Strong fluorescence emission occurred in sea urchin larvae when irradiated with blue light under a fluorescence microscope. The blue light irradiation first broke red granules in the pigment cells, releasing green fluorescent substance(s) into the cytoplasm of the pigment cells. The released and dispersed fluorescent substance(s) then made the entire pigment cell emit strong green fluorescence. With prolonged blue light irradiation, the pigment cell itself bursted, dispersing the fluorescent substance(s) into the body cavity or seawater. This resulted in “explosive emission” of the fluorescence. Cells that can emit such fluorescence first appeared at the late-blastula stage, proliferated with development, and changed into the red-pigmented cells. Similar fluorescence emission was observed in the larvae of Clypeaster japonicus, Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus, of which C. japonicus larvae displayed the strongest fluorescence.
Organisms that can emit fluorescence were reported in various species from bacteria to fish (Haneda, 1985). In Echinodermata, strong fluorescence emission was reported with sea star, sea lily, and sea cucumber. However, no such examples have been reported in the class Echinoidea. The egg and embryo of many kinds of sea urchin are known to display auto-fluorescence, although it is usually weak. We were surprised to observe an unusually strong, explosive fluorescence emission in the pluteus larvae of a sea urchin, Clypeaster japonicus, upon irradiation with blue light under a fluorescence microscope. Here we described this novel discovery.
MATERIALS AND METHODS
Clypeaster japonicus, Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus were collected from the local coast of Toyama Bay and kept in running seawater aquaria at 13–25°C. Shedding of gametes was induced by injecting 0.5 M KCl into the coelomic cavity. Sperm were kept ‘dry’ in the refrigerator until use; eggs were shed into filtered seawater, and washed three times by sedimentation. Fluorescence emission was observed by an Olympus BH2 microscope equipped with epifluorescence optics. Micrographs were taken on Fujicolor Super HG 1600 film or Kodak TriX pan film. The emission was measured using a PM-10 microscope spectrometer (Hamamatsu Photonics K.K., Hamamatsu, Japan).
RESULTS AND DISCUSSION
When we observed some pluteus larvae of the sea urchin, C. japonicus, under a fluorescence microscope, we found an unusually strong, explosive fluorescence emission (Fig. 1). Under the B excitation mode (450–490 nm), the emission had a maximum at about 540 nm (Fig. 2). About 1-2 minutes after the onset of excitation in our system, sporadic “explosive emission” of strong green fluorescence occurred at various parts on the body surface and the tips of the postoral arms (Figs. 1B,C). The fluorescence emission in one explosion lasted for a few seconds, repeating several hundred times over a period of >30 min. The emission was observed at the highest frequency at the tips of the postoral arms. Similar but somewhat weaker fluorescence emission was observed in the larvae of Hemicentrotus pulcherrimus, Anthocidaris crass-ispina and Pseudocentrotus depressus. In adult C. japonicus also, fluorescence emission occurred in the spines and the epidermis (data not shown).
The fluorescence was apparently emitted by red granules in the pigment cells. The change in the emission pattern suggests that blue light irradiation first disrupted the red granules, releasing and dispersing green fluorescent substance(s) into the cytoplasm of the pigment cell (Fig.3B). The dispersed fluorescent substance(s) caused the entire pigment cell to emit strong green fluorescence (Figs. 1B, 3B). Prolonged blue light irradiation, then broke the pigment cell itself, causing the fluorescent substance(s) to disperse into the body cavity or sea-water (Figs. 1C-1I). This results in “explosive emission” of the fluorescence. In C. japonicus, pigment cells were often broken down simultaneously with the breakdown of the red granules they contained, and then they rapidly emitted strong fluorescence. Each glow sphere of fluorescence seen in Figs. 1E-1I may possibly correspond to the breakdown of a single cell.
Developmentally, immature fluorescent pigment cells first appeared in the vegetal basement of late blastula or early gastrula in C. japonicus. In an early stage, they apparently have no pigment granules and emitted only weak fluorescence (Fig. 4A′). These immature pigment cells increased the number (Fig. 4) and display amoeboid movements in the blastocoel and blastoderm, wherein they become mature pigment cells with well-developed pseudopodia. Those cells emitted bright fluorescence (Figs. 4B′, C′). These observations suggest that the pigment cells identified by their fluorescence are similar to those studied previously using a specific monoclonal antibody (Gibson and Burke, 1985).
The function of the pigment cells has not been established, although several studies have suggested that they may function in photoreception, myogenesis, or some nervous activity (Reyberg, 1979; Reyberg and Lundgren, 1979; Gras and Weber, 1977). In this study, we observed mature pigment cells have well-developed pseudopodia, and distributed on the tips of the postoral arms at high density. We therefore speculated that the pigment cells are involved in some nervous activity or morphogenesis.
To our knowledge, such a strong fluorescence as we found has not been reported before in sea urchin. The chemical and photochemical nature of the fluorescent pigment, as well as the mechanism by which blue light breaks open the pigment granules and pigment cells remains to be studied.
We would like to thank Dr. Ritsu Kamiya (Tokyo University) for his helpful comments and critical reading of this paper.
S. Nakamura et al.