BULLETIN OF MARINE SCIENCE, 37(2): 556-566, 1985 EVIDENCE FOR A SOLUBLE METAMORPHIC INDUCER IN PHESTILLA: 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 nudibranch Phestilla sibogae. 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.