the available literature based on multi-gene analyses. Our study supports the simultaneous use of sequence-structure data to reconstruct accurate and robust phylogenetic trees.

Methods for Paramecium tetraurelia ciliary membrane protein identification and function.

In this chapter we provide some tools to study the ciliary proteins that make it possible for Paramecium cells to swim by beating their cilia. These proteins include many ion channels, accessory proteins, peripheral proteins, structural proteins, rootlets of cilia, and enzymes. Some of these proteins are also found in the soma membrane, but their distinct and critical functions are in the cilia. Paramecium has 4000 or more cilia per cell, giving it an advantage for biochemical studies over cells that have one primarily cilium per cell. Nonetheless, a challenge for studies of many ciliary proteins in Paramecium is their low abundance. We discuss here several strategies to overcome this challenge and other challenges such as working with very large channel proteins. We also include for completeness other techniques that are critical to the study of swimming behavior, such as genetic crosses, recording of swimming patterns, electrical recordings, expression of very large channel proteins, RNA Interference, among others.

Toxicological impacts and likely protein targets of bisphenol a in Paramecium caudatum.

Bisphenol A (BPA) is a widely used plasticizer agent and a well-known ubiquitous endocrine disruptor, which is frequently associated with a series of reproductive, developmental, and transgenerational effects over wildlife, livestocks, and humans. Although extensive toxicological data is available for metazoans, the impact of BPA over unicellular eukaryotes, which represents a considerable proportion of eukaryotic diversity, remains largely overlooked. Here, we used acute end-point toxicological assay and an inverted virtual-screening (IVS) approach to evaluate cellular impairments infringed by BPA over the cosmopolitan ciliated protist, Paramecium caudatum. Our data indicate a clear time-dependent effect over P. caudatum survival, which seems to be a consequence of disruptions to multiple core cellular functions, such as DNA and cell replication, transcription, translation and signaling pathways. Finally, the use of this ciliate as a biosensor to monitor BPA within environments and the relevance of bioinformatic methods to leverage our current knowledge on the impacts of emerging contaminants to biological systems are discussed.

Developmental mRNA clearance by PIWI-bound endo-siRNAs in Paramecium.

The clearance of untranslated mRNAs by Argonaute proteins is essential for embryonic development in metazoans. However, it is currently unknown whether similar processes exist in unicellular eukaryotes. The ciliate Paramecium tetraurelia harbors a vast array of PIWI-clade Argonautes involved in various small RNA (sRNA) pathways, many of which have not yet been investigated. Here, we investigate the function of a PIWI protein, Ptiwi08, whose expression is limited to a narrow time window during development, concomitant with the start of zygotic transcription. We show that Ptiwi08 acts in an endogenous small interfering RNA (endo-siRNA) pathway involved in the clearance of untranslated mRNAs. These endo-siRNAs are found in clusters that are strictly antisense to their target mRNAs and are a subset of siRNA-producing clusters (SRCs). Furthermore, the endo-siRNAs are 2'-O-methylated by Hen1 and require Dcr1 for their biogenesis. Our findings suggest that sRNA-mediated developmental mRNA clearance extends beyond metazoans and may be a more widespread mechanism than previously anticipated.

Morphological diversity and molecular phylogeny of five Paramecium bursaria (Alveolata, Ciliophora, Oligohymenophorea) syngens and the identification of their green algal endosymbionts.

Paramecium bursaria is a mixotrophic ciliate species, which is common in stagnant and slow-flowing, nutrient-rich waters. It is usually found living in symbiosis with zoochlorellae (green algae) of the genera Chlorella or Micractinium. We investigated P. bursaria isolates from around the world, some of which have already been extensively studied in various laboratories, but whose morphological and genetic identity has not yet been completely clarified. Phylogenetic analyses of the SSU and ITS rDNA sequences revealed five highly supported lineages, which corresponded to the syngen and most likely to the biological species assignment. These syngens R1-R5 could also be distinguished by unique synapomorphies in the secondary structures of the SSU and the ITS. Considering these synapomorphies, we could clearly assign the existing GenBank entries of P. bursaria to specific syngens. In addition, we discovered synapomorphies at amino acids of the COI gene for the identification of the syngens. Using the metadata of these entries, most syngens showed a worldwide distribution, however, the syngens R1 and R5 were only found in Europe. From morphology, the syngens did not show any significant deviations. The investigated strains had either Chlorella variabilis, Chlorella vulgaris or Micractinium conductrix as endosymbionts.

Chromatin remodeling is required for sRNA-guided DNA elimination in Paramecium.

Small RNAs mediate the silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. The unicellular ciliate Paramecium is a model to study dynamic genome organization in eukaryotic cells, given its unique feature of nuclear dimorphism. Here, the formation of the somatic macronucleus during sexual reproduction requires eliminating thousands of transposon remnants (IESs) and transposable elements scattered throughout the germline micronuclear genome. The elimination process is guided by Piwi-associated small RNAs and leads to precise cleavage at IES boundaries. Here we show that IES recognition and precise excision are facilitated by recruiting ISWI1, a Paramecium homolog of the chromatin remodeler ISWI. ISWI1 knockdown substantially inhibits DNA elimination, quantitatively similar to development-specific sRNA gene knockdowns but with much greater aberrant IES excision at alternative boundaries. We also identify key development-specific sRNA biogenesis and transport proteins, Ptiwi01 and Ptiwi09, as ISWI1 cofactors in our co-immunoprecipitation studies. Nucleosome profiling indicates that increased nucleosome density correlates with the requirement for ISWI1 and other proteins necessary for IES excision. We propose that chromatin remodeling together with small RNAs is essential for efficient and precise DNA elimination in Paramecium.

Paramecium epigenetics in development and proliferation.

The term epigenetics is used for any layer of genetic information aside from the DNA base-sequence information. Mammalian epigenetic research increased our understanding of chromatin dynamics in terms of cytosine methylation and histone modification during differentiation, aging, and disease. Instead, ciliate epigenetics focused more on small RNA-mediated effects. On the one hand, these do concern the transport of RNA from parental to daughter nuclei, representing a regulated transfer of epigenetic information across generations. On the other hand, studies of Paramecium, Tetrahymena, Oxytricha, and Stylonychia revealed an almost unique function of transgenerational RNA. Rather than solely controlling chromatin dynamics, they control sexual progeny's DNA content quantitatively and qualitatively. Thus epigenetics seems to control genetics, at least genetics of the vegetative macronucleus. This combination offers ciliates, in particular, an epigenetically controlled genetic variability. This review summarizes the epigenetic mechanisms that contribute to macronuclear heterogeneity and relates these to nuclear dimorphism. This system's adaptive and evolutionary possibilities raise the critical question of whether such a system is limited to unicellular organisms or binuclear cells. We discuss here the relevance of ciliate genetics and epigenetics to multicellular organisms.

Paramecium Polycomb repressive complex 2 physically interacts with the small RNA-binding PIWI protein to repress transposable elements.

Polycomb repressive complex 2 (PRC2) maintains transcriptionally silent genes in a repressed state via deposition of histone H3K27-trimethyl (me3) marks. PRC2 has also been implicated in silencing transposable elements (TEs), yet how PRC2 is targeted to TEs remains unclear. To address this question, we identified proteins that physically interact with the Paramecium enhancer-of-zeste Ezl1 enzyme, which catalyzes H3K9me3 and H3K27me3 deposition at TEs. We show that the Paramecium PRC2 core complex comprises four subunits, each required in vivo for catalytic activity. We also identify PRC2 cofactors, including the RNA interference (RNAi) effector Ptiwi09, which are necessary to target H3K9me3 and H3K27me3 to TEs. We find that the physical interaction between PRC2 and the RNAi pathway is mediated by a RING finger protein and that small RNA recruitment of PRC2 to TEs is analogous to the small RNA recruitment of H3K9 methylation SU(VAR)3-9 enzymes.

Distinction of Paramecium strains by a combination method of RAPD analysis and multiplex PCR.

Paramecium is employed as a valuable model organism in various research fields since a large number of strains with different characteristics of size, morphology, degree of aging, and type of conjugation can be obtained. It is necessary to determine a method for the classification and simple identification of strains to increase their utility as a research tool. This study attempted to establish a polymerase chain reaction (PCR)-based method to differentiate strains of the same species. Genomic DNA was purified from several strains of P. caudatum, P. tetraurelia, and P. bursaria used for comparison by the random amplified polymorphic DNA (RAPD)-PCR method. In P. tetraurelia and P. bursaria, it was sufficiently possible to distinguish specific strains depending on the pattern of random primers and amplification characteristics. For the classification of P. caudatum, based on the sequence data obtained by RAPD-PCR analysis, 5 specific primer sets were designed and a multiplex PCR method was developed. The comparative analysis of 2 standard strains, 12 recommended strains, and 12 other strains of P. caudatum provided by the National BioResource Project was conducted, and specific strains were identified. This multiplex PCR method would be an effective tool for the simple identification of environmental isolates or the management of Paramecium strains.

GC content, but not nucleosome positioning, directly contributes to intron splicing efficiency in Paramecium.

Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the more than 90,000 introns of Paramecium tetraurelia stand among the shortest introns reported in eukaryotes. The mechanisms specifying the correct recognition of these tiny introns remain poorly understood. Splicing can occur cotranscriptionally, and it has been proposed that chromatin structure might influence splice site recognition. To investigate the roles of nucleosome positioning in intron recognition, we determined the nucleosome occupancy along the P. tetraurelia genome. We show that P. tetraurelia displays a regular nucleosome array with a nucleosome repeat length of ∼151 bp, among the smallest periodicities reported. Our analysis has revealed that introns are frequently associated with inter-nucleosomal DNA, pointing to an evolutionary constraint favoring introns at the AT-rich nucleosome edge sequences. Using accurate splicing efficiency data from cells depleted for nonsense-mediated decay effectors, we show that introns located at the edge of nucleosomes display higher splicing efficiency than those at the center. However, multiple regression analysis indicates that the low GC content of introns, rather than nucleosome positioning, is associated with high splicing efficiency. Our data reveal a complex link between GC content, nucleosome positioning, and intron evolution in Paramecium.

Broad domains of histone marks in the highly compact Paramecium macronuclear genome.

The unicellular ciliate Paramecium contains a large vegetative macronucleus with several unusual characteristics, including an extremely high coding density and high polyploidy. As macronculear chromatin is devoid of heterochromatin, our study characterizes the functional epigenomic organization necessary for gene regulation and proper Pol II activity. Histone marks (H3K4me3, H3K9ac, H3K27me3) reveal no narrow peaks but broad domains along gene bodies, whereas intergenic regions are devoid of nucleosomes. Our data implicate H3K4me3 levels inside ORFs to be the main factor associated with gene expression, and H3K27me3 appears in association with H3K4me3 in plastic genes. Silent and lowly expressed genes show low nucleosome occupancy, suggesting that gene inactivation does not involve increased nucleosome occupancy and chromatin condensation. Because of a high occupancy of Pol II along highly expressed ORFs, transcriptional elongation appears to be quite different from that of other species. This is supported by missing heptameric repeats in the C-terminal domain of Pol II and a divergent elongation system. Our data imply that unoccupied DNA is the default state, whereas gene activation requires nucleosome recruitment together with broad domains of H3K4me3. In summary, gene activation and silencing in Paramecium run counter to the current understanding of chromatin biology.

N-glycans from Paramecium bursaria chlorella virus MA-1D: Re-evaluation of the oligosaccharide common core structure.

Paramecium bursaria chlorella virus MA-1D is a chlorovirus that infects Chlorella variabilis strain NC64A, a symbiont of the protozoan Paramecium bursaria. MA-1D has a 339-kb genome encoding ca. 366 proteins and 11 tRNAs. Like other chloroviruses, its major capsid protein (MCP) is decorated with N-glycans, whose structures have been solved in this work by using nuclear magnetic spectroscopy and matrix-assisted laser desorption ionization-time of flight mass spectrometry along with MS/MS experiments. This analysis identified three N-linked oligosaccharides that differ in the nonstoichiometric presence of three monosaccharides, with the largest oligosaccharide composed of eight residues organized in a highly branched fashion. The N-glycans described here share several features with those of the other chloroviruses except that they lack a distal xylose unit that was believed to be part of a conserved core region for all the chloroviruses. Examination of the MA-1D genome detected a gene with strong homology to the putative xylosyltransferase in the reference chlorovirus PBCV-1 and in virus NY-2A, albeit mutated with a premature stop codon. This discovery means that we need to reconsider the essential features of the common core glycan region in the chloroviruses.

'Candidatus Gromoviella agglomerans', a novel intracellular Holosporaceae parasite of the ciliate Paramecium showing marked genome reduction.

Holosporales are an alphaproteobacterial lineage encompassing bacteria obligatorily associated with multiple diverse eukaryotes. For most representatives, little is known on the interactions with their hosts. In this study, we characterized a novel Holosporales symbiont of the ciliate Paramecium polycaryum. This bacterium inhabits the host cytoplasm, frequently forming quite large aggregates. Possibly due to such aggregates, host cells sometimes displayed lethal division defects. The symbiont was also able to experimentally stably infect another Paramecium polycaryum strain. The bacterium is phylogenetically related with symbionts of other ciliates and diplonemids, forming a putatively fast-evolving clade within the family Holosporaceae. Similarly to many close relatives, it presents a very small genome (<600 kbp), and, accordingly, a limited predicted metabolism, implying a heavy dependence on Paramecium, thanks also to some specialized membrane transporters. Characterized features, including the presence of specific secretion systems, are overall suggestive of a mild parasitic effect on the host. From an evolutionary perspective, a potential ancestral trend towards pronounced genome reduction and possibly linked to parasitism could be inferred, at least among fast-evolving Holosporaceae, with some lineage-specific traits. Interestingly, similar convergent features could be observed in other host-associated lineages, in particular Rickettsiales among Alphaproteobacteria.

Comparative Analysis Between Paramecium Strains with Different Syngens Using the RAPD Method.

Paramecium spp. are a genus of free-living protists that live mainly 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.