Amino acid composition of early stages of cephalopods and effect of amino acid dietary treatments on Octopus vulgaris paralarvae

Aquaculture 242 (2004) 455 – 478 www.elsevier.com/locate/aqua-online

Amino acid composition of early stages of cephalopods and effect of amino acid dietary treatments on Octopus vulgaris paralarvae R. Villanueva a,*, J. Riba a, C. Ruı´z-Capillas b, A.V. Gonza´lez a, M. Baeta a a

Institut de Cie`ncies del Mar (CSIC), Passeig Marı´tim 37-49, E-08003 Barcelona, Spain b Instituto del Frı´o (CSIC), Ciudad Universitaria s/n, 28040 Madrid, Spain

Received 30 January 2004; received in revised form 7 April 2004; accepted 7 April 2004

Abstract During the present study, we aimed to provide a first look at the amino acid composition of the early stages of cephalopods and follow possible effects of certain dietary treatments. Amino acid profiles of cuttlefish Sepia officinalis, squid Loligo vulgaris and octopus, Octopus vulgaris hatchlings and wild juveniles of L. vulgaris and O. vulgaris were analysed. Cephalopod hatchlings showed high fractions of non-protein nitrogen (NPN), from 25% to 38% of the dry weight. Lysine, leucine and arginine represented half of the total content of essential amino acids (EAA), and glutamate and aspartate represented also nearly half of the non-essential amino acids (NEAA). In O. vulgaris, a general tendency for a decrease in the level of EAA from mature ovary and eggs to hatchlings was observed. Hatchlings after 4 days of fasting lost 28% of their dry weight and the level of EAA and NEAA decreased in both the total content and free forms. Free proline after 2 days of fasting and free tyrosine at 4 days of fasting were not detected. Comparison of the total EAA profiles of preys showed few differences between enriched Artemia nauplii and hatching crab zoeae (Pagurus prideaux and Maja squinado). The enriched Artemia nauplii EAA profiles showed no differences with the EAA profiles of O. vulgaris paralarvae during first 10 days of culture, except for histidine. Present results confirm the positive capacity for amino acid uptake from seawater by early stages of cephalopods. In the three species analysed, radiolabelled phenylalanine was incorporated in inverse relation to body size. After 10 days of culture, O. vulgaris paralarvae showed a tendency to increase the levels of total and free amino acids in the groups receiving a daily amino acids solution. At 20 days of age, the O. vulgaris cultures that received the amino acids solution had survivals that on average were three times that of the control group. However, the supposed beneficial effects of the

* Corresponding author. Tel.: +34-932-309-500; fax: +34-932-309-555. E-mail address: [email protected] (R. Villanueva). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.04.006

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amino acids solution remained unclear, as the dry weight of these paralarvae was equal or lower to that of paralarvae from the control group. In wild O. vulgaris juveniles, the percentage of protein and total amino acids increased with the dry weight of the individuals. These juvenile biochemical changes were associated with strong morphometric changes in body proportions after settlement with the development of the muscular, protein-rich arm crown. D 2004 Elsevier B.V. All rights reserved. Keywords: Cephalopods; Amino acids; Larvae; Sepia; Loligo; Octopus

1. Introduction All living cephalopods are carnivores and proteins are the major organic materials in cephalopod tissue. Analysis from wild stomach contents of planktonic paralarvae indicates that they also select a carnivorous diet (Passarella and Hopkins, 1991; Vecchione, 1991) and the high protease activity recorded from the digestive enzymes of wild individuals during their early life also suggests a proteinaceous diet from first feeding (Boucaud-Camou and Roper, 1995). Due to the rapid paralarval and juvenile growth, there is a large amino acid requirement for maintaining optimal growth and to supply the fuel for energy in this group of animals that have a vigorous protein and amino acid metabolism (Lee, 1994). The present knowledge of the amino acid composition of this group of molluscs only comes from subadult and adult forms and it is focused on selected organs or body portions, such as mantle and arms (Florkin and Bricteux-Gre´goire, 1972; Jhaveri et al., 1984; Iwasaki and Harada, 1985; Rosa et al., 2002), branchial heart (Nakahara and Shimizu, 1985), ovary (O’Dor and Wells, 1973), eggs (Rossi et al., 1985), nervous system (D’Aniello et al., 1995), lens (Siezen and Shaw, 1982), retina (Seidou et al., 1988), ink (Shirai et al., 1997), beak, shell and radula (Hunt and Nixon, 1981). In adult cephalopods, direct mobilization of muscle protein provides metabolic energy during periods of starvation and the direct use of protein as an energy reserve may account for the lack of major glycogen or lipid reserves in cephalopod tissues (Storey and Storey, 1983; O’Dor et al., 1984). In fact, early stages of cephalopods have low lipid content of 11 –13% of dry weight in hatchlings (Navarro and Villanueva, 2000). However, studies on their lipid composition have shown that their lipid requirements are high in phospholipids, cholesterol and especially, polyunsaturated fatty acids, particularly docosahexaenoic acid (DHA) for the maintenance of the structural and functional properties of the cell membranes during fast early growth (Navarro and Villanueva, 2000, 2003). On the other hand, as far as we know, no data on the amino acid composition of the early stages has been published. Consequently, the amino acid requirements of early stages of cephalopods are practically unknown. Semi-purified diets enriched with amino acids have been used only with limited success to feed subadult cephalopods such as Sepia officinalis (Castro et al., 1993; Castro and Lee, 1994). These studies concluded that more information on the amino acid requirements for cephalopods is necessary before artificial diets could be developed. Amino acid profiles are usually the first consideration in formulating test or commercial feeds and

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the amino acid composition of whole fishes has been used as a starting point for the determination of the amino acid requirements of fishes and invertebrates (Wilson, 1989). During the present study we aimed to provide a first look at the amino acid composition in the early stages of cephalopods and follow the possible effects of dietary treatments. In this way, two objectives were proposed. First, to analyse the amino acid profiles of the mature ovary, eggs in different stages of development, hatchlings and wild juveniles in order to obtain a ‘‘natural’’ amino acid profile of the common octopus Octopus vulgaris Cuvier, 1797. To complete this objective, hatchlings of the European cuttlefish S. officinalis Linnaeus, 1758 and common squid Loligo vulgaris Lammarck, 1798 hatchlings and wild juveniles were analysed to determine possible common trends in the amino acid composition of early cephalopod stages. These three cephalopod species represent the three main cephalopod orders, all of which have high commercial interest. The second objective was to determine the effects of amino acid enriched diets on O. vulgaris paralarvae during the first month of culture. Artemia nauplii enriched with crystalline methionine (Tonheim et al., 2000) was used as food during paralarval rearings and compared with control experiments. Methionine was chosen, since it is the essential amino acid that has the lowest relative content in Artemia nauplii and zooplankton (Helland et al., 2000, 2003). Methionine and lysine supplementation in artificial diets has been shown to promote significant growth in subadults of S. officinalis (Domingues, 1999) and a high methionine content has been reported in the lens proteins of the squid Nototodarus gouldi (Siezen and Shaw, 1982). To complete this objective, amino acid profile of natural preys of the cephalopod paralarvae was also analyzed as a first approximation to determine the paralarval amino acid requirements, as observed in cultured fish larvae (Conceicßao et al., 2003). With this aim, decapod crab zoeae used previously with success as live prey on cephalopod paralarval cultures as Pagurus prideaux (Villanueva, 1994, 2000) and Maja squinado (Carrasco et al., 2003; Iglesias et al., in press) were analyzed. In addition, the effects of amino acid uptake from seawater by O. vulgaris paralarvae were explored by the daily use of a crystalline amino acids solution during two rearing experiments. Amino acid uptake from seawater plays a vital role in the biology of soft-bodied marine invertebrates and, generally, supplies the nutritional needs of the epidermis and active superficial structures, which commonly form less than 10% of the total metabolic needs in adults (Ferguson, 1982). These metabolic rates increase in early stages and the rates of amino acid uptake in larval forms of molluscs from a concentration of 1.6 AM in seawater could supply sufficient energy to account for 39 –70% of the rates in veliger larvae of Haliotis rufescens (Manahan, 1990). In cephalopods, the first observations on this capacity were recorded in planktonic hatchlings of Octopus spp. and benthic hatchlings of O. joubini, which showed uptake of amino acids and hexoses from seawater (Castille and Lawrence, 1978). In this group of soft-bodied molluscs, the epithelial cells and microvilli of the integument of the body, arms and tentacles possess the classical features of absorptive epithelia (Budelmann et al., 1997), and the capacity for amino acid uptake from seawater, as recorded for European cuttlefish S. officinalis adults (de Eguileor et al., 2000).

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2. Materials and methods 2.1. Collection of material 2.1.1. Cephalopod hatchlings and wild juveniles Egg masses of S. officinalis and L. vulgaris attached to fishery lines were collected off Barcelona (NW Mediterranean) by means of the artisanal trap fishery and they were transported to the laboratory the same days during April and May 2001. Eggs were incubated at ambient temperature (range: 15.4– 18.4 jC), using an open circuit of filtered seawater at the Institut de Cie`ncies del Mar (ICM), Barcelona. Egg masses of O. vulgaris were obtained from a broodstock maintained as described in Navarro and Villanueva (2000). During June 2001, samples of active, healthy individuals of all three species were preserved during the first 24 h after hatching in the laboratory. The samples were collected using a hand net, washed in tap water, then placed on blotting paper to remove the excess water and weighed using an Ohaus Analytical Plus AP250D-O microbalance. Samples were frozen at 80 jC and freeze-dried overnight. The dry weight was obtained from the freeze dried samples, which were then stored again at 80 jC for subsequent nitrogen and amino acid analysis (see below). In addition, the weight of the shell in S. officinalis hatchlings was determined, in order to quantify this highly calcified portion of the body, which is very low in proteins and amino acids. As a result, the shells were dissected out and weighed separately for five groups of five hatchling S. officinalis individuals. Five L. vulgaris juveniles (2.0 – 2.8 g wet weight) were collected from the local trawl fishery off Barcelona during June 2001. Five benthic O. vulgaris juveniles (3.5 – 14.2 g wet weight) were captured from the wild by scuba diving at depths between 10 and 15 m off l’Estartit (NW Mediterranean) from December 1998 to June 1999 (the same specimens reported for lipid and fatty acid composition in Table 2 of Navarro and Villanueva, 2003). All juveniles were weighed and freeze-dried upon arrival in the laboratory. 2.1.2. Rearing experiments of O. vulgaris paralarvae An egg mass of O. vulgaris, obtained from a broodstock maintained as described in Navarro and Villanueva (2000), was used for the rearing experiments conducted in the open-circuit seawater system of the ICM, Barcelona. Two 700-l semi-closed seawater systems (see Villanueva et al., 2002 for system details) connected to the open circuit of the ICM were used for the paralarval rearing experiments, which were conducted during June and July 2001. During the present study, four experimental treatments were used (see below) and each treatment was conducted in quadruplicate. Paralarvae were reared for 30 days using cylindrical 25-l volume PVC tanks, of 65 cm height and 24 cm diameter. Water flow was 80 l h 1. A total of 1200 freshly hatched O. vulgaris paralarvae (density equivalent to 48 paralarvae l 1) were counted and placed in each rearing tank. All paralarvae used were from the same egg mass. The temperature ranged from 19.2 to 21.1 jC (mean 20.4 jC). In all experiments, illumination was constant for 24 h day 1. During experiments, tanks were cleaned daily and any dead animals were removed. In all experiments, on day 20, all individuals in each rearing tank were counted and transferred

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to an identical clean tank. The percentage survival (S) was calculated as S = 100S(I B) 1, where S was the number of surviving individuals on day x, I was the initial number of individuals in the culture, and B was the total number of individuals killed for sampling purposes to day x. To determine growth and for subsequent analysis (see below), samples were collected every 5 days, from day 0 to day 30. Six samples (groups of 18– 106, mean = 31 paralarvae) of fresh paralarvae (total sample fresh weight ranging from 60 to 230, mean = 81 mg) were consecutively collected from each rearing experiment on the sampling days. Paralarval samples were collected 2 h after the first daily food addition (see below), washed in tap water, placed over a plastic mesh on blotting paper to remove excess water, stored in Eppendorf tubes and weighed using an Ohaus Analytical Plus AP250D-O microbalance. Samples were frozen at 80 jC and freeze-dried overnight. The dry weight was obtained from the freeze-dried paralarvae, which were stored again at 80 jC for subsequent nitrogen and amino acid analysis (see below). 2.1.3. Feeding and amino acid treatments of O. vulgaris paralarvae All treatments were fed enriched Artemia nauplii (AF, INVE Aquaculture) 450 Am in length, which were provided from day 0 to day 20 at a ration of 6 –7 nauplii ml 1 day 1, decreasing to 4 nauplii ml 1 day 1 thereafter until an age of 30 days, due to a decrease in paralarval survival and density (see Results). Artemia nauplii were enriched in seawater for 24 h at 28 jC with one of the following enrichment diets: (a) Diet SS: DC Super Selco (INVE) 0.6 g l 1 and (b) Diet MET: DC Super Selco 0.6 and 0.8 g l 1 of L-methionine (Sigma Products). Artemia nauplii were added twice daily, at approximately 10:00 and 20:00 h, with half of the total daily ration. Nauplii for the second daily feed were stored in aerated seawater at 4 jC to prevent energetic losses before addition. Remains of live Artemia nauplii in the rearing tanks from the previous day were removed as far as possible before the first daily addition. To test the influence of the presence of amino acids in seawater, essential L-amino acids in crystalline form (Sigma Products) were added to the rearing tanks. An amino acid mixture was formulated equivalent in composition to the total essential amino acid profile plus tyrosine profile of O. vulgaris hatchlings, which had been previously obtained specifically for this purpose. The crystalline amino acids were dissolved previously in 500ml seawater and then added directly to the rearing tank. The final concentration (mg l 1) of each amino acid in the rearing tank was: arginine 2.1, histidine 2.1, isoleucine 1.3, leucine 1.3, valine 1.2, lysine 1.8, phenylalanine 1.7, methionine 1.5, threonine 1.2 and tyrosine 1.8, leading to an amino acid solution of 10 AM. This amount represented the daily addition of a solution of 400 mg of crystalline amino acids to each 25-l rearing tank. After the amino acid addition, the water flow was closed and gentle aeration using air pumps was maintained for 1 h. After this period the water flow was restored. The amino acids solution was added once a day, approximately between 0900 and 1000 h, before O. vulgaris were fed their first daily ration of enriched Artemia nauplii. During the present study four treatments were tested. (1) Control group: paralarvae were fed Artemia nauplii enriched with Diet SS; (2) MET group: paralarvae were fed Artemia nauplii enriched with Diet MET; (3) AA group: paralarvae were fed Artemia nauplii enriched with Diet SS and they also received a daily amino acids solution in the rearing tank; and (4) METAA group: paralarvae were fed Artemia nauplii enriched with

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Diet MET and they also received a daily amino acids solution in the rearing tank. An unfed group was also maintained from the hatchling stage to day 4. 2.1.4. Paralarval preys The amino acid compositions of Artemia nauplii from the Diet SS and Diet MET groups, as well as the amino acid composition of recently hatched zoeae of the hermit crab P. prideaux and the spider crab M. squinado were analysed. These zoeae (not used as food during the present study) have been used previously with success as a food resource for rearing L. vulgaris (Villanueva, 2000) and O. vulgaris (Villanueva, 1995; Villanueva et al., 1995; Carrasco et al., 2003; Iglesias et al., in press) during the first 2 months of paralarval life. Ovigerous females of P. prideaux were collected from the by-catch of the artisanal fishery off Vilanova i la Geltru´ (Barcelona, NW Mediterranean) and ovigerous females of M. squinado were collected from the Galician coast (NW Spain, East Atlantic). Ovigerous females were transported to the laboratory, maintained as described in Villanueva (1994) and the recently hatched zoeae were used for analysis. Prey samples were collected and preserved as described for the cephalopod paralarvae, frozen at 80 jC and freeze-dried overnight. 2.1.5. Mature ovary and eggs of O. vulgaris Ovary samples were obtained from a wild female, 2900 g total fresh weight, collected off Barcelona, NW Mediterranean. Eggs of stages I– II and X – XII (Naef, 1928) were collected from egg masses obtained in the laboratory. 2.2. Analytical 2.2.1. Amino acid and nitrogen profiles The amount of total nitrogen was determined using a Thermo-Finningan NA2100 organic analyser by measuring oxygen combustion (Pella et al., 1984). The total protein was calculated from this total nitrogen amount using a conversion factor (N  6.25). The determination of non-protein nitrogen (NNP) was done according to the method recommended by the AOAC (1995), using 7.5% tricloroacetic acid to precipitate the protein that was removed by filtration. Nitrogen content of the filtrate was determined by the Kjeldahl method. All amino acid analyses were made from at least three replicates. In the present study, the nine amino acids considered as essentials (EAA) were: leucine, lysine, arginine, threonine, valine, isoleucine, phenylalanine, histidine and methionine. To determine the total amino acids content, freeze-dried samples were hydrolysed with 6 N HCl under nitrogen atmosphere at 105 jC for 24 h. Each sample contained 25 Al norleucine as an internal standard (Sigma Products, ref. no. N-6752). Amino acid analyses were performed using an automatic amino acid analyser (Amersham Pharmacia Biotech, UK). The chromatographic runs were made in a cation exchange high performance column Lithium 4151 (Biochrom, Cambridge, UK; ref. no. 80-2038-26) with 7 –10 Am particle size, 200  4.6 mm column size, lithium citrate buffers (Biochrom) and a thermal gradient from 34 to 80 jC. The amino acids were determined and measured using ninhydrin (Biochrom, ref. no. 80-2110-76) derivative reagent to develop the colour, which was detected with a colorimeter with

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beam splitter that allows to obtain the absorbance at 440 and 570 nm (two channels) that are added in order to obtain an unique signal that was recorded using an integrator (Waters 743 Data Module, Waters chromatography, Milford, MA, USA). Amino acid standards were used for hydrolizates (Sigma Products, ref. no. A2908) and for free amino acids (Sigma Products, ref. no. A9906). To determine the free amino acids, 15 mg of the freeze-dried samples were homogenised with 2 ml of 0.05N HCl followed by centrifugation at 12,000  g for 15 min to precipitate the protein. Then, to 1.4 ml of the supernatant 100 Al of norleucine 2000 AM were added as internal standard, and an aliquot of the obtained solution was filtered by using low binding regenerated cellulose Ultrafree-MC filters, 10,000 Da cut-off (Millipore, Bedford, MA), using a centrifuge (Hettich, Mikroliter, Germany) at 5000  g for 15 min. Then, 100 Al of the filtered solution were injected into the amino acid analyser as described before for the determination of total amino acid. 2.2.2. Amino acid uptake from seawater by cephalopod paralarvae To determine the amino acid uptake from seawater, the radiolabelled amino acid L[2,6-3H]phenylalanine, specific activity 48 Ci/nmol, was used (Amersham Pharmacia Biotech). An initial 2 AM solution of 2,6-3H phenylalanine was prepared in filtered seawater and diluted 1:2 in filtered seawater containing the cephalopod paralarvae. Four replicates of each sampling were performed using four cylindrical glasses of 50 ml and 40 mm diameter, with a final volume of 25 ml on each glass, at 1 AM of the final concentration. A control group without the radiolabelled solution was also maintained for each species at age 0. Three (for S. officinalis) or fifteen (for L. vulgaris and O. vulgaris) hatchling individuals were placed in each glass. In addition, markings for O. vulgaris were also undertaken with 15 individuals aged 7, 14 and 21 days. These individuals were obtained from cultures performed during August 2002 using the same rearing protocol as for the control group described previously. Radiolabelled markings were carried out at 19.5 –21 jC for 6 h, usually between 1100 and 1700 h. After this period individuals were collected on a plastic mesh, rinsed in tap water to eliminate radiolabelled material, placed in Eppendorf tubes and frozen in liquid nitrogen. The samples were homogenised in 0.5 M perchloric acid and centrifuged at 4 jC and 12,000 rpm. Protein content was determined (Lowry et al., 1951) after solubilization in 0.2 M NaOH. For each species, the specific activity of protein bound phenylalanine (dpm nmol 1 phenylalanine) was calculated as the quotient between liquid scintillation counts in solubilized protein and the mg of protein added to the scintillation vial divided by the number of nmol of phenylalanine contained in 1 mg of protein (Conceicß ao et al., 1997). A Beckman LS 6500 scintillation counter was used. 2.3. Data treatment Mean values (after log-transformation for weight data, and arcsinus-transformation for survival data) were compared by the Student’s t-test and analysis of variance, followed by the Tukey –Kramer HSD test. Differences were considered significant when P < 0.05. Data were assessed using the JMP statistical package.

Hatchlings Sepia Loligo officinalis vulgaris

Wild juveniles

Octopus vulgaris

Loligo vulgaris

Octopus vulgaris

2.1 F 0.1 0.3 F 0.0

1993 394

2051 421

2068 413

2637 559

2779 571

3505 814

5012 1128

5763 1380

10,960 2189

14,188 3671

Amino acid Arg His Ile Leu Val Lys Phe Met Thr

3.3 F 0.4 1.0 F 0.3 2.6 F 0.3 4.0 F 0.4 1.8 F 1.4 3.8 F 0.5 1.8 F 0.3 1.2 F 0.2 2.9 F 0.1

3.9 F 0.3 1.3 F 0.1 3.3 F 0.1 4.8 F 0.1 3.3 F 0.1 4.9 F 0.2 2.2 F 0.1 1.3 F 0.2 3.1 F 0.1

3.2 F 0.1 1.2 F 0.0 2.1 F 0.0 3.5 F 0.1 2.1 F 0.0 3.5 F 0.1 2.1 F 0.0 0.8 F 0.1 2.2 F 0.1

4.1 F 0.4 1.2 F 0.1 2.7 F 0.2 4.6 F 0.3 2.7 F 0.1 4.7 F 0.2 2.4 F 0.2 1.6 F 0.1 2.5 F 0.3

4.4 F 0.1 1.2 F 0.1 2.7 F 0.2 4.6 F 0.2 2.7 F 0.2 4.8 F 0.1 2.4 F 0.1 1.1 F 0.4 2.7 F 0.4

4.0 F 0.7 1.2 F 0.2 2.6 F 0.4 4.3 F 0.6 2.5 F 0.3 4.4 F 0.6 2.3 F 0.3 1.3 F 0.2 2.4 F 0.4

4.6 F 0.2 1.4 F 0.0 2.7 F 0.1 4.6 F 0.0 3.0 F 0.0 5.1 F 0.2 2.7 F 0.0 1.8 F 0.1 2.5 F 0.2

3.8 F 0.2 1.2 F 0.1 2.5 F 0.5 4.6 F 0.4 2.6 F 0.2 4.8 F 0.3 2.4 F 0.2 1.6 F 0.1 2.5 F 0.3

3.0 F 1.0 1.1 F 0.1 2.1 F 0.1 3.6 F 0.0 1.8 F 0.2 3.6 F 0.2 2.0 F 0.1 1.2 F 0.1 2.1 F 0.1

4.5 F 0.2 1.0 F 0.0 2.1 F 0.1 3.7 F 0.1 1.8 F 0.2 3.6 F 0.2 2.0 F 0.1 1.2 F 0.1 2.1 F 0.1

4.5 F 0.3 1.2 F 0.1 2.3 F 0.1 4.0 F 0.2 2.2 F 0.1 4.0 F 0.2 2.2 F 0.1 1.2 F 0.2 2.2 F 0.2

4.5 F 0.5 1.3 F 0.1 2.5 F 0.4 4.0 F 0.4 2.2 F 0.3 4.1 F 0.5 2.2 F 0.2 1.4 F 0.2 2.4 F 0.3

Ala Asp Cys Glu Gly HxPro Pro Ser Tyr

2.2 F 0.3 5.5 F 0.5 0.3 F 0.2 7.9 F 0.3 1.5 F 0.2 – 1.6 F 0.8 2.7 F 0.1 2.2 F 0.3

3.0 F 0.2 5.4 F 0.1 0.5 F 0.0 9.0 F 0.2 2.4 F 0.2 – 3.2 F 1.4 2.5 F 0.1 2.6 F 0.1

2.2 F 0.0 4.3 F 0.0 0.7 F 0.0 6.4 F 0.1 2.5 F 0.0 – 2.0 F 0.1 2.5 F 0.1 2.3 F 0.0

3.4 F 0.2 6.1 F 0.3 0.5 F 0.0 9.6 F 0.7 2.7 F 0.2 – 6.1 F 0.7 2.3 F 0.2 2.9 F 0.2

3.3 F 0.1 6.1 F 0.2 0.4 F 0.1 9.9 F 0.3 2.7 F 0.1 – 4.0 F 1.1 2.3 F 0.1 2.9 F 0.2

3.1 F 0.4 3.5 F 0.2 5.2 F 0.7 5.7 F 0.5 0.4 F 0.0 0.7 F 0.1 9.3 F 1.2 10.1 F 1.1 2.5 F 0.3 2.7 F 0.1 – – 3.0 F 0.6 3.1 F 0.2 2.1 F 0.3 2.3 F 0.2 2.8 F 0.3 3.3 F 0.0

3.7 F 0.1 6.1 F 0.7 0.5 F 0.1 9.1 F 1.7 2.7 F 0.2 – 6.1 F 1.3 2.1 F 0.4 2.5 F 0.8

2.7 F 0.1 5.0 F 0.2 0.5 F 0.1 8.2 F 0.4 2.8 F 0.2 0.2 F 0.0 1.8 F 0.1 2.5 F 0.1 2.1 F 0.1

2.7 F 0.1 5.1 F 0.3 0.7 F 0.3 8.4 F 0.5 2.8 F 0.1 0.2 F 0.0 2.0 F 0.2 2.5 F 0.1 2.1 F 0.1

2.9 F 0.2 5.3 F 0.5 0.8 F 0.1 9.0 F 0.7 3.0 F 0.3 0.4 F 0.1 2.0 F 0.1 2.6 F 0.2 2.3 F 0.1

3.1 F 0.4 3.2 F 0.2 5.7 F 0.6 6.0 F 0.4 0.6 F 0.2 0.6 F 0.1 9.8 F 1.1 10.1 F 0.4 3.7 F 0.5 3.4 F 0.2 0.5 F 0.1 0.2 F 0.2 2.3 F 0.3 2.3 F 0.2 2.8 F 0.4 3.0 F 0.2 2.3 F 0.2 2.5 F 0.3

EAA NEAA Protein

4.3 F 0.3 1.4 F 0.1 2.5 F 0.2 4.4 F 0.2 2.3 F 0.2 4.4 F 0.3 2.4 F 0.2 1.5 F 0.1 2.5 F 0.2

22.7 F 3.7 28.1 F 1.2 20.8 F 0.3 26.3 F 1.6 26.5 F 0.8 25.0 F 3.4 28.5 F 0.5 26.0 F 2.0 20.5 F 1.4 22.2 F 0.8 23.7 F 1.4 24.6 F 3.0 25.7 F 1.6 23.9 F 0.6 28.6 F 2.3 22.9 F 0.1 33.7 F 2.4 31.7 F 1.7 28.3 F 3.9 31.4 F 1.9 32.9 F 3.8 25.8 F 1.2 26.4 F 1.5 28.4 F 2.1 30.8 F 3.8 31.4 F 1.9 62.6 F 0.0 73.5 F 1.1 72.7 F 0.7 74.3 F 0.9 74.6 F 0.2 73.7 F 0.1 75.6 F 0.5 75.7 F 1.5 69.5 F 1.8 68.8 F 0.5 73.5 F 1.2 71.3 F 1.1 76.1 F 1.7

Amino acid analysis were made from at least three replicates. EAA, essential amino acids; NEAA, non-essential amino acids. Protein as N  6.25. Wet and dry weights (in mg ind 1) in hatchling correspond to the means F S.D.; 0.0 are values below 0.05.

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Wet weight 82.1 F 5.3 3.5 F 0.1 Dry weight 20.8 F 1.0 0.8 F 0.0

Wild juveniles

462

Table 1 Means F S.D. of the total amino acid content (in mg/100 mg of dry weight) of S. officinalis, L. vulgaris and O. vulgaris hatchlings, and five wild juveniles of L. vulgaris and O. vulgaris

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3. Results 3.1. Nitrogen and amino acid composition of cephalopod hatchlings and wild juveniles In hatchlings of S. officinalis, L. vulgaris and O. vulgaris, the lowest percentage protein corresponded to S. officinalis (Table 1). This was probably enhanced by the presence of the calcareous shell in the cuttlefish, where shell weight represented 2.1 F 0.5% of the wet weight and 6.0 F 0.7% of the dry weight of the animal. The non-protein nitrogen (NPN) levels in hatchlings of the three species were 24.8 F 1.1, 38.1 F 0.6 and 37.3 F 0.4 mg/100 mg of the dry weight, respectively. The lowest levels of NPN in S. officinalis were in relation to the lowest percentage protein (Table 1). Lysine, leucine and arginine represented 49%, 48% and 49% of the essential amino acids (EAA) of S. officinalis, L. vulgaris and O. vulgaris hatchlings, respectively. Glutamate and aspartate represented 56%, 50% and 47% of the non-essential amino acids (NEAA) for each species, respectively (Table 1). In wild O. vulgaris juveniles, the levels of EAA and NEAA were

Table 2 Means F S.D. of the total amino acid content (in mg/100 mg of dry weight) of mature ovary, spawned eggs at stages I – II and X – XII, hatchlings, and hatchlings fasted 2 and 4 days of O. vulgaris Mature ovary

Eggs stage I – II

Eggs stage X – XII

Dry weight Amino acid Arg His Ile Leu Val Lys Phe Met Thr

4.0 F 0.2a 1.5 F 0.1ab 3.5 F 0.1a 6.4 F 0.3a 3.6 F 0.2a 5.4 F 0.3a 2.1 F 0.1a 1.2 F 0.0a 3.5 F 0.2ab

Hatchlings 0 day

Hatchlings fasted 2 days

Hatchlings fasted 4 days

337.9 F 10.1a

284 F 4.3b

244 F 5.9c

3.9 F 0.4a 1.6 F 0.1a 3.7 F 0.2a 6.7 F 0.2a 3.7 F 0.2a 5.5 F 0.2a 2.0 F 0.1a 1.1 F 0.2a 3.7 F 0.2a

4.0 F 0.2a 1.4 F 0.0b 2.9 F 0.1b 5.0 F 0.1b 2.8 F 0.0b 4.7 F 0.1b 2.0 F 0.0a 1.1 F 0.1a 3.2 F 0.1b

3.2 F 0.1a 1.2 F 0.0a 2.1 F 0.0a 3.5 F 0.1a 2.1 F 0.0a 3.5 F 0.1a 2.1 F 0.0a 0.8 F 0.1a 2.2 F 0.1a

3.1 F 0.0a 1.1 F 0.0b 1.9 F 0.0b 3.3 F 0.1ab 1.9 F 0.0b 3.4 F 0.0b 2.0 F 0.1a 0.9 F 0.0a 2.0 F 0.0b

3.0 F 0.1a 1.0 F 0.0b 1.9 F 0.1b 3.2 F 0.2b 1.7 F 0.1c 3.2 F 0.1b 2.0 F 0.1a 0.9 F 0.0a 1.8 F 0.1c

2.0 F 0.0b 4.0 F 0.0b 0.7 F 0.0a 6.0 F 0.1ab 2.4 F 0.0a 1.8 F 0.0a 2.2 F 0.0b 2.2 F 0.1a

2.0 F 0.1b 4.0 F 0.2b 0.6 F 0.1a 5.6 F 0.3b 2.5 F 0.1a 1.9 F 0.1a 2.1 F 0.1b 2.1 F 0.2a

Ala Asp Cys Glu Gly Pro Ser Tyr

2.3 F 0.2a 5.6 F 0.3a 1.2 F 0.0a 10.5 F 0.8a 1.5 F 0.1a 2.5 F 0.1a 3.6 F 0.2a 2.2 F 0.1a

2.2 F 0.1a 5.6 F 0.2a 1.3 F 0.1ab 10.4 F 0.4a 1.4 F 0.1a 2.5 F 0.1a 3.6 F 0.1a 2.1 F 0.1a

2.3 F 0.1a 5.4 F 0.1a 1.5 F 0.0b 8.8 F 0.2b 1.9 F 0.1b 2.3 F 0.1a 3.2 F 0.2a 2.2 F 0.0a

2.2 F 0.0a 4.3 F 0.0a 0.7 F 0.0a 6.4 F 0.1a 2.5 F 0.0a 2.0 F 0.1a 2.5 F 0.1a 2.3 F 0.0a

EAA NEAA

33.1 F 1.4a 29.3 F 1.7a

31.9 F 1.5a 29.2 F 1.2a

27.1 F 0.6b 27.6 F 0.5a

20.8 F 0.3a 22.9 F 0.1a

19.5 F 0.3b 21.3 F 0.3b

18.8 F 0.5b 20.7 F 1.0b

Dry weights (in Ag ind 1) in hatchlings correspond to the means F S.D. Amino acid analysis were made from at least three replicates. Means F S.D. with same superscript letters for ovary and eggs, and for hatchlings and fasted for dry weight and for the same amino acid denote no statistical differences within the group ( P>0.05). EAA, essential amino acids; NEAA, non-essential amino acids; 0.0 are values below 0.05.

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Fig. 1. Mean and S.D. of the total amino acid content (in mg/100 mg of dry weight) of mature ovary, spawned eggs at stages I – II and X – XII, hatchlings, and hatchlings fasted 2 and 4 days of O. vulgaris. EAA, essential amino acids; NEAA, non-essential amino acids.

R. Villanueva et al. / Aquaculture 242 (2004) 455–478

Fig. 1 (continued).

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positively correlated with dry weight and all EAA increased, with the exception of arginine that did not correlate with weight. Lysine, leucine and arginine represented 52% of the EAA and glutamate and aspartate represented 51% of the NEAA in O. vulgaris juveniles. In these wild juveniles, NEAA were also positively correlated with dry weight, except for cysteine and hydroxyproline. In wild L. vulgaris juveniles, the short weight range of the individuals did not allow any changes associated with size to be detected. For juvenile L. vulgaris, lysine, leucine and arginine represented 51% of the EAA, and glutamate and aspartate represented 49% of the NEAA (Table 1). 3.2. Amino acid composition of mature ovary, eggs and effects of fasting on O. vulgaris hatchlings A general tendency for a decrease in the level of total EAA, except for phenylalanine, from mature ovary and eggs to hatchlings was observed. Levels of NEAA did not change from ovary to eggs, but they decreased to hatchlings (Table 2). When fasting for 4 days after hatching, the total content of EAA decreased, except for arginine, methionine and phenylalanine. The total content of NEAA also decreased from hatching, whereas cysteine, glycine, proline and tyrosine did not vary and the remaining NEAA decreased (Fig. 1). The total levels of amino acids (EAA + NEAA) decreased. When comparing free amino acid levels, proline after 2 days of fasting and tyrosine after 4 days were not detected (Table 3). All free EAA decreased, except for leucine, phenylalanine and methionine that did not change, resulting in a decrease of the total level of the free EAA pool. After 4 days of fasting the levels of free NEAA decreased to nearly half of hatching levels, with the exception of cysteine. 3.3. Amino acid composition of O. vulgaris paralarvae and prey during paralarval culture An increase of the free EAA was observed from hatching to an age of 25 days. Ten days after hatching, the total EAA were higher for the METAA group. In comparison with the control group, all total EAA reached higher levels in METAA, excluding arginine, which showed no difference (Table 4). The free EAA were also higher for METAA. In comparison with the control group, all free EAA reached higher levels in METAA, excluding arginine, lysine and threonine, where no differences were observed (Table 3). At age 25 days, levels of total amino acids showed reduced differences between treatments; however, groups that received the amino acids solution (AA and METAA) showed higher free EAA levels (Table 3). At 25 days, glutamate was the most abundant free amino acid, followed by arginine and aspartate. These amino acids, with leucine and lysine, were also the most abundant in the total content, although glutamate had the highest levels. Regarding the free EAA, it should be pointed out that there were high levels of arginine observed in all groups analysed (Table 3), which represented nearly half of the free EAA pool for hatchlings, 55% after 4 days fasting, 38 –59% at 10 days and 32 – 45% at 25 days. Total EAA and NEAA showed no differences between both types of Artemia used. The total EAA showed few differences between enriched Artemia nauplii and the decapod crab zoeae, where M. squinado zoeae showed the lower EAA level (Table 5). Comparisons between the EAA profiles of hatchling and 10 days old O. vulgaris paralarvae versus the

Table 3 Composition of the free pool amino acids (in mg/g of dry weight) of hatchlings, hatchlings fasted 2 and 4 days, and paralarval rearings of O. vulgaris at the ages of 10 and 25 days in four treatments: Control, MET, AA and METAA (see Materials and methods) Amino acid

Hatchlings Hatchlings 0 day

10 days Fasted 2 days

Fasted 4 days

Control

25 days MET

AA

METAA

Control

MET

AA

METAA

2.9 F 0.2a 0.5 F 0.4a 0.1 F 0.0a 0.5 F 0.0ab 0.3 F 0.1a 0.5 F 0.0a 0.5 F 0.0a 0.2 F 0.0ab 0.4 F 0.0a

2.7 F 0.0ab 0.6 F 0.0a 0.1 F 0.0a 0.5 F 0.1a 0.3 F 0.1a 0.4 F 0.1a 0.5 F 0.1a 0.3 F 0.1a 0.2 F 0.0b

2.5 F 0.1b 0.5 F 0.1a 0.1 F 0.0b 0.3 F 0.0b 0.2 F 0.0a 0.3 F 0.0b 0.3 F 0.0a 0.2 F 0.0b 0.1 F 0.0c

3.8 F 0.1a 0.3 F 0.0a 0.2 F 0.0a 0.6 F 0.0a 0.3 F 0.0a 0.4 F 0.0a 0.5 F 0.0a 0.2 F 0.0a 0.3 F 0.0a

4.1 F 0.5a 0.4 F 0.0a 0.2 F 0.0a 0.6 F 0.1a 0.3 F 0.0a 0.4 F 0.1a 0.5 F 0.1a 0.3 F 0.1a 0.2 F 0.1a

3.7 F 0.3a 0.5 F 0.0b 0.3 F 0.0b 0.5 F 0.1a 0.6 F 0.1b 0.3 F 0.0a 0.7 F 0.1ab 1.1 F 0.0b 0.3 F 0.0a

3.8 F 0.1a 0.5 F 0.0b 0.9 F 0.1c 1.1 F 0.2b 1.0 F 0.1c 0.3 F 0.1a 0.8 F 0.1b 1.2 F 0.2b 0.2 F 0.2a

3.9 F 0.3a 0.3 F 0.0a 0.3 F 0.0a 1.3 F 0.1a 0.5 F 0.0a 0.6 F 0.0a 0.9 F 0.2a 0.5 F 0.2a 0.4 F 0.0a

3.2 F 0.1a 0.3 F 0.0a 0.3 F 0.0a 1.3 F 0.0a 0.5 F 0.1a 0.6 F 0.1a 0.9 F 0.1ab 0.5 F 0.1ab 0.4 F 0.0a

3.8 F 0.3a 0.4 F 0.1ab 0.8 F 0.1b 1.6 F 0.2a 0.9 F 0.1b 0.5 F 0.1a 1.2 F 0.1bc 0.8 F 0.1bc 0.5 F 0.1ab

3.5 F 0.5a 0.5 F 0.1b 0.8 F 0.0b 1.7 F 0.1a 0.9 F 0.1b 0.6 F 0.1a 1.3 F 0.0c 1.1 F 0.1c 0.5 F 0.0b

Ala Asp Cyst Glu Gly Pro Ser Tyr

1.7 F 0.3a 2.1 F 0.1a 0.1 F 0.1a 4.8 F 0.4a 0.5 F 0.0a 0.5 F 0.1a 0.3 F 0.0a 0.3 F 0.0a

1.0 F 0.3b 1.7 F 0.1b 0.1 F 0.0a 3.0 F 0.3b 0.3 F 0.0b 0.0 F 0.0b 0.3 F 0.1ab 0.2 F 0.0b

0.6 F 0.0b 1.7 F 0.2b 0.1 F 0.0a 2.4 F 0.2b 0.3 F 0.0b 0.0 F 0.0b 0.2 F 0.0b 0.0 F 0.0c

1.2 F 0.1a 1.6 F 0.1a 0.1 F 0.0a 4.0 F 0.2a 0.2 F 0.0a 1.2 F 0.1a 0.3 F 0.0a 0.5 F 0.0a

1.2 F 0.2ab 1.6 F 0.2a 0.1 F 0.0ab 4.1 F 0.5a 0.2 F 0.0a 1.2 F 0.2a 0.3 F 0.1a 0.5 F 0.1a

1.6 F 0.1b 1.2 F 0.4ab 0.1 F 0.0a 4.4 F 0.2a 0.1 F 0.0a 1.1 F 0.4a 0.3 F 0.1a 0.7 F 0.1b

1.4 F 0.2ab 0.9 F 0.2b 0.1 F 0.0b 4.2 F 0.1a 0.2 F 0.0a 1.1 F 0.1a 0.3 F 0.1a 0.9 F 0.1b

1.6 F 0.5a 1.5 F 0.0a 0.1 F 0.0a 4.3 F 0.5a 0.3 F 0.0a 1.6 F 0.4a 0.4 F 0.1a 0.7 F 0.1a

1.8 F 0.3a 1.4 F 0.1ab 0.1 F 0.0a 4.5 F 0.4a 0.2 F 0.1a 1.6 F 0.5a 0.4 F 0.1a 0.8 F 0.0ab

1.7 F 0.2a 1.2 F 0.1b 0.1 F 0.0a 4.4 F 0.2a 0.2 F 0.0a 1.8 F 0.1a 0.4 F 0.1a 1.0 F 0.1bc

1.8 F 0.1a 1.2 F 0.1b 0.1 F 0.0a 4.6 F 0.1a 0.2 F 0.0a 1.6 F 0.1a 0.4 F 0.0a 1.1 F 0.1c

EAA NEAA

5.9 F 0.6a 10.3 F 0.7a

5.7 F 0.6a 6.6 F 0.6b

4.5 F 0.1b 5.4 F 0.3b

6.7 F 0.2a 9.0 F 0.3a

6.9 F 1.0a 9.1 F 1.3a

7.9 F 0.4a 9.7 F 0.8a

9.9 F 0.7b 9.1 F 0.4a

8.7 F 0.4a 10.4 F 1.4a

8.0 F 0.5a 10.8 F 1.3a

10.5 F 0.6b 10.9 F 0.8a

10.9 F 0.7b 11.1 F 0.2a

Tau GABA Orn

54.2 F 3.2a 0.4 F 0.1a 0.2 F 0.0a

48.9 F 3.2a 0.3 F 0.1a 0.3 F 0.1a

53.5 F 4.8a 0.3 F 0.1a 0.3 F 0.0a

15.6 F 2.5a 0.4 F 0.0a 0.5 F 0.1a

15.2 F 4.3a 0.3 F 0.1a 0.4 F 0.1a

31.6 F 6.0b 0.3 F 0.2a 0.4 F 0.1a

10.1 F 1.3a 0.4 F 0.0a 0.4 F 0.1a

17.2 F 3.4a 0.3 F 0.1a 0.5 F 0.1a

16.4 F 3.0a 0.2 F 0.1a 0.5 F 0.1a

10.8 F 1.2b 0.3 F 0.0a 0.5 F 0.1a

11.8 F 0.8ab 0.3 F 0.0a 0.4 F 0.0a

467

Amino acid analysis were made from at least three replicates. Means F S.D. with same superscript letters for each of the three groups (hatchlings + fasted, 10 days, 25 days) and amino acid denote no statistical differences within the group ( P>0.05). EAA, essential amino acids; NEAA, non-essential amino acids; 0.0 are values below 0.05.

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Arg His Ile Leu Val Lys Phe Met Thr

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Table 4 Means F S.D. of the total amino acid content (in mg/100 mg of dry weight) during rearing experiments of O. vulgaris at the ages of 10 and 25 days in four treatments: Control, MET, AA and METAA (see Materials and methods for details) Amino 10 days acid Control

25 days MET

AA

METAA

Control

MET

AA

METAA

Arg His Ile Leu Val Lys Phe Met Thr

2.9 F 0.3a 1.0 F 0.1a 1.9 F 0.2a 3.0 F 0.2a 1.9 F 0.2a 3.2 F 0.3a 1.8 F 0.1a 0.9 F 0.0a 1.8 F 0.2a

3.2 F 0.1a 1.1 F 0.0ab 2.0 F 0.0ab 3.2 F 0.0ab 2.1 F 0.0bc 3.5 F 0.0a 2.0 F 0.0ab 1.0 F 0.1a 2.0 F 0.0ab

3.1 F 0.0a 1.1 F 0.0ab 2.0 F 0.1ab 3.2 F 0.2ab 2.0 F 0.0ab 3.4 F 0.0ab 2.1 F 0.0b 1.0 F 0.1ab 2.1 F 0.1ab

3.3 F 0.1a 1.2 F 0.0b 2.2 F 0.1b 3.4 F 0.1b 2.3 F 0.1c 3.6 F 0.1b 2.1 F 0.1b 1.1 F 0.1b 2.1 F 0.1b

3.2 F 0.1a 1.1 F 0.0a 2.0 F 0.0a 3.2 F 0.0a 2.0 F 0.0a 3.4 F 0.0a 2.1 F 0.0a 1.1 F 0.1a 2.0 F 0.0a

3.2 F 0.0a 1.2 F 0.0ab 2.1 F 0.0a 3.3 F 0.0a 2.0 F 0.1a 3.5 F 0.0a 2.2 F 0.0a 1.2 F 0.0a 2.0 F 0.0a

3.3 F 0.1a 1.2 F 0.0ab 2.2 F 0.0a 3.5 F 0.1b 2.3 F 0.1b 3.6 F 0.1a 2.1 F 0.1a 1.1 F 0.0a 2.1 F 0.1a

3.3 F 0.2a 1.3 F 0.1b 2.2 F 0.1a 3.3 F 0.1a 2.2 F 0.1ab 3.7 F 0.3a 2.4 F 0.2a 1.1 F 0.0a 2.1 F 0.2a

Ala Asp Cys Glu Gly Pro Ser Tyr

2.2 F 0.1a 3.9 F 0.2a 0.7 F 0.2a 5.7 F 0.4a 2.2 F 0.1a 1.8 F 0.2a 2.0 F 0.2a 2.0 F 0.1a

2.3 F 0.2ab 4.2 F 0.0ab 0.9 F 0.1a 6.2 F 0.0ab 2.3 F 0.0ab 1.9 F 0.0ab 2.2 F 0.0ab 2.2 F 0.0ab

2.1 F 0.0a 4.0 F 0.1ab 0.7 F 0.0a 6.0 F 0.0ab 2.2 F 0.0ab 1.9 F 0.0ab 2.2 F 0.0ab 2.3 F 0.0b

2.5 F 0.1b 4.4 F 0.2b 0.9 F 0.1a 6.5 F 0.2b 2.4 F 0.1b 2.0 F 0.1b 2.3 F 0.1b 2.3 F 0.1b

2.1 F 0.0a 4.1 F 0.1a 0.8 F 0.0ab 5.8 F 0.1a 2.2 F 0.0a 2.0 F 0.0a 2.2 F 0.0a 2.3 F 0.0a

2.2 F 0.0a 4.4 F 0.0a 0.7 F 0.1a 6.1 F 0.0ab 2.3 F 0.0a 2.0 F 0.0a 2.3 F 0.0a 2.4 F 0.0a

2.5 F 0.1b 4.5 F 0.1a 0.9 F 0.0ab 6.6 F 0.2b 2.4 F 0.1a 2.1 F 0.1a 2.3 F 0.1a 2.4 F 0.1a

2.3 F 0.2ab 4.4 F 0.3a 1.0 F 0.2bc 6.2 F 0.4ab 2.4 F 0.2a 2.1 F 0.2a 2.3 F 0.2a 2.5 F 0.2a

EAA 18.5 F 1.5a 20.1 F 0.3ab 20.0 F 0.1ab 21.3 F 0.8b 20.1 F 0.3a 20.6 F 0.2a 21.5 F 0.6a 21.7 F 1.3a NEAA 20.4 F 1.2a 22.2 F 0.2ab 21.5 F 0.1ab 23.3 F 0.9b 21.4 F 0.4a 22.4 F 0.3a 23.6 F 0.7a 23.3 F 1.8a Amino acid analysis were made from at least three replicates. Means F S.D. with same superscript letters for the same age and amino acid denote no statistical differences within the group ( P>0.05). EAA, essential amino acids; NEAA, non-essential amino acids; 0.0 are values below 0.05.

EAA profiles of the preys are showed in Fig. 2. This comparison indicates that enriched Artemia nauplii (Artemia SS) shown only low values for histidine, when compared to the O. vulgaris total EAA profile during first 10 days of rearing. 3.4. Survival, growth and amino acid uptake from seawater At 20 days, supplementation with methionine did not improve weight or survival of O. vulgaris (Table 6). In contrast, at an age of 20 days, the higher survival was obtained for the cultures receiving the amino acids solution (AA and METAA). Mean survival was 12.6 F 3.2 for the control, 17.2 F 11.2 for MET, 41.2 F 9.9 for AA and 54.1 F 4.6 for METAA. No differences were obtained between the control and MET, or between the AA and METAA groups. After this age, mortality increased as Artemia nauplii became an unsuitable prey for O. vulgaris (Villanueva et al., 2002) and at 30 days survival decreased to 0%, 2.8%, 7.8% and 12.5% for the control, MET, AA and METAA treatments, respectively. Growth in weight was not positively affected by the amino acids solution and the dry weight was lower in METAA, which was the group with higher survival (Table 6). The uptake of radiolabelled phenylalanine in hatchlings of S. officinalis, L. vulgaris and O.

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Table 5 Means F S.D. of the total amino acid content (in mg/100 mg of dry weight) of Artemia nauplii (used as food during the present study for cultures of O. vulgaris paralarvae) and hatchling decapod crab zoeae of P. prideaux and M. squinado (used as food for cephalopod paralarvae in previous studies) Amino acid

Pagurus prideaux zoeae

Maja squinado zoeae 2.9 F 0.1 1.1 F 0.0a 1.6 F 0.1b 2.9 F 0.1a 2.1 F 0.1a 3.6 F 0.1a 1.7 F 0.0b 0.9 F 0.0a 1.9 F 0.1a

3.0 F 0.2 1.0 F 0.1a 2.1 F 0.1a 3.2 F 0.2a 2.3 F 0.1a 3.5 F 0.2a 2.1 F 0.1ac 0.8 F 0.1a 2.1 F 0.1a

3.2 F 0.1a 1.0 F 0.1a 2.2 F 0.1a 3.4 F 0.2a 2.5 F 0.1a 3.8 F 0.2a 2.3 F 0.1c 1.8 F 0.1b 2.3 F 0.1a

Ala Asp Cys Glu Gly Pro Ser Tyr

3.4 F 0.4a 4.3 F 0.5a 0.4 F 0.0a 8.0 F 1.0a 4.2 F 0.5a 3.9 F 1.2a 1.9 F 0.3ab 2.0 F 0.6a

2.9 F 0.1ab 2.1 F 0.1b 0.4 F 0.1a 6.4 F 0.2a 3.1 F 0.1b 3.2 F 0.1a 1.8 F 0.1a 1.8 F 0.0a

2.6 F 0.2b 3.9 F 0.3a 0.7 F 0.1b 6.8 F 0.7a 2.1 F 0.1c 2.4 F 0.1a 2.2 F 0.1ab 2.3 F 0.1a

2.5 F 0.2b 4.0 F 0.3a 0.7 F 0.1b 6.7 F 0.3a 2.3 F 0.1c 2.4 F 0.1a 2.3 F 0.1b 2.5 F 0.1a

20.1 F 1.0ab 23.0 F 1.8a

22.4 F 1.1a 23.3 F 1.3a

18.6 F 0.6b 21.8 F 0.8a

a

Artemia MET nauplii

3.5 F 0.5 1.2 F 0.2a 2.2 F 0.3a 3.1 F 0.4a 2.5 F 0.3a 3.6 F 0.4a 1.8 F 0.2ab 0.6 F 0.4a 2.3 F 0.6a

20.8 F 2.2ab 28.1 F 4.3a

a

Artemia SS nauplii

Arg His Ile Leu Val Lys Phe Met Thr

EAA NEAA

a

Artemia SS was enriched only with SuperSelco; Artemia MET was enriched with SuperSelco plus methionine (see Materials and methods). Amino acid analysis was made from at least three replicates. Means F S.D. with same superscript letters for the same amino acid denote no statistical differences within the group ( P>0.05). EAA, essential amino acids; NEAA, non-essential amino acids; 0.0 are values below 0.05.

vulgaris was 3915 F 529, 7900 F 1161 and 10426 F 2446 nmol of phenylalanine per mg of protein, respectively. This uptake, in reared O. vulgaris paralarvae, at ages of 7, 14 and 21 days was 9621 F 773, 10720 F 1292 and 8891 F 2135 nmol of labelled phenylalanine per mg of protein, respectively.

4. Discussion 4.1. Nitrogen and amino acid composition The total nitrogen in the muscular mantle and arms of subadult and adult cephalopods is divided into two fractions. One is the protein nitrogen, composed mainly of myofibrillar (64.8 –79%), sarcoplasmic (11.5– 15.2%) and stroma (3– 16%) proteins represented by protein amino acids (Kariya et al., 1986). The second fraction is composed of non-protein nitrogen compounds (NPN). The cephalopod hatchlings analysed showed high fractions of NPN, in agreement with the percentages reported for subadult and adult cephalopods where NPN constitutes from 20 to 50% of the total nitrogen (Sikorski and Kolodziejska, 1986; Iida et al., 1992; Ruı´z-Capillas et al., 2002). The major components in this fraction

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are volatile bases such as ammonia and trimethylamine oxide, creatine, free amino acids, nucleotides, purine bases and urea. The lowest levels of NPN in S. officinalis hatchlings were related to the observed lowest percentage of proteins and were probably enhanced by the presence of the calcareous shell (that represents 6% of the hatching dry weight), in contrast to the small and chitinous shells of squids and octopus. In the three cephalopod species studied, lysine, leucine and arginine represented nearly half of the total EAA, and glutamate and aspartate represented also nearly half of the NEAA. The high level of lysine, leucine and arginine in the body composition indicates that these amino acids could be limiting essential amino acids in their diets. In addition, arginine is the EAA that reaches higher levels in the free form in O. vulgaris paralarvae. It would thus be of interest to test these amino acids in future experimental diets to detect the nutritional amino acid requirements for early stages of cephalopods. Arginine is vigorously metabolized in cephalopods (Hochachka et al., 1983). During anaerobic work, arginine phosphate is hydrolyzed leading to increased availability of the arginine for condensation with glucose-derived pyruvate to form octopine, the main anaerobic end product that accumulates in adult cephalopods during periods of exercise and stress (Hochachka et al., 1976; Storey et al., 1979). In adult cuttlefish S. officinalis, arginine-derived carbon appears in CO2, urea, ornithine, glutamate, citrulline, alanine, octopine and proline (Hochachka et al., 1983). In fact, in the current study, proline was the first free amino acid not detected after 2 days of fasting in hatchlings of O. vulgaris. This amino acid is involved in oxidative metabolism in cephalopods and, during exercise, the adult squid Loligo pealeii show a drop in mantle muscle proline concentrations of 2 Amol g 1 wet weight (Storey and Storey, 1978). O. vulgaris use the mantle for swimming continuously by a jet propulsion system during planktonic life (Villanueva et al., 1995, 1996), and it is a mode of locomotion that needs high metabolic rates in comparison with the ondulatory swimming of fishes, although few data exist for early stages (O’Dor and Webber, 1986; O’Dor et al., 1986; Parra et al., 2000). In fact, during the present study hatchlings lost 28% of their dry weight after 4 days of fasting and the level of essential amino acids decreased in both the total content and free forms. After this period of fasting free tyrosine was not detected. Tyrosine has been considered non-essential in studies of other molluscs, such as abalone (Allen and Kilgore, 1975; Mai et al., 1994) and the particular needs of this amino acid in cephalopods needs further research. Present results show that planktonic O. vulgaris have high levels of taurine (Table 3). This richness has been reported previously in cephalopod nervous tissue (D’Aniello et al., 1995) and squid ink (Shirai et al., 1997). Taurine is mainly an intracellular component and high concentrations of taurine are a common feature of marine molluscs, including O. vulgaris adults, since taurine is involved in osmoregulation (Florkin and Bricteux-Gre´goire, 1972).

Fig. 2. Comparison of the total essential amino acids (EAA) profiles of O. vulgaris paralarvae of hatchling (left column) and 10 days (right column) posthatching (from the Control treatment) and the total EAA profiles of Artemia nauplii (enriched with Diet SS) and hatchling zoeae of P. prideaux and M. squinado. Points above the line of slope 1 and intercept 0 suggest deficiencies for that amino acid in the prey. Amino acids with significantly different ( P < 0.05) levels in the paralarvae and prey are indicated (*). Each point represents a mean of three replicates. DW, dry weight.

472

Age (days)

0 2 4 5 10 15 20 25 30

WW

DW

Fasting

Control

MET

AA

METAA

Fasting

Control

MET

AA

METAA

2.1 F 0.0a 1.7 F 0.1 1.5 F 0.1

2.1 F 0.0a

2.1 F 0.0a

2.1 F 0.0a

2.1 F 0.0a

337.9 F 10.1a 284.8 F 4.3 244.2 F 5.9

337.9 F 10.1a

337.9 F 10.1a

337.9 F 10.1a

337.9 F 10.1a

2.2 F 0.1a 2.3 F 0.3a 2.9 F 0.0ab 3.4 F 0.1a 3.8 F 0.3a

2.3 F 0.2a 2.6 F 0.1a 2.7 F 0.2a 3.5 F 0.1a 3.7 F 0.1a 4.0 F 0.2a

2.2 F 0.1a 2.6 F 0.1a 3.0 F 0.1b 3.2 F 0.2b 3.5 F 0.3ab 3.9 F 0.2a

2.3 F 0.1a 2.5 F 0.2a 2.7 F 0.1a 2.8 F 0.2c 3.2 F 0.1b 3.6 F 0.2a

400.5 F 9.2a 474.5 F 16.5a 606.6 F 7.0a 682.8 F 15.4a 712.3 F 27.1a

426.3 F 8.3b 486.1 F 17.9a 538.8 F 43.5b 681.6 F 28.4a 718.1 F 25.3a 812.3 F 36.2a

388.1 F 5.9c 480.2 F 30.1a 596.0 F 10.5a 653.6 F 20.7a 692.4 F 32.6ab 762.0 F 19.1ab

395.1 F 6.3ac 479.7 F 10.3a 498.4 F 23.4b 566.2 F 24.5b 647.3 F 28.3b 718.9 F 26.5b

Six samples (groups of 18 – 106, mean = 31 paralarvae) of paralarvae were consecutively collected from each rearing experiment on the sampling days. At age 30 days no paralarval survived for the Control treatment. Means F S.D. with same superscript letters for the same age denote no statistical differences within the group ( P>0.05); 0.0 are values below 0.05.

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Table 6 Mean wet weight (WW, mg) and dry weight (DW, Ag) of O. vulgaris during fasting and four treatments: Control, MET, AA and METAA (see Materials and methods)

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The amount of total amino acids increased with dry weight in wild O. vulgaris juveniles. These biochemical changes associated with juvenile growth are related to morphometric changes in body proportions. The development of the protein-rich muscular arm crown during the juvenile stage is remarkable, since juveniles have arm lengths four to five times smaller than subadult and adult octopuses (Villanueva, 1995). This increment in protein is related to the decrease in total lipid content observed in the same individuals as the octopuses grow (see Table 2 in Navarro and Villanueva, 2003), due to the relative decrease of the visceral mass, where lipids are abundant (O’Dor et al., 1984). The relative increase in protein as octopuses grow implies changes in the energetic reserves for this group of animals due to the predominance of their amino acid metabolism. It should be considered that on the basis of ATP equivalents per gram of muscle, the mantle and arms are similar, but when total muscle weights are considered, ATP production is at least 10fold greater in the arms than in the mantle, as recorded in adults of the blue-ringed octopus Hapalochlaena maculosa (Baldwin and England, 1980). In subadult O. vulgaris, Houlihan et al. (1990) showed that individuals growing at 3% day 1 synthesized 0.54 g of protein, with 0.43 g of this protein retained as growth, whereas over 90% of the protein was retained as growth when animals reached growth rates of 6% day 1. High mobilization of amino acids exists in mature octopuses and, in contrast to vertebrates, ovarian yolk proteins in O. vulgaris are formed within the ovary rather than being synthesized elsewhere and transported through the blood (O’Dor and Wells, 1973). The results obtained show that total amino acids represented 62% of the mature ovary dry weight, which decreased to 44% in the hatchling paralarvae (Table 2). It would be particularly interesting to investigate in future experiments whether the high amounts of total amino acids observed in ovary and eggs of O. vulgaris corresponded to a possible high proportion of free compounds that were fuelled before hatching (Fig. 1; Table 2). To this respect should be considered that the free amino acid pool may serve as fuel in some early stages of fishes, depending on the species (Ronnestad and Fyhn, 1993). The total EAA showed few differences between enriched Artemia nauplii and hatching decapod crab zoeae. Comparisons between these EAA profiles and that of the cephalopod paralarvae should consider that nauplii and zoeae amino acid profile obtained during present study correspond to the whole crustaceans, including the exoskeleton. It is known that during the external digestion, O. vulgaris paralarvae do not ingest the carapace of the crustacean zoeae, leaving a whole and empty exoskeleton (Herna´ndez-Garcı´a et al., 2000), in similar way as adult octopuses when they eat crabs (Nixon, 1984; Grisley et al., 1999). The enriched Artemia nauplii EAA profiles showed no differences with the EAA profiles of O. vulgaris paralarvae during first 10 days of culture, except for histidine (Fig. 2). As the use of Artemia as single prey for O. vulgaris paralarval culture results in poor growth and survival (Boletzky and Hanlon, 1983; Villanueva et al., 2002), present results seem to support the hypothesis that nutritional deficiencies of Artemia when used as food for O. vulgaris are mainly from lipidic origin (Navarro and Villanueva, 2000, 2003). 4.2. Amino acid uptake from seawater The present results confirm the positive capacity for amino acid uptake from seawater by early stages of cephalopods (Castille and Lawrence, 1978), which has also been

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observed in adult forms (de Eguileor et al., 2000). The ability of cephalopods to absorb amino acids by the integument fluids also exists in other molluscs (Manahan, 1990) and may represent a phylogenetic remnant for this group of exclusively carnivorous animals. In the three species analysed, radiolabelled Phe was incorporated in inverse relation to body size (i.e. lower for the large-sized S. officinalis and higher for the small-sized O. vulgaris). Tentatively, it can be hypothesized that the surface– volume relationship can influence the different absorption. The participation of microorganisms on the absorption of radiolabelled phenylalanine could not be ruled out and during the present study the percentage of radiolabelled uptake done by the bacteria present in the seawater during the radiolabelled markings is unknown. The present results indicate that after 10 days of culture, O. vulgaris paralarvae show a tendency for increased levels of total and free amino acids in the groups receiving a daily amino acids solution. At an age of 25 days, differences between groups were minimized and only the essential free amino acids were observed to be higher in the groups receiving the amino acids solution. At an age of 20 days, the O. vulgaris cultures that received the amino acids solution reached survivals that were on average three times that of the groups without the amino acids solution. However, the supposed beneficial effect of the amino acids solution remains unclear since the dry weight of these paralarvae was equal to or lower than the control group. The present results support previous observations made on cephalopod paralarval cultures. For example, cultures of the squid Loligo forbesi reared without food from the hatchling stage and kept in a seawater culture medium with a high concentration (10 mg C l 1) of dissolved organic material (DOM), registered positive growth, as well as development of the digestive gland and a survival percentage at 10 days higher than groups receiving food (Vecchione and Hand, 1989). However, it is not known how the nutrients obtained from seawater are transformed. In this respect, high alkaline phosphatase activity has been recorded in the skin of Octopus sp. paralarvae by BoucaudCamou and Roper (1995). These authors suggested that the presence of this digestive enzyme indicates active absorption through the skin from seawater during the paralarval period. Nevertheless, it should also be considered that, in addition to nutrition, the absorptive properties of amino acids by epidermal cells can provide osmolytes for the regulation of the internal environment of the cephalopod (de Eguileor et al., 2000). The levels of amino acid concentration in seawater for maximum uptake by cephalopod paralarvae have not been determined. The amino acid transport systems by invertebrate larvae show saturation with increasing substrate concentration that can be described by Michaelis– Menten kinetics and different species have different transport rates for different classes of amino acids (Manahan, 1990). In addition to the direct amino acid uptake from seawater by the cephalopod paralarvae, changes in the chemotactic behaviour influenced by the presence of dissolved amino acids in the rearing tank cannot be excluded, since O. vulgaris paralarvae have well-developed chemosensory organs on their skin (Lenz et al., 1995; Lenz, 1997; Wildenburg, 1997). In this way, amino acids in seawater produce attractant or arrestant effects in adult octopuses (Lee, 1992) and for subadult O. vulgaris, glycine and glutamate elicited locomotion in the direction of the amino acid source (Chase and Wells, 1986). This effect on cephalopod paralarvae has not yet been determined and may be able to change food-searching behaviour when crystalline amino acids are added to the rearing tank. The skin of cephalopods is a very active tissue containing chromato-

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phores within the intrinsic musculature, nerves, gland cells and blood vessels (Packard, 1988) and a high amount of oxygen is obtained by transcutaneous means (Po¨rtner, 1994). For example, subadult O. vulgaris cutaneous respiration can provide 41% of the total oxygen requirement of an animal at rest and 33% during exercise (Madan and Wells, 1996). All of these epidermal properties, probably enhanced in small animals by the high surface– volume relationship, suggest that in early cephalopod stages the acquisition of small organic molecules can be of significant interest to the metabolic needs during the first feeding period. Future research is needed in this field. It will be of interest to determine the effects on paralarval survival of the presence of different amino acids in the seawater, as well as at different amino acid concentrations.

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