Larvae of marine invertebrates undergo metamorphosis in response to environmental cues (Chia & Burke, 1978). In sea urchins, free fatty acids (Kitamura et al., 1993), dibromomethane (Taniguchi et al., 1994), pheromonal peptides (Burke, 1984) and L-glutamine (Yazaki & Harashima, 1994; Yazaki, 1995) have been known as metamorphosis-inducing substances. The mechanisms by which cells respond to these cues and how the larval tissues are absorbed have not been clear, however. In the present study, we used L-glutamine (Gln) and a natural cue, green algae (Ulvella sp.), to induce metamorphosis of Hemicentrotus pulcherrimus and Anthocidaris crassispina, and investigated the intracellular changes during metamorphosis.

After being subjected to 10−5–10−3 M Gln for 10–24 h, larvae cease swimming, settle, begin to retract their larval arms, extrude the primary podia and finally evert their echinus rudiment (ER). In H. pulcherrimus, larvae retracted their arms from 6 h to 24 h after the start of Gln treatment and then everted the ER. A. crassispina larvae underwent similar processes to those of H. pulcherrimus. The larval surface is composed of squamous epithelium and columnar epithelium. The epithelium of the ciliary bands or epaulets is columnar.

In the squamous epithelium, the nuclear chromatin in the larval arms and body, and in the oesophagus, markedly condensed after treatment with Gln for 24 h. Electron microscopy revealed swelling of both nuclei and mitochondria, while their membranes seemed to be intact. In the cytoplasm, lipid-like structures and electron-dense substances appeared. A further 24 h after Gln treatment, the chromatin condensation had progressed. Most nuclei in which chromatin had condensed were positive to the TUNEL assay, which detects DNA fragmentation. These results suggest that cell death in the squamous epithelium is apoptotic rather than necrotic.