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.

Sugar analysis

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).

Fig. 1

Mating reactivity rhythms of strain Ok2. Mating reactivities of green Ok2 cells and Chlorella-free white cells (Ok2w) were tested in LL (A) and DD (B) every 3 hr by mixing them with “tester” cells of a complementary mating type. Ok2 cells (•) showed circadian mating reactivity rhythms in LL and DD, but Ok2w cells (○) showed almost no expression of mating reactivity in LL. (C): When Ok2w cells were given a dark pulse for 9 hr, they showed one cycle of mating reactivity rhythm.

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.

Fig. 2

Effects of infected Chlorella on the expression of mating reactivity of Ok2w cells in LL. (A) Mating reactivity rhythms of Ok2w cells (○) and OkwS cells (•) infected with Chlorella isolated from Sj2. Ok2w cells showed almost no expression of mating reactivity in LL. OkwS cells infected with Chlorella showed mating reactivity rhythm in LL. (B) Mating reactivity of Ok2w cells mixed with sonicated Sj2w cells. They showed no mating reactivity in LL.

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.

Fig. 3

Effects of a photosynthesis inhibitor on the expression of mating reactivity in Ok2. (A) Mating reactivity of Ok2 cells (•) and cells treated with 5 μg/ml of paraquat (○). Mating reactivity of the treated cells declined gradually and finally disappeared, as was seen in Ok2w cells. (B) Mating reactivity of Sj2w cells that show a normal mating reactivity rhythm in LL treated with paraquat. These cells showed a normal mating reactivity rhythm in LL. Thus, paraquat itself has no effect on the expression of mating reactivity.

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.

Table 1

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.

Fig. 4

Mating reactivity rhythms of Ok2w cells treated with sugars. To 20 ml cell suspensions were added 0.5 ml of 10^-2 M sugars every 3 hr during subjective day phases (0-12, 24-36 and 48-60 hr) in LL. The lower bar indicates subjective day and night phases that are the light/dark cycle given in previous to LL. Closed circles (•) show mating reactivity rhythms of Ok2w cells treated with glucose (A), maltose (B), maltotriose (C) and maltotetraose (D). Open circles (○) show mating reactivity rhythms of untreated Ok2w cells as controls. Maltose and maltotriose induced a mating reactivity rhythm in Ok2w cells. Glucose and maltotetraose had no effects on the expression of mating reactivity.