Galaxea, Journal of Coral Reef Studies 12: 77-81（2010）

Note

Aboral localization of responsiveness to a metamorphic neuropeptide in the planula larva of Acropora tenuis

Kanae MATSUSHIMA, Masato KIYOMOTO, and Masayuki HATTA*

Graduate School of Humanities and Sciences, and Tateyama Marine Laboratory, Marine and Coastal Research Center, Ochanomizu University, Tokyo 112-8610, Japan

* Corresponding author: M. Hatta E-mail: [email protected]

Communicated by Michio Hidaka (Editorial Board Member)

Abstract Planula larvae of the coral genus Acropora ini­ marine substrates on which they settle (Morse et al. 1996). tiate metamorphosis in response to microorganisms on the Microorganisms on the substrates trigger a sequence of undersea substrate. Since planulae move forward in the metamorphosis and settlement, and calcareous algae direction of the aboral side, it is supposed that initial (Morse et al. 1996) and a bacterium (Negri et al. 2001) metamorphic signaling localizes on this side of planula. have been identified as environmental inducers for meta­ We dissected planulae of Acropora tenuis into fragments morphosis of acroporids’ larvae. Following reception of perpendicularly to the oral-aboral axis, and tested their external cues, internal signal cascades are supposed to response to a metamorphosis-inducing neuropeptide. Abo- perform metamorphic reactions, including cell differenti­ ral 1/3 fragments metamorphosed in response to the neu­ ation, in larvae. ropeptide with high efficiency while most oral 2/3 frag­ In Cnidaria, the marine hydrozoa Hydractinia echinata ments did not. The results suggest that the tissue in the has long been investigated focusing on metamorphosis aboral 1/3 region receives the metamorphic neuropeptide mechanisms. Planula larvae of H. echinata settle on shells and releases downstream signals to complete metamor­ of hermit crabs, and a bacterium Pseudoalteromonas (Al­ phosis of the oral side in A. tenuis larvae. In addition, oral teromonas) espejiana has been identified as an envi­ron­ fragments gained a metamorphic ability in several days, mental inducer for settlement and metamorphosis (Leitz revealing regeneration of the aboral tissue. and Wagner 1993). It is thought that sensation of the bacterium by sensory neurons is followed by secretion of Keywords Acropora, GLWamide, metamorphosis, neuro­ neuropeptides of the GLWamide family to initiate meta­ peptide morphosis (Schmich et al. 1998). The GLWamide neuro­ peptide was first isolated from an anthozoan sea anemone to induce metamorphosis of H. echinata (Leitz et al. 1994), and was later found in H. echinata itself (Gajewski Introduction et al. 1996), a freshwater hydra (Takahashi et al. 1997, Leviev et al. 1997), and another sea anemone species Planula larvae of the reef-building coral genus Acropora (Leviev and Grimmelikhuijzen 1995). Thus GL­Wamide have a strict preference to microenvironments of sub­ neuropeptides are common in Cnidaria. In corals, one of 78 Matsushima et al.: Aboral metamorphosis by peptide in Acropora the hydra GLWamide members, Hym248, induces meta­ Planula dissection and peptide assay morphosis of acroporids’ larvae with 100% efficiency Planula larvae, approximately 1 mm long, were dissected (Iwao et al. 2002, Hatta and Iwao 2003, Erwin and Szmant in a plastic culture dish filled with autoclaved 0.22 µm- 2010). The hydra peptide is thought to have similar struc­ filtered seawater using a thin glass needle, which was tures to the coral endogenous counterpart and to mimic its made by stretching the tip of a glass bar soon after melting action. In H. echinata, some cations and drugs, such as it using a burner. The peptide, Hym248 (EPLPIGLWa,

＋ ＋ Cs , NH4 , diacylglycerol, and tumor-promoting phorbol Takahashi et al. 1997), was purchased from OPERON ester, have also been known to be involved in metamor­ Biotechnologies, Japan. In each well of 24-well multiplates phosis signaling pathways (for review, Frank et al. 2001). for cell culture, 3-5 larval fragments were put with 0.5 ml These ions and drugs are thought to act on the PI-PKC of autoclaved 0.22 µm-filtered seawater, and the peptide pathway resulting in the secretion of GLWamide neuro­ was added at a concentration of 1×10−6 M or 2×10−6 M peptides, the endogenous metamorphosis-inducing hor­ following Iwao et al. (2002). The number of metamor­ mone. phosed specimens was counted 24 hrs after peptide ad­ Planulae move forward in the direction of the aboral ministration. Metamorphosis was defined as the flattened (anterior) side by beating cilia, and the aboral side of shape or septation of the body tissue (Hatta and Iwao planula corresponds to the basal part by which the animal 2003). attaches to the substrate during and after metamorphosis. These features give rise to an idea that the initial signaling pathway for metamorphosis is localized in the aboral side Results and discussion of planula, since it is reasonable to sense and respond to environmental cues by anterior tissues. Indeed, in H. First, we tested the metamorphic response of dissected echinata, the aboral half metamorphosed in response to halves of A. tenuis planulae. The dissected fragments moved Cs＋, but the oral (posterior) half did not, when planulae around as intact larvae, and mortality was not led by were dissected into halves (Seipp et al. 2007). In this dissection itself. The results revealed a clear difference in study, we investigated localization of responsiveness to a response to the metamorphic neuropeptide, Hym248; the GLWamide in planula larvae of Acropora tenuis. aboral half planulae metamorphosed with high effi­ciency (14/16 individuals by 1×10−6 M of Hym248 on 5.5 days post fertilization (dpf), 11/12 individuals by 2×10−6 M on Materials and methods 11 dpf) whereas the oral half did not respond to the peptide at all (0/17 individuals by 1×10−6 M on 5.5 dpf, 0/10 in­ Gamete collection and larval rearing dividuals by 2×10−6 M on 11 dpf). Thus, res­ponsiveness Coral larvae were raised from gametes collected in the to the peptide was restricted to the aboral half. To narrow field during the annual mass-spawning event off Maja down the corresponding part, planulae were dissected into beach, Aka Island, Okinawa, Japan (2611N, 12717E) 1/3 and 2/3 fragments and treated with Hym248 (Fig. 1). on June 8, 2009. Gamete-collecting devices were set above The aboral 1/3 fragment still responded with high effi­ each individual colony of A. tenuis (described in detail in ciency as the intact planula (Iwao et al. 2002). Unlike the Hatta et al. 2004). Bundles of gametes taken from 6 results of the oral 1/3 fragment and oral half (0% meta­ colonies were immediately brought to Akajima Marine morphosis), a small fraction (18.1%) of the oral 2/3 frag­ Science Laboratory, and mixed to allow fertilization as ments revealed metamorphic responses. The dissecting described in Iwao et al. (2002). Larvae were daily position might, on occasion, shift to the aboral side a bit transferred into fresh 0.22 µm-filtered seawater, and were more than expected, and some oral 2/3 fragments would kept at about 26℃. have contained aboral tissues responsible for the peptide sensitivity. The high percentage metamorphosis of the aboral 1/3 fragments indicates that metamorphosis of Matsushima et al.: Aboral metamorphosis by peptide in Acropora 79

thin, perforated structures. Tight attachment of the meta­ morphosed aboral fragments to the bottom and wall of culture wells represents formation of the basal tissues of the primary polyp. In contrast, metamorphosed oral 2/3 fragments appeared a thick, well-septated body without attaching to the culture wells (Fig. 2D), suggesting the lack of basal tissues required for attachment. These results reveal that tissue identity along the oral-aboral axis in the planula is directly reflected in the tissue organization of the primary polyp, in other words, the transition of tissue identity, or transdifferentiation, rarely happens during Fig. 1 Metamorphosis percentage of dissected fragments metamorphosis processes, as in H. echinata (Spindler and of planulae. Planula larvae (10.5-13 days post fertilization) Müller 1972). were dissected into 1/3 and 2/3 fragments as the schematic Next, we investigated regeneration of dissected planula drawings, and treated with Hym248 at 2×10−6 M after 1-3 hours of healing. The number of tested specimens is shown fragments, since oral fragments of H. echinata planulae in the brackets regenerate to recover metamorphic competence 3 days after dissection (Schwoerer-Böhning et al. 1990). Figure 3 represents the percentage of metamorphosis of dissected fragments, which were induced to metamorphose by these fragments was not affected by the presumed dif­ Hym248 after 0-6 days of recuperation post dissection. ferences in cutting position. These results suggest that the The dissected fragments continued swimming even after peptide-receiving tissues are spreading over the aboral 1/3 6 days, and no changes were observed until the peptide region rather than localized at the aboral tip. The dis­ treatment. Aboral 2/3 fragments maintained high percent­ tribution of neurons producing the metamorphic GL­ ages of metamorphosis throughout the experiments. How­ Wamide peptide would be a future subject of interest. ever, the metamorphosis percentage decreased in aboral Actually, GLWamide-positive neurons are restricted to 1/3 fragments after 5 and 6 days of recuperation prior to the aboral side of planula in H. echinata (Schmich et al. peptide administration. Since the aboral side of planula is 1998). Note that the intact planula realizes metamorphic thicker than the oral side in Acropora as the schematic responses over the entire body from the aboral tip to the drawings in Fig. 1, the aboral 1/3 fragment might need to oral tip. Then, metamorphic responses on the oral side, recruit relatively a large proportion of tissue for wound which is insensitive to the peptide, should require signals healing of a wide area of cut surface. A counteraction from the aboral side to undergo metamorphic responses might occur in regeneration from small-sized tissues following exposure to the peptide. This is the first study accompanying reorganization of tissue identity (morphal­ suggesting the existence of signals downstream of the laxis) resulting in recruitment of excessive aboral tissues GLWamide peptide in cnidarian metamorphosis signaling and disappearance of responsiveness to the peptide. On cascades. the other hand, oral fragments began to acquire the ability Representative morphologies of metamorphosed frag­ of metamorphosis after 3 or 4 days of recuperation, and ments are shown in Fig. 2. Metamorphosed individuals of the percentage of metamorphosis increased to more than the aboral 2/3 fragments revealed a similar morphology to 80% gradually as the recuperation days became longer. In those of intact planulae, but the body was flatter and oral addition, those metamorphosed specimens from oral (central) structures were missing (Fig. 2B). Aboral 1/3 fragments frequently attached to the culture wells (data fragments transformed into thin disks, and pores often not shown). These results suggest epimorphic regeneration appeared in the tissues (Fig. 2C). Morphogenesis from an of the aboral tissues, which are sensitive to the peptide insufficient amount of tissues might have resulted in the and correspond to the basal attachment tissues after meta­ 80 Matsushima et al.: Aboral metamorphosis by peptide in Acropora

Fig. 2 Representative morphologies of metamorphosed fragments. Metamorphosed specimen derived from A control intact planulae; B aboral 2/3 fragment; C aboral 1/3 fragment; D oral 2/3 fragment, 1 day after peptide administration. A-C top view, D diagonal side view. Oral 2/3 fragments resulted in floating specimens due to the lack of basal tissues for attachment. Scale bar＝0.5 mm

morphosis, in the oral fragments during a few days. In conclusion, the planula larva of the coral A. tenuis have special tissues localized strictly in the aboral 1/3 part, which respond to the metamorphic neuropeptide and release downstream signals for metamorphic reactions of oral tissues. The palnula larva also has the ability of regeneration of aboral tissues within several days.

Acknowledgments

We thank Akajima Marine Science Laboratory, and Fig. 3 Percentage of metamorphosis of dissected planula Drs. T. Hayashibara and G. Suzuki at Seikai National fragments after represented days of recuperation following Fisheries Research Institute, for sampling support. This dissection. About 40 larvae were dissected everyday for a work was supported by the Sasakawa Scientific Research week, and the fragments were kept until the day of peptide Grant from the Japan Science Society to KM, and by the treatment. All fragments were treated with Hym248 at 2×10−6 M on the same day (13 days post fertilization) to Grant-in-Aid for Scientific Research (C) from MEXT adjust the larval age at the peptide treatment. n＝36 for each Japan to MH. point, in total 1008 specimens Matsushima et al.: Aboral metamorphosis by peptide in Acropora 81

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