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Sexually mature planarians produce sex-inducing substances that induce postembryonic development of hermaphroditic reproductive organs in asexual freshwater planarians. Although the sex-inducing substances may be useful for elucidating the mechanism underlying this reproductive switch, the available information is limited. The potency of sex-inducing activity is conserved, at least at the order level. Recently, we showed that the sex-inducing activity in the land planarian Bipalium nobile was much higher than that in freshwater planarians. In the present study, we performed bioassay-guided fractionation of the sex-inducing substances produced by B. nobile and propose that crucial sex-inducing activity that triggers complete sexualization for asexual worms of the freshwater planarian Dugesia ryukyuensis is produced by additive and/or synergetic effects of various sex-inducing substances involved in ovarian development. The current study provided an isolation scheme for the minimum-required combination of sex-inducing substances for producing crucial sex-inducing activity.

Many flatworms can alternate between asexual and sexual reproduction. This is a powerful reproductive strategy enabling them to benefit from the features of the two reproductive modes, namely, rapid multiplication and genetic shuffling. The two reproductive modes are enabled by the presence of pluripotent adult stem cells (neoblasts), by generating any type of tissue in the asexual mode, and producing and maintaining germ cells in the sexual mode. In the current study, RNA sequencing (RNA-seq) was used to compare the transcriptomes of two phenotypes of the planarian Dugesia ryukyuensis: an asexual OH strain and an experimentally sexualized OH strain. Pathway enrichment analysis revealed striking differences in amino acid metabolism in the two worm types. Further, the analysis identified serotonin as a new bioactive substance that induced the planarian ovary de novo in a postembryonic manner. These findings suggest that different metabolic states and physiological conditions evoked by sex-inducing substances likely modulate stem cell behavior, depending on their different function in the asexual and sexual reproductive modes. The combination of RNA-seq and a feeding assay in D. ryukyuensis is a powerful tool for studying the alternation of reproductive modes, disentangling the relationship between gene expression and chemical signaling molecules.

Sexually mature planarians produce sex-inducing substances that induce postembryonic development of hermaphroditic reproductive organs in asexual freshwater planarians. Although the sex-inducing substances may be useful for elucidating the mechanism underlying this reproductive switch, the available information is limited. The potency of sex-inducing activity is conserved, at least at the order level. Recently, we showed that the sex-inducing activity in the land planarian

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Background 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. Results 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. Conclusions 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. Electronic supplementary material The online version of this article (10.1186/s40851-018-0096-9) contains supplementary material, which is available to authorized users.

Table S2. Dry weight of fractions derived from the cytosolic fraction. (PDF 69 kb)

Some freshwater planarians (Platyhelminthes, Turbellaria, Seriata, and Tricladida) reproduce asexually by transverse fission and subsequent regeneration. Depending on environmental conditions, some asexual worms also develop complex hermaphroditic reproductive organs from planarian pluripotent stem cells, or neoblasts. Acquired sexual worms then mate, and eventually lay a cocoon filled with several fertilized eggs and a large number of yolk gland cells. The mechanisms underlying the switch from an asexual to a sexual state and the differentiation of germ cells from adult stem cells are of interest because they represent fundamental aspects of both reproductive biology and developmental biology. To study this mechanism, an experimental system was established in a triploid asexual strain, the OH strain of Dugesia ryukyuensis (Kobayashi et al. 1999). In this assay system, asexual worms acquire sexuality and cease asexual reproduction by transverse fission when experimentally dosed with sex-inducing substances produced by sexually mature planarians. Acquired sexual worms are then able to produce sex-inducing substances by themselves to maintain a sexual state, without the intake of sex-inducing substances. Interestingly, triploid acquired sexual worms sexually produce four types of offspring, namely, diploid asexual worms, diploid innate sexual worms, triploid asexual worms, and triploid innate sexual worms. Although the relationship between sexuality and ploidy has not yet been clarified, innate sexuality noticeably differs to acquired sexuality because worms never convert to an asexual state. This review discusses insights obtained from the study of these intricate biological phenomena to help elucidate the mechanisms used for reproductive strategies in planarians, including that of switching from an asexual to a sexual state.

Table S1.. Relationship between test worm and food in the experimental sexual induction. (PDF 71 kb)

Table S3. Weight of precipitates and EtOAc layers. (PDF 69 kb)

Figure S1. Preparation of foods for the bioassay on Fr. M0 and M10 of Bd. brunnea treated with Actinase E. (PDF 303 kb)

Many metazoans start germ cell development during embryogenesis, while some metazoans possessing pluripotent stem cells undergo postembryonic germ cell development. The latter reproduce asexually but develop germ cells from pluripotent stem cells or dormant primordial germ cells when they reproduce sexually. Sexual induction of the planarian Dugesia ryukyuensis is an important model for postembryonic germ cell development. In this experimental system, hermaphroditic reproductive organs are differentiated in presumptive gonadal regions by the administration of a crude extract from sexual planarians to asexual ones. However, the substances involved in the first event during postembryonic germ cell development, i.e., ovarian development, remain unknown. Here, we aimed to identify a bioactive compound associated with postembryonic ovarian development. Bioassay-guided fractionation identified ʟ-tryptophan (Trp) on the basis of electrospray ionization–mass spectrometry, circular dichroism, and nuclear magnetic resonance spectroscopy. Originally masked by a large amount of ʟ-Trp, d-Trp was detected by reverse-phase high-performance liquid chromatography. The ovary-inducing activity of d-Trp was 500 times more potent than that of ʟ-Trp. This is the first report describing a role for an intrinsic d-amino acid in postembryonic germ cell development. Our findings provide a novel insight into the mechanisms of germ cell development regulated by low-molecular weight bioactive compounds.

Nanos (Nos) is an evolutionarily conserved protein essential for the maintenance of primordial germ cells (PGCs). In Drosophila, the PGCs or pole cells express head involution defective (hid), which is required for caspase activation, but its translation is repressed by maternal Nos. In the absence of Nos activity, translation of hid mRNA into protein induces apoptosis in pole cells. However, it remains unclear how hid mRNA is regulated in pole cells. Here, we report that hid expression requires eiger (egr), a tumor necrosis factor ligand (TNF) homologue, which is induced in pole cells by decapentaplegic (dpp). In addition, we demonstrate that p53 and loki (lok), a damage-activated kinase known to be required for p53 phosphorylation, are both required for hid expression in pole cells. Since maternal lok mRNA is enriched in pole cells, it is possible that ubiquitously distributed p53 is activated in pole cells by maternal Lok. We propose that hid expression is activated in a pole cell-specific manner by loki/p53 and dpp/egr during embryogenesis.

The planarian Dugesia ryukyuensis reproduces both asexually and sexually, and can switch from one mode of reproduction to the other. We recently developed a method for experimentally switching reproduction of the planarian from the asexual to the sexual mode. We constructed a cDNA library from sexualized D. ryukyuensis and sequenced and analyzed 8,988 expressed sequence tags (ESTs). The ESTs were analyzed and grouped into 3,077 non-redundant sequences, leaving 1,929 singletons that formed the basis of unigene sets. Fifty-six percent of the cDNAs analyzed shared similarity (E-value

In certain planarian species that are able to switch between asexual and sexual reproduction, determining whether a sexual has the ability to switch to the asexual state is problematic, which renders the definition of sexuals controversial. We experimentally show the existence of two sexual races, acquired and innate, in the planarian Dugesia ryukyuensis. Acquired sexuals used in this study were experimentally switched from asexuals. Inbreeding of acquired sexuals produced both innate sexuals and asexuals, but inbreeding of innate sexuals produced innate sexuals only and no asexuals. Acquired sexuals, but not innate sexuals, were forced to become asexuals by ablation and regeneration (asexual induction). This suggests that acquired sexuals somehow retain asexual potential, while innate sexuals do not. We also found that acquired sexuals have the potential to develop hyperplastic and supernumerary ovaries, while innate sexuals do not. In this regard, acquired sexuals were more prolific than innate sexuals. The differences between acquired and innate sexuals will provide a structure for examining the mechanism underlying asexual and sexual reproduction in planarians.