INTEGR. COMP. BIOL., 43:242–246 (2003)
Switch from Asexual to Sexual Reproduction in the Planarian Dugesia ryukyuensis1
MOTONORI HOSHI,*,2,3 KAZUYA KOBAYASHI,†,2 SACHIKO ARIOKA,*,2 SUMITAKA HASE,‡
AND
MIDORI MATSUMOTO*,2
*Department of Chemistry and Center for Life Science and Technology, Keio University, Hiyoshi, Yokohama 223-8522, Japan
†Form and Function, PRESTO, Japan Science and Technology Corporation, Saitama 332-0012, Japan
‡Department of Biological Sciences, Tokyo Institute of Technology, Nagatsuta, Yokohama 226-8501, Japan
SYNOPSIS. Many metazoans convert the reproductive modes presumably depending upon the environmental conditions and/or the phase of life cycle, but the mechanisms underlying the switching from asexual to
sexual reproduction, and vice versa, remain unknown. We established an experimental system, using an
integrative biology approach, to analyze the mechanism in the planarian, Dugesia ryukyuensis (Kobayashi
et al., 1999). Worms of exclusively asexual clone (OH strain) of the species gradually develop ovaries, testes
and other sexual organs, then copulate and eventually lay cocoons filled with fertilized eggs, if they are fed
with sexually mature worms of Bdellocephala brunnea (an exclusively oviparous species). This suggests the
existence of a sexualizing substance(s) in sexually mature worms. Random inbreeding of experimentally
sexualized worms (acquired sexuals) produces an F1 population of spontaneous sexuals (innate sexuals) and
asexuals in a ratio of approximately 2:1. All regenerants from various portions of innate sexuals become
sexuals. In the case of acquired sexuals, head fragments without sexual organs regenerated into asexuals
though regenerants from other portions became sexuals. Thus, we conclude that neoblasts, the totipotent
stem cells in the planarians, of acquired sexuals remain ‘‘asexual’’ and the worms require external supply
of a sexualizing substance for the differentiation of sexual organs and gametes. On the other hand, some, if
not all, neoblasts in innate sexuals are somehow ‘‘sexual’’ and do not require external supply of a sexualizing
substance for the eventual differentiation of themselves and/or other neoblasts into sexual organs and gametes. It is also shown that sexuality in acquired sexuals is maintained by the putative sexualizing substance(s)
of their own. The sexualization is closely coupled with cessation of fission, and the worms seem to have an
unknown way of controlling the karyotype. Our integrative approach integrates multiple fields of study,
including classic breeding, regeneration, and genetics experiments, as well as karyotyping, and biochemical
and molecular biological analyses; none of which would have revealed much about the intricate mechanisms
that regulate sex and fission in these animals.
INTRODUCTION
Asexual reproduction in a strict sense, namely amixis, is the reproduction without meiosis and syngamy.
This type of reproduction is widespread in many animal phyla including Porifera, Placozoa, Mesozoa, Cnidaria, Ctenophora, Platyhelminthes, Nemertea, Entoprocta, Rotifera, Sipuncula, Annelida, Arthropoda,
Phoronida, Ectoprocta, Echinodermata, Hemichordata
and Chordata (Bell, 1982; Pearse et al., 1989). Most
of those that can asexually reproduce convert their reproductive modes from asexual to sexual reproduction,
and vice versa, sometime in their life or in some phase
of life cycle, and those like bdelloid rotifers that reproduce exclusively asexually are very rare (Welch
and Meselson, 2000). However, the mechanisms underlying the switch of reproductive modes in metazoans remain hardly known.
It is known that some planarians, like those in the
genus Dugesia, comprise at least three races with respect to the reproductive mode: asexual race, sexual
race, and physiological race (Jenkins, 1967). In the
asexual race, worms reproduce by fission without sexual organs. In the sexual race, worms have hermaphroditic sexual organs, and copulate and then lay cocoons filled with several fertilized eggs. They are not
likely to fission. In the physiological race, worms convert between asexual and sexual reproduction seasonally. In these planarians, the asexual race switches
from asexual to sexual reproduction by being fed with
sexually mature worms of the same, as well as different, species (Grasso and Benazzi, 1973; Benazzi and
Grasso, 1977; Sakurai, 1981). This result suggests that
sexually mature planarians contain a sexualizing substance(s) of poor species-specificity.
EXPERIMENTAL SEXUALIZATION
The OH strain of Dugesia ryukyuensis is an exclusively asexual clone established by Dr. S. Ishida of
Hirosaki University from a single worm collected in
Okinawa, Japan in 1986. We established an experimental system for the sexualization in the OH strain
by feeding them with sexually mature worms of Bdellocephala brunnea, an exclusively oviparous species
(Kobayashi et al., 1999). In this system, practically all
the worms are fully sexualized within a few weeks.
The process of experimental sexualization is divided
into five distinct stages by anatomical and histological
changes as summarized in Figures 1 and 2 (Kobayashi
et al., 1999; Kobayashi and Hoshi, 2002). Although
the OH strain worms were believed not to have any
1 From the Symposium The Promise of Integrative Biology presented at the Annual Meeting of the Society for Integrative and
Comparative Biology, 2–6 January 2002, at Anaheim, California.
2 Present Address of M. Hoshi, K. Kobayashi, S. Arioka and M.
Matsumoto is Department of Biosciences and Informatics, and Center for Life Science and Technology, Keio University, Hiyoshi, Yokohama 223-8522, Japan.
3 E-mail: [email protected]
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FIG. 1. Five distinct stages along with the sexualization. The development and topological position of reproductive organs are shown. Red
region: ovary; blue region: testis and seminal duct; yellow region: yolk gland and oviduct; green region; a copulatory apparatus with a genital
pore. It had been revealed by histological search that worms at stage 0 (the OH strain) have a pair of undeveloped ovaries. Briefly, at stage
1, the ovaries become larger enough to be externally apparent, yet neither oocytes nor other sexual organs were detectable. At stage 2, oocytes
appear in the ovaries. At stage 3, the primordial testes and a copulatory apparatus emerge, and at stage 4, a genital pore in the copulatory
apparatus become externally apparent, primordial yolk glands develop and spermatocytes appear in the testes. At stage 5, mature yolk glands
are formed and many mature spermatozoa are detectable in the testes. (Reproduced with permission from Kobayashi and Hoshi, 2002).
sexual organs or their remnants, in fact they have two
aggregates of a few cells which develop into a pair of
ovaries during sexualization. In the first stage, the ovaries become large enough to be externally apparent;
the oocytes appear first at stage 2; the primordial testes
emerge at stage 3; a genital pore opens, yolk gland
primordia develop and spermatocytes appear at stage
4; and finally at stage 5 mature spermatozoa and yolk
glands are formed and the worms mate and eventually
lay cocoons filled with fertilized eggs. Experimentally
sexualized worms in this way are named acquired sexuals.
Further work found that the sexualization has a
point-of-no-return between stages 2 and 3. Before the
point, the worms return to the asexual condition unless
feeding of sexually mature worms of B. brunnea is
maintained, whereas after the point they spontaneously
develop into sexually mature worms even if the feeding on sexually mature B. brunnea is interrupted. A
yolk gland gene named Dryg, which has no significant
homology with any known genes, is expressed at stage
3 onwards (Hase et al., 2003). Thus we use it as a
useful marker of the point-of-no-return (Kobayashi et
al., 2002a).
It may be worthy to note that experimental sexualization by feeding experiments depends upon the population density of worms. As shown in Figure 3, sexualization seems to proceed faster if the population
density is lower at least in the cases summarized in
this figure. All the worms in the lower density population opened the genital pore by week six, whereas
none did so in the higher density population. However,
histological examination revealed that all the worms
even in the higher density population reached stage 3,
in other words the sexualization proceeded beyond the
point-of-no-return by week six, because they had developed oocytes in the ovaries and formed primordial
testes (K.K., S.A., and M.H., unpublished data).
Another observation to be noted is that the offspring
of acquired sexuals by random inbreeding is a heterogeneous population consisting of both asexual (fissiparous) worms and those that develop spontaneously
into sexual worms (innate sexuals) in a ratio of approximately 2:1 (Kobayashi et al., in preparation). The
asexual offspring are similarly sexualized as OH strain
worms by being fed with sexually mature worms of
B. brunnea. Although acquired sexuals and innate sexuals are hardly distinguishable by appearances, they
are quite different in nature. The ovaries of acquired
sexuals are much larger than those of innate sexuals,
and acquired sexuals, but not innate sexuals, form supernumerary ovaries if they are kept feeding on sexually mature worms of B. brunnea (Kobayashi et al.,
in preparation). These results suggest that regulation
of differentiation and development of ovaries is not so
much strict in acquired sexuals as in innate ones.
SEXUALIZING SUBSTANCE AND MAINTENANCE
OF SEXUALITY
The apparent activity of sexualizing substance in B.
brunnea worms shows a seasonal change, high in winter and low in summer, in accordance with the annual
change of reproductive activity (Kobayashi et al.,
2002b). A fraction of the homogenate of sexually mature worms of B. brunnea collected in winter has the
activity of sexualization of OH strain worms. The putative sexualizing substance appears to be a small,
heat-stable, papain-resistant, and rather hydrophilic
molecule (K.K. and M.H., unpublished data). Acquired
sexuals and innate sexuals derived from OH strain also
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ET AL.
FIG. 2. Development of gonads along with the sexualization. Testes: stage 0 (A), stage 1 (B), stage 2 (C), stage 3 (G), stage 4 (H) and stage
5 (I). Ovaries: stage 0 (D), stage 1 (E), stage 2 (F), stage 3 (J), stage 4 (K) and stage 5 (L). In the image (I), yolk glands are also shown. O,
ovary; og, oogonia; oc, oocytes; T, testis; Y, yolk gland. Scale bar, 100 mm. (Reproduced with permission from Kobayashi et al., 1999).
SWITCH
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FIG. 4. Scheme of the surgical ablation of the sexualized worms.
By the surgical ablation, three different fragments were obtained
with respect to the topological position of the sexual organs; H
(Head) fragment has no sexual organ; P (Prepharyngeal) fragment
has a pair of ovaries, testes and yolk glands; T (Tail) fragment has
testes, yolk glands and a copulatory apparatus. Colored regions correspond with the sexual organs described in the figure legend of
Figure 1. (Reproduced with permission from Kobayashi et al.,
2002a).
FIG. 3. Effects of population density on the sexualization. Worms
with a pair of ovaries (A) and with a genital pore (B) are scored.
Solid circles, high population density (5 worm/ml); open squares,
low population density (0.17 worm/ml). Means and standard deviations (vertical bars) of three groups (fifty worms each) are shown.
ular differences in neoblasts between acquired sexuals
and innate ones.
SEXUALIZATION WITH CESSATION OF
FISSION
It is observed that the worms gradually stop fission
as sexualization proceeds, and the fully sexualized
worms do not fission at all, suggesting the coupling of
sexualization with cessation of fission, namely apparent loss of fissiparous capacity. It is well known that
fission is triggered in asexual worms by decapitation
(Best et al., 1969). Thus we examined whether effect
of decapitation changes along with the process of sexualization (Kobayashi and Hoshi, 2002). All worms at
stages 0–4 started fission within ten days after decapitation, but fully sexualized worms never fissioned
even after decapitation. Then the reproductive mode
of the offspring derived from decapitated worms were
examined. All the offspring of the worms decapitated
at stages 0–2 were asexual. Both asexual and sexual
offspring were obtained if the worms are decapitated
at stages 3–4. All the worms decapitated at stage 5
became sexual (Kobayashi and Hoshi, 2002). These
results suggest that the sexualization and the cessation
of fission are closely coupled through unknown mechanisms.
COUPLING
contain a similar sexualizing substance(s) (Kobayashi
et al., 2002a).
If the acquired sexuals are surgically cut into three
pieces as shown in Figure 4, head fragments without
sexual organs regenerate into asexuals though regenerants from other portions became sexuals. If similar
surgical ablation is applied upon innate sexuals, all
regenerants become sexuals (Kobayashi et al., 2002a).
Sakurai (1981) reported that head region of sexually
mature worms lacks the putative sexualizing substance
necessary for complete sexualization. Thus, it is likely
that head fragments without sexual organs do not contain enough of an amount of putative sexualizing substance. In other words, the sexuality of acquired sexuals is maintained by the sexualizing substance produced by themselves.
From the surgical ablation experiments, we reach
the more important conclusion that neoblasts, the totipotent stem cells in the planarians, of acquired sexuals remain ‘‘asexual’’ and the worms require external
supply of a sexualizing substance for the differentiation of sexual organs and gametes. On the other hand,
some, if not all, neoblasts in innate sexuals are somehow ‘‘sexual’’ and do not require external supply of a
sexualizing substance for the eventual differentiation
of themselves and/or other neoblasts into sexual organs and gametes. Now we are looking for the molec-
OF
KARYOTYPE
Although a bisexually reproducing all-triploid toad
was recently discovered (Stock et al., 2002), it is generally believed that animals of triploid biotype repro-
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M. HOSHI
duce by parthenogenesis (pseudogamy), but not by bisexual reproduction. Gremigni et al. (1980) reported
that the planaria Dugesia lugubris s.l., which has triploid somatic cells, diploid male germ cells and hexaploid female germ cells, makes embryos of pseudogamic clone of the female. Since OH strain is a triploid
biotype (3n 5 21), we wondered whether the offspring
of acquired sexuals are produced by pseudogamy or
by truly bisexual reproduction. Meiotic images we
have observed so far in the gonads of acquired sexuals
indicate that both eggs and sperm are derived from
diploid cells. However, both diploid worms and triploid worms are found in the offspring and there is no
correlation between the ploidy and sexuality: Namely,
diploid innate sexuals, triploid innate sexuals, diploid
asexuals and triploid asexuals are all found in the offspring (Arioka et al., in preparation). This preliminary
result raises another important question on the behavior of chromosomes during meiosis and fertilization.
FURTHER INTEGRATIVE APPROACH
Our integrative approach integrates multiple fields
of study, including classic breeding, regeneration, and
genetics experiments, as well as karyotyping, and biochemical and molecular biological analyses; none of
which would have revealed much about the intricate
mechanisms that regulate sex and fission in these animals. Going on this line, we would hopefully be able
to identify the sexualizing substance and understand
the mode of its action. Then we have to connect the
mechanism that we would reveal in the experimental
systems with what happened in nature. Since the selection of a reproductive mode has such a remarkable
significance in ecology, we have to pursue ecological
approaches as well for the better understanding of reproduction and its evolution.
ACKNOWLEDGMENTS
We thank Dr. Sachiko Ishida, of Hirosaki University
for her kind gift of the OH strain. Our thanks also go
to Ms. Yukako Shirato for her invaluable assistance in
collecting worms from the field. This work was supported in part by a Grant-in-Aid for Scientific Re-
ET AL.
search (No. 100082) to MH from the Ministry of Science, Culture, Sports and Education, Japan.
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