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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.

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.

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.

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 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.

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.

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.

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.

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.

Summary 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 Ca2+, 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 Ca2+ 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 Ca2+-dependent contraction of the myonemal network.

Intracellular rearrangements of chloroplasts with respect to the direction of light, e.g. as found in Vaucheria, Adiantum, Mougeotia and Mesotaenium , are model systems for investigating the processing of light signals from perception through transduction to the terminal response. Two photosensory pigments, namely blue-/UV-absorbing pigment and — in a few exceptional species — the red/far-red reversible phytochrome system, are localized in the cytoplasm outside the chloroplasts. Dichroic orientation of the pigments is important for the detection of light direction via generation of an intracellular absorption gradient. Chloroplast movement, resulting in altered positioning, is based on the actomyosin system. Light-governed reorganization of the cortical and/or subcortical actin cytoskeleton results in biased chloroplast translocation. Besides polymerization or anchoring of actin to cell structures, the activity of myosin may be a point of control in some species. Local changes in membrane properties, including transient modulation of the membrane potential, appear to be the earliest steps in signal tranduction. The role of calcium and calmodulin, obviously involved in some species, is not clear as yet.

The effect of the singlet oxygen quencher crocetin on the photomotile responses and photodynamic killing of the ciliated protozoan Blepharisma japonicum was investigated. The percentage of cells surviving photodynamic killing is higher in the presence of the water-soluble carotenoid derivative than in control samples. However, no significant difference is observed in the photobehavior of the control and crocetin-containing samples. These results indicate that blepharismin-sensitized photodynamic reactions occur in Blepharisma, but do not play any meaningful role in initiating photosensory processes.

Summary Nuclear phenomena at conjugation of the multimicronucleate ciliate Blepharisma japonicum were investigated by using fluorescence microscopy. Several hours after cells united in pairs, micronuclei differentiated into meiotic micronuclei and “somatomicronuclei”. Meiotic micro-nuclei participated in conventional sexual processes producing new micro- and macronuclei through meiosis, karyogamy and postkaryogamic mitoses. On the other hand, somatomicronuclei participated in characteristic asexual processes in which each micronucleus differentiated into a macronucleus without undergoing any nuclear division and karyogamy. These two paths of macronucleus differentiation, one sexual and the other asexual, proceeded side by side in each cell, but eventually one path dominated the other. The sexual path regularly dominated in crossing conjugation which was induced by mixing clones of complementary mating types I and II. However, if macronuclear anlagen in the sexual path were removed, the asexual path took over to form macronuclei, indicating that the asexual path serves as a reserve path of macronucleus formation. The asexual path regularly dominated in intraclonal conjugation of high-frequency selfers, suggesting that it functions in reducing the genetic effect of inbreeding. These findings not only clarify nuclear phenomena at conjugation in Blepharisma , but also provide a new opportunity for the study of macronucleus differentiation.

Summary 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.

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.

Optical spectroscopy of photoreceptor pigments can substantially contribute to our understanding of the molecular processes which are the basis of photoreception and sensory transduction in photomotile microorganisms. The main spectroscopic techniques are briefly illustrated, together with the most significant types of progress that can be achieved. A few “case examples” are discussed in some detail: Halobacterium , with particular attention to the contribution of flash photolysis studies to the identification and characterization of sensory rhodopsins; Euglena , and the role of in vivo microspectrofluorometry in confirming the flavin nature of its photoreceptor pigment; the first suggestions on the rhodopsin-like nature of the Chlamydomonas photosensing system; Stentor and Blepharisma and the contribution of static and time-resolved fluorescence studies to a molecular model of the primary events in their photoreceptor pigments (stentorin and blepharismin) and systems.

The light-induced avoiding reaction of Blepharisma Japonicum was investigated by means of microscope videorecording techniques. The percentage of photoreacting cells and the lag time between stimulus onset and reaction were measured as a function of the fluence rate and of the light stimulus wavelength. Action spectra indicate that the red pigment blepharismin is the photoreceptor pigment for this photoresponse. The fluorescence lifetimes and quantum yields of ethanol-extracted pigments and cold-extruded pigment granules were measured by means of a phase-modulation spectrofluorometer. The fluorescence lifetime and quantum yield of the pigment in granules are half those of pigments in ethanol solution. This could be in agreement with the hypothesis that blepharismin is the photoreceptor pigment for the observed photobehaviour.

The ciliate Blepharisma persicinum Perty has been found to be positively geotaxic. Statistical analyses suggest that time of day and CO 2 or O 2 stress have a significant influence on the geotaxic behavior of the animals. The swimming rate was also determined experimentally to be 320 μm · s −1 . The force exerted in swimming at this speed by an average animal was calculated to be 4.1 × 10 −5 dynes.

Summary Cell body of Blepharisma was found to elongate by means of mechanical or light stimulation. Mean length of the normal vegetative cells was 320-330 μm when the cells were adapted to an experimental solution containing 1 mM CaCl2, 1 mM KCl and 5 mM Tris-HCl (pH 7.2) for 24 h. After the cells were stimulated by pipetting or light irradiation (5 × 103 lx), they elongated gradually to reach a maximum level; 459 μm by pipetting and 482 μm by light stimulation. Elongation of the cell body was found to occur strikingly in the posterior region, while the anterior region could hardly elongate. Recovery duration or process of the elongated cells caused by light application or mechanical stimulation was different from each other. The mechanically stimulated cells gradually shortened to their initial lengths, while the cells applied by the light stimulation rapidly recovered. Moreoever, the electron microscopical observations revealed that the three fibrous components (Km fiber, myoneme and vacuole-associated microtubules) might be involved with cell elongation and/or contraction of Blepharisma.

Summary Meiosis and other nuclear changes in conjugation of Blepharisma japonicum regularly occur when cells of complementary mating types I and II unite (heterotypic union). But no nuclear changes occur if unions are induced between cells of the same mating type (homotypic union). Similarly, chains of homotypically united cells, induced by treating type II cells of a doublet strain (mutant with two attachment points) with gamone of mating type I, do not undergo nuclear changes. However, if a type I cell unites at one end of such a chain, the nuclear changes of conjugation occur not only in the doublet to which the type I cell unites but also in other doublets in the same chain. We examined the mode of propagation of nuclear activation by surgically separating all cells in the chain and observing the subsequent occurrence of nuclear changes in these isolated cells. The nuclear activation began at the site of heterotypic union and propagated from cell-to-cell without skipping. In chains of a given length, the propagation slowed down as it proceeded in the chain. If compared at the corresponding site of the chain, the propagation was slower in longer chains. We conclude that meiosis and other nuclear changes in conjugation are initiated by a substance originating at the site of heterotypic union and transferable to other cells through the united regions of the cells.

Local functional differences of the cell surface were investigated in the formation of cell unions in conjugation of Blepharisma intermedium . When cells of this ciliate are treated by the gamone (the conjugation-inducing substance) of the complementary mating type, they first unite by cilia and then more intimately by the direct contact of cell bodies. The ciliary union was found to be formed by a specific contact between two morphologically distinguishable surfaces, the adoral zone of membranelles (AZM) of one cell and the anterior extension of the undulating membrane (extUM) of the other. The AZM gains the capacity to unite first and the extUM next. The extUM of gamone-treated cells sticks not only to the AZM but also to glass and some other artificial substrata under some conditions. These stickings are inhibited by cycloheximide which also inhibits the ciliary union. The role of specific contact between AZM and extUM in the cell recognition is discussed.

Summary 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.

1. 1. The effect of selected dosages of monochromatic ultraviolet light of wavelengths 238, 245, 253, 265, 280, 302, 313, 366 mμ were studied on the following: 1) regeneration of cut Blepharisma ; 2) division rate; 3) conjugation; 4) survival of colored and bleached individuals. 2. 2. No evidence for photoreactivation could be demonstrated in the ultraviolet injured animals. 3. 3. Fission was most delayed at wavelength 280 mμ at the first division, but a somewhat longer delay was observed at 254 and 265 mμ for the third division. 4. 4. The action spectrum for survival after large dosages of ultraviolet light best fits a curve that is a composite for a mixture of nucleic acid and protein, rather than either substance alone.

1. 1. Hydrostatic pressure ruptures postperistomal pieces of Blepharisma very rapidly at 15,000 pounds per square inch (psi) and more slowly at lower pressures. 2. 2. At 4000 and 3000 psi blepharismas do not cytolyze but they fail to regenerate while under pressure, although they regenerate normally when brought to atmospheric pressure. 3. 3. Regeneration occurs with considerable delay at 2500 and 2000 psi; at 1500 to 1000 psi regeneration occurs with relatively little delay. 4. 4. Brief exposure of 10 to 15 sec to 15,000 psi damages postperistomal pieces of Blepharisma to some extent, inasmuch as regeneration at atmospheric pressure thereafter is slightly delayed, the time for 50 per cent regeneration being increased by 20 to 40 per cent. 5. 5. Whole blepharismas are about as sensitive as postperistomal pieces: death occurs rapidly at 15,000 psi. 6. 6. Brief exposure to 15,000 psi, insufficient to cause disintegration, alters the whole blepharismas to some extent inasmuch as they shorten and become larger in circumference. However, the division rate of such individuals implanted into culture medium is comparable to that of controls. 7. 7. Under high hydrostatic pressure (3000–4000 psi), which just prevents regeneration, incorporation of 14C-uridine continues at a somewhat lower rate than in the control. 8. 8. Pressure which stops regeneration also considerably delays the macronuclear changes which occur during regeneration. As soon as pressure is released the macronucleus continues its changes in shape, and regeneration is completed.

Macromolecular syntheses were studied by incorporation of 14 C-labeled thymidine, 14 C-uridine and 14 C-amino acids, and by use of specific inhibitors of RNA, DNA, and protein synthesis upon the rate of regeneration of post-peristomial pieces of Blepharisma intermedium . The effect of these inhibitors on the macronuclear cycle which occurs during regeneration was also determined. Mitomycin, naladixic acid, and pheleomycin—inhibitors of DNA synthesis—if in relatively high concentration and applied for a long time, delayed regeneration. 14 C-Thymidine was incorporated into most fragments of unsynchronized cells slowly during regeneration, although it was incorporated actively into blepharismas just before division. Naladixic acid and phleomycin reduced 14 C-thymidine incorporation during regeneration. 14 C-Thymidine was not incorporated into macronuclei of cells cut soon after division (synchronized cells). 14 C-Uridine was incorporated actively immediately after removal of the peristome and continued throughout regeneration. The label at first was strongest in or over the macronucleus and near it; it was weak in the cytoplasm, becoming stronger with lapse of time after cutting. Actinomycin D, which retards regeneration most actively immediately after cutting, reduced incorporation of 14 C-uridine. 14 C-Histidine, 14 C-leucine and 14 C-amino acids (mixture) were incorporated into regenerating blepharismas. Puromycin retarded regeneration of Blepharisma most actively 1 to 2 h after cutting. Puromycin of the concentration which retards regeneration reduced the incorporation of 14 C-amino acids. The results suggest that cutting Blepharisma incites active RNA synthesis (mRNA?) in the macronucleus. The spread of RNA in the cytoplasm is accompanied by protein synthesis. The experiments suggest that macronuclear DNA synthesis is not essential to regeneration, but probably occurs when the cells are cut at the time they are in or near the synthetic stage.

1. 1. The regeneration of irradiated fragments of Blepharisma undulans var. americanus , is delayed by X-irradiation, the delay being greater the larger the dose, the effect increasing at a rate greater than linear. Only the posterior fragments were used in these experiments and the reappearance of the food vacuoles was taken as the end point of reformation of functional mouth parts. 2. 2. The delay in regeneration of X-irradiated fragments is parallel to a delay in macronuclear reorganization which always accompanies regeneration. 3. 3. Subsequent division of the regenerated fragments is also delayed similarly to delay of regeneration. 4. 4. Decreasing the oxygen tension by flushing the culture with nitrogen decreases the effectiveness of a given dose of radiations but does not alter the qualitative nature of the results. Fragments irradiated in pure oxygen were no more retarded than those irradiated in air. 5. 5. Fragments of starved Blepharisma proved to be more susceptible to X-rays than fragments of well-fed animals, as indicated by more prolonged regeneration-delay as well as division delay. Again, only a quantitative difference in susceptibility to X-rays between experiments carried out in air and in nitrogen was observed. 6. 6. The delay in regeneration produced by X-rays is greatest when the irradiation occurs three hours after cutting, indicating a period of radiation sensitivity at this time. When the fragments are irradiated four and a half hours after cutting, regeneration is delayed very much less. 7. 7. The delay of division of the regenerated fragments referred to in #6 is much the same regardless of the time after cutting when irradiation occurs. 8. 8. Regeneration of fragments cut after X-irradiation is most delayed when the animals are cut soon after irradiation; the delay decreases progressively until in four to six hours after irradiation the delay is very small and in eight to twelve hours almost nil. 9. 9. Cutting the irradiated Blepharisma was found to accelerate recovery from division-retarding effects of X-rays.