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Dynamics of fatty acids in the larval development, metamorphosis and post-metamorphosis of Ostrea edulis (L.)
Comparative Biochemistry and Physiology Part A 123 (1999) 249 – 254 www.elsevier.com/locate/cbpa
Dynamics of fatty acids in the larval development, metamorphosis and post-metamorphosis of Ostrea edulis (L.) U. Labarta a,*, M.J. Ferna´ndez-Reiriz a, A. Pe´rez-Camacho b a
CSIC, Instituto de In6estigaciones Marinas, c/Eduardo Cabello, 6.36208 Vigo, Spain b Instituto Espan˜ol de Oceanografı´a, La Corun˜a, Spain
Received 21 April 1998; received in revised form 26 March 1999; accepted 30 March 1999
Abstract We analysed the composition of fatty acids during the larval development of Ostrea edulis in view of the role played by essential fatty acids (i.e. 20:5n–3 and 22:6n–3) during the three most important stages in the development, with particular emphasis on metamorphosis, of this mollusc. In the first stage (larval period) all fatty acids increase. During metamorphosis (50% eyed, 72 h) there is still an increase but smaller than during the larval period, for all major fatty acids with the exception of polienoic, n–3PUFAs (polyunsaturated fatty acid) and C20 and C22 non-methylene-interrupted dienoic (NMID). The juvenile shows the highest gain for all the main fatty acids. Changes in the PUFAs composition were marked by an increase in the 22:6n – 3/20:5n–3 ratio during the period of larval development, followed by a decrease in this ratio until 6 days after the onset of metamorphosis, and finally a further increase during the post-metamorphosis stage. The losses in 22:6n – 3 for the period between the end of metamorphosis and 6 days after its onset would seem to indicate a specific function for each of these two fatty acids, with a structural role for 22:6n–3 and an energy-providing role for 20:5n – 3. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Fatty acids; Ostrea edulis; Larvae; Metamorphosis; Post-metamorphosis
1. Introduction Fatty acids are the fundamental structural components of practically all forms of lipids. The study of fatty acids of Ostrea edulis has been focused principally on juveniles and adults and therefore very little exists in the literature on other stages of development. The studies carried out on larvae of O. edulis refer to fatty acids in 1- and 10-day-old larvae, and to the fatty acid composition of early non-feeding larvae [11,17]. This last work [11] establishes the preferential utilization of the polyunsaturated fatty acids (PUFAs) of the neutral lipids, and at the same time of the phospholipid PUFAs. It is also indicated that the larvae conserve certain phospholipid PUFAs such as 20:4n – 6 and * Corresponding author. Tel.: +34-986-231930; fax: + 34-86292762. E-mail address: [email protected] (U. Labarta)
22:6n–3, but nevertheless use 20:5n–3 in the fractioning of both polar lipids and neutral lipids. The importance of lipids in larval development and metamorphosis of O. edulis [12,14] could indicate an important physiological role of fatty acids, and specifically of PUFAs, in the said developmental stages, as has been observed in the embryogenetic processes of other molluscs [6,18], and in larvae and adults of O. edulis [17], where the higher degree of unsaturation in the 10-day-old and adult compared to the 1-day-old larvae was due exclusively to the proportion of 22:6n–3 and 20:5n–3, respectively, and not to a generalized increase in unsaturation. In this context and in that of the essentiality established for the PUFAS and the nutritional requirements from the diet, this work studies the fatty acid dynamics of total lipids in larval and postlarval development of O. edulis. Special attention has been paid to the metamorphosis process, analysing the role played by some fatty acids which can be essential in this process.
1095-6433/99/$ - see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 ( 9 9 ) 0 0 0 5 4 - 9
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2. Material and methods
2.1. Lar6al culture The larvae of O. edulis (L.), immediately after release from the paleal cavity of the mother oysters, were cultured in two replicate 150-l tanks, in 1-mm filtered and UV sterilised sea water. Culturing took place at 16°C, 4°C lower than the optimal temperature for larval development [3] in order to slow down the consumption of reserves by reducing the metabolic rate and thus allow detailed observation of the differential evolution, if any, of the different components during the various stages studied. Culturing was carried out over a 27-day period. The water was renewed totally every 2 days and food was added at a concentration of 50 cells/ml of Isochrysis galbana and Tetraselmis suecica. The algal cells were counted with a Coulter Counter TA II. The morphological development of the larvae and recently settled seed was inspected every 2 days under a microscope. Samples were taken for lipid analysis in two stages, the first when the larvae were expelled from the paleal cavity until they settled, and the second at 10 days subsequent to settling. During the first stage samples were taken on days 1, 7, 11 and 17. On day 17 the eyespot was present in more than 50% of the larvae, which indicated that they were ready for settling, and with this in mind PVC collecting plates were placed in the culture tanks, and renewed every 12 h. The first major peak of settling was observed 1 day (24 h) after the collecting plates were first placed in the culture vessels. The collecting plates were therefore removed and placed in another identical tank, at the same temperature and with the same diet as in the larval period. Samples for biochemical analysis were taken during this second stage at 24 and 72 h, and at 6 and 10 days, after settling. The sample size taken for determining weight and for lipid analysis varied, according to the weight of the larvae and postlarvae, between 100 000 larvae for the first sample and 1000 postlarvae in the final sample. The samples were freeze-dried in a Telstar freeze-drier, and stored at −30°C until analysis. Dry weights per individual were taken after the larvae had been rinsed in distilled water and stove-dried at 110°C for 3 h. Weight was measured on a Sartorius M3P electronic microscale.
2.2. Fatty acids 2.2.1. Analytical methods Lipids were first extracted with chloroform:methanol (1:2) and after centrifugation at 3246× g, re-extracted
from the precipitate with chloroform:methanol (2:1). Both supernatants were then washed with chloroform:methanol:water (8:4:3) as described previously [9]. The solvents contain 0.05% butylated hydroxytoluene. Total lipids were determined gravimetrically by evaporating 200 ml of lipidic extract on preweighed aluminium plates on a slide warmer (60–80°C). Storage until further processing was carried out under nitrogen at −70°C. Fatty acids from total lipids were transesterified to methyl esters with methanolic hydrogen chloride as previously described [4] and subsequently analysed on a gas chromatograph (Perkin-Elmer, 8500), equipped with a fused silica capillary column (Supelco, SP-2330; 30 m length, 0.25 mm i.d.), PTV cold injector (Perkin-Elmer) operated in the solvent elimination mode. The injector temperature was 275°C and the column temperature increased from 140 to 210°C at a rate of 1.0°C/min. Nitrogen was used as the carrier gas at 10 psi. Nonadecanoic acid was used as an internal standard and a response factor was calculated for each fatty acid in order to perform quantitative analyses. Since there is no absolute method for identifying fatty acids by GLC, a series of tentative identification procedures was applied to the lipid mixtures studied. This approach included co-injecting the sample along with standard mixtures of established composition and GC-MS (Hewlett-Packard 5971 Mass Detector). All solvents, reagents and fatty acid standards used in this work were of analytic grade (E. Merck, Darmstadt and Sigma). Differences between means were analysed by oneway analysis of variance (ANOVA) followed by Tukey’s multiple comparison [19].
3. Results
3.1. Fatty acid composition of the diet (I. galbana + T. suecica) The main fatty acids found in the diet (composed of I. galbana + T. suecica) are 16:0; 18:0; 18:1n–9; 18:4n– 3; 18:3n–3; 14:0; 20:5n–3 and 22:6n–3. Other fatty acids identified are only present in very small amounts. The main group of fatty acids are saturated fatty acids (60.2 mg/mg dry weight), polyunsaturated fatty acids (54.6 mg/mg dry weight) and monounsaturated fatty acids (28.6 mg/mg dry weight). The n–3PUFAs content is 9.5 mg/mg dry weight. The ratio n–3/n–6 is 2.9, whilst the ratio n–6/n–3 is 0.5. The ratio 22:6n–3/ 20:5n–3 is 0.7.
U. Labarta et al. / Comparati6e Biochemistry and Physiology, Part A 123 (1999) 249–254
3.2. Fatty acid composition of O. edulis Table 1 shows the composition of the fatty acids of the larval and postlarval cultures of O. edulis (the data are given in mg/mg dry weight). Total fatty acid content rises significantly (P B 0.05) from the start of the culture period until 24 h after onset of metamorphosis, when the significantly greatest (P B 0.05) fatty acid content is reached (39.1 mg/mg dry weight). From this point, the content decreases signifi-
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cantly (PB 0.05) until the end of the culture period. The behaviour described above also applies to saturated, monounsaturated and polyunsaturated fatty acids (including n–6, n–9 and n–3PUFAs), except for the content monounsaturated fatty acid at the end of the culture period which is not significantly different (P\ 0.05) from that of the larvae. In the case of n–6 fatty acid, there are no significant differences between initial larvae and 7-day-old larvae and larvae 72 h after the onset of metamorphosis (P\ 0.05).
Table 1 Fatty acid composition (mg/mg dry weight) of O. edulis †
14:0 15:0 16:0 16:1n–9 16:1n–7 17:0 17:1n–9 18:0 18:1n–9 18:1n–7 18:2n–6 18:3n–4 18:3n–3 18:3n–6 18:4n–3 20:0 20:1n–11 20:1n–9 20:1n–7 20:2NMID1 20:2NMID2 20:2n–6 20:3n–6 20:4n–6 20:5n–3 22:2NMID1 22:2NMID2 22:4n–6 22:5n–3 22:6n–3 S Saturated S Monoenoic S Polyenoic S Total FA S n–6 S n–9 S n–3PUFAs S NMID n–3/n–6 22:6n–3/20:5n–3 NMID/PUFA Lipids‡
Initial
7 days
11 days
50% eyed
24 h
72 h
6 days
10 days
0.4 90.0 0.2 9 0.0 2.7 90.2 0.2 90.0 0.4 90.0 0.3 9 0.0 0.4 9 0.0 2.2 90.2 0.7 90.0 0.5 90.0 0.4 90.0 0.2 90.0 0.2 90.0 0.2 90.0 0.2 90.0 0.1 9 0.0 0.3 90.0 0.1 90.0 0.6 9 0.0 0.3 90.0 0.9 90.0 0.3 90.0 0.4 9 0.0 0.5 90.0 1.6 9 0.0 0.2 9 0.0 0.6 90.0 0.2 90.0 0.1 90.0 1.4 90.2 5.9 9 0.4a 3.2 90.0a 7.7 9 0.2a 16.8 9 0.6a 2.0 9 0.0a 1.4 9 0.0a 3.3 9 0.2a 2.0 9 0.0 1.7 9 0.1 0.9 90.1 0.6 90.0 4.5 9 0.1a
0.59 0.1 0.1 90.0 3.5 9 0.3 0.6 9 0.0 0.2 9 0.0 0.390.0 0.39 0.5 2.490.1 2.7 9 0.9 0.790.1 0.79 0.1 0.2 9 0.0 1.3. 90.4 0.290.0 2.0 9 0.9 0.1 90.0 0.290.1 0.3 9 0.1 0.5 90.1 0.290.1 1.3 9 0.3 0.19 0.1 0.4 90.1 0.39 0.0 1.59 0.5 0.2 90.1 0.490.0 0.59 0.3 0.090.0 2.0 9 0.4 6.99 0.5b 5.59 1.8b 11.393.3b 23.795.6b 2.290.6a 3.991.5b 4.59 1.8b 2.19 0.5 3.1 91.8 1.39 0.7 0.5 9 0.3 5.89 0.3b
0.890.2 0.29 0.1 5.3 90.9 0.5 90.0 0.39 0.1 0.39 0.0 0.5 90.3 3.29 0.4 3.991.5 1.09 0.3 1.090.3 0.3 90.1 1.99 0.7 0.39 0.0 3.191.4 0.29 0.0 0.39 0.1 0.490.1 0.79 0.1 0.39 0.0 1.4 90.2 0.1 9 0.2 0.69 0.1 0.59 0.0 1.790.3 0.39 0.0 0.59 0.0 0.790.1 0.19 0.0 2.690.2 10.091.6c 7.69 2.5c 15.495.1c 33.099.2c 3.29 0.7b 5.39 1.9c 7.59 1.9c 2.590.2 2.99 1.5 1.590.4 0.39 0.1 7.79 0.1c
0.7 9 0.2 0.2 90.1 5.5 9 1.5 0.5 90.0 0.4 9 0.1 0.4 9 0.0 1.2 91.4 3.4 90.4 5.1 90.8 1.2 90.1 1.0 90.0 0.3 90.0 2.1 90.1 0.5 90.3 3.8 90.1 0.1 9 0.1 0.3 90.0 0.4 90.0 0.9 9 0.1 0.4 90.2 0.4 9 0.3 0.3 9 0.1 0.5 90.1 0.6 90.2 2.1 90.6 0.4 9 0.1 0.6 90.2 0.8 901 0.2 90.0 3.6 90.9 10.3 9 2.3d 10.0 9 2.5d 17.6 9 3.3d 37.9 9 8.1d 3.7 9 0.8c 7.2 92.2d 9.7 91.6d 1.8 90.9 3.2 9 1.1 1.7 90.9 0.2 90.1 8.0 90.1c
0.7 9 0.0 0.3 90.0 4.6 9 1.0 0.5 90.1 0.4 9 0.0 0.5 9 0.2 1.4 90.8 3.3 9 0.7 5.9 90.8 1.2 90.3 1.2 90.4 0.4 90.0 2.3 90.6 0.2 9 0.2 4.1 90.2 0.0 9 0.0 0.3 9 0.2 0.6 90.1 1.0 90.2 0.3 9 0.0 0.2 9 0.0 0.2 9 0.1 0.5 90.0 0.6 90.1 2.4 90.6 0.4 9 0.1 0.7 90.2 0.9 90.1 0.2 9 0.0 3.8 90.4 9.49 1.9d 11.3 9 2.5d 18.4 9 3.0d 39.1 9 7.4d 3.6 90.9c 8.4 91.8d 10.5 9 1.2d 1.6 9 0.3 3.6 9 1.4 1.6 90.6 0.2 9 0.0 5.7 90.1b
0.5 9 0.0 0.29 0.0 3.4 9 0.2 0.4 9 0.0 0.2 9 0.0 0.2 9 0.0 0.2 9 0.3 2.3 9 0.0 3.9 9 1.0 0.7 9 0.1 0.7 9 0.1 0.3 9 0.0 1.3 9 0.5 0.2 9 0.0 2.1 9 0.5 0.0 9 0.0 0.2 9 0.0 0.4 9 0.1 0.69 0.1 0.2 9 0.0 0.1 9 0.1 0.1 9 0.0 0.59 0.1 0.3 9 0.1 1.3 9 0.4 0.2 9 0.1 0.3 9 0.1 0.4 9 0.1 0.1 9 0.1 1.6 9 0.6 6.6 90.2e 6.6 91.6e 9.7 92.8e 22.9 9 4.6e 2.2 90.4a 4.9 91.4e 5.1 91.6e 0.8 90.3 2.9 9 1.5 1.2 90.8 0.2 9 0.1 3.4 90.0d
0.3 9 0.1 0.1 9 0.0 2.1 9 0.4 0.2 9 0.0 0.2 9 0.0 0.2 9 0.0 0.0 9 0.1 1.3 9 0.4 2.0 9 0.5 0.4 9 0.1 0.3 9 0.1 0.2 9 0.0 0.6 9 0.1 0.1 9 0.0 1.0 9 0.3 0.0 9 0.0 0.1 9 0.0 0.2 9 0.0 0.3 9 0.0 0.0 9 0.0 0.1 9 0.0 0.1 9 0.0 0.3 9 0.1 0.2 9 0.1 0.5 9 0.3 0.1 9 0.0 0.2 9 0.0 0.2 9 0.1 0.0 9 0.0 0.5 9 0.3 4.0 90.9f 3.4 90.7a 4.4 91.4f 11.8 9 3.0f 1.2 90.4d 2.4 90.6f 2.0 9 0.9f 0.4 9 0.0 2.1 9 1.5 1.0 91.2 0.2 9 0.1 2.4 90.1e
0.2 90.1 0.1 90.0 1.7 90.2 0.2 90.0 0.2 90.0 0.1 90.0 0.1 90.1 1.1 90.2 2.0 90.3 0.3 90.0 0.5 90.1 0.2 90.0 0.5 90.1 0.1 90.1 1.0 90.2 0.0 90.0 0.1 90.0 0.2 90.0 0.2 90.0 0.0 90.0 0.1 90.0 0.2 90.2 0.3 90.0 0.2 90.1 0.5 90.1 0.1 90.0 0.1 90.0 0.2 90.0 0.1 90.0 0.8 90.2 3.2 90.5f 3.3 9 0.4a 4.9 9 1.1f 11.4 9 2.0f 1.5 90.5d 2.5 90.4f 2.4 90.5f 0.3 9 0.0 1.9 9 1.0 1.6 9 0.7 0.1 90.0 2.1 9 0.0e
P-value
* * * * * * *
***
FA, total fatty acid; NMID, non-methylene interrupted dienoic fatty acid; PUFA, polyunsaturated fatty acid. Mean 9S.D. of two group replicates. Mean values within the same rows with different superscript letters are significantly different (PB0.05). ‡ % of dry weight. * PB0.05; *** PB0.001. †
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At the beginning of the experiment the main fatty acids found in oyster larvae are 16:0; 18:0; 20:5n –3 and 22:6n–3. Other fatty acids are only present in small amounts. Although variations in the amounts of the different fatty acids identified were observed during the development of O. edulis, palmitic acid (16:0) was consistently the main fatty acid. During the first 7 days of growth 18:1n–9; 18:3n – 3; 18:4n – 3 and 20:2NMID2 (NMID, non-methylene-interrupted dienoic) appear in large amounts. These fatty acids appear in large amounts until the end of metamorphosis, after which point there is a significant decrease (P B 0.05) in amounts of these main fatty acids which is maintained until the end of the experimental period. Nevertheless it should be noted that at 10 days after onset of metamorphosis, the decrease in the main fatty acids has ceased, while 22:6n–3 and 18:2n – 6 show a significant recovery (P B 0.05). The rates (gains or losses), in ng/ind./day, of each of the main fatty acids can be seen in Table 2. The changes for the three main stages of the development period were also analysed. During larval development, the rate increased significantly (PB 0.05) until the onset of metamorphosis, and this increase continued until 24 h after this process had begun. However, negative values appeared for all the fatty acids analysed for the period starting 24 h after the onset of metamorphosis until the end of this process (72 h). From the end of metamorphosis to the beginning of postlarval stage (6 days after the onset of metamorphosis), the main fatty acids showed a positive rate but the 22:6n–3 rate remained negative. The final period of the experiment, from 6 to 10 days after the onset of metamorphosis, showed an increase for all main fatty acids.
4. Discussion The PUFAs 20:5n – 3 and 22:6n – 3 are important lipid components in larval mollusc development [16], it having been shown that levels of 20:5n – 3 and 22:6n–3 contribute significantly to growth of O. edulis [8]. The composition of fatty acids in the diet influences the composition of bivalves fed on this diet [2,7,21]. The chosen diet contains a balanced composition of fatty acids [9] according to the needs defined as essential in bivalves, for example the presence of n – 3PUFAs (i.e. 20:5n–3 and 22:6n– 3) and a n – 6/n – 3 ratio of around 0.5. Many bivalves lack the ability to produce PUFAs by chain elongating and/or desaturating dietary precursors [5]. However in other bivalve species it has been established that they can synthesize PUFAs (C20 – C22) from the shorter chain (C18) fatty acids in the diet [15,20]. The need for certain fatty acids seems to be species
dependent. Tapes semidecussata and Mercenaria mercenaria need 22:6n–3 while Crassostrea sp. shows a fundamental need for 20:5n–3 [10]. Both 20:5n–3 and/or 22:6n–3 can alternatively meet the requirements of bivalves for n–3PUFA [16]. Whether supplied by the diet or synthesized by the bivalve it is clear that PUFAs are necessary. The function of these fatty acids could be as an energy source or as precursors to produce eicosanoids or prostaglandins [1]. During embryonic development of P. yessoensis, a linear decrease is observed in the content of the n– 3PUFAs, with the exception of 22:6n–3 [22]. The constant level of 22:6n–3 throughout embryogenesis indicates a major structural function as opposed to the energy-providing function of 20:5n–3. In the transition of larvae to juveniles of Crassodoma gigantea there was an increase in 22:6n–3 [23] a fatty acid that is usually associated with membrane structures [16]. In Pecten maximus [6] it was established that although poor results in the settlement of postlarvae may be due to other factors apart from the PUFAs, this effect is nevertheless directly associated with a low level of 22:6n–3 in the neutral lipids of pediveliger larvae. In the present work it can be seen that 22:6n–3 (a fatty acid deemed to be essential and which follows the same pathway of formation as 20:5n–3 from 18:3n–3) behaves differently from 20:5n–3. Its behaviour should not only be attributed to the amount present in the diet (the ratio 22:6n–3/20:5n–3 is 0.7), but rather may be explained by the facts expounded [6,23], which assign a separate role to 22:6n–3 during two stages of the development: settlement and metamorphosis of bivalve. On this basis, our results may also allow a similar interpretation of the structural role of 22:6n–3 and energy-providing role of 20:5n–3, in the metamorphosis and postlarval development of O. edulis like that proposed by the aforementioned authors in the embryogenesis and settlement at postlarvae stage in the other bivalves [6,22,23]. With regard to the C20 and C22 non-methylene-interrupted dienoic (NMID) fatty acids it is known that these fatty acids are preferably found in the polar lipids of O. edulis and other molluscs [13]. It seems probable that these are membrane lipids structural components rather than the basis of eicosanoids, the role ascribed below to certain polyunsaturated fatty acids [1]. Napolitano et al. [17], working with larvae of O. edulis, indicate that the origin of the NMIDs is endogenous (from monoenoic fatty acids), and these fatty acids, which are the precursors of the NMIDs, are present in significant amounts in oysters. In the present study it can be seen that the NMIDs, specifically 20:2NMID, can be found in similar amounts to those of some PUFAs in the larval development stage, but start to lose their relative importance from the onset of metamorphosis, and are only present during the postlarval stage in residual quantities.
16:0 18:0 18:2n–6 18:3n–3 18:4n–3 20:5n–3 22:6n–3 S Saturated S Monoenoic S Polienoic S n–3 PUFA S NMID S Total FA a
Initial 7 days
7–11 days
11 days, 50% eyed
50% eyed, 24 h
24–72 h
72 h–6 days
6–10 days
Initial, 50% eyed
50%,72 h
72 h–10 days
0.6 0.4 0.1 0.3 0.5 0.2 0.4 1.1 1.0 2.1 1.1 0.1 4.3
2.9 1.6 0.6 1.0 1.8 0.8 1.3 5.3 4.1 8.1 4.0 0.4 17.6
2.6 1.7 0.4 1.0 2.1 1.1 2.2 4.8 5.9 9.0 5.5 0.6 19.7
1.5 4.3 2.9 5.2 8.6 5.4 7.1 9.6 25.2 34.6 22.1 2.5 69.4
0.3 1.1 1.0 2.2 5.4 2.5 6.6 2.5 9.2 21.2 14.7 1.5 33.0
4.1 2.5 0.5 0.4 1.2 0.3 0.2 7.8 4.5 5.0 1.3 0.3 17.3
12.9 7.6 5.0 4.1 9.2 4.8 8.7 23.5 27.8 46.7 23.2 2.1 98.0
1.8 1.1 0.3 0.7 1.3 0.7 1.2 3.4 3.5 6.0 3.3 0.3 12.9
0.3 0.7 0.3 0.2 0.7 0.2 2.0 1.5 2.3 2.6 2.4 0.1 1.2
7.6 4.6 2.3 1.9 4.4 2.1 3.3 14.1 13.8 21.7 10.1 1.0 49.6
The increase represent the difference compare to the preceding stage. The values in bold are negative. See Table 1 for abbreviations.
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Table 2 Increases of the main fatty acids (ng/ind. per day) of O. edulis a
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U. Labarta et al. / Comparati6e Biochemistry and Physiology, Part A 123 (1999) 249–254
Acknowledgements We would like to thank J.L. Garrido, A. Ayala, L. Nieto and B. Gonza´lez for biochemical analyses and C. Ferna´ndez-Pena for helpful technical assistance in the algal and larvae cultures. This work was funded by CICYT-CSIC.IEO project I+ D number MAR900821-CO2–2.
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1987;62:281 – 8. [11] Helm MM, Holland DL, Utting SD, East J. Fatty acid composition of early non-feeding larvae of the European flat oyster, Ostrea edulis. J Mar Biol Assoc UK 1991;71:691 – 705. [12] Holland DL, Spencer BE. Biochemical changes in fed and starved oysters, Ostrea edulis L. during larval development, metamorphosis and early spat growth. J Mar Biol Ass UK 1973;53:287 – 98. [13] Irazu CE, Pollero RJ, Brenner RR. Occurrence of a 22:2 nonmethylene interrupted dienoic fatty acid and its seasonal distribution among lipids and tissues of the fresh water bivalve Diplodon delodontus from an isolated environment. Lipids 1984;19:649 – 55. [14] Labarta U, Ferna´ndez-Reiriz MJ, Perez-Camacho A. Energy, biochemical substrates and growth in the larval development, metamorphosis and postlarvae of Ostrea edulis (L.). J Exp Mar Biol Ecol 1999 238:227 – 44. [15] Laing I, Child AR, Janke A. Nutritional value of dried algae diets for larvae of Manila clam (Tapes philippinarum). J Mar Biol Assoc UK 1990;70:1 – 12. [16] Langdon CJ, Waldock MJ. The effect of algal and artificial diets on the growth and fatty acid composition of Crassostrea gigas spat. J Mar Biol Assoc UK 1981;61:431 – 48. [17] Napolitano GE, Ratnayake WMN, Ackman RG. Fatty acid components of larval Ostrea edulis (L.): importance of triacylglycerols as a fatty acid reserve. Comp Biochem Physiol 1988;90B:875 – 83. [18] Paradis M, Ackman RG. Potential for employing the distribution of anomalous non-methylene-interrupted dienoic fatty acids in several marine invertebrates as part of food web studies. Lipids 1977;12:170 – 6. [19] Sokal RR, Rohlf FJ. Biometry. New York: Freeman, 1969. [20] Waldock MJ, Holland DL. Fatty acid metabolism in young oyster, Crassostrea gigas: polyunsaturated fatty acids. Lipids 1984;19:332 – 6. [21] Watanabe T, Ackman RG. Lipids and fatty acids of the American (Crassostra 6irginica) and European flat (Ostrea edulis) oyster from a common habitat, and after one feeding with Dicrateria inornata or Isochrysis galbana. J Fish Res Board Can 1974;31:403 – 9. [22] Whyte JNC, Bourne N, Ginther NG. Biochemical and energy changes during embryogenesis in the rock scallop Crassadoma gigantea. Mar Biol 1990;106:239 – 44. [23] Whyte JNC, Bourne N, Ginther NG, Hodgson CA. Compositional changes in the larvae to juvenile development of the scallop Crassadoma gigantea. J Exp Mar Biol Ecol 1992;163:13– 29.
Dynamics of fatty acids in the larval development, metamorphosis and post-metamorphosis of Ostrea edulis (L.) U. Labarta a,*, M.J. Ferna´ndez-Reiriz a, A. Pe´rez-Camacho b a
CSIC, Instituto de In6estigaciones Marinas, c/Eduardo Cabello, 6.36208 Vigo, Spain b Instituto Espan˜ol de Oceanografı´a, La Corun˜a, Spain
Received 21 April 1998; received in revised form 26 March 1999; accepted 30 March 1999
Abstract We analysed the composition of fatty acids during the larval development of Ostrea edulis in view of the role played by essential fatty acids (i.e. 20:5n–3 and 22:6n–3) during the three most important stages in the development, with particular emphasis on metamorphosis, of this mollusc. In the first stage (larval period) all fatty acids increase. During metamorphosis (50% eyed, 72 h) there is still an increase but smaller than during the larval period, for all major fatty acids with the exception of polienoic, n–3PUFAs (polyunsaturated fatty acid) and C20 and C22 non-methylene-interrupted dienoic (NMID). The juvenile shows the highest gain for all the main fatty acids. Changes in the PUFAs composition were marked by an increase in the 22:6n – 3/20:5n–3 ratio during the period of larval development, followed by a decrease in this ratio until 6 days after the onset of metamorphosis, and finally a further increase during the post-metamorphosis stage. The losses in 22:6n – 3 for the period between the end of metamorphosis and 6 days after its onset would seem to indicate a specific function for each of these two fatty acids, with a structural role for 22:6n–3 and an energy-providing role for 20:5n – 3. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Fatty acids; Ostrea edulis; Larvae; Metamorphosis; Post-metamorphosis
1. Introduction Fatty acids are the fundamental structural components of practically all forms of lipids. The study of fatty acids of Ostrea edulis has been focused principally on juveniles and adults and therefore very little exists in the literature on other stages of development. The studies carried out on larvae of O. edulis refer to fatty acids in 1- and 10-day-old larvae, and to the fatty acid composition of early non-feeding larvae [11,17]. This last work [11] establishes the preferential utilization of the polyunsaturated fatty acids (PUFAs) of the neutral lipids, and at the same time of the phospholipid PUFAs. It is also indicated that the larvae conserve certain phospholipid PUFAs such as 20:4n – 6 and * Corresponding author. Tel.: +34-986-231930; fax: + 34-86292762. E-mail address: [email protected] (U. Labarta)
22:6n–3, but nevertheless use 20:5n–3 in the fractioning of both polar lipids and neutral lipids. The importance of lipids in larval development and metamorphosis of O. edulis [12,14] could indicate an important physiological role of fatty acids, and specifically of PUFAs, in the said developmental stages, as has been observed in the embryogenetic processes of other molluscs [6,18], and in larvae and adults of O. edulis [17], where the higher degree of unsaturation in the 10-day-old and adult compared to the 1-day-old larvae was due exclusively to the proportion of 22:6n–3 and 20:5n–3, respectively, and not to a generalized increase in unsaturation. In this context and in that of the essentiality established for the PUFAS and the nutritional requirements from the diet, this work studies the fatty acid dynamics of total lipids in larval and postlarval development of O. edulis. Special attention has been paid to the metamorphosis process, analysing the role played by some fatty acids which can be essential in this process.
1095-6433/99/$ - see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 ( 9 9 ) 0 0 0 5 4 - 9
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2. Material and methods
2.1. Lar6al culture The larvae of O. edulis (L.), immediately after release from the paleal cavity of the mother oysters, were cultured in two replicate 150-l tanks, in 1-mm filtered and UV sterilised sea water. Culturing took place at 16°C, 4°C lower than the optimal temperature for larval development [3] in order to slow down the consumption of reserves by reducing the metabolic rate and thus allow detailed observation of the differential evolution, if any, of the different components during the various stages studied. Culturing was carried out over a 27-day period. The water was renewed totally every 2 days and food was added at a concentration of 50 cells/ml of Isochrysis galbana and Tetraselmis suecica. The algal cells were counted with a Coulter Counter TA II. The morphological development of the larvae and recently settled seed was inspected every 2 days under a microscope. Samples were taken for lipid analysis in two stages, the first when the larvae were expelled from the paleal cavity until they settled, and the second at 10 days subsequent to settling. During the first stage samples were taken on days 1, 7, 11 and 17. On day 17 the eyespot was present in more than 50% of the larvae, which indicated that they were ready for settling, and with this in mind PVC collecting plates were placed in the culture tanks, and renewed every 12 h. The first major peak of settling was observed 1 day (24 h) after the collecting plates were first placed in the culture vessels. The collecting plates were therefore removed and placed in another identical tank, at the same temperature and with the same diet as in the larval period. Samples for biochemical analysis were taken during this second stage at 24 and 72 h, and at 6 and 10 days, after settling. The sample size taken for determining weight and for lipid analysis varied, according to the weight of the larvae and postlarvae, between 100 000 larvae for the first sample and 1000 postlarvae in the final sample. The samples were freeze-dried in a Telstar freeze-drier, and stored at −30°C until analysis. Dry weights per individual were taken after the larvae had been rinsed in distilled water and stove-dried at 110°C for 3 h. Weight was measured on a Sartorius M3P electronic microscale.
2.2. Fatty acids 2.2.1. Analytical methods Lipids were first extracted with chloroform:methanol (1:2) and after centrifugation at 3246× g, re-extracted
from the precipitate with chloroform:methanol (2:1). Both supernatants were then washed with chloroform:methanol:water (8:4:3) as described previously [9]. The solvents contain 0.05% butylated hydroxytoluene. Total lipids were determined gravimetrically by evaporating 200 ml of lipidic extract on preweighed aluminium plates on a slide warmer (60–80°C). Storage until further processing was carried out under nitrogen at −70°C. Fatty acids from total lipids were transesterified to methyl esters with methanolic hydrogen chloride as previously described [4] and subsequently analysed on a gas chromatograph (Perkin-Elmer, 8500), equipped with a fused silica capillary column (Supelco, SP-2330; 30 m length, 0.25 mm i.d.), PTV cold injector (Perkin-Elmer) operated in the solvent elimination mode. The injector temperature was 275°C and the column temperature increased from 140 to 210°C at a rate of 1.0°C/min. Nitrogen was used as the carrier gas at 10 psi. Nonadecanoic acid was used as an internal standard and a response factor was calculated for each fatty acid in order to perform quantitative analyses. Since there is no absolute method for identifying fatty acids by GLC, a series of tentative identification procedures was applied to the lipid mixtures studied. This approach included co-injecting the sample along with standard mixtures of established composition and GC-MS (Hewlett-Packard 5971 Mass Detector). All solvents, reagents and fatty acid standards used in this work were of analytic grade (E. Merck, Darmstadt and Sigma). Differences between means were analysed by oneway analysis of variance (ANOVA) followed by Tukey’s multiple comparison [19].
3. Results
3.1. Fatty acid composition of the diet (I. galbana + T. suecica) The main fatty acids found in the diet (composed of I. galbana + T. suecica) are 16:0; 18:0; 18:1n–9; 18:4n– 3; 18:3n–3; 14:0; 20:5n–3 and 22:6n–3. Other fatty acids identified are only present in very small amounts. The main group of fatty acids are saturated fatty acids (60.2 mg/mg dry weight), polyunsaturated fatty acids (54.6 mg/mg dry weight) and monounsaturated fatty acids (28.6 mg/mg dry weight). The n–3PUFAs content is 9.5 mg/mg dry weight. The ratio n–3/n–6 is 2.9, whilst the ratio n–6/n–3 is 0.5. The ratio 22:6n–3/ 20:5n–3 is 0.7.
U. Labarta et al. / Comparati6e Biochemistry and Physiology, Part A 123 (1999) 249–254
3.2. Fatty acid composition of O. edulis Table 1 shows the composition of the fatty acids of the larval and postlarval cultures of O. edulis (the data are given in mg/mg dry weight). Total fatty acid content rises significantly (P B 0.05) from the start of the culture period until 24 h after onset of metamorphosis, when the significantly greatest (P B 0.05) fatty acid content is reached (39.1 mg/mg dry weight). From this point, the content decreases signifi-
251
cantly (PB 0.05) until the end of the culture period. The behaviour described above also applies to saturated, monounsaturated and polyunsaturated fatty acids (including n–6, n–9 and n–3PUFAs), except for the content monounsaturated fatty acid at the end of the culture period which is not significantly different (P\ 0.05) from that of the larvae. In the case of n–6 fatty acid, there are no significant differences between initial larvae and 7-day-old larvae and larvae 72 h after the onset of metamorphosis (P\ 0.05).
Table 1 Fatty acid composition (mg/mg dry weight) of O. edulis †
14:0 15:0 16:0 16:1n–9 16:1n–7 17:0 17:1n–9 18:0 18:1n–9 18:1n–7 18:2n–6 18:3n–4 18:3n–3 18:3n–6 18:4n–3 20:0 20:1n–11 20:1n–9 20:1n–7 20:2NMID1 20:2NMID2 20:2n–6 20:3n–6 20:4n–6 20:5n–3 22:2NMID1 22:2NMID2 22:4n–6 22:5n–3 22:6n–3 S Saturated S Monoenoic S Polyenoic S Total FA S n–6 S n–9 S n–3PUFAs S NMID n–3/n–6 22:6n–3/20:5n–3 NMID/PUFA Lipids‡
Initial
7 days
11 days
50% eyed
24 h
72 h
6 days
10 days
0.4 90.0 0.2 9 0.0 2.7 90.2 0.2 90.0 0.4 90.0 0.3 9 0.0 0.4 9 0.0 2.2 90.2 0.7 90.0 0.5 90.0 0.4 90.0 0.2 90.0 0.2 90.0 0.2 90.0 0.2 90.0 0.1 9 0.0 0.3 90.0 0.1 90.0 0.6 9 0.0 0.3 90.0 0.9 90.0 0.3 90.0 0.4 9 0.0 0.5 90.0 1.6 9 0.0 0.2 9 0.0 0.6 90.0 0.2 90.0 0.1 90.0 1.4 90.2 5.9 9 0.4a 3.2 90.0a 7.7 9 0.2a 16.8 9 0.6a 2.0 9 0.0a 1.4 9 0.0a 3.3 9 0.2a 2.0 9 0.0 1.7 9 0.1 0.9 90.1 0.6 90.0 4.5 9 0.1a
0.59 0.1 0.1 90.0 3.5 9 0.3 0.6 9 0.0 0.2 9 0.0 0.390.0 0.39 0.5 2.490.1 2.7 9 0.9 0.790.1 0.79 0.1 0.2 9 0.0 1.3. 90.4 0.290.0 2.0 9 0.9 0.1 90.0 0.290.1 0.3 9 0.1 0.5 90.1 0.290.1 1.3 9 0.3 0.19 0.1 0.4 90.1 0.39 0.0 1.59 0.5 0.2 90.1 0.490.0 0.59 0.3 0.090.0 2.0 9 0.4 6.99 0.5b 5.59 1.8b 11.393.3b 23.795.6b 2.290.6a 3.991.5b 4.59 1.8b 2.19 0.5 3.1 91.8 1.39 0.7 0.5 9 0.3 5.89 0.3b
0.890.2 0.29 0.1 5.3 90.9 0.5 90.0 0.39 0.1 0.39 0.0 0.5 90.3 3.29 0.4 3.991.5 1.09 0.3 1.090.3 0.3 90.1 1.99 0.7 0.39 0.0 3.191.4 0.29 0.0 0.39 0.1 0.490.1 0.79 0.1 0.39 0.0 1.4 90.2 0.1 9 0.2 0.69 0.1 0.59 0.0 1.790.3 0.39 0.0 0.59 0.0 0.790.1 0.19 0.0 2.690.2 10.091.6c 7.69 2.5c 15.495.1c 33.099.2c 3.29 0.7b 5.39 1.9c 7.59 1.9c 2.590.2 2.99 1.5 1.590.4 0.39 0.1 7.79 0.1c
0.7 9 0.2 0.2 90.1 5.5 9 1.5 0.5 90.0 0.4 9 0.1 0.4 9 0.0 1.2 91.4 3.4 90.4 5.1 90.8 1.2 90.1 1.0 90.0 0.3 90.0 2.1 90.1 0.5 90.3 3.8 90.1 0.1 9 0.1 0.3 90.0 0.4 90.0 0.9 9 0.1 0.4 90.2 0.4 9 0.3 0.3 9 0.1 0.5 90.1 0.6 90.2 2.1 90.6 0.4 9 0.1 0.6 90.2 0.8 901 0.2 90.0 3.6 90.9 10.3 9 2.3d 10.0 9 2.5d 17.6 9 3.3d 37.9 9 8.1d 3.7 9 0.8c 7.2 92.2d 9.7 91.6d 1.8 90.9 3.2 9 1.1 1.7 90.9 0.2 90.1 8.0 90.1c
0.7 9 0.0 0.3 90.0 4.6 9 1.0 0.5 90.1 0.4 9 0.0 0.5 9 0.2 1.4 90.8 3.3 9 0.7 5.9 90.8 1.2 90.3 1.2 90.4 0.4 90.0 2.3 90.6 0.2 9 0.2 4.1 90.2 0.0 9 0.0 0.3 9 0.2 0.6 90.1 1.0 90.2 0.3 9 0.0 0.2 9 0.0 0.2 9 0.1 0.5 90.0 0.6 90.1 2.4 90.6 0.4 9 0.1 0.7 90.2 0.9 90.1 0.2 9 0.0 3.8 90.4 9.49 1.9d 11.3 9 2.5d 18.4 9 3.0d 39.1 9 7.4d 3.6 90.9c 8.4 91.8d 10.5 9 1.2d 1.6 9 0.3 3.6 9 1.4 1.6 90.6 0.2 9 0.0 5.7 90.1b
0.5 9 0.0 0.29 0.0 3.4 9 0.2 0.4 9 0.0 0.2 9 0.0 0.2 9 0.0 0.2 9 0.3 2.3 9 0.0 3.9 9 1.0 0.7 9 0.1 0.7 9 0.1 0.3 9 0.0 1.3 9 0.5 0.2 9 0.0 2.1 9 0.5 0.0 9 0.0 0.2 9 0.0 0.4 9 0.1 0.69 0.1 0.2 9 0.0 0.1 9 0.1 0.1 9 0.0 0.59 0.1 0.3 9 0.1 1.3 9 0.4 0.2 9 0.1 0.3 9 0.1 0.4 9 0.1 0.1 9 0.1 1.6 9 0.6 6.6 90.2e 6.6 91.6e 9.7 92.8e 22.9 9 4.6e 2.2 90.4a 4.9 91.4e 5.1 91.6e 0.8 90.3 2.9 9 1.5 1.2 90.8 0.2 9 0.1 3.4 90.0d
0.3 9 0.1 0.1 9 0.0 2.1 9 0.4 0.2 9 0.0 0.2 9 0.0 0.2 9 0.0 0.0 9 0.1 1.3 9 0.4 2.0 9 0.5 0.4 9 0.1 0.3 9 0.1 0.2 9 0.0 0.6 9 0.1 0.1 9 0.0 1.0 9 0.3 0.0 9 0.0 0.1 9 0.0 0.2 9 0.0 0.3 9 0.0 0.0 9 0.0 0.1 9 0.0 0.1 9 0.0 0.3 9 0.1 0.2 9 0.1 0.5 9 0.3 0.1 9 0.0 0.2 9 0.0 0.2 9 0.1 0.0 9 0.0 0.5 9 0.3 4.0 90.9f 3.4 90.7a 4.4 91.4f 11.8 9 3.0f 1.2 90.4d 2.4 90.6f 2.0 9 0.9f 0.4 9 0.0 2.1 9 1.5 1.0 91.2 0.2 9 0.1 2.4 90.1e
0.2 90.1 0.1 90.0 1.7 90.2 0.2 90.0 0.2 90.0 0.1 90.0 0.1 90.1 1.1 90.2 2.0 90.3 0.3 90.0 0.5 90.1 0.2 90.0 0.5 90.1 0.1 90.1 1.0 90.2 0.0 90.0 0.1 90.0 0.2 90.0 0.2 90.0 0.0 90.0 0.1 90.0 0.2 90.2 0.3 90.0 0.2 90.1 0.5 90.1 0.1 90.0 0.1 90.0 0.2 90.0 0.1 90.0 0.8 90.2 3.2 90.5f 3.3 9 0.4a 4.9 9 1.1f 11.4 9 2.0f 1.5 90.5d 2.5 90.4f 2.4 90.5f 0.3 9 0.0 1.9 9 1.0 1.6 9 0.7 0.1 90.0 2.1 9 0.0e
P-value
* * * * * * *
***
FA, total fatty acid; NMID, non-methylene interrupted dienoic fatty acid; PUFA, polyunsaturated fatty acid. Mean 9S.D. of two group replicates. Mean values within the same rows with different superscript letters are significantly different (PB0.05). ‡ % of dry weight. * PB0.05; *** PB0.001. †
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At the beginning of the experiment the main fatty acids found in oyster larvae are 16:0; 18:0; 20:5n –3 and 22:6n–3. Other fatty acids are only present in small amounts. Although variations in the amounts of the different fatty acids identified were observed during the development of O. edulis, palmitic acid (16:0) was consistently the main fatty acid. During the first 7 days of growth 18:1n–9; 18:3n – 3; 18:4n – 3 and 20:2NMID2 (NMID, non-methylene-interrupted dienoic) appear in large amounts. These fatty acids appear in large amounts until the end of metamorphosis, after which point there is a significant decrease (P B 0.05) in amounts of these main fatty acids which is maintained until the end of the experimental period. Nevertheless it should be noted that at 10 days after onset of metamorphosis, the decrease in the main fatty acids has ceased, while 22:6n–3 and 18:2n – 6 show a significant recovery (P B 0.05). The rates (gains or losses), in ng/ind./day, of each of the main fatty acids can be seen in Table 2. The changes for the three main stages of the development period were also analysed. During larval development, the rate increased significantly (PB 0.05) until the onset of metamorphosis, and this increase continued until 24 h after this process had begun. However, negative values appeared for all the fatty acids analysed for the period starting 24 h after the onset of metamorphosis until the end of this process (72 h). From the end of metamorphosis to the beginning of postlarval stage (6 days after the onset of metamorphosis), the main fatty acids showed a positive rate but the 22:6n–3 rate remained negative. The final period of the experiment, from 6 to 10 days after the onset of metamorphosis, showed an increase for all main fatty acids.
4. Discussion The PUFAs 20:5n – 3 and 22:6n – 3 are important lipid components in larval mollusc development [16], it having been shown that levels of 20:5n – 3 and 22:6n–3 contribute significantly to growth of O. edulis [8]. The composition of fatty acids in the diet influences the composition of bivalves fed on this diet [2,7,21]. The chosen diet contains a balanced composition of fatty acids [9] according to the needs defined as essential in bivalves, for example the presence of n – 3PUFAs (i.e. 20:5n–3 and 22:6n– 3) and a n – 6/n – 3 ratio of around 0.5. Many bivalves lack the ability to produce PUFAs by chain elongating and/or desaturating dietary precursors [5]. However in other bivalve species it has been established that they can synthesize PUFAs (C20 – C22) from the shorter chain (C18) fatty acids in the diet [15,20]. The need for certain fatty acids seems to be species
dependent. Tapes semidecussata and Mercenaria mercenaria need 22:6n–3 while Crassostrea sp. shows a fundamental need for 20:5n–3 [10]. Both 20:5n–3 and/or 22:6n–3 can alternatively meet the requirements of bivalves for n–3PUFA [16]. Whether supplied by the diet or synthesized by the bivalve it is clear that PUFAs are necessary. The function of these fatty acids could be as an energy source or as precursors to produce eicosanoids or prostaglandins [1]. During embryonic development of P. yessoensis, a linear decrease is observed in the content of the n– 3PUFAs, with the exception of 22:6n–3 [22]. The constant level of 22:6n–3 throughout embryogenesis indicates a major structural function as opposed to the energy-providing function of 20:5n–3. In the transition of larvae to juveniles of Crassodoma gigantea there was an increase in 22:6n–3 [23] a fatty acid that is usually associated with membrane structures [16]. In Pecten maximus [6] it was established that although poor results in the settlement of postlarvae may be due to other factors apart from the PUFAs, this effect is nevertheless directly associated with a low level of 22:6n–3 in the neutral lipids of pediveliger larvae. In the present work it can be seen that 22:6n–3 (a fatty acid deemed to be essential and which follows the same pathway of formation as 20:5n–3 from 18:3n–3) behaves differently from 20:5n–3. Its behaviour should not only be attributed to the amount present in the diet (the ratio 22:6n–3/20:5n–3 is 0.7), but rather may be explained by the facts expounded [6,23], which assign a separate role to 22:6n–3 during two stages of the development: settlement and metamorphosis of bivalve. On this basis, our results may also allow a similar interpretation of the structural role of 22:6n–3 and energy-providing role of 20:5n–3, in the metamorphosis and postlarval development of O. edulis like that proposed by the aforementioned authors in the embryogenesis and settlement at postlarvae stage in the other bivalves [6,22,23]. With regard to the C20 and C22 non-methylene-interrupted dienoic (NMID) fatty acids it is known that these fatty acids are preferably found in the polar lipids of O. edulis and other molluscs [13]. It seems probable that these are membrane lipids structural components rather than the basis of eicosanoids, the role ascribed below to certain polyunsaturated fatty acids [1]. Napolitano et al. [17], working with larvae of O. edulis, indicate that the origin of the NMIDs is endogenous (from monoenoic fatty acids), and these fatty acids, which are the precursors of the NMIDs, are present in significant amounts in oysters. In the present study it can be seen that the NMIDs, specifically 20:2NMID, can be found in similar amounts to those of some PUFAs in the larval development stage, but start to lose their relative importance from the onset of metamorphosis, and are only present during the postlarval stage in residual quantities.
16:0 18:0 18:2n–6 18:3n–3 18:4n–3 20:5n–3 22:6n–3 S Saturated S Monoenoic S Polienoic S n–3 PUFA S NMID S Total FA a
Initial 7 days
7–11 days
11 days, 50% eyed
50% eyed, 24 h
24–72 h
72 h–6 days
6–10 days
Initial, 50% eyed
50%,72 h
72 h–10 days
0.6 0.4 0.1 0.3 0.5 0.2 0.4 1.1 1.0 2.1 1.1 0.1 4.3
2.9 1.6 0.6 1.0 1.8 0.8 1.3 5.3 4.1 8.1 4.0 0.4 17.6
2.6 1.7 0.4 1.0 2.1 1.1 2.2 4.8 5.9 9.0 5.5 0.6 19.7
1.5 4.3 2.9 5.2 8.6 5.4 7.1 9.6 25.2 34.6 22.1 2.5 69.4
0.3 1.1 1.0 2.2 5.4 2.5 6.6 2.5 9.2 21.2 14.7 1.5 33.0
4.1 2.5 0.5 0.4 1.2 0.3 0.2 7.8 4.5 5.0 1.3 0.3 17.3
12.9 7.6 5.0 4.1 9.2 4.8 8.7 23.5 27.8 46.7 23.2 2.1 98.0
1.8 1.1 0.3 0.7 1.3 0.7 1.2 3.4 3.5 6.0 3.3 0.3 12.9
0.3 0.7 0.3 0.2 0.7 0.2 2.0 1.5 2.3 2.6 2.4 0.1 1.2
7.6 4.6 2.3 1.9 4.4 2.1 3.3 14.1 13.8 21.7 10.1 1.0 49.6
The increase represent the difference compare to the preceding stage. The values in bold are negative. See Table 1 for abbreviations.
U. Labarta et al. / Comparati6e Biochemistry and Physiology, Part A 123 (1999) 249–254
Table 2 Increases of the main fatty acids (ng/ind. per day) of O. edulis a
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U. Labarta et al. / Comparati6e Biochemistry and Physiology, Part A 123 (1999) 249–254
Acknowledgements We would like to thank J.L. Garrido, A. Ayala, L. Nieto and B. Gonza´lez for biochemical analyses and C. Ferna´ndez-Pena for helpful technical assistance in the algal and larvae cultures. This work was funded by CICYT-CSIC.IEO project I+ D number MAR900821-CO2–2.
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