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The cyst walls of the ciliatesBlepharisma undulans andPseudomicrothorax dubius were examined ultrastructurally and by postembedding labeling with wheat germ agglutinin (WGA)-gold conjugate. Different methods of fixation and embedding were performed. In all procedures, WGA-gold binds selectively to material of the cyst wall. Pretreatment of the sections with chitinase inhibits labeling. The cyst walls of both species contain 3 nm fibrils, which are supposed to be of chitinous nature. In the cyst wall ofB. undulans, several thin layers of WGA-binding fibrils are interspaced with thick layers of other material. InP. dubius, WGA-binding sites are mainly concentrated in the mesocyst, where the microfibrils appear to represent the major component. These results obtained from two phylogenetically distant species confirm that chitin synthesis is an ancestral feature of ciliated protozoa. The amount and distribution of the chitin fibrils may play an important role in the properties and functions of the wall of the resting cyst.

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.

SYNOPSIS. Conjugation involving two Japanese strains of Blepharisma, Niigata and Nara, which are considerably different from each other in external morphology, but are rather similar in nuclear configuration, was induced experimentally. Compared with normal conjugations of the two strains, both the Niigata- and Nara-interbred exconjugants show a strikingly high mortality. The possibilities of the occurrence of autogamy and cross-fertilization are discussed on the basis of the results obtained from observations on living and fixed material.

— –The intracellular pigment of the ciliate protozoan Blepharisma in the presence of oxygen sensitizes the cells to bright visible light (2700 foot candles (fc)). Illumination of the cells with dim visible light (200 fc) changes the color of the pigment to blue-gray; such cells are no longer sensitive to bright visible light. The pigment which exists in granules can be extruded by cold treatment and is slowly regenerated. The suspension of red cells, the extruded pigment from them and an ethanol extract of the pigment all have very similar absorption spectra. Illumination of the red pigment in ethanol converts it to the blue form of the pigment but only if oxygen is present, indicating a photooxidation. The pigment can be oxidized in darkness to the blue form by ozonation. A suspension of blue cells, the extruded pigment from them and an ethanol extract from them, all have similar absorption spectra. The pigments in red and blue form are very similar spectrophotometrically and in solubility in three species of Blepharisma studies: B. americanum, B. intermedium and B. japonicum. The purified pigment has strong absorption in the far (200–300 nm) ultraviolet (u.v.) and may serve as a screen against damaging U.V. radiation, especially as Blepharisma shows poor photoreactivation.

SYNOPSIS. An analysis of the pigments from the protozoan Blepharismn undtulans is reported. Methods of isolation and separation by paper chromatography are described. along with subsequent spectrophotometric and chemical analysis. Since the two major pigments were available only in amounts insufficient for thorough chemical analysis, these pigments were compared with the known pigment hypericin. Although similarities in the visible spectra indicate that the B. unduluns pigments possess the same mesodianthrone structure as hypericin, infrared spectra and chemical reactions disclose differences in the position, and possibly in the number, of the functional groups. A tentative structure is proposed.

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.

SYNOPSIS. Autoradiographic studies on DNA-dependent RNA synthesis in isolated macronuclei of Blepharisma intermedium in vitro are described. Isolated macronuclei incubated in a medium containing RNA precursors incorporated cystidine and uridine into RNA. Uptake of cystidine was greater than that of uridine. Addition of ATP did not appreciably affect the uptake of precursors into RNA. Actinomycin D (5 μg/ml) blocked incorporation of cystidine and uridine. It is concluded that in isolated macronuclei of Blepharisma, RNA precursors are 1st converted into triphosphates and then incorporated into RNA thru DNA-dependent RNA polymerase.

1. A brei of fragmented Blepharisma contains some substance which is quite toxic to Paramecium and a variety of other protozoans and to sea urchin larvae, but it is not toxic to Blepharisma itself.2. Paramecia suspended in a dense culture of Blepharisma are unaffected by the mere presence of Blepharisma.3. Blepharisma is eaten by Actinosphaerium; therefore the toxin does not protect it from attack and use as food, but it is not eaten by Woodruffia, Podophrya or Didinium.4. In the presence of Blepharisma, paramecia grow at the same rate as they do alone, indicating that no toxin is secreted during growth.5. Brei of Blepharisma bleached by light is not toxic to paramecia.6. The pigment of Blepharisma extracted in alcohol and after drying re-extracted in alcohol and, after another drying, re-extracted in water is highly toxic to paramecia.7. The tentative conclusion is drawn that the toxin is the pigment or something very closely associated with it.

SYNOPSIS. An account of conjugation in an American species of Blepharisma is given. A comparison is also made with the available knowledge of the two Indian species of this genus. In the conjugating pair, the condensed macronucleus shows Feulgen negative regions. Before the conjugants separate, the macronuclear and micronuclear anlagen become distinguishable. The species is characterized by a number of striking features which become noticeable after syngamy. The synkaryon divides thrice giving rise to 8 bodies. Of these, 3 to 7 become the macronuclear anlagen and the rest, the micronuclei. The resorption of the old macronucleus occurs much later, just before the exconjugant attains the vegetative form. No metagamic fissions occur in this species and each exconjugant becomes a vegetative animal in 5 or 6 days. During this period, the macronuclear anlagen arrange themselves in a series and develop slender connections with one another to produce the moniliform macronucleus of the vegetative animal. The micronuclear anlagen, on the other hand, divide by mitosis to attain the vegetative number. In this species 40% to 45% of the exconjugants are viable and the rest die.

SYNOPSIS. Cannibal giant Blepharisma undulans americanus, NYU strain, grown in mass culture, showed increased numbers of macronuclear lobes and greater variability in their number, compared to the bacteria-fed form. Feulgen and methyl greenpyronin preparations stained both the DNA and RNA of the cannibals and of the ingested forms within the food vacuoles. During the first 3 to 6 hours of digestion, macronucleus and deoxyribonucleic acid disappear from the food vacuole; during the last 6 hours the ribonucleic acid disappears from the food vacuole; DNA and RNA are therefore presumed to be digested and absorbed.

SYNOPSIS. Regeneration of different stocks of Blepharisma representing several species is retarded to different degrees by identical doses of UV radiation (2654 A). UV susceptibility of regeneration is greater in some small-sized stocks than in some large-sized stocks and in stocks with a lesser amount of pigment than in the more deeply pigmented ones. For the same dose regeneration is retarded more by low-intensity than by high-intensity short wavelength UV radiation. The UV action spectrum for regeneration delay in the Rao A stock, determined with UV of similar intensity at various short UV wavelengths, resembles absorption by nucleoprotein more than ahsorption by other cell constituents. Post-regeneration division is also retarded by UV, and the aotion spectrum for division delay resembles that for regeneration delay, except that 2801 A is relatively more effective in retarding division than regeneration. Possible explanations for the results are considered.

SYNOPSIS. The following four subgenera. I based chiefly on the form of the macronucleus, are proposed for thr genus Blepharisma Perty 1852: 1) Compactum, having a single compact interphase macronucleus. Type species B Compactum hyalinum Perty. 2) Filijormis, having a vermiform or tubular elongate interphase macronuclcus. occasionally doubled or even fragmented in individuals. Type spcies B. (Filiformis) intermedium Bhandary. 3) Blepharisma, having an interphase macronucleus with two or more nodes usually connected by a very thin strand between the nodes. Type species B. (Blepharisma) Perisicinum Perty. 4) Halteroides, having an interphase macronucleus in two nodes connected by a thick strand. Type species B. (Halteroides) tropicum Bhandary. Differences between the subgenera also can be found in the size, pigmentation, presence or absence of cysts and conjugaion 26 species are presented. While integradations between the subgenera and species exist, it is felt that separation of the four groups is warranted and may indicate possible evolutionary trends within the genus.

SYNOPSIS. Nucleic acid hybridisation involving DNA samples was used to study relationships between various protozoa. The most hybridisation or nucleic acid homology was always in the homologous reaction between 2 DNA samples from the same source. It was concluded that all the protozoa tested have nucleotide sequences or genes in common with Paramecium aurelia. The ciliates could be placed in a sequence of decreasing homology relative to Paramecium: Tetrahymena, Colpidium, Stentor, Didinium, Dileptus, Blepharisma. Actinosphaerium had fewer sequences than any of the ciliates and the flagellate Euglena had the fewest sequences in common. The bacterium Aerobacter had none. Similar relationships were inferred from competitive hybridisation experiments; these relationships were also in general mirrored by morphologic relationships and overall G + C base compositions which ranged from 46% for Euglena to 32% for P. aurelia. These experiments, it is hoped, will contribute to studies on origins of the metazoa.

SYNOPSIS. The regeneration of three species of Blepharisma—americanum, undulans and japonicum (grown in conslant yellow light in a constant-temperature room at 25dC) from specimens of which the hypostome had been removed, was studied under a variety of conditions. The reliability of the methods was checked by an extensive series of experiments with B. americanum and B. japonicum under normal conditions. The time of regeneration of 50% of the individuals was taken as a convenient means for comparing action of various agents. No diurnal rhythm of rate of regeneration was observed. Regeneration is readily affected by the state of nutrition of the cells, starved ones regenerating more slowly than well-fed ones. Regeneration is accelerated by maintaining the cells at 30d but retarded at 35d. Even brief exposures to 40d may retard regeneration. Regeneration is slowed by maintenance at temperatures below 25d, and stopped at 10d. Cut Blepharisma placed at 5d lose pigment in a “shock” reaction and many die. The injury is more marked in lettuce medium than in balanced salt solution. Between 13 and 30d the rate of regeneration increases with rise in temperature, the Q10′s for the ranges 13 to 18d, 18 to 25d and 25 to 30d being, respectively, 6.92, 3.93 and 1.56. The effect of temperature upon division of Blepharisma was found to be similarly sensitive to temperature, the Q10's for the ranges 13 to 18d, 18 to 25d and 25 to 30d being 3.87, 2.92 and 1.59, respectively. High and low temperature limits for division were also found to be similar to those for regeneration. Preliminary evidence indicates some degree of thermal adaptation in Blepharisma to both higher and lower temperatures than those usually tolerated. The studies indicate a general similarity in sensitivity of regeneration and division to environmental influences in the various races of the species studied, with some individual variations.

SYNOPSIS. A full account of the nuclear changes during binary fission and conjugation in a local race of Blepharisma is presented in this paper. The macronucleus consists of 2 nodes connected by a strand. Number of micronuclei varies from 6 to 18. During binary fission, condensation of macronucleus is followed by elongation and thinning of the middle region which finally breaks. Daughter nuclei later attain the typical vegetative form. Notably, during binary fission some micronuclei appear to complete their mitoses by the time the macronucleus attains the condensed form, while others lag behind and exhibit practically every stage of mitosis. During conjugation, from 6 to 10 micronuclei undergo the first pregamic division, the same number through the second division, and two products of the second division take part in the third division. The rest degenerate. Division products of the nuclei in the paraoral region take part in synkaryon formation. The synkaryon undergoes either 2 or 3 divisions. In the former case, of the 4 products, 2 become the macronuclear anlagen, one the micronucleus and the fourth degenerates. In the latter case, of the 8 products, 3 to 4 become the macronuclear anlagen and the rest become micronuclei. Chromatin elimination has been observed during the division of the macronuclear anlage, followed by an extra metagamic fission of the cell. Comparison with two other races from India and an American race indicates considerable diversity in the structure and behaviour of the nuclear apparatus in different races of Blepharisma undulans.

The mitotic, micronuclear division of the heterotrichous genus Blepharisma has been studied by electron microscopy. Dividing ciliates were selected from clone-derived mass cultures and fixed for electron microscopy by exposure to the vapor of 2% osmium tetroxide; individual Blepharisma were encapsulated and sectioned. Distinctive features of the mitosis are the presence of an intact nuclear envelope during the entire process and the absence of centrioles at the polar ends of the micronuclear figures. Spindle microtubules (SMT) first appear in advance of chromosome alignment, become more numerous and precisely aligned by metaphase, lengthen greatly in anaphase, and persist through telophase. Distinct chromosomal and continuous SMT are present. At telophase, daughter nuclei are separated by a spindle elongation of more than 40 µ, and a new nuclear envelope is formed in close apposition to the chromatin mass of each daughter nucleus and excludes the great amount of spindle material formed during division. The original nuclear envelope which has remained structurally intact then becomes discontinuous and releases the newly formed nucleus into the cytoplasm. The micronuclear envelope seems to lack the conspicuous pores that are typical of nuclear envelopes. The morphology, size, formation, and function of SMT and the nature of micronuclear division are discussed.

1. Blepharisma undulans, a protozoan with a reddish pigment, shows increased oxygen consumption under the influence of light. 2. If the light intensity is high, the animals are killed during a burst of oxygen consumption. 3. If the blepharismas are first bleached by exposure to light of low intensity they show only slightly increased oxygen consumption under the influence of light and they are not killed. 4. A preparation in which the animals are killed by heat still shows the increase in oxygen consumption on illumination with brilliant light. The supernatant solution does so as well, as does an alcohol extract of the dye. 5. The conclusion is drawn that the blepharismas are killed during photooxidation of the pigment, but the mechanism of action is not clear. Several possibilities are considered in the discussion.

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. Two ciliates, Blepharisma undulans and Paramecium caudatum, were studied with reference to form stability and integrity (resistance to cytolysis) under varying conditions of hydrostatic pressure (up to 10,000 lbs./in.2) and of temperature (12°-25° C.).2. At lower pressures the specimens retained their elongate form, but at higher levels, depending on temperature, species, and age of the cloned cultures, the cells gradually become shorter and more rounded. Following this form change, cytolysis occurred in a varying percentage of the specimens. Older cloned cultures showed a greater and more variable susceptibility to the pressure-temperature effects, so that selected younger cultures were used for the quantitative evaluations.3. For Blepharisma, the critical pressure, which gave 50 per cent cytolysis within a 15-minute compression period, displayed a distinct temperature dependence, being 8000 psi. at 12° C., 8700 at 15° C., 9200 at 20° C., and 9300 at 25° C. Paramecium, in contrast, showed a distinctly...

SYNOPSIS. From characteristics of binary fission, conjugation, size and number of micronuclei, body size and incidence of giantism, a Blepharisma isolate hitherto called B. undulans is classified as B. dawsoni sp. nov. Binary fission in B. wardsi differs from fission in B. dawsoni in that the strand connecting the macronuclear nodes is severed; in B. dawsoni the strand persists.

SYNOPSIS. A spontaneous albino mutant of the red heterotrichous ciliate Blepharisma intermedium has been isolated and studied. In addition to its apparent lack of pigment, the albino differs from the parental stock in its manner and frequency of conjugation, high incidence of cannibalism and reduced intensity of micronuclear stainability. The conjugation pattern of the albino is compared to that of the pigmented form as described in the literature. The albinistic character thus far has bred true. Alcoholic extracts of the albino yielded only a trace of pigment. Absorption spectra of alcoholic extracts of the pigmented, the artificially bleached, and the albino organisms were compared. As far as is known, no conjugation or other type of internal nuclear reorganization was observed preceding the phenotypic expression of the mutation; this raises several questions pertaining to the site of mutation and means of its expression. These questions are discussed.

SYNOPSIS. Blepharisma seculum sp. nov. is described as a small-sized blepharisma with a compact, spheroid macronucleus and a distinct curvature of the body anteriorly. A comparison of this form with other species of the genus Blepharisma subgenus (Compactum) is given.

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.

SYNOPSIS. Electron-microscope studies have revealed the ciliature and infraciliature of Blepharisma undulans to include tubular filaments about 24 mμ in diameter. As these filaments pass from the kinetosomes into the surrounding cytoplasm they appear to divide into double helical subfilaments, each with a diameter of about 12 mμ. In the somatic region the ciliary rows are composed of series of paired kinetosomes. Connected with the anterior member of a pair are the cilium and transverse filaments, the posterior member giving rise to the posterior kinetodesmal fiber. From between the two kinetosomes arises the anterior kinetodesmal fiber. The kinetodesmal fiber system is associated with the subpellicular fibril system which lines the area just beneath the pellicle of the animal. In the oral region a series of these filaments extends from the proximal portion of the adoral zone of membranelle kinetosomes into the cytoplasm. They traverse the oral area to connect with the undulating membrane on the right margin of the mouth. Also present are fibers comparable to the kinetodesmal fibers and transverse fibers of the somatic region. The functional significance of this system is discussed. The nucleus and nuclear and cytoplasmic inclusions are described, including pigment granules, storage products, the Golgi apparatus, mitochondria and vacuole systems.

SYNOPSIS. An initial rise in temperature increases the reproductive rate in Blepharisma intermedium; after it reaches the maximum, it gradually declines and finally fission is blocked. Cytochemical investigations made on Blepharisma acclimated to 28 C—the temperature at which the reproductive rate is maximum—show that conditions are optimum for cell division. Requisite quantities of glycogen, basic proteins, unsaturated lipids, enzymes alkaline and acid phosphatases are present. At 38 C the cell metabolism is altered, there is a decrease in the quantity of glycogen reserves, proteins and lipids, the enzyme activity diminishes, the free amino acid pattern changes and cell division is blocked. However, this block is transitory. Upon return to room temperature and when fresh culture medium is added, after a lag of 3 days, the cell metabolism becomes normal and the ciliates resume fission.

Suspensions of Blepharisma intermedium were fed latex particles for 5 min and then were separated from the particles by filtration. Samples were fixed at intervals after separation and incubated to demonstrate acid phosphatase activity. They were subsequently embedded and sectioned for electron microscopy. During formation of the food vacuole, the vacuolar membrane is acid phosphatase-negative. Within 5 min, dumbbell-shaped acid phosphatase-positive bodies, possibly derived from the the acid phosphatase-positive Golgi apparatus, apparently fuse with the food vacuole and render it acid phosphatase-positive. A larger type of acid phosphatase-positive, vacuolated body may also fuse with the food vacuole at later stages. At about 20 min after formation, acid phosphatase-positive secondary pinocytotic vesicles pinch off from the food vacuoles and approach a separate system of membrane-bounded spaces. By 1 hr after formation, the food vacuole becomes acid phosphatase-negative, and the undigested latex particles are voided into the membrane-bounded spaces. The membrane-bounded spaces are closely associated with the food vacuole at all stages of digestion and are generally acid phosphatase-negative. Within the membrane-bounded spaces, dense, pleomorphic, granular bodies are found, in which are embedded mitochondria, paraglycogen granules, membrane-limited acid phosphatase-containing structures, and Golgi apparatuses. The granular bodies may serve as vehicles for the transport of organelles through the extensive, ramifying membrane-bounded spaces.

1. The respiration of Amoeba proteus was measured. 10 c. mm. of cells were found to use about 1.6 mm.3 of oxygen per hour at 20°C. The respiratory quotient was found to be nearly unity. 2. No anaerobic metabolism was found for Amoeba. 3. The respiration of Blepharisma was found to be from 3 to 7 mm.3 oxygen per hour for 10 mm.3 cells. The respiratory quotient was about 1. 4. Blepharisma was shown to have a definite anaerobic metabolism. 80 mm.3 cells caused the evolution of 12.5 mm.3 carbon dioxide per hour at 20°C. in the presence of bicarbonate.

The same dosage of ultraviolet (UV) radiation retards division of several protozoans more effectively when the light is intermittent than when it is continuous, and especially at temperatures of 25–35°C. At lower temperatures the difference between the effects of intermittent and continuous radiations is less marked. Somewhat similar results were obtained with the ciliates Paramecium caudatum, Blepharisma japonicum, and Colpidium colpoda, the disparity between intermittent and continuous light decreasing in the order given. The data are taken to indicate that thermochemical dark reactions succeed the absorption of UV radiations by the cells. In Blepharisma, besides initial delay in division, the cells stop dividing after one or two divisions, a "stasis" ensuing. Stasis is marked when the cells are irradiated at higher temperatures but is slight when they are irradiated at low temperatures, as if the temperature-sensitive reaction involved stasis (in all cases cultures are grown at 25°C). The data are related to findings in the literature.

SYNOPSIS. Feeding activity of Homalozoon vermiculare on Paramecium and Blepharisma and on a number of non-living materials was compared in order to understand its feeding behavior and some of the factors governing it. During a given period it involves a series of feeding responses, each response being completed in 5 stages: (i) contact of prey or non-living particles with the oral rim of Homalozoon, (ii) ejection of toxin by Homalozoon, if the food is a living organism, (iii) paralysis of the prey, (iv) grasping, and (v) swallowing. The feeding activity of Homalozoon differs from one food to another and also on any one food at different times. These differences are governed by (i) the physiologic condition of Homalozoon and (ii) certain food characteristics. Its starvation, but not its developmental stage, markedly influences its feeding activity on any food. The food characteristics most important for feeding by Homalozoon are: (i) concentration of prey or non-living particles in the medium, insuring their adequate contact with the organism's mouth, (b) susceptibility of living organisms to paralysis by the toxin, (c) suitability of particle size for accommodation in the organism's mouth, and (d) suitability of the food material to stimulate the grasping and swallowing responses. Homalozoon much prefers Paramecium to Blepharisma on account of (i) a greater susceptibility of Paramecium to paralysis by the toxin, and (ii) certain differences in the chemical characteristics of the certain proteins, particularly albumins, hemoglobin, etc., of the 2 organisms.

FOR conjugation of Blepharisma intermedium to take place interaction of soluble gamones with the cells is essential for union between complementary mating types I and II (refs 1 and 2). Type I cells excrete gamone 1 which transforms type II cells so that they can unite. Type II cells excrete gamone 2 which similarly transforms type I cells. Moreover, each gamone promotes the production of the other. Gamone 1, blepharmone, is a glycoprotein of molecular weight 20,000 (refs 3 and 4). Gamone 2, blepharismone, is calcium 3-(2′-formylamino-5′-hydroxybenzoyl) lactate5.

(1881). On the vitality of the germs of Artemia salina and Blepharisma lateritia. Annals and Magazine of Natural History: Vol. 8, No. 48, pp. 456-458.

CAN a cell which has already entered on division go on to divide even when the presumptive or visible fission line or furrow is removed? This question can be answered conclusively for the large ciliate Stentor coeruleus, which is remarkably suitable for micro-surgical operations1. One might expect that the ciliate would then be uable to divide. Uhlig2 reported this finding after deleting the fission line from dividing S. coeruleus, and when Suzuki3 excised mid-cell disks bearing the visible fission line from dividing Blepharisma, these ciliates also failed to divide, although removal of the presumptive fission line did not prevent division, as I have confirmed in Stentor.

SUMMARY. Isolated Blepharisma undulans were studied for the following: (1) fission rate, (2) mating behavior, (3) giant formation, (4) effects of daily transections. The organisms were maintained at 22°C. and isolated daily for 60 days. Fission rate varied from two divisions a day to one in 29 days. The major factor producing the variation appeared to be the type or amount of food. Frequency of conjugation also varied greatly. Daughters of a single organism will mate within 24 hours after division. Conjugation followed by one division, re-isolation, then conjugation within 24 hours following a second division occurred within a 5 day period. Giants were produced within 3 days by feeding with Khawkinea, Chilomonas and Paramecium. Paramecium alone did not produce giants. Exposure to 10−m, 10−4M colchicine did not accelerate this process. Daily transections into approximately equal halves were made. The anterior halves containing the mouth membranelles were followed. Fission rate was not affected by cutting. A progressive decrease in size occurred. All ultimately lysed, most on the 7th day. If cutting was suspended on any day, recovery and fission followed. If cutting was re-instituted, lysis would again occur on or about the 7th day. Daily amputation of the posterior tip for 2 weeks did not affect either size, fission rate, or longevity.

Gamone 1 of Blepharisma intermedium was isolated, identified as a slightly basic glycoprotein (mizoleclular weight, 2 x 10(5)), and designated as blepharmnone. At the concentration of 6 x 10(-8) milligram per milliliter, it specifically transforms matinig type 2 cells, so that they can conjugate in about 2 hours.

1. 1. After exposure to far-ultraviolet (UV) radiation (265 nm), an equally high degree of photoreactivation (PR), about 80 to 90% of the radiation-induced division retardation, was obtained with the albino mutant of Blepharisma japonicum as with other ciliates previously tested, contrary to an earlier report. 2. 2. Maximal PR was observed only when PR light of low intensity was used and the dose was optimum. If too large a dose was applied, apparent PR was correspondingly reduced. 3. 3. Violet (405 nm) and blue (436 nm) light were most photoreactive; near-UV (366 nm) somewhat less so, and green (540 nm) and yellow light (570 nm) only slightly so. This is similar to what has been found for many other organisms. 4. 4. Under appropriate conditions similar degrees of PR were obtained for red wild-type B. japonicum as for the albino, though higher doses of PR light (longer exposures) were required. 5. 5. Under restricted conditions, which were different as to dose of PR light required, comparable PR was obtained in B. americanum, B. stoltei and B. japonicum. A high degree of PR is therefore probably characteristic of the genus, not just the albino. 6. 6. In the light of the present study, the previously reported low degree of PR of far-UV induced division delay, which had been considered exceptional among ciliates, appears to be an artifact, a result of (a) the use of logarithmic cultures which show less PR; (b) exposure to yellow light during handling and microscopic observation, which, though non-photoreactive to other ciliates, photoreactives far-UV damage in Blepharisma, and so reduces apparent PR measured; (c) supraoptimal light dosages which themselves induce division delay and so reduce the measurable PR. 7. 7. Blepharisma is much more sensitive to visible light in the PR spectral range than is Colpidium colpoda, which tolerated very much larger doses without injury.

Germ nuclei of Blepharisma japonicum begin meiosis within a few hours when cells of complementary mating types conjugate. We synchronized the onset of conjugation and treated cells in different stages of meiosis with 10 micrograms/ml cycloheximide which strongly inhibits protein synthesis in this ciliate. Cycloheximide arrested meiosis at six stages: I, between pairing of cells and swelling of germ nuclei; II, leptotene; III, zygotene; IV, pachytene; XI, interkinesis; XII, prometaphase II. Five of these arrests were reversible. Puromycin (250-500 micrograms/ml) also inhibited the progress of meiosis, though to lesser extents. We propose that the progression of meiosis of B. japonicum requires at least six proteins which are synthesized sequentially during meiosis.

With the discovery of the existence of mating types in sibling species of the P. aurelia complex by Sonneborn in 1937, the genetics of Paramecium and other ciliates entered a new phase. With this important fact in the hands of biologists, it now became possible to actually cross-breed stocks of diverse genetic ancestry just as in the higher multicellular animals. To date, Paramecium, Euplotes, and Tetrahymena have been extensively exploited. With the exception of Blepharisma (Miyake, 1974; Nanney, 1980), the great wealth of material in other ciliates is as yet untouched. The existence of mating types in other ciliates may demonstrate more effectively the answers to some of the problems in genetics, sexuality, and cytogenetics.