An Analysis of Macrostome Production in Tetrahymena vorax Strain V2S-Type*

SYNOPSIS. Tetrahymena vorax is a small polymorphic holotrichous ciliate capable of transforming to macrostomes when microstomes are washed and suspended with prey in distilled water. Extrinsic factors having an effect on this transformation were examined; maximum yields of macrostomes (in excess of 90%) were obtained under the following conditions: populations of both prey, T. pyriformis, and potential predator, T. vorax microstomes, were grown on Loefer's medium for 48 hours prior to washing in distilled water. The density of the prey was adjusted to 300,000 cells/ml and the predator density to 2,000–3,000 cells/ml. Five ml of prey suspension and 5 ml of T. vorax microstome suspension were mixed together in a large petri dish because a high surface-volume ratio is important for high yields of macrostomes. The pH was adjusted to 6.0 and the petri dish was placed at 20 C for 12 hr. Macrostomes then appeared about 6 hr after addition of the prey. A dialyzable, heat stable substance released by the prey which can induce the microstome-macrostome transformation was isolated. This material was effective after being stored for weeks in the cold; its activity was not affected by the protein digesting enzymes pepsin or trypsin. This factor was called stomatin because its first visible effect in producing microstome-macrostome transformation appeared to be to incite reorganization of the oral structures.     

A Comparison of Cortical Proteins in Tetrahymena vorax Microstomes and Macrostomes1

Proteins of surface membranes and surface-related cytoskeletons in Tetrahymena vorax microstomes and macrostomes were compared by one-dimensional SDS polyacrylamide gel electrophoresis to see if protein differences could be detected that correlate with the transformation from one phenotype to the other. Some differences were observed. However, these alterations appear to result from the heat-shock procedure used to synchronize the microstome-to-macrostome transition. The apparent lack of transformationspecific changes in cortical proteins is discussed. Similarities and differences between cytoskeletal proteins of T. pyriformis GL-C and T. vorax are also noted and discussed.     

Microstome-Macrostome Transformation in Tetrahymena vorax Strain V2 Type S Induced by a Transforming Principle, Stomatin

SYNOPSIS. Microstome →macrostome transformation in Tetrahymena vorax was induced by suspending microstomes in a transforming principle, stomatin, released by a potential prey, T. pyriformis. It was found that 70–90% of the microstomes formed macrostomes within 7 hours following suspension in this transforming principle. Macrostome formation occurred by the process of oral replacement. This process involved resorption of the microstome oral apparatus and its replacement with a larger (macrostome) one, which arose from an anarchic field that formed behind the resorbing oral area. Ninety-five percent of those microstomes which were destined to form macrostomes were in some stage of oral replacement 195 minutes after their suspension in stomatin. Several commercially produced products were tested over a wide range of concentrations to determine their ability to act as an inducer of macrostomes. Only 2, Trypticase and Bactocasitone, had any activity, and it was too small to be considered really effective. An attempt was also made to destroy the activity of stomatin by using enzymes. RNAse was effective but only in very high concentrations, so it was suggested that this activity might be related to the destruction of RNA within the transforming cell and not related to hydrolysis of stomatin. None of the other enzymes tested had any effect in reducing the activity of stomatin.     

The Formation and Fate of Tetrahymena patula Cryptostomes

A reliable method for producing reproductive cysts in Tetrahymena patula is described. The procedure involves the isolation of macrostomes without cytopharyngeal pouches in microdrops of distilled water under oil. The study of silver-impregnated specimens has shown that a complex pattern of oral resorption and reformation occurs within the cyst that leads to the formation of a group of small cells with recessed oral apparatuses. These cells, called “cryptostomes,” swim very rapidly on excystment and are incapable of either feeding or reproducing. They are presumably dispersal forms. Oral morphogenesis during the transformation of excysted cryptostomes into microstomes and macrostomes is also described.     

Morphology and ontogenesis of Platyophrya bromelicola nov. spec., a new macrostome-forming colpodid (Protists,Ciliophora) from tank bromeliads of Jamaica

Platyophrya bromelicola nov. spec. was discovered in tanks of bromeliads from Jamaica. Its morphology, ontogenesis, and small-subunit rDNA were studied using standard methods. Platyophrya bromelicola differs from its congeners mainly by the pyriform, unflattened body (vs. reniform and flattened); the free-swimming (planktonic) habit (vs. biofilm creepers); and the unique ability to form two distinct morphs, i.e., small, bacteriophagous microstomes and large, predaceous macrostomes. Microstomes and macrostomes can be distinguished not only by body size and feeding preferences but also by the postoral pseudomembrane composed of two vs. three to four dikinetids per kinety. The ability to form macrostomes is considered as an adaptation to the highly competitive habitat. Ontogenesis closely resembles that of other members of the family. Platyophrya bromelicola is distinct not only morphologically but also genetically (3.7% in the small-subunit rDNA) from P. vorax, a common, cosmopolitan moss and soil species.     

Studies of Macrostome Formation of Low-Transforming Tetrahymena vorax. Transformation Enhancers,Generation Time,and Membrane Fluidity1

ABSTRACT. Periodically, stocks of Tetrahymena vorax, which normally yield 70–90% macrostomes when subjected to heat shock or other induction methods, become low-transformers and yield ≥30% macrostomes. The addition to the post-heat-shock wash buffer (pH 6.8) of 2.7 × 10^-4 M Fe³⁺, 1.6 × 10^-5 M Cu²⁺, 1 × 10^-4 M retinol palmitate or the adjustment of the buffer to a pH of 4 to 5 boosts transformation significantly over controls in inorganic medium alone. The addition of Fe²⁺ or Cu¹⁺ has a similar, but less pronounced effect on transformation. Ferric ion (2.7 × 10^-4 M) will significantly increase transformation in starved non-heat-shocked cells, whereas Fe²⁺, copper, retinol palmitate, and hydrogen ion concentration have no effect. The agents that boost transformation appear to act by delaying cell division in pre-transformants. Membrane fluidity, as inferred by fluorescence polarization measurements of 1,6-diphenyl-1,3,5-hexatriene, is altered in a consistent manner by starvation and heat shock. Enhancing agents, including compounds previously shown to boost heat-shock-induced macrostome formation, produce diverse shifts in membrane fluidity. Their effect on transformation of these low-transforming cells therefore appears to be attributable to some mechanism or mechanisms other than a direct alteration of membrane physical properties.     

The Effect of the Lipogenic Inhibitor Cerulenin, on Macrostomal Cell Formation in Tetrahymena vorax

ABSTRACT. Macrostomal cell formation is blocked by the antibiotic cerulenin at levels of 15 μg/ml or higher. Inhibition can be reversed up to 4 h following cerulenin addition by washing and resuspending cells in new, noncerulenin-treated transforming principle. In these latter cases, additional time equal to the time spent in the inhibitor, is needed for cells to reach control values of transformation. Neither the addition of saturated or unsaturated fatty acids, cholesterol added alone or in combination with stearic acid, nor a mixture of lipids extracted from Tetrahymena vorax reversed the cerulenin effect. Radioisotope incorporation data showed while protein synthesis was reduced by the end of 1 h and tetrahymanol synthesis by the end of 2 h, little or no effect of this inhibitor occurred on RNA or fatty acid synthesis during these times. One interpretation of these results is that cerulenin, by preventing first protein synthesis and later tetrahymanol synthesis, interferes with synthesis and formation of membranes required for the microstome to macrostome transition.     

Electrophysiological Properties of the Microstome and Macrostome Morph of the Polymorphic Ciliate Tetrahymena vorax

Polymorphic ciliates, like Tetrahymena vorax, optimize food utilization by altering between different body shapes and behaviours. Microstome T. vorax feeds on bacteria, organic particles, and solutes, whereas the larger macrostome cells are predators consuming other ciliates. We have used current clamp and discontinuous single electrode voltage clamp to compare electrophysiological properties of these morphs. The resting membrane potential was approximately ?30 mV in both morphs. The input resistance and capacitance of microstomes were approximately 350 MΩ and 105 pF, whereas the corresponding values for the macrostomes were 210 MΩ and 230 pF, reflecting the larger cell size. Depolarizing current injections elicited regenerative Ca²⁺ spikes with a maximum rate of rise of 7.5 Vs^?1 in microstome and 4.7 Vs^?1 in macrostome cells. Depolarizing voltage steps from a holding potential of ?40 mV induced an inward Ca²⁺ ‐current (I_ca) peaking at ?10 mV, reaching approximately the same value in microstome (?1.4 nA) and macrostome cells (?1.2 nA). Because the number of ciliary rows is the same in microstome and macrostome cells, the similar size of I_Ca in these morphs supports the notion that the voltage‐gated Ca²⁺ channels in ciliates are located in the ciliary membrane. In both morphs, hyperpolarizing voltage steps revealed inward membrane rectification that persisted in Na⁺‐free solution and was only partially inhibited by extracellular Cs⁺. The inward rectification was completely blocked by replacing Ca²⁺ with Co²⁺ or Ba²⁺ in the recording solution, and is probably due to Ca²⁺ ‐activated inward K⁺ current secondary to Ca²⁺ influx through channels activated by hyperpolarization.     

The Effects of Purine and Pyrimidines Upon Transformation in Tetrahymena vorax,Strain V2S1

Column chromatography with Biogel P2 (molecular exclusion of 1800 daltons) indicates that the transforming principle causing microstomes to become macrostomes is a small molecule. Absorbance tests show that only those fractions with high absorbance at 260 nm have biological activity, indicating that the active principle is a component of nucleic acids. Tests of purines and pyrimidines show that purines are active, with hypoxanthine having the highest activity. The combination of hypoxanthine with uridine shows a synergistic reaction. As these two compounds are the natural catabolic excretory products from nucleic acids in Tetrahymena, the fact that they induce transformation in concentrated, starving cells may be a survival mechanism allowing cannibalism to be induced when nutrients are depleted, thereby allowing the survival of the transformed cells until such time as adequate nutritional conditions are restored.     

Preuves Cinétiques de l'Existence d'une Période de Compétence pour la Transformation Microstome-Macrostome dans le Cycle Cellulaire de Tetrahymena paravorax

RÉSUMÉ. Des expérimentations portant sur des cellules isolées ont montré que 77% des microstomes de Tetrahymena paravorax, prélevés au hasard dans des cultures en phase logarithmique de croissance, se transforment directement en macrostomes en présence de “stomatine.” Ces macrostomes apparaissent à des moments variés entre 1,5 et 9 h après l'addition de la stomatine (“point 50% de transformations” vers 2,5 h). La compétence pour le remplacement oral est en relation avec la position dans le cycle cellulaire. Les pourcentages de transformation les plus élevés sont observés avec les populations testées pendant la première moitié de la période moyenne d'interfission. La formation des macrostomes est d'autant plus rapide que l'ǎge initial des microstomes est plus proche du point médian du cycle cellulaire (“point de compétence”). Dans la seconde moitié de ce cycle, le temps moyen de transformation reste à peu près constant, mais le pourcentage de divisions augmente: le “point de transition” (50% de divisions) se trouve au début d'une phase terminale représentant 19–20% de la durée totale du cycle. La transformation des produits de bipartition antérieur et postérieur est nettement asynchrone: dans la majorité des paires cellulaires, l'opisthe se transforme avant le proter. Les cellules-soeurs se divisent aussi de manière asynchrone: le temps de génération du proter est plus long que celui de l'opisthe. Le problème de l'acquisition de la compétence pour le changement de phénotype est discuté en envisageant les corrélations éventuelles avec certains processus majeurs du cycle cellulaire. SYNOPSIS. Seventy-seven percent of the microstomes of Tetrahymena paravorax taken from random samples of log-phase cultures transform directly into macrostomes in the presence of “stomatin.” These macrostomes appear between 1.5 and 9 h after addition of stomatin (“50% transformation point,”～ 2.5 h). Competence for oral replacement is related to the position in the cell cycle. The highest percentages of transformation are observed in populations tested during the first half of the mean interfission period. Formation of macrostomes is more rapid when the initial age of the microstomes is nearer to the midpoint of the cell cycle (“competence point”). In the 2nd half of this cycle, the mean transformation time remains approximately constant, but the percentage of cells undergoing division is increasing. The “transition point” (50% divided cells) is found at the beginning of a terminal phase which accounts for 19–20% of the cell cycle. Transformation of anterior and posterior fission products is fairly asynchronous; in the majority of individual pairs, the opisthe is transformed before the proter. The daughter cells also divide asynchronously, the generation time of the proter being longer than that of the opisthe. The problem of acquisition of competence for phenotypic change is discussed in light of possible correlations with certain major processes of the cell cycle.     

2-mercapto-1-( -4-pyridethyl) benzimidazole, an inhibitor of RNA synthesis: a reevaluation

The effect of 2-mercapto-1-(β-4-pyridethyl) benzimidazole (MPB) was determined in fetal rat liver explants. MPB exerted little effect upon the uptake of ¹⁴C-labeled orotic acid by the fetal liver, but exerted a very pronounced inhibition of the incorporation of the precursor into TCA-insoluble, alkalilabile material. These studies justify in fetal rat liver systems the use of MPB as an inhibitor of RNA synthesis unmediated by permeability effects.     

Polypeptide Synthesis with Microsomes from Pressure-Treated Tetrahymena1

SYNOPSIS High hydrostatic pressure is known to interfere with mitosis, cytokinesis and synthesis of DNA, RNA and protein. In Tetrahymena, incorporation of phenylalanine and formation of polysomes are known to be pressure-sensitive. Microsomal preparations from Tetrahymena pyriformis GL can incorporate [¹⁴C]-phenylalanine into polypeptides. Incorporation was enhanced by addition of supernatant fraction and 14.5 mM Mg⁺⁺ An energy-generating system and exogenous messenger (poly U) were essential for polypeptide biosynthesis, Microsomes from pressurized cells (14,000 psi for 5 min) incorporated [¹⁴C]phenylalanine as efficiently as control microsomes. Microsomal function was not grossly damaged by pressure in a test system containing exogenous messenger, crude microsomal preparation, exogenous energy-generating system and supernatant fraction containing activating enzymes.     

Transmission of broad bean stain virus and Echtes Ackerbohnenmosaik-Virus to field beans (Viciajaba) by weevils

Sitona lineatus and Apion vorax were the two most common species of weevil on field beans (Vicia faba minor) at Rothamsted between 1970 and 1974. In glasshouse tests, A. vorax was a much more efficient vector than 5. lineatus of broad bean stain virus (BBSV) and Echtes Ackerbohnenmosaik-Virus (EAMV), and both species transmitted EAMV more often than BBSV. Five other species of Apion transmitted the viruses infrequently or not at all. S. lineatus adults transmitted no more often after 8–16 days on infected plants than after 1–2 days. Some A. vorax adults transmitted EAMV, but not BBSV, after feeding on infected leaves for a few minutes. After 4 days on infected plants, A. vorax sometimes remained infective for the following 8 days. No A. vorax collected from woodland plants in spring was infective with BBSV or EAMV, but 4% from bean crops containing seed-borne infection carried BBSV and 17% carried EAMV. BBSV and EAMV were recovered from triturated weevils, but not from weevil haemolymph. Possibly the viruses are transmitted as contaminants of the mouthparts or by regurgitation during feeding, but A. vorax was observed to regurgitate only when anaesthetized. BBSV and EAMV were not transmitted by aphids (Aphis fabae and Acyrthosiphon pisum), nor by pollen beetles {Meligethes spp.). Field observations suggest that infected seed is the main source of BBSV and EAMV in spring-sown crops, and that crops grown from virus-free seed, and isolated from infected crops by 250–500 m, remain free of infection for most of the season.     

Food Vacuoles During Heat-Shock-Induced Transformation from the Microstomal to the Macrostomal Cell Type of Tetrahymena vorax1

ABSTRACT. The heat-shock method for induction of the macrostomal form of Tetrahymena vorax involves the transfer of cells to reduced nutrient medium and the application of a series of elevated temperature shocks followed by washing the protozoa into inorganic medium. The component of the procedure that had the greatest effect on food vacuoles was the heat shocks. At the end of the heat shocks, cells formed vacuoles at a lower rate than non-heat-shocked cells, but the size of the vacuoles formed was larger and the protozoa contained an increased number of vacuoles and total vacuolar membrane. The rate was further reduced by washing cells into nonnutrient medium. In the absence of the heat shocks, the medium had little effect on the capacity of the cells to form vacuoles although after 7.5 h in inorganic medium, the vacuoles formed were smaller and the protozoa possessed fewer vacuoles and therefore less vacuolar membrane. The amount of membrane required to form the cytopharyngeal pouch of the macrostomal cell type was equivalent to the surface area of food vacuoles present in cells prior to the onset of the heat shocks, but the number and surface area of vacuoles decline between the time of oral resorption and pouch development.     

Efficiency of Filter Feeding in Two Species of Tetrahymena*

SYNOPSIS. Rates of removal of suspended India ink particles from the surrounding medium by 2 ciliates, Tetrahymena pyriformis strain GL and Tetrahymena vorax strain V₂S, have been measured. It is evident from the results that the food vacuoles concentrate the suspended particles, clearing a volume of the surrounding suspension fluid 500 × greater than the total volume of food vacuoles made during the same period of time.     

Poly(A)+ RNA from Tetrahymena: Stimulation of Protein Synthesis in Vitro*†

SYNOPSIS Cell-free synthesis of high molecular weight polypeptides, programmed by RNA from Tetrahymena pyriformis strain W is reported, and methods for preparation of the RNA are described. The RNA was extracted by the SDS-phenol-chloroform-isoamyl alcohol technic. The bulk of extracted RNA was ribosomal and on sucrose gradients peaked at -17S and 25S. After heat denaturation all the 25S RNA was converted to 17S. indicating the presence of hidden breaks, possibly the result of nuclease activity during extraction. Nevertheless, when poly(A)–RNA was collected using oligo-(dT)-cellulose column chromatography, it promoted a 15–fold increase in incorporation of [35S] methionine into TCA-precipitable material. Slab-gel electrophoresis and autoradiography of the product revealed 12 different major polypeptides, varying in weight from 28.000 to 65,000 Daltons. A method for preparation of translatable RNA from Tetrahymena will make possible the comparison of messenger RNAs associated with specific cell structures and with different developmental events.     

5S and 5.8S ribosomal RNA evolution in the suborder tetrahymenina (Ciliophora: Hymenostomatida)

Summary The nucleotide sequences of the 5S and 5.8S rRNAs of eight strains of tetrahymenine ciliates have been determined. The sequences indicate a clear distinction betweenTetrahymena paravorax and its suggested conspecificT. vorax, but leave the taxonomic distinction betweenT. vorax andT. leucophrys in doubt. The rRNA sequences of sixTetrahymena species and of three other species of the suborder Tetrahymenina have been used to deduce evolutionary schemes in which ancestral rRNA sequences and changes are proposed. These schemes suggest the predominant acceptance of GA and CT transitions in the 5S rDNA during the evolution of the suborder.     

The Incorporation of Phenylalanine and Uridine in Tetrahymena: A Pressure Study1

SYNOPSIS. The action of pressure was studied on the incorporation of labelled phenylalanine and uridine, and on the synthesis of water-soluble proteins in heat-synchronized Tetrahymena pyriformis. Incorporation of [¹⁴C] phenylalanine and [³H] uridine into TCA insoluble fractions was markedly decreased in pressuretreated Tetrahymena. Moreover, the incorporation was dependent upon the age of the cells. Water-soluble protein synthesis was unaffected. These results appear consistent with the proposal that pressure-induced division-delays occur by inhibiting the accumulation of “division proteins” which are essential for cell division.