J. Cell Set. 41, 177-191 (1980)
Printed in Great Britain © Company of Biologists Limited igSo
177
CLONAL DEATH ASSOCIATED WITH THE
NUMBER OF FISSIONS IN
PARAMECIUM CAUDATUM
YOSHIOMI TAKAGI AND MICHIKO YOSHIDA
Department of Biology, Nara Women's University, Nara 630, Japan
SUMMARY
Contrary to an earlier report suggesting clonal immortality in Paramecium caudatum, the
clones of the same species in this study terminated inevitably with a maximal life-span of 658
fissions and showed, prior to clonal death, decreased fission rate, increased probability of the
appearance of non-dividing or dead cells after cell division, and increased frequency of
morphological abnormality and of division asynchrony. Clonal senescence and death after a
limited number of fissions was reproducible even if subclones derived from the original clone
of a known fission age were examined again after a lag of 468 days. These results indicate that
clones of P. caudatum are mortal and that they use fissions, not days, to measure life-span.
Possible causes for the discrepancies between the earlier report and the present one are
discussed.
INTRODUCTION
We have attempted to test the hypothesis that clones of Paramecium caudatum age
and die. This hypothesis cannot be considered a priori correct. First, although the
reality of clonal aging was demonstrated in Paramecium aurelia by Sonneborn (1954)
and has been suggested in P. bursaria (Jennings, 1944; Siegel, 1967) and P. jenningsi
(Miyake & Miyake, 1967), senescence in other paramecia species remains still to be
studied. Second, some species of Tetrahymena, except some genetic strains, are
regarded as essentially immortal (Nanney, 1959, 1974; Nanney & McCoy, 1976).
There are some other species, such as Uroleptus mobilis (Calkins, 1919), Euplotes
crassus (Heckmann, 1967), E. patella (Katashima, 1971) and E. woodruffi (Kosaka,
1974), in which clonal senescence was suggested. Third, more directly, old work by
Galadjieff & Metalnikow (1933) on P. caudatum suggested a limitless capacity for
cell division, and this claim has remained unchallenged.
The present study indicates, if not proves, the reality of clonal aging terminated
by death in P. caudatum and suggests the importance of fission age rather than
calendar age in the determination of clonal life-span. The results have partly been
reported in abstracts (Takagi & Yoshida, 1977; Yoshida & Takagi, 1978).
178
Y. Takagi and M. Yoshida
MATERIALS AND METHODS
Stocks
Stocks Ksy-i (mating type V), dNi4a (VI), 34 (V) and 39 (VI) of Paramecium caudatum,
syngen 3 were the sources of the clones studied. Several hundred clones were obtained from
Ksy-i x dNi4a and from S4 x so,, but those derived through macronuclear regeneration and
those dying soon after conjugation were discarded. Among the rest, 6 vigorous clones that
reached the 150th fission were used to estimate the clonal life-span. Of these 6, clones 7a,
16a and 17a were derived from the cross Ksy-i x dNi4a, and clones D6a, D6b and Mi8a
were derived from the cross 34 x 39. D6a and D6b were sister clones originating from a pair
of exconjugants. All of the clones except 17a were mating type V.
Culture conditions
The culture medium was 2 % lettuce juice in 2 mM sodium phosphate buffer solution,
pH 68, inoculated 1 day before use with Aerobacter aerogenes. The temperature was kept at
about 25 CC throughout the study unless otherwise stated.
Maintenance of cell lines
Cell lines were maintained in serial reisolation cultures: single cells were put in slide
depressions (3 depressions per slide) containing 0-3-0-5 ml culture medium, the number of
daughter cells was counted, and one of them was reisolated with a micropipette under a
dissecting microscope into fresh medium. This procedure was repeated every alternate day in
Experiment I, and daily in Experiment II.
Experiment I
Experiment I was designed to estimate the clonal life-span. Conjugation indicated zero time.
At a given number of fissions after conjugation, 3 cells were selected randomly to expand 3
lines: this was done at the age of 5 or 6 fissions in 7a, 16a, 17a and Mi8a; at the age of 14
fissions in D6a and D6b. Lines were thereafter maintained by alternate-day isolations. The
cells remaining after reisolation were supplied with food to give rise to surplus cultures
consisting of about io3 cells: they were used for replacement of lines, for testing mating
reactivity to determine the onset of maturity, and for inspecting abnormal cells and selfers
(intraclonal conjugation). To check selfers, not only were selfing pairs looked for under a
dissecting microscope but also samples of 200-300 cells in stationary phase were stained with
acetocarmine to determine whether cells with fragmented macronuclei characterizing the
completion of selfing conjugation were included. Evidence for selfing was never detected in the
surplus cultures throughout the experiment. The surplus cultures were, with intervals of
about 50 fissions, further grown to mass cultures of about 1-6 X io4 cells in 18-ml test tubes,
which were kept at 17 °C as stock cultures and maintained by replacing half of the culture with
fresh culture medium about every 3 weeks.
Each clone was, as a rule, maintained in a set of 3 reisolation lines. Cells for reisolation were
usually selected at random; but, if the number of fission-products deviated from 2" {it: integer)
or if visibly abnormal cells were present, the most rapidly dividing and visibly normal cell
was selected. Lines which became extinct were replaced whenever possible by isolating cells
from other lines of the set or from the most recent surplus culture. Isolations from the surplus
cultures were done only when the surplus cultures were still in logarithmic phase of growth.
The number of lines per set was increased to 6 by replicating the set of 3 lines when replacements were needed frequently in later stages of clonal life. The time when this was done
differed from clone to clone. The sum of the number of fissions in the longest-lived line of the
set was taken as the life-span of the clone.
Experiment II
Experiment II was designed to make the closed life history revealed in Experiment I more
certain. From one of the stock cultures of Mi8a, which had been kept at 17 °C for 468 days
Clonal death in Paramecium
179
since the 178th fission, 10 cells were isolated randomly and allowed to divide twice to initiate
10 subclones each consisting of a set of 3 lines (30 daily reisolation lines in total). The number
of fissions during the stock culture was estimated to be 60 from growth curves of 20 serial subcultures produced for 468 days. Thus the fission age when 30 daily reisolation lines were initiated was estimated to be 240 (178 fissions during the period from conjugation, to the transfer
to the stock culture, plus 60 fissions during the stock culture at 17 °C for 468 days, plus 2
fissions for the expansion to 3 lines).
Ten subclones or 30 lines were halved and maintained by Y. T. and M. Y., independently.
In Experiment II, the total number of reisolation lines was fixed to 15 for each person; the
number of lines of some subclones was increased from 3 to 6, 9 and so on, after the other
subclones died out.
Other experimental procedures were the same as in Experiment I. Again, no selfing was
observed in the surplus cultures throughout Experiment II.
Evaluation of division potential
As shown diagrammatically below, daily reisolation lines are composed of branch lines
produced by the expansion or replacement (in this diagram, 3 daily reisolation lines are
composed of 6 branch lines).
X = dead or non-dividing cell
Evaluation of division potential was made in 2 ways. First, the frequency of non-dividing or
dead cells in the set of lines was designated as % replacement; the number of reisolations
(arrows) ending in X was divided by the number of all the reisolations during a given interval
(5/3° o r 1&'7% ' n 10-day period of this diagram). Second, the longevity of each branch line
measured in fissions was summed and averaged during a given interval.
RESULTS
Estimate of clonal life-span
In Experiment I, clonal life-span was estimated with 6 clones derived from 2
crosses (Table 1). Although the clonal life-spans varied greatly from clone to clone,
progeny clones from S4 x so, lived longer than those from Ksy-i x dNi4a. Mi8a
lived longest; the clonal life-span was 478 fissions if represented by a set of 3 lines or
527 fissions if represented by a set of 6 lines.
Changes in mean fission rates and in the frequency of replacements for successive
1 o-day periods throughout the life history in these clones are shown in Fig. 1.
The first 10-day means of fission rate were generally low and were followed by a
rapid or gradual increase to the peak level. A fission rate at near the peak was maintained for a long time in 16a, D6a and D6b, while the peak of fission rate was followed
by a sudden or gradual decline in 7a, 17a and Mi8a.
Replacement of lines was not common during about the first 100 fissions, but was
necessary continuously and increasingly during about the last 100 fissions of the
180
Y. Takagi and M. Yoshida
life history. It was, however, continuously necessary throughout the life cycle of
clone 17a.
The first appearance of visibly abnormal cells in 7a and 16a was at 87 and 152
fissions, respectively. In the other clones, it was roughly the same as that of the first
replacement.
Table 1. Life-spans of 6 clones studied
Maximal life-span in
fissions when the clone
is represented by
Cross
Ksy x dNi4a
34 x 89
1
Clone
Mating
type
7a
V
192
192
16a
17a
V
337
395
VI
V
V
V
157
213
402
402
445
480
478
527
D6a
D6b
Mi8a
3 lines
6 lines
Each clone was cultivated in a set of 3 lines of serial alternate-day isolation cultures. The
number of lines per set was later increased to 6 by replicating the initial set of 3 lines. Replacements were allowed within the set of lines if some lines failed to continue. The total number of
fissions from conjugation to death in the longest-lived line in the set was taken as the life-span
of the clone.
Reproducibility of the fission life-span
In Experiment II, one of the 6 clones, Mi8a, was examined again regarding
longevity. Ten subclones each consisting of a set of 3 lines were started at the parental
age of 240 fissions after the surplus culture of the 178th fission had been kept at
17 °C for 468 days.
The results are summarized in Table 2. Residual life-spans in fissions of 30 lines
ranged from 10 to 230 with a mean of 141-9 (Ax) or total life-spans ranging from 250
to 470 with a mean of 381-9 (A2). The mean total life-span of 10 subclones was 422-9
fissions if represented by the longest-lived line in the non-replaceable set of 3 lines
(A3); 478-1, 543-4 and 626 fissions if represented by the longest-lived line in the
replaceable set of 3 (B), 6 (C) and 15 lines (D), respectively. The difference of the
life-span between subclones maintained by Y.T. (Y1-Y5) and those maintained by
M.Y. (M1-M5) was insignificant, even if comparisons were made with A2 data
(380-6 + 60-5 vs. 383-3 ±56-3) or A3 data (431-4122-4 vs. 414-4137-5) or B data
(489-0 ± 50-8 vs. 467-2 + 56-7). This suggests that the termination of lines reflects not
a technical artefact but some intrinsic process. Although the maximal life-span of
Mi8a was finally lengthened to 658 fissions, the initially estimated value of 478
fissions or 527 fissions (p. 179) was reproducible, if comparisons were made in the
corresponding scale of the set of lines: 478 vs. 478-1 + 52-0 or 527 vs. 543-4 + 21-2.
This good agreement indicates that clones of P. caudatum use fissions or something
correlated with fissions, not days, to measure duration of life history.
Clonal death in Paramecium
181
r-100
-60
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-100
-60
-20
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-100
-60
-20
£
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-60
2ft
-20
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84 100
* i \_r--r
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i
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1 i
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i
400
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-100
E
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3211 2 3 4 5 6 7 8 9 10 11121314 1516171819 2021222324 2526272829
10-day periods
Fig. i. Changes in mean fission rates (solid line) and percent replacements (dotted
line) for successive 10-day periods throughout the life history of 6 clones: A, 7a;
B, 16a; c, 17a; D, D6a; E, D6b; F, MI8a. Each clone was subcultured to 6 lines, which
were maintained in alternate day-isolation cultures and allowed to replace one another
if some line became extinct. The clone was represented by the longest-lived line.
Every iooth fission and the fission age at the onset of maturity are marked off by
arrows and open arrows, respectively.
Table
2.
Life-spans of
10
subclotres or
30
lines infisions
+
Ten subclones (30 lines) were initiated from the clone M18a 240 fissions old. They were halved and maintained by Y. T. (YI-Yg) and
M. Y. (MI-Mg) independently. Each line was cultivated in daily reisolation cultures until termination without any replacement; the number
of fissions during this period is shown in A,; the total number of fissions since conjugation (A, 240) in A,; the largest number of fissions
in each set of 3 lines in A,. After termination of any one of the lines, replacements were allowed within the set of 3 lines. T h e number of
lines per subclone was increased to 6-15 in later periods. The life-span of subclones is also shown as the total number of fissions since conjugation in the longest-lived line in a set of 3 (El) or 6 (C) or 15 lines (D).
S.D. = standard deviation.
184
Y. Takagi and M. Yoshida
On the basis that clonal life-span varied greatly among lines and could be lengthened
by an increase in the number of lines, a question may be raised whether an indefinite
elongation of clonal life history may be possible. One will never be able to answer to
this question strictly, since it is impossible to follow the fate of all the cell lines
produced during 658fissionsor more. But indirect evidence against clonal immortality
is given in Fig. 2, which shows that replacements in later stages resulted in branch
£
Y4a
Y4b
Y4c
Y4-
No. of fissions
40
80
160
120
200
240
280
320
360
Fig. 2. Longevity in fissions of branch lines of the subclone Y4, which was derived
from Mi8a 240 fissions old, here corresponding to o fission, and maintained in daily
reisolation cultures.
Table 3. Mean longevity in fissions of branch lines produced
at different ages of subclones
Subclone
Y4
Clonal age when
branch lines
were made
' 240
400—500
5OI-55O
55i-56o
561-570
571-580
581-590
591-600
/
Collectively"
2
4°
250-300
301-400
401-450
451-500
501-550
S51-600
". All the subclones except Y4 are included.
No. of
branch
lines
3
12
29
Longevity of
branch lines,
mean ± s.D.
I8I-O±6'9
32-9126-7
12-3 ±9-9
11
5-6 ± 6 3
12
39±3'3
3-7±3'9
i'4±i-8
19
30
6
27
3
29
ss
65
129
122
o-5±o-8
1376 ±59-0
883 ±66-5
37-0 ±45-7
io-2± 15-7
8-8±i5-i
6-2±7-9
4'0±4'8
Clonal death in Paramecium
185
lines with shorter longevity than replacements in earlier stages. This was not specific
for the subclone Y4, but a general trend in all the subclones as summarized in Table 3.
Some characteristics of clonal senescence
In addition to a decrease in division potential with age (Fig. 2, Table 3), some agecorrelated deteriorative changes were observed.
The starting age of 240 fissions in the 10 subclones can be considered to have been
at the period of peak fission rate (Fig. 1 F). Gradual decline in fission rate from the
peak level was again observed in the 10-day means of fission rates of 30 lines (Fig. 3).
Mean fission rate of 30 lines was higher than 3 (fission/day) for the first two 10-day
periods but decreased to less than 2 after the 7th 10-day period: the difference is
statistically highly significant (f-test; P < o-oi).
* 2{
2*
4X
5
Q
T
%
$
W
W ' 12 13 14 ' 15 16" 17 ' 18 ' 19 ' 20
10 • day periods
Fig. 3. Changes in mean fission rates for successive 10-day periods in 30 lines of 10
subclones, which were derived from Mi8a 240 fissions old and maintained in daily
reisolation cultures. Standard deviations are indicated by vertical bars.
Since the number of daughter cells produced from every reisolated cell was
recorded in Experiment II, the proportion of each daughter cell-number was calculated
every 50 fissions in all the reisolation lines. Changes in the frequency distribution of
the daughter cell-number with age are shown in Fig. 4. During the first 50fissions,a
majority of the daughter cell-number was 8. But with age the proportion of 8 and
those larger than 8 decreased gradually and, instead, the proportion of 2, 3, and 4
increased gradually. Fig. 4 shows also a gradual increase in frequency of non-dividing
and dead cells (black areas) with age.
Among the spectra of the daughter cell-numbers from 3 to 16, the numbers 4, 8
and 16 may be regarded as the products of synchronous cell divisions. If their
proportion is regarded as a tentative index of division synchrony, the index decreases
linearly with fission age (Fig. 5).
One of the most conspicuous properties of aged clones was the increased appearance
of morphologically abnormal cells: cells with cytoplasmic protrusions or vacuoles;
fat or round or tiny or twiggy cells; cells resulting from delay or complete failure of
cell divisions, which produced a chain of 2-8 cells, or L-shaped cells, or various
kinds of amorphous monsters, etc. Some abnormalities appearing in aged surplus
cultures older than 350 fissions are shown in Fig. 6. Uncoupling of cytokinesis with
karyokinesis was often observed (e.g. Fig. 6 c, E). Typical abnormal cells rarely, if
ever, appeared in surplus cultures of 30 lines by about 350 fissions and, if they did,
13
cm
41
Y. Takagi and M. Yoshida
i86
Distribution of daughter cell no.
No. of fissions
9101 1131415
5 6
i
0-50| 4
7
8
• • * • « « :
51-100 B_2_N
12
16
.-=_
-~*T,>V,,.
56
i
101-150
2
4
3
5 6
151-200
7
'II
8
5 6 7
-
•
»
^
201-250
2
251-300
3
4
5 6
i
R
Fig. 4. Changes in the frequency distribution of daughter cell-numbers for successive
50-fission periods in 30 lines of 10 subclones, which were derived from Mi8a 240
fissions old, here corresponding to o fission, and maintained in daily reisolation
cultures. All the reisolated cells were classified according to the number of daughter
cells produced in a day; the frequency of each daughter cell-number is represented
by the fraction area. Black fractions indicate the frequencies of the number of o and 1,
i.e. the frequency of dead and non-dividing cells.
50
51
I
100
101
151
201
150
200
250
251
)
300
Fission periods
Fig. 5. Changes in a tentative index of division synchrony for successive 50-fission
periods. On the basis of the data in Fig. 4, the percent fractions of 4, 8 and 16 cells
among those of more than 3 cells was regarded tentatively as an index of division
synchrony.
Fig. 6. Examples of abnormal cells appeared in old surplus cultures, x 150. A,
unequally dividing cell, stained with acetocarmine. B, single cell with protrusion at a
vestibular region, living, c, non-disjunctive dividing cell with unequal macronuclear
distribution, stained, D, non-disjunctive dividing cell with equal macronuclear distribution, stained. E, chained cells with unequal macronuclear distribution, stained.
The single cell (lower) had been united before staining to the end of the binuclear cell.
F, chained cells, living. G-l, monsters, living. J-L, giant monsters, stained.
Clonal death in Paramecium
187
13-2
188
Y. TakagiandM. Yoshida
they could produce normal cells after cell divisions. But subsequently they appeared
increasingly and produced either abnormal or dead cells by cell divisions with increasing probability with age. At last almost all the cells in surplus cultures were
more or less morphologically abnormal.
DISCUSSION
In order to estimate clonal life-span, the process of asexual reproduction must
never be interrupted by the occurrence of sexual reproduction. The classical work by
Woodruff (see Wichterman, 1953), which was interpreted as showing that clones of
P. aurelia have an unlimited life-span, was demonstrated to be erroneous by the
discovery of periodic autogamy (Sonneborn, 1954). The constant presence of excess
food in daily reisolation cultures was the method used to avoid occurrence of autogamy
and conjugation in P. aurelia. In P. caudatum, the method of reisolation on alternate
days appears to be adequate for this purpose, since fission rate is less than two thirds
of that in P. aurelia and there is no natural autogamy. The possibility of occurrence
of the sole sexual process, i.e. selfing, can be ruled out in the present study, since (1)
replacements either from the set of lines or from surplus cultures were made only
when the cultures were still in the logarithmic phase of growth, and (2) selfing was
in fact never detected in any surplus cultures used for replacements.
Galadjieff & Metalnikow (1933) reported the occurrence of 8704 fissions without
conjugation during the course of 22 years and 5 months in P. caudatum. This report
has remained unchallenged although there are now reasons to question its validity.
The study was performed before the discovery of mating type in Paramecium
(Sonneborn, 1937a) and before the establishment of modern concepts concerning
various sexual processes. At that time, endomixis was believed to occur in P. caudatum
as well as in P. aurelia (see, for example, Sonneborn, 19376, pp. 484-485). Although
originally described as a nuclear reconstruction process without meiosis, endomixis
was later demonstrated cytologically (Diller, 1936) and genetically (Sonneborn, 1939,
1947) to be autogamy. Present' knowledge excludes the occurrence of both endomixis and natural autogamy in P. caudatum (Hiwatashi, 1969). To examine their
results we may suppose that Galadjieff and Metalnikow isolated cells from the surplus
cultures containing exconjugants originated from selfing conjugation. They would
undoubtedly regard the cells as vegetative products, since nuclear changes in single
cells, if detected, would be regarded as endomixis. A careful reading of their report
led us to suspect that they made an oversight of selfing that could occur in the starved
surplus cultures they used for replacements.
Selfing in P. caudatum occurs seldom in the clone of odd-numbered mating type
(Hiwatashi, 1968; Myohara & Hiwatashi, 1975). Although the present clones are,
except 17a, all mating type V, we made a careful examination for selfing in various
test-tube cultures or flask cultures raised from stock cultures of known ages. We
found that selfing could take place in all the clones studied here under specific
conditions, i.e. in stationary phase of growth in old clones. In Mi8a, for example,
selfing was observed at a frequency of less than 1%, but only in a part of the cultures
Clonal death in Paramecium
189
that were older than 274 fissions. Judging from its low frequency and late expression,
selfing in the odd-numbered mating type appears to be under a different control
mechanism from selfing in the even-numbered mating type, which comes to expression
in a high frequency at an early period of maturity (Myohara & Hiwatashi, 1975):
the former phenomenon appears similar to that reported in Tetrahymena canadensis
(Outka, 1961) or Euplotes (Heckman, 1967; Katashima, 1971), in which selfing occurs
only in old clones. Anyway, this finding made us discard experimental data on 10
subclones (30 lines) of Mi8a reinitiated at the parental age of 450 fissions. In fact a
discrepant distribution of total life-spans in these subclones suggested the occurrence
of selfing in 4 subclones during the long period of periodic starvation in stock culture:
4 subclones showed a total life-span of 770 ± 70 fissions on average, ranging from
699 to 854, while the other 6 showed a total life-span of 546 + 36 fissions on average,
ranging from 478 to 579. Thus the former 4 subclones might involve 2 generations
including selfing progeny that would live for at least 249-414 fissions (699 or 854
minus 450).
The maximum life-span revealed in this study was 658 fissions. However, this
could be further lengthened by selection even if selfing was not allowed to occur.
The possibility of selecting more vigorous cells will increase as the population from
which cells are transferred to the next culture increases in size. This was shown in
this study as an effect of lengthening the life-span by an increase in the number of
lines from 3 to 6, 9 and so on. Even more effective selection would be realized in
mass cultures. However, the fact that, among 45 stocks of P. caudatum collected in
nature 30 years ago, only 1 stock which is a selfer has remained (K. Hiwatashi,
personal communication) suggests that clonal death is inevitable even in mass
cultures. We estimate that the maximum life-span of his stocks did not exceed
1000 fissions.
Changes associated with clonal senescence in P. aurelia include: (1) decline in
fission rate and in viability after sexual and asexual reproduction (Sonneborn, 1954;
Sonneborn & Schneller, i960; Fukushima, 1975; Rodermel & Smith-Sonneborn,
1977); (2) drift of sexual pattern from conjugation to autogamy (Sonneborn, 1957);
(3) increased frequency of cytological abnormality (Mitchison, 1955; Dippell, 1955;
Sonneborn, 1957; Sonneborn & Dippell, i960); (4) increased sensitivity to ultraviolet
(Smith-Sonneborn, 1971) and caffeine (Smith-Sonneborn, 1974); and (5) reduction
in DNA content (Schwartz & Meister, 1973; Klass & Smith-Sonneborn, 1976),
RNA synthesis, DNA template activity (Klass & Smith-Sonneborn, 1976) and
endocytic capacity (Smith-Sonneborn & Rodermel, 1976). In the present study,
some of these changes were also encountered in old clones: decline in fission rate,
increased probability of the appearance of non-dividing or dead cells after cell
division and increased frequency of morphological abnormality and of division
asynchrony.
The results in this study thus show it is very likely that clonal death in P. caudatum
is inevitable, if selfing is avoided, after about 600-700 fissions (1000 fissions as a
maximum) since the previous conjugation, although we cannot absolutely deny the
exceptional existence of an immortal cell line.
190
Y. Takagi and M. Yoshida
An additional finding in this study is that clonal life-span appears to be related to
number of fissions rather than to days elapsed. This association is consistent with the
results in P. aurelia (Smith-Sonneborn & Reed, 1976). In paramecia fission age, not
calendar age, is associated with the length of immaturity (Kroll & Barnett, 1968;
Takagi, 1970, 1974; Miwa & Hiwatashi, 1970). Although it is not yet clear whether
paramecia use a common cellular clock for both the timing of clonal maturity and
clonal death, the following observations are suggestive: (1) Both duration of immaturity (Siegel, 1961) and life-span (Smith-Sonneborn, Klass & Cotton, 1974) can be
shortened by increasing the parental age; and (2) both duration of immaturity
(Takagi, 1974) and life-span (Fukushima, 1974) can be shortened by u.v. and X-ray,
respectively. In this connexion, it would be interesting to study whether early
maturity mutants in P. caudatum (Myohara & Hiwatashi, 1978) have a shorter
life-span.
We would like to thank Dr Joan Smith-Sonneborn (University of Wyoming) and Dr Koichi
Hiwatashi (Tohoku University) for their helpful suggestions in preparation of this manuscript.
Gratitude is also expressed to Dr Tracy M. Sonneborn (Indiana University) for correcting the
English of our original manuscript and for his encouragement.
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DIPPELL, R. V. (1955). Some cytological aspects of aging in variety 4 of Paramecium aurelia.
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FUKUSHIMA, S. (1974). Effect of X-irradiations on the clonal life-span and fission rate in
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FUKUSHIMA, S. (1975). Clonal age and the proportion of defective progeny after autogamy in
Paramecium aurelia. Genetics, Princeton 79, 377-381.
GALADJIEFF, M. A. & METALNIKOW, S. (1933). L'immortalit£ de la cellule. Vingtdeux ans de
culture d'infusoires sans conjugaison. Archs Zool. exp. ght. 75, 331-352.
HECKMANN, K. (1967). Age-dependent intraclonal conjugation in Euplotes crassus. J. exp. Zool.
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(Received 26 May 1979 - Revised 9 August 1979)
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