J. Cell Sci. 38, 193-199 (1979)
193
Printed in Great Britain © Company of Biologists Limited 1979
IMMATURITY SUBSTANCES IN PARAMECIUM
PRIMAURELIA AND THEIR SPECIFICITY
ISOJI MIWA
Biological Laboratory, College of General Education,
Ibaraki University, Mito 310, Japan
SUMMARY
Cells of'Paramecium primaurelia in the immaturity period of the life cycle contain, substances
which repress the expression of mating ability when introduced into mature cells. When the
immature cytoplasm of P. primaurelia was injected into mating-reactive cells of P. tetraurelia,
P. caudatum and P. multimicronucleatum, immaturity was also induced in them. But the
immature cytoplasm of P. primaurelia was ineffective when injected into mature cells of
P. bursaria. When the immature cytoplasm of P. bursaria was injected into mating-reactive
cells of P. primaurelia and P. tetraurelia, no difference from the control was observed. When
the immature cytoplasm of P. caudatum was injected into mating-reactive cells of P. primaurelia and P. tetraurelia, immaturity was induced in cells of the latter 2 species.
INTRODUCTION
An exconjugant clone of Paramecium, as with a variety of other ciliates, has a
well-defined life cycle, consisting of the periods of immaturity, maturity and senility.
During the immaturity period, Paramecium is unable to mate even under appropriate
conditions. The duration of the immaturity period is measured in terms of the
number of fissions after conjugation (Sonneborn, 1957; Miwa & Hiwatashi, 1970,
and others). As already reported, cells of P. caudatum in the immaturity period
contain immaturity substances which repress the expression of mating ability when
introduced into mature cells by microinjection (Miwa, Haga & Hiwatashi, 1975).
Immature cells of P. bursaria also contain immaturity substances but with different
specificity to those of P. caudatum (Miwa, 1979). 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 reported in Tetrahymena thermophila (Bleyman & Simon, 1967) and in P. caudatum (Myohara &
Hiwatashi, 1978). Thus, the phenomenon of immaturity in Paramecium provides one
of the best systems for studying the genetics of biological timing because of the
possibility of identifying its material basis through immaturity substances.
In the present article, I will show that the immature cells of P. primaurelia contain
immaturity substances which are effective in inducing immaturity when injected
into P. tetraurelia, P. caudatum and P. multimicronucleatum but are ineffective in
P. bursaria.
194
!• Miwa
MATERIALS AND METHODS
Immature cells used as donor for the microinjection of cytoplasm were progeny from the
cross between stocks 16 and 513, which belong to mating types I and II respectively in P.
primaurelia (one of the sibling species in the P. aurelia complex (Sonneborn, 1975)). The
stocks used as recipients and testers for mating reactivity were as follows. Stocks hrd of mating
types VII and VIII in P. tetraurelia (another sibling species in P. aurelia complex) were
highly mating-reactive mutants derived from stock 51 (Sonneborn, 1974) supplied by
S. Koizumi. Stocks d m -2i (mating type V) and dm-23 (mating type VI) belonging to syngen
3 of P. caudatum were F 3 progeny from a cross between natural stocks Kok 1 and Koj. Stocks
53B (mating type III) and 49B (mating type IV) of syngen 2 in P. multimicronucleatum were
acyclic strains obtained from Y. Takagi. Stocks Bw and Cw (complementary mating types
belonging to an unknown syngen) in P. bursaria were Chlorella-free variants induced by
rapid growth in the dark from natural stocks Bs and O collected by Y. Tsukii.
All stocks in the present experiment were cultured in lettuce medium which was 1-25 % (vv/v)
fresh lettuce juice in Dryl's solution (Dryl, 1959), pH 7-0, inoculated with Klebsiella pneumoniae 1 day before use (Hiwatashi, 1968). Cultures were kept at 25 °C.
Microinjection of cytoplasm was performed with a micromanipulator of de Fonbrune type
(Narisige, Tokyo) with 2 needles after the method of Knowles (1974). Recipient cells after
injection were kept in the cell-free culture supernatant from the stationary phase culture
of the recipient for 3 h and then grown in a microcapillary (Drummond) with a certain
volume of culture medium, and mating reactivity was observed in depression slides by adding
about 100 highly reactive living cells of the complementary mating type.
RESULTS
Injection of cytoplasm from immature cells into mature cells in P. primaurelia
About 5xio 3 /tm 3 cytoplasm from immature cells (8 fissions after conjugation
between stock 16 and 513) were injected into mating-reactive cells of stock 16
(mating type I), which had undergone 20fissionssince the last autogamy. As a control,
the same volume of mature cytoplasm from mating-reactive cells of stock 16 (20
fissions after autogamy) was injected into cells of the same stock at the same stage.
About 3 h after injection, each recipient cell was transferred into 1 /tl of fresh
culture medium in a microcapillary. Each injected cell undergoes 1-2 fissions 1 day
after injection, and 3 fissions 2 days after injection in the microcapillary. Mating
reactivity of each recipient clone was tested 2 days after injection in a depression
slide by adding 10/tl of mating-reactive culture (contains about 100 cells) of stock
513 (mating type II). The number of mating-reactive cells in the recipient clone,
which usually contains 8 cells, was calculated. The results are shown in Table 1.
In 19 out of 32 clones injected with immature cytoplasm, no cells expressed mating
reactivity when tested 3fissionsafter injection, but in the control, which was injected
with mature cytoplasm, all clones showed mating reactivity. The immature cytoplasm of P. primaurelia can induce immaturity when injected into mature cells.
Injection of immature cytoplasm from P. primaurelia into cells of P. tetraurelia
In order to know whether immature cytoplasm of P. primaurelia has an ability to
induce immaturity in the mating-reactive cells of P. tetraurelia, which is known to
have no immaturity period after conjugation, stock hr d of P. tetraurelia (mating type
VII) was used as the recipient cells. Stock hr d cells are suitable for such experiments
Immaturity substances in Paramecium
195
since mating reactivity of this stock is strong and is retained for a long time. About
5xio 3 /tm 3 cytoplasm from immature cells (8 fissions after conjugation between
stock 16 and 513) were injected into mating-reactive cells of stock hi d (VII) 5-6
fissions after autogamy. The injected cells were transferred into 1 /tl fresh culture
medium after being kept for 3 h in the cell-free culture supernatant and were grown
for 2 days, during which the cells divided 3 times. Mating reactivity of each recipient
clone was then tested by adding 10 /.tl mating-reactive culture of stock hr d (VIII)
Table 1. Loss of mating reactivity in recipient clones grown in a capillary after immature
cytoplasm of P. primaurelia was injected into mature cells of the same species
Frequency distribution, of recipient clones which show
various ratios of mating-reactive cells*
Donor
Immature cellsf
Mature cellsj
(control)
0/8
1/8
2/8
19
0
5
0
6
0
3/8
2
4
4/8
0
4
5/8
0
6
0
5
6/8
0
7
7/8
0
2
8/8
Total
clones
32
28
* Reactivity in recipient clones (stock 16) 3 fissions after injection was tested with 10 /tl of
tester (stock 513).
f Progenies of a cross between stock 16 and 513, 8 fissions after conjugation.
X Mating-reactive cells of stock 16, 20 fissions after autogamy.
6-7 fissions after autogamy. In the control, mating-reactive cells of hr d (VII) 5-6
fissions after autogamy were injected with the mature cytoplasm from mating-reactive
cells of stock 16 in P. primaurelia. Mating reactivity of the control was tested in the
same way. As seen in Table 2, cells of P. tetraurelia which have no immaturity
period after conjugation showed immaturity after the injection of immature cytoplasm
from P. primaurelia. The results suggest that the effect of immature cytoplasm of
the P. aurelia complex has no species specificity within the complex.
Injection of immature cytoplasm from P. primaurelia into mature cells of P. caudatum,
P. multimicronucleatum and P. bursaria
To examine further the species specificity of the effect of immature cytoplasm in
P. primaurelia, immature cytoplasm of P. primaurelia was injected into mature cells
of P. caudatum, P. multimicronucleatum and P. bursaria. Immature cells 8 fissions
after conjugation between stock 16 and 513 (P. primaurelia) were used as donors
and mating-reactive cells of dm-23 (P. caudatum), 49B (P. multimicronucleatum) and
Bw (P. bursaria) were used as the recipients. About 1 x io 4 /tm 3 cytoplasm from the
immature cells of P. primaurelia were injected into mating-reactive cells of the
recipient stocks mentioned above. About 3 h after injection, each recipient cell
was transferred into 2 /tl fresh culture medium in a microcapillary and was grown
for 2 days, during which period most cells divided twice. Mating reactivity of each
recipient clone was tested 2 days after injection by adding 20 /tl mating-reactive
cultures of complementary mating types, stocks d m -2i, 53B and Cw respectively.
In the controls, mating-reactive cells of d m -2i, 53B and B w were injected with
/ . Miwa
196
cytoplasm from mating-reactive cells of stock 16. The results are shown in Table 3.
Many recipient clones of P. caudatum and P. multimicronucleatum which were
injected with P. primaurelia immature cytoplasm failed to express mating reactivity
when tested 2 fissions after injection, while normal mating reactivity was observed
Table 2. Loss of mating reactivity in clones derived from mating-reactive cells of
P. tetraurelia injected with cytoplasm from immature cells of P. primaurelia
Frequency distribution of recipient clones which show
various ratios of mating-reactive cells*
Total
clones
A
Donor
Immature cellsf
Mature cellsj
(control)
0/8
1/8
2/8
3/8
4/8
5/8
6/8
7/8
8/8
18
4
2
0
0
0
0
0
0
0
0
1
0
3
0
S
6
14
28
25
* Reactivity in recipient clones (stock hrd mating type VII) 3 fissions after injection was
tested with 10 [A of tester (stock hrd mating type VIII).
ft As in Table 1.
Table 3. Mating reactivity in recipient clones of j different species 2 fissions after
injection of immature cytoplasm from P. primaurelia
Frequency of recipient clones
which show various ratios of
mating-reactive cells*
A
Donor
Recipient
{P- primaurelia)
(Immature cellsf
•! Mature cellsj
[ (control)
r Immature cellsf
P. multimicronucleatum \ Mature cellsj
( (control)
(Immature cellsf
P. bursaria
J, Mature cellsj
1 (control)
P. caudatum
0/4
3/4
4/4
Total
i/4
2/4
21
6
3
0
0
30
0
0
1
7
26
34
13
10
3
2
0
28
0
0
0
4
23
27
0
1
S
22
3
b
9
6
7
0
10
25
* Reactivity in recipient clones (dm-23, 49B, Bw) 2 fissions after injection was tested with
testers (d m -2i, 53B, O ) .
ft As in Table 1.
in the controls. In P. bursaria, however, no significant difference was observed
between the experimental and the control groups. The immature cytoplasm of
P. primaurelia is effective in inducing immaturity when injected into P. caudatum
and P. multimicronucleatum but ineffective in P. bursaria.
Immaturity substances in Paramecium
197
Injection of immature cytoplasm from P. caudatum into cells of P. primaurelia and
P. tetraurelia
Next, experiments were performed to discover the reciprocal effects of immature
cytoplasm. Immature cells 15 fissions after conjugation between d m -2i and dm-23
of P. caudatum were used as the donor. Mating-reactive cells of stock 16 (mating
type I) 20 fissions after autogamy in P. primaurelia and of hrd (VII) 5-6 fissions
after autogamy in P. tetraurelia were used as the recipients. The cells injected with
about 5 x io 3 /tm 3 donor cytoplasm were kept in the cell-free supernatant for about
3 h and then grown in 1 /tl fresh culture medium for 2 days, during which period
most cells divided 3 times. Mating reactivity of each recipient clone was tested
2 days after injection by adding 10 /i\ mating-reactive culture of stock 513 (mating
type II) in P. primaurelia or of hr d (VIII) in P. tetraurelia. In the control, matingreactive cells of stocks 16 and hr d (VII) were injected with cytoplasm from matingreactive cells of d m -23. The results are shown in Table 4. As seen in the table, many
clones not only of P. primaurelia but also of P. tetraurelia failed to express mating
reactivity when tested 3 fissions after injecting immature cytoplasm of P. caudatum,
while no significant loss of mating reactivity was observed in the controls. The
immature cytoplasm of P. caudatum is effective in inducing immaturity when injected
into P. primaurelia and P. tetraurelia.
Injection of immature cytoplasm from P. bursaria into cells of P. primaurelia and
P. tetraurelia
Almost the same experiment was done with immature cells of P. bursaria as the
donor. About 5 x io3/^m3 cytoplasm from immature cells of P. bursaria (15 fissions
after conjugation between B w and Cw) were injected into mating-reactive cells of
stock 16 (mating type I) in P. primaurelia 20 fissions after autogamy and of stock hr d
(VII) in P. tetraurelia 5-6 fissions after autogamy. In the control, mature cytoplasm
of stock B w was used for injection. About 3 h after injection, each recipient cell
was grown in 1 /tl fresh culture medium for 2 days, during which period cells
underwent 3 fissions. Mating reactivity of each clone was tested 2 days after injection
by adding 10/tl mating-reactive culture of stock 513 (mating type II) in P. primaurelia
or of stock hrd (VIII) in P. tetraurelia. The results are shown in Table 5. Essentially
normal mating reactivity was observed in either the experimental or in the control
groups. The immature cytoplasm of P. bursaria is ineffective in inducing immaturity
in P. primaurelia or in P. tetraurelia.
DISCUSSION
In the previous paper (Miwa, 1979), I reported that the immature cells of P.
caudatum and P. bursaria contain immaturity substances that repress the mating
ability when introduced into mature cells by microinjection, and that a difference
in specificity of the immaturity substances exists between those 2 species. The
present experiments extend those observations to the P. aurelia complex. Immature
\
f
I5
o
o
10
3
o
I
o
2
o
o
3
9
o
2
o
2
o
3
5
4
3
5/8
h
418
318
218
o
118
20
018
o
7
o
9
6/8
9
o
o
6
2
o
3
818
o
718
Frequency of recipient clones which show various ratios of mating-reactive cells*
\
29
27
30
32
Total
- - --
*
t
(contiwi)
P. e t a r e a 'Mature cellsf
\ ccontron
o
o
o
o
3
3
3 18
o
o
o
5
418
I
o
6
3
518
5
4
7
8
618
As in Table 4.
Progenies of a cross between Bw and Cw, 15 fissions after conjugation.
f Mating-reactive cells of Bw.
o
o
o
o
2
o
218
/8
Immature cellst
I
o
o
018
A
13
9
5
9
718
Frequency of recipient clones which show various ratios of mating-reactive cells*
o
o
I
Immature cellst
Donor
(P. bursaria)
--
P. primourelin Mature cells1
Recipient
- -
14
I I
5
4
818
\
28
29
27
33
Total
Table j . Maintenance of mating reactivity in recipient clones when immature cytoplasm of P. bursaria was injected into
mature cells of P. primaurelia and P. tetraurelia
t
Reactivity in recipient clones (stock 16, hr* VII) 3 fissions after injection was tested with testers (stock 5 1 3 , hrd VIII).
Progenies of a cross between d m - 2 1 and dm-23, 15 fissions after conjugation.
f Mating-reactive cells of dm-23.
*
\
Immature cellst
P. primaurelia 'Mature cells1
(control)
Immature cellst
P. tetraurelia 'Mature cells1
(control)
Recipient
Donor
(P. caudatum)
Table 4:. Loss of mating reactivity in recipient clones when immature cytoplasm of P. caudatzm was injected into mature cells
of P. primaurelia and P. tetraurelia
U
sa
;s:
?(
\D
01
Immaturity substances in Paramecium
199
cells of P. primaurelia also contain an immaturity substance and its specificity is the
same as that of P. caudatum; immature cytoplasms of the 2 species are reciprocally
effective in inducing immaturity in mature cells. Though reciprocal injections were
unable to be done with P. multimicronucleatum, because immature cells were not
obtained in the available stocks of this species, the available evidence suggests that
the specificity of the immaturity substances is common in 3 species complexes of
the 'aurelia group': P. caudatum, P. aurelia and P. multimicronucleatum. Only one
species, P. bursaria, among those in the ' bursaria group' was tested and its immaturity
substance is different from that of the 'aurelia group'. Other species of the 'bursaria
group' should also be examined.
P. tetraurelia is a sibling species in P. aurelia complex which has no immaturity
period after conjugation but nevertheless responds to immaturity substances from
P. primaurelia and from P. caudatum. The cells of P. tetraurelia probably share with
P. primaurelia and P. caudatum a common receptor to the substances.
In the late part of the period of maturity in the P. aurelia complex, autogamy is
induced in response to food depletion (see Sonneborn, 1974). Whether the induction
of autogamy is delayed by the injection of the immaturity substances is an interesting
problem which is now under investigation.
The author wishes to thank Dr K. Hiwatashi for help in preparation of the manuscript.
This work was supported by a grant from the Japanese Ministry of Education, Science and
Culture.
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{Received 23 January 1979)
BLEYMAN,