Characterization of the active substances in water conditioned by the coralline red alga Corallina pilulifera as inducers of metamorphosis in larvae of the sea urchin Anthocidaris crassispina

Water conditioned by the coralline red alga Corallina pilulifera (C. pilulifera-conditioned water) in combination with the periphytic diatom Navicula ramosissima was found to induce metamorphosis in larvae of the sea urchin Anthocidaris crassispina. Bioassay-guided characterization of the active substances in C. pilulifera-conditioned water revealed that relatively heat-stable, non-volatile, polar substances are inducers of larval metamorphosis in A. crassispina. The molecular weights were estimated to be about 100 or less. Furthermore, fractionation by ODS column chromatography (0 to 100%, four gradients of MeOH) revealed that the activity was eluted mainly in the 0% and 70% fraction, although the activity was much lower than that of the control (C. pilulifera-conditioned water). When all four fractions (F1, F2, F3, and F4) were combined, the metamorphosis-inducing activity was recovered to almost the same level as that of the control. These results indicate that more than two metamorphosis-inducing substances may be present in C. pilulifera-conditioned water. Several authentic reagents such as phloroglucin and bromophenol did not show the metamorphosis-inducing activity even when combined with the diatom N. ramosissima.