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Carbohydrate and fatty acid composition of the rotifer, Brachionus plicatilis, fed monospecific diets of yeast or phytoplankton
263
Aquaculture, 89 ( 1990) 263-212 Elsevier Science Publishers B.V., Amsterdam
Carbohydrate and fatty acid composition of the rotifer, Bra&onus plicatilis, fed monospecific diets of yeast or phytoplankton John N.C. Whyte and Warren D. Nagata Department of Fisheries and Oceans, Biological Sciences Branch, Pacific Biological Station, Nanaimo, B.C., V9R 5K6 (Canada) (Accepted 20 January 1990)
ABSTRACT Whyte, J.N.C. and Nagata, W.D., 1990. Carbohydrate and fatty acid composition of the rotifer, Brachionusplicatilis, fed monospecific diets of yeast or phytoplankton. Aquaculture, 89: 263-212. Rotifers fed yeast, Saccharomyces cerevisiae, and four phytoplanktons, Thalassiosira pseudonana, Zsochrysis aff. galbana (T-iso clone), Tetraselmis suecica, and Chlorella saccharophila, contained carbohydrate composed of 61430% glucose, 9-18% ribose and 0.8-7.0% of galactose, mannose, deoxyglucose, fucose and xylose, with relative proportions of each being influenced by diet. Glucose was present as glycogen which was characterized, for the first time in a rotifer, by the absorbance maximum of the iodine complex and the action of amylolytic enzymes on the isolated polysaccharide. Detailed fatty acid profiles of rotifers and diets illustrated the transfer and storage of specific fatty acids from the diets to the rotifers. The diatom T. pseudonana was the only diet to produce rotifers with the required complement of n3 polyunsaturated fatty acids suitable for larval fish rearing.
INTRODUCTION
The nutritional quality of cultured Brachionus plicatilis for rearing larval fish depends on the transfer of dietary components from phytoplankton or yeast to the rotifer. Although proximate chemical composition of the rotifer and its diet may not be similar, constituent units of dietary macronutrients, particularly fatty acids of lipids, are known to be transferred (Watanabe et al., 1983). Carbohydrate contents in 3. plicatilis fed phytoplankton and yeasts have been reported by Scott and Baynes ( 1978), and Ben-Amotz et al. ( 1987); yet no information on the sugar constituents of this carbohydrate component has been published. Fatty acid composition of rotifers is extremely important because n3 polyunsaturated fatty acids, particularly 20 : 5n3 and 22 : 6n3 acids, are essential for early larval growth of many fish species (Scott and Middleton, 1979; Watanabe et al., 1983; Ben-Amotz et al., 1987; Dendrinos and 0044-8486/90/$03.50
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B.V.
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J.N.C. WHYTE AND W.D. NAGATA
Thorpe, 1987; James and Abu-Rezeq, 1988). Rotifers are capable of synthesizing n3 polyunsaturated fatty acids, but tissue levels are insufficient to meet the demands for normal growth and development of fish larvae (Lubzens et al., 1985). Tissue accumulation of these acids by rotifers is therefore dependent on exogenous supplies from phytoplankton, and in this study, species which have not been used before were examined as dietary sources of fatty acids for rotifers. Sugar constituents of rotifers fed Saccharowlyces cerevisiae, Chlorella saccharophila, Isochrysis aff. galbana (T-iso clone), Tetraselmis suecica, and Thalassiusira pseudonana were investigated. Dietary influence on the presence and quantity of essential fatty acids in rotifers used in feeding larval fish was also examined. MATERIALS AND METHODS
Rotifer growth A clone of B. plicatilis isolated from the Seto Inland Sea was used. C. saccharophila was cultured on Yashima medium (SISFFA, 1964) and I. aff. galbana (T-iso clone), T. suecica, and T. pseudonana (3H clone) were batch
cultured in filtered ( 1 pm) sterilized seawater (28 2 l%o) enriched with nutrients (Harrison et al., 1980). All algae were grown at 20 5 2 oC under continuous light at 1 klux. S. cerevisiae was moist baker’s yeast (Fleishmann’s Yeast Ltd.) stored at 5°C. A 20-l stock culture of B. plicatilis was fed a mixture of the diets for a 2-week acclimation period, then subdivided into 15 groups, each placed into a 2-l Erlenmeyer flask. Three replicate flasks were supplied with each of the five experimental diets over a 15-day experimental period. The rotifers were maintained at 20 + 0.5 ‘C, 20%0 salinity, and 1 klux light intensity on a 16 : 8 h L: D photoperiod. Algal cultures were centrifuged and resuspended in 1 1 filtered seawater (20%0) to the desired concentration. These suspensions were fed to the rotifers daily following removal of 1 1 of the culture medium. No aeration was provided, but the flasks were swirled twice daily. Feeding levels were scaled on a dry weight basis and equivalent food rations were provided throughout the experiment. Initially food was provided to a level of 22 pg dry wt./ml medium as established from previous measurements on C. saccharophila (Nagata, 1985 ) . As the rotifer populations increased, the amount of food provided was increased proportionally if none remained in suspension. The rotifers were collected on a 40-pm screen, washed with cold 3% ammonium formate solution, concentrated by centrifugation, frozen at - 80 ’ C, and lyophilized for subsequent analysis.
COMPOSITION
OF BZUCHZONUS
PLZCATZLZSFED YEAST OR PHYTOPLANKTON
265
Analysis of neutral sugars Dry rotifers (25 mg) were washed successively on a fused fiberglass filter with chloroform and acetone (50 ml each) and the defatted residue hydrolyzed with 0.5 M H2S04 at 100°C for 16 h. The hydrolysate was neutralized with barium carbonate to yield parent sugars which were derivatized to corresponding alditol acetates (Sawardeker et al., 1965 ) . Derivatives were identified by comparison with standard alditol acetates and percentage composition determined by gas-liquid chromatography on glass columns ( 1.8 m x 0.32 cm) of (a) 3% Silar 1OC and (b) 3% ECNSS-M on Gas Chrom Q ( loo-120 mesh) operating at 205 and 195 ‘C, respectively, with a nitrogen flow rate of 30 ml/min. Mean values presented are from replicate samples and had coefficients of variance 25%. Isolation and analysis of polysaccharides Dry rotifers ( 1.O g) fed T. pseudonana were extracted with a homogeneous mixture of chloroform : methanol : water (2 : 4 : 1; v/v/v, 4 x 50 ml portions) using a cell disrupter for mixing (Whyte et al., 1987 ) . The sample, freed from lipid and low-molecular weight compounds, was then extracted with 200 ml water at 60’ C for 30 min and the mixture centrifuged. Extraction of the residue with two further 50-ml portions of water at 60°C yielded a combined supernatant which was concentrated by rotary evaporation to about 50 ml and cooled to 0 “C in an ice bath. Trichloroacetic acid ( lo%, 20 ml, 0’ C ) was added, the solution stirred and allowed to stand for 4 min. Filtration of the resultant mixture through’a GF/C filter removed proteinaceous material and the filtrate was poured into 4 volumes of 95% ethanol. The precipitated polysaccharide was allowed to settle and then isolated by centrifugation. Further purification by dissolution in distilled water, clarification by filtration and reprecipitation in ethanol, gave a polysaccharide which was dialyzed against cold-distilled water for 2 days, and freeze-dried to yield glucan, 59 mg ( 5.9%). Polysaccharide (5 mg) was hydrolyzed and sugars derivatized to alditol acetates and examined by gas-liquid chromatography as described previously. Purity of the polysaccharide was determined using the phenol-sulphuric acid reagent of Dubois et al. ( 1956) with D-glucose as the calibration standard. To prepare the iodine-polysaccharide complex, polysaccharide (5 mg) was dissolved initially in 6 ml water, then HCl (3 M, 0.02 ml) was added, followed by iodine solution (0.2 ml, containing 2 mg iodine and 20 mg KI per ml) and the resultant solution diluted to 10 ml. Maximum absorbance was measured in a 1-cm cell against iodine solution in the reference cell. Oyster glycogen was treated in an identical manner and absorbance of the iodine complex recorded as a control. Polysaccharide ( 12 mg) was dissolved in water (25 ml, containing 0.2 ml of 0.2 M acetate buffer at pH 7.0) and porcine pancreas cx-amylase (2 mg, Sigma VI-A) added and the solution stirred slowly at 2 1 oC. Similarly, poly-
266
J.N.C. WHYTE AND W.D. NAGATA
saccharide ( 12 mg) was dissolved in water (25 ml, containing 0.2 ml 0.2 M acetate buffer at pH 4.9) and barley fi-amylase (2 mg, Sigma II-B) added. Samples ( 10 ml) of the enzyme hydrolysates were removed after 1 and 16 h and evaporated to dryness on a rotary evaporator. The hydrolysate residues were extracted into 80% aqueous ethanol, evaporated to dryness, redissolved in water, deionized by cation exchange resin, and evaporated to dryness. Oyster glycogen was treated by both enzymes in exactly the same manner as controls. Qualitative analysis of sugar hydrolysates used paper chromatography on Whatman No. 1 paper in the solvent systems (a) ethyl acetate-pyridinewater, 10 : 4 : 3, (b) ethyl acetate-acetic acid-formic acid-water, 18 : 3 : 1: 4 and (c) n-butanol-ethanol-water, 3 : 1: 1. Sugars were detected with alkaline silver nitrate reagent (Trevelyan et al., 1950). Lipid contents andfatty acid profiles Total lipids in the rotifers were extracted and determined by the procedures described by Whyte et al. ( 1987). Dry rotifers and diets (50 mg) were saponified and methylated in a Reactivial according to the procedure described by Whyte ( 1988). Analyte solutions were separated on a Supelcowax 10 fused silica capillary column ( 30 m x 0.32 mm ID, 0.25-pm film). Fatty acids were identified by comparison with standard mixtures or ECLs and assigned in accord with data presented by Ackman ( 1986 ). Peaks of less than 0.2% were not included in the profiles, and the mean values presented from replicate samples had coefficients of variance 55%. RESULTS
Glucose formed 6 l-80% of the total sugar constituents of rotifers, indicating the presence of a storage glucan which varied with diet (Table 1) . A high percentage, 9- 18%, of ribose suggested a considerable incorporation of riboTABLE
1
Percent composition phytoplankton
of neutral
sugars in Brachionus plicatiiis fed yeast and four species
of
Sugars
Yeast
Thalassiosira pseudonana (3H clone)
Isochrysis galbana (T-iso clone)
Tetraselmis suecica
ChlorelIa saccharophila
Fucose Ribose Xylose Deoxyglucose Mannose Galactose Glucose
3.89 17.93 0.80 0.79 7.40 1.42 67.77
6.73 16.11 3.73 3.34 5.12 3.73 61.24
5.50 12.87 1.65 1.60 4.95 2.95 71.28
4.92 11.29 2.42 3.20 3.91 3.16 71.10
3.43 9.30 1.99 1.63 2.72 1.39 79.54
COMPOSITION OF BRACHIONUS
PLICATILIS
FED YEAST OR PHYTOPLANKTON
267
nucleotide units from nucleic acids in all rotifers. Lesser amounts, ranging from 0.8 to 7.0% of galactose, mannose, 2-deoxyglucose, fucose and xylose formed the remainder of the neutral sugar constituents in the rotifers. Hot-water extraction of defatted rotifers fed T. pseudomna yielded 5.9% of a purified polysaccharide. Acid hydrolysis, and gas-liquid chromatographic analysis of the derived sugar alditol acetates revealed that only glucose was present. The glucan contained 98.4% glucose and exhibited a maximum absorbance for the corresponding iodine complex at 480 nm. This value was consistent with absorbance maxima of 420-490 nm for iodine complexes of glycogens from many sources rather than the 500-550~nm absorbances of starches (Percival and McDowell, 1967). The iodine complex of oyster glycogen exhibited a maximum absorbance at 467 nm. Treatment of the glucan with a-amylase for 1 h yielded mainly glucose and maltose with minor amounts of maltotriose, as shown by paper chromatography in three solvent systems. An increase in glucose, a decrease in maltose, but no maltotriose was evident in the hydrolysate when the enzyme treatment was continued for 16 h. The action of fi-amylase on the glucan for 1 h showed mainly maltose on paper chromatography with minor amounts of glucose, the latter indicating the presence of a second amylolytic enzyme presumed to be cu-amylase in the commercial enzyme product. Similar depolymerization products were obtained by the action of both enzymes on oyster glycogen. The enzymatic depolymerization was consistent with a glycogen structure for the glucan. Total lipids in rotifers fed S. cerevisiae, T. pseudonana, I. gaZbana (T-iso ) , T. suecica and C. saccharophila were 9.8 + 0.2%, lO.O+ 0.3%, 15.8 -+0.4%, 13.12 0.3%, and 9.8 5 0.1% of the dry weight, respectively. The relative percentages of fatty acids in the total lipids of the rotifers and phytoplankton fed to the rotifers are presented in Table 2. Major fatty acids in S. cerevisiae and corresponding rotifers fed this yeast were 16 : 0, 16 : 1n7, and 18 : 1n9, and in T. pseudonana and rotifers were 14:0, 16:0, 16: 1~17, 18:4n3, 20:5n3 and 22:6n3. 1. galbana (T-iso) and rotifers that consumed this flagellate contained 14:0, 16:0, 18: ln9, 18:2n6, 18:3n3,18:4n3, and 22:6n3 as themajor acids, whereas T. suecica and the corresponding rotifers contained 16 : 0, 16:4n3, 18: ln9, 18: ln7, 18:2n6, 18:3n3, 18:4n3 and 20:5n3. The major acids in C. saccharophila and corresponding rotifers contained predominantly 16:0, 16: ln7,18: ln9, 18:2n6 and 18:3n6 acids. Lipids of C. succharophila, S. cerevisiae and rotifers fed these diets contained the highest level of saturated and monoethylenic fatty acids, respectively (Table 2). In general, the levels of polyethylenic fatty acids in rotifers were similar to levels in the corresponding diets. The level of polyethylenic fatty acids in lipids of all algae was 10 times that of the yeast, and n3 polyunsaturated fatty acids constituted over 50% of the polyethylenic fraction of all algae but C. saccharophila. Eicosapentaenoic acid, 20: 5n3, comprised 50%,
J.N.C.WHYTE AND W.D. NAGATA
268 TABLE 2
Fatty acids in the total lipids of Brachionus plicatilis fed yeast and four species of phytoplankton and in the total lipids of the five diets Fatty acids composition (% of total)
14:o 14: ln5 15:o 16:0 16: ln9 16: ln7 16:2n6 16:2n4 17:o 16:3n4 16:3n3 16:3nl 16:4n3 16:4nl 18:0 18:lnll 18: ln9 18: ln7 18:2n7 18:2n6 18:2n4 18:3n6 18:3n3 18:4n3 20: lnll 20: ln9 20: ln7 20:2n6 20: 3n6 20 : 4n6 20: 3n3 20:4n3 20:5n3 22: ln9 21:5n3 22:4n6 22: 5n6 22:5n3 22:6n3 Saturated Monoethylenic Polyethylenic X4-6n3/poly. 20: 5n3/poly. 22: 6n3/poly.
Yeast
Thalassiosira pseudonana
Zsochysis galbana (T-iso)
Tetraselmis suecica
Chlorella saccharophila
Rotifer
Diet
3.56 0.64 0.69 19.72 0.98 7.70 0.25 1.69 2.25
0.27 0.42 40.77 1.42 4.48 0.33 0.35 0.84
1.07
0.31 0.73
1.25
3.00 4.63 5.10 3.53 6.24 12.87 1.25 3.88 1.01 0.72
0.73 2.67 0.84 19.32 15.59 2.98 1.03 -
0.99
0.98 7.03 5.48
0.38 0.28 -
0.35 5.98
0.73 0.68 0.50
0.20 -
_
1.78
-
1.84
-
(3H) Rotifer
Diet
Rotifer
Diet
Rotifer
Diet
Rotifer
1.94 0.52 0.59 7.26 0.62 23.58
0.68 0.20 8.41 39.14
6.53 0.51 0.56 11.44 3.34 25.13 2.73 3.74
7.85 0.29 0.29 10.56 0.48 3.36
0.73 0.62
0.31 0.71
4.61 0.51 0.73 12.78 13.18 1.38 1.99 2.71
15.47 0.31 0.20 8.33 2.84 3.77 0.40 0.73 -
1.12 0.28 0.32 13.26 0.98 1.75 0.44 1.41 1.11
4.07
0.88 1.94
3.97 40.92 -
2.55 1.13
2.4 -
1.59 1.28
0.78 0.24
2.83
1.28 2.40 0.98 0.20 0.93
0.20
0.28 0.49
8.89 1.63
3.64 2.98 5.29 4.86
0.29
7.17
8.72
9.47
7.48 -
6.47 12.26
1.16 7.72 18.55
1.07 14.44 4.38 0.75 2.65 0.51 0.32 0.79 2.70 0.72 4.44 4.94 0.97
0.88 11.69 9.43
0.25 6.09
3.14
0.96
0.45 0.99 1.70 _
-
23.33 -
0.78 14.65 66.26 11.59 49.18 0.00 6.73
5.95 2.22 1.16
_
_
13.31 80.50 3.96 19.70 0.00 0.00
6.65 6.02 22.00 22.50 48.77 78.55 42.11 12.34
1.02 1.03 11.49 -
2.50 0.86 15.25 3.04
1.41
_
20.54 0.95
14.23 6.67 1.60 0.47 2.27
4.42
1.75 3..88 1.36 5.34 1.40 1.25 -
0.39 0.63
0.41 -
0.56 4.13 2.50 28.46 6.76
Diet
4.30 18.53 29.75 46.24 72.92 50.45 9.30
2.07 1.40 8.53 21.20 27.58 48.44 67.57 4.58 17.61
_ 0.24 0.78 0.83 _ 0.25 2.21 10.84 24.20 17.44 52.84 57.19 1.57 20.51
,
2.23 1.39 19.45 21.02 53.16 50.51 9.29 2.61
0.20 11.80 2.20 -
14.82 22.90 53.28 53.00 11.22 0.00
28.66 24.81 46.34 9.32 1.47 0.00
42.12 9.83 41.99 4.66 0.00 0.00
COMPOSITION OF 3RACHfOh’US PLICATILIS
FED YEAST OR PHYTOPLANKTON
269
2%, and 11% of the polyethylenic fraction of T. pseudonana, I. galbana (Tiso), and T. suecica, respectively and was absent in both C. saccharophitaand S. cerevisiae. Docosahexaenoic acid, 22: 6n3, was present only in T. pseudonana and I. galbana (T-iso) at 9% and 2 1% of the polyethylenic fraction, respectively. DISCUSSION
The value of rotifer carbohydrate as a dietary energy reserve when fed to crustacean or fin-fish larvae is unknown, as sugars present in rotifers have never been identified. Results indicated that glucose formed 61-80% of the rotifer sugars which consisted mainly as a glucan characterized as glycogen, the ubiquitous source of energy, via the citric acid cycle, in most animals. Although carbohydrate in general forms a minor component of the marine food chain, its use as an energy source “sparing” protein for tissue synthesis has been recognized in nutritional studies of aquaculture species (Enright et al., 1986; Alava and Pascual, 1987; Degani and Viola, 1987; Whyte et al., 1989). Assimilation of rotifer glycogen by larval fish is probable because digestion of starches, considerably more insoluble than glycogen, has served as a source of energy in fish (Bergot and Breque, 1983 ) . Glycogen formed the major portion of carbohydrates in rotifers fed different diets in this study, and therefore weight gain of larval fish from this energy source would probably be comparable regardless of diet. The fatty acid profiles of rotifers and corresponding diets fed to the rotifers indicated transferral and storage of major fatty acid constituents in the feeding process. Similar results have been reported for B. plicatilisfed other species of phytoplankton (Scott and Middleton, 1979; Ben-Amotz et al., 1987). The presence of fatty acids, such as 18 : 1n 11,22 : 1n9,22 : 5n3 and 22 : 6n3 in the rotifers but not in corresponding diets, suggests endogenous synthesis of these acids and/or assimilation and concentration of minor amounts which were not evident in the profiles of the diets. Similarly, the active accumulation of 18 : 0,18 : 1n7,18 : 1n 1 I, and 20: 1n9 fatty acids by the rotifers, regardless of diet supply, implied a need for these acids during growth and development. Marine fish have been reported to be unable to synthesize de novo polyunsaturated fatty acids of either the n6 or n3 series (Sinnhuber, 1969; Cowey and Sargent, 1972; Kanazawa et al., 1979) and rotifers have been found to synthesize only minor amounts of n3 polyunsaturated acids (Lubzens et al., 1985 ). Therefore, these acids must be provided by the rotifer diet to meet the possible requirement of larval fish. In this laboratory provision of polyunsaturated fatty acids to rotifers was crucial to their use as food for larval sablefish, Anaplopoma fimbria. Eggs of the sablefish have been found to contain almost 90% 4-6n3 fatty acids in the total polyethylenic acid fraction, and
270
J.N.C. WHYTE AND W.D. NAGATA
86% of the total 4-6n3 acids consisted of approximately equal amounts of 20 : 5n3 and 22 : 6n3 acids (Whyte, 1988 ). The essential requirement for both these acids in fish has been reviewed by Watanabe et al. ( 1983), but in addition, dietary 18 : 3n3 has been reported to promote growth of turbot (Gate-soupe et al., 1977) and sole (Dendrinos and Thorpe, 1987). A level of approximately 12% of 20: 5n3 in rotifers was considered normal for feeding marine fish larvae (Watanabe et al., 1983 ). Rotifers fed T. pseudonana, I. galbana (T-iso) and T. suecica contained lO.O%, 15.8% and 13.1% total lipids, which included in their fatty acid profiles 20.5%, 2.2% and 4.9%, 20: 5n3 acid, 6.0%, 8.5% and 1.4%, 22: 6n3 acid and 1.3%, 6.5% and 14.4% 18 : 3n3 acid, respectively. T. pseudonana was the only algal diet that produced rotifers with sufficient tissue levels of essential fatty acids. The diatom is easy to mass-culture, and its potential to provide a complement of essential fatty acids in the rotifers suitable for larval fish growth is recorded for the first time. Levels of essential acids were minimal or absent in the 9.8% total lipids in rotifers fed S. cerevisiaeor C. saccharophilu.Although this deficiency has been recognized in strains of yeast (Dendrinos and Thorpe, 1987 ) , highly variable levels of these acids, O-20+%, have been recorded in rotifers fed species of ChZoreZZa (Teshima et al., 1983, 1987; Ben-Amotz et al., 1987; James and Abu-Rezeq, 1988 ) . The variance in fatty acid profiles in species of ChZoreZZa has been attributed to culture conditions but may also reflect taxonomic inaccuracies (Fukusho, 1985; James and Abu-Rezeq, 1988). These results illustrated that among all the diets examined only T. pseudonana was capable of supplying the required composite of n3 polyunsaturated fatty acids. Predictable culture of larval fish will not be fully realized without the controlled culture of nutrient-specific rotifers used as food for the early and critical stages of larval development. In this study on rotifers, diet changed the carbohydrate constituents slightly, but glycogen remained the dominant component which would be available as an energy source for developing fish larvae. Diet was more crucial in formation of lipid constituents, and only the rotifers fed T. pseudonana provided the complement of fatty acids considered essential for culture of marine organisms. ACKNOWLEDGEMENT
W.D. Nagata expresses his thanks to the Natural Science and Engineering Research Council of Canada who provided funding through an NSERC visiting fellowship. REFERENCES A&man, R.G., 1986. WCOT (capillary) gas-liquid chromatography. J.B. Rossell (Editors), Analysis of Oils and Fats. Elsevier Applied New York, NY, pp. 137-206.
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COMPOSITION OF BRACHIONUS
PLICATILIS
FED YEAST OR PHYTOPLANKTON
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Teshima, S., Yamasaki, S. and Hirata, H., 1983. Effects of water temperature and salinity on eicosapentaenoic acid level of marine Chlorella. Bull. Jpn. Sot. Sci. Fish., 49: 805. Teshima, S., Kanazawa, A., Horinoushi, K_, Yamasaki, S. and Hirata, H., 1987. Phospholipids of the rotifer, prawn, and larval fish. Nippon Suisan Gakkaishi, 53: 609-6 15. Trevelyan, W.E., Procter, D.P. and Harrison, J.S., 1950. Detection of sugars on paper chromatograms. Nature (London), 166: 444-445. Watanabe, T., Kitajima, C. and Fujita, S., 1983. Nutritional values of live organisms used in Japan for mass propagation of fish: a review. Aquaculture, 34: 115- 143. Whyte, J.N.C., 1988. Fatty acid profiles from direct methanolysis of lipids in tissue of cultured species. Aquaculture, 75: 193-203. Whyte, J.N.C., Boume, N. and Hodgson, C.A., 1987. Assessment of biochemical composition and energy reserves in larvae of the scallop Patinopecten yessoensis. J. Exp. Mar. Biol. Ecol., 113: 113-124. Whyte, J.N.C., Boume, N. and Hodgson, C.A., 1989. Influence of algal diets on biochemical composition and energy reserves in Patinopecten yessoensis (Jay) larvae. Aquaculture, 78: 333-347.
Aquaculture, 89 ( 1990) 263-212 Elsevier Science Publishers B.V., Amsterdam
Carbohydrate and fatty acid composition of the rotifer, Bra&onus plicatilis, fed monospecific diets of yeast or phytoplankton John N.C. Whyte and Warren D. Nagata Department of Fisheries and Oceans, Biological Sciences Branch, Pacific Biological Station, Nanaimo, B.C., V9R 5K6 (Canada) (Accepted 20 January 1990)
ABSTRACT Whyte, J.N.C. and Nagata, W.D., 1990. Carbohydrate and fatty acid composition of the rotifer, Brachionusplicatilis, fed monospecific diets of yeast or phytoplankton. Aquaculture, 89: 263-212. Rotifers fed yeast, Saccharomyces cerevisiae, and four phytoplanktons, Thalassiosira pseudonana, Zsochrysis aff. galbana (T-iso clone), Tetraselmis suecica, and Chlorella saccharophila, contained carbohydrate composed of 61430% glucose, 9-18% ribose and 0.8-7.0% of galactose, mannose, deoxyglucose, fucose and xylose, with relative proportions of each being influenced by diet. Glucose was present as glycogen which was characterized, for the first time in a rotifer, by the absorbance maximum of the iodine complex and the action of amylolytic enzymes on the isolated polysaccharide. Detailed fatty acid profiles of rotifers and diets illustrated the transfer and storage of specific fatty acids from the diets to the rotifers. The diatom T. pseudonana was the only diet to produce rotifers with the required complement of n3 polyunsaturated fatty acids suitable for larval fish rearing.
INTRODUCTION
The nutritional quality of cultured Brachionus plicatilis for rearing larval fish depends on the transfer of dietary components from phytoplankton or yeast to the rotifer. Although proximate chemical composition of the rotifer and its diet may not be similar, constituent units of dietary macronutrients, particularly fatty acids of lipids, are known to be transferred (Watanabe et al., 1983). Carbohydrate contents in 3. plicatilis fed phytoplankton and yeasts have been reported by Scott and Baynes ( 1978), and Ben-Amotz et al. ( 1987); yet no information on the sugar constituents of this carbohydrate component has been published. Fatty acid composition of rotifers is extremely important because n3 polyunsaturated fatty acids, particularly 20 : 5n3 and 22 : 6n3 acids, are essential for early larval growth of many fish species (Scott and Middleton, 1979; Watanabe et al., 1983; Ben-Amotz et al., 1987; Dendrinos and 0044-8486/90/$03.50
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B.V.
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J.N.C. WHYTE AND W.D. NAGATA
Thorpe, 1987; James and Abu-Rezeq, 1988). Rotifers are capable of synthesizing n3 polyunsaturated fatty acids, but tissue levels are insufficient to meet the demands for normal growth and development of fish larvae (Lubzens et al., 1985). Tissue accumulation of these acids by rotifers is therefore dependent on exogenous supplies from phytoplankton, and in this study, species which have not been used before were examined as dietary sources of fatty acids for rotifers. Sugar constituents of rotifers fed Saccharowlyces cerevisiae, Chlorella saccharophila, Isochrysis aff. galbana (T-iso clone), Tetraselmis suecica, and Thalassiusira pseudonana were investigated. Dietary influence on the presence and quantity of essential fatty acids in rotifers used in feeding larval fish was also examined. MATERIALS AND METHODS
Rotifer growth A clone of B. plicatilis isolated from the Seto Inland Sea was used. C. saccharophila was cultured on Yashima medium (SISFFA, 1964) and I. aff. galbana (T-iso clone), T. suecica, and T. pseudonana (3H clone) were batch
cultured in filtered ( 1 pm) sterilized seawater (28 2 l%o) enriched with nutrients (Harrison et al., 1980). All algae were grown at 20 5 2 oC under continuous light at 1 klux. S. cerevisiae was moist baker’s yeast (Fleishmann’s Yeast Ltd.) stored at 5°C. A 20-l stock culture of B. plicatilis was fed a mixture of the diets for a 2-week acclimation period, then subdivided into 15 groups, each placed into a 2-l Erlenmeyer flask. Three replicate flasks were supplied with each of the five experimental diets over a 15-day experimental period. The rotifers were maintained at 20 + 0.5 ‘C, 20%0 salinity, and 1 klux light intensity on a 16 : 8 h L: D photoperiod. Algal cultures were centrifuged and resuspended in 1 1 filtered seawater (20%0) to the desired concentration. These suspensions were fed to the rotifers daily following removal of 1 1 of the culture medium. No aeration was provided, but the flasks were swirled twice daily. Feeding levels were scaled on a dry weight basis and equivalent food rations were provided throughout the experiment. Initially food was provided to a level of 22 pg dry wt./ml medium as established from previous measurements on C. saccharophila (Nagata, 1985 ) . As the rotifer populations increased, the amount of food provided was increased proportionally if none remained in suspension. The rotifers were collected on a 40-pm screen, washed with cold 3% ammonium formate solution, concentrated by centrifugation, frozen at - 80 ’ C, and lyophilized for subsequent analysis.
COMPOSITION
OF BZUCHZONUS
PLZCATZLZSFED YEAST OR PHYTOPLANKTON
265
Analysis of neutral sugars Dry rotifers (25 mg) were washed successively on a fused fiberglass filter with chloroform and acetone (50 ml each) and the defatted residue hydrolyzed with 0.5 M H2S04 at 100°C for 16 h. The hydrolysate was neutralized with barium carbonate to yield parent sugars which were derivatized to corresponding alditol acetates (Sawardeker et al., 1965 ) . Derivatives were identified by comparison with standard alditol acetates and percentage composition determined by gas-liquid chromatography on glass columns ( 1.8 m x 0.32 cm) of (a) 3% Silar 1OC and (b) 3% ECNSS-M on Gas Chrom Q ( loo-120 mesh) operating at 205 and 195 ‘C, respectively, with a nitrogen flow rate of 30 ml/min. Mean values presented are from replicate samples and had coefficients of variance 25%. Isolation and analysis of polysaccharides Dry rotifers ( 1.O g) fed T. pseudonana were extracted with a homogeneous mixture of chloroform : methanol : water (2 : 4 : 1; v/v/v, 4 x 50 ml portions) using a cell disrupter for mixing (Whyte et al., 1987 ) . The sample, freed from lipid and low-molecular weight compounds, was then extracted with 200 ml water at 60’ C for 30 min and the mixture centrifuged. Extraction of the residue with two further 50-ml portions of water at 60°C yielded a combined supernatant which was concentrated by rotary evaporation to about 50 ml and cooled to 0 “C in an ice bath. Trichloroacetic acid ( lo%, 20 ml, 0’ C ) was added, the solution stirred and allowed to stand for 4 min. Filtration of the resultant mixture through’a GF/C filter removed proteinaceous material and the filtrate was poured into 4 volumes of 95% ethanol. The precipitated polysaccharide was allowed to settle and then isolated by centrifugation. Further purification by dissolution in distilled water, clarification by filtration and reprecipitation in ethanol, gave a polysaccharide which was dialyzed against cold-distilled water for 2 days, and freeze-dried to yield glucan, 59 mg ( 5.9%). Polysaccharide (5 mg) was hydrolyzed and sugars derivatized to alditol acetates and examined by gas-liquid chromatography as described previously. Purity of the polysaccharide was determined using the phenol-sulphuric acid reagent of Dubois et al. ( 1956) with D-glucose as the calibration standard. To prepare the iodine-polysaccharide complex, polysaccharide (5 mg) was dissolved initially in 6 ml water, then HCl (3 M, 0.02 ml) was added, followed by iodine solution (0.2 ml, containing 2 mg iodine and 20 mg KI per ml) and the resultant solution diluted to 10 ml. Maximum absorbance was measured in a 1-cm cell against iodine solution in the reference cell. Oyster glycogen was treated in an identical manner and absorbance of the iodine complex recorded as a control. Polysaccharide ( 12 mg) was dissolved in water (25 ml, containing 0.2 ml of 0.2 M acetate buffer at pH 7.0) and porcine pancreas cx-amylase (2 mg, Sigma VI-A) added and the solution stirred slowly at 2 1 oC. Similarly, poly-
266
J.N.C. WHYTE AND W.D. NAGATA
saccharide ( 12 mg) was dissolved in water (25 ml, containing 0.2 ml 0.2 M acetate buffer at pH 4.9) and barley fi-amylase (2 mg, Sigma II-B) added. Samples ( 10 ml) of the enzyme hydrolysates were removed after 1 and 16 h and evaporated to dryness on a rotary evaporator. The hydrolysate residues were extracted into 80% aqueous ethanol, evaporated to dryness, redissolved in water, deionized by cation exchange resin, and evaporated to dryness. Oyster glycogen was treated by both enzymes in exactly the same manner as controls. Qualitative analysis of sugar hydrolysates used paper chromatography on Whatman No. 1 paper in the solvent systems (a) ethyl acetate-pyridinewater, 10 : 4 : 3, (b) ethyl acetate-acetic acid-formic acid-water, 18 : 3 : 1: 4 and (c) n-butanol-ethanol-water, 3 : 1: 1. Sugars were detected with alkaline silver nitrate reagent (Trevelyan et al., 1950). Lipid contents andfatty acid profiles Total lipids in the rotifers were extracted and determined by the procedures described by Whyte et al. ( 1987). Dry rotifers and diets (50 mg) were saponified and methylated in a Reactivial according to the procedure described by Whyte ( 1988). Analyte solutions were separated on a Supelcowax 10 fused silica capillary column ( 30 m x 0.32 mm ID, 0.25-pm film). Fatty acids were identified by comparison with standard mixtures or ECLs and assigned in accord with data presented by Ackman ( 1986 ). Peaks of less than 0.2% were not included in the profiles, and the mean values presented from replicate samples had coefficients of variance 55%. RESULTS
Glucose formed 6 l-80% of the total sugar constituents of rotifers, indicating the presence of a storage glucan which varied with diet (Table 1) . A high percentage, 9- 18%, of ribose suggested a considerable incorporation of riboTABLE
1
Percent composition phytoplankton
of neutral
sugars in Brachionus plicatiiis fed yeast and four species
of
Sugars
Yeast
Thalassiosira pseudonana (3H clone)
Isochrysis galbana (T-iso clone)
Tetraselmis suecica
ChlorelIa saccharophila
Fucose Ribose Xylose Deoxyglucose Mannose Galactose Glucose
3.89 17.93 0.80 0.79 7.40 1.42 67.77
6.73 16.11 3.73 3.34 5.12 3.73 61.24
5.50 12.87 1.65 1.60 4.95 2.95 71.28
4.92 11.29 2.42 3.20 3.91 3.16 71.10
3.43 9.30 1.99 1.63 2.72 1.39 79.54
COMPOSITION OF BRACHIONUS
PLICATILIS
FED YEAST OR PHYTOPLANKTON
267
nucleotide units from nucleic acids in all rotifers. Lesser amounts, ranging from 0.8 to 7.0% of galactose, mannose, 2-deoxyglucose, fucose and xylose formed the remainder of the neutral sugar constituents in the rotifers. Hot-water extraction of defatted rotifers fed T. pseudomna yielded 5.9% of a purified polysaccharide. Acid hydrolysis, and gas-liquid chromatographic analysis of the derived sugar alditol acetates revealed that only glucose was present. The glucan contained 98.4% glucose and exhibited a maximum absorbance for the corresponding iodine complex at 480 nm. This value was consistent with absorbance maxima of 420-490 nm for iodine complexes of glycogens from many sources rather than the 500-550~nm absorbances of starches (Percival and McDowell, 1967). The iodine complex of oyster glycogen exhibited a maximum absorbance at 467 nm. Treatment of the glucan with a-amylase for 1 h yielded mainly glucose and maltose with minor amounts of maltotriose, as shown by paper chromatography in three solvent systems. An increase in glucose, a decrease in maltose, but no maltotriose was evident in the hydrolysate when the enzyme treatment was continued for 16 h. The action of fi-amylase on the glucan for 1 h showed mainly maltose on paper chromatography with minor amounts of glucose, the latter indicating the presence of a second amylolytic enzyme presumed to be cu-amylase in the commercial enzyme product. Similar depolymerization products were obtained by the action of both enzymes on oyster glycogen. The enzymatic depolymerization was consistent with a glycogen structure for the glucan. Total lipids in rotifers fed S. cerevisiae, T. pseudonana, I. gaZbana (T-iso ) , T. suecica and C. saccharophila were 9.8 + 0.2%, lO.O+ 0.3%, 15.8 -+0.4%, 13.12 0.3%, and 9.8 5 0.1% of the dry weight, respectively. The relative percentages of fatty acids in the total lipids of the rotifers and phytoplankton fed to the rotifers are presented in Table 2. Major fatty acids in S. cerevisiae and corresponding rotifers fed this yeast were 16 : 0, 16 : 1n7, and 18 : 1n9, and in T. pseudonana and rotifers were 14:0, 16:0, 16: 1~17, 18:4n3, 20:5n3 and 22:6n3. 1. galbana (T-iso) and rotifers that consumed this flagellate contained 14:0, 16:0, 18: ln9, 18:2n6, 18:3n3,18:4n3, and 22:6n3 as themajor acids, whereas T. suecica and the corresponding rotifers contained 16 : 0, 16:4n3, 18: ln9, 18: ln7, 18:2n6, 18:3n3, 18:4n3 and 20:5n3. The major acids in C. saccharophila and corresponding rotifers contained predominantly 16:0, 16: ln7,18: ln9, 18:2n6 and 18:3n6 acids. Lipids of C. succharophila, S. cerevisiae and rotifers fed these diets contained the highest level of saturated and monoethylenic fatty acids, respectively (Table 2). In general, the levels of polyethylenic fatty acids in rotifers were similar to levels in the corresponding diets. The level of polyethylenic fatty acids in lipids of all algae was 10 times that of the yeast, and n3 polyunsaturated fatty acids constituted over 50% of the polyethylenic fraction of all algae but C. saccharophila. Eicosapentaenoic acid, 20: 5n3, comprised 50%,
J.N.C.WHYTE AND W.D. NAGATA
268 TABLE 2
Fatty acids in the total lipids of Brachionus plicatilis fed yeast and four species of phytoplankton and in the total lipids of the five diets Fatty acids composition (% of total)
14:o 14: ln5 15:o 16:0 16: ln9 16: ln7 16:2n6 16:2n4 17:o 16:3n4 16:3n3 16:3nl 16:4n3 16:4nl 18:0 18:lnll 18: ln9 18: ln7 18:2n7 18:2n6 18:2n4 18:3n6 18:3n3 18:4n3 20: lnll 20: ln9 20: ln7 20:2n6 20: 3n6 20 : 4n6 20: 3n3 20:4n3 20:5n3 22: ln9 21:5n3 22:4n6 22: 5n6 22:5n3 22:6n3 Saturated Monoethylenic Polyethylenic X4-6n3/poly. 20: 5n3/poly. 22: 6n3/poly.
Yeast
Thalassiosira pseudonana
Zsochysis galbana (T-iso)
Tetraselmis suecica
Chlorella saccharophila
Rotifer
Diet
3.56 0.64 0.69 19.72 0.98 7.70 0.25 1.69 2.25
0.27 0.42 40.77 1.42 4.48 0.33 0.35 0.84
1.07
0.31 0.73
1.25
3.00 4.63 5.10 3.53 6.24 12.87 1.25 3.88 1.01 0.72
0.73 2.67 0.84 19.32 15.59 2.98 1.03 -
0.99
0.98 7.03 5.48
0.38 0.28 -
0.35 5.98
0.73 0.68 0.50
0.20 -
_
1.78
-
1.84
-
(3H) Rotifer
Diet
Rotifer
Diet
Rotifer
Diet
Rotifer
1.94 0.52 0.59 7.26 0.62 23.58
0.68 0.20 8.41 39.14
6.53 0.51 0.56 11.44 3.34 25.13 2.73 3.74
7.85 0.29 0.29 10.56 0.48 3.36
0.73 0.62
0.31 0.71
4.61 0.51 0.73 12.78 13.18 1.38 1.99 2.71
15.47 0.31 0.20 8.33 2.84 3.77 0.40 0.73 -
1.12 0.28 0.32 13.26 0.98 1.75 0.44 1.41 1.11
4.07
0.88 1.94
3.97 40.92 -
2.55 1.13
2.4 -
1.59 1.28
0.78 0.24
2.83
1.28 2.40 0.98 0.20 0.93
0.20
0.28 0.49
8.89 1.63
3.64 2.98 5.29 4.86
0.29
7.17
8.72
9.47
7.48 -
6.47 12.26
1.16 7.72 18.55
1.07 14.44 4.38 0.75 2.65 0.51 0.32 0.79 2.70 0.72 4.44 4.94 0.97
0.88 11.69 9.43
0.25 6.09
3.14
0.96
0.45 0.99 1.70 _
-
23.33 -
0.78 14.65 66.26 11.59 49.18 0.00 6.73
5.95 2.22 1.16
_
_
13.31 80.50 3.96 19.70 0.00 0.00
6.65 6.02 22.00 22.50 48.77 78.55 42.11 12.34
1.02 1.03 11.49 -
2.50 0.86 15.25 3.04
1.41
_
20.54 0.95
14.23 6.67 1.60 0.47 2.27
4.42
1.75 3..88 1.36 5.34 1.40 1.25 -
0.39 0.63
0.41 -
0.56 4.13 2.50 28.46 6.76
Diet
4.30 18.53 29.75 46.24 72.92 50.45 9.30
2.07 1.40 8.53 21.20 27.58 48.44 67.57 4.58 17.61
_ 0.24 0.78 0.83 _ 0.25 2.21 10.84 24.20 17.44 52.84 57.19 1.57 20.51
,
2.23 1.39 19.45 21.02 53.16 50.51 9.29 2.61
0.20 11.80 2.20 -
14.82 22.90 53.28 53.00 11.22 0.00
28.66 24.81 46.34 9.32 1.47 0.00
42.12 9.83 41.99 4.66 0.00 0.00
COMPOSITION OF 3RACHfOh’US PLICATILIS
FED YEAST OR PHYTOPLANKTON
269
2%, and 11% of the polyethylenic fraction of T. pseudonana, I. galbana (Tiso), and T. suecica, respectively and was absent in both C. saccharophitaand S. cerevisiae. Docosahexaenoic acid, 22: 6n3, was present only in T. pseudonana and I. galbana (T-iso) at 9% and 2 1% of the polyethylenic fraction, respectively. DISCUSSION
The value of rotifer carbohydrate as a dietary energy reserve when fed to crustacean or fin-fish larvae is unknown, as sugars present in rotifers have never been identified. Results indicated that glucose formed 61-80% of the rotifer sugars which consisted mainly as a glucan characterized as glycogen, the ubiquitous source of energy, via the citric acid cycle, in most animals. Although carbohydrate in general forms a minor component of the marine food chain, its use as an energy source “sparing” protein for tissue synthesis has been recognized in nutritional studies of aquaculture species (Enright et al., 1986; Alava and Pascual, 1987; Degani and Viola, 1987; Whyte et al., 1989). Assimilation of rotifer glycogen by larval fish is probable because digestion of starches, considerably more insoluble than glycogen, has served as a source of energy in fish (Bergot and Breque, 1983 ) . Glycogen formed the major portion of carbohydrates in rotifers fed different diets in this study, and therefore weight gain of larval fish from this energy source would probably be comparable regardless of diet. The fatty acid profiles of rotifers and corresponding diets fed to the rotifers indicated transferral and storage of major fatty acid constituents in the feeding process. Similar results have been reported for B. plicatilisfed other species of phytoplankton (Scott and Middleton, 1979; Ben-Amotz et al., 1987). The presence of fatty acids, such as 18 : 1n 11,22 : 1n9,22 : 5n3 and 22 : 6n3 in the rotifers but not in corresponding diets, suggests endogenous synthesis of these acids and/or assimilation and concentration of minor amounts which were not evident in the profiles of the diets. Similarly, the active accumulation of 18 : 0,18 : 1n7,18 : 1n 1 I, and 20: 1n9 fatty acids by the rotifers, regardless of diet supply, implied a need for these acids during growth and development. Marine fish have been reported to be unable to synthesize de novo polyunsaturated fatty acids of either the n6 or n3 series (Sinnhuber, 1969; Cowey and Sargent, 1972; Kanazawa et al., 1979) and rotifers have been found to synthesize only minor amounts of n3 polyunsaturated acids (Lubzens et al., 1985 ). Therefore, these acids must be provided by the rotifer diet to meet the possible requirement of larval fish. In this laboratory provision of polyunsaturated fatty acids to rotifers was crucial to their use as food for larval sablefish, Anaplopoma fimbria. Eggs of the sablefish have been found to contain almost 90% 4-6n3 fatty acids in the total polyethylenic acid fraction, and
270
J.N.C. WHYTE AND W.D. NAGATA
86% of the total 4-6n3 acids consisted of approximately equal amounts of 20 : 5n3 and 22 : 6n3 acids (Whyte, 1988 ). The essential requirement for both these acids in fish has been reviewed by Watanabe et al. ( 1983), but in addition, dietary 18 : 3n3 has been reported to promote growth of turbot (Gate-soupe et al., 1977) and sole (Dendrinos and Thorpe, 1987). A level of approximately 12% of 20: 5n3 in rotifers was considered normal for feeding marine fish larvae (Watanabe et al., 1983 ). Rotifers fed T. pseudonana, I. galbana (T-iso) and T. suecica contained lO.O%, 15.8% and 13.1% total lipids, which included in their fatty acid profiles 20.5%, 2.2% and 4.9%, 20: 5n3 acid, 6.0%, 8.5% and 1.4%, 22: 6n3 acid and 1.3%, 6.5% and 14.4% 18 : 3n3 acid, respectively. T. pseudonana was the only algal diet that produced rotifers with sufficient tissue levels of essential fatty acids. The diatom is easy to mass-culture, and its potential to provide a complement of essential fatty acids in the rotifers suitable for larval fish growth is recorded for the first time. Levels of essential acids were minimal or absent in the 9.8% total lipids in rotifers fed S. cerevisiaeor C. saccharophilu.Although this deficiency has been recognized in strains of yeast (Dendrinos and Thorpe, 1987 ) , highly variable levels of these acids, O-20+%, have been recorded in rotifers fed species of ChZoreZZa (Teshima et al., 1983, 1987; Ben-Amotz et al., 1987; James and Abu-Rezeq, 1988 ) . The variance in fatty acid profiles in species of ChZoreZZa has been attributed to culture conditions but may also reflect taxonomic inaccuracies (Fukusho, 1985; James and Abu-Rezeq, 1988). These results illustrated that among all the diets examined only T. pseudonana was capable of supplying the required composite of n3 polyunsaturated fatty acids. Predictable culture of larval fish will not be fully realized without the controlled culture of nutrient-specific rotifers used as food for the early and critical stages of larval development. In this study on rotifers, diet changed the carbohydrate constituents slightly, but glycogen remained the dominant component which would be available as an energy source for developing fish larvae. Diet was more crucial in formation of lipid constituents, and only the rotifers fed T. pseudonana provided the complement of fatty acids considered essential for culture of marine organisms. ACKNOWLEDGEMENT
W.D. Nagata expresses his thanks to the Natural Science and Engineering Research Council of Canada who provided funding through an NSERC visiting fellowship. REFERENCES A&man, R.G., 1986. WCOT (capillary) gas-liquid chromatography. J.B. Rossell (Editors), Analysis of Oils and Fats. Elsevier Applied New York, NY, pp. 137-206.
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PLICATILIS
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Alava, V.R. and Pascual, F.P., 1987. Carbohydrate requirements of Penaeus monodon (Fabricius) juveniles. Aquaculture, 61: 211-217. Ben-Amotz, A., Fishler, R. and Schneller, A., 1987. Chemical composition of dietary species of marine unicellular algae and rotifers with emphasis on fatty acids. Mar. Biol., 95: 3 l-36. Bergot, F. and Breque, J., 1983. Digestibility of starch by rainbow trout: effects of the physical state of starch and of the intake level. Aquaculture, 34: 203-2 12. Cowey, C.B. and Sargent, J.R., 1972. Fish nutrition. Adv. Mar. Biol., 10: 383-492. Degani, G. and Viola, S., 1987. The protein sparing effect of carbohydrate in the diet of eels (Anguilla anguilla). Aquaculture, 64: 283-291. Dendrinos, P. and Thorpe, J.P., 1987. Experiments on the artificial regulation of the amino acid and fatty acid contents of food organisms to meet the assessed nutritional requirements of larval, post-larval and juvenile Dover sole (Solea solea L. ). Aquaculture, 6 1: 12 l-l 54. Dubois, M., Gillies, K.A., Hamilton, JK, Rebers, P.A. and Smith, F., 1956. Calorimetric method for the determination of sugars and related substances. Anal. Chem., 28: 350-356. Entight, C.T., Newkirk, G.F., Craigie, J.S. and Castell, J.D., 1986. Growth of juvenile Ostrea edulis L. fed Chaetoceros gracilis Schutt of varied chemical composition. J. Exp. Mar. Biol. Ecol., 96: 15-26. Fukusho, K, 1985. Present status and problems in culture of the rotifer Brachionusplicatilis for fry production of marine fishes in Japan. Symposium Intemacional de Acuacultura, Coquimbo, Chile, September 1983, pp. 361-374. Gatesoupe, F.J., Leger, C., Boudon, M., Metailler, R. and Luquet, P., 1977. Alimentation lipidique du Turbot (Scophthalmus maximus L.). II. Influence de la supplCmentation en esters methyliques de l’acide linolenique et de la complementation en acides gras de la s&e n3 sur la croissance. Ann. Hydrobiol., 8: 247-254. Harrison, P.J., Waters, R.E. and Taylor, F.J.R., 1980. A broad spectrum artificial seawater medium for coastal and open ocean phytoplankton. J. Phycol., 16: 28-35. James, C.M. and Abu-Rezeq, T.S., 1988. Effect of different cell densities of Chlorellu capsuluta and a marine Chlorella sp. for feeding the rotifer Brachionus plicatilis. Aquaculture, 69: 4356. Kanazawa, A., Teshima, S.-I. and Ono, K., 1979. Relationship between essential fatty acid requirements of aquatic animals and the capacity for bioconversion of linolenic acid to highly unsaturated fatty acids. Comp. Biochem. Physiol. B, 63: 295-298. Lubzens, E., Marko, A. and Tietz, A., 1985. De novo synthesis of fatty acids in the rotifer Brachionusplicatilis. Aquaculture, 47: 27-37. Nagata, W.D., 1985. Long-term acclimation of a parthenogenetic strain of Bruchionusplicutilis Muller to subnormal temperatures. II. Effect on clearance and ingestion rates. Bull. Fat. Fish. Hokkaido Univ., 36: l-l 1. Percival, E. and McDowell, R.H., 1967. Chemistry and Enzymology of Marine Algal Polysaccharides. Academic Press, London, 157 pp. (cf., p. 76). Sawardeker, J.S., Sloneker, J.H. and Jeanes, A., 1965. Quantitative determination of monosaccharides as their alditol acetates by gas-liquid chromatography. Anal. Chem., 37: 1602- 1604. Scott, A.P. and Baynes, S.M., 1978. Effect of algal diet and temperature on the biochemical composition of the rotifer, Brachionusplicatilis. Aquaculture, 14: 247-260. Scott, A.P. and Middleton, C., 1979. Unicellular algae as a food for turbot (Scophthalmus rnaximus L.) larvae - the importance of dietary long-chain polyunsaturated fatty acids. Aquaculture, 18: 227-240. Sinnhuber, R.O., 1969. The role of fats. In: O.W. Neuhaus and J.E. Halver (Editors), Fish in Research. Academic Press, New York, NY, pp. 245-26 1. SISFFA (Set0 Inland Sea Farming Fisheries Association), 1964. Cultivation of live food organisms at the Yashima Station. Newsletter of Saibai Gyogyo, 2-4, 4 pp (in Japanese).
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