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.

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 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.

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 S120, L120, S123, L123, S126 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 (LD12:12), 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

  3. Short day (LD 12:12), 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 0·1 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.

Sexual reproduction of experimental groups is summarized in Table 1 and Fig. 1, asexual reproduction in Table 2.

Table 1.

Sexual reproduction of sexually derived parent populations (L and S) and five generations of their asexually derived offspring L(1) to L(5), S(1) to S(5))

Sexual reproduction of sexually derived parent populations (L and S) and five generations of their asexually derived offspring L(1) to L(5), S(1) to S(5))
Sexual reproduction of sexually derived parent populations (L and S) and five generations of their asexually derived offspring L(1) to L(5), S(1) to S(5))
Table 2.

15-Day means of asexual reproduction of all groups in percent population

15-Day means of asexual reproduction of all groups in percent population
15-Day means of asexual reproduction of all groups in percent population
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 %.

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 %.

At 20 °C reproduction was largely sexual preceded by very limited fission. At 23 °C prolonged fission periods were followed by cocoon deposition, but 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.

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 régimes, 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.

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. 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 régime 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.

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 (1940a, b, 1941, 1967) has long pointed out 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.

Benazzi
,
M.
(
1940a
).
Nuove osservazioni sul determinism e sulla ereditarietà della riproduzione asessuale in una razza di Dugesia (Euplanarid) gonocephala
.
Boll. Zool.
11
,
25
31
.
Benazzi
,
M.
(
1940b
).
Sulla sterilità degli esemplari ex-scissipari di Dugesia (Euplanaria) gonocephala
.
Boll. Zool.
11
,
143
145
.
Benazzi
,
M.
(
1941
).
La sterilità 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 sessualità
.
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
.
J. Insect Physiol.
3
,
92
117
.
Lees
,
A. D.
(
1960
).
The role of photoperiod and temperature in the determination of parthenogenetic and sexual forms in the aphid Megoura viciae Buckton. II. The operation of the ‘interval timer’ in young clones
.
J. Insect Physiol.
4
,
154
175
.
Lees
,
A. D.
(
1963
).
The role of photoperiod and temperature in the determination of parthenogenetic and sexual forms in the aphid Megoura viciae Buckton. III. Further properties of the maternal switching mechanism in apterous aphids
.
J. Insect Physiol.
9
,
153
164
.
Vowinckel
,
C.
(
1970
).
Stimulation of germ cell proliferation in the planarian Dugesia tigrina (Girard)
.
J. Embryol. exp. Morph.
23
,
407
418
.
Vowinckel
,
C.
,
Wolfson
,
N.
&
Marsden
,
J. R.
(
1970
).
A crowding factor causing spontaneous decapitation in cultures of the planarian Dugesia tigrina.
Can. J. Zool.
48
,
1059
1062
.
Vowinckel
,
C.
&
Marsden
,
J. R.
(
1971
).
Reproduction of Dugesia tigrina under short-day and long-day conditions at different temperatures. I. Sexually derived individuals
.
J. Embryol. exp. Morph.
26
,
587
598
.