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.     

R-body-producing bacteria.

Until 10 years ago, R bodies were known only as diagnostic features by which endosymbionts of paramecia were identified as kappa particles. They were thought to be limited to the cytoplasm of two species in the Paramecium aurelia species complex. Now, R bodies have been found in free-living bacteria and other Paramecium species. The organisms now known to form R bodies include the cytoplasmic kappa endosymbionts of P. biaurelia and P. tetraurelia, the macronuclear kappa endosymbionts of P. caudatum, Pseudomonas avenae (a free-living plant pathogen), Pseudomonas taeniospiralis (a hydrogen-oxidizing soil microorganism), Rhodospirillum centenum (a photosynthetic bacterium), and a soil bacterium, EPS-5028, which is probably a pseudomonad. R bodies themselves fall into five distinct groups, distinguished by size, the morphology of the R-body ribbons, and the unrolling behavior of wound R bodies. In recent years, the inherent difficulties in studying the organization and assembly of R bodies by the obligate endosymbiont kappa, have been alleviated by cloning and expressing genetic determinants for these R bodies (type 51) in Escherichia coli. Type 51 R-body synthesis requires three low-molecular-mass polypeptides. One of these is modified posttranslationally, giving rise to 12 polypeptide species, which are the major structural subunits of the R body. R bodies are encoded in kappa species by extrachromosomal elements. Type 51 R bodies, produced in Caedibacter taeniospiralis, are encoded by a plasmid, whereas bacteriophage genomes probably control R-body synthesis in other kappa species. However, there is no evidence that either bacteriophages or plasmids are present in P. avenae or P. taeniospiralis. No sequence homology was detected between type 51 R-body-encoding DNA and DNA from any R-body-producing species, except C. varicaedens 1038. The evolutionary relatedness of different types of R bodies remains unknown.     

R-body-producing bacteria.

Until 10 years ago, R bodies were known only as diagnostic features by which endosymbionts of paramecia were identified as kappa particles. They were thought to be limited to the cytoplasm of two species in the Paramecium aurelia species complex. Now, R bodies have been found in free-living bacteria and other Paramecium species. The organisms now known to form R bodies include the cytoplasmic kappa endosymbionts of P. biaurelia and P. tetraurelia, the macronuclear kappa endosymbionts of P. caudatum, Pseudomonas avenae (a free-living plant pathogen), Pseudomonas taeniospiralis (a hydrogen-oxidizing soil microorganism), Rhodospirillum centenum (a photosynthetic bacterium), and a soil bacterium, EPS-5028, which is probably a pseudomonad. R bodies themselves fall into five distinct groups, distinguished by size, the morphology of the R-body ribbons, and the unrolling behavior of wound R bodies. In recent years, the inherent difficulties in studying the organization and assembly of R bodies by the obligate endosymbiont kappa, have been alleviated by cloning and expressing genetic determinants for these R bodies (type 51) in Escherichia coli. Type 51 R-body synthesis requires three low-molecular-mass polypeptides. One of these is modified posttranslationally, giving rise to 12 polypeptide species, which are the major structural subunits of the R body. R bodies are encoded in kappa species by extrachromosomal elements. Type 51 R bodies, produced in Caedibacter taeniospiralis, are encoded by a plasmid, whereas bacteriophage genomes probably control R-body synthesis in other kappa species. However, there is no evidence that either bacteriophages or plasmids are present in P. avenae or P. taeniospiralis. No sequence homology was detected between type 51 R-body-encoding DNA and DNA from any R-body-producing species, except C. varicaedens 1038. The evolutionary relatedness of different types of R bodies remains unknown.     

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.     

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.     

Micronucleus‐Specific Bacterium Holospora elegans Irreversibly Enhances Stress Gene Expression of the Host Paramecium caudatum

ABSTRACT. The bacterium Holospora is an endonuclear symbiont of the ciliate Paramecium. Previously, we reported that paramecia bearing the macronuclear‐specific symbiont Holospora obtusa survived better than symbiont‐free paramecia, even under high temperatures unsuitable for growth. The paramecia with symbionts expressed high levels of hsp70 mRNAs even at 25 °C, a usual growth temperature. We report herein that paramecia bearing the micronuclear‐specific symbiont Holospora elegans also acquire the heat‐shock resistance. Even after the removal of the bacteria from the hosts by treatment with penicillin, the resulting aposymbiotic paramecia nevertheless maintained their heat shock‐resistant nature for over 1 yr. Like symbiotic paramecia, these aposymbiotic paramecia also expressed high levels of both hsp60 and hsp70 mRNAs even at 25 °C. Moreover, analysis by fluorescent in situ hybridization with a probe specific for Holospora 16S rRNA revealed that the 16S rRNA of H. elegans was expressed around the nucleoli of the macronucleus in the aposymbiotic cells. This result suggests the possible transfer of Holospora genomic DNA from the micronucleus into the macronucleus in symbiotic paramecia. Perhaps this exogenous DNA could trigger the aposymbiotic paramecia to induce a stress response, inducing higher expression of Hsp60 and Hsp70, and thus conferring heat‐shock resistance.     

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.     

The utilization of paramecia by the carnivorous plant Utricularia gibba

Summary A method has been devised to ensure capture of large numbers of live paramecia within a short period of time under controlled conditions by the bladders of Utricularia gibba. The method permits a direct evaluation of the role of entrapped animals in the nutrition of this carnivorous plant. Paramecia captured by the bladders of plants growing in a near optimal inorganic medium do not cause an increase in number or length of internodes. In contrast, feeding paramecia to plants grown in a poorly balanced or incomplete medium does result in an increase in both number and length of internodes produced. Feeding paramecia to Utricularia also results in an increase in number of bladders.This study was supported in part by an Undergraduate Research Participant stipend from Public Health Service, grant number 2-TIHE 5 303-09, to the first author and in part by Agricultural Research Service, U.S. Department of Agriculture, Grant No. 12-14-100-7981 (34) to the second author, administered by Crops Research Division, Beltsville, Maryland.     

Interspecies transfer of mitochondria in Paramecium aurelia.

Summary Erythromycin-resistant mitochondria from species 1, 5 and 7 of P. aurelia were injected into erythromycin-sensitive paramecia of each of the same three species. Mitochondria from species 1 and 5 were successfully transferred to all three species, but species 7 mitochondria failed to develop in species 1 and 5. Minor differences were indicated in the frequency of successful transfers of species 1 mitochondria into species 1 and 5 cells. From studies on the transferability of mitochondria from hybrid cells, containing mitochondria from one species and nuclei from another, it was concluded that mitochondrial compatibility was mainly under control of the nuclear genome, with a possible minor control also by the mitochondrial genome.Dedicated to T.M. Sonneborn on the occasion of his seventieth birthday. This paper is part of a Sonneborn Festchrift most of which will appear in Genetical Research in 1976.     

Computer simulation of Paramecium chemokinesis behavior

A computer program was designed to simulate the distribution of paramecia in a T-maze assay for chemo-accumulation and dispersal. Simulated values of chemokinesis are compared to experimental values for normal and mutant paramecia. The roles of components of swimming behavior (turning frequency and swimming speed), adaptation, and reaction at the border of solutions are examined.     

The Relationship of the Nutritive State of the Prey Organism Paramecium aurelia to the Growth and Encystment of Didinium nasutum

SYNOPSIS. Only well fed Paramecium aurelia , grown either monoflorally or on a mixture of 2 species of bacteria, are adequate to maintain optimal fission rates and encystment rates for Didinium nasutum. Progressive starvation of paramecia prior to their being fed to didinia leads to decreased fission rates, the appearance of abnormal cells and a loss of ability to encyst by the didinia. This depression can be fully overcome by allowing the didinia to feed again upon well nourished paramecia. A minimum of 45 well-fed paramecia is required daily for each Didinium if maximal fission rates are to be maintained. Encystment and fission appear to be mutually exclusive processes, but encystment rates are related to fission rates and seem to be exclusive of the density of the didinial culture.     

Green paramecia as an evolutionary winner of oxidative symbiosis: a hypothesis and supportive data

A single cell of the green paramecia (Paramecium bursaria) harbors several hundreds of endo-symbiotic Chlorella-like algae in its cytoplasm. Removal of algae from the host organism and re-association of ex-symbiotic host paramecia with ex-symbiotic algae can be experimentally demonstrated in the laboratory. However, the mechanism precisely governing the alga-protozoan association is not fully understood, and the origin of symbiosis in the evolutionary view has not been given. Here, we propose the possible biochemical models (models 1 and 2) explaining the co-evolution between Paramecium species and algal symbionts by pointing out that algal photosynthesis in the host paramecia plays a dual role providing the energy source and the risk of oxidative damage to the host. Model 1 lays stress on the correlation between the (re)greening ability of the paramecia and the tolerance to oxidative stress whereas model 2 emphasizes the cause of evolutionary selection leading to the emergence of Paramecium species tolerant against reactive oxygen species.     

Effect of hydrophobic mismatch on phase behavior of lipid membranes

We investigate the competing effects of hydrophobic mismatch and chain stretching on the morphology and evolution of domains in lipid membranes via Monte Carlo techniques. We model the membrane as a binary mixture of particles that differ in their preferred lengths, with the shorter particles mimicking unsaturated nonraft lipids and the longer particles mimicking saturated raft lipids. We find that phase separation can be induced upon increasing either the ratio J/kappa of the hydrophobic surface tension J to the compressibility modulus kappa. J/kappa determines the decay length for thickness changes. When this decay length is larger than the system size the membrane remains mixed. Furthermore, increasing the thickness relaxation time can induce transient phase separation.     

Responses of Paramecium to Temperature Change

SYNOPSIS. The effect of temperature on the swimming velocity of Paramecium was investigated. When paramecia cultured at 25 C were transferred to various temperatures, their swimming velocity was increased immediately and then decreased exponentially with time to a new steady velocity. The relaxation time was about 1 min, independent of the new temperature. At a constant temperature the steady velocity was inversely proportional to viscosity. The velocity acceleration was observed when the sudden temperature change was larger than ± 1 C. Its magnitude became constant when the temperature change was greater than several degrees. The steady velocity as a function of temperature had a sharp maximum at the culture temperature and decreased on both sides of this temperature. Incubation of paramecia at 30 C for several hr after cultivation at 25 C shifted the maximum temperature of the steady velocity to 30 C. The temperature at which paramecia gathered in a temperature gradient cell correlated closely with the temperature of the maximum steady velocity.     

A computer method for the evaluation of Paramecium motor activity using video records of their movement

A method for the evaluation of Paramecium caudatum motility was proposed as a tool for the investigation of magnetobiological as well as other physical and chemical effects. The microscopically observed movement of paramecia is recorded and processed using a special software program. The protozoan motility is determined as a function of their mean velocity in a definite time. The main advantages of the method are that it is easily modified for determining various characteristics of the motor activity of paramecia and that the video data obtained can be reused.     

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     

Isolation and composition of bacteriophage-like particles from kappa of killer Paramecia

Summary Phage-like particles from kappa of stock 562 of Paramecium aurelia have been isolated by CsCl density gradient centrifugation. Analyses show that the particles contain about 1.6×10¹⁶g DNA and 2.0×10^-16g protein. Their buoyant density is approximately 1.47. DNA from the particles has a buoyant density very close to that of whole kappa DNA. The presence of DNA in the particles has been confirmed by a cytochemical technique. The results support the conclusion that kappa contains a bacteriophage.     

Flow cytometry as a strategy to study the endosymbiosis of algae in Paramecium bursaria

BACKGROUND: The stable symbiotic association between Paramecium bursaria and algae is of interest to study such mechanisms in biology as recognition, specificity, infection, and regulation. The combination of algae-free strains of P. bursaria, which have been recently established by treating their stocks of green paramecia with herbicide paraquat (Hosoya et al.: Zool Sci 12: 807-810, 1995), with the cloned symbiotic algae isolated from P. bursaria (Nishihara et al.: Protoplasma 203: 91-99, 1998), provides an excellent clue to gain fundamental understanding of these phenomena. METHODS: Flow cytometry and light microscopy have been employed to characterize the algal cells after they have been released from the paramecia by ultrasonic treatment. Algal optical properties such as light scattering and endogenous chlorophyll fluorescence intensity have been monitored for symbiotic and free-living strains, and strains at stages of interaction with a host. RESULTS: Neither algal morphology nor chlorophyll content has been found to be altered by sonication of green paramecia. This fact allows to interpret in adequate degree changes in the optical properties of symbiont that just has been released from the association with a host (decreased forward light scatter and chlorophyll fluorescence signals). Optical characterization of both symbiotic and free-living algal strains with respect to their ability to establish symbioses with P. bursaria showed that chlorophyll content per cell volume seems to be a valuable factor for predicting a favorable symbiotic relationship between P. bursaria and algae. CONCLUSIONS: Flow cytometry combined with algae-free paramecia and cloned symbiotic algae identifies algal populations that may be recognized by host cells for the establishment of symbioses.     

Interfacial and lipid transfer properties of human phospholipid transfer protein: implications for the transfer mechanism of phospholipids

In circulation the phospholipid transfer protein (PLTP) facilitates the transfer of phospholipid-rich surface components from postlipolytic chylomicrons and very low density lipoproteins (VLDL) to HDL and thereby regulates plasma HDL levels. To study the molecular mechanisms involved in PLTP-mediated lipid transfer, we studied the interfacial properties of PLTP using Langmuir phospholipid monolayers and asymmetrical flow field-flow fractionation (AsFlFFF) to follow the transfer of 14C-labeled phospholipids and [35S]PLTP between lipid vesicles and HDL particles. The AsFlFFF method was also used to determine the sizes of spherical and discoidal HDL particles and small unilamellar lipid vesicles. In Langmuir monolayer studies high-activity (HA) and low-activity (LA) forms of PLTP associated with fluid phosphatidylcholine monolayers spread at the air/buffer interphase. Both forms also mediated desorption of [14C]dipalmitoylphosphatidylcholine (DPPC) from the phospholipid monolayer into the buffer phase, even when it contained no physiological acceptor such as HDL. After the addition of HDL3 to the buffer, HA-PLTP caused enhanced lipid transfer to them. The particle diameter of HA-PLTP was approximately 6 nm and that of HDL3 approximately 8 nm as determined by AsFlFFF analysis. Using this method, it could be demonstrated that in the presence of HA-PLTP, but not LA-PLTP, [14C]DPPC was transferred from small unilamellar vesicles (SUV) to acceptor HDL3 molecules. Concomitantly, [35S]-HA-PLTP was transferred from the donor to acceptor, and this transfer was not observed for its low-activity counterpart. These observations suggest that HA-PLTP is capable of transferring lipids by a shuttle mechanism and that formation of a ternary complex between PLTP, acceptor, and donor particles is not necessary for phospholipid transfer.     

Role of Non-Surface Antigens in Controlling Paramecium Surface Antigen Synthesis*

SYNOPSIS. Paramecium aurelia exposed to antisera prepared against cells of a different surface antigenic type are often induced to transform to a new serotype. One possible explanation is that paramecia that are so affected have antigens related to the ciliary antigens, but not accessible to immobilizing antibodies. It is these secondary antigens that are bound by the antibodies, thereby forcing the cells to alter their pattern of antigen synthesis. Examination of affected paramecia has disclosed that secondary antigens are often present but the specificity of these antigens cannot account for the activity of the antisera. It is therefore proposed that antibodies directed against substances other than the immobilization antigens may induce transformation. Two kinds of antiserum, neither of which contains immobilizing antibodies of any sort, are able to markedly alter the expression of the serotypes. One was obtained by immunizing rabbits with culture fluid in which paramecia had been growing. The 2nd was made by injecting rabbits with normal sera from other rabbits.