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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 carbohydrate fraction was sub-estimated, since only the glycogen content was quanti-fied.

Fig. 1 Octopus vulgaris, O. defilippi. Protein content (% dry wt) inthe gonad, digestive gland and muscle of males and females atdifferent stages of gonad development (Im. immature; Mat.mature). Means (SD) marked with different letters representsignificant differences (P


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Statistical analysis

Data were analysed using an ANOVA when comparingmultiple groups (k>3). Prior to analysis, normality andhomogeneity of variances were verified by KolmogorovSmirnov and Bartlett tests, respectively. Having dem-onstrated a significant difference somewhere among the

groups with ANOVA, we applied the Tukey test to findout where those differences were (Zar 1996).

Results

Biological data

The mean mantle length, total weight, gonadosomaticand digestive gland indices of Octopus vulgaris andO. defilippiare shown in Table 1. The ML, TW and GSIincreased during oogenesis and spermatogenesis of bothspecies. A similar trend was observed in DgI, with the

exception of O. vulgaris males.

Protein and amino acid content

The protein content in the gonad, digestive gland andmuscle of O. vulgaris and O. defilippi is presented inFig. 1. Both species showed an increase of these nitrogencompounds in the gonad throughout sexual maturation,being most significant in oogenesis (from 49.89% to63.37% dry wt in O. vulgaris and from 56.78% to

60.48% dry wt in O. defilippi) (F7,16=16.38, Tukey testP


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(namely oogenesis), and higher values were alwaysattained in males and females of O. defilippi(F7,16=31.77, Tukey test P


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(MUFA) content as 18:1 and 20:1 and polyunsaturated

fatty acid (PUFA) content as arachidonic acid (ARA;20:4n-6), eicosapentaenoic acid (EPA; 20:5n-3) anddocosahexaenoic acid (DHA; 22:6n-3). Among thedifferent tissues analysed, the highest lipid and FAlevels were obtained in the digestive gland, and thelowest were in the muscle. In the digestive gland, asignificant increase in the lipid and FA content wasobserved during spermatogenesis and oogenesis (Fig. 2;Table 6; lipids and FA: F7,16=24.61 and 30.26, Tukeytest P


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feeding grounds, and there was no evidence for theutilisation of either digestive gland or mantle tissues tosupply energy for the gonads.

The onset of the maturation process in O. vulgarisresults from increased secretion of a gonadotropin bythe optic glands (Wells and Wells 1959, 1972, 1975);activation of the glands inhibits protein synthesis and

enhances the breakdown of muscle proteins, flooding thebloodstream with free amino acids, which are funda-mental in forming yolk proteins (ODor and Wells1978). The yolk of the oocytes of O. vulgaris is rich inneutral glycoproteins, sulphidric and thiolic proteins andproteins rich in tyrosine and tryptophan residues (Bo-lognari et al. 1976). Moreover, Octopus gonadial growthis associated with a threefold increase in the concentra-tion of many amino acids and proteins in the urine(Wells and Clarke 1996). In the present study, bothoctopus species studied showed an increase in the gonad

amino acid and protein content throughout sexualmaturation (namely in oogenesis), but allocation ofthese nitrogen compounds from the muscle (proteincatabolism) was not evident.

With regards to carbon compounds, there was littleevidence of accumulated lipid storage products beingused for egg production. A significant increasing trend in

the lipid and FA contents in the gonad, digestive glandand muscle was observed. It seems that for egg pro-duction both Octopus species use energy directly fromfood, rather than from stored products. This directacquisition has also been proposed by other authors fortemperate (Guerra and Castro 1994; Collins et al. 1995)and tropical squid (Moltschaniwskyj and Semmens2000) and differs greatly from the previously describedmodel for O. vulgaris proposed by ODor and Wells(1978). Some lipid classes, e.g. phospholipids, play animportant role in the production of gametes (Pollero and

Table 5 Octopus vulgaris, O.defilippi. Fatty acid composition (lg mg1 dry wt) in the gonad of males and females at different stages ofgonad development.Different superscript letters within rows for each species represent significant differences (P


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Iribarne 1988), because they are probably involved inthe synthesis of the vitellus, which is a yolk phospho-lipoprotein (Fujii 1960). Octopus gonad was mainlyconstituted by PUFA and HUFA from the n-3 series,especially DHA and EPA. These fatty acids have beenidentified in the last few decades as essential nutrients formarine animals (Sargent et al. 1999). EPA and ARA are

important as structural components of cell membranesand as precursors of prostaglandins (Lilly and Bottino1981; Bell and Sargent 2003). DHA may be importantfor the correct development and survival of fast-grow-ing, phospholipid-rich cephalopod paralarvae (Navarroand Villanueva 2000, 2003). Therefore, the function oflipids in the Octopus life cycle must be the supply ofessential fatty acids and membrane components, such asphospholipids and cholesterol.

The digestive gland showed significantly higher lipid,FA and cholesterol levels than gonad and muscle. The

digestive gland is a site of digestive absorption andintracellular digestion (Boucher-Rodoni et al. 1987;Semmens 2002), and the differences between O. vulgarisand O. defilippi are probably related to different feedingecologies. In fact, a comparison of stomach content andlipid analysis confirmed that lipid from the digestivegland is very likely to be derived from the diet, with little

or no modification prior to deposition (Hayashi et al.1990; Phillips et al. 2001).

Voogt (1973) reported that cephalopods are able tosynthesise sterols, though molluscs generally only ap-pear to be able to carry out this biosynthesis slowly(Goad 1978). According to Kanazawa (2001), cepha-lopods seem to incorporate acetate and mevalonateinto sterols poorly and then require dietary sources ofsterol for growth and survival. Octopuses are activecarnivores, and, if their component sterols are of adietary origin, considerable variation in the cholesterol

Table 6 Octopus vulgaris, O.defilippi. Fatty acid composition (lg mg1 dry wt) in the digestive gland of males and females at differentstages of gonad development. Different superscript letters within rows for each species represent significant differences (P


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content of the digestive gland of O. vulgaris andO. defilippi might be expected on the basis of the sterolcomposition of their prey. In fact, Apsimon and Bur-nell (1980) and Ballantine et al. (1981) found anexogenous rather than endogenous origin for thecomponent sterols of Spirula spirula and Illex illece-brosus, respectively.

In the present study, the cholesterol content in thegonad, digestive gland and muscle of both O. vulgarisand O. defilippi exhibited variations that do not seem tobe correlated with the maturation process. However, it isknown that cholesterol is an important precursor ofsteroid hormones, moulting hormones, bile salts andvitamin D (Kanazawa 2001). In other invertebrates,namely crustaceans, it has been demonstrated thatexogenous cholesterol is converted into sex hormonessuch as progesterone, 17a-hydroxyprogesterone, andro-stenedione and testosterone (Kanazawa 2000). Signifi-

cant differences in the cholesterol content were obtainedbetween genders, suggesting that perhaps there is agreater physiological demand for cholesterol duringspermatogenesis than oogenesis.

In relation to carbohydrate metabolism, the presentknowledge is mainly confined to studies involving mus-cle work (see Storey and Storey 1983). Cephalopods

have the ability to utilise carbohydrate, lipid and proteinas metabolic substrates, with lipid and protein fuellingroutine aerobic metabolism and carbohydrate providingenergy for burst (anaerobic) activity (Wells and Clarke1996). During maturation, the glycogen reserves in-creased significantly in the gonad and decreased signifi-cantly in the digestive gland and muscle of O. vulgaris(these trends were not evident in O. defilippi). Therefore,as in other invertebrates, glycogen may be important forthe maturation process and embryogenesis. Carbohy-drates are precursors of metabolic intermediates in the

Table 7 Octopus vulgaris, O.defilippi. Fatty acid composition (lg mg1 dry wt) in the muscle of males and females at different stages ofgonad development.Different superscript letters within rows for each species represent significant differences (P


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production of energy and non-essential amino acids, and

as a component in ovarian pigments (Harrison 1990).It was evident that sexual maturation had a signifi-

cant effect upon the gonad energy content of O. vulgarisand O. defilippi. In fact, gonadal maturation is associ-ated with the increased biosynthetic work that supportsthe lecithotrophic strategy of para-larvae. The lack ofsignificant energy variation in the digestive gland andmuscle revealed that there was no evidence that storagereserves were transferred from one organ to another, asthe digestive gland, gonad and muscle constituents var-ied independently of one another. The biochemical

composition of the digestive gland and muscle may not

be influenced by sexual maturation, but rather by otherbiotic factors, such as feeding activity, food availability,spawning and brooding. Moreover, in experiments withSepia officinalis, Castro et al. (1992) showed that therapid decrease in DgI during starvation was related withprotein and lipid catabolism in the digestive gland. Atthe end of the reproductive cycle (spawning andbrooding), due to food deprivation in octopus females(only rarely feed while guarding the eggs), there is adecrease in the content of the major myofibrillar pro-teins (myosin, actin and paramyosin) (Rosa et al. 2002),

Fig. 3 Octopus vulgaris, O. defilippi. Cholesterol content (% drywt) in the gonad, digestive gland and muscle of males and femalesat different stages of gonad development (Im. immature; Mat.mature). Means (SD) marked with different letters representsignificant differences (P


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due to the enhancement of the protease activity in themuscle and also a decrease in digestive gland lipids (Tait

1986). Male octopuses also die at the same time as theirmates, because they too cease to feed during the last fewweeks of their lives (van Heukelem 1973). Protein andlipid depletion due to long periods of feed deprivation,which results in tissue depletion in muscle, has also beenobserved in other marine animals.

Acknowledgements The Portuguese Foundation for Science andTechnology (FCT) supported this study through a doctoral grantto the first author. Gratitude is due to P. da Conceicao for histechnical assistance and to Dr. J. Pereira for his help obtaining thespecimens.

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