22 Comparative studies on the nutrition of two species of abalone, Haliotis tuberculata L. and Haliotis discus hannai Ino. IV. Optimum dietary protein level for growth

Aquaculture 252 (2006) 225 – 233 www.elsevier.com/locate/aqua-online Effect of diatom diets on growth and survival of the abalone Haliotis discus hannai postlarvae Nurit Gordon a, Amir Neori a,*, Muki Shpigel a, John Lee b, Sheenan Harpaz c a Israel Oceanographic and Limnological Research, National Center for Mariculture, P.O Box 1212, Eilat 88112, Israel b Department of Biology, City College of City University of New York, New York, NY 10031, USA c Department of Aquaculture, Agricultural Research Organization, The Volcani Center, P.O Box 6, Bet Dagan 50250, Israel Received 12 January 2004; received in revised form 17 June 2005; accepted 20 June 2005 Abstract Growth and survival of postlarval abalone Haliotis discus hannai Ino fed different diatom diets were examined for one month from settlement Two diatoms, Amphora luciae Cholnoky and Navicula cf lenzii Hustedt, supported high postlarval growth and survival, especially when supplied in combination A third species, Nitzschia laevis Hustedt, did not support survival for more than two weeks as a unialgal diet and had limited value in mixed diets Diatom mixtures were superior to single-species diets as of the first week after settlement The mixture of N cf lenzii and A lucia supported the highest survival, up to 50%, and growth rate up to 36Am of shell length per day, reaching a size of 1.4mm 30 days after settlement The three diatom species contained high levels of total lipids (6.4%–14.5% of dry weight) and fatty acids (16%–22% of lipids); from 39% to 48% of fatty acids were polyunsaturated (PUFA) The three diatoms were richer in n-3 PUFA than in n-6 PUFA The content of the essential fatty acid 20:5n-3 (EPA) was highest among the PUFAs and higher, though not significantly, in the two diatom species A luciae and N cf lenzii that produced the better results Among the free amino acids, arginine was dominant in N laevis, proline in N cf lenzii, and both free amino acids plus glutamic acid were equally dominant in A luciae The suitability of A luciae and N cf lenzii for enhancing growth and survival of postlarvae was attributed to their complementary balanced nutritional properties D 2005 Elsevier B.V All rights reserved Keywords: Abalone; Postlarvae; Biochemical composition; Growth; Survival; Diets; Diatoms; Fatty acids; Amino acids Introduction * Corresponding author Tel.: +972 6361445; mobile: +972 50 5993746; fax: +972 6375761 E-mail address: aneori@shani.net (A Neori) 0044-8486/$ - see front matter D 2005 Elsevier B.V All rights reserved doi:10.1016/j.aquaculture.2005.06.034 Benthic diatoms are the principal food source for postlarval abalone in hatcheries (Kawamura, 1996, Kawamura et al., 1998a) In spite of the increasing number of studies on the nutrition of newly settled abalone larvae (Kawamura and Takami, 1995; Kawa- 226 N Gordon et al / Aquaculture 252 (2006) 225–233 mura, 1996; Kawamura et al., 1998a,b; Daume et al., 1999, 2000; Roberts et al., 1999; Searcy-Bernal et al., 2001), growth and survival rates during the early postlarval stages as reported in the literature are variable and generally low (Searcy-Bernal et al., 1992) Poor and unpredictable performance is related to variability in food (different diatoms and their composition), as well as to abalone species and the growing conditions in hatcheries (Kawamura et al., 1998a) To improve growth and survival of abalone postlarval stages in a specific growing system using specific diatom species, a better understanding of their basic diet requirements is necessary Cell density, digestion efficiency, ingestibility, extra-cellular products, and associated bacteria are known to affect food value of diatoms in early postlarval stages (Kawamura and Takami, 1995; Kawamura et al., 1995, 1998a,b; Roberts et al., 1999; Searcy-Bernal et al., 2001) The biochemical composition of algal cells is another important factor (Dunstan et al., 1994), but its effect has been examined mostly in juvenile abalone (Mercer et al., 1993; Mai et al., 1994, 1995a,b, 1996) rather than in newly settled postlarvae The biochemical composition of the diet is most important once the postlarvae acquire the capability to digest and benefit from diatom cell content (Kawamura et al., 1998a) According to them, the diatom diet has little impact on growth rates during the first two weeks after settlement Diet-dependent postlarval growth rates diverge at 800 Am SL, when the postlarvae begin digesting and utilizing the cell content According to Daume et al (1999), differences in growth rates by postlarvae fed different diatoms can already be observed earlier, a week following settlement The nutritional value of microalgae as a feed is influenced to a great extent by the fatty acid composition of their lipids (Brown et al., 1997; Renaud et al., 1999) and to a lesser extent by sugar composition (Chu et al., 1982) The protein amino acid composition of microalgae is generally conserved (Brown et al., 1996) and is unlikely to account for major differences in the nutritional value of a particular species (Brown, 1991; Brown and Jeffrey, 1995; Brown et al., 1997) Free amino acids (FAA) may constitute a significant proportion of the total amino acids in the algal cell (Dortch et al., 1984; Brown, 1991) and their composition does vary among algal species (Derrien et al., 1998) FAA are easily absorbed by postlarvae (Manahan and Jaeckle, 1992), a fact that is especially important in very early life stages, before the complete development of the gut enzymes involved in protein digestion (Takami et al., 1998) For this reason the diatom composition phase of the present study has focused on FAA and fatty acids Diatoms, as a class, offer high levels of lipids and PUFAs, especially the essential PUFA 20:5(n-3) (Dunstan et al., 1994; Brown et al., 1997), and therefore may fulfill the nutritional requirements of abalone postlarvae better than other algae Polyunsaturated fatty acids (PUFA) of both n-3 and n-6 families are essential for growth of juvenile Haliotis discus hannai (Mai et al., 1996) Their primary function is considered to be structural (Mai et al., 1995a; Floreto et al., 1996) Among PUFAs, 20:5(n-3) seems to contribute the most to faster growth of juvenile H discus hannai (Mai et al., 1996) The aim of this research was to investigate growth and survival of H discus hannai postlarvae fed different diets of diatoms (including local species), which had previously been shown to induce larval settlement (Gordon et al., 2004), and verify whether these could be correlated to the diatoms’ nutritional quality Materials and methods 2.1 Preparation of abalone postlarvae Larvae of H discus hannai were obtained from an indoor abalone hatchery in Eilat (Red Sea, Israel) Adults were induced to spawn using ultraviolet light (Kikuchi and Uki, 1974) Fertilized eggs were collected and transferred into 20-L aquaria at a concentration of 12 eggs/ml To control bacterial growth, an antibiotic (Rafamycin) was added at a concentration of 1.5 mg/L Larvae were kept at 22 8C with a 12 L:12 D photo cycle (60–70 Amol photons mÀ sÀ 1), for 4–5 days, until reaching competence Larval competency on day was assessed by observing the swimming behavior, as described by Seki and Taniguchi (1996) Competent larvae were used for the growth experiments 2.2 Diatom cultures Benthic diatoms were isolated from the Red Sea (Eilat, Israel) and from the Atlantic Ocean (Massachu- N Gordon et al / Aquaculture 252 (2006) 225–233 setts, USA) (Table 1) Axenic cultures were prepared as described in Gordon et al (2004) The diatoms were cultured in 1-L Erlenmeyer flasks filled with f/2 medium (Guillard and Ryther, 1962), enriched with silica (Na2SiO3) and aerated with CO2 Temperature was maintained at 22 8C and light intensity was 60–70 Amol photons mÀ sÀ throughout the growth experiment 2.3 Growth experiments with abalone postlarvae Competent larvae of 280 F 12 Am in size were transferred to 90-mm petri dishes, filled with 32 ppt Red Sea water (diluted with DDW from 40 ppt) To reduce handling damage to the larvae, the number of larvae transferred was calculated according to samples taken from culture bottles Each petri dish was stocked with 82 F 17 larvae Settlement was induced by adding AM gamma-amino butyric acid (GABA) (Morse, 1992), to petri dishes, whose media included 50 mg/l (each) penicillin and streptomycin (Sigma) (Morse and Morse, 1984) This approach was preferred over natural settlement induction by the diatoms, to obtain a better reproducibility of larval settlement across diatom treatments After 24 h, GABA was rinsed out of the dishes and algae, as monocultures or mixtures, were added (Table 1) Five replicate dishes were made for each diet Algal cell concentration was adjusted to obtain a similar cell volume rather than cell number throughout all the experiments Water in the dishes was exchanged daily, and algae were replaced with fresh cultures once a week During the week algae were added to each dish once clear patches (consumed diatoms) developed around the postlarvae, to keep them supplied Larval survival was measured as a percentage of 227 postlarvae surviving from all the larvae introduced into the petri dishes at the beginning of the experiment Larval shell length (SL) was measured once a week for all postlarvae that were on the bottom of the dish, with the aid of a calibrated ocular micrometer, and then averaged Daily growth rate (DGR) was calculated according to the formula: L f À L i / t, where L f = final shell length (Am), L i = initial shell length (Am) and t = time in days The duration of each experiment was 31 days 2.4 Biochemical analysis of algal cells Fatty acid analysis was carried out for the three algal species (Nitzschia laevis, Amphora luciae and Navicula cf lenzii) on batches harvested during the late logarithmic growth phase Centrifugally concentrated algal cells were lyophilized and the lipids were extracted (Folch et al., 1957) The lipid extracts were then transmethylated to fatty acids methyl esters (FAME) by acidified methylation overnight at 50 8C in 2% H2SO4 in methanol The resulting FAME was concentrated in hexane and injected into an on-column Chrompack CP9001 gas chromatograph (Koven et al., 2001) For free amino acid analysis, late logarithmic phase cells from the three diatom species were centrifuged and homogenized with an ultrasonic cell disruptor (Microson) for Cell disruption was confirmed microscopically Free amino acid analysis was carried out with an HPLC (Biotronik LC-5000 Amino Acid Analyzer) as described by Moor and Stein (1951) Dry matter was calculated from weight loss after drying for 24 h at 105 8C Crude protein was calculated from Kjeldahl nitrogen multiplied by 6.25 Crude lipid was measured gravimetrically after homogeniza- Table Details of the diatoms in the seven diets used in this study Diet # Species Source Cell dimensions Length (Am) Width (Am) Navicula cf lenzii Nitzschia laevis Amphora luciae Mixture + + Mixture + Mixture + Mixture + Sediment pond, IOLR, Eilat, Israel Sediment pond, IOLR, Eilat, Israel Lake Tashmoo, Martha’s Vineyard Island, MA, USA 24 8–10 10 5 Initial density (total cells/cm2) 5.3 Â 103 2.5 Â 104 1.9 Â 104 1.2 Â 104 1.3 Â 104 9.7 Â 103 1.2 Â 104 N Gordon et al / Aquaculture 252 (2006) 225–233 tion of the sample in chloroform-methanol (2:1), separation and vacuum drying (Folch et al., 1957) Ash content was calculated from weight loss after incineration of samples in a muffle furnace for 24 h at 550 8C Carbohydrates were calculated as: Carbohydrates ¼ dry matter À ðcrude protein þ lipid þ ashÞ: 2.5 Statistical analyses The data were compared using ANOVA (one way) with Duncan’s multiple range tests The results, in percentages, were arc-sine transformed prior to ANOVA analysis to homogenize variances (Sokal and Rohlf, 1969) Results 3.1 Survival of abalone postlarvae Survival of larvae/postlarvae during the first month (Table 2) varied from as low as 4% when fed on a diet of N laevis (diet 2) to a high of 49% when fed on a diet of N cf lenzii and A luciae (diet 6) Survival of postlarvae with a diet of N laevis was significantly lower than with all the other diets ( P b 0.05); these postlarvae were excluded from later data analysis 3.2 Growth of abalone postlarvae With the exception of the diet based on N laevis, the postlarvae grew steadily on all the diets offered Diets N cf lenzii N laevis A luciae Mix 1+2+3 Mix 1+2 1500 Shell length (microns) 228 Mix 1+3 Mix + 1000 a a b c b c d e d 500 12 24 36 Time from larval introduction (days) Fig Growth of postlarval shell length (SL F SE) during 31 days post settlement More information on the diets is provided in Table Significant differences (ANOVA, Duncan’s multiple range test, p b 0.05) between data points are letter-labeled only on days 24 and 31, to reduce clutter The highest growth rates of postlarvae were obtained on a mixture diet of N cf lenzii and A luciae (diet 6) and a mixture of the three diatoms (diet 4) After one month in culture, the postlarvae fed these diets reached mean SL of 1.4 mm and 1.3 mm, respectively (Fig 1) Growth rates in these treatments were significantly higher ( P b 0.05) than in all the other diets The SL of the postlarvae increased by an average of 35.5 F 1.1 Am dayÀ on the diet of A luciae and N cf lenzii and 33.2 F Am dayÀ on the diet of the three algae combined In the first 12 days, postlarvae grew faster when fed the two-diatom mixture of A luciae and N laevis (diet 7) than those fed most other diets (Table 2) Diet also supported the largest ( P b 0.05) postlarval Table Survival rates and daily growth rates (DGR based on shell length) of the postlarvae fed the seven different diets of diatom listed in Table DGR, % dayÀ F SE (*) Diet # Initial individuals per dish (# F SD) Survival rate at 31 days (% F SE*) 1–12 days 13–24 days Total 31 days 69 F 16 84 F 30 66 F 74 F 15 70 F 103 F 24 109 F 17 39.8 F 4.75a 4.4 F 1.9b 42.6 F 5.5a 39.6 F 10.8a 42.4 F 5.2a 49.2 F 6.1a 31.4 F 14.1a 19.8 F 1.4d 15.0 F 1.4e 23.2 F 1.1cd 25.5 F 1.2bc 20.1 F 0.5d 28.4 F 1.9ab 32 F 1.5a 23.8 F 2c – 30.4 F 3.1bc 48 F 4.9a 26 F 2.9bc 45.4 F 1.7a 34 F 2.8b 20.6 F 0.9c – 23.7 F 1.3bc 33.2 F 2a 22.8 F 2.8bc 35.5 F 1.1a 27.3 F 2b * Data with the same letter indicate treatments that are not significantly different from each other within columns (ANOVA, Duncan’s multiple range test, critical p = 0.05) N Gordon et al / Aquaculture 252 (2006) 225–233 SL for the first weeks (Fig 1) However, later, diet and diet became the significantly better diets with respect to both DGR and SL of the postlarvae, while the performance of diet deteriorated by day 24 and became even worse by day 31 (Fig 1) Growth of the postlarvae on the single alga N laevis (diet 2) was worst ( P b 0.05) of all diets after the first week (Fig 1) The diet of A luciae (diet 3) was best of the singlediatom diets, yet significantly ( P b 0.05) inferior to all but one of the mixed-diatom diets 3.3 Lipid content and composition of diatoms Lipids comprised between 6.4% and 14.5% of the dry weight of the diatoms analyzed (Table 3) Polyunsaturated fatty acids (PUFA) constituted the largest fraction (between 41% and 47%) of the total fatty acids (TFA) The proportion of the various PUFAs varied among the diatom species Although all of the analyzed diatoms had significant quantities (between 14% and 21% of TFA) of 20:5n-3 (EPA), N laevis had slightly lower percentages of this fatty acid and higher quantities (9.7% of TFA) of 20:4n-6 arachidonic acid (AA) Shorter chain PUFAs, 16:2n-4 and 16:3n-4, were also present in significant quantities (4.2–8.5% of TFA) in all three diatoms (Table 4) 3.4 Amino acid content and composition of diatoms The three diatoms varied in their total free amino acid (TFAA) composition (Table 5) Proline was the main free amino acid (2.8 fmol cellÀ 1, 49% of TFAA) in N cf lenzii but only a minor constituent (0.13 fmol cellÀ 1, 8% of TFAA) in N laevis The proline content of A luciae was intermediate (0.8 fmol cellÀ 1, 30% of TFAA) The share of arginine in the TFAA fluctuated even more than proline among the analyzed species In N laevis arginine was the main free amino acid (0.7 fmol cellÀ 1, 43% of TFAA), while in N cf lenzii it was only 0.18 fmol cellÀ (3% of TFAA) In A 229 Table The content of specific fatty acids as fractions of total fatty acids (TFA) in the three diatoms used in this study (n = in a, n = in b) Type of fatty acid Diatom species N laevis a (% F SD) A luciae a (% F SD) N cf lenzii b (% F SD) Saturated 14:0 15:0 16:0 18:0 Sum 6.0 F 0.4 0.9 F 0.5 14.7 F 0.5 1.4 F 1.5 23.0 F 1.3 10.4 F 2.5 0.9 F 0.3 13.7 F 0.9 1.6 F 1.8 26.6 F 1.9 2.5 F 0.0 0.5 F 0.1 13.9 F 0.1 2.4 F 0.5 19.3 F 0.4 Monounsaturates 15:1n-8 16:1n-7 16:1n-9 18:1n-7 18:1n-9 Sum 0.3 F 0.3 22.4 F 1.6 2.7 F 0.9 1.0 F 0.7 2.7 F 2.6 29.1 F 3.7 0.1 F 0.2 18.4 F 2.4 2.8 F 0.3 0.8 F 0.8 3.6 F 2.6 25.7 F 0.7 0.0 F 0.0 18.8 F 0.4 3.5 F 0.1 1.5 F 0.2 4.5 F 0.0 28.2 F 0.3 Polyunsaturates 16:2n-4 18:2n-6 16:3n-4 18: 3n-3 18:3n-4 18:3n-6 18:4n-3 20:2n-6 20:3n-3 20:4n-6 (AA) 20:4n-3 20:5n-3 (EPA) 22:1n-9 22:5n-3 22:6n-3 (DHA) Sum PUFA Sum n-3 PUFA Sum n-6 PUFA 4.2 F 0.2 1.7 F 0.7 8.1 F1.7 0.6 F 0.4 0.3 F 0.4 0.8 F 1.1 0.0 F 0.0 0.4 F 0.5 0.3 F 0.6 9.7 F 1.3 0.0 F 0.0 14.3 F 3.5 0.0 F 0.0 0.4 F 0.5 1.6 F 0.2 42.4 F 3.4 17.2 F 2.9 12.6 F 0.9 4.2 F 0.6 3.4 F 0.1 5.4 F 0.9 0.5 F 0.2 0.1 F 0.3 0.8 F 1.2 1.1 F 0.9 0.5 F 0.7 0.0 F 0.0 5.0 F 1.4 0.3 F 0.4 18.5 F 2.9 0.0 F 0.0 0.8 F 0.2 0.5 F 0.6 41.2 F 2.6 21.7 F 3.2 9.7 F 3.1 5.6 F 0.4 1.5 F 0.1 8.5 F 0.5 0.9 F 0.6 0.0 F 0.0 1.8 F 0.0 0.0 F 0.0 0.7 F 0.1 0.5 F 0.1 2.8 F 0.1 0.0 F 0.0 21.2 F 0.2 0.7 F 0.85 F 1.8 F 0.9 46.6 F 1.1 25.1 F1.9 6.8 F 0.1 luciae arginine appeared in between these extremes (0.5 fmol cellÀ 1, 22% of TFAA) The content of glutamic acid in N laevis was lesser in the other two species but it contained glutamine, which was Table Biochemical composition of the three diatoms used in this study Diatom Protein (% in DW) Lipids (% in DW) Nitzschia laevis Navicula cf lenzii Amphora luciae 38.32 32.00 32.65 11.25 14.55 6.43 a A missing value (technical reason) Fatty acids (% in Lipids) Carbohydrates (% in DW) Ash (% in DW) 16.48 17.55 25.09 19.36 32.88 28.36 41.56 a 21.6 230 N Gordon et al / Aquaculture 252 (2006) 225–233 Table The content of free amino acids (FAA) in cells of the three diatoms used in this study Amino acid Diatom species N laevis fmol cellÀ Aspartic acid Glutamic acid Glutamine Proline Glycine Alanine Valine Ornithine Lysine Arginine Total FAA A luciae % fmol cellÀ N cf lenzii % fmol cellÀ % 0.12 7.11 0.21 9.56 0.59 10.43 0.21 12.69 0.45 20.14 1.08 19.24 0.24 0.13 0.02 0.16 0.02 0.01 0.04 0.71 1.66 14.21 7.87 1.02 9.90 1.27 0.76 2.28 42.89 0.00 0.68 0.02 0.21 0.04 0.06 0.06 0.50 2.24 0.00 30.38 1.02 9.56 1.71 2.73 2.73 22.18 0.00 2.79 0.10 0.66 0.10 0.07 0.07 0.18 5.63 0.00 49.46 1.80 11.69 1.80 1.26 1.26 3.24 not found in the other species The protein content was the same in N cf lenzii and A luciae (32% of DW) and higher in N laevis (38% of DW) (Table 3) The carbohydrate content was lowest in N laevis (18%) and highest in N cf lenzii (25%) The ash content was highest in A luciae (41%) Discussion 4.1 Growth and survival of abalone postlarvae The growth experiments with H discus hannai postlarvae fed the different diatom diets, together with biochemical analysis of these diatoms, established that those diatoms that are attractive for the larval settlement usually also support postlarval growth and survival In our previous study (Gordon et al., 2004) we found that the three diatom species used in this research, N laevis, N cf lenzii and A luciae, induced settlement of H discus hannai larvae We examined the suitability of these diatoms to support early postlarval growth, concurring with other studies, where conditions that induced a good larval settlement were usually followed by high growth rates and survival of the settled postlarvae (Daume et al., 1999) However, in the present study, the three dsettlement-inductiveT diatoms differed greatly in their nutritive value A luciae and N lenzii were indeed highly nutritious A luciae was the best among unialgal diets and, combined with N cf lenzii in a two-diatom diet, supported the best postlarval growth and survival in this study On the other hand, N laevis, the preferred species for settlement (Gordon et al., 2004), was unsuitable as a sole diet for early postlarval growth or survival This finding is reminiscent of the results with the diatom Cocconeis scutellum var parva, when fed to postlarvae of H discus hannai by Takami et al (1997) Both diatoms induced good larval settlement, but were poor food for newly settled postlarvae The latter authors attributed their observations to the scarce mucus secretion and the highly adhesive strength of C scutellum var parva These properties made it an unsuitable diet for the postlarvae, which eventually starved In contrast, N laevis, being small, poorly silicified and attached only weakly, is probably more easily edible Yet it did not support growth, and 96% of the postlarvae began to avoid the algae after several days and eventually died within weeks This observation suggests that the abalone postlarvae not like N laevis as a sole food for extended periods, for reasons yet to be determined It could be that N laevis secretes unfavorable or toxic substances that gradually accumulate in the dishes (Wen and Chen, 2002) A good growth of the postlarvae was apparently related to a wholesome diet, as indicated by the synergism between the two bbestQ diatoms, A luciae and N cf lenzii, in the sustenance of the fastest growth rates and largest SL when administered together in this study, as also suggested by Epifanio (1979) However, Kawamura et al (1998a) showed the benefit of a good diet is not necessarily steady and sustained, as we have shown with the mixture of A luciae and N laevis An inconstant nutritional value of this mixture may reflect a dual function, wherein N laevis apparently provides the postlarvae feeding stimulation right after settlement, while A luciae provides the required balanced nutrition for more sustained growth This diet combination could therefore support the best growth only for the first weeks after settlement Afterwards, however, the same reasons that caused the postlarvae to avoid feeding on a uni-algal N laevis diet after several days apparently came into play, making this mixture unsuitable for further growth N Gordon et al / Aquaculture 252 (2006) 225–233 Nutritional developments with postlarval age seem to include increases in the rate and efficiency of feeding and digestion, leading to the acceleration in the postlarval growth rates with most diets during days 13–24 post-settlement; this phenomenon was already noted by other investigators (Martinez-Ponce and Searcy-Bernal, 1998; Kawamura et al., 1998a; Roberts et al., 1999) The reduced postlarval growth rates in the fourth week of the experiment, when they were already over mm long, probably resulted from excessive biomass densities (Kawamura et al., 1998a) reached in the petri dishes 4.2 Nutritional value of diatoms to abalone postlarvae The three diatoms contained protein levels that were deemed optimal for juvenile abalone (Mai et al., 1995b) They were probably also similar to each other in their high protein quality (Brown and Jeffrey, 1995) Since the diatoms were grown under similar conditions (nutrients, light and temperature) and harvested in the same phase of growth, it can be assumed they had similar nutritional value with respect to their amino acid profiles (Brown and Jeffrey, 1995; Brown et al., 1997) Conversely, as in Martin-Jezequel et al (1988), De Roeck-Holtzhauer et al (1993) and Derrien et al (1998), the composition of free amino acid (FAA) varied between the diatoms and was dominated in each diatom by different FAAs In A luciae, our bbestQ unialgal diet, three predominant FAAs (proline, arginine and glutamic acid) were in equal amounts In N cf lenzii, which supported moderate yet steady growth of the postlarvae, proline was predominant Proline is considered an essential amino acid for molluscs (Harrison, 1975) and a major component in the FAA pool in the tissues of early developmental stages of abalone (Litaay et al., 2001) However, arginine, an often limiting essential amino acid for abalone postlarvae (Mai et al., 1994), was the predominant FAA in N laevis, which was not as good a diet as the other two species Good lipid complements also contribute to the nutritional value of a diatom In the three diatoms studied, total lipid content was about double the value reported to be required for maximal growth of juvenile abalone (Uki et al., 1986; Mai et al., 1995a) 231 The relative PUFA content in our three diatoms was high in comparison to diatoms of similar size (Volkman et al., 1989; Renaud et al., 1999) and so was the ratio of n-3 to n-6 PUFA Both of these fatty acid groups are considered essential for the growth of H discus hannai juveniles (Uki et al., 1986; Mai et al., 1996) The essential fatty acid 20:5n-3 (EPA) was the predominant n-3 PUFA in the diatoms studied here, as in other diatoms (Dunstan et al., 1994) This PUFA is reported to promote a fast growth in H discus hannai juveniles (Mai et al., 1995a, 1996; Dunstan et al., 1996) Indeed, our two dbetterT diatoms, N cf lenzii and A luciae, contained larger fractions of n-3 PUFA, especially EPA, than N laevis (though the latter still had a higher EPA content than other Nitzschia species reported for instance by Renaud et al., 1999) On the other hand, in our worst diatom, N laevis, PUFAs were dominated by n-6 and particularly by arachidonic acid 20:4n-6; these PUFAs are important for larval stages of fish but have no special reported importance in abalone The fatty acid 22:6n-3 (DHA), which is low in abalone tissue and therefore presumed of a lesser quantitative importance (Dunstan et al., 1996; Fleming et al., 1996; Mai et al., 1996), was also low in the three diatoms studied here Carbohydrate content in our three diatoms was high compared to other studied diatoms (Brown and Jeffrey, 1995; Renaud et al., 1999; Simental-Trinidad et al., 2001) and within the range needed for juvenile abalone diet (Mercer et al., 1993) The fact that no gross composition nor single chemical component (fatty acid or free amino acid) could be decisively correlated with postlarval growth or survival was, as suggested by others (Chu et al., 1982; Mai et al., 1996; Brown et al., 1997), probably due to the multitude of components that determine the nutritional value of a diatom Conclusions Carefully controlled, mixed and administered diets of selected diatoms have provided consistently good growth and survival of abalone postlarvae during their most critical stage of life, when mortalities are highest The results presented here substantiate the nutritional basis proposed for low performance of abalone postlarvae in their natural habitat and in certain arti- 232 N Gordon et al / Aquaculture 252 (2006) 225–233 ficial settings; the biochemical composition of the diatoms has been shown to affect their suitability as feed for abalone postlarvae Differences in n-3 PUFA and in FAA composition of diets used in this study can partly explain differences in diatom nutritional value, as reflected in postlarval growth and survival The results can be of practical help in the reproduction of abalone in culture Acknowledgements This work was supported by the Israeli Ministry for National Infrastructures (N.G., M.S and A.N.), by several grants from the European Commission (M.S and A.N.) and NIH /NIGMS 08168-22 (J.J.L.) 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0.00 30.38 1.02 9.56 1.71 2.73 2.73 22.1 8 0.00 2.79 0.10 0.66 0.10 0.07 0.07 0.18 5.63 0.00 49.46 1.80 11.69 1.80 1.26 1.26 3.24 not