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Ciliophora are one of the most diverse phyla of unicellular eukaryotes. Members of this group are widely distributed and have been found in several environments including those that are anaerobic. However, the paucity of sufficient omics data for the major classes of the phylum Ciliophora, especially the anaerobic ciliate lineages, has been a serious impediment to research thus far. In this study, omics data were expanded to include representatives of 16 ciliate classes (excluding Cariacotrichea and Copemetopea) using single‐cell sequencing techniques. Using omics data of 57 ciliates and two apicomplexans, we conducted a comparative genomics study. Phylogenomic analysis based on 156 orthologous genes showed that: there are two superclades within the subphylum Intramacronucleata, i.e., Colpodea + Oligohymenophorea + Nassophorea + Plagiopylea + Prostomatea + Phyllopharyngea (CONThreeP), and Spirotrichea + Armophorea + Litostomatea + Odontostomatea + Muranotrichea + Parablepharismea (SALOMP); with the exception of Armophorea, all ciliate classes are monophyletic; neither Metopus nor Plagiopyla is monophyletic; Paraspathidium has a close affiliation with prostomateans. The results of the divergence time estimations indicated that: the radiation of Ciliophora occurred ca. 1067 million years ago (Mya); the SALOMP and CONThreeP lineages diverged from the intramacronucleatean common ancestor ca. 892 Mya and 851 Mya, respectively; the split between Muranotrichea and Parablepharismea occurred ca. 712 Mya; Odontostomatea are the youngest class, with a crown age estimated at ca. 19 Mya. In addition, we profiled stop codon usage and programmed ribosomal frameshifting (PRF) events. Our analyses revealed that: most of the species investigated use either TAA or TGA as the biased stop codon; stop codon usage in Paraspathidium sp. resembles that of Prostomatea; almost all ciliates included in the analyses have prevalent +1 and −1 PRF events. Finally, through GO enrichment analysis for group‐shared orthogroups of five anaerobic ciliate classes, we successfully identified the biological process and molecular function GO terms that were related to the adaptation of this particular group of ciliates to anaerobic environments. Together, these findings provide new insights into the evolutionary history of the Ciliophora and deepen our understanding of the adaptation of anaerobic ciliate groups to anaerobic environments from an evolutionary perspective. [ABSTRACT FROM AUTHOR]

Most eukaryotes have one nucleus and nuclear genome per cell. Ciliates have instead evolved distinct nuclei that coexist in each cell: a silent germline vs. transcriptionally active somatic nuclei. In the best-studied model species, both nuclei can divide asexually, but only germline nuclei undergo meiosis and karyogamy during sex. Thereafter, thousands of DNA segments, called internally eliminated sequences (IESs), are excised from copies of the germline genomes to produce the streamlined somatic genome. In Loxodes, however, somatic nuclei cannot divide but instead develop from germline copies even during asexual cell division, which would incur a huge overhead cost if genome editing was required. Here, we purified and sequenced both genomes in Loxodes magnus to see whether their nondividing somatic nuclei are associated with differences in genome architecture. Unlike in other ciliates studied to date, we did not find canonical germline-limited IESs, implying Loxodes does not extensively edit its genomes. Instead, both genomes appear large and equivalent, replete with retrotransposons and repetitive sequences, unlike the compact, gene-rich somatic genomes of other ciliates. Two other hallmarks of nuclear development in ciliates--domesticated DDE-family transposases and editing-associated small RNAs--were also not found. Thus, among the ciliates, Loxodes genomes most resemble those of conventional eukaryotes. Nonetheless, base modifications, histone marks, and nucleosome positioning of vegetative Loxodes nuclei are consistent with functional differentiation between actively transcribed somatic vs. inactive germline nuclei. Given their phylogenetic position, it is likely that editing was present in the ancestral ciliate but secondarily lost in the Loxodes lineage. [ABSTRACT FROM AUTHOR]

Understanding the relative importance of the factors that drive global patterns of biodiversity is among the major topics of ecological and biogeographic research. In freshwater bodies, spatial, temporal, abiotic, and biotic factors are important structurers of these ecosystems and can trigger distinct responses according to the facet of biodiversity considered. The objective was to evaluate how different facets of β-diversity (taxonomic, functional, and phylogenetic) based on data from the planktonic ciliate community of a Neotropical floodplain, are influenced by temporal, spatial, abiotic, and biotic factors. The research was conducted in the upper Paraná River floodplain between the years 2010 and 2020 in different water bodies. All predictors showed significant importance on the facets of β-diversity, except the abiotic predictors on species composition data, for the taxonomic facet. The functional and phylogenetic facets were mostly influenced by abiotic, biotic, and spatial factors. For temporal predictors, results showed influence on taxonomic (structure and composition data) and functional (structure data) facets. Also, a fraction of shared explanation between the temporal and abiotic components was observed for the distinct facets. Significant declines in β-diversity in continental ecosystems have been evidenced, especially those with drastic implications for ecosystemic services. Therefore, the preservation of a high level of diversity in water bodies, also involving phylogenetic and functional facets, should be a priority in conservation plans and goals, to ensure the maintenance of important ecological processes involving ciliates. [ABSTRACT FROM AUTHOR]

Learning is usually associated with a complex nervous system, but there is increasing evidence that life at all levels, down to single cells, can display intelligent behaviors. In both natural and artificial systems, learning is the adaptive updating of system parameters based on new information, and intelligence is a measure of the computational process that facilitates learning. Stentor coeruleus is a unicellular pond-dwelling organism that exhibits habituation, a form of learning in which a behavioral response decreases following a repeated stimulus. Stentor contracts in response to mechanical stimulation, which is an apparent escape response from aquatic predators. However, repeated low-force perturbations induce habituation, demonstrated by a progressive reduction in contraction probability. Here, we introduce a method for quantifying Stentor habituation using a microcontroller board-linked apparatus that can deliver mechanical pulses at a specified force and frequency, including methods for building the apparatus and setting up the experiment in a way that minimizes external perturbations. In contrast to the previously described approaches for mechanically stimulating Stentor, this device allows the force of stimulation to be varied under computer control during the course of a single experiment, thus greatly increasing the variety of input sequences that can be applied. Understanding habituation at the level of a single cell will help characterize learning paradigms that are independent of complex circuitry.

Although learning is often viewed as a unique feature of organisms with complex nervous systems, single-celled organisms also demonstrate basic forms of learning. The giant ciliate Stentor coeruleus responds to mechanical stimuli by contracting into a compact shape, presumably as a defense mechanism. When a Stentor cell is repeatedly stimulated at a constant level of force, it will learn to ignore that stimulus but will still respond to stronger stimuli. Prior studies of habituation in Stentor reported a graded response, suggesting that cells transition through a continuous range of response probabilities. By analyzing single cells using an automated apparatus to deliver calibrated stimuli, we find that habituation occurs via a single step-like switch in contraction probability within each cell, with the graded response in a population arising from the random distribution of switching times in individual cells. This step-like response allows Stentor behavior to be represented by a simple two-state model whose parameters can be estimated from experimental measurements. We find that transition rates depend on stimulus force and also on the time between stimuli. The ability to measure the behavior of the same cell to the same stimulus allowed us to quantify the functional heterogeneity among single cells. Together, our results suggest that the behavior of Stentor is governed by a two-state stochastic machine whose transition rates are sensitive to the time series properties of the input stimuli.

Unicellular ciliates like Tetrahymena are best known as free-living bacteriovores, but many species are facultative or obligate parasites. These "histophages" feed on the tissues of hosts ranging from planarian flatworms to commercially important fish and the larvae of imperiled freshwater mussels. Here, we developed a novel bioinformatics pipeline incorporating the nonstandard ciliate genetic code and used it to search for Ciliophora sequences in 34 publicly available Platyhelminthes EST libraries. From 2,615,036 screened ESTs, we identified nearly 6,000 high-confidence ciliate transcripts, supporting parasitism of seven additional flatworm species. We also cultured and identified Tetrahymena from nine terrestrial and freshwater planarians, including invasive earthworm predators from the genus Bipalium and the widely studied regeneration models Dugesia japonica and Schmidtea mediterranea. A co-phylogenetic reconstruction provides strong evidence for the coevolution of histophagous Ciliophora with their Platyhelminthes hosts. We further report the antiprotozoal aminoglycoside paromomycin expels Tetrahymena from S. mediterranea , providing new opportunities to investigate the effects of this relationship on planarian biology. Together, our findings raise the possibility that invasive flatworms constitute a novel dispersal mechanism for Tetrahymena parasites and position the Platyhelminthes as an ideal model phylum for studying the ecology and evolution of histophagous ciliates. [ABSTRACT FROM AUTHOR]

The present investigation deals with the accounts of a novel species of ciliate parasite belonging to the genus Paratrichodina namely, Paratrichodina indiana n. sp. identified from the respiratory organ of goldfish Carassius auratus at Nadia district of West Bengal, India. 276 fish have been collected for parasitological examination from various fish farm of West Bengal, India. The parasitological investigation was conducted using the AgNO3 impregnation technique, which was supplemented by a taxonomic description, morphological variations and data comparison with previously documented closely related species. Paratrichodina indiana n. sp. is a small-sized trichodinid, measures uniquely 19.21 ± 1.6 µm in body diameter, with prominent adhesive disc measure 15.55 ± 1.22 µm. The current investigation supports the first record of the genus from C. auratus and adds one new species to the genus Paratrichodina, as well as report of a new host, region and prevalence rates. [ABSTRACT FROM AUTHOR]

Cellular components are non-randomly arranged with respect to the shape and polarity of the whole cell.1-4 Patterning within cells can extend down to the level of individual proteins and mRNA.5,6 But how much of the proteome is actually localized with respect to cell polarity axes? Proteomics combined with cellular fractionation7-11 has shown that most proteins localize to one or more organelles but does not tell us how many proteins have a polarized localization with respect to the large-scale polarity axes of the intact cell. Genome-wide localization studies in yeast12-15 found that only a few percent of proteins have a localized position relative to the cell polarity axis defined by sites of polarized cell growth. Here, we describe an approach for analyzing protein distribution within a cell with a visibly obvious global patterning-the giant ciliate Stentor coeruleus.16,17 Ciliates, including Stentor, have highly polarized cell shapes with visible surface patterning.1,18 A Stentor cell is roughly 2 mm long, allowing a "proteomic dissection" in which microsurgery is used to separate cellular fragments along the anterior-posterior axis, followed by comparative proteomic analysis. In our analysis, 25% of the proteome, including signaling proteins, centrin/SFI proteins, and GAS2 orthologs, shows a polarized location along the cell's anterior-posterior axis. We conclude that a large proportion of all proteins are polarized with respect to global cell polarity axes and that proteomic dissection provides a simple and effective approach for spatial proteomics.

The giant ciliate Stentor coeruleus is a classical model system for studying regeneration and morphogenesis in a single cell. The anterior of the cell is marked by an array of cilia, known as the oral apparatus, which can be induced to shed and regenerate in a series of reproducible morphological steps, previously shown to require transcription. If a cell is cut in half, each half regenerates an intact cell. We used RNA sequencing (RNAseq) to assay the dynamic changes in Stentor's transcriptome during regeneration, after both oral apparatus shedding and bisection, allowing us to identify distinct temporal waves of gene expression including kinases, RNA -binding proteins, centriole biogenesis factors, and orthologs of human ciliopathy genes. By comparing transcriptional profiles of different regeneration events, we identified distinct modules of gene expression corresponding to oral apparatus regeneration, posterior holdfast regeneration, and recovery after wounding. By measuring gene expression after blocking translation, we show that the sequential waves of gene expression involve a cascade mechanism in which later waves of expression are triggered by translation products of early-expressed genes. Among the early-expressed genes, we identified an E2F transcription factor and the RNA-binding protein Pumilio as potential regulators of regeneration based on the expression pattern of their predicted target genes. RNAi-mediated knockdown experiments indicate that Pumilio is required for regenerating oral structures of the correct size. E2F is involved in the completion of regeneration but is dispensable for earlier steps. This work allows us to classify regeneration genes into groups based on their potential role for regeneration in distinct cell regeneration paradigms, and provides insight into how a single cell can coordinate complex morphogenetic pathways to regenerate missing structures.

BackgroundWound healing is one of the defining features of life and is seen not only in tissues but also within individual cells. Understanding wound response at the single-cell level is critical for determining fundamental cellular functions needed for cell repair and survival. This understanding could also enable the engineering of single-cell wound repair strategies in emerging synthetic cell research. One approach is to examine and adapt self-repair mechanisms from a living system that already demonstrates robust capacity to heal from large wounds. Towards this end, Stentor coeruleus, a single-celled free-living ciliate protozoan, is a unique model because of its robust wound healing capacity. This capacity allows one to perturb the wounding conditions and measure their effect on the repair process without immediately causing cell death, thereby providing a robust platform for probing the self-repair mechanism.ResultsHere we used a microfluidic guillotine and a fluorescence-based assay to probe the timescales of wound repair and of mechanical modes of wound response in Stentor. We found that Stentor requires ~ 100-1000 s to close bisection wounds, depending on the severity of the wound. This corresponds to a healing rate of ~ 8-80 μm2/s, faster than most other single cells reported in the literature. Further, we characterized three distinct mechanical modes of wound response in Stentor: contraction, cytoplasm retrieval, and twisting/pulling. Using chemical perturbations, active cilia were found to be important for only the twisting/pulling mode. Contraction of myonemes, a major contractile fiber in Stentor, was surprisingly not important for the contraction mode and was of low importance for the others.ConclusionsWhile events local to the wound site have been the focus of many single-cell wound repair studies, our results suggest that large-scale mechanical behaviors may be of greater importance to single-cell wound repair than previously thought. The work here advances our understanding of the wound response in Stentor and will lay the foundation for further investigations into the underlying components and molecular mechanisms involved.

La bioseguridad en la acuicultura se ocupa de la gestión de la sanidad de los animales acuáticos y la reducción de los riesgos a la salud pública asociados con la producción y el consumo de productos acuícolas. Particularmente, las enfermedades son un tema de atención en todo sistema de cría y producción de animales. En la acuicultura cubana, una de las especies ícticas de mayor importancia es el pez gato africano Clarias gariepinus. Esta especie introducida es afectada por protozoos ciliados parásitos de la familia Trichodinidae durante los primeros estadios de vida. El presente trabajo se trazó como objetivo, identificar y caracterizar las especies de tricodínidos que afectan el cultivo de C. gariepinus en la estación de alevinaje La Juventud, Provincia Pinar del Río, Cuba. Dichas especies identificadas y caracterizadas fueron T. heterodentata, T. acuta y T. merciae, las cuales son consideradas peligros biológicos para el cultivo de este pez. Estos resultados constituyen el primer paso para desarrollar un análisis de riesgo que garantice un manejo adecuado de la bioseguridad en las estaciones acuícolas.

Abstract Background The emergence of multi-resistant pathogens have increased dramatically in recent years, becoming a major public-health concern. Among other promising antimicrobial molecules with potential to assist in this worldwide struggle, cysteine-stabilized αβ (CS-αβ) defensins are attracting attention due their efficacy, stability, and broad spectrum against viruses, bacteria, fungi, and protists, including many known human pathogens. Results Here, 23 genomes of ciliated protists were screened and two CS-αβ defensins with a likely antifungal activity were identified and characterized, using bioinformatics, from a culturable freshwater species, Laurentiella sp. (LsAMP-1 and LsAMP-2). Although any potential cellular ligand could be predicted for LsAMP-2; evidences from structural, molecular dynamics, and docking analyses suggest that LsAMP-1 may form stably associations with phosphatidylinositol 4,5-bisphosphates (PIP2), a phospholipid found on many eukaryotic cells, which could, in turn, represent an anchorage mechanism within plasma membrane of targeted cells. Conclusion These data stress that more biotechnology-oriented studies should be conducted on neglected protists, such ciliates, which could become valuable sources of novel bioactive molecules for therapeutic uses.

Periphyton communities in freshwater systems play an essential role in biogeochemical processes, but knowledge of their structure and dynamics lags far behind other environments. We used eDNA metabarcoding of 16S and 18S rRNA markers to investigate the formation and establishment of a periphytic community, in addition to a morphology-based approach for peritrich ciliate determinations, its most abundant group. We sampled two nearby sites within a large Neotropical lake at four time points, aiming to assess whether periphyton establishment can be replicated on this local scale. Producers and denitrifiers were abundant in the community, illustrating the relevant role of biofilms in freshwater nutrient recycling. Among microeukaryotes, peritrich ciliates dominated the community, with genera Epistylis and Vorticella being the most abundant and showing a clear succession at both sites. Other ciliates were morphologically identified and, in some cases, their occurrence was strongly related to bacterial abundance. The structure of both prokaryotic and eukaryotic components of periphyton was not different, while the turnover dynamics differed between the two sites, in spite of their adjacent locations and similar abiotic properties. This indicates that the establishment of these communities can vary even on a local scale within a lake ecosystem. [ABSTRACT FROM AUTHOR]

Marine sediments are one of the largest carbon reservoir on Earth, yet the microbial communities, especially the eukaryotes, that drive these ecosystems are poorly characterised. Here, we report implementation of a sampling system that enables injection of reagents into sediments at depth, allowing for preservation of RNA in situ. Using the RNA templates recovered, we investigate the ribosomally active eukaryotic diversity present in sediments close to the water/sediment interface. We demonstrate that in situ preservation leads to recovery of a significantly altered community profile. Using SSU rRNA amplicon sequencing, we investigated the community structure in these environments, demonstrating a wide diversity and high relative abundance of stramenopiles and alveolates, specifically: Bacillariophyta (diatoms), labyrinthulomycetes and ciliates. The identification of abundant diatom rRNA molecules is consistent with microscopy-based studies, but demonstrates that these algae can also be exported to the sediment as active cells as opposed to dead forms. We also observe many groups that include, or branch close to, osmotrophic-saprotrophic protists (e.g. labyrinthulomycetes and Pseudofungi), microbes likely to be important for detrital decomposition. The sequence data also included a diversity of abundant amplicon-types that branch close to the Fonticula slime moulds. Taken together, our data identifies additional roles for eukaryotic microbes in the marine carbon cycle; where putative osmotrophic-saprotrophic protists represent a significant active microbial-constituent of the upper sediment layer.

The phenomenon of ciliary coordination has garnered increasing attention in recent decades and multiple theories have been proposed to explain its occurrence in different biological systems. While hydrodynamic interactions are thought to dictate the large-scale coordinated activity of epithelial cilia for fluid transport, it is rather basal coupling that accounts for synchronous swimming gaits in model microeukaryotes such as Chlamydomonas. Unicellular ciliates present a fascinating yet understudied context in which coordination is found to persist in ciliary arrays positioned across millimetre scales on the same cell. Here, we focus on the ciliate Stentor coeruleus, chosen for its large size, complex ciliary organization, and capacity for cellular regeneration. These large protists exhibit ciliary differentiation between cortical rows of short body cilia used for swimming, and an anterior ring of longer, fused cilia called the membranellar band (MB). The oral cilia in the MB beat metachronously to produce strong feeding currents. Remarkably, upon injury, the MB can be shed and regenerated de novo. Here, we follow and track this developmental sequence in its entirety to elucidate the emergence of coordinated ciliary beating: from band formation, elongation, curling and final migration towards the cell anterior. We reveal a complex interplay between hydrodynamics and ciliary restructuring in Stentor, and highlight for the first time the importance of a ring-like topology for achieving long-range metachronism in ciliated structures. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

Ciliates, a eukaryotic clade that is over 1 billion years old, are defined by division of genome function between transcriptionally inactive germline micronuclei and functional somatic macronuclei. To date, most analyses of gene family evolution have been limited to cultivable model lineages (e.g., Tetrahymena, Paramecium, Oxytricha, and Stylonychia). Here, we focus on the uncultivable Karyorelictea and its understudied sister class Heterotrichea, which represent two extremes in genome architecture. Somatic macronuclei within the Karyorelictea are described as nearly diploid, while the Heterotrichea have hyperpolyploid somatic genomes. Previous analyses indicate that genome architecture impacts ciliate gene family evolution as the most diverse and largest gene families are found in lineages with extensively processed somatic genomes (i.e., possessing thousands of gene-sized chromosomes). To further assess ciliate gene family evolution, we analyzed 43 single-cell transcriptomes from 33 ciliate species representing 10 classes. Focusing on conserved eukaryotic genes, we use estimates of transcript diversity as a proxy for the number of paralogs in gene families among four focal clades: Karyorelictea, Heterotrichea, extensive fragmenters (with gene-size somatic chromosomes), and non-extensive fragmenters (with more traditional somatic chromosomes), the latter two within the subphylum Intramacronucleata. Our results show that (i) the Karyorelictea have the lowest average transcript diversity, while Heterotrichea are highest among the four groups; (ii) proteins in Karyorelictea are under the highest functional constraints, and the patterns of selection in ciliates may reflect genome architecture; and (iii) stop codon reassignments vary among members of the Heterotrichea and Spirotrichea but are conserved in other classes.IMPORTANCE To further our understanding of genome evolution in eukaryotes, we assess the relationship between patterns of molecular evolution within gene families and variable genome structures found among ciliates. We combine single-cell transcriptomics with bioinformatic tools, focusing on understudied and uncultivable lineages selected from across the ciliate tree of life. Our analyses show that genome architecture correlates with patterns of protein evolution as lineages with more canonical somatic genomes, such as the class Karyorelictea, have more conserved patterns of molecular evolution compared to other classes. This study showcases the power of single-cell transcriptomics for investigating genome architecture and evolution in uncultivable microbial lineages and provides transcriptomic resources for further research on genome evolution.

Quantitative analysis of the giant ciliate Stentor roeselii shows that a single cell can make decisions, based on the ability to switch between several different behaviors in a non-random order.

Nearly ubiquitous and usually speciose in most aquatic habitats, the meiofaunal-sized gastrotrichs are recognized as an important component of marine and freshwater ecosystems. The common observations that gastrotrichs feed on bacteria, microalgae and biodetritus strongly imply that they play a relevant role in linking the microbial loop to the higher trophic levels. Which are the organisms that in turn prey on gastrotrichs is, however, a substantially unexplored question. Inspecting meiofauna samples collected from shallow sites of the Tyrrhenian coast, we had the chance to spot a wild case of a macrodasyidan gastrotrich predated by a dileptid ciliate. This case is documented here with a set of in-vivo photos, jointly with an unequivocal taxonomic identification of the preyed gastrotrich with Paraturbanella teissieri and a tentative identification of the predator ciliate with Pseudomonilicaryon marinus.

The detection of Giardia duodenalis and Cryptosporidium spp. was performed, along with the identification of the ciliated protozoa biodiversity, to evaluate the correlation between these protozoa in freshwater quality monitoring. Water and sediment samples from two sites in the Atibaia River (Campinas, São Paulo, Brazil) were collected monthly for 2 years (n = 96). Pathogenic protozoa in water and sediment were detected by using immunomagnetic separation, followed by visualization by immunofluorescence assay (IFA). All positive aliquots in IFA were subjected to DNA extraction and subsequently nested PCR. Qualitative (in vivo observation and silver impregnation) and quantitative (in vivo enumeration) analyses were performed for the ciliated protozoa. Giardia cysts were detected in 62.5% of the surface water samples and Cryptosporidium spp. in 25.0%. In the sediment, cysts were detected in 35.4% samples and oocysts in 16.6%. A total of 57 samples positive for Giardia cysts were subjected to sequencing, 40 of which were harboring G. duodenalis (24 were characterized as sub-assemblage AII). For ciliated protozoa, 73 taxa belonging to 53 genera were identified over the period of the study. These results revealed a high degree of contamination by waterborne protozoa in the main water source which supplies drinking water for more than one million people in Campinas (São Paulo), highlighting the need for continuous monitoring of this catchment site. In addition, the present study provides important data regarding the sources of the water body degradation, i.e., fecal contamination of human origin, in addition to the survey of the ciliated protozoa. [ABSTRACT FROM AUTHOR]

Background: The emergence of multi-resistant pathogens have increased dramatically in recent years, becoming a major public-health concern. Among other promising antimicrobial molecules with potential to assist in this worldwide struggle, cysteine-stabilized αβ (CS-αβ) defensins are attracting attention due their efficacy, stability, and broad spectrum against viruses, bacteria, fungi, and protists, including many known human pathogens. Results: Here, 23 genomes of ciliated protists were screened and two CS-αβ defensins with a likely antifungal activity were identified and characterized, using bioinformatics, from a culturable freshwater species, Laurentiella sp. (LsAMP-1 and LsAMP-2). Although any potential cellular ligand could be predicted for LsAMP-2; evidences from structural, molecular dynamics, and docking analyses suggest that LsAMP-1 may form stably associations with phosphatidylinositol 4,5-bisphosphates (PIP2), a phospholipid found on many eukaryotic cells, which could, in turn, represent an anchorage mechanism within plasma membrane of targeted cells. Conclusion: These data stress that more biotechnology-oriented studies should be conducted on neglected protists, such ciliates, which could become valuable sources of novel bioactive molecules for therapeutic uses. [ABSTRACT FROM AUTHOR]

Phytoplankton and associated microbial communities provide organic carbon to oceanic food webs and drive ecosystem dynamics. However, capturing those dynamics is challenging. Here, an in situ, semi-Lagrangian, robotic sampler profiled pelagic microbes at 4 h intervals over ~2.6 days in North Pacific high-nutrient, low-chlorophyll waters. We report on the community structure and transcriptional dynamics of microbes in an operationally large size class (>5 μm) predominantly populated by dinoflagellates, ciliates, haptophytes, pelagophytes, diatoms, cyanobacteria (chiefly Synechococcus), prasinophytes (chiefly Ostreococcus), fungi, archaea, and proteobacteria. Apart from fungi and archaea, all groups exhibited 24-h periodicity in some transcripts, but larger portions of the transcriptome oscillated in phototrophs. Periodic photosynthesis-related transcripts exhibited a temporal cascade across the morning hours, conserved across diverse phototrophic lineages. Pronounced silica:nitrate drawdown, a high flavodoxin to ferredoxin transcript ratio, and elevated expression of other Fe-stress markers indicated Fe-limitation. Fe-stress markers peaked during a photoperiodically adaptive time window that could modulate phytoplankton response to seasonal Fe-limitation. Remarkably, we observed viruses that infect the majority of abundant taxa, often with total transcriptional activity synchronized with putative hosts. Taken together, these data reveal a microbial plankton community that is shaped by recycled production and tightly controlled by Fe-limitation and viral activity.

Nearly ubiquitous and usually speciose in most aquatic habitats, the meiofaunal-sized gastrotrichs are recognized as an important component of marine and freshwater ecosystems. The common observations that gastrotrichs feed on bacteria, microalgae and biodetritus strongly imply that they play a relevant role in linking the microbial loop to the higher trophic levels. Which are the organisms that in turn prey on gastrotrichs is, however, a substantially unexplored question. Inspecting meiofauna samples collected from shallow sites of the Tyrrhenian coast, we had the chance to spot a wild case of a macrodasyidan gastrotrich predated by a dileptid ciliate. This case is documented here with a set of in-vivo photos, jointly with an unequivocal taxonomic identification of the preyed gastrotrich with Paraturbanella teissieri and a tentative identification of the predator ciliate with Pseudomonilicaryon marinus. [ABSTRACT FROM AUTHOR]

Hydrothermal vents are critical to marine geochemical cycling and ecosystem functioning. Although hydrothermal vent-associated megafauna and chemoautotrophic prokaryotes have received extensive dedicated study, smaller hydrothermal vent-associated eukaryotes such as meiofauna and nanobiota have received much less attention. These communities comprise critical links in trophic flow and carbon cycling of other marine habitats, so study of their occurrence and role in hydrothermal vent ecosystems is warranted. Further, an understudied vent habitat is the thin sediment cover at the base of hydrothermal vent chimneys. An initial study revealed that sediments at the base of vent chimneys of the Izu-Ogawasara Arc system (western North Pacific) support metazoan meiofauna, but very little is known about the taxonomic composition and abundance of the meiobenthic protists and nanobiota, or their millimeter-scale distributions. Using the Fluorescently Labeled Embedded Coring method (FLEC), we describe results on meiofaunal and nanobiota higher-level identifications, life positions and relative abundances within sediments from three habitats (base of vent chimneys, inside caldera but away from chimneys, and outside caldera) of the Myojin-Knoll caldera and vicinity. Results suggest that the chimney-base community is unique and more abundant compared to non-chimney associated eukaryotic communities. Supporting evidence (molecular phylogeny, scanning and transmission electron microscopy imaging) documents first known hydrothermal-vent-associated occurrences for two protist taxa. Collectively, results provide valuable insights into a cryptic component of the hydrothermal vent ecosystem.

Although we often think of cells as small, simple building blocks of life, in fact they are highly complex and can perform a startling variety of functions. In our bodies, cells are programmed by complex differentiation pathways and are capable of responding to a bewildering range of chemical and physical signals. Free-living single-celled organisms, such as bacteria or protists, have to cope with varying environments, locate prey and potential mates, and escape from predators - all of the same tasks that a free-living animal is faced with. When animals face complex behavioral challenges, they rely on their cognitive abilities - the ability to learn from experience, to analyse a situation and choose an appropriate course of action. This ability is essential for survival and should, in principle, be a ubiquitous feature of all living things regardless of the complexity of the organism.

The emergence of robust single-cell 'omics techniques enables studies of uncultivable species, allowing for the (re)discovery of diverse genomic features. In this study, we combine single-cell genomics and transcriptomics to explore genome evolution in ciliates (a > 1 Gy old clade). Analysis of the data resulting from these single-cell 'omics approaches show: 1) the description of the ciliates in the class Karyorelictea as "primitive" is inaccurate because their somatic macronuclei contain loci of varying copy number (i.e., they have been processed by genome rearrangements from the zygotic nucleus); 2) gene-sized somatic chromosomes exist in the class Litostomatea, consistent with Balbiani's (1890) observation of giant chromosomes in this lineage; and 3) gene scrambling exists in the underexplored Postciliodesmatophora (the classes Heterotrichea and Karyorelictea, abbreviated here as the Po-clade), one of two major clades of ciliates. Together these data highlight the complex evolutionary patterns underlying germline genome architectures in ciliates and provide a basis for further exploration of principles of genome evolution in diverse microbial lineages.

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Separate germline and somatic genomes are found in numerous lineages across the eukaryotic tree of life, often separated into distinct tissues (e.g., in plants, animals, and fungi) or distinct nuclei sharing a common cytoplasm (e.g., in ciliates and some foraminifera). In ciliates, germline-limited (i.e., micronuclear-specific) DNA is eliminated during the development of a new somatic (i.e., macronuclear) genome in a process that is tightly linked to large-scale genome rearrangements, such as deletions and reordering of protein-coding sequences. Most studies of germline genome architecture in ciliates have focused on the model ciliates Oxytricha trifallax, Paramecium tetraurelia, and Tetrahymena thermophila, for which the complete germline genome sequences are known. Outside of these model taxa, only a few dozen germline loci have been characterized from a limited number of cultivable species, which is likely due to difficulties in obtaining sufficient quantities of "purified" germline DNA in these taxa. Combining single-cell transcriptomics and genomics, we have overcome these limitations and provide the first insights into the structure of the germline genome of the ciliate Chilodonella uncinata, a member of the understudied class Phyllopharyngea Our analyses reveal the following: (i) large gene families contain a disproportionate number of genes from scrambled germline loci; (ii) germline-soma boundaries in the germline genome are demarcated by substantial shifts in GC content; (iii) single-cell omics techniques provide large-scale quality germline genome data with limited effort, at least for ciliates with extensively fragmented somatic genomes. Our approach provides an efficient means to understand better the evolution of genome rearrangements between germline and soma in ciliates.IMPORTANCE Our understanding of the distinctions between germline and somatic genomes in ciliates has largely relied on studies of a few model genera (e.g., Oxytricha, Paramecium, Tetrahymena). We have used single-cell omics to explore germline-soma distinctions in the ciliate Chilodonella uncinata, which likely diverged from the better-studied ciliates ~700 million years ago. The analyses presented here indicate that developmentally regulated genome rearrangements between germline and soma are demarcated by rapid transitions in local GC composition and lead to diversification of protein families. The approaches used here provide the basis for future work aimed at discerning the evolutionary impacts of germline-soma distinctions among diverse ciliates.

The diversity of prokaryotic symbionts in Ciliophora and other protists is fascinatingly rich; they may even include some potentially pathogenic bacteria. In this review, we summarize currently available data on biodiversity and some morphological and biological peculiarities of prokaryotic symbionts mainly within the genera Paramecium and Euplotes. Another direction of ciliate symbiology, neglected for a long time and now re‐discovered, is the study of epibionts of ciliates. This promises a variety of interesting outcomes. Last, but not least, we stress the new technologies, such as next generation sequencing and the use of genomics data, which all can clarify many new aspects of relevance. For this reason, a brief overview of achievements in genomic studies on ciliate's symbionts is provided. Summing up the results of numerous scientific contributions, we systematically update current knowledge and outline the prospects as to how symbiology of Ciliophora may develop in the near future. [ABSTRACT FROM AUTHOR]

We assessed genetic diversities among Ichthyophthirius multifiliis (Ich) field isolates collected from farmed rainbow trout (Oncorhynchus mykiss) in Turkey. The overall prevalence of Ich was 35.3% (634/1798). Five novel Ich genotypes (ImulTR1 and ImulTR3‒ImulTR6) were described based on mitochondrial cox‐1 and nad1_b genes. The remaining genotype ImulTR2 was identical to the previously reported NY3 (or Ark9 and TW7) genotype from the United States and South Asia. Phylogenetic analysis indicated Turkish Ich isolates separated genetically into at least four distinct groups. Our study presents the first data on the genotypes of Ich in Turkey. We also provide evidence for the wide distribution of the NY3 genotype (or Ark9 and TW7) from the United States and South Asia to Turkey. Genetic diversities within the mitochondrial genes provided adequate resolution for describing novel genotypes and identifying the known genotype within Turkish Ich isolates. Description of the Ich genotypes allows for tracking of pathogen genotypes worldwide. Thus, we can better understand the connections between Ich outbreaks in the fisheries aquaculture. [ABSTRACT FROM AUTHOR]

A survey of gill parasites was conducted in July 2019 on a small sample of cypress minnow, Hybognathus hayi Jordan, from Calion Lake, Union County, Arkansas (Ouachita River drainage). Six of 9 (67%) specimens harbored parasites, including ciliates Ichthyophthirius multifiliisFouquet, 1876, and an undetermined Trichodina sp., a myxozoan, Myxobolus sp., and a monogenean, Dactylogyrus nuchalisWood and Mizelle, 1957, in 2 (22%). Dactylogyrus hankinsoniHanek, Molnar, and Fernando, 1975, was found in 3 (33%) of the host specimens. All 5 taxa of these gill-inhabiting parasites are reported for the first time from H. hayi, and the monogeneans are documented as new geographic distribution records in Arkansas. [ABSTRACT FROM AUTHOR]

Wound repair is a key feature distinguishing living from nonliving matter. Single cells are increasingly recognized to be capable of healing wounds. The lack of reproducible, high-throughput wounding methods has hindered single-cell wound repair studies. This work describes a microfluidic guillotine for bisecting single Stentor coeruleus cells in a continuous-flow manner. Stentor is used as a model due to its robust repair capacity and the ability to perform gene knockdown in a high-throughput manner. Local cutting dynamics reveals two regimes under which cells are bisected, one at low viscous stress where cells are cut with small membrane ruptures and high viability and one at high viscous stress where cells are cut with extended membrane ruptures and decreased viability. A cutting throughput up to 64 cells per minute-more than 200 times faster than current methods-is achieved. The method allows the generation of more than 100 cells in a synchronized stage of their repair process. This capacity, combined with high-throughput gene knockdown in Stentor, enables time-course mechanistic studies impossible with current wounding methods.

Many organisms and tissues display the ability to heal and regenerate as needed for normal physiology and as a result of pathogenesis. However, these repair activities can also be observed at the single-cell level. The physical and molecular mechanisms by which a cell can heal membrane ruptures and rebuild damaged or missing cellular structures remain poorly understood. This Review presents current understanding in wound healing and regeneration as two distinct aspects of cellular self-repair by examining a few model organisms that have displayed robust repair capacity, including Xenopus oocytes, Chlamydomonas, and Stentor coeruleus Although many open questions remain, elucidating how cells repair themselves is important for our mechanistic understanding of cell biology. It also holds the potential for new applications and therapeutic approaches for treating human disease.

Ciliates such as Tetrahymena thermophila have two distinct nuclei within one cell: the micronucleus that undergoes mitosis and meiosis and the macronucleus that undergoes amitosis, a type of nuclear division that does not involve a bipolar spindle, but still relies on intranuclear microtubules. Ciliates provide an opportunity for the discovery of factors that specifically contribute to chromosome segregation based on a bipolar spindle, by identification of factors that affect the micronuclear but not the macronuclear division. Kinesin-14 is a conserved minus-end directed microtubule motor that cross-links microtubules and contributes to the bipolar spindle sizing and organization. Here, we use homologous DNA recombination to knock out genes that encode kinesin-14 orthologues (KIN141, KIN142) in Tetrahymena. A loss of KIN141 led to severe defects in the chromosome segregation during both mitosis and meiosis but did not affect amitosis. A loss of KIN141 altered the shape of the meiotic spindle in a way consistent with the KIN141's contribution to the organization of the spindle poles. EGFP-tagged KIN141 preferentially accumulated at the spindle poles during the meiotic prophase and metaphase I. Thus, in ciliates, kinesin-14 is important for nuclear divisions that involve a bipolar spindle.

The giant, single-celled organism Stentor coeruleus has a long history as a model system for studying pattern formation and regeneration in single cells. Stentor [1, 2] is a heterotrichous ciliate distantly related to familiar ciliate models, such as Tetrahymena or Paramecium. The primary distinguishing feature of Stentor is its incredible size: a single cell is 1 mm long. Early developmental biologists, including T.H. Morgan [3], were attracted to the system because of its regenerative abilities-if large portions of a cell are surgically removed, the remnant reorganizes into a normal-looking but smaller cell with correct proportionality [2, 3]. These biologists were also drawn to Stentor because it exhibits a rich repertoire of behaviors, including light avoidance, mechanosensitive contraction, food selection, and even the ability to habituate to touch, a simple form of learning usually seen in higher organisms [4]. While early microsurgical approaches demonstrated a startling array of regenerative and morphogenetic processes in this single-celled organism, Stentor was never developed as a molecular model system. We report the sequencing of the Stentor coeruleus macronuclear genome and reveal key features of the genome. First, we find that Stentor uses the standard genetic code, suggesting that ciliate-specific genetic codes arose after Stentor branched from other ciliates. We also discover that ploidy correlates with Stentor's cell size. Finally, in the Stentor genome, we discover the smallest spliceosomal introns reported for any species. The sequenced genome opens the door to molecular analysis of single-cell regeneration in Stentor.

Assessing functional diversity of communities is an efficient method to link community composition to ecosystem quality. Still, studies using functional traits of microeukaryote ciliate communities in biological wastewater treatment plants are lacking. The present work explores the functional diversity of the ciliate protist community in a wastewater treatment plant (WWTP) operating with a combined UASB-activated sludge system, and specifically to: 1) investigate the taxonomic and functional composition of the ciliate communities over time; 2) compare taxonomic and functional diversity indices with regard to its applicability in WWPS; 3) assess the relationship between the ciliate community’s functional composition and the WWTPs temporal conditions; and 4) investigate the potential use of functional diversity as an indicator of WWTP efficiency. Totally, we recorded 21 ciliate species throughout 37 samplings. The number of species was low compared to other plants. Bacterivorous and flake-forming species were the main functional strategies found in the samples. The correlation between taxonomic and functional richness was significant, indicating a functionally redundant community. There was a correlation between the Simpson and Rao’s quadratic entropy indexes suggesting that loss of taxonomic diversity leads to a loss of functional diversity. The homogeneity of the measured physical and chemical data led to functional homogenization and redundancy (homogenous CWM) of the ciliate community. The functional diversity is positively correlated with parameters of removal efficiency, indicating a promising application in WWTPs. Future studies will broaden knowledge on functional diversity in biological wastewater treatment systems, this being a first step with the unprecedented application of this methodology in artificial ecosystems.

[Display omitted] • Phylogenomics, divergence time estimation and macroevolutionary analyses are conducted on 105 ciliate species and 247 genes. • A robust reference tree for the phylum Ciliophora. • The radiation of Ciliophora occurred around 1175 Ma in the middle of the Proterozoic Eon. • Most classes diverged from their sister lineages during the latter half of the Proterozoic Eon. • The variation in species richness among classes is primarily attributed to net diversification rates rather than age. Ciliophora, an exceptionally diverse lineage of unicellular eukaryotes, exhibits a remarkable range of species richness across classes in the ciliate Tree of Life. In this study, we have acquired transcriptome and genome data from 40 representative species in seven ciliate classes. Utilizing 247 genes and 105 taxa, we devised a comprehensive phylogenomic tree for Ciliophora, encompassing over 60 % of orders and constituting the most extensive dataset of ciliate species to date. We established a robust phylogenetic framework that encompasses ambiguous taxa and the major classes within the phylum. Our findings support the monophyly of each of two subphyla (Postciliodesmatophora and Intramacronucleata), along with three subclades (Protocruzia, CONTHREEP, and SAPML) nested within Intramacronucleata, and elucidate evolutionary positions among the major classes within the phylum. Drawing on the robust ciliate Tree of Life and three constraints, we estimated the radiation of Ciliophora around 1175 Ma during the middle of the Proterozoic Eon, and most of the ciliate classes diverged from their sister lineage during the latter half of this period. Additionally, based on the time-calibrated tree and species richness pattern, we investigated net diversification rates of Ciliophora and its classes. The global net diversification rate for Ciliophora was estimated at 0.004979 species/Ma. Heterogeneity in net diversification rates was evident at the class level, with faster rates observed in Oligohymenophorea and Spirotrichea than other classes within the subclades CONTHREEP and SAPML, respectively. Notably, our analysis suggests that variations in net diversification rates, rather than clade ages, appear to contribute to the differences in species richness in Ciliophora at the class level. [ABSTRACT FROM AUTHOR]

Rustic aquaculture systems represent a sustainable production alternative in the world, providing appropriate conditions for the establishment of epibiotic ciliates on crayfish. However, the impact of epibiotic ciliates on the aquaculture production has been insufficiently studied hitherto. The objective of this work was to identify the epibiotic ciliates on the surface of the crayfish Procambarus (Austrocambarus) sp. and analyze whether there is any correlation between seasonality and environmental conditions in an artificial pond in Chiapas, Mexico. The crayfish were collected during dry and rainy seasons to investigate their ciliate epibionts. Some physical and chemical parameters of the sampled water were measured and correlation matrix analyses were applied to detect possible relationships between the presence of ciliates and environmental parameters. Thirteen species belonging to the subclasses Peritrichia and Suctoria were identified. Significant differences were found for some environmental parameters and species recorded in both seasons. The present data could help to better understand the relationship bewteen the epibiotic ciliate community on crayfish and some environmental parameters in aquaculture systems. [ABSTRACT FROM AUTHOR]

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Ciliates have a long history of being central in evolutionary and ecological studies on eukaryotic microorganisms. Although thousands of species have been discovered, their total diversity still remains unknown. Here, we will discuss two unsolved problems that hinder the further exploration of ciliate diversity at the species level, and potential solutions to these problems are proposed. First, ciliate morphospecies are difficult to identify because the different silver stains are not scalable (they do not represent high-throughput methods) and basic supplies are lacking (e.g., protargol); a solution may be the development of fluorescent staining techniques. Second, ciliate phylogenetic species are difficult to identify because of extensive paralogy in nuclear-protein-coding genes; a solution may be to concentrate on sequencing mitochondrial genomes. These two approaches could be integrated into a high-throughput fluorescent-single-cell sorting and mitochondrial genomes sequencing process that would enable the observation and better understanding of ciliate species on a massive scale. [ABSTRACT FROM AUTHOR]

The spatial patterns of planktonic ciliate communities were studied from May to June 2019 in the Nile Delta's Damietta region, southeastern Mediterranean. The ciliate communities were sampled from twenty-five sites of five stressed domains with spatial gradients of environmental status. A total of 32 ciliate taxa with six dominant species were identified, comprising 21 tintinnids and 11 aloricate ciliates. The abundance and richness of each ciliate group varied geographically and were most strongly influenced by salinity variations; tintinnid ciliates attained high abundance and richness at high salinity sites in the harbour and coastal region and decreased within the estuary upstream. Aloricate ciliates were poorly represented at most sites but were a substantial proportion of upstream estuarine sites. Multivariate/univariate analyses demonstrated that spatial patterns of the ciliate communities were significantly correlated with environmental variables, especially salinity, chlorophyll-a, and nutrients, either alone or in combination with one another. These results indicate that the ciliates can be useful bioindicators in stressed environments while also allowing the detection of impacts on short time scales by rapidly responding to environmental variations., (© 2024. The Author(s).)

Ciliated protozoa (ciliates) are an ecologically important group of microeukaryotes that play roles in the flow of energy and nutrients in aquatic and terrestrial ecosystems. The community distribution and diversity of soil ciliates in the Nianchu River Basin were investigated by sampling four major habitats, i.e., grassland, farmland, wetland and sea buckthorn forest during May, August and October 2020. Cultivation identification and enumeration of soil ciliates were performed by the non-submerged culture method, in vivo observations and protargol silver staining, and direct counting methods, respectively. A total of 199 species were identified representing, 89 genera, 67 families, 31 orders and 11 classes. Haptorida was the dominant group with 35 species, accounting for 17.59% of the total. The results showed that the α and β diversity indices of soil ciliate communities in the Nianchu River Basin varied significantly in spatial distribution, but not in temporal distribution. Mantel test showed that soil water content, total nitrogen and organic matter were significantly correlated with soil ciliates. Soil water content was the main environmental factor driving the spatial distribution of soil ciliates. Co-occurrence network analysis showed that soil ciliate species in the Nianchu River Basin depend on each other in the relationship of solidarity and cooperation or ecological complementarity. Thus maintaining or enhancing the diversity and stability of the community. Community assembly shows that randomness process was an important ecological process driving soil ciliate community construction in the Nianchu River Basin., Competing Interests: The authors declare no competing interests., (Copyright: © 2024 Zhu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Ciliate protozoa are key members of the microbial community of the rumen. Their study is important to the health and productivity of cattle, which are their hosts. However, there have been persistent challenges in culturing this microbial group in the laboratory. This review will sum up recent advances along with these persistent challenges. Protozoa have been maintained in three types of cultures (ex vivo, in vitro batch, in vitro continuous). Ex vivo cultures are prepared readily from rumen contents by washing away contaminating cells (e.g., bacteria). They have been useful in making basic observations of metabolism, such as which types of fermentation products protozoa form. However, these cultures can be maintained for only short periods (minutes or hours). In vitro batch and in vitro continuous cultures can be used in longer experiments (weeks or longer). However, it is not currently possible to maintain protozoa in these cultures unless bacteria are also present. We conclude the review with a protocol for preparing ex vivo cultures of protozoa. Our protocol has been standardized and used successfully across animal diets, users, and institutions. We anticipate this review will prepare others to culture rumen ciliate protozoa and reach new insights into this important microbial group., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

[Display omitted] • The last common ancestor for Ciliophora was likely a free-living organism. • Symbiotic relationships are recent in the evolution of Ciliophora phylum. • The parasitism emerged several times and independently within Ciliophora phylum. • The parasite ciliates originated from free-living and mutualists ancestral ciliates. • Ciliates associated host increase the rate of diversification in Ciliophora. Ciliophora is a phylum that is comprised of extremely diverse microorganisms with regard to their morphology and ecology. They may be found in various environments, as free-living organisms or associated with metazoans. Such associations range from relationships with low metabolic dependence such as epibiosis, to more intimate relationships such as mutualism and parasitism. We know that symbiotic relationships occur along the whole phylogeny of the group, however, little is known about their evolution. Theoretical studies show that there are two routes for the development of parasitism, yet few authors have investigated the evolution of these characteristics using molecular tools. In the present study, we inferred a wide dated molecular phylogeny, based on the 18S rDNA gene, for the entire Ciliophora phylum, mapped life habits throughout the evolutionary time, and evaluated whether symbiotic relationships were linked to the variation in diversification rates and to the mode of evolution of ciliates. Our results showed that the last common ancestor for Ciliophora was likely a free-living organism, and that parasitism is a recent adaptation in ciliates, emerging more than once and independently via two distinct routes: (i) a free-living ciliate evolved into a mutualistic organism and, later, into a parasitic organism, and (ii) a free-living ciliate evolved directly into a parasitic organism. Furthermore, we have found a significant increase in the diversification rate of parasitic and mutualistic ciliates compared with their free-living conspecifics. The evolutionary success in different lineages of symbiont ciliates may be associated with many factors including type and colonization placement on their host, as well as physical and physiological conditions made available by the hosts. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine if rumen protozoa could form large amounts of reserve carbohydrate compared to the amounts formed by bacteria when competing for glucose in batch cultures. We separated large protozoa and small bacteria from rumen fluid by filtration and centrifugation, recombined equal protein masses of each group into one mixture, and subsequently harvested (reseparated) these groups at intervals after glucose dosing. This method allowed us to monitor reserve carbohydrate accumulation of protozoa and bacteria individually. When mixtures were dosed with a moderate concentration of glucose (4.62 or 5 mM) (n = 2 each), protozoa accumulated large amounts of reserve carbohydrate; 58.7% (standard error of the mean [SEM], 2.2%) glucose carbon was recovered from protozoal reserve carbohydrate at time of peak reserve carbohydrate concentrations. Only 1.7% (SEM, 2.2%) was recovered in bacterial reserve carbohydrate, which was less than that for protozoa (P < 0.001). When provided a high concentration of glucose (20 mM) (n = 4 each), 24.1% (SEM, 2.2%) of glucose carbon was recovered from protozoal reserve carbohydrate, which was still higher (P = 0.001) than the 5.0% (SEM, 2.2%) glucose carbon recovered from bacterial reserve carbohydrate. Our novel competition experiments directly demonstrate that mixed protozoa can sequester sugar away from bacteria by accumulating reserve carbohydrate, giving protozoa a competitive advantage and stabilizing fermentation in the rumen. Similar experiments could be used to investigate the importance of starch sequestration.

Integral membrane transport proteins homologous to those found in the Transporter Classification Database (TCDB; www.tcdb.org) were identified and bioinformatically characterized by transporter class, family, and substrate specificity in three ciliates, Paramecium tetraurelia (Para), Tetrahymena thermophila (Tetra), and Ichthyophthirius multifiliis (Ich). In these three organisms, 1,326 of 39,600 proteins (3.4%), 1,017 of 24,800 proteins (4.2%), and 504 out of 8,100 proteins (6.2%) integral membrane transport proteins were identified, respectively. Thus, an inverse relationship was observed between the % transporters identified and the number of total proteins per genome reported. This surprising observation provides insight into the evolutionary process, giving rise to genome reduction following whole genome duplication (as in the case of Para) or during pathogenic association with a host organism (Ich). Of these transport proteins in Para and Tetra, about 41% were channels (more than any other type of organism studied), 31% were secondary carriers (fewer than most eukaryotes) and 26% were primary active transporters, mostly ATP-hydrolysis driven (more than most other eukaryotes). In Ich, the number of channels was selectively reduced by 66%, relative to Para and Tetra. Para has four times more inorganic anion transporters than Tetra, and Ich has nonselectively lost most of these. Tetra and Ich preferentially transport sugars and monocarboxylates while Para prefers di- and tricarboxylates. These observations serve to characterize the transport proteins of these related ciliates, providing insight into their nutrition and metabolism.

The presence of parasitic ciliates of the hymenostome genus Tetrahymena was examined in 150 mollusks belonging to six bivalve and 13 gastropod species in Slovakia, Central Europe. Tetrahymenids were detected only in two species, viz., in the invasive Lusitanian slug (Arion vulgaris) and in the native swollen river mussel (Unio tumidus). Although only 10.52% of the examined mollusk taxa were positive, their Tetrahymena infections were very intensive accounting for several hundreds of ciliates per host. Phylogenetic analyses of the 16S and 18S rRNA genes as well as of the barcoding region of the gene encoding for cytochrome c oxidase subunit I revealed that both isolates represent new taxa, T. foissneri sp. n. and T. unionis sp. n. The former species belongs to the 'borealis' clade and its nearest relative is T. limacis, a well-known parasite of slugs and snails. Besides molecular data, T. foissneri can be distinguished from T. limacis also morphologically by the body shape of the parasitic-phase form, dimensions of micronuclei, and the silverline system. On the other hand, T. unionis was classified within the 'paravorax' clade along with T. pennsylvaniensis, T. glochidiophila, and T. nigricans. Although these four species are genetically distinct, T. unionis could be morphologically separated only from T. nigricans by body shape and size. The present study suggests that both aquatic and terrestrial mollusks represent interesting hosts for the discovery of novel Tetrahymena lineages. [ABSTRACT FROM AUTHOR]

Morphogenesis and pattern formation are vital processes in any organism, whether unicellular or multicellular. But in contrast to the developmental biology of plants and animals, the principles of morphogenesis and pattern formation in single cells remain largely unknown. Although all cells develop patterns, they are most obvious in ciliates; hence, we have turned to a classical unicellular model system, the giant ciliate Stentor coeruleus. Here we show that the RNA interference (RNAi) machinery is conserved in Stentor. Using RNAi, we identify the kinase coactivator Mob1--with conserved functions in cell division and morphogenesis from plants to humans-as an asymmetrically localized patterning protein required for global patterning during development and regeneration in Stentor. Our studies reopen the door for Stentor as a model regeneration system.