Field sampling

Individuals of M. nipponica were collected on the intertidal and subtidal zone at a beach in Oshoro Bay, in front of the Oshoro Marine Station, Hokkaido University, which is located in Otaru, Hokkaido (Fig. 2A, B). Especially, at low tide, kelp roots or Rhodophyta (red algae) were collected from shallow rocky reefs (Fig. 2C, D). These algae were washed in plastic trays, where the focal species of syllids was released and collected (Fig. 2E). The species was identified according to previous studies (e.g., San Martín et al., 2014). Collected individuals were kept in small plastic containers (84 × 57 × 44 mm) and brought to the laboratory (Fig. 2F). The number of collected individuals was counted at every sampling date, and the average numbers of sampled syllid individuals were normalized by the number of collectors who participated in each sampling.

Laboratory rearing

In the laboratory, 10–20 individuals were put in the small containers with filtered sea water (FSW) (c.a. 50 ml/case). Six small containers were further put into a larger container with moistened paper towels to prevent desiccation. According to the previous studies of other species such as Syllis prolifera (Franke, 1980, 1983; Massa Gallucci and Gambi, 2014), the syllids were maintained under constant rearing conditions (21 ± 1°C, 16L8D) without aeration in an incubator (Fig. 2G). In S. prolifera, synchronous stolonization is induced by moonlight (Franke, 1985; Massa Gallucci and Gambi, 2014); we therefore exposed M. nipponica individuals to moonlight for one week per month. As food for adult individuals, purchased spinach powder (Mikasa Sangyo, Tokyo) was dissolved in FSW and provided to the syllid cultures (ca. 10 mg/case) every 2 days (Fig. 2H). Fecal pellets were removed with seawater from containers, using a plastic pipette, and clean FSW was added when water level became low. Additionally, fish diet (Hikari Premium Megabite Green, Kyorin Co., Ltd., Himeji), containing fish and shrimp meats, was also supplied to adult specimens (c.a. 5–10 mg/ case; Fig. 2I; See also Results). For larvae and juveniles, marine algal plankton, Nannochloropsis sp. (ca. 200 µl of Nannochloropsis Green Water, Institute of Environmental Biotechnology, Inc., Yaizu) was mainly supplied (∼200 µl/case), since it has been previously considered an optimal food for invertebrate larvae because of its small size and high nutrient content (Antia and Cheng, 1982).

Observations of postembryonic development

After the start of rearing, the number of surviving individuals and the degree of stolonization were recorded daily. When the detachment of stolons occurred, they were transferred to another plastic case. Immediately after encountering stolons of the opposed sex, they started swimming and spawning. Observations of eggs and sperms were carried out. Spawned eggs were maintained, the process of postembryonic development was observed and dates of developmental events were recorded.

Fig. 1.

The focal species Megasyllis nipponica. (A) Mature adult individual with a well-developed stolon. The stolon is indicated by a bracket, and the eyes of the stolon are indicated by an arrowhead. (B) A male and a female stolon, which can be distinguished by their body color.

Fig. 2.

Field sampling and rearing system of M. nipponica. (A) Location of Oshoro Marine Station, which belongs to Hokkaido University. (B) Oshoro Marine Station and field sampling site. (C) Individuals of M. nipponica inhabit in Rhodophyta algae. (D) The focal species can easily be distinguished by body color and white and black bands at its phalynx. (E) Collected individuals were taken back to the laboratory in plastic containers. In the laboratory, 10–20 individuals were kept in each plastic case (F), and put in an incubator under constant conditions (21 ± 1°C, 16L:8D) (G). Spinach powder (H) and fish food (I) were supplied as food.

Postembryonic stages were classified as follows; EG (egg stage from spawning to hatching), MT (metatrochophore stage from hatchling to juvenile with visible proventricle), JV (juvenile stage, when the proventricle is visible but before the appearance of a black band on the pharynx), BS (black-spotted stage, after the appearance of the black band). The morphology of BS individuals was almost the same as that of adults, so they were treated as adults. The periods required for the different postembryonic stages were recorded daily. It was difficult to track each individual, hence the dates of the first appearance of focal stages in a culture of the syllids were recorded.

Species identification

The selected specimens fully agreed with the available descriptions of M. nipponica (Imajima, 1966, 1996; San Martín et al., 2014). Firstly, the species were easily identified as Megasyllis because of the presence of segments with several annuli and a strong alternation in cirri length (Fig. 1A). Then, the animals were subsequently identified as M. nipponica because of the following features: (1) antennae and dorsal cirri distinctly annulated (Fig. 3A); (2) posterior compound chaetae with distinct subdistal serration on shafts without long filiform subdistal spines (Fig. 3B); (3) tri-annulated segments (Fig. 3C); (4) a pharynx with 10 soft papillae; (5) live specimens with a green body color (sometimes yellowish brown) and a black spot at the posterior part of whitish pharynx (Fig. 1A).

Habitat and seasonality

The specimens were sampled at Oshoro Bay on 13 and 28 October and 2 December 2 2016, 22 February, 28 March, 11 April, 9 May, 26–27 June, and 16–17 September 2017, and 9 June, 14 July, 19 August, 22 September, and 10 October 2018 ( Supplementary Table S1 (10.2108.zsj.36.372.s1.pdf), Fig. 4). Megasyllis nipponica appears to be one of the most common syllid species at the sampling site. They dwell among kelp roots or Rhodophyta algae, by coiling their bodies onto them. As the distribution of the focal species is known to range from Hokkaido to Shikoku Island (Imajima, 1996), the authors sampled the same species also from Muroran, Tateyama, Misaki, and Manazuru (data not shown).

Fig. 3.

Key characters for the identification of M. nipponica. (A) Head morphologies show the characteristics of Megasyllis, namely, eyes, prostomium, palps and tentacles. (B) Posterior compound chaetae show distinctly subdistal serration on shafts. (C) Body segments are tri-annulated.

The number of collected individuals clearly showed a seasonal trend, although the survey was not precisely quantitative. Namely, many individuals were found from August to October, while few individuals were collected from December to July. Therefore, the best sampling season for M. nipponica at Oshoro Bay was from August to October (Fig. 4). A previous survey on the annual transition of water temperature at the area reveals that the temperature range from August to October is approximately 16–22°C (Nakata et al., 2001). The largest number of sampled individuals was in September, probably as a response to the temperature increase in August.

Feeding habits

Although very little is known about syllid feeding habits (Jumars et al., 2014), some species in Autolytinae and Syllinae have been considered carnivorous, while certain species in Exogoninae and Eusyllinae are supposed to be herbivorous or detritivorous (Malaquin, 1893; Allen, 1921; Okada, 1928; Korringa, 1951; Hammond, 1969; Hughes, 1975; Fauchald and Jumars, 1979). A recent extensive study on fecal pellet contents of syllid species revealed that many of the syllid species were omnivorous or detritivorous (Giangrande et al., 2000). As most of Syllinae species, our observations suggest that M. nipponica is omnivorous in nature, since they fed not only on plant tissues like spinach powder or Nannochroropsis, but also on dead bodies of other individuals and fish food.

Table 1.

Periods required for the postembryonic development in Megasyllis nipponica. EG: egg, MT: metatrochophore, JV: juvenile, BS: black-spotted individual; see text for details.

Fig. 4.

Seasonal transitions of the number of M. nipponica individuals (red line) that were collected at Oshoro Bay. The number of sampled individuals was normalized by by the number of collectors who participated in each sampling (See Materials and Methods for details). The average temperature transition of seawater during three years of collection at Oshoro Bay (from 2016 to 2018) is also shown (dotted blue line). The sampling results suggest that the seasonal dynamics of the syllid population largely depends on water temperature.

Stolonization process

The stolonization process in laboratory conditions of M. nipponica was recorded (Fig. 5A–F). The presence of a stolon at the time when the specimens were collected seemed to depend on the seasons, being more abundant during warmer months. However, when reared in laboratory conditions, most individuals developed stolons and released them regularly.

Almost all individuals fed well on spinach powder and fish diet. Their digestive tracts full of food materials were clearly visible through their transparent body walls. The posterior part of the digestive tract was constricted and kinked in some adult individuals that have not developed gonads and stolons yet (Fig. 5A). This constriction was seen in the posterior part of the body, suggesting that this is a previous stage to stolonization. However, the precise causal relationship with stolonization is unraveled.

Stolonization process started with the development of gonads. In adult specimens, around 100 segments, a pair of gonads can be seen in each of the last ∼30 segments (Fig. 5B). The size of gonads was smaller at the beginning, but became larger, almost filling the segment (Fig. 5C). Simultaneously with the gonad development, newly-formed eyes were observed at the most anterior segment of the gonad-developing segments (Fig. 5D). During stolonization, the stolon head and gonads develop as well as the modification of swimming chaetae (Franke, 1999). Our observations indicated that the gonad development occurs before the formation of eyes (Fig. 5B–D), as has been observed in other species (Aguado et al., 2015b). The position where the gonad development started, i.e., both sides of each segment, would indicate the location of gonad primordia at both sides of each segment (Fig. 5B). In annelids, the proliferation of peritoneum cells is suggested to lead the formation of gametogonia (primary gametocytes), which are released in the coelom (e.g., Anderson and Huebner, 1968; Eckelbarger, 1979). These primary gametocytes are suggested to form the gonad primordia on both sides of future stolon segments.

Fig. 5.

The transformation of posterior segments during stolonization. (A) Before the beginning of stolonization, a kinked structure is seen in the digestive tract, relatively at the posterior region. (B) A pair of gonad primordia is seen in each of the posterior segments. (C) Even when gonads develop, the digestive tract inside the stolon still works, so that fecal pellets were expelled from the posterior part of the stolon. (D) Eyes are then formed, and the border between the stock individual and the stolon becomes apparent. (E) Sexes of fully developed stolons can easily be distinguished by their body color: female stolons are greenish and males are yellowish. (F) Prior to the detachment of the stolon, the new stock tail has already been formed under the stolon head. (G, H) Abnormal individuals sometimes appear. The most frequent type is the individuals with numerous eyes in all the posterior segments.

When eyes started to develop (Fig. 5C), the digestive tract inside the developing stolon is still connected with that of the stock individual, since fecal pellets came out from the tail of the developing stolon. The digestive tube in the stolon gradually became smaller, as the coelum became occupied by gonads. At the end of gonad development, the sex of stolons could easily be distinguished by the color of testis and ovary tissues (Fig. 5E): male stolons were yellowish white while females were more greenish with visible eggs (Figs. 1B, 5E). Interestingly, before the stolon detachment, a new tail (pygidium and posterior segments) of the stock already started to be formed under the head of the stolon, i.e., on the ventral side of the stolon head (Fig. 5F). The onset process of posterior regeneration is different from the normal regeneration process which is initiated by amputation of body parts (Bely and Nyberg, 2010; Brockes and Kumar, 2008; Ribeiro et al., 2018). This situation has been previously reported in Megasyllis and Alcyonosyllis species (Aguado and San Martín, 2009; Aguado et al., 2012).

Among cultured individuals, abnormal individuals occasionally appeared. One of the most frequent morphotype showed numerous orange-reddish lateral spots in the posterior segments, which resembled eyes (Fig. 5G, H). The possible development of lateral eyes suggests that every posterior segment possesses the potential to become a stolon head. These individuals appeared when environmental conditions were poor, suggesting that some physiological regulations are involved in the stolonization process; however, careful and extensive investigations would be required to prove this.

The number of male and female stolons produced after sampling (28 October 2016) were counted ( Supplementary Figure S1 (10.2108.zsj.36.372.s1.pdf)). Although we could not distinguish the first, second, or third production of stolons from each individual, the results suggest that a maximum of three rounds of stolonization per stock individual occurred. Male stolons were produced at 77.64 ± 21.35 days (n = 126) after sampling, while female stolons were produced at 73.03 ± 22.48 days (n = 123). There was no significant difference between the sexes (t-test; P = 0.098).

Spawning and fertilization

When the opposite sex stolons encountered, they began to actively vibrate to spawn eggs and sperms (Fig. 6A). Eggs were spawned as a cluster or spread on the bottom of the container (Fig. 6A, B). Female stolons often held the eggs under the laboratory condition, and also male stolons occasionally did so. The diameter of an egg was about 100 µm, and the color was whitish (Fig. 6B, C). The sperm head was oval-shaped and the sperm was strait shaped and ca. 40–50 µm in length (Fig. 6D). Parthenogenesis is not likely, since unfertilized eggs did not undergo embryogenesis (data not shown). Furthermore, artificial fertilization was attempted by dissecting mature detached stolons, but none of the eggs hatched thereafter. Some activation processes appear to be required at spawning.

Fig. 6.

Spawned eggs and sperms. (A) Stolons were located alongside the spawned eggs. Both male and female stolons sometimes held the egg mass. (B) Eggs were often laid on the bottom of the plastic case. (C, D) By DIC microscopic observations, eggs and sperms were recognized.

Postembryonic development: Metatrochophore

The process of postembryonic development was observed in detail, and the period required for each developmental stage was also recorded (Table 1). After spawning, embryogenesis immediately started. Female stolons sometimes spawned eggs even without male stolons, although those eggs did not develop. It took 2.67 ± 1.25 days (n = 87) after fertilization until hatching. After hatching, metatrochophore larvae crawled on the bottom (Fig. 7A, B). Although the detailed process of embryogenesis was not observed in this study, the larvae hatched from eggs were not planktonic ones, but rather benthic metatrochophores, exhibiting direct development, as previously described in other species, such as Typosyllis prolifera (Franke, 1980). Only a few syllid species, such as Typosyllis pulchra (Heacox, 1980), have been reported to have swimming trochophore larvae. In M. nipponica, the developmental phase of trochophore seems to take place inside the extraembryonic membrane, and the hatched metatrochophores immediately crawl by ventral ciliation.

Postembryonic development: Juvenile

The metatrochophore period (MT) until the appearance of a complete digestive tract (including the proventricle) was 2.69 ± 2.07 days (n = 71). After the appearance of the digestive tract, these individuals fed on spinach powder and Nannochloropsis, which were visible inside the digestive tract (Fig. 7C, D). These individuals were termed “juveniles (JV),” as they did not possess some of the adult characters, such as the black spot. Later, the body size and the number of segments increased (Fig. 7E). The JV stage with a clearly detectable digestive tract before the appearance of a black spot was relatively long (23.26 ± 11.36 days: n = 38).

Fig. 7.

Postembryonic stages. (A, B) Metatrochophore larvae. (C, D) Juveniles with apparent digestive tracts. (E) Large juveniles in which a black spot is about to appear. (F, G) Black-spotted individual which possesses adult-like characters. Arrows indicate the black spot. (H) Lateral view of the head of a black-spotted individual. The black spot is actually a band located at the posterior end of the pharynx.

Table 2.

Periods from appearance of black-spot to stolon detachment.

Postembryonic development: Appearance of black spot

The end of the juvenile stage was defined as the stage at the appearance of a black spot, which is actually a black ring surrounding the pharynx (Fig. 7F–H). The black-spotted individuals (BS) possessed almost the same body structure as adult individuals. The total duration from spawning until the BS stage (EG-BS) was 26.49 ± 15.02 days (n = 45). After the appearance of the black spot, the individuals were maintained to measure the period until stolonization. The black spot was one of the key identifying traits of this species, but the adaptive significance of the pigmentation is totally unknown. When feeding, the black spot moves back and forth, showing that it surrounds the pharynx and moved by muscle contraction.

The period from the beginning of the BS stage until the first production of male stolons was 34.87 ± 15.03 days (n = 15), and until the first production of female stolons was 26.27 ± 13.05 days (n = 15) (Table 2). A significant difference between these two periods was not detected (t-test, P = 0.394).

Conclusion: M. nipponica as a study material

Based on the observation results, the life cycle of M. niponica is documented (Fig. 8). Under the rearing conditions (21°C, 16L:8D), postembryonic development up to the BS stage requires less than one month (∼25 days). Then, another month is required until the detachment of a stolon (∼35 days). In total, two months are suggested to be required for a generation, under favorable conditions. We have successfully cultured the focal strain in the laboratory up to four offspring generations (three full life cycles through four stolonizations; data not shown). The necessities for culturing this material species are food supply, maintenance of water quality, and collection of stolons for successful reproduction. Although they sometimes require careful handling, this species may be a suitable experimental animal that can be easily maintained in the laboratory. Since this animal possesses general biological features shared by Syllinae species, future developmental studies using M. nipponica will contribute to unraveling the shared mechanisms underlying the fascinating reproductive mode that has been acquired in this animal lineage.

Fig. 8.

Life cycle of Megasyllis nipponica. Number of days required for each postembryonic stage under the laboratory rearing conditions is indicated. The generation time for the focal species is suggested to be about two months.