Ž .Aquaculture 182 2000 301–315www.elsevier.nlrlocateraqua-online

The nutritional value of algae grown under differentculture conditions for Mytilus edulis L. larvae

Nikos Leonardos 1, Ian A.N. Lucas )

School of Ocean Sciences, Menai Bridge, North Wales LL59 5EY, UK

Received 4 January 1999; received in revised form 19 July 1999; accepted 19 July 1999

Abstract

Continuous cultures of Skeletonema costatum, Chaetoceros muelleri, Rhinomonas reticulataand PaÕloÕa lutheri were subjected to phosphorus or nitrogen limitation at two light intensities inorder to manipulate their biochemical composition. They were then fed to Mytilus edulis larvaeover a 2-week period, and the larval growth and mortality were assessed. All monospecific dietssupported growth, sometimes equal to or better than a control diet containing a mixture of speciesŽ .R. reticulata and P. lutheri . Survival was not affected by the diets but significantly better larvalgrowth was obtained with low-light, nitrogen-limited or non-nutrient-limited cells of S. costatumand high-light, nitrogen-limited or low-light cells of C. muelleri grown under no limitation ornitrogen limitation. High-light phosphorus-limited or low-light non-nutrient-limited R. reticulatacells and high- light phosphorus-limited cells of P. lutheri were also superior in relation to theircounterparts. A novel computer-aided image analysis technique was used for measuring the lengthof the larvae. A multidimensional model was used in an effort to correlate algal biochemicalcomponents with larval growth. Some fatty acids were found to be significant in determining thealgal nutritional value, with protein and carbohydrate playing a secondary ‘‘modifying’’ role. In

Ž .P. lutheri, the 16:0 and saturated fatty acids SAFA were significantly positively correlated withlarval growth while the contrary was found for dietary ny3 fatty acids, suggesting their strongnegative effect on larval growth. Similar results were found in R. reticulata, although in this case,both protein and carbohydrate content were found to determine the algal nutritional valueconcurrently with some fatty acids. In the other two diatoms, S. costatum and C. muelleri, noconsistent relationship could be established, thus suggesting that either there is a species-specific

) Corresponding author. Telefax: q44-1248-382871; E-mail: oss066@bangor.ac.uk1 E-mail: nikos.leonardos@tuebingen.mpg.de.

0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.Ž .PII: S0044-8486 99 00269-0

( )N. Leonardos, I.A.N. LucasrAquaculture 182 2000 301–315302

relationship or that other components are important in determining the algal nutritional value.q 2000 Elsevier Science B.V. All rights reserved.

Keywords: Chaetoceros muelleri; Fatty acids; Light; Larvae; Mytilus edulis; Nutrients; PaÕloÕa lutheri;Rhinomonas reticulata; Skeletonema costatum

1. Introduction

Many marine animals rely on phytoplankton as a food source especially in their earlystages of development. For a number of bivalve molluscs which are of commercialinterest, live phytoplankton cultures are routinely used as food for the larval and adultstages. From the plethora of phytoplankton species found in nature, only a small number

Ž .have been used as food sources De Pauw et al., 1984 . In early research, the nutritionalvalue of phytoplankton cultures in terms of biochemical composition was not consideredŽ .Walne, 1963 . Instead, palatability and digestibility were suggested as being moresignificant. However, later studies, benefitting from advances in analytical methods,emphasized the indisputable role of the algal biochemical composition in determining its

Ž .nutritional value Wikfors et al., 1984; Enright et al., 1986b . Factors influencing algalŽ .biochemical composition have been described by Leonardos 1998 .

There is only general agreement on which biochemical constituents of phytoplanktoncells are most significant in determining their food value for planktonic herbivores.Numerous reports have examined biochemical classes of components, primarily protein,carbohydrate and lipid and have reached different conclusions about the significance of

Žgross biochemical composition Epifanio, 1979; Webb and Chu, 1983; Enright et al.,.1986a,b; Wikfors et al., 1992; Thompson et al., 1994 . However, since the observation

Ž .that certain long-chain polyunsaturated fatty acids PUFA are essential to many marineorganisms, considerable research has focused on the availability and the role of PUFA in

Ž . Žhatcheries Watanabe et al., 1983 and in the natural environment Sargent and Whittle,.1981 . But there is remarkably little experimental evidence that diets containing more

PUFA are superior for herbivores and there is now growing evidence that the impor-Žtance of dietary PUFA may have been overgeneralized Dickey-Collas and Geffen,

.1992; Thompson et al., 1993, 1994 . This confusion may reflect true differences in thenutritional requirements of various bivalve species. However, even minor changes in theculturing conditions employed in different laboratories may lead to different morpholog-

Žical, physiological andror biochemical properties of the algae in question Gallager and.Mann, 1981; Thompson et al., 1993 . In most hatcheries and laboratory feeding trials

concerning the nutritional value of algae which essentially use the larvae as bioassayorganisms, the algal biochemical composition is not constant over the whole time rangeof the feeding trials. Thus, direct comparisons of results from different laboratories may

Žbe misleading. Although this conflict is commonly recognized by phycologists Baars,.1981 , its potential importance to mariculture, in general, has received comparatively

little attention.The present report tries to overcome some of these problems by employing continu-

ous algal culturing techniques which, in theory, should produce algal cells of constant

( )N. Leonardos, I.A.N. LucasrAquaculture 182 2000 301–315 303

biochemical composition, thus rendering the experiments internally comparable; how-ever, in practice, some variation in the algal biochemical composition is to be expected.Using monospecific diets, the growth performance of the larvae can be correlated withalgal biochemical properties, without introducing differences in digestibility, cell sizeand palatability which may have compromised previous studies using multispecies diets.Special emphasis was placed on algal fatty acids due to ever increasing evidencesuggesting the important role of these compounds as diet components of particular

Ž .nutritional value Thompson et al., 1996 .

2. Materials and methods

2.1. Algal cultures, chemical and biochemical analyses

The algal species diet used as food for the larvae of Mytilus edulis were PaÕloÕaŽlutheri obtained from Culture Centre of Algae and Protozoa, Oban, Scotland, CCAP. Ž . Ž .931r1 , Chaetoceros muelleri CCAP 1010r3 , Rhinomonas reticulata CCAP 995r2

and Skeletonema costatum, the strain of which was isolated by the first author, from aŽ .sample taken from Syros island Greece . The algae were grown using a continuous

culture regime under a light–dark cycle of 14–10 h, at 208"18C. Two light intensitiesŽ y1 y2were employed high-light, HLs890–950 kphotons s m and low-light, LLs

y1 y2 .275–337 kphotons s m , and three nutrient conditions were used in each lightŽ . Ž . Ž .intensity: no nutrient limitation fr2 , phosphorus P and nitrogen N limitation. The

phosphorus-limited medium and nitrogen-limited medium were prepared by adding aquarter of the amount of the respective salt. The density of the cultures was determinedtwice daily and adjustments to the flow rate were made on a daily basis to keep the celldensity constant. Typically, the cultures were maintained in the selected cell density"10% throughout the feeding trials. The collector flasks were emptied regularly andalgae, which were to be used either as food or for samples for cell density determinationand for the subsequent biochemical analyses, were left to stand for no more than 3 h inthat vessel. The algae used in the control diet were cultured in 20 l glass round flasks ata light intensity of 715–827 kphotons sy1 my2 using a semi-continuous batch culturingsystem and Conway medium under constant illumination. All cultures were maintainedin uni-algal condition. Chemical and biochemical methods and techniques are described

Ž .in Leonardos and Lucas 2000a and the biochemical results are expressed in a per cellbasis since this was the unit of standardization for the feeding trials. Three samplesunderwent protein and carbohydrate analyses while the fatty acid profile was determinedin two samples.

2.2. Mussels spawning and feeding trials

Ripe mussels, M. edulis, collected from the Conwy estuary, N. Wales, in the springŽ .of every experimental year 1995–1997 , were used as broodstock for the trials. These

were kept at 68"18C in flowing seawater until required for spawning. Mussels of ca. 60

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mm in length were selected and epifauna and byssals threads were removed. SpawningŽ .was induced as in Loosanoff and Davis 1963 . Fertilized eggs were incubated, unfed at

148C for 4 days and at the D-stage larval pool, the percentage of normality andŽ Ž .abnormality according to the developmental stages described by Bayne 1965 as well

as the total number of larvae were calculated. A small sub-sample of larvae was takenŽfrom this group and photographed or videotaped see later part of the section for image

.analysis under a microscope for later calculation of the initial mean length of the larvae.Ž .All batches of larvae used had a small -10% percentage of abnormal larvae.

An appropriate volume was taken from the larval D-stage group and diluted to aconcentration of 30,000 larvae ly1. These were transferred into sufficient plastic 1-lbeakers to give three replicates for each treatment. To each beaker, the respectivespecies and quantities of algae were added and then filled to 1 l and left at 148"18C.Each diet was tested in triplicates. Feeding densities of 50 cells mly1 were the same forall diets; the control diet was a mixture of P. lutheri and R. reticulata in a 4:1 ratio. Achange of the 0.2 mm filtered water and feeding of the larvae was carried out everysecond day with the respective algal food added to a final concentration of 50 cellsmly1. For the high-light and low-light experiments of the two diatoms, S. costatum andC. muelleri, two different batches of larvae were used for each light intensity; hence,

Ž .results are compared against two sets of controls control diet and unfed larvae . Thehigh- and low-light experiments of R. reticulata and P. lutheri were carried out withthe same batch of larvae; therefore, only one set of controls was needed for each trial.

After 2 weeks, samples were taken from the larval cultures to calculate the totalŽ .number of live larvae as well as percentage of normal individuals Bayne, 1965 ; the

samples were stored in a glass vial and fixed with few drops of Lugol’s iodine solution.Each sample was, at a later stage, decanted into a sedimentation chamber and countedusing an inverted microscope.

Growth was estimated on the basis of length increase of the larvae. To measurelength, two image analysis techniques were used. The larvae were measured at the

Ž .beginning of every experiment while at D-stage and at the end, after a 2-week period.The first technique was based on measuring photographic negatives of the animals andwas used for the S. costatum and C. muelleri feeding trials, while the second was basedon processing videotaped information of the larvae and was employed in the P. lutheriand R. reticulata experiments. The first steps for both techniques were the same: thelarvae from each container were concentrated by pouring its contents into a 45-mmnylon sieve. A small sub-sample was taken with a pipette and placed onto a slide.

In the photographic technique, the mean length of the larvae was estimated fromŽ .negatives taken with a Nikon FM2 camera; 60 normal Bayne, 1965 , clearly focused,

larvae were measured from the negative using the same microscope fitted with aneyepiece scale. The length of the larvae was calculated using a magnification factorcomputed from an image of a standard graticule.

In the videotape technique, the slide with the larvae was placed on a Nikon invertedŽmicroscope with a microscope video camera Hitachi MOS Colour Video Camera,

.VK-C150ED attached. The resulting picture was recorded with a video recorderŽ .Panasonic, NV-J35HQ . The recorded images were later transferred to an IBM-compat-ible computer, equipped with a Miro PCTV video card. Images of the larvae in clear

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Ž .focus were grabbed with the aid of Microsoft’s Media Manager Video capture 32 andthe resulting still images were saved as bitmap images on the computer’s hard disk.

Ž .Then an image analysis software package Jandel Scientific Sigma Scan was used tomeasure the length of 60 larvae. This technique was compared with the photographictechnique by measuring the same slide of larvae. The mean values obtained by the twotechniques did not differ by more than 5%.

2.3. Statistical analysis

Before applying any parametric test each data set was tested for normality andhomogeneity of variances, with a normal probability plot and with Barlett’s test,respectively. Significance was tested at the 95% level.

Logarithmic transformation of data resulted in more data sets having a normaldistribution; however, in no instance were all the distributions of all data sets normal.Even when one of the assumptions necessary for applying parametric tests was violated

Ž .in a small part of the data set typically one or two sets of larval lengths , parametrictests were used. The experimental design was such that nested ANOVA was used in allexperiments to assess whether significant differences existed between diets. This testalso reveals whether there is significant variation between the replicates of the same diet.

ŽIn order to identify the groups that demonstrated significant differences 95% confidence. X Žlimit , the multiple comparisons between means of T -method was used Sokal and

.Rohlf, 1987 . All tests were carried out with the aid of the statistical software package,Minitabw , while preliminary calculations and graphs were made in Quattrow Pro. Toidentify any relationship between algal biochemical component and larval growth,correlation analysis was used. This type of analysis was used instead of ordinaryregression analysis because the assumptions of model I regression analysis do not hold

Ž .true with this type of data Sokal and Rohlf, 1987 .

3. Results

The gross biochemical composition of the algal species grown under various nutrientŽ .light and nutrient conditions is taken from Leonardos and Lucas 2000a and reproduced

here in Table 1 for their protein, carbohydrate and proportions of some of the major fattyacids. The percentage of change in per cell protein content in S. costatum, C. muelleri

Žand P. lutheri as a result of the various culture conditions employed was smaller one-.to twofold change than in R. reticulata where protein content varied from 4.17 pg

y1 y1 Ž .cell to 18.27 pg cell fourfold change . Protein levels of the two diatoms and of R.reticulata were increased under low light. S. costatum exhibited a peak of proteincontent under phosphorus limitation in both light intensities, while R. reticulata for eachcorresponding nutrient condition should protein increase at high-light condition; at thelow-light nutrient conditions, protein content increased under nitrogen limitation. Proteinlevels of C. muelleri were not greatly affected under the culture conditions employed,although they decreased to some extent with nutrient limitation and increased lightintensity. The change in protein content of P. lutheri was not consistent.

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Table 1Responses of S. costatum, C. muelleri, R. reticulata and P. lutheri to various growth conditions

Ž .HL shigh light; LL s low light; fr2s fr2 medium no nutrient limitation ; Psphosphorus-limited medium,Nsnitrogen-limited medium. Fatty acids are expressed as percentages of TIFA. SAFA ssaturated fatty acids;PUFA spolyunsaturated fatty acids. BDsbelow detection levels. Reproduced from Leonardos and LucasŽ .2000a . Three samples were analyzed for their protein and carbohydrate content while two samples underwentfatty acid analysis. Numbers in parentheses indicate standard deviation.

Treatment Protein Carbohydrate 16:0 18:3ny3 20:5ny3 SAFA PUFA ny3 FAsy1 y1Ž . Ž .pg cell pg cell

Ž . Ž .S. costatum HL fr2 3.8 1.03 1.22 0.07 8.47 1.26 20.07 23.62 56.81 25.98Ž . Ž .HL P 5.8 0.87 2.71 0.57 8.16 0.79 19.9 26.1 53.9 25.1Ž . Ž .HL N 6.3 0.30 1.79 0.22 9.10 0.54 19.8 29.1 51.4 26.5Ž . Ž .LL fr2 6.4 0.08 2.07 0.09 11.57 2.42 15.66 18.91 46.42 24.09Ž . Ž .LL P 6.3 0.23 2.56 0.01 12.98 1.26 12.52 29.91 34.42 17.60Ž . Ž .LL N 3.9 0.88 1.06 0.16 15.91 1.12 12.65 30.00 37.79 18.31Ž . Ž .C. muelleri HL fr2 9.2 1.80 3.19 0.81 11.10 0.86 17.79 24.33 50.99 22.44Ž . Ž .HL P 8.6 0.74 5.60 0.22 17.22 0.24 14.38 29.93 42.08 17.57Ž . Ž .HL N 7.2 0.45 4.85 0.47 23.41 B.D. 16.51 32.20 37.11 19.85Ž . Ž .LL fr2 12.5 1.5 3.20 0.30 10.75 0.49 21.45 23.22 52.02 25.11Ž . Ž .LL P 10.5 2.0 2.61 0.17 22.69 B.D. 9.35 42.71 29.53 13.11Ž . Ž .LL N 10.3 1.0 2.88 0.39 14.44 0.95 13.59 27.99 37.87 17.28Ž . Ž .R. reticulata HL fr2 25.9 3.49 4.2 0.78 7.08 15.92 11.67 17.04 69.71 59.77Ž . Ž .HL P 29.0 3.94 7.6 1.43 11.04 15.07 3.87 24.12 53.36 34.33Ž . Ž .HL N 36.2 2.12 18.3 0.8 25.09 13.43 2.08 49.64 33.21 27.32Ž . Ž .LL fr2 25.4 1.68 8.0 2.31 6.10 12.31 9.95 12.93 78.09 52.44Ž . Ž .LL P 28.2 2.89 4.7 0.04 8.37 17.44 8.63 12.33 75.39 59.01Ž . Ž .LL N 32.8 3.66 6.3 0.25 13.03 12.36 8.10 23.16 66.24 42.64Ž . Ž .P. lutheri HL fr2 3.7 1.25 0.51 0.06 18.50 0.99 27.88 27.71 55.71 43.61Ž . Ž .HL P 6.6 0.51 0.94 0.09 25.20 1.48 16.51 34.55 40.84 28.18Ž . Ž .HL N 4.1 0.2 0.45 0.17 17.62 1.88 17.66 27.11 57.40 39.69Ž . Ž .LL fr2 4.5 0.54 0.55 0.02 14.15 1.31 22.18 22.97 64.07 49.00Ž . Ž .LL P 5.7 1.93 0.54 0.15 17.75 2.44 25.63 26.01 59.84 42.81Ž . Ž .LL N 7.1 0.74 0.74 0.08 16.29 1.53 27.45 24.07 62.53 47.24

In all four algal species used, carbohydrate content per cell was not greatly affectedby the culture conditions showing one- to twofold changes. Carbohydrate content of S.costatum was generally increased with decreased irradiance except for the nitrogen-limited cultures where carbohydrate content remained practically unchanged at bothirradiance levels. R. reticulata, on the other hand, showed a different response withconservative changes in carbohydrate content; at the higher-light intensity, carbohydratecontent was increased only under phosphorus limitation and at the lower-light undernitrogen limitation. The carbohydrate content of C. muelleri was affected more bynutrient limitation at the higher-light intensity while at low-light, there was little change.In contrast, P. lutheri showed little change in its carbohydrate content under theconditions employed.

With regard to the fatty acid content of the algal species used, it appeared thatŽ .saturated fatty acids SAFA were increased as a proportion of the total identifiable fatty

Ž .acids TIFA under the higher-light intensity in both diatom species. A more complex

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Fig. 1. Length of D-stage larvae as well as final length of M. edulis larvae after the 14-day feeding trial usingthe Control, the S. costatum and P. lutheri as food cultured under various light and nutrient conditions.Results shown are average of three replicate cultures and vertical lines indicate "standard deviation.

Ž .fr2s fr2 medium no nutrient limitation ; Psphosphorus-limited medium, Nsnitrogen-limited medium.

Fig. 2. Length of D-stage larvae as well as final length of M. edulis larvae after the 14-day feeding trial usingthe Control, the C. muelleri and the R. reticulata diets as food cultured under various light and nutrientconditions. Results shown are average of three replicate cultures and vertical lines indicate "standard

Ž .deviation. fr2s fr2 medium no nutrient limitation ; Psphosphorus-limited medium, Nsnitrogen-limitedmedium.

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Ž .picture emerged for the PUFA and the ny3 series of fatty acids ny3 . In S.costatum, PUFA and ny3 decreased with nutrient limitation under the higher-lightintensity, but under low-light, PUFA proportions increased in the phosphorus-limitedcells and decreased in the nitrogen-limited cells. C. muelleri increased its SAFAproportion under nutrient stress at both light intensities, while ny3 FA decreased underthe same conditions. A similar decrease occurred with PUFA, except with phosphoruslimitation at low light, where its proportions were approximately the same as with the

Ž .non-limiting nutrient fr2 conditions. The proportion of PUFA and ny3 in R.

Table 2Percent survival of larvae after the S. costatum, R. reticulata, C. muelleri and P. lutheri feeding trials, foreach of the triplicate cultures

Ž .Numbers in parentheses indicate standard deviation. fr2s fr2 medium no nutrient limitation ; Psphosphorus-limited medium; Nsnitrogen-limited medium.

Diet High-light trial Low-light trial High-light trial Low-light trial

S. costatum R. reticulata

Ž . Ž . Ž . Ž .Control 1 74.4 5.67 47.2 13.2 39.8 5.79 39.8 5.79Ž . Ž . Ž . Ž .Control 2 92.8 16.9 44.4 11.0 38.0 4.61 38.0 4.61Ž . Ž . Ž . Ž .Control 3 88.9 15.1 35.6 2.08 39.2 7.89 39.2 7.89Ž . Ž . Ž . Ž .Unfed 1 87.2 3.42 30.0 9.81 7.0 1.43 7.0 1.43Ž . Ž . Ž . Ž .Unfed 2 52.8 2.08 55.0 4.91 9.4 0.83 9.4 0.83Ž . Ž . Ž . Ž .Unfed 3 90.0 10.6 38.9 4.37 14.6 5.03 14.6 5.03Ž . Ž . Ž . Ž .fr2 1 98.9 14.1 28.9 3.14 31.0 5.79 34.5 4.14Ž . Ž . Ž . Ž .fr2 2 91.1 10.9 38.9 0.79 35.1 4.30 32.8 5.96Ž . Ž . Ž . Ž .fr2 3 81.1 6.98 30.6 2.08 32.2 5.79 25.1 0.83Ž . Ž . Ž . Ž .P 1 91.7 1.36 18.3 2.72 40.9 2.98 37.4 3.61Ž . Ž . Ž . Ž .P 2 93.3 10.6 18.9 1.57 28.7 5.79 32.8 2.19Ž . Ž . Ž . Ž .P 3 77.2 8.31 17.2 2.08 26.9 0.83 39.2 7.21Ž . Ž . Ž . Ž .N 1 87.2 4.16 28.9 3.42 19.3 1.43 29.8 4.96Ž . Ž . Ž . Ž .N 2 86.1 5.15 15.0 3.60 19.9 2.98 30.4 2.19Ž . Ž . Ž . Ž .N 3 84.4 12.6 12.2 5.67 28.7 5.96 35.7 3.31

C. muelleri P. lutheriŽ . Ž . Ž . Ž .Control 1 74.4 5.67 47.2 13.2 35.0 2.36 35.0 2.36Ž . Ž . Ž . Ž .Control 2 92.8 16.9 44.4 11.0 40.0 7.20 40.0 7.20Ž . Ž . Ž . Ž .Control 3 88.9 15.1 35.6 2.1 43.3 3.60 43.3 3.60Ž . Ž . Ž . Ž .Unfed 1 87.2 3.42 30.0 9.8 36.7 1.36 36.7 1.36Ž . Ž . Ž . Ž .Unfed 2 52.8 2.08 55.0 4.9 29.4 1.57 29.4 1.57Ž . Ž . Ž . Ž .Unfed 3 90.0 10.6 38.9 4.4 35.6 4.16 35.6 4.16Ž . Ž . Ž . Ž .fr2 1 82.8 8.20 20.6 5.5 43.3 8.28 37.2 6.71Ž . Ž . Ž . Ž .fr2 2 84.4 8.20 25.0 6.2 38.9 8.75 44.4 3.14Ž . Ž . Ž . Ž .fr2 3 93.3 16.0 32.8 5.2 35.6 3.42 32.8 4.37Ž . Ž . Ž . Ž .P 1 73.3 13.0 23.9 3.9 36.1 5.50 39.4 3.42Ž . Ž . Ž . Ž .P 2 87.8 4.37 42.2 6.3 27.2 4.37 37.8 13.2Ž . Ž . Ž . Ž .P 3 74.4 9.26 19.4 4.2 27.2 0.79 37.8 2.83Ž . Ž . Ž . Ž .N 1 85.0 8.28 19.4 4.4 26.7 3.60 42.2 12.9Ž . Ž . Ž . Ž .N 2 81.7 10.8 23.3 3.6 33.3 4.91 31.1 9.06Ž . Ž . Ž . Ž .N 3 85.6 8.31 22.2 2.1 33.3 4.71 40.6 6.43

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reticulata showed smaller changes under low irradiance, whereas at the high light, therewas a peak of PUFA when nutrients were not limited. It was also noticeable that therewas a marked increase in the SAFA proportion in R. reticulata grown under nitrogenlimitation at the higher-light conditions. For P. lutheri, there was little change in overallSAFA components with the various treatments. The exception occurred under high-lightphosphorus limitation where the SAFA proportion was considerably increased and therewas a corresponding reduction in the PUFA and ny3 proportions.

The final length of M. edulis larvae achieved after the 14-day feeding trial, as well asthe corresponding length of the control unfed and at the initial D-stage, is illustrated inFig. 1, when the larvae were fed either on S. costatum or P. lutheri and in Fig. 2, whenfed on C. muelleri or R. reticulata. With the aid of nested ANOVA and analysis ofmeans, the S. costatum diets were ranked, in decreasing order of exhibited larval

Ž .growth, with ‘‘) ’’ indicating significant difference 95% , as follows: LL NsLLfr2)Control)HL fr2sHL PsHL N)LL P. The C. muelleri diets were ranked,in decreasing quality, in the high-light experiment as: HL N)Control)HL fr2sHLP, and for the low-light experiment, LL fr2sLL N)ControlsLL P. The R.reticulata diets were ranked as: HL NsLL fr2sControl)LL N)HL fr2sHLPsLL P. Finally, the P. lutheri diets were ranked, again in decreasing quality order as:HL P)HL fr2sHL N)LL NsLL P)LL fr2sControl.

Ž .It becomes clear that survival varied between experiments Table 2 , which werecarried out using different batches of larvae, while in the same experiment, survivalcould not be correlated with the algal diet. This argument is supported by the variation

Žin survival between triplicates of the same diet showing significant differences P-.0.05 .

Fig. 3. Relationship between proportion of dietary fatty acids of P. lutheri and length of the M. edulis larvaeachieved after feeding on the corresponding diets. SAFA s total saturated fatty acids. PUFA s total poly-unsaturated fatty acids.

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Ž .Fig. 4. Relationship between 16:0 proportion as percentage of total identifiable fatty acids, TIFA , protein andcarbohydrate content of R. reticulata and larval length achieved after feeding for 2 weeks on this species.Protein content is shown colour-coded.

There were few occasions that a coherent relationship between algal biochemicalcomponents and larval size could be established. In P. lutheri, there was very clear

Ž .Fig. 5. Relationship between 18:3ny3 proportion as percentage of total identifiable fatty acids, TIFA ,protein and carbohydrate content of R. reticulata and larval length achieved after feeding for 2 weeks on thisspecies. Protein content is shown colour-coded.

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Ž .Fig. 6. Relationship between total ny3 FA proportion as percentage of total identifiable fatty acids, TIFA ,protein and carbohydrate content of R. reticulata and larval length achieved after feeding for 2 weeks on thisspecies. Protein content is shown colour-coded.

positive correlation between the 16:0 and SAFA, with growth and negative correlationsŽ .existing between PUFA and ny3 FA with larval size Fig. 3 , with correlation values

of 0.97, 0.98, y0.98 and y0.97, respectively. Protein and carbohydrate content werenot significantly correlated with larval growth, with correlation values of 0.19 and 0.57,respectively. However, protein, carbohydrate and some fatty acids of R. reticulata werecorrelated with larval growth. Generally, lower correlation values were calculated with0.73 and 0.48 for protein and carbohydrate content and 0.55, y0.91, 0.56, y0.6 for16:0, 18:3ny3, SAFA and ny3 FA, respectively. Four-dimensional plots of larvallength, algal protein, carbohydrate and one of these algal fatty acids components areshown in Figs. 4–6 for the 16:0, 18:3ny3 and ny3 FA, respectively. Collectively,these graphs, together with the correlation analysis, indicate that there is a positivecorrelation between algal 16:0 and SAFA with larval growth while algal 18:3ny3 andny3 FA were negatively correlated with larval growth. Despite careful examination ofthe data for C. muelleri and S. costatum, no consistent relationship could be established.

4. Discussion

Culturing live algae for food in hatcheries is essential, especially given the limitedŽ .success, up to now, of artificial feeds Laing, 1987 ; hence, optimizing the nutritional

value of any alga for a specific animal should be of critical importance. EnvironmentalŽconditions have been shown to influence algal biochemical composition Leonardos and

.Lucas, 2000a . The optimization of the algal nutritional value in this investigation wasfound to be of significant importance, with algal culturing conditions increasing larvalsize from 10 to 30%, depending on the algal species.

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S. costatum is a species used extensively in hatcheries mainly because of its ability tothrive in a variety of environments while also supporting good growth of animalsŽ .O’Connor et al., 1992 . It was found to be the second highest-ranked diatom for Ostrea

Ž .edulis growth by Enright et al. 1986a among the species tested and one of the bestŽ .diets for the hard clam, Mercenaria mercenaria Wikfors et al., 1992 . C. muelleri has

Žbeen shown to have good potential in a subtropical greenhouse bivalve hatchery Nelson.et al., 1992 . A good nutritional value record is also reported for this diatom for the

Ž .Sydney rock oyster, Saccostrea commercialis by O’Connor et al. 1992 and its highnutritional value is further confirmed here with M. edulis larvae. It appears that thisChaetoceros species is no exception from the generally good nutritional value record

Žthat this genus has been shown to demonstrate Chu, 1989; O’Connor and Heasman,. Ž .1997 . Of all the species tested here, R. reticulata previously Rhodomonas baltica is

the least studied, although other Cryptomonads, described as Rhodomonas species, haveŽ .been found to be good diets for O. edulis Enright et al., 1986a . On the basis of the

very high nutritional value found here, the apparent lack of extensive usage of thisspecies in mariculture is difficult to substantiate. P. lutheri has an ambiguous record ofnutritional value in which it appears to be a very good food for larvae of several

Žbivalves, e.g., doughboy scallops, Mimachlamys asperrima O’Connor and Heasman,. Ž1997 , American and European oysters, Crassostrea Õirginica and O. edulis Walne,

.1963; Chu and Dupuy, 1980 , but mediocre and poor for some others like the JapaneseŽ .scallop, Patinopecten yessoensis Thompson et al., 1994 and the Pacific oyster, C.

Ž .gigas Langdon and Waldock, 1981 . The present work classifies this species as anexcellent food for M. edulis larvae since in almost all of its biochemical compositions, itperformed better than the control diet. This control two-species diet was expected to beof superior value, because it was considered to contain the greater diversity ofbiochemical constituents to satisfy most nutritional requirements for growth than any

Ž .one monospecific diet Widdows, 1991 .Ž .Thompson et al. 1996 correlated positively the proportion of dietary 16:0 and

negatively the dietary 20:5ny3 of P. lutheri with larval mortality in C. gigas; boththese results were unequivocally confirmed here with the use of correlation analysis. Itappears that in PUFA and ny3 FA, in general, rather than specifically, the 20:5ny3was negatively correlated with larval growth. Hence, it is not clear whether the positiveand negative correlations of 16:0 and 20:5ny3, respectively, are due to their specificrole or is a reflection of the positive and negative correlations of SAFA and ny3 FA,generally.

The use of a multidimensional model enables a more comprehensive inclusion ofdietary components that have been shown in the past to be important in determining thealgal nutritional value, overcoming the problematic usage of multilinear regression

Ž .analysis Wikfors et al., 1992 . Although the FA content appears to be the main factorinfluencing algal nutritional value in some cases, the protein and carbohydrate content isalso modifying the alga’s dietary value; increased carbohydrate and protein contentwithin an algal species enhances its nutritional value. As shown in the four dimensionalgraphs, optimal larval growth is achieved when the alga contains the highest proportionof 16:0 and SAFA as well as increased protein and carbohydrate content; singly any ofthese components cannot sufficiently explain the resulting variation of larval growth.

( )N. Leonardos, I.A.N. LucasrAquaculture 182 2000 301–315 313

Again, the decrease of nutritional value was found when higher proportions of 18:3ny3and ny3 fatty acids, in general, were observed. The descriptive nature of this approachdoes not provide an exact measure by which the contributions to the nutritional value ofany algal component can be determined, but it provides a means of demonstrating how

Ždietary components interact to determine the overall dietary value Enright et al.,. Ž .1986a,b . However, the comments of Dickey-Collas and Geffen 1992 and Thompson

Ž .et al. 1996 about the positive effects of increased 16:0 and SAFA and the negativeeffects of increased PUFA and more specifically, the ny3 fatty acids are generallyconfirmed here. It would seem that in the cases where such a clear-cut relationship isfound, with dietary fatty acid being the main factor in determining nutritional value, theother biochemical components are of minor importance. In other cases, there is apositive effect of increased protein and carbohydrate content, with fatty acids alsocontributing to the nutritional value.

ŽThese results are apparently in conflict with the widely publicized held view e.g., Su.et al., 1988 that increased dietary PUFA enhance the dietary value of an algal diet, not

least because larval PUFA have been shown to be a positive growth index for M. edulisŽ . Ž .Leonardos and Lucas, 2000b . Langdon and Waldock 1981 demonstrated the impor-tance of PUFA, especially the 20:5ny3, to C. gigas. Many researchers in theaquacultural field, largely because of this strong correlation of the larval 20:5ny3 fattyacid and PUFA, in general, with growth, use the occurrence and quantity of these

Ž .compounds as ‘‘a rule of thumb’’ guide to rank algal diets Su et al., 1988 . Otherworkers have indirectly further substantiated this relationship by trying to explain thenutritional deficiency of particular algal species in terms of the lack of some essential

Ž .fatty acids, such as some of the ny3 groups De Pauw et al., 1984 . From the currentfindings, this is a misleading overgeneralization to make. A clear distinction should bemade between the nutritional elements in the diet and the way that they influence larvalgrowth and development on one hand and the biochemical composition of the larvaethemselves. A low level or lack of one dietary component, e.g., a particular fatty acid,will not necessarily be reflected by a deficiency in the same component in the larvae,since the animals may transform almost all of the components that they digest, throughtheir metabolic pathways; a deficiency in the input element will translate into adeficiency of the end product inside the animal cells. Therefore, it is not entirely correctto substantiate the correlation of a larval fatty acid with larval growth by observing theeffect that it has if it is excluded or inadequately present in the diet, or vice versa. Thesole significance is related to the nutritional quality of the diet for that given organismand not as to the importance as a growth index for the animals themselves.

The optimal nutritional value was obtained at low-light, nitrogen-limited or non-limited nutrient cells for S. costatum and C. muelleri, for R. reticulata at high-lightconditions under phosphorus limitation or low-light without nutrient limitation and forP. lutheri at high-light conditions under phosphorus limitation. Given the simplicity ofthe modification of the culture conditions, this approach is a cost-effective way ofincreasing the nutritional value of the cultured algae and should be further explored forother algae. Although algal fatty acids were shown to be of particular importance indetermining the algal nutritional value, overall, they were found to be species-specific.Therefore, future research not only should be directed towards examining the way in

( )N. Leonardos, I.A.N. LucasrAquaculture 182 2000 301–315314

which environmental conditions determine the nutritional value of an alga, but the rangeof species and clones investigated must also be widened. Further research should aim atenlightening the interactive way in which discrete algal biochemical components deter-mine the nutritional value of the species, with emphasis given to their fatty acid profile.

Acknowledgements

The first author would like to thank Andy Beaumont for his helpful commentsthroughout this work and J. East for his invaluable help with the fatty acid analyses. Thepresent work is part of a PhD research thesis funded by the Greek State Scholarship

Ž .Foundation I.K.Y. whose assistance is acknowledged.

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