Fish Physiol Biochem (2008) 34:373384 DOI 10.

1007/s10695-007-9197-7

Development of digestive enzyme activity in larvae of spotted sand bass Paralabrax maculatofasciatus. 1. Biochemical analysis
C. A. Alvarez-Gonzalez F. J. Moyano-Lopez R. Civera-Cerecedo vez J. L. Ortiz-Galindo S. Dumas V. Carrasco-Cha

Received: 14 August 2007 / Accepted: 16 December 2007 / Published online: 7 February 2008 Springer Science+Business Media B.V. 2008

Abstract Spotted sand bass Paralabrax maculatofasciatus is a potential aquaculture species in Northwest Mexico. In the last few years it has been possible to close its life cycle and to develop larviculture technology at on pilot scale using live food, however survival values are low (11%) and improvements in growth and survival requires the study of the morpho-physiological development during the initial ontogeny. In this research digestive activity of several enzymes were evaluated in larvae, from hatching to 30 days after hatching (dah), and in live prey (rotifers and Artemia), by use of biochemical and
C. A. Alvarez-Gonzalez (&) DACBIOL Laboratorio de Acuacultura, Universidad Juarez Autonoma de Tabasco, Carretera Villahermosa Cardenas km 0.5, 86139 Villahermosa, Tabasco, Mexico e-mail: [email protected] F. J. Moyano-Lopez Departamento de Biologa Aplicada, Escuela Politecnica Superior, La Canada de San Urbano, Universidad de Almera, 04120 Almeria, Spain R. Civera-Cerecedo Laboratorio de Nutricion Acucola, Centro de Investigaciones Biologicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, 23090 La Paz, BCS, Mexico C. A. Alvarez-Gonzalez V. Carrasco-Chavez J. L. Ortiz-Galindo S. Dumas Unidad Piloto de Maricultivos, CICIMAR-IPN. Av. IPN s/n, Col. Playa Palo de Santa Rita, 23096 La Paz, BCS, Mexico

electrophoretic techniques. This paper, is the rst of two parts, and covers only the biochemical analysis. All digestive enzyme activities were detected from mouth opening; however the, maximum activities varied among different digestive enzymes. For alkaline protease and trypsin the maximum activities were detected from 12 to 18 dah. Acid protease activity was observed from day 12 onwards. The other digestive enzymes appear between days 4 and 18 after hatching, with marked uctuations. These activities indicate the beginning of the juvenile stage and the maturation of the digestive system, in agreement with changes that occur during morpho-physiological development and food changes from rotifers to Artemia. All enzymatic activities were detected in rotifers and Artemia, and their contribution to enhancement the digestion capacity of the larvae appears to be low, but cannot be minimised. We concluded that the enzymatic equipment of P. maculatofasciatus larvae is similar to that of other marine sh species, that it becomes complete between days 12 and 18 after hatching, and that it is totally efcient up to 25 dah. Keywords Amylase Lipase Ontogeny Paralabrax maculatofasciatus Phosphatases Proteases Spotted sand bass Introduction Rearing of marine sh larvae is based primarily on the use of live prey during a variable period lasting

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374 Table 1 Feeding schedule during rearing of spotted sand bass larvae Food item Microalgae Non-enriched rotifers (NER) Enriched rotifers (ER)a Non-enriched Artemia nauplii (AN)b Enriched Artemia meta-nauplii (EAMN)a, Artemia juveniles (AJ)
a b c b, c b

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Species Nannochloropsis oculata Brachionus plicatilis Artemia sp.

Quantity 300,000 cells ml-1 0.72 prey ml-1 210 prey ml-1 12 prey ml-1 212 prey ml-1 0.10.2 prey ml-1

Period (dah) 18 13 415 1215 1530 2030

The enrichment used in this experiment was Super HUFA enrich (Salt Creek, Salt Lake City, Utah, USA) The Artemia were obtained from Microest Artemia Cyst (Burn Philip Food, Oklahoma, USA) Fed with Chaetoceros calcitrans

from two to several weeks. In recent years, infrastructure, human effort, and energy costs of maintaining live food cultures in hatcheries have increased with the demand for a greater number of juveniles generated by the growing facilities. Moreover, the dependence of hatcheries on a few Artemia suppliers is becoming a bottleneck for the development of sh rearing. In addition, it is well known that traditional live foods (rotifers and Artemia) do not quite cover the nutrient requirements of larvae (Versichelle et al. 1989). The development of articial diets is considered an important alternative. However, the use of these feeds may be seriously limited by several factors, one of the most important being the ability of the larvae to efciently digest the principal nutrients. As a result of the difculty of performing digestibility studies with larvae, the most popular approach to evaluate their potential ability to digest food has been to study their digestive biochemistry. These studies have focused on quantifying the activity of the principal enzymes and assessing their variability throughout the development of the larvae. Several species have been studied, including turbot (Scophthalmus maximus), gilthead seabream (Sparus aurata), winter ounder (Pleuronectes americanus), Atlantic halibut (Hippoglossus hippoglossus), European sea bass (Dicentrarchus labrax), Senegal sole (Solea senegalensis), white seabream (Diplodus sargus), Japanese eel (Anguilla japonica), California halibut (Paralichthys californicus), and kingsh (Seriola lalandi) (Cousin et al. 1987; Moyano et al. 1996; Baglole et al. 1998; Martnez et al. 1999; Gawlicka et al. 2000; Zambonino-Infante and Cahu 2001; Ribeiro et al. 2002; Cara et al. 2003; Pedersen

et al. 2003; Alvarez-Gonzalez et al. 2005; Chen et al. 2006). The spotted sand bass (P. maculatofasciatus) is a serranid that appears to be suitable for aquaculture. It is adaptable to environmental changes, has low territoriality, and market prices are good. Reproduction of this species may be induced almost year-round under controlled laboratory conditions. The possibility of using articial feed for the larval stages has been tested by Aviles-Quevedo et al. (1995) and Anguas-Velez et al. (2000), who recorded low success with a survival below 2.5%. However, better survival of 11% was obtained by Alvarez-Gonzalez et al. (2001) in larval culture. The morphology of the digestive system during ontogeny has been described using live prey (Pena-Martnez et al. 2003). Considering this information, the purpose of this study was to evaluate the development of the activity of several digestive enzymes and the effect of digestive enzymes from live prey during the early ontogeny of this species by use of biochemical techniques.

Materials and methods Rearing and sampling of larvae Larvae were obtained from a spontaneous spawning of P. maculatofasciatus broodstock in the Laboratorio de Biologa Experimental, CICIMAR-IPN. One-dayold larvae were stocked at a density of 50 larvae l-1 in 12 glass ber tanks (140 l) connected to a recirculat ing system, as described by Alvarez-Gonzalez et al. (2001). The feeding schedule applied to the rearing of larvae is detailed in Table 1. The water parameters in

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the experiment included salinity: 35.8 1.1 ppt, dissolved oxygen: 6.4 0.3 mg l-1, and temperature: 23.1 1.1C. Individual dry weight of larvae (lg individual-1) was calculated by counting and from the weight of each pooled sample of freeze-dried larvae taken on each day, by use of an analytical balance (Sartorious, Gottingen, Germany; precision of 1 9 10-4 g). Thirteen samples of feed larvae were taken from different tanks, in triplicate (the numbers in parentheses are the numbers of larvae sampled per replicate) on day 0 (embryos, 1,000), 1 (860), 2 (440), 3 (420), 4 (380), 5 (290), 7 (230), 9 (230), 12 (90), 15 (60), 18 (50), 25 (40), and 30 (20) after hatching. Additionally, ve samples of starved larvae were obtained on day 0 (embryos, 950), 1 (830), 2 (370), 3 (440), and 4 (460) after hatching. All feed larvae were sampled at 7:00 am before feeding to avoid food contamination. Finally, four samples of live prey were takennon-enriched rotifers (NER), enriched rotifers (ER), enriched Artemia meta-nauplii (EAMN), and Artemia juveniles (AJ). Larvae were anesthetized with tricaine methanesulfonate (MS 222), and all samples were rinsed with distilled water, frozen at -50C, freeze-dried, and stored at 50C for later analysis.

Biochemical analyses Three pooled samples of fed and starved larvae taken on each day, and live prey (rotifers and Artemia) were homogenized (30 mg ml-1) in cold 50 mmol l-1 TrisHCl 20 mmol l-1 CaCl2 buffer, pH 7.5. Supernatant obtained after centrifugation (16,000g for 15 min at 5C) was stored at -20C to be used for enzyme analysis. The concentration of soluble protein in pooled samples was determined by the Bradford (1976) method using bovine serum albumin as a standard. Digestive enzyme activity was expressed as U mg protein-1; U larvae-1 or U prey-1 was estimated using the number of individuals in each pooled sample. Total alkaline protease activity was measured using casein (0.5%) in 50 mmol l-1 TrisHCl buffer, pH 9.0, following the method of Kunitz (1947), modied by Walter (1984). Acid protease activity was evaluated according to Anson (1938) using 0.5% hemoglobin in 0.1 mmol l-1 glycine

HCl, pH 2.0. One unit of enzyme activity was dened as 1 lg tyrosine released per minute. Chymotrypsin activity in extracts was determined using BTEE (N-benzoyl-L-tyrosine ethyl ester) according to Asgeirsson and Bjarnason (1991). One unit of enzyme activity was dened as 1 lmol BTEE hydrolyzed per minute. The amidase activity of alkaline proteases (trypsin amidase activity) was assayed using BAPNA (N-a-benzoyl-DL-arginine 4nitroanilide hydrochloride) as substrate according to Erlanger et al. (1961). One unit of enzyme activity was dened as 1 lmol p-nitroaniline released per minute. Leucine aminopeptidase was determined using leucine p-nitroanilide (0.1 mmol l-1 in DMSO) as substrate, according to Maraux (1973). One unit of enzyme activity was dened as 1 lg nitroanilide released per minute. Determination of a-amylase activity was carried out following the Somoyi-Nelson procedure described by Robyt and Whelan (1968). One unit of activity was dened as the amount of enzyme able to produce 1 lg maltose per minute. Lipase activity was quantied using b-naphthyl caprylate according to Versaw et al. (1989). One unit of activity was dened as 1 lg naphthol released per minute. Acid and alkaline phosphatases were assayed using 4-nitrophenyl phosphate in acid citrate buffer (pH 5.5) or glycineNaOH buffer (pH 10.1) according to Bergmeyer (1974). One unit was dened as 1 lg nitrophenyl released per minute. All assays were performed by triplicate at 37C.

Statistical analysis Growth of larvae was determined with an exponential model Y = aebX, with previous data transformation using logarithm to the base 10, and the parameters of the model were calculated by use of the least-squares technique. A MannWhitney U-test was used to compare digestive enzyme activity between starved larvae, fed larvae at 2, 3, and 4 dah, and live foods. A KruskalWallis test was used to compare the enzyme activity between fed larvae and live foods. A nonparametric Nemenyi test was used when signicant differences were detected. All tests were carried out with the software Statistica v7.0. (StatSoft, Tulsa, OK, USA).

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Results Growth during larviculture was observed to be exponential. The weights of the fed and the starved larvae were statistically different (P \ 0.05) at days 4 and 5 after hatching, with fed larvae larger than starved larvae (Fig. 1). Changes in the activity of the different proteases recorded throughout larval development are detailed in Figs. 2aj. Alkaline proteases and trypsin specic activity (Figs. 2a, e) showed similar patterns where two main peaks were detected, a small one on day 4 after hatching and the maximum peak between days 12 and 18 after hatching. Chymotrypsin specic activity increased rapidly from day 4 and continued to increase, with uctuation, until day 18 after hatching, when maximum activity was detected, then decreased gradually on days 25 and 30 after hatching (Fig. 2g). Acid protease specic activity (Fig. 2c) was detected at 12 dah, remained constant until 25 dah, and reached a maximum at 30 dah. Leucine aminopeptidase specic activity (Fig. 2i) had its maximum activity on day 12, and decreased rapidly from day 15 onwards. On the other hand, activity of all the individual alkaline proteases (including trypsin, chymotrypsin, and leucine aminopeptidase) increased exponentially from days 3 and 4 after hatching to a maximum value at 30 dah. Finally, individual acid protease activity showed a progressive increase, also reaching a maximum value at 30 dah (Fig. 2d). Lipase specic activity (Fig. 3a) reached a maximum (P \ 0.05) 7 dah, followed by a gradual decrease and then an increase to reach a second peak

Fig. 1 Mean dry weight (lg larva-1 std. dev., n = 3 pooled larvae) of spotted sand bass larvae

on day 30 after hatching. The specic amylase activity prole (Fig. 3c) showed two peaks, the rst on day 4 after hatching and the second peak (maximum) on 18 dah. The specic activity of the acid phosphatases was greater than that of the alkaline phosphatases during the rst day. The activity decreased for both phosphatases from 4 to 7 dah, whereas a sharp increase was detected at 12 dah when maximum activity was recorded for both digestive enzymes (Fig. 3e). Finally, the individual activity of lipase, amylase, and phosphatase were exponential (Figs. 3b, d, f), with statistically signicant maximum activity recorded 30 dah. Comparison of all enzymatic activity between starved and the fed larvae on days 2, 3, and 4 after hatching (Table 2) showed signicant differences (P \ 0.05). From day 2 onwards, specic lipase, amylase, trypsin, chymotrypsin, leucine aminopeptidase, and acid phosphatase activity was higher in the fed larvae. However, signicant differences were only detected on day 3 after hatching for the specic alkaline protease and alkaline phosphatase activity; again, activity was maximum in fed larvae. On the other hand, individual trypsin, chymotrypsin, and lipase showed signicant differences at day 2 after hatching, when fed larvae had the maximum values. For individual alkaline protease, leucine aminopeptidase, amylase, and alkaline phosphatase showed signicant differences at day 3 after hatching, when activity was highest for fed larvae. Finally, individual acid phosphatase activity did not show signicant differences. Digestive enzyme activity in live prey was the same as for the spotted sand bass larvae. Signicant differences were detected (Table 3) for alkaline protease, amylase, trypsin, and leucine aminopeptidase between different organisms as follows: Artemia juveniles (AJ) [ enriched rotifers (ER) [ nonenriched rotifers (NER) [ enriched Artemia metanauplii (EAMN). Acid protease and acid phosphatase activity were higher in ER and NER than in EAMN and AJ. A higher lipase activity was detected for AJ, followed by NER, ER, and, nally, EAMN, while chymotrypsin activity was higher in NER followed by AJ and then EAMN. Lower activity was detected for ER. Calculating the values of digestive enzyme activity per prey, the maximum statistically signicant value was obtained for Artemia juveniles (AJ) follow by enriched Artemia meta-nauplii (EAMN),

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Fish Physiol Biochem (2008) 34:373384 Fig. 2 Digestive enzyme activity during spotted sand bass larviculture (mean SD, n = 3 pooled larvae). (a) Specic alkaline protease activity, (b) individual alkaline protease activity, (c) specic acid protease activity, (d) individual acid protease activity, (e) specic trypsin activity, (f) individual trypsin activity, (g) specic chymotrypsin activity, (h) individual chymotrypsin activity, (i) specic leucine aminopeptidase activity, (j) individual leucine aminopeptidase activity. NER: non-enriched rotifers, ER: enriched rotifers, AN: Artemia nauplii, EAMN: enriched Artemia metanauplii, AJ: Artemia juvenile

377

non-enriched rotifers (NER), and enriched rotifers (ER). Using the data of rotifer and Artemia individual weight (Theilacker and Kimball 1984; Gara-Ortega et al. 1998) and consumption during the larviculture, we calculated the percentage of exogenous alkaline and acid proteases (4.3 and 2.3% from rotifers, and 1.7 and 3.2% from Artemia respectively), trypsin (1.3% from rotifers and 0.2% from Artemia), chymotrypsin (0.5% from rotifers and 0.2% from Artemia), leucine aminopeptidase (1.8% from rotifers and 1.2% from Artemia), acid and alkaline phosphatases (9.9 and 6.0% from rotifers, and 1.8 and 3.5%

from Artemia, respectively), lipase (5.3% from rotifers and 2.1% from Artemia), and amylase (9.7% from rotifers and 2.2% from Artemia).

Discussion Protease activity The presence of alkaline protease activity was detected from 2 dah onwards in P. maculatofasciatus, which is considered early compared with other

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378 Fig. 3 Digestive enzyme activity during spotted sand bass larviculture (mean SD., n = 3 pooled larvae). (a) Specic lipase activity, (b) individual lipase activity, (c) specic amylase activity, (d) individual amylase activity, (e) specic acid and alkaline phosphatase activity, (f) individual acid and alkaline phosphatase activity. NER: non-enriched rotifers, ER: enriched rotifers, AN: Artemia nauplii, EAMN: enriched Artemia meta-nauplii, AJ: Artemia juvenile

Fish Physiol Biochem (2008) 34:373384

species such as S. auratus and D. labrax (Zambonino-Infante and Cahu 1994; Moyano et al. 1996). Since the prole obtained for total alkaline protease activity coincides with that obtained for trypsin, it is possible that this enzyme is responsible for most of the total activity. Both trypsin and trypsinogen have been identied during the early stages of larval development in other species (Gawlicka et al. 2000; Cuvier-Peres and Kestemont 2002; Alvarez-Gonzalez et al. 2005; Kvale et al. 2007). In contrast with the sharp variations in the specic activity of trypsin observed during the rst 15 days of life, chymotrypsin activity was more constant from 3 dah onwards. In fact, this activity may represent the main alkaline protease secreted during the rst week after hatching, a result already reported for the Dover sole (Solea solea) (Clark et al. 1986). The variations observed in the specic activity of the alkaline proteases of P. maculatofasciatus during larval development have been reported for many other species including S. aurata, D. sargus, and S. lalandi (Cara et al. 2003; Chen et al. 2006). These

uctuations may be explained by the combination of three different factors: 1. Variable induction as a response to changes in the amount and composition of the live food provided. An increase in activity was detected in 12day-old larvae when Artemia nauplii were added. This increase may be explained by the different nutritional composition of rotifers and Artemia (Moyano et al. 1996; Morais et al. 2004). Changes in the amount of soluble protein in the larvae as a result of growth that may affect the expression of activity in relation to this fraction. As reported for D. sargus (Cara et al. 2003), the pronounced decrease in the activity observed from day 20 may be partly explained by this factor. Changing expression of different enzymes as a response to variations in larval metabolism, for example yolk resorption and stomach development. It has been suggested that alkaline proteases are responsible for protein digestion

2.

3.

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Table 2 Comparison of digestive enzyme activity between starved and fed larvae of spotted sand bass (from embryos until day 4 after hatching) (mean std. dev.) Yolk-sac larvae Starved larvae 1 2 Fed larvae Starved larvae 3 Fed larvae Starved larvae 4 Fed larvae

Days after hatching (dah) Embryo 0

Alkaline protease 1.1 0.3 12.7 3.7 0.8 0.5 0.0 0.0 143.2 10.6 1.1 0.1 1.3 0.5 0.0 0.0 0.0 0.0 0.1 0.1 0.0 0.0b 1.9 0.4b 2.5 0.4a 3.6 0.7b 10.0 0.8a 0.1 0.0a 0.6 0.1
b

U mg prot-1 2.1 0.3 17.8 2.2 0.4 0.1b 0.0 0.0 73.2 8.0b 0.7 0.1
a b

1.6 0.2 12.7 1.6 0.6 0.1a 0.1 0.0 123.0 18.7a 0.6 0.1
b a

2.0 0.4 14.9 1.9 0.4 0.1b 0.0 0.0 100.8 15.0b 2.3 0.1
a b b

2.4 0.3b 93.0 2.8 1.3 0.1a 0.1 0.0 324.9 12.0a
a a

14.1 0.4a 12.6 1.7 0.5 0.2b 0.0 0.0

2.9 0.4b

16.4 0.4a 129.5 2.9a 1.2 0.1a 0.1 0.0a 251.7 16.2a

mU larvae

-1

15.2 2.1

Trypsin

mU 9 10-3 mg prot-1

0.5 0.4

mU 9 10

-3

larvae

-1

0.0 0.0

Chymotrypsin 76.5 10.6b 0.4 0.1

Fish Physiol Biochem (2008) 34:373384

mU 9 10-3 mg prot-1

133.5 11.7

mU 9 10

-3

larvae

-1

1.3 0.1

2.7 0.2a 2.7 0.7b 0.0 0.0b 8.8 1.6a 0.1 0.0a

Leucine aminopeptidase

mU 9 10-3 mg prot-1

1.1 0.3

mU 9 10-3 larvae-1

0.0 0.0

Lipase U mg prot-1 25.9 0.4 45.0 11.4 0.5 0.1 5.9 0.4 0.1 0.0 16.2 1.2 0.2 0.0 0.1 0.0 14.7 1.2b 0.1 0.0 7.6 1.0 10.9 1.0 0.1 0.0 22.7 0.6a 0.1 0.0 1.3 0.1 1.3 0.2 155.1 6.5b 195.5 36.6a 34.5 4.4b 51.1 4.7a 48.7 0.5b 316.7 32.7b 1.9 0.2b 8.0 0.3b 0.0 0.0
b

1040.9 86.7 2182.7 33.7

4106.1 529.2b 7986.7 734.5a 7835.1 82.5b 11911.7 53.2a 4621.7 644.4b 15478.1 1437.8a 78.5 0.4a 970.7 35.8a 6.4 0.2a 19.1 1.8a 0.1 0.0 11.0 1.2b 0.1 0.0
a

U larvae

-1

9.9 0.8

20.0 2.8b 236.8 46.8b 1.0 0.2b 8.5 1.7b 0.0 0.0 13.9 0.6a 0.1 0.0 12.7 0.7 0.1 0.0
b

122.0 11.3a 882.0 82.6a 7.0 1.1a 17.6 1.7a 0.1 0.0a 12.6 0.6 0.1 0.0

Amylase

U mg prot-1

59.0 11.8

mU larvae-1

0.6 0.1

Alkaline phosphatase

U mg prot-1

3.5 0.2

mU larvae

-1

0.0 0.0

Acid phosphatase

U mg prot-1

22.7 1.1

mU larvae

-1

0.2 0.0

Different superscripts in columns show signicant differences (P \ 0.05) between starved and fed larvae on days 2, 3, and 4 after hatching

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Table 3 Digestive enzyme activity from live food used in spotted sand bass larviculture (mean std. dev.) Digestive activity Alkaline protease (U mg protein-1) (mU prey ) Acid protease (U mg protein-1) (mU prey ) Trypsin (mU 9 10-3 mg protein-1) (mU 9 10-6 prey-1) Chymotrypsin (mU 9 10-3 mg protein-1) (mU 9 10
-3 -1 -1

NER

ER

EAMN

AJ

3.1 0.3b 0.09 0.01 4.2 0.4a 0.12 0.01 1.2 0.2b 0.02 0.00c 265.1 33.1a 0.01 0.00 34.6 2.0b 0.6 0.0
c c d c

4.3 0.3b 0.07 0.01 4.7 0.8a 0.08 0.01 1.5 0.2b 0.02 0.00c 114.0 10.6d 0.01 0.00 35.7 2.1b 0.6 0.0
c c c c

2.3 0.3b 1.9 0.2

23.4 0.7a 137.2 4.2a 3.0 0.4b 17.5 2.1a 8.0 0.6a 77.6 5.8a 201.2 7.0b 2.0 0.1a 44.7 3.5a 436.5 34.3a 13397.2 675.6a 78.5 9.8a 1442.4 94.2a 8.5 0.6a 4.9 1.2c 28.5 6.7a 12.6 2.3a 73.6 13.3a

2.3 0.1bc 1.9 0.0

0.5 0.1c 0.3 0.0b 172.3 17.5c 0.09 0.01 7.1 2.1c 3.9 1.1
b b

prey )

-1

Leucine aminopeptidase (mU 9 10-3 mg protein-1) (mU 9 10 Lipase (U mg protein-1) (U prey ) Amylase (U mg protein-1) (U prey ) Acid phosphatase (mU mg protein-1) (mU 9 10
-3 -1 -1 -6

prey )

-1

6736.7 669.6b 0.20 0.02

5666.2 404.7b 0.10 0.01

420.3 33.1c 0.4 0.0

645.1 34.9b 0.02 0.00 13.0 0.3b 0.40 0.01 10.2 1.0a 0.30 0.03
c c c

774.1 47.0b 0.01 0.00 21.5 0.7a 0.40 0.01 11.3 1.3a 0.20 0.02
c c c

49.4 11.9c 0.4 0.1

1.7 0.6d 1.4 0.1

prey )

-1

Alkaline phosphatase (mU mg protein-1) (mU 9 10

4.8 0.6b 3.9 0.5

prey )

-1

NER: non-enriched rotifers, ER: enriched rotifers, EAMN: Enriched Artemia meta-nauplii, AJ: Artemia juvenile. Different superscripts in columns show signicant differences (P \ 0.05)

until the stomach is fully developed (Ribeiro et al. 2002). A decrease in the activity of the alkaline proteases may be related to an increase in the functionality of this organ (Cahu and Zam bonino-Infante 1994; Baglole et al. 1998; Kvale et al. 2007). Histological studies have shown that the stomach of P. maculatofasciatus larvae is fully functional earlier than in other species (Pena-Martnez et al. 2003). Pepsin activity was detected in this study as early as 10 dah. Activity increased and was very high by 25 dah. In the case of the enzymes involved in parietal digestion, a progressive increase should be expected together with the progressive maturation of enterocytes. Leucine aminopeptidase activity was initially

low but increased as a response to the presence of food. Similar results have been reported for other species such as S. maximus (Cousin et al. 1987) and D. labrax (Cahu and Zambonino-Infante 1997). The pattern of activity coincided with that of other brushborder enzymes which should indicate a maturation of enterocytes (Zambonino-Infante and Cahu 2001; Hakim et al. 2007), while the clear drop in all activity from 12 dah onwards may be related to the onset of stomach activity. True lipase activity Several studies have evaluated the activity of lipases during the larval stage of marine sh (Martnez et al.

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1999; Zambonino-Infante and Cahu 1999; Gawlicka et al. 2000; Hoehne-Reitan et al. 2001; Sidell and Hazel 2002). Lipase activity was detected in P. maculatofasciatus larvae from 0 to 7 dah, after which it decreased markedly. Early activity of lipases in embryos and yolk-sac larvae of some marine sh species has been reported by several authors (Ozkizilcik et al. 1996; Ribeiro et al. 2002). Initial high lipase activity followed by a marked decrease has also been reported. Martnez et al. (1999) recorded a maximum lipase activity 10 dah in S. senegalensis larvae and related it to the development of the pancreas, while Cousin et al. (1987) observed lipase activity only at 15 dah in S. maximus larvae. These authors explained that lipid catabolism during the rst day (before stomach formation) can only be carried out by esterases. When the digestive system is fully developed, lipid digestion is supported by true lipases. It is now widely accepted there are two types of bile salt-dependent lipase; the rst type should be activated during the embryonic period and helps in the digestion of yolk lipids (Diaz et al. 2002) whereas the second type should be directly secreted by the pancreas to digest lipids from food (Buchet et al. 2000). The use of only one type of substrate (a derivative of a short-chain fatty acid) in this study may have provided a limited picture of the evolution of lipid digestion, because the progressive appearance of pancreatic lipases may have been better evidenced by the use of more suitable substrates.

Phosphatase activity As previously mentioned, the importance of the parietal digestive enzymes (leucine aminopeptidase, and phosphatases) indicates maturation of the enterocytes. In this sense, phosphatases have two major functions. The rst is in the hydrolysis of inorganic phosphates to be used for energy production; the second is in the transportation of nutrients through membranes into cells (absorption process). When enterocytes reach their maximum capacity, phosphatase activity increases and is related to the decay of exopeptidase activity. Other enzymes such as phosphatase are also indirectly involved in digestion, facilitating the activity of other enzymes by modifying phosphate side-chains of amino acids, thereby promoting nutrient absorption by Ca2+ transport from lumen into cells (Copeland 1996). When the brushborder membrane enzymes, for example phosphatase, abruptly increase in activity during the course of the larval stage, this indicates the time the intestine matures and attains a more adult character with improved luminal digestive capacity (Ribeiro et al. 2002; Zambonino Infante and Cahu 2007). This increment was detected at day 12 after hatching for P. maculatofasciatus, which coincides with a decrease of aminopeptidase activity and the beginning of acid protease activity.

Digestive enzymes in starved larvae and live prey Amylase activity The specic activity of amylase in P. maculatofasciatus larvae showed an increase in yolk absorption from 2 dah until 18 dah, when a sharp decrease was noted. A high activity followed by a pronounced reduction has also been reported for other species (Zambonino-Infante and Cahu 1994; Moyano et al. 1996; Martnez et al. 1999; Buchet et al. 2000 Cuvier-Peres and Kestemont 2002; German et al. 2004). It has been suggested that early expression of amylase may be genetically determined (Peres et al. 1996) but is maintained if the composition of the diet makes it necessary (Munilla-Moran and SaboridoRey 1996). In the case of P. maculatofasciatus larvae, the absence of carbohydrates in the diet should result in a decrease in secretion. All enzymes were present in the starved larvae of P. maculatofasciatus, although activity was low compared with in fed larvae. The early appearance of digestive enzyme activity before the addition of food has also been detected in D. labrax larvae (Zambonino-Infante and Cahu 2001). Some studies have reported the participation of exogenous enzymes from live prey. Pedersen and Andersen (1992) studied C. harengus larvae fed different foods (copepods) and microspheres, and demonstrated that the pancreatic trypsinogen content, food size, and prey dimension are the most important factors in trypsinogen secretion, especially in stomach-less larvae. In this sense, the contribution of exogenous enzymes from the live prey could be signicant in improving the digestion of the larvae. For P. maculatofasciatus, the contribution in general is low for all digestive

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Fish Physiol Biochem (2008) 34:373384 (CONACyT) Project 31666B, by the Universidad de Almera, and by the Centro de Investigaciones Biologicas del Noroeste (CIBNOR). We also want to thank the Comision de Operacion y Fomento de Actividades Academicas (COFAA-IPN) and the Programa Institucional para la Formacion de Investigadores (PIFI-IPN).

enzymes (from 0.2% for Artemia trypsin to 9.7% for rotifer amylase). This is not in total agreement with results obtained by Gawlicka et al. (2000) who found that the exogenous contribution of amylase to H. hippoglossus larvae was of 50%, although participation in the activity of trypsin, lipase, and alkaline phosphatase from Artemia was only 10%. Additionally, Perez-Casanova et al. (2006) reported a contribution from live prey to haddock (Melanogrammus aeglenus) and Atlantic cod (Gadus morhua) of 13 and 16.4%, respectively, for proteases and 100% for amylase activity provided by rotifers. On the other hand, it has been reported that although the activity of enzymes increases signicantly after addition of live food, participation of the exogenous enzymes might be worthless (Moyano et al. 1996; Morais et al. 2004) such as observed in this research. In fact, larvae have their own genetically programmed enzymatic equipment, which is expressed in the rst day of life. Kurokawa et al. (1998) worked with Sardinops melanoticus larvae and reported that the contribution of proteases from rotifers to digesting capacity was approximately 0.6% of total protease activity. Thus, the contribution of exogenous enzymes is negligible for some species whereas for others it is vital. However, it seems to be necessary to increase the innate pancreatic enzyme secretion of the larvae in both cases. In addition, rotifers and Artemia present some auto-hydrolysis after being swallowed by larvae, resulting in their nutrients becoming available. This has been demonstrated for S. aurata and D. dentex larvae (Daz et al. 1997). It is concluded that P. maculatofasciatus larvae are precocious organisms with some digestive activity from the time of hatching and yolk absorption. Their digestive enzymatic equipment is complete on day 12 after hatching when the stomach becomes functional and all morpho-physiological changes are concluded (Pena-Martnez et al. 2003). By this time, the larvae have a monogastric digestive system characterized by the digestion of food in the stomach and nutrient absorption in the intestine. On the basis of these results, early weaning at day 12 after hatching could be possible.
Acknowledgements This research was supported by the institutional projects: CGPI 998022, 20010825 and 20020357, by the Consejo Nacional de Ciencia y Tecnologa, Mexico

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