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1 Aquaculture 300 (2010) Contents lists available at ScienceDirect Aquaculture journal homepage: High DHA content in Artemia is ineffective to improve Octopus vulgaris paralarvae rearing Pedro Seixas a,b, Manuel Rey-Méndez b, Luísa M.P. Valente c, Ana Otero a, a Dpto. de Microbiología y Parasitología, CIBUS, Campus Sur, Universidad de Santiago de Compostela, Santiago de Compostela, Spain b Grupo de Sistemática Molecular de la Universidad de Santiago de Compostela (Unidad Asociada al CSIC), Dpto. de Bioquímica y Biología Molecular, CIBUS, Campus Sur, Santiago de Compostela, Spain c CIIMAR Centro Interdisciplinar de Investigação Marinha e Ambiental and ICBAS, Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Rua dos Bragas, 177, Porto, Portugal article info abstract Article history: Received 29 October 2009 Received in revised form 11 December 2009 Accepted 19 December 2009 Keywords: Octopus Paralarvae Growth Survival Artemia Microalgae DHA We analyzed the performance and biochemical composition of Octopus vulgaris paralarvae fed on three different diets, consisting of Artemia juveniles ( mm) differing in their highly unsaturated fatty acid (HUFA) composition and protein/lipid (P/L) ratio. The live prey were supplied twice a day at equal proportions, with the first daily meal being common to all groups: Artemia enriched with a mixture of the microalgae Rhodomonas lens and Isochrysis galbana (70:30 dry-weight basis). In the second daily meal, the control group (AR+I) was supplied with the same Artemia enriched with microalgae; group AGOLD was fed with Artemia enriched with Ori-Gold (Skretting), a commercial product very rich in docosahexaenoic acid (DHA, 22:6n-3); and group AGOPEL was fed with Artemia enriched with a manually prepared diet rich in protein and in HUFAs. A trend for better survival was observed in group AR+I at 25 days of rearing, though no statistical differences were found among groups. The dry weight (DW) and total length (TL) of 15-day post hatch (dph) and 25-dph paralarvae from groups AR+I and AGOPEL were significantly higher than values found for paralarvae from AGOLD (Pb0.05). A significant and positive linear correlation was found between paralarval DW and dietary protein/lipid ratio (Pb0.01), while no beneficial effects of higher DHA content in Artemia were observed. Regarding the fatty acid (FA) composition of paralarvae, a remarkable drop in DHA was observed in all groups with respect to hatchlings. However, this decrease was more pronounced in paralarvae from AR+I (Pb0.05) than in those from groups AGOLD and AGOPEL. Despite this evidence, the best growth and survival rates were observed in group AR+I, which was the only one where 35-dph paralarvae were attained. These data question the nutritional essentiality of DHA for paralarvae, and suggest that in order to sustain a good performance of paralarvae a minimum dietary P/L ratio should be attained before the HUFA composition of the diet may play a significant role Elsevier B.V. All rights reserved. 1. Introduction Zoeae of some crustacean species have been found to represent appropriate live prey for the rearing of planktonic Octopus vulgaris paralarvae until they settle and become benthic juveniles (Itami et al., 1963; Villanueva, 1994, 1995; Iglesias et al., 2004; Carrasco et al., 2006). In contrast, the supply of enriched Artemia (nauplii or juvenile) alone, or in co-feeding regimes with inert diets for the first feeding of octopus paralarvae failed in most cases (Iglesias et al., 2000; Navarro and Villanueva, 2000, 2003; Villanueva et al., 2002, 2004; Okumura et al., 2005; Kurihara et al., 2006). Previous works with Sepia officinalis hatchlings have also shown better growth results when using mysid shrimp as prey items than with adult Artemia (Domingues et al., Corresponding author. Tel.: x16913; fax: address: anamaria.otero@usc.es (A. Otero). 2001). Only Hamazaki et al. (1991) reported the achievement of benthic O. vulgaris juveniles by using Artemia (1.5 2 mm) as sole prey in a large-scale tank. Despite some successful rearing experiments of octopus paralarvae have been described, their unpredictable mortality is still the major bottleneck of octopus culture. Marine zooplankton is known to represent a better prey for marine fish larvae than rotifers or Artemia, mainly due to differences in some key nutritional components (Sargent et al., 1999; Bell et al., 2003; Støttrup, 2003). Zooplankton is naturally rich in phospholipids, cholesterol, and n-3 highly unsaturated fatty acids (HUFAs) such as docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6) (Navarro and Villanueva, 2000; Bell et al., 2003), which are of major importance as structural components for membrane biogenesis and as precursors of physiologically active molecules (Sargent et al., 1999; Tocher et al., 2008). Natural zooplankton also contains higher amounts of free amino acids than Artemia nauplii, readily available for absorption /$ see front matter 2009 Elsevier B.V. All rights reserved. doi: /j.aquaculture

2 P. Seixas et al. / Aquaculture 300 (2010) (Helland et al., 2003). Limitations to the use of copepods and other zooplankton arise principally from the difficulty in providing sufficient live prey and the risks of disease that extensive culture methods imply (Bell et al., 2003). For this reason, the improvement of the biochemical composition of Artemia and the development of inert microdiets have been pointed as key issues to overcome the high mortalities often encountered in octopus paralarval rearing (Iglesias et al., 2007). Although limited progress has been achieved in the rearing of this species, important information about the body composition of early life stages of O. vulgaris has been published by many authors (Navarro and Villanueva, 2000, 2003; Villanueva et al., 2004, 2009; Okumura et al., 2005; Villanueva and Bustamante, 2006; Seixas et al., 2008; Villanueva and Norman, 2008), allowing many questions on the hypothetical nutritional requirements of octopus paralarvae to be addressed. An important subject is related to changes in the lipid and fatty acid (FA) profiles of reared paralarvae. The effect of the FA composition of food is evident in paralarvae after a few days of rearing, and noticeable changes have been described in comparison with hatchlings or wild juveniles (Navarro and Villanueva, 2000, 2003; Kurihara et al., 2006). The high mortalities and poor growth observed were in general associated with lipid and FA imbalances, such as low DHA content or DHA/EPA ratio b1.5 (Navarro and Villanueva, 2000, 2003; Villanueva and Norman, 2008). We have recently observed similar changes in the FA composition of paralarvae fed with juvenile Artemia enriched with either microalgae (Rhodoand Isochrysis galbana) or Easy DHA-Selcoc (Seixas, 2009). In the present work, three dietary treatments based on combinations of enriched Artemia juveniles were tested to feed octopus paralarvae, with the aim of supplying different essential nutrients. Therefore, for the enrichment of Artemia, different products/compounds were used: Ori-Gold (Skretting) in order to increase its DHA content, a mixture of microalgae (Rhodomonas lens and Isochrysis galbana 70:30 dry-weight basis) to achieve optimal protein levels and moderate in HUFAs (Seixas et al., 2008), or a manually prepared diet rich in protein and in DHA. Survival and growth of paralarvae were recorded, as well as their proximate composition and fatty acid profiles. 2. Material and methods 2.1. Production and enrichment of juvenile Artemia sp. Artemia nauplii (AF, INVE, Belgium) were initially grown with Rhodomonas lens in 12-l plastic tanks at 26.5±0.5 C. Two-day old Artemia were finally enriched for 24 h with a mixture of Rhodomonas lens and Isochrysis galbana (70:30 dry-weight basis) which were cultured semi-continuously in nutrient saturated conditions and with a 30% daily renewal rate, as described elsewhere (Seixas et al., 2009). These enriched Artemia juveniles ( mm) were supplied as the first daily meal to all groups of paralarvae. In the second meal, paralarvae were fed with 3-day old Artemia ( 2.1 mm) enriched for 6 h according to the following procedures: the same mixture of microalgae (AR+I control group); a commercial product rich in DHA (Ori-Gold, Skretting) at a concentration of 0.4 g l 1 (AGOLD); or with a manually prepared diet (AGOPEL). This diet consisted of pellets for turbot (Sorgal, Portugal) devoid of fish-oil, which were extensively grinded to attain very small particle-sizes and supplemented with 10% Ori-Gold (wet weight basis) to increase DHA levels. A ration of 1.5 g of this prepared diet was used for the enrichment of Artemia in a 700- ml glass flask. Samples of Artemia juveniles were collected in three different days over the course of the experiment, washed with distilled water and immediately frozen at 18 C for later biochemical analysis Rearing experiment of Octopus vulgaris paralarvae One day post-hatching (-dph) octopus paralarvae from two brooding females kept isolated in a 1-m 3 plastic-tank at the Spanish Oceanographic Institute (IEO) of Vigo (Spain), were carefully transferred to a 35-l container in July 2008, and transported within 1 h to the University of Santiago de Compostela (Spain). Upon arrival, homogeneous groups of paralarvae were distributed among nine 50-l conical fibre glass-tanks with 0.5 m diameter and white walls. Paralarvae were individually counted to attain an initial density of 10 indiv l 1. The tanks were connected to a semi-closed seawater circuit equipped with particle retention meshes and biological filters. The total volume of the circuit was 600 l and a 10% daily renewal rate was established. Before entering the circuit, seawater passed through 1-µm filtercartridges and was disinfected with UV lamps. Water temperature was kept at 19.5±0.5 C in a stable climate room using thermo-heaters RENA 100 W. Fluorescent day-light lamps (OSRAM L36W/10) were placed 40 cm above water surface with a 14 h light/10 h darkness photoperiod (light period from 10:00 h until 24:00 h). The diet provided to paralarvae consisted of enriched Artemia juveniles ( mm) distributed twice a day (11:00 h and 17:00 h) at half of the daily ration, established at 0.05 Artemia ml 1 day 1. This amount was enough to guarantee prey availability throughout the day with the advantage that few Artemia juveniles were left over the next day. As the mortality of paralarvae increased, the amount of prey supplied to tanks was adjusted to lower levels, especially from day 15 onwards. In any case, Artemia left in the tanks from the previous days were removed as far as possible during the siphoning operations. Three dietary treatments were set in triplicate, each receiving a different combination of Artemia juveniles. The first meal was equal for all groups and consisted of Artemia enriched with a mixture of microalgae, whereas the second meal differed as follows: the same diet for control group (AR+I), Artemia enriched with Ori-Gold (AGOLD); and Artemia enriched with a manually prepared diet (AGOPEL). Total length and dorsal mantle length of paralarvae were measured as described by Villanueva (1995), under a stereoscope provided with a calibrated ocular micrometer (n=40 hatchlings and 20 paralarvae per replicate on each sampling day). To determine paralarvae dry weight (DW), samples of 10 individuals per replicate were collected, washed with distilled water, dried in a stove for 24 h at 100±1 C and individually weighed. Daily growth index (DGI, % day 1 ) was calculated as follows: 100 [(DW f 1/3 DW i 1/3 )/(T f T i )], where DW f and DW i are the dry weight of paralarvae at sampling days T f and T i. Samples of hatchlings and of 15-dph, 25-dph and 35-dph paralarvae (60 to 80 individuals) were collected prior to supplying the first daily meal (i.e. animals starved for at least 12 h), briefly washed with distilled water and immediately frozen at 18 C for later biochemical composition analyses Biochemical composition analysis Biochemical composition analyses included determination of total protein, total lipid, carbohydrate, ash content and fatty acids (FAs). C N H analyses were determined by combustion using an autoanalyzer (Fisons Model EA 1108). Protein content was derived from total nitrogen using the conversion formula N Lipid levels were calculated gravimetrically after extraction of total lipids with chloroform/methanol (2:1 v/v) according to Bligh and Dyer (1959), and carbohydrate was determined by the phenol/sulphuric acid method (Kochert, 1978). Ash content was determined in a muffle furnace heated at 550 C for 24 h. FA profiles were determined by submitting total lipid extracts to methanolysis (5% HCl in methanol) at 85 C during 2.5 h (Sato and Murata, 1988), followed by extraction of the methyl esters with hexane. Identification and quantification of FAs was made using a GC-MS (Fisons Instruments, MD-800) equipped with a column Omegawax 250 (Supelco) 30 m 0.25 mm and using helium as gas carrier. Triheptadecanoin (Sigma, St. Louis, Mo.) was used as internal standard. All biochemical analyses were carried out in triplicate.

3 158 P. Seixas et al. / Aquaculture 300 (2010) Statistical analysis Statistical analyses were performed using the software SPSS V statistical package (SPSS, Inc.). Total length and mantle length of paralarvae were compared by analysis of variance (ANOVA) followed by Tukey Kramer HSD tests for post-hoc multiple comparisons, at a significance level of After log-transformation of dryweight data and arcsine- transformation of biochemical composition and survival percentages, the same statistical tests were carried out (Zar, 1999). 3. Results 3.1. Gross composition and fatty acid profile of juvenile Artemia Protein content in juvenile Artemia (% of dry weight) ranged between 63 and 65% (Table 1), with group AGOPEL showing significantly higher levels (Pb0.05). The highest lipid levels were observed in group AGOLD (16%), being significantly lower in AR+I (12%, Pb0.05); whereas carbohydrate content ranged between 9 and 10% in groups AR+I and AGOPEL, being lower in group AGOLD (7%, Pb0.05). The protein/lipid (P/L) ratios among Artemia treatments (Table 1) decreased from AR+I (5.4) to AGOLD (3.9, Pb0.05). Regarding the FA composition of Artemia (Table 2), juveniles from group AGOLD had the highest level of DHA (8%) and ARA (1.8%); whereas, juveniles from group AGOPEL contained the highest level of EPA (15.4%). The levels of saturated FAs were nearly the same in all groups (25 28%); whereas, monoenes in group AR+I were about half of the values found in AGOLD and AGOPEL. Polyunsaturated fatty acid (PUFA) levels were higher in AR+I, mainly due to the high levels of 18:3n-3 and 18:4n-3. Table 2 Fatty acid (FA) composition (% of total FA) and total FA (% of dry weight) of the Artemia juveniles enriched with different diets that were supplied to octopus paralarvae. Fatty acid Artemia ( mm) AR+I AGOLD AGOPEL 14:0 1.9±0.0 a 1.3±0.1 b 1.8±0.0 a 16: ±0.2 a 12.7 ±0.4 b 12.8±0.3 b 16:1n-7 2.5±0.0 c 3.7±0.9 b 7.5±0.1 a 18: ±0.2 a 10.1 ±0.1 b 9.5±0.1 c 18:1n-9 3.1±0.1 b 14.7 ±0.2 a 14.2±0.2 a 18:1n-7 9.1±0.2 b 8.0±1.1 b 10.6±0.2 a 18:2n-6 1.0±0.2 c 10.1 ±0.5 a 8.0±0.1 b 18:3n ±0.2 a 9.7±1.5 b 5.1±0.1 c 18:4n ±0.3 a 4.1±0.2 b 4.1±0.1 b 20:1n-9 0.4±0.2 b 0.6±0.3 a 1.0±0.1 a 20:2n-6 0.4± ± ±0.0 20:4n-6 0.4±0.1 b 1.8±0.8 a 1.4±0.0 a 20:3n-3 1.0±0.0 a 0.7±0.0 b 0.2±0.0 c 20:4n-3 2.2±0.1 a 0.9±0.1 c 1.4±0.1 b 20:5n ±0.6 b 11.2 ±0.3 c 15.4±0.2 a 22:1 n.f. n.f. 0.2±0.0 22:5n-6 n.f. 0.5±0.2 n.f. 22:5n-3 n.f. 0.4± ±0.1 22:6n-3 1.6±0.2 c 8.0±0.7 a 5.7±0.1 b saturated 28.0 ±0.4 a 25.2 ±0.3 b 24.8±0.4 b monoenes 15.8 ±0.3 c 27.1 ±0.2 b 33.5±0.1 a PUFA 56.2 ±0.5 a 47.8 ±0.5 b 41.7±0.4 c n ±0.7 a 35.0 ±1.2 b 32.2±0.4 c n-6 1.4±0.3 c 12.8 ±0.8 a 9.5±0.1 b DHA/EPA EPA/ARA Total FA 2.5±0.2 b 3.4±0.1 a 3.1±0.3 a saturated also include the FA 15:0 and 22:0. Abbreviations of AR+I, AGOLD and AGOPEL are like in Table 1. Data are means±s.d. (n=3, sampled on three different days). n.f.: not found. Different superscript letters within the same line indicate significant differences among groups (α=0.05) Survival, growth and biochemical composition of paralarvae Survival of paralarvae at the end of 15 days ranged between 35 and 53% (Fig. 1) and between 7 and 20% at day 25. Although there was a certain trend for better survival in the AR+I group, differences were not statistically significant due to high standard deviations in this group (PN0.05). After sampling 25-dph paralarvae for biometric and biochemical composition analyses, the number of paralarvae remaining in some of the tanks was very low or none, and thus it was decided to stop comparisons among groups. However, due to the higher survival observed in AR+I, it was possible to follow this group until day 35 in one tank (Fig. 1). Regarding the dry weight (DW) of 15-dph and 25-dph paralarvae, higher values were observed in groups AR+I and AGOPEL, in comparison with group AGOLD (Pb0.05, Fig. 2). When correlating the DW of paralarvae from the different groups (15-dph and 25-dph) with the P/L of the corresponding dietary treatment, positive linear correlations could be found at a significance level of Pb0.01 (Fig. 3). Total length (TL) and dorsal mantle length (ML) of paralarvae confirmed the results found for the DW, as significant differences were detected among groups (Table 3). The TL of paralarvae from groups AR+I and AGOPEL was higher than values found for AGOLD at both sampling days. As for ML significant differences were only observed between groups AR+I and AGOLD (Pb0.05) for both 15-day old and 25-day old paralarvae. Table 1 Proximate composition (% of dry weight) of the Artemia juveniles ( mm) enriched with different diets that were used to feed Octopus vulgaris paralarvae. AR+I AGOLD AGOPEL Protein (N 6.25) 63.8 ±0.9 b 63.4±0.4 b 65.5 ±0.3 a Lipid 11.8 ±0.8 b 16.2±1.2 a 14.6 ±1.2 a Carbohydrate 9.9±0.5 a 6.8±0.2 b 8.9±0.8 a Ash 16.2 ±1.0 a 14.6±1.2 a,b 13.9 ±1.1 b P:L ratio 5.4±0.2 a 3.9±0.3 b 4.5±0.2 a,b C:N ratio (atoms) 4.8±0.08 b 5.1±0.03 a 5.0±0.02 b AR+I: Artemia enriched with microalgae (R. lens and I. galbana); AGOLD: Artemia enriched with Ori-Gold; AGOPEL: Artemia enriched with grinded pellets and Ori-Gold at 10% (wet weight). Data are means±s.d. (n=3, sampled on three different days). Different superscript letters within the same line indicate significant differences among groups (α=0.05). Fig. 1. Survival (%) of octopus paralarvae in the course of the experiment. AR+I: treatment Artemia enriched with R. lens and I. galbana; AGOLD: treatment Artemia enriched with Ori-Gold ; AGOPEL: treatment Artemia enriched with grinded pellets and Ori-Gold. Data are means ±S.D. (n=3). Survival of group AR+I at days 30 and 35 correspond to a single tank.

4 P. Seixas et al. / Aquaculture 300 (2010) Fig. 2. Dry weight of octopus paralarvae (μg paralarva 1 ) in the course of the experiment. Abbreviations are like in Fig. 1. Data are means ±S.D. (n=3, 10 paralarvae per replicate were individually weighed). Data of 35-dph paralarvae from group AR+I correspond to a single tank (n=15 individuals). Fig. 3. Correlations between the dry weight of 15-dph and 25-dph paralarvae and the protein/lipid ratios of the different diets. 15-dph (white) and 25-dph (black). Regarding the proximate composition of paralarvae, we found a protein content of circa 68% in hatchlings, which decreased slightly to 64 66% in 15-dph and 25-dph paralarvae from all groups (Table 3). As for lipid, no significant differences in 15-dph paralarvae were found among groups. In contrast, the lipid content of 25-dph paralarvae from group AGOLD was significantly higher than levels observed for the other groups (Pb0.05). The results of the C:N ratio found for paralarvae in general supported the obtained proximate composition data (i.e., the proportion of protein to lipid found in each group). As for the FA composition of hatchlings and paralarvae (Table 4), results showed that DHA and palmitic acid (16:0) were the major FAs found in hatchlings (both accounting for 20% of total FAs), followed by stearic acid (18:0) and EPA (15% and 13%, respectively). ARA represented 6.6% of total FAs. Total PUFA in hatchlings represented circa 48% of total FAs, remaining nearly unchanged in paralarvae along time. In contrast, monoene levels increased in paralarvae with respect to hatchlings, at the expense of a decrease in saturated FAs. The most striking differences between hatchlings and paralarvae were related with HUFA levels. Whereas DHA and ARA decreased considerably with time, a progressively increase of EPA was observed in all groups. However, paralarvae from groups AGOLD and AGOPEL contained significantly higher DHA levels than paralarvae from AR+I (Pb0.05). Interestingly, the levels of DHA in paralarvae from AR+I stabilized between days 25 and 35 of rearing; whereas, EPA levels still increased slightly (Table 4). 4. Discussion The importance of HUFAs, especially DHA and EPA, for the early development of paralarvae was previously suggested by some authors (Navarro and Villanueva, 2000, 2003; Okumura et al., 2005; Kurihara et al., 2006; Villanueva and Norman, 2008) after observing high levels of these FAs in the composition of octopus early life stages. Due to the fast growth of these carnivore animals (Villanueva et al., 1995), and very well developed neural and vision systems, it is expected that they have high requirements for DHA and EPA and other structural lipids as well. In the present work, despite considerable differences in the levels of DHA of juvenile Artemia could be obtained through the different enrichment procedures, no beneficial effects of higher DHA could be observed on O. vulgaris paralarvae growth or survival, even if the levels of DHA in the reared paralarvae were significantly higher in group AGOLD (fed with Artemia with the highest DHA content). On the contrary, the DW of 15-dph and of 25-dph paralarvae was well correlated with the dietary P/L ratio, since an increase in paralarval DW was observed with increasing P/L ratio in Artemia. Moreover, it should be kept in mind that the first daily meal was equal to all groups and consisted of Artemia with the highest P/L ratio (AR+I), which means that higher differences in paralarval growth could probably be obtained if instead of Artemia combinations only monodiets had been used. Previous experiments in which marine fish larvae were fed live prey having low P/L ratios, or containing inadequate lipid levels (either quantitative or qualitative) have also shown poor growth or low survival results, or abnormal larval digestion and absorption abilities (Øie et al., 1997; Olsen et al., 2000; Morais et al., 2005a,b). In studies with juvenile cuttlefish (Sepia officinalis) and abalone (Haliotis Table 3 Total length (mm), dorsal mantle length (mm), daily growth index (DGI 100, %day 1 ) and proximate composition (% of dry weight) of octopus hatchlings and of 15-dph, 25-dph and 35-dph paralarvae fed on three different dietary treatments. Hatchlings 15-dph paralarvae 25-dph paralarvae 35-dph AR+I AGOLD AGOPEL AR+I AGOLD AGOPEL AR+I* Total length 2.77 ± ±0.14 a 3.71±0.21 b 3.77 ±0.23 b 4.52 ±0.32 x 4.13±0.26 z 4.33±0.30 y 5.45 ±0.24 Mantle length 2.01 ± ±0.10 a 2.46±0.15 b 2.49 ±0.16 a,b 2.96 ±0.20 x 2.81±0.16 y 2.94±0.16 x,y 3.56 ±0.13 DGI 6.8±0.3 a 4.5±0.6 b 6.4±0.5 a 10.3 ±0.2 x 8.5±0.2 y 9.6±0.1 x 2.6 Protein (N 6.25) 67.8± ± ± ± ± ± ± ±0.1 Lipid 14.7 ± ± ± ± ±0.7 y 11.8±0.5 x 10.7±0.4 y 10.0 ±0.6 C:N ratio (atoms) 4.59 ± ±0.08 b 4.83±0.08 a 4.70 ±0.06 b 4.87 ±0.05 y 4.91±0.05 x,y 4.95±0.07 x 4.69 ±0.01 AR+I: paralarvae fed with Artemia enriched with R. lens and I. galbana; AGOLD: paralarvae fed with Artemia enriched with Ori-Gold; AGOPEL: paralarvae fed with Artemia enriched with grinded pellets plus Ori-Gold at 10% wet weight. Data are means±s.d. (n=3, sampled on three different days). *Analyses of 35-dph paralarvae are from a single tank (triplicate analysis). Different superscript letters within the same line indicate significant differences among groups (Pb0.05).

5 160 P. Seixas et al. / Aquaculture 300 (2010) Table 4 Fatty acid (FA) composition (% of total FA) and total FA (% of dry weight) of Octopus vulgaris hatchlings and of 15-dph, 25-dph and 35-dph paralarvae fed on three different dietary treatments. Fatty acid Hatchlings 15-dph paralarvae 25-dph paralarvae 35-dph paralarvae AR+I AGOLD AGOPEL AR+I AGOLD AGOPEL AR+I* 14:0 1.7± ± ± ± ± ± ± ±0.0 15:0 0.5± ± ± ± ± ± ± ±0.0 16: ± ± ± ± ±0.6 x,y 14.2±0.2 y 12.7±0.6 x 14.0 ±0.7 16:1n-7 1.2± ± ± ± ± ± ± ±0.4 18: ± ± ± ± ±1.0 x 15.1±0.8 x,y 13.6±0.4 y 14.5 ±1.0 18:1n ±0.2 n.f. n.f. n.f. n.f. n.f. n.f. 2.2 ±0.5 18:1n-9 3.0± ± ± ± ± ± ± ±0.5 18:1n-7 1.8± ± ± ± ± ± ± ±0.6 18:2n-6 1.2± ± ± ± ±0.0 x 2.8±0.9 y 3.1±0.3 y 2.3 ±1.0 18:3n-3 n.f. 5.2± ± ± ±0.2 x 4.8±0.2 y 5.4±0.6 x,y 5.0 ±0.3 18:4n-3 n.f. 2.6±0.3 a 1.7±0.2 b 1.8±0.4 b 3.1±0.1 x 2.2±0.1 y 2.6±0.4 x,y 2.4 ±0.1 20:0 n.f. 0.4± ± ± ± ± ± ±0.0 20:1n ± ± ± ± ± ± ± ±0.3 20:2n-6 0.7± ±0.0 a 0.7±0.0 b 0.7±0.0 b 0.6±0.0 x 0.8±0.1 y 0.8±0.1 y 0.8 ±0.1 20:4n-6 6.6± ±0.4 a 3.8±0.2 b 3.5±0.1 a,b 2.7± ± ± ±0.4 20:3n-3 1.8± ±0.1 a 1.4±0.1 b 1.4±0.1 b 1.6± ± ± ±0.1 20:4n-3 n.f. 1.7±0.4 a 1.2±0.1 a,b 1.0±0.1 b 2.1±0.1 x 1.5±0.1 y 1.6±0.1 y 1.7 ±0.1 20:5n ± ± ± ± ± ± ± ±2.2 22:0 n.f. 0.9± ± ± ± ± ± ±0.2 22:1 1.5± ± ± ± ± ± ± ±0.2 22:4n-6 1.6± ± ± ± ± ± ± ±0.2 22:5n-6 0.7± ± ± ± ± ± ± ±0.2 22:5n-3 2.2± ± ± ± ± ± ± ±0.2 22:6n ± ±1.0 b 13.6±0.7 a 12.4 ±0.1 a 7.2±0.5 y 10.0±0.3 x 9.6±0.6 x 8.0 ±0.7 saturated 38.1 ± ± ± ± ± ± ± ±1.4 monoenes 13.6 ± ± ± ± ± ± ± ±1.0 PUFA 48.4 ± ± ± ± ± ± ± ±0.5 n ± ± ± ± ± ± ± ±1.4 n ± ±1.0 a,b 10.2±0.0 a 9.3±0.3 b 5.7±0.2 y 7.7±1.1 x 8.1±0.6 x 7.1 ±1.9 DHA/EPA FA (% of DW) 8.6± ± ± ± ± ± ± ±0.2 Abbreviations are like in Table 3. Data are means ±S.D. (n=3, sampled on three different days). *Analyses of 35-day old paralarvae are from a single tank of group AR+I (triplicate analysis). n.f.: not found. Different superscript letters within the same day indicate significant differences among groups (Pb0.05). fulgens) the best growth rates were achieved with maximum dietary protein/energy ratios (Lee, 1994; Gómez-Montes et al., 2003). Cephalopods are carnivorous species that show very high growth rates, with protein making up from 70 to 85% of their body composition, and amino acids are used as main energy fuel, even for routine metabolism (Lee, 1994). Therefore, it is not surprising that the level of protein in the diet for octopus paralarvae, or perhaps more importantly, the P/L ratio in the diet, is a more important factor than for marine fish larvae. In fact, the P/L ratios that promoted best growth results in early life stages of marine fish species ( ) reported by other authors (Øie et al., 1997; Morais et al., 2001, 2005a,b), were lower than the best ratio described in this study (5.4 in AR+I). In a recent study we analyzed the proximate composition of wild Maja brachydactyla zoeae and observed a P/L ratio of 7.1 for this prey (Seixas, 2009), that has been pointed out in previous work as appropriate to rear octopus paralarvae (Iglesias et al., 2004; Carrasco et al., 2006). The DW of 25-dph paralarvae found in this study ( mg) was within the same range of values reported previously by other authors when feeding paralarvae with Artemia alone or in co-feeding regime with microdiets (Navarro and Villanueva, 2000; Moxica et al., 2002; Villanueva et al., 2002, 2004; Okumura et al., 2005). However, these values were lower than those reported by other authors ( mg) when using crustacean zoeae alone or combined with Artemia (Itami et al., 1963; Villanueva, 1995; Iglesias et al., 2004; Carrasco et al., 2006). In all cases establishing direct comparison of DW values among different studies is always risky and implies a certain degree of erroneous conclusions, as many key rearing factors vary considerably, such as temperature, prey density, tank dimensions and the use or not of green-water conditions. The protein content observed in O. vulgaris hatchlings (68%) was very similar to values reported by Villanueva et al. (2004) for hatchlings or wild juveniles (69 to 76%). In this study, although a slight decrease in the protein content was observed from hatchlings to 15-dph paralarvae, values remained unchanged in 25-dph and in 35-dph paralarvae. Lipid levels found in octopus hatchlings (14.7%) were slightly higher than values reported by other authors (10 13%, Navarro and Villanueva, 2000; Okumura et al., 2005; Kurihara et al., 2006). The higher lipid levels found in 25-dph paralarvae from AGOLD in comparison with the remaining groups (Pb0.05), reflected the higher lipid content of the supplied diet, i.e. Artemia enriched with Ori-Gold (16% lipid), a product that contains 85% lipid (data not shown). Navarro and Villanueva (2000, 2003) have also described an increase in the lipid levels of reared paralarvae (14 25%) fed with high-lipid Artemia in comparison with hatchlings, a tendency that these authors found to be in disagreement with the progressive reduction of lipid levels in wild octopus juveniles with increasing weight (from 12% to circa 7%). The FA composition of octopus hatchlings observed in this work was similar to results described by other authors (Navarro and Villanueva, 2000, 2003; Okumura et al., 2005; Kurihara et al., 2006), with an overall abundance of DHA (20%), EPA (13%) and the saturated FA 16:0 and 18:0 (20 and 16%, respectively). Following the trend of previous data reported by Navarro and Villanueva (2000, 2003) and Kurihara et al. (2006), we have also found remarkable changes in the PUFA profile of reared paralarvae with respect to hatchlings. DHA levels dropped significantly in paralarvae from all groups and differences were already clear after 15 days of rearing, decreasing further in 25-dph paralarvae. In contrast, Okumura et al. (2005) reported a certain stabilization of DHA levels in paralarvae fed with Artemia nauplii complemented with a diet naturally rich in DHA (sandeel flakes). In the present work we observed that a diet poor in DHA (AR+I) was not a limiting factor for paralarvae development. In fact, despite this Artemia contained the lowest DHA levels among the different treatments, paralarvae fed with this diet displayed the best growth and survival and were the only ones to attain

6 P. Seixas et al. / Aquaculture 300 (2010) dph. Moreover, paralarvae from this group presented the most pronounced reduction of DHA levels in its body composition, but no correlations with poor growth or higher mortality were observed. As in the present work, Kurihara et al. (2006) did not found significant differences in the growth of paralarvae being fed with enriched Artemia alone (poor in DHA) or Artemia plus sandeel flakes (rich in DHA) in the first month of rearing. However, the best results of paralarvae performance after 42 days of rearing obtained by those authors were attained with the mixed diet. Rearing trials with marine fish larvae have also shown that Senegal sole (Solea senegalensis) fed with Artemia nauplii with no DHA content, but containing other n-3 PUFA, could survive and grow as well as larvae fed nauplii with increasing DHA levels (Morais et al., 2004; Villalta et al., 2005). Other studies conducted with seabream (Sparus aurata)orturbot(scophtalmus maximus) showedno relation between larval survival and n-3 HUFAs levels in supplied diets (Koven et al., 1990; Rainuzzo et al., 1994; Reitan et al., 1994). However, several other studies with red seabream (Pagrus major), seabream (Sparus aurata) and Atlantic halibut (Hippoglossus hippoglossus) showed positive relationships between n-3 HUFAs and the performance of fish larvae (Watanabe, 1993; Salhi et al., 1994; Ibeas et al., 1994; Hamre and Harboe, 2008). Reaching the accurate n-3 HUFA requirements in early life stages of marine species is a hard task requiring considerable experimental work, due to the complexity of determining the exact balance and quantity of n-3 HUFAs to be included in the diet, as reported for seabream (Ibeas et al., 1996, 1997, 2000). Specific work to determine n-3 HUFA requirements of octopus paralarvae is still needed, both at quantitative and qualitative levels. Present results do not completely exclude the importance of DHA for the successful rearing of paralarvae, but indicate that before carrying out this kind of study, it is crucial to optimize the proportion of protein to lipid in the diet that will allow the best growth and survival of paralarvae. The importance and requirements of other lipids such as cholesterol and phospholipids should also be investigated as previously pointed out (Iglesias et al., 2007; Villanueva and Norman, 2008). In conclusion, dietary P/L ratio was shown to influence paralarval growth and seemed to improve survival as well. The supply of Artemia containing very low DHA levels was not a limiting factor to promote good performance of paralarvae, as Artemia containing four-times more DHA did not improve growth or survival. The fine tuning of Artemia biochemical composition is required to improve paralarvae performance and should continue to be a matter of research in order to progress in octopus culture. Interestingly, it has been shown that the enhancement of important nutritional components such as phospholipids, vitamins and free amino acid in live prey is very effective in short-term boosting (Barr et al., 2005; Monroig et al., 2007). These techniques might be useful to improve the nutritional composition of Artemia juveniles for the first feeding of octopus paralarvae, in order to overcome the bottleneck of octopus rearing. Acknowledgments We are thankful to José Iglesias from the Spanish Oceanographic Institute (IEO) of Vigo for the supply of octopus paralarvae and to José Luis Sánchez, Director of the Aquaculture Institute (IA) of the University of Santiago de Compostela, for kindly authorizing the use of the IA facilities. Pedro Seixas was financed through a Ph.D. grant (Ref.:SFRH/BD/16419/2004) by the Science and Technology Foundation (FCT) of Portugal, in the period This work was cofinanced by JACUMAR Secretaría General de Pesca Marítima (Spain). 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