Marine Biology (2003) 143: 327-338DOl 1O.1007/s00227-003-1076-x

N. Nevejan . V. Courtens . M. Hauva . G. GajardoP. Sorgeloos

Effectof lipidemulsions on productionand fatty acid compositionof eggs of the scallop Argopecten purpuratus

Received: 29 October 2002/ Accepted: 21 March 2003/Published online: 17 May 2003@ Springer-Verlag 2003

Abstract The impact of supplementing lipid emulsionsrich in eicosapentaenoic acid (EmEPA), docosahexae-noic acid (EmDHA) or saturated fatty acids (EmCOCO)to a standard algal diet [3:1mixture of Isochrysisgalbana(T-iso) and Chae/oceros neogracile, St-diet] on Argo-pecten purpura/us broodstock was evaluated. Brood-stock fecundity was compared as well as the egg qualityin terms of lipid content, fatty acid composition andlipid class distribution. Fecundity was defined as thenumber of eggs released in the spawning process, sincespawning was virtually complete. Results indicated thatthe total lipid content of the eggs of A. purpura/us wasdiet independent. A greater energy reserve was spent ona larger number of oocytes and not on bigger sized 00-cytes with a higher lipid content. The lipids suppliedthrough the emulsions were at least partially allocated tothe eggs, demonstrating that the fatty acid compositionof the eggs could be manipulated, especially the neutrallipid fraction. Levelsof EPA changed more rapidly thanDHA levels, supporting the observation that they ful-filled an energetic and structural role, respectively. TheSt-diet supplemented with 50%EmCOCO resulted in asignificantly higher fecundity compared to the algal dietsupplemented with 25%EmEPA + 25%EmDHA andthe non-supplemented algal diet. It would seem thatsaturated fatty acids (SAFA) were more easily or pref-erentially incorporated in the female gonads of A. pur-pura/us. The relative content of SAFA and 18:2(n-6)inthese eggs rose significantly. The relative content of thehighly unsaturated fatty acids, EPA and DHA, on theother hand was substantially lower in the neutral lipid

Communicated by O. Kinne, Oldendorf/Luhe

N. Nevejan (121). M. Hauva . G. GajardoUniversidad de Los Lagos, Laboratory of Genetics & Aquaculture,P.O. Box 933, Osorno, ChileE-mail: Nancy.Nevejan@rug.ac.beFax: + 32-9-2644193

N. Nevejan . V. Courtens . P. SorgeloosGhent University, Laboratory of Aquaculture & Artemia ReferenceCenter, Rozier 44, 9000 Ghent, Belgium

fraction, but hardly affected in the polar lipid fraction. Itappeared that the maintenance of an adequate DHA/EPA ratio (approximately 1.2)was more important thanthe absolute levels of the two fatty acids, as long as athreshold value was reached.

Introduction

The ChileanfPeruvian scallop Argopec/en purpura/us(Lamarck 1819) has become an important aquaculturespecies in Chile. Because the high fishing pressureendangered the existence of the natural banks (Wolffand Alarcon 1993),extraction has been forbidden, andits culture has developed extensively during the lastdecade (Martinez et al. 2000).The production increased16-fold in 10years, from 1,182 tonne (in 1990) to19,038tonne (in 2000) (SERNAPESCA 2001). Thescallop is a functional hermaphrodite and exhibits con-tinuous gametogenic activity with two main spawningpeaks, one in late sUmmerand a minor one in autumn(Wolff 1988; Martinez 1991). A great variability inspawning success and larviculture results in hatcheriesstimulated researchers to study the factors that influencethe reproductive process of A. purpura/us (Martinezet al. 1992,2000; Farias et al. 1997; Caers et al. 1999b,2000; Navarro et al. 2000; Farias and Uriarte 2001).

Marine bivalves undergo a seasonal cycle of energystorage and utilisation that is linked to the annualreproductive cycle,which is regulated by exogenous andendogenous factors. Energy substrates stored duringnon-reproductive periods are used subsequently: (1) tosupport maintenance requirements when food is scarce,(2) to replace body mass lost at other times of the year,(3) to produce a net increase in mass over the year and(4) to produce gametes (Barber and Blake 1985b;Bayne1985).

Bivalvebroodstock conditioning in hatcheries aims atmaximising fecundity of the parent animals, whilemaintaining egg quality and larval viability (Utting and

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VLAAMS INSTITUUT VOOR DE ZEl

FLANDERS MARINE INSTITUTEOostende - Belgium

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Millican 1997). The quality of the diets given to thebroodstock explains a great portion of the variabilityexperienced during bivalve rearing as it affects gametecomposition and larval quality. Wolff (1988) suggestedthat, for A. purpuratus, high temperature, althoughfavouring maturation and spawning, might be lesscritical for successful spawning than food availability.Devauchelle and Mingant (1991) found that, althoughtemperature initiates the process of gametogenesis,fecundity of Pecten maximus and the quality of the eggsproduced were enhanced when algal supplements weregIven.

During oogenesis, the oocytes acquire their lipid re-serves from three main sources: (1) the adductor muscle,whereby muscle glycogen is converted to lipid (Gabbott1975;Bayne et al. 1982;Barber and Blake 1985a,1985b);(2) lipid transfer from reserves in the digestive gland tothe female gonad (Vassalo 1973; Barber and Blake1981,1985a);and (3) directly from food when adults areput under nutritional stress (Gallager and Mann 1986;Soudant et al. 1996b).

Stored egg reserves are catabolised initially duringembryonic development from fertilised egg to straight-hinge veligers. Further catabolism occurs to maintainthe larvae during the transitional stage of exogenousfeeding (Whyte et al. 1990). Survival of Mercenariamercenaria and Crassostrea virginica larvae is highlycorrelated with the total lipid reserves laid down in theeggs during vitellogenesis (Gallager and Mann 1986).The early development of Myti/us edulis larvae is cor-related with the lipid level in unfertilised eggs (Bayneet al. 1975), and the viability of Ostrea edulis larvae isrelated to the lipid content at the time of liberation,particularly the neutral lipid fraction (Helm et al. 1973).

Conditioning broodstock with diets of different lipidcontent and quality showed the effects of specificfattyacid deficiencies in bivalve eggs and larvae (Utting andDoyou 1992; Millican and Helm 1994; Soudant et al.1996a). Based on these findings, the aim of this studywas to examine whether the supplementation of differentlipid emulsions to the algal diet would enhance brood-stock fecundity and to what extent it would alter thelipid content and composition of the spawned eggs.Three different emulsions were tested, either rich in22:6(n-3) (EmDHA), 20:5(n-3) (EmEPA) or saturatedfatty acids (EmCOCO).

Materialsand methods

Diets

Two algal species used as standard feed, Isochrysis ga/bana (T-iso)and Chaetoceros neograci/e, were grown in 12-1or 16-1plastic bags(batch system) with aerated, filtered (0.5 jlm) and UV-treated sea-water enriched with the commercial fertiliser Bayfolan. Previous toinoculation with the algae, water in the bags was additionallysterilised with chlorine (0.1 ml r1) for 24 h and then neutralisedwith thiosulphate (0.1 g I-I) and aeration. The algae were grownunder a 24-h light regime at a temperature of 26:!: 1°C and wereharvested in the exponential phase.

Experimental enrichment emulsions (INVE Aquaculture NVBaasrode, Belgium) contained 62% lipid on a wet weight basis, inaddition to water, vitamins, emulgators, antioxidants and preser-vatives. Lipids were present mainly as triglycerides. The two lipidemulsions named EmEPA and EmDHA contained approximately30% n-3 highly unsaturated fatty acids (HUFAs) on total fattyacids and had a DHA/EPA ratio of 0.6 and 4, respectively. Thelipid emulsion EmCOCO consisted mainly of saturated lipids, inparticular 12:0, 14:0 and 16:0. It contained hardly any n-3 poly-unsaturated fatty acids (PUFAs) (ICES 1994). The percentage oflipid supplement was calculated on basis of the dry weight (DW) ofthe administered algae, being 21 and 70 pg cell-I for I. ga/bana(T-iso) and C. neogracile, respectively.

Experiments

The scallops of the two experiments were sent from Coquimbo(region IV) to the field laboratory of the Universidad de Los Lagosin Calbuco (region X). The animals belonging to one treatmentwere divided evenly into two tanks, each with 50 I of filtered(0.5 jlm) and UV-treated seawater. Aeration was provided bymeans of aquarium pumps. The temperature was maintained at17°C (:!: 1°C). Water was changed with a flow-through system twicea week and completely renewed once a week. When changedcompletely, the tanks were washed with soap and diluted chlorine.The temperature of the fresh seawater was always one degree lowerthan the temperature in the tanks, to avoid undesired spawning.Towards the end of the conditioning period, when the animalsshowed developed gonads, only half the ration of algae was fedwhen water was changed completely. The other half was compen-sated for the following day by giving 1.5 times the ration. Thescallops were fed 6 days a week, and food was distributed with adrip system. Antibiotic (chloramphenicol) was added with everywater change at a concentration of 4 mg I-I.

The animals in experiment I (expt I) had been kept in thelaboratory for nearly 2 months before the experiment started,receiving algae ad libitum. They were stimulated to spawn prior tothe experiment, and those exhibiting completely empty gonads wereselected. The experiment took place during the winter months (endJuly-end August). The animals had an average shell height of78.3 mm (:!:3.0 mm). Groups of ten animals were fed three dif-ferent diets. Each animal received 3xlO9 alga cells daily when fedthe standard diet (St) or the St-diet supplemented with 50%EmDHA (St + 50dha). The St-diet consisted of a mixture of1. ga/bana (T-iso) and C. neograci/e (3:1, based on cell counts) thatwas considered to be a good maturation diet (Farias et al. 1997;Martinez et al. 2000). A third diet consisted of only half the amountof algae, maintaining the same proportion between the two algalspecies, supplemented with 50%EmDHA (I/2St + 50dha). After33 days, nine animals belonging to different treatments spawnedspontaneously. The remaining animals were stimulated to spawnthe next day.

The animals in experiment 2 (expt 2) had an average shellheight of 66.6 mm (:!:2.5 mm). They arrived straight from thenorth and had just gone through a long and severe winter. Theywere devoid of any gonadal tissue. The experiment took placeduring springtime (early September-end November). Four dietswere tested, and each treatment consisted of 14 animals. TheSt-diet served as the control diet. The other three diets consistedof the St-diet supplemented with lipid emulsions, eitherwith 25%EmDHA+25%EmEPA (St+50hufa), 50%EmEPA(St+50epa), or with 50%EmCOCO (St+50coco). All animalsreceived 2.5x109 alga cells daily. A higher ration, as in expt I, wasnot used since it led to the production of pseudo-faeces. Duringthe last 20 days of the conditioning period, however, the rationwas increased to 3xlO9 alga cells daily without any production ofpseudo-faeces.

The two tanks of the St + 50epa treatment and one tank ofthe St-treatment spawned spontaneously after 62 days. Thereleased eggs were recovered by passing the water through a sieveof 37 jlm, while emptying the tanks, and preserved for lipid

analysis. It was impossible however to measure all other eggcharacteristics. No animals were left to represent the St + 50epa-diet, and only eight animals of the St-diet were taken into furtherconsideration. On day 82, all remaining animals were stimulatedto spawn.

Analysis

Eggs of each individual animal were diluted in a known volume ofseawater. Four subsamples were taken to count the eggs. Fecunditywas defined as the number of eggs released in the spawning process,since spawning was virtually complete. The diameter of 50 eggsfrom each spawning animal was measured to determine the averagesize. A total of 200,000 eggs were filtered on pre-combusted andpre-tared GFC-filters to determine DW. Three filters per animalwere dried at 60°C for 48 h.

Samples of eggs for lipid analysis were washed twice with 0.5 Mammonium formate to remove salts. They were freeze-dried for48 h and subsequently stored at -30°C under nitrogen. Samples ofalgae for lipid analysis were concentrated by using a refrigeratedcentrifuge and submitting subsamples of 40 ml to a rotation speedof 8,000 rpm (c. neograci/e) or 9,000 rpm (I. go/bona); between 3and 4 I of the algal culture was concentrated in this way. Tubeswith the concentrated algae were frozen for 30 min at -70°C, thenthe algal paste was removed and washed in 0.5 M ammoniumformate and kept at -70°C in a tube with a Teflon cap untilanalysis.

Total lipids (TL) were extracted as described by Folch et at.(1957), with some small modifications. They were extracted withthe solvent mixture chloroform:methanol (2:1, v/v). Followingsolvent evaporation under nitrogen, lipids were determinedgravimetrically. TL were dissolved again in the solvent mix at aconcentration of 10 mg fat ml-I solvent mix. A total of 200 JlIwastaken to determine the fatty acid methyl esters (FAME) and di-methyl acetals (DMA), and another 200 JlI, to separate neutralfrom polar lipids. Esterification of fat was accomplished by theaddition of freshly prepared acetylchloride-methanol (1:20) andleaving the reaction to take place for I h in a boiling water bath.Once cooled, FAME and DMA were extracted with hexane. Forthe calculation of the percentage of individual DMAs andFAMEs, "total fatty acids" refers to the sum of all DMA andFAME present in the sample. Neutrallipids (NL) and polar lipids(PL) were separated over a silicagel-microcolumn according to themethod of Marty et at. (1992). FAMEs of the NL and PL frac-tions were prepared by transmethylation with a mixture of sul-phuric acid and methanol (1:100, v/v) for 16 h at 50°C.Quantitative determination of FAME and DMA was done by aChrompack CP9001 gas chromatograph equipped with an auto-sampler and a temperature-programmable, on-column injector.Injections were performed on-column into a polar, 50 m capillarycolumn, BPX70 (SGE Australia) with a diameter of 0.32 mm anda layer thickness of 0.25 Jlm connected to a 2.5 m methyl-deacti-vated pre-column. The carrier gas was hydrogen. Identificationwas based on standard reference mixtures (Nu-Chek-Prep, USA).Integration and calculations were done with the software programMaestro (Chrompack). Lipid class analysis was performed with anIATROSCAN MK-5. The neutral solvent system existed of850 parts hexane, 150 parts ether and 0.4 parts formic acid. Thepolar solvent system existed of 700 parts chloroform, 350 partsmethanol and 35 parts water. Each sample was run three times,and identification was based on a standard reference mixture(l8-5C of Nu-Chek-Prep, USA).

Data analysis

The statistical package STATISTICA 5.0 was used for dataanalysis. All data were log-transformed before applying any sta-tistics, while values in percentage were arcsine-square-root trans-formed (Sokal and Rohlf 1995). The homogeneity of variances of

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means was tested by univariate tests (Cochran, Hartley, Bartlett).Significant differences were detected using one-way ANOV A, Pbeing set at 0.05. The Newman-Keuls' test was used for post hoccomparison. In cases when data were not homogeneously distrib-uted, the Mann-Whitney V-test was used to determine differencesbetween each set of pairs.

Results

Diets

The major fatty acids in Isochrysisgalbana (clone T-iso)were 14:0, 16:0, 18:I(n-9), 18:3(n-3), 18:4(n-3) and22:6(n-3), whilst in Chaetoceros neogracile they were14:0, 16:0, 16:1(n-7) and 20:5(n-3) (Table I). The fattyacids 16:0and 16:I(n-7) accounted for about 50% of thetotal fatty acids (TFA) of C. neogracile. This alga alsohad a relatively high amount of arachidonic acid (ARA).The emulsions EmEPA and EmDHA had, respectively,17% and 8% EPA and Il% and 29% DHA. The sat-urated fatty acid (SAFA) content for both emulsionsrepresented about 25% of TF A. EmCOCO on the otherhand contained 81% SAFA, of which nearly half wasmade up of lauric acid (12:0). The content in 14:0 wasabout four times higher than in EmEPA or EmDHA.Hardly any (n-3)PUFAs were present in the emulsionEmCOCO. The three lipid emulsions had more or lessthe same FAME content, being 600-750 mg FAME g-JDW.

Experiment I

No mortality was observed during the experiment.After 33 days, all animals fed the St-diet spawnedsperm and eggs, while 40% of the broodstock spawnedeggs when fed the St + 50dha-diet and 30% when fedthe 1/2St+ 50dha-diet (Fig. lA). Egg size and DW didnot differ significantly among the treatments (Fig: IB,C). There was however a significant difference inthe number of eggs produced by the differenttreatments (F(2,J4)= 5.05, P <0.05) (Fig. 1D). Brood-stock animals receiving 1/2St+ 50dha producedsignificantly less eggs than the broodstock fedSt+ 50dha [2.2(::f:1.8)xl06 and 8.6(::f:3.4)xI06 eggsanimal-I, respectively].There was no difference betweenthe latter and the St-diet [5.4(::f:2.5)xl01, although thelipid supplementation of 50%EmDHA tended tofavour egg production.

Sixty percent of the fatty acid composition of eggswas made up of 16:0, 16:1(n-7),18:I(n-9), 20:5(n-3)and22:6(n-3) (Table 2). This corresponded well with thehigh levels of these fatty acids provided by the diets tothe parent animals. The high content of 14:0in the algaldiet, however, was not reflected in the egg composition.The addition of 50%EmDHA to the diet clearly affectedthe DHA content in eggs. Eggs spawned by animalsconditioned with the diets St + 50dha and 1/2St+ 50dha

Fig. 1 Argopecten purpuratus.Egg-spawning results ofexperiments I and 2(abbreviations for diets, see"Materials and methods"; DWdry weight

Percentage broodstoc:k spawning eggsIn Experiment 1and 2

Egg size (I1m) In experiment 1 and 2

1008060

.,. 4020o

6058

13.:5250

rJN (nglegg) of eggs spawnedIn Experiment 1 and 2

Number of eggs (x E61anlmal)releasedin experiment 1 and 2

possessed 33% and 54% more DHA, respectively, thanthe eggs released by St-diet-fed animals (Table 2). Thisled to a significantly higher DHA/EPA ratio and ahigher content of (n-3)PUFA. The EPA and ARAcontents were not affected by the differences in theparental diet. The DMAs were dominated by 18:0. No

significant difference in (18:0)DMA and total DMA wasnoticed among the different diets. The three broodstockdiets did not lead to significant differences in SAFA and(n-6)PUFA levels in the eggs. The animals fed with theSt-diet released eggs richest in MUF A, in particular16:1(n-7) and 18:1(n-7).

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Table 1 lsochrysis galbana,Fatty acid I. galbana C. neogracile EmEPA

Chaetoceros neogracile. FattyEmDHA EmCOCO

acid composition (% ofTFA, (n=5) (n = 5) (n = 6) (n = 5) (n=2)

total fatty acids) of the algaeand emulsions (EmEPA, 12:0 0.5:i:0.0 0.4:i:0.5 tr tr 38.3:i: 0.0

EmDHA, EmCOCO) used in 14:0 16.2:i:3.8 11.9:i: 1.0 6.5:i:0.2 7.1 :i:3.7 24.2:i: 0.0

this study. Values are means 16:0 13.5:i:4.4 19.2:i:7.8 15.4:i:0.5 12.8:i:5.2 14.1:i:0.0

(:i:SD) (SAFA saturated fatty 18:0 1.7:i: 1.5 1.7:i:0.2 3.5:i: 1.2 3.7:i: 1.4 4.4 :i:0.0

acids; MUFA monounsaturated SAFA 33.H9.0 35.5:i:8.1 25.4:i: 1.1 23.7:i:3.9 81.1 :1:0.0

fatty acids; PUFA 16:I(n-7) 5.4:i:0.8 32.8:i: 3.6 7.8:1:0.4 2.8:i: 1.7 0.1 :1:0.0

polyunsaturated fatty acids; 18:I(n-9) 14.1:1:4.7 0.9:i:0.3 11.4:1:0.7 11.6 :1:2.0 10.3:1:0.0

DHA docosahexaenoic acid; 18:I(n-7) 2.8:i:0.4 2.5 :i: 1.4 3.7 :1:0.5 1.8:i:0.5 0.3:1:0.0

EPA eicosapentaenoic acid; MUFA 23.2:1:5.4 38.4:i:2.4 22.9:i:0.7 16.3 :i:3.7 10.8:1:0.0

FAME fatty acid methyl esters; 18:2(n-6) 5.0:i: 1.3 1.0:1:0.3 5.l:!: 0.3 6.1 :i:0.6 7.2:1:0.0

DW dry weight; n number of 18:3(n-6) 0.4:i:0.2 0.8:i:0.3 0.2:1:0.0 0.1 :i:0.0 tr

samples; tr trace) 20:3(n-6) tr tr 0.1:i:0.0 tr tr20:4(n-6) 0.1 :1:0.1 3.7:i:0.6 1.0:1:0.0 1.1:i:0.6 tr22:5(n-6) 1.8:i: 1.2 tr 0.4:1:0.1 1.2:i:0.5 tr

(n-6)PUFA 7.6:i:2.0 5.H 1.0 6.HO.3 8.4:i:0.7 7.2:1:0.018:3(n-3) 7.6:i: 1.4 0.2 :i:0.1 1.3 :1:0.2 1.0:i:0.1 0.6:1:0.018:4(n-3) 16.1:1:6.2 0.6:i:0.4 1.9:i:0.1 0.7:i:0.1 tr20:3(n-3) tr tr 0.1 :1:0.0 tr tr20:4(n-3) tr tr O.HO.O 0.4:i:0.1 tr20:5(n-3) 0.9:1:0.4 11.2:i:7.3 17.0:1:1.6 8.0:i: 1.9 tr22:5(n-3) 1.0:i: 1.7 tr 2.5:1:0.1 3.6:i:2.7 tr22:6(n-3) 8.4:i: 4.2 1.2:i:0.4 11.3:1:0.7 29.4:i:5.0 tr

(n-3)PUFA 34.1:i: 13.5 13.3 :1:8.1 34.7:i:1.7 43.0:i:8.6 0.6:i:0.0DHA/EPA 8.8:i:4.2 0.1:i:0.1 0.7:i:0.1 3.7 :i:0.4 trn-3/n-6 4.5:i: 1.9 2.4 :I:1.2 5.2:i:0.4 5.2:i: 1.4 0.08 :i:0.0mg FAME g-I DW 146.1 184.7 754.3:1: 169.6 748.1:i: 154.3 608.7:1: 11.0

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Generally, the NL of the eggs were evenly dividedbetween SAFA (30%), MUFA (30%) and (n-3)PUFA(25%) (Table 3). The remaining 15% were present as(n-6)PUFA (10%) and OMA (5%). The PL fraction, on

Table 2 Argopecten purpuratus. Experiment I: fatty acid composi-tion (% of TFA and DMA) of eggs produced by the broodstockunder three different nutritional conditions (abbreviations for diets,see "Materials and methods"). Values are means (:i:SD). Values inone row followed by a different letter are significantly different(Newman-Keuls' test, P<0.05) (DMA dimethyl acetal; otherabbreviations, see Table I)

Fatty acid St (n= 10) St+50dha (n=4) 1/2St+5Odha (n=3)

331

the contrary, was dominated by (n-3)PUFA (35%),SAFA (25%) and OMA (20%). MUFA and (n-6)PUFArepresented about 10% of the PLs. EPA was present inalmost equal amounts in the NL and PL fractions, whilethe OHA content in the PL fraction was at least twice ashigh as in the NL fraction.

The EPA content in the NL and PL fractions was notaffected by the diet, while the relative content of OH A inboth fractions increased significantly when lipid emul-sion was supplemented. As a consequence, the OHAIEPA ratio differed significantly amongst the diets, withthe highest value observed for 1/2St + 50dha. In the PLfraction, the (n-3)PUFA content and the n-3In-6 ratiowere also significantly higher when lipids were supple-mented, while OHA and ARA were preferentiallyincorporated in this fraction. Replacing 50% of thealgae with lipid emulsion led to a significant decrease inMUF A content in the NL and PL fractions, comparedto the 100% algal diet.

Experiment 2

No mortality was observed during the experiment. Therewere no significant differences in egg size (52-54 Jtm) oregg OW (24-27 ng egg-I) among the diets, while the eggquantity varied significantly (Fig. 1B, C, 0). Animalsthat received the 50%EmCOCO supplement performedbest. They released on average 7.8(:1::2.7)x106eggsindividually, which is significantly better than thebroodstock animals of the St- and the St + 50hufa-dietthat produced 2.8(:1::1.0)xl06 and 4.9(:!::3.0)xI06 eggs,

14:0 204:i:0.2a 2.2:i: 0.2ab 1.9:i:OAb16:0 18.6:i: 1.0 19.0:i:OA 19.1:i:0.918:0 4.2:i:0.5 4.3:i:0.3 4.5:i:0.3

SAFA 28.0:i: 1.5 28.2:i:0.6 28.9:i: 1.216:I(n-7) 13.2:i:0.9a 11.2:i:0.5b 8.5:f:0.4c18:I(n-9) 7.6:i:0.5a 9.6:i:0.3b 9.8:f:0.3b18:I(n-7) 3.6:i:0.3a 3.2:i:0.2b 2.9:i: 0.1b

MUFA 27.8:i: 1.5a 26.6:i:0.8a 23.3:i:0.3b18:2(n-6) 2.3:i:0.la 2.6:i:O.lb 2.9:f:0.lc20:4(n-6) 3.9:i:0.5 3.6:i:0.2 3A:i:0.222:5(n-6) 2.2:i:0.3 2.2 :i:0.1 2.3:i:0.2

(n-6)PUFA 1O.3:i:1.0 9.5:i:0.3 9.HOA18:3(n-3) 2.0:i:0.2a 1.5:i:0.1b 1.4:f:0.1b18:4(n-3) 3.6:i:0.5a 2.9:i:0.2b 2.5:f:0.2b20:5(n-3) 8.7:i:0.6 8.HO.2 9.0:i:0.622:5(n-3) 0.3:i:0.la 0.5:i: O.lab 0.6:f:0.1b22:6(n-3) 12.2:i: 1.1a 16.2:i:0.9b 18.8:i:0.8c

(n-3)PUFA 27.7:i:2.la 30.9:i: 0.9b 32.7:i:0.lb(18:0)DMA 3.2:i:0.1 3A:i: 0.2 3.3:i:0.2

Total DMA 5.3:i:0.2 5.2:i:0.2 5A:f:0.3DHA/EPA 1.4:i:0.la 1.9:i:0.1b 2.1 :f:O.Ocn-3/n-6 2.7:i: 0.5 3.2:i:0.1 3.4:f:0.8

Table 3 Argopecten purpuratus. Experiment I: fatty acid composi- means (:i:SD). Values in one row belonging to each lipid categorytion (% of TFA and DMA) of neutral (NL) and polar (PL) lipids followed by a different letter are significantly different (Newman-of eggs produced under three different nutritional conditions Keuls' test, P < 0.05) (other abbreviations, see Table I)(abbreviations for diets, see "Materials and methods"). Values are

Fatty acid NL PL

St (n=9) St+50dha (n=4) 1/2St+ 50dha (n = 3) St(n=9) 8t + 50dha (n = 4) I/2St + 50dha (n = 3)

14:0 3.2:i: 0.2a 2.6 :i:0.2b 2.2:i:0.7c 1.1:i:0.7 0.7:i:0.3 00416:0 21.0 :i:0.6 20.S:!:0.2 20A:i:0.3 12.1 :i: 1.9a 12.7:i: 1.4ab 8Ab18:0 3.7:i:0.5 4.0:i:0.3 4.6:i: 0.1 7.5:i:0.7 7.1 :i:0.9 7.3

SAFA 30.7:i: 0.5 30.5 :i:0.2 30.9:i: 0.9 25.6:i: 204 25.0:i:1.7 21.616:I(n-7) 16.9:i: 1.4a 13A:i:0.8b 1O.2:i: O.Oc 2.S:!: 1.4 1.9:i:0.3 0.918:1(n-9) 9.5:i: OAa 11.5:i:0.5b 12A:i:0.lc 2.5:i: 1.2 3.0:i:0.7 1.818:I(n-7) 4A:i:OAa 3.7 :i:0.3b 3.3:i:0.1b 1.2:i:OAa 1.0:i:0.1a 0.5b

MUFA 34.0:i: 2.0a 31.7:i: 1.2a 28.S:!: 0.1 b 1O.2:i:204 9.0:i:0.6 5.818:2(n-6) 2A:i:0.la 2.9 :i:0.4b 3A:i:0.Oc 0.8:i:0.2 0.9:i:0.1 0.720:4(n6) 3.1 :i:0.3 3.1 :i:0.2 3.1 :i:0.3 7.2:i:0.8 6.1 :i:0.9 7.022:5(n-6) 1.5:i:0.1a 1.6:i:O.lb 1.8:i:O.Oc 4.3:i:0.5 4.2:i:0.3 3.8

(n-6)PUFA 8.5:i: O.5a 9.1 :i:OAab 9.6:f:0.3b 12.S:!:0.9 12.0:i: 1.1 11.518:3(n-3) 2.1:i:0.la 1.7:i:0.2b 1.6:i:0.lb 0.6:i:0.2a OA:i:0.2ab 0.2b18:4(n-3) 4.3:i: 0.5a 3A:i:0.5b 2.5 :i:O.Oc 1.S:!:0.5 1.6:i:0.1 1.520:5(n-3) 8.7:i:0.7 8A:i:OA 9.1 :i:0.1 7.S:!:0.7 7.3:i:0.6 8.022:5(n-3) 0.2:i:0.1a 0.3:i:0.lb 0.5 :i:O.Oc 0.3:i: O.la 0.6:i:0.1b 0.3a22:6(n-3) 8.7:i: 1.0a 11.l:!: 2.3b 14.3:i:0.8c 21.3:i: 1.4a 26.3:i: l.5b 27.7b

(n-3)PUFA 24A:i: 2.2 25.3:i:2.1 28.5:i: 0.7 32.0:i: 1.8a 36A:i: 1.9b 37.7b(18:0)DMA 1.6:i:0.2 1.8:i:0.2 1.4:f:0.1 1l.5 :i:2.5 11.1:i: 1.0 15.7

Total DMA 2.2:i:0.3 2.3:i:0.2 2.0:i:0.7 19.0:i:304 17.5:i: 1.3 23.6DHA/EPA 1.0:i:0.1a 1.3 :i:0.2b l.5 :i:O.lc 2.7:i:0.3a 3.6:i: 0.3b 3.5bn-3/n-6 2.9 :i:OA 2.7:i:0.2 3.0:i:0.3 2.5:i: 0.2a 3.0:i:OAb 3.3b

respectively. No significant difference was noticedbetween the latter two (P=0.08).

The addition of EmCOCO supplement led to eggswith the highest SAFA content in TL, i.e. 12:0, 14:0and18:0(Table 4). Eggs of these parent animals also showedthe highest levels of 18:2(n-6),reflecting the high contentof this fatty acid in EmCOCO. They had the lowestlevels in EPA and DHA, and consequently in(n-3)PUFA. Broodstock animals fed on St + 50epa andSt + 50hufa released eggs with a clear increase in 20:5(n-3) and 22:5(n-3) content. EPA content increased from10.1% (St) to 15.4% and 13.2%, respectively, and22:5(n-3) content increased from 0.2% to 1.4% and1.0%, respectively. The DHA content on the other handwas not affected.

The distribution of the fatty acids over the differentlipid groups was the same as found in expt 1 (Table 5).In the NL fraction, SAFA, MUF A and (n-3)PUFArepresented each 30%, while (n-6)PUFA representedonly 10%. The importance of MUFA in PL on the otherhand was much less (10%), while DMA represented21%.

The same differences in the fatty acid (FA) compo-sition of the TL were found again in the FA compositionof the NL fraction. Eggs of the St + 50coco-diet havesignificantly more SAFA and (n-6)PUFA and less(n-3)PUFA than the eggs of the other diets. The dietsSt + 50epa and St + 50hufa led to a higher content inEPA and in EPA and DHA, respectively, than the St-diet. The eggs resulting from the St-diet had the highestMUF A content. With regard to the PL fraction, therewas no significant difference in SAFA and MUF Aamong the diets. The (n-6)PUFA fraction was alsosimilar among the diets, except for the St + 50epa-eggswith a significant lower content. The diets St + 50epaand St + 50hufa resulted in eggs with a higher

--

(n-3)PUFA content (more specifically EPA) in PL thanthe diets St and St + 50coco. The PL fraction had at leasttwice as much 22:6n-3and at least seven times as muchDMA as the NL fraction.

Lipid class analysis

Despite the addition of lipid emulsions during the con-ditioning of the adults, no significant increase in totallipid content was observed in the eggs compared to theeggs released by the animals fed with the St-diet (Ta-ble 6). The NL fraction represented roughly 85% of thetotal egg lipids and was dominated by triglycerides(TAG, roughly 90%). Free sterols and sterol esters madeup the remaining 10%. The most important PL class wasphosphatidylcholine (PC), representing about 71%(range 66-79%) of the PLs. The other 29% corre-sponded to phosphatidylethanolamine (PE, roughly21%) and phosphatidylserine + phosphatidylinositol(PS+ PI, roughly 6%).

Reducing the algal ratio by half Cl/2St + 50dha, expt1)led to a significantlylower PS + PI content in the eggs.The lower TAG content in eggs from animals fed withthe St-diet in expt 2 led to a significantly lower contentin total NL and a relatively higher content in PL; thiswas not the case for expt 1.

Discussion

The fatty acid profiles of the microalgae (Isochrysisgalbana (T-iso), Chaetoceros neogracile) used in thiswork were similar to those previously reported (Napo-litano et al. 1990;Delaunay et al. 1993;Thompson et al.1993;Tzovenis et al. 1997).C. neogracilehad a relatively

332

Table 4 Argopectenpurpuratus.Fatty acid St (n=6) St + 50epa (n = 2) St + 50hufa (n = 9)Experiment 2: fatty acid St+ 50coco (n = 12)

composition (% ofTFA and12:0DMA) of totallipids of eggs O.O::l:O.Oa O.O::l:O.Oa 0.1 ::I:O.Ob 1.7::1:0.2c

produced under four different 14:0 3.3::1:0.3a 3.3::1: O.Oa 3.0::1:0.2b 4.2::1:0.2cnutritional conditions 16:0 17.8::1:0.2ab 18.3 ::I:O.3a 17.6::1:0.4b 18.4::1: O.4a

(abbreviations for diets, see 18:0 3.7::1:0.3a 4.1::I:0.3b 4.1::I:0.2b 5.0::l:0.2c"Materials and methods"). SAFA 27.6::1: 0.3a 27.6::1:0.6a 27.3 ::I:O.5a 31.4::1:0.7bValues are means (::I:SD). 16: I(n-7) 11.7::1:O.4a 8.2::1:O.4b 8.7:!:0.6b 8.2::1:0.3bValues in one row followed by a 18: I(n-9) 6.6::1:0.3a 9.4::1:0.3b 9.0::l:0.4b \0.1 ::I:O.5c

different letter are significantly 18:I(n-7) 4.1 ::1:0.la 3.3::1: O.Ob 3.5 ::I:O.2b 2.6::1:0.lcdifferent (Newman-Keuls' test, MUFA 25.3::1: 0.5a 24.0::l:0.8b 24.0::1:0.9b 23.7::1: 0.9bP<0.05) (other abbreviations, 18:2(n-6) 2.3::1:0.la 2.5::1:0.2b 2.8 ::I:O.2c 5.6::1:0.2dsee Table I) 20:4(n6) 3.6::1:O.4a 2.3::1: 0.1 b 3.0::l:0.lc 2.3 ::I:O.2b

22:5(n-6) 2.6::1:0.la 1.6::1: O.Ob 2.1 ::I:O.1c 1.9::1:O.1d(n-6)PUF A 10.0 ::I:O.5a 7.5::I:0.Ob 9.3::1:0.3a 12.5::1: L8c

18:3(n-3) 2.1 ::1:0.la 1.3::1:0.lb 1.5::1:0.1c 2.5 ::I:0.9d18:4(n-3) 5.0::1:0.3a 2.7::1:0.3b 3.1 ::I:O.3c 5.8 ::I:O.3d20:5(n-3) 10.1 ::I:O.2a 15.4::1: O.Ob 13.2::1:0.7c 7.8::1:0.5d22:5(n-3) 0.2 ::I:O.1a 1.4::1:0.0b 1.0 ::I:O.1c 0.2::1: O.Od

22:6(n-3) 12.8::1:0.4a 14.7::1:0.5a 12.1 ::I:O.5a 8.8::1:2.5b(n-3)PUFA 31.3 ::I:O.6a 36.4::1: 1.0b 34.5::1: 1.3c 26.6::1: 1.0d

(18:0)DMA 3.3::1:0.5 3.0::1:0.7 3.2::1:0.2 3.5::1:0.3Total DMA 5.4::1:O.4a 4.4::1:0.3b 4.8::1:0.7ab 5.3::1:O.4aDHA/EPA 1.3 ::I:O.Oa 0.9::1:0.3b 1.1 ::I:O.Oc 1.2::1:O.lan-3/n-6 3.1 ::I:O.2a 4.8::1:0.lb 3.7::1:0.2c 2.2::1:0.3d

Table 6 Argopecten purpuratus. Lipid classes of eggs spawned inexperiments I and 2 (abbreviations for diets, see "Materials andmethods"). Values are means (:J:SD). Values in one row belongingto the column experiment I or experiment 2 followed by a differentletter are significantly different (Newman-Keuls' test, P<0.05).Significant differences for total NL and total PL were determined

Experiment I

by Mann-Whitney U-test because data were not homogeneous(Chol.ester cholesterol esters; TAG triglycerides; FFA free fattyacids; Chol cholesterol; DAG diglycerides; NL neutral lipids; PEphosphatidylethanolamine; PA phosphatic acid; PS phosphatidyl-serine; PI phosphatidylinositol; PC phosphatidylcholine; PL polarlipids; TL totallipids)

Experiment 2

St (n=6) St+50dha (n=4) 1/2St+dha (n= 1,*n=2) St (n=6) St+epa (n=2) St+hufa (n=9) St+coco (n= 12)

high amount of arachidonic acid, which also corre-sponded with the findings of Napolitano et al. (1990).

Egg size

Both experiments demonstrated that the broodstock dietdid not affect the size of the eggs or the DW of Argo-pecten purpuratus. Even the diet with only half theamount of algae (1/2St + 50dha) resulted in equally sized

eggs. This is in agreement with Caers et al. (2002), whofound no difference in egg size of Crassostrea gigasamong the mixed algal control diet, a single diet ofDunalie/la tertiolecta and a single diet of D. tertiolectasupplemented with an emulsion rich in EPA and DHA.Bayne and Newell (1983)and Oevauchelle and Mingant(1991) concluded that egg size is a species-specificcharacteristic. Farias and Uriarte (2001) found that theegg size of A. purpuratus fed high and normal proteindiets was similar and concluded that the additional

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Table 5 Argopecten purpuratus. Experiment 2: fatty acid composi- means (:J:SD). Values in one row belonging to each lipid categorytion (% ofTFA and DMA) of neutral (NL) and polar (PL) lipids followed by a different leller are significantly different (Newman-of eggs produced under four different nutritional conditions Keuls' test, P<0.05) (other abbreviations, see Table I)(abbreviations for diets, see "Materials and methods"). Values are

Fatty acid NL PL

St (n=6) St + 50epa St + 50hufa St + 50coco St (n=6) St + 50epa St + 50hufa St + 50coco(n=2) (n=9) (n= 12) (n = 2) (n = 9) (n = 12)

12:0 0.1 :J:O.la 0.1 :J:O.Oa 0.1 :J:0.2a 1.9:J:0.3b 0.2:J:0.I 0.2:J:0.I 0.1 :J:0.2 0.1 :J:O.I14:0 3.9:J:O.4a 3.7:J:0.la 3.5:J:0.3a 5.H 0.2b 1.0:J:0.4 I.O:J:O.I 0.9:J:0.3 \.I :J:0.416:0 18.7 :J:0.5a 20.0 :J:O.Ob 18.3:J:1.0a 20.HO.5b II.O:J:2.8 12.4:J:1.3 12.U 1.6 12.H 1.818:0 3.6:J:0.3a 4.2:J:0.lb 3.9:J:0.2a 4.8:J:0.2c 4.9:J: 1.3a 4.8 :J:0.8ab 6.0 :J:O.4b 7.2:J:0.9c

SAFA 29.1 :J:0.6a 30.3:J:0.la 28.6:J: 1.6a 34.2:J:0.6b 21.0 B.O 21.5:J:2.7 22.0:J: 2.2 23.6:J: 1.916:I(n-7) 13.7:J:0.7a 9.5:J:0.4b 9.9 :i:O.5b 9.H 0.6b 2.6:J: 1.8 2.0:J:0.3 2.0:J:0.7 1.9:J:0.518:I(n-9) 8.0:J:0.3a 10.9 :J:0.3cd 1O.5:J:0.6c II.HO.6d 2.5:J: 1.6a 3.0:J:0.3ab 3.6:J: \.I ab 4.4:J:0.8b18:I(n-7) 4.7:J:0.la 3.8:J:0.lb 4.IHO.2b 3.2:J:0.2c 1.7:J:0.7 I.HO.l 1.6:J:0.5 1.2:J:0.2

MUFA 29.6:J:0.9a 27.2 :i:O.8b 28.4 :J:1.2b 28.1 :J:0.9b 10.7:J:4.0 9.HO.7 10.6:J:2.1 10.7:J: 1.518:2(n-6) 2.7 :J:O.la 2.7 :J:O.Oa 3.35:J:0.3b 6.3:J:0.3c 0.8:J:O.4a 0.9:J:0.la 1.2:J:0.6a 2.7:J: 1.0b20:4(n6) 3.1 :J:O.la 2.1 :J:O.lb 2.54:J:0.lc I.8:i:O.ld 5.1:J: \.la 3.1:J:0.lb 4.3 :J:0.5a 4.4:J:0.8a22:5(n-6) 2.0:J:0.2a 1.2:J:0.Ob 1.6:J:0.2c I.HO.lb 5.0:J:0.5a 3.0:J:O.lb 3.7:J:0.3c 4.4:J:0.3d

(n-6)PUFA 9.6:J:0.5a 7.2:J:0.lb 8.9:J:0.4c 11.5:J:0.3d 12.0:J:0.6a 8.6:J: 1.63b 10.8 :J:2.3a 12.7 :J:0.8a18:3(n-3) 2.5:J:0.la 1.4:J:0.lb 1.6:J:0.lc 2.HO.ld O.H 0.6 0.4:J:0.1 0.6:J:0.2 1.7:J:1.318:4(n-3) 5.4:J:0.4a 2.7 :J:0.3b 3.3 :J:O.4c 6.1 :J:0.2d 2.3 :J:0.5a 1.8:J:0.3a 1.7:J:0.3a 3.1 :J:0.5b20:5(n-3) 10.1 :J:0.3a 15.HO.lb 13.1:J:I.Oc 7.4:J:0.4d 8.6:J:0.8a 12.0:J:0.3b 10.6 :J:0.9c 8.0:J:0.8a22:5(n-3) 0.2:J:0.la \.l:J:O.lb 0.8:J:0.lc 0.1 :J:O.Od 0.3 :J:O.la 1.8:i:0.3b 1.3:J:0.4c 0.4:J:0.la22:6(n-3) 9.9:J:0.6a 11.8:J:0.3b 12.0:J:1.0b 6.3 :J:0.6c 22.2:J: 1.6a 23.2:J:0.5a 23.5:J: 1.6a 19.8:J: 1.9b

(n-3)PUFA 28.8 :J:0.5a 33.2 :J:0.8b 31.2:J: 1.8c 23.5:J: 1.0d 34.8 :J:2.la 4O.0:J:I.5b 38.6 :J:2.3b 33.6:J:2.la(l8:0)DMA 1.8:J:0.5 \.l5:J:0.1 1.6:J:0.2 1.6:J:0.4 12.0:J:4.5 13.7:J:1.2 11.8:J:2.0 13.3:J: 1.3

Total DMA 2.4:J:0.7 1.85:J:0.1 2.5:J:0.3 2.5:J:0.6 2 \.I H.I 20.H 2.1 18.8 :J:3.3 19.0:J: 1.7DHA/EPA 1.0 :J:0.1a 0.8:J:0.3b 0.9 :J:O.Oac 0.8 :J:O.lbc 2.6:J:O.4a I.H O.Ob 2.2 :J:0.3ab 2.5:J:0.2an-3/n-6 3.0:J:0.2a 5.5:J:O.lb 3.5:J:0.3c 2.0:J:O.ld 2.9:J:0.2a 4.8:i: \.I b 3.7:J:0.6c 2.7 :J:0.2a

Cho1.ester 3.3:J:0.6 3.6:J:0.6 2.9 4.3:J: 1.0 2.9:J:0.7 3.4:J: 1.0 2.9:J:0.8TAG 76.8 :J:2.9 78.4:J:0.6 76.9 70.:J:5.9a 78.0:J:2.6b 77.8:J:4.3b 78.6:J: 3.0bFFA 0.1 :J:0.2 0.0:J:0.6 0.0 1.2:J:1.6a 3.2:J: I.5b 0.5 :J:0.8a 0.7 :J:0.8aChol + DAG 3.4:J:0.7 3.9:J:0.6 4.2 5.8:J:2.6 5.0:J:0.5 3.2:J:0.6 3.6:J: 1.2Total NL 83.7:J: 1.8 86.0:J: 1.2 84.1 81.4:J:2.4a 89.1 :J:0.2b 85.0:J:4.4bc 85.9:J: 1.6cPE+ PA 3.7:J:0.7 2.7:J:0.6 2.8 4.5 :J:0.6a 2.5:J:0.9b 3.H2.4b 3.2:J:0.6abPS + PI 1.3:J:0.3a 0.7:J:0.6ab 0.5b \.I :J:0.4 0.8 :J:0.4 0.9:J:0.4 0.7:J:0.3PC I 1.3:J:\.I 10.6:J:0.6 12.6 12.7:J:1.6a 7.2:J: I.Ib 10.4:J:2.0c 10.1 :J: 1.2acTotal PL 16.3:J: 1.8 14.0:J: 1.2 15.9 18.5:J:2.4a 10.9:J:0.2b 15.0:J:4.4bc 14.1:J:1.6cTL 24.6:J:1.7 24.6 :J:0.6 24.0:J: 1.1* 21.1:J:2.6 24.4:J:0.I 23.4:J:2.4 22.4:J: 3.4

334

protein in the high protein diet is mainly used for theproduction of a larger number of eggs and not for largereggs. The same observation was made for Placopectenmagellanicus when grown in poor environmental con-ditions (Napolitano et al. 1992). Nevertheless, otherauthors have observed that environmental conditionscan affect the size and subsequently the viability of eggs(Bayne 1985; Gallager and Mann 1986).

Although the broodstock animals of expt I wereconsiderably larger in size than those of expt 2 (78.3and66.6 mm, respectively), the egg sizewas very similar andvaried between 52 and 56 ILm.This would indicate thatthe egg size of A. purpuratus was independent of the sizeof fully mature parent animals. The average egg sizeobserved in this study was rather low in comparison toother studies with the same species: 66.8 (Farias et al.1998), 61.3 and 62.4 ILm(Uriarte et al. 1996).

Fecundity

Millican and Helm (1994) showed that broodstock diethad an impact on the fecundity of Ostrea edu/is. Laingand Lopez-Alvarado (1994) found that the higher lipidreserves in clams fed live diets released more eggs perfemale. Higher numbers of eggs per female were col-lected from oysters fed on algal and lipid supplementscompared to those from non-fed C. gigas kumamoto(Robinson 1992). Our study demonstrated that supple-mentation of emulsions rich in HUF A to the St-dietimproved fecundity of A. purpuratus, though not sig-nificantly (161% for St + 50dha and 173% forSt + 50hufa when St = 100%). This is in agreement withthe findings of Caers et al. (1999b). Supplementationwith 50%EmCOCO in expt 2, however, led to signifi-cantly higher egg production compared to the St-diet(278%) and St + 50hufa-diet (161%). Saturated lipidsseem more effective in increasing fecundity than poly-unsaturated fatty acids. If energy supply is the key factorduring gametogenesis, then bioodstock animals were inneed of an accessible energy source. Since energy of fatsis more efficiently released via p-oxidation of saturatedthan unsaturated fats (Langdon and Waldock 1981;Lehninger 1982, cited in Thompson et al. 1993),adultsmay have benefited more from the extra supply in short-chained fatty acids and may have built up the greaterenergy reserves necessary for gamete production. Theemulsion EmCOCO was also rich in 18:2(n-6),the levelsof which increased significantly in the egg lipids of theNL and PL fractions when broodstock was fedSt + 50coco. This may be important since Santiago andReyes (1993)demonstrated the impact of linoleicacid onfecundity for the Nile tilapia (Oreochromisnilotica).

When the algal diet was reduced to half(1/2St+ 50dha), fecundity dropped sharply. TheEmDHA could not replace the efficiencyof live algae atsuch a high replacement percentage. Low fecundity waspossibly caused by resorption of oocytes, since theanimals were put under nutritional stress. Such phe-

nomena have been observed in A. purpuratus by Marti-nez et al. (1992) and in O. edu/is by Lane (1989).Another possibility, however, is that the scallops re-duced the clearance rate because chlorophyll concen-trations were depleted below a certain threshold(threshold feeding response), as observed for Argopectenirradiansirradians(Rheault and Rice 1996)and Mytilusedu/is (Thompson and Bayne 1972). The findings ofMartinez et al. (2000)support this idea, since they foundmore spawning animals when the scallops were fed thelipid-supplemented algal diet (70% microal-gae + 30%EmDHA) compared to the pure algal diet,only when the feeding ratio was set at 6% of dry bio-mass per day and not at 3%. The latter was consideredto be an insufficient food supply.

A significant difference in fecundity was recordedbetween the St-diet groups of the two experiments, i.e.broodstock of expt 1 spawned an average of5.36x106eggs individually, while the animals of expt 2spawned only 2.81x106eggs. The size of the parentanimals is very likely to have played an important role.Avendano (1993, cited in Le Pennec et al. 1998)showeda positive correlation between the shell height ofA. purpuratus and the number of eggs spawned. A sub-stantial difference in condition and energy reservesbetween the two groups is also a possible reason, sincethe animals of expt 2 had just gone through a severewinter period under "natural" conditions (lantern net inTongoy Bay), while the animals of expt I had receivedalgae at libitum in the laboratory for 2 months previousto the experiment. The impact of the season on fertilityin our study was negligible in comparison to the impactof the nutritional condition, since lower fecundity wasexpected for the "winter experiment (expt 1)", while theopposite occurred.

Egg totallipids and fatty acids

Lipids play an important role during embryogenesisprior to the development of a functional mechanism forparticulate feeding. A study of the rock scallop Cras-sadoma gigantea demonstrated that 56.9% of the totalenergy of the egg was expended during embryogenesisand that lipid, protein and carbohydrate were catabo-lised linearly to contribute 46.7%, 43.5% and 9.8% ofthe total energy used, respectively (Whyte et al. 1990).Several authors believe that lipids are also an essentialfactor in the exotrophic phase and determine larvalgrowth. This hypothesis seems to be validated inM. edu/is (Bayne et al. 1975),O. edu/is (Helm et al. 1973)and Ostrea chilensis (Wilson et al. 1996). However, itdoes not hold true for Crassostreagigas (Gallager et al.1986), Crassostreavirginica(Gallager and Mann 1986),Mercenaria mercenaria (Gallager and Mann 1986) andPecten maximus (Delaunay et al. 1992).

The total lipid content of A. purpuratus eggs wasapproximately 23.5% on a DW basis or 5.5 ng egg-I.This value is comparable to the 24.8% found by Caers

et al. (1999b) for eggs from A. purpuratus broodstockfed pure algae and in agreement with the 5.1 ng egg-1found by Farias et al. (1998). The different maturationdiets in our study did not change the total lipid contentof the eggs, which is in disagreement with Caers et al.(1999b). They found that lipid supplementation resultedin a significant increase of the TL content in A. purpu-ratus eggs. Similarly, Gallager and Mann (1986) foundthat variations in the broodstock-conditioning protocolof M. mercenaria and C. virginica induced large fluctu-ations in egg lipid levels. Caers et al. (2002), however,demonstrated that the lipid content of C. gigas is dietindependent, and Utting and Millican (1997) suggestedthat bivalves adjust their fecundity in order to maintaina consistent lipid level in the eggs. Millican and Helm(1994)and Laing and Lopez-Alvarado (1994)found thatO. edu/is and Tapes philippinarum maintain their eggproduction of a particular quality rather than produce alarge number of eggs.

The essential fatty acid composition of the gonad andegg lipids in bivalves is related to the fatty acids in themicroalgae fed to broodstock during hatchery condi-tioning (Whyte et al. 1990, 1991; Marty et al. 1992;Napolitano et al. 1992;Utting and Millican 1997;Caerset al. 1999b, 2000, 2002). Soudant et al. (1996a) dem-onstrated that the lipid composition of the eggs ofP. maximus reflects the fatty acid profile of the gonad,and no change in fatty acid pattern occurs betweengametogenesis and spawning. In the present work, theabundance of 16:0, 16:1(n-7) and 18:I(n-9) in the TL ofthe eggs reflected the high percentage of these fatty acidsin the algae, but substantial amounts of algal 14:0werenot fully expressed in the fatty acid profile of the eggs.The significant increase of20:5(n-3), 22:6(n-3)and 12:0inthe eggs of brood stock fed the lipid emulsions EmEPA,EmDHA and EmCOCO, respectively, illustrated thatrecently ingested fatty acids did significantly contributeto the lipid accumulation process in the maturing femalegonad. This confirms the findings ofCaers et al. (1999b).Although the 'lipid emulsions altered the fatty acidcomposition of the eggs, the relative abundance of thedifferent lipid groups, i.e, SAFA, MUFA, (n-3)PUFAand (n-6)PUFA, was maintained in NL and PL fractions.

The high percentage in the PL fraction of 20:4(n-6)and especially 22:6(n-3) (twice as much as in the NLfraction) supports the idea of an important functionalrole for these fatty acids. It also indicates that the scal-lops could at least partially control which fatty acidswere incorporated in the PL fraction. DHA would beimportant during egg differentiation, while ARA isimportant for the process of oogenesis (Soudant et al.1996b). ARA is also the precursor to prostaglandins,which play an active role in water transport and osmo-regulation in marine invertebrates (Freas and Grollman1980, cited in Whyte et al. 1991).

The addition of 50%EmDHA (expt 1) led to a sig-nificant increase in DHA content in the TL (33%), NL(27%) and PL (23%) fractions of the released eggs, ascompared to the St-diet eggs. The EPA content was not

335

affected, in contrast to the findings of Caers et al.(1999b), who found a significantly lower EPA contentwhen 50%EmDHA was supplemented to the algal dietof A. purpuratus broodstock. The 1/2St+ 50dha-dietresulted in eggs with significantly less 16:1(n-7), due tothe fact that less C. neogracilewas offered to the parentanimals. The loss in 16:I(n-7) content was compensatedfor by an increase in relative DHA content, originatingfrom the emulsion.

Incorporation of additional EPA in eggs (expt 2)increased steadily in line with higher amounts offered tothe parent animals. EPA levels increased by 30% and23% in the NL and PL fraction, respectively, when25%EmEP A (St + 50hufa) was supplemented and by51% and 39%, respectively, when 50%EmEPA(St + 50epa) was added. This is in disagreement with thefindings of Caers et al. (1999b), who found that therelative content of 20:5(n-3) in the PL fraction ofA. purpuratus eggs was not changed when 50%EmEPAwas supplemented to the algal diet.

When the conditioning diet contained high EPAlevels, DHA was also being selectivelyincorporated intothe NL fraction. This was illustrated by an increase of19% and 21% in DHA content when the broodstockwas offered the St + 50epa- and St + 50hufa-diet,respectively. It would seemthat the DHA levelin the NLfraction varied in order to maintain an appropriateDHA/EPA ratio of approximately 1.2 for TL, while thePL fraction was only affected when very high levels ofadditional DRA were offered (St + 50dha). In general,the EPA concentration in eggs was more readily affectedby dietary (n-3)HUFA than DHA. This could be relatedto the fact that EPA is used as an energy source duringembryogenesis (Whyte et al. 1990, 1991).

Compared to the St-diet, the supplementation ofEmCOCO (expt 2), rich in 12:0 and 18:2(n-6),led to asignificant increase of these fatty acids in the eggs andhence to higher SAFA (117%) and (n-6)PUFA (120%)contents in the NL fraction. The increase in these lipidfractions was compensated for by a decrease in EPA(73% of St-diet eggs) and DHA (63% of St-diet eggs).Such a trade-off was not seen in the PL fraction of theeggs, which maintained basically the same relativecomposition as the PL of the St-diet eggs. The DHA/EPA ratio in the TL fraction was maintained at 1.2. Itwould seem that the DHA/EPA ratio was more impor-tant than the absolute levelsofDHA and EPA as long asa minimum threshold level was maintained.

The high percentage of 20:5(n-3)and 22:6(n-3) in theeggs of scallops fed solely algae (St-diet) cannot fully beexplained by the algal content. It has been well describedthat fatty acids of the NL fraction of the digestive glandare transferred to the female gonad during gametogen-esis (Vassallo 1973;Taylor and Venn 1979;Barber andBlake 1981, 1985b; Napolitano and Ackman 1993).Caers et al. (2000) suggested that because of the simi-larities between the fatty acid composition of the femalegonad and the digestive gland of A. purpuratus, lipids

336

were transferred from the lipid-rich digestive gland tothe female gonad.

Molluscs in general are rich in plasmalogens, whichare primarily found in the PL fraction (PE and PC) andto a lesser extent in the NL fraction (Sargent 1989).Ourdata confinn that plasmalogens represented roughly20% of the FAMEs in the polar fraction of A. purpu-ratus eggs. Comparable high levelsof DMA in the polarlipids have been found in eggs of C. gigas (Caers et al.2002). The fatty acid 18:0 is the most important alkenylether chain, regardless of the broodstock diet. Caerset al. (1998, I999b) and Sargent (1989) also found that18:0 was dominant in A. purpuratus, C. gigas andT. philippinarum, independent from organ (gonads,digestive gland, adductor muscle, gills, or mantle) ordevelopment stage (egg, spat, adult).

The exact function of plasmalogen and the impor-tance of the fatty acid chain composition are not veryclear yet, but they probably have a critical role inmembrane integrity (Chapelle 1987; Caers et al. 1999a)and/or are involved in some type of transmembranetransport process (Rapport 1961).A role as endogenousantioxidant to protect polyunsaturated fatty acidsagainst peroxyl-radical-promoted oxidation has beenproposed by Hahnel et al. (1999) in mammalian cells.

Egg lipid classes

In general, the diets did not change the relative contentof the lipid classes, except for expt 2, where the St-dietresulted in eggs with less TAG compared to the lipid-supplemented diets. This was not true for expt I. Fur-ther confinnation is needed to prove that this differencewas related to the broodstock diet.

Pecten maximus has a NL/PL ratio of 2-2.5 (Soudantet al. I996a), while eggs of Crassadoma gigantea andPatinopectenyessoensis have a value of 3-4 (Whyteet al.1990, 1991).Napolitano et al. 1992found a value of 1.7-1.9 in Placopectenmage/lanicus.The eggsof A. purpuratusin our study had a ratio of 5-6, which is quite high incomparison to the above-mentioned values for otherscallops. The high levelof the NL fraction (about 85% ofTL) in the A. purpuratus eggs was dominated by TAG(86-92%) and may imply a very high energy demandduring embryogenesis. Several researchers have con-finned the findings of Gallager and Mann (1986) that,although phospholipids are partially catabolised, TAG isthe predominant fuel for the process of embryogenesis(Bayne et al. 1975;Whyte et al. 1990).PC (71%) and to aminor degree PE (22%) were the quantitatively mostimportant polar lipid classes. Caers et al. (2002) foundthat PC and PE were the dominant lipid classes in Cras-sostreagigaseggs,each representing about 30% ofthe PL.

Although there was no follow-up of the fertilisationrate and D-larval survival in this study, Martinez et al.(2000)did not find a difference for these two parameterswhen comparing eggs from A. purpuratus broodstockfed the pure microalgal diet with those from broodstock

fed the diet consisting of 70%algae + 30%EmDHA. Thiswould be in line with our findings, since no difference inTL content was observed in the eggs resulting from non-supplemented and lipid-supplemented diets.

Conclusions

Results indicated that the total lipid content of the eggsof A. purpuratus was diet independent. A greater energyreserve was spent in a larger number of oocytes and notin bigger sized oocytes with a higher lipid content. Thelipids supplied through the emulsions were at leastpartially allocated to the eggs, demonstrating that thefatty acid composition of the eggs could be manipulated,especiallythe NL fraction. Levels of EPA changed morerapidly than DHA levels, supporting the observationthat they fulfilled an energetic and structural role,respectively. The St-diet supplemented with 50%EmCOCO resulted in significantly higher fecunditycompared to the algal diet supplemented with 25%Em-EPA + 25%EmDHA and the non-supplemented algaldiet. It would seem that SAFAs were more easily orpreferentially incorporated in the female gonads ofA. purpuratus.The relative content of SAFA and 18:2(n-6) in these eggs rose significantly. The relative content ofthe highly unsaturated fatty acids EPA and DHA, onthe other hand, was substantially lower in the NL frac-tion, but hardly affected in the PL fraction.

Acknowledgements This study was carried out in the framework ofthe R&D project INCO (ICI8-Cf97-0188) and the bilateralFlemish Government project (grant BIL98/03) with Chile. N.Nevejan acknowledges a grant from the Flemish Organization forDevelopment and Technical Assistance (VVOB). The authors wishto express their gratitude to the company Cultivos Marinos Inter-nacionales (CMI) for kindly providing us with adult scallops. Theauthors declare that the experiments comply with the current lawsof Chile.

References

Avendaiio M (1993) Donnees sur la biologie de Argopecten pur-puratus (Lamarck, 1819), Mollusques Bivalves du ChilL Thesed'Universite, Universite de Bretagne Occidentale, Brest

Barber BJ, Blake NJ (1981) Energy storage and utilization inrelation to gametogenesis in Argopecten irradians concentricus.J Exp Mar Bioi Ecol 52:121-134

Barber 81, Blake NJ (1985a) Intra-organ biochemical transfor-mations associated with oogenesis in the bay scallop Argopectenirradians concentricus (Say) as indicated by 14C incorporation.Bioi Bull (Woods Hole) 168:39-49

Barber BJ, Blake NJ (1985b) Substrate catabolism related toreproduction in the bay scallop Argopecten irradians concen-tricus, as determined by O/N and RQ physiological indexes.Mar Bioi 87:13-18

Bayne BL (1985) Ecological consequences of stress. In: Bayne BL,Brown DA, Burns K, Dixon DR, Ivanovici A, Livingstone DR,Lowe DM, Moore MN, Stebbing ARD, Widdows J (eds) Theeffects of stress and pollution on marine animals. Praeger, NewYork, pp 141-157

Bayne BL, Newell RC (1983) Physiological energetics of marinemolluscs. In: Saleuddin ASM, Wilbur KM (eds) The Mollusca4(1). Academic Press, New York, pp 407-515

Bayne BL, Gabbott PA, Widdows J (1975) Some effects of stress inthe adult on the eggs and larvae of Mytilus edu/is L. J Mar BioIAssoc UK 55:675-689

Bayne BL, Bubel A, Gabbott PA, Livingstone DR, Lowe DM,Moore MN (1982) Glycogen utilization and gametogenesis inMytilus eduJis L. Mar BioI Lett 3:89-105

Caers M, Coutteau P, Lombeida P, Sorgeloos P (1998) The effect oflipid supplementation on growth and fatty acid composition ofTapes philippinarum (L.) spat. Aquaculture 162:287-299

Caers M, Coutteau P, Cure C, Morales V, Gajardo G, Sorgeloos P(I999a) The Chilean scallop Argopecten purpuratus (Lamarck,1819). I. Fatty acid composition and lipid content of six organs.Comp Biochem Physiol 123:89-96

Caers M, Coutteau P, Cure C, Morales V, Gajardo G, Sorgeloos P(1999b) The Chilean scallop Argopecten purpuralus (Lamarck,1819).11. Manipulation of the fatty acid composition and lipidcontent of the eggs via lipid supplementation of the broodstockdiet. Comp Biochem Physiol 123:97-103

Caers M, Coutteau P, Sorgeloos P, Gajardo G (2000) The Chileanscallop Argopeclen purpuratus (Lamarck, 1819). IV. Impact ofalgal diets and emulsions on the fatty acid composition andcontent of selected tissues of adult broodstock. In: Caers M:Lipid and fatty acid requirements of bivalves. PhD thesis,Ghent University, Ghent

Caers M, Utting SD, Coutteau P, Millican PF, Sorgeloos P (2002)Impact of the supplementation of a docosahexaenoic acid-richemulsion on the reproductive output of oyster broodstock,Crassoslrea gigas. Mar BioI 140:1157-1166

Chapelle S (1987) Plasmalogens and O-alkylglycerophospholipidsin aquatic animals. Comp Biochem Physiol 88:1-6

Delaunay F, Marty Y, Moal J, Samain J-F (1992) Growth andlipid class composition of Pecten maximlls (L.) larvae grownunder hatchery conditions. J Exp Mar BioI Ecol 163:209-219

Delaunay F, Marty Y, Moal J, Samain J-F (1993) The effect ofmonospecific algal diets on growth and fatty acid composition ofPecten maximus (L.) larvae. J Exp Mar Bioi EcoI173:163-179

Devauchelle N, Mingant C (1991) Review of the reproductivephysiology of the scallop, PeClen maximus, applicable tointensive aquaculture. Aquat Living Res 4:41-51

Farias A, Uriarte I (2001) Effect of microalgae protein on the go-nad development and physiological parameters for the scallopArgopeclen purpuralus (Lamarck, 1819). J Shellfish Res 20:97-105

Farias A, Uriarte I, Varas P (1997) Estudio de los requerimientosnutricionales del osti6n del norte Argopeclen purpuralus (La-marck, 1819) durante el acondicionamiento reproductivo. RevBioI Mar Ocean 32:127-136

Farias A, Uriarte I, Castilla JC (1998) A biochemical study of thelarval and postlarval stages of the Chilean scallop Argapeclenpurpuratus. Aquaculture 166:37-47

Folch JM, Lee M, Sloane-Stanley GH (1957) A simple method forthe isolation and purification of totallipids from animal tissues.J BioI Chem 266:497-509

Freas W, Grollman S (1980) Ionic and osmotic influences onprostaglandin release from the gill tissue of a marine bivalve,Modiolus demissus. J Exp BioI 84:169-185

Gabbott PA (1975) Storage cycles in marine bivalve molluscs. Ahypothesis concerning the relationship between glycogenmetabolism and gametogenesis. In: Proceedings of the 9thEuropean Marine Biology Symposium. Aberdeen UniversityPress, Aberdeen, pp 191-211

Gallager SM, Mann R (1986) Growth and survival of larvae ofMercenaria mercenaria (L.) and Crassoslrea virginica (Gmelin)relative to broodstock conditioning and lipid content of eggs.Aquaculture 56:105-121

Gallager SM, Mann R, Sasaki G (1986) Lipid as an index ofgrowth and viability in three species of bivalve larvae. Aqua-culture 56:81-103

Hahnel D, Beyer K, Engelmann B (1999) Inhibition of peroxylradical-mediated lipid oxidation by plasmalogen phospholipidsand IX-tocopherol-implications for the oxidized low densitylipoprotein hypothesis. Free Radic Bioi Med 27:1094-1098

337

Helm MM, Holland DL, Stephenson RR (1973) The effect ofsupplementary algal feeding of a hatchery breeding stock ofOstrea edulis L. on larval vigour. J Mar BioI Assoc UK 53:673-684

ICES (International Council for the Exploration of the Sea)(1994) Report of ICES working group on mass rearingof juvenile marine fish. Int Coun Explor Sea Comm MeetI994jF:6

Laing I, Lopez-Alvarado J (1994) Effect of dried algae diets onconditioning and fecundity of Manila clam, Tapes philippina-rum (Adams and Reeve). Aquacult Fish Manag 25:157-166

Lane A (1989) The effect of a microencapsulated fatty acid diet onlarval production in the European flat oyster Ostrea edulis L.In: De Pauw N, Jaspers E, Ackefors H, Wilkins N (eds)Aquaculture-a biotechnology in progress. European Aqua-culture Society, Bredene, Belgium

Langdon Cl, Waldock MJ (1981) The effect of algal and artificialdiets on the growth and fatty acid composition of Crassostreagigas spat. J Mar BioI Assoc UK 61:431-448

Lehninger AL (1982) Principles of biochemistry. Worth, New YorkLe Pennec M, Robert R, Avendafio M (1998) The importance

of gonadal development on larval production in pectinids.J Shellfish Res 17:97-101

Martinez G (1991) Seasonal variation in biochemical compositionof threesizeclassesof theChileanscallopArgopectenpurpu-ratus Lamarck, 1819. Veliger 34:335-343

Martinez G, Torres M, Uribe E, Diaz MA, Perez H (1992) Bio-chemical composition of broodstock and early juvenile Chileanscallops, Argopecten purpuratus Lamarck, held in two differentenvironments. J Shellfish Res 11:307-313

Martinez G, Aguilera C, Mettifogo L (2000) Interactive effects ofdiet and temperature on reproductive conditioning of Argo-pectenpurpuratus broodstock. Aquaculture 183:149-159

Marty Y, Delaunay F, Moal J, Samain J-F (1992) Changes in thefatty acid composition of Pecten maximus (L.) during larvaldevelopment. J Exp Mar BioI EcoI163:221-234

MilIican PF, Helm MM (1994) Effects of nutrition on larvae pro-duction in the European flat oyster, Ostrea edulis. Aquaculture123:83-94

Napolitano GE, Ackman R (1993) Fatty acid dynamics in seascallops Placopeclen magellanicus (Gmelin, 1791) from GeorgesBank, Nova Scotia. J Shellfish Res 12:267-277

Napolitano GE, Ackman RG, Ratnayake WMN (1990) Fatty acidcomposition of three cultured algal species (Isochrysis galbana,Chaetoceros gracilis and Chaetoceros calcitrans) used as foodfor bivalve larvae. J World Aquacult Soc 21:122-130

Napolitano GE, MacDonald BA, Thompson RI, Ackman RG(1992) Lipid composition of eggs and adductor muscle in giantscallops (Placopecten magellanicus) from different habitats. MarBioI 113:71-76

Navarro JM, Leiva GE, Martinez G, Aguilera C (2000) Interactiveeffects of diet and temperature on the scope for growth of thescallop Argopectenpurpuratus during reproductive condition-ing. J Exp Mar BioI Ecol 247:67-83

Rapport MM (1961) The IX,p-unsaturated ether (plasmalogen)content of the tissues of several mollusks. BioI Bull (WoodsHole) 121:376--377

Rheault RB, Rice MA (1996) Food-limited growth and conditionindex in the eastern oyster, Crassostrea virginica (Gmelin, 1791),and the bay scallop, Argopecten irradians irradians (Lamarck,1819). J Shellfish Res 15:271-283

Robinson A (1992) Dietary supplements for reproductive condi-tioning of Crassostrea gigas kumamoto (Thunberg). I. Effects ongonadal development, quality of ova and larvae throughmetamorphosis. J Shellfish Res 11:437-441

Santiago CB, Reyes OS (1993) Effect of dietary lipid source onreproductive performance and tissue lipid levels of Nile tilapiaOreochromisniloticus (Linnaeus) broodstock. J Appl Ichthyol9:33-40

Sargent JR (1989) Ether linked glycerides in marine animals. In:Ackman RG (ed) Marine biogenic lipids, fats and oils. CRCPress, Boca Raton, Aa., pp 175-197

338

SERNAPESCA (Servicio Nacional de Pesca) (2001) Anuario es-tadistico de pesca 2000. Ministerio de Economia, Fomento yReconstrucci6n, Chile

Sokal RR, Rohlf FJ (1995) Biometry. Principles and practice ofstatistics in biological research. Freeman, San Francisco

Soudant P, Marty Y, Moal J, Robert R, Quere C, Le Coz JR,Samain JF (1996a) Effect of food fatty acid and sterol qualityon Pecten maxim us gonad composition and reproduction pro-cess. Aquaculture 143:361-378

Soudant P, Marty Y, Moal J, Samain JF (1996b) Fatty acids andegg quality in great scallop. Aquacult Int 4:191-200

Taylor AC, Venn TJ (1979) Seasonal variation in weight and bio-chemical composition of the tissues of the queen scallop,Ch/amys opercu/aris, from the Clyde Sea area. J Mar Bioi AssocUK 59:605-621

Thompson PA, Guo M, Harrison P J (1993) The influence ofirradiance on the biochemical composition of three phyto-plankton species and their nutritional value for larvae of thePacific oyster (Crassostrea gigas). Mar Bioi 117:259-268

Thompson RJ, Bayne BL (1972) Active metabolism associated withfeeding in the mussel, Myti/us edu/is L. J Exp Mar Bioi Ecol9:111-124

Tzovenis I, De Pauw N, Sorgeloos P (1997) Effect of different lightregimes on the docosahexaenoic acid (DHA) content oflsochrysis aff. ga/bana (clone T-Iso). Aquacult Int 5:489-507

- --- - - - --

Uriarte I, Farias A, Muiioz C (1996) Cultivo en hatchery y pre-engordadelosti6ndelnorte,Argopectenpurpuratus(Lamarck,1819) en el sur de Chile. Rev BioI Mar 31:81-90

Utting SD, Doyou J (1992) The increased utilization of egg lipidreserves following induction of triploidy in the Manila clam(Tapesphilippinarum). AquacuIture 103:17-28

Utting SD, Millican PF (1997) Techniques for the hatchery con-ditioning of bivalve broodstocks and the subsequent effect onegg quality and larval viability. Aquaculture 155:45-54

Vassallo MT (1973) Lipid storage and transfer in the scallopCh/amys hericia Gould. Comp Biochem Physiol 44: 1169-1175

Whyte JNC, Bourne N, Ginther NG (1990) Biochemical changesduring embryogenesis in the rock scallop Crassadoma gigantea.Mar BioI 106:239-244

Whyte JNC, Bourne N, Ginther NG (1991) Depletion of nutrientreserves during embryogenesis in the scallop Patinopectenyessoensis (Jay). J Exp Mar Bioi Eeol 149:67-79

Wilson JA, Chaparro OR, Thompson RJ (1996) The importance ofbroodstock nutrition on the viability of larvae and spat in theChilean oyster Ostrea chi/ensis. Aquaculture 139:63-75

Wolff M (1988) Spawning and recruitment in the Peruvian scallopArgopecten purpuratus. Mar Ecol Prog Ser 42:213-217

Wolff M, Alarcon E (1993) Structure of a scallop Argopecten pur-puratus (Lamarck, 1819)dominated subtidal macro-invertebrateassemblage in northern Chile. J Shellfish Res 12:295-304