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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., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

We aimed to freeze-dry the ciliate Spirostomum ambiguum, obtained from water, without fixation and observe it using scanning electron microscopy (SEM). Living cells were placed on a specimen stub and frozen upon contact with a Cu block kept at either -80 °C or -100 °C. Samples were then freeze-dried and observed by SEM. In most cases, no damage to the specimen due to ice crystal formation was observed. Because of the instantaneous freezing, the metachronal wave of cilia on the body surface of the ciliate was well preserved. Approximately 70-80% of cells were preserved in the contracted state due to inevitable exposure to mechanical vibration immediately before freezing. The remaining samples were preserved in a fully-extended state. Morphometric measurements of the cell surface showed that in the extended state, ciliary rows were almost parallel to the long axis of the cell, whereas in the contracted state, they were twisted in a left-handed helix at an angle of 45-65°. The distance between adjacent cilia along a ciliary row was 1.88 ± 0.43 μm in the extended state and 1.32 ± 0.41 μm in the contracted state (mean ± SD). However, the spacing between adjacent ciliary rows remained unchanged., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

The genus Stentor is a relatively well-known ciliate owing to its lucid trumpet shape. Stentor pyriformis represents a green, short, and fat Stentor, but it is a little-known species. We investigated 124 ponds and wetlands in Japan and confirmed the presence of S. pyriformis at 23 locations. All these ponds were noticeably oligotrophic. With the improvement of oligotrophic culture conditions, we succeeded in long-term cultivation of three strains of S. pyriformis. The cytoplasm of S. piriformis contains a large number of 1-3 μm refractive granules that turn brown by Lugol's staining. The granules also show a typical Maltese-cross pattern by polarization microscopy, strongly suggesting that the granules are made of amylopectin-rich starch. By analyzing the algal rDNA, it was found that all S. pyriformis symbionts investigated in this study were Chlorella variabilis. This species is known as the symbiont of Paramecium bursaria and is physiologically specialized for endosymbiosis. Genetic discrepancies between C. variabilis of S. pyriformis and P. bursaria may indicate that algal sharing was an old incident. Having symbiotic algae and storing carbohydrate granules in the cytoplasm is considered a powerful strategy for this ciliate to withstand oligotrophic and cold winter environments in highland bogs.

Freshwater protists often harbor unicellular green algae within their cells. In ciliates, possibly because of large host cell sizes and the small size of algal coccoids, a single host cell typically contains more than a hundred algal cells. While surveying such algae-bearing protists on Minami Daito Jima Island in Japan, we found a green Loxodes ciliate (Loxodida, Karyorelictea) that contained one or two dozens of very large coccoid algae. We isolated one of these algae and analyzed its characteristics in detail. A small subunit (SSU) rDNA phylogeny indicated Pseudodidymocystis species (Scenedesmaceae, Chlorophyceae) to be the taxon closest to the alga, although it was clearly separated from this by 39 or more different sites (inclusive of gaps). SSU rRNA structure analyses indicated that these displacements included eight compensatory base changes (CBCs) and seven hemi-CBCs. We therefore concluded that this alga belongs to a separate genus, and described it as Pediludiella daitoensis gen. et sp. nov. The shape of the isolated and cultured P. daitoensis was nearly spherical and reached up to 30 µm in diameter. Chloroplasts were arranged peripherally and often split and elongated. Cells were often vacuolated and possessed a net-like cytoplasm that resembled a football (soccer ball) in appearance, which was reflected in the genus name.

Most ciliates use a deviant genetic code. Eukaryotic release factor (eRF1) appears to play an important role in the process of reassignment of stop codons. Although the precise site on eRF1 for recognition of stop codons remains obscure, studies have suggested that the tip region NIKS and its adjacent YxCxxxF motifs in domain 1 are important for stop codon recognition. Litostomatea is a class of ciliate that appears to use the standard genetic code. We used Dileptus (Litostomatea) eRF1 in this study to identify key residues located in or near the YxCxxxF motif. We predicted sites involving stop codon recognition by computational calculation of RNA-protein interaction propensity. We introduced mutations at the predicted sites of Dileptus eRF1 and examined the activity of the mutated Dileptus eRF1 using in vivo assay systems. The results show that the single mutation R128I (Dileptus eRF1 numbering) in the YxCxxxF motif converted the omnipotent recognition of Dileptus eRF1 to Euplotes-type dualpotent eRF1. Our results indicate that R128 is one of the key residues preserving the ability to recognize all three stop codons, especially UGA, in Dileptus. We discuss a possible advantage that ciliates from the Litostomatea class may gain from using the standard genetic code., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

In contrast to most ciliates, meiosis and successive nuclear changes during conjugation occur only in heterotypic pairs in Blepharisma. It has been suggested that homotypic pairs are ready for conjugation, but lack a trigger to initiate the nuclear changes, and the conjugation process is arrested before the onset of meiosis. To explore the possible nature of the trigger, we previously identified the genes BjCdk1 (homologous to cdk1/cdc2), Bj4HPPD (4-hydroxy-phenylpyruvate dioxygenase) and BjCks (cyclin dependent kinase regulatory subunit) whose expression is up-regulated in gamone1-treated type II cells. In this study, we investigated the molecular structures of these three genes, and compared their expression patterns in homotypic and heterotypic pairs, finding remarkable differences. BjCdk1, Bj4HPPD and BjCks were expressed specifically in gamone1-treated type II cells, but not in gamone2-treated type I cells. In heterotypic pairs, the expression of these genes stayed at the same level or gradually decreased throughout the entire process of conjugation, but it rapidly decreased and ceased after 10hours in homotypic pairs. These results indicate that some genes are expressed in a mating-type specific manner. Alternative gene expression in mating type I and type II cells and merging of individual factors in a heterotypic pair may induce nuclear changes including meiosis., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Recent multigene phylogenetic analyses have contributed much to our understanding of eukaryotic phylogeny. However, the phylogenetic positions of various lineages within the eukaryotes have remained unresolved or in conflict between different phylogenetic studies. These phylogenetic ambiguities might have resulted from mixtures or integration from various factors including limited taxon sampling, missing data in the alignment, saturations of rapidly evolving genes, mixed analyses of short- and long-branched operational taxonomic units (OTUs), intracellular endoparasite and ciliate OTUs with unusual substitution etc. In order to evaluate the effects from intracellular endoparasite and ciliate OTUs co-analyzed on the eukaryotic phylogeny and simplify the results, we here used two different sets of data matrices of multiple slowly evolving genes with small amounts of missing data and examined the phylogenetic position of the secondary photosynthetic chromalveolates Haptophyta, one of the most abundant groups of oceanic phytoplankton and significant primary producers. In both sets, a robust sister relationship between Haptophyta and SAR (stramenopiles, alveolates, rhizarians, or SA [stramenopiles and alveolates]) was resolved when intracellular endoparasite/ciliate OTUs were excluded, but not in their presence. Based on comparisons of character optimizations on a fixed tree (with a clade composed of haptophytes and SAR or SA), disruption of the monophyly between haptophytes and SAR (or SA) in the presence of intracellular endoparasite/ciliate OTUs can be considered to be a result of multiple evolutionary reversals of character positions that supported the synapomorphy of the haptophyte and SAR (or SA) clade in the absence of intracellular endoparasite/ciliate OTUs.

Preconjugant interactions between complementary mating-type cells in ciliates occur before sexual reproduction. The interactions include retardation of swimming behaviour, courtship dancing, chemoattraction, nuclear activation, cell division, or cell agglutination, depending on ciliate species. In Blepharisma japonicum, chemoattraction of mating-type I by mating-type II has been reported previously. It has been shown that chemoattraction here is caused by a conjugation-inducing substance called gamone 2 secreted by mating-type II cells. In this study, we show that mating-type II cells accumulate near the site where gamone 1 secreted by mating-type I cells is present at a high concentration. We also show that the behaviour of individual cells changes when exposed to the complementary mating-type gamone; cells begin to rotate and swim slowly, thus shortening their minimum path length (final displacement of a cell from its origin). These results suggest that gamones 1 and 2 induce behavioural changes in type II and I cells, respectively, and that gamone-stimulated cells may accumulate at the site with the highest activity of the complementary gamone, after repetition of swimming changes in the gradient of gamone concentration. This reciprocal induction of the changes in behaviour may increase the probability of sexual encounters for conjugation., (Copyright (c) 2010 Elsevier GmbH. All rights reserved.)

A ciliated protozoan, Halteria grandinella, swam backward rapidly with a migration distance per second attaining 100 times the cell size. This high swimming velocity was accompanied by a high frequency of ciliary beating. Recordings with a high-speed digital video (10(3) frames/s) revealed that the frequency during forward and backward swimming was, respectively, 105 +/- 10 Hz and 260 +/- 30 Hz. These frequencies are the highest among cilia and flagella reported to date. Electron microscopic observation of the ciliary structure confirmed normal 9 + 2 arrangements of the axoneme except that cilia for migration are bundled into membranelles. Ciliary beating of saponin-treated cells was reactivated by the addition of Mg2+ -ATP, although the beating amplitude was smaller than that of intact cells. Kinetic analysis of the ATP-dependent increase of beating frequency revealed that the maximal frequency in the presence of free Ca2+ and 0.9 microM Ca2+ was approximately 60 and 110 Hz, respectively. A possible mechanism to increase beating frequency with Ca2+ is discussed., (Copyright 2005 Wiley-Liss, Inc.)

Internal Ca2+ levels control the pattern of ciliary and flagellar beating in eukaryotes. In ciliates, ciliary reversal is induced by a rise in intra-ciliary Ca2+, but the mechanism by which Ca2+ induces reversal is not known. We injected the fluorescent Ca2+ indicator Calcium Green into a ciliate Didinium nasutum and observed the intra-ciliary Ca2+ level during the initial reversed stroke preceding spontaneous cyclic reversed beating. In D. nasutum, Ca2+ rose throughout the length of the cilia undergoing initial reversed stroke. Electron microscopy with a combined oxalate-pyroantimonate method showed Ca2+ deposits distributed throughout the reversed cilia. We injected caged Ca2+ into D. nasutum and irradiated the base or mid region of the cilia with UV to locally increase Ca2+ concentration. Uncaging Ca2+ in the middle of the cilia produced reversal distally, but not proximally to the site of Ca2+ release. These results strongly suggest that not only Ca2+ influx sites, but also Ca2+ binding sites and vectoral bending machineries for ciliary reversal, are distributed throughout the cilium., (Copyright 2004 Wiley-Liss, Inc.)

An electrode sensitive to tetraphenyl phosphonium (TPP+) was applied to monitor the changes in the membrane potential of the ciliate Blepharisma japonicum during the process of cell elongation. Uptake of TPP+ by Blepharisma cells was observed when they were incubated in a medium containing 2 x 10(-6)M TPP+. Subsequent light stimulation (3,000 lux) led to an efflux of accumulated TPP+ from the cells, indicating that depolarization of the plasma membrane occurred with the concomitant cell elongation. TPP+ efflux was always observed whenever cell elongation was induced in response to various stimuli: valinomycin (10(-6)M), K+ (30 mM), Co2+ (5 mM), and Ca2+ (5 mM). Furthermore, elongation was also evoked by electrical stimulation. These observations indicate that membrane depolarization is involved in the process of cell elongation in Blepharisma.