Paramecium bursaria shows many kinds of circadian rhythms, including a mating reactivity rhythm. P. bursaria cells normally contain several hundred Chlorella in the cytoplasm as endosymbionts. We found an interesting mutant strain (Ok2) from nature. Chlorella-contain green cells (Ok2) showed a normal mating reactivity rhythm in a constant light condition (LL) or constant darkness (DD). However, Chlorella-free white cells (Ok2w) derived from Ok2 did not display a mating reactivity rhythm in LL. In DD, they showed a normal mating reactivity rhythm. When Ok2w cells were infected with Chlorella isolated from green cells that show normal mating reactivity rhythms, they exhibited a circadian mating rhythm in LL. The green Ok2 cells reverted to the non-reactive state in LL by treatment with the herbicide paraquat. Sugar components in the cytosol of Ok2 and Ok2w were analyzed by HPLC, and four kinds of sugars were identified in Ok2 cells of day time. When maltose and maltotriose were added to Ok2w cell culture, the mating reactivity rhythms appeared in white cells in LL. These results suggest that the photosynthetic products of symbiotic Chlorella are closely related to the expression of circadian rhythms in a mutant strain of P. bursaria.
Circadian rhythms are found in all levels of organisms, including both eukaryotes and prokaryotes (Kondo et al., 1993), as basic adaptive behavior to periodical changes in the global environment. Circadian rhythms are controlled by one or more endogenous oscillators, often called the “circadian clock.” Circadian clock mechanisms have recently been investigated in molecular genetic studies (Gekakis et al., 1998; Rutila et al., 1998; Dunlap, 1998); however, the essential nature of the mechanisms has not yet been elucidated.
The unicellular ciliate Paramecium bursaria is an interesting model for the study of a coexisting system in intracellular symbiosis. The cells of P. bursaria collected from nature normally contain several hundred cells of the green alga Chlorella established in the cytoplasm as endosymbionts (Loefer, 1936). Chlorella-free white cells can be produced easily from natural green cells by rapid growth in constant darkness (DD). Chlorella can be isolated from host cells and re-infected easily to Chlorella-free white cells. Cloning culture of endsymbiotic algae isolated from P. bursaria was already accomplished (Nishihara et al., 1998). Since both green and white cells display many kinds of circadian rhythms, including mating reactivity and photoaccumulation rhythms (Miwa et al., 1987; Johnson et al., 1989), they are also suitable materials to analyze the cellular mechanisms of biological rhythms.
The sexual interaction of Paramecium, called mating reaction, occurs upon mixing of cells of complementary mating types when they are in the stationary phase and are sexually mature (Sonneborn, 1957; Hiwatashi, 1981). P. bursaria cells exhibit high mating reactivity in the light period and low reactivity in the dark period when they were exposed to a light/dark cycle (LD 12:12 hr). This rhythm is endogenously generated and continues for several days as a circadian rhythm in constant light (LL) (Ehret, 1953; Miwa et al., 1987). However, Chlorella-containing green cells of some strains show a normal mating reactivity rhythm in LL but a low mating reactivity in DD (Tanaka and Miwa, 1996).
We previously reported that symbiotic Chlorella forced the Paramecium cells to lengthen the period and to shift the phase of photoaccumulation rhythms in LL (Miwa et al., 1996). Furthermore, symbiotic Chlorella rescued the mating reactivity rhythms of arrhythmic mutant cells in LL but not in DD (Tanaka and Miwa, 1996). In this study, we found another interesting mutant strain (Ok2) from nature and their mating reactivity rhythms were characterized. Chlorella-free white cells (Ok2w) did not show a mating reactivity rhythm in LL. When Ok2w cells were infected with Chlorella isolated from green cells that showed normal mating reactivity rhythms, they exhibited a circadian mating rhythm in LL. We discuss the relationship between photosynthetic products of symbiotic Chlorella and the expression of circadian rhythms of Ok2w cells in LL.
MATERIALS AND METHODS
Strains and Culture
Two strains of Paramecium bursaria (syngen 1) were used in the experiments. Strain Ok2 (mating type III, collected in Okazaki, Japan) and Sj2 (mating type I, collected in Shimane, Japan) contain Chlorella symbionts and are thus designated green. Chlorella-free white cells, Ok2w and Sj2w cells, were induced from natural green cells of Ok2 and Sj2, respectively, by rapid growth in DD.
All strains were cultured in a 1.25% fresh lettuce juice medium in K-DS solution (0.6 mM KH2PO4, 1.4 mM Na2HPO4, 2 mM Na3C6H5O7, 1.5 mM CaCl2, pH 7.0), that had been inoculated with Klebsiella pneumoniae one day before use (Hiwatashi 1968). Cultures were kept at 25°C under a light/dark cycle (LD 12:12 hr, 1,500 lux of cool-white fluorescent light).
Test of mating reactivity
The mating reactivity of green Ok2 cells was tested by mixing them with white “tester” cells of a complementary mating type. When the white cells (Ok2w) were tested, green cells were used as “tester” cells. Mating reactivity in cell populations was measured every 3 hr as follows: 10 cells were placed in each of 6 different wells of a depression glass plate, and about 100 highly reactive tester cells were added to each well. After 5 min, the percentage of mating reactive cells clumping with tester cells was counted under a binocular microscope.
To prepare tester cells with high reactivity, four groups of green and white cells were entrained to four light/dark cycles (LD 12:12 hr, staggered by 6 hr). Each group of tester cells was used twice in the mating reactivity tests conducted at 3-hr consecutive intervals. Since each group of tester cells showed high reactivity for at least 6 hr in a day, tester cells were always highly reactive in every test.
Re-infection of Chlorella
Chlorella cells were prepared for re-infection into Ok2w cells as follows. Green Sj2 cells in a 100-ml culture were concentrated by low speed centrifugation. One ml of cell suspension was put into a micro test tube and then sonicated with an ultra-sonicator for 10 sec. Chlorella cells were collected through a 15-μm opening nylon mesh and washed with K-DS solution. About 4×107 Chlorella cells/ml were mixed with about 2×103 /ml of Ok2w cells.
Treatment with an inhibitor
Five μg/ml of the herbicide paraquat (1,1′-Dimethyl-4,4′-bipyridinium; methyl viologen) was added to the culture of green Ok2 cells to inhibit the photosynthetic activity of symbiotic Chlorella. The same amount of paraquat was added to the culture of white cells (Sj2w) as control. The cells treated with paraquat were tested for mating reactivity rhythms over a period of about 60 hr in LL.
Homogenates made from Ok2 and Ok2w cells by ultra-sonication were analyzed for their sugar components.
Each sample was centrifuged at 14,000 rpm, 4°C for 20 min to separate free-Chlorella and cell fragments. The supernatant was dried under vacuum at 40°C, and then a dried powder was dissolved in a little ultra-pure water. After centrifugation again at 1,4000 rpm, 4°C for 20 min, the extract was separated from nucleic acid, polysaccharides and free-peptides by an ultrafiltration. Finally, it was lyophilized. The lyophilized fractionation was analyzed for sugar components by HPLC using an amidosilica column.
Treatment with maltose
To investigate the effects of photosynthetic products of symbiotic Chlorella, cells were treated with maltose and maltose-type sugars. Ok2w cells in a 20-ml culture were added 0.5 ml of 10-2 M sugar every 3 hr during 12 hr of subjective day phase (0–12, 24–36 and 48–60 hr) in LL. Sugar amount is calculated at 3 mM final concentration after 60 hr. The same amounts of glucose were also tested as controls. Treated cells were assayed for mating reactivity rhythms in LL.
Characteristics of a mutant strain
It is generally thought that Chlorella-free white cells derived from natural green cells show a normal mating reactivity rhythm in LL or DD and that Chlorella-containing green cells of many strains also show a normal mating reactivity rhythm in LL but a low mating reactivity in DD. In the strain Ok2 corrected from nature, unusual mating reactivity rhythms were observed. White Ok2 cells (Ok2w) showed no circadian rhythm of mating reactivity in LL except for the first cycle (Fig. 1A) and green Ok2 cells showed a normal mating reactivity rhythm in DD (Fig. 1B). After the Ok2w cells had been exposed to a dark pulse for 9 hours, they expressed one cycle of mating reactivity rhythm (Fig. 1C).
Mating reactivity rhythms appeared by infection with Chlorella
Ok2w cells were infected with Chlorella isolated from natural green cells (Sj2) that exhibited a normal rhythm in LL. The cells that had become green (OkwS cells) were tested for mating reactivity every 3 hr. It has been reported that a period of about five days is needed for infected Chlorella to be established as endosymbionts (Nishihara et al., 1998). However, we previously showed that infected Chlorella affected the expression of circadian photoaccumulation rhythms in P. bursaria at three days after infection (Miwa et al., 1996). Moreover, the effects of infected Chlorella on expression of mating reactivity rhythms appeared 24 hr after infection (Tanaka and Miwa, 1996). Therefore, Ok2w cells were infected with Chlorella one day before the beginning of measurements in this experiment. On the following day, the cells contained many Chlorella cells and were clearly designated green, and they were assayed for mating reactivity rhythms. It was observed that cells of Chlorella began to propagate in the host cells two days after infection. The OkwS cells expressed a circadian rhythm of mating reactivity in LL (Fig. 2A). To exclude the possibility that the expression of mating reaction was caused by sonicated cytoplasm and nuclei of donor cells (Sj2), Ok2w cells were infected with sonicated white cells (Sj2w). As seen in Fig. 2B, they did not show a mating reactivity rhythm in LL. These results show that the expression of a mating reactivity rhythm in the recipient cells is caused by infected Chlorella.
Inhibition of photosynthetic activity in symbiotic Chlorella
To determine whether the photosynthetic activity of Chlorella is related to the expression of a mating reactivity rhythm, Ok2 cells were treated with paraquat. Their mating reactivity rhythms disappeared in LL, as was the case with Ok2w cells (Fig. 3A). In a previous study (Miwa et al., 1996), DCMU was used to inhibit the effects of Chlorella on the period length of photoaccumulation rhythm. However, it was found that DCMU was toxic for expression a mating reactivity in P. bursaria. Therefore paraquat was used in this experiment. Paraquat forms free radical in plant cells and destroys chloroplast. Thus it was used as photosynthetic inhibitor in this experiment. To confirm that paraquat has no effects on the ability of mating reaction in P. bursaria, Chlorella-free white cells (Sj2w) that showed normal mating reactivity rhythm in LL were treated with paraquat. As shown in Fig. 3B, they showed a normal mating reactivity rhythm in LL. Paraquat, therefore, affects only the photosynthetic activity of symbiotic Chlorella. These results suggest that the photosynthetic activity of symbiotic Chlorella in Ok2 cells has relation to the expression of mating reactivity rhythms of Ok2w cells in LL.
Sugar analysis in the cytosol
It is known that symbiotic Chlorella affects the circadian rhythms in many natural strains and mutant strain (MC1w) in LL but not in DD (Miwa et al., 1996; Tanaka and Miwa, 1996). This fact suggests that the photosynthesis of symbiotic Chlorella is related to the expression of circadian rhythms. To clarify that symbiotic Chlorella cells release photosynthetic products in the cytoplasm of Ok2 cells, sugar components in the cytosol were analyzed by HPLC. For the analysis, the following three kinds of samples were prepared: 1) Ok2 cells in light phase (day cells), 2) Ok2 cells in dark phase (night cells), 3) Ok2w cells in light phase (day cells). The results are shown in Table 1. Glucose, maltose, and unidentified tri-saccharide and tetra-saccharide were detected in the cytosol of Ok2 day cells, while only glucose was detected in Ok2 night cells and Ok2w day cells. These results suggest that symbiotic Chlorella cells release maltose in Ok2 cells according to the LD cycle. Unidentified tri-saccharide and tetra-saccharide were thought to be maltose-type sugars, maltotriose and maltotetraose, respectively. These sugars were also released in Ok2 cells in the light phase.
Sugar components in the cytosols of Ok2 and Ok2w cells analyzed by HPLC.
Effects of maltose on the expression of mating reactivity rhythm
The effects of sugar components in Ok2 cells on the expression of mating reactivity rhythms were investigated. Glucose, maltose, maltotriose and maltotetraose were added to a culture medium of Ok2w cells every 3 hr during the subjective day phases (0–12, 24–36, and 48–60 hr), and their mating reactivity rhythms were assayed in LL. The results are shown in Fig. 4. Mating reactivity rhythms of Ok2w cells were induced by the supply of both maltose and maltotriose in LL. However, the other sugars, glucose and maltotetraose, had no effects on the induction of mating reactivity in Ok2w. Thus, the secretion of maltose and maltotriose from symbiotic Chlorella has close connection with the expression of circadian rhythms in Ok2w cells.
P. bursaria shows many kinds of circadian rhythms, including mating reactivity and photoaccumulation (Miwa et al., 1987; Johnson et al., 1989). In this study, we showed the correlation of photosynthetic products of symbiotic Chlorella, maltose and maltose-type sugar, with the expression of mating reactivity rhythms in a mutant strain (Ok2) of P. bursaria.
Ok2 is an interesting natural strain. Chlorella-containing Ok2 cells showed a normal mating reactivity rhythm in LL or in DD. Chlorella-free Ok2w cells displayed normal mating reactivity rhythms in DD but they did not show mating reactivity in LL. This fact indicates that Ok2w cells have a normal circadian oscillator and that it shows ordinary functions irrespective of the presence of symbiotic Chlorella. A continuous light condition may inhibit the output route from a circadian oscillator to the expression of mating reactivity rhythms. On the other hand, Ok2w cells displayed a normal mating reactivity rhythm in LL after infection of Chlorella. Thus, it was suggested that infected Chlorella could affect the output process in the circadian systems of Ok2w cells. Ok2w cells could be suitable materials for investigating the output systems from a circadian oscillator. Nishihara et al. (1998) reported a method for establishing cloned culture of algae from heterogeneous symbiotic algae in P. bursaria cells, and they did their infection experiments using cloned symbiotic algae. In the present study, we used uncloned Chlorella for the re-infection experiments. It is important to investigate whether cloned symbiotic Chlorella induce a mating reactivity rhythm in Ok2w cells in LL.
In our previous paper, we reported that green cells of two strains (T316 and Sj2) showed low or no mating reactivity in DD (Tanaka and Miwa, 1996). It was thought that these results were due to the digestion of endosymbiotic Chlorella in DD. In the present study, however, Ok2 cells displayed normal mating reactivity rhythms in DD. It is possible that the interaction between host cells and symbiotic Chlorella varies according to the strain. When the mating reactivity rhythm of Ok2 cells was observed after 60 hr in DD, their mating reactivity became to low gradually. It would be interesting to clarify the transition of density of symbiotic Chlorella of Ok2 in DD.
When Ok2 cells were treated with paraquat, their mating reactivity disappeared in LL. It has been reported that symbiotic Chlorella was destroyed and that green cells changed into Chlorella-free white cells when green cells were treated with paraquat for a period of 4–10 days (Hosoya et al., 1995). However, in the present study, Ok2 cells treated with paraquat remained green during the period of measurement of mating reactivity (about 60 hr). Furthermore, paraquat itself had no effect on the expression of mating reactivity in Chlorella-free white cells (Sj2w). It is thought that the disappearance of mating reactivity in Ok2 cells is caused by interruption of the supply of photosynthetic products from symbiotic Chlorella.
It has been reported that symbiotic Chlorella cells provide their photosynthetic products, maltose, to the host cells as living energy (Weis, 1979, 1980). We analyzed the sugar components in the cytosol of Ok2 and Ok2w cells by HPLC, and we found maltose and maltose-type sugars in Ok2 day cells. These sugars were not detected in the cytosol of Ok2 night cells and Ok2w day cells. When the Ok2w cells were exposed to maltose or maltotriose, they showed mating reactivity rhythms in LL. However, glucose and maltotetraose had no effect on the expression of mating reactivity rhythms. These results are possibly due to the recognition range of sugar receptors in P. bursaria. It is generally thought that the sugar receptor (lectin) on cell-surface recognizes disaccharide and trisaccharide but that it is difficult for the receptor to recognize monosaccharide, tetrasaccharide. In the present study, since Ok2w cells induced mating reactivity after infection of Chlorella in LL, it is considered that the cells also have a sugar receptor like a lectin in cytoplasm. It is interesting to make sure the receptor of sugars in P. bursaria cells.
The circadian system in higher organisms may involve several oscillators, as a hierarchical combination of a master and slave oscillators, or as coupled circadian oscillators, each of which drives different rhythms (Pittendrigh, 1981). Furthermore, it has been reported that the unicellular alga Gonyaulax polyedra has two oscillators (Roenneberg and Morse, 1993). Analysis of the effects of intracellular interaction between P. bursaria and their symbiotic Chlorella on the expression of circadian rhythms is valuable as a model for the coupling system of plural oscillators in higher organisms. In a previous paper (Tanaka and Miwa, 1996), we proposed that symbiotic Chlorella might regulate the mating reactivity rhythms via mitochondria in P. bursaria. On the basis of the results of the present study, we propose another hypothesis that maltose and maltotriose released from symbiotic Chlorella might be a trigger to express a mating reactivity rhythm in Ok2w cells. However, we have no evidence how the sugar signal is recognized in Ok2w cells. Further experiments are needed to clear the process of intracellular signal transduction of sugars in P.bursaria cells.
We thank Dr. Tadao Saito of Tohoku University for his technical support in the HPLC analysis. This research was supported by a grant from the Ministry of Education, Science and Culture of Japan (No. 10640660) to I. Miwa.