/. Embryol. exp. Morph. Vol. 26, 3, pp. 599-609, 1971
Printed in Great Britain
599
Reproduction of Dugesia tigrina under short-day
and long-day conditions at different temperatures
II. Asexually derived individuals
By C. VOWINCKEL 1 AND J. R. MARSDEN 1
From the Department of Biology, McGill University
SUMMARY
Five successive generations of asexually derived populations of D. tigrina and their sexually
derived parental population were reared under each of six combinations of 20, 23 and 26 °C
with 12 and 16 h of daylight. Asexual and sexual reproduction were recorded for each population.
Peaks of asexual reproduction occurred increasingly earlier in consecutive generations to
finally disappear or occur later again.
Peaks of sexual reproduction likewise occurred increasingly earlier in consecutive generations to be retarded again or disappear in later generations. Retardation occurs in earlier
generations under short-day conditions than under long-day conditions.
All generations later than the third, asexually derived, were sterile.
The implications of these results are discussed and it is suggested that the time of cocoon
deposition of all asexually derived offsprings is determined regardless of intervening generations during early development of the sexually derived parent. Attention is drawn to the
similarity between the suggested mechanism and environmental control of aphid reproduction.
INTRODUCTION
Planarian reproduction is profoundly influenced by daily illumination and
temperature. We have pointed out earlier (Vowinckel, 1970) that the effectiveness
of critical environmental factors varies with the previous history of the animal,
namely the pathway - sexual or asexual - through which the individual was
derived.
In a companion paper (Vowinckel & Marsden, 1971) we have explored the
effect of long and short days, at three temperature levels, on the reproduction
of sexually derived (i.e. cocoon hatched) individuals of Dugesia tigrina. We came
to the conclusion that constant temperatures play a permissive non-inductive
role. High temperatures (26 °C) exclude sexual reproduction but permit asexual
reproduction. Conversely, low temperatures (20 °C) permit sexual reproduction
and limit asexual reproduction. At 23 °C both pathways struggle for expression.
The daily photoperiod, on the other hand, has a regulatory function. Constant
1
Authors' address: Department of Biology, McGill University, P.O. Box 6070, Montreal
101, Canada.
600
C. VOWINCKEL AND J. R. MARSDEN
short-day conditions (LD 12:12) [L = light; D = dark] were correlated with
shorter generation lengths than long days (LD 16:8) and it appears that the
period of earliest development within the maternal body, before enclosure in the
cocoon, is the time span of an individual's life most implicated as the period
sensitive to photoperiod induction of generation length. Daylength also influences
the number of worms which hatch from a cocoon. Twice as many worms
hatched from cocoons laid under short days than under long days.
In the present paper, then, we explore the reproductive pathways and success
of individuals which did not hatch from cocoons but were derived asexually
(and under the same environmental conditions) from the populations used in the
study on reproduction of sexually derived individuals.
MATERIALS AND METHODS
The experiments discussed below were all executed simultaneously with and
in the same incubators as those dealing with cocoon hatched animals. Greater
details of material, methods and record collection are given in the companion
paper.
Material. Sexually mature individuals of D. tigrina were collected from the
St Lawrence River and laid cocoons under daily illuminations of LD12:12
(On from 8 a.m. to 8 p.m. EST - eastern standard time) and LD 16:8 (On from
6 a.m. to 10 p.m. EST) both at 20 °C. These cocoons were distributed to form
one population in each of six incubators representing all possible variations of
the three temperature levels and two photoperiods. The six populations hatching
from these cocoons are here termed the parental populations and are identical to
populations S^O, L]20, S ^ , Li23, SX26 and L26 of the companion paper.
Experimental design. Asexually derived populations were established in the
following manner: fission products were collected from the parental populations
and reared separately under the same conditions and in the same incubator as
the parents. They were termed the first-generation fission products. They in turn
gave rise asexually to the second-generation fission products which again were
separately maintained. Establishment of further asexually derived generations
continued up to and including the fifth generation for each of the six different
parental populations leading to the following serial arrangements:
1. Short day (LD 12:12), 20 °C
S20: parental population
S(l)20: first-generation fission products
S(2): 20: second-generation fission products
S(3):20: third-generation fission products
S(4)20: fourth-generation fission products
S(5): 20: fifth-generation fission products
2. Long day (LD 16:8), 20 °C
L20, L(l)20, L(2)20, L(3)20, L(4)20, L(5)20
Reproduction o/Dugesia tigrina. / /
601
3. Short day (LD12:12), 23 °C
S23, S(l)23, S(2)23, S(3)23, S(4)23, S(5)23
4. Long day (LD 16:8), 23 °C
L23, L(l)23, L(2)23, L(3)23, L(4)23, L(5)23
5. Short day (LD 12:12), 26 °C
S26, S(l)26, S(2)26, S(3)26, S(4)26, S(5)26
6. Long day (LD 16:8), 26 °C
L26, L(l)26, L(2)26, L(3)26, L(4)26, L(5)26
Fission products of each population were collected from the start of asexual
reproduction (in that population) until approximately 60 individuals of the next
generation were accumulated. Later fission products were recorded and discarded. This led to populations of relatively uniform age. At 20 °C asexual
reproduction is very limited, many later generations therefore only formed small
populations.
All cocoons laid by the experimental populations were separately maintained
under room temperature and natural daylight. The number of worms hatching
from these cocoons was recorded.
Maintenance. All series were maintained in incubators at controlled temperatures and photoperiods. Both temperature and light were continuously monitored by a multi-channel a.c.-d.c. recorder. Photoperiod was automatically
regulated. The light source was a shaded 9 in (22-8 cm) fluorescent light of less
than 750 lux intensity at 10 cm distance. Diurnal temperature variations stayed
below ±0-5 °C. Due to the long span of the experiment (over 1 year) and
consequent slow changes of the room temperature the overall variation stayed
only just below ±1-0 °C. Since these changes affected all incubators in a parallel
fashion we do not think that our results were much influenced by them.
Animals were maintained in glass containers at population densities not above
01 worm/ml (Vowinckel, Wolfson & Marsden, 1970) in non-chlorinated river
water. All containers were covered with non-translucent materials. Beef liver
was fed to all series 4-5 times weekly on an all or none basis. Cultures were
cleaned on the same day and water replaced at the same temperature. Fission
products and cocoons were recorded daily except weekends and removed. All
losses from the population were likewise recorded. The latter resulted mostly
from animals that crawled under the lid and dried up. Animals that fissioned
anterior to the pharynx were removed and counted as losses.
RESULTS
Sexual reproduction of experimental groups is summarized in Table 1 and
Fig. 1, asexual reproduction in Table 2.
At 20 °C reproduction was largely sexual preceded by very limited fission.
At 23 °C prolonged fission periods were followed by cocoon deposition, but
602
C. VOWINCKEL AND J. R. MARSDEN
these cocoons were infertile with the exception of the long-day first-generation
group. At 26 °C no sexual development was observed in any population. After
high initial fission rates lasting for 2 months the long-day populations showed
signs of exhaustion. After another month of decreased and irregular fissioning
worms began to die. At this point all 26 °C populations were shifted to 20 °C to
see if they were still capable of sexual reproduction.
I
50
100
150
I
I
I
200
I
I
I
I
I
250
I
I
I
I
I
300
Age of population (days)
Fig. 1. Asexual and sexual reproduction of parental populations (L and S) and
successive generations of their asexually derived offsprings at 20 °C in percent of
population producing a cocoon or fissioning. Asexual reproduction, in white, is
expressed as 5-day means and sexual reproduction, in black, as 5-day sums. All L
under long-day, all S under short-day illumination. S(4), S(5) and L(5) did not reproduce sexually and are omitted. Figures to the right: population size. Asterisk: end of
record. Black bars added above white bars. All values below 1 % entered as 1 %.
L(2)
L(3)
L(4)
L(5)
S
S(l)
S(2)
S(3)
S(4)
S(5)
L(D
L
Population
60
54
58
39
17
12
40
24
7
6
2
2
94
37
23
9
12
0
34
24
18
2
0
0
43
3
13
11
—
—
62
—
22
100
—
—
2-2
20
1-3
20
—
—
4-9
—
3-3
1-5
—
—
Total
Popu- cocoons
%
Mean,
lation investi- fertile worms/
size N gated cocoons cocoon
20 °C
±0-84
—
—
—
—
—
±0-94
—
—
—
—
—
S.D.
of
mean
S(2)
S(3)
S(4)
S(5)
SO)
S
L(2)
L(3)
L(4)
L(5)
L
UD
36
48
51
56
58
52
52
62
47
43
62
48
2
23
4
11
0
0
15
0
0
0
0
0
10
Total
Popu- cocoons
%
Mean,
Popu- lation investi- fertile worms/
lation size N gated cocoons cocoon
23 °C
—
S.D.
of
mean
All L under long day, all S under short day.
S(2)
S(3)
S(4)
S(5)
S(D
S
L(2)
L(3)
L(4)
L(5)
UD
L
30
39
39
34
24
13
27
51
58
49
56
21
14
84
52
1
0
5
7
9
0
0
0
0
7
32
50
10
3-8
3-3
Total
Popu- cocoons
%
Mean,
Popu- lation investi- fertile worms/
lation size TV gated cocoons cocoon
26-20 °C
Table 1. Sexual reproduction of sexually derived parent populations {L and S) and five
generations of their asexually derived offspring (L(l) to L(5), S(l) to S(5))
—
±1-51
±1-17
S.D.
of
mean
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Reproduction o/Dugesia tigrina. / /
605
Sexual reproduction (Fig. 1 and Table 1) presents several features that are of
interest:
(1) At 20 °C successive generations reproduced sexually according to a definite
pattern (Fig. 1). The peak of reproductive activity was first shifted forward with
regard to age. This antecedence was most pronounced in the long-day generations.
In later generations the reproductive peak always was delayed again. This
regression included more generations under short-day than under long-day
conditions. Thus long days elicited three generations where the peak was
advanced followed by one generation where it regressed again. Short days only
elicited one generation with a preceded peak followed by two where it regressed.
At 23 °C and long days this scheme was only weekly maintained. The first
and third generations bred increasingly early but the second generation straggled.
Only very few cocoons were laid in every case. Under short-day conditions at
this temperature no sexual reproduction at all took place in the asexually
derived series.
(2) There is a quick and pronounced decrease in fertility observable in all
lines of descent. Under short-day regimes, asexually derived populations at 23
and 26-20 °C are completely sterile. At 20 °C four fertile cocoons are laid each
by the long- and short-day lines of descent. However, the four cocoons laid under
short days at 20 °C are the only fertile cocoons ever laid by short-day asexually
derived generations. Things are not quite as barren in the long-day generations.
Two fertile cocoons were laid at 23 °C and fertility comparable to that of the
parental population is retained in the first two generations of long-day populations shifted from 26 to 20 °C. We, therefore, find greater fertility of asexually
derived generations under long-day conditions.
(3) The number of successive generations that lay cocoons are higher under
long-day illumination than under short days at all temperatures.
(4) Under long-day conditions at 26 °C shifted to 20 °C the parental cocoon
hatched population shows smaller cocoon production and less fertility than the
asexually derived first and second generation. This is also expressed at 23 °C
though fertility is very low.
(5) The exhaustion of populations at 26 °C occurred only under long-day
conditions. Short-day populations were quite unaffected.
Asexual reproduction (Table 2) complements sexual reproduction and one
further feature of interest emerges:
(6) We find a shift of peaks of reproductive activity - this time with respect to
fission - comparable to that observed earlier for cocoon production (see (1)
above). Again the period of greatest reproductive activity shifts in successive
filial populations of the same line first towards increasingly earlier ages and later
recedes and/or disappears. This is true, both, for long- and short-day illumination.
606
C. YOWINCKEL AND J. R. MARSDEN
DISCUSSION
Our results show that the reproductive pattern of asexually derived populations differs from that of sexually derived populations in several ways. It differs
first of all in the timing. Asexually derived generations breed in a definite
pattern which in some way seems related to their distance in time or in the
number of intervening asexual generations from the sexually derived parent
population. It differs furthermore in fertility. Asexually derived generations
under constant conditions of temperature and light patterns are highly infertile
when compared with the sexually derived parental populations. In contrast the
long-day generations L(l) and L(2), which were shifted from 26 to 20 °C,
approach normal fertility.
Our results therefore bear out the earlier suggestion we made that the effectiveness of critical environmental factors varies depending on the pathway through
which the individual was derived.
How, then, could reproduction be regulated in planarian populations consisting of sexually and asexually derived members ? In the companion paper
we concluded that in sexually derived individuals photoperiod controls the
timing and other factors of reproduction. In particular we suggested that a
period of sensitivity to photoperiod induction ought to be found very early
during the existence of an individual. The environmental factor, we assumed,
acts at this time on and through the maternal body which would, via some factor,
determine the future reproductive pattern of the emerging individual in the
developing ovum or very early embryo.
In asexually derived individuals quite obviously neither the time in the
maternal ovary nor in the cocoon can be considered as a phase sensitive to
inductive stimuli since they do not exist. Nor would it be easy to establish that
a later period during development represents the 'sensitive period'. It would
then have to be assumed that the same period acts differently to the same
stimulus, depending on the time or number of generations which have intervened since the cocoon-hatched ancestor. Any attempt to explain the accelerated
reproduction in succeeding generations on the basis of individual induction,
being renewed in every generation, must for this reason become quite complicated.
It seems simpler to explain our results by invoking a timing mechanism which
seems to be, in most respects, analogous to one found in aphids. We would,
therefore, suggest the following interpretation which owes much to the papers
by Lees mentioned below: the time of future cocoon production may be determined only once, namely in the cocoon-hatched ancestor which, then, gives rise
to a small clone of several generations of asexually derived descendants during
the summer. One would have to assume that a factor is passed on from this
ancestral individual through the successive fission products. This factor remains
latent. It becomes active only at a set time after its induction in the ancestor.
Reproduction o/Dugesia tigrina. II
607
It becomes active in all clone members at the same time, regardless of the
number of intervening generations, leading to increasingly earlier reproductive
activity in the life cycle of successive generations. It leads to approximately
synchronized cocoon deposition in a clone.
In aphids we find an arrangement where the switch-over from parthenogenetic
to sexual reproduction in a population seems regulated by just such a factor,
an 'interval-timer' as Lees (1959, 1960, 1963) has termed it. This is induced in
the fundatrices of clones, that is the first animals to hatch from eggs in spring
which are, themselves, derived through sexual reproduction. The factor, passed
on through succeeding parthenogenetic generations, becomes active at the same
time in all descendants of the fundatrix, regardless of the number of intervening
generations.
How do our results and observations from the natural population fit such a
scheme ? Cocoons, under natural conditions which are of course never constant,
are laid during early July. This means that in our latitude they receive approximately 16 h of daylight. In our experimentation, therefore, the long-day regime
most closely approaches natural inductive conditions. It is in the long-day, 20 °C
generations that we find the best example of antecedence of sexual reproduction.
It extends down to and probably includes the third generation. In the natural
habitat sexually derived animals begin to hatch around the fifteenth of July.
The time span between the start of successive generations is approximately
3 weeks at 23 °C and 2 weeks at 26 °C if we accept our experimental data as
representative.
Now, even if we calculate on a 2-week basis the third generation could hardly
arise before the first of September. It is about this time that we observe the
termination of fissioning in the natural population. This would mean that the
production of more than three successive generations of asexually derived
individuals per season would be a rare event. It is therefore these first three
generations for which a timing mechanism has evolved in the material with which
we are working. Later experimental generations need not necessarily be expected
to conform. In other words, the factor which, as we have suggested, acts as
timer would be set to become active in such a way as to include the third but no
later generations if induced in the ancestor under a photoperiod of LD16:8.
In later generations, artificially generated, the timing mechanism then might
become active too early for the worm to respond, leading to retardation of sexual
reproduction possibly through continued deactivation or dilution of the factor.
The same argument would lead us to expect this retardation much earlier
under short-day conditions. The shorter generation time would demand a much
earlier activation of the 'interval timer'. The earliest developmental age at which
individuals are capable to respond would, therefore, be encountered in earlier
generations. From Fig. 1 we note that already the first short-day generation
responds as early or even earlier than the third generation under long-day
conditions and retardation begins already with the second generation.
608
C. VOWINCKEL AND J. R. MARSDEN
The shifting peaks in asexual reproduction give us a further indication of the
number of generations on which the factor can act before its timing ceases to
coordinate with development. At 20 °C and short days antecedence occurs up to
and including the second generation in both sexual and asexual reproduction.
Under long days the second and third generation start sexual reproduction at
the same age but the peak of asexual reproduction shows that the third generation is already somewhat retarded.
At 23 °C generations succeed each other faster and antecedence gives way to
retardation only in the fifth generation under both daylengths. At 20 °C
retardation begins already in the third generation. This difference is the only
indication we have that the interval timer may not depend on the number of
generations evolved since the sexually derived ancestor, but on the actual time
passed since the ancestors induction.
It emerges from our experiments that a successive line of asexually derived
generations inevitably appears to become sexually sterile. These results have
been quite uniform with our stock. The process can be retarded by a shift to
lower temperatures but does not seem to be preventable. Not only do successive
generations lay fewer cocoons but the fertility of these cocoons decreases even
more rapidly. In the companion paper we pointed out the frequent presence of
supernumerary gonopores in the parental populations and suggested that they
might be responsible, in part, for the number of sterile cocoons laid. This would
be much less true for asexually derived populations since excess gonopores
became fewer in later generations. Fifth generations showed no excess gonopores
at all.
The decrease in fertility of cocoons is most pronounced under short-day
conditions where almost complete or complete infertility results at all temperature levels although at 20 °C cocoons are deposited by the first three asexually
derived generations. Under long-day conditions the fertility of cocoons is
exhausted less rapidly but Table 1 shows that from the third generation on
cocoons are all sterile. This effect is not altered by a shift of water temperatures
from 26 to 20 °C. While the percentage of fertile cocoons in the first two
generations rises sharply under such conditions the temperature change does not
increase the fertility of later generations as compared with those kept at 20 °C
without any temperature shift. However, the sharp rise in fertility in the first
two generations is of special interest since we pointed out earlier (Vowinckel,
1970) that a drop in water temperature stimulates germ-cell proliferation. It also
indicates that in the natural population asexually derived individuals probably
are fully functional members of the breeding population. Sudden temperature
changes of 6 °C and more have been recorded in the natural habitat during
August and we already pointed out above that only about three asexually
derived generations could be expected per season under natural conditions.
The final sterility of asexually derived planarians is not peculiar to our
experimental arrangement. Benazzi (1940#, b, 1941, 1967) has long pointed out
Reproduction
of Dugesia tigrina. / /
609
this phenomenon and, by crossing, showed that it is the female gametes which
become sterile first in asexually derived planarians. This, then, would be in
contrast to the possible failure of sperm survival which, in the companion paper,
we suggested as a reason for the general sterility of cocoons at 23 °C.
We thus find under short-day conditions parental populations which lay
cocoons that have a higher percentage fertility and hatch a greater number of
worms/cocoon than populations under long-day conditions. At the same time
we find that the generations derived asexually from these more fertile short-day
parent populations are even more liable to be sterile than the long-day generations. To us this suggests the exhaustion, earlier in the case of short-day generations and later in the case of long-day generations, of a limited supply of germ
cells.
The widely accepted theory of the totipotency of the adult planarian's neoblast
cell would be somewhat in disagreement with such a suggestion. If neoblasts
were totipotent it seems to us that sterile generations should not arise. However,
since we want to take up this question in a separate publication we limit ourselves
here to pointing out the problem.
We are indebted to Professor K. G. Davey for drawing our attention to A. D. Lees' work.
This investigation was supported by a grant from the National Research Council of Canada.
REFERENCES
BENAZzr, M. (1940a). Nuove osservazioni sul determinismo e sulla ereditarieta della riproduzione asessuale in una razza di Dugesia {Euplanaria) gonocephala. Boll. Zool. 11, 25-31.
BENAZzr, M. (19406). Sulla sterilita degli esemplari ex-scissipari di Dugesia {Euplanaria)
gonocephala. Boll. Zool. 11, 143-145.
BENAZZI, M. (1941). La sterilita degli esemplari ex-scissipari di Dugesia {Euplanaria) gonocephala Duges dipende dal gamete femminile. Boll. Zool. 12, 143-148.
BENAZZI, M. (1967). Considerazioni sui rapporti tra moltiplicazione agamica e sessualita.
Accad. Naz. dei Line. (Ser. 8), 42, 742-746.
LEES, A. D. (1959). The role of photoperiod and temperature in the determination of parthenogenetic and sexual forms in the aphid Megoura viciae Buckton. I. The influence of these
factors on apterous virginoparae and their progeny. /. Insect Physiol. 3, 92—117.
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(Manuscript received 13 May 1971)