A comprehensive comparison of sex-inducing activity in asexual worms of the planarian Dugesia ryukyuensis: the crucial sex-inducing substance appears to be present in yolk glands in Tricladida
- Haruka Nakagawa†1,
- Kiyono Sekii†1,
- Takanobu Maezawa2,
- Makoto Kitamura3,
- Soichiro Miyashita1,
- Marina Abukawa1,
- Midori Matsumoto4 and
- Kazuya Kobayashi1Email authorView ORCID ID profile
© The Author(s). 2018
Received: 21 November 2017
Accepted: 11 May 2018
Published: 12 June 2018
Turbellarian species can post-embryonically produce germ line cells from pluripotent stem cells called neoblasts, which enables some of them to switch between an asexual and a sexual state in response to environmental changes. Certain low-molecular-weight compounds contained in sexually mature animals act as sex-inducing substances that trigger post-embryonic germ cell development in asexual worms of the freshwater planarian Dugesia ryukyuensis (Tricladida). These sex-inducing substances may provide clues to the molecular mechanism of this reproductive switch. However, limited information about these sex-inducing substances is available.
Our assay system based on feeding sex-inducing substances to asexual worms of D. ryukyuensis is useful for evaluating sex-inducing activity. We used the freshwater planarians D. ryukyuensis and Bdellocephala brunnea (Tricladida), land planarian Bipalium nobile (Tricladida), and marine flatworm Thysanozoon brocchii (Polycladida) as sources of the sex-inducing substances. Using an assay system, we showed that the three Tricladida species had sufficient sex-inducing activity to fully induce hermaphroditic reproductive organs in asexual worms of D. ryukyuensis. However, the sex-inducing activity of T. brocchii was sufficient only to induce a pair of ovaries. We found that yolk glands, which are found in Tricladida but not Polycladida, may contain the sex-inducing substance that can fully sexualize asexual worms of D. ryukyuensis.
Our results suggest that within Tricladida, there are one or more common compounds or functional analogs capable of fully sexualizing asexual worms of D. ryukyuensis; namely, the crucial sex-inducing substance (hydrophilic and heat-stable, but not a peptide) produced in yolk glands.
Metazoans occasionally switch their mode of reproduction on the basis of environmental changes, life cycle phase, or both. However, the mechanisms underlying the switch from an asexual to a sexual mode of reproduction and vice versa are poorly understood. Scyphozoan cnidarian, Aurelia aurita, seasonally switches their life cycle between asexual polyps and sexual medusae . Under laboratory conditions, the switch from polyp to medusa can be induced by lowering the water temperature. The mechanism controlling the switch consists of retinoic signaling and temperature-sensitive signaling by secreted protein CL390, which encodes the precursor of a putative peptide hormone . The administration of 9-cis-RA or the deduced peptide hormone from CL390 to the polyps (the asexual state) triggers the metamorphosis to the medusa (the sexual state). Therefore, the compounds that control this switch from an asexual to a sexual state will possibly provide clues to help elucidate the molecular mechanism for the reproductive switch. We call such a compound a sex-inducing substance.
Some freshwater planarians (Platyhelminthes, Turbellaria, Tricladida, and Continenticola) can reproduce asexually as well as sexually. Sexual worms have hermaphroditic reproductive organs. In contrast, asexual worms regenerate lost body parts after fission without developing reproductive organs . Therefore, when asexual worms switch to a sexual state, i.e., sexual induction based on environmental stimuli [3–6], they differentiate hermaphroditic reproductive organs from pluripotent stem cells called neoblasts [7–15]. The existence of a planarian sex-inducing substance(s) was suggested by an experimental sexual induction by “feeding” [16–20]. If asexual planarians are fed minced sexually mature worms of the same or different freshwater planarian species, they develop reproductive organs without having been exposed to the environmental stimuli that typically induce this switch (Additional file 1). This suggests that a sex-inducing substance(s) contained in sexually mature worms is a common compound(s) or functional analog(s) in freshwater planarians.
Turbellaria comprise two macroturbellarians (Tricladida and Polycladida) and nine microturbellarians [24, 25]. Microturbellarians are not quantitatively suitable as sources of putative sex-inducing substances in our assay system. In this study, to narrow down the phylogenetic range of species with sex-inducing activity toward asexual worms of D. ryukyuensis, where possible, we used the land planarian Bipalium nobile (Tricladida, Continenticola, Bipaliidae) and marine flatworm Thysanozoon brocchii (Polycladida), with D. ryukyuensis and Bd. brunnea as sources of a sex-inducing substance (Fig. 1d, e). A slug, Ambigolimax valentianus (Mollusca), a natural food source for Bi. nobile, was also used (Fig. 1f). To examine the potency of their sex-inducing activity toward asexual worms of D. ryukyuensis, we compared sex-inducing activity in four fractions from the five species obtained by a fractionation method using this assay system. Here, we report that the crucial sex-inducing substance may be a common compound or functional analog that is produced in the yolk glands in Tricladida.
An exclusively asexual strain, the OH strain, of the freshwater planarian D. ryukyuensis (Fig. 1a) was maintained at 20 °C in dechlorinated tap water and fed chicken liver once a week. Worms of this strain were used as test animals for sexual induction. Sexual worms of D. ryukyuensis (Fig. 1b) were obtained by feeding worms of the OH strain with sexual worms as described previously . The sexual worms of D. ryukyuensis were cut and allowed to regenerate. They were maintained at 20 °C in dechlorinated tap water and fed chicken liver once a week until maturity. They then began to lay cocoons constantly. The sexually mature worms and the fresh cocoons were collected within a day of deposition were stored at − 80 °C for use as a source of the sex-inducing substance. Sexually mature populations of the freshwater planarian Bd. brunnea (Fig. 1c), land planarian Bi. nobile (Fig. 1d), marine flatworm T. brocchii (Fig. 1e), and slug A. valentianus (Fig. 1f) were collected near Yamagata City, Shinjuku-ku, Tokyo, the Misaki Marine Station of Tokyo University, and Chofu City, Tokyo, respectively, in Japan. The fresh cocoons of Bd. brunnea were collected within a day of deposition. They were also frozen in liquid nitrogen and stored at − 80 °C for use as a source of the sex-inducing substance.
Preparation of foods for the bioassay of sexual induction
Bioassay and estimation of sex-inducing activity
In this study, we set the standard dose of each sample for the bioassay at 3.9 mg dry weight to compare sex-inducing activity. To produce the test food for the bioassay, we mixed 3.9 mg of each dried sample with 200 μL of chicken liver homogenate, which is used as a food for planarian maintenance, and then freeze-dried the mixture. Freeze-dried chicken liver homogenate was used as a negative (vehicle) control. Thirty test worms were fed a piece of food daily for 4 weeks.
This experimental sexual induction has a point-of-no-return between stages 2 and 3. Worms at stages 1 and 2 return to the asexual state if the administration of the sex-inducing substance is stopped, whereas from stages 3 onward worms will continue to develop sexual organs, even if the administration of the sex-inducing substance is stopped, which suggests reversible and irreversible phases as evidenced by the point-of-no-return from external observations (Fig. 3). In the present study, a crucial sex-inducing substance means a compound responsible for overcoming the point-of-no-return.
Digestion of Fr. M0 and M10 derived from Bd. brunnea by Actinase E
Foods were prepared for bioassay of the Fr. M0 and M10 fractions of Bd. brunnea treated with Actinase E (KAKEN PHARMACEUTICAL CO., LTD.), which is a powerful enzyme for the elimination of peptides/proteins (Additional file 4). The Fr. M0 and M10 fractions from approximately 8 g wet weight of sexually mature worms of Bd. brunnea were prepared according to the fractionation procedure shown in Fig. 2. Actinase E was added to each solution containing Fr. M0 or M10 from approximately 4 g wet weight at a final concentration of 0.1% (w/v in water). The reaction solutions were incubated at 37 °C for 16 h, and then boiled for 15 min to deactivate Actinase E. As a control (− Actinase E), the solutions containing the Fr. M0 or M10 from approximately 4 g wet weight and 0.1% Actinase E solution were independently incubated and boiled, and finally mixed. To produce the test food for the bioassay, we mixed each dried four sample (Fr. M0 + Actinase E, Fr. M0 – Actinase E, Fr. M10 + Actinase E and Fr. M10 – Actinase E) with 200 μL of chicken liver homogenate, and then freeze-dried the mixture. Thirty test worms were fed a piece of food daily for 4 weeks.
Test worms were relaxed in cold 2% (v/v) HCl in 5/8 Holtfreter’s solution  for 5 min and then fixed in 4% paraformaldehyde and 30% ethanol in 5/8 Holtfreter’s solution for 3 h at room temperature. The fixed specimens were dehydrated through an ethanol series, cleared in xylene, and embedded in Paraplast Plus embedding medium (Sigma-Aldrich Co., St. Louis, MO, USA). The embedded specimens were cut into 4 μm thick sections and stained with hematoxylin and eosin.
Data pertaining to the occurrence of worms at stages 1–2, worms from stage 3 onward, and worms at stages 4–5 (Fig. 3b) were analyzed using chi-square or Fisher’s tests.
Comparison of sex-inducing activity on asexual D. ryukyuensis
According to the fractionation method for the sex-inducing substance , we homogenized 4 g of worms in phosphate-buffered saline (PBS) and then obtained the cytosolic fraction of the supernatant (Supernatant-2) and two fractions of the precipitates (Precipitate-1 and -2) after a two-step centrifugation (Fig. 2). Compounds that are more hydrophilic must be extracted into the cytosolic fraction, whereas compounds that are more hydrophobic must be contained in the precipitates. In the present study, each cytosolic fraction from the five species was applied to a commercial octadecylsilane (ODS) column and eluted stepwise by changing the methanol concentration of the eluent (0, 10, and 100% (v/v)) (Fig. 2 and Additional file 2). Each precipitate was extracted with ethanol. To reliably remove the residual hydrophilic compounds in the precipitates, the extractions were partitioned between water and ethyl acetate. Since 1 g NaCl was added to the partitioned solutions to facilitate better partitioning, the test worms could not eat the water layer owing to a high salt concentration. Consequently, compounds that are more hydrophobic must be recovered in the ethyl acetate layer (EtOAc layer) (Fig. 2 and Additional file 3).
Summary of the stages by histological changes in sexual induction
Stage of sexual induction
Comparative analysis of sex-inducing activity after the point-of-no-return
Number of worms at stage 3 onwards
Number of worms at stages 4–5
3 / 30
0 / 30
4 / 30
0 / 30
30 / 30
13 / 30
7 / 29
0 / 29
20 / 25
2 / 25
21 / 30
0 / 30
The ovary-inducing activity in the cytosolic fractions from Thysanozoon brocchii and Ambigolimax valentianus
Number of worms at stage 1–2
Chicken liver (Control)
0 / 30
14 / 30
30 / 30
10 / 28
4 / 30
0 / 30
0 / 29
The slug, A. valentianus (Mollusca), did not have significant sex-inducing activity, although a pair of small ovaries became visible in a few asexual worms of D. ryukyuensis in this assay (Figs. 4 and 5, Table 3). In D. ryukyuensis, primordial ovaries were histologically identified even in asexual worms, although they were barely visible externally  (Fig. 3). The ovarian morphology in the worms fed with the test food containing the Fr. M0 of A. valentianus was nearly identical to that of the primordial ovaries (Fig. 5u, v).
Is the hydrophilic crucial sex-inducing substance a peptide?
Feeding experiment with cocoons laid by freshwater planarians
In some orders of turbellarians, worms have yolk glands, a reproductive organ filled with nurse cells; namely, yolk gland cells. Their eggs are ectolecithal (cocoons), which have several fertilized eggs and numerous yolk gland cells . In D. ryukyuensis (Tricladida), the existence of intact yolk gland cells in fresh cocoons collected within a day of deposition has been suggested by quantitative reverse transcription polymerase chain reaction analysis of a yolk gland marker gene .
Turbellarian species generally have pluripotent stem cells called neoblasts (i.e., Catenulidae , Macrostomida [29–31], Polycladida ). They undergo homeostatic regulation of their body size by “cell turnover,” which requires neoblasts , and have the capacity for regeneration owing to these neoblasts. Furthermore, they can post-embryonically produce germ line cells from neoblasts. Some low-molecular-weight compounds are involved in post-embryonic germ cell development, yet little information about them exists. Owing to these characteristics, some turbellarian species can switch between an asexual and a sexual state in nature. They may use the low-molecular-weight compounds involved in post-embryonic germ cell development as sex-inducing substances when they alternate from an asexual to a sexual state. Thus, sex-inducing substances are important from the aspects of both developmental and reproductive biology.
A feeding assay system using asexual test worms (the OH strain) of the freshwater planarian D. ryukyuensis (Tricladida, Continenticola, Dugesiidae)  is useful in evaluating sex-inducing activity. In D. ryukyuensis, sexual induction has a point-of-no-return between stages 2 and 3 (Fig. 3) . Worms at stages 1 and 2 return to the asexual condition if feeding with a test food containing a sex-inducing substance is stopped. In contrast, worms at stage 3 and beyond keep developing sexual organs even if feeding with the test food is stopped. Recently, we identified the ability of d-Trp to induce stage 2 ovaries in asexual worms of D. ryukyuensis . However, d-Trp does not induce the other reproductive organs. The crucial sex-inducing substance required to overcome the point-of-no-return has not yet been identified. Previous studies suggested that the crucial sex-inducing substance is evolutionarily conserved in, at least freshwater planarians (Additional file 1).
In this study, to further estimate a phylogenetic relationship of species containing the crucial sex-inducing substance, a comprehensive comparison of sex-inducing activity in asexual worms of D. ryukyuensis was carried out using the freshwater planarians D. ryukyuensis and Bd. brunnea, land planarian Bi. nobile (Tricladida, Continenticola, Bipaliidae), and marine flatworm T. brocchii (Polycladida) as sources of the sex-inducing substance. A slug Ambigolimax valentianus (Mollusca) was also used.
The present study clearly showed that in the cytosolic fractions, the probability of conspecific worms displaying sex-inducing activity was always lower than that of Bd. brunnea and Bi. nobile (Table 2). In particular, the Fr. M0 from the land planarian Bi. nobile showed the highest sex-inducing activity in the cytosolic fractions among four turbellarian species (Figs. 4 and 5, Tables 1 and 2). Molecular phylogenetic analysis of freshwater and land planarians has suggested that in terms of phylogenetic distance, freshwater planarians in the family Dugesiidae and land planarian in the family Bipaliidae are more closely related than freshwater planarians in the family Dendrocoelidae and those in the family Planariidae . The ability to produce crucial sex-inducing activity in asexual planarians in the family Dugesiidae has been confirmed in sexual planarians of the families Dendrocoelidae, Planariidae, and Dugesiidae (Additional file 1). The ability of the land planarian Bi. nobile (Bipaliidae) to produce strong sex-inducing activity in asexual worms of D. ryukyuensis (Dugesiidae) may be consistent with the aforementioned phylogenetic relationship.
In contrast, insufficient sex-inducing activity to overcome the point-of-no-return was found in the cytosolic fraction of a marine flatworm T. brocchii (Fig. 4), although the induced ovaries were extraordinarily large and contained oocytes (Fig. 5p, q). It was noted that of all the species, only the Fr. M100 of T. brocchii showed significant sex-inducing activity (Fig. 4 and Table 3). The marine flatworm T. brocchii may possess an analog with the extremely low sex-inducing activity, or only an ovary-inducing substance like d-Trp. Additionally, there is possibly a compound unique to the sex-inducing activity in Fr. M100. In gastropod mollusks containing A. valentianus, the tripeptide l-Asn-d-Trp-l-Phe-NH2 (NdWFamide) acts as a neuropeptide [36–40]. Thus, A. valentianus must contain free d-Trp as a degradant of this neuropeptide. In the fractionation procedure, d-Trp is recovered primarily in Fr. M0 . It may be reasoned that a few asexual worms of D. ryukyuensis fed with the test food containing the Fr. M0 of A. valentianus developed a pair of ovaries (Figs. 4 and 5u, v). These results suggest that there might be a common compound or a functional analog as the hydrophilic crucial sex-inducing substance in Tricladida, but not in Polycladida.
In the present study, the sex-inducing activity of more hydrophobic compounds recovered in EtOAc layer was examined. The administration of the EtOAc layer in D. ryukyuensis, Bd. brunnea, and T. brocchii induced only a pair of ovaries, even though asexual worms of D. ryukyuensis were fed about ten times the standard dose of the EtOAc layer (about 39 mg dry weight) (Fig. 4). This suggests that there is a hydrophobic ovary-inducing substance in these species. However, approximately ten times the dry weight of the EtOAc layer from Bi. nobile resulted in enough sex-inducing activity required to overcome the point-of-no-return (Figs. 4 and 6). The existence of a hydrophobic crucial sex-inducing substance in Bi. nobile may be associated with terrestrial organisms.
There is much debate on the identity of the organs or tissues responsible for producing the crucial sex-inducing substance. One theory is that the putative hormone produced by the testes is responsible for the development of the copulatory apparatus . Indeed, it was suggested that sexual worms of D. ryukyuensis lacking testes after treatment with the RNAi of Dr-nanos and Dr-piwi1 could not maintain their acquired sexuality [42, 43]. The other theory is that they are derived from the neurosecretion responsible for gonad maturation as described above [44–46]. Interestingly, neuropeptide NPY-8 is specifically associated with testicular differentiation in the freshwater planarian Schmidtea mediterranea . The RNAi knockdown of npy-8 in sexually mature worms results in the regression of the testes, which acts to maintain planarian sexuality. However, to date, the yolk gland has not been a candidate for the source of the crucial sex-inducing substance.
Together, the results in the present study suggest that turbellarians possess a compound(s) with the sex-inducing activity in asexual worms of D. ryukyuensis. Furthermore, the crucial sex-inducing substance needed to overcome the point-of-no return in asexual worms of D. ryukyuensis may be contained in worms of Tricladida, but not those of Polycladida. An anatomically crucial difference between Tricladida and Polycladida is the presence or absence of yolk glands. Immediately after the point-of-no-return (stage 3), primordial yolk glands emerged in D. ryukyuensis (Fig. 3). Recently, we also found that a large amount of l-Trp is incorporated and pooled in the yolk glands, resulting in the accumulation of d-Trp that is involved in the ovarian development of asexual worms as a sex-inducing substance . Motivated by these findings, we fed the asexual worms of D. ryukyuensis with fresh cocoons of D. ryukyuensis and Bd. brunnea containing numerous yolk gland cells, resulting in full sexual induction (Fig. 8). Besides, the sex-inducing activity of Fr. M0 and M10 from Bd. brunnea did not decrease with treatment with Actinase E, which is a powerful enzyme causing the elimination of peptides/proteins in a solution (Fig. 7). We concluded that the crucial sex-inducing substance in the asexual worms of D. ryukyuensis is present in yolk glands and is not a peptide.
A slug, A. valentianus, is a food for the land planarian Bi. nobile in nature. There were no fractions from A. valentianus that produced significant sex-inducing activity in asexual worms of D. ryukyuensis (Fig. 4). The crucial sex-inducing substance could be de novo synthesized in the yolk glands of Tricladida. In the present study, we used worms from two orders (Tricladida and Polycladida) in Turbellaria, namely macroturbellarians as sources of a sex-inducing substance. As worms in the other nine orders in Turbellaria, namely microturbellarians, are small, we abandoned using them as sources of a sex-inducing substance. However, six microturbellarians produce ectolecithal eggs (cocoons) like Tricladida , meaning that they also have yolk glands (−like organs). They also may contain the crucial sex-inducing substance in the asexual worms of D. ryukyuensis. In the near future, we will seek to identify the crucial sex-inducing substance on the basis of the results of the present study.
Certain low-molecular-weight compounds found in sexually mature animals act as sex-inducing substances during post-embryonic germ cell development when the animals alternate from an asexual to a sexual state (sexual induction). The crucial sex-inducing substance responsible for the sexual induction of freshwater planarians has not yet been identified. An assay system that involves feeding asexual worms of the freshwater planarian D. ryukyuensis is useful for evaluating this type of sex-inducing activity. In the present study, to estimate a phylogenetic range of species that may possess compounds with sex-inducing activity in asexual worms of D. ryukyuensis, we carried out a comprehensive comparison of the sex-inducing activity containing worms in two orders (Tricladida and Polycladida) in Turbellaria as sources of a sex-inducing substance. Using this assay system, we showed that the three species in Order Tricladida have strong sex-inducing activity and can fully sexualize asexual worms of D. ryukyuensis. Interestingly, the sex-inducing activity displayed by the conspecific sexual worms was not higher than that of the freshwater planarian Bd. brunnea or land planarian Bi. nobile, which belong to the same order. In contrast, the sex-inducing activity displayed by the marine flatworm T. brocchii, which belongs to Order Polycladida, was extremely low. On the basis of these results, we found that yolk glands, which exist in Tricladida but not Polycladida, possibly contain the crucial sex-inducing substance (hydrophilic and heat-stable, but not a peptide) that can fully sexualize asexual worms of D. ryukyuensis. The results obtained in this study will contribute to the identification of the crucial sex-inducing substance.
We thank Dr. Yuni Nakauchi’s group at Yamagata University and the staff of the Misaki Marine Biological Station (The University of Tokyo), for their invaluable assistance in collecting Bd. brunnea and T. brocchii, respectively. We thank Dr. Takashige Sakurai for invaluable advice on sexual induction by cocoon feeding. Our thanks also go to Mr. Masaki Ishikawa for his invaluable assistance in drawing the illustrations.
This work was supported in part by a Grant-in-Aid for Scientific Research (Nos. 16086209 [MM], 15770147, 15K07121 [KK], 20116007 [KK], 26114501 [KK] and 16H01249 [KK]) from the Ministry of Science, Culture, Sports, and Education, Japan.
Availability of data and materials
Data sharing not applicable to this article as no datasets were generated during the current study.
KK and MK fractionated the predicted sex-inducing fractions from crude samples and prepared test foods. KK, KS and HN performed bioassays. HN performed histological analysis. SM and MA performed the additional experiment in Additional file 4. TM, KS, and MM supervised the project and discussed the results. KK and HN conducted and designed the experiments. KK wrote the manuscript. All authors read and approved the final manuscript.
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