Correlation of Sugar Release and Concanavalin A Agglutinability with Infectivity of Symbiotic Algae from Paramecium bursaria for Aposymbiotic P. bursaria

Synopsis.
Eighteen strains of algae, including 17 formerly symbiotic with Paramecium bursaria , were tested for capacity to release sugar. Detectable amounts of sugar were found in the supernatant fluids from 10 strains, including 6 strains infective for aposymbiotic P. bursaria syngen 2. The other 4 sugar-releasing strains were noninfective and released ˜26–46 g sugar/mg dry cell weight compared to ˜90–175 g sugar/mg dry cell weight for infective strains. This relationship of infectivity with capacity to release sugar supplements data that indicate a relationship of infectivity with resistance to Con A agglutination. The correlation is completed if we assume that resistance to Con A agglutination and capacity for sugar release must both be present in an algal strain for infectivity. The data thus strongly suggest that these 2 characteristics must be present for infectivity by any algal strains for aposymbiotic P. bursaria syngen 2.     

Effect of Exposure Period and Algal Concentration on the Frequency of Infection of Aposymbiotic Ciliates by Symbiotic Algae from Paramecium bursaria

The effect of exposure period and concentration of algae on the frequency of infection of aposymbiotic ciliates by algae obtained from the same clone of Paramecium bursaria syngen 2, was studied. The frequency of infection was roughly proportional to the algal concentration and to the exposure time of ciliates to algae. The relationship of algal concentration to infection frequency closely fitted the Poisson distribution curve for N = 1, suggesting that the minimum number of algae required to infect a single ciliate is 1. However, the data also strongly suggested that the average number of algae required to initiate infection of an average ciliate was ? 1,000. Three possible resolutions of this situation are: (a) the selection by the ciliate of a rare infective variant from a heterogeneous population: (b) the rare escape of an alga from digestion by the ciliate; and (c) the requirement for a large number of algae-ciliate contacts to induce susceptibility in the ciliate. Splitting the exposure of ciliates to algae into 2 periods of 0.5 h, separated by 5 h in the absence of algae, produced a much higher frequency of infection than a single l-h exposure, supporting the suggestion that the large number of algae is required to induce susceptibility in the ciliate which can then be infected by as few as a single algal cell.     

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.     

Digestion of Added Homologous Algae by Chlorella-Bearing Paramecium bursaria

SYNOPSIS. Endosymbiotic algae from Paramecium bursaria when added to the culture medium are ingested by Chlorella -bearing P. bursaria at a rate of 2,000 algae/organism/day. That the ingested algae are digested and assimilated by the ciliates is suggested by the more rapid growth of Paramecium when algae are added to the medium ( G = 40 hr with algae compared to 190 hr without). The digestion by the ciliates of exogenous algae contrasts with the survival of these algae under normal growth conditions. It is suggested that the protection of the endogenous algae is related to their location in peripheral perialgal vacuoles.     

Transfer of Photosynthetically Produced Carbohydrate from Endosymbiotic Chlorellae to Paramecium bursaria*

Paramecium bursaria (Ehrenberg) and an endozoic zoochlorella Chlorella conductrix (Brandt) live in a symbiotic relationship. Uptake of NaH¹⁴CO₃ was studied to determine if carbohydrate products of photosynthesis are transferred to the host paramecium. Paramecium bursaria containing the algal symbionts took up NaH¹⁴CO₃ but those without the algal symbionts did not. Radioactive maltose, glucose, fructose and malate were identified from the ethanolic extract of paramecia. Transfer of materials from Paramecium to Chlorella and the transfer of other materials from Chlorella to Paramecium, led to the conclusion that this is a mutualistic relationship, both organisms benefiting from the relationship.     

Phylogenetic position of endosymbiotic green algae in Paramecium bursaria Ehrenberg from Japan

Endosymbiotic green algae of Japanese Paramecium bursaria were phylogenetically analyzed based on DNA sequences from the ribosomal DNA operon (18S rDNA, ITS1, 5.8S rDNA, and ITS2). Phylogenetic trees constructed using 18S rDNA sequences showed that the symbionts belong to the Chlorella sensu stricto (Trebouxiophyceae) group. They are genetically closer to the C. vulgaris Beijerinck group than to C. kessleri Fott et Nováková as proposed previously. Branching order in C. vulgaris group was unresolved in 18S rDNA trees. Compared heterogeneities of 18S rDNA, ITS1, 5.8S r, and ITS2 among symbionts and two Chlorella species, indicated that the ITS2 region (and probably also ITS1) is better able to resolve phylogenetic problems in such closely related taxa. All six symbiotic sequences obtained here (approximately 4000-bp sequences of 18S rDNA, ITS1, 5.8S rDNA, and ITS2) were completely identical in each, strongly suggesting a common origin.     

Life Cycle of Paramecium bursaria Syngen 1 in Nature

ABSTRACT. Studies were completed on the natural population density of Paramecium bursaria syngen 1 and on the life cycle stages to which the individuals belonged. Green paramecia were collected from two streams once every 20 days for over one year: 413 individuals on 26 collection dates in Mikumarikyo stream and 83 individuals on 23 collection dates in Momijidani-gawa stream. Individuals in nature did not maintain at a steady density but fluctuated greatly depending on the month. It seems that conjugation occurred from April to June in the Mikumarikyo stream and from May to June in the Momijidani-gawa stream. The appearance of individuals with mating ability might be related closely to increasing population so that sexual reproduction probably occurred near the peak of the population density. The 413 individuals from Mikumarikyo stream were examined to determine their position within the life cycle; 309 (74%) were immature, 55 (13%) were adolescent, and 49 (12%) were mature. No senile individuals were observed. The fraction of individuals with mating ability was generally less than 30% at any collection. Four mating types were observed occurring with about equal frequencies in mature individuals. The results show the frequencies of the recessive genes for mating types (a and b) are higher than for dominant genes (A and B). Of 83 individuals from Momijidani-gawa stream, 44 (52%) were immature, 21 (25%) were adolescent, and 18 (21%) were mature. Again, no senile individuals were observed. Because only two mating types were found, II and III (genotypes aaB- and aabb), it seems possible that the dominant gene A was rare or absent in the Momijidani-gawa population.     

Mechanism of Photoaccumulation in Paramecium bursaria

Responses of Paramecium bursaria to light intensity changes were investigated. The resting paramecia show a direction changing response (photophobic response) to a sudden decrease of light intensity, whereas no response was shown to an increase in intensity. The critical intensity decrease dI_c necessary to show the response was measured at various values of initial light intensity, and the ratio dI_c/I was found to be equal to ～0.15. The swimming paramecia show different behavior depending on their swimming direction in the spatial gradient of light intensity. Paramecia show direction change more frequently when they are swimming down the gradient than in the opposite direction. This difference in the rate of direction changing is 13–17%. These results may offer an explanation for the mechanism of photoaccumulation.     

Effect of Japanese Paramecium bursaria extract on photosynthetic carbon fixation of symbiotic algae

To investigate the relationship between the Japanese Paramecium bursaria host and its symbiont, we studied the effect of a host cell-free extract on carbon fixation and photosynthate release of the symbiont. The host extract enhanced symbiotic algal carbon fixation about 3-fold at an increased concentration; however, release of photosynthate hardly changed. Since the enhancing effect was not affected by elimination of carbon dioxide from the host extract, the existence of a host factor that stimulates algal carbon fixation was made clear. The host factor is a heat-stable, low molecular weight substance. In relation to the pH dependence, the extract improved carbon fixation at acidic and neutral pH and showed almost no effect at pH 9.0. Therefore, the stimulation of carbon fixation by the host factor is unlikely to be caused by intracellular pH change. The extract also improved carbon fixation of several Chlorella species, symbiotic and free-living, and apparently exhibited no species specificity. Therefore, the host seems to regulate the photosynthesis of the symbiont via a specific compound.     

The effect of host Chlorella NC64A carbon : phosphorus ratio on the production of Paramecium bursaria Chlorella Virus-1

1. We used the freshwater alga Chlorella NC64A (Division Chlorophyta) and its virus Paramecium bursaria Chlorella virus‐1 (PBCV‐1) as a model system to test for potential stoichiometric constraints on a virus–host interaction. 2. Media phosphorus concentrations were manipulated to create Chlorella NC64A host cells with low (91 ± 23) or high (453 ± 246) C : P ratio. In contrast, the C : P ratio of PBCV‐1, calculated from its biochemical composition, was 17 : 1. 3. Stoichiometric theory predicts that infection success and postinfection viral production should be depressed in high C : P cultures due to insufficient intracellular P for production of P‐rich viral particles. 4. Consistent with this hypothesis, viral production was strongly affected by host C : P ratio. While host C : P ratio did not affect viral attachment or the percentage of new viral particles that were infectious, in the low C : P Chlorella NC64A treatment, nine times more viruses were produced per infected cell than in the high C : P treatment (158 ± 138 versus 18 ± 18), indicating that the low C : P cells were higher quality for PBCV‐1 proliferation. 5. This result implies that the stoichiometric quality of algal cells can have a major effect on host–virus population dynamics.     

Multiple origins of the symbioses in Paramecium bursaria

Many organisms have symbioses with photosynthetic algae as typified by corals, clams, lichens, and some protozoa. Paramecium bursaria contains green algal symbionts and this unicellular ciliate is a textbook example used for microscopic observation in junior high school science projects. We have determined molecular phylogenies for the green algal symbionts. The symbiotic algae are the main constituent of the Paramecium cytoplasm, and we have recognized a total of four species, of which two were newly discovered in the present study. One should be regarded genetically as Chlorella vulgaris, and it belongs phylogenetically to the Chlorella clade (Chlorellaceae, Trebouxiophyceae) as well as "American" and "European" groups, which we previously introduced. Their genetic dissimilarities are 0.50-0.83% in 18S rDNA comparisons, but those of the internal transcribed spacer 2 (ITS2) reach an unambiguous level (22.6-26.6%). These dissimilarities suggest that they are equivalent to discrete species derived from multiple origins as paramecian symbionts. Another newcomer was clearly separated from the Chlorellaceae, and this alga clustered with Coccomyxa spp. in ITS2 analyses. These symbiotic relations indicate multiple origins of symbionts.     

Correlation between circadian periods and cellular activities in Paramecium bursaria

Paramecium bursaria shows a circadian rhythm of photoaccumulation: photoaccumulation is stronger during the day than at night. We obtained five strains of P. bursaria having different circadian periods under continuous light conditions, ranging from 20.9 to 27.9 h. Various physiological activities were compared in the cells of these strains. The periods of contractile vacuole contraction were in the range 10–15 s, which was almost proportional to the periods of the circadian rhythm in each strain. Swimming velocities were inversely proportional to the circadian period; i.e. swimming velocities were high in strains whose circadian periods were short. Resting membrane potential was more depolarized in strains with longer circadian periods. Finally, the membrane resistance of the resting state was reduced in proportion to the increase of the circadian period. Such correlation between the cellular properties and the circadian period suggests that the circadian clock mechanism is associated with various physiological activities of the cell.     

Genetic evidence of "American" and "European" type symbiotic algae of Paramecium bursaria Ehrenberg

Paramecium bursaria is composed of a "host" ciliate and a "symbiont" green alga. Based upon physiology, DNA hybridization and virus infection, two types of symbionts, called "American" type and "European" type, have been reported to date. Here, we determined the 18S rDNA and internal transcribed spacer 2 (ITS2) regions for both "American" and "European" types. Sequence features clearly separated into two lineages; NC64A (USA), Syngen 2-3 (USA), Cs2 (Chinese), MRBG1 (Australian), and Japanese strains belong to the "American", whereas PB-SW1 (German) and CCAP 1660/11 (British) strains belong to the "European". In "American" 18S rDNA, three introns were inserted in the same positions as for previously described Japanese symbionts. In "European" 18S rDNA, a single intron occurred in a different position than in the "American". Between the types, sequence differences were seven or eight nucleotides (0.39 %) in the 18S rDNA exon, and more than 48 nucleotides (19.2 %) in ITS2 regions. We subsequently sequenced the host 18S rDNA. As a result, two groups: Cs2, MRBG1, and Japanese strains, and PB-SW1 and CCAP 1660/11 strains, were separated (with 23 substitutions and 4 insertions or deletions between the groups). The congruent separations between hosts and symbionts may imply that the type of symbiont depends on the host type.     

Chlorella variabilis and Micractinium reisseri sp. nov. (Chlorellaceae,Trebouxiophyceae): Redescription of the endosymbiotic green algae of Paramecium bursaria (Peniculia,Oligohymenophorea) in the 120th year

Symbiotic algae of the ciliate Paramecium bursaria (Ehrenberg) Focker are key species in the fields of virology and molecular evolutionary biology as well as in the biology of symbiotic relationships. These symbiotic algae were once identified as Zoochlorella conductrix Brandt by the Dutch microbiologist, Beijerinck 120 years ago. However, after many twists and turns, the algae are today treated as nameless organisms. Recent molecular analyses have revealed several different algal partners depending on P. bursaria strains, but nearly all P. bursaria contains a symbiont belonging to either the so‐called ‘American’ or ‘European’ group. The absence of proper names for these algae is beginning to provoke ill effects in the above‐mentioned study areas. In the present study, we confirmed the genetic autonomy of the ‘American’ and ‘European’ groups and described the symbionts as Chlorella variabilis Shihira et Krauss and Micractinium reisseri Hoshina, Iwataki et Imamura sp. nov., respectively (Chlorellaceae, Trebouxiophyceae).     

Quaternary assembly and crystal structure of GDP-D-mannose 4,6 dehydratase from Paramecium bursaria Chlorella virus

GDP-D-mannose 4,6 dehydratase is the first enzyme in the de novo biosynthetic pathway of GDP-L-fucose, the activated form of L-fucose, a monosaccharide found in organisms ranging from bacteria to mammals. We determined the three-dimensional structure of GDP-D-mannose 4,6 dehydratase from the Paramecium bursaria Chlorella virus at 3.8A resolution. Unlike other viruses that use the host protein machinery to glycosylate their proteins, P. bursaria Chlorella virus modifies its structural proteins using many glycosyltransferases, being the first virus known to encode enzymes involved in sugar metabolism. P. bursaria Chlorella virus GDP-D-mannose 4,6 dehydratase belongs to the short-chain dehydrogenase/reductase protein superfamily. Accordingly, the family fold and the specific Thr, Tyr, and Lys catalytic triad are well conserved in the viral enzyme.     

Identification of Chlorella viruses in Paramecium bursaria clones by pulse-field electrophoresis

The ciliates Paramecium bursaria contain endosymbiotic green algae Chlorella spp. in their cytoplasm. The algae isolated from P. bursaria are sensitive to large DNA-containing viruses of the family Phycodnaviridae. The type virus of this family is PBCV-1 (Paramecium bursaria Chlorella virus). Investigation of the total DNA of P. bursaria clones by pulse-field electrophoresis (PEGE) revealed a pronounced band on PEGE profiles of some P. bursaria clones; the band was formed by DNA molecules of approx. 300 kb. This band probably contained the DNA of Chlorella virus. Two approaches were used in the present work to confirm this hypothesis. Microbiological tests were used to scan a collection of P. bursaria clones for specific types of viruses; the 300-kb band was revealed only in the PEGE profiles of virus-containing clones. Blot hybridization of P. bursaria total DNA separated by pulse-field electrophoresis with the virus-specific probe revealed that the band under study was formed by the DNA of a Chlorella virus. Paramecium clones were shown to contain approx. 10⁵ copies of nonintegrated viral DNA.     

Evidence of de novo synthesis of maltose excreted by the endosymbiotic Chlorella from Paramecium bursaria

The endosymbiotic Chlorella sp. from Paramecium bursaria excretes maltose both in the light and in the dark. Experiments on photosynthetic ¹⁴CO₂ fixation and ¹⁴CO₂ pulse-chase experiments show that maltose is synthesized in the light directly from compounds of the Calvin cycle, whereas in the dark it results from starch degradation.     

Photobehaviour of Paramecium bursaria infected with different symbiotic and aposymbiotic species of Chlorella

The endosymbiotic unit of Paramecium bursaria and Chlorella spec. shows two types of photobehaviour: 1) A step-up photophobic response which possibly depends on photosensitive agents in the ciliate cell itself — as is also shown by alga-free Paramecium bursaria - and can be drastically enhanced by photosynthetic activity of symbiotic algae; and 2) a step-down photophobic response. The step-down response leads to photoaccumulation of green paramecia. Both types of photobehaviour in Paramecium bursaria do not depend on any special kind of algal partners: The infection of alga-free Paramecium bursaria with different Chlorella species results in new ciliatealgae-associations. They are formed not only by combination of the original symbiotic algae with their host, but also by infection with other symbiotic or free-living (aposymbiotic) chlorellae, respecitively. Systems with other than the original algae are not permanently stable — algae are lost under stress conditions — but show the same types of photobehaviour. Photoaccumulation in general requires algal photosynthesis and occurs only with ciliates containing more than fifty algae/cell. It is not mediated by a chemotactic response to oxygen in the medium, since it occurs at light fluence rates not sufficient for a release of oxygen by the symbiotic system, e.g., below its photosynthetic compensation point. Photoresponses can be inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Sensory transduction does not depend on any special symbiotic features of the algae, e.g., sugar excretion. The participation of oxygen in the Paramecium cell, of its cytoplasmic pH and of ions released or taken up by endosymbiotic algae in sensory transduction is discussed.