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Nuclear dimorphism is a fundamental feature of ciliated protozoa, which have separate somatic and germline genomes in two distinct organelles within a single cell. The transcriptionally active somatic genome, contained within the physically larger macronucleus, is both structurally and functionally different from the silent germline genome housed in the smaller micronucleus. This difference in genome architecture is particularly exaggerated in Oxytricha trifallax, in which the somatic genome comprises tens of thousands of gene-sized nanochromosomes maintained at a high and variable ploidy, while the germline has a diploid set of megabase-scale chromosomes. To examine the compositional differences between the nuclear structures housing the genomes, we performed a proteomic survey of both types of nuclei and of macronuclear histones using quantitative mass spectrometry. We note distinct differences between the somatic and germline nuclei, with many functional proteins being highly enriched in one of the two nuclei. To validate our conclusions and the efficacy of nuclear separation, we used protein localization through a combination of transformations and immunofluorescence. We also note that the macronuclear histones strikingly display only activating marks, consistent with the conclusion that the macronucleus is the hub of transcription. These observations suggest that the compartmentalization of different genome features into separate structures has been accompanied by a similar specialization of nuclear components that maintain and facilitate the functions of the genomes specific to each nucleus.

Cleavage of dimethylsulfoniopropionate (DMSP) can deter herbivores in DMSP-producing eukaryotic algae; however, it is unclear whether a parallel defence mechanism operates in marine bacteria. Here we demonstrate that the marine bacterium Puniceibacterium antarcticum SM1211, which does not use DMSP as a carbon source, has a membrane-associated DMSP lyase, DddL. At high concentrations of DMSP, DddL causes an accumulation of acrylate around cells through the degradation of DMSP, which protects against predation by the marine ciliate Uronema marinum. The presence of acrylate can alter the grazing preference of U. marinum to other bacteria in the community, thereby influencing community structure. The marine bacterium Puniceibacterium antarcticum SM1211 can produce acrylate from dimethylsulfoniopropionate (DMSP) cleavage by the DMSP lyase, DddL, which protects against grazing by a ciliate predator.

Fossil microeukaryotes are key elements for understanding ancient ecosystems at microscopic level and improving the knowledge on the diversification of microbial life as a whole. We describe Palaeohypothrix bahiensis gen. et sp. nov., an exceptionally well-preserved Lower Cretaceous (Berriasian–Barremian; 145–125 Mya) amber-entrapped microeukaryote, identified as a spirotrich ciliate. The preservation of structures interpreted as the nuclear apparatus and remains of the ciliature revealed a novel ground plan, not found in modern Spirotrichea, thus representing a putatively extinct higher taxon lineage, viz. the Palaeohypotricha nov. tax. Based on cladistic analysis, the new taxon is hypothesized as phylogenetically related to the Protohypotrichia.

Our knowledge of ciliate endosymbiont diversity greatly expanded over the past decades due to the development of characterization methods for uncultivable bacteria. Chlamydia-like bacteria have been described as symbionts of free-living amoebae and other phylogenetically diverse eukaryotic hosts. In the present work, a systematic survey of the bacterial diversity associated with the ciliate Euplotes octocarinatus strain Zam5b-1 was performed, using metagenomic screening as well as classical full-cycle rRNA approach, and a novel chlamydial symbiont was characterized. The metagenomic screening revealed 16S rRNA gene sequences from Polynucleobacter necessarius, three previously reported accessory symbionts, and a novel chlamydia-like bacterium. Following the full-cycle rRNA approach, we obtained the full-length 16S rRNA gene sequence of this chlamydia-like bacterium and developed probes for diagnostic fluorescence in situ hybridizations. The phylogenetic analysis of the 16S rRNA gene sequences unambiguously places the new bacterium in the family Rhabdochlamydiaceae. This is the first report of chlamydia-like bacterium being found in Euplotes. Based on the obtained data, the bacterium is proposed as a new candidate genus and species: "Candidatus Euplotechlamydia quinta."

The objective of this research was to establish loop-mediated isothermal amplifications (LAMP) that could be used to detect parasitic ciliate Ichthyophthirius multifiliis (I. multifiliis) in freshwater cyprinid fish. Primers were developed from the distinguishing fragments of 18S ribosomal RNA of I. multifiliis and the LAMP test was then used to evaluate and optimize various concentrations of chemicals, time and temperature. The results indicated that LAMP required 1.6 μM of FIP primers and BIP primers, 0.2 μM of F3 and B3, 2 mM of Mg2+, 1 M of Betaine, and 0.6 mM of dNTP. This assay was able to detect parasite DNA within a 40 min period of incubation and at a constant optimal temperature of 64oC. The positive sample appeared as a clear ladder like pattern on gel electrophoresis, while a yellowish green color appeared with SYBR Green I under ultraviolet light with the use of a heating block. The LAMP test was determined to be more sensitive than conventional PCR in the detection of I. multifiliis. In conclusion, we have presented a sensitive and specific rapid detection system for I. multifiliis based on isothermal DNA amplification. Importantly, this system could then be employed as an alternative and effective diagnostic method in place of other molecular techniques.

The aetiological agents of red sore disease (RSD) reportedly comprise a taxonomically ambiguous stalked ciliate (a species of Epistylis) and Aeromonas hydrophila. The taxonomic identity of each pathogen remains provisional: using supra-specific morphological features for the ciliate and culture-based methods that cannot delineate bacterial strain. On 7 and 9 November 2017 and 28 May 2020, biologists and anglers reported a local epizootic (Hiwassee and Chattahoochee river basins; Georgia) wherein some moribund fish presented RSD-like lesions. The ciliates were assigned to Epistylis by morphology. The ciliate is regarded as Epistylis cf wuhanensis, as nucleotide sequences from its small subunit ribosomal DNA were identical to those of Epistylis wuhanensis. The bacterium was identified as Aeromonas hydrophila by phenotypic markers and nucleotide sequences from the DNA gyrase subunit B; our sequences comprised 3 strains and phylogenetically were recovered sister to strains of Eurasian origin. Histological sections of lesions revealed effacement or partial deterioration of the epithelium covering scales, scale loss, haemorrhaging, necrosis, oedema, and extensive inflammatory infiltrate in the dermis. This is the first nucleotide sequence information for the symbionts implicated in RSD.

Background Heterotrichous ciliates are common members of microeukaryote communities which play important roles in both the transfer of material and the flow of energy in aquatic food webs. This group has been known for over two centuries due to their large body size and cosmopolitan distribution. Nevertheless, species identification and phylogenetic relationships of heterotrichs remain challenging due to the lack of accurate morphological information and insufficient molecular data. Results The morphology and phylogeny of two heterotrichous ciliates, namely Gruberia foissneri spec. nov. and Linostomella vorticella (Ehrenberg, 1833) Aescht in Foissner et al., 1999, were studied using rigorous methods (living morphology, stained preparations, and small subunit rDNA sequence data). Gruberia foissneri spec. nov. is morphologically very similar to G. uninucleata Kahl, 1932, however, it can be distinguished from the latter by having more ciliary rows (about 32 vs. about 20) and macronuclear shape (sausage-shaped vs. ellipsoid). Based on a combination of previous and present studies, an improved diagnosis of L. vorticella is supplied and several taxonomic anomalies are clarified. In addition, phylogenetic analyses based on SSU rDNA sequence data support the generic assignment of these two species. Conclusions Modern ciliate taxonomy should be performed by means of detailed living observation, stained preparations and molecular information. For those species that have been reported in previous studies, it is necessary to provide as much useful information as possible using state-of-the-art methods in order to resolve taxonomic anomalies.

The spirotrichean ciliate Stylonychia notophora has previously been recorded in India although the descriptions are lacking in detail. It has been suggested several times that the Indian population, S. notophora sensu Sapra and Dass, 1970 collected along the Delhi stretch of the River Yamuna, is identical to Tetmemena pustulata, but this has never been confirmed due to insufficient data for the former. The present study includes detailed descriptions (classical and molecular) of populations of Tetmemena isolated from six locations along the River Yamuna, India. These include four from the Delhi stretch including that from which Sapra and Dass, 1970 isolated their population of S. notophora. Due to the lack of a sufficiently detailed description, the taxonomic status of S. notophora sensu Sapra and Dass, 1970 was not clear. Comparisons among the populations isolated in the present study with previous descriptions of T. pustulata and S. notophora sensu Sapra and Dass, 1970 show only minor differences in morphometry, morphogenesis and in 18S rDNA sequences. The 18S rDNA sequences of all six populations had 99% similarity to both T. pustulata and S. notophora. These findings support the contention that S. notophora sensu Sapra and Dass, 1970 was misidentified and is a population of T. pustulata. This study supports the need for adopting an integrative approach based on morphological, morphogenetic and molecular data in order to understand species delimitation in ciliated protists.

Intraflagellar transport (IFT) represents a bidirectional dynamic process that carries cargo essential for cilia building and the maintenance of ciliary function, which is important for the locomotion of single cells, intracellular and intercellular signalling transduction. Accumulated evidence has revealed that defects in IFT cause several clinical disorders. Here, we determined the role of IFT80, an IFT-B protein that is mutated in Jeune asphyxiating thoracic dystrophy. Using the RNAi method in the ciliate Paramecium as model, we found that loss of IFT80 prevents cilia biogenesis and causes strong cell lethality. A specific antibody against IFT80 was also prepared in our study, which labelled IFT80 in cilia of Paramecium. GFP fusion experiments were performed to illustrate the dynamic movement of IFT-A and IFT-B proteins in cilia of Paramecium; then, we found that the depletion of IFT80 in cells prevents IFT-A and IFT-B proteins from entering the cilia. Our results showed the distribution change of other IFT proteins in cells that were depleted of IFT80, and we discuss the possible roles of IFT80 in Paramecium.

Tintinnid ciliates build loricae, whose structure, shape, and size still largely represent the basis for taxonomy and classification, although genetic analyses demonstrated their limited utility for inferring evolutionary relationships. The textures of the lorica walls, however, result from the chemical and physical properties of the forming material, which is supposed to be rather conserved in closely related taxa, viz., congeners and confamilial genera. Within a particular texture, small deviations in the chemical composition might affect the wall's stickiness and accordingly its capability to adhere foreign particles, explaining the intertwining of tintinnids with hyaline and agglutinated loricae in phylogenetic inferences. In a comprehensive comparative study, the lorica textures were electron microscopically and morphometrically analyzed in 21 species from 17 genera and more than nine families together with literature data. Most species were investigated for the first time, and the taxa cover a substantial portion of the molecular genealogy. The phylogeny-aware analysis of the lorica-related features provides a preliminary hypothesis on lorica evolution. Eventually, this conspectus suggests the dominance of hard lorica walls with an alveolar texture and proposes different modes of lorica formation.

Calcium (Ca2+) is a universal second messenger that plays a key role in cellular signaling. However, Ca2+ signals are transduced with the help of Ca2+-binding proteins, which serve as sensors, transducers, and elicitors. Among the collection of these Ca2+-binding proteins, calmodulin (CaM) emerged as the prototypical model in eukaryotic cells. This is a small protein that binds four Ca2+ ions and whose functions are multiple, controlling many essential aspects of cell physiology. CaM is universally distributed in eukaryotes, from multicellular organisms, such as human and land plants, to unicellular microorganisms, such as yeasts and ciliates. Here, we review most of the information gathered on CaM in Paramecium, a group of ciliates. We condense the information here by mentioning that mature Paramecium CaM is a 148 amino acid-long protein codified by a single gene, as in other eukaryotic microorganisms. In these ciliates, the protein is notoriously localized and regulates cilia function and can stimulate the activity of some enzymes. When Paramecium CaM is mutated, cells show flawed locomotion and/or exocytosis. We further widen this and additional information in the text, focusing on genomic data. Junta de Andalucía - BIO-210 Gobierno de España -Plan Estatal 2017-2020 PID2019-103850GB-I00 Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid - B2017/BMD-36

In the genus Blepharisma, reproductive isolation between different species appears to be established at least by two barriers: (1) a mating pheromone, i.e., gamone 1, and (2) a factor involved in pair formation. Using four species, we experimentally investigated other potential barriers to interspecific conjugation in Blepharisma, as well as the first and second barriers. Cell-free fluid from type I cells (CFF1) of B. americanum had no effect on B. undulans, B.japonicum, or B.stoltei. Type II cells of B. americanum responded to CFF1 from B. americanum but not to CFF1 from B. undulans, B.japonicum, or B.stoltei. Gamone 1, therefore, seems to be the first reproductive barrier (with the inclusion of B. americanum species [megakaryotype 3]) as reported previously. In pretreated cells with complementary gamones in B. undulans and B.americanum, inter-species pair formation was rare, but pair formation between B. americanum and B.japonicum and between B. americanum and B. stoltei occurred at relatively high frequency. Most of the inter-species B.americanum–B. stoltei pairs underwent nuclear changes specific to conjugation. No significant difference was observed between the intra- and inter-species pairs over the time course of the nuclear changes, but the percentage of abnormal cells was higher in inter-species pairs than in intra-species pairs, and no progenies were produced by inter-species pairs. These results suggest a third barrier or step, in addition to the first and second ones, in nuclear changes after pair formation that prevents interspecific conjugation in Blepharisma.

Relationships between ribotypic and phenotypic traits of protists across life cycle stages remain largely unknown. Herein, we used single cells of two soil and two marine ciliate species to examine phenotypic and ribotypic traits and their relationships across lag, log, plateau, cystic stages and temperatures. We found that Colpoda inflata and Colpoda steinii demonstrated allometric relationships between 18S ribosomal DNA (rDNA) copy number per cell (CNPC), cell volume (CV), and macronuclear volume across all life cycle stages. Integrating previously reported data of Euplotes vannus and Strombidium sulcatum indicated taxon-dependent rDNA CNPC-CV functions. Ciliate and prokaryote data analysis revealed that the rRNA CNPC followed a unified power-law function only if the rRNA-deficient resting cysts were not considered. Hence, a theoretical framework was proposed to estimate the relative quantity of resting cysts in the protistan populations with total cellular rDNA and rRNA copy numbers. Using rDNA CNPC was a better predictor of growth rate at a given temperature than rRNA CNPC and CV, suggesting replication of redundant rDNA operons as a key factor that slows cell division. Single-cell high-throughput sequencing and analysis after correcting sequencing errors revealed multiple rDNA and rRNA variants per cell. Both encystment and temperature affected the number of rDNA and rRNA variants in several cases. The divergence of rDNA and rRNA sequence in a single cell ranged from 1% to 10% depending on species. These findings have important implications for inferring cell-based biological traits (e.g., species richness, abundance and biomass, activity, and community structure) of protists using molecular approaches. IMPORTANCE Based on phenotypic traits, traditional surveys usually characterize organismal richness, abundance, biomass, and growth potential to describe diversity, organization, and function of protistan populations and communities. The rRNA gene (rDNA) and its transcripts have been widely used as molecular markers in ecological studies of protists. Nevertheless, the manner in which these molecules relate to cellular (organismal) and physiological traits remains poorly understood, which could lead to misinterpretations of protistan diversity and ecology. The current research highlights the dynamic nature of cellular rDNA and rRNA contents, which tightly couple with multiple phenotypic traits in ciliated protists. We demonstrate that quantity of resting cysts and maximum growth rate of a population can be theoretically estimated using ribotypic trait-based models. The intraindividual sequence polymorphisms of rDNA and rRNA can be influenced by encystment and temperature, which should be considered when interpreting species-level diversity and community structure of microbial eukaryotes.

Very few studies exist on the description of protozoan ciliates from industrially contaminated sites. In this study, we report a description of a novel hypotrich ciliate isolated from water samples collected from an industrially contaminated outlet in Onsan, Ulsan, South Korea. The oxytrichid ciliate, Histriculus tolerans n. sp., was investigated using live observation and protargol impregnation. The morphology, morphogenesis, and molecular phylogeny inferred from small-subunit (SSU) rRNA gene sequences were studied. The new species is mainly characterized by a cell size of about 70 × 40 μm in vivo, two elongate ellipsoidal macronuclear nodules and one or two micronuclei, adoral zone of about 51% of body length with 32 membranelles on average, about 34 cirri in the right and 24 cirri in the left marginal row, 18 frontoventral transverse cirri, six dorsal kineties including two dorsomarginal rows, and dorsal kinety 1 with 26 bristles. Morphogenesis is similar to that of the type species, i.e., Histriculus histrio, except that oral primordium does not contribute to anlage II of the proter. Phylogenetic analyses, based on small-subunit rRNA gene sequences, consistently place the new species within the family Oxytrichidae, clustering with H. histrio.

The morphology and ciliature of a new freshwater ciliate, Frontonia apoacuminata sp. nov., isolated from an artificial pond in Qingdao, PR China, were investigated using live observation and silver staining methods. The main features separating F. apoacuminata sp. nov. from its congeners are as follows: a broad elliptical body that is slightly pointed at the posterior end, four ophryokineties, one or two spherical micronuclei of a ‘compact’ type, a dorsally positioned contractile vacuole, and peniculi 1–3 each with five kinetosome rows though the left-most two rows in peniculus 3 are extremely shortened (with only two or three kinetosomes each). Additionally, an improved diagnosis of F. acuminata is provided. Phylogenetic analyses based on the small subunit ribosomal RNA (SSU rRNA) gene show that F. apoacuminata sp. nov. clusters with F. atra, F. minuta, F. acuminata and F. terricola. These five species group with Disematostoma, Marituja and Stokesia rather than with other Frontonia species, causing polyphyly of the genus Frontonia.

A new species of Protocruzia, isolated from the deep-sea Pacific Ocean (>3,000-m depth) in the vicinity of the Mariana Trench, is described based on morphological and molecular data. The systematic status of the ciliate genus Protocruzia has long been highly ambiguous, and Protocruzia species have been assigned to an independent class until recently. In the present study, we described Protocruzia marianaensis sp. n. as a small (25–32 × 14–17 μm in vivo) drop-shaped ciliate, with longitudinal furrows along the ciliary rows on the right side, six adoral membranelles, eight somatic kineties, and one macronucleus comprising 7–11 nuclear globules. Phylogenetic analyses inferred from small subunit rRNA gene sequences revealed that seven Protocruzia species in the phylogenetic tree formed a fully supported clade representing an independent class. Protocruzia marianaensis sp. n. was established to be most closely related to Protocruzia adhaerens, with a sequence similarity of 96.64%, and was found to be able to survive at both atmospheric pressure and hydrostatic pressure of 320 bar, thereby indicating effective barotolerance.

Tetrahymena rostrata is a free-living ciliated protozoan and is a facultative parasite of some species of terrestrial mollusks. It is a potential biopesticide of pest slugs, such as the grey field slug, which cause considerable damage to crops. T. rostrata has several developmental forms. Homogeneous preparations of the feeding stage cells (trophonts) and excysted stage cells (theronts) were compared for their ability to infect and kill Deroceras reticulatum slugs. Theronts were more effective and remained viable and infective, even after prolonged starvation.

The Heterotrichea Stein, 1859 are a group of ciliated protists (single-celled eukaryotes) that occur in a wide variety of aquatic habitat where they play important roles in the flow of nutrients and energy within the microbial food web. Many species are model organisms for research in cytology and regenerative biology. In the present study, the morphology and phylogeny of two heterotrich ciliates, namely, Linostomella pseudovorticella n. sp. and Peritromus kahli Villeneuve-Brachon, 1940, collected from subtropical wetlands of China, were investigated using morphological and molecular methods. L. pseudovorticella n. sp. differs from its only known congener, Linostomella vorticella Ehrenberg, 1833 Aescht in Foissner et al., 1999, by having more ciliary rows (48–67, mean about 56 vs. 26–51, mean about 42) and its small-subunit (SSU) rDNA sequence, which shows a 15-bp divergence. Although P. kahli has been reported several times in recent decades, its infraciliature has yet to be described. A redescription and improved diagnosis of this species based on a combination of previous and present data are here supplied. Phylogenetic analyses based on SSU rDNA sequences revealed that the genus Linostomella is positioned within Condylostomatidae, and Peritromidae is sister to Climacostomidae with relatively low support, and the family Spirostomidae is the root branch of the class Heterotrichea.

Ciliates are abundant microplankton that are widely distributed in the ocean. In this paper, the distribution patterns of ciliate diversity in the South China Sea (SCS) were analyzed by compiling community data from previous publications. Based on morphological identification, a total of 592 ciliate species have been recorded in the SCS. The ciliate communities in intertidal, neritic and oceanic water areas were compared in terms of taxonomy, motility and feeding habit composition, respectively. Significant community variation was revealed among the three areas, but the difference between the intertidal area and the other two areas was more significant than that between neritic and oceanic areas. The distributions of ciliates within each of the three areas were also analyzed. In the intertidal water, the community was not significantly different among sites but did differ among habitat types. In neritic and oceanic areas, the spatial variation of communities among different sites was clearly observed. Comparison of communities by taxonomic and ecological traits (motility and feeding habit) indicated that these traits similarly revealed the geographical pattern of ciliates on a large scale in the SCS, but to distinguish the community variation on a local scale, taxonomic traits has higher resolution than ecological traits. In addition, we assessed the relative influences of environmental and spatial factors on assembly of ciliate communities in the SCS and found that environmental selection is the major process structuring the taxonomic composition in intertidal water, while spatial processes played significant roles in influencing the taxonomic composition in neritic and oceanic water. Among ecological traits, environmental selection had the most important impact on distributions.

Understanding the magnitude and causes of isotopic fractionation between organisms and their dietary resources is crucial for gaining knowledge on stable isotope ecology. However, little is known regarding the diet-tissue fractionation values of marine ciliates, which play a critical role in the reconstruction of microbial food webs. In the present study, we conducted experiments on two benthic (Pseudokeronopsis pararubra and Protocruzia labiata) and two pelagic (Strombidium sulcatum and Uronemella filificum) marine ciliates, where they were fed with isotopically constant foods (Chaetoceros calcitrans and Isochrysis galbana) under laboratory culture conditions to determine their carbon and nitrogen isotopic fractionation values (Δ13C and Δ15N). The stable isotope values (δ13C and δ15N) of ciliates for all experiments rapidly increased after the initial feeding, with half-lives ranging from 6.1 to 23.0h for δ13C and from 3.1 to 24.9h for δ15N. The Δ13C and Δ15N for all ciliates represented significantly positive enrichments, with overall mean fractionations of 0.6±0.2 and 1.2±0.4, respectively. Irrespective of the dietary type, both Δ13C and Δ15N were very similar for the same ciliate species. These results suggest that Δ13C and Δ15N for marine ciliates are similar to those found in common marine organisms with very little food-dependent variation. Overall, quantifying the specific isotopic fractionation of marine ciliates is expected to provide fundamental information on the trophic transfer of carbon, nitrogen, and energy flow through the microbial pathway in marine ecosystems.

Balantidium coli (B. coli) is a trophic ciliate of low virulence which can cause dysentery in humans. The organism is cosmopolitan in nature and can be found wherever pigs are present. Disease is mostly seen in developing countries, where water sources are frequently contaminated with porcine or human feces. B. coli can become an opportunistic parasite in individuals with immunosupression in urban environments, where pigs do not play a role in infection. The parasites can be easily identified by their characteristic morphology and rapid spiraling motility.

We investigated how Legionella pneumophila (Lp) JR32 interacts with Anteglaucoma CS11A and Colpoda E6, two ciliates that we isolated from sewage and sink trap sludge, respectively, using a handmade maze device containing a 96-well crafting plate. Our 18S rDNA-based phylogenetic analysis showed that Anteglaucoma CS11A and Colpoda E6 formed distinct clades. Scanning electron microscopy showed that Anteglaucoma CS11A had a bigger-sized body than Colpoda E6 and, unlike Tetrahymena IB (the reference strain), neither ciliate produced pellets, which are extracellular vacuoles. Fluorescence microscopic observations revealed that although the intake amounts differed, all three ciliates rapidly ingested LpJR32 regardless of the presence or absence of the icm/dot virulence genes, indicating that they all interacted with LpJR32. In co-cultures with Anteglaucoma CS11A, the LpJR32 levels were maintained but fell dramatically when the co-culture contained the LpJR32 icm/dot deletion mutant instead. Anteglaucoma CS11A died within 2 days of co-culture with LpJR32, but survived co-culture with the deletion mutant. In co-cultures with Colpoda E6, LpJR32 levels were maintained but temporarily decreased independently of the virulence gene. Concurrently, the Colpoda E6 ciliates survived by forming cysts, which may enable them to resist harsh environments, and by diminishing the sensitivity of trophozoites to Lp. In the Tetrahymena IB co-cultures with LpJR32 or Δicm/dot, the Lp levels were maintained, albeit with temporal decreases, and the Tetrahymena IB levels were also maintained. We conclude that unlike Tetrahymena IB with pellet production, Anteglaucoma CS11A can be killed by LpJR32 infection, and Colpoda E6 can resist LpJR32 infection through cyst formation and the low sensitivity of trophozoites to Lp. Thus, the two ciliates that we isolated had different susceptibilities to LpJR32 infection.

This study shows that flagellate epibionts channel nutrient flows towards their large diatom host.

Tunicothrix halophila n. sp. was discovered in a hypersaline marine sample from Jeju Island, Korea. It is characterized by the highly reduced number of dorsal bristles. In addition, the main character of the genus Tunicothrix (e.g., alveolar layer) is absent/indistinct. To figure out its identity and phylogenetic relationship, we examined the new species based on modern morphological methods and molecular phylogenetic analyses. Since the parabirojimids are of basal position to core hypotrichs and a smaller data set could show incorrect phylogenetic relationships among the hypotrichs, we used a huge data set composed of 1,460 DNA sequences to infer the phylogenetic tree. The reduction of dorsal bristles is very likely a secondarily evolved character in hypotrichs, resulting in the independent phenotypic adaptation in the hypersaline ecosystems as shown in other hypersaline hypotrichs. Furthermore, the so-called right marginal row 1 in other congeners is found to produce a pretransverse and transverse cirrus and thus we recommend using the term frontoventral row. Based on our data, we can justify Tunicothrix halophila n. sp. as a new species; however, despite the phenotypic distinctiveness, we refrain to establish a new genus because of the missing data and the non-monophyly of Tunicothrix.

The morphology and the regulation of cortical pattern associated with the cell size, division, and phylogenetic position of a new hypotrichous ciliate, Quadristicha subtropica n. sp. collected from a freshwater pond in southern China, were investigated. Quadristicha subtropica n. sp. is characterized as follows: size in vivo 60–115 μm × 25–45 μm; 19–21 adoral membranelles; buccal cirrus near anterior end of endoral and paroral; cirrus IV/3 at about level of buccal vertex; right marginal row begins ahead of buccal vertex; 11–16 right and 12–19 left marginal cirri; and dorsal cilia about 5 μm long. The basic morphogenetic process in Q. subtropica n. sp. is consistent with that of the type species, Quadristicha setigera. Phylogenetic analyses based on small subunit ribosomal DNA sequence data reveal that the systematic position of Q. subtropica n. sp. is rather unstable with low support values across the tree and the genus Quadristicha is not monophyletic.

This study aims to perform population analysis of the rumen ciliated protozoa of the free-living European bison (wisent, Bison bonasus, Linnaeus). The samples of the rumen fluid from the 18 bison subjected to the controlled culls within the free-ranging population in the Bialowieza primeval forest in Poland were collected and examined. The examined ciliates population consisted of the species of the families Isotrichidae and Ophryoscolecidae. There were 12 genera (Isotricha, Dasytricha, Diplodinium, Elytroplastron, Entodinium, Eodinium, Epidinium, Eremoplastron, Eudiplodinium, Metadinium, Ophryoscolex, and Ostracodinium) and 32 morphospecies of the ciliates. We observed the prevalence of a type B protozoan population (56% animals) with the typical Epidinium and Eudiplodinium genera members. Other examined animals possessed the mixed A–B population with Ophryoscolex genus, distinct for type A ciliate population. The average total ciliates count was 2.77 ± 1.03 × 105/ml (mean ± SD). The most abundant genera were Entodinium, 83%, and Dasytricha, 14%. The abundance of other genera was Entodinium species determined, the most abundant species was Entodinium nanellum (16.3% of total ciliates count). The average Shannon–Wiener diversity index was 2.1 ± 0.39, evenness was 0.7 ± 0.11, and species richness was 24 ± 3.0 (mean ± SD). Our study is the first report on the population composition and diversity of rumen ciliates of European bison. The composition and counts of ciliate genera and species were similar to the composition and counts of the rumen ciliated protozoa of American bison and many other kinds of free-living and domestic ruminants. Our European bison ciliate population analysis has shown medium ciliate density and high diversity typical for large free-living ruminants with mixed feeding behavior.

The skin mucus is the fish primary defense barrier protecting from infections via the skin epidermis. In a previous study, we have investigated the proteome of common carp (Cyprinus carpio) skin mucus at two different time points (1 and 9 days) post-exposure to Ichthyophthirius multifiliis. Applying a nano-LC ESI MS/MS technique, we have earlier revealed that the abundance of 44 skin mucus proteins has been differentially regulated including proteins associated with host immune responses and wound healing. Herein, in skin mucus samples, we identified six proteins of I. multifiliis associated with the skin mucus in common carp. Alpha and beta tubulins were detected in addition to the elongation factor alpha, 26S proteasome regulatory subunit, 26S protease regulatory subunit 6B, and heat shock protein 90. The identified proteins are likely involved in motility, virulence, and general stress during parasite growth and development after parasite attachment and invasion. Two KEGG pathways, phagosome and proteasome, were identified among these parasite proteins, mirroring the proteolytic and phagocytic activities of this parasite during host invasion, growth, and development, which represent a plausible host invasion strategy of this parasite. The results obtained from this study can support revealing molecular aspects of the interplay between carp and I. multifiliis and may help us understand the I. multifiliis invasion strategy at the skin mucus barrier. The data may advance the development of novel drugs, vaccines, and diagnostics suitable for the management and prevention of ichthyophthiriosis in fish.

In September 2020 when we were investigating about opportunistic free living amoeba in patients with meningitis in Thi –Qar province south of Iraq, throughout examination the CSF which was cultured on NN Agar medium. It was surprisingly we found the ciliates protozoa (Orchitophryidae) in both stages, vegetative and cystic, after that the ciliates were isolated and cultured in vitro and then morphological, molecular and virulence characterizations were studies. The results of morphological study revealed that these ciliates were similar to Mesanophrys sp. whereas the sequence of rDNA of studied ciliate with accession numbers being given. in NCBI LC621170.1 was show (86.04%) homology identity to Mesanophrys sp. 18S rRNA gene (accession number MN260367.1). but show (98.56%) and (97.92%) homology identity to Orchitophrydiae 18S rRNA gene (accession number EF023919.1 and EF023549.1), that may be this ciliates in our study is a new species for Mesanophrys. In addition experimental infection confirmed that Mesanophrys like ciliates was the pathogen that infected human. In summary this study represent the first record of the ciliophora Orchitophryidae (Mesanophrys like ciliates) as a human pathogen from Thi-Qar province / Iraq.

Organic Lake in Antarctica is a marine-derived, cold (−13∘C), stratified (oxic-anoxic), hypersaline (>200 gl–1) system with unusual chemistry (very high levels of dimethylsulfide) that supports the growth of phylogenetically and metabolically diverse microorganisms. Symbionts are not well characterized in Antarctica. However, unicellular eukaryotes are often present in Antarctic lakes and theoretically could harbor endosymbionts. Here, we describe Candidatus Organicella extenuata, a member of the Verrucomicrobia with a highly reduced genome, recovered as a metagenome-assembled genome with genetic code 4 (UGA-to-Trp recoding) from Organic Lake. It is closely related to Candidatus Pinguicocccus supinus (163,218 bp, 205 genes), a newly described cytoplasmic endosymbiont of the freshwater ciliate Euplotes vanleeuwenhoeki (Serra et al., 2020). At 158,228 bp (encoding 194 genes), the genome of Ca. Organicella extenuata is among the smallest known bacterial genomes and similar to the genome of Ca. Pinguicoccus supinus (163,218 bp, 205 genes). Ca. Organicella extenuata retains a capacity for replication, transcription, translation, and protein-folding while lacking any capacity for the biosynthesis of amino acids or vitamins. Notably, the endosymbiont retains a capacity for fatty acid synthesis (type II) and iron–sulfur (Fe-S) cluster assembly. Metagenomic analysis of 150 new metagenomes from Organic Lake and more than 70 other Antarctic aquatic locations revealed a strong correlation in abundance between Ca. Organicella extenuata and a novel ciliate of the genus Euplotes. Like Ca. Pinguicoccus supinus, we infer that Ca. Organicella extenuata is an endosymbiont of Euplotes and hypothesize that both Ca. Organicella extenuata and Ca. Pinguicocccus supinus provide fatty acids and Fe-S clusters to their Euplotes host as the foundation of a mutualistic symbiosis. The discovery of Ca. Organicella extenuata as possessing genetic code 4 illustrates that in addition to identifying endosymbionts by sequencing known symbiotic communities and searching metagenome data using reference endosymbiont genomes, the potential exists to identify novel endosymbionts by searching for unusual coding parameters.

Many protists form cell colonies. Among them several are filter-feeders depending on suspended food particles such as bacteria. It has been suggested that the formation of colonies enhances feeding efficiency and implied that - in the case of colonial choanoflagellates - it was an adaptive trait that led to the evolution of metazoans. Here it is shown experimentally - for a colonial peritrich ciliate and for a choanoflagellate - that colony-formation does not enhance the efficiency of filter-feeding relative to solitary cells and that the adaptive significance of cell colony-formation must have some other explanation.

Ampullofolliculina Hadži, 1951 is a monotypic genus, the only species being A. lageniformis which was first discovered in estuarine habitats in the U.S. and has never been redescribed. In the present study, we investigated the taxonomy and phylogeny of A. lageniformis Hadži, 1951 based on analyses of a population collected from a brackish wetland in Ningbo, China. The main characteristics of this species are as follows: trophont about 450-700 μm long in vivo with two short, rounded peristomial lobes of equal size; lorica flask-shaped, transparent and smooth with a short, wide neck at the base of which two transparent valves are asymmetrically inserted; cortex with brownish to reddish cortical granules and greenish pigment granules; about 80 somatic kineties evenly arranged; moniliform macronucleus with 4-8 ellipsoidal nodules; swarmer dark green, vermiform in shape, about 200-350 μm long in vivo, with about 60 adoral membranelles and 85 somatic kineties, no mouth nor paroral membrane. Phylogenetic analyses inferred from SSU rDNA sequences show that A. lageniformis is closely related to Folliculina and Eufolliculina which nest within a large clade that comprises five families, i.e. Stentoridae, Blepharismidae, Fabreidae, Maristentoridae, and Folliculinidae.

A novel hypotrichous ciliate, Neogastrostyla aqua nov. gen., nov. spec. isolated from River Yamuna, Delhi, India, is described using morphological, morphometric and morphogenetic characters from live and stained cells supplemented with derivation of phylogenetic relationships using SSU rRNA gene. Neogastrostyla aqua nov. gen., nov. spec., is characterized by light yellow cortical granules present singly or in clusters of two to five, distributed randomly; parental adoral zone of membranelles retained unchanged for proter; Oxytricha-pattern of paroral and endoral; 25–27 frontal-ventral-transverse cirri including a distinct bipartite frontoventral row of composite origin; three dorsal kineties with no fragmentation of the third kinety, two dorsomarginal kineties, three caudal cirri, two macronuclear nodules and two or three micronuclei. Though Neogastrostyla aqua nov. gen., nov. spec. has a frontoventral row like Gastrostyla species, it differs distinctly in the combination of characters from Gastrostyla and other genera with Gastrostyla-like ventral ciliature (Protogastrostyla, Hemigastrostyla, Apogastrostyla and Pseudogastrostyla) particularly in the colour and distribution of cortical granules as well as arrangement and formation of ciliature. According to the phylogenetic analyses, Neogastrostyla aqua nov. gen., nov. spec. clustered consistently with Gastrostyla sp. Y2 (no description available), Oxytricha granulifera and Architricha indica as close neighbours.

Ciliates are highly divergent unicellular eukaryotic organisms with nuclear dualism and a highly specialized ciliary pattern. They inhabit all biotopes and play crucial roles in regulating microbial food webs as they prey on bacteria, protists and even on microscopic animals. Nevertheless, subtle morphological differences and tiny sizes hinder proper species identification for many ciliates. In the present review, an attempt has been made to elaborate the various approaches used by modern day ciliate taxonomists for species identification. The different approaches involved in taxonomic characterization of ciliates such as classical (using live-cell observations, staining techniques, etc.), molecular (involving various marker genes) and statistical (delimitation of cryptic species) methods have been reviewed. Ecological and behavioural aspects in species identification have also been discussed. In present-day taxonomy, it is important to use a ‘total evidence’ approach in identifying ciliates, relying on both classical and molecular information whenever possible. This integrative approach will help in the mergence of classical methods with modern-day tools for comprehensive species description in future.

The morphology, morphogenesis and molecular phylogeny of the oxytrichid ciliate, Monomicrocaryon euglenivorum euglenivorum (Kahl, 1932) Foissner, 2016, isolated from freshwater in a seaside park, Guangzhou, China, were investigated. Monomicrocaryon euglenivorum euglenivorum can be recognized as follows: caudal cirri in midline of body; dorsal kinety 1 without a one-kinetid-wide gap; transverse cirri acicular or rod-shaped with a fringed distal end; right marginal row commences at level of buccal vertex or anterior to buccal vertex. The main events during binary fission are as follows: (1) the proter retains the parental adoral zone of membranelles entirely; (2) frontoventral-transverse cirral anlagen I-VI are segmented in the ordinary pattern 1:3:3:3:4:4 from left to right, which form three frontal, four frontoventral, one buccal, three postoral ventral, two pretransverse ventral and five transverse cirri, respectively; (3) dorsal morphogenesis is in the typical Oxytricha-pattern, but fragmentation of dorsal kinety 3 is indistinct; and (4) three caudal cirri are formed, one at the posterior end of each of dorsal kineties 1, 2 and 4. Phylogenetic analyses based on SSU rDNA sequences showed that M. euglenivorum euglenivorum clustered with Kleinstyla dorsicirrata and Heterourosomoida lanceolata rather than with its congener M. elegans. The genus Monomicrocaryon is not monophyletic in this study; however, its monophyly is not rejected by the AU test.

Morphogenesis of the soil hypotrich ciliate, Holostichides chardezi Foissner, 1987, collected from southeastern China, was investigated using the protargol staining method. The main morphogenetic events follow a similar process with that of its congeners. Phylogenetic analyses based on the SSU rDNA sequence data indicate that Holostichides is nonmonophyletic; H. chardezi, the type species of Holostichides, clusters with H. heterotypicus, while H. terrae is distinctly separate from these species. H. terrae can be distinguished from H. chardezi (type species of Holostichides) and H. heterotypicus by undulating membranes relatively long and distinctly curved (vs. relatively short and straight), pharynx with (vs. without) rod-shaped structure, and two (vs. more than two) frontoterminal cirri. Therefore, a new genus, Anteholostichides nov. gen., has been proposed for H. terrae. Further, the diagnosis of the genus Holostichides is improved.