The effect of microalgal diets on growth, biochemical composition, and fatty acid profile of Crassostrea corteziensis (Hertlein) juveniles

Aquaculture 263 (2007) 199 – 210 www.elsevier.com/locate/aqua-online

The effect of microalgal diets on growth, biochemical composition, and fatty acid profile of Crassostrea corteziensis (Hertlein) juveniles Susana Rivero-Rodríguez a , Andy R. Beaumont a , María Concepción Lora-Vilchis b,⁎ b

a School of Ocean Sciences, University of Wales. Bangor, Menai Bridge, Gwynedd, LL59 5AB. Wales, UK Centro de Investigaciones Biológicas del Noroeste. Mar Bermejo, 195.Col. Playa de Santa Rita. P.O. 128. La Paz, Baja California Sur, México

Received 2 June 2006; received in revised form 28 September 2006; accepted 28 September 2006

Abstract The culture of the mangrove oyster Crassostrea corteziensis is a promising enterprise on the Pacific coast of Mexico, yet little is known about the diet and essential nutrients required to maximize growth during culture. C. corteziensis juveniles were grown for 22 days under hatchery conditions and using monospecies or binary diets of the microalgal species Chaetoceros calcitrans, Chaetoceros muelleri, Isochrysis galbana clone T-iso, Phaeodactylum tricornutum, and Tetraselmis suecica. The monospecies diet of C. calcitrans provided a superior diet for C. corteziensis juveniles, yielding a growth rate up to 272-μm shell length day− 1. The nutritional value of the microalgae tested was in the order C. calcitrans N C. muelleri N I. galbana clone T-iso N P. tricornutum N T. suecica. The reasons for the different nutritional values of the algae investigated were not related to carbohydrate, protein, or lipid content. The HUFA composition of oysters was related to their diets. C. calcitrans is characterized by high levels of arachidonic acid (ARA; 20:4n-6). The monospecies diet of T. suecica, which yielded the lowest growth rate (107-μm shell length day− 1) lacked docosahexaenoic acid (DHA; 22:6n-3) together with having low levels of ARA and eicosapentaenoic acid (EPA; 20:5n-3) that could explain the poor performance of this diet. There were significant differences in the fatty acid composition of the oysters fed different diets but the only correlation with growth was in the case of ARA. We conclude that C. calcitrans supplies the appropriate nutrient balance that C. corteziensis needs at this juvenile stage. © 2006 Elsevier B.V. All rights reserved. Keywords: Crassostrea corteziensis; Juvenile; Oyster; Nutrition; Fatty acid

1. Introduction Populations of Crassostrea corteziensis (Hertlein), the pleasure oyster, have declined drastically in recent years (Chávez-Villalba, 2004), but this oyster has shown a high potential for culture that could assist its development on the Mexican Pacific coast. The Japanese oyster (Crassostrea gigas), which is the second most ⁎ Corresponding author. Tel.: +52 612 12 3 84 84x3311; fax: +52 612 125 3625. E-mail address: [email protected] (M.C. Lora-Vilchis). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.09.038

important marine species in volume of production in México, has shown significant problems in culture because of the lack of seed-production strategies, such as a selected broodstock. For these reasons, there is an increasing interest in the potential commercial culture of C. corteziensis under hatchery conditions. Hatchery rearing of bivalve molluscs depends on the production of live algae, which generally accounts for about 30% of the total seed-production cost. With juveniles representing the largest biomass in the hatchery and demanding the highest weight-specific rations, large volumes of algal culture are needed for this stage of the

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life cycle (Coutteau and Sorgeloos, 1995). Even a small improvement in the diet could result in substantial savings in a bivalve nursery. A precise knowledge of microalgal composition is essential to provide juvenile oysters with the correct diet to support effective growth. Among the most common species used in aquaculture to feed juvenile oysters are Chaetoceros muelleri Lemmerman, Chaetoceros calcitrans (Paulsen) Takano, Skeletonema costatum (Greville) Cleve, Isochrysis galbana Parke clone T-iso, Thalassiosira pseudonana (Hustedt) Hastle and Heimdal, Pavlova lutheri Droop, and Tetraselmis suecica (Kylin) Butcher (Enright et al., 1986; Laing and Millican, 1986; O'Connor et al., 1992). Phytoplankton, the primary food source for bivalves, provides essential sterols and highly unsaturated fatty acids (HUFAs), such as 20:4n-6 (arachidonic acid, ARA), 20:5n-3 (eicosapentaenoic acid, EPA), and 22:6n-3 (docosahexaenoic acid, DHA), although the amount of these components vary with species and seasons (Soudant et al., 2000). Lipids in general, and specifically n-3 HUFAs, have an essential role in bivalve mollusc nutrition. Langdon and Waldock (1981) demonstrated an essential fatty acid requirement in bivalve molluscs and identified the deficiency of the HUFAs EPA and DHA in Dunaliella tertiolecta as a growth-limiting factor in C. gigas spat. Enright et al. (1986) showed that nutritional requirements in juvenile oysters are highest for essential fatty acids, especially DHA and EPA, with lower requirements for carbohydrates and then amino acids or proteins. Generally, the ability to synthesize ARA, EPA, DHA, and sterols in bivalves is limited (Soudant et al., 2000). Therefore, the gross chemical composition of phytoplankton can be a defining factor when choosing an algal species as the food source for bivalve culture. Semicontinuous dilution regimes in marine microalgal cultures produces biomass of a stable biochemical composition at a selected growth phase (Brown et al., 1993) and is therefore the best method of algal culture for the determination of bivalve nutritional requirements. Defining the exact dietary requirements of essential fatty acids (EFAs) in animals requires consideration not only of the relative and the absolute amounts of individual fatty acids in the animals' diets, but also of the innate ability of the animal to metabolize these fatty acids, whether anabolically or catabolically (Sargent et al., 2002). In bivalve nutrition the main HUFAs generally considered are ARA with its metabolic precursor 18:2n-6 (linoleic acid), and EPA and DHA with their metabolic precursor 18:3n-3 (linolenic acid). It has been suggested that the (n-3)/(n-6) ratio has a great influence on the quality of the diet (Soudant et al., 1999).

In our study we examined the dietary contribution of 5-algal species (C. calcitrans, C. muelleri, I. galbana clone T-iso, P. tricornutum, and T. suecica) provided as mono- or bi-algal diets to C. corteziensis juveniles. We related the biochemical characteristics of the diets to the growth, nutritional condition, lipid and fatty acid concentration, and composition of the juvenile oysters. 2. Materials and methods 2.1. Microalgal cultures The microalgal species used were T. suecica (Prasinophyceae), I. galbana clone T-iso (Prymnesiophyceae), P. tricornutum (Bacillariophyceae), C. muelleri (Bacillariophyceae), and C. calcitrans (Bacillariophyceae). Cultures of T. suecica and C. muelleri were from the Provasoli–Guillard National Center for Culture of Marine Phytoplankton (CCMP), I. galbana and P. tricornutum were from The Culture Collection of Algae at the University of Texas at Austin (UTEX), and C. calcitrans cultures were from the French Research Institute for Exploitation of the Sea, IFREMER. The microalgae were grown as semicontinuous, single-species cultures in filtered (1 μm) and sterilized seawater with salinity of 34 PSU and temperature of 22 ± 1 °C. The enriched f/2 medium (Guillard and Ryther, 1962) was supplied to all algal cultures with silicate solution (NaSiO3U9H2O, 30 g/L in H2O) added to diatom cultures only. Light intensity was 135 ± 10 μmol m−2 s− 1 and with a cycle of 12:12 h L:D. Microalgae were harvested during the exponential phase. Cellular density was determined daily by using a Neubauer haemocytometer before feeding the oysters. 2.2. Oyster culture and dietary conditioning C. corteziensis broodstock was obtained from Bahia de Ceuta, Culiacan, Sinaloa, Mexico. A mixture of six oysters were spawned to produce veliger larvae with the survival from metamorphosis to seed 90%. The juveniles were used 5 weeks after settlement. They were maintained in hatchery-reared conditions and fed on I. galbana clone T-iso and C. calcitrans (50/50 mixture) and were graded with mesh sieves to obtain spat of a uniform size. Groups of 30 specimens were blotted with an absorbent cloth to remove excess surface water and weighed. The shell length was measured to the nearest 0.1 mm from a lifesize photocopy of spat spread on a petri dish. At the start of the experiment the mean weight (± s) of 30 individuals was 0.360 ± 0.046 g and the average individual spat length (± s) was 4.6 ± 0.1 mm.

S. Rivero-Rodríguez et al. / Aquaculture 263 (2007) 199–210 Table 1 Algal diets used for the oyster feeding trial Single species diet C. calcitrans C. muelleri I. galbana clone T-iso P. tricornutum T. suecica

Mixed species diet (C) C. calcitrans + C. muelleri (CM) (M) C. calcitrans + I. galbana clone T-iso (CI) (I) C. calcitrans + P. tricornutum (CP) (P) (T)

C. calcitrans + T. suecica C. muelleri + I. galbana clone T-iso C. muelleri + P. tricornutum C. muelleri + T. suecica I. galbana clone T-iso + P. tricornutum I. galbana clone T-iso + T. suecica P. tricornutum + T. suecica

(CT) (MI) (MP) (MT) (IP) (IT) (PT)

Oyster juveniles were placed in down-welling silos in separate aerated 4-L containers with seawater at 40 ppt salinity and 25 °C, 30 per silo. They were fed diets composed of 15 different combinations of five species of algae (C. calcitrans, C. muelleri, I. galbana clone T-iso, P. tricornutum, and T. suecica) over 22 days with three replicates of each diet used. Filtered seawater (1 μm) was changed every morning just before feeding. Fresh food was supplied daily as 1.5% of the oyster live weight in algal organic weight (Urban et al., 1983). The ration was divided into two doses, one in the morning and the other in late afternoon. Oyster survival, shell length, and live weight were monitored weekly and the ration adjusted accordingly. At the end of the trial the oysters were (as a group of 30) weighed. Four replicates of pooled oysters (five

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oysters/replicate) were sampled and later analyzed for carbohydrate, protein, lipid, and fatty acids. Additional oyster juveniles were dried and ashed to determine dry and ash-free dry weight (AFDW) according to LoraVilchis and Doctor (2001). 2.3. Experimental diets Both monospecies and mixed-species (50/50 mixture equal organic weight) algal diets of C. calcitrans, C. muelleri, I.galbana clone T-iso, P. tricornutum, and T. suecica were assessed (Table 1). For dry weight and ash-free dry weight (AFDW) determinations a volume (30 to 40 mL) of algal culture of known concentration was filtered through Whatman GF/C filter paper. Algal residues were washed with 4-mL 0.5 M ammonium formate to remove sea salts and then dried at 65 °C to a constant weight (48 h). The filters were then ashed at 500 °C for 6 h. Large quantities of algae were harvested for compositional analyses and stored at − 40 °C after centrifugation and lyophilization. 2.4. Biochemical analyses Biochemical analyses were determined on 4 to 6 mg of lyophilized algal cells and 200 to 250 mg of frozen oyster tissue. Protein and total carbohydrate were determined by the methods of Lowry et al. (1951) and Dubois et al. (1956). Total lipid and fatty acids were extracted by a

Table 2 Growth rate calculated in length (μm day−1), in live weight (mg day−1 30 oysters−1), in ash-free dry weight (mg day−1 30 oysters−1); live weight per 30 oysters (g·30 oysters−1), percentage of dry weight relative to live weight, and percentage of ash-free dry weight AFDW relative to dry weight of juvenile C. corteziensis after 22 days of feeding on various diets (Mean, s, n = 3) Diet C I M P T IC IM IT IP CM CP CT MP MT TP

Growth rate length a

272.1 ± 3.8 142.9 ± 6.4c 163.7 ± 14.0b 111.9 ± 5.2c 107.5 ± 5.2c 218.8 ± 31.6a 222.0 ± 58.4a 129.5 ± 9.9c 136.3 ± 21.3c 232.9 ± 22.1a 230.0 ± 31.8a 214.3 ± 34.8a 175.5 ± 18.1b 174.6 ± 11.9b 134.0 ± 4.2c

Growth rate live weight a

186.1 ± 14.5 75.9 ± 3.0abc 93.8 ± 11.6abc 48.1 ± 2.8c 51.4 ± 1.9bc 134.4 ± 11.6abc 93.2 ± 16.1abc 61.2 ± 14.2abc 63.6 ± 2.3abc 159.1 ± 41.5ab 130.0 ± 29.6abc 153.8 ± 28.7abc 82.7 ± 8.7abc 92.7 ± 6.3abc 60.3 ± 2.2abc

Growth rate AFDW a

0.28 ± 0.06 0.13 ± 0.02abc 0.13 ± 0.08abc 0.05 ± 0.01c 0.08 ± 0.04bc 0.19 ± 0.02ab 0.14 ± 0.04abc 0.08 ± 0.01bc 0.14 ± 0.05abc 0.19 ± 0.03ab 0.21 ± 0.07ab 0.18 ± 0.05ab 0.18 ± 0.06abc 0.14 ± 0.06abc 0.09 ± 0.01bc

Live weight a

4.24 ± 0.32 1.97 ± 0.07c 2.33 ± 0.31b 1.38 ± 0.08c 1.46 ± 0.07c 3.15 ± 0.26a 2.31 ± 0.37b 1.65 ± 0.37c 1.67 ± 0.05c 3.71 ± 0.96a 3.59 ± 0.66a 3.09 ± 0.69a 2.33 ± 0.16b 2.09 ± 0.24b 1.62 ± 0.04c

Dry weight

AFDW

60.7 ± 2.0 62.5 ± 1.1 64.8 ± 1.9 65.1 ± 1.1 63.4 ± 4.2 63.0 ± 2.1 60.6 ± 1.7 63.2 ± 2.6 63.5 ± 0.7 63.3 ± 1.1 61.4 ± 1.3 62.4 ± 3.3 65.6 ± 1.5 64.8 ± 0.4 62.1 ± 1.7

8.0 ± 0.1 9.2 ± 1.5 7.6 ± 0.3 7.7 ± 0.1 8.0 ± 0.9 8.3 ± 0.7 7.8 ± 1.4 8.6 ± 0.4 9.1 ± 0.9 7.4 ± 0.6 8.0 ± 1.2 8.0 ± 0.7 7.6 ± 0.2 8.9 ± 1.4 7.9 ± 0.5

For diet abbreviations, see Table 1. s: standard deviation, n: number of replicates. Values within the same column sharing a common superscript letter are not different ( p N 0.05).

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Table 3 Biochemical composition of the microalgae used as food for juveniles of C. corteziensis Microalgae

Carbohydrate

Protein

Total lipid

C. calcitrans I. galbana C. muelleri P. tricornutum T. suecica

11.4 ± 0.2 13.8 ± 0.5 15.6 ± 0.2 32.0 ± 4.8 17.7 ± 1.6

40.5 ± 7.6 38.4 ± 0.8 34.4 ± 4.7 35.2 ± 7.0 40.9 ± 8.7

11.4 ± 2.2 17.9 ± 2.2 11.1 ± 1.1 13.3 ± 2.0 16.1 ± 3.1

Data are expressed as a percentage of AFDW (Mean, s, n = 3). s: standard deviation, n: number of replicates.

modification of the Bligh and Dyer (1959) method. The samples were extracted with 2:1 methanol–chloroform mixture at 4 °C for 24 h and lipids were analyzed by the Marsh and Weinstein (1966) method. The neutral and polar lipid fractions were derivatized with HCl–CH3OH (5%, v/v) and treated according to Sato and Murata (1988). The fatty acid methyl ester (FAME)-containing fraction was resuspended in hexane and collected in tapered vials containing tricosanoic acid 23:0 as an internal standard. The FAMEs were analyzed using a Hewlett Packard G1800B gas mass chromatograph equipped with a oncolumn injector and a silica capillary column (omega wax TM 250 Supelco) internally covered by polyethylene glycol (30 m × 0.25 mm, 0.25 μm film thickness) and with an electron ionization detector. Helium of high purity was used as the carrier gas with a flow of 0.9 mL/min. The temperature of the oven was programmed from 110 °C to 165 °C at 30 °C min− 1 and from 165 °C to 209 °C at 2.2 °C min− 1. The detector and injector temperature were 260 °C and 250 °C. Fatty acids were identified by comparison of their retention time with 29 standards and with the data bases NIST 2000, NIST 98, and NB575K. They were verified by using a mass spectral library with the search program (NIST–EPA–NIH, Data Version: NIST'02, Software Version: 2.0). Electron-impact mass spectra were acquired and processed with a G107 4B Computer Workstation. 2.5. Statistical analysis Comparison of length, weight, and biochemical composition between diets was made by an ANOVA after arcsine transforming of rates and percentages and checking normality and homogeneity of variance by Anderson–Darling and Bartlett or Levene tests and, when significant ( P b 0.05), a Tukey test (95% confidence interval) was used.

The percentages of the fatty acids of the oysters were arcsine transformed to ensure normality and then a Principal Component Analysis was developed using the covariate matrix. 3. Results 3.1. Oyster growth Oyster growth varied significantly as a function of the diet supplied. Performance of the oyster juveniles was assessed in terms of growth rate based on length, live weight, and ash-free dry weight (AFDW). Total live weight, dry weight, and AFDW at the end of the trial were also evaluated. Growth rates, live weight, dry weight, and AFDW for C. corteziensis juveniles grown under fifteen different algal diets are shown in Table 2. The oysters fed on C. calcitrans showed the greatest growth rates with 272.1 ± 3.8 μm day− 1 length, 186.1 ± 14.5 mg day− 1 30 oysters− 1 live weight, and 0.28 ± 0.06 mg day− 1 30 oysters− 1 AFDW, and the greatest live weight (4.24 ± 0.32 g 30 oysters− 1). The oysters that showed the poorest performance were those fed on T. suecica and P. tricornutum monospecies algal diets with 107.5 ± 5.2 μm day− 1 and 111.9 ± 5.2 μm day− 1 1 in length growth rate respectively, and 0.08 ± 0.04 mg day− 1 30 oysters − 1 and 0.05 ± 0.01 mg day− 1 30 oysters− 1 for

Table 4 Biochemical composition of C. corteziensis juveniles at the beginning and at the end of the feeding trial Treatment

Carbohydrate

Initial 0.31 ± 0.05 After 22 days of culture C 0.40 ± 0.03a I 0.37 ± 0.01ab M 0.26 ± 0.03c P 0.34 ± 0.04abc T 0.25 ± 0.03c IC 0.32 ± 0.03abc IM 0.31 ± 0.04abc IT 0.32 ± 0.02abc IP 0.31 ± 0.03abc CM 0.27 ± 0.04c CP 0.30 ± 0.03bc CT 0.25 ± 0.04c MP 0.25 ± 0.05c MT 0.27 ± 0.01c TP 0.27 ± 0.02bc

Protein

Total lipid

2.06 ± 0.12

0.28 ± 0.01

1.96 ± 0.01 1.66 ± 0.21 2.03 ± 0.15 1.96 ± 0.04 1.89 ± 0.17 1.96 ± 0.20 1.74 ± 0.31 1.98 ± 0.37 2.01 ± 0.03 2.16 ± 0.30 1.94 ± 0.23 1.97 ± 0.19 1.86 ± 0.47 1.96 ± 0.32 1.94 ± 0.14

0.25 ± 0.04 0.26 ± 0.05 0.20 ± 0.04 0.28 ± 0.09 0.20 ± 0.00 0.26 ± 0.03 0.26 ± 0.02 0.25 ± 0.00 0.28 ± 0.07 0.21 ± 0.02 0.24 ± 0.02 0.22 ± 0.01 0.21 ± 0.03 0.18 ± 0.01 0.24 ± 0.03

Data are expressed as a percentage of live weight (Mean, s, n = 3). For diet abbreviations, see Table 1. s: standard deviation, n: number of replicates. Values within the same column sharing a common superscript letter are not different (P N 0.05).

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Table 5 Relative fatty acid composition of microalgae used as food for juveniles of C. corteziensis

14:0 14:1n-3 15:0 16:0 16:1n-9 16:1n-7 16:1n-5 16:2n-6 16:2n-4 16:3n-6 17:0 16:3n-3 16:4 17:1 18:0 18:1n-9 18:1n-7 18:2n-6 18:3n-6 18:3n-3 18:4n-3 18:4 20:1n-9 20:3 20:4n-6 (ARA) 20:3n-6 20:4n-3 20:5n-3 (EPA) 22:0 22:5n-6 24:0 22:6n-3 (DHA) ∑ saturated ∑ monoenoic ∑ PUFA ∑ HUFA ∑ n-3 ∑ n-6 (n-3) / (n-6) ARA/ DHA DHA/EPA

C. calcitrans

C. muelleri

I. galbana

P. tricornutum

T. suecica

6.8 – 0.2 3.9 – 17.4 1.2 2.4 6.1 – – 14.5 1.8 – 0.4 0.7 0.5 2.0 – 1.3 0.6 – – – 11.3 – – 26.3 0.2 – 0.1 2.3 11.7 19.7 28.7 39.9 45.1 15.1 2.9 4.9 0.09

5.9 – 0.3 7.1 – 13.9 0.7 2.8 5.6 – 0.1 14.4 0.4 – 1.5 1.0 0.7 0.5 0.8 0.8 0.6 – – – 3.0 – – 36.8 0.2 – 0.1 2.6 15.3 16.4 26.0 42.4 55.3 7.1 7.8 1.1 0.07

11.5 0.5 0.1 4.7 0.1 3.6 – 0.3 0.6 – – – – 0.1 0.3 8.7 0.6 5.9 1.0 7.9 18.8 3.8 – – – – – 0.9 – 3.8 – 26.8 16.6 13.6 38.3 31.4 54.8 11.0 5.0 – 29.8

5.9 – 0.2 11.8 0.1 16.1 0.8 0.3 3.6 – – 11.1 0.5 – 0.3 3.1 1.3 2.1 0.8 0.4 0.6 – – 1.3 0.9 0.7 0.2 35.0 0.1 – 1.1 1.5 19.4 21.4 19.4 39.8 48.8 4.8 10.4 0.6 0.04

0.2 – 0.1 13.7 1.2 0.2 0.9 0.6 – 4.1 0.1 1.1 17.7 – 1.4 7.8 1.4 10.3 1.2 15.4 8.5 1.9 1.1 – 2.1 – 0.6 8.4 – – – – 15.4 12.8 60.7 11.1 33.9 18.2 1.9 – –

Data expressed as percentage of total fatty acids, n = 3. n: number of replicates. The standard deviation was omitted for clarity. PUFA: polyunsaturated fatty acids, HUFA: highly unsaturated fatty acids.

growth rate relative to AFDW, respectively. The dry weight was between 58% and 68% and AFDW between 6% and 11% of live weight (Table 2). Diets containing C. calcitrans (C, CM, CP, CT, and IC) and diet IM produced significantly more growth than other diets. Oysters fed diets I, IP, IT, P, T, and TP grew slower than oysters fed other diets whereas oysters fed diets M, MP, and MT showed intermediate growth. Diets containing C. calcitrans (C, CM, CP, CT, and IC) yielded the highest growth rates for length and AFDW

and the highest live weight. C. calcitrans alone yielded the highest growth rates and live weight although not significantly different (P N 0.05) from its bi-algal combinations. Live weight attained with bi-algal diets including C. muelleri (IM, MP, and MT) and C. muelleri alone was intermediate. Oysters grown on I, IP, IT, P, T, and TP had similar final weights all of which were lower than those obtained with other diets. No significant effects of diet on dry weight or AFDW of the oysters were seen.

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Table 6 Fatty acid content of microalgae (μg mg−1 dry weight) used as food for juveniles of C. corteziensis FAME

C. calcitrans

C. muelleri

I. galbana

P. tricornutum

T. suecica

18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3

1.51 ± 0.17 1.16 ± 0.33 8.02 ± 0.11 19.83 ± 1.91 2.02 ± 0.59

0.30 ± 0.01 0.51 ± 0.04 1.77 ± 0.31 26.14 ± 2.09 1.78 ± 0.03

10.37 ± 2.65 10.36 ± 1.34 0 1.32 ± 0.12 32.92 ± 7.31

3.38 ± 1.57 0.92 ± 0.66 1.22 ± 0.29 49.44 ± 15.48 1.92 ± 0.37

9.21 ± 0.35 13.79 ± 0.48 1.54 ± 0.50 7.84 ± 0.24 0

Data averaged for microalgae at the beginning and at the end of the experiment (mean ± s, n = 6). s: standard deviation, n: number of replicates. FAME: fatty acid methyl esters.

3.2. Biochemical composition 3.2.1. Microalgae The data of biochemical composition in Table 3 show carbohydrate, protein, and total lipid content of microalgae expressed as percentage of AFDW. The gross biochemical composition of the microalgae did not appear to be related in any obvious way with their effectiveness as diets for oysters. 3.2.2. Oysters Table 4 shows the biochemical composition from C. corteziensis juveniles at the beginning and at the end of the feeding trial. The percentage of carbohydrate present in the oysters fed on each of the diets varied from 0.43% (C) to 0.21% (CT) of live weight. There were significant differences in carbohydrate content between oysters, although the relation with final live weight was weak. Protein is the main component of the live weight and was 1.51% (I) to 2.37% (CM). The protein content of the oysters fed on each of the 15 different diets was similar and is not related to final live weight. Lipids account for the smallest proportion of the oysters, with values between 0.16% (M) and 0.37% (P) of live weight. The diets assessed showed a nonsignificant effect on total lipid content of oysters. 3.3. Fatty acid profile 3.3.1. Microalgae The relative fatty acid composition of microalgae expressed as a percentage of total fatty acids is given in Table 5. Chaetoceros sp. were characterized by the predominance of 14:0, 16:1n-7, 16:3n-3, and EPA fatty acids, which accounted for 65% in C. calcitrans and 71% in C. muelleri of total fatty acids. These species were similar in both classes and proportions of fatty acids. Their main difference lies in the proportion of ARA that in C. calcitrans accounts for 11% of the total fatty acids but in C. muelleri is just 3% of total fatty acids. C. muelleri was richer in EPA than C. calcitrans.

The I. galbana clone T-iso had a different composition with 14:0, 18:1n-9, 18:2n-6, 18:3n-3, 18:4n-3, and DHA as the major fatty acids, with the DHA the most abundant fatty acid in this microalga and an average value of 27%. P. tricornutum had high proportions of 16:0, 16:1n-7, 16:3n-3, and EPA fatty acids, accounting altogether for 74% of total fatty acids. T. suecica was characterized by the predominance of 16:0, 16:4, 18:2n6, and 18:3n-3 fatty acids and a notably lower amount of highly unsaturated fatty acids (HUFAs) than other species. The n-3/n-6 ratio was considerably higher for P. tricornutum (10.4) and C. muelleri (7.8) than for the other species. The ARA/DHA ratio was low or nonexistent for all algal strains apart from C. calcitrans, which showed a ratio of 4.9. The DHA/EPA ratio for all microalgae assessed except I. galbana was low, b1. The fatty acid content for the major FAMEs in μg mg− 1 of algal dry weight, obtained from the chromatograms, is shown in Table 6. The levels of 18:2n-6 and Table 7 Major FAME (fatty acid methyl ester) contents (as percentage of total fatty acids) of juvenile oysters at the start (initial) and at the end of the trial when fed on uni-algal or bi-algal diets Diet

18:2n-6

18:3n-3

20:4n-6

20:5n-3

22:6n-3

Initial C I M P T IC IM IT IP CM CP CT MP MT TP

1.9 0.5 4.0 0.6 1.1 5.9 2.0 2.5 3.9 2.3 0.7 1.0 2.3 0.5 2.5 2.7

1.0 0.4 1.7 0.2 0.2 5.8 0.8 1.3 2.0 1.6 0.0 0.3 1.1 0.8 1.9 2.4

9.5 12.7a 3.4e 8.2b 2.3e 7.3c 8.4b 5.0d 4.9d 2.7e 11.4a 8.8b 11.7a 4.8d 6.9c 3.8e

7.0 15.3c 3.7f 19.1b 27.9a 14.5c 8.4e 8.6e 4.7f 11.7d 15.8c 19.8b 14.4c 22.6b 17.9b 23.5b

21.6 8.7c 32.7a 15.5b 8.2c 9.0c 22.4a 27.5a 30.1a 26.7a 11.1c 9.2c 9.8c 11.3c 12.6b 10.3c

For diet abbreviations, see Table 1. 20:4n-6 = ARA, 20:5n-3= EPA, 22:6n-3= DHA. Values are means of 3 replicates. Values within the same column sharing a common superscript letter are not different ( P N 0.05).

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18:3n-3 were both higher in the flagellate microalgae tested (I. galbana clone T-iso and T. suecica) than those of diatoms (C. calcitrans, C. muelleri, and P. tricornutum). C. calcitrans is the microalga with the highest content of ARA, 8.02 μg mg− 1 of dry weight, in contrast to I. galbana clone T-iso characterized by a complete lack of this fatty acid. Both C. muelleri, 26.14 μg. mg− 1 dry weight, and P. tricornutum, 49.44 μg mg− 1 dry weight, showed a high content of EPA. I. galbana clone T-iso stands out because of its content of DHA, 15 times higher than that of any other microalgae. The paucity of n-6 fatty acids in this microalga is noteworthy. The absence of DHA in T. suecica samples was its main feature. 3.3.2. Oysters The fatty acid profile of oysters, regardless of diet, were characterized by the predominance of ARA, EPA, and DHA as the major fatty acids although proportions of these fatty acids varied among oysters as a function of the diet on which they were fed (Table 7). The total HUFA content varied from 47% (T) to 84% (I) with EPA and DHA as the most important contributors. The content of n-3 fatty acids in oysters was from 29% (C) to 47% (IP) (Table 8). The ARA/ DHA ratio was low, less than 1 in all cases apart from those diets containing C. calcitrans (C, CM, CP, and CT) and excluding the combination with I. galbana clone T-iso. The DHA/EPA ratio was higher for I. galbana and for the combination diets with this microalga (IC, IM, IT, and IP). Table 8 Total HUFA, total n-3 and n-6 fatty acids, and n-3/n-6, ARA/DHA, DHA/EPA ratios of C. corteziensis juveniles at the end of the feeding trial Diet ∑ HUFA ∑ n-3 ∑ n-6 n-3/ n-6 ARA/DHA DHA/EPA C I M P T IC IM IT IP CM CP CT MP MT TP

47.8 84.0 55.2 51.9 46.5 50.0 51.8 51.8 51.7 49.9 49.0 49.3 51.2 51.2 49.7

28.4 44.3 39.2 46.4 35.2 37.5 42.7 43.1 47.2 32.3 35.0 30.4 42.7 38.1 43.2

19.1 14.5 16.3 7.6 18.8 18.0 14.2 15.9 11.2 19.6 16.1 21.3 10.7 15.2 11.7

1.4 3.0 2.4 6.1 1.9 2.1 3.0 2.7 4.2 1.6 2.2 1.4 4.0 2.5 3.7

1.5 0.1 0.5 0.3 0.8 0.4 0.2 0.2 0.1 1.0 1.0 1.2 0.4 0.5 0.4

0.6 8.9 0.8 0.3 0.6 2.7 3.2 6.4 2.3 0.7 0.5 0.7 0.5 0.7 0.4

Data are expressed as percentage of total fatty acids and are means of 3 replicates.

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Fig. 1. Plot for the multipliers of the first two principal components of PCA analysis of the fatty acid profile of juvenile oysters after 22 days grown on uni-algal and bi-algal diets. For diet abbreviations, see Table 1.

A Principal Component Analysis (PCA) using the covariate matrix was made to explore any differences in the fatty acid profile of oysters as a function of the diet (Fig. 1). Both PC 1 and PC 2 together explained 73% of the differences in the fatty acid profile of the oysters. In PC 1 the main differences came from 18:4n-3, EPA, and DHA fatty acids. In PC 2 the major fatty acids causing differences were 14:0, 18:3n-3, ARA, and EPA. The plot PC 2 versus PC 1 (using their multipliers as the variable) showed a clear pattern (Fig. 1). The circle symbols (corresponding to diets including C. calcitrans) were gathered at the bottom of the plot on the left (highly negative both for PC 1 and PC 2) apart from IC (oyster fed on a mixture of C. calcitrans and I. galbana clone T-iso) around the zero point on PC 1 but still negative on PC 2. Similarly, the triangle symbols (corresponding to diets including I. galbana clone Tiso) were spread across the bottom of the plot on the right (highly positive). Oysters fed exclusively on T. suecica were by themselves at the top right indicating a different fatty acid profile from any of the other oysters. The rest of the sampled oysters, M, MP, MT, P, and TP, did not show a clear pattern and were all clumped together in the upper left region of the plot. Besides the obvious effect of diet on the fatty acid profile, interest resides on specific fatty acids involved in a better growth response of oysters as a function of the diet. Table 7 shows ARA, EPA, and DHA content as percentage of total fatty acids for the oysters. An ANOVA demonstrated a significant effect of diet on ARA, EPA, and DHA content (F = 177.31, P b 0.001; F = 130.64, P b 0.001; F = 115.67, P b 0.001 respectively). There was a significant and highly positive correlation (r = 0.767, P b 0.001) between the content of ARA and the final live weight of oysters, unlike for the EPA and DHA content that showed no significant correlation (r = 0.014, P = 0.929 EPA; and r = − 0.276, P = 0.067 DHA) with final live weight.

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Table 9 Total amount in μg 30 oysters− 1 in dry weight of the main FAMEs given through the diet and incorporated (Inc) and the percentages of incorporation (%) by the oysters over the entire experiment Diet

C I M P T IC IM IT IP CM CP CT MP MT TP

18:2n-6

18:3n-3

20:4n-6

20:5n-3

22:6n-3

Given

Inc

%

Given

Inc

%

Given

Inc

%

Given

Inc

%

Given

Inc

%

741 2844 116 907 2566 1972 1541 2354 1751 427 1071 2073 663 1576 1665

20 113 2 14 70 74 61 52 89 15 30 70 10 34 47

3 4 2 2 3 4 4 2 5 3 3 3 1 2 3

569 2841 196 247 3844 1899 1577 3364 1401 394 465 2877 256 2370 1948

20 46 0 0 74 29 30 26 80 6 8 31 9 27 44

3 2 0 0 2 2 2 1 6 2 2 1 3 1 2

3936 0 679 326 428 1614 322 228 172 2304 2112 1946 548 573 364

698 50 128 0 45 301 87 24 61 371 318 363 147 68 27

18 0 19 0 11 19 27 10 36 16 15 19 27 12 8

9731 363 10009 13268 2186 4203 4998 1331 7210 10859 15138 5585 13554 5996 7558

865 84 383 568 155 316 203 35 465 549 791 473 854 282 452

9 23 4 4 7 8 4 3 6 5 5 8 6 5 6

993 9032 683 514 0 5703 5306 4200 4309 898 876 417 671 321 253

392 885 219 56 0 828 653 359 1017 287 263 229 321 97 94

39 10 32 11 0 15 12 9 24 32 30 55 48 30 37

Data are means (n = 3) with standard deviations omitted for clarity.

For the fatty acid data, the total amount of food given to the oysters over the entire experiment was calculated (data not shown) to account for possible differences in oyster growth because of inability to provide enough of the main FAME suspected to have an important role in bivalve nutrition (18:2n-6, 18:3n-3, ARA, EPA, and DHA). There are large differences between the amount of food provided in each treatment. These are probably related to differences in ingestion, digestion, and absorption rates, which caused oyster live-weight differences between treatments that accumulated over the entire experiment and were reflected in the food ration that was readjusted weekly. The total amount of the major FAMEs (18:2n-6, 18:3n-3, ARA, EPA, and DHA) in μg 30 oysters− 1 dry weight given through the diet were calculated. The incorporation of the FAMEs by the oysters were also calculated from the difference between that quantified from chromatograms and the initial content. It is presented in μg 30 oysters− 1 dry weight and in percentage (Table 9). Accumulation of 18:2n-6 in oyster tissues was proportional to the content of the former fatty acid provided by the diet. In general, the retention of 18:2n-6 by the oysters was between 1% and 5% of the dietary fatty acid. In spite of the high levels of 18:3n-3 provided through the diet, in general little of this fatty acid was incorporated as such by the oysters. There were two diets, M and P, in which no 18:3n-3 was incorporated as part of their tissues. The content of ARA in oysters clearly reflects that provided by the diet. Oysters with a higher content of

ARA were those fed on C. calcitrans as a monospecies or mixed diet with values ranging from 698 μg 30 oysters− 1 (C) to 301 μg 30 oysters− 1 (IC), whereas the ARA content for any of the other treatments did not exceed 146 μg 30 oysters− 1. Oysters fed exclusively on I. galbana clone Tiso produced an interesting result. From Table 9 the amount of ARA provided by the diet was zero, however the content of ARA recorded in oysters fed I. galbana was 50 μg 30 oysters− 1. This fatty acid cannot be synthesized de novo by marine bivalves and the synthesis from the metabolic precursor 18:2n-6 is limited. Other possibilities can be a great accumulation of the small quantity of ARA supplied by diet and a retroconversion from 22:5 n-6 when it is in high concentration in the diet. This has been reported in rotifers (Koven et al., 2001). In Artemia fed diets with DHA a large amount is retroconverted to EPA (Han et al., 2001). The lack of ARA introduced through the diet could be a growth-limiting factor in oysters fed on I. galbana clone T-iso. There is a trend in the EPA incorporation by the oysters in which between 3% and 9% of the EPA offered through the diet was incorporated. This is true for all apart from the oysters fed exclusively on I. galbana clone T-iso in which the incorporation of EPA was up to 23% of that supplied (Table 9). This is concomitant with a failure by these oysters to incorporate its metabolic precursor 18:3n-3, which could suggest as for the ARA fatty acid, that part of the EPA measured in the oysters fed I. galbana clone T-iso can be related to a great retention of the small quantities provided by this diet. The absence of DHA in T. suecica could have been a growth-limiting factor in C. corteziensis juveniles. The

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results in Table 9 showed that oysters fed on a monospecies or mixed diet of T. suecica could not meet their DHA demand completely from their diet. The amount of DHA incorporated exceeded that given by the diet in the T. suecica diet or its combinations CT, MT, and TP, except with the IT diet in which the contribution of I. galbana to the mixed diet is high enough to meet their demand. For ARA and EPA fatty acids, DHA cannot be synthesized de novo by marine bivalves therefore the greater concentrations of DHA in oyster tissues than that provided by the diet can be interpreted as selective retention of DHA and -or evidence of biosynthesis of DHA from its metabolic precursor 18:3n-3 or from EPA. Indeed, the incorporation of 18:3n-3 as such was low for oysters fed on a monospecies or mixed diet of T. suecica. Oysters fed exclusively on I. galbana clone T-iso were those with the highest input of DHA, 9032 μg per 30 oysters, though their incorporation of DHA was similar to that of oysters fed on I.galbana clone T-iso and C. calcitrans (IC) with an input of only 5703 μg per 30 oysters. 4. Discussion 4.1. Oyster growth This study has identified the monospecies diet C. calcitrans as a superior diet for C. corteziensis juveniles versus the other 14 diets tested. The C. calcitrans diet yielded growth rates twice as great as those for some of the poorest diets. The increase in oyster live weight promoted with the C. calcitrans diet was greater than that with any of the other diets, producing a 14% increase relative to the C. calcitrans and C. muelleri mixed diet, the second best diet in terms of live weight increase. In general, the oysters did moderately well when fed on any combination of C. calcitrans and any of the other microalgae tested. The poorest growth was associated with I. galbana, T. suecica, and P. tricornutum microalgae, and their bi-algal combinations. Our results agree with those obtained by Laing and Millican (1986) on O. edulis spat. They reported the best growth (increase in live weight) of oysters fed exclusively on C. calcitrans, whereas oysters fed on T. suecica did poorly. C. calcitrans was considered a complete, or almost complete, food for O. edulis spat because the spat attained their maximum growth with this species and none of the mixtures provided gave significantly better growth. Indeed, Chaetoceros spp. were reported to be the best diets for O. edulis juveniles (Enright et al., 1986) among 16 phytoplankton species tested. The limited oyster growth obtained with P.

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tricornutum in this study, noted by other authors for other species of bivalves (Epifanio et al., 1981; Laing et al., 1987; Albentosa et al., 1996) could be explained because this microalga is difficult to digest. Epifanio et al. (1981) reported it to be indigestible to spat of O. edulis and C. virginica and suggested that the nutritional inadequacy of P. tricornutum is possibly caused by either its indigestibility or its lack of tryptophan. Several studies (Romberger and Epifanio, 1981; O'Connor et al., 1992; Brown et al., 1998) have reported that mixed species diets generally produce greater oyster growth than monospecies diets. Epifanio (1979) uses the term “nonadditive” to define growth supported by combined diets when the growth was not equal to that predicted by summing the growth responses of oysters fed individual components of the diets. Equally, the term “synergistic” was used to define the growth response of oysters fed a mixed diet with that diet promoting a greater growth than that produced by either of the dietary components. In this study, both nonadditive and synergistic growth effects were observed and were in accordance with those described for O. edulis by Laing and Millican (1986). This suggests that the relative food value of the microalgal species tested is mainly caused by whether or not they contain certain essential nutrients and at what concentration these nutrients are present. 4.2. Biochemical composition The significant differences in protein content of oysters did not appear to be related to oyster final live weight in any obvious way. Determining relationships between oyster growth and the gross composition of algal diets is complicated by diet-specific factors such as effects of trace nutrients, digestibility, or limitation of a specific diet component. Gross composition may not always correlate directly with nutritional value (Webb and Chu, 1982) but when other specific essential nutrients (e.g. essential fatty acids, EFAs, sterols, vitamins, minerals) are in adequate proportion the differences may become important. Carbohydrates are reported to be the primary energy source of juvenile oysters (O. edulis; Holland and Hannant, 1974; C. gigas; Knuckey et al., 2002). Therefore carbohydrate content is an important factor not to be overlooked in feeding trials of juvenile oysters given that at this stage of life the energy requirements for growth are high. Dietary carbohydrates balance the use of protein and lipids in bivalve biosynthesis against their catabolism for energy (Whyte et al., 1989). In agreement with our results, protein and lipid content were not significantly related to growth of the hard clam Mercenaria mercenaria (Wikfors et al., 1992).

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Dietary gross-biochemical composition does not explain the high growth performance of oysters fed on C. calcitrans. This species was characterized by the lowest carbohydrate content, moderate content of protein, but with total lipids that did not differ significantly from that of the other microalgae. This suggests that the factor contributing to observed differences in oyster growth with diets might be the presence or absence of other components provided by the diet. 4.3. Fatty acid composition Although some bivalve molluscs have the ability to elongate and desaturate linolenic acid (18:3n-3) to C20 and C22 HUFAs (De Moreno et al., 1976; Waldock and Holland, 1984; Chu and Greaves, 1991), it is not sufficient to support optimum growth. Therefore, these mollusks rely on dietary contributions of these HUFAs (ARA, EPA, and DHA) to fulfill their requirements. The sterol and polyunsaturated fatty acids (PUFA) composition of microalgae used in hatcheries and nurseries significantly influenced the fatty acid and sterol composition of the reared larvae, spat, and broodstock of C. virginica, C. gigas, and P. maximus (Soudant et al., 2000). The microalga I. galbana clone T-iso completely lacked the ARA in its fatty acid composition. Despite this lack, oysters fed exclusively on this microalga had 3.4% of ARA in their tissues, which corresponds to 36% of the ARA present in oyster tissues at the beginning of the experiment. In oysters fed P. tricornutum the percentage of EPA increased from the initial 7% of total fatty acids to 28%. Similarly, oysters fed on T. suecica showed 9% of DHA in their tissues despite the absence of this fatty acid in T. suecica. This amount is 42% of DHA measured in oyster tissues at the beginning of the experiment. The biosynthesis of the long-chain PUFAs ARA, EPA, and DHA from their metabolic precursors 18:2n-6 and 18:3n-3 has not been demonstrated in some studied bivalves as C. gigas (Waldock and Holland, 1984) or Mesoderma mactroides (De Moreno et al., 1976). Our results present an evidence of the selective retention of DHA in oyster tissues fed T. suecica, but for those oysters fed I. galbana, a highly probable retroconversion from 22:5n-6 may largely explain the presence of ARA as reported in rotifers and Artemia (Koven et al., 2001; Han et al., 2001). Our experiment did not have the purpose of studying the essential fatty acid requirements for C. corteziensis juveniles, however the correlation between ARA content and the good performance of C. calcitrans alone or in bi-

algal combinations is suggestive of an important role of this HUFA in the nutrition of these oysters. The nutritional requirements of bivalves for fatty acids vary from species to species. Bivalve nutrition studies have mainly focused on n-3 HUFAs whereas the n-6 PUFAs have received little attention. The importance of n-3 HUFAs, such as EPA and DHA, has been identified in several bivalve species such as C. gigas (Langdon and Waldock, 1981), O. edulis (Enright et al., 1986), and P. maximus (Marty et al., 1992), whereas n-6 PUFAs such as 18:2n-6 are essential for some bivalves (Webb and Chu, 1982; Delaunay et al., 1993). Our work has identified C. calcitrans as a superior diet for C. corteziensis juveniles and suggests that the high nutritional value of the C. calcitrans diet may be related to different things; the high concentration of ARA (see results section fatty acid profile); a higher ingestion, digestion, and absorption; and other components not measured in this study, can also be related. In relation to ARA content, diets with a low input of ARA promoted less growth than diets with a higher input. The low levels of EPA in the flagellate microalgae (I. galbana clone T-iso and T. suecica) associated with the absence (I. galbana clone T-iso) or low levels (T. suecica) of ARA were related to poor performance in this experiment. The requirements of DHA elucidated in this experiment for C. corteziensis juveniles were low. Oysters exclusively fed on C. calcitrans (the best performing diet) had 9% of DHA in their tissues, as opposed to oysters fed I. galbana with 33% of DHA in their tissues. This supports the hypothesis of the existence of a threshold level beyond which more DHA will not improve growth or survival (Thompson and Harrison, 1992; Caers et al., 2000). Thompson et al. (1993) suggested that EPA and DHA fatty acids are required at a low threshold level, less than 2% of total fatty acids for each, for C. gigas larvae. Similarly, McCausland et al. (1999) suggested that this may be true for C. gigas juveniles because in their experiment the requirements for EPA and DHA were met by the seston in the flowing unfiltered sea water providing approximately 2% of fatty acids. A relative decrease in the ARA/DHA ratio between diet and oyster tissues was measured, indicating either selective DHA retention or selective use of ARA in their tissues. The DHA/EPA ratio in oyster tissues showed a large increase relative to that of the diet, indicative of selective DHA incorporation or selective use of EPA in the tissues. These results support previous findings where preferential incorporation of DHA at the expense of EPA has been reported in the polar lipids of P. maximus larvae (Soudant et al., 1998) and in postlarval

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sea scallop P. magellanicus (Milke et al., 2004). Decreasing levels of ARA could be associated with eicosanoid production because ARA is converted into other bioactive compounds (Delaunay et al., 1993). DHA is an essential structural component of cell membranes assimilated specifically in some phospholipid classes (Soudant et al., 2000), which may therefore explain the preferential retention of DHA. The EPA is considered to be an energetic rather than a structural component during embryogenesis and larval growth in P. maximus (Marty et al., 1992). The relative decrease in the n-3/n-6 ratio of fatty acids between diets and tissues of C. corteziensis juveniles measured in the experiment suggests that n-6 fatty acids play an important role in this organism. Evidence of a requirement for n-6 fatty acids has also been described for P. maximum larvae (Marty et al., 1992) and for postlarval sea scallop P. magellanicus (Milke et al., 2004). Therefore, we can conclude that the C. calcitrans is the best diet for C. corteziensis juveniles and its effects are probably related to ARA levels, however we cannot to ensure this to be true because other characteristics of the diets; ingestion, digestion, assimilation, and other biochemical components, were not measured. This still remains unclear and further research is needed. 5. Conclusion The high production costs of microalgae remain a constraint to many bivalve hatcheries. Identification of a cost-effective algal diet that maximizes survival and growth of the cultured species is a top priority for aquaculturists. A general practice within the bivalve aquaculture sector is the use of algal diets consisting of more than one species. These are believed to provide a better nutritional balance of essential nutrients than monoalgal diets and consequently a better growth response. However, in this study C. calcitrans on its own was identified as the best diet for C. corteziensis juveniles yielding oyster growth rates superior to any other monospecies or mixed algal diet tested. This finding could prove beneficial to the developing hatchery industry of C. corteziensis on the Pacific coast of Mexico providing a cheaper and better performing alternative to the standard diet used so far, a combination of C. calcitrans and I. galbana clone T-iso. The use of a single algal species instead of two could favor reduced costs of labor and space and more effort could be diverted to produce algae of a higher quality under specific culture conditions. The fatty acid content and its proportion in the biomass are controlled by growth rate of the culture with

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the fatty acid profile being richer in HUFAs at higher growth rates (Molina-Grima et al., 1999). A semicontinuous culture of C. calcitrans with high renewal rates to ensure high HUFA concentrations appears to be a good and more economical alternative to a mixed diet composed of C. calcitrans and I. galbana clone T-iso for the rearing of mangrove oyster juveniles. Acknowledgements We thank Minerva Cerro for the algal inocula, Armando Monge and Pablo Monsalvo for their technical assistance at different stages of the study, and Jose Luis Ramirez who provided the oyster juveniles and contributed useful ideas for the experimental design. We would like to extend sincere thanks to Laura Carreón and Jorge del Angel for their valuable guidance on fatty acid data analysis. Thanks to Dr. Bertha Arredondo who supported part of the fatty acid analysis. Thanks also to Dr. Ellis Glazier for editing this English-language text. This study has been supported by SAGARPA 2003-02-035 project, DESARROLLO DE UNA BIOTECNOLOGIA ALTERNA PARA LA PRODUCCION DE OSTION C. corteziensis: DOMESTICACION Y CONFORMACION DE PIE DE CRIA, PRODUCCION DE TRIPLOIDES Y TETRAPLOIDES, the Responsible Reseacher is Dr. Ana Ibarra, and the European Social Funding (Postgraduate Fellowship to Susana Rivero Rodríguez). References Albentosa, M., Pérez-Camacho, A., Labarta, V., Fernández-Reiriz, M.J., 1996. Evaluation of live microalgal diets for the seed culture of Ruditapes decussatus using physiological and biochemical parameters. Aquaculture 148, 11–23. Bligh, E.C., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Brown, M.R., Garland, C.D., Jeffrey, S.W., Jameson, I.D., LeRoi, J.M., 1993. The gross and amino acid compositions of batch and semicontinuous cultures of Isochrysis (clone T-Iso), Pavlova lutheri and Nannochloropsis oculata. J. Appl. Phycol. 5, 285–296. Brown, M.R., McCausland, M.A., Kowalski, K., 1998. The nutritional value of four Australian microalgal strains fed to Pacific oyster Crassostrea gigas spat. Aquaculture 165, 281–293. Caers, M., Coutteau, P., Sorgeloos, P., 2000. Incorporation of different fatty acids, supplied as emulsions or liposomes, in the polar and neutral lipids of Crassostrea gigas spat. Aquaculture 186, 157–171. Chávez-Villalba, J., 2004. Estudios sobre el ostión Crassostrea corteziensis en Sonora, México: aprovechamiento del recurso. Memorias del I Congreso Regional de Ciencias Ambientales. Cd. Obregon, Sonora, México. Chu, F.L.E., Greaves, J., 1991. Metabolism of palmitic, linoleic, and linolenic acids in adult oysters, Crassostrea virginica. Mar. Biol. 110, 229–236. Coutteau, P., Sorgeloos, P., 1995. Algae replacement/supplementation in intensive rearing of bivalve mollusks. In: Calderón, J., Sorgeloos, P.

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