Animals

An exclusively fissiparous strain, the OH strain of the planarian Dugesia ryukyuensis presented by Dr. S. Ishida of Hirosaki University was maintained at 20°C in dechlorinated tap water by being fed with chicken liver. After starvation for two to three weeks, the worms were used as asexual recipients in a feeding experiment. Wild populations of Bdellocephala brunnea, an exclusively oviparous planaria, were collected in the vicinities of Yamagata City, Japan. They were used as the food to sexualize the worms of D. ryukyuensis.

The feeding experiment was performed as previously described (Kobayashi et al., 1999). Three groups of fifty worms of the OH strain were fed with the sexually mature worms of B. brunnea for six weeks. External observation was carried out under a binocular microscope every week to examine whether or not the recipients develop sexual organs (a pair of ovaries and a genital pore). Fission in the population was examined everyday. In case of fission in the recipients, the tail fragments were removed to keep the population density (five worms in about 30 ml of dechlorinated tap water), because the population density effects the process of sexualization (Kobayashi and Hoshi, unpublished data).

Regeneration experiment

The recipients were cut at the prepharyngeal level with a razor (Fig. 2). The head fragments were fixed with 10% formalin in phosphate-buffered saline (34 mM NaCl, 7 mM KCl, 2.5 mM Na₂HPO₄, 4.5 mM KH₂PO₄, pH 7.4). The fixed specimens were dehydrated through ethanol series, cleared in xylene and embedded in Paraplast Plus™ (Sherwood Medical, St. Louis, MO). The embedded specimens were cut into sections at 4 μm thickness and stained with hematoxylin and eosin. The stages of sexualization in the recipients were determined by the results of the histological examination of the head fragments according to Kobayashi et al. (1999) (Fig. 1).

Fig. 2

Scheme of surgical ablation in the recipients. Surgical ablation was performed at the prepharyngeal level. The illustration of a planaria arranged to the anterior on the left. The two larger black dots represented a pair of ovaries; a white bar, a black bar and a white dot represented a pharynx, a copulatory apparatus and a genital pore, respectively. In case of the fully sexualized worms, the ovaries and the copulatory apparatus with a genital pore were apparent.

Additionally, each tail fragment was placed in a plastic dish of 35 mm in diameter and allowed to regenerate by being fed on chicken liver once a week. External observation was carried out every week for 6 months. When the tail regenerants developed sexual organs without fissioning, we regarded them as sexual worms. On the contrary, when they fissioned, we continued to maintain them. We regarded the fissioned offspring as asexual worms, when they fissioned further without developing sexual organs.

Fission in the recipients during sexualization

We divided the process of sexualization into five distinct stages by external observation and histological examination (Fig. 1, Kobayashi et al., 1999). In this experiment, most worms at each week of feeding treatment approximately corresponded with stages 1 to 5 in order. Fig. 3A shows the number of fissioned worms along with the progression of sexualization. Worms fed on B. brunnea did not fission by Week 1, because the body size was not large enough to fission yet, and then they started to fission after about one week of feeding treatment. At almost the same time, a pair of ovaries were recognized externally in a half of the worms (Fig. 3B). From two to three weeks of the treatment, consequently, the worms with a pair of ovaries actively fissioned (Fig. 3A), though some of them developed a copulatory apparatus at the postpharyngeal region without fissioning. A few worms with copulatory apparatus, as well as a pair of ovaries fissioned. After three weeks of the treatment, fission was not observed (Fig. 3A). The worms that had not fissioned opened a genital pore on the ventral at Week 3 (Fig. 3C). Finally, the worms that had experienced fission also developed a copulatory apparatus and a genital pore after Week 4.

Fig. 3

The number of fissioned worms (A), the number of worms with a pair of ovaries (B) and the number of worms with a genital pore (C) along with the sexualization. Three groups of fifty worms of the OH strain were fed with the sexually mature worms of B. brunnea for six weeks. During the feeding experiment, second fission in the fissioned worms was not observed. Thus, total number of fission in the populations is expressed as the cumulative number of the fissioned worms. Means and standard errors (vertical bar) of the three groups were shown.

Regeneration experiment

In order to examine the fissiparous capacity and the degree of the sexualization in the recipients, we carried out the regeneration experiment as described in MATERIALS AND METHODS. Table 1 summarizes the fissiparous capacity of the recipients corresponding with the stages of sexualization (Fig. 1; Kobayashi et al., 1999). In the case of the worms at stages 0–4, all the decapitated worms started to fission within ten days after surgery. The appearance of sexual offspring in the decapitated worms at stages 3 and 4 was followed by dramatic decrease in the frequency of fission (Table 2; as described below in detail). On the other hand, the decapitated worms at stage 5 never fissioned.

Table 1

The degree of sexualization and the fissiparous capacity in the recipients

As a result of these cycles of fission, many offspring were obtained. We also examined their reproductive mode (Table 2). All the offspring at stages 0–2 became asexual. Both asexual and sexual offspring were observed in the decapitated worms at stages 3 and 4. The decapitated worms at stage 5 became sexual. When the decapitated worms at stages 3 and 4 fissioned, the head offspring always became sexual. However, the tail offspring followed quite a different fate along with the process of sexualization (Fig. 4): in type 1, the tail offspring produced two sexual worms in the same manner as the head offspring in the first cycle of the asexual reproduction, though it produced an asexual worm after two cycles of the asexual reproduction; in type 2, after a cycle of the asexual reproduction in the tail offspring, the head and tail regenerants became sexual and asexual worms respectively; in type 3, the tail offspring became asexual; in type 4, although the tail offspring underwent a cycle of the asexual reproduction, both the head and the tail regenerants became sexual; in type 5, the tail off-spring became sexual. The decapitated worms at stage 3 followed the fate of types 1–3, while those at stage 4 followed mainly the fate of types 4 and 5, though a few of them followed the fate of types 2 and 3 (Table 3).

Table 2

The reproductive mode of the offspring from the decapitated recipients^a

Fig. 4

Illustration of the different fates in decapitated worms at stages 3 and 4. Planarian illustrations painted with dark gray represented sexual offspring that developed full sexual organs without fissioning. Planarian illustrations painted with light gray represented asexual offspring that continued to fission without developing the sexual organs.

Fissiparous capacity during sexualization

Mostly, freshwater planarians (triclads) can reproduce asexually without sexual organs (Pearse et al., 1987). In such planarians, some populations may alternate between asexual and sexual reproduction (Curtis, 1902; Hyman, 1939; Jenkins, 1967). They cannot seem to fission, namely the asexual reproduction, whenever they keep a sexual state; that is, they not only develop hermaphroditic sexual organs but also cease to fission, when they switch from asexual to sexual reproduction (sexualization). However, the cessation of fission, a significant event in sexualization, is not described in detail.

We established an assay system for sexualization in the OH strain of Dugesia ryukyuensis by feeding with the putative sexualizing substance contained in Bdellocephala brunnea and divided the process of the sexualization into five distinct stages by histological changes (Fig. 1) (Kobayashi et al., 1999). The experimental sexualization has a point-of-no-return between stages 2 and 3. The worms at stages 3 onward no longer need feeding on B. brunnea to maintain the sexuality. We first examined fissioned worms along with the process of sexualization. External observation revealed that worms developing a pair of ovaries can fission, then they seem to cease the fission around the time they differentiate copulatory apparatus, and eventually they never fission after the differentiation of a genital pore (Fig. 3). Appearance of genital pore indicates that the worms were sexualized by stage 4. Thus, the cessation of fission is performed immediately after the point-of-no-return under the experimental conditions. We also observed that the worms after the point-of-no-return were fully sexualized without fissioning, even if feeding on B. brunnea was stopped (data not shown). These results suggest that, after the point-ofno-return, downstream mechanisms induced by the putative sexualizing substance become responsible for the cessation of fission. In other words, the sexualizing substance contained in B. brunnea would not be directly responsible for the cessation of fission, since the worms before the point-ofno-return are able to fission in spite of the progression of the sexualization. Grasso and Benazzi (1973) suggested that the putative sexualizing substance in sexual specimens serves to cease fission before the differentiation of sexual organs. The discrepancy between the two suggestions may be attributed to the difference in the experimental conditions.

Fissiparous capacity after decapitation

In order to estimate the fissiparous capacity of the recipients intensively and extensively, we carried out decapitation of the recipients by surgical ablation, because decapitation of asexual planarians facilitates the fission, suggesting that the cephalic system (brain) controls fission (Best et al., 1969; Best et al., 1975). The decapitation induced fission in all the worms at stages 0–4, while it did not in those at stage 5 (Table 1). Interestingly, the decapitated worms at stages 3 and 4 fissioned, though the intact worms did not. This suggests that mechanisms responsible for the cessation of fission, which are induced by the sexualizing substance in B. brunnea, are associated with the cephalic system. On the contrary, in the worms after stage 5, namely, fully sexualized worms, other mechanisms independent of cephalic control may accomplish the cessation of fission, because the worms at stage 5, even decapitated ones, never fissioned.

Change of reproductive mode during cycles of fission

As a result of the regeneration experiment, many off-spring were obtained by fission from the decapitated worms. The decapitated worms at stages 1 and 2 produced only asexual offspring, while those at stages 3 onward always produced sexual ones in spite of the lack of the putative sexualizing substance in B. brunnea (Table 2). This result supports the point-of-no-return between stages 2 and 3 (see described above; Kobayashi et al., 1999).

We also found that the decapitated worms at stages 3 and 4 followed five distinct fates (Fig. 4, Table 3). During several cycles of fission, the head offspring always became sexual, while tail ones produced asexual offspring (Fig. 4; Types 1–3). In the decapitated worms at stage 4, almost all the offspring became sexual, though they underwent a few cycles of fission (Fig. 4; Types 4 and 5). It is notable that frequency of fission and appearance of asexual offspring dramatically decreased after the point-of-no-return (Table 2). Additionally, all the head fragments regenerated to become sexual without fissioning, if worms at stages 3 onward in which a copulatory apparatus became externally visible were cut as described in Fig. 2 (data not shown). This suggests that, within a planarian body, the acquisition of sexuality first takes place, and then the region unable to fission is committed from head toward tail gradually. In this paper, we conclude that the cessation of fission is determined after the acquisition of sexuality in the individual as well as at tissue level.

Table 3

Number in each type of the decapitated recipients*