A circadian rhythm of mating type reversals in Paramecium multimicronucleatum, syngen 2, and its genetic control

Two new alleles, C and c, involved in mating type expression were demonstrated. A dominant allele, C(cycler), must be present for the expression of the rhythm involving a sequential alternation of the two complementary mating types (III and IV). Cultures can be entrained with light-dark cycles. The phase of each clone can be characterized by its III to IV and IV to III transitions in relation to the zero hour of a given light-dark cycle. Phase is a stable phenotypic trait during asexual reproduction, but following sexual reproduction it does not display Mendelian segregation. Instead phase is determined through nuclear differentiation, i.e., the trait is controlled by differently determined macronuclear alagen (caryonidal inheritance) which normally segregate at the second cell division after conjugation. The phase of a clone within its genetic limits is a function of the photofractions and the light intensities used in the entraining treatment. By examining a number of clones a variety of phase angles between the mating type cycle and the entraining light-dark cycle are found. Dividing cells which are sexually unreactive and therefore do not express the rhythm can be entrained and following entrainment, phase is inherited through repeated cell replications at a rate greater than one fission a day in continuous darkness or continuous dim light. This result unique to this system indicates that the cellular processes underlying the phase and period of this circadian rhythm persist (unexpressed: sexual reactivity requires slight starvation) through repeated cell replications even when the division cycle is considerably shorter than the expressed circadian period. The rhythm has a circadian period in continuous darkness or light (tested for six days) of less than 24 hours. The reversal of mating type ceases in continuous light at higher intensities. Cells homozygous for the recessive allele, c(acyclic), do not reverse mating type but are either mating type III or IV, again as a consequence of nuclear differentiation. Since individual cells with the dominant allele express both mating types, differentiation for mating type can not involve the absence in the macronucleus of mating type determining factors.     

The rhythm of mating in Paramecium aurelia, syngen 3

The periodic mating behavior of some stocks of Paramecium aurelia, syngen 3, exhibits features typical of circadian rhythms. In the most extensively studied stock (37^p), rhythmicity persists at least four days in continuous darkness, but disappears rapidly in continuous illumination (200 foot-candles). The period of the free-running rhythm is 22.2 hours, and relatively insensitive to ambient temperature. Cycles of illumination and temperature can regulate the mating rhythm. Changes in illumination at specified times in the circadian cycle will induce shifts in the phase of the rhythm. Stock differences with respect to the persistence of the rhythm and the environmental control of its phase have been observed.     

Feeding and Mating Reactivity in Paramecium multimicronucleatum

SYNOPSIS. Paramecium multimicronucleatum produced abnormal (L- and Δ-shaped) cells when cultivated in the presence of 3 mM adenine. These abnormal cells were unable to form food vacuoles in the presence of bacteria in the culture. In bacteria-rich culture, mating reactivity was not expressed in normal Paramecium ; however, it was expressed in the adenine-treated abnormal cells even in the presence of excess bacteria. The expression of mating reactivity in Paramecium was not affected by ingestion of polystyrene latex particles. These results show that the inhibition of mating reactivity in bacterized culture medium is caused by absorption of nutrients from bacteria digested in food vacuoles.     

Chemical Induction of Autogamy in Paramecium multimicronucleatum,Syngen 21

The combination of KCl + acriflavine + Ca²⁺-poor condition, known as conjugation-inducing-chemicals, was found to be autogamy-inducing-chemicals as well. Suspension of a single cell of Paramecium multimicronucleatum, syngen 2 in this medium for three hours or more resulted in autogamy that was evidenced by cytological similarity to conjugation.     

Effect of Adenine on Paramecium multimicronucleatum*

SYNOPSIS. The addition of adenine to the culture medium of Paramecium multimicronucleatum caused an inhibition of conjugation and fission and also an increase in the number of food vacuoles. The inhibition of conjugation was observed at the stage of adhesion of cell membranes. With adenine, the duration of fission was prolonged and L-shaped cells were formed. Adenine apparently repressed the excretion of food vacuoles and thus caused an accumulation of food vacuoles within the cells. All these effects were observed at similar concentrations of adenine higher than 2 mM. The addition of uridine to the adenine-containing medium reversed the inhibition of fission; conjugation and vacuole accumulation remained unaffected.     

Rhythmicity of swimming activity in an axenic population of Paramecium multimicronucleatum

A unicellular organism, Paramecium, exhibits circadian rhythm activities in many physiological phenomena, i.e., mating reactivity, photoaccumulation in Paramecium bursaria and mating type reversals in Paramecium multimicronucleatum. In this study, we used an image-processing system to analyze swimming activity in a population of Paramecium multimicronucleatum cultured axenically under 12 h-light/12 h-dark cycles (LD 1212). Swimming behavior was recorded both under LD 1212 and constant darkness and images tracing the tracks of Paramecium were produced every 4 min. Swimming activity was represented by the occupied area by the tracks relative to the total observed area. It is high during daytime and low at night and exhibits a freerunning rhythm in constant darkness. Furthermore, criteria for two major components of swimming behavior, straight and circle swimming, were established and analyzed. The results indicate that swimming behavior alters depending on the time of day: straight swimming increased during the day and circling was dominant around dusk both under LD 1212 and constant darkness.Abbreviations ZT Zeitgeber Time - LD 1212 12h- light/12h-dark cycles - TF transversing frequency     

Effects of pH and Viscosity on Surface Membranes in Paramecium multimicronucleatum

SYNOPSIS. Paramecium multimicronucleatum was grown in a buffered desiccated lettuce medium maintained at pH 7.5, with Aerobacter aerogenes the main bacterial source. The maximum expansion in length and width was at pH 6.0; the minimum expansion in length and width at pH 8.5. The paramecia were usually stouter at pH levels < 7.0 except at pH 5.5 and generally more slender > pH 7.0.
The pH of the medium affected the size of food vacuoles as indicated by the fact that the maximum food vacuolar diameter was at pH 6.0, the minimum at 7.0.
The fastest and greatest expansion of food vacuole diam. was at a relative viscosity of 8; the minimum food vacuolar diam. was in controls (without gum arabic) having a viscosity of 1.3.     

Helical Nature of the Ciliary Beat of Paramecium multimicronucleatum*

SYNOPSIS. Phase and interference cinemicrographs of cilia of Paramecium multimicronucleatum, immersed 3–24 hours in 1.0% methyl cellulose, revealed that 1) in swimming Paramecium the cilia beat with a traveling helical wave from base to tip rather than with the back and forth movement usually assumed, 2) during ciliary reversal the cilia merely change direction, but continue to beat with a traveling helical wave, and 3) in stationary Paramecium the beat is conicoidal. The traveling wave appears as an undulatory wave about 1 1/4 wave lengths long in both surface and profile views, and therefore must be helical. Envelope of the wave is cylindrical except near the base. Observations were confirmed in media without methyl cellulose by means of high speed cinemicrography, up to 4000 frames/sec. The back and forth movement, as described in all textbooks and monographs, is based mostly on 1) analogy to the abfrontal cilia (cirri) of Mytilus, which do beat with a back and forth movement, and 2) conclusions drawn from fixed preparations which do not represent what actually happens in a living animal. In a stationary Paramecium the envelope of the beat is conicoidal as seen in profile, but probably is a spiral wave, i.e., similar to a helix but increasing in diameter from base to tip. This change in wave form could be caused by the increase in resistance of the water in a stationary organism over one that is moving. Cilia and flagella (also ciliates and flagellates) are usually distinguished on the basis of wave form, but the present observations, together with previous data on flagella, show that such distinctions are untenable.     

Relation Between Cell Union and Nuclear Reorganization in Triplet Conjugants of Paramecium caudatum and Paramecium multimicronucleatum

SYNOPSIS Triplet conjugants of Paramecium caudatum which appeared naturally in mating mixtures and those of Paramecium multimicronucleatum which were produced by conjugation-inducing chemicals were isolated. Triplet conjugants lasting for more than 3 h were stained to examine macronuclear events. In P. caudatum , only 2 triplets among 182 (1%) contained macronuclear fragmentation in all 3 members. The most frequently occurring triplets (79%) were those producing 1 cell without and 2 cells with macronuclear fragments. There were also triplets (17%) producing 1 cell with, and 2 without macronuclear fragments, and some (3%) with 3 cells that contained no fragments. The length of persistence of the triplet was not responsible for the occurrence of macronuclear fragmentation in the 3rd cell of the triplet. In P. multimicronucleatum , the same 4 classes of triplets occurred, but the most frequently occurring class was that consisting of 3 cells (91%) with macronuclear fragments. Induction of nearly 100% of triplets with 3 such cells was possible by isolating the triplets' from a culture which was treated chemically at about 24 h after the last feeding. Treatment with chemicals in starved cultures resulted in triplets with incompletely fragmented or nonfragmented macronuclei. Further, in P. multimicronucleatum , chemicallyinduced triplets involving only holdfast pairs to which the 3rd cells were uniting often produced 3 cells with fragmented macronuclei.     

Genetic analysis of mating type differentiation in Paramecium tetraurelia. III. A mutation restricted to mating type E and affecting the determination of mating type

In P. tetraurelia each cell is determined to express only one of the two complementary mating types, O and E. This determination is under cytoplasmic control and seems to be achieved only by the commitment or noncommitment to the expression of mating type E. All the previously known mutations affecting the differentiation of mating type prevent the expression of the E mating type (O-restricted mutations) without affecting the determination process. An E-restricted mutation was obtained: mtF^E. Its phenotypic properties indicate that the mutation affects the determination process itself. When an O cell becomes mtF^E/mtF^E it acquires the E mating type and an E-determining cytoplasm. We propose that this constitutive determination for the E mating type is due to the inefficiency of a factor which is normally active in an O cell. This factor would act like a repressor and stabilize the E functions under an inactive state.     

Adenosine Triphosphate Acceleration of the Nephridial Apparatus of Paramecium multimicronucleatum*

SYNOPSIS. Paramecium multimicronucleatum was exposed to various external concentrations of adenosine triphosphate (Na₂ATP) to determine the effects thereof on the cycling rate of the nephridial apparatus. Normal rate was found to vary from 3.46 to 4.28 cycles/min with a mean rate of 3.85 cycles/min at 20 C. Concentrations of ATP of less than 5 × 10⁻⁴ M caused only slight, very temporary acceleration of the cycling rate. At 5 × 10⁻⁴ M the cycling rate was accelerated less than 15%. At 3 × 10⁻³ M cycling rate was accelerated, varying from 5.35 to 7.24 cycles/min, with a mean accelerated rate of 6.25 cycles/min, a mean aoceleration of 88.3%. Changes in rate after addition of 5 × 10⁻³ M ATP ranged from a decrease of 6.2% to an increase of 1.8%, with the nephridial apparatus ultimately stopping. At higher concentrations, stoppage was almost immediate. Paramecium is rapidly dehydrated by the ATP-accelerated cycling of its nephridial apparatuses, with a net loss of 27% of its volume in 6 minutes in the 3 × 10⁻³ M ATP.