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

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 metamor- phose 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 oper- culum, 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 in- vestigations 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) 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 develop- mental 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 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 micro- graph: 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 meta- morphic 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 compe- tent), 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% aceto- nitrile. 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). Filtration and Ultrafiltration: the extract is passed serially through filters of pore sizes ranging from 0.45 ~m to 1,000 molecular weight. Desalting and concentration: (a) the filtered extract is passed onto a column of XAD-4 resin; (b) the column is washed with distilled water to remove salts; (c) the organic materials are eluted from the column with acetonitrile and evaporated to dryness. HPLC: (a) the extract is redissolved in distilled water and injected onto a C-18, reverse-phase column; (b) the column is eluted with a 0-100% aqueous-acetonitrile gradient; (c) the active fraction is determined by bioassay and reinjected; (d) the process is repeated to purity. quantity for structural elucidation. The mass spectroscopy data confirm earlier estimates of molecular size based on ultrafiltration (Hadfield and Scheuer, 1985). These investigations have revealed a number of characteristics of the coral product that is such a potent morphogen for Phestilla sibogae. It is a small mol- ecule, with a molecular weight between 200 and 500. It is a fairly polar substance, being very soluble in water, insoluble in methylene chloride, and eluting from C-18 columns at less than 50% acetonitrile. It is also a very potent substance. One 60-70 liter extract yielded about 10 J.Lgof dried material after the fifth HPLC pass with the bioactive fraction isolated after each pass. If we assume that there were five compounds in equal proportions in the 10 J.Lg, then there were about 2 micrograms of inducer in the original ~65 liters of extract. Assuming also that the molecular weight of the substance is 300, a compromise between 200 and 500, we calculate the effective molarity of the original extract at 10-10 M. If th{: effective concentration is calculated instead from bioassays with the mostly pur·· ified end-product, we obtain a value of 5 x 10-10 M (i.e., the maximum effectiv{: 1 dose of the material was about 0.007 J.Lg·ml- ; times 0.2 [our estimate of the fraction of inducer in the HPLC purified material], yields an effective concentra-, tion of 1.4 mg per liter; divided by 300 [our estimate of molecular weight of the inducer] gives us an effective dosage at 5 x 10-9 M). Either way, the inducer molecule is very potent. The coral product that induces metamorphosis in P. sibogae is a small, polar, and very potent molecule. Work continues toward purification of a sufficient quantity of the inducer to complete its structural identification because it is of considerable interest to understand the structure of a molecule with such potent biological activity. It is also possible that knowledge of its structure may tell more about its mode of activity. Structural characterization of the molecule will be a major step toward its artificial synthesis, and thus its general availability for experimental-developmental studies, and the probability of adding a radio-tag for use in locating the receptor for the inducer molecule on the larva.

PROBLEMS WITH THE PHESTlLLA INDUCER STUDIES One of the major problems of the search for the coral-produced molecule that induces metamorphosis in larvae of P. sibogae has been the inconsistency of results from one trial to the next. Probably there really is variation from coral head to coral head, but more likely the differences in extraction results lie in variations in the degree to which we avoid mucus production during extraction and free the active product from mucous substances during later processing. 460 BULLETIN OF MARINE SCIENCE, VOL. 46, NO.2, 1990

Table 2. Effects offiltration on metamorphic induction activity of a coral extract, measured as percent oflarvae metamorphosing within 48 hours of exposure to the extract. Controls include metamorphosis in the presence of living coral and in artificial seawater (MBL) alone

Filter pore size % Metamorphosis (mean ± 95% CI) % Activity lost to filtration

0.45 11m 94 ± 6 o 0.22 11m 87 ± 3 o 10,000 MW 59 ± 14 40 1,000 MW 17 ± 14 83 Coral 89 ± 7 MBL 2±2

However, another possibility that has been impossible to rule out absolutely is that we are dealing with more than one substance. For instance, if there were two, (1) they could be equal inducers with individual larvae variably more sensitive to one or the other, or (2) they could be synergists, active alone but far more potent together. Activity could even vary with the relative ratios of two substances present in an extract. What evidence is there that there is more than one inductive substance in the coral extracts? The only somewhat convincing evidence is the simple fact that when living coral is present, almost invariably 100% of larvae metamorphose, while even when concentrated many thousand fold, most extracts do not yield more than about 70% metamorphosis. This is especially true with the HPLC-purified material; even though activity elutes only during a small part of a run, the most active fractions typically induce only 10-50% of larvae to metamorphose, even at high concentrations. This could be due to structural changes in the inducer molecule that occur during purification; a simple oxidation could lead to decreased activity. In a recent reinvestigation of this problem, we obtained the following results. A fresh extract of coral was prepared and passed through a 0.45 micron filter and bioassayed; nearly 100% of the larvae metamorphosed. We then ran a filtration series, assaying for percent metamorphosis after each successive filtration (Table 2). While we saw no significant retention of activity by the 0.45 or 0.22 .urn filters, filtrate from the 10,000 molecular-weight cutoff ultrafilter showed about 40% less inductive activity. After passing through the 1,000 molecular-weight filter, activity had declined a total of nearly 90%. To determine if the loss of activity we had seen in this experiment was due to filtration removal of a second, larger inducer molecule, we ran a dilution series on the original, 0.45 micron-filtered material and tested it for metamorphic in- duction. The resultant data, shown in Figure 4, indicate that there was no decrease in activity down to 50% dilution. When diluted by 99%, metamorphosis ap- proached 10%, the value we had obtained after ultrafiltration of the same material. While proving nothing about the possibility of a second inducer, these data tend to show that the best explanation for the filtration-loss of metamorphic stimulation was progressive removal ofa single molecular entity. The 10,000 molecular-weight filter removed about 95% of the inducer, and the 1,000 MW filter removed another 4%, summing in about 99% loss of the molecule. This is consistent with the idea that the persistent problem of loss of activity during inducer purification results from adsorption of the low molecular weight inducer onto larger proteoglycans of varying sizes, and thus the variable loss of inducer on different filters. This leaves unanswered the question of why we can't regain maximal inducer activity by concentrating the material that has passed through all of the filters HADFIELD AND PENNINGTON: METAMORPHIC INDUCTION IN A NUDIBRANCH 461

100 1xl0-1 ~ z Vl o iii o ... I II: ~-= \x 10-1 II: o W "-::;; U oZ ~ 4() U ::;; ...-' tt Z 20 II: W 1xlQ-J >- •...• 10-5 M ~ •....•. 10-4 M o •....• 10-3 M o 20 40 60 80 100 24 48 72 96 % OF FULL STRENGTH TIME (hours) Figure 4 (left). Percent of larvae (mean ± SD) undergoing metamorphosis when exposed to various: dilutions of an extract of Porites compressa in artificial seawater. Less than maximal response was, observed only in samples diluted more than 50%. Figure 5 (right). Uptake of I4C-labelled choline (mean concentration ± SD) by larvae of Phesti/la sibogae as a function of time at three external concentrations, 10-5, 10-4 and 10-3 molar. Internal concentration is dose dependent; it plateaus after 48-72 h exposure.

and columns. This problematical question could probably be answered if we knew the chemical structure of the inducer, something we're still trying to determine.

How DOES THE INDUCER ACT ON THE LARVA? How Is THE SIGNAL RECEIVED?

It is clear that this small water-soluble inducer molecule can stimulate meta- morphosis in solution; in earlier work (Hadfield and Scheuer, 1985) we showed that water drawn from coral heads in the field induces metamorphosis in Phestilla larvae in the laboratory. We have demonstrated also that larvae will metamor- phose several hours after removal from an inducer preparation (Hadfield, 1978). This appears to separate the metamorphic stimulus of Phestilla sibogae from those of many other marine invertebrates which are detected as adsorbed layers on the benthos (Crisp, 1974). Only for the sand-dollar Dendraster excentricus have sim- ilar decisive data been presented demonstrating metamorphosis in response to a dissolved substance (Burke, 1984). We assume that the inducer acts on an external, cellular receptor somewhere on the larva. Bonar (1978) suggested that the cephalic (apical) ciliary organ, a clearly sensory structure lying at the top of the head within the velar ciliary loop, fits both structural and topographical criteria for the receptor ofthe metamorphic signal. However, other sensory structures and areas exist on the larva, and as yet there is no direct evidence that the apical organ is the receptor of interest. Wherever the receptor happens to lie, its neurological nature is strongly sug- gested by its potassium dependence. With potassium ion reduced or absent in the medium, larvae do not respond to the coral product and metamorphose. If po- tassium concentration is reduced by 40% in an artificial seawater containing coral extract, resultant metamorphosis is reduced by 80%. By contrast, a 15 mM increase in potassium concentration stimulates metamorphosis in the absence of inducer (Y001 et al., 1986). Potassium induction of metamorphosis, first recorded by Baloun and Morse (1984) for abalone larvae, has now been found in a wide variety of invertebrate larvae (Yool et al., 1986; Pechenik and Heyman, 1987). 462 BULLETIN OF MARINE SCIENCE, VOL. 46, NO.2, 1990

Table 3. Some biological roles of choline

Extracellular In high concentration: activates acetylcholine receptors (Krnjevic' and Reinhardt, 1979). Intracellular Increased synthesis and release of acetylcholine (Blusztajn and Wurtman, 1983). In rat brain: stimulates synthesis and release of epinephrine (Dlus and Wurtman, 1976). In adrenal gland: stimulates synthesis of epinephrine and norepinephrine (at 3-10 mM choline) (Holz and Stenter, 1981; Blusztajn and Wurtman, 1983). Participates in phosphatidylcholine biosynthesis in membranes (Stryer, 1988). Serves as a methyl donor in, e.g., creatine synthesis (Stryer, 1988).

INTERNAL ACTIVATION OF METAMORPHOSIS It is probably the nervous system that activates and coordinates the many processes of metamorphosis in larvae of P. sibogae. The bases for this assertion follow. Metamorphosis in response to natural inducer is all-or-none. There is no graded response to different inducer concentrations among the different parts of a larva that undergo independent transformations (Hadfield, 1978). Instead, there is merely a change in the percentage of larvae that metamorphose. This is in distinct contrast to hormone-mediated metamorphoses (e.g., in insects and am- phibians), where different tissues respond to different hormone titres in the blood- stream. Metamorphic induction in P, sibogae is a relatively rapid process seemingly allowing little time for de novo syntheses of one or more internal messengers. Furthermore, metamorphosis was found to proceed normally in the presence of inhibitors of RNA and protein synthesis (Hadfield, 1978). Finally, the evidence from so-called "artificial" inducers of metamorphosis points towards an inductive role for known neurotransmitters in all or some of the tissue transformations. Of particular significance have been the inductive powers of choline chloride and epinephrine. Choline induces complete meta- morphosis in competent larvae, but only at doses greater than millimolar and after about 48 hours exposure (Bonar, 1976; Hadfield, 1978; Hadfield and Scheuer, 1985; Hirata and Hadfield, 1986). Furthermore, we have shown that choline does not compete with the natural inducer and that precompetent larvae do not habituate to choline as they do to the natural inducer, both indicating that the site of choline action is separate from the receptor for the coral product (Hirata and Hadfield, 1986). In recent studies (Hadfield and Plantenberg, in prep.), we have learned that larvae are capable of actively taking up large quantities of choline from seawater (Fig. 5). At micromolar concentrations of choline chloride in the external medium, larvae accumulate choline to millimolar internal concentrations, a concentration factor of about 1,000. It takes approximately 48 hours for the internal concen- tration of choline to achieve a plateau, and for internal concentration to rise above centimolar, external dosage must be greater than millimolar (Fig. 5). Only at such very high internal concentrations does metamorphosis occur. These uptake data are consistent with our published data relative to required dose and latency for choline-induced metamorphosis in P. sibogae (Hadfield and Scheuer, 1985; Hirata and Hadfield, 1986). What role could choline play in the translation of the metamorphic process, accepting that it does not act on the external receptor for the natural inducer? In work on vertebrates, mainly mammals, many different biological roles have been HADAELD AND PENNINGTON: METAMORPHIC INDUCTION IN A NUDIBRANCH 463 faund far chaline; they are summarized in Table 3. Selecting fram these knawn activities af chaline, the mast likely ane far the effects af high cancentratians an Phestilla larvae is in generating nervaus activity, either (1) by acting directly an acetylchaline receptars, (2) by fueling acetylchaline biasynthesis, ar (3) by stirn·· ulating synthesis and build-up af catechalamines. Bath high cancentratians and latency af effect are cansistent between chaline affects an the vertebrate nervaus system and aur abservatians an Phestilla. The ather chaline functians catalagued in Table 3 are hard to.recancile with a regulatary rale far the many changes that metamarphasis entails. We have knawn far a number af years that same metamarphic changes accur in respanse to. epinephrine (Hadfield, 1984). Namely, the larvae lase their velar labes, but praceed no.further in metamarphasis. Recently, we have been attempt- ing to.lacate age-specific sites af catechalamine biasynthesis ar accumulatian with immunacytachemistry (Kempf et a1., in prep.). Thus far, we have been able to. laak directly anly far dapamine and narepinephrine because antibadies are cam- mercially available far these twa catechalamines ar enzymes unique to. their syntheses. Pasitive results with campetent larvae expased to. antibadies far da- pamine indicate that this appraach will be fruitfu1.

SUMMARY The majar problem remaining to. be salved in arder to.better understand the develapmental bialagy afmetamarphic inductian in campetent larvae af Phestilla sibogae is the structural elucidatian af the caral malecule that induces metamar- phasis. Knawledge af this structure may, in itself, tell samething af its made af activity. Certainly, having the substance in hand will apen avenues far lacating the receptar far this inducer. Electran micrascapic studies can then be used to. characterize its cellular nature. Prabably the methads af neurabialagy hald greatest pramise far explaring the mechanisms by which the inducer-receptar interactian stimulates the rapid tissue lasses and transfarmatians that culminate in meta- marphasis in larvae af P. sibogae. However, to understand stimulation of cellular metamarphic changes at the level of the cell, it may be necessary to. use prepa- ratians that da nat include the entire larva. Eventually, it may be possible to determine, with the techniques of molecular bialagy, what specific subcellular processes are stimulated during metamorphosis.

ACKNOWLEDGMENTS

We are most grateful to numerous colleagues who have assisted us on various aspects of the Phestilla- metamorphosis project over many years, including S. Grau, S. Miller and L. Ho Iseke. The NMR and mass spectroscopy data were made available through the courtesy of Drs. T. Carroll and P. Scheuer, Department of Chemistry, University of Hawaii; we are in their debt for these efforts. Research reported here has been supported by NSF grants to M.G.H., the most recent of which is No. DCB- 8602149 and by NIH grant No. GI2-RRO-306\.

LITERATURE CITED

Baloun, A. and D. E. Morse. 1984. Ionic control of settlement and metamorphosis in larval Ha/iotis rufescens (). Brain Res. Bull. 167: 124-138. Blusztajn, J. K. and R. J. Wurtman. 1983. Choline and cholinergic neurons. Science 221: 614-620. Bonar, D. B. 1976. Molluscan metamorphosis: a study in tissue transformation. Am. Zool. 16: 573- 59\. --. 1978. Ultrastructure of a cephalic sensory organ in larvae of the gastropod Phestilla sibogae (Aeolidiacea, Nudibranchia). Tissue Cell. 10: 153-165. -- and M. G. Hadfield. 1974. Metamorphosis of the marine gastropod Phestil/a sibogae. I. Light 464 BULLETINOFMARINESCIENCE,VOL.46, NO.2, 1990

and electron microscopic analysis oflarval and metamorphic stages. J. Exp. Mar. BioI. Ecol. 16: 1-29. Burke, R. D. 1984. Pheromonal control of metamorphosis in the Pacific sand dollar, Dendraster excentricus. Science 225: 442-443. Crisp, D. J. 1974. Factors influencing the settlement of marine invertebrate larvae. Pages 177-265 in P. T. Grant and A. M. Mackie, eds. Chemoreception in marine organisms. Academic Press, London. Hadfield, M. G. 1977. Chemical interactions in larval settling of a marine gastropod. Pages 403- 413 in D. J. Faulkner and W. H. Fenical, eds. Marine natural products chemistry. Plenum, New York. ---. 1978. Metamorphosis in marine molluscan larvae: an analysis of stimulus and response. Pages 165-175 in F.-S. Chia and M. E. Rice, eds. Settlement and metamorphosis of marine invertebrate larvae. Elsevier. ---. 1984. Settlement requirements of molluscan larvae: new data on chemical and genetic roles. Aquaculture 39: 283-298. ---. 1986. Settlement and recruitment of marine invertebrates: a perspective and some proposals. Bull. Mar. Sci. 39: 418-425. --- and D. Scheuer. 1985. Evidence for a soluble metamorphic inducer in Phestilla sibogae: ecological, chemical and biological data. Bull. Mar. Sci. 37: 556-566. --- and M. F. Switzer-Dunlap. 1984. Reproduction in Opisthobranchs. Pages 209-350 in K. Wilbur, ed. The biology of molluscs, Yol. 6. Academic Press, New York. Harris, L. G. 1975. Studies on the life history of two coral-eating nudibranchs of the genus Phestilla. BioI. Bull. 149: 539-550. Highsmith, R. T. 1982. Induced settlement and metamorphosis of sand dollar (Dendraster excen- tricus) larvae in predator-free sites: adult sand dollar beds. Ecology 63: 329-337. Hirata, K. Y. and M. G. Hadfield. 1986. The role of choline in metamorphic induction of Phestilla (Gastropoda: Nudibranchia). J. Compo Biochem. Physiol. 84C: 15-21. Holz, R. W. and R. A. Stenter. 1981. Choline stimulates nicotinic receptors on adrenal medullary chromaffin cells to induce catecholamine secretion. Science 214: 466-468. Kempf, S. C. and M. G. Hadfield. 1985. Planktotrophy in the lecithotrophic larvae of a nudibranch, Phestilla sibogae (Gastropoda). BioI. Bull. 169: 119-130. Krnjevic', K. and W. Reinhardt. 1979. Choline excites cortical neurons. Science 206: 1321-1323. Miller, S. E. and M. G. Hadfield. 1986. Ontogeny of phototaxis and metamorphic competence in larvae of the nudibranch Phestilla sibogae Bergh (Gastropoda: Opisthobranchia). J. Exp. Mar. BioI. Ecol. 96: 1-18. Morse, D. E. 1990. Recent progress in larval settlement and metamorphosis: closing the gaps between molecular biology and ecology. Bull. Mar. Sci. 46: 465-483. Pawlik, J. R. 1990. Natural and artificial induction of metamorphosis of Phragmatopoma lapidosa californica (Polychaeta: Sabellariidae) with a critical look at the effects of bioactive compounds on marine invertebrate larvae. Bull. Mar. Sci. 46: 512-536. Pechenik, J. A. and W. D. Heyman. 1987. Using KCI to determine size at competence for larvae of the marine gastropod Crepidulafornicata (L.). J. Exp. Mar. BioI. Ecol. 112: 27-38. Stryer, L. 1988. Biochemistry, third ed. W. H. Freeman, New York. 1089 pp. Ulus, I. I. and R. J. Wurtman. 1976. Choline administration: activation of tyrosine hydroxylase in dopaminergic neurons of rat brain. Science 194: 1060-1061. Yool, A. J., S. M. Grau, M. G. Hadfield, R. A. Jensen, D. A. Markell and D. E. Morse. 1986. Excess potassium induces larval metamorphosis in four marine invertebrate species. BioI. Bull. 170: 255-266.

DATEACCEPTED: April 24, 1989.

ADDRESS: Kewalo Marine Laboratory, University of Hawaii, 41 Ahui St., Honolulu, Hawaii 96813.