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Heterotrichous ciliates play an important role in aquatic ecosystem energy flow processes and many are model organisms for research in cytology, regenerative biology, and toxicology. In the present study, we combine both morphological and molecular data to infer phylogenetic relationships at family-genus level and propose new evolutionary hypotheses for the class Heterotrichea. The main results include: (1) 96 new ribosomal DNA sequences from 36 populations, representing eight families and 13 genera, including three poorly annotated genera, Folliculinopsis, Ampullofolliculina and Linostomella; (2) the earliest-branching families are Spirostomidae in single-gene trees and Peritromidae in the concatenated tree, but the family Peritromidae probably represents the basal lineage based on its possession of many "primitive" morphological characters; (3) some findings in molecular trees are not supported by morphological evidence, such as the family Blepharismidae is one of the most recent branches and the relationship between Fabreidae and Folliculinidae is very close; (4) the systematic positions of Condylostomatidae, Climacostomidae, and Gruberiidae remain uncertain based either on morphological or molecular data; and (5) the monophyly of each genus included in the present study is supported by the molecular phylogenetic trees, except for Blepharisma in the SSU rDNA tree and Folliculina in the ITS1-5.8S-ITS2 tree.

Adaptation to life at different oxygen tensions plays a role in protozoan ecology and controls the distribution of different species in anoxic habitats. The ciliate genusSpirostomuminhabiting fresh or low salinity water globally where these species are considered as bioindicators. Under anaerobic or low oxygen conditions, the rhodoquinol-dependent pathway has been reported in the species from the class Heterotrichea. With the help of RNA sequencing (RNAseq) data,Spirostomumspp., are suitable for deep molecular investigations onrquAfor rhodoquinone (RQ) biosynthesis. In this study,Spirostomum ambiguum,Spirostomum subtilis, andSpirostomum terescollected from densely vegetated freshwater habitat in Fuzhou, China, explored the evidence ofrquA. Based on transcriptome analysis, two to three RquA proteins were identified inS. ambiguum,S. teres, andS. subtilis, respectively. The presence of a key Motif-I of RquA and mitochondrial targeting signals (MTS), also confirmed the identity of these as RquA. Furthermore,SpirostomumRquA proteins could be sorted into two groups based on their conserved amino acid (CAA) residues. Phylogenetic analysis also exhibited RquA division into two subclades contained RquA1 and RquA2/RquA3 and supports two to three paralogs ofrquAgenes in the genomesSpirostomumspp. Additional transcriptomes and genomes analysis ofBlepharismaspp., andStentorspp., respectively, also revealed at least two paralogs ofrquAin members of the class Heterotrichea. The present study provides evidence for the presence of RquA and rhodoquinol dependent fumarate reduction pathway inSpirostomumspecies potentially use to respire in the oxygen-depleted habitats and two to three diverserquAgenes.

Blepharisma hyalinum Perty, 1849 Distribution. VENEZUELA: South margin of the Henri Pittier National Park, Biological Station Rancho Grande (Foissner 2016)., Published as part of Dur��n-Ram��rez, Carlos Alberto, Dias, Roberto J��nio Pedroso & Estrada, Rosaura May��n-, 2020, Checklist of ciliates (Alveolata: Ciliophora) that inhabit in bromeliads from the Neotropical Region, pp. 1-36 in Zootaxa 4895 (1) on page 8, DOI: 10.11646/zootaxa.4895.1.1, http://zenodo.org/record/4321437, {"references":["Foissner, W. (2016) Terrestrial and semiterrestrial ciliates (Protozoa, Ciliophora) from Venezuela and Galapagos. Denisia, 35, 1 - 912."]}

Blepharisma steini Kahl, 1932 Distribution. MEXICO: Coffee plantation La Onza, near Coatepec (Dur��n-Ram��rez et al. 2015), (Th); Coffee plantation Virginia Armand, near Coatepec (Dur��n-Ram��rez et al. 2015), (Th)., Published as part of Dur��n-Ram��rez, Carlos Alberto, Dias, Roberto J��nio Pedroso & Estrada, Rosaura May��n-, 2020, Checklist of ciliates (Alveolata: Ciliophora) that inhabit in bromeliads from the Neotropical Region, pp. 1-36 in Zootaxa 4895 (1) on page 8, DOI: 10.11646/zootaxa.4895.1.1, http://zenodo.org/record/4321437, {"references":["Duran-Ramirez, C. A., Garcia-Franco, J. G., Foissner, W. & Mayen-Estrada, R. (2015) Free living ciliates from epiphytic tank bromeliads in Mexico. European Journal of Protistology, 51, 15 - 33. https: // doi. org / 10.1016 / j. ejop. 2014.09.002"]}

Blepharismins are photodynamic hypericin-like dianthrones produced as a variable pigment blend in Blepharisma ciliates and mostly studied in the Afro-Asiatic Blepharisma japonicum. The present work describes the bioactivity of pigments from the Brazilian Blepharisma sinuosum. Comparative analyses showed that the pigments from both species can trigger photo-induced modifications in phospholipids, but different redox properties and biological activities were assigned for each pigment blend. Stronger activities were detected for B. sinuosum pigments, with the lethal concentration LC50 10 × lower than B. japonicum pigments in light-irradiated tests against Bacillus cereus and less than half for treatments on the human HeLa tumor cells. HPLC showed B. sinuosum producing a simpler pigment blend, mostly with the blepharismin-C (~ 70%) and blepharismin-E (~ 30%) types. Each blepharismin engaged a specific dose-response profile on sensitive cells. The blepharismin-B and blepharismin-C were the most toxic pigments, showing LC50 ~ 2.5-3.0 µm and ~ 100 µm on B. cereus and HeLa cells, respectively, after illumination. Similarity clustering analysis compiling the bioactivity data revealed two groups of blepharismins: the most active, B and C, and the less active, A, D and E. The B. sinuosum pigment blend includes one representative of each clade. Functional and medical implications are discussed.

The morphology of Blepharisma sinuosum resting cysts and the dynamics of pigmentation at different stages of encystment are presented for the first time. Cyst morphometrics are similar to other Blepharisma species, with three-wall layers, bacteria surrounding the ectocyst, a conical plug, and wrinkly surface toward the plug in mature stages. The vegetative moniliform macronucleus changes to a horseshoe shape, and at early stages, the cystic cytoplasm is homogeneously pigmented, comprising a contractile vacuole; later, pigments polarize toward the plug, decorate the cortical layer, and become brownish. This work reinforces the potential role of pigment dynamics on cyst biology.

It is known that the freshwater heterotrich ciliate Blepharisma japonicum uses five pigments called blepharismins, stored in its extrusive pigment granules, for both light perception and chemical defense against predators. In this work we focused our attention on the defensive strategies of two additional pigmented species of Blepharisma, B. stoltei and B. undulans. In particular: (1) we observed the predator–prey interactions of B. stoltei or B. undulans against one multicellular and two unicellular predators; (2) we clarified the nature of B. stoltei and B. undulans pigments by means of High Performance Liquid Chromatography (HPLC) and Liquid Chromatography Mass Spectrometry (LC-MS); and (3) we demonstrated and compared the toxicity of the purified pigments on a panel of ciliated protists, and against one metazoan predator. The results indicate that the chemical defense mechanism present in B. stoltei and B. undulans is mediated by the same five blepharismins previously characterized for B. japonicum, although produced in different proportions.

Using standard methods, I describe Heterometopus meisterfeldi nov. gen., nov. spec., a new armophorean ciliate from dry soil of the bed of the Fogg Dam in northern Australia. It appeared in the non-flooded Petri dish culture three weeks after soil re-wetting, i.e., when the bottom zone of the culture became anaerobic. The new genus is characterized by a cylindroid body and a short, slightly oblique, J-shaped adoral zone extending only ventrally and about one third of body length. The species has an average size of 140×30μm and has 23 ciliary rows, 30 adoral polykinetids, and 25 false kineties in the perizonal stripe. Metopus palaeformis, as redescribed by Foissner et al. (2002) and in the present paper, shows very similar characteristics and is thus transferred to the new genus: Heterometopus palaeformis (Kahl, 1927) nov. comb. Heterometopus meisterfeldi and H. palaeformis differ by body size (∼140×30 vs. 100×20 in vivo), the shape of the macronucleus (ellipsoid, ∼2:1 vs. elongate ellipsoid, ∼3:1), the width of the side-stripe (12μm vs. 4μm), the number of false kineties in the perizonal stripe (25 vs. 14), and the number of adoral polykinetids (∼30 vs. ∼20). Very likely, several species from Kahl's Metopus groups II and III belong to Heterometopus but a transfer should await detailed redescriptions. Heterometopus meisterfeldi belongs very likely to the family Metopidae. The short, J-shaped adoral zone of polykinetids highly resembles the blepharismid genera Pseudoblepharisma and Blepharisma.

Among protists, pheromones have been identified in a great variety of algal species for their activity in driving gamete-gamete interactions for fertilization. Analogously in ciliates, pheromones have been identified for their activity in inducing the sexual phenomenon of conjugation. Although this identification was pioneered by Kimball more than fifty years ago, an effective isolation and chemical characterization of ciliate pheromones has remained confined to species of Blepharisma, Dileptus and Euplotes. In Euplotes species, in which the molecular structures have been determined, pheromones form species-specific families of structurally homologous helical, cysteine-rich, highly-stable proteins. Being structurally homologous, they can bind cells in competition with one another, raising interesting functional analogies with the families of growth factors and cytokines that regulate cell differentiation and development in higher organisms. In addition to inducing conjugation by binding cells in heterologous fashion, Euplotes pheromones act also as autocrine growth factors by binding to, and promoting the vegetative reproduction of the same cells from which they originate. This autocrine activity is most likely primary, providing a concrete example of how the original function of a molecule can be obscured during evolution by the acquisition of a new one.

We investigated mating pair formation between three Blepharisma species Blepharisma undulans, Blepharisma japonicum, and Blepharisma stoltei to determine whether their respective gamones (mating pheromones) effectively induce mating pairs between different species. Cell-free fluid from type II cells (CFF2) of B. undulans (megakaryotype II) induced pairing of B. japonicum and B. stoltei type I cells (megakaryotype IV), and CFF2 of B. japonicum and B. stoltei induced pairing of B. undulans type I cells. Cell-free fluid from B. undulans type I cells (CFF1) did not induce pairing of B. japonicum and B. stoltei type II cells, and CFF1 of B. japonicum and B. stoltei failed to induce pairing of B. undulans. CFF1 from B. japonicum and B. stoltei mutually induced pairing, as previously reported. These results indicate that gamone 2 is common among megakaryotypes II and IV, and that gamone 1 appears to be at least megakaryotype-specific. When cells belonging to megakaryotypes II and IV are separately pre-treated with effective gamones and mixed, mating pairs between megakaryotypes rarely form. Taken together, these results suggest at least two barriers, a gamone and a factor involved in pair formation, that prevent conjugation between different megakaryotypes of Blepharisma.

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing enzyme that catalyses the oxidative cleavage of L-Trp. The ciliate Blepharisma stoltei has four IDO genes (IDO-I, -II, -III and -IV), which seem to have evolved via two sequential gene duplication events. Each IDO enzyme has a distinct enzymatic property, where IDO-III has a high affinity for L-Trp, whereas the affinity of the other three isoforms for L-Trp is low. IDO-I also exhibits a significant catalytic activity with another indole compound: 5-hydroxy- l -tryptophan (5-HTP). IDO-I is considered to be an enzyme that is involved in the biosynthesis of the 5-HTP-derived mating pheromone, gamone 2. By analysing a series of chimeric enzymes based on extant and predicted ancestral enzymes, we identified Asn131 in IDO-I and Glu132 in IDO-III as the key residues responsible for their high affinity for each specific substrate. These two residues were aligned in an identical position as the substrate-determining residue (SDR). Thus, the substrate affinity and specificity are regulated mostly by a single amino acid residue in the Blepharisma IDO-I and IDO-III enzymes.

Mating pheromones (gamone 1 and gamone 2) in the ciliate Blepharisma are biologically active substances that trigger sexual reproduction (conjugation) under starvation conditions. Gamone 1 is a glycoprotein secreted by type I cells, and gamone 2 is a tryptophan (Trp)-derivative compound secreted by type II cells. Both gamones stimulate complementary mating type cells to promote each gamone production and induce pair formation. To elucidate the biosynthetic pathway of gamone 2, we investigated the enzymes involved in the pathway and the specificity of the enzymes. An RNA-seq analysis revealed that Blepharisma stoltei (Heterotrichea) possesses four indoleamine 2,3-dioxygenase (IDO) genes showing distinct expression patterns. Along with results from real-time PCR, these findings demonstrated that each IDO gene has different expression patterns that depend on the cellular conditions. Expression of IDO-I was correlated with the intensity of gamone 2 expression, and the recombinant IDO-I protein showed catalytic activity for 5-hydroxy-L-Trp (5-HTP) but very weak activity for L-Trp. Our results indicate that IDO-I is an enzyme evolutionary specialized to gamone 2 production in Blepharisma, and that the biosynthetic pathway for gamone 2 uses 5-HTP as an intermediate.

We collected a heterotrich ciliate from hypersaline waters on the Cape Verde island Sal. After living observation and protargol-staining, it closely matched Blepharisma halophilum Ruinen, 1938. However, because (i) Ruinen most likely described several different species as B. halophilum, and (ii) he provided only very scarce diagnostic features, we redescribe this species with state-of-the-art methods according to modern standards. This will facilitate an accurate identification of this ciliate species, globally distributed in hypersaline water bodies: halophile Blepharisma, about 100-250μm in vivo; 32-42 adoral membranelles; 13-17 somatic kineties, including 2-4 anterior kinety fragments; single elongated macronucleus; cortical granules colourless. Its SSU rRNA gene sequence is very similar (99.7%) to an uncultured organism (environmental sequence) obtained from the hypersaline Lake Tyrrell in Australia. In phylogenetic analyses, these two sequences form a cluster, which with nearly full support (ML/BI, 99/1.00) branches as sister to the Fabreidae, showing for the first time that Blepharisma is yet another non-monophyletic ciliate genus.

Heterotrichs are generally larger than ciliates of other groups with a seemingly cosmopolitan distribution, and recent studies have demonstrated that they exhibit great biodiversity. In the present work, we investigated the morphology and small subunit rRNA (SSU rRNA) gene sequences of three heterotrichous species, including that of a new one, Anigsteinia paraclarissima spec. nov. The new organism is morphologically very similar to A. clarissima, however, it can be distinguished from the latter by the larger dimensions and more somatic kineties (25–32 vs. 18–26), and its sequence similarity of SSU rRNA gene is 97.14% which indicate that it is a distinct species. Detailed morphological and molecular data for Blepharisma bimicronucleatum are supplied together for the first time in this study. In addition, the morphology of a poorly known species, Spirostomum yagiui is redescribed and an improved species diagnosis is provided. Finally, based on phylogenetic analyses of SSU rRNA gene sequences data, the Spirostomum group contains two main clades based on the type of the macronucleus. Spirostomum yagiui was newly sequenced here and clustered with the other S. yagiui populations and positioned within the Spirostomum assemblage. The Anigsteinia clade, including A. paraclarissima and A. clarissima, clustered within the family Spirostomidae.

The morphology and morphometric data of seven populations of Blepharisma sinuosum from southeastern Brazil were investigated. The description is based on live observations, protargol impregnation, and scanning electron microscopy. Blepharisma sinuosum measures 75–255 μm in length and 25–93 μm in width and has a spindle-shaped body, pink color, a single contractile vacuole located at the posterior end, 50 adoral membranelles, a conspicuous paroral, 17–35 somatic kineties, a moniliform macronucleus with 2–7 connected nodules, and 3–20 micronuclei. Morphological comparisons with similar species were performed and suggest that B. americanum is the junior synonym of B. sinuosum. The 18S rDNA gene sequence of B. sinuosum was obtained and compared with that of other Blepharisma species. The length and GC content of the obtained sequence is 1652 bp and 47.03%, respectively, and has a very high structural similarity (99.9%) with the B. undulans sequence. The validity of the classification of Blepharisma species in morphonuclear subgenera is also discussed.

Ciliates communicate via diffusible signaling pheromones that have been identified in association with genetic mechanisms of mating types, either binary or multiple, that control the ciliate-specific sexual phenomenon of conjugation. Except for a glycoprotein and a tryptophan-related molecule acting as pheromones in Blepharisma, the other pheromones are cysteine-rich proteins varying in length among Euplotes species from 32 to 109 amino acid residues. These proteins are all structurally homologous sharing a common three-helix bundle fold, which allows them to bind cells in competition with one another in close analogy with the families of protein growth factors and cytokines of multicellular organisms. Although identified in the extracellular environment for their mating-induction activity, pheromones act also, and presumably primarily, as autocrine growth factors that bind to, and promote the reproduction of the same cells from which they are constitutively released.

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 10 hours 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.

The coloured ciliate Blepharisma japonicum changes swimming velocity (positive photokinesis) and elongates its body in response to a prolonged illumination. We have recently proposed that alterations in the phosphorylation level of the ciliate phosducin (Pdc) may be involved in light-induced cell elongation, which in turn affects the interaction of βγ-dimer of G-proteins (Gβγ) with β-tubulin and subsequent cytoskeletal remodelling. The cellular mechanism that governs the photokinetic effect in this ciliate has not been elucidated. In the present study, we utilise real-time PCR to demonstrate that the levels of ciliate Pdc mRNA are significantly reduced in Pdc-RNAi-treated cells compared to cells fed with bacteria carrying the empty vector (control cells). Using western immunoblotting, we confirmed that these cells treated with Pdc-RNAi expressed a substantially lower level of the Pdc protein. The assay also revealed that in ciliates treated with Pdc-RNAi and exposed to light, the cytosolic level of Gβ (~36 kDa) was reduced, whereas the level of Gβ localized to the membrane (~32 kDa) was increased compared to control cells. In addition, behavioural analysis of the cells indicated a substantial reduction of photokinesis. The findings in this study provide additional characterization of the functional properties of the ciliate Pdc protein and we discuss a likely role for this phosphoprotein in the photokinetic phenomenon of the ciliate protist Blepharisma.

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.

The unicellular eukaryote, Blepharisma japonicum, is a light-sensitive ciliated protozoa. It possesses a photoreceptor pigment called blepharismin that plays critical roles in defensive behavior against predators and step-up photophobic response. In addition, the pigment generates reactive oxygen species such as singlet oxygen and hydroxyl radicals which contribute to photodynamic action. Previous studies reported that intense light (>300 W m−2) induced rapid photodynamic killing (necrosis) characterized by cell swelling and plasma efflux, while moderate light (3–30 W m−2) only induced pigment extrusion and photooxidation. We have found that moderate light (5 W m−2) induced apoptosis-like cell death. Microscopically it was found that >3 h of moderate light irradiation induced macronuclear condensation and plasma efflux without cell swelling. Single cell gel electrophoresis assay showed that DNA fragmentation occurred between 1 and 3 h of irradiation, and the condensed macronuclei contained quite fragmented DNA. Macronuclear DNA extracted from light-irradiated cells contained DNA fragments of 180–200 and 360–400 bp, which were seen as apoptosis ladders.

Two ciliate species of Blepharisma were collected from the riverine wetland and mountain peak soil in Korea, Blepharisma undulans Stein, 1867 and B. steini Kahl, 1932, which are reported for the first time from Korea. The description was based on the observations of live and protargol impregnation specimens. Diagnostics of these species (Blepharisma undulans vs. B. steini) are as follows. Body length/width ratio in vivo about 6/1 vs. 2/1, shaped in spindle vs. pyriform to ellipsoid and colored in pinkish vs. reddish. Macronuclei 2 vs. 1, with 4-9 vs. 4-7 spherical micronuclei. Adoral zone of membranelles extends from anterior end to 1/3 vs. 1/2-2/3 of body length, with 47-56 vs. 33-45 adoral membranelles. The numbers of somatic kineties are 22-27 vs. 18-22.

The systematically poorly known ciliate genus Condylostoma was erected by Vincent in 1826. About 10 morphotypes have been reported, but any molecular investigations concerning this group so far are lacking. In this work, the small subunit ribosomal RNA (SS rRNA) gene of three marine Condylostoma species was sequenced, by which the phylogenetic trees were constructed by distance-matrix, maximum parsimony and Bayesian inference methods. The results show that (1) all the trees have similar topologies with high supports; (2) Condylostoma is mostly related to the genus Condylostentor ; and (3) three Condylostoma species as well as Condylostentor auriculatus cluster together and form a sister group with other heterotrichs. This is moderately consistent with the assessment of phylogenetic relationships of Condylostoma -related heterotrichs from the morphological information. The phylogenetic relationship of some other related heterotrichs, Peritromus, Folliculina, Stentor and Blepharisma , has been also discussed.

application/pdf, In the absence of O2, the step-up photophobic response of Blepharisma japonicum is suppressed. To evaluate the possibility that reactivated oxygen species (ROS) produced by blepharismin photosensitization are involved in the primary transduction for the step-up photophobic response of B. japonicum, the effects of OH radical scavengers and the singlet oxygen quencher on the photophobic response were examined. The reagents used in the present assays hardly suppressed the step-up photophobic response, even though OH radical scavengers were rescued from the photodynamic killing of the cells. Alternatively, in the presence of an electron acceptor, the latency of the step-up photophobic response was prolonged. In addition, oxyblepharismin fluorescence was quenched slightly in the presence of free amino acids. These results support a hypothesis proposed by Lenci’s research group, that electron transfer from the first excited singlet state of blepharismin (or oxyblepharismin) to associated proteins may be involved in the primary phototransduction for the step-up photophobic response.

In the ciliated protozoan Blepharisma, step-up photophobic response is believed to be mediated by a novel type of photosensory pigment known as "blepharismins" (BL) that are contained in the pigment granules located just beneath the plasma membrane. We examined the ultrastructure of the pigment granules by freeze-fracture and thin-section electron microscopy and proposed a schematic diagram showing the granules' three-dimensional inner membranous structure. Some of the BL are suggested to be associated with 200 kDa membrane protein. High-pressure liquid chromatography analysis of pigment species associated with 200 kDa protein obtained from blue forms of Blepharisma (oxyblepharisma) revealed that the 200 kDa protein was associated with five types of oxyblepharismin. The fluorescence intensity was increased when the pigments were dissociated from the 200 kDa protein. The result supports the hypothesis that the pigment-200 kDa complex is able to transduce light energy into signals mediating the photobehavior of Blepharisma.

As in animals and other multicellular organisms, protozoan ciliates communicate via diffusible signalling pheromones. These pheromones have been identified in the culture supernatant of various species. However, their isolation and definitive structural characterization have been achieved only in species of Blepharisma and Euplotes. With the exception of the two B. japonicum pheromones represented by a tryptophan-related molecule and a glycoprotein, all the other pheromones isolated from Euplotes species are proteins that vary in extension between 38 and 109 amino acids. They form species-specific families of structurally homologous members, in full accord with their multi-allelic determination at an apparently single genetic locus. The determination of the Nuclear Magnetic Resonance (NMR) solution structures of E. raikovi and E. nobilii pheromones has provided direct evidence that, although structurally unique, Euplotes pheromones adopt a common, species-specific three-helix folding. This close s...

The hepatic genera Colura and Pleurozia show trap-like structures in the water sacs of their leaves. The function of these structures usually has been interpreted as water storage, although zoophagy has also been suggested. The experimental evidence that a species of Colura was able to trap protozoa was presented some years ago. Therefore the trap-like structure in the water sacs of Pleurozia purpurea has been studied. It is described and illustrated by light and electron scanning microscopy. Feeding experiments revealed that the structures in the water sacs of Pleurozia purpurea are able to trap individuals of Blepharisma americana (Ciliata). This provides evidence that the lobules of this second hepatic genus function in zoophagy. Other experiments have shown that individuals of Blepharisma are attracted by plants of Pleurozia, although the exact reason for this behavior is not known. Observations in the field revealed a high percentage of lobules filled with various small animals and suggest t...

Several recent studies suggest that community composition can take on alternate, apparently stable, states. For the most part, these studies are observational in nature and have not shown that alternate states arise from the order of species arrival during community assembly. Previous studies also provide little insight into the mechanisms that create alternate states, especially when direct species interactions, rather than environmental feedback, control their formation. We conducted experiments in identical physical environments to show that differences in the sequence of community assembly can create alternate species compositions that persist for many generations. Assembly sequence determined whether experimental food webs contained a single intraguild predator, or two intraguild predators. When the intraguild predator, Blepharisma americanum, became established in communities before the introduction of Tetrahymena vorax, combined effects of resource depletion and intraguild predation prevented the establishment of Tetrahymena. In contrast, when Tetrahymena preceded Blepharisma, both species frequently coexisted. Supporting experiments describe the exploitative competitive abilities of both intraguild predators, document the population dynamics of each species in less complex community modules, and determine the ability of Tetrahymena to invade Blepharisma cultures in the absence of competing prey. These results show that both competition and predation contribute to the formation of two alternate community states and confirm that one stable state resists transformation to the other.

Blepharismins are polycyclic quinones found in the pigment granules of the ciliated protozoan, Blepharisma. Exposure to purified blepharismins results in lethal damage to several other ciliates. We here report that, at cytotoxic concentrations, blepharismins formed cation-selective channels in planar phospholipid bilayer membranes. The channels formed in a diphytanoylphosphatidylcholine bilayer had a K + /Cl 3 permeability ratio of 6.6:1. Single channel recordings revealed the conductance to be quite heterogeneous, ranging from 0.2 to 2.8 nS in solutions containing 0.1 M KCl, possibly reflecting different states of aggregation of blepharismin. Our observations suggest that channel formation is a cytotoxic mechanism of blepharismin's action against predatory protozoa. fl 2001 Fed- eration of European Biochemical Societies. Published by Else- vier Science B.V. All rights reserved.

Predation among species that compete for resources is widespread. Theory suggests a counterintuitive consequence of such intraguild predation: enrichment of the shared resource, for which the intraguild prey must be the superior competitor, reduces the population size of the intraguild prey and may even cause its extinction. Experiments with bacteria (shared resource) and the ciliates Tetrahymena (intraguild prey) and Blepharisma (intraguild predator) confirmed these expectations. Tetrahymena was the better resource competitor. Grown separately, it persisted at lower levels of enrichment and drove bacterial densities to lower levels than did Blepharisma. When grown together with Blepharisma, Tetrahymena declined in density with increasing enrichment, whereas bacteria and Blepharisma increased. The data thus support the theoretically suggested mechanism as the cause of this pattern: with enrichment, increasing densities of bacteria sustained an increasing predation pressure from Blepharisma on Tetrahymena, which overrode the direct positive effect of bacteria as a food source of Tetrahymena. Generalizing from these results, we speculate that staggered invasions of omnivores followed by extinctions of intermediate prey should contribute to the frequently observed unimodal or neutral responses of species richness and food chain length to increasing productivity.

In the ciliated protozoan Blepharisma, step-up photophobic response is mediated by a novel type of photosensory complex of pink-colored pigment “blepharismins” and 200-kDa membrane protein contained in the pigment granules located just beneath the plasma membrane. We found that the fluorescence intensity of isolated blepharismins decreased prominently with a decrease of H+ concentration in the surrounding medium. In the present study, therefore, we utilized the endogenous pigment blepharismins as the pH indicator. Light stimulation evoked a sudden decrease in fluorescence intensity in a photosensitive anterior portion of the cell, suggesting that a drop in H+ concentration occurred in the anterior region. The result indicates that the photosignal is transduced into cytoplasmic signaling of H+ translocation across the outer membrane surrounding the pigment granules, so that cytosolic H+ concentration in the vicinity of plasma membrane might be increased. J. Exp. Zool. 289:467–471, 2001. © 2001 Wiley-Liss, Inc.

We investigate the rate of increase of protists introduced at low density into experimental communities comprising different sets of coexisting species. The experiment separates (I) the main effect of six introduced species. (2) the main effect of eleven resident communities, and (3) the introduction × community interaction. Introduced species differ markedly in their capacity to invade, and only one, Paramecium, invades all of the communities. Most of the introduced species invade some communities, and there is a large introduction × community interaction that comes about from bringing together particular combinations of species. For example, the rate of increase of Amoeba is greater when introduced into communities containing Paramecium than when introduced into communities in which Paramecium is absent. The omnivore Blepharisma increases faster in the presence of one of its prey Tetrahymena, notwithstanding the fact that it is also a potential competitor with this prey for bacteria. There is little indication that the species richness of resident communities (over the limited range available) affects the success of invasion. The results suggest that understanding invasions depends as much on detailed knowledge of idiosyncratic biological interactions as on general properties of community structure.

Although omnivory (the consumption of resources from more than one trophic level) is widespread, this fundamental limitation to the applicability of food chain theory to real communities has received only limited treatment. We investigated effects of enrichment (increasing carrying capacity, K, of the resource) on a system consisting of a resource (R), an intermediate consumer (N), and an omnivore (P) using a general mathematical model and tested the relevance of some of its predictions to a laboratory system of mixed bacteria (=R) and the ciliates Tetrahymena (=N) and Blepharisma (=P). The model produced six major predictions. First, N may facilitate or inhibit P. Enrichment may revert the net effect of N on P from facilitation to inhibition. Second, along a gradient of K, up to four regions of invasibility and stable coexistence of N and P may exist. At the lowest K, only R is present. At somewhat higher K, N can coexist with R. At intermediate K, either N and P coexist, or either consumer excludes the other depending on initial conditions. At the highest K, N may be excluded through apparent competition and only R and P can coexist. The pattern of persistence of Tetrahymena and Blepharisma along an enrichment gradient conformed fairly well to the scenario allowing coexistence at intermediate K. Third, for stable equilibria of the omnivory system, R always increases and N always decreases with K. The abundances of bacteria and Tetrahymena were suggestive of such a pattern but did not allow a strict test because coexistence occurred at only one level of enrichment. Fourth, an omnivore can invade an R-N system at a lower K than an otherwise identical specialist predator of N. Fifth, an omnivore can always invade a food chain with such a specialist predator. Sixth, over ranges of K where both omnivory systems and otherwise identical three-level food chains are feasible, N is always less abundant in the omnivory system, whereas the relative abundances of R and P in omnivory systems compared to food chains may change with K. It is thus possible that total community biomass at a given K is lower in an omnivory system than in a food chain. Both the model and the experimental results caution that patterns of trophic-level abundances in response to enrichment predicted by food chain theory are not to be expected in systems with significant omnivory.

Theory predicts that productivity and the relative efficiency of resource utilization determine the outcome of interactions between intraguild predators and prey. Interactions between two freshwater protists, Colpidium striatum (the intraguild prey) and Blepharisma americanum (the intraguild predator), in laboratory microcosms support the predictions of intraguild predation theory. Colpidium competitively excluded Blepharisma when bacterial production was low, even though Blepharisma had a potential advantage conferred by its ability to consume Colpidium. In contrast to competitive exclusion at low productivity, Colpidium and Blepharisma coexisted for many generations at higher levels of bacterial production. Theory predicts a similar transition from competitive exclusion to coexistence as productivity increases, if intraguild predators use resources less efficiently than intraguild prey/competitors. Other experiments showed that Colpidium depresses bacterial densities more than Blepharisma, a result consistent with greater efficiency of resource utilization by Colpidium. These results indicate that simple mathematical models can provide important insights into the population dynamics of intraguild predators and prey under different levels of productivity.

— The toxic and phototoxic properties of blepharismin and oxyblepharismin that were purified from the pigments of a ciliated protozoan, Blepharisma japonicum, by thin-layer chromatography, were investigated in detail. The toxicity was tested against the ciliated protozoan Dileptus margaritifer, which is relatively sensitive to blepharismin. Although oxyblepharismin has been believed to be neither toxic nor phototoxic, it was found that oxyblepharismin is toxic in the dark and is also phototoxic. This shows that oxyblepharismin can act as a photosensitizer. The toxicity and phototoxicity of these pigments were compared with those of hypericin, which is known to be a typical, strong photosensitizer from plants. It was concluded that blepharismin and oxyblepharismin have strong intrinsic toxicities in the dark compared with hypericin, but their phototoxicities are slightly weaker than that of hypericin. This strong intrinsic toxicity supports our proposal that blepharismin acts as a defensive device against predators in the dark as well as in the light. The decrease in the defensive ability and the increase in the resistance to photokilling of Blepharisma concomitant with its color change from red to blue-purple in response to weak illumination can be explained by the decrease in toxicity and phototoxicity of the pigment itself and by the decrease in amount of the pigment.

Summary The defensive function of pigment granules in Blepharisma japonicum against the predatory ciliate Dileptus margaritifer was further investigated by 1) observing the discharge of pigment granules of Blepharisma as a response to the attack by the predator and 2) measuring the toxicity of purified blepharismin, the red pigment localized in pigment granules. When a Blepharisma was attacked by the toxicysts-bearing proboscis of a Dileptus , the Blepharisma instantly released a mass of reddish material at the attacked site. The Dileptus retreated and the Blepharisma swam away. The observation suggested that the Blepharisma discharged pigment granules as a response to the attack. This assumption was confirmed by scanning-electronmicroscopic observations; many pigment granules near the attacked site were discharged at the moment of the attack. Purified blepharismin was highly toxic to Dileptus and several other ciliates, but was not toxic to Blepharisma . We also showed that blepharismin is toxic in the dark. These results strongly support the previously presented hypothesis that the defensive function of pigment granules in B. japonicum against D. margaritifer is based on the discharge of blepharismin as a response to the attack by the predator. We conclude that pigment granules of B. japonicum are extrusomes (extrusive organelles in protists) for chemical defense.

Summary Phototaxis describes the directed movement of motile microorganisms with respect to the direction of light. In ciliates, phototactic orientation has been reported for more than a dozen species. Some, like Stentor and Blepharisma , arc brightly coloured due to their numerous pigment granules. In Chlamydodon , a stigma appears in conjunction with a behavioural change from negative to positive phototaxis. The histophagous ciliates Ophryoglena and Porpostoma , which either possess a watchglass organelle or a composed crystalline organelle, display different phototactic behaviour at different stages of their life cycles. It has been suggested that the characteristic pigments and cell organelles of phototactic ciliates play important roles in the detection of the light direction. Bleached cells with a reduced number of pigment granules, transparent cells without a stigma, and cells that lacked a watchglass organelle due to micromanipulation were found to be little sensitive to light, or showed inaccurate or no phototactic orientation. While the pigment granules of the heterotrichs presumedly contain the photoreceptor molecules mediating photo-orientation, the stigma of Chlamydodon , the watchglass organelle of Ophryoglena , and the composed crystalline organelle of Porpostoma are suspected shading or light reflecting structures. Interacting with the sites of sensory transduction, which should be located in the cell membrane nearby, but are unidentified, conspicuous organelles could be used by the cells as direction-sensitive light detectors.

Summary During conjugation of Blepharisma, only some micronuclei enter meiosis. Other micronuclei, called somatomicronuclei, do not degenerate but differentiate directly into macronuclear anlagen (secondary anlagen) without meiosis and karyogamy. Normal (primary) anlagen develop from synkaryon derivatives. We observed these processes at the ultrastructural level. In early conjugation (0–2 hours after pair formation), all micronuclei swell. This correlates with decondensation of the micronuclear chromatin. At 3 hours, the micronuclei differentiate into somatic and meiotic (leptotene) ones: the latter develop bundles of microtubules. The somatomicronuclei remain homogeneous and lack microtubules. At 8–9 hours meiotic micronuclei display synaptonemal complexes and thus are in pachytene; at the same time, structures in form of loose chromatin patches first appear in somatomicronuclei. The patches gradually condense and become conspicuous at 10–12 hours (stages from diplotene to metaphase I of meiosis). At about 12 hours, the meiotic micronuclei are in metaphase I and display acentric intranuclear spindles with blunt poles and homogenous polar caps; the bivalents have prominent kinetochores. At 16 hours, the somatomicronuclei contain numerous chromatin patches which are possibly subchromosomes, and first nucleoli appear in them. At 16–18 hours, the stage of pronuclei is reached; and other meiotic products start degenerating. The migratory pronuclei show concentration of the chromatin at the centre of the nucleus. At 20, 26, 28 and 34 hours, the fine structure of somatomicronuclei (secondary anlagen) changes little. Their size remains constant (about 5–6 μm). However the nucleoli enlarge at 34 hours. The first division of the synkaryon has protruding poles and no polar caps, unlike meiosis I; though, the nuclear envelope remains intact even at the poles. The synkaryon divisions give rise to new micronuclei and primary macronuclear anlagen. The primary (meiotic) macronuclear anlagen differentiate in number of 2–4 at 22–24 hours. They are much larger than secondary anlagen (up to 20 μm) and, at early stages of development, their chromatin is so strongly decondensed that the anlagen look “empty”. However later (by 34 hours) loose chromatin patches, small bodies of condensed chromatin and nucleolar primordia appear in them, like in somatomicronuclei, and the primary anlagen at 34 hours show additionally a karyosome-like central condensation of the chromatin.

— Photosensitivity for the step-up photophobic response of Blepharisma is localized in the anterior 1/5 of the cell body. Blepharismin pigment, which is believed to be a photoreceptor pigment mediating the step-up photophobic response of the cells, was separated into five types of blepharismin (BL-1, -2, -3, -4 and -5). Blepharismin-1, -3, -4 and -5 were localized in the posterior 4/5, while BL-2 was located over the entire cell body; the anterior end, which is the photosensitive region, contained only BL-2. The results indicate that a functional photoreceptor pigment mediating the step-up photophobic response is BL-2. Hydroxylapatite chromatography revealed that BL-2 was bound to a 200 kDa membrane protein. We concluded that a photoreceptor mediating the step-up photophobic response was a BL-2/200 kDa protein complex.

The aim of the research presented here was to determine whether low concentrations (1 μg/ml) of a highly specific microtubule depolymerizing agent, nocodazole (NOC), affects the meiosis ac tivation process in conjugating pairs of the ciliated protozoan Blepharisma japonicum. In order to identify and quantify differen ces in meiosis, both NOC treated and untreated coeval pairs were stained and examined for their meiotic phases. A clear‐cut delay in the progression of meiosis in treated pairs was found. The findings indicate that NOC reversibly inhibits meiosis induction and that treated pairs need both continuous cell union and a period of recovery in order to progress through meiosis after NOC is with drawn. These results suggest that cytoplasmic microtubule dynamics may be involved in the meiotic activation processs of Blepharisma conjugating pairs.

To study how organism responds to periodic stimulation is meaningful since it may be an approach to an elementary capacity of time memory and learning in chronological events. We reported here that the ability of time memory found in true slime mold Physarum was also found in a protozoan ciliate, Blepharisma japonicum and a green plant Chara. Stimulation of temperature or light was repeated several times in a regular period, and the creature anticipated the next timing of stimulation. After the anticipatory behavior disappeared some time later, another single stimulation triggered recalling of periodicity of the previous stimulation. We discuss that the observed capacity is expected to be common in a range of species as the similar capacity has been reported in true slime mold Physarum. The observed responses were, however, dependent of individual of organism and a wide range of different responses was observed. We need an extensive study of both experimental characterization and mathematical modeling of ethological dynamics. abstract environment.

The quinone pigment blepharismin isolated from Blepharisma, which is believed to be a photoreceptor pigment mediating photobehaviour (P. Scevoli, F. Bisi, G. Colombetti, F. Ghetti, F. Lenci and V. Passarelli, Photomotile responses of Blepharisma japonicum. I. Action spectra determination and time-resolved fluorescence of photoreceptor pigments, J. Photochem. Photobiol. B: Biol., 1 (1987) 75–84; T. Matsuoka, S. Matsuoka, Y. Yamaoka, T. Kuriu, Y. Watanabe, M. Takayanagi, Y. Kato and K. Taneda, Action spectra for step-up photophobic response in Blepharisma, J. Protozool., 39 (1992) 498–502), killed mammalian cells (L cells) when activated by light. Electron spin resonance (ESR) spectroscopy demonstrated that light-activated blepharismin generated hydroxyl radicals (.OH). Light-activated blepharismin pigment promoted lipid peroxidation of L cells, which was partially suppressed in the presence of the singlet oxygen quencher sodium azide (NaN3). The results suggest that the photodynamic action of blepharismin is correlated with lipid peroxidation which may be caused by hydroxyl radicals and/or singlet oxygen (1O2) produced via reactions sensitized by the pigment.

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In the ciliated protozoan, Blepharisma japonicum, the pink-colored pigment (blepharismin) contained in the pigment granules is believed to be the photoreceptor pigment responsible for the step-up photophobic response. When the cells partially bleached by extrusion of the pigment granules caused by cold shock were subsequently cultured under illuminated conditions, the pigment-less granules regenerated and the cells were further bleached (pigment content below 0.5%). The photosensitivity of such colorless cells disappeared completely. In contrast, the blepharismin pigment regenerated gradually when such colorless cells were transferred to darkness. The photosensitivity of the cells also recovered with regeneration of the pigment. We found that blepharismin pigment was not photobleached in the absence of O2. The step-up photophobic response was also completely repressed in the absence of O2. These results strongly confirm that blepharismin is a photoreceptor pigment mediating photobehavior of Blepharisma and that O2 is required for the early step in the phototransduction of the light-excited pigment.

Blepharismin is an endogenous photosensitizing pigment found in the protozoan Blepharisma . This pigment inhibited the generation of Superoxide anion (O 2 − ) in neutrophils not only via a diacylglycerol-induced protein kinase C (PKC)-dependent reaction but also by an arachidonate-induced PKC-independent reaction. The inhibition was light and concentration dependent for both reactions. Light-activated inhibition was strong at wavelengths between 520 and 570nm but not above 610nm. PKC activity in neutrophils and from rat brain was inhibited by blepharismin in a light- and concentration dependent manner. Moreover, arachidonate-activated NADPH oxidase activity in a cell-free system was also inhibited by the pigment in a light- and concentration-dependent manner. These results suggest that blepharismin inhibits NADPH oxidase activation through the non-specific inhibition of various membrane-bound enzymes and that this inhibition may also be correlated with that of PKC.

Summary The defensive function of pigment granules in the red heterotrichous ciliate Blepharisma japonicum was studied by comparing normally-pigmented red cells, albino mutant cells and light-bleached cells (a phenocopy of the albino mutant) as prey for a carnivorous ciliate Dileptus margaritifer. Albino and bleached cells were eaten more readily than red cells by dilepti. Under certain conditions, dilepti were killed by red cells, but not by albino and light-bleached cells. The cell-free fluid of red cells, but not of albino and bleached cells, was toxic to dilepti. We conclude that one of the functions of pigment granules in B. japonicum is defense against predators. The offense-defense interaction between D. margaritifer and B. japonicum is, therefore, mediated by their extrusomes, toxicysts in the former and pigment granules in the latter. Other extrusomes of unknown function might also participate in offense-defense cell-cell interaction in protists.

The red pigment granule of Belpharisma japonicum is believed to be a photoreceptor organelle mediating photodispersal. Freeze-fracture and thin section electron microscopy revealed that the pigment granules contained a honeycomb-like structure constructed of folded membranes. In the fracture face of the honeycomb-like structure, small membrane particles were observed, which might correspond to pigment—protein complexes. The pigment granules were isolated and detergent-solubilized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the pigment granules mainly contained a 200 kDa membrane protein. The complex of red pigment and 200 kDa protein was isolated by gel-filtration chromatography of the detergent-solubilized components, and the protein was subjected to SDS-PAGE and amino acid analysis. The 200 kDa protein could not be dissociated into subunits by 2-mercaptoethanol, indicating that the protein is composed of a single polypeptide chain. Hydrophobic amino acids contained in the 200 kDa protein were not dominant, suggesting that only partial domains may extend across the membrane of the honeycomb-like structure.

When the cells of Blepharisma cultured in lettuce juice were transferred to media containing lettuce juice with temperature gradient of 20–30°C, they accumulated in a region corresponding to about 25°C. The cells washed in a saline solution, however, accumulated in the region above 30°C. The results indicate that certain components contained in lettuce juice change the thermosensitivity of the cells. When the temperature was suddenly changed, a transient decrease or increase in the frequency of ciliary reversal was observed. The response of the cells incubated in a saline solution without lettuce juice was different from those in the saline solution containing lettuce juice above 25°C. Above 25°C, the cells incubated in a saline solution without lettuce juice responded by increased frequency of ciliary reversal only to step-down in temperature and by repression of ciliary reversal to a step-up in temperature, while the cells incubated in the same saline solution containing lettuce juice responded by increased frequency of ciliary reversal to a step-up in temperature and by repression of ciliary reversal to a step-down in temperature. The thermal response was examined in bisected fragments of the cells. The difference in response between the saline solution without lettuce juice and the solution containing lettuce juice was detected only in posterior fragments above 25°C. Above 25°C, the posterior fragments incubated in the solution without lettuce juice did not respond to sudden temperature changes, whereas the fragments kept in the medium containing lettuce juice responded (step-up thermophobic response and step-down repression of ciliary reversal) in the presence of lettuce juice.

—Blepharisma japonicum exhibits a step-up photophobic response when subjected to an increase in light stimulus intensity. This response is characterized by the stop reaction after a period of delay followed by backward swimming (lateral rotation). The latency of the stop response decreased and duration of the lateral rotation increased as the intensity of light stimuli was raised. A step-increase in light intensity elicited a graded membrane depolarization (photic receptor potential), as measured by intracellular microelectrode. When the amplitude of receptor potential exceeded a threshold depolarization for membrane excitation (15–25 mV), an all-or-none action potential of 50–65 mV in amplitude was evoked which also occurred with some latency. Light stimuli of higher intensity (suprathreshold) elicited action potential which was followed by a membrane after-depolarization. Increasing the intensity of stimuli caused generation of an action potential with shorter lag period and prolonged after-depolarization. The action spectra for the latency of stop reaction, receptor potential amplitude and cell photoresponsiveness showed maxima at 460, 530 and 580 nm. The analysis of temporal relationships between the electrophysiological responses and the motile events showed that latency of an action potential, induced by the receptor potential, correlates well with the latency of a cell stop response. Also the duration of membrane after-depolarization resembled the time period of the cell's backward swimming (cell rotation). The data obtained indicate that the primary reaction initiated by light absorption in the photoreceptor pigment (blepharismin) is converted into the observed electrical potential changes, which in turn results in the photomotile response of Blepharisma cells.