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Preliminary observations on the nutritional value of ‘Tahiti Isochrysis’ to bivalve larvae
Aquaculture, 62 (1987) 281-288 Elsevier Science Publishers B.V., Amsterdam
281 -
Printed
in The Netherlands
Preliminary Observations on the Nutritional Value of ‘Tahiti Isochrysis’ to Bivalve Larvae M.M. HELM and I. LAING Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fisheries Experiment Station, Conwy, Gwynedd LL32 8UB (Great Britain) (Accepted
5 January
1987)
ABSTRACT Helm, M.M. and Laing, I., 1987. Preliminary observations Isochrysis’ to bivalve larvae. Aquaculture, 62: 281-288.
on the nutritional
value of ‘Tahiti
The tropical flagellate, Zsochrysis aff. galbana (Clone T-ISO) , and the small, centric diatom, Chaetoceros calcitrans (Paulsen) Takano were fed singly and in mixture to larvae of the Pacific oyster, Crassostreagigas, the mangrove oyster, Crassostrea rhizophorae, the American hard-shell clam, Mercenaria mercenaria and the Manila clam, Tapes semidecussata. Trials started at the Dveliger stage and continued until larvae were about to metamorphose. C. cakitrans supported good growth of each of these bivalves but T-IS0 was only good for the clams. T-IS0 seemed deficient in at least one nutritionally important component to Crassostrea spp. larvae which led to growth retardation beyond 6-day-old veligers. Enhanced growth effects were observed in all of the bivalves, except for M. mercenaria, in response to the mixed diet. Results are compared with previous published data of the food value of Isochrysis galbana Parke where some evidence suggests a similar deficiency to that in T-IS0 for Crassostrea larvae. Polyunsaturated fatty acid composition of the algae tested is considered.
INTRODUCTION
The tropical flagellate Isochrysis aff. gulbunu (Clone T-ISO), commonly referred to as ‘Tahiti Isochrysis’ or ‘T-ISO’, has largely replaced I. galbana Parke as a food species in the culture of bivalve larvae. This is because of its advantageous culture characteristics, particularly its ability to withstand higher culture temperatures. Comparisons of the food value of these apparently similar flagellates to larvae of Crussostreu uirginicu Gmelin suggested close nutritional similarity when both were tested in combination with the small centric diatom Thulussiosiru pseudonanu Clone 3H (Ewart and Epifanio, 1981) . Early trials at this laboratory, in which both flagellates were offered as single-species diets to newly liberated Ostreu edulis L. larvae, showed no significant differences in nutritive value. Since then T-IS0 has replaced I. gulbunu
Crown
Copyright
reserved.
282
in the standard mixed algae diet used at this laboratory, reported by Helm and Millican (1977). It is only recently that we had reason to question its value in the nutrition of larvae of certain bivalves. This doubt arose during comparative nutrition trials with larvae of four commercially important species including the Pacific oyster, Crassostrea gigas Thunberg, the mangrove oyster, Crussostrea rhizophorue Guilding, the American hard-shell clam, Mercenariu mercenaria L. and the Manila clam, Tapes semidecussutu Reeve. Results and their possible significance in the identification of differences in nutritional requirements between these bivalves are reported. Comparisons are made with previously published data on the food value of I. gulbunu. MATERIALS AND METHODS
Clam larvae were obtained from temperature stimulated spawnings of hatchery conditioned breeding stock and larvae of the Crussostreu spp. by pipetting gametes from opened mature oysters as described by Helm and Millican (1977). 0. edulis larvae were from broods liberated by hatchery maintained stocks (Helm et al., 1973). Trials of 4-days duration were duplicated in l-l borosilicate glass beakers with 400-600 larvae per treatment. Longer duration trials, in which larvae were reared to metamorphosis, were in 125-1 tapered, cylindrical, polyethylene vessels. These initially contained 12 000-16 000 larvae/l in 2-pm filtered, ultra-violet light sterilized sea water ( Walne, 1974). The culture temperature in all trials was 24.0 2 0.5 “C. Salinity was adjusted to 25%0 with filtered fresh water for Crussostreu spp. larvae and was at 30-33%0, the ambient, for other species. All vessels were aerated and complete water changes were made three times each week. I. gulbunu (Parke, Plymouth) and T-IS0 (Clone T-ISO, University of Delaware) were cultured in 20-l flasks as described by Walne (1966,1974). Twentyfive per cent of the culture was harvested three times each week, while in the active growth phase, and the volume restored with freshly prepared culture medium. Cell densities at harvest ranged from 12 000 to 20 OOO/pl and, in the case of T-ISO, the ash-free dry weight per lo6 cells averaged 20.8 ,ug. Chuetoceros culcitruns (Paulsen) Takano (Imai, Sendai) , used as the control diet, was cultured in 2-l batches in autoclaved sea water medium (Laing, 1979). Cell densities at harvest ranged from 45 000 to 65 OOO/pl and the mean ashfree dry weight per lo6 cells was 7.2 pg. Approximately equal rations of single-species diets of T-IS0 and C. culcitruns and a mixture of the two were provided daily in long duration trials. Rations were based on the ash-free dry weights of the two algae such that larvae were fed either 100 cells/p1 of T-ISO, 250 cells/p1 of C. culcitruns or half the quantity of each in the mixture. These rations were increased as the larvae grew and required greater quantities of food. Samples of the diets were taken for analysis of fatty acids. Analyses were made by D. L. Holland, UCNW,
283 TABLE 1 The growth of newly liberated larvae of Ostrea edulis (mean initial length 186.6 pm) in a 4-day period when fed different rations by cell density of T-IS0 and I. galbana (95% confidence intervals of final mean shell length are given) Diet
Cells (j&l )
Final shell length (pm )
1. gutbuna
40 80 120
232.8 k 1.9 226.9 Z!I2.9 223.8 + 2.3
T-IS0
40 80 120
231.Ok2.0 234.7k2.1 231.3k2.1
Marine Science Laboratories, Menai Bridge, using the methods Waldock and Holland (1984 ) .
described
by
RESULTS
A 4-day trial in l-1 glass beakers compared the value of T-IS0 and 1. g&bona to newly liberated larvae of 0. e&is. Both algae were fed at densities of 40,80 and 120 cells/pi. The larvae were 186.6 pm mean shell length initially and their mean sizes at the completion of the trial are given in Table 1. T-IS0 at the higher rations supported greater growth of larvae than at equivalent cell densities of I. galbana but there was no significant difference in growth at the 95% confidence level between the best cell density of each alga, i.e., 40 cells/p1 I. gulbunu, 80 cells/p1 T-ISO. Growth rates of Crussostreu spp. and clam larvae supported by single-species diets of T-ISO, C. culcitruns and the two-species combination are shown in Fig. 1. Values given are mean shell lengths from measurements of 50-100 individuals sampled at intervals throughout the pelagic growth phase until larvae were about to metamorphose. This was recognised by the development of eye spots in oyster larvae and pediveligers in clams. Day 0 denotes the time of spawning and fertilization. In both Crussostrea spp. the initial growth rate of larvae fed with T-IS0 alone was slightly lower than with the other diets (Table 2). After 4-6 days, varying with the oyster species, growth rates diverged. Small daily growth increments after the first 6-8 days with T-IS0 (Fig. 1) were partly the effect of a differential mortality of the smaller larvae. Larvae fed T-IS0 showed > 60% mortality by day 13 compared with 25%-35% with the other diets. Larvae fed C. culcitrans or the two-species diet grew strongly and reached settlement in 16 days and 15 days in C. gigus and C. rhizophorae, respectively. Enhanced growth effects were observed, the two-species mixture supporting significantly greater growth (PC 0.05) than when the algae were fed alone (Fig. 1).
-g
140
=I .c GI 3 i -E f : 0 I
60
0
2
4
6
6
10
12 14 16
260
601
0
2
4
6
-2
610
0
2
4
6
Days
Fig. 1, The growth of (A) C. gigas, (B ) C. rhizophorae, (C ) M. mercenaria and (D ) T. semidecusata larvae fed T-IS0 (closed circles), C. calcitrans (open circles) and the two-species diet (triangles). Vertical bars indicate the 95% confidence intervals of mean shell length.
A4. mercenaria grew as well when fed T-IS0 as with either of the other diets and high yields of post-metamorphosis juveniles were obtained from each treatment. Results were less conclusive from the T. semidecussata trial because all larvae were fed C. calcitrans between days 1 and 2. From day 2, when the three diet treatments were started, larvae grew rapidly to the onset of metamorphosis on each diet. Enhanced growth effects were observed in T. semidecussata in response to the two-species diet but not with A4. mercenaria. Data on the polyunsaturated fatty acid composition of T-IS0 and C. calcitrans sampled during the reported trials are given in Table 3. Values are compared with previously published data for these species and with I. galbana.
285 TABLE 2 Mean shell lengths + 95% confidence intervals of early veligers of C. gigas and C. rhizophorae fed different diets. Daily rations of the diets as algae cells (,I&‘) are given. C. gigas larvae were 73.4 pm initially and C. rhizophorae 64.2 pm Diet (cells/pi)
Mean shell length (pm) C. rhizophorae
C.gigas
T-IS0 (100) C. calcitrans (250) T-IS0 + C. calcitrans (50 + 125)
Day 4
Day 6
Day 6
Day 8
90.2 * 2.7 93.4 rt 2.0 97.Ok2.7
101.4 & 4.3 120.4 + 3.2 128.2 f 3.8
102.6 i 2.5 107.4? 3.2 109.4 * 3.2
103.4k3.1 127.8 4 3.0 137.0? 3.1
DISCUSSION
T-IS0 can be beneficial as a constituent species of a mixed algal diet as shown by significantly improved growth of the larvae of three of the four bivalves tested. However, only M. mercenaria grew and developed as rapidly with the mono-species diet of T-IS0 as with the other diets tested. In T. semidecussatu, larvae grew to metamorphosis on the T-IS0 diet but by day 12 mean shell length was significantly smaller than in those fed only C. culcitruns or the twospecies combination. For both C. gigas and C. rhizophorue T-IS0 was a poor food; growth and development slowed beyond day 6 and larvae failed to reach settlement within the trial period. These results suggest that T-IS0 is lacking in a component (or components ) vital to normal growth and development in Crussostreu spp. larvae. It is possible that larvae carry sufficient of this component over from parentally derived reserves, via the eggs, to support early TABLE 3 Polyunsaturated fatty acid composition of T-IS0 (Pillsbury, 1985)) I. galbana ( Waldock and Nascimento, 1979) and C. calcitrans (Webb and Chu, 1983). Data in parentheses refer to cultures from this laboratory, taken during the trials and analysed by D. L. Holland” (Marine Science Laboratories, Menai Bridge). Values are percentages of total fatty acids Fatty acid
T-IS0
18: 206 18: 303 18: 4~3 20: 5w3 22: 603
4.0 5.6 19.4 -( 7.9
’ See Waldock and Holland
( 6.9 ) ( 4.3 ) ( 8.7 ) 0.71) (11.4 )
(1984) for methods
used.
I. galbana
C. calcitrans
4.3 11.0 9.2 3.6 18.9
0.2 3.0 0.5 15.4 1.9
( 1.8 ) ( 1.5 ) ( 0.6 ) (22.9 ) ( 0.97)
development. The growth of early veligers fed on T-IS0 was similar to the growth when fed with the two other diets (Table 2). It can be assumed that the larvae are unable to biosynthesise or have a limiting biosynthesis rate for this component to permit normal development. A similar pattern of larval growth on a single-species diet of T-IS0 was reported by Pillsbury (1985) working with the queen conch, Strombus gigas Linne. The initial rate of growth after hatching was equally as rapid as with the best diet tested but then slowed so that by day 20 mean size was not significantly different from the size 6 days earlier. Pillsbury (1985) quotes the fatty acid profile of T-IS0 which, when compared with profiles for I. galbana ( Waldock and Nascimento, 1979; Webb and Chu, 1983)) shows that T-IS0 lacks the polyunsaturated fatty acid, 205~3 while 22:6w3 is present in both. In contrast, T-IS0 cultured at Conwy contained 20~503 but as a lower percentage of total fatty acids than found in I. galbana by Waldock and Nascimento (1979) (Table 3 ) . Both of these fatty acids are regarded as important in the nutrition of crustacean, molluscan and fish larvae (see reviews in Kanazawa, 1983; Webb and Chu, 1983; Pillsbury, 1985). The total lipid content of I. galbana is similar to that of T-ISO. Utting (1985) quotes values of 16.9%-22.0% of dry weight for I. galbana depending on nitrogen availability in the culture medium, and Pillsbury (1985) a value of 23.0% of dry weight in T-ISO. In the work reported here T-IS0 was cultured in similar conditions to those used previously for I. galbana and its total lipid content was 21.1% +-3.6% (standard deviation) of ash-free dry weight, equivalent to 19.0% of dry weight. Generally, I. galbana has been regarded as a satisfactory food for bivalve larvae (Loosanoff and Davis, 1963; Walne, 1965). Certainly, it is one of the better of the food species tested for 0. edulis, supporting rapid rates of growth in larvae at all stages in development (Helm, 1977; Wilson, 1979). However, it was fully tested at this laboratory only as a constituent of a mixed algal diet in the culture of C. gigas larvae (Helm and Millican, 1977). Work by Utting (1979) and Nascimento (1980) with C. gigus straight-hinged, D-veligers demonstrated the nutritional superiority of C. calcitrans over I. galbana but these trials were of insufficient duration to identify possible longterm deficiencies arising with the latter species. Long duration nutrition trials by Wilson (1978) provided information which points to a nutritional deficiency developing in C. gigas larvae fed solely on I. galbana. In a series of six trials he grew veligers from the D-stage for 12 days. All growth data were converted to growth coefficients and, in the absence of quoted values of initial mean shell lengths, it is difficult to interpolate accurately the sizes reached by larvae as the trials progressed. Assuming that larvae were 75 pm mean shell length at the beginning of each trial (from Helm and Millican, 1977) calculations from the mean K4 values given by Wilson (1978) show that they reached 92.5 pm after 4 days, 111.9 ,um after 8 days, and 133.9 ,um after 12 days. These
287
estimates suggest a growth performance similar to that with T-IS0 but not as good as with C. calcitruns. (Wilson found that better growth was obtained with the diatom Phaeodactylum tricornutum Bohlin. ) Evidence, therefore, suggests that T-IS0 and I. gulbunu are both poor foods for C. gigus larvae and probably for all Crassostreu spp. T-IS0 was of poor value to C. rhizophorae here and I. gulbunu did not rank among the better foods for larvae of C. uirginicu in feeding trials by.Davis (1953). In contrast, both flagellates seem good foods for 0. edulis and the various clam species mentioned. C. culcitruns is shown to be a very good food for Crussostreu spp. larvae as well as for the clams (Fig. 1) . Waldock and Nascimento (1979) quote its fatty acid content as 9.7% of dry weight compared with 12.5% for I. gulbunu. Of this total, 15.4% was 20x503 compared with 3.6% in I. gulbanu, while values for 22:6@3 were 1.9% and l&9%, respectively (Table 3). Similarly high values of 20:5co3 and low values of 22:6~3 were determined in C. calcitruns cultured for the present trials. Waldock and Nascimento (1979) show that larval growth rate was not correlated with the amount of lipid in the diet nor were differences in fatty acid composition solely responsible for growth rate differences between the diets. Pillsbury (1985) reached similar conclusions. Nevertheless, such marked differences in larval growth rates between bivalve species with diets of C. calcitruns compared with I. gulbuna and T-IS0 provide a useful starting point to evaluate precise nutritional requirements. It is possible that the solution may be not only in the presence or absence of certain essential components in the diet but in the correct balance between them, which may differ for the various bivalves so far investigated. The possibility arises in this present work that Crassostrea spp. larvae have a high specific requirement for 20:5~3 met by feeding them with C. culcitruns but not with I. gaZbana or T-ISO, where limited quantities of this polyunsaturate were available. The species of clam tested appear to have no similar requirement for 20:51x3, growing equally as well with all diets offered. Since all these diets contained 22:6w3 a specific requirement may be indicated and needs to be tested. Alternatively, the relative food value of the different algae to different bivalves may be related to other nutritionally important factors.
REFERENCES Davis, H. C., 1953. On food and feeding of larvae of the American oyster, C. uirginica. Biol. Bull., 104: 334-350. Ewart, J. W. and Epifanio, C. E., 1981. A tropical flagellate food for larval and juvenile oysters, Crassostrea virginica Gmelin. Aquaculture, 22: 297-300. Helm, M. M., 1977. Mixed algal feeding of Ostrea edulis larvae with Zsochrysisgalbana and Tetraselmis suecica. J. Mar. Biol. Assoc. U.K., 57: 1019-1029. Helm, M. M. and Millican, P. F., 1977. Experiments in the hatchery rearing of Pacific oyster larvae (Crussostreagigas Thunberg). Aquaculture, 11: 1-12.
Helm, M. M., Holland, D. L. and Stephenson, R. R., 1973. The effect of supplementary algal feeding of a hatchery breeding stock of Ostrea edulis L. on larval vigour. J. Mar. Biol. Assoc. U.K., 53: 673-684. Kanazawa, A., 1983. Penaeid nutrition. In: G.D. Pruder, C. Langdon and D. Conklin (Editors), Biochemical and Physiological Approaches to Shellfish Nutrition, Proc. 2nd Int. Conf. Aquaculture Nutr. World Maricult. Sot. Spec. Publ. No. 2, Louisiana State University, LA, pp. 87-105. Laing, I., 1979. Recommended procedures for the culture of Chaetoceros calcitrans. Fish. Res. Tech. Rep., MAFF Direct. Fish. Res., Lowestoft, No. 53: 18 pp. Loosanoff, V. L. and Davis, H. C., 1963. Rearing of bivalve molluscs. Adv. Mar. Biol., 1: 1-136. Nascimento, I. A., 1980. Growth of the larvae of Crassostrea gigas Thunberg, fed with different algal species at high cell concentrations. J. Cons. Int. Explor. Mer, 39 (2) : 134-139. Pillsbury, K. S., 1985. The relative food value and biochemical composition of five phytoplankton diets for queen conch, Strombus gigas (Linne) larvae. J. Exp. Mar. Biol. Ecol., 90: 221-231. Utting, S. D., 1979. The biochemical content of Chaetoceros calcitrans, Tetraselmis suecica and Zsochrysisgalbana in relation to the growth of Crassostreagigas larvae and spat. Ph. D. Thesis, University College of North Wales, Marine Science Laboratories, Menai Bridge, 89 pp. Utting, S. D., 1985. Influence of nitrogen availability on the biochemical composition of three unicellular marine algae of commercial importance. Aquacultural Eng., 4: 175-190. Waldock, M. J. and Holland, D. L., 1984. Fatty acid metabolism in young oysters, Crassostrea gigas: polyunsaturated fatty acids. Lipids, 19 (5) : 332-336. Waldock, M. J. and Nascimento, I. A., 1979. The triacylglycerol composition of Crassostrea gigas larvae fed on different diets. Mar. Biol. Lett., 1: 77-86. Walne, P. R., 1965. Observations on the influence of food supply and temperature on the feeding and growth of the larvae of Ostrea edulis L. Fishery Invest., Lond. Ser. 2,24 (1): l-45. Walne, P. R., 1966. Experiments in the large-scale culture of the larvae of Ostrea edulis L. Fishery Invest., Lond. Ser. 2,25 (4) : l-53. Walne, P. R., 1974. Culture of Bivalve Molluscs, 50 Years’ Experience at Conwy. Fishing News (Books), West Byfleet, 173 pp. Webb, K. L. and Chu, F. L., 1983. Phytoplankton as a food source for bivalve larvae. In: G. D. Pruder, C. Langdon and D. Conklin (Editors), Biochemical and Physiological Approaches to Shellfish Nutrition. Proc. 2nd Int. Conf. Aquaculture Nutr. World Mar&cult. Sot. Spec. Publ. No. 2, Louisiana State University, LA, pp. 272-291. Wilson, J. H., 1978. The food value of Phaeodactylum tricornutum Bohlin to the larvae of Ostrea edulis L. and Crassostrea gigas Thunberg. Aquaculture, 13: 313-323. Wilson, J. H., 1979. Observations on the grazing rates and growth of Ostrea edulis L. larvae when fed algal cultures of different ages. J. Exp. Mar. Biol. Ecol., 38: 187-199.
281 -
Printed
in The Netherlands
Preliminary Observations on the Nutritional Value of ‘Tahiti Isochrysis’ to Bivalve Larvae M.M. HELM and I. LAING Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fisheries Experiment Station, Conwy, Gwynedd LL32 8UB (Great Britain) (Accepted
5 January
1987)
ABSTRACT Helm, M.M. and Laing, I., 1987. Preliminary observations Isochrysis’ to bivalve larvae. Aquaculture, 62: 281-288.
on the nutritional
value of ‘Tahiti
The tropical flagellate, Zsochrysis aff. galbana (Clone T-ISO) , and the small, centric diatom, Chaetoceros calcitrans (Paulsen) Takano were fed singly and in mixture to larvae of the Pacific oyster, Crassostreagigas, the mangrove oyster, Crassostrea rhizophorae, the American hard-shell clam, Mercenaria mercenaria and the Manila clam, Tapes semidecussata. Trials started at the Dveliger stage and continued until larvae were about to metamorphose. C. cakitrans supported good growth of each of these bivalves but T-IS0 was only good for the clams. T-IS0 seemed deficient in at least one nutritionally important component to Crassostrea spp. larvae which led to growth retardation beyond 6-day-old veligers. Enhanced growth effects were observed in all of the bivalves, except for M. mercenaria, in response to the mixed diet. Results are compared with previous published data of the food value of Isochrysis galbana Parke where some evidence suggests a similar deficiency to that in T-IS0 for Crassostrea larvae. Polyunsaturated fatty acid composition of the algae tested is considered.
INTRODUCTION
The tropical flagellate Isochrysis aff. gulbunu (Clone T-ISO), commonly referred to as ‘Tahiti Isochrysis’ or ‘T-ISO’, has largely replaced I. galbana Parke as a food species in the culture of bivalve larvae. This is because of its advantageous culture characteristics, particularly its ability to withstand higher culture temperatures. Comparisons of the food value of these apparently similar flagellates to larvae of Crussostreu uirginicu Gmelin suggested close nutritional similarity when both were tested in combination with the small centric diatom Thulussiosiru pseudonanu Clone 3H (Ewart and Epifanio, 1981) . Early trials at this laboratory, in which both flagellates were offered as single-species diets to newly liberated Ostreu edulis L. larvae, showed no significant differences in nutritive value. Since then T-IS0 has replaced I. gulbunu
Crown
Copyright
reserved.
282
in the standard mixed algae diet used at this laboratory, reported by Helm and Millican (1977). It is only recently that we had reason to question its value in the nutrition of larvae of certain bivalves. This doubt arose during comparative nutrition trials with larvae of four commercially important species including the Pacific oyster, Crassostrea gigas Thunberg, the mangrove oyster, Crussostrea rhizophorue Guilding, the American hard-shell clam, Mercenariu mercenaria L. and the Manila clam, Tapes semidecussutu Reeve. Results and their possible significance in the identification of differences in nutritional requirements between these bivalves are reported. Comparisons are made with previously published data on the food value of I. gulbunu. MATERIALS AND METHODS
Clam larvae were obtained from temperature stimulated spawnings of hatchery conditioned breeding stock and larvae of the Crussostreu spp. by pipetting gametes from opened mature oysters as described by Helm and Millican (1977). 0. edulis larvae were from broods liberated by hatchery maintained stocks (Helm et al., 1973). Trials of 4-days duration were duplicated in l-l borosilicate glass beakers with 400-600 larvae per treatment. Longer duration trials, in which larvae were reared to metamorphosis, were in 125-1 tapered, cylindrical, polyethylene vessels. These initially contained 12 000-16 000 larvae/l in 2-pm filtered, ultra-violet light sterilized sea water ( Walne, 1974). The culture temperature in all trials was 24.0 2 0.5 “C. Salinity was adjusted to 25%0 with filtered fresh water for Crussostreu spp. larvae and was at 30-33%0, the ambient, for other species. All vessels were aerated and complete water changes were made three times each week. I. gulbunu (Parke, Plymouth) and T-IS0 (Clone T-ISO, University of Delaware) were cultured in 20-l flasks as described by Walne (1966,1974). Twentyfive per cent of the culture was harvested three times each week, while in the active growth phase, and the volume restored with freshly prepared culture medium. Cell densities at harvest ranged from 12 000 to 20 OOO/pl and, in the case of T-ISO, the ash-free dry weight per lo6 cells averaged 20.8 ,ug. Chuetoceros culcitruns (Paulsen) Takano (Imai, Sendai) , used as the control diet, was cultured in 2-l batches in autoclaved sea water medium (Laing, 1979). Cell densities at harvest ranged from 45 000 to 65 OOO/pl and the mean ashfree dry weight per lo6 cells was 7.2 pg. Approximately equal rations of single-species diets of T-IS0 and C. culcitruns and a mixture of the two were provided daily in long duration trials. Rations were based on the ash-free dry weights of the two algae such that larvae were fed either 100 cells/p1 of T-ISO, 250 cells/p1 of C. culcitruns or half the quantity of each in the mixture. These rations were increased as the larvae grew and required greater quantities of food. Samples of the diets were taken for analysis of fatty acids. Analyses were made by D. L. Holland, UCNW,
283 TABLE 1 The growth of newly liberated larvae of Ostrea edulis (mean initial length 186.6 pm) in a 4-day period when fed different rations by cell density of T-IS0 and I. galbana (95% confidence intervals of final mean shell length are given) Diet
Cells (j&l )
Final shell length (pm )
1. gutbuna
40 80 120
232.8 k 1.9 226.9 Z!I2.9 223.8 + 2.3
T-IS0
40 80 120
231.Ok2.0 234.7k2.1 231.3k2.1
Marine Science Laboratories, Menai Bridge, using the methods Waldock and Holland (1984 ) .
described
by
RESULTS
A 4-day trial in l-1 glass beakers compared the value of T-IS0 and 1. g&bona to newly liberated larvae of 0. e&is. Both algae were fed at densities of 40,80 and 120 cells/pi. The larvae were 186.6 pm mean shell length initially and their mean sizes at the completion of the trial are given in Table 1. T-IS0 at the higher rations supported greater growth of larvae than at equivalent cell densities of I. galbana but there was no significant difference in growth at the 95% confidence level between the best cell density of each alga, i.e., 40 cells/p1 I. gulbunu, 80 cells/p1 T-ISO. Growth rates of Crussostreu spp. and clam larvae supported by single-species diets of T-ISO, C. culcitruns and the two-species combination are shown in Fig. 1. Values given are mean shell lengths from measurements of 50-100 individuals sampled at intervals throughout the pelagic growth phase until larvae were about to metamorphose. This was recognised by the development of eye spots in oyster larvae and pediveligers in clams. Day 0 denotes the time of spawning and fertilization. In both Crussostrea spp. the initial growth rate of larvae fed with T-IS0 alone was slightly lower than with the other diets (Table 2). After 4-6 days, varying with the oyster species, growth rates diverged. Small daily growth increments after the first 6-8 days with T-IS0 (Fig. 1) were partly the effect of a differential mortality of the smaller larvae. Larvae fed T-IS0 showed > 60% mortality by day 13 compared with 25%-35% with the other diets. Larvae fed C. culcitrans or the two-species diet grew strongly and reached settlement in 16 days and 15 days in C. gigus and C. rhizophorae, respectively. Enhanced growth effects were observed, the two-species mixture supporting significantly greater growth (PC 0.05) than when the algae were fed alone (Fig. 1).
-g
140
=I .c GI 3 i -E f : 0 I
60
0
2
4
6
6
10
12 14 16
260
601
0
2
4
6
-2
610
0
2
4
6
Days
Fig. 1, The growth of (A) C. gigas, (B ) C. rhizophorae, (C ) M. mercenaria and (D ) T. semidecusata larvae fed T-IS0 (closed circles), C. calcitrans (open circles) and the two-species diet (triangles). Vertical bars indicate the 95% confidence intervals of mean shell length.
A4. mercenaria grew as well when fed T-IS0 as with either of the other diets and high yields of post-metamorphosis juveniles were obtained from each treatment. Results were less conclusive from the T. semidecussata trial because all larvae were fed C. calcitrans between days 1 and 2. From day 2, when the three diet treatments were started, larvae grew rapidly to the onset of metamorphosis on each diet. Enhanced growth effects were observed in T. semidecussata in response to the two-species diet but not with A4. mercenaria. Data on the polyunsaturated fatty acid composition of T-IS0 and C. calcitrans sampled during the reported trials are given in Table 3. Values are compared with previously published data for these species and with I. galbana.
285 TABLE 2 Mean shell lengths + 95% confidence intervals of early veligers of C. gigas and C. rhizophorae fed different diets. Daily rations of the diets as algae cells (,I&‘) are given. C. gigas larvae were 73.4 pm initially and C. rhizophorae 64.2 pm Diet (cells/pi)
Mean shell length (pm) C. rhizophorae
C.gigas
T-IS0 (100) C. calcitrans (250) T-IS0 + C. calcitrans (50 + 125)
Day 4
Day 6
Day 6
Day 8
90.2 * 2.7 93.4 rt 2.0 97.Ok2.7
101.4 & 4.3 120.4 + 3.2 128.2 f 3.8
102.6 i 2.5 107.4? 3.2 109.4 * 3.2
103.4k3.1 127.8 4 3.0 137.0? 3.1
DISCUSSION
T-IS0 can be beneficial as a constituent species of a mixed algal diet as shown by significantly improved growth of the larvae of three of the four bivalves tested. However, only M. mercenaria grew and developed as rapidly with the mono-species diet of T-IS0 as with the other diets tested. In T. semidecussatu, larvae grew to metamorphosis on the T-IS0 diet but by day 12 mean shell length was significantly smaller than in those fed only C. culcitruns or the twospecies combination. For both C. gigas and C. rhizophorue T-IS0 was a poor food; growth and development slowed beyond day 6 and larvae failed to reach settlement within the trial period. These results suggest that T-IS0 is lacking in a component (or components ) vital to normal growth and development in Crussostreu spp. larvae. It is possible that larvae carry sufficient of this component over from parentally derived reserves, via the eggs, to support early TABLE 3 Polyunsaturated fatty acid composition of T-IS0 (Pillsbury, 1985)) I. galbana ( Waldock and Nascimento, 1979) and C. calcitrans (Webb and Chu, 1983). Data in parentheses refer to cultures from this laboratory, taken during the trials and analysed by D. L. Holland” (Marine Science Laboratories, Menai Bridge). Values are percentages of total fatty acids Fatty acid
T-IS0
18: 206 18: 303 18: 4~3 20: 5w3 22: 603
4.0 5.6 19.4 -( 7.9
’ See Waldock and Holland
( 6.9 ) ( 4.3 ) ( 8.7 ) 0.71) (11.4 )
(1984) for methods
used.
I. galbana
C. calcitrans
4.3 11.0 9.2 3.6 18.9
0.2 3.0 0.5 15.4 1.9
( 1.8 ) ( 1.5 ) ( 0.6 ) (22.9 ) ( 0.97)
development. The growth of early veligers fed on T-IS0 was similar to the growth when fed with the two other diets (Table 2). It can be assumed that the larvae are unable to biosynthesise or have a limiting biosynthesis rate for this component to permit normal development. A similar pattern of larval growth on a single-species diet of T-IS0 was reported by Pillsbury (1985) working with the queen conch, Strombus gigas Linne. The initial rate of growth after hatching was equally as rapid as with the best diet tested but then slowed so that by day 20 mean size was not significantly different from the size 6 days earlier. Pillsbury (1985) quotes the fatty acid profile of T-IS0 which, when compared with profiles for I. galbana ( Waldock and Nascimento, 1979; Webb and Chu, 1983)) shows that T-IS0 lacks the polyunsaturated fatty acid, 205~3 while 22:6w3 is present in both. In contrast, T-IS0 cultured at Conwy contained 20~503 but as a lower percentage of total fatty acids than found in I. galbana by Waldock and Nascimento (1979) (Table 3 ) . Both of these fatty acids are regarded as important in the nutrition of crustacean, molluscan and fish larvae (see reviews in Kanazawa, 1983; Webb and Chu, 1983; Pillsbury, 1985). The total lipid content of I. galbana is similar to that of T-ISO. Utting (1985) quotes values of 16.9%-22.0% of dry weight for I. galbana depending on nitrogen availability in the culture medium, and Pillsbury (1985) a value of 23.0% of dry weight in T-ISO. In the work reported here T-IS0 was cultured in similar conditions to those used previously for I. galbana and its total lipid content was 21.1% +-3.6% (standard deviation) of ash-free dry weight, equivalent to 19.0% of dry weight. Generally, I. galbana has been regarded as a satisfactory food for bivalve larvae (Loosanoff and Davis, 1963; Walne, 1965). Certainly, it is one of the better of the food species tested for 0. edulis, supporting rapid rates of growth in larvae at all stages in development (Helm, 1977; Wilson, 1979). However, it was fully tested at this laboratory only as a constituent of a mixed algal diet in the culture of C. gigas larvae (Helm and Millican, 1977). Work by Utting (1979) and Nascimento (1980) with C. gigus straight-hinged, D-veligers demonstrated the nutritional superiority of C. calcitrans over I. galbana but these trials were of insufficient duration to identify possible longterm deficiencies arising with the latter species. Long duration nutrition trials by Wilson (1978) provided information which points to a nutritional deficiency developing in C. gigas larvae fed solely on I. galbana. In a series of six trials he grew veligers from the D-stage for 12 days. All growth data were converted to growth coefficients and, in the absence of quoted values of initial mean shell lengths, it is difficult to interpolate accurately the sizes reached by larvae as the trials progressed. Assuming that larvae were 75 pm mean shell length at the beginning of each trial (from Helm and Millican, 1977) calculations from the mean K4 values given by Wilson (1978) show that they reached 92.5 pm after 4 days, 111.9 ,um after 8 days, and 133.9 ,um after 12 days. These
287
estimates suggest a growth performance similar to that with T-IS0 but not as good as with C. calcitruns. (Wilson found that better growth was obtained with the diatom Phaeodactylum tricornutum Bohlin. ) Evidence, therefore, suggests that T-IS0 and I. gulbunu are both poor foods for C. gigus larvae and probably for all Crassostreu spp. T-IS0 was of poor value to C. rhizophorae here and I. gulbunu did not rank among the better foods for larvae of C. uirginicu in feeding trials by.Davis (1953). In contrast, both flagellates seem good foods for 0. edulis and the various clam species mentioned. C. culcitruns is shown to be a very good food for Crussostreu spp. larvae as well as for the clams (Fig. 1) . Waldock and Nascimento (1979) quote its fatty acid content as 9.7% of dry weight compared with 12.5% for I. gulbunu. Of this total, 15.4% was 20x503 compared with 3.6% in I. gulbanu, while values for 22:6@3 were 1.9% and l&9%, respectively (Table 3). Similarly high values of 20:5co3 and low values of 22:6~3 were determined in C. calcitruns cultured for the present trials. Waldock and Nascimento (1979) show that larval growth rate was not correlated with the amount of lipid in the diet nor were differences in fatty acid composition solely responsible for growth rate differences between the diets. Pillsbury (1985) reached similar conclusions. Nevertheless, such marked differences in larval growth rates between bivalve species with diets of C. calcitruns compared with I. gulbuna and T-IS0 provide a useful starting point to evaluate precise nutritional requirements. It is possible that the solution may be not only in the presence or absence of certain essential components in the diet but in the correct balance between them, which may differ for the various bivalves so far investigated. The possibility arises in this present work that Crassostrea spp. larvae have a high specific requirement for 20:5~3 met by feeding them with C. culcitruns but not with I. gaZbana or T-ISO, where limited quantities of this polyunsaturate were available. The species of clam tested appear to have no similar requirement for 20:51x3, growing equally as well with all diets offered. Since all these diets contained 22:6w3 a specific requirement may be indicated and needs to be tested. Alternatively, the relative food value of the different algae to different bivalves may be related to other nutritionally important factors.
REFERENCES Davis, H. C., 1953. On food and feeding of larvae of the American oyster, C. uirginica. Biol. Bull., 104: 334-350. Ewart, J. W. and Epifanio, C. E., 1981. A tropical flagellate food for larval and juvenile oysters, Crassostrea virginica Gmelin. Aquaculture, 22: 297-300. Helm, M. M., 1977. Mixed algal feeding of Ostrea edulis larvae with Zsochrysisgalbana and Tetraselmis suecica. J. Mar. Biol. Assoc. U.K., 57: 1019-1029. Helm, M. M. and Millican, P. F., 1977. Experiments in the hatchery rearing of Pacific oyster larvae (Crussostreagigas Thunberg). Aquaculture, 11: 1-12.
Helm, M. M., Holland, D. L. and Stephenson, R. R., 1973. The effect of supplementary algal feeding of a hatchery breeding stock of Ostrea edulis L. on larval vigour. J. Mar. Biol. Assoc. U.K., 53: 673-684. Kanazawa, A., 1983. Penaeid nutrition. In: G.D. Pruder, C. Langdon and D. Conklin (Editors), Biochemical and Physiological Approaches to Shellfish Nutrition, Proc. 2nd Int. Conf. Aquaculture Nutr. World Maricult. Sot. Spec. Publ. No. 2, Louisiana State University, LA, pp. 87-105. Laing, I., 1979. Recommended procedures for the culture of Chaetoceros calcitrans. Fish. Res. Tech. Rep., MAFF Direct. Fish. Res., Lowestoft, No. 53: 18 pp. Loosanoff, V. L. and Davis, H. C., 1963. Rearing of bivalve molluscs. Adv. Mar. Biol., 1: 1-136. Nascimento, I. A., 1980. Growth of the larvae of Crassostrea gigas Thunberg, fed with different algal species at high cell concentrations. J. Cons. Int. Explor. Mer, 39 (2) : 134-139. Pillsbury, K. S., 1985. The relative food value and biochemical composition of five phytoplankton diets for queen conch, Strombus gigas (Linne) larvae. J. Exp. Mar. Biol. Ecol., 90: 221-231. Utting, S. D., 1979. The biochemical content of Chaetoceros calcitrans, Tetraselmis suecica and Zsochrysisgalbana in relation to the growth of Crassostreagigas larvae and spat. Ph. D. Thesis, University College of North Wales, Marine Science Laboratories, Menai Bridge, 89 pp. Utting, S. D., 1985. Influence of nitrogen availability on the biochemical composition of three unicellular marine algae of commercial importance. Aquacultural Eng., 4: 175-190. Waldock, M. J. and Holland, D. L., 1984. Fatty acid metabolism in young oysters, Crassostrea gigas: polyunsaturated fatty acids. Lipids, 19 (5) : 332-336. Waldock, M. J. and Nascimento, I. A., 1979. The triacylglycerol composition of Crassostrea gigas larvae fed on different diets. Mar. Biol. Lett., 1: 77-86. Walne, P. R., 1965. Observations on the influence of food supply and temperature on the feeding and growth of the larvae of Ostrea edulis L. Fishery Invest., Lond. Ser. 2,24 (1): l-45. Walne, P. R., 1966. Experiments in the large-scale culture of the larvae of Ostrea edulis L. Fishery Invest., Lond. Ser. 2,25 (4) : l-53. Walne, P. R., 1974. Culture of Bivalve Molluscs, 50 Years’ Experience at Conwy. Fishing News (Books), West Byfleet, 173 pp. Webb, K. L. and Chu, F. L., 1983. Phytoplankton as a food source for bivalve larvae. In: G. D. Pruder, C. Langdon and D. Conklin (Editors), Biochemical and Physiological Approaches to Shellfish Nutrition. Proc. 2nd Int. Conf. Aquaculture Nutr. World Mar&cult. Sot. Spec. Publ. No. 2, Louisiana State University, LA, pp. 272-291. Wilson, J. H., 1978. The food value of Phaeodactylum tricornutum Bohlin to the larvae of Ostrea edulis L. and Crassostrea gigas Thunberg. Aquaculture, 13: 313-323. Wilson, J. H., 1979. Observations on the grazing rates and growth of Ostrea edulis L. larvae when fed algal cultures of different ages. J. Exp. Mar. Biol. Ecol., 38: 187-199.