Fig. 4.

Reducing conditions do not preserve Heliocidaris larvae. (A and B) H. erythrogramma. (A) Normal 2-day larva focused to show the internal pentameral adult rudiment (arrow); large lipid droplets that serve as “yolk” are also visible (19). (Metamorphosis would occur at day 4.) (B) Larva from a culture killed at 2 days by treatment with 1% ammonium for 20 min, then placed in seawater containing 100 mM β-ME, photographed after 2 days. Larvae become fragile, lose structural integrity, and disintegrate. In the specimen shown, adult rudiment structure is lost (arrow) and pigmented epithelium (arrowheads) is detaching. (C–J) H. tuberculata. (C) Normal 48-h pluteus; skeletal rods in arms and body are visible within the transparent pigmented epithelium. (D) Normal 36-h pluteus, starting stage for experiments in E–J. Photographed with Nomarski optics to display internal skeletal structure; hindgut is visible in body of larva. (E–I) Embryos from the same culture as D, killed by treatment with 1% ammonium for 20 min, then placed in seawater containing 100 mM (E) or 10 mM β-ME (F and G) or returned to normal seawater (H and I), photographed after 1 day. (E) In strong reducing conditions, structural integrity and morphology are rapidly lost; skeletal elements rapidly disappear. (In sample shown, no skeletal elements were detected under polarizing optics.) (F) In less stringent reducing conditions, structural integrity and pigmentation disappear more slowly. Pluteus morphology and skeletal organization are lost, but skeletal elements (arrow) are stable. (G) Same field as F under polarizing optics to display skeletal elements. (H) In normal seawater, cytoplasm is subject to both internal and external decay processes, including predation by protists, and rapidly disappears. Skeletal organization is lost, but skeletal elements are preserved for prolonged periods. (I) Same field as H under polarizing optics. (J) Normal 3-day pluteus from untreated controls under polarizing optics. (Scale bar: 200 μm.).