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Ciliate cells are separated from each other during most of their life cycle, but under certain conditions two cells temporarily unite to form a bicellular complex (conjugant pair) and undergo a series of developmental processes which are collectively called conjugation.

Three protein kinases (EC 2.7.1.37) were detected in Blepharisma and partially purified. The enzymes were most active with histone as substrate protein. The stability of the bond between phosphate and protein acceptor showed the characteristics of seryl- or threonylphosphate. Protein kinase I was solubilized by ultrasonication or freezing and thawing, while the enzymes II and III were readily solubilized by mild homogenization. Protein II and III were noticeably activated by cAMP and cGMP, while protein kinase I was inhibited by cAMP. Associated with protein kinase II and III activity was the ability to bind labeled cAMP. The following molecular weights were determined: 90000 for enzyme I, 280000 for enzyme II, and 95000 for enzyme III. Various apparent Michaelis constants were estimated.

At the outset, it should be mentioned that only very few protozoan photoreceptors are described in this review. The limited scope of the present review is chiefly dictated by the fact that the chemical identity and molecular properties of the isolated photoreceptors of only a few protozoan organisms have been characterized to any great degree. In particular, the photoreceptor systems of the ciliates Stentor and Blepharisma, the flagellate Euglena, and the filamentous alga Mougeotia,l are the subject of this review. The photoreceptors of Paramecium and selected photosynthetic algae are also discussed briefly.

Blepharisma (Figs. 1A and 1B) is a genus of ciliate protozoans in which most of the 47 listed species contain a pink pigment named blepharismin that renders them sensitive to glass-transmitted visible and nearultraviolet (UV) radiation. Initially, the pigment was called zoopurpurin by Arcichovskij (1905), who first determined its absorption spectrum. The name was changed to blepharismin to conform to the terminology of hypericin-like pigments, using the genus name as the basis of nomenclature: hypericin from Hypericum, stentorin from Stentor, fabrein from Fabrea, and blepharismin from Blepharisma (Giese, 1973).

Photobiological processes in the heterotrich ciliate Blepharisma japonicum have been extensively and intensively investigated (e.g., [6,7] and reference therein); in particular, similarly to several other microorganisms (e.g., [9]), Blepharisma has been shown to be able to respond to environmental illumination conditions, clustering in shadowed regions [7].

SYNOPSIS. Observations made with tritiated thymidine uptake indicate that Blepharisma cannot utilize this precursor directly from the organic medium. An intermediary, such as a food organism (bacteria or Tetrahymena, is needed. These food organisms are not only capable of incorporating thymidine from the medium, but must also break it down or prepare it in a form that is utilizable for Blepharisma. DNA synthesis phase in Blepharisma occurs 17 hours after division. Maximum incorporation is obtained 18 and 19 hours postdivision and lasts thru 24 hours. This is followed by a 1–2 hour postsynthesis phase and division.

Publisher Summary This chapter discusses the macronuclear variability of Blepharisma associated with growth. Ten different strains of the genus Blepharisma were selected for comparative studies of their nuclear and cytoplasmic growth from the twenty different strains in culture in the laboratory. A cyst-producing strain from Hingham, Massachusetts, was included among the ten strains. The different species, when grown at 18°C and fed upon bacteria, are morphologically distinguishable as species. The two strains of B. undulans, while they are distinctively different physiologically, are not sufficiently morphologically different to separate them at present into different species. Studies of the effects of different types of nutrition, combined with exposures to 5°C and 36°C, showed that at the high and low temperatures, although there were at first differences in reaction, ultimately all of the species were so drastically altered that they became morphologically indistinguishable. Organisms that were exposed to the 5°C again exhibited their characteristic morphology when returned to 18°C. Adaptation to 36°C of some of the organisms of one species fed on Tetrahymena occurred. That species was B. seshachari. The adapted individuals maintained their species characteristics.

One of the gamones (gamone II) which are effective for the induction of conjugation in Blepharisma intermedium has been isolated in a crystalline form and designated as blepharismin. From the result of chemical and spectroscopic investigations, in which x-ray crystallographic analysis was used as a definitive tool, blepharismin has been found to have the structure of calcium 3-(2'-formylamino-5'-hydroxybenzoyl)lactate.

Regeneration of Blepharisma undulans variety japonicus from which the hypostome has been removed is retarded by dosages of 3000 to 4600 ergs/mm.2 at wavelength 2654A most strongly when the fragment is exposed soon after cutting. Dosages greater than 4600 ergs/mm.2 prevent regeneration. Regeneration is also retarded strongly when the Blepharisma are cut soon after irradiation. Starvation retards regeneration and potentiates the effect of ultraviolet radiations. Division after regeneration of Blepharisma is also retarded by ultraviolet radiations about equally, regardless of when the Blepharisma are cut indicating a more lasting effect of the radiations upon the cells. Blepharisma cut after irradiation usually recover from the effects of the radiations sooner than uncut individuals given the same dosage. Retardation of division by ultraviolet radiation is subject to photoreversal by visible light, especially in a nitrogen atmosphere, provided the ultraviolet dose is not excessive. Visible light alone if prolonged, retards regeneration or may even kill the cut fragments of Blepharisma.

X-rays and ultraviolet radiations delay regeneration and division of Blepharisma undulans . Irradiated blepharisma which have not been cut divide once or twice fairly soon after the controls then they cease dividing for a period of time (stasis) which may last for many hours or even days. Once they recover from stasis they divide at a rate comparable to controls. Cut irradiated blepharisma on the other hand having to replace lost parts start to divide later than the uncut irradiated individuals but little if any stasis occurs. As a consequence although irradiated with the same dosage cut individuals recover a normal rate of division sooner than uncut ones.

1. 1. Studies were made on sodium and potassium regulation in Blepharisma intermedium. Ionic levels were expressed in terms of μM/g protein. Sodium and potassium were measured with a Beckman DU spectrophotometer adapted for use as a flame photometer. Protein was measured by the Lowry method. 2. 2. The ionic levels of Blepharisma in different concentrations of a modified Taylor-Strickland balanced salt solution were studied. High internal potassium levels existed in all concentrations studied. Blepharisma maintained low internal sodium levels up to 20 × balanced salt solution. Above this concentration, internal sodium levels increased with external levels although the internal level was always maintained about 37 mM/1 below the environment. 3. 3. Blepharisma placed at 5 °C gain sodium and lose potassium in about equal amounts. Sodium is extruded and potassium accumulated when the animals are brought back to room temperature. Recovery in a K-free solution slows the rate of sodium extrusion. 4. 4. Inhibitors of oxidative metabolism (sodium azide, dinitrophenol, and sodium cyanide) and of glycolysis (sodium fluoride) produce an increase in internal sodium levels. Usually these inhibitors have little effect on the internal potassium levels but occasionally produce a slight, unexpected increase in internal potassium. 5. 5. Ouabain, an inhibitor of the sodium pump, produced a large increase in internal sodium and a small increase in internal potassium. 6. 6. Gum tragacanth and tetrodotoxin have little effect on sodium and potassium levels. 7. 7. Blepharisma was immobilized in salt solutions containing high sodium concentrations but not in solutions containing high potassium chloride, magnesium chloride, magnesium sulfate, calcium chloride, or sucrose. Sodium azide and dinitrophenol immobilize Blepharisma at low inhibitor concentrations. Other metabolic inhibitors immobilize Blepharisma at concentrations at which internal sodium has increased.

Regeneration of postperistomial pieces of Blepharisma intermedium was retarded by increasing doses of UV 265 nm; a dose of 3 200 ergs/mm −2 usually doubled the time for 50 % regeneration while one greater than 5 600 ergs/mm −2 generally stopped regeneration. However, a dose of 4 000 ergs/mm −2 just detectably reduced incorporation of 14 C-uridine into RNA (RNAase-removable) in and near the macronucleus, doses of 4 400 ergs/mm −2 and more produced progressively greater effects, reduction of tracer incorporation being evident both in degree of labeling of individual cells and number of cells strongly labeled. Incorporation of 14 C-thymidine into DNA (DNAase-removable) in and around the macronucleus of those postperistomial pieces which showed any incorporation of Blepharisma was reduced by UV doses of 4 000–5 000 ergs mm −2 , as was incorporation of 14 C-amino acids into proteins. Growth in bromodeoxyuridine did not sensitize fragments of Blepharisma to UV. Regeneration was retarded most by UV in cells irradiated immediately after cutting. However, incorporation of 14 C-uridine into postperistomial pieces continued throughout regeneration and was about equally sensitive to UV 4 h after cutting as immediately afterwards. Regeneration was also retarded more if irradiated whole cells were cut soon after exposure than if cut later. However, incorporation of 14 C-uridine continued throughout regeneration and was about as sensitive to UV in those cut later as in those cut earlier. The macronuclear condensation cycle which accompanies regeneration was retarded by UV, the delay increasing with dose.

A cytological and experimental study was carried out on conjugations involving more than two individuals of Blepharisma undulans. Attachment of the participants in a conjugating group occurs in a number of characteristic patterns. Regardless of the attachment pattern, nuclear changes occur in all members of a multiconjugant system. These changes are identical in nature and duration to those observed in normal paired conjugation, and occur synchronously in all participants of the system. The stocks of B. undulans used are found to be selfing, physiologically not differentiated into mating types. Multiconjugation can be induced by varying food supply and population density. The probability of autogamous processes in connection with multiconjugation is discussed.

1. 1. The effects of selected dosages of colchicine (10 −2 to 10 −13 M ) were studied in a red-pigmented, heterotrichous ciliate Blepharisma undulans on the following: (1) division, (2) regeneration of cut Blepharisma , (3) conjugation, (4) survival of whole Blepharisma , regenerating and non-regenerating fragments. 2. 2. Concentrations below 10 −4 M were ineffective on all processes studied. Higher concentrations (10 −2 M and above) were cytolytic to Blepharisma . 3. 3. Reconstruction of the red pigment in bleached forms was unaffected by colchicine. The response of bleached forms to colchicine was the same as unbleached organisms. Cannibal-giants were less affected by colchicine on the processes studied than normal forms. 4. 4. The effect on division indicated that in ciliate protozoa where there is an amitotically dividing nucleus, the macronucleus, the division of the cytoplasm is apparently unaffected by disruption of the mitotically dividing micronuclei. 10 −4 M produced micronuclear mitotic inhibition without affecting cytoplasmic division, 10 −3 M affected both processes. 5. 5. Conjugating whole Blepharisma or conjugant fragments were more affected by lower concentrations (10 −4 M ) than non-conjugating forms. In some instances conjugating organisms exposed during conjugation did not recover from colchicine exposure. The inability to recover may possibly be due to a disruption of the new nucleus. 6. 6. The process of regeneration in Blepharisma was more affected by lower concentrations (10 −4 M ) than was division. The effect on regeneration may possibly be due to a disruption of the ciliary fibrillar systems. 7. 7. An explanation is offered for the change from the normal-sized individual to the cannibal-giant. This change may be due to the production of a polyploid individual.

1. A pigment can be extracted from Blepharisma undulans by heat treatment of a concentrated suspension of the deeply pigmented animals. 2. In the presence of this pigment, various colorless protozoans are sensitized to light and killed if exposed long enough. The protozoans show a differential sensitivity, some being much more sensitive than others. 3. Bleached colorless blepharismas are not sensitized to their own pigment even after prolonged exposure in the most concentrated solutions available. 4. Blepharisma is also less sensitive than any of the protozoans tested to such photodynamic dyes as rose bengal. 5. The pigment is not extracted from wet blepharismas by non-polar solvents, but is readily extracted into such polar organic solvents as the alcohols. 6. When the alcohol extract is dried, the amorphous residue is readily soluble in a variety of organic solvents, but not in the most non-polar. 7. The pigment is highly stable in alcohol extract and has been kept in the dark for years. 8. The pigment is bleached by light in a photooxidation. 9. Absorption maxima are found at wave lengths 5800, 5400, 4800, and 3300 A, the latter being the largest. Similar peaks are found in alcohol and water solutions, although the heights are not exactly the same. In both alcoholic and aqueous solutions pH had an effect on the absorption spectrum. Heat has little effect but illumination with intense visible light or exposure to ultraviolet light bleaches the pigment with decreases in the characteristic peaks. 10. Preliminary absorption column experiments indicate a single pigment in the alcohol extract. 11. Experiments on the migration of zoopurpurin in agar and gelatin gels indicate that it diffuses at about the same rate as eosin and is therefore probably not a large molecule.

Cells of B. intermedium differentiate into complementary mating types I and II. Type I cells excrete into the medium gamone I which transforms type II cells to pair-forming cells and at the same time induces them to produce and excrete gamone II. This gamone transforms type I cells to pair-forming cells. Transformed cells can unite in all three possible combinations but only heterotypic pairs (type I–type II) undergo nuclear changes characteristic of conjugation. Gamone I was concentrated and partially purified. The applicability of the system to the study of the regulation mechanism of the cell is suggested.

Synopsis. A simplified method for obtaining axenic cultures of Blepharisma involves the use of penicillin, streptomycin, and tetracycline. Cytologic examination disclosed no significant effect of these antibiotics on the structure of the ciliates.

Blepharisma: The Biology of a Light-sensitive Protozoan. By Arthur C. Giese. Pp. x + 366. (Stanford University Press: Stanford, California; Oxford University Press: London, July 1973.) £8.75.

Chromatographic analysis of acid hydrolyzates of the asexual and sexual stages of Blepharisma indicated the presence of 14 amino acids in the asexual animals (I) and 18, including all 14 found in I, in the conjugants (II). The total N content was 2.5 mg./ml. of protein for II and 1.5 mg./ml. for I. The presence of proline, hydroxyproline, threonine, and histidine only in II is significant, but no biochem. significance can be assigned at present. [on SciFinder(R)]

Studies on the life history of Blepharisma undulans have been in progress for over five and a half years, all the animals employed being the progeny of a single individual isolated November, 1921. The work was planned to determine, in particular, the viability of this infusorian in the absence of fertilization, and the effects of fertilization on the rate of reproduction, in an attempt to broaden the results from our earlier studies on Paramecium aurelia1 and Spathidium spatula.2 A preliminary statement of the characteristics of this race of Blepharisma undulans was presented in 1922.4Whereas earlier workers on this organism found that conjugation occurred rather infrequently and invariably was followed by death, conjugation was readily induced in our pedigree cultures, and the exconjugants proved to be viable in nearly every case. Accordingly excellent material was afforded for the problem at hand.Two conclusions may be emphasized from the data secured to date:(1) The animals of this race of Blepharisma ...

In a study of regeneration of form and function at various phases in the life history of the infusorians Spathidium spathula and Blepharisma undulans, it has been determined that the power of restoring lost parts and of continuing normal existence is in all cases dependent upon the same factor, the nuclear content of the fragment. Individuals of known pedigree were cut transversely, and the behavior or the fragments observed until regeneration and division or regeneration and death had occurred. During the vegetative state, regenerative power is highly developed in both ciliates, but because of the more distributed condition of the nucleus in Spathidium than in Blepharisma relatively smaller fragments from either extremity of the former are capable of complete regeneration. All fragments which continue to divide contain both macro and micronuclear material. It has been impossible to produce an amicronucleate race by artificial means.1In Spathidium, where the two nuclear elements extend throughout the cell...

Studies on Blepharisma undulans and Blepharisma lateritia Stein, have emphasized the impossibility or great difficulty of observing the micronuclei, and also the inevitable death of ex-conjugants without dividing. Thus Butschli1 was unable to identify micronuclei in vegetative specimens of B. lateritia though he found them in conjugants. Exconjugants invariably died. Calkins2 did not find micronuclei in non-dividing B. undulans, but discovered them during division within the macronuclear membrane, and described their emergence and activities during conjugation. He likewise was unsuccessful in obtaining a single viable exconjugant.During the past five months we have had under observation a pedigree culture of Blepharisma undulans which emphasizes racial differences within the species. The animals of this race possess from four to fourteen relatively conspicuous micronuclei, all of which are free in the cytoplasm during every stage of the life of the cell; and all the exconjugants thus far secured proved to...

From a study of the normal and regenerating contractile vacuoles in vivo, evidence was obtained that the normal contractile vacuole in Blepharisma undulans is not a permanent cell organ, but a system of temporary, potentially independent fluid vacuoles.1 In view of the fact that conflicting opinions concerning the permanence of contractile vacuoles have arisen from the failure of investigators to study both living and stained preparations, the above conclusions have been tested by a study of fixed material, derived from the pedigreed cultures. The Nassanov methods and the original Kolatschev technique were employed, not only because of their proved success in demonstrating contractile vacuole structure, but also because by their use the Blepharisma vacuole could be examined in the light of the Nassanov homology.In the several series of preparations which were made, Paramecia, mixed with the Blepharisma prior to fixation and treated identically step by step, were used as controls. Although the contractile ...

BLEYMAN, L. K. 1975. Mating type and sexual maturation in Blepharisma. Genetics, 80 (Suppl.): s14. BURCHILL, B. R. 1967. Conjugation in Stentor coeruleus. J. Protozool., 14: 683-687. BURCHILL, B. R., GEORGE, S. B., LINDBERG, R. E. & SIMS, J. P. 1974. Conjugation in Stentor multiformis. Trans. Amer. Micros. Soc., 93: 134-135. DE TERRA, N. 1974. Cortical control of cell division. Science, 184: 530-537. HARDEN, C. M. & HOLLAND, T. 1968. The effects of conjugation on Stentor coeruleus. J. Protozool., 15: 246-251. RAPPORT, D., BERGER, J. & REID, D. B. W. 1972. Determination of food preference of Stentor coeruleus. Biol. Bull., 142: 103-109. TARTAR, V. 1961. The Biology of Stentor. Pergamon Press, London and New York. 413 pp. WEBB, T. L. & FRANCIS, D. 1969. Mating types in Stentor coeruleus. J. Protozool., 16: 758-763.

The most rapidly cleared particle size has been reported to increase with the body size of the ciliate, a relationship attributed to changes in the morphological features of the oral apparatus. This study examined the possibility that the spacing of the adoral zone of membranelles (AZM) in Blepharisma japonicum could change or adapt to different sizes of prey. To test this hypothesis, specimens of B. japonicum were cultured on the bacterium Proteus vulgaris, the small ciliate Tetrahymena pyriformis, and smaller individuals of B. japonicum, each of which represented a different size of prey. After completing the culture period, a portion of the AZM was viewed by scanning electron microscopy and measurements of intermembranellar spacing were made from the micrographs. Significant differences (P = 0.01) in intermembranellar spacing were found between treatments: the smaller the prey size, the greater the spacing between the cilia of adjacent membranelles. The relationship between body size and particle size in Blepharisma cannot be explained by the inverse relationship already noted. The possibility remains that other oral structures (e.g., the "gullet") may govern this relationship. In natural waters, the range of food particle sizes often changes seasonally. Therefore, filter feeding organisms would benefit by being adaptable to different particle sizes. Copepods have been shown to shift their grazing pressure from one size of particle to another in order to take advantage of peak particle concentration (Poulet, 1974). Nival & Nival (1976) formulated an equation for filter feeding efficiency from a number of quantitative setal characteristics including setal spacing. They were able to show that the most efficiently filtered particle size increased throughout the maturation of the copepod. Richman et al. (1977) suggested that the flexibility in copepod feeding behavior cannot be explained solely by the mechanism of a fixed sieve, but they admitted not being able to offer an alternative explanation until the kinetics of filter feeding become more clearly understood. Ciliates, like copepods, are suspension (filter) feeders, but unlike copepods, possess cilia rather than setae for filtering. Fenchel (1980a,b) studied suspension feeding organelles and suspension feeding efficiency of ciliates in relation to particle size. He found: (1) that the free space between membranellar cilia determined the minimum size of particle retained; and (2) that the size of particle most rapidly cleared increased with the size class of Blepharisma americanum (see Fenchel, 1980b). He attributed the latter relationship to changes in the morphological properties of the oral apparatus. The oral apparatus of Blepharisma consists of both oral ciliature and cytopharyngeal struc1 We thank David Montagnes, Guy Gilron, and Stephen Wickham for their enthusiastic support and helpful criticism throughout this investigation. Research supported by the Department of Zoology and by N.S.E.R.C. Canada Operating Grant A-6544 awarded to D. Lynn. 2 Person to whom reprint requests should be addressed. TRANS. AM. MICROSC. SOC., 104(4): 333-340. 1985. ? Copyright, 1985, by the American Microscopical Society, Inc. This content downloaded from 157.55.39.27 on Sat, 26 Nov 2016 04:15:15 UTC All use subject to http://about.jstor.org/terms TRANS. AM. MICROSC. SOC. tures (Giese, 1973). The two major components of the oral ciliature are: (1) the adoral zone of membranelles (AZM), which functions to set up a vortex and draw in food particles from the surrounding water; and (2) the undulating membrane, which directs the particles into the cytostome (Giese, 1973; Sleigh & Barlow, 1982). Since the AZM functions to concentrate the food particles, it also may function in the selection of particle size (Fenchel, 1980a). In the present study, intermembranellar spacing was chosen as a possible site of long-term adaptation. The membranelles of the AZM are anchored by microtubular rootlets interconnected with microfilaments (Dembitzer & Hirshfield, 1966; Giese, 1973). By contraction of the filamentous components, the ciliate may be able to change intermembranellar spacing. However, Fenchel's (1980b) study assumed that intermembranellar spacing is more or less constant for a species. We assumed, then, for the purposes of this study, that the intermembranellar spacing is constant and does not change or adapt to prey size. Blepharisma were fed natural prey organisms to test for the possibility of adaptation of intermembranellar spacing to size of prey after several weeks of exposure to prey. Exposure to actual prey represents a more plausible ecological situation than using latex beads (Fenchel, 1980b) since the prey organisms chosen are known to support growth of Blepharisma (see Giese, 1973). Since a ciliate is more likely to encounter a monospecific patch of prey organisms in the natural environment, Blepharisma has been grown with one prey species at a time. MATERIALS AND METHODS

In mass cultures of several different races of Blepharisma undulans, including one which has been cultured by the writer for several years and used in connection with other investigations, cannibalism has occurred frequently. This phenomenon has not been very generally noted among ciliate protozoans, as the writer found in reporting a similar case in the Hypotrichida,1 and there are certain aspects of this process in Blepharisma which promise to add to the knowledge of digestive processes in ciliate protozoa.Cannibalism takes place in mass cultures in which growth has been rapid and large numbers of Blepharisma are present. The animal about to become a cannibal is always considerably larger than the individual that is ingested. The process of ingestion is relatively slow, requiring several minutes for completion. Ingested animals are not readily killed by the cannibal but show movements for varying periods, in one case as high as 8 hours. Ingestion is not always followed by digestion of ingested individua...