Growth and kinetics of lipids and fatty acids of the clam Venerupis pullastra during larval development and postlarvae

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1 Growth and kinetics of lipids and fatty acids of the clam Venerupis pullastra during larval development and postlarvae M.J. FERNA NDEZ-REIRIZ,A.PÉREZ-CAMACHO, L.G. PETEIRO & U. LABARTA Consejo Superi Investigaciones Cientificas, Instituto de Investigaciones Marinas, Eduardo Cabello, Vigo, Spain; Centro Oceanogra fico de La Cun a, IEO, Muelle de Animas, La Cun a, Spain Abstract This study examines the larval development, metamphosis and postlarval stage of Venerupis pullastra in relation to growth, lipids content and fatty acid composition, specifically those believed to be essential f most bivalves (i.e. 0:n- and :n-). Clam larvae were fed with two species of microalgae supplied individually mixed Isochrysis galbana and Tetraselmis suecica species nmally used in bivalve hatcheries. Larvae fed with showed a progressive accumulation of lipids and fatty acids but did not survive to metamphosis. Contrarily, larvae fed with mixed diet showed a progressive decline in lipids and essential fatty acids (0:n- and :n-) from the pediveliger stage onwards, and a survival rate of % until the start of metamphosis. The lower content in n- and the absence of :n- in diet might contribute to the massive mtality observed f larvae fed with this diet. That diet seems to fail in the supply of some particular nutrient that allows energetic transfmation of reserves f growth and metamphosis. Nevertheless, larvae fed on mixture diet showed higher weight growth values at postlarval stage than those larvae fed on diet. : clam, fatty acids, growth, larval development, lipidõs kinetic, microalgal diets, Venerupis pullastra Received March 00, accepted June 00 Crespondence: M.J. Ferna ndez-reiriz, Consejo Superi de Investigaciones Cientı ficas, Instituto de Investigaciones Marinas, Eduardo Cabello,. 0 Vigo, Spain. mjreiriz@iim.csic.es Introduction A N U 0 B Larval development and survival is determined by the energy reserves sted during two stages of development. One cresponds to embryonic development and is mainly governed by Dispatch:..0 Journal: ANU CE: Anusha Journal Name Manuscript No. Auth Received: No. of pages: PE: Ilamathi Aquaculture Nutrition Ó 00 The Auths doi: 0./j x the endogenous reserves supplied to the eggs from the parents (Bayne ). The next stage is a previous period until metamphosis when sted energy reserves are essential, and depends on the feed value of the diets supplemented f larval growth (Whyte et al., 0). Recently, an in-depth revision of lipid metabolism, Sewell (00), has discussed the imptance of the lipid matrix during bivalve larval development. Utting () and Whyte et al. (, 0), on the other hand, noted the imptance of the protein content of the diet f strong larval growth up to metamphosis, as well as the requirement of a diet sufficiently balanced in proteins, lipids and carbohydrates. Labarta et al. () evaluated the growth and processes of energy acquisition in Ostrea edulis during larval development, as well as the role of lipids, proteins and carbohydrates from an energetic and structural perspective, and showed that lipids were the main source of metabolic energy f O. edulis throughout larval development. The imptance of lipids as a dietary requirement has been extensively studied f many species of bivalves over recent decades (Albentosa et al., ; Caers et al. ; Fernández-Reirı z et al. ; Soudant et al. ; Pernet et al. 00). The fatty acid composition has been described f some bivalve species (Watanabe & Ackman ; Holland ; Waldock & Holland ; Hendriks et al. 00; Milke et al. 00). Nonetheless knowledge of fatty acid composition in V. pullastra during larval and postlarval development is lacking. Existing results showed that long-chain (n-) and (n-) PUFA were imptant f mollusc larvae (Delaunay et al. ; Leonardos & Lucas 000), similar to many marine species. These criteria, as well as acceptability and digestibility, may help explain their nutritional value (Albentosa et al., ; Fernández-Reirı z et al. ). Waldock & Holland () investigated the metabolism of fatty acids in Crassostrea gigas juveniles. This auth pointed out that C. gigas has some capacity f elongating and Journal compilation Ó 00 Blackwell Publishing Ltd

2 M. J. Fernández-Reiriz et al desaturating n- fatty acids to produce n-hufa, although too low to sustain optimum growth. The same results were obtained by Chu & Greaves () f Crassostrea virginica where C-labelled 0:n- and :n- were not detected from labelled :n-. These findings are comparable to those observed by Albentosa et al. (, ) in V. pullastra spat and Ruditapes decussatus spat. The requirement f certain fatty acids appears to be species dependent. Tapes semidecussatus and Mercenaria mercenaria require :n-, while Crassostrea sp. shows a fundamental requirement f 0:n- (Helm & Laing ). Both 0:n- and/ :n- can meet the bivalve requirements f n- PUFA (Ferna ndez-reirı z et al. ). Nonetheless, Pernet et al. (00) repted that the mussel seemed better able that the oyster to selectively incpate 0:n- fatty acid. The NMID fatty acids are preferably found in the polar lipids of mollusks (Ackman & Hooper ; Irazu et al. ; Kraffe et al. 00). Pathways f the biosynthesis of 0:NMI and :NMI fatty acids have been repted in the bivalve mollusks Scapharca broughtoni and Mytilus edulis (Zhukova ). These results indicated that mollusks have active fatty acid elongation and desaturation systems that allow synthesis of these NMI fatty acids. The NMIDs, specifically 0:NMID, were observed in similar amounts to some PUFAs in the larval development stage of O. edulis, but became relatively less imptant from the onset of metamphosis. Furtherme, these acids were only present in residual quantities during the postlarval stage (Labarta et al. ). This study investigates the larval development, metamphosis and postlarval stage of V. pullastra in relation to growth, kinetic response of lipids and the fatty acid composition, with regard to three experimental diets. Materials and methods Larval cultivation Venerupis pullastra (L.) larvae were obtained from broodstock conditioned at the Instituto Espan ol de Oceanografı a (A Cun a, NW Spain). Spawning and larval culture were carried out following Pérez-Camacho et al. (). Larvae were maintained f days until attaining the postlarval stage. After days of incubation of the eggs in 00 L glass fibre containers, D-veliger larvae stage was attained, showing a mean length of. lm. These larvae were transferred to 00 L tanks maintained at C with filtered water ( lm) and 0 cells ll ) of clon T-ISO as diet concentration. The water and food was renewed every days. After days under these conditions, the larvae reached a mean length of. lm (umbonate larvae), which allowed them to capture cells with the size of Tetraselmis suecica (about. lm). At this point, the experiment with different diets was initiated. Two tanks f each experimental diet were deployed. The experiment was carried out within 00 L fibre glass tanks with filtered sea water ( lm) at C with a larval density of five larvae ml ). The larvae were fed with two species of microalgae ( and ) supplied individually (00 cells ll ) f and its equivalent volume, 0 cells ll ) f ) mixed (0 cells ll ) and five cells ll ) f ). The water and food were renewed every days. Larval samples f biochemical analysis were taken at day (D larva), day (umbonate larvae) day (pediveliger larvae), day (start of metamphosis) and day (postlarvae). Size was determined using a binocular microscope with ocular micrometer (model SMZ-0, Nikon Instruments Europe, Amstelveen, The Netherlands). Individual dry weights (DW) were measured on glass microfibre filters (Cat Nº -0, Whatman International Ltd, Maidstone, UK) after washing the larvae with distilled water and dried in an oven at 0 C f h. Organic weight (OW) was determined by the difference between the dry and ashed weight following combustion (0 C f h). The weight was measured using an electronic microbalance (model MP; Sartius AG, Goettingen, Germany). Analysis of lipids and fatty acids Lipids were first extracted with chlofm:methanol (:; VWR International S.A.S., Briare, France) and, after centrifugation ( g), the precipitate was re-extracted with chlofm:methanol (:). Both supernatants were subsequently washed with chlofm:methanol:water (::) as described previously (Ferna ndez-reirı z et al. ). The solvents contained 0.0% butylated hydroxytoluene (Merck Schuchardt OHG, Hochenbrunn, Germany). To quantify total lipids, the method described by Marsh & Weinstein () was used with a tripalmitine standard (Sigma Aldrich Inc., Buchs, Switzerland). Samples were sted under nitrogen at 0 C until further processing. The results of lipids were transfmed into their energy equivalent (kj ind ) 0 ) ) following Beukema & De Bruin (). Fatty acids from total lipids were trans-esterified to methyl esters with methanolic hydrogen chlide (VWR. Ó 00 The Auths Journal compilation Ó 00 Blackwell Publishing Ltd Aquaculture Nutrition

3 V. pullastra larvae: growth, lipids and fatty acids International S.A.S., Briare, France) following Christie (). The acids were subsequently analysed on a gas chromatograph (model 00; Perkin-Elmer Inc., MA, USA) equipped with a fused silica capillary column (0-m length, 0. mm i.d.; model SP-0, Supelco, PA, USA) and a PTV cold inject (Perkin-Elmer Inc.) operated in the solvent elimination mode. The inject temperature was C and the column temperature was increased from 0 to 0 Cat a rate of.0 C min ), with N carrier gas (0.0 Pa = 0 psi). Non-adecanoic acid (Sigma-Aldrich Inc., Buchs, Switzerland) was used as an internal standard and a response fact was calculated f each fatty acid f quantitative analyses. A combination of analytical procedures (GC-MS; gas chromatograph model HP0 and mass detect model, Agilent Technologies Inc., CA, USA) was required f conclusive structure determination of non-methylene-interrupted dienoic (NMID) fatty acids. Statistical analysis Homogeneity of variance was tested with the Bartlett test. When non-homogeneity, data were modified using logarithmic transfmation. The differences between means of growth and lipid content over time were analysed using ANOVA and a Tukey test at a significance level of P < 0.0 (Snedec & Cochran 0; Zar ). Crelations between clam growth and fatty acid contents were examined by PearsonÕs crelation coefficients. Results Growth and survival The survival rate of larvae fed with and the mixture of and was % until the start of metamphosis (day ). The larvae fed exclusively with had a mtality rate above 0% at day, which increased to 00% over the following two days, at which point the culture was ended. The D larvae displayed significantly lower lengths and dry weights (. ±. lm and 0. ± 0.0 lg) than the umbonate larvae (. ±. lm and 0. ± 0.0 lg; ANOVA, P < 0.00). Significant changes were also observed in ganic (0.0 ± 0.00 and 0. ± 0.00 lg OW f D and umbonate larvae respectively; ANOVA, P < 0.00) and lipidic content (0.0 ± 0.0 and 0.0 ± 0.0 lg lg DW ) f D and umbonate larvae, respectively; ANOVA, P < 0.00). Differences in growth parameters were observed between diets in some stages (Table ; ANOVA, P < 0.0). Larvae fed Table Average and standard deviation of growth parameters (size, dry weight (DW) and ganic weight (OW)) and lipidic content (expressed in dry weight and ganic weight basis and in individual content in weight (lg ind ) ) and in energetic equivalents (KJ ind ) 0 ) )) during Venerupis pullastra larvae development days days days days days days days days days days D Larvae Umbonate Pediveliger Metamphosis Postlarvae Pediveliger Metamphosis Pediveliger Metamphosis Postlarvae Size (lm). ±.. ±. 0. ±.0 a. ± 0. d.0 ±. g 0. ±. a 0. ± 0. e. ±. a. ±. d. ±. g DW (lg) 0. ± ± ± 0. a. ± 0.0 d. ±.0 g 0. ± 0. b. ± 0.0 e 0. ± 0.0 a. ± 0. d. ±, h OW (lg) 0.0 ± ± ± 0.0 a 0. ± 0.0 d. ± 0.0 g 0. ± 0.0 b 0. ± 0.0 e 0. ± 0.0 a 0. ± 0. d. ± 0. h Lipids lg lg DW ) 0.0 ± ± ± 0.0 a 0.0 ± 0.0 d 0.0 ± 0.0 g 0.0 ± 0.0 a 0. ± 0.0 b 0.0 ± 0.0 a 0.0 ± 0.00 d 0.0 ± 0.0 g lg lg OW ) 0. ± ± ± 0.0 a 0. ± 0.0 d 0.0 ± 0.0 g 0. ± 0.0 a 0. ± 0.0 b 0.0 ± 0.0 a 0. ± 0.0 d 0. ± 0.0 g lg ind ) 0.0 ± ± ± 0.0 a 0.0 ± 0.0 d 0. ± 0. g 0.0 ± 0.0 a 0. ± 0.0 b 0.0 ± 0.0 a 0.0 ± 0.0 d 0. ± 0.0 h kj ind ) 0 ) 0. ± 0.0. ± 0.0. ± 0. a. ±. d. ±. g. ± 0. a. ± 0. b. ± 0. a. ± 0. d. ±. h Different letters represent significant differences (ANOVA ANOVA; P < 0.0) between experimental diets in each larval stage [pediveliger (a, b, c), metamphosis (d, e, f) and postlarvae (g, h)]. Ó 00 The Auths Journal compilation Ó 00 Blackwell Publishing Ltd Aquaculture Nutrition

4 M. J. Fernández-Reiriz et al on showed significant lower weight values than the other diets at day whereas no differences in length were observed between diets (Table ; ANOVA P < 0.0). At the onset of metamphosis (day ), larvae fed with showed significantly lower shell length and dry weight (0. ± 0. lm and. ± 0.0 lg; ANOVA P < 0.0) than larvae fed with (. ± 0. lm and a weight of. ± 0.0 lg) the mixture diet. ±. lm and. ± 0. lg). After metamphosis, the postlarvae fed with presented similar lengths than those fed on mixture diet (.0 ±. and. ±. lm, respectively) but lower weight values (. ±.0 and. ±. lg f and mixture diets, respectively; ANOVA P < 0.0). The highest increase in growth rates (length weight) were observed between metamphosis and postlarval stage (Fig. ). Nonetheless, no significant differences were detected in length weight growth rates between diets in any of the larval stages (Fig. ). The lipid content of larvae fed with increased over the day experimental period (onset of the metamphosis). However, with the other diets the lipid content showed the maxima values at the pediveliger stage and a decrease onwards, showing the lowest content in the postlarval stage (day ; ANOVA P < 0.0). In the pediveliger stage, the energy content of the lipids was similar (ANOVA; P > 0.0) in the larvae fed with the three diets (. kj ind ) 0 ), Table ). At the onset of metamphosis, the larvae fed with showed significantly higher lipid content (. kj ind ) 0 ), ANOVA, P < 0.0) although they did not survive metamphosis. The largest lipid content (ANOVA, P < 0.00) in the postlarval stage was found in the larvae fed with the mixed diet (. kj ind ) 0 ) ) due to their higher weight values (Table ). Equations were derived to describe the evolution of lipid content, dry and ganic weight in their energetic equivalents (kj g ), dry weight, basis, Fig. ) from the onset of the dietary experience. Larvae fed with showed a linear exponential increase along the development in lipid content and weight values (Fig. ; Appendix ) whereas larvae fed with the mixed diet showed a maximum in the pediveliger stage and a progressive decline in lipid content and weight (Fig. ; Appendix ). Fatty acids Fatty acid composition of the diets Table shows the composition of fatty acids of the three different experimental diets. (a) Length GR (µm day ) (b) DW GR (µg day ) (c) OW GR (µg day ) D-U U-P P-M M-Post D-U U-P P-M M-Post F, = 0.; P = 0. F, = 0.; P = 0. F, =.; P = 0.0 F, =.; P = 0.0 F, = 0.; P = 0. F, = 0.0; P = 0. F, = 0.0; P = 0. F, = 0.0; P = 0. F, = 0.; P = 0. D-U U-P P-M M-Post Figure Length growth rate (lm day ) ) (a), dry weight growth rate (lg day ) ) (b) and ganic growth rate (lg day ) ) (c) over Venerupis pullastra larval development (D U: growth rate from veliger D to umbonate veliger; U P: growth rate from umbonate veliger to pediveliger; P M: growth rate from pediveliger to metamphosis, M-Post: growth rate from metamphosis to postarvae) with ANOVA results f differences between diets. The main fatty acids found in diet were :0, :0, :0, :n-, :n- and :n-. The total fatty acid content was. lg mg DW- (.,. and. lg mg DW- f saturated, monoenoic and polyenoic fatty acids, respectively). The content of n- fatty acids was. lg mg DW- and. lg mg DW- f n- PUFA. The n-:n- and n-:n- ratios were. and 0., respectively. In diet the main fatty acids recded were :0, :0, :n-, :n-, and 0:n-. The total fatty acids. Ó 00 The Auths Journal compilation Ó 00 Blackwell Publishing Ltd Aquaculture Nutrition

5 V. pullastra larvae: growth, lipids and fatty acids (a) Lipid (kj g ) Time (days) (b) T-ISO OW (kj g ) DW (kj g ) Time (days) (c) Time (days) Figure Fits on the evolution of lipid content (a) dry (b) and ganic weight (c) (expressed in energy equivalents (kj g ) ) during larval development of Venerupis pullastra fed with different diets. content was three times lower than in the other experimental diets,. lg mg DW- (.,. and. lg mg DW- f saturated, monoenoic and polyenoic fatty acids, respectively). n- fatty acid content was. lg mg DW- and f n- PUFA was. lg mgdw ). The n-:n- ratio was., and 0. f n-:n-. The main fatty acids found in the diet composed of + were :0, :0, :n-, :n-, :n-, :0, 0:n- and :n-. The main groups were saturated fatty acids (0. lg mgdw ) ), polyunsaturated fatty acids (. lg mgdw ) ) and monounsaturated fatty acids (. lg mgdw ) ). The n-pufas content was Ó 00 The Auths Journal compilation Ó 00 Blackwell Publishing Ltd Aquaculture Nutrition. lg mgdw ). The n-:n- ratio was., and 0. f n-:n-. Fatty acid composition of the clam Table shows the main fatty acids content and groups of fatty acids (lg mgdw ) ) along the larval development with different experimental diets. From a developmental point of view, we observed f some fatty acids (:0 and :0) a progressive increase until metamphosis with a sharp decline in postlarvae stage f the three experimental diets. In the case of :n-, 0:NMID and :n-, we observed an opposite trend, with a progressive decrease f the three experimental diets until postlarvae stage, but with significant lower values in larvae fed with (Table ; ANOVA, P < 0.0). Another group of fatty acids (:n-, :n-, :n-, 0:n- and :n-) showed the latter trend f and mixture diets but a continuous accumulation in larvae fed on (Table ; ANOVA, P < 0.0). With regard to 0:n-, although we observed the later trend, changes in content during development were not significant f any experimental diet. From a diet point of view, the 0:n- content at pediveliger stage was significantly higher in larvae fed on. Similarly the 0:n- was significantly higher in larvae fed on, whereas the :n- was higher in the larvae fed with and mixed diets in all the developmental stages (Table ; ANOVA, P < 0.0). The larvae fed with showed a progressive increase on the total fatty acids content over development (P < 0.0), reaching significantly greater quantities at metamphosis (Table ; ANOVA, P < 0.0). In the other two diets the total fatty acid content decreased significantly during the development (P < 0.0), reaching postlarval stage with the lowest content. The behavi described above was also observed f the saturated, monoenoic and polyenoic fatty acids (Table ). Relationship between growth and fatty acids No significant crelation was observed between weight growth values and dietary fatty acids (data not shown). Crelation analysis between fatty acids content in larvae and weight growth during larval and postlarval development of V. pullastra revealed various positive and negative crelations with fatty acids with their ratios (Table ). In the first larval stages (D and umbonates larvae; larvae fed with ) larval weight was positively related to

6 M. J. Fernández-Reiriz et al some fatty acids (among others :n-, n:n ratio and total fatty acids content; Table A) and negatively related to :0, 0:n-, 0:NMID, R Saturated and R n-. In the successive developmental stages (from pediveliger to postlarvae) all the significant crelations indicated a negative relationship between weight growth of larvae fed with and the content of main fatty acids (including 0:n-, 0:NMID and :n-; Table B). Nonetheless, growth of larvae fed with, showed positive relationships with six fatty acids and only one negative crelation with :n- content (Table C). With the mixture diet, weight growth were significantly and negatively related to five fatty acids, none of which were the essential fatty acids 0:n- :n-, whereas a significant relationship was observed with the 0:NMID content (Table D). Table Fatty acid composition of experimental diets lg mgdw ) lgmgdw ) lgmgdw ) :0. ± ± ± 0. :0. ± ± ± 0.0 :0. ± 0.0. ± 0.. ± 0. :n-. ± ± ± 0. :n-.0 ± ± ± 0. :n- 0. ± 0.0 nd nd :0.0 ± ± 0.0. ± 0.0 :n- 0.0 ± 0.0 nd nd :0. ± 0.0. ± 0.. ± 0. :n-. ± 0.. ± 0.0. ± 0. :n-.0 ± ± 0.0. ± 0.0 :n-. ± 0.0. ± 0..0 ± 0. :n- 0.0 ± 0.00 nd. ± 0.0 :n-.0 ± ± 0..0 ± 0. :n-. ± 0.0 nd. ± 0. :n-. ± ± 0.00 nd 0:0 0.0 ± ± 0.0 nd 0:n- nd 0. ± 0.0 nd 0:n-.0 ± 0.0 nd 0. ± 0.0 0:n-.0 ± 0, 0. ± ± 0.0 0:n-.0 ± 0.0 nd nd 0:n- 0. ± ± ± 0. :n-. ± 0,0 nd. ± 0. :n- 0. ± 0.0 nd nd :n-. ± 0.0 nd. ± 0.0 R Saturated.. 0. R Monoenoic... R Polyenoic... R Total FA... Rn Rn-... Rn Rn-... Rn- PUFA.0.0. n-:n-..0. n-:n Discussion In general, bivalves fed with multi-specific microalgal diets show higher growth than those fed with mono-specific diets (Albentosa et al. ; Milke et al. 00). In the present study, diets comprised of and the mixture diets ( and ) showed higher growth values in length and weight during larval development of V. pullastra than those fed with. Furtherme, only these diets led to survival past metamphosis to the postlarval stage. Larvae fed with mixture diet showed higher growth values at postlarval stage than larvae fed on diet. Numerous studies have examined the nutritional value of microalgal species f bivalve mollusc culture (Webb & Chu ; Ferreiro et al. 0; Albentosa et al., ; Delaunay et al. ; Fernández-Reirı z et al., 00; Pe rez-camacho et al. ; Milke et al. 00). The size and cellular volume of is greater than (. lm and. lm compared to.0 lm and.0 lm, respectively). However, both can be efficiently retained by bivalve filtration system (Albentosa et al., ). The digestibility of the microalgal cells may be another key fact f growth. Romberger & Epifanio () repted 0 times lower assimilation efficiency of than cells by C. virgıńica. In our study, significant relationships were observed between weight growth and fatty acid composition of larvae, but no crelation was observed between growth and dietary fatty acids. In agreement, Leonardos & Lucas (000) observed significant crelation between certain larval fatty acids (i.e. n- fatty acids) and growth in M. edulis larvae. However, the latter could not be extrapolated directly to similar relationships between dietary fatty acids and larval growth (Leonardos & Lucas 000). Despite of the nutritional imptance of the n- group that includes 0:n- and :n-, PearsonÕs crelation only showed significant negative crelation with growth and n- group when larvae were cultivated with. This diet was used from the beginning of the experimentation. However, feeding with the other two diets was initiated at the umbonate phase which can suggest that fatty acids are not only transferred but accumulated in the food web. Results also showed a negative crelation between growth and the 0:NMID fatty acid content in larvae fed with and mixed diet. Latter results suggest that this fatty acid have a significant role in determining the weight of the larvae V. pullastra despite of their low content. Although little is known about the function of the NMID fatty acids, the pathways f their biosynthesis in mollusc have been. Ó 00 The Auths Journal compilation Ó 00 Blackwell Publishing Ltd Aquaculture Nutrition

7 V. pullastra larvae: growth, lipids and fatty acids Table Fatty acid composition (average ± SD expressed in lg mgdw ) ) during Venerupis pullastra larval development days days days days days days days days days days D Larvae Umbonate Pediveliger Metamphosis Postlarvae Pediveliger Metamphosis Pediveliger Metamphosis Postlarvae Fatty acid :0. ± 0.0. ± 0.0. ± 0.0 a. ± 0.00 d. ± 0.00 g. ± 0.0 b. ± 0.0 e. ± 0. c. ± 0. f.0 ± 0. h :0 0. ± ± ± ± ± ± ± ± ± ± 0.0 :0. ± 0.. ± 0.. ± 0. a 0. ± 0.00 d. ± 0.00 g. ± 0. b. ± 0. e 0. ± 0. a 0. ± 0. d. ±.00 h :n. ± ± ± ± ± ± 0.. ± ± 0.. ± ± 0. :n.0 ± 0.0. ± 0.0. ± 0.0. ± ± ± 0.0. ± 0.0. ± 0.0. ± ± 0. :0 0. ± ± ± ± ± ± ± ± ± ± 0.0 :n. ± 0..0 ± ± ± ± ± ± 0..0 ± ± ± 0.0 :0. ± 0.0. ± 0.. ± 0.0 a. ± 0.00 d.0 ± 0.00 g. ± 0. a. ± 0. d. ± 0. a. ± 0.0 e. ± 0. h :n. ± 0.. ± 0..0 ± 0. a.0 ± 0.00 d. ± 0.00 g 0. ± 0. b 0. ± 0. e. ± 0. c. ± 0. f. ± 0.0 h :n. ± 0.0. ± 0.0. ± ± ± ± 0.0. ± 0.0. ± 0.0. ± 0..0 ± 0. :n 0, ± 0.0. ± 0.0. ± 0.0 a.0 ± 0.00 b 0. ± 0.00 g 0. ± 0.0 b.0 ± 0.0 d.0 ± 0.0 a 0. ± 0.0 d 0. ± 0.0 h :n 0. ± ± ± ± ± ± ± ± ± ± 0.0 :n 0. ± ± ± ± ± ± ± ± ± ± 0.0 :n 0. ± 0.. ± 0.0. ± 0. a. ± 0.00 d.0 ± 0.00 g.0 ± 0.0 b.0 ± 0.0 e. ± 0. c. ± 0.0 d. ± 0. h :n 0. ±.0. ± 0..0 ±.0 a. ± 0.00 d. ± 0.00 g. ± 0. b. ± 0.0 e. ± 0. a. ± 0.0 e. ± 0.0 h 0:n 0. ± ± ± ± ± ± ± ± ± ± :n 0. ± 0.0. ± ± ± ± ± ± 0.0. ± 0.0. ± ± 0. 0:n 0.0 ± ± ± ± ± ± ± ± ± ± 0.0 0:n 0. ± ± ± ± ± ± ± ± ± ± 0.0 0:n 0. ± ± ± ± ± ± 0.0. ± ± ± ± 0.0 0:n 0. ± ± ± 0. a 0. ± 0.00 d 0. ± 0.00 g 0. ± 0.0 a 0. ± 0.0 e 0. ± 0.0 a 0. ± 0.0 d 0. ± 0.0 g 0:n 0. ± ± ± ± ± ± 0.0. ± 0..0 ± ± ± 0. 0:n. ± ± ± 0.0 a 0. ± 0.00 d 0. ± 0.00 g. ± 0. b. ± 0.0 e. ± 0. c 0.0 ± 0.0 f 0. ± 0. h :n 0. ± ± ± ± ± ± ± ± ± ± 0.0 0:NMID 0. ± ± ± 0.0 ab 0. ± 0.00 d 0. ± 0.00 g 0. ± 0.0 a 0.00 ± 0.00 e 0. ± 0.0 b 0. ± 0.0 f 0.0 ± 0. h :n 0.0 ± ± ± ± ± ± 0.. ± ± ± ± 0. :n 0. ± ± ± ± ± ± ± ± ± ± 0.00 :n 0. ± ± ± 0. a 0. ± 0.00 d 0.0 ± 0.00 g 0.0 ± 0.0 b 0.0 ± 0. d 0. ± 0.0 c 0. ± 0.0 e 0. ± 0.0 h :n 0. ± 0.. ± 0.0. ± 0.. ± ± ± ± 0.0. ± ± ± 0. :n 0. ± ± ± ± ± ± ± ± ± ± 0.00 :n. ±.. ± ±. a. ± 0.00 d. ± 0.00 g. ± 0.0 b. ± 0.0 e. ± 0. c. ± 0.0 f. ± 0. h R Saturated. ± 0.. ± 0.. ± 0. a. ± 0.00 d. ± 0.00 g. ± 0. a. ± 0. e. ± 0. a.00 ± 0. f. ±. h R Monoenoic. ± 0.. ± 0.. ± 0. a. ± 0.00 d. ± 0.00 g. ± 0. b. ± 0. e.0 ± 0. c. ± 0. f. ± 0.0 g R Polyenoic.0 ±.. ± 0.. ±. a. ± 0.00 d. ± 0.00 g. ± 0. b.00 ± 0. e. ± 0. a.0 ± 0. f. ±.0 g Rn-. ±.. ± 0.0. ±.. ± ± ± 0.0. ± 0.. ± 0.. ± 0.. ± 0. Rn-. ± 0.. ± 0.. ± 0.. ± ± ± 0.. ± 0.. ± ± ± 0. Rn-. ± 0.. ± ± 0.. ± ± ± 0.0. ± 0..0 ± 0.. ± 0..0 ± 0. Rn-. ± 0.. ± 0.. ± 0..0 ± ± ± 0.. ± 0.. ± 0..0 ± 0.. ± 0. Rn- 0. ± ± ± ± ± ± ± ± ± ± 0.00 R PUFA n-.0 ±.. ± 0.0. ±. a. ± 0.00 d. ± 0.00 g. ± 0.0 b. ± 0. e. ± 0.0 a. ± 0.0 f. ± 0. h Ó 00 The Auths Journal compilation Ó 00 Blackwell Publishing Ltd Aquaculture Nutrition

8 M. J. Fernández-Reiriz et al Table (Continued) days days days days days days days days days days D Larvae Umbonate Pediveliger Metamphosis Postlarvae Pediveliger Metamphosis Pediveliger Metamphosis Postlarvae Fatty acid n-/n-. ± 0..0 ± 0.0. ± 0. a. ± 0.00 d. ± 0.00 g. ± 0.0 b.0 ± 0. e. ± 0. a. ± 0.0 f. ± 0. h n-/n- 0. ± ± ± 0.0 a 0.0 ± 0.00 d 0. ± 0.00 g 0. ± 0.0 a 0. ± 0.0 d 0. ± 0.00 a 0. ± 0.0 e 0. ± 0.0 h NMID 0. ± ± ± ± ± ± ± ± ± ± 0. Total FA. ±. 0.0 ± 0.. ±. a. ± 0.00 d. ± 0.00 g. ± 0. a. ±. e.0 ±. b. ±.0 f. ±. g FA, total fatty acid; NMID, non-methhylene interrupted dienoic fatty aci; PUFA, polyunsaturated fatty acid. Different numbers represent significant differences (ANOVA ANOVA; P < 0.0) in fatty acid content between developmental stages fed with the same experimental diet. Different letters represent significant differences (ANOVA ANOVA; P < 0.0) in fatty acids content between experimental diets in each larval stage [pediveliger (a, b, c), metamphosis (d, e, f) and postlarvae (g, h)]. established (Zhukova ) and, since this group of fatty acids are not detected in the algal diets may have been entirely synthesized by the V. pullatra larvae. Alkanani et al. (00) wking with adults of M. edulis showed that although n- were significantly crelated with growth, stepwise regression did not find n- in combination with other variables to be an imptant growth predict. However, the stepwise regression showed that 0:NMID fatty acid explained the maj percentage of the variance of the mussel growth and consequently this fatty acid is considered as a maj predict f mussel growth. The survival during larval development may depend on the ability to develop new structures, including shells, while reserves are being consumed (Labarta et al. ; Veniot et al. 00). The absence of 0:NMID could be related to the failure of metamphosis in larvae fed with. Larvae fed with reached day (pediveliger stage) with an intermediate size compared to the other two diets and dry weight values significantly lower than larvae fed with and mixed diet. At day, majity of larvae are close to metamphosis it has been already initiated. Table PearsonÕs crelation coefficient between larval fatty acids and weight growth values (lg DW indiv ) ) f V. pullastra. (A) befe the onset of the experimental diets and f larvae fed with Isochrysis (B), Tetraselmis (C) (D) diets Fatty acid (A) (B) (C) (D) DW DW DW DW :0 0.** )0.** 0.** )0.* :0 ns )0.** 0.* )0.0* :0 )0.* )0.* ns )0.* :n- 0.** )0.** 0.* )0.0* :n- 0.* )0.** 0.* ns :n- 0.* )0.** 0.* ns :n- 0.** )0.* )0.0* ns 0:n- ns ns ns ns 0:n- )0.** )0.** ns ns 0:NMID )0.* )0.** ns )0.* :n- ns )0.** ns ns :n- 0.** )0.* ns ns R Saturated )0.** )0.** ns )0.0* R Monoenoic.000** )0.** ns )0.0* R Polyenoic.000** )0.** 0.* ns Rn-PUFA.000** )0.0* ns ns Rn- Ns )0.* ns ns Rn- Ns )0.* ns ns Rn- Ns )0.** ns ns Rn- 0.** )0.** ns ns Rn- ).000** ns ns ns Rn-/Rn-.000** ns ns ns Total fatty acids 0.** )0.** ns ns *P < 0.0; **P < 0.00; ns, no significant.. Ó 00 The Auths Journal compilation Ó 00 Blackwell Publishing Ltd Aquaculture Nutrition

9 V. pullastra larvae: growth, lipids and fatty acids At this point, larvae fed with showed lower weight growth that those fed with and mixed diet but higher lipid content (Table ). Similarly, total fatty acids content showed higher values f larvae fed with, although this diet showed the lowest content in total fatty acids. The latter could indicate that larvae fed with were lacking of some particular nutrient that prevent an adequate utilization of energetic stes in growth, whereas larvae fed with and mixed diet utilized their reserves to increase their growth and development. The dietary fatty acids profiles were comparables to previously described results (Albentosa et al. ). In agreement with Soudant et al. (), we observed that dietary fatty acids composition influences the fatty acid profile of the larvae V. pullastra, as highlighted the significant differences observed between diets in the content of different groups of fatty acids (Table ). These results suggest a limited capacity f de novo synthesis of longchain PUFA in bivalves, as was previously repted (Delaunay et al. ; Caers et al. 00). PUFAs sted during larval development are used during metamphosis to provide the energetic requirements f the synthesis of new structures (Delaunay et al. ). Beside other nutrients, lack of :n- fatty acid in diet might contribute to the higher mtality observed f larvae fed on the latter diet. Delaunay et al. () repted that this fatty acid is partially replaced by 0:n- in polar lipids of larvae fed on Chaetoceros calcitrans with no apparent negative effects on growth. Nonetheless fewer pediveliger larvae were able to settle in comparison to those which accumulated primarily :n- (Delaunay et al. ). Other deficient fatty acids in diet, were long-chain n-pufa. Delaunay et al. () showed that Pecten maximus larvae need also n-pufa as previously demonstrated f adult oysters (Trider & Castell 0). Although no significant relationships between the 0:n- content and growth was observed, the imptant metabolic role of 0:n- fatty acid as a precurs of prostaglandins (Smith & Murphy 00) may result in a high turnover and requirement f this fatty acid. In summary, the higher content of 0:n- in diet compared to the other diets apparently was not enough to compensate the absence of 0:n-. In addition, the lower n:n ratio pointed out deficiencies in the n- group, also imptant f larval growth. Those dietary deficiencies might prevent an adequate use of energetic reserves that were continuously accumulated in larvae and consequently preclude an adequate development and survival. Nonetheless when is combined with diet in mixture Ó 00 The Auths Journal compilation Ó 00 Blackwell Publishing Ltd Aquaculture Nutrition diet, nutritional deficiencies might be compensated as pointed out the survival rate. In addition, larvae fed on mixture diet reached postlarval stage with weight growth values higher than those fed on, as was expected f multi-specific algal diets (Albentosa et al. ; Milke et al. 00). Acknowledgements We thank B. Gonza lez and L. Nieto in the biochemical analyses f their helpful technical assistance in the algal and larvae cultures. This wk was funded by MEC. AGL00-00-C0-0/ACU. References Ackman, R.G. & Hooper, S.N. () Non-methylene-Interrupted fatty acids in lipids of shallow-water marine invertebrates. A comparison of two molluscs (Littina littea and Lunatia triserita) with the sand shrimp (Crangon septumspinosus). Comp. Biochem. Phys., B,. Albentosa, M., Pérez-Camacho, A., Labarta, U., Beiras, R. & Ferna ndez-reiríz, M.J. () Nutritional value of algal diets to the clam spat Venerupis pullastra. Mar. Ecol. Prog. Ser.,,. Albentosa, M., Labarta, U., Pérez-Camacho, A., Fernández-Reiríz, M.J. & Beiras, R. () Fatty acid composition of Venerupis pullastra spat fed on different microalgae diets. Comp. Biochem. Physiol., 0A,. Albentosa, M., Labarta, U., Fernández-Reiríz, M.J. & Pérez- Camacho, A. () Fatty acid composition of Ruditapes decussatus spat fed on different microalgae diets. Comp. Biochem. Physiol., A,. Alkanani, T., Parrish, C.C., Thompson, R.J. & McKenzie, C.H. (00) Role of fatty acids in cultured mussels, Mytilus edulis, grown in Notre Dame Bay, Newfoundland. J. Exp. Mar. Biol. Ecol.,,. Bayne, B.L. () Aspects of the metabolism of Mytilus edulis during starvation. Netherlands J. Sea Res.,,. Beukema, J.J. & De Bruin, W. () Calic vaues of the soft parts of the tellinid bivalve Macoma baltica (L.)as detemined by two methods. J. Exp. Mar. Biol. Ecol.,, 0. Caers, M., Coutteau, P., Lombeira, P. & Sgeloos, P. () The effect of lipid suplementation on the growth and fatty acid composition of Tapes philippinarum. Aquaculture,,. Caers, M., Couteau, P., Sgellos, P. & Gajardo, G. (00) Impact of algal diets and emulsions on the fatty acid composition and content of selected tissues of adult broodstock of the Chilean scallop Argopecten purpuratus (Lamarck, ). Aquaculture,,. Christie, E.E. () Lipid Analysis: Isolation, Separation, Identification and Structural Analysis of Lipids. Pergamon Press, Oxfd, UK. Chu, F.E. & Greaves, J. () Metabolism of palmitic, linoleic, and linolenic acids in adult oyster, Crassostrea virginica. Mar. Biol., 0,. Delaunay, F., Marty, Y., Moal, J. & Samain, J.F. () The effect of monospecific algal diets on growth and fatty acid composition

10 0 M. J. Fernández-Reiriz et al of Pecten maximus (L.) larvae. J. Exp. Mar. Biol. Ecol.,,. Fernández-Reiríz, M.J., Pérez-Camacho, A., Ferreiro, M.J., Blanco, J., Planas, M., Campos, M.J. & Labarta, U. () Biomass production and variation in the biochemical profile (Total protein, carbohydrates, RNA, lipids and fatty acids) of seven species of marine microalgae. Aquaculture,,. Fernández-Reiríz, M.J., Labarta, U., Albentosa, M. & Pérez- Camacho, A. () Effect of microalgal diets and commercial wheatgerm flours on the lipid profile of Ruditapes decussatus spat. Comp. Biochem. Physiol., A,. Fernández-Reiríz, M.J., Labarta, U., Albentosa, M. & Pérez- Camacho, A. () Lipid profile and growth of the clam spat, Ruditapes decussatus (L.), fed with microalgal diets and cnstarch. Comp. Biochem. Physiol., B, 0. Fernández-Reiríz, M.J., Labarta, U., Albentosa, M. & Pérez- Camacho, A. (00) Lipid composition of Ruditapes philippinarum spat: Effect of ration and diet quality. Comp. Biochem. Physiol. Part B,,. Ferreiro, M.J., Perez-Camacho, A., Labarta, U., Beiras, R., Planas, M. & Fernandez-Reiriz, M.J. (0) Changes in the biochemical composition of Ostrea edulis larvae fed on different food regimes. Mar. Biol., 0, 0. Helm, M.M. & Laing, I. () Preliminary observations on the nutritional value of Tahiti Isochrysis to bivalve larvae. Aquaculture,,. Hendriks, I.E., Van Duren, L.A. & Herman, P. (00) Effect of dietary polyunsaturated fatty acids on reproductive output and larval growth of bivalves. J. Exp. Mar. Biol. Ecol.,,. Holland, D.L. () Lipid reserves and energy metabolism in the larvae of benthic marine invertebrates. In: Biochemical and Biophysical Perspectives in Marine Biology, Vol. (Malins, D.C. & Sargent, J.R. eds), pp.. Academic Press, New Yk. Irazu, C.E., Pollero, R.J. & Brenner, R.R. () Occurrence of a : non-methylene interrupted dienoic fatty acid and its seasonal distribution among lipids and tissues of the fresh water bivalve Diplodon delodontus from an isolated environment. Lipids,,. Labarta, U., Ferna ndez-reiríz, M.J. & Pe rez-camacho, A. () Energy, biochemical substrates and growth in the larval development, metamphosis and postlarvae of Ostrea edulis (L.). J. Exp. Mar. Biol. Ecol.,,. Leonardos, N. & Lucas, I.A.N. (000) The use of larval fatty acid as an index of growth in Mytilus edulis L. larvae. Aquaculture,,. Marsh, J.B. & Weinstein, D.B. () Simple charring method f determination of lipids. J. Lipid Res.,,. Milke, L.M., Bricelj, V.M. & Parrish, C.C. (00) Growth of postlarval sea scallops, Placopecten magellanicus, on microalgal diets, with emphasis on the nutritional role of lipids and fatty acids. Aquaculture,,. Pérez-Camacho, A., Román, G. & Tre Cervigo n, M. () Experiencias en cultivos de larvas de tres especies de moluscos bivalvos: Venerupis pullastra (Montagu), Venerupis decussata (Linnaeus) y Ostrea edulis (Linnaeus). Bol. Inst. Esp. Ocean., III,,. Pérez-Camacho, A., Albentosa, M., Fernández-Reiríz, M.J. & Labarta, U. () Effect of microalgal and inert diets on the growth perfmance and biochemical composition of Ruditapes decussatus seed: cnmeal and cnstarch. Aquaculture, 0, 0. Pernet, F., Bricelj, V.M. & Cartier, S. (00) Lipid class dynamics during ontogeny of sea scallops, Placopecten magellanicus, in relation to metamphic success and response to antibiotics. J. Exp. Mar. Biol. Ecol.,, 0. Pernet, F., Trembaly, R., Comeau, L. & Guderley, H. (00) Temperature adaptation in two bivalve species from different thermal habitats: energetics and remodelling of membrane lipids. J. Exp. Biol., 0, 0. Romberger, H.P. & Epifanio, C.E. () Comparative effects of diets consisting of one two algal species upon assimilation efficiencies and growth of juvenile oysters, Crassostrea virginica (Gmelin). Aquaculture,,. Sewell, M.A. (00) Utilization of lipids during early development of the sea urchin Evechinus cloticus. Mar. Ecol. Prog. Ser., 0,. Smith, W.L. & Murphy, R.C. (00) The eicosanoids: cyclooxygenase, lipoxygenase and epoxygenase pathways. In: Biochemistry of lipids, lipoproteins and membranes. Vol. (Vance, D.E. & Vance, J.E. eds.), pp.. Elsevier Science, Amsterdam. Snedec, G.W. & Cochran, W.G. (0) Statistical Methods. Iowa State University Press, Ames, Iowa. Soudant, P., Van Ryckeghem, K., Marty, Y., Moal, J., Samain, J.F. & Sgeloos, P. () Comparison of the lipid class and fatty acid composition between a reproductive cycle in nature and standard hatchery conditioning of the pacific oyster Crassotrea gigas. Comp. Bioch. Physiol., B, 0. Trider, D.J. & Castell, J.D. (0) Effect of dietary lipids on growth, tissue composition and metabolism of the oyster (Crassostrea virginica). J. Nutr., 0, 0 0. Utting, S.D. () A preliminary study on growth of Crassostrea gigas larvae and spat in relation to dietary protein. Aquaculture,,. Veniot, A., Bricelj, V.M. & Beninger, P.G. (00) Ontogenetic changes in gill mphology and potential significance f food acquisition in the scallop Placopecten magellanicus. Mar. Biol.,,. Waldock, M.J. & Holland, D.L. () Fatty acid metabolism in young oyster, Crassostrea gigas: polyunsaturated fatty acids. Lipids,,. Watanabe, T. & Ackman, R.G. () Lipids and fatty acids of the American (Crassostrea virginica) and European flat (Ostrea edulis) oysters from a common habitat, and after one feeding with Dicrateria innata Isochrysis galbana. J. Fish. Res. Board Can.,, 0 0. Webb, K.L. & Chu, F.E. () Phytoplankton as food source f bivalve larvae. In: Proceedings of the nd International Conference on Aquaculture Nutrition: Biochemical and Physiological Approaches to Shellfish Nutrition (Pruder, G.D., Langdon, C. & Conklind, D. eds), pp.. Wld Mariculture Society, Washington, D.C. Special Publication number. Whyte, J.N.C., Bourne, N. & Hodgson, C.A. () Influence of algal diets on biochemical composition and energy reserves in Patinopecten yessoensis (Jay) larvae. Aquaculture,,. Whyte, J.N.C., Bourne, N. & Ginther, N.G. (0) Biochemical and energy changes during embryogenesis in the rock scallop Crassadoma gigantea. Mar. Biol., 0,. Zar, J.H. () Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, New Jersey. Zhukova, N.V. () The pathway of the byosynthesis of Non- Methylene. Interrupted dienoic fatty acid Zhukova Zhukovas in mollusks. Comp. Biochem. Phys., 00, 0 0. Appendix Equations to describe the evolution of dry weight (DW), ganic weight (OW) and lipid content, expressed in their. Ó 00 The Auths Journal compilation Ó 00 Blackwell Publishing Ltd Aquaculture Nutrition

11 V. pullastra larvae: growth, lipids and fatty acids energy equivalents (kj g ) ) during larval development f the three experimental diets. DW (kj g ) ) =.e 0.0 time (r = 0., P < 0.00) OW (kj g ) ) =.e 0.0 time (r = 0., P < 0.00) Lipids (kj g ) ) = 0.0 time+. (r = 0., P < 0.0) DW (kj g ) )=)0.00 time + 0. time +. (r = 0., P < 0.00) OW (kj g ) )=)0.00 time time+.0 (r = 0., P < 0.00) Lipids DW OW Lipids (kj g ) )=)0.00 time + 0. time+. (r = 0., P < 0.00) (kj g ) )=)0.00 time + 0. time+. (r = 0., P < 0.00) (kj g ) )=)0.00 time time+. (r = 0., P < 0.00) (kj g ) )=)0.0 time + 0. time+. (r = 0., P < 0.00) Ó 00 The Auths Journal compilation Ó 00 Blackwell Publishing Ltd Aquaculture Nutrition

12 Auth Query Fm Journal: ANU Article: 0 Dear Auth, During the copy-editing of your paper, the following queries arose. Please respond to these by marking up your proofs with the necessary changes/additions. Please write your answers on the query sheet if there is insufficient space on the page proofs. Please write clearly and follow the conventions shown on the attached crections sheet. If returning the proof by fax do not write too close to the paper s edge. Please remember that illegible mark-ups may delay publication. Many thanks f your assistance. Query reference Query Remarks AUTHOR: Please provide % values in g kg ) AUTHOR: Please provide city name f Perkin-Elmer Inc. AUTHOR: Please provide city name f Agilent Technologies Inc.

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