Budding, bud morphogenesis, and regeneration in Carybdea marsupialis Linnaeus, 1758 (Cnidaria: Cubozoa)

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1 Hydrobiologia 530/531: , D.G. Fautin, J.A. Westfall, P. Cartwright, M. Daly & C.R. Wyttenbach (eds), Coelenterate Biology 2003: Trends in Research on Cnidaria and Ctenophora. Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands. 331 Budding, bud morphogenesis, and regeneration in Carybdea marsupialis Linnaeus, 1758 (Cnidaria: Cubozoa) Ayako B. Fischer 1 & Dietrich K. Hofmann 1,2, * 1 Department of Developmental Biology Unit, Ruhr-University Bochum, Bochum, Germany 2 Present address: Department of Zoology and Neurobiology, Ruhr-University Bochum, Bochum, Germany (*Author for correspondence: Tel.: , Fax: , Dietrich.K.Hofmann@ruhr-uni-bochum.de) Key words: Cnidaria, Cubozoa, Carybdea marsupialis, budding, regeneration Abstract The cubopolyp of the box jelly Carybdea marsupialis (L.) reproduces asexually by lateral budding, and by almost total transformation of the polyp into a cubomedusa. Buds, one or more at a time, emerge laterally from the body column and separate from the parent as secondary polyps bearing hypostome and some tentacle anlagen. We describe at the light microscopic level the consecutive steps of bud formation and propose to discriminate a series of eight developmental stages. Once detached, these buds elongate and pass through a vagile phase, creeping over the substratum with the tentacular region ahead. The vagile time period appears to depend on the culture condition; it ends with the firm attachment of the young polyp which then develops the full complement of tentacles along with further growth. The budding rate is positively correlated with the frequency of feeding, in contrast to conditions leading to medusa formation, and is enhanced in the dark. The creeping polyp stage reveals a high regeneration potential: both oral and aboral fragments, obtained by midbody transection, regenerate the ablated portion within 72 h. Regeneration in fragments excised from the body column restores also polyp morphology according to the original polar organization. Results of pilot experiments involving excision of the tentacle and hypostomial portion of adult budding and non-budding cubopolyps lead us to discuss a possible interplay between regeneration of oral structures and bud formation. Introduction Asexual reproduction of the polyp stage by budding off motile propagules which eventually settle and develop into secondary polyps is a common feature in several cnidarian taxa. Many species within the Limnohydrina (e.g. Gonionemus spp., Craspedacusta sowerbyi) produce vermiform, nonciliated, creeping frustules which develop polyp features only after attachment to the substratum (Tardent, 1978; Werner, 1984, for review). A particular type of larva-like, ciliated propagule emerging from the lower part of the polyp s calyx is restricted to species of the scyphozoan order Rhizostomeae (Hofmann & Crow, 2002, for review). Life-cycle studies in species of the cubozoan genera Tripedalia, Carybdea and Chironex (e.g. Werner et al., 1971; Cutress & Studebaker, 1973; Yamaguchi & Hartwick, 1980) have briefly shown that cubopolyps, apart from total or almost total transformation into cubomedusae, reproduce asexually by lateral budding of propagules which, after detachment from the parent, develop into secondary polyps. These propagules share the creeping mode of locomotion with the frustules of the Limnohydrina, but with reversed polarity. They develop, however, like buds in Hydra spp., already a hypostome and some tentacles at the

2 332 time of detachment, and are able to feed. They were termed the creeping polyp stage. The present account aims at providing a more detailed description of the budding process in Carybdea marsupialis Linnaeus 1758 at the light-microscopical level, thereby proposing to discriminate eight successive stages. It further provides, for the first time, data on budding rates under different feeding and light regimes, complementing the staging and experimental induction of medusa formation in this species published on by Stangl et al. (2002). The study mentions briefly the further development of the bud, and describes results of a set of transection and regeneration experiments performed on both adult, budding, and non-budding polyps, and on the creeping polyp stage. Material and methods Stock cultures of Carybdea marsupialis polyps (kindly provided by R. Golz) were maintained in glass and styrene dishes filled with 200 (or 500 ml respectively) of pasteurized natural sea water from the North Sea, and propagated asexually in the laboratory at 23 C. The cubopolyps were fed Artemia salina nauplii twice a week. Containers were cleaned and the sea water replaced about 4 h after feeding. The light regime was adjusted to long-day conditions (16 h light: 8 h dark). For some experiments (see below) cultures were protected from light by dark covers, and/or fed more frequently. Transection experiments on adult polyps or on detached buds were performed using sharpened tungsten needles on individuals collected from the cultures, washed three times in antibiotic-containing seawater (ABS, as detailed below), and immobilized in Ca 2+ -free artificial, antibiotic-containing seawater for min. Fragments were placed individually in wells of 24- well tissue culture plates each in 1 ml of ABS made up with 100 mg penicillin, 100 mg streptomycin, and 130 mg neomycin per liter pasteurized sea water. Development was scored under a dissecting microscope daily, sometimes at shorter intervals, for 2 7 days. Microphotographs were taken using either an Axiophot (Zeiss) microscope, an Olympus IMT2 inverted microscope, or an Olympus OM2n camera mounted on an Olympus dissecting microscope, on Kodak professional color slide film. A non-parametric v 2 test was applied to analyse data from the dissection experiments. Results Bud formation Buds, sometimes more than one at a time, emerge from the upper part of the polyp s column (Fig. 1). We propose to classify bud formation according to the sequence of eight developmental stages depicted in Figure 2: Stage 1: Onset of budding recognizable as a slight protrusion of the endo- and ectoderm. Stage 2: Protrusion more pronounced, angle between bud and body column >130. Stage 3: Bud assumes cuboidal shape, angle between bud and body column <130. Stage 4: Angle between bud and body column decreases to 90 or less; specialized future hypostomal cells become visible at the slightly pointed distal end of the bud. Stage 5: The terminal area of future hypostome cells becomes larger; axial extension of bud. Stage 6: Length of bud exceeds it s basal diameter; hypostomal cells start forming oral opening; usually five tentacle primordia Figure 1. Carybdea marsupialis. Cubopolyp with bud ready to detach (stage 8). Scale bar, 100 lm (Photograph courtesy of Barbara Simmes).

3 333 Figure 2. Carybdea marsupialis. Eight consecutive stages of bud formation, details described in the text. All scale bars, 100 lm. appear; first autonomous contractions of the bud. Stage 7: Bud diameter increased in the midgastric portion, a constriction appears at the basis; hypostome prominent with functional oral opening; tentacle primordia elongated. Stage 8: Bud length increased, diameter decreased; bud-parent connection reduced to a narrow strand; a weak, necklike constriction separates gastric column from the ring of tentacle primordia encircling the bulbous hypostome. Bud ready to detach. Under our laboratory conditions the entire budforming process may take 3 5 days. The fully differentiated buds separate actively by strong

4 334 contractions and by turns around the main body axis. Final bud length was found to vary between 500 and 1500 lm; larger parent polyps tend to produce larger buds. The budding rate is positively correlated with the feeding frequency: Whereas a group of 30 polyps, fed once a week, produced a total of 25 buds within 12 days (i.e. an average of 2.1 buds per day), a second group of 30 polyps, fed 5 a week, developed a total of 156 buds (i.e. an average of 13 buds per day). The budding rate is negatively correlated with illumination. The following observations were made on polyps already adapted to the different conditions: When exposed to the artificial light in the laboratory for 16 h, 60 polyps, fed once a week, produced 36 buds within 12 day (i.e. an average of 3 buds per day). The other group of 60 polyps, also fed once a week, was protected from light and propagated 63 buds (i.e. an average of 5.2 buds per day). Further development of the buds Once detached from the parent polyps, buds start creeping around, the oral end with the tentacles ahead ( creeping polyp stage, Fig. 3a and b). Turns appear to be performed through stronger contractions and extensions. The vagile phase may last for some hours or up to several days, after which the individuals settle, contract, and firmly attach through adhesive material secreted by cells at the aboral pole (pedal disc). Then the young polyps become erect, resume growth, and increase the number of tentacles. We have followed up the development of one particular bud from separation through attachment until reaching the erect stage within a time period of 48 h (photomicrographs not shown). Active feeding usually starts with separation of the creeping polyp, but stage 7 Figure 3. Carybdea marsupialis. The creeping polyp stage of a bud recently detached from the parent, photographed in two phases of movement. Scale bars, 100 lm. and 8 buds were occasionally observed to catch and ingest live prey. Though not analyzed statistically, observations of C. marsupialis in culture indicated that the time period until attachment of the creeping polyps tends to be considerably longer and to take several days in large, clean dishes provided with pasteurized seawater. However crowded conditions, very small containers, and dense biofilms on the bottom of the culture dishes promote settlement within hours. The asexual offspring settles often near parent polyps, thus creating small colonies (i.e. clones) of secondary polyps. Furthermore, creeping polyps seem to settle faster in the light than in the dark. This circumstantial evidence should prompt detailed investigations into presettlement behavior and into induction of irreversible attachment and polyp formation. Regeneration When adult, non-budding polyps were deprived of the oral part by sectioning below the tentacle crown, all fragments (n ¼ 30) regenerated the ablated oral portion. Different results were obtained following transection of adult, budding polyps during early phases of bud formation (n ¼ 50 in each experiment): Decapitation Budding stage 1 2 Budding stage 2 3 Oral part regenerated, bud fully developed and separated 32% 78% Oral part regenerated, bud developed but not separated 42% 22% (Y-shaped, double-headed polyp) Oral part failed to regenerate, bud developed but not separated 26% 0%

5 335 The newly released creeping polyps exibited remarkable regeneration capacities: Within a group of 30 individuals bisected in the midgastric region, all 30 oral fragments regenerated the ablated portion either attached to the substratum or not. Of the 30 aboral fragments, two degenerated, but 28 regenerated an oral portion conforming with the original polarity. Within a group of 30 creeping polyps sectioned into three fragments of similar size (oral, midgastric, and aboral), all of the oral, tentacle-bearing fragments regenerated the ablated aboral portion. All of the 30 gastric fragments regenerated both the oral, tentacular region and the aboral part. However only 17 posterior isolates showed regeneration of the oral structures; the other 13 fragments degenerated. Discussion Previous studies performed on Carybdea marsupialis as well as on other cubozoans have focussed mainly on life-cycle features, on morphology, histology, ecology, taxonomy, and evolutionary relationships (e.g. Werner, 1984, for review). Special attention was paid to nematocytes because some of the medusae called box jellies range among the most harmful cnidarians (Burnett, 1992; Golz, 1993; Avian et al., 1997; Simmes, 2002; Stangl et al., 2002). Brief remarks on budding in species of the cubozoan genera Tripedalia, Carybdea and Chironex have been included in the papers by Werner et al. (1971), Cutress & Studebaker (1973), and Yamaguchi & Hartwick (1980). It appears that the budding mode is very similar: That of Carybdea alata (Arneson & Cutress, 1976) matches well with features described in this paper for C. marsupialis. The present account on bud morphogenesis and regeneration offers an approach to foster C. marsupialis as an experimental system for developmental studies. Easy to propagate in large numbers in the laboratory, most, potentially all, life-cycle stages are accessible. The proposed staging of the budding process, now added to the nine stages of medusa formation described in C. marsupialis by Stangl et al. (2002), may facilitate both future structural and developmental studies, and work applying molecular genetic techniques. The solitary cubopolyps form buds in an apparently similar way and with the same polar organization as seen in species of the fresh-water hydrozoan Hydra, a traditional model organism for developmental studies (Galliot & Schmid, 2002, for review), thus offering the possibility for detailed comparative studies. Whereas Hydra spp. buds are released as fully differentiated young polyps readily attaching to the substratum, a stage named the creeping polyp is intercalated in C. marsupialis and other cubozoans. The type of locomotion is very similar to that observed in the non-ciliated frustules produced by budding of polyps in the taxon Limnohydrina. This creeping mode has been studied in Gonionemus murbachi in detail by Werner (1950). Subject of controversies for a long time, Werner (1950 for G. murbachi) and Reisinger (1957 for Craspedacusta sowerbii) demonstrated that frustules move with the future aboral pole of the polyp ahead, in contrast to the creeping polyps of the cubozoans which creep with the hypostome and tentacle anlagen in front. Creepers of Tripedalia cystophora thereby stretch out, like an antenna, one extremely long tentacle as the leading structure (Werner, 1984, Fig. 74B). The impact of feeding on budding and on transformation into medusae respectively is remarkably different: whereas the budding rate increased with more frequent feeding (which was not unexpected), Stangl et al. (2002) observed that polyp metamorphosis into medusae is induced by food deprivation and can be prevented by daily feeding under otherwise permissive culture conditions. Once detached from the parent polyps, it appears that some culture parameters, e.g. crowding, dense biofilms, speed up settlement of the creeping polyps, and final morphogenesis of the cubopolyp. Hartwick (1991) noted that creepers of Chironex fleckeri attached to various hard substrata with a bias towards vertical surfaces, indentations, and corners, suggesting a certain rugophila. However studies under strict axenic in vitro conditions to assess the possible role of surface texture, and of external biogenic cues for settlement, possibly emanating from biofilms on surfaces are not available yet, neither for C. marsupialis nor for any other cubozoan species (see Mu ller & Leitz, 2002 for a state of the art review on control of metamorphosis in cnidarians).

6 336 The ability of creeping polyp fragments to regenerate both the ablated oral and aboral portion matches well with the high bipolar regeneration capacities of frustules of Vallentinia gabriellae (Limnohydrina) reported by Auberson & Tardent (1980). It is, however, in obvious contrast to the strictly unidirectional restitution or regeneration of oral structures in fragments excised from larvalike propagules and polyps of the scyphozoan Cassiopea spp. (Curtis & Cowden, 1971; Neumann, 1977). Many experimental studies have been addressed to head regeneration (i.e. hypostome and ring of tentacles) induced concomitant with budding in Hydra spp. Mu ller (1996) points out that these processes are mutually exclusive and subject to a postulated secondary head forming/ inhibiting system (see also Galliot & Schmid, 2002, for review). Our observations on cubopolyps at early budding stages, which had been deprived of the head region, point to the possibility that concurrent regeneration of the oral portion and bud formation (including separation) in Carybdea marsupialis are governed by a mutual control system as well. The pilot results presented here warrant extended comparative studies on both the experimental and theoretical level. Acknowledgments We thank M. Rudschewski for skilled technical assistance, S. Sturma for the expert digital processing, and two anonymous reviewers for valuable comments. DKH extends particular thanks to K. P. Hoffmann for providing generous working facilities in his department. References Arneson, A. C. & C. E. Cutress, Life history of Carybdea alata Reynaud., 1830 (Cubomedusae). In Mackie, G. O. (ed.), Coelenterate Ecology and Behavior. Plenum, New York: Auberson, B. & P. Tardent, The regeneration of polyps and frustules of Vallentinia gabriellae Mendes, 1948 (Limnomedusae). In Tardent, P. & R. Tardent (eds), Developmental and Cellular Biology of Coelenterates. Elsevier/ North Holland Biomedical Press, Amsterdam: Avian, M., N. Budri & L. Rottini Sandrini, The nematocysts of Carybdea marsupialis Linnaeus, 1758 (Cubozoa). In Proceedings of the Sixth International Conference on Coelenterate Biology. Nationaal Naturhistorisch Museum, Leiden: Burnett, J. W., Human injuries following jellyfish stings. Maryland Medical Journal 41: Curtis, S. K. & R. R. Cowden, Normal and experimentally modified development of buds in Cassiopea. Acta Embryologiae Experimentalis 3: Cutress, C. E. & J. P. Studebaker, Development of the cubomedusae, Carbydea marsupialis. In Proceedings of the Association of the Islands Marine Laboratories of the Caribbean 9: 25. Galliot, B. & V. Schmid, Cnidarians as model systems for understanding evolution and regeneration. International Journal of Developmental Biology 46: Golz, R., Anchorage and retraction of nematocytes in the tentacles of the cubopolyp Carybdea marsupialis are mediated by a species-specific mesogloeal support. Cell & Tissue Research 274: Hartwick, R. F., Distributional ecology and behavior of the early life stages of the box-jellyfish Chironex fleckeri. Hydrobiologia 216/217: Hofmann, D. K. & G. Crow, Induction of larval metamorphosis in the tropical scyphozoon Mastigias papua: striking similarity with upside down-jellyfish Cassiopea spp. (with notes on related species). Vie et Milieu 52: Mu ller, W. A., Pattern formation in the immortal Hydra. Trends in Genetics 12: Mu ller, W. A. & T. Leitz, Metamorphosis in the Cnidaria. Canadian Journal of Zoology 80: Neumann, R., Polyp morphogenesis in a scyphozoan: evidence for a head inhibitor from the presumptive foot end in vegetative buds of Cassiopea andromeda. Wilhelm Roux Archives 183: Reisinger, E., Zur Entwicklungsgeschichte und Entwicklungsmechanik von Craspedacusta (Hydrozoa, Limnotrachylina). Zeitschrift fu r Morphologie und Ökologie der Tiere 45: Simmes, B., Entwicklungs- und zellbiologische Untersuchungen zur Nematogenese bei Carybdea marsupialis (Cubozoa, Cnidaria) und Mastigias papua (Scyphozoa, Cnidaria). Diploma Thesis, Ruhr-University Bochum, Germany. Stangl, K., L. V. Salvini-Plawen & T. W. Holstein, Staging and induction of medusa metamorphosis in Carybdea marsupialis (Cnidaria, Cubozoa). Vie et Milieu 52: Tardent, P., Coelenterata, Cnidaria. In Seidel, F. (ed.), Morphogenese der Tiere. Lieferung 1: A-I. Fischer Verlag, Jena/Stuttgart: Werner, B., Weitere Beobachtungen u ber das Auftreten der Meduse Gonionemus murbachi Mayer im Sylter Wattenmeer und ihre Entwicklungsgeschichte. Verhandlungen der Deutschen Zoologischen Gesellschaft, Tu bingen: Werner, B., Stamm Cnidaria. In Kaestner, A. (ed.), Lehrbuch der Speziellen Zoologie, 4th edn. by H. E. Gruner. Band 1, Teil 2. VEB Gustav Fischer Verlag, Jena:

7 337 Werner, B., C. E. Cutress & J. P. Studebaker, Life cycle of Tripedalia cystophora Conant (Cubomedusae). Nature 232: Yamaguchi, M. & R. Hartwick, Early life history of the sea wasp Chironex fleckeri (Class Cubozoa). In Tardent, P. & R. Tardent (eds), Developmental and Cellular Biology of Coelenterates. Elsevier/North Holland Biomedical Press, Amsterdam: