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The first scleractinians, progenitors of modern corals, began to appear 240 million years ago; by the late Jurassic (150 Ma) most families of modern corals had evolved and begun forming reefs (1, 2). Mechanisms controlling the recruitment of new corals to sustain these structures are, however, poorly understood (3). Corals, like many marine invertebrates, begin life as soft-bodied larvae that are dispersed in the plankton (3, 4). As the first step in developing a calcified coral colony, the larva must settle out of the plankton onto a suitable substratum and metamorphose to the single calcified polyp stage cemented to the reef (3, 5). Our analyses of the metamorphic requirements of larvae in divergent coral families surprised us by revealing the existence of a common chemosensory mechanism that is required to bring larvae out of the plankton and onto the reef. This mechanism appears to be quite old, predating both the phylogenetic divergence of these coral families and the development of different modes of coral reproduction.

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The leaf coral Agaricia humilis occurs mainly on the undersides of surfaces in shallow water, a distribution different from the vast majority of corals at our study site in Bonaire, Netherlands Antilles. A series of hypotheses were tested for specific mechanisms that could cause the observed distributions of Agaricia humilis. We found that a suite of larval swimming and settling behaviors, in large part, drives the adult distribution of the species. These behaviors include: (1) swimming behavior that causes larvae to position themselves in shallow water, (2) orientation behavior during settlement that causes larvae to preferentially settle on the undersides of surfaces, and (3) settlement behavior where chemosensory recognition of morphogenic molecules associated with the cell walls of specific crustose red algae is required for induction of settlement and metamorphosis. The consequences of atypical larval behavior are severe and include decreased survivorship, growth, and ability to reproduce sexually.

Larvae of the common Caribbean scleractinian coral, Agaricia humilis, are induced to settle and metamorphose by contact with specific crustose (nongeniculate) coralline red algae. This requirement for an exogenous trigger of settlement and metamorphosis has been shown to control the distribution of recruits of this coral in the natural environment. Results reported here demonstrate that the stringency and specificity of this larval requirement persist for at least 30 days following the planktonic release of the brooded larvae, thus enhancing both the capacity for dispersal of the larvae and the substratum specificity of their metamorphosis and recruitment. The inducer of metamorphosis is shown to be associated with an insoluble macromolecular carbohydrate. This molecule is found with the partially purified cell walls obtained from a morphogenetic crustose red alga, Hydrolithon boergesenii, or its associated microflora. Because two non-inductive crustose red algal species also lack the cell wall-associated inducer, the substratum specificity of metamorphosis is probably the result of larval recognition of this molecule. In procedures that should prove widely applicable to other systems, purified and highly specific enzymes were used to cleave the inductive cell wall-associated polysaccharides and to solubilize the active morphogen. Enzymes were also used as probes with which to identify essential structural features required for the morphogenetic activity. These enzymatic and related biochemical studies show that the morphogen is associated with, and may itself contain, a sulfated glycosaminoglycan that includes multiple N-acetylglucosamine and galactose residues. The larval receptors that recognize this complex carbohydrate cue may thus be related to lectins. The solubilized morphogen induces normal settlement, attachment, and the metamorphosis of A. humilis and A. tenuifolia larvae on clean polystyrene surfaces, and the larvae seem to have no other requirement. This effect is apparently specific for larvae of species induced to settle by the intact alga; larvae of the sympatric coral Tubastraea aurea are not induced by this chemical, or by the intact algal surface. A wide variety of other natural and synthetic sulfated polysaccharides and related polymers have little or no inductive effect on the A. humilis larvae, suggesting that the larval receptors involved in substratum recognition are highly specific. A similar high specificity of lectin- and sulfated polysaccharide-mediated recognition, and the resulting control of differentiation, has been observed in a wide variety of biological systems.

Addition of hydrogen peroxide to seawater causes synchronous spawning in gravid male and female abalones, and certain other mollusks as well. This effect is blocked by exposure of the animals to aspirin, an inhibitor of the enzyme catalyzing oxidative synthesis of prostaglandin endoperoxide. Hydrogen peroxide activates this enzymatic reaction in cell-free extracts prepared from abalone eggs (a very rich source of the prostaglandin endoperoxide synthetase); this effect appears to reveal a fundamental property of prostaglandin endoperoxide synthesis. Applicability of these findings to both mariculture and medical purposes is suggested.

Larvae of the scleractinian coral Agaricia humilis settle and metamorphose in response to chemosensory recognition of a morphogen on the surfaces of Hydrolithon boergesenii and certain other crustose coralline red algae. The requirement of the larva for this inducer apparently helps to determine the spatial pattern of recruitment in the natural environment. Previous research showed that the inducer is associated with the insoluble cell wall fraction of the recruiting algae or their microbial epibionts, and that a soluble but unstable fragment of the inducing molecule can be liberated by limited hydrolysis, either with alkali or with enzymes specific for cell wall polysaccharides. We now show that the parent morphogen can be solubilized by gentle decalcification of the algal cell walls with the chelators EGTA or EDTA, suggesting that the morphogen may be a component of the calcified recruiting alga itself, rather than a product of any noncalcified microbial epibionts. The solubilized inducer is subsequently purified by hydrophobic-interaction and DEAE chromatography. The purified, amphipathic morphogen retains activity when tightly bound to beads of a hydrophobic-interaction chromatography resin, and this activity (tested with laboratory-reared larvae) is identical in the ocean and the laboratory. We have attached the purified, resin-bound inducer to surfaces coated with a silicone adhesive and thus produced a potent artificial recruiting substratum--i.e., a morphogen-based chemical "flypaper" for A. humilis larvae. This material should prove useful in resolving the role of chemosensory recognition of morphogens in the control of substratum-specific settlement, metamorphosis, and recruitment and in the maintenance of species isolation mechanisms in the natural environment.

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