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Notice: ©1991 Elsevier B.V. The final published version of this manuscript is available at http://www.sciencedirect.com/science/journal/00448486 and may be cited as: Gustafson, R. G., Creswell, R. L., Jacobsen, T. R., & Vaughan, D. E. (1991). Larval biology and mariculture of the angelwing clam, Cyrtopleura costata. Aquaculture, 95(3-4), 257-279. doi:10.1016/0044-8486(91)90092-L Aquaculture, 95 (1991) 257-279 257 Elsevier Science Publishers B.V .. Amsterdam
Larval biology and mariculture ofthe angelwing clam, Cyrtopleura costata
KG. Gustafson"·l, R.L. Creswell", T.R. Jacobsen" and D.E. Vaughan" "Division olCoastal. Environmental and .iquacultural SCiCIICCS, Harbor Branch Oceanographic Institution, 5600 Old DixlC Highway, Fort Pierce, FL 34946, U,)',I "Department otMarinc and Coastal SCiCIICCS, Rutgers Shellfish Research l.aboratorv. New Jcrscv .tgnrultural Evperimcnt Station, Rutgers University, Port Norris, NJ 08349, USI (Accepted 7 November 1990)
ABSTRACT
Gustafson. R.G .. Creswell, R.L. Jacobsen. T.R. and Vaughan. D.E .. 1991. Larval biology and mari culture of the angclwing clam. Cvrtoplcura costata. Aquaculture, 95: 25 7~2 79.
The deep-burrowing angclwing clam. Cvrtoplcura costata (Family Pholadidac ). occurs in shallow water from Massachusetts. USA. to Brazil and has been a commercially harvested food product in Cuba and Puerto Rico. This study examines its potential for commercial aquaculture development. The combined effects of salinity and temperature on survival and shell growth to metamorphosis of angclwing larvae were studied using a 5 X 5 factorial design: salinities ranged from 15 to 35%0 S. in 5OftlO S intervals. and temperatures ranged from 15 to 35C in 5 C intervals. Greatest larval shell growth occurred at 30"C and 20OftlO S over the first 8 days and at 30"C and 25°1<., S over the entire 16 day larval period. Substantial survival (at least 70% of maximal ) occurred at all temperature-salinity combinations below 30'C and 35°1<., S over the first 8 days: and at 15.20 and 25'T combined with all salinities. as well as at 3(YC combined with 20°1<., Sand 25°l INTRODUCTION Indigenous bivalve populations of the family Pholadidae are harvested for food in various parts of the world, although none are cultured on a commer cial scale. The early Romans used the piddock, Pholas dactylus L., as food (Turner, 1954) and this species is still gathered for human consumption on the Normandy coast (Rogers, 1951), in Brittany, in the Channel Islands 'Current address: Institute of Marine and Coastal Sciences. Rutgers Shellfish Research Labo ratory. New Jersey Agricultural Experiment Station. Rutgers University. P.O. Box 687. Port Norris. NJ 08349. USA. 0044-8486/91/$03.50 C<) 1991 - Elsevier Science Publishers B.V. 258 R.G. CiUSTAFSON ET AL. (Davidson, 1980) and in Italy (Palombi and Santarelli, 1969). P. chi/oensis Molina is harvested recreationally in parts of Chile (Turner, 1954; P. Chan ley, pers. comm., 1984), whereas P. (Monothyra) orientalis Gmelin is sold either fresh or dried in markets in Thailand (hoy pim), Malaysia (siput selat batu), Hong Kong (haw chung), and the Philippines (diwal) (Ablan, 1938; Davidson, 1976; Saraya, 1982; Young and Serna, 1982; Tokrisana et aI., 1985; Amornjaruchit, 1988). The angelwing clam Cyrtopleura costata (Linne 1758) was at one time a staple food item in the markets of Havana, Cuba (Arango y Molina, 1878; Rogers, 1951; Turner, 1954) and was commercially har vested in Puerto Rico (Warmke and Abbott, 1975). The deep-burrowing habit and fragile shell of these edible pholadids have to date prevented their use in aquaculture (Alston et al., 1983). Preliminary studies have shown the angelwing clam, Cyrtopleura costata, to be an excellent candidate for aquaculture (Chanley, 1984; Creswell and Schilling, 1985; Kraeuter and Castagna, 1989). Angelwings burrow in sandy mud and are found in shallow water from southern Massachusetts, USA, to Brazil (Turner, 1954; Abbott, 1974; Rios, 1975). Larvae of the angelwing clam have been successfully reared in a commercial-scale hatchery and expe rience with post-larvae and juveniles indicates that angelwings are capable of reaching market size (5-7 cm ) in 4-6 months (Creswell and Schilling, 1985). Tan Tui et al. (1989) have investigated growth and survival of angelwing larvae in response to known rations of various warm-water-adapted phyto plankton strains. In this paper, we examine: growth of angelwing larvae and ju veniles to market size; the combined effects of temperature and salinity on larval angelwing survival and shell growth; and methods used to induce larval angelwing metamorphosis in the hatchery. MATERIALS AND METHODS , /,' Spawning stock was obtained from indigenous populations in the Banana River Lagoon and Indian River Lagoon, Florida, USA. Mature clams were collected by hand, encircled with an elastic rubber band to maintain lateral pressure on the valves, dry-packed in damp seaweed, and transported to the mollusc hatchery of the Division of Coastal, Environmental and Aquacul tural Sciences, Harbor Branch Oceanographic Institution (HBOI), Fort Pierce, Florida. Spawning ofmature angelwings was induced by thermal stim ulation or, rarely, by injection of 0.4 ml of a 2 mM serotonin solution (crys talline 5-hydroxytryptamine, creatine sulfate complex, Sigma Chemical Co. ). Hatchery, nursery andfield techniques In the case of batch cultures, fertilized eggs were washed in 5 Jim filtered and UV-treated seawater on a 20 Jim screen and placed in 500-1 conical fiber glass tanks. After 24-48 h, density of straight-hinge larvae was adjusted to 7 lARVAL BIOLOGY AND MARlCliLTliRE OF THE AN(iELWING CLAM 259 larvae/rnl. Cultures were gently aerated and seawater was exchanged either every day or every other day, at which time mixed rations of unicellular algal cultures [Nannoehloris oculata Butcher, Thalassiosira pseudonana Hassel et Heimdal (Husdedt) (clone 3H), Dunaliella tertiolecta Butcher, and Isochry sis aff. galbana Green (clone T-ISO) ] were provided at a nominal combined initial density of25000 cells/rnl, which was increased to 50 000 cells/rnl after 72 h. Growth rates were determined from a sample of 100 randomly selected larvae taken at each water change. In early experiments (1983), larvae were maintained at ambient salinity and temperature, which fluctuated between 22-32%0 Sand 22-30°e. In later cultures (1987), larvae were maintained at the optimum salinity-temperature combination of25%0 Sand 30°e. In early experiments (1983), chloramphenicol (6 mg /l ) was added to cultures to re tard bacterial growth. When larvae reached pediveliger stage, shallow fiberglass cylinders (20.3 em diameterX 2.5 ern depth) with mesh bottoms and containing graded sili con sand (300-500 zzm ) to a depth of 2 em, were suspended in the conical tanks to collect competent larvae. In other cases, competent larvae were in duced to metamorphose upon exposure for I h to a 10- 3 M solution of epi nephrine dissolved in natural seawater. These treated larvae were then resus pended within upwellers in the conical tanks until metamorphosis was complete. In early experiments ( 1983), newly settled juveniles were stocked in mesh lined, sand-filled "Nestier" trays (56 X 56 X 5 cm ) at a density of4000 clams/ tray (12755 clams/m') and suspended on racks in concrete raceways (2.1 X 0.72 X 0.53 m). Mixed phytoplankton assemblages [lsoehrysis aff. gal bana (clone T-ISO) and unidentified local diatoms], cultured in 50 000 I semi-cylindrical aluminum tanks, were combined with filtered seawater and continuously supplied to the raceways. Juveniles were placed in the field at a mean shell length of 11.6 mm in shallow, sand-filled trays ( 1.2 X2.4 X0.1 m) at a density of 10000 clams/trr', and submerged in < 1.5 m water depth. Shell growth was monitored monthly from a sample of 50 individuals from each tray. In subsequent experiments (1987-1988), newly settled juveniles were placed directly in a sand-filled tray ( 1.2 X 2.4 X 0.1 m) at a density of 100000 clams/m'; supplied with a combination of filtered seawater and cultured al gae iIsochrvsis aff. galbana (clone T-ISO) ] of varying cell density. Survivors were subsequently field-planted, without protection, when 10-12 mm in shell length. Temperature-salinity combination A 5 X 5 factorial design was utilized for temperature-salinity experiments. Salinities ranged from 15 to 35%0Sin 5%0S intervals and temperatures ranged from 15 to 35 0 C in SOC intervals. Two complete temperature-salinity exper- 260 R.G. GUSTAFSON ET AL. iments were conducted on separate spawnings of Cyrtopleura costata. Dupli cate static cultures of 18-h-old, straight-hinge larvae were established at each of the 25 different temperature-salinity combinations in I-I polystyrene beakers at an initial density of 5 larvae/rnl. Salinities were adjusted with NANOpure (Barnstead/Thermolyne Corp.) water or Instant Ocean (Aquar ium Systems, Inc.) sea salts and maintained within ± 1.0%0S of the desired levels. Beakers were immersed to above their water line in constant tempera ture ( ± 1.0 0 C) water baths; cultures were lightly aerated and exchanged with 0.45 /lm filtered natural seawater at 2-day intervals. Larvae were fed a ration of 40000 cells/rnl Isochrysis aff. galhana (clone T-ISO), subsequent to each water change. A 100-ml sample was taken (after thorough agitation) for analysis of sur vival and shell growth rate at 8 and 16 days (the initial day of non-induced natural settlement) after fertilization from each temperature-salinity com bination, preserved in 5% buffered formalin, and examined under a com pound microscope. Growth and survivorship were determined on a random sample of 50 larvae from each culture container. Length (measured to the nearest 5 /lm with a calibrated ocular micrometer) was defined as the maxi mum length of the shell parallel to the hinge line. For survivorship determi nations, both live larvae and empty, hinged shells had an equal chance of being selected. The pair of cultures with the highest mean survival or growth rate for the total ofthe two experiments, on a specified sampling day, was designated the 100% value. All other values were calculated as a relative percentage of these 100% values. Growth, or the mean increase in shell length, was calculated relative to the mean size ofstraight-hinge larvae at the beginning of each ex periment (58.1 zzm, Experiment 1; 57.8/lm, Experiment 2). Data were analysed using response surface methodology, a standard tech nique used to examine the response of an organism over levels of two vari , . (J ables. A standard regression analysis is used in this technique to fit the re sponse surface to biological data (Alderdice, 1972; Kleinbaum and Kupper, 1978; Zar, 1984). The arcsin transformation was performed on all propor tional data to normalize the distribution. Analysis of variance is facilitated by normalizing the distribution of the data (Zar, 1984). After employing transformations of the response or environmental variables, an analysis of variance usually suggests that a significant portion of the total variance is explained by treatment effects (Alderdice, 1972). Transformed tempera ture-salinity combination data were applied to a stepwise multiple-regression computer analysis to produce a series of regression coefficients and analysis of variance tables incorporating the F-statistic to determine the level of sig nificance for the linear and quadratic effects of temperature and salinity and the interactive effect of temperature and salinity on the observed response (statistical tables available on request from the first author) (Zar, 1984). LARVAL BIOl()(;Y AND MARICliLTliRE OF THE ANGELWING CLAM 261 When significant (P= 0.0 I ), these regression coefficients were substituted into a full quadratic equation where all possible values for both independent vari ables (temperature and salinity) were utilized to obtain the predicted re sponse values (relative percentage growth or survival) and plotted on a three dimensional graph and a response surface (Alderdice, 1972). The form ofthe equation was: 2+b 2+b Y=bo+b, T+b25+bJ T 45 s TX5 where Y is the arcsin square root of relative percentage survival or growth; b., is a constant; b 1, b2 , b-; bs. and b, are the regression coefficients; T and 5 are the linear effects of temperature and salinity, respectively; T 2 and 52 are the quadratic effects of temperature and salinity, respectively; and Tx5 is the interaction effect of temperature and salinity. Analysis of variance deter mined the significance of the linear and quadratic effect of temperature and salinity and the interaction effect oftemperature and salinity on the response values (relative growth or survival). This 2nd order polynomial model was chosen to explain the observed effects as it is the model traditionally used to explain data ofthis type (Alderdice, 1972) and provides a basis for compar ison with the studies ofDavis and Calabrese ( 1964 ), Calabrese ( 1969), Hrs Brenko and Calabrese (1969), Cain (1973), Lough, (1973, 1975), Lough and Gonor (1973a,b), Davenport et al. (1975), Lehnberg and Theede ( 1979), Macinnes and Calabrese ( 1979), Siddall ( 1979), and Tettelbach and Rhodes (1981 ). Induction ofmetamorphosis All metamorphic induction experiments were performed [in accordance with the methods of Coon et al. (1985)] with metamorphically competent larvae in plastic tissue culture plates (24-well; Falcon No. 3047) using 0.45 .urn filtered natural seawater at 25%0 Sand 30°e. All treatments in each of three experiments were conducted in triplicate. Approximately 24 larvae were exposed in each tissue culture well to a test compound for either 1 or 18 h, then removed, rinsed and placed in fresh seawater to be scored for metamor phosis under a dissecting microscope after 24-48 h. Metamorphosis was deemed to have occurred if the velum was noticeably resorbed and the disso conch had begun to be secreted. The test compounds were prepared immedi ately before use as lOX stock solutions in glass-distilled water and diluted to the necessary concentration with seawater. Conditions tested included expo sure to specific concentrations of gamma amino-n-butyric acid (GABA), L 3,4-dihydroxyphenylalanine (L-DOPA), (- j-epinephrine, (-)-norepineph rine, 5-hydroxytryptamine (serotonin), KCI, acetylcholine chloride, acetyl choline iodide (all Sigma Chemical Co.), native sand, clean sand, adult an gelwing tissue extract, water filtered by adult angelwings, extract of the red alga Laurentia sp., and seawater. 262 R.G. GUST AFSON ET AL. RESULTS Hatchery-, nursery- andfield-rearing Adult angelwing clams spawned in the hatchery throughout most of the year, with greatest success occurring from September to November and from March to May. Adult clams held in raceways could be conditioned to ripeness during all but the warmest months. Fertilized eggs measured 42.7 .urn ( ± 0.8.um s.d., n =25) in diameter (Fig. I) and subsequent stages exhibited normal spiral clea vage. At 30°C , the first polar body was extruded within 30 min of fertil ization, with the first unequal cleavage occurring by 40-50 min (Fig. 2). Sec ond cleavage was complete within 60 min and an 8-cell stage was produced by 85 min . Polar lobes and a prominent fertilization membrane were not ob served. Swimming gastrulae were obtained by 4.5 h after fertilization and the straight-hinge veliger stage (58-66.um in shell length ) was reached by 18 h. Subsequent development was typical ofpholadids (Fig. 3) , with young shells exhibiting a faint pink coloration at the umbo. Shell growth was rapid until day 12, under the initial growth conditions (1983); however, larvae did not metamorphose until 16 to 21 days following fertilization and at an average shell length of 317 .urn (Fig. 4). Competent pedi veliger larvae possessed an excurrent siphon and two or three gill filaments. In initial experiments (1983 ), clams that metamorphosed in sand-filled collectors remained free of fouling organisms (Fig. 5) , whereas juveniles placed on screens or sand-coated surfaces became fouled by benthic microal gae, bryozoans, and Capitella sp., resulting in high mortalities ( > 80%). Clams in deep sand trays were free of biofouling and grew rapidly, reaching an av- - CD Fig. I. Eggof Cyrtopleura costata spawned in the hat chery. Th e germi nal vesicle (GV) is visible in the center ofthe egg. Scale bar= 10 Ji m. LA RVAL BIOL OGY AND M ARI CULT UR E OF T HE ANGELWING CLAM 263 CD Fig. 2. Unequal two-cell stage of Cyrtopleura costata embryo showing a sma ller (AS) and larger (C D) blastom ere. Scale bar= 10 ,urn. CD Fig. 3. Eight-d ay-old larvae of Cyrtopleura costata. Scale bar= 250 Jim . erage shell length of 5.6 mm ( ± 1.4 mm s.d., n = 50) after 30 days . Sixty days after metamorphosis, clams measuring 11.6 mm in shell length and of 0.2 g live weight (n =50) were removed from the land-based nursery and placed in sand-filled trays in the Indian River Lagoon. Growth rates were high during spring months (Fig. 6) and declined in summer when temperature ap proached 30°C and salinity was high. In the later experiments ( 1987-1988), post-larval angelwings grew rapidly in sand-filled trays in the nursery, obtain- 264 R.G. GU STAFSON ET AL. 400 ~ 350 til c 2 300 .~ -S 250 :r: I- 200 t:> Z w --' 150 --' --'w 100 :r: (j') 50 0 0 2 4 6 8 10 12 14 16 18 20 DAYS Fig. 4. Shell growth of hatchery-rcared Cyrtopleura costata larvae. Vertical bars represent one s.d. abo ut the mean . II = 50. Fig. 5. Early ju venile angelwing, Cyrtopleura costata, showing the extended foot (F) and the inhalent (IS) and exhalent ( ES ) siphons. Scale bar = 100 ,urn . ing an average shell length of II.7 mm (± 1.5 mm s.d., n = 150) in 2 months (Fig. 7). Adult angelwings were unable to rebury themselves if removed from the sediment, therefore it was important to determine when this ability to rebury was lost. Of 150 angelwing juveniles with a mean shell length of 11 .7 mm , 55% had reburied themselves within 5 h, whereas 81% had reburied by 24 h. Angelwing juveniles larger than approximately 15 mm in shell length , were Fig. 6.Weekly mean temp erature (A) and salinity ( B) in the Indi an River Lagoon , Florida during field-growth period depicted in part C. ( C) Gr owth of hatchery-raised angelwing clams , Cyrtopleura costata, in sand-filled trays in the Indian River Lagoon, Florid a, du ring 1983. Error bars repres ent one s.d., II = 50. LARVAL BIOLOGY AND MARIClJLTlJRE OF THE ANGELWINCi CLAM 265 30 A E 25 W a: ....j c( 20 a: w ll.. ::I w.... 15 10 34 B 32 ii So 30 >.... Z ::; 28 c( Ul 26 24 40 C I 30 :I:.... o Z ....w 20 .... W :I: Ul 10 FEBRUARY MARCH APRIL MAY JUNE 266 R.G. GUSTAFSON ET AL. Fig. 7. Sixt y-day-old Cyrtopleura costata (11.7 mm average length) j ust prior to field-planting. Scale bar = 5 mm . TA BLE I Relative percentage surviva l of larvae through 8 days after fertilizatio n under 25 different combina tions ofsalinity and temperature. Percentages are calculated relative to mean value for dup licate cul tures from a total oftwo replicate experiments Salinity Tempera ture ( Oe) (%0) 15.0 20.0 25.0 30.0 35.0 15.0 100.0 100.0 100.0 100.0 8.2 20.0 99.6 98.8 99.7 94.8 3.8 25.0 100.0 100.0 92.4 96.6 0.5 30.0 100.0 98.6 92.9 93.4 0.0 35.0 99.6 98.2 93.1 57.0 0.0 unabl e to rebury themselves and had to be manually placed beneath the sed iment during field-planting. In the later experiments ( 1987-1988), surviving angelwings that had been placed in the field without artificial protection were at a commercially harvestable size of approximately 50 mm in shell length within 5 months after fertilization. Temperature-salinity combinations Larvae sur vived from 1 to 8 days after fertilization in all cultures except those at 35°C and 30 and 35%0 S (Table 1). Maximal survival occurred at 15%0 S at all temperatures except 35°C; at 25%0 Sand 15 and 20°C; and at 30%0 Sand 15°C. However, substantial survival (70% or more) occurred at LARVAL HIOL()(iY AND M ,\RlCliLTI IRE 01 THE AN(;[,LWIN(i CLAM 267 all temperature-salinity combinations below the level of30 0e at 35%0S. Lar val survival was generally lower at high temperature (35°e), regardless of salinity, than at lower temperatures (30 0 e or less). The response surface con tour and three-dimensional plot for survival data from I to 8 days after fertil ization are presented in Fig. 8. The linear and quadratic effects of tempera ture (Tand T 2) were highly significant (P< 0.001 ), whereas effects of salinity (S and S2) and temperature-salinity (TXS) were not significant in account ing for the variations in observed response and are not incorporated in the polynomial expression. Statistical analysis of 8-day survival data produced a polynomial expres sion incorporating all the variables and accounting for 90.0% ofthe variation, where Y is the arcsin square root of relative mean percentage 8-day survival: 2 Y = - 1.37067 +0.28902 (T) - 0.00649 (T ) The maximal mean increase in length over the first 8 days of growth oc curred at 30°C and 20°/()0 S (Table 2). Growth of the larval shell was not observed at all conditions where there was survival (35 0 Cat 15,20 and 25%0 S ). Substantial growth (near 70% of maximal) occurred at temperatures of 20 G e,25° C, and 30° e at salinities of20, 25 and 30%0S. The response surface contour and three-dimensional plot for growth data from I to 8 days after 2 fertilization are presented in Fig. 9. The quadratic effect oftemperature (T ) in the statistical analysis was highly significant (P< 0.00 I ), whereas the qua dratic effect ofsalinity (S2) was significant at the level ofP<0.05. The linear and interactive effects of temperature and salinity (T, S, and TXS) were not significant and are not incorporated in the polynomial expression. Statistical analysis of 8-day growth data produced a polynomial expression incorporat ing all the variables and accounting for 77.2% ofthe variation, where Y is the arcsin square root of relative mean percentage 8-day growth: Y=-5.48710-0.00797(T2)-0.00310 (S2) Maximal survival of larvae through the initial day of metamorphosis (16 days after fertilization) occurred at 25°C and 15%0 S (Table 3). Mortality was complete at 35°C in all salinities tested. The response surface contour and three-dimensional plot ofsurvival data through 16 days post-fertilization are presented in Fig. 10. The linear and quadratic effects of temperature (T and T 2) were again highly significant (P< 0.001) in accounting for varia tions in observed response in survival to metamorphosis, while effects of sal inity (S and S2) and temperature-salinity (TX S) were not significant and are not incorporated in the polynomial expression. Statistical analysis of 16 day survival data produced a polynomial expression incorporating all the variables and accounting for 94.2% of the variation, where Y is the arcsin 268 R.C;. GUSTAFSON ET AL. A 20 20 25 30 35 Temperature Fig. 8. Response-surface contour (A) and three-dimensional diagram (B) for relative percent age survival of Cyrtopleura costata larvae through 8 days after fertilization in response to 25 different temperature-salinity combinations. TABLE 2 Relative percentage growth of larval shell length through 8 days after fertilization under 25 different combinations of salinity and temperature. Percentages arc calculated relative to mean value for du plicate cultures from a total of two replicate experiments LARVAL BIOLOGY AND MARICULHIRE OF THE ANGELWING CLAM 269 A Fig. 9. Response-surface contour (A) and three-dimensional diagram (B) for relative percent age growth of Cyrtopleura costata larvae through 8 days fertilization in response to 25 different temperature-salinity combinations. TABLE 3 Relative percentage survival oflarvae through initial day ofsetting (16 days after fertilization) under 25 different combinations of salinity and temperature. Percentages are calculated relative to mean value for duplicate cultures from a total of two replicate experiments Salinity Temperature ( C) (%0) 15.0 20.0 25.0 30.0 35.0 15.0 99.4 98.9 100.0 38.4 0.0 20.0 96.8 95.4 97.2 72.3 0.0 25.0 96.5 97.8 87.2 86.4 0.0 30.0 99.3 95.8 90.7 35.4 0.0 35.0 98.2 93.6 85.9 19.0 0.0 270 R(;. (;lISTAFSON ET AL. 35 A 30 20 1515 20 25 30 Temperature B ..., 10 ~ 0 .~ ~ "Is ~ •q ~ So ~ Fig. 10. Response-surface contour (A) and three-dimensional diagram (B) for relative per centage survival of Cyrtoplcura costata larvae through initial day ofsetting ( 16 days after fertil ization) in response to 25 different temperature-salinity combinations. TABLE 4 Relative percentage growth of larval shell length through initial day ofsetting ( 16 days after fertiliza tion) under 25 different combinations ofsalinity and temperature. Percentages are calculated relative to mean value for duplicate cultures from a total of two replicate experiments LARVAL BIOLOCiY AND MARICliLTliRE OF THE ANCiELWINCi CLAM 271 square root of relative mean percentage 16-day survival: 2 Y=-0.77947+0.2l432 (T)-0.00558 (T ) The maximal mean increase in length over the first 16 days of growth oc curred at 30°C and 25%0 S (Table 4). Substantial growth (> 70% of maxi mal) occurred only at 30°C combined with salinities of 20,25 and 30%0 S. The response surface contour and three-dimensional plot of growth data for the first 16 days after fertilization are presented in Fig. II. The quadratic 2 effect of temperature (T ) in the statistical analysis was highly significant (P A 35 30 t> :§ 25 OJ Ul 20 B 100 t 0 G ~5 ~ ~ c ~ 50 ~ ~ :.:J 25 ~ "," ..-~ ~ "'"0 ~$~ ....?'a o$'~i;,,> V"o ....~ Fig. 11. Response-surface contour (A) and three-dimensional diagram (B) for relative per centage growth of err/lip/cum cos/a/a larvae through initial day of setting ( 16 days after fertil ization) in response to 25 different temperature-salinity combinations. 272 R.G. GUSTAFSON ET AL. and accounting for 64.2% of the variation, where Y is the arcsin square root 2 of relative mean percentage 16-day growth: Y= -4.81605 -0.00728 (T ). Induction ofmetamorphosis Compounds that induced some degree of metamorphosis ofcompetent lar vae at various concentrations are listed in Table 5. GABA and L-DOPA were either toxic or induced a low percentage oflarvae to metamorphose. Epineph rine and norepinephrine induced a consistently high percentage of larvae to metamorphose at exposures of 10- 3 M for 1 hand 18 h, respectively. An exposure of 1 h in 10- 3 M epinephrine was designated the optimum meta morphic inducer for competent hatchery-reared angelwing larvae. Exposure 2 3 3,10-4 to various concentrations ofKCl (10- and 10- M), serotonin (10- , 5 6 3 4 5 6 10- and 10- M), acetylcholine chloride (10- , 10- , 10- and 10- M), 3 4 5 6 acetylcholine iodide (10- , 10- , 10- and 10- M), native sand, clean sand, adult angelwing tissue extract, water filtered by adult angelwings, extract of the red alga Laurentia sp. (used to stimulate metamorphosis in queen conch TABLE 5 Effects of selected compounds on metamorphosis of Cvrtoplcura costata. Percent metamorphosis scored after 24-h recovery Compound Molar Percent metamorphosis concentration I h 18 h GABA \O-} 0.6 16.5 \0,,4 1.0 9.0 10- 5 1.4 2.5 \0-6 .~I /.,i: L-DOPA \O-} a \0-4 a \0- 5 0.6 \0-6 (- )-Epinephrine \O-} 96.4 a \0-4 17.6 85.0 \0-5 3.8 0.9 \0-6 (- )-Norepinephrine \O-} 24.5 97.4 \0-4 a 10- 5 \0-6 -. no effects. a, toxic. LARVAL BIOLOGY AND MARICliLTlIRE OF THE ANCiELWINCi CLAM 273 Strombus gigas L. ), and seawater controls for I or 18 h did not elicit meta morphosis ofcompetent larvae. DISCUSSION The rapid growth rates of post-larval and juvenile angelwings during the "nursery stage" reported in this study are at the high end of the scale ofclam growth rate as reviewed by Malouf and Bricelj (1989, their table 2.11 ). Al though growth rates vary with season and with size and age of the organisms, a comparison ofangelwing growth with reported rates ofgrowth ofother clam species is worthwhile. Over the first 30 days following metamorphosis, an gelwings grew from 0.3 mm to 5.6 mm or 0.175 mm/day at ambient temper ature. Over the next 30 days, juvenile angelwings grew from 5.6 mm to 11.6 mm or 0.20 I mrrr/ day at ambient temperature (Fig. 6). The reported mean growth of 0.1 90 mrrr/ day over the first 60 days following metamorphosis of juvenile angel wings in this study has been eclipsed in the aquaculture litera ture only twice [Spisula solidissima (Dillwyn), 0.421 mnr/day and Mya ar enaria (L.), 0.255 mm/day (Maloufand Bricelj, 1989)]. Rapid growth rate is characteristic of pholadid clams in the genus Martesia, where shell growth rates of0.383 mm/day over the first 30 days and 0.222 mrrr/day over the first 6 months of life have been recorded (Turner, 1954). The rapid post-larval growth ofCyrtopleura costata indicates that market-size clams (5-7 em) could be grown in Florida in 6 months, more rapidly than reported for any other North American clam species (Maloufand Bricelj, 1989, their table 2.10). Chanley and Andrews ( 1971 ) reported a spawning period of May through September for Cyrtopleura costata from Virginia, while specimens in this study from subtropical Florida were ripe in all but the summer months of June through August. Larval development of C. costata in this study was in agree ment with that originally described for this species by Chanley and Andrews ( 1971 ). As reported by these authors, the early larval shell was pinkish in color around the umbo and dorsal margin, and angelwing pediveligers fre quently possessed an excurrent siphon and several gill filaments. A pink to purple coloration in the larval umbo region has been reported for the pholad ids Parapholas quadrizonata Spenger (Miyazaki, 1935), Zirphaea crispata (L.) (Jorgensen, 1946), Barnea candida (L.) (Zakhvatkina, 1959), and B. truncata Say (Chanley, 1965; Chanley and Andrews, 1971) although this co loration may be related to type of food (Loosanoff and Davis, 1963; Boyle and Turner, 1976). Siphon formation has also been reported in pelagic pho ladid larvae ofPholas sp. (Kandler, 1926), B. candida (Jorgensen, 1946), Z. crispata (Werner, 1939), and Xylophaga atlantica Richards (Culliney and Turner, 1976). Chanley and Andrews (1971) stated that angelwing larvae metamorphosed at a shell length of 300 zzrn, which agrees favorably with our estimate of317 usn. Tan Tui et al. ( 1989) investigated the capacity ofvarious 274 R.G. GUSTAFSON ET AL. microalgal species, alone and in combination, to support survival, growth, and metamorphosis of C. costata larvae. Although some combination diets supported growth and survival of angelwing larvae equal to or slightly better than a unialgal diet ofIsochrysis aff. galbana, no single algal species was found that could surpass the ability of I. aff. galbana (the larval food used in the present study) to promote growth and survival (Tan Tui et al., 1989). Temperature-salinity combinations The combined effects of temperature and salinity on survival and growth provide a better indication of reaction to environmental factors than the ef fect of either factor alone (Kinne, 1964; Bayne, 1983). Analysis of survival and shell length at 8 and 16 days results in a better definition of varying en vironmental requirements over time. More frequent collection of data may reveal beneficial factor combinations, which would be deemed unfavorable or lethal by analysis of data collected only from terminal samples. For in stance, culturing ofangelwing larvae at 30 0 C and a salinity approaching 20%0 S for the first 8 days oflarval life followed by a change to a salinity of 25%0S for the next 8 days will optimize growth, whereas use of the most favorable terminal factor combination for the entire larval period will sacrifice a sub stantial growth benefit during the first 8 days of life (Tables 2 and 4). In addition, analysis of survival and growth at both 8 and 16 days allows for some intriguing ecological speculation. Angelwing larvae become less tolerant of extremes in temperature and salinity with age as reflected in their nar rowed limits for substantial survival and growth. Siddall (1979) reported similar temporal changes in temperature and salinity requirements for tropi cal mussel larvae. The slow growth exhibited by angelwing larvae at high (35%0) and low (15%0) salinity suggests that larvae drifting either too far seaward or too far up the estuary away from the common adult mid-estuarine ~i and inshore habitat (Turner, 1954; Castagna and Chanley, 1973) would not , ' ,',I likely survive to metamorphosis. Castagna and Chanley ( 1973) reported that adult Cyrtopleura costata were not found in nature below 10%0 S, whereas adults were able to acclimate to salinities from 7.5-30%0 S. The failure of larvae exposed to a temperature of 35°C to survive or grow at any salinity beyond day 8 equates well with the fact that spawning ofadult angelwings was virtually non-existent in the hot summer months ofJune-August. Survival of angelwing larvae to both 8 and 16 days post-fertilization was enhanced at low temperatures (15-25 0 C); however, growth rate at these temperatures was negligible compared to that at 30°e. A reduction in rate of larval growth was evident at both low (15%0) and high (35%0) salinities at all temperatures, where survival occurred, in both 8- and 16-day cultures. Similar reductions in growth at low salinities at all temperatures tested have been reported for larvae ofMercenaria mercenaria (L.), Crassostrea virginica (Gmelin) (Davis, 1958; Davis and Calabrese, 1964), Ostrea edulis L. (Davis LARVAL HI Induction ofmetamorphosis The use ofepinephrine to induce metamorphosis in bivalve larvae was first introduced by Coon et al. (1985) for Crassostrea gigas and is now widely used in oyster hatcheries. Although the natural inducer of metamorphosis in Cyrtopleura costata is unknown, l-h exposure ofcompetent larvae to 10- 3 M epinephrine (dissolved in natural seawater) was very effective in inducing metamorphosis. Although this concentration was reported toxic to C. gigas larvae (Coon et aI., 1985), more dilute solutions had minimal effect on an gelwing metamorphosis (Table 5). Later experiments in our hatchery (HBOI) with similar levels of epinephrine demonstrated toxic effects, suggesting the potential of epinephrine interaction with certain seasonally variable compo nents of natural seawater. It is therefore advisable to test various concentra tions of epinephrine with a subset of competent larvae prior to its general application. In summary, the following procedures are suggested for large-scale rearing of Cyrtopleura costata: (I) larvae should be grown at 30°C and 20%0 S for 276 R.G. GUSTAFSON ET AL. the first 8 days and 30°C and 25%0S subsequently, following otherwise stan dard bivalve hatchery procedures; (2) when competent, larvae should be ex posed to a 10- 3 M solution of epinephrine in seawater for 1 h (previously tested for toxic effects); (3) exposed larvae should be allowed to metamor phose in collectors over a fine layer ofsand; (4) post-larvae should be placed in sand-filled trays and supplied with large amounts of cultured algae; and (5) juveniles should be placed in the field prior to their reaching 15 mm in shell length. Further studies are needed to develop methods for: (1) land based, commercial-scale production of planting-size ( > 15 mm) juvenile an gelwings; (2) bringing these juveniles to harvest size in the field; and (3) commercial harvest. Once economical field production and harvesting techniques are devel oped, the angel wing clam has great potential as a cultured product. Substan tial markets exist in the United States for steamed and raw soft-shell, Mya arenaria, and geoduck, Panope generosa (Gould), clams and in Asia for dried pholadid clams (Philippines, Thailand). Investigations into spawning and hatchery techniques for the Thai angelwing, Pho/as (Monothyra) orientalis, are underway (Sahavacharin et al., 1988). The excellent flavor of angelwing clams, the attractive white shell, the very fast growth to market size, and a history of use as a food product in the Caribbean (FAa, 1978) are positive attributes for its adoption by the aquaculture industry. The major drawback to marketing of Cyrtop/eura costata is its relatively short shelf-life, which could be offset by limiting this product to local markets and by adapting to the mar ket for dried clams in the Orient. ACKNOWLEDGEMENTS We thank the staffofthe Division ofCoastal, Environmental and Aquacul tural Sciences, Harbor Branch Oceanographic Institution (HBOI) and par ticularly Mary Schilling for assistance in collecting and culturing angelwings; Drs. S.R. Fegley and R.B. Laughlin, Jr. for assistance with things statistical; and K. Chalermwat for certain Thai references. The comments of K. Chal errnwat, Dr. S.R. Fegley, Dr. Roger Mann, and one anonymous reviewer helped improve early drafts. This is Harbor Branch Oceanographic Institu tion Contribution No. 794, New Jersey Agricultural Experiment Station Pub lication No. D32406-z89 and Contribution No. 90-27 of the Institute of Ma rine and Coastal Sciences, Rutgers University, supported by HBOI and Rutgers University Postdoctoral Fellowships to the first author. REFERENCES Abbott, R.T., 1974. American Seashells, 2nd edn. Van Nostrand Reinhold, New York. NY, 663 pp. LARVAL BIOLOGY AND MARIClILTliRE OF TilE ANGELWINCi CLAM 277 Ablan, G.J., 1938. 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