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The evaluation of fresh algae and stored algal concentrates as a food source for Sydney rock oyster, Saccostrea commercialis (Iredale & Roughley), larvae
Aquaculture, 99(1991)277-284
277
Elsevier Science Publishers B.V., Amsterdam
‘Theevaluation of fresh algae and stored algal concentrates as a food source for Sydney rock oyster, Succostrea commercialis (Iredale & Roughley), larvae John A. Nell and Wayne A. O’Connor N.S. ib! Agriculiure & Fisheries, Brackish WaterFish Culture Research Station, Salamander Bay, N.S. W., 2301, Australia
(Accepted 27 March ! 99 1)
ABSTRACT Nell, J.A. and O’Connor, W.A., I99 1. The evaluation of fresh algae and stored algal concentrates as a food source for Sydney rock oyster, Saccostrea commercialis (Iredale i3;Rolrghley ), !arva:. .4quacultwr . 99: 277-284. Six algal species were fed singly and in combination with Pavlovalutheri, in both fresh and concentrated form, to l-day-old Sydney rock oyster, Saccostrea commercialis, larvae for 6 days. Although a number of algal species produced high larval length increases. the best fresh diet consisted oi P. lutheri and Isochr.wisaff. galbana (T-iso). However, when concentrated to a paste and stored for ?- I4 days at 4°C. a combination of P. lutheri and Chaetoceroscalcitrans produced greater length increases than any other fresh or stored, single or combined diet tested. Centrifugation and stcrage of algae prior to feeding had varied effects on the algal species tested, but it had no detected deleterious effects on any of the diatoms tested.
INTRODUCTION
.A nclmber of studies have been conducted to assess the value of algal species as a food source to oysters and oyster larvae. Davis and Guiliard ( 1958 j tested ten algal species with iarvae of the American oyster ( Crassostreu virgiutica Gmelin ), whilst Walne ( 1970 ) tested 19 algal species as a food source for juvenik flat oysters ( Ostrea edulis L. ). However, despite having been reared in hatcheries for over 10 years, the value of the various a,lgaI species used to feed Sydney rock oyster, Saxostrm mmm~~cialis (Ircdalc & vae has not been determined. The two algal species ?avlova lutheri (Droop) Green and Isocizvg;sisaff. galbana Green (clone T-iso; termed Tahitian Isochrysis) are wideEy used in overseas hatcheries (Holliday, 1985 ) and have been used routinely for feeding Sydney rock oyster larvae (Numaguchi and 0044"8486/91j$O3.50 0 I991 Eisevier Science Publishers B.V. All rights recerved.
278
J.A. NELL AND W.A. O’CONNOR
Nell, 199 1). Tahitian Isuchrysiscontains a high concentration of the 22:6n-3
fatty acids, while P. Zuthericontains high concentrations of both the 20:5n-3 and the 22:6n-3 long-chain unsaturated fatty acids (Brown et al., 1989). These two fatty acids have been found to be important for growth of 0. edufisspat (Enright et al., 1986a,b). Concentrated algae have been used to feed penaeid shrimp (Fox, 1983), rotifers (Stewart et al., 1987), Brachionusplicatilis(Muller), and have also been incorporated in the diet of marron (Somme,r, 1988), Cherax tenuimanus Smith. Algae concentrated into pastes or sl$krrieshave been used to feed oyster larvae in Taboratories (Ukeles, 1975) and in commercial oyster hatcheries (Watson, 1986; Watson et al,, 1986; Donaldson, 1989). This technique allows algae to be stored for several days in the dark at 10°C (Ukeles, 1975), or for a week at 4°C (Watson et al., 1986) before the food value may beg,G to deteriorate. Watson et al. ( 1986) reported that Thadassiosira pseudonnna ( Hustedt ) Hasle and Heimdal and Chaetoceroscalcitrans(Paulsen) Takano could be readily concentrated by centrifugation without damage to their cell walls, whereas P. lutheriand T. I. galbanawere prone to eel1damage. A series of experiments was designed to determine the effect on the growth of Sydney rock oyster larvae of the feeding of fresh or concentrated algal species (listed in Table i ), either singly or in combination with I? lutheri. MATERL4LSANDMETHODS
Oysterlarvae Oyster larvae ranging in initial length ,:+orn69.5 to 7 1.3 pm were obtained from oysters spawned according ricrte&&ques described by Holliday ‘( 1985). One-day-old D-stage oyster larvae were stocked in 8-l non-aerated aquaria at a density of 5 larvae ml- I. Water in the aquaria was maintained at 26 “C and changed every 48 h with the larvae being retained on a 45 pm diagonal screen. Larvae were supplied daily with 0.475 mg 1-l (dry weight) of the algal species, or combination of species, listed in Tables 2 and 3. Algal culture growth was monitored to ensure the cultures used were in logarithmic growth phase. When a combination of algal species was used (experiment 2), they were fed on an equal dry weight basis. Both experiments (with four replicates per treatment) were terminated after 6 days and the larvae were preserved in seawater containing 10% formahn. The length (greatest distance parallel to the hinge) of r 50 preserved larvae per aquarium was measured using a microscope and a micrometer slide ( + 5.0 lrn ). For all experiments, the percentage of dead larvae was less than 5% at the time of termination. Oceanic seawater (35Yi) was filtered using 1 pm (nominal ) cartridge filters before use to reduce the background level of potential oyster food items
ALGAE As FOOD FOR SYDNEY ROCK OYSTER
in the water. Levels of suspended solids in seawater were s 0.6 mg lweight), eq:?l to 63% of the weight of the algal supplements.
279 ’
(dry
Algalculture techniques The algal species used are shown in Table 1. Algal cultures were produced axenically in 5-l borosilicate glass flasks or 20-I polycarbonate carboys. All species were batch cultured in oceanic water (salinity 34-35%0) using f/2 beta growth medium (Guillard, 1983 ) at 2 12 1“C with a 16 : 8 h light: dark q~le and were illuminated with cool white fluorescent tubes to an intensity of 4000 lux at the container surface. Dry weightdeterminationof algalcelis Algal cultures were filtered through a 35 pm (diagonal) mesh screen to remove any debris or clumps of algal cells and the cell concentration determined with a Coulter counter. A known volume ( 50- 150 ml ) was filtered through a 0.7 pm pore size glass fibre filter to collect the algal cells. The filter was washed with 100 ml of 0.5 it4 ammonium formate to remove seasalts ( Epifanio, 1979 ) and dried at 100 ‘C. The-dry weight of suspended solids in the seawater used for the experiments was also determined in this way. Centrifugationand storageof algae Algal cells were concentrated using a cream separator. The concentrated algae were stored at 4 “C for a minimum of 7 and a maximum of f 4 days before use. Experimental design _ Tvm preliminary feeding exye:%men;s SncnTorating the algal species 1isterI in Table 1, were conducted to determine those species most suited to use v&Gth l-day-old Sydney rock oyster larvae. For experiment 1, the six species prc++ moting the highest length increase in the preliminary studies were fed to larvae singly, in fresh and concentrated form (Table 2 ) and in experiment 2, larvae were supplied a diet of the alga P. lutheri in combination with one of the other algal species used previously. These five diets were provided in both fresh and concentrated form. Both experiments included an unfed control. Statisticalanalysis Homogeneity of variance was confirmed using the Cochrsn test (Wirier, 1971 ), The data were analysed with a two-factor an&G of variance to exa.mine the effects of species and processing (fresh or concentrated) and to determine if there was a significant interaction between the two factors. AS $ificant interactions (P< 0.05 j were present for both experiments, the data f&m all treatment combinations for each of these experiments were then subj’ectcd to a single-factor analysis of variance following Unde,!wood ( 1981) * In
J.A.NELL AND W.A.O’CONNOR
280
both experiments mean values were compared using Tukey’s w (Sokal and Rohlf, 1981). RESULTS
The average dry weight of algal cells ranged from 8 pg for Th. pseudonona to 96 pg for Tetraselmis chui Butcher (Table 1). The length/breadth ratio of all species measured had a narrow range from 1.1 to 1.7 except for PhaeodactyEumtricornutum Boh!in which had a ratio of 6.4 (Table 1). Microscopic examination of concentrated feeds indicated great variCon in the tolerance of the various species to centrifugation. The diatoms Ph rricornutum and Ch. calcitrans appeared to be unaffected by centrifugation All the flagellates showed an increase in the number of clumped cells when resuspended following centrifugation. Despite centrifugation, Z suecka (Kylin) retained a high level of motility which was still present after refrigeration for 7 days, in contrast to P. lutheri and Tahitian Psochrysiswhich suflered reductions in motility. Exjleriment I
The effects of both algal species and processing (fresh or coccentrated) were significant (P
Size (pm)*
n
xfs.d.
Length xfs.d.
4 4 4 4
202 3.8 119f3.0 15k3.3 81t 1.3
7.4k1.2 5.4+1.1 1.4 ,23.8+2.9 3.7kO.8 6.4 4.OkO.9 3.1kO.6 1.3 5.7fl.O 4.6f 1.0 1.2
6 6
96+ 17.9 83k21.1 6Clf 1.4 52? 11.0 23f 4.1 21f 4.8
Breadth x+s.d.
Length/ breadth ratio
Diatoms Chaetoceros
gracilis Schiitt I%sc~$~r(vl~c,ntvirwwutun~ Bohlin Chaetocem cuicitram ( Pauisen) Takano Tha!miosira pseudonana Hasle and Heimdal Flagellates Tetrmelmis chui Butcher Durdiella tertiolecta Butcher Chroomonas sdiw ( Wislouch ) Butcher %rzc/,vr!s arecfca ( Kylin ) Pavlov~~ Iutheri (Droop) Green . &nnochloris atmus Butcher Tahitian Ksochrysis aff. galbanu Green
_... ‘I pg=lxlO-‘“g. %22= 30.
4
6 5 5
4 l--“._._.._.......____....... . ................”
i9f
4.3
13.8+ 1.3 10.2* 1.5 11.4& I.5 9.7f1.4 6.9k1.4 5.021.1 8.5f 1.6
9.1 I!I1.3 8.3+_1.8 6.9* 1.2 7.lkO.9 4.9kO.8 4.5kO.9 5.4fl.l
1.5 1.2 1.7 1.4 1.4 1.1 1.6 .--
ALGAE
AS FOOD FOR SYDNEY
ROCK OYSTE?
281
TABLE 2 Comparison of the food value of fresh and concentrated algal species fed to I-day-old Sydney rock oyster (Succostreu commercialis) larvae for 6 days: experiment I Algal species
Average length increase after 6 days (pm ) ’ Fresh
D”..I,,,”
l...h”..:
Concentrated and stored
32.5 + 1.5”
l&Of. 1.3d’
Chaetoceroscalcitrans Phaeodactylumtricornutum EI=nn&S%-is=:r NlG Tetraselmissuecica
30.3 f 1*gab 28.0+2.1b 14.8k 0 9ef 13.5+0:3’ 12.1k 1.2’
20.6 f 1.6cd 27.8 f 2.4b
Unfed control
15.4+ l.lef
1
UY‘“IU
.&%,,,sz,*
Tahitian isochrysis
24.Of. 6.7’
15.4Ik~Y’ 14.7-+09ef - .
‘Values are means+s.d. For both columns means with a common superscript do not differ significantly (P> 0.05 ). Initial average length of l-day-old larvae was 71.3 + 2.3 pm.
Tahitian fsochrysis and Ch. cafcitrans produced greater length increases when fed fresh, although Ph. tricornutum, Nannockloris atomus But&r and T. suecica produced greater length increases when fed following concentration. Single-factor analysis of variance showed that species differences were significant (P< 0.05) for P. lutheri, Tahitian Isochrysis and Ph. tricornutum. P. lutheri promoted the greatest larval length increase and was significantly (P< 0.05) better than all other diets except Tahitian kotThrysis,which was similar (P> 0.05 ) to Ch, calcitrans, and significantly (P-C0.05 ) better than all other diets. In terms of larval length increase, the poorest diets were Ph. tricornutum, N. atomus and T. suecica (Table 2 j. Experiment 2
The effects of both aigal species and processing were sign&ant (P< 0.05) and there was a significant interaction (P~0.05) between the two factors. This interaction was caused by the combination of P. lutheri and T. seucica producing significantly (PC 0.05 ) greater length increases when fed fresh as opposed to the concentrated stored form, whilst the combinaticin of P. lutheri and -Ch.calcitrans produced significantly (P< 0,05 ) greater length increases when fed in the concentrated form (Table 3). Single-factor analysis of variance showed no significant (P> 0.05 ) diflerence between the combinations of species used, although a combination of P. lutheri and Tahitian Isuchrysis produced greater length increases than any other fresh diet. When fed in concyntrated stored form, the combination of P. lutheri and Ch. calcitrans produced significantly (P~0.05 ) greater length increases than any other combination.
J.A.NELL AND W.A.O’CONNOR
282 TABLE 3
Comparison of ihe food value of fresh and concentrated algal species in combination with Pavlova iurheri to I -day-old rock oyster (Saccostrea commercialis) larvae for 6 days: experiment 2 Algal species
Avfzragelength increase after 6 days (pm ) ’ -
Fresh
Concentrated and stored
Panbva Mheri/Tahitian Isochrysis Pavijva IuthrMNannochloris atomus Pavlow Iwheri/TetraseCmissuecica Pavlovalurheri/Phaeodacty/um trirornutum PavlovaIuth~WChaetoceroscalcitrans
40.8 + 6.7ab 32.0f5.1bC 31.5+ 1.9bC 33.0f 1.7bC 33.9k 1.6””
37.2+3.Sb 2 1.6 k 7.0Cd
Unfed control
21.0+ 1.3d
16.5+2.7d 31.0$5,1bC 48.1k1.3”
‘Values arc means+s.d. For both columns means with a common superscript do not differ significantly (P> 0 05 ). initial average iength of i-day-old larvae was 69.5 f 2.4 pm. DISCUSSION
Practical experience in the rearing of Sydney rock oysters has, over a period of 10 years, selected three species, P. lutheri, Tahitian lsochrysisand Ch. cal~&XUIS, a.!;superior larval feeds (L. Goard, pers. commun., 1990). Gur experimentation supported the practical observations as one, or a combination, of these species produced the greatest length increase in larvae whether fed singly, or in combination with P. futheri, in fresh or concentrated stored form. These thre,e species contain high levels of one or both of the long-chain unsaturated falty acids, 20:5+3 and 22:6n-3 (Brown et al., 1989), which individually or in combination have been shown to be important in obtaining high growth rates in Pacific oyster spat (Langdon and Waldock, I98 i ) and in flat oyster spat (Enright et al., 1986a,b). However, it has been suggested (Whyte et al., 1990 ) that oyster larvae have a relatively low requirement for 20:5n-3 and 22:6n-3 fatty acids and once this is met, carbohydrate level is the most important factor governing the food value of an algal species. The similarities between algal species protein levels (Enright et a,l., 1986a) and amino acid levels (Webb and Chu, 1983) indicate that they are unlikely to be important
factors. Several algal species (Ph. tricornuturn,IV.atoms and r suecim )
used as the sole food did not produce growth increases sjgnificantty geater (p> 0.05 ) than unfed controls (Table 2), Centrifugation an‘d storage of algae prior in feeding had varied effects on the algal species tested. When fed singly, fresh I? !a!!hM produced significantly (PC 0.05 ) greater length increases than when fed in the concentrated form. whilst C>. cdcitrans performed equally well in the fresh and concentrated form (Table 2 ). The combination of concentrated stored F. /usheri md Ch. c&tram produced the greatest larval length increase of any diet tested, fresh or concentrated (Table 3); the reason whep.
ALGAE AS FOOD FOR SYDNEY ROCK OYSTER
283
for the synergetic effect between stored P. h&w-i and 01. calcitranscould not be explained. According to Watson ( 1986), the poor performance of P. lutheri as a single concentrated feed may be due to the clumping and cell disruption observed after centrifugation. Watson ( 1986) also observed ;hat the degree of damage incurred during centrifugation varied from batch to batch. It is possible that the batch of centrifuged stored P. lutheri used in experiment 2 had suffered less cellular disruption or, when used in combination with Ch. caicitrans, fewer intact P. lutheri cells were required to promote high growth. Centrifugation and storage of Ph. tricorvzutumsignificantly (PcO.05; Table 2) increased the food value of this species, and indeed had no deleterious effects on the food value of any of the diatoms tested. The performance of various algal species in relation to one another varied from one experiment to another. Growth rates of algae produced under routine culture conditions differ from time to time, and this may have altered their food value. Differences in nutrient reserves of the 1-day-old larvae used may also have affected the relative performance of the algal species in different ex.periments. The amount of naturai%od present in filtered oceanic water (0.6 mg 1-l dry weight of suspended solids) is also an important, but often overlooked factor influencing the growth of o:gster hrvae. In summary, when algae were supplied fresh to I-7-day-old Sydney rock oyster larvae, a wide range of algal species in combination with P. lutheri produced high length increases (Tables 2 and 3). However, in the case of concitntrated stored algae, a diet of P. Zutheriwith Ch. calcitrans produced the bes? results (Table 3). The successful centrifugation and storage of algae could have great implications for aquacultural research (Ukeles, 1975 ) and commercial hatcheries. Centralised algal production units could supply a number of hatcheries and research institutions. in addition, hatcheries could store rather than discard excess algal production for their own use or to supply farmers who use remote settling techniques (Holliday et al., I99 1) . ACKNOWLEDGEMENTS
We thank the staff of the Brackish Water Fish Culture Research Station for their assfstanc e, in particular Ken Frankish and Lindsay Goard for providing larvae, and Joanae Luck and John Diemar for assistance with algae. Thanks are also due to Geoff Allan and Stephen Battaglene for critical review of the manuscript. F&al funding for ?his research was provided by the Fishing In:~~ztEentCc I>S& dr:stry Researc’s and Dr.%<; REFERENCES Brown, M.R., Jeffrey, S.W. and Garland, CD., 1989. Nutritional aspects of microalgae used in maricu’lture; a literature review. CSIRO Mar. Lab. Rep., Hobart, Tas., 44 pp.
284
J.A.NELL 9ND W.A. O’CONNOR
Davis, H.C. and Guillard, R.R., 1958. Relative value of ten genera Of micro-OrganiSmS as fhd for oyster and clam larvae. Fish. Bull., Fish. Wildl. Serv. US, 58: 293-304. Donaldson, J., 1989.Coast Oyster Company. Aquaculture Digest, February, PP. I-5. Enright, CT., Newkirk, G.F., Craigie, J.S and Caste& J.D., 1986a. Evaluation Ofphytoplankton as diets for juvenile Ostreu edufis L. J. Exp. Mar. Biol. Ecol., 96: 1-l 3. Enright, CT., Newkirk, G.F., Craigie, J.S. and Castell, J.D., 1986b. Growth of juvenile Ostreu ed&s L. fed Chaemems grucilis Schiitt of varied chemical composition. J. Exp. Mar. Biol. Ecol., 96: 15-26. Epifanio, C.E., 1979. Growth in bivalve mohuscs: nutritional effects of two or more species of algae in diets fed to the American oyster Crusmt~eu virginicu (Gmelin ) and the hard clam Mercenuriu ( L). Aquaculture, 18: I - 12. Fox, J.M., 1983. Intensive algal culture techniques. In: J:P. McVey (Editor), Handbook of Mariculture. CRC Press, Boca Raton, FL, pp. 15-4 1. Guillard, R.L., 1983. Culture of phytoplankton for feeding marine invertebrates. In: C.J. Berg, Jr. (Editor), Culture of Marine Invertebrates. Hutchinson Ross Publishing Company, Stroudberg, PA, pp. 108-l 32. Holliday, J.E., 1985. International developments in oyster hatchery technology. Misc. Rep. No. 1, Dep. Agric. N.S.W., Brackish Water Fish Culture Research Station, Salamander Bay, N.S.W., !Ol pp. Holliday, J.E., Allan, G.L. and Frances, J., 199 1. Cold storage effects on setting of larvae of the Sydney rock oyster Succostreu colmmerciulis and the Pacific oyster Crussostreu gigus. Aquacuhure, 92: 179- 185. Langdon, C.J. and Waldock, M-J., 1981. The effect of algal and artificial diets on the growth and fatty acid composition of Crussostreu gigus spat. J. Ma.r. Biol. Assoc. UK., 61: 431-448. Numaguchi, K. and Nell, J.A., 199 I. Effects of gelatin-acacia microcapsule and algal meal supplementation of algal diets on gowth rates of Sydney rock oyster, Succostreu commerckdis (Irkzdale & Roughley ), larvae. Aquaculture 94.65-?8. Sokal. R.R. and Rohlf, F.J., 198 I. Biometry. W.H. Freeman, New York, NY, 895 pp. Sommer, T., 1988. Algal mass culture technology: application for shellfish production. In: L.H. Evans and D.O’Sullivan (Editors), Proc. First Aust. Shellfish Aquaculture Conf., Curt:.a Univ. Technol., Perth, W.A., pp. 226-230. Stewart, M., Barlow, C.G. and Longhurst, C., 1987. Reliable food production essential for rearing of barramundi larvae. Aust. Fish., 46 (7 >: 22-24. t Ikeles, R., 1975. Views on bivalve nutrition. Iii: K.S. Price, Jr., W.N. Shaw and KS. Danberg (Editors), Proc. First. Int. Conf. Aquaculture Nutr. Univ. of Delaware, Newark, DE, pp. 127-162. Underwoo%, A.J.. I98 1. Techniques of analysis of variance in experimental marine biology and ecology. Oceanogr. Mar. Biol. Annu. Rev., 19: 5 13-605. Walne, P.R., 1970. Studies on the food value of nineteen genera of algae to juvenile bivalves of ,the genera ~Ostreu. ‘Crmsostreu, Mercenurh and &‘yil’!dis.Fisher> Invest. Lond., Ser. !I, 26 (5 ): 62 p;c. Watson, R.H., 1986. Instant food for bivalve hatcheries. Aquacuhure Digest, September: pp. 67
Watson, R.H .+Jones, G.G. and Jones, B.L., 1986: !Jsing centrifuged algae fcr feeding oyster larvae. J. Shellfish Res., 5: I36 (abstract 1. Webb, K.L. and Chu, F,-L.E., 1983. Phytoplankton as a food source for bivalve larvae: In: G.D. Pruder, C. Langdon and D. Conklin (Editors), Proceedings of the Second International Conference on Aquaculture Nutrition: Biochemical and Physiological Approaches to Shellfish Nutrition. World Maricult. Sot. Spec. Publ., 2: 272-291. Whyte. J.N.C.. Bourne, N. and Hodgson, C.A., 1990. Nutritional condition of rock scallop, Crassado?rra gigunteu (Grey ), larvae fed mixed algal diets. Aquaculture, 86: 25-40. Wirier, B.J., h97 I. Statistical Principles in Experimental Design. McGraw-Hill Kogakusha, Tokyo, pp. 20%2 lij.
277
Elsevier Science Publishers B.V., Amsterdam
‘Theevaluation of fresh algae and stored algal concentrates as a food source for Sydney rock oyster, Succostrea commercialis (Iredale & Roughley), larvae John A. Nell and Wayne A. O’Connor N.S. ib! Agriculiure & Fisheries, Brackish WaterFish Culture Research Station, Salamander Bay, N.S. W., 2301, Australia
(Accepted 27 March ! 99 1)
ABSTRACT Nell, J.A. and O’Connor, W.A., I99 1. The evaluation of fresh algae and stored algal concentrates as a food source for Sydney rock oyster, Saccostrea commercialis (Iredale i3;Rolrghley ), !arva:. .4quacultwr . 99: 277-284. Six algal species were fed singly and in combination with Pavlovalutheri, in both fresh and concentrated form, to l-day-old Sydney rock oyster, Saccostrea commercialis, larvae for 6 days. Although a number of algal species produced high larval length increases. the best fresh diet consisted oi P. lutheri and Isochr.wisaff. galbana (T-iso). However, when concentrated to a paste and stored for ?- I4 days at 4°C. a combination of P. lutheri and Chaetoceroscalcitrans produced greater length increases than any other fresh or stored, single or combined diet tested. Centrifugation and stcrage of algae prior to feeding had varied effects on the algal species tested, but it had no detected deleterious effects on any of the diatoms tested.
INTRODUCTION
.A nclmber of studies have been conducted to assess the value of algal species as a food source to oysters and oyster larvae. Davis and Guiliard ( 1958 j tested ten algal species with iarvae of the American oyster ( Crassostreu virgiutica Gmelin ), whilst Walne ( 1970 ) tested 19 algal species as a food source for juvenik flat oysters ( Ostrea edulis L. ). However, despite having been reared in hatcheries for over 10 years, the value of the various a,lgaI species used to feed Sydney rock oyster, Saxostrm mmm~~cialis (Ircdalc & vae has not been determined. The two algal species ?avlova lutheri (Droop) Green and Isocizvg;sisaff. galbana Green (clone T-iso; termed Tahitian Isochrysis) are wideEy used in overseas hatcheries (Holliday, 1985 ) and have been used routinely for feeding Sydney rock oyster larvae (Numaguchi and 0044"8486/91j$O3.50 0 I991 Eisevier Science Publishers B.V. All rights recerved.
278
J.A. NELL AND W.A. O’CONNOR
Nell, 199 1). Tahitian Isuchrysiscontains a high concentration of the 22:6n-3
fatty acids, while P. Zuthericontains high concentrations of both the 20:5n-3 and the 22:6n-3 long-chain unsaturated fatty acids (Brown et al., 1989). These two fatty acids have been found to be important for growth of 0. edufisspat (Enright et al., 1986a,b). Concentrated algae have been used to feed penaeid shrimp (Fox, 1983), rotifers (Stewart et al., 1987), Brachionusplicatilis(Muller), and have also been incorporated in the diet of marron (Somme,r, 1988), Cherax tenuimanus Smith. Algae concentrated into pastes or sl$krrieshave been used to feed oyster larvae in Taboratories (Ukeles, 1975) and in commercial oyster hatcheries (Watson, 1986; Watson et al,, 1986; Donaldson, 1989). This technique allows algae to be stored for several days in the dark at 10°C (Ukeles, 1975), or for a week at 4°C (Watson et al., 1986) before the food value may beg,G to deteriorate. Watson et al. ( 1986) reported that Thadassiosira pseudonnna ( Hustedt ) Hasle and Heimdal and Chaetoceroscalcitrans(Paulsen) Takano could be readily concentrated by centrifugation without damage to their cell walls, whereas P. lutheriand T. I. galbanawere prone to eel1damage. A series of experiments was designed to determine the effect on the growth of Sydney rock oyster larvae of the feeding of fresh or concentrated algal species (listed in Table i ), either singly or in combination with I? lutheri. MATERL4LSANDMETHODS
Oysterlarvae Oyster larvae ranging in initial length ,:+orn69.5 to 7 1.3 pm were obtained from oysters spawned according ricrte&&ques described by Holliday ‘( 1985). One-day-old D-stage oyster larvae were stocked in 8-l non-aerated aquaria at a density of 5 larvae ml- I. Water in the aquaria was maintained at 26 “C and changed every 48 h with the larvae being retained on a 45 pm diagonal screen. Larvae were supplied daily with 0.475 mg 1-l (dry weight) of the algal species, or combination of species, listed in Tables 2 and 3. Algal culture growth was monitored to ensure the cultures used were in logarithmic growth phase. When a combination of algal species was used (experiment 2), they were fed on an equal dry weight basis. Both experiments (with four replicates per treatment) were terminated after 6 days and the larvae were preserved in seawater containing 10% formahn. The length (greatest distance parallel to the hinge) of r 50 preserved larvae per aquarium was measured using a microscope and a micrometer slide ( + 5.0 lrn ). For all experiments, the percentage of dead larvae was less than 5% at the time of termination. Oceanic seawater (35Yi) was filtered using 1 pm (nominal ) cartridge filters before use to reduce the background level of potential oyster food items
ALGAE As FOOD FOR SYDNEY ROCK OYSTER
in the water. Levels of suspended solids in seawater were s 0.6 mg lweight), eq:?l to 63% of the weight of the algal supplements.
279 ’
(dry
Algalculture techniques The algal species used are shown in Table 1. Algal cultures were produced axenically in 5-l borosilicate glass flasks or 20-I polycarbonate carboys. All species were batch cultured in oceanic water (salinity 34-35%0) using f/2 beta growth medium (Guillard, 1983 ) at 2 12 1“C with a 16 : 8 h light: dark q~le and were illuminated with cool white fluorescent tubes to an intensity of 4000 lux at the container surface. Dry weightdeterminationof algalcelis Algal cultures were filtered through a 35 pm (diagonal) mesh screen to remove any debris or clumps of algal cells and the cell concentration determined with a Coulter counter. A known volume ( 50- 150 ml ) was filtered through a 0.7 pm pore size glass fibre filter to collect the algal cells. The filter was washed with 100 ml of 0.5 it4 ammonium formate to remove seasalts ( Epifanio, 1979 ) and dried at 100 ‘C. The-dry weight of suspended solids in the seawater used for the experiments was also determined in this way. Centrifugationand storageof algae Algal cells were concentrated using a cream separator. The concentrated algae were stored at 4 “C for a minimum of 7 and a maximum of f 4 days before use. Experimental design _ Tvm preliminary feeding exye:%men;s SncnTorating the algal species 1isterI in Table 1, were conducted to determine those species most suited to use v&Gth l-day-old Sydney rock oyster larvae. For experiment 1, the six species prc++ moting the highest length increase in the preliminary studies were fed to larvae singly, in fresh and concentrated form (Table 2 ) and in experiment 2, larvae were supplied a diet of the alga P. lutheri in combination with one of the other algal species used previously. These five diets were provided in both fresh and concentrated form. Both experiments included an unfed control. Statisticalanalysis Homogeneity of variance was confirmed using the Cochrsn test (Wirier, 1971 ), The data were analysed with a two-factor an&G of variance to exa.mine the effects of species and processing (fresh or concentrated) and to determine if there was a significant interaction between the two factors. AS $ificant interactions (P< 0.05 j were present for both experiments, the data f&m all treatment combinations for each of these experiments were then subj’ectcd to a single-factor analysis of variance following Unde,!wood ( 1981) * In
J.A.NELL AND W.A.O’CONNOR
280
both experiments mean values were compared using Tukey’s w (Sokal and Rohlf, 1981). RESULTS
The average dry weight of algal cells ranged from 8 pg for Th. pseudonona to 96 pg for Tetraselmis chui Butcher (Table 1). The length/breadth ratio of all species measured had a narrow range from 1.1 to 1.7 except for PhaeodactyEumtricornutum Boh!in which had a ratio of 6.4 (Table 1). Microscopic examination of concentrated feeds indicated great variCon in the tolerance of the various species to centrifugation. The diatoms Ph rricornutum and Ch. calcitrans appeared to be unaffected by centrifugation All the flagellates showed an increase in the number of clumped cells when resuspended following centrifugation. Despite centrifugation, Z suecka (Kylin) retained a high level of motility which was still present after refrigeration for 7 days, in contrast to P. lutheri and Tahitian Psochrysiswhich suflered reductions in motility. Exjleriment I
The effects of both algal species and processing (fresh or coccentrated) were significant (P
Size (pm)*
n
xfs.d.
Length xfs.d.
4 4 4 4
202 3.8 119f3.0 15k3.3 81t 1.3
7.4k1.2 5.4+1.1 1.4 ,23.8+2.9 3.7kO.8 6.4 4.OkO.9 3.1kO.6 1.3 5.7fl.O 4.6f 1.0 1.2
6 6
96+ 17.9 83k21.1 6Clf 1.4 52? 11.0 23f 4.1 21f 4.8
Breadth x+s.d.
Length/ breadth ratio
Diatoms Chaetoceros
gracilis Schiitt I%sc~$~r(vl~c,ntvirwwutun~ Bohlin Chaetocem cuicitram ( Pauisen) Takano Tha!miosira pseudonana Hasle and Heimdal Flagellates Tetrmelmis chui Butcher Durdiella tertiolecta Butcher Chroomonas sdiw ( Wislouch ) Butcher %rzc/,vr!s arecfca ( Kylin ) Pavlov~~ Iutheri (Droop) Green . &nnochloris atmus Butcher Tahitian Ksochrysis aff. galbanu Green
_... ‘I pg=lxlO-‘“g. %22= 30.
4
6 5 5
4 l--“._._.._.......____....... . ................”
i9f
4.3
13.8+ 1.3 10.2* 1.5 11.4& I.5 9.7f1.4 6.9k1.4 5.021.1 8.5f 1.6
9.1 I!I1.3 8.3+_1.8 6.9* 1.2 7.lkO.9 4.9kO.8 4.5kO.9 5.4fl.l
1.5 1.2 1.7 1.4 1.4 1.1 1.6 .--
ALGAE
AS FOOD FOR SYDNEY
ROCK OYSTE?
281
TABLE 2 Comparison of the food value of fresh and concentrated algal species fed to I-day-old Sydney rock oyster (Succostreu commercialis) larvae for 6 days: experiment I Algal species
Average length increase after 6 days (pm ) ’ Fresh
D”..I,,,”
l...h”..:
Concentrated and stored
32.5 + 1.5”
l&Of. 1.3d’
Chaetoceroscalcitrans Phaeodactylumtricornutum EI=nn&S%-is=:r NlG Tetraselmissuecica
30.3 f 1*gab 28.0+2.1b 14.8k 0 9ef 13.5+0:3’ 12.1k 1.2’
20.6 f 1.6cd 27.8 f 2.4b
Unfed control
15.4+ l.lef
1
UY‘“IU
.&%,,,sz,*
Tahitian isochrysis
24.Of. 6.7’
15.4Ik~Y’ 14.7-+09ef - .
‘Values are means+s.d. For both columns means with a common superscript do not differ significantly (P> 0.05 ). Initial average length of l-day-old larvae was 71.3 + 2.3 pm.
Tahitian fsochrysis and Ch. cafcitrans produced greater length increases when fed fresh, although Ph. tricornutum, Nannockloris atomus But&r and T. suecica produced greater length increases when fed following concentration. Single-factor analysis of variance showed that species differences were significant (P< 0.05) for P. lutheri, Tahitian Isochrysis and Ph. tricornutum. P. lutheri promoted the greatest larval length increase and was significantly (P< 0.05) better than all other diets except Tahitian kotThrysis,which was similar (P> 0.05 ) to Ch, calcitrans, and significantly (P-C0.05 ) better than all other diets. In terms of larval length increase, the poorest diets were Ph. tricornutum, N. atomus and T. suecica (Table 2 j. Experiment 2
The effects of both aigal species and processing were sign&ant (P< 0.05) and there was a significant interaction (P~0.05) between the two factors. This interaction was caused by the combination of P. lutheri and T. seucica producing significantly (PC 0.05 ) greater length increases when fed fresh as opposed to the concentrated stored form, whilst the combinaticin of P. lutheri and -Ch.calcitrans produced significantly (P< 0,05 ) greater length increases when fed in the concentrated form (Table 3). Single-factor analysis of variance showed no significant (P> 0.05 ) diflerence between the combinations of species used, although a combination of P. lutheri and Tahitian Isuchrysis produced greater length increases than any other fresh diet. When fed in concyntrated stored form, the combination of P. lutheri and Ch. calcitrans produced significantly (P~0.05 ) greater length increases than any other combination.
J.A.NELL AND W.A.O’CONNOR
282 TABLE 3
Comparison of ihe food value of fresh and concentrated algal species in combination with Pavlova iurheri to I -day-old rock oyster (Saccostrea commercialis) larvae for 6 days: experiment 2 Algal species
Avfzragelength increase after 6 days (pm ) ’ -
Fresh
Concentrated and stored
Panbva Mheri/Tahitian Isochrysis Pavijva IuthrMNannochloris atomus Pavlow Iwheri/TetraseCmissuecica Pavlovalurheri/Phaeodacty/um trirornutum PavlovaIuth~WChaetoceroscalcitrans
40.8 + 6.7ab 32.0f5.1bC 31.5+ 1.9bC 33.0f 1.7bC 33.9k 1.6””
37.2+3.Sb 2 1.6 k 7.0Cd
Unfed control
21.0+ 1.3d
16.5+2.7d 31.0$5,1bC 48.1k1.3”
‘Values arc means+s.d. For both columns means with a common superscript do not differ significantly (P> 0 05 ). initial average iength of i-day-old larvae was 69.5 f 2.4 pm. DISCUSSION
Practical experience in the rearing of Sydney rock oysters has, over a period of 10 years, selected three species, P. lutheri, Tahitian lsochrysisand Ch. cal~&XUIS, a.!;superior larval feeds (L. Goard, pers. commun., 1990). Gur experimentation supported the practical observations as one, or a combination, of these species produced the greatest length increase in larvae whether fed singly, or in combination with P. futheri, in fresh or concentrated stored form. These thre,e species contain high levels of one or both of the long-chain unsaturated falty acids, 20:5+3 and 22:6n-3 (Brown et al., 1989), which individually or in combination have been shown to be important in obtaining high growth rates in Pacific oyster spat (Langdon and Waldock, I98 i ) and in flat oyster spat (Enright et al., 1986a,b). However, it has been suggested (Whyte et al., 1990 ) that oyster larvae have a relatively low requirement for 20:5n-3 and 22:6n-3 fatty acids and once this is met, carbohydrate level is the most important factor governing the food value of an algal species. The similarities between algal species protein levels (Enright et a,l., 1986a) and amino acid levels (Webb and Chu, 1983) indicate that they are unlikely to be important
factors. Several algal species (Ph. tricornuturn,IV.atoms and r suecim )
used as the sole food did not produce growth increases sjgnificantty geater (p> 0.05 ) than unfed controls (Table 2), Centrifugation an‘d storage of algae prior in feeding had varied effects on the algal species tested. When fed singly, fresh I? !a!!hM produced significantly (PC 0.05 ) greater length increases than when fed in the concentrated form. whilst C>. cdcitrans performed equally well in the fresh and concentrated form (Table 2 ). The combination of concentrated stored F. /usheri md Ch. c&tram produced the greatest larval length increase of any diet tested, fresh or concentrated (Table 3); the reason whep.
ALGAE AS FOOD FOR SYDNEY ROCK OYSTER
283
for the synergetic effect between stored P. h&w-i and 01. calcitranscould not be explained. According to Watson ( 1986), the poor performance of P. lutheri as a single concentrated feed may be due to the clumping and cell disruption observed after centrifugation. Watson ( 1986) also observed ;hat the degree of damage incurred during centrifugation varied from batch to batch. It is possible that the batch of centrifuged stored P. lutheri used in experiment 2 had suffered less cellular disruption or, when used in combination with Ch. caicitrans, fewer intact P. lutheri cells were required to promote high growth. Centrifugation and storage of Ph. tricorvzutumsignificantly (PcO.05; Table 2) increased the food value of this species, and indeed had no deleterious effects on the food value of any of the diatoms tested. The performance of various algal species in relation to one another varied from one experiment to another. Growth rates of algae produced under routine culture conditions differ from time to time, and this may have altered their food value. Differences in nutrient reserves of the 1-day-old larvae used may also have affected the relative performance of the algal species in different ex.periments. The amount of naturai%od present in filtered oceanic water (0.6 mg 1-l dry weight of suspended solids) is also an important, but often overlooked factor influencing the growth of o:gster hrvae. In summary, when algae were supplied fresh to I-7-day-old Sydney rock oyster larvae, a wide range of algal species in combination with P. lutheri produced high length increases (Tables 2 and 3). However, in the case of concitntrated stored algae, a diet of P. Zutheriwith Ch. calcitrans produced the bes? results (Table 3). The successful centrifugation and storage of algae could have great implications for aquacultural research (Ukeles, 1975 ) and commercial hatcheries. Centralised algal production units could supply a number of hatcheries and research institutions. in addition, hatcheries could store rather than discard excess algal production for their own use or to supply farmers who use remote settling techniques (Holliday et al., I99 1) . ACKNOWLEDGEMENTS
We thank the staff of the Brackish Water Fish Culture Research Station for their assfstanc e, in particular Ken Frankish and Lindsay Goard for providing larvae, and Joanae Luck and John Diemar for assistance with algae. Thanks are also due to Geoff Allan and Stephen Battaglene for critical review of the manuscript. F&al funding for ?his research was provided by the Fishing In:~~ztEentCc I>S& dr:stry Researc’s and Dr.%<; REFERENCES Brown, M.R., Jeffrey, S.W. and Garland, CD., 1989. Nutritional aspects of microalgae used in maricu’lture; a literature review. CSIRO Mar. Lab. Rep., Hobart, Tas., 44 pp.
284
J.A.NELL 9ND W.A. O’CONNOR
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