Host Paramecium Research Articles

Comparative Analyses of the Symbiotic Associations of the Host Paramecium bursaria with Free-Living and Native Symbiotic Species of Chlorella.

Each symbiotic Chlorella variabilis associated with the ciliate Paramecium bursaria is enclosed in a symbiosome called the perialgal vacuole. Various potential symbionts, such as bacteria, yeasts, other algae, and free-living Chlorella spp., can infect P. bursaria. However, the detailed infection process of each of them in algae-free P. bursaria is unknown. Here, we aimed to elucidate the difference of the infection process between the free-living C. sorokiniana strain NIES-2169 and native symbiotic C. variabilis strain 1N. We investigated the fate of ingested algae using algae-free P. bursaria exposed separately to three types of algal inocula: NIES-2169 only, 1N only, or a mixture of NIES-2169 and 1N. We found that (1) only one algal species, preferably the native one, was retained in host cells, indicating a type of host compatibility and (2) the algal localization style beneath the host cell cortex varied between different Chlorella spp. showing various levels of host compatibilities, which was prospectively attributable to the difference in the formation of the perialgal vacuole membrane.

A 63-kDa Periplasmic Protein of the Endonuclear Symbiotic Bacterium Holospora obtusa Secreted to the Outside of the Bacterium during the Early Infection Process Binds Weakly to the Macronuclear DNA of the Host Paramecium caudatum.

The Gram-negative bacterium Holospora obtusa is a macronucleus-specific symbiont of the ciliate Paramecium caudatum. It is known that an infection of this bacterium induces high level expressions of the host hsp60 and hsp70 genes, and the host cell acquires both heat-shock and high salt resistances. In addition, an infectious form of H. obtusa-specific 63-kDa periplasmic protein with a DNA-binding domain in its amino acid sequence is secreted into the host macronucleus after invasion into the macronucleus and remain within the nucleus. These facts suggest that binding of the 63-kDa protein to the host macronuclear DNA causes changes in the host gene expressions and enhances an environmental adaptability of the host cells. This 63-kDa protein was renamed as periplasmic region protein 1 (PRP1) to distinguish it from other proteins with similar molecular weights. To confirm whether PRP1 indeed binds to the host DNA, SDS-DNA PAGE and DNA affinity chromatography with calf thymus DNA and P. caudatum DNA were conducted and confirmed that PRP1 binds weakly to the P. caudatum DNA with a monoclonal antibody raised for the 63-kDa protein.

Autolysis of Chlorella variabilis in Starving Paramecium bursaria Help the Host Cell Survive Against Starvation Stress.

The endosymbiosis between Paramecium bursaria and Chlorella spp. is mutualistic. Symbiotic algae localize beneath the host Paramecium cell cortex compete for their attachment sites with preexisting organelle trichocysts. To examine the relationship between P. bursaria trichocysts and their symbiotic algae, algae-bearing or alga-free P. bursaria were starved for several days and the changes in the number of Chlorella sp. and presence or absence of trichocysts were evaluated. We conducted an indirect immunofluorescence microscopy with an anti-trichocyst monoclonal antibody against P. bursaria cells. Indirect immunofluorescence microscopy demonstrated that under starvation and darkness conditions, the immunofluorescence of trichocysts in alga-free P. bursaria decreased much faster than that in the normal algae-bearing P. bursaria. In the latter case, our observations proposed the possibility that the nutrition obtained from symbiotic algal digestion may promote trichocysts synthesis. This algal digestion mechanism may permit host P. bursaria cells to survive for a longer time under starvation condition. To the best of our knowledge, this may be a new benefit that host P. bursaria gain from harboring symbiotic algae.

Among-Strain Variation in Resistance of Paramecium caudatum to the Endonuclear Parasite Holospora undulata: Geographic and Lineage-Specific Patterns.

Resistance is a key determinant in interactions between hosts and their parasites. Understanding the amount and distribution of variation in this trait between strains can provide insights into (co)evolutionary processes and their potential to shape patterns of diversity in natural populations. Using controlled inoculation in experimental mass cultures, we investigated the quantitative variation in resistance to the bacterial parasite Holospora undulata across a worldwide collection of strains of its ciliate host Paramecium caudatum. We combined the observed variation with available information on the phylogeny and biogeography of the strains. We found substantial variation in resistance among strains, with upper-bound values of broad-sense heritability >0.5 (intraclass correlation coefficients). Strain estimates of resistance were repeatable between laboratories and ranged from total resistance to near-complete susceptibility. Early (1 week post inoculation) measurements provided higher estimates of resistance heritability than did later measurements (2–3 weeks), possibly due to diverging epidemiological dynamics in replicate cultures of the same strains. Genetic distance (based on a neutral marker) was positively correlated with the difference in resistance phenotype between strains (r = 0.45), essentially reflecting differences between highly divergent clades (haplogroups) within the host species. Haplogroup A strains, mostly European, were less resistant to the parasite (49% infection prevalence) than non-European haplogroup B strains (28%). At a smaller geographical scale (within Europe), strains that are geographically closer to the parasite origin (Southern Germany) were more susceptible to infection than those from further away. These patterns are consistent with a picture of local parasite adaptation. Our study demonstrates ample natural variation in resistance on which selection can act and hints at symbiont adaptation producing signatures in geographic and lineage-specific patterns of resistance in this model system.

Photosynthetic Endosymbionts Benefit from Host's Phagotrophy, Including Predation on Potential Competitors.

In many endosymbioses, hosts have been shown to benefit from symbiosis, but it remains unclear whether intracellular endosymbionts benefit from their association with hosts [1, 2]. This makes it difficult to determine evolutionary mechanisms underlying cooperative behaviors between hosts and intracellular endosymbionts, such as mutual exchange of vital resources. Here, we investigate the fitness effects of symbiosis on the ciliate hostParamecium bursaria and on the algal endosymbiont Chlorella [3, 4], using experimental microcosms that include the free-living alga Chlamydomonas reinhardtii to mimic ecologically realistic conditions. We demonstrate that both host ciliate and the endosymbiotic algae gain fitness benefits from the symbiosis when another alga C.reinhardtii is present in the system. Specifically, the endosymbiotic Chlorella can grow as the host ciliate feeds and grows on C.reinhardtii, whereas the growth of free-living Chlorella is reduced by its competitor, C.reinhardtii. Thus, we propose that the endosymbiotic algae benefit from the host's phagotrophy, which allows the endosymbiont to access particulate nutrient sources and to indirectly prey on the potential competitors competing with its free-living counterparts. Even though the ecological contexts in which each partner receives its benefits differ, both partners would gain net fitness benefits in an ecological timescale. Thus, the cooperative behaviors can evolve through fitness feedback (partner fidelity feedback) between the host and the endosymbiont, without need for special partner control mechanisms. The proposed ecological and evolutionary mechanisms provide a basis for understanding cooperative resource exchanges in endosymbioses, including many photosynthetic endosymbioses widespread in aquatic ecosystems.

A putative TetR-type transcription factor AZC_3265 from the legume symbiont Azorhizobium caulinodans represses the production of R-bodies that are toxic to eukaryotic cells

ABSTRACTAzorhizobium caulinodans ORS571 is a microsymbiont of the legume Sesbania rostrata, which forms nitrogen-fixing nodules on stems and roots. This bacterium harbors a reb operon, which is associated with R-body production. R-bodies are large proteinaceous ribbons and were first observed in Caedibacter species, which are obligate bacterial endosymbionts in paramecia. R-body-producing Caedibacter species released from their host paramecia are toxic to the symbiont-free paramecia. R-body-producing cells of A. caulinodans mutants are also toxic to the plant host cells. To maintain harmonic symbiosis with S. rostrata, A. caulinodans has to repress the expression of the reb operon. To date, it has been revealed that the PraR transcription factor and Lon protease repress reb operon expression, in direct and indirect manners, respectively. In this study, we carried out transposon-based mutagenesis screening, and found that the AZC_3265 (locus tag on the genome) gene encoding a putative TetR-type transcription factor was involved in the repression of reb operon expression. The AZC_3265 gene deletion mutant showed high levels of reb operon expression and R-body formation, and this strain formed stem nodules defective in nitrogen-fixing activity. Systematic evolution of ligands by exponential enrichment (SELEX) experiment revealed that AZC_3265 protein could bind to the consensus palindromic sequence TTGC-N6-GCAA. However, this consensus sequence was not found in the reb operon promoter region. Additionally, an electrophoretic mobility shift assay (EMSA) also revealed that AZC_3265 could not bind to the reb operon promoter region. These results suggested that AZC_3265 repressed the expression of the reb operon in an indirect manner. In conclusion, the present data demonstrated that multiple regulators participate in the regulation of expression of the reb operon. The presence of multiple mechanisms for regulating the expression of the reb operon suggested that its expression was controlled in response to multiple biological and environmental factors.

Stringent Expression Control of Pathogenic R-body Production in Legume Symbiont Azorhizobium caulinodans.

ABSTRACTR bodies are insoluble large polymers consisting of small proteins encoded by reb genes and are coiled into cylindrical structures in bacterial cells. They were first discovered in Caedibacter species, which are obligate endosymbionts of paramecia. Caedibacter confers a killer trait on the host paramecia. R-body-producing symbionts are released from their host paramecia and kill symbiont-free paramecia after ingestion. The roles of R bodies have not been explained in bacteria other than Caedibacter. Azorhizobium caulinodans ORS571, a microsymbiont of the legume Sesbania rostrata, carries a reb operon containing four reb genes that are regulated by the repressor PraR. Herein, deletion of the praR gene resulted in R-body formation and death of host plant cells. The rebR gene in the reb operon encodes an activator. Three PraR binding sites and a RebR binding site are present in the promoter region of the reb operon. Expression analyses using strains with mutations within the PraR binding site and/or the RebR binding site revealed that PraR and RebR directly control the expression of the reb operon and that PraR dominantly represses reb expression. Furthermore, we found that the reb operon is highly expressed at low temperatures and that 2-oxoglutarate induces the expression of the reb operon by inhibiting PraR binding to the reb promoter. We conclude that R bodies are toxic not only in paramecium symbiosis but also in relationships between other bacteria and eukaryotic cells and that R-body formation is controlled by environmental factors.

Intracellular symbiosis of algae with possible involvement of mitochondrial dynamics

Algal endosymbiosis is widely present among eukaryotes including many protists and metazoans. However, the mechanisms involved in their interactions between host and symbiont remain unclear. Here, we used electron microscopy and three-dimensional reconstruction analyses to examine the ultrastructural interactions between the symbiotic zoochlorella and the organelles in the host Paramecium bursaria, which is a model system of endosymbiosis. Although in chemically fixed samples the symbiotic algae show no direct structural interactions with the host organelles and the perialgal vacuole membrane (PVM), in cryofixed P. bursaria samples the intimate connections were identified between the host mitochondria and the symbiotic algae via the PVM. The PVM was closely apposed to the cell wall of the symbiotic algae and in some places it showed direct contacts to the host mitochondrial membrane and the cell wall of the symbiotic algae. Further, the PVM-associated mitochondria formed a mitochondrial network and were also connected to host ER. Our observations propose a new endosymbiotic systems between the host eukaryotes and the symbionts where the benefiting symbiosis is performed through intimate interactions and an active structural modification in the host organelles.

Fitness Impact of Obligate Intranuclear Bacterial Symbionts Depends on Host Growth Phase.

According to text book definition, parasites reduce the fitness of their hosts whereas mutualists provide benefits. But biotic and abiotic factors influence symbiotic interactions, thus under certain circumstances parasites can provide benefits and mutualists can harm their host. Here we addressed the question which intrinsic biotic factors shape a symbiosis and are crucial for the outcome of the interaction between the obligate intranuclear bacterium Holospora caryophila (Alphaproteobacteria; Rickettsiales) and its unicellular eukaryotic host Paramecium biaurelia (Alveolata; Ciliophora). The virulence of H. caryophila, i.e., the negative fitness effect on host division and cell number, was determined by growth assays of several P. biaurelia strains. The performances of genetically identical lines either infected with H. caryophila or symbiont-free were compared. Following factors were considered as potentially influencing the outcome of the interaction: (1) host strain, (2) parasite strain, and (3) growth phases of the host. All three factors revealed a strong effect on the symbiosis. In presence of H. caryophila, the Paramecium density in the stationary growth phase decreased. Conversely, a positive effect of the bacteria during the exponential phase was observed for several host × parasite combinations resulting in an increased growth rate of infected P. biaurelia. Furthermore, the fitness impact of the tested endosymbionts on different P. biaurelia lines was not only dependent on one of the two involved strains but distinct for the specific combination. Depending on the current host growth phase, the presence of H. caryophila can be harmful or advantageous for P. biaurelia. Thus, under the tested experimental conditions, the symbionts can switch from the provision of benefits to the exploitation of host resources within the same host population and a time-span of less than 6 days.

Simultaneous Evaluation of Life Cycle Dynamics between a Host Paramecium and the Endosymbionts of Paramecium bursaria Using Capillary Flow Cytometry.

Endosymbioses are driving forces underlying cell evolution. The endosymbiosis exhibited by Paramecium bursaria is an excellent model with which to study symbiosis. A single-cell microscopic analysis of P. bursaria reveals that endosymbiont numbers double when the host is in the division phase. Consequently, endosymbionts must arrange their cell cycle schedule if the culture-condition-dependent change delays the generation time of P. bursaria. However, it remains poorly understood whether endosymbionts keep pace with the culture-condition-dependent behaviors of P. bursaria, or not. Using microscopy and flow cytometry, this study investigated the life cycle behaviors occurring between endosymbionts and the host. To establish a connection between the host cell cycle and endosymbionts comprehensively, multivariate analysis was applied. The multivariate analysis revealed important information related to regulation between the host and endosymbionts. Results show that dividing endosymbionts underwent transition smoothly from the division phase to interphase, when the host was in the logarithmic phase. In contrast, endosymbiont division stagnated when the host was in the stationary phase. This paper explains that endosymbionts fine-tune their cell cycle pace with their host and that a synchronous life cycle between the endosymbionts and the host is guaranteed in the symbiosis of P. bursaria.

Symbiotic Chlorella variabilis strain, 1 N, can influence the digestive process in the host Paramecium bursaria during early infection

The association between the ciliate Paramecium bursaria and symbiotic Chlorella spp. is mutually beneficial. However, this relationship is facultative mutualism because both the host and the symbiotic algae can grow by themselves. This association is easily re-established by mixing the two species together. Following algal mixing, some algae become enclosed in the digestive vacuole membrane of the paramecia to which both acidosomes and lysosomes fuse. To establish endosymbiosis, some algae acquire temporal resistance to the host lysosomal enzymes in the digestive vacuoles. We examined whether the algae influence the differentiation of the host digestive process using LysoSensor staining to evaluate the acidification of the digestive vacuoles. Furthermore, to assess lysosomal fusion with the digestive vacuole, Gomori’s staining was conducted. Acidification and lysosomal fusion occurred later in digestive vacuoles containing living algae than in those containing boiled algae or latex spheres. This phenomenon was observed when the living algae were maintained under a constant light condition. These results suggest that the algae release some unknown factor in response to light exposure, and the factor may be associated with the alteration of the host digestive process, indicating that the living algae can influence the host digestive processes during early algal infection.

Shining a Light on Exploitative Host Control in a Photosynthetic Endosymbiosis

Endosymbiosis allows hosts to acquire new functional traits such that the combined host and endosymbiont can exploit vacant ecological niches and occupy novel environments [1, 2]; consequently, endosymbiosis affects the structure and function of ecosystems [3, 4]. However, for many endosymbioses, it is unknown whether their evolutionary basis is mutualism or exploitation [5-9]. We estimated the fitness consequences of symbiosis using the interaction between the protist host Paramecium bursaria and the algal symbiont Chlorella sp. [10]. Host fitness was strongly context dependent: whereas hosts benefited from symbiosis at high light intensity, carrying endosymbionts was costly to hosts in the dark and conferred no benefit over growing autonomously at intermediate light levels. Autonomous Chlorella densities increased monotonically with light intensity, whereas per-host symbiont load and symbiont abundance peaked at intermediate light levels and were lowest at high light intensity. This suggests that hosts controlled the costs of symbiosis by manipulating symbiont load according to light intensity. Photosynthetic efficiency was consistently lower for symbiotic compared to autonomous algae, suggesting nutritional constraints upon algae in symbiosis. At intermediate light levels, we observed the establishment of small populations of free-living algae alongside the hosts with endosymbionts, suggesting that symbionts could escape symbiosis, but only under conditions where hosts didn't benefit from symbiosis. Together, these data suggest that hosts exerted strong control over endosymbionts and that there were no conditions where this nutritional symbiosis was mutually beneficial. Our findings support theoretical predictions (e.g., [5, 9]) that controlled exploitation is an important evolutionary pathway toward stable endosymbiosis.

Synchronous Induction of Detachment and Reattachment of Symbiotic Chlorella spp. from the Cell Cortex of the Host Paramecium bursaria

Paramecium bursaria harbor several hundred symbiotic Chlorella spp. Each alga is enclosed in a perialgal vacuole membrane, which can attach to the host cell cortex. How the perialgal vacuole attaches beneath the host cell cortex remains unknown. High-speed centrifugation (> 1000×g) for 1min induces rapid detachment of the algae from the host cell cortex and concentrates the algae to the posterior half of the host cell. Simultaneously, most of the host acidosomes and lysosomes accumulate in the anterior half of the host cell. Both the detached algae and the dislocated acidic vesicles recover their original positions by host cyclosis within 10min after centrifugation. These recoveries were inhibited if the host cytoplasmic streaming was arrested by nocodazole. Endosymbiotic algae during the early reinfection process also show the capability of desorption after centrifugation. These results demonstrate that adhesion of the perialgal vacuole beneath the host cell cortex is repeatedly inducible, and that host cytoplasmic streaming facilitates recovery of the algal attachment. This study is the first report to illuminate the mechanism of the induction to desorb for symbiotic algae and acidic vesicles, and will contribute to the understanding of the mechanism of algal and organelle arrangements in Paramecium.

Characterization of Apoptotic Mechanisms Involved in R Body Induced Death inParamecia tetraurelia

Abstract. Paramecium tetraurelia is a unicellular eukaryotic ciliate. Certain strains of paramecia have been shown to contain endosymbiotic bacteria. Furthermore, some of these endosymbiotic bacteria confer a killing ability on the host paramecia, known as killer paramecia, because the endosymbiont can be lethal to other paramecia, known as sensitive paramecia, if it is released into the environment. This death has been connected to a protein found within the P. tetraurelia known as an R body. Interestingly, the R body-related-death resembles apoptosis in that it causes membrane blebbing, a characteristic of apoptosis. Furthermore, paramecia contain another marker of apoptosis, a mechanism to degrade DNA which is used in normal paramecia conjugation. Therefore, exposure to the R body and toxin may be activating some existing pathways in the paramecia, leading to apoptosis. To determine whether the mode of cell death induced by the R bodies is apoptotic, a flow cytometer was used to examine DNA fragmentati...

Genetics of the relationship between the ciliate Paramecium bursaria and its symbiotic algae

Abstract. Paramecium bursaria, a freshwater protozoan, typically harbors hundreds of symbiotic algae (Chlorella sp.) in its cytoplasm. The relationship between host paramecia and symbiotic algae is stable and mutually beneficial in natural environments. We recently collected an aposymbiotic strain of P. bursaria. Infection experiments revealed that the natural aposymbiotic strain (Ysa2) showed unstable symbiosis with Chlorella sp. The algae aggregated at the posterior region of the host, resulting in aposymbiotic cell production after cell division. Cross‐breeding analyses were performed to determine the heritability of the aposymbiotic condition. In crosses of Ysa2 with symbiotic strains of P. bursaria, F1 progeny were able to form stable symbioses with Chlorella sp. However, unstable symbiosis, resembling Ysa2 infection, occurred in some F2 progeny of sibling crosses between symbiotic F1 clones. Infection experiments using aposymbiotic F2 cells showed that these F2 subclones have limited ability to reestablish the symbiosis. These results indicate that the maintenance of stable symbiosis is genetically controlled and heritable, and that Ysa2 is a mutant lacking the mechanisms to establish stable symbiosis with Chlorella sp.

Sequence, Transcription Activity, and Evolutionary Origin of the R-BodyCoding Plasmid pKAP298 from the Intracellular Parasitic BacteriumCaedibacter taeniospiralis

We isolated the intracellular parasitic bacterium Caedibacter taeniospiralis from cultures of the freshwater ciliate Paramecium tetraurelia strain 298. Plasmid pKAP298 as well as the total RNA were isolated from the bacteria. pKAP298 was totally sequenced (49.1 kb; NCBI accession number AY422720). From southern blots of pKAP-fragments and Digoxigenin-labeled cDNA of the Caedibacter-RNA, we generated transcription maps of pKAP298. The observed transcription activity indicated functions of the plasmid besides the synthesis of the R-body, a complex protein inclusion associated with toxic effects of Caedibacter cells on host paramecia. We identified 63 potential protein coding regions on pKAP298, and a novel transposon as well as known transposons were characterized. A group II intron was identified. Homologies with putative phage genes were detected on pKAP298 that direct to the evolution of pKAP298 from a bacteriophage. This original phage most probably belonged to the Caudovirales. Hints on a toxin coding region of pKAP298 are given: a protein with homology to the Soj-/ParA-family also has homologies to a membrane associated ATPase, which is involved in eukaryotic ATPase dependent ion carriers and may be associated with toxic effects on paramecia ingesting this protein.

The Toxic Symbiont Caedibacter caryophila in the Cytoplasm of Paramecium novaurelia.

Endosymbiotic bacteria were observed to inhabit the cytoplasm of the freshwater ciliate Paramecium novaurelia. Transmission electron microscopy and toxicity tests with sensitive paramecia showed that the endosymbionts belong to the genus Caedibacter. The bacteria conferred a killer trait to their host paramecia. The production of a proteinaceous inclusion body ("R-body") in the bacterial cell makes them toxic to other paramecia after they become enclosed in food vacuoles. R-bodies of Caedibacter sp were associated with phages, which are known in most other Caedibacter species to code for the R-body proteins. The killer-effect of P. novaurelia on sensitive P. caudatum strains was of the "paralysis" type, which is a characteristic of the symbiont species Caedibacter caryophila. Until now C. caryophila was known to inhabit the macronucleus of Paramecium caudatum only. Sequencing of the 16S rRNA-gene proved that Caedibacter sp from the cytoplasm of P. novaurelia belongs to the species C. caryophila as well. The rDNA-sequence of 1695 bp length differed in a total of only 1 bp from the corresponding gene in C. caryophila from the macronucleus of P. caudatum. The results indicate that the infection of specific host cell compartments may depend on host genes, but not on different traits of the infecting symbiont species. The occurrence of killer and sensitive paramecia strains together in one pond is discussed with respect to the competitive advantage of the killer trait.

Surface proteins of the gram-negative bacterium Holospora obtusa bind to macronuclear proteins of its host Paramecium caudatum

Summary Holospora obtusa is a gram-negative bacterium living inside the macronucleus of the ciliate Paramecium caudatum . The bacteria leave the food vacuole after having been ingested, are transported through the cytoplasm, enter the nucleus, and start dividing. In order to analyze protein-protein interactions in the infection process, bacterial and macronuclear proteins were isolated. Binding of labeled macronuclear proteins was observed on semi-thin sections of bacteria. Using an overlay technique, blotted bacterial proteins were incubated with labeled host proteins and two bacterial proteins were found binding to nuclear proteins.

Characterization of genetic determinants for R body synthesis and assembly in Caedibacter taeniospiralis 47 and 116.

Caedibacter taeniospiralis, an obligate bacterial endosymbiont of Paramecium tetraurelia, confers a killing trait upon its host paramecium. Type 51 R bodies (refractile inclusion bodies) are synthesized by these endosymbionts and are required for expression of the killing trait. The nucleotide sequence of the genetic determinants for type 51 R body synthesis and assembly was determined for C. taeniospiralis 47 and 116. Three independently transcribed genes (rebA, rebB, and rebC) were characterized. To date these are the only genes from C. taeniospiralis to be sequenced and characterized. DNA regulatory regions are recognized by Escherichia coli, and codon usage appears similar to that in E. coli. A fourth open reading frame with appropriate regulatory sequences was found within the reb locus, but no evidence was obtained to suggest that this putative gene is expressed in E. coli. The R body-encoding sequences from both strains are identical. Two-dimensional gel electrophoresis of deletion derivatives shows that two polymerization events are involved in R body assembly. One polymerization event requires only RebB and RebC; the other requires all three proteins. Expression of RebC is necessary for the posttranslational modification of RebA and RebB into species with three and two different molecular weights, respectively. In the presence of RebC, each species of RebB with a different molecular weight has six different isoelectric points.

Bacterial Endosymbiosis in Paramecium aurelia: Bacteriophage-like Inclusions in a Kappa Symbiont

SUMMARY: The bacterial endosymbiont kappa of stock A35 of Paramecium aurelia (syngen 2) carries virus-like inclusions after it has developed a refractile or R body which is necessary for kappa to exert the killing effect. In exponentially growing Paramecium, the proportion of symbionts bearing R bodies and virus-like inclusions is very low. On starvation, R bodies and virus-like inclusions reappear, and the proportion builds up to between one-quarter and one-half of the total number of symbionts in a cell. Thus the development of R bodies, virus-like inclusions and killing action is influenced by the environment of the host Paramecium cell.