in fresh C medium 2.5 × 10
3
cells per ml. Four 0.1-ml aliquots containing about 250 cells each
of each cell suspension were separately inoculated into respective petri dishes which contained 25 ml
of C medium. Subsequently, the inocula were in- cubated for 4, 11, 17 or 22 days under illumination
of 2000 lx, and at the end of each culture period, cells of each strain were harvested. To obtain cells
at pregamete and gamete stages, vegetative cells of each strain grown for 17 days in C medium were
collected and mixed together in the cell number noted above. The mixed cells were washed twice
with MIH medium, and then inoculated into a petri dish containing 25 ml of MIH medium 300
cells per ml. Subsequently, the inoculum was in- cubated for 4 days with illumination of 3000 lx
and then harvested. To count the number of pyrenoidscell, pyrenoids were stained according
to Rosowski and Hoshaw [11] Fig. 1L and M, with the exception that 50 propionic acid which
contained 4 ferric ammonium sulfate was used as the mordant, followed by 3-D counting under
an Olympus BX50 microscope. To determine pyrenoidal size, ten or more cells were selected for
each stage and photographed under the micro- scope at 100 × . For each stage, the diameters of
500 pyrenoids were measured on microphoto- graphs enlarged at a final magnification of 400 × .
To investigate the effects of DNA synthesis inhibitors on pyrenoid production and pyrenoidal
size, vegetative cells of each strain were grown for 17 days, collected and inoculated separately into
petri dishes which contained 25 ml of C medium 300 cells per ml with or without the test chemi-
cals. The cells were incubated for 5 days with illumination of 3000 lx and then harvested. This
was followed by measurement of pyrenoidscell number and pyrenoid diameter.
2
.
4
. Statistical analysis Experiments to investigate the frequency of
zygote formation and growth rate per day were performed in triplicate. Experiments on pyrenoid
number and size were repeated more than twice, and the same tendencies were observed. To com-
pare treatment means with a control mean, statis- tical analyses were carried out using Student’s
t-test, and the degree of significance was expressed in terms of P-values.
3. Results
3
.
1
. Induction of zygote formation by DNA synthesis inhibitors
When two heterothallic strains of C. ehrenbergii are mixed, zygotes are induced Fig. 1K. In the
present study, the effect that inhibitors of DNA synthesis have on the frequency of zygote forma-
tion in the alga was investigated Table 1. In- hibitors of prokaryotic-type DNA synthesis, EB,
AO and NB, which are considered to prevent cytDNA synthesis, increased the frequency of
Fig. 1. C. ehrenbergii cells. Photomicrographs shown in A – J and K – M are enlarged at the same magnification, respec-
tively. Scale bars, 100 mm. Arrows indicate pyrenoids. A – D Vegetative cells of an m
+
strain during asexual reproduction. E – H Vegetative cells of an m
−
strain during asexual repro- duction. Vegetative cells were grown for 4 days A and E, 11
days B and F, 17 days C and G and 22 days D and H after inoculation in C medium. Each cell comprised two
identical semicells and the nucleus was localized in the central region of the cell. I A pair of pregametes. J A pair of
gametes. K A pair of normal zygotes. They were almost spherical and had thick walls. L – M M
+
strain cells treated according to Rosowski and Hoshaw [11] to stain pyrenoids.
Before treatment, the cells were grown for 5 days in C medium in the absence L or presence M of 2.5 × 10
− 6
M of EB. Pyrenoids are stained.
Table 1 Induction of zygote formation in C. ehrenbergii by the administration of DNA synthesis inhibitors
a
Ratio Inhibitors effective concentration, M
Growth rate per day Zygote formation frequency
Ratio Treated b
ba
b
Untreated c Treated d
dc
b
Untreated a Prokaryote type
EB 2.5×10
− 6
N
+
34.1 9 4.3 67.1 9 1.4
c
1.97 1.95 9 0.11
1.33 9 0.05
c
0.68 56.4 9 5.2
c
1.65 1.95 9 0.11
34.1 9 4.3 1.35 9 0.08
b
7.5×10
− 6
N
+
0.69 68.5 9 8.7
0.89 2.5×10
− 6
N
−
77.0 9 4.4 69.5 9 2.5
c
0.9 77.0 9 4.4
7.5×10
− 6
N
−
AO 60.2 9 14.7
c
1.63 1.78 9 0.01
3×10
− 7
N
+
1.79 9 0.05 36.9 9 11.2
1.01 54.8 9 12.7
1.49 1.78 9 0.01
36.9 9 11.2 1.61 9 0.02
c
1×10
− 6
N
+
0.9 79.9 9 11.8
3×10
− 7
N
−
82.2 9 6.3 1.03
1×10
− 6
N
−
64.7 9 12.6 79.9 9 11.8
0.81 NB
47.1 9 10.0 3×10
− 6
N
+
1.18 39.8 9 1.0
1.68 9 0.19 1.75 9 0.02
1.04 1×10
− 5
N
+
52.5 9 6.0
c
39.8 9 1.0 1.32
1.68 9 0.19 1.74 9 0.00
1.04 76.9 9 3.0
1.09 70.5 9 9.0
3×10
− 6
N
−
72.7 9 8.0 1.03
1×10
− 5
N
−
70.5 9 9.0 Both type
MC 2.9×10
− 5
N
+
53.4 9 3.0
c
35.6 9 2.0 1.50
1.85 9 0.04 1.64 9 0.02
c
0.89 8.7×10
− 5
N
+
35.6 9 2.0 46.2 9 4.4
c
1.30 1.85 9 0.04
1.33 9 0.20
c
0.72 77.7 9 2.0
c
1.13 68.6 9 3.4
2.9×10
− 5
N
−
68.6 9 3.4 8.7×10
− 5
N
−
64.0 9 6.9 0.93
HU 29.7 9 6.3
1.13 1.83 9 0.03
26.4 9 8.3 1.82 9 0.06
3×10
− 6
N
+
0.99 26.4 9 8.3
1×10
− 5
N
+
27.7 9 2.6 1.05
1.83 9 0.03 1.68 9 0.08
0.92 56.1 9 3.7
3×10
− 6
N
−
64.4 9 5.8
c
1.15 66.7 9 4.6
c
1.19 56.1 9 3.7
1×10
− 5
N
−
Ara-C 35.7 9 6.7
c
1×10
− 4
N
+
1.63 21.9 9 9.1
1.75 9 0.01 1.86 9 0.03
c
1.06 28.8 9 9.6
1.32 1.75 9 0.01
21.9 9 9.1 1.74 9 0.03
3×10
− 4
N
+
0.99 1×10
− 4
N
−
58.2 9 7.8 54.3 9 6.8
1.07 52.1 9 2.6
0.96 54.3 9 6.8
3×10
− 4
N
−
Eukaryote type APC
d
1×10
− 6
N
+
48.7 9 5.3 41.5 9 3.7
1.17 1.95 9 0.08
1.82 9 0.01
c
0.93 50.5 9 3.9
c
1.22 1.95 9 0.08
41.5 9 3.7 1.58 9 0.04
c
3×10
− 6
N
+
0.81 81.0 9 4.5
c
1.10 1×10
− 6
N
−
73.9 9 2.6 52.6 9 12.3
c
0.71 73.9 9 2.6
3×10
− 6
N
− a
All measurements were made at the end of 5 days of culture. Values represent the mean 9 S.D. of triplicates. N
+
, nitrate present. N
−
, nitrate absent.
b
Treatment meannon-treatment mean.
c
PB0.05 vs. untreated cultures.
d
Since APC was dissolved in dimethyl sulfoxide DMSO, DMSO was added to the medium at a final concentration of 0.03 in the experiments with APC.
zygote formation in the presence of nitrate. The increases were 97, 63 and 32 of the values for
non-treated cultures at the most effective concen- trations, 2.5 × 10
− 6
, 3 × 10
− 7
and 10
− 5
M, re- spectively. However, in the absence of nitrate, the
chemicals did not increase the frequency at any concentration. By contrast, an inhibitor of eukary-
otic-type DNA synthesis, APC, which is consid-
ered to block nDNA synthesis, increased the zygote formation frequency in both the presence
and absence of nitrate. The frequency increases at the most effective concentrations were 22 and 10
of non-treated values in the presence and absence of nitrate, respectively. On the other hand, in-
hibitors of both prokaryotic and eukaryotic DNA synthesis, MC, HU and Ara-C, increased the fre-
quencies in the presence andor absence of nitrate. Namely, at the most effective concentration, 2.9 ×
10
− 5
M, MC elevated the frequencies by 50 and 13 in the presence and absence of nitrate, respec-
tively. HU at 3 × 10
− 6
M increased the frequency by 15 in the absence of nitrate, and Ara-C at
10-4 M by 63 in its presence. The effects of these inhibitors on the growth
rate per day in the alga were tested Table 1. NB, HU and Ara-C little affected the growth rate at
concentrations effective for zygote formation. By contrast, EB, AO, APC and MC decreased it by
10 – 30 at these concentrations.
3
.
2
. Numbers of pyrenoidscell and pyrenoid diameters at asexual and sexual reproduction
stages To investigate the numbers of pyrenoidscell
and pyrenoid diameters during asexual reproduc- tion, vegetative m
+
and m
−
strain cells of C. ehrenbergii which were grown for 4 days early-log
phase, Fig. 1A and E, 11 days mid-log phase, Fig. 1B and F, 17 days late-log phase, Fig. 1C
and G and 22 days stationary phase, Fig. 1D and H in C medium were observed. In these cells,
pyrenoids were almost spherical and distributed randomly.
In early-log phase, the average number of pyrenoidscell was 57.5 9 12.8 n = 57 and 42.3 9
9.5 n = 63 for m
+
and m
−
vegetative cells, re- spectively. This number decreased gradually with
the advance of stage in both strains; the averages were 58.6 9 13.9 n = 76 and 45.7 9 10.4 n = 70
in m
+
cells at mid-log and late-log phases, respec- tively, and 38.1 9 8.8 n = 63 and 35.6 9 8.1 n =
50 in m
−
cells at mid-log and late-log phases, respectively. And, they reached 39.5 9 11.0 n =
47 and 34.5 9 6.9 n = 48 in the respective cells at the stationary phase. On the other hand, in early-
log phase, the averages of pyrenoid diameters were 5.3 9 1.2 mm n = 500 and 5.2 9 1.4 mm n = 500
in m
+
and m
−
vegetative cells, respectively. These diameters increased gradually as the stage pro-
gressed in both strains of cells; the averages were 5.8 9 1.4 mm n = 500 and 6.3 9 1.8 mm n = 500
in m
+
cells at mid-log and late-log phases, respec- tively, and 5.6 9 1.4 mm n = 500 and 6.5 9 1.8
m m n = 500 in m
−
cells at mid-log and late-log phases, respectively. They reached 6.9 9 1.8 mm
n = 500 and 7.0 9 1.8 mm n = 500 in the respec- tive cells at stationary phase.
During sexual reproduction, the number of pyrenoidscell and pyrenoid diameters were deter-
mined at pregamete Fig. 1I and gamete Fig. 1J stages. At these stages, pyrenoids were also almost
spherical and distributed randomly within cells.
The average number of pyrenoidscell was 38.6 9 10.8 n = 47 and 20.1 9 6.7 n = 48 at the
pregamete and gamete stages, respectively. There were no significant differences between the average
number of pyrenoids per cell at the pregamete stage and vegetative stage in either strain of cells
grown for 17 days in C medium late-log phase, which were mixed to induce zygote formation.
Moreover, the average number per cell at the gamete stage was half of that at the pregamete
stage. These results imply that pyrenoid produc- tion occurs neither during pregametogenesis nor
during gametogenesis. On the other hand, at the pregamete and gamete stages, the pyrenoid diame-
ters were greatly increased compared to those during asexual reproduction; the average value
being 10.6 9 2.4 mm n = 500 at pregamete and gamete stages. The result that the averages of
pyrenoid diameters at the pregamete and gamete stages were the same indicates an increase of
pyrenoidal size during pregametogenesis.
3
.
3
. Modulation of pyrenoid production and pyrenoidal size by DNA synthesis inhibitors
To investigate whether inhibition of DNA syn- thesis is involved in the modulation of pyrenoid
production and pyrenoidal size during pregameto- genesis, vegetative m
+
and m
−
strain cells of C. ehrenbergii were incubated separately with DNA
inhibitors, and the number of pyrenoidscell and pyrenoid diameters were determined Table 2 and
Figs. 2 and 3. Comparison of the average number of pyrenoidscell and pyrenoid diameters between
untreated cultures and cultures treated with EB 2.5 × 10
− 6
M indicated that EB caused arrest of pyrenoid
production and
enlargement of
pyrenoidal size. Treatment with AO and NB also caused both a decrease in pyrenoid numbers and
an increase in diameter in both strains at concen- trations effective for zygote formation. By con-
trast, APC caused only the pyrenoid diameter to increase: it actually evoked an increase in pyrenoid
numbers at concentrations sufficient to decrease the growth rate. MC, HU and Ara-C at the effec-
tive concentrations caused either the number of pyrenoidscell to decrease or the pyrenoid diame-
ter to increase in both strains of cell. That is, MC and HU increased the diameter, and Ara-C de-
creased the number. In addition, MC and HU caused the pyrenoid number to increase at higher
concentrations, 8.7 × 10
− 5
and 10
− 5
M, respec- tively, like APC. These results indicate that the
inhibition of nDNA andor cytDNA synthesis is closely involved in the modulation of pyrenoid
production and pyrenoidal size.
4. Discussion
