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Preliminary assessment of a microencapsulated diet for larval culture of the Sydney rock oyster, Saccostrea commercialis (Iredale & Roughley)
Aquaculture, 105 (1992) 345-352 Elsevier Science Publishers B.V., Amsterdam
34s
AQUA 5000I
ccostrea corn Paul C. Southgatea, Peter S. Lee” and John A. Nellb ‘Zoology Department,James Cook Universityof North Queensland,Townsville,Qld., Australia bNSW Agricultureand Fisheries,Brackish Water Fish Culture ResearchStation, SalamanderBay, N.S. W., Australia (Accepted 12 November 1991)
ABSTRACT Southgate, P.C., Lee, P.S. and Nell, J.A., 1992. Preliminary assessment of a microencapsulated diet for larval culture of the Sydney rock oyster, Saccostrea commercialis ( Iredale & Roughley ). Aquaculture, 105: 345-352. A microencapsulated artificial diet was assessed against a standard diet of microalgae in rearing larvae of the Sydney rock oyster, Saccostreacommercialis (Iredale & Roughley ). Unfed control larvae did not develop beyond the straight-hinge stage during the &day experiment, whereas those receiving exogenous nutrients, either microalgae or the artificial diet, reached umbone stage. The artificial diet supported larval growth in excess of 80% that of algae-fed larvae in terms of shell length increment; however, there was no significant difference in ash-free dry weight increment between larvae fed the artificial diet and those fed microalgae. Dissolved yeast extract did not enhance larval growth when used as a supplement to the microcapsules. The relative growth rates obtained are the best to date for oyster larvae reared on an artificial diet. The artificial diet used will serve as a benchmark for future nutritional studies with S. commercialis larvae, allowing further development of artificial diets for this species.
INTRODUCTION
Larval culture of bivalves has traditionally relied on the provision of live microalgae as food. Algal culture is technically demanding and expensive, and may represent between 30% and 50% of hatchery operating costs (Jeffrey and Garland, 1987). Algal culture also requires specialised equipment and facilities which contribute significantly to the capital costs of hatchery construction. Clearly, the development of a suitable artificial diet to replace microalCorrespondence tv: Dr. P.C. Southgate, Zoology Department, James Cook University of North Queensland, Townsville, Qld. 48 11, Australia.
0044~8486/92/$05.00
0 1992 Elsevier Science Publishers B.V. All rights reserved.
346
P.C.SOUTHGATEETAL.
gae would be a major advance in hatchery culture of bivalves, resulting in considerably reduced hatchery establishment and running costs. Several studies have used non-algal diets for Sydney rock oysters (Nell and Wisely, 1983, 1984; Nell, 1985). Although such diets were useful in improving the condition of adult oysters, they were unsuitable for larval rearing. Other studies have investigated the suitability of various types of microencapsulated diets for bivalve larvae as partial replacements or supplements for microalgae (Teshima et al., 1982; Numaguchi and Nell, 199 1) or as a total replacement (Chu et al., 1982, 1987; Southgate, 1988; P.C. Southgate and R.D. Braley, unpubl., 1989). Chu et al. ( 1987 ) achieved metmorphosis of American oyster (Crassustrea virginica) larvae fed on microcapsules; however, growth and survival of these larvae were inferior to that of algae-fed larvae. More recently, a biphasic artificial diet, consisting of microcapsules and dissolved yeast extract, was used successfully for mass-culture of giant clam larvae and yielded almost 4.5 times the number of juveniles at harvest (2.5 months) than a standard algal diet (P.C. Southgate and R.D. Braley, unpubl., 1989). This study was undertaken as a preliminary assessment of the suitability of similar artificial diets for larval culture of the Sydney rock oyster, Saccostrea commercialis( Iredale & Roughley ) . MATERIALS AND METHODS
One-day-old D-stage Saccostreacommercialislarvae were stocked in lightly aerated 8-l culture vessels at a density of 5 larvae ml- ‘. Each vessel contained l-pm filtered sea water with a salinity of 35%0,maintained at 25 + 1OC.Four replicates of each of four dietary treatments were randomly allocated the 16 rearing vessels. The four feeding regimes used for the study were microalgae, microcapsules, microcapsules plus dissolved yeast extract, and unfed controls. The microalgal diet consisted of a mixture of equal quantities (by dry weight) of Isochrysisaff. galbana (clone T-Iso), Chaetoceroscalcitransand Pavlovaiutheri.This algal diet is now standard for larvae of S. commercialis and is highly nutritious (Nell and O’Connor, 199 1) . Microalgae were administered at a feeding rate of 0.237 mg l- ’ and 0.284 mg l- * per day for days 2 and 3, respectively, and 0.475 mg I- ’ per day for the remainder of the feeding trial. Protein-walled microcapsules containing a mixture of whole chicken egg, ovalbumin, homogenised marine fish roes and cod liver oil, were manufactured by a modification of the method described by Langdon ( 1989). The modified method included the use of adipoyl chloride as the cross-linking agent. The encapsulation reaction was terminated by dilution of the reaction mixture with cyclohexane containing 2% w/v crude soy lecithin. The microcapsules were allowed to settle from suspension and the cyclohexane de-
MICROENCAPSULATED
DIET FOR LARVAL SYDNEY
ROCK OYSTER
347
canted. Freshly manufactured microcapsules were taken up in aqueous suspension and vacuum-filtered through 10.pm nylon mesh prior to use. The stock suspension was maintained under refrigeration for the duration of the feeding trial. The dry weight of microencapsulated diet per unit volume of stock suspension was determined from known volumes of the suspension which were oven-dried at 50°C in pre-weighed containers. The proximate composition of the microencapsulated diet was analysed using freeze-dried microcapsules; protein was determined by the biuret method (Layne, 1957) using bovine serum albumin as the standard, carbohydrate was determined by the method of Dubois et al. ( 1956 ) using glucose as the standard, and total lipid content was determined gravimetrically following extraction by the method of Folch et al. ( 1957 ). The diet contained 68.2 g protein, 3.7 g carbohydrate and 16.1 g lipid per 100 g dry diet. The microencapsulated diet was fed at the same dry weight as the algal diet. The yeast extract ( VegemiteT”, Kraft Foods Ltd., Melbourne, Vie. ) was used at 1 mg I- ‘; vitamins in the yeast extract provided the cultures with thiamin (0.1 I lugl- ’ ), riboflavin (0.16 ,ug1-l ) and nicotinamide ( 1.1 pg I- ’ ). Feeding was commenced 2 days after fertilisation. Food was added to the vessels daily and water was changed every 48 h by sieving off the larvae. The trial was terminated after 8 days ( 10 days post-fertilisation), which is sufficient to show growth differences between experimental diets in Sydney rock oyster larvae (Nell and O’Connor, 199 1; Numaguchi and Nell, 199 1) . Larvae from each rearing vessel were concentrated into a small glass beaker containing 50 ml sea water and surviva s estimated from the number of larvae present in triplicate l.O-ml sub-sp pies. A small portion of the larvae were fixed in 4% formalin solution for s uent shell length measurements of 50 larvae. Remaining larvae were retained on 53-pm nylon mesh briefly with distilled water; rapid draining of the water was achiev ing the mesh on tissue paper. Larvae were then oven-dried at 50°C and dry weights were determined on a Cahn 21 Electrobalance from three replicate determinations of approximately 200 larvae. Ash content of larvae was determined as that remaining after heating at 500” C for 4 h (His and Maurer, 1988) and ash-free dry weight (AFDW) was calculated as the difference between larval dry weight and larval ash weight. Larval survival data were arcsin transformed prior to statistical analysis. Homoscedasticity of data was confirmed using Bartlett’s test (Zar, 1984); data were then analysed using nested ANOVA and significantly different means were determined using the Tukey test (Zar, 1984). RESULTS
Larvae which received exogenous nutrients, either algae or the artificial diets, had developed to umbone stage at the termination of the feeding trial.
PC. SOUTHGATE ET AL.
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TABLE I Morphometric parameters and survival (mean + s.d. ) of Saccostrea commerciaiis larvae at the end of the feeding trial’ Diet ALGAE MED5 MED/DN” UNFED
Shell length2 (pm) 130.78 (k6.4) 119.2b (k4.3) 120.0b (kS.Sj 88.7’ (k4.1)
Dry weight3 (ng)
AFDW’ (ng)
Survival (%)
644.9” (k46.1) 498.2b (k33.4) 476.4b ( k 47.2) 278.9’ (k63.6)
131.01a (kl6.8) 99.45=-b (kl8.1) 92.21b ( Z!I20.7) 42.60’ ( f 8.6)
95.9a*b (k2.7) 99.1a ( f 1.3) 89.S0sb (27.2) 85.6b (26.7)
‘Means in columns sharing a common superscript were not significantly different (P> 0.05). ‘Initial mean shell length 73.3 ( + 3.28) pm. ‘Initial mean dry weight 73.46 ( f 16.4) ng. 41nitial mean AFDW 23.86 ( f. 5.3) ng. 5MED= Microencapsulated diet. 6DNI Dissolved nutrients (yeast extract ) .
700
600
d 0 600
rzJ’ a I=-+=
400
JO0
3’ c) -I 21 ii
200
T 55
100
0
Diet Fig. 1. Mean ( f s.d. ) increase in shell length, dry weight and AFDW of Saccosrrea commercialis larvae over a growth trial with four diets. I&gED=Microencapsulateddiet; DN =dissolved nutrients (yeast extract). m, Shell length; 0, dry weight: and m, ash-free dry weight.
In contrast, unfed larvae did not develop beyond the straight-hinge stage. Survival, shell length, total dry weight, and AFDW of larvae at the end of the feeding trial are shown in Table 1. Highest mean percent survival was shown by microcapsule-fed larvae and only this treatment gave significantly higher survival than unfed larvae. There was no significant difference (P> 0.05 ) between other treatment means.
MICROENCAPSULATED DIET FOR LARVAL SYDNEY ROCK OYSTER
349
Mean increases in shell length, total dry weight and AF W over the feeding trial are shown in Fig. 1. The algal diet supported niticantly greater (P-=0.05) increase in shell length and dry weight than either of the artificial diets. Although the algal diet supported a greater mean increase in AF (i.e. organic content) than either of the artificial diets, there was no significant difference (P> 0.05) in the AFDW content of algae-fed larvae and those receiving microcapsules alone. No significant difference (P> 0.05 ) in shell length, dry weight or AFDW was found between the two artificial diets. Unfed larvae showed significantly less (PC 0.05 ) shell and tissue growth than larvae in all other treatments. DISCUSSION
Numaguchi and Nell ( 199 1) studied the potential of lipid-containing microcapsules as a diet for S. commercialis larvae. They found that the microcapsules were a good partial replacement or supplement for microalgae, but were inadequate as a complete diet. This study has shown that microcapsules containing a more suitable diet are able to support high growth rates of S. commercialis larvae. Chu et al. ( 1987) were the first to achieve metamorphosis of oyster (Crassostrea virginicu) larvae fed on a microencapsulated diet; however, microcapsule-fed larvae showed considerably slower growth than algae-fed larvae. For example, in the first of their two experiments, shell length increases of loday-old larvae were approximately 165 pm and 60 pm for algae-fed and microcapsule-fed larvae, respectively. The microcapsules, therefore, supporte approximately 36% of the growth of algae-fed larvae to this point. In the second experiment, the best microencapsulated diet supported a growth rate of approximately 46% that of algae-fed larvae up to 10 days. In this study, 1Oday-old S. commerciaks larvae fed microcapsules and microcapsules with dissolved nutrients had growth rates in shell length of 80% and 81.3% that of algae-fed larvae, respectively. Although shell length is a convenient measure of growth in bivalve larvae, it may be an unreliable indicator of larval tissue growth ( ayne, 1983; Widdows, 1991). This is illustrated here, where larvae fed the crocapsules alone were significantly smaller than algae-fed larvae in terms of shell length and . The microcapsules total dry weight, but did not differ significantly in AF were clearly of high nutritional value for S. commerc It is notable that unfed larvae increased in shell length, dry weight and AFDW during the feeding trial (Table 1, Fig. 1) . Young oyster larvae possess maternally derived lipid reserves which are utilised during early larval growth (Gallager et al., 1986). Catabolism of such reserves may have partially contudy; however, this tributed to shell growth of unfed larvae in the pre ) which must result cannot account for the increase in organic mass (A
350
P.C. SOUTHGATE ET AL.
from uptake of exogenous nutrients. It is likely that bacteria and/or dissolved nutrients present in the culture water were the source of this nutrition. In a similar study, unfed larvae of the American oyster Crassostrea virginica were able to maintain shell growth for up to 25 days (Chu et al., 1987) and the authors suggested that larvae wtilised bacteria as a nutrient source. Although Saccostrea commercialis adults readily take up dissolved organic nutrients (Nell et al., 1983; Nell and Dunkley, 1984) no similar studies have been conducted with S. commercialis larvae. Larvae of other oyster species, namely, Ostrea edulis (Rice et al., 1980; Manahan and Crisp, 1982) and Crassostrea gigas (Fankboner and De Burgh, 1978; Manahan and Crisp, 1982; Stephens and Manahan, 1984; Welbom and Manahan, 1990) have been shown to utilise dissolved organic nutrients. Langdon ( 1980) demonstrated that a dissolved organic supplement to an artificia! diet enhanced growth of Pacific oyster (Crassostrea gigas) larvae. In a similar study, dissolved yeast extract promoted the growth of giant clam ( Tridacna gigas) larvae when used as a supplement to a microencapsulated diet (Southgate, 1990). In contra.st, supplemental yeast extract, used here at the same concentration as Southgate ( 1990), failed to significantly enhance growth of S. commercialis larvae. This may reflect differences in the levels of dissolved nutrients naturally present in sea water or may indicate that the ability of bivalve larvae to utilise dissolved nutrients varies between species. The growth of unfed larvae in this study may reflect substantial leveis of dissolved nutrients already present in the culture water, thus reducing the beneficial effects of supplementary dissolved nutrients. Recent estimates of the cost of hatchery produced microalgae range from S$250 (Walsh et al., 1987) to IJS$322 (PC. Southgate and R.D. Bk?aley, unpubl., 1989) per kg dry weight, with production costs varying according to the site and the scale of production. In contrast, the cost of microcapsules used in this study was approximately $USl59 pen !cgdq! vzight when made at a laboratory scale. This figure assumes the recc+ery by redistillation, and subsequent reuse, of 90% of the cyclohexane used during microcapsule manufacture. A major advantage of microcapsules is that they are made when required and, unlike microalgae, require no additional maintenance. Clearly, replacing microalgae with such a diet would sigr.;k-tcantlyreduce lrc?tcheryoperating costs. The aim of this study was to assess the potenrial of a microencapsulated diet as a replacement for microalgae in hatchery r::aring of S. commercialis larvae. The microcapsules had suitable physical characteristics for larval ingestion, they were digestible and highly nutritious and supported greater relative growth rates than achieved in previous studies with oyster larvae. As such, this study has been a valuable step in the development of an “off the shelf” diet for oyster larvae. Further studies are necessary to determine more thenutritional requirements of Sydney rock oyster larvae, to optimise the
of
MICROENCAPSULATED DIET FOR LARVAL SYDNEY ROCK OYSTER
351
composition of artificial diets, and to establish whether an artificial diet will support growth and development of larvae through metamorphosis. The microencapsulated diet used here will serve as a benchmark for such future studies. ACKNOWLEDGMENTS
This study was funded by the Australian Research Council (Project No. AA8931821 ) and was conducted at the Brackish Water Fish Culture Research Station (BWFCRS), Salamander Bay, New South les. We would like to thank the staff of BWFCRS, in particular r. Lindsay Coard, Cassie Rose and Mr. John Diemar, for their assistance. We are grateful to Associate Professor J.S. Lucas of James Cook University for his comments on an earlier manuscript.
REFERENCES Bayne, B.L., 1983. Physiological ecology of marine molluscan larvae. In: N.H. Verdonk and J.A.M. Van den Biggelaar (Editors), The Mollusca, Vol. 3, Development. Academic Press, London, pp. 299-343. Chu, F.-L.E., Webb, K.L., Hepworth, D. and Roberts, M., 1982. The acceptability and digestibility of inicrocapscles by larvae of Crassostrea virginicu. J. Shellfish Res., 2: 29-34. Chu, F.-L.E., Webb, K., Hepworth, D. and Casey, B.B., 1987. Metamorphosis of larvae of Crassosrrea virginicu fed microencapsulated diets. Aquaculture, 64: 185- 197. Dubois, M., Gillies, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F., 1956. Calorimetric method for the determination of sugars and related substances. Anal. Chem., 28: 350-356. Fankboner, P.V. and De Burgh, M.E., 1978. Comparative rates of dissolved organic carbon accumulation by juveniles and pediveligers of the Japanese oyster Crassoutreu gigm Thunberg. Aquaculture, 13: 205-2 12. Folch, J., Lees, M. and Sloane-Stanlev, G.H., 1957. A simple method for the isolation and purification of total lipid from animal tissues. J. Biol. Chem., 226: 497-509. Gallager, S.M., Mann, R. and Sasaki, G.C., 1986. Lipid as an index of growth and viability of three species of bivalve larvae. Aquaculture, 56: 8 1- 103. His, E. and Maurer, D., 1988. Shell growth and gross biochemical composition ofoyster larvae (Crussostreu gigus) in the field. Aquaculture, 69: 185-l 94. Jeffrey, S.W. and Garland, C.D., 1987. Mass culture of microalgae essential for mariculture hatcheries. Aust. Fish., 46( 5 ): 24- 18. Langdon, C.J., 1980. The nutrition of Crussostreu gigus (Thunberg) larvae and spat fed on artificial diets. Ph.D. thesis, University College North Wales, Bangor, WK, 209 pp. Langdon, C.J., 1989. Preparation and evaluation of protein microcapsules for a marine suspension feeder, the Pacific oyster Crmostreu gigus. Mar. Biol., 102: 2 17-224. Layne, E., 1957. Spectrophotometric and turbidimetric methods for measuring protein. In: S.P. Colowick and N.O. Kaplan (Editors), Methods in Enzymology, Volume 3. Academic Press, New York,NY, pp. 447-466. Manahan, D.T. and Crisp, D.J., 1982. The role of dissolved organic materials in the nutrition of pelagic larvae: amino acid uptake by bivalve veligers. Am. Zool., 22: 635.-646.
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Nell, J.A., 1985. Comparison of some single cell proteins in the diet of the Sydney rock oyster
(Saccostreu commercialis). Prog. Fish-Cult., 47: I I O-l 13. Nell, J.A. and Dunkley, P.R., 1984. Effects of temperature, nutritional factors and salinity On the uptake of L-methionine by the Sydney rock oyster Succostreu commercialis.Mar. Biol., 80: 335-339. Nell, J.A. and O’Connor, W.A., 199 I. The evaluation of fresh algae and stored algal concentrates as a food source for Sydney rock oyster Succostreucommercialis(Iredale & Roughley ) larvae. Aquaculture, 99: 277-284. Nell, J.A. and Wisely, B., 1983. Experimental feeding of Sydney rock oysters (Succostreucommerciufis). II. Protein supplementation of artificial diets for adult oysters. Aquaculture, 32: l-9. Nell, J.A. and Wisely, B., 1984. Experimental feeding of Sydney rock oysters (S’uccostreucommerciulis). 111.Food concentration and fattening procedures. Aquaculture, 37: 197-208. Nell, J.A., Skeel, M.E. and Dunkley, P., 1983. Uptake of some dissolved organic nutrients by the Sydney rock oyster Succosfreucommercialis.Mar. Biol., 74: 3 13-3 18. Numaguchi, K. and Nell, J.A., 199 1. Effects of gelatin acacia microcapsules and algal meal supplementation of algal diets on growth rates of Sydney roC;t oyster Succosrreucommercialis (Iredale & Roughley ) larvae. Aquaculture, 94: 65-78. Rice, M.A., Wallis, K. and Stephens, G.C., 1980. Influx and net flux of amino acids into larval and juvenile flat oysters, Osrreuedulis (L.). J. Exp. Mar. Biol. Ecol., 48: 5 l-59. Southgate, P.C., 1988. Use of microencapsulated diets in the culture of giant clam larvae. In: J.W. Copland and J.S. Lucas (Editors), Giant Clams in Asia and the Pacific. ACIAR, Canberra, Australia, pp. I 55- 160. Southgate, PC., 1990. Aspects of the nutrition of giant clam larvae with emphasis on the development of a microencapsulated artificial diet. Ph.D. thesis, James Cook University of North Queensland, Australia, 282 pp. Stephens, G.C. and Manahan, D.T., 1984. Technical advances in the study of nutrition of marine molluscs. Aquaculture, 39: I 55- 164. Teshima, S., Kanazawa, A. and Sakamoto, M., 1982. Micro-partieulate diets for the larvae of aqualic animals. Mini Rev. Data File Fish. Res., Kagoshima Univ., 2: 67-86. Walsh, BUT.,Withstandly, C,A., Kraus, R.A. and Petrovits, E.J., 1987. Mass culture of selected marine microalgae for the nursery production of bivalve seed. J. Shellfish Res., 6: 7 l-77. Welborn, J.R. and Manahan, D.T., 1990. Direct measurement of sugar uptake from sea water into molluscan larvae. Mae. Ecol. Prog. Ser., 65: 233-239. Widdows, J., 199 I. Physiological ecology of mussel larvae. Aquaculture, 94: 147- 163. Zar, J.H., 1984. Biostatistical Analysis. Prentice-Hall International, Inc., London, 7 18 pp.
34s
AQUA 5000I
ccostrea corn Paul C. Southgatea, Peter S. Lee” and John A. Nellb ‘Zoology Department,James Cook Universityof North Queensland,Townsville,Qld., Australia bNSW Agricultureand Fisheries,Brackish Water Fish Culture ResearchStation, SalamanderBay, N.S. W., Australia (Accepted 12 November 1991)
ABSTRACT Southgate, P.C., Lee, P.S. and Nell, J.A., 1992. Preliminary assessment of a microencapsulated diet for larval culture of the Sydney rock oyster, Saccostrea commercialis ( Iredale & Roughley ). Aquaculture, 105: 345-352. A microencapsulated artificial diet was assessed against a standard diet of microalgae in rearing larvae of the Sydney rock oyster, Saccostreacommercialis (Iredale & Roughley ). Unfed control larvae did not develop beyond the straight-hinge stage during the &day experiment, whereas those receiving exogenous nutrients, either microalgae or the artificial diet, reached umbone stage. The artificial diet supported larval growth in excess of 80% that of algae-fed larvae in terms of shell length increment; however, there was no significant difference in ash-free dry weight increment between larvae fed the artificial diet and those fed microalgae. Dissolved yeast extract did not enhance larval growth when used as a supplement to the microcapsules. The relative growth rates obtained are the best to date for oyster larvae reared on an artificial diet. The artificial diet used will serve as a benchmark for future nutritional studies with S. commercialis larvae, allowing further development of artificial diets for this species.
INTRODUCTION
Larval culture of bivalves has traditionally relied on the provision of live microalgae as food. Algal culture is technically demanding and expensive, and may represent between 30% and 50% of hatchery operating costs (Jeffrey and Garland, 1987). Algal culture also requires specialised equipment and facilities which contribute significantly to the capital costs of hatchery construction. Clearly, the development of a suitable artificial diet to replace microalCorrespondence tv: Dr. P.C. Southgate, Zoology Department, James Cook University of North Queensland, Townsville, Qld. 48 11, Australia.
0044~8486/92/$05.00
0 1992 Elsevier Science Publishers B.V. All rights reserved.
346
P.C.SOUTHGATEETAL.
gae would be a major advance in hatchery culture of bivalves, resulting in considerably reduced hatchery establishment and running costs. Several studies have used non-algal diets for Sydney rock oysters (Nell and Wisely, 1983, 1984; Nell, 1985). Although such diets were useful in improving the condition of adult oysters, they were unsuitable for larval rearing. Other studies have investigated the suitability of various types of microencapsulated diets for bivalve larvae as partial replacements or supplements for microalgae (Teshima et al., 1982; Numaguchi and Nell, 199 1) or as a total replacement (Chu et al., 1982, 1987; Southgate, 1988; P.C. Southgate and R.D. Braley, unpubl., 1989). Chu et al. ( 1987 ) achieved metmorphosis of American oyster (Crassustrea virginica) larvae fed on microcapsules; however, growth and survival of these larvae were inferior to that of algae-fed larvae. More recently, a biphasic artificial diet, consisting of microcapsules and dissolved yeast extract, was used successfully for mass-culture of giant clam larvae and yielded almost 4.5 times the number of juveniles at harvest (2.5 months) than a standard algal diet (P.C. Southgate and R.D. Braley, unpubl., 1989). This study was undertaken as a preliminary assessment of the suitability of similar artificial diets for larval culture of the Sydney rock oyster, Saccostrea commercialis( Iredale & Roughley ) . MATERIALS AND METHODS
One-day-old D-stage Saccostreacommercialislarvae were stocked in lightly aerated 8-l culture vessels at a density of 5 larvae ml- ‘. Each vessel contained l-pm filtered sea water with a salinity of 35%0,maintained at 25 + 1OC.Four replicates of each of four dietary treatments were randomly allocated the 16 rearing vessels. The four feeding regimes used for the study were microalgae, microcapsules, microcapsules plus dissolved yeast extract, and unfed controls. The microalgal diet consisted of a mixture of equal quantities (by dry weight) of Isochrysisaff. galbana (clone T-Iso), Chaetoceroscalcitransand Pavlovaiutheri.This algal diet is now standard for larvae of S. commercialis and is highly nutritious (Nell and O’Connor, 199 1) . Microalgae were administered at a feeding rate of 0.237 mg l- ’ and 0.284 mg l- * per day for days 2 and 3, respectively, and 0.475 mg I- ’ per day for the remainder of the feeding trial. Protein-walled microcapsules containing a mixture of whole chicken egg, ovalbumin, homogenised marine fish roes and cod liver oil, were manufactured by a modification of the method described by Langdon ( 1989). The modified method included the use of adipoyl chloride as the cross-linking agent. The encapsulation reaction was terminated by dilution of the reaction mixture with cyclohexane containing 2% w/v crude soy lecithin. The microcapsules were allowed to settle from suspension and the cyclohexane de-
MICROENCAPSULATED
DIET FOR LARVAL SYDNEY
ROCK OYSTER
347
canted. Freshly manufactured microcapsules were taken up in aqueous suspension and vacuum-filtered through 10.pm nylon mesh prior to use. The stock suspension was maintained under refrigeration for the duration of the feeding trial. The dry weight of microencapsulated diet per unit volume of stock suspension was determined from known volumes of the suspension which were oven-dried at 50°C in pre-weighed containers. The proximate composition of the microencapsulated diet was analysed using freeze-dried microcapsules; protein was determined by the biuret method (Layne, 1957) using bovine serum albumin as the standard, carbohydrate was determined by the method of Dubois et al. ( 1956 ) using glucose as the standard, and total lipid content was determined gravimetrically following extraction by the method of Folch et al. ( 1957 ). The diet contained 68.2 g protein, 3.7 g carbohydrate and 16.1 g lipid per 100 g dry diet. The microencapsulated diet was fed at the same dry weight as the algal diet. The yeast extract ( VegemiteT”, Kraft Foods Ltd., Melbourne, Vie. ) was used at 1 mg I- ‘; vitamins in the yeast extract provided the cultures with thiamin (0.1 I lugl- ’ ), riboflavin (0.16 ,ug1-l ) and nicotinamide ( 1.1 pg I- ’ ). Feeding was commenced 2 days after fertilisation. Food was added to the vessels daily and water was changed every 48 h by sieving off the larvae. The trial was terminated after 8 days ( 10 days post-fertilisation), which is sufficient to show growth differences between experimental diets in Sydney rock oyster larvae (Nell and O’Connor, 199 1; Numaguchi and Nell, 199 1) . Larvae from each rearing vessel were concentrated into a small glass beaker containing 50 ml sea water and surviva s estimated from the number of larvae present in triplicate l.O-ml sub-sp pies. A small portion of the larvae were fixed in 4% formalin solution for s uent shell length measurements of 50 larvae. Remaining larvae were retained on 53-pm nylon mesh briefly with distilled water; rapid draining of the water was achiev ing the mesh on tissue paper. Larvae were then oven-dried at 50°C and dry weights were determined on a Cahn 21 Electrobalance from three replicate determinations of approximately 200 larvae. Ash content of larvae was determined as that remaining after heating at 500” C for 4 h (His and Maurer, 1988) and ash-free dry weight (AFDW) was calculated as the difference between larval dry weight and larval ash weight. Larval survival data were arcsin transformed prior to statistical analysis. Homoscedasticity of data was confirmed using Bartlett’s test (Zar, 1984); data were then analysed using nested ANOVA and significantly different means were determined using the Tukey test (Zar, 1984). RESULTS
Larvae which received exogenous nutrients, either algae or the artificial diets, had developed to umbone stage at the termination of the feeding trial.
PC. SOUTHGATE ET AL.
348
TABLE I Morphometric parameters and survival (mean + s.d. ) of Saccostrea commerciaiis larvae at the end of the feeding trial’ Diet ALGAE MED5 MED/DN” UNFED
Shell length2 (pm) 130.78 (k6.4) 119.2b (k4.3) 120.0b (kS.Sj 88.7’ (k4.1)
Dry weight3 (ng)
AFDW’ (ng)
Survival (%)
644.9” (k46.1) 498.2b (k33.4) 476.4b ( k 47.2) 278.9’ (k63.6)
131.01a (kl6.8) 99.45=-b (kl8.1) 92.21b ( Z!I20.7) 42.60’ ( f 8.6)
95.9a*b (k2.7) 99.1a ( f 1.3) 89.S0sb (27.2) 85.6b (26.7)
‘Means in columns sharing a common superscript were not significantly different (P> 0.05). ‘Initial mean shell length 73.3 ( + 3.28) pm. ‘Initial mean dry weight 73.46 ( f 16.4) ng. 41nitial mean AFDW 23.86 ( f. 5.3) ng. 5MED= Microencapsulated diet. 6DNI Dissolved nutrients (yeast extract ) .
700
600
d 0 600
rzJ’ a I=-+=
400
JO0
3’ c) -I 21 ii
200
T 55
100
0
Diet Fig. 1. Mean ( f s.d. ) increase in shell length, dry weight and AFDW of Saccosrrea commercialis larvae over a growth trial with four diets. I&gED=Microencapsulateddiet; DN =dissolved nutrients (yeast extract). m, Shell length; 0, dry weight: and m, ash-free dry weight.
In contrast, unfed larvae did not develop beyond the straight-hinge stage. Survival, shell length, total dry weight, and AFDW of larvae at the end of the feeding trial are shown in Table 1. Highest mean percent survival was shown by microcapsule-fed larvae and only this treatment gave significantly higher survival than unfed larvae. There was no significant difference (P> 0.05 ) between other treatment means.
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Mean increases in shell length, total dry weight and AF W over the feeding trial are shown in Fig. 1. The algal diet supported niticantly greater (P-=0.05) increase in shell length and dry weight than either of the artificial diets. Although the algal diet supported a greater mean increase in AF (i.e. organic content) than either of the artificial diets, there was no significant difference (P> 0.05) in the AFDW content of algae-fed larvae and those receiving microcapsules alone. No significant difference (P> 0.05 ) in shell length, dry weight or AFDW was found between the two artificial diets. Unfed larvae showed significantly less (PC 0.05 ) shell and tissue growth than larvae in all other treatments. DISCUSSION
Numaguchi and Nell ( 199 1) studied the potential of lipid-containing microcapsules as a diet for S. commercialis larvae. They found that the microcapsules were a good partial replacement or supplement for microalgae, but were inadequate as a complete diet. This study has shown that microcapsules containing a more suitable diet are able to support high growth rates of S. commercialis larvae. Chu et al. ( 1987) were the first to achieve metamorphosis of oyster (Crassostrea virginicu) larvae fed on a microencapsulated diet; however, microcapsule-fed larvae showed considerably slower growth than algae-fed larvae. For example, in the first of their two experiments, shell length increases of loday-old larvae were approximately 165 pm and 60 pm for algae-fed and microcapsule-fed larvae, respectively. The microcapsules, therefore, supporte approximately 36% of the growth of algae-fed larvae to this point. In the second experiment, the best microencapsulated diet supported a growth rate of approximately 46% that of algae-fed larvae up to 10 days. In this study, 1Oday-old S. commerciaks larvae fed microcapsules and microcapsules with dissolved nutrients had growth rates in shell length of 80% and 81.3% that of algae-fed larvae, respectively. Although shell length is a convenient measure of growth in bivalve larvae, it may be an unreliable indicator of larval tissue growth ( ayne, 1983; Widdows, 1991). This is illustrated here, where larvae fed the crocapsules alone were significantly smaller than algae-fed larvae in terms of shell length and . The microcapsules total dry weight, but did not differ significantly in AF were clearly of high nutritional value for S. commerc It is notable that unfed larvae increased in shell length, dry weight and AFDW during the feeding trial (Table 1, Fig. 1) . Young oyster larvae possess maternally derived lipid reserves which are utilised during early larval growth (Gallager et al., 1986). Catabolism of such reserves may have partially contudy; however, this tributed to shell growth of unfed larvae in the pre ) which must result cannot account for the increase in organic mass (A
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from uptake of exogenous nutrients. It is likely that bacteria and/or dissolved nutrients present in the culture water were the source of this nutrition. In a similar study, unfed larvae of the American oyster Crassostrea virginica were able to maintain shell growth for up to 25 days (Chu et al., 1987) and the authors suggested that larvae wtilised bacteria as a nutrient source. Although Saccostrea commercialis adults readily take up dissolved organic nutrients (Nell et al., 1983; Nell and Dunkley, 1984) no similar studies have been conducted with S. commercialis larvae. Larvae of other oyster species, namely, Ostrea edulis (Rice et al., 1980; Manahan and Crisp, 1982) and Crassostrea gigas (Fankboner and De Burgh, 1978; Manahan and Crisp, 1982; Stephens and Manahan, 1984; Welbom and Manahan, 1990) have been shown to utilise dissolved organic nutrients. Langdon ( 1980) demonstrated that a dissolved organic supplement to an artificia! diet enhanced growth of Pacific oyster (Crassostrea gigas) larvae. In a similar study, dissolved yeast extract promoted the growth of giant clam ( Tridacna gigas) larvae when used as a supplement to a microencapsulated diet (Southgate, 1990). In contra.st, supplemental yeast extract, used here at the same concentration as Southgate ( 1990), failed to significantly enhance growth of S. commercialis larvae. This may reflect differences in the levels of dissolved nutrients naturally present in sea water or may indicate that the ability of bivalve larvae to utilise dissolved nutrients varies between species. The growth of unfed larvae in this study may reflect substantial leveis of dissolved nutrients already present in the culture water, thus reducing the beneficial effects of supplementary dissolved nutrients. Recent estimates of the cost of hatchery produced microalgae range from S$250 (Walsh et al., 1987) to IJS$322 (PC. Southgate and R.D. Bk?aley, unpubl., 1989) per kg dry weight, with production costs varying according to the site and the scale of production. In contrast, the cost of microcapsules used in this study was approximately $USl59 pen !cgdq! vzight when made at a laboratory scale. This figure assumes the recc+ery by redistillation, and subsequent reuse, of 90% of the cyclohexane used during microcapsule manufacture. A major advantage of microcapsules is that they are made when required and, unlike microalgae, require no additional maintenance. Clearly, replacing microalgae with such a diet would sigr.;k-tcantlyreduce lrc?tcheryoperating costs. The aim of this study was to assess the potenrial of a microencapsulated diet as a replacement for microalgae in hatchery r::aring of S. commercialis larvae. The microcapsules had suitable physical characteristics for larval ingestion, they were digestible and highly nutritious and supported greater relative growth rates than achieved in previous studies with oyster larvae. As such, this study has been a valuable step in the development of an “off the shelf” diet for oyster larvae. Further studies are necessary to determine more thenutritional requirements of Sydney rock oyster larvae, to optimise the
of
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composition of artificial diets, and to establish whether an artificial diet will support growth and development of larvae through metamorphosis. The microencapsulated diet used here will serve as a benchmark for such future studies. ACKNOWLEDGMENTS
This study was funded by the Australian Research Council (Project No. AA8931821 ) and was conducted at the Brackish Water Fish Culture Research Station (BWFCRS), Salamander Bay, New South les. We would like to thank the staff of BWFCRS, in particular r. Lindsay Coard, Cassie Rose and Mr. John Diemar, for their assistance. We are grateful to Associate Professor J.S. Lucas of James Cook University for his comments on an earlier manuscript.
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Nell, J.A., 1985. Comparison of some single cell proteins in the diet of the Sydney rock oyster
(Saccostreu commercialis). Prog. Fish-Cult., 47: I I O-l 13. Nell, J.A. and Dunkley, P.R., 1984. Effects of temperature, nutritional factors and salinity On the uptake of L-methionine by the Sydney rock oyster Succostreu commercialis.Mar. Biol., 80: 335-339. Nell, J.A. and O’Connor, W.A., 199 I. The evaluation of fresh algae and stored algal concentrates as a food source for Sydney rock oyster Succostreucommercialis(Iredale & Roughley ) larvae. Aquaculture, 99: 277-284. Nell, J.A. and Wisely, B., 1983. Experimental feeding of Sydney rock oysters (Succostreucommerciufis). II. Protein supplementation of artificial diets for adult oysters. Aquaculture, 32: l-9. Nell, J.A. and Wisely, B., 1984. Experimental feeding of Sydney rock oysters (S’uccostreucommerciulis). 111.Food concentration and fattening procedures. Aquaculture, 37: 197-208. Nell, J.A., Skeel, M.E. and Dunkley, P., 1983. Uptake of some dissolved organic nutrients by the Sydney rock oyster Succosfreucommercialis.Mar. Biol., 74: 3 13-3 18. Numaguchi, K. and Nell, J.A., 199 1. Effects of gelatin acacia microcapsules and algal meal supplementation of algal diets on growth rates of Sydney roC;t oyster Succosrreucommercialis (Iredale & Roughley ) larvae. Aquaculture, 94: 65-78. Rice, M.A., Wallis, K. and Stephens, G.C., 1980. Influx and net flux of amino acids into larval and juvenile flat oysters, Osrreuedulis (L.). J. Exp. Mar. Biol. Ecol., 48: 5 l-59. Southgate, P.C., 1988. Use of microencapsulated diets in the culture of giant clam larvae. In: J.W. Copland and J.S. Lucas (Editors), Giant Clams in Asia and the Pacific. ACIAR, Canberra, Australia, pp. I 55- 160. Southgate, PC., 1990. Aspects of the nutrition of giant clam larvae with emphasis on the development of a microencapsulated artificial diet. Ph.D. thesis, James Cook University of North Queensland, Australia, 282 pp. Stephens, G.C. and Manahan, D.T., 1984. Technical advances in the study of nutrition of marine molluscs. Aquaculture, 39: I 55- 164. Teshima, S., Kanazawa, A. and Sakamoto, M., 1982. Micro-partieulate diets for the larvae of aqualic animals. Mini Rev. Data File Fish. Res., Kagoshima Univ., 2: 67-86. Walsh, BUT.,Withstandly, C,A., Kraus, R.A. and Petrovits, E.J., 1987. Mass culture of selected marine microalgae for the nursery production of bivalve seed. J. Shellfish Res., 6: 7 l-77. Welborn, J.R. and Manahan, D.T., 1990. Direct measurement of sugar uptake from sea water into molluscan larvae. Mae. Ecol. Prog. Ser., 65: 233-239. Widdows, J., 199 I. Physiological ecology of mussel larvae. Aquaculture, 94: 147- 163. Zar, J.H., 1984. Biostatistical Analysis. Prentice-Hall International, Inc., London, 7 18 pp.