Sessile Organisms 21 (1): 1-6 (2004) The Sessile Organisms Society of Japan Combination of macroalgae-conditioned water and periphytic diatom Navicula ramosissima as an inducer of larval metamorphosis in the sea urchins Anthocidaris crassispina and Pseudocentrotus depressus Jing-Yu Li1)*, Siti Akmar Khadijah Ab Rahimi1), Cyril Glenn Satuito 1)and Hitoshi Kitamura2)* 1) Graduate School of Science and Technology, Nagasaki University, 1-14 Bunkyo, Nagasaki 852-8521, Japan 2) Faculty of Fisheries, Nagasaki University, 1-14 Bunkyo, Nagasaki 852-8521, Japan *correspondingauthor (JYL) e-mail:[email protected] (Received June 10, 2003; Accepted August 7, 2003) Abstract The induction of larval metamorphosis in the sea urchins Anthocidaris crassispina and Pseudocentrotus depressus was investigated in the laboratory, using waters conditioned by 15 different macroalgae com- bined with the periphytic diatom Navicula ramosissima. Larvae of P. depressus did not metamorphose, but larvae of A. crassispina showed a high incidence of metamorphosis, especially in waters conditioned by coralline red algae or brown algae. High inductive activity for larval metamorphosis was detected in Corallina pilulifera-conditioned water during a 2.5-year investigation, but the activity was relatively low in February or March and in September, the off growth seasons of the alga. By contrast, Ulva pertusa-con- ditioned water did not show metamorphosis-inducing activity except in spring or early summer. These re- sults indicate that during their growth phase, red and brown algae release into their environment active sub- stances that are involved in the larval metamorphosis of A, crassispina. Key words: Macroalgae-conditioned water, Navicula ramosissima, metamorphosis, Anthocidaris crassispina, Pseudocentrotus depressus Introduction franciscanus, Loxechinus albus, Lytechinus pictus, and Macroalgae and microbial films are known to play Arbacia punctulata has been reported to be induced by important roles as inducers of metamorphosis in larvae microbial films (Cameron and Hinegardner, 1974; of sea urchins (Pawlik, 1992; Wieczorek and Todd, Cameron and Schroeter, 1980; Gonzalez et al., 1987). 1998; Hadfield and Paul, 2001). Laboratory-reared lar- During mass production of the economically im- vae of the sea urchin Strongylocentrotus droebachiensis portant sea urchin species Pseudocentrotus depressus, metamorphose upon contact with coralline red algae, Anthocidaris crassispina, and Hemicentrotus brown algae, and green algae (Pearce and Scheibling, pulcherrimus, diatom-based film grown on plastic 1990, 1991). Field observations have shown that newly plates, consisting of Navicula, Achnanthes, Amphora, settled sea urchins (S. purpuratus and S. franciscanus) and Nitzschia species, is used as an inducer of larval can form dense populations on rocky areas covered metamorphosis (Tani and Ito, 1979; Ito,1984; Ito et al., with crustose coralline red algae (Rowley, 1989). 1991). Mass-produced sea urchin larvae are also in- Metamorphosis of larvae of S purpuratus, S. duced to metamorphose by the brown algae Hizikia 2 Sessile Organisms, vol. 21, no. 1, 2004 fusiformis and Sargassum thunbergii (Ito, 1984). Sargassum horneri), and three green algae These studies led us to infer that macroalgae and (Monostroma nitidum, Ulva pertusa, and Codiumfrag- microbial films possess chemical cues that induce meta- ile) were employed in the study. morphosis in sea urchin larvae. The aqueous cues from These algae were collected from a rocky shore in the red alga Delisea pulchra appear to be a complex of Nomo Bay, Nagasaki, Japan, between March, 1996 and sugar floridoside and isethionic acid (Williamson et al., April, 2001. In addition, C. pilulifera and U. pertusa 2000). The lipophilic cues from the red alga Corallina were collected every month from February, 1993 to pilulifera and the green alga Ulvella lens have been July, 1995. They were brought to the laboratory, identified as eicosapentaenoic acid (Kitamura et al., cleaned by removing epiphytes and epifauna attached to 1993) and glycoglycerolipids (Takahashi et al., 2002), their surfaces, and then placed in flasks filled with fil- respectively. Dibromomethane is another supposed cue tered seawater (FSW, Whatman glass fiber filter, detected from several coralline red algae (Taniguchi et GF/C). The quantity of alga immersed was 5g per 100 al., 1994). However, chemical cues associated with mi- ml FSW (Ito et al., 1991). Flasks were incubated at 22- crobial films remain unclear. 24℃ for 24hr in a dark room. After the 24-hr incuba- Ito et al. (1991) reported a unique method of induc- tion, the alga was removed and the conditioned water ing metamorphosis in mass-produced sea urchin larvae, was filtered through GF/C and stored in a freezer at - using water conditioned by the brown alga Hizikia 30℃ until it was used in assays. In this study, we chose fusiformis to enhance metamorphosis in P. depressus to filter conditioned water through GF/C for practical larvae exposed to diatom-based film. He reported that reasons: this is the procedure that is used during mass diatom-based film grown in a 15t tank usually yielded production of juvenile sea urchins. only 30-40% metamorphosis in P. depressus larvae, but A unialgal culture of the periphytic diatom that this proportion almost doubled (to 60-80% meta- Navicula ramosisstma (apical×transapical length: 15 morphosis) when water conditioned by the brown alga ×8μm) was used for the experiments instead of the H. fusiformis was added to the tanks, even though con- mixed-species diatom-based film usually used in mass ditioned water alone did not induce metamorphosis. production. The diatom was cultured in 200-ml Pyrex The nature of this synergistic effect of macroalgae- beakers containing 100ml of modified Erd-Schreiber conditioned water and periphytic diatoms on larval medium (Kitamura and Hirayama, 1984) at 23℃ under metamorphosis, as well as any seasonal variation in the a 14L: 10D light cycle for 5 days. The medium was re- synergistic action, remains unknown and requires fur- placed with 50% diluted medium on the second day, ther investigation. and with filtered seawater on the fourth day. This was In this study, we investigated the synergistic effects done in order to minimize any possible effect of the cul- of waters conditioned by 15 species of macroalgae in ture medium during assays, although it was not con- combination with the periphytic diatom Navicula firmed whether larval assay results were any different ramosissima on the metamorphosis of laboratory- when the culture medium was diluted. The density of cultured larvae of Pseudocentrotus depressus and the diatoms in culture was 0.8-1.0×106 cells/cm2 on the Anthocidaris crassispina. In addition, we examined the fifth day. seasonal variation in metamorphosis-inducing activity The metamorphosis-inducing activity of the waters of the macroalga-conditioned waters. conditioned by the 15 macroalgae combined with the diatom N. ramosissima was assayed for larvae of A. Materials and Methods crassispina and P, depressus as follows. Beakers Fifteen species of macroalgae including eight red filmed with N ramosissima were filled with 100ml of algae (four coralline: Corallina pilulifera, Corallina the water conditioned by an alga as described above. officinalis, Amphiroa zonata, and Marginisporum Subsequently, 30 8-armed competent larvae of A. crassissimum; four non-coralline: Gelidium amansii, crassispina or P. depressus were placed into each Lomentaria catenata, Laurencia undulata, and beaker. These beakers were kept in a dark room at Gloiopeltis furcata), four brown algae (Padina 22℃ for A. crassispina and at 18℃ for P. depressus. arborescens, H. fusiformis, Sargassum thunbergii, and After 24hr, the percentage of individuals that had Combination of macroalgae-conditioned water and periphytic diatom 3 metamorphosed to the juvenile form was checked under without the periphytic diatom Navicula ramosissima, a stereoscopic microscope. Metamorphosis-inducing nor the diatom without conditioned water, induced lar- activities of the conditioned waters diluted logarithmi- val metamorphosis in the sea urchins Anthocidaris cally to 32% and 56% (equivalent to 1.6 and 2.8g alga crassispina and Pseudocentrotus depressus. Initially, /100ml FSW) were also assayed. The larval assay for metamorphosis-inducing activities of the waters condi- each sample was replicated six to nine times, using lar- tioned by 15 macroalgae combined with N. vae from more than two different cultures. ramosissima were tested at three concentration levels Assays were also conducted to evaluate the meta- 1.6, 2.8, and 5.0g/100ml, and 2.8g/100ml was morphosis-inducing activities of the conditioned water found to have the best metamorphosis-inducing activity only, the diatom in FSW, and FSW only. In the assay for all tested algae and was used generally in the pre- using water conditioned by C. pilulifera, metamorpho- sent results. In P. depressus, none of the conditioned sis was also checked after 1, 2, 3, 6, and 12hr. Simul- waters prepared from the 15 macroalgae induced larval taneously, the activity of the fresh alga (0.5g/beaker) metamorphosis when combined with the diatom N. with no diatom film was also observed. ramosissima. Hence, the combined effect of the brown Larvae of A. crassispina and P. depressus used in alga Hizikia fusiformis on P. depressus reported by Ito all assays were cultured in the laboratory to the 8-armed et al. (1991) was not confirmed in the present study. stage with