Aquacult Int (2010) 18:11211135 DOI 10.

1007/s10499-010-9328-5

Growth and fatty acid composition of Octopus vulgaris paralarvae fed with enriched Artemia or co-fed with an inert diet
sa M. P. Valente Jorge Dias Pedro Seixas Ana Otero Lu ndez Manuel Rey-Me

Received: 22 October 2009 / Accepted: 2 February 2010 / Published online: 20 February 2010 Springer Science+Business Media B.V. 2010

Abstract The rearing of Octopus vulgaris paralarvae during its planktonic life stage is a major challenge, as mortality is currently very high and unpredictable. In this study, we examined the survival and growth rates, as well as the fatty acid composition, of O. vulgaris paralarvae fed on three different dietary treatments: group ArDHA was offered juvenile Artemia enriched with a lipid emulsion (Easy DHA-Selco); group ArMA was fed with juvenile Artemia enriched with a mixture of microalgae (Rhodomonas lens and Isochrysis galbana); and group ArMA?ID received the same Artemia as group ArMA complemented with an inert diet. Dietary treatments were tested in triplicate with homogenous groups of paralarvae (25 individuals l-1) established in 50-l tanks, and the experiment was conducted for 15 days. The survival rate of 15-day post-hatch (-dph) paralarvae from groups ArMA (20 8%) and ArMA?ID (17 4%) tended to be higher than in group ArDHA (13 5%), though these differences were not statistically different. The dry weight (DW) of 15-dph paralarvae increased by almost 60% in groups ArMA and ArMA?ID, and nearly 40% in group ArDHA, with respect to hatchlings. The fatty acid (FA) composition of paralarvae revealed a remarkable drop of docosahexaenoic acid (22:6n-3, DHA) from hatchlings to 15-dph paralarvae of all groups (P \ 0.05). However,
ndez (&) P. Seixas M. Rey-Me tica Molecular de la Universidad de Santiago de Compostela (Unidad Asociada al Grupo de Sistema mica y Biolog a Molecular, CIBUS, Campus Sur, Universidad de Santiago de CSIC), Dpto. de Bioqu Compostela, 15782 Santiago de Compostela, Spain e-mail: manuel.rey.mendez@usc.es P. Seixas A. Otero a y Parasitolog a, CIBUS, Campus Sur, Universidad de Santiago de Compostela, Dpto. de Microbiolog 15782 Santiago de Compostela, Spain L. M. P. Valente o Marinha e Ambiental and ICBAS, CIMAR/CIIMAR, Centro Interdisciplinar de Investigac a ncias Biome dicas de Abel Salazar, Universidade do Porto, Rua dos Bragas, 177, Instituto de Cie 4050-123 Porto, Portugal J. Dias ncias do Mar do Algarve, Universidade do Algarve, CIMAR/CCMAR, Centro de Cie Campus de Gambelas, 8005-139 Faro, Portugal

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paralarvae from group ArDHA contained higher levels of DHA than those from ArMA and ArMA?ID (P \ 0.05). Despite Artemia enriched with DHA-Selco contained three-times more DHA than Artemia enriched with microalgae, no benecial effects of this dietary treatment were observed on the performance of paralarvae. Keywords Artemia DHA Fatty acids Growth Octopus Paralarvae

Introduction The rearing of Octopus vulgaris Cuvier, 1797 paralarvae during its planktonic life stage is still a major bottleneck in the development of this species for aquaculture (Iglesias et al. 2007). As far as we know, Itami et al. (1963) were the rst authors to successfully rear O. vulgaris paralarvae until the benthic stage, using as live prey zoeae of the shrimp Palaemon serrifer. More recent works in which zoeae of decapod crustaceans alone, or in combination with enriched Artemia (14 mm), were used as food items have also been successful, though high mortality of paralarvae was observed (Villanueva 1994, 1995; Iglesias et al. 2004; Carrasco et al. 2006). Yet, the constant supply of decapod zoeae to feed paralarvae is limited and uncertain, becoming difcult to use this prey beyond the experimental scale (Navarro and Villanueva 2000). So far, only one work has reported the rearing of O. vulgaris paralarvae until the benthic stage when using Artemia as single live prey, through the use of a large-scale tank and green water conditions (Hamazaki et al. 1991). In contrast, previous works in which Artemia alone (nauplii or juveniles) enriched with either microalgae or commercial enrichment products, or in co-feeding regimen with inert diets, were used to feed octopus paralarvae resulted in mass mortalities after few weeks of rearing (Iglesias et al. 2000; Navarro and Villanueva 2000; Villanueva et al. 2002). Advances in paralarvae rearing were recently reported combining enriched Artemia nauplii with frozen-akes of pacic sandeel (Ammodytes personatus) as feeding strategy (Okumura et al. 2005). One of the main research topics about O. vulgaris paralarvae rearing has been the importance of the preys lipid composition, especially their fatty acid (FA) prole, and its effects on the performance and body composition of paralarvae (Navarro and Villanueva 2000, 2003; Okumura et al. 2005). Comparisons of the lipid classes and FA proles of early life stages of wild octopus with reared paralarvae fed with enriched Artemia revealed some lipid imbalances, both qualitative and quantitative (Navarro and Villanueva 2000, 2003). The essentiality of n-3 highly unsaturated fatty acids (HUFAs) in diets for many marine sh and crustacean larvae is well known (Coutteau et al. 1997; Sargent et al. 1999; Tocher et al. 2008). Several works have reported poor growth, malformations or high mortalities in a variety of marine sh larvae such as gilthead seabream (Sparus aurata), red seabream (Pagrus major), turbot (Scophtalmus maximus) and Senegalese sole (Solea senegalensis) resulting from dietary lipid imbalances or deciencies (Mourente et al. 1993; Watanabe guez et al. 1994; Este vez et al. 1999; Morais et al. 2005; Izquierdo 2006). 1993; Rodr Despite the common use of both enrichment products available in the market and microalgae to enrich Artemia for the rst feeding of O. vulgaris paralarvae, no comparative studies of the effects that these two strategies have on paralarval performance were carried out before. The aim of this study was therefore to analyse the effects of feeding O. vulgaris paralarvae with Artemia juveniles enriched with either a lipid emulsion rich in DHA (Easy DHA-Selco, INVE) or with microalgae of optimal and controlled biochemical composition. From previous works related to the improvement of the nutritional composition of

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Artemia juveniles enriched with different microalgal species (Seixas et al. 2008), a mixture of Rhodomonas lens and Isochrysis galbana (70:30 dry weight basis) was chosen. R. lens was selected due to its very high protein content and moderate levels of HUFAs, whereas I. galbana because of its high levels of HUFAs. An experimental high-protein inert diet for O. vulgaris paralarvae was also formulated, taking into account previous data about the body composition of octopus early life stages (Navarro and Villanueva 2000; Villanueva et al. 2004), and was tested in co-feeding regime with the Artemia enriched with microalgae.

Materials and methods Formulation of an inert diet for paralarvae A high-protein inert diet (crude protein: 63% DM; crude fat: 16% DM) was formulated with high quality feed ingredients (Table 1), and manufactured by Aphytec (France). The pellets (1 mm) were cylindrical in shape and had a moisture content of nearly 9%, being light brown in colour. Such pellets were used in combination with Artemia enriched with microalgae as a feeding treatment. The proximate composition and the fatty acid prole of the inert diet are shown in Tables 1 and 2, respectively. Growth and enrichment of Artemia juveniles as live prey Non-axenic cultures of Rhodomonas lens and Isochrysis galbana were carried out semicontinuously in at-bottomed asks containing 5 l cultures, with a 30% daily renewal rate to obtain biomass of constant and controlled composition. Nutrients were added at a nal concentration of 2 mM NaNO3 in I. galbana culture and of 4 mM in R. lens culture, in order to ensure nutrient saturation conditions (Seixas et al. 2009), which was conrmed by 2 determination of the NO3 concentration (Clesceri et al. 1989) in the out-ow cultures. The daily harvested biomass was used for the on-growing and enrichment of Artemia juveniles. Newly hatched Artemia nauplii (AF, INVE, Belgium) were initially grown with R. lens in 12-l plastic tanks in seawater (34%) at 26.5 0.5C. The two-day-old Artemia (circa 1.3 mm) were then enriched for 4 h (to feed paralarvae that same day at 3:00 p.m.) or for 24 h (supplied at 9:30 a.m. in the following day) with one of the following compounds: either Easy DHA-Selco (group ArDHA) at a concentration of 0.3 g l-1 or with a mixture of R. lens and I. galbana (group ArMA) in a proportion of 70:30 (dry weigh basis). The concentration of DHA-Selco used to enrich Artemia juveniles was reduced to half of that recommended by the manufacturer for nauplii enrichment (0.6 g l-1), as high mortality of Artemia was observed in the 24-h enrichment using this concentration (data not shown). The length of Artemia juveniles was measured under a stereoscope using a calibrated ocular micrometre (n = 40). Samples of juveniles enriched for 4 and 24 h were taken in three different days in the course of the experiment, washed with distilled water and immediately frozen at -18C for later biochemical analysis. Octopus vulgaris paralarvae rearing experiment Several O. vulgaris individuals with nearly 1.5 kg were kept in captivity in optimal conditions inside an 8 9 7 9 2 m cage in the coast of Viana do Castelo (Portugal), which is

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Table 1 Formulation of the experimental inert diet (ID) and proximate composition (% of dry weight) of the ID and of the Artemia juveniles (1.52.3 mm) enriched with either Easy DHA-Selco (ArDHA) or with microalgae (ArMA), used to feed Octopus vulgaris paralarvae Ingredients of the ID LT shmeal (Norvik 70) CPSP G1 Squid protein concentrate Yellow pea Fish oil Egg yolk phospholipids Choline chloride Vitamin and mineral premix Lutavit C35 (BASF) Lutavit E50 (BASF) Bethaine Proximate composition Protein (%) Lipid (%) Carbohydrate (%) Protein/lipid ratio* Protein/energy ratio* ID 62.5 2.9a 16.4 1.0 6.9 0.0 3.8 0.2 27.9 0.4
b b a a

% 13.0 30.15 40.0 10.0 5.0 1.0 0.1 0.5 0.05 0.1 0.1 ArDHA 46.2 4.7b 22.4 1.6
a b b b

ArMA 50.7 1.7b 12.6 0.9c 8.9 0.2a 4.0 0.4a 27.4 0.8a

5.8 1.1 2.1 0.2 22.2 0.8

1 che, France). Data are means SD (triplicate CPSP G: Fish soluble protein concentrate (Soprope analyses for the ID; n = 6 for Artemia juveniles, three samples from each enrichment period: 4 and 24 h). Different superscript letters within the same line indicate signicant differences. ANOVA followed by TukeyKramer HSD tests for post hoc multiple comparisons (a = 0.05)

* Non-parametric test of KruskalWallis (P \ 0.05)

exposed to daily tides. Octopuses were fed every 2 days ad libitum on either sh (Trachurus trachurus) or crabs (Carcinus maenas) at equal proportions, until females started to spawn. In August 2007, a female octopus with an egg-mass was transported to the University of Santiago de Compostela (Spain) and kept isolated in a 200-l plastic tank, in a closed water circuit, at 1820C. Once the bulk of paralarvae started to hatch (late September), newly hatchlings from the same day were individually counted and transferred to 50-l conical bre glass tanks with 50 cm diameter and white walls. Illumination was provided by daylight lamps placed 40 cm above the water surface, establishing an 18 h light/6 h dark photoperiod. Tanks were provided with gentle aeration, and 25% of the water was renewed every 3 days. Before entering the tanks, seawater (salinity of 34%) was ltered through 1-lm cartridge lters and disinfected with UV lamps. Water temperature was kept within the range 1718C in a stable climatized room. Paralarvae density was established at 25 ind l-1 and food was provided since the rst day. Three dietary treatments, each in triplicate, were tested: group ArDHA was fed with juvenile Artemia enriched with DHA-Selco; group ArMA was fed with juvenile Artemia enriched with the mixture of microalgae; and group ArMA?ID was fed the same amount of Artemia as group ArMA plus an inert diet, which was distributed automatically for 15 min, every 3 h, during the light period (3 g of pellets tank-1 day-1). In all groups, Artemia juveniles were

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Table 2 Fatty acid (FA) composition (% of total FA) and total FA (% of dry weight) of the Artemia juveniles enriched with either Easy DHA-Selco (ArDHA) or with microalgae (ArMA), and of the inert diet (ID) which were supplied to Octopus vulgaris paralarvae Fatty acid Juvenile Artemia (1.52.3 mm) ArDHA 14:0 15:0 16:0 16:1n-7 16:4n-3 18:0 18:1n-9 18:1n-7 18:2n-6 18:3n-6 18:3n-3 18:4n-3 20:1n-9 20:4n-6 20:3n-3 20:4n-3 20:5n-3 22:1 22:5n-3 22:6n-3 P Saturated P Monoenes P PUFA P n-3 P n-6 DHA/EPA Total FA 2.8 0.3 0.7 0.0 6.2 0.8 n.f. 8.8 0.4b 16.7 1.8a 6.3 0.4 6.3 0.3 n.f. 7.3 1.9b 4.4 1.5c 1.6 0.3 1.3 0.3 0.4 0.1 9.8 0.7 n.f. 6.7 0.6b 33.1 3.3 30.9 2.3a 36.0 5.6 6.6 0.8 0.7 9.9 1.4a
a b b a a b b a b a a

ArMA 2.1 0.2 0.4 0.0 2.8 0.6 n.f. 11.9 1.0a 3.2 0.1b 9.0 1.1 1.7 0.1 n.f. 18.6 1.9a 14.3 1.8a 0.5 0.1 0.4 0.1 0.9 0.1
b b a a c c c b

ID 6.2 0.4a 0.6 0.0b 18.9 0.9a 8.8 0.1a 1.1 0.1 5.1 0.3c 16.2 1.7a 3.6 0.4c 2.8 0.1b 0.9 0.1 1.7 0.1c 7.8 0.6b 0.3 0.0c 0.9 0.0a n.f. 0.6 0.0c
a

18.0 1.0
b

15.2 1.0
c

1.2 0.3b
b b

2.6 0.2a 13.0 0.8 0.7 0.1 n.f. 2.0 0.3c 29.5 2.2 16.9 2.2b 53.5 4.4 51.5 4.2 2.1 0.1 0.2 6.9 0.8b
c a a b

9.6 0.2b 4.0 0.3a 1.2 0.0 9.0 0.1a 30.8 1.5 32.9 1.8a 35.6 0.6a 30.9 0.6b 4.6 0.2b 0.9 8.8 0.4a

0.8 0.4

29.4 4.8

Data are means SD (triplicate analyses for the ID; n = 6 for Artemia juveniles, three samples from each enrichment period: 4 and 24 h). Different superscript letters within the same line indicate signicant differences (a = 0.05)

supplied in two meals, at 9:30 a.m. and at 3:00 p.m., being the total daily ration 0.1 Artemia ml-1 day-1. Octopus paralarvae were measured under a stereoscope using a calibrated ocular micrometre (n = 30 hatchlings and n = 20 paralarvae per replicate at days 10 and 15 of rearing). Total length and dorsal mantle length were measured as described by Villanueva (1995). The dry weight (DW) of paralarvae was determined by weighing samples of 10 individuals (n = 5 per replicate) which were collected randomly, washed with distilled water and dried at 100 1C for 24 h. Specic growth rate (SGR, % day-1) was calculated as follows: 100 9 [(lnDW2 - lnDW1)/t2 - t1], where DW2 and DW1 represent the DW of paralarvae at sampling days t2 and t1, and ln the natural logarithm.

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Samples of hatchlings and of 10- and 15-dph paralarvae (7090 individuals) collected before providing the rst daily meal (i.e. animals starved for at least 12 h) were briey washed with distilled water and immediately frozen at -18C for later biochemical composition analyses (total lipid and fatty acid proles). Biochemical composition analysis Protein content was determined by the Folin-phenol method (Lowry et al. 1951), after hydrolysis with 1.0 M NaOH at 95C for 1 h, whereas carbohydrate was determined by the phenol/sulphuric acid method (Kochert 1978). Lipid was determined gravimetrically after extraction of total lipids with chloroform/methanol (2:1 v/v) according to Bligh and Dyer (1959). Energy was calculated using the caloric values for protein, lipid and carbohydrate proposed by the National Research Council (1993). The fatty acid composition of Artemia juveniles, inert diet and paralarvae was determined by submitting lipid extracts to methanolysis (5% HCl in methanol) at 85C during 2.5 h (Sato and Murata 1988), followed by extraction of methyl esters with hexane, and analysed in a GCMS (Fisons Instruments, MD-800) using a column Omegawax 250 (Supelco) 30 m 9 0.25 mm and helium as gas carrier. Triheptadecanoin (Sigma, St. Louis, MI) was used as internal standard. All biochemical analyses were carried out in triplicate. Statistical analysis Statistical analyses were carried out with the software SPSS V 14.0.1 (SPSS, Inc.). Total length and mantle length of paralarvae were compared by analysis of variance (ANOVA) followed by TukeyKramer HSD tests for post hoc multiple comparisons, at a signicance level of 0.05. After log-transformation of dry weight data and arcsine-H transformation of survival and biochemical composition percentages, the same statistical tests were carried out (Zar 1999). Statistical comparisons of dietary protein/lipid (P/L) and protein/energy (P/E) ratios and of SGR among groups were carried out by the non-parametric test of KruskalWallis (Zar 1999).

Results Biochemical composition of the diets The inert diet contained the highest protein level (62%, P \ 0.001) of all diets (Table 1). Despite different protein contents were observed in Artemia juveniles from groups ArMA and ArDHA, no statistically signicant differences were found. Lipid levels were considerably higher in Artemia from ArDHA (22%) than in the inert diet (16%) or in Artemia from ArMA (13%, P \ 0.001), whereas carbohydrate was higher in ArMA (P \ 0.01) than in ArDHA or in the inert diet (Table 1). The protein/energy and protein/lipid ratios of both ArMA and the inert diet were found to be higher than in Artemia from ArDHA (Table 1). The fatty acid (FA) composition of Artemia juveniles revealed important differences between groups, as well as in comparison with the inert diet (Table 2). Similar levels of total saturated FA, monoenes and polyunsaturated fatty acids (PUFA) were found in Artemia from ArDHA and the experimental pellets, whereas juveniles from ArMA contained higher levels of total PUFA, at the expense of a decrease in monoenes. The higher

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content of PUFA in Artemia from ArMA was mainly due to the high percentages of 18:3n3 and 18:4n-3 found in this group. The highest level of DHA was found in pellets (9%), being lower in Artemia from ArDHA (6.8%) and substantially lower in ArMA (2%, P \ 0.001). In turn, the highest levels of EPA and of arachidonic acid (20:4n-6) were found, respectively, in ArMA (13%, P \ 0.001) and in ArDHA (1.3%, P \ 0.05). As found for the total lipid levels, the highest amount of FA (% of DW) was found in ArDHA juveniles, followed by the inert diet and nally in Artemia from ArMA (Table 2). Survival, growth and fatty acid composition of O. vulgaris paralarvae After 15 days of rearing, a tendency for higher survival of paralarvae from groups ArMA and ArMA?ID (20 and 17%, respectively) was observed in comparison with group ArDHA (13%), though such differences did not prove statistically differences (Table 3). Due to the low number of paralarvae remaining in tanks after sampling 15-dph paralarvae, the experiment was ceased that same day. Paralarvae were found to grow regularly in the course of the experiment, increasing in both DW and size (Fig. 1; Table 3). Results showed that 15-dph paralarvae from groups ArMA and ArMA?ID increased their DW by almost 60% with respect to hatchlings, whereas this increase in group ArDHA was nearly 40%. Signicant differences in the DW of paralarvae among groups could already be noticed at day 10 (P \ 0.01, Fig. 1), and the same trend was found in 15-dph paralarvae, though statistically signicant differences were only found between groups ArMA?ID and ArDHA (P \ 0.05). Regarding the total length (TL) and the dorsal mantle length (ML) of paralarvae (Table 3), higher values of TL and ML were found in groups ArMA and ArMA?ID in comparison with individuals from ArDHA. Ten-dph paralarvae from ArMA?ID and ArMA showed signicantly higher SGR values than those from ArDHA. Despite a similar tendency could be observed for 15-dph paralarvae, the high weight dispersion observed at this stage did not allowed a statistical differentiation among the various treatments. Concerning the acceptance of food, paralarvae were seen to actively attack juvenile Artemia as soon as this prey was supplied to tanks. In contrast, the ingestion of the inert diet was not observed during the distribution period, mainly due to its fast sinking in the water column. Even if paralarvae were seen to display pursuing behaviour towards the pellets, once they touched the bottom of tanks paralarvae would lose interest on them. However, pellets were left in the bottom of tanks until the next day, when cleaning by siphoning was carried out. Regarding the body composition of paralarvae, a slight decrease of lipid levels was observed with time in octopus from groups ArMA and ArMA?ID with respect to hatchlings (12% of DW) (Table 4). An inverse tendency was observed in 10- and 15-dph paralarvae from group ArDHA, which showed slightly higher lipid levels. The lipid content of 15-dph paralarvae from ArDHA was signicantly higher than values found for groups ArMA and ArMA?ID (P \ 0.05). The saturated palmitic acid 16:0 was the major FA found in hatchlings (Table 4), representing 28% of the total FA, followed by DHA (19.5%) and EPA (14.5%). The sum of saturated FA and of PUFA was very similar (nearly 43% each), whereas monoenes accounted for nearly 13% of the total FA. In general, the changes observed in the FA composition of the reared paralarvae, with respect to the FA composition of hatchlings, reected the FA composition of the dietary treatment (Table 4). The percentage of DHA dropped in 10-dph paralarvae from all groups, whereas EPA remained stable in group ArDHA and increased slightly in groups ArMA and ArMA?ID. After 15 days of rearing,

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15-dph paralarvae ArMA 59.4 9.7 3.30 0.16b 2.20 0.12 3.2 0.2
b b

Table 3 Survival (S, %), total length (TL, mm), dorsal mantle length (ML, mm) and specic growth rate (SGR 9 100, % day-1) of Octopus vulgaris paralarvae fed on three different dietary treatments

Hatchlings ArMA?ID 51.5 7.3 3.40 0.16c 2.23 0.14 3.4 0.4
b b

10-dph paralarvae ArDHA 13.3 4.7 3.40 0.13x 2.24 0.13 2.5 1.6
x

ArDHA

ArMA 19.7 7.5 3.57 0.13y 2.28 0.11 3.2 0.7

x,y

ArMA?ID 17.3 4.0 3.61 0.22y 2.34 0.13y 3.3 0.2

49.3 2.6

TL

3.00 0.13

3.20 0.13a

ML

1.97 0.10

2.13 0.09

SGR*

2.2 0.1

ArDHA: group fed with Artemia enriched with Easy DHA-Selco; ArMA: group fed with Artemia enriched with microalgae; ArMA?ID: group fed with the same Artemia as group ArMA plus the inert diet (ID). Data are means SD (n = 3 tanks). Different superscript letters within the same day indicate signicant differences among groups. ANOVA followed by TukeyKramer HSD tests for post hoc multiple comparisons (P \ 0.05) Aquacult Int (2010) 18:11211135

* Non-parametric test of KruskalWallis (P B 0.05)

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700

Dry weight (g paralarva -1)

600 500

ArDHA ArMA ArMA+ID

b a b

x,y x

400 300 200 100 0

Day 0 Day 10 Day 15

Fig. 1 Dry weight of Octopus vulgaris paralarvae fed on three different dietary treatments. ArDHA: group fed with juvenile Artemia enriched with Easy DHA-Selco; ArMA: group fed with Artemia enriched with microalgae; ArMA?ID: group fed with the same Artemia as group ArMA plus the inert diet. Data are means SD (n = 3 tanks, ve samples per replicate). Different superscript letters within the same day among groups indicate signicant differences (P \ 0.05)

the levels of DHA continued to decrease further in all groups, but paralarvae from group ArDHA evidenced an higher percentage of DHA (12.6%) than paralarvae from groups ArMA and ArMA?ID (9.910.6%, P \ 0.05). A signicant drop in the ratio DHA/EPA was observed from hatchlings to 10-dph paralarvae, decreasing further in 15-dph paralarvae (Table 4). However, this decrease was less evident in paralarvae from ArDHA than in paralarvae from the remaining groups. The levels of arachidonic acid (ARA, 20:4n-6) remained similar to initial values in group ArDHA, decreasing in groups ArMA and ArMA?ID. The sum of monounsaturated FA increased in paralarvae from all groups at the expense of slight decreases in both saturated FA and PUFA.

Discussion The gross composition of the Artemia juveniles (1.52.3 mm) enriched with the mixture of R. lens and I. galbana (ArMA) was in the same range as values reported previously for 1.52.0 mm Artemia juveniles enriched with monocultures of R. lens, I. galbana or other microalgal species (Seixas et al. 2008). Additionally, the FA composition of the Artemia juveniles also reected the ingestion of the microalgal mixture, showing high EPA levels (13% of total FA) but low DHA (2%). The lipid content found in Artemia juveniles enriched with DHA-Selco (22%) was similar to values reported by Navarro and Villanueva (2000) for 13 mm Artemia enriched with Super-Selco (25% lipid), but slightly higher percentages of DHA and EPA (7 and 10%, respectively) were achieved in the present study, supporting the effectiveness of increasing the levels of these HUFA when using DHA-Selco. The survival observed for 15-dph paralarvae in this study was similar or lower than values reported by other authors when feeding paralarvae with Artemia as sole prey (Iglesias et al. 2000; Villanueva et al. 2002, 2004; Okumura et al. 2005), but this could be due to differences in paralarval density, volume of tanks, water-circuit system and prey density. Other reasons that could have inuenced the mortality of paralarvae were related with the daily task of siphoning the bottom of tanks, which was found to be stressful for paralarvae.

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Table 4 Fatty acid composition (% of total FA), total lipid (% of dry weight) and fatty acid (% of dry weight) of Octopus vulgaris hatchlings and of 10- and 15-dph paralarvae fed on three different dietary treatments Fatty acid Hatchlings 10-dph paralarvae ArDHA 14:0 15:0 16:0 16:1n-7 18:0 18:1n-11 18:1n-9 18:1n-7 18:2n-6 18:3n-3 18:4n-3 20:1n-9 20:2n-6 20:4n-6 20:3n-3 20:5n-3 22:1 22:4n-6 22:5n-6 22:5n-3 22:6n-3 P Saturated P 3.1 0.3 0.6 0.0 1.2 0.3 n.f. 3.4 0.2 1.8 0.2 0.7 0.1 n.f. n.f. 5.5 0.5 0.7 0.0 3.4 0.1 1.4 0.0 1.3 0.2 1.0 0.1 0.5 0.1 1.5 0.2 1.5 0.2 0.5 0.0a 2.1 0.1a 1.0 0.1 6.2 0.4 5.1 0.6 1.6 0.2 1.1 0.2 0.3 0.1 3.6 0.1 0.6 0.0a 3.8 0.1a 1.3 0.1
b a b b a a

15-dph paralarvae ArMA?ID 1.9 0.2 0.5 0.0a,b 26.5 0.5 1.4 0.4b 14.6 0.6 1.2 0.1 3.3 0.2 0.8 0.1 2.1 0.5 0.9 0.1
a,b b

ArMA 1.7 0.1 0.4 0.0b 25.7 0.8 1.3 0.1b 14.6 0.1 1.2 0.0 3.1 0.1 0.8 0.0 2.5 0.2 0.8 0.0
b b

ArDHA 1.5 0.1 0.3 0.1 23.1 1.8 1.7 0.6x 14.6 0.7 0.7 0.1 8.1 1.0 2.1 0.4 2.1 0.5 0.5 0.1 0.6 0.0 3.5 0.1x 1.0 0.1
y y y x y x y y y

ArMA 1.4 0.0 0.3 0.0 23.0 0.7 1.2 0.1y 17.4 0.3 1.1 0.1 3.6 0.1 8.0 0.1 0.8 0.0 3.6 0.3 0.9 0.1
x y x y x x x

ArMA?ID 1.5 0.3 0.4 0.0 22.8 1.1 1.4 0.1y 16.9 0.8x 1.1 0.0x 3.6 0.3y 8.3 0.8x 0.9 0.1y 3.9 0.8x 1.2 0.2x 2.9 0.2 0.5 0.0 2.9 0.0z 1.3 0.1x
x

28.0 1.0 25.2 0.3 12.1 0.2 13.5 0.9

5.0 0.1
b a a

4.9 0.8
b a a

6.6 0.7

3.7 0.2 0.4 0.1b 2.3 0.6b 1.2 0.4 16.0 0.7 1.4 0.1 1.3 0.1 0.7 0.0 1.4 0.0 14.5 0.9 42.4 0.7 15.7 0.2 41.8 0.7 36.4 1.3 5.4 0.7 0.9 3.4 0.1
a a

3.7 0.5 0.5 0.0a 3.3 0.3a 1.4 0.2 15.4 0.7 1.3 0.2 1.1 0.2 0.5 0.1 1.2 0.3 13.7 0.3 43.4 0.3 15.8 0.6 40.8 0.3 34.6 0.6 6.2 0.6 0.9 3.1 0.5
a,b a,b

3.1 0.4

3.2 0.2 0.5 0.0 2.8 0.0y 1.4 0.1

x

14.5 0.8 14.2 0.6 1.3 0.1 1.1 0.1 0.5 0.1 1.1 0.1
b

14.6 0.5 1.1 0.2 0.4 0.1 0.9 0.2

16.4 0.5 1.1 0.2 0.4 0.0 1.1 0.2

17.6 0.7x 0.9 0.1 0.6 0.1y 0.3 0.0 0.8 0.1 41.7 1.6 18.3 0.8 40.1 1.1 34.7 1.0 5.3 0.2 0.6 3.2 0.2y

0.8 0.1x,y 0.9 0.1x

19.5 1.3 14.4 0.4 43.7 0.9 40.7 1.4 19.3 0.6 40.0 0.9 32.5 1.0 7.5 0.1 1.0 3.6 0.3

12.6 0.7x 10.6 0.2y 9.9 0.5y 39.5 2.6 21.3 1.1 39.2 1.5 31.9 1.2 7.3 0.3 0.9 4.2 0.4x 42.2 0.5 18.3 0.4 39.5 0.2 34.0 0.2 5.5 0.1 0.6 3.1 0.1y

13.2 0.2 Monoenes P PUFA 43.0 1.1 P n-3 36.9 1.1 P n-6 6.2 0.2 DHA/EPA FA content (% of DW) Total lipid (% of DW) 1.3 3.9 0.2

11.9 1.2 12.6 0.5

11.6 1.2

10.3 1.2

12.4 0.7x 10.3 0.4y 10.6 0.6y

Abbreviations of ArDHA, ArMA and ArMA?ID are like in Table 3. Data are means SD (n = 3). Values coded as 0.0 were below 0.05. Different superscript letters within the same day of paralarvae sampling indicate signicant differences among groups (P \ 0.05)

The DW of paralarvae obtained in the present work was in general slightly inferior to values reported by other authors (Iglesias et al. 2000; Villanueva et al. 2002, 2004), which could be explained by the different rearing temperatures used in this work (1718C) and

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that of those experiments (mean temperature of 20C). Indeed, temperature is a major factor inuencing cephalopod growth rates when food is not a limiting issue (Leporati et al. 2007). Thus, the best DW of 10- and 15-dph paralarvae obtained in this work (431 lg ind-1 and circa 500 lg ind-1, respectively) was inferior to values reported previously by other authors for paralarvae fed with enriched Artemia (nauplii or juvenile) or co-fed with microdiets or with spider crab Maja brachydactyla zoeae (470820 lg ind-1 for 10-dph paralarvae, and from 540 to 880 lg ind-1 for 15-dph paralarvae) (Iglesias et al. 2000; Villanueva et al. 2002, 2004; Carrasco et al. 2006). Protein is the major component of cephalopods body composition (7085% of DW), and in comparison to shes they contain overall 20% more protein and 50100% less lipid and carbohydrate (Lee 1994). The high requirement that cephalopods have for protein is primarily fullled by high ingestion rates and high digestion efciency (Forsythe and Van Heukelem 1987; Lee 1994). The enrichment of Artemia juveniles with microalgae cultured in controlled conditions, or co-fed with the inert diet, was shown to promote better growth and survival of paralarvae than juveniles enriched with DHA-Selco. This could be related with the higher P/E ratio, or high P/L ratio, found in the dietary treatment of groups ArMA and ArMA?ID. Similarly, it has been shown that diets containing maximum P/E ratios promoted higher growth rates of Sepia ofcinalis juveniles (Lee 1994), as well as in other mez-Montes et al. 2003). Since the molluscs such as the green abalone Haliotis fulgens (Go carbohydrate content in Artemia of the different treatments ranged between 6 and 9%, and this source of energy is of minor importance for cephalopods (Lee 1994), the P/L ratio of the diet could be used instead of the P/E ratio to compare the effects that different dietary treatments have on paralarval performance, with the extra advantage of being more easily calculated. However, comparisons of different dietary treatments using the P/L ratio index could only be used whenever carbohydrate levels are similar and do not represent a major dietary component. Previous works with marine-sh larvae have also shown that low dietary P/L ratio or inadequate quantitative or qualitative lipid levels would promote poor performance or could interfere with larval digestion and absorption ability (ie et al. 1997; Olsen et al. 2000; Morais et al. 2005). Regarding the composition of paralarvae, total lipid found in O. vulgaris hatchlings (12% of DW) was similar to values previously reported (1113.4%, Navarro and Villanueva 2000; Okumura et al. 2005). In this study, the lipid content of the reared paralarvae reected somehow the dietary lipid level, as a slightly increase in lipid was observed in individuals from group ArDHA with respect to initial values found in hatchlings, whereas in paralarvae from groups ArMA and ArMA?ID a slightly decrease in lipid was observed. Navarro and Villanueva (2003) have also reported increased lipid levels in paralarvae fed with high-lipid Artemia in comparison with hatchlings, besides diverging with the general tendency for a progressive reduction of lipid in wild juvenile octopus. Certain PUFAs such as 18:3n-3 and 18:4n-3, initially not found in hatchlings, tended to increase in the reared paralarvae due to its presence in the supplied Artemia juveniles, indicating the incorporation of these PUFAs into paralarval body composition. Despite the remarkable drop of DHA in paralarvae from all groups, 15-dph individuals from group ArDHA contained higher levels of DHA than individuals from the remaining groups, and conserved EPA levels similar to values found in hatchlings. However, no benecial effects derived from this better FA prole were observed, as the growth rate of paralarvae from ArDHA was the lowest of all groups and the survival rate tended to be inferior. Navarro and Villanueva (2003) had previously described that the levels of DHA decreased signicantly in paralarvae with enriched Artemia nauplii alone or in combination with microdiets rich in DHA, suggesting that the poor growth and high mortality of paralarvae

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could be related with imbalances in the dietary lipid composition, especially in its FA prole. The decrease of DHA observed in paralarvae could be related with inadequate levels of this FA in the supplied prey, as DHA levels were still low in comparison with octopus hatchlings, but results drive us to speculate if in fact the presentation mode of DHA is a key factor for its correct absorption by paralarvae. In contrast to crustacean zoeae and copepods, which contain HUFA mainly within phospholipids, in Artemia they are located predominantly in the triglyceride fraction (Navarro and Villanueva 2000; Bell et al. 2003). In fact, this could be one of the reasons why crustacean zoeae have been previously used with success in the rearing of octopus paralarvae until settlement, whereas Artemia alone failed in most cases. Previous works with marine-sh larvae (gilthead seabream, red seabream, turbot and Senegalese sole) have shown poor larval growth or deciencies related with qualitative lipid imbalances in the diets (Mourente et al. 1993; Watanabe guez et al. 1994; Este vez et al. 1999; Morais et al. 2005; Izquierdo 2006). 1993; Rodr However, other works showed the ineffectiveness of increasing n-3 HUFA levels in enriched rotifers and Artemia to improve the survival and growth of turbot larvae (Rainuzzo et al. 1994; Reitan et al. 1994). Similarly, experiments carried out with Senegalese sole (Solea senegalensis) larvae, on which Artemia was enriched with graded levels of DHA and EPA to feed larvae, failed to improve the survival and growth of larvae, in comparison with a group fed with non-enriched Artemia (Morais et al. 2004; Villalta et al. 2005). Moreover, Villalta et al. (2005) found that Senegalese sole larvae fed with Artemia without any DHA content, could grow until 36-dph at growth and survival rates as good as larvae fed with Artemia containing medium to high DHA levels. In the present work, despite Artemia enriched with DHA-Selco contained three-times more DHA than Artemia enriched with microalgae, we did not observe any clear positive effects on the growth and survival of paralarvae. The formulated inert diet resulted ineffective for the proposed objective due to inadequate physical properties, and therefore future tests should comprise oating or slowsinking inert diets to match O. vulgaris paralarvae feeding behaviour. However, even if paralarvae from this group were not seen to ingest the inert pellets, a slightly higher DW of these individuals was observed in comparison with group ArMA. This difference could be due to increased dissolved organics in the water leaching from pellets that could be directly absorbed through the skin of paralarvae, as described for cephalopod hatchlings (Lee 1994). We could also speculate if nutrient leaching from uneaten pellets could somehow have stimulated additional Artemia intake. Further work in the eld of inert diets development for paralarvae should be undertaken, as previous works have shown active capture of microdiets by O. vulgaris paralarvae (Villanueva et al. 2002), and reasonable growth rates were achieved when feeding Sepia ofcinalis juveniles with semi-puried diets (Castro et al. 1993; Castro and Lee 1994). Another subject that should deserve more attention in future trials of paralarvae rearing is the effects of green-waters (either as blooms or by adding microalgae to tanks) versus clear-waters. The use of green-waters was shown to improve the survival and growth of more than 40 species in comparison with clear-waters, and although the reasons for these positive effects are not fully understood, several hypothesis such as the stabilisation or improvement of the water quality, the continuous enrichment of prey, and the regulation of lleropportunistic bacteria and antibacterial or probiotic action have been pointed out (Mu Feuga et al. 2003). In this work, the use of the mixture of Rhodomonas/Isochrysis to enrich Artemia originated better results than DHA-Selco to rear paralarvae, and therefore the addition of these microalgae to tanks might be benecial. In fact, some of the few works

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reporting the achievement of benthic octopuses were carried out in large-scale tanks to which microalgae were added (Hamazaki et al. 1991; Iglesias et al. 2004). In conclusion, in order to understand the role or the implications that the levels of dietary DHA may have in O. vulgaris paralarvae development and nutritional requirements, specic studies varying the levels and the source of DHA in Artemia or in inert feeds are needed. It would be important to investigate if the supply of DHA within triglycerides or in phospholipids affects paralarval performance. Tailoring the biochemical composition of Artemia juveniles through the use of microalgae of controlled composition, or with other enrichment products/puried compounds, to feed octopus paralarvae (as combinations of Artemia or in co-feeding treatments) is a key subject of paralarval rearing in order to provide all essential nutrients. It should also be kept in mind that the dietary P/L ratio may be a fundamental nutritional parameter to improve paralarval growth.
Lu s Sa nchez Lo pez, Director of the Aquaculture Acknowledgments We would like to thank Jose Institute of the University of Santiago de Compostela, for kindly authorising the use of the Institute facilities to carry out the octopus rearing trials. Pedro Seixas was supported by a PhD grant (Ref.: SFRH/BD/16419/ ncia e a Tecnologia, Portugal), in the period 20042008. This o para a Cie 2004) conceded by FCT (Fundac a a General de Pesca Mar tima (Spain). work was co-nanced by JACUMARSecretar

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