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.

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Partial sequences of 12S and 16S rRNA genes were collected from four thoracican barnacle genera, Capitulum, Chthamalus, Megabalanus, and Tetraclita, to investigate their phylogenetic relationships. Both neighbor joining and maximum parsimony analyses and combined data showed monophyly of the four genera with high bootstrap values, congruent with classification of barnacles based on morphology. The three genera, Chthamalus, Megabalanus and Tetraclita, formed a monophylic group, and the pedunculate genus, Capitulum, formed a paraphylic group with them. The phylogenetic relationships among the three sessile barnacles based on the 12S rRNA gene differed from those based on the 16S rRNA gene or a combination of the 12S and 16S rRNA genes. Based on the 12S rRNA gene the two sessile barnacle genera, Megabalanus and Chthamalus, formed one group, whereas Tetraclita formed another group. However, based on the 16S rRNA gene and combined data of the 12S and 16S rRNA genes, Megabalanus and Tetraclita were the closest relatives among the three sessile barnacles. This was congruent with the results based on larval characters and the nuclear 18S rRNA gene reported previously, but differed from morphological classification. Thus, the 16S rRNA gene appears to be more reliable than the 12S rRNA gene in elucidating the phylogenetic relationships of these four genera within the thoracican barnacles.

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