Comparative Biochemistry and Physiology Part B 124 (1999) 309–318

Lipid profile and growth of the clam spat, Ruditapes decussatus(L), fed with microalgal diets and cornstarch

M.J. Fernandez-Reiriz a,*, U. Labarta a, M. Albentosa b, A. Perez-Camacho b

a Consejo Superior In6estigaciones Cientificas, Instituto de In6estigaciones Marinas, Eduardo Cabello, 6, E-36208 Vigo, Spainb Instituto Espanol de Oceanografıa, Centro Oceanografico de la Coruna, La Coruna, Spain

Received 4 February 1999; received in revised form 26 July 1999; accepted 3 August 1999

Abstract

The influence of the lipid composition of microalgal diets and cornstarch on the lipid classes and fatty acids of Ruditapesdecussatus spat was studied. These aspects of the nutritional value of the diets are discussed in relation to the growth of the spat.Cornstarch is rich in carbohydrate, but is deficient in protein and lipids. The microalgal cells contains lipid as well as protein andcarbohydrate. Tahitian Isochrysis cells contain phospholipids and triacylglycerols as majority lipids, whereas in the cornstarchparticles the more abundant lipids are free fatty acids and phospholipids.The n-3 fatty acids were the most abundant acids in themicroalgae whereas the n-6 fatty acids were in the cornstarch. The n-3 PUFA were not detected in cornstarch. Diets with a lipidcomposition high in phospholipids and triacylglycerols and in essential fatty acids produce good growth rates in spat of R.decussatus. However these elements alone are not sufficient to explain the quality of the diet for R. decussatus. In fact, the lipidcontent of the diet affects growth, but does not explain it totally and it is the carbohydrates content that explains the differencesin growth found when we use diets with the same composition in lipids. © 1999 Elsevier Science Inc. All rights reserved.

Keywords: Fatty acid; Lipid classes (sterol ester+waxes, sterols, free fatty acids, triacylglycerols, phospholipids); Total lipid; Growth; Microalgaldiets; Cornstarch; Clam spat

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1. Introduction

The clam Ruditapes decussatus is a species of signifi-cant commercial interest in Spanish aquaculture, but itsdevelopment in production has hitherto been limited bythe scarcity of naturally-produced spat and the highcost of producing spat in industrial hatcheries, mainlyowing to the expense of producing the phytoplanktonused in the diet [13].

In recent years, several authors have attempted tofind a substitute for live microalgae in the diet ofbivalve molluscs, and experiments have been carriedout with alternative foods such as spin- and freeze-driedmicroalgae [7], yeast and bacteria [25], modified yeasts[1,12], and microcapsules [22]. Cornstarch has beenused as a natural complement to seawater in order toimprove the index of condition and the glycogen con-

tent of adult oysters [14]. The results of the presentstudy, together with others obtained by our workinggroup [8,17,26] are the first referring the use of cereal-based products in growth-enhancing diets for the spatof bivalves.

In spite of the many attempts made to correlate thedifferences in the growth rate of molluscs with thenutrient composition of their diet [15], their nutritionalneeds, both in quantative and qualitative terms, have asyet not been definitely established. In reference tolipids, the literature indicates that diet componentsshould supply not only energy for growth, maintenanceand reproduction, (lipids, e.g. triacylglycerols) but alsocompounds that cannot be synthesized by the animal[23]. Many studies [3,4,31] show that the nutritionalvalue of the diet depends on the presence of certainfatty acids deemed to be essential (e.g. 20:5n-3 and22:6n-3). These criteria, in addition to others such asacceptability and digestibility, can be used to explainthe nutritional value of a diet [6].

* Corresponding author. Tel.: +34-86-231930; fax:+34-86-292762.

E-mail address: mjreiriz@iim.csic.es (M.J. Fernandez-Reiriz)

0305-0491/99/$ - see front matter © 1999 Elsevier Science Inc. All rights reserved.PII: S 0 3 0 5 -0491 (99 )00129 -7

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The aim of this work was to assess the influence ofthe lipid contribution of microalgal diets and commer-cial flours (e.g. cornstarch) diets on the composition ofthe lipid classes and fatty acids of the spat of R.decussatus and their relation to the growth rates ob-tained in the spat. In addition, other aspects of thenutritional value of the diet, such as carbohydrate andprotein content and their relation to the growth ratesobtained in the spat, are discussed.

2. Materials and methods

2.1. Spat

Experimental trials were carried out with clam spatR. decussatus obtained from broodstock conditioned atthe Instituto Espanol de Oceanografıa. From the avail-able stock, spat of 2.10 mm with an initial live weight(LW) of about 2.5 mg per spat, an individual dryweight (DW) of about 1.6 mg per ind and an organiccontent of 12.5% of the ash-free dry weight (AFDW)were selected and randomly distributed between theexperimental cultures.

2.2. Experimental cultures

Spat was placed on the bottom of 6 l plastic vessels.To avoid food sedimentation, each experimental culturevessel was equipped with an air point and each diet wassupplied in six daily doses, as follows: the daily foodration was suspended in 4 l of seawater and placed inflasks with strong aeration to keep the food in suspen-sion; these flasks were connected to the culture vesselsby a multi-channel dosing pump (Ismatec) which wasactivated for 15 min every 4 h by means of a timingdevice. Each diet was assayed in triplicate. The seawa-ter temperature in the culture vessels was maintained at2091oC, and the experimental cultures were main-tained for 6 weeks.

Microalgae were cultured in 6 l glass flasks in atemperature-controlled chamber at 18oC with continu-ous illumination at 9900 lux. The culture medium em-ployed was that described by Walne [30], and themicroalgae were harvested during the initial stationaryphase of growth.

2.3. Diets

Different diets composed of several ratios of themicroalgae Isochrysis galbana (clone T-ISO, termedTahitian Isochrysis) and cornstarch were evaluated. Thecornstarch was manufactured by Casa Santiveri S.A.,Spain. The food rations were suspended in seawaterusing a household food blender, and the suspensionwas then passed through a 25 mm sieve.

Three different diets were supplied at a daily ration,expressed as organic weight, of 2% of the spat liveweight [6]. One of these, the optimal microalgal diet(Diet A) was composed exclusively of microalgae, whilethe other two diets were composed of differing propor-tions of microalgae and cornstarch (1:1 and 0.5:1.5microalgae/cornstarch, diets C and E, respectively). Toassess the effect of cornstarch on the diet, two controlcultures were carried out with 50% and 25% of theoptimal ration of microalgae (1 and 0.5% of live spatweight/day, diets B and D, respectively).

The organic weight of microalgal cells was calculatedby filtration of a volume of the algal cultures throughWhatman GF/C glass fiber filters previously ashed andrinsed with a 0.5 M ammonium formate solution. Thefilters were dried to constant weight at 100°C and ashedat 450°C in a muffle furnace. A Multisizer CoulterCounter was used to determine the concentrations ofthe microalgal cultures. Water and organic content ofthe processed cornstarch were estimated at 100 and450°C, respectively.

Microalgae samples were collected throughout theexperiment for biochemical analysis. Samples of theculture were centrifuged, resuspended with 0.5 M am-monium formate solution, freeze-dried and stored at−30°C until analysed. Samples of the processed corn-starch were also collected for different analysis.

2.4. Analytical methods

Proteins were quantified following the method de-scribed by Lowry et al. [24], after hydrolysis withNaOH 0.5 N for 24 h at 30°C. Carbohydrates werequantified as glucose by the phenol–sulphuric acidmethod [28].

Lipids were first extracted with chloroform:methanol(1:2), and after centrifugation (3246×g), the precipi-tate was again extracted with chloroform:methanol(2:1). Both supernatants were pooled and washed withchloroform:methanol:water (8:4:3) as described by Fer-nandez-Reiriz et al. [16]. The solvents contained 0.05%butylated hydroxytoluene (BHT). Total lipids were de-termined gravimetrically by evaporating the solvent of200 ml of purified extract on preweighed aluminiumplates on a slide warmer (60–80°C). Storage until fur-ther processing was carried out under nitrogen at −70°C.

Fatty acids from total lipids were transesterified tomethyl esters with methanolic hydrogen chloride asdescribed by Christie [11] and subsequently analysed ona gas chromatograph (Perkin-Elmer, 8500) equippedwith a fused silica capillary column (Supelco, SP-2330)(30 m length, 0.25 mm i.d.) and a PTV cold injector(Perkin-Elmer) operated in the solvent eliminationmode. The injector temperature was 275°C and thecolumn temperature increased from 140 to 210°C at a

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rate of 1.0°C min−1. Nitrogen was used as the carriergas at 0.069 pascals (10 psi). Nonadecanoic acid wasused as an internal standard and a response factor wascalculated for each fatty acid in order to performquantitative analyses.

Since there is no absolute method for identifyingfatty acids by GLC, a series of tentative identificationprocedures was applied to the lipid mixtures studied.This approach involved co-injecting the sample alongwith standard mixtures of established composition andGC-MS (Hewlett-Packard 5971 Mass Detector). Lipidclasses were studied by thin-layer chromatography(TLC)-densitometry. Silica gel 60W plates (Merck16486) measuring 20×20 cm with a layer thickness of0.25 mm were used. Samples were applied by an auto-matic TLC sampler (Camag 27220). The plates werestained according to Freeman and West [18], with a10% CuSO4 solution in 0.85% H3PO4 by heating to180°C [10]. For the quantitative analyse, the standardsused for sterol ester+waxes, sterols, free fatty acidsand triacylglycerols were cholesteryl palmitate andarachidonic acid palmityl ester, cholesterin, oleic acidand cod liver oil (CLO, Sigma), respectively. For phos-pholipids, a standard obtained from clam (R. decussa-tus) was used. The plates were scanned with aShimadzu CS9000 densitometer, using a monochro-matic 370 nm beam of 0.4×0.4 mm working in thezigzag mode, reading the whole spot, with automaticautozero for baseline correction.

All solvents, reagents and fatty acid standards usedin this work were of analytic grade (E. Merck, Darm-stadt or Sigma Chemical Co.).

Differences between mean results were analysed byone-way analysis of variance (ANOVA) followed byTukey’s multiple comparison [27]. Correlation analysesbetween dietary versus growth of the spat were alsoperformed.

3. Results

3.1. Growth

The growth rate of the spat differs depending on thediet. The dry weight of the spat at the end of theexperiment varies between 14.0991.11 mg dry weight(DW) ind−1, by spat fed exclusively on the optimalmicroalgal diet (diet A), and 5.5190.24 mg DW ind−1

for spat fed on the diet D. The growth rate for the spatfed on diet C was lower than that for the spat fed ondiet A. Spat fed on diet B gave a final dry weightcomparable to that obtained with diet E and signifi-cantly higher than that obtained with diet D (ANOVA,PB0.05, Table 1).

3.2. Chemical composition of the components of thediets

The chemical composition is shown in Table 2. Themain component of cornstarch is carbohydrate. Thetotal lipid content of Tahitian Isochrysis cells was con-siderably higher than that of cornstarch.

3.3. Composition of lipid classes and fatty acids of thecomponents of the diets

3.3.1. Lipid classesThe major lipids found in Tahitian Isochrysis are

phospholipids and triacylglycerols, while other lipidsare only present in small amounts. In the case ofcornstarch, free fatty acids account for the majority oflipids; the phospholipids are present in small amountsand the other lipids only in trace amounts (ANOVA,PB0.05, Table 3).

3.3.2. Fatty acidsThe content of each fatty acid is expressed as a

relative % of the total fatty acids and as mg mg DW−1

(Table 4). Tahitian Isochrysis has the higher content oftotal fatty acids: 148.9 mg mg DW−1, whilst the totalfatty acid content of cornstarch is 0.1 mg mg DW−1.

In the case of the n-3 PUFA, Tahitian Isochrysiscontain 13.0 mg mg DW−1, while these acids were notdetected in cornstarch. The content of n-6 fatty acids incornstarch was 0.1 mg mg DW−1 (61.1% of total fattyacids) whereas in Tahitian Isochrysis, it was 11.3 mg mgDW−1 (7.6% of total fatty acids).

Table 1Growth rate of R. decussatus spat fed on different diets*

End of theInitialexperiment(mg DW ind−1)(mg DW ind−1)

Diet A (optimal mi- 14.0991.1a1.5890.04a

croalgal diet, 2%of live spat weight)

1.6090.03aDiet B (50% optimal 8.6590.33b

rations of microal-gal diet)

10.8090.31c1.5890.01aDiet C (50% microal-gal diet+50% corn-starch)

1.6290.02aDiet D (25% optimal 5.5190.24d

rations of microal-gal diet)

Diet E (25% microal- 8.0890.05b1.6090.00a

gal diet+75% corn-starch)

* Mean values within the same columns with different superscriptare significantly different (PB0.05). Mean9SD of three replicategroups.

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Table 2Chemical composition of Isochrysis aff. Galbana (clone T-ISO) and cornstarch tested as food for R. decussatus spata

Carbohydrates (mg mg DW−1)Proteins (mg mg DW−1) Lipids (mg mg DW−1)

235.1918.8293.9915.2 298.4914.5I. galbana (T-ISO)Cornstarch 5.091.0 993.090.2 2.090.0

a Mean9SD of three replicate groups.

Table 3Lipid class content (% of total lipids and mg mg DW−1) of Isochrysis aff. galbana (clone T-ISO) and cornstarch tested as food for R. decussatus spat*

SterolsPhospholipids Sterol ester+waxes Triacylglycerols Free fatty acids

mg mg DW−1 % mg mg DW−1 % mg mg DW−1%%mg mg DW−1 mg mg DW−1%

53.995.2 44.699.0 132.1910.0b 0.790.2 2.090.5c 0.890.2 2.590.8c159.499.1a trI. galbana (T-ISO) trtr tr tr 73.398.0 1.190.2b tr trCornstarch 26.794.5 0.490.1a tr

* Mean values within the same rows with different superscript are significantly different (PB0.05). Mean9SD of three replicate groups.

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Table 4Fatty acid composition (% of total fatty acids and mg/mg DW−1) ofIsochrysis aff. galbana (clone T-ISO) and cornstarch tested as foodfor R. decussatus spata

Isochrysis galbana Cornstarch(T-ISO)

mg mg% % mg mgDW−1DW−1

21.390.0 0.690.0 tr14:0 14.390.11.490.0 0.490.00.990.0 tr15:025.490.4 20.990.516:0 tr17.090.42.190.0 0.990.01.490.0 tr16:1n-9

2.790.016:1n-7 4.190.0 0.390.0 tr0.290.0 n.d.0.290.0 n.d.16:4n-3

0.990.017:0 1.490.0 n.d. n.d.0.590.0 n.d. n.d.17:1n-7 0.390.014.890.2 1.790.09.990.1 tr18:026.290.3 7.890.518:1n-9 tr17.690.31.790.0 0.790.01.190.0 tr18:1n-7

2.890.018:2n-6 4.190.0 59.592.0 0.190.00.490.0 n.d.0.290.0 n.d.18:3n-6

3.290.018:3n-3 4.890.0 5.290.5 tr17.490.0 n.d.18:4n-3 n.d.11.790.11.690.2 n.d.1.190.1 n.d.18:5n-30.690.0 n.d.20:0 n.d.0.490.0n.d. 0.690.1n.d. tr20:1

2.390.020:3n-6 3.590.1 n.d. n.d.1.290.9 n.d.0.890.6 n.d.20:4n-6

0.890.120:4n-3 1.190.2 n.d. n.d.0.590.020:5n-3 0.890.0 n.d. n.d.

n.d. 1.6n.d. tr22:4n-62.290.0 n.d.22:5n-6 n.d.1.590.00.490.0 n.d.0.390.0 n.d.22:5n-3

8.090.022:6n-3 11.890.1 n.d. n.d.

43.5Saturated 64.8 23.3 tr34.5 10.3� Monoenoic tr23.249.6 66.333.3 0.1� Polyenoic148.9a� Total FA 0.1b

38.1 5.225.3 tr� n-37.6� n-6 11.3a 61.1 0.1b

6.2 1.04.2 tr� n-719.0� n-9 28.3 8.7 tr

� n-3 PUFA 8.8 13.0 n.d. n.d.

a Total fatty acid (FA); polyenoic fatty acid (PUFA); trace value(tr); not detected (n.d.). Mean9SD of three replicate groups. Meanvalues within the same rows with different superscript are signifi-cantly different (PB0.05).

those fed on diets C (50% microalgae+50% corn-starch) and E (25% microalgae+75% cornstarch).The spat fed on diets B and D (50% and 25% opti-mal microalgal diet) have the lowest lipid content(ANOVA, PB0.05, Table 6).

3.4.1. Lipid classesPhospholipid were the main lipids present in the

clam spat. Other lipid classes, detected in significantlower amounts, included triacylglycerols, free fattyacids and sterols. The sterol ester+waxes were de-tected in trace amounts.

The spat fed on diet A (100% Tahitian Isochrysis)show the highest content of phospholipids.The signifi-cantly highest content in triacylglycerols is found inspat fed on diets A and C.There is no significantdifference in the triacylglycerol content of diets B(50% optimal ration of microalgae) and E (25% mi-croalgae+75% cornstarch). The spat fed on diet C(50% microalgae+50% cornstarch) show the highestcontent in free fatty acids. There are no significantdifferences in the contents of sterols of the spat fedon diets A, B, C and E, but significant differenceswere observed between these spat and those fed ondiet D (25% optimal ration of microalgae), (ANOVA,PB0.05, Table 5).

3.4.2. Fatty acidsThe main fatty acids present in the spat of R. de-

cussatus were 16:0, 18:1n-9 and 22:6n3, followed bysignificantly lower contents of 18:4n-3 and 18:1n-7.The essential fatty acid 20:5n-3 is present in smallamounts. The polyenoic fatty acids were present inthe largest amounts, followed by the monoenoic andsaturated acids (ANOVA, PB0.05, Table 6).

Differences were observed in the total fatty acidcontent according to the diet supplied. Diets A and Cshow a significantly higher content of fatty acids,with significant differences between the two. The low-est values were those shown by the diets B, D and E(ANOVA, PB0.05, Table 6).

Significant differences were observed in the contentsof the different groups of fatty acids according to thediet supplied. The significantly highest contents of thesaturated, monoenoic and polyenoic fatty acids werefound in the spat fed on diets A and C. The spat fedon diets D and E show the lowest contents.

The content of n-3 PUFA, n-9, n-6 and NMIDfatty acids differed significantly (PB0.05) accordingto the diet supplied. Spat fed on diet A (100% mi-croalgae) shows the significantly highest content inn-3 PUFA and n-9 fatty acids.The n-3 PUFA contentof the spat fed on mixed diet C (50% microalgae+50% cornstarch) is significantly higher (PB0.05) thanthat for diets B, D and E.

3.4. Composition of lipid classes and fatty acids of thespat

The composition of lipid classes, fatty acids andtotal lipids present in the spat fed on the differentdiets is shown in Tables 5 and 6.

The spat fed on diet A (optimal microalgal diet)shows the highest total lipid content followed by

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Table 5Lipid class content (% of total lipids and mg/mg DW−1) of R. decussatus spat fed on different diets*

Diet C fed Diet D fed Diet E fedInitial Diet A fed Diet B fed

% mg mg % mg mg % mg mg% % mg mgmg mgmg mg%DW−1DW−1DW−1 DW−1DW−1 DW−1

7.590.2b 44.995.1 6.691.4c 70.698.9 4.890.3d11.091.0a 45.9910.368.291.8 5.191.1e67.1910.19.692.060.899.2Phospholipidstr tr tr tr tr tr tr tr trtrSterol ester+waxes tr tr

1.590.0b 14.392.3 2.190.8a 7.493.9 0.590.0c 14.495.613.690.3 1.690.6b17.192.3 2.890.6aTriacylglycerols 23.498.0 3.791.09.191.07.692.0 1.090.1a,c 31.391.9 4.692.0b 11.893.6 0.890.0c 30.692.3 3.490.8d1.290.5 7.991.0 1.390.4aFree fatty acids

1.390.1a 9.191.0 1.090.1a 9.590.2 1.490.7a 10.392.6 0.790.0b 9.090.9 1.090.2aSterols 8.293.0 1.390.2 7.990.9

* Mean values within the same rows with differnt superscript are significantly different (PB0.05). Mean9SD of three replicate groups. Diet A: optimal microalgal diet; diet B: 50% optimalrations of microalgal diet; diet C: 50% microalgal diet+50% cornstarch; diet D: 25% optimal rations of microalgal diet; and diet E: 25% microalgal diet +75% cornstarch.

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Table 6Fatty acid composition (% of total fatty acid and mg/mg DW−1) of R. decussatus spat fed on different diets*

Diet C fed Diet D fed Diet E fedInitial Diet A fed Diet B fed

mg mg DW−1 % mg mg DW−1 % mg mg DW−1 %% mg mg DW−1% mg mg DW−1%mg mg DW−1

0.590.1 5.190.8 0.290.0 3.790.2 0.290.0 3.490.3 0.190.0 3.591.2 0.190.014:0 0.390.04.390.6 5.690.50.090.0 0.790.1 0.090.0 1.590.2 0.090.0 2.390.21.190.4 0.190.01.490.215:0 0.190.11.090.10.190.0

17.490.418.590.7 0.790.2 15.991.6 0.990.0 17.690.4 0.590.1 18.191.3 0.590.11.390.1 17.692.2 1.490.116:00.190.0 1.890.2 0.190.016:1n-9 2.290.81.490.0 0.190.0 4.292.0 0.190.10.190.0 1.290.1 0.190.1 1.690.20.190.0 3.890.3 0.290.0 3.890.1 0.190.0 5.090.13.090.1 0.190.016:1n-7 0.290.33.090.30.390.03.890.10.090.0 0.990.2 0.090.0 1.690.0 0.090.0 1.490.217:0 0.090.00.790.1 0.190.0 0.790.2 0.190.0 1.090.10.190.0 3.690.6 0.290.0 4.890.1 0.190.0 4.590.02.190.4 0.190.017:1n-7 0.190.11.190.30.290.02.790.4

4.090.24.590.2 0.290.0 4.390.7 0.290.0 5.490.0 0.190.0 6.190.2 0.290.00.390.0 3.190.5 0.390.318:00.590.2 11.291.3 0.690.1 10.190.3 0.390.0 9.591.014.490.2 0.290.013.791.218:1n-9 1.390.216.391.01.090.1

5.990.24.990.2 0.290.1 5.990.4 0.39 0.0 7.390.3 0.290.0 6.790.4 0.290.00.490.0 5.290.5 0.490.218:1n-70.190.0 2.390.4 0.19 0.018:2n-6 1.390.11.890.2 0.090.0 2.390.7 0.190.00.190.0 1.991.0 0.290.3 1.890.00.090.0 0.590.2 0.0 90.0 0.690.1 0.090.0 0.590.10.490.0 0.090.0tr18:2n-4 0.690.0 0.190.0 0.390.00.190.0 1.890.1 0.19 0.0 1.390.2 0.090.0 1.390.218:3n-3 0.090.02.390.7 0.290.1 2.690.4 0.290.1 1.990.10.090.0 0.390.0 0.090.0 0.690.1 0.090.0 0.690.30.690.2 0.090.018:3n-6 0.190.00.590.10.190.00.990.0

5.190.15.390.3 0.290.1 4.790.5 0.390.0 3.190.1 0.090.0 2.690.1 0.190.00.490.1 7.090.9 0.690.118:4n-30.090.0 0.290.0 0.090.0 0.190.2 0.090.0 0.290.30.390.0 0.090.00.290.020:0 tr0.190.0tr

0.590.00.690.1 0.090.0 0.590.1 0.090.0 0.790.1 0.090.0 0.490.5 0.090.00.190.0 0.190.0 tr20:1n-110.190.0 2.590.3 0.190.0 2.890.2 0.190.0 2.390.820:1n-9 0.190.02.990.1 0.290.0 3.690.3 0.390.0 3.290.10.090.0 0.690.1 0.090.0 1.090.1 0.090.0 0.790.10.790.0 0.090.020:1n-7 0.190.10.690.20.190.00.790.10.090.0 1.490.1 0.190.0 1.090.0 0.090.0 1.290.120:2NMID1 0.090.01.090.3 0.190.1 1.190.8 0.190.1 1.190.00.090.0 0.890.1 0.090.0 0.790.0 0.090.0 1.090.00.590.0 0.090.020:2NMID2 0.190.10.590.10.190.10.690.3

1.490.11.490.6 0.190.0 1.890.2 0.190.0 1.590.0 0.090.0 1.990.1 0.190.00.19 .0.0 1.490.5 0.190.020:2n-60.090.0 0.790.2 0.090.0 0.090.0 0.090.0 0.890.10.390.1 0.090.0tr20:3n-6 0.290.0 tr0.190.0

1.690.11.790.1 0.190.0 1.290.3 0.190.0 1.690.4 0.090.0 1.690.1 0.090.00.190.0 1.590.3 0.190.020:4n-6

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Table 6 (Continued)

Diet D fedInitial Diet E fedDiet A fed Diet B fed Diet C fed

mg mg DW−1 % mg mg DW−1 % mg mg DW−1 %% mg mg DW−1mg mg DW−1 % mg mg DW−1 %

0.090.0 1.090.2 0.190.0 0.390.0 0.090.00.190.0 0.790.120:4n-3 0.090.00.590.10.790.30.190.00.790.14.790.1 1.190.1 0.090.0 0.990.1 0.190.0 1.190.0 0.090.0 1.290.5 0.090.00.390.0 1.190.220:5n-3 0.190.1

0.090.0 0.690.0 0.090.0 0.490.022:1n-11 0.090.00.590.1 0.790.2 0.090.0tr 0.590.2 tr 0.690.10.190.1 2.691.0 0.290.1 4.890.3 0.190.0 4.691.23.690.3 0.190.02.990.9 0.290.022:2NMID1 3.190.2 0.290.0

1.890.32.290.2 0.190.0 1.290.4 0.190.0 2.890.0 0.190.0 3.090.4 0.190.00.290.0 0.990.2 0.190.022:2NMID20.090.0 1.390.1 0.190.0 1.090.0 0.090.0 0.790.022:4n-6 0.090.01.090.1 0.190.0 1.490.3 0.190.1 1.190.10.090.0 1.090.1 0.190.0 0.390.0 0.090.0 1.190.90.590.3 0.090.00.890.022:5n-3 0.190.10.490.00.190.0

16.091.010.990.5 0.690.2 20.395.6 1.190.5 15.690.8 0.490.0 9.691.4 0.290.00.890.1 16.091.9 1.390.122:6n-3

1.1b 25.7 1.3b 29.6 0.7d2.4a 31.6� Saturated 0.9d28.928.12.129.631.2 32.0 1.1b 30.5 1.5c 33.1 0.9d 34.0 0.8d2.4 31.6� Monoenoic 2.5a

1.4b 43.8 2.6c 37.6 0.6d� Polyenoic 34.739.2 0.7d3.1 40.3 3.5a 39.33.6b 5.4c 2.2d 2.4d� Total FA 8.47.6

6.87.0 0.3 7.6 0.4 6.0 0.0 7.9 0.20.5 6.8 0.6� n-60.7b� n-9 15.518.0 0.8b 15.1 0.5c 16.0 0.4c1.3 21.1 1.7a 19.20.0 1.1 0.0 1.1 0.0 1.11.1 0.00.6 tr� n-11 1.1 0.1

23.222.4 0.8b 27.9 1.7c 20.4 0.4d 15.2 0.3d1.7 25.2 2.2a� n-3 PUFA0.3� NMID 6.06.9 0.4 9.3 0.2 9.8 0.20.6 5.4 0.5 7.011.0b 15.8c 7.1d 13.1e18.3aTotal lipids 17.4

* Mean values within the same rows with different superscript are significantly different (PB0.05). Mean9SD of three replicate groups. Diet A: optimal microalgal diet; diet B: 50% optimalrations of microalgal diet; diet C: 50% microalgal diet+50% cornstarch; diet D: 25% optimal rations of microalgal diet and diet E: 25% microalgal diet +75% cornstarch. Total fatty acid (FA);Polyunsaturated fatty acid (PUFA); Nonmethylene interrupted dienoic fatty acid (NMID).

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4. Discussion

Our study shows the different nutritional values of thefive diets used to feed the spat of R. decussatus. Thereplacement of 50% of the optimal microalgal diet bycornstarch, produces a growth rate, in dry weight, of 77%of to that obtained with the microalgal diet. The sub-op-timal diet, consisting of 25% of the optimal microalgalration, gave the lowest growth rate, 39% of that obtainedwith the optimal microalgal diet. The 25% microalgae+75% cornstarch diet produced a growth rate equivalentto 57% of that of the spat fed on the microalgal diet.Although a substitution of 50% of the microalgae withcornstarch is more advantageous for achieving growth(Table 1), it may well be more profitable on certainoccasions to employ diets with substitution levels of 75%of microalgae with cornstarch, bearing in mind thereduction in costs attributable to the production ofmicroalgae.

Comparable growth rates to those obtained in thepresent work were achieved in a previous study [17]where wheatgerm was used as a substitute food. How-ever, although the substitution of 50% of the optimalmicroalgal diet with wheatgerm instead of cornstarchproduces higher growth rates, the use of cornstarchshould not be rejected, since the use of wheatgerm is moreexpensive.

Phospholipids and triacylglycerols play an importantrole as a structural base and a source of energy in larvae,juveniles and adults of bivalve molluscs [20,21,29].Beninger [9] observed that both triacylglycerols andphospholipids contributed to maintenance energy re-quirements in two species of adult clam (R. decussatusand R. philippinarum). Certain specific differences havebeen observed: R. philippinarum contains more triacyl-glycerols, while R. decussatus contains more phospho-lipids.

In our studies R. decussatus was observed to containmore phospholipids than triacylglycerols, coincidingwith the observations for this species recorded byBeninger [9]. Also, spat fed on a diet containing asignificantly higher content of phospholipids and triacyl-glycerols is that which returned the highest growth rates(diet A), echoing Beninger’s conclusions on the impor-tant energetic and structural role performed by thesecompounds.

Nevertheless, it is necessary to indicate that diets withsimilar phospholipid and triacylglycerol contents pro-duce significantly different growth rates: this is the casefor diets B and C so as diets D and E, with the highergrowth rates in both cases corresponding to the diets thatwere supplemented with cornstarch (993.0 mg/mg DW−1

of carbohydrates).Another significant finding about carbohydrate re-

quirements in the spat clam diets, is that diets B and Eachieve similar growth rates (P\0.05), although diet B

contains 50% more phospholipids and triacylglycerolsthan diet E. This can be explained by the fact that dietB only contains 11.75% DW of carbohydrates while dietE contains 80.35% DW of carbohydrates.

The need for certain fatty acids seems to be speciesdependent. Tapes semidecussata and Mercenaria merce-naria need 22:6n-3 while Crassostrea sp. shows a funda-mental need for 20:5n-3 [19]. Both 20:5n-3 and/or 22:6n-3can alternatively meet the requirements of bivalves forn-3 PUFA [23]. Fernandez-Reiriz et al. [17] also confirmthe essential nature of 20:5n-3 and/or 22:6n-3 in the spatof R. decussatus, and observe that a decrease in theamount of 22:6n-3 present in the diet has a negative effecton the growth rate of the spat. Our study confirms theessential and alternative nature of 20:5n-3 and/or 22:6n-3. Nevertheless, as is the case with phospholipids andtriacylglycerols, diets with identical contents of 22:6n-3show significantly different growth rates (PB0.05).

Positive and highly significant correlations (PB0.001)were observed between the lipid composition and spatgrowth (i.e. phospholipids and triacylglycerols and foressential fatty acids: 20:5n-3, 22:6n-3 and n-3PUFA).

In previous research [2,5] our group has stablished thatR. decussatus has low requirements in proteins and lipidsand high nutritional requirements in carbohydrates andthis can explain the results of the present work. In fact,the lipid content of the diet affects growth, but does notexplain it totally and it is the carbohydrates content thatexplains the differences in growth found when we usediets with the same composition in lipids. However, thisfactor probably does not have the same effect in others clams. Particularly, this seems to be the case for V.pullastra, with its higher nutritional requirements inproteins and lipids than in carbohydrates according toour results. These differences in nutritional requirementsbetween the two species of clams (R. decussatus and V.pullastra) can be due to their different habitats. R.decussatus is a intertidal species with periods of aerobicand anaerobic metabolism, while V. pullastra is subtidaland its metabolism is aerobic.

Acknowledgements

We would like to thank Dr J.L. Garrido, B. Gonzalez,L. Nieto, and A. Ayala in the biochemical analyses andC. Fernandez Pena and H. Regueiro for their helpfultechnical assistance in the algal and spat cultures Thiswork was funded by CICYT-CSIC.IEO projet I+Dnumber AGF95-1003-CO2.

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