Aquaculture 300 (2010) 194–205

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Effect of age-at-weaning on digestive capacity of white seabream (Diplodus sargus)

Inês Guerreiro a, Mahaut de Vareilles a, Pedro Pousão-Ferreira b, Vera Rodrigues a,Maria Teresa Dinis a, Laura Ribeiro a,b,⁎a Centro de Ciências do Mar, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugalb INRB I.P./IPIMAR, Av. 5 de Outubro s/n, 8700-305 Olhão, Portugal

⁎ Corresponding author. INRB I.P./IPIMAR, Av. 5 dePortugal. Tel.: +351 289715346; fax: +351 289715579

E-mail addresses: mribeiro@ualg.pt, lribeiro@cripsul

0044-8486/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.aquaculture.2009.11.019

a b s t r a c t

a r t i c l e i n f o

Article history:Received 31 March 2009Received in revised form 16 November 2009Accepted 19 November 2009

Keywords:Diplodus sargusDigestive enzymesWeaningOntogenyFish larvaeDigestive capacity

White seabream (Diplodus sargus) is today recognized as a potential species for Mediterranean aquaculture.Still, scarce information exists on weaning practices in order to reduce the live food period. This study aimsto evaluate the effect of an early weaning on fish larvae digestive enzymes activities. In order to accomplishthis, larvae were weaned with an inert diet at 20 days after hatching (DAH) (feeding regime W20) and acontrol group was weaned at 27 DAH (feeding regime W27). Before weaning, the onset and development ofthe main digestive enzymes were studied. The pattern of variation of digestive enzymes activities wereanalyzed at 0, 2, 9, 13 and 20 DAH, and from then on at 0, 1 and 3 weeks after diet introduction; that is, at 20,27 and 41 days for feeding regime W20, and days 27, 34 and 48 days for feeding regime W27.Trypsin, amylase, lipase, acid protease, alkaline and acid phosphatase, aminopeptidase and leucine–alaninepeptidase were analyzed on whole larvae until 20 DAH and on the abdominal cavity and purified brushborder during weaning trial period.Until 20 DAH, trypsin, alkaline phosphatase, leucine–alanine, aminopeptidase and acid phosphatase specificactivities increased significantly while acid protease, amylase and lipase kept relatively constant.Early-weaned larvae (W20) exhibited a lower growth compared with normally weaned larvae (W27) butthe pattern of Relative Growth Rate (RGR) variation was identical for both feeding regimes. Amylase andlipase activities increased swiftly after weaning in both groups as a result of an adaptation to the inert dietcomposition. During this period acid protease increased as a result of a functional stomach (pepsin activity)rather than inert diet introduction. Results obtained with alkaline phosphatase and aminopeptidasesuggested that an early weaning did not affect intestinal maturation. Moreover, results obtained with thebrush border could indicate that an early weaning had a positive role in intestinal maturation.Nonetheless, no significant differences were observed on the majority of fish larvae digestive enzymatic activitybetween treatments 3 weeks after inert diet introduction, and although larvae from W20 feeding regime weremore affected initially, theywere able to recover to similar levels of activity. This study suggests that an inert dietcan be included in the feeding regime of white seabream as early as 20 DAH (360 degree-days).

Outubro s/n, 8700-305 Olhão,..ipimar.pt (L. Ribeiro).

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© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Mediterranean aquaculture is mainly based on gilthead seabream(Sparus aurata) and sea bass (Dicentrarchus labrax) species, whoseoverproduction leads to reduced profits. Consequently, diversificationof aquaculture species and production systems has become a priorityfor European aquaculture (Basurco and Lovatelli, 2003). Whiteseabream (Diplodus sargus) meets several characteristics that makeit a species with potential for Mediterranean aquaculture, namely itshigh market value and demand and its easy adaptation to captivity(Abellan and Basurco, 1999).

The increase of marine aquaculture production in species otherthan D. labrax and S. aurata is hampered by a lack of knowledge ondigestive physiology and nutritional requirements during the larvalperiod, among others, which in turn seems to restrain juvenileproduction (Zambonino-Infante et al., 2008).

As food is the main source of energy and nutrients for larval growthand development, it is extremely important to adapt feeding protocolsto the fish larvae digestive and assimilation capacities. Moreover, thelarval period is a transition betweenhatching and juvenile stages,whereorganisms are submitted to intense and complex modifications of thedigestive system, amongother systems, and differences on the digestivecapacities during development are expected to occur.

Several studies have analyzed the ontogeny and digestive enzymeactivities in order to understand the larvae digestive process so as tosynchronize feeding protocols with larvae physiological stages and to

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define adequate age-specific nutritional protocols, thereby optimizinglarval production (see review Zambonino-Infante et al., 2008).

Changes in the diet are not the main reason for changes in theenzymatic activity during ontogeny, but they determine the plateaulevel of enzymes, as fish are able to modulate their enzymatic activityaccording to changes in the characteristics of feed (Cahu andZambonino-Infante, 1994).

Indeed, one of the main objectives of aquaculture is a completesubstitution of live feed by inert diets, although this was onlyaccomplished with D. labrax larvae (Zambonino-Infante et al., 2008).

Gisbert et al. (2009) sum up the main steps considered importantfor the maturation of the digestive functions of fish larvae. The firststep consists in acquiring the pancreas secretion function, the secondis the onset of the brush border membrane enzymes in the intestineand the third is the development of the stomach and onset of acidicdigestion, although this event is considered less relevant than theprevious two, since the pancreas and the intestine are also involved inprotein digestion.

Aspects such as lipid and fatty acid composition of broodstock,eggs and larvae (Cejas et al., 2003, 2004a,b;), nutritional studies(Saavedra et al., 2006, 2008;) and stock enhancement (D' Anna et al.,2004) have already been studied for D. sargus. Nonetheless, D. sargusfarming still exhibits several bottlenecks, such as the lack ofinformation on nutritional requirements, digestive capacities andweaning procedures. Thus, efforts must be made to contribute toincrease knowledge on D. sargus in order to develop adequate rearingprocedures. Cara et al. (2003) studying digestive enzymes ontogenyfor D. sargus, observed a peak of activity of the studied digestiveenzymes around 22 days after hatching (DAH). According to theseauthors, this pattern could indicate the onset of acidic digestion.Moreover, Saavedra et al. (2006) reported the ability of this species touse microdiets at 25 DAH in a co-feeding regime. The present studyaims to analyze the effect of age-at-weaning on the activities ofdigestive enzymes in D. sargus so as to contribute to a reduction of thelive food period and enhance the knowledge on D. sargus' digestivephysiology.

2. Materials and methods

2.1. Eggs and larvae rearing and sampling

Eggs were obtained from natural spawning of wild whiteseabream (Diplodus sargus) broodstock stocked in 10.6 m3 tankskept at the Aquaculture Research Center of the IPIMAR (Olhão,Portugal) under controlled temperature (18±1 °C) and naturalphotoperiod conditions. Eggs were incubated in 220 L incubators at18±1 °C temperature and 35‰ salinity. Newly hatched larvae werereared, according to standard rearing protocols for this species atAquaculture Research Center (Pousão-Ferreira et al., 2005), in 300 Lcylindrical round bottom tanks in a semi-closed circuit with an initialdensity of 80 individuals L−1 until 17 DAH. After that age fish larvaewere randomly divided in six tanks (300 L) and reared until 48 DAH.The rearing tanks were connected to a recirculation unit equippedwith mechanical, biological and UV-filters. Water parameters weremonitored daily, with temperature stabilized at 18.6±1.1 °C, salinityat 35±1‰ and dissolved oxygen above 90% of saturation.

Until 33 DAH (varied with the feeding regime) fish larvae were fedlive prey, namely from4until 26 DAHwithenriched rotifers (Brachionussp) (Protein Selco; Inve, Belgium) at a quantity ranging from 6×105 to6×106 individuals per tank. Microalgae (Nannochloropsis oculata andIsochrysis galbana, at a density of 300,000 cells L−1) were added to thelarval rearing tanks as longas rotiferswereusedas live prey. Between13to 19 DAH fish larvae were fed with Artemia sp AF (“Artemia franciscanagrade”) nauplii, from 4.5×102 to 75×103 nauplii per tank. At 16 DAH,Artemia sp EG (“Artemia sp enrichment grade”) metanauplii wasintroduced in the larval feeding regime at a quantity of 60×103 until

33 DAH. Artemia metanauplii were enriched with Rich Advanced®(Rich SA, Greece).

At 20 DAH an inert diet was introduced in a co-feeding regime for5 days with rotifers and Artemia EG - feeding regime W20; and at27 DAH an inert diet was introduced in a co-feeding regime for5 days with Artemia EG - feeding regime W27. After 5 days fishlarvae fromboth treatmentswere fed only on inert diet (Lucky Star®1 (100–200 micra, μm) and 2 (200–400 micra, μm), Hung Kuo,Taiwan) that was provided until satiation. Both feeding regimeswere followed in triplicate.

Fish larvae were sampled for dry weight and digestive enzymesanalysis at 0, 2, 9, 13 and 20 DAH. From this age on, for length andweight determination, fish larvae were sampled at days 27, 34,41 DAH for both treatments and at 48 DAH for treatment W27. Fordigestive enzymes analysis fish larvae were sampled at the introduc-tion of the inert diet and 1 and 3 weeks afterwards. Sampling wasalways done before feed distribution. Larvae for weight measurementwere used first for length measurement (only during the weaningtrial) and then rinsedwith 3% ammonium formiate and distilled waterprior to frozen in liquid nitrogen for dry weight analysis.

Weights were then used to calculate relative growth rate (RGR)using the formulae RGR=(eg–1)*100, g=[(ln final weight)–(ln initialweight)]/[(final day)–(initial day)] (Ricker, 1958).

Survival rate was determined based on information on the initialnumber and the final number of larvae in the tanks at 17 DAH. Duringthe weaning trial dead larvae were counted daily.

2.2. Preparation of extracts and analytical methods for enzymes

After being sampled larvae were rinsed with distilled water toremove salts, immediately frozen in liquid nitrogen, and stored at−80 °C until being assayed. Whole body homogenates were used forenzymatic analysis in larvae younger than 20 DAH. Older larvae weredissected on a glassmaintained at 0 °C, in order to obtain the abdominalcavity. The samples were homogenized in 15 volumes (w/v) of ice-colddistilled water, centrifuged at 3300×g at 4 °C for 3 min, and thesupernatant was sonicated for 10 s. For the purification of the brushborder membranes, abdominal cavity segments were homogenized in30 volumes (w/v) of ice-coldManitol (50 mM), Tris-HCl buffer (2 mM),pH 7.0. Purified brush border membranes from the abdominal cavitysegment homogenate were obtained according to a method developedfor fish larvae (Cahu and Zambonino-Infante, 1994).

Trypsin (E.C.3.4.21.4) activity was measured at 25 °C using BAPNA(Nα-Benzoyl-DL-arginine-p-nitroanilide) as substrate in trizma-CaCl2buffer (20 mM), pH 8.2 as described by Holm et al. (1988). Amylase(E.C.3.2.1.1) activity was assayed using starch as the substrate dissolvedin NaH2PO4 buffer (0.07 M), pH 7.4 (Métais and Bieth, 1968). Lipase(E.C.3.1.1) activity was measured using p-nitrophenyl myristate(0.53 mM) as a substrate dissolved in Tris-HCl buffer (0.25 M, pH 9.0),2-methoxyethanol (0.25 mM), and sodiumcholate (5 mM)asdescribedby Iijima et al. (1998). The reaction was stopped using a solution ofacetone and n-heptane (1:1), centrifuged at 6080×g at 4 °C for 2 min,before reading at 405 nm. Acid protease activitywasdetermined at pH2using bovine haemoglobin as substrate (Anson, 1938) dissolved in HCl(1 M). The reaction was stopped using TCA 5%. Alkaline phosphatase(E.C.3.1.3.1) activity was assayed using pNPP 5 mM(p-nitrophenylpho-sphate) as substrate in a solution of carbonate buffer (30 mM), pH 9.8(Bessey et al., 1946). Aminopeptidase N (E.C.3.4.11.2) activity wasdetermined, using L-leucinep-nitroanilide (0.1 M) as substrate in bufferphosphate (80 mM), pH 7.0 as described by Maroux et al. (1973). Acidphosphatase (E.C.3.1.3.2) activity was determined according to Terraet al. (1979) using pNPP 5.5 mM(p-nitrophenylphosphate) as substratemade in a solution of citrate buffer 0.1 M (citric acid and sodiumcitrate),pH 4.8. Leucine–alanine peptidase (E.C.3.4.11) activity was measuredusing leucine–alaninepeptidase (0.01 M) as substrate in LAOR(L-aminooxidase, horseradish peroxidase, o-dianisine) and was dissolved in a

Table 1Diplodus sargus growth during weaning. Inert diet was introduced at 20 DAH and27 DAH (week 0), respectively for feeding regimes W20 and W27 and maintained for3 weeks. Fish larvae were co-fed for 5 days. Values are presented as means±standarddeviation (n=30).

Treatments

W20 W27

Week 0Dry weight (mg) 0.4a±0.05 1.3b±0.5Total length (mm) 7.1a±0.6 9.8b±1.0RGR (% d−1) 12.8 12.7

Week 1Dry weight (mg) 1.2a±0.3 3.1b±1.2Total length (mm) 9.5a±0.8 12.2b±1.4RGR (% d−1) 7.3 8.5

Week 3Dry weight (mg) 4.1a±2.2 11.4b±5.4Total length (mm) 13.2a±1.8 17.1b±2.3RGR (% d−1) 11.2 11.0

Means with different superscripts indicate significant differences between means.

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Tris-HCl 50 mM buffer, pH 8.0 as described by Nicholson and Kim(1975).

Enzyme activities were calculated as micromoles of substratehydrolysed per minute (i.e., U) at 37 °C for alkaline phosphatase,aminopeptidase and leucine–alanine, and 25 °C for trypsin. Amylaseactivity was expressed as the equivalent enzyme activity which wasrequired to hydrolyse 1 mg of starch in 30 min at 37 °C. Pepsin activitywas expressed as specific activity with 1 U representing 1 mMequivalent of tyrosine liberated per minute per mg of protein at 37 °C.One unit of lipase activity was defined as 1 µmoL of p-nitrophenolreleased per minute at 30 °C. Protein was determined by the Bradfordmethod (Bradford, 1976). Enzyme activities were expressed as specificactivities, i.e., U/mg protein or mU/mg protein, and as segmentalactivities, i.e., total activity per larvae segment.

Enterocyte maturation index was calculated as the ratio betweenbrush border enzyme total activity (alkaline phosphatase and amino-peptidase) and leucine–alanine peptidase total activity, according toZambonino-Infante et al. (1997).

Since fish larvae received the compound diet at different develop-mental stages, it was important to calculate the relative increase indigestive enzymatic capacity after the beginning of weaning based onthe formula RI=final total activity (week 1 and 3)/initial total activity(week 0).

2.3. Statistical analysis

Data obtained in this study was presented as means±standarddeviation (SD). Homogeneity of variance was verified using Bartlett'stest. Data for length, dry weight and digestive enzyme activitiesduring larval period were compared using one-way ANOVA, followedby Tukey–Kramer Multiple comparison test when significant differ-ences were found at α=0.05 level. Student's t-test was used whencomparing data obtained during weaning trial (GraphPad InStat 3(GraphPad software, California, U.S.A.).

3. Results

3.1. Larval growth and survival

Until 20 DAH, two periods of growth were identified during D.sargus development (Fig. 1). Initially, a smooth increase until 9 DAHfollowed by a more pronounced growth at 13 DAH that coincidedwith the introduction of Artemia in the feeding regime.

At the beginning of weaning, fish larvae from feeding regime W20were almost 4 times lighter than fish larvae from feeding regimeW27(Table 1), and although this difference decreased by the end of theexperimental period (3 times), fish larvae from feeding regime W27were still heavier than fish larvae from feeding regimeW20 (Pb0.05).In order to compare dry weight at 48 DAH for both feeding regimes,dry weight of feeding regimeW20 fish larvae was calculated based on

Fig. 1. Growth as dry weight of Diplodus sargus during larval development. Verticalarrows indicate the moment of introduction of each type of live prey, and horizontallines, for how long they were given/fed. Values are presented as means±SD (n=30).

the growth curve equation. Thus, at 48 DAH fish larvae from feedingregime W20 would have weighed 9.5 mg, which is still a lower valuethan the 11.4 mg dry weight observed for fish larvae from feedingregime W27. Despite this difference, feeding regimes exhibitedsimilar values for RGR as well as a similar variation pattern. Moreover,diet introduction affected both feeding regimes equally since RGRdecreased after one week of weaning. Although fish larvae fromfeeding regime W20 were more affected than W27 fish larvae, theywere able to recover, and presented an RGR identical to the oneobserved at the beginning of weaning and higher than feeding regimeW27.

High mortalities were observed during the first 7–8 days. Thoughless intense afterwards, this resulted in a 6% survival rate at 17 DAH.During the weaning period, survival rates were identical betweentreatments (PN0.05), respectively 93.3% and 97.4% for feedingregimes W20 and W27.

3.2. Digestive enzymes activities during larval development

Trypsin specific activity (Fig. 2) exhibited a slight decrease until9 DAH (41.32 mU/mg protein), followed by a significant increase upto 99.53 mU/mg protein at 20 DAH. In fact, trypsin specific activityvalues doubled at 13 and 20 DAH.

Amylase specific activity (Fig. 2) exhibited a slight increase at2 DAH (2.36 U/mg protein), and decreased at 9 DAH (0.71 U/mgprotein) to a value that remained quite stable until 20 DAH.

Lipase activity (Fig. 2) was only detected at 9 DAH, with fish larvaeexhibiting 0.5 U/mg protein of activity, increasing significantly after-wards until 20 DAH (Pb0.05). Acid protease activity (Fig. 2) wasdetected at hatching (3.67×10−5 U/mg protein), where it showed itshighest level, followed by a decrease to negligible values at 2 DAH. Fromthis age onwards acid protease activity increased steadily until 20 DAH.Due to the high variability observed at hatching no significantdifferences were observed during this developmental period.

Alkaline phosphatase specific activity (Fig. 2) significantly in-creased up to 280.72 mU/mg protein at 20 DAH, and except for 9 and13 DAH, all other ages differed significantly from each other.

Aminopeptidase specific activity (Fig. 2) kept amore or less constantlevel of activity until 9 DAH, increasing afterwards to a value 2.25 timeshigher at 20 DAH (45.75 mU/mg protein). Fish larvae exhibited a ratherconstant level of acid phosphatase specific activity (Fig. 2) until 9 DAH(199.41 mU/mg protein), followed by a steep increase at 13 DAH(379.86 mU/mg protein) and 20 DAH (492.98 mU/mg protein)(Pb0.05).

Leucine–alanine specific activity (Fig. 2) was negligible at 2 DAH,at 9 DAH exhibited a value of 116.97 U/mg protein, followed by a

Fig. 2. Specific activities of digestive enzymes during Diplodus sargus larvae development. Enzymes were assayed in whole larvae. Results are expressed as means±SD (n=3).Different superscripts indicate significant differences between means.

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significant increased at 13 DAH to a level kept rather constant until20 DAH. The specific activity at 9 DAH was 2.2 times lower than at 13and 20 DAH.

Amylase, lipase, acid protease and alkaline phosphatase totalactivities (Fig. 3) increased with fish larvae development, (Pb0.05),whereas trypsin, aminopeptidase, acid phosphatase and leucine–alanine peptidase total activities increased significantly (Pb0.05) onlyafter 9 DAH.

3.3. Digestive enzymes activities at weaning

Trypsin specific activity (Fig. 4) of fish larvae from feeding regimeW20 decreased from 99.53 mU/mg protein at beginning of weaning (0)to 34.48 mU/mg protein at week 1 (after inert diet introduction),followed by a slight increase at week 3 (51.54 mU/mg protein). Incontrast, trypsin specific activity of larvae from feeding regime W27increased slightly at week 1 (53.98 mU/mg protein) to a levelmaintained until the end of week 3. Thus, before diet introductiontrypsin specific activity of fish larvae from feeding regimeW20 was 2.3

times higher than fish larvae from feeding regimeW27, whereas by theend of week 1 fish larvae from feeding regimeW20 exhibited 1.6 timesless trypsin activity then fish larvae from feeding regimeW27.

Amylase specific activity (Fig. 4) of fish larvae from feeding regimeW20 presented a slight decrease 1 week after diet introduction (0.44 U/mgprotein), followedby a strong increase by the endofweek 3 (2.02 U/mg protein). For fish larvae from feeding regimeW27, amylase specificactivity increased significantly during the experimental period (0.55 to1.55 U/mgprotein), presenting a value2.4 times higher thangroupW20by the end of week 1.

Lipase specific activity (Fig. 4) increased throughout the experi-mental period, reaching a value of 1.52 and 2.20 U/mg protein,respectively for fish larvae from feeding regime W20 and W27. Atweek 1 fish larvae from feeding regimeW27 exhibited 2.2 times moreactivity than fish larvae from feeding regime W20.

Fish larvae from feeding regime W20 exhibited a decrease of acidprotease specific activity (Fig. 4) by the end ofweek 1 (9.52×10−6 U/mgprotein), followed by a strong increase at week 3 (7.31×10−5 U/mgprotein), whereas fish larvae from feeding regime W27 exhibited a

Fig. 3. Total activities of digestive enzymes during Diplodus sargus larvae development. Enzymes were assayed in whole larvae. Results are expressed as means±SD (n=3).Different superscripts indicate significant differences between means.

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steady increase in acid protease specific activity during the experimentalperiod (9.43×10−5 U/mg protein). Before inert diet introduction fishlarvae from feeding regimeW20presented ahigher level of acid proteasespecific activity (Pb0.05) than larvae from feeding regime W27, thoughthis pattern was significantly reversed by the end of week 1.

Alkaline phosphatase and aminopeptidase activities of (Fig. 4) ofD. sargus fish larvae exhibited a similar pattern of activity after inert dietintroduction. The specific activities of larvae from feeding regimeW27, forboth enzymes, increased after week 1 (356.43 and 46.32 mU/mg proteinrespectively for alkaline phosphatase and aminopeptidase), followed by adecrease at week 3 (208.80 and 37.91 mU/mg protein, respectively, foralkaline phosphatase and aminopeptidase); whereas fish larvae fromfeeding regime W20 exhibited relatively constant values during theexperiment (mean value of 252.99 and 43.70 mU/mg protein, respec-tively, for alkaline phosphatase and aminopeptidase). Fish larvae fromboth feeding regimes exhibited significantly different values of alkalinephosphatase andaminopeptidase specific activities at thebeginningof thetrial, which were respectively, 1.6 and 1.2 times higher in larvae fromfeeding regime W20 than in larvae from feeding regime W27.

Leucine–alanine peptidase specific activity (Fig. 4) stayed rela-tively constant for both feeding regimes. Fish larvae from feedingregime W20 presented an activity within a range of 255.4–171.3 U/mg protein, corresponding to week 0 and 1 respectively and larvaefrom W27 between 198.1–153.0 U/mg protein, corresponding toweek 1 and 3 respectively.

Acid phosphatase specific activity (Fig. 4) was significantly higherfor feeding regime W20 fish larvae (492.98 mU/mg protein) whencompared with feeding regime W27 (219.72 mU/mg protein), at themoment of inert diet introduction. However, this difference disap-peared 1 week later and both feeding regimes exhibited similar valuesuntil the end of the experiment.

Alkaline phosphatase and aminopeptidase specific activities deter-mined on fish larvae brush border for both feeding regimes (W20 andW27) (Fig. 5), were rather stable at week 1 and 3. Fish larvae fromfeeding regime W20 exhibited aminopeptidase specific activity 1.4times higher than fish larvae from feeding regimeW27 only at week 3.

Values of total activities of all the enzymes studied increased afterinert diet introduction (Fig. 6). An exception was observed for fish

Fig. 4. Specific activities of digestive enzymes of Diplodus sargus after weaning. Inert diet was introduced at 20 DAH and 27 DAH (week 0), respectively, for feeding regime W20 andW27 andmaintained for 3 weeks. Fish larvae were co-fed for 5 days. Enzymes were assayed in the dissected abdominal cavity. Results are expressed as means±SD (n=3). Differentsuperscripts indicate significant differences between treatments means.

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larvae of feeding regime W20 where trypsin and acid phosphatasetotal activities maintained similar values at week 1 after weaning.

During the weaning trial, fish larvae from feeding regime W27exhibited higher levels of enzyme activities when compared with fishlarvae from treatment W20, except for amylase, acid protease andleucine–alanine peptidase at the beginning of weaning (week 0).

Since fish larvae from feeding regime W27 were bigger, highervalues of digestive enzymes total activitieswere expected. Therefore, forcomparative purposes with fish larvae from feeding regime W20, theincrease of digestive enzyme total activity in relation to the valueobtained at time 0 for each treatment was calculated, in order tominimize the influence of size. The results obtained (Table 2) coincidedwith the pattern observed for digestive enzymes total activities, where

fish larvae from feeding regime W27 exhibited a higher increase inactivity than fish larvae from feeding regime W20, after inert dietintroduction. Nonetheless, despite the greater increase in activitypresented by fish larvae from feeding regime W27, differences weremore pronounced between pancreatic enzymes than intestinalenzymes.

Alkaline phosphatase and aminopeptidase total activities deter-mined on the brush border (Fig. 7) increased significantly for bothfeeding regimes until the end of the experiment (Pb0.05), althoughfish larvae weaned at 27 DAH exhibited higher values of activitieswhen compared with early-weaned fish larvae (W20).

The enterocyte maturation index (Fig. 8) followed a pattern ofvariation similar to alkaline phosphatase and aminopeptidase activities.

Fig. 5. Specific activities of digestive enzymes of Diplodus sargus after weaning. Inert diet was introduced at 20 DAH and 27 DAH (week 0), respectively for feeding regime W20 andW27 and maintained for 3 weeks. Fish larvae were co-fed for 5 days. Enzymes were assayed in the brush border segment. Results are expressed as means±SD (n=3). Differentsuperscripts indicate significant differences between treatments means.

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Although fish larvae from feeding regimeW27 exhibited a higher indexvalue at week 1, by the end of the period studied (week 3), bothexhibited similar indexes of enterocyte maturation.

4. Discussion

4.1. Larval development and digestive enzymes activities

During the larval period, D. sargus growth was identical to thegrowth previously reported for this species (Saavedra et al., 2006). D.sargus larvae showed an exponential growth, typical of the larvalperiod, as observed for other species such as common pandora(Pagellus erythrinus), red porgy (Pagrus pagrus), blackspot seabream(Pagellus bogaraveo), sharpsnout seabream (Diplodus puntazzo), reddrum (Sciaenops ocellatus), sea bass (Dicentrarchus labrax) andcommon dentex (Dentex dentex) (Cahu and Zambonino-Infante,1994; Suzer et al., 2006; Lazo et al., 2007; Suzer et al., 2007a,b;Ribeiro et al., 2008; Gisbert et al., 2009). Cara et al. (2003) observedthat from 3 to 20 DAH the weight of D. sargus larvae increased 21times, whereas in this study at 20 DAH larvaeweight had increased byonly 16 times.

The survival rate observed in this study was low (6%), but near theminimum value of the range reported for this species (8–18%;Saavedra et al., 2006; Saavedra, 2008). Studies with other Sparidaespecies obtained survival rates between 21.2% and 21.7% (Suzer et al.,2006, 2007a). However Crespo et al. (2001) working with D. dentex,obtained survival rates ranging from 4.78% to 15.43% by day 17 andfrom 0.5% to 1.55% by 36 DAH. Low survival rates are often observedwhen studying new species, which indicate the need for research onlarval rearing requirements, on egg quality, broodstock condition, etc.In this study, the low survival rate was probablymore due to technical(mechanical/water flux) rather than biological factors.

The general pattern observed for digestive enzymes agrees withthe one seen for D. puntazzo, P. bogaraveo and D. sargus (Cara et al.,2003; Suzer et al., 2007a; Ribeiro et al., 2008).

As seen in other fish species, the majority of digestive enzymesstudied exhibited activity before first feeding (Ribeiro et al., 1999; Lazoet al., 2007; Gisbert et al., 2009), indicating that enzyme activity is undergenetic control and not induced by feed ingestion (Zambonino-Infanteet al., 2008).

Trypsin is a pancreatic enzyme specialized in degrading peptidesinto small sizes (Ronestad and Morais, 2008). In this study, thespecific activity of trypsin in white seabream increased until 20 DAH.This was similar with the pattern described for D. labrax, D. puntazzo,P. erythrinus, P. pagrus and D. dentex, whose trypsin activity peakedaround 20 DAH, (Cahu and Zambonino-Infante, 1994; Suzer et al.,2006, 2007a,b; Gisbert et al., 2009), but different for species like S.ocellatus and Senegalese sole, Solea senegalensis, in which trypsinactivity decreased with age, reaching the lowest values around20 DAH (Ribeiro et al., 1999; Lazo et al., 2007). These differencesseem to be related to the growth strategy of species and not just with

rearing temperature, as reported before (Ribeiro et al., 2008), since D.sargus was reared at temperatures identical to S. senegalensis.Moreover, differences in trypsin activity between species might berelated to the biology of each species and specific protein require-ments. Therefore a trypsin peak might indicate a turning point duringlarval growth since a strong correlation between trypsin activity,growth rate and food conversion efficiency was reported for Atlanticcod (Gadus morhua) (Lemieux et al., 1999). More recently, studieswith Coho salmon (Oncorhynchus kisutch) suggest that growthlimitation is not affected by digestive enzyme activities but may beassociated with the trypsin/chymotrypsin ratio (Blier et al., 2002).Differences in trypsin activity can also be explained by differentabiotical factors such as light, salinity and pH (Suzer et al., 2007a).Nonetheless, since different light conditions affect fish larvaeingestion rate (Rocha et al., 2008), the differences observed in trypsinactivity might also be a result of a different feed intake.

Amylase specific activity peaked at 2 DAH and then decreased,suggesting D. sargus' ability to use carbohydrates at mouth opening, asobserved for other marine fish species (Ribeiro et al., 1999, 2008; Lazoet al., 2007; Suzer et al., 2007a; Gisbert et al., 2009). Although thedecrease in amylase activity after hatching seems to be under geneticcontrol, amylase mRNA levels decreased independently of the dietaryglucide concentration (Zambonino-Infante et al., 2008), leading someauthors to hypothesize that it could be related to a low carbohydratecontent of live prey (Cara et al., 2003). Studies with rainbow trout(Oncorhynchus mykiss) support the theory that there is a nutritionalprogramming in amylase activity since the submission of O. mykiss toa short hyperglucidic stimulus at an early life stage influencedpermanently carbohydrate digestion (Geurden et al., 2007).

Lipase is one of the main lipolitic enzymes and is responsible forthe catalysis of carboxy-ester bonds of molecules like triacylglycerols,cholesterol esters or fat-soluble vitamin esters (Cahu and Zambonino-Infante, 2001). In fishes this enzyme is commonly known as bile-saltdependent lipase being located in pancreas. In this study, the decreaseof lipase specific activity after 13 DAH might be a consequence of dietchanges, since Artemia nauplii were introduced at this age in thefeeding plan of the fish larvae. Changes in enzymatic activity can be aresponse to changes in the composition of live feed or a result ofgrowth and development of tissues and organs (Martínez et al., 1999).A maximal lipase activity was reported for S. senegalensis at 10 DAH,probably related to the development of the exocrine pancreas(Martínez et al., 1999), which could explain the high activity detectedat 13 DAH in white seabream larvae.

The acid protease activity observed during the larval period of thisstudy was attributed to cathepsins activity, since an acidic mode ofdigestion was only described for this species between 25 and 30 DAHwhen the stomach was fully developed (Ortiz-Delgado et al., 2003).Cathepsins are normally associated to lysosomes that provide the acidicpH conditions for the efficient activity of these enzymes. Among otheractivities cathepsins are also involved in yolk degradation, whichmightexplain the high activity observed at hatching when larvae still

Fig. 6. Total activities of digestive enzymes of Diplodus sargus after weaning. Inert diet was introduced at 20 DAH and 27 DAH (week 0), respectively for feeding regime W20 andW27 andmaintained for 3 weeks. Fish larvae were co-fed for 5 days. Enzymes were assayed in the dissected abdominal cavity. Results are expressed as means±SD (n=3). Differentsuperscripts indicate significant differences between treatments means.

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exhibited some endogenous reserves. Carnevali et al. (2001) studyingsea bass reported the highest level of cathepsins activities at hatching,when comparing with other embryonic stages. Moreover, during larvalperiod, fish larvae undergo striking and complex tissue differentiation,which seems to involve tissuemobilization andmight justify the activityof cathepsines. Conceição et al. (1997) reported high protein turnoverduring turbot (Schopthalmus maximus) larvae development. Similarprocesses might occur with other species thus explaining the observedacid protease activities. High values of acid protease also observed for S.senegalensis before stomach development were attributed to proteinmobilization from body tissues by cathepsines (Fernández-Díaz et al.,2001). In fact, Lazo et al. (2007) remarked that lysosomal cathepsinesactivitymight dissimulate pepsin activitywhenusingwhole larval body.

Aminopeptidase and alkaline phosphatase are intestinal enzymesinvolved in the digestion of small peptides and assimilation of nutrients,respectively. Thus, the steady increase observed during larval period,probably indicates an improvement of the intestine digestive capacity

along larval development. Similar values were observed for D. puntazzo(Suzer et al., 2007a), which exhibited a steady increase in theseintestinal enzymes activities until 50 DAH. In contrast, for some speciesan earlier peak of activity was described for these enzymes, namely at6 DAH forD. dentex (Gisbert et al., 2009) and at 9 DAH forD. sargus (Caraet al., 2003; only alkaline phosphatase). Fluctuations of the specificactivity of digestive enzymes are normally observed during fish larvaedevelopment as a result of different metabolic and physiological events(Zambonino-Infante et al., 2008).

In the present study, acid phosphatase specific activity increasedthroughout larval development to reach its peak at 20 DAH, whereasCara et al. (2003) reported a higher variability on the level of thisenzyme activity along development, that peaked around 22 DAH. Theslight temporal difference might be related with different develop-mental rate and/ or larval performance of fish larvae from bothstudies. Nonetheless, the pattern of acid phosphatase activity for bothstudies might reflect the use of pynocitosis during the first 3 weeks of

Table 2Relative activity increase of total activities of digestive enzymes for Diplodus sarguslarvae after weaning. Inert diet was introduced at 20 DAH and 27 DAH (week 0),respectively for feeding regimes W20 andW27 and maintained for 3 weeks. Fish larvaewere co-fed for 5 days. Enzymes were assayed in the dissected abdominal cavity.Determination based on the formula RI=final total activity (week 1 and 3)/ initial totalactivity (week 0).

Treatments

Relative increase activity Weeks W20 W27

Total activity (mU/larva)Trypsin 1 0.7 2.0

3 3.1 7.3Amylase (U) 1 1.3 4.9

3 16.3 27.3Pepsin 1 1.2 8.2

3 26.0 82.0Lipase (U) 1 2.3 5.3

3 12.5 26.1Alkaline phosphatase 1 1.7 3.4

3 4.8 7.8Aminopeptidase 1 1.8 2.1

3 5.8 6.7Leucine–alanine (U) 1 1.1 1.6

3 3.4 5.4Acid phosphatase 1 0.8 1.7

3 2.6 6.2

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larval development, whereas the consequent decrease might be aresult of a higher intestinal activity indicating the beginning of theenterocyte maturation.

Other metabolic and physiological processes occurring duringlarval development might influence, directly or indirectly, the level ofactivity of digestive enzymes. For instances, alkaline and acidphosphatase, enzymes responsible for the hydrolysis of phosphateesters, beyond their important role on the digestive process, exhibitsimilar activities on other tissues like bone, respectively on bonemineralization and bone formation and resorption (Piattelli et al.,1997).

Leucine–alanine peptidase is a hydrolytic enzyme located inenterocyte cytoplasm, which activity is normally related withpynocitotic activity. In this study, leucine–alanine specific activitytended to increase during larval development reaching a plateauaround 20 DAH. A similar pattern was described by Cara et al. (2003)studying the same species. However, Gisbert et al. (2009) observed apeak of activity of leucine–alanine peptidase in D. Dentex between 6and 12 DAH followed by a decrease to a plateau level of activity,whereas for D. puntazzo the peak occurred at 6 DAH (Suzer et al.,2007a). Changes in larval metabolism and physiology might justifythis decrease, though Gisbert et al. (2009) suggested an earlymaturation of enterocyte, since the decrease in cytosolic activitymight indicate a lower dependence of larvae on pynocitotic activity.However, the dietary importance of this route for macromolecularabsorption has been questioned, since this route has normally been

Fig. 7. Total activities of digestive enzymes of Diplodus sargus after weaning. Inert diet wasW27 and maintained for 3 weeks. Fish larvae were co-fed for 5 days. Enzymes were assayesuperscripts indicate significant differences between treatments means.

related to antigen sampling (Ronestad and Morais, 2008). Furtherstudies are needed to provide insights on larval intestinal function andphysiology.

Six days without sampling at early life stages of development,represent a huge loss of information for different aspects related withlarval development, digestive physiology included. Since this studyaimed to focus on the effect of weaning rather than on the ontogeny ofdigestive enzymes, and pressured by using a unique spawn, samplingeffort was direct towards later stages of development. Consequently,in this study between 2 and 9 DAH the activity of digestive enzymesmight have had a pattern of variation more similar to the abovementioned studies that normally mentioned an early peak of activity,justifying the rather constant values of activity at early life stages.

Total activity of digestive enzymes increased with age for all theenzymes studied, indicating that larvae enhanced their digestivecapacity throughout development. This is a general pattern observedfor other marine fish species such as D. puntazzo, S. senegalensis,P. bogaraveo, S. occellatus (Ribeiro et al., 1999, 2008; Lazo et al., 2007;Suzer et al., 2007a).

4.2. Weaning at different stages of development

As expected fish larvae from the feeding regime W27 exhibitedhigher growth values by the end of the experimental period whencompared to feeding regimeW20.Nonetheless, the introduction of inertdiet at different developmental stages affected growth identically, sincethe pattern of RGR variation was identical for both feeding regimes.Moreover, the higher RGR value of feeding regime W20 after 3 weekshighlights the growth potential of these younger larvae, and based ondry weight estimation, values were not so different at 48 DAH for bothfeeding regimes.

In this study, fish larvae from both feeding regimes exhibitedhigher dry weight values at 48 DAH (observed — W27; estimated —

W20) than the ones reported by Cara et al. (2003) for the same species(about 8 mg), which were fed with microdiets at 30 DAH. Comparedwith fish of the same genera, D. sargus exhibited a specific growth rateof 4.2%d−1, which was lower than the 5.9%d−1 specific growth rateobtained for D. puntazzo (Suzer et al., 2007a,b), despite the fact thatprevious studies indicated D. sargus as a faster growing species(Pousão-Ferreira et al., 2005). However, in spite of the lower growthrate D. sargus were bigger than D. puntazzo at 50 DAH, respectively,11.4 mg dry weight, 17.1 mm total length and 8.0 mg dry weight (20%dry matter),14 mm total length.

During the weaning trial high survival rates were observed for bothfeeding regimes,withvalues above 90%. The selectivepressure observedduring larval period might justify the high survival values. In additionthe fast adaptation ofD. sargus to themicrodiet (5 day co-feeding)musthave contributed to the observed survival rates. Reduced adaptation tomicrodiet was identified by Suzer et al. (2006) as the cause for highmortalities for P. erythrinus at weaning.

introduced at 20 DAH and 27 DAH (week 0), respectively for feeding regime W20 andd in the brush border segment. Results are expressed as means±SD (n=3). Different

Fig. 8. Enterocyte maturation index (%) determined using the total activity of brush border enzymes and leucine–alanine peptidase, 1 and 3 weeks after weaning of Diplodus sarguslarvae. Inert diet was introduced at 20 DAH and 27 DAH (week 0), respectively for feeding regime W20 and W27 and maintained for 3 weeks. Fish larvae were co-fed for 5 days.Results are expressed as means±SD (n=3). Different superscripts indicate significant differences between treatments means.

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The early weaning affected fish larvae digestive capacity, since fishlarvae from theW20 feeding regime exhibited a decrease in the specificactivities of the digestive enzymes, whereas older larvae kept ratherconstant values, after diet introduction. However, when comparing thefeeding regimes it seems that in addition to the diet type, somebiological factors might have influenced the pattern of digestiveenzymes activities. Until 20 DAH fish larvae from both feeding regimes(W20 andW27)were fed the samediet and identical digestive enzymesactivities were expected at that age. Thus, when comparing the valuesobtained at 20 DAH for feeding regimeW20 and 27 DAH for fish larvaefrom feeding regime W27 (before diet introduction) a decrease in theactivity of digestive enzymes was also observed. A similar pattern ofactivity was described in other studies after 20 DAH for D. sargus (Caraet al., 2003) and P. pagrus (Suzer et al., 2007b), before inert dietintroduction. Therefore, inert diet introduction and probably biologicalfactors contributed to a temporary reduction on the digestive capacity offish larvae fed the feeding regimeW20. However, these larvaewere ableto recover reaching values identical to fish larvae from feeding regimeW27after threeweeks of inert diet. According to Suzer et al. (2007a) thedecrease in trypsin activity observed for D. puntazzo, was coincidentwith changes in feeding regime and brush border maturation, but forother sparids like P. erythrinus and P. pagrus (Suzer et al., 2006, 2007b)metamorphosis started around this age being a potential biologicaloccurrence.

In general, pancreatic enzymes and acid protease activities weremore affected than intestinal enzymes activities 1 week after inertdiet introduction, especially fish larvae from feeding regimeW20. Thisobservation suggested that at 20 DAH pancreatic mechanisms stillexhibited some difficulties in adapting to different molecular forms ofdiet ingredients.

Specific activity of pancreatic enzymes for larvae from treatmentW27 tended to increase until the end of the experiment except fortrypsin which stayed relatively constant.

Trypsin and pepsin are responsible for the extracellular digestionof proteins. During early life stages alkaline proteases, like trypsin, areresponsible for protein digestion. With the development of gastricglands and the acquisition of an acidic mode of digestion, pepsin startsto be secreted enhancing fish larval ability to digest different types ofproteins. In this study, the rather stable level of trypsin activity andthe increase in acid proteases activity at 27 DAH, indicated that pepsinprobably started to be secreted from this age onwards. A constantlevel of trypsin activity was related to the beginning of gastricdigestion, in P. bogaraveo (Ribeiro et al., 2008). In addition, accordingto Ortiz-Delgado et al. (2003) D. sargus gastric glands were abundantat 22–23 DAH, and a fully developed stomach was attained at 30 DAHlarvae, reinforcing the hypothesis that after 27 DAH the acid proteaseactivity might be attributed to pepsin. Moreover, for P. erythrinus andP. pagrus a decline in trypsin activity was observed after 20 DAH,whereas pepsin increased at 25 DAH peaking around 30 DAH,although gastric glands were observed at 27 DAH and a stomachwas completely developed after 30 DAH (Suzer et al., 2006, 2007b).

The identical pattern described in this study for D. sargus supports thehypothesis that pepsin started to be secreted around 27 DAH. Inaddition, pepsin activity provides more substrates (peptides) for theactivity of other enzymes, resulting in higher enzymatic activities(Cara et al., 2003), as observed for some of the enzymes analyzed inthis study.

The increase in amylase and lipase specific activity for fish larvaefrom both feeding regimes seems to reflect the inert diet introduction,that is, an adaptation to carbohydrates and lipids present in the diet,as was already described for other Sparidae species (Suzer et al., 2006,2007a; Ribeiro et al., 2008).

In this study alkaline phosphatase and aminopeptidase exhibited arather constant level of activity after weaning for both feedingregimes. A similar pattern has been previously described for D. sargusand D. dentex weaned at 30 DAH (Cara et al., 2003; Gisbert et al.,2009). These observations supports that the early introduction of inertdiet did not affected the pattern of activity, since both enzymesmaintained similar hydrolytic activities after weaning.

In fishes, an adult mode of intestinal digestion is achieved when thehydrolysis of dietary components is mainly performed by enzymeslocated in the brush border membranes of enterocyte (Cahu andZambonino-Infante, 2001). In this study, alkaline phosphatase andaminopeptidase activities on the brush bordermembranes followed thesame pattern of variation observed for the intestinal homogenate butwith higher magnitude, which confirms the adequate brush bordermembranes purification. Brush border enzymes maintained a similarlevel of activity after inert diet introduction, except for aminopeptidasein larvae of feeding regime W20 that significantly increased activity3 weeks after inert diet introduction, indicating that an early weaningdid not affect intestinal maturation.

Acid phosphatase decreased its activity between larval period andweaning trial, suggesting a relative decrease of the cytosolic digestion(pynocitosis). In fact, Ortiz-Delgado et al. (2003) observed acidophilicsupranuclear inclusions related to pynocitosis of proteins until 25 DAH.During the weaning period the pattern of activity of this enzyme wasrather stable for both treatments, contrasting with the pattern presentby Cara et al. (2003) where acid phosphatase increased after dietintroduction, probably caused by a higher activity of lysosomes.

Leucine–alanine peptidase, a peptidase located in the cytoplasm ofenterocytes, kept rather constant values of activity after inert dietintroduction. A similar pattern of activity was described for Atlantichalibut (Hippoglossus hippoglossus) andG. morhua (Kvåle et al., 2007), incontrast with the abrupt decrease observed for D. labrax andS. senegalensis between 20 and 30 DAH (Ribeiro et al., 1999; Cahu andZambonino-Infante, 2001). The normal maturation of the enterocytesinvolves a decline of cytosolic enzymes in developing larvae (Zambo-nino-Infante et al., 2008). Kvåle et al. (2007) studying H. hippoglossussuggested that the inexistence of a decrease in leucine–alanine peptidaseactivity could be due to the effect of other tissues present in the intestinalhomogenate that masked the changes in ontogenetic leucine–alaninedetection. Is it possible that a less pronounced decline in cytosolic

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enzymes activitymight be related to species specific digestive strategies?Is this cytosolic activity related with the role of the intestine in immunedefense as mentioned before? Further research is required to providemore insights on the functions of the intestine during larvaldevelopment.

As referred previously, the typical process of intestine maturationis characterized by a progressive increase in the specific activities ofthe brush border membrane enzymes (alkaline phosphatase, amino-peptidase) and a concomitant decrease in the activities of thecytosolic enzymes (leucine–alanine peptidase) (Cahu and Zambo-nino-Infante, 2001). This pattern of variation was observed forS. senegalensis and D. puntazzo (Ribeiro et al., 1999; Suzer et al.,2007a). A less clear pattern of variation was obtained in the presentstudy for D. sargus, but also for others species, such as P. bogaraveo, D.dentex where alkaline phosphatase activity remained rather constant(Ribeiro et al., 2008; Gisbert et al., 2009), considering that intestinalenzymes followed the same pattern of variation of brush borderenzymes although with 10 times less magnitude (Ribeiro et al., 2008)This different pattern of intestinal enzymes activities might resultfrom differences in intestinal maturation and/or different physiolog-ical responses as a consequence of inert diet introduction (Zambo-nino-Infante et al., 2008).

Still, the enterocyte maturation index obtained in this studyindicated that the introduction of inert diet earlier in the feedingregime of D. sargus did not hinder enterocyte maturation as observedfor P. bogareveo (Ribeiro et al., 2008). In fact, 3 weeks after inert dietintroduction, fish larvae from treatmentW20 kept a rather stable levelof enterocyte maturation whereas fish larvae from feeding regimeW27 exhibited a slight decrease, though not significantly.

Three weeks after the introduction of inert diet no significantdifferences were observed between an early or late introduction for D.sargus indicating that the early weaning did not affect fish larvae'sdigestive capacity. Furthermore, it indicated that D. sargus larvaewereable to modulate their digestive enzyme activity, as reported forD. labrax weaned at various times after hatching: 10, 15, 20 and25 days (Cahu and Zambonino-Infante, 1994).

Fish larvae from feeding regime W27 exhibited higher totalactivity values of digestive enzymes after the beginning of treatmentsfor all the enzymes analyzed, but this higher activity could be aconsequence of larval size rather than larval digestive ability. In fact,fish larvae from feeding regime W27 began to eat inert diet later, sothey were older and more developed than larvae from feeding regimeW20 throughout the experiment. The bigger size and consequently anincrease in enzyme activity per individual, were reflected in thehigher total activity as expected, since during development fish larvaeincrease their digestive capacity (Cahu and Zambonino-Infante,2001). Comparing the relative increase of enzyme activities afterinert diet introduction, the results supported the ones obtained withtotal activity. Results showed that total enzymatic activities fromlarvae of feeding regime W27 increase faster than in larvae fromfeeding regimeW20. Thus, total activity may reflect a higher digestiveenzymatic capacity in larvae from W27, but it cannot be forgottenthat, per mg of protein, larvae from both feeding regimes tended tohave the same digestive ability.

5. Conclusions

Enzymes and their level of activity determined in this experimentfor D. sargus followed a similar pattern of activity observed for otherSparidae species studied to date, and reflected the ability of thisspecies to digest food at early life stages. The pattern of digestiveenzymes activity was related to organogenesis and the type of foodused at different developmental stages. Data obtained for growth andenzymatic activity after the introduction of the inert diet suggestedthat D. sargus might be weaned from earlier stages, since they wereable to recover from this early introduction without affecting

digestive capacities. Further multidisciplinary studies are needed toreally understand the pattern of enzymatic activity on D. sargus and tooptimize larval feeding protocols in order to enhance digestivecapacity. The possibility and effects of introducing inert diet earlierthan 20 DAH in D. sargus feeding plan need further investigation.

References

Abellan, E., Basurco, B.E., 1999. Marine Finfish Diversification: Current Situation andProspects inMediterranean Aquaculture. OptionsMediterraneennes, No 24. CIHEAM,Zaragoza, Spain.

Anson, M.L., 1938. The estimation of pepsin, trypsin, papain, and cathepsin withhemoglobin. J. Gen. Physiol. 22, 79–89.

Basurco, B., Lovatelli, A., 2003. The aquaculture situation in the Mediterrenean Sea.Predictions for the future.

Bessey, O.A., Lowry, O.H., Brock, M.J., 1946. Rapid coloric method for determination ofalkaline phosphatase in five cubic millimetres of serum. J. Biol. Chem. 164, 321–329.

Blier, P.U., Lemieux, H., Devlin, R.H., 2002. Is the growth rate of fish set by digestiveenzymes or metabolic capacity of the tissues? Insight from transgenic CohoSalmon. Aquaculture 209, 379–384.

Bradford, M.M., 1976. A rapid sensitive method for the quantitation of microgramquantities of protein utilising the principle of protein–dye binding. Anal. Biochem.72, 248–254.

Cahu, C.L., Zambonino-Infante, J.L., 1994. Early weaning of sea bass (Dicentrarchuslabrax) larvae with a compound diet: effect on digestive enzymes. Comp. Biochem.Physiol. 109A, 213–222.

Cahu, C.L., Zambonino-Infante, J.L., 2001. Substitution of live food by formulated diets inmarine fish larvae. Aquaculture 200, 161–180.

Cara, J.B., Moyano, F.J., Cárdenas, S., Fernández-Dias, C., Yúferas, M., 2003. Assessment ofdigestive enzyme activities during larval development of white bream. J. Fish Biol.63, 48–58.

Carnevali, O., Mosconi, G., Cambi, A., Ridolfi, S., Zanuy, S., Polzonetti-Magni, A.M., 2001.Changes of lysosomal enzyme activities in sea bass Dicentrarchus labrax eggs anddeveloping embryos. Aquaculture 202, 249–256.

Cejas, J.R., Almansa, E., Villamandos, J.E., Badía, P., Bolaños, A., Lorenzo, A., 2003. Lipidand fatty acid composition of ovaries from wild fish and ovaries and eggs fromcaptive fish of white sea bream (Diplodus sargus). Aquaculture 216, 299–313.

Cejas, J.R., Almansa, E., Jérez, S., Bolaños, A., Samper, M., Lorenzo, A., 2004a. Lipid and fattyacid composition ofmuscle and liver fromwild and captivemature female broodstocksof white seabream, Diplodus sargus. Comp. Biochem. Physiol. B 138, 91–102.

Cejas, J.R., Almansa, E., Jérez, S., Bolaños, A., Felipe, B., Lorenzo, A., 2004b. Changes inlipid class and fatty acid composition during development in white seabream(Diplodus sargus) eggs and larvae. Comp. Biochem. Physiol. B 139, 209–216.

Conceição, L.E.C., van der Meeren, T., Verreth, J.A.J., Evjen, M.S., Houlihan, D.F., Fyhn, H.J.,1997. Amino acid metabolism and protein turnover in larval turbot (Scophthalmusmaximus) fed natural zooplankton or Artemia. Mar. Biol. 129, 255–265.

Crespo, S., Marín de Mateo, M., Santamaría, C.A., Sala, R., Grau, A., Pastor, E., 2001.Histopathological observations during larval rearing of common dentex Dentexdentex L. (Sparidae). Aquaculture 192, 121–132.

D' Anna, V.M.G., Giacalone, V.M., Badalamenti, F., Pipitone, C., 2004. Releasing ofhatchery-reared juveniles of the white seabream Diplodus sargus (L.1758) in theGulf of Castellammare Artificial Reef Area (NW Sicily). Aquaculture 233, 251–268.

Fernández-Díaz, C., Yúfera, M., Cañavate, J.P., Moyano, F.J., Alarcón, F.J., Díaz, M., 2001.Growth and physiological changes during metamorphosis of Senegal sole reared inthe laboratory. J. Fish Biol. 58, 1086–1097.

Geurden, I., Aramendi, M., Zambonino-Infante, J., Panserat, S., 2007. Early feeding ofcarnivorous rainbow trout (Oncorhynchus mykiss) with a hyperglucidic diet duringa short period: effect on dietary glucose utilization in juveniles. Am. J. Physiol.Regul. Integr. Comp. Physiol. 292, R2275–R2283.

Gisbert, E., Giménez, G., Fernández, I., Kotzamanis, Y., Estévez, A., 2009. Development ofdigestive enzymes in common dentex Dentex dentex during early ontogeny.Aquaculture 287, 381–387.

Holm, H., Hanssen, L.E., Krogdahl, A., Florholmen, J., 1988. High and low inhibitorsoybean meals affect human duodenal proteinase activity differently: in vivocomparison with bovine serum albumin. J. Nutr. 118, 515–520.

Iijima, N., Tanaka, S., Ota, Y., 1998. Purification and characterization of bile salt-activatedlipase from the hepatopancreas of red sea bream, Pagrus major. Fish Physiol. Biochem.18, 59–69.

Kvåle, A., Mangor-Jensen, A., Moren, M., Espe, M., Hamre, K., 2007. Development andcharacterisation of some intestinal enzymes in Atlantic cod (Gadus morhua L.) andAtlantic halibut (Hippoglossus hippoglossus L.) larvae. Aquaculture 264, 457–468.

Lazo, J.P., Mendoza, R., Holt, G.J., Aguilera, C., Arnold, C.R., 2007. Characterization ofdigestive enzymes during larval development of red drum (Sciaenops ocellatus).Aquaculture 256, 194–205.

Lemieux, H., Blier, P., Dutil, J.-D., 1999. Do digestive enzymes set a physiological limit ongrowth rate and food conversion efficiency in the Atlantic cod (Gadus morhua)?Fish Physiol. Biochem. 20, 293–303.

Maroux, S., Louvard, D., Baratti, J., 1973. The aminopeptidase fromhog-intestinal brushborder. Biochim. Biophys. Acta 321, 282–295.

Martínez, I., Moyano, F.J., Fernández-Díaz, C., Yúfera, M., 1999. Digestive enzyme activityduring larval development of the Senegal sole (Solea senegalensis). Fish Physiol.Biochem. 21, 317–323.

Métais, P., Bieth, J., 1968. Détermination de l'a-amylase par une microtechnique. Ann.Biol. Clin. 26, 133–142.

205I. Guerreiro et al. / Aquaculture 300 (2010) 194–205

Nicholson, J.A., Kim, Y.S., 1975. A one-step L-amino acid oxidase assay for intestinalpeptide hydrolase activity. Anal. Biochem. 63, 110–117.

Ortiz-Delgado, J.B., Darias, M.J., Cañavate, J.P., Yúfera, M., Sarasquete, C., 2003.Organogenesis of the digestive tract in the white seabream, Diplodus sargus.Histological and histochemical approaches. Histol. Histopathol. 18 (4), 1141–1154.

Piattelli, A., Piattelli, M., Scarano, A., 1997. Simultaneous demonstration of alkaline andacid phosphatase activity in bone, at bone-implant interfaces and at the epiphysealgrowth plate in plastic-embedded undemineralized tissues. Biomoterials 18,545–549.

Pousão-Ferreira, P., Gonçalves, C.C., Dores, E., 2005. Larval rearing of four sparidaespecies. European Aquaculture Society. Special Publication No. 36.

Ribeiro, L., Zambonino-Infante, J.L., Cahu, C., Dinis, M.T., 1999. Development of digestiveenzymes in larvae of Solea senegalensis, Kaup 1858. Aquaculture 179, 465–473.

Ribeiro, L., Couto, A., Olmedo, M., Álvarez-Blázquez, B., Linares, F., Valente, L.M.P., 2008.Digestive enzyme activity at different developmental stages of blackspot seabream,Pagellus bogaraveo (Brunnich 1768). Aquacult. Res. 39, 339–346.

Ricker, W.E., 1958. Handbook of Computations for Biological Statistics of FishPopulations. Queen's printer and controller of stationary, Ottawa.

Rocha, R.J., Ribeiro, L., Costa, R., Dinis, M.T., 2008. Does the presence of microalgaeinfluence fish larvae prey capture? Aquacult. Res. 39, 362–369.

Ronestad, I., Morais, S., 2008. Digestion. In: Finn, R.N., Kapoor, B.G. (Eds.), Fish LarvalPhysiology. Science Publishers, Inc, Enfield, USA, pp. 201–262.

Saavedra, M., 2008. Aminoacid requirements of white seabream (Diplodus sargus)larvae: effects on growth and performance. Dissertation for the degree of doctor atthe University of Algarve.

Saavedra, M., Conceição, L.E.C., Pousão-Ferreira, P., Dinis, M.T., 2006. Amino acid profilesof Diplodus sargus (L.1758) Larvae: implications for feed formulation. Aquaculture261, 587–593.

Saavedra, M., Conceição, L.E.C., Helland, S., Pousão-Ferreira, P., Dinis, M.T., 2008. Effectof lysine and tyrosine supplementation in the amino acid metabolism of Diplodussargus larvae fed rotifers. Aquaculture 284, 180–184.

Suzer, C., Fırat, K., Saka, Ş., 2006. Ontogenic development of the digestive enzymes incommon pandora, Pagellus erythrinus, L. larvae. Aquac. Res. 37, 1565–1571.

Suzer, C., Aktülün, S., Çoban, D., Kamacı, H.O., Saka, Ş., Fırat, K., Alpbaz, A., 2007a.Digestive enzyme activities in larvae of sharpsnout seabream (Diplodus puntazzo).Comp. Biochem. Physiol. A 148, 470–477.

Suzer, C., Kamaci, H.O., Çoban, D., Saka, Ş., Firat, K., Özkara, B., Özkara, A., 2007b.Digestive enzyme activity of the red porgy (Pagrus pagrus, L.) during larvaldevelopment under culture conditions. Aquac. Res. 38, 1778–1785.

Terra, W.R., Ferreira, C., De Bianchi, A.G., 1979. Distribution ofdigestive enzymes amongthe endo-and ectoperitrophic spaces and midgut cells of Rhynchosciara and itsphysiological significance. J. Insect Physiol. 25, 487–494.

Zambonino-Infante, J.L., Cahu, C.L., Péres, A., 1997. Partial substitutionofdi- and tripeptidesfor native proteins in sea bass diet improvesDicentrarchus labrax larval development.J. Nutr. 127, 608–614.

Zambonino-Infante, J.L., Gisbert, E., Sarasquete, C., Navarro, I., Gutiérrez, J., Cahu, C.L.,2008. Ontogeny and physiology of the digestive system of marine fish larvae. In:Cyrino, J.E.O., Bureau, D., Kapoor, B.G. (Eds.), Feeding and Digestive Functions ofFish. Science Publishers, Inc, Enfield, USA, pp. 277–344.