<I>Phestilla Sibogae</I>

BULLETIN OF MARINE SCIENCE, 46(2): 455-464, 1990 CHEMICAL FACTORS SYMPOSIUM NATURE OF THE METAMORPHIC SIGNAL AND ITS INTERNAL TRANSDUCTION IN LARVAE OF THE NUDIBRANCH PHESTILLA SIBOGAE Michael G. Hadfield and J. T. Pennington ABSTRACT The veliger larvae of the coral-eating nudibranch mollusc Phestilla sibogae have provided an excellent model for the study of chemical-induction of metamorphosis. They metamorphose only in response to a water-soluble metabolite that escapes from the coral prey of the adult nudibranchs. Metamorphosis, occurring 18-20 h after larvae are exposed to coral, is decisive: larvae attach to a substratum, shed their velar-swimming organs, shell and operculum, and undergo major morphological reorganization. Extraction and HPLC purification of the coral product show it to be a small (< 500 MW), polar, water-soluble molecule that is probably effective in inducing metamorphosis at concentrations of \0-10 M or less. The rapidness and cascade nature of metamorphic induction, coupled with the partial or complete inductive action of potassium ions, choline and epinephrine, point to the larval nervous system in the detection of the coral product and the internal mediation of metamorphosis. Problems associated with the isolation and concentration of the coral inducer hamper investigations of the larval receptor and its mode of action. While largely aware that many-probably most-benthic marine invertebrates settle and metamorphose in response to general environmental signals, including illumination, physical texture and chemical cues (Crisp, 1974), many students of invertebrate development have found it useful to study metamorphic induction in organisms with highly specific triggering cues. Among the more highly specific settlers are: (1) sessile organisms that need members of their own species nearby with which to mate and that often settle gregariously (e.g., barnacles); and (2) animals with very specific prey, usually only one or a few closely related species, that are usually nonmotile and patchy in the environment (Hadfield, 1986). Study of such stenophagous animals provides an opportunity to look at the developmental consequences of specific-species interactions. The species interactions are often translated into specific chemical interactions at settlement. Many of the species most studied in recent years are highly specific settlers, including abalones (Morse, 1990), gregarious polychaetes (Pawlik, 1990), and the Pacific sand dollar Dendraster excentricus (Highsmith, 1982; Burke, 1984). Opis- thobranch molluscs have long been recognized as an important component of this category, principally because oftheir very narrow prey specificities (reviewed by Hadfield and Switzer-Dunlap, 1984). Sea hares restricted to a small taxonomic group of algae and nudibranchs that feed on a single prey species are common. Among the latter is the wide-spread tropical nudibranch Phestilla sibogae, a pred- ator on hermatypic corals of the genus Porites (Harris, 1975). More than 20 years of investigation of Phestilla sibogae have shown it to be an excellent model for the study of chemically specific induction of a complex metamorphosis which includes ecological, anatomical and physiological trans- formations. Phestilla sibogae is a good model for the study of metamorphosis because: (1) its life-history is relatively short, and it is easily reared; (2) it has a generation time of about 30 days, including a 6-day embryonic period, a 3-day pre-competent larval period, and a 2-3 week juvenile stage; and (3) it is intensely prey specific, a specificity that is similarly imposed on larval settlement and 455 456 BULLETIN OF MARINE SCIENCE, VOL. 46, NO.2, 1990 Figure I. A metamorphically competent veliger larva of Phestil/a sibogae. Scanning electron micrograph: F, foot; S, shell; Y, velum (bar = 50 J.lm). metamorphosis. It has also proved useful that extracts of the prey provide a very specific chemical inducer of metamorphosis. Phestilla sibogae has been used for examination of (1) metamorphic morpho- genesis (Bonar and Hadfield, 1974; Bonar, 1976; 1978; Hadfield, 1978); (2) the chemical nature of the coral-produced inducer (Hadfield, 1977; 1978; 1984; Had- field and Scheuer, 1985); (3) the developmental significance of metamorphic com- petence (Hadfield, 1978; Hirata and Hadfield, 1986; Miller and Hadfield, 1986); (4) the importance of external food sources during extended larval development (Kempf and Hadfield, 1985); and (5) the biological nature of the induction process and internal activation of metamorphosis (Hadfield, 1984; Hirata and Hadfield, 1986; Yool et aI., 1986). In this paper we briefly review prior work and then provide a summary progress report on several current lines of research on metamorphic induction in P. sibogae, particularly the nature of the inducer, the mech- anism of induction, and the internal control system. BRIEF REVIEW OF METAMORPHOSIS IN PHESTILLA SIBOGAE Larvae hatch from gelatinous egg ribbons 5 to 7 days after the eggs are laid. At hatching the larvae are not capable of metamorphosis (i.e., they are not competent), a developmental state they achieve 2-4 days later. They do not have to feed to become metamorphically competent. A competent larva of P. sibogae is shown in Figure 1. Competent larvae can survive up to 3 weeks if unfed, and up to 6 weeks if supplied with phytoplankton (Kempf and Hadfield, 1985). HADFIELD AND PENNINGTON: METAMORPHIC INDUCTION IN A NUDIBRANCH 457 Figure 2. A newly metamorphosed juvenile of Phestilla sibogae observed in dorsal view. Scanning electron micrograph:A, anterior end; R, rhinophore (bar = 50 /Lm). Competent larvae of P. sibogae settle and metamorphose if they come into contact with: (1) corals of the genus Porites; (2) water in which coral has been standing for a few hours, even if filtered; or (3) aqueous extracts of coral. Early and late metamorphic stages are illustrated in Figures 2 and 3 (see also Hadfield, 1978). Metamorphosis occurs within 18-20 h of exposure to coral. It involves attach- ment to the substratum, velar loss, evacuation from the shell and operculum, and re-organization of the body (Figs. 2, 3). The swimming larva was an herbivore, feeding on phytoplankton; the metamorphosed juvenile is a carnivore, devouring coral flesh. CHEMICAL NATURE OF THE METAMORPHIC INDUCER Our investigations into the chemical nature of the coral product that induces metamorphosis in larvae of P. sibogae have extended over a long time and have been time consuming and expensive. Progress has been slow, mainly due to problems associated with isolating and concentrating a water-soluble marine nat- ural product. Through largely trial-and-error efforts we have developed a meth- odology that, at times, gives a relatively good yield and purification (Table 1). The procedure is as follows: Porites compressa, a common Hawaiian "finger coral," is broken into small sections and soaked 18-20 h in tris-buffered (pH 8.3), 0.56 molar sodium chloride. This extraction step is carried out in large shallow bowls into which are placed the coral, extraction medium and a pipet delivering a stream of air bubbles. The large surface area and bubbling are provided in an effort to reduce coral decay 458 BULLETIN OF MARINE SCIENCE, VOL. 46, NO.2, 1990 Figure 3. Juvenile of Phestilla sibogae about 24 hours after metamorphosis; right lateral view. Scan- ning electron micrograph:A, anterior end; R, rhinophore (bar = 50 ~m). and mucus production. The extraction ratio is approximately 1.3 kg of coral per liter of extraction medium. The extract is decanted from the coral and filtered sequentially through 0.45 J.£m, 0.22 J.£m, 10,000 MW, and 1,000 MW filters. Very recently, a Millipore- Pellicon ®, tangential-flow filtration system has greatly facilitated this process. Various of the intermediate filters can be omitted, but if the material isn't initially filtered at 0.22 to 0.45 J.£m, the mucous substances can quickly clog the finer filters. The ultrafiltrate, now containing only water-soluble molecuies with molecular weights below 1,000, is passed through a column packed with Amberlite® XAD-4 resin, a styrene-type polymer of large surface area and porosity. The advantage of XAD-4 is the broad spectrum of organic molecules that adsorb to it. The column is flushed with several volumes of distilled and deionized water to remove NaCI and other inorganic salts, and then is eluted with acetonitrile. Most of the coral products, including the inductive substance, pass off the column with the solvent front. This fraction is collected and evaporated to dryness at 70- 80°C. The yield of dried material at this point is approximately 0.65 g per 100 liters ofIiquid coral extract (= 130 kg of coral). The dried extract, now consisting of the desalted, water-soluble coral products with molecular weights below 1,000, is dissolved in a minimal volume of water and subjected to repeat high-performance liquid chromatography on a C-18, reverse-phase column. The mobile phase for the first one or two runs is a 0-100% acetonitrile gradient. Subsequent passes are isocratic elutions at 12-20% acetonitrile. With an ultraviolet detector set at 225 or 290 nm, a great number of compounds is detected on the first several passes through the C-18 HPLC column. Even at the fourth pass, up to 30 peaks are visible at 225 nm. In one separation, on the fifth HPLC pass we recorded about 7 peaks at 225 nm, with activity, determined by bioassay of eluate fractions, confined to a small area not coinciding with one of the major peaks. Subsequent examination of this active fraction with NMR and mass spectros- copy showed it to contain at least five different molecular species, none in sufficient HADFIELD AND PENNINGTON: METAMORPHIC INDUCTION IN A NUDIBRANCH 459 Table I. Methods for extraction and purification of the water-soluble coral product that induoes metamorphosis in larvae of Phestilla sibogae Extraction: the coral Porites compressa is extracted for 18-20 h at 1.3 kg of coral per liter of 0.56 M tris-buffered NaCI (pH 8.2).

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