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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 genus Spirostomum inhabiting 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, Spirostomum spp., are suitable for deep molecular investigations on rquA for rhodoquinone (RQ) biosynthesis. In this study, Spirostomum ambiguum, Spirostomum subtilis, and Spirostomum teres collected from densely vegetated freshwater habitat in Fuzhou, China, explored the evidence of rquA. Based on transcriptome analysis, two to three RquA proteins were identified in S. ambiguum, S. teres, and S. 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, Spirostomum RquA 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 of rquA genes in the genomes Spirostomum spp. Additional transcriptomes and genomes analysis of Blepharisma spp., and Stentor spp., respectively, also revealed at least two paralogs of rquA in members of the class Heterotrichea. The present study provides evidence for the presence of RquA and rhodoquinol dependent fumarate reduction pathway in Spirostomum species potentially use to respire in the oxygen-depleted habitats and two to three diverse rquA genes.

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 genus Spirostomum inhabiting 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, Spirostomum spp., are suitable for deep molecular investigations on rquA for rhodoquinone (RQ) biosynthesis. In this study, Spirostomum ambiguum , Spirostomum subtilis , and Spirostomum teres collected from densely vegetated freshwater habitat in Fuzhou, China, explored the evidence of rquA. Based on transcriptome analysis, two to three RquA proteins were identified in S. ambiguum , S. teres , and S. 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, Spirostomum RquA 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 of rquA genes in the genomes Spirostomum spp. Additional transcriptomes and genomes analysis of Blepharisma spp., and Stentor spp., respectively, also revealed at least two paralogs of rquA in members of the class Heterotrichea. The present study provides evidence for the presence of RquA and rhodoquinol dependent fumarate reduction pathway in Spirostomum species potentially use to respire in the oxygen-depleted habitats and two to three diverse rquA genes. [ABSTRACT FROM AUTHOR]

In consumer communities, intra‐guild predation (IGP) is a commonly observed interaction that is widely believed to increase resource density. However, some recent theoretical work predicts that resource density should first decrease, and then increase as the strength of IGP increases. This occurs because weak to intermediate IGP increases the IG predator density more than it reduces the IG prey density, so that weak to intermediate IGP leads to the lowest resource density compared to weak or strong IGP. We test this prediction that basal resource density would first decrease and then increase as the strength of IGP increase. We used a well‐studied system with two protozoa species engaged in IGP and three bacteria species as the basal resources. We experimentally manipulated the percentage of the IG prey population that was available to an IG predator as a proxy for IGP strength. We found that bacterial density first decreased (by ~25%) and then increased (by ~30%) as the strength of IGP increased. Using a modified version of a published IGP model, we were able to explain ~70% of the variation in protozoa and bacterial density. Agreement of the empirical results with model predictions suggests that IGP first increased the IG predator density by consuming a small proportion of the IG prey population, which in turn increased the summed consumer density and decreased the bacterial resource density. As IGP strength increased further, the IG predator became satiated by the IG prey, which then freed the bacterial resource from predation and thus increased bacterial density. Consequently, our work shows that IGP can indeed decrease or increase basal resource density depending on its strength. Consequently, the impacts of IGP on resource density is potentially more complex than previously thought. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Blepharisma americanum, a member of the understudied ciliate class Heterotrichea, has a moniliform somatic macronucleus that resembles beads on a string. Blepharisma americanum is distinguishable by its pink coloration derived from the autofluorescent pigment blepharismin and tends to have a single somatic macronucleus with 3–6 nodes and multiple germline micronuclei. We used fluorescence confocal microscopy to explore the DNA content and amplification between the somatic and germline nuclei of B. americanum through its life cycle. We estimate that the DNA content of the macronucleus and micronucleus are 43 ± 8 Gbp and 83 ± 16 Mbp respectively. This correlates with an approximate DNA content difference of 500‐fold from micronucleus to macronucleus and a macronuclear ploidy of ~1,100 N as compared to the presumably diploid micronucleus. We also investigate a previously reported macronuclear inclusion, which is present sporadically across all life cycle stages; this inclusion looks as if it contains blepharismin based on its fluorescent properties, but its function remains unknown. We also provide additional detail to our understanding of life cycles changes in B. americanum by analyses of fluorescent images. Overall, the data analyzed here contribute to our understanding of the diversity of nuclear architecture in ciliates by providing details on the highly polyploid somatic macronucleus of B. americanum. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The step-up photophobic response of the heterotrich ciliate Blepharisma japonicum is mediated by a hypericinic pigment, blepharismin, which is not present in any of the known six families of photoreceptors, namely rhodopsins, phytochromes, xanthopsins, cryptochromes, phototropins, and BLUF proteins. Upon irradiation, native cells become light-adapted (blue) by converting blepharismin into the photochemically stable oxyblepharismin (OxyBP). So far, OxyBP has been investigated mainly from a photophysical point of view in vitro, either alone or complexed with proteins. In this work, we exploit the vivid fluorescence of OxyBP to characterize its lifetime emission in blue B. Japonicum cells, on account of the recognized role of the fluorescence lifetime to provide physicochemical insights into the fluorophore environment at the nanoscale. In a biological context, OxyBP modifies its emission lifetime as compared to isotropic media. The phasor approach to fluorescence lifetime microscopy in confocal mode highlights that fluorescence originates from two excited states, whose relative balance changes throughout the cell body. Additionally, Cilia and kinetids, i.e., the organelles involved in photomovement, display lifetime asymmetry between the anterior and posterior part of the cell. From these data, some hypotheses on the phototransduction mechanism are proposed. [ABSTRACT FROM AUTHOR]

Oxyblepharismin is the photo-oxidized form of blepharismin, the chromophore responsible for the photophobic response of heterotrich ciliate Blepharisma japonicum, and represents a nice model for the study of photo-transduction. In this work, we focused on the photophysical characterization of OxyBP, in view of highlighting the main features related to excitation and emission. By a combined experimental and computational approach we identified the main features of absorption and fluorescence emission of the molecule in solvents of different properties, identifying the nature of transitions as well as the possible heterogeneity at ground/excited state. The thorough photophysical characterization of OxyBP is meant to provide the starting point for the elucidation of the photo-transduction pathway in vivo . [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 100–250 μm 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. [ABSTRACT FROM AUTHOR]

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 ofBlepharismaandEuplotes. With the exception of the twoB. japonicumpheromones represented by a tryptophan-related molecule and a glycoprotein, all the other pheromones isolated fromEuplotesspecies 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 ofE. raikoviandE. nobiliipheromones has provided direct evidence that, although structurally unique,Euplotespheromones adopt a common, species-specific three-helix folding. This close structural homology among members of the same family well accounts for the pheromone capacity to compete with one another in cell receptor binding reactions and elicit context-dependent, autocrine (growth-promoting) or paracrine (mating-inducing) cell responses. [ABSTRACT FROM PUBLISHER]

Synopsis Objective Mascara is a mild irritant that causes a range of medical problems. Animal models to predict ocular irritation have, however, been questioned at a number of levels, and there is a continued need to develop in vitro testing methods. Methods We assess changes in an easily quantifiable attribute, ciliated protozoan growth rate, as a sensitive, sublethal measure. Specifically, we test six, randomly chosen, commercial mascara products against a control (as treatments) and reveal through ANOVA ( n = 6, α = 0.05) significant differences in the specific growth rate to treatments (for both protozoa). Results We provide evidence that two easily cultured protozoa ( Paramecium caudatum, Blepharisma japonicum) should be considered as models to assess ocular irritancy (and possibly cosmetics in general) and establish the groundwork for such studies to be applied at a more commercial level. We do this by developing a bioassay for mascara toxicity and indicate the low cost (after equipment is purchased, on the order of $100s) and the ease of performing such tests (able to be conducted by undergraduate students), as a consideration for their future commercial application. We first examined dose dependence of responses, revealing that there was a need to conduct preliminary work to determine appropriate levels for sublethal responses. We then show that some products resulted in mortality at high concentrations, others decreased growth rate by >50% (compared with the control), whereas others had no significant effect, compared with the control. Conclusion We have provided a novel, quick and inexpensive means to assess mascara; the next step is to validate these ciliate bioassays by comparison with animal testing and epidemiological studies, which is beyond the scope of this fundamental 'proof-of-concept' study. [ABSTRACT FROM AUTHOR]

Abstract: 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 75–255μm in length and 25–93μm 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 1652bp 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

In species with variant genetic codes, one or two stop codons encode amino acid residues and are not recognized by the intrinsic class I translation termination factor (eRF1). Ciliata include a large number of species with variant genetic codes. The stop codon specificity of the Blepharisma japonicum translation termination factor eRF1 was determined in an in vitro eukaryotic translation system and in an in vivo assay (a dual reporter system). It was shown that eRF1 of B. japonicum retained specificity to all three stop codons, although the efficiency of peptydyl-tRNA hydrolysis in the presence of UGA was reduced in the in vitro assay. Since Heterotrichea (including B. japonicum) are the earliest diverged lineage in the phylogenetic tree of ciliates, B. japonicum probably possesses a universal genetic code similar to the putative ciliate ancestor group. [ABSTRACT FROM AUTHOR]

The coloured ciliate Blepharisma japonicumchanges 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 PdcmRNA 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 Gβlocalized 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. [ABSTRACT FROM AUTHOR]

Blepharisma japonicumciliates display reversible cell elongation in response to lasting bright illumination. This light-induced phenomenon has been ascribed to the active sliding of the cortical microtubules of the ciliate. The detailed intracellular signaling pathway that activates the microtubule network in response to light, resulting in cell elongation, is unknown. We have previously reported that light stimulation initiates sequential molecular events consisting of a decrease in the phosphorylation of ciliate Pdc, followed by increased binding of Pdc to membrane-localised Gβγand the subsequent translocation of the Pdc-Gβγcomplex to the cytoplasm. In this study, we used selected agents known to influence protein phosphorylation to test whether alterations in Pdc phosphorylation levels by light affect ciliate shape. Behavioural analysis indicated that cell treatment with okadaic acid, an inhibitor of protein phosphatase activity, heavily abolished the effect of light on cell elongation, whereas the presence of H-89, a specific inhibitor of cAMP-dependent protein kinase (PKA) activity, had no appreciable effect on the cell length. Phosphorylation assays showed that cell incubation with H-89 mimicked light by promoting Pdc dephosphorylation and its colocalization with Gβγ. However, as demonstrated by FRET-AP, Pdc-Gβγcomplex formation and changes in the length of the cell did not occur under the same conditions. Moreover, fluorescence microscopy showed localization of Gβγand β-tubulin in the same cell compartment and demonstrated that a direct interaction between these proteins occurs in cells adapted to darkness or exposed to prolonged illumination (≥10 min). In contrast, an opposite effect, i.e.a transient decrease in the interaction between Gβγand β-tubulin and distinct Pdc dephosphorylation, was observed in cells illuminated for short time. Under these conditions, Pdc preferentially occupies the cell submembrane region and interacts with Gβγ. In cells illuminated for a longer time (≥10 min) and despite the constant light intensity, Pdc was progressively rephosphorylated and then dissociated from Gβγ, relocalizing within the cell cytoplasm. The results obtained in this study suggest that alterations in Pdc phosphorylation may be involved in light-induced elongation of the Blepharismacell body, which affects the interaction of Gβγwith β-tubulin and cell cytoskeleton remodelling. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

Abstract: We present a picosecond transient circular dichroism study of OBIP, the putative photoreceptor protein involved in the photophobic response of Blepharisma japonicum. The probe wavelength was chosen at 230nm. The results are compared to those of the isolated chromophore, OxyBP, in solution. The CD changes in OBIP and OxyBP do not show the same dynamics: OBIP’s signal relaxes in a few ps whereas no such decay is obtained for OxyBP. This observation brings support to the formerly evoked existence of a fast photoinduced reaction in the chromoprotein, and demonstrates the implication of local geometrical changes that accompany this process. [Copyright &y& Elsevier]

Abstract: 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 (>300Wm−2) induced rapid photodynamic killing (necrosis) characterized by cell swelling and plasma efflux, while moderate light (3–30Wm−2) only induced pigment extrusion and photooxidation. We have found that moderate light (5Wm−2) induced apoptosis-like cell death. Microscopically it was found that >3h 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 3h of irradiation, and the condensed macronuclei contained quite fragmented DNA. Macronuclear DNA extracted from light-irradiated cells contained DNA fragments of 180–200 and 360–400bp, which were seen as apoptosis ladders. [Copyright &y& Elsevier]

Previous studies of communities implicate many potential mechanisms that can create alternate stable states. These include density-dependent foraging behavior, size refuges reached by early colonists, environmental feedback following disturbance, and different initial densities of intraguild predators. Previous work shows that alternate states of varying stability can occur in food webs containing the intraguild predators Blepharisma americanum and Tetrahymena vorax. Differences in colonization history could create the alternate states, consisting of dominance by either Blepharisma or Tetrahymena, but it was unclear whether results depended on effects of initial density or only on changes in the resource base. We manipulated initial densities of both species to determine if density effects alone could create alternate stable states. Convergence of these communities over time indicated that differences in initial density did not create alternate stable states. By default, other factors influenced by colonization history, such as resource availability, may produce alternate states. Models of alternate stable-state phenomena should incorporate differences in resource availability in addition to direct competitive and predatory interactions to provide a more complete depiction of the causes of differences in community composition in otherwise similar habitats. [ABSTRACT FROM AUTHOR]

We present new femtosecond transient-absorption and picosecond fluorescence experiments performed on OBIP, the oxyblepharismin-binding protein believed to trigger the photophobic response of the ciliate Blepharisma japonicum. The formerly identified heterogeneity of the sample is confirmed and rationalized in terms of two independent populations, called rOBIP and nrOBIP. The rOBIP population undergoes a fast photocycle restoring the initial ground state in less than 500 ps. Intermolecular electron transfer followed by electron recombination is identified as the excited-state decay route. The experimental results support the coexistence of the oxyblepharismin (OxyBP) radical cation signature with a stimulated-emission signal at all times of the evolution of the transient-absorption spectra. This observation is interpreted by an equilibrium being reached between the locally excited state and a charge-transfer state on the ground of a theory developed by Mataga and co-workers to explain the fluorescence quenching of aromatic hydrogen-bonded donor−acceptor pairs in nonpolar solvents. OxyBP is supposed to bind to an as yet unknown electron acceptor by a hydrogen-bond (HB) and the coordinate along which forward and backward electron transfer proceed is assumed to be the shift of the HB proton. The observed kinetic isotope effect supports this interpretation. Protein relaxation is finally proposed to accompany the whole process and give rise to the highly multiexponential observed dynamics. As previously reported, the fast photocycle of rOBIP can be interpreted as an efficient sunscreen mechanism that protects Blepharisma japonicumfrom continuous irradiation. The nrOBIP population, the transient-absorption of which strongly reminds that of free OxyBP in solution, might be proposed to actually trigger the photophobic response of the organism through excited-state deprotonation of the chromophore occurring in the nanosecond regime. Additional femtosecond transient-absorption spectra of OxyBP and peri-deprotonated OxyBP are also reported and used as a comparison basis to interpret the results on OBIP. [ABSTRACT FROM AUTHOR]

Immunoblotting of isolated cell membrane fractions from ciliates Blepharisma japonicum and Stentor coeruleus with a polyclonal antibody raised against rhodopsin revealed one strong protein band of about 36 kDa, thought to correspond to protozoan rhodopsin. Inspection of both ciliates labeled with the same antibody using a confocal microscope confirmed the immunoblotting result and demonstrated the presence of these rhodopsin-like molecules localized within the cell membrane area. Immunoblot analysis of the ciliate membrane fractions resolved by two-dimensional gel electrophoresis identified two distinct 36 kDa spots at pIs of 4.5 and 7.0 for Blepharisma, and three spots at pIs of 4.4, 5.0 and 6.0 for Stentor, indicating a possible mixture of phosphorylated rhodopsin species in these cells. The obtained results suggest that both Blepharisma and the related ciliate Stentor contain within the cell membrane the rhodopsin-like proteins, which may be involved as receptor molecules in the sensory transduction pathway mediating motile photoresponses in these protists as in other species of lower eukaryota. [ABSTRACT FROM AUTHOR]

Immunological techniques and high-resolution FRET analysis were employed to investigate the in vivo colocalization and interaction of phosducin (Pdc) with the βγ-subunits of G-protein (Gβγ) in the ciliate Blepharisma japonicum. Immunological techniques revealed that illumination of cells resulted in a decrease in phosphorylation levels of Pdc and its colocalization with Gβγ. The observed light-induced Pdc dephosphorylation was also accompanied by significant enhancement of Gβγ binding by this molecule. Possible formation of the Pdc-Gβγ complex in cells exposed to light was corroborated by FRET between these proteins. Treatment of cells with okadaic acid, an inhibitor of phosphatase activity, entirely prevented Pdc dephosphorylation by light, colocalization of this phosphoprotein with Gβγ and generation of the Pdc-Gβγ complex. Cell fractionation and immunoblotting revealed that in cells exposed to light, the formation of Pdc-Gβγ complex and its translocation into the cytoplasm occur simultaneously with a change in the gel migration of Gβ. Moreover, a 33 kDa immunoanalog of 14-3-3 protein was identified and we showed that this protein is bound by phosphorylated Pdc in a cell adapted to darkness. The results of this study provide additional detailed characterization of the functional properties of the ciliate Pdc. The likely functional role of Pdc in Blepharisina is discussed. [ABSTRACT FROM AUTHOR]

Abstract: Blepharismins, toxic pigments of the ciliate Blepharisma japonicum, are polycyclic ring-condensed compounds. Assignment of 13C NMR signals for blepharismin C, a major constituent of blepharismins, was achieved by analyses of the HMQC, HMBC, and INADEQUATE spectra of 13C-enriched samples obtained by feeding experiments using sodium [1-13C], [2-13C], and [1,2-13C2]acetates. [Copyright &y& Elsevier]

Abstract: Blepharismone (gamone 2) is a mating-inducing pheromone of the ciliate Blepharisma japonicum. N-Pyrenylbutyryl-blepharismone and N-biphenylacetyl-blepharismone, which are fluorescent derivatives of blepharismone, were synthesized as molecular probes for the gamone 2 receptor. Further, we proved that they have inhibitory activities against the blepharismone-induced monotypic pairing of B. japonicum. [Copyright &y& Elsevier]

Abstract: The primary photoprocesses in the photoreceptor for the step-up photophobic response of the light-adapted cells of Blepharisma japonicum (OBIP, OxyBlepharismin bInding Protein) have been studied by ultrafast UV–vis transient spectroscopy. The results are rationalized in terms of heterogeneity of the OBIP sample. Two independent classes of chromoprotein are proposed: a “reactive” species, which presents a specific 680-nm band decaying in 4 and 56ps and a “non-reactive” one, which behaves like the free chromophore (OxyBP) in solution. A bimolecular photooxidation of OxyBP in the presence of 1,4-benzoquinone was performed to record the absorption spectrum of the OxyBP radical cation. Comparison with reactive OBIP suggests that an electron transfer could be involved in the primary photoprocesses of OBIP and possibly trigger the sensory transduction chain of B. japonicum. In addition, the specificity of the chromophore–protein interaction was investigated by studying the artificial complex that OxyBP forms with human serum albumin (HSA). OxyBP–HSA happens to be spectroscopically much closer to free OxyBP than to OBIP. This highlights the specific nature of the interaction between OxyBP and its native protein partner and further supports the proposal that OBIP is the actual photoreceptor for the photophobic response of B. japonicum. [Copyright &y& Elsevier]

To determine the phylogenetic position of Stentor within the Class Heterotrichea, the complete small subunit rRNA genes of three Stentor species, namely Stentor polymorphus, Stentor coeruleus, and Stentor roeseli, were sequenced and used to construct phylogenetic trees using the maximum parsimony, neighbor joining, and Bayesian analysis. With all phylogenetic methods, the genus Stentor was monophyletic, with S. roeseli branching basally. [ABSTRACT FROM AUTHOR]

Abstract: Both enantiomers of blepharismone, a mating inducing pheromone produced by type II cells of Blepharisma japonicum, were synthesized via the Stille cross-coupling reaction of [4-(tert-butyl-dimethyl-silanyloxy)-2-trimethylstannanyl-phenyl]-carbamic acid tert-butyl ester with an acid chloride derived from (S)- and (R)-malic acid as a key reaction. The mating inducing activity of synthetic (S)-blepharismone was as effective as that of the natural one. The enantiomer (R)-blepharismone showed no mating inducing activity. [Copyright &y& Elsevier]

We experimentally investigated effects of nutrient enrichment and trophic structure in a microbial food web consisting of mixed bacteria, two bacterivorous ciliates ( Tetrahymena sp. and Colpidium sp.) and an omnivorous ciliate ( Blepharisma sp.) feeding on both trophic levels. We assembled all possible food webs including one or more of the ciliate species and cross-classified them with four levels of enrichment of the bacterial medium. The qualitative outcome of food web interactions was independent of enrichment and always the same: Tetrahymena strongly depressed or excluded Colpidium, and Blepharisma strongly depressed or excluded both bacterivores. Consequently, in all sub-webs only the dominant ciliate species responded positively to enrichment. The total density of bacteria increased with enrichment irrespective of food web composition. In contrast, the response of single-celled bacteria to enrichment depended on food web composition and was only weakly positive in most food webs with the omnivore. Enrichment had a positive effect on the relative success of (presumably more defended) bacterial aggregates. The outcome of interspecific interactions among ciliates could not be predicted from monoculture experiments and deviated from earlier experiments in which each bacterivore coexisted separately with the omnivore. As a potential explanation we suggest that changes in experimental protocol reduced spatial heterogeneity and increased attack rates. A simple, dynamical model shows that increased attack rates can indeed greatly decrease the upper limit and range of enrichment over which intermediate consumers can coexist with omnivores. [ABSTRACT FROM AUTHOR]

Abstract: Blepharisma japonicum and Stentor coeruleus are related ciliates, conspicuous by their photosensitivity. They are capable of avoiding illuminated areas in the surrounding medium, gathering exclusively in most shaded places (photodispersal). Such behaviour results mainly from motile photophobic response occurring in ciliates. This light-avoiding response is observed during a relatively rapid increase in illumination intensity (light stimulus) and consists of cessation of cell movement, a period of backward movement (ciliary reversal), followed by a forward swimming, usually in a new direction. The photosensitivity of ciliates is ascribed to their photoreceptor system, composed of pigment granules, containing the endogenous photoreceptor – blepharismin in Blepharisma japonicum, and stentorin in Stentor coeruleus. A light stimulus, applied to both ciliates activates specific stimulus transduction processes leading to the electrical changes at the plasma membrane, correlated with a ciliary reversal during photophobic response. These data indicate that both ciliates Blepharisma japonicum and Stentor coeruleus, the lower eukaryotes, are capable of transducing the perceived light stimuli in a manner taking place in some photoreceptor cells of higher eukaryotes. Similarities and differences concerning particular stages of light transduction in eukaryotes at different evolutional levels are discussed in this article. [Copyright &y& Elsevier]

Abstract: The karyorelictid ciliate Loxodes striatus has pigment granules which are similar in size, structure and distribution to the pigmentocysts in the heterotrich ciliates, Blepharisma japonicum and Stentor coeruleus, which are known to be extrusomes for chemical defence against predators. We examined whether the pigment granules of L. striatus are also defensive organelles. We showed that: (1) pigment granules of L. striatus are extrusive organelles; (2) bleached cells of L. striatus produced by inducing a massive discharge of pigment granules are more vulnerable than normally pigmented cells to the raptorial ciliate Dileptus margaritifer and the turbellarian Stenostomum sphagnetorum, while they are indistinguishable from intact cells in external morphology and the capacity to grow; (3) the cell-free fluid (CFF) which contains the pigment discharged from pigment granules of L. striatus induced in D. margaritifer behavioural and pathological reactions which are essentially the same as those observed in the interaction with L. striatus, and this effect of the CFF disappeared when the pigment was bleached by light. We conclude that pigment granules of L. striatus are extrusomes for chemical defence against predators, and that the defence is based on the toxic pigment contained in these organelles. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

A cDNA clone that is inducible by light stimulation was cloned by a differential screening method from a cDNA library of the protozoan Blepharisma japonicum, and the light-dependent expression was checked by semi-quantitative reverse transcription polymerase chain reaction analysis. Sequence analysis showed that the cDNA encodes a glutathione S-transferase (GST) that has not been characterized in the protozoa. Multiple alignment of B. japonicum GST (BjGST1), known protozoan, and mammalian α-, μ-, π-, σ-, θ-, ζ-, κ-, and ω-class GSTs suggested that the BjGST1 may be a novel class GST. Furthermore, highly conserved amino acid residues among the GSTs and the substrate specificity of recombinant BjGST1 showed that BjGST1 is related to α-, μ-, π-, and σ-class GSTs rather than the other class of GSTs. [Copyright &y& Elsevier]

The heterotrich ciliate Climacostomum virens has colourless cortical granules morphologically similar to pigmentocysts (extrusive pigment granules) in the two coloured heterotrichs, Blepharisma japonicum and Stentor coeruleus, in which toxic pigments in pigmentocysts are used for chemical defence against predators. We examined whether cortical granules in C. virens are also defensive organelles. We showed: 1) cortical granules in C. virens are toxin-containing extrusive organelles; 2) artificially-induced cortical-granules-deficient cells in C. virens are more vulnerable than normal cells to the raptorial ciliate Dileptus margaritifer, while they are indistinguishable from intact cells in their capacity to grow; 3) cortical granules contain the colourless resorcinolic toxin climacostol, 1,3-dihydroxy-5-[(Z)-2′-nonenyl]benzene, which was recently isolated from C. virens; and 4) chemically-synthesized climacostol induces in D. margaritifer behavioural and pathological reactions which are essentially the same with those observed in the interaction with C. virens. We conclude that cortical granules of C. virens are extrusomes (extrusive organelles in protists) for chemical defence against predators and that they carry out the defence by discharging colourless toxins including climacostol. The function of chemical defence in extrusomes of heterotrich ciliates is, therefore, independent of pigmentation. [Copyright &y& Elsevier]

Background: The rapid expansion in the availability of genome and DNA sequence information has opened up new possibilities for the development of methods for detecting free-living protozoa in environmental samples. The protozoan Blepharisma japonicum was used to investigate a rapid and simple detection system based on polymerase chain reaction amplification (PCR) from organisms immobilised on FTA paper. Results: Using primers designed from the a-tubulin genes of Blepharisma, specific and sensitive detection to the equivalent of a single Blepharisma cell could be achieved. Similar detection levels were found using water samples, containing Blepharisma, which were dried onto Whatman FTA paper. Conclusion: This system has potential as a sensitive convenient detection system for Blepharisma and could be applied to other protozoan organisms. [ABSTRACT FROM AUTHOR]

Reports on the identification, characterization and complete amino acid sequence of the conjugation-inducing glycoprotein in the ciliate Blepharisma japonicum. Mechanism of induction of conjugation in Blepharisma japonicum; Estimation of sugar arrangement of gamone 1; Cloning and sequence determination of gamone 1 cDNA.

Presents several abstracts of studies on cell biology and morphology, presented at the 72nd annual meeting of the Zoological Society of Japan. 'Effects of Waveform on Swimming Speed of a Spermatozoon,' by S. Ishijima and M. Hara; 'The Study of Early Stage of Conjugation in Blepharisma Japonicum,' by C. Yoshimura and T. Harumoto; 'Potassium Independent Sperm Motility Initiation in Chum Salmon,' by Masaya Morita and Makoto Okuno.

It was found that some ciliates, Stentor, Spirostomum and Blepharisma, which can contract rapidly like the stalks of Vorticellidae, have Ca-binding proteins that are very similar to spasmins, in the immunological sense. The presence of spasmins in other Protozoa and in some Metazoa was also investigated. [ABSTRACT FROM AUTHOR]