Heterotrich Ciliate Research Articles

Defensive function of trichocysts in Paramecium tetraurelia against heterotrich ciliate Climacostomum virens

Summary Defensive function of trichocysts in Paramecium tetraurelia was tested against the heterotrophic heterotrich ciliate Climacostomum virens , which ingests various sizes of prey by means of a well-developed buccal apparatus. Trichocystnon-discharge mutant and wild-type cells were compared as prey for this predator. The mutant cells were much more vulnerable than wild-type cells. Wild-type cells entering the buccal tube of Climacostomum often escaped by discharging trichocysts. We also studied the possible defensive function of backward swimming, which often follows Climacostomum-Paramecium interaction. The mutant incapable of backward swimming and wild-type cells escaped from C. virens in about the same frequency. Defensive function of trichocysts in Paramecium has been verified against the haptorian ciliates, Dileptus margaritifer and Monodinium balbiani , which have toxicysts as offensive organelles. In this work, we showed defensive function of trichocysts against the heterotrich ciliate C. virens , which does not have toxicysts. This work suggests that the trichocyst discharge may defend Paramecium from a variety of predators.

Multiple acquisition of methanogenic archaeal symbionts by anaerobic ciliates.

Anaerobic heterotrichous ciliates (Armophoridae and Clevelandellidae) possess hydrogenosomes that generate molecular hydrogen and ATP. This intracellular source of hydrogen provides the basis for a stable endosymbiotic association with methanogenic archaea. We analyzed the SSU rRNA genes of 18 heterotrichous anaerobic ciliates and their methanogenic endosymbionts in order to unravel the evolution of this mutualistic association. Here, we show that the anaerobic heterotrichous ciliates constitute at least three evolutionary lines. One group consists predominantly of gut-dwelling ciliates, and two to three, potentially four, additional clades comprise ciliates that thrive in freshwater sediments. Their methanogenic endosymbionts belong to only two different taxa that are closely related to free-living methanogenic archaea from the particular ecological niches. The close phylogenetic relationships between the endosymbionts and free-living methanogenic archaea argue for multiple acquisitions from environmental sources, notwithstanding the strictly vertical transmission of the endosymbionts. Since phylogenetic analysis of the small-subunit (SSU) rRNA genes of the hydrogenosomes of these ciliates indicates a descent from the mitochondria of aerobic ciliates, it is likely that anaerobic heterotrichous ciliates hosted endosymbiotic methanogens prior to their radiation. Therefore, our data strongly suggest multiple acquisitions and replacements of endosymbiotic methanogenic archaea during their host's adaptation to the various ecological niches.

Climacostol, a defense toxin of the heterotrich ciliate Climacostomum virens against predators

A toxic substance (climacostol) of the protozoan ciliate Climacostomum virens against the predatory ciliate Dileptus margaritifer was established as 1,3-dihydoxy-5-[(Z)-2′-nonenyl]benzene. The structure was rigorously confirmed by the total synthesis.

Evolution of anaerobic ciliates from the gastrointestinal tract: phylogenetic analysis of the ribosomal repeat from Nyctotherus ovalis and its relatives.

The 18S and 5.8S rDNA genes and the internal transcribed spacers ITS-1 and ITS-2 of ciliates living in the hindgut of frogs, millipedes, and cockroaches were analyzed in order to study the evolution of intestinal protists. All ciliates studied here belong to the genus Nycrotherus. Phylogenetic analysis revealed that these ciliates from a monophyletic group that includes the distantly related anaerobic free-living heterotrichous ciliates Metopus palaeformis and Metopus contortus. The intestinal ciliates from the different vertebrate and invertebrate hosts are clearly divergent at the level of their rDNA repeats. This argues for the antiquity of the associations and a predominantly vertical transmission. This mode of transmission seems to be controlled primarily by the behavior of the host. The different degrees of divergence between ciliates living in different strains of one and the same cockroach species most likely reflect the different geographical origins of the hosts. In addition, host switches must have occurred during the evolution of cockroaches, since identical ciliates were found only in distantly related hosts. These phenomena prevent the reconstruction of potential cospeciation events.

Cell Body Contraction of Splrostomum Does Not Involve Shortening of Inter-Kinetosomal Distance along Ciliary Lines

Abstract A heterotrichous ciliate Spirostomum is known to show characteristic rapid contraction of the cell body accompanied by twisting of ciliary lines which run longitudinally on the cell surface. The ciliary lines of Stentor, a closely related heterotrichous ciliate, are known to become shorter by themselves when the cell contracts, resulting in sliding between adjacent longitudinal microtubular sheets (LMSs) running just beneath the ciliary lines. In Spirostomum, in contrast, there is controversy over whether lengths of the ciliary lines alter or not. In this study, we examined changes of the ciliary line lengths by scanning electron microscopy, to measure the distance between the proximal ends of two neighboring cilia which are lining up along each ciliary line (inter-kinetosomal distances). We found that the inter-kinetosomal distance remained constant regardless of cell contraction and elongation, indicating that the cell body contraction of Spirostomum results from a cellular twisting motion with...

Effect of Mg 2+ on Ca 2+-dependent contraction of a Spirostomum cell model

Summary A detergent-extracted cell model of the heterotrichous ciliate Spirostomum ambiguum showed ATP-independent contraction on addition of > 10−7 M Ca2+, which is caused by contraction of the myoneme. To further understand the mechanism of cell contraction, the effects of Mg2+ and other cations were examined. Among various kinds of cations tested, Ba2+, Mn2+, Sr2+ and Cd2+ induced slight contraction, but they required higher concentrations (> 10−3 M). Contraction of the cell model was not induced by Mg2+. However, it shifted the threshold of Ca2+ concentration for inducing contraction to higher levels. The Hill constant, denoted as the number of calcium ions cooperatively bound to a contractile element in the myoneme, was 2–3 at 0–1 mM Mg2+, while it decreased with increasing Mg2+ concentration. The Ca2+-induced contraction was not influenced by changing ionic strength of the reactivation medium. These results suggest that Mg2+ ions interact with the Ca2+-binding sites which induce contraction of the myoneme.

Insights into the evolution of nuclear dualism in the ciliates revealed by phylogenetic analysis of rRNA sequences.

The small subunit rRNA gene sequences of the karyorelictean ciliates, Loxodes striatus and Protocruzia sp., and the heterotrichian ciliates, Climacostomum virens and Eufolliculina uhligi, were used to test the evolution of nuclear dualism in the Phylum Ciliophora. Phylogenies derived using a least squares distance method, neighbour joining, and maximum parsimony demonstrate that the karyorelictean ciliates sensu Small and Lynn, 1985 do not form a monophyletic group. However, Loxodes and the heterotrich ciliates form the first branch in the ciliate lineage, and Protocruzia branches, in distance methods, basal to the spirotrich lineage. It is proposed that Protocruzia be removed from the Class Karyorelictea, and placed in closer taxonomic association with the spirotrich lineage. The distribution of nuclear division types along the phylogenetic tree is consistent with the notion that macronuclei incapable of division represent a derived rather than a primitive or "karyorelictid" character trait.

Identification of calcium and phosphorus binding sites in the heterotrich ciliate Blepharisma japonicum

The fine structure of Blepharisma japonicum has been widely described in the literature and several authors report thepresence of many very electron-dense granules with dimensions ranging from 5 to 50 nm. These granules are present almost everywhere in the cell, but localized primarily on the ciliary membrane. We have performed an Electron Spectroscopic Imaging (ESI) analysis to detect the presence of calcium, phosphorus and magnesium in the granules. Our results reveal the presence of calcium and phosphorus, but not magnesium, within the limits of detection of ESI technique. Moreover, our results show no detectable difference between ciliary and cytoplasmic granules. Comparisons with other organisms suggest that these granules are found not only in ciliates, but that they are probably very common in a large variety of organisms.

The Infraciliature of two Sapropelic Heterotrich Ciliates, Cirranter mobilis and Saprodinium dentatum (Ciliophora, Heterotrichida)

The infraciliature of two sapropelic heterotrich ciliates, Cirronter mobilis and Saprodinium dentatum , is described. The somatic infraciliature of C. mobilis is constituted by a pcrizonal zone and one kinety of the bell. The perizonal zone is formed by numerous short longitudinal kinelies with 4 kinetids each (dikinetids). Complex longitudinal fibrillar systems connect the kinetosomes of each kinety. The kinety of the bell is composed of two rows of kinetosomes with long fibrillar derivates. In the oral zone, the paroral membrane is formed by one row of kinetosomes, and the adoral zone of membranelles is constituted by numerous membranelles each one with two rows of kinetosomes. Ihese membranelles are connected one another through transversal microfibrillar tracts. Our observations concerning the somatic infraciliature of S. dentatum indicate that the perizonal zone (PZ), the longitudinal somatic kineties (LK) and the kineties of the spines (SK) are closely related between them. The right extremes of each kinetosomal row of the PZ continuate with somatic longitudinal kinetics, which lack of fibrillar derivates. Moreover, the SK constitute the modified posterior extremes of some of LK. At the ventral margin of ciliate, the most posterior row of kinetosomes of the PZ is connected through fibrillar bundles with two short rows of kinetosomes (additional kineties) with a complex cytoskeletal array. Groups of 2–4 kinetosomes without fibrillar associates constitute the infraciliature of the bell kinety. The oral infraciliature ol this species is composed by 9–10 membranelles each one with 2–3 kinetosomal rows of different length and 2–3 argentophilic rows, which as in C. mobilis , have not been described until now.

Ultrastructure and calcium-dependent contraction of the myonemal network in a heterotrich ciliate, Blepharisma japonicum

This laboratory has previously demonstrated that shortening of the cell body of a heterotrich ciliate, Blepharisma japonicum, could be induced as a step-down photophobic response. Here, we examined the structure and contractility of the myonemes in detergent-extracted cell models and in isolated cortical fragments. Ultrastructural observation showed that the myoneme was connected to the basal ends of the posterior kinetosomes and constructed a systematic network as a whole. Shortening of the cell model was induced by > 10(-4) M Ca(2+), while the rounded cell model did not re-elongate even when it was washed in a calcium-free solution either with or without addition of ATP. Fluffy fibrils, which were tentatively identified as aggregated bundles of the myonemes, were isolated with the kinetosomal complex and showed calcium-dependent and ATP-independent contraction. The minimum concentration of Ca(2+) required for inducing contraction was at the level of 10(-6) M. These results suggest that the cell body shortening in Blepharisma is caused by the Ca(2+)-dependent contraction of the myonemal network.

Studies on the mechanism of cell elongation in Blepharisma japonicum: 4. Three dimensional construction of the kinetosomal complex and its functional role on cell elongation

The kinetosomal complex of a heterotrich ciliate Blepharisma japonicum was investigated at ultrastructural level, with special reference to its cell elongation in response to light stimulation. In serial sections, vacuole-associated microtubules were found to be originated from both anterior fiber sheet and left surface of the anterior kinetosome. These microtubules form a bundle and extend toward the anterior end of the organism. The postciliary microtubular sheet is attached to the proximal half of the posterior kinetosome. Two types of transverse microtubules are present: the anterior 8-9 microtubules arising at the base of the anterior kinetosome and the posterior 2-3 microtubules arising between the paired kinetosomes. Based on these results, a three dimensional model of the kinetosomal complex was proposed and the mechanism of cell elongation in Blepharisma was discussed.

Studies on the mechanism of cell elongation in Blepharisma japonicum: 3. Cytoplasmic calcium ions may correlate to cell elongation in calmodulin-dependent manner

Among many heterotrichous ciliates, Blepharisma japonicum especially demonstrates negative phototaxis in response to light stimulation, which is attributed to be caused by swimming acceleration accompanied by cell elongation and ciliary reversal. When Blepharisma cells were treated with 0.1 mM EGTA, cell elongation gradually decreased in its degree as the adaptation time lapsed. Calmodulin inhibitors such as W-7 (10(-5) M) and chlorpromazine (10(-5) M) inhibited cell elongation. These results suggest that a rise in cytoplasmic free Ca(2+) concentration might cause cell elongation through the Ca-calmodulin system. On the other hand, Ca(2+)-channel blockers (La(3+), Co(2+), Cd(2+), Zn(2+), Mn(2+) and verapamil) did not inhibit cell elongation. Ca2+ localization examined by calcium pyroantimonate cytochemistry suggests that Ca(2+) ions required for cell elongation might be supplied from the vacuoles located in the cortical region of the cell instead of Ca(2+) influx from the surrounding medium.

The fine structure of the encysting salt marsh heterotrich ciliate Fabrea salina.

Wall and internal organelles of the encysting salt marsh heterotrich ciliate Fabrea salina were examined by bright field and Nomarski interference contrast microscopy and by transmission and scanning electron microscopy. A mucoid sheath believed to be derived from bacteria covers the ectocyst. The possible bacterial origin of this sheath has been demonstrated experimentally by its removal and reappearence after 24 hours. Control ectocyst maintained in sterile seawater did not replace the sheath. The ectocyst has a crinkled appearance. The endocyst is composed of two layers. The inner endocyst material is continuous with that of the plug located at one end of the cyst. The two membranes are separated from one another by an interwall space filled with fibrous material. The cytoplasm, covered by an outer double membrane pellicle, contains mucocysts, pigment granules, microtubules, non-ciliated kinetosomes, Golgi complexes, ribosome-studded endoplasmic reticulum, and mitochondria. The endoplasm contains numerous autophagosomes, mitochondria, and food reserve materials. The macronucleus is centrally located in the cytoplasm of the encysting organism.

Three-dimensional construction of the kinetosomal complex in a ciliate Blepharisma, revealed by isolation technique and serial sectioning

In a heterotrichous ciliate, Blepharisma japonicum, pairs of basal bodies (kinetosomes) and their fibrous associates containing microtubular sheets and bundles are closely related to cell elongation in response to light stimulation. Up to date, however, three dimensional construction of them has not been clarified because of their own complexity and difficulty of establishment of their isolation technique. By applying the newly devised isolation medium, however, they were found to be isolated fruitfully from the cell bodies. The present isolation medium was composed of 10 mM MgSO4, 10 mM EGTA, 1 mM dithiothreitol, 32 mM sucrose, AH Triton X-100, 20% glycerol, 100 mM HEPES and 20 mM PIPES adjusted to pH 7.2 with KOH. The isolated complexes were mounted onto Formvar-coated grid, fixed briefly with 2% glutai-aldehyde (GA) buffered with 0.1 mM sodium cacodylate at pH 7.2, rinsed with deionized distilled water and finally stained with 1% aqueous uranyl acetate (UA). For thin sectioning, on the other hand, the organisms were fixed for 10 min with a mixture of 2.5% GA and 1% OsO4 in cacodylate buffer (pH 7.2) at room temperature.

Defensive function of pigment granules in Blepharisma japonicum

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.

Evidence for intermediate-like filaments in a heterotrichous ciliate

In the heterotrichous ciliate Nyctotherus ovalis, a meshwork of 10 nm filaments present in the cell cortex surrounds cytostome and cytoproct and is connected by thin strands to a perinuclear coat. Like intermediate filaments (If) of higher organisms the filaments are insoluble in non-ionic detergent, and paracrystals can be formed after dissolution in 8 M urea. The cross-reactions of these filaments with monoclonal anti-vimentin and anti-cytokeratin antibodies suggest that they share common epitopes with both types of If proteins.

Nouvelles Données sur l’Infraciliature Somatique et Buccale de Caenomorpha uniserialisLevander, 1894 (Ciliophora, Heterotrichida)

Summary This paper describes the somatic and the buccal infraciliature of the heterotrich ciliate Caenomorpha uniserialis using the pyridinated silver carbonate method of Fernandez-Galiano (1976). The somatic infraciliature of this species is located in three zones: a) the perizonal zone, composed by 100–120 short oblique kineties, each one with 10–15 paired kinetosomes, b)the bell kinety, formed by 90–100 kinetosomes which are disposed in a zigzag pattern, and c) three or four short kineties placed at the base of the longer spine. The buccal infraciliature consists of one adorai zone of membranelles, each one with three rows of kinetosomes, and of a paroral kinety composed by two parallel rows of kinetosomes.

Studies on the mechanism of cell elongation in Blepharisma japonicum I. A physiological mechanism how light stimulation evokes cell elongation

In a heterotrichous ciliate, Blepharisma japonicum, longitudinal elongation of the cell body is induced by light stimulation (1000 to 3000 lux). This light-induced response was inhibited under the existence of cyclic mononucleotide phosphodiesterase (PDE) antagonists such as papaverine (10(-4)M), theophylline (10(-3) M), dibutyril-cAMP (10(-4)M), 3-isobutyl-1-methyl-xanthine (10(-4) M) and dibutyril-cGMP (10(-4) M). Microinjection of cyclic mononucleotides, especially cGMP, inhibited cell elongation. These observations suggest that the cell elongation was mediated by intracellular cyclic mononucleotide. K(+) specific ionophore valinomycin (10(-8)-10(-7) M) enhanced light-induced cell elongation. This effect of valinomycin became more remarkable when valinomycin coexisted with PDE antagonists, while it was diminished under high K+ conditions. Moreover, the K(+) channel blockers tetraethylammonium (TEA) and CsCl inhibited cell elongation. These observations suggest that the cell elongation is also mediated by K(+) hyperpolarization, and that this electrical change is probably elicited after the intracellular concentration of cyclic mononucleotide decreased.

Ultrastructure of the Isolated Microtubules and Intermicrotubular Bridges in a Heterotrichous Ciliate, <italic>Spirostomum ambiguum</italic>

Free cytoplasmic microtubules and postciliary longitudinal microtubular sheets (LMS) of a heterotrichous ciliate, Spirostomum ambiguum, could be isolated from the cell body by using the isolation medium composed of 1 mM MgSO 4 , 40 mM EGTA, 2.5% DMSO, 0.1% Triton X-100, and 0.5 M HEPES (pH 6.9). After being negatively stained, three-dimensional arrays of globular subunits composing the microtubular wall were analyzed by optical diffraction and filtration technique, comparing free cytoplasmic microtubules with compact ones of the LMS. And then, ultrastructure of the intermicrotubular bridges were examined by means of both the translational reinforcement of electron micrographs and the optical diffraction and filtration. Three-dimensional arrays of tubulin molecules were thus clarified and the bridges were found to arrange periodically at the interval of 20 to 22 nm, to be inclined by about 30 degrees against the vertical line to the longitudinal axis of every microtubule, and to be composed of three subdomains in each bridge.

Cell model contraction in the ciliate spirostomum

AbstractAs the species name indicates, the large heterotrichous ciliate Spirostomum ambiguum is characterized by a twisting contraction of the cell body that is easily triggered by various kinds of external stimuli. On the basis of morphological studies, contraction and extension of this organism have been considered to result from antagonistic actions of myoneme and microtubular ribbons. After many trials, we have succeeded in preparing cell models to examine induced contractions and extensions of the cell body. The contraction of this model was induced by increasing the free Ca2+ concentrations even in the absence of Mg‐ATP and was reversed by adding Mg‐ATP without Ca2+. Using dynein ATPase inhibitors such as vanadate and ATP analogs, furthermore, the experiments revealed that the ATPase that generated the force between the two neighboring microtubular ribbons might be a dynein‐like ATPase.