Strain and Culture

The strains of Paramecium bursaria, syngen 1 used in the experiment of measuring the period length of photoaccumulation rhythm were collected at different places in Japan and China. Most of these strains were obtained from Dr. Kosaka at Hiroshima University. We used especially following strains for cross: Kz1 (mating type III, collected at Kouzan in Hirosima), Sj2 (mating type I, collected at Sinji in Shimane), T316 (mating type IV, collected at Tsukuba in Ibaraki), UK1 (mating type I, collected at Urizura in Ibaraki). Strain Sj2w (Chiorella-free white cells) have been induced from Sj2 (Chlorella-containing stock) by rapid growth in the dark. Strain E2 was an early mature mutant induced from Kz1 by treatment with nitrosoguanidine (2 μg/ml for 5 hours).

All strains were cultured in fresh lettuce juice medium, which had been inoculated with Klebsiella pneumoniae one day before use [6]. Cultures were kept at 25°C under the light/dark cycle (LD 12:12 hr, 1,000 lux of cool-white fluorescent light).

Photoaccumulation rhythm

The photoaccumulation rhythm was measured in constant light (LL) as described in the previous paper [9]. The apparatus used to assay this rhythm of Paramecium was modified from that described by Bruce [2]. Assay of this rhythm, expressed as the decrease of transmittance in percent of the initial transmittance, is easily automated by a microcomputer-assisted data collection apparatus. The trough or peak of the photoaccumulation rhythm was used as the phase reference point. The time of the phase reference point was calculated by a computer program that was made by Kondo [11], which fitted a parabola to the data of each peak and trough. For calculation of period, the time of the phase reference points were subjected to a least-squares regression.

Mating reactivity rhythm

Mating reactivity of the green cells was tested by mixing them with white “tester” cells of complementary mating type as described in the previous paper [14]. Cells of P. bursaria are usually green, containing symbiotic Chlorella in the cell. Chlorella-free white cells can be derived from green cells. White cells were used as tester cells, since the green cells were easily distinguished from the white tester cells in the mating clumps.

Induction of short period mutant

Concentrated log phase cells of Kz1 (green cells, mating type III) were treated with 2 μg/ml nitrosoguanidine for 5 hours. Cells were washed 3 times with K-DS solution. After about 10 fissions, cells were mixed with Sj2w (white cells, mating type I) for induction of cytogamy to get homozygotes. Cytogamy can be induced with osmotic shock by treatment with 1% Methyl Cellulose for 2 hours at the 3rd micronuclear meiotic division [18]. The conjugation pairs were collected by nylon mesh. At first barium resistant green cells were selected with 9 mM BaCl₂ for 15 minutes 5–6 fissions after cytogamy. Then barium resistant cells were assayed for a photo-accumulation rhythm.

Determination of the duration of immaturity

The number of fissions after conjugation was calculated by using the daily isolation culture in depression slides [13]. The cells remaining in the depression slide after daily isolation were transferred into small test tubes containing 1 ml of culture medium, then 2 ml and 4 ml of fresh culture medium were added to the test tubes for two days successively. The cells divide 6 times during the culture of these test tubes. Tests of mating reactivity were performed every day with the cells in the test tubes starved for two days. The cells were mixed with four different mating types (I–IV) of tester cells. We excluded clones that showed the same mating type to parents at the first mating reactivity test (immature test). We used clones whose mating ability did not appear at the first test as a true progeny. When the cells of progeny get to adolescence they can mate with cells of two mating types, and when they get to maturity they can mate with cells of three mating types (all except their own mating type).

Genetic analysis of the period of photoaccumulation rhythm

The photoaccumulation rhythms of 30 strains collected at different places in Japan and China were measured automatically for 7 days in LL. Although most of the strains showed 23–26 hour periods of the photoaccumulation rhythm, two strains of conspicuously different period length were isolated, one strain UK1 showing shorter period (21.8 hr) and another strain T316 showing longer period (28.7 hr), as shown in Fig. 1. To know the period of another circadian rhythm, the mating reactivity rhythm of these two different strains was assayed in LL. As shown in Fig. 2, these two strains exhibited a similar period length for mating reactivity as for the photoaccumulation rhythm.

Fig. 1

Photoaccumulation rhythms of short period strain (UK1, 21.8 hr) and long period strain (T316, 28.7 hr) in LL at 25°C. (A): photoaccumulation (ordinate) was assayed every hour automatically by a microcomputer assisted data collection apparatus, and was expressed as the decrease of transmittance in percent of the initial transmittance. (B): The period of rhythm was calculated by a computer program that performs a least-squares regression using the phase reference points of peak values. Points of peaks are presented as a double plot.

Fig. 2

Mating reactivity rhythms of the short period strain (UK1, A) and the long period strain (T316, B) in LL. Mating reactivity of cell population was tested every 3 hours by mixing them with white tester cells of complementary mating type. The percentage of mating reactive green cells clumping with white tester cells was assayed.

Next we isolated 32 F₁ progeny from a cross of UK1 and T316, and their photoaccumulation rhythm was assayed after the immature test. As shown in Table 1, they showed about 24.7 hours mean period. The range from the shortest to the longest period was 22.5–26.8 hours. Then we isolated 28 F2 progeny from the clones showing a period of medium duration (about 24 hours). Mean period of F2 progeny was 23.8 hours. These period lengths did not segregate into two or three major groups, and they were distributed almost equally between 21.8 to 26.7 hours. In the back cross between F1 progeny and parents, the two groups of period were not segregated and the range of periods spread equally. However, the mean period in the progeny from back cross with T316 was longer than that from the back cross with UK1 (Table 2). These results suggest that the difference of periods between UK1 and T316 is controlled polygenetically or by the ecotype that is influenced by the ecosystem.

Table 1

Period of F1 and F2 progeny from a cross between a short period strain and a long period strain in photoaccumulation rhythm

Table 2

Period of photoaccumulation rhythm in the progeny from backcross between F1 and parents

Induction of mutation

To get a typical mutant clone of the circadian rhythm, we treated cells of Kz1 with nitrosoguanidine (MNNG). Cells were mutagenized as described in Fig. 3 and were screened with 9 mM BaCl₂ for 15 minutes. We could obtain 24 barium resistant cells after performing this protocol 5 times. The photoaccumulation rhythms of these clones were measured. Almost all clones showed a period of about 26.0 hours, which is nearly the same as the period of Kz1. But one clone (E2) showed a short period rhythm of 21.8 hr (Fig. 4), and we found no clones which exhibited a long period rhythm or an arrhythmic pattern. As listed in Table 3, the progeny from a cross between E2 and UK1 showed a short period, and that from a cross between E2 and T316 showed a slightly longer period. But the range of period length of these progeny did not so wide. Progeny from E2 and Sj2w showed an intermediate period length. During this experiment, we noticed that the progeny of E2 matured early.

Fig. 3

Schedule of mutagenesis with nitrosoguanidine (MNNG) in P. bursaria, stock Kz1.

Fig. 4

Period length of photoaccumulation rhythms in natural strain Kz1 (A) and short period strain E2 (B) that was induced from Kz1 by treatment of nitrosoguanidine. The phase reference points of peak were analyzed as in Fig. 1.

Table 3

Period of photoaccumulation rhythm in the progeny of the short period strain (E2) induced with nitrosoguanidine

Genetic analysis of early mature mutant

An exconjugant clone of Paramecium has a period of immaturity, during which cells cannot mate even when they enter the stationary phase. The length of immaturity depends upon the number of fissions undergone since conjugation. In the case of P. bursaria, exconjugants become adolescent after about 40 fissions and mature about 55 fissions after conjugation. They never show mating reactivity after less than 30 fissions. For genetic analysis of the early maturation character, E2 (mating type III) was mated with Sj2w (mating type I). Almost all progeny were able to mate about 25 fissions after conjugation and showed mating type I or IV. The appearance of maturity of F2 progeny clones (from SE11 × SE14, which are F1 progeny from Sj2 × E2) segregated into three groups, about 15, 22, 52 fissions after conjugation (Fig. 5). Number of F2 progeny is too small for χ²-statistical test, but its ratio (3:10:4) was close to 1:2:1 (Table 4). It seems that strain E2 is early mature mutant. The length of adolescence of the two early maturation groups was shorter than that of late maturation group (Fig. 5). The period length of the circadian photoaccumulation rhythm in LL of these three groups of F2 clones was, on the average, 20.8 ± 0.5 (range 20.3–21.2 hr), 22.0 ± 0.9 (range 20.9–23.6 hr) and 25.6 ± 1.0 (range 24.2–26.7 hr) hours. The early maturation groups showed a short period rhythm.

Fig. 5

Maturation curves of F2 progeny clones from a cross between early maturation strain E2 and natural strain Sj2w. The length of F2 progeny clones segregated into three groups (A-C) as shown in Fig. 4. A included 3 clones, B included 10 clones, C included 4 clones. Mating reactivity of progeny clones was tested every day after growth in a small test tube by mixing with four mating types tester cells (I–IV). Onset of adolescence was determined the day that cells mate with two mating type tester cells. When the cells mate with three mating types tester cells (all except their own mating type), they became mature. ○—○: onset of adolescence, •—•: onset of maturity.

Table 4

Segregation of early maturation clones and mating types in the F1 and F2 progeny from a cross between E2 and Sj2W

Further, to investigate the correlation between the period length of circadian rhythms and the length of immaturity, E2 (early mature mutant) and Kz1 (wild type) were mated with short period strain (UK1) and long period strain (T316) in photoaccumulation rhythm, respectively. As shown in Fig. 6, the progeny of short period strains became mature earlier than that of longer period strains. The length of adolescence of the progeny from the early mature strain (E2) was shorter than that of the progeny from the wild type strain (Kz1).

Fig. 6

Maturation curves of the progeny clones from crosses between various strains. Mating reactivity of progeny clones was tested as in Fig. 5. A: early maturation strain E2 (III) crossed with short period strain UK1 (I). B: E2 (III) crossed with Song period strain T316 (IV). C: natural strain Kz1 (III) crossed with UK1 (I). D: Kz1 (III) crossed with T316. ○—○: onset of adolescence, •—•: onset of maturity.