Search

Pleurostomatid ciliates, as a highly diverse and widely distributed unicellular eukaryote group, play a crucial role in the cycling of nutrients and energy in microbial food webs. However, research on pleurostomatids remains insufficient, resulting in a paucity of molecular information and substantial gaps in knowledge of their phylogenetic relationships. In recent years, we investigated pleurostomatid diversity in various Chinese habitats, including their systematic relationships and taxonomic circumscriptions, which were comprehensively analyzed using an integrative morphomolecular approach. Results revealed that: (1) pleurostomatids can be categorized into two suborders, Protolitonotina subord. nov. and Amphileptina Jankowski, 1967; (2) Protolitonotina subord. nov. represents the ancestral pleurostomatid group and includes two genera, Protolitonotus and Heterolitonotus gen. nov. , characterized by right kineties progressively shortened along rightmost full kineties and absence of a left dorsolateral kinety; (3) Heterolitonotus gen. nov. represents an orphan lineage and is defined by an oral slit extending to its dorsal margin; (4) " Protolitonotus clampi " does not group with congeners but instead represents an orphan lineage, thus Novilitonotus gen. nov. is established to which P. clampi is transferred as Novilitonotus clampi comb. nov. ; (5) three new species, Apoamphileptus paraclaparedii sp. nov. , Heterolitonotus rex gen. nov., sp. nov. , and Loxophyllum apohelus sp. nov. , are described; and (6) helices 21es6a to 21es6d within the V4 region of small subunit ribosomal RNA (SSU rRNA) may serve as a useful tool for discriminating pleurostomatids. The evolutionary relationships among all main lineages of pleurostomatids are discussed and a key to the identification of pleurostomatid genera is provided.

The endosymbiont Candidatus Azoamicus ciliaticola was proposed to generate ATP for its eukaryotic host, an anaerobic ciliate of the Plagiopylea class, fulfilling a function analogous to mitochondria in other eukaryotic cells. The discovery of this respiratory endosymbiosis has major implications for both evolutionary history and ecology of microbial eukaryotes. However, with only a single species described, knowledge of its environmental distribution and diversity is limited. Here we report four complete, circular metagenome assembled genomes (cMAGs) representing respiratory endosymbionts inhabiting groundwater in California, Ohio, and Germany. These cMAGs form two lineages comprising a monophyletic clade within the uncharacterized gammaproteobacterial order UBA6186, enabling evolutionary analysis of their key protein complexes. Strikingly, all four cMAGs encode a cytochrome cbb 3 oxidase, which indicates that these endosymbionts have the capacity for aerobic respiration. Accordingly, we detect these respiratory endosymbionts in diverse habitats worldwide, thus further expanding the ecological scope of this respiratory symbiosis., (© 2024. The Author(s).)

Cryptocaryon irritans, a protozoan parasite that infects marine fish, is characterized by a complex life cycle that includes a cyst-forming reproductive phase. However, the composition of the cyst wall and mechanism of its formation remain unclear. In this study, we identified chitin as a key component of the cyst wall using calcofluor white and wheat germ agglutinin, with Fourier-transform infrared spectroscopy confirming its β-form structure. Two chitin synthase genes, CHS1 and CHS2, were identified as being expressed throughout the life cycle and show close phylogenetic relationships with chitin synthase from ciliates. Incubation with specific anti-CHS1 and -CHS2 antibodies significantly reduced both the thickness and chitin content of the cyst wall, highlighting the critical role of these enzymes in chitin biosynthesis. Treatment with benzoylureas, which inhibit chitin synthesis, caused thinning of the cyst wall and downregulation of CHS gene expression, resulting in an 84 % reduction in the hatching rate after treatment with 0.01 mM CuSO 4 compared with control tomonts. Western blot analysis demonstrated that recombinant CHS proteins are immunogenic, and tomonts from CHS-immunized grouper exhibited reduced size. These findings bridge a crucial knowledge gap in understanding of the C. irritans cyst wall and highlight promising targets for infection prevention and control strategies., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Prostomateans, as common inhabitants in diverse aquatic environments, are among the simplest ciliate lineages, and serve as trophic links in food webs. However, only a few members are well-known and thoroughly studied, and the diversity of this group remains elusive. The unique genus Plagiocampa has a long history of research, but few studies have been performed using up-to-date methods. In the present work, Plagiocampa longis Kahl, 1927 and Plagiocampa minima Kahl, 1927, collected from Chinese coastal habitats, were investigated based on microscopical observation, protargol staining, and SSU rRNA gene sequencing. Their ciliature and morphometric data as well as gene sequences are documented. Phylogenetic analyses revealed that the family Plagiocampidae is likely monophyletic and has a closer relationship with parasitic Cryptocaryon., 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 Elsevier GmbH. All rights reserved.)

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., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Protists are diverse single-celled eukaryotes found in various habitats. They exhibit a wide range of forms and functions, representing a significant portion of the eukaryotic tree of life, which also includes animals, plants, and fungi. Due to their high sensitivity to environmental changes, these organisms are widely used as biological indicators of organic pollution., Methods and Results: We investigated the molecular diversity of ciliate protists at seven strategic points along the Sapucaí River (Itajubá, Minas Gerais State, Brazil), to assess the impact of urban pollution on the richness, abundance, and diversity indexes of these communities. For each sampling point, values of physicochemical parameters were also recorded. DNA sequences were obtained by high-throughput sequencing (HTS) and analyzed using the V4 18S-rRNA molecular marker, employing the DNA metabarcoding method. We recorded 125 ciliate taxonomic units (OTUs), with nearly 80% corresponding to the classes Spirotrichea, Oligohymenophorea, and Litostomatea. At the genus level, 54 OTUs (43.2%) were identified, spanning 28 genera., Conclusions: The composition of ciliates varied significantly along the river's course, from upstream to downstream of Itajubá city. Samples collected from the urban area displayed the lowest richness and diversity, corroborating the influence of the pollution gradient on these communities. The physicochemical parameters showed little variation among the samples and were not linked to the observed changes in ciliate communities, revealing that these organisms are strongly affected by environmental changes and respond more sensitively to these disturbances than physicochemical parameters, emphasizing their potential as bioindicators., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Microeukaryotes are key contributors to marine carbon cycling. Their physiology, ecology, and interactions with the chemical environment are poorly understood in offshore ecosystems, and especially in the deep ocean. Using the Autonomous Underwater Vehicle Clio, microbial communities along a 1050 km transect in the western North Atlantic Ocean were surveyed at 10-200 m vertical depth increments to capture metabolic signatures spanning oligotrophic, continental margin, and productive coastal ecosystems. Microeukaryotes were examined using a paired metatranscriptomic and metaproteomic approach. Here we show a diverse surface assemblage consisting of stramenopiles, dinoflagellates and ciliates represented in both the transcript and protein fractions, with foraminifera, radiolaria, picozoa, and discoba proteins enriched at >200 m, and fungal proteins emerging in waters >3000 m. In the broad microeukaryote community, nitrogen stress biomarkers were found at coastal sites, with phosphorus stress biomarkers offshore. This multi-omics dataset broadens our understanding of how microeukaryotic taxa and their functional processes are structured along environmental gradients of temperature, light, and nutrients., (© 2024. The Author(s).)

Background: Tubulins are major components of the eukaryotic cytoskeletons that are crucial in many cellular processes. Ciliated protists comprise one of the oldest eukaryotic lineages possessing cilia over their cell surface and assembling many diverse microtubular structures. As such, ciliates are excellent model organisms to clarify the origin and evolution of tubulins in the early stages of eukaryote evolution. Nonetheless, the evolutionary history of the tubulin subfamilies within and among ciliate classes is unclear., Results: We analyzed the evolutionary pattern of ciliate tubulin gene family based on genomes/transcriptomes of 60 species covering 10 ciliate classes. Results showed: (1) Six tubulin subfamilies (α_Tub, β_Tub, γ_Tub, δ_Tub, ε_Tub, and ζ_Tub) originated from the last eukaryotic common ancestor (LECA) were observed within ciliates. Among them, α_Tub, β_Tub, and γ_Tub were present in all ciliate species, while δ_Tub, ε_Tub, and ζ_Tub might be independently lost in some species. (2) The evolutionary history of the tubulin subfamilies varied. Evolutionary history of ciliate γ_Tub, δ_Tub, ε_Tub, and ζ_Tub showed a certain degree of consistency with the phylogeny of species after the divergence of ciliate classes, while the evolutionary history of ciliate α_Tub and β_Tub varied among different classes. (3) Ciliate α- and β-tubulin isoforms could be classified into an "ancestral group" present in LECA and a "divergent group" containing only ciliate sequences. Alveolata-specific expansion events probably occurred within the "ancestral group" of α_Tub and β_Tub. The "divergent group" might be important for ciliate morphological differentiation and wide environmental adaptability. (4) Expansion events of the tubulin gene family appeared to be consistent with whole genome duplication (WGD) events in some degree. More Paramecium-specific tubulin expansions were detected than Tetrahymena-specific ones. Compared to other Paramecium species, the Paramecium aurelia complex underwent a more recent WGD which might have experienced more tubulin expansion events., Conclusions: Evolutionary history among different tubulin gene subfamilies seemed to vary within ciliated protists. And the complex evolutionary patterns of tubulins among different ciliate classes might drive functional diversification. Our investigation provided meaningful information for understanding the evolution of tubulin gene family in the early stages of eukaryote evolution., (© 2024. The Author(s).)

The morphology and molecular phylogeny of a new ciliate, Conchophthirus sinanodontae n. sp., which was discovered in the freshwater mussel Sinanodonta woodiana (Lea, 1834) from the Chilsancheon River, Buyeo-gun, South Korea, were investigated. The new species was characterized and could be distinguished from congeners by a combination of characters including the ovate body outline, four to six oral polykinetids deeply embedded in the upper wall of the buccal cavity, six to ten vestibular kineties, 34-49 ventral and 36-53 dorsal somatic kineties. The genetic differences among C. sinanodontae n. sp. and other congeners with available 18S rDNA sequences further support its distinctness. Moreover, the phylogenetic analyses based on the 18S rDNA sequences show that the new species clusters with other congeners, corroborating the monophyly of the genus Conchophthirus. The Conchophthirus clade nests within the cluster of Dexiotricha spp., Loxocephalus luridus, and Haptophrya spp., 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 Elsevier GmbH. All rights reserved.)

Coleps is a common genus of pelagic ciliates in freshwater and brackish water habitats. Classification and phylogeny of Coleps species are, however, still full of confusion. In this study, we investigated Coleps shanghaiensis n. sp., collected from a river in Shanghai, China, by living observation, protargol staining, and molecular methods. Coleps shanghaiensis is about 70-90 µm × 35-55 µm in size, has a barrel-shaped body with three posterior spines, and possesses 21-24 ciliary rows, each composed of two perioral dikinetids and 19-22 monokinetids, and six caudal cilia. In SSU rRNA gene phylogenies, C. shanghaiensis fell within the clade of subspecies of Levicoleps biwae, which questions the validity of the genus Levicoleps. Furthermore, the biogeography of the genus Coleps is discussed., 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 Elsevier GmbH. All rights reserved.)

Ciliate protozoa are an integral part of the rumen microbial community involved in a variety of metabolic processes. These processes are thought to be in part the outcome of interactions with their associated prokaryotic community. For example, methane production is enhanced through interspecies hydrogen transfer between protozoa and archaea. We hypothesize that ciliate protozoa are host to a stable prokaryotic community dictated by specific functions they carry. Here, we modify the microbial community by varying the forage-to-concentrate ratios and show that, despite major changes in the prokaryotic community, several taxa remain stably associated with ciliate protozoa. By quantifying genes belonging to various known reduction pathways in the rumen, we find that the bacterial community associated with protozoa is enriched in genes belonging to hydrogen utilization pathways and that these genes correspond to the same taxonomic affiliations seen enriched in protozoa. Our results show that ciliate protozoa in the rumen may serve as a hub for various hydrogenotrophic functions and a better understanding of the processes driven by different protozoa may unveil the potential role of ciliates in shaping rumen metabolism., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Spirostomum is a genus of large ciliates, and its species are distributed worldwide. However, there has been limited research conducted on their geographical distribution and genomics. We obtained nine samples of ciliates from eight regions in Liaoning Province, China, and conducted a study on their geographical distribution and characteristics. Morphological and second-generation high-throughput sequencing methods were applied to identify the species, and a phylogenetic tree was established to gain a deeper understanding of the geographical distribution and evolutionary relationships of Spirostomum in Northeast China. The results identified Spirostomum yagiui and Spirostomum subtilis as a newly recorded species in Northeast China region. There are now five species of Spirostomum that have been recorded in China, and new details on the genomic characteristics of Spirostomum yagiui were provided. In addition, this study also identified the main branches of Spirostomum teres and Spirostomum minus in northern China, and provided a theoretical basis for the existence of hidden species. Spirostomum yagiui is the first species in the family Spirostomidae to have undergone mitochondrial genome sequencing., 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 Elsevier GmbH. All rights reserved.)

The subclass Trichostomatia (Ciliophora, Litostomatea) constitutes a well-supported monophyletic group, which includes ciliates exclusively found as symbionts of vertebrates, primarily herbivorous mammals. Recent molecular analyses reinforce the subclass monophyly, though almost all orders, suborders, families, and genera are found to be non-monophyletic. Here, we reconstructed the evolutionary history of the subclass Trichostomatia using a phylogenomic approach and discussed some systematic inconsistencies. We propose a new Ophryoscolecidae genus, Dagostonium, to include Diplodinium polygonale. Monoposthium cynodontum is transferred to the genus Cycloposthium., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

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., 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. Published by Elsevier Inc.)

Large yellow croaker (L. crocea) is a productive species in marine aquaculture with great economic value in China. However, the sustainable development of large yellow croaker is hampered by various diseases including cryptocaryonosis caused by Cryptocaryon irritans. The genetic regulation processes for cryptocaryonosis in large yellow croaker are still unclear. In this present study, we analyzed differential alternative splicing events between a C. irritans resistance strain (RS) and a commercial strain (CS). We identified 678 differential alternative splicing (DAS) events from 453 genes in RS and 719 DAS events from 500 genes in CS. A set of genes that are specifically alternatively spliced in RS was identified including mfap5, emp1, and trim33. Further pathway analysis revealed that the specifically alternative spliced genes in RS were involved in innate immune responses through the PRR pathway and the Toll and Imd pathway, suggesting their important roles in the genetic regulation processes for cryptocaryonosis in large yellow croaker. This study would be helpful for the studies of the pathogenesis of cryptocaryonosis and dissection of C. irritans resistance for L. crocea., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cryptocaryoniasis, caused by Cryptocaryon irritans, is a significant threat to marine fish cultures in tropical and subtropical waters. However, controlling this disease remains a challenge. Fish infected with C. irritans acquires immunity; however, C. irritans is difficult to culture in large quantities, obstructing vaccine development using parasite cells. In this study, we established a method for expressing an arbitrary protein on the surface of Tetrahymena thermophila, a culturable ciliate, to develop a mimetic C. irritans. Fusing the signal peptide (SP) and glycosylphosphatidylinositol (GPI) anchor sequences of the immobilization antigen, a surface protein of C. irritans, to the fluorescent protein, monomeric Azami-green 1 (mAG1) of the stony coral Galaxea fascicularis, allowed protein expression on the surface and cilia of transgenic Tetrahymena cells. This technique may help develop transgenic Tetrahymena displaying parasite antigens on their cell surface, potentially contributing to the development of vaccines using "mimetic parasites"., (© 2023. The Author(s).)

Lactococcus garvieae has recently been identified and listed as one of the causative agents of hyperacute hemorrhagic sepsis in fish. In intensive recirculating aquaculture systems where there are high fish densities and minimal water changes, not only will it be conducive to the growth of bacteria, but Cryptocaryon irritans as a marine protozoan fish parasite is also prone to appear. This study reports the disease status of Trachinotus ovatus in an aquaculture area in Yangjiang City, Guangdong Province. Through the diagnosis of clinical symptoms of the diseased fish, identification of specific primers, 16s rRNA sequences phylogenetic tree analysis, physiological and biochemical identification, and observation of histopathological sections, the result of the experiment is that the mass death of T. ovatus is caused by a mixture of L. garvieae and C. irritants infections. Subsequently, regression infection experiments were performed to verify Koch's law. It was confirmed that the pathogen had strong virulence to T. ovatus. This is the first time that the co-infection of L. garvieae and C. irritans to T. ovatus was found in South China. The research results of this experiment have certain enlightenment significance for the epidemic trend of fish diseases in relevant sea areas., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Huang 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.)

Mixing of entire microbial communities represents a frequent, yet understudied phenomenon. Here, we mimicked estuarine condition in a microcosm experiment by mixing a freshwater river community with a brackish sea community and assessed the effects of both environmental and community coalescences induced by varying mixing processes on microeukaryotic communities. Signs of shifted community composition of coalesced communities towards the sea parent community suggest asymmetrical community coalescence outcome, which, in addition, was generally less impacted by environmental coalescence. Community stability, inferred from community cohesion, differed among river and sea parent communities, and increased following coalescence treatments. Generally, community coalescence increased alpha diversity and promoted competition from the introduction (or emergence) of additional (or rare) species. These competitive interactions in turn had community stabilizing effect as evidenced by the increased proportion of negative cohesion. The fate of microeukaryotes was influenced by mixing ratios and frequencies (i.e. one-time versus repeated coalescence). Namely, diatoms were negatively impacted by coalescence, while fungi, ciliates, and cercozoans were promoted to varying extents, depending on the mixing ratios of the parent communities. Our study suggests that the predictability of coalescence outcomes was greater when the sea parent community dominated the final community, and this predictability was further enhanced when communities collided repeatedly., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

α,α-trehalose is a well-known sugar that plays a key role in establishing tolerance to environmental stresses in many organisms, except unicellular eukaryotes. However, almost nothing is known about α,β-trehalose, including their synthesis, function, and even presence in living organisms. In this study, we identified α,β-trehalose in the resting cyst, a dormancy cell form characterized by extreme tolerance to environmental stresses, of the ciliated protist Colpoda cucullus, using high-performance liquid chromatography (HPLC), and a proton nuclear magnetic resonance ( 1 H NMR). Gene expression analysis revealed that the expression of trehalose-6-phosphate synthase (TPS), glycosyltransferase (GT), alpha-amylase (AMY), and trehalose transporter 1 (TRET1), were up-regulated in encystment, while the expression of α-glucosidase 2 (AG2) and trehalase (TREH) was up-regulated in excystment. These results suggest that α,β-trehalose is synthesized during encystment process, while and contributes to extreme tolerances to environmental stressors, stored carbohydrates, and energy reserve during resting cyst and/or during excystment., 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 Elsevier Inc. All rights reserved.)

Several hundred ciliate species live in animals' guts as a part of their microbiome. Among them, Muniziella cunhai (Trichostomatia, Pycnotrichidae), the largest described ciliate, is found exclusively associated with Hydrochoerus hydrochaeris (capybara), the largest known rodent reaching up to 90 kg. Here, we present the sequence, structural and functional annotation of this giant microeukaryote macronuclear genome and discuss its phylogenetic placement. The 85 Mb genome is highly AT rich (GC content 25.71 %) and encodes a total of 11 397 protein-coding genes, of which 2793 could have their functions predicted with automated functional assignments. Functional annotation showed that M. cunhai can digest recalcitrant structural carbohydrates, non-structural carbohydrates, and microbial cell walls, suggesting a role in diet metabolization and in microbial population control in the capybara's intestine. Moreover, the phylogenetic placement of M. cunhai provides insights on the origins of gigantism in the subclass Trichostomatia.

While metopids (Armophorea: Metopida) represent the most species-rich group of free-living anaerobic ciliates thriving in hypoxic environments, our understanding of their true diversity remains incomplete. Most metopid species are still characterized only morphologically. Particularly, the so-called IAC clade (named in the past after some of the taxa included, Idiometopus, Atopospira, and Clevelandellida), comprising free-living members as well as the endosymbiotic ones (order Clevelandellida), is in serious need of revision. In our study, we establish a new free-living genus in the IAC clade, Pidimetopus n. gen., with descriptions of two new species, P. nanus n. sp., and P. permonicus n. sp., using up-to-date molecular and morphologic methods. The genus is characterized by small cells (up to 75 μm long), not more than 10 adoral membranelles and eight somatic kineties, and usually, four long caudal cilia that can stiffen. In addition to morphologic and molecular characterizations, we also conducted a statistical morphotype analysis of the polymorphic species P. nanus n. sp. We discuss the relevance of the earlier morphologically described species Metopus minor as a putative collective taxon for several small metopids less than 50 μm long., (© 2024 International Society of Protistologists.)

Microorganisms play a central role in sustaining soil ecosystems and agriculture, and these functions are usually associated with their complex life history. Yet, the regulation and evolution of life history have remained enigmatic and poorly understood, especially in protozoa, the third most abundant group of organisms in the soil. Here, we explore the life history of a cosmopolitan species- Colpoda steinii . Our analysis has yielded a high-quality macronuclear genome for C. steinii , with size of 155 Mbp and 37,123 protein-coding genes, as well as mean intron length of ~93 bp, longer than most other studied ciliates. Notably, we identify two possible whole-genome duplication events in C. steinii , which may account for its genome being about twice the size of C. inflata's , another co-existing species. We further resolve the gene expression profiles in diverse life stages of C. steinii , which are also corroborated in C. inflata . During the resting cyst stage, genes associated with cell death and vacuole formation are upregulated, and translation-related genes are downregulated. While the translation-related genes are upregulated during the excystment of resting cysts. Reproductive cysts exhibit a significant reduction in cell adhesion. We also demonstrate that most genes expressed in specific life stages are under strong purifying selection. This study offers a deeper understanding of the life history evolution that underpins the extraordinary success and ecological functions of microorganisms in soil ecosystems.IMPORTANCE Colpoda species, as a prominent group among the most widely distributed and abundant soil microorganisms, play a crucial role in sustaining soil ecosystems and promoting plant growth. This investigation reveals their exceptional macronuclear genomic features, including significantly large genome size, long introns, and numerous gene duplications. The gene expression profiles and the specific biological functions associated with the transitions between various life stages are also elucidated. The vast majority of genes linked to life stage transitions are subject to strong purifying selection, as inferred from multiple natural strains newly isolated and deeply sequenced. This substantiates the enduring and conservative nature of Colpoda 's life history, which has persisted throughout the extensive evolutionary history of these highly successful protozoa in soil. These findings shed light on the evolutionary dynamics of microbial eukaryotes in the ever-fluctuating soil environments. This integrative research represents a significant advancement in understanding the life histories of these understudied single-celled eukaryotes., Competing Interests: The authors declare no conflict of interest.

Ciliates of the class Plagiopylea play a vital role in various anaerobic environments as consumers of prokaryotes. Yet, the diversity and phylogeny of this group of ciliates, especially marine representatives, remain poorly known. In this study, three Parasonderia species, viz., Parasonderia elongata spec. nov., and the already known P. cyclostoma and P. vestita, discovered in anaerobic sediments from various intertidal zones in China, were investigated based on their living morphology, infraciliature, and small subunit ribosomal rRNA gene sequences. Parasonderia elongata can be recognized by its larger body size, elongated body shape, oval oral opening, number of oral kineties, and significantly shortened leftmost postbuccal polykineties on the cell surface. Improved diagnosis and redescription of P. cyclostoma is provided for the first time, including data on infraciliature and molecular sequence. Phylogenetic analyses revealed that the three species cluster together and with the sequence of a Chinese population of P. vestita already present in the GenBank database, forming a robust clade., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The relationships of the mainly free living, obligately anaerobic ciliated protists belonging to order Metopida continue to be clarified and now comprise three families: Metopidae, Tropidoatractidae, and Apometopidae. The most species-rich genus of the Metopidae, Metopus has undergone considerable subdivision into new genera in recent years as more taxa are characterized by modern morphologic and molecular methods. The genus, Castula, was established to accommodate setae-bearing species previously assigned to Metopus: C. setosa and C. fusca, and one new species, C. flexibilis. Another new species, C. specialis, has been added since. Here we redescribe another species previously included in Metopus, using morphologic and molecular methods, and transfer it to Castula as C. strelkowi n. comb. (original combination Metopus strelkowi). We also reassess the monotypic genus, Pileometopus, which nests within the strongly supported Castula clade in 18S rRNA gene trees and conclude that it represents a morphologically divergent species of Castula., 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 Elsevier GmbH. All rights reserved.)

The Rodrigo de Freitas Lagoon is a highly eutrophic lacustrine system and has one of the longest histories of exploration and anthropic alteration in Brazil. Despite its relevance, limited studies explored the diversity of micro-eukaryotes in the lagoon. Ciliates (Alveolata, Ciliophora) are overlooked in environmental microbiology, especially in tropical and subtropical ecosystems, resulting in limited knowledge about their diversity and functional relevance in South American habitats, particularly in coastal lagoons. To fill this gap, here we investigated the diversity of ciliates in a brackish coastal lagoon in an urban area of Rio de Janeiro, Brazil, applying and comparing the performance of morphological and metabarcoding approaches. The metabarcoding analysis, based on high-throughput sequencing of the hipervariable region V4 of the 18S rRNA genes detected 37 molecular operational taxonomic units (MOTUs) assigned to Ciliophora, representing only about a half (56.9%) of the diversity detected by microscopy, which counted 65 ciliate morphotypes. The most representative classes in both approaches were Spirotrichea and Oligohymenophorea. The metabarcoding analysis revealed that 35.3% of the ciliate MOTUs had less than 97% similarity to available sequences in the NCBI database, indicating that more than one-third of these MOTUs potentially represents still not represented or undescribed ciliate species in current databases. Our findings indicate that metabarcoding techniques can significantly enhance the comprehension of ciliate diversity in tropical environments, but the scarcity of reference sequences of brackish ciliates in molecular databases represents a challenge to the taxonomic assignment of the MOTUs. This study provides new insights into the diversity of ciliates in a threatened coastal lagoon, revealing a vast array of still unknown and rare ciliate taxonomic units in tropical environments., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

The morphology and morphogenesis of Lamtostyla paravitiphila nov. spec., a novel soil hypotrichous ciliate collected from eastern China, were investigated based on live observations and protargol-stained specimens. The new species is morphologically characterized as follows: seven to twelve macronuclear nodules, cortical granules absent, 19-26 adoral membranelles, three or four frontoventral cirri, the amphisiellid median cirral row extends to about mid-body and composed of 12-18 cirri, two or three transverse cirri, 27-39 left and 30-41 right marginal cirri, three almost bipolar dorsal kineties. Morphogenetically, it is characterized by the initial formation of six frontal-ventral-transverse cirral anlagen as primary primordia. Notably, the amphisiellid median cirral row and the posterior frontoventral cirrus (or cirri) contribute to the development of the frontal-ventral-transverse cirral anlagen, while the buccal cirrus may not participate in this process. Phylogenetic analyses based on small subunit ribosomal DNA sequence data indicate that the Lamtostyla species with available molecular data do not form a monophyletic group., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Background: The eukaryotic-bacterial symbiotic system plays an important role in various physiological, developmental, and evolutionary processes. However, our current understanding is largely limited to multicellular eukaryotes without adequate consideration of diverse unicellular protists, including ciliates., Results: To investigate the bacterial profiles associated with unicellular organisms, we collected 246 ciliate samples spanning the entire Ciliophora phylum and conducted single-cell based metagenome sequencing. This effort has yielded the most extensive collection of bacteria linked to unicellular protists to date. From this dataset, we identified 883 bacterial species capable of cohabiting with ciliates, unveiling the genomes of 116 novel bacterial cohabitants along with 7 novel archaeal cohabitants. Highlighting the intimate relationship between ciliates and their cohabitants, our study unveiled that over 90% of ciliates coexist with bacteria, with individual hosts fostering symbiotic relationships with multiple bacteria concurrently, resulting in the observation of seven distinct symbiotic patterns among bacteria. Our exploration of symbiotic mechanisms revealed the impact of host digestion on the intracellular diversity of cohabitants. Additionally, we identified the presence of eukaryotic-like proteins in bacteria as a potential contributing factor to their resistance against host digestion, thereby expanding their potential host range., Conclusions: As the first large-scale analysis of prokaryotic associations with ciliate protists, this study provides a valuable resource for future research on eukaryotic-bacterial symbioses. Video Abstract., (© 2024. The Author(s).)

Background: Predation is a fundamental mechanism for organisms to acquire energy, and various species have evolved diverse tools to enhance their hunting abilities. Among protozoan predators, raptorial Haptorian ciliates are particularly fascinating as they possess offensive extrusomes known as toxicysts, which are rapidly discharged upon prey contact. However, our understanding of the genetic processes and specific toxins involved in toxicyst formation and discharge is still limited., Results: In this study, we investigated the predation strategies and subcellular structures of seven Haptoria ciliate species and obtained their genome sequences using single-cell sequencing technology. Comparative genomic analysis revealed distinct gene duplications related to membrane transport proteins and hydrolytic enzymes in Haptoria, which play a crucial role in the production and discharge of toxicysts. Transcriptomic analysis further confirmed the abundant expression of genes related to membrane transporters and cellular toxins in Haptoria compared to Trichostomatia. Notably, polyketide synthases (PKS) and L-amino acid oxidases (LAAO) were identified as potentially toxin genes that underwent extensive duplication events in Haptoria., Conclusions: Our results shed light on the evolutionary and genomic adaptations of Haptorian ciliates for their predation strategies in evolution and provide insights into their toxic mechanisms., (© 2024. The Author(s).)

The phylogenetic and taxonomic affinities of lineages currently assigned to the non-monophyletic ciliate order Loxocephalida Jankowski (1980) within subclass Scuticociliatia Small (1967) remain unresolved. In the current study, we redescribe the morphology of the type species, Loxocephalus luridus Eberhard (1862) based on two Czech populations and include the first scanning and transmission electron microscopy images of the species. We provide the first 18S rRNA gene sequences for L. luridus and consider its phylogenetic position. Our results support the separation of Dexiotricha from Loxocephalus; however, the former genus is recovered as non-monophyletic. The monophyly of genus Dexiotricha and that of Loxocephalus + Dexiotricha is rejected. Loxocephalus luridus, together with Dexiotricha species, nests within a fully supported clade with Conchophthirus species, long presumed to belong to the Pleuronematida. Haptophrya is recovered as sister to this clade. The monophyly of the Astomatia Schewiakoff (1896) including Haptophrya is rejected. No clear morphologic synapomorphy is identified for the fully supported clade consisting of Haptophrya, Dexiotricha, Loxocephalus, and Conchophthirus., (© 2024 International Society of Protistologists.)

Ciliates are unicellular eukaryotes, regularly involved in symbiotic associations. Symbionts may colonize the inside of their cells as well as their surface as ectosymbionts. Here, we report on a new ciliate species, designated as Zoothamnium mariella sp. nov. (Peritrichia, Sessilida), discovered in the northern Adriatic Sea (Mediterranean Sea) in 2021. We found this ciliate species to be monospecifically associated with a new genus of ectosymbiotic bacteria, here proposed as Candidatus Fusimicrobium zoothamnicola gen. nov., sp. nov. To formally describe the new ciliate species, we investigated its morphology and sequenced its 18S rRNA gene. To demonstrate its association with a single species of bacterial ectosymbiont, we performed 16S rRNA gene sequencing, fluorescence in situ hybridization, and scanning electron microscopy. Additionally, we explored the two partners' cultivation requirements and ecology. Z. mariella sp. nov. was characterized by a colony length of up to 1 mm. A consistent number of either seven or eight long branches alternated on the stalk in close distance to each other. The colony developed three different types of zooids: microzooids ("trophic stage"), macrozooids ("telotroch stage"), and terminal zooids ("dividing stage"). Viewed from inside the cell, the microzooids' oral ciliature ran in 1 ¼ turns in a clockwise direction around the peristomial disc before entering the infundibulum, where it performed another ¾ turn. Phylogenetic analyses assigned Z. mariella sp. nov. to clade II of the family Zoothamnidae. The ectosymbiont formed a monophyletic clade within the Gammaproteobacteria along with two other ectosymbionts of peritrichous ciliates and a free-living vent bacterium. It colonized the entire surface of its ciliate host, except for the most basal stalk of large colonies, and exhibited a single, spindle-shaped morphotype. Furthermore, the two partners together appear to be generalists of temperate, oxic, marine shallow-water environments and were collectively cultivable in steady flow-through systems., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kendlbacher 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.)

During a survey of Korean marine ciliates, Trochilia sigmoides, the type species of the genus Trochilia, was collected and examined using in vivo observation and protargol impregnation. Moreover, scanning electron microscopy and 18S rRNA gene sequencing have been applied for the first time to study this species. Morphologically, T. sigmoides is characterized by the small body size, the oval body outline, and the spiral dorsal ridges. The Korean population of T. sigmoides shows only minute differences to other populations reported in the literature, mainly in body size and the number of dorsal ridges. Phylogenetic analyses based on 18S rRNA gene sequences show that T. sigmoides and T. petrani are placed together with two members of the family Kyaroikeidae, causing the family Dysteriidae to be non-monophyletic. The present new data increase the knowledge about the morphology and phylogeny of the genus Trochilia and would assist in understanding the phylogenetic relationship between the free-living Dysteriidae and the parasitic Kyaroikeidae., 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 Elsevier GmbH. All rights reserved.)

Ciliates play a key role in most ecosystems. Their abundance in natural samples is crucial for answering many ecological questions. Traditional methods of quantifying individual species, which rely on microscopy, are often labour-intensive, time-consuming and can be highly biassed. As a result, we investigated the potential of digital polymerase chain reaction (dPCR) for quantifying ciliates. A significant challenge in this process is the high variation in the copy number of the taxonomic marker gene (ribosomal RNA [rRNA]). We first quantified the rRNA gene copy numbers (GCN) of the model ciliate, Paramecium tetraurelia, during different stages of the cell cycle and growth phases. The per-cell rRNA GCN varied between approximately 11,000 and 130,000, averaging around 50,000 copies per cell. Despite these variations in per-cell rRNA GCN, we found a highly significant correlation between GCN and cell numbers. This is likely due to the coexistence of different cellular stages in an uncontrolled (environmental) ciliate population. Thanks to the high sensitivity of dPCR, we were able to detect the target gene in a sample that contained only a single cell. The dPCR approach presented here is a valuable addition to the molecular toolbox in protistan ecology. It may guide future studies in quantifying and monitoring the abundance of targeted (even rare) ciliates in natural samples., (© 2024 The Authors. Environmental Microbiology published by John Wiley & Sons Ltd.)

Many ciliated protists prey on other large microbial organisms, including other protists and microscopic metazoans. The ciliate class Litostomatea unites both predatory and endosymbiotic species. The evolution of predation ability in ciliates remains poorly understood, in part, due to a lack of genomic data. To fill this gap, we acquired the transcriptome profiles of six predatory litostomateans using single-cell sequencing technology and investigated their transcriptomic features. Our results show that: (1) in contrast to non-predatory ciliates, the predatory litostomateans have expanded gene families associated with transmembrane activity and reactive oxidative stress response pathways, potentially as a result of cellular behaviors such as fast contraction and extension; (2) the expansion of the calcium-activated BK potassium channel gene family, which hypothetically regulates cell contractility, is an ancient evolutionary event for the class Litostomatea, suggesting a rewired metabolism associated with the hunting behavior of predatory ciliates; and (3) three whole genome duplication (WGD) events have been detected in litostomateans, with genes associated with biosynthetic processes, transmembrane activity, and calcium-activated potassium channel activity being retained during the WGD events. In addition, we explored the evolutionary relationships among 17 ciliate species, including eight litostomateans, and provided a rich foundational dataset for future in-depth phylogenomic studies of Litostomatea. Our comprehensive analyses suggest that the rewired cellular metabolism via expanded gene families and WGD events might be the potential genetic basis for the predation ability of raptorial ciliates., 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 Elsevier GmbH. All rights reserved.)

Individual organisms can host multiple species of parasites (or symbionts), and one species of parasite can infect different host species, creating complex interactions among multiple hosts and parasites. When multiple parasite species coexist in a host, they may compete or use strategies, such as spatial niche partitioning, to reduce competition. Here, we present a host–symbiont system with two species of Selenidium (Apicomplexa, Gregarinida) and one species of astome ciliate co-infecting two different species of slime feather duster worms (Annelida, Sabellidae, Myxicola ) living in neighbouring habitats. We examined the morphology of the endosymbionts with light and scanning electron microscopy (SEM) and inferred their phylogenetic interrelationships using small subunit (SSU) rDNA sequences. In the host ‘ Myxicola sp. Quadra’, we found two distinct species of Selenidium ; S . cf. mesnili exclusively inhabited the foregut, and S . elongatum n. sp. inhabited the mid to hindgut, reflecting spatial niche partitioning. Selenidium elongatum n. sp. was also present in the host M . aesthetica , which harboured the astome ciliate Pennarella elegantia n. gen. et sp. Selenidium cf. mesnili and P . elegantia n. gen. et sp. were absent in the other host species, indicating host specificity. This system offers an intriguing opportunity to explore diverse aspects of host–endosymbiont interactions and competition among endosymbionts.

High infestations of epibiont ciliates on vertebrates or invertebrates are normally related to aquaculture tanks or similar environments, and the importance of this relationship in natural habitats is often disregarded. Here, we describe the first record of high infestation of ciliates on Aegla serrana in South America and conduct a brief morphological and phylogenetic characterization of these ciliates. Our findings confirm that cases of high infestation of ciliates on metazoans can indeed occur in natural environments.

Background: Encystment is an important survival strategy extensively employed by microbial organisms to survive unfavorable conditions. Single-celled ciliated protists (ciliates) are popular model eukaryotes for studying encystment, whereby these cells degenerate their ciliary structures and develop cyst walls, then reverse the process under more favorable conditions. However, to date, the evolutionary basis and mechanism for encystment in ciliates is largely unknown. With the rapid development of high-throughput sequencing technologies, genome sequencing and comparative genomics of ciliates have become effective methods to provide insights into above questions., Results: Here, we profiled the MAC genome of Pseudourostyla cristata, a model hypotrich ciliate for encystment studies. Like other hypotrich MAC genomes, the P. cristata MAC genome is extremely fragmented with a single gene on most chromosomes, and encodes introns that are generally small and lack a conserved branch point for pre-mRNA splicing. Gene family expansion analyses indicate that multiple gene families involved in the encystment are expanded during the evolution of P. cristata. Furthermore, genomic comparisons with other five representative hypotrichs indicate that gene families of phosphorelay sensor kinase, which play a role in the two-component signal transduction system that is related to encystment, show significant expansion among all six hypotrichs. Additionally, cyst wall-related chitin synthase genes have experienced structural changes that increase them from single-exon to multi-exon genes during evolution. These genomic features potentially promote the encystment in hypotrichs and enhance their ability to survive in adverse environments during evolution., Conclusions: We systematically investigated the genomic structure of hypotrichs and key evolutionary phenomenon, gene family expansion, for encystment promotion in ciliates. In summary, our results provided insights into the evolutionary mechanism of encystment in ciliates., (© 2024. The Author(s).)

Colpoda are cosmopolitan unicellular eukaryotes primarily inhabiting soil and benefiting plant growth, but they remain one of the least understood taxa in genetics and genomics within the realm of ciliated protozoa. Here, we investigate the architecture of de novo assembled mitogenomes of six Colpoda species, using long-read sequencing and involving 36 newly isolated natural strains in total. The mitogenome sizes span from 43 to 63 kbp and typically contain 28 - 33 protein-coding genes. They possess a linear structure with variable telomeres and central repeats, with one Colpoda elliotti strain isolated from Tibet harboring the longest telomeres among all studied ciliates. Phylogenomic analyses reveal that Colpoda species started to diverge more than 326 million years ago, eventually evolving into two distinct groups. Collinearity analyses also reveal significant genomic divergences and a lack of long collinear blocks. One of the most notable features is the exceptionally high level of gene rearrangements between mitochondrial genomes of different Colpoda species, dominated by gene loss events. Population-level mitogenomic analysis on natural strains also demonstrates high sequence divergence, regardless of geographic distance, but the gene order remains highly conserved within species, offering a new species identification criterion for Colpoda species. Furthermore, we identified underlying heteroplasmic sites in the majority of strains of three Colpoda species, albeit without a discernible recombination signal to account for this heteroplasmy. This comprehensive study systematically unveils the mitogenomic structure and evolution of these ancient and ecologically significant Colpoda ciliates, thus laying the groundwork for a deeper understanding of the evolution of unicellular eukaryotes.IMPORTANCE Colpoda , one of the most widespread ciliated protozoa in soil, are poorly understood in regard to their genetics and evolution. Our research revealed extreme mitochondrial gene rearrangements dominated by gene loss events, potentially leading to the streamlining of Colpoda mitogenomes. Surprisingly, while interspecific rearrangements abound, our population-level mitogenomic study revealed a conserved gene order within species, offering a potential new identification criterion. Phylogenomic analysis traced their lineage over 326 million years, revealing two distinct groups. Substantial genomic divergence might be associated with the lack of extended collinear blocks and relaxed purifying selection. This study systematically reveals Colpoda ciliate mitogenome structures and evolution, providing insights into the survival and evolution of these vital soil microorganisms., Competing Interests: The authors declare no conflict of interest.

The morphology, morphogenesis, and molecular phylogeny of Heterometopus palaeformis (Kahl, 1927) Foissner, 2016 were studied using microscopical observations on live and protargol-stained specimens as well SSU rRNA gene sequencing. The morphogenetic data for the genus are presented for the first time. Compared to other metopids, the morphogenesis of H. palaeformis is distinct since its (1) perizonal stripe rows 4 and 5 are involved in the formation of the opisthe's adoral polykinetids; (2) perizonal stripe rows 3-5 and two adjacent preoral dome kineties contribute to most of the opisthe's paroral membrane while perizonal stripe rows 1 and 2 contribute very little; (3) four kinety rows are formed to the left of the opisthe's adoral zone of polykinetids. The Chinese population resembles the original and neotype populations well in terms of general morphology - characterized by a life size of 55-120 × 10-20 μm, an elongate ellipsoidal body with a hardly spiralized flat preoral dome, about 18 somatic kineties and 20 adoral polykinetids. The SSU rDNA sequence of the present population exhibits a disparity of 1.33%-2.22% divergence from sequences of other populations. Nevertheless, phylogenetic analysis reveals that populations of H. palaeformis form a separate, stable cluster within the paraphyletic Metopidae clade., 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 © 2023 Elsevier GmbH. All rights reserved.)

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., Competing Interests: Conflict of Interest: The authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The morphology, morphogenesis, and molecular phylogeny of a new ciliate, Aspidisca koreana n. sp., discovered in the eastern coast of South Korea, were investigated. The morphological description is based on the observation of living cells, 4'-6-diamidino-2-phenylindole (DAPI) and silver-stained specimens (e.g., protargol, silver nitrate), and scanning electron micrographs. The new species is characterized by having a small body size (17-25 × 15-18 μm in vivo), a distinct peristomial spur on the posterior portion of left margin, seven frontoventral cirri in "polystyla-arrangement", and the arrangement of the anterior portion of adoral zone of membranelles, i.e., anteriormost membranelle is distinctly separated from the other three membranelles. The morphogenesis follows the typical pattern of this genus. Phylogenetic analyses, using the 18S rDNA sequence, also support the establishment of a new species., 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 © 2023 Elsevier GmbH. All rights reserved.)

The morphology and morphogenesis of a new urostylid, Holostichides (Extraholostichides) eastensis tianjinensis subgen. nov. subspec. nov. were analyzed. The new subspecies differs from the nominotypical subspecies H. (Extraholostichides) eastensis eastensis Wang et al., 2022 by the relatively long frontoterminal row (about 60% vs. 30% of body length), colorless cortical granules (vs. dark brown), two (vs. one) parabuccal cirri, and usually an extra cirrus behind the first midventral pair (vs. lacking). Based on the difference in the frontal ciliature, we split Holostichides into two subgenera: H. (Extraholostichides) subgen. nov. (type species Holostichides eastensis Wang et al., 2022; with a short cirral row behind the middle frontal cirrus) and H. (Holostichides) Foissner, 1987 (type species Holostichides chardezi Foissner, 1987; lacking this short row). The main morphogenetic characters of the new subspecies are very similar to those of H. (Extraholostichides) eastensis eastensis except for some minor differences. Phylogenetic analyses based on SSU rDNA sequences indicate that H. (Extraholostichides) subgen. nov. is monophyletic and nested within the monophyletic genus Holostichides, which is sister to Eschaneustyla lugeri., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

The living morphology, infraciliature, and molecular phylogeny of a new soil ciliate, Hemiurosomoida koreana n. sp., discovered in a sample collected from a mountain in the northeast of South Korea, were investigated. The new species possesses the characteristics of the genus Hemiurosomoida, i.e., a reduced number of frontal-ventral-transverse cirri, three dorsal kineties of which kineties 1 and 2 each bears a caudal cirrus, and a single dorsomarginal kinety. It is distinguishable from congeners and other similar species by at least one distinct qualitative or quantitative character including the body size, the presence and arrangement of cortical granules, the number of adoral membranelles, marginal cirri, and dorsal dikinetids, or by the arrangement of transverse cirri. Phylogenetic analyses based on 18S rRNA gene sequences also support the assignment of the new species to the non-monophyletic genus Hemiurosomoida. In addition, the living morphology, infraciliature, and the 18S rRNA gene sequence of a Korean population of Nothoholosticha flava were studied., 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 © 2023 Elsevier GmbH. All rights reserved.)

Anaerobes have emerged in several major lineages of ciliates, but the number of independent transitions to anaerobiosis among ciliates is unknown. The APM clade (Armophorea, Muranotrichea, Parablepharismea) represents the largest clade of obligate anaerobes among ciliates and contains free-living marine and freshwater representatives as well as gut endobionts of animals. The evolution of APM group has only recently started getting attention, and our knowledge on its phylogeny and genetics is still limited to a fraction of taxa. While ciliates portray a wide array of alternatives to the standard genetic code across numerous classes, the APM ciliates were considered to be the largest group using exclusively standard nuclear genetic code. In this study, we present a pan-ciliate phylogenomic analysis with emphasis on the APM clade, bringing the first phylogenomic analysis of the family Tropidoatractidae (Armophorea) and confirming the position of Armophorida within Armophorea. We include five newly sequenced single cell transcriptomes from marine, freshwater, and endobiotic APM ciliates - Palmarella salina, Anteclevelandella constricta, Nyctotherus sp., Caenomorpha medusula, and Thigmothrix strigosa. We report the first discovery of an alternative nuclear genetic code among APM ciliates, used by Palmarella salina (Tropidoatractidae, Armophorea), but not by its close relative, Tropidoatractus sp., and provide a comparative analysis of stop codon identity and frequency indicating the precedency to the UAG codon loss/reassignment over the UAA codon reassignment in the specific ancestor of Palmarella. Comparative genomic and proteomic studies of this group may help explain the constraints that underlie UAR stop-to-sense reassignment, the most frequent type of alternative nuclear genetic code, not only in ciliates, but eukaryotes in general., 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 © 2023 Elsevier Inc. All rights reserved.)

The evolution of lineage-specific gene families remains poorly studied across the eukaryotic tree of life, with most analyses focusing on the recent evolution of de novo genes in model species. Here we explore the origins of lineage-specific genes in ciliates, a ~1 billion year old clade of microeukaryotes that are defined by their division of somatic and germline functions into distinct nuclei. Previous analyses on conserved gene families have shown the effect of ciliates' unusual genome architecture on gene family evolution: extensive genome processing-the generation of thousands of gene-sized somatic chromosomes from canonical germline chromosomes-is associated with larger and more diverse gene families. To further study the relationship between ciliate genome architecture and gene family evolution, we analyzed lineage specific gene families from a set of 46 transcriptomes and 12 genomes representing x species from eight ciliate classes. We assess how the evolution lineage-specific gene families occurs among four groups of ciliates: extensive fragmenters with gene-size somatic chromosomes, non-extensive fragmenters with "large'' multi-gene somatic chromosomes, Heterotrichea with highly polyploid somatic genomes and Karyorelictea with 'paradiploid' somatic genomes. Our analyses demonstrate that: 1) most lineage-specific gene families are found at shallow taxonomic scales; 2) extensive genome processing (i.e., gene unscrambling) during development likely influences the size and number of young lineage-specific gene families; and 3) the influence of somatic genome architecture on molecular evolution is increasingly apparent in older gene families. Altogether, these data highlight the influences of genome architecture on the evolution of lineage-specific gene families in eukaryotes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Maurer-Alcalá 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.)

The genus Rhabdostyla is one of the oldest of the family Epistylididae, it was described over 120 years ago and has more than 40 species considered valid. This group went through several systematic changes throughout its existence and due to the lack of taxonomic information, said to be essential, for most species that compose it, some of these changes are still questioned. In order to better understand and validate the species and relationships identified for this genus, the present work makes a brief review for the genus Rhabdostyla and its related genera, Opisthostyla and Orborhabdostyla. A 18S-rDNA phylogenetic analysis was also performed to understand the relationship between these groups. As a result of this review, 45 diagnoses were compiled and reorganized for the current model, accompanied by new graphical representations for the species considered valid for the genus Rhabdostyla and its associates. The major changes that the genus underwent, such as synonymy and separation to a new genus, were discussed in light of new phylogenetic information, but despite this, other information is still needed for a better validation of these changes.

The association between anaerobic ciliates and methanogenic archaea has been recognized for over a century. Nevertheless, knowledge of these associations is limited to a few ciliate species, and so the identification of patterns of host-symbiont specificity has been largely speculative. In this study, we integrated microscopy and genetic identification to survey the methanogenic symbionts of 32 free-living anaerobic ciliate species, mainly from the order Metopida. Based on Sanger and Illumina sequencing of the 16S rRNA gene, our results show that a single methanogenic symbiont population, belonging to Methanobacterium, Methanoregula, or Methanocorpusculum, is dominant in each host strain. Moreover, the host's taxonomy (genus and above) and environment (i.e. endobiotic, marine/brackish, or freshwater) are linked with the methanogen identity at the genus level, demonstrating a strong specificity and fidelity in the association. We also established cultures containing artificially co-occurring anaerobic ciliate species harboring different methanogenic symbionts. This revealed that the host-methanogen relationship is stable over short timescales in cultures without evidence of methanogenic symbiont exchanges, although our intraspecific survey indicated that metopids also tend to replace their methanogens over longer evolutionary timescales. Therefore, anaerobic ciliates have adapted a mixed transmission mode to maintain and replace their methanogenic symbionts, allowing them to thrive in oxygen-depleted environments., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

As unicellular predators, ciliates engage in close associations with diverse microbes, laying the foundation for the establishment of endosymbiosis. Originally heterotrophic, ciliates demonstrate the ability to acquire phototrophy by phagocytizing unicellular algae or by sequestering algal plastids. This adaptation enables them to gain photosynthate and develop resistance to unfavorable environmental conditions. The integration of acquired phototrophy with intrinsic phagotrophy results in a trophic mode known as mixotrophy. Additionally, ciliates can harbor thousands of bacteria in various intracellular regions, including the cytoplasm and nucleus, exhibiting species specificity. Under prolonged and specific selective pressure within hosts, bacterial endosymbionts evolve unique lifestyles and undergo particular reductions in metabolic activities. Investigating the research advancements in various endosymbiotic cases within ciliates will contribute to elucidate patterns in cellular interaction and unravel the evolutionary origins of complex traits., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Phenotypic plasticity, which involves phenotypic transformation in the absence of genetic change, may serve as a strategy for organisms to survive in complex and highly fluctuating environments. However, its reaction norm, molecular basis, and evolution remain unclear in most organisms, especially microbial eukaryotes. In this study, we explored these questions by investigating the reaction norm, regulation, and evolution of phenotypic plasticity in the cosmopolitan marine free-living ciliates Glauconema spp., which undergo significant phenotypic changes in response to food shortages. This study led to the de novo assembly of macronuclear genomes using long-read sequencing, identified hundreds of differentially expressed genes associated with phenotypic plasticity in different life stages, validated the function of two of these genes, and revealed that the reaction norm of body shape in response to food density follows a power-law distribution. Purifying selection may be the dominant evolutionary force acting on the genes associated with phenotypic plasticity, and the overall data support the hypothesis that phenotypic plasticity is a trait maintained by natural selection. This study provides novel insight into the developmental genetics of phenotypic plasticity in non-model unicellular eukaryotes and sheds light on the complexity and long evolutionary history of this important survival strategy., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Ciliates are a diverse group of protists known for their ability to establish various partnerships and thrive in a wide variety of oxygen-depleted environments. Most anaerobic ciliates harbor methanogens, one of the few known archaea living intracellularly. These methanogens increase the metabolic efficiency of host fermentation via syntrophic use of host end-product in methanogenesis. Despite the ubiquity of these symbioses in anoxic habitats, patterns of symbiont specificity and fidelity are not well known. We surveyed two unrelated, commonly found groups of anaerobic ciliates, the Plagiopylea and Metopida, isolated from anoxic marine sediments. We sequenced host 18S rRNA and symbiont 16S rRNA marker genes as well as the symbiont internal transcribed spacer region from our cultured ciliates to identify hosts and their associated methanogenic symbionts. We found that marine ciliates from both of these co-occurring, divergent groups harbor closely related yet distinct intracellular archaea within the Methanocorpusculum genus. The symbionts appear to be stable at the host species level, but at higher taxonomic levels, there is evidence that symbiont replacements have occurred. Gaining insight into this unique association will deepen our understanding of the complex transmission modes of marine microbial symbionts, and the mutualistic microbial interactions occurring across domains of life., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

The phylum Ciliophora (ciliates) comprises about 2600 symbiotic and over 5500 free-living species. The inclusion of symbiotic ciliates in phylogenetic analyses often challenges traditional classification frameworks due to their morphological adaptions to the symbiotic lifestyle. Conchophthirus is such a controversial obligate endocommensal genus whose affinities to other symbiotic and free-living scuticociliates are still poorly understood. Using uni- and multivariate morphometrics as well as 2D-based molecular and phylogenetic analyses, we attempted to test for the monophyly of Conchophthirus, study the boundaries of Conchophthirus species isolated from various bivalves at mesoscale, and reveal the phylogenetic relationships of Conchophthirus to other scuticociliates. Multidimensional analyses of morphometric and cell geometric data generated the same homogenous clusters, as did phylogenetic analyses based on 144 new sequences of two mitochondrial and five nuclear molecular markers. Conchophthirus is not closely related to 'core' scuticociliates represented by the orders Pleuronematida and Philasterida, as assumed in the past using morphological data. Nuclear and mitochondrial markers consistently showed the free-living Dexiotricha and the mouthless endosymbiotic Haptophrya to be the nearest relatives of Conchophthirus. These three highly morphologically and ecologically dissimilar genera represent an orphan clade from the early radiation of scuticociliates in molecular phylogenies., 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 © 2023 Elsevier Inc. All rights reserved.)