The effect of nutritional state on photoreversal of ultraviolet injuries in Didinium nasutum

1. The effect of the nutritional state of Didinium nasutum on its resistance to short ultraviolet (UV) radiation (2654 A) and its recovery from the injury following illumination with visible light (4350 A, blue) was studied. 2. The resistance of a didinium to UV is considerably increased by feeding it a paramecium 15 to 60 minutes before exposure to UV. If fed just before exposure to UV, the resistance is less than that of an unfed control. 3. Photoreversal is only slightly greater in didinia fed after irradiation with UV but before exposure to visible light as compared to those fed after exposure to visible light. 4. Irradiated paramecia are eaten by didinia, provided they have not started to cytolyze. Didinia fed on irradiated paramecia divide at about the same rate as controls or slightly faster. 5. The available stock of Didinium declines in vigor with lapse of time after excystment, as measured by the time required for division. The sensitivity of Didinium to UV did not change essentially during the 5 month period over which tests were made. 6. The theoretical implications of the results are considered.     

Excystment of Didinium nasutum

SYNOPSIS. Excystment of Didinium nasutum is solely dependent upon dense bacterial populations and is independent of the type of medium in which the bacteria have been grown. Beers' hypothesis of the need for a factor from proteose-peptone is thereby not confirmed, but the need for living, intact bacteria as postulated by him is fully shown. The process of excystment is initiated by bacteria, but their presence is not required throughout the excystment. Axenically grown paramecia, shown to be adequate as food organisms for didinia, will not induce excystment. Five types of bacteria including Gram-positive, Gram-negative, aerobic and anaerobic all suffice to induce excystment, suggesting that the initiation of this process is caused by some general metabolic process or product of bacteria.     

Spontaneous and heat shock-induced irregularities in the cell structure of Didinium cf. nasutum

Deviations of the normal morphology and arrangement of the ciliary girdles and of the dorsal brushes of Didinium cf. nasutum are documented by SEM. Additionally, irregular fission stages such as symmetrical doublets which possess two parallel or antiparallel probosces are found in normal cultures. These ciliates are relatively long-lived (about one month). The didinia with oppositely oriented probosces feed continuosly on paramecia and produce normal cells which are indistinguishable from type-cells under a light microscope . Didinia with three or four probosces were not viable for longer than one to two weeks. All types of irregular forms could be induced experimentally by a 15-min exposure at 30 °C of cells cultured at 18 °C.     

Competitive advantages of Caedibacter-infected Paramecia

Intracellular bacteria of the genus Caedibacter limit the reproduction of their host, the freshwater ciliate Paramecium. Reproduction rates of infected strains of paramecia were significantly lower than those of genetically identical strains that had lost their parasites after treatment with an antibiotic. Interference competition occurs when infected paramecia release a toxic form of the parasitic bacterium that kills uninfected paramecia. In mixed cultures of infected and uninfected strains of either P tetraurelia or of P novaurelia, the infected strains outcompeted the uninfected strains. Infection of new host paramecia seems to be rare. Infection of new hosts was not observed in either mixtures of infected with uninfected strains, or after incubation of paramecia with isolated parasites. The competitive advantages of the host paramecia, in combination with their vegetative reproduction, makes infection of new hosts by the bacterial parasites unnecessary, and could be responsible for the continued existence of "killer paramecia" in nature. Caedibacter parasites are not a defensive adaptation. Feeding rates and reproduction of the predators Didinium nasutum (Ciliophora) and Amoeba proteus (Amoebozoa, Gymnamoebia) were not influenced by whether or not their paramecia prey were infected. Infection of the predators frequently occurred when they preyed on infected paramecia. Caedibacter-infected predators may influence competition between Paramecium strains by release of toxic parasites into the environment that are harmful to uninfected strains.     

Specific Incorporation of Precursors into DNA by Feeding Labeled Bacteria to Paramecium aurelia

SYNOPSIS. Studies were carried out on the introduction of labeled precursors into the DNA of Paramecium aurelia (syngen 4, stock 51) by way of the bacteria that are used for food. A thymine-requiring strain of Escherichia coli (15 T⁻) was labeled by growth in either H³-methyl thymidine or 2-C¹⁴ bromouracil, washed free of the exogenous label, and fed to the paramecia. The tritium label from the bacteria was incorporated almost exclusively into the DNA of the paramecia, whereas it was much less specifically incorporated when introduced directly from the medium. The Cu label from bromouracil was also incorporated mainly into the DNA of the paramecia although a small amount appeared in RNA. The formation of labeled food vacuoles was followed. Food vacuoles were formed at a nearly constant rate, with the total number of vacuoles increasing throughout the cycle. The lifetime of the vacuoles was about 2.5 hours. Incorporation of the label into the DXA of the paramecia begins within a few minutes of the formation of the first labeled vacuole. DNA synthesis begins about 1.5 hr after the previous fission (total cell cycle about 5.8 hr) and progresses at a nearly constant rate throughout the remainder of the cycle.     

Clonal Aging In Two Species of Spathidium (Ciliophora: Gymnostomatida)*

Thirty newly excysted Spathidium spathula (Müller) and 60 newly excysted Spathidium muscicola (Kahl) were selected as progenitors of clonal daily reisolation lines that omitted both conjugation and encystment. Daily division rates were determined for each clone either until it died or until the end of the 170 days of reisolation. Both species had reduced fission rates as they accumulated fissions in the absence of macronuclear reorganization. Spathidium spathula had a significant reduction of daily fission rate after 100-120 fissions and S. muscicola after 20-30 fissions. Older clones of both species contained a noticeable proportion of abnormal organisms. A significant increase (10.5%) in daily fission rate occurred in aged sublines of S. spathula following conjugation (selfing) and its concomitant nuclear reorganization. Spathidium muscicota did not conjugate, but recently excysted sublines, compared to aged lines, had an increased daily fission rate.     

Alpha, an Infectious Macronuclear Symbiont of Paramecium aurelia

SYNOPSIS. A spiral, rod- or crescent-shaped symbiont here designated alpha, is present in the macronucleus of killer stock 562, syngen 2 of Paramecium aurelia. This stock has a cytoplasmic symbiont, kappa, as well as alpha. Lines were obtained which had only alpha, others which had only kappa, and some which had neither. It was possible to purify and separate both kinds of symbiont from homogenates of stock 562 using an ECTEOLA column. The killing action of this stock is due to kappa, not alpha. Observations on the structure of alpha with the electron microscope indicate that alpha, like the cytoplasmic symbionts in this species, is a bacterium. Alpha is never seen in the micronucleus, is rarely found in the cytoplasm, but abounds in the macronucleus. If paramecia are allowed to grow slowly after autogamy, alpha passes from the old macronuclear fragments, infects the new macronucleus, and all animals retain alpha. In exautogamous paramecia growing at maximum fission rate, however, alpha often does not infect the new macronucleus and is lost from many lines when the old macronuclear fragments disappear. In mixed cultures containing alpha-bearing and alphafree paramecia, it has been found that alpha readily invades the macronucleus of paramecia of susceptible stocks. Homogenates of alpha-bearing cultures are also infective. Infection is highly specific, occurring in only 6 of the 44 stocks of P. aurelia in which infection was attempted, and these 6 are all syngen 2. It is suggested that the short rod or crescent form of alpha is the reproductive form, while the elongated spiral form is probably the invasive motile form.     

Levels of social organization and male-female bonding in the genus Papio

Pair-bonding in the genus Papio seems to be the result of fusion of troops as well as fission. When troop segments regularly split and join again, males who permanently maintain exclusive access to a few females may have an advantage over males who compete for all females who are in estrus. When two or more troops regularly fuse, this advantage may be greatly increased.     

Entamoeba invadens: the requirement for galactose ligands during encystment

During periods of stress, trophozoites of Entamoeba invadens (strain IP-1) undergo a process of differentiation (encystment) that results in a dormant cyst with a chitin-containing cyst wall. Encystment can be induced by resuspension of trophozoites from growth medium into a diluted glucose-free medium (47% LG) containing 5% adult bovine serum (ABS). ABS is thought to be a source of gal-terminated ligands that are required for high levels of encystment. After resuspension of trophozoites in 47% LG, encystment cultures were examined every 2h for responses to the (i) addition of 10mM free-galactose, (ii) resuspension of cells to serum-free medium, (iii) and dilution of encysting cultures to cell densities below that known to support full encystment (from 5 x 10(5) to 1 x 10(4)cells/ml). The role of serum components (and the gal-terminated ligand asialofetuin; ASF) adsorbed onto the surface upon which encystment proceeds, and their effect on the multi-cellular aggregation patterns formed during encystment, were also investigated. The addition of free-galactose reduced the levels of encystment (compared with the control) even when added at 10h after resuspension of trophozoites in 47% LG. The requirement for the presence of ABS during encystment was lost within 6h, with levels of encystment of cells washed free of serum reaching 80% of the control. The ability of cells to encyst when diluted to a cell density below that normally thought to support encystment reached over 50% by 8h. Efficient encystment could be obtained in 47% LG in the absence of ABS or ASF using pre-treated glass culture tubes. Encystment (47% LG; 5% ABS) using ultra low attachment plates was poor, suggesting attachment of cells to a surface via gal-terminated ligands was important for efficient encystment. The results suggest that ABS is probably not the only source of gal-terminated ligands necessary for high levels of encystment in 47% LG. While serum may provide a source of ligands which enhance the levels of encystment initially, other gal-terminated ligands possibly released by the encysting cells are still required for the completion of the encystment process and the formation of mature cysts. In addition, the gal-terminated ligands necessary for encystment efficiency may be adsorbed onto the glass surface of culture tubes and aid the initial aggregation process, as well as be involved in cell signaling during the encystment process.     

Induction of Synchronized Fissions in Telotrochidium henneguyi

SYNOPSIS. Cultures of Telotrochidium henneguyi , begun with logarithmic phase cells, were employed in an effort to produce synchronized fission by heat treatment. The cells tolerated a temperature range of 20–50 C; temperatures above 50 were lethal. When cells were exposed to a single shock for 30 min, 30–40 produced 0–50% encystment with total excystment after 10 min exposure to room temperature (heat shock range). No encystment occurred between 20–30 (intershock range). Encystment and excystment time varied directly with temperature between 40–50.
The most effective procedure for inducing synchronized fission consisted of 6 cycle program of 38/28 C (shock temperature/intershock temperature) administered for 15/15 (shock/intershock duration in min). Division indices (DI = cells dividing/total population X 100 =%) ranged from 12–66% with a mean of 37.25%. In control cells, division indices ranged from 2–20% with an average of 12%. Inferences from these independently derived findings are discussed.     

Synchronized development of gametophytic germlings of the aquatic PhycomyceteAllomyces macrogynus

Summary Gametophytic germlings ofAllomyces macrogynus are examined with regard to the sequence of developmental events which have been suggested to be effected by long-lived messenger RNA. During encystment of the meiospore, the cytoplasmic triplet microtubules are depolymerized, the gamma bodies are mobilized, the nuclear cap envelope is fragmented and the cap ribosomes are dispersed, the side body complex breaks into its components, and a cyst wall is deposited. All these processes take place prior to the appearance of significant amounts of protein synthesis. The first fission of the mitochondria occurs shortly after the onset of protein synthesis but before detectable RNA synthesis. The developmental sequence of gametophytic and sporophytic germlings was found to be very similar.     

Ectosymbiotic role of food bacteria for paramecium: bacterial detoxification of paramecia-killing toxin contained in wheat grass powder

Bacterized plant infusion is a popular culture medium for Paramecium, using Klebsiella pneumoniae for the bacterium and Wheat Grass Powder (WGP) for the plant. It has been thought that WGP plays a role in the growth of bacteria, which in turn serve as the direct food for paramecia. However, we found that bacteria suspended in saline solution were unable to support the growth of paramecia. WGP including no bacteria was able to support neither the growth nor the survival of paramecia; instead, it killed paramecia. The killing effect of the WGP-derived substance(s), estimated to be of molecular weight less than 1,000, was abolished when bacteria were once grown in the WGP and then eliminated, suggesting that bacteria might change the toxic substance into an inactive form. This inactivation of the toxic substance may be caused either by metabolization inside of the bacteria or by neutralization by means of bacteria-derived substance outside of the bacteria. The second alternative is likely, because paramecia were able to survive and grow in the WGP medium containing a sufficient amount of dead bacteria killed by formalin or kanamycin. Dead bacteria killed by autoclaving were ineffective, probably because bacterial contents were lost. These findings revealed an ectosymbiotic role of bacteria; they confer benefits upon paramecia not only as food but also as machinery to detoxicate a plant toxin.     

Food web structure and the strength of transient indirect effects

The relative importance of direct and indirect effects in ecological communities remains unresolved. Indirect effects may diminish as they propagate through highly reticulate food webs. We tested this hypothesis by assembling replicate food webs of different complexity in laboratory microcosms, and comparing the transmission of indirect effects through these webs. By providing the top predator ( Didinium ) with either one ( Paramecium ) or two ( Paramecium and Colpidium ) species of protists as prey, we created linear or reticulate food webs where we could examine the transient response of predators to an indirect effect. Addition of Chlamydomonas , a small alga consumed by Paramecium , but not by Colpidium , perturbed the system and generated an indirect effect on Didinium . We expected the proportional response of Didinium to Chlamydomonas addition would be smaller in the reticulate web containing alternative, unperturbed prey ( Colpidium ). We measured predator response as predator yield, the maximum number of predators produced prior to overexploitation of prey and subsequent predator decline. The ratio of yield in perturbed bottles to yield in unperturbed bottles measures the proportional response of Didinium to Chlamydomonas addition. We expected this ratio to be smaller with Colpidium present. Contrary to expectations, alternative prey enhanced rather than diminished predator response to the perturbation. This resulted from competition between the prey species, a factor ignored in some simple verbal arguments. Food web complexity may have unanticipated consequences for the strength of indirect effects.     

Microsurgical analysis of the clonal age and the cell-cycle stage required for the onset of autogamy in Paramecium tetraurelia

When autogamy was induced in competent cells of Paramecium tetraurelia by depriving them of food, the onset of autogamy was preceded by a critical fission which occurred in the starvation medium. When the cells were fed again immediately after the fission, they did not undergo autogamy. However, they did undergo autogamy when they were fed later than 1 hr after the critical fission. The irreversible differentiation for autogamy seems to be at about 1 hr after the critical fission. This procedure thus provides the opportunity to induce autogamy synchronously. The result of macronuclear transplantation demonstrated that autogamy was under the control of macronucleus. Moreover, the clonal age required for autogamy was found to be shortened by repetitive elimination of a part of the macronucleus. The result can be explained by the hypothesis that clonal age is measured in rounds of chromosome replication or DNA synthesis rather than cell divisions.     

Nucleolar apparatus in the macronucleus of Didinium nasutum (Ciliata): EM and 3D reconstruction

The three-dimensional (3D) organization of nucleoli in the somatic nuclei (macronuclei) of recently fed and starved Didinium nasutum was reconstructed on the basis of serial ultra-thin sections. It was shown that nucleoli, looking on the single sections like individual separate structures, appeared to be parts of the large complicated branchy nucleolar networks. A 30 h starvation did not lead to disintegration of this network, but stimulated formation of numerous vacuoles in the granular component of nucleoli, which becomes more condensed. Unlike starved D. nasutum, in fed ciliates numerous holes appeared in the fibrillar component located at the periphery of nucleoli. These holes may presumably serve as channels for transporting newly synthesized rRNA. To our knowledge, this is the first report of a 3D reconstruction of the nucleolar apparatus in ciliates.     

Adenine inhibits microcyst formation inPhysarum flavicomum and affects the cellular level of S-adenosylmethionine

Summary The effect of various purines, pyrimidines and nucleosides on the encystment of haploid cells ofPhysarum flavicomum was determined. Of the compounds tested guanine, guanosine, cytidine, cytosine, 5-methylcytosine and uracil had no effect on encystment. Adenosine, thymine, uridine and 3-methyladenine only slightly delayed encystment and protein degradation. Adenine and, to a lesser extent, hypoxanthine produced a significant inhibition of encystment and greatly increased rates of autolytic protein and RNA degradation, which eventually led to about 75% cell death in the adenine-exposed cells. The inhibition of microcyst formation by adenine was concentration dependent. The incubation of cells with adenine resulted initially in elevated intracellular levels of S-adenosylmethionine up to 3.5 times the level of untreated control cells.     

Phagosomal acidification in Paramecium: effects on lysosomal fusion

Phagosomes of paramecia and amoeba and endosomes of fibroblasts and other mammalian cells are acidified prior to lysosomal fusion. The question, whether the phagosomal acidification process in paramecia is required for phagosome-lysosome fusion, was studied using ionophores, weak bases, and cytochalasin B (CB) in combination with monoclonal antibodies, acid phosphatase (AcPase) cytochemistry, and lysosome morphometry. Digestive vacuoles (DVs) of known ages were treated and examined. In untreated cells, lysosome binding to the membrane of the acidified DV increased linearly with age and reached a maximum before lysosome-DV fusion. When the fusion of the acidosomes with the very young DVs was prevented by CB, causing a block in the normal vacuole-pH drop, lysosome binding to the DVs as well as the rate and extent of lysosome-DV fusion were all greatly reduced. These effects of CB were reversible. When present prior to acidification, three ionophores and two weak bases did not inhibit the acidosome-DV fusion but raised the phagosomal pH and reduced both the rates of DV acidification and of lysosome-DV fusion. However, when added after acidification but prior to lysosome-DV fusion, five of the six perturbants studied did not inhibit this fusion but prolonged the period when DVs remained AcPase positive. Lastly, lysosome-DV fusion rates were found to be related to acidification rates. We conclude that an inhibition in acidosome-DV fusion or a reduction in both the acidification rate and vacuolar-pH drop would inhibit lysosome-DV fusion.     

Toxic effects of antimalarial drugs in Paramecium : role of calcium channels

The antimalarial drugs, quinacrine, quinine and mefloquine, as well as the structurally-similar compound, W-7, inhibit calcium-dependent backward swimming and calcium currents in Paramecium calkinsi. These drugs are also toxic to paramecia at high concentrations. Therefore, one site of toxic action of the drugs may be the calcium channel. To test this hypothesis, the toxicity of the antimalarials and W-7 was compared in paramecia with and without calcium channels. Since calcium channels are located on the cilia, calcium channels were removed from the paramecia by deciliating the cells. Deciliated cells were found to be less susceptible to the lethal effects of the antimalarials and W-7 than their ciliated counterparts. Moreover, Pawns, mutants of P. tetraurelia that possess cilia but lack functional calcium channels, were also less susceptible to the antimalarials than wild-type cells. Thus, calcium channels may be one site of toxic action of the antimalarial drugs in paramecia and perhaps in other protists. Accepted: 27 December 1996     

Rapid Growth of Acanthamoeba In Defined Media; Induction of Encystment By Glucose-Acetate Starvation

Defined media are described that support 14-20 h generation times for Acanthamoeba castellanii and A. rhysodes in monolayer cultures. the media differ in minor ways from previously described media, but the growth rates are greatly improved over previously reported values. Maximum growth rates were observed for A, castellanii in a complex medium containing 21 amino acids, but near-maximum rates could be achieved in relatively simple media containing 9 amino acids. Growth occurred with 6 amino acids, as reported by others, but generation times exceeded 30 h. Amitosis was a common problem during early subcultures in defined media, but became infrequent after repeated transfers. Synchronous encystment resulting in 70-80% cyst formation could be induced in the defined media by glucose and acetate starvation. the rate of encystment varied with cell density at the time of starvation and was optimal at initial densities of 400-800 amebae/mm2.     

Initiation of Encystment in the Mycetozoan Protostelium mycophaga

SYNOPSIS. Protostelium was cultured monoxenically with the yeast Rhodotorula mucilaginosa on either corn meal agar or in liquid corn meal medium. Nearly synchronous encystment was induced in amoebae by washing them free of yeast and incubating them in an encystment medium devised by Neff et al. (1964) for Acanthamoeba.
The extrinsic requirements for encystment were investigated by replacing various components of the encystment medium with other substances. The results indicate that encystment depends on a high concentration of inorganic monovalent cations and that it occurs best at a basic pH. The effect of the ions does not appear to be strictly osmotic.