BULLETIN OF MARINE SCIENCE, 37(2): 556-566, 1985

EVIDENCE FOR A SOLUBLE METAMORPHIC INDUCER IN : ECOLOGICAL, CHEMICAL AND BIOLOGICAL DATA

Michael G. Hadfield and Deborah Scheuer

ABSTRACT A natural product of the coral Porites compressa induces larval metamorphosis in a predator of the coral, the . Seawater removed from coral heads in the field induces metamorphosis in these larvae. Concentrated "coral seawater" prepared in the laboratory is active in metamorphic induction after filtration through a 2,OOO-m.w. ultrafilter, but activity is at least partially retained by a 300-500-m. w. ultrafilter. This seawater contains twice as much dissolved organic carbon and eight times as much dissolved organic nitrogen as seawater standards. Larvae of P. sibogae exposed to coral-produced, metamorphic inducer at any time before achieving metamorphic competence do not metamorphose as long as they remain in the inducer. This habituation to inducer is reversed in competent larvae by 1-5 h removal to clean seawater before re-exposure to the coral product. The data imply that upwardly diffusing substances could influence the behavior of planktonic larvae so as to bring about site-specific settlement.

Larvae of the coral-eating nudibranch Phestilla sibogae metamorphose in re- sponse to the presence of the adult prey, the stony coral Porites compressa. We have previously shown that competent larvae of P. sibogaewill also metamorphose in seawater which had contained P. compressa and in seawater containing redis- solved, lyophilized, distilled-water extract of P. compressa (Hadfield, 1977). This interaction has both ecological and developmental significance. It assures that the nudibranch larvae will settle in locales appropriate for post-metamorphic survival. Larvae withheld from exposure to Porites compressa or its extracts continue to swim for at least 2 weeks, but eventually die without metamorphosing. The coral product serves as a specific trigger for the massive morphological and physiological transformations of metamorphosis (Hadfield, 1978), and thus its function is anal- ogous to that of a hormone. Crisp (1974; 1977) has noted for many marine invertebrate groups that specific settlement stimuli must be encountered as adsorbed layers on benthic substrata, and he has implied that this is a necessary prerequisite for all such settlement stimuli. That this is not universally the case is supported by data presented here. First, we show that water samples taken from coral heads in the field and then filtered are capable of inducing metamorphosis in larvae of P. sibogae. Secondly, we present data showing that seawater made metamorphically inductive by storing dense amounts of coral in it for 18 h, retains its inductive capacity after passage through ultrafilters; such seawater shows significant quantities of dissolved organic carbon and nitrogen. Finally, we present data supporting the hypothesis that larvae are affected by components of the coral product before they are competent to settle and meta- morphose. Such swimming, pre competent larvae become refractory to the in- ductive capacity of the coral product if exposed to it before they become com- petent, a process we have previously dubbed "habituation" (Hadfield, 1980). Here we present data on age at competence and its relationship to habituation and on the process of dehabituation to inducer.

556 HADFIELD AND SCHEUER: METAMORPHIC INDUCTION IN A NUDIBRANCH 557

METHODS

Populations of Phestilla sibogae are maintained in tanks receiving a continuous supply of unfiltered seawater at the Kewalo Marine Laboratory, Honolulu, Hawaii. Living heads of the coral Porites compressa are collected weekly from Kaneohe Bay, Oahu, Hawaii, to replace those which have been eaten by Phestilla. This coral also adds to the stock of Phestilla as 3-5 minute, newly metamorphosed nudibranchs usually occur on any coral head larger than about 10 cm diameter taken from the patch reefs of Kaneohe Bay. Newly laid egg masses were collected daily from the stock tanks of adult P. sibogae and transferred to plastic screen baskets that were suspended in a tank receiving a continuous flow of 5.0-!!m-filtered seawater. One day prior to their spontaneous hatching date (which varies from about 5 to 8 days depending on seasonal seawater temperature), the egg masses were removed to bowls of filtered seawater and mechanically hatched with fine forceps. The larvae were transferred to new medium (0.22-~m- filtered seawater containing 60 !!glml Penicillin G and 50 ~glml streptomycin sulfate) every 2-3 days as needed. Field Sampling of "Coral Water. "-Swimmers using masks and snorkels collected water samples from within heads of Porites compressa, in the field, with large, plastic syringes. Similar samples were collected about 2-3 cm away from the surfaces oflive coral heads. Sampling was done on four different occasions. Water conditions in the field varied from calm to highly turbulent on different days, and thus the relative flushing of coral heads varied from one collection to the next. In the laboratory, the samples were passed through a 43-~m mesh sieve and then centrifuged at 1,800 rpm for 20 min to rid them of particulate matter. Bioassays were conducted in 62-mm stender- dishes containing 25 ml of test seawater and 20 larvae; 4-10 replicate assays were run on each test solution. Controls included (I) filtered seawater drawn from the laboratory's seawater system to test for the occurrence of spontaneous metamorphosis, and (2) seawater containing 0.1 % by weight of a lyophilized, distilled water extract of the coral (Hadfield, 1977; we refer to this preparation routinely as "Crude Inducer") to test for their competence to metamorphose. All larvae used in any assay were maternal siblings derived from a single egg mass. Percent metamorphosis was determined after 24 and 48 h. Characterization of Metamorphosis-eliciting "Coral-seawater. "-Glass beakers (I liter) were filled to capacity with broken tips of freshly collected Porites compressa, and filtered seawater was added to capacity. Air was bubbled through the coral from a pasteur pipette inserted centrally to the bottom of each beaker. The beakers were maintained at room temperature (ca. 24"C) for 18 h. Subsequently, the coral was discarded and the seawater was serially filtered through paper, a 0.22-!!m Millipore® filter, and a IO,OOO-m.w. Amicon® ultrafilter. On several occasions, the seawater was also passed through a 2,000-m.w. Amicon filter and a third Amicon filter (code YC05) with size-dependent rejection in the range of 300-500 m.w. The coral-steeped seawater was tested for its metamorphosis- inducing capacity after each filtration, the assays being carried out as described above. Because the assays showed that metamorphosis-inducing activity passed through all filters except the 300-500-m.w. filter, and that it provided only partial retention, coral-seawater samples that had passed through 10,000- and 300-500-m.w. ultrafilters, as well as those that were retained above the 300-500-m.w. filter, were compared to similarly filtered clean seawater with regard to the following components: ammonia, inorganic phosphate, total dissolved phosphate, inorganic nitrate and nitrite combined, total dissolved nitrogen and total dissolved organic carbon. Dissolved organic nitrogen was determined by extrapolation. Determinations were carried out by Analytical Services, Inc. (Honolulu, Hawaii). Habituation Studies. - To explore the possibility that larvae exposed to metamorphic inducer prior to competence become refractory to the effect of inducer (i.e., are habituated to it), egg masses were mechanically hatched a day or two prior to their anticipated hatching age and the larvae were established in the standard culture conditions. On the day of hatching and daily thereafter, batches oflarvae were transferred from the stock culture to dishes containing the maximum effective dose of lyophilized coral extract (0.1% Crude Inducer; Hadfield, 1977). At 24-h intervals after the larvae were placed in inducer solution, the cultures were checked and the percentage oflarvae having metamorphosed was recorded. These experiments revealed both the age at which larvae became competent and the degree to which habituation had occurred. Habituation results in larvae that do not undergo metamorphosis at the time they normally would. Therefore, habituation was measured by assessing how many ad- ditionallarvae within a particular time period metamorphosed in cultures exposed for only 24 h when compared to the number that metamorphosed under conditions of continuous exposure to inducer. In these experiments, the counts made just 24 h after induction are assumed to represent the proportion of potentially competent larvae on any given day; in reality, it probably underestimates this quantity because, as will be shown below, maximum metamorphosis under a given set of conditions usually occurs between 28 and 32 h after exposure to inducer. 558 BULLETINOFMARINESCIENCE,VOL.37, NO.2, 1985

TIMINGOF HABITUATION.Experiments were run to determine more precisely when, following ex- posure to inducer, habituation takes place. To do this, sibling cultures were established at a time when, by previous observation, it had been determined that most larvae would become competent, in this case between the beginnings of the seventh and eighth days. From stock cultures, sibling larvae were transferred to inducer solution at 4-h intervals beginning at 0800 on day 7 and ending at 0800 on day 8. The numbers that had metamorphosed in these induced cultures were determined on the mornings of days 8, 9 and 10 (i.e., 24, 48, and 72 h after the onset of the experiment). Thus at first count (day 8), the larvae had been in inducer 0, 4, 8, 12, 16, 20, and 24 h. At the second count (day 9) they varied from 24 to 48 h exposure, and when assayed on day 10, between 48 and 72 h exposure. HABITUAnON INTHEEGGMASS.At cooler temperatures more larvae are competent to metamorphose at the time of hatching. To determine whether or not habituation can occur while larvae are still inside the egg coverings, egg masses were put into beakers of either aerated seawater or aerated seawater containing 0.1 % Crude Inducer 24 h prior to their normal, expected hatching time. Upon hatching the following day, larvae from both groups were tested for their metamorphic responsiveness to inducer. These experiments were done at temperatures below 24"C, when hatching occurred at about day 8 after egg laying. Comparison of percent metamorphosis on day 9 between the cultures established from larvae exposed and not exposed to inducer prior to hatching provided a measure of prehatching habituation to inducer. DEHABITUATION.The ability oflarvae to dehabituate was investigated by removing habituated larvae from the inducer solution and transferring them to clean seawater where they remained for 24 h. They were then reintroduced to inducer solution and assayed the following day for metamorphic response. Because these experiments demonstrated that larvae could regain their responsiveness to inducer after removal from inducer solutions, the duration of removal necessary to allow dehabituation was studied. These experiments were conducted during late summer when sea temperatures are at their highest (over 27"C), and as a consequence, some egg masses were hatching prior to day 5. Thus egg masses were hatched on day 4, and larvae were immediately placed in seawater containing 0.1 % Crude Inducer. About 150 larvae were retained in seawater to determine by subsequent daily new exposures of larvae to inducer when maximum competence was occurring (between days 8 and 9 in this series). The constantly exposed, habituated larvae were then removed to clean seawater and reintroduced to inducer solution at each subsequent hourly interval for 5 h. Percent metamorphosis was determined 26-33 h later. To test for the possibility that dehabituation was simply due to the mechanical disturbance of being transferred, a similar experiment was done wherein larvae were pipetted at regular intervals to new inducer solution, left for 24 h, and assayed. Habituated larvae were also stirred on a magnetic stirrer, while in inducer solution, for 1-2 min and checked 24 h later for metamorphosis. In all experiments, the number of larvae that metamorphosed was expressed as the percentage of the total number oflarvae tested under any given set of conditions. All percentage data were subjected to arcsine transformation before calculation of means, a procedure recommended for normalizing proportional data lying towards the ends of distribution possibilities (Sokal and Rolf, 1981). Standard errors were calculated from untransformed data.

RESULTS Assay of Inductive Capacity of Seawater Taken from Coral Heads in the Field. - Seawater extracted from heads of Porites compressa in the field contained factors that induce metamorphosis in larvae of P. sibogae (Fig. 1). The amount of such activity varied and could be roughly predicted from the amount of water move- ment observed at the time of sampling. When water motion was high (Fig. I, sample I), the coral heads appeared to be flushed clean of most inductive activity. At times when waters were relatively calm (Fig. 1, sample II), coral-head water induced metamorphosis at levels close to those of our standardized Crude Inducer. Water taken 2-3 em above coral heads (labelled 0 and 0' in Fig. 1) showed detectable, but very low activity. Metamorphic Induction by Coral-seawater Prepared in the Laboratory. -Seawa- ter from beakers filled with live coral tips and bubbled for 18 h or more, always showed strong metamorphic induction in P. sibogae larvae. Furthermore, the inductive activity passed through all filters with exclusions greater than the 300- SOO-m.w. range ofthe Amicon YCOS. The data in Table I indicate that the coral product responsible for metamorphic induction lies near the range of exclusion HADFIELD AND SCHEUER: METAMORPHIC INDUCTION IN A NUDIBRANCH 559

80

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I ]I m Figure 1. Induction of metamorphosis in larvae of Phestilla sibogae by samples of water drawn from coral heads in the field. Each set of bar graphs (I, II, III, and IV) represents water samples collected on a different day and from a different location in Kaneohe Bay, Oahu, Hawaii. C and C', samples from within coral heads taken the same day; 0 and 0', samples taken 2-3 em from the coral heads sampled as C and C'; S, filtered seawater from the laboratory supply; I, 0.1% Crude Inducer. All assays were counted 48 hours after larvae were exposed to the "coral seawater." of the Amicon YC05 filter. Results with this filter were equivocal, and the amount of activity seen in the filtrate appeared to be a function of the degree to which the retentate was concentrated. For example, in one run the early filtrate produced 20% metamorphosis on bioassay; as the concentration factor approached seven- fold, the filtrate produced nearly 80% metamorphosis. The results of chemical analysis of coral-seawater are presented in Table 2. For comparison, values for seawater from the same source, but not exposed to coral, are included in the table. Analyzed were 1O,000-m.w. and YCOS (300-500-m.w.) filtrates and the YCOS retentate. About 47% ofthe volume applied to the YCOS filter was passed through the filter, concentrating excluded solute about 2.2 times in the retentate; the salinity of the YCOS retentate had increased from 340/00to 40%0, while that of the filtrate was reduced to 280/00.The significant results here are the clear demonstration of relatively high levels of dissolved organic carbon and dissolved organic nitrogen in the coral-seawater filtrates. Most of this material was retained on the YC05 filter along with the metamorphosis-inducing activity. Habituation Studies. -Data presented in Figures 2 and 3 clearly demonstrate inhibition of metamorphosis in larvae exposed to inducer prior to competence. Because ages of hatching and competence vary with temperature, the oldest age at which larvae can be exposed to inducer and become habituated to it also varies with temperature (compare the two sets of data in Table 4). In two series of 560 BULLETIN OF MARINE SCIENCE, VOL. 37, NO.2, 1985

Table 1. Induction of metamorphosis in Phestilla sibogae by "coral seawater" after successive fil- trations (Results are percent of larvae metamorphosed after 48-h exposure)

% Metamorphosed Treatment Trial: A B C D E

0.22-~m filtrate 64 47 100 72 83 10,000-m.w. filtrate 65 70 95 88 2,000-m.w. filtrate 57 55 300-500-m.w. filtrate 7 15 38 17 14 experiments performed to ascertain how long prior to competence larvae must be exposed to inducer in order to habituate, sibling groups were selected that would be in their most rapid phase of increasing competence at the start of the experiment; in this instance, it was during day 7 after fertilization. Larvae were exposed to inducer at successive 4-h intervals beginning at 0800 on day 7 and concluding at 0800 on day 8; counts of the numbers metamorphosed were made at 0800 on days 8, 9 and 10. Results of the two sets are presented in Table 3. They show that competence in this group of larvae increased from the onset of the experiment over the ensuing 12-16 h. The habituation effect persisted over the same period of time that competence was increasing. These data strongly suggest that almost any exposure to inducer prior to competence will bring about habituation. Note that in both sets of experiments 10-12% of the larvae were either competent at the time the experiments began or were resistant to the habituation effect. The fact that some larvae always metamorphosed, even in cultures exposed to inducer for several days prior to competence (Figs. 2 and 3), indicates that resistance to habituation at least partly accounts for the fraction of first exposed larvae that metamorphosed in these experiments. The data in Table 3 demonstrate another interesting fact. In any given exper- iment (i.e., bowl of larvae exposed to inducer), the maximum response occurs somewhere between 28 and 32 h after exposure to inducer if habituation has not occurred. Thus the last cultures established had not been exposed sufficiently long on day 9 to see the full response; all had been by day 10. Habituation in the egg mass is shown by data in Table 4. Here larvae exposed to inducer for 24 h prior to hatching, as well as the 24 h after hatching, failed to metamorphose (i.e., in part B they were exposed continuously from day 7, hatched on day 8 and counted on day 9). Batches of larvae first exposed after hatching

Table 2. Chemical analysis of "coral seawater"

NO,& % Treatment PO, NO, NH, TDP' DOP" TDN" DON" TOC" Meta. Coral seawater IO,OOO-m.Wofiltrate 0037 19014 3.66 4.69 4032 102.67 79.87 44.75 58 300-500-mow. filtrate 0.21 18026 3.88 0.62 0.41 51.39 29.25 28.35 a 300-500-mow. retentate 0.89 17.96 0.15 9.83 8.94 201.13 183.03 49084 55 Untreated seawater IO,OOO-m.w. filtrate 0.21 0.38 0.82 0.44 0.22 10.49 9.29 22.83 0 Seawater was steeped in coral and bubbled for 18 h, filtered through IO,OOO-m.w.and 300-500-m.w. Amieon" filters and analyzed. Control data represent seawater not treated with coral. Bioassay results of metamorphosis oflarvae of Phestilla sibogae in paired samples of coral seawater are included . • TDP, total dissolved phosphorus; DOP, dissolved organic phosphorus; TDN, total dissolved nitrogen; DON, dissolved organic nitrogen; TOe, total dissolved organic carbon. Units are micTomoles per liter for all except carbon which is recorded as milligrams carbon per liter. HADFIELD AND SCHEUER: METAMORPHIC INDUCTION IN A NUDIBRANCH 561

60 11 80

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10 5 10 6 7 6 0 0 6 7 8 9 10 11 6 7 8 9 10 11 12 13 Age (days post-fertiliz;ation) Age (days post-fertilization) Figure 2. (Left) Habituation in larvae of Phesti/la sibogae. The percent of larvae which metamor- phosed after initial 24-h exposure to 0.1 % Crude Inducer is traced by the dashed line. Solid lines follow percent responses after the initial day of exposure in larvae continuously exposed to Crude Inducer. Numbers at the ends of lines indicate the age (in days) of the larvae when initially exposed to inducer. No assays were set up on day 9. Plotted are means of8-1O assays; bars indicate ± I standard error. Water temperature: 25-26°C. Figure 3. (Right) Habituation in larvae of Phesti/la sibogae. The percent of larvae which metamor- phosed after initial 24-h exposure to 0.1 % Crude Inducer is traced by the dashed line. Solid lines follow percent responses after the initial day of exposure in larvae that remained in inducer throughout the experiment. Numbers at the ends of the lines indicate the age (in days) of larvae when initially exposed to Crude Inducer. Plotted are means of 5 assays; bars indicate ± I standard error. Water temperature: 23-24"C. showed 37% metamorphosis by the following day. Therefore, significant numbers of larvae were competent at hatching, but had habituated within the egg masses. Larvae habituated in the egg mass are as readily dehabituated as those first exposed to inducer after hatching. Dehabituation by removal of habituated larvae from inducer and subsequently re-exposing them is demonstrated in a number of experiments (Table 4). In these cases, the period of removal from inducer was 24 h. To learn how long larvae must be free of inducer to lose the refractoriness to it, larvae that had been hatched at day 4 and continuously exposed to inducer until day 9, were removed from inducer for periods of 1-5 h, replaced in 0.1 % Crude Inducer solution, and ex- amined 24 h later. The results of this experiment, presented in Figure 4, show that removals as brief as 1 h allow some larvae to express metamorphic com- petence, and that the responsiveness of treated sibling groups increases over the ensuing 5 h of de-exposure. We have no explanation at this time for the apparent depression in the dehabituation effect seen at 2 h in Figure 4. Neither transferring habituated larvae from one inducer solution to another, no~ mechanically stirring them resulted in significant increases in metamorphosis. 562 BULLETIN OF MARINE SCIENCE, VOL. 37, NO.2, 1985

Table 3. Percent metamorphosis as a function of larval age in hours, beginning the morning of day 7. In experiment 1, data are presented as means of three tests with separate sets of sibling larvae; in experiment 2, data are means of two tests

Day 7

No. h Day 8 Day 9 Day 10 after 1st ex- No. h No. h No. h Time at exposure posure exposed x% meta. ± SE exposed x% meta. ± SE exposed ii'll>meta. ± SE Experiment 1 0800 0 24 8.6 ± 6.2 48 16.7 ± 7.1 72 18.3 ± 8.2 1200 4 20 9.6 ± 5.0 44 18.5 ± 10.6 68 18.8 ± 11.2 1600 8 16 7.9 ± 1.2 40 25.1 ± 8.3 64 25.6 ± 7.1 2000 12 12 3.5 ± 5.6 36 31.4 ± 5.4 60 33.9 ± 10.9 2400 16 8 1.1 ± 5.8 32 37.7 ± 12.6 56 38.9 ± 12.5 Day 8; 0400 20 4 0 28 24.1 ± 4.0 52 39.6 ± 22.7 Day 8; 0800 24 0 24 21.1 ± 5.5 48 35.2 ± 10.6 Experiment 2 0800 0 24 13.5 48 29.3 ± 4.4 72 33.2 ± 2.8 1200 4 20 30.0 44 54.0 ± 5.7 68 57.4 ± 4.4 1600 8 16 13.2 40 55.3 ± 13.7 64 57.2 ± 8.2 2000 12 12 2.5 36 75.0 ± 1.3 60 76.2 ± 1.3 2400 16 8 0 32 76.6 ± 12.7 56 73.3 ± 15.8 Day 8; 0400 20 4 0 28 51.4 ± 17.6 52 66.5 ± 7.4 Day 8; 0800 24 0 0 24 49.0 ± 20.8 48 71.3 ± 0.1

The dehabituation process requires that larvae be removed from the presence of the coral product.

DISCUSSION Larvae of the nudibranch Phestilla sibogae are induced to metamorphose by a soluble substance released into the surrounding seawater by Porites compressa, the coral prey of the adult P. sibogae. This material is released into the water within coral heads in the field, but is rapidly diluted as it diffuses from the corals. The inducer substance has a molecular weight in the range of 300 to 500, or possibly less, as determined by ultrafiltration. Seawater made metamorphically active by Porites contains significant concentrations of dissolved organic carbon and nitrogen. The soluble, diffusible nature of the coral-produced metamorphic' inducer of Phestilla larvae distinguishes it from the settlement stimulating sub-' stances of barnacles (Crisp, 1974; 1977), abalones (Morse and Morse, 1984; Morse et al., 1980), oysters (Crisp, 1967), and polychaetes (Kirchman et al., 1982; Wilson, 1968; 1970), and the "primary films" responsible for settlement stimulation in forms as diverse as cnidarians, bryozoans and sea urchins (Brancato and Wool- lacott, 1982; Burke, 1983; Cameron and Hinegardner, 1974; Cameron and Schroe- ter, 1980). It is not, however, the only soluble metamorphic inducer recognized. Thompson (1958) demonstrated diffusion of a bryozoan factor that stimulates metamorphosis in the nudibranch Ada/aria proxima; Culliney (1973) described a soluble, habitat-related factor that stimulates metamorphosis in shipworm lar- vae; and Highsmith (1982) reported on a soluble substance released by adult sand dollars that induces metamorphosis in their larvae, an observation subsequently confirmed by Burke (1984). Not all opisthobranchs respond to specific or soluble prey-related metamorphic cues (Hadfield and Switzer-Dunlap, 1984). The degree of specificity of the meta- HADFIELD AND SCHEUER: METAMORPHIC INDUCTION IN A NUDIBRANCH 563

Table 4. Habituation in the egg mass. CI, crude inducer; SW, seawater

Day 7 DayS Day 9 Day 10 Day II A: 23.3"C % Meta- Eggs Hatched & morphosed ± % Meta- exposed to: exposed to: transfer to: morpho sed in: 0% 0% ~CI CI CI 71% SW 00/0I "SW 0% 37% 67% ~CI SW SW 0% 0%

B: 24.2°C % Meta- Eggs Hatched & morphosed ± % Meta- exposed to: exposed to: transfer to: morphosed in: 0% 12.5% ~CI CI CI 85%

sw 0°\ SW 0% 37% 49% ~CI SW SW 0% 0%

morphic stimulus in marine invertebrate larvae can often be predicted on the basis ofthe nature and specificity of their post-metamorphic habitats and trophic requirements. To some degree the adult habitat is found through specific behav- ioral patterns exhibited by settling larvae. Larvae of many sessile filter-feeders probably respond to light in such a way that they find an appropriate depth or intertidal level, and then settle in the presence of suitable substrata. Predators that feed on a variety of motile or non-motile prey tend to settle in response to the types of substratum where prey are most likely to occur. This is probably true for a number of opisthobranchs reported to settle in the absence of a specific chemical stimulus (Bickell and Kempf, 1983; Harrigan and Alkon, 1978; Kempf and Willows, 1977). Many opisthobranchs, including nudibranchs, anaspideans and sacoglossans, are very specific in their prey, and for many the choice food is sessile: sponges, cnidarians, barnacles and bryozoans for nudibranchs; and specific algae for an- aspideans and most sacoglossans (Bickell, 1978; Chia and Koss, 1978; Perron and Turner, 1977; Switzer-Dunlap and Hadfield, 1981; Todd, 1981; West et aI., 1984). For few or none of these has there been specific examination of the interaction to determine if soluble factors are present. Diffusible, soluble, metamorphosis-inducing substances are probably rapidly diluted below their effective concentrations within short distances of their bio- logical sources. Yet, they do offer the potential for at least short-range chemotaxis in swimming larvae, an as yet unexplored possibility. The delicate detection of 564 BULLETIN OF MARINE SCIENCE, VOL. 37, NO.2, 1985

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0 I I I I 0 1 234 5 6 Hours Removed from Inducer Figure 4. Dehabituation of Phestilla sibogae larvae. Larvae which had been continuously exposed to inducer from day 4 were removed for periods of 1-5 h on day 9, then re-exposed to Crude Inducer. Plotted are mean percentages (± I standard error) of larvae that had metamorphosed 24 h after re- exposure to inducer. Three replicate experiments were averaged to produce the dashed line, two replicates to produce the solid line. salinity differences noted for crustacean larvae and their accompanying behavioral changes (Harges and Forward, 1982) demonstrate a potential for chemically me- diated behavior in many minute larval forms. The habituation response of precompetent Phestilla larvae to the inducer sub- stance has apparent ecological significance. Since some larvae may be competent at hatching, the accumulation of inducer in the coral heads, discussed above, assures that these larvae will not settle immediately after hatching in the coral head containing the adults that produced them. This mechanism imposes a min- imal dispersal period. The data on dehabituation demonstrate that this period need not be very long, perhaps only as long as it takes to reach a second coral head. The significance of habituation at the cellular level is potentially great. If we perceive the larval-inducer interaction in P. sibogae as one wherein a specific HADFIELD AND SCHEUER: METAMORPHIC INDUCTION IN A NUDIBRANCH 565 chemical entity, the coral-produced substance, acts on the membrane ofa specific cellular receptor and elicits the larval metamorphic response, then habituation represents a special kind of transmitter-receptor interaction. It has the nature of receptor regulation, noted in a number of endocrine and neuroendocrine systems (Lee et aI., 1983). The implication is that the transmitter (the metamorphic in- ducer), or possibly a second substance accompanying it, acts on the immature receptor in such a way as to prevent its maturation; the receptor is "down regu- lated." The process is readily and rapidly reversed; there is even some evidence for enhanced (i.e., "up-regulated") responsiveness in dehabituated larvae (Ta- ble 4). Metamorphic induction by soluble substances is significant in its implication that swimming, planktonic larvae might be induced to change their behavior (settle) and undergo metamorphosis in response to materials diffusing upward from the benthos. The habitat might thus have a more active role in recruitment of larvae from the plankton than has been traditionally envisioned.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the energetic, competent and cheerful assistance, both in the field and in the lab, of S. Grau, M. Switzer-Dunlap and C. Uetake Ford. We also thank S. Miller for his contribution to the statistical analyses and graphics. This material is based upon work supported by the National Science Foundation under Grant No. PCM-8215552.

LITERATURE CITED

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DATEACCEPTED: March 20, 1985.

ADDRESSES:(M.G.H.) Kewalo Marine Laboratory, Pacific Biomedical Research Center, University of Hawaii, 41 Ahui St., Honolulu, Hawaii 96813; (D.S.) Department of PhYSiology, School of Medicine, University of California at San Francisco, San Francisco, California 94143.