Effect of Sexual Maturation on the Tissue Biochemical Composition of Octopus Vulgaris

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    R E S E A R C H A R T I C L E

    R. Rosa P. R. Costa M. L. Nunes

    Effect of sexual maturation on the tissue biochemical composition

    of Octopus vulgaris and O. defilippi(Mollusca: Cephalopoda)

    Received: 14 November 2003 / Accepted: 23 February 2004/ Published online: 1 April 2004 Springer-Verlag 2004

    Abstract Changes in the protein, lipid, glycogen, cho-lesterol and energy contents, total amino acid and fattyacid profiles of Octopus vulgaris and O. defilippi tissues(gonad, digestive gland and muscle) during sexual mat-

    uration (spermatogenesis and oogenesis) were investi-gated. Both species showed an increase of amino acidsand protein content in the gonad throughout sexualmaturation (namely in oogenesis), but allocation ofthese nitrogen compounds from the digestive gland andmuscle was not evident. The major essential amino acidsin the three tissues were leucine, lysine and arginine. Themajor non-essential amino acids were glutamic acid,aspartic acid and alanine. With respect to carbon com-pounds, a significant increasing trend (P

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    simultaneous terminal spawning (formerly known assemelparity), i.e. monocyclic spawning with synchro-nous ovulation, and egg-laying occurs in a very shortperiod at the end of the animals life (Rocha et al. 2001).Biological information in O. defilippi is scarce, and itsreproductive strategy is still unknown.

    Like in many marine invertebrates, sexual maturationand reproduction are the most energy-intense periods ofthe cephalopods life cycle. Somatic production exceedsgamete production during early life, but is later exceededby gamete production, which eventually dominates tis-sue growth (Rodhouse 1998). During the paralarval andjuvenile exponential growth phase there are high rates ofprotein synthesis, high efficiencies of retention of syn-thesised protein and, therefore, little protein degradation(Houlihan et al. 1990). Cephalopods have a vigorousprotein and amino acid metabolism, and, therefore, ahigh amino acid requirement exists to maintain optimalgrowth and to supply energy (Lee 1994). The direct useof protein as an energy reserve may account for the lackof major glycogen and lipid reserves in cephalopod tis-sues (Storey and Storey 1983; ODor et al. 1984). Nev-

    ertheless, it is worth noting that lipids in the digestivegland have been mentioned as a possible metabolicsubstrate and site for energy storage in cephalopods(ODor and Wells 1978; ODor and Webber 1986;Moltschaniwskyj and Semmens 2000).

    The production of gonads is fuelled by increased foodintake as well as mobilization of previously stored re-serves (Clarke et al. 1994). The rate at which these re-serves are used is an important factor in reproductivestrategies. The complete dedication of energy to repro-duction results in a terminal spawning event, whilepartial allocation, both before and during reproduction,will allow individuals to spawn repeatedly throughout

    their adult life (Calow 1979).The metabolic substrate used, its transfer between

    organs during sexual maturation, as well as yolk syn-thesis and sequestration have not been thoroughly de-scribed yet. In the present study, we have examined, indetail, the changes in the biochemical composition(protein, lipid, glycogen, cholesterol and energycontents, total amino acid and fatty acid profiles) ofO. vulgaris and O. defilippi tissues (gonad, digestivegland and muscle) during sexual maturation (sper-matogenesis and oogenesis).

    Materials and methods

    Samples

    Octopus vulgaris specimens were collected on the Por-tuguese west coast (Peniche) by commercial vessels(trawls, clay pots and traps) in February, March andMay 2002. Octopus defilippi specimens were collected on

    the south coast (Algarve), off the Instituto de Investi-gaca o das Pescas e do Mar (IPIMAR), during severalcruises aboard the R.V. Noruega and R.V. Capric-o rnio in February, April and June 2002. For eachanimal the following parameters were recorded: mantlelength (ML, mm), total weight (TW, g), gonad weight(GW, g), digestive gland weight (DgW, g) and catchingdepth range (m) (see Table 1). For both species, matu-rity stages were determined following Quetglas et al.(1998). Gonadosomatic index (GSI; gonad wet weight/body wet weight, 100) and digestive gland index (DgI;digestive gland weight/body wet weight, 100) were alsodetermined. All the tissues collected (gonad, digestive

    gland and muscle) were pooled after freeze-drying in aSavant VP100. The biochemical analyses were per-formed in triplicate in these tissues.

    Protein and amino acid analyses

    Protein concentration was determined (with 100 mg ofwet tissue) on the washed TCA (trichloroacetic acid)precipitate solubilised in 1 M NaOH (sodium hydrox-ide) for 24 h, as described by Lowry et al. (1951), usingthe Bio-Rad protein assay (BIO-RAD). Bovine gammaglobulin (BIO-RAD) was used as a standard.

    In order to determine the total amino acid profile,proteins were hydrolysed with 6 N hydrochloric acid(containing 0.1% phenol) in a MLS-1200 Mega Micro-wave System (Milestone), at 800 W and 160C for10 min. The hydrolysis was performed under inert andanaerobic conditions to prevent oxidative degradationof amino acids. The hydrolysates were filtered and dis-solved in sodium citrate buffer (pH 2.2). Amino acidswere separated by ion exchange liquid chromatographyin an automatic analyser (Biochrom 20; AmershamBiosciences), equipped with a column filled with a

    Table 1 Octopus vulgaris, O.defilippi. Number of individuals, mantle length (ML), total weight (TW), gonad weight (GW), digestive gland

    weight (DgW), gonadosomatic index (GSI), digestive gland index (DgI) and catching depth range (m)

    Species Gender Maturationstage

    n ML (mm) TW (g) GW (g) DgW (g) GSI DgI Catching depthrange (m)

    Octopusvulgaris

    Males Immature 9 123.3327.04 751.25189.94 4.571.42 23.4412.90 0.540.19 3.340.95 1070Mature 37 188.8922.56 2474.66806.27 18.425.84 60.9225.79 0.880.13 2.930.76 1070

    Females Immature 18 130.003.54 626.1596.60 1.090.12 19.203.95 0.180.03 3.191.13 1070Mature 13 215.0010.80 3051.26882.85 92.6245.72 124.3856.72 3.302.06 3.981.23 1070

    Octopusdefilippi

    Males Immature 7 91.257.50 154.5428.42 3.311.27 3.340.88 2.120.35 2.150.28 2545Mature 4 101.254.79 189.8135.05 5.201.39 4.661.12 2.720.43 4.591.07 4045

    Females Immature 15 88.0015.86 222.3162.27 4.342.12 9.934.07 1.130.55 3.301.12 1050Mature 10 106.0012.47 264.7586.36 9.800.64 11.211.87 2.201.63 3.710.95 2550

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    polysulphonated resin (2504.6 mm), using three so-dium citrate buffers (pH 3.20, 4.25 and 6.45; AmershamBiosciences) and three different temperatures (50C,58C and 95C). The detection of amino acids was doneat 440 and 570 nm after reaction with ninhydrin(Amersham Biosciences). Amino acids were identified bycomparison of their retention time with those of specificstandards (Sigma) and were quantified with the softwareEZChrom Chromatography Data System, version 6.7(Scientific Software), using norleucine (Sigma) as inter-nal standard.

    Total lipids and fatty acid analyses

    Total lipids were extracted by the Bligh and Dyer(1959) method. Determination of the fatty acid profilewas based on the experimental procedure of Lepageand Roy (1986), modified by Cohen et al. (1988). Thefatty acid methyl esters were analysed in a Varian 3400gas chromatograph, equipped with an auto-samplerand fitted with a flame ionisation detector. The sepa-ration was carried out with helium as carrier gas in afused silica capillary column Chrompack CPSil/88(50 m0.32 mm i.d.), programmed from 180C to200C a t 4C min1, held for 10 min at 200C andheated to 210C for 14.5 min, with a detector at250C. A split injector (100:1) at 250C was used.Fatty acid methyl esters were identified by comparisonof their retention time with those of chromatographicSigma standards. Peak areas were determined usingVarian software.

    Cholesterol analyses

    The quantification of cholesterol content was based onthe experimental procedure of Naemmi et al. (1995),modified by Oehlenschla ger (2000). Cholesterol wasanalysed in a Hewlett Packard 5890 gas chromatograph.The separation was carried out with helium as carriergas in a column HP5 (30 m0.5 mm i.d.). The temper-atures of the oven, injector and detector were 280C,285C and 300C, respectively. Cholesterol was identi-fied and quantified by comparison with standards (Sig-ma), from which a standard curve was prepared.

    Glycogen analysis and bioenergetic calculation

    Glycogen concentrations were determined according tothe method described by Viles and Silverman (1949).Tissue samples were boiled with 1 ml of 33% potassiumhydroxide for 15 min. After cooling, 50 ll of a saturatesodium sulphate solution and 2 ml of 96% ethanol wereadded. Samples were placed in an ice bath for precipi-tation ($30 min). Following centrifugation, the precip-itate was dissolved in 0.5 ml of distilled water, againprecipitated with 1 ml of ethanol and redissolved in0.4 ml of distilled water. Glycogen was then measuredby the anthrone-reagent method (72 ml of sulphuric acid

    concentrated was added to 28 ml of distilled water,0.05 g anthrone and 0.05 g of thiurea; the mixture washeated at 90C for 20 min) and the absorbance read at620 nm. A calibration curve was prepared with a gly-cogen (Sigma) standard.

    The energy content was estimated according toWinberg (1971) using factors of 12.6, 12.1 and 39.3 Jmg1 for protein, carbohydrate and lipid, respectively. Itis worth noting that the car