Echinoidea: Clypeasteroida)

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 metamorphosed 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 metamorphosis on the survival and growth of postmetamorphic juveniles has not been investigated. The few studies that have been conducted on postsettlement mortality (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 metamorphosis may have an effect on juveniles. As D. excentricus larvae delay metamorphosis, 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/rudiment region tended to become clearer and less yellow. In view of these observations, 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 competence 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 pseudonana 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. This method was used for the remainder of the work because it produced uniformly rapid results. If the test animals metamorphosed, the first set of experimental animals was stimulated to metamorphose and the amount of time that metamorphosis was delayed in the other larvae in the culture was calculated from this date. To assure that competence to metamorphose was uniform, 50-100 larvae in each culture vessel were examined with a dissecting microscope and fewer than 5% of the larvae did not have well- developed rudiments with spines and tubefeet. Subsequent groups of 60-100 larvae were stimulated to metamorphose at 7-day intervals until the larval cultures were spent due to spontaneous metamorphosis, mortality and failure of the surviving larvae to respond. Newly metamorphosed juveniles were placed in I75-rni custard dishes. Usually 10 juveniles were placed in each dish and a small amount of sand from the sand dollar bed was added. The juveniles' test and total (spine tip to spine tip) diameters were measured weekly for the first 4 weeks and then every other week for 4 weeks with a dissecting microscope at 40 x. Water (unfiltered) was changed once per week. Daily, 2 ml of D. tertio[ecta and 2 ml of T. pseudonana were centrifuged, resuspended, and added to half the culture dishes for each settlement date. The algal cell concentrations were not determined. Juveniles in the other dishes were not fed and had to depend on material in the sand or in the unfiltered water for any external nourishment they may have obtained. The experiments were terminated 8 weeks after settlement. Temperature was measured daily during both the larval and juvenile phases of the study.

RESULTS Dendraster excentricus: Larval Studies. - Larvae gained metamorphic competence 5 weeks after fertilization and those remaining in the culture were still competent 6 weeks later (Figs. 1 and 2). Spontaneous metamorphosis increased gradually during the first 3 weeks of competence, peaked sharply in the fourth week, and then declined to approximately 20% for each of the remaining 3 weeks (Fig. 1). Animals that had attached to the culture vessel and had the rudiment partially everted but still retained the preoral hood and larval arms on the aboral surface were counted as partially metamorphosed because it is possible that some of these individuals could detach and re-enter the water column. The high per- 350 BULLETIN OF MARINE SCIENCE, VOL. 39, NO.2, 1986

Table 1. Dendraster excentricus. Mean ann lengths of larvae I, 3 and 7 weeks after becoming competent to metamorphose. Underscore joins means that are not significantly different (Student- Newman-Keuls test, P < 0.05). A minimum of 10 larvae were measured per ann type per week

Mean arm lengths u.m) Arm type Week 1 Week 3 Week 7 Anterolateral 293 256 201 Preoral 272 257 225 Posterodorsal 327 143 125 Postoral 318 145 144

centage in the fourth week should be interpreted with caution because there was a power outage that resulted in the culture not being stirred for several hours and also in warming of the water. Whether either or both factors had an effect on the number of larvae that metamorphosed is not known. The mean number oflarvae out of 10 per bowl that metamorphosed within 24 h after exposure to the inducing substance was 6.5 the first week but increased to greater than 8.0 per bowl for all remaining weeks (Fig. 2). There is a significant difference among the means (one-way ANOYA, F = 6.76, P = 0.001) and a multiple comparison test indicates that the mean for the first week is different from the others (Student-Newman-Keuls test, P < 0.05) but the means for the last 6 weeks are not significantly different (SNK P > 0.05). The low average in the first week was probably due to variability in the onset of competence. The lack of difference among the last 6 weeks indicates that the larval population had reached a uniform level of competence and maintained the ability to metamorphose during this period. With prolonged competence, the larval arms became shorter (Fig. 3, Table 1). During the first 3 weeks, there was a significant decrease of approximately 200 JLm in the length of the postoral and posterodorsal arms, but no significant change during the remaining 4 weeks. Shortening of the preoral and anterolateral arms was more gradual and occurred over most of the 7-week period. The anterolateral arms became significantly shorter between weeks 1 and 3 and between weeks 3 and 7. The preoral arms were not significantly different between weeks 1 and 3, but did decrease in length significantly between weeks 3 and 7. At both weeks 3 and 7, each of the echinus rudiments examined had 17 interambulacral spines, all of which were larger than the ambulacral spines. The length of the interambulacral spines did not change between the third and seventh weeks (Fig. 3; t-test, t = 0.277, P = 0.78). Although the larvae were preserved in buffered formalin, the rudiment spines for animals preserved in week 1 dissolved and, therefore, could not be measured. Aristotle's lantern was present in the rudiments of both the 3-week and 7-week larvae. This indicates that developmental events continue in the rudiment after competence is reached because Aristotle's lantern is not present usually in larvae that have just reached competence (R. B. Emlet, unpubl. obs.). Juvenile Growth and Mortality. -Growth rates of juveniles from weeks 2 and 4 that were maintained at 20°C with and without sand (Fig. 4) indicate that juveniles grew more rapidly if sand was present (ANOY A, F = 158.4, P = 0.00 1). Growth rates of juveniles from weeks 2 and 4 were not significantly different (ANOY A, F= 3.77, P = 0.516). Because the final test length measurements were not made an equal number of days after metamorphosis, the lengths for week 4 juveniles HIGHSMITH AND EMLET: DELAYED METAMORPHOSIS IN SAND DOLLARS 351

400 IIEIIJlASTEA ~ 000 DENDAASTER 350 LARVAL ARM LEN6THS -= 300 DC 700 JUVENILE ""'"" £ ~••• !---j 250 ~ 600 .=:z: ~ 200 -< t.O z c SOO ••• 150 ~ ... Ul ••• 100 ~ 400 /~-! c SO 2 3 4 5 6 7 o 10 20 30 40 SAMPLE WEEK DAYS AFTER METAMORPHOSIS

D PREORAL <> POSTEROIlORSAl D WEEK 2. SAND <> WEEK ". SAND • AHTEAIIl..JoTERAL • POSTOAAL • WEEK 2. NO SAND • WEEK ". NO SAND X IUJIIEHT SPINES Figure 3. (Left) Dendraster excentricus. Mean length of larval arms and echinus rudiment spines over 6-week period from onset of competence. Vertical bars indicate standard deviation (Table I). Figure 4. (Right) Dendraster excentricus. Mean growth rates of juveniles that metamorphosed 2 weeks and 4 weeks after gaining competence and that were maintained at 20·C in bowls with and without sand. Vertical bars indicate standard deviation. were estimated for day 35 by interpolating growth between days 20 and 41, assuming linear growth during this interval. Statistical analysis was performed on the interpolated data. For all fed treatments, time from metamorphosis to first mortality was inversely correlated with time from onset of competence to metamorphosis (Table 2).

Similarly, time from metamorphosis to 25% mortality (LD25) was inversely correlated with time from start of competence to metamorphosis (Table 2). Time to 25% mortality was estimated by interpolating mortality between days where sur- vivors were counted. Mortality rates were assumed to be linear. In the "starved" treatments, mortality rates ranged from 0 to 12%, much lower than the rates for the corresponding fed treatments (Table 3). Echinarachnius parma: Development. - The development of E. parma was de- scribed in detail by Fewkes (1886) and Gordon (1929). Because Fewkes was not able to rear larval cultures through metamorphosis and temperatures were not reported by either author, we include these notes on the time of development of

Table 2. Dendraster excentricus. Time from metamorphosis to first mortality and 25% mortality

(LD2S) for fedjuveniles from larvae that metamorphosed at weekly intervals after becoming competent. Both mortality measures are inversely correlated with time from competence to metamorphosis (Spear- man rank correlation)

Group (weeks) 1st monality (days) LD" (days)

I 30 41 2 35 57 3 29 36 4 20 3i.5 5 26 30.5 6 22 23 7 20 Correlation: Rs -0.795 -0.97 p 0.Q25 0.01 352 BULLETIN OF MARINE SCIENCE, VOL. 39, NO.2, 1986

Table 3. Dendraster excentricus. Percent mortality of fed and "starved" juveniles (End of the experiment indicated as number of days after metamorphosis)

Group (week) Fed Starved Days

I 75% 12% 64 3 49% 10% 48 6 40% 0% 29

various stages of two E. parma cultures (temperatures given in Table 4): 0 h = fertilization, egg diam. ~ 125 ~m; 3 h = 2-cell, a few 4-cell and 8-cell; 16 h = blastula; 24 h = hatched, start of gastrulation; 44 h = gastrula; 52 h = prism, some early 2-armed larvae; 68 h = 2-armed and 4-armed larvae; 90 h = 4-armed; 6-8 days = start of 3rd pair of arms; 8-9 days = 6-armed; 8-11 days = start of 4th pair of arms; 10-12 days = 8-armed, stomodeal invagination visible, 13-14 days = echinus rudiment begins to develop; 16 days = rudiment small, clear; 18 days = rudiment enlarging, more opaque; 26 days = rudiment prominent; 35 days = spines and tubefeet visible on rudiment; 37-40 days = competent to metamorphose; time from settlement to loss of arm spicules ~ 1 h. As the focus ofthis study was on postmetamorphic events, the information given here is taken from observations made as time permitted and does not represent an attempt to precisely document development rate. In the two cultures that we reared at lO- 11°C (Table 4), the larvae first became competent after 37 days in one culture and 40 days in the other. Gordon (1929) had one culture reach competence in 22 days and another reached competence in the fifth week. Growth rate of the larvae (Fig. 5) was determined by measuring the distance from the posterior end of the pluteus to the tip of the postoral arms (through 6-armed stage) or preoral arms (8-armed stage). In contrast to D. excentricus, the larval arms did not become shorter during the competent period. Temperatures during development up to metamorphosis for the various delay groups are given in Table 4. Juvenile Survival and Growth. -Growth rates for postsettlementjuveniles derived from larval cultures fertilized 27 September (Trial 1) and 8 October (Trial 2) are shown in Figures 6 and 7, respectively. Eight weeks after settlement, the juveniles from the second larval culture were significantly larger than those from the earlier fertilization in both fed (t = 7.78, df = 212, P < 0.0005) and non-fed (t = 6.42, df = 252, P = 0.0003) treatments (Table 5). There was no significant difference in the size of the juveniles immediately following metamorphosis (Table 5; fed:

Table 4. Echinarachnius parma. Temperatures eC) during development of two larval cultures (Trial 1 and Trial 2). N = number of temperature measurements

Trial 1 Trial 2

ii SD N ii SD N

Fertilization to: Competence 10.9 0.4 17 10.7 1.0 28 Competence + 7 days 10.8 0.5 22 10.5 1.1 38 Competence + 14 days 10.8 1.0 36 10.6 1.1 44 Competence + 21 days 10.6 1.0 48 10.6 1.0 53 Competence + 28 days 10.5 1.0 64 HIGHSMITH AND EM LET: DELAYED METAMORPHOSIS IN SAND DOLLARS 353

1000 ECHINARACHNIUS + + 900 LARVAL GROHTH + 800 ~ 3 700 •....:x: 600 t.!) z UJ 500 Y = 281.9 + 20.2X -.J 2 400 R = 0.935 300 200 0 5 10 15 20 25 30 35 40

TIME (DAYS) Figure 5. Echinarachnius parma. Larval growth rate. Measurements were made from the posterior tip of the body to the tip of the postoral arms through the 6-armed stage and to the tip of the preoral arms in 8-armed larvae. Means shown are for 3 to 9 larvae per date (median = 5). t = 1.628, df= 240, 0.06> P> 0.05; unfed: t = 0.25, df= 247, P = 0.4). Mean temperatures during the 8-week growth periods are included in Figures 6 and 7. Feeding techniques were worked out with the first larval culture which, conse- quently, suffered considerable mortality. Feeding was better adjusted for the second larval culture which appeared healthier and experienced less mortality. Per- haps the condition of the larvae prior to metamorphosis affected the growth rate of post settlement juveniles, although other factors, such as genetic variability, could be responsible for the different growth rates. Juveniles in the fed treatments were significantly larger than those in the unfed treatments after 8 weeks (Table 6). Variability in lengths tended to be greater in fed treatments than in nonfed and in delayed than in non-delayed animals (Figs. 6, 7). As in D. excentricus (Table 3), percent mortality was greater in the fed treatments than in the unfed treatments (Wilcoxon matched-pairs, signed ranks test, P < 0.025). Mortality rates were positively correlated with length of delay of settlement in the fed treatments (Spearman rank correlation, rs = 0.911, P < 0.01), but there was no apparent pattern in the unfed treatments (Table 6). As in D. excentricus, most of the juvenile mortalities occurred in just a few bowls. Sixty- two percent of the E. parma juveniles that died did so in just 5 of the 60 bowls. In these cases, the spine tips of the animals became fouled and eventually all or nearly all of the juveniles in the bowl died. Through an oversight, the water was not changed between weeks 6 and 8 for one group of juveniles (Fig. 6B) and the animals in 3 of the 4 bowls died. These mortalities are in addition to the 5 bowls mentioned above. We suspect that the cause of mortality was elevated salinity due to evaporation rather than fouling of the water. Test lengths were compared between juveniles from larvae that metamorphosed at 0 days and juveniles from larvae that metamorphosed at 7, 14, 21, and 28 days 354 BULLETIN OF MARINE SCIENCE, VOL. 39, NO.2. 1986

1.2 B I 1.1 1.2 A 1 i1.1 ~ ~ .9 ~ :J: .8 1; .9 z :;l UJ .7 ...J .B ~ ~ .8 TEMP. X-l0.3C tl.8 •...::; .7 TEMP. ~·10.3C tl.S .6 10 20 ao 40 50 so .5 0 10 20 30 40 50 80 .61 + FED I .58 • UNFED ! .56 .51 :J: i .51 ~ ..s UJ Iii ~ .46 ••••... '"UJ .41 t- .41

10 20 ao 40 50 so 60 DAYS AFTER METAMORPHOSIS DAYS AFTER METAMORPHOSIS

1.2 1.2 _ 1.1 Z 1.1 D ! ,!; i!: :J: ~ .9 '" .9 z ffi UJ ...J .8 ...J .B ...J ~ .7 ~ .7 •...o •...o .8 TEMP. ~·SO.3C tl.0 .6 TEMP. ~-10.6C tl.2 .5 .5 o 10 20 30 40 50 so o 10 20 30 40 50 so .81 .61 + FED • UNfED I .58 i .56 :J: i .51 i .51 UJ ~ .48 •...... J .46 en iii UJ ~ .41 t- .41

BO 40 80

DAYS AFTER METAMORPHOSIS DAYS AFTER METAMORPHOSIS Figure 6. Echinarachnius parma. Mean test length and total length of fed (+) and unfed (.) juveniles from Trial I larvae. A. Juveniles that metamorphosed at beginning of competence. B. Juveniles that metamorphosed 4 days after start of competence. C. Juveniles that metamorphosed 14 days after onset of competence. D. Juveniles that metamorphosed 21 days into competent period. Vertical bars indicate standard deviation (Table 6 for treatment N's). after gaining competence. For juveniles from Trial I larvae, many of the O-day larvae did not actually metamorphose for up to 2 weeks, presumably due to an insufficient settlement cue concentration (the effect of lower temperature water had not been discovered at this time so adult sand was used). However, in one HIGHSMITH AND EMLET: DELAYED METAMORPHOSIS IN SAND DOLLARS 355

l.2 l.2 1.2 1'" I l.l it.! , I , ~ i!O i .9 !I! .9 ~ .9 ~ .9 ~ .9 ~ .B ;/ ;/ ;/ .7 e .7 ~ ~ .7 .9 TE•••• ~-IO.6C .1.2 .6 TEll'. ~-10.6C tI.7

. , 0 .5 • 10203040~BO 10 20 30 .cO 50 80 10 20 30 40 ~ 80 .9' .6' I .58 1.56 i'" .49 e .4'

eo DAYS #.FlER METAMORPHOSIS DAYS AFTER METAMOfFHOSIS 0,\ YS AFTER METAMORPHOSIS

l.2 l.2 1 l.1 I., E I ! i!O .9 i!O .9

~ .8 ~ .B ;/ e .7 ;! .7 TEll'. ~-10.7C t1.3 l'! .8 .B TEMP. X-10."C H.B

.5 .5 o • •• 20 30 40 50 eo .9' .B' I .58 1.58 % i.51 .5' ~ e .4e .tB 0 w .41 .41

60 80

DAYS AFTER METAMORPHOSIS DAYS AFTEA MET4MORPHOSIS Figure 7. Echinarachnius parma. Mean test length and total length of fed (+) and (.) unfed juveniles from Trial 2 larvae. A. Juveniles that metamorphosed at beginning of competent period. B. Juveniles that metamorphosed 7 days after becoming competent. C. Juveniles that metamorphosed 14 days after start of competent period. D. Juveniles that metamorphosed 21 days after start of competent period. E. Juveniles that metamorphosed 28 days after start of competent period. Vertical bars indicate standard deviation (Table 6 for treatment N's). bowl each of the fed and unfed treatments, all 10 larvae metamorphosed on day zero. Therefore, to make valid comparisons with respect to actual length of delay, it was necessary to combine data from these two bowls with the 4-day delay data. This combination was used as the "No Delay" group for Trial I in Table 7. At

Table 5. Echinarachnius parma mean test lengths within 1-2 days after metamorphosis and 8 weeks after metamorphosis for juveniles from 2 different larval cultures (Trials)

Melamorphosis (mm) 8 weeks (mm) it SD N SD N Trial I Fed 0.367 0.026 61 0.492 0.074 74 Unfed 0.370 0.019 64 0.439 0.051 88 Trial 2 Fed 0.372 0.020 181 0.591 0.095 140 Unfed 0.370 0.020 185 0.482 0.050 166 356 BULLETIN OF MARINE SCIENCE, VOL. 39, NO.2, 1986

... vi§:- ... ·0 Zo V

•....""'00v ""'v 00000 "'" "'"

••..•vO0\ 000"'" '" •...."'" ""'-v •.... '0 . '-0 ... r/lo ... r/lo ZC! ZC! o o •....•....00\ ""''

o ... o ... o V

•....""'0N

000"'" '" •....00 v"'"

vi ... ioo vi8 o ·0 o Zo V V

•....v '"*•.... 000""'v •....r-:'" v"'"

;; '1: f-< HIGHSMITH AND EMLET: DELAYED METAMORPHOSIS IN SAND DOLLARS 357

Table 7. Echinarachnius parma. Comparison (t-tests) of test lengths between juyeniles from larvae that metamorphosed with no delay and those from larvae that metamorphosed 7, 14, 21 and 28 days after becoming competent [Statistical tests were made on test lengths shortly after metamorphosis (start) and at end of the experiment (finish)]

Start P Finish· P Tria] ] Fed: No delay ys. ]4 day ]4> 0 0.007 0> ]4 <0.000] Fed: No delay ys. 21 day 21> 0 0.0]2 o > 2] <0.0001 Unfed: No delay Ys. 14 day 14> 0 0.03 0> 14 <0.05 Unfed: No delay ys. 2] day N.S. N.S. Tria] 2 Fed: No delay ys. 7 day 7> 0 0.04 0>7 0.Q2 Fed: No delay ys. 14 day N.S. N.S. Fed: No delay Ys. 2] day N.S. N.S. Unfed: No delay vs. 7 day 7> 0 0.04 7> 0 <0.0002 Unfed: No delay YS. ]4 day N.S. N.S. Unfed: No delay ys. 2] day N.S. o > 2] <0.004 Unfed: No delay vs. 28 day N.S. 0> 28 <0.0001

.•Trial I: 6 weeks after metamorphosis (the a delay animals died due to a maintenance problem before the last measurement). Trial 2: 8 weeksaftermetamorphosis. the finish of the experiment, juveniles in the a-day group were larger than delay groups in 6 cases, were not different from delay groups in 4 cases and were smaller than juveniles in a delay group in one instance (Table 7). Similar comparisons made for test lengths shortly after metamorphosis show that for six cases there was no difference in size between the a-day group and the other groups and that in five cases, the delay groups were even larger than the a-day group (Table 7). Although there were exceptions, these data suggest that juveniles from larvae that metamorphosed soon after gaining competence tended to grow more rapidly than juveniles from larvae that delayed metamorphosis. These differences in growth rate occurred in both fed and unfed treatments.

DISCUSSION Between D. excentricus and E. parma, over 1,000 postlarval juveniles were studied for up to 8 weeks with respect to potential effects of delayed metamorphosis on growth and survival. For the hypothesis that utilization of stored energy reserves by larvae during an extended competent period would reduce juvenile survival and growth rates, we found that: (1) Development of the rudiment continues after competence is reached and, in contrast to Clypeaster rosaceus (Emlet, 1984), the rudiment spines are not resorbed or reduced during delay of metamorphosis. In D. excentricus, the larval arms became significantly shorter during the competent period (Fig. 3). (2) In fed treatments, juveniles from larvae that delayed metamorphosis tended to have lower growth rates than non-delayed juveniles. (3) Fed juveniles from larvae that delayed metamorphosis had higher mortality rates than fed juveniles from larvae that did not delay metamorphosis. (4) Unfed juveniles grew although growth rates were significantly lower than for fed animals. (5) Unfed juveniles, including those from larvae that had been competent for I to 7 weeks before metamorphosing, not only survived but generally had much lower mortality rates than fed juveniles of the same age. In regard to points I and 2, larvae of D. excentricus do not appear to reach a stasis in form or differentiation at competence; the arms become increasingly 358 BULLETIN OF MARINE SCIENCE, VOL. 39, NO.2, 1986

shorter and the rudiment continues to develop, Also, the initial postmetamorphic test length of E, parma juveniles was often significantly larger in individuals that had delayed metamorphosis (Table 7). In D. excentricus, approximately 70% of the ciliated feeding band is located on the larval arms at the time of rudiment formation (McEdward, 1984). The resorption of the larval arms (Fig. 3) indicates a 25-30% reduction in ciliated band length and presumably a commensurate reduction in feeding capability (Strathmann, 1971). Though we did not find a point beyond which larvae were unable to metamorphose during the 6-week period competent larvae were maintained, continued arm length reduction suggests such a possibility. It seems paradoxical that larvae that delay metamorphosis for a few weeks have better developed echinus rudiments and Aristotle's lanterns than larvae that do not delay, but the resulting juveniles have lower growth rates than juveniles from non-delayed larvae. In addition to possible depletion of energy reserves, perhaps Aristotle's lantern is not utilized or at least not critical to feeding in the first several weeks of juvenile life and hence, its state of development has no effect on initial juvenile growth rates. Chia and Burke (1978) found that approximately 7 days were required to complete metamorphosis of the digestive tract. Whether reduced lipid stores would delay completion of the gut is unknown. Both larval and adult sand dollars are apparently capable of utilizing dissolved organic matter (Stephens et al., 1978; De Bergh and Burke, 1983; Davis and Stephens, 1984) and our data for unfed and "starved" juveniles show they can survive or even grow somewhat with little or no particulate food. The higher growth rate of the non-delayed juveniles may have ecological sig- nificance with respect to reduced predation. There are numerous small predators in soft-bottom habitats and rapid growth would reduce the period of time needed to gain a refuge in size. Juvenile D. excentricus have an escape in size from the tanaid, Leptochelia dubia, at about 1.5 mm total length (Highsmith, 1982) and older juvenile D. excentricus may have a potential size refuge from juvenile crabs (Highsmith and Emlet, in prep.). Our findings suggest that delay of metamorphosis may result in juveniles that have a somewhat longer period of susceptibility to these very abundant small predators. During the course of this study, it was found that competent E. parma larvae could be dependably and quickly induced to metamorphose by changing their water with:::: 2IhoCcolder water (see Methods). Although it is possible that handling or salinity differences between the old and new water might trigger metamorphosis, temperature change seems the most likely cause. Spontaneous metamorphosis increased dramatically in chiton larvae transferred from 10-1 2°C to 16°C (Peche- nik, 1984a). In the D. excentricus work reported above (Fig. 1), spontaneous metamorphosis was highest during a week in which the culture experienced a temperature increase (amount undetermined) due to a power failure. Another D. excentricus culture of ours that had been competent for 4 weeks metamorphosed entirely, presumably due to warming, when the seawater bath was unexpectedly turned offby a maintenance crew. Pechenik (1984a) speculated that the response to higher temperatures might be adaptive for intertidal animals as the larvae may come into contact with warmer water near the end of their larval phase. Because E. parma responded to colder water (warmer water was not tested), perhaps larvae will respond to a temperature "shock" in either direction which would be more difficult to interpret from an adaptive standpoint. Perhaps temperature differences between water masses, such as streams entering the sea, are indicators of suitable settlement sites. In the protected bays of the Puget Sound region, D. excentricus tends to occur in intertidal locations with freshwater seeps or small streams (unpubl. obs.), but this species also occurs subtidally along the open Pacific Coast of HIGHSMITH AND EMLET: DELAYED METAMORPHOSIS IN SAND DOLLARS 359 the U.S. (Merrill and Hobson, 1970) and E. parma also occurs from the intertidal to deep water (Stanley and James, 1971). Evidence for larval delay of spontaneous metamorphosis in the field is rare. Hadfield (1978) found that larvae of the enteropneustPtychoderaflava may remain competent in the plankton for as long as 4 to 5 months. On two separate occasions during a field study, Emlet (1985) collected competent larvae of D. excentricus with shortened larval arms, possibly indicating that they had delayed metamorphosis. Laboratory-reared larvae that delay metamorphosis tend to settle less selectively with time (Wilson, 1948; Highsmith, 1982). Although D. excentricus has a specific settlement cue produced by adults (Highsmith, 1982; Burke, 1984), spontaneous metamorphosis reached an asymptote of approximately 20% per week in our larval culture and those that remained unmetamorphosed still responded to the specific cue. Spontaneous metamorphosis by delayed larvae may explain why juveniles occasionally are found at some distance from adult aggre- gations (Cameron and Rumrill, 1982; B. Lemon, pers. comm.). The only published growth rates for postlarval clypeasteroids appear to be those of Gordon (1929) for E. parma and Highsmith (1982) for D. excentricus. Gordon (1929) reported a test length of 1.0 mm and a total length of 1.5 mm for E. parma juveniles 4 months after metamorphosis. Sizes at intermediate times were not given. Sizes of D. excentricus without sand (Fig. 4) and unfed E. parma (Figs. 6 and 7) are similar to those (0.45 mm test length at 60 days) reported by Highsmith (1982) for juvenile D. excentricus from non-delayed larvae that were maintained in beach sand but were not fed other than whatever material was introduced during water (unfiltered) changes. D. excentricus with sand (Fig. 4) and fed E. parma (Figs. 6, 7) tended to have a mean size at a given age approximately 50% greater than that found by Highsmith. However, the D. excentricus with sand were maintained at 20°C, several degrees warmer than ambient seawater temperatures (12-14°C) in Highsmith's study. It is not clear why there was such a difference in growth rates between fed D. excentricus with and without sand (Fig. 4). The sand was washed in fresh,,·~t~i' and presumably did not harbor any food items. Perhaps the sand provided substrate for development of organic films that were consumed by the juveniles or possibly some of the algal food was retained among the sand grains during water changes. In any event, this result suggests that substratum is an important variable that must be controlled for in juvenile growth experiments. Another puzzling result in this study was that even though fed juveniles had higher growth rates than unfed animals, they also had significantly higher mortality rates. The mortalities were random and not associated with any particular feeding event. In E. parma, most of these mortalities occurred between the fourth and eighth weeks and, as stated in the Results section, the majority of mortalities occurred in just a few bowls in which all or nearly all of the juveniles died. This "bowl effect" probably accounts for the mortality of delayed juveniles in the preliminary work mentioned in the introduction. Because of this unusual pattern, it does not appear that mortality was due directly to delay per se, but may indicate that delayed juveniles were more susceptible to some pathogen or destructive agent that, by chance, gets into some bowls during water changes and whose deleterious effect is dependent upon the presence of the algae, algal metabolic products or perhaps the small amount of nutrient medium introduced with the centrifuged and resuspended algae. The fact that this type of mortality occurred in two different species at locations over 2,000 km apart suggests it is a general phenomenon and that large numbers of replicate bowls, including filtered-water controls, should be used in studies on postlarval echinoids. Further, non-fed 360 BULLETIN OF MARINE SCIENCE, VOL. 39, NO.2, 1986 juveniles from delayed larvae survived for 8 weeks with very low mortality rates so the hypothesized juvenile mortality directly attributable to larval utilization of critical energy reserves did not occur. Within fed treatments, however, mortality rates were correlated with delayed metamorphosis so the question remains, "Does delay of metamorphosis result in higher mortality rates in the field?" In summary, juveniles from larvae that did not delay metamorphosis had higher growth rates and, within fed treatments, lower mortality rates than juveniles from larvae that delayed metamorphosis. Juveniles fed unicellular algae had higher growth rates and, surprisingly, higher mortality rates than unfed juveniles. Lower growth rates may result in higher predation rates on the juveniles. The cause of increased mortality in juveniles from larvae that delayed metamorphosis was not determined, but it does not appear to be starvation due to utilization of energy reserves by the larvae during the prolonged competent period.

ACKNOWLEDGMENTS

We are grateful to the Friday Harbor Laboratories, Kasitsna Bay Laboratory and Seward Marine Center for use of facilities. T. Pennington graciously shared a larval culture with us. J. Marks and P. Shoemaker provided invaluable help during the course of this work. We thank R. A. Cameron and anonymous reviewers for helpful comments on the manuscript. Financial support for R.B.E. was provided by NSF grant OCE-8400818 to Dr. R. Strathmann and for R.C.H. by the Institute of Marine Science, University of Alaska-Fairbanks. Transportation expenses for R.C.H. to present this paper at the Larval Biology Workshop, Friday Harbor Laboratories, were provided by an Andrew W. Mellon Professional Travel Grant through the University of Alaska.

LITERATURE CITED

Bayne, B. L. 1965. Growth and the delay of metamorphosis of the larvae of Mytilus edulis (L.). Ophelia 2: 1-47. Birkeland, C., F. Chia and R. R. Strathmann. 1971. Development, substratum selection, delay of metamorphosis and growth in the seastar, Mediaster aequalis Stimpson. Bio!' Bull. 141: 99-108. Burke, R. D. 1984. Pheromonal control of metamorphosis in the Pacific sand dollar, Dendraster excentricus. Science 225: 442-443. Caldwell, J. W. 1972. Development, metamorphosis, and substrate selection of the larvae of the sand dollar, Mellita quinquesperforata (Leske, 1778). Unpub!. M.S. Thesis, University of Florida, Gainesville, Florida. 64 pp. Cameron, R. A. and S. S. Rumrill. 1982. Larval abundance and recruitment of the sand dollar Dendraster excentricus in Monterey Bay, California. Mar. Bio!. 71: 197-202. Chia, F. S. and R. D. Burke. 1978. Echinoderm metamorphosis: fate oflarval structures. Pages 219- 234 in F. S. Chia and M. E. Rice, eds. Settlement and metamorphosis of marine invertebrate larvae. Elsevier, New York. 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, New York. Davis, J. P. and G. C. Stephens. 1984. Uptake of free amino acids by bacteria-free larvae of the sand dollar Dendrasterexcentricus. Am. J. Physio!. 247: R733-R739. De Bergh, M. E. and R. D. Burke. 1983. Uptake of dissolved amino acids by embryos and larvae of Dendraster excentricus (Eschscholtz) (Echinodermata: Echinoidea). Can. J. Zoo!. 61: 349-354. Emlet, R. B. 1984. Facultative planktotrophy in a tropical sea biscuit, Clypeaster rosaceus: advantages of larval feeding. Am. Zoo!. 24: 45A. --. 1985. Functional morphology and ecology oflarvae of c1ypeasteroid echinoderms and other ciliated larvae. Ph.D. Dissertation, University of Washington, Seattle, Washington. 152 pp. Fewkes, J. W. 1886. Preliminary observations on the development of Ophiopholis and Echinarach- nius. Bull. Museum Compo Zoo!. 12: 120-145, plates II-VII. Gordon, I. 1929. V. Skeletal development in Arbacia, Echinarachnius and Leptasterias. Phi!. Trans. R. Soc. London (B) 217: 289-326. Grosberg, R. K. 1981. Competitive ability influences habitat choice in marine invertebrates. Nature 290: 700-702. Hadfield, M. G. 1978. Growth and metamorphosis of planktonic larvae of Ptychoderajlava. Pages HIGHSMITHANDEMLET:DELAYEDMETAMORPHOSISINSANDDOLLARS 361

247-254 in F. S. Chia and M. E. Rice, eds. Settlement and metamorphosis of marine invertebrate larvae. Elsevier, New York. Highsmith, R. C. 1977. Larval substrate selection, metamorphosis and mortality in the sand dollar, Dendraster excentricus. Am. Zool. 17: 935. ---. 1982. Induced settlement and metamorphosis of sand dollar (Dendraster excentricus) larvae in predator-free sites: adult sand dollar beds. Ecology 63: 329-337. Hinegardner, R. T. 1969. Growth and development of the laboratory cultured sea urchin. BioI. Bull. 137: 465-475. Holland, D. L. and G. Walker. 1975. The biochemical composition of the cypris larva of the barnacle Balanus balanoides. J. Cons. Int. Explor. Mer 36: 162-165. ---, R. Tantanasiriwong and P. J. Hannant. 1975. Biochemical composition and energy reserves in the larvae and adults of the four British periwinkles Littorina Iittorea, L. Iittoralis, L. saxatilis and L. neritoides. Mar. BioI. 33: 235-239. Kempf, S. C. 1981. Long-lived larvae of the gastropod Aplysia juliana: do they disperse and metamorphose or just slowly fade away? Mar. Ecol. Prog. Ser. 6: 61-65. Keough, M. J. and B. J. Downes. 1982. Recruitment of marine invertebrates: the role of active larval choices and early mortality. Oecologia (Berl.) 54: 348-352. Lucas, M. I., G. Walker, D. L. Holland and D. J. Crisp. 1979. An energy budget for the free- swimming and metamorphosing larvae of Balanus balanoides (Crustacea: Cirripedia). Mar. BioI. 55: 221-229. Luckenback, M. W. 1984. Settlement and early post-settlement survival in the recruitment of Mulinia lateralis (Bivalvia). Mar. Ecol. Prog. Ser. 17: 245-250. McEdward, L. R. 1984. Morphometric and metabolic analysis of the growth and form of an echino- pluteus. J. Exp. Mar. BioI. Ecol. 82: 259-287. Pechenik, J. A. 1980. Growth and energy balance during the larval lives of three prosobranch gastropods. J. Exp. Mar. BioI. Ecol. 44: 1-28. ---. 1984a. Influence of temperature and temperature shifts on the development of chiton larvae, Mopalia muscosa. Int. J. Invert. Reprod. Dev. 7: 3-12. ---. 1984b. The relationship between temperature, growth rate, and duration of planktonic life for larvae of the gastropod Crepidula fornicata (L.). J. Exp. Mar. BioI. Ecol. 74: 241-257. Stanley, D. J. and N. P. James. 1971. Distribution of Echinarachnius parma (Lamarck) and associated fauna on Sable Island Bank, Southeast Canada. Smithsonian Cont. Earth Sci. 6: 1-24. Stephens, G. C., M. J. Volk, S. H. Wright and P. S. Backlund. 1978. Transepidermal accumulation of naturally occurring amino acids in the sand dollar, Dendraster excentricus. BioI. Bull. 154: 335-347. Strathmann, R. R. 1971. The feeding behavior of planktotrophic echinoderm larvae: mechanisms, regulation, and rates of suspension feeding. J. Exp. Mar. BioI. Ecol. 6: 109-160. --. 1975. Larval feeding in echinoderms. Am. Zool. 15: 717-730. ---. 1978. Larval settlement in echinoderms. Pages 235-246 in F. S. Chia and M. E. Rice, eds. Settlement and metamorphosis of marine invertebrate larvae. Elsevier, New York. ---, T. L. Jahn and J. R. C. Fonseca. 1972. Suspension feeding by marine invertebrate larvae: clearance of particles by ciliated bands of a rotifer, pluteus, and trochophore. BioI. Bull. 142: 505- 519. Thorson, G. 1950. Reproductive and larval ecology of marine bottom invertebrates. BioI. Rev. 25: 1-45. Wilson, D. P. 1948. The relation of the substratum to the metamorphosis of Ophelia larvae. J. Mar. BioI. Ass. U.K. 27: 723-760. Young, C. M. and F. S. Chiao 1984. Microhabitat-associated variability in survival and growth of subtidal solitary ascidians during the first 21 days after settlement. Mar. BioI. 81: 61-68.

DATEDACCEPTED: January 15, 1986.

ADDRESSES:(R.C.H.) Seward Marine Center, Institute of Marine Science, University of Alaska. P.O. Box 730, Seward, Alaska 99664; (R.B.E.) Friday Harbor Laboratories, University of Washington, P.O. Box 459, Friday Harbor, Washington 98250.