BULLETIN OF MARINE SCIENCE, 39(2): 347-361,1986 LARVAL INVERTEBRATE WORKSHOP DELAYED METAMORPHOSIS: EFFECT ON GROWTH AND SURVIVAL OF JUVENILE SAND DOLLARS (ECHINOIDEA: CLYPEASTEROIDA) Raymond C. Highsmith and Richard B. Emlet ABSTRACT Delay of metamorphosis is an important aspect of the larval biology and ecology of many marine invertebrates. In most studies on delay of metamorphosis, the focus has been on the length of time that larvae could remain competent to metamorphose. The potential effects of delayed metamorphosis on postlarval juveniles have not been investigated previously. Results of studies on the sand dollars Dendraster excentricus and Echinarachnius parma, both of which have planktotrophic larvae, indicate that juveniles from larvae that meta- morphosed soon after becoming competent tended to have higher growth rates than juveniles from larvae that metamorphosed after a prolonged period of competence. Juveniles that were fed cultured algae had higher growth rates but also higher mortality rates than unfed juveniles. Mortality rates in fed juveniles were correlated with length of delay of metamorphosis, but some mortality may have been an artifact of feeding. Unfed juveniles from larvae that had delayed metamorphosis for 4 to 7 weeks had low mortality rates suggesting that either the larvae had not utilized a significant amount of the lipid energy reserves needed by the juveniles to survive until they become self-sufficient or that energy reserves accumulated by larvae are not critical to early juvenile survival. Larvae of most marine invertebrates are capable of delaying metamorphosis (Thorson, 1950; Crisp, 1974; Strathmann, 1978). The length of time that larvae can maintain competence to metamorphose in the absence of an appropriate settlement cue has been the subject of numerous studies (Wilson, 1948; Hine- gardner, 1969; Birkeland et al., 1971; Caldwell, 1972; Kempf, 1981). Other studies have examined larval growth and metabolism during competence (Lucas et al., 1979; Pechenik, 1980; 1984a; 1984b). However, the effect of delayed metamor- phosis on the survival and growth of postmetamorphic juveniles has not been investigated. The few studies that have been conducted on postsettlement mor- tality (Grosberg, 1981; Keough and Downes, 1982; Luckenback, 1984; Young and Chia, 1984) have dealt with mortality due to such ecological factors as sedi- mentation, competition and predation rather than to the condition of the larvae. On two separate occasions in earlier work with Dendraster excentricus, juveniles from larvae that had delayed settlement all died within 30 days of metamorphosis, but juveniles from larvae from the same culture that metamorphosed soon after becoming competent did not die. Test lengths were measured in one case, and the juveniles from the non-delayed larvae grew more rapidly than juveniles from the delayed larvae. In both instances there were no replicate containers so the results could only be considered a preliminary indication that delay of meta- morphosis may have an effect on juveniles. As D. excentricus larvae delay meta- morphosis, their arms tend to become shorter (unpubl. obs.; this paper) resulting in a shorter ciliated band length and presumably reduced feeding capability (Strathmann, 1971; 1975; Strathmann et al., 1972). Concurrently, the gut/rudi- ment region tended to become clearer and less yellow. In view of these obser- vations, we hypothesized that morphological changes in larvae during prolonged competence result in reduced feeding capability and eventual resorption of lipid energy reserves needed for growth and/or survival during the extended transition period from larva to feeding juvenile (Bayne, 1965; Holland et al., 1975; Holland 347 348 BULLETIN OF MARINE SCIENCE, VOL. 39, NO.2, 1986 and Walker, 1975; Chia and Burke, 1978). Use of the storage product would result in reduced initial growth rate of the juvenile and if the larva utilized too much of the storage product, the juvenile would not have sufficient reserves to survive. To test this hypothesis we conducted experiments with two sand dollar species, D. excentricus (Eschscholtz) and Echinarachnius parma (Lamarck). We followed morphological changes in the larvae of D. excentricus during prolonged compe- tence and for both species, we followed growth and survivorship of juveniles that delayed metamorphosis for differing lengths of time in replicate fed and unfed treatments. METHODS Dendraster excentricus. - This portion of the research was conducted at the Friday Harbor Labora- tories, University of Washington, San Juan Island, Washington, U.S.A. Adult D. excentricus and some sand were collected intertidally at nearby Orcas Island. Gametes were obtained and fertilized using standard methods (Highsmith, 1982) to produce a culture of approximately 1,500 sibling larvae. The culture was maintained in a 3-liter container at 8-IO°C with continuous stirring and the water was changed twice per week. The culture was fed Dunaliel/a tertiolecta Butcher and Thalassiosira pseu- donana Hasle and Heimdal at each water change. The algae were centrifuged for 10 min, decanted and resuspended in filtered seawater before being added to the larval culture. Algal cell concentrations were not determined. After the larvae became competent, the water in the larval culture was changed and food was added once per week, at the time larvae were collected for induction of metamorphosis. At each water change, larvae that had metamorphosed spontaneously in the culture were counted and removed. Periodically, larvae were collected and preserved to examine the development and condition of the echinus rudiment. The arms of the larvae were measured from the arm base to the tip and then the tissues were dissolved in dilute commercial bleach so that the skeletal elements ofthe rudiment could be observed and the juvenile spines on the rudiment measured. Competent larvae can be induced to metamorphose by a chemical cue produced by adult sand dollars and which is retained in sand occupied by adults (Highsmith, 1977; 1982; Burke, 1984). For the work reported here, a concentrated extract of the cue was used to induce metamorphosis. The extract was prepared by mixing approximately 500 ml of wet sand from the sand dollar bed with 500 ml of distilled water in a blender for 3 min. The resulting liquid was decanted, frozen and lyophilized to produce a whitish powder. A concentration of 0.9 mg powder/ml seawater, 70 ml per bowl, was used to induce metamorphosis. Once per week, starting at the onset of competence (5 weeks after fertilization) and continuing for a total of 7 weeks, 50 randomly selected larvae were placed in bowls (lOlbowl) with the inducing substance. In weeks 1 and 3, 80 larvae were placed in 8 bowls. As an assay of larval competence, the bowls were checked for percent metamorphosis 24 h after introduction of the larvae. Five bowls for each of weeks 1,3,5, and 7 and 2 bowls for week 6 were maintained without sand at 8-IO°C, fed :::::4 ml D. tertiolecta and T. pseudonana total per week (cell concentrations were not determined), and given water changes biweekly. The algae were centrifuged and resuspended in seawater prior to being fed to the juveniles. The bowls were checked for juvenile survival approximately once per week for periods of 20 days (week 7 bowls) up to 64 days (week I bowls). Three bowls each from sample weeks 1,3 and 6 were "starved," i.e., not fed and maintained in l/lm-filtered seawater which was changed every 3 days to reduce bacterial build-up. To examine the effect of temperature and substratum on survival and growth, two groups ofjuveniles, sample weeks 2 and 4, were kept in a 20°C incubator and sand cleaned in fresh water was added to half the bowls. The juveniles in both treatments were fed as above and test lengths were measured periodically. Echinarachnius parma.-Adult E. parma and some associated sand were collected in late September 1984, at McDonald Spit, Kachemak Bay, Alaska, using the facilities of the Kasitsna Bay Laboratory. The animals were transported to the Seward Marine Center where the study was conducted. Standard methods were used to obtain and fertilize gametes (Highsmith, 1982). The cultures were maintained in I,OOO-ml beakers until the eight-armed larval stage was well developed and then transferred to 300-ml custard dishes so that the larvae could be examined easily with a dissecting microscope to evaluate them for competence to metamorphose. The cultures were stirred for several seconds at least twice per day using a glass probe. Water was changed every 3-4 days. The water was filtered with either Whatman No.3 filter paper or a 0.4 /lm Millipore apparatus. After gastrulation, daily feeding was initiated using D. tertiolecta and T. pseudonana. The algae were centrifuged for 10 min and resuspended in filtered sea water before addition to the larval cultures. HIGHSMITH AND EMLET: DELAYED METAMORPHOSIS IN SAND DOLLARS 349 lIEIIIlA5TEA :;0 SPlINT ANElIU5 METAIIlAPHlISIS 10 P ~STEA 0l::;;~~l I I I I 2 3 4 5 6 7 2 IfOJCED METAMORPHOSIS SAI1PLE WEEK I • • I I I I o 1554 1215 lin 922 469 2119 ISS I 2 345 6 1 NUI1BER OF LARVAl SAMPLE WEEK • TOTAU. Y METAIllJIIIHlISED o PARTlAU.Y + TOTALLY METAIllJIIIHlISED • EllPERlMEHT AlS Figure 1. (Left) Dendraster excentricus. Percentage spontaneous metamorphosis in the larval culture during an extended period of competence beginning (Sample Week 1) at the end of 5 weeks after fertilization. Figure 2. (Right) Dendraster excentricus. Mean number out of 10 larvae per bowl that metamorphosed within 24 h of exposure to the inducing substance as a function of time after becoming competent. Vertical bars indicate standard deviation. When ready to metamorphose, as judged by the appearance of well developed spines and tubefeet on the echinus rudiment, a few larvae were placed in a bowl containing adult sand (Highsmith, 1982) to see if they would metamorphose. During the course of the study, it was discovered that a water change with"" 2'hOC colder water would induce metamorphosis within approximately 1 h.