Sessile Organisms 21 (1): 1-6 (2004) The Sessile Organisms Society of Japan

Combination of macroalgae-conditioned water and periphytic 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 Strongylocentrotus droebachiensis portant sea urchin 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 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 fully developed urchin rudiments (Kitamura Larvae of A, crassispina responded differently to et al., 1993). In brief, swimming pyramid-shaped lar- the 15 macroalgae-conditioned waters when combined vae obtained 24hr after fertilization were transferred to with N. ramosissima (Fig. 1). Larval metamorphosis 30-l tanks filled with 25l of filtered seawater (final lar- was induced by waters conditioned by red and brown val density was 0.8 larva/ml) and fed with a diet of algae (at a dilution equivalent to 2.8g/100ml ), while the planktonic diatom Chaetoceros gracilis. Culture green algal species did not induce larval metamorphosis tanks were continuously aerated (200ml/min) in a except for the low metamorphosis observed in Ulva dark room. Seawater was partially renewed every day, pertusa-conditioned water. The brown alga H. by removing half of the culture water and replacing it fusiformis induced 53% metamorphosis, while the other with the same amount of filtered seawater. The culture three species of brown algae induced 63-96% metamor- period for A. crassispina to the 8-armed larval stage phosis. On the other hand, C. pilulifera and C. was 2 weeks at 22-24℃, while that for P. depressus officinalis induced more than 90% metamorphosis, was 3 weeks at 17-19℃. Cultures of A. crassispina while the other two coralline red algae induced 52-76% were maintained from May to August, and those of P. metamorphosis. Except for G. amansii, which induced depressus, from October to March. Larval assays were 72% metamorphosis, non-coralline red algae induced conducted during these periods. Significant differences among the metamorphosis- inducing activity of the conditioned water combined with the diatom, the conditioned water only, the diatom in FSW, and FSW only were analyzed with 1-way ANOVA followed by Tukey's test (P <0.05). Signifi- cant differences among the metamorphosis-inducing ac- tivity of waters conditioned by 15 macroalgae were also analyzed with the same method. Differences between the activity of C. pilulifera-conditioned water combined with the diatom and the activity of fresh C. pilulifera alga was analyzed with Student's t-test (P<0.05) (Zar, 1999). Fig. 1 Percent metamorphosis of A. crassispina larvae in re- sponse to water conditioned by macroalgae (2.8g/100 ml) combined with the periphytic diatom N. Results ramosissima. Error bars denote standard deviations of Throughout the study, neither conditioned water six replicates. 4 Sessile Organisms, vol. 21, no. 1, 2004

lower metamorphosis (23-36%) then did coralline red combined with N. ramosissima showed no metamor- algae (Tukey's test, P<0.05). phosis within the first 2hr, but 42% metamorphosed Metamorphosis of A. crassispina larvae was ob- after 6hr and the proportion increased to nearly 100% served at different time intervals in response to C. after 24hr. pilulifera-conditioned water (2.8 g/100ml) combined Seasonal variation in the activities of C. pilulifera- with N ramosissima and to the fresh alga (0.5g/ and U. pertusa-conditioned waters combined with N. beaker) (Fig. 2). In the case of the fresh alga, 16% lar- ramosissima, with regard to inducing larval metamor- val metamorphosis was observed within the first 2hr, phosis in A. crassispina, was evident (Fig. 3). During increasing to 82% after 6hr, and reaching 99% within more than two years of investigation, U. pertusa exhib- 12hr from the commencement of the assay. By con- ited no activity except in spring or early summer, while trast, larvae exposed to C. pilulifera-conditioned water C. pilulifera usually induced metamorphosis ranging from 50 to 90% during the months between April and August.

Discussion Combinations of waters conditioned by red or brown algae and the periphytic diatom Navicula ramosissima exhibited metamorphosis-inducing activi- ties in larvae of Anthocidaris crassispina (Fig. 1), but none of the algae induced larval metamorphosis in Pseudocentrotus depressus. The reason for the differ- ent responses of larvae of the two species of sea urchin Fig. 2 Percent metamorphosis of A. crassispina larvae at dif- may be related to the environment of their natural habi- ferent time intervals, when exposed to C. pilulifera- conditioned water combined with N. ramosissima (col- tat. That is, juveniles of A. crassispina usually inhabit umn, 2.8g/100ml) and to fresh alga (○, 0.5g/ coralline flats (Imai, 1980a; Imai, 1980b; Tsuji et al., beaker). Error bars denote standard deviations of six rep- 1989), while those of P. depressus tend to inhabit licates. filmed stone fields (Imai and Arai, 1994). The present finding that larvae of A. crassispina respond to macroalgae while those of P. depressus do not, is con- sistent with their behavior in their natural environment. On the other hand, our findings on P, depressus were not consistent with the report of Ito et al. (1991): diatom-based film alone also supported larval metamor-

phosis of P, depressus, they found, but this was not the case in our investigation. A reason for the difference in our results may be the different manner of preparation of the diatom-based film. Ito et al. (1991) used multi- species (Navicula sp. and Amphora sp. being dominant) diatom-based films cultured for 30 days, while our films were uni-algal (N ramosissima) and cultured in the laboratory for only 5 days. Our finding that N. ramosissima film alone did not induce larval metamor- phosis in either of the two sea urchin species was also Fig. 3 Seasonal variation in the activity of water conditioned not consistent with the finding of Ito (1984). However, by C. pilulifera and U. pertusa (2.8g/100ml) combined Ito (1984) used multi-species diatom-based films cul- with N. ramosissima in inducing larval metamorphosis of tured from diatoms grown in mass production tanks for A. crassispina. Error bars denote standard deviations of sea urchin juveniles, while we used uni-algal diatom- six to nine replicates. Combination of macroalgae-conditioned water and periphytic diatom 5

based films cultured from uni-algal stocks maintained Hadfield, M.G. and V.J. Paul (2001). Natural chemical cues in our laboratory. for settlement and metamorphosis of marine-invertebrate larvae. In: Marine Chemical Ecology, ed. J.B. The present investigation also showed that larvae McClintock and B.J. Baker, CRC Press, Boca Raton, pp. of A, crassispina responded more readily to Corallina 431-461. pilulifera than to its conditioned water (Fig. 2). It is Imai, T. (1980a). On the sea urchins off Miura city. The study possible that the active substance in the water condi- of distribution, environment, growth and gonads in tioned by C. pilulifera may have transformed to a less Jogashima. Bull. Kanagawa Pref. Fish. Exp. Stn., 1, 35- 79 (in Japanese). active form, although there is no evidence to confirm Imai, T. (1980b). On the sea urchins off Miura city. The study this. This problem requires further investigation. of distribution, environment, growth and gonads from The green alga Ulva pertusa induced larval meta- Hatsuse to Kamimiyata. Bull. Kanagawa Pref. Fish. Exp. morphosis in A. crassispina only in spring or early sum- Stn., 2, 27-36 (in Japanese). mer and showed no metamorphosis-inducing activity Imai, T. and S. Arai (1994). Local peculiarities as living for the red sea urchin Pseudocentrotus depressus of during the other seasons (Fig. 3). At present, we have bishamon waters off southern Miura Peninsula, no evidence as to whether U pertusa produced the Kanagawa Prefecture, Japan. Suisanzoshoku (The same active substance released by the red and brown Aquaculture), 42, 307-313 (in Japanese). algae, or whether these algae produced different active Ito, S., J. Kobayakawa, Y. Tani and N. Nakamura (1991). The substances. 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