Development of Tripedalia Binata Moore, 1988 (Cubozoa

Home , Carybdea, Carybdeida, Copula sivickisi, Tamoya, Tripedalia cystophora

Hydrobiologia (2017) 792:37–51 DOI 10.1007/s10750-016-3022-1

PRIMARY RESEARCH PAPER

Development of Tripedalia binata Moore, 1988 (Cubozoa: Carybdeida: Tripedaliidae) collected from the eastern Gulf of Thailand with implications for the phylogeny of the Cubozoa

Sho Toshino . Hiroshi Miyake . Khwanruan Srinui . Nattawut Luangoon . Vorathep Muthuwan . Saowapa Sawatpeera . Shiho Honda . Haruka Shibata

Received: 17 March 2016 / Revised: 27 September 2016 / Accepted: 8 October 2016 / Published online: 28 October 2016 Ó Springer International Publishing Switzerland 2016

Abstract Box jellyfishes are considered among the of budding. Complete metamorphosis of a whole most dangerous sea creatures due to the lethal polyp into a single medusa occurred. Newly detached poisonous stings to humans. In order to predict the medusae were distinguished from those of other occurrences of box jellyfishes, it is necessary to cubozoans by the pattern of nematocyst warts on the understand their ecology and life cycle. The small box exumbrella and red chromatophores. The develop- jellyfish Tripedalia binata was collected from eastern mental features of T. binata resemble most closely Thailand, in order to observe its life history, to those of T. cystophora and Copula sivickisi. The compare its morphological characters with other similarities in all early life cycle stages of those cubozoans, and to discuss ecology and phylogeny in species support the close relationship of these species the class Cubozoa. Fertilization occurred internally, in the family Tripedaliidae. blastulae developed into planulae. Planulae were bred in the gastral pocket of the female medusa and Keywords Copulation Á Cyst Á Polyp formation Á released into the water. Free swimming planulae Medusa Á Metamorphosis settled and metamorphosed into polyps. Adult polyps formed cysts at temperatures below 20°C water deterioration or starvation. Budding occurred in adult polyps, and buds were released after commencement Introduction

Box jellyfishes are considered among the most dan- Handling editor: Jo¨rg Dutz gerous sea creatures due to the lethal poisonous stings to humans (Cunningham & Goetz, 1996; Fenner & S. Toshino (&) Á H. Miyake Á S. Honda Á H. Shibata Graduate School of Fisheries Sciences, Kitasato Williamson, 1996). In order to predict to occurrences University, 1-15-1 Kitasato, Sagamihara, of box jellyfishes, it is necessary to understand their Kanagawa 252-0373, Japan ecology and life cycle. Research on the development e-mail: [email protected] of Cubozoa was first reported by Conant (1898). He S. Toshino found mature eggs of Tripedalia cystophora Conant, Kuroshio Biological Research Foundation, Nishidomari, 1897, in stomach pouches of the female medusa which Otsuki, Hata, Kochi 788-0333, Japan developed into swimming planulae. The planulae settled and developed into small polyps with three to K. Srinui Á N. Luangoon Á V. Muthuwan Á S. Sawatpeera Institute of Marine Science, Burapha University, Mueang, five tentacles. Unfortunately, the polyps died after Chon Buri, Chon Buri 20131, Thailand three weeks in aquaria without undergoing further 123 38 Hydrobiologia (2017) 792:37–51 development. Okada (1927) observed the segmenta- suggested that T. binata likely shows copulation tion of eggs of Carybdea brevipedalia Kishinouye, behavior because both sexes possess stomach purses, 1891 (as Carybdea rastonii), and reared the resulting and a male specimen was shown to have sper- planulae to three-tentacled polyps but they died of matophores in its purse (Straehler-Pohl et al., 2014). starvation within a few weeks. Tripedalia binata has been reported from eastern Werner et al. (1971) first succeeded rearing T. India and northern Australia (Moore, 1988; Under- cystophora and showed an entire life cycle of this wood et al., 2013). Medusae of this species were found species. Cubozoa has alternation of generations, in creeks, aquaculture pond, and sandy beaches, near between a sexually planktonic medusa and an asex- the mangroves during the rainy season (Moore, 1988; ually benthic polyp (Werner et al., 1971). Cubopolyps Underwood et al., 2013). However, the life cycle of possess a clear radial-symmetrical body that lacks all this species has never been studied. The present paper inner structures, including the four gastric septae, four describes its life history from fertilized eggs to polyp gastric pockets, four longitudinal muscle strands, and formation to metamorphosis into a medusa and four septal funnels present in scyphopolyps (Werner, compares morphological characters between other 1973, 1976, 1993; Chapman, 1978). Additionally, cubozoans to discuss ecology and phylogeny in the solitary cubopolyp metamorphose completely into a class Cubozoa. single juvenile medusa without any remaining regenerative residuum on the substrata (Werner et al., 1971; Cutress & Studebaker, 1973; Arneson & Cutress, Materials and methods 1976; Yamaguchi & Hartwick, 1980; Straehler-Pohl & Jarms, 2011; Carrette et al., 2014; Toshino et al., Tripedalia binata medusae (Fig. 1A, B) were col- 2014). Werner (1973) established the class Cubozoa lected using an underwater fish-luring lamp (YF-500, based on these unique features. However, cubopolyps Hapyson, Japan) at Mangrove swamp, Trat Province, of Carybdea marsupialis (Linnaeus, 1758) and Mor- the eastern Gulf of Thailand (Fig. 2), between 21:00 bakka virulenta (Kishinouye, 1910) have been shown and 23:30 on March 17, 2014 (water temperature to undergo something akin to a modified strobilation 32.3°C, salinity 21.8). Two mature medusae, one (Straehler-Pohl & Jarms, 2005; Toshino et al., 2015). female and one male, were taken by a dip net (mesh The family Tripedaliidae currently comprises three size 0.2 mm). Male and female medusae were kept in a species in two genera, Tripedalia and Copula (Bent- bottle (diameter 72 mm, height 97 mm, water volume lage et al., 2010; Straehler-Pohl et al., 2014). 1000 ml) with fresh seawater (salinity 33) at about 25 Tripedaliids are characterized by their display of to 30°C in the laboratory at Burapha University, sexual dimorphism of the gonads, production of Thailand. These medusae were fed with mysid or 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 phyloge- netic 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 Fig. 1 Tripedalia binata Moore, 1988, live, lateral view, in laboratory. A Mature female, B mature male. GF gastric mature female possess butterfly-shaped ovaries filaments, P pedalium, Rh rhopalium, T tentacle. Scale bars (Straehler-Pohl et al., 2014). Additionally, it was 5mm 123 Hydrobiologia (2017) 792:37–51 39

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 Fig. 2 Map of the sampling site, Trat, Eastern Thailand 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 Artemia nauplii on a daily basis. Rearing water was cavity. Planulae (Fig. 3C, D) were about 100 lmin replaced with fresh seawater twice or thrice a week. diameter and about 130 lm in length and had about Planulae obtained from these medusae in the twenty dark reddish larval ocelli at the part of aboral. laboratory were incubated in petri-dishes (diameter Two to four days after planula formation, planulae 75 mm, height 45 mm) filled with filtered seawater settled on the bottom of petri-dishes or water surface (0.22 lm filter pore size) at 30°C (temperature of the (Fig. 4A). Planulae developed into primary polyps, sampling site) in an incubator. Primary polyps were and larval ocelli faded out within 2 days. Primary transferred to petri-dishes (diameter 78 mm, height polyps were either settled (Fig. 4B–D) or actively 24 mm) filled with filtered seawater (1 lm filter pore detached to start a creeping phase (Fig. 4B, E). The size) and kept at 30°C. Chopped Artemia nauplii were shape of the settled primary polyps resembled a pouch fed directly to polyps using a fine needle on a daily with a very short stalk, with one to four tentacles basis. Rearing water was completely replaced with protruding from the ovoid calyx around the mouth filtered seawater (1 lm filter pore size) about 3 h after cone. At a body length of about 0.28 mm the mouth feeding. During metamorphosis from polyp to disc diameter of the polyps was about 0.08 mm. The medusa, the cultures were not fed nor the water primary creeping polyps had elastic worm-shaped changed. Several polyps were separated and exposed body with one to four outstretched tentacles. They to a wide range of temperatures (15 to 30°C) to crept to change location using their body and tentacles. observe polyp reaction. At a body length of about 0.44 mm, the mouth disc Newly detached medusae were kept in a disposable diameter of the polyps was about 0.07 mm. Both polypropylene cup (water volume 1000 ml) with settled and creeping primary polyps bore five to ten filtered seawater (1 lm filter pore size) at 30°C. nematocysts (American football-shaped p-rhopaloids) Artemia nauplii were fed to the medusae on a daily in the tip of their tentacles (Fig. 5A). basis. Culture water was replaced with fresh seawater The primary polyps developed into adult polyps about three hours after feeding. within 49 days. The settled adult polyps (Fig. 4C, D) 123 40 Hydrobiologia (2017) 792:37–51

Fig. 3 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 lm

Fig. 4 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 lm, C, D 0.2 mm, E 0.5 mm

were able to detach and creep in order to change polyps in creeping phase: mean body length 0.63 mm, locations. When the polyps began to creep, a peris- mean mouth disc diameter 0.16 mm). Both settled and tomal cup was left behind. The shape of the adult creeping polyps had seven to eight tentacles and each polyps was almost identical to the primary polyp tip of the tentacle bore about 15 heterotrichous stage, but they were larger (settled polyps: mean body euryteles and two American football-shaped p-rhopa- length 0.52 mm, mean mouth disc diameter 0.36 mm; loids (Fig. 5B, C). The polyps (N = 3) possessed three

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Fig. 5 Nematocysts of Tripedalia binata. A Nematocysts of AFPR American football-shaped p-rhopaloid, HE heterotric- tentacle tip of primary polyp, B nematocysts of tentacle tip of hous eurytele, OHME ovoid heterotrichous microbasic eurytele, adult polyp, C nematocysts of adult polyp, D nematocysts of OI ovoid isorhiza, RC Red chromatophore, SHI spherical exumbrella warts, ovoid heterotrichous microbasic euryteles holotrichous isorhiza, SPR spherical p-rhopaloid. Scale bars A– and spherical holotrichous isorhizas, E nematocysts of tentacle, C 50 lm, D, E 20 lm American football-shaped p-rhopaloids, and ovoid isorhizas. types of nematocysts, heterotrichous euryteles (31%), Cysts American football-shaped p-rhopaloids (44%), and spherical p-rhopaloids (25%) in their body (Fig. 5C). Below temperatures of 20°C, culturing water quality Further developmental stages, i.e., budding and meta- deterioration, or starvation, adult polyps formed morphosis took only place in settled polyps. resting stages (Fig. 4D, 7A). The polyps contracted into a ball and were encapsulated by a soft layer, Asexual polyp reproduction forming cysts. The cysts were yellowish, about 0.45 mm in diameter and fixed to the substrate. After Asexual reproduction was observed when the 3-ten- the temperature was raised above 23°C or improve- tacled stage was reached. Bud formation occurred at ment of water condition and nutritional condition of the middle part of the calyx (Fig. 6A). One day after cysts, polyps regenerated within two weeks, excysting the start of bud formation, two tentacles and mouth from the capsules (Fig. 7B, C). were formed on the new bud (Fig. 6B). Three days after the start of bud formation, a secondary polyp Metamorphosis (bud) was released from the parent polyp (Fig. 6C). The buds settled near the parent polyp at first and Metamorphosis occurred at temperatures between detached later on actively to perform a creeping phase 23 and 30°C. Metamorphosis from a single 8-ten- (Fig. 6D) like the one observed in the adult polyp. The tacled polyp into a medusa was observed (Table 1). creeping phase lasted for one day until the creeping The first evidence of medusa development was the bud settled on the bottom of the petri dish and grew out formation of four temporary vertical furrows in the into an adult polyp within 60 days. The settled buds hypostome of the polyp (Phase 1). The bases of resembled the parent polyps (Fig. 6E, F). The buds different numbers of the polyp tentacles (one to were 0.19 mm in diameter and had three to four three) coalesced randomly on the four corners of tentacles. the peristomal edge. Two days after the initiation of

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Fig. 6 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

Fig. 7 The process of excystment of polyp. A cyst, B just after excysted, C 1 week later after excysted. Scale bars 0.5 mm metamorphosis, the bases of the tentacles fused and into darker yellowish-brown shades. Four days after thickened to bulbs, and the free distal parts were the initiation of metamorphosis, transformation of absorbed (Phase 2). Pigmentation marks appeared the polyp tentacles into four rhopalia and the on the hypostome-facing side of these tentacle base hypostome into a manubrium was complete. Upper bulbs. One medusa tentacle formed between each two-thirds of calyx changed into medusoid tissue. tentacle base bulbs, for a total of four tentacles. Five days after the initiation of metamorphosis, the Three days after the initiation of metamorphosis, medusa opened the lips within the subumbrella the pigmented marks transformed into two eyes (Phase 4). All of the calyx changed into medusoid with lenses and four ocelli, while statocysts tissue. Six days after the initiation of metamorpho- emerged on the opposite side of the bulbs (Phase sis, the medusa bell enlarged considerably and 3). A shallow horizontal groove appeared on the nematocyst clusters appeared on the exumbrella lateral sides of the polyp, dividing the calyx into a (Phase 5). The medusae began pulsating. The metamorphosing upper half with plate like medu- following day it detached from the substrate and soid tissue and a polypoid lower part with cylin- transformed into a single medusa without leaving drical cells. The yellow color of the polyps changed any regenerative remnants. 123 Hydrobiologia (2017) 792:37–51 43

Table 1 The process of metamorphosis in the polyp of Tripedalia binata

HG horizontal groove, MTe medusa tentacle, MTi medusoid tissue, Pe periderm, PT polyp tentacle, St statocyst, TBB tentacle base bulb, Rh rhopalium, VF vertical furrow

Medusa development pedalium buds appeared at the bases of the pedalium. The primary pedalium elongated and thickened. The Newly detached medusae had a tetrameric, pyramid- border between tentacle and pedalium was obscure. A like bell with a rounded top and were yellowish-brown downwardly directed gelatinous roof formed above to yellowish in color (Fig. 8A–C). They were about the sensory organ. Four-day-old medusae were about 1.2 mm in umbrella height, about 1.3 mm in umbrella 2.1 mm in height and about 2.7 mm in width. Ovoid width, and all had one pedalium and one tentacle per gonad appeared at the middle part of interradial interradius. Red chromatophores were scattered in the furrows. The secondary tentacles developed which bell tissue (Fig. 5D). The exumbrella was sprinkled bore four to six nematocyst rings. Upper half of the with very small (0.01–0.05 mm) round nematocyst primary pedalium thickened. The border between clusters, consisting mainly of spherical holotrichous tentacle and pedalium became distinct. Seven-day-old isorhizas and ovoid heterotrichous microbasic euryte- medusae were about 3.3 mm in height and about les (Fig. 5D), aligned along radial furrows. The 3.9 mm in width. Full part of the primary and manubrium had four lips and was about 30% of the secondary pedalium thickened. The primary pedalia umbrella height. One to two gastric filaments per was about twice as long as the secondary ones. The corner were visible through the apex of the umbrella. sensory roof arch deepened. Gonads enlarged. Ten- The sensory niches were shallow and still roofless day-old medusae were about 3.3 mm in height and (Table 2). Tentacles appeared as a ‘‘string of pearls’’ about 4.0 mm in width. The gonads elongated and (Fig. 8D) with about twenty spherical white nemato- became rod-shaped. Two gastric filaments per corner cyst batteries consisting of American football-shaped were visible through the apex of the umbrella. The p-rhopaloids and ovoid isorhizas (Fig. 5E). When pedalium developed knife-shaped. Dome-shaped rho- completely extended, the tentacles were up to 3 mm in paliar niches were formed. Twenty-day-old medusae length. were about 4.5 mm in height and about 6.2 mm in Two-day-old medusae were about 1.4 mm in height width. Two to three gastric filaments were observed. and about 1.8 mm in width (Table 2). Secondary The sensory roof almost surrounded rhopalium, and 123 44 Hydrobiologia (2017) 792:37–51

Fig. 8 Newly detached medusae of Tripedalia binata. A lateral view, B apical view, C oral view, D tentacle. Arrows indicate nematocyst wart. Scale bars 1mm

dome-shaped rhopaliar niche developed. The primary Fertilized eggs of T. binata developed into planulae and secondary pedalium developed knife-shaped in the gastral pockets of the female medusa. This habit which length of both pedalium was equal. Gonads had been reported in T. cystophora (Werner et al., were rod-shaped. 1971). In case of the tripedaliid C. sivickisi, blastulae developed into planulae in the gelatinous strands which were released from the female medusae Discussion (Hartwick, 1991a; Lewis & Long, 2005; Lewis et al., 2008; Toshino et al., 2014). In other cubozoans, From our laboratory rearing experiments, the early life internal fertilization occurred in C. marsupialis cycle of T. binata was clarified (Table 3; Fig. 9). Both (Studebaker, 1972), Alatina moseri (Mayer, 1906) male and female medusae possess stomach purses, collected from Puerto Rico (Arneson & Cutress, 1976; especially, spermatophores were found in the purses of as Carybdea alata), while external fertilization the male medusae (Straehler-Pohl et al., 2014). On the occurred in Chironex fleckeri Southcott, 1956 (Yam- night of collection, spermatophores were not observed aguchi & Hartwick, 1980), M. virulenta (Toshino in the stomach purse of both sexes, and gonads of et al., 2013) and A. moseri from Hawaii and Australia female and male medusae were still transparent. In (Carrette et al., 2014). Breeding behavior and embryo case of C. sivickisi, ovaries of the female medusae strand seem to be systems that protect blastulae and soon became cloudy after spermatophores transfer planulae against predators (Toshino et al., 2014). (Lewis & Long, 2005). The embryo release occurred Cubozoan planulae bear larval ocelli which have 24 h after the female captured the first spermatophore been described as a character common to all species (Lewis et al., 2008; Toshino et al., 2014). In our except M. virulenta (Widersten, 1968; Nordstro¨m laboratory, fertilized eggs were observed in female et al., 2003; Toshino et al., 2013). Ocelli are distributed medusae three days after collection. It is seems likely in the middle part of planulae in C. brevipedalia that copulation and spermatophores transfer was (Okada, 1927; as Carybdea rastonii), C. marsupialis conducted after the time of medusae collection. collected from Puerto Rico (Studebaker, 1972), A.

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Table 2 The process of young medusae development

BD bell diameter, BH bell height, G gonad, IF interradial furrow, R roof, SP secondary pedalium

Table 3 Chronology of developmental events in Tripedalia binata Stage/event Cumulative Length or height (mm) Diameter (mm) No. of interval tentacles (day) Mean Range SD Mean Range SD

Fertilization 0 – – – – – – – Planulae released (in female) 3 – – – – – – – Planulae released (out of female) 5 0.13 0.11–0.16 7.2 0.10 0.08–0.11 10.7 – Planulae settled 7 – – – 0.10 0.08–0.11 10.7 – Primary polyps 9 0.32 0.24–0.53 0.08 0.10 0.08–0.13 0.01 1–4 Bud formation 16 – – – 0.24 0.21–0.27 0.02 3–4 Adult polyps 58 0.52 0.38–0.64 0.08 0.36 0.33–0.41 0.03 8–9 Metamorphosis 68 0.52 0.38–0.64 0.08 0.36 0.33–0.41 0.03 8–9 Newly released medusae 75 1.2 1.0–1.5 0.13 1.3 1.2–1.4 0.06 1 9 4 20-day-old medusae 95 4.5 – – 6.2 – – 2 9 4 moseri collected from Puerto Rico (Arneson & virulenta (Toshino et al., 2013). Planulae of T. binata Cutress, 1976;asC. alata) and C. sivickisi (Hartwick, had about twenty dark reddish larval ocelli in the aboral 1991a; as Carybdea sivickisi, Toshino et al., 2014), region. This distribution pattern of larval ocelli is up to across the whole planula except the oral and aboral now unique within the Cnidaria. ends in Chironex yamaguchii (Lewis & Bentlage, Fully developed polyps of T. binata can be distin- 2009) (Toshino, unpublished data), and lacking in M. guished from other cubozoan polyps by their shape and 123 46 Hydrobiologia (2017) 792:37–51

Fig. 9 Composite image of the life cycle of T. binata, based on laboratory and ﬁeld surveys. Inter-stage durations are also shown.

size, and arrangement and number of the tip of tentacles recruitment to adequate solid substrates such as roots nematocyst (Table 4). Other cubozoan polyps are flask- of mangrove trees. shaped like C. marsupialis collected from Puerto Rico The polyps of T. binata formed cysts. Encystment (Studebaker, 1972; Straehler-Pohl & Jarms, 2011)and and excystment of polyps has been observed in T. A. moseri (Straehler-Pohl & Jarms, 2011 as Alatina cystophora (Werner, 1975; Straehler-Pohl, 2001; mordens; Carrette et al., 2014), amphora-shaped like 2009), C. fleckeri (Hartwick, 1991b), C. marsupialis Alatina morandinii (Straehler-Pohl & Jarms, 2011) collected from Puerto Rico (Straehler-Pohl, 2001; (Straehler-Pohl & Jarms, 2011 as Carybdea moran- 2009), A. moseri collected from Hawaii and Australia dinii), and tulip-shaped like M. virulenta (Toshino et al., (Straehler-Pohl, 2001, 2009 as A. mordens, Carrette 2013). Polyps of tripedaliids (T. binata, T. cystophora, et al., 2014), A. morandinii (Straehler-Pohl & Jarms, and C. sivickisi) had a pouch-shaped body and were 2011), and C. sivickisi (Toshino et al., 2014). Condi- smaller than other cubozoans. Tip of tentacles contain a tions causing encystment were drastic salinity changes single stenotele in A. morandinii, A. moseri, C. marsu- in T. cystophora and C. fleckeri, water temperature pialis,andC. fleckeri multiple in tripedaliids and M. changes above 27°C or below 15°CinA. morandinii virulenta but different arrangements showed which (Straehler-Pohl & Jarms, 2011) and below 20°CinC. were vertically in Tripedaliids (Fig. 5A, B), radially in sivickisi (Toshino et al., 2014) below 19°C or when M. virulenta (Toshino et al., 2013). salinity was increased to 42 or feeding ceased for a The polyps of T. binata had the ability to settle or period of four to five weeks in A. moseri (Carrette creep and displayed budding of creeping polyps. This et al., 2014). Excystment occurred after favorable flexibility appears well suited for the muddy condi- environmental conditions returned in A. morandinii, tions of their native habitat in mangrove swamps. raising temperature above 28°CinC. sivickisi, and Substrate for attachment of the polyps is scarce in feeding in A. moseri. Polyps of T. binata encysted at mangrove swamps. If adult T. binata medusae low temperature (20°C) and salinity 33, culturing spawned planulae over soft bottoms such as in our water quality deterioration or starvation, and excysted sampling site, polyps might get buried in the sediment. at a higher temperature (23°C), or improvement of Under these conditions, the creeping polyps of T. water condition and nutritional condition of cysts. The binata may play a key role in assuring successful sampling site was a semi-closed mangrove swamp

123 yrbooi 21)792:37–51 (2017) Hydrobiologia Table 4 Size (mm) and morphology of fully developed cubozoan polyps in previous and the present studies Total Mouth Number Hypostome Calyx length Stalk Nematocyst Bud type Metamorphosis References body disk of length proportion length types no. and type length diameter tentacles proportion shape proportion arrangenment Culturing condition

Tripedalia binata 0.38–0.64 0.33–0.41 7–8 0.09–0.24 0.22–0.43 0.03–0.06 HE, AFPR Creeping Complete Present study Mean: Mean: Mean: 7 Mean: 0.16 Mean: 0.32 Mean: 0.04 13 ? 2 polyp metamorphosis 0.52 0.36 31% 61% 8% Vertical Stable (23–30°C) Pouch-shaped Tripedalia cystophora 0.52–1.02 0.17–0.31 7–13 0.12–0.22 0.36–0.70 0.04–0.08 HE Creeping Complete Straehler- Mean: Mean: Mean: 9 Mean: 0.18 Mean: 0.58 Mean: 0.07 20–40 polyp metamorphosis Pohl and 0.83 026 2–3 Stable (23–28°C) Jarms 22% 70% 8% Vertical (2011) Pouch-shaped Copula sivickisi 0.28–0.97 0.15–0.20 2–7 0.09–0.14 0.22–0.79 0.02–0.05 AFPR Settled Complete Toshino et al. Mean: Mean: Mean: 4 Mean: 0.12 Mean: 0.35 Mean: 0.04 5–9 polyp 3 metamorphosis (2014) 0.50 0.17 35% 64% 1% Vertical Increase (from 23 Toshino et al. to 28°C) Pouch-shaped (unpublished Stable (23–30°C) data) Carybdea marsupialis from Puerto 1.35–2.78 0.40–0.91 19–26 0.20–0.44 0.72–1.50 0.37–0.81 ST Creeping Complete Straebler- Rico Mean: Mean: Mean: 24 Mean: 0.33 Mean: 112 Mean: 0.60 1 polyp metamorphosis Pohl and 2.08 0.62 4–6 Strobilation Jarms 16% 54% 29% Vertical (2005) Flask-shaped stable (23–25°C) Morbakka virulenta 2.25–4.66 0.49–1.08 8–17 0.27–0.62 1.05–1.89 0.60–2.71 TR, SSPR Swimming Strobilation Toshino et al. Mean: Mean: Mean: 13 Mean: 0.48 Mean: 1.49 Mean: 1.52 More than 30 polyp 2 Increase (from 15 (2013) 3.50 0.65 14% 44% 41% Radial to 20°C) Toshino et al. (2015) Tulip-shaped Alatina morandinii (identiﬁed as 0.83–1.80 0.34–0.74 9–18 0.16–0.36 0.43–0.86 0.22–0.54 ST Creeping Complete Straebler- Carybdea morandinii) Mean: Mean: Mean: 13 Mean: 0.26 Mean: 0.61 Mean: 0.39 1 polyp metamorphosis Pohl and 4–6 Jarms 1.28 0.51 21% 45% 31% Vertical Stable (23–25°C), day light (2011) Amphora- Straebler- shaped Pohl and Jarms (2015) Alatina moseri (identiﬁed as 1.43–1.63 0.41–0.46 11–19 0.19–0.22 0.87–0.94 0.36–0.41 ST Creeping Complete Straehler- Alatina mordens or Alatina cf. Mean: Mean: Mean: 16 Mean: 0.21 Mean: 0.91 Mean: 0.40 1 polyp metamorphosis Pohl and moseri, respectively) 4–6 Jarms 1.52 0.43 14% 60% 26% Vertical Stable (25–29°C) 123 (2011) Flask-shaped Carrette et al. (2014) 47 48 Hydrobiologia (2017) 792:37–51

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 regu- 1950 and Hamrick ( Yamaguchi References larly. Under these conditions, water temperature and salinity of the swamp change drastically. Cysts of T. C) ° binata seem to play an important role to survive in these drastic conditions. Metamorphosis in T. binata was complete and condition metamorphosis Culturing Complete Stable (27–28 type 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 polyp 4–8 Creeping Bud type Metamorphosis

trirhopaloids induced by stable temperature in T. cystophora, C.

TR morandinii, C. marsupialis, A. moseri (Straehler-Pohl & Jarms, 2011), and C. ﬂeckeri (Yamaguchi & Hartwick, 1980), increasing temperature in M. virulenta (Toshino

stenotele, et al., 2015) and C. sivickisi (Toshino et al., 2014)

ST (Table 4). Metamorphosis of T. binata was induced by Nematocyst types no. and arrangenment 1 Vertical 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., -rhopaloids, p Stalk length proportion 2013) when water temperatures change from approxi- mately 24–29°C (Australian Government, Bureau of Meteorology, 2015). Under these conditions, the polyps of T. binata may produce medusae. small spherical Calyx length proportion shape Newly detached medusae of T. binata can be distinguished from hitherto described medusae of

SSPR 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 Hypostome length proportion 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 Number of tentacles with four long pearl string-like tentacles and nemato-

heterotrichous euryteles, cyst warts lining the radial furrows (Fig. 8B), very

HE closely resembling the characters of T. cystophora and Mouth disk diameter C. sivickisi. Alatina morandinii by contrast shows ﬁliform tentacles; C. marsupialis shows only two tentacles; and A. moseri and M. virulenta shows four -rhopaliods, – 0.75 40–45 – – – – Total body length p 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 simi- continued

American football-shaped larities support the hypothesis that T. binata, T. cystophora, and C. sivickisi are closely related as part Chironex ﬂeckeri Table 4 The proportion is the ratioAFPR of the mean length of that structure to the mean total body length in the family Tripedaliidae that was established based 123 yrbooi 21)792:37–51 (2017) Hydrobiologia Table 5 Size (mm) and morphology of cubozoan medusae in previous and the present studies Umbrella Umbrella Manubrium No. of Nematocyst Nematocyst warts (exumbrella) Nematocyst References height diameter length tentacles types types proportion tentacle (tentacles) (exumbrella) shape

Tripedalia binata 1.1–1.5 1.2–1.4 0.23–0.41 4 AFPR Very small (0.01–0.05 mm), SHE Present study Mean: 1.2 Mean: 1.3 Mean: 0.32 Pearl OI round, alined along radial OHME furrows 30% string- like Tripedalia cystophora 1.0–1.4 1.3–1.6 0.50–0.30 4 OHME (two Medium large (0.06, 0.08 or 0.1 OHI Straehler-Pohl and Mean: 1.2 Mean: 1.5 Mean: 0.60 Pearl size mm), round, alined along radial Jarms (2011) classes) furrows 50% string- like Copula sivickisi 1.0–1.3 0.7–1.0 0.37–0.48 4 OHME Very small (0.03–0.06 mm) round, OI Toshino et al. 2014 Mean: 1. Mean: Mean: 0.43 Pearl OI alined two rows along radial OHME furrow and two con-spicuous 15 0.90 40% string- SE like nematocysts over sense organs Carybdea marsupialis from 1.0–1.4 0.9–1.2 0.30–0.40 2 OHME Large (0.14 9 0.06 mm), ovoid, OHI Straehler-Pohl and Puerto Rico Mean: 1.2 Mean: 1.2 Mean: 0.36 Pearl (two size ambilateral of the interradial SOAI Jarms (2005) furrow 30% string- classes) like Morbakka virulenta 2.3–2.8 1.7–2.7 0.41–0.91 4 ME Small (0.05–0.10 mm), OI Toshino et al. (2015) Mean: Mean: Mean: 0.77 Capitate OI innumerable circular shaped, ME scattered in a dense regular 2.46 2.17 30% pattern over the whole exumbrella Alatina morandinii (identified 0.6–0.9 0.4–0.6 0.18–0.27 4 OHME Very small (0.03–0.04 mm), RHI Straehler-Pohl and as Carybdea morandinii) Mean: 0.7 Mean: 0.5 Mean: 0.21 Filiform (single) circular shaped, irregularly OHME Jarms (2011) scattered over whole exumbrella 30% AI Straehler-Pohl and HI Toshino (2015) Alatina moseri (identified as 1.2–1.6 1.1–1.5 0.24–0.30 4 OHME Small (0.06 mm), innumerable, ME Straehler-Pohl and Alatina mordens or Alatina Mean: Mean: Mean: 0.27 Pearl circular shaped, scattered in a SHI Jarms (2011) cf moseri, respectively) dense, regular pattern over the 1.35 1.24 20% string- Carrette et al. (2014) like whole exumbrella The proportion is the ratio of the mean length of the manubrium to the mean umbrella height 123 AFPR American football-shaped p-rhopaliods, AI atrichous isorhizas; SOAI small ovoid atrichous isorhizas; OHI ovoid holotrichous isorhizas; RHI round holotrichous isorhizas; SHI spherical holotrichous isorhizas; SE sub-spherical euryteles; ME microbasic euryteles; OHME ovoid heterotrichous microbasic euryteles; OI ovoid isorhizas 49 50 Hydrobiologia (2017) 792:37–51 on adult medusa morphology and a molecular phylo- Hartwick, R. F., 1991a. Observations on the anatomy, beha- genetic analysis by Bentlage et al. (2010) and viour, reproduction and life cycle of the cubozoan Caryb- dea sivickisi. Hydrobiologia 216(217): 171–179. Straehler-Pohl et al. (2014). Research on the life cycle Hartwick, R. F., 1991b. Distributional ecology and behavior of elucidates the connection between benthic polyp and the early life stages of the box jellyfish Chironex fleckeri. planktonic medusa. Research on the cubozoan life Hydrobiologia 216(217): 181–188. cycles is important to further understand the evolu- Moore, S. J., 1988. A new species of cubomedusan (Cubozoa: Cnidaria) from northern Australia. The Beagle, Records of tionary history and ecology of Cubozoa. the Northern Territory Museum of Arts and Sciences 5(1): 1–4. Acknowledgements We would like to express our sincere Lewis, C. & T. A. F. Long, 2005. Courtship and reproduction in thanks to staffs of the Institute of Marine Science, Burapha Carybdea sivickisi (Cnidaria: Cubozoa). Marine Biology University (Supattra Taleb and Siriwan Choosri) for providing 147: 477–483. specimens and helping in the field work in Thailand. The Lewis, C., S. Kubota, A. E. Migotto & A. G. 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