Fatty Acid Composition of Three Cultured Algal Species (Isochvysis galbana, Chaetoceros gracilis and Chaetoceros calcitrans) Used as Food for Bivalve Larvae

JOURNAL OF THE WORLD AQUACULTURE SOCIETY

Vol. 21, No. 2 June, 1990

Fatty Acid Composition of Three Cultured Algal Species (Isochvysis galbana, Chaetoceros gracilis and

Chaetoceros calcitrans) Used as Food for Bivalve Larvae GUILLERMO E. NAPOLITANO

Department of Oceanography, Dalhousie University, Hal&x, Nova Scotia B3H 4JI Canada

ROBERT G. ACKMAN' AND WALISUNDARA M. N. RATNAYAKE~ Canadian Institute of Fisheries Technology, Technical University of Nova Scotia,

P.O. Box 1000, Halifar. Nova Scotia B3J2X4 Canada

Abstract Fatty acids of three cultured unicellular algae wldely used in aquaculture (Isochrysis galbanrr,

clone T-iso, Chaetoceros cdcitrans, and Chaetoceros gracilis) were analyzed in detail by gas-liquid chromatography and mass spectrometry. Fatty acid compositions of the individual species, harvested at monthly intervals during a four month period, showed a very consistent pattern. Shorter chain (C16) fatty acids accounted for more than 70% of total fatty acids in the diatoms but less than 20% in I. gulbana. Medium chain (C18) and the long chain 226w3 unsaturated fatty acids represented more than 50% of the total fatty acids in I. gulbana but less than 10% of the total In the Chaeroceros spp. However, in the latter, 205w3 replaced 226w3 at an equivalent level. Fatty acid profiles of c . cukitrans and c. gracilis were similar, except that the latter contained lower levels of 1 6 2 ~ 4 , 1 6 3 ~ 4 and 1 6 4 ~ 1 , and higher levels of 1 8 2 6 and l8:36. In particular C. gracilis contained a higher proportion of the biochemically important fatty acid 2 0 4 6 (arachldonic acid). One of the salient features of I. galbana lipids was the presence of the fatty acid 185~3. This fatty acid is not generally reported in algae or invertebrate lipid analyses, but is a typical component of I. gulbana and of other marine photosynthetic flagellates. Its potential for utilization by aquatic organisms is unknown.

Brine shrimp and rotifers are the most frequently used live food for cultured larval marine fishes and crustaceans, and their culture depends partially on the production of unicellular algae @e Pauw and Pruder 1986; Millamena et al. 1988). Microalgae, specif- ically, are required for rearing bivalve molluscs (Langdon and Waldock 198 l) and pe- naeid shrimp (Shigueno 197 S), at least until suitable artificial diets are developed.

In larval diets, lipids have been regarded primarily as sources of energy, with relatively little attention paid to the inclusion of essential fatty acids in the diets. Microal-

Corresponding author. * Current address: Food Directorate, Sir Frederick

G. Banting Research Centre, Department of National Health and Welfare Canada, Ottawa, Ontario KIA OL2 Canada.

gal lipid composition has been suggested as more critical than protein and carbohydrate composition for promoting optimal growth and development in bivalve larvae (Hol- land 1978; Webb and Chu 1983). Marine animals have clearly shown essential fatty acid requirements (Langdon and Waldock 1981; Watanabe 1982). Like proteins that can be broken down to amino acids and like carbohydrates that can be degraded to basic monosaccharides, lipids yield fatty acids. Saturated and monounsaturated fatty acids are generally not essential, but certain polyunsaturated fatty acids (PUFA) are essential dietary nutrients for reasons associated with their specific structures (Ackman 1983; Lands 1986).

Evaluation of the food value of several single species of microalgae, measured as daily bivalve larval growth, shows Chae-

0 Copyright by the World Aquaculture Society 1990

122

FAMY ACIDS OF CULTURED MICROALGAE 123

toceros gracilis to be one of the best food for larvae, followed by C. calcitrans (Enright et al. 1986a). Higher larval growth was obtained with mixed algal species containing Z. galbana (Enright et al. 1986a).

Most recent attention has been given to essential fatty acid requirements of marine animals, focusing on the w3 or linolenic family of fatty acids, primarily 2 0 5 ~ 3 (eicosapentaenoic acid or EPA) and 22:6w3 (docosahexaenoic acid or DHA) (Cowey et al. 1976; Waldock and Holland 1984). However, comparative studies have shown biosynthesis and activity of the eicosanoids (oxygenated and biologically active prod- ucts of C20 fatty acids known in higher animals (Marcus 1984)) derived from both the w3 and w6 series of fatty acids in econom- ically important bivalve molluscs and in other marine invertebrates (Christ and Van Dorp 1972; Nomura and Ogata 1976; Rug- geri and Thoroughgood 1985).

Traditionally, the analysis of fatty acids in marine samples by gas-liquid chromatography (GLC) on packed columns has given satisfactory but rather incomplete com- positional results. Capillary GLC provides a more exhaustive analysis of algal fatty acids, including those minor and unusual phytoplanktonic fatty acids normally ig- nored, misidentified or not resolved from other components. The objective of this work is to present detailed information on the fatty acid constituents of three cultured microalgae widely used in aquaculture. At- tention has been paid to the occurrence of PUFA of both the w3 and w6 series and to their incorporation into the lipids of two species of bivalve larvae.

Materials and Methods Cultures

The microalgae used in the present study were Zsochrysis galbana (Parke) clone T-iso (Pry mnesioph yceae) , Chaet oceros gracilis Schutt (Bacillariophycea) and Chaetoceros calcitrans Takano.

Algal cultures were obtained from the Bi-

ology Department of Dalhousie University. Cultures were developed under the routine conditions for feeding oyster larvae described by Enright (1 984) and Enright et al. (1 986). Axenic stock cultures were maintained at 15 C in a culture chamber. Ap- proximately 50 mL of stock culture were used in the sterile inoculation of an autoclaved 20 L carboy filled with filtered (1 pm filter) and autoclaved seawater. Cultures were grown in the f/2 nutrient mixture of Guillard and Ryther (1 962), under a max- imum irradiance of 300 mE/m2/s in the spectrum between 400 and 700 nm. Aera- tion and C02 were supplied by gas cylinders. The pH of the algal cultures was maintained between 7.9 and 8.4. Algal cultures were monitored monthly for contamination using a microscope magnification of 1,000 x for a four month period.

Three to seven replicates of approximately 200,000 larvae of both the European oyster Ostrea edulis and the sea scallop Pla- copecten magellanicus were used in each feeding trial. Larvae were reared in 100 L PVC tanks with filtered seawater (1 pm), at temperature of 24 C (approximate density of 5 individuals/mL), with gentle aeration. Food of the developing larvae consisted of equal amounts of I . galbana and C. gracilis (or C. calcitrans, depending on availability) offered every second day at a final concentration in the rearing tank of 100 cells/pL. The progenitors of the larvae used in this work were fed the same algae as described above. The general conditions for lipid analysis of bivalve larvae have been described elsewhere (Napolitano et al. 1988a).

Cell Concentration and Fatty Acid Recovery

Algal cultures, 4 to 10 L, were centrifuged in a continuous centrifuge or at 2,000 rpm for 5 minutes in 50 mL tubes. Yields ofalgae obtained by these procedures were approximately 1 g/L. Total fatty acids were obtained by direct saponification of the algal pellets instead of using a conventional solvent extraction. This was done to maximize

124 NAPOLITANO ET AL.

fatty acid recovery. In some algae, fatty acids may be bound. in lipids that could be pro- tected from solvent extraction by the cellular wall (Ackman et al. 1970). Fatty acid extracts were either immediately subjected to GLC or were stored intact at -30 C in screw-capped (Teflon-lined) glass centrifuge tubes under a nitrogen atmosphere.

Fatty Acid Identijkation Fatty acid methyl esters (FAME) were

prepared using 7% BF3-methanol by the method described by Momson and Smith (1964). Analytical GLC of the FAME was camed out in a Perkin-Elmer Model 9 10 equipped with a flame ionization detector (FID) and a flexible fused silica capillary column (30 m in length x 0.25 mm internal diameter) with bonded Carbowax-20M as the liquid phase (SUPELCOWAX- 10, Su- pelco Inc., Bellefonte, Pennsylvania). This liquid phase (polyglycol Carbowax-20M in “bonded” form in capillary columns) has been proposed as especially suitable for natural marine lipid systems, and retention data have been published (Ackman 1984, 1986, 1987; Christie 1988). It is being tested in proposed standard methods for the GLC of fatty acids of marine origin (Firestone 1986). Additional GLC was carried out with a fused silica column with a similar bonded liquid phase (DB-WAX; J and W Scientific Inc., Folsom, California).

Fatty acids were identified using the fol- lowing methods: co-injection of the sample along with authentic standards of FAME mixtures of established composition, cata- lytic hydrogenation of the sample over Pt02, and hydrazine reduction and/or silver ni- trate-thin layer chromatography (AgN03- TLC) followed by GLC of fractions. The molecular weights of most of the fatty acids were confirmed by gas-liquid chromatography/mass spectrometry (GUMS) using a Finnigan MAT 700 ITD (Ion Trap Detec- tor, Finnigan MAT, San Jose, California). The procedures for AgN03-TLC of the orig- inal esters, the hydrazine reduction of the isolated bands, and the comparative GLC

have been described elsewhere (Ackman et al. 1974).

Data were treated statistically by a one way analysis of variance. Prior to this analysis, fatty acid percentages were normalized through (arcsine %)-2 transformation (Snedecor and Cochran 1980).

Results and Discussion Chaetoceros spp.

The 20 L cultures used for lipid analysis were maintained under bacterial-controlled but not axenic conditions. Bacterial lipids contain primarily saturated and monounsaturated fatty acids which do not interfere when studying the biochemistry of polyunsaturated algal fatty acids (Gillan et al. 1988).

Reproducible patterns of fatty acid composition were obtained for both C. gracilis and C. calcitrans (Table 1). These species were apparently similar in both classes and proportions of fatty acids. In general, the qualitative and quantitative data for major fatty acids presented here (Table 1) agree with those reported in previous studies of the same or closely related species (Ackman et al. 1968; Chuecas and Riley 1969; Boutry and Bordes 1979; Waldock and Nascimento 1979; Enright et al. 1986a, 1986b).

Approximately 10 fatty acids accounted for 90% of the total in C. gracilis and C. calcitrans. The important saturates were 14:O and 16:0, while 16: lw7 was the major monounsaturate. PUFA consisted primarily of 16:2w7, 16:3w4, 16:4wl (in C. calcitrans but not in C. gracilis), 20:5w3 and 22:6w3. It is important to note that 16:lw7 and 20:5w3 accounted for about 50% of the total.

The comparison between the two Chae- toceros species showed that C. calcitrans was richer in C16 PUFA, while C. gracilis contained larger amounts of 18:2w6, 18:3w6 and 20:4w6 (Table 1). Analysis of four replicate samples of each of two diatoms showed significant differences between species (P < 0.05) in the proportions of the above fatty acids.

In fatty acids of cultured marine phyto-

FATTY ACIDS OF CULTURED MICROALGAE 125

TABLE 1. Fatty acids (weight percent of total fatty acids f SO) from batch cultures of three unicellular algae. A-A, B-B, significant differences (P < 0.05) of unsaturated fatty acids discussed in the text.

C. calcitrans C. gracilis 1. galbana (N = 4) (N = 4) (N = 4)

12:o 14:O 15:O

Anteiso-16:0 16:O 16:lw7 16:lw5 16:2w7 16:2w4 16:3w4 16:4w 1 17:O 18:O 18:lw9 18:lw7 18:lwS 18:2w6 18:2w4 18:3w6 18:3w3 18:4wl 18:4w3 l8:5w3 20:o 20:3w6 20:4w6 20:4w3 20:5w3 21:5w3 22:o 22:5w6 22:5w3 22:6w3 Total saturated Total PUFAc

Is0-16:O

0.21 f 0.19 13.43 f 0.10 0.35 f 0.28 0.59 k 0.12 tra 7.13 f 0.21

21.40 f 0.25 A 0.42 f 0.18 5.20 f 0.01 A 6.23 f 0.13 A

10.80 f 0.45 A 2.70 f 0.13 A tr 0.17 f 0.07 0.11 f 0.11 0.66 + 0.09 0.19 f 0.1 0.06 k 0.06 A 0.27 k 0. I3 0.08 f 0.04 A 0.24 k 0.16 A 0.04 k 0.01 0.91 f 0.05 A nd 0.32 k 0.12 tr 0.17 f 0.05 0.13 k 0.03

21.53 f 1.10A 0.09 k 0.03 0.17 f 0.05 0.07 f 0.06 0.03 f 0.01 1.16 k 0.11 A

22.4 47.0

0.36 f 0.26 11.10 f 1.10 0.99 f 0.43 0.51 f 0.33 0.57 * 0.12

11.70 f 6.05 29.70 k 1.53A 0.40 k 0.12 3.10 * 0.63 A 1.92 f 0.90A 5.87 k 1.03A 0.18 f 0.07 A 0.08 f 0.01 0.60 k 0.34 0.19 f 0.81 0.92 f 1.29 0.15 k 0.09 1.02 f 0.24 A 0.06 f 0.03 1.21 k 0.06A 0.13 * 0.07 B 0.04 f 0.01 0.85 f 0.07 B nd 0.09 * 0.09 0.20 f 0.08 3.50 k 0.52 0.09 f 0.05

18.39 k 5.96 B 0.12 f 0.11 0.15 k 0.05 0.16 k 0.06 0.16 * 0.09 2.67 f 0.07 B

26.1 39.4

0.04 k 0.03 15.80 f 3.90 0.31 f 0.21 tr tr

10.81 k 1.48 2.71 f 0.80 A 0.20 f 0.20 0.62 f 0.78 A 0.53 f 0.11 A 0.21 f 0.24A ndb 0.17 k 0.13 0.36 f 0.14 11.0 f 2.93 1.00 f 0.49 0.15 k 0.17 3.94 f 0.99 A 0.06 f 0.01 0.51 f 0.13 A 5.40 * 0.46 AB 0.05 f 0.01

14.31 f 2.01 AB 2.86 f 0.4 0.2 f 0.07

0.12 f 0.06 0.22 f 0.08 0.04 f 0.03 0.89 f 0.16 AB 0.09 k 0.03 0.21 f 0.13 3.11 f 0.59 0.29 f 0.02

20.47 k 4.90 AB 27.9 53.6

a Trace amount. Not detected. Polyunsaturated fatty acid.

plankton, arachidonic acid (AA) normally is a minor component of the total fatty acid composition (Ackman et al. 1968; Chuecas and Riley 1969), and with the exception of Rhodophyceae (Ackman et al. 1968; Takagi et al. 1985) AA is rarely important in marine algae in general. An extraordinary fatty acid composition showing high proportions of triarachidonin and diarachidonoyl phos- phatidylcholine has been reported in the

marine red alga Gracilaria verrucosa (Ki- noshita et al. 1986). It has been suggested that these fatty acids may lead to production of prostaglandins active in man (Fusetani and Hashimoto 1984; Moghaddam et al. 1989). Arachidonic acid has been reported in significant amounts only in the diatoms, Skeletonema costaturn (- 1 .O% of total fatty acids (Ackman et al. 1964; Chuecas and Ri- ley 1969)) and Asterionela japonica (6.0%

126 NAF'OLITANO ET AL.

TABLE 2. Linoleic, gamma-linolenic, dihomo-gamma-linolenic and arachidonic acid contents (percent of total fatty acids) reported for some microalgal species used as food for the culture of marine invertebrates.

Species 18:2w6 18:3w6 203w6 204w6

Chaetoceros calcitrans (lyl 0.2 1.4 tr tr C. gracilis (3) 1.4 nr nr 1.9 Isochrysis galbana (1) 4.3 0.2 nd 0.2

(2) 4.0 0.8 nr nr (5) 5.2 0.3 nr nr

Emiliania huxleyi (2) 1.7 0.3 nr nr (5) 2.3 tr nr nr

Dunaliella tertiolecta (2) 6.1 4.1 nr nr (8) 7.1 4.0 nr nd

Chlorella saccharophila (3) 1 .5b 0.8 nr 3.9 C. minutissima (4) 4.4 nd 0.8 3.9c Chlorella spp. (6) 4.7 0.4 1.1 4.1

(7)d 14.2 nd nr nd Tetraselmis suecica (8) 1.6 tr nr 0.8 Tetraselmis tetrahele (6) 6.5 0.1 0.2 2.4 Pseudoischrysis paradoxa (7Id 3.3 nr nr tr Pyramimonas virginica (1) 2.6 0.9 nd nd

(7)d 0.8 nr nr 8.6

* References: (1) Waldock and Nascimento 1979; (2) F'illsbury 1985; (3) Teshima et al. 1987; (4) %to et al. 1984; ( 5 ) Volkman et al. 1981; (6) Fukusho et al. 1984; (7) Chu and Dupuy 1981; (8) Waldock and Holland 1984. b All isomers.

Contains 22:O. 20 day old culture.

tr = trace amount, nr = not reported, nd = not detected.

(Chuecas and Riley 1969)). However, the normal proportions for this fatty acid in centric diatoms, including some other ex- amples of the genus Chaetoceros, do not exceed 0.5% of total fatty acids (Chuecas and Riley 1969; Kayama et al. 1989).

In order to confirm the relatively high content of AA in C. gracilis. FAME were subjected to GC/MS. The mass spectrum of the peak in question was not specific for bond position but was similar to those obtained for other PUFA and showed the protonated molecular ion appearing at mass/ charge (m/z) 3 19 (monoisotopic molecular weight of 20:4w6 methyl ester is 318). In ITD GUMS of FAME the molecular ion usually appears in the protonated (MH+) form (Ratnayake et al. 1986). The advan- tages of high resolution GLC for identifi- cations through retention time are such that other 20:4 structures are precluded and the

confirmation of mass is satisfactory proof of AA (Ackman 1987).

The difference in the C 1 6/C 1 8 PUFA ra- tio of C. calcitrans and C. gracilis deserves comment. The peculiar C16 wl , w4 and w7 series (Ackman et al. 1964; Ackman 1989) of fatty acids have no known metabolic function and are only extended to higher chain lengths such as 18:2w4 and 18:4wl. The high proportion of C16 PUFA in C. calcitrans (24.9Oh of the total vs. 1.6% of C18) suggests that these fatty acids may be as effective in membranes in diatoms as are the C18 PUFA in higher plants (commonly 18:2w6, 18:3w6 and 18:3w3). C. gracilis had twice as much C18 PUFA (3.3%) as calcitrans, but only half as much C16 PUFA

Table 2 shows the proportions of the metabolically important fatty acids of the w6 series: linoleic, gamma-linolenic, dihomo-

(1 1 .OO/o).


TABLE 3. Selected fatty acid content (weight per cent of total fatty acids * SD)from total lipids and phospholipids of laboratory reared oyster (0. edulis) and scallop (P. magellanicus) larvae fed C. gracilis, C. calcitrans and I. galbana. A-A, B-B, signifzcant diferences (P < 0.05) of unsaturated fatty acids discussed in the text.

Oyster larvae Scallop larvae

Phospholipids Total lipids Phospholipids Total lipids (N = 4) (N = 4) (N = 3) (N = 3)

~

14:O 16:O 16:lw7 18:O 18:lw9 18:lw7 18:2w6 18:3w6 18:3w3 18:4w3 20:4w6 20:5w3 22:5w6 22:5w3 22:6w3

1.75 f 0.40 11.05 f 1.06 2.71 f 1.80 4.54 f 0.30 7.60 f 4.97 0.89 f 0.66 2.38 f 0.74 0.35 f 0.20 0.67 f 0.41 0.99 ? 0.38 A 6.55 f 2.70 A

12.83 f 7.52 A 3.40 f 0.86 1.06 f 0.32

17.35 f 4.85

2.77 f 0.51 12.51 f 1.22 3.09 f 1.06 5.49 f 2.59 5.92 f 0.86 3.34 f 1.38 4.03 f 1.24 0.20 f 0.17 0.98 * 0.44 0.40 f 0.25 A 3.97 f 0.99 A 7.25 f 1.46 A 2.93 f 0.89 1.21 * 0.25 19.8 f 4.36

2.56 f 0.74 16.74 f 1.50 3.31 -C 2.36 5.71 f 1.67 4.58 f 0.77 4.71 f 1.67 1.56 f 0.39 0.15 f 0.07 1.14 f 0.32 3.74 f 2.03 B 2.49 k 0.40 B 7.46 f 3.54 B 3.47 f 1.80 0.75 f 0.22

19.74 f 4.66

5.44 f 2.81 17.27 f 0.78

5.4 f 5.08 3.94 f 0.48 5.93 * 1.67 4.52 f 1.06 2.26 f 0.47 0.25 f 0.09 2.27 + 1.47 4.65 f 1.80 B 1.98 f 0.90 B 3.65 f 2.31 B 2.30 f 1.30 0.45 f 0.21

15.51 f 5.95

gamma-linolenic and AA from recent analyses of microalgae used as food for marine invertebrates (Webb and Chu 1983). Chlo- rella spp., Tetraselmis spp., C. gracilis and Pyrarnimonas virginica contain relatively high proportions of AA. In the other species AA is a very minor component or it is not reported.

Isochrysis galbana Table 1 shows the relative amounts of the

fatty acids averaged from four samples of I. galbana. About 45 fatty components were identified in four replicate samples. Major constituents included saturated (14:O and 16:0), monounsaturated (1 6: 1 w7 and 18: 1 w9) and polyunsaturated (1 8:2w6, 18: 4w3, 1 8 5 ~ 3 , 2 2 5 ~ 6 and22:6~3)fattyacids (90% of the total fatty acids). It is important to note that 22:6w3 (or DHA) was the most abundant fatty acid in this microalga, reach- ing an average value of 20%, followed by 18:4w3 at 14%.

One of the salient features of I . galbana lipids was the presence of the unusual 1 8 5 ~ 3 fatty acid. Current interest in w3- PUFA in human health (Lands 1986; Leaf

and Weber 1988) and in aquaculture led us to pay special attention to the presence of 1 8 5 ~ 3 . In a previous report (Napolitano et al. 1988b) the chromatographic behavior of 18:5w3 and its poor resolution from other typical marine fatty acids were illustrated. The fact that 1 8 5 ~ 3 does not accumulate in marine invertebrates (Mayzaud et al. 1976) strongly suggests immediate conver- sion to biochemically useful EPA by addi- tion of an acetate unit.

Comparing the fatty acid profiles obtained for I . galbana and the Chaetoceros species, a marked difference in the carbon number of the dominant fatty acids in each group of algae is apparent. The C16 acids account for more than 50% of the total in the diatoms while they account for less than 20% of the total in I. galbana. In contrast, the C18 acids represent about 40% of the total in I. galbana and only about 6% in the Chaetoceros spp.

Bivalve Larvae Table 3 shows the proportions of major

and metabolically important fatty acids in the total lipids and in the polar lipid fraction of larval European oysters Ostrea edulis and


sea scallops Placopecten magellanicus fed C. gracilis, C. calcitrans and I . galbana.

Since phospholipids represent a large proportion of the total lipids in both European oyster (- 38Oh) (Napolitano et al. 1988a) and sea scallop larvae (- 36%) (Napolitano and Ackman, unpublished results), it is possible to observe a significant enrichment of AA (P < 0.05) in the polar lipid fractions in comparison to the total lipid fatty acids.

Fatty acid analyses of many molluscs (Jo- seph 1982, 1989; Napolitano et al. 1988a) have shown that AA is selectively accumulated in phospholipids, indicating a specific role in cellular membranes. Arachi- donic acid is also a precursor of prostaglandins and other eicosanoids (Rug- geri and Thoroughgood 1985), but there is no evidence conclusively showing that AA is an essential fatty acid in marine invertebrates.

Interestingly, not all PUFA are preferentially accumulated in the animal polar lipids. Table 3 indicates a net accumulation of 20:5w3 in polar lipids of both oyster and scallop larvae, but on the other hand, I8:4w3 is depleted in the polar lipid fraction. This fatty acid, typical of I. galbana (Table 1) and other phytoflagellates, has no obvious metabolic significance for bivalves. There- fore, unlike most other highly unsaturated fatty acids, it is not accumulated in the phospholipid fraction and is preferentially found associated with the neutral lipids (Ta- ble 3; Napolitano et al. 1988b). The ex- pected enrichment in 22:6w3 in the polar lipid fraction of the oyster larvae is not apparent, probably due to the high amounts of 2 0 5 ~ 3 fatty acid.

Many marine invertebrates have been shown to include a number of polyunsaturated C20 fatty acids with an isolated ethyl- enic bond in the A5 position (Takagi et al. 1986). This indicates fatty acid A5 desatu- rase activity that should lead to AA and/or EPA if suitable precursors are available. Such abnormal PUFA lack the methylene- interrupted structures of AA and EPA and should be biochemically inactive. The at-

tempts by these invertebrates to biosyn- thesize potentially active C20 PUFA sug- gest that these compounds are needed and in the wild are not readily available in some circumstances. In this study such unusual fatty acids were not observed in the larval bivalves under study, and it is concluded that the high food value of Chaetoceros spp. and mixtures containing I. galbana (Enright et al. 1986b) results from a fatty acid composition which provides the metabolically important fatty acids of both the w3 and w6 series.

Fatty acids clearly require more consid- eration as dietary factors for marine invertebrates, since these animals constitute an important food resource for human con- sumption. The biochemical mechanisms regulating their growth and reproduction are of fundamental importance for their indus- trial cultivation.

Acknowledgments G. E. Napolitano was supported by the

Consejo Nacional de Investigaciones Cien- tificas y Tecnicas (CONICET, Argentina) and by grants to Dr. P. J. Wangersky. The cooperation of Dr. G. Newkirk in supplying the algal culture and M. A. Silva in supplying the scallop larvae is greatly acknowl- edged. The Natural Science Research and Engineering Council of Canada provided support for R. G. Ackman and P. J. Wan- gersky.

Literature Cited Ackman, R. G. 1983. Fatty acid metabolism in bi-

valves. Pages 358-376 in G. D. Pruder et al., editors. Biochemical and physiological approaches to shellfish nutrition. Proceedings of the 2nd International Conference on Aquaculture Nu- trition. Louisiana State University, Baton Rouge, Louisiana, USA.

Ackman, R. G. 1984. Straight-chain fatty acids. Pages 95-240 in CRC handbook of chromatography. CRC Press, Boca Raton, Florida, USA.

Ackman, R. G. 1986. WCOT (capillary) gas-liquid chromatography. Pages 137-206 in R. G. Ham- ilton and J. B. Rossell, editors. Analysis of oils and fats. Elsevier, New York, USA.

Ackman, R. G. 1987. Simplification of analyses of


fatty acids in fish lipids and related lipid samples. Acta Medica Scandinavica 222:99-103.

Ackman, R. G. 1989. Fatty acids. Pages 103-137 in R. G. Ackman, editor. Marine biogenic lipids, fats and oils. CRC Press, Boca Raton, Florida, USA.

Ackman, R. G., P. M. Jangaard, R. J. Hoyle and H. Brockerhoff. 1964. Origin of marine fatty acids. I. Analyses of the fatty acids produced by the dia- tom Skeletonema costalum. Journal of the Fish- eries Research Board of Canada 21:747-756.

Ackman, R. C., C. S. Tocher and J. McLachlan. 1968. Marine phytoplankter fatty acids. Journal of the Fisheries Research Board ofCanada 25: 1603-1620.

Ackman, R. G., R. F. Addison, S. N. Hooper and A. Prakash. 1970. Halosphaera viridis: fatty acid composition and taxonomical relationships. Jour- nal of the Fisheries Research Board of Canada 27:

Ackman, R. G., A. Manzer and J. Joseph. 1974. Ten- tative identification of an unusual naturally-oc- cuning polyenoic fatty acid by calculations from precision open-tubular GLC and structural ele- ment retention data. Chromatographia 7: 107-1 14.

Boutry, J-L. and M. Bordes. 1979. La diatomee marine Chaetoceros simplex calcitrans Paulsen at son environnement. VII. Aspects dynamiques des echanges en lipides entre la diatomee et son milieu. Journal of Experimental Marine Biology and Ecol-

Christ, E. J. and D. A. Van Dorp. 1972. Comparative aspects of prostaglandin biosynthesis in animal tissues. Biochimica et Biophysica Acta 270:537- 545.

Christie, W. W. 1988. Equivalent chain-lengths of methyl ester derivatives of fatty acids on gas chromatography. A :eappraisal. Journal of Chroma- tography 447:305-3 14.

Chu, F-L. E. and J. L. Dupuy. 1981. The fatty acid composition of three unicellular algal species used as food sources for larvae of the American oyster (Crassostrea virginica). Lipids 15:356-364.

Chuecas, L. and J. P. Riley. 1969. Component fatty acids of the total lipids of some marine phytoplankton. Journal of the Marine Biological Asso- ciation of the United Kingdom 49:97-116 1.

Cowey, C. B., J. W. Adron, J. M. Owen and R. J. Zoberts. 1976. The effect of different dietary oils in tissue fatty acids and tissue pathology in turbot, Scophthalmus maximus. Comparative Biochem- istry and Physiology 53B:399-403.

De Pauw, N. and G. Pruder. 1986. Use and production of microalgae as food in aquaculture: prac- tices, problems and research needs. Pages 77-101 in M. Bilio, H. Rosenthal and C. J. Sindermann, editors. Realism in aquaculture: achievements, constraints, perspectives. European Aquaculture Society Bredene, Belgium.

Enright, C. T. 1984. Determination of the relative value or phytoplankton for feeding the juvenile

25 1-255.

ogy 40:223-233.

oyster, Ostrea edulis. Doctoral thesis. Dalhousie University, Halifax, Canada.

Enright, C. T., G. F. Newkirk, J. S. Craigie and J. D. Castell. 1986a. Evaluation of phytoplankton as diets for juvenile Ostrea edulis L. Journal of Ex- perimental Marine Biology and Ecology 96:l-13.

Enright, C. T., G. F. Newkirk, J. S. Craigie and J. D. Castell. 1986b. Growth ofjuvenile Ostrea edulis L. fed Chaetoceros gracilis Shutt of varied chem- ical composition. Journal of Experimental Marine Biology and Ecology 96: 15-26.

Firestone, D. 1986. Report on oil and fats. Journal of the Association of Official Analytical Chemists

Fukusho, K., M. Okauchi, S. Nurani, A. Tsujigado and T. Watanabe. 1984. Food value of a rotifer Brachionus plicatilis cultured with Tetraselmis tetrahele for larvae of the red seabrean Pagrus major. Bulletin of the Japanese Society of Scientific Fish- eries 50: 1439-1444.

Fusetani, N. and K. Hashimoto. 1984. Prostaglandin E2: a candidate for causative agent of “ogonori” poisoning. Bulletin of the Japanese Society of Sci- entific Fisheries 50:465469.

Gillan, F. T., I. L. Stoilov, J. E. Thompson, R. W. Hogg, C. R. Wilkinson and C. Djerassi. 1988. Fatty acids as biological markers for bacterial sym- bionts in sponges. Lipids 23: 1 139-1 145.

Guillard, R. R. and J. H. Ryther. 1962. Studies on marine plankton diatoms. I. Cyclotella nana Hu- stedt and Detonula ConJervacea (Cleve). Canadian Journal of Microbiology 8:229-239.

Holland, D. L. 1978. Lipid reserves and energy metabolism in the larvae of benthic marine invertebrates. Pages 85-123 in D. C. Malins and J. R. Sargent, editors. Biochemical and biophysical perspectives in marine biology. Academic Press, New York, USA.

Joseph, J. D. 1982. Lipid composition of marine and estuarine invertebrates. Part 11. Mollusca. Progress in Lipid Research 21:109-153.

Joseph, J. D. 1989. Marine invertebrates. Pages 49- 143 in R. G. Ackman, editor. Marine biogenic lipids, fats and oils. CRC Press, Boca Raton, Flor- ida, USA.

Kayama, M., S. Araki and S. Sato. 1989. Lipids of marine plants. Pages 4-48 in R. G. Ackman, editor. Marine biogenic lipids, fats and oils. CRC Press, Boca Raton, Florida, USA.

1986. Triarachidonin and diarachidonoylphosphatidyl- choline in “ogonori” Gracilaria verrucosa. Bulletin of the Japanese Society of Scientific Fisheries 52: 757.

Lands, W. E. M. 1986. Fish and human health. Ac- ademic Press, Orlando, Florida, USA.

Langdon, C. J. and M. J. Waldock. 1981. The effect of algal and artificial diets on the growth and fatty acid composition of Crassostrea gigas spat. Jour-

68:249-25 1.

Kinoshita, K., K. Takahashi and K. Zama.


nal of the Marine Biological Association of the United Kingdom 61:43 1448.

Leaf, A. and P. C. Weber. 1988. Cardiovascular effects of n-3 fatty acids. New England Journal of Medicine 318:549-557.

Marcus, A. J. 1984. The eicosanoids in biology and medicine. Journal of Lipid Research 25: 15 1 1- 1516.

Mayzaud, P., C. A. Eaton and R. G. Ackman. 1976. The Occurrence and distribution of octadecapen- taenoic acid in a natural plankton population. A possible food chain index. Lipids 11:858-862.

Millamena, 0. M., R. F. Bombeo, N. A. Jumalon and K. L. Simpson. 1988. Effects of various diets on the nutritional value ofArtemia sp. as food for the prawn Penaeus monodon. Marine Biology 98:2 17- 221.

Moghaddam, M. F., W. H. Gerwick and D. L. Ballan- tine. 1989. Discovery of 12-(S)-hydroxy- 5,8,10,14-icosatetraenoic acid [ 12-(S)-HETE] in the tropical red alga Platysiphonia miniata. Pros- taglandins 37:303-308.

Morrison, W. R. and L. M. Smith. 1964. Preparation of fatty acids, methyl esters and dimethylacetals from lipids with boron fluoride-methanol. Journal of Lipid Research 5:600-608.

Napolitano, G. E., W. M. N. Ratnayake and R. G. Ackman. 1988a. Fatty acid components of larval Ostrea edulis (L.): importance of triacylglycerols as a fatty acid reserve. Comparative Biochemistry and Physiology 90B875-883.

Napolitano, G. E., W. M. N. Ratnayake and R. G. Ackman. 1988b. All-cis-3,6,9,12,15-0ctadeca- pentaenoic acid: a problem of resolution in the GC analysis of marine fatty acids. Phytochemistry

Nomura, T. and H. Ogata. 1976. Distribution of prostaglandins in the animal kingdom. Biochim- ica et Biophysica Acta 431:127-131.

Pillsbury, K. S. 1985. The relative food value and biochemical composition of five phytoplankton diets for queen conch, Strombus gigas (Linne) larvae. Journal of Experimental Marine Biology and

Ratnayake, W. M. N., A. Timmins, T. Ohshima and R. G. Ackman. 1986. Mass spectra of fatty acid derivatives, of isopropylidenes of novel glyceryl ethers of cod muscle, and of phenolic acetates ob-

27~1751-1755.

Ecology 90~221-231.

tained with the Finnigan Mat Ion Trap Detector. Lipids 21:5 18-524.

Ruggeri, B. and C. A. Thoroughgood. 1985. Prosta- glandins in the aquatic fauna: a comprehensive review. Marine Ecology Progress Series 23:301- 306.

Seto, A., H. L. Wang and C. W. Hesseltine. 1984. Culture conditions affect eicosapentaenoic acid content of Chlorella rninutissima. Journal of the American Oil Chemists’ Society 6 133924394.

Shigueno, K. 1975. Shrimp culture in Japan. Trading Corporation, San Francisco, California, USA.

Snedecor, G. W. and W. G. Cochran. 1980. Statistical methods. Iowa State University Press, Ames, Iowa, USA.

Takagi, T, M. &hi and Y. Itabashi. 1985. Fatty acid composition of twelve algae from Japanese waters. Yukagaku 34: 12-16.

Takagi, T., M. Kaneniwa, Y. Itabashi and R. G. Ack- man. 1986. Fatty acids in Echinoidea: unusual cis-5 olefinic acids as distinctive lipid components in sea urchins. Lipids 21558-565.

Tesbima, S., A. Kanazawa and R. Shimamoto. 1987. Effects of algal diets on the sterol and fatty acid compositions of the pearl oyster Pinctada fucata. Nippon Suisan Gakkaishi 53: 1663-1 767.

Volkman, J. K., D. J. Smith, G. Eglinton, T. E. Fors- berg and D. S. Corner. 1981. Sterol and fatty acid compositions of four marine Haptophycean algae. Journal of the Marine Biological Associa- tion of the United Kingdom 6 1509-5 17.

Waldock, M. J. and D. L. Holland. 1984. Fatty acid metabolism in young oysters, Crassostrea gigas: polyunsaturated fatty acids. Lipids 19:332-336.

Waldock, M. J. and L. A. Nascimento. 1979. The triacylglycerol composition of Crassostrea gigas larvae fed on different algal diets. Marine Biology Letters 1:77-86.

Watanabe, T. 1982. Lipid nutrition in fish. Com- parative Biochemistry and Physiology 73B:3-15.

Webb, K. L. and F-L. E. Cbu. 1983. Phytoplankton as a food source for bivalve larvae. Pages 272-29 1 in G. D. Pruder et al., editors. Biochemical and physiological approaches to shellfish nutrition. Proceedings of the 2nd International Conference on Aquaculture Nutrition. Louisiana State Uni- versity, Baton Rouge, Louisiana, USA.

Documents

Fatty Acid Composition of Three Cultured Algal Species (Isochvysis galbana, Chaetoceros gracilis and Chaetoceros calcitrans) Used as Food for Bivalve Larvae