J. Cell Sci. 36, 253-260 (1979) 253
Printed in Great Britain © Company of Biologists Limited J979
ISOJI MIWA
Biological Laboratory, College of General Education, Ibaraki University
Mito 310, Japan
SUMMARY
Cytoplasm of Paramecium caudatum syngen 3 in the period of sexual immaturity was effective in inducing immaturity when injected into mating-reactive cells of other syngens of
P . caudatum. When immature cytoplasm of P. caudatum was injected into mature cells of P . vndtimicronucleatum, immaturity was also induced in the latter cells. When cytoplasm of
P . bursaria in the immaturity period was injected into mature cells of the same species, the latter became immature. But immature cytoplasm of P. caudatum was ineffective when injected into mature cells of P . bursaria. When immature cytoplasm of P . bursaria was injected into mature cells of P . caudatum, no difference from the control was observed. These results suggest that a difference in specificity of the immaturity substances exists between paramecia of the 'aurelia' group and those of the 'bursaria' group.
INTRODUCTION
An exconjugant clone of Paramecium, as with many other ciliates, has no ability to mate during the immaturity period after conjugation, even under appropriate conditions. The duration of the immature period is measured in terms of the number of fissions, because even when environmental conditions cause the immature period to be shortened or lengthened, the number of fissions through which a given clone passes during the immature period is almost the same (Sonneborn, 1957; Kroll & Barnett,
1968; Takagi, 1970; Miwa & Hiwatashi, 1970; Bleyman, 1971). The duration of the immature period has been shown to be controlled genetically in P. primaurelia (Siegel,
1961) and some mutants with altered length of immaturity have been discovered in
Tetrahymena pyriformis (Bleyman & Simon, 1967) and in P. caudatum (Myohara &
Hiwatashi, 1978).
In a previous paper (Miwa, Haga & Hiwatashi, 1975) we reported that in syngen 3 of P. caudatum, cells in the immature period contain immaturity substances which repress the expression of mating ability when introduced into mature cells by microinjection. The immaturity substances are contained in the soluble fraction of immature cells and have the properties of heat-labile macromolecules (about 10000 mol.
wt or under). In order to test the specificity of these immaturity substances, attempts were made to inject immature cytoplasm of P. caudatum syngen 3 into other syngens of
P. caudatum and also into two other species of Paramecium, P. multimicronucleatum and P. bursaria, since these two species are known to have long immaturity periods after conjugation (Sonneborn, 1957).
In the present article, I will show that the immaturity substances of P. caudatum
17 CEL 36

254 I- Miwa syngen 3 are effective in inducing immaturity when injected into other syngens of
P. caudatum and into P. multimicronucleatum but are ineffective in P. bursaria.
MATERIALS AND METHODS
Immature cells used as donor for the microinjection of cytoplasm were progeny from the cross between stocks d m - n and d m - i 3 which belong to mating type V and VI respectively in syngen 3 of P . caudatum. Those stocks were F
2
progeny from a cross between natural stocks
Kok 1 and Koj. The stocks used as recipients and testers for test of mating reactivity were as follows. Stocks AaA 2 (mating type I) and AsA 3 (mating type II) belonging to syngen 1 of
P . caudatum were natural stocks collected by Y. Tsukii in Asamushi. Stocks Isn 4 (mating type
XXIII) and Ikz 1 (mating type XXIV) of syngen 12, and stocks Ak 1 (mating type XXV) and
Hj 2 (mating type XXVI) of syngen 13 in P . caudatum were obtained from Dr K. Hiwatashi's laboratory. Stock d m
-24 of P . caudatum was also used for some experiments. This results from a cross between stocks d m
- i 1 and d m
- i 3 and belongs to mating type VI. Stocks CH 313 (mating type III) and CH 312 (mating type IV) belonging to syngen 2 of P . multimicronucleatuvi were
F t
segregants from CH 101, which was Fx hybrid between stock 11 and stock 30 (Miyake,
1968). Stocks B w
and C w
in P . bursaria were Chlorella-free variants induced by rapid growth in the dark from natural stocks B g
and C g
collected by Mr Tsukii.
All stocks in the present experiment were cultured in lettuce medium which was 1-25 % (w/v) fresh lettuce juice in Dryl's solution (Dryl, 1959), pH 7-0 and inoculated with Klebsiella
pnewnoniae one day before use (Hiwatashi, 1968). Cultures were kept at 25
C
C.
Microinjection of cytoplasm or brei of cells was performed with a Chamber's micromanipulator with two needles after Knowles (1974). Mating reactivity of the recipient cells 3 and 7 h after the injection was observed by adding mating-reactive formalin-killed cells of complementary mating type, and that of the clones grown after the injection was observed on 3 successive days in the stationary phase by adding living cells of the complementary mating type
(tester). The method of carrying out the mating reactivity test with formalin-killed cells and that of preparing the brei for the injection are described in a previous paper (Miwa et al. 1975).
RESULTS
Injection of immature cytoplasm from syngen 3 of P. caudatum into cells of different syngens
In order to know whether injected immature cytoplasm of P. caudatum syngen 3 has any effect upon the mature cells of other syngens, stocks AsA 3, Ikz 1 and Hj 2 which belong to syngen 1, 12 and 13 of P. caudatum respectively, were used as the recipient cells. About 5X io
3
/tm
3
of immature cytoplasm from immature cells (15 fissions after conjugation between d m
- n and
m
-i3) were injected into mating-reactive cells of stocks AsA 3 (mating type II), Ikz 1 (mating type XXIV) and Hj 2 (mating type XXVI). As a control, the same volume of mature cytoplasm from mating-reactive cells of d m
-i3, mating type VI was injected into cells of the stocks described above.
The injected cells were kept in the cell-free culture supernatant from the stationary phase culture of the recipient. About 3 h after injection, each injected cell was transferred into fresh culture medium and grown as a clone. The test of mating reactivity was made 6 or 7 fissions after injection. Samples of each clone were starved and tested for mating reactivity on 3 successive days by adding mating-reactive cells of stocks
AsA 2 (mating type I), Isn 4 (mating type XXIII) and Ak 1 (mating type XXV). The results are shown in Table 1. In all 3 syngens used as recipient, many clones injected with immature cytoplasm of syngen 3 did not express mating reactivity when tested

Specificity of immaturity substances 255
6 to 7 fissions after injection, but no loss of mating reactivity was observed in controls which were injected with mature cytoplasm. The results indicate that the effect of immature cytoplasm from P. caudatum syngen 3 has no syngen specificity, at least qualitatively.
Table 1. Loss of mating reactivity in recipient clones when immature cytoplasm of
P. caudatum syngen 3 was injected into mature cells of other different syngens
Mating reactivity
Recipient Donor (Syngen 3) Reactive Non-reactive
Syngen 1
Syngen 12
Immature cellsf
Mature cellsj (control)
Immature cellsf
Mature cellsj (control)
Immature cellsf
Mature cellsj (control)
1 1
3
1 1
25
10
0
18
0
Syngen 13
7
24
18
0
• Reactivity was tested 7 fissions after injection when syngens 1 and 12 were used as recipient and 6 fissions after injection when syngen 13 was recipient.
t Progenies of a cross between d
X Mating-reactive cells of d m
-i3.
m
-n and d ra
-i3, 15 fissions after conjugation.
Injection of immature cytoplasm from P. caudatum into cells of P. multimicronucleatum
Immature cytoplasm of P. caudatum syngen 3 was injected into mature cells of
P. multimicronucleatum syngen 2. Immature cells 20 fissions after conjugation between d m
- n and d m
-i3 were used as the donor and mating-reactive cells of CH 312, mating type IV, were used as the recipient. Mating-reactive cells of CH 313, mating type III, killed with formalin (10% v/v) were used for testing mating reactivity of the injected cells. The cells injected with about i x i o ' / i m
3
donor cytoplasm were kept in the cell-free culture supernatant from the stationary phase culture of the recipient.
Mating reactivity of the injected cells was tested 3 and 7 h after injection by adding the formalin-killed tester. After the tests, the cells were transferred into fresh culture medium to establish clones. After 7 fissions of growth, mating reactivity was again tested with living test cells. In the control, mating-reactive cells of CH 312 were injected with the mature cytoplasm from mating-reactive cells of d m
-24. Mating reactivity of injected cells was tested in the same way. The results are shown in Table 2.
As seen in the Table, no loss of mating reactivity was observed in both the experimental and the control cells until 3 h after the injection, but 7 h after the injection more loss of reactivity was observed in the experimental cells than in the control.
Seven fissions after the injection, half the clones in the experiment showed no mating reactivity while all clones in the control showed reactivity.
The results when the brei of immature cells of P. caudatum was injected into mature cells of P. multimicronucleatum are shown in Table 3. The brei of immature cells was made from cells which had undergone 25 fissions after conjugation between
17-2

256 / . Miwa d m
- n and d m
-i3. The brei was filtered through a 0-45-/6111 Millipore filter and injected into mating-reactive cells of CH 312. The volume of the injected filtrate was
1 x io
4
/tm
3
. In the control, the brei of mating-reactive cells of d m
-24 prepared in the same way was used for injection. Mating reactivity of the injected cells was tested
Table 2. Loss of mating reactivity in mature cells of P. multimicronucleatum injected with cytoplasm from immature cells of P. caudatum
Donor
Hours or no. of fissions after injection
Mating reactivity in recipients*
Reactive Non-reactive
Dead or no growth
Immature cellsf
Mature cellsj (control)
3 h
7 h
7 fissions
7
3h h
7 fissions
10
13
9
13
4
6
5
1 1
0
6
2
6
0
0
2
0
3
0
• Reactivity in recipients (P. multimicromicleatttm, stock CH 312) 3 and 7 h after injection was tested with formalin-killed tester (stock CH 313); in recipients 7 fissions after injection, with living tester (CH 313).
t Progenies of a cross between d
J Mating-reactive cells of d m
-24.
m
- n and d m
-i3, 20 fissions after conjugation.
Table 3. Loss of mating reactivity in clones derived from mature cells of P. multi- micronucleatum injected with a brei of immature cells of P. caudatum*
Donor
No. of fissions after injection
Reactivity in recipient clonesf
Reactive Non-reactive
Immature cellsj
Mature cells§ (control)
8
15
8
15
6 14
18 2
18 0
18 0
• Breis were filtered through o-4S-/tm Millipore filter before use.
f Reactivity in recipient clones (CH 312) was tested with living tester (CH 313).
X, § As in Table 2.
8 and 15 fissions after injection using living test cells. As seen in Table 3, the injection of the brei of immature cells showed almost the same effect as that of immature cytoplasm. These results indicate that the immaturity substances of P. caudatum can induce immaturity in mating-reactive cells of P. multimicronucleatum.
Injection of immature cytoplasm between P. caudatum and P. bursaria
Almost the same experiment was done with cells of P. bursaria as the recipient.
About 5 x io
3
fim
3
of cytoplasm from immature cells of P. caudatum (15 fissions after

Specificity of immaturity substances 257 conjugation between d m
- u and d m
-i3) were injected into mating-reactive cells of stock B w
in P. bursaria. In the control, mature cytoplasm of d m
-24 was used for injection. Mating reactivity tests of each injected cell 3 and 7 h after injection need formalin-killed highly reactive tested cells of P. bursaria, but they were not obtainable.
For this reason, injected cells were grown without the 3 and 7 h tests and the test for mating reactivity was made 7 and 14 fissions after injection. The results are shown in
Table 4. Maintenance of mating reactivity in recipient clones when immature cytoplasm of P. caudatum was injected into mature cells of P. bursaria
Donor
No. of fissions after injection
Reactivity in recipient clones*
Reactive Non-reactive
Immature cellsf
14
7 11 0
11 0
Mature cellsj (control)
14
7 10 0
10 0
• Reactivity in recipient clones (P. bursaria, B w f Progenies of a cross between d
% Mating-reactive cells of d m
-24.
m
-n and d m
) was tested with living tester (C
-i3, 15 fissions after conjugation.
w
).
Table 5. Mating reactivity in recipients when immature cytoplasm of
P. bursaria was injected into mature cells of P. caudatum
Donor
Hours or no. of nOOIAnO QrTPf injection
Mating reactivity in recipients*
Reactive Non-reactive
1 | p n n f\r lyCUU UT no growth
Immature cellsf 3 h
7 h
7 fissions
2 6
2 1
2 4
2
0
7
0
0
4
Mature cellsj (control) 3 h
7 h
7 fissions
2 4
IS
2 0
1
0
9
0
1
5
• Reactivity in recipients (d m tester ( d m
-24) 3 and 7 h after injection was tested with formalin-killed
- n ) ; in recipients 7 fissions after injection, with living tester (d f Progenies of a cross between B w
% Mating-reactive cells of B .
w
and C w m
, 15 fissions after conjugation.
-ii).
Table 4. No loss of mating reactivity was observed in both experimental and control clones. The result suggests several possibilities: the immaturity substances from
P. caudatum have no effect upon the mating-reactive cells of P. bursaria, the amount injected was insufficient, or 7 fissions after injection was too late to find the effect.
Experiments were next performed to determine whether immature cytoplasm of
P. bursaria is effective when introduced into mature cells of P. caudatum. Immature cells 15 fissions after conjugation between B w
and C w
of P. bursaria were used as the

258 /. Miwa donor and mating-reactive cells of d m
-24, mating type VI in P. caudatum, were used as the recipient. The volume of injected immature cytoplasm was about 5 x io
3
/tm
3
.
Mating reactivity of the injected cells was tested 3 and 7 h after injection by adding the formalin-killed tester (d m
-n, mating type V) and with the living tester after 7 fissions of growth. As control, mature cytoplasm of B w
was injected into d m
-24. The results are shown in Table 5. Though some non-reactive cells were observed both in the experiment and in the control 7 h after injection, all surviving clones in the experiment and the control showed high reactivity 7 fissions after injection. The results suggest that injection of immature cytoplasm from P. bursaria to mating-reactive cells of P.
caudatum induces no immaturity in the latter.
Table 6. Loss of mating reactivity in clones derived from mature cells of
P. bursaria injected with immature cytoplasm of the same species
No. of
Reactivity in recipient clones*
Donor injection Reactive Non-reactive
Immature cellsf
Mature cellsf (control)
7
14
7
1 4
2 13
6 9
12 0
12 0 t
I m As in Table 4.
Progenies of a cross between B
Mating-reactive cells of B w
.
w and C w
, 15 fissions after conjugation.
Injection of immature cytoplasm into mature cells in P. bursaria
As shown in the previous experiment, injection of immature cytoplasm into mature cells between P. caudatum and P. bursaria was ineffective. This raises the question whether the immaturity substances such as are found in P. caudatum are not present in P. bursaria or the substances in P. bursaria have a different specificity from those in
P. caudatum. To answer these questions, injection of immature cytoplasm of P.
bursaria into mature cells of the same species was performed. About 5 x io
3
/tm
3
of cytoplasm from immature cells (15 fissions after conjugation between B w
and C w
) was injected into mating-reactive cells of B w
. As control, the same volume of mature cytoplasm from mating-reactive cells of B w
was injected into other cells of the same culture. Injected cells were grown and the test for mating reactivity was made 7 and
14 fissions after injection. The results are shown in Table 6. As seen in the Table, loss of mating reactivity was observed in 13 clones out of 15, 7 fissions after injection, and in 9 out of 15 even 14 fissions after injection. In the control, all clones showed high reactivity 7 and 14 fissions after injection. The results show that the immature cytoplasm of P. bursaria represses the expression of mating ability when introduced into mature cells by injection. This suggests that the immaturity substances exist in the immature cells of P. bursaria but that they have different specificity from those of
P. caudatum.

Specificity of immaturity substances 259
DISCUSSION
As shown in the present experiments, the immature cytoplasm of P. caudatum syngen 3 was effective in inducing immaturity when injected into mature cells of other syngens. Though sexual isolation by mating reaction is complete and no intersyngenic mating reaction is observed among syngens 1, 3, 12 and 13, the immaturity substances of P. caudatum display no qualitative syngen specificity, though quantitative specificity has not been examined. When immature cytoplasm of P. caudatum was injected into mature cells of P. multimicronucleatum, which is known to have a long immaturity period after conjugation, immaturity was induced in the latter species. But immature cytoplasm of P. caudatum was ineffective when injected into mature cells of P. bursaria which also have a long immaturity period after conjugation. The ineffectiveness was the same when immature cytoplasm of P. bursaria was injected into mature cells of
P. caudatum. However, when immature cytoplasm of P. bursaria was injected into mature cells of the same species, the effect was positive. This shows that cells of
P. bursaria in the immaturity period contain immaturity substances but with different specificity. Whether the immaturity substances of P. caudatum are capable of inducing immaturity when injected into cells of P. tetraurelia, which is known to have no immaturity period after conjugation, and whether immaturity substances exist in the immature cells of P. primaurelia, which has an immaturity period after conjugation, are interesting problems which are now under investigation.
The author wishes to thank Dr K. Hiwatashi for help in preparation of the manuscript.
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{Received 20 June 1978)