Description of Conchophthirus sinanodontae n. sp. (Ciliophora: Scuticociliatia), a new endocommensal ciliate of the freshwater mussel Sinanodonta woodiana from Korea.

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.

Taxonomy and molecular phylogeny of genus Plagiocampa (Ciliophora, Prostomatea), with redescriptions of two poorly known species.

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.

Coleps shanghaiensis n. sp. challenges the validity of the genus Levicoleps (Ciliophora, Prostomatida).

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.

Phylogenomics corroborates morphology: New discussions on the systematics of Trichostomatia (Ciliophora, Litostomatea).

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.

Curved crease origami and topological singularities enable hyperextensibility of L. olor.

Fundamental limits of cellular deformations, such as hyperextension of a living cell, remain poorly understood. Here, we describe how the single-celled protist Lacrymaria olor, a 40-micrometer cell, is capable of reversibly and repeatably extending its necklike protrusion up to 1200 micrometers in 30 seconds. We discovered a layered cortical cytoskeleton and membrane architecture that enables hyperextensions through the folding and unfolding of cellular-scale origami. Physical models of this curved crease origami display topological singularities, including traveling developable cones and cytoskeletal twisted domain walls, which provide geometric control of hyperextension. Our work unravels how cell geometry encodes behavior in single cells and provides inspiration for geometric control in microrobotics and deployable architectures.

Bit by bit toward the diversity of metopids: Description of the genus Pidimetopus n. gen. (Ciliophora: Armophorea).

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.

Morphology, morphogenesis and molecular phylogeny of a new soil ciliate, Lamtostyla paravitiphila nov. spec. (Ciliophora, Hypotrichia).

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.

Morphology and molecular phylogeny of three Parasonderia species including a new species (Ciliophora, Plagiopylea).

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.

Reductitherus cryptostomus n. gen., n. sp. (Ciliophora: Armophorea: Clevelandellida), a remarkable new nyctotherid from an Australian cockroach, Parapanesthia gigantea (Blaberidae: Panesthiinae).

Ciliates of the family Nyctotheridae (Armophorea: Clevelandellida) are frequent intestinal symbionts of various invertebrates and some poikilotherm vertebrates. Depending on the classification scheme, there are between 15 and 18 recognized genera of Nyctotheridae, the majority of which exhibit a rather uniform morphology. They have round to ellipsoidal cells with an adoral zone of membranelles that begins anteriorly in an adoral groove and continues posteriorly into the buccal cavity where it extends deep into the cell in the peristomial funnel. The taxonomy of the Nyctotheridae is primarily based on the number and location of kinetal sutures. The only known divergence from the relatively conservative nyctortherid body plan are the bizarre symbionts of Panesthiinae cockroaches, ciliates of the family Clevelandellidae, which forms a clade nested within the Nyctotheridae genus Nyctotherus. In this study we report another ciliate that diverges morphologically from the canonical Nyctotheridae body plan, and which is also found in Panesthiinae hosts. The novel ciliate Reductitherus cryptostomus n. gen., n. sp. differs from the rest of Nyctotheridae by absence of the anterior adoral groove, a shortened adoral zone completely enclosed in a notably small buccal cavity, and two strongly reduced kinetal sutures, one left anterodorsal and the other right posterodorsal.

Redescription and molecular phylogeny of the freshwater metopid, Castula strelkowi (Jankowski, 1964) from the Czech Republic and synonymization of Pileometopus with Castula.

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.

Morphological reconstruction during cell regeneration in the ciliate Spirostomum ambiguum.

When the ciliate Spirostomum ambiguum is transected into two pieces, both fragments regenerate and proliferate. In the anterior fragments, which have lost their contractile vacuoles due to transection, new contractile vacuoles were formed at their posterior ends in a few minutes. When the cells were cut into three pieces, new contractile vacuoles were formed in the anterior and middle fragments, both at their posterior ends. Thus, the anterior-posterior axis of S. ambiguum was maintained after transection. Morphological repair, including the formation of the contractile vacuole, was also observed when only the anteriormost portion was transected to cut out a small fragment that did not contain part of the macronucleus. Scanning electron microscopy was performed to observe changes in the shape of the cleavage surface of S. ambiguum during the wound healing process. Within minutes after cutting, the cut surface was covered with a cilia-free membrane, preventing leakage of cytoplasmic contents. The surface of the cut area then rounded with time and was covered with cilia, completing the repair of the cut area in about one day.

Morphology and molecular phylogeny of a new brackish pleurostomatid ciliate, Loxophyllum paludosum sp. n. (Ciliophora, Litostomatea, Haptoria), from a mangrove wetland in China.

A new ciliate species of the genus Loxophyllum Dujardin, 1841, Loxophyllum paludosum sp. n., is described from a mangrove wetland near Daya Bay in Guangdong Province, southern China, based on morphological and molecular analyses. The new species can be distinguished from its congeners by a combination of the following characters: (1) 12-14 right kineties and 4-6 left kineties; (2) two macronuclear nodules and one micronucleus; (3) a single contractile vacuole located terminally; (4) extrusomes bar-shaped, evenly spaced along entire ventral margin, and clustered to form 5-7 warts along dorsal margin; and (5) presence of three ridges on the left side of cell. The new species is divergent from its congeners from 0.4% to 6.7% (5-104 nucleotide sites) based on the small subunit (SSU) rRNA gene sequence data. The validity of the new species is also supported by molecular phylogenetic trees inferred from SSU rRNA gene sequences.

Signaling in temperature-induced resting cyst formation in the ciliated protozoan Colpoda cucullus.

The terrestrial ciliated protozoan Colpoda cucullus inhabits soil. When the habitat conditions become unfavorable, the vegetative cells of C. cucullus quickly transform into resting cysts. C. cucullus culture is established in our laboratory, and encystment is routinely induced by the addition of Ca2+ to overpopulated vegetative cells. However, an increase in Ca2+ concentration and overpopulation of vegetative cells do not always occur in natural. We investigated the effect of temperature and found that cyst formation was induced by a rapid increase of 5 °C within 2 min but not by a decrease. Moreover, an increase in intracellular Ca2+ concentrations is essential, but Ca2+ inflow does not necessarily occur during encystment. Ca2+ image analysis showed that Ca2+ is stored in vesicular structures and released into the cytoplasm within 60 s after temperature stimulation. Multiple signaling pathways are activated after the release of Ca2+ from vesicles, and cAMP is a candidate second messenger with a crucial role in the process of temperature-induced encystment. Further studies are needed to clarify the mechanism underlying the sensing of temperature and release of Ca2+ from vesicles.

An integrative approach sheds new light onto the systematics and ecology of the widespread ciliate genus Coleps (Ciliophora, Prostomatea).

Species of the genus Coleps are one of the most common planktonic ciliates in lake ecosystems. The study aimed to identify the phenotypic plasticity and genetic variability of different Coleps isolates from various water bodies and from culture collections. We used an integrative approach to study the strains by (i) cultivation in a suitable culture medium, (ii) screening of the morphological variability including the presence/absence of algal endosymbionts of living cells by light microscopy, (iii) sequencing of the SSU and ITS rDNA including secondary structures, (iv) assessment of their seasonal and spatial occurrence in two lakes over a one-year cycle both from morphospecies counts and high-throughput sequencing (HTS), and, (v) proof of the co-occurrence of Coleps and their endosymbiotic algae from HTS-based network analyses in the two lakes. The Coleps strains showed a high phenotypic plasticity and low genetic variability. The algal endosymbiont in all studied strains was Micractinium conductrix and the mutualistic relationship turned out as facultative. Coleps is common in both lakes over the whole year in different depths and HTS has revealed that only one genotype respectively one species, C. viridis, was present in both lakes despite the different lifestyles (mixotrophic with green algal endosymbionts or heterotrophic without algae). Our results suggest a future revision of the species concept of the genus Coleps.

Anaerobic endosymbiont generates energy for ciliate host by denitrification.

Mitochondria are specialized eukaryotic organelles that have a dedicated function in oxygen respiration and energy production. They evolved about 2 billion years ago from a free-living bacterial ancestor (probably an alphaproteobacterium), in a process known as endosymbiosis1,2. Many unicellular eukaryotes have since adapted to life in anoxic habitats and their mitochondria have undergone further reductive evolution3. As a result, obligate anaerobic eukaryotes with mitochondrial remnants derive their energy mostly from fermentation4. Here we describe 'Candidatus Azoamicus ciliaticola', which is an obligate endosymbiont of an anaerobic ciliate and has a dedicated role in respiration and providing energy for its eukaryotic host. 'Candidatus A. ciliaticola' contains a highly reduced 0.29-Mb genome that encodes core genes for central information processing, the electron transport chain, a truncated tricarboxylic acid cycle, ATP generation and iron-sulfur cluster biosynthesis. The genome encodes a respiratory denitrification pathway instead of aerobic terminal oxidases, which enables its host to breathe nitrate instead of oxygen. 'Candidatus A. ciliaticola' and its ciliate host represent an example of a symbiosis that is based on the transfer of energy in the form of ATP, rather than nutrition. This discovery raises the possibility that eukaryotes with mitochondrial remnants may secondarily acquire energy-providing endosymbionts to complement or replace functions of their mitochondria.

Taxonomy, phylogeny, and geographical distribution of the little-known Helicoprorodon multinucleatum Dragesco, 1960 (Ciliophora, Haptorida) and key to species within the genus.

A little-known haptorid ciliate, Helicoprorodon multinucleatum Dragesco, 1960, was found in a sandy beach at Qingdao, China. Its morphology was studied based on microscopic observations of live and protargol-stained specimens and morphometrics, and the phylogeny was analyzed using SSU rRNA gene sequences. Helicoprorodon multinucleatum is characterized by the combination of the following features: (i) a very narrowly worm-like body with a size of about 300-1500 µm × 30-60 µm in vivo, and two circles of horn-like protuberances around the head; (ii) 50-160 spherical macronuclear nodules scattered throughout the body; (iii) rod-shaped, 10-50 µm long extrusomes gathered into several bunches, which are randomly distributed beneath pellicle; and (iv) 42-88 somatic kineties, including four oralized kineties and two dorsal brush rows. Phylogenetic analyses reveal that both the family Helicoprorodontidae and the genus Helicoprorodon might be monophyletic. In addition, we provide an illustrated key to the species and the geographical distribution of the genus Helicoprorodon.

Anteholosticha foissneri n. sp., a marine hypotrich ciliate (Ciliophora: Spirotrichea) from Vietnam: Morphology, morphogenesis, and molecular phylogeny.

In a study of marine ciliate diversity, we collected an Anteholosticha monilata-like population from Vietnam. To identify this population, we analyzed its morphology, some morphogenetic stages, and molecular phylogeny. Based on these data, we conclude that the Vietnamese population is new to science. Anteholosticha foissneri n. sp. resembles Anteholosticha monilata-like species considering (1) the number and arrangement of macronuclear nodules and micronuclei; (2) the presence of cortical granules; and (3) the saline habitat. However, the new species can be easily distinguished from these species by the arrangement, color, and shape of the cortical granules. The divisional morphogenesis commences with the de novo proliferation of basal bodies as a single longitudinal patch left of the posteriormost midventral cirral pair. This character state has not been reported before in Anteholosticha (based on check of the available data) and probably reflects a distinct clade within the nuclear small subunit ribosomal RNA gene tree.

The Function and Evolution of Motile DNA Replication Systems in Ciliates.

DNA replication is a ubiquitous and conserved cellular process. However, regulation of DNA replication is only understood in a small fraction of organisms that poorly represent the diversity of genetic systems in nature. Here we used computational and experimental approaches to examine the function and evolution of one such system, the replication band (RB) in spirotrich ciliates, which is a localized, motile hub that traverses the macronucleus while replicating DNA. We show that the RB can take unique forms in different species, from polar bands to a "replication envelope," where replication initiates at the nuclear periphery before advancing inward. Furthermore, we identify genes involved in cellular transport, including calcium transporters and cytoskeletal regulators, that are associated with the RB and may be involved in its function and translocation. These findings highlight the evolution and diversity of DNA replication systems and provide insights into the regulation of nuclear organization and processes.

Two Anaerobic Ciliates (Ciliophora, Armophorea) from China: Morphology and SSU rDNA Sequence, with Report of a New Species, Metopus paravestitus nov. spec.

The morphology and phylogeny of two metopid ciliates, collected from anaerobic habitats in China, were investigated using live observation, protargol staining method, and SSU rDNA sequencing. The new species Metopus paravestitus nov. spec. can be distinguished by a combination of the following features: oblong cell with densely arranged ectobiotic prokaryotes perpendicular to cell surface, filiform intracytoplasmic structures packed in the anterior portion of the cell. Our work also demonstrates the wide geographical distribution of Metopus es (Müller, 1776) Lauterborn, 1916. The order Metopida is consistently depicted as a paraphylum in SSU rDNA phylogeny. Metopus paravestitus nov. spec. is closely related to its marine congeners than to freshwater forms. The present study confirms once again the non-monophyly of the genus Metopus and genus Metopidae.

Does the Gonostomum-patterned oral apparatus in hypotrichia carry a phylogenetic signal? Evidence from morphological and molecular data based on extended taxon sampling using three nuclear genes (Ciliophora, Spirotrichea).

The classification of hypotrichs based on the gonostomatid oral structure is widely accepted, but the phylogenetic signal of this character is unknown. Here, we infer the species phylogeny of those gonostomatids for which molecular data are available, plus 26 new sequences of SSU-rDNA, ITS1-5.8S-ITS2 and LSU-rDNA genes. The results indicate that: (i) the endoral is more phylogenetically informative than the paroral; (ii) the structure of the endoral and the Gonostomum-pattern adoral zone of membranelles are plesiomorphies for the hypotrichs sensu stricto; (iii) the group of species possessing these features is monophyletic in all our phylogenetic analyses, except that for the SSU-rDNA; (iv) Schmidingerotrichidae is monophyletic in all trees, suggesting that it is a well-defined family; (v) the Gonostomatidae is polyphyletic in the SSU-rDNA and ITS1-5.8S-ITS2 trees, with Gonostomum, Cladotricha, Cotterillia, Metagonostomum, Paragonostomum and Wallackia distributed among separate clades, but monophyletic in the LSU-rDNA and concatenated trees; (vi) higher hypotrich taxa such as core urostyloids and core sporadotrichids/stichotrichids might have evolved from species that possessed a gonostomatid oral apparatus.