Introduction

Box jellyfishes are considered among the most dangerous sea creatures due to the lethal poisonous stings to humans (Cunningham & Goetz, 1996; Fenner & Williamson, 1996). In order to predict to occurrences of box jellyfishes, it is necessary to understand their ecology and life cycle. Research on the development of Cubozoa was first reported by Conant (1898). He found mature eggs of Tripedalia cystophora Conant, 1897, in stomach pouches of the female medusa which developed into swimming planulae. The planulae settled and developed into small polyps with three to five tentacles. Unfortunately, the polyps died after three weeks in aquaria without undergoing further development. Okada (1927) observed the segmentation of eggs of Carybdea brevipedalia Kishinouye, 1891 (as Carybdea rastonii), and reared the resulting planulae to three-tentacled polyps but they died of starvation within a few weeks.

Werner et al. (1971) first succeeded rearing T. cystophora and showed an entire life cycle of this species. Cubozoa has alternation of generations, between a sexually planktonic medusa and an asexually benthic polyp (Werner et al., 1971). Cubopolyps possess a clear radial-symmetrical body that lacks all inner structures, including the four gastric septae, four gastric pockets, four longitudinal muscle strands, and four septal funnels present in scyphopolyps (Werner, 1973, 1976, 1993; Chapman, 1978). Additionally, solitary cubopolyp metamorphose completely into a single juvenile medusa without any remaining regenerative residuum on the substrata (Werner et al., 1971; Cutress & Studebaker, 1973; Arneson & Cutress, 1976; Yamaguchi & Hartwick, 1980; Straehler-Pohl & Jarms, 2011; Carrette et al., 2014; Toshino et al., 2014). Werner (1973) established the class Cubozoa based on these unique features. However, cubopolyps of Carybdea marsupialis (Linnaeus, 1758) and Morbakka virulenta (Kishinouye, 1910) have been shown to undergo something akin to a modified strobilation (Straehler-Pohl & Jarms, 2005; Toshino et al., 2015).

The family Tripedaliidae currently comprises three species in two genera, Tripedalia and Copula (Bentlage et al., 2010; Straehler-Pohl et al., 2014). Tripedaliids are characterized by their display of sexual dimorphism of the gonads, production of spermatophores, and males and females possessing subgastral sacs, pockets, or purses which function as seminal vesicles or spermathecae (Straehler-Pohl et al., 2014). In addition, recent molecular phylogenetic analyses suggest that Copula sivickisi (Stiasny, 1926) is more closely related to T. cystophora than any other species (Bentlage et al., 2010).

Tripedalia binata Moore, 1988, is a small species in the class Cubozoa, with a maximum bell height of 11 mm and maximum bell diameter (interpedalial distance) of 14.5 mm, that possesses two pedalia per corner of the bell margin instead of the three characteristic of T. cystophora (Moore, 1988). This species displays sexual dimorphism of the gonads in which mature males possess stick-shaped testes while mature female possess butterfly-shaped ovaries (Straehler-Pohl et al., 2014). Additionally, it was suggested that T. binata likely shows copulation behavior because both sexes possess stomach purses, and a male specimen was shown to have spermatophores in its purse (Straehler-Pohl et al., 2014).

Tripedalia binata has been reported from eastern India and northern Australia (Moore, 1988; Underwood et al., 2013). Medusae of this species were found in creeks, aquaculture pond, and sandy beaches, near the mangroves during the rainy season (Moore, 1988; Underwood et al., 2013). However, the life cycle of this species has never been studied. The present paper describes its life history from fertilized eggs to polyp formation to metamorphosis into a medusa and compares morphological characters between other cubozoans to discuss ecology and phylogeny in the class Cubozoa.

Materials and methods

Tripedalia binata medusae (Fig. 1A, B) were collected using an underwater fish-luring lamp (YF-500, Hapyson, Japan) at Mangrove swamp, Trat Province, the eastern Gulf of Thailand (Fig. 2), between 21:00 and 23:30 on March 17, 2014 (water temperature 32.3°C, salinity 21.8). Two mature medusae, one female and one male, were taken by a dip net (mesh size 0.2 mm). Male and female medusae were kept in a bottle (diameter 72 mm, height 97 mm, water volume 1000 ml) with fresh seawater (salinity 33) at about 25 to 30°C in the laboratory at Burapha University, Thailand. These medusae were fed with mysid or Artemia nauplii on a daily basis. Rearing water was replaced with fresh seawater twice or thrice a week.

Fig. 1
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Tripedalia binata Moore, 1988, live, lateral view, in laboratory. A Mature female, B mature male. GF gastric filaments, P pedalium, Rh rhopalium, T tentacle. Scale bars 5 mm

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Fig. 2
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Map of the sampling site, Trat, Eastern Thailand

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Planulae obtained from these medusae in the laboratory were incubated in petri-dishes (diameter 75 mm, height 45 mm) filled with filtered seawater (0.22 μm filter pore size) at 30°C (temperature of the sampling site) in an incubator. Primary polyps were transferred to petri-dishes (diameter 78 mm, height 24 mm) filled with filtered seawater (1 μm filter pore size) and kept at 30°C. Chopped Artemia nauplii were fed directly to polyps using a fine needle on a daily basis. Rearing water was completely replaced with filtered seawater (1 μm filter pore size) about 3 h after feeding. During metamorphosis from polyp to medusa, the cultures were not fed nor the water changed. Several polyps were separated and exposed to a wide range of temperatures (15 to 30°C) to observe polyp reaction.

Newly detached medusae were kept in a disposable polypropylene cup (water volume 1000 ml) with filtered seawater (1 μm filter pore size) at 30°C. Artemia nauplii were fed to the medusae on a daily basis. Culture water was replaced with fresh seawater about three hours after feeding.

For nematocyst identification in polyps and medusae, fresh tissue was squashed under a cover slip and examined under an optical microscope (CX 21, Olympus, Japan). Nematocysts were identified according to Gershwin (2006) and Collins et al. (2011). For determination of the respective abundance of nematocyst types in multiple areas on the tentacles of polyps and medusae, at least 200 nematocysts were counted.

Results

Fertilization and polyp formation

On the night of collection, gonads of female and male medusae were transparent. Spermatophores were not observed in the stomach purse of both male and female medusae. Three days after medusae were collected, and fertilization was observed in the laboratory. The gonads of female became yellow, while male testis withered. Three days after fertilization, hundreds of free-swimming planulae (Figs. 1A, 3A, B) were bred in gastric cavity of the female. Two days later, all planulae were released from the female’s gastric cavity. Planulae (Fig. 3C, D) were about 100 µm in diameter and about 130 µm in length and had about twenty dark reddish larval ocelli at the part of aboral.

Fig. 3
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Early embryogenesis of Tripedalia binata. A Planulae swimming in gastric cavity of female medusa, B planulae swimming in velarial canals of female medusa, C planula, lateral view, note eye spots, D planula, vertical view, note eye spots. Arrows indicate velarial canal. Scale bars A, B 1 mm, C, D 100 µm

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Two to four days after planula formation, planulae settled on the bottom of petri-dishes or water surface (Fig. 4A). Planulae developed into primary polyps, and larval ocelli faded out within 2 days. Primary polyps were either settled (Fig. 4B–D) or actively detached to start a creeping phase (Fig. 4B, E). The shape of the settled primary polyps resembled a pouch with a very short stalk, with one to four tentacles protruding from the ovoid calyx around the mouth cone. At a body length of about 0.28 mm the mouth disc diameter of the polyps was about 0.08 mm. The primary creeping polyps had elastic worm-shaped body with one to four outstretched tentacles. They crept to change location using their body and tentacles. At a body length of about 0.44 mm, the mouth disc diameter of the polyps was about 0.07 mm. Both settled and creeping primary polyps bore five to ten nematocysts (American football-shaped p-rhopaloids) in the tip of their tentacles (Fig. 5A).

Fig. 4
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Polyps of Tripedalia binata. A Just after settled planulae and primary polyps on water surface, B primary polyps; left creeping polyp; right settled polyp, C adult polyp, lateral view, D adult polyp with cysts, oral view; upper just after excysted bud; lower cyst, E creeping adult polyp. Scale bars A 1 mm, B 100 µm, C, D 0.2 mm, E 0.5 mm

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Fig. 5
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Nematocysts of Tripedalia binata. A Nematocysts of tentacle tip of primary polyp, B nematocysts of tentacle tip of adult polyp, C nematocysts of adult polyp, D nematocysts of exumbrella warts, ovoid heterotrichous microbasic euryteles and spherical holotrichous isorhizas, E nematocysts of tentacle, American football-shaped p-rhopaloids, and ovoid isorhizas. AFPR American football-shaped p-rhopaloid, HE heterotrichous eurytele, OHME ovoid heterotrichous microbasic eurytele, OI ovoid isorhiza, RC Red chromatophore, SHI spherical holotrichous isorhiza, SPR spherical p-rhopaloid. Scale bars A–C 50 µm, D, E 20 µm

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The primary polyps developed into adult polyps within 49 days. The settled adult polyps (Fig. 4C, D) were able to detach and creep in order to change locations. When the polyps began to creep, a peristomal cup was left behind. The shape of the adult polyps was almost identical to the primary polyp stage, but they were larger (settled polyps: mean body length 0.52 mm, mean mouth disc diameter 0.36 mm; polyps in creeping phase: mean body length 0.63 mm, mean mouth disc diameter 0.16 mm). Both settled and creeping polyps had seven to eight tentacles and each tip of the tentacle bore about 15 heterotrichous euryteles and two American football-shaped p-rhopaloids (Fig. 5B, C). The polyps (N = 3) possessed three types of nematocysts, heterotrichous euryteles (31%), American football-shaped p-rhopaloids (44%), and spherical p-rhopaloids (25%) in their body (Fig. 5C). Further developmental stages, i.e., budding and metamorphosis took only place in settled polyps.

Asexual polyp reproduction

Asexual reproduction was observed when the 3-tentacled stage was reached. Bud formation occurred at the middle part of the calyx (Fig. 6A). One day after the start of bud formation, two tentacles and mouth were formed on the new bud (Fig. 6B). Three days after the start of bud formation, a secondary polyp (bud) was released from the parent polyp (Fig. 6C). The buds settled near the parent polyp at first and detached later on actively to perform a creeping phase (Fig. 6D) like the one observed in the adult polyp. The creeping phase lasted for one day until the creeping bud settled on the bottom of the petri dish and grew out into an adult polyp within 60 days. The settled buds resembled the parent polyps (Fig. 6E, F). The buds were 0.19 mm in diameter and had three to four tentacles.

Fig. 6
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Asexual reproduction of polyp. Arrow indicates bud. A just after start of budding, B 1 day later, C 2 days later, D newly released creeping polyp, E newly released settled polyp, lateral view, F newly released settled polyp, oral view. Scale bars A–D 0.5 mm, E, F 0.2 mm

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Cysts

Below temperatures of 20°C, culturing water quality deterioration, or starvation, adult polyps formed resting stages (Fig. 4D, 7A). The polyps contracted into a ball and were encapsulated by a soft layer, forming cysts. The cysts were yellowish, about 0.45 mm in diameter and fixed to the substrate. After the temperature was raised above 23°C or improvement of water condition and nutritional condition of cysts, polyps regenerated within two weeks, excysting from the capsules (Fig. 7B, C).

Fig. 7
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The process of excystment of polyp. A cyst, B just after excysted, C 1 week later after excysted. Scale bars 0.5 mm

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Metamorphosis

Metamorphosis occurred at temperatures between 23 and 30°C. Metamorphosis from a single 8-tentacled polyp into a medusa was observed (Table 1). The first evidence of medusa development was the formation of four temporary vertical furrows in the hypostome of the polyp (Phase 1). The bases of different numbers of the polyp tentacles (one to three) coalesced randomly on the four corners of the peristomal edge. Two days after the initiation of metamorphosis, the bases of the tentacles fused and thickened to bulbs, and the free distal parts were absorbed (Phase 2). Pigmentation marks appeared on the hypostome-facing side of these tentacle base bulbs. One medusa tentacle formed between each tentacle base bulbs, for a total of four tentacles. Three days after the initiation of metamorphosis, the pigmented marks transformed into two eyes with lenses and four ocelli, while statocysts emerged on the opposite side of the bulbs (Phase 3). A shallow horizontal groove appeared on the lateral sides of the polyp, dividing the calyx into a metamorphosing upper half with plate like medusoid tissue and a polypoid lower part with cylindrical cells. The yellow color of the polyps changed into darker yellowish-brown shades. Four days after the initiation of metamorphosis, transformation of the polyp tentacles into four rhopalia and the hypostome into a manubrium was complete. Upper two-thirds of calyx changed into medusoid tissue. Five days after the initiation of metamorphosis, the medusa opened the lips within the subumbrella (Phase 4). All of the calyx changed into medusoid tissue. Six days after the initiation of metamorphosis, the medusa bell enlarged considerably and nematocyst clusters appeared on the exumbrella (Phase 5). The medusae began pulsating. The following day it detached from the substrate and transformed into a single medusa without leaving any regenerative remnants.

Medusa development

Newly detached medusae had a tetrameric, pyramid-like bell with a rounded top and were yellowish-brown to yellowish in color (Fig. 8A–C). They were about 1.2 mm in umbrella height, about 1.3 mm in umbrella width, and all had one pedalium and one tentacle per interradius. Red chromatophores were scattered in the bell tissue (Fig. 5D). The exumbrella was sprinkled with very small (0.01–0.05 mm) round nematocyst clusters, consisting mainly of spherical holotrichous isorhizas and ovoid heterotrichous microbasic euryteles (Fig. 5D), aligned along radial furrows. The manubrium had four lips and was about 30% of the umbrella height. One to two gastric filaments per corner were visible through the apex of the umbrella. The sensory niches were shallow and still roofless (Table 2). Tentacles appeared as a “string of pearls” (Fig. 8D) with about twenty spherical white nematocyst batteries consisting of American football-shaped p-rhopaloids and ovoid isorhizas (Fig. 5E). When completely extended, the tentacles were up to 3 mm in length.

Fig. 8
figure 8

Newly detached medusae of Tripedalia binata. A lateral view, B apical view, C oral view, D tentacle. Arrows indicate nematocyst wart. Scale bars 1 mm

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Two-day-old medusae were about 1.4 mm in height and about 1.8 mm in width (Table 2). Secondary pedalium buds appeared at the bases of the pedalium. The primary pedalium elongated and thickened. The border between tentacle and pedalium was obscure. A downwardly directed gelatinous roof formed above the sensory organ. Four-day-old medusae were about 2.1 mm in height and about 2.7 mm in width. Ovoid gonad appeared at the middle part of interradial furrows. The secondary tentacles developed which bore four to six nematocyst rings. Upper half of the primary pedalium thickened. The border between tentacle and pedalium became distinct. Seven-day-old medusae were about 3.3 mm in height and about 3.9 mm in width. Full part of the primary and secondary pedalium thickened. The primary pedalia was about twice as long as the secondary ones. The sensory roof arch deepened. Gonads enlarged. Ten-day-old medusae were about 3.3 mm in height and about 4.0 mm in width. The gonads elongated and became rod-shaped. Two gastric filaments per corner were visible through the apex of the umbrella. The pedalium developed knife-shaped. Dome-shaped rhopaliar niches were formed. Twenty-day-old medusae were about 4.5 mm in height and about 6.2 mm in width. Two to three gastric filaments were observed. The sensory roof almost surrounded rhopalium, and dome-shaped rhopaliar niche developed. The primary and secondary pedalium developed knife-shaped which length of both pedalium was equal. Gonads were rod-shaped.

Discussion

From our laboratory rearing experiments, the early life cycle of T. binata was clarified (Table 3; Fig. 9). Both male and female medusae possess stomach purses, especially, spermatophores were found in the purses of the male medusae (Straehler-Pohl et al., 2014). On the night of collection, spermatophores were not observed in the stomach purse of both sexes, and gonads of female and male medusae were still transparent. In case of C. sivickisi, ovaries of the female medusae soon became cloudy after spermatophores transfer (Lewis & Long, 2005). The embryo release occurred 24 h after the female captured the first spermatophore (Lewis et al., 2008; Toshino et al., 2014). In our laboratory, fertilized eggs were observed in female medusae three days after collection. It is seems likely that copulation and spermatophores transfer was conducted after the time of medusae collection.

Table 1 The process of metamorphosis in the polyp of Tripedalia binata
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Table 2 The process of young medusae development
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Table 3 Chronology of developmental events in Tripedalia binata
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Fig. 9
figure 9

Composite image of the life cycle of T. binata, based on laboratory and field surveys. Inter-stage durations are also shown.

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Fertilized eggs of T. binata developed into planulae in the gastral pockets of the female medusa. This habit had been reported in T. cystophora (Werner et al., 1971). In case of the tripedaliid C. sivickisi, blastulae developed into planulae in the gelatinous strands which were released from the female medusae (Hartwick, 1991a; Lewis & Long, 2005; Lewis et al., 2008; Toshino et al., 2014). In other cubozoans, internal fertilization occurred in C. marsupialis (Studebaker, 1972), Alatina moseri (Mayer, 1906) collected from Puerto Rico (Arneson & Cutress, 1976; as Carybdea alata), while external fertilization occurred in Chironex fleckeri Southcott, 1956 (Yamaguchi & Hartwick, 1980), M. virulenta (Toshino et al., 2013) and A. moseri from Hawaii and Australia (Carrette et al., 2014). Breeding behavior and embryo strand seem to be systems that protect blastulae and planulae against predators (Toshino et al., 2014).

Cubozoan planulae bear larval ocelli which have been described as a character common to all species except M. virulenta (Widersten, 1968; Nordström et al., 2003; Toshino et al., 2013). Ocelli are distributed in the middle part of planulae in C. brevipedalia (Okada, 1927; as Carybdea rastonii), C. marsupialis collected from Puerto Rico (Studebaker, 1972), A. moseri collected from Puerto Rico (Arneson & Cutress, 1976; as C. alata) and C. sivickisi (Hartwick, 1991a; as Carybdea sivickisi, Toshino et al., 2014), across the whole planula except the oral and aboral ends in Chironex yamaguchii (Lewis & Bentlage, 2009) (Toshino, unpublished data), and lacking in M. virulenta (Toshino et al., 2013). Planulae of T. binata had about twenty dark reddish larval ocelli in the aboral region. This distribution pattern of larval ocelli is up to now unique within the Cnidaria.

Fully developed polyps of T. binata can be distinguished from other cubozoan polyps by their shape and size, and arrangement and number of the tip of tentacles nematocyst (Table 4). Other cubozoan polyps are flask-shaped like C. marsupialis collected from Puerto Rico (Studebaker, 1972; Straehler-Pohl & Jarms, 2011) and A. moseri (Straehler-Pohl & Jarms, 2011 as Alatina mordens; Carrette et al., 2014), amphora-shaped like Alatina morandinii (Straehler-Pohl & Jarms, 2011) (Straehler-Pohl & Jarms, 2011 as Carybdea morandinii), and tulip-shaped like M. virulenta (Toshino et al., 2013). Polyps of tripedaliids (T. binata, T. cystophora, and C. sivickisi) had a pouch-shaped body and were smaller than other cubozoans. Tip of tentacles contain a single stenotele in A. morandinii, A. moseri, C. marsupialis, and C. fleckeri multiple in tripedaliids and M. virulenta but different arrangements showed which were vertically in Tripedaliids (Fig. 5A, B), radially in M. virulenta (Toshino et al., 2013).

Table 4 Size (mm) and morphology of fully developed cubozoan polyps in previous and the present studies
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Table 5 Size (mm) and morphology of cubozoan medusae in previous and the present studies
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The polyps of T. binata had the ability to settle or creep and displayed budding of creeping polyps. This flexibility appears well suited for the muddy conditions of their native habitat in mangrove swamps. Substrate for attachment of the polyps is scarce in mangrove swamps. If adult T. binata medusae spawned planulae over soft bottoms such as in our sampling site, polyps might get buried in the sediment. Under these conditions, the creeping polyps of T. binata may play a key role in assuring successful recruitment to adequate solid substrates such as roots of mangrove trees.

The polyps of T. binata formed cysts. Encystment and excystment of polyps has been observed in T. cystophora (Werner, 1975; Straehler-Pohl, 2001; 2009), C. fleckeri (Hartwick, 1991b), C. marsupialis collected from Puerto Rico (Straehler-Pohl, 2001; 2009), A. moseri collected from Hawaii and Australia (Straehler-Pohl, 2001, 2009 as A. mordens, Carrette et al., 2014), A. morandinii (Straehler-Pohl & Jarms, 2011), and C. sivickisi (Toshino et al., 2014). Conditions causing encystment were drastic salinity changes in T. cystophora and C. fleckeri, water temperature changes above 27°C or below 15°C in A. morandinii (Straehler-Pohl & Jarms, 2011) and below 20°C in C. sivickisi (Toshino et al., 2014) below 19°C or when salinity was increased to 42 or feeding ceased for a period of four to five weeks in A. moseri (Carrette et al., 2014). Excystment occurred after favorable environmental conditions returned in A. morandinii, raising temperature above 28°C in C. sivickisi, and feeding in A. moseri. Polyps of T. binata encysted at low temperature (20°C) and salinity 33, culturing water quality deterioration or starvation, and excysted at a higher temperature (23°C), or improvement of water condition and nutritional condition of cysts. The sampling site was a semi-closed mangrove swamp leading to the sea, with sea water flowing into the swamp during rising tides. Moreover, during the wet seasons (June–October in Thailand), it rained regularly. Under these conditions, water temperature and salinity of the swamp change drastically. Cysts of T. binata seem to play an important role to survive in these drastic conditions.

Metamorphosis in T. binata was complete and without any residuum as in the majority of known cubozoan life cycles, with C. marsupialis from Puerto Rico and M. virulenta as exceptions (Straehler-Pohl & Jarms, 2005; Toshino et al., 2015). Metamorphosis was induced by stable temperature in T. cystophora, C. morandinii, C. marsupialis, A. moseri (Straehler-Pohl & Jarms, 2011), and C. fleckeri (Yamaguchi & Hartwick, 1980), increasing temperature in M. virulenta (Toshino et al., 2015) and C. sivickisi (Toshino et al., 2014) (Table 4). Metamorphosis of T. binata was induced by stable temperatures between 23 and 30°C. In Australia, young and adult medusae of T. binata occurred between March and October (Moore, 1988; Underwood et al., 2013) when water temperatures change from approximately 24–29°C (Australian Government, Bureau of Meteorology, 2015). Under these conditions, the polyps of T. binata may produce medusae.

Newly detached medusae of T. binata can be distinguished from hitherto described medusae of other cubozoans by the shape and cnidome of tentacles, pattern of nematocyst warts on the exumbrella (Table 5). The shape of the medusae resembles more closely those of T. cystophora and C. sivickisi than C. morandinii, C. marsupialis, M. virulenta, and A. moseri (Table 5). T. binata has a spherical umbrella with four long pearl string-like tentacles and nematocyst warts lining the radial furrows (Fig. 8B), very closely resembling the characters of T. cystophora and C. sivickisi. Alatina morandinii by contrast shows filiform tentacles; C. marsupialis shows only two tentacles; and A. moseri and M. virulenta shows four extremely short capitate tentacles. T. binata has characteristic tentacular nematocysts (American football-shaped p-rhopaloids) and red chromatophores in the exumbrella.

Our results show that the developmental features of T. binata resemble more closely those of T. cystophora and C. sivickisi than any other cubozoan. The similarities support the hypothesis that T. binata, T. cystophora, and C. sivickisi are closely related as part in the family Tripedaliidae that was established based on adult medusa morphology and a molecular phylogenetic analysis by Bentlage et al. (2010) and Straehler-Pohl et al. (2014). Research on the life cycle elucidates the connection between benthic polyp and planktonic medusa. Research on the cubozoan life cycles is important to further understand the evolutionary history and ecology of Cubozoa.