Novel insights into molecular mechanisms of vegetative cell cycle and resting cyst formation in Apodileptus cf. visscheri (Alveolata, Ciliophora).

Ciliates usually with big cell sizes, complex morphological structures, and diverse life cycles, are good model organisms for studying cell proliferation regulation of eukaryotes. Up to date, the molecular regulation mechanisms for the vegetative cell cycle and encystment of these ciliates are poorly understood. Here, transcriptomes of Apodileptus cf. visscheri, which has an asexual vegetative cell cycle and is apt to encyst when environmental conditions become unfavorable, were sequenced to enrich our related knowledge. In this study, three replicates were sequenced for each of four cell stages, including initial period of growth, morphogenesis, cell division, and resting cyst. The significant transcription differences, involving cell cycle, biosynthesis, and energy metabolism pathways, were revealed between the resting cyst and vegetative cell cycle. Further investigations showed that the cell cycle pathway was enriched during morphogenesis stage and cell division stage. Compared to the initial period of growth stage, the differentially expressed genes involved in cellular components and molecular function were significantly enriched during cell division stage, while cellular components and biological processes were significantly enriched during morphogenesis stage. These provide novel insights into a comprehensive understanding at the molecular level of the survival and adaptive mechanism of unicellular eukaryotes.

Morphology and SSU rRNA gene-based phylogeny of three peniculid ciliates (Ciliophora, Oligohymenophorea) from China, including a new Frontonia species.

In the present study, a new freshwater peniculid species, Frontonia apoelegans sp. nov., and two other peniculid species, Frontonia atra (Ehrenberg, 1833) Bütschli, 1889 and Stokesia vernalis Wenrich, 1929, were isolated from Lake Weishan wetland, northern China. Their morphology and infraciliature are described based on live observations and silver staining methods. The SSU rRNA gene sequences are also provided. Frontonia apoelegans sp. nov. is recognized by the following combination of characteristics: two contractile vacuoles located right-dorsally, without collecting canals; peniculi 1 and 2 four-rowed, peniculus 3 three-rowed with leftmost row containing only one kinetosome; 62-76 somatic kineties; three ophryokineties; and four or five postoral kineties. We also provide improved diagnoses for Frontonia atra and Stokesia vernalis based on current and previous reports. Comparisons with sequences of morphologically similar species clearly support the validity of the new species. Phylogenetic analyses based on SSU rRNA gene sequence data reveal that Frontonia species with two contractile vacuoles cluster in a single clade, indicating these species may have a common origin. The family Frontoniidae is non-monophyletic whereas the family Stokesiidae remains monophyletic according to our analyses.

New contribution to the peritrichous genus Ophrydium (Protista, Ciliophora) with notes on the morphology, taxonomy, and phylogeny of a well-known species Ophrydium crassicaule Penard, 1922.

The peritrichous genus Ophrydium is relatively distinctive since its colonies have a gelatinous lorica. In the present work, one morphologically well-described species, Ophrydium crassicaule, was collected from a freshwater habitat in China. Both morphological characters and sequence data for SSU rDNA, ITS1-5.8S-ITS2 region and LSU rDNA were obtained. Morphologically, O. crassicaule is characterized by its elongated vase-shaped zooid, single-layered peristomial lip, dichotomously branched stalk, colony with gelatinous lorica, and infundibular polykinety 3 (P3) containing one short inner row and two long outer rows. Phylogenetic analyses revealed that the genus Ophrydium is monophyletic and nests within the family Vorticellidae. In addition, we briefly revise the nominal congeners for aiding the species identification in future studies.

Integrative studies on three new freshwater Amphileptus species (Ciliophora, Pleurostomatida) discovered in northern China.

The morphology and molecular phylogeny of freshwater pleurostomatid ciliates are insufficiently explored. In the present study, we investigated three new Amphileptus species discovered in Lake Weishan and its vicinity, northern China, using standard alpha-taxonomic methods. Amphileptus paracarchesii sp. nov. is characterized by a lateral fossa (groove) in the posterior body portion, four macronuclear nodules, contractile vacuoles distributed along the dorsal margin, and 4-6 left and 44-50 right somatic kineties. Amphileptus pilosus sp. nov. differs from congeners by having 4-14 macronuclear nodules, numerous contractile vacuoles scattered throughout the cytoplasm, and 22-31 left and 35-42 right somatic kineties. Amphileptus orientalis sp. nov. is characterized by two ellipsoidal macronuclear nodules, three ventral contractile vacuoles, and about four left and 31-35 right somatic kineties. Phylogenetic analyses of nuclear small subunit ribosomal DNA (SSU rDNA) sequences indicate that the family Amphileptidae might be monophyletic while the genus Amphileptus is paraphyletic, as Pseudoamphileptus macrostoma robustly groups with Amphileptus sp. Although deep phylogenetic relationships of amphileptids are poorly resolved, multiple well-delimited species groups are recognizable within the genus Amphileptus. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-022-00143-0.

The micronuclear histone H3 clipping in the unicellular eukaryote Tetrahymena thermophila.

Clipping of the histone H3 N-terminal tail has been implicated in multiple fundamental biological processes for a growing list of eukaryotes. H3 clipping, serving as an irreversible process to permanently remove some post-translational modifications (PTMs), may lead to noticeable changes in chromatin dynamics or gene expression. The eukaryotic model organism Tetrahymena thermophila is among the first few eukaryotes that exhibits H3 clipping activity, wherein the first six amino acids of H3 are cleaved off during vegetative growth. Clipping only occurs in the transcriptionally silent micronucleus of the binucleated T. thermophila, thus offering a unique opportunity to reveal the role of H3 clipping in epigenetic regulation. However, the physiological functions of the truncated H3 and its protease(s) for clipping remain elusive. Here, we review the major findings of H3 clipping in T. thermophila and highlight its association with histone modifications and cell cycle regulation. We also summarize the functions and mechanisms of H3 clipping in other eukaryotes, focusing on the high diversity in terms of protease families and cleavage sites. Finally, we predict several protease candidates in T. thermophila and provide insights for future studies. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-022-00151-0.

Case Study of the Response of N6-Methyladenine DNA Modification to Environmental Stressors in the Unicellular Eukaryote Tetrahymena thermophila.

Rediscovered as a potential epigenetic mark, N6-methyladenine DNA modification (6mA) was recently reported to be sensitive to environmental stressors in several multicellular eukaryotes. As 6mA distribution and function differ significantly in multicellular and unicellular organisms, whether and how 6mA in unicellular eukaryotes responds to environmental stress remains elusive. Here, we characterized the dynamic changes of 6mA under starvation in the unicellular model organism Tetrahymena thermophila. Single-molecule, real-time (SMRT) sequencing reveals that DNA 6mA levels in starved cells are significantly reduced, especially symmetric 6mA, compared to those in vegetatively growing cells. Despite a global 6mA reduction, the fraction of asymmetric 6mA with a high methylation level was increased, which might be the driving force for stronger nucleosome positioning in starved cells. Starvation affects expression of many metabolism-related genes, the expression level change of which is associated with the amount of 6mA change, thereby linking 6mA with global transcription and starvation adaptation. The reduction of symmetric 6mA and the increase of asymmetric 6mA coincide with the downregulation of AMT1 and upregulation of AMT2 and AMT5, which are supposedly the MT-A70 methyltransferases required for symmetric and asymmetric 6mA, respectively. These results demonstrated that a regulated 6mA response to environmental cues is evolutionarily conserved in eukaryotes. IMPORTANCE Increasing evidence indicated that 6mA could respond to environmental stressors in multicellular eukaryotes. As 6mA distribution and function differ significantly in multicellular and unicellular organisms, whether and how 6mA in unicellular eukaryotes responds to environmental stress remains elusive. In the present work, we characterized the dynamic changes of 6mA under starvation in the unicellular model organism Tetrahymena thermophila. Our results provide insights into how Tetrahymena fine-tunes its 6mA level and composition upon starvation, suggesting that a regulated 6mA response to environmental cues is evolutionarily conserved in eukaryotes.

The completed macronuclear genome of a model ciliate Tetrahymena thermophila and its application in genome scrambling and copy number analyses.

The ciliate Tetrahymena thermophila has been a powerful model system for molecular and cellular biology. However, some investigations have been limited due to the incomplete closure and sequencing of the macronuclear genome assembly, which for many years has been stalled at 1,158 scaffolds, with large sections of unknown sequences (available in Tetrahymena Genome Database, TGD, http://ciliate.org/ ). Here we completed the first chromosome-level Tetrahymena macronuclear genome assembly, with approximately 300× long Single Molecule, Real-Time reads of the wild-type SB210 cells-the reference strain for the initial macronuclear genome sequencing project. All 181 chromosomes were capped with two telomeres and gaps were entirely closed. The completed genome shows significant improvements over the current assembly (TGD 2014) in both chromosome structure and sequence integrity. The majority of previously identified gene models shown in TGD were retained, with the addition of 36 new genes and 883 genes with modified gene models. The new genome and annotation were incorporated into TGD. This new genome allows for pursuit in some underexplored areas that were far more challenging previously; two of them, genome scrambling and chromosomal copy number, were investigated in this study. We expect that the completed macronuclear genome will facilitate many studies in Tetrahymena biology, as well as multiple lines of research in other eukaryotes.

A distinct class of eukaryotic MT-A70 methyltransferases maintain symmetric DNA N6-adenine methylation at the ApT dinucleotides as an epigenetic mark associated with transcription.

Rediscovered as a potential eukaryotic epigenetic mark, DNA N6-adenine methylation (6mA) varies across species in abundance and its relationships with transcription. Here we characterize AMT1-representing a distinct MT-A70 family methyltransferase-in the ciliate Tetrahymena thermophila. AMT1 loss-of-function leads to severe defects in growth and development. Single Molecule, Real-Time (SMRT) sequencing reveals that AMT1 is required for the bulk of 6mA and all symmetric methylation at the ApT dinucleotides. The detection of hemi-methylated ApT sites suggests a semi-conservative mechanism for maintaining symmetric methylation. AMT1 affects expression of many genes; in particular, RAB46, encoding a Rab family GTPase involved in contractile vacuole function, is likely a direct target. The distribution of 6mA resembles H3K4 methylation and H2A.Z, two conserved epigenetic marks associated with RNA polymerase II transcription. Furthermore, strong 6mA and nucleosome positioning in wild-type cells is attenuated in ΔAMT1 cells. Our results support that AMT1-catalyzed 6mA is an integral part of the transcription-associated epigenetic landscape. AMT1 homologues are generally found in protists and basal fungi featuring ApT hyper-methylation associated with transcription, which are missing in animals, plants, and true fungi. This dichotomy of 6mA functions and the underlying molecular mechanisms may have implications in eukaryotic diversification.

RNAi-dependent Polycomb repression controls transposable elements in Tetrahymena.

RNAi and Polycomb repression play evolutionarily conserved and often coordinated roles in transcriptional silencing. Here, we show that, in the protozoan Tetrahymena thermophila, germline-specific internally eliminated sequences (IESs)-many related to transposable elements (TEs)-become transcriptionally activated in mutants deficient in the RNAi-dependent Polycomb repression pathway. Germline TE mobilization also dramatically increases in these mutants. The transition from noncoding RNA (ncRNA) to mRNA production accompanies transcriptional activation of TE-related sequences and vice versa for transcriptional silencing. The balance between ncRNA and mRNA production is potentially affected by cotranscriptional processing as well as RNAi and Polycomb repression. We posit that interplay between RNAi and Polycomb repression is a widely conserved phenomenon, whose ancestral role is epigenetic silencing of TEs.

Morphology, morphogenesis and molecular phylogeny of a new brackish water subspecies, Neourostylopsis flava paraflava nov. subsp. (Ciliophora, Hypotrichia, Urostylidae), with redefinition of the genus Neourostylopsis.

The morphology, morphogenesis and small subunit (SSU) rRNA gene-based phylogeny of Neourostylopsis flava paraflava nov. subsp. were investigated. Neourostylopsis flava paraflava nov. subsp. was separated from N. flava flava nov. stat. by habitat (brackish water vs. fresh water), pretransverse cirri (absent in all specimens vs. present in 15% of specimens), the numbers of frontal cirri (8-15 vs. 6-8) and left marginal cirral rows (6-9 vs. 4-5). The main morphogenetic features of N. flava paraflava nov. subsp. are as follows: (1) streaks I-VI (or I-VIII, deduced from morphological data) produce the bicorona; (2) the oral primordium and frontoventral-transverse cirral anlagen in the opisthe are formed de novo on the cell surface; (3) the numerous macronuclear nodules fuse into a branch-like mass; (4) two pretransverse ventral cirri are formed initially but disappear in the later stages; and (5) some of the frontoventral-transverse cirral anlagen develop in a primary mode. Based on SSU rDNA sequence data, phylogenetic analyses show a close relationship between N. flava paraflava nov. subsp., N. flava flava nov. stat. and other Neourostylopsis species. An improved diagnosis for Neourostylopsis is provided: Urostylidae with five or more frontal cirri which form an indistinct or distinct bicorona; pretransverse cirri present or absent; transverse cirri present; buccal cirri present; two frontoterminal cirri; midventral complex composed of midventral pairs only; more than one row of marginal cirri on each side which derive from individual anlagen within each parental row; caudal cirri lacking; three dorsal kineties; endoral and paroral rather long, endoral straight, paroral distinctly curved anteriorly.

Phylogenetic relationship analyses of complicated class Spirotrichea based on transcriptomes from three diverse microbial eukaryotes: Uroleptopsis citrina, Euplotes vannus and Protocruzia tuzeti.

Ciliates are one of the eukaryotic unicellular organisms which are thought to be the oldest life forms, and widely geographically distributed. For a variety of reasons, some groups of ciliates have attracted more attention than others, such as the class Spirotrichea and related species with its complicated evolutionary relationships. In this study, we obtained the transcriptome data of three typical ciliates, Uroleptopsis citrina, Euplotes vannus, Protocruzia tuzeti using high throughput sequencing. The genetic relationships were revealed by phylogenomic analysis of 109 genes comprising of 34,882 amino acid residues, and analyses based on SSU rDNA of 55 species, as well as the comparison of gene content among spirotricheans and related species. Our phylogenomic analyses show the Spirotrichea is monophyletic when Protocruzia is excluded, in which four subclasses: Oligotrichia, Choreotrichia, Hypotrichia and Euplotia also formed momophyletic groups respectively. The Hypotrichia was placed as a sister branch to the assemblage, in which two oligotrichs clustered with two choreotrichs. In addition to this, the Protocruziidia was placed in an independent lineage status out of the Spirotrichea. Together with its high binding-related gene content compared to other species and the significant variation in morphological characters, these findings support the removal of Protocruzia from the class Spirotrichea.

GPSit: An automated method for evolutionary analysis of nonculturable ciliated microeukaryotes.

Microeukaryotes are among the most important components of the microbial food web in almost all aquatic and terrestrial ecosystems worldwide. In order to gain a better understanding their roles and functions in ecosystems, sequencing coupled with phylogenomic analyses of entire genomes or transcriptomes is increasingly used to reconstruct the evolutionary history and classification of these microeukaryotes and thus provide a more robust framework for determining their systematics and diversity. More importantly, phylogenomic research usually requires high levels of hands-on bioinformatics experience. Here, we propose an efficient automated method, "Guided Phylogenomic Search in trees" (GPSit), which starts from predicted protein sequences of newly sequenced species and a well-defined customized orthologous database. Compared with previous protocols, our method streamlines the entire workflow by integrating all essential and other optional operations. In so doing, the manual operation time for reconstructing phylogenetic relationships is reduced from days to several hours, compared to other methods. Furthermore, GPSit supports user-defined parameters in most steps and thus allows users to adapt it to their studies. The effectiveness of GPSit is demonstrated by incorporating available online data and new single-cell data of three nonculturable marine ciliates (Anteholosticha monilata, Deviata sp. and Diophrys scutum) under moderate sequencing coverage (~5×). Our results indicate that the former could reconstruct robust "deep" phylogenetic relationships while the latter reveals the presence of intermediate taxa in shallow relationships. Based on empirical phylogenomic data, we also used GPSit to evaluate the impact of different levels of missing data on two commonly used methods of phylogenetic analyses, maximum likelihood (ML) and Bayesian inference (BI) methods. We found that BI is less sensitive to missing data when fast-evolving sites are removed.

N6-adenine DNA methylation is associated with the linker DNA of H2A.Z-containing well-positioned nucleosomes in Pol II-transcribed genes in Tetrahymena.

DNA N6-methyladenine (6mA) is newly rediscovered as a potential epigenetic mark across a more diverse range of eukaryotes than previously realized. As a unicellular model organism, Tetrahymena thermophila is among the first eukaryotes reported to contain 6mA modification. However, lack of comprehensive information about 6mA distribution hinders further investigations into its function and regulatory mechanism. In this study, we provide the first genome-wide, base pair-resolution map of 6mA in Tetrahymena by applying single-molecule real-time (SMRT) sequencing. We provide evidence that 6mA occurs mostly in the AT motif of the linker DNA regions. More strikingly, these linker DNA regions with 6mA are usually flanked by well-positioned nucleosomes and/or H2A.Z-containing nucleosomes. We also find that 6mA is exclusively associated with RNA polymerase II (Pol II)-transcribed genes, but is not an unambiguous mark for active transcription. These results support that 6mA is an integral part of the chromatin landscape shaped by adenosine triphosphate (ATP)-dependent chromatin remodeling and transcription.

A comparative study of genome organization and epigenetic mechanisms in model ciliates, with an emphasis on Tetrahymena, Paramecium and Oxytricha.

As a group of unicellular eukaryotes, ciliates offer a unique system to explore epigenetic regulation, mostly due to their nuclear dualism. Ciliates launched a successful radiation after their early evolutionary branching, therefore harboring an unexpectedly rich pool of diverse biological functions and mechanisms. In this review, we compare distinct features of different ciliates in mating type determination, genome organization, DNA methylation, and removal of internal eliminated sequences (IES), with emphasis on Tetrahymena, Paramecium and Oxytricha. Firstly, we review studies on mating type determination in Paramecium, one of the foundational phenomena that defined the field of epigenetics, and compare this process with that in Tetrahymena. Secondly, we showcase the high diversity in genome structure of several ciliates, such as genome size, gene copy numbers, genome rearrangement, etc. Thirdly, we present a brief description of features and potential functions of 5-methylcytosine (5mC) and N6-methyladenine (6mA) in ciliates so far studied. Fourthly, we describe both the initial and the continuously optimized scan RNA (scnRNA) model for IES elimination in Tetrahymena and contrast it with that in Paramecium and Oxytricha. Finally, we discuss the importance of integrative approaches to the study of epigenetic diversity in ciliates and provide possible directions for future research.

N6-methyladenine DNA modification in the unicellular eukaryotic organism Tetrahymena thermophila.

N6-methyladenine DNA (6mA) modification exists in both prokaryotes and eukaryotes in which it plays a crucial role in regulating numerous biological processes. In prokaryotes, 6mA is a marker to distinguish self from foreign DNA. Its role in eukaryotes, however, remains elusive. Ciliates were among the first eukaryotes reported to contain 6mA. In the model organism T. thermophila, cellular localization and features of 6mA have been extensively studied, especially in ribosomal DNA (rDNA) molecules. Here, we summarize the features and potential functions of 6mA in Tetrahymena thermophila and other ciliates, and the major findings and contributions of the Tetrahymena model in studies of 6mA methylation. We also discuss other questions in order to improve understanding of the function and evolution of 6mA in eukaryotes.